Increased Glial Fibrillary Acidic Protein Expression Research Articles

Ferroptosis contributes to diabetes-induced visual pathway neuronal damage via iron accumulation and GPX4 inactivation

The damage of the diabetic visual pathway is one of the main causes of blindness in diabetic patients. Visual pathways include anatomic parts from the retina to the occipital lobe. This study investigated the involvement of ferroptosis, a planned cell death brought on by the buildup of free iron in cells, in the impairment of visual pathways in diabetes mellitus. Streptozotocin (STZ) was used to construct a diabetic rat model. Pathological and ultrastructural changes of the occipital lobe, retina, and optic nerve were observed by Hematoxylin–Eosin (HE) staining and transmission electron microscopy (TEM). The expressions of Neuronal nuclei (NeuN), Glial fibrillary acidic protein (GFAP), and Glutathione Peroxidase 4 (GPX4) in the occipital lobe and retina were detected by immunofluorescence, and Western Blotting was used to identify the NeuN GFAP and GPX4 expressions in the occipital lobe. Iron content in the occipital lobe and retina was detected by Iron Assay Kit. The success rate of the diabetic rat model was 93.3%. In the diabetic group, the cells of the occipital lobe and retina were arranged disorderly, and the boundaries were unclear. The membrane of the occipital lobe, retina, and optic nerve was broken, some vacuoles were observed, mitochondrial morphology was changed, swelling was observed, and the mitochondrial ridge disappeared. There was a large increase in GFAP expression and iron concentration and a significant decrease in the expression of NeuN, and GPX4 in the retina and occipital lobe. Ferroptosis plays an important role in visual pathway damage in diabetes, and GPX4 regulates this process.

Fluorocitrate inhibition of astrocytes reduces nicotine self-administration and alters extracellular levels of glutamate and dopamine within the nucleus accumbens in male wistar rats

Emerging evidence suggests an important role of astrocytes in mediating behavioral and molecular effects of commonly misused drugs. Passive exposure to nicotine alters molecular, morphological, and functional properties of astrocytes. However, a potential involvement of astrocytes in nicotine reinforcement remains largely unexplored. The overall hypothesis tested in the current study is that astrocytes play a critical role in nicotine reinforcement. Protein levels of the astrocyte marker glial fibrillary acidic protein (GFAP) were examined in key mesocorticolimbic regions following chronic nicotine intravenous self-administration. Fluorocitrate, a metabolic inhibitor of astrocytes, was tested for its effects on behaviors related to nicotine reinforcement and relapse. Effects of fluorocitrate on extracellular neurotransmitter levels, including glutamate, GABA, and dopamine, were determined with microdialysis. Chronic nicotine intravenous self-administration increased GFAP expression in the nucleus accumbens core (NACcr), but not other key mesocorticolimbic regions, compared to saline intravenous self-administration. Both intra-ventricular and intra-NACcr microinjection of fluorocitrate decreased nicotine self-administration. Intra-NACcr fluorocitrate microinjection also inhibited cue-induced reinstatement of nicotine seeking. Local perfusion of fluorocitrate decreased extracellular glutamate levels, elevated extracellular dopamine levels, but did not alter extracellular GABA levels in the NACcr. Fluorocitrate did not alter basal locomotor activity. These results indicate that nicotine reinforcement upregulates the astrocyte marker GFAP expression in the NACcr, metabolic inhibition of astrocytes attenuates nicotine reinforcement and relapse, and metabolic inhibition of astrocytes disrupts extracellular dopamine and glutamate transmission. Overall, these findings suggest that astrocytes play an important role in nicotine reinforcement and relapse, potentially through regulation of extracellular glutamate and dopamine neurotransmission.

The Effect of Prenatal and Neonatal Fluoride Exposure to Morphine-Induced Neuroinflammation.

Physical dependence is associated with the formation of neuroadaptive changes in the central nervous system (CNS), both at the molecular and cellular levels. Various studies have demonstrated the immunomodulatory and proinflammatory properties of morphine. The resulting neuroinflammation in drug dependence exacerbates substance abuse-related behaviors and increases morphine tolerance. Studies prove that fluoride exposure may also contribute to the development of neuroinflammation and neurodegenerative changes. Morphine addiction is a major social problem. Neuroinflammation increases tolerance to morphine, and neurodegenerative effects caused by fluoride in structures related to the development of dependence may impair the functioning of neuronal pathways, change the concentration of neurotransmitters, and cause memory and learning disorders, which implies this element influences the development of dependence. Therefore, our study aimed to evaluate the inflammatory state of selected brain structures in morphine-dependent rats pre-exposed to fluoride, including changes in cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) expression as well as microglial and astroglial activity via the evaluation of Iba1 and GFAP expression. We provide evidence that both morphine administration and fluoride exposure have an impact on the inflammatory response by altering the expression of COX-1, COX-2, ionized calcium-binding adapter molecule (Iba1), and glial fibrillary acidic protein (GFAP) in brain structures involved in dependence development, such as the prefrontal cortex, striatum, hippocampus, and cerebellum. We observed that the expression of COX-1 and COX-2 in morphine-dependent rats is influenced by prior fluoride exposure, and these changes vary depending on the specific brain region. Additionally, we observed active astrogliosis, as indicated by increased GFAP expression, in all brain structures of morphine-dependent rats, regardless of fluoride exposure. Furthermore, the effect of morphine on Iba1 expression varied across different brain regions, and fluoride pre-exposure may influence microglial activation. However, it remains unclear whether these changes are a result of the direct or indirect actions of morphine and fluoride on the factors analyzed.

Protective effects of neuropeptide Y treatment on retina in a mouse model of experimental glaucoma

Purpose: Neuropeptide Y (NPY) is an endogenous 36‐amino acid linear peptide with tyrosine residues on both ends of the molecule. Cumulative evidence suggests that NPY and its receptors in the CNS may be a potential neuroprotective targets in various degenerative conditions. This study aimed to investigate the protective effects of NPY in the retina in an experimentally induced glaucoma animal model.Methods: Weekly intracameral microbead injections were performed to induce sustainably increased intraocular pressure (IOP) and NPY was administered intravitreally for 2 months. Wild‐type C57BL/6J mice (n = 40) were categorized into four major groups i) control, ii) control + NPY, iii) high IOP model, & iv) high IOP + NPY. Inner retinal function was evaluated by using positive scotopic threshold response (pSTR) amplitudes. Eye and optic nerve sections were then immunostained with pNFH (phosphorylated neurofilament heavy chain), GFAP (glial fibrillary acidic protein) and Iba‐1(Ionized calcium‐binding adaptor molecule 1) antibodies to understand the protective effects of NPY treatment.Results: IOP elevation was observed in the microbead model (control, 10.5 ± 0.75; microbead, 27.35 ± 1.92 mmHg). Functional data revealed a significant decline in the pSTR amplitudes in high IOP conditions (p < 0.0001, n = 10) compared to the control group, and a significant increase in pSTR was observed in the high IOP + NPY group (p < 0.001, n = 10). Optic nerves stained with pNFH revealed a significant protective effect of NPY treatment against high IOP‐induced axonal damage (p < 0.01, n = 4). Increased GFAP expression observed in high IOP condition was significantly decreased with NPY treatment both in the retina (p < 0.001, n = 4) and optic nerves (p < 0.01, n = 4). Similarly, microglial activation evaluated by Iba‐1 expression was also significantly decreased by NPY treatment both in the retina (p < 0.01, n = 4) and optic nerves (p < 0.01, n = 4).Conclusions: NPY treatment significantly reduced inner retinal functional loss and optic nerve damage against high IOP‐induced glaucomatous injury. NPY also reduced the microglial activation and reactive gliosis in experimental glaucoma. Our ongoing investigation will reveal specific NPY receptors activation and biochemical mechanism underlying these protective effects in the retina.

Characterisation of GFAP-Expressing Glial Cells in the Dorsal Root Ganglion after Spared Nerve Injury.

Satellite glial cells (SGCs), enveloping primary sensory neurons' somas in the dorsal root ganglion (DRG), contribute to neuropathic pain upon nerve injury. Glial fibrillary acidic protein (GFAP) serves as an SGC activation marker, though its DRG satellite cell specificity is debated. We employed the hGFAP-CFP transgenic mouse line, designed for astrocyte studies, to explore its expression within the peripheral nervous system (PNS) after spared nerve injury (SNI). We used diverse immunostaining techniques, Western blot analysis, and electrophysiology to evaluate GFAP+ cell changes. Post-SNI, GFAP+ cell numbers increased without proliferation, and were found near injured ATF3+ neurons. GFAP+ FABP7+ SGCs increased, yet 75.5% of DRG GFAP+ cells lacked FABP7 expression. This suggests a significant subset of GFAP+ cells are non-myelinating Schwann cells (nmSC), indicated by their presence in the dorsal root but not in the ventral root which lacks unmyelinated fibres. Additionally, patch clamp recordings from GFAP+ FABP7-cells lacked SGC-specific Kir4.1 currents, instead displaying outward Kv currents expressing Kv1.1 and Kv1.6 channels specific to nmSCs. In conclusion, this study demonstrates increased GFAP expression in two DRG glial cell subpopulations post-SNI: GFAP+ FABP7+ SGCs and GFAP+ FABP7- nmSCs, shedding light on GFAP's specificity as an SGC marker after SNI.

#3587 INTERMITENT RECURRENT HYPONATREMIA: A NEW EXPERIMENTAL MODEL – A STUDY ON ITS EFFECTS ON WATER BALANCE AND CENTRAL NERVOUS SYSTEM

Abstract Background and Aims Hyponatremia is the most common electrolyte disturbance in clinical care. Even mild presentations are associated with poor prognosis and increased mortality, in spite of which there is a trend to minimize the importance of small variations in natremia, that have historically been dismissed as not having negative consequences despite growing evidence against it. In this regard, it has not been studied to date whether an intermittent but recurrent hyponatremia is relevant. There are clinical scenarios where this condition could occur and be overlooked, such as cirrhosis or heart failure. Method Different rat models have been used to study the effects of different hypotonic situations on the electrolyte balance and central nervous system: intermittent recurrent hyponatremia (intraperitoneal (i.p.) daily dose of desmopressin acetate (ddAVP) and a water dose equivalent to 2.5% of the animal's body weight in hyposodic diet fed animals), acute on intermittent recurrent hyponatremia (i.p. water overload equivalent to 10% of the animal's body weight in animals subjected to intermittent recurrent hyponatremia) and acute hyponatremia (i.p. administration of a 10% of the animal's body weight water overload in chow fed animals). Apparent diffusion coefficient (ADC) obtained from diffusion weighted images (DWI) acquired through magnetic resonance (7T Bruker Biospec) was used to study content and distribution of brain water, and immunohistochemistry was used to examine glial fibrillary acidic-protein (GFAP), astrocyte marker, and myelin basic protein (MBP), oligodendrocyte and myelin marker. Results In the intermittent recurrent hyponatremia model, mild and transient hyponatremia was induced (baseline Nap 136.50±1.73mEq/L vs 4h post-medication 129.44±1.20mEq/L, p<0.001), which was recovered 24h after treatment (141.25±0.96mEq/L, NS compared to baseline). However, this situation was repeated over a 7 day period. This translated into a lower ADC value in the whole brain (WB) compared to chow fed animals (25.07±1.71 vs 26.71±2.40*10−3mm2/s, p = 0.05) after this 7 day period, suggesting an increase in total brain water in this situation. There was also an increase in GFAP expression in the gray matter (GM) compared to chow fed animals (33.22±5.25 vs 25.07±2.31au, p = 0.031), although no significant changes in MBP’s expression were seen. Acute on intermittent recurrent hyponatremia induced hypotonic hyponatremia (116.00±1.16mEq/L, p<0.001 compared to baseline). In this situation, a progressive decrease in ADC values in the WB was seen, and it was less pronounced compared to chow fed animals (slope -0.11±0.02 vs -0.26±0.006, p = 0.014). When GM and white matter (WM) were analysed separately, they both showed a progressive increase in ADC values, more evident in the WM (slope WM 0.19±0.04, p<0.05; slope GM 0.05±0.02, p = 0.002). The water overload increased GFAP and MBP’s expression in the WM (GFAP 23.17±6.37 vs 16.83±5.17au, p = 0.001; MBP 45.20±8.32 vs 28.76±7.03au, p<0.001), but no changes were seen in the GM, similarly to what had been observed in the acute hyponatremia model. Conclusion Intermittent recurrent hyponatremia is a novel animal model that suggests there can be significant water retention after only a few hours of hyponatremia a day, provided this situation is repeated over time. Such water retention translates into greater brain water accumulation and astroglial activation in the GM. These animals's response to an additional water overload does not show big variations compared to what is observed in acute hyponatremia. This study highlights the importance of slight fluctuations in natremia, which, if maintained over time, can translate underlying water retention with consequences at the central nervous system level.

Clinical utility of serum glial fibrillary acidic protein in glial neoplasm.

Glial fibrillary acidic protein (GFAP) is a member of the cytoskeletal protein family and is widely expressed in astroglial and neural stem cells, also in glial tumors such as astrocytoma and Glioblastoma (GBM). Increased GFAP expression and disruption of the blood-brain barrier are the characteristic features of GBM. Higher serum GFAP levels can help differentiate GBM from GBM mimics (such as primary central nervous system lymphoma, metastasis, or demyelinating lesions). This prospective study was carried out in a tertiary care center in the department of neurosurgery on newly diagnosed glioma patients who underwent surgery from January 2018 to July 2019, excluded patients with history of the previous surgery for glioma, traumatic brain injury, and ischemic or hemorrhagic stroke. The blood sample was obtained at admission before undergoing invasive procedure. Pathological examination of the tumor biopsy sample was carried out using classical hematoxylin-eosin and immunohistochemical staining. All statistical analyses were performed using SPSS version 24.0. The mean preoperative tumor volume was 40 cm3 (range 17.19-65.57 cm3; standard deviation [SD] = 9.99 cm3) which showed 98.25% mean reduction in volume postsurgery (mean tumor volume = 0.7 cm3; SD = 0.19 cm3). Preoperative serum GFAP measurements show higher levels (spearman's rho coefficient = 0.610 with P = 0.000) with increasing grade of tumor. GFAP levels also demonstrated higher value with increasing preoperative tumor volume. Increasing serum GFAP levels in the preoperative period correlate with higher tumor grade, especially grade III and grade IV tumors. The serum GFAP levels showed relation to tumor volume, both before and after surgery.

Engineering a 3D hydrogel system to study optic nerve head astrocyte morphology and behavior

In glaucoma, astrocytes within the optic nerve head (ONH) rearrange their actin cytoskeleton, while becoming reactive and upregulating intermediate filament glial fibrillary acidic protein (GFAP). Increased transforming growth factor beta 2 (TGF β2) levels have been implicated in glaucomatous ONH dysfunction. A key limitation of using conventional 2D culture to study ONH astrocyte behavior is the inability to faithfully replicate the in vivo ONH microenvironment. Here, we engineer a 3D ONH astrocyte hydrogel to better mimic in vivo mouse ONH astrocyte (MONHA) morphology, and test induction of MONHA reactivity using TGF β2. Primary MONHAs were isolated from C57BL/6J mice and cell purity confirmed. To engineer 3D cell-laden hydrogels, MONHAs were mixed with photoactive extracellular matrix components (collagen type I, hyaluronic acid) and crosslinked for 5 minutes using a photoinitiator (0.025% riboflavin) and UV light (405-500 nm, 10.3 mW/cm2). MONHA-encapsulated hydrogels were cultured for 3 weeks, and then treated with TGF β2 (2.5, 5.0 or 10 ng/ml) for 7 days to assess for reactivity. Following encapsulation, MONHAs retained high cell viability in hydrogels and continued to proliferate over 4 weeks as determined by live/dead staining and MTS assays. Sholl analysis demonstrated that MONHAs within hydrogels developed increasing process complexity with increasing process length over time. Cell processes connected with neighboring cells, coinciding with Connexin43 expression within astrocytic processes. Treatment with TGF β2 induced reactivity in MONHA-encapsulated hydrogels as determined by altered F-actin cytoskeletal morphology, increased GFAP expression, and elevated fibronectin and collagen IV deposition. Our data sets the stage for future use of this 3D biomimetic ONH astrocyte-encapsulated hydrogel to investigate astrocyte behavior in response to injury.

Reactive medullary astrocytes are associated with RVLM neuroinflammation in heart failure rats

Heart failure with preserved ejection fraction (HFpEF) is a complex clinical syndrome. Brain inflammation has been suggested to play a key role in HFpEF progression by modulating the activity of several brainstem circuits encompassing cardiovascular and respiratory regulation. Indeed, we recently reported RVLM‐C1 catecholaminergic neurons hyperactivity in rats with HFpEF. However, the precise mechanism underpinning changes in C1 activity in HFpEF remains to be determined. Interestingly, glial cell activation within the RVLM (particularly astrocytes) has been shown to modulate C1 neuron activity. Despite this evidence whether astrocytes may become activated in HFPEF has not been previously studied. Accordingly, we aimed to determine changes in astrocyte activity within the RVLM of HFpEF rats and its relation with RVLM neuroinflammatory status. Adult male Sprague‐Dawley rats (250 g) underwent volume overload to induce HFpEF. Eight weeks after surgical induction of HFpEF, rats underwent respiratory function recordings in freely moving conditions using whole‐body plethysmography. At the end of the physiological experiments, brains were harvested, fixed in 4% PFA and processed for immunohistochemical analysis. Immunofluorescence for tyrosine hydroxylase and glial fibrillary acidic protein (GFAP) were performed in 25 μm free‐floating brainstem sections containing the RVLM. Sholl analysis was performed to evaluate astrocytic morphological differences between HFpEF and Sham rats (total length of processes, # of processes, maximum intersection radius, # of intersections). RVLM neuroinflammatory status was measured in micropunches by immunoblot (GFAP) and qPCR (TNF‐α, IL‐1β). Compared to Sham rats, HFpEF animals showed clear signs of astrocyte reactivity evidenced by increases in the number of astrocyte processes (11.27 ± 1.096 vs. 18.64 ± 1.333; HFpEF vs. Sham, respectively), length of astrocyte processes (267.5 ± 17.92μm vs. 392.8 ± 22.53μm; HFpEF vs. Sham, respectively) and number of intersections (3.410 ± 0,25 vs. 4.538 ± 0.317; HFpEF vs. Sham, respectively). Consistently, we found an increased GFAP expression within the RVLM of HFpEF rats compared with Sham rats (99.9 ± 5.9 vs. 135.3 ± 15.8; HFpEF vs. Sham, respectively. In addition, we found that TNF‐α and IL‐1β were both augmented in HFpEF (~7 and ~3 fold, respectively) compared to Sham. Notably, respiratory dysfunction observed in HFpEF were significantly and positively correlated with the degree of activation of RVLM astrocytes. Indeed, respiratory irregularity score (R2 = 0.98; p<0.05), short‐term variability of the breath‐to‐breath interval (R2 = 0.98; p<0.05), and apneas/hypopneas index (R2 = 0.94; p<0.05)) were all related to GFAP expression in HFpEF rats. Our findings showed the presence of reactive astrocytes within the RVLM of HFpEF animals and that the degree of astrocyte activity was associated with RVLM neuroinflammation. Together our results suggest that increases in astrocyte reactivity in HFpEF may help to induce/maintain a pro‐iflammatory niche within the RVLM that ultimately lead to C1 neuron activity alterations.

Baicalin treats cerebral ischemia reperfusion-induced brain edema in rats by inhibiting TRPV4 and AQP4 of astrocytes

This study aims to explore the pharmacodynamic effect of baicalin on rat brain edema induced by cerebral ischemia reperfusion injury and discuss the mechanism from the perspective of inhibiting astrocyte swelling, which is expected to serve as a refe-rence for the treatment of cerebral ischemia with Chinese medicine. To be specific, middle cerebral artery occlusion(suture method) was used to induce cerebral ischemia in rats. Rats were randomized into normal group, model group, high-dose baicalin(20 mg·kg~(-1)) group, and low-dose baicalin(10 mg·kg~(-1)) group. The neurobehavior, brain index, brain water content, and cerebral infarction area of rats were measured 6 h and 24 h after cerebral ischemia. Brain slices were stained with hematoxylin and eosin(HE) for the observation of pathological morphology of cerebral cortex after baicalin treatment. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the content of total L-glutathione(GSH) and glutamic acid(Glu) in brain tissue, Western blot to measure the content of glial fibrillary acidic protein(GFAP), aquaporin-4(AQP4), and transient receptor potential vanilloid type 4(TRPV4), and immunohistochemical staining to observe the expression of GFAP. The low-dose baicalin was used for exploring the mechanism. The experimental results showed that the neurobehavioral scores(6 h and 24 h of cerebral ischemia), brain water content, and cerebral infarction area of the model group were increased, and both high-dose and low-dose baicalin can lower the above three indexes. The content of GSH dropped but the content of Glu raised in brain tissue of rats in the model group. Low-dose baicalin can elevate the content of GSH and lower the content of Glu. According to the immunohistochemical staining result, the model group demonstrated the increase in GFAP expression, and swelling and proliferation of astrocytes, and the low-dose baicalin can significantly improve this situation. The results of Western blot showed that the expression of GFAP, TRPV4, and AQP4 in the cerebral cortex of the model group increased, and the low-dose baicalin reduce their expression. The cerebral cortex of rats in the model group was severely damaged, and the low-dose baicalin can significantly alleviate the damage. The above results indicate that baicalin can effectively relieve the brain edema caused by cerebral ischemia reperfusion injury in rats, possibly by suppressing astrocyte swelling and TRPV4 and AQP4.

Oridonin Alleviates LPS-Induced Depression by Inhibiting NLRP3 Inflammasome via Activation of Autophagy.

Objective: Oridonin (Ori) is a diterpene compound that has multiple biological properties. Here, our study was conducted to observe the therapeutic effect of Ori on depression as well as to uncover the mechanism.Methods: Lipopolysaccharide (LPS)-induced depression models were established both in C57BL/6 mice and primary astrocytes, which were treated with Ori, autophagy agonist Rapamycin (Rap) and autophagy inhibitor 3-Methyladenine (3-MA). The depressive-like behaviors were assessed with behavioral tests. Autophagy was evaluated in the hippocampus and astrocytes by investigating autophagosomes under transmission electron microscope (TEM) and detecting LC3II/I, Beclin1 and P62 through western blotting. Astrocyte marker glial fibrillary acidic protein (GFAP) was investigated by immunofluorescence. NLRP3 inflammasome activation was evaluated by detecting IL-1β, NLRP3, ASC and Caspase-1 expression and reactive oxygen species (ROS) accumulation was quantified via DCFH-DA probe. Autolysosomes, autophagosomes and mitophagy were separately observed through mTag-Wasabi-LC3 plasmid, MitoTracker Deep Red staining, and TEM.Results: Our results showed that Ori administration alleviated LPS-induced depressive-like behaviors and increased GFAP expression in the hippocampus. Furthermore, Ori treatment promoted autophagy activation and cell viability as well as weakened NLRP3 inflammasome activation and ROS accumulation both in LPS-induced mice and astrocytes. Ori promoted the autophagic flux unblocked through enhancing fusion of autophagosomes with lysosomes as well as enhanced mitophagy in LPS-treated astrocytes. The therapeutic effect of Ori was enhanced by Rap and weakened by 3-MA.Conclusion: Collectively, our findings provided a promising antidepressant drug and uncovered that Ori alleviated LPS-induced depression by inhibiting NLRP3 inflammasome through activation of autophagy.

Extracellular cardiolipin regulates select immune functions of astrocytes in a toll-like receptor (TLR) 4-dependent manner.

Cardiolipin (CL) is a mitochondrial phospholipid that may be released extracellularly from damaged or dying cells where it can act as a signaling molecule by interacting with the surrounding cells. Previous research demonstrates that extracellular CL regulates select immune functions of microglia in a toll-like receptor (TLR) 4-dependent manner. However, the effects of CL on astrocyte functions are unknown. Astrocytes are essential participants in diverse physiological brain functions; however, they become overactivated in chronic neuroinflammatory states, including Alzheimer's disease, contributing to damage and death of surrounding tissues and cells. Since astrocytes express TLR 4, we hypothesized that, by interacting with this receptor, extracellular CL modulates the secretion of cytokines and cytotoxins by astrocytes, as well as their phagocytic activity. We studied the effects of CL added to the culture media of primary murine astrocytes and human U118 MG astrocytic cells, which were used as astrocyte models. Fluorescent latex microspheres and synaptosomes were used to investigate the phagocytic activity of cells. The secretion of inflammatory cytokines was measured by the enzyme-linked immunosorbent assay or immunoblotting. U118 MG astrocyte supernatants were transferred to human SH-SY5Y neuronal cells to assess the secretion of cytotoxins. The expression of TLR 4 and glial fibrillary acidic protein (GFAP) by U118 MG astrocytic cells was quantified by immunostaining and immunoblotting, respectively. Extracellular CL alone upregulated the phagocytic activity of human U118 MG astrocytic cells and their secretion of monocyte chemoattractant protein (MCP)-1 and interferon (IFN)-β. Extracellular CL upregulated the phagocytic activity of primary murine astrocytes. TLR 4-specific antagonist TAK-242 inhibited CL-induced secretion of MCP-1 by human U118 MG cells. CL alone upregulated the cell-surface expression of TLR 4 by the same cell type. Extracellular CL inhibited the lipopolysaccharide (LPS)-induced increase in GFAP expression as well as the secretion of cytotoxins by U118 MG astrocytic cells. Extracellular CL may be used to modulate select functions of astrocytes, which become dysregulated in chronic neuroinflammatory states. Therefore, CL, or liposomes containing this phospholipid, could be used to ameliorate the chronic neuroinflammation and adverse astrocyte activation associated with Alzheimer's disease.

Protective multi‑target effects of DL‑3‑n‑butylphthalide combined with 3‑methyl‑1‑phenyl‑2‑pyrazolin‑5‑one in mice with ischemic stroke.

DL-3-n-butylphthalide (NBP) and 3-methyl-1- phenyl-2-pyrazolin-5-one (edaravone) are acknowledged neuroprotective agents that protect against ischemic stroke. However, the underlying mechanisms of a combination therapy with NBP and edaravone have not yet been fully clarified. The aim of the present study was to explore whether the co-administration of NBP and edaravone had multi-target protective effects on the neurovascular unit (NVU) of mice affected by ischemic stroke. Male C57BL/6 mice were randomly divided into the following three groups: i) Sham operation control, ii) middle cerebral artery occlusion (MCAO) and reperfusion, iii) and MCAO/reperfusion with the co-administration of NBP (40 mg/kg) and edaravone (6 mg/kg) delivered via intraperitoneal injection at 0 and 4 h after reperfusion (NBP + edaravone). After ischemia and reperfusion, infarct volumes and neurological deficits were evaluated. The immunoreactivity of the NVU, comprising neurons, endothelial cells and astrocytes, was determined using immunofluorescence staining of neuronal nuclei (NeuN), platelet and endothelial cell adhesion molecule 1 (CD31) and glial fibrillary acidic protein (GFAP). Western blotting was used to detect the expression levels of apoptosis-related proteins. The infarct volume, neurological function scores and cell damage were increased in the MCAO group compared with the sham operation group. Furthermore, the MCAO mice had reduced NeuN and CD31 expression and increased GFAP expression compared with the sham group. By contrast, the NBP + edaravone group exhibited reduced cell damage and consequently lower infarct volume and neurological deficit scores compared with the MCAO group. The NBP + edaravone group exhibited increased NeuN and CD31 expression and decreased GFAP expression compared with the MCAO group. Furthermore, the expression levels of Bax and cleaved caspase-3 in the NBP + edaravone group were decreased significantly compared with the MCAO group, while the expression levels of Bcl-2 and mitochondrial cytochrome c were increased. In conclusion, the results of the present study demonstrated that NBP and edaravone effectively prevented ischemic stroke damage with multi-target protective effects. In addition, NBP + edaravone may be a promising combination therapy for ischemic stroke.

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.

Glial decline and loss of homeostatic support rather than inflammation defines cognitive aging.

Glial decline and loss of homeostatic support rather than inflammation defines cognitive aging.

Pexidartinib treatment in Alexander disease model mice reduces macrophage numbers and increases glial fibrillary acidic protein levels, yet has minimal impact on other disease phenotypes

BackgroundAlexander disease (AxD) is a rare neurodegenerative disorder that is caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), an intermediate filament that is primarily expressed by astrocytes. In AxD, mutant GFAP in combination with increased GFAP expression result in astrocyte dysfunction and the accumulation of Rosenthal fibers. A neuroinflammatory environment consisting primarily of macrophage lineage cells has been observed in AxD patients and mouse models.MethodsTo examine if macrophage lineage cells could serve as a therapeutic target in AxD, GFAP knock-in mutant AxD model mice were treated with a colony-stimulating factor 1 receptor (CSF1R) inhibitor, pexidartinib. The effects of pexidartinib treatment on disease phenotypes were assessed.ResultsIn AxD model mice, pexidartinib administration depleted macrophages in the CNS and caused elevation of GFAP transcript and protein levels with minimal impacts on other phenotypes including body weight, stress response activation, chemokine/cytokine expression, and T cell infiltration.ConclusionsTogether, these results highlight the complicated role that macrophages can play in neurological diseases and do not support the use of pexidartinib as a therapy for AxD.

Nitric Oxide/Cyclic GMP-Dependent Calcium Signalling Mediates IL-6- and TNF-\u03b1-Induced Expression of Glial Fibrillary Acid Protein

Astrocyte activation is characterized by hypertrophy with increased glial fibrillary acidic protein (GFAP), whose expression may involve pro-inflammatory cytokines. In this study, the effects of pro-inflammatory IL-6 and TNF-α and anti-inflammatory cytokines IL-4 and IL-10 on nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signalling, intracellular calcium concentration ([Ca2+]i) and GFAP expression were investigated. In human glioblastoma astrocytoma U-373 MG cells, IL-6 and TNF-α, but not IL-4 or IL-10, increased iNOS, cGMP, [Ca2+]i and GFAP expression. The inhibitors of iNOS (1400 W), soluble guanylyl cyclase (ODQ) and IP3 receptors (ryanodine and 2-APB) reversed the increase in cGMP or [Ca2+]i, respectively, and prevented GFAP expression. In rat striatal slices, IL-6 and TNF-α, at variance with IL-4 and IL-10, promoted a concentration-dependent increase in Ca2+ efflux, an effect prevented by 1400 W, ODQ and RY/2APB. These data were confirmed by in vivo studies, where IL-6, TNF-α or the NO donor DETA/NO injected in the striatum of anaesthetised rats increased cGMP levels and increased GFAP expression. The present findings point to NO/cGMP-dependent calcium signalling as part of the mechanism mediating IL-6- and TNF-α-induced GFAP expression. As this process plays a fundamental role in driving neurotoxicity, targeting NO/cGMP-dependent calcium signalling may constitute a new approach for therapeutic interventions in neurological disorders.

Sex differences in T cell immune responses, gut permeability and outcome after ischemic stroke in aged mice

IntroductionStroke is a disease that presents with well-known sex differences. While women account for more stroke deaths, recent data show that after adjusting for age and pre-stroke functional status, mortality is higher in men. Immune responses are key determinants of stroke outcome and may differ by sex. This study examined sex differences in central and peripheral T cell immune responses, systemic effects on gut permeability and microbiota diversity and behavioral outcomes after stroke in aged mice. We hypothesized that there are sex differences in the immune response to stroke in aged animals. MethodsC57BL/6CR mice (20–22 months) were subjected to 60 min middle cerebral artery occlusion, or sham surgery. T cells were quantified in brain and blood at 3, 7 and 15 days (d) post-stroke by flow cytometry. Peripheral effects on gut permeability and microbiota diversity, as well as neurological function were assessed up to 14 d, and at 21 d (cognitive function) post-stroke. Brain glial fibrillary acidic protein (GFAP) expression was evaluated at 42 d post-stroke. Results and discussionMortality (50% vs 14%, p < 0.05) and hemorrhagic transformation (44% vs 0%) were significantly higher in males than in females. No difference in infarct size at 3d were observed. Peripherally, stroke induced greater gut permeability of FITC-dextran in males at d3 (p < 0.05), and non-reversible alterations in microbiota diversity in males. Following the sub-acute phase, both sexes demonstrated a time-dependent increase of CD4+ and CD8+ T cells in the brain, with significantly higher levels of CD8+ T cells and Regulatory T cells in males at d15 (p < 0.01). Aged males demonstrated greater neurological deficits up to d5 and impaired sensorimotor function up to d15 when assessed by the corner asymmetry test (p < 0.001 and p < 0.01, respectively). A trend in greater cognitive decline was observed at d21 in males. Increased GFAP expression in the ischemic hemisphere, indicating astroglial activation and gliosis, was demonstrated in both males and females 42d post-stroke. Our findings indicate that despite a similar initial ischemic brain injury, aged male mice experience greater peripheral effects on the gut and ongoing central neuroinflammation past the sub-acute phase after stroke.

Zinc, but not paracetamol, prevents depressive-like behavior and sickness behavior, and inhibits interferon-gamma and astrogliosis in rats

Considering all mental and addictive disorders, depression is the most responsible for years of life lost due to premature mortality and disability. Antidepressant drugs have limited effectiveness. Depression can be triggered by immune/inflammatory factors. Zinc and paracetamol interfere with immune system and have demonstrated beneficial effects on depression treatment when administered concomitant with antidepressant drugs. The objective of this study was to test zinc and/or paracetamol as treatments of depressive-like behavior, sickness behavior, and anxiety in rats, as well as to understand the central and peripheral mechanisms involved. Sickness behavior and depressive-like behavior were induced in rats with repetitive lipopolysaccharide (LPS, 1 mg/kg for two consecutive days) administrations. Rats received zinc and/or paracetamol for three consecutive days. Sickness behavior (daily body weight and open field general activity); anxiety (light-dark test); depressive-like/antidepressant behavior (forced swim test); plasma corticosterone and interferon (IFN)-gamma levels; and glial fibrillary acidic protein (GFAP) and tyrosine hydroxylase (TH) brain expression were evaluated. LPS induced sickness behavior and depressive-like behavior, as well as elevated IFN-gamma levels and increased GFAP expression. Zinc prevented both behavioral and biochemical impairments. Paracetamol and zinc + paracetamol association induced only slight beneficial effects. Anxiety, corticosterone, and TH do not seem be related with depression and the other behavioral and neuroimmune changes. In conclusion, zinc treatment was beneficial for sickness behavior and depressive-like behavior without concomitant administration of antidepressants. IFN-gamma and GFAP were linked with the expression of sickness behavior and depressive-like behavior and were also involved with the antidepressant effects. Therefore, zinc, IFN-gamma, and GFAP pathways should be considered for depression treatment.

The effects of prenatal radiation of mobile phones on white matter in cerebellum of rat offspring

To evaluate the effects of prenatal radiation of 850～1 900 MHz mobile phone on white matter in cerebellum of adult rat offspring. Pregnant rats were randomly divided into short term maternal radiation group, long term maternal radiation group and control group. Rats in short term and long term maternal radiation group were exposed to 6 h/d and 24 h/d mobile phone radiation during 1-17 days of pregnancy, respectively. The cerebellums of offspring rats at the age of 3 month(n＝8)were taken. Cell morphology in cerebellum was studied by hematoxylin-eosin (HE) staining. The expressions of myelin basic protein (MBP), neurofilament-L (NF-L) and glial fibrillary acidic protein (GFAP) in cerebellum of rat offspring were detected by immunohistochemistry and Western blot. Compared to control group, the morphological changes of purkinje cells in cerebellum were obvious in rat offspring of short term and long term maternal radiation group. Compared to control group, decreased MBP and NF-L expressions and increased GFAP expression were observed in long term maternal radiation group(all P＜0.05). Compared to short term radiation group, the expressions of MBP and NF-L were down-regulated (all P＜0.05) and the expression of GFAP was up- regulated(P＜0.05) in long term radiation group. Prenatal mobile phone radiation might lead to the damage of myelin and axon with activity of astrocytes in cerebellum of male rat offspring, which is related to the extent of radiation.