The NADPH oxidase Nox2 regulates VEGFR1/CSF‐1R‐mediated microglial chemotaxis and promotes early postnatal infiltration of phagocytes in the subventricular zone of the mouse cerebral cortex

The phagocyte NADPH oxidase Nox2 generates superoxide ions implicated in the elimination of microorganisms and the redox control of inflammatory signaling. However, the role of Nox2 in phagocyte functions unrelated to immunity or pathologies is unknown. During development, oriented cell migrations insure the timely recruitment and function of phagocytes in developing tissues. Here, we have addressed the role of Nox2 in the directional migration of microglial cells during development. We show that microglial Nox2 regulates the chemotaxis of purified microglia mediated by the colony stimulating factor‐1 receptor (CSF‐1R) and the vascular endothelial growth factor receptor‐1 (VEGFR1). Stimulation of these receptors triggers activation of Nox2 at the leading edge of polarized cells. In the early postnatal stages of mouse brain development, Nox2 is activated in macrophages / microglial cells in the lateral ventricle or the adjacent subventricular zone (SVZ). Fluorescent microglia injected into the lateral ventricle infiltrate the dorso‐caudal SVZ through a mechanism that is blocked by pretreatment of the injected cells with an irreversible Nox inhibitor. Infiltration of endogenous microglia into the caudal SVZ of the cerebral cortex is prevented by (1) Nox2 gene deficiency, (2) treatment with a Nox2 inhibitor (apocynin), and (3) invalidation of the VEGFR1 kinase. We conclude that phagocytes move out of the lateral ventricle soon after birth and infiltrate the cortical SVZ through a mechanism requiring microglial Nox2 and VEGFR1 activation. Nox2 therefore modulates the migration of microglia and their development.     

Microglial repopulation resolves inflammation and promotes brain recovery after injury

Microglia mediate chronic neuroinflammation following central nervous system (CNS) disease or injury, and in doing so, damage the local brain environment by impairing recovery and contributing to disease processes. Microglia are critically dependent on signaling through the colony‐stimulating factor 1 receptor (CSF1R) and can be eliminated via administration of CSF1R inhibitors. Resolving chronic neuroinflammation represents a universal goal for CNS disorders, but long‐term microglial elimination may not be amenable to clinical use. Notably, withdrawal of CSF1R inhibitors stimulates new microglia to fully repopulate the CNS, affording an opportunity to renew this cellular compartment. To that end, we have explored the effects of acute microglial elimination, followed by microglial repopulation, in a mouse model of extensive neuronal loss. Neuronal loss leads to a prolonged neuroinflammatory response, characterized by the presence of swollen microglia expressing CD68 and CD45, as well as elevated levels of cytokines, chemokines, complement, and other inflammatory signals. These collective responses are largely resolved by microglial repopulation. Furthermore, microglial repopulation promotes functional recovery in mice, with elevated plus maze performance matching that of uninjured mice, despite the loss of 80% of hippocampal neurons. Analyses of synaptic surrogates revealed increases in PSD95 and synaptophysin puncta with microglial repopulation, suggesting that these cells sculpt and regulate the synaptic landscape. Thus, our results show that short‐term microglial elimination followed by repopulation may represent a clinically feasible and novel approach to resolve neuroinflammatory events and promote brain recovery.     

P2X7 receptor differentially modulates astroglial apoptosis and clasmatodendrosis in the rat brain following status epilepticus

Recently, it has been reported that astroglial loss/dysfunction plays a role in epileptogenesis. In addition, astroglial loss is accompanied by up‐regulation of P2X7 receptor expression in microglia. Therefore, we investigated whether P2X7 receptor is involved in astroglial damages induced by status epilepticus (SE). In the present study, astroglial loss showed the regional‐specific manner and the differential responses to P2X7 receptor functions. Both OxATP and brilliant blue G (P2X7 receptor antagonists) infusion prevented apoptotic astroglial loss in the molecular layer of the dentate gyrus and the frontoparietal cortex, while it promoted clasmatodendrosis in the CA1 region as compared to saline treatment. In contrast, BzATP (a P2X7 receptor agonist) treatment exacerbated apoptotic astroglial loss in the molecular layer of the dentate gyrus and the frontoparietal cortex, but alleviated SE‐induced astroglial swelling in the CA1 region. Astroglial loss in the piriform cortex was not affected by P2X7 receptor agonist‐ or antagonist‐infusion. These findings suggest that P2X7 receptor function differently modulates SE‐induced astroglial loss in distinct brain regions. © 2010 Wiley Periodicals, Inc.     

In vitro proliferation of axotomized rat facial nucleus-derived activated microglia in an autocrine fashion

Transection of rat adult facial nerve leads to an increase in the number of activated microglia in the facial nucleus (FN), with a peak in proliferation 3 days after transection. To investigate the characteristics of these activated microglia, we isolated the cells with high purity from axotomized FN (axFN) 3 days after transection according to the previously reported procedure for explant culture. The isolated microglia exhibited immunocytochemical properties similar to those in vivo, and their numbers increased approximately five- to sevenfold over a period of 10 days without the addition of any mitogens, suggesting that self-reproduction was occurring. Actually, the microglia actively incorporated bromodeoxyuridine (BrdU) and strongly expressed an S-phase-specific protein marker, proliferating cell nuclear antigen (PCNA). To examine the mechanism underlying this proliferation, the expression of the mitogens and specific receptors of the microglia were analyzed in conditioned medium (CM) and cells. Macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage-CSF (GM-CSF) were detected in the CM as well as in the cells. Their specific receptor proteins, c-Fms and GMCSFRalpha, were also detected in the cell homogenate. These proliferating microglia were not found to produce deleterious factors for neurons. In summary, the microglia isolated from the axFN were found to be proliferative in an autocrine fashion and to have some cellular properties in common with those observed in vivo.     

Microglial cell activation and proliferation precedes the onset of CNS autoimmunity

Microglial cells are central nervous system (CNS) resident cells that are thought to become activated and contribute to the inflammation that occurs in the human autoimmune disease multiple sclerosis (MS). This has never been proven, however, because microglial cells cannot be phenotypically distinguished from peripheral macrophages that accumulate in MS inflammatory lesions. To study the kinetics and nature of microglial cell activation in the CNS, we used the animal model of MS, experimental autoimmune encephalomyelitis (EAE), and induced EAE in bone marrow (BM) chimera mice generated using major histocompatibility complex (MHC)-mismatched donor BM, allowing the separation of microglial cells and peripheral monocytes/macrophages. We found that microglial cell activation was evident before onset of disease symptoms and infiltration of peripheral myeloid cells into the CNS. Activated microglial cells underwent proliferation and upregulated the expression of CD45, MHC class II, CD40, CD86, and the dendritic cell marker CD11c. At the peak of EAE disease, activated microglial cells comprised 37% of the total macrophage and dendritic cell populations and colocalized with infiltrating leukocytes in inflammatory lesions. Our findings thus definitively demonstrate that during EAE, microglial cells become activated early in EAE disease and then differentiate into both macrophages and dendritic-like cells, suggesting they play an active role in the pathogenesis of EAE and MS.     

Caspase-mediated death of newly formed neurons in the adult rat dentate gyrus following status epilepticus

A large proportion of cells that proliferate in the adult dentate gyrus under normal conditions or in response to brain insults exhibit only short-term survival. Here, we sought to determine which cell death pathways are involved in the degeneration of newly formed neurons in the rat dentate gyrus following 2 h of electrically induced status epilepticus. We investigated the role of three families of cysteine proteases, caspases, calpains, and cathepsins, which can all participate in apoptotic cell death. Status epilepticus increased the number of bromodeoxyuridine (BrdU)-positive proliferated cells in the subgranular zone of the dentate gyrus. At the time of maximum cell proliferation, immunohistochemical analyses revealed protein expression of active caspase-cleaved poly (ADP-ribose) polymerase (PARP) in approximately 66% of the BrdU-positive cells, while none of them expressed cathepsin B or the 150-kDa calpain-produced fodrin breakdown product. To evaluate the importance of cysteine proteases in regulating survival of the newly formed neurons, we administered intracerebroventricular infusions of a caspase inhibitor cocktail (zVAD-fmk, zDEVD-fmk and zLEHD-fmk) over a 2-week period, sufficient to allow for neuronal differentiation, starting 1 week after the epileptic insult. Increased numbers of cells double-labelled with BrdU and neuron-specific nuclear protein (NeuN) marker were detected in the subgranular zone and granule cell layer of the caspase inhibitor-treated rats. Our data indicate that caspase-mediated cell death pathways are active in progenitor cell progeny generated by status epilepticus and compromise survival during neuronal differentiation.     

The effects of C5aR1 on leukocyte infiltration following pilocarpine‐induced status epilepticus

This study aimed to determine the role C5aR1 plays in mediating immune responses acutely after pilocarpine‐induced status epilepticus (SE), specifically those of brain‐infiltrating leukocytes. Three days following pilocarpine SE, we determined by flow cytometry the brain immune cell phenotypes and measured key proinflammatory and antiinflammatory cytokine expression by infiltrating leukocytes and microglia in C5aR1‐deficient and wild‐type mice. Absence of C5aR1 reduced by 47% the numbers of Ly6G⁺ neutrophils in the brains of No‐SE mice and decreased neutrophil entry after SE to levels found in wild‐type brains that did not undergo SE (No‐SE). Moreover, C5aR1‐deficient mice showed increased interleukin (IL)‐4 expression in infiltrating leukocytes, but not in microglia. Increases in IL‐4 expression in infiltrating leukocytes coupled with decreased neutrophil invasion in C5aR1‐deficient mice after SE is likely to contribute to the reduced neuronal loss previously found in these mice compared to their wild‐type littermates. Although other SE models need to be investigated to substantiate our findings, this study provides further evidence that C5aR1 is an inflammatory mediator and may play a role in epileptogenesis.     

Resistance of immature hippocampus to morphologic and physiologic alterations following status epilepticus or kindling.

Seizures in adult rats result in long-term deficits in learning and memory, as well as an enhanced susceptibility to further seizures. In contrast, fewer lasting changes have been found following seizures in rats younger than 20 days old. This age-dependency could be due to differing amounts of hippocampal neuronal damage produced by seizures at different ages. To determine if there is an early developmental resistance to seizure-induced hippocampal damage, we compared the effects of kainic acid (KA)-induced status epilepticus and amygdala kindling on hippocampal dentate gyrus anatomy and electrophysiology, in immature (16 day old) and adult rats. In adult rats, KA status epilepticus resulted in numerous silver-stained degenerating dentate hilar neurons, pyramidal cells in fields CA1 and CA3, and marked numerical reductions in CA3c pyramidal neuron counts (-57%) in separate rats. Two weeks following the last kindled seizure, some, but significantly less, CA3c pyramidal cell loss was observed (-26%). Both KA status epilepticus and kindling in duced mossy-fiber sprouting, as evidenced by ectopic Timm staining in supragranular layers of the dentate gyrus. In hippocampal slices from adult rats, paired-pulse stimulation of perforant path axons revealed a persistent enhancement of dentate granule-cell inhibition following KA status epilepticus or kindling. While seizures induced by KA or kindling in 16-day-old rats were typically more severe than in adults, the immature hippocampus exhibited markedly less KA-induced cell loss (-22%), no kindling-induced loss, no detectable synaptic rearrangement, and no change in dentate inhibition. These results demonstrate that, in immature rats, neither severe KA-induced seizures nor repeated kindled seizures produce the kind of hippocampal damage and changes associated with even less severe seizures in adults. The lesser magnitude of seizure-induced hippocampal alterations in immature rats may explain their greater resistance to long-term effects of seizures on neuronal function, as well as future seizure susceptibility. Conversely, hippocampal neuron loss and altered synaptic physiology in adults may contribute to increased sensitivity to epileptogenic stimuli, spontaneous seizures, and behavioral deficits.     

Biolistic expression of the macrophage colony stimulating factor receptor in organotypic cultures induces an inflammatory response

The receptor for macrophage colony-stimulating factor (M-CSFR; c-fms) is expressed at increased levels by microglia in Alzheimer's disease (AD) and in mouse models for AD. Increased expression of M-CSFR on cultured microglia results in a strong proinflammatory response, but the relevance of this cell culture finding to intact brain is unknown. To determine the effects of increased microglial expression of M-CSFR in a complex organotypic environment, we developed a system for biolistic transfection of microglia in hippocampal slice cultures. The promoter for the Mac-1 integrin alpha subunit CD11b is active in cells of myeloid origin. In the brain, CD11b expression is restricted to microglia. Constructs consisting of the promoter for CD11b and a c-fms cDNA or an enhanced green fluorescent protein (EGFP) cDNA were introduced into monotypic cultures of microglia, neurons, and astrocytes. Strong CD11b promoter activity was observed in microglia, whereas little activity was observed in other cell types. Biolistic transfection of organotypic hippocampal cultures with the CD11b/c-fms construct resulted in expression of the c-fms mRNA and protein that was localized to microglia. Furthermore, biolistic overexpression of M-CSFR on microglia resulted in significantly increased production by the hippocampal cultures of the proinflammatory cytokines interleukin (IL)-1alpha macrophage inflammatory protein (MIP-1alpha), and trends toward increased production of IL-6 and M-CSF. These findings demonstrate that microglial overexpression of M-CSFR in an organotypic environment induces an inflammatory response, and suggest that increased microglial expression of M-CSFR could contribute to the inflammatory response observed in AD brain.     

Induction of neonatal sodium channel II and III alpha-isoform mRNAs in neurons and microglia after status epilepticus in the rat hippocampus

Sodium channels (NaChs) regulate neuronal excitability in both physiological and pathological conditions, including epilepsy and are therefore an important target for antiepileptic drugs. In the present study, we examined the distribution of mRNAs encoding neonatal NaChs II and III alpha-isoforms in control rat hippocampus and after electrically-induced status epilepticus (SE), using nonradioactive in situ hybridization (ISH). Only weak expression of neonatal NaCh II and III mRNAs was observed in control hippocampus. By contrast, increased expression of neonatal NaCh II and III mRNAs was observed 4 h after the induction of SE in neurons of CA1-CA3 and the dentate granule cell layer. These changes were detected only in rats in which SE was successfully induced and persisted, although less intense, for up to 3 months, when rats display spontaneous seizures. Strong expression of neonatal NaCh alpha-isoforms was observed 1 week after SE in microglial cells, as confirmed by double labelling, combining ISH with immunocytochemistry for microglia markers. The increased expression of neonatal isoforms of the NaCh in both neurons and microglial cells may represent a critical mechanism for modulation of neuronal excitability, glial function and pharmacological response to antiepileptic drugs in the course of epileptogenesis.     

Memory impairment following status epilepticus in immature rats: time-course and environmental effects

Status epilepticus (SE) has a high mortality and morbidity rate in children. Disturbances in learning and memory are frequently associated with SE although it is not clear when the cognitive deficits occur. If cognitive dysfunction occurs immediately following the seizure, the window of opportunity for therapeutic intervention is limited. The first goal of this study was to determine the timing of cognitive dysfunction following SE in weanling rats. As there is evidence that enriching the environment can improve cognitive and motor deficits following brain injury, our second goal was to determine whether environmental enrichment improves cognitive function following SE. Rats underwent lithium-pilocarpine-induced SE at postnatal (P) day 20 and were then tested for visual-spatial memory in the water maze at P22, P25, P30, or P50. Rats with SE performed significantly worse in the water maze than control rats at all time points. Once the time-courses of visual-spatial memory deficits were determined, a second group of P20 rats were subjected to SE and were then placed in an enriched environment (enriched group) or remained in standard cages in the vivarium (nonenriched group) for 28 days. Following environmental manipulation, the animals were tested in the water maze. Rats housed in an enriched environment following the SE performed substantially better in the water maze than rats housed in standard cages. However, no differences were found between the enriched and nonenriched groups in EEG or histological evaluation. Although SE results in cognitive impairment within days of the seizure, housing in an enriched environment after SE has a beneficial effect on cognitive performance in rats.     

Antisense oligonucleotide to GABAA receptor γ2 subunit induces limbic status epilepticus

γ-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the brain. A deficiency of GABAergic inhibition mediated via the GABA_A receptor complex has for a long time been suspected to be a central factor in epileptogenesis. Status epilepticus is a condition of sustained and prolonged excitation of neuronal circuits, as detected by epileptiform discharges in the electroencephalogram (EEG). Reduction of GABA_A receptor-mediated hippocampal inhibition has been implicated in the development of status epilepticus. The present study provides direct evidence of a link between the GABA_A receptor and epilepsy. We show that selective inhibition of the expression of the GABA_A receptor γ2 subunit in the rat hippocampus by means of antisense oligonucleotides leads to spontaneous electrographic seizures that evolve into profound limbic status epilepticus, ultimately resulting in severe neurodegenerative changes. Concurrent treatment with diazepam prevents the development of status epilepticus and markedly reduces neuronal cell loss. These findings strongly support the hypothesis that the GABA_A receptor is critically involved in the pathogenesis of seizures and status epilepticus. J. Neurosci. Res. 54:863–869, 1998. © 1998 Wiley-Liss, Inc.     

脑炎后癫痫持续状态进展为难治性癫痫持续状态及超级难治性癫痫持续状态的早期预测因素

目的 探讨脑炎后癫痫持续状态(SE)进展为难治性SE(RSE)及超级RSE(SRSE)的早期预测因素.方法 根据疾病进展情况将89例脑炎后SE患者分为非RSE组、RSE组及SRSE组.比较各组临床资料.结果 非RSE组、RSE组及SRSE组年龄、SE严重程度评分量表(STESS)评分、基于流行病学SE病死率评分(EMS...     

Spatiotemporal characteristics of astroglial death in the rat hippocampo-entorhinal complex following pilocarpine-induced status epilepticus

Recently we reported that astroglial loss and subsequent gliogenesis in the dentate gyrus play a role in epileptogenesis following pilocarpine-induced status epilepticus (SE). In the present study we investigated whether astroglial damages in the hippocampo-entorhinal complex following SE are relevant to pathological or electrophysiological properties of temporal lobe epilepsy. Astroglial loss/damage was observed in the entorhinal cortex and the CA1 region at 4 weeks and 8 weeks after SE, respectively. These astroglial responses in the hippocampo-entorhinal cortex were accompanied by hyperexcitability of the CA1 region (impairment of paired-pulse inhibition and increase in excitability ratio). Unlike the dentate gyrus and the entorhinal cortex, CA1 astroglial damage was protected by conventional anti-epileptic drugs. alpha-Aminoadipic acid (a specific astroglial toxin) infusion into the entorhinal cortex induced astroglial damage and changed the electrophysiological properties in the CA1 region. Astroglial regeneration in the dentate gyrus and the stratum oriens of the CA1 region was found to originate from gliogenesis, while that in the entorhinal cortex and stratum radiatum of the CA1 region originated from in situ proliferation. These findings suggest that regional specific astroglial death/regeneration patterns may play an important role in the pathogenesis of temporal lobe epilepsy.     

Astroglial loss and edema formation in the rat piriform cortex and hippocampus following pilocarpine-induced status epilepticus

In the present study we analyzed aquaporin-4 (AQP4) immunoreactivity in the piriform cortex (PC) and the hippocampus of pilocarpine-induced rat epilepsy model to elucidate the roles of AQP4 in brain edema following status epilepticus (SE). In non-SE-induced animals, AQP4 immunoreactivity was diffusely detected in the PC and the hippocampus. AQP4 immunoreactivity was mainly observed in the endfeet of astrocytes. Following SE the AQP4-deleted area was clearly detected in the PC, not in the hippocampus. Decreases in dystrophin and α-syntrophin immunoreactivities were followed by reduction in AQP4 immunoreactivity. These alterations were accompanied by the development of vasogenic edema and the astroglial loss in the PC. In addition, acetazolamide (an AQP4 inhibitor) treatment exacerbated vasogenic edema and astroglial loss both in the PC and in the hippocampus. These findings suggest that SE may induce impairments of astroglial AQP4 functions via disruption of the dystrophin/α-syntrophin complex that worsen vasogenic edema. Subsequently, vasogenic edema results in extensive astroglial loss that may aggravate vasogenic edema.     

Granulocyte macrophage colony stimulating factor stimulates in vitro proliferation of astrocytes derived from simian mature brains

In the brain, granulocyte-macrophage colony stimulating factor (GM-CSF) may be released by infiltrated cells of the immune system including T and B lymphocytes and mononuclear phagocytes, but also by nervous system resident cells such as microglia and astrocytes. Astrocyte-secreted GM-CSF may play an important role in enhancing the local inflammatory response to central nervous system (CNS) injury and in recruting microglia and activated macrophages. In this study, we demonstrated that GM-CSF, as TNFα and IL 6, stimulates in vitro proliferation of simian astrocytes in primary cultures. Results were confirmed by blocking experiments performed with a specific neutralizing mAb directed against GM-CSF. Furthermore, we demonstrated that GM-CSF mediates its effect on these cells through the α subunit of the GM-CSF receptor which is constitutively expressed at the membrane of the cultured simian astrocytes as assessed by immunofluorescence. GM-CSF effects on astrocytes could be involved in astrocytosis, a hallmark of various neurological injuries and in inflammatory processes in an autocrine manner. © 1996 Wiley-Liss, Inc.     

Reactive microglia are the major source of tumor necrosis factor alpha and contribute to astrocyte dysfunction and acute seizures in experimental temporal lobe epilepsy

Extensive microglia reactivity has been well described in human and experimental temporal lobe epilepsy (TLE). To date, however, it is not clear whether and based on which molecular mechanisms microglia contribute to the development and progression of focal epilepsy. Astroglial gap junction coupled networks play an important role in regulating neuronal activity and loss of interastrocytic coupling causally contributes to TLE. Here, we show in the unilateral intracortical kainate (KA) mouse model of TLE that reactive microglia are primary producers of tumor necrosis factor (TNF)α and contribute to astrocyte dysfunction and severity of status epilepticus (SE). Immunohistochemical analyses revealed pronounced and persistent microglia reactivity, which already started 4 h after KA-induced SE. Partial depletion of microglia using a colony stimulating factor 1 receptor inhibitor prevented early astrocyte uncoupling and attenuated the severity of SE, but increased the mortality of epileptic mice following surgery. Using microglia-specific inducible TNFα knockout mice we identified microglia as the major source of TNFα during early epileptogenesis. Importantly, microglia-specific TNFα knockout prevented SE-induced gap junction uncoupling in astrocytes. Continuous telemetric EEG recordings revealed that during the first 4 weeks after SE induction, microglial TNFα did not significantly contribute to spontaneous generalized seizure activity. Moreover, the absence of microglial TNFα did not affect the development of hippocampal sclerosis but attenuated gliosis. Taken together, these data implicate reactive microglia in astrocyte dysfunction and network hyperexcitability after an epileptogenic insult.     

Cerebral hemiatrophy and hemiparesis following hemiclonic status epilepticus in Dravet syndrome

Dravet syndrome is currently considered as an developmental and epileptic encephalopathy and, recently, mandatory, alert, and exclusionary criteria have been proposed. Here, we describe three patients with Dravet syndrome with the typical early presentation including febrile and afebrile alternating hemiclonic seizures due to loss-of-function SCN1A variants. Subsequently, they developed episodes of febrile focal status epilepticus (SE) associated with hemiparesis and cerebral hemiatrophy with posterior focal seizures, as a consequence of Dravet syndrome. This sequence of events has been previously published in patients with Dravet syndrome and does not contradict the recent classification by the International League Against Epilepsy (ILAE). The ILAE guidance identifies “Focal neurological findings” as alert criteria and “MRI showing a causal focal lesion” as exclusionary criteria for making an initial diagnosis of Dravet syndrome at presentation. Our three patients would correspond to a severe phenotype, similar to the well-known presentation of generalized atrophy following prolonged status epilepticus. Common genetic findings in cases of diffuse and unilateral brain involvement may help explain these clinical presentations. Further genotype–phenotype studies may provide additional insights into this electroclinical behavior.