REVIEW ARTICLE

Biology of Microglia in the Developing Brain

Charanjit Kaur, PhD, Gurugirijha Rathnasamy, PhD and Eng-Ang Ling, PhD, DSc

AbstractMicroglia exist in different morphological forms in the develop-

ing brain. They show a small cell body with scanty cytoplasm with

many branching processes in the grey matter of the developing

brain. However, in the white matter such as the corpus callosum

where the unmyelinated axons are loosely organized, they appear in

an amoeboid form having a round cell body endowed with copious

cytoplasm rich in organelles. The amoeboid cells eventually trans-

form into ramified microglia in the second postnatal week when the

tissue becomes more compact with the onset of myelination. Micro-

glia serve as immunocompetent macrophages that act as neuropath-

ology sensors to detect and respond swiftly to subtle changes in the

brain tissues in pathological conditions. Microglial functions are

broadly considered as protective in the normal brain development as

they phagocytose dead cells and sculpt neuronal connections by

pruning excess axons and synapses. They also secrete a number of

trophic factors such as insulin-like growth factor-1 and transforming

growth factor-b among many others that are involved in neuronal

and oligodendrocyte survival. On the other hand, microglial cells

when activated produce a plethora of molecules such as proinflam-

matory cytokines, chemokines, reactive oxygen species, and nitric

oxide that are implicated in the pathogenesis of many pathological

conditions such as epilepsy, cerebral palsy, autism, and perinatal

hypoxic-ischemic brain injury. Although many studies have investi-

gated the origin and functions of the microglia in the developing

brain, in-depth in vivo studies along with analysis of their transcrip-

tome and epigenetic changes need to be undertaken to elucidate their

full potential be it protective or neurotoxic. This would lead to a bet-

ter understanding of their roles in the healthy and diseased develop-

ing brain and advancement of therapeutic strategies to target

microglia-mediated neurotoxicity.

Key Words: Antigen presentation, Developing brain, Hypoxia-is-

chemia, Inflammatory cytokines, Microglia, Phagocytosis.

INTRODUCTIONMicroglia are gaining tremendous recognition in recent

years because they are implicated in different neurodegenera-tive diseases and neurological disorders (1, 2). In the maturerodent brain, they contribute 5%–12% of the total glial popula-tion, and in reference to other glial cells, the frequency appearsto vary with regions, for example, in the cerebral cortex or cor-pus callosum (3). The prevailing view regarding roles ofmicroglia is that they act as resident immunocompetent phago-cytic cells in the disease processes in infectious, traumatic, in-flammatory, ischemic, and degenerative conditions of the cen-tral nervous system (CNS) (4). As far as can be ascertained,the existence of microglia as a separate cellular entity in thenormal brain or in pathological conditions was first docu-mented by Del Rio-Hortega (5) in the early part of the last cen-tury; yet compared with other glial types in the brain, it wouldappear that microglia had attracted little attention by the con-temporary authors until decades later (6, 7). It is possible thatthis might have been hindered by the lack of a reliable stainingmethod for identification of microglia during that period. Theweak silver carbonate staining method routinely used then forthis purpose was described to be capricious, often yielding un-satisfactory results. Despite the technical limitations, someearly authors had attempted to investigate the origin, develop-ment and roles of microglia. The study was later extended tothe ultrastructural identification of microglia in which the sil-ver carbonate staining was adapted for transmission electronmicroscopy (6, 7). The seminal report on the characterizationof microglia by electron microscopy was documented in acomprehensive review by Ling (8). Adding to this was the firstultrastructural evidence indicating that microglia shared fea-tures of phagocytes (9).

Another step forward was a subsequent experimentalstudy that demonstrated that microglia in the developing brainrepresent nascent brain macrophages; more importantly, theywere derived from infiltrated blood monocytes (8, 10). Theview that microglia in the developing brain were monocytederived had since opened a new vista on the roles of microgliain the normal and diseased brain (11). This was followed bythe immunophenotypic characterization of microglia usingspecific markers common to tissue macrophages (12–15).Since then, studies on microglia specifically their roles inneuropathology have been extensively explored using differ-ent experimental models or paradigms (16–18). The past 3decades have seen an upsurge in microglia research and, in-deed, an exponential growth in our knowledge on microgliaespecially in connection with their roles in various

From the Department of Anatomy, Yong Loo Lin School of Medicine, Na-tional University of Singapore, Singapore (CK, E-AL); and Departmentof Ophthalmology and Visual Sciences, School of Medicine and PublicHealth, University of Wisconsin, Madison, Wisconsin (GR)

Send correspondence to: Charanjit Kaur, PhD, Department of Anatomy,Yong Loo Lin School of Medicine, Blk MD10, 4 Medical Drive, NationalUniversity of Singapore, Singapore 117594; E-mail:antkaurc@nus.edu.sg

This study was supported by research grants R-181-000-148-750, R-181-000-162-733, and R-181-000-173-112 from the National UniversityHealth System (NUHS), Singapore.

The authors have no conflicts of interest to declare.

1VC 2017 American Association of Neuropathologists, Inc. All rights reserved.

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neurodegenerative diseases and neurological disorders. Un-doubtedly, the roles of microglia are now better clarified andamplified, although they appear to be extremely complex ei-ther as neuroprotective or neurotoxic (4, 19).

ORIGIN OF MICROGLIAMicroglial cells have been reported to originate from

different mesodermal sources such as the embryonic mesen-chymal cells in the pia, fetal macrophages of the yolk sac orthe fetal liver depending on the embryonic age (20). The con-cept of mesodermal origin was first postulated by Del Rio-Hortega (5) based on congregation of pial cells stained withsilver carbonate in the superior tela choroidea, pia coveringthe cerebral peduncles and inferior tela choroidea in the em-bryonic brain. From these concentrations the cells theninvaded the brain and transformed into the round amoeboidmicroglial cells distributed throughout the brain. The pial(mesodermal) origin of microglial cells was supported bymany authors (21–25). Later studies supported further themesodermal origin of microglia using histochemical stainingwith isolectin Griffonia simplicifolia (GSA I-B4) and Ricinuscommunis agglutinin-1 (RCA-1) (26–28). Other studies havesuggested that precursor cells in the yolk sac give origin tomicroglia during the embryonic period (29). In the embryos ofchick and quail, macrophages in the CNS were reported to ori-ginate from the yolk sac before the circulation was established(30). It has been demonstrated that microglia in the avian em-bryonic brain were derived by invasion and proliferation ofmacrophages from the pial surface (31).

As opposed to the above views some authors, however,had argued in favor of a neuroectodermal lineage of microglia(32–35). Thus, the subependymal cells (glioblasts) in the sub-ventricular zone of lateral ventricles were thought to be theprecursors of microglia (32, 36, 37). With the use of RCA-1and monoclonal antibodies that recognize tissue macrophagesand microglia, it was proposed that microglia originate in thegerminal matrix rather than in the pial mesenchyme. However,others authors had contended that microglial precursors withinthe neuroepithelium were cells traversing the neuroepitheliumfrom the cerebral ventricles to enter the nervous parenchymaand settle as microglia (38).

The hypothesis that microglia are derived from circulat-ing monocytes was proposed by other workers including thepresent authors based on experimental animal studies (9, 10,39–42). However, the possibility that microglia are derivedfrom other sources, including the bone marrow or the myeloidtissue (43), cannot be excluded.

MICROGLIA IN THE DEVELOPING HUMANBRAIN

There is only a modicum of information addressing theissue of origin of microglia in the human brain. Almost allstudies regarding microglial origin and differentiation inhumans have been carried out in the fetal CNS tissue collectedat the time of elective or spontaneous pregnancy terminations(44–46). Microglia have been reported to colonize the humanbrain and spinal cord before 12th week of gestation (47, 48),

with limited numbers of amoeboid microglia being present inthese regions (45, 49). By using RCA-1 or antibodies such asCD 68 (EBM-11) that recognize human tissue macrophagesand microglia, it had been shown that the greatest number oflabeled cells between 13 and 18 weeks of gestation wasobserved in the germinal matrix (44) and in the white matter(50). During this period of development, the morphology ofmicroglia was of the amoeboid type in the germinal matrix,whereas they progressively differentiate into ramified cellswith the growth of the brain (44, 50) and disperse throughoutthe CNS. Andjelkovic et al had used immunoperoxidase,RCA-1, Lycopersicon esculentum (tomato lectin) and CD68 tolabel microglia in the brain tissue of human embryos andfetuses ranging from 4.5 to 13.5 gestational weeks of age (45).Based on the staining patterns, these authors believed that 2populations of microglia exist in the human brain during earlydevelopment and these may arise from 2 different sources—monocytes and the yolk sac—and progressively develop typicalmicroglia morphology. Ramified microglial cells have beenreported to be the predominant type in the infant brain (48).

AMOEBOID MICROGLIA AND RAMIFIEDMICROGLIA

In the search of the origin and mode of formation ofmicroglia in the corpus callosum in the mature brain (32), inwhich the cells were first identified and characterized ultra-structurally (6, 7), attention was first drawn to the investiga-tion of microglia in the same region of the pre (41) andpostnatal (9, 33, 51) rat brain (Fig. 1). Interestingly, microgliaas identified by their small cell body and scanty cytoplasmwith branching processes in the mature brain and termed“ramified microglia” were absent in the corpus callosum; in-stead, the same area composed of loosely organized andunmyelinated axons was occupied by a large number of roundand amoeboid cells that were also stained by the weak silvercarbonate staining (Fig. 2) (33). These cells termed“amoeboid microglia” exhibited cytochemical characteristicssuch as staining with acid phosphatase and nonspecific ester-ase shared by tissue macrophages (51, 52), thus alluding tothe possibility of them being monocytic in nature as in othertissue macrophages. With the progress of brain maturation,the amoeboid microglia transformed into ramified microglia(Fig. 2) within 2 weeks postnatally as evidenced by using afluorescent tracer, rhodamine isothiocynate (53), and specificmicroglia markers such as the antibody OX42 (which recog-nizes complement type 3 receptors) (14) and lectins (GSA I-B4, RCA-1, and tomato lectin) (54, 55) (Fig. 2). The trans-formation of amoeboid microglia into ramified microglia is aprocess that is coincident with the onset of myelination ofaxons in the corpus callosum (11, 56, 57). Electron micro-scopic studies have shown that amoeboid cells in 1- to 5-day-old animals possess a round nucleus with marginal chromatinclumps and abundant cytoplasm displaying lysosomal densegranules, vacuoles and a well-developed Golgi apparatus(58). Most of the cells in older animals were elongated andbranched, showing a flattened nucleus and scanty cytoplasmcontaining a few lysosomal granules (58) (Fig. 3). These cellswere immunoreactive with the antibody OX42 and showed

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labeling with lectin (Fig. 4) It is to be noted that most of thein vitro studies of primary microglia in later years by us aswell as by others have been based on microglia harvestedfrom neonatal rats, which presumably would represent pre-dominantly the amoeboid microglia (59, 60). Notwithstand-ing, in view of their developmental relationship of having thesame cell lineage and the existence of transitional forms, theyare regarded as and simply referred to as microglia in the pre-sent description.

MICROGLIA AS A NEUROPATHOLOGY SENSORIn the developing brain, microglia notably those prepon-

derant in the white matter, respond vigorously to lipopolysac-charide (LPS) and interferon-c (IFN-c) (61–63) injected intra-peritoneally into postnatal rats. They were induced to expressmajor histocompatibility complex II (MHC II) antigens withsome of the cells closely associated with the blood capillaries,indicating that they serve as immunocompetent cells in thedeveloping brain, which might interact with the circulatinglymphocytes. Microglia in the postnatal brain are also ex-tremely sensitive to hypoxic exposure. It was argued that theymay contribute to the periventricular white matter damage inthe developing brain in hypoxia (64). Either in the developingor mature brain, microglia act as a neuropathology sensor thatcan detect and respond swiftly to subtle changes in the braintissues, be it acute or chronic (11).

FUNCTIONS OF MICROGLIAAccumulating evidence in recent years support that

microglia are immune-regulatory cells that play importantroles in the healthy and diseased CNS. They help to maintainthe homoestasis of the brain environment under normal condi-tions but display robust reaction in response to inflammatorystimuli, injury, hypoxia-ischemia and other adverse conditionsaffecting the normal function of the brain. The activatedmicroglia assume an amoeboid phenotype, proliferate and mi-grate to the site of injury or damage where they execute pro-tective function involved in removal of dead cells and cellulardebris (65, 66). On the other hand, overactivation of microgliawith excess production of inflammatory mediators may resultin neurotoxic consequences (67, 68).

Protective RolesPhagocytosis

Microgia are active phagocytes that help to eliminatedegenerating axons and cells during early CNS developmentas well as during infections, injuries and other pathologicalconditions. They share many common features of peripheraltissue macrophages. The phagocytic nature microglia espe-cially on activation was evidenced in the developing brain asmany of these cells were found to contain phagosomes at theultrastructural level (Fig. 3). Additional evidence of their mac-rophagic nature was provided by the demonstration of hydro-lytic enzymes including acid phosphatase, aryl phosphatase,nonspecific esterase and 50-nucleotidase localized in them (52,69). Remarkably, microglia in the developing brain exhibitnonspecific esterase known to be specific to blood monocytes(52), thus alluding to for the first time their cytochemical linkto monocytes. The above-mentioned enzymes were found tobe localized in the lysosomes. These cells avidly engulfed ex-ogenous substances such as rhodamine isothiocyanate andhorseradish peroxidase (HRP) that leaked into the brain tissuewhen administered intraperitoneally or intravenously (70, 71)and biotinylated dextran administered directly into the brain(72). Ingestion of HRP by the microglial cells occurred fol-lowing injection of HRP in the lumbosacral region of the spi-

FIGURE 1. (A) A schematic diagram showing a representativecoronal section of the postnatal rat brain at the level of theoptic chiasma. Note the concentration of amoeboid microglia(circled area) in the corpus callosum (cc) above the lateralventricle (LV). Ramified microglia (squared area) aredistributed mainly in the cerebral cortex (cx). (B) A confocalimage of a coronal section of the brain of a 3-day-old ratstained with tomato lectin. The amoeboid microglia in thecorpus callosum (cc) are stained (green). Blood vessels in thecerebral cortex (cx) and other parts of the brain are alsostained (green). LV, lateral ventricle.

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nal cord (73). E coli injected intracerebrally into the neonatalbrain were internalized by the microglial cells in<3 hours fol-lowing the injection (74). Recent investigations in the devel-oping cerebellum have provided further evidence for thephagocytic nature of these cells. Microglial cells were foundto engulf varying amounts of cellular debris in the neonatalcerebellum until postnatal day 17 (75). Microglial cells wereshown to limit the production of cortical neurons in the cere-bral cortex of prenatal and postnatal macaques and rats byphagocytosing the neural precursor cells (76). In our own stud-ies, we have encountered phagocytosis of dead cells and non-myelinated axons in the normal developing corpus callosumby electron microscopy (33, 58).

In fetal and postnatal rat brains, the microglial cellswere found to phagocytose necrotic and apoptotic cells as wellas degenerating axons following a hypoxic insult (77, 78).They tend to accumulate near dead neurons and clear dead ordying cells from the neonatal hippocampus following injury,thereby helping to limit secondary injury during the criticalearly time point following excision of hippocampal slices (79,80). In inflammatory lesions and neonatal stroke, microglialcells were found to be engaged in phagocytosis (81).

Microglial cells in the developing brain express com-plement type 3 receptors (CR3), which are known to beinvolved in endocytosis (14), similar to tissue macrophages.CR3-expressing microglial cells appeared to phagocytose a

FIGURE 2. (A) Amoeboid microglia in the corpus callosum of a 1-day-old rat are evidently labeled by the weak silver carbonatestaining. The cells are rich in cytoplasm, which appears to be vacuolated. The cells appear round or amoeboidic bearing shortprocesses. (B) Two ramified microglia in the cerebral cortex of a 5-day-old rat brain stained by weak silver carbonate stain. Notethe long branching processes. (C) OX42-labeled round amoeboid microglial cells in the corpus callosum of a 2-day old rat. (D)OX 42-labeled ramified cells in the cerebral cortex of a 2-day-old rat. (E) Lectin-labeled round, amoeboid microglial cells in thecorpus callosum of a 1-day-old rat. Blood vessels (asterisks) are also labeled. (F) Lectin-labeled ramified microglial cells in thecerebral cortex of 7-day-old rat.

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large number of apoptotic cells following an X-ray inducedinjury in the neonatal rat brain (82). Upregulation of CR3was observed on the microglial cells following intracerebralE. coli administration in postnatal rats (74). Besides CR3,microglia are known to express triggering receptorexpressed on myeloid cells-2 (TREM2), which is thought tobe involved in phagocytosis (83, 84). Recent studies in the

developing brain have reported that TREM2 was expressedon a subpopulation of microglial cells during first postnatalweek in grey and white matter (85), suggesting that it maybe related to phagocytosis of apoptotic cells. In the whitematter of the developing cerebellum, microglia express gan-glioside GD3 (86), which has been reported to play a partin phagocytosis of oligodendrocytes during CNS develop-ment. Toll-like receptors (TLRs) have also been shown tobe involved in microglial clearance of axonal debris ofdegenerating axons in microglial-axon coculture (87), and

FIGURE 3. (A) Electron micrograph of an amoeboid microgliain the loosely structured corpus callosum of a 5-day-old rat.Note the widely spaced unmyelinated axons. The abundantcytoplasm shows profiles of rough endoplasmic reticulum(rER), Golgi apparatus (G), lysosomes (Ly), a large phagosome(P) and some vacuoles (V). (B) Electron micrograph of aramified microglia in the corpus callosum of a 20-day-old rat.Note the closely packed axons some of them are evidentlymyelinated (Ax). The cell shows a small amount of cytoplasmwith a paucity of organelles and inclusions, which include afew lysosomes (Ly), Golgi apparatus (g) and profiles of roughendoplasmic reticulum (arrow).

FIGURE 4. (A) Electron micrograph of an amoeboid microglialcell showing OX42 immunostaining reaction products on theplasma membrane (arrows) in the corpus callosum of a 5-day-old rat. The cell shows a reniform nucleus (N), Golgi apparatus(G) and a phagosome (P). (B) Electron micrograph shows anelongated microglial cell whose plasma membrane is outlinedby the lectin GSA I-B4 staining (arrows) in the corpus callosumof a 10-day old rat. The cell contains a nucleus (N) bearingcoarse chromatin masses and some lysosomes (Ly).

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their deficiency impairs phagocytosis of degenerating axonsby microglial cells. We have reported enhanced TLR4 ex-pression in microglia in neonatal rat brain following a hyp-oxic injury (88). Although the expression was related toneuroinflammation in response to hypoxia, it may also beinvolved in phagocytosis of degenerating axons and apop-totic cells which are seen frequently in different parts ofthe developing brain such as the hippocampus, cerebellum,and the corpus callosum (78, 89) (Fig. 5).

Antigen Presentation

Macrophages, dendritic, and other cells belonging to theimmune system capture foreign antigens, process them andpresent them to T lymphocytes during initial stages of an im-mune response. Major histocompatibility complex I (MHC I)and MHC II molecules on the surface of macrophages andother cells (90–92) mediate antigen presentation.

The CNS has been considered historically as an im-munologically privileged site for a long time based on thepresence of blood-brain barrier (BBB), lack of lymphatics andan absence of antigen presenting cells. This concept has beenchanged in recent years in view of the expression of MHC Iantigens, involved in presentation of foreign antigens to cyto-toxic T lymphocytes, in microglial cells in the developingbrain (15). Expression of MHC II antigens responsible forpresentation of a foreign antigen to helper T lymphocytes,however, was not found on these cells under normal condi-tions. When stimulated with LPS, IFN- c, or E-coli, the ex-pression of MHC II antigens was induced on microglial cellsin the developing brain (61, 62, 74). Expression of MHC II hasalso been reported on microglia in the normal and pathologicalhuman fetal spinal cord (93). MHC I/II antigen expression onmicroglial cells has also been reported following perinatalhypoxia (94). The expression of these molecules providesevidence for the protective role of microglial cells in thedeveloping brain as BBB is immature and leaky and, indeed,vulnerability to a potential immune threat is feasible. Disrup-tion of the BBB has been reported in neonatal meningitis (95),hypoxic-ischemic injuries (96) and in excitotoxic lesions (97),and increased numbers of CD4-positive T cells in the brainhave been noted in the white matter of brains of mice sufferingfrom chronic perinatal hypoxia (98).

Synaptic Pruning and Remodeling

It has been suggested that microglia play an importantrole in regulating synaptic function through elimination ofsynaptic connections and their involvement in maturation ofsynapses in the developing brain. Studies have shown thatmicroglia are involved in synaptic pruning and remodelingduring postnatal brain development (99, 100) and impairedmicroglia function results in delayed maturation of synapticcircuits in certain regions of the brain such as the hippocampus(99). Complement 3 (C3) molecules are localized on thedeveloping synapses and microglia specific phagocytic path-way, CR3/C3-dependent signaling, is believed to be one of theunderlying mechanisms in synaptic pruning and remodeling

of developing synapses by microglia (100, 101). In vitro stud-ies suggested that interleukin-10 signaling may be involved inpromoting synapse formation by microglia (102). Depletion ofmicroglia from the mouse brain had resulted in alterations insynaptic protein levels and glutamatergic synaptic function(103). Besides CR3/C3 cascade, MHC class I molecules mayalso be involved in developmental microglia-mediated prun-ing or refinement of synapses (104–107).

Role in Development of Various Cells in the Brain

Microglia can regulate the number of neurons in thedeveloping brain by phagocytosis of dead or dying cells andprovide trophic support to the neural progenitor cells for theirproliferation and maturation (108–110). In vitro studies have

FIGURE 5. (A) Electron micrograph of an apoptotic cell (Ap) inthe hippocampus of a 2-day-old hypoxic rat showing crescentshaped chromatin condensation at the periphery of thenucleus. (B) An amoeboid microglial (Am) cell in the corpuscallosum of a 2-day-old hypoxic rat showing lysosomes (Ly)and some vacuoles (asterisks) in the cytoplasm. The amoeboidmicroglial cell is in the process of phagocytosing an apoptotic(Ap) cell with electron dense nuclear chromatin condensation.Reproduced from Kaur and You (89), with permission fromElsevier.

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shown that depletion of microglia or a deficiency of the che-mokine receptor, CX3C chemokine receptor 1 (CX3CR1),also known as fractalkine receptor, in microglia increases thenumber of apoptotic neurons in the cerebral cortex in normalbrain (111). Microglia-derived insulin-like growth factor-1(IGF)-1 was also identified as a trophic factor involved inmaintenance of neuronal survival. Production of factors suchas IGF-1, nuclear factor-kappaB (NF-jB), interleukin (IL)-1b,and IL-6 by microglia is thought to play a part in the survival,differentiation and maturation of oligodendrocytes (112–114).Using cultures of neural progenitor/stem cells obtained fromrat embryonic day 16 subventricular zone and microglial cellsfrom I day old rat cortex, Nakanishi et al (115) have shownthat microglia-derived IL-6 and leukemia inhibitory factor areessential molecules for differentiation of neural progenitorcells into astrocytes.

In addition to the above, microglial cells in the develop-ing CNS have also been suggested to regulate vascularization(110, 116) and influence myelination (117).

Angiotensin and Angiotensin Receptors

The renin angiotensin system (RAS) regulates bloodpressure and electrolyte homeostasis.

Renin produced by the juxtaglomerular cells of the kid-ney converts angiotensinogen to angiotensin I, which is thenconverted to angiotensin II (Ang II) by the angiotensin-converting enzyme. Ang II is the most active peptide of theRAS and binds to 2 receptors, Ang II type 1 (AT1R) and type2 (AT2R). Most of the physiological or pathophysiologicalfunctions of Ang II are carried out through its binding withthese receptors (118, 119). Several studies have reported theexistence of various components of the RAS in the brain (120)and have also reported that this system is involved in the regu-lation of fetal cardiovascular responses, body fluid balance,and neuroendocrine regulation (121). We have reported thatAng II, AT1R, and AT2R are expressed on microglial cells inthe neonatal rat and the expression is sustained in the maturebrain (122). The expression of Ang II and AT1R was reducedfollowing a hypoxic injury whereas the expression of AT2Rwas increased (122). Since hypoxic insults to the brain areknown to reduce the blood flow to the various regions of thebrain and induce BBB dysfunction (123, 124), decreased AngII and AT1R expression may be helpful in restoring the bloodflow in such conditions. AT1R has also been reported to playa part in in apoptosis, oxidative stress, and neuroinflammation,and its suppression with AT1R blockers reduces inflammation(125) and suppresses apoptosis (126). An enhanced expressionof AT2R in the microglial cells following a hypoxic injurywas suggested to be neuroprotective (122).

Production of Neuroprotective FactorsEndothelins

The family of endothelins (ETs), considered tradition-ally as potent vasoconstrictors, consists of 3 isopeptides, ieendothelin-1 (ET-1), endothelin-2, and endothelin-3 (127,128), which bind to 2 specific G-protein-coupled receptors

subtypes, ET-A and ET-B receptors (129, 130), to carry outtheir actions. Endothelial cells, monocytes and macrophagesare important sources of circulating ET-1 (131). In addition toits vasoconstrictor action, ET-1 exerts mitogenic and anti-apoptotic actions (132, 133). Microglia in the periventricularwhite matter in the developing brain express ET-1 (134),which was suggested to be involved in the proliferation anddifferentiation of glial precursors under normal conditions.Hypoxic injury resulted in a decreased production of ET-1 bythe microglial cells with implications that its reduction mayadversely affect the development of glial cells (134). How-ever, ET-1 appears to be a double-edged sword with severalstudies reporting its beneficial effects as mentioned abovewhereas others argued that its increased expression iscorrelated with vasogenic edema formation via BBBdisruption (135).

Insulin-Like Growth Factors

Microglial cells produce IGF-1 and -2 (136, 137) thatare known to regulate the development of the nervous system(138) by promoting cell proliferation and differentiation (139)especially oligodendrocyte survival and myelination (140–142). In a coculture of primary microglia and neurons,microglia-derived IGF-1 favored the survival of neurons(111). IGF-1 is known to play a significant role in recoveryfrom hypoxic-ischemic insults (143) by blocking tumor necro-sis factor (TNF)-a-induced apoptosis and promoting prolifer-ation/differentiation and survival of oligodendrocyte precur-sors (144, 145). However, when proinflammatory cytokinesare overexpressed the secretion of IGF-1 by microgliadecreases, which could cause deleterious effects (146).

Transforming Growth Factor-b

Transforming growth factor-b (TGF-b) family plays animportant role in embryogenesis determining the right-left or-ganization, axis formation and tissue patterning. All 3 iso-forms of TGF-b, (TGF-b1-3), are expressed in the brain andare essential for neuronal and glial differentiation (147, 148).Following hypoxic exposure, TGF-b1 mRNA expression wasincreased in the periventricular white matter of neonatal rats.In these animals, TGF-b1 expression was localized in themicroglial cells (149). In TGF-b1 knockout mice there wasincreased neuronal loss and microglial activation (150), sug-gesting a role for TGF-b1 in regulating microglial activationstatus. Interestingly the microglial cells also expressed recep-tors for TGF-b (149), which supports the idea of TGF-b-medi-ated regulation of microglial functions. This could include theability of TGF-b to decrease the expression of MHCII, inter-cellular cell adhesion molecule 1, vascular cell adhesion mol-ecule and TNF-a (151), which are known to be expressed inactivated microglia. TGF-b is known to induce the productionof nerve growth factors by acting through its receptorsexpressed by the brain parenchymal cells (152). It is highlypossible that TGF-b could do the same in microglia by actingin an autocrine manner. Moreover, TGF-b1 was recently dem-

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onstrated to have a role in the synapse formation in developingcerebellum (153).

Neurotrophins

Microglia also serve as a source for neurotrophins suchas nerve growth factor (NGF), brain-derived neurotrophic fac-tor (BDNF) and neurotrophin-3 (NT3) (154–157). While un-stimulated cultured microglia could express mRNAs for allthese neurotrophic factors mentioned above, stimulation withLPS up-regulates the mRNA expression of these factors (158).These neurotrophins are essential for the survival, differenti-ation and proliferation of the brain parenchymal cells duringpostnatal development (159), and they exhibit their action byeither activating the tyrosine kinase B (TrkB) receptors or thep75 neurotrophin receptors (160–162). While activation ofTrkB receptors could result in enhanced survival, activation ofp75 neurotrophin receptor has been linked to neuronal apop-tosis (163). In cocultures of microglia and dorsal root ganglionneurons, microglia-derived BDNF promotes neurite outgrowthand terminal contacts of dorsal root ganglion neurons (164).BDNF derived from ethanol treated microglia was shown toprevent apoptosis of hypothalamic neurons through mecha-nisms modulating reactive oxygen species (ROS) and cAMPresponsive element binding protein pathway (165). Addition ofconditioned medium from neurons to the microglial culturesfavors the production of neurotrophins in microglia (157). Inmicroglia cell lines, BV2 and N9, BDNF and NGF enhancetheir proliferation in a concentration dependent manner (166).Consistent with this, under inflammatory conditions, intranasaladministration of BDNF was found to increase the number ofactivated and phagocytic microglia at the site of injury (167).Similar to TGF-b, these neurotrophins could inhibit the expres-sion of MHC II in the activated microglial cells via the p75 neu-rotrophin receptor (160). Taken together these studies putforward the premise that brain injury could trigger neurotrophinproduction in microglia in order to enhance the survival of neu-rons. In turn, the neurotrophins could act in an autocrine fashionon microglia and inhibit inflammation providing a conduciveenvironment for the survival of neurons at the injured site.

Neurotoxic RolesBesides their neuroprotective functions, microglial cells

are also known to play detrimental roles under various patho-logical conditions when they produce a plethora of moleculessuch as proinflammatory cytokines, chemokines, reactive oxy-gen species (ROS), and nitric oxide (NO), thus causingneurotoxicity.

Role in Neuroinflammation

Proinflammatory molecules are expressed at higher lev-els in the developing than in the mature brain in the absence ofany pathology. Thus, it is suggested that they may have an im-portant role in CNS development. Involvement of cytokinessuch as TNF-a and interleukin (IL)-1b in developmental proc-esses such as neural cell migration, proliferation, differenti-ation, and death has been reported (168), whereas IL-6 has

been shown to contribute to development of the vasculature(169, 170). In CNS injury, microglial cells release augmentedamounts of proinflammatory cytokines which when sustainedover a long period can cause damage to the developing braincells. TNF-a and IL-1b are released in varying amounts bymicroglial cells in different areas of the developing brain fol-lowing a hypoxic injury (67, 68, 171, 172). Upregulation ofTNF-a and IL-1b in microglial cells was coupled with expres-sion of their respective receptors TNF-R (1) and IL-1R (1) onoligodendrocytes in the periventricular white matter and thePurkinje neurons in the developing cerebellum (67, 68). Thebinding of these cytokines to their respective receptors wassuggested as a mechanism leading to cell death. In neonatalrats subjected to a hypoxic exposure iron accumulation wasincreased in the microglial cells and this was shown to mediateaugmented production of TNF-a, IL-1b, and ROS (172, 173).In addition to the above, cyclooxygenase-1 (COX-1),cyclooxygenase-2 (COX-2), microsomal prostaglandin-E syn-thase, E-prostanoid receptor 2, and prostaglandin E2 (PGE2)expression was increased in microglia in the hypoxic develop-ing brain (174). In connection with this, PGE2 was found to beinvolved in the regulation of TNF-a and IL-1b production.Increased accumulation of microglial cells occurred in theperiventricular white matter in hypoxic neonatal rats throughexpression of monocyte chemoattractant protein (MCP)-1 andaugmented the inflammatory response (175). Macrophagecolony-stimulating factor (M-CSF) is another cytokinederived from microglia that promoted proinflammatory cyto-kine production by other glial cells such as the astrocytes inthe periventricular white matter following hypoxic exposure,which added to the white matter damage induced bymicroglia-derived inflammatory cytokines (176). TLRs havebeen reported to play a role in immune responses. Microgliaalso express TLR4 whose expression is enhanced in hypoxicinjuries and mediates neuroinflammation via NF-jB signalingpathway through production of TNF-a, IL-1b, inducible nitricoxide synthase (iNOS), ROS, and NO (88).

Microglia in the developing brain have also been dem-onstrated to be activated by alcohol in conditions such as thefetal alcohol syndrome. In this regard, ethanol triggers produc-tion of ROS and inflammatory cytokines as well as phagocyt-osis through activation of TLR2 and TLR4 signaling inmicroglia (177–179). Although alcohol-induced abnormalitiesin glial cells have been implicated in the adverse effects ofalcohol on the developing brain (180), more work is clearlydesirable to unravel the underlying molecular mechanisms.

Following a maternal injection of the teratogen cyclo-phosphamide to induce neural tube defects, the microglialcells in the fetal brain showed a marked increase in the levelsof TNF-a and TGF-b expression (181, 182). It was suggestedthat upregulation of proinflammatory cytokines caused bycyclophosphamide may be the underlying cause of increasedrate of neural tube defects. Increased expression of M-CSF bythe microglia and a concomitant increased expression of M-CSF receptor in these cells following the teratogen-inducedneuronal injury suggested that microglia are capable ofresponding to self-derived M-CSF in an autocrine fashion thatresults in cell proliferation and a proinflammatory response(183). This observation is supported by several studies that

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have reported that M-CSF receptor expression induces micro-glial proliferation, cytokine expression, and a paracrine in-flammatory response in brain pathologies (184, 185).

Accumulation of glutamate in hypoxic conditions in thedeveloping brain results in activation of N-methyl D-aspartatereceptor (NMDAR) and a-amino-3-hydroxy-5-methyl-4-iso-xazolepropionic acid receptors (AMPAR) on microglial cells(137, 186). Increased accumulation of glutamate enhanced theproduction of TNF-a and IL-1b by microglia (137). Further-more, NMDAR expression in microglia resulted in TNF-a andIL-1b release via NF-kB signaling pathway in the periven-tricular white matter of neonatal rats following hypoxia (186).In addition, activation of glutamate receptors also led to theproduction of excessive amounts of NO through the iNOS iso-form in the microglial cells (186). Activation of the microglialNMDAR triggers inflammation and plays a pivotal role inneuronal death in the perinatal and mature brain (187)

Notch signaling pathway is a key regulator of neurogen-esis in the CNS (188) and administration of Notch ligands inthe brain after ischemic injury has been reported to expandstem cell numbers and improve motor skills (189, 190). On theother hand, activation of Notch signaling and Notch pathwayhas been reported to be associated with exaggerated inflamma-tion in many tissues (191), and inhibition of this pathway hasbeen shown to ameliorate the severity of conditions such asautoimmune encephalomyelitis (192) and experimental auto-immune uveoretinitis (193). We have shown that microglia inthe developing brain express Notch-1 receptor, which wascolocalized with its ligands, Jagged-1, and Delta-1 (194).Notch-1 receptor expression was increased in the microglialcells in postnatal rats challenged with LPS and its suppressionresulted in increased levels of TNF-a released from microglialcells, suggesting that Notch signaling pathway plays an im-portant role in neuroinflamnation (194).

Apart from cytokines, activated microglia also secretechemokines that mediate the infiltration of leukocytes to thesite of injury. LPS treatment increases the levels of chemo-kines such as interferon-c-inducible protein-10, regulated onactivation normal T expressed and secreted (RANTES), MCP-1, macrophage inflammatory protein (MIP)-1a (MIP-1a, alsocalled CCL3), and MIP-1b in human fetal microglia (195,196). In neonatal rat, following hypoxic ischemic injury, therewas increased expression of MIP-1a/CCL3 in microglia(197). Hypoxic microglia in the presence of syndecan-2released excessive amounts of CCL2/MCP-1, CXCL12 (alsocalled stromal derived factor-1 [SDF-1]) (198). Infection ofthe prenatal rodent brains in utero with cytomegalovirus alsoupregulated the expression of chemokines such as CCL2/MCP1, CCL3, CCL4/MIP-1b, CCL7/MCP-3, and CCL12/MCP-5 in microglia (199), and all of the above-mentionedchemokines have been demonstrated to attract peripheralmonocytes into the brain and exacerbate the injury. In additionto secreting chemokines, microglia also express chemokinereceptors that influence their activation. One of the chemokinereceptors expressed by microglia during development includeCX3CR1, a receptor for neuronal chemokine fractalkine(200). The fractalkine-CX3CR1 signaling is implicated in thedevelopmental events carried out by microglia, such as phago-cytosis and synaptic pruning (201). Similarly, microglial

migration during cortical development is also dependent onthe chemokine CXCL12/SDF-1. This is facilitated in micro-glia through the expression of SDF-1 receptor, CXCR4. Che-mokine signaling in an injured brain could also be detrimental.For instance, following hypoxic-ischemic injury in neonatalrodents, the neurons at the site of injury express MCP-1 that isreported to facilitate the migration of microglial cells to theinjury location (202), which might exaggerate the injury byproducing inflammatory mediators. In these animals ,the ex-pression of MIP-1a and MIP-1b, in the infarcted region of thebrain, preceded the microglia/monocyte accumulation (203).

Role in Oxidative Stress

Microglia-derived free radicals or ROS is a common ob-servance following an injury to the brain. Free radicals such assuperoxide, hydrogen peroxide, etc. are unstable moleculesthat could initiate oxidative stress. Under physiological condi-tions, they are neutralized by the antioxidant defense system.However, in the immature brains there is a lack of antioxidantsystem to combat the excessive oxidants (204, 205). Hence ex-cessive generation of free radicals in the developing braincould cause damage to the susceptible cells (ie oligodendro-cytes [206] and neurons) by causing lipid peroxidation (207)and by damaging the myelin sheath (208). As aforementioned,microglial activation could also lead to excess production ofiNOS and subsequent NO, which is also a free radical. NO hasmultiple roles depending on the cellular source of origin, andNO produced by iNOS is considered detrimental. The mostharmful effect of NO is observed when it reacts with super-oxide to produce the highly reactive peroxynitrite, which couldresult in DNA strand breaks, lipid peroxidation, and protein ni-tration. In developing periventricular white matter, followinghypoxic exposure the expression of iNOS was predominant inthe microglia, suggesting them to be the source of NO (78). Incultured microglia, hypoxic exposure increases the generationof reactive oxygen as well as reactive nitrogen species (172),including NO (186), and their subsequent release into the cul-ture medium. In primary oligodendrocytes treated with condi-tioned medium from hypoxic microglia there was increasedlipid peroxidation with a parallel reduction in the glutathionecontent (172). However, antioxidant drugs such as edaravonecould inhibit free radical production by microglia and renderprotection to the immature brains (198). Taken together, all ofthese studies provide converging evidence for the damagingrole of activated microglia in an injured immature brain.

SIGNALLING PATHWAYS IN MICROGLIAAssociated with the production of various proinflamma-

tory mediators, activated microglia showed upregulated ex-pression of JNK and p38 MAPK pathways (209). Moreover,increased NF-jB/p65 expression was a consistent feature(210), which was to be expected given the fact that the cellsproduce significant amount of iNOS and NO. JAK-STATpathway is also implicated in the production of pro-inflammatory molecules, iNOS and NO in N9 microglialcells (211). In addition, activation of JAK/STAT1 pathwaywas reported to favor migration of microglia towards the

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injury site in a stab wound mouse model (212). Hence, it ispossible that depending upon the STAT subunit that is acti-vated, it might have an influence on the functions of microglia.The most unexpected finding was the detection of Notch-1signaling in activated amoeboid microglia including its down-stream elements when challenged with LPS both in vivo andin vitro (88, 193, 213, 214). Even more striking was the dem-onstration of a reciprocal transactivation between Notch-1 andNF-jB in activated amoeboid microglia in regulation of pro-duction of proinflammatory mediators (209, 215). Microgliaalso produce pro-inflammatory molecules through activationof receptors for recognizing molecular patterns that are associ-ated with pathogens or any type of danger. TLRs are one ofsuch receptors expressed by amoeboid microglia and activa-tion of TLRs might lead to inflammation either via the NF-jBpathway (88) or the interferon regulatory-3 (IRF3) signalingpathway (216). The other pattern recognition receptors thatare not well studied in amoeboid microglia include thenucleotide-binding oligomerization domain (nod)-like recep-tors, and the retinoic acid-inducible gene-1-like receptors.Similar to TLR, activation of these pathways could also leadto inflammation by converging at either the NF-jB or IRF3signaling pathways; however, further studies are required toconfirm this in microglia in the developing brain. TREM2 sig-naling pathway is a pathway through which microglial innateimmune response is controlled in the postnatal brain (85) anda favorable environment is ensured for myelination and nor-mal development (217). Suffice it to say that the above mayrepresent only a few of the many signaling pathways that regu-late microglial activation (4).

RELEASE OF GLUTAMATE AND GLUTAMATERECEPTOR EXPRESSION

Glutamate is the most predominant and vital excitatoryneurotransmitter in the immature brain, that exerts its actionby binding through its receptors, which are either ionotropic(AMPAR, NMDAR, kainate receptors) or metabotropic recep-tors (mGluR) (218–220). In the developing brain, it aids in theearly development by signaling for proliferation, differenti-ation and migration of neurons. However, in the injured imma-ture brains, excessive levels of glutamate are detrimental andactivated microglia seem to be one of the sources of glutamate(137, 221–223). For instance, production of glutamate was ap-parently increased in cultured microglia treated with eitherLPS or sodium arsenite or infected with virus (222, 224). Thesame was observed even in microglial cultures subjected tohypoxia (137). In addition to releasing glutamate, microglialcells express receptors for glutamate (185, 225). In the whitematter of neonatal rat brain following hypoxic injury, micro-glial cells were found to concomitantly overexpress bothAMPAR (GluR2-4) and NMDAR subunits (NR1, NR2A-D)(137, 186). Overactivation of these receptors instigates theproduction of pro-inflammatory cytokines such as TNF-a andIL-1b along with other toxic factors such as NO and Fas L,which are known to cause oligodendrocyte and neuronal apop-tosis. Addition of glutamate or kainate to the microglialcultures enhances the release of TNF-a through activation ofeither AMPAR or kainate receptors (226, 227). Along with

TNF-a and IL-1b, hypoxia-induced activation of microglialNMDAR increases the expression of iNOS and subsequentproduction of NO (186). Even activation of mGluR2expressed in microglia could enhance the release of cytokinesand Fas ligand. All of these toxic factors act upon the highlysusceptible cells of the brain, such as the oligodendrocytes andneurons, and cause their death by activating caspase-3 (172,186, 228). While activated microglia could also produceneurotrophic factors such as IGF-I and IGF-II, excess glutam-ate suppressed IGF-I levels and delayed the repair mecha-nisms (137). The proinflammatory cytokines released frommicroglia could inhibit the glutamate reuptake by astrocytes(229), further complicating the entire injury mechanism. Thusexcess glutamate released by activated microglia could be det-rimental to oligodendrocytes and neurons, either by directbinding to their receptors expressed on these cells, or indir-ectly by enhancing the release of toxic factors and suppressingthe release of neurotrophic factors from microglia.

MICROGLIAL ACTIVATION IN VARIOUSDISORDERS OF THE DEVELOPING BRAIN

Periventricular White Matter DamageMicroglia in the amoeboid form accumulate preferen-

tially in the periventricular region in the corpus callosumabove the lateral ventricles and the subependyma (33, 53),cavum septum pellucidum (230, 231), and also the subpendy-mal cysts associated with the ventricular system (231, 232).The significance of this remains speculative, but it was sug-gested that microglia in these areas may be involved in clear-ance of cellular debris resulting from spontaneous cell deathor remodeling of callosal axons (33) or early physical expan-sion of the developing brain (231). In view of their closeassociation with the fiber tracts, it is conceivable that whenactivated by hypoxia (64), ischemia or septicemia (233), ex-cess amounts of proinflammatory mediators released by themwould affect the structural and functional integrity of the fibertracts. There is evidence supporting the involvement of acti-vated microglia in aggravating death of immature oligoden-drocytes and degeneration of axons in the periventricularwhite matter in neonatal rats following a hypoxic insult (64,78). We have demonstrated that the pathogenesis of periven-tricular white matter damage is multifactorial involving fac-tors such as inflammation, excitotoxicity, excess production ofNO, iron related oxidative stress, and vascular changes amongmany others (64, 67, 78, 172, 175, 186). The involvement ofsome of the above factors in periventricular white matter dam-age in hypoxic injuries has received support from other studies(223, 234–238).

Cerebral PalsyCerebral palsy is a motor disorder due to brain white

matter damage that appears in infancy or in early childhoodand affects movement, muscle tone, coordination, and bal-ance. Most cases are congenital as the damage to thebrain white matter occurs before birth. Intrauterine infection/inflammation, premature birth, and low birth weight have beensuggested as the underlying causes leading to cerebral palsy. Pre-

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clinical studies subjecting animals to hypoxia–ischemia or LPShave shown axonal damage and oligodendrocyte death in thecerebral white matter as well as neuronal damage in many regionsof the developing brain (78, 239–241). Microglia are present inlarge numbers in the developing white matter tracts and arebelieved to regulate the development of the white matter byremoving excessive axons in the developing brain (242). Postmor-tem studies in the human newborns have implicated cytokinessuch as TNF-a and its receptors TNFR1 and TNFR2 as the majorplayers in causing damage (243, 244). Activation of microglialcells in the white matter and other regions of the brain andenhanced release of TNF-a, IL-1b, and other proinflammatorymolecules such as release of NO through iNOS by them has beenreported in various animal models (67, 172, 186, 245, 246). Be-sides their inflammatory role, microglia may also have a protect-ive role in early stages by phagocytizing dead cells and degenerat-ing axons (78). Microglial activation and accompanying COX-1enzyme expression have also been implicated in overproductionof proinflammatory mediators resulting in white matter damageand development of cerebral palsy (247). Excitotoxic mechanismshave also been suggested to play a role in neuronal death (248)and in axonal damage in the developing brain and this is sup-ported by expression of NMDAR on premyelinated axons inhuman mid-term fetuses (249) and on the microglial cells in theneonatal rat brains (186).

EpilepsyEpilepsy is a disorder in which seizures develop due to an

imbalance between cerebral excitability and inhibition resultingin uncontrolled excitability. Seizures have been reported to de-velop more readily in the immature brain than in the maturebrain. Many developmental processes such as cell division, mi-gration, expression of receptors, formation, and stabilization ofsynapses are affected by seizures (250, 251). Temporal andfrontal lobes are frequently involved. Premature birth or lowbirth weight, birth trauma, fever or infections are some of theunderlying causes of epilepsy in newborns and infants. Glial ac-tivation and neuronal damage occurring in seizures has beenreported in brain regions such as the hippocampus (252). Severalstudies have shown that proinflammatory molecules influencesusceptibility to seizures (253–255). Enhanced levels of TNF-aand IL-1b were reported to play a role in the pathophysiology ofepilepsy (256). Microglial activation in the postnatal mousehippocampus was linked to heightened seizure susceptibility(257). Upregulation of TNF-a and IL-1b in microglia was read-ily detected after the induction of acute seizures (257, 258).Binding of IL-1b to its receptor in the hippocampus has beensuggested to result in increased synaptic excitability (259). TNF-a affects neural circuits by preventing refinement and elimin-ation of synapses occurring during normal brain developmentthat may lead to increased connectivity and epilepsy (260).

Autism Spectrum DisordersAutism spectrum disorders (ASD) are neurodevelop-

mental disorders with disabilities in communication and socialskills and repetitive behaviors. Genetic factors, prenatal drugs,or chemical exposures are some of the risk factors for develop-

ment of autism. Involvement of microglia due to their role insynaptic refinement during development has been suggestedin these disorders (261). Deficient synaptic pruning due to areduction in microglial numbers has been associated withweak synaptic transmission and decreased functional brainconnectivity (262) that may be involved in autism and otherneuropsychiatric disorders. Cerebellar dysfunction and subse-quent thalamic hyperactivation in early childhood may berelated to the development of ASD (263). Thalamic hyperacti-vation is thought to be induced by microglia-mediated neuro-inflammation (263), and neuroinflammation is considered asan important factor in the pathogenesis of neuropsychiatricdisorders (264, 265). Microglia activation and proliferation,along with increased expression of TNF-a, IL-1b, IL-6, andIL-17 in the brain and cerebrospinal fluid of ASD patients, hasbeen observed (266, 267). Focal inflammation due to abnor-mal microglial activation and proliferation seems to affectnormal synaptic activity in brains of patients with ASD (268–270). Along with increased microglia activation, decreasedneuronal activity was observed in human brain cortical tissuesamples from ASD patients (267). The above findings pointtowards the important role that microglia play in the pathogen-esis of ASD (271, 272). Interactions between mast cells andmicroglia in the brain have been reported to occur as evi-denced by induction of microglia activation and proliferationby mast cell-derived histamine (273, 274) that amelioratedwith suppression of mast cells (275).

Perinatal StrokePerinatal stroke occurs in newborns in the first few days

after birth due to a disturbance in the blood supply to the brainresulting in hypoxia and leads to significant morbidity andlong-term neurological and cognitive deficits. Blood clottingdisorders, maternal infection, preeclampsia, maternal diabetesand smoking are some of the risk factors that can lead to aperinatal stroke. In the rodent model of neonatal stroke aftertransient middle cerebral artery occlusion, microglial cellshave been reported to have a neuroprotective function. Deple-tion of microglia by intracerebral injection of liposome-encapsulated clodronate in rats at postnatal day 5 has beenshown to trigger hemorrhages at 24 hours after transient mid-dle cerebral artery occlusion (276). Along with this, increasedlevels of several cytokines and chemokines already elevatedby ischemia-reperfusion and increase in the severity and vol-ume of injury were demonstrated in animal models of neonatalstroke following depletion of microglia (81). These findingspoint towards a protective role exerted by microglia during thesubacute injury phase. However, other studies have reportedthat infiltration and activation of microglia in the brain of post-natal rats after neonatal stroke occurs over several days result-ing in neuroinflammation and cell death (277).

EPIGENETICS AND TRANSCRIPTOME ANALYSISA recent search for the interacting noncoding RNAs,

genes, and their epigenetic changes, (epigenetics refers to theheritable changes in the mechanisms that regulate the gene ex-pression without altering the underlying genetic code), has

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brought into light several unknown aspects of microglia,including their involvement in disorders that were thought tobe microglia independent. By cDNA microarray analysis,amoeboid microglia were found to express proliferation- anddifferentiation-related genes, Sox4, Sox11, and Runx1t1 andalso those genes involved in cell cycle process and migration(278). Interestingly, with the ramification of the cells withage, the expression of various genes was altered and this maybe related to the specific functions of the 2 phenotypes. Micro-glia also express a neuroprotective and inflammatory pheno-type, which depends on the environmental cues. For instance,transcriptomic analysis in postmortem brain samples from aut-istic children revealed the transformation of microglia towardsthe inflammatory phenotype (279). Genes of immune path-way such as C1qA, C3, CR3, and TNF-a, which are expressedby microglia and transcription factors such as IRF8 andSPI1, that are essential for development of microglia werefound to be methylated in autistic brains (280). Additionalevidence for the epigenetic regulation in microglia and its in-fluence in the childhood disorders comes from microglia spe-cific methyl cytosine binding protein 2 (MeCP2) knockoutmice. Of note, MeCP2 regulates gene transcription by bind-ing to the methylated cytosine and guanine rich sites (CpGislands), and its reduced expression has a role in the etiologyof autism (281). Deletion of MeCP2 in mice contributed tothe dysregulation of extracellular glutamate levels and aber-rant neuronal dendrites (282). In primary cultures of micro-glia from neonatal rodents, hypoxia mediated reduction inthe microRNA21 expression accounted for the increased pro-duction of Fas ligand that induces neuronal injury (283).microRNA124 is specifically expressed in microglia and itsuppresses the expression of MHCII antigens. Loss of micro-RNA124 precedes the onset of experimental autoimmune en-cephalitis (284), which explains the enhanced autoimmunityobserved. Prenatal stress has been shown to increase themethylation of genes such as those for glucocorticoid recep-tors elsewhere (285); however, at this juncture there is noclear evidence if the same is seen in microglia of prenatalbrains, which could be linked to the increased inflammatoryphenotype that is observed under injury conditions. All thesestudies converge to a point, that epigenetic regulations occur-ring in microglia could have a greater impact on the normalbrain development as well as various disorders of the devel-oping brain.

Concluding RemarksMicroglia in the developing brain exist in 2 different

phenotypes: amoeboid microglia and ramified microglia.Amoeboid microglia are monocyte-derived brain macro-phages, that evolve to become the ramified microglia in thecourse of brain development. Microglia play an indispensablerole in building the normal brain histoarchitecture includingphagocytosis of apoptotic neurons and axons, and pruning ofunwanted synapses in brain remodeling. They secrete neuro-trophic factors such as IGF-1, NT-3, BDNF, and NGF; henceare neuroprotective. Overt microglia activation due to perturb-ation of the microenvironment can lead to increased release ofglutamate and proinflammatory mediators including TNF-a,

IL-1b, NO, ROS, etc., which in turn would exacerbate braindamage. Microglia-mediated neuroinflammation is implicatedin different neurological diseases and disorders such as peri-ventricular white matter damage, cerebral palsy, autism spec-trum disorders, epilepsy, and perinatal stroke. Microglia acti-vation in the developing brain is controlled through a complexregulatory mechanism and multiple signaling pathways suchas NF-kB, Notch-1, JNK, and p38 MARK that are linked toproduction of proinflammatory mediators. While the func-tional roles of microglia in the developing brain are greatlyamplified in recent years, there remain many issues to be fullyexplored such as transcriptome and epigenetics changes inmicroglial activation. This may pave way for designing appro-priate therapeutic strategies in which the neurotoxic effects ofmicroglial activation may be eliminated to mitigate the injuryin the developing brain.

ACKNOWLEDGMENTThe technical assistance provided by Ms. Suat Hoon

Tan, Ms. Yee Gek Chan, Mrs. Eng Siang Yong, Mrs. GeokLan Ng, Mr. Tuck Yong Yick, Dr. Qiong Cao, and Dr. YajunWu is gratefully acknowledged.

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