Glial fibrillary acidic protein in regenerating teleost spinal cord

Immunohistological and ultrastructural studies were carried out on normal and regenerating spinal cord of the gymnotid Sternarchus albifrons, and in the brain and spinal cord of the goldfish Carassius auratus, to examine the distribution of glial fibrillary acidic protein (GFAP) in these tissues. Sections of normal goldfish brain and spinal cord exhibited positive staining for GFAP. In normal Sternarchus spinal cord, electron microscopy has revealed filament-filled astrocytic processes; however, such astrocytic profiles were more numerous in regenerated cord. Likewise, while normal Sternarchus spinal cord showed only a small amount of GFAP staining, regenerated cords were strongly positive for GFAP. Positive staining with anti-GFAP was observed along the entire length of the regenerated cord in Sternarchus, and was especially strong in the transition zone between regenerated and unregenerated cord. Both regeneration of neurites and production of new neuronal cell bodies occur readily in such regenerating Sternarchus spinal cords (Anderson MJ, Waxman SG: J Hirnforsch 24: 371, 1983). These results demonstrate that the presence of GFAP and reactive astrocytes in Sternarchus spinal cord does not prevent neuronal regeneration in this species.     

Resident macrophages in the cochlear blood-labyrinth barrier and their renewal via migration of bone-marrow-derived cells

A large population of perivascular cells was found to be present in the area of the blood-labyrinth barrier in the stria vascularis of normal adult cochlea. The cells were identified as perivascular resident macrophages (PVMs), as they were positive for several macrophage surface molecules including F4/80, CD68, and CD11b. The macrophages, which were closely associated with microvessels and structurally intertwined with endothelial cells and pericytes, constitutively expressed scavenger receptor classes A₁ and B₁ and accumulated blood-borne proteins such as horseradish peroxidase and acetylated low-density lipoprotein. The PVMs were demonstrated to proliferate slowly, as evidenced by the absence of 5-bromo-2-deoxyuridine (BrdU)-positive PVMs at 3–14 days in normal mice injected with BrdU. However, in irradiated mice, the majority of the PVMs turned over via bone-marrow-cell migration within a 10-month time-frame. The existence of PVMs in the vascular wall of the blood-labyrinth barrier might therefore serve as a source for progenitor cells for postnatal vasculogenesis and might contribute to the repair of damaged vessels in the context of a local inflammatory response.     

Glial Fibrillary Acidic Protein Expression in Rat Brain and in Radial Glia Culture Is Delayed by Prenatal Ethanol Exposure

Abstract: The alterations in astrocyte proliferation and differentiation induced by prenatal exposure to alcohol (PEA) suggest that ethanol exposure affects the radial glial cells, the main astrocytic precursors. We have investigated the effects of ethanol on the early stages of astrogliogenesis by analyzing the developmental pattern of vimentin and glial fibrillary acidic protein (GFAP) immunoreactivity and their mRNA levels during embryonic/fetal brain development and in radial glia in primary culture. GFAP appeared late in gestation and at day 5 of culture of radial glial, whereas GFAP mRNA was first detected on fetal day 15 and increased in content on fetal day 21. In contrast, the levels of vimentin and its mRNA were high at fetal day 15 but decreased on day 21. Alcohol exposure delays the appearance of GFAP and its mRNA and significantly decreases the GFAP expression in fetal brain and in primary culture of radial glial. In addition, some morphological alterations were observed in PEA glial cells in culture. These results demonstrate that astroglial precursor cells are damaged by prenatal exposure to ethanol and suggest that abnormalities in the astrogliogenesis may underlie the disruption in neuronal migration and other CNS alterations observed after prenatal ethanol exposure.     

Specific in vivo staining of astrocytes in the whole brain after intravenous injection of sulforhodamine dyes

Fluorescent staining of astrocytes without damaging or interfering with normal brain functions is essential for intravital microscopy studies. Current methods involved either transgenic mice or local intracerebral injection of sulforhodamine 101. Transgenic rat models rarely exist, and in mice, a backcross with GFAP transgenic mice may be difficult. Local injections of fluorescent dyes are invasive. Here, we propose a non-invasive, specific and ubiquitous method to stain astrocytes in vivo. This method is based on iv injection of sulforhodamine dyes and is applicable on rats and mice from postnatal age to adulthood. The astrocytes staining obtained after iv injection was maintained for nearly half a day and showed no adverse reaction on astrocytic calcium signals or electroencephalographic recordings in vivo. The high contrast of the staining facilitates the image processing and allows to quantify 3D morphological parameters of the astrocytes and to characterize their network. Our method may become a reference for in vivo staining of the whole astrocytes population in animal models of neurological disorders.     

Demonstration of glial fibrillary acidic (GFA) protein by electron immunocytochemistry in the granular cells of a choristoma of the neurohypophysis

The origin of the nests of granular cells comprising choristomas of the infundibular process and the stalk of the pituitary gland is controversial. Using electron microscopic immunocytochemistry, the astrocytic marker, glial fibrillary acid protein (GFAP), has been demonstrated diffusely in the cytoplasm of some of the granular cells, but not within the granules or cellular organelles of some of the granular cells. Cytoplasmic filaments were not detected in these granular cells, but cells with abundant filaments extended processes between the granular cells. These filament-rich cells stained much more intensely for GFAP than the positively staining granular cells. The expression of GFAP by the granular cells and the filament-containing cells between them in the pituitary implies an astrocytic origin for both cell types, but the absence of filaments in the granular cells suggests that the GFAP is in an unpolymerized (soluble) form. The granular cell is likely to represent a transitional cell type of astrocytic origin in which the glial filaments have undergone partial or complete degradation.     

Early Activation of STAT3 Regulates Reactive Astrogliosis Induced by Diverse Forms of Neurotoxicity

Astrogliosis, a cellular response characterized by astrocytic hypertrophy and accumulation of GFAP, is a hallmark of all types of central nervous system (CNS) injuries. Potential signaling mechanisms driving the conversion of astrocytes into “reactive” phenotypes differ with respect to the injury models employed and can be complicated by factors such as disruption of the blood-brain barrier (BBB). As denervation tools, neurotoxicants have the advantage of selective targeting of brain regions and cell types, often with sparing of the BBB. Previously, we found that neuroinflammation and activation of the JAK2-STAT3 pathway in astrocytes precedes up regulation of GFAP in the MPTP mouse model of dopaminergic neurotoxicity. Here we show that multiple mechanistically distinct mouse models of neurotoxicity (MPTP, AMP, METH, MDA, MDMA, KA, TMT) engender the same neuroinflammatory and STAT3 activation responses in specific regions of the brain targeted by each neurotoxicant. The STAT3 effects seen for TMT in the mouse could be generalized to the rat, demonstrating cross-species validity for STAT3 activation. Pharmacological antagonists of the neurotoxic effects blocked neuroinflammatory responses, pSTAT3^tyr705 and GFAP induction, indicating that damage to neuronal targets instigated astrogliosis. Selective deletion of STAT3 from astrocytes in STAT3 conditional knockout mice markedly attenuated MPTP-induced astrogliosis. Monitoring STAT3 translocation in GFAP-positive cells indicated that effects of MPTP, METH and KA on pSTAT3^tyr705 were localized to astrocytes. These findings strongly implicate the STAT3 pathway in astrocytes as a broadly triggered signaling pathway for astrogliosis. We also observed, however, that the acute neuroinflammatory response to the known inflammogen, LPS, can activate STAT3 in CNS tissue without inducing classical signs of astrogliosis. Thus, acute phase neuroinflammatory responses and neurotoxicity-induced astrogliosis both signal through STAT3 but appear to do so through different modules, perhaps localized to different cell types.     

Maternal high-fat diet affects Msi/Notch/Hes signaling in neural stem cells of offspring mice

Numerous research have begun to reveal the importance of maternal nutrition in offspring brain development. Particularly, the maternal obesity or exposure to high-fat diet has been strongly suggested to exert irreversible impact on the structure and function of offspring's brain. However, it remains obscure about whether neonatal neural stem cells (NSCs) in offspring's brain are susceptible to maternal exposure to high-fat diet. Here we focused on the alternation in the Notch signaling in NSCs derived from neonatal mice, which had been given birth by female mice with a high-fat diet and found that, in fact, the high-fat diet administration imposed effects on not only maternal mice, indicated by the accumulation of viscera fat as well as the increase in body weight and serum total cholesterol, but also NSCs in the offspring’s brain, where significant increase was observed in the expression of genes, either downstream of Notch signaling or regulating this pathway, which have been shown essential for the maturation of NSCs. Therefore, our data provided the first evidence for the potential effect of maternal exposure to the high-fat diet on the Notch signaling pathway in offspring’s NSCs, indicating this altered signaling response might contribute to a profound change in offspring’s brains as a result of maternal high-fat diet prior to and during gestation.     

Use of monoclonal antibodies to glial fibrillary acidic protein in the cytologic diagnosis of brain tumors

Monoclonal antibodies were used to immunocytochemically demonstrate glial fibrillary acidic protein (GFAP) in 174 smear preparations of brain tumor tissue in order to investigate the presence and distribution of GFAP in a variety of intracranial tumors and to evaluate the value of this technique in the cytodiagnosis of brain tumors. GFAP-positive cells were found in the astrocytic tumors and in some of the oligodendrogliomas, ependymomas and medulloblastomas. In contrast, schwannomas, meningiomas, a primary lymphoma, a hemangiopericytoma pituitary adenomas, germinomas and metastatic tumors were negative for GFAP. The cytodiagnostic accuracy of the 174 brain tumors was raised from 90.8% to 97.1% when GFAP-immunoperoxidase staining was employed to aid the routine cytologic diagnosis. These findings indicate that immunoperoxidase staining for GFAP can be successfully applied to cytologic specimens and is a useful adjunct to routine cytologic diagnosis.     

产前850-1900MHz手机暴露对子代大鼠齿状回PCNA和DCX表达的影响

目的：探讨产前手机暴露对子代大鼠海马齿状回增殖细胞核抗原（PCNA）和双皮质素（DCX）表达的影响。方法：构建孕鼠手机射频暴露模型,分为对照组、短时暴露组和长时暴露组（n=6）,短时和长时暴露组于孕第1-17天分别给予6 h/d和24 h/d的手机通话暴露,观察孕鼠的孕期长短、孕期体重增长和各组的胎儿数、胎儿出生体重。1月龄子代大鼠行焦油紫染色观察海马齿状回细胞形态,免疫组化观察齿状回PCNA和DCX表达,Western blot检测DCX和脑源性神经营养因子（BDNF）表达。结果：各组孕鼠的孕期、妊娠期体重增长和各组的胎儿数、胎儿出生体重无显著差异,长时暴露组子代大鼠的齿状回多形细胞层锥形细胞和DCX阳性细胞出现形态改变。与对照组、短时暴露组比较,长时暴露组子代大鼠齿状回PCNA阳性细胞和DCX、BDNF表达均明显减少（P<0.05）。结论：产前长时手机暴露可能通过改变子代大鼠海马BDNF而影响齿状回的PCNA和DCX表达。     

Demonstration of glial fibrillary acidic (GFA) protein by electron immunocytochemistry in the granular cells of a choristoma of the neurohypophysis

Summary The origin of the nests of granular cells comprising choristomas of the infundibular process and the stalk of the pituitary gland is controversial. Using electron microscopic immunocytochemistry, the astrocytic marker, glial fibrillary acid protein (GFAP), has been demonstrated diffusely in the cytoplasm of some of the granular cells, but not within the granules or cellular organelles of some of the granular cells. Cytoplasmic filaments were not detected in these granular cells, but cells with abundant filaments extended processes between the granular cells. These filament-rich cells stained much more intensely for GFAP than the positively staining granular cells. The expression of GFAP by the granular cells and the filament-containing cells between them in the pituitary implies an astrocytic origin for both cell types, but the absence of filaments in the granular cells suggests that the GFAP is in an unpolymerized (soluble) form. The granular cell is likely to represent a transitional cell type of astrocytic origin in which the glial filaments have undergone partial or complete degradation.     

Phenotypic and gene expression modification with normal brain aging in GFAP-positive astrocytes and neural stem cells

Astrocytes secrete growth factors that are both neuroprotective and supportive for the local environment. Identified by glial fibrillary acidic protein (GFAP) expression, astrocytes exhibit heterogeneity in morphology and in the expression of phenotypic markers and growth factors throughout different adult brain regions. In adult neurogenic niches, astrocytes secrete vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) within the neurogenic niche and are also a source of special GFAP-positive multipotent neural stem cells (NSCs). Normal aging is accompanied by a decline in CNS function and reduced neurogenesis. We asked whether a decreased availability of astrocyte-derived factors may contribute to the age-related decline in neurogenesis. Determining alterations of astrocytic activity in the aging brain is crucial for understanding CNS homeostasis in aging and for assessing appropriate therapeutic targets for an aging population. We found region-specific alterations in the gene expression of GFAP, VEGF, and FGF-2 and their receptors in the aged brain corresponding to changes in astrocytic reactivity, supporting astrocytic heterogeneity and demonstrating a differential aging effect. We found that GFAP-positive NSCs uniquely coexpress both VEGF and its key mitotic receptor Flk-1 in both young and aged hippocampus, indicating a possible autocrine/paracrine signaling mechanism. VEGF expression is lost once NSCs commit to a neuronal fate, but Flk-1-mediated sensitivity to VEGF signaling is maintained. We propose that age-related astrocytic changes result in reduced VEGF and FGF-2 signaling, which in turn limits NSC and progenitor cell maintenance and contributes to decreased neurogenesis.     

大鼠产前手机辐射对子代小脑白质的影响

目的:探讨大鼠产前850～1 900 MHz手机辐射对成年子代小脑白质的影响。方法:孕鼠随机分为短时产前手机辐射组、长时产前手机辐射组和对照组,短时和长时辐射组于孕期第1～17日分别进行每天6 h和24 h手机辐射,各组雄性子代大鼠(n＝8)于3月龄取小脑组织,进行苏木精-伊红(HE)染色观察小脑皮质细胞形态,免疫组化和Western blot检测髓鞘碱性蛋白(MBP)、神经微丝-L(NF-L)和胶质纤维酸性蛋白(GFAP)表达。结果:与对照组比较,短时程和长时程产前手机辐射组子代大鼠小脑浦肯野细胞出现形态学改变;与对照组比较,长时程辐射组MBP、NF-L表达明显减少(P均＜0.05),而GFAP表达明显增多(P＜0.05);与短时程辐射组比较,长时程组子代大鼠小脑MBP、NF-L表达明显减少(P均＜0.05),而GFAP表达明显增多(P＜0.05)。结论:产前手机辐射会导致雄性子代大鼠小脑髓鞘和轴突的损害,以及星形胶质细胞的活化,且这种改变与产前手机辐射的时程相关。     

Meningeal and perivascular macrophages of the central nervous system play a protective role during bacterial meningitis

Meningeal (MM) and perivascular macrophages (PVM) constitute major populations of resident macrophages in the CNS that can be distinguished from microglial cells. So far, there is no direct evidence that demonstrates a possible role of MM and PVM in the CNS during normal or pathologic conditions. To elucidate the role of the MM and PVM during CNS inflammation, we have developed a strategy using a single intraventricular injection of mannosylated clodronate liposomes, which results in a complete and selective depletion of the PVM and MM from the CNS. Depletion of the MM and PVM during experimental pneumococcal meningitis resulted in increased illness, which correlated with higher bacteria counts in the cerebrospinal fluid and blood. This was associated with a decreased influx of leukocytes into the cerebrospinal fluid, which occurred despite an elevated production of relevant chemokines (e.g., macrophage-inflammatory protein-2) and a higher expression of vascular adhesion molecules (e.g., VCAM-1). In contrast, the higher bacterial counts correlated with elevated production of local and systemic inflammatory mediators (e.g., IL-6) indicating enhanced local leukocyte and systemic immune activation, and this may explain the worsening of the clinical signs. These findings show that the PVM and MM play a protective role during bacterial meningitis and suggest that a primary action of these macrophages is to facilitate the influx of leukocytes at the blood-brain barrier. More in general, we demonstrate for the first time that the PVM and MM play a crucial role during inflammation in the CNS.     

Expression of catalase mRNA and protein in adult rat brain: detection by nonradioactive in situ hybridization with signal amplification by catalyzed reporter deposition (ISH-CARD) and immunohistochemistry (IHC)/immunofluorescence (IF).

Catalase, the classical peroxisomal marker enzyme, decomposes hydrogen peroxide and is involved in the antioxidant defense mechanisms of mammalian cells. In addition, catalase can oxidize, by means of its peroxidatic activity, a variety of substrates such as methanol and ethanol, producing the corresponding aldehydes. The involvement of brain catalase in the oxidation of ethanol is well established, and severe afflictions of the CNS in hereditary peroxisomal diseases (e.g., Zellweger syndrome) are well known. Whereas the distribution of catalase in the CNS has been investigated by enzyme histochemistry and immunohistochemistry (IHC), very little is known about the exact localization of catalase mRNA in brain. Here we report the application of a tyramine/CARD (catalyzed reporter deposition)-enhanced nonradioactive in situ hybridization (ISH) protocol for detection of catalase mRNA in sections of perfusion-fixed, paraffin-embedded rat brain. Catalase mRNA could be demonstrated in a large number of neurons throughout the rat brain as a distinct cytoplasmic staining signal with excellent morphological resolution. Compared to our standard ISH protocol, the CARD-enhanced protocol for catalase mRNA detection in rat brain showed higher sensitivity and significantly better signal-to-noise ratio. In parallel IHC experiments, using an antigen retrieval method consisting of combined trypsin digestion and microwave treatment of paraffin sections, the catalase antigen was found as distinct cytoplasmic granules in most catalase mRNA-positive neurons. In addition, catalase-positive granules, presumably peroxisomes, were found by confocal laser scanning microscopy in glial cells, which were identified by double labeling immunofluorescence for GFAP and CNPase for astroglial cells and oligodentrocytes, respectively. The excellent preservation of morphology and sensitive detection of both mRNA and protein in our preparations warrant the application of the protocols described here for systematic studies of catalase and other peroxisomal proteins in diverse pathological conditions such as Alzheimer's disease and aging.     

Occurrence of ommochrome-containing pigment granules in the central nervous system of the silkworm, Bombyx mori

Dark-red pigment granules were found in the brain and ganglion of the normal strain of the silkworm, Bombyx mori, by light microscopy. No other pigmentation was seen in the brain or ganglia. Electron microscopy showed that the granules were electron-dense. The granules were similar to the ommochrome-containing pigment granules that are present in the epidermal cells of the quail mutant, as previously reported. The pigment in the larval central nervous system (CNS) of the normal silkworm was identical to the ommin standard with respect to the absorption spectrum, the infrared spectrum, and the Rf value in thin-layer chromatography (TLC). After acid hydrolysis of the pigment, 3-hydroxykynurenine was detected by TLC. The pigment granules in the CNS contained mainly ommin. An ommochrome-binding protein was also detected in the CNS by in vitro binding studies and Western blotting. The ommochrome granules may have an important function in the CNS of the silkworm.     

Increased Nitric Oxide Production and GFAP Expression in the Brains of Influenza A/NWS Virus Infected Mice

The cause of influenza to the brain was investigated using the A/NWS/33 influenza virus infected BALB/c mouse model. NOS-2 mRNA levels in the infected mouse brain was greater than in control mice in all brain regions examined, particularly in the olfactory bulb and hippocampus by 1 day p.i. On the contrary, no differences in NOS-1 or NOS-3 mRNA levels were found between infected and control mice. There was also a marked increase in the levels of metabolites of nitric oxide in the olfactory bulb and hippocampus. Immunohistochemistry showed positive staining for anti-NOS-2 primarily in the hippocampus of infected mice. Further, anti-NOS-2 and GFAP staining was mostly found around capillary blood vessels of the hippocampus starting early in the course of the disease. These results indicate that the NWS enhances the activation of astrocytes and NOS-2 expression which in turn enhances NO production and the expansion of capillary blood vessels.     

Inhibition of cadmium-induced oxidative injury in rat primary astrocytes by the addition of antioxidants and the reduction of intracellular calcium

Exposure of the brain to cadmium ions (Cd(2+)) is believed to lead to neurological disorders of the central nervous system (CNS). In this study, we tested the hypothesis that astrocytes, the major CNS-supporting cells, are resistant to Cd(2+)-induced injury compared with cortical neurons and microglia (CNS macrophages). However, treatment with CdCl(2) for 24 h at concentrations higher than 20 microM substantially induced astrocytic cytotoxicity, which also resulted from long-term exposure to 5 microM of CdCl(2). Intracellular calcium levels were found to rapidly increase after the addition of CdCl(2) into astrocytes, which led to a rise in reactive oxygen species (ROS) and to mitochondrial impairment. In accordance, preexposure to the extracellular calcium chelator EGTA effectively reduced ROS production and increased survival of Cd(2+)-treated astrocytes. Adenovirus-mediated transfer of superoxide dismutase (SOD) or glutathione peroxidase (GPx) genes increased survival of Cd(2+)-exposed astrocytes. In addition, increased ROS generation and astrocytic cell death due to Cd(2+) exposure was inhibited when astrocytes were treated with the polyphenolic compound ellagic acid (EA). Taken together, Cd(2+)-induced astrocytic cell death resulted from disrupted calcium homeostasis and an increase in ROS. Moreover, our findings demonstrate that enhancement of the activity of intracellular antioxidant enzymes and supplementation with a phenolic compound, a natural antioxidant, improves survival of Cd(2+)-primed astrocytes. This information provides a useful approach for treating Cd(2+)-induced CNS neurological disorders.