[Frontiers in Bioscience S3, 884-900, June 1, 2011]

Role of microglia in the process of inflammation in the hypoxic developing brain

Yi Yu Deng 1, Jia Lu2, Eng-Ang Ling1, Charanjit Kaur

1Department of Anatomy, Yong Loo Lin School of Medicine, Blk MD10, 4 Medical Drive, National University of Singapore, Singapore 117597,  2Defence Medical and Environmental Research Institute, DSO National Laboratories,27 Medical drive, Singapore 117510

TABLE CONTENTS

1. Abstract
2. Introduction
3. Etiology and risk factors of hypoxic brain injury
4. Pathological changes of the hypoxic developing brain
4.1. Neuronal loss
4.2. Oligodendrocyte injury and myelination delay
4.3. Axon injury
4.4. Microglia activation and astrogliosis
4.5. Blood-Brain Barrier damage
5. Inflammation in the hypoxic developing brain
6. Roles of microglia in the neuroinflammatory response in the hypoxic developing brain
6.1. Distribution and morphology of microglia
6.2. Phagocytosis
6.3. Antigen presentation
6.4. Proliferation
6.5. Migration
6.6. Release of cytokines and chemokines
6.6.1. Tumor necrosis factor-α and its receptors
6.6.2. Interleukin-1 and its receptors
6.6.3. Macrophage colony stimulating factor
6.6.4. Monocyte chemoattractant protein-1
6.7. Expression of ion channels
6.7.1. Kv1.2 .and Kv1.1 .
6.7.2. Sodium channels
6.8. Syndecan-2
6.9. Generation of reactive oxygen species and nitrogen intermediates
7. Conclusions
8. Acknowledgements
9. References

1. ABSTRACT

The developing brain is susceptible to hypoxic damage because of its high oxygen and energy requirements. Hypoxia-induced inflammatory response has been recognized as one of the main culprits in the development of hypoxic brain injury. In this regard, a hallmark feature is microglial activation which results in overproduction of inflammatory cytokines, free radicals and nitric oxide. Concomitantly, activated microglia exhibit enhanced expression of ion channels such as Kv1.2, Kv1.1 and Nav which further promote the release of inflammatory cytokines, chemokines and reactive oxygen species. Through the above-mentioned inflammatory mediators, activated microglia induce neuronal loss, axonal damage and oligodendroglial death along with myelination disturbances. Our recent studies have extended that tumor necrosis factor-α, interleukin-1β, monocyte chemoattractant protein-1 and macrophage colony stimulating factor produced by activated microglia are linked to the pathogenesis of periventricular white matter damage in the hypoxic brain. It is envisaged that a better understanding of the interactions between microglia and neurons, axons and oligodendrocytes is key to the development of effective preventive and therapeutic strategies for mitigation of hypoxic brain injury.

2. INTRODUCTION

A regular supply of oxygen is required for the structural and functional integrity of brain (1). The brain is particularly vulnerable to hypoxic conditions because of its high energy requirements (2). The human brain represents only 2% of the body weight but accounts for 20% of the total body oxygen utilization (3, 4). Hypoxia-ischemia affects the normal development and maturation of the brain (5, 6) resulting in long-term neurological deficits (7). Although damage to selected regions of the brain is caused by hypoxia-ischemia at different ages, the white matter peripheral to the lateral ventricles called periventricular white matter (PWM) is selectively vulnerable to damage in premature infants (8, 9, 10, 11, 12). Hypoxia results in direct brain injury which occurs in the acute hypoxic phase and indirect brain injury occurring after reoxygenation which is mainly attributed to neuro-immunological activation in the central nervous system (CNS) (13). Inflammatory response induced by hypoxia plays a crucial role in indirect hypoxic brain injury (14).

Neuroinflammatory response in the CNS is a complicated process which involves numerous damage signals, cellular responses and alterations in the microenvironment (15, 16). Following the neuroinflammatory response, a complex cascade of cellular events occurs. Microglia, which are resident immune cells in the CNS and belong to the mononuclear phagocyte system, have been recognized as sensors of brain injury and main cytokine producers (17, 18). The prevailing concept is that they are mainly derived from blood monocytes and/or their hematopoietic precursors although another school of thought has maintained that they arise from the mesoderm or neuroectoderm (19). The first mentioned view states that monocytes invade the brain during early postnatal development to become amoeboid microglial cells (AMC) bearing features of monocytes or monocyte-derived macrophages (19, 20). As a consequence of pathological events such as hypoxia/ischemia, stroke or trauma, microglia are activated and increase in cell numbers through proliferation or migration (21, 22). The microglia resemble macrophages in their ability to secrete factors, scavenge, engulf and clear cellular debris in and around the injury site (21, 22, 23). In the developing brain, AMC, considered to be nascent form of microglia, are present in large numbers in the PWM (20). It is well documented that under hypoxic condition, AMC are activated and the ensuing modulated inflammatory response leads to PWM damage (PWMD) in neonatal rats (10, 24).

The pathogenesis of hypoxic brain injury is complex and is not fully elucidated. Neuronal loss, white matter damage and immune cell activation in the CNS have been described as hallmark features of hypoxic brain injury (12, 25). Over the past decade, the molecular mechanisms underlying hypoxic brain injury, especially in the developing brain, have been the focus of many studies. To this end, this review provides a brief overview on the role of microglia in the neuroinflammatory response elicited by hypoxia with special reference to the PWM in the developing brain.

3. ETIOLOGY AND RISK FACTORS OF HYPOXIC BRAIN INJURY

Hypoxia-ischemia has been reported to be a leading cause of morbidity and mortality in the perinatal period (26).The developing brain is extremely sensitive to oxygen deprivation (27) resulting in neuronal loss (28) and PWMD (29, 30, 31, 32). Seizures, mental retardation, epilepsy and cerebral palsy are some of the consequences of a cerebral hypoxic/ischemic insult suffered during the perinatal period (30, 33). Many maternal causes such as diabetes, asthma, anemia and smoking and intrapartum events such as prolonged labor are associated with fetal hypoxia (34, 35, 36). In the neonates, pulmonary or cardiac dysfunction and neonatal stroke result in hypoxia (37). Hypoxia can also occur during the antepartum or intrapartum periods due to placental insufficiency, cord prolapse, prolonged or decreased maternal oxygenation (38). Premature babies are at high risk of a variety of complications, such as respiratory distress syndrome, bronchopulmonary dysplasia (due to immaturity of lungs) and bradycardia among many others (39). These complications result in oxygen deprivation to the brain tissue.

4. PATHOLOGICAL CHANGES IN THE HYPOXIC DEVELOPING BRAIN

Following hypoxic-ischemic injuries, a significant cell death is known to occur in the neonatal brain (40, 41, 42) in many regions such as the hippocampus, cerebellum, thalamus (43, 44, 45). Neurons in the hippocampus and cerebral cortex and Purkinje cells are the most frequently injured in term infants whereas in preterm infants the oligodendrocytes in the PWM are most susceptible to hypoxic-ischemic damage (30, 46). Besides oligodendrocyte death, hypoxic-ischemic injuries in the neonatal brain are known to result in axonal degeneration (31) and disturbances in myelination (32, 47). Other changes occurring as a result of hypoxic injury in the neonatal brain are activation of microglia, astrogliosis and blood - brain barrier (BBB) damage.

4.1. Neuronal loss

Neuronal loss is known to occur following hypoxic-ischemic injuries (48, 49). Hypoxia, especially when associated with ischemia, initially results in neuronal damage due to failure of Na+ and K+ pumps which leads to membrane depolarization, failure of synaptic transmission and release of excess glutamate (50). Activation of calcium permeable N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy 5-methyl-4-isoxazolepropionic acid (AMPA) receptors by glutamate causes massive Ca2+ influx into neurons (51, 52). Excessive extracellular glutamate release and calcium influx lead to neuronal death by necrosis or apoptosis (53, 54) apoptosis being a more prominent mode of neuronal death following hypoxic-ischemia in the neonates compared to the adult brain (55, 56).

Other mechanisms of neuronal injury include free radical formation, mitochondrial dysfunction and inflammation (25). Several pathways of free radical generation including cyclooxygenase, lipooxygenase, xanthine oxidase and nitric oxide synthase (NOS) which lead to membrane lipid peroxidation resulting in neuronal cell damage may be initiated by increased intracellular calcium in hypoxia (52). Acute hypoxic episodes result in neuronal loss in the neonatal brain. Following this, secondary brain damage, which is attributed to neuroimmunological cell activation, excess excitatory amino acid release and overproduction of free radicals occurs (31).

4.2. Oligodendrocytes injury and myelination defects

Oligodendrocytes are the myelin-forming cells in the CNS and originate from neuroepithelial cells of the ventricular zones at very early stages of embryonic life (57, 58). Oligodendrocyte progenitors migrate to the developing white matter tracts and undergo substantial proliferation before their final differentiation into myelin-forming cells (58). Mature oligodendrocyte processes spiral around the axon to form myelin sheaths (59).

Some studies have demonstrated that immature oligodendrocytes during a specific prenatal window were vulnerable to hypoxia, which is a significant underlying factor in the pathogenesis of PWMD (60). Myelination deficits have also been observed in the brains of animals following perinatal asphyxia (61). Hypoxic-ischemic injury in the neonatal brain is known to result in death of oligodendrocyte progenitors, disruption of myelin gene expression and disturbances in myelination (62, 63). Reduced myelin basic protein (MBP) immunoreactivity was observed in the PWM in hypoxic rats (31).

At present the specific mechanisms responsible for oligodendrocyte injury have not been fully elucidated. But glutamate excitotoxicity and cytokine-mediated injury are believed to be the major contributors (24, 31). In addition, developing oligodendrocytes are liable to be damaged by oxidative stress because of lack of anti-oxidant enzymes (64, 65). Likewise, activation of cytokine receptors has been involved in oligodendroglial death not only at various stages of development but also in the pathogenesis of adult demyelinating disorders (66). The activation of cytokine receptors on the surface of oligodendrocytes can cause their death through cross-talk between ligand and receptors, which activate intracellular signaling pathways related to apoptosis and energy metabolism disruption (66). Our previous study has shown that under hypoxic conditions, the AMC in the PWM in the neonatal brain produce inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta via MAPK signaling pathway (24). The possible interaction between AMC and oligodendrocytes via the above-mentioned proinflammatory cytokines and their respective receptors might lead to damage of the latter cell type (24). This was evidenced by the common occurrence of apoptotic oligodendrocytes following hypoxia (24). It was suggested that the decrease in the number of oligodendrocytes would result in myelination deficits, a hallmark in PWMD (24).

4.3. Axon injury

The axon is an elongated fiber that extends from the neuronal body to the terminal endings and transmits the neural signal. The larger the axon, the faster it transmits information (59). Some axons are covered with a fatty substance called myelin that acts as an insulator (59). These myelinated axons transmit information much faster than other non-myelinated neurons. In normal myelinated axons, Na+ channels are concentrated at nodes of Ranvier, which accelerate signal transduction through allowing the action potential to rapidly jump from node to node (59). The axons are non-myelinated in the neonatal brain. In hypoxic injuries, axons are vulnerable to energy depletion, loss of myelin (59) and hypoxia-induced inflammatory response (31). In our recent study, the most conspicuous finding was the occurrence of swollen and degenerating axons when neonatal rats were subjected to a hypoxic exposure (31). Quantification of the degenerating axons in the PWM showed a significantly higher number of degenerating axons in the hypoxic rats as compared to the controls (31). Occasional axons were observed to be myelinated in 7-day-old rats, and the myelin sheaths of these axons appeared distorted (31). This phenomenon of axonal degeneration in the hypoxic rats was especially conspicuous in the vicinity of the blood vessels (31). The most striking structural alteration at 14 days after the hypoxic exposure was dilatation of a variable number of axons which appeared club shaped in sections and distortion of myelin sheaths (67). Many of the dilated axons contained a large number of vacuoles. In some sectional profiles, myelin-like figures appeared to be internalized in the axoplasm (67).

Mechanisms underlying axon injury are complex and remain unclear. Some studies have demonstrated that premyelinated white matter axons in isolated rodent optic nerve were highly resistant to hypoxic-ischemic injury, whereas early and late myelinating white matter axons were increasingly vulnerable (68). So the vulnerability of white matter axons to hypoxic-ischemic injury varies during development. Similarly, mechanisms of hypoxic-ischemic axon injury may also be different during development (68). Energy depletion during hypoxia-ischemia leads to failure of energy-dependent extracellular and intracellular ionic balance, resulting in axonal Ca2+ overload, conduction damage, and structural injury (69). Excessive glutamate receptor activation, or excitotoxicity, has been reported to result in ischemic white matter axon injury (70). This observation is supported by intracerebral injection of AMPA which induced axon injury (70). AMPA/kainate receptors participate in ischemic injury to myelinated axons in vivo, but not to isolated axons. Studies have shown that ionotropic glutamate receptor agonists did not cause damage to isolated axons, nor did glutamate receptors antagonists protected isolated axons from oxygen-glucose deprivation (71). Therefore, glutamate induces white matter axons injury by causing damage to myelinating oligodendrocytes. It has been reported in some studies that axonal injury in myelinated white matter results from oligodendrocyte excitotoxicity and can be prevented by blockade of oligodendrocyte AMPA/kainate receptors (71). The mechanisms linking oligodendrocyte injury to axon damage might include release of toxic substances from injured oligodendrocytes, loss of trophic support to axons, or loss of glial homeostatic functions (72). Attenuation of oligodendrocyte-myelin-axon interactions in myelinated white matter decreases axonal injury after AMPA injection (72).

Some reports have suggested that oligodendrocyte excitotoxicity does not result in axonal damage in premyelinated white matter (71), where the axons are non-myelinated, as interaction between these axons and pre-oligodendrocytes is weak (72). Axons in premyelinated white matter are vulnerable to hypoxic-ischemic injury through a non-excitotoxic way. The mechanism of axonal injury likely includes failure of ionic homeostasis and intra-axonal Ca2+ overload, as mentioned above (71). The downstream pathways of Ca2+-induced axonal injury remain unclear.

Activation of cytokine receptors has also been reported to be involved in axonal injury. The activation of cytokine receptors on the surface of axons can cause their death through cross-talk between ligand and receptors, which activate intracellular signaling pathways related to energy metabolism disruption (73). Our recent study has shown an upregulated TNF-R1 and IL-1R1 expression on the axons in the PWM of hypoxic neonatal rats (67). This was coupled with the disruption of MBP positive processes of oligodendrocytes and associated neurofilament-200 positive axons in the PWM (67). Therefore, it was postulated that overproduction of local TNF-alpha and IL-1beta may damage axons and delay their myelination via binding to their respective receptors (67).

4.4. Microglial activation and astrogliosis

It is well established that microglial cells are activated in hypoxic injuries in the developing brain (74, 75, 76). Our in vitro study has shown that the mRNA and protein expression of TNF- alpha and IL-1 beta in cultured microglia were upregulated significantly after hypoxic exposure for 4 hours (24). This indicated that hypoxia could activate isolated microglia to produce inflammatory mediators (24). Activated microglia can proliferate, potentiate phagocytosis, actively migrate to the site of injury and release a variety of factors including cytokines, chemokines and nitric oxide (NO) thereby causing an inflammatory response (77, 78, 79).

Under physiological conditions, astrocytes are closely associated with vascular development, formation of synapses and BBB within specific regions including PWM (10). However, astrocytes may produce reactive responses following various kinds of injury or in neurodegenerative diseases (80). This reactive process, known as astrogliosis or reactive gliosis, is characterized by cellular hypertrophy, hyperplasia, and an increase in immunodetectable glial fibrillary acidic protein (GFAP) (81). It has been reported that astrocytic reactivity occurs within the deep white matter in the hypoxic developing brain, which is coupled with widespread axonal damage and production of the pro-inflammatory cytokines (10, 82). Astrocytic responses may help to repair the injured CNS, but excessive astrogliosis may be detrimental and contribute to neuronal injury. Reactive gliosis has the potential to overproduce free radicals and inflammatory mediators such as TNF-alpha and IL-1 beta that are toxic to oligodendrocyte progenitors or axons (10). Reactive gliosis contributes to scar-formation at the injury site, which can form a local biochemical and physical barrier to prevent axonal regeneration and re-establishment of new synapses (83, 84).

4.5. Blood- brain barrier damage

The integrity and proper functioning of the BBB play a crucial role in maintaining the homeostasis of the brain microenvironment and so-called "immune-privileged" status of the brain by preventing the entry of T lymphocytes (74, 85). In vitro studies have demonstrated that hypoxia and hypoxia/reoxygenation may increase permeability of BBB and result in disruption of BBB tight junction (86, 87). Our in vivo studies have confirmed that permeability of the cerebral blood vessels is increased after a hypoxic injury (88). Some tracers such as rhodamine isothiocyanate (RhIC) and horseradish peroxidase (HRP) injected intravenously can extravasate from the blood vessels into the brain parenchyma following such injuries (88) suggesting that the BBB was damaged. This was further evidenced by alterations in the endothelial cells such as increased pinocytotic vesicles and derangement of the tight junction proteins (88). Overproduction of vascular endothelial growth factor (VEGF), NO and inflammatory cytokines in hypoxic conditions were reported to be responsible for the disruption and increased permeability of the BBB (85, 86). Recently increased gene and protein expression of VEGF in the PWM and its specific localization in the astrocytes in the neonatal brain was reported following a hypoxic exposure (31). Many VEGF positive astrocytes were observed in close association with the blood vessels (31). This increased VEGF expression may link hypoxia and vascular leakage which lead to the formation of cerebral oedema (31). Indeed increased leakage of tracers such as RhIC was observed in the PWM of hypoxic neonatal rats (89).

5. INFLAMMATION IN THE HYPOXIC DEVELOPING BRAIN

It is well known that hypoxia initiates an inflammatory response with enormous production of inflammatory mediators (90, 91). We reported recently a strong and persistent inflammatory response with production of inflammatory cytokines namely, TNF-alpha and IL-1 beta in the PWM of neonatal rats after hypoxic insults (24). NO levels were also significantly higher in hypoxic rats (31). Overproduction of proinflammatory cytokines TNF-alpha, IL-1 beta, IL-6 and IL-2 together with adhesion molecules such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 is coupled with apoptosis of oligodendrocyte progenitors, myelination delay and axonal degeneration (92, 93, 94). A concomitant expression of the corresponding receptors of above-mentioned proinflammatory cytokines was upregulated on the oligodendrocyte progenitors and axons (24) suggesting that inflammatory mediators were involved in PWMD in the hypoxic developing brain (24). Microglial cells are present in large numbers in the developing PWM (20). Being active brain macrophages, they would be the main source of inflammatory mediators in the developing brain; other sources of proinflammatory cytokine production in the PWM may be the astrocytes. Our recent study has shown that up regulated TNF-alpha and IL-1 beta expression was specifically detected in AMC at early time points whereas in the reactive astrocytes at late time points in the PWM in hypoxic injuries (67). This suggested that microglia may be responsible for the early phase of inflammatory response and astrocytes may contribute to it at later stages.

6. ROLE OF MICROGLIA IN THE NEUROINFLAMMATORY RESPONSE IN THE HYPOXIC DEVELOPING BRAIN

Microglial cells are known to be active macrophages removing cellular debris during normal development as well as in pathological conditions. Although microglial cells are necessary for phagocytic functions in the normal developing brain, their activation can cause bystander injury to other CNS cells. There is ample evidence to suggest that microglia not only initiate PWMD, but also contribute to the growth of lesions within the white matter (10).

6.1. Distribution and morphology of microglia

Microglia constitute 5-20% of all glial cells in the CNS (95, 96, 97). In the developing brain, they are present in large numbers in the PWM (20); are more sparsely distributed in the cortex, hippocampus and thalamus (98). It is still not clear why microglial cells are distributed unevenly in the developing brain, but from a speculative point of view, it is possible that they might execute some specific physiological functions in the local environment in different brain regions.

Two microglial phenotypes have been described: AMC and ramified microglial cells (19). In the developing brain, the preponderant AMC exhibit a round cell body with some processes (19). These cells transform into ramified microglial cells with the development and maturity of the brain (19); the latter exist as resting form under normal conditions. However, under pathological conditions such as trauma, infection and hypoxia-ischemia, ramified microglia retract their processes and assume an amoeboidic form (99, 100). It has been reported that AMC in the PWM begin to ramify at 7 days of age and persist as ramified cells thereafter through the adulthood (19). It is striking that all AMC remained amoeboidic in 7 days hypoxic rats (101), suggesting that hypoxia contributes to delaying the morphological transformation of AMC. A possible explanation for this may be that hypoxia being a stimulus for microglia had induced their activation which was sustained over a protracted duration.

6.2. Phagocytosis

The phagocytic nature of AMC has been shown by various methods and observations, including the localization of hydrolytic enzymes, ultrastructural features shared by tissue macrophages, uptake of exogenous substances, and the activation of surface receptors and antigens related to phagocytosis (102). Indeed, direct phagocytosis of carbon particles (103) and the bacteria Escheria coli (E coli) by AMC was observed through ultrastructural studies (102). In the fetal and postnatal rat brains, AMC were observed to engulf dead cells after transient maternal hypoxia (104, 105). In neonatal rats exposed to hypoxia, AMC in the PWM were engaged in the phagocytosis of apoptotic cells and degenerating axons (24, 105). It is evident from these observations that AMC in the developing brain are in constant surveillance to scavenge local cellular debris and exogenous substances that may gain access into the brain tissues; hence, their protective role at a stage when the BBB is not fully established.

6.3. Antigen presentation

Although the CNS, under normal conditions, has been considered an immunologically privileged site for a long time, our studies showed for the first time that major histocompatibility class (MHC) I antigens were expressed by AMC (106, 107). MHC antigens are surface molecules required for the participation of macrophages in the activation of T lymphocytes by presenting certain antigens to them. MHC I antigens serve as restriction elements for cytotoxic/suppressor lymphocytes. The expression of these antigens on AMC was related to their phagocytic activity. The presence of MHC I antigens on AMC also suggested that these cells were ready to interact with infiltrating lymphocytes as the BBB is immature in the developing brain and a potential immune damage in early development may be present (108).

MHC class II (MHC II) antigens, required for interaction with helper/inducer T lymphocytes, on the other hand, are not expressed by AMC under normal conditions. The expression of MHC II is induced under pathological and experimental conditions (106). For example, these antigens are expressed when the cells are challenged with lipopolysaccharide (LPS) or with interferon-gamma (IFN-gamma) (106). MHC II expression on AMC was also induced on the introduction of live E coli in their vicinity (102, 108). The expression of these antigens on AMC under pathological conditions suggests that they have the capability of interacting with helper/inducer cells to mount a potential immune response.

6.4. Proliferation

Although microglial population remains relatively static in the adult brain (57), its population has a remarkable capacity to expand in response to injury or neurological disease processes (109, 110). This expansion has been considered predominantly to be due to proliferation of activated microglia and to a lesser extent migration of microglia from adjacent brain areas (109, 110). Indeed, proliferating microglia have been implicated in the onset and/or progression of a number of CNS pathological conditions such as trauma (111), ischemia (112), Parkinson's disease (113) and demyelination disorders (114). These cells respond to injury may participate in brain repair and functional recovery by phagocytosis of debris. However, they may also contribute to glial scar formation and excess release of cytotoxic factors (115). A variety of molecules such as IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, and colony stimulating factor (CSF)-1  have been demonstrated to be potent stimuli for microglia proliferation in vitro (116). Very interestingly, for all of these cytokines, microglia can express the corresponding receptors (116). In addition, all of the growth factors mentioned above can be produced locally in the CNS (117). Therefore, under pathological conditions, microglial proliferation may be induced by some cytokines mentioned above through autocrine and paracrine modes. Several in vivo studies using cell proliferation markers such as Ki67 and BrdU, have confirmed the hypothesis that microglia can proliferate in a developing brain environment (117).

6.5. Migration Microglia are capable of migrating toward damaged neural tissue to clear the debris of injury site when necessary (118). It has been reported that rapid increase in number of microglial cells at injury sites is partly due to recruitment from blood monocytes or migration from other CNS regions (119). The mechanisms inducing microglial migration to the injury sites are complex and poorly understood. Migration of microglia is modulated by chemokines released by themselves through autocrine or paracrine fashion during injury and infection (118). Cytoskeleton proteins such as actin and tubulin are closely related to cellular migration. Microglia have a perinuclear distribution of actin and tubulin in the resting state and lack membrane localization of these proteins (120). When cells are treated with a chemotactic stimulus, rearrangement of actin and tubulin occurs to facilitate the process of attachment, protrusion and traction that allows microglia to migrate (120). Chemokine receptors can also be redistributed when microglia acquire a migratory phenotype (121). Our studies have shown that the cell number of AMC significantly increased in the PWM of hypoxic postnatal rats (101). Very interestingly, there was no significant difference in the proliferation rate of microglia after hypoxic exposure when compared with the corresponding control rats (101). The results suggested that the increase in AMC numbers soon after hypoxic exposure was primarily due to the migration of AMC from the neighboring areas of the brain or from invasion of their precursor cells, namely, circulating monocytes into the postnatal hypoxic brain (101).

6.6. Release of cytokines and chemokines

A plethora of cytokines and chemokines is usually expressed or secreted by microglia under inflammatory conditions. They are implicated in microglial communication and effector network system with other cell types (122). Cytokines and chemokines secreted by microglia participate in innate defense mechanisms, help initiation of immune responses, modulate the recruitment of leukocytes into the CNS, and contribute to tissue repair and recovery (122).

6.6.1. Tumor necrosis factor-alpha and its receptors

TNF-alpha is one of the most important proinflammatory cytokines with pleiotropic functions, secreted by microglia as well as blood-derived macrophages during CNS inflammation (123). In microglia culture, synthesis and release of TNF-alpha is induced by pathogens or pathogen components such as LPS and IFN-gamma (78). TNF- alpha can amplify CNS inflammatory response by inducing expression of chemokines and adhesion molecules in cerebrovascular endothelial cells and astrocytes, which help in recruitment of leukocytes into the CNS (124). In addition, TNF- alpha is a multipotent inflammatory cytokine that initiates various responses such as apoptosis in some cells and proliferation in others through activation of its different receptors (124). Therefore, it is possible that TNF- alpha plays a wide variety of roles in brain damage and repair.

All known effects of TNF- alpha are mainly completed by binding to one of two different receptors, designated TNF-R1 (p55) and TNF-R2 (p75), which are differentially expressed on various cell types in physiological and pathological conditions (125). The extracellular ligand-binding domains of TNF receptors show sequence homology containing cysteine-rich subdomains (126). On the contrary, the intracellular domains of TNF receptors have no sequence homology and are in lack of intrinsic enzyme activity, and transduce distinct biological signals by interaction with different cytosolic protein complexes (126). The TNF-R1 contains death domain, but TNF-R2 does not. The distinction in TNF-R1 and TNF-R2 constitution leads to their diverse functions. In general, the cell apoptotic process and pro-inflammatory response are largely mediated through TNF-R1. Therefore, the activation of TNF-R1 is frequently associated with tissue or organ injury (126). The consequences of TNF-R2 activation are not fully known, but it is known to mediate cell proliferation and promote tissue repair and angiogenesis (126). It has been found that the AMC produce TNF- alpha via the MAP kinase signaling pathway and expression of TNF-R1 in the oligodendrocytes increased significantly in the PWM in the hypoxic neonatal brain (24). This may be correlated to increased oligodendrocyte apoptosis via TNF-R1 in hypoxic injuries (24).

6.6.2. Interleukin-1 and its receptors

IL-1 includes two mediators, IL-1 alpha and IL-1 beta, which have less than 30% sequence homology but similar three-dimensional structure and biological functions (127). The action of IL-1 is modulated by different mechanisms including a true receptor (IL-1RI), a decoy (IL-1RII), and a specific receptor antagonist (IL-1ra) (127). Although IL-1 alpha and IL-1beta can bind equally to type I and type II receptors, signal transduction across the plasma membrane has been reported to be accomplished only via the type I receptor (127). At present, there is no evidence that a signal can be transmitted through the type II receptor. In the CNS, IL-1 is expressed at high levels during prenatal and postnatal development, and its expression declines to low constitutive levels in the normal adult and markedly increases after injury (128). Astroglia and microglia possess IL-1 receptors and are the main intrinsic sources of IL-1 in the CNS, but oligodendroglial cells also produce IL-1 and express IL-1 receptors (128). IL-1 induces activation and secretion of multiple factors by microglia and astrocytes and it has been reported by a recent study that remyelination is dramatically reduced in the absence of IL-1beta, but the effects of this cytokine on the oligodendroglial lineage remain to be elucidated (128). We have demonstrated that AMC expressed IL-1beta and the expression of IL-1RI on the oligodendrocytes was increased significantly in hypoxic rats (24). Unlike TNF- alpha, IL-1beta was reported as being non toxic to oligodendrocyte lineage cells as oligodendrocyte apoptosis was not induced through this receptor. However, some studies have demonstrated that IL-1beta can block oligodendrocyte proliferation at the late progenitor/pro-oligodendrocyte stage (128). Based on these results, it is postulated that IL-1beta produced by AMC in hypoxic conditions may delay the white matter development and recovery in hypoxic conditions via inhibition of oligodendrocyte progenitor proliferation (24).

6.6.3. Macrophage colony-stimulating factor

The macrophage colony-stimulating factor (M-CSF), also designated as CSF-1, is a hematopoietic cytokine that induces proliferation, migration, and activation of microglia and is released mainly by macrophages, T cells, B cells and microglia (129). M-CSF has also been identified as an important mediator of the inflammatory response and can also regulate the release of proinflammatory cytokines from macrophages (130). M-CSF exerts its pleiotropic effects by binding to its high-affinity receptor CSF-1R that is encoded by the c-fms proto-oncogene (131). Our recent study has shown that M-CSF produced by AMC in the early phase following hypoxic injury may activate astrocytes and regulate the release of proinflammatory cytokines from them (67). This may then sustain and exacerbate the inflammatory response in the PWM of hypoxic neonatal rats in the late phase.

6.6.4. Monocyte chemoattractant protein-1

Among various chemokines, monocyte chemoattractant protein-1 (MCP-1), (also known as CCL2), a member of beta-chemokine subfamily, mediates the migration of microglia, monocytes and lymphocytes to the inflammation sites in the CNS (132, 133, 134). It is produced mainly by microglia and astrocytes (135). MCP-1 acts on its targets by binding to its receptor, CCR2 which is a seven-transmembrane domain G-protein coupled receptor. The expression of MCP-1 and CCR2 has been shown to be induced following diverse CNS insults, including ischemia (136, 137), Alzheimer's disease (138), HIV type-1-associated dementia (139), multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE) (140, 141, 142). In EAE animal model, MCP-1 induced the recruitment and activation of endogenous microglia and blood-derived macrophages to demyelinated areas, promoting myelin phagocytosis (143). Moreover, the functional antagonism of MCP-1 attenuates leukocyte infiltration and decreases the severity of CNS injury (144, 145). In murine stroke model, MCP-1 deficiency has been shown to have a protective role in acute infarct growth (146). Our previous study has shown that under hypoxic conditions, activated microglia generate MCP-1 that would induce the migration of microglial cells themselves either in an autocrine or paracrine fashion to PWM in the neonatal rats, since these cells express the MCP-1 receptor, namely, CCR2 (101). It has been reported that through binding with its receptor CCR2, MCP-1 induces changes in actin polymerization and subsequent reorganization of the actin cytoskeleton, formation of focal adhesions and pseudopod extension which contributes to the microglial migration when activated (147).

6.7. Expression of ion channels

It is well documented that microglia can express a large number of cell surface receptors and ion channels when they are activated under pathological conditions (148, 149). These ion channels or receptors can transduce extracellular stimuli into an intracellular cascade response through activation of signaling pathways (150, 151). Microglia express several types of ion channels such as Na+, K+, Cl- and Ca2+ channels, which confer different functions on these cells.

6.7.1. Kv1.2 and Kv1.1

Microglia are known to exhibit only inwardly rectifying K+ channels (152). The existence of delayed rectifier channels in microglial cells has been demonstrated in cultured cells derived from different species (153). The outward potassium currents caused by delayed rectifying channels appears to be highly correlated with the activation of the microglial cells (154). We reported recently that Kv1.2 is vigorously expressed in the AMC at 1-7 postnatal days but it is barely detected in the ramified microglial cells at 14 postnatal days under physiological conditions (154). Arising from this, it was suggested that AMC are endowed with an electrophysiological characteristic distinct from the ramified microglia. It was speculated that the vigorous expression of Kv1.2 in AMC in early postnatal period may be related to their active secretion of proinflammatory cytokines and other factors such as insulin-like growth factors and endothelins (155, 156). The diminution of Kv1.2 expression in ramified microglia suggested that they may be less active in their secretory function.

It has been reported that Kv1 channel transcription is influenced by oxygen supply (157). Kv1.2 is an isoform of the delayed rectifier potassium channel and its α-subunit is sensitive to oxygen (158). Therefore, it was suggested that Kv1.2 in microglia is also sensitive to oxygen supply and it may take part in the hypoxia induced outward potassium rectifier especially during cell activation (158). This is supported by the fact that Kv1.2 expression in microglia was augmented in postnatal rats and in BV-2 cells (a murine microglia cell line) subjected to hypoxia (154). In this connection, increase in Kv1.2 expression was accompanied by a decline in intracellular potassium coupled by an increased expression of TNF-alpha and IL-1beta (154). Blockade of Kv1.2 with Tityustoxin (TsTx) reversed the above-mentioned process but remarkably it also depressed the intracellular ROS (154). It is, therefore, suggested that Kv1.2 constitutively expressed in microglia in the hypoxic developing brain may regulate the release of proinflammatory cytokines and reactive oxygen species (ROS) production through changing the intracellular potassium concentration. Likewise, a recent study demonstrated that Kv1.1, constitutively expressed by microglia, is elicited by hypoxia and this may be linked to production of proinflammatory cytokines, endothelins and NO (159). In the light of the above, it was proposed that Kv1.2 and Kv1.1 play an important role in inflammatory response elicited by activated microglia in the hypoxic developing brain.

6.7.2. Sodium channels

Voltage-gated sodium channels are traditionally viewed as being expressed on some excitable cells such as neurons and muscle cells (160). They are mainly responsible for the initiation and propagation of action potential (160). However, it has been proven that sodium channels are also expressed in some nonexcitable cells and contribute to cellular functions (161). As mentioned above, microglia are activated under pathological conditions, which result in morphological transformation, proliferation, enhanced migration, phagocytosis, secretion of inflammatory mediators and antigen presentation (122, 162). At present, it has been found that tetrodotoxin (TTX)-sensitive Nav1.1, Nav1.6 and TTX-resistant Nav1.5 are detected in primary microglia in vitro (163); on the other hand, Nav1.2, Nav1.3, Nav1.7, Nav1.8 and Nav1.9 are not present in microglia. Sodium channel blockade with phenytoin (40uM) and TTX (0.3uM) significantly attenuated the phagocytic activity of microglia by 50-60% when they were activated with LPS (163). Sodium channel blockade with phenytoin significantly reduced IL-1alpha, IL-1beta, TNF-alpha secretion by LPS-activated microglia. Phenytoin and TTX also significantly decreased adenosine triphosphate-induced migration of microglia by 50% (163). Furthermore, Nav1.6 plays a prominent role in microglial migration. These studies demonstrate that the activity of sodium channels contributes to phagocytosis, migration and secretion of inflammatory mediators by activated microglia (163). It remains to be ascertained if the expression of sodium channels in microglia is upregulated in the hypoxic developing brain and, if so, whether it would contribute to above-mentioned processes.

6.8. Syndecan-2

Syndecans (Sdcs) are transmembrane heparan sulphate proteoglycans consisting of syndecan-1,-2,-3, and -4 (Sdc-1,-2,-3 and -4). Heparan sulphate glycosaminoglycan side chains, which have affinity for a wide variety of secreted molecules and extracellular matrix components, mediate the extracellular functions of Sdcs (164, 165, 166). Sdc-2 is a cell surface heparan sulphate proteoglycan consisting of a short cytoplasmic domain, a single transmembrane domain and a large ectodomain with three covalently attached heparan sulphate chains close to the N-terminus (167). It is expressed on fibroblasts (168), endothelial cells (169), primary osteoblasts (170) and in activated macrophages (171). The various members of the Sdc family have been implicated in a number of biological processes such as cell adhesion and signal transduction (172, 173). It is well documented that heparan sulphate chains interact with many growth factors, cytokines, chemokines, and extracellular matrix molecules relevant to vascular development and repair, hypoxia, angiogenesis, and immune cell function. It also regulates key mechanisms of the host inflammatory response (174).

A number of studies have documented that hypoxia activates macrophages and other inflammatory cells (175) and induces inflammatory responses in many tissues (176). Macrophages regulate many processes associated with injury and repair which can affect the synthesis of a wide variety of extracellular matrix proteins such as fibronectins (177) and proteoglycans (178). Both monocytes and macrophages express Sdc-1, Sdc-4, (179) and Sdc-2 (171) and secrete a variety of cytokines such as IL-1, transforming growth factor-beta and TNF-alpha. Changes in expression of sulphated proteoglycans are associated with activation of macrophages (180). Sdcs have been implicated as key modulators of inflammation in many diseases reflecting the capacity of heparan sulphate proteoglycans to regulate a wide variety of inflammatory mediators and their processes (174). A recent study has shown that the expression of Sdc-2 was increased significantly in the AMC in the PWM of the rat developing brain following a hypoxic exposure (181). Furthermore, it has been further shown that Sdc-2 could augment the hypoxia-induced proinflammatory mediators secretion and ROS production (181). Therefore, a proinflammatory role of Sdc-2 in the hypoxic developing brain was suggested as it potentiated activated microglia in releasing more cytokines, chemokines and ROS (181).

6.9. Generation of reactive oxygen species and nitrogen intermediates

Microglia can generate ROS when activated by various stimuli such as hypoxia and LPS. Nicotinamide adenine dinucleotide phosphate hydrogenase (NADPH) oxidase, which is involved in production of microglial-derived ROS (182), is a pivotal enzyme which catalyses the production of superoxide from the oxygen. In addition to the production of ROS, NADPH oxidase is associated with microglial signaling pathway related to inflammatory response (183). NADPH oxidase-generated intracellular ROS can augment the inflammatory response through effects on kinase cascades and transcription factor activation (183). ROS can also strengthen phagocytosis by microglia by supporting the degradation of the ingested antigens and cellular debris (183). However, excessive intracellular ROS leads to microglial apoptosis. Some studies have shown that the overactivation of NADPH oxidase and the dysregulation of intracellular ROS in microglia are involved in the pathogenesis of some neurodegenerative diseases (184).

NO, an important free radical, is synthesized by the enzyme NOS from L-arginine. NOS from neurons (nNOS) and endothelial cells (eNOS) are constitutively expressed enzymes, the activities of which are stimulated by increase in intracellular calcium. Inducible NOS (iNOS) is calcium-independent, and NO generated from this isoform is known to mediate immune functions. NO possesses a diverse array of physiologic functions, such as muscle relaxation, immune modulation, and neuronal activity (185). NO directly reacts with guanylate cyclase, cytochrome P450, cyclooxygenase, and hemoglobin, to modulate their functions. In addition, NO can react with membrane lipids and induce lipid peroxidation. Indirectly, the combination of NO and superoxide can form highly reactive intermediates, such as peroxynitrite, that can induce DNA strand breaks, lipid peroxidation, and protein nitration (186). In the postnatal brain, iNOS is not normally expressed but can be expressed by microglia in response to hypoxic stimuli (31, 187). As described above, activated microglial cells in the PWM may contribute to perinatal brain injury through secretion or production of proinflammatory cytokines such as TNF-alpha and IL-1beta. Excessive production of NO from iNOS has been reported to be toxic to the oligodendrocytes and may be of significance in the development of PWMD as it has been implicated in damaging myelin-producing oligodendrocytes (188) and, hence, delayed myelination. In vitro studies have shown that NO produced by the activated microglial cells is highly damaging to the oligodendrocytes causing their lysis (189).

7. CONCLUSIONS

Hypoxia induces severe and persistent inflammatory response in the developing brain. Overproduction of inflammatory cytokines, free radicals and NO has been implicated in this inflammatory response that results in neuronal loss, damage to the axons and death of oligodendrocytes along with myelination disturbances (Fig.1). Microglial cells are the main cellular source of the inflammatory mediators and culprits of hypoxia-induced injury to the oligodendrocytes and axons. Our recent studies have shed some light on the cross-talk between microglial cells, astrocytes, oligodendrocytes and axons, which may be involved in the pathogenesis of hypoxic brain injury in the neonatal period. Undoubtedly, a better understanding of the above processes will help to develop potential preventive and therapeutic strategies for mitigation of hypoxia induced brain injury.

8. ACKNOWLEDGEMENTS

This study was supported by research grant (R181-000-065-112) from the National University of Singapore

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Abbreviations: AMC: amoeboid microglial cells,  AMPA: alpha-amino-3-hydroxy 5-methyl-4-isoxazolepropionic acid,  BBB: blood - brain barrier,  CNS: central nervous system,  CSF-1: colony stimulating factor-1,  CSF-1R: colony stimulating factor-1 receptor,  EAE: experimental autoimmune encephalomyelitis,  eNOS: endothelial nitric oxide synthase,  GFAP: glial fibrillary acidic protein,  GM-CSF: granulocyte-macrophage colony stimulating factor,  IFN- gamma: interferon- gamma, IL-1beta: interleukin-1ß, IL-1R1: IL-1 receptor 1,  IL-2: interleukin-2, IL-6: interleukin-6, iNOS: inducible nitric oxide synthase,  LPS: lipopolysaccharide,  MBP: myelin basic protein, MHC: major histocompatibility class,  MCP-1: monocyte chemoattractant protein-1,  M-CSF: macrophage colony-stimulating factor,  NADPH: nicotinamide adenine dinucleotide phosphate hydrogenase,  NMDA: N-methyl-d-aspartate,  nNOS: neuronal nitric oxide synthase,  NO: nitric oxide, PWM: periventricular white matter,  PWMD: periventricular white matter damage,  ROS: reactive oxygen species,  Sdcs: syndecans, TNF-alpha: tumor necrosis factor-alpha,  TNF-R1: TNF receptor 1,  VEGF: vascular endothelial growth factor 

Key Words: Hypoxia, Periventricular White Matter Damage, Microglia, Inflammation, Review

Send correspondence to: Charanjit Kaur, Department of Anatomy, Yong Loo Lin School of Medicine, MD Blk 10, 4 Medical Drive, National University of Singapore, Singapore 117597, Tel: 65-65163209, Fax: 65-67787643, E-mail:antkaurc@nus.edu.sg