Aging and Age Related Ocular Diseases

Aging and Age-Related Ocular Diseases
Guest Editor
E. Ltjen-Drecoll, Erlangen-Nrnberg
55 figures and 10 tables, 2000
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Vol. 214, No. 1, 2000
Contents
Preface
Ltjen-Drecoll, E. (Erlangen)
Alpha-B-Crystallin in Neuropathology
van Rijk, A.F.; Bloemendal, H. (Nijmegen)
13
Alpha-B-Crystallin Expression in Tissues Derived from Different Species in Different Age Groups
Oertel, M.F.; May, C.A. (Erlangen); Bloemendal, H. (Nijmegen); Ltjen-Drecoll, E. (Erlangen)
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Vascular and Glial Changes in the Retrolaminar Optic Nerve in Glaucomatous Monkey Eyes
Furuyoshi, N.; Furuyoshi, M.; May, C.A. (Erlangen); Hayreh, S.S. (Iowa City, Iowa); Alm, A. (Uppsala); Ltjen-Drecoll, E. (Erlangen)
33
Regulation of Trabecular Meshwork Contractility

Stumpff, F.; Wiederholt, M. (Berlin)
54
Full-Thickness Retinal Transplants: A Review

Ghosh, F.; Ehinger, B. (Lund)
70
Ultrastructure of Retinal Cells Transplanted to the Rabbit Choroid

Ltjen-Drecoll, E. (Erlangen); Bergstrm, A.; Ehinger, B. (Lund)
78
Oxidative Stress and Age-Related Cataract

Ottonello, S.; Foroni, C.; Carta, A.; Petrucco, S.; Maraini, G. (Parma)
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The Ageing Lens

Bron, A.J. (Oxford); Vrensen, G.F.J.M. (Amsterdam); Koretz, J. (Troy, N.Y.); Maraini, G. (Parma); Harding, J.J. (Oxford)
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Ophthalmologica 2000;214:5
Preface
The phenomenon of aging is characterized by various degenerative changes which differentially affect the highly specialized structures within the eye, such as the purely cellular lens, the brain-derived retina and the connective tissue of the uvea and sclera. The eye can therefore serve as an excellent model system to study age-related degenerative diseases. Glaucoma is a common age-related ocular disease. Studies in glaucomatous eyes have shown that there is a correlation between optic nerve fiber loss and changes in the outflow tissues, with the latter being presumably responsible for increased intraocular pressure (IOP). Another finding in glaucoma is that increased expression of the stress protein B-crystallin occurs in TM of glaucomatous eyes. B-crystallin is one of the proteins normally protecting lens fiber proteins from unfolding due to various stress factors. In this volume, an overview article deals with the molecular biology of this molecule, and original articles give further insights into the distribution and possible functional significance of nonlenticular Bcrystallin. A better understanding of the role of protective molecules in the eye might open new perspectives in the treatment of degenerative diseases. The commonest causes of blindness are the tapetoretinal degenerations and age-related macular degeneration.
Retinal transplantation studies have been undertaken that may ultimately lead to therapy for these diseases. New kinds of laminated transplants with promising survival times have been implanted and studied using different immunohistochemical methods. Further, an attempt has been made to investigate factors that influence the development of the retinal tissues, since less differentiated embryonic retina has the potential to be well integrated into degenerated retina. In the essentially cellular lens, it is difficult to define when development ends and ageing begins. An overview article examines cells of the deep lens fibers. These cells lose organelles and finally undergo denucleation, even though they remain viable for the life of the lens. The membranous channel system of the lens fibers plays a role in retaining transparency of the lens. In vitro, oxidative damage is one factor responsible for inducing changes in these lenticular ion channels. Protection against oxidative damage might therefore protect against cataract formation. The studies were kindly supported by the European Commission as part of the Biomed project BMH4-CT961593 (Brussels). We thank especially Mr. Kallasvaara for his help and his patience. E. Ltjen-Drecoll, Erlangen
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Prof. E. Ltjen-Drecoll Anatomisches Institut II Universittsstrasse 19 D91054 Erlangen (Germany) Tel. +49 9131 8522865, Fax +49 9131 8522862
A.F. van Rijk H. Bloemendal
Faculty of Science, Department of Biochemistry, University of Nijmegen, The Netherlands
Key Words B-Crystallin W Neurodegenerative diseases W Heat shock protein
Abstract B-Crystallin, which has homology with the small heat shock proteins, is the basic subunit of -crystallin, a major component of the vertebrate eye lens. These crystallins have for a long time been thought to be absolutely lens specific. However, about a decade ago B-crystallin has been detected extralenticularly in many tissues among which the central nervous system. Under pathological conditions the expression level of B-crystallin frequently increases. For this reason it is considered to be a useful marker in a variety of neurodegenerative diseases. In this mini-review, a number of typical neurodegenerative disorders is dealt with in which B-crystallin may play a role.
Copyright 2000 S. Karger AG, Basel
Introduction
B-Crystallin was discovered as the basic subunit of -crystallin, a major structural protein found in the eye lens of vertebrates [1, 2]. -Crystallin is an aggregate with a molecular mass of about 800 kD that is composed of two closely related subunits, A- and B-crystallin, both hav-
ing a molecular mass of approximately 20 kD. These subunits which share the ability to form protein aggregates with each other or on their own show a 57% sequence homology. For a long time it has been thought that crystallins were the prototype of organ-specific proteins. However, this idea became doubtful when these proteins were found occasionally outside the lens [35]. The real extralenticular detection of B-crystallin, the basic subunit of -crystallin, came when this protein was discovered in muscle, heart, brain and kidney [6]. Also A-crystallin, the acidic subunit of -crystallin, was detected extralenticularly in brain, spleen, thymus and retina, albeit to a far lesser extent [7]. In order to explain the occurrence of crystallins outside the lens, it is now assumed that during evolution normal metabolic proteins were recruited to function as structural proteins of the eye lens in order to maintain its transparency. Ingolia and Craig [8] showed for the first time that crystallin belongs to the large family of small heat shock proteins (sHSPs). Heat shock proteins (HSPs) are preferentially synthesized in organs exposed to heat or other types of physiological stress. These proteins are classified as several families: HSP100, HSP90, HSP70, HSP60, respectively, and the sHSP family that enjoy a wide phylogenetic distribution. The sHSPs, including B-crystallin, prevent protein unfolding or aggregation, a property called chaperone-like activity, while others are involved in refolding or proteo-
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lytic destruction. HSPs play an important role because they mediate many cellular processes. The chaperone property of -crystallin has been discovered by Horwitz [9]. In most if not all cells HSPs exist in low, sometimes extremely low, concentrations. As a rule the level of expression is enhanced following different kinds of stress. This holds true too for B-crystallin, which in contrast to A-crystallin is stress inducible. However, both proteins contain the so-called -crystallin domain that is shared by all members of the sHSP family [10] and that presumably is responsible for the chaperone-like activity. sHSPs as well as B-crystallin appeared to be involved in intracellular changes during disease progression [11, 12]. Besides, in disease progression, B-crystallin is also involved in reactive processes of astrocytes and oligodendrocytes in the central nervous system [13], development of astrocytic tumors [14] and in the development of benign tumors associated with tuberous sclerosis. B-Crystallin and HSP27 are expressed at elevated levels in the brains of patients suffering from Alexanders [15] and Alzheimers disease [16]. Therefore, B-crystallin is indeed a useful biochemical marker for studying the pathogenesis of various types of human neurodegenerative disorders and brain tumors [17]. Moreover, this protein is also a major component of ubiquitinated inclusion bodies in human degenerative diseases [18]. In this mini-review we shall restrict ourselves to the discussion of the expression of B-crystallin in some selected neurodegenerative diseases.
B-Crystallin in Patients with Alexanders Disease
Alexanders disease [19] is a rare sporadic nonfamilial leukodystrophy occurring in early childhood. There is no evidence of inherited enzyme deficiency or other genetic factors [2023]. Megalencephaly, psychomotor retardation, spastic paresis and epileptic seizures are the clinical symptoms. Also extensive proliferation of abnormal astrocytes, the formation of inclusions in astrocytes and Rosenthal fibers (RFs) are typical of this disease. They are predominantly found in perivascular, subependymal and subpial regions [2426]. The disease already manifests itself during the first months of life and progresses during the next 2 years, culminating in death. A definite diagnosis can only be assessed after death by brain biopsy. The disease is characterized by a diffuse leukodystrophy, which histopathologically can be seen as subcortical de-
myelination involving the brainstem, cerebellum and spinal cord together with the presence of RFs, osmiophilic dense abnormal inclusions in astrocytes. Recently it has been shown that RFs which are characteristic of Alexanders disease, are composed of glial acidic fibrillary protein (GFAP), B-crystallin (some of which is ubiquitinated) and the sHSP HSP27, which is closely related to B-crystallin [15, 27]. Since B-crystallin and HSP27 are stress proteins, they might be induced in response to stress caused by the disease per se. Indeed it is assumed that the formation of RFs is due to a chronic stress to a yet unknown stimulus [28]. The overproduction of B-crystallin in astrocytes as a response to stress may also be the cause of RF accumulation in some neurological disorders other than Alexanders disease. The interaction of Bcrystallin with other sHSPs, GFAP or ubiquitin may play an important role in RF accumulation. Ubiquitin is a highly conserved protein 76 amino acids long that is involved in the nonlysosomal degradation of proteins in eukaryotic cells by forming conjugates with abnormal proteins [29]. Since it has been shown by immunocytochemical analysis that the RF matrix reacts with antibodies to B-crystallin and GFAP and in addition with antibodies to ubiquitin it might well be that all those proteins together form the RFs [15]. Goldman and Corbin [30] provided evidence that RFs contain mono- and polyubiquitinated conjugates of Bcrystallin. The binding of the two proteins together could be shown by using both the antibodies to B-crystallin and ubiquitin on identical Western brain samples from diseased people. B-Crystallin contains a number of lysines that are potential substrates for isopeptide bond formation with ubiquitin. The finding of ubiquitin-B-crystallin conjugates in pathological inclusions raises a number of questions. One is whether or not B-crystallin normally becomes ubiquitinated, possibly as an intermediate in the turnover of the protein, or whether the conjugates in RFs are only seen as part of a pathological process. But if ubiquitin conjugation plays a role in the metabolism of B-crystallin, then it seems paradoxical that the conjugates accumulate. Several explanations are possible for the accumulation. Either B-crystallin might be altered by posttranslational modifications in a way that it becomes a poor substrate for proteolytic enzymes, or B-crystallin in RFs might be more stable than soluble B-crystallin. Another option is that the proteolytic system in astrocytes is changed. Although ubiquitin-B-crystallin complexes are formed, most of the B-crystallin in RFs is not ubiquitinated. This might be due to overloading of the ubiquitin conjugating system or to B-crystallin not being accessible
van Rijk/Bloemendal
for conjugation. By definition, RFs are abnormal inclusions accumulating in cells because they are not degraded efficiently. Ubiquitin has been implicated in the breakdown of proteins [31], but despite the ubiquitination of B-crystallin in RFs, the affected astrocytes still accumulate B-crystallin and its ubiquitinated conjugates [30].
Amyotrophic Lateral Sclerosis
Amyotrophic lateral sclerosis (ALS) is a human progressive neurodegenerative lethal disease with the onset in midlife. It is characterized by the degeneration of large motor neurons of the spinal cord, brainstem and motor cortex. Death of these neurons leads to weakness, atrophy and spasticity [3234]. ALS is one of the most devastating neurodegenerative disorders since it leads to death in less than 5 years. The etiology of ALS consistently shows an early axonal degeneration, increased remyelination and a preponderance of fibers with smaller diameters [35]. Motor neurons in the motor cortex, spinal cord and brainstem are targets, with death ensuing once respiratory functions are paralyzed. ALS occurs both sporadically and in familial forms, which are clinically and pathologically similar [36]. Mutations in the Cu/Zn-dependent superoxide dismutase (SOD-1) gene are associated with the latter type of the disease and are found in approximately 20% of patients with familial ALS [37, 38]. This means that 80% of familial ALS cases may be associated with a genetic locus yet to be identified [39]. SOD has been considered to be an excellent buffer against free concentrations of copper ions in the cytosol. Unbound copper ions have the potential to cause oxidative injury to tissues or to initiate an apoptotic process in motor neurons [40]. A defective copper binding site in SOD-1 could cancel or limit buffering capacity. For example, the H4R6 mutation in SOD-1 from ALS patients has been shown to impair the binding of copper to SOD [41]. ALS belongs to the group of neurodegenerative disorders which are characterized by the occurrence of socalled ballooned (swollen) neurons. A comparative study has recently been reported on the expression of B-crystallin, other stressresponse proteins and phosphorylated neurofilament protein. B-Crystallin was expressed in the ballooned neurons of Picks disease (a rare dementing disorder) and Creutzfeldt-Jakob disease, but not in those of ALS and other cases of neuropathology. In contrast, phosphorylated neurofilament protein was detected in most abnormal neurons [42]. Skein or skein-like inclusions in
motor neurons detected by ubiquitin immunohistochemistry are characteristic of ALS. A first report on these inclusions has been published recently. The results strongly support the notion that ALS is a multisystem disease and not simply a pathology of the motor neurons [43]. Eosinophilic fibrillary neuronal inclusions have been described in patients with sporadic ALS. Aberrant phosphorylation of neurofilament protein paralleled by induction of B-crystallin was shown to exist in ballooned neurons bearing eosinophilic fibrillary neuronal inclusions [44].
Alzheimers Disease
Alzheimers disease (AD) is the most common form of dementia and results in the loss of intellectual abilities in an age-dependent manner. Pathologically, AD is defined by extracellular cortical amyloid deposits called senile plaques and intraneuronal bundles of paired helical filaments referred to as neurofibrillary tangles [45]. The main proteins of the amyloid plaques are the so-called -amyloid peptides (Ab140 amino acids), proteolytic fragments of the -amyloid precursor protein (APP). Those peptides are normal products of the APP metabolism. However, in patients with familial forms of AD either the overall production of Ab peptides or the generation of elongated peptides (Ab142 and Ab143 amino acids) is increased [4649]. Expression of B-crystallin in normal human brain is found in oligodendroglia and subpial astrocytes [13, 16]. On the other hand in AD patients, B-crystallin expression was found to be increased. The protein was detected in reactive astrocytes, microglia and oligodendrocytes, indicating that all types of glia overexpress B-crystallin in response to stress associated with AD. However, in comparison to the large increase in the number of GFAPpositive astrocytes the increase in B-crystallin-expressing cells seems minor [50]. Nevertheless it may be concluded that under pathological conditions the expression of B-crystallin increases, suggesting that this protein together with GFAP may serve as a marker for gliosis in neurodegenerative diseases. B-Crystallin might also be a marker for neurons at the edge of areas of cerebral infarction, since it is found in cells regenerating upon damage. The presence of B-crystallin in those ballooned neurons and its close association with filaments suggests that Bcrystallin may be involved in aggregation and remodeling of neurofilaments in this particular disease. In families in which AD is inherited as an autosomal dominant trait,
four genes that are identified with the development of the disease have been characterized [51]. Mutations in three genes account for virtually all early-onset familial AD: APP on chromosome 21, presenilin 1 on chromosome 14 and presenilin 2 on chromosome 1. One allele of apolipoprotein E on chromosome 9 is associated with late-onset AD, in patients with and without a strong family history [52]. Another example for the connection of B-crystallin with AD stems from patients with Downs syndrome (DS). Those people over 40 years develop features of dementia of AD [53]. The idea behind it is not fully understood, but it is probably a genetic effect. The APP gene seems very important since people with DS have trisomy for the chromosome containing the APP gene. Disturbance of the organization of microtubules might underlie both AD and DS [54]. The neuropathological similarities and biochemical changes in people with DS and people suffering from AD suggest a common mechanism underlying the degenerative process in the two conditions [55, 56]. The expression of B-crystallin in people with DS appears to be related to the presence of dementia and AD neuropathological structures. Virtually no B-crystallin could be found in DS people without AD. As in people without DS, the B-crystallin in AD brains was found in reactive glia [57]. The significance of the expression of B-crystallin in AD brains is not yet known. The elevation of the protein as a response to stress might influence the progression of AD. It might protect other cells from stressful attacks or influence the toxicity of the Ab peptides [58]. Recently a scheme to screen out cases of AD, dementia with Lewy bodies, has been developed in order to discriminate between nonAD degenerative dementias and AD. Besides ubiquitin and protein, B-crystallin plays a key role in the diagnosis assessed by immunochemical detection of these marker proteins [59].
B-Crystallin in Other Neurodegenerative Diseases
The results of an ultrastructural and immunohistochemical study on ballooned cortical neurons in Creutzfeldt-Jakob disease suggested that the expression of phosphorylated neurofilament proteins and synaptophysin may reflect axonal impairment and that the presence of B-cystallin in addition to HSP27 and ubiquitin might be related to the degenerative processes that neurons undergo in Creutzfeldt-Jakob disease [60].
By immunochemical analysis ubiquitinated B-crystallin has been found in glial cytoplasmic inclusions obtained from the brains of patients with multiple system atrophy [61]. Multiple sclerosis (MS) is a major but poorly understood neurological disease of young adults in the Western world; in fact, it is a demyelinating disorder. In a review van Noort [62] discussed functional and therapeutic implications of his finding that B-crystallin is a single immunodominant myelin antigen to human T cells expressed at enhanced levels in MS-affected myelin [63]. Using immunohistochemical techniques, oligodendrocytes as well as astrocytes were shown to express B-crystallin in MS lesions. Different regulatory pathways for Bcrystallin have been suggested in either type of glia cells [64]. It seems that alterations of the normal myelin structure or normal oligodendrocytes might be the primary events that affect the susceptibility to MS [65]. Once triggered the immune system attacks and damages myelinforming cells. Oligodendrocytes respond to the attack of the immune cells and their secreted products, through modulation of their metabolism and gene expression. As part of this process, induction of HSPs, including B-crystallin, may take place in the oligodendrocytes as a kind of survival response. Corticobasal degeneration is another example of a neuropathological aberration. In this disease there is a marked neuronal loss and gliosis in the substantia nigra. Again the ballooned neurons are positively stained for phosphorylated neurofilament protein, synaptophysin and B-crystallin [66]. Ballooned neurons are also present in the cerebral cortex of these patients. It is concluded that the cytopathology of the subcortical gray matter and brainstem in corticobasal degeneration patients resembles that of progressive supranuclear palsy [67]. These swollen neurons which were positive for phosphorylated neurofilament protein and B-crystallin appeared to be abundant in 2 out of 6 patients. Cortical degeneration was observed in the precentral cortex. Ballooned neurons are a constant feature of amygdaloid nuclei in patients with argyrophilic grain disease [68]. Those ballooned neurons appeared to be strongly labeled with antibodies against B-crystallin and phosphorylated and neurofilament protein. In contrast nonballooned neurons that were immunoreactive with anti- remained consistently unstained with the crystallin antibody. The conclusion is drawn that two different pathological mechanisms may be operative in limbic neurons. As final example of a neurodegenerative disorder we want to mention Parkinsons disease that is the second
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van Rijk/Bloemendal
largest neurodegenerative disorder progressively destroying part of the brain which regulates coordinated motion. In Europe the number of patients suffering from this disease amounts to more than 1 million persons. With increasing numbers of aged people a substantial increase in Parkinsons disease has to be anticipated. Major symptons are resting tremor, rigidity, gait disturbance, postural instability, vegetative and psychiatric aberrations. A prominent feature of Parkinsons disease is a gradual loss of dopaminergic neurons in the midbrain. The role of Bcrystallin in the development and progression of this
pathology is far from being clear, and the amount of sound data in the literature is not overwhelming. It is reported that a familial parkinsonism is accompanied by the presence of ballooned neurons and B-crystallin [69]. A similar expression pattern of sHSPs as observed in AD has also been described for Parkinsons disease, a disorder without dementia [70]. At any rate the putative role of enhanced B-crystallin production in the cases discussed is still not much more than a matter of speculation.
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35 Heads T, Pollock M, Robertson A, Sutherland WH, Allpress S: Sensory nerve pathology in amyotrophic lateral sclerosis. Acta Neuropathol 1991;82:316320. 36 Horton WA, Eldridge R, Brody JA: Familial motor neuron disease: Evidence for at least three different types. Neurology 1976;26:460 465. 37 Deng HX, Hentati A, Tainer JA, Iqbal Z, Cayabyab A, Hung WY, Getzoff ED, Hu P, Herzfeldt B, Roos RP, Warner C, Deng G, Soriano E, Smyth C, Parge HE, Ahmed A, Roses AD, Hallewell RA, Perick-Vance MA, Siddique T: Amyotrophic lateral sclerosis and structural defects in Cu/Zn superoxide dismutase. Science 1993;261:10471051. 38 Roses Dr, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, Donaldson D, Goto J, ORegan JP, Deng HX, Rahmani Z, Krizus A, McKenna-Yasek D, Cayabyab A, Caston SM, Berger R, Tanzi RE, Halperin JJ, Herzfeldt B, Van Den Bergh R, Hung WY, Bird T, Deng G, Mulder DW, Smyth C, Laing NG, Soriano E, Pericak-Vance MA, Haines J, Rouleau GA, Gusella JS, Horvitz H, Brown RH Jr: Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 1993;362:5962. 39 Brown RH Jr: Superoxide dismutase and oxidative stress in amyotrophic lateral sclerosis; in Oxidative Stress and the Molecular Biology of Antioxidant Defenses. Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 1997, pp 569586. 40 Rabizadeh S, Gralla E, Borchelt D, Gwinn R, Valentine JS, Sisodia S, Wong P, Lee M, Hahn H, Bredesen DE: Mutations associated with amyotrophic lateral sclerosis convert superoxide dismutase from an antiapoptotic gene to a proapoptotic gene: Studies in yeast and neural cells. Proc Natl Acad Sci USA 1995;92:3024 3028. 41 Carri MT, Battistoni A, Polizio F, Desideri A, Rotilio G: Impaired copper binding by the H46R mutant of Cu/Zn superoxide dismutase involved in amyotrophic lateral sclerosis. FEBS Lett 1994;356:314316. 42 Kato S, Hirano A, Umahara T, Kato M, Herz F, Ohama E: Comparative histochemical study on the expression of B-crystallin, ubiquitin and stress response protein 27 in ballooned neurons in various disorders. Neuropathol Appl Neurobiol 1992;18:335340. 43 Kawashima T, Kikuchi H, Takita M, Dohura K, Ogomori K, Oda M, Iwaki T: Skein-like inclusions in the neostriatum from a case of amyotrophic lateral sclerosis with dementia. Acta Neuropathol 1998;117:541545. 44 Arima K, Ogawa M, Sunohara N, Nishio T, Shimomura Y, Hirai S, Eto K: Immunohistochemical and ultrastructural characterization of ubiquitinated eosinophilic fibrillary neuronal inclusions in sporadic amyotrophic lateral sclerosis. Acta Neuropathol Berl 1998;96:75 85.
45 Selkoe DJ: Cell biology of the beta-protein precursor and the mechanism of Alzheimers disease. Annu Rev Cell Biol 1994;10:373403. 46 Johnston JA, Cowburn RF, Norgren S, Wiehager B, Venizelos N, Winblad B, Vigo Pelfrey C, Schenk D, Lannfelt L, ONeill C: Increased -amyloid release and levels of amyloid precursor protein (APP) in fibroblast cell lines from family members with the Swedish Alzheimers disease APP670/671 mutation. FEBS Lett 1994;354:274278. 47 Scheuner D, Eckman C, Jensen M, Song X, Citron M, Suzuki N, Bird TD, Hardy J, Hutton M, Kukull W, Larson E, Levy-Lahad E, Viitanen M, Peskind E, Poorkaj P, Schellenberg G, Tanzi R, Wasco W, Lannfelt L, Selkoe D, Younkin S: Secreted amyloid beta-protein similar to that in the senile plaques of Alzheimers disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimers disease. Nat Med 1996;2:864 870. 48 Jarrett JT, Berger EP, Lansbury PT Jr: The carboxy terminus of the -amyloid protein is critical for the seeding of amyloid formation: Implications for the pathogenesis of Alzheimers disease. Biochemistry 1993;32:46934697. 49 Lansbury PT Jr: Consequences of the molecular mechanism of amyloid formation for the understanding of the pathogenesis of Alzheimers disease and the development of therapeutic strategies. Arzneimittel Forschung 1995;45: 432434. 50 Renkawek K, de Jong WW, Merck KB, Frenken CW, van Workum FP, Bosman GJCGM: B-Crystallin is present in reactive glia in Creutzfeldt-Jakob disease. Acta Neuropathol Berl 1992;83:324327. 51 Rubinsztein DC: The genetics of Alzheimers disease. Prog Neurobiol 1997;52:447454. 52 Stege GJJ, Bosman GJCGM: The Biochemistry of Alzheimers disease. Drugs Aging 1999; 14:437446. 53 Wisniewski KE, Wisniewski HM, Wen GY: Occurrence of neuropathological changes and dementia of Alzheimers disease in Downs syndrome. Ann Neurol 1985;17:278282. 54 Heston LL: Alzheimers disease, trisomy 21 and myeloproliferative disorders: Associations suggesting a genetic diathesis. Science 1977; 196:322323. 55 Ball MJ, Nuttall K: Neurofibrillary tangles, granulovacuolar degeneration, and neuron loss in Down syndrome: Quantitative comparison with Alzheimers dementia. Ann Neurol 1980; 7:462465. 56 Yates CM, Simpson J, Gordon A: Regional brain 5-hydroxytryptamine levels are reduced in senile Downs syndrome as in Alzheimers disease. Neurosci Lett 1986;65:189192.
57 Renkawek K, van Workum FAP, de Jong WW: Immunohistochemical expression of B-crystallin and heat shock protein 27 in the brain in Down syndrome. Dev Brain Dysfunct 1995;8: 3539. 58 Stege GJJ, Renkawek K, Overkamp PSG, Verschuure P, Van Rijk AF, Reijnen-Aalbers A, Boelens WC, Bosman GJCGM, de Jong WW: The molecular chaperone alpha B-crystallin enhances amyloid beta neurotoxicity. Biochem Biophys Res Comm 1999;262:152156. 59 Lowe J: Establishing a pathological diagnosis in degenerative dementias. Brain Pathol 1998;8: 403406. 60 Kato S, Hirano A, Umahara T, Llena JF, Herz F, Ohama E: Ultrastructural and immunohistochemical studies on ballooned cortical neurons in Creutzfeldt-Jakob disease: Expression of B-crystallin, ubiquitin and stress-response protein 27. Acta Neuropathol Berl 1992;84: 443448. 61 Tamaoka A, Mizusawa H, Mori H, Shoji S: Ubiquitinated B-crystallin in glial cytoplasmic inclusions from the brain of a patient with multiple system atrophy. J Neurol Sci 1995; 129:192198. 62 van Noort JM: Multiple sclerosis: An altered immune response or an altered stress response. J Mol Med 1996;74:285296. 63 van Noort JM, van Sechel AC, Bajramovic JJ, Elouagmiri M, Polman CH, Lassmann H, Ravid R: The small heat-shock protein B-crystallin as candidate autoantigen in multiple sclerosis. Nature 1995;375:798801. 64 Bajramovic JJ, Lassmann H, van Noort JM: Expression of B-crystallin in glia cells during lesional development in multiple sclerosis. J Neuroimmunol 1997;78:143151 65 Boccaccio GL, Steinman L: Multiple sclerosis From a myelin point of view. J Neurosci Res 1996;45:647654. 66 Matsumoto S, Udaka F, Kameyama M, Kusaka H, Ito H, Imai T: Subcortical neurofibrillary tangles, neuropil threads, and argentophilic glial inclusions in corticobasal degeneration. Clin Neuropathol 1996;15:209214. 67 Mori H, Oda M, Mizuno Y: Cortical ballooned neurons in progressive supranuclear palsy. Neurosci Lett 1996;209:109112. 68 Tolnay M, Probst A: Ballooned neurons expressing B-crystallin as a constant feature of the amygdala in argyrophilic grain disease. Neurosci Lett 1998;246:165168. 69 Mizutani T, Inose T, Nakajima S, Kakimi S, Uchigata M, Ikeda K, Gambetti P, Takasu T: Familial parkinsonism and dementia with ballooned neurons, argyrophilic neuronal inclusions, atypical neurofibrillary tangles, tau-negative astrocytic fibrillary tangles, and Lewy bodies. Acta Neuropathol Berl 1998;95:1527. 70 Renkawek K, Bosman GJCGM, de Jong WW: Expression of the small heat shock protein HSP27 in reactive gliosis in Alzheimer disease and other types of dementia. Acta Neuropathol 1994;87:511519.
12
van Rijk/Bloemendal
Alpha-B-Crystallin Expression in Tissues Derived from Different Species in Different Age Groups
Markus F. Oertel a Christian Albrecht May a Hans Bloemendal b Elke Ltjen-Drecoll a
a Department
of Anatomy II, Friedrich Alexander University, Erlangen, Germany; b Department of Biochemistry, University of Nijmegen, The Netherlands
Key Words Immunohistochemistry W Anterior eye segment W Optic nerve W Heart muscle W Dot blot W B-Crystallin
Abstract B-Crystallin is constitutively expressed in a variety of tissues including the nervous system, the eye, heart and striated muscles and the kidney. The functional significance of the protein in the different cell populations is not yet known. Experimental data indicate that mechanical stress to the cells might play a role but that there is also a close correlation with markers of oxidative activity. Increased expression of B-crystallin is seen in a number of age-related degenerative diseases. Whether aging per se induces expression of the protein has not been investigated yet. In this study tissue samples of the anterior eye segment, optic nerve, heart muscle and thyroid gland from mouse, rat, pig, cow and human donors of different age groups were investigated with immunohistochemical methods. B-Crystallin levels in heart muscle and optic nerve samples from different species and different age groups were investigated using protein immunoblotting (dot blot) and the mRNA levels using semiquantitative PCR methods. The results showed that neither in heart muscle known to show constitutively high
amounts of the protein nor in nonlenticular eye tissues with variations in staining intensity of different cell populations or in glandular cells studied for the first time, there were significant age-related staining differences. Dot blot methods as a quantitative evaluation method gave similar results. There were, however, species differences. In the eye these differences could be due to functional differences related to the development of a fovea centralis and an accommodative system in primates. In addition, in all mouse tissues there was less protein expression than in the other species. Differences in the absolute life span might be a factor involved in B-crystallin expression. In summary the findings show that an increase in B-crystallin with age may occur but is not a general phenomenon in tissues constitutively expressing this protein.
Introduction
Until recently it has been assumed that B-crystallin occurs exclusively in ocular lenses forming part of the structural -crystallins [1]. However, in the last decade the presence of the protein has also been demonstrated in a variety of nonlenticular cells in the body. Immuno- and
ABC
Table 1. Immunohistochemical demonstration of B-crystallin in normal nonlenticular tissues
Tissue Central nervous system glia Peripheral nervous system glia Heart muscle Skeletal muscle Ciliary muscle Vascular smooth muscle Myoepithelial cells Skin Kidney Lung Colon Placenta Thyroid Eye (not lens) Inner ear Olfactory bulb
Staining ++ +++ +++ +++ +++ + + ++ ++ + + ++ ++ ++ +++ ++
Species rat, dog, human rat, monkey, human mouse, rat, pig, cow, human mouse, rat, pig, cow, human monkey, human human human breast human mouse, rat, human mouse, rat human rat human rat, cow, monkey, human rat, monkey, human rat
References 8, 25, 33, 34, 39 25, 33, 34, 35 7, 8, 9, 14, 25, 36, 37, 38 8, 10, 14, 25, 33, 34, 36 26 34 40 34 14, 25, 27, 33, 34, 36 8, 28, 36 34 33 34 23, 26, 33, 41 35 25
+++ = All; ++ = some; + = some cells stain infrequently. The descriptions in the literature do not allow statements about species differences in distribution or intensity of B-crystallin staining.
Northern blotting have been used to identify the protein and its corresponding mRNA in heart and skeletal muscle, in Schwann cells of the peripheral nervous system, in skin, kidney, lung and inner ear (table 1). The functional significance of the protein in these different cell populations is not yet known. In vitro B-crystallin gene expression can be induced by heat, chemical and osmotic stress, consistent with the idea that B-crystallin is a functional heat shock protein [24]. Horwitz [5] was the first to demonstrate that Bcrystallin has chaperone-like properties protecting other proteins against heat aggregation and aid chemically denatured proteins to renature properly [for a review, see 6]. In vivo, B-crystallin was mainly found in desmin preparations from heart muscle. Immunohistochemical staining indicates that it might be involved in the organization of cytoskeletal filaments of the Z lines [7]. Previous studies have already revealed a high concentration of Bcrystallin in both fetal and adult hearts with similar amounts of the protein [8, 9]. This finding may be related to the early functional maturation of this tissue. In adult rats, passive tension increased B-crystallin expression in fast and slow skeletal muscles, whereas denervation increased expression of the protein in fast but decreased it in slow muscles [10]. These data indicate that mechanical stress to the cells might be one of the factors explaining the
constitutive presence of the stress protein. However, other factors may also play a role. In muscle cells and in some of the epithelial cells which constitutively express B-crystallin, Iwaki et al. [11] found a close correlation with markers of oxidative activity. During early development, in chicken B-crystallin one of the proteins is selectively present in cells undergoing cytomorphological reorganization [12]. Hence it might be involved in cellular morphogenesis [13]. On the other hand, increased expression of B-crystallin is also seen in age-related degenerative diseases. Rosenthal fibers of astrocytes of patients with Alexanders disease express B-crystallin [14, 15]. The protein has also been found in glial cells in a number of other neurodegenerative diseases [1619]. In fibroblasts, which normally do not express the protein, B-crystallin has been detected in patients with Werners syndrome, a disease characterized by inherited premature aging [20]. In senescence-accelerated (SAMP) mice, B-crystallin expression in the brain cortex and cerebellum was higher than in the control group [21]. Whether aging per se induces expression of the protein has not been investigated yet. We therefore studied the distribution of Bcrystallin in the anterior eye segment, in the retrolaminar optic nerve and the heart muscle in various age groups of rats and mice, as well as in some age groups of pigs, cows and primates. In addition, quantitative immunoblotting and semiquantitative PCR methods were used to study
14
Oertel/May/Bloemendal/Ltjen-Drecoll
Table 2. Number of tissues in different
age groups investigated by immunohistochemistry
Animal
Age
Anterior eye segment
Optic nerve 1 3 2 1
Heart muscle 1 1 2 1 2 2 1 3 3 2 2 2 2 1 3 2
Glandular tissue
Mouse 4 weeks Mouse 3 months Mouse 12 months Mouse 19 months Rat 9 days Rat 6 weeks Rat 3 months Rat 7 months Rat 14 months Rat 23 months Pig 5 months Pig 6 years Cow 7 months Cow 6 years Human 1940 years Human 4060 years Human 6095 years
3 2
2 1 2 1 4 2 1
1 5 2
3 4 1 3 1 3 1 2 4 4
5 5 3 7 12
2 1
the concentration and mRNA levels of B-crystallin in the retrolaminar optic nerve and heart muscle in different age groups of the same species.
antibody was added (dilution 1:800; Dianova, Hamburg, Germany) for 1 h. The slides were rinsed again in TBS and mounted with Kaisers glycerin gelatine. An Aristoplan fluorescence microscope (Leitz, Wetzlar, Germany) was used to examine the staining reaction. Immunoelectron Microscopy Paraformaldehyde-perfused small heart muscle tissue samples of 3 Wistar rats were immersion fixed in a mixture of 4% paraformaldehyde and 30% dimethylformamide (DMF) for 1 h on ice. Cryoprotection was performed by washing in 30% DMF twice for 30 min on ice. The samples were then incubated in methylbutane for 30 min and rapidly frozen in liquid nitrogen using an EM-CPC workstation (Leica). Low-temperature embedding was performed using Lowicryl HM20 according to the instruction manual of the manufacturer (Polysciences Europe, Eppelheim, Germany). After UV polymerization, ultrathin sections were mounted on Ni-grids, air dried and preincubated with 2% bovine serum albumin in phosphate-buffered saline (PBS, pH 7.4) for 20 min. Incubation with the primary antibody (rabbit anti-B-crystallin, 1:100) followed for 60 min. The grids were then washed and labeled using goat antirabbit IgG and 10-nm gold particles. Uranyl-stained sections were viewed with a Zeiss EM 902 (Zeiss, Oberkochen, Germany). Protein Immunoblotting (Dot Blot) Frozen unfixed heart muscle and retrolaminar optic nerve tissue samples were homogenized with 2 ! sample buffer (20% glycerol, 6% sodium dodecyl sulfate in 0.12 M Tris, pH 6.8) and five times freeze-and-thaw lysed, using liquid nitrogen and a 37 C waterbath, respectively. Following spinning at 13,000 g for 10 min, the protein content of the supernatant was measured using BCA protein assay reagent (Pierce, Rockford, Ill., USA). The samples were diluted with 2 ! sample buffer to equalize protein concentration and further diluted with 20% methanol to a working dilution of 10 g protein/ 100 l. A standard of purified B-crystallin was prepared with bovine serum albumin to reach the same protein concentration as the tissue samples. Methanol-presoaked nitrocellulose transfer mem-
Material and Methods

Tissue samples of the anterior eye segment, retrolaminar optic nerve, heart muscle and thyroid gland were collected from mouse, rat, pig, cow and human donors. From our own breedings, C57/ Black6 mice (3 weeks to 23 months of age), SAMP accelerated-aging mice (1112 months of age), Wistar rats (3 weeks to 12 months of age) and pink-eyed rdy+ Royal College of Surgeons (RCS) rats (9 days to 22 months of age) were used. From the local slaughterhouse tissue samples of pigs (5, 6 months and 6 years of age) and cows (7 months and 5, 6 years of age) were obtained (tables 2, 3). Human tissue was obtained from 19- to 87-year-old donors (postmortem time listed in table 5). Immunohistochemistry Anterior eye segments, optic nerve, heart muscle and gland tissue samples were fixed in 4% paraformaldehyde for 46 h, then rinsed in Tris-buffered saline (TBS, pH 7.4). Frozen, 14-m-thick sections through the heart and glands, sagittal and tangential sections through the retrolaminar optic nerve and the anterior eye segment were mounted on poly-L-lysine-covered glass slides. In addition, whole mounts of the anterior eye segment of rat eyes were stained free floating. After preincubation with Blottos dry milk solution [22] for 30 min to reduce nonspecific background staining, incubation with a polyclonal rabbit anti-B-crystallin antibody (dilution 1:400; antibody described in Flgel et al. [23]) was performed overnight in a moist chamber at room temperature. Sagittal sections through the optic nerve were also stained with a polyclonal rabbit anti-GFAP antibody (1:200; Bio Genex Laboratories, San Ramon, Calif., USA). After rinsing in TBS, a fluorescein-conjugated goat antirabbit Cy3
B-Crystallin and Age
15
Table 3. Number of tissues in different
age groups investigated by protein immunoblotting (dot blot) and polymerase chain reaction (PCR)
Animal
Age
Retrolaminar optic nerve dot blot PCR
Heart muscle dot blot 1 1 1 1 1 1 1 1 1 2 4 1 2 2 1 2 2 2 2 1 2 2 2 2 2 3 PCR
Mouse (Black6) 3 weeks Mouse (Black6) 2 months Mouse (Black6) 13 months Mouse (Black6) 18 months Mouse (Black6) 19 months Mouse (Black6) 21 months Mouse (Black6) 23 months Mouse (SAMP) 11 months Mouse (SAMP) 12 months Rat (Wistar) 3 weeks Rat (Wistar) 1 month Rat (Wistar) 1.5 months Rat (Wistar) 2 months Rat (Wistar) 3 months Rat (Wistar) 4 months Rat (Wistar) 6 months Rat (Wistar) 10 months Rat (RCS) 12 months Rat (RCS) 1 12 months Rat (RCS) 3 months Rat (RCS) 6 months Rat (RCS) 9 months Rat (RCS) 12 months Rat (RCS) 15 months Rat (RCS) 22 months Pig 5 months Pig 6 months Pig 6 years Cow 7 months Cow 2 years Cow 5 years Cow 6 years Human (for ages, see table 5)
1 1
1 1 1 1 4 2 2 1 2 1 1 1
1 1 1 1 1 1 1 1 1 1
1 1
1 1 1 3 1 1 3 4 2 1 11
3 2 1 15
branes (Schleicher and Schuell, Dassel, Germany) were fixed in a 96well dot blot apparatus (Minifold I SRC 96 D, Schleicher and Schuell) and 100 l of the standard and probes were added. After vacuum filtering and washing in 20% methanol the transfer membrane was air dried. For immunostaining, the membrane was incubated with PBS containing 0.05% Tween 20 and 1% milk powder for 1 h. The primary antibody (B-crystallin diluted 1:20,000) was then added and allowed to react overnight at room temperature. After washing twice in PBS + Tween and once in alkaline phosphate buffer (0.1 M NaCl, 5 mM MgCl, 0.1 M Tris; pH 9.5), the membrane was incubated with an alkaline-phosphatase-conjugated goat antirabbit IgG antibody (dilution 1:10,000; Promega, Madison, Wisc., USA) for 2 h. Visualization was achieved using fast NBT/BCIP buffered substrate tablets (Sigma, St. Louis, Mo., USA). The reaction was stopped after 10 min with 20 mM EDTA in PBS and the membrane was air dried. For densitometric quantitation of B-crystallin the membranes were scanned with a Hewlett Packard Scanner (Scan Jet IIcx) and analyzed using TINA (ray test) and EXCEL software.
Semiquantitative Polymerase Chain Reaction Total RNA of rat and human optic nerve specimens and rat heart muscle was isolated by the guanidinium thiocyanate phenol-chloroform single-step extraction (RNA isolation kit; Stratagene, La Jolla, Calif., USA). The structural integrity of the total RNA samples was analyzed by 1.2% agarose gel electrophoresis with ethidium bromide staining. To remove traces of contaminating genomic DNA, all RNA samples were treated with 3 units of RQ RNase-free DNase (Promega) for 35 min at 37 C. Samples were then extracted with phenolchloroform (1:1) and precipitated by addition of ethanol. First-strand complementary DNA preparation and polymerase chain reaction (PCR) amplification were performed with the same methods and conditions described previously [24] using a specific primer for glyceraldehyde-3-phosphate dehydrogenase, which served as an internal standard. The primers were purchased from Roth (Karlsruhe, Germany). Semiquantitative evaluation was performed using a LumiImager workstation (Boehringer Mannheim, Mannheim, Germany).
16
Results
Immunohistochemistry Anterior Eye Segment. Staining for B-crystallin in the anterior eye segment showed clear differences between the various species investigated. Mouse anterior eye segments revealed a bright B-crystallin staining only in the corneal endothelium and epithelium. A small number of nonpigmented ciliary epithelial (NPE) cells were stained particularly in the apical region of the most anterior pars plicata; the epithelial cells of the pars plana remained unstained. No positive staining for B-crystallin was found in the trabecular meshwork and outflow pathways, iris or ciliary muscle. In the rat, the corneal endothelial cells showed slight B-crystallin staining that continued into the anterior portion of the trabecular meshwork. Intense staining for Bcrystallin was seen in iris, ciliary and retinal pigmented epithelial (RPE) cells but with regional differences. In the iris, the entire layer of the pigmented epithelium stained for B-crystallin. In the anterior part of the pars plicata, some NPE cells were intensely stained, whereas in the posterior pars plicata only slight B-crystallin staining was seen in some pigmented epithelial cells (PE). Staining intensity increased in the pars plana region. The RPE cells stained for B-crystallin in a scattered way, showing unstained RPE cells next to intensely stained ones (fig. 1a). Porcine eyes showed an intense B-crystallin staining of the corneal endothelium and clear staining in the trabecular meshwork. The scleral spur cells and the ciliary muscle cells remained unstained. The iris epithelium and almost all epithelial cells of the pars plicata showed positive staining for B-crystallin with pronounced labeling in the apical region. In the pars plana, staining was present in NPE and PE cells but was much less distinct. Staining of the anterior eye segment of bovine and primate eyes confirmed our results of previous studies. In bovine eyes the corneal endothelium, iris and ciliary body NPE, PE and RPE cells were stained positively for Bcrystallin. In primate eyes, intense staining was seen in the corneal endothelium in the iris and ciliary epithelium, especially at the tips of the pars plicata. In addition, staining for B-crystallin was found in the ciliary muscle and in the trabecular meshwork but was mainly restricted to the outer portion adjacent to Schlemms canal. In none of the five species investigated were age-related differences seen in staining intensity or principal distribution of stained cells (table 4). The individual variations were greater than the age-related differences.
b
Fig. 1. Immunohistochemical demonstration of B-crystallin in the eye. a Whole mount of the anterior retinal pigmented epithelium of a rat eye. The RPE cells show scattered staining for B-crystallin. ! 140. b Sagittal section through the rat retrolaminar optic nerve. Intense staining is present in glial cells within the individual nerve fiber bundles, whereas the astrocyte processes separating the bundles from the connective tissue septa remain unstained. ! 180.
Optic Nerve. In the mouse and rat optic nerve, only glial cells stained for B-crystallin (fig. 1b). Astrocyte processes forming a rim separating the nerve fiber bundles from the connective tissue septa appeared unstained. Considerably more staining was found in optic nerve of the pig and cow. Numerous glial cells with their cytoplasmic processes appeared B-crystallin positive, including the astrocyte processes of the nerve fiber bundle rim. The staining intensity was more pronounced in the peripheral part of the optic nerve and less intense in the central part. The distribution of B-crystallin in the primate optic nerve was comparable to that described for the porcine and bovine optic nerve. The slightly larger variability in staining intensity of human optic nerve specimens might be explained by the greater variability of the postmortal time. In none of the species studied were age-related differences found in staining intensity or distribution of staining.
17
Heart Muscle. The distribution of B-crystallin within the heart muscle was similar in all animals studied. Using immunohistochemistry, the antibody against B-crystallin labeled almost exclusively the region of the Z band of cardiac myocytes (fig. 2a). In addition, Schwann cells also stained positive for B-crystallin. Ultrastructural studies of the rat heart muscle revealed that most of the gold particles were located in the Z band region, but a number of gold particles was also scattered in the adjacent cytoplasm (fig. 2b). These particles were not adhering to cytoskeletal filaments or cell organelles. The staining intensity varied greatly between the different species studied. Weak staining was found in heart muscle cells of the mouse, clear staining in that of rats and intense staining in the pig, cow and human heart muscle. Investigation of animals in different age groups did not show clear-cut differences in staining intensity. Thyroid Gland. In the thyroid gland of all species studied (mouse, rat, pig, human), follicular epithelial cells showed bright staining for B-crystallin (fig. 3a). The staining was not evenly distributed within the circumference of the follicles and varied in different parts of the gland (fig. 3b). The colloid of the follicle remained unstained as did the parafollicular cells of the stroma. The intracellular distribution of B-crystallin was regular and showed no preference for any compartment. The mouse thyroid gland revealed less intense staining than the thyroid of the other three species investigated. No age-related staining differences were found. Protein Immunoblotting (Dot Blot) and PCR Optic Nerve. In rat eyes, quantitative densitometric evaluation of B-crystallin in the dot blots (fig. 4a) showed that there were no differences in the protein expression with increasing age (fig. 5a). The interindivid-
b
Fig. 2. Ultrastructural demonstration of B-crystallin in the heart
muscle. Gold particles showing localization of B-crystallin are scattered in the cytoplasm adjacent to the Z bands. a ! 13,300. b ! 48,000.
Table 4. Distribution of B-crystallin in
the anterior eye segment of various animals
Species
Corneal Ciliary epithelium endothelium

pars plana
pars plicata
Iris epithelium (+) ++ ++ ++ ++
Trabecular meshwork 0 (+) + 0 ++
Ciliary muscle 0 0 0 0 +
Mouse Rat Pig Cow1 Primate1
+ + ++ ++ ++
0 + ++ ++ ++
(+) + ++ ++ ++
++ = Intense; + = Mild; (+) = weak; 0 = no staining reaction. Data are in agreement with Flgel et al. [23] and Siegner et al. [26].
18
b
Fig. 3. Immunohistochemical demonstration of B-crystallin in human (a) and rat (b) thyroid gland. The thyroidal follicular cells are intensely stained throughout the entire cytoplasm. There are, however, staining differences in cells within single follicles. The endocrine cells of the parathyroid gland (right half of b) are unstained. Arrows indicate positively stained Schwann cells. a ! 80. b ! 200. Fig. 4. Dot blot analysis of B-crystallin in retrolaminar optic nerve (a) and heart muscle (b) of different species. The standard for quantitative evaluation is shown in lanes 1 and 2. a Optic nerve samples
show different amounts of the protein when comparing mouse (lane 3), rat (lanes 4 and 5), pig (lane 6), cow (lanes 7 and 8) and human samples (lanes 9 and 10). b B-Crystallin in heart muscle of mouse (lane 10), rat (lanes 69), pig (lane 5, bottom), cow (lane 5, top) and human samples (lanes 2, bottom, 3 and 4).
ual differences were predominant with B-crystallin levels varying between 90 and 310 ng/mg protein. In mouse eyes of different age groups, B-crystallin levels varied between 210 ng/mg protein in a 3-week-old mouse to 100 ng/mg protein in a 21-month-old animal (fig. 6a). In 2 SAMP mice, 11 and 12 months old, only 70 ng/mg protein were detected.
The bovine optic nerves were derived from animals 7 months, 2, 5 and 6 years old (maximal life span of cows is about 15 years). Individual differences ranged between 790 and 2,250 ng/mg protein (fig. 7a). In porcine optic nerves only 5- to 6-month-old animals and one 6-year-old animal could be investigated (maximal life span of pigs is about 10 years). In the younger group, 2 animals had lev-
19
350 300 250 200 150 100 50
a
aB-Crystallin (ng/mg protein) s
250
aB-Crystallin (ng/mg protein)
200
s s s s
150
100
50
0 0 3 6 Age (months) 9 12
3 weeks
13 months Age
21 months
14,000 12,000 10,000 8,000 6,000 4,000 2,000 0 0
b
aB-Crystallin (ng/mg protein) s
600 500 400 300 200 100 0
s s s s s sss s s s s 5
s s s ss s 10 Age (months) s s s 15 20 s
ee
on
on
on
on
on
13
18
19
21
Age
Fig. 5. Densitometric quantitation of B-crystallin (ng/mg total protein) in the dot blots of rat optic nerve (a) and heart muscle (b). There
Fig. 6. Densitometric quantitation of B-crystallin (ng/mg total protein) in the dot blots of mouse optic nerve (a) and heart muscle (b).
are individual but no age-related differences in the amount of Bcrystallin in both tissues.
The amount of B-crystallin in the optic nerve is lower in the old animals compared to the young mouse. The protein expression in the heart muscle shows no age-related differences.
els of around 500 ng/mg protein, whereas 2 other animals had B-crystallin levels of approximately 2,000 ng/mg protein. The old animal had 550 ng/mg protein B-crystallin (fig. 8). In human eyes there was no age-related decrease or increase in the amount of B-crystallin in the optic nerve either, but individual differences ranging between 470 and 2,450 ng/mg protein were observed (table 5). These data are in the range of those obtained from porcine and bovine optic nerves, but the postmortem time of the human tissues was between 4 h and 3 days, compared to the immediately obtained tissue of the animals.
PCR analysis was performed with optic nerve tissues derived from rat and human donors. In the 5 human optic nerves (donor age 4187 years) and in the rat optic nerves, derived from 8 animals (1.515 months old), no obvious differences in B-crystallin mRNA were seen (fig. 9). Heart. In the rat heart muscle the mean concentration of B-crystallin was 1,570 ng/mg protein with no changes in protein expression during aging (fig. 5b). In the heart muscle of mice protein concentrations of B-crystallin were between 260 and 580 ng/mg protein (fig. 6b). The amount of B-crystallin remained nearly at the same level in all age groups.
20
23
on
s s
ks
s th
th
th
th
th
th
2,500
a
2,500
2,000
2,000
1,500
1,500
1,000
1,000
500
500
0 0 7 7 7 24 24 24 24 Age (months) 60 60 72 5 5 5 6 Age (months) 72
9,000 8,000 aB-Crystallin (ng/mg protein) 7,000 6,000 5,000 4,000 3,000 2,000 1,000 0
2 Age (years)
9
Fig. 7. Densitometric quantitation of B-crystallin (ng/mg total protein) in the dot blots of bovine optic nerve (a) and heart muscle (b). Fig. 8. Densitometric quantitation of B-crystallin (ng/mg total pro-
The amount of B-crystallin in the optic nerve does not change with age. In the heart muscle, levels of the protein in 5- and 6-year-old animals are higher than in the 2-year-old animals.
In the bovine heart muscle higher values of B-crystallin (mean concentration of 3,210 ng/mg protein) were measured. In 2-year-old animals the values varied between 370 and 2,530 ng/mg protein, in the 5- and 6-yearold animals values increased towards 2,0408,540 ng/mg protein (fig. 7b). In pigs only heart muscle tissue from three 5-month-old animals was available. B-Crystallin levels were 820, 970 and 1,000 ng/mg protein, respectively. The human heart, even if obtained more than 12 h postmortem, still showed concentrations of up to 10,650
tein) in the dot blot of porcine optic nerve. The amount of B-crystallin in the optic nerve shows individual but no age-related differences. Fig. 9. Agarose gel electrophoresis and ethidium bromide staining of PCR products amplified from human (a) and rat (b) optic nerve tissue with primers specific for B-crystallin. Glyceraldehyde-3-phosphate dehydrogenase (white arrow) shows equal amounts of total cDNA preparation. All human samples show similar amounts of Bcrystallin mRNA (black arrow). In the rat, an increase in B-crystallin mRNA is seen with increasing age of the animals.
ng/mg protein (mean concentration 2,040 ng/mg protein; table 5). The B-crystallin mRNA levels obtained from 11 rat heart muscles of different age groups showed no age-related differences (data not shown).
21
Table 5. Quantitative measurement (ng/mg protein) of B-crystallin in the retrolaminar optic nerve and heart muscle of human donors by dot blotting
Age, years 19 34 35 36 41 46 53 63 65 68 70 75 79 80 87 87
Sex
Postmortem time 47 h 2 days 50 h 3 days 2 days 2 days 15 h 26 h 18 h 20 h 48 h 1 day 40 h 28 h 30 h 4h
Optic nerve
Heart muscle 10,650 560 1,190 1,400 1,280 450 680 5,780 1,580 660 1,080 3,060 480 1,360 450
F F F M M M M M F F M M F F M M
1,350 1,190 840 500 1,080 1,210 1,190 470 1,510 2,450 1,040
Discussion
In the present study, the distribution of B-crystallin staining was investigated in the heart muscle, known to show constitutively high amounts of the protein [25], in nonlenticular eye tissues with variations in staining intensity of different cell populations [23, 26] and in a number of glands not studied before. In none of these tissues were significant age-related changes in B-crystallin staining found. Using dot blot methods as a semiquantitative evaluation method, no increase in B-crystallin was detected in rat heart muscle, but there was more B-crystallin in 5- to 6-year-old than in 2-year-old cows. A tendency towards an increase in B-crystallin mRNA with age was also seen in rat optic nerve tissues. Age-related increases in B-crystallin expression in rat heart muscle and kidney have been described by Bhat and Nagineni [8], Kato et al. [25] and Iwaki et al. [27]. However, in these studies only developmental stages up to 20 days of life were investigated. Katoh-Semba and Kato [21] found a tendency towards increased amounts of B-crystallin in mouse brain tissue with increasing age. In that study, the oldest age group of normal animals was 8 months. Unfortunately, we were not able to obtain tissues from various age groups of pigs and cows, but the values obtained from young and middle-aged porcine and bovine optic nerves showed no differences in B-crystallin expression. In hu-
man tissues derived from middle-aged and old donors, there were no age-related differences in the protein expression either, but the data are of limited value as the material was derived from donors with different postmortem times. On the other hand, in all species studied pronounced interindividual differences were found. If a molecule functions as a stress protein, individual differences might well reflect differences in individual stress situations. Immunohistochemical staining for B-crystallin also revealed differences in the distribution and amount of the protein in the five species investigated. These differences may be due to differences in the specificity of the antibody. On the other hand, the DNA sequence of B-crystallin shows great homologies between all species investigated [28, 29]. The same primer could be used for the detection of B-crystallin mRNA in the various species so that the staining differences might in fact be due to species differences in B-crystallin expression. In the eye, positive staining of the ciliary muscle and localized staining of the outer part of the trabecular meshwork were only seen in primates [23, 26]. It is well established that in the mammalian species there is a well-developed fovea centralis and accommodation system only in higher monkeys and human eyes [for a review, see 30]. It is therefore tempting to speculate that the characteristic staining pattern in the primate ciliary muscle and outer part of the trabecular meshwork with its connections to the ciliary muscle tendons might be related to mechanical stress during accommodation [31]. Species differences were also seen in semiquantitative evaluation of the protein using the dot blot method. The values in rat, porcine and bovine heart muscles were in the same range as those described in the literature [25, 32]. In all mouse tissues evaluated there was less protein expression than in the other species. In human tissues, the values measured were the same as those found in porcine and bovine tissues even if the material was obtained after longer postmortem times. It is possible that the values in the living tissue are much higher. Differences in the absolute life span of the species might be another factor involved in the observed species differences. However, this hypothesis cannot be proven by our studies due to lack of material from species with longer life spans. In summary, our studies show that an increase in Bcrystallin with age may occur but is not a general phenomenon in tissues constitutively expressing this protein.
22
References
1 Bloemendal H: Molecular and Cell Biology of the Eye Lens. New York, Wiley, 1981. 2 Ingolia TD, Craig EA: Four small Drosophila heat shock proteins are related to each other and to mammalian -crystallin. Proc Natl Acad Sci USA 1982;79:23602364. 3 Klemenz R, Frohli E, Aoyama A, Hoffmann S, Simpson RJ, Moritz RL, Schafer R: B-Crystallin accumulation is a specific response to Ha-ras and v-mos oncogene expression in mouse NIH 3T3 fibroblasts. Mol Cell Biol 1991;11:803812. 4 Dasgupta S, Hohman TC, Carper D: Hypertonic stress induces B-crystallin expression. Exp Eye Res 1992;54:461470. 5 Horwitz J: Alpha-crystallin can function as a molecular chaperone. Proc Natl Acad Sci USA 1992;89:1044910453. 6 Piatigorsky J: The twelfth Frederick H. Verhoeff lecture: Gene sharing in the visual system. Trans Am Ophthalmol Soc 1993;91:283 297. 7 Longoni S, Lattonen S, Bullock G, Chiesi M: Cardiac alpha-crystallin. II. Intracellular localization. Mol Cell Biochem 1990;97:121128. 8 Bhat SP, Nagineni CN: B-Subunit of lens-specific protein -crystallin is present in other ocular and non-ocular tissues. Biochem Biophys Res Commun 1989;158:319325. 9 Leach IH, Tsang ML, Church RJ, Lowe J: BCrystallin in the normal human myocardium and cardiac conducting system. J Pathol 1994; 173:255260. 10 Atomi Y, Yamada S, Strohman R, Nonomura Y: B-Crystallin in skeletal muscle: Purification and localization. J Biochem 1991;110: 812822. 11 Iwaki T, Iwaki A, Goldman JE: B-crystallin in oxidative muscle fibers and its accumulation in ragged-red fibers: A comparative immunohistochemical and histochemical study in human skeletal muscle. Acta Neuropathol 1992;85: 475480. 12 Scotting P, McDermott H, Mayer RJ: Ubiquitin-protein conjugates and B-crystallin are selectively present in cells undergoing major cytomorphological reorganization in early chicken embryos. FEBS Lett 1991;285:7579. 13 Quinlan RA: Assembly of intermediate filament proteins is modulated by the chaperone activity of alpha-crystallins. Invest Ophthalmol Vis Sci 1993;34:989. 14 Iwaki T, Kume-Iwaki A, Liem RKH, Goldman JE: B-Crystallin is expressed in non-lenticular tissues and accumulates in Alexanders disease brain. Cell 1989;57:7178. 15 Head MW, Corbin E, Goldman JE: Overexpression and abnormal modification of the stress proteins B-crystallin and HSP27 in Alexander disease. Am J Pathol 1993;143:1743 1753. 16 Iwaki T, Tateishi J: Immunohistochemical demonstration of alphaB-crystallin in hamartomas of tuberous sclerosis. Am J Pathol 1991; 139:13031308. 17 Iwaki A, Iwaki T, Goldman JE, Ogomori K, Tateishi J, Sakaki Y: Accumulation of B-crystallin in brains of patients with Alexanders disease is not due to an abnormality of the 5)flanking and coding sequence of the genomic DNA. Neurosci Lett 1992;140:8992. 18 Renkawek K, deJong WW, Merck KB, Frenken CWGM, vanWorkum FPA, Bosman GJCGM: B-Crystallin is present in reactive glia in Creutzfeldt-Jakob disease. Acta Neuropathol 1992;83:324327. 19 Renkawek K, Voorter CEM, Bosman GJCGM, vanWorkum FPA, deJong WW: Expression of B-crystallin in Alzheimers disease. Acta Neuropathol 1994;87:155160. 20 Murano S, Thweatt R, Reis RJS, Jones RA, Moerman EJ, Goldstein S: Diverse gene sequences are overexpressed in Werner syndrome fibroblasts undergoing premature replicative senescence. Mol Cell Biol 1991;11: 39053914. 21 Katoh-Semba R, Kato K: Age-related changes in levels of the -subunit of nerve growth factor in selected regions of the brain: Comparison between senescence-accelerated (SAM-P8) and senescence-resistant (SAM-R1) mice. Neurosci Res 1994;20:251256. 22 Duhamel RC, Johnson DA: Use of nonfat dry milk to block nonspecific nuclear and membrane staining by avidin conjugates. J Histochem Cytochem 1985;33:711714. 23 Flgel C, Liebe S, Voorter C, Bloemendal H, Ltjen-Drecoll E: Distribution of B-crystallin in the anterior segment of primate and bovine eyes. Curr Eye Res 1993;12:871876. 24 Welge-Lssen U, Eichhorn M, Bloemendal H, Ltjen-Drecoll E: Classification of human scleral spur cells in monolayer culture. Eur J Cell Biol 1998;75:7884. 25 Kato K, Shinohara H, Kurobe N, Inaguma Y, Shimizu K, Ohshima K: Tissue distribution and developmental profiles of immunoreactive B-crystallin in the rat determined with a sensitive immunoassay system. Biochem Biophys Acta 1991;1074:201208. 26 Siegner A, May CA, Welge-Lssen U, Bloemendal H, Ltjen-Drecoll E: B-Crystallin in the primate ciliary muscle and trabecular meshwork. Eur J Cell Biol 1996;71:165169. 27 Iwaki T, Iwaki A, Liem RKH, Goldman JE: Expression of B-crystallin in the developing rat kidney. Kidney Int 1991;40:5256. 28 Frederiksen PH, Dubin RA, Piatigorsky J: Structure and alternate tissue-preferred transcription of the murine B-crystallin gene. Nucleic Acids Res 1994;22:56865694. 29 Liao JH, Hung CC, Lee JS, Wu SH, Chiou SH: Characterization, cloning, and expression of porcine B-crystallin. Biochem Biophys Res Commun 1998;244:131137. 30 Rohen JW: The evolution of the primate eye in relation to the problem of glaucoma; in LtjenDrecoll E (ed): Basic Aspects of Glaucoma Research. Stuttgart, Schattauer, 1982, pp 333. 31 Welge-Lssen U, May CA, Eichhorn M, Bloemendal H, Ltjen-Drecoll E: B-Crystallin in the trabecular meshwork is inducible by transforming growth factor-. Invest Ophthalmol Vis Sci 1999;40:22352241. 32 Inaguma Y, Hasegawa K, Goto S, Ito H, Kato K: Induction of the synthesis of hsp27 and Bcrystallin in tissues of heat-stressed rats and its suppression by ethanol or an 1-adrenergic antagonist. J Biochem 1995;117:12381243. 33 Iwaki T, Kume-Iwaki A, Goldman JE: Cellular distribution of B-crystallin in non-lenticular tissues. J Histochem Cytochem 1990;38:31 39. 34 Lowe J, McDermott H, Pike I, Spendlove I, Landon M, Mayer RJ: B-Crystallin expression in non-lenticular tissues and selective presence in ubiquinated inclusion bodies in human disease. J Pathol 1992;166:6168. 35 May CA, Arnold B, Welge-Lssen U, Arnold W, Bloemendal H, Ltjen-Drecoll E: B-Crystallin in the mammalian inner ear. Otorhinolaryngology 1998;60:121125. 36 Dubin RA, Wawrousek EF, Piatigorsky J: Expression of the murine B-crystallin gene is not restricted to the lens. Mol Cell Biol 1989;9: 10831091. 37 Gopal-Srivastava R, Haynes JI, Piatigorsky J: Regulation of the murine B-crystallin/small heat shock protein gene in cardiac muscle. Mol Cell Biol 1995;15:70817090. 38 Bhat SP, Horwitz J, Srinivasan A, Ding L-L: B-Crystallin exists as an independent protein in the heart and in the lens. Eur J Biochem 1991;102:775781. 39 Iwaki T, Wisniewski T, Iwaki A, Corbin E, Tomokane N, Tateishi J, Goldman JE: Accumulation of B-crystallin in central nervous system glia and neurons in pathologic conditions. Am J Pathol 1992;140:345356. 40 Pinder SE, Balsitis M, Ellis IO, Landon M, Mayer RJ, Lowe J: The expression of B-crystallin in epithelial tumours: A useful tumour marker? J Pathol 1994;174:209215. 41 Nishikawa S, Ishiguro S, Kato K, Tamai M: A transient expression of B-crystallin in the developing rat retinal pigment epithelium. Invest Ophthalmol Vis Sci 1994;35:41594164.
23
Vascular and Glial Changes in the Retrolaminar Optic Nerve in Glaucomatous Monkey Eyes
N. Furuyoshi a M. Furuyoshi a Ch.A. May a S.S. Hayreh b A. Alm c E. Ltjen-Drecoll a
a Department
of Anatomy II, Friedrich Alexander University Erlangen-Nrnberg, Erlangen, Germany; of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, USA; c Department of Ophthalmology, University Hospital, Uppsala, Sweden
b Department
Key Words Optic nerve W Glaucoma W B-crystallin W Glial fibrillary acidic protein W Vessels
Abstract Vascular and glial changes of the retrolaminar optic nerve were studied in monkey eyes with increased intraocular pressure (IOP) from 1 to 4 years and with different stages of optic nerve atrophy. In histological crosssections of retrolaminar optic nerves of 11 rhesus and 6 cynomolgus monkeys the entire area, number of axons and vessels and area of pial septa were quantitated and three different kinds of nerve degeneration classified. Ultrathin sections of these different stages were performed and the number of open and occluded vessels was determined. In addition, in cynomolgus monkey optic nerves immunohistochemical staining for B-crystallin, glial fibrillary acidic protein (GFAP) and vimentin was performed. Even in animals with the same duration of glaucoma and comparable mean IOP values the axon degeneration varied considerably. Independently of axon loss the number of capillaries in the rhesus mon-
keys remained constant, whereas there was a slight decrease in the cynomolgus monkeys. Some of the vessels, especially in the most severely damaged regions, were occluded. The density of glial cells increased whereas the total number remained nearly constant. In control sections all astrocytes stained for GFAP and Bcrystallin. In the glaucomatous optic nerves the density of B-crystallin- and GFAP-positive cells was significantly increased. The vascular reaction in the retrolaminar glaucomatous optic nerves differs from that described in the prelaminar region. We assume that in the postlaminar region in areas with diminished nutritional needs vessels occlude and finally degenerate.
Introduction
It is generally assumed that the axonal insult in glaucomatous optic neuropathy is located at or near the scleral lamina cribrosa, a tissue presumed to be easily affected by elevated intraocular pressure (IOP). Histopathological investigations of primate eyes with acute elevation of IOP to
ABC
very high levels have shown abnormal accumulation of cell organelles and localized swellings of optic nerve fiber axons within the scleral lamina cribrosa, reflecting blocking of axoplasmic flow [15]. In monkey eyes with laserinduced chronic glaucoma, it has been reported that in the prelaminar region of the optic nerve the amount of axonal tissue decreases and is partly replaced by glial tissue [5, 6]. Morphologically no changes in the number of capillaries in the optic nerve head were found in eyes with elevated IOP for up to 4 months [5]. In later stages of optic nerve atrophy, the proportion of capillaries in the optic disk remained constant [7]. Detailed descriptions of morphological changes in the retrolaminar region of glaucomatous optic nerves are rare. In owl monkey eyes, in which an acute high IOP was induced by injection of -chymotrypsin into the posterior chamber and maintained for up to 1 week, axonal degeneration and reactive axonal enlargement as well as degenerative changes in oligodendrocytes, an increase in phagocytosing cells and an activation of astrocytes have been described [1]. In monkey eyes with laser-induced chronic ocular hypertension, histological studies revealed that as in glaucomatous human eyes axons in the upper and lower temporal quadrant start to atrophy [4, 8]. In a previous study, we found that in monkey eyes with laserinduced chronic glaucoma a significant nerve fiber loss developed in the retrolaminar region of the optic nerve but the nerve fiber counts varied greatly between individual eyes [9]. The reason for the interindividual differences is unclear. As ocular hypertension lasted more than 1 year in all eyes and the mean IOP was almost similar, differences in nerve fiber loss could be due to any of the following: differences in variations of IOP not measured, varying duration and IOP in different eyes, structural differences in the lamina cribrosa, differences in the susceptibility of the vasculature or differences in reactivity of the glial cell system. Regeneration studies of the optic nerve in the retrolaminar region in fish and rat eyes have revealed that the glial environment which surrounds an axon influences its capacity to regenerate after injury [10]. Factors derived from mature oligodendrocytes [1113] and extracellular matrix produced by astrocytes have been discussed in this context [1417]. Whether these factors that favor regeneration might also enhance the ability to survive the initial insults of damage to the nerve fibers has not been studied. Since myelinization of the optic nerve fibers starts only in the retrolaminar region of the optic nerve and the proportion of oligodendrocytes and astrocytes also changes markedly in this region, the mechanism of optic nerve
fiber damage in the retrolaminar region could be different from that seen in the prelaminar portion of the optic nerve. In the present study we investigated the retrolaminar part of the optic nerve in experimentally induced chronic high-pressure glaucoma in monkey eyes, trying to correlate ultrastructural changes, immunohistochemical changes and vascular changes with the individual degree of glaucomatous optic neuropathy.
Materials and Methods

We performed this study in 24 optic nerves 12 from rhesus monkeys (Macaca mulatta) and 12 from cynomolgus monkeys (Macaca fascicularis) as indicated in table 1. Eight rhesus monkey optic nerves and 6 from cynomolgus monkeys were from eyes with experimental chronic high-pressure glaucoma, induced by the method described in a previous paper [9]. The remaining eyes (4 of rhesus monkeys and 6 of cynomolgus monkeys) were normal, healthy fellow eyes of these animals, with normal IOP, and these acted as control. All experiments were performed in accordance with the ARVO Resolution for the Use of Animals in Ophthalmic and Vision Research and the local university rules for experiments on primates. Tissue Samples from Iowa City (S.S. Hayreh) The data and clinical course of 8 rhesus monkey eyes with chronic elevated IOP have been described previously [9]. The duration of the elevated IOP varied between 14 and 43 months, and the IOP values ranged from 22 to 42 mm Hg in the treated eyes compared to 17 19 mm Hg in the normal fellow eyes (table 1). Before enucleation, the animals were perfusion fixed with 4% paraformaldehyde transcardially as described previously [9]. The optic nerve was cut directly behind the lamina cribrosa and at the level of the entrance of the central retinal artery into the optic nerve. This cylindrically shaped optic nerve specimen was then transversally cut into three to four 1to 2-mm-wide disks and postfixed in Itos solution [18] for at least 1 week. Tissue Samples from Uppsala (A. Alm) The data and clinical course of 6 of the cynomolgus monkey eyes have been described previously [19]. The duration of glaucoma was 4 years. In these eyes IOP measurements were performed only in the first 9 months after treatment and at the end of the experiment. The mean IOP ranged between 29 and 52 mm Hg after treatment and between 7 and 24 mm Hg at the time of sacrifice. As in these animals microspheres had been injected for measuring blood flow, the animals were not perfusion fixed before enucleation [19]. After enucleation the retrolaminar optic nerve was divided into 2 pieces. The portion between lamina cribrosa and entrance of the central retinal artery was placed in 4% paraformaldehyde for immunohistochemical investigations, the optic nerve posterior to the entering central retinal artery was immersion fixed in Itos solution for light- and electron-microscopic examination. Histological and Electron-Microscopic Investigations Thin slices of all Ito-fixed specimens were postfixed in OsO4, dehydrated in an ascending series of alcohol and acetone, and
Optic Nerve in Monkeys with Laser Glaucoma
25
Table 1. Animal specification, clinical data and various quantitative measurements of optic nerve cross-sections of laser-induced glaucoma-
tous eyes
Eye number Animal type and age, years Glaucoma duration Mean IOP mm Hg 18 19 17 19 36 35 32 42 37 34 27 27 Maximum IOP mm Hg 20 23 19 22 62 68 68 75 80 54 56 60 Nerve fiber count Crosssection area, mm2 4.89 6.54 5.76 4.32 3.82 2.34 2.09 2.41 2.34 2.21 3.37 2.0 8.2 5.5 5.49 6.44 5.31 8.64 2.17 5.7 1.42 3.32 2.84 1.41 Total number of vessels 590 540 483 431 599 412 645 648 688 570 739 495 692 548 567 494 388 848 285 58 5 320 302 362 409 Total number of glial cells 2,385 3,179 2,790 2,328 2,105 1,682 2,245 1,123 1,857 2,998 3,227 1,683 1,399 1,278 1,524 2,170 1,500 2,521 1,405 n.e. 936 612 518 561 Pial arteriolar changes 0 0 0 0 1 0 2 2 0 1 0 2 0 0 0 0 0 0 2 0 2 0 0 2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
rhesus, 17 rhesus, 20 rhesus, 20 rhesus, 20 rhesus, 13 rhesus, 17 rhesus, 20 rhesus, 13 rhesus, 20 rhesus, 20 rhesus, 20 rhesus, 20 cynomolgus cynomolgus cynomolgus cynomolgus cynomolgus cynomolgus cynomolgus cynomolgus cynomolgus cynomolgus cynomolgus cynomolgus
control control control control 14 months 17 months 18 months 18 months 19 months 36 months 40 months 43 months control control control control control control 4 years 4 years 4 years 4 years 4 years 4 years
30 31 38 41 42 52
45 33 44 50 43 64
913,381 1,409,580 1,322,799 850,833 362,366 241,925 22,431 911 153,146 120,856 73,068 52,579 1,089,878 867,570 1,085,926 1,220,432 1,100,955 1,150,353 70,039 839,325 5,715 259,657 352,575 3,306
Pial arteriolar changes: 0 = normal; 1 = mild; 2 = clear intima thickening. n.e. = Not evaluable.
embedded in Epon. Semithin sections were stained with toluidine blue and examined by light microscopy. The entire area of the optic nerve was evaluated using a Quantimet 500 computer (Leica, Cambridge, UK). To determine the total number of axons, each specimen was divided into 4 sectors and 2 regions. In each of the 8 fields an area 1,000 m2 in size was selected at random, the number of myelinated axons determined and converted into the number of the corresponding area. Three different grades of axonal loss were defined if there was a diffuse nerve fiber loss: mild (more than 800,000 nerve fibers; grade 1), moderate (100,000800,000 nerve fibers; grade 2) and severe axonal loss (less than 100,000 nerve fibers; grade 3). In optic nerve cross-sections with focal nerve fiber loss, grading was performed for each affected area separately: an area with mild damage histologically appeared nearly unchanged; areas with severe damage were characterized by almost complete loss of nerve fibers which were replaced by glial cells. In areas with moderate damage there were still some nerve fibers present. The classification was confirmed by four of the investigators (N.F., M.F., C.A.M., E.L.D.) independently. The total amount of glial cells was calculated by multiplying the glial cell density (number of glial cell nuclei, counted in a defined measuring field of 0.115 mm2) with the entire area of nervous tissue in the cross-section. The areas occupied by nervous tissue and pial septa were measured with the working plate of the Quantimet computer. The pial septum area was calculated as percentage of the total optic nerve cross-section area minus the complete area occupied by neuronal tissue.
The total number of blood vessels was counted on the optic nerve cross-sections with a magnification of ! 400 and the location of each vessel was recorded on a schematic drawing. Vessel density was calculated by dividing the total number of vessels through the entire cross-section area of the optic nerve. From optic nerves with different forms of degeneration (grades 13 as defined above) within the cross-section, at least two ultrathin sections of each of the different affected regions were examined by transmission electron microscopy. The nuclei of astrocytes, oligodendrocytes and microglial cells (cell types identified according to the descriptions in Kettenmann and Ransom [20]) were counted in a single measuring field of 0.028 mm2, representing approximately 25% of the total neural area. Twenty capillaries within the pial septa were selected at random to determine the capillary occlusion rate. Capillaries with a slit-like lumen or no lumen were rated as being occluded. Immunohistochemistry For immunohistochemical staining, antibodies against glial fibrillary acidic protein (GFAP), vimentin and B-crystallin were used. GFAP was selected as a general marker for astrocytes in the optic nerve [21, 22]. Vimentin, as a general marker of mesoderm-derived tissue, was selected to study the changes of vimentin-positive glial cells such as activated microglial cells and a subgroup of astrocytes [23, 24]. B-Crystallin, a member of the small heat shock proteins with chaperone properties, has been demonstrated in oligodendrocytes of brain tissues [25]. Astrocytes in the brain only occasionally
26
Furuyoshi/Furuyoshi/May/Hayreh/Alm/ Ltjen-Drecoll
Table 2. Vessel occlusion rate, mean area of pial septa, mean glial
density, number of oligodendrocytes, astrocytes and microglial cells in a measuring field of 0.28 mm2 in relation to different stages of glaucomatous optic neuropathy Nerve damage 0 (n = 10) 1 (n = 6) 2 (n = 10) 3 (n = 12) Vessel occlusion rate, % 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 0, 0, 10, 10, 15, 25 0, 0, 5, 5, 10, 10, 15, 25, 30, 60 0, 0, 5, 10, 10, 10, 10, 10, 35, 50, 50, 85 Mean area of pial septa 7.4% (4.415) 15% (521) 19.4% (8.836) 29% (1946) Mean glial density, % 398 794 944 1,324
Figures in parentheses indicate minimum-maximum. Optic nerve damage; 0 = normal appearance (control sections); 1 = mild; 2 = moderate; 3 = severe degeneration of myelinated nerve fibers; n = number of areas used for quantitative measurements.
Immunohistochemical staining for B-crystallin and GFAP revealed an almost identical staining pattern. The periphery of the nerve fiber bundles adjacent to the pial septa stained intensely for both antibodies. A delicate network of stained processes was evenly distributed throughout the nerve, whereas cells surrounding the single axons were unstained (fig. 1a, c). In contrast, vimentin staining was seen only in single star-shaped cells within the nerve fiber bundles and in the fibroblasts and vascular cells of the pial septa (fig. 1e). Stained glial cells were found in all nerve fiber bundles but were located mainly centrally. Several fine processes reached towards the marginal zones of the nerve fiber bundles. The periphery of the bundles and the cells surrounding the nerve fibers were unstained. The number of vimentin-positive glial cells in the normal optic nerve varied in the individual monkeys. Optic Nerves of Eyes with Chronically Elevated IOP In the glaucoma eyes, the severity of optic nerve fiber degeneration varied from nearly normal to complete loss of nerve fibers (fig. 2). In general, diffuse damage to axons was seen throughout the optic nerve in all glaucoma cases, while several optic nerves revealed prominent axonal loss only in some quadrants. The entire cross-section area was significantly smaller in the glaucoma eyes, as was the number of axons (table 1). Based on the severity of axonal loss, glaucomatous neuropathy was graded into mild, moderate and severe stages. Mild Glaucomatous Optic Neuropathy. This group included cases No. 20 for the entire optic nerve and No. 5 and 6 for parts of the optic nerve cross-section. On light microscopy, the nerve fiber bundle structure appeared nearly normal, but with the exception of case No. 20, the number of axons was decreased (table 1). On electron microscopy, in some regions of the nerve the myelin sheath was disarranged and partly destroyed. The pial septal architecture was well preserved occasionally showing slight thickening of the connective tissue septa. Glial cells were observed at places of pronounced nerve fiber lesions, showing an increase in rough endoplasmic reticulum. Inclusion bodies of degenerative myelin, indicating phagocytosis, were seen in astrocytes but not microglial cells. Immunohistochemistry revealed findings almost similar to those in the control optic nerve sections. While no changes occurred in the amount or distribution of Bcrystallin and GFAP staining, a mild increase in vimentin-positive cell processes, located mainly in the center of the nerve, could be observed in the glaucomatous optic nerves compared to their contralateral controls.
show expression of B-crystallin [26, 27]; however, an increase in B-crystallin in astrocytes has been described under pathological conditions [26, 28, 29]. As an increase in B-crystallin was found in the trabecular meshwork of human glaucomatous eyes [30], staining for this protein was included to investigate whether B-crystallin is also increased in glial cells of optic nerves with glaucomatous optic neuropathy. The paraformaldehyde-fixed specimens were rinsed in Tris-buffered saline (TBS, pH 7.27.4) and cryoprotected in TBS containing 20% sucrose. Frozen sections were cut at a thickness of 14 m and placed on poly-L-lysine-coated glass slides. After preincubation with Blottos dry milk solution [31] the sections were incubated with the primary antibody overnight at room temperature. As primary antibody, we used rabbit anti-B-crystallin (provided by H. Bloemendal, Nijmegen, the Netherlands; dilution 1:400), rabbit anti-GFAP (Bio Genex Laboratories, San Ramon, Calif., USA; dilution 1:200) and mouse antivimentin antibody (Dako, Glostrup, Denmark; dilution 1:50). After washing in TBS the sections were covered with a Cy3-fluorescein-conjugated secondary antibody (Dianova, Hamburg, Germany; dilution 1:500) for 2 h at room temperature. After washing again, the sections were mounted with Kaisers glycerin jelly and viewed with a Leitz Aristoplan microscope (Wetzlar, Germany). Negative control experiments were performed using TBS or rabbit preimmune serum, substituted for the primary antibody.
Results
Optic Nerves of Normal Control Eyes All retrolaminar optic nerve sections of the untreated fellow eyes appeared normal with large bundles of myelinated nerve fibers, surrounded by delicate pial septa. The optic nerve cross-section area, axon count and area covered by pial septa are shown in tables 1 and 2.
27
a,b
Fig. 1af. Immunohistochemical staining
of cross-sections through the postlaminar optic nerve of cynomolgus monkeys. a, c, e Control nerves. b, d, f Severely damaged glaucomatous nerves. a, b Staining for Bcrystallin. c, d Staining for GFAP. e, f Staining for vimentin. In the control optic nerve, several cells within and bordering the nerve fiber bundles stain for B-crystallin and GFAP, whereas only single cells within the bundle as well as fibroblasts and vascular cells within the pial septa stain for vimentin. In glaucomatous nerves, the density of cells staining for B-crystallin and GFAP increases significantly as does the number of vimentin-positive cells. ! 40.
c,d
e,f
Moderate Glaucomatous Optic Neuropathy. This group included cases No. 22 and 23 for the entire optic nerve and cases No. 5, 6, 912 and 19 for parts of the optic nerve cross-section. On light microscopy, this stage of optic neuropathy showed extensive degeneration of axons and their myelin sheaths (fig. 2a, c), which was either diffuse throughout the entire cross-section or focal within some areas of the optic nerve. The pial septa were clearly thickened with increased amounts of collagen fibers. Both astrocytes and microglial cells showed activation indicated by an increase in rough endoplasmic reticulum and myelin inclusion bodies. In this stage of neuropathy, however, phagocytosis was more prominent in the microglial cells than in the astrocytes.
Immunohistochemistry showed an increase and thickening of B-crystallin- and GFAP-positive glial processes throughout the areas of pronounced optic neuropathy. The periphery of the bundles adjacent to the pial septa was heavily stained, whereas the remaining cells surrounding nerve fibers were unstained. In addition, at some places numerous focal thickenings of processes stained for GFAP but not for B-crystallin. The number of vimentin-positive glial cells in these areas was increased and their processes formed a dense network in the remaining neuronal tissue. Severe Glaucomatous Optic Neuropathy. This group included cases No. 7, 8, 21 and 24 for the entire optic nerve and cases No. 912 and 19 for parts of the optic nerve cross-section.
28
Fig. 2ad. Semithin sections through the
postlaminar region of the optic nerve, stained with toluidine blue. a Case No. 5: more than half of the nerve fibers are degenerated. b Case No. 13: nearly the entire nervous tissue is replaced by glial cells and pial septa. c Case No. 7: in areas with especially thick pial septa several vessels appear occluded (arrows). d Case No. 10: in regions with almost total gliosis the vessels show an open lumen (arrows). a, b ! 30. c, d ! 300.
c d
On light microscopy, severe optic neuropathy was characterized by an almost complete loss of nerve fibers, replaced by glial tissue (table 1, fig. 2b, d). On electron microscopy, the density of astrocytes had increased markedly, forming glial scars. The total number of astrocytes was, however, the same as or even smaller than in the control optic nerves. The density of microglial cells also increased in all but one (case No. 8) of the severely damaged optic nerves. Oligodendroglial cells had almost com-
pletely disappeared. A large number of lipid spherules and remnants of myelin were seen throughout these areas. In some cases the pial septa were much thicker than in normal nerves; the connective tissue at places appeared hyalinized. Other sections showed only a little thickening of connective tissue in individual pial septa. The cytoplasm of the astrocytes and microglial cells contained myelin debris and lipid spherules but only a modest amount of rough endoplasmic reticulum.
29
a
Fig. 3a, b. Electron micrograph of a capillary in the pial septum of a normal (a) and a glaucomatous (b) optic nerve. In the latter, the endothelial cells are enlarged and no lumen is visible. a ! 4,000. b ! 16,000.
Immunohistochemically, nearly all cells between the connective tissue septa stained for GFAP and B-crystallin (fig. 1b, d). In addition, processes were seen extending into the septa. These processes stained for GFAP but not for B-crystallin. In contrast to the ubiquitous distribution of GFAP and B-crystallin in the gliotic tissue, only single vimentin-positive cells were present within the remaining nerve fiber bundles, but they appeared more numerous than in the control eyes (fig. 1f). Vascular Reaction In the contralateral controls of the glaucoma eyes, the localization and morphology of the vessels were the same as described in normal optic nerves. The capillaries in the connective tissue septa showed normal height of endothelial cells and were nearly completely surrounded by pericytes (fig. 3a). In rhesus monkey eyes with increased IOP maintained for up to 4 years, the density of vessels within the retrolaminar region of the optic nerve increased significantly, whereas the absolute number remained nearly constant. In cynomolgus monkey eyes with an increased IOP induced 4 years earlier but with no IOP measurements during the last 3 years prior to the experiment, the
density of vessels also increased slightly, but the absolute number decreased when compared to the contralateral control eyes (table 1). The differences in the two series of animals may be due to the fact that the optic nerve sections in the rhesus monkeys were cut distal from the entrance of the central retinal artery while in cynomolgus monkeys sections proximal from the entrance were investigated. However, these findings were independent of the severity of the nerve fiber damage. Electron-microscopic investigation revealed that in the control optic nerves the lumina of all capillaries were wide open, whereas in glaucoma optic nerves some of the lumina were partly or completely occluded (fig. 3b). Most occluded vessels were seen in thickened pial septa (fig. 2c) and were therefore more common in severely damaged optic nerves. On the other hand, there were still regions in the optic nerves with severely damaged nerve fibers, where the vessels were not occluded (fig. 2d). In those capillaries the endothelium appeared flatter than in the controls. None of these vessels showed fenestrations, and pericytes were still present. The central retinal artery within the optic nerve showed focal thickening of the intima in all but 1 (case
30
No. 9) glaucomatous optic nerves, and 2 of the control optic nerves (cases No. 1 and 3) also showed mild intimal thickening. There was no correlation between the intimal changes in the central retinal artery and age of the animals, duration of glaucoma, IOP or severity of optic neuropathy. Pial Arteries In the control eyes the arterioles in the periphery of the optic nerve cross-sections showed a complete internal elastic membrane and an intima of regular thickness. In most glaucoma eyes (8 of 14), some of the arterioles showed ultrastructural changes. The intima of single vessels was thickened due to an increase in connective tissue in the intimal layer, and some smooth muscle cells had invaded the inner elastic membrane. Correlation of Morphological Changes with IOP In the rhesus monkeys, there was no correlation between the severity of optic nerve damage and either last IOP measurement, IOP peaks or mean IOP. In cynomolgus monkeys with increased IOP for 4 years, loss of nerve fibers accompanied by loss of oligodendrocytes and loss of capillaries was more pronounced than in the rhesus monkey eyes with glaucoma for 1443 months. This may be due to higher levels of sustained IOP in the latter group than in the former in the former group a sustained relatively lower level of IOP was maintained by antiglaucoma medication.
Discussion
The majority of our findings agree with what one would expect to occur in atrophy of nervous tissue [21]. Axons are destroyed and the space filled by astrocytes and collapsed connective tissue. The cellular reactions occurring in glaucomatous optic nerve atrophy were similar to those described for optic nerves undergoing Wallerian degeneration: staining for GFAP [22] and vimentin increases [23, 24], while the total number of astrocytes remains constant [32]. Accumulation of GFAP and vimentin also occurs in white matter astrocytes after axonal injury [33, 34]. In human open-angle glaucoma, changes in the extracellular material and in immunohistochemical staining of astrocytes for GFAP and neural cell adhesion molecule have been described [35, 36]. So far, B-crystallin and vimentin have not been investigated in glaucomatous optic nerves. In contrast to what is seen in the brain, in the normal optic nerve all glial cells which stained for
GFAP also stained for B-crystallin, indicating that optic nerve astrocytes express this small stress protein. We do not know whether B-crystallin is increased in these cells in glaucomatous eyes, because the staining in the normal eyes was already so bright that differences could not be detected morphologically. Due to fixation the material could not be used for further biochemical investigations. We found that in glaucomatous optic nerves some vessels were patent while others were partly or completely occluded. Most occluded vessels were seen in thickened pial septa and were therefore more numerous in severely damaged optic nerves. On the other hand, there were still regions in the optic nerves with severely damaged nerve fibers, whose vessels were not occluded; however, in those capillaries the endothelium appeared flatter than in the controls. Quigley and co-workers reported that there were no morphological changes in the number of capillaries in the optic nerve head in eyes with elevated IOP for up to 4 months [5], and in later stages of optic nerve atrophy the proportion of capillaries in the optic disk remained constant [7]. They did not specify whether, in the latter case, the capillaries seen by them were patent or occluded. Morphological demonstration of presence of capillaries gives no information whatsoever about the state of the circulation in them, which is of prime importance in proper nutrition of the optic nerve. We assume, however, that the occluded vessels were the branches of the few abnormal pial vessels in the periphery of the optic nerve. When nutritional needs are diminished in a region of the retrolaminar optic nerve, it is possible that some vessels become closed and others remain open. From the available information we cannot know whether the differences in morphology between pre- and retrolaminar glaucomatous optic nerves reflect a difference between white and gray matter in general. We can conclude, at least, in glaucomatous optic neuropathy that in the retrolaminar region of the optic nerve there are not only regional differences in loss of nerve fibers but also in vascular and glial changes.
Acknowledgement
The authors wish to thank Marco Gsswein for the excellent preparation of the photographs. This research was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 539 to E.L.D.), Akademie der Wissenschaften, Mainz (to E.L.D.), European Community (Biomed to E.L.D.), US National Institute of Health (EY 01576 to S.S.H.) and an unrestricted grant for research by Research to Prevent Blindness (to S.S.H.).
31
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Glia 1993;8:111. 16 Bastmeyer M, Beckmann M, Schwab ME, Stuermer CAO: Growth of regenerating goldfish axons is inhibited by rat oligodendrocytes and CNS myelin but not by goldfish optic nerve tract oligodendrocyte-like cells and fish CNS myelin. J Neurosci 1991;11:626650. 17 Battisti WP, Shinar Y, Schwartz M, Levitt P, Murray M: Temporal and spatial pattern of expression of laminin, chondroitin sulphate proteoglycan and HNK-1 immunoreactivity during regeneration in the goldfish optic nerve. J Neurocytol 1992;21:557573. 18 Ito S, Karnovsky MJ: Formaldehyde-glutaraldehyde fixatives containing trinitro compounds. J Cell Biol 1968;39:168a169a. 19 Alm A, Lambrou GN, Mepea O, Nilsson SFE, Percicot C: Effects on ocular flow and optic nerve morphology of experimental glaucoma in cynomolgus monkeys. Ophthalmologica 1997; 211:178182. 20 Kettenmann H, Ransom BR: Neuroglia. New York, Oxford University Press, 1995. 21 Bignami A, Dahl D: Gliosis; in Kettenmann H, Ransom BR (eds): Neuroglia. 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Acta Neuropathol (Berl) 1992;83:324327. 30 Ltjen-Drecoll E, May CA, Polansky JR, Johnson DH, Bloemendal H, Nguyen TD: Localization of the stress protein B-crystallin and trabecular meshwork inducible glucocorticoid response protein in normal and glaucomatous trabecular meshwork. Invest Ophthalmol Vis Sci 1998;39:517525. 31 Duhamel RC, Johnson DA: Use of nonfat dry milk to block nonspecific nuclear and membrane staining by avidin conjugates. J Histochem Cytochem 1985;33:711714. 32 Skoff RP, Vaughn JE: An autoradiographic study of cellular proliferation in degenerating rat optic nerve. J Comp Neurol 1971;141:133 156. 33 Mansour H, Asher R, Dahl D, Labkovsky B, Perides G, Bignami A: Permissive and nonpermissive reactive astrocytes: Immunofluorescence study with antibodies to the glial hyaluronate-binding protein. J Neurosci Res 1990; 25:300311. 34 Oblinger MM, Singh LD: Reactive astrocytes in neonate brain upregulate intermediate filament gene expression in response to axonal injury. Int J Dev Neurosci 1993;11:149156. 35 Varela HJ, Hernandez MR: Astrocyte responses in human optic nerve head with primary open-angle glaucoma. J Glaucoma 1997;6: 303313. 36 Hernandez MR, Pena JDO: The optic nerve head in glaucomatous optic neuropathy. Arch Ophthalmol 1997;115:389395.
32

Friederike Stumpff Michael Wiederholt
Institut fr Klinische Physiologie, Universittsklinikum Benjamin Franklin, Freie Universitt Berlin, Deutschland
Key Words Glaucoma W Trabecular meshwork W Smooth muscle W Aqueous humor outflow W Contractility
Introduction
Abstract Ample evidence supports the theory that trabecular meshwork possesses smooth-muscle-like properties. Trabecular meshwork cells express a large number of transporters, channels and receptors, many of which are known to regulate smooth-muscle contractility. It has been shown that trabecular meshwork can be induced to contract and relax in response to pharmacological agents. In the model of the bovine eye, confirmed in some cases by experiments on primates, agents that contract trabecular meshwork reduce outflow. On the cellular level, this is coupled with depolarization and a rise in intracellular calcium. Relaxation of trabecular meshwork, on the other hand, appears to be coupled to a stimulation of the maxi-K channel, inducing hyperpolarization and a closure of L-type calcium channels. No significant differences between cells from a human and a bovine source emerged, either in classical measurements of membrane voltage, in measurements of intracellular calcium or patch-clamp experiments. Thus, pharmacological agents that relax trabecular meshwork seem promising candidates for further research the ultimate goal being an improvement of glaucoma therapy in humans.
The major route for the outflow of aqueous humor is via the trabecular meshwork into Schlemms canal [14]. The form and area of the spaces which are enclosed by the trabecular beams are important for aqueous drainage. The composition of the extracellular material in these spaces seems to be mainly responsible for outflow resistance [5]. In glaucoma, outflow is reduced due to an increase in different forms of extracellular material deposited within the cribriform layer of the trabecular meshwork, correlating with the loss of axons in the optic nerve [5]. In the traditional concept, trabecular meshwork is an inert tissue, with no regulatory properties of its own. In this concept, regulation of outflow resistance is determined by the ciliary muscle. Connected to the trabecular meshwork by fibers called zonules, contraction of the ciliary muscle passively distends the trabecular meshwork, increasing intratrabecular spaces [3, 4]. The same mechanism determines the accommodative state of the lens. Recent evidence speaks for an additional involvement of the scleral spur, a contractile structure containing myofibroblasts and projecting like a shelf into the trabecular meshwork from its posterior margin [4, 6, 7]. Physiologically, the regulation of two such massively different parameters as accommodation and intraocular pressure by the tone of the same muscle seems surprising.
ABC
Dr. Friederike Stumpff Institut fr Klinische Physiologie, Freie Universitt Berlin Hindenburgdamm 30 D12200 Berlin (Germany) Tel. +49 30 8445 2791, Fax +49 30 8445 4239, E-Mail stumpff@zedat.fu-berlin.de
Work done during the last decade has established that, in addition to being passively distended by the ciliary muscle, the trabecular meshwork has contractile properties of its own [1], and that the contraction and relaxation of this structure may influence ocular outflow in the sense that relaxation reduces intraocular pressure. It seems possible that administration of smooth-muscle-relaxing substances might lower intraocular pressure, via relaxation of trabecular meshwork, while simultaneously improving retinal circulation by vasodilation of retinal capillaries. Trabecular meshwork thus appears as an interesting target tissue for new approaches in glaucoma therapy. Barany [8] was the first to advance a hypothesis that trabecular meshwork possesses contractile properties of its own. Subsequently, a number of studies have been performed to investigate this theory. Histologically, extensive innervation of the trabecular meshwork has been shown [4, 9]. Using electron microscopy, Ringvold [10] observed cytoplasmic filaments in meshwork cells of monkeys. Using a histochemical technique, he demonstrated that these filaments consist of actin material. Various scientists have shown that the microfilaments present in trabecular meshwork are smooth-muscle actin [1115] and that there may be smooth-muscle myosin in the human trabecular meshwork [16, 17]. Morphologically, data from various authors show that muscarinergic agonists such as pilocarpine directly affect the trabecular meshwork of the eye of humans and various animal species and vary their form in culture [8, 1821]. Measurements of intracellular calcium underscore the notion that trabecular meshwork possesses characteristics of smooth-muscle cells. Shade et al. [22] and Llobet et al. [23] have shown that, as in smooth muscle, substances that contract trabecular meshwork elevate cytosolic calcium. Thus, it can by now be seen as evident that trabecular meshwork resembles smooth muscle, with the potential for regulating intraocular pressure. The search for specific pharmacological agents that interact with channels, transporters, receptors or other proteins in the signalling cascade leading to changes in trabecular meshwork contractility is thus a worthwhile undertaking, potentially leading to a better and more specific pharmacological control of intraocular pressure.
not been performed before we attempted to obtain direct measurements of isolated trabecular meshwork strips. While the ciliary muscle extends into the trabecular meshwork of higher primates making it hard to isolate strips of trabecular meshwork, the bovine eye is well suited for contractility experiments. In this species, the ciliary muscle is more posteriorly located and can easily be removed from the trabecular meshwork. For the contractility measurements, bovine eyes were dissected and strips of trabecular meshwork and ciliary muscle prepared according to established methods [29]. Using a purpose-built force-length transducer similar to that described by Brutsaert et al. [30], the isometric force of the strips could be monitored for a period of several hours after an equilibration period of approximately 1 h [29, 3134]. Depolarization by High External K+ As the membrane potential of the vast majority of cells depends to a large extent on the potassium gradient, raising external potassium has a profound influence on this potential, leading to depolarization. Application of highpotassium external solution is a standard procedure for inducing smooth-muscle contraction [35, 36]. In trabecular meshwork, application of 120 mmol/l KCl evoked a contraction that was biphasic but corresponded to only 19% of the response attainable by acetylcholine [29]. Part of the KCl response was blockable by atropine. This can be explained by the fact that in many tissues, depolarization induces a release of acetylcholine from cholinergic nerve terminals. This indicates that only a fraction of the entire contractile response of trabecular meshwork can be attributed to depolarization of the cell membrane per se. However, this result has to be taken with a grain of salt as the application of such a high-potassium solution has to be seen as a very unphysiological maneuver. We would also like to point out that this experiment does not allow conclusions on the voltage dependence of the signalling pathway leading to relaxation or about the response of trabecular meshwork to hyperpolarization. Cholinergic Agents Cholinergic agents induced strong, reproducible contractions in trabecular meshwork cells (fig. 1), which were inhibitable by atropine, demonstrating the presence of muscarinic receptors [29, 31]. The induction of force could be increased by application of physostigmin, a reversible anticholinesterase inhibitor, demonstrating the presence of acetylcholinesterase in trabecular meshwork cells. Experiments using muscarinic antagonists of the M1
Contractility Measurements
While the contractility of ciliary muscle has been studied in various mammalian species including man [2427], measurements of trabecular meshwork contractility had
34
Stumpff/Wiederholt
Fig. 1. Typical recording of a contractility
experiment with a strip of bovine trabecular meshwork. The muscarinergic agent carbachol contracts the strip in a dose-dependent way; part of this contractile response can be blocked by the M3 receptor blocker diphenylacetoxy-N-methylpiperidine methiodide.
and M3 type [37] suggest that both in human and in bovine trabecular meshwork, functional receptors are of the M3 type [31, 38] (fig. 1). However, other muscarinic subtypes have not been excluded. In addition, it has been shown that, in primates, receptor subtype antagonists are modulators of aqueous humor outflow [39]. Adrenergic Agents Human trabecular meshwork cells have been shown to express - and -adrenergic receptors, especially of the 2adrenergic subtype [4043]. In addition, agonists like epinephrine have been shown to reduce outflow resistance and to increase outflow facility through direct actions on trabecular meshwork and via the uveoscleral route in a primate model [2, 44, 45]. The interpretation of this fact is complicated, however, due to the fact that epinephrine is nonspecific and dose-dependently acts on both - and -receptors. In contractility experiments, both 1- and 2-adrenergic agonists contracted the trabecular meshwork strips with approximately 20% of the potency of carbachol [31]. The effect of 2-agonists was greater than that of 1-agonists and the effects could be blocked by specific antagonists. In contrast, -agonists such as isoproterenol significantly relaxed the tissue precontracted by carbachol, an effect that could be blocked by metipranolol. Application of a blocker on the unstimulated tissue had no effect [31]. In the concentration used to reduce intraocular pressure (10 4 to 10 3 mol/l), epinephrine contracted strips of
trabecular meshwork. Additional application of metipranolol, a -blocker, further increased trabecular meshwork tone, indicating blockage of the relaxing effect of the component of epinephrine. It seems that the net effect of epinephrine on trabecular meshwork contractility should depend on the balance of - and -adrenergic receptors in the tissue of the species observed. Low External Ca2+ While relaxation of precontracted ciliary muscle was total when external calcium was removed, only a part of the contractile response of trabecular meshwork depended on the presence of external calcium, with 42% of the response to carbachol and 23% of the response to endothelin remaining after removal of this ion [46]. It seems that in trabecular meshwork, both a calciumdependent and a calcium-independent mechanism of contractile response exists. Similar effects have been reported for other tissues, pointing towards a regulation of myosin activity via protein kinases [47, 48]. Blockers of Ca2+ Channels Blockage of calcium channels is one of the new therapeutic approaches in glaucoma therapy, aimed at reducing intraocular pressure and, simultaneously, improving retinal circulation. While verapamil has been reported to enhance ocular outflow in humans [49], other calcium blockers have been reported to affect the rate of inflow [50]. Different blockers of calcium channels had varying effects on trabecular meshwork contractility [1, 32, 46].
35
While the highly specific calcium antagonist nifedipine in the low dosage of 10 5 mol/l only had a relaxing effect on about 10% of the total contractile response, verapamil, which is reported to block a number of other channels including potassium channels and chloride channels [51 53], blocked more than 70% of the response. Ni2+, an inorganic calcium blocker which has been shown to inhibit low-threshold (T-type) Ca2+ channels [54] and Na+/Ca2+ exchange [55], had a relaxing effect both on the contractile response to endothelin (86%) and carbachol (41%). In summary, it appears that external calcium is needed for a part but not all of the contractile response of trabecular meshwork to carbachol and endothelin. Endothelin Endothelin-like immunoreactivity is 23 times higher in aqueous humor of human and bovine eyes than in the corresponding plasma [32, 56], and an elevation of endothelin-like immunoreactivity in the aqueous humor of glaucoma patients has been reported [57]. It seems that endothelin, the release of which has been shown to be stimulated by stretch and fluid flow rate [58], might be an important hormone regulating aqueous humor production and/or outflow. The finding of higher endothelin levels in glaucoma patients indicates a possible dysfunction involving an altered production of endothelin [57]. This theory is underscored by the fact that endothelin evokes a strong contractile response in isolated trabecular meshwork strips which is in the range of the carbachol effect if equal concentrations are applied (fig. 2). 77% of this contractile response was dependent on extracellular calcium [32, 46]. Nitric Oxide Nitric oxide has been implicated in the physiology of aqueous humor dynamics [5961], and the use of nitrovasodilatators in the therapy of glaucoma is being discussed. Thus, NO synthase could be detected in the outflow pathway of the bovine and human eye, and NO synthase immunoreactivity was reduced in patients with primary open-angle glaucoma [59]. NO, nitrovasodilatators and nonnitrates like sodium nitroprusside have been shown to increase cyclic GMP [62, 63]. Application of membrane-permeable cGMP (8bromo-cGMP) relaxed precontracted strips of trabecular meshwork to 41% of the tone under carbachol [64] (fig. 3). The organic nitrovasodilatators like ISDN (isosorbide dinitrate) and 5-isosorbide mononitrate, too, were able to relax trabecular meshwork. The most potent relaxants were nonnitrates like SNP (sodium nitroprusside) and
Fig. 2. Recording showing the contractile response of bovine tra-
becular meshwork to endothelin. Again, the contractile force depends on the dose of endothelin applied.
SNAP (S-nitroso-N-acetylpenicillamine), reducing trabecular meshwork tone in response to carbachol by over 60%. Interestingly, inhibition of NO synthase by L-nitroarginine (L-NAG) increased carbachol-induced contraction significantly, while ISDN and SNP also significantly relaxed uncontracted trabecular meshwork. These findings indicate a continuous release of NO by trabecular meshwork both under contraction and under resting conditions. Prostaglandins Prostaglandins (PGs) represent a new class of topically effective ocular antihypertensive drugs [65, 66]. PGF2 and its analogues [e.g. PhXA34 (latanoprost)] have been shown to enhance outflow facility [67, 68], although most of the effect seems to have concerned the uveoscleral route. However, the existence of PGF2 receptors in human trabecular meshwork has been demonstrated using the RT-PCR technique [69]. Mediated by a number of different prostanoid receptor subtypes, PGs have been shown to have differing effects on various types of smooth-muscle tissue. Trabecular meshwork, too, responded to the various PGs in different ways [34]. Sulprostone and the thromboxane mimetic U46619 caused contraction (fig. 4). These effects could be blocked by the TP thromboxane activated receptor antagonist SQ-29548. In contrast, PGF2 and 17-phenyl PGF2 had no effect, while the nonselective EP (E prostanoid)
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Fig. 3. The membrane-permeable analogue of cGMP, 8-bromo-cGMP, relaxes precontracted bovine trabecular meshwork. The same effect could be observed after application of nitrate and nonnitrate vasodilatators that function by elevating cytosolic pH.
agonist 11-deoxy PGE1 and the specific EP2 (E2 prostanoid) agonist AH-13205 significantly relaxed precontracted trabecular meshwork strips. We conclude that trabecular meshwork possesses both TP and EP2 receptors the activation of which causes opposite effects. Cyclooxygenase Inhibitors It has been shown that in smooth-muscle tissue, muscarinic stimulation initiates a well-described cascade of second-messenger signalling involving, among other things, the release of PGs. Cyclooxygenase is the enzyme responsible for the production of prostanoids. In contraction experiments [70], application of the cyclooxygenase inhibitor indomethacin (5 W 10 6 mol/l) on trabecular meshwork and ciliary muscle strips precontracted by carbachol resulted in an additional contractile response to almost 140% of the uninfluenced contraction by carbachol. These results suggest that carbachol induces the production of relaxing PG in trabecular meshwork and ciliary muscle, the inhibition of which results in contraction. Interestingly, in the monkey eye, the effect of decreasing outflow resistance is partly inhibited by indomethacin [71]. Diuretics Recent research indicates that the function of the Na+2Cl -K+ cotransporter is altered in glaucomatous eyes and that this should affect the cell volume of trabecular meshwork cells [72, 73]. This transporter is sensitive to the loop
Fig. 4. Prostaglandins had varying effects on trabecular meshwork, ranging from relaxation to contraction. This recording shows the strong, contractile response observable after application of the thromboxane mimetic U-46619.
diuretics bumetanide and furosemide, and to the rather unspecific agent ethacrynic acid, which is known to inhibit not only this transporter, but also sodium-dependent anion transporters and to modulate the cytoskeleton. However, contractility of trabecular meshwork was not altered when this transporter was blocked by bumetanide [32, 74]. The diuretic hydrochloride, which blocks the Na+Cl cotransporter [32], had no effect on contractility.
37
Fig. 5. Trace demonstrating the relaxation of precontracted trabecular meshwork by flufenamic acid and ethacrynic acid. The effects were additive.
Fig. 6. Trabecular meshwork was relaxed by application of genistein, a tyrosine kinase inhibitor. Tyrphostin 51, a synthetic tyrosine kinase inhibitor, had similar effects.
On the other hand, ethacrynic acid relaxed trabecular meshwork. Studies on primates have shown that systemic or local application of various diuretics including bumetanide had no effect on aqueous humor dynamics and intraocular pressure [74], while local application of ethacrynic acid increased outflow facility in the human eye [75]. Flufenamic Acid Flufenamic acid, a therapeutically used antirheumatic agent [7678], relaxed trabecular meshwork strips precontracted by carbachol or endothelin 1 [32] (fig. 5). These relaxing effects were independent of the relaxing effects of ethacrynic acid and isosorbide dinitrate. While in many tissues, the blockage of nonselective cation channels by flufenamic acid has been reported [79, 80], it appears that in trabecular meshwork, the maxi-K channel is stimulated. Modifiers of the Cytoskeleton Both ethacrynic acid and cytochalasin D relaxed trabecular meshwork [32] (fig. 5). Both agents are thought to disrupt microtubules which help constitute the cytoskeleton [8183]. Protein Kinase Inhibitors Phosphorylation of cellular proteins controls various cellular functions such as mitogenesis but also contractili-
ty [8487]. Recently, it has been demonstrated that protein tyrosine kinase pathways are involved in the regulation of smooth muscle contractility [88, 89]. In trabecular meshwork, stimulation of the EGF (epidermal growth factor) receptor with tyrosine kinase activity by EGF (100 g/l) caused relaxation of tissue strips precontracted by carbachol (10 6 mol/l) [33]. Application of tyrosine kinase inhibitors like tyrphostin 51 and genistein in concentrations of 5 W 10 5 mol/l produced relaxation (fig. 6). Inhibition of PKA-PKG by H-8 (5 W 10 6 mol/l) and of the serine threonine kinase PKC by chelerythrine or NPC-15437 (both at 10 6 mol/l) was also able to relax trabecular meshwork; these effects were additive to the effects of the inhibition of tyrosine kinase [33]. Recently, it has been shown that trabecular facility is indeed increased after application of the nonselective serine threonine protein kinase inhibitors H-7 and staurosporine [27, 90, 91]. Interestingly, there were marked differences in the response of trabecular meshwork and ciliary muscle [33]. In contrast to the relaxing effect of EGF on trabecular meshwork, ciliary muscle was contracted by application of EGF. The effect of inhibiting tyrosine kinase was more pronounced in trabecular meshwork. Inhibiting PKC and PKA-PKG had no effect on the contractility of ciliary muscle.
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110 perfused anterior segment (bovine) 40 min
Relative outflow (%)
100 n = 10
90
Fig. 7. Summary of 10 experiments measuring ocular outflow in the model of the perfused anterior segment of the bovine eye in which the ciliary muscle has been removed. The muscarinergic agonist pilocarpine, which contracts trabecular meshwork, reduced ocular outflow.
80
pilocarpine (105 mol/l)
Cellular Contractions
On a cellular level using cultivated trabecular meshwork cells, changes in shape indicating contractile processes have also been observed. Agents that have been shown to decrease the area of trabecular meshwork cells include the vasoactive compounds bradykinin [92] and acetylcholine [21], as well as ethacrynic acid, colchicine and vinblastine [81, 93, 94], all of which are known to disrupt the cytoskeleton.
The Perfused Anterior Segment
The isolated, perfused anterior segment of primate and bovine eyes is an established method for studying aqueous humor outflow [28, 92, 9597]. Perfusion of the anterior segment of bovine eyes with detached iris, ciliary body and ciliary muscle at a pressure of 8.8 mm Hg yielded a constant outflow rate of 68 l/min with an outflow facility of 0.87 l W mm Hg/min and an outflow resistance of 1.15 mm Hg W min/l. The perfusion rate remained constant for up to 3 h; no washout occurred. Elevation of the pressure in the outflow chamber increased outflow resistance in a linear fashion. Relative outflow in this model could be influenced by a variety of drugs [28]. Carbachol reduced outflow by a maximum of 37% with a half-maximal effective concentration of 3 W 10 8 mol/l; the effect of the drug could be completely blocked by atropine. Pilocarpine was some-
what less effective with a reduction in outflow of 15% (fig. 7). Endothelin 1, a potent vasoactive agent that contracts trabecular meshwork strips [46], also reduced ocular outflow. Bradykinin is generated by the action of the kallikrein-kinin system and known for its vasoactive properties. Intracameral administration of bradykinin is known to increase intraocular pressure [98] and to contract smooth-muscle tissue; in accordance with this, a drop in outflow facility in the model of the perfused anterior segment was observed [92]. As in the contractility experiments, the net effect of epinephrine depended on the concentration used [28]. At 10 5 mol/l, outflow was reduced, while at a lower concentration (10 6 mol/l), outflow in the bovine eye increased, an effect also reported for the human eye. This increase in the perfusion rate could be blocked by application of the -blocker metipranolol. While in the higher concentration range the effect of epinephrine on -adrenergic receptors predominates, it seems that in the lower concentration range, the net effect can be explained by the stimulation of -adrenergic receptors. Disruption of the cytoskeleton by cytochalasin D, which led to a relaxation of trabecular meshwork cells in contractility experiments, also increased outflow facility [32]. In conclusion, it is possible to say that substances that contract trabecular meshwork strips reduce outflow in the model of the perfused anterior segment, while substances that relax trabecular meshwork induce an increase in the outflow rate.
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Fig. 8. Measurement of intracellular calcium using the fura method. Endothelin increased cytosolic calcium concentration in human trabecular meshwork cells.
Intracellular Calcium
It is a well-established fact that smooth-muscle contractility is regulated by the concentration of intracellular calcium [35, 99, 100]. Using fura-2-loaded bovine trabecular meshwork cells, we measured the intracellular calcium concentration by recording the fluorescence ratio of calcium-bound to calcium-free dye [101]. In accordance with findings by Shade et al. [22], we found a basal resting calcium concentration of 4080 nmol/l. Cytosolic calcium levels could be elevated both by depolarizing the cells using high-potassium solution and by application of the opener of L-type calcium channels, Bay K 8644 (5 mol/l). Application of endothelin 1, which contracts trabecular meshwork [46] and induces a reduction in the outflow rate [28], caused a biphasic increase in the level of cytosolic calcium [28, 102] (fig. 8); the same could be observed when acetylcholine was applied. This biphasic response is well known from other preparations of smooth muscle tissue [35, 99, 100]. The initial peak is thought to be due to a release of calcium from cytosolic stores via an established second-messenger pathway involving G proteins and inositol triphosphate. The subsequent plateau phase is due to calcium influx from the outside through various, tissue-dependent influx pathways [26]. In trabecular meshwork, these include voltage-dependent calcium channels [101, 103]. Thus, an increase in cytosolic calcium could be obtained by depolarization with highpotassium solution and by application of a specific opener of L-type calcium channels, Bay K 8644 [103]. The participation of nonselective cation channels [32] and of cal-
cium-release-activated calcium currents [104, 105] is also discussed. Measurements on cultured human trabecular meshwork cells have shown that the response to muscarinergic agents is almost identical in the human and the bovine species both in terms of absolute concentrations of cytosolic calcium reached, and in terms of the biphasic profile observed [22, 101]. The use of different muscarinic receptor subtype antagonists has revealed that the M3 receptor subtype is the most important receptor involved [22]. In addition, it was possible to show that phosphoinositide production was stimulated as a result of the stimulation of muscarinic receptors, with subsequent activation of the phospholipase C system. Subsequent research demonstrates a rise in calcium in response to the application of various neuropeptides (neuropeptide Y, substance P, bombesin, calcitonin gene-related peptide, vasoactive intestinal peptide) in trabecular meshwork cells [106]. Neuropeptide Y proved to be a particularly potent elevator of cytosolic calcium levels and phosphoinositide turnover. Bradykinin also elevated cytosolic calcium levels [92], as did the application of PGF2 [69]. Both atrial natriuretic peptide and C type natriuretic peptide increased the accumulation of cGMP, leading to a suppression of carbacholinduced calcium mobilization [107]. Elevation of hydraulic pressure also induced a rise in cytosolic calcium [108], in accordance with the observation that trabecular facility decreases with rising intraocular pressure [1, 96]. Endothelin 1 and histamine [38, 101, 102, 109] also elevated internal calcium in trabecular meshwork, while angiotensin II had no effect [102].
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Regulation of Intracellular pH
Intracellular pH (pHi) is a key factor in determining the activity of many cellular enzymes [110, 111]. Transporters involved in pHi regulation have been identified in many cell types and have been found to participate in cell homeostasis, transmembrane transport, transepithelial transport, growth factor activation and cell proliferation. In order to demonstrate the existence of transporters that regulate pHi in trabecular meshwork, bovine trabecular meshwork cells were loaded with 5(6)carboxy-4),5)dimethylfluorescein and pHi was monitored by measuring the pH-dependent absorbance of this dye [1, 112]. In physiological, bicarbonate-containing Ringers solution, pHi averaged 7.02. When the CO2/HCO buffer was replaced 3 (ensuring that external pH remained at a constant value of 7.4 by addition of HEPES), pHi dropped significantly, a first indication of bicarbonate-dependent transporters regulating trabecular meshwork pHi. We were able to demonstrate the existence of three independent pH-regulating transporters. In bicarbonate-free medium, both maintaining steady-state pHi and recovery after an acid load was Na+ dependent (fig. 9). The underlying mechanism could be totally blocked by amiloride. These data point to the existence of an Na+/H+ exchanger in trabecular meshwork, a transport process responsible for eliminating acid equivalents from the cytosolic compartment that can be found in a number of cells. The existence of this exchanger was confirmed by another study [113]. When cells were acidified in bicarbonate-containing medium, pHi recovery continued even after the Na+/H+ exchanger had been blocked with amiloride. Additional blockage with DIDS (4,4)-diisothiocyanatostilbene-2,2)disulfonate) or pyridoxal 5)-phosphate, both blockers of bicarbonate-dependent anion exchangers, eliminated the recovery process. Replacing Cl in the extracellular solution was also able to eliminate recovery from an acid load and led to alkalinization instead. The recovery was sodium dependent and blockable by ethacrynic acid, a blocker of the sodium-dependent chloride-bicarbonate exchanger. In a further series of experiments, recovery after an alkaline load was tested. This recovery was sensitive to removal of external bicarbonate. Removal of external chloride reversed the direction of regulation. Removal of sodium had no impact, while DIDS blocked alkaline extrusal. These experiments point to the existence of two additional pHi-regulating transporters in trabecular meshwork the sodium-dependent and the sodium-independent chloride-bicarbonate exchangers.
Fig. 9. Measurement of pHi using bovine trabecular meshwork.
After an acidifying prepulse with NH4Cl, the pHi returns to the resting level. This process is dependent on the presence of extracellular sodium, pointing towards the Na+/H+ exchanger as the underlying transporter.
The pHi-regulating transporters of other ocular tissues such as the cornea and ciliary epithelium have been described in more detail [110, 111, 114, 115]. In these tissues, the Na+/H+ exchanger is the dominant force after acidification of the cell, the sodium-independent chloride-bicarbonate exchanger is activated by alkalinization, while the sodium-dependent chloride-bicarbonate exchanger maintains pH at the basal level. Further work is needed to determine if these exchangers have housekeeping function only or if they influence other aspects of cellular functioning. Interestingly, trabecular meshwork cells express active receptors for growth factors, the stimulation of which involves changes of cytosolic pH [116]. In addition, endothelin has been shown to increase pHi in trabecular meshwork cells [102]. Considering that endothelin contracts trabecular meshwork cells, it is possible that pHi is involved in the signalling cascade leading to contraction of trabecular meshwork, in parallel to observations on other smooth muscle cells [117, 118].
Electrophysiology of Trabecular Meshwork
The electrophysiology of trabecular meshwork has been investigated using both traditional measurement of membrane voltage and the patch-clamp technique in the
41
Fig. 10. In classical measurements of membrane voltage, application of high-potassium solution evoked depolarization, the extent of which was dependent on the concentration of potassium applied (bovine trabecular meshwork).
hopes of further illuminating signal transduction pathways leading to trabecular meshwork contractility. All electrophysiological experiments were carried out using cultured trabecular meshwork cells of either human or bovine origin [14, 101, 119122]. Bovine trabecular meshwork cells were all from the third passage. Human trabecular meshwork cells were obtained from donor eyes and cultivated for up to the eighth passage; for the cell puncture experiments, human cell lines were used. Resting Membrane Voltage Classical measurements of membrane voltage using cell puncture demonstrated the existence of three different cell types in cultures of bovine trabecular meshwork cells: a spindle cell type, with a membrane voltage of 71 B 2 mV (n = 48), an epithelial cell type with a membrane voltage of 50 B 1 mV (n = 143) and a mixed type with a voltage of 55 B 1 mV (n = 191) [14]. Five different cell lines of human trabecular meshwork displayed membrane voltages in the range of 44 to 63 mV [120], cell puncture experiments on cells from a primary culture of human cells fell within that range. All of these voltages are more positive than the equilibrium potential for potassium due to the flux of other ions like sodium, calcium or chloride across the cell membrane. This fact was confirmed by changing the concentration of extracellular potassium. This maneuver resulted in a depolarization of the cells which depended on the concentration of potassium applied (fig. 10). Subsequently, the relative K+ permeability could be calculated,
showing that potassium ions only account for 5070% of the total potassium conductance. No significant differences between human and bovine cells emerged [14, 101, 119, 120]. Excitability One of the key features of smooth muscle cells is their excitability pattern, the predominant feature of which are spontaneous oscillations of membrane potential called abortive action potentials or spikes which can either occur spontaneously or can be induced by such maneuvers as applying extracellular Ba2+ [123125]. In cell puncture experiments, both human and bovine meshwork exhibited such behavior [14, 120] so that electrophysiologically, they react like smooth muscle cells (fig. 11). Smooth muscle cell spikes are typically insensitive to tetrodotoxin, an inhibitor of fast Na+ channels; this behavior could also be observed in trabecular meshwork. Instead, the voltage oscillations depended on the presence of extracellular calcium and could be blocked by application of nifedipine, a highly selective Ca2+ channel blocker [101] (fig. 11). The exact sequence of events leading to the spikes has not been clarified [126]. However, it is known that the application of barium blocks a number of potassium channels and should therefore depolarize the cells. Depolarization in turn opens voltage-operated (nifedipine-sensitive) calcium channels; driven by the concentration gradient, Ca2+ ions flow in, leading to further depolarization. The rise in cytosolic calcium, especially in the compart-
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ment immediately under the cell membrane, should then activate calcium-dependent potassium channels, leading to repolarization in form of the downward stroke of the spike. This theory is validated by the fact that trabecular meshwork cells do indeed express calcium-dependent potassium channels in the form of maxi-K channels [121]. Interestingly, it could be shown that in trabecular meshwork, these channels are indeed insensitive to the application of external barium and should thus not be blocked by the barium necessary to evoke the spike. Electrical Characteristics of Ion Channels and Transporters In a series of experiments, we tried to determine what ionic channels and transporters are involved in regulating the membrane potential of trabecular meshwork cells and what pharmacological agents are able to modify this parameter. Potassium Channels. We have already indicated that potassium conductance is the major factor contributing to the resting membrane voltage of trabecular meshwork cells. Accordingly, an elevation of external potassium to 135 mmol/l depolarized both human and bovine trabecular meshwork cells by preventing efflux of potassium [14, 119, 120]. The same effect could be achieved by blocking potassium channels with Ba2+. More detailed analysis of the potassium channels involved was obtained using the patch-clamp technique; the results will be discussed below. Calcium Channels. While removal of calcium did not have an impact on resting membrane voltage, this maneuver lowered the excitability suppressing the formation of spikes (see above). The same effect was observed when L-type channels were blocked by the specific calcium channel blocker nifedipine or by the somewhat less specific verapamil [101, 103]. Sodium Channels. Tetrodotoxin, a blocker of fast sodium channels, had no effect on resting voltage [14, 119, 120]. However, this does not rule out a participation of these channels in the response to pharmacological stimulation [127]. Transporters. Ouabain, a blocker of the Na+/K+ATPase, depolarized trabecular meshwork [14, 120]. Low sodium and low bicarbonate induced a DIDS-sensitive depolarization in human trabecular meshwork cells. These effects can be explained by the presence of an electrogenic Na+-HCO symport, which mediates a flux of 3 bicarbonate coupled to sodium into the cell [14, 120]. This symport should also influence pHi and has been described in bovine corneal endothelium and human cil-
Fig. 11. In human and bovine trabecular meshwork, application of
barium resulted in typical spikes of membrane voltage. Spiking could be blocked by application of nifedipine, a specific blocker of L-type channels.
iary muscle [14, 120]. Interestingly, this transporter was absent in trabecular meshwork cells of bovine origin [14, 120]. Muscarinic, Adrenergic and Endothelin Receptors. Trabecular meshwork cells are known to express muscarinic receptors [22, 31, 128]. In measurements of membrane voltage on human trabecular meshwork, the voltage response observed upon application of acetylcholine exhibited the pattern typical of muscarinic receptors coupled to a phospholipase-C-dependent second-messenger system [120, 129] (fig. 12). This cascade generally leads to a release of calcium from cytosolic stores and to an influx of extracellular calcium. This elevation in calcium is thought to activate calcium-activated potassium channels, resulting in a transient hyperpolarization which we were able to observe in our preparation [130, 131]. However, the complete picture is far from clear [132] and also involves suppression of potassium channels. The predominant effect of muscarinergic stimulation, however, is that of a long, sustained depolarization (fig. 12), the exact cause of which has yet to be determined. Possibly, it is due to the opening of nonselective cation channels [32, 132]. The entire voltage response of trabecular meshwork cells to acetylcholine could be blocked by the application of atropine [120].
43
Fig. 12. Acetylcholine reversibly depolar-
ized human and bovine trabecular meshwork cells. Note the transient hyperpolarization prior to the prolonged depolarization.
endothelin A receptors in both tissues. The response to endothelin seems to follow a mechanism similar to that of other receptor-mediated signalling cascades. In summary, it appears that agents that contract trabecular meshwork depolarize trabecular meshwork cells. This corresponds to the classical, well-described mechanism known for other preparations of smooth-muscle tissue. However, it must be pointed out that contractility experiments using high-potassium solution imply that only part of the contractile response of trabecular meshwork is mediated by changes in membrane potential [29], an important part apparently being due to non-voltagemediated, pharmacomechanical coupling [36, 133].
Patch-Clamp Measurements
Fig. 13. Endothelin, too, depolarized trabecular meshwork cells of
both species in measurements of membrane voltage.
Application of isoproterenol, too, caused trabecular meshwork cells to depolarize [120]. The response was sensitive to metipranolol. Other authors, too, have described the existence of -adrenergic receptors in trabecular meshwork, mainly of the 2-subtype. As in ciliary muscle cells, endothelin dose-dependently depolarized cultured bovine and human trabecular meshwork cells [101] (fig. 13). This indicates the presence of
Measurements of membrane voltage have the advantage of leaving cells close to their native state. However, in order to determine if particular channels are indeed involved in determining the resting membrane voltage level of the cell or in its response to agents that alter membrane voltage, more information is needed than provided by a measurement of membrane potential. Using the patch-clamp technique, it is possible to determine both voltage and current during a measurement and to alter the ionic constituents on both sides of the cytosolic membrane. The drawback of this method is that voltage, which by definition is clamped using this technique, cannot fluc-
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Fig. 14. In patch-clamp experiments, human trabecular meshwork cells express an inward current, part of which depends on the presence of external sodium.
tuate freely as in physiological situations. The solution used to fill the cell via the pipette allows an exact determination of the reversal potentials to be expected but alters the protein content of the cell, making complex secondmessenger cascades virtually impossible. An alternative to exchanging the entire cytosolic fluid is the perforatedpatch modification of the patch-clamp technique [134], by which the cell membrane is perforated using the ionophore nystatin, allowing potassium (and voltage) to freely equilibrate on both sides of the membrane. This leaves the other ionic and proteinoid constituents of the cytosolic compartment untouched. Trabecular meshwork cells of both human and bovine origin were investigated using the patch-clamp technique. Inward Rectifier Potassium Channel Bovine trabecular meshwork cells exhibited an inward current at hyperpolarizing voltages that could be totally blocked by barium, tetraethylammonium chloride and withdrawal of potassium. As with the chloride currents, various pharmacological maneuvers had no impact on this conductance so that, until further evidence emerges, we are inclined to see the function of this channel in the domain of cell homeostasis [unpubl. observation]. Sodium Conductance An Na+-selective current has been described in human trabecular meshwork cells by Rich et al. [127], which we were also able to detect [103] (fig. 14). This current can be stimulated by melatonin (5 W 10 8 to 10 4 mol/l) [127]. Interestingly, both intraocular pressure and physiological
melatonin levels show a circadian rhythm [135]. However, more research is needed before a definite link between these two observations can be established. Outwardly Rectifying Potassium Conductance Trabecular meshwork cells of both human and bovine origin display a strong, outwardly rectifying current upon depolarization [121] (fig. 15). The outward current level was greatly reduced from 0.5 B 0.1 nA (n = 9) in solutions containing potassium to 0.05 B 0.01 nA (n = 4) in potassium-free solutions and blocked almost completely by application of tetraethylammonium chloride (n = 4). To assess precisely which potassium conductances are involved, various specific potassium channel blockers were applied. ATP-Dependent Potassium Channels ATP-dependent potassium channels, which shut down when levels of cytosolic ATP rise, have been identified in a number of tissues and are believed to play an important role in regulating smooth-muscle contractility [130]. Derivatives of sulfonylurea, like glibenclamide, are generally believed to be highly specific blockers of this channel. Bovine trabecular meshwork cells did not show a significant alteration in current when glibenclamide (10 5 mol/l, n = 7) was applied [122]. Although it cannot be ruled out that ATP-dependent potassium channels are activated in trabecular meshwork by some pharmacological maneuver, resting current shows no contribution of these channels.
45
Fig. 15A, B. Human and bovine trabecular
meshwork cells express a strong, outward current in patch-clamp experiments.
Small-Conductance Calcium-Activated Potassium Channels Another channel often described in smooth muscle tissue is the small-conductance calcium-activated potassium channel [130]. This channel is activated by elevation of cytosolic calcium, has a single channel conductance of below 100 pS and is specifically blocked by a compound from bee venom, apamin. In bovine preparations of trabecular meshwork, apamin (10 6 mol/l, n = 4) had no effect on resting current [122]. Again, this does not rule out measurable participation of these channels when the cells are in an activated state. Large-Conductance Calcium-Activated Potassium Channels Large-conductance calcium-activated potassium channels, also known as maxi-K channels, feature three properties: large conductance of over 200 pS, activation by elevation of cytosolic calcium (fig. 16A, B) and activation by an increase in resting voltage [136]. Charybdotoxin and, especially, iberiotoxin, both won from scorpion venom, are highly specific blockers of this channel [137] (fig. 16C, 17). Given in a dosage of 10 7 mol/l, iberiotoxin blocked current to 22 B 6% (n = 4, p ! 0.001) of current to outward current at 80 mV in bovine trabecular meshwork cells, while charybdotoxin blocked initial 71 B 16% (n = 4, p ! 0.001) of outward current [121], reflecting the greater potency of iberiotoxin for blocking this channel [138]. In human trabecular meshwork, the values were 42 B 8% (n = 5) for charybdotoxin (10 7 mol/l), while iberiotoxin (10 7 mol/l) blocked 44 B 6% (n = 9) of the total outward current. Statistically, no significant differences emerged between the species, either in the absolute levels of out-
ward current or in the contribution of the maxi-K channel to this current. Current voltage relationships were also identical. In human trabecular meshwork cells, elevation of cytosolic calcium by applying the calcium ionophore (10 5 mmol/l) led to a dramatic increase in the total outward current to 645 B 45% (ppaired ! 0.005, n = 3, V = 80 mV) of the original value; charybdotoxin blocked 37 B 6% (ppaired ! 0.001, n = 3, V = 80 mV) of this current. Subsequent single-channel measurements demonstrated the existence of high-conductance potassium channels with a conductance level of 326 B 4 pS (n = 10; bovine) and 302 B 13 pS (n = 4; human) in symmetrical potassium solution (135 mmol/l); again, significant differences between the bovine and the human species did not emerge. Both in human and in bovine cells, elevating cytosolic calcium from 10 7 to 10 6 mol/l had no significant effect on open probability; however, further elevation to 10 5 mol/l and higher greatly increased channel activity in excised inside-out patches. These values are in good accordance with values reported by other groups for other tissues. Cytosolic application of ATP (1 mmol/l) also enhanced open probability, a first indication of the fact that while maxi-K channels in trabecular meshwork are indeed stimulated by calcium, calcium is not the only and perhaps not even the physiologically most important stimulant. The effect of barium depended on the side to which it was applied: given from the cytosolic side, a block of outward current through maxi-K channels could be observed, while outside application of barium showed no effect on conductance through these channels. Possibly, blockage of maxi-K channels by barium occurs via the ball-andchain model.
46
Stumpff/Wiederholt
Fig. 16. Both single-channel measurements (A, B) and measurements in the whole-cell configuration (C) confirmed the existence of
maxi-K channels in bovine and human trabecular meshwork cells. Traces A and B demonstrate how these channels are stimulated when cytosolic calcium is elevated from 107 mol/l to 105 mol/l. The trace in C demonstrates a reduction in outward current by the specific blocker charybdotoxin.
Fig. 17. Experiment demonstrating the
stimulating effect of the tyrosine kinase inhibitor genistein on maxi-K channels. Throughout the experiment, cells were exposed to acetylcholine, in mimicry of contractility experiments on precontracted strips of trabecular meshwork. Outward current was significantly reduced by application of the specific blocker of maxi-K channels, iberiotoxin. In the presence of this blocker, genistein only evokes a small response. Only when iberiotoxin was withdrawn, could the full, reversible effect of genistein on outward current be observed. Both human and bovine trabecular meshwork cells responded in this way.
Stimulation of Outward Current by cGMP In contractility experiments, substances that elevate cyclic GMP, like the organic nitrovasodilatators ISDN and 5-ISMN or the nonnitrates SNP and SNAP, were shown to relax trabecular meshwork [64]. Direct application of the membrane-permeable cGMP analogue 8-bromo-cGMP to strips of trabecular meshwork evoked the same response [64]. In patch-clamp experiments, superfusion of bovine trabecular meshwork cells with a solution containing 8-bromo-cGMP (10 3 mol/l) evoked a stimulation of outward current to 290 B 57% (n = 4, p ! 0.05) that was sensitive to charybdotoxin (107 mol/l). These data indicate that cyclic GMP stimulates maxi-K chan-
nels. Extrusion of potassium is thus enhanced, leading to hyperpolarization. In the classical concept of the regulation of smooth-muscle contractility, this hyperpolarization should lead to a reduction in cytosolic calcium through a shutdown of L-type calcium channels and a reduced emission of calcium from stores [103]. Thus, relaxation of trabecular meshwork by substances that elevate cGMP involves stimulation of maxi-K channels. Stimulation of Outward Current by Tyrosine Kinase Inhibitors Tyrosine kinase inhibitors have been shown to relax trabecular meshwork cells [33]. Application of the tyro-
47
Fig. 18. Flufenamic acid stimulates outward potassium current in trabecular meshwork. The experiment demonstrates the stimulation of maxi-K channels by this compound, in analogy to the experiment depicted in figure 17.
sine kinase inhibitor genistein (5 W 10 5 mol/l) on bovine trabecular meshwork cells stimulated with acetylcholine resulted in a reversible increase in outward current to 578 B 154% (n = 16) of the initial current level [122]. In human trabecular meshwork, the effect was comparable (fig. 17). The effect of genistein was dosage dependent. Reversal potential was hyperpolarized by 15 B 3 mV (n = 9). Tyrphostin 51, a synthetic inhibitor of tyrosine kinases, had the same effect (433 B 46%; n = 7). Daidzein, a nonactive structural analogue of genistein, had no effect (n = 4). The stimulation of outward current by tyrosine kinase inhibitors could be blocked by substitution of potassium by tetraethylammonium ions, while the potassium channel blockers glibenclamide (K-ATP) and apamin (small-conductance calcium-activated potassium channel) had no effect. Blockage of the large-conductance calcium-activated potassium channel (maxi-K) by charybdotoxin or iberiotoxin (10 7 mol/l) suppressed 86 B 18% (n = 4) of the response. Depleting the cells of calcium did not have an effect on the current stimulated by genistein. In the excised inside-out configuration, open probability increased to 417 B 39% (n = 3) after exposure to genistein. It appears that both in human and in bovine trabecular meshwork, inhibition of tyrosine kinase stimulates maxi-K channels through a mechanism not involving changes in cytosolic calcium. As discussed above, this leads to hyperpolarization due to efflux of potassium and explains the relaxation observed in the contractility experiments.
Stimulation of Outward Current by Flufenamic Acid Flufenamic acid relaxes trabecular meshwork [32], reportedly by blocking nonselective cation channels. We tested the impact of flufenamic acid (10 5 mol/l) on membrane currents of trabecular meshwork cells. No impact on inward current was observed, as would have been the case if nonselective cation channels had been affected by flufenamic acid in a major way. Instead, we observed a strong rise in outward current, the major part of which was attributable to maxi-K channels [139] (fig. 18). It appears that in trabecular meshwork, flufenamic acid hyperpolarizes the cell by stimulating potassium efflux. Subsequently, calcium influx through L-type channels and other voltage-dependent influx pathways is reduced, intracellular calcium levels decline and the cell relaxes [32]. Calcium Channels Both human and bovine trabecular meshwork cells expressed L-type channels with an inactivation time constant of 157 B 76 ms (n = 8; bovine trabecular meshwork) and 194 B 167 ms (n = 9; human trabecular meshwork) in a solution containing 10 mmol/l Ca2+ [103]. No significant differences between the two types of tissue emerged. When calcium was substituted by barium, the inactivation constant typically increased to much larger values (1,582 B 440 and 1,449 B 396 ms, respectively; fig. 19A, B). Application of Bay K 8644 (10 5 mol/l) resulted in a significant increase in inward Ba2+ current to 141 B 10%
48
Stumpff/Wiederholt
Fig. 19. Both human and bovine trabecular meshwork cells express L-type calcium channels. A Trace in the presence of 10 mmol/l calcium. B Typically, inward current is enhanced by application of barium. C A further stimulation of inward current can be observed after exposure to the specific opener of L-type channels, Bay K 8644.
Transporters
2 K+ Na+
Na+ HCO3 HCO3
Na+ Cl K+
Na+ glucose 3 Na+
Channels
3 Na+ H+ Cl Cl
Signaling cascades second messengers Ca2+ (L-type) K+ Nonselective Na+ cation channel Ca2+ H2O (aquaporin)
s
[Ca2+]i
G-proteins Tyrosine kinases Serine/threonine kinase cGMP cAMP IP3
Membrane voltage
Relaxation/ contraction
Muscarinic M3>>M1
abAdrenergic
Endothelin (ETA)
Prostaglandins (TP, EP2)
Neuro- Melatonin Histamine peptides
Receptors
Fig. 20. Schematic representation of trabecular meshwork cell. A large number of transporters, channels and recep-
tors have been identified in these cells, many of which are involved in regulating smooth-muscle contractility.
ss s
ss s
Trabecular meshwork cell
Ca2+ K+ KIr (Inward rectifier)

s
K+
Maxi-K (KCa)
Na2+
49
(n = 13) of control value in bovine trabecular meshwork cells and to 150 B 2% (n = 7) of control value in human trabecular meshwork cells (fig. 19C). Application of nifedipine (10 3 mol/l) led to a significant decrease in inward Ba2+ current in bovine trabecular meshwork cells to 48 B 13% of control value with a recovery to 96 B 5% (n = 5). In human trabecular meshwork cells, the values were 64 B 4 and 106 B 24% (n = 4), respectively. The existence of voltage-activated L-type calcium channels in trabecular meshwork is further evidence of the smooth-muscle-like characteristics that allow trabecular meshwork to respond to agents that alter membrane voltage by contraction and relaxation.
Functional Antagonism between Trabecular Meshwork and Ciliary Muscle
From the above, it can be concluded that trabecular meshwork is indeed contractile and that trabecular meshwork contractility regulates ocular outflow in the sense that relaxation of trabecular meshwork enhances ocular outflow. Contractility experiments have demonstrated that differences exist between the regulation of the contractility of ciliary muscle and trabecular meshwork. Ideally, it should be possible to find a substance with a maximal relaxing effect on trabecular meshwork but only a minimal effect on ciliary muscle. Such a compound might lead to a new approach in glaucoma therapy.
Much work still needs to be done on clarifying the signalling cascade that eventually leads to the contractile response of trabecular meshwork. However, a large number of transporters, channels and receptors have been identified in trabecular meshwork, many of which are known to regulate smooth-muscle contractility (fig. 20). In particular, we have outlined how substances that relax trabecular meshwork stimulate the maxi-K channel, leading to hyperpolarization and ultimately affecting cytosolic calcium. Measurements of contractility demonstrate that a second, voltage- and calcium-independent pathway also leads to an alteration of trabecular meshwork tone. It is still not clear how these processes lead to a change in the shape of trabecular meshwork cells, but it appears that ultimately, these pathways would tend to alter the conformation of the cytoskeletal proteins actin, myosin and tubulin [27, 90, 140, 141]. Finally, we should point out that recent research indicates that a third smooth-muscle-like structure seems to be involved in regulating ocular outflow, namely the scleral spur [57, 16, 17].
Acknowledgements
The authors want to thank M. Boxberger for expert technical assistance and O. Strauss, K. Steinhausen, H. Thieme, Y. Que and R. Rosenthal for helpful discussions. This work was supported by DFG grant Wi 328/19 and Biomed 2 grant BMH4-CT96-1593.
References
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112 Coroneo M, Rssner A, Lepple-Wienhues A, Wiederholt M: Cytoplasmic pH regulation in cultured bovine trabecular meshwork cells. Invest Ophthalmol Vis Sci 1992;33:1165. 113 Chu TC, Keith C, Green K: Intracellular pH regulation by a Na+/H+ exchanger in cultured bovine trabecular cells. Acta Ophthalmol (Copenh) 1992;70:772779. 114 Helbig H, Korbmacher C, Stumpff F, CocaPrados M, Wiederholt M: Role of HCO in 3 regulation of cytoplasmic pH in ciliary epithelial cells. Am J Physiol 1989;257:C696 C705. 115 Helbig H, Korbmacher C, Stumpff F, CocaPrados M, Wiederholt M: Na+/H+ exchange regulates intracellular pH in a cell clone derived from bovine pigmented ciliary epithelium. J Cell Physiol 1988;137:384389. 116 Wordinger RJ, Clark AF, Agarwal R, Lambert W, McNatt L, Wilson SE, Qu Z, Fung BK: Cultured human trabecular meshwork cells express functional growth factor receptors. Invest Ophthalmol Vis Sci 1998;39: 15751589. 117 Smith GL, Austin C, Crichton C, Wray S: A review of the actions and control of intracellular pH in vascular smooth muscle. Cardiovasc Res 1998;38:316331. 118 Tepel M, Jankowski J, Ruess C, Steinmetz M, van der Giet M, Zidek W: Activation of Na+, H+ exchanger produces vasoconstriction of renal resistance vessels. Am J Hypertens 1998;11:12141221. 119 Lepple-Wienhues A, Stahl F, Wunderling D, Willner U, Schneider U, Wiederholt M: Membrane properties of trabecular meshwork endothelial cells Action of barium and endothelin. Pflgers Arch 1991;418:R76. 120 Lepple-Wienhues A, Rauch R, Clark AF, Grassmann A, Berweck S, Wiederholt M: Electrophysiological properties of cultured human trabecular meshwork cells. Exp Eye Res 1994;59:305311. 121 Stumpff F, Strauss O, Boxberger M, Wiederholt M: Characterization of maxi-K channels in bovine trabecular meshwork and their activation by cyclic guanosine monophosphate. Invest Ophthalmol Vis Sci 1997;38:1883 1892.
122 Stumpff F, Que Y, Boxberger M, Strauss O, Wiederholt M: Stimulation of maxi-K channels in trabecular meshwork by tyrosine kinase inhibitors. Invest Ophthalmol Vis Sci 1999;40:14041417. 123 Yamamoto Y, Fukuta H, Nakahira Y, Suzuki H: Blockade by 18beta-glycyrrhetinic acid of intercellular electrical coupling in guinea-pig arterioles. J Physiol (Lond) 1998;511:501 508. 124 Knot HJ, de Ree MM, Gahwiler BH, Ruegg UT: Modulation of electrical activity and of intracellular calcium oscillations of smooth muscle cells by calcium antagonists, agonists, and vasopressin. J Cardiovasc Pharmacol 1991;18(suppl 10):S7S14. 125 Hermsmeyer K: Ba2+ and K+ alteration of K+ conductance in spontaneously active vascular muscle. Am J Physiol 1976;230:10311036. 126 Korbmacher C, Helbig H, Haller H, Erickson-Lamy KA, Wiederholt M: Endothelin depolarizes membrane voltage and increases intracellular calcium concentration in human ciliary muscle cells. Biochem Biophys Res Commun 1989;164:10311039. 127 Rich A, Farrugia G, Rae JL: Effects of melatonin on ionic currents in cultured ocular tissues. Am J Physiol 1999;276:C923C929. 128 Gupta N, Drance SM, McAllister R, Prasad S, Rootman J, Cynader MS: Localization of M3 muscarinic receptor subtype and mRNA in the human eye. Ophthalmic Res 1994;26: 207213. 129 Coroneo MT, Erickson-Lamy KA, Wiederholt M: Membrane voltage recordings in a cell line (H1TM) derived from human trabecular meshwork. Invest Ophthalmol Vis Sci 1991; 32:942. 130 Nelson MT, Quayle JM: Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol 1995;268: C799C822. 130 Nelson MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot HJ, Lederer WJ: Relaxation of arterial smooth muscle by calcium sparks. Science 1995;270:633637.
131 Kotlikoff MI, Dhulipala P, Wang YX: M2 signaling in smooth muscle cells. Life Sci 1999; 64:437442. 132 Somlyo AP, Wu X, Walker LA, Somlyo AV: Pharmacomechanical coupling: The role of calcium, G-proteins, kinases and phosphatases. Rev Physiol Biochem Pharmacol 1999; 134:201234. 134 Horn R, Marty A: Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol 1988;92: 145159. 135 Liu JH, Dacus AC: Endogenous hormonal changes and circadian elevation of intraocular pressure. Invest Ophthalmol Vis Sci 1991; 32:496500. 136 Kaczorowski GJ, Knaus HG, Leonard RJ, McManus OB, Garcia ML: High-conductance calcium-activated potassium channels: Structure, pharmacology and function. J Bioenerg Biomembr 1996;28:255267. 137 Garcia ML, Knaus HG, Munujos P, Slaughter RS, Kaczorowski GJ: Charybdotoxin and its effects on potassium channels. Am J Physiol 1995;269:C1C10. 138 Gribkoff VK, Lum-Ragan JT, Boissard CG, Post-Munson DJ, Meanwell NA, Starrett JE, Jr, Kozlowski ES, Romine JL, Trojnacki JT, McKay MC, Zhong J, Dworetzky SI: Effects of channel modulators on cloned large-conductance calcium-activated potassium channels. Mol Pharmacol 1996;50:206217. 139 Stumpff F, Que Y, Boxberger M, Steinhausen K, Strauss O, Wiederholt M: Flufenamic acid stimulates maxi-K channels in trabecular meshwork cells. Pflgers Arch 1999;437: R79. 140 Epstein DL, Rowlette LL, Roberts BC: Actomyosin drug effects and aqueous outflow function. Invest Ophthalmol Vis Sci 1999;40: 7481. 141 Peterson JA, Tian B, Bershadsky AD, Volberg T, Gangnon RE, Spector I, Geiger B, Kaufman PL: Latrunculin-A increases outflow facility in the monkey. Invest Ophthalmol Vis Sci 1999;40:931941.
53
Full-Thickness Retinal Transplants: A Review

Fredrik Ghosh Berndt Ehinger
Department of Ophthalmology, Lund University Hospital, Lund, Sweden
Key Words Immunocytochemistry W Major histocompatibility complex W Neural development W Rabbit W Retina W Retinal degeneration W Transplant W Ultrastructure W Vitrectomy
Abstract Embryonic full-thickness rabbit neuroretinal sheets were transplanted to the subretinal space of adult hosts. This was accomplished by using a new transplantation technique involving vitrectomy and retinotomy. The grafts were followed from 10 to 306 days after surgery and were then examined by different histological techniques. In the light microscope, the transplants were seen to develop the normal retinal lamination and fusion with the host retina, especially after long survival times. Ultrastructurally, normal photoreceptor outer segments, well integrated with the host retinal pigment epithelium, were found. Growth cones were present in the zone of fusion between graft and host retina. Immunohistochemical labeling revealed many of the normal retinal components not previously found in retinal transplants, and graft-host connections between neurons in the rod pathway were seen. The morphology of vibratome-sectioned neuroretinal sheets as well as adult full-thickness grafts was also examined. These transplantation types showed
less of the normal morphology compared with embryonic full-thickness grafts. The immunogenicity of embryonic full-thickness and fragmented grafts was compared using major histocompatibility complex immunolabeling. Fragmented grafts elicited a response from the host immune system similar to a chronic transplant rejection. This reaction was absent in the full-thickness grafts which is in accordance with their good long-term survival.
Introduction
Retinal Transplants The main aim of retinal transplantation is to find a cure for degenerative retinal disease, primarily the group of diseases collectively called retinitis pigmentosa. The diseases within this group are very heterogeneous with varying onsets, severities and other general clinical manifestations, but they all have two things in common: (1) they manifest degeneration of photoreceptor cells, and (2) there is no effective treatment available, except for very rare cases where special diets may be beneficial [1, 2]. In recent years, much knowledge on the pathogenesis of
ABC
Fredrik Ghosh Department of Ophthalmology Lund University Hospital S22185 Lund (Sweden) Tel. +46 46 17 24 84, Fax +46 46 17 27 21, E-Mail fredrik.ghosh@oft.lu.se
these disorders has been gained. Many different mutations in genes which control the retinal biochemical machinery have been found, and many of them have also been correlated with defective protein synthesis [3]. The possibility of correcting defective genes is appealing, and experimental studies are in progress [4]. There are, however, both practical and theoretical problems to this approach, such as how to transfer genes specifically to diseased cells and when to perform the procedure. The rationale of retinal transplantation is to replace instead of repair the degenerating cells and thus provide a definite cure. During the second half of the 1980s, the field of experimental retinal transplantation expanded greatly, following in the footsteps of other successful neuronal transplantation experiments [5, 6]. Three different forms of retinato-retina transplantation techniques were soon developed. The first, described by Turner and Blair in 1986 [7], involved donor tissue in the form of embryonic retinas which were drawn into a syringe and thereby fragmented into small pieces. When placed in the subretinal space of the host, these grafts do not display the normal retinal appearance but develop into so-called rosettes, earlier seen in tumor-transformed retina, e.g. retinoblastoma. The rosette is a sphere of retinal tissue with layering similar to the adult retina, but with photoreceptor outer segments in the center and inner layers more peripherally. The fragment grafts contain many of the normal retinal cells [810] and have also been reported to sprout fibers towards the host retina [11] and to possess light-transducing properties [12]. Small pieces of a fragmented graft can survive for extended periods [13], but the major part loses its organization and degenerates after 45 months [14, 15]. A second transplantation technique was developed by del Cerro et al. [16], who used enzymatically dissolved retinal cell suspensions. These grafts show less organization when compared to fragmented counterparts [17] but have been reported to restore vision in light-blinded rats [18]. Cell suspension transplants have recently been performed in humans, but the results have not been encouraging so far [19]. The third method, which has also reached human trials, was developed by Silverman and Hughes [20]. Their concept was to replace only the cells most affected by degenerative disease, namely the photoreceptors. They developed a technique where the retinal graft was embedded in gelatin and then shaved with a vibratome so that only the photoreceptor cells remained. Initially, good results were reported, but they have not been reproduced.
In human experiments, the technique has been reported to be safe, but no improvement of vision has been established in operated patients [21]. Many important facts such as the superiority of embryonic versus adult tissue, the good differentiation of embryonic grafts and the lack of acute graft rejection have been established by work involving the methods mentioned above. They do, however, leave room for improvement, especially in the areas of transplant morphology, long-time survival and graft-host interactions.
Immunological Aspects of Retinal Transplants The eye is one of the immunologically privileged sites [22] where the immune response to foreign antigen is actively downregulated, thereby minimizing destruction of the surrounding tissue. The presence of the atypical low-keyed immune response in the eye, often named anterior-chamber-associated immune deviation [23], is clearly relevant for the survival of retinal grafts, even though recent investigations indicate that the privileged status of the subretinal space is not perfect [24]. Syngeneic embryonic fragmented grafts are well tolerated by the host, but their allogeneic counterparts display an upregulation of major histocompatibility complex (MHC) molecules, announcing an interaction and possibly a rejection by the host immune system [25]. This might explain why fragment transplants diminish considerably and lose their organization after 45 months [14]. Conversely, even xenogeneic fragmented transplants can survive for at least 41 weeks in immunosuppressed hosts [26]. The immunological status of the host as well as the immunogenicity of the graft obviously play important roles in the survival of neuronal transplants.
Neural Development This review describes a new procedure of neuroretinal transplantation [27]. The concept on which it relies is very dependent on basal mechanisms governing neural development, and a brief analysis of this subject is therefore motivated. In normal embryonic development, the cells of the body go through extensive changes resulting in a multitude of different tissues, all originating from the fertilized egg cell. Embryonic cells possess an immense plasticity, which is evident from the fact that each cell in the very early mammalian embryo can differentiate into any cell
Full-Thickness Retinal Transplants
55
type, depending on its environment [28]. This dependency is very prominent and crucial in neural development, where each cell needs to take part in a highly refined network. To achieve this, the developing neuron goes through two important phases. In the first, the cell after final mitosis migrates to its appropriate location. In this process, glial cells function as a scaffold, guiding the cell. The importance of this scaffold is well exemplified in the developing retina which will form rosettes instead of laminated layers if the prime glial cells, the Mller cells, are disturbed [29]. The second characteristic of neuronal development is axonal and dendritic pathfinding, a process governed by surrounding cells and extracellular matrix. A multitude of adhesion molecules, intercellular signals and growth factors guide the growing neurite towards its target. In the retina, the Mller cells are again important, providing both positional information as well as chemical cues for correct neurite growth [3032]. The target of the developing neurite also plays an important role in the construction of the neuronal network by releasing neurotrophic factors [33]. This is illustrated by the fact that embryonic retinal cells can be grafted to the brain and form connections with visual centers [34]. Together, these findings suggest that developing neurons are dependent on the integrity of their surroundings and on stimulation from their targets.
Vibratome-Sectioned Transplants To remove inner retinal elements [39], some adult and embryonic full-thickness grafts were embedded in gelatin and sectioned on a vibratome using the method described by Silverman and Hughes [20]. These embedded partial transplants were trimmed to a rectangular shape measuring approximately 1.5 ! 3 mm. Fragment Transplants The fragment transplants [36] were obtained by drawing an E19 embryonic neuroretina into a syringe.
Hosts
Pigmented mixed-strain rabbits, aged 35 months, were used as hosts in all experiments. In order to determine whether the animals might be syngeneic, 3 rabbits (litter-mates) from our local breeder received skin transplants from each other. All skin transplants were rejected after 7 days, suggesting a low degree of inbreeding among the animals.
Surgical Procedures
Preoperative Preparations The right eye of the recipient rabbit was instilled with cyclopentolate (1%) and phenylephrine (10%) 30 min prior to surgery. General anesthesia was provided with ketamine (40 mg/kg) and xylazine (5 mg/kg) intramuscularly. Topical tetracaine (0.5%) was applied just before surgery. Operation The conjunctiva was incised limbally 180 from 10 to 4 oclock with a vertical incision at 12 oclock, creating two flaps. A 20-gauge infusion cannula was sutured to the sclera in the 4-oclock position 1 mm posterior to the limbus and a balanced salt solution (Endosol; Allergan Medical Optics) was infused, containing adrenaline (5.5 ! 10 9 M ) for pupil dilatation and heparin (5 IU/ml) to minimize postoperative inflammation. A metal ring for support of a contact lens was sutured in place with limbal sutures at 3 and 9 oclock and sclerotomies were made in the 10- and 2-oclock positions. A vitrectomy contact lens was placed on the cornea with 2% methyl cellulose (Methocel ) as contact medium. As much vitreous as possible was removed, using a vitreous cutter (Ocutome ) and an intraocular illuminator. The large rabbit lens made it impossible to remove more than 5060% of the vitreous, and due to firm vitreoretinal adhesion the posterior hyaloid could not be removed with certainty. To detach a small area of neuroretina, a thin flexible polyethylene capillary (outer diameter: 0.6 mm, inner diameter: 0.4 mm) attached to a 1.0-ml syringe filled with Ames solution and supported by a blunt and slightly bent 20-gauge metal cannula was introduced through the sclerotomy at 10 oclock. Penetrating the retina 34 mm inferior to the optic nerve, it was positioned in the subretinal space. The fluid from the syringe was carefully infused, creating a limited circular retinal detachment with a diameter of approximately 3 mm. A second, smaller retinotomy was then made inferior to the first. The donor tissue was drawn into a glass cannula (outer diameter: 1.2 mm, inner diameter: 1.0 mm; fig. 1). In some experiments, the cannula had a slightly conical shape with the narrow end measuring 0.8 mm in outer and 0.6 mm in inner diameter. The cannula was
Donor Tissue
Embryonic Transplants Time-mated female pigmented rabbits were killed with 5 ml intravenous sodium pentobarbital (60 mg/ml), and the embryos were collected and used as donors in all embryonic transplants. Tissue from stage E15 to E19 (1519 days after conception) was used [27, 3539]. The eyes were enucleated, and the neuroretinas carefully dissected out and trimmed at the edges, forming an elliptical sheet of approximately 2 ! 3 mm. This sheet was kept in +4 C Ames solution [40] and used either as a full-thickness graft or was further processed by vibratome sectioning or fragmentation (see below). Adult Transplants Pigmented rabbits aged 35 months were used as donors for adult transplants [39]. The animals were killed with 5 ml sodium pentobarbital (60 mg/ml) intravenously, and both eyes were enucleated. The anterior segments were cut away with dissecting scissors. Using a 2mm circular biopsy punch (Stiefel), 46 disks of full-thickness retina were cut out from each eye. The neuroretinas were gently separated from the retinal pigment epithelium by infusing Ames solution subretinally. The neuroretinal disks were kept in +4 C Ames solution and were used either as full-thickness grafts or were further processed by vibratome sectioning (see below).
56
Ghosh/Ehinger
Fig. 1. Important steps in the transplantation procedure. A The full-thickness em-
bryonic grafts are stored in Ames solution. Prior to actual transplantation, the neuroretina and its accompanying fluid is drawn into a glass cannula. B A thin flexible polyethylene capillary is introduced through the vitreous. Penetrating the retina 34 mm inferior to the optic nerve, it is positioned in the subretinal space. Fluid from an attached syringe is infused, creating a limited circular retinal detachment (by permission from the journal Retina). C A second, smaller retinotomy is created (by permission from the journal Retina ). D The transplant is now introduced subretinally by means of the glass cannula. The fluid flows out of the second retinotomy, but the transplant stays in place due to its relatively larger diameter (by permission from the journal Retina ).
connected to a 1.0-ml syringe via a polyethylene tube, allowing the graft together with a small amount of the surrounding solution to be drawn in. As it entered the cannula, it adopted a curved shape. The graft was then carefully moved towards the end of the cannula by gently pushing the plunger of the syringe. The cannula was now introduced into the eye through the 10-oclock sclerotomy and its end was positioned against the superior retinotomy. The graft was pushed out of the cannula into the subretinal bleb, and while the accompanying fluid passed out into the vitreous space through the inferior retinotomy, the transplant stayed securely in place subretinally. On ejection, varying degrees of flattening of the transplant occurred. The donor retina was kept with its inner (vitreal) surface up throughout the procedure to maintain correct polarity. When kept in Ames solution, it had a tendency to fold, adopting its originally slightly curved state. This was favorable, as it needed to adopt this shape inside the glass cannula and also made the drawing in of the graft more atraumatic. In the cannula, the transplant was visualized and the correct orientation kept by twisting the instrument. The vibratome-sectioned transplants [39] were placed subretinally using a custom-made injector, consisting of a piece of flattened plastic tubing with a plastic piston. This injector had the same dimensions as the one used by Silverman and Hughes [20], with an inner width of 1.5 mm, an outer width of 2.0 mm and a height of 1.0 mm. The injector, being larger than the glass cannula, required a larger sclerotomy and retinotomy for the transplant procedure. Finally, the vitrectomy lens and its supporting ring were removed, the sclerotomies were sutured and 0.51.0 ml of air was injected into the eye to avoid hypotonia. After suturing the conjunctiva, 25 mg gentamicin and 2 mg betamethasone were injected subconjunctivally. The operation times averaged 35 min. The time from sacrifice of the mother to actual transplantation was noted and ranged from 60 to 460 min. For fragment transplants, 810 l of the retinal solution was placed in the subretinal space of the host using a polyethylene catheter inserted through the sclera and vitreous [36].
Postoperative Management and Follow-Up

No postoperative treatment was given. Ophthalmoscopic examinations were made on postoperative days 1 and 7, weekly for 1 month and thereafter monthly. The animals were killed 10306 days after transplantation.
Tissue Preparation
Light Microscopy For light microscopy, the eyes were enucleated and fixed for 30 min in formaldehyde (4%, generated from paraformaldehyde) at pH 7.4 in a 0.1 M Srensens phosphate buffer. The anterior segment was then removed and the posterior eyecup postfixed in the same solution for 4 h. Tissue specimens were obtained as approximately 2.5- to 4-mm-wide pieces, including the area of the transplant together with parts of the myelinated fibers and optic nerve. After fixation, most specimens were washed with Srensens phosphate buffer (0.1 M, pH 7.4) and then either washed again using the same solution with added sucrose of rising concentrations (5, 10, 15, 20%) or sectioned at 12 m on a cryostat. The remaining specimens were dehydrated, embedded in paraffin and sectioned at 7 m. The slides were then stained with hematoxylin and eosin. Cryostat sections were used in order to allow immunohistochemical analysis (see below). The vertical length of the transplants was measured directly in the light microscope, whereas their horizontal length was calculated from consecutive sections. Electron Microscopy Two transplants with postoperative times of 92 and 141 days were prepared for electron microscopy [37]. These tissue specimens were fixed for 4 h (4% paraformaldehyde mixed with 1% glutaraldehyde in 0.1 M, pH 7.2, phosphate buffer containing 0.15 mM CaCl2 at 4 C). They were then washed overnight in phosphate-buffered
57
Table 1. Antigen/antibody specifications
Antigen
Antibody name
Retinal target
Poly/ monoclonal
Concentration Source or working dilution 1:10,000 1:200 1:500 5 g/ml 1:100 1:100 1:100 1:10,000 Dr. K.R. Fry, The Woodlands, Tex., USA Sigma Chemical Co., St. Louis, Mo., USA Chemicon International Inc., Temecula, Calif., USA Boehringer Mannheim, Germany Dr. B. Wiggert Spring Valley Laboratories Inc., Woodbine, Md., USA Spring Valley Laboratories Inc., Woodbine, Md., USA Sigma Chemical Co., St. Louis, Mo., USA Sigma Chemical Co., St. Louis, Mo., USA Chemicon International Inc., Temecula, Calif., USA Boehringer Mannheim, Germany
AB5 (bovine brain extract) Calbindin (chicken gut calbindin 28 kD) ChAT (human placental enzyme) GFAP (purified glia filament) IRBP MHC class I (rabbit thymocytes) MHC class II (rabbit thymocytes) NF 160 kD (porcine spinal cord) Parvalbumin (carp muscle) PKC (human 80-kD PKC) Vimentin (bovine lens)
AB5 calbindin D ChAT G-A-5 IRBP MAb 73.2 MAb 45-3 NN 18
ganglion cells cone bipolar cells, horizontal cells cholinergic amacrine cells Mller cells IRBP ganglion cell axons, horizontal cell processes amacrine AII cells rod bipolar cells Mller cells
mono. mono. poly. mono. poly. mono. mono. mono.
parvalbumin human protein kinase C Vim 3B4
mono. poly. mono.
1:1,000 1:3,000 5 g/ml
ChAT = Choline acetyltransferase; GFAP = glial fibrillary acidic protein; IRBP = interphotoreceptor retinoid binding protein; MAb = monoclonal antibody; NF = neurofilament.
saline (PBS) and then in 0.15 M sodium cacodylate buffer, postfixed in 1% osmium tetroxide in 0.15 M sodium cacodylate buffer for 1 h at 4 C, washed in the buffer, dehydrated through a graded series of alcohol and embedded in Araldite (Fluka Chemie AG, Buchs, Switzerland). Ultrathin sections were cut on an LKB Ultrotome (Bromma, Sweden) and contrasted with uranyl acetate and lead citrate according to standard electron-microscopic procedures. The grids were examined in a Jeol 1200 EX electron microscope. Semithin sections were cut and stained with a mixture of methylene blue and azur II blue for preliminary analysis in the light microscope. Immunohistochemistry The sections were thawed and washed in 0.1 M sodium PBS with 0.25% Triton X-100 (PBS/Triton, pH 7.2). For labeling with the ABC kit, the sections were incubated with the appropriate normal serum at room temperature for 30 min and then with the primary antibody diluted in PBS/Triton with 1% bovine serum albumin (BSA) at +4 C [35]. After 1618 h, the sections were washed in PBS/Triton for 2 ! 15 min, incubated with the secondary antibody diluted in PBS/Triton with 1% BSA for 30 min,
washed in PBS/Triton for 2 ! 15 min, and reacted with an immunohistochemical avidin-biotin-peroxidase system (Elite ABC kit standard; Vector Laboratories). They were then washed in PBS/Triton for 2 ! 15 min, developed in 3,3)-diaminobenzidine solution (DAB kit, Vector Laboratories) for 210 min, washed in distilled water, dehydrated and mounted in Permount. For lectin demonstration, sections were incubated in biotinylated peanut agglutinin (Vector Laboratories) at 75 g/ml for 1 h after rinsing in PBS with 0.5% BSA. After incubation, the sections were rinsed and processed with the ABC kit. For single-labeling fluorescence immunohistochemistry, the sections were incubated with the primary antibody for 1618 h [35, 36, 38]. They were then rinsed, incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:100) for 30 min, rinsed again and finally mounted in custom-made antifading mounting media. For parvalbumin and protein kinase C (PKC) double labeling, the tissue was incubated with the parvalbumin antibodies for 1820 h, rinsed in PBS/Triton and then incubated with the PKC antibodies for 1820 h [38]. After rinsing in PBS/Triton, the tissue was incu-
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bated for 45 min in darkness with a mixture of the secondary antibodies conjugated with two different fluorophores (antirabbit FITC and antimouse Texas red). The dilution of each secondary antibody was 1:100. For calbindin and AB5 double labeling, a different approach was chosen since these antibodies were made in the same species (mouse) [38]. The sections were first incubated with the calbindin antibody for 1820 h. They were then rinsed in PBS/Triton and incubated with antimouse Texas red. After another thorough rinse in PBS/Triton, the AB5 immunolabeling was performed in a similar manner but with antimouse FITC as secondary antibody. This secondary antibody now recognized all mouse antigen in the tissue and, consequently, the FITC fluorescence was present at both anticalbindin and AB5-positive sites. To differentiate the AB5 and calbindin labeling, separate digital images of the tissue activity of the fluorophores were superimposed on each other. In this composite image, calbindin-positive sites were yellow (green + red) while AB5 was seen as green. Antibodies and dilutions are listed in table 1. Control experiments included labeling of normal adult rabbit retinas as well as labeling procedures without the primary antibodies on sections from operated animals. All proceedings and animal treatment were in accordance with the guidelines and requirements of the Government Committee on Animal Experimentation at Lund University and with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
found in the vitreous and shrunk with time. Adult grafts generally also diminished considerably after the first postoperative week.
Morphology of Full-Thickness Embryonic Grafts Light Microscopy In all series, approximately 1025% of the full-thickness grafts developed into rosettes without distinct lamination (fig. 2). The remainder were all in the form of laminated retinas with layers parallel to the host RPE. Their laminated length measured from 0.5 to 3.2 mm. In the smaller ones, disorganized cells in the form of rosettes were found at the edges of the transplant. In laminated transplants, the inner and outer segments appeared to be of normal length, facing the host RPE in the normal fashion. In transplants up to 3 months postoperatively, the outer nuclear layer was often thick and contained a large number of perikarya. In older ones (410 months), this layer was thinner and more comparable to the normal adult outer nuclear layer. The outer plexiform layer appeared normal. All inner retinal layers including the ganglion cell layer (GCL) and nerve fiber layer (NFL) were present in the majority of transplants, but the cells of the GCL were often smaller than normal ganglion cells. In young transplants, the GCL and NFL were often present throughout the major part of the transplant. In 4- to 10month specimens, these layers were found in a small section of the transplant if at all present. Minimal defects in the host RPE were seen in some cases. There were no signs of inflammatory cell infiltration in any of the specimens. Electron Microscopy Both long-term transplants examined in the electron microscope displayed long photoreceptor outer segments with the normal arrangement of stacked disks [37]. The inner segments appeared normal and contained the usual organelles. Well-developed photoreceptor terminals with horizontal and bipolar cell process invaginations were seen in the outer plexiform layer. The inner plexiform layer (IPL) was thinner than normal in the young transplants (92 days postoperatively) but of a more normal thickness in the older ones (141 days postoperatively). Bipolar and amacrine cell synapses were present in this layer but were not as common as in the normal rabbit retina.
Results Surgical Complications
Peroperatively, areas of missing host retinal pigment epithelium (RPE) were often seen after the bleb formation. Other peroperative complications in full-thickness transplantations included limited choroidal bleeding in approximately 10% of the cases and lens touch in another 10%. In a few cases, these complications led to an inflammation of the vitreous and retina with massive epiretinal membrane formation. In eyes receiving vibratome-sectioned grafts, complications were more serious and common [39]. Of these eyes, 30% displayed a significant vitreal hemorrhage which often led to epiretinal membrane formation and retinal tractional detachment on postoperative examinations.
Macroscopic Findings The embryonic full-thickness transplants were easily located at the operation site, under the host retina. Initially, they had the shape of a gray disk, approximately the same size as the optic disk. In time they became more transparent and often included small spots of hyperpigmentation. Vibratome-sectioned grafts were sometimes
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Fig. 2. Transplant morphology. A Embryonic E16 transplant (T)
10 days postoperatively. The cells form a neuroblastic cell mass with 1015 layers and a thin ganglion cell layer. No outer segments are seen. The transplant is folded with the part not against the host RPE forming rosettes. The host retina (H) has degenerated extensively, leaving only a thin inner retinal layer (by permission from the journal Retina ). Paraffin section. HE. Scale bar = 200 m. B E19 transplant (T) 35 days postoperatively. The major part of the graft faces the host RPE and is laminated with correct polarity. The host retina (H) is seen to straddle the transplant and is degenerating in this area (by permission from the journal Retina). Paraffin section. HE.
Scale bar = 200 m. C E19 transplant (T) 86 days postoperatively. The transplant displays all normal retinal layers including photoreceptor outer segments facing the host RPE. The host (H) outer retina has degenerated. Disorganized cells are present between the laminated parts of the host and transplant. Cryostat section. HE. Scale bar = 50 m. D E19 transplant (T) 309 days postoperatively. The laminated structure of the graft can still be appreciated, as well as the well-developed photoreceptors. Fusion of transplant and host has taken place at the level of the inner plexiform layer of the transplant. Cryostat section. HE. Scale bar = 50 m.
Immunohistochemistry Interphotoreceptor Matrix. Peanut agglutinin labeling of the laminated transplants showed segmentally arranged labeling at regular intervals in the photoreceptor outer segment layer [35]. When compared with the normal retina, the labeling was more compressed, i.e. with increased numbers of labeled structures in a given area, presumably reflecting a higher density of photoreceptor cells. In rosettes, PNA labeling was associated with most or all photoreceptors, as previously described [41]. In 5 of 6 grafts, specific IRBP labeling of the photoreceptor outer segment region was seen. When compared with the host, labeling was somewhat more intense in 4 of these transplants. Rod Bipolar Cells. In all 14 long-term transplants, PKC-labeled rod bipolar cell bodies were identified in their normal position in the scleral part of the inner
nuclear layer [37]. In the 3-month grafts, they seemed to be as common as in the normal rabbit retina, although no precise cell count was made. In 4- or 10-month grafts, their number had decreased moderately. The cells extended vertical axons with terminal bulbs in the vitreal part of the transplant IPL. The axons in some cases were longer than in the normal retina. Large and well-labeled structures consisting of minute fibers were seen in a zone more to the vitreal side of the transplant IPL. They were most common in the 3-month specimens and were often in contact with bipolar cell axons in the graft. The structures were interpreted as collections of growth cones [42]. The PKC-labeled rod bipolar cells extended dendritic branches to the most scleral part of the outer plexiform layer in the normal manner.
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In the host retina, PKC-labeled rod bipolar cells were often found but were not as common and not as strictly organized as in the normal retina. The cells extended axons with terminals in the most vitreal part of the host IPL. Dendrites extending towards the transplant were seen in specimens of all ages. These dendrites often displayed growth cones with minute filopodium-like processes at their tips. Cone Bipolar Cells. Calbindin-labeled cone bipolar and horizontal cells were found in all long-term transplants [37]. The cone bipolar cells were identified by their small size and their thinner, more vertical processes. These processes often ended in a network of fibers on the vitreal side of the transplant IPL. The cells were not as common as in the normal rabbit retina. In the host, labeled cone bipolar cells with processes terminating in the host IPL were seen, although these cells were not as numerous as in the normal retina. Some remaining horizontal cell processes were also noted in the host retina. AII Amacrine Cells. Parvalbumin-labeled AII amacrine cells in the 3-month specimens were often organized in 3 layers: (1) in the transplant, with cell bodies in the inner nuclear layer and GCL, and with processes in the IPL, (2) in an intermediate plexiform layer on the vitreal side of the transplant IPL and (3) in the host inner nuclear layer with processes in the host IPL [37]. In the 4- to 10month specimens, the AII amacrine cells around the transplant IPL were fewer, and more labeled cell bodies and processes were seen in the intermediate plexiform layer. The AII amacrine cells in the host retina of these specimens displayed branches in the host IPL. There was no apparent decrease in the total number of labeled cells in the oldest specimens (10 months postoperatively) when compared to younger ones. Cholinergic Amacrine Cells. Cells displaying cholineacetyltransferase-like immunoreactivity were found along both margins of the IPL of the transplant and host in a similar fashion [35]. When compared with the same section stained with hematoxylin and eosin, the majority of cells in the GCL of the transplant had not been labeled. Ganglion Cells. AB5-positive cell bodies were found in the GCL of 2 out of 6 short-term transplants, 31 and 49 days postoperatively [35]. The cells were large and showed branching like host retinal ganglion cells. AB5labeled fibers in the NFL were found in one additional graft. NF 160 kD labeling revealed labeled fibers in the NFL of all 6 grafts with horizontal or oblique fibers, either single or in clusters. In long-term specimens, a few AB5-
labeled large cells were seen in the GCL of 2 out of 14 transplants (86 and 126 days postoperatively) [38]. In the host retina, NF-160-kD-labeled fibers were found in the NFL of short-term specimens. AB5-labeled ganglion cells were seen in the GCL of the host in all 18 specimens investigated (110 months postoperatively). Their number seemed to be relatively constant in all specimens and they displayed branches in the host IPL as well as axons in the NFL. Mller Cells. Vimentin-labeled, straight and normally arranged Mller cells extending from the NFL to the outer nuclear layer, were seen in 6 out of 6 short-term transplants [35]. Normal, branched endfeet were seen forming a thin inner limiting membrane. The host Mller cell labeling was moderately stronger. Mller cells labeled by glial fibrillary acidic protein were found in 3 out of 6 specimens in the host retina [35]. In these specimens glial fibrillary acidic protein could also be detected in the Mller cells of the transplant. Their morphology was normal, but the labeling intensity was less than in the host. In 3 animals, no activity was found in either host or transplant. Graft-Host Integration Host Retina. The host retina appeared normal in all eyes, except for the part covering the graft where the outer layers had degenerated completely and the inner layers were affected in varying degrees. The inner nuclear layer, IPL, GCL and NFL of the host were preserved in specimens up to 3 months postoperatively but tended to thin out in older ones (410 months postoperatively). Photoreceptor-RPE Integration. In laminated transplants, photoreceptor outer segments were seen facing the host RPE in the normal manner. Small defects in the host RPE were noted in some of these specimens. In the electron microscope, transplanted photoreceptor outer segments were seen to integrate well with microprocesses extending from the host RPE [37]. Inner Retinal Integration. In specimens up to 3 months postoperatively, an array of disorganized cells, often in the form of rosettes, was evident between the laminated parts of the host and graft. These cells could not with certainty be defined as belonging either to graft or host, and prevented contact between laminated parts of the two. In 4- to 10-month specimens, the disorganized cells were few, and long segments without remaining GCL and NFL in the transplants were evident. This allowed for close contact between host and graft, and fusion was often evident at the level of the IPL of the transplant.
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The border between transplant and host retina could be identified more readily in the electron microscope and was seen in the vitreal part of the transplant IPL. Along this border, processes derived from grafted Mller cells arranged in a lamellar pattern were seen, but there was no sign of a limiting membrane with tight junctions. At regular intervals along the host side of the border, bundles of fimbriae of approximately 46 m were also present. These fimbriae were interpreted as Mller cells by their content of glycogen granules. On the vitreal side of these fimbriae, aggregates of mitochondria were seen in the Mller cell cytoplasm. Also closely associated with the fimbriae, bundles of nerve fibers were visible on both sides of the border. They contained mature nerve cell processes, identified by their contents of microtubuli, as well as irregular larger structures filled with clear vesicles of different sizes. These structures lacked vacuoles and waste products and were interpreted as growth cones [43]. Bundles of nerve cell fibers were also present closer to the host IPL, passing the host inner nuclear cells, in close contact with Mller cell processes. Graft-Host Connections In short-term grafts, NF-160-kD-labeled fibers running from the transplant vertically towards the host were seen [35]. In 8 of the 14 long-term transplants, double labeling with PKC and parvalbumin revealed numerous small PKC- and parvalbumin-labeled processes closely together in a thick intermediate plexiform layer located between host and graft [37]. These processes seemed to be derived from both host and graft rod bipolar and AII amacrine cells. In all these specimens, rod bipolar cells in the host extended sprouting dendrites towards the intermediate plexiform layer. Sprouting fibers from host AII amacrine cells were also common. Direct contacts between rod bipolar cells in the transplant and AII amacrine cells in the host were seen in 3 of the specimens. AII amacrine cells in the transplant were sometimes seen to extend fibers terminating on rod bipolar dendrites in the host. Contacts between calbindin-labeled cone bipolar cells in the graft and AB5-labeled ganglion cells were not apparent. In one specimen, 126 days postoperatively, a few AB5-labeled fibers from the host came very close to calbindin-labeled cone bipolar axons from the graft, but no definite contact was seen. In the 10-month specimens, the distance between ganglion cell processes from the host and cone bipolar cell processes from the transplant was short, yet even then, no direct contacts were seen.
Morphology of Other Transplants Adult Full-Thickness Grafts One out of 4 adult full-thickness transplants could be identified in the light microscope [39]. This specimen was obtained 14 days after transplantation and the graft measured 0.6 mm in its vertical extent. It displayed laminated layers, but with a reduction in number of all cell types. Embryonic Vibratome-Sectioned Grafts Nine out of 14 embryonic vibratome-sectioned grafts survived the postoperative period of 1127 days. They all displayed abnormal morphology with rosettes and sometimes also laminated retinas with reversed polarity [39]. The laminated segments often included many inner retinal cells, but these were always adjacent to the host RPE or Bruchs membrane. Adult Vibratome-Sectioned Grafts A total of 5 out of 20 adult vibratome-sectioned grafts could be identified with certainty [39]. These grafts displayed normal lamination and in all parts included the inner retina, suggesting that the vibratome sectioning in these cases had been incomplete. The photoreceptor outer segments faced the host RPE but were shorter than normal. No rosettes were seen. Four of the surviving transplants were from postoperative day 1516 and measured from 0.4 to 1.3 mm in their vertical extent. One was from day 29 and measured 0.6 mm. Embryonic Fragment Transplants The fragment transplants were all in the form of rosettes, often with a certain degree of differentiation, and with photoreceptor outer segments facing in towards the lumen [36]. In the majority of specimens, large defects in the host RPE were seen. There was no apparent invasion of lymphocytes in any of the specimens, but in 2 out of 5 fragment transplants (49 days postoperatively), large clusters of cells which did not have the morphology of inflammatory cells were seen in the choroid and graft.
Immunogenicity MHC Expression Normal Tissue and Controls In normal adult and embryonic retina, no MHC-classI- or -class-II-labeled structures were seen [36]. The choroid of adult specimens displayed single MHC-class-Iand -class-II-labeled cells. The MHC-class-I-expressing cells were round and had no processes. The MHC-class-
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II-expressing cells had an elongated appearance and often displayed 1 or 2 dendritic processes. There was no MHC class I or class II labeling in the negative controls. Full-Thickness Transplants None of the 7 full-thickness grafts displayed any MHC class I and class II labeling, and there was no sign of increased MHC expression in the choroid when compared to normal specimens. Fragment Transplants Cells expressing MHC class I were found in 2 animals (31 and 49 days postoperatively). In the younger transplant, a few clusters of labeled cells were found in the rosettes. The older one displayed an array of numerous labeled cells in an area corresponding to the host choroid and scleral part of the graft. In this area, no RPE cells could be seen. Cells expressing MHC class II were found in 4 of the 5 specimens. In 2 of these specimens (31 days postoperatively), clusters of a few labeled cells in the graft were present in areas with small RPE defects. In the other 2 (49 days postoperatively), large aggregates of MHC-class-IIexpressing cells in the choroid and scleral part of the graft were seen in areas where the RPE was completely missing. The labeled cells in the choroid were mostly concentrated in the part adjacent to the transplant where they were considerably more frequent than in the normal rabbit. Their number decreased and returned to normal at some distance from the graft. Many of the cells labeled by MHC class I and class II had relatively large, elongated cell bodies with long dendritic processes, but the ones found inside rosettes were more round and lacked processes. There were no MHCexpressing cells in the vitreal part of the grafts or in the host retina. In the fifth specimen (31 days postoperatively), no MHC class I or II labeling was found.
and finally held in place by gas (20% sulfur hexafluoride) after a gas-fluid exchange. This procedure allowed for a more complete vitrectomy than the current one, and the graft was kept flat and in position. All of these cases, however, developed serious epiretinal membranes and eventually a traction detachment. To avoid epiretinal membranes, the procedure was simplified in the manner described above. Initially, only the first retinotomy was included, and in 50% of the operations, the graft ended up in the vitreous due to fluid reflux through the one existing retinotomy. After the introduction of the second retinotomy, this was no longer a problem. Other groups have succeeded in transplanting sheets through only one retinotomy [21], but we found the second retinotomy to be essential. Vitrectomy Vitrectomy was for several reasons used in the transplantation procedure. The established technique with an internal light source gives good access to the retina which enables the delicate microsurgery. The actual removal of vitreous also makes it easier for the retina to lift, allowing a localized bleb of retinal detachment to be created, and facilitates the positioning of the transplant subretinally. Although a complete removal of the vitreous is not possible with the lens in place, a central vitrectomy including the area of transplantation is a sine qua non for the success of the procedure. Other groups have tried to perform full-thickness transplantation without the three-port vitrectomy technique [44, 45]. The results, however, indicate a comparatively low frequency of successful grafts. Vitrectomies have been safely performed in rabbit eyes for many years [4648], but complications do occur and are well known. The size of the rabbit lens is considerably greater than its human counterpart, and navigating the vitrectomy instruments between this large lens and the retina can be a perilous task since neither tissue accepts maltreatment. The rabbit lens responds to operation trauma by cataract formation and inflammation leading to epiretinal membranes. Removing the lens altogether is not a good option, since lensectomy is in itself well known to precipitate inflammation leading to traction detachment [49]. Epiretinal membranes also develop in response to vitrectomy [50], but in our procedure this was effectively counteracted by using heparin in the infusion solution [51]. Retinal detachment is perhaps the most serious complication of vitrectomy but is usually caused by vitreous traction associated with lensectomy and did not occur in our series once we had omitted the lensectomy.
Discussion
Surgical Considerations Development of the Transplantation Technique The surgical procedure described for full-thickness transplantation evolved from a series of pilot experiments. The earliest technique involved lensectomy, vitrectomy and a 3-mm retinectomy. The transplant was then positioned in the defect produced by the retinectomy
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Placing the Donor Tissue The most delicate part in the transplant procedure was positioning the graft flat and with correct polarity. The tendency of the donor tissue to arch enabled it to be drawn in with little trauma into the glass cannula. When placed in the subretinal space, it unfolded to a certain degree, but the margins of the graft were often elevated. Judged by the flat appearance in the light microscope, the combination of transplant growth and the diminishing bleb with the host retina pressing down on the transplant is evidently enough to flatten the bulk of the tissue. Embedding retinal sheets in gelatin keeps them flat throughout the transplant procedure but has some disadvantages compared with the glass cannula used for fullthickness transplantation. Silverman and Hughes [20] worked with rats and mice and used a transcorneal approach to place vibratome-sectioned sheets subretinally. This procedure involved detachment of the retina from the ora serrata to the posterior pole. To avoid such trauma to the host retina we favored the transvitreal approach also used by Kaplan et al. [21] for our gelatin-embedded grafts [39]. This method requires an eye of approximately the same size as that of humans but minimizes detachment of the host retina. The instrument used for gelatin-embedded grafts is wider than the glass cannula and consequently demands a larger sclerotomy and retinotomy. This enhances the risk of per- and postoperative hemorrhage due to scleral and choroidal damage. In addition, a large sclerotomy makes the intraocular pressure more difficult to maintain at a constant level. In our material, blood in the vitreous was evident in approximately 30% of the eyes receiving gelatin-embedded grafts. Most of these eyes also developed a total retinal detachment. Approximately 10% of the eyes receiving full-thickness grafts with the glass capillary displayed a vitreal hemorrhage, and retinal detachment was rare. A large retinotomy, apart from causing more damage, also makes the bleb of the host retina more unstable. Kaplan et al. [21] were forced to use hyaluronate sodium to maintain the height of the bleb, which in 1 out of 2 cases led to incomplete healing of the retinotomy. Another complication of the large retinotomy is that the transplant does not stay in place. In our full-thickness grafts approximately 5% of the grafts ended up in the vitreous, while 10% of the vibratome-sectioned ones did so. To conclude, our results suggest that full-thickness grafts can be placed accurately with a standardized, safe operation technique. Gelatin-embedded grafts are difficult to transplant for physical reasons, and this procedure is, at least in our hands, associated with complications such as vitreous hemorrhage and retinal detachment.
Transplant Morphology Organization and Survival Good long-term photoreceptor survival has previously been reported in microaggregate (fragment) transplants [13]. However, the success rate was comparatively low and could not be influenced easily, as small pieces of donor tissue were placed at random subretinally. One important conclusion from the work on fragment transplants is that grafted photoreceptors will only survive if they are oriented correctly, i.e. facing the host RPE. The majority of our embryonic full-thickness transplants displayed a laminated morphology and they extended over areas of more than one disk diameter. The photoreceptors showed well-developed outer segments facing the host RPE even after long survival times. The laminated flat morphology of the full-thickness grafts thus seems advantageous for photoreceptor cell survival. The grafts contained many of the normal retinal components, and their organization in most cases mirrored the one found in the normal rabbit retina. Specifically labeled ganglion cells, however, were not common, even though a GCL was often seen. In short-time transplants we found cells in the GCL labeled with AB5, a specific ganglion cell marker [52], and also NF-160-kD-labeled fibers in the NFL, indicating that ganglion cells were present. Many cells in the GCL, however, were not labeled either by AB5 or choline acetyltransferase, suggesting that a number of the cells were not standard ganglion or amacrine cells. Rosette Formation Fragment transplants are well known to form rosettes with an initial degree of layering [8]. A small fraction of our full-thickness transplants developed into rosettes which indicates that other factors than the donor tissue integrity may be important in rosette formation of neuroretinal grafts. Why rosettes appeared in these transplants is not quite clear. All transplants were mechanically separated from the RPE in the donor eye, and it is possible that excessive force, enough to cause an abnormal development, was used in these cases. Another cause of rosette development of full-thickness grafts may be reversed polarity, either peroperatively or during the first postoperative days when then host retinal bleb was still present. Two of our long-term grafts developed into rosettes and displayed a degenerated morphology which is in accordance with the fate of other rosetted transplants [8]. The fact that transplants rich in rosettes as well as disorganized parts of otherwise laminated transplants tend to
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degenerate, again confirms that a normal organization of the transplanted retina is important for its survival. Embryonic versus Adult Donor Tissue The good survival of immature retinal transplants is well documented [13, 16, 20], while studies on adult transplants are not as common. Our results indicate that embryonic transplants survive better than their adult counterparts. In our relatively small group of adult fullthickness grafts, only one survived, and this graft showed severe signs of degeneration [39]. Adult retina is very sensitive to ischemia, which is evident by the rapid degenerative changes occurring post mortem [53]. Differences in tolerance to drastic environmental changes, such as dissection and transplantation may in part be responsible for the poor survival of adult grafts. In our 5 (out of 20) surviving adult vibratome-sectioned grafts, large parts of the inner retina remained which of course indicates that vibratome sectioning in these grafts was not complete and that the surviving transplants were in effect almost full thickness. The implication of this observation is interesting however adult transplants can survive, at least for short periods, if they are gelatin embedded and the inner retina remains. The finding is consistent with those of Silverman et al. [54] who in their vibratome-sectioned transplants observed a better preservation of the photoreceptors when portions of the inner retina were included in the transplant. In conclusion the embryonic full-thickness transplants develop well and survive for at least 10 months. From photoreceptor outer segments to IPL their morphology is comparable to the normal retina.
Integration Two factors prevented graft-host fusion in the shortterm specimens: (1) the well-developed inner retinal layers in the graft and (2) the presence of disorganized cells without proper lamination between the laminated layers of graft and host. Longer surviving times led to a loss of the innermost layers of the transplant and to a decrease in the number of intervening nonlaminated cells. In long-term specimens, complementary elements from host and graft together almost reconstructed the normal retinal appearance with all layers present. Connections In the 2 specimens examined in the electron microscope, we were able to identify and characterize the border between graft and host. Fiber bundles containing mature nerve fibers as well as growth cones on both sides of this border were found. The close relationship of these neurites with Mller cell fimbriae and mitochondria at regular intervals along the border suggests that neuron sprouting might be guided by these cells. This is comparable to normal retinal neuron development in which Mller cells are known to play an essential role [29, 32, 55]. Growth cones are usually associated with developing neurites [42, 56] but are also present in adult retinal regenerating neurons, especially when stimulated by an embryonic environment [57] or retinal detachment [58]. Using immunohistochemistry, the growth cones of our specimens were found to be both host and graft derived, originating predominantly from rod bipolar and AII amacrine cells. The sprouting activity seemed directed towards the formation of an intermediate plexiform layer which most likely represented a vitreally displaced transplant IPL fused with inner layers of the host. The abundance of neurites from both host and graft in this layer suggests that it might function as a switchboard between neurons of graft and host. The intermediate plexiform layer was not organized in sublaminae, and the order of neurons was not always clear. The direct connections found between grafted rod bipolar cells and host AII amacrine cells however indicate that connections between interneurons normally found in the rod pathway [59] can be established. The AB5 and calbindin double-label experiments showed that both cone bipolar and ganglion cells survived for at least 10 months in the host, and that they often retained their normal contacts in the host IPL. This finding is of great importance since they constitute the last two elements in the rod pathway and thus are critical for any graft-host transmission of visual information. In severe
Graft-Host Integration and Connections Degeneration of the Host Retina A prerequisite for good graft host integration of fullthickness transplants is degeneration of the host outer retina. The host retina, even in our short-time grafts, very much resembled a retina affected by degenerative disease with an almost complete loss of the outer nuclear layer. The retina of the rabbit is merangiotic, i.e. vessels are confined to a horizontal band at the myelinated nerve fibers, and this makes it vulnerable when separated from the RPE. Degeneration of the outer nuclear layer of the host has also been reported in fragment transplants to holangiotic (completely vascularized) retina [17], indicating that other mechanisms than separation from the RPE may be involved in this phenomenon.
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retinitis pigmentosa, 70% of the ganglion cells perish, possibly because of transsynaptic degeneration [60]. This can be compared with our specimens, where the outer retina of the host rapidly degenerates but where most of the ganglion cells seem to survive long postoperative times. Whether host ganglion cells survive because of input from the transplant remains to be investigated. Very few of the cone bipolar cells found in the transplant made direct contact with ganglion cell processes from the host. The cone pathway is difficult to analyze since only a fraction of the cone bipolar cells can be labeled with commercially available antibodies. In the normal rabbit retina, however, contacts between calbindin-labeled cone bipolar and AB5-labeled ganglion cell processes were readily seen, and these results indicate that graft-host connections in the cone pathway of our specimens are not common.
Immunology of Neuroretinal Transplants The results of our immunological study indicate a difference in immune response depending on donor tissue integrity. The lack of any detectable expression of MHC in the full-thickness transplants suggests a low degree of immunogenicity in these grafts which is encouraging and in accordance with their good long-time survival. The different state of the fragmented donor tissue does not readily explain why these grafts display an enhanced MHC expression, and a further analysis of this phenomenon is therefore necessary. The literature on host immune responses to neuroretinal grafts is limited, but more knowledge based on other types of transplants is available. A classic graft rejection is triggered by allogeneic MHC molecules [61], and when cells expressing these antigens are grafted to an incompatible host, an immune response leading to rejection ensues [62, 63]. The response is characterized by MHC upregulation in graft-derived cells followed by upregulation in host cells and subsequent T cell infiltration [64, 65]. The immune response seen in our fragment transplants differs from the one seen in classic rejection in two ways: first, as will be further discussed, most cells that expressed MHC appeared to be host derived; secondly, the reaction was limited to an increase in MHC-expressing cells without any obvious infiltration of lymphocytes, indicating that a full-blown graft rejection was not at hand.
The Origin of MHC-Expressing Cells The tight junctions of the RPE constitute the outer blood-retina barrier which prevents the passage of large molecules and cells to and from the retina. This barrier was broken in both full-thickness and fragment transplantation procedures by the injection of fluid into the subretinal space which swept away many of the RPE cells. In the fragment transplants, the RPE defects appeared to have healed only partially and were often associated with an increase in MHC-labeled cells, many of which had a dendritic morphology. The concentration of labeled cells in the choroid and scleral part of the graft and the presence of cells with similar morphology in the normal choroid support the assumption that they were host derived. The breakdown of the blood-retina barrier in fragment transplants thus seems to permit dendritic cells from the host choroid to invade the graft. The delayed healing of RPE defects associated with these grafts as compared with normal RPE healing [66] indicates that the fragmented donor tissue affects the host RPE, either directly or through a response from the host immune system. The RPE defects in full-thickness grafts on the other hand healed well, and the remaining small breaks were never associated with any increase in MHC-expressing cells. This suggests that full-thickness grafts do not affect the host RPE in the same manner as their fragmented counterparts and that a disturbance in the blood-retina barrier alone is not enough to provoke a reaction from the host immune system. The labeled cells found in the lumen of rosettes in the graft were presumed to be macrophages as previously described [67]. Whether these cells were host or graft derived is difficult to ascertain, but they probably represent transformed RPE cells [68] which have not been removed during the dissection of the donor neuroretina. The Host Immune Response We found no MHC expression in normal embryonic retinas suggesting that triggers of the immune response in fragment transplants involved predominantly minor instead of major histocompatibility antigens [69]. The apparent absence of lymphocytes and the abundance of dendritic cells in the grafts suggest that the rejection process may be different from the one seen in classic graft rejection. Dendritic cells are known for their constitutive expression of MHC class II antigens [70, 71] and have been found in a variety of tissues including the choroid where they can increase in number in response to experimental uveitis [72]. To our knowledge, they have not been described in retinal transplants previously, but host-
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derived dendritic cells have been implicated in experimental brain transplants [73] and have also been found in human renal grafts [74]. Their exact role in the rejection process is uncertain, but one possible function is to extract antigens from the graft and present them to host T cells [75]. Fragmenting the graft may in this setting increase antigen exposure to dendritic cells, which would help to explain the difference in the immune response between fragmented and full-thickness transplants.
Conclusions
Future Considerations of Neuroretinal Transplants The important question regarding the ability of embryonic full-thickness grafts to transfer visual information to the host has not yet been answered. One disadvantage with our model is that the recipient rabbits have normal retinas, which makes electrophysiological and behavioral testing difficult. To get more information on graft-host transmission of useful visual information, animals suitable for vitrectomy also displaying retinal degeneration will probably have to be used in the future. Another important issue is how grafts are to be obtained in a future clinical setting. Our experiments confirm earlier findings on the superiority of embryonic grafts over adult ones, which makes access to immature tissue desirable. Human retinal cells from aborted fetuses have already been used in clinical trials involving cell suspension transplants [19], and fetal brain transplants have been used for a number of years in surgical treatment of parkinsonian syndromes [6]. Examining other sources of donor tissue is important, however, since the amount of human fetal tissue is limited. Possible sources include tissue from cell cultures and xenogeneic grafts [26], in which the low immunogenicity of full-thickness grafts may prove favorable.
Full-thickness embryonic retinas can be transplanted into adult rabbits with a minimum of complications. The transplants develop into laminated retinas with layers parellel to the host RPE and display most of the normal retinal morphology. The survival time of the transplants is at least 10 months, after which they still retain their laminated appearance and show no signs of photoreceptor degeneration. In long-term specimens, a graft-host adaptation occurs where neurons from both entities coalesce to form an intermediate plexiform layer. Graft-host connections exist, and the participating neuronal types are the ones seen in the normal rod pathway of the retina. Fragmented neuroretinal transplants trigger a host immune response characterized by an increase in MHCexpressing cells which is not present in full-thickness grafts, indicating that the integrity of the donor tissue is important for favorable host integration.
Acknowledgements
This work was supported by a BioMed2 EU grant, the Swedish Medical Research Council (project 2321), the Faculty of Medicine at the University of Lund, the Segerfalk Foundation, the Crafoord Foundation, the H. and L. Nilsson Foundation, the Royal Physiographic Society in Lund, the Clas Groschinskys Memorial Foundation, the Swedish Society for Medical Research, the Edwin Jordan Foundation for Ophthalmic Research and Crown Princess Margaretas Foundation for Blind Children.
References
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Ultrastructure of Retinal Cells Transplanted to the Rabbit Choroid

Elke Ltjen-Drecoll a Anders Bergstrm b Berndt Ehinger b
a Department
of Anatomy II, University of Erlangen-Nrnberg, Germany; b Department of Ophthalmology, University Hospital, Lund, Sweden
Key Words Rabbit W Choroid W Retinal cell transplants W Ultrastructure
Abstract Purpose: Allogenic rabbit-to-rabbit retinal cell transplants survive in the choroid, which is not as expected because it has not been shown that this is an immuneprivileged site. We have therefore examined the ultrastructure of such transplants, looking for features that might explain the phenomenon. Methods: Rabbit retinal tissue fragment transplants were produced with previously described methods. The donor age was 15 days and the transplants were examined by standard electron microscopy when the transplants were 12 months (3 transplants) or 34 months old, of postconception age (3 transplants). Results: The transplants survived and developed as expected from previous observations. Rosettes were seen, but they were not as common as in transplants produced with the same technique in the subretinal space of rabbits. Photoreceptor outer segments were not seen in the transplants. At 1 month, there was an incomplete sheath of Mller cells around the transplants, and a complete one at 34 months. There was also a well-developed basement membrane around the transplant at 34 months, but less so at 1 month. Blood vessels did not enter the transplant. The fenestrations in the choriocapillaris were not affected as long as the pigment epithelium was normal. Conclusions: The
enclosure of the transplants by Mller cells might help to insulate them from the immune system of the host, but it is a late phenomenon and it is not likely to have much effect for the first few weeks after the transplantation. We suspect that either the rabbit choroid is an immune-privileged site, even though there is no previous direct evidence for this, or that the retinal tissue itself is responsible for the prolonged survival at this site.
Introduction
Fetal retinal cells can be transplanted to the interior of the eye [for reviews, see 14] where they continue to develop, differentiating into mature cells. It is not necessary for the transplant to have the same cell recognition antigens as the host, and it is generally enough that the transplant comes from the same species as the host. The interior of the eye thus appears to be immunologically relatively inert. The reasons for the special immune-privileged status of the interior of the eye are not fully known and are likely to be complex. One of them is that there is an active downregulation of the immune responses in the interior of the eye (the ACAID system [5]), making it immunologically privileged. The system appears to be present in mammals only [6]. It is regarded as a dynamic state in which the immune response to ocular antigens is modi-
ABC
Dr. B. Ehinger Department of Ophthalmology Lund University Hospital S221 85 Lund (Sweden) Tel. +46 46 17 16 90, Fax +46 46 211 50 74, E-Mail berndt.ehinger@oft.lu.se
fied by the eye itself. The immune deviation involves the appearance of unique immunoregulatory factors in the aqueous humor. Antigen-presenting cells in the eye pick up some antigen and leave the eye through Schlemms canal to enter the blood stream. In the spleen, they activate a unique spectrum of antigen-specific T and B cells. When returning to the eye these cells induce a specific ocular immune tolerance to the antigen. There is no systemic delayed hypersensitivity nor do any complementfixing antibodies appear in the circulation. This immune privilege may be the eyes way of protecting its vital functions from immunopathogenic injury. The cells of the pigment epithelium of the eye are connected by tight junctions, which prevent the free movement of water and solutes across the epithelium. The endothelial cells of the retinal blood vessels also constitute a barrier for most substances. This is often referred to as the blood-retina barrier or the blood-aqueous barrier. The choroid is thus walled off from the retina by the pigment epithelium, and since it is highly vascularized and since its capillaries are fenestrated, blood components have rapid access to it [7]. Transplants with cell and tissue recognition antigens different from those of the host might therefore be expected to survive poorly. Preliminary experimental results in the monkey by Eichhorn et al. [8] suggest that when the uveoscleral outflow is increased with prostaglandin F2 isopropyl ester, the ACAID reaction is no longer inducible. This may be a direct effect of the prostaglandin, or the uvea may not be an immuneprivileged site in this species. In rabbits, on the other hand, Bergstrm [9] noted that transplants develop and survive also in the choroid, suggesting that the environment is for some reason more protective in this species than in, for instance, rats in which transplants have not been seen to survive [Ehinger and Larsson, unpubl. data]. We have therefore studied the ultrastructure of retinal cell transplants to the rabbit choroid, examining possible anatomical features that might protect them. There is no previous such study available. We have found certain anatomical barriers that appear to play a role, but it seems unlikely that they are the only factors involved.
Transplants were produced in outbred pigmented rabbits as previously described by Bergstrm et al. [10, 11]. The procedure is a development of the ones described by Lopez et al. [12], Gouras et al. [13] and by Turner and Blair [14]. All animals were purpose-bred for experimental laboratory use and were obtained from certified local breeders. They were of a mixed rural brown strain. Six transplants have been examined in the electron microscope, all produced with
tissue obtained from fetuses taken 15 days after conception. Four fetal retinas were used for each transplantation. Three transplants were obtained 34 weeks after the transplantation (28, 28 and 21 days) and three after 34 months (109, 116 and 123 days). The host rabbits were sacrificed with an overdose of barbiturates. The transplanted eye was enucleated and bisected a little anteriorly of the equator. Small pieces of the entire posterior segment containing sclera, choroid and retina were fixed according to Ito and Karnowsky [15]. The tissue pieces were embedded in Epon, sectioned and stained with uranyl acetate according to standard electronmicroscopic procedures. Sections taken from different parts of the transplants and surrounding tissues were examined in a Zeiss 902 EM microscope. The animals were treated according to the ARVO convention for the use of animals in ophthalmic and vision research and according to pertinent Swedish laws and regulations. Appropriate permits for the work were given by the Swedish government committee on animal experimentation ethics.
Results
Transplants were readily identified in the choroid, having a morphology that is distinctly different from that of the choroid. They were composed of tightly packed cells with immature, usually oval nuclei, as has been previously described by e.g. Bergstrm et al. [16]. The three 1-month transplants were in places thicker than the regular choroid, leaving only narrow strips of it on either side. In 2 cases the transplants partly extended into the subretinal space. The 4-month transplants were thinner. In 1 case, the transplant was seen to reach into the vitreous through the retinotomy made at the time of surgery. In 2 cases, parts of the transplant extended from the choroid into the subretinal space. The overall morphology of the transplants was the same irrespective of their location in the choroid. Rosettes were present in the choroidal transplants, but they were less prominent than what e.g. Sharma et al. [3] and others have usually seen in standard 2- or 4-week subretinal transplants. The cells tended to form whorls and small sheets, but no regular lamination was apparent (fig. 1). It was not the purpose of this study to examine the different cell types that might be present in the transplant. Nevertheless, it was clear from the morphological observations that photoreceptors, amacrine cells and bipolar cells had developed, because the synaptic structures and other markers for such cells were easily and repeatedly observed (fig. 24). In both 1-month and 4-month transplants, neuropil regions were regularly seen with nerve cell processes of a morphology similar to that seen in the regular inner plexiform layer (fig. 4). Photoreceptor outer segments were rudimentary and not as common as in
Choroidal Retinal Cell Transplants
71
Fig. 1. Rabbit-to-rabbit choroidal trans-
plants, 116 days postconceptional age. The transplant is not vascularized and there is no inflammatory reaction in the choroid at the transplant. Semithin section, toluidine blue. Scale bar = 50 m.
a
Fig. 2a, b. Low-power micrograph (a) of a rosette in a choroidal rabbit-to-rabbit transplant, 46 days after conception,
b 31 days after transplantation. Dark photoreceptor nuclei (Ph) surround the central lumen (L), which in this case appears like a cleft. Junctional complexes corresponding to the outer limiting membrane (arrowheads) surround the lumen. Photoreceptor outer segments have not developed. A mitotic cell is evident (*). Higher magnifications of the junctional complexes are seen in b. Scale bars = 10 and 0.5 m.
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Ltjen-Drecoll/Bergstrm/Ehinger
Fig. 3. Photomontage of the edge of a choroidal rabbit-to-rabbit transplant, 131 days after conception, 116 days after transplantation. At this stage, rosettes are usually not present and the transplant is ensheathed by Mller cells. Note the quiescence of the surrounding tissue. Scale bar = 10 m. Fig. 4. Region resembling the inner plexiform layer in a choroidal transplant. Its age was 131 days after conception, 116 days after transplantation. There are several conventional-type synapses, characteristic of amacrine cells (arrowheads). M is a pigmentcontaining cell. Scale bar = 1 m.
73
a
Fig. 5. Low- and medium-power micrographs of a rabbit-to-rabbit transplant obtained 124 days after conception, 109 days after transplantation. b Magnification of a part of a. Note the continuous sheet of Mller cells at the edge of the transplant (M in a) and the basement membrane at their surface (BM in b). Scale bars = 20 and 0.2 m.
standard subretinal transplants, but connecting cilia were common. Clusters of tight junctions of the kind that form the outer limiting membrane were common in regions where connecting cilia were also seen. Cells identifiable as microglia were uncommon. Amacrine and bipolar cells were identified by means of their general morphology and the presence of the characteristic synapses. Mller cells were identified on the basis of the appearance of their cytoplasm, the density of the nucleus and the distribution of its chromatin. We did not try to classify each cell, but only to obtain an overview of the cell types that were present. There was a well-developed basement membrane around the transplants at 4 months (fig. 5), but it was less well developed in 1-month transplants. Mller cells formed a continuous sheath around the transplants at 4 months, usually more than one cell layer deep. In one of the 4-month transplants, the Mller cells were the dominating cell type. They did not show the pillar-like shape that is evident in the normal retina and were connected by junctional complexes morphologically resembling tight junctions and desmosomes (fig. 6b). In 1-month transplants, Mller cells were less prominent than in the 4month transplants and did not form any continuous sheath around the transplant, even though they were easily found. Capillaries were occasionally seen at the surface of the transplants. They were never fenestrated and most often showed a comparatively thick endothelium (fig. 6a). However, capillaries facing the normal host pigment epi-
thelium remained fenestrated, also when the transplant was only a few tens of micrometers away in the choroid (fig. 6b). In the 2 cases where transplants were found in the subretinal space, the epithelium had degenerated, showing a loss of microvilli as well as lateral membrane infoldings. In these cases, there were no fenestrations in the capillaries facing the pigment epithelium (fig. 7). There was hardly any cellular reaction in the choroid around the transplant and no signs of scar formation. Macrophages and plasma cells have occasionally been observed, but are no regular constituent at the transplants. There was no sign of any immunological cell attack on the transplant. On the other hand, pyknotic cells and cells which looked like apoptotic cells were frequent at 1 month. Dying cells were infrequent at 4 months, if present at all. Mitoses were regularly observed in 1-month transplants (fig. 2), but were not detectable at 4 months.
Discussion
It is now well established that fetal retinal cells can be transplanted to epiretinal or subretinal sites in mammalian eyes [14]. Survival up to 3 or 4 months has been seen in a number of instances, and transplants with the longest survival times so far (about 40 weeks) were obtained by Ehinger et al. [17, 18] with human fetal retinal cells transplanted to rats. There is also a previous report by Bergstrm [9] that rabbit-to-rabbit retinal cell transplants to the choroid survive and differentiate, and the present
74
6a
6b
Fig. 6 a, b. Capillaries in the vicinity of a choroidal transplant obtained 124 days after conception, 109 days after transplantation. a There is a capillary (C) very close to a transplant (T). Note that the endothelium of the vessel is not fenestrated. b A photomicrograph taken only a few tens of micrometers away from a shows a capillary (C) with fenestrations in the vessel wall facing Bruchs membrane (b). Scale bars = 5 and 0.5 m. Fig. 7. Rabbit-to-rabbit transplant, 43 days after conception, 28 days after transplantation. The transplant was placed in the choroid, but a part of it (T) entered the subretinal space, as seen in the picture. The retinal pigment epithelium (RPE) has reacted and has no microvilli and no lateral membrane infoldings. Note that the capillary next to the pigment epithelium (C) no longer has any fenestrations. Scale bar = 10 m.
75
study corroborates this observation. It may be noted that the experimental animals were well outbred. As seen in this study, retinal transplants are able to survive in the choroid of outbred rabbits for extended periods of time, much longer than needed for rejection responses to develop. When comparing with transplants located between the host photoreceptors and the choroid, there is possibly a reduction in the number of neurons in comparison with the glial Mller cells, and the organization is less exact and without many rosettes. However, the important point is that the normal complement of different constituents remained in the transplants. There can be no doubt that fetal rabbit retinal neurons survive, develop and differentiate also when placed in the well-vascularized choroid of rabbits. Further, mitotic figures and apoptotic reactions appeared only in the 1-month transplants and not in the older ones. It is apparent that there is no conventional rejection response against the retinal transplants in the rabbit choroid. It may further be noted how little microglial reaction and few other signs of scar formation there were. It has previously been shown by Bergstrm [9] that serum proteins enter the transplant for several days after the transplantation, but subsequently, this passage is blocked. There are no vessels within the transplants at this stage, but there are numerous fenestrated capillaries in its immediate vicinity, and they are the likely source of serum proteins. These capillaries remain fenestrated, also when only a few tens of micrometers away from the transplant. This suggest that the transplant does not release factors that influence the fenestration of the capillaries. Instead, such factors seem to be released by the retinal pigment epithelium as suggested by May et al. [19], and in the present study, it was also seen that the fenestrations were absent if the retinal pigment epithelium had been disturbed (fig. 7). From the present study it can be seen that the glial Mller cells form a cellular barrier around the transplant, and it appears likely that to a large extent they wall it off from its host so that the immune system does not attack it. However, there was no complete wall in the 1-month translants, only in the 3- to 4-month ones. There were no blood vessels within the transplant at either 1 or 4 months. Since the rabbit retina is not normally vascularized, this was as expected. Tight junctions are not seen in the normal retina, only in the retinal pigment epithelium. In previous studies Eichhorn et al. [20] have shown that pigmented ciliary epithelium cells in vitro also form tight junctions if the nonpigmented epithelium that would normally form these junctions is absent. We assume that sim-
ilar to the events in the cultured ciliary epithelium, transplanted Mller cells develop tight junctions when the pigment epithelium cells that would normally do so are absent. The ACAID system has been well investigated in rodents and primates and reviewed by Streilein et al. [5] and Streilein [21, 22]. It has been demonstrated in the anterior segment and the retina, but Eichhorn et al. [20] have shown that it may not be present in the primate choroid. In pilot experiments with rat eyes, we have also failed to see surviving transplants so that the choroid is perhaps not immunoprivileged in these animals. The ACAID system has not been well examined in rabbits, whose eyes are special in several respects. For instance, there is a rapid and easily elicited liberation of prostaglandins in the anterior segment, resulting in an acute inflammatory reaction and breakdown of the blood-retina and blood-aqueous barriers. Among other things, this makes it difficult to assess whether or not there is an ACAID system in the rabbit, even though Mondino et al. [23] presented indirect evidence from results with intravitreal injections of bacteria that an ACAID type of reaction may be present. Fenestrated capillaries occur in the choriocapillaris at a distance of only about 50100 m for at least 34 months after the transplantation and probably for much longer. In the loose meshes of the choroid, this is not a very long distance for serum globulins to diffuse or for immunocompetent blood cells to move. Further, the Mller cell encapsulation is only partial for several weeks after the transplantation. We therefore suggest that strictly anatomical structures are not likely to be solely responsible for the protection of the transplants in the rabbit choroid, but also other factors may participate, such as the ACAID immunosuppressing system [for reviews, see 5, 21, 22]. We consequently suspect that the ACAID or a similar system is active in the rabbit choroid, although direct proof for this is currently not available, or that the rabbit retina in a yet unknown way protects itself from rejection.
Acknowledgements
We are grateful to Ms. Elke Kretschmer and Ms. Karin Arnr for expert technical assistance and to Mr. Marco Gsswein for preparing the photographs. This work was supported by grants to B.E. from the EU BioMed2 program, the RP Foundation, the Segerfalk Foundation, the H. and L. Nilssons Stiftelse, the T. and R. Sderberg Foundation, the Swedish Medical Research Council (project 14X-2321), the Riksbankens Jubileumsfond and the Faculty of Medicine at the University of Lund and by grants to E.L.D. from the IZKF.
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References
1 del Cerro M: Retinal transplants. Prog Retinal Res 1990;9:229272. 2 del Cerro M, Gash DM, Notter MFD, Rao GN, Wiegand SJ, Jiang LQ, del Cerro C: Transplanting strips of immature retinal tissue and suspensions of dissociated retinal cells into normal and extensively damaged eyes. Ann NY Acad Sci 1995;112:692695. 3 Sharma RK, Bergstrm A, Ehinger B: Retinal cell transplants. Prog Retinal Eye Res 1995;15: 197230. 4 Ehinger B, Sharma RK, Ghosh F, Bruun A, Arnr K: Development in retinal cell transplants; in Andrasson S, Ehinger B, Ponjavic V (eds): Tapetoretinal Degenerations. Experimental Neurobiology and Treatment Possibilities. Eric K Fernstrm Symposium June 14, 1997. Boston, Digital Journal of Ophthalmology, 1998. 5 Streilein JW, Ksander BR, Taylor AW: Immune deviation in relation to ocular immune privilege. J Immunol 1997;158:35573560. 6 Jiang LQ, Streilein JW, McKinney C: Immune privilege in the eye: An evolutionary adaptation. Dev Comp Immunol 1994;18:421431. 7 Trnquist P, Alm A, Bill A: Permeability of ocular vessels and transport across the bloodretinal-barrier. Eye 1990;4:303309. 8 Eichhorn M, Horneber M, Streilein JW, Ltjen-Drecoll E: Anterior chamber-associated immune deviation elicited via primate eyes. Invest Ophthalmol Vis Sci 1993;34:2926 2930. 9 Bergstrm A: Embryonic rabbit retinal transplants survive and differentiate in the choroid. Exp Eye Res 1994;59:281289. 10 Bergstrm A, Ehinger B, Wilke K, Zucker CL, Adolph AR, Aramant R, Seiler M: Transplantation of embryonic retina to the subretinal space in rabbits. Exp Eye Res 1992;55:2937. 11 Bergstrm A: Experimental Retinal Cell Transplants; MD/PhD thesis, University of Lund, 1994, pp 1182. 12 Lopez R, Gouras P, Brittis M, Kjeldbye H: Transplantation of cultured rabbit retinal epithelium to rabbit retina using a closed-eye method. Invest Ophthalmol Vis Sci 1987;28: 11311137. 13 Gouras P, Lopez R, Kjeldbye H, Sullivan B, Brittis M: Transplantation of retinal epithelium prevents photoreceptor degeneration in the RCS rat. Prog Clin Biol Res 1989;314:659 671. 14 Turner JE, Blair JR: Newborn rat retinal cells transplanted into a retinal lesion site in adult host eyes. Brain Res 1986;391:91104. 15 Ito S, Karnowsky MJ: Formaldehyde-glutaraldehyde fixatives containing trinitro compounds. J Cell Biol 1968;39:168. 16 Bergstrm A, Zucker CL, Wilke K, Adolph A: Electron microscopy of rabbit retinal transplants. Neuroophthalmology 1994;14:247 257. 17 Ehinger B, Bergstrm A, Seiler M, Aramant RB, Zucker CL, Gustavii B, Adolph AR: Ultrastructure of human retinal cell transplants with long survival times in rats. Exp Eye Res 1991; 53:447460. 18 Ehinger B, Zucker C, Bergstrm A, Seiler M, Aramant R, Adolph A: Electron microscopy of human first trimester and rat mid-term retinal cell transplants with long development time. Neuroophthalmology 1992;12:103114. 19 May CA, Horneber M, Ltjen-Drecoll E: Quantitative and morphological changes of the choroid vasculature in RCS rats and their congenic controls. Exp Eye Res 1996;63:7584. 20 Eichhorn M, Bermbach G, Dermietzel R, Ltjen-Drecoll E: Characterization of bovine ciliary pigmented epithelial cells in monolayer culture: An ultrastructural, enzyme histochemical and immunohistochemical study. Graefes Arch Clin Exp Ophthalmol 1993;231:2128. 21 Streilein JW: Tissue barriers, immunosuppressive microenvironments, and privileged sites: The eyes point of view. Reg Immunol 1993;5: 253268. 22 Streilein JW: Immune privilege as the result of local tissue barriers and immunosuppressive microenvironments. Curr Opin Immunol 1993;5:428432. 23 Mondino BJ, Adamu SA, Pitchekian Halabi H: Antibody studies in a rabbit model of corneal phlyctenulosis and catarrhal infiltrates related to Staphylococcus aureus. Invest Ophthalmol Vis Sci 1991;32:18541863.
77

Simone Ottonello a Chiara Foroni a Arturo Carta b Stefania Petrucco b Giovanni Maraini b
a Istituto di Scienze Biochimiche, Faculty of Sciences, and b Institute of Ophthalmology, Faculty of Medicine, University of Parma, Italy
Key Words Cataract W Oxidative stress W Lens
Abstract The authors review the available evidence supporting the possible role of oxidative stress in cataract formation from an epidemiological and a clinical point of view. They discuss in more detail what is presently known about the molecular mechanisms of response of the mammalian lens to an oxidative insult and report unpublished data on gene modulation upon oxidative stress in a bovine lens model. Main research endeavors that seem to be a most promising source of new insights into the problem of age-related cataract formation are briefly discussed.
Introduction and Background
Age-related cataract is the worlds most frequent cause of curable blindness and population projections suggest that the number of cataract-blind persons could reach
Supported by Biomed grant BMH4-CT96-1593.
close to 40 million by the year 2025 [1]. The disease entails a progressive reduction of the transparency of the eye lens which deteriorates the quality of the optic image formed on the retina and reduces visual acuity eventually causing blindness. Three main types of lens opacity are described in age-related cataract (nuclear, cortical and posterior subcapsular). Pure forms of cataract (with only one type of opacity present) are found more frequently in early less advanced forms of the disease but, as the cataract becomes severer, different types of opacity frequently coexist in the same lens producing the so called mixed type of cataract. Although cortical cataract is probably the most frequently observed type of opacity, nuclear and posterior subcapsular cataracts (which primarily affect the optical axis of the eye lens) are certainly the types of cataract that more frequenly cause visual deterioration and lead patients to surgery [2]. As for other types of age-related degenerative eye diseases, the oxidative insult suffered by eye tissues during the lifelong exposure to visual/UV light radiation has been the subject of extensive investigation over the past decades as a possible determinant of cataract formation. Reactive oxygen species, like the superoxide anion, are formed by photochemical reactions of O2 in the presence of electron donors such as riboflavin and are converted to hydrogen peroxide via ascorbic-acid-mediated reduction.
ABC
Giovanni Maraini Institute of Ophthalmology, University of Parma via Gramsci 14, I43100 Parma (Italy) Tel +39 0521 259098, Fax +39 0521 994820 E-Mail maraini@ipruniv.cce.unipr.it
Hydrogen peroxide is present in the aqueous humor of the mammalian eye [3] and has been reported to be increased in the aqueous humor of eyes with age-related cataract [4]. Many of the protein and membrane alterations observed in cataractous human lenses are of oxidative origin, and incubation of animal lenses in the presence of hydrogen peroxide (or other oxidants) reproduces many of these changes and may lead to lens opacification. The high concentration of reduced glutathione (GSH) protects the lens from the damaging effect of hydrogen peroxide. The resulting oxidized glutathione (GSSG) is then converted to GSH in situ by a redox system involving glutathione reductase and reduced nicotinamide-adenine dinucleotide phosphate generated by the pentose monophosphate shunt. GSH is therefore crucial in the lens to protect protein SH groups preventing soluble protein cross-linking and the formation of insoluble protein aggregates, as well as to preserve normal permeability and transport function of cell membranes [5, 6]. Blocking GSH regeneration in cultured lenses enhances the toxic effect of hydrogen peroxide. GSH levels in the lens progressively decrease with age and are drastically reduced in cataractous lenses. In keeping with this view, epidemiological evidence from observational studies supports the possibility that persons with a higher dietary intake of antioxidants are at a lower risk of developing age-related cataract than individuals with a lower intake, and preliminary results from some intervention studies suggest that treatment with antioxidant vitamins might delay progression of age-related lens opacities. In this paper we shall briefly review the available evidence supporting the role of oxidative stress in cataract formation from an epidemiological and clinical point of view and will discuss in more detail what is presently known about the molecular mechanisms of response of the mammalian lens to oxidative insults.
Epidemiological Evidence: Observational and Intervention Studies
Experimentally the lens has been shown to be damaged by exposure to ultraviolet B radiation [7, 8]. It is therefore not surprising that the association between sunlight, UVB exposure and cataract formation has been investigated in several observational studies. First suggested by Hiller et al. [9], an association between UVB exposure and cortical cataract has been found by several studies which also revealed an association between an increasing average annual UVB light exposure and risk of cataract [10], and a dose-response relationship between sunlight exposure and
cortical opacities [odds ratio, OR = 2.26 (95% confidence interval [CI] 1.144.46) for high vs. very low exposure] after adjusting for potential confounders [11]. A positive association between UVB exposure and increasing odds of cortical opacities (OR = 1.10; 95% CI, 1.041.30) was reported also by West et al. [12] and between UVB and posterior subcapsular cataract by Bochow et al. [13]. On the same note the greater prevalence of cortical opacities in the lower nasal quadrant of the lens also provides indirect support to a possible role of sunlight in cataract etiology [14]. Other studies however reported negative or inconclusive results [1518]. These results may be explained, at least in part, by the high percentage of nuclear cataracts in the corresponding study samples, by different ranges of sunlight exposure among participants, by a different population mobility during lifetime or by residual confounding. Certainly the main difficulty of this type of assessment appears to be the limitation of self-reported assessment of lifetime exposure which can only be partially controlled by the use of detailed questionnaires. Overall, however, the available evidence indicates a dose-related association between sunlight (and the inherent UVB exposure) and cortical and, perhaps, posterior subcapsular cataracts. Since 1991 several studies have explored the possible role of known antioxidants in preventing the development of age-related lens opacities. This issue has been addressed in observational studies which evaluated the antioxidant status of participants by assessing dietary intake or measuring plasma levels of vitamins C, E and carotenoids or use of multivitamin supplements. Despite some inconsistencies regarding the specific cataract type or nutrient, many of these studies produced data suggesting that persons with higher antioxidant plasma levels or intake are at lower risk of developing age-related lens opacities [1922]. Other studies found negative or inconclusive results [23, 24]. Besides possible limitations inherent to the methods of nutritional assessments, cataract definition and geographic characteristics, a lower range of variability of nutrition in a given population with respect to another might explain some of the observed inconsistencies between studies which used similar design and assessment methods [21, 23]. Obviously, final proof of this antioxidant hypothesis will only come from randomized clinical trials comparing the effectiveness of multivitamin supplementation in preventing the development of cataract or in delaying its progression. Two large ongoing trials sponsored by the National Eye Institute are currently testing this possibility. At the moment the only available data are those derived
79
from two large and controlled cancer prevention trials that have been conducted in China using vitamin-mineral supplements. One of these studies enrolled 2,141 patients with esophageal dysplasia (dysplasia trial) randomized to placebo or to a mineral-multivitamin supplemention reinforced with -carotene. The second study (general population trial) enrolled 3,249 participants from the general population who were randomized to one of 4 possible treatments (retinol/zinc; riboflavin/niacin; ascorbic acid/ molybdenum; selenium/-tocopherol/ carotene) or to placebo. At the end of the follow-up, the treatment group of the first study and the group treated with riboflavin/ niacin of the second study showed, for the age range between 65 and 74 years, a significant reduction of the prevalence of N cataract with respect to controls (OR = 0.57, 95% CI 0.360.90 for the first study; OR = 0.45, 95% CI 0.310.64 for the second study) [25].
The Oxidative Insult Cataract Model: Oxidative Stress Response Mechanism in the Lens
The application of oxidative stress conditions to in vitro cultured lenses, either by incubation in the presence of H2O2 or by a photochemical insult in the presence of a photosensitizer (e.g. riboflavin), forms the basis of one of the most widely used models of artificially induced lens opacification. Under organ culture conditions the opacification starts in the equatorial region, is subepithelial in location and eventually spreads to the entire lens cortex. Loss of lens transparency is associated with the alteration of critical biochemical and transport parameters that can be prevented by an H2O2-decomposing enzyme like catalase. The sequence of events leading to the loss of lens transparency has been investigated in detail by Spector et al. [26], who studied the effect on the cultured rat lens of a short photochemically induced oxidative insult resulting in H2O2 levels in the culture medium not higher than 100 M. These authors found that the oxidative insult is associated with an epithelial cell damage, which precedes lens opacification, develops slowly in the postinsult period and is related to the intensity and duration of the stress. The main changes observed in the epithelial cell layer are a progressive loss of cell viability, as revealed by an increased Trypan blue staining and, upon a longer stress time, DNA fragmentation and decreased 3H-thymidine incorporation [26, 27]. Interestingly, by comparing human noncataractous eye bank lens epithelial cells to human cataractous samples, Kleiman and Spector [28] found that the proportion of cells showing DNA single-
strand breaks is significantly increased in cataractous lenses. Similarly, a comparison of lens epithelial cells from a group of cataract patients with those of 8 normal human lenses of comparable age revealed an increased number of apoptotic cells in cataractous samples [29]. These results, however, have not been confirmed by Harocopos et al. [30]. In fact, a comparison of cataractous epithelial specimens obtained at surgery with specimens obtained from transparent and cataractous eye bank eyes led these authors to conclude that apoptotic cells are not present in cataractous epithelia if they are immediately fixed after surgery. Dead epithelial cells, only found when the samples are allowed to remain for some time in culture media, are produced by a necrotic, rather than an apoptotic, process and are probably secondary to the surgical trauma. The same authors did not find any evidence for a significant decrease in lens epithelial cell density during cataract formation and ageing. In the photo-oxidative cataract model, epithelial damage leads to a reduced uptake and an increased efflux of rubidium, reduced cellular levels of ATP and increased lens wet weight. Another important landmark of lens oxidative damage is the progressive decrease in the GSH/GSSG ratio, reflecting a drastic change in the redox set point which almost completely recovers upon stress removal. An upregulation, which can reach 38-fold for c-jun, 72-fold for c-fos and 5-fold for c-myc, has also been reported for these protooncogenes to occur in a rabbit lens epithelial cell line and in cultured rat lenses exposed to H2O2 levels ranging from 25 to 200 M [31, 32]. The resulting accumulation of cjun and c-fos, the two subunits of the transcription factor AP-1, may in turn promote the activation of a number of target genes, thus determining a large reprogramming of lens gene expression. The presence of an AP-1 binding site upstream of the gene has further led to the proposal that this gene may be one of the targets of the transactivator AP-1. Unfortunately, however, no evidence that H2O2 can influence -A-crystallin expression or that c-jun and c-fos are upregulated in lens epithelia from human cataractous lenses has been obtained so far. A slight, H2O2induced upregulation of mRNA levels of the antioxidative stress genes of glutathione peroxidase and catalase [31] and the posttranscriptional upregulation of an ubiquitin-activating enzyme [33] have also been reported in the mammalian lens. An alternative approach to identify mammalian lens genes modulated in response to a severe oxidative stress is differential display (DD), a nontargeted gene-searching procedure that does not require any prior knowledge of either gene function or identity. One may utilize mRNA
80
Ottonello/Foroni/Carta/Petrucco/Maraini
Fig. 1. Representative example of a DD analysis. The autoradiogram shows the results of a typical DD experiment comparing mRNAs in control bovine lenses () with those expressed in lenses exposed to 25 mM H2O2 for 2.5 h. The random primers AP-H, AP-6, AP-7, AP-10 were used in combination with the anchored primer (T)12 MG (GenHunter, Brookline, Mass., USA). Amplified cDNA fragments (amplicons) corresponding to differentially modulated transcripts are indicated with arrows.
DD analysis to investigate the mechanism of response of the mammalian lens to a severe oxidative stress. In a preliminary analysis, Ottonello et al. [34] have utilized DD to investigate the in vitro response of bovine lenses exposed for 2.5 h to a severe H2O2 stress that reduced GSH levels to about 80% of controls. Focusing on mRNA modulation during the acute phase of oxidative stress, total RNA, extracted from epithelium/capsule preparations from treated and control lenses, was sub-
jected to mRNA DD analysis using 7 different combinations of primers. A total of 1,400 cDNA amplicons (corresponding to approx. 10% of the estimated mRNA lens population) was visualized in this way. Thirty putatively differential amplicons, 23 upregulated and 7 downregulated, were identified and eluted from display gels (fig. 1). Following reamplification and intermediate hybridization screening, 14 cDNA fragments representative of putative differentially modulated mRNAs (12 upregu-
81
Table 1. Differential amplicons identified
in bovine lenses incubated for 2.5 h in the presence of 25 mM hydrogen peroxide
Differential amplicons 1/G4 2/G4 3/G4 1/G6 2/G6 2/G7 3/G7 4/G7 1/G10 2/G10 1/A2 2/A2 2/A2 1/A5
Size bp 562 477 310 510 370 502 406 306 450 430 900 654 359 258
Homology
Modulation
subunit F1-ATPase, bovine human cDNA clone human cDNA clone, pineal gland human cDNA clone structural protein of human chromatin human cDNA clone, pancreas islets arginyl-tRNA synthase, human protein binding the releasing factor of human corticotropin transglutaminase (chicken) latexin (mouse) none none ubiquitin-activating enzyme none
upregulated upregulated upregulated upregulated upregulated upregulated upregulated upregulated upregulated upregulated upregulated
lated and 2 downregulated), were cloned and sequenced. Thirteen of them were found to share significant similarities with known mammalian cDNAs or expressed sequence tag sequences, but only one, corresponding to transglutaminase, has previously been reported to increase in cultured bovine lenses after UVA irradiation (table 1). It has been impossible, so far, to confirm the differential expression of mRNAs identified by these amplicons in hybridization experiments carried out with either RNase protection or Northern assays. Because these hybridization experiments have been carried out on mRNA samples other than those originally utilized for DD analysis, and since the apparent rate of false-positive results is much higher than that observed for other systems in our laboratory, we have considered the possibility that an uncontrolled source of variability (possibly related to the severity of the oxidative stress) is present in our system. We thus utilized the c-fos transcript as an additional indicator of oxidative stress. Quite surprisingly, in two independent Northern hybridization experiments, c-fos was found to be strongly downregulated following exposure to severe oxidative stress conditions (i.e. incubation in the presence of 0.35 mM H2O2 as in previous DD experiments) which nevertheless allowed a prompt recovery of control GSH levels following H2O2 removal (fig. 2). This result thus seems to indicate that the mechanism of response of the lens qualitatively depends on the intensity of the stress, as reflected by the GSH level and the GSH/GSSG ratio, and may suggest that a c-fos-
independent response mechanism does exist in the mammalian lens. The same DD approach has recently been utilized by Kantorow et al. [35] in what, to the best of our knowledge, is at the moment the only published investigation performed with this technique on human lenses. These authors used one combination of primers to compare pooled epithelia obtained from transparent or cataractous human lenses. They displayed 32 cDNA fragments and identified 15 putative differentially expressed amplicons, 4 of which were upregulated and 11 downregulated in cataractous as compared to noncataractous samples. One upregulated and 1 downregulated amplicon were further analyzed and found to correspond, respectively, to metallothionein IIa (METII) and protein phosphatase 2A regulatory subunit. The expression of their mRNAs was then examined in epithelial/capsule samples obtained from 6 individual transparent and 14 cataractous lenses. Although with a considerable degree of variability between individual samples, the results of this analysis were overall consistent with those obtained from pooled epithelium preparations. A certain heterogeneity of cataract type might be responsible for differences observed among individual samples. METII is a low-molecular-weight, cysteine-rich protein with metal binding and antioxidant activity. In other tissues, it has been reported to be involved in the cellular response to different types of stress, such as X irradiation [36] and UV-induced DNA damage [37]. When overexpressed, METII protects pulmonary artery endothelial cells against a variety of pro-
82
Fig. 2. c-fos mRNA expression analysis. Equal amounts of total
RNA extracted from bovine lenses exposed to 25 mM H2O2 for 2.5 h (+) or control lenses () were hybridized under high-stringency conditions to a 32P-labelled, bovine c-fos DNA probe (275 bp); mRNAs, shown below each lane, were visualized on the wet gel by ethidium bromide staining and were used as size markers.
oxidant stimuli by preventing lipid peroxidation [38] and suppresses spontaneous mutagenesis as well as mutagenesis induced by oxygen-radical-generating compounds in mammalian cells [39]; its scavenging activity towards hydroxyl radicals has been reported to be 50 times greater than that of GSH on a molar basis [40].
Conclusions
Available experimental evidence indicates that the oxidative insult cataract model reproduces a number of the biochemical changes that characterize age-related cataract. The epithelial layer of the lens is the main target of the oxidative insult and some of the mechanisms of response to this stress have been partially elucidated. Both transcriptional and posttranscriptional mechanisms have been documented. Along with preliminary data pointing to the opposite effects elicited by H2O2 on c-fos modulation, this indicates that multiple and diverse response pathways operate in the lens. Also attesting to this multi-
plicity of responses is the variety of putatively involved genes that have recently been identified by DD and other types of molecular approaches. Many of the genes that we and others [41] have identified by DD analysis have no obvious implication for the maintenance of lens transparency. The randomized nature of this approach combined with poor knowledge of the repertoire and the mode of regulation of genes involved in lens protection probably explains this current limitation. This situation is best exemplified by osteonectin/BM40 (SPARC), a seemingly lens-unrelated gene that has recently been found to be upregulated in human cataracts by DD analysis [35] and independently shown to cause a severe age-related cataract in knockout mice [42, 43]. Another, previously unsuspected link with cataract formation regards iron homeostasis. In this case, known as hereditary hyperferritinemia cataract syndrome, increased plasma levels of L-ferritin lead to a bilateral congenital or early-onset cataract [44, 45]. Interestingly, in this syndrome, hyperferritinemia is caused by heterogeneous mutations in the iron-regulatory element of the ferritin Light-chain mRNA which by interacting with the iron-regulatory protein plays a crucial role in the posttranscriptional negative regulation of ferritin synthesis. Considering that one regulatory protein, ironregulatory protein 1, is rapidly activated by H2O2 [46, 47] and that iron toxicity strongly relies on Fenton chemistry (i.e. the generation of highly reactive hydroxyl radicals from the reaction of H2O2 with Fe2+ [48]), the regulatory causal relationship between iron metabolism, oxidative stress and cataract formation appears particularly important to understand. At present, two main research endeavors thus seem to be a most promising source of new insights into the problem of age-related cataract formation. On the one hand, it will be important to establish whether the most significant changes in gene expression that are induced in vitro upon exposure to H2O2 are also present in human cataractous lenses. The availability of techniques allowing the molecular analysis of tissue samples only containing a very limited number of cells, such as capsulorhexis specimens obtained during surgical phacoemulsification, should make this goal attainable. Careful classification of cataract type is an essential prerequisite of such studies, as considerable evidence indicates that morphologically distinct types of opacity have different biochemical characteristics, possibly identifying different pathogenetic mechanisms.The other main target of future studies is the acquisition of more detailed information on genes which, when mutated, contribute to the development of agerelated cataract. Such mutations, though by themselves
83
not sufficient to cause cataract, may contribute to cataract development in the presence of other mutant genes or environmental insults. A number of candidate genes implicated in human congenital cataracts or in animal models or differentially expressed in cataractous lenses has
been identified. Genetic epidemiology studies aimed to compare the mutation rates of these candidate genes in individuals affected by age-related cataract with respect to the normal population are greatly needed and might considerably add to our understanding of this disease.
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Invest Ophthalmol Vis Sci 1977;16:932 939. 8 Sderberg PG: Na and K in the lens after exposure to radiation in the 300 nm wavelength region. J Photochem Photobiol 1991;8:279 294. 9 Hiller R, Sperduto RD, Ederer F: Epidemiological associations with nuclear, cortical and posterior subcapsular cataracts. Am J Epidemiol 1986;124:916925. 10 Taylor H, West SK, Rosenthal FS, Muoz B, Newland HS, Abbey H, Emmett EA: Effect of ultraviolet radiation on cataract formation. N Engl J Med 1988;319:14291433. 11 Rosmini F, Stazi MA, Milton RC, Sperduto RD, Pasquini P, Maraini G, and the ItalianAmerican Cataract Study Group: A dose-response effect between a sunlight index and agerelated cataracts. Ann Epidemiol 1994;4:266 270. 12 West SK, Duncan DD, Muoz B, Rubin GS, Fried LP, Bandeen-Roche G, Schein OD: Sunlight exposure and risk of lens opacities in a population based study: The Salisbury Eye Evaluation Project. JAMA 1998;280:714718. 13 Bochow TW, West SK, Azar A, Muoz B, Sommer A, Taylor HR: Ultraviolet light exposure and risk of posterior subcapsular cataracts. Arch Ophthalmol 1989;107:369372. 14 Graziosi P, Rosmini F, Bonacini M, Ferrigno L, Sperduto RD, Milton RC, Maraini G: Location and severity of cortical opacities in different regions of the lens in age-related cataract. Invest Ophthalmol Vis Sci 1996;37:1698 1703. 15 Dolezal JM, Perkins ES, Wallace RB: Sunlight, skin sensitivity, and senile cataract. Am J Epidemiol 1989;129:559568. 16 Colman GW, Shore DL, Shy CM, Checkoway H, Luria AS: Sunlight and other risk factors for cataracts: An epidemiologic study. Am J Publ Health 1988;78:14591462. 17 Wong L, Ho SC, Coggon D, Cruddas AM, Hwang CH, Ho CP, Robertshaw AM, MacDonald DM : Sunlight exposure, antioxidant status, and cataract in Hong Kong fishermen. J Epidemiol Community Health 1993;47:46 49. 18 Mitchell P, Cumming RG, Attebo H, Panchapakesan J: Prevalence of cataract in Australia: The Blue Mountains eye study. Ophthalmology 1997;4:581588. 19 Jaques PF, Chylack LT Jr, McGandy RB, Hartz SC: Antioxidant status in persons with and without senile cataract. Arch Ophthalmol 1988;106:337340. 20 Robertson J, Donner AP, Trevithick JR: A possible role for vitamins C and E in cataract prevention. Am J Clin Nutr 1991;53:364S351S. 21 Leske MC, Chylack LT Jr, Wu S : The lens opacities case control study: Risk factors for cataract. Arch Ophthalmol 1991;109:244251. 22 Rouhianinen P, Rouhianinen H, Salonen JT: Association between low plasma vitamin E concentration and progression of early cortical lens opacities. Am J Epidemiol 1995;144:496 500. 23 Italian-American Cataract Study Group: Risk factors for age-related cortical, nuclear, and posterior subcapsular cataracts. Am J Epidemiol 1991;133:541553. 24 Vitale S, West SK, Hallfrisch H, Alston C, Wang F, Moorman C, Muller D, Singh V, Taylor HR: Plasma antoxidant and risk of cortical and nuclear cataract. Epidemiology 1993;4: 195203. 25 Sperduto RD, Hu TS, Milton RC, Zhao JL, Everett DF, Cheng QF, Blot WJ, Bing L, Taylor PR, Jun-Yao L, Dawsey S, Guo W: The Linxian cataract studies: Two nutritional intervention trials. Arch Ophthalmol 1993;111:1246 1253. 26 Spector A, Wang GM, Wang RR, Li W, Kuszak JR: A brief photochemically induced oxidative insult causes irreversible lens damage and cataract. I. Transparency and epithelial cell layer. Exp Eye Res 1995;60:471481. 27 Spector A, Wang GM, Wang RR, Li W, Kleiman NJ: A brief photochemically induced oxidative insult causes irreversible lens damage and cataract. II. Mechanism of action. Exp Eye Res 1995;60:483493. 28 Kleiman NJ, Spector A: DNA single strand breaks in human lens epithelial cells from patients with cataract. Curr Eye Res 1993;12: 423431. 29 Li WC, Kuszak JR, Dunn K, Wang RR, Wang GM, Spector A, Leib M, Cotliar AM, Weiss M, Espy J, Howard G, Farris RL, Auran J, Donn A, Hofeldt A, Mackay C, Merriam J, Mittl R, Smith TR: Lens epithelial cells apoptosis appears to be a common cellular basis for noncongenital cataract development in humans and animals. J Cell Biol 1995;130:169181. 30 Harocopos GJ, Alvares KM, Kolker AE, Beebe DC: Human age-related cataract and lens epithelial cell death. Invest Ophthalmol Vis Sci 1998;39:26962706. 31 Li WC, Wang GM, Wang RR, Spector A: The redox active components of H2O2 and N-acetyl-L-cysteine regulate expression of c-jun and c-fos in lens systems. Exp Eye Res 1994;59: 179190. 32 Li DW, Spector A: Hydrogen peroxide-induced expression of the proto-oncogenes, c-jun, c-fos and c-myc in rabbit lens epithelial cells. Mol Cell Biochem 1997;173:5969. 33 Shang F, Gong X, Taylor A : Activity of ubiquitin-dependent pathway in response to oxidative stress. J Biol Chem 1997;272:23086 23093. 34 Ottonello S, Foroni C, Petrucco S, Maraini G: Differential display analysis of mRNAs modulated in bovine lenses exposed to toxic levels of hydrogen peroxide (ARVO suppl). Invest Ophthalmol Vis Sci 1998;39:S523. 35 Kantorow M, Kays T, Horwitz J, Huang Q, Sun J, Piatigorsky J, Carper D: Differential display detects altered gene expression between cataractous and normal human lenses. Invest Ophthalmol Vis Sci 1998;39:23442354. 36 Shiraishi N, Aono K, Utsumi K: Increased metallothionein content in rat liver by X-irradiation and exposure to high oxygen tension. Radiat Res 1983;95:298302.
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37 Fornace AJ Jr, Schalch H, Alamo I Jr: Coordinate induction of metallothionein I and II in rodent cells by UV-irradiation. Mol Cell Biol 1988;8:47164720. 38 Pitt BR, Schwarz M, Woo ES, Yee E, Wasserloos K, Tran S, Weng W, Mannix RJ, Watkins SA, Tyurina YY, Tyurin VA, Kagan VE, Lazo JS: Overexpression of metallothionein decreases sensitivity of pulmonary endothelial cells to oxidant injury. Am J Physiol 1997;273: 856865. 39 Rossman TG, Goncharova EI: Spontaneous mutagenesis in mammalian cells is caused mainly by oxidative events and can be blocked by antioxidants and metallothionein. Mutat Res 1998;402:103110. 40 Miura T, Muraoka S, Ogiso T: Antioxidant activity of metallothionein compared with reduced glutathione. Life Sci 1997;60:301309.
41 Carper DA, Sun JK, Iwata T, Zigler JS Jr., Ibaraki N, Lin LR, Reddy V: Oxidative stress induces differential gene expression in a human lens epithelial cell line. Invest Ophthalmol Vis Sci 1999;40:400406. 42 Gilmour DT, Lyon GJ, Carlton MBL, Sanes JR, Cunningham JM, Anderson JR, Hogan BLM, Evans MJ, Colledge WH: Mice deficient for the secreted glycoprotein SPARC/osteonectin/BM40 develop normally but show severe age-onset cataract formation and disruption of the lens. EMBO J 1998;17:18601870. 43 Norose K, Clark JI, Syed NA, Basu A, HeberKatz E, Sage EH, Howe CC: SPARC deficiency leads to early-onset cataractogenesis. Invest Ophthalmol Vis Sci 1998;39:26742680. 44 Girelli D, Corrocher R, Bisceglia L, Olivieri O, Zelante L, Panozzo G, Gasparini P: Hereditary hyperferritinemia-cataract syndrome caused by a 29-base pair deletion in the iron responsive element of ferritin L-subunit gene. Blood 1997;90:20842088.
45 Cazzola M, Bergamaschi G, Tonon L, Arbusini E, Grasso M, Vercesi E, Barosi G, Bianchi PE, Caairo G, Arosio P: Hereditary hyperferritinemia-cataract syndrome: Relationship between phenotypes and specific mutations in the iron-responsive element of ferritin light-chain mRNA. Blood 1997;90:814821. 46 Pantopoulos K, Hentze MW: Activation of regulatory protein-1 by oxidative stress in vitro. Proc Natl Acad Sci USA 1998;95:10559 10563. 47 Menotti E, Henderson BR, Khn LC: Translational regulation of mRNAs with distinct IRE sequences by iron regulatory proteins 1 and 2. J Biol Chem 1998;273:18211824. 48 Halliwell B, Gutteridge JMC: Role of free radicals and catalytic metal ions in human disease: An overview. Methods Enzymol 1990;186:1 85.
85
The Ageing Lens

A.J. Bron a G.F.J.M.Vrensen b
a Nuffield
J. Koretz c G. Maraini d J.J. Harding a
Laboratory of Ophthalmology, University of Oxford, UK; b Department of Morphology, Netherlands Ophthalmic Research Institute, Amsterdam, The Netherlands; c Biology/Biophysics, Rensselaer Polytech Institute, Science Centre, Troy, N.Y., USA; and d Ophthalmology, University of Parma, Italy
Key Words Ageing lens W Lens epithelium W Lens fibres W Sutures W Crystallins W Presbyopia W Cataract
Abstract The human lens grows by a process of epithelial cell division at its equator and the formation of generations of differentiated fibre cells. Despite the process of continuous remodelling necessary to achieve growth within a closed system, the lens can retain a high level of light transmission throughout the lifetime of the individual, with the ability to form sharp images on the retina. Continuous growth of the lens solves the problem imposed by terminal differentiation within a closed, avascular system, from which cells cannot be shed. The lens fibre tips arch over the equator to meet anteriorly and posteriorly and form branching sutures of increasing complexity. The stages of branching may create the optical zones of discontinuity seen on biomicroscopy. The lens is exposed to the cumulative effects of radiation, oxidation and postranslational modification. These later proteins and other lens molecules in such a way as to impair membrane functions and perturb protein (particularly crystallin) organisation, so that light transmission and image formation may be compromised. Damage is min-
imised by the presence of powerful scavenger and chaperone molecules. Progressive insolublisation of the crystallins of the lens nucleus in the first five decades of life, and the formation of higher molecular weight aggregates, may account for the decreased deformability of the lens nucleus which characterises presbyopia. Additional factors include: the progressive increase in lens mass with age, changes in the point of insertion of the lens zonules, and a shortening of the radius of curvature of the anterior surface of the lens. Also with age, there is a fall in light transmission by the lens, associated with increased light scatter, increased spectral absorption, particularly at the blue end of the spectrum, and increased lens fluorescence. A major factor responsible for the increased yellowing of the lens is the accumulation of a novel fluorogen, glutathione-3-hydroxy kynurenine glycoside, which makes a major contribution to the increasing fluorescence of the lens nucleus which occurs with age. Since this compound may also cross-link with the lens crystallins, it may contribute to the formation of high-molecular-weight aggregates and the increases in light scattering which occur with age. Focal changes of microscopic size are observed in apparently transparent, aged lenses and may be regarded as precursors of cortical cataract formation.
ABC
A.J. Bron Nuffield Laboratory of Ophthalmology, University of Oxford Walton Street Oxford OX2 6AW (UK) Tel. +44 1865 723 485, Fax +44 1865 794 508
Introduction
Ageing and Senescence Generally, the term ageing implies a functional loss which accumulates with time. The implications differ according to whether the concept is applied to the whole organism or to its individual organs. Thus, in youth, there may be a time of peak physiological performance for the individual, which may be quite different for discrete organs. Even within an organ, ageing is unlikely to be a homogeneous process, and thus it is that in the lens, said to be derived from but a single cell type, its component parts respond asymmetrically to the passage of time. In the lens, which grows throughout life, it is difficult to define where development ends and ageing begins. It may even be unwise to identify such a watershed, except for operational reasons. This paper will therefore describe a sequence of events which lead to ageing, but will not attempt to define a point of transition. However, it is worth directing attention at the outset to one landmark in the temporal history of the lens, specified by the deepest fibres of the lens bow, where terminally differentiated fibres lose their organelles at the time of denucleation. Fibres located superficial to this point have passed through their life cycle. Fibres deep to this point, are retained for the life of the lens and are greatly depleted in metabolic capacity. Although the term senescence is often used synonymously with the term ageing, it is also used in the more specific sense of cell senescence, determined as the number of population doublings of a cell cultured in ideal conditions of growth, the so-called Hayflick limit [1, 2]. This is characteristic of a given cell type and correlates with telomere length and the stepwise shortening of the telomere which occurs at each cell division. Little information of this sort is currently available for the lens though the area is developing. The lens grows throughout the human lifespan. None of its cells are cast off. The attraction of studying lens ageing is therefore that it is like a clock, in which time is represented spatially across its profile. Its component cells are added to as time goes by. Those at the centre of the lens are as old as the individual in whom they reside. New cells arising for instance in an octagenarian by division at the lens equator, are required to function much as those laid down in early life. New fibres at the surface of an 80-year-old lens exhibit a high quality of optical performance, reflecting the stability of the lens genome with ageing. One of the most interesting questions to ask, is how such fibres, formed at this age, differ from infantile fibres
in their form, content, connectivity and function, and how the germinal cells giving rise to these fibres are maintained over this extended period. In this account we describe the effects of ageing on lens growth, organisation and remodelling, mechanics and optical performance and consider the molecular events associated with these changes. For the purposes of this paper, the lens nucleus will be defined as that mass of fibres which is laid down prenatally, and the term cortex refers to all those fibres laid down after birth.
Lens Growth
The lens grows by the regular addition of fibres to its fibre mass. In the human lens this occurs across the life span, whereas in other species, such as rat and rabbit, growth rates level off in later life [3]. Growth rate is not uniform throughout the human life span and is greatest in fetal life. Fetal lens mass increases at about 181 mg/year and drops 100-fold after birth, becoming 1.3 mg/year between 10 and 90 years [4, 5]. There is concomitantly a steady increase in postnatal volume, while estimates of average lens density suggest that the protein concentration remains relatively fixed at around 33% of the wet weight over the age span [6]. However, there is a gradient of density across the superficial cortex, and this influences the overall refractive index of the lens.
Lens Dimensions
Lens dimensions change in a complex manner with lens growth. In early fetal life, the lens is almost perfectly spherical, but by birth its sagittal profile is ellipsoidal, as equatorial growth outstrips growth in the sagittal plane. At birth (though information is fragmentary), the equatorial lens diameter is about 6.5 mm, while the sagittal width is about 3 mm. By the ninth decade, this changes to about 910 mm in the equatorial plane and 56 mm in the sagittal plane (fig. 1). It is worth emphasising that while a sagittal thickness of 3 mm is acquired in utero over a period of 7 months, the additional 3 mm are added over a period of 8 decades [4, 7]. In the first 2030 postnatal years, there is little increase in thickness in the sagittal plane and possibly even a decrease [7, 8]. Growth continues to be concentrated in the equatorial plane over this period [4]. A reinterpretation of the data of Willekens et al. [4], where the ratio of equatorial to sagittal width was plotted (for each of 111
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5
s
s s s s s ss s
Sagittal width (mm)
Collins Saunte von Helmholz s Raeder Jansson s Sorsby Larsen Dub (full)
2 0 20 40 Age (years) 60 80
Fig. 1. Composite graphs depicting growth of the lens in the sagittal plane. With permission [7] (see original for
references).
fixed lenses) suggests that the ratio rises from the age of 1030 years (when equatorial growth predominates) and then falls thereafter, as sagittal growth resumes and equatorial growth plateaus (fig. 2). Equatorial expansion in the first, but not later decades, can be attributed in part to the continued action of zonular forces, with growth of the anterior segment in the equatorial plane [9]. Failure to grow in the sagittal plane might be explained by the process of compaction, in which it is envisaged that there is a loss of volume in established fibres, as a result of the simultaneous removal of both water and protein, with no net change in density. It is likely that compaction occurs in both the nucleus and cortex. Compaction is the term employed by Brown [10] to explain why subepithelial opacities such as glaucomflecken recede from the surface at a faster rate than the sagittal growth rate, suggesting that fibres deep to the level of the lesion become compacted. Support for nuclear compaction has come from the study of a small group of patients
with lamellar cataract, in whom shrinkage of the lamellar shell of opacity was demonstrated, particularly in the sagittal plane [11]. The concept of compaction is also supported by the study of Koretz et al. [12], who reported that (1) sagittal lens thickness remains constant until the end of the second decade; (2) nuclear thickness in the sagittal axis decreases approximately linearly with time, and therefore (3) increase in cortical thickness along the sagittal axis balances compaction almost exactly. This cortical growth rate is reduced, but remains roughly linear along the sagittal axis with age, from about age 20 onward. From the third decade, it appears, on the basis of a study of the anterior segment by magnetic resonance imaging, that equatorial growth is approximately zero [13]. This confirms earlier proposals that continued lens growth in the adult eye is primarily in the sagittal direction. It is possible that, upon the completion of nuclear compaction by the end of the second decade, there is a fundamental
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Bron/Vrensen/Koretz/Maraini/Harding
Fig. 2. Change in equatorial diameter of the lens with age (fixed specimens). Note that the increase demonstrated in the first two to three decades of life levels off in later decades. With permission [4].
Fig. 3. Refractive index profile in the equatorial plane of 3 human
lenses. With permission [15].
change in factors influencing lens growth leading to a transition from development to maintenance of lens function. This transition leads inevitably to a shortening of the anterior lens radius. Although this would lead to an increase in optical power of the lens if acting in isolation over this period, in fact, in the normal population, there is a shift from the hypermetropia of childhood towards emmetropia in the second decade, which is maintained or replaced by hypermetropia in later life. The factors regulating this process are obscure, and no reason is known why it should affect the sagittal more than the equatorial plane. The implication is that fibre cytoplasm is preferentially displaced towards the equator as new fibres are added. Brown coined the term lens paradox to describe the failure of curvature changes to affect lens power [14]. It implies a compensatory refractive shift within the lens. For a given lens curvature, the dioptric power of the lens is dependent on the refractive gradient (and hence changes in protein concentration and/or state) across the substance of the lens. Figure 3 shows the rise in refractive index which occurs on passing across the superficial cor-
tex to the lens nucleus along the equatorial axis [15]. In parallel with this, Siebinga et al. [16], using confocal Raman microspectroscopy, have shown a rapid decrease in water content across the superficial cortex, on passing towards the nucleus (fig. 4). Several pieces of evidence suggest that this gradient decreases with increasing age. Pierscionek et al. [15], supported by experimental work and by ray tracing studies [17], suggested that the increase in curvature could be compensated by changes in the cortical refractive index gradient, which would require small, regional, age-related variations in protein and water content. Raman microspectroscopy, however, suggests that the cortical gradient is stable, but that nuclear water rises with age [16]. Whichever event is dominant, the effect is to decrease the net refractive index of the lens over time by a shallowing of the overall gradient, which offsets the optical effect of a shortened anterior radius of curvature. Alternatively, the change in the pattern of lens growth between the youthful and adult lens that leads to shortened anterior lens radius of curvature may function to offset age-related changes in the state of the lens proteins.
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100
80
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a
ac 1 2 Axial position (mm) 3 pc
100
Postnatally, the Y sutures give way to sutures with increasing numbers of branch points. In the mature lens, the suture length into which the fibres insert, increases steadily in relation to the space they occupy at the equator, and in keeping with this, the fibre tips curve and flare as they insert. The direction of curve is in the opposite sense anteriorly and posteriorly. The overlap of the fibre tips at their insertion and the near perfect registration of the sutures over one another in the sagittal plane increase scatter in the region of the sutures [20]. The number of suture braches increases and becomes more disordered in the adult lens. As the circumference of the lens increases, there is a requirement to increase either the number or equatorial width of the lens fibres. In the rabbit, increased fibre width is the major change-accommodating equatorial expansion [21]. Epithelium and Capsule The lens capsule is composed chiefly of type IV collagen, with small amounts of types I and III. Other extracellular matrix materials include laminin, fibronectin, heparan sulphate proteoglycan and entactin [22]. The anterior lens capsule is synthesised by lens epithelium, and its thickness increases with age [2326]. However, posterior capsular thickness remains relatively constant with age, and it is not clear whether posterior subcapsular fibres make any contribution to capsular synthesis. This raises a number of points about lens remodelling. With increasing lens volume, there is an increase in the peripheral extent of both the anterior and posterior capsule and a peripheral expansion of the epithelial plate. Epithelial cell numbers rise with age, while density falls [27]. This may imply that increased cell numbers do not keep pace with increasing surface area, or that cells are lost with time. Although it has been suggested that epithelial cells are regularly lost by apoptosis [28, 29], this view has been contested [30]. Increase in extent of the anterior capsule is probably achieved by additional epithelial synthesis, but extension of the posterior capsule implies either that the peripheral capsule may be spun out by the equatorial epithelium, or that the superficial cortical fibres have a synthetic role. This information is not available. There are also questions as to how capsule thickness is regulated centrifugally during the lifespan, since the peripheral capsule is generally held to be no thinner than the central capsule. It is not clear to what extent the remodelling process affects the zonular insertions, but the observation that the points of insertion move away from the equator in old age
Water content (mass %)
Water content (mass %)
80
60
b
ec 1 2 Axial position (mm) 3 pc
Fig. 4. Absolute water content along the optic (a) and equatorial (b)
axis of an 8-year-old human lens. ac = Anterior capsule; pc = posterior capsule; ec = equatorial capsule [16].
Organisation and Remodelling in the Lens
Growth of the lens implies a constant remodelling of its component parts, the fibres, the capsule and the epithelium. Fibres The complexity of lens fibre organisation increases with age and has been clearly summarised by Kuszak et al. [18]. The Y sutures of the fetal lens are created by fibres arising over a 60 span of the lens circumference inserting into suture branches measuring some 30 so that the fibres are wider at the equator than in the sutural region [19].
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Fig. 5. Load strain (a) and stress strain (b)
curves for the rings of anterior lens capsules of different ages. The load strain curves reflect the mechanical function of the lens capsule in situ, while the stress strain curves express the material properties of the tissue (force per unit cross-sectional area). With permission [26].
Fig. 6. Fall in extensibility (ultimate strain)
of anterior capsular rings with age. With permission [26].
[31, 32] suggests that postnatally, capsular remodelling occurs peripheral to the zonular insertions, except at the equator itself. The observation of Sakabe et al. [33] that the zonule-free zone of the lens capsule decreases with age postnatally could be an artefact of measurement, since their experiments involved removing lens substance from the capsular bag by a phaco technique and refilling with a viscoelastic material prior to measurement. It may be that the more extensible capsule of the young lens allowed greater expansion of the capsular bag than in the old lens. The effect of ageing on capsular biomechanics has been studied in detail by Krag et al. [26], who assessed the mechanical properties of a ring of anterior capsule taken
from a zone corresponding to the site of a standard capsulorhexis. Unlike Fisher [34, 35], they found a non-linear response to increasing load across the age span, with a relatively flat strain response to the lowest loads and a more acute rise with higher loads. The load/strain and stress/ strain curves shifted increasingly to the left with increasing age, with a shortening of the toe region at the lowest loads (fig. 5). Their data showed that ultimate tensile strength decreased by a factor of 5 during the life span, while extensibility decreased by a factor of 2 (fig. 6). In youth, extensibility was as high as 100%, while in old age it dropped to
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responding to the steep, linear part of the curve, may relate to a realignment of the meshwork of collagen, to load bearing and to the development of a resistance to further extension before rupture. Some of the biophysical changes which occur with age could be the result of increased cross-linking of matrix molecules, due in part to glycation [39, 40]. Lens Zones An optical section of the living lens, as provided by slit-lamp biomicroscopy or Scheimpflug photography, demonstrates the presence of several zones of optical discontinuity across its layers, due to changes in the lightscattering properties of the fibres on passing from one zone to the next [41]. Goldmann [42] concluded that the zones of discontinuity were generated every 4 years and Weale [9] has observed the same cycle for the refraction of schoolchildrens eyes. Huggert [43] attributed their presence to changes in the refractive index between the zones, and in keeping with this, Fagerholm et al. [44, 45], using X-ray microdensitometry, reported increases in protein concentration corresponding to the interfaces. This was not supported, however, by the studies of Yaroslavsky et al. [46], who found no correlation between changes in light scatter and protein concentration along the visual axis of the lens, supporting an earlier study by this group using Raman spectroscopy, suggesting that optical changes were due to fluctuations in protein conformation [47]. The Oxford system of lens zoning is simpler than that of Goldmann [7] (fig. 7). If the nucleus of the lens is defined as that fibre mass which is present at the time of birth, then the cortex represents all those fibres which are added postnatally. This simple nomenclature avoids the need to employ terms such as infantile nucleus and juvenile nucleus when referring to the perinuclear regions of the lens in the adult. Using the Oxford system, all fibres laid down postnatally are referred to as cortex. In the adult lens, the cortex can be divided into four zones of discontinuity. The most superficial zone, C1, is a subepithelial clear zone of constant sagittal width (about 125 m), which shows minimal reduction in thickness with age. It represents about 4 or 5 years of growth and about 20 generations of fibres. Its posterior part (C1) is a light-scattering zone which separates C1 from C2. C2 is a clear zone which increases in thickness with age. The transition from C1 to C2 implies a change in the optical behaviour of fibres, 4 years or so old, over a period of one or two generations. A possible explanation for this increased scattering is the process of denucleation of the
Fig. 7. Zones of optical discontinuity of the lens, labelled according to the Oxford system. See text for details. With permission [128].
4060%. The breaking load of the capsules of older donors was only one quarter of that observed for younger donors, despite the fact that older capsules were almost twice as thick. These studies support the clinical experience that the young capsule is strong and highly extensible, and explain why it is more difficult to open surgically. It has a relatively high fracture toughness and a high breaking strength. The old capsule is more fragile and brittle, less extensible and with a markedly reduced breaking strength. It appears that the structural strength of the capsular proteins decreases with age, so that the capsule weakens despite a progressive increase in thickness. This may correlate with the known chemical and morphological changes which occur in the capsule with age. There is an increase in non-collagenous proteins in the capsule [36], and the capsule accumulates linear densities [25, 37] and loses its laminated structure [25, 38]. Krag et al. [26] suggest that the shape of the load/strain curve is explained by the composition of the capsule. The high extensibility of the capsule with small loads at the toe end of the load/ strain curve is thought to reflect a phase of reorientation of type IV collagen molecules in the direction of stretching, while the ultimate elastic stiffness of the capsule, cor-
92
fibre associated with the formation of the nuclear bow, and the concident breakdown of the mitochondria [48 51]. These events, which lead to the dispersal of particulate material and Ca2+ within the fibre, can be conceived as giving rise to, first a rise, and then a fall in the lightscattering properties of the cytoplasm [52]. Zones C3 and C4 form the deep, perinuclear cortex. Zone C3 is a light-scattering zone, whose scattering properties increase with age, while C4 is relatively transparent. The basis for the increased scattering at the level of C3 is not known, but it may reflect the compaction of lens fibres occurring at this level and a consequent increase in fibre membrane density. Some support comes from the presence of so-called relief patterns, observed at this depth by specular microscopy, in which the suture branches are seen to be elevated in relation to the neighbouring fibre mass. This implies a disproportionate compaction of fibre bodies in relation to the fibre tips at the sites of the sutures. An alternative explanation for the zones of discontinuity also comes from data based on Scheimpflug slit-lamp photography of the human lens. Since all of the zones are in the cortical region of the lens, and since the number of zones increases with age, it should be possible to correlate zone location with the age at which their component lens fibres were laid down [12]. Using combined data sets on cortical and nuclear thicknesses in the human lens covering a period from about the age of 8 years to 70 years (Forbes et al. [53] for the young-eye data and Cook et al. [54] for the adult data) and measuring zone location from the central, clear region of the nucleus, it was possible to assign ages to these zones appropriately. These ages correlate very well with the average ages at which new and progressively more complex patterns of suture shells, are initiated. It suggests a causal relationship between the two. Their locations as determined by Koretz et al. [12] were well correlated with locations assigned to the zones using the Oxford system of zone classification [55], and both studies demonstrated a bimodal increase in scattering by the innermost zones with increasing age [54].
Fig. 8. Change in accommodative amplitude with age in females (open symbols) and males (closed symbols). a Subjective measurement. b Objective measurement by refractometry [60].
Presbyopia
Presbyopia is the age-related, symptomatic loss of the ability to focus on a near target. Loss of accommodative power is a lens-related problem which starts in infancy. In youth, accommodation for near is achieved by ciliary muscle contraction, which relaxes zonular tension and allows the lens to become more globular, due mainly to
shortening of its anterior curvature. This is achieved by a forward and inward movement of the ciliary body apex during contraction [56]. The lens thickens axially, and the lens centre and anterior surface move slightly forward. Thickening of the lens is accounted for entirely by a change in nuclear shape, with the cortical thickness remaining unaltered [5759]. Koretz et al. [59] suggested, on the basis of this, that accommodation involves a centrifugal redistribution of fibre cytoplasm in the nuclear fibres. With age, there is a steady loss of accommodative power, which is completed by the age of about 50 years and is termed presbyopia (fig. 8) [60]. The ability to restore focus from near to far is also affected. The factors responsible for presbyopia are multiple [7]:
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Fig. 9. Changes in lens thickness during far-
near (FN) and near-far (NF) accommodation in three subjects, measured by continuous ultrasonic biometry. Responses plotted according to a biomechanical model of the mechanism of accommodation. With permission [61].
(1) There is a forward shift of the internal apical part of the ciliary body with age which reduces the functional reserve of the muscle [56]. However, this does not appear to affect the curvature of the nucleus in the non-accommodated state. (2) There is a major increase in the connective tissue content of the ciliary muscle with age, particularly in the region of the posterior elastic tendon, which may reduce compliance and dysaccommodative recoil. There are minor changes in muscle structure and a reduced responsiveness to neural stimulation.
(3) There is an increase in stiffness of the cortex, and more particularly in the nucleus of the lens, with age. Koretz et al. [59] suggested that presbyopia might result from a restriction of centrifugal, cytoplasmic movement in the nuclear fibres during accommodation, and there is some support for this view (see below). An increase in thickness of the cortex, due to growth, could also reduce the ability of the changes in nuclear curvature to influence surface curvatures. (4) Increased curvature (shortened radius) of the anterior lens surface reduces the ability of ciliary contraction to cause further change; altered zonular insertion loca-
94
tions as a result of increased lens volume, increased sagittal thickness and altered location of the lens centre of mass relative to the ciliary body reduce zonular tension in the unaccommodated eye. The relative contribution of these changes to the presbyopic state is not established. Beers and van der Heijde [61] studied the far-near and near-far dynamics of accommodation by continuous ultrasound biometry and fitted the results to a biomechanical model (fig. 9). They found that the speed and extent of accommodation diminished with time. Like other authors, they showed a significant dcrease in the maximum accommodative amplitude with age and, in addition, a slowing down of the process, as indicated by a lengthening of the far-near and near-far time constants, which increased at an approximately equal rate of 7 ms/year B 1.8 SEM and 7 ms/year B 1.6 SEM, respectively. With age, the lens changes in size and in elastic properties [34, 35, 6266], the choroid changes in elastic properties [66, 67], the ciliary muscle shows hypertrophic changes [68] and loss of elastic tissue at its choroidal attachment [69] while the elastic properties of the zonules do not change [65, 66]. Beers and van der Heijde [61] argued that since the far-near time constant is dependent on lens properties, while the near-far time constant depends on lens, zonule and choroidal properties, then since the values change equally with age, most of the changes must be due to changes in lens properties, rather than to changes in choroidal or zonular stiffness. In keeping with their proposition, the damping coefficient of the lens increased 20-fold between the ages of 15 and 55, while the spring constant increases about 6-fold. Beers and van der Heijde [61] suggest that an increase in the number of fibres with age could explain the increase in both. They concluded that presbyopia is caused by changes in lenticular elastic properties and that the decrease in accommodative speed is caused by changes in lenticular viscoelastic properties. The elastic and viscous properties of the lens are dependent on the properties of the fibre membranes, cytoskeleton [61,70] and crystallins. The lens fibre membranes are characterised by an extremely high cholesterolto-phospholipid ratio of between 3 and 5. It has been shown that with age the 3--OH cholesterol decreases, due to formation of cholesterol esters and 5- hydroxy cholesterol [71]. This might affect the deformability of the membranes. Loss of lens deformability with age has also been attributed to increased binding of lens proteins to the cell membranes with age [7274]. It has been suggested too, that actin could impart a contractile tone within the fibres
that resists deformation and restores the lens to its nearaccommodated shape [75, 76]. It is also assumed that since the lens fibres are interlocked, the change in shape of the lens is achieved by a redistribution of cytoplasm within individual fibres [59], and of relevance to this view is the change in composition of the nuclear proteins of the lens which takes place with age. In the young lens, under 15 years of age, nearly half of the nuclear soluble protein is -crystallin. By the mid twenties, this has dropped to 25% of the void volume fraction, and by the mid forties, crystallin represents less than 5% of the water-soluble protein of the nuclear void volume, while there has been a steady increase in an aggregated component, rich in and mainly -crystallin [77]. It is not clear to what extent such aggregates exist in an insoluble form in the intact lens, but it may be, as McFall-Ngai et al. [77] suggest, that they restrict the redistribution of nuclear fibre cytoplasm during attempted accommodation. Finally, it has been shown that with age the nuclear water content decreases, giving rise to a lower refractive index [16].
Age-Related Changes in Lens Proteins and Scavenger Molecules
The lens crystallins (, and ) make up over 90% of the lens proteins and account for the high refractive index of the lens. Other proteins include cytoskeletal and membrane proteins including transporter and channel proteins, junctional proteins and proteins concerned with cell communication, such as the gap junctional proteins (i.e. connexins: 1-connexin 43 in the epithelium, and 3- and 8-connexins 46 and 50 in the cortical fibres). There are also many enzymes concerned with lens metabolism. The lens membranes are rich in cholesterol and phospholipids [78]. Although crystallins are found in all lens cells, they are not evenly distributed. While fibre cells contain each of the crystallins in varying proportion, the lens epithelium contains only -crystallin. The proportions of , and in the lens nucleus change during intra-uterine development (table 1). In the first 119 days of prenatal life, -crystallin is the predominant form (49%), with and present in almost equal proportions [79]. From 119 to 231 days of fetal life, there is a small increase in - and a corresponding reduction in - and -crystallin content. Of the -crystallins, at birth [80], or by about the age of 6 years [81], all but the synthesis of S-crystallin has ceased. In the adolescent lens, about 42% of the soluble fraction of the total lens crystallins, is -crystallin, about 35% -crystallin and
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Table 1. Change in fetal crystallin concentration with age in the human lens [Ahrend et al., 1987]
Crystallin Total Total (1, 2, 3) S
119 days, % 23.5 49.3 25.6 6.1
231 days, % 19.6 42.6 29.3 7.2
13% -crystallin. In older lenses, there is a rise in - at the expense of - and -crystallin due to the incorporation of these crystallins into higher-molecular-weight complexes. Certain lens proteins are degraded over time as part of the process of fibre differentiation, while others are completely degraded, truncated or modified by post-translational events; - and -tubulin are expressed in the fibre during fibre elongation and are lacking in the fully differentiated fibre. In the chick, the basal membrane complex of elongating fibres inserts into a lateral fibre membrane rich in N-cadherin; the complex disappears after elongation is completed, but N-cadherin persists throughout the lens substance. Neural cellular adhesion molecule (NCAM) is found only in the epithelium and superficial cortex. The beaded filament proteins phakenin and filensin are expressed in the superficial cortex, but only in truncated form in the deep cortex, whereas the intermediate filament protein vimentin is expressed throughout the cortex, but not in the lens nucleus. Sodium-potassium activated ATPase is found in a non-functional form in the deeper cortex and nucleus. In some species, there is a decrease in epithelial ATPase activity in older lenses. By pooling data obtained in three different laboratories, which included electrical measurements, ion analyses and flux measurements, Duncan et al. [82] were able to characterise some of the changes in membrane physiology which occur in the transparent human lens with increasing age. Membrane potential and resistance progressively declined with age from 50 mV at age 20 to about 30 mV at age 60, together with an increase in internal Na+ and free Ca2+ and a concomitant stimulation of Na+ and K+ transmembrane fluxes. These data have been interpreted as suggesting that ageing is associated in the human lens with an increasing contribution to membrane ion traffic from channels, allowing Na+, K+ and Ca2+ to pass. Relative permeability (PNa/PK) computed from the Goldman equation shows a 6-fold increase from age 20 to 80 years. The fact that the changes in lens
Na+ and K+ permeability ratio with age are congruent with the changes which occur in lens optical density over the same period of time, suggests either a common mechanism for the two phenomena or a cause and effect relationship. This indicates that the major ionic events which occur in cortical cataract are initiated in the transparent lens as a result of ageing. This also raises the possibility that part of the increase in sagittal lens dimensions that occurs with age and which is interpreted as lens growth is actually due to a degree of cortical swelling. Also, major intrinsic protein (MIP26) of the lens fibres, representing 50% of its membrane proteins and corresponding to the water channel protein CHIP28, exists increasingly in older lenses in a truncated form (MIP22), shortened at its C-terminal end and lacking the calmodulin binding site necessary for calcium-activated closure of the channel [83]. Such a modification could render the lens vulnerable to the spread of ion/water decompensation from fibre to fibre in response to local stress or injury. This increase in vulnerability could lead to the spread of opacification. The machinery for synthesis of MIP26 and cholesterol is confined to the superficial 510% of the lens, although some synthetic enzymes persist into the lens nucleus [84]. Cholesterol concentration peaks in the most superficial cortex when studied by filipin Raman microscopy and falls in the nucleus while the cholesterol/phospholipid ratio, higher in the lens than in any other tissue, is invariant with age along the optic axis. There is a relative fall in cholesterol levels in the anterior versus the posterior cortex with age which has been mooted to be due to a loss of cholesterol through UV-induced lipid peroxidation [85]. The fibre crastallins are modified postnatally in the mature lens fibre. All of the crystallins are glycated to some degree, and the proportion of crystallins modified glycation increases with age. In addition, A- and B-crystallins are truncated from the C-terminal end, leading to the production of 10 or more different subunits of , which are encountered in the cortex and nucleus of older lenses. There is a shift of crystallins from -low (lowmolecular weight) to -high (high-molecular weight), from cortex to nucleus and an increase in -high in the cortex and nucleus with age. -High probably represents the formation of soluble -crystallin aggregates with other crystallins. These events correspond to earlier findings in which -crystallin almost disappears from the soluble extracts of the nucleus with age, with over 80% of these proteins leaving the soluble fraction [15, 77] or, more probably, entering the high-molecular weight fraction. McFall-Ngai et al. [77] observing the steady insolubilisa-
96
Fig. 10. Loss of -crystallin (!) and highmolecular-weight proteins ([) in the human lens with age. !! = Percentage of total soluble nuclear protein as -crystallin; [ [ = percentage of the total area of the nuclear protein chromatogram peaks represented as the void volume peak. The difference between the curves reflects a gradual incorporation of primarily -crystallins into the void volume fraction [88].
tion of higher-molecular-weight species in the lens nucleus, particularly between 3545 years of age (fig. 10), remarked on the temporal similarity of these events to those of presbyopia. They inferred that the increasing stiffness (or hardness) of the -crystallin aggregates might cause a restricted flow of nuclear fibre cytoplasm during attempted accommodation, which could explain the reduced deformability of the lens nucleus that occurs with age. These observations support the original proposal of Koretz et al. [59]. Derham and Harding [86] found no age-dependent loss of the chaperone activity of both -crystallin fractions in the cortex of the rabbit lens, while nuclear -high showed a greatly compromised chaperoning ability. It is likely that these latter changes are due to post-translational modifications of -crystallin. In older human lenses, no -low is found in the soluble fraction of the crystallins [87]. The -crystallins become more polydisperse with age. There is an age-related fall in B2-crystallin in the soluble fraction, while some is found there in a truncated form due to cleavage at the N-terminal end of the molecule [88, 89]. --Monomers become more acidic with age and show an accumulation of S-crystallin [90]. There is evidence of N- and C-terminal modification of the -crystallins with time, most of which occurs by the age of 60 years
[89], and D and S fragments, cleaved between their domains, have been identified in the ageing lens. Deamidation of crystallins is a significant age-related event occurring in the lens [91]. Lampi et al. [92] found that the majority of protein changes occurred before the age of 17 years, and included a decrease in B1, B3, A3, C and D and the appearance of new species, namely the deamidated forms of B1 and A3/A1, deamidated at their N-terminal extensions. The importance of deamidation was also noted by Ma et al. [93] using electrospray ionisation mass spectrometry, who also found an increase in the ratios of B/A and S/C. There has been less emphasis in recent times on the formation of insoluble, high-molecular-weight aggregates, but soluble complexes of -crystallin, with and located in the centre of the complex have been described [94]. The relative lack of crystallin oxidation in cataract-free, aged lenses speaks for the integrity of those systems which protect the lens from oxidative stress, but hydrogen peroxide is present in human aqueous humour and has been invoked to explain human cataract formation [95a]. Glutathione is present in the young lens at levels up to 4 mmol [78], and there is a steady fall in reduced glutathione with age. But the level is still in the region of 2 mmol in the 80-year-old lens. The reason for the fall in GSH is not certain, but some loss is accounted for by the formation of
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adducts with proteins, such as the crystallins, or with 3OH-kynurenine glucoside, to form an important fluorophore (see below) [95b]. There is a fall in the glutathione synthetic enzymes glutamyl cysteine synthetase and in glutamine synthase with age [96], but it has also been suggested that availability of cysteine may be a limiting factor. Glutathione peroxidase, an enzyme involved in scavenging hydrogen peroxide, is at a low level in the newborn lens, rises to a peak in the teens and then falls, from the age of 40 years, to low levels once more. Information about the effects of ageing on the activity of catalase, superoxide dismutase and vitamin E in the human lens are sparse, but we may conclude that although the integrity of scavenger systems within the lens may extend well into old age, the lens must become increasingly susceptible to the possibility of oxidation, partly because its proteins are becoming more vulnerable, and partly because its scavenger systems are less secure. It must also be supposed, since we necessarily are presented with averaged values for tissue levels of scavenger molecules, that there will be asymmetries of distribution making some cells more vulnerable to attack than others.
Scatter, Absorption and Fluorescence
The young human lens is colourless and transmits almost 100% of the incident light. With age, there is increased scatter and absorption of optical radiation by the lens, and it becomes yellow and fluorescent. Scatter increases in all zones of the lens, with the least change in the lens centre. Scheimpflug photography shows a dip in light-scattering in this region, at the so-called nuclear dip or sulcus, which is centred on the embryonic nucleus, and exhibits the least level of scattering throughout the age span. The greatest increase in scattering over time is encountered in zone C3 of the deep cortex [53, 97] and then the nucleus. Increased scattering has been attributed to the accumulation of high-molecular-weight crystallin aggregates within the lens [98]. Random fluctuations in protein density, leading to aggregates of 50 ! 106 g/mol or more were proposed as sufficient to cause lens turbidity. The presence of such aggregates was quickly confirmed by studies by Spector et al. [99] and Jedziniak et al. [100], and attributed to the presence of disulphide cross-linking [101]. Since studies by Yu et al. [102] have failed to show the presence of such cross-links in clear human lenses up to and greater than 65 years in age, it would appear that the
exponential increase in back scattering observed in cataract-free lenses (for instance by quasi-elastic light scattering) [103] is due, for instance in the nucleus of the lens, to the presence of complexes, referred to earlier, formed between -crystallin and unfolded - and, to a lesser extent, -crystallins. Unfolding is caused by post-translational modifications brought about by glycation, carbamylation, methionine oxidation and racemisation. Of these, glycation (by sugars and by ascorbate derivatives) is probably the most important. Paradoxically, the hexoselysine adduct does not increase in the human lens [104], presumably because the initial adducts are converted into more stable forms, such as carboxymethyl-lysine and caroxyethyl-lysine), which accumulate in the lens at least up to the age of 80 years [105]. The lens possesses three major tryptophan-derived fluorophores, or UV filter compounds, 3-hydroxykynurenine glucoside [106] (3-OHKG), 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid)-O-glucoside (AHBG) [107] and a newly discovered compound which is the glutathione adduct of GSH with a deaminated form of 3OHKG (termed GSH-3-OHKG) [95b]. Unlike the first two compounds, whose concentrations either decline or remain constant during adult life [108110], this latter compound increases with age. Increasing yellowing of the lens and absoprtion of wavelengths at the blue end of the spectrum play a protective role against the effects of optical radiation on the macula. The novel fluorogen GSH-3-OHKG, has absorption maxima at 260 and 365 nm and is fluorescent (Ex360 nm/ Em500 nm), It has been suggested that GSH-3-OHKG (fig. 11) is generated via the formation of an -unsaturated carbonyl derivative of 3-OHKG, resulting from a deamidation of the kynurenine side chain. This could occur slowly at physiological pH, and its feasibility has been demonstrated in vitro [95b]. An enzymic mechanism of formation has also been proposed. The enzyme, kynurenine transaminase, forms various xanthenuric acid derivatives, which may contribute to lens fluorescence [111]. Its concentration in the lens increases with age. Reopening of the xanthurenic ring, perhaps by a glutathionyl radical (as occurs in other systems) has been proposed as an alternative route to GSH-3-OHKG formation [95b]. GSH-3-OHKG may be the major contributor to fluorescence in the ageing lens. The absorption of optical radiation by the lens rises exponentially with age [112115] inversely more for all wavelengths of the visible spectrum and in the ultraviolet. The rise in absorption is highest for wavelengths at the blue end of the spectrum, around 460470 nm, and, as
98
noted, this has been attributed to the postnatal accumulation of yellow chromophores [116]. Mellerio [117] found that the optical density of the lens at 400 nm was about equal in the cortex and nucleus of the young lens, but became higher in the nucleus in the older lens. Weale [118], recalculating the data of van Heyningen [119] for the, probably, 3-OH-kynurenine glucoside, found it to be higher for the nucleus than the cortex by an order of magnitude. Of the various fluorescent species found in the cataract-free lens [120], one is a perinuclear, green fluorescence, less prominent in the nuclear region, excited at a wavelength of 490 nm and with an emission at 530 nm [115, 120, 121]. The other is a blue fluorescence, excited at 340 nm, with an emission at 420 nm. This is associated with an age-related insolubilisation of protein. Some of the fluorogens responsible for this category of fluorescence change in concentration with age, with an upward shift in fluorescence towards longer wavelengths. A shift in the shorter blue wavelengths is seen in the nucleus and in the longer blue wavelengths in the cortex [121]. Yu et al. [122] identified a deep red fluorescence (672 nm) in many, but not all, clear, old lenses of 75 years and over. Various proposals have been made as to how these fluorogens can interact with and modify lens proteins and thus play a damaging role which may lead to cataract formation [107, 111, 123125]. In addition to the changes in protein structure and conformation, and the increase in light scatter, absorption and fluorescence which occur with age, membrane breaks become evident in the fourth decade of life which correlate with the presence of tiny, focal opacities (fig. 12a) [126]. Vrensen et al. [126] and Willekens et al. [4], studying clear human post-mortem lenses between the ages of 23 and 82 years found three age-related, ultrastructural changes which were confined to the fibres of the superficial, equatorial cortex and absent from the anterior and posterior cortex, supranuclear equatorial cortex and nucleus. These were: (1) membrane ruptures, (2) water vacuoles and (3) multilamellar bodies. Membrane ruptures (fig. 12b) were rare below the age of 40 years, but by the eighth and ninth decades they were frequent, so that 10 might be observed in one low-power field (!5001,000). Water vacuoles were also found in the superficial equatorial cortex, often in large numbers, covering multiple fibers over long distances (fig. 12c). They were sometimes accompanied by membrane holes, and were infrequent in young and middle-aged eyes. Multilamellar bodies were found within otherwise perfectly normal fibres. They ranged in appearance from simple
O HOOC H2 C N H O
6 5 6 1
COOH H N C O H2 C C H2 CH NH2
C aCH bCH2 S
H
4
CH2OH
5
4 3
8 CH 10 C 9 COOH H2 NH2
C
7
HO HO
3
H H
O H
1 2
OH H
Fig. 11. Proposed structure of GSH-3-OHKG. With permission
[95b].
vacuoles surrounded by a single or double membrane, to multilamellar whirls. An intermediate form comprised concentric arrangements of electron-dense, membranebound structures, apparently in a process of evolution. Globular elements were also seen occasionally, filling degenerative foci in a small part of one or a few fibres, and associated with dust-like opacities on biomicroscopy (fig. 12d). A prevalence of such features of 50% was found in the age group 4560 years [4]. Their presence within a lens which is otherwise (functionally, optically) effective is a reminder of the fact that the transition between ageing events in the lens and those which characterise the onset of cataract may be regional rather than global, and that the lens possesses mechanisms which can maintain a spatiotemporal separation between normally functioning and disordered regions. The focal opacities referred to above are restricted to the superficial equatorial cortex of the lens. Bron and Brown [127] suggested on clinical grounds that they are stationary or slowly progressive features segregated from unaffected parts of involved fibres by a process of annealing (sealing off of disordered regions from healthy regions; fig. 12e), and form adjacent, healthy fibres by a loss of functional communicating junctions (fig. 12). This was confirmed by Vrensen et al. [126], who demonstrated by electron microscopy that these structures are globular masses, whose membranes are studded with numerous
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c
Fig. 12. a Dark-field picture of human lens with small cortical opacities (white arrows). Dark arrows indicate peripheral shades. With permission [85]. b Scanning electron microscopy (SEM) of superficial membrane ruptures in a clear lens. With permission [126]. c SEM to show large (arrow) and small water vacuoles in a group of adjacent lens fibres. Holes (asterisks) are much larger than the sockets of ball and socket junctions (small arrows). With permission [126]. d SEM to show an accumulation of globular elements involving contiguous fibres. From a clear lens showing dust particles biomicroscopically. With permission [126].
100
g
Fig. 12. e SEM to show a flake-like body (asterisk) comprising segments extending across nearly 20 fibres and
segregated from them. Inset shows cortical position of these opacities (asterisk). Dark field. e = Equator; p = periphery. With permission [85]. f Transmission electron microscopy (TEM) of free-fractured membranes inside and at the margin of a small opacity containing globular elements. With permission [85]. g A higher-power view of f showing square arrays (arrowed). With permission [85]. h TEM of a lens opacity demonstrating the localisation of loosely bound and free calcium (arrows). With permission [85].
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square arrays (fig. 12f, g). This suggests that the membranes are non-leaking and would prevent the export of cataract-inducing factors. Electron microscopy (fig. 12h) also showed that the opacities are filled with membrane-bound vesicles, containing elevated levels of free Ca2+. Confocal Raman imaging [85] proved that the vesicles are rich in cholesterol and contain altered phospholipids. The proteins in the opacities have decreased levels of aromatic amino acids
and elevated levels of disulfide bridges. These tiny opacities are considered to be the precursors of spherical, cortical opacities and ultimately to mature cataracts in old age.
Acknowledgments
Our thanks are due to Dr. Brett Garner for a critical review of the manuscript.
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Aging and Age Related Ocular Diseases

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Aging and Age-Related Ocular Diseases

55 figures and 10 tables, 2000

Vol. 214, No. 1, 2000

Regulation of Trabecular Meshwork Contractility

Full-Thickness Retinal Transplants: A Review

Ultrastructure of Retinal Cells Transplanted to the Rabbit Choroid

Oxidative Stress and Age-Related Cataract

The Ageing Lens

2000 S. Karger AG, Basel 00303755/00/21410005$17.50/0 Accessible online at: www.karger.com/journals/oph

Key Words B-Crystallin W Neurodegenerative diseases W Heat shock protein

2000 S. Karger AG, Basel 00303755/00/21410007$17.50/0 Accessible online at: www.karger.com/journals/oph

B-Crystallin in Patients with Alexanders Disease

Amyotrophic Lateral Sclerosis

B-Crystallin in Other Neurodegenerative Diseases

2000 S. Karger AG, Basel 00303755/00/21410013$17.50/0 Accessible online at: www.karger.com/journals/oph

Table 1. Immunohistochemical demonstration of B-crystallin in normal nonlenticular tissues

Staining ++ +++ +++ +++ +++ + + ++ ++ + + ++ ++ ++ +++ ++

Table 2. Number of tissues in different

age groups investigated by immunohistochemistry

Anterior eye segment

Material and Methods

B-Crystallin and Age

Table 3. Number of tissues in different

Retrolaminar optic nerve dot blot PCR

Heart muscle dot blot 1 1 1 1 1 1 1 1 1 2 4 1 2 2 1 2 2 2 2 1 2 2 2 2 2 3 PCR

B-Crystallin and Age

Table 4. Distribution of B-crystallin in

the anterior eye segment of various animals

Corneal Ciliary epithelium endothelium

Iris epithelium (+) ++ ++ ++ ++

Trabecular meshwork 0 (+) + 0 ++

Mouse Rat Pig Cow1 Primate1

B-Crystallin and Age

350 300 250 200 150 100 50

aB-Crystallin (ng/mg protein)

14,000 12,000 10,000 8,000 6,000 4,000 2,000 0 0

600 500 400 300 200 100 0

aB-Crystallin (ng/mg protein)

aB-Crystallin (ng/mg protein)

0 0 7 7 7 24 24 24 24 Age (months) 60 60 72 5 5 5 6 Age (months) 72

B-Crystallin and Age

Postmortem time 47 h 2 days 50 h 3 days 2 days 2 days 15 h 26 h 18 h 20 h 48 h 1 day 40 h 28 h 30 h 4h

B-Crystallin and Age

2000 S. Karger AG, Basel 10153755/00/21410024$17.50/0 Accessible online at: www.karger.com/journals/oph

Materials and Methods

Optic Nerve in Monkeys with Laser Glaucoma

Optic Nerve in Monkeys with Laser Glaucoma

Fig. 1af. Immunohistochemical staining

Fig. 2ad. Semithin sections through the

Optic Nerve in Monkeys with Laser Glaucoma

Optic Nerve in Monkeys with Laser Glaucoma

Regulation of Trabecular Meshwork Contractility

2000 S. Karger AG, Basel 00303755/00/21410033$17.50/0 Accessible online at: www.karger.com/journals/oph

Fig. 1. Typical recording of a contractility

Regulation of Trabecular Meshwork Contractility

Fig. 2. Recording showing the contractile response of bovine tra-

Regulation of Trabecular Meshwork Contractility

110 perfused anterior segment (bovine) 40 min

Relative outflow (%)

pilocarpine (105 mol/l)

The Perfused Anterior Segment

Regulation of Trabecular Meshwork Contractility

Fig. 9. Measurement of pHi using bovine trabecular meshwork.

Electrophysiology of Trabecular Meshwork