NG2 and NG2‐positive cells delineate focal cerebral infarct demarcation in rats

Focal cerebral ischemia induces cellular responses that may result in secondary tissue damage. We recently demonstrated multi‐facetted spatial and temporal patterns of neuroinflammation by multimodal imaging. In the present study, we especially focus on the separation of vital and necrotic tissue, which enabled us to define a demarcation zone. Focal cerebral ischemia was induced via macrosphere embolization of the middle cerebral artery in Wistar rats. Subsequent cellular processes were investigated immunohistochemically from 3 to 56 days after onset of ischemia. We detected several infarct subareas: a necrotic infarct core and its margin adjacent to a nerve/glial antigen 2 (NG2)+ zone delineating it from a vital peri‐infarct zone. Initially transition from necrotic to vital tissue was gradual; later on necrosis was precisely separated from vital tissue by a narrow NG2+ belt that was devoid of astrocytes, oligodendrocytes or neurons. Within this demarcation zone NG2+ cells associate with ionized calcium binding adaptor molecule 1 (Iba1) but not with GFAP, neuronal nuclear antigen (NeuN) or 2′, 3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase). During further infarct maturation NG2 seemed to be positioned in the extracellular matrix (ECM) of the demarcation zone, whereas Iba1+ cells invaded the necrotic infarct core and GFAP+ cells built a gliotic containing belt between the lesion and NeuN+ unaffected tissue. Overall, our data suggested that NG2 proteoglycan expression and secretion hallmarked demarcation as a process that actively separated necrosis from vital tissue and therefore decisively impacts secondary neurodegeneration after ischemic stroke.     

Astrocytic transforming growth factor‐beta signaling reduces subacute neuroinflammation after stroke in mice

Astrocytes limit inflammation after CNS injury, at least partially by physically containing it within an astrocytic scar at the injury border. We report here that astrocytic transforming growth factor‐beta (TGFβ) signaling is a second, distinct mechanism that astrocytes utilize to limit neuroinflammation. TGFβs are anti‐inflammatory and neuroprotective cytokines that are upregulated subacutely after stroke, during a clinically accessible time window. We have previously demonstrated that TGFβs signal to astrocytes, neurons and microglia in the stroke border days after stroke. To investigate whether TGFβ affects astrocyte immunoregulatory functions, we engineered “Ast‐Tbr2DN” mice where TGFβ signaling is inhibited specifically in astrocytes. Despite having a similar infarct size to wildtype controls, Ast‐Tbr2DN mice exhibited significantly more neuroinflammation during the subacute period after distal middle cerebral occlusion (dMCAO) stroke. The peri‐infarct cortex of Ast‐Tbr2DN mice contained over 60% more activated CD11b⁺ monocytic cells and twice as much immunostaining for the activated microglia and macrophage marker CD68 than controls. Astrocytic scarring was not altered in Ast‐Tbr2DN mice. However, Ast‐Tbr2DN mice were unable to upregulate TGF‐β1 and its activator thrombospondin‐1 2 days after dMCAO. As a result, the normal upregulation of peri‐infarct TGFβ signaling was blunted in Ast‐Tbr2DN mice. In this setting of lower TGFβ signaling and excessive neuroinflammation, we observed worse motor outcomes and late infarct expansion after photothrombotic motor cortex stroke. Taken together, these data demonstrate that TGFβ signaling is a molecular mechanism by which astrocytes limit neuroinflammation, activate TGFβ in the peri‐infarct cortex and preserve brain function during the subacute period after stroke. GLIA 2014;62:1227–1240     

Radixin expression in microglia after cortical stroke lesion

Stroke induces extensive tissue remodeling, resulting in the activation of several cell types in the brain as well as recruitment of blood‐borne leucocytes. Radixin is part of a cytoskeleton linker protein family with the ability to connect transmembrane proteins to the actin cytoskeleton, promoting cell functions involving a dynamic cytoskeleton such as morphological changes, cell division and migration which are common events of different cell types after stroke. In the healthy adult brain radixin is expressed in Olig2⁺ cells throughout the brain and in neural progenitor cells in the subventricular zone. In the current study, we detected a 2.5 fold increase in the number of radixin positive cells in the peri‐infarct cortex two weeks after the induction of cortical stroke by photothrombosis. Similarly, the number of Olig2⁺ cells increased in the peri‐infarct area after stroke; however, the number of radixin⁺/Olig2⁺ cells was unchanged. Neural progenitor cells maintained radixin expression on their route to the infarct. More surprising however, was the expression of radixin in activated microglia in the peri‐infarct cortex. Seventy percent of Iba1⁺ cells expressed radixin after stroke, a population which was not present in the control brain. Furthermore, activation of radixin was predominantly detected in the peri‐infarct region of oligodendrocyte progenitors and microglia. The specific location of radixin⁺ cells in the peri‐infarct region and in microglia suggests a role for radixin in microglial activation after stroke.     

Down‐regulation of microglia and NG2‐positive cells reaction in trimethyltin‐injured hippocampus of rats treated with myelin basic protein‐reactive T cells: Possible contribution to the neuroprotective effect of T cells

In our previous investigations, we demonstrated that CD4⁺ antimyelin basic protein (MBP) T cells protect hippocampal neurons against trimethyltin‐induced damage. We hypothesized involvement of T cells, interacting with the various glial populations activated during the neurodegeneration process. In this study, we employ immunocytochemical methods to investigate the influence of administration of T cells on the response of microglia and of NG2⁺ cells to trimethyltin (TMT)‐induced damage. Female Lewis rats were treated with anti‐MBP CD4⁺ T cells (4 million per animal, i.v) 24 hr after TMT (8 mg/kg, i.p) intoxication. TMT caused degeneration of CA4 hipppocampal neurons and evoked an abundant reaction of microglial and NG2⁺ cells in the injured region. The cells changed morphology into the activated state, and the number of OX42⁺ and NG2⁺ cells increased about 4.5‐fold and 3‐fold, respectively, relative to controls as assessed on day 21 after TMT treatment. Additionally, the cells of ameboid morphology, which expressed NG2 or microglial antigens, appeared in the zone of neurodegeneration. Furthermore, certain cells of ameboid phenotype shared both antigens. In rats treated with T cells, down‐regulation of the activation of both glial classes and reduction of formation of their ameboid forms was observed. The number of the total OX42⁺ and NG2⁺ cells decreased by 21% and 54%, respectively, and the number of their ameboid forms decreased by 46% and 73%, respectively. Our data suggest that the diminished activation of microglia and NG2⁺ cells, particularly the reduced number of their ameboid forms, may contribute to the neuroprotective effect of T cells. © 2009 Wiley‐Liss, Inc.     

Transforming growth factor‐β1 primes proliferating adult neural progenitor cells to electrophysiological functionality

The differentiation of adult neural progenitors (NPCs) into functional neurons is still a limiting factor in the neural stem cell field but mandatory for the potential use of NPCs in therapeutic approaches. Neuronal function requires the appropriate electrophysiological properties. Here, we demonstrate that priming of NPCs using transforming growth factor (TGF)‐β1 under conditions that usually favor NPCs' proliferation induces electrophysiological neuronal properties in adult NPCs. Gene chip array analyses revealed upregulation of voltage‐dependent ion channel subunits (Kcnd3, Scn1b, Cacng4, and Accn1), neurotransmitters, and synaptic proteins (Cadps, Snap25, Grik4, Gria3, Syngr3, and Gria4) as well as other neuronal proteins (doublecortin [DCX], Nrxn1, Sept8, and Als2cr3). Patch‐clamp analysis demonstrated that control‐treated cells expressed only voltage‐dependent K⁺‐channels of the delayed‐rectifier type and the A‐type channels. TGF‐β1‐treated cells possessed more negative resting potentials than nontreated cells owing to the presence of delayed‐rectifier and inward‐rectifier channels. Furthermore, TGF‐β1‐treated cells expressed voltage‐dependent, TTX‐sensitive Na⁺ channels, which showed increasing current density with TGF‐β1 treatment duration and voltage‐dependent (+)BayK8644‐sensitive L‐Type Ca²⁺ channels. In contrast to nontreated cells, TGF‐β1‐treated cells responded to current injections with action‐potentials in the current‐clamp mode. Furthermore, TGF‐β1‐treated cells responded to application of GABA with an increase in membrane conductance and showed spontaneous synaptic currents that were blocked by the GABA‐receptor antagonist picrotoxine. Only NPCs, which were treated with TGF‐β1, showed Na⁺ channel currents, action potentials, and GABAergic currents. In summary, stimulation of NPCs by TGF‐β1 fosters a functional neuronal phenotype, which will be of relevance for future cell replacement strategies in neurodegenerative diseases or acute CNS lesions. GLIA 2013;61:1767–1783     

Long‐term accumulation of microglia with proneurogenic phenotype concomitant with persistent neurogenesis in adult subventricular zone after stroke

Neural stem cells (NSCs) in the adult rat subventricular zone (SVZ) generate new striatal neurons during several months after ischemic stroke. Whether the microglial response associated with ischemic injury extends into SVZ and influences neuroblast production is unknown. Here, we demonstrate increased numbers of activated microglia in ipsilateral SVZ concomitant with neuroblast migration into the striatum at 2, 6, and 16 weeks, with maximum at 6 weeks, following 2 h middle cerebral artery occlusion in rats. In the peri‐infarct striatum, numbers of activated microglia peaked already at 2 weeks and declined thereafter. Microglia in SVZ were resident or originated from bone marrow, with maximum proliferation during the first 2 weeks postinsult. In SVZ, microglia exhibited ramified or intermediate morphology, signifying a downregulated inflammatory profile, whereas amoeboid or round phagocytic microglia were frequent in the peri‐infarct striatum. Numbers of microglia expressing markers of antigen‐presenting cells (MHC‐II, CD86) increased in SVZ but very few lymphocytes were detected. Using quantitative PCR, strong short‐ and long‐term increase (at 1 and 6 weeks postinfarct) of insulin‐like growth factor‐1 (IGF‐1) gene expression was detected in SVZ tissue. Elevated numbers of IGF‐1‐expressing microglia were found in SVZ at 2, 6, and 16 weeks after stroke. At 16 weeks, 5% of microglia but no other cells in SVZ expressed the IGF‐1 protein, which mitigates apoptosis and promotes proliferation and differentiation of NSCs. The long‐term accumulation of microglia with proneurogenic phenotype in the SVZ implies a supportive role of these cells for the continuous neurogenesis after stroke. © 2008 Wiley‐Liss, Inc.     

Interaction of NG2+ glial progenitors and microglia/macrophages from the injured spinal cord

Spinal cord contusion produces a central lesion surrounded by a peripheral rim of residual white matter. Despite stimulation of NG2⁺ progenitor cell proliferation, the lesion remains devoid of normal glia chronically after spinal cord injury (SCI). To investigate potential cell–cell interactions of the predominant cells in the lesion at 3 days after injury, we used magnetic activated cell sorting to purify NG2⁺ progenitors and OX42⁺ microglia/macrophages from contused rat spinal cord. Purified NG2⁺ cells from the injured cord grew into spherical masses when cultured in defined medium with FGF2 plus GGF2. The purified OX42⁺ cells did not form spheroids and significantly reduced sphere growth by NG2⁺ cells in co‐cultures. Conditioned medium from these OX42⁺ cells, unlike that from normal peritoneal macrophages or astrocytes also inhibited growth of NG2⁺ cells, suggesting inhibition by secreted factors. Expression analysis of freshly purified OX42⁺ cells for a panel of six genes for secreted factors showed expression of several that could contribute to inhibition of NG2⁺ cells. Further, the pattern of expression of four of these, TNFα, TSP1, TIMP1, MMP9, in sequential coronal tissue segments from a 2 cm length of cord centered on the injury epicenter correlated with the expression of Iba1, a marker gene for OX42⁺ cells, strongly suggesting a potential regional influence by activated microglia/macrophages on NG2⁺ cells in vivo after SCI. Thus, the nonreplacement of lost glial cells in the central lesion zone may involve, at least in part, inhibitory factors produced by microglia/macrophages that are concentrated within the lesion. © 2009 Wiley‐Liss, Inc.     

Distribution of mRNAs encoding transforming growth factors‐β1, ‐2, and ‐3 in the intact rat brain and after experimentally induced focal ischemia

Transforming growth factors‐β1 (TGF‐β1), ‐2, and ‐3 form a small group of related proteins involved in the regulation of proliferation, differentiation, and survival of various cell types. Recently, TGF‐βs were also demonstrated to be neuroprotective. In the present study, we investigated their distribution in the rat brain as well as their expression following middle cerebral artery occlusion. Probes were produced for all types of TGF‐βs, and in situ hybridization was performed. We demonstrated high TGF‐β1 expression in cerebral cortex, hippocampus, central amygdaloid nucleus, medial preoptic area, hypothalamic paraventricular nucleus, substantia nigra, brainstem reticular formation and motoneurons, and area postrema. In contrast, TGF‐β2 was abundantly expressed in deep cortical layers, dentate gyrus, midline thalamic nuclei, posterior hypothalamic area and mamillary body, superior olive, areas of monoaminergic neurons, spinal trigeminal nucleus, dorsal vagal complex, cerebellum, and choroid plexus, and a high level of TGF‐β3 mRNA was found in cerebral cortex, hippocampus, basal amygdaloid nuclei, lateral septal nucleus, several thalamic nuclei, arcuate and supramamillary nuclei, superior colliculus, superior olive, brainstem reticular formation and motoneurons, area postrema, and inferior olive. Focal brain ischemia induced TGF‐βs with markedly different expression patterns. TGF‐β1 was induced in the penumbral region of cortex and striatum, whereas TGF‐β2 and ‐β3 were induced in different layers of the ipsilateral cortex. The expression of the subtypes of TGF‐βs in different brain regions suggests that they are involved in the regulation of different neurons and bind to different latent TGF‐β binding proteins. Furthermore, they might have subtype‐specific functions following ischemic attack. J. Comp. Neurol. 518:3752–3770, 2010. © 2010 Wiley‐Liss, Inc.     

TGF‐β2 and TGF‐β3 from cultured β‐amyloid‐treated or 3xTg‐AD‐derived astrocytes may mediate astrocyte–neuron communication

Astrocytes participate in the development and resolution of neuroinflammation in numerous ways, including the release of cytokines and growth factors. Among many, astrocytes release transforming growth factors beta (TGF‐β) TGF‐β1, TGF‐β2 and TGF‐β3. TGF‐β1 is the most studied isoform, while production and release of TGF‐β2 and TGF‐β3 by astrocytes have been poorly characterized. Here, we report that purified cultures of hippocampal astrocytes produce mainly TGF‐β3 followed by TGF‐β2 and TGF‐β1. Furthermore, astrocytes release principally the active form of TGF‐β3 over the other two. Changes in release of TGF‐β were sensitive to the calcineurin (CaN) inhibitor FK506. Starvation had no effect on TGF‐β1 and TGF‐β3 while TGF‐β2 mRNA was significantly up‐regulated in a CaN‐dependent manner. We further investigated production and release of astroglial TGF‐β in Alzheimer's disease‐related conditions. Oligomeric β‐amyloid (Aβ) down‐regulated TGF‐β1, while up‐regulating TGF‐β2 and TGF‐β3, in a CaN‐dependent manner. In cultured hippocampal astrocytes from 3xTg‐AD mice, TGF‐β2 and TGF‐β3, but not TGF‐β1, were up‐regulated, and this was CaN‐independent. In hippocampal tissues from symptomatic 3xTg‐AD mice, TGF‐β2 was up‐regulated with respect to control mice. Finally, treatment with recombinant TGF‐βs showed that TGF‐β2 and TGF‐β3 significantly reduced PSD95 protein in cultured hippocampal neurons, and this effect was paralleled by conditioned media from Aβ‐treated astrocytes or from astrocytes from 3xTg‐AD mice. Taken together, our data suggest that TGF‐β2 and TGF‐β3 are produced by astrocytes in a CaN‐dependent manner and should be investigated further in the context of astrocyte‐mediated neurodegeneration.     

The 6‐hydroxydopamine‐induced nigrostriatal neurodegeneration produces microglia‐like NG2 glial cells in the rat substantia nigra

Neuron/glial 2 (NG2)‐expressing cells are often referred to as oligodendrocyte precursor cells. NG2‐expressing cells have also been identified as multipotent progenitor cells. However, microglia‐like NG2 glial cells have not been fully examined in neurodegenerative disorders such as Parkinson's disease (PD). In the present study, we chose two rat models of PD, i.e., intranigral or intrastriatal injection of 6‐hydroxydopamine (6‐OHDA), since the cell bodies of dopamine (DA) neurons, which form a nigrostriatal pathway, are in the substantia nigra pars compacta (SNpc) while their nerve terminals are in the striatum. In the nigral 6‐OHDA‐injected model, activated NG2‐positive cells were detected in the SNpc but not in the striatum. In contrast, in the striatal 6‐OHDA‐injected model, these cells were detected in both the SNpc and the striatum. In both models, activated NG2‐positive cells were located close to surviving tyrosine hydroxylase (TH)‐positive neurons in the SNpc. In addition, activated NG2‐positive cells in the SNpc coexpressed ionized calcium‐binding adaptor molecule 1 (Iba1), a microglia/macrophage marker. Interestingly, these double‐positive glial cells coexpressed glial cell line‐derived neurotrophic factor (GDNF). These results suggest that microglia‐like NG2 glial cells may help protect DA neurons and may lead to new therapeutic targets in PD. © 2010 Wiley‐Liss, Inc.     

Deficiency of NG2+ cells contributes to the susceptibility of stroke-prone spontaneously hypertensive rats

Aims: The purpose of this study is to investigate whether the NG2⁺ cells, a class of oligodendrocyte progenitor cells, is involved in the pathophysiology of stroke in stroke‐prone spontaneously hypertensive rat (SHR‐SP). Methods: SHR‐SP, SHR, Wistar‐Kyoto rats (WKY), and C57BJ/6 mice were used. Immunohistochemistry was conducted to evaluate the number of NG2⁺ cells in frozen brain sections. Demyelination was evaluated by Sudan black staining and serum level of myelin basic protein. Middle cerebral artery occlusion (MCAO) was performed to prepare experimental stroke model. Results: The number of NG2⁺ cells was significantly decreased in infarct core and increased in penumbra in WKY rats after MCAO. In brain sections of 6‐month‐old SHR‐SP, the number of NG2⁺ cells was significantly (P < 0.01) less than that in age‐matched SHR and WKY rats. However, this phenomenon was not observed in 3‐month‐old rats. Demyelination was found in 6‐month‐old SHR‐SP but not in 3‐month‐old SHR‐SP. Pharmacological treatment of cuprizone in mice induced demyelination and enlargement of cerebral infarction after MCAO. Conclusion: The decline of NG2⁺ cells may cause demyelination and contribute to the susceptibility of SHR‐SP to ischemic brain injury.     

Impaired glutamate recycling and GluN2B‐mediated neuronal calcium overload in mice lacking TGF‐β1 in the CNS

Transforming growth factor β1 (TGF‐β1) is a pleiotropic cytokine expressed throughout the CNS. Previous studies demonstrated that TGF‐β1 contributes to maintain neuronal survival, but mechanistically this effect is not well understood. We generated a CNS‐specific TGF‐β1‐deficient mouse model to investigate the functional consequences of TGF‐β1‐deficiency in the adult mouse brain. We found that depletion of TGF‐β1 in the CNS resulted in a loss of the astrocyte glutamate transporter (GluT) proteins GLT‐1 (EAAT2) and GLAST (EAAT1) and decreased glutamate uptake in the mouse hippocampus. Treatment with TGF‐β1 induced the expression of GLAST and GLT‐1 in cultured astrocytes and enhanced astroglial glutamate uptake. Similar to GLT‐1‐deficient mice, CNS‐TGF‐β1‐deficient mice had reduced brain weight and neuronal loss in the CA1 hippocampal region. CNS‐TGF‐β1‐deficient mice showed GluN2B‐dependent aberrant synaptic plasticity in the CA1 area of the hippocampus similar to the glutamate transport inhibitor DL‐TBOA and these mice were highly sensitive to excitotoxic injury. In addition, hippocampal neurons from TGF‐β1‐deficient mice had elevated GluN2B‐mediated calcium signals in response to extrasynaptic glutamate receptor stimulation, whereas cells treated with TGF‐β1 exhibited reduced GluN2B‐mediated calcium signals. In summary, our study demonstrates a previously unrecognized function of TGF‐β1 in the CNS to control extracellular glutamate homeostasis and GluN2B‐mediated calcium responses in the mouse hippocampus.     

Novel NG2‐CreERT2 knock‐in mice demonstrate heterogeneous differentiation potential of NG2 glia during development

NG2 (nerve/glia antigen‐2) is a type I transmembrane glycoprotein and also known as chondroitin sulfate proteoglycan 4. In the parenchyma of the central nervous system, NG2‐expressing (NG2⁺) cells have been identified as a novel type of glia with a strong potential to generate oligodendrocytes (OLs) in the developing white matter. However, the differentiation potential of NG2 glia remained controversial, largely attributable to shortcomings of transgenic mouse models used for fate mapping. To minimize these restrictions and to more faithfully mimic the endogenous NG2 expression in vivo, we generated a mouse line in which the open reading frame of the tamoxifen‐inducible form of the Cre DNA recombinase (CreERT2) was inserted into the NG2 locus by homologous recombination. Results from this novel mouse line demonstrate that at different developmental stages of the brain, NG2⁺ cells either stayed as NG2 glia or differentiated into OLs during the whole life span. Interestingly, when Cre activity was induced at embryonic stages, a significant number of reporter⁺ astrocytes could be detected in the gray matter after birth. However, in other brain regions, such as olfactory bulb, brain stem, and cerebellum, all of the NG2 glia was restricted to the OL lineage. In addition, tamoxifen‐sensitive and NG2 gene locus‐dependent gene recombination could be detected in a small, but persistent population of cortical NeuN⁺ neurons starting from the second postnatal week. GLIA 2014;62:896–913     

Expression of MCP‐1 and fractalkine on endothelial cells and astrocytes may contribute to the invasion and migration of brain macrophages in ischemic rat brain lesions

Some macrophages expressing NG2 chondroitin sulfate proteoglycan (NG2) and the macrophage marker Iba1 accumulate in the ischemic core of a rat brain subjected to transient middle cerebral artery occlusion (MCAO) for 90 min. These cells are termed BINCs (for brain Iba1⁺/NG2⁺ cells) and may play a neuroprotective role. Because BINCs are bone marrow‐derived cells, they are able to invade ischemic tissue after the onset of an ischemic insult. In this study, chemokine‐based mechanisms underlying the invasion of BINCs or their progenitor cells were investigated. We found that isolated BINCs expressed mRNA encoding CCR2 and CX3CR1 at high levels. Cultured astrocytes expressed mRNA encoding their ligands, MCP‐1 and fractalkine. Recombinant MCP‐1 and/or fractalkine, as well as astrocytes, induced the migration of BINCs in vitro. mRNA for MCP‐1, fractalkine, CCR2, and CX3CR1 was expressed in the ischemic core during the acute phase of the ischemic event. Immunohistochemical studies revealed that vascular endothelial cells and astrocytic endfeet expressed MCP‐1 and fractalkine, respectively, in the ischemic core during the acute phase. CCR2⁺/Iba1⁺ monocytes attached to the inside of the vascular wall at 1 day postreperfusion (dpr), and there were CCR2⁺/CX3CR1⁺ macrophage‐like cells in the parenchyma in the ischemic lesion core at 2 dpr, which may be the progenitors for BINCs. These results suggest that CCR2⁺ monocytes are first attracted to the ischemic lesion by MCP‐1⁺ endothelial cells and migrate toward fractalkine⁺ astrocytic endfeet through the disrupted blood–brain barrier. Thus, chemokines may play a critical role in the accumulation of neuroprotective BINCs. © 2013 Wiley Periodicals, Inc.     

Dynamic cell type‐specific expression of Nrf2 after traumatic brain injury in mice

Nrf2 plays a pivotal role in antioxidant response and anti‐inflammation after traumatic brain injury (TBI), and its deletion aggravates TBI‐induced brain damage. Previous studies have demonstrated that Nrf2 is activated post TBI, but dynamic changes in expression and cell type‐specific characteristics remain unclear. In this study, the Feeney weight‐drop contusion model was conducted to mimic TBI, and the ipsilateral cerebral cortex was collected at 1, 3, 7 and 14 days post TBI (dpi). Nrf2 protein levels were observed by western blot. Cell type‐specific localization of Nrf2 after TBI was detected at different time intervals by double immunofluorescence staining. NeuN, GFAP, IBA1 and NG2 were used as cell type‐specific markers to neurons, astrocytes, microglia and NG2 glia, respectively. After TBI, Nrf2 protein levels peaked at 1 dpi. Robust transient Nrf2 accumulation was co‐localized with neurons, which was predominant at 1 dpi. Continuous weak Nrf2 expression was detected in activated astrocytes, and the number of double positive cells peaked at 7 dpi. Inducible widespread immunostaining of Nrf2 was observed in the nucleus of the microglia, and the number of Nrf2+ microglia peaked at 7 dpi. In addition, we also explored colocalization of Nrf2 in NG2 glia, in which the percentage of Nrf2+ in NG2 glia reached a climax at 3 dpi. This study reveals that the accumulation of endogenous Nrf2 might mediate different pathophysical roles in neurons and glias after TBI, the cell‐type specific and time‐dependent expression provide insights to explain the roles of Nrf2 in different neural cells.     

Long‐term impact of neonatal inflammation on demyelination and remyelination in the central nervous system

Perinatal inflammation causes immediate changes of the blood‐brain barrier (BBB) and thus may have different consequences in adult life including an impact on neurological diseases such as demyelinating disorders. In order to determine if such a perinatal insult affects the course of demyelination in adulthood as “second hit,” we simulated perinatal bacterial inflammation by systemic administration of lipopolysaccharide (LPS) to either pregnant mice or newborn animals. Demyelination was later induced in adult animals by cuprizone [bis(cyclohexylidenehydrazide)], which causes oligodendrocyte death with subsequent demyelination accompanied by strong microgliosis and astrogliosis. A single LPS injection at embryonic day 13.5 did not have an impact on demyelination in adulthood. In contrast, serial postnatal LPS injections (P0‐P8) caused an early delay of myelin removal in the corpus callosum, which was paralleled by reduced numbers of activated microglia. During remyelination, postnatal LPS treatment enhanced early remyelination with a concomitant increase of mature oligodendrocytes. Furthermore, the postnatal LPS challenge impacts the phenotype of microglia since an elevated mRNA expression of microglia related genes such as TREM 2, CD11b, TNF‐α, TGF‐β1, HGF, FGF‐2, and IGF‐1 was found in these preconditioned mice during early demyelination. These data demonstrate that postnatal inflammation has long‐lasting effects on microglia functions and modifies the course of demyelination and remyelination in adulthood. GLIA 2014;62:1659–1670     

Impaired microglia process dynamics post‐stroke are specific to sites of secondary neurodegeneration

Stroke induces tissue death both at the site of infarction and at secondary sites connected to the primary infarction. This latter process has been referred to as secondary neurodegeneration (SND). Using predominantly fixed tissue analyses, microglia have been implicated in regulating the initial response at both damage sites post‐stroke. In this study, we used acute slice based multiphoton imaging, to investigate microglia dynamic process movement in mice 14 days after a photothrombotic stroke. We evaluated the baseline motility and process responses to locally induced laser damage in both the peri‐infarct (PI) territory and the ipsilateral thalamus, a major site of post‐stroke SND. Our findings show that microglia process extension toward laser damage within the thalamus is lost, yet remains robustly intact within the PI territory. However, microglia at both sites displayed an activated morphology and elevated levels of commonly used activation markers (CD68, CD11b), indicating that the standardly used fixed tissue metrics of microglial “activity” are not necessarily predictive of microglia function. Analysis of the purinergic P₂Y₁₂ receptor, a key regulator of microglia process extension, revealed an increased somal localization on nonresponsive microglia in the thalamus. To our knowledge, this is the first study to identify a non‐responsive microglia phenotype specific to areas of SND post‐stroke, which cannot be identified by the classical assessment of microglia activation but rather the localization of P₂Y₁₂ to the soma.