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High mobility group box 1 (HMGB1) is a classic damage-associated molecular pattern that has an important role in the pathological inflammatory response. In vitro studies have demonstrated that the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway is involved in the regulation of HMGB1 expression, mediating the inflammatory response. Therefore, the purpose of the present study was to evaluate JAK2/STAT3 pathway involvement in the subarachnoid hemorrhage (SAH)-dependent regulation of HMGB1, using an in vivo rat model. A SAH model was established by endovascular perforation. Western blotting, immunohistochemistry and immunofluorescence were used to analyze HMGB1 expression after SAH. In addition, the effects of AG490 after SAH on JAK2/STAT3 phosphorylation, HMGB1 expression and brain damage were evaluated. The results of the present study demonstrated that JAK2/STAT3 was significantly phosphorylated (P<0.05) and the total HMGB1 protein level was significantly increased (P<0.05) after SAH. In addition, the cytosolic HMGB1 level after SAH demonstrated an initial increase followed by a decrease to the control level, while the nuclear HMGB1 level after SAH demonstrated the opposite trend, with an initial decrease and subsequent increase. AG490 administration after SAH significantly inhibited JAK2/STAT3 phosphorylation (P<0.05), suppressed the expression and translocation of HMGB1, reduced cortical apoptosis, brain edema and neurological deficits. These results demonstrated the involvement of the JAK2/STAT3 pathway in HMGB1 regulation after SAH. [ABSTRACT FROM AUTHOR]

A rapid increase in matrix metalloproteinase-9 (MMP-9) expression by stimulated leukocytes is common in many diseases. Recent evidence suggests that the beneficial effects of statins are mediated in part by the suppression of MMP-9 release. In this study, we investigated the effect of statin on MMP-9 expression and its antagonist, tissue inhibitor of metalloproteinase-1 (TIMP-1) in LPS-stimulated leukocytes. Rat neutrophils and monocytes were stimulated with lipopolysaccharide (LPS) in the presence of simvastatin. MMP-9 secretion and mRNA expression were analyzed using ELISA and RT-PCR, respectively. Total MMP-9 protein production was measured by Western blot analysis. Potential signal transduction pathways responsible for MMP-9 production were investigated using luciferase reporter assays (NF-κB), pull-down assays (RhoA), and pharmacological inhibition. Our data show that MMP-9 and TIMP-1 expression are differentially induced by LPS in neutrophils and monocytes. We showed that rapid MMP-9 release occurred mainly via secretion from intracellular stores. Moreover, we showed that statin significantly suppressed LPS-induced MMP-9 release and mRNA expression in a time- and concentration-dependent manner. We also evaluated that simvastain postponed the rapid LPS-induced MMP-9 release for about 20 min. In conclusion, we demonstrated that the suppressive effect of simvastatin on LPS-stimulated MMP-9 release does not occur via the NF-κB pathway and the MAPKs pathway, but via the RhoA/ROCK pathway. [ABSTRACT FROM AUTHOR]

Ischemic postconditioning refers to controlling reperfusion blood flow during reperfusion after ischemia, which can induce an endogenous neuroprotective effect and reduce ischemia-reperfusion injury. Activation of endogenous neuroprotective mechanisms plays a key role in protecting against brain ischemia-reperfusion injury. The mechanisms of cerebral ischemic postconditioning are not completely clear, and the following aspects may be involved: downregulation of oxidative stress, attenuating mitochondrial dysfunction, attenuating endoplasmic reticulum stress, accelerating the elimination of glutamate, increasing rCBF, inhibiting apoptosis, inhibiting autophagy, and regulating signal transduction. [ABSTRACT FROM AUTHOR]

Objective To clarify the effect of cortical spreading depression (CSD) on cortical cerebral blood flow and apoptosis following subarachnoid hemorrhage (SAH) in rats. Methods SAH was induced in male Wistar rats by autologous blood injection into the cisterna magna and CSD was induced in rats with KCl. The cortical blood flow was measured by laser Doppler flowmeter and apoptosis were detected by TUNEL staining. Results In SAH groups the cortical cerebral blood flow was significantly reduced and cortical cell apoptosis was increased compared with those in blank and normal saline groups. After CSD, the cortical cerebral blood flow and apoptosis in SAH groups were more obvious than in blank and normal saline groups. Conclusion CSD can reduce cortical cerebral blood flow and aggravate apoptosis after SAH. It may be one of the mechanisms of delayed ischemic neurologic deficit. [ABSTRACT FROM AUTHOR]

Objective To study the role of neuropeptide Y (NPY) in vasospasm after subarachnoid hemorrhage (SAH) in vitro cultured rat cerebral vascular smooth muscle cells (VSMCs) by observing the proliferation and contraction protein expression of VSMCs. Methods Male or female SD rats weighing 150 g were killed by blood loss. After infusion with saline, the circle of Willis artery and its branches were harvested and primary VSMCs were cultured. Then in vitro model of SAH was established by exposure to oxyhemoglobin of 10-6 mol/L. Then the cells were treated with NPY or BIB03304 (a commonly used blocker of NPY receptor), or NPY + BIB03304. After culturing 1,3,5 and 7 days, immunofluorescence was used to measure the expression of α-actin. IPP software was used to measure the fluorescence intenty and SPSS16.0 statistical software was used for data analysis. Results VSMCs were successfully cutured whli digettion method. The fifth generation of visible cells showed a typical "valley-peak"-like growth. In NPY and NPY + SAH groups, 3-day cell intervention significantly increased the fluorescence intensity of α-actin (P<0. 05), while BIBO 3304 treatment reduced the expression of α-actin, the intensity of which was as weak as that of the blank group (P>0.05). Conclusion NPY increases α-actin expression in VSMCs while BIBO 3304 blocks the up-regulation of α-acin by NPY. NPY plays an important role in delayed cerebral vasospasm after SAH. [ABSTRACT FROM AUTHOR]

Objective To investigate changes of AdipoR in the central nervous system and concentration of serum adiponectin after subarachnoid hemorrhage (SAH) in rats. Methods Fifty-four male Sprague-Dawley rats were randomly divided into three groups: normal group (n = 12), sham-operation group(n = 12), and SAH model group (n = 30). An endovascular perforation model was used to induce SAH in the rats. The expression of AdipoR in the central nervous system was detected by Western blot. The concentration of serum adiponectin was detected by ELISA. Results Compared with that in normal group and sham-operation group, the concentration of serum adiponectin was decreased signíficantly at day 1, 2 and 3 after SAH (P<0.05). Compared with that in normal group and sham-operation group, the expression of AdipoR1 in the cerebral cortex and the hippocampus was up-regulated sharply after SAH (P<0.05), but the expression of AdipoR2 did not change significantly (P>0.05). Conclusion Adiponectin may be involved in the pathophysiological process of early brain injury after SAH. The decreased concentration of serum adiponectin and the up-regulation of AdipoR1 expression are both important pathological mechanisms leading to early brain injury after SAH. [ABSTRACT FROM AUTHOR]

Objective To investigate the possible role of canonical trasnient receptor potential channels (TRPCs) in calcium overload of neurons after subarachnoid hemorrhage (SAH) and discuss the mechanism of TRPCs involved in delayed neuronal death (DND) after SAH. Methods Rat models of SAH were established by double-blood injection via the citerna magna. SAH intervention group was established by using SKF96365 intracerebroventricular injection. Intracellular calcium concentration was detected by Fura-2AM. The dynamic expressions of TRPCs in the cortex and basilar artery were determined by Western blot. The mRNA levels of TRPCs in the cortex and basilar artery were determined by RT-PCR. The results were compared with those in DMSO control model group, saline control group and normal group. The apoptosis of cortical neurons was detected by TUNEL. One-way ANOVA was used to compare within each group. Results The protein expressions and mRNA levels of TRPC1 in the cortex and the basilar artery continuourly increased after SAH and reached the peak on day 5 while intracellular calcium concentration and TUNEL-positive cell counts also reached the peak. After injection of SKF96365, a TRPCs blocker, the protein expressions and mRNA levels of TRPC1 were inhibited, intracellular calcium concentration and TUNEL-positive cell counts decreased. Meanwhile, the scores of rat neurological functions were improved. The expressions of TRPC3 in the cortex and the basilar artery showed no significant change. Conclusion TRPC1 induced calcium overload and CVS after SAH are the major causes of delayed neuronal death. Suppressing calcium influx induced by TRPC1 could delay neuronal death and improve neurological functions. [ABSTRACT FROM AUTHOR]

Objective To explore the dynamic expression of high mobicity group protein B1(HMGB1) in rats brain tissue after subarachnoid hemorrhage (SAH) and its significance. Methods Rats SAH models were established by perforation of anterior cerebral artery, and randomly divided into 5 subgroups (control group, 6 h, 1, 3, 7 d). ELISA was used to determine the plasm HMGB1 levels after SAH; immunohistochemical staining and western blot were used to survey the dynamic expression of HMGB1 in brain tissue after SAH. Results Plasm HMGB1 levels significantly increased after SAH, and continued to rise from 6 hto 7 d. Immunohistochemical staining indicated that HMGB1 positive cells significantly decreased within the brain tissue in early stage (6 h, 1 d) after SAH, in late stage (3, 7 d) it began to rise and translocated to cytoplasm; Western blot results also showed the expression of HMGB1 decreased first and then increased in the frontal lobe cortex after SAH. Conclusion HMGB1 expression significantly increased not only in brain tissue but also in plasma. HMGB1 may play a important role in inflammatory brain damage after SAH. [ABSTRACT FROM AUTHOR]

Early brain injury (EBI) and delayed cerebral injury following subarachnoid hemorrhage (SAH) are closely associated with poor prognois in patients. Studies on the molecular mechanisms of EBI have revealed several pathological mechanisms involved in neurovascular dysfunction. These mechanisms include microvascular changes, altered ionic homeostaiis, blood-brain barrier disruption, inflammation, oxidative stress, and cell death pathway activation. These studies also provide multiple possible intervention targets for protecting neurological function after SAH, in which calcium channel blockers, endothelin-1 antagonism, anti-platelet agents, anti-inflammatory agents, and antioxidants may have neuroprotective effects agaimt EBI. Further elaborating on the molecular mechanisms of SAH-induced hypoxic-ischemic brain injury, neuroinflammation, cortical spreading depression, neuronal cell death and revealing the evolvement of these complex pathophysiological proceises are of great significance for reducing brain injury after SAH, promoting nerve repair, and ameliorating patients' quahty of life and overall mortahty. [ABSTRACT FROM AUTHOR]

Objective To investigate the trends of peripheral blood leucocyte counts and MMP-9/TIMP-1 ratio in acute experimental subarachnoid hemorrhage model in rats and their relationship wíth early brain injury. Methods A rat single cisterna magna injection model was used. Leukocytes were counted by blood counting chamber, and MMP-9 and TIMP-1 were measured by ELISA. The dry-wet weight method was used to measure brain water content. The inside diameter was measured on an HE stained basilar artery. Results The brain water content increased significantly at 6 h after subarachnoid hemorrhage and peaked at 24 h, so did the leukocyte count as well as MMP-9 and TIMP-1 contents. Both the leukocyte count and MMP-9/TIMP-1 ratio were related to early brain injury after subarachnoid hemorrhage. Conclusion The leukocyte count and MMP-9/TIMP-1 ratio increase significantly in the early stage of subarachnoid hemorrhage and return to normal after 72 h. The trend is related to early brain injury after subarachnoid hemorrhage. The MMP-9/TIMP-1 ratio may be used as a predictor of the appearance of early brain injury. [ABSTRACT FROM AUTHOR]

Objective To investigate the activation of necroptosis pathway and explore the role and molecular mechanism of necroptosis in early brain injury after subarachnoid hemorrhage (SAH). Methods Forty adult male SD rats were divided into four groups ( n = 10 in each): sham, SAH, and SAH treatment plus Nec-1 or dimethyl sulfoxide (DMSO) . SAH model was established by endovascular perforation technique. In SAH+Nec-1 and SAH+ DMSO groups, Nec-1 or DMSO was injected intracerebroventsicularly 30 min prior to SAH establishment. Animals were then sacrificed at 24 h after modeling in each group. The neurological severity scores and brain water content were determined; immunohistochemical method was used in detecting the expression of receptor interacting protein 3 (RIP3). Results Neurological dysfunction, the number of necrotic neuronal cells, brain water content and the expression of RIP3 were significantly increased in the brain tissues of SAH rats (P<0.01). Neurological dysfunction, the number of necrotic neuronal cells, brain water content and the expression of RIP3 were significantly reduced after Nec-1 treatment (P<0.01). Conclusion Necroptoiis is activated and involved in the pathophysiological mechanism of early brain injury after SAH. RIP3 may play a crucial role in necroptosis pathway. Inhibiting necroptosis may be of great importance for protecting neuronal cells and reducing edema in early brain injury after SAH. [ABSTRACT FROM AUTHOR]

Objective To investigate the possible role of store-operated calcium channels (SOCC) in early brain injury (EBI) after subarachnoid hemorrhage (SAH) and to explore the relationship between SOCC and calcium overload induced delayed neuronal death (DND) after SAH. Methods Rabbits models of SAH were established using the single-blood injection method via the cisterna magna. SAH intervention group was established by intracerebroventricular injection of 2-aminoethyldiphenyl borate (2-APB). Intracellular calcium concentration was detected by Fura-2AM. The dynamic expression of SOCC was determined by Western blot. The apoptosis of cortical neurons was detected by TUNEL. One-way ANOVA was used to compare between experimental, normal control and sham-operation groups. Results The protein expressions of STIM1 and ORAI1 in the cortex increased continuously after SAH and reached the peak at day 3; intracellular calcium concentraron also reached the peak. After injection of SOCC blocker, the protein expressions of STIM1 and ORAI1 were inhibíted and intracellular calcium concentration decreased. Meanwhile, the scores of neurological functions. [ABSTRACT FROM AUTHOR]

Objective To explore the effects of necrostatin-1 on cortical necroptosis, autophagy and necrosis after subarachnoid hemorrhage (SAH). Methods Totally 72 SD rats were randomly divided into sham operation group, model group, vehicle group and necrostatin-1 group. Rats in vehicle group were given 2 μL of dimethyl sulfoxide by intracerebroventricular injection while those in necrostatin-1 group were given 5.2 μg of necrostatin-1 dissolved in 2 μL of dimethyl suboxide. Subarachnoid hemorrhage models were induced by intravascular puncture. In each group 9 rats were used to observe the expressions of receptor interacting protein 3 and microtubule associated protein 1 light chain 3 in the cerebral cortex by Western blot; 9 rats were injected intraperitoneally with propidium iodide to observe necroiis in each group. Loeffler neurological score was recorded for all rats at 6, 12 and 24 h. Results Necrostatin-1 suppressed receptor interacting protein 3 and microtubule associated protein 1 iight chain 3 in the cerebral cortex (P<0.05), decreased the number of propidium iodide positive celis in the cortex (P<0.05), and improved Loeffler neurological scores at 12 and 24 h (P<0.05), which showed that necrostatin-1 could improve the prognosis after SAH. Conclusion Necrostatin-1 might suppress the level of autophagy and necrosis when inhibiting post-SAH necroptosis in cerebral cells. [ABSTRACT FROM AUTHOR]

Objective To investigate the activation of AMP-activated protein kinase (AMPK) and Bim in rat cortex after subarachnoid hemorrhage (SAH) and to explore the role of AMPK in cortical apoptosis in early brain injury after SAH. Methods SAH model was established by an endovascular perforation technique. The immunohistochemical method was used to detect the localization of AMPK and phosphorylated AMPK. RT-PCR was performed to detect the dynamic mRNA expression of AMPKα, and Western blot was used to detect the protein leveis of phosphorylated AMPK and apoptosis-related proteins (Bim and caspase-3) in the cortex. After AICAR and compound C were administered via i. c. v, the change of p-AMPK level was examined, and the effects of AICAR and compound C on neurological behavior and apoptosis-related protein expressions were observed. Results Experimental SAH increased the expressions of AMPKα mRNA and phosphorylated AMPK. Bim and caspase-3 were also significantiy increased. Wth compound C treatment, the phosphorylation level of AMPK and the up-regulation of Bim were modfied. However, AICAR administration exacerbated cortical apoptosis and neurological deficit through up-regulation of Bim. Conclusion AMPK is involved in the pathophysiological process of neuronal apoptosis in early brain injury after SAH. The mechanism may be related to the regulation of transcription of Bim. Inhibition of AMPK signaling pathway can provide neuroprotection by reducing cortical apoptosis after SAH. [ABSTRACT FROM AUTHOR]

Highlights: [•] The first study on serum MMP-9 and TIMP-1 expression in acute stage of acute cerebral infarction (ACI) in a rat model. [•] Our results indicate that brain edema induced by ACI is associated with increased MMP-9 response and MMP-9/TIMP-1 ratio in serum. [•] No relationship could be found between TIMP-1 and brain edema. [Copyright &y& Elsevier]

Highlights: [•] Allicin attenuates mechanical trauma induced neuronal injury. [•] Allicin differentially regulates NOS expression levels. [•] Allicin induced protection is dependent on NOS regulation. [•] Allicin induced regulation of NOS is mediated by Akt and ERK. [ABSTRACT FROM AUTHOR]