Astrocyte-Derived Exosomes Contribute to Pathologies of Neuromyelitis Optica Spectrum Disorder in Rodent Model.

OBJECTIVE: Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelinating disease that leads to severe disability. A large proportion of NMOSD patients are seropositive for aquaporin-4 autoantibodies (AQP4-IgG, named as NMO-IgG) targeting AQP4, which is selectively expressed on astrocytes in the central nervous system. This study tests the hypothesis that in response to NMO-IgG, the pathogenic astrocyte-derived exosomes are released and injure the neighboring cells. METHODS: IgG purified from serum of either NMOSD patients or healthy controls was used to generate astrocyte-derived exosomes (AST-ExosNMO vs AST-ExosCON ) in cultured rat astrocytes. The exosomes were respectively delivered to cultured rat oligodendrocytes in vitro, tissue culture of rat optic nerve ex vivo, and rat optic nerve in vivo to evaluate the pathogenic roles of AST-ExosNMO . The microRNA (miRNA) sequencing of AST-Exos and verification were performed to identify the key pathogenic miRNA. The custom-designed adeno-associated virus (AAV) antagonizing the key miRNA was evaluated for its therapeutic effects in vivo. Moreover, the serum levels of the key exosomal miRNA were measured between NMOSD patients and healthy controls. RESULTS: AST-ExosNMO led to notable demyelination in both cultured oligodendrocytes and optic nerve tissue. Exosomal miR-129-2-3p was identified as the key miRNA mediating the demyelinating pathogenesis via downstream target gene SMAD3. AAV antagonizing miR-129-2-3p protected against demyelination in an NMOSD rodent model. The serum exosomal miR-129-2-3p level was significantly elevated in NMOSD patients and correlated with disease severity. INTERPRETATION: Astrocytes targeted by NMO-IgG release pathogenic exosomes that could potentially be used as therapeutic targets or disease monitoring biomarkers in NMOSD. ANN NEUROL 2023;94:163-181.

Regulatory T cells alleviate myelin loss and cognitive dysfunction by regulating neuroinflammation and microglial pyroptosis via TLR4/MyD88/NF-κB pathway in LPC-induced demyelination.

Demyelination occurs in multiple central nervous system (CNS) disorders and is tightly associated with neuroinflammation. Pyroptosis is a form of pro-inflammatory and lytic cell death which has been observed in CNS diseases recently. Regulatory T cells (Tregs) have exhibited immunoregulatory and protective effects in CNS diseases. However, the roles of Tregs in pyroptosis and their involvement in LPC-induced demyelination have not been explicated. In our study, Foxp3-diphtheria toxin receptor (DTR) mice treated with diphtheria toxin (DT) or PBS were subjected to two-site lysophosphatidylcholine (LPC) injection. Immunofluorescence, western blot, Luxol fast blue (LFB) staining, quantitative real-time PCR (qRT-PCR) and neurobehavior assessments were performed to evaluate the severity of demyelination, neuroinflammation and pyroptosis. Pyroptosis inhibitor was further used to investigate the role of pyroptosis in LPC-induced demyelination. RNA-sequencing was applied to explore the potential regulatory mechanism underlying the involvement of Tregs in LPC-induced demyelination and pyroptosis. Our results showed that depletion of Tregs aggravated microgliosis, inflammatory responses, immune cells infiltration and led to exacerbated myelin injury as well as cognitive defects in LPC-induced demyelination. Microglial pyroptosis was observed after LPC-induced demyelination, which was aggravated by Tregs depletion. Inhibition of pyroptosis by VX765 reversed myelin injury and cognitive function exacerbated by Tregs depletion. RNA-sequencing showed TLR4/myeloid differentiation marker 88 (MyD88) as the central molecules in Tregs-pyroptosis pathway, and refraining TLR4/MyD88/NF-κB pathway alleviated the aggravated pyroptosis induced by Tregs depletion. In conclusion, our findings for the first time indicate that Tregs alleviate myelin loss and improve cognitive function by inhibiting pyroptosis in microglia via TLR4/MyD88/NF-κB pathway in LPC-induced demyelination.

Involvement of purinergic P2Y1R in antidepressant-like effects of electroacupuncture treatment on social isolation stress mice.

Depression is a common neuropsychiatric disorder with high incidence and disability. Electroacupuncture (EA) is effective in the treatment of depression. However, the underlying mechanisms are not fully understood. Social isolation stress during post-weaning period can impair purinergic signaling in the brain of rodents and has emerged as a major risk factor for depression. The purpose of this study was to investigate the involvement of P2Y1 receptor (P2Y1R) in the antidepressant-like effects of EA. In this study, C57BL/6 mice were randomly assigned to group-housed (GH) or social isolated (SI) groups at post-natal day 21. After 6 weeks of social isolation, EA was performed on acupoints "Bai-hui" (GV20) and "Yin-tang" (GV29), or non-acupoints for 4 weeks. The SI mice received either intracerebroventricular injection of a selective P2Y1R agonist, MRS2365 (1 nmol); or a selective P2Y1R antagonist, MRS2179 (2 µmol), before and after EA. We found that SI mice exhibited depression-like behaviors accompanied with anxiety-like behaviors. The expressions of P2Y1R were well co-localized with GFAP-positive astrocytes and increased in the prefrontal cortex and hippocampus of SI mice. After treated with MRS2179, the depression-like behaviors of SI mice were attenuated, but not with MRS2365. Meanwhile, we found that EA could attenuate social isolation caused depression- and anxiety-like behaviors, and inhibited the up-regulation of P2Y1R in the prefrontal cortex and hippocampus of SI mice. Notably, the positive effects of EA on depression-like behaviors of SI mice could be reversed by MRS2365, while MRS2365 had no effect on the anxiolytic-like effects of EA. Therefore, we provide new evidence that EA could ameliorate depression- and anxiety-like behaviors in social isolation stress mice, and P2Y1R was involved in the antidepressant-like effects of EA.

MiR196a-5p in extracellular vesicles released from human nasopharyngeal carcinoma enhance the phagocytosis and secretion of microglia by targeting ROCK1.

The microenvironment of the brain has become increasingly recognized as an essential regulator in metastatic and primary brain tumors. Recent studies demonstrate that circulating tumor-derived exosomes are critical for the brain tumor microenvironment. Nasopharyngeal carcinoma (NPC), a malignant tumor of the head and neck, often invades the skull base but infrequently extends to brain parenchyma. Neurobiological communication between microglia and tumor-derived extracellular vesicles (EVs) has been extensively studied, but how NPC cells regulate the immune microenvironment in the brain remains unknown. Here, we report that NPC derived EVs lead to increased microglial phagocytosis and proliferation, and heightened levels of IL-6, IL-8, CXCL1 and TGF-ß1. Analysis of microRNAs in EVs reveal that miR196a-5p is the major effector microRNA. Moreover, we demonstrate an enrichment of miR196a-5p in the plasmatic EVs of NPC patients. Further investigation demonstrated that miR196a-5p was transferred to microglia and regulated microglial structure and functions by downregulating the expression of ROCK1. Therefore, these data indicate that NPC-derived EVs are potent modulators of microglial functions in brain microenvironment. Regardless of brain colonization, EVs-mediated functional changes in microglia may be a universal phenomenon that results in the alteration of the tumor host's microenvironment in the brain.

Depletion of regulatory T cells exacerbates inflammatory responses after chronic cerebral hypoperfusion in mice.

Vascular cognitive impairment is the second most common cause of dementia which can be induced by chronic cerebral hypoperfusion. Regulatory T cells (Tregs) have been proven to provide beneficial effects in several central nervous system (CNS) diseases, but the roles of Tregs in chronic cerebral hypoperfusion-induced white matter damage have not been explored. In this study, Foxp3-diphtheria toxin receptor (DTR) mice treated with diphtheria toxin (DT) and wild type C57BL/6 mice treated with anti-CD25 antibody were subjected to bilateral carotid artery stenosis (BCAS). Flow cytometry analysis showed Tregs were widely distributed in spleen whereas barely distributed in brain under normal conditions. The distribution of lymphocytes and Tregs did not change significantly in spleen and brain after BCAS. Depletion of Tregs decreased the numbers of mature oligodendrocytes and anti-inflammatory microglia at 14 days and 28 days following BCAS. And pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and interferon-γ (IFN-γ) showed higher expression after Tregs depletion. In contrast, Tregs depletion did not change the overall severity of white matter injury as shown by the expression of myelin-associated glycoprotein (MAG), myelin basic protein (MBP), luxol fast blue (LFB) staining and electron microscopy assay. Moreover, Tregs depletion had marginal effect on cognition defects after BCAS revealed by Morris water maze and novel object recognition examination at 28 days after BCAS. In summary, our results suggest an anti-inflammatory role of Tregs with marginal effects on white matter damage in mice after BCAS-induced chronic cerebral hypoperfusion.

Exosomes derived from astrocytes after oxygen-glucose deprivation promote differentiation and migration of oligodendrocyte precursor cells in vitro.

BACKGROUND: Excessive release of glutamate, oxidative stress, inflammation after ischemic brain injury can lead to demyelination. Astrocytes participate in the maturation and differentiation of oligodendrocyte precursor cells (OPCs), and play multiple roles in the process of demyelination and remyelination. Here, we studied the role of Astrocyte-derived exosomes (AS-Exo) under ischemic conditions in proliferation, differentiation and migration of OPCs in vitro. METHODS AND RESULTS: Exosomes were collected from astrocytes supernatant by differential centrifugation from control astrocytes (CTexo), mild hypoxia astrocytes (O2R24exo) which were applied oxygen-glucose deprivation for 2 h and reperfusion for 24 h (OGD2hR24h) and severe hypoxia astrocytes (O4R24exo) which were applied oxygen-glucose deprivation for 4 h and reperfusion for 24 h (OGD4hR24h). Exosomes (20 µg/ml) were co-cultured with OPCs for 24 h and their proliferation, differentiation and migration were detected. The results showed that AS-Exo under severe hypoxia (O4R24exo) inhibit the proliferation of OPCs. Meanwhile, all exosomes from three groups can promote OPCs differentiation and migration. Compared to control, the expressions of MAG and MBP, markers of mature oligodendrocytes, were significantly increased in AS-Exo treatment groups. AS-Exo treatment significantly increased chemotaxis for OPCs. CONCLUSIONS: AS-Exo improve OPCs' differentiation and migration, whereas AS-Exo with severe hypoxic precondition suppress OPCs' proliferation. AS-Exo may be a potential therapeutic target for myelin regeneration and repair in white matter injury or other demyelination related diseases.

[The application of percutaneous transhepatic cholangioscopy in the treatment of benign bilioenteric anastomotic stricture:a report of 9 cases].

Objective: To investigate the feasibility and effectiveness of percutaneous transhepatic cholangioscopy(PTCS) in the treatment of bilioenteric anastomotic stricture after choledochojejunostomy. Methods: From April 2016 to April 2020, the clinical data of 9 patients (7 males and 2 females, aged 40-76 years) who underwent percutaneous transhepatic cholangioscopy(PTCS) for stricture expansion and lithotomy at Department of Hepatobiliary and Pancreatic Surgery, Second Affiliated Hospital of Zhejiang University School of Medicine were retrospectively analyzed. The operation was divided into two stages. In the first stage, ultrasound-guided percutaneous intrahepatic bile duct puncture was performed, and the sheath tube was inserted and fixed. In the second stage, percutaneous choledochoscopy was used for anastomotic stricture after sinus formation.The clinical outcome was evaluated by related biochemical indexes. Results: The operation time was (53.3±31.0)minutes(range:15-120 minutes).The postoperative hospital stay was (4.4±2.3)days(range:2-9 days).After systematic treatment, the preoperative symptoms, such as abdominal pain, jaundice, fever and shivering, disappeared in 8 patients. The range of alkaline phosphatase was 122-1 334 U/L before operation and 85-702 U/L after operation. The range of gamma glutamyl transpeptidase was 44-1 219 U/L before operation and 46-529 U/L after operation. Conclusion: PTCS is a safe and effective option for minimally invasive treatment of bilioenteric anastomotic stricture.

Microglial Hv1 proton channels promote white matter injuries after chronic hypoperfusion in mice.

Microglia are critical in damage/repair processes during ischemic white matter injury (WMI). Voltage-gated proton channel (Hv1) is expressed in microglia and contributes to nicotinamide adenine dinucleotide phosphate oxidase complex-dependent production of reactive oxygen species (ROS). Recent findings have shown that Hv1 is involved in regulating luminal pH of M1-polarized microglial phagosomes and inhibits endocytosis in microglia. We previously reported that Hv1 facilitated production of ROS and pro-inflammatory cytokines in microglia and enhanced damage to oligodendrocyte progenitor cells from oxygen and glucose deprivation. To investigate the role of Hv1 in hypoperfusion-induced WMI, we employed mice that were genetically devoid of Hv1 (Hv1-/- ), as well as a model of subcortical vascular dementia via bilateral common carotid artery stenosis. Integrity of myelin was assessed using immunofluorescent staining and transmission electron microscopy, while cognitive impairment was assessed using an eight-arm radial maze test. Hv1 deficiency was found to attenuate bilateral common carotid artery stenosis-induced disruption of white matter integrity and impairment of working memory. Immunofluorescent staining and western blotting were used to assay changes in oligodendrocytes, OPCs, and microglial polarization. Compared with that in wild-type (WT) mice, Hv1-/- mice exhibited reduced ROS generation, decreased pro-inflammatory cytokines production, and an M2-dominant rather than M1-dominant microglial polarization. Furthermore, Hv1-/- mice exhibited enhanced OPC proliferation and differentiation into oligodendrocytes. Results of mouse-derived microglia-OPC co-cultures suggested that PI3K/Akt signaling was involved in Hv1-deficiency-induced M2-type microglial polarization and concomitant OPC differentiation. These results suggest that microglial Hv1 is a promising therapeutic target for reducing ischemic WMI and cognitive impairment.

Microglial Hv1 exacerbates secondary damage after spinal cord injury in mice.

The pathological process of spinal cord injury (SCI) is complex, particularly during secondary damage that triggers a multiphasic glial reaction consisting of both detrimental and beneficial effects. Deletion of a novel voltage-gated proton channel (Hv1) functionally expressed in microglia has been shown to confer neuroprotection during ischemic stroke. Here, we hypothesized that microglial Hv1 may also participate in the process of SCI through modulating glial responses. To test this hypothesis, we employed an SCI model in Hv1-knockout (Hv1-/-) and wild type (WT) mice and assessed resulting microglial polarization, accumulation of pro-inflammatory cytokines, astrocytic activation, oligodendrocytic apoptosis, lesion sizes, and demyelinated areas. Compared with post-SCI results in WT mice, post-SCI Hv1-/- mice exhibited an M2-dominant microglial polarization, decreased accumulation of microglia, and reduced production of pro-inflammatory factors such as tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1ß). Additionally, Hv1-/- mice had significantly attenuated reactive astrogliosis and reduced expression of chondroitin sulphate proteoglycans (CSPGs) after SCI. Furthermore, Hv1 deficiency reduced SCI-induced oligodendrocytic apoptosis, demyelinated areas, and cavity formation. Collectively, our results provide the first evidence suggesting that microglial Hv1 may be a multi-mechanism therapeutic target for the treatment of SCI.

Deficiency of the microglial Hv1 proton channel attenuates neuronal pyroptosis and inhibits inflammatory reaction after spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) causes neurological dysfunction with devastating consequences. SCI pathogenesis is accompanied by inflammasome activation and neuronal damage. But the spatial pattern and the time course of neuronal pyroptosis and apoptosis after SCI should be further elucidated. The microglial voltage-gated proton channel (Hv1) is implicated in reactive oxygen species (ROS)-induced neuronal damage following ischemic stroke. However, there is a lack of quantification on the neuronal pyroptosis and apoptosis associated with microglial Hv1 after SCI. METHODS: We analyzed spatial and temporal characteristics of neuronal pyroptosis and apoptosis following SCI and investigated the effects of Hv1 deficiency on neuronal pyroptosis and the nod-like receptor 3 (NLRP3) inflammasome pathway by using a mouse model of SCI. We tested the effects of Hv1-deficient microglia on ROS production in vivo and examined the relationship between ROS and neuronal pyroptosis in vitro. RESULTS: We observed that apoptosis was detected closer to the injury core than pyroptosis. The incidence of neuronal apoptosis peaked on day 1 after SCI and occurred before pyroptosis. Hv1 deficiency reduced neuronal apoptosis and NLRP3-inflammasome-mediated pyroptosis, improved axonal regeneration, and reduced motor deficits. SCI led to elevated ROS levels, whereas Hv1 deficiency downregulated microglial ROS generation. In vitro, ROS upregulated neuronal pyroptosis and activated the NLRP3 inflammasome pathway, both of which were reversed by addition of a ROS scavenger. Our results suggested that microglial Hv1 regulated neuronal apoptosis and NLRP3-induced neuronal pyroptosis after SCI by mediating ROS production. CONCLUSION: Following SCI, neuronal pyroptosis lasted longer and occurred farther away from the injury core compared with that of neuronal apoptosis. Microglial Hv1 deficiency downregulated microglial ROS generation and reduced apoptosis and NLRP3-induced neuronal pyroptosis. Our findings may provide novel insights into Hv1-associated mechanisms underlying neuronal damage after SCI.

Deficiency of TREK-1 potassium channel exacerbates blood-brain barrier damage and neuroinflammation after intracerebral hemorrhage in mice.

BACKGROUND: Intracerebral hemorrhage (ICH) is a devastating medical emergency with high mortality and severe neurological deficit. ICH-related poor outcomes are due to a combination of pathological processes that could be complicated by secondary insults. TWIK-related K+ channel 1 (TREK-1) is a two-pore-domain potassium channel that is highly expressed in the mammalian nervous system. Previous studies have shown that TREK-1 channels play important roles in various central nervous system diseases. However, its role in the secondary injuries after intracerebral hemorrhage remains unknown. In this study, we explored the function of TREK-1 in secondary blood-brain barrier injuries and neuroinflammation after intracerebral hemorrhage in mice. METHODS: Adult male TREK-1-/- mice and WT mice were subjected to a collagenase-induced ICH model. Immunostaining, western blot, and enzyme-linked immunosorbent assay were used to assess inflammatory infiltration and neuronal death. Blood-brain barrier compromise was assessed using electron microscopy and Evans Blue dye injection on days 1 and 3 after intracerebral hemorrhage. Magnetic resonance imaging and behavioral assessments were conducted to evaluate the neurologic damage and recovery after intracerebral hemorrhage. RESULTS: Genetic deficiency of TREK-1 channel exacerbated blood-brain barrier impairment and promoted cerebral edema after intracerebral hemorrhage. Meanwhile, TREK-1 deficiency aggravated focal inflammatory featured by the increased recruitment of microglia and neutrophils, the enhanced secretion of proinflammatory factors interleukin-1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), and cell adhesion molecules (CAMs). Furthermore, TREK-1 deficiency promoted neuronal injury and neurological impairment. CONCLUSIONS: These results establish the first in vivo evidence for the protective role of TREK-1 in blood-brain barrier injury and neuroinflammation after intracerebral hemorrhage. TREK-1 may thereby be harnessed to a potential therapeutical target for the treatment of intracerebral hemorrhage.

Molecular and clinical relationship between live-attenuated Japanese encephalitis vaccination and childhood onset myasthenia gravis.

OBJECTIVE: The incidence of childhood onset myasthenia gravis (CMG) in China is higher than that in other countries; however, the reasons for this are unclear. METHODS: We investigated the clinical and immunological profiles of CMG, and assessed the potential precipitating factors. For the mouse studies, the possible implication of vaccination in the pathogenesis was explored. RESULTS: In our retrospective study, 51.22% of the 4,219 cases of myasthenia gravis (MG) were of the childhood onset type. The cohort study uncovered that the pathophysiology of CMG was mediated by immune deviation, rather than through gene mutations or virus infections. The administration of the live-attenuated Japanese encephalitis vaccine (LA-JEV), but not the inactivated vaccine or other vaccines, in mice induced serum acetylcholine receptor (AChR) antibody production, reduced the AChR density at the endplates, and decreased both muscle strength and response to repetitive nerve stimulation. We found a peptide (containing 7 amino acids) of LA-JEV similar to the AChR-α subunit, and immunization with a synthesized protein containing this peptide reproduced the MG-like phenotype in mice. INTERPRETATION: Our results describe the immunological profile of CMG. Immunization with LA-JEV induced an autoimmune reaction against the AChR through molecular mimicry. These findings might explain the higher occurrence rate of CMG in China, where children are routinely vaccinated with LA-JEV, compared with that in countries, where this vaccination is not as common. Efforts should be made to optimize immunization strategies and reduce the risk for developing autoimmune disorders among children. Ann Neurol 2018;84:386-400.

Hv1 proton channel facilitates production of ROS and pro-inflammatory cytokines in microglia and enhances oligodendrocyte progenitor cells damage from oxygen-glucose deprivation in vitro.

The contribution of microglial activation to oligodendrocyte precursor cell (OPC) damage in the brain is considered to be a principal pathophysiological feature of periventricular leukomalacia (PVL). Nicotinamide adenine dinucleotide phosphate oxidase (NOX)-dependent reactive oxygen species (ROS) produced in microglia has been shown to be significantly toxic to OPCs. The voltage-gated proton channel Hv1 is selectively expressed in microglia and is essential for NOX-dependent ROS production in the central nervous system. This study aimed to investigate the effects of microglial Hv1 deficiency on the protection of OPCs from oxygen-glucose deprivation (OGD)-induced injury in vitro. In the present study, the levels of OGD-induced ROS and pro-inflammatory cytokine production were dramatically lower in Hv1-deficient microglia (Hv1-/-) than in wild-type (WT) microglia. Following OGD, OPCs co-cultured with WT microglia had increased apoptosis and decreased proliferation and maturation, while those co-cultured with Hv1-/- microglia had attenuated apoptosis and greater proliferation and differentiation. Furthermore, the attenuated damage and enhanced regeneration of OPCs were associated with decreases in extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase phosphorylation. These results indicate that the protective effects of Hv1 deficiency on OPCs are due to the suppression of ROS and pro-inflammatory cytokine production in microglia. We thus suggest that the microglial proton channel Hv1 may be a potential therapeutic target in PVL.

Deficiency of TREK-1 potassium channel exacerbates secondary injury following spinal cord injury in mice.

Spinal cord injury (SCI) involves complex pathological process which can be complicated by secondary injury. TREK-1 is a member of the two-pore domain potassium (K2P) channel family, which can be modulated by a number of physiological and pathological stimuli. Recent studies suggest that TREK-1 plays an active role in depression, pain and neuroprotection. However, its role in the pathological process after SCI remains unclear. In this study, we tested the expression and function of TREK-1 in spinal cord of mice after traumatic SCI. TREK-1 was widely expressed in mice spinal cord, including astrocytes and neurons. Deficiency of TREK-1 significantly exacerbated focal inflammatory responses as indicated by the increased accumulation of microglia/macrophage as well as pro-inflammatory factor interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha expression. Meanwhile, TREK-1 knockout mice showed enhanced reactive astrogliosis, chondroitin sulphate proteoglycans (CSPGs) production and decreased glutamate transporter-1 expression compared to the wide-type mice after SCI. Furthermore, TREK-1 deficiency promoted neurons and oligodendrocytes apoptosis, aggravated demyelination, cavity formation and retarded motor recovery. In summary, our findings provide the first in vivo evidence suggesting that TREK-1 may thereby constitute a promising therapeutic target to treat acute SCI.

Blocking transient receptor potential vanilloid 2 channel in astrocytes enhances astrocyte-mediated neuroprotection after oxygen-glucose deprivation and reoxygenation.

Astrocytes play important roles in homeostatic regulation in the central nervous system and are reported to influence the outcome of ischemic injury. Regulating Ca2+ signaling of astrocytes is a promising strategy for stroke therapy. Herein, we report for the first time that transient receptor potential vanilloid 2 (TRPV2), a Ca2+ -permeable channel that is important in osmotic balance regulation, expresses in rat cortical astrocytes by immunofluorescence. Moreover, oxygen-glucose deprivation and reoxygenation (OGD/R) treatment enhanced the expression. The TRPV2 is functional because Ca2+ imaging showed that activating the TRPV2 channel in cultured astrocytes increased intracellular Ca2+ level and the increment of intracellular Ca2+ level expanded when astrocytes were treated with OGD/R. Staining with 5-ethynyl-2'-deoxyuridine (EdU) revealed that while blocking the TRPV2, it promoted the proliferation of astrocytes. Additionally, blocking the TRPV2 in astrocytes increased the synthesis of nerve growth factor (NGF) mRNA and the secretion of NGF by real-time PCR and enzyme-linked immunosorbent assay respectively. We further found that the increased secretion of NGF could be reversed by c-JunN-terminalkinase (JNK) inhibitor and blocking the TRPV2 caused the phosphorylation of JNK. These indicated that blocking the TRPV2 induced NGF secretion via the mitogen-activated protein kinase (MAPK)-JNK signaling pathway. As the promoted proliferation of astrocytes and secretion of NGF were reported to have neuroprotective effects in the early stage of stroke, we concluded that targeting the TRPV2 channel in astrocytes might be a potential new therapeutic strategy in ischemic stroke.

Elevated Homocysteine Level Related to Poor Outcome After Thrombolysis in Acute Ischemic Stroke.

BACKGROUND Hyperhomocysteinemia (HHcy) is a well-known risk factor for ischemic stroke. However, whether HHcy can influence the treatment outcome of acute ischemic stroke (AIS) patients has yet to be fully determined. In this study, we investigated the relationship between serum homocysteine (Hcy) level and prognosis in AIS patients who received tissue plasminogen activator (tPA) treatment. MATERIAL AND METHODS Patients were recruited according to the research criteria and grouped by their serum Hcy levels. Neurological outcome was evaluated by National Institute of Health Stroke Scale (NIHSS) score system before and 1 week after treatment, and functional outcome was evaluated by modified Rankin Scale (MRS) score system after 3 months. All patients took CT/MRI examination to detect cerebral hemorrhage in 24 hours after tPA treatment. Receiver operating characteristic curve (ROC) was employed to assess if serum homocysteine level can be used as an index to predict the outcome after tPA treatment. RESULTS The mean (±SD) serum Hcy level of 194 patients was 22.62±21.23 µmol/L. After 1-week tPA treatment, the NIHSS scores of high Hcy level group were significantly higher than those of low level group (p<0.05), meantime the high Hcy group showed obvious symptomatic intracerebral hemorrhage risk after 24 hours (p<0.05). Poor outcome was presented in mRS score results after 3 months in high Hcy level group, which compared with low Hcy level group (p<0.01). The ROC showed that Hcy level was a moderately sensitive and specific index to predict the prognosis with an optimal cut-off value at 19.95 µmol/L (sensitivity [58.2%], specificity [80.3%]). CONCLUSIONS High serum homocysteine level could potentially predict poor prognosis in acute ischemic stroke patients after tPA treatment.

Ephrin-A3 reverse signaling regulates hippocampal neuronal damage and astrocytic glutamate transport after transient global ischemia.

Increasing evidence indicates that the Eph receptors and their ephrin ligands are involved in the regulation of interactions between neurons and astrocytes. Moreover, astrocytic ephrin-A3 reverse signaling mediated by EphA4 receptors is necessary for controlling the abundance of glial glutamate transporters. However, the role of ephrin-A3 reverse signaling in astrocytic function and neuronal death under ischemic conditions remains unclear. In the present study, we found that the EphA4 receptor and its ephrin-A3 ligand, which were distributed in neurons and astrocytes, respectively, in the hippocampus showed a coincident up-regulation of protein expression in the early stage of ischemia. Application of clustered EphA4 decreased the expressions of astrocytic glutamate transporters together with astrocytic glutamate uptake capacity through activating ephrin-A3 reverse signaling. In consequence, neuronal loss was aggravated in the CA1 region of the hippocampus accompanied by impaired hippocampus-dependent spatial memory when clustered EphA4 treatment was administered prior to transient global ischemia. These findings indicate that EphA4-mediated ephrin-A3 reverse signaling is a crucial mechanism for astrocytes to control glial glutamate transporters and prevent glutamate excitotoxicity under pathological conditions. Astrocytic ephrin-A3 reverse signaling mediated by EphA4 receptor is necessary for controlling the abundance of glial glutamate transporters under physiological conditions. However, the role of ephrin-A3 reverse signaling in astrocytic function and neuronal death under ischemic conditions remains unclear. We found EphA4-mediated ephrin-A3 reverse signaling to be a crucial mechanism for astrocytes to control glial glutamate transporters and protect hippocampal neurons from glutamate excitotoxicity under ischemic conditions, this cascade representing a potential therapeutic target for stroke.

Linolenic acid provides multi-cellular protective effects after photothrombotic cerebral ischemia in rats.

Alpha-linolenic acid (LIN) has been shown to provide neuroprotective effects against cerebral ischemia. LIN is a potent activator of TREK-1 channel and LIN-induced neuroprotection disappears in Trek1-/- mice, suggesting that this channel is directly related to the LIN-induced resistance of brain against ischemia. However, the cellular mechanism underlying LIN induced neuroprotective effects after ischemia remains unclear. In this study, using a rat photochemical brain ischemia model, we investigated the effects of LIN on the protein abundance of astrocytic glutamate transporter and AQP4, microglia activation, cell apoptosis and behavioral recovery following ischemia. Administration of LIN rescued the protein abundance of astrocytic glutamate transporter GLT-1, decreased the protein abundance of AQP4 and brain edema, inhibited microglia activation, attenuated cell apoptosis and improved behavioral function recovery. Meanwhile, TREK-1 was widely distributed in the cortex and hippocampus, primarily localized in astrocytes and neurons. LIN could potentiate the TREK-1 mediated astrocytic passive conductance and hyperpolarize the membrane potential. Our results suggest that LIN provides multiple cellular neuroprotective effects in cerebral ischemia. TREK-1 may serve as a promising multi-mechanism therapeutic target for the treatment of stroke.

Different responses of cell cycle between rat vascular smooth muscle cells and vascular endothelial cells to paclitaxel.

Although previous reports showed drug-eluting stent (DES) could effectively inhibit neointima formation, in-stent restenosis (ISR) remains an important obstacle. The purpose of this study was to investigate different effects of paclitaxel on proliferation and cell cycle regulators between vascular smooth muscle cells (VSMCs) and vascular endothelial cells (VECs) of rats in vitro. The cultured VSMCs and VECs of rats from the same tissues were examined by using immunohistochemistry, flow cytometry and Western blotting in control and paclitaxel-treated groups. The results showed paclitaxel could effectively inhibit proliferation of VSMCs and VECs. However, as compared with VECs, proliferation of VSMCs in paclitaxel-treated group decreased less rapidly. The percentage of cells in G0-G1 and G2-M phases was reduced, and that in S phase increased after treatment for 72 h. The expression of cyclin D1 and B1, p27 and PCNA in VSMCs of paclitaxel-treated group was up-regulated, but that of p21 down-regulated as compared with VECs. It is concluded that there are significant differences in the expression of cell cycle regulators and proliferation rate between paclitaxel-treated VSMCs and paclitaxel-treated VECs, suggesting that the G1-S checkpoint regulated by paclitaxel may play a critical role in the development of complications of DES, which provides new strategies for treatments of ISR.