Bioelectromagnetics 23:488^495 (2002)

Calcite Microcrystals in the Pineal Gland of the Human Brain: First Physical and Chemical Studies
Simon Baconnier,1 Sidney B. Lang,1* Maria Polomska,2 Bozena Hilczer,2 Garry Berkovic,3 and Guilia Meshulam3
Department of Chemical Engineering, Ben-Gurion University of the Negev, Beer Sheva, Israel 2 Institute of Molecular Physics, Polish Academy of Sciences, Poznan, Poland 3 Soreq NRC,Yavne, Israel
A new form of biomineralization has been studied in the pineal gland of the human brain. It consists of small crystals that are less than 20 mm in length and that are completely distinct from the often observed mulberry-type hydroxyapatite concretions. A special procedure was developed for isolation of the crystals from the organic matter in the pineal gland. Cubic, hexagonal, and cylindrical morphologies have been identied using scanning electron microscopy. The crystal edges were sharp whereas their surfaces were very rough. Energy dispersive spectroscopy showed that the crystals contained only the elements calcium, carbon, and oxygen. Selected area electron diffraction and near infrared Raman spectroscopy established that the crystals were calcite. With the exception of the otoconia structure of the inner ear, this is the only known nonpathological occurrence of calcite in the human body. The calcite microcrystals are probably responsible for the previously observed second harmonic generation in pineal tissue sections. The complex texture structure of the microcrystals may lead to crystallographic symmetry breaking and possible piezoelectricity, as is the case with otoconia. It is believed that the presence of two different crystalline compounds in the pineal gland is biologically signicant, suggesting two entirely different mechanisms of formation and biological functions. Studies directed toward the elucidation of the formation and functions, and possible nonthermal interaction with external electromagnetic elds are currently in progress. Bioelectromagnetics 23:488495, 2002. 2002 Wiley-Liss, Inc. Key words: microcrystals; second harmonic generation; piezoelectricity; scanning electron microscopy
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INTRODUCTION The pineal gland is a neuroendocrine transducer that converts a neuronal signal into an endocrine output. The neural signal originates in the suprachiasmatic nuclei of the hypothalamus [Reiter, 1991]. The pineal gland is located close to the anatomical center of the human brain, near the third ventricle, and between the superior colliculi. The pineal gland is a highly active organ that secretes a number of important products, the best known of which is melatonin. It has a several important physiological effects that have been studied by a large number of researchers. Among recent reviews are works by Reiter [1991], Karasek [1999], and Pevet [2000]. Pineal calcications have been found in numerous animals and in humans. They have been given numerous names in the literature, including corpora arenacea, acervuli, psammoma bodies, and brain sand [Welsh, 1985; Vigh et al., 1998]. Two major forms of pineal calcications have been observed: (i) polycrystalline
2002 Wiley-Liss, Inc.

complexes with dimensions of the order of hundreds of micrometers, often called mulberry-like structures or concretions [Vigh et al., 1998], and (ii) small, well dened crystals having long dimensions of the order of 1020 mm [Lang et al., 1996]. The mulberry-like structures consist of a mineral component, hydroxyapatite, and protein and glycoprotein organic components
Contract grant sponsor: Cooperant du Service National Grant; Contract grant sponsor: The Israel Science Foundation; Contract grant number: 54/98. *Correspondence to: Sidney B. Lang, Department of Chemical Engineering, Ben-Gurion University of the Negev, 84105 Beer Sheva, Israel. E-mail: lang@bgumail.bgu.ac.il Received for review 29 January 2002; Final revision received 16 April 2002 DOI 10.1002/bem.10053 Published online in Wiley InterScience (www.interscience.wiley.com).

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[Krstic, 1976; Michotte et al., 1977; Ostrowski et al., 1980; Kodaka et al., 1994; Nakamura et al., 1995]. Chemical methods have been used to identify a number of elements in the pineal concretions, including Ca, P, Cu, Mn, Zn, Fe, Si, Al, Na, Mg, Cr, K, Sr, Ti, Co, and Ni [Michotte et al., 1977; Humbert and Pevet, 1991, 1996]. Various authors, using a number of different physical techniques including light microscopy, X-ray diffraction, electron probe analysis, and scanning and transmission electron microscopy, have analyzed human pineal concretions. The physiopathological role of pineal acervuli is unknown. The mechanism of mineralization of these structures is also not clear [Schmid, 1993]. Although the concretions have been studied extensively, no experiments have been done on the newly observed calcications that are also found in the pineal gland [Humbert and Pevet, 1991, 1996]. In a recent study, Lang et al. [1996] have observed weak second harmonic generation (SHG) in pineal tissue slices. These authors proposed that the SHG was due to the presence of a crystalline component. SHG is normally found only in materials having a noncentrosymmetric crystallographic point group [Kurtz and Dougherty, 1978]. Because piezoelectricity is also only found in noncentrosymmetric crystals, it was further suggested that the tissues might respond to electromagnetic elds through a piezoelectric transducer mechanism. The aim of this work was to determine the physical characteristics and chemical composition of the microcrystals and to ascertain if the microcrystals were responsible for the SHG. In this research, the microcrystals were studied by a large number of techniques. The crystalline nature of the pineal deposits was rst conrmed with optical microscopy using a polarizing microscope. Scanning electron microscopy (SEM) was used to characterize the morphology of the crystals and the different shapes of the mineral deposits. Elemental analysis was performed with the energy dispersive spectroscope (EDS) of the SEM. High-resolution transmission electron microscopy (HRTEM) coupled with selected area electron diffraction (SAED) was used to determine the composition of the crystals. The SAED results were conrmed by Raman spectroscopy. SHG was used to show that calcite rather than hydroxyapatite was the source of the SHG source observed previously. MATERIALS AND METHODS Pineal Glands and Crystal Isolation Human pineal glands were supplied by the Institute of Pathology of the Soroka Medical Centre in Beer Sheva, Israel, and by the Anatomopathology Service of

the CHU Michalon in Grenoble, France. The pineal glands were xed in 10% formalin (approximately 4% formaldehyde). A total of 20 glands from subjects ranging in age from 15 to 68 years were studied. The microcrystals were isolated from the pineal glands using a procedure developed by Weiner and Price [1986]. This technique has been reviewed by Tomazic et al. [1993]. Small pieces of the pineal gland (about 10 mg) were placed in a microcentrifuge tube containing 1.5 ml of 2.5% sodium hypochlorite (commercial bleach diluted twice, is a convenient source) and sonicated for 15 min. After allowing the sample to settle for 1 min, the supernatant liquid was transferred to a second microcentrifuge tube and centrifuged at approximately 9000g for 1 min. The pellet containing the solids was immediately washed twice with 95% ethanol and then resuspended in approximately 50 ml of 100% ethanol. It should be emphasized that at no point, did any of the samples come into contact with solutions containing calcium ions. Microcrystals were found in every gland in quantities ranging from 100 to 300 crystals/mm3 of gland. No attempt was made to correlate the quantity of crystals with either the age of the subject or pathological details. SEM Samples The isolated crystal samples were prepared by sonicating the resuspended crystals to break up agglomerates and to homogenize the suspension. Then TEM grids were dipped into the suspension and allowed to dry overnight. The samples were mounted on SEM stubs using double sided carbon tape. They were gold coated with a Polaron SEM Coating Unit E5100. The observations were carried out with a JEOL JSM 5600 SEM. Microanalysis studies were performed with a NORAN EDS Analyzing System. HRTEM and SAED Samples Because the microcrystals were too thick for HRTEM observation, they were rst crushed between two glass slides and the powder was suspended in 50 ml of 100% ethanol. The suspension was sonicated for 2 min. TEM carbon-coated copper grids were immediately dipped into the suspension. Excess ethanol was bled off from the edge and the samples were dried for 2 h. Samples were studied in a JEOL-2010 transmission electron microscope equipped with an analytical ISIS system for energy dispersive X-ray spectroscopy (EDS). Near Infrared Raman Spectroscopy Samples Near infrared Raman spectra of isolated crystals and pure calcite were obtained with a Bruker IFS 66 FTIR spectrometer equipped with an FRA 106 Raman

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module and a Ramanscope microscope. Measurements were made with a 40 objective (spot size $25 mm). The spectral resolution was 2 cm1. The samples were excited at 1064 nm using a diode pumped Nd:YAG laser at about 5 mW power. SHG Samples SHG measurements were made on four types of materials: powdered concretions, pure hydroxyapatite powder, microcrystals, and pure calcite crystals. Concretion powder and hydroxyapatite powder samples were mounted on glass slides with imaging chambers. Microcrystals and pure calcite crystals were mounted directly on glass slides. The concretions had been separated previously in the rst stage of the microcrystal isolation, and then were crushed to a powder. The SHG equipment was similar to that used by Kurtz and Dougherty [1978] and Lang et al. [1996]. The exciting laser was a Nd:YAG laser that produced 7 ns pulsed radiation at 1064 nm, and the detected SHG was at 532 nm. It was necessary to separate SHG at 532 nm from spurious background interference due to instrument noise, luminescence, or thermally excited processes. This was achieved by inserting a 540 nm band pass lter that could be tilted about an axis to allow passage of specic wavelength bands in the range 520540 nm as has been described by Morrison et al. [1996]. RESULTS The SEM studies of single microcrystals permitted high quality morphological analysis. The results conrmed that the microcrystal isolation protocol was completely satisfactory. Three different shapes of crystals were observed: cubic, hexagonal, and cylindrical. Typical SEM photographs of the three types are shown in Figure 1a,b,c. Length dimensions of the crystals varied from 23 to about 20 mm. The most common morphology was a cylindrical body with sharp extremities. This form comprised about 95% of the samples observed. Edges were usually very sharp. The surface, which was very rough on the body and very smooth on the extremity edges, was a common feature on most crystals. Examples of a complete crystal and a magnied portion of one are shown in Figures 2a,b, respectively. The roughness may indicate crystallization steps and the smoothness, a sign of the presence of a highly organized material. As a reference, SEM photographs were taken of the mulberry-like concretions as well. Two general sizes were observed as shown in Figure 3a,b. Their outer structure was similar to those observed by others [Michotte et al., 1977; Kodaka et al., 1994].

Fig. 1. SEM photos of isolated pineal microcrystals on a Formvarcovered TEM grid. The three different crystal shapes observed were (A) cubic, (B) hexagonal, and (C) cylindrical.

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Fig. 2. SEM photos of isolated crystal on a Formvar-covered TEM grid. (A) Microcrystal with rough surface and sharp edges, (B) detail of the rough surface of a crystal.

Fig. 3. SEM photos of mulberry-like concretions in cryofractured pineal tissue. (A) Small concretions with lobes, (B) large conglomerate.

The EDS analyzer coupled to the SEM was used to determine the composition of the crystals. The principal elements identied were calcium, carbon, and oxygen, with less than 0.5 wt% each of silicon, aluminum, sodium, and magnesium. An SEM photograph of a crystal and the corresponding EDS results are shown in Figure 4a,b,c. It was especially signicant that no phosphorus was detected. It was not possible to determine the carbon and oxygen quantitatively because of their presence in the sample holders double-sided carbon tape and the Formvar-covered copper grid. Among biominerals containing the detected atoms, only calcite (calcium carbonate) and calcium oxalate are potential candidates. Electron diffraction patterns were used to identify the microcrystals. Because the crystals were too thick

for electron penetration, the studies were carried out on small particles from isolated crystals. HRTEM observation showed that only a few particles were thin enough for analysis, despite the high power of the microscope (200 keV). A typical diffraction pattern is shown in Figure 5. The electron diffraction patterns taken from these particles were indexed in terms of a hexagonal unit cell with lattice parameters a 4.989 nm, c 17.062 nm, and a 120 8. The dimensions are the same as those of calcite. The Raman frequencies of the observed lines correspond to main lines in calcite Raman spectra. Nakamoto [1978] reported calcite peaks at 1434, 1088, 714, 283, and 156 cm1. These peaks are in good agreement with our spectra: 1450, 1085.1, 710.9, 299.9, and 154.2 cm1. As an additional check, Raman spectra

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Fig. 5. Indexed SAED pattern from fragment of microcrystal.

Fig. 4. Elemental energy dispersive spectra (0 ^10 keV) showing the composition of isolated microcrystals. (A) SEM of microcrystal, (B) EDS measured at location1, and (C) EDS measured at location 2.

were measured on pure calcite powder in the same Raman apparatus. Those results and the measurements on pineal microcrystals are shown in Figure 6. The peaks match perfectly. The additional peaks at 962 and 1283 cm1 may have come from another chemical substance present in the crystal, such as a protein. These results and the electron diffraction measurements denitely prove that the microcrystals are calcite. Lang et al. [1996] have observed SHG in dried thin slices of pineal gland. The observed SHG signals were about 103 times that of a standard urea powder. An example of their results is shown in Figure 7. In the present study, SHG measurements were made to determine the origin of the previous observations. The crystalline materials in the pineal tissue were hydroxyapatite concretions and calcite microcrystals. Both hydroxyapatite and calcite are centrosymmetric and would not be expected to show SHG by the usual dipolar mechanism [Kurtz and Dougherty, 1978]. However, although calcite has a centrosymmetric crystal structure, it has been shown to exhibit SHG, albeit far weaker than in SHG-active noncentrosymmetric crystals [Terhune et al., 1962; Bjorkholm and Siegman, 1967; Dinev et al., 1978]. The SHG in calcite is quadrupolar in nature, and is preferentially along a specic crystal direction due to birefringence. For a genuine powdered sample of calcite, we measured an SHG intensity that was 4 orders of magnitude weaker than a urea powder standard. Hydroxyapatite is also centrosymmetric. However, we were unable to detect SHG in either pure hydroxyapatite powder (<5 106 times

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Fig. 6. Raman spectra of pineal gland microcrystals (A) and pure calcite powder (B).

that of urea) or in the large pineal concretions. SHG could not be detected in a small sample of isolated pineal microcrystals as discussed below. DISCUSSION Crystalline deposits in the pineal gland have been observed for many years [Welsh, 1985]. All of the prior research concerned investigations of the large concretions of the mulberry-type. Recently, a study described the presence of small geometric shapes associated with the concretion globules [Humbert and Pevet, 1991], but detailed investigations were not carried out. The technique developed here for isolation of the crystals gave

Fig. 7. SHG results on pineal tissue slices from six subjects. The ordinate is a logarithmic scale giving the mean and the standard deviation of the SHG photon counts detected by the photomultiplier during the operating period of the laser.The numbers on the bars are the total numbers of measurements at different locations on the tissues. In every case, the number of photon counts at the 532-nm SHG wavelength was statistically significant using an unpaired Students t-test at the P <.05 level. Figure adapted from [Lang et al.,1996].

us access to large enough quantities for a study of this new mineral structure, which we have tentatively named myeloconia (brain dust in Greek). The strikingly different compositions of the calcite microcrystals and the hydroxyapatite mulberry-like concretions is especially signicant. Consequently, it is very unlikely that the microcrystals are either degradation products of the mulberry-like concretions or precursor structures. The lack of an SHG response with the microcrystals can be attributed to several causes. The quantity of microcrystals was very small and the crystals were preferentially aligned with their long axes parallel to the glass slide on which they were mounted. In the pure calcite powder sample, there were very large numbers of randomly oriented crystals, so that some were properly aligned to allow the SHG to reach the optical detector. The conditions of large numbers of crystals and alignment were not met with the microcrystal sample. The complete absence of an SHG response in the hydroxyapatite concretions and the existence of a weak SHG response in pure calcite strongly suggest that the calcite microcrystals were the source of the SHG in the tissue samples [Lang et al., 1996]. Reasons for the formation of the crystals and their possible biological signicance are not known at present. However, some of the SEM photographs, such as the multilayered structure in Figure 8, suggest a growth mechanism for the crystals. The crystal appears to consist of a stack of thin rhombohedrons with their at faces normal to the long axis of the crystal. A sketch of this type of structure is shown in Figure 9. The sharp edges and rough body could be explained in this way. Twinning is a very common phenomenon in mineralogical calcite, and complex patterns are frequently observed [Dana, 1951]. The microcrystals bear a striking resemblance to the calcite crystals that form the otoconia of the inner ear. Otoconia have been studied extensively in a number of species, including human beings [Ross and Pote, 1984; Sanchez-Fernandez and Rivera-Pomar, 1984; Sanchez-Fernandez et al., 1989]. Their growth stages are very similar to those of the pineal microcrystals. The structure and chemical composition of the microcrystals are also similar to those found in the biomineralized crystals of sea urchin spines and sponge spicules [Aizenberg et al., 1997]. Crystal growth in the sea urchin spine and sponge spicules may be regulated by an acidic molecule recently identify as an acidic glycoprotein [Albeck et al., 1996]. A supposition is that the microcrystal structure could result from the action of the same type of molecule. It seems that organic material may be located on the surface of the biomineralized microcrystals because Raman lines that can be ascribed to proteins are

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Fig. 8. SEM photos of an isolated pineal microcrystal on a Formvar-covered TEM grid showing the multilayer structure.

superimposed on the Raman spectrum characteristic of the calcite polymorph of calcium carbonate. We believe that the presence of two different crystalline compounds in the same organ is biologically signicant, suggesting two entirely different mechanisms of formation and biological functions. The calcite in otoconia has been shown to exhibit piezoelectricity [Morris and Kittleman, 1967], a property that is normally forbidden in centrosymmetric crystals by symmetry requirements. Both the otoconia and the pineal microcrystals appear to consist of a stack of thin rhombohedrons with their at faces normal to the long axis of the crystal. These complex structures can be classied using the texture point group nomenclature of Shubnikov et al. [1958]. The textures may be noncen-

trosymmetric, even though the single crystals do have a center of symmetry. This symmetry breaking would allow both SHG and piezoelectricity. If piezoelectricity were to exist, an electromechanical coupling mechanism to external electromagnetic elds may be possible. The possibility of nonthermal coupling of electromagnetic radiation to biological systems has been considered recently [Kirschvink, 1992]. Reiter [1993] has reviewed the literature on the possible effects of static and low frequency electromagnetic elds on the production of melatonin by the pineal gland. A study by de Seze [1998, 1999] showed no inuence of microwave frequency radiation on melatonin secretion. However, Kirschvink et al. [1992] and Kirschvink [1996] have shown the presence of minute crystals of magnetite in the human brain and have suggested a mechanism for coupling of microwave radiation to them. Additional research on the nonthermal effects of microwave radiation is denitely warranted. In conclusion, we believe that even a very small risk of possible nonthermal coupling of radiation to microcrystals in the pineal gland merits further detailed study. Our future research will address these questions. ACKNOWLEDGMENTS We thank Prof. Jean-Francois Lebas for his special interest in this project. We also thank Dr. Poech who provided the pineal bodies; Dr. Isebrand Prinsloo and Prof. Joel Bernstein for their valuable advice; and Valodian Ezersky, Aviva Kiriaty, and Olga Nabutovsky for their advice and their skillful operation of instrumentation. We thank Prof. Amalia Konsta for suggesting the name myleconia for the microcrystals. We are indebted to Prof. A.A. Marino for originally suggesting the research and for his contributions in the earlier stages of the work. This study was supported by grants from Cooperant du Service National Grant to Simon Baconnier and The Israel Science Foundation to Sidney B. Lang.

REFERENCES
Aizenberg J, Hanson J, Koetzle TF, Weiner S, Addadi S. 1997. Control of macromolecule distribution within synthetic and biogenic single calcite crystals. J Am Chem Soc 119:881 886. Albeck S, Addadi I, Weiner S 1996. Regulation of calcite crystal morphology by intracrystalline acidic proteins and glycoproteins. Connect Tissue Res 35:365370. Bjorkholm HE, Siegman AE. 1967. Accurate cw measurements of optical second-harmonic generation in ammonium dihydrogen phosphate and calcite. Phys Rev 154:851860. Dana ES. 1951. Textbook of mineralogy. New York: John Wiley & Sons.

Fig. 9. Schematic drawing showing possible twinned texture of microcrystals.

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de Seze R, Fabbro-Peray P, Miro L. 1998. GSM radiocellular telephones do not disturb the secretion of antepituitary hormones in humans. Bioelectromagnetics 19:271278. de Seze R, Ayoub J, Peray P, Miro L, Touitoo Y. 1999. Evaluation in human of the effects of radiocellular telephones on the circadian patterns of melatonin secretion, a chronobiological rhythm marker. J Pineal Res 27:237242. Dinev SG, Saltiel SM, Stamenov KV, Stankov KA, Tunkin VG. 1978. Measurement of the quadrupole nonlinear coefcient dispersion in calcite crystal. Opt Commun 24:225230. Humbert W, Pevet P. 1991. Calcium content and concretions of the pineal glands of young and old rats. Cell Tissue Res 263: 593596. Humbert W, Pevet P. 1996. Electron probe X-ray microanalysis of the elemental composition of the pineal gland of young and aged rats. J Pineal Res 20:3944. Karasek M. 1999. Melatonin in humansWhere we are 40 years after its discovery. Neuroendocrinol Lett 20:179188. Kirschvink JL. 1992. Comment on Constraints on biological effects of weak extremely-low-frequency electromagnetic elds. Phys Rev A 46:21782184. Kirschvink JL, Kobayashi-Kirschivink A, Woodford BJ. 1992. Magnetite biomineralization in the human brain. Proc Natl Acad Sci USA 89:76837687. Kirschvink JL. 1996. Microwave absorption by magnetite: A possible mechanism for coupling nonthermal levels of radiation to biological systems. Bioelectromagnetics 17:187194. Kodaka T, Mori R, Debari K, Yamada M. 1994. Scanning electron microscopy and electron probe microanalysis studies of human pineal concretions. J Electron Microsc (Tokyo) 43:307317. Krstic R. 1976. A combined scanning and transmission electron microscopic study and electron probe microanalysis of human pineal acervuli. Cell Tissue Res 174:129137. Kurtz SK, Dougherty JP. 1978. Methods for detection of noncentrosymmetry in solids. Syst Mater Anal 4:269327. Lang SB, Marino AA, Berkovic G, Fowler M, Abreo KD. 1996. Piezoelectricity in the human pineal gland. Bioelectrochem Bionenerg 41:191195. Michotte Y, Lowenthal A, Knaepen L, Collard M, Massart DL. 1977. A morphological and chemical study of calcication of the pineal gland. J Neurol 215:209219. Morris RW, Kittleman LR. 1967. Piezoelectric property of otoliths. Science 158:368370.

495

Morrison ID, Denning RG, Laidlaw WM, Stammers MA. 1996. Measurement of rst hyperpolarizabilities by hyper-Rayleigh scattering. Rev Sci Instrum 67:14451453. Nakamoto K. 1978. Infrared and Raman spectra of inorganic and coordination compounds. New York: John Wiley & Sons. Nakamura KT, Nakahara H, Nakamura M, Tokioka T, Kiyomura H. 1995. Ultrastructure and X-ray microanalytical study of human pineal concretions. Ann Anat 177:413419. Ostrowski K, Dziedzic-Goclawska A, Michalik J, Stachowicz W, Mazur S. 1980. Crystallinity of human pineal calcospherulites. Calcif Tissue Int 30:179182. Pevet P. 2000. Melatonin and biological rhythms. Biol Signals Recept 9:203212. Reiter RJ. 1991. Pineal gland: Interface between the photoperiodic environment and the endocrine sytem. Trends Endocrinol Metab 2:1319. Reiter RJ. 1993. Static and extremely low frequency electromagnetic eld exposure: Reported effects on the circadian production of melatonin. J Cell Biochem 51:394403. Ross MD, Pote KG. 1984. Some properties of otoconia. Philos Trans R Soc Lond B 304:445452. Sanchez-Fernandez JM, Rivera-Pomar JM, Tello MJ. 1989. Human otoconial crystal growth. ORL J Otorhinolaryngol Relat Spec 51:108115. Sanchez-Fernandez JM, Rivera-Pomar JM. 1984. A scanning electron microscopy study on human otoconia genesis. Acta Otolaryngol 97:479488. Schmid HA. 1993. Decreased melatonin biosynthesis, calcium ux, pineal gland calcication and aging: A hypothetical framework. Gerontology 39:189199. Shubnikov AV, Zheludev IS, Konstantinova VP, Silvestrova IM. 1958. Etudes des textures piezoelectriques. Paris: Dunod. Terhune RW, Maker PD, Sagage CM. 1962. Optical harmonic generation in calcite. Phys Rev Lett 8:404406. Tomazic BB, Brown WE, Eanes ED. 1993. A critical evaluation of the purication of biominerals by hypochlorite treatment. J Biomed Mater Res 27:217225. Vigh B, Szel A, Debreceni K, Fejer Z, Manzano e Silva MJ, VighTeichmann I. 1998. Comparative histology of pineal calcication. Histol Histopathol 13:851870. Weiner S, Price PA. 1986. Disaggregation of bone into crystals. Calcif Tissue Int 39:365375. Welsh MG. 1985. Pineal clacication: Stuctural and functional aspects. Pineal Res Rev 3:4168.