Chloride Co-transporter NKCC1 Inhibitor Bumetanide Enhances Neurogenesis and Behavioral Recovery in Rats After Experimental Stroke

Bumetanide, a selective Na⁺-K⁺-Cl^?-co-transporter inhibitor, is widely used in clinical practice as a loop diuretic. In addition, bumetanide has been reported to attenuate ischemia-induced cerebral edema and reduce neuronal injury. This study examined whether bumetanide could influence neurogenesis and behavioral recovery in rats after experimentally induced stroke. Adult male Wistar rats were randomly assigned to four groups: sham, sham treated with bumetanide, ischemia, and ischemia treated with bumetanide. Focal cerebral ischemia was induced by injection of endothelin-1. Bumetanide (0.2 mg/kg/day) was infused into the lateral ventricle with drug administration being initiated 1 week after ischemia and continued for 3 weeks. Behavioral impairment and recovery were evaluated by tapered/ledged beam-walking test on post-stroke days 28. Then, the rats were perfused for BrdU/DCX (neuroblast marker), BrdU/NeuN (neuronal marker), BrdU/GFAP (astrocyte marker), and BrdU/Iba-1 (microglia marker) immunohistochemistry. The numbers of neuroblasts in the subventricular zone (SVZ) were significantly increased after the experimentally induced stroke. Bumetanide treatment increased migration of neuroblasts in the SVZ towards the infarct area, enhanced long-term survival of newborn neurons, and improved sensorimotor recovery, but it did not exert any effects on inflammation. In conclusion, our results demonstrated that chronic bumetanide treatment enhances neurogenesis and behavioral recovery after experimentally induced stroke in rats.     

Erythropoietin Amplifies Stroke-Induced Oligodendrogenesis in the Rat

Background

Erythropoietin (EPO), a hematopoietic cytokine, enhances neurogenesis and angiogenesis during stroke recovery. In the present study, we examined the effect of EPO on oligodendrogenesis in a rat model of embolic focal cerebral ischemia.

Methodology and Principal Findings

Recombinant human EPO (rhEPO) at a dose of 5,000 U/kg (n = 18) or saline (n = 18) was intraperitoneally administered daily for 7 days starting 24 h after stroke onset. Treatment with rhEPO augmented actively proliferating oligodendrocyte progenitor cells (OPCs) measured by NG2 immunoreactive cells within the peri-infarct white matter and the subventricular zone (SVZ), but did not protect against loss of myelinating oligodendrocytes measured by cyclic nucleotide phosphodiesterase (CNPase) positive cells 7 days after stroke. However, 28 and 42 days after stroke, treatment with rhEPO significantly increased myelinating oligodendrocytes and myelinated axons within the peri-infarct white matter. Using lentivirus to label subventricular zone (SVZ) neural progenitor cells, we found that in addition to the OPCs generated in the peri-infarct white matter, SVZ neural progenitor cells contributed to rhEPO-increased OPCs in the peri-infarct area. Using bromodeoxyuridine (BrdU) for birth-dating cells, we demonstrated that myelinating oligodendrocytes observed 28 days after stroke were derived from OPCs. Furthermore, rhEPO significantly improved neurological outcome 6 weeks after stroke. In vitro, rhEPO increased differentiation of adult SVZ neural progenitor cells into oligodendrocytes and enhanced immature oligodendrocyte cell proliferation.

Conclusions

Our in vivo and in vitro data indicate that EPO amplifies stroke-induced oligodendrogenesis that could facilitate axonal re-myelination and lead to functional recovery after stroke.     

Microglial Hv1 proton channels promote white matter injuries after chronic hypoperfusion in mice

Microglia are critical in damage/repair processes during ischemic white matter injury (WMI). Voltage-gated proton channel (Hv1) is expressed in microglia and contributes to nicotinamide adenine dinucleotide phosphate oxidase complex-dependent production of reactive oxygen species (ROS). Recent findings have shown that Hv1 is involved in regulating luminal pH of M1-polarized microglial phagosomes and inhibits endocytosis in microglia. We previously reported that Hv1 facilitated production of ROS and pro-inflammatory cytokines in microglia and enhanced damage to oligodendrocyte progenitor cells from oxygen and glucose deprivation. To investigate the role of Hv1 in hypoperfusion-induced WMI, we employed mice that were genetically devoid of Hv1 (Hv1^-/-), as well as a model of subcortical vascular dementia via bilateral common carotid artery stenosis. Integrity of myelin was assessed using immunofluorescent staining and transmission electron microscopy, while cognitive impairment was assessed using an eight-arm radial maze test. Hv1 deficiency was found to attenuate bilateral common carotid artery stenosis-induced disruption of white matter integrity and impairment of working memory. Immunofluorescent staining and western blotting were used to assay changes in oligodendrocytes, OPCs, and microglial polarization. Compared with that in wild-type (WT) mice, Hv1^-/- mice exhibited reduced ROS generation, decreased pro-inflammatory cytokines production, and an M2-dominant rather than M1-dominant microglial polarization. Furthermore, Hv1^-/- mice exhibited enhanced OPC proliferation and differentiation into oligodendrocytes. Results of mouse-derived microglia-OPC co-cultures suggested that PI3K/Akt signaling was involved in Hv1-deficiency-induced M2-type microglial polarization and concomitant OPC differentiation. These results suggest that microglial Hv1 is a promising therapeutic target for reducing ischemic WMI and cognitive impairment.     

Long-Term Upregulation of Inflammation and Suppression of Cell Proliferation in the Brain of Adult Rats Exposed to Traumatic Brain Injury Using the Controlled Cortical Impact Model

The long-term consequences of traumatic brain injury (TBI), specifically the detrimental effects of inflammation on the neurogenic niches, are not very well understood. In the present in vivo study, we examined the prolonged pathological outcomes of experimental TBI in different parts of the rat brain with special emphasis on inflammation and neurogenesis. Sixty days after moderate controlled cortical impact injury, adult Sprague-Dawley male rats were euthanized and brain tissues harvested. Antibodies against the activated microglial marker, OX6, the cell cycle-regulating protein marker, Ki67, and the immature neuronal marker, doublecortin, DCX, were used to estimate microglial activation, cell proliferation, and neuronal differentiation, respectively, in the subventricular zone (SVZ), subgranular zone (SGZ), striatum, thalamus, and cerebral peduncle. Stereology-based analyses revealed significant exacerbation of OX6-positive activated microglial cells in the striatum, thalamus, and cerebral peduncle. In parallel, significant decrements in Ki67-positive proliferating cells in SVZ and SGZ, but only trends of reduced DCX-positive immature neuronal cells in SVZ and SGZ were detected relative to sham control group. These results indicate a progressive deterioration of the TBI brain over time characterized by elevated inflammation and suppressed neurogenesis. Therapeutic intervention at the chronic stage of TBI may confer abrogation of these deleterious cell death processes.     

Adult neurogenic process in the subventricular zone‐olfactory bulb system is regulated by Tau protein under prolonged stress

ObjectivesThe area of the subventricular zone (SVZ) in the adult brain exhibits the highest number of proliferative cells, which, together with the olfactory bulb (OB), maintains constant brain plasticity through the generation, migration and integration of newly born neurons. Despite Tau and its malfunction is increasingly related to deficits of adult hippocampal neurogenesis and brain plasticity under pathological conditions [e.g. in Alzheimer''s disease (AD)], it remains unknown whether Tau plays a role in the neurogenic process of the SVZ and OB system under conditions of chronic stress, a well‐known sculptor of brain and risk factor for AD.Materials and methodsDifferent types of newly born cells in SVZ and OB were analysed in animals that lack Tau gene (Tau‐KO) and their wild‐type littermates (WT) under control or chronic stress conditions.ResultsWe demonstrate that chronic stress reduced the number of proliferating cells and neuroblasts in the SVZ leading to decreased number of newborn neurons in the OB of adult WT, but not Tau‐KO, mice. Interestingly, while stress‐evoked changes were not detected in OB granular cell layer, Tau‐KO exhibited increased number of mature neurons in this layer indicating altered neuronal migration due to Tau loss.ConclusionsOur findings suggest the critical involvement of Tau in the neurogenesis suppression of SVZ and OB neurogenic niche under stressful conditions highlighting the role of Tau protein as an essential regulator of stress‐driven plasticity deficits.     

The response of NG2-expressing oligodendrocyte progenitors to demyelination in MOG-EAE and MS

Remyelination of primary demyelinated lesions is a common feature of experimental models of multiple sclerosis (MS) and is also suggested to be the normal response to demyelination during the early stages of MS itself. Many lines of evidence have shown that remyelination is preceded by the division of endogenous oligodendrocyte precursor cells (OPCs) in the lesion and its borders. It is suggested that this rapid response of OPCs to repopulate the lesion site and their subsequent differentiation into new oligodendrocytes is the key to the rapid remyelination. Antibodies to the NG2 chondroitin sulphate proteoglycan have proved exceedingly useful in following and quantitating the response of endogenous OPCs to demyelination. Here we review the literature on the response of NG2-expressing OPCs to demyelination and provide some new evidence on their response to the chronic inflammatory demyelinating environment seen in recombinant myelin oligodendrocyte glycoprotein (MOG) induced experimental allergic encephalomyelitis (EAE) in the DA rat. NG2-expressing OPCs responded to the inflammatory demyelination in this model by becoming reactive and increasing in number in a very focal manner. Evidence of NG2⁺OPCs in lesioned areas beginning to express the oligodendrocyte marker CNP was also seen. The response of OPCs appeared to occur following successive relapses but did not always lead to remyelination, with areas of chronic demyelination observed in the spinal cord. The presence of OPCs in the adult human CNS is clearly of vital importance for repair in multiple sclerosis (MS). As in rat tissue, the antibody labels an evenly distributed cell population present in both white and grey matter, distinct from HLA-DR⁺microglia. NG2⁺cells are sparsely distributed in the centre of chronic MS lesions. These cells apparently survive demyelination and exhibit a multi-processed or bipolar morphology in the very hypocellular environment of the lesion.     

Brain ischemia, neurogenesis, and neurotrophic receptor expression in primates

Generation of new neurons persists in the normal adult mammalian brain, with neural stem/progenitor cells residing in at least two brain regions: the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG). Adult neurogenesis is well documented in the rodent, and has also been demonstrated in vivo in nonhuman primates and humans. Brain injuries such as ischemia affect neurogenesis in adult rodents as both global and focal ischemic insults enhance the proliferation of progenitor cells residing in SGZ or SVZ. We addressed the issue whether an injury triggered activation of endogenous neuronal precursors also takes place in the adult primate brain. We found that the ischemic insult increased the number of progenitor cells in monkey SGZ and SVZ, and caused gliogenesis in the ischemia-prone hippocampal CA1 sector. To better understand the mechanisms regulating precursor cell division and differentiation in the primate, we analyzed the expression at protein level of a panel of potential regulatory molecules, including neurotrophic factors and their receptors. We found that a fraction of mitotic progenitors were positive for the neurotrophin receptor TrkB, while immature neurons expressed the neurotrophin receptor TrkA. Astroglia, ependymal cells and blood vessels in SVZ were positive for distinctive sets of ligands/receptors, which we characterized. Thus, a network of neurotrophic signals operating in an autocrine or paracrine manner may regulate neurogenesis in adult primate SVZ. We also analyzed microglial and astroglial proliferation in postischemic hippocampal CA1 sector. We found that proliferating postischemic microglia in adult monkey CA1 sector express the neurotrophin receptor TrkA, while activated astrocytes were labeled for nerve growth factor (NGF), ligand for TrkA, and the tyrosine kinase TrkB, a receptor for brain derived neurotrophic factor (BDNF). These results implicate NGF and BDNF as regulators of postischemic glial proliferation in adult primate hippocampus.     

Fractalkine-induced activation of the phosphatidylinositol-3 kinase pathway attentuates microglial activation in vivo and in vitro

Several neurodegenerative disorders are associated with evidence of inflammation, one feature of which is increased activation of microglia, the most likely cellular source of inflammatory cytokines like interleukin-1β. It is now recognized that interaction of microglia with other cells contributes to maintenance of microglia in a quiescent state and the complementary distribution of the chemokine, fractalkine (CX₃CL1) on neurons and its receptor (CX₃CR1) on microglia, suggests that this interaction may play a role in modulating microglial activation. Here we demonstrate that both soluble and membrane-bound fractalkine attenuate lipopolysaccharide-induced microglial activation in vitro. We also show that fractalkine expression is reduced in the brain of aged rats and this is accompanied by an age-related increase in microglial activation. Treatment of aged rats with fractalkine attenuates the age-related increase in microglial activation and the evidence indicates that fractalkine-induced activation of the phosphatidylinositol-3 kinase pathway is required to maintain microglia in a quiescent state both in vivo and in vitro .     

Inflammation mediates varying effects in neurogenesis: relevance to the pathogenesis of brain injury and neurodegenerative disorders

Brain inflammation is a complex cellular and molecular response to stress, injury or infection of the CNS in attempt to defend against insults, clear dead and damaged neurons and return the CNS to a normal state. Inflammation in the CNS is driven by the activation of resident microglia, astrocytes and infiltrating peripheral macrophages, which release a plethora of anti- and pro-inflammatory cytokines, chemokines, neurotransmitters and reactive oxygen species. This inflammatory state inadvertently causes further bystander damage to neurons and produces both detrimental and favorable conditions for neurogenesis. Inflammatory factors have varying effects on neural progenitor cell proliferation, migration, differentiation, survival and incorporation of newly born neurons into the CNS circuitry. The unique profile of inflammatory factors, which depends on the severity of inflammation, can have varying consequences on neurogenesis. Inflammatory factors released during mild acute inflammation usually stimulate neurogenesis; where as the factors released by uncontrolled inflammation create an environment that is detrimental to neurogenesis. This review will provide a summary of current progress in this emerging field and examine the potential mechanisms through which inflammation affects neurogenesis during neurological complications.     

Increased Plp1 gene expression leads to massive microglial cell activation and inflammation throughout the brain

PMD (Pelizaeus–Merzbacher disease) is a rare neurodegenerative disorder that impairs motor and cognitive functions and is associated with a shortened lifespan. The cause of PMD is mutations of the PLP1 [proteolipid protein 1 gene (human)] gene. Transgenic mice with increased Plp1 [proteolipid protein 1 gene (non-human)] copy number model most aspects of PMD patients with duplications. Hypomyelination and demyelination are believed to cause the neurological abnormalities in mammals with PLP1 duplications. We show, for the first time, intense microglial reactivity throughout the grey and white matter of a transgenic mouse line with increased copy number of the native Plp1 gene. Activated microglia in the white and grey matter of transgenic mice are found as early as postnatal day 7, before myelin commences in normal cerebra. This finding indicates that degeneration of myelin does not cause the microglial response. Microglial numbers are doubled due to in situ proliferation. Compared with the jp (jimpy) mouse, which has much more oligodendrocyte death and hardly any myelin, microglia in the overexpressors show a more dramatic microglial reactivity than jp, especially in the grey matter. Predictably, many classical markers of an inflammatory response, including TNF-α (tumour necrosis factor-α) and IL-6, are significantly up-regulated manyfold. Because inflammation is believed to contribute to axonal degeneration in multiple sclerosis and other neurodegenerative diseases, inflammation in mammals with increased Plp1 gene dosage may also contribute to axonal degeneration described in patients and rodents with PLP1 increased gene dosage.     

Neurogenesis response of middle-aged hippocampus to acute seizure activity

Acute Seizure (AS) activity in young adult age conspicuously modifies hippocampal neurogenesis. This is epitomized by both increased addition of new neurons to the granule cell layer (GCL) by neural stem/progenitor cells (NSCs) in the dentate subgranular zone (SGZ), and greatly enhanced numbers of newly born neurons located abnormally in the dentate hilus (DH). Interestingly, AS activity in old age does not induce such changes in hippocampal neurogenesis. However, the effect of AS activity on neurogenesis in the middle-aged hippocampus is yet to be elucidated. We examined hippocampal neurogenesis in middle-aged F344 rats after a continuous AS activity for >4 hrs, induced through graded intraperitoneal injections of the kainic acid. We labeled newly born cells via daily intraperitoneal injections of the 5'-bromodeoxyuridine (BrdU) for 12 days, commencing from the day of induction of AS activity. AS activity enhanced the addition of newly born BrdU+ cells by 5.6 fold and newly born neurons (expressing both BrdU and doublecortin [DCX]) by 2.2 fold to the SGZ-GCL. Measurement of the total number of DCX+ newly born neurons also revealed a similar trend. Furthermore, AS activity increased DCX+ newly born neurons located ectopically in the DH (2.7 fold increase and 17% of total newly born neurons). This rate of ectopic migration is however considerably less than what was observed earlier for the young adult hippocampus after similar AS activity. Thus, the plasticity of hippocampal neurogenesis to AS activity in middle age is closer to its response observed in the young adult age. However, the extent of abnormal migration of newly born neurons into the DH is less than that of the young adult hippocampus after similar AS activity. These results also point out a highly divergent response of neurogenesis to AS activity between middle age and old age.     

Hippocampal neurogenesis and neuroinflammation after cranial irradiation with (56)Fe particles

Exposure to heavy-ion radiation is considered a potential health risk in long-term space travel. In the central nervous system (CNS), loss of critical cellular components may lead to performance decrements that could ultimately compromise mission goals and long-term quality of life. Hippocampal-dependent cognitive impairments occur after exposure to ionizing radiation, and while the pathogenesis of this effect is not yet clear, it may involve the production of newly born neurons (neurogenesis) in the hippocampal dentate gyrus. We irradiated mice with 0.5-4 Gy of (56)Fe ions and 2 months later quantified neurogenesis and numbers of activated microglia as a measure of neuroinflammation in the dentate gyrus. Results showed that there were few changes after 0.5 Gy, but that there was a dose-related decrease in hippocampal neurogenesis and a dose-related increase in numbers of newly born activated microglia from 0.5-4.0 Gy. While those findings were similar to what was reported after X irradiation, there were also some differences, particularly in the response of newly born glia. Overall, this study showed that hippocampal neurogenesis was sensitive to relatively low doses of (56)Fe particles, and that those effects were associated with neuroinflammation. Whether these changes will result in functional impairments or if/how they can be managed are topics for further investigation.     

The NKCC1 ion transporter modulates microglial phenotype and inflammatory response to brain injury in a cell-autonomous manner

The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has remained unclear to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology, process recruitment to the site of injury, and adaptation to changes in cellular volume in a cell-autonomous manner via regulating membrane conductance. In addition, microglial NKCC1 deficiency results in NLRP3 inflammasome priming and increased production of interleukin-1β (IL-1β), rendering microglia prone to exaggerated inflammatory responses. In line with this, central (intracortical) administration of the NKCC1 blocker, bumetanide, potentiated intracortical lipopolysaccharide (LPS)-induced cytokine levels. In contrast, systemic bumetanide application decreased inflammation in the brain. Microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema and worse neurological outcome. Thus, NKCC1 emerges as an important player in controlling microglial ion homeostasis and inflammatory responses through which microglia modulate brain injury. The contribution of microglia to central NKCC1 actions is likely to be relevant for common neurological disorders.

This study shows that inflammatory responses of microglial cells are markedly influenced by the ion transporter, NKCC1; blockade or genetic deletion of microglial NKCC1 has broad cell-autonomous effects, leading to changes in morphology, membrane conductance, process recruitment after injury, and cytokine production, with worsened neurological outcome after stroke.     

Indomethacin treatment reduces microglia activation and increases numbers of neuroblasts in the subventricular zone and ischaemic striatum after focal ischaemia

Neuroblasts from the subventricular zone (SVZ) migrate to striatum following stroke, but most of them die in the ischaemic milieu and this can be related to exacerbated microglial activation. Here, we explored the effects of the non-steroidal anti-inflammatory indomethacin on microglial activation, neuronal preservation and neuroblast migration following experimental striatal stroke in adult rats. Animals were submitted to endothelin-1 (ET-1)-induced focal striatal ischaemia and were treated with indomethacin or sterile saline (i.p.) for 7 days, being perfused after 8 or 14 days. Immunohistochemistry was performed to assess neuronal loss (anti-NeuN), microglial activation (anti-Iba1, ED1) and migrating neuroblasts (anti-DCX) by counting NeuN, ED1 and DCX-positive cells in the ischaemic striatum or SVZ. Indomethacin treatment reduced microglia activation and the number of ED1⁺ cells in both 8 and 14 days post injury as compared with controls. There was an increase in the number of DCX⁺ cells in both SVZ and striatum at the same survival times. Moreover, there was a decrease in the number of NeuN⁺ cells in indomethacin-treated animals as compared with the control group at 8 days but not after 14 days post injury. Our results suggest that indomethacin treatment modulates microglia activation, contributing to increased neuroblast proliferation in the SVZ and migration to the ischaemic striatum following stroke.     

Beneficial Effects of Minocycline on Cuprizone Induced Cortical Demyelination

In this study, we investigated the potential of minocycline to influence cuprizone induced demyelination in the grey and white matter. To induce demyelination C57BL/6 mice were fed with cuprizone for up to 6 weeks and were analysed at different timepoints (week 0, 4, 5, 6). Mice treated with minocycline had less demyelination of the cortex and corpus callosum compared with sham treated animals. In the cortex decreased numbers of activated and proliferating microglia were found after 6 weeks of cuprizone feeding, while there were no significant effects for microglial infiltration of the corpus callosum. In addition to the beneficial effects on demyelination, minocycline prevented from motor coordination disturbance as shown in the beam walking test. For astrogliosis and the numbers of OPC and oligodendrocytes no treatment effects were found. In summary, minocycline treatment diminished the course of demyelination in the grey and white matter and prevented disturbances in motor coordination.     

Nitric oxide regulates antagonistically phagocytic and neurite outgrowth inhibiting capacities of microglia

Traumatic injury or the pathogenesis of some neurological disorders is accompanied by inflammatory cellular mechanisms, mainly resulting from the activation of central nervous system (CNS) resident microglia. Under inflammatory conditions, microglia up‐regulate the inducible isoform of NOS (iNOS), leading to the production of high concentrations of the radical molecule nitric oxide (NO). At the onset of inflammation, high levels of microglial‐derived NO may serve as a cellular defense mechanism helping to clear the damaged tissue and combat infection of the CNS by invading pathogens. However, the excessive overproduction of NO by activated microglia has been suggested to govern the inflammation‐mediated neuronal loss causing eventually complete neurodegeneration. Here, we investigated how NO influences phagocytosis of neuronal debris by BV‐2 microglia, and how neurite outgrowth of human NT2 model neurons is affected by microglial‐derived NO. The presence of NO greatly increased microglial phagocytic capacity in a model of acute inflammation comprising lipopolysaccharide (LPS)‐activated microglia and apoptotic neurons. Chemical manipulations suggested that NO up‐regulates phagocytosis independently of the sGC/cGMP pathway. Using a transwell system, we showed that reactive microglia inhibit neurite outgrowth of human neurons via the generation of large amounts of NO over effective distances in the millimeter range. Application of a NOS blocker prevented the LPS‐induced NO production, totally reversed the inhibitory effect of microglia on neurite outgrowth, but reduced the engulfment of neuronal debris. Our results indicate that a rather simple notion of treating excessive inflammation in the CNS by NO synthesis blocking agents has to consider functionally antagonistic microglial cell responses during pharmaceutic therapy. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 566–584, 2016     

Microglial cells of the rat brain in postnatal period (comparative immunocytochemical analysis)

In the course of the brain’s development, distribution of microglial cells was studied in rats using immunocytochemical detection. To identify the microglial cells, antibodies to lipocortin 1 (LC1) and phosphotyrosine (PT) were used. On postnatal day 1, LC1-positive microglial cells of an ameboid shape were distributed mainly in the subventricular zone; their mean density was 31±8 cells/mm² (counted across the total area of frontal sections). On postnatal day 7, microglial cells of an intermediate type were located throughout the whole brain; their density was 54±15 cells/mm². On the 15th day, LC1-positive cells were of a ramified shape, and their density reached 104±20 cells/mm² (the microglial cell density in the mature normal brain was 103±3 cells/mm²). On postnatal day 7, PT-positive cells were similar in their morphology to LC1-positive cells of an intermediate type, while their mean density was 32 cells/mm². In the mature brain, the density of PT-positive microglia was 53±5 cells/mm²; the shape of the cells in the white and gray matter of the brain was, on the whole, similar to that of LC1-positive microglia. Therefore, LC1 is a specific marker for different types of microglial cells in the developing brain. Our data about 3D distribution and morphological peculiarities of microglial cells at different stages of postnatal development are consistent with the hypothesis on the neuroectodermal origin of microglia.     

Upregulation of Vascular Endothelial Growth Factor Receptor-3 in the Spinal Cord of Lewis Rats with Experimental Autoimmune Encephalomyelitis

We investigated the spatiotemporal expression of vascular endothelial growth factor receptor–3 (VEGFR-3) in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. VEGFR-3 mRNA and protein were constitutively expressed in gray matter neurons and in a few white matter astrocytes. Induction of VEGFR-3 occurred predominantly in perivascular infiltrated macrophages in the spinal cord white matter during the inductive phase of EAE. VEGFR-3 expression was also induced in activated microglial cells in the gray and white matter, mainly in the peak phase. In addition, reactive astrocytes in the white matter, but not in the gray matter, expressed VEGFR-3 as disease severity increased. These data suggest that VEGFR-3 is involved in the recruitment of monocytic macrophages and in glial reactions during EAE.     

Effects of Intra-Amniotic Lipopolysaccharide and Maternal Betamethasone on Brain Inflammation in Fetal Sheep

Rationale

Chorioamnionitis and antenatal glucocorticoids are common exposures for preterm infants and can affect the fetal brain, contributing to cognitive and motor deficits in preterm infants. The effects of antenatal glucocorticoids on the brain in the setting of chorioamnionitis are unknown. We hypothesized that antenatal glucocorticoids would modulate inflammation in the brain and prevent hippocampal and white matter injury after intra-amniotic lipopolysaccharide (LPS) exposure.

Methods

Time-mated ewes received saline (control), an intra-amniotic injection of 10 mg LPS at 106d GA or 113d GA, maternal intra-muscular betamethasone (0.5 mg/kg maternal weight) alone at 113d GA, betamethasone at 106d GA before LPS or betamethasone at 113d GA after LPS. Animals were delivered at 120d GA (term=150d). Brain structure volumes were measured on T2-weighted MRI images. The subcortical white matter (SCWM), periventricular white matter (PVWM) and hippocampus were analyzed for microglia, astrocytes, apoptosis, proliferation, myelin and pre-synaptic vesicles.

Results

LPS and/or betamethasone exposure at different time-points during gestation did not alter brain structure volumes on MRI. Betamethasone alone did not alter any of the measurements. Intra-amniotic LPS at 106d or 113d GA induced inflammation as indicated by increased microglial and astrocyte recruitment which was paralleled by increased apoptosis and hypomyelination in the SCWM and decreased synaptophysin density in the hippocampus. Betamethasone before the LPS exposure at 113d GA prevented microglial activation and the decrease in synaptophysin. Betamethasone after LPS exposure increased microglial infiltration and apoptosis.

Conclusion

Intra-uterine LPS exposure for 7d or 14d before delivery induced inflammation and injury in the fetal white matter and hippocampus. Antenatal glucocorticoids aggravated the inflammatory changes in the brain caused by pre-existing intra-amniotic inflammation. Antenatal glucocorticoids prior to LPS reduced the effects of intra-uterine inflammation on the brain. The timing of glucocorticoid administration in the setting of chorioamnionitis can alter outcomes for the fetal brain.