Immunohistochemical analysis of GFAP expression in the experimental sepsis-associated encephalopathy

Pathophysiology of sepsis-associated encephalopathy (SAE) is linked to blood-brain barrier breakdown, neuroinflammation and neurotransmitter imbalance in the brain. Astroglia, the most abundant cell population within the brain, plays the critical role in control of all kinds of homeostatic processes, thereby regulating the adaptive reactions of the brain to various challenges. Astroglia are highly heterogenous across the brain regions, therefore, damaging factors stimulate heterogenous astroglial reactivity and response in different brain regions. The aim of this study was determining immunohistochemical features of GFAP expression in various brain regions in the model of rodent experimental sepsis. Materials and methods. The experiment was performed in Wistar rats: control group of 5 sham-operated rats and the main group of 20 rats subjected to cecum ligation and puncture (CLP) procedure. The immunohistochemical study of GFAP expression in the sensorimotor cortex, subcortical white matter, hippocampal, thalamic and caudate nucleus/putamen regions was performed from 20 to 48 hours of the postoperative period. Results. Starting from the 12th hour after CLP, animals began display progressive increase in signs of periorbital exudation, piloerection, fever-/hypothermia, diarrhea, social isolation, lethargy, and respiratory impairment. In the period of 20–38 hours, 9 animals showed expressed previously listed symptoms and were euthanized (CLP-B – lethal group), 11 rats survived until 48 hours of the experiment (CLP-A – survived group). In the lethal group, starting from 20 to 38 hours after the CLP procedure, a significant (relative to control) regionally-specific dynamic increase in the level of GFAP expression was observed in the brain: in the cortex – by 465 %, in the subcortical white matter – by 198 %, in the hippocampus – by 250 %, from the 23rd hour – in the caudate nucleus/putamen by 18 %. In the thalamus, no significant changes in the level of GFAP expression were observed. In the cortex and hippocampus of survived animals, 48 h after CLP, higher values of GFAP expression were observed comparing to the group of non-survived animals. Conclusions. Under conditions of the experimental SAE, an early dynamic increase in the astroglial reactivity was observed in the cortex, hippocampus, white matter, and caudate nucleus/putamen of the brain with the most significant increase of indicators in the cortex and hippocampus, which potentially indicates relatively more vulnerable areas of the brain to damaging factors, as well as places of the most active intercellular interaction in the condition of systemic inflammation. Higher values of GFAP expression in the cortex and hippocampus of survived animals at 48 hours of the experiment, compared with indicators of non-survived group, indicate increased astroglial reactivity in these brain regions at the noted time period, accompanied by relatively more favorable clinical course of the disease.

Reduced GFAP Expression in Bergmann Glial Cells in the Cerebellum of Sigma-1 Receptor Knockout Mice Determines the Neurobehavioral Outcomes after Traumatic Brain Injury

Neuroprotective effects of Sigma-1 receptor (S1R) ligands have been observed in multiple animal models of neurodegenerative diseases. Traumatic brain injury (TBI)-related neurodegeneration can induce long-lasting physical, cognitive, and behavioral disabilities. The aim of our study was to evaluate the role of S1R in the development of neurological deficits after TBI. Adult male wild-type CD-1 (WT) and S1R knockout (S1R-/-) mice were subjected to lateral fluid percussion injury, and behavioral and histological outcomes were assessed for up to 12 months postinjury. Neurological deficits and motor coordination impairment were less pronounced in S1R-/- mice with TBI than in WT mice with TBI 24 h after injury. TBI-induced short-term memory impairments were present in WT but not S1R-/- mice 7 months after injury. Compared to WT animals, S1R-/- mice exhibited better motor coordination and less pronounced despair behavior for up to 12 months postinjury. TBI induced astrocyte activation in the cortex of WT but not S1R-/- mice. S1R-/- mice presented a significantly reduced GFAP expression in Bergmann glial cells in the molecular layer of the cerebellum compared to WT mice. Our findings suggest that S1R deficiency reduces TBI-induced motor coordination impairments by reducing GFAP expression in Bergmann glial cells in the cerebellum.

Investigating the Effects of Allium sativum on the Prefrontal Cortex in Lithium Chloride Pilocarpine-Induced Epilepsy in Wistar Rat

The prefrontal cortex (PFC), mediating executive brain functions is impaired in epilepsy. Allium sativum (AS) anti-seizure potential on the PFC of experimentally-induced epilepsy was investigated. Forty-eight male Wistar rats (200-250g) were randomized into six groups. Control (2mL/kg distilled water); AS only (100mg/kg); LiCl+PC (lithium chloride, 127mg/kg, and pilocarpine, 30mg/kg); LiCl+PC+AS100mg/kg and LiCl+PC+AS300mg/kg received LiCl+PC and 100mg/kg AS and 300mg/kg AS respectively; LiCl+PC+SV received LiCl+PC and sodium valproate (10mg/kg). Treatments lasted for 21 days, behavioural tests then preceded sacrifice. Brain tissues were excised, fixed in 10% neutral buffered formalin for demonstration of PFC cytoarchitecture and glial fibrillary acidic protein (GFAP) expression. Neurotransmitters were also assayed. Walling and rearing frequencies reduced significantly (p<0.05) in the LiCl+PC group compared to control. Glutamate and acetylcholine levels increased in all groups except AS only, while gamma-aminobutyric acid, dopamine, serotonin and norepinephrine levels increased in the LiCl+PC+AS100mg/kg, LiCl+PC+AS300mg/kg and LiCl+PC+SV groups compared to the control. Cytochrome C oxidase and glucose-6-phosphate dehydrogenase activities significantly increased (p<0.05) in all groups, while nitric oxide levels increased in the LiCl+PC+AS300mg/kg and LiCl+PC+SV groups compared to the control. Cytoarchitecturally, the LiCl+PC PFC showed neurodegenerative features, increased GFAP expression, while the treated groups showed preserved neurons and mild astrogliosis. Conclusively, AS showed neuroprotective potentials against LiCl+PC-induced neuronal degeneration, mitigated reactive PFC astrogliosis. However, AS did not lower glutamate and other neurotransmitter levels.

Caffeine, Nicotine and MDMA Effects on Retina suggestive of Potential Functional Consequences

The implications of the use of psychoactive substances, that are usually abused on the retina are yet to be adequately explored. The retina is neural in nature. Most investigations on psychoactive agents have only studied their effects on the brain and behaviour. The mechanisms employed by these agents in producing their effects on the brain suggest that the retina, being neural, might also be significantly affected by the use of the substances. This research investigated the effects of the prolonged use of caffeine, nicotine and 4-methylenedioxymethamphetamine [MDMA] on the retina. Juvenile male experimental Wistar were grouped and administered the lower and higher dose of each agent while a reference group remained as the Group A. Groups B and C received the lower [30mg/kg body weight]and the higher [50mg/kg body weight]doses of caffeine respectively; Groups D and E received the lower [10mg/kg body weight] and higher [20mg/kg body weight] doses of nicotine respectively while Groups F and G received the lower [20 mg/kg body weight] and the higher [40 mg/kg body weight] doses of MDMA respectively. The substances had effects on the thickness of the retina with higher doses in each instance causing reductions in retina thickness; the patterns of GFAP expression were also aberrant with the MDMA treated groups being most aberrant. There was no sign of extensive loss of any type of retinal cells. Rhodopsin expression generally demonstrated active rods and provided insight into relatively heathy cones. There is evidence that these agents altered retina thickness and GFAP expression but without extensive disruptions to serve as pathological hallmarks of retina degeneration. The consequences of these might be further investigated.

Neuroprotective Effects of Berberine Hydrochloride on Cognitive Dysfunction-Induced by Methamphetamine: Immunohistochemical and Behavioral Studies in Rats

Introduction: Methamphetamine (MA) as an addictive psychostimulant drug affects the central nervous system. The present research aimed at evaluating the impact of Berberine-hydrochloride on cognitive function Improvement and neuroprotective effects in MA Addicted Rats. Methods: In this study, 27 Male Wistar rats were randomly assigned to three groups, including Control, MA addiction and MA addiction with Berberine Hydrochloride (100 mg/kg/day) per oral during the three-week period of withdrawal. Two groups received inhaled MA self-administration for two weeks (up to 10 mg/kg). Following the experimental procedures, Morris Water Maze (MWM) and shuttle box were used to assess memory and hippocampal sections from the animals were examined for caspase-3, ki-67 and GFAP expression. Results: The obtained results from MWM showed that Berberine Hydrochloride decreases (p<0.01) the distance moved and spent time to reach the hidden platform in four-day learning trails phase and there were significant differences in the distance moved, spent time and frequency of motion in target quadrant on probe test day between groups. Berberine Hydrochloride reduced also the latency to enter animals into the dark chamber in the treated group in comparison with the control group (p<0.05). A significant decrease in activation of caspases-3, higher percentages of Ki67 expression and increase in GFAP expression of cells in Addicted group was found to compare with Berberine-treated and control groups (p<0.05). Conclusions: Berberine Hydrochloride administration for 3 weeks improves cognition function in MA addiction and it has a potential for neuroprotective efficacy.

Mingmu Xiaomeng Tablets Restore Autophagy and Alleviate Diabetic Retinopathy by Inhibiting PI3K/Akt/mTOR Signaling

Objective: To investigate the effect of Mingmu Xiaomeng tablets (MMXM) on the expression of phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR)-related proteins in a diabetic rat model.Methods: Thirty-two male Sprague Dawley rats were randomly divided into four groups: normal control (NC), diabetic model (DM) control, MMXM, and calcium dobesilate (CD) Rats injected with streptozotocin (STZ) were used as an experimental diabetes model. After 14 weeks, autophagy and PI3K/Akt/mTOR signaling pathway proteins were detected by western blot. Glial fibrillary acidic protein (GFAP) expression in Müller cells was examined by immunohistochemistry. Retinal function was evaluated with electroretinography, and retinal ultrastructure was observed by transmission electron microscopy. Serum cytokine levels were detected with protein chip technology.Results: MMXM restored autophagy by decreasing the protein expression of LC3-II and p62 and reducing the phosphorylation of PI3K, Akt, and mTOR, thus promoting autophagy. MMXM decreased GFAP expression in retinal Müller cells; restored electrophysiology indexes and retinal ultrastructures; and reduced serum levels of interleukin (IL)-1β, IL-4, IL-6, tumor necrosis factor-α, and vascular endothelial growth factor.Conclusion: MMXM may protect the diabetic retina by inhibiting PI3K/Akt/mTOR signaling and enhancing autophagy.

In vitro Characterization of the Regional Binding Distribution of Amyloid PET Tracer Florbetaben and the Glia Tracers Deprenyl and PK1195 in Autopsy Alzheimer’s Brain Tissue

Background: Emerging evidence indicates a central role of gliosis in Alzheimer’s disease (AD) pathophysiology. However, the regional distribution and interaction of astrogliosis and microgliosis in association with amyloid-β (Aβ) still remain uncertain. Objective: Here we studied the pathological profiles in autopsy AD brain by using specific imaging tracers. Methods: Autopsy brain tissues of AD (n = 15, age 70.4±8.5 years) and control cases (n = 12, age 76.6±10.9) were examined with homogenate binding assays, autoradiography for Aβ plaques (3H-florbetaben/3H-PIB), astrogliosis (3H-L-deprenyl), and microgliosis (3H-PK11195/3H-FEMPA), as well as immunoassays. Results: In vitro saturation analysis revealed high-affinity binding sites of 3H-florbetaben, 3H-L-deprenyl, and 3H-PK11195/3H-FEMPA in the frontal cortex of AD cases. In vitro 3H-florbetaben binding increased across cortical and subcortical regions of AD compared to control with the highest binding in the frontal and parietal cortices. The in vitro 3H-L-deprenyl binding showed highest binding in the hippocampus (dentate gyrus) followed cortical and subcortical regions of AD while the GFAP expression was upregulated only in the hippocampus compared to control. The in vitro 3H-PK11195 binding was solely increased in the parietal cortex and the hippocampus of AD compared to control. The 3H-florbetaben binding positively correlated with the 3H-L-deprenyl binding in the hippocampus and parietal cortex of AD and controls. Similarly, a positive correlation was observed between 3H-florbetaben binding and GFAP expression in hippocampal AD and control brain. Conclusion: The use of multi-modal imaging tracers revealed different regional pattern of changes in autopsy AD brain with respect to amyloid plaque pathology versus astrogliosis and microgliosis.

miR-31 promotes neural stem cell proliferation and restores motor function after spinal cord injury

This study aims to examine whether miR-31 promotes endogenous NSC proliferation and be used for spinal cord injury management. In the present study, the morpholino knockdown of miR-31 induced abnormal neuronal apoptosis in zebrafish, resulting in impaired development of the tail. miR-31 agomir transfection in NSCs increased Nestin expression and decreased ChAT and GFAP expression levels. miR-31 induced the proliferation of mouse NSCs by upregulating the Notch signaling pathway, and more NSCs entered G1; Notch was inhibited by miR-31 inactivation. Injection of a miR-31 agomir into mouse models of spinal cord injury could effectively restore motor functions after spinal cord injury, which was achieved by promoting the proliferation of endogenous NSCs. After the injection of a miR-31 agomir in spinal cord injury mice, the expression of Nestin and GFAP increased, while GFAP expression decreased. In conclusion, the zebrafish experiments prove that a lack of miR-31 will block nervous system development. In spinal cord injury mouse models, miR-31 overexpression might promote spinal cord injury repair.

RGC-32 Regulates Generation of Reactive Astrocytes in Experimental Autoimmune Encephalomyelitis

Astrocytes are increasingly recognized as critical contributors to multiple sclerosis pathogenesis. We have previously shown that lack of Response Gene to Complement 32 (RGC-32) alters astrocyte morphology in the spinal cord at the peak of experimental autoimmune encephalomyelitis (EAE), suggesting a role for RGC-32 in astrocyte differentiation. In this study, we analyzed the expression and distribution of astrocytes and astrocyte progenitors by immunohistochemistry in spinal cords of wild-type (WT) and RGC-32-knockout (KO) mice with EAE and of normal adult mice. Our analysis showed that during acute EAE, WT astrocytes had a reactive morphology and increased GFAP expression, whereas RGC-32 KO astrocytes had a morphology similar to that of radial glia and an increased expression of progenitor markers such as vimentin and fatty acid binding protein 7 (FABP7). In control mice, GFAP expression and astrocyte density were also significantly higher in the WT group, whereas the number of vimentin and FABP7-positive radial glia was significantly higher in the RGC-32 KO group. In vitro studies on cultured neonatal astrocytes from WT and RGC-32 KO mice showed that RGC-32 regulates a complex array of molecular networks pertaining to signal transduction, growth factor expression and secretion, and extracellular matrix (ECM) remodeling. Among the most differentially expressed factors were insulin-like growth factor 1 (IGF1), insulin-like growth factor binding proteins (IGFBPs), and connective tissue growth factor (CTGF); their expression was downregulated in RGC-32-depleted astrocytes. The nuclear translocation of STAT3, a transcription factor critical for astrogliogenesis and driving glial scar formation, was also impaired after RGC-32 silencing. Taken together, these data suggest that RGC-32 is an important regulator of astrocyte differentiation during EAE and that in the absence of RGC-32, astrocytes are unable to fully mature and become reactive astrocytes.