Astrocytic Glial Fibrillary Acidic Protein Research Articles

Effects of wind-dispelling drugs and deficiency-nourishing drugs of Houshiheisan compound prescription on astrocyte activation and inflammatory factor expression in the corpus striatum of cerebral ischemia rats.

This study explored protective effects of Houshiheisan and its compound prescription of wind-dispelling drugs and deficiency-nourishing drugs on cerebral ischemia in terms of astrocyte activation and inflammatory factor expression. Results suggested that Houshiheisan lessened neuronal degeneration in the corpus striatum on the ischemic side of rats following cerebral ischemia/reperfusion injury, contributed to astrocyte activation and glial fibrillary acidic protein expression in the corpus striatum and decreased the levels of interleukin-2, interleukin-6, interleukin-1β and tumor necrosis factor-α. Factor analysis results demonstrated that deficiency-nourishing drugs were more beneficial in protecting neurons and upregulating glial fibrillary acidic protein expression than wind-dispelling drugs. However, wind-dispelling drugs were more effective in increasing the number of glial fibrillary acidic protein-positive cells and reducing inflammatory factor expression than deficiency-nourishing drugs. These indicate that different ingredients of Houshiheisan suppress cerebral ischemic injury by promoting astrocyte activation and diminishing inflammatory factor expression.

The structure of the optic nerve after the administration of interferon α-2a in adult male albino rats and the role of α-lipoic acid supplementation

Introduction Interferon alpha (IFN-α) therapy is used considerably in Egypt because of a high prevalence rate of chronic hepatitis C virus infection. α-Lipoic acid (ALA) has been found to play a neuroprotective role in many insults. The aim of this study is to observe the histological structure of the optic nerve of rats after an injection of IFN-α and to determine the role of ALA supplementation. Materials and methods Forty adult male albino rats were divided equally into four groups. Group I served as the control group. Group II included rats that received ALA alone (100mg/kg/day, intraperitoneally). Group III included rats that received IFN-α alone (100000 IU/kg/three times/week, intraperitoneally). Group IV included rats that received both IFN-α and ALA. After 8 weeks, the optic nerves were extirpated and processed for light and electron microscope examination. Results Optic nerves of the group that received IFN-α showed nerve damage manifested as axonal damage and changes in the myelin sheath. Neuroglia showed vacuolation in their cytoplasm and heterochromatic nuclei. Morphometric and statistical analyses showed a significant increase in the surface area of positive glial fibrillary acidic protein astrocytes, indicating reactive astrogliosis. Blood capillaries were distorted with ill-defined walls and protrusion of the endothelial cells into their lumina. These changes were limited by concomitant ALA supplementation with IFN-α. Conclusion IFN-α exerted a deleterious effect on the histological structure of the optic nerve in rats and ALA supplementation minimized these effects.

Cellular, synaptic, and biochemical features of resilient cognition in Alzheimer's disease

Although neuritic plaques and neurofibrillary tangles in older adults are correlated with cognitive impairment and severity of dementia, it has long been recognized that the relationship is imperfect, as some people exhibit normal cognition despite high levels of Alzheimer's disease (AD) pathology. We compared the cellular, synaptic, and biochemical composition of midfrontal cortices in female subjects from the Religious Orders Study who were stratified into three subgroups: (1) pathological AD with normal cognition (“AD-Resilient”), (2) pathological AD with AD-typical dementia (“AD-Dementia”), and (3) pathologically normal with normal cognition (“Normal Comparison”). The AD-Resilient group exhibited preserved densities of synaptophysin-labeled presynaptic terminals and synaptopodin-labeled dendritic spines compared with the AD-Dementia group, and increased densities of glial fibrillary acidic protein astrocytes compared with both the AD-Dementia and Normal Comparison groups. Further, in a discovery-type antibody microarray protein analysis, we identified a number of candidate protein abnormalities that were associated with a particular diagnostic group. These data characterize cellular and synaptic features and identify novel biochemical targets that may be associated with resilient cognitive brain aging in the setting of pathological AD.

Ameliorative effects of Diammonium Glycyrrhizinate on inflammation in focal cerebral ischemic-reperfusion injury

The present study investigated the neuroprotective potential of Diammonium Glycyrrhizinate (DG) in focal cerebral ischemic-reperfusion (IR) injury in mice. The middle cerebral artery occlusion (MCAO) model of the mouse was used. Mice were treated with DG (20mg/kg per day, intraperitoneal injection) or saline as control, from the beginning of the reperfusion to 7days. The focal cerebral IR injury resulted in significant neurological deficits, infarct size, and brain water content (BWC) at 1day, 3days and 7days after MCAO. A significant increase in various inflammatory mediators like interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and nuclear factor-κB (NF-κB) and astrocytic glial fibrillary acidic protein (GFAP) was also observed in the IR challenged brains. The DG treatment significantly improved neurofunction, decreased infarct size, and suppressed edema in the focal cerebral IR injury. The neuroprotective effect of DG was found to be associated with significant reduction in the IL-1, TNF-α, COX-2, iNOS, NF-κB and GFAP levels. In summary, this study suggested that DG has a neuroprotective effect on cerebral IR injury and this effect is likely related to DG's anti-inflammatory function.

The Role of Attenuated Astrocyte Activation in Infantile Neuronal Ceroid Lipofuscinosis

Infantile neuronal ceroid lipofuscinosis (INCL) is an inherited neurodegenerative disorder affecting the CNS during infancy. INCL is caused by mutations in the CLN1 gene that lead to a deficiency in the lysosomal hydrolase, palmitoyl protein thioesterase 1 (PPT1). A murine model of INCL, the PPT1-deficient (PPT1(-/-)) mouse, is an accurate phenocopy of the human disease. The first pathological change observed in the PPT1(-/-) brain is regional areas of glial fibrillary acidic protein (GFAP) upregulation, which predicts future areas of neurodegeneration. We hypothesized that preventing GFAP and vimentin upregulation in reactive astrocytes will alter the CNS disease. To test this hypothesis, we generated mice simultaneously carrying null mutations in the GFAP, Vimentin, and PPT1 genes (GFAP(-/-)Vimentin(-/-)PPT1(-/-)). Although the clinical and pathological features of the GFAP(-/-)Vimentin(-/-)PPT1(-/-) mice are similar to INCL, the disease appears earlier and progresses more rapidly. One mechanism underlying this accelerated phenotype is a profound neuroinflammatory response within the CNS. Thus, our data identify a protective role for intermediate filament upregulation during astrocyte activation in INCL, a model of chronic neurodegeneration.

Brain-derived neurotrophic factor–activated astrocytes produce mechanical allodynia in neuropathic pain

Neuropathic pain management is challenging for physicians and a vexing problem for basic researchers. Recent studies reveal that activated spinal astrocytes may play a vital role in nerve injury-induced neuropathic pain, although the mechanisms are not fully understood. We have found increased glial fibrillary acidic protein (GFAP) expression, a hallmark of reactive gliosis, and elevated brain-derived neurotrophic factor (BDNF) expression in the dorsal horn in a rat model of allodynia induced by spinal nerve ligation (SNL). The high GFAP expression and mechanical allodynia that SNL induces were prevented by the intrathecal injection of the BDNF-sequestering fusion protein TrkB/Fc. Additionally, mechanical allodynia and GFAP overexpression was induced by the spinal administration of exogenous BDNF to naive rats, and exogenous BDNF given together with fluorocitrate, an astrocytic metabolism inhibitor, inhibited allodynia and GFAP upregulation. Exogenous BDNF also activated the astrocytes directly when tested in vitro. Furthermore, intrathecal administration of BDNF-stimulated astrocytes also induced mechanical allodynia in naive rats. All of these results indicate that astrocytes activated by BDNF might contribute to mechanical allodynia development in neuropathic pain in rats.

Lipopolysaccharide modulates astrocytic S100B secretion: a study in cerebrospinal fluid and astrocyte cultures from rats

BackgroundInflammatory responses in brain are primarily mediated by microglia, but growing evidence suggests a crucial importance of astrocytes. S100B, a calcium-binding protein secreted by astrocytes, has properties of a neurotrophic or an inflammatory cytokine. However, it is not known whether primary signals occurring during induction of an inflammatory response (e.g. lipopolysaccharide, LPS) directly modulate S100B.MethodsIn this work, we evaluated whether S100B levels in cerebrospinal fluid (CSF) and serum of Wistar rats are affected by LPS administered by intraperitoneal (IP) or intracerebroventricular (ICV) injection, as well as whether primary astrocyte cultures respond directly to lipopolysaccharide.ResultsOur data suggest that S100B secretion in brain tissue is stimulated rapidly and persistently (for at least 24 h) by ICV LPS administration. This increase in CSF S100B was transient when LPS was IP administered. In contrast to these S100B results, we observed an increase in in TNFα levels in serum, but not in CSF, after IP administration of LPS. In isolated astrocytes and in acute hippocampal slices, we observed a direct stimulation of S100B secretion by LPS at a concentration of 10 μg/mL. An involvement of TLR4 was confirmed by use of specific inhibitors. However, lower levels of LPS in astrocyte cultures were able to induce a decrease in S100B secretion after 24 h, without significant change in intracellular content of S100B. In addition, after 24 h exposure to LPS, we observed a decrease in astrocytic glutathione and an increase in astrocytic glial fibrillary acidic protein.ConclusionsTogether, these data contribute to the understanding of the effects of LPS on astrocytes, particularly on S100B secretion, and help us to interpret cerebrospinal fluid and serum changes for this protein in neuroinflammatory diseases. Moreover, non-brain S100B-expressing tissues may be differentially regulated, since LPS administration did not lead to increased serum levels of S100B.

Electroacupuncture attenuates mechanical and warm allodynia through suppression of spinal glial activation in a rat model of neuropathic pain

Neuropathic pain remains one of the most difficult clinical pain syndromes to treat. It is traditionally viewed as being mediated solely by neurons; however, glial cells have recently been implicated as powerful modulators of pain. It is known that the analgesic effects of electroacupuncture (EA) are mediated by descending pain inhibitory systems, which mainly involve spinal opioid, adrenergic, dopaminergic, serotonergic, and cholinergic receptors. However, studies investigating the suppressive effects of EA on spinal glial activation are rare. In the present study, we assessed the cumulative analgesic effects of EA on mechanical and warm allodynia in a rat model of neuropathic pain. We investigated the clinical efficacy of EA as long-term therapy and examined its effects on spinal glia, matrix metalloproteinase (MMP)-9/MMP-2, proinflammatory cytokines and serum immunoglobulin G (IgG) concentration. Rats were randomly divided into four groups as follows: the operation group (OP), operation with EA-non acupoint (EA-NA), operation with EA-ST36 acupoint (EA-ST36), and sham operation (shamOP). Following neuropathic or sham surgery, repeated EA was performed every other day after the behavioral test. On day 53 after the behavioral test, rats were perfused for immunohistochemistry and Western blot analysis to observe quantitative changes in spinal glial markers such as OX-42, astrocytic glial fibrillary acidic protein (GFAP), MMP-9/MMP-2, and proinflammatory cytokines. Allodynia and OX-42/GFAP/MMP-9/MMP-2/tumor necrosis factor (TNF)-α/interleukin (IL)-1β activity in the EA-ST36 group was significantly reduced, compared to the OP and EA-NA groups, and IgG in EA-ST36 rats significantly increased. Our results suggest that the analgesic effect of EA may be partly mediated via inhibition of inflammation and glial activation and repeated EA stimulation may be useful for treating chronic pain clinically.

Cathepsin K deficiency in mice induces structural and metabolic changes in the central nervous system that are associated with learning and memory deficits

BackgroundCathepsin K is a cysteine peptidase known for its importance in osteoclast-mediated bone resorption. Inhibitors of cathepsin K are in clinical trials for treatment of osteoporosis. However, side effects of first generation inhibitors included altered levels of related cathepsins in peripheral organs and in the central nervous system (CNS). Cathepsin K has been recently detected in brain parenchyma and it has been linked to neurobehavioral disorders such as schizophrenia. Thus, the study of the functions that cathepsin K fulfils in the brain becomes highly relevant.ResultsCathepsin K messenger RNA was detectable in all brain regions of wild type (WT) mice. At the protein level, cathepsin K was detected by immunofluorescence microscopy in vesicles of neuronal and non-neuronal cells throughout the mouse brain. The hippocampus of WT mice exhibited the highest levels of cathepsin K activity in fluorogenic assays, while the cortex, striatum, and cerebellum revealed significantly lower enzymatic activities. At the molecular level, the proteolytic network of cysteine cathepsins was disrupted in the brain of cathepsin K-deficient (Ctsk-/-) animals. Specifically, cathepsin B and L protein and activity levels were altered, whereas cathepsin D remained largely unaffected. Cystatin C, an endogenous inhibitor of cysteine cathepsins, was elevated in the striatum and hippocampus, pointing to regional differences in the tissue response to Ctsk ablation. Decreased levels of astrocytic glial fibrillary acidic protein, fewer and less ramified profiles of astrocyte processes, differentially altered levels of oligodendrocytic cyclic nucleotide phosphodiesterase, as well as alterations in the patterning of neuronal cell layers were observed in the hippocampus of Ctsk-/- mice. A number of molecular and cellular changes were detected in other brain regions, including the cortex, striatum/mesencephalon, and cerebellum. Moreover, an overall induction of the dopaminergic system was found in Ctsk-/- animals which exhibited reduced anxiety levels as well as short- and long-term memory impairments in behavioral assessments.ConclusionWe conclude that deletion of the Ctsk gene can lead to deregulation of related proteases, resulting in a wide range of molecular and cellular changes in the CNS with severe consequences for tissue homeostasis. We propose that cathepsin K activity has an important impact on the development and maintenance of the CNS in mice.

Induction of Ischemic Tolerance Protects the Retina From Diabetic Retinopathy

Diabetic retinopathy is a leading cause of acquired blindness. Available treatments are not very effective. We investigated the effect of a weekly application of retinal ischemia pulses (ischemic conditioning) on retinal damage induced by experimental diabetes. Diabetes was induced by an intraperitoneal injection of streptozotocin. Retinal ischemia was induced by increasing intraocular pressure to 120 mmHg for 5 minutes; this maneuver started 3 days after streptozotocin injection and was weekly repeated in one eye, whereas the contralateral eye was submitted to a sham procedure. Diabetic retinopathy was evaluated in terms of i) retinal function (electroretinogram and oscillatory potentials), ii) integrity of blood-retinal barrier (by albumin-Evans blue complex leakage and astrocyte glial fibrillary acidic protein IHC), iii) optical and electron microscopy histopathologic studies, and iv) vascular endothelial growth factor levels (using Western blot analysis and IHC). Brief ischemia pulses significantly preserved electroretinogram a- and b-wave and oscillatory potentials, avoided albumin-Evans blue leakage, prevented the decrease in astrocyte glial fibrillary acidic protein levels, reduced the appearance of retinal edemas, and prevented the increase in vascular endothelial growth factor levels induced by experimental diabetes. When the application of ischemia pulses started 6 weeks after diabetes onset, retinal function was significantly preserved. These results indicate that induction of ischemic tolerance could constitute a fertile avenue for the development of new therapeutic strategies for diabetic retinopathy treatment.

Brain Abnormalities and Glioma-Like Lesions in Mice Overexpressing the Long Isoform of PDGF-A in Astrocytic Cells

BackgroundDeregulation of platelet-derived growth factor (PDGF) signaling is a hallmark of malignant glioma. Two alternatively spliced PDGF-A mRNAs have been described, corresponding to a long (L) and a short (S) isoform of PDGF-A. In contrast to PDGF-A(S), the PDGF-A(L) isoform has a lysine and arginine rich carboxy-terminal extension that acts as an extracellular matrix retention motif. However, the exact role of PDGF-A(L) and how it functionally differs from the shorter isoform is not well understood.Methodology/Principal FindingsWe overexpressed PDGF-A(L) as a transgene under control of the glial fibrillary acidic protein (GFAP) promoter in the mouse brain. This directs expression of the transgene to astrocytic cells and GFAP expressing neural stem cells throughout the developing and adult central nervous system. Transgenic mice exhibited a phenotype with enlarged skull at approximately 6-16 weeks of age and they died between 1.5 months and 2 years of age. We detected an increased number of undifferentiated cells in all areas of transgene expression, such as in the subependymal zone around the lateral ventricle and in the cerebellar medulla. The cells stained positive for Pdgfr-α, Olig2 and NG2 but this population did only partially overlap with cells positive for Gfap and the transgene reporter. Interestingly, a few mice presented with overt neoplastic glioma-like lesions composed of both Olig2 and Gfap positive cell populations and with microvascular proliferation, in a wild-type p53 background.ConclusionsOur findings show that PDGF-A(L) can induce accumulation of immature cells in the mouse brain. The strong expression of NG2, Pdgfr-α and Olig2 in PDGF-A(L) brains suggests that a fraction of these cells are oligodendrocyte progenitors. In addition, accumulation of fluid in the subarachnoid space and skull enlargement indicate that an increased intracranial pressure contributed to the observed lethality.

The Unique Transcriptional Activation Domain of Nuclear Factor-I-X3 Is Critical to Specifically Induce Marker Gene Expression in Astrocytes

Transcription factors of the nuclear factor 1 (NFI) family regulate normal brain development in vertebrates. However, multiple splice variants of four NFI isoforms exist, and their biological functions have yet to be elucidated. Here, we cloned and analyzed human NFI-X3, a novel splice variant of the nfix gene, which contains a unique transcriptional activation (TA) domain completely conserved in primates. In contrast to previously cloned NFI-X1, overexpression of NFI-X3 potently activates NFI reporters, including glial fibrillary acidic protein (GFAP) reporter, in astrocytes and glioma cells. The GAL4 fusion protein containing the TA domain of NFI-X3 strongly activates the GAL4 reporter, whereas the TA domain of NFI-X1 is ineffective. The expression of NFI-X3 is dramatically up-regulated during the differentiation of neural progenitors to astrocytes and precedes the expression of astrocyte markers, such as GFAP and SPARCL1 (Secreted Protein, Acidic and Rich in Cysteines-like 1). Overexpression of NFI-X3 dramatically up-regulates GFAP and SPARCL1 expression in glioma cells, whereas the knockdown of NFI-X3 diminishes the expression of both GFAP and SPARCL1 in astrocytes. Although activation of astrocyte-specific genes involves DNA demethylation and subsequent increase of histone acetylation, NFI-X3 activates GFAP expression, in part, by inducing alterations in the nucleosome architecture that lead to the increased recruitment of RNA polymerase II.

Blood-Brain Barrier Disruption and Oxidative Stress in Guinea Pig after Systemic Exposure to Modified Cell-Free Hemoglobin

Systemic exposure to cell-free hemoglobin (Hb) or its breakdown products after hemolysis or with the use of Hb-based oxygen therapeutics may alter the function and integrity of the blood-brain barrier. Using a guinea pig exchange transfusion model, we investigated the effect of a polymerized cell-free Hb (HbG) on the expression of endothelial tight junction proteins (zonula occludens 1, claudin-5, and occludin), astrocyte activation, IgG extravasation, heme oxygenase (HO), iron deposition, oxidative end products (4-hydroxynonenal adducts and 8-hydroxydeoxyguanosine), and apoptosis (cleaved caspase 3). Reduced zonula occludens 1 expression was observed after HbG transfusion as evidenced by Western blot and confocal microscopy. Claudin-5 distribution was altered in small- to medium-sized vessels. However, total expression of claudin-5 and occludin remained unchanged except for a notable increase in occludin 72 hours after HbG transfusion. HbG-transfused animals also showed increased astrocytic glial fibrillary acidic protein expression and IgG extravasation after 72 hours. Increased HO activity and HO-1 expression with prominent enhancement of HO-1 immunoreactivity in CD163-expressing perivascular cells and infiltrating monocytes/macrophages were also observed. Consistent with oxidative stress, HbG increased iron deposition, 4-hydroxynonenal and 8-hydroxydeoxyguanosine immunoreactivity, and cleaved caspase-3 expression. Systemic exposure to an extracellular Hb triggers blood-brain barrier disruption and oxidative stress, which may have important implications for the use of Hb-based therapeutics and may provide indirect insight on the central nervous system vasculopathies associated with excessive hemolysis.

Cell Proliferation and Interleukin-6–Type Cytokine Signaling Are Implicated by Gene Expression Responses in Early Optic Nerve Head Injury in Rat Glaucoma

In glaucoma, the optic nerve head (ONH) is the principal site of initial axonal injury, and elevated intraocular pressure (IOP) is the predominant risk factor. However, the initial responses of the ONH to elevated IOP are unknown. Here the authors use a rat glaucoma model to characterize ONH gene expression changes associated with early optic nerve injury. Unilateral IOP elevation was produced in rats by episcleral vein injection of hypertonic saline. ONH mRNA was extracted, and retrobulbar optic nerve cross-sections were graded for axonal degeneration. Gene expression was determined by microarray and quantitative PCR (QPCR) analysis. Significantly altered gene expression was determined by multiclass analysis and ANOVA. DAVID gene ontology determined the functional categories of significantly affected genes. The Early Injury group consisted of ONH from eyes with <15% axon degeneration. By array analysis, 877 genes were significantly regulated in this group. The most significant upregulated gene categories were cell cycle, cytoskeleton, and immune system process, whereas the downregulated categories included glucose and lipid metabolism. QPCR confirmed the upregulation of cell cycle-associated genes and leukemia inhibitory factor (Lif) and revealed alterations in expression of other IL-6-type cytokines and Jak-Stat signaling pathway components, including increased expression of IL-6 (1553%). In contrast, astrocytic glial fibrillary acidic protein (Gfap) message levels were unaltered, and other astrocytic markers were significantly downregulated. Microglial activation and vascular-associated gene responses were identified. Cell proliferation and IL-6-type cytokine gene expression, rather than astrocyte hypertrophy, characterize early pressure-induced ONH injury.

Restraining Stem Cell Niche Plasticity: A New Talent of Eph Receptors

Multiple Eph receptor tyrosine kinases and ephrin ligands are expressed in stem cells and their niches, where their complex roles are under intense investigation. Nomura et al. (2010) reveal an unexpected function of Eph signaling in suppressing cell fate plasticity of niche cells in the adult brain.

The Role of TNFα in the Periaqueductal Gray During Naloxone-Precipitated Morphine Withdrawal in Rats

Tolerance and dependence are common complications of long-term treatment of pain with opioids, which substantially limit the long-term use of these drugs. The mechanisms underlying these phenomena are poorly understood. Studies have implicated the midbrain periaqueductal gray (PAG) in the pathogenesis of morphine withdrawal, and recent evidence suggests that proinflammatory cytokines in the PAG may play an important role in morphine withdrawal. Here we report that chronic morphine withdrawal-induced upregulation of glial fibrillary acidic protein (GFAP), tumor necrosis factor alpha (TNFα) and phosphorylation of ERK1/2 (pERK1/2) in the caudal ventrolateral PAG (vlPAG). Microinjection of recombinant TNFα into the vlPAG followed by intraperitoneal naloxone resulted in morphine withdrawal-like behavioral signs, and upregulation of pERK1/2, expression of Fos, and phosphorylation of cAMP response element binding (pCREB) protein. We used a herpes simplex virus (HSV)-based vector expressing p55 soluble TNF receptor (sTNFR) microinjected into the PAG to examine the role of the proinflammatory cytokine TNFα in the PAG in the naloxone-precipitated withdrawal response. Microinjection of HSV vector expressing sTNFR into the PAG before the start of morphine treatment significantly reduced the naloxone-precipitated withdrawal behavioral response and downregulated the expression of GFAP and TNFα in astrocytes of the PAG. TNFR type I colocalized with neuronal pERK1/2. Microinjection of HSV vector expressing sTNFR into the PAG also significantly reduced the phosphorylation of both ERK1/2 and CREB, and reduced Fos immunoreactivity in neurons of the PAG following naloxone-precipitated withdrawal. These results support the concept that proinflammatory cytokines expressed in astrocytes in the PAG may play an important role in the pathogenesis of morphine withdrawal response.

Hydrocephalus induces dynamic spatiotemporal regulation of aquaporin-4 expression in the rat brain.

BackgroundThe water channel protein aquaporin-4 (AQP4) is reported to be of possible major importance for accessory cerebrospinal fluid (CSF) circulation pathways. We hypothesized that changes in AQP4 expression in specific brain regions correspond to the severity and duration of hydrocephalus.MethodsHydrocephalus was induced in adult rats (~8 weeks) by intracisternal kaolin injection and evaluated after two days, one week and two weeks. Using magnetic resonance imaging (MRI) we quantified lateral ventricular volume, water diffusion and blood-brain barrier properties in hydrocephalic and control animals. The brains were analysed for AQP4 density by western blotting and localisation by immunohistochemistry. Double fluorescence labelling was used to study cell specific origin of AQP4.ResultsLateral ventricular volume was significantly increased over control at all time points after induction and the periventricular apparent diffusion coefficient (ADC) value significantly increased after one and two weeks of hydrocephalus. Relative AQP4 density was significantly decreased in both cortex and periventricular region after two days and normalized after one week. After two weeks, periventricular AQP4 expression was significantly increased. Relative periventricular AQP4 density was significantly correlated to lateral ventricular volume. AQP4 immunohistochemical analysis demonstrated the morphological expression pattern of AQP4 in hydrocephalus in astrocytes and ventricular ependyma. AQP4 co-localized with astrocytic glial fibrillary acidic protein (GFAP) in glia limitans. In vascular structures, AQP4 co-localized to astroglia but not to microglia or endothelial cells.ConclusionsAQP4 levels are significantly altered in a time and region dependent manner in kaolin-induced hydrocephalus. The presented data suggest that AQP4 could play an important neurodefensive role, and may be a promising future pharmaceutical target in hydrocephalus and CSF disorders.

Activation of astrocytes in the spinal cord contributes to the development of bilateral allodynia after peripheral nerve injury in rats

Activation of spinal cord microglia and astrocytes after peripheral nerve injury contributes to the development of behavioral hypersensitivity. Suppression of spinal cord glial activation attenuates the development of nerve injury-induced allodynia. The contribution of spinal cord glia to existing allodynia, however, is not known. We investigated whether intrathecally administered propentofylline, a glial inhibitor, reverses existing allodynia after nerve injury. Male Sprague–Dawley rats underwent L5 spinal nerve transection, and mechanical allodynia was assessed by measuring hind paw withdrawal thresholds bilaterally using von Frey filaments. Rats received either saline or propentofylline (1, 3, and 10 μg/d) for 7 days (days 0–7) by intrathecal infusion with an osmotic minipump. Other groups of rats received either intrathecal infusion of saline or propentofylline (10 μg/d) for 7 days on days 14–21 or 60–67 after surgery. After completing the intrathecal infusion, lumbar spinal cord sections were assessed for immunostaining of astrocytic glial fibrillary acidic protein and microglial OX-42. Propentofylline infusion on days 0–7 suppressed development of allodynia in both the ipsilateral and contralateral hind paws in a dose-dependent manner. Propentofylline treatment on days 14–21 or 60–67 did not reverse existing allodynia. Propentofylline infusion (10 μg/d) inhibited astrocytic activation bilaterally on days 0–7, 14–21, and 60–67 and inhibited microglial activation on days 14–21 but not on days 0–7 and 60–67. These results suggest that activation of spinal glia, especially astrocytes, dominantly contributes to the development of neuropathic pain and also to mirror-image pain.

Involvement of p300 in constitutive and HIV‐1 Tat‐activated expression of glial fibrillary acidic protein in astrocytes

HIV-1 Tat protein is an important pathogenic factor in HIV-1-associated neurological diseases. One hallmark of HIV-1 infection of the central nervous system (CNS) is astrocytosis, which is characterized by elevated glial fibrillary acidic protein (GFAP) expression in astrocytes. We have shown that Tat activates GFAP expression in astrocytes [Zhou et al., (2004) Mol Cell Neurosci 27:296-305] and that GFAP is an important regulator of Tat neurotoxicity [Zou et al., (2007) Am J Pathol 171:1293-1935]. However, the underlying mechanisms for Tat-mediated GFAP up-regulation are not understood. In this study, we reported concurrent up-regulation of adenovirus E1a-associated 300 kDa protein p300 and GFAP in Tat-expressing human astrocytoma cells and primary astrocytes. We showed that p300 was indeed induced by Tat expression and HIV-1 infection and that the induction occurred at the transcriptional level through the cis-acting elements of early growth response 1 (egr-1) within its promoter. Using siRNA, we further showed that p300 regulated both constitutive and Tat-mediated GFAP expression. Moreover, we showed that ectopic expression of p300 potentiated Tat transactivation activity and increased proliferation of HIV-1-infected astrocytes, but had little effect on HIV-1 replication in these cells. Taken together, these results demonstrate for the first time that Tat is a positive regulator of p300 expression, which in turn regulates GFAP expression, and suggest that the Tat-Egr-1-p300-GFAP axis likely contributes to Tat neurotoxicity and predisposes astrocytes to be an HIV-1 sanctuary in the CNS.

GLIAL TRANSCRIPTS AND IMMUNE-CHALLENGED GLIA IN THE SUPRACHIASMATIC NUCLEUS OF YOUNG AND AGED MICE

Biological rhythms are frequently disturbed with advancing age, and aging-related changes of glia in the hypothalamic suprachiasmatic nucleus (SCN), the master circadian pacemaker, require special attention. In particular, astrocytes contribute to SCN function, and aging is associated with increased inflammatory activity in the brain, in which microglia could be especially implicated. On this basis, we investigated in the SCN of young and old mice glial transcripts and cell features, and the glial cell response to a central inflammatory challenge. Quantitative real-time reverse transcriptase–polymerase chain reaction (RT-PCR) was used to analyze the expression of mRNAs encoding the astrocytic glial fibrillary acidic protein and the microglial antigen CD11b. Both these transcripts, here investigated in the SCN for the first time, were significantly increased in the old SCN. Glial cell phenotyping with immunohistochemistry revealed hypertrophic and intensely stained astrocytes and microglia in the aged SCN. In both age groups, microglia were scattered throughout the SCN and astrocytes were prominent in the ventral portion, where retinal fibers are densest; in the aged SCN, astrocytes were also numerous in the dorsal portion. After intracerebroventricular injections of a mixture of interferon-γ and tumor necrosis factor-α, or phosphate-buffered saline as control, immunolabeling was evaluated with stereological cell counts and confocal microscopy. Phenotypic features of astrocyte and microglia activation in response to cytokine injections were markedly enhanced in the aged SCN. Subregional variations in glial cell density were also documented in the aged compared to the young SCN. Altogether, the findings show increases in the expression of glial transcripts and hypertrophy of astrocytes and microglia in the aged SCN, as well as age-dependent variation in the responses of immune-challenged SCN glia. The data thus point out an involvement of glia in aging-related changes of the biological clock. (Author correspondence: marina.bentivoglio@univr.it)