GFAP Content Research Articles

COMBINED USE OF NOVEL MR IMAGING AND FLUID BIOMARKERS REVEAL DETERMINANTS OF MYELIN AND AXONAL LOSS WITH AGING

Abstract Cerebral white matter damage is a feature of Alzheimer’s disease, vascular dementia, and other neurodegenerative conditions, yet little is known about the molecular underpinnings of key features of white matter pathology. In a series of studies we have used targeted assays and large-scale proteomics platforms to determine how biomarkers of Alzheimer’s disease pathology (Aß42/40 ratio; pTau181), neuronal injury (NfL), reactive astrogliosis (GFAP), and other disease processes relate to state-of-the-art diffusion-based MRI measures of brain myelin content and axonal density in cognitively unimpaired older adults. Using data from the Baltimore Longitudinal Study of Aging (BLSA) and the GESTALT study, we measured plasma biomarkers using the Quanterix Simoa assay (targeted measurement of 4 proteins) and the SomaScan proteomic platform (untargeted measurement of 7000 proteins). Additionally, participants underwent 3T MRI, from which measure of myelin content (defined by myelin water fraction [MWF]) and axonal density (defined by neurite density index [NDI]) were derived. We found an association between higher plasma GFAP and lower myelin content in temporal brain regions. Additionally, higher levels of NfL and GFAP were associated with lower total brain axonal density. Using untargeted proteomics, we identified a set of 155 plasma proteins that were associations with myelin content (P <0.05). Using these findings, we applied machine learning to construct a plasma protein-based predictor of brain myelin content, which we plan to validate in a cohort of individuals with multiple sclerosis: a condition characterized by demyelination.

Curcumin modulates astrocyte function under basal and inflammatory conditions

Curcumin is a pleiotropic molecule with well-known anti-inflammatory effects. This molecule has attracted attention due to its capacity to pass the blood–brain–barrier and modulate central nervous system (CNS) cells, such as astrocytes. Astrocytes are the most numerous CNS cells, and play a pivotal role in inflammatory damage, a common feature in neurodegenerative diseases such as Alzheimer’s Disease. Although the actions of curcumin have been studied extensively in peripheral cells, few studies have investigated the effect of curcumin on astrocytes under basal and inflammatory conditions. The aim of this study was to characterize the effect of curcumin on astrocytic function (glutamatergic metabolism, GFAP and S100B), and investigate a possible synergic effect with another molecule, piperine. For this purpose, we used primary cultured astrocytes; our results showed that curcumin increases GSH and GFAP content, but decreases S100B secretion under basal conditions. Under inflammatory conditions, provoked by lipopolysaccharide (LPS), curcumin and piperine reversed the LPS-induced secretion of TNF-α, and piperine reverted the LPS-induced upregulation of GFAP content. Interestingly, curcumin decreases S100B secretion even more than LPS. These results highlight important context-dependent effects of curcumin and piperine on astrocytes. Although we did not observe synergic effects of co-treatment with curcumin and piperine, their effects were complementary, as piperine modulated GFAP content under inflammatory conditions, and curcumin modulated S100B secretion. Both curcumin and piperine had important anti-inflammatory actions in astrocytes. We herein provide new insights into the actions of curcumin in the CNS that may aid in the search for new molecular targets and possible treatments for neurological diseases.

Astrocyte culture models: Molecular and function characterization of primary culture, immortalized astrocytes and C6 glioma cells

The understanding of the physiology of astrocytes and their role in brain function progresses continuously. Primary astrocyte culture is an alternative method to study these cells in an isolated system: in their physiologic and pathologic states. Cell lines are often used as an astrocyte model, since they are easier and faster to manipulate and cost less. However, there are a few studies evaluating the different features of these cells which may put into question the validity of using them as astrocyte models. The aim of this study was to compare primary cultures (PC) with two cell lines - immortalized astrocytes and C6 cells, in terms of protein characterization, morphology and metabolic functional activity. Our results showed, under the same culture condition, that immortalized astrocytes and C6 are positive for differentiated astrocytic markers (eg. GFAP, S100B, AQP4 and ALDH1L1), although expressing them in less quantities then primary astrocyte cultures. Glutamate metabolism and cell communication are reduced in proliferative cells. However, glucose uptake is elevated in C6 lineage cells in comparison with primary astrocytes, probably due to their tumorigenic origin and high proliferation rate. Immortalized astrocytes presented a lower growth rate than C6 cells, and a similar basal morphology as primary astrocytes. However, they did not prove to be as good reproductive models of some of the classic astrocytic functions, such as S100B secretion and GFAP content, especially while under stimulation. In contrast, C6 cells presented similar results in comparison to primary astrocytes in response to stimuli. Here we provide a functional comparison of three astrocytic models, in an attempt to select the most suitable model for the study of astrocytes, optimizing the research in this area of knowledge.

Interleukin 4 Leads to Sustained Phosphorylation of the STAT6 and ERK Pathways in the Retina and Disrupts Subcortical Visual Circuitry in Rodents

Objective: Interleukin 4 (IL-4) is an anti-inflammatory cytokine related to different aspects of central nervous system development such as survival, proliferation, and differentiation, among others. Our goals were to investigate the effect of intravitreous treatment with IL-4 on the activation of downstream signaling pathways in the retina and the distribution of retinal axons within the superior colliculus (SC). Material and Methods: Lister hooded rats were submitted to an intravitreous injection of either IL-4 (5 U/µL) or PBS (vehicle) at postnatal day 10 (PND10). At PND11 or PND14, retinas were processed for Western blot or immunohistochemistry. At PND13, a group of animals received an intraocular injection of an anterograde tracer in the left (untreated) eye in order to label the uncrossed retinotectal axons. Results: Our data revealed that intravitreous treatment with IL-4 at PND10 leads to a decrease in GFAP content and a sustained increase in the phosphorylation of STAT6 and ERK levels in the retina. IL-4 also increases retinal axonal arbors within the SC, compared to control groups. Conclusions: These data suggest that a single in vivo treatment with IL-4 during the early stages of development modulates signaling pathways in the retina, resulting in altered binocular subcortical visual connectivity.

Effects of dexamethasone on the Li-pilocarpine model of epilepsy: protection against hippocampal inflammation and astrogliosis

BackgroundTemporal lobe epilepsy (TLE) is the most common form of partial epilepsy and is accompanied, in one third of cases, by resistance to antiepileptic drugs (AED). Most AED target neuronal activity modulated by ionic channels, and the steroid sensitivity of these channels has supported the use of corticosteroids as adjunctives to AED. Assuming the importance of astrocytes in neuronal activity, we investigated inflammatory and astroglial markers in the hippocampus, a key structure affected in TLE and in the Li-pilocarpine model of epilepsy.MethodsInitially, hippocampal slices were obtained from sham rats and rats subjected to the Li-pilocarpine model of epilepsy, at 1, 14, and 56 days after status epilepticus (SE), which correspond to the acute, silent, and chronic phases. Dexamethasone was added to the incubation medium to evaluate the secretion of S100B, an astrocyte-derived protein widely used as a marker of brain injury. In the second set of experiments, we evaluated the in vivo effect of dexamethasone, administrated at 2 days after SE, on hippocampal inflammatory (COX-1/2, PGE2, and cytokines) and astroglial parameters: GFAP, S100B, glutamine synthetase (GS) and water (AQP-4), and K+ (Kir 4.1) channels.ResultsBasal S100B secretion and S100B secretion in high-K+ medium did not differ at 1, 14, and 56 days for the hippocampal slices from epileptic rats, in contrast to sham animal slices, where high-K+ medium decreased S100B secretion. Dexamethasone addition to the incubation medium per se induced a decrease in S100B secretion in sham and epileptic rats (1 and 56 days after SE induction). Following in vivo dexamethasone administration, inflammatory improvements were observed, astrogliosis was prevented (based on GFAP and S100B content), and astroglial dysfunction was partially abrogated (based on Kir 4.1 protein and GSH content). The GS decrease was not prevented by dexamethasone, and AQP-4 was not altered in this epileptic model.ConclusionsChanges in astroglial parameters emphasize the importance of these cells for understanding alterations and mechanisms of epileptic disorders in this model. In vivo dexamethasone administration prevented most of the parameters analyzed, reinforcing the importance of anti-inflammatory steroid therapy in the Li-pilocarpine model and possibly in other epileptic conditions in which neuroinflammation is present.

Glial-associated changes in the cerebral cortex after collagenase-induced intracerebral hemorrhage in the rat striatum.

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.

The impact of chronic unpredictable stress on cuprizone-induced demyelination and the occurrence of depressive-like behavior in C57BL/6 mice

Objective To investigate whether cuprizone-induced demyelinating mice is more susceptible to developing depressive-like behaviors induced by chronic unpredictable stress (CUS), and to examine the underlying neuropathological changes. Methods C57BL/6 mice were randomly divided into four groups: control (n=10), stress (n=12), cuprizone (n=12) and cuprizone+ stress group (n=12). Mice in stress group were exposed to CUS for 3 weeks and mice in cuprizone group were fed with 0.3% cuprizone-containing food for 3 weeks. Mice in cuprizone+ stress group were exposed to both stress and cuprizone, and mice in control group were fed with normal food without exposure to stress. After 3 week exposure, they were subjected to behavioral tests including spontaneous activity, anxiety level and depressive-like behaviors and their brains were possessed for Western blot to test myelin marker MBP and astrocyte activation marker GFAP. Results CUS did not change the locomotor activity, anxiety levels and sucrose preference of cuprizone-induced demylinating mice, but did significantly increase their immobile time in forced swimming test (control (109.3±8.0)s, stress (111.3±21.8)s, cuprizone (90.5±8.9)s, cuprizone+ stress (155.0±9.1)s). Western blot revealed that CUS further decreased MBP expression (control (1.014±0.06), stress(1.088±0.104), cuprizone(0.436±0.071), cuproznie+ stress(0.150±0.041), (P<0.05)) and increased GFAP content (control (1.026±0.045), stress(0.846±0.078), cuprizone(1.736±0.215), cuprizone+ stress(2.428±0.314), (P<0.05)) in cuprizone-induced demyelinating mice. Conclusion Our findings suggest that demyelinating pathology increases individual susceptibility to chronic stress, which may induce depressive behaviors through damage of oligodendrocyte/myelin. Key words: Chronic unpredictable stress; Cuprizone; Demyelination; Myelin basic protein; Glial frillary acidic protein; Major depressive disorder

In vivo intracerebral administration of L-2-hydroxyglutaric acid provokes oxidative stress and histopathological alterations in striatum and cerebellum of adolescent rats

Patients affected by L-2-hydroxyglutaric aciduria (L-2-HGA) are biochemically characterized by elevated L-2-hydroxyglutaric acid (L-2-HG) concentrations in cerebrospinal fluid, plasma, and urine due to a blockage in the conversion of L-2-HG to α-ketoglutaric acid. Neurological symptoms associated with basal ganglia and cerebelar abnormalities whose pathophysiology is still unknown are typical of this neurometabolic disorder. In the present study we evaluated the early effects (30min after injection) of an acute in vivo intrastriatal and intracerebellar L-2-HG administration on redox homeostasis in rat striatum and cerebellum, respectively. Histological analyses of these brain structures were also carried out 7 days after L-2-HG treatment (long-term effects). L-2-HG significantly decreased the concentrations of reduced (GSH) and total glutathione (tGS), as well as of glutathione peroxidase (GPx) and reductase (GR) activities, but did not change the activities of superoxide dismutase and catalase in striatum. Furthermore, the concentrations of oxidized glutathione (GSSG) and malondialdehyde (MDA), as well as 2′,7′-dichlorofluorescein (DCFH) oxidation and hydrogen peroxide (H2O2) production, were increased, whereas carbonyl formation and nitrate plus nitrite concentrations were not altered by L-2-HG injection. It was also found that the melatonin, ascorbic acid plus α-tocopherol, and creatine totally prevented most of these effects, whereas N-acetylcysteine, the noncompetitive glutamate NMDA antagonist MK-801, and the nitric oxide synthase inhibitor L-NAME were not able to normalize the redox alterations elicited by L-2-HG in striatum. L-2-HG intracerebellar injection similarly provoked a decrease of antioxidant defenses (GSH, tGS, GPx, and GR) and an increase of the concentrations of GSSG, MDA, and H2O2 in cerebellum. These results strongly indicate that the major accumulating metabolite in L-2-HGA induce oxidative stress by decreasing the antioxidant defenses and enhancing reactive oxygen species in striatum and cerebellum of adolescent rats. Regarding the histopathological findings, L-2-HG caused intense vacuolation, lymphocyte and macrophage infiltrates, eosinophilic granular bodies, and necrosis in striatum. Immunohistochemistry revealed that L-2-HG treatment provoked an increase of GFAP and a decrease of NeuN immunostaining, indicating reactive astroglyosis and reduction of neuronal population, respectively, in striatum. Similar macrophage infiltrates, associated with less intense vacuolation and lymphocytic infiltration, were observed in cerebellum. However, we did not observe necrosis, eosinophilic granular bodies, and alteration of GFAP and NeuN content in L-2-HG-teated cerebellum. From the biochemical and histological findings, it is presumed that L-2-HG provokes striatal and cerebellar damage in vivo possibly through oxidative stress induction. Therefore, we postulate that antioxidants may serve as adjuvant therapy allied to the current treatment based on a protein-restricted diet and riboflavin and L-carnitine supplementation in patients affected by L-2-HGA.

Green tea (−)epigallocatechin-3-gallate reverses oxidative stress and reduces acetylcholinesterase activity in a streptozotocin-induced model of dementia

Alzheimer's disease (AD) is the most prevalent form of dementia. Intracerebroventricular (ICV) infusion of streptozotocin (STZ) provides a relevant animal model of chronic brain dysfunction that is characterized by long-term and progressive deficits in learning, memory, and cognitive behavior, along with a permanent and ongoing cerebral energy deficit. Numerous studies on green tea epigallocatechin gallate (EGCG) demonstrate its beneficial effects on cognition and memory. As such, this study evaluated, for the first time, the effects of sub-chronic EGCG treatment in rats that were submitted to ICV infusion of STZ (3mg/kg). Male Wistar rats were divided into sham, STZ, sham+EGCG and STZ+EGCG groups. EGCG was administered at a dose of 10mg/kg/day for 4 weeks per gavage. Learning and memory was evaluated using Morris’ Water Maze. Oxidative stress markers and involvement of the nitric oxide (NO) system, acetylcholinesterase activity (AChE) and glucose uptake were evaluated as well as glial parameters including S100B content and secretion and GFAP content. Our results show that EGCG was not able to modify glucose uptake and glutathione content, although cognitive deficit, S100B content and secretion, AChE activity, glutathione peroxidase activity, NO metabolites, and reactive oxygen species content were completely reversed by EGCG administration, confirming the neuroprotective potential of this compound. These findings contribute to the understanding of diseases accompanied by cognitive deficits and the STZ-model of dementia.

Short- and long-term treatment with estradiol or progesterone modifies the expression of GFAP, MAP2 and Tau in prefrontal cortex and hippocampus

AimsWe analyzed the effects of the short- and long-term administration of estradiol (E2) or progesterone (P4) after ovariectomy on the expression of MAP2, Tau and GFAP in prefrontal cortex and hippocampus. Main methodsSprague Dawley rats were ovariectomized and immediately treated with E2 or P4 for 2 or 18weeks. At the end of treatments, hippocampus and prefrontal cortex were excised, proteins were extracted and MAP2, Tau and GFAP were analyzed by Western blot. Key findingsMAP2 and Tau content was not modified by E2 in the prefrontal cortex. On the contrary, P4 decreased MAP2 content after a short-term treatment, while it increased that of MAP2 and TAU in this brain region after a long-term treatment. E2 increased MAP2 content in hippocampus. In this region, short-term administration of P4 increased that of MAP2. GFAP content was diminished after a long-term administration of P4 in hippocampus. SignificanceCurrent data emphasize the importance of short- and long-term sex steroid treatment on neuronal and glial cytoskeletal proteins expression.

Insulin treatment prevents diabetes-induced alterations in astrocyte glutamate uptake and GFAP content in rats at 4 and 8 weeks of diabetes duration

Rat astrocyte function is changed by diabetes mellitus relative to the nondiabetic state and we believe that altered function contributes to the central nervous system symptoms manifested by individuals with diabetes. We report here a comparison of astrocyte glutamate uptake and GFAP expression in streptozotocin-induced type 1 diabetic rats and insulin-treated diabetic rats at 4 and 8 weeks following diabetes onset. In glial plasmalemmal vesicle (GPV) preparations from treated rats, insulin prevented the increase observed in untreated, diabetic rats of both sodium-dependent and sodium-independent glutamate uptake. We determined by immunoblotting and immunohistochemistry that insulin treatment prevented the decrease of GFAP expression detected in the cerebral cortex, hippocampus, and cerebellum of untreated, diabetic rats. These observations indicate that insulin effects on astrocyte function are significant in managing diabetes-induced central nervous system pathology.

Neuroinflammation and astrocytic reaction in the course of Phoneutria nigriventer (armed-spider) blood–brain barrier (BBB) opening

Phoneutria nigriventer spider venom (PNV) causes uneven BBB permeability throughout different cerebral regions. Little is known about cellular and molecular responses which course with the PNV-induced BBB opening. We investigate by immunohistochemistry (IHC) and Western blotting (WB), the GFAP, S100, IFN-γ and TNF-α proteins expression in hippocampus and cerebellum after different time-points from venom or saline intravenous injection. All proteins variably altered its expression temporally and regionally. WB showed increased GFAP content at 15–45 min followed by a shift below the control level which was less pronounced in hippocampus. IHC showed reactive gliosis during all the trial period. In cerebellum, GFAP was mostly immunodetected in astrocytes of the molecular layer (Bergmann glia), as was S100 protein. The maximum S100 immunolabeling was achieved at 5 h. IFN-γ and TNF-α, expressed mostly by hippocampal neurons, increased along the trial period, suggesting a role in BBB permeability. In envenomed animals, closer contacts astrocyte–astrocyte, granule cells–granule cells and astrocytes-Purkinje cells were observed in cerebellum. Closer contacts between neurons–neurons–astrocytes–astrocytes were also seen in hippocampus. PNV contains serotonin, histamine, Ca 2+ channels-blocking toxins, some of which affect glutamate release. The hypothesis that such substances plus the cytokines generated, could have a role in BBB permeability, and that calcium homeostasis loss and disturbance of glutamate release are associated with the marked GFAP/S100 reaction in Bergmann glia is discussed. The existence of a CNS mechanism of defense modulated differentially for fast synthesis and turnover of GFAP, S100, IFN-γ and TNF-α proteins was evident. A clear explanation for this differential modulation is unclear, but likely result from regional differences in astrocytic/neuronal populations, BBB tightness, and/or extent/distribution of microvasculature and/or ion channels density/distribution. Such differences would respond for transient characteristics of BBB disruption. This in vivo model is useful for studies on drug delivery throughout the CNS and experimental manipulation of the BBB.

Postnatal and diet‐dependent increases in enteric glial cells and VIP‐containing neurones in preterm pigs

A mature enteric nervous system (ENS) is required to ensure a normal pattern of intestinal motility in order to regulate digestion after birth. We hypothesized that neuronal and glial components of the ENS would mature during the first postnatal days in preterm pigs that are a sensitive animal model of food intolerance and necrotizing enterocolitis (NEC). Stereological volume densities of the general neuronal population [assessed by betaIII-tubulin immunoreactivity (IR)] and subsets of neuronal (VIP-IR and nitrergic IR) and glial cells (GFAP-IR and S100-IR) were determined in the small intestine of newborn preterm piglets (93% gestation), after 3 days of receiving total parenteral nutrition (TPN) and after 3 days of TPN plus 2 days of enteral feeding with sow's colostrum or milk formula. Following TPN, VIP in the myenteric and inner submucous plexus and GFAP in the inner submucous plexus increased, while the relative volume of the total neuronal population remained constant. Introduction of enteral food induced variable degrees of food intolerance and NEC, especially after formula feeding, a diet that gave rise to a higher myenteric VIP and GFAP content in the inner submucous plexus than colostrum feeding. However, the ENS seemed unaffected by the presence of NEC-like intestinal lesions. Nevertheless, this study shows that the ENS is highly plastic during the first days after premature birth and adapts in an age- and diet-dependent manner. The observed postnatal adaptation in enteric VIP and GFAP may help to maintain intestinal homeostasis during suboptimal feeding regimens in preterm neonates.

Disorders in the Cytoskeleton of Astroglia and Neurons in the Rat Brain Induced by Long-Lasting Exposure to Ethanol and Correction of These Shifts by Hydrated Fullerene С60

Using an immunoblotting technique, we examined the content of proteins of intermediate filaments of the cytoskeleton of neurons and astroglial cells and also changes in the polypeptide composition of these proteins in different brain regions of rats subjected to long-term (12 weeks) alcoholization. The sensitivity of these indices to the effect of ethanol in different cerebral structures was in the following sequence: hippocampus > cerebral cortex > cerebellum. The greatest changes in a marker of the astrocyte cytoskeleton (glial fibrillary acidic protein, GFAP) were observed in the hippocampus of alcoholized animals, where the GFAP level was by 72% lower with respect to the control values. In this cerebral region, the content of the neurofilament 210-kdalton subunit also sharply dropped (by 76% with respect to the control). A positive correlation between a decrease in the GFAP content and loss of the neurofilament 210-kdalton subunit was demonstrated. These data show that the organization of the intracellular filamentary system of neurons and gliocytes is disturbed under experimental conditions, and this is one of the probable reasons for cell death in the nerve tissue induced by chronic consumption of ethanol. The use of a hydrated form of fullerene С60 (its molecular/colloid solution) for antioxidant correction of the pathological state of the CNS induced by the above-mentioned toxicant removed, to a considerable extent, negative modifications of cytoskeletal structures and protected astroglial and nerve cells from degeneration.

Astrogliosis in the hippocampus and cortex and cognitive deficits in rats with streptozotocin-induced diabetes: Effects of melatonin

We examined, using a Western blot technique, the contents and compositions of a specific neuronal protein, NCAM, and of an astrocyte marker, GFAP, in the hippocampus and cortex of rats with streptozotocin (STZ)-induced diabetes and compared these indices with those in control (intact) animals and STZ-diabetic rats treated with melatonin. Behavioral cognitive indices manifested in the passive avoidance test (PAT) and Morris water maze (MWM) learning performance were also estimated in the above groups of animals. As was found, STZ-diabetic rats demonstrated clear cognitive deficits according to the values of the retention latency in the PAT and time of reaching the escape platform in the MWM performance. In these animals, the GFAP content was elevated, and the amount of degraded products of this protein increased, as compared with the control. Simultaneously, considerable down-regulation of the NCAM expression and modifications of NCAM isoform composition were found in diabetic animals. In addition, significantly increased levels of lipid peroxidation (according to the amounts of malondialdehyde + 4-hydroxyalkenals) were measured in the cortex and hippocampus of rats with stable diabetic hyperglycemia. All the above-mentioned shifts were significantly smoothed or even nearly completely compensated in the case of treatment of STZ-diabetic rats with melatonin (10 mg/kg per day). The role of diabetes-related changes in the amount and composition of specific neural and glial proteins in the development of cognitive deficits, the involvement of oxidative stress in the mechanisms of the respective shifts, and possible mechanisms of the neuroprotective effect of melatonin with respect to diabetes-related pathological biochemical and behavioral shifts are discussed.

Ebselen increases cytosolic free Ca 2+ concentration, stimulates glutamate release and increases GFAP content in rat hippocampal astrocytes

We have investigated the effect of the seleno-organic compound and radical scavenger ebselen on rat hippocampal astrocytes in culture. Throughout our study we carried out determinations of [Ca 2+] c in fura-2-loaded cells by single cell imaging, glutamate secretion employing an enzymatic-based assay and GFAP expression, which was monitorized by immunocytochemistry and confocal microscopy. Our results show that ebselen (1–20 μM) dose dependently increases [Ca 2+] c, stimulates glutamate release and increases GFAP content, a hallmark of astrocyte reactivity. Ebselen did not alter significantly cell viability as assayed by determination of LDH release into the extracellular medium. Ebselen-evoked glutamate release and increase in GFAP content were Ca 2+-dependent, because incubation of astrocytes in the absence of extracellular Ca 2+ (medium containing 0.5 mM EGTA) and in the presence of the intracellular Ca 2+ chelator BAPTA (10 μM) significantly reduced ebselen-evoked changes in these parameters. The effects of ebselen we have observed may underline various signalling pathways which are important for cell proliferation, differentiation and function. However, aberrations in astroglial physiology could significantly compromise brain function, due to their role as modulators of neuron activity. Therefore, we consider that careful attention should be paid when employing ebselen as a prophylactic agent against brain damage.

Ethanol stimulates ROS generation by mitochondria through Ca 2+ mobilization and increases GFAP content in rat hippocampal astrocytes

We have employed rat hippocampal astrocytes in culture to investigate the effect of ethanol on reactive oxygen species (ROS) production as well as its effect on [Ca 2+] c and GFAP expression. Cells were loaded with the fluorescent probes fura-2 and H 2DCFDA for the determination of changes in [Ca 2+] c and ROS production respectively, employing spectrofluorimetry. GFAP content was determined by immunocytochemistry and confocal scanning microscopy. Our results show ROS production in response to 50 mM ethanol, that was reduced in Ca 2+-free medium (containing 0.5 mM EGTA) and in the presence of the intracellular Ca 2+ chelator BAPTA (10 μM). The effect of ethanol on ROS production was significantly reduced in the presence of the alcohol dehydrogenase inhibitor 4-methylpyrazole (1 mM), and the antioxidants resveratrol (100 μM) or catalase (300 U/ml). Preincubation of astrocytes in the presence of 10 μM antimycin plus 10 μM oligomycin to inhibit mitochondria completely blocked ethanol-evoked ROS production. In addition, ethanol led to a sustained increase in [Ca 2+] c that reached a constant level over the prestimulation values. Finally, incubation of astrocytes in the presence of ethanol increased the content of GFAP that was significantly reduced in the absence of extracellular Ca 2+ and by resveratrol and catalase pretreatment. The data obtained in the present study suggest that astrocytes are able to metabolize ethanol, which induces two effects on intracellular homeostasis: an immediate response (Ca 2+ release and ROS generation) and later changes involving GFAP expression. Both effects may underline various signaling pathways which are important for cell proliferation, differentiation and function.

Ketogenic diet fed rats have low levels of S100B in cerebrospinal fluid

Ketogenic diets have been used to treat seizure disorders of children resistant to conventional anti-epileptic drug treatment. The mechanism of action of this diet, however, is unknown. Gliosis is a very common characteristic in tissues associated with epileptogenesis and glial cytokines may be involved in the pathology of seizure disorders. We investigate herein, whether ketogenic diet fed rats demonstrate changes in the immunocontent of S100B, an astrocyte-derived cytokine elevated in the temporal lobe of refractory epilepsy. Lower levels of S100B were observed in cerebrospinal fluid with no significant changes in S100B and GFAP content in brain tissue. Ketogenic fed rats presented a lower seizure severity induced by pentylenetetrazole and no change in cerebrospinal fluid S100B after pentylenetetrazole administration. These results support the concept that the ketogenic diet is neuroprotective in seizure disorders. Since S100B has an extracellular activity in neuronal excitability and synaptic plasticity, it would be reasonable to conceive that a decrease in the S100B could be involved in the mechanism of action of the ketogenic diet. However, it is not possible to establish a direct link between reduced CSF S100B and decreased severity of PTZ-induced attacks at present moment. Regardless of this, CSF S100B could be proposed as an index of efficacy of ketogenic diet for seizure disorders.

A delayed increase in hippocampal proliferation following global asphyxia in the neonatal rat

Adult neurogenesis has been shown to be upregulated following a wide variety of brain injury paradigms. During the first weeks of postnatal life there is around 50 fold more neurogenesis occurring than in the adult CNS, yet little is known regarding the effect of neonatal brain injury on this developmental proliferation. We have investigated the effect of a global perinatal birth asphyxia on postnatal proliferation at 2, 5, 8, 11, 15, 21 and 28 days after birth (injury) using a 3H-thymidine tracer study. We found a specific upregulation of proliferation at 5 days after the injury within the injured hippocampus only, with an associated increase in hippocampal mass and without any changes in GFAP content at any timepoint. Perinatal asphyxia did not alter proliferation within the cerebellum, sub ventricular zone, olfactory bulb, cervical or thoracic spinal cord. Similarly, no changes in corticosterone levels were induced by the injury. Since there were no changes in GFAP content we hypothesize that this increased proliferation is likely neurogenetic, similar to what is seen in the adult brain following injury. Further we show that the dramatic increase in corticosterone at the end of the stress hyporesponsive period is not responsible for the equally dramatic decrease in postnatal proliferation within the CNS.

Long-term microglial and astroglial activation in the hippocampus of trimethyltin-intoxicated rat: stimulation of NGF and TrkA immunoreactivities in astroglia but not in microglia

In the present study we investigated the microglial and astroglial response after trimethyltin (TMT) exposure over a prolonged period of time. Male Wistar rats were given a single dose of TMT (8 mg/kg, i.p.) and survived 4, 7, 21, 60 and 180 days after the administration of the toxin. Histochemistry (Griffonia simplicifolia lectin staining) and immunocytochemistry for GFAP were applied to identify micro- and astroglial cells, respectively. To assess the trophic response of glial cells (NGF and TrkA expression), single or double staining experiments were performed. In addition, the biochemical evaluation of GFAP and NGF were carried out at chosen timepoints using immunoblotting technique and ELISA, respectively. The main findings of our study were as follows. (1) A protracted activation of microglia (at least up to 2 months posttreatment). (2) A long-lasting expression of GFAP immunoreactivity (at least up to 6 months posttreatment) and a steady increase in GFAP content (at least up to 2 months posttreatment). (3) The appearance of enormously enlarged, round-shape astrocytes exclusively localized to CA1 and observed 2 months posttreatment. (4) The stimulation of NGF and TrkA expression in reactive astrocytes. (5) The strongest activation of micro- and astroglia coincided with the most prominent neurodegeneration in the hippocampus, i.e., in CA4/CA3c and CA1. It is tempting to assume that the activation of glial cells in the hippocampal areas particularly vulnerable to TMT may affect neuronal fate after neurotoxic insult.