[G protein-coupled estrogen receptor alleviates cerebral ischemia-reperfusion injury through inhibiting endoplasmic reticulum stress].

The aim of this study was to investigate whether G protein-coupled estrogen receptor (GPER) could alleviate hippocampal neuron injury under cerebral ischemia-reperfusion injury (CIRI) by acting on endoplasmic reticulum stress (ERS). The CIRI animal model was established by middle cerebral artery occlusion (MCAO). Female ovariectomized (OVX) Sprague-Dawley (SD) female rats were randomly divided into 4 groups: control, ischemia-reperfusion injury (MCAO), vehicle (MCAO+DMSO), and GPER-specific agonist G1 (MCAO+G1) groups. The neurobehavioral score was assessed by the Longa score method, the morphological changes of the neurons were observed by the Nissl staining, the cerebral infarction was detected by the TTC staining, and the neural apoptosis in the hippocampal CA1 region was detected by TUNEL staining. The distribution and expression of GRP78 (78 kDa glucose-regulated protein 78) in the hippocampal CA1 region were observed by immunofluorescent staining. The protein expression levels of GRP78, Caspase-12, CHOP and Caspase-3 were detected by Western blot, and the mRNA expression levels of GRP78, Caspase-12, and CHOP were detected by the real-time PCR. The results showed that the neurobehavioral score, cerebral infarct volume, cellular apoptosis index, as well as GRP78, Caspase-12 and CHOP protein and mRNA expression levels in the MCAO group were significantly higher than those of control group. And G1 reversed the above-mentioned changes in the MCAO+G1 group. These results suggest that the activation of GPER can decrease the apoptosis of hippocampal neurons and relieve CIRI, and its mechanism may involve the inhibition of ERS.

Neuritin attenuates early brain injury in rats after experimental subarachnoid hemorrhage.

OBJECTIVES: Early brain injury (EBI) is central to the pathological progress of subarachnoid hemorrhage (SAH). In this study, we determined if neuritin protects the brain against EBI in rats and discussed the role of apoptosis pathway mediated by endoplasmic reticulum stress in this neuroprotective route. METHODS: A total of 96 male Sprague Dawley rats were divided into control, sham, SAH and SAH + neuritin groups. The rat SAH model was induced by injection 0.3 mL of nonheparinized arterial blood into the prechiasmatic cistern. Mortality assay, neurological scores, brain water content measurement, Evans blue dye assay, TUNEL stain assay and Western blot analysis were performed. RESULTS: Neuritin significantly improved the neurological scores, brain water content, blood-brain barrier (BBB) and apoptosis compared with the control and sham groups within 24 h after SAH. TUNEL staining assay results demonstrated that apoptosis was ameliorated, MMP-9 expression was reduced, whereas GRP78, CHOP, caspase-12 and ASK1 levels were markedly preserved after neuritin application. CONCLUSIONS: Our study demonstrated that neuritin plays a neuroprotective role on EBI after SAH by attenuating BBB disruption, brain edema and apoptosis.

A Study on the Efficacy of Thrombectomy in Patients with Atherosclerotic and Cardioembolic Basilar Artery Occlusion.

Background Studies on basilar artery occlusion are relatively few compared with those of anterior circulation stroke. The aim of the present study was to compare the efficacy of endovascular therapy (EVT) in patients with basilar artery occlusion classified as large artery atherosclerosis (LAA) and cardioembolism (CE), and to analyze the independent risk factors affecting the prognosis of EVT. Methods A total of 123 people were assigned to the LAA and CE groups (97 to the LAA and 26 to the CE). The primary outcome was a modified Rankin Scale (mRS) score of 2 or lower at 90 days. The primary safety outcome was mortality at 90 days. Secondary safety endpoints included the rates of symptomatic intracranial hemorrhage and reinfarction. Multiple logistic regression was used to screen out independent risk factors for EVT prognosis of the LAA and CE groups. Results In the analysis, the patients with LAA stroke had better collateral circulation (American Society of Interventional and Therapeutic Neuroradiology/Society of Interventional Radiology [SIR] score of 2-4; 61.9 vs. 19.2%, p = 0.000), and higher angioplasty rate (32.0 vs. 3.8%, p = 0.002). The proportions of patients with a 90-day mRS score of 0 to 2 and 90-day mortality were not found to be statistically significant between the two groups. Multivariate logistic regression analysis indicated that age, SIR, white blood cell, blood glucose, and modified thrombolysis in cerebral infarction were independent risk factors for the poor prognosis of EVT in the LAA group. Conclusion Although there were differences in clinical characteristics and imaging features between LAA and CE, there was no evidence of a significant difference in prognosis after EVT. In addition, the National Institutes of Health Stroke Scale score was not among the independent risk factors affecting the prognosis of the LAA group.

Down-regulation of CX43 expression by miR-1 inhibits the proliferation and invasion of glioma cells.

Background: Connexin (CX) 43 makes glioblastoma resistant to temozolomide, the first-line chemotherapy drug. However, targeting CX43 is very difficult because the mechanisms underlying CX43-mediated resistance remain unclear. CX43 is highly expressed in glioblastoma, which is closely associated with poor prognosis and chemotherapy resistance. The present study was to analyze the mechanism of microRNA (miR)-1 in regulating the proliferation and invasion of glioma cells. Methods: The effects of knockdown of miR-1 on the growth of glioma cell lines were observed by establishing blank, miR-1 inhibitor, and miR-1 mimic groups. Cell proliferation was detected using a Cell Counting Kit-8 (CCK-8) assay, cell apoptosis was detected by flow cytometry, and protein expression was detected by western blot. We used the Student's t-test to assess continuous data between the two groups and the Kruskal-Wallis test was adopted for multiple group comparisons. Results: Compared with the mimics normal control (NC) group, the apoptosis rate of the miR-1-3p mimics group was decreased, while that of the miR-1-3p inhibitor group was increased compared to the inhibitor NC group. In addition, the miR-1-3p mimics model of U251 cells exerted an inhibitory effect on the invasion ability of cells, whereas the miR-1-3p inhibitor model of U251 cells showed an invasion-promoting effect. The dual-luciferase assay showed that miR-1-3p had a targeted relationship with the CX43 gene. Conclusions: Down-regulation of CX43 expression by miR-1 inhibited the infiltration and growth of glioma cells and further promoted the apoptosis of glioma cells by regulating CX43 expression.

Correlation between Arteriole Membrane Potential and Cerebral Vasospasm after Subarachnoid Hemorrhage in Rats.

OBJECTIVES: Microvessel constriction plays an important role in delayed cerebral ischemia after aneurismal subarachnoid hemorrhage (SAH). This constriction has been demonstrated in both animal model and clinical operation. The present study examined the time-related membrane potential (Em) alteration of arterioles isolated from SAH model rats and the correlation between the potential alteration of arterioles and the diameter of basilar artery. MATERIALS AND METHODS: Sprague-Dawley rats (n = 90), weighing 300 g to 350 g, were divided into t control, sham, and SAH groups. In the SAH group, blood was injected into the prechiasmatic cistern of the rats. The Em of arterioles and basilar artery diameter was measured using whole-cell clamp recordings and pressure myograph, respectively, 1, 3, 5, 7, and 14 days after SAH. The correlation was evaluated using Pearson correlation coefficients. RESULTS: The Em of arterioles in the SAH group depolarized on days 3, 5, and 7, and peaked on day 7. The diameters significantly decreased on days 1, 3, 5, 7, and 14, and the smallest diameter was observed on day 7. A significant correlation between potential alteration of arterioles and diameter of basilar artery was found. CONCLUSIONS: Similar to the artery, arteriole constriction is also involved in the pathophysiological events of delayed cerebral ischemia.

Expression and effect of sodium-potassium-chloride cotransporter on dorsal root ganglion neurons in a rat model of chronic constriction injury.

Sodium-potassium-chloride cotransporter 1 (NKCC1) and potassium-chloride cotransporter 2 (KCC2) are associated with the transmission of peripheral pain. We investigated whether the increase of NKCC1 and KCC2 is associated with peripheral pain transmission in dorsal root ganglion neurons. To this aim, rats with persistent hyperalgesia were randomly divided into four groups. Rats in the control group received no treatment, and the rat sciatic nerve was only exposed in the sham group. Rats in the chronic constriction injury group were established into chronic constriction injury models by ligating sciatic nerve and rats were given bumetanide, an inhibitor of NKCC1, based on chronic constriction injury modeling in the chronic constriction injury + bumetanide group. In the experiment measuring thermal withdrawal latency, bumetanide (15 mg/kg) was intravenously administered. In the patch clamp experiment, bumetanide (10 µg/µL) and acutely isolated dorsal root ganglion neurons (on day 14) were incubated for 1 hour, or bumetanide (5 µg/µL) was intrathecally injected. The Hargreaves test was conducted to detect changes in thermal hyperalgesia in rats. We found that the thermal withdrawal latency of rats was significantly decreased on days 7, 14, and 21 after model establishment. After intravenous injection of bumetanide, the reduction in thermal retraction latency caused by model establishment was significantly inhibited. Immunohistochemistry and western blot assay results revealed that the immune response and protein expression of NKCC1 in dorsal root ganglion neurons of the chronic constriction injury group increased significantly on days 7, 14, and 21 after model establishment. No immune response or protein expression of KCC2 was observed in dorsal root ganglion neurons before and after model establishment. The Cl- (chloride ion) fluorescent probe technique was used to evaluate the change of Cl- concentration in dorsal root ganglion neurons of chronic constriction injury model rats. We found that the relative optical density of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (a Cl- fluorescent probe whose fluorescence intensity decreases as Cl- concentration increases) in the dorsal root ganglion neurons of the chronic constriction injury group was significantly decreased on days 7 and 14 after model establishment. The whole-cell patch clamp technique revealed that the resting potential and action potential frequency of dorsal root ganglion neurons increased, and the threshold and rheobase of action potentials decreased in the chronic constriction injury group on day 14 after model establishment. After bumetanide administration, the above indicators were significantly suppressed. These results confirm that CCI can induce abnormal overexpression of NKCC1, thereby increasing the Cl- concentration in dorsal root ganglion neurons; this then enhances the excitability of dorsal root ganglion neurons and ultimately promotes hyperalgesia and allodynia. In addition, bumetanide can achieve analgesic effects. All experiments were approved by the Institutional Ethics Review Board at the First Affiliated Hospital, College of Medicine, Shihezi University, China on February 22, 2017 (approval No. A2017-169-01).

17ß-Estradiol Attenuates Neuropathic Pain Caused by Spared Nerve Injury by Upregulating CIC-3 in the Dorsal Root Ganglion of Ovariectomized Rats.

17ß-estradiol plays a role in pain sensitivity, analgesic drug efficacy, and neuropathic pain prevalence, but the underlying mechanisms remain unclear. Here, we investigated whether voltage-gated chloride channel-3 (ClC-3) impacts the effects of 17ß-estradiol (E2) on spared nerve injury (SNI)-induced neuropathic pain in ovariectomized (OVX) female Sprague Dawley rats that were divided into OVX, OVX + SNI, OVX + SNI + E2, OVX + SNI + E2 + DMSO (vehicle, dimethyl sulfoxide), or OVX + SNI + E2+Cltx (ClC-3-blocker chlorotoxin) groups. Changes in ClC-3 protein expression were monitored by western blot analysis. Behavioral testing used the paw withdrawal threshold to acetone irritation and paw withdrawal thermal latency (PWTL) to thermal stimulation. Immunofluorescence indicated the localization and protein expression levels of ClC-3. OVX + SNI + E2 rats were subcutaneously injected with 17ß-estradiol once daily for 7 days; a sheathed tube was implanted, and chlorotoxin was injected for 4 days. Intrathecal Cltx to OVX and OVX + SNI rats was administered for 4 consecutive days (days 7-10 after SNI) to further determine the contribution of ClC-3 to neuropathic pain. Patch clamp technology in current clamp mode was used to measure the current threshold (rheobase) dorsal root ganglion (DRG) neurons and the minimal current that evoked action potentials (APs) as excitability parameters. The mean number of APs at double-strength rheobase verified neuronal excitability. There was no difference in behaviors and ClC-3 expression after OVX. Compared with OVX + SNI rats, OVX + SNI + E2 rats showed a lower paw withdrawal threshold to the acetone stimulus, but the PWTL was not significantly different, indicating increased sensitivity to cold but not to thermal pain. Co-immunofluorescent data revealed that ClC-3 was mainly distributed in A- and C-type nociceptive neurons, especially in medium/small-sized neurons. 17ß-estradiol administration was associated with increased expression of ClC-3. 17ß-estradiol-induced increase in ClC-3 expression was blocked by co-administration of Cltx. Cltx causes hyperalgesia and decreased expression of ClC-3 in OVX rats. Patch clamp results suggested that 17ß-estradiol attenuated the excitability of neurons induced by SNI by up-regulating the expression of ClC-3 in the DRG of OVX rats. 17ß-estradiol administration significantly improved cold allodynia thresholds in OVX rats with SNI. The mechanism for this decreased sensitivity may be related to the upregulation of ClC-3 expression in the DRG.