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The relation of astrocytic endfeet to the vasculature plays a key functional role in the neuro-glia-vasculature unit. We characterize the spatial organization of astrocytes and the structural aspects that facilitate their involvement in molecular exchanges. Using double transgenic mice, we performed co-immunostaining, confocal microscopy, and three-dimensional digital segmentation to investigate the biophysical and molecular organization of astrocytes and their intricate endfoot network at the micrometer level in the isocortex and hippocampus. The results showed that hippocampal astrocytes had smaller territories, reduced endfoot dimensions, and fewer contacts with blood vessels compared with those in the isocortex. Additionally, we found that both connexins 43 and 30 have a higher density in the endfoot and the former is overexpressed relative to the latter. However, due to the limitations of the method, further studies are needed to determine the exact localization on the endfoot. The quantitative information obtained in this study will be useful for modeling the interactions of astrocytes with the vasculature., (© 2024 The Author(s). GLIA published by Wiley Periodicals LLC.)

How aging affects cells of the human brain active milieu remains largely unknown. Here, we analyze astrocytes and neurons in the neocortical tissue of younger (22-50 years) and older (51-72 years) adults. Aging decreases the amount of reduced mitochondrial cytochromes in astrocytes but not neurons. The protein-to-lipid ratio decreases in astrocytes and increases in neurons. Aged astrocytes show morphological atrophy quantified by the decreased length of branches, decreased volume fraction of leaflets, and shrinkage of the anatomical domain. Atrophy correlates with the loss of gap junction coupling between astrocytes and increased input resistance. Aging is accompanied by the upregulation of glial fibrillary acidic protein (GFAP) and downregulation of membrane-cytoskeleton linker ezrin associated with leaflets. No significant changes in neuronal excitability or spontaneous inhibitory postsynaptic signaling is observed. Thus, brain aging is associated with the impaired morphological presence and mitochondrial malfunction of cortical astrocytes, but not neurons., (© 2023. The Author(s).)

Background: Astrocytes are considered key players in neuroimmunopathological processes, and they play a certain role in neuroinflammation. Rodent primary astrocyte cultures are commonly used in the study of human neuroinflammation. However, gene sequence homologies are closer between humans and dogs than between humans and rodents., New Method: We established protocols to isolate astrocytes from the canine forebrain. Cerebral hemispheres of 3-4-week-old dogs were used. The isolation procedure included the use of the Neural Tissue Dissociation Kit P, demyelination by the magnetic bead method, and separation and preparation by differential adhesion., Results: We found a 96% astrocyte purification rate after isolation by differential adhesion. Purified canine astrocytes increased the secretion of interleukin-1β, interleukin-6, and tumor necrosis factor-alpha, and increased the expression of glial fibrillary acidic protein after lipopolysaccharide stimulation. We sequenced the transcriptome of the purified canine astrocytes and analyzed the differentially expressed genes among the rodent, human, and canine astrocytes. Transcriptome profiling and gene ontology analysis of the genes co-expressed in humans and canines indicate that human and canine astrocytes may be different from their rodent counterparts in terms of mediated interactions with metals., Compared With the Existing Methods: The cells prepared by our method allow for the rapid separation of astrocytes with a relatively small resource scheme. The method also retains the cell phenotype and has an in vitro culture lifetime of approximately 2-3 months., Conclusion: We established a method for preparing canine astrocytes with high purity, which can be used to study the biological function of astrocytes in vitro., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Fetalization associated with a delay in development and the preservation of the features of the embryonic structure of the brain dominates the ontogeny of salmonids. The aim of the present study was to comparatively analyze the distribution of the glial-type aNSC markers such as vimentin and glial fibrillar acidic protein (GFAP) and the migratory neuronal precursors such as doublecortin in the telencephalon subpallium of juvenile masu salmon, Oncorhynchus masou , in normal conditions and at 1 week after an injury to the dorsal pallium. Immunohistochemical labeling of vimentin, GFAP, and doublecortin in the pallium of intact juvenile masu salmon revealed single cells with similar morphologies corresponding to a persistent pool of neuronal and/or glial progenitors. The study of the posttraumatic process showed the presence of intensely GFAP-labeled cells of the neuroepithelial type that form reactive neurogenic zones in all areas of the subpallial zone of juvenile masu salmon. A comparative analysis of the distribution of radial glia in the dorsal, ventral, and lateral zones of the subpallium showed a maximum concentration of cells in the dorsal part of subpallium (VD) and a minimum concentration in the lateral part of subpallium VL. An essential feature of posttraumatic immunolabeling in the masu salmon subpallium is the GFAP distribution patterns that are granular intracellular in the apical periventricular zone (PVZ) and fibrillar extracellular in the subventricular (SVZ) and parenchymal zones (PZ). In contrast to those in intact animals, most of the GFAP+ granules and constitutive neurogenic niches in injured fish were localized in the basal part of the PVZ. With the traumatic injury to the subpallium, the number of Vim+ cells in the lateral and ventral regions significantly increased. At 1 week post-injury, the total immunolabeling of vimentin cells in the PVZ was replaced by the granular pattern of Vim immunodistribution spreading from the PVZ to the SVZ and deeper parenchymal layers of the brain in all areas of the subpallium. A significant increase in the number of DC+ cells was observed also in all areas of the subpallium. The number of cells increased both in the PVZ and in the SVZ, as well as in the deeper PZ. Thus, at 1 week after the injury to the dorsal pallium, the number of DC, Vim, and GFAP expressing cells of the neuroepithelial type in the subpallium of juvenile masu salmon increased, and additionally GFAP+ radial glia appeared in VD, which was absent from intact animals.

Numerous beneficial effects of food restriction on aging and age-related pathologies are well documented. It is also well-established that both short- and long-term food restriction regimens induce elevated circulating levels of glucocorticoids, stress-induced hormones produced by adrenal glands that can also exert deleterious effects on the brain. In the present study, we examined the effect of long-term food restriction on the glucocorticoid hormone/glucocorticoid receptor (GR) system in the cortex during aging, in 18- and 24-month-old rats. Corticosterone level was increased in the cortex of aged ad libitum-fed rats. Food restriction induced its further increase, accompanied with an increase in the level of 11β-hydroxysteroid dehydrogenase type 1. However, alterations in the level of GR phosphorylated at Ser 232 were not detected in animals on food restriction, in line with unaltered CDK5 level, the decrease of Hsp90, and an increase in a negative regulator of GR function, FKBP51. Moreover, our data revealed that reduced food intake prevented age-related increase in the levels of NFκB, gfap , and bax , confirming its anti-inflammatory and anti-apoptotic effects. Along with an increase in the levels of c-fos , our study provides additional evidences that food restriction affects cortical responsiveness to glucocorticoids during aging.

Irreversible neuron loss caused by central nervous system injuries usually leads to persistent neurological dysfunction. Reactive astrocytes, because of their high proliferative capacity, proximity to neuronal lineage, and significant involvement in glial scarring, are ideal starting cells for neuronal regeneration. Having previously identified several small molecules as important regulators of astrocyte-to-neuron reprogramming, we established herein that miR-124, ruxolitinib, SB203580, and forskolin could co-regulate rat cortical reactive astrocyte-to-neuron conversion. The induced cells had reduced astroglial properties, displayed typical neuronal morphologies, and expressed neuronal markers, reflecting 25.9% of cholinergic neurons and 22.3% of glutamatergic neurons. Gene analysis revealed that induced neuron gene expression patterns were more similar to that of primary neurons than of initial reactive astrocytes. On the molecular level, miR-124-driven neuronal differentiation of reactive astrocytes was via targeting of the SOX9-NFIA-HES1 axis to inhibit HES1 expression. In conclusion, we present a novel approach to inducing endogenous rat cortical reactive astrocytes into neurons through co-regulation involving miR-124 and three small molecules. Thus, our research has potential implications for inhibiting glial scar formation and promoting neuronal regeneration after central nervous system injury or disease.

Blast exposures are a hallmark of contemporary military conflicts. We need improved preclinical models of blast traumatic brain injury for translation of pharmaceutical and therapeutic protocols. Compared with rodents, the ferret brain is larger, has substantial sulci, gyri, a higher white to gray matter ratio, and the hippocampus in a ventral position; these attributes facilitate comparison with the human brain. In this study, ferrets received compressed air shock waves and subsequent evaluation of glia and forms of tau following survival of up to 12 weeks. Immunohistochemistry and Western blot demonstrated altered distributions of astrogliosis and tau expression after blast exposure. Many aspects of the astrogliosis corresponded to human pathology: increased subpial reactivity, gliosis at gray-white matter interfaces, and extensive outlining of blood vessels. MRI analysis showed numerous hypointensities occurring in the 12-week survival animals, appearing to correspond to luminal expansions of blood vessels. Changes in forms of tau, including phosphorylated tau, and the isoforms 3R and 4R were noted using immunohistochemistry and Western blot in specific regions of the cerebral cortex. Of particular interest were the 3R and 4R isoforms, which modified their ratio after blast. Our data strongly support the ferret as an animal model with highly translational features to study blast injury., (2021 American Association of Neuropathologists, Inc. This work is written by US Government employees and is in the public domain in the US.)

Interlaminar astrocytes (ILAs) are a subset of cortical astrocytes that reside in layer I, express GFAP, have a soma contacting the pia, and contain long interlaminar processes that extend through several cortical layers. We studied the prenatal and postnatal development of ILAs in three species of primates (rhesus macaque, chimpanzee, and human). We found that ILAs are generated prenatally likely from radial glial (RG) cells, that ILAs proliferate locally during gestation, and that ILAs extend interlaminar processes during postnatal stages of development. We showed that the density and morphological complexity of ILAs increase with age, and that ILAs express multiple markers that are expressed by RG cells (Pax6, Sox2, and Nestin), specific to inner and outer RG cells (Cryab and Hopx), and astrocyte markers (S100β, Aqp4, and GLAST) in prenatal stages and in adult. Finally, we demonstrated that rudimentary ILAs in mouse also express the RG markers Pax6, Sox2, and Nestin, but do not express S100β, Cryab, or Hopx, and that the density and morphological complexity of ILAs differ between primate species and mouse. Together these findings contribute new information on astrogenesis of this unique class of cells and suggest a lineal relationship between RG cells and ILAs., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cortical superficial siderosis is an established haemorrhagic neuroimaging marker of cerebral amyloid angiopathy. In fact, cortical superficial siderosis is emerging as a strong independent risk factor for future lobar intracerebral haemorrhage. However, the underlying neuropathological correlates and pathophysiological mechanisms of cortical superficial siderosis remain elusive. Here we use an in vivo MRI, ex vivo MRI, histopathology approach to assess the neuropathological correlates and vascular pathology underlying cortical superficial siderosis. Fourteen autopsy cases with cerebral amyloid angiopathy (mean age at death 73 years, nine males) and three controls (mean age at death 91 years, one male) were included in the study. Intact formalin-fixed cerebral hemispheres were scanned on a 3 T MRI scanner. Cortical superficial siderosis was assessed on ex vivo gradient echo and turbo spin echo MRI sequences and compared to findings on available in vivo MRI. Subsequently, 11 representative areas in four cases with available in vivo MRI scans were sampled for histopathological verification of MRI-defined cortical superficial siderosis. In addition, samples were taken from predefined standard areas of the brain, blinded to MRI findings. Serial sections were stained for haematoxylin and eosin and Perls' Prussian blue, and immunohistochemistry was performed against amyloid-β and GFAP. Cortical superficial siderosis was present on ex vivo MRI in 8/14 cases (57%) and 0/3 controls (P = 0.072). Histopathologically, cortical superficial siderosis corresponded to iron-positive haemosiderin deposits in the subarachnoid space and superficial cortical layers, indicative of chronic bleeding events originating from the leptomeningeal vessels. Increased severity of cortical superficial siderosis was associated with upregulation of reactive astrocytes. Next, cortical superficial siderosis was assessed on a total of 65 Perls'-stained sections from MRI-targeted and untargeted sampling combined in cerebral amyloid angiopathy cases. Moderate-to-severe cortical superficial siderosis was associated with concentric splitting of the vessel wall (an advanced form of cerebral amyloid angiopathy-related vascular damage) in leptomeningeal vessels (P < 0.0001), but reduced cerebral amyloid angiopathy severity in cortical vessels (P = 0.048). In terms of secondary tissue injury, moderate-to-severe cortical superficial siderosis was associated with the presence of microinfarcts (P = 0.025), though not microbleeds (P = 0.973). Collectively, these data suggest that cortical superficial siderosis on MRI corresponds to iron-positive deposits in the superficial cortical layers, representing the chronic manifestation of bleeding episodes from leptomeningeal vessels. Cortical superficial siderosis appears to be the result of predominantly advanced cerebral amyloid angiopathy of the leptomeningeal vessels and may trigger secondary ischaemic injury in affected areas., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In the brain capillaries, endothelial cells, pericytes, astrocytes and microglia form a structural and functional complex called neurovascular unit (NVU) which is critically involved in maintaining neuronal homeostasis. In the present study, we applied a comprehensive immunohistochemical approach to investigate the structural alterations in the NVU across different Alzheimer's disease (AD) neuropathological stages. Post-mortem human cortical and hippocampal samples derived from AD patients and non-demented elderly control individuals were immunostained using a panel of markers representing specific components of the NVU including Collagen IV (basement membrane), PDGFR-β (pericytes), GFAP (astrocytes), Iba1 (microglia), MRC1 (perivascular macrophages) and lectin as an endothelial cell label. Astrocytes (GFAP) and microglia (Iba1) were quantified both in the whole visual-field and specifically within the NVU, and the sample set was additionally analyzed using anti-tau (AT8) and three different anti-Aβ (clones G2-10, G2-11, 4G8) antibodies. Analyses of lectin labeled sections showed an altered vascular distribution in AD patients as revealed by a reduced nearest distance between capillaries. Within the NVU, a Braak-stage dependent reduction in pericyte coverage was identified as the earliest structural alteration during AD progression. In comparison to non-demented elderly controls, AD patients showed a significantly higher astrocyte coverage within the NVU, which was paralleled by a reduced microglial coverage around capillaries. Assessment of perivascular macrophages moreover demonstrated a relocation of these cells from leptomeningeal arteries to penetrating parenchymal vessels in AD patients. Collectively, the results of our study represent a comprehensive first in-depth analysis of AD-related structural changes in the NVU and suggest distinct alterations in all components of the NVU during AD progression., (© 2020 International Society of Neuropathology.)

Background: A high-fat diet (HFD) is correlated with a higher risk of metabolic syndrome. The effect of HFD on neuroinflammation and apoptosis in acute stage after traumatic brain injury (TBI) in rats is not well known., Materials and Methods: Five-week-old male Sprague-Dawley (SD) rats were fed a HFD or normal diet (ND) for 8 weeks. Anesthetized male SD rats were divided into two subgroups: sham-operated and TBI. Motor function was measured using an inclined plane. The numbers of apoptotic neurons (markers Neu-N, TUNEL, caspase-3), activated astrocytes (marker GFAP) and microglia (marker OX42), and TNF-α expression in microglia and astrocytes in the ischemic cortex were investigated using an immunofluorescence assay. All of the parameters were measured on the 3rd day after TBI., Results: Rats fed a HFD for 8 weeks had higher body weight, serum cholesterol, and triglycerides. TBI-induced motor deficits, neuronal decrease, neuronal apoptosis, astrocyte and microglia activation, and TNF-α expression in microglia and astrocytes in the ischemia cortex were significantly increased in ND and HFD rats compared to sham rats. However, these parameters were not significantly different between the ND and HFD TBI groups, except motor deficit., Conclusions: HFD has no significant effects on neuronal apoptosis or neuroinflammation in acute stage compared with ND for 8 weeks after moderate TBI in experimental rats.

To understand the cellular basis for the neurodevelopmental effects of intrauterine growth restriction (IUGR), we examined the global and regional expression of various cell types within murine (Mus musculus) fetal brain. Our model employed maternal calorie restriction to 50% daily food intake from gestation day 10-19, producing IUGR offspring. Offspring had smaller head sizes with larger head:body ratios indicating a head sparing IUGR effect. IUGR fetuses at embryonic day 19 (E19) had reduced nestin (progenitors), β-III tubulin (immature neurons), Glial fibrillary acidic protein (astrocytes), and O4 (oligodendrocytes) cell lineages via immunofluorescence quantification and a 30% reduction in cortical thickness. No difference was found in Bcl-2 or Bax (apoptosis) between controls and IUGR, though qualitatively, immunoreactivity of doublecortin (migration) and Ki67 (proliferation) was decreased. In the interest of examining a potential therapeutic peptide, we next investigated a novel pro-survival peptide, mouse Humanin (mHN). Ontogeny examination revealed highest mHN expression at E19, diminishing by postnatal day 15 (P15), and nearly absent in adult (3 months). Subanalysis by sex at E19 yielded higher mHN expression among males during fetal life, without significant difference between sexes postnatally. Furthermore, female IUGR mice at E19 had a greater increase in cortical mHN versus the male fetus over their respective controls. We conclude that maternal dietary restriction-associated IUGR interferes with neural progenitors differentiating into the various cellular components populating the cerebral cortex, and reduces cerebral cortical size. mHN expression is developmental stage and sex specific, with IUGR, particularly in the females, adaptively increasing its expression toward mediating a pro-survival approach against nutritional adversity., (© 2019 Wiley Periodicals, Inc.)

Brahma-related gene 1 (Brg1) is one of the two mutually exclusive catalytic subunits of the SWItch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex. Several roles of Brg1 have been described including acting as a tumor suppressor but also functioning in neural stem cell (NSC) maintenance, neural crest development, or differentiation of oligodendrocytes and Schwann cells. Here, we generated human glial fibrillary acidic protein (hGFAP)-cre::Brg1fl/fl mice to analyze the function of Brg1 in multipotential NSCs during late stages of neural development. hGFAP-cre::Brg1fl/fl mice died approximately 2 weeks after birth. Macroscopic examination revealed a severe hydrocephalus and a decreased brain weight caused by the loss of Brg1. The cerebellum of hGFAP-cre::Brg1fl/fl mice displayed disorganized cortical layers as well as a massive hypoplasia due to a dramatically reduced number of granule neurons. The cerebrum presented with less proliferative and more apoptotic precursor cells in the subventricular zone (SVZ). Furthermore, the cerebral cortex stood out with significantly thinned upper layers and with impressive dendrite pathology. Finally, the hippocampus was severely underdeveloped with only a sparse number of detectable neurons. We conclude that NSCs depend on Brg1 to give rise to major essential brain structures including the cerebellum, the cerebral cortex, and the hippocampus., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

The identification of cerebral microinfarctions with magnetic resonance imaging (MRI) and histological methods remains challenging in aging and dementia. Here, we matched pathological changes in the microvasculature of cortical cerebral microinfarcts to MRI signals using single 100 μm-thick histological sections scanned with ultra-high-resolution 11.7 T MRI. Histologically, microinfarcts were located in superficial or deep cortical layers or transcortically, compatible with the pattern of layer-specific arteriolar blood supply of the cerebral cortex. Contrary to acute microinfarcts, at chronic stages the core region of microinfarcts showed pallor with extracellular accumulation of lipofuscin and depletion of neurons, a dense meshwork of collagen 4-positive microvessels with numerous string vessels, CD68-positive macrophages and glial fibrillary acidic protein (GFAP)-positive astrocytes. In MRI scans, cortical microinfarcts at chronic stages, called chronic cortical microinfarcts here, gave hypointense signals in T1-weighted and hyperintense signals in T2-weighted images when thinning of the tissue and cavitation and/or prominent iron accumulation were present. Iron accumulation in chronic microinfarcts, histologically verified with Prussian blue staining, also produced strong hypointense T2*-weighted signals. In summary, the microinfarct core was occupied by a dense microvascular meshwork with string vessels, which was invaded by macrophages and astroglia and contained various degrees of iron accumulation. While postmortem ultra-high-resolution single-section imaging improved MRI-histological matching and the structural characterization of chronic cortical cerebral microinfarcts, miniscule microinfarcts without thinning or iron accumulation could not be detected with certainty in the MRI scans. Moreover, string vessels at the infarct margin indicate disturbances in the microcirculation in and around microinfarcts, which might be exploitable in the diagnostics of cortical cerebral microinfarcts with MRI in vivo.

Multiple sclerosis (MS) is a chronic neurodegenerative disorder with clinical symptoms of neuroinflammation and demyelination in the central nervous system. Recently, herbal medicines are clinically effective against MS as the current disease-modifying drugs have limited effectiveness. Hence, the present study evaluated the therapeutic potential of Ocimum basilicum essential oil (OB) in ethidium bromide (EB)-induced cognitive deficits in the male rats. Further, the effect of OB (50, 100 and 200 μL/kg) was evaluated on EB-induced neuroinflammation, astrogliosis and mitochondrial dysfunction in the pre-frontal cortex (PFC) of the animals. The EB was injected through bilateral intracerebroventricular route into hippocampus to induce MS-like manifestations in the rats. OB (100 and 200 μL/kg) and Ursolic acid (UA) significantly reduced the EB-induced cognitive deficits in Morris water maze and Y-maze test paradigms. OB (100 and 200 μL/kg) and UA significantly attenuated the EB-induced neuroinflammation in terms of increase in the levels of pro-inflammatory cytokines (TNF-alpha and IL-6) in the rat PFC. Further, OB (100 and 200 μL/kg) and UA significantly attenuated the EB-induced astrogliosis in terms of increase in the levels of GFAP (Glial fibrillary acidic protein) and Iba-1 (Ionized calcium binding adaptor molecule-1) in the rat PFC. In addition, OB (100 and 200 μL/kg) and UA significantly attenuated the EB-induced decrease in the mitochondrial function, integrity, respiratory control rate and ADP/O in the PFC of the rodents. Moreover, OB (100 and 200 μL/kg) and UA significantly reduced the EB-induced mitochondria-dependent apoptosis in the PFC of the rat. Hence, it can be presumed that OB could be a potential alternative drug candidate in the pharmacotherapy of MS.

Mucopolysaccharidosis type I (MPS I) is caused by genetic deficiency of α-l-iduronidase and impairment of lysosomal catabolism of heparan sulfate and dermatan sulfate. In the brain, these substrates accumulate in the lysosomes of neurons and glial cells, leading to neuroinflammation and neurodegeneration. Their storage also affects lysosomal homeostasis-inducing activity of several lysosomal proteases including cathepsin B (CATB). In the central nervous system, increased CATB activity has been associated with the deposition of amyloid plaques due to an alternative pro-amyloidogenic processing of the amyloid precursor protein (APP), suggesting a potential role of this enzyme in the neuropathology of MPS I. In this study, we report elevated levels of protein expression and activity of CATB in cortex tissues of 6-month-old MPS I ( Idua -/- mice. Besides, increased CATB leakage from lysosomes to the cytoplasm of Idua -/- cortical pyramidal neurons was indicative of damaged lysosomal membranes. The increased CATB activity coincided with an elevated level of the 16-kDa C-terminal APP fragment, which together with unchanged levels of β-secretase 1 was suggestive for the role of this enzyme in the amyloidogenic APP processing. Neuronal accumulation of Thioflavin-S-positive misfolded protein aggregates and drastically increased levels of neuroinflammatory glial fibrillary acidic protein (GFAP)-positive astrocytes and CD11b-positive activated microglia were observed in Idua -/- cortex by confocal fluorescent microscopy. Together, our results point to the existence of a novel CATB-associated alternative amyloidogenic pathway in MPS I brain induced by lysosomal storage and potentially leading to neurodegeneration.

Baicalin is a plant-derived flavonoid that has anti-inflammatory and anti-oxidative effects. We investigated an anti-inflammatory effect of baicalin against lipopolysaccharide (LPS)-induced damage in cerebral cortex. Adult mice were divided into control, LPS-treated, and LPS and baicalin co-treated animals. LPS (250 µg/kg/day) and baicalin (10 mg/kg/day) were intraperitoneally injected for 7 days. LPS treatment induced histopathological changes in cerebral cortex, whereas baicalin protected neuronal cells against LPS toxicity. Moreover, baicalin treatment attenuated LPS-induced increases of reactive oxygen species and oxidative stress in cerebral cortices. Ionized calcium binding adaptor molecule-1 (Iba-1) and glial fibrillary acidic protein (GFAP) are known as markers of activated microglia and astrocyte, respectively. Results of Western blot and immunofluorescence staining showed that LPS exposure induces increases of Iba-1 and GFAP expressions, whereas baicalin alleviates LPS-induced increases of these proteins. Baicalin also prevented LPS-induced increase of nuclear factor kappa B (NF-κB). LPS treatment led to increases of pro-inflammatory factors including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Increases of these pro-inflammatory mediators were attenuated in baicalin co-treated animals. These results demonstrated that baicalin regulates neuroglia activation and modulates inflammatory factors in LPS-induced neuronal injury. Thus, our findings suggest that baicalin exerts a neuroinflammatory effect against LPS-induced toxicity through decreasing oxidative stress and inhibiting NF-κB mediated inflammatory factors, such as IL-1β and TNF-α.

WW domain-containing oxidoreductase (Wwox) is a putative tumor suppressor. Several germline mutations of Wwox have been associated with infant neurological disorders characterized by epilepsy, growth retardation, and early death. Less is known, however, about the pathological link between Wwox mutations and these disorders or the physiological role of Wwox in brain development. In this study, we examined age-related expression and histological localization of Wwox in forebrains as well as the effects of loss of function mutations in the Wwox gene in the immature cortex of a rat model of lethal dwarfism with epilepsy ( lde/lde ). Immunostaining revealed that Wwox is expressed in neurons, astrocytes, and oligodendrocytes. lde/lde cortices were characterized by a reduction in neurite growth without a reduced number of neurons, severe reduction in myelination with a reduced number of mature oligodendrocytes, and a reduction in cell populations of astrocytes and microglia. These results indicate that Wwox is essential for normal development of neurons and glial cells in the cerebral cortex.

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by silencing of the FMR1 gene and subsequent loss of its protein product, fragile X retardation protein (FMRP). One of the most robust neuropathological findings in post-mortem human FXS and Fmr1 KO mice is the abnormal increase in dendritic spine densities, with the majority of spines showing an elongated immature morphology. However, the exact mechanisms of how FMRP can regulate dendritic spine development are still unclear. Abnormal dendritic spines can result from disturbances of multiple factors during neurodevelopment, such as alterations in neuron numbers, position and glial cells. In this study, we undertook a comprehensive histological analysis of the cerebral cortex in Fmr1 KO mice. They displayed significantly fewer neuron and PV-interneuron numbers, along with altered cortical lamination patterns. In terms of glial cells, Fmr1 KO mice exhibited an increase in Olig2-oligodendrocytes, which corresponded to the abnormally higher myelin expression in the corpus callosum. Iba1-microglia were significantly reduced but GFAP-astrocyte numbers and intensity were elevated. Using primary astrocytes derived from KO mice, we further demonstrated the presence of astrogliosis characterized by an increase in GFAP expression and astrocyte hypertrophy. Our findings provide important information on the cortical architecture of Fmr1 KO mice, and insights towards possible mechanisms associated with FXS.

Braided multi-electrode probes (BMEPs) for neural interfaces comprise ultrafine microwire bundles interwoven into tubular braids. BMEPs provide highly flexible probes and tethers, and an open lattice structure with up to 24 recording/stimulating channels in precise geometries, currently all within a [Formula: see text] diameter footprint. This paper compares the long-term tissue effects of BMEPs ( [Formula: see text] wires) versus single conventional 50- [Formula: see text] wires, by testing nearby chronic immune response and neural survival in rat cortex. Four different types of electrodes were implanted in cortex in each of eight rats: 1) BMEP with tether; 2) tethered 50- [Formula: see text] wire; 3) BMEP without a tether; and 4) untethered 50- [Formula: see text] wire. Quantitative immunohistological statistical comparisons after eight weeks using GFAP, ED1, and NeuN staining clearly showed that both BMEP implants had significantly less tissue immune response and more neuronal survival than either of the 50- [Formula: see text] wires ( ) in each of the eight rats. Data strongly indicate that BMEP tissue responses are superior, and that BMEP designs partly alleviate chronic tissue inflammatory responses and neural losses. The flexible body, tether and open braid lattice, and finer wire diameters of BMEP designs may all contribute to reducing the biological long-term response.

RASopathies are a family of related syndromes caused by mutations in regulators of the RAS/Extracellular Regulated Kinase 1/2 (ERK1/2) signaling cascade that often result in neurological deficits. RASopathy mutations in upstream regulatory components, such as NF1, PTPN11/SHP2, and RAS have been well-characterized, but mutation-specific differences in the pathogenesis of nervous system abnormalities remain poorly understood, especially those involving mutations downstream of RAS. Here, we assessed cellular and behavioral phenotypes in mice expressing a Raf1L613V gain-of-function mutation associated with the RASopathy, Noonan Syndrome. We report that Raf1L613V/wt mutants do not exhibit a significantly altered number of excitatory or inhibitory neurons in the cortex. However, we observed a significant increase in the number of specific glial subtypes in the forebrain. The density of GFAP+ astrocytes was significantly increased in the adult Raf1L613V/wt cortex and hippocampus relative to controls. OLIG2+ oligodendrocyte progenitor cells were also increased in number in mutant cortices, but we detected no significant change in myelination. Behavioral analyses revealed no significant changes in voluntary locomotor activity, anxiety-like behavior, or sociability. Surprisingly, Raf1L613V/wt mice performed better than controls in select aspects of the water radial-arm maze, Morris water maze, and cued fear conditioning tasks. Overall, these data show that increased astrocyte and oligodendrocyte progenitor cell (OPC) density in the cortex coincides with enhanced cognition in Raf1L613V/wt mutants and further highlight the distinct effects of RASopathy mutations on nervous system development and function., Competing Interests: The authors have declared that no competing interests exist.

Longitudinal studies using two-photon fluorescence microscopy (TPFM) are critical for facilitating cellular scale imaging of brain morphology and function. Studies have been conducted in the mouse due to their relatively higher transparency and long term patency of a chronic cranial window. Increasing availability of transgenic rat models, and the range of established behavioural paradigms, necessitates development of a chronic preparation for the rat. However, surgical craniotomies in the rat present challenges due to craniotomy closure by wound healing and diminished image quality due to inflammation, restricting most rat TPFM experiments to acute preparations. Long-term patency is enabled by employing sterile surgical technique, minimization of trauma with precise tissue handling during surgery, judicious selection of the size and placement of the craniotomy, diligent monitoring of animal physiology and support throughout the surgery, and modification of the home cage for long-term preservation of cranial implants. Immunohistochemical analysis employing the glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule-1 (Iba-1) showed activation and recruitment of astrocytes and microglia/macrophages directly inferior to the cranial window at one week after surgery, with more diffuse response in deeper cortical layers at two weeks, and amelioration around four weeks post craniotomy. TPFM was conducted up to 14 weeks post craniotomy, reaching cortical depths of 400 µm to 600 µm at most time-points. The rate of signal decay with increasing depth and maximum cortical depth attained had greater variation between individual rats at a single time-point than within a rat across time.

Proteins and microRNAs (miRNAs) within the axon locally regulate axonal development. However, protein profiles of distal axons of cortical neurons have not been fully investigated. In particular, networks of genes encoding axonal proteins and their related miRNAs in sub compartments of neurons such as axons remain unknown. Using embryonic cortical neurons cultured in a microfluidic device and proteomic approaches, we found that distal axons contain 883 proteins. Bioinformatics analysis revealed that 94 out of these 883 proteins are related to regulating axonal growth. Of the 94 genes encoding these proteins, there were 56 candidate genes that can be putatively targeted by axon-enriched 62 miRNAs with 8mer sites that exactly match these target genes. Among them, we validated 11 proteins and 11 miRNAs, by means of western blot and RT-PCR, respectively. Treatment of distal axons with chondroitin sulfate proteoglycans (CSPGs) that inhibit axonal growth elevated miR-133b, -203a, -29a, and -92a, which were associated with reduced protein level of AKT, MTOR, PI3K, DPYSL2, MAP1B, and PPP2CA. In contrast, reduction of miR-128, -15b, -195, -26b, -34b, -376b, and -381 by CSPGs was accompanied by increased EZR, KIF5A, DCX, GSK3B, and ROCK2 proteins. In silico pathway analysis revealed an interconnected network of these miRNAs and protein coding genes that is highly related to regulating axonal growth. Our data provide new insights into networks of miRNAs and their related proteins in distal axons in mediating axonal growth.

Glutamate-ammonia ligase (glutamine synthetase; Glul) is enriched in astrocytes and serves as the primary enzyme for ammonia detoxification and glutamate inactivation in the brain. Loss of astroglial Glul is reported in hippocampi of epileptic patients, but the mechanism by which Glul deficiency might cause disease remains elusive. Here we created a novel mouse model by selectively deleting Glul in the hippocampus and neocortex. The Glul deficient mice were born without any apparent malformations and behaved unremarkably until postnatal week three. There were reductions in tissue levels of aspartate, glutamate, glutamine and GABA and in mRNA encoding glutamate receptor subunits GRIA1 and GRIN2A as well as in the glutamate transporter proteins EAAT1 and EAAT2. Adult Glul-deficient mice developed progressive neurodegeneration and spontaneous seizures which increased in frequency with age. Importantly, progressive astrogliosis occurred before neurodegeneration and was first noted in astrocytes along cerebral blood vessels. The responses to CO 2 -provocation were attenuated at four weeks of age and dilated microvessels were observed histologically in sclerotic areas of cKO. Thus, the abnormal glutamate metabolism observed in this model appeared to cause epilepsy by first inducing gliopathy and disrupting the neurovascular coupling., (Copyright © 2018. Published by Elsevier Ltd.)

Objectives: Osteopontin (OPN) is an inflammatory cytokine inducer involved in cell proliferation and migration in inflammatory diseases or tumors. To investigate the function of OPN in astrocyte activation during brain injury, we compared OPN-deficient (OPN/KO) with wild-type (WT) mouse brains after stab wound injury and primary culture of astrocytes., Methods: Primary cultures of astrocytes were prepared from either WT or OPN/KO postnatal mouse brains. Activation efficiency of astrocytes in primary culture was accessed using Western blotting by examining the protein levels of glial fibrillary acidic protein (GFAP) and tenascin-C (TN-C), which are markers for reactive astrocytes, following lipopolysaccharide (LPS) stimulation. Furthermore, the stab wound injury on the cerebral cortex as a brain traumatic injury model was used, and activation of astrocytes and microglial cells was investigated using immunofluorescent analysis on fixed brain sections., Results: Primary cultures of astrocytes prepared from WT or OPN/KO postnatal mouse brains showed that only 25% of normal shaped astrocytes in a flask were produced in OPN/KO mice. The expression levels of both GFAP and TN-C were downregulated in the primary culture of astrocytes from OPN/KO mice compared with that from WT mice. By the immunofluorescent analysis on the injured brain sections, glial activation was attenuated in OPN/KO mice compared with WT mice., Discussion: Our data suggest that OPN is essential for proper astrocytic generation in vitro culture prepared from mouse cerebral cortex. OPN is indispensable for astrocyte activation in the mouse brain injury model and in LPS stimulated primary culture., Abbreviations: AQP4: aquaporin 4; BBB: blood brain barrier; BrdU: bromo-deoxy uridine; CNS: central nervous system; GFAP: glial fibllirary acidic protein; IgG: immunoglobulin G; LPS: lipopolysaccharide; OPN: osteopontin; OPN/KO: osteopontin-deficient; TN-C: tenascin-C.

Nuclear factor-kappaB (NF-κB) is activated in neural progenitor cells in the developing murine cerebral cortex during the neurogenic phase, when it acts to prevent premature neuronal differentiation. Here we show that NF-κB activation continues in mouse neocortical neural progenitor cells during the neurogenic-to-gliogenic switch. Blockade of endogenous NF-κB activity during neocortical gliogenesis leads to the formation of supernumerary committed gliogenic progenitors and premature glial cell differentiation. Conversely, forced NF-κB activation during the neocortical neurogenic-to-gliogenic transition causes delayed gliogenic commitment and decreased astroglial gene expression. NF-κB activation continues in neocortical gliogenic progenitors following commitment and is important to inhibit the differentiation of oligodendrocyte precursor cells and to maintain persistent expression of glial fibrillary acidic protein in maturing astrocytes. These results reveal a number of previously uncharacterized roles for NF-κB during different phases of neocortical gliogenesis and identify NF-κB as an inhibitor of early oligodendrocyte development in the cerebral cortex., (© 2018 Wiley Periodicals, Inc.)

We investigated astroglial cells in several areas of the telencephalic cortex of the lesser hedgehog tenrec (Echinops telfairi). Compared to other mammals, the cortex of the tenrec has a relatively large paleocortex and a low encephalization index. We stained sections from tenrec forebrains with structural and functional glia markers focusing on selected cortical areas, the paleocortex, rhinal cortex, neocortex and the dentate gyrus of the hippocampal formation. We found that in all parts of the tenrec forebrain cortex, radial processes exist which are positive for glial fibrillary acidic protein (GFAP) although with differential localization: in the rhinal cortex and neocortical region radial glial fibers are located in the subventricular regions, whereas in the dentate gyrus and paleocortex they appear to arise from the cells in the respective granular layers. The relatively high abundance of the radial fibers in layer III of the paleocortex was very conspicuous. Only few of these radial processes were also co-labeled with doublecortin (DCX), yet most of the DCX-positive cells were negative for GFAP. The GFAP-positive radial fibers were in turn neither positive for glutamine synthetase, nor did they show immunoreactivity for the astroglia-specific water channel aquaporin-4 (AQP4). Star-shaped astrocytes, however, displayed the typical perivascular and subpial expression patterns for AQP4. We conclude that the radial glia in the adult tenrec represents an immature form of astroglia that persists in these animals throughout life.

Previous studies have shown that estradiol reduces reactive gliosis after a stab wound injury in the cerebral cortex. Since the therapeutic use of estradiol is limited by its peripheral hormonal effects, it is of interest to determine whether synthetic estrogenic compounds with tissue-specific actions regulate reactive gliosis. Tibolone is a synthetic steroid that is widely used for the treatment of climacteric symptoms and/or the prevention of osteoporosis. In this study, we have assessed the effect of tibolone on reactive gliosis in the cerebral cortex after a stab wound brain injury in ovariectomized adult female mice. By 7 days after brain injury, tibolone reduced the number of glial fibrillary acidic protein (GFAP) immunoreactive astrocytes, the number of ionized calcium binding adaptor molecule 1 (Iba1) immunoreactive microglia, and the number of microglial cells with a reactive phenotype in comparison to vehicle-injected animals. These effects on gliosis were associated with a reduction in neuronal loss in the proximity to the wound, suggesting that tibolone exerts beneficial homeostatic actions in the cerebral cortex after an acute brain injury.

Background: Loss of UBE3A causes Angelman syndrome, whereas excess UBE3A activity appears to increase the risk for autism. Despite this powerful association with neurodevelopmental disorders, there is still much to be learned about UBE3A, including its cellular and subcellular organization in the human brain. The issue is important, since UBE3A's localization is integral to its function., Methods: We used light and electron microscopic immunohistochemistry to study the cellular and subcellular distribution of UBE3A in the adult human cerebral cortex. Experiments were performed on multiple tissue sources, but our results focused on optimally preserved material, using surgically resected human temporal cortex of high ultrastructural quality from nine individuals., Results: We demonstrate that UBE3A is expressed in both glutamatergic and GABAergic neurons, and to a lesser extent in glial cells. We find that UBE3A in neurons has a non-uniform subcellular distribution. In somata, UBE3A preferentially concentrates in euchromatin-rich domains within the nucleus. Electron microscopy reveals that labeling concentrates in the head and neck of dendritic spines and is excluded from the PSD. Strongest labeling within the neuropil was found in axon terminals., Conclusions: By highlighting the subcellular compartments in which UBE3A is likely to function in the human neocortex, our data provide insight into the diverse functional capacities of this E3 ligase. These anatomical data may help to elucidate the role of UBE3A in Angelman syndrome and autism spectrum disorder., Competing Interests: All procedures regarding human tissue were performed with the approval of the UCSF Committee on Human Research and the University of Maryland Institutional Review Board.All authors read and approved the final manuscript.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Lead (Pb) is a neurotoxic heavy metal that largely affects the developing nervous system. The present study examined the temporal effect of perinatal Pb exposure on neurogenesis and cortical neuronal morphology. Wistar pregnant rats were exposed to 0.5% lead acetate throughout pregnancy and to postnatal day (PD) 28. Offspring were grouped as gestational day (GD) 18 and 21 and PD 7, 14, 21, and 28 in both control and experimental groups. Brain sections were processed for immunohistological staining with anti-proliferating cell nuclear antigen (PCNA) or glial fibrillary acidic protein (GFAP). Brains from 14, 21, and 28 PDs pups were processed for Golgi-Cox stain. Pb exposure significantly increased PCNA-positive nuclei in the ventricular and subventricular zones of the lateral ventricle at 18 and 21 GDs. Postnatally, the Pb-treated groups showed a significant decrease in PCNA-positivity and neuron density compared to control. This reduction was associated with an increase in damaged or apoptotic cell profiles in the experimental groups. At PD 21, there was a significant increase in GFAP immunoreactivity in Pb-exposed groups compared with control. Furthermore, the total apical and basal dendritic length of pyramidal neurons in layer 2-3 of the Golgi-Cox stained sensorimotor cortex was comparable in both control and Pb-exposed groups. Spine density per 10 µm was significantly increased at PD 14 and 21 on the apical dendrites but not basal dendrites of Pb-treated groups. In conclusion, developmental Pb exposure in rats induces a toxic effect on neurogenesis and on cortical neurons, which may be related to cognitive disabilities observed in children exposed to lead.

Astrocytes play a pivotal role in neuronal survival in the setting of post-ischemic brain inflammation, but the astrocyte-derived mediators of ischemic brain injury remain to be defined. 2-(2-Benzofu-ranyl)-2-imidazoline (2-BFI) is a newly discovered ligand for high-affinity imidazoline I2 receptors (I2Rs) mainly located on the mitochondrial outer membrane in astrocytes. We previously reported that in a rat model of cerebral ischemia-reperfusion injury, 2-BFI limits infarct volume, reduces neurological impairment scores, and inhibits neuronal apoptosis in the ischemic penumbra. This study was performed to clarify the underlying mechanism in an astrocyte oxygen-glucose deprivation (OGD) model. The results show that 2-BFI reduces lipid peroxidation and inhibits mitochondria apoptotic pathways., (Copyright © 2017. Published by Elsevier B.V.)

Objective: To investigate the effects of ketamine on autophagy and apoptosis of astrocytes in the cerebral cortex of rats, and determine whether nuclear factor-κB (NF-κB) pathway is involved in the regulation of autophagy and apoptosis of astrocytes., Materials and Methods: A total of 36 male Sprague-Dawley (SD) rats were randomly divided into 3 groups: control group (Group C: intraperitoneal injection of equal amount of normal saline), glutamic acid group (Group G: intraperitoneal injection of 1 mg/kg glutamic acid) and glutamic acid + ketamine group (Group GK: intraperitoneal injection of 1 mg/kg glutamic acid and then injection of 5 mg/kg ketamine after 30 min). The cerebral cortex of rats in each group was taken after successive administration for 5 d. The number of glial fibrillary acidic protein (GFAP)-positive cells in the cerebral cortex of rats in each group was detected via immunofluorescence. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells (apoptotic cells) in the cerebral cortex was detected via TUNEL staining. The levels of inflammatory factors were detected using the enzyme-linked immunosorbent assay (ELISA) kit. Moreover, the expressions of autophagy-related proteins and apoptosis-related proteins in the cerebral cortex were detected via Western blotting, and the expressions of IκB-a and NF-κBp65 were also detected., Results: The results of immunofluorescence showed that the number of GFAP-positive cells in the cerebral cortex of rats in Group G was significantly increased compared with that in Group C (p<0.01), and it was significantly decreased in Group GK compared with that in Group G (p<0.01). The results of TUNEL staining revealed that the number of TUNEL-positive cells in the cerebral cortex in Group G was significantly larger than that in Group C, and it was significantly smaller in Group GK than that in Group G (p<0.01). Results of ELISA demonstrated that compared with those in Group C, the contents of interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) in Group G were significantly increased (p<0.01), but the content of IL-10 was significantly decreased (p<0.01). Compared with those in Group G, the contents of IL-6 and TNF-a in Group GK were significantly decreased (p<0.01), but the level of IL-10 was statistically elevated (p<0.01). Compared with those in Group C, the levels of LC3 II/I and cleaved caspase-3 in the cerebral cortex in Group G were significantly increased (p<0.01), but the p62 level and B-cell lymphoma-2/Bcl-2 associated X protein (Bcl-2/Bax) ratio were significantly decreased (p<0.01). In Group GK, the levels of LC3 II/I and cleaved caspase-3 were reduced, but the p62 level and Bcl-2/Bax ratio were increased. The expressions of IκB-α and NF-κBp65 in Group G were significantly decreased compared with those in Group C (p<0.01), and they were significantly higher in Group GK than those in Group G (p<0.01)., Conclusions: Ketamine can reduce the glutamic acid-induced activation of astrocytes in the cerebral cortex, inhibit the autophagy and alleviate the apoptosis of astrocytes, the process of which is mediated by the NF-κB pathway, which provides the new molecular basis of ketamine in protecting astrocytes.

Sonic Hedgehog (Shh) plays an instrumental role in brain development, fine-tuning processes such as cell proliferation, patterning, and fate specification. Although, mutations in the SHH pathway in humans are associated with various neurodevelopmental disorders, ranging from holoprosencephaly to schizophrenia, its expression pattern in the developing human brain is not well established. We now determined the previously not reported wide expression of SHH in the human fetal cerebral cortex during most of the gestation period (10-40 gestational weeks). This spatiotemporal distribution puts Shh in a position to influence the fundamental processes involved in corticogenesis. SHH expression increased during development, shifting from progenitor cells in the proliferative zones to neurons, both glutamatergic and GABAergic, and astrocytes in upper cortical compartments. Importantly, the expression of its downstream effectors and complementary receptors revealed evolutionary differences in SHH-pathway gene expression between humans and rodents.

Deletion of the phosphatase and tensin (PTEN) gene in neonatal mice leads to enlargement of the cell bodies of cortical motoneurons (CMNs) in adulthood (Gutilla et al., 2016). Here, we assessed whether PTEN deletion in adult mice would trigger growth of mature neurons. PTEN was deleted by injecting AAV-Cre into the sensorimotor cortex of adult transgenic mice with a lox-P flanked exon 5 of the PTEN gene and Cre-dependent reporter gene tdTomato. PTEN-deleted CMN's identified by tdT expression and retrograde labeling with fluorogold (FG) were significantly enlarged four months following PTEN deletion, and continued to increase in size through the latest time intervals examined (12-15 months post-deletion). Sholl analyses of tdT-positive pyramidal neurons revealed increases in dendritic branches at 6 months following adult PTEN deletion, and greater increases at 12 months. 12 months after adult PTEN deletion, axons in the medullary pyramids were significantly larger and G-ratios were higher. Mice with PTEN deletion exhibited no overt neurological symptoms and no seizures. Assessment of motor function on the rotarod and cylinder test revealed slight impairment of coordination with unilateral deletion; however, mice with bilateral PTEN deletion in the motor cortex performed better than controls on the rotarod at 8 and 10 months post-deletion. Our findings demonstrate that robust neuronal growth can be induced in fully mature cortical neurons long after the developmental period has ended and that this continuous growth occurs without obvious functional impairments., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Although many studies show the toxic effects of proline, recently it has been reported some anti-inflammatory effect of this amino acid. Our principal objective was to investigate the effects of proline on the alterations caused by LPS (lipopolysaccharide) administration in the cerebral cortex and cerebellum of young Wistar rats. The animals were divided into four groups: control (0.85% saline); proline, (12.8 μmol of proline/g body weight from day 7 to 13; 14.6 μmol of proline/g body weight from day 14 to 17 and 16.4 μmol of proline/g body weight from day 18 to 21); LPS (1 mg/g body weight); LPS plus proline. The animals were killed at 22 days of age, 12 h after the last injection, by decapitation without anesthesia. The brain cortex and cerebellum were separated for chemical determinations. The effects of proline and LPS in the cerebral cortex and cerebellum on the expression of S100B and GFAP, oxidative stress parameters, enzymes of phosphoryl transfer network activity, and mitochondrial respiration chain complexes were investigated. Two-way ANOVA showed that the administration of proline did not alter the analyzed parameter in cerebral cortex and cerebellum. On the other hand, LPS administration caused a change in these parameters. Besides, the co-administration of proline and LPS showed the ability of Pro in preventing the effects of LPS. These results indicated that LPS induces inflammation, oxidative stress, and alters energy parameters in cerebral cortex and cerebellum of the rats. Moreover, co-administration of Pro was able to prevent these harmful effects of LPS.

Human mesial temporal lobe epilepsy (MTLE) features subregion-specific hippocampal neurodegeneration and reactive astrogliosis, including up-regulation of the glial fibrillary acidic protein (GFAP) and down-regulation of glutamine synthetase (GS). However, the regional astrocytic expression pattern of GFAP and GS upon MTLE-associated neurodegeneration still remains elusive. We assessed GFAP and GS expression in strict correlation with the local neuronal number in cortical and hippocampal surgical specimens from 16 MTLE patients using immunohistochemistry, stereology and high-resolution image analysis for digital pathology and whole-slide imaging. In the cortex, GS-positive (GS+) astrocytes are dominant in all neuronal layers, with a neuron to GS+ cell ratio of 2:1. GFAP-positive (GFAP+) cells are widely spaced, with a GS+ to GFAP+ cell ratio of 3:1-5:1. White matter astrocytes, on the contrary, express mainly GFAP and, to a lesser extent, GS. In the hippocampus, the neuron to GS+ cell ratio is approximately 1:1. Hippocampal degeneration is associated with a reduction of GS+ astrocytes, which is proportional to the degree of neuronal loss and primarily present in the hilus. Up-regulation of GFAP as a classical hallmark of reactive astrogliosis does not follow the GS-pattern and is prominent in the CA1. Reactive alterations were proportional to the neuronal loss in the neuronal somatic layers (stratum pyramidale and hilus), while observed to a lesser extent in the axonal/dendritic layers (stratum radiatum, molecular layer). We conclude that astrocytic GS is expressed in the neuronal somatic layers and, upon neurodegeneration, is down-regulated proportionally to the degree of neuronal loss., (© 2018 Wiley Periodicals, Inc.)

Intracerebroventricular injection of streptozotocin (ICV-STZ) in rodents leads to cognitive impairments and several pathological changes like Alzheimer's disease (AD). However, there is hardly any research about the effect of ICV-STZ on regional cerebral glucose metabolism in rodents. Previous studies have demonstrated that intranasal insulin improves cognition in AD patients. However, the underlying mechanism remains elusive. Here, we treated the ICV-STZ rats with daily intranasal delivery of insulin (2 U/day) for 6 consecutive weeks, then monitored 18 F-fluorodeoxyglucose ( 18 F-FDG) uptake using a high-resolution small-animal positron emission tomography (microPET) and studied the expression of neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) using immunohistochemical staining. We observed that 18 F-FDG uptake decreased significantly at the prefrontal cortex, cingulate cortex, striatum, hippocampus, and entorhinal cortex in ICV-STZ rats as compared with the control rats. Intranasal insulin restores the cerebral glucose metabolism in prefrontal and cingulate cortex and attenuates astroglia activation and neuronal loss in the hippocampus of ICV-STZ rats. These findings provide the mechanistic basis for treating AD patients with intranasal insulin.

Bergmann glia (BG) are important in the inward type of radial migration of cerebellar granule neurons (CGNs). However, details regarding the functions of Cdc42 and Rac in BG for radial migration of CGN are unknown. To examine the roles of Cdc42 and Rac in BG during cerebellar corticogenesis, mice with a single deletion of Cdc42 or Rac1 and those with double deletions of Cdc42 and Rac1 under control of the glial fibrillary acidic protein (GFAP) promoter: GFAP-Cre;Cdc42 flox/flox (Cdc42-KO), GFAP-Cre;Rac1 flox/flox (Rac1-KO), and GFAP-Cre; Cdc42 flox/flox ;Rac1 flox/flox (Cdc42/Rac1-DKO) mice, were generated. Both Cdc42-KO and Rac1-KO mice, but more obviously Cdc42-KO mice, had disturbed alignment of BG in the Purkinje cell layer (PCL). We found that Cdc42-KO, but not Rac1-KO, induced impaired radial migration of CGNs in the late phase of radial migration, leading to retention of CGNs in the lower half of the molecular layer (ML). Cdc42-KO, but not Rac1-KO, mice also showed aberrantly aligned Purkinje cells (PCs). These phenotypes were exacerbated in Cdc42/Rac1-DKO mice. Alignment of BG radial fibers in the ML and BG endfeet at the pial surface of the cerebellum evaluated by GFAP staining was disturbed and weak in Cdc42/Rac1-DKO mice, respectively. Our data indicate that Cdc42 and Rac, but predominantly Cdc42, in BG play important roles during the late phase of radial migration of CGNs. We also report here that Cdc42 is involved in gliophilic migration of CGNs, in contrast to Rac, which is more closely connected to regulating neurophilic migration., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objectives: Severe grey and white matter volume reductions were found in patients with anorexia nervosa (AN) that were linked to neuropsychological deficits while their underlying pathophysiology remains unclear. For the first time, we analysed the cellular basis of brain volume changes in an animal model (activity-based anorexia, ABA)., Methods: Female rats had 24 h/day running wheel access and received reduced food intake until a 25% weight reduction was reached and maintained for 2 weeks., Results: In ABA rats, the volumes of the cerebral cortex and corpus callosum were significantly reduced compared to controls by 6% and 9%, respectively. The number of GFAP-positive astrocytes in these regions decreased by 39% and 23%, total astrocyte-covered area by 83% and 63%. In neurons no changes were observed. The findings were complemented by a 60% and 49% reduction in astrocyte (GFAP) mRNA expression., Conclusions: Volumetric brain changes in ABA animals mirror those in human AN patients. These alterations are associated with a reduction of GFAP-positive astrocytes as well as GFAP expression. Reduced astrocyte functioning could help explain neuronal dysfunctions leading to symptoms of rigidity and impaired learning. Astrocyte loss could constitute a new research target for understanding and treating semi-starvation and AN.

Prematurely born children often develop neurodevelopmental delay that has been correlated with reduced growth and microstructural alterations in the cerebral cortex. Much research has focused on apoptotic neuronal cell death as a key neuropathological features following preterm brain injuries. How scattered apoptotic death of neurons may contribute to microstructural alterations remains unknown. The present study investigated in a rat model the effects of targeted neuronal apoptosis on cortical microstructure using in vivo MRI imaging combined with neuronal reconstruction and histological analysis. We describe that mild, targeted death of layer IV neurons in the developing rat cortex induces MRI-defined metabolic and microstructural alterations including increased cortical fractional anisotropy. Delayed architectural modifications in cortical gray matter and myelin abnormalities in the subcortical white matter such as hypomyelination and microglia activation follow the acute phase of neuronal death and axonal degeneration. These results establish the link between mild cortical apoptosis and MRI-defined microstructure changes that are reminiscent to those previously observed in preterm babies., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Available evidence suggests that the magnitude of the foreign body response (FBR) to implants placed in cortical brain tissue is affected by the extent of vasculature damage following device insertion and the magnitude of the ensuing macrophage response. Since the extracellular matrix (ECM) serves as a natural hemostatic and immunomodulatory agent, we examined the ability of an FDA-approved neurosurgical hemostatic coating and an ECM coating derived from primary rat astrocytes to reduce the FBR surrounding a penetrating microelectrode array chronically implanted in rat cortex. Using quantitative methods, we examined various components of the FBR in vitro and after implantation. In vitro assays showed that both coatings accelerated coagulation in a similar fashion but only the astrocyte-derived material suppressed macrophage activation. In addition, the ECM coating derived from astrocytes, also decreased the astrogliotic response 8 weeks after implantation. Neither coating had a significant influence on the intensity or spatial distribution of FBR biomarkers 1 week after implantation or on degree of macrophage activation or neuronal survival at the later time point. The results show that microelectrode coatings with similar hemostatic properties but different immunomodulatory characteristics differentially affect the FBR to an anchored, single-shank, silicon microelectrode array. The results also support the concept that divergent biological pathways affect the various components of the FBR in the CNS and suggests that decreasing its impact will require a multifaceted approach., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

An essential feature of Alzheimer's disease (AD) is implicated in brain energy metabolic impairment that is considered underlying pathogenesis of cognitive impairment. Therefore, therapeutic interventions to allay cognitive deficits that target energy metabolism may be an efficacy strategy in AD. In this study, we found that electroacupuncture (EA) at the DU20 acupoint obviously increased glucose metabolism in specific brain regions such as cortex, hippocampus, cingulate gyrus, basal forebrain septum, brain stem, and cerebellum in APP/PS1 transgenic mice by animal 18 F-Fluoro-2-deoxy-D-Glucose ( 18 F-FDG)/positron emission tomography (PET) imaging, accompanied by cognitive improvements in the spatial reference learning and memory and memory flexibility and novel object recognition performances. Further evidence shown energy metabolism occurred in neurons or non-neuronal cells of the cortex and hippocampus in terms of the co-location of GLUT3/NeuN and GLUT1/GFAP. Simultaneously, metabolic homeostatic factors were critical for glucose metabolism, including phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and AKT serine/threonine kinase. Furthermore, EA-induced phosphorylated AMPK and AKT inhibited the phosphorylation level of the mammalian target of rapamycin (mTOR) to decrease the accumulation of amyloid-beta (Aβ) in the cortex and hippocampus. These findings are concluded that EA is a potential therapeutic target for delaying memory decline and Aβ deposition of AD. The AMPK and AKT are implicated in the EA-induced cortical and hippocampal energy metabolism, which served as a contributor to improving cognitive function and Aβ deposition in a transgenic mouse model of AD., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Striatum and the cerebral cortex are regions susceptible to secondary injury after intracerebral hemorrhage (ICH) and glial cells in tissue adjacent to the hematoma may modulate cellular vulnerability after brain damage. Nonetheless, while the glial- associated changes occurring in the cerebral cortex after ICH may be important in maximizing brain recovery, they are not fully understood. The aim of this study was to evaluate the temporal profile of glial-associated changes in the cerebral cortex after ICH. First, the motor consequences of ICH and its relation to the lesion volume were analyzed. Secondly, glial cell proportion (GFAP+ and S100B+ astrocytes, CD11+ microglia) in the ipsilesional sensorimotor cortex and striatum, using flow cytometry were evaluated. ELISA was used to measure GFAP and S100B content in these structures as well as S100B levels in serum and cerebral spinal fluid. Main results revealed that ICH induced a delayed increase in GFAP+ cells in the sensorimotor cortex, as compared to the striatum, although the pattern of GFAP expression was similar in both structures. Interestingly, the time-curve patterns of both S100B and CD11+ microglial cells differed between the cortex and striatum. Altogether, these results suggest a different dynamics of glial-associated changes in the cerebral cortex, suggesting it is a vulnerable structure and undergoes an independent secondary process of reactive glial plasticity following intracerebral hemorrhage., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Repetitive transcranial magnetic stimulation (rTMS), a non-invasive form of brain stimulation, has shown experimental and clinical efficacy in a range of neuromodulatory models, even when delivered at low intensity (i.e. subthreshold for action potential generation). After central nervous system (CNS) injury, studies suggest that reactive astrocytes and microglia can have detrimental but also beneficial effects; thus modulating glial activity, for example through application of rTMS, could potentially be a useful therapeutic tool following neurotrauma. Immunohistochemistry was used to measure the effect of low intensity rTMS (LI-rTMS) on GFAP (astrocyte), IBA1 (microglial), and CS56 (proteoglycan) expression in a unilateral penetrating cortical stab injury model of glial scarring in young adult and aged male and female C57BL6/J mice. Mice received contralateral low frequency, ipsilateral low frequency, ipsilateral high frequency or sham LI-rTMS (4-5mT intensity), for two weeks following injury. There was no significant difference in the overall volume of tissue containing GFAP positive ( + ) astrocytes, IBA1 + microglia, or proteoglycan expression, between sham and LI-rTMS-treated mice of all ages and sex. Importantly however, the density of GFAP + astrocytes and IBA1 + microglia immediately adjacent to the injury was significantly reduced following ipsilateral low and high frequency stimulation in adult and aged females (p≤0.05), but was significantly increased in adult and aged males (p≤0.05). LI-rTMS effects were generally of greater magnitude in aged mice compared to young adult mice. These results suggest that sex differences need to be factored into therapeutic rTMS protocols. In particular, more work analyzing frequency and intensity specific effects, especially in relation to age and sex, is required to determine how rTMS can best be used to modify glial reactivity and phenotype following neurotrauma., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Lamin B1, a key component of the nuclear lamina, plays an important role in brain development. Ablation of endogenous Lamin B1 (Lmnb1) in the mouse strongly impairs embryonic brain development and corticogenesis. However, the mechanisms underlying these neurodevelopmental effects are unknown. Here, we report that Lamin B1 levels modulate the differentiation of murine neural stem cells (NSCs) into neurons and astroglial-like cells. In vitro, endogenous Lmnb1 depletion favors NSC differentiation into glial fibrillar acidic protein (GFAP)-immunoreactive cells over neurons, while overexpression of human Lamin B1 (LMNB1) increases the proportion of neurons. In Lmnb1-null embryos, neurogenesis is reduced, while in vivo Lmnb1 silencing in mouse embryonic brain by in utero electroporation of a specific Lmnb1 sh-RNA results in aberrant cortical positioning of neurons and increased expression of the astrocytic marker GFAP in the cortex of 7-day old pups. Together, these results indicate that finely tuned levels of Lamin B1 are required for NSC differentiation into neurons, proper expression of the astrocytic marker GFAP and corticogenesis.

After the initial insult in traumatic brain injury (TBI), secondary neurodegeneration occurs that is intimately associated with neuroinflammation. Prostaglandin (PG) synthases and cyclooxygenase (COX) 1 and 2 may contribute to inflammation in the brain. Temporal and spatial expression features of PG and COX1 and 2 following trauma may guide the development of antineuroinflammation strategies. Here, we examined PG synthase signaling and COX1 and 2 gene expression levels and COX-1- and 2-positive cell types and their temporal localization in TBI-induced brain in an effort to reveal their participation in the disease's evolving neuroinflammation. Using brain samples from the cerebral cortex of rats subjected to TBI model of lateral moderate fluid percussion injury (FPI), we sought to characterize the temporal (subacute TBI) and spatial (lateral cortical lesion) brain alterations accompanying the disease progression. Temporal gene expression changes of PG synthase signaling were compared between sham-operated and TBI-treated rats using microarray pathway analysis. Moreover, we examined COX1 and 2 expression patterns and their intracellular distribution in sham-operated and TBI-treated rats by immunohistochemistry. After FPI, COX1 and 2 gene expression levels, and PGE 2 synthase increased while PGD 2 synthase decreased, suggesting that PGE 2 and PGD 2 afforded contraindicative effects of inflammation and anti-inflammation, respectively. Immunohistochemical analyses showed that both COX1 and COX2 increased in a time-dependent manner in the brain, specifically in degenerating neurons of the cortex. Interestingly, the expression of COX cell type was cell-specific, in that COX1 was particularly increased in degenerating neurons while COX2 was expressed in macrophages. In view of the dynamic temporal and spatial expression of PG, COX1 and 2 gene expression and localization in the injured brain regulating PG synthase and COX1 and 2 activity will require a careful disease-specific tailoring of treatments to abrogate the neuroinflammation-plagued secondary cell death due to TBI.

The early release of adenosine following traumatic brain injury (TBI) suppresses seizures and brain inflammation; thus, it is important to elucidate the cellular sources of adenosine following injurious stimuli triggered by TBI so that therapeutics for enhancing the early adenosine-release response can be optimized. Using mass spectrometry with 13 C-labeled standards, we investigated in cultured rat neurons, astrocytes, and microglia the effects of oxygen-glucose deprivation (OGD; models energy failure), H 2 O 2 (produces oxidative stress), and glutamate (induces excitotoxicity) on intracellular and extracellular levels of 5'-AMP (adenosine precursor), adenosine, and inosine and hypoxanthine (adenosine metabolites). In neurons, OGD triggered increases in intracellular 5'-AMP (2.8-fold), adenosine (2.6-fold), inosine (2.2-fold), and hypoxanthine (5.3-fold) and extracellular 5'-AMP (2.2-fold), adenosine (2.4-fold), and hypoxanthine (2.5-fold). In neurons, H 2 O 2 did not affect intracellular or extracellular purines; yet, glutamate increased intracellular adenosine, inosine, and hypoxanthine (1.7-fold, 1.7-fold, and 1.6-fold, respectively) and extracellular adenosine, inosine, and hypoxanthine (2.9-fold, 2.1-fold, and 1.6-fold, respectively). In astrocytes, neither H 2 O 2 nor glutamate affected intracellular or extracellular purines, and OGD only slightly increased intracellular and extracellular hypoxanthine. Microglia were unresponsive to OGD and glutamate, but were remarkably responsive to H 2 O 2 , which increased intracellular 5'-AMP (1.6-fold), adenosine (1.6-fold), inosine (2.1-fold), and hypoxanthine (1.6-fold) and extracellular 5'-AMP (5.9-fold), adenosine (4.0-fold), inosine (4.3-fold), and hypoxanthine (1.9-fold)., Conclusion: Under these particular experimental conditions, cultured neurons are the main contributors to adenosine production/release in response to OGD and glutamate, whereas cultured microglia are the main contributors upon oxidative stress. Developing therapeutics that recruit astrocytes to produce/release adenosine could have beneficial effects in TBI., (© 2017 International Society for Neurochemistry.)

The potent neurotoxic agent acrylamide (ACR) is formed during Maillard reaction in food processing. Epigallocatechin-3-gallate (EGCG), a major bioactive component of green tea, is an antioxidant, but its effects on ACR-induced neurotoxicity are unclear. Here, we investigated the neuroprotective effects of EGCG against ACR-induced apoptosis and astrogliosis in the cerebral cortex. Rats were pretreated with EGCG for 4 d and then co-administered ACR for 14 d. Immunohistochemical analysis of glial fibrillary acidic protein and 8-hydroxy-2'-deoxyguanosine indicated that EGCG attenuated astrogliosis and DNA damage in ACR-treated rats. Analysis of DNA fragmentation and protein expression of Bax, Bcl-2, caspase 3, and cytochrome c revealed that EGCG inhibited ACR-induced apoptosis. Furthermore, EGCG inhibited oxidative stress by enhancing the activity of antioxidant enzymes and glutathione levels and reducing the formation of reactive oxygen species and lipid peroxidation. Taken together, our data demonstrate that EGCG inhibits ACR-induced apoptosis and astrogliosis in the cerebral cortex.

Introduction: Tuberous sclerosis is associated with three central nervous system pathologies: cortical/subcortical tubers, subependymal nodules (SENs), and subependymal giant cell astrocytomas (SEGAs). Tubers are associated with epilepsy, which is often medication-resistant and often leads to resective surgery. Recently, mammalian target of rapamycin inhibitors (mTORi) have been shown to be effective reducing seizure burden in some patients with tuberous sclerosis complex (TSC)-related refractory epilepsy. mTORi have also been shown to be an alternative for surgery treating SEGAs. We describe several cases of resected tubers that contained SEGA tissue without an intraventricular SEGA., Methods: After institutional review board (IRB) protocol approval, we retrospectively reviewed the surgical-pathological data for all TSC patients who underwent cortical resections for treatment of refractory epilepsy at NYU Langone Medical Center and Tel Aviv Medical Center between 2003 and 2013. Data included demographics, epilepsy type, MRI characteristics, epilepsy outcome, and histopathological staining., Results: We reviewed cortical resections from 75 patients with complete pathological studies. In three patients, cortical lesions demonstrated histopathological findings consistent with a SEGA within the resected tuber tissue, with no intraventricular SEGA. All lesions were cortically based and none had any intraventricular extension. No patient had been treated before surgery with an mTORi. Two of the three patients remain Engel grade I-II. All lesions stained positive for glial fibrillary acidic protein (GFAP), synaptophysin, and neuronal nuclear antigen (NeuN)., Conclusion: This is the first description of cortical tubers harboring SEGA tissue. This observation though preliminary may suggest a subgroup of patients with intractable epilepsy in whom mTORi may be considered before surgical intervention.