The International Journal of Biochemistry & Cell Biology 42 (2010) 15801585

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The International Journal of Biochemistry & Cell Biology

journal homepage: www.elsevier.com/locate/biocel

Cells in focus

Microglia: Activation in acute and chronic inammatory states and in response to cardiovascular dysfunction
Emilio Badoer
School of Medical Sciences and Health Innovations Research Institute, RMIT University, PO Box 71, Bundoora 3083, Melbourne, Victoria, Australia

a r t i c l e

i n f o

a b s t r a c t
Microglia are the resident immune cells in the central nervous system and are constantly monitoring their environment. After an insult, they are activated and secrete both pro- and anti-inammatory mediators. Thus, they can have both detrimental and protective actions. Microglia are activated in many conditions that involve chronic inammation such as Alzheimers and Parkinsons diseases and in neuropathic pain. Following cerebral ischemia and stroke, microglia are activated and acutely contribute to neuronal loss and infarct damage. Chronically, in this condition, neuroprotective actions of activated microglia include clearance of the dead cells and secretion of neurotrophins. Of great interest is the recent observation that following myocardial infarction, there is increased inammation within the hypothalamus and a marked increase in activated microglia. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 26 February 2010 Received in revised form 5 June 2010 Accepted 8 July 2010 Available online 16 July 2010 Keywords: Myocardial infarction Inammation Hypothalamus

Cell facts Microglia in the resting state appear highly branched with a small cell soma. Resting microglia are motile and constantly survey their environment. Microglia undergo dramatic changes in morphology and phenotype following activation. Upon activation microglia can up-regulate their expression of various receptors and pro- and anti-inammatory mediators. Microglia may contribute to the inammatory processes occurring in chronic conditions like Alzheimers and Parkinsons diseases and neuropathic pain. Microglia may also have detrimental and protective actions in cardiovascular conditions like cerebral ischemia and following myocardial infarction.

1. Introduction Microglia are the brains resident immune cells involved in the surveillance of the microenvironment. Microglia are abundant within the brain and comprise up to approximately 20% of the total glial population. They are found in both the gray and white mat-

Tel.: +61 3 9925 7081; fax: +61 3 9925 7063. E-mail address: emilio.badoer@rmit.edu.au. 1357-2725/$ see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2010.07.005

ter but are not uniformly distributed, and their density can vary considerably between brain regions. The greatest concentrations are found in areas that include the hippocampus, basal ganglia, and substantia nigra. The lowest density of microglia is found in areas that include the brainstem and cerebellum (Savchenko et al., 1997). Microglia were rst observed by Nissl staining and were subsequently characterized as glial cells by Ramon y Cajal. It was Ramon y Cajals student, Rio-Hortega, who focussed on microglia and identied them as a separate cell population, distinct from the other glial cells, and capable of phagocytosis. From his work he correctly hypothesised microglia were derived from myeloid mononuclear cells. At rest in the adult brain, microglia have a small soma with little perinuclear cytoplasm. Arising from the soma are several main processes from which emanate numerous extensively branched ne processes that are long thin and nger-like, a morphology termed ramied (Fig. 1). The processes of microglia can contact astrocytes, neurons and blood vessels but do not appear to contact other microglia, and there can be 5060 m between individual microglia. Recent work using time lapse photography combined with two-photon microscopy has shown that microglia at rest are in fact highly motile with their ne processes undergoing cycles of retractions and elongations that suggests sampling of the microenvironment (Nimmerjahn et al., 2005; Lambertsen et al., 2009; Wake et al., 2009). Calculations of the speed of the motility of the processes and the volume of extracellular space in the brain suggest that the brain parenchyma can be completely sampled in a few hours (Nimmerjahn et al., 2005). Microglia are kept in this resting state by stimulatory and inhibitory signals nely balanced in the microenvironment.

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Upon an insult to the brain tissue, such as an injury, or ischemia, microglia become activated, markedly changing their morphology and this is reviewed in detail by several recent reviews (Colton and Wilcock, 2010; von Bernhardi et al., 2010). The changes include an enlarged soma and thicker, shorter processes that have a stubby appearance (Fig. 1). Some activated microglia become ameboid, capable of phagocytosing debris and proliferation (Fig. 1).

3. Functions The fundamental functions of the microglia involve protecting the central nervous system by detecting and acting upon invading organisms, injury or other insults. As discussed earlier, in normal conditions, microglia are believed to constantly survey their environment using their motile ne nger-like processes. When there is an insult, infection, injury to the tissue, or stimulatory signals are present in the microenvironment the microglia become activated and can undergo phenotypic and morphological changes. These changes enable the microglia to mount a localised inammatory response by (i) up-regulation of receptors and the production and secretion of mediators involved in inammation, (ii) migration to the injured region, (iii) phagocytosis, and (iv) antigen presentation. Many receptors on resting ramied microglia may be expressed at low levels or even not at all, but upon activation there can be dramatic increases in their expression. Receptors found on microglia include those activated by purines, glutamate, GABA, catecholamines, opioids, cannabinoids, cytokines, prostaglandins, and chemokines (see Pocock and Kettenmann, 2007 for an extensive discussion). Activated microglia also dramatically increase the expression of peripheral benzodiazepine receptors located on mitochodria and ligands targetting these have been used as markers to image inammation in the brain in humans (Winkeler et al., 2010). Neurochemicals released by activated microglia can have both neuroprotective effects and apoptotic or neurotoxic actions. Neuroprotective effects may be mediated by neurotrophins (e.g. NGF, BDNF see Fig. 1) and interleukins (e.g. IL-3 and IL-6 which may be neuroprotective in some cases, see Fig. 1) (Inoue and Tsuda, 2009; Napoli and Neumann, 2009). Apoptotic effects and neurotoxic actions may be mediated by TNF , glutamate and the generation of reactive oxygen species such as NO, superoxide and hydrogen peroxide (Lambertsen et al., 2009) (Fig. 1). Activated microglia can also proliferate and this involves the actions of colony stimulating factors (CSFs), in particular monocyte CSF and granulocyte CSF,

2. Cell origin and plasticity Microglia retain the ability to divide, and to undergo DNA synthesis so that they can maintain the population of microglia in the central nervous system without reliance on peripheral, bone marrow haematopoietic cell production. Microglia are derived from haematopoietic progenitor cells but the exact lineage and derivation of microglia is not known. Unlike astrocytes, oligodendrocytes and neurons which are derived from the neuroectoderm, microglia are believed to derive from peripheral myeloid progenitor cells. Emerging evidence suggests that both extravascular and circulating routes are used by progenitor monocytes to inltrate the developing central nervous system primarily in embryonic and fetal stages of development. Perinatal microglia are predominantly in the ameboid phagocytic form which transform into the resting ramied morphology postnatally (Chan et al., 2007). During development of the central nervous system, microglia may be responsible for the dramatic reduction in neuron numbers via apoptotic processes and later in development microglia may also be involved in the removal of unwanted synapses (Napoli and Neumann, 2009). Complement proteins may be involved in this process, however, the evidence is only circumstantial and based on data that shows complement components are up-regulated on activated microglia and that complement proteins can opsonize unwanted synapses and thereby reduce neuronal connections (Stevens et al., 2007).

Fig. 1. Schematic representation of resting ramied microglia which upon activation transform morphologically and can ultimately become ameboid phagocytic cells. The reverse process may also occur when the stimulatory/inammatory signals have subsided. Ramied microglia are constantly surveying their environment and are kept in this state by a balance of inhibitory and excitatory signals. Microglia contain receptors or binding sites for many neurochemicals which when stimulated can activate or propagate the activation of microglia. Receptors on microglia that are excitatory include those activated by purines (ATP and adenosine), glutamate (glu), interleukins (IL)-1 , IL-6, IL-2, IL-4, IL-5, lysophosphatidic acid (LPA), tumor necrosis factor (TNF) , lipopolysaccharide (LPS), toll like receptor (TLR) activation, macrophage inammatory protein (MIP)-1, monocyte chemoattractant protein (MCP)-1, chemokines (CC), substance P (Sub P), bradykinin (BK), prostaglandins (PGs) and the surrounding ionic milieu. Inhibitory substances include chemokine-3-ligand 1 (CX3CL1), and possibly cannabinoids (CB) and catecholamines (CAT). GABA can inhibit LPS induced IL production by microglia.

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which appear to be critical. These CSFs originate from astrocytes. Additionally, IL-2, IL-4 and IL-5 may contribute to the proliferation process (Inoue and Tsuda, 2009) (Fig. 1). Various neurochemicals that regulate the migration of microglia have been identied (Pocock and Kettenmann, 2007). These include lysophosphatidic acid, ATP by activating P2Y12 and P2X4 receptors, opioids acting on mu-opioid receptors, substance P acting on NK1 receptors, cannabinoids acting on CB 2 receptors, and some chemokines and trophic factors (Pocock and Kettenmann, 2007). Fig. 1 schematically shows changes occurring during activation and includes potential mediators. It is critical that pro-inammatory signals are turned off as required, since the failure to do so may lead to the negative impacts seen in chronic inammation as discussed below in Section 4. Anti-inammatory mediators that may play an important role in counteracting the actions of pro-inammatory mediators include IL-10 and fractaline (Inoue and Tsuda, 2009). These can also emanate from microglia. Cannabinoids, GABA and catecholamines may also reduce cytokine release from microglia (Pocock and Kettenmann, 2007). The receptors and neurochemicals synthesised by activated microglia may vary between areas in the brain and according to the state of activation of microglia (Carson et al., 2007). Thus, it is becoming increasingly clear that the detection of a single receptor or neurochemical as a marker of activated microglia may not provide sufcient information on the state of activation of microglia. In the future it is likely that molecular characterization of microglia including high throughput microarrays will make substantial contributions to our understanding of the changes that occur in microglia during inammation in the central nervous system (Gebicke-Haertler, 2005). Microglia are the major phagocytic cells in the central nervous system involved in the removal of microbes, protein degradation products, apoptotic cells and other cellular debris. Phagocytic microglia move toward an injury, the size of which determines the number of activated microglia attracted. Exactly how this is achieved is unclear but it is likely to involve various markers/signals that interact with membrane phospholipids on cells and receptors on microglia. Receptors on microglia that contribute to the phagocytosis of microbes include toll like receptors, complement receptors, Fc receptors and scavenger receptors (Napoli and Neumann, 2009). Stimulation of these receptors can also contribute to the increased production of pro-inammatory cytokines by microglia. Phagocytosis of apoptotic cells involve receptors on microglia that include P2Y6, which is activated by uridine tri-phosphate released by neurons, TREM2 (triggering receptor expressed on myeloid cells 2) and receptors that recognize phosphatidyl serine residues (Napoli and Neumann, 2009). Some microglia upon activation become capable of antigen presentation to inltrating T-cells and this involves the up-regulation of MHC class I and II proteins and complement proteins (Yang et al., 2010).

4.1. Cardiovascular conditions: stroke/cerebral ischemia Following a stroke or cerebral ischemia there are changes in the morphology and phenotype of microglia, as discussed earlier. Within minutes of the onset of ischemia there is activation of microglia which peaks at approximately 72 h later, and can be maintained for weeks after the initial insult (Napoli and Neumann, 2009). Expression of adhesion molecules on endothelial cells, leucocytes and microglia contributes to the subsequent inltration of the damaged tissue by peripheral leucocytes, including neutrophils, macrophages and T-cells (Napoli and Neumann, 2009). These contribute to the increased production of cytokines including TNF , particularly from activated microglia, which are the major producers of this cytokine following ischemia (Lambertsen et al., 2009). Acutely after a stroke or cerebral ischemia, the inammatory process appears detrimental to the survival of neurons. The activation of microglia has been correlated with the increase in neuronal loss that occurs with occlusion of the middle cerebral artery in rodents (Liu et al., 2007). Additionally, it has been shown that in normal healthy brain microglial processes make contact with neuronal synapses lasting on average approximately 5 min, and the frequency of contacts (once per hour) decreases with reduced neuronal activity. During ischemia of the brain, there is a dramatic increase in the duration of the contact time (approximately 80 min) that is associated with an increased loss of synapses (Wake et al., 2009), suggesting that the enhanced contact time with neurons is detrimental. The mechanisms involved in these harmful effects are not clear. TNF may contribute since loss of function of this cytokine soon after the onset of ischemia reduces the infarct damage, neuronal loss and suppresses neurogenesis. Activation of microglia after stroke may also have neuroprotective actions and this may also involve TNF (Lambertsen et al., 2009). The actions of TNF are mediated through two subtypes of receptor. Some studies suggest that activation of the TNF-p55 receptor elicits detrimental actions and activation of the TNF-p75 receptor subtype induces protective actions. The nature of the ischemic insult and its duration most likely induces different environmental conditions in the ischemic area and this may markedly inuence the ultimate responses. For example, recent studies have shown that activation of the TNF-p55 receptor may be neuroprotective after ischemia induced by permanent occlusion of the middle cerebral artery in mice (Lambertsen et al., 2009). Whatever the mechanisms, there is some consensus in the literature suggesting that immediate activation of microglia after an ischemic insult results in neuronal toxicity and increased infarct damage. In the longer term, activation of microglia appears to be neuroprotective by promoting clearance of the dead cells and secretion of neurotrophins to promote survival of neurons. These opposing actions occurring in inammation may explain why in the clinical setting, inhibition of inammation in the brain following stroke has not led to successful clinical outcomes (Enlimomab Acute Stroke Trial Investigators, 2001). 4.2. Cardiovascular conditions: myocardial infarction

4. Associated pathologies Activation of microglia has been observed in many conditions of chronic inammation within the central nervous system. Below I briey discuss Alzheimers and Parkinsons diseases and neuropathic pain. Before doing so, we discuss the potential roles of microglia in cardiovascular conditions, rstly in a condition in which there is an initial acute injury or insult to the brain (i.e. stroke/cerebral ischemia) and secondly, a cardiovascular condition in which there is no direct injury to the brain (i.e. following myocardial infarction) but in which recent evidence highlights there is activation of microglia.

The studies of inammation following the injury to the brain tissue that occurs with stroke or ischemia have, naturally, focussed on the roles of microglia in the acute and chronic survival of neurons. Recent studies show that even in the absence of damage to the brain, such as following myocardial infarction elicited by coronary artery occlusion, there is an increase in pro-inammatory cytokine levels in the hypothalamus within 24 h post-myocardial infarct. They are also elevated at the time heart failure is established, approximately 68 weeks after a myocardial infarct in the rat (Francis et al., 2004). Pro-inammatory cytokines, such as TNF and IL-1 , induce the production of reactive oxygen species raising the possibility that reactive oxygen species may mediate some of

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the inammatory actions of TNF and IL-1 in the hypothalamus in heart failure induced by myocardial infarction. Microinjection of pro-inammatory cytokines, e.g. TNF , into the hypothalamus elicits increases in renal sympathetic nerve activity (Kang et al., 2008), most likely through the activation of hypothalamic neurons that project to the spinal cord and the rostral ventrolateral medulla (Shafton et al., 1998). These sympatho-excitatory effects of the pro-inammatory cytokines may contribute to the abnormally elevated renal sympathetic nerve activity seen in chronic heart failure. The mechanisms that lead to an increase in pro-inammatory cytokines in the hypothalamus following myocardial infarction are not known. One hypothesis has suggested a critical role of the pro-inammatory cytokines which are released into the bloodstream by the damaged heart. These may cross the bloodbrain barrier and induce local cytokine production or induce PGE2 production in endothelial cells in the cerebral blood vessels (Felder et al., 2003; Yu et al., 2007). PGE2 is known to increase cytokine production. Another potential mechanism includes the activation of the reninangiotensinaldosterone system, which occurs following myocardial infarction. Angiotensin II and aldosterone may act either directly or via induction of reactive oxygen species to initiate the inammatory response (Felder et al., 2003; Guggilam et al., 2007; Lindley et al., 2004). Recently published work suggests that activation of microglia in the hypothalamic paraventricular nucleus (PVN) may also contribute to the increase in pro-inammatory cytokines (Rana et al., 2010). In that study it was found that activation of microglia was observed in the PVN at 2 weeks and at 5 weeks following myocardial infarction. Echocardiography and haemodynamic parameters at these times after the myocardial infarction indicated reduced left ventricular function but congestive heart failure had not developed, suggesting that reduced left ventricular function was sufcient to induce activation of microglia. Furthermore, the activation of microglia did not occur in the ventral hypothalamus adjacent to the PVN nor in the cortex (Rana et al., 2010). This suggests that there was not a wholesale breakdown of the bloodbrain barrier that could account for the activation of microglia, which differs from other peripheral inammatory conditions where inammation in the brain occurs such as inammatory bowel disease. The mechanism resulting in the activation of microglia in the PVN and not in adjacent nuclei following myocardial infarction is not known. It is possible that changes in the neurochemical and ionic milieu elicited by an increase in the neuronal activity in the PVN that occurs following a myocardial infarction (Patel, 2000), could stimulate microglia, possibly via glutamatergic or purinergic receptor activation (Taylor et al., 2005; Hide et al., 2000). NMDA receptors are up-regulated in the PVN in heart failure induced by myocardial infarction in rats and this mediates an enhanced sympathetic nerve response to local NMDA receptor activation (Li et al., 2003). Interestingly, activation of microglia following restraint stress involves activation of NMDA receptors (Nair and Bonneau, 2006). The recent nding that the activation of microglia by stimulation of P2X7 purinoceptors can occur in the absence of injury or insult to tissue (Monif et al., 2009) suggests that future investigations into the role of ATP acting on the P2X7 purinoceptors in the activation of microglia following myocardial infarction would be interesting. Activation of microglia in areas in which neuronal activity has been elevated by a physiological stimulus and in the absence of local tissue damage has been previously described (Ayoub and Salm, 2003). Thus, it is speculated that activated microglia could contribute to the increased local production of pro-inammatory cytokines observed in the hypothalamic paraventricular nucleus following myocardial infarction and resulting reduced left ventricular function. The activation of microglia induces secretion of

Fig. 2. A schematic representation showing that activated microglia may contribute to the increased local production of pro-inammatory cytokines observed in the hypothalamic paraventricular nucleus (PVN) following myocardial infarction. The mechanisms responsible for the activation of microglia that occurs after a myocardial infarction are unknown but may involve elevated plasma levels of angiotensin II, aldosterone, and cytokines, or changes in the local milieu within the paraventricular nucleus such as increased glutamate (Glu) and ATP. Activation of microglia induces secretion of pro-inammatory cytokines, such as TNF and IL-1 , can activate hypothalamic paraventricular neurons that project to the spinal cord where sympathetic preganglionic (SPN) motor neurons are located. Increased activity of these neurons increases sympathetic nerve activity. In heart failure there is increased sympathetic nerve activity to the kidneys (RSNA). Thus, it is speculated that chronic activation of microglia may contribute to the elevated sympathetic nerve activity seen in chronic heart failure. Abbreviations: III, third ventricle; PGN, post-ganglionic neuron.

pro-inammatory cytokines that may activate hypothalamic paraventricular neurons that regulate sympathetic nerve activity. This may contribute to the elevated sympathetic nerve activity seen in chronic heart failure. The mechanisms responsible for the activation of microglia are unknown but the presence of glutamate and ATP may be involved (Fig. 2). 4.3. Alzheimers disease Alzheimers disease is a debilitating neurodegenerative disease that causes cognitive impairment in patients. The common characteristics of Alzheimers disease is the formation of extracellular amyloid plaques and intracellular neurobrillary tangles. Amyloid is cleaved from membrane bound precursors via and secretases to produce soluble and insoluble isoforms. Amyloid can activate microglia resulting in increased secretion of inammatory cytokines, reactive oxygen species and phagocytosis. Microglia migrate to amyloid plaques (Bolmont et al., 2008), and since the expression of CD14 receptors on microglia increases in inammation (this receptor has been implicated in amyloid removal) this could lead to an increased phagocytosis of amyloid plaques. This may involve toll like receptors (Richard et al., 2008). Enhanced expression of complement proteins in activated microglia have also been implicated in the removal of amyloid . However, increased expression of pro-inammatory cytokines and other proteins such as prostaglandin and cyclo-oxygenase may actually enhance amyloid deposition. Thus, activated microglia in Alzheimers disease may have both positive and negative effects. The current view is that the useful actions of activated microglia occur acutely following activation of microglia but with continued chronic activation of microglia, as occurs in Alzheimers disease, the detrimental actions of activated microglia predominate in this disease (Schlachetzki and Hull, 2009).

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4.4. Parkinsons disease Parkinsons disease affects mainly older individuals and as the population ages, it is expected that the incidence of this disease will increase. It is characterized by loss of dopaminergic neurons in the substantia nigra and affects motor control that can result in tremors, rigidity and loss of balance (Long-Smith et al., 2009). Accompanying the loss of dopaminergic neurons, there is increased activation of microglia, increased production of pro-inammatory cytokines and inammatory mediators (Long-Smith et al., 2009). Inammatory processes are believed to play an important role in the aetiology and progression of Parkinsons disease since epidemiological studies show that regular use of non-steroidal anti-inammatory agents is associated with reduced incidence of Parkinsons disease. The increase in activated microglia is observed in human post-mortem brain tissue and in the brains of animals in which Parkinsons disease symptoms have been induced (e.g. with MPTP, LPS) (Liu, 2006). Activated microglia are found mainly in the substantia nigra but are not restricted to that brain region. Animal studies have been important in correlating the activation of microglia and damage to dopaminergic neurons. In common models of Parkinsons disease, microglia are activated rapidly and maximum numbers of activated microglia are observed within days corresponding to the increasing damage of dopaminergic neurons (Liu, 2006). Various pro-inammatory mediators are increased in the brains of patients who suffered from Parkinsons disease, and in animal models. These mediators include TNF and IL-1 , whose receptors are up-regulated in dopaminergic neurons suggesting these contribute to the damage and subsequent loss of those neurons. 4.5. Neuropathic pain Neuropathic pain is a chronic pain occurring following sensory nerve injury and is often accompanied by tactile allodynia. Neuropathic pain is not relieved satisfactorily by currently available analgesics and non-steroidal anti-inammatory agents. Neuropathic pain is characterized by hyper-excitability of dorsal horn neurons. Activation of microglia occurs within hours of nerve injury and is increased 24-fold within days. There are many signals that have been reported to be involved including purines (Burnstock, 1997). In allodynia the P2X4 purinoceptor appears to be particularly important (Burnstock, 1997; Inoue and Tsuda, 2009). The expression of this receptor is increased markedly in microglia in dorsal root ganglia following peripheral nerve injury. Activation of this receptor is necessary and sufcient for neuropathic pain and tactile allodynia (Inoue and Tsuda, 2009). Other purinoceptor subtypes that are up-regulated and may contribute to the symptoms of neuropathic pain include P2X7 and P2Y12. Chemokines including CCL2, CX3CR1, and receptors including TLRs and cannabinoid B2 are also increased on microglia following nerve injury (Inoue and Tsuda, 2009). 5. Conclusion Microglia are the resident immune cells in the central nervous system and are constantly monitoring their environment. At the rst signs of an insult or injury to the tissue, they become activated and secrete both pro- and anti-inammatory mediators. Thus they can have both detrimental and protective actions. Microglia are activated in many conditions that involve chronic inammation such as Alzheimers and Parkinsons diseases and in neuropathic pain. Following cerebral ischemia and stroke, microglia are activated and acutely contribute to neuronal toxicity and infarct damage. Chronically, protective actions of activated microglia

include clearance of the dead cells and secretion of neurotrophins. Of great interest is the recent observation that following myocardial infarction, there is increased inammation within the hypothalamus and a marked increase in activated microglia. It remains to be established as to whether this contributes to the dysregulation of sympathetic nerve activity observed in chronic heart failure following myocardial infarction. The imaging of microglia in vivo will provide greater insight into the processes involved in their activation in neuropathological and neuroinammatory conditions, and the expression of different receptors and an understanding of the signalling cascade mediating the activation of microglia are likely to contribute to the development of novel therapies for neuropathological conditions such as Alzheimers disease and cardiovascular diseases like stroke and heart failure. References
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