Glial Fibrillary Acidic Protein Immunoreactivity Research Articles

Meningeal Damage and Interface Astroglial Scarring in the Rat Brain Exposed to a Laser-Induced Shock Wave(s).

In the past decade, signature clinical neuropathology of blast-induced traumatic brain injury has been under intense debate, but interface astroglial scarring (IAS) seems to be convincing. In this study, we examined whether IAS could be replicated in the rat brain exposed to a laser-induced shock wave(s) (LISW[s]), a tool that can produce a pure shock wave (primary mechanism) without dynamic pressure (tertiary mechanism). Under certain conditions, we observed astroglial scarring in the subpial glial plate (SGP), gray-white matter junctions (GM-WM), ventricular wall (VW), and regions surrounding cortical blood vessels, accurately reproducing clinical IAS. We also observed shock wave impulse-dependent meningeal damage (dural microhemorrhage) in vivo by transcranial near-infrared (NIR) reflectance imaging. Importantly, there were significant correlations between the degree of dural microhemorrhage and the extent of astroglial scarring more than 7 days post-exposure, suggesting an association of meningeal damage with astroglial scarring. The results demonstrated that the primary mechanism alone caused the IAS and meningeal damage, both of which are attributable to acoustic impedance mismatching at multi-layered tissue boundaries. The time course of glial fibrillary acidic protein (GFAP) immunoreactivity depended not only on the LISW conditions but also on the regions. In the SGP, significant increases in GFAP immunoreactivity were observed at 3 days post-exposure, whereas in the GM-WM and VW, GFAP immunoreactivity was not significantly increased before 28 days post-exposure, suggesting different pathological mechanisms. With the high-impulse single exposure or the multiple exposure (low impulse), fibrotic reaction or fibrotic scar formation was observed, in addition to astroglial scarring, in the cortical surface region. Although there are some limitations, this seems to be the first report on the shock-wave-induced IAS rodent model. The model may be useful to explore potential therapeutic approaches for IAS.

Alcohol exacerbates psychosocial stress-induced neuropsychiatric symptoms: Attenuation by geraniol

Adaptation to psychosocial stress is psychologically distressing, initiating/promoting comorbidity with alcohol use disorders. Emerging evidence moreover showed that ethanol (EtOH) exacerbates social-defeat stress (SDS)-induced behavioral impairments, neurobiological sequelae, and poor therapeutic outcomes. Hence, this study investigated the effects of geraniol, an isoprenoid monoterpenoid alcohol with neuroprotective functions on EtOH escalated SDS-induced behavioral impairments, and neurobiological sequelae in mice. Male mice chronically exposed to SDS for 14 days were repeatedly fed with EtOH (2 g/kg, p. o.) from days 8–14. From days 1–14, SDS-EtOH co-exposed mice were concurrently treated with geraniol (25 and 50 mg/kg) or fluoxetine (10 mg/kg) orally. After SDS-EtOH translational interactions, arrays of behavioral tasks were examined, followed by investigations of oxido-inflammatory, neurochemicals levels, monoamine oxidase-B and acetylcholinesterase activities in the striatum, prefrontal-cortex, and hippocampus. The glial fibrillary acid protein (GFAP) expression was also quantified in the prefrontal-cortex immunohistochemically. Adrenal weights, serum glucose and corticosterone concentrations were measured. EtOH exacerbated SDS-induced low-stress resilience, social impairment characterized by anxiety, depression, and memory deficits were attenuated by geraniol (50 and 100 mg/kg) and fluoxetine. In line with this, geraniol increased the levels of dopamine, serotonin, and glutamic-acid decarboxylase enzyme, accompanied by reduced monoamine oxidase-B and acetylcholinesterase activities in the prefrontal-cortex, hippocampus, and striatum. Geraniol inhibited SDS-EtOH-induced adrenal hypertrophy, corticosterone, TNF-α, IL-6 release, malondialdehyde and nitrite levels, with increased antioxidant activities. Immunohistochemical analyses revealed that geraniol enhanced GFAP immunoreactivity in the prefrontal-cortex relative to SDS-EtOH group. We concluded that geraniol ameliorates SDS-EtOH interaction-induced behavioral changes via normalization of neuroimmune-endocrine and neurochemical dysregulations in mice brains.

Changes in Immunoreactivity of Vimentin and GFAP in the Hippocampus of Patients with Drug-Resistant Epilepsy

Epilepsy is a serious neurological disorder that can greatly impact a person’s quality of life and even result in disability. Unfortunately, despite the use of various antiepileptic drugs, more than 30% of patients develop a form of the disease that is resistant to medication, leaving surgery as the only treatment option. In order to better understand and potentially treat epilepsy, there has been a focus on the role of glial mechanisms and the potential for pathogenetic treatment. Specifically, researchers have looked at the expression of cytoskeletal proteins, such as Glial Fibrillary Acidic Protein (GFAP) and vimentin, in nervous tissue. Our study examined the expression of these proteins in the hippocampus of patients with different types of hippocampal sclerosis and Drug-Resistant Epilepsy (DRE). We found a significant increase in the expression of GFAP and vimentin in these structures, suggesting a potential role in the development of reactive gliosis. However, we did not find any correlations between these proteins in all areas studied. This suggests that the activation of these cytoskeletal proteins may occur independently and ultimately contribute to the development of reactive gliosis. These findings provide valuable insights into the mechanisms of epilepsy and may help improve diagnostic and treatment approaches.

Impaired amygdala astrocytic signaling worsens neuropathic pain-associated neuronal functions and behaviors.

Introduction: Pain is a clinically relevant health care issue with limited therapeutic options, creating the need for new and improved analgesic strategies. The amygdala is a limbic brain region critically involved in the regulation of emotional-affective components of pain and in pain modulation. The central nucleus of amygdala (CeA) serves major output functions and receives nociceptive information via the external lateral parabrachial nucleus (PB). While amygdala neuroplasticity has been linked causally to pain behaviors, non-neuronal pain mechanisms in this region remain to be explored. As an essential part of the neuroimmune system, astrocytes that represent about 40-50% of glia cells within the central nervous system, are required for physiological neuronal functions, but their role in the amygdala remains to be determined for pain conditions. In this study, we measured time-specific astrocyte activation in the CeA in a neuropathic pain model (spinal nerve ligation, SNL) and assessed the effects of astrocyte inhibition on amygdala neuroplasticity and pain-like behaviors in the pain condition. Methods and Results: Glial fibrillary acidic protein (GFAP, astrocytic marker) immunoreactivity and mRNA expression were increased at the chronic (4weeks post-SNL), but not acute (1week post-SNL), stage of neuropathic pain. In order to determine the contribution of astrocytes to amygdala pain-mechanisms, we used fluorocitric acid (FCA), a selective inhibitor of astrocyte metabolism. Whole-cell patch-clamp recordings were performed from neurons in the laterocapsular division of the CeA (CeLC) obtained from chronic neuropathic rats. Pre-incubation of brain slices with FCA (100µM, 1h), increased excitability through altered hyperpolarization-activated current (Ih) functions, without significantly affecting synaptic responses at the PB-CeLC synapse. Intra-CeA injection of FCA (100µM) had facilitatory effects on mechanical withdrawal thresholds (von Frey and paw pressure tests) and emotional-affective behaviors (evoked vocalizations), but not on facial grimace score and anxiety-like behaviors (open field test), in chronic neuropathic rats. Selective inhibition of astrocytes by FCA was confirmed with immunohistochemical analyses showing decreased astrocytic GFAP, but not NeuN, signal in the CeA. Discussion: Overall, these results suggest a complex modulation of amygdala pain functions by astrocytes and provide evidence for beneficial functions of astrocytes in CeA in chronic neuropathic pain.

Ghrelin ameliorates neuronal damage, oxidative stress, inflammatory parameters, and GFAP expression in traumatic brain injury

ABSTRACT Objective This study investigated the effects of ghrelin on oxidative stress, working memory, inflammatory parameters, and neuron degeneration. Methods TBI was produced with the weight-drop technique. Rats in the G+TBI and TBI+G groups received ghrelin for 7 or 2 days, respectively. The control group received saline. On the 8th day of the study, the brain and blood tissue were taken under anesthesia. Results A significant increase in brain GSH-PX, MDA, IL-1β, TGF-β1, and IL-8 levels and a significant decrease in CAT levels were found in the TBI group compared to the control. Serum MDA, GSH, IL-1β, and IL-8 levels were increased with TBI. Ghrelin treatment after TBI significantly increased the serum GSH, CAT, GSH-PX, and brain GSH and CAT levels, while it significantly decreased the serum MDA, IL-1β, and brain MDA, TGF-β1, and IL-8 levels. Histological evaluations revealed that ghrelin treatment led to a reduction in inflammation, while also significantly ameliorating TBI-induced neuron damage and vascular injuries. Immunohistochemistry staining showed that GFAP staining intensity was significantly increased in the cortex and hippocampus in TBI, and GFAP immunoreactivity was decreased with ghrelin treatment. Conclusion The results from this study suggested that ghrelin may have curative effects on TBI.

AAV8 vector induced gliosis following neuronal transgene expression.

Expression of light sensitive ion channels by selected neurons has been achieved by viral mediated transduction with gene constructs, but for this to have therapeutic uses, for instance in treating epilepsy, any adverse effects of viral infection on the cerebral cortex needs to be evaluated. Here, we assessed the impact of adeno-associated virus 8 (AAV8) carrying DNA code for a soma targeting light activated chloride channel/FusionRed (FR) construct under the CKIIa promoter. Viral constructs were harvested from transfected HEK293 cells in vitro and purified. To test functionality of the opsin, cultured rodent neurons were transduced and the light response of transduced neurons was assayed using whole-cell patch-clamp recordings. In vivo expression was confirmed by immunofluorescence for FR. Unilateral intracranial injections of the viral construct were made into the mouse neocortex and non-invasive fluorescence imaging of FR expression made over 1-4 weeks post-injection using an IVIS Spectrum system. Sections were also prepared from injected mouse cortex for immunofluorescence staining of FR, alongside glial and neuronal marker proteins. In vitro, cortical neurons were successfully transduced, showing appropriate physiological responses to light stimulation. Following injections in vivo, transduction was progressively established around a focal injection site over a 4-week period with spread of transduction proportional to the concentration of virus introduced. Elevated GFAP immunoreactivity, a marker for reactive astrocytes, was detected near injection sites associated with, and proportional to, local FR expression. Similarly, we observed reactive microglia around FR expressing cells. However, we found that the numbers of NeuN+ neurons were conserved close to the injection site, indicating that there was little or no neuronal loss. In control mice, injected with saline only, astrocytosis and microgliosis was limited to the immediate vicinity of the injection site. Injections of opsin negative viral constructs resulted in comparable levels of astrocytic reaction as seen with opsin positive constructs. We conclude that introduction of an AAV8 vector transducing expression of a transgene under a neuron specific promotor evokes a mild inflammatory reaction in cortical tissue without causing extensive short-term neuronal loss. The expression of an opsin in addition to a fluorescent protein does not significantly increase neuroinflammation.

Dorsal hippocampal astrocytes mediate the development of heroin withdrawal-enhanced fear learning.

There is a significant co-occurrence of opioid use disorder (OUD) and post-traumatic stress disorder (PTSD) in clinical populations. However, the neurobiological mechanisms linking chronic opioid use, withdrawal, and the development of PTSD are poorly understood. Our previous research has shown that proinflammatory cytokines, expressed primarily by astrocytes in the dorsal hippocampus (DH), play a role in the development of heroin withdrawal-enhanced fear learning (HW-EFL), an animal model of PTSD-OUD comorbidity. Given the role of astrocytes in memory, fear learning, and opioid use, our experiments aimed to investigate their involvement in HW-EFL. Experiment 1 examined the effect of withdrawal from chronic heroin administration on GFAP surface area and volume, and identified increased surface area and volume of GFAP immunoreactivity in the dentate gyrus (DG) following 24-hour heroin withdrawal. Experiment 2 examined astrocyte morphology and synaptic interactions at the 24-hour withdrawal timepoint using an astroglial membrane-bound GFP (AAV5-GfaABC1D-lck-GFP). Although we did not detect significant changes in surface area and volume of GfaABC1D-Lck-GFP labelled astrocytes, we did observe a significant increase in the colocalization of astrocyte membranes with PSD-95 (postsynaptic density protein 95) in the DG. Experiment 3 tested if stimulating astroglial Gi signaling in the DH alters HW-EFL, and our results demonstrate this manipulation attenuates HW-EFL. Collectively, these findings contribute to our current understanding of the effects of heroin withdrawal on astrocytes and support the involvement of astrocytes in the comorbid relationship between opioid use and anxiety disorders.

Oleuropein Has Modulatory Effects on Systemic Lipopolysaccharide-Induced Neuroinflammation in Male Rats

BackgroundNeuroinflammation induced by systemic inflammation is a risk factor for developing chronic neurologic disorders. Oleuropein (OLE) has antioxidant and anti-inflammatory properties; however, its effect on systemic inflammation-related neuroinflammation is unknown. ObjectivesThis study aimed to determine whether OLE protects against systemic lipopolysaccharide (LPS)-induced neuroinflammation in rats. MethodsSix-wk-old Wistar rats were randomly assigned to 1 of the following 5 groups: 1) control, 2) OLE-only, 3) LPS + vehicle, 4) OLE+LPS (O-LPS), and 5) a single-dose OLE + LPS (SO-LPS group). OLE 200 mg/kg or saline as a vehicle was administered via gavage for 7 d. On the seventh day, 2.5 mg/kg LPS was intraperitoneally administered. The rats were decapitated after 24 h of LPS treatment, and serum collection and tissue dissection were performed. The study assessed astrocyte and microglial activation using glial fibrillary acidic protein (GFAP) and CD11b immunohistochemistry, nod-like receptor protein-3, interleukin (IL)-1β, IL-17A, and IL-4 concentrations in prefrontal and hippocampal tissues via enzyme-linked immunosorbent assay, and total antioxidant/oxidant status (TAS/TOS) in serum and tissues via spectrophotometry. ResultsIn both the O-LPS and SO-LPS groups, LPS-related activation of microglia and astrocytes was suppressed in the cortex and hippocampus (P < 0.001), excluding cortical astrocyte activation, which was suppressed only in the SO-LPS group (P < 0.001). Hippocampal GFAP immunoreactivity and IL-17A concentrations in the dentate gyrus were higher in the OLE group than those in the control group, but LPS-related increases in these concentrations were suppressed in the O-LPS group. The O-LPS group had higher cortical TAS and IL-4 concentrations. ConclusionsOLE suppressed LPS-related astrocyte and microglial activation in the hippocampus and cortex. The OLE-induced increase in cortical IL-4 concentrations indicates the induction of an anti-inflammatory phenotype of microglia. OLE may also modulate astrocyte and IL-17A functions, which could explain its opposing effects on hippocampal GFAP immunoreactivity and IL-17A concentrations when administered with or without LPS.

Entopallium Lost GFAP Immunoreactivity during Avian Evolution: Is GFAP a “Condition Sine Qua Non”?

Introduction: The present study demonstrates that in the same brain area the astroglia can express GFAP (the main cytoskeletal protein of astroglia) in some species but not in the others of the same vertebrate class. It contrasts the former opinions that the distribution of GFAP found in a species is characteristic of the entire class. The present study investigated birds in different phylogenetic positions: duck (Cairina moschata domestica), chicken (Gallus gallus domesticus), and quails (Coturnix japonica and Excalfactoria chinensis) of Galloanserae; pigeon (Columba livia domestica) of a group of Neoaves, in comparison with representatives of other Neoaves lineages, which emerged more recently in evolution: finches (Taeniopygia guttata and Erythrura gouldiae), magpie (Pica pica), and parrots (Melopsittacus undulatus and Nymphicus hollandicus). Methods: Following a perfusion with 4% buffered paraformaldehyde, immunoperoxidase reactions were performed with two types of anti-GFAP: monoclonal and polyclonal, on floating sections. Results: The entopallium (formerly “ectostriatum,” a telencephalic area in birds) was GFAP-immunopositive in pigeon and in the representatives of Galloanserae but not in songbirds and parrots, which emerged more recently in evolution. The lack of GFAP expression of a brain area, however, does not mean the lack of astroglia. Lesions induced GFAP expression in the territory of GFAP-immunonegative entopallia. It proved that the GFAP immunonegativity is not due to the lack of capability, but rather the suppression of GFAP production of the astrocytes in this territory. In the other areas investigated besides the entopallium (optic tectum and cerebellum), no considerable interspecific differences of GFAP immunopositivity were found. It proved that the immunonegativity of entopallium is due to neither the general lack of GFAP expression nor the incapability of our reagents to detect GFAP in these species. Conclusion: The data are congruent with our proposal that a lack of GFAP expression has evolved in different brain areas in vertebrate evolution, typically in lineages that emerged more recently. Comparative studies on GFAP-immunopositive and GFAP-immunonegative entopallia may promote understanding the role of GFAP in neural networks.

A study on placental expression of neuronal markers in intrauterine growth restriction

Introduction and Aim: The restriction of intrauterine growth (IUGR) has a 20% recurrence rate and is one of the leading causes of postnatal illness and death. The diagnosis of intrauterine retardation refers to the infant's increased risk of neurological issues over an extended period, neonatal morbidity, and mortality, and in utero death. Materials and Methods: One hundred placenta samples were collected and divided into cases and controls. Clearance from the ethics committee was taken from the institute prior to the commencement of this study. Exclusion criteria include the patients with multiple pregnancies, unknown gestational age, gestational diabetes, and HIV. The inclusion criteria are the singleton pregnancy, normal and cesarean section, maternal age between 18-35 years and GA between 34 – 41 weeks. Standard immunohistochemistry protocols were followed for the study and glial fibrillary acidic protein (GFAP), neuron specific enolase (NSE) markers were used as neuronal markers. Results: Strong immunoreactivity of glial fibrillary acidic protein and neuron specific enolase was observed in fetal growth restriction placenta indicating perinatal brain damage of neonate. Conclusion: In our study we observed strong positive immunoreactivity of GFAP and NSE in IUGR only. This study suggests that these markers are used to predict brain damage in IUGR neonates.

Regulation of microglial responses after pediatric traumatic brain injury: exploring the role of SHIP-1.

Severe traumatic brain injury (TBI) is the world's leading cause of permanent neurological disability in children. TBI-induced neurological deficits may be driven by neuroinflammation post-injury. Abnormal activity of SH2 domain-containing inositol 5' phosphatase-1 (SHIP-1) has been associated with dysregulated immunological responses, but the role of SHIP-1 in the brain remains unclear. The current study investigated the immunoregulatory role of SHIP-1 in a mouse model of moderate-severe pediatric TBI. SHIP-1+/- and SHIP-1-/- mice underwent experimental TBI or sham surgery at post-natal day 21. Brain gene expression was examined across a time course, and immunofluorescence staining was evaluated to determine cellular immune responses, alongside peripheral serum cytokine levels by immunoassays. Brain tissue volume loss was measured using volumetric analysis, and behavior changes both acutely and chronically post-injury. Acutely, inflammatory gene expression was elevated in the injured cortex alongside increased IBA-1 expression and altered microglial morphology; but to a similar extent in SHIP-1-/- mice and littermate SHIP-1+/- control mice. Similarly, the infiltration and activation of CD68-positive macrophages, and reactivity of GFAP-positive astrocytes, was increased after TBI but comparable between genotypes. TBI increased anxiety-like behavior acutely, whereas SHIP-1 deficiency alone reduced general locomotor activity. Chronically, at 12-weeks post-TBI, SHIP-1-/- mice exhibited reduced body weight and increased circulating cytokines. Pro-inflammatory gene expression in the injured hippocampus was also elevated in SHIP-1-/- mice; however, GFAP immunoreactivity at the injury site in TBI mice was lower. TBI induced a comparable loss of cortical and hippocampal tissue in both genotypes, while SHIP-1-/- mice showed reduced general activity and impaired working memory, independent of TBI. Together, evidence does not support SHIP-1 as an essential regulator of brain microglial morphology, brain immune responses, or the extent of tissue damage after moderate-severe pediatric TBI in mice. However, our data suggest that reduced SHIP-1 activity induces a greater inflammatory response in the hippocampus chronically post-TBI, warranting further investigation.

THU124 High Fat Diet Disrupts Blood-Brain Barrier Integrity In Adult Males And Females

Abstract Disclosure: J.A. Sheng: None. R.J. Handa: None. S.A. Tobet: None. Background: The blood-brain barrier (BBB) protects the brain from the influx of harmful compounds in the blood. It is comprised of multiple cells, including endothelial cells sharing tight junctions, pericytes, and astrocyte endfeet. The paraventricular nucleus of the hypothalamus (PVN) is 3-5 times more vascularized than surrounding regions in the brain. The current study focuses on the BBB in the PVN as a function of stress. Recent data further suggests a high fat diet (HFD) disrupts the integrity of the BBB and leads to impairment of brain function (Li et al, 2021), suggesting a potential mechanism that may influence stress-related diseases. Methods: Adult male and female mice were placed on standard mouse chow (2918; Tekland) or a HFD (TD.06414, Tekland) for 6 weeks and further divided into control and stressed groups. Control mice were euthanized directly out of their home cage and stressed mice were euthanized 60-min after a 20-min acute restraint stress. Mice were perfused with fluorescein isothiocyanate in phosphate buffered saline followed by fixation with 4% paraformaldehyde to visualize blood vessel integrity (FITC leakage; Frahm &amp; Tobet, 2015). Immunolabeled Glial Fibrillary Acidic Protein (GFAP; astrocytic end feet) and IBA-1 (microglia) were used to further assess BBB integrity and neuroinflammation. Results: Results showed ∼40% more FITC leakage in the PVN vasculature in adult HFD females (p&amp;lt;0.0001) but not males. Similar leakage was not noted in the region lateral to the PVN. IBA-1 immunoreactivity (microglia) showed a HFD-related 50% increase in raw cell counts (p&amp;lt;0.001) and a HFD x restraint decrease in fluorescence intensity (p&amp;lt;0.02) in both sexes. GFAP immunoreactivity (astrocyte end feet) additionally showed 2-fold increase in cell counts in both sexes by HFD (p&amp;lt;0.0001) and a female-specific HFD-induced increase in area (∼40%; p&amp;lt;0.001). Conclusions: Data suggest a chronic high fat diet impairs BBB integrity in PVN vasculature that can be exacerbated further by a brief exposure to stress in adult mice. This effect is being further studied at the cellular level in astrocytes and microglia. Such changes in these BBB components could indicate critical roles for the uniquely dense PVN vasculature, increasing risk of damage to neural functions that could include obesity, cardiovascular autonomic regulation, or depression-like behaviors. Supported by ORWH-NIMH U54 MH118919 SCORE.

Adult human neural cells in culture following traumatic brain injury

Objective: The present study aims to evaluate the viability of adult human neural cells in culture obtained from traumatized brain tissues collected in emergency surgery procedures. Methods: Exploratory, descriptive, quantitative and cross-sectional study evaluating samples obtained from patients who underwent traumatic brain injury with extrusion of brain tissue submitted to cell culture in a standardized medium, being preserved during 168h. After observation under phase contrast microscopy and immunohistochemical processing for neuronal (MAP-2) and glial (GFAP) markers, morphometric parameters of neural cells (cell body area, dendritic field length and fractal dimension) were evaluated using ImageJ software, with data obtained after 24, 72 and 168h being compared using non-parametric Kruskal Wallis test, followed by Dunn’s post hoc test. Results: The explant of the nervous tissue revealed a consolidated pattern of cell migration into the culture medium. Cell proliferation, upon reaching confluence, presented an aspect of cellular distribution juxtaposed along the culture medium at all time points analyzed. Both neurons and glial cells remained viable after 168h in culture, with their morphologies not varying significantly throughout the time points evaluated. Immunohistochemistry for MAP-2 showed a relatively well-preserved cytoskeletal organization. GFAP immunoreactivity revealed activated astrocytes especially at the later time point. Conclusions: Our results point out the viability of cell culture from traumatized human nervous tissue, opening up perspectives for the use of substances of natural origin that may contribute neuroprotectively to neuronal maintenance in culture, allowing future translational approach.

A hypothermia mimetic molecule (zr17-2) reduces ganglion cell death, gliosis, and electroretinogram distortion in male rats subjected to perinatal asphyxia.

Introduction: Perinatal asphyxia (PA) represents a major problem in perinatology and may cause visual losses, including blindness. We, and others, have shown that hypothermia prevents retinal symptoms associated to PA. In the present work, we evaluate whether a hypothermia mimetic small molecule, zr17-2, has similar effects in the context of PA. Methods: Four experimental groups were studied in male rats: Naturally born rats as controls (CTL), naturally born rats injected s.c. with 50µL of 330nmols/L zr17-2 (ZR), animals that were exposed to PA for 20min at 37°C (PA), and rats that were exposed to PA and injected with zr17-2 (PA-ZR). Forty-five days after treatment, animals were subjected to electroretinography. In addition, morphological techniques (TUNEL, H&E, multiple immunofluorescence) were applied to the retinas. Results: A reduction in the amplitude of the a- and b-wave and oscillatory potentials (OP) of the electroretinogram (ERG) was detected in PA animals. Treatment with zr17-2 resulted in a significant amelioration of these parameters (p < 0.01). In PA animals, a large number of apoptotic cells was found in the GCL. This number was significantly reduced by treatment with the small molecule (p < 0.0001). In a similar way, the thickness of the inner retina and the intensity of GFAP immunoreactivity (gliosis) increased in PA retinas (p < 0.0001). These parameters were corrected by the administration of zr17-2 (p < 0.0001). Furthermore, injection of the small molecule in the absence of PA did not modify the ERG nor the morphological parameters studied, suggesting a lack of toxicity. Discussion: In conclusion, our results indicate that a single s.c. injection of zr17-2 in asphyctic neonates may provide a novel and efficacious method to prevent the visual sequelae of PA.

NAG-1/GDF-15 Transgenic Female Mouse Shows Delayed Peak Period of the Second Phase Nociception in Formalin-induced Inflammatory Pain.

Non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1), also known as growth differentiation factor-15 (GDF-15), is associated with cancer, diabetes, and inflammation, while there is limited understanding of the role of NAG-1 in nociception. Here, we examined the nociceptive behaviors of NAG-1 transgenic (TG) mice and wild-type (WT) littermates. Mechanical sensitivity was evaluated by using the von Frey filament test, and thermal sensitivity was assessed by the hot-plate, Hargreaves, and acetone tests. c-Fos, glial fibrillary acidic protein (GFAP), and ionized calcium binding adaptor molecule-1 (Iba-1) immunoreactivity was examined in the spinal cord following observation of the formalin-induced nociceptive behaviors. There was no difference in mechanical or thermal sensitivity for NAG-1 TG and WT mice. Intraplantar formalin injection induced nociceptive behaviors in both male and female NAG-1 TG and WT mice. The peak period in the second phase was delayed in NAG-1 TG female mice compared with that of WT female mice, while there was no difference in the cumulative time of nociceptive behaviors between the two groups of mice. Formalin increased spinal c-Fos immunoreactivity in both TG and WT female mice. Neither GFAP nor Iba-1 immunoreactivity was increased in the spinal cord of TG and WT female mice. These findings indicate that NAG-1 TG mice have comparable baseline sensitivity to mechanical and thermal stimulation as WT mice and that NAG-1 in female mice may have an inhibitory effect on the second phase of inflammatory pain. Therefore, it could be a novel target to inhibit central nervous system response in pain.

Systemic Administration of Porphyromonas Gingivalis Lipopolysaccharide Induces Glial Activation and Depressive-Like Behavior in Rats.

Periodontitis is one of the most common chronic inflammatory disorders in adults. Although clinical studies have suggested a causal relationship between periodontitis and major depression (MD), the biological mechanisms by which periodontitis instigates MD are unknown. We investigated whether a systemic administration of lipopolysaccharide (LPS) from Porphyromonas gingivalis (Pg), a major Gram-negative pathogen of periodontitis, causes depressive-like behavior and glial activation in the hippocampus and the prefrontal cortex (PFC), which are MD-related brain regions. Eight-week-old male Sprague Dawley rats were randomly divided into a behavioral test group and an immunohistochemistry group. The rats in each group were further assigned to the sham injection (saline) and Porphyromonas gingivalis-lipopolysaccharide (Pg-LPS) injection protocols. The rats received an intraperitoneal injection of saline or Pg-LPS with gradually increasing doses (day 1: 0.5, day 2: 0.5, day 3: 0.75, day 4: 0.75, day 5: 1.0, day 6: 1.0, and day 7: 1.0 mg/kg of body weight) for seven consecutive days. After the systemic administration, the behavior test group underwent the forced swimming test (FST) and Y-maze test. For the immunohistochemistry group, we quantified the immunoreactivity for microglial Iba-1 (ionized calcium-binding adapter molecule 1) and astrocytic glial fibrillary acidic protein (GFAP) in the hippocampus (dentate gyrus [DG], cornu ammonis [CA1 and CA3]) and PFC (prelimbic [PrL] and the infralimbic [IL]) areas. The FST immobility time in the Pg-LPS group was significantly longer than that in the sham group. In the Y-maze test, a significant decline in spontaneous alternation behavior was observed in the Pg-LPS group compared to the sham group. The peripheral administration of Pg-LPS significantly increased the immunoreactivity for Iba-1 in the CA3 and PrL. Pg-LPS injection significantly increased the immunoreactivity for GFAP in the DG, CA1, and CA3. The major result of this study is that a repeated systemic administration of Pg-LPS caused depressive-like behavior and both microglial and astrocytic activation in rats. This finding may comprise biological evidence of a causal relationship between periodontitis and MD.

HLA-II-Dependent Neuroimmune Changes in Group A Streptococcal Necrotizing Fasciitis.

Streptococcus pyogenes (Group A Streptococcus, GAS) bacteria cause a spectrum of human diseases ranging from self-limiting pharyngitis and mild, uncomplicated skin infections (impetigo, erysipelas, and cellulitis) to highly morbid and rapidly invasive, life-threatening infections such as streptococcal toxic shock syndrome and necrotizing fasciitis (NF). HLA class II allelic polymorphisms are linked with differential outcomes and severity of GAS infections. The dysregulated immune response and peripheral cytokine storm elicited due to invasive GAS infections increase the risk for toxic shock and multiple organ failure in genetically susceptible individuals. We hypothesized that, while the host immune mediators regulate the immune responses against peripheral GAS infections, these interactions may simultaneously trigger neuropathology and, in some cases, induce persistent alterations in the glial phenotypes. Here, we studied the consequences of peripheral GAS skin infection on the brain in an HLA-II transgenic mouse model of GAS NF with and without treatment with an antibiotic, clindamycin (CLN). Mice expressing the human HLA-II DR3 (DR3) or the HLA-II DR4 (DR4) allele were divided into three groups: (i) uninfected controls, (ii) subcutaneously infected with a clinical GAS strain isolated from a patient with GAS NF, and (iii) GAS-infected with CLN treatment (10 mg/kg/5 days, intraperitoneal). The groups were monitored for 15 days post-infection. Skin GAS burden and lesion area, splenic and hippocampal mRNA levels of inflammatory markers, and immunohistochemical changes in hippocampal GFAP and Iba-1 immunoreactivity were assessed. Skin GAS burden and hippocampal mRNA levels of the inflammatory markers S100A8/A9, IL-1β, IL-33, inflammasome-related caspase-1 (Casp1), and NLRP6 were elevated in infected DR3 but not DR4 mice. The levels of these markers were significantly reduced following CLN treatment in DR3 mice. Although GAS was not detectable in the brain, astrocyte (GFAP) and microglia (Iba-1) activation were evident from increased GFAP and Iba-1 mRNA levels in DR3 and DR4 mice. However, CLN treatment significantly reduced GFAP mRNA levels in DR3 mice, not DR4 mice. Our data suggest a skin-brain axis during GAS NF, demonstrating that peripherally induced pathological conditions regulate neuroimmune changes and gliotic events in the brain.

Therapeutic effect of thymoquinone on brain damage caused by nonylphenol exposure in rats.

Nonylphenol (NP), causes various harmful effects such as cognitive impairment and neurotoxicity. Thymoquinone (TQ), has antioxidant, anti-inflammatory, and neuroprotective properties. In this study, our aim is to investigate the effects of TQ on the brain damage caused by NP. Corn oil was applied to the control group. NP (100 mg/kg/day) was administered to the NP and NP + TQ groups for 21 days. TQ (5 mg/kg/day) was administered to the NP + TQ and TQ groups for 7 after 21 days. At the end of the experiment, the new object recognition test was applied to the rats and the rats were killed and their brain tissues were removed. Sections taken from brain tissues were stained with hematoxylin-eosin for histopathological evaluation. In addition, neuronal nuclei (NeuN), glial fibrillary acidic protein(GFAP), Cas-3, and nervegrowth factor (NGF) immunoreactivities were evaluated in brain tissue sections. In addition,malondialdehyde (MDA), superoxide dismutase(SOD), and catalase(CAT) activities were determined. Comet assay was applied to determine DNA damage in cells. The results of our study showed that NP, caused behavioral disorders and damage to the cerebral cortex in rats. This damage in the form of neuron degeneration seen in the cortex was associated with apoptosis involving Cas-3 activation, increased DNA damage, and free oxygen radicals. NP, SOD, and CAT caused a decrease in enzyme activities. In addition, the cellular protein NeuN was decreased, astrocytosis-associated GFAP was increased, and growth factor NGF was decreased. When all our evaluations are taken together, treatment with TQ showed an ameliorative effect on the behavioral impairment and brain damage caused by NP exposure.

Unravelling the Role of Habenula Subnuclei on Avoidance Response: Focus on Activation and Neuroinflammation.

Understanding the mechanisms responsible for anxiety disorders is a major challenge. Avoidance behavior is an essential feature of anxiety disorders. The two-way avoidance test is a preclinical model with two distinct subpopulations-the good and poor performers-based on the number of avoidance responses presented during testing. It is believed that the habenula subnuclei could be important for the elaboration of avoidance response with a distinct pattern of activation and neuroinflammation. The present study aimed to shed light on the habenula subnuclei signature in avoidance behavior, evaluating the pattern of neuronal activation using FOS expression and astrocyte density using GFAP immunoreactivity, and comparing control, good and poor performers. Our results showed that good performers had a decrease in FOS immunoreactivity (IR) in the superior part of the medial division of habenula (MHbS) and an increase in the marginal part of the lateral subdivision of lateral habenula (LHbLMg). Poor performers showed an increase in FOS in the basal part of the lateral subdivision of lateral habenula (LHbLB). Considering the astroglial immunoreactivity, the poor performers showed an increase in GFAP-IR in the inferior portion of the medial complex (MHbl), while the good performers showed a decrease in the oval part of the lateral part of the lateral complex (LHbLO) in comparison with the other groups. Taken together, our data suggest that specific subdivisions of the MHb and LHb have different activation patterns and astroglial immunoreactivity in good and poor performers. This study could contribute to understanding the neurobiological mechanisms responsible for anxiety disorders.

Astrocyte expression in the extended amygdala of C57BL/6J mice is sex-dependently affected by chronic intermittent and binge-like ethanol exposure.

Excessive ethanol drinking is a major problem within the United States, causing alterations in brain plasticity and neurocognitive function. Astrocytes are glial cells that regulate neurosynaptic plasticity, modulate neurochemicals, and monitor other homeostatic roles. Astrocytes have been found to play a part in modulating excessive ethanol consumption. The basolateral amygdala (BLA), central amygdala (CeA), and bed nucleus of the stria terminalis (BNST) are brain regions that process stress, anxiety, and reward; they are also implicated in modulating ethanol intake. Little is understood, however, about how astrocyte expression in each region is modulated by chronic and binge-like ethanol drinking patterns. In the present report, we utilized two separate animal models of excessive drinking: chronic intermittent ethanol (CIE) and "Drinking-in-the-dark" (DID). Following these paradigms, animal brains were processed through immunohistochemistry (IHC) and stained for glial fibrillary acidic protein (GFAP). Collected data illustrated a sex-dependent relationship between ethanol intake and GFAP immunoreactivity (IR) in the BLA and BNST, but not in the CeA. Specifically, CIE and DID ethanol drinking resulted in blunted GFAP-IR (specifically via GFAP-positive cell count) in the BLA and BNST, particularly in males. These findings may implicate sex-dependent ethanol-induced changes in BLA and BNST astrocytes, providing a potential therapeutic target for anxiety and stress disorders.