Vascular endothelial growth factor A promotes platelet adhesion to collagen IV and causes early brain injury after subarachnoid hemorrhage.

The role of vascular endothelial growth factor A in platelet adhesion in cerebral microvessels in the early stage of subarachnoid hemorrhage remains unclear. In this study, the endovascular puncture method was used to produce a rat model of subarachnoid hemorrhage. Then, 30 minutes later, vascular endothelial growth factor A antagonist anti-vascular endothelial growth factor receptor 2 antibody, 10 µg, was injected into the right ventricle. Immunohistochemistry and western blot assay were used to assess expression of vascular endothelial growth factor A, occludin and claudin-5. Immunohistochemical double labeling was conducted to examine co-expression of GP Ia-II integrin and type IV collagen. TUNEL was used to detect apoptosis in the hippocampus. Neurological score was used to assess behavioral performance. After subarachnoid hemorrhage, the expression of vascular endothelial growth factor A increased in the hippocampus, while occludin and claudin-5 expression levels decreased. Co-expression of GP Ia-II integrin and type IV collagen and the number of apoptotic cells increased, whereas behavioral performance was markedly impaired. After treatment with anti-vascular endothelial growth factor receptor 2 antibody, occludin and claudin-5 expression recovered, while co-expression of GP Ia-II integrin and type IV collagen and the number of apoptotic cells decreased. Furthermore, behavioral performance improved notably. Our findings suggest that increased vascular endothelial growth factor A levels promote platelet adhesion and contribute to early brain injury after subarachnoid hemorrhage. This study was approved by the Biomedical Ethics Committee, Medical College of Xi'an Jiaotong University, China in December 2015.

AG490 ameliorates early brain injury via inhibition of JAK2/STAT3-mediated regulation of HMGB1 in subarachnoid hemorrhage.

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.

Protection of FK506 against neuronal apoptosis and axonal injury following experimental diffuse axonal injury.

Diffuse axonal injury (DAI) is the most common and significant pathological features of traumatic brain injury (TBI). However, there are still no effective drugs to combat the formation and progression of DAI in affected individuals. FK506, also known as tacrolimus, is an immunosuppressive drug, which is widely used in transplantation medicine for the reduction of allograft rejection. Previous studies have identified that FK506 may play an important role in the nerve protective effect of the central nervous system. In the present study, apoptosis of neuronal cells was observed following the induction of experimental DAI. The results demonstrated that it was closely related with the upregulation of death­associated protein kinase 1 (DAPK1). It was hypothesized that FK506 may inhibit the activity of DAPK1 by inhibiting calcineurin activity, which may be primarily involved in anti­apoptosis following DAI induction. Through researching the expression of nerve regeneration associated proteins (NF­H and GAP­43) following DAI, the present study provides novel data to suggest that FK506 promotes axon formation and nerve regeneration following experimental DAI. Therefore, FK506 may be a potent therapeutic for inhibiting nerve injury, as well as promoting the nerve regeneration following DAI.

Rosiglitazone ameliorates diffuse axonal injury by reducing loss of tau and up-regulating caveolin-1 expression.

Rosiglitazone up-regulates caveolin-1 levels and has neuroprotective effects in both chronic and acute brain injury. Therefore, we postulated that rosiglitazone may ameliorate diffuse axonal injury via its ability to up-regulate caveolin-1, inhibit expression of amyloid-beta precursor protein, and reduce the loss and abnormal phosphorylation of tau. In the present study, intraperitoneal injection of rosiglitazone significantly reduced the levels of amyloid-beta precursor protein and hyperphosphorylated tau (phosphorylated at Ser(404)(p-tau (S(404))), and it increased the expression of total tau and caveolin-1 in the rat cortex. Our results show that rosiglitazone inhibits the expression of amyloid-beta precursor protein and lowers p-tau (S(404)) levels, and it reduces the loss of total tau, possibly by up-regulating caveolin-1. These actions of rosiglitazone may underlie its neuroprotective effects in the treatment of diffuse axonal injury.

Role of the AMPK signaling pathway in early brain injury after subarachnoid hemorrhage in rats.

BACKGROUND: AMP-activated protein kinase (AMPK) is a key metabolic and stress sensor/effector. Few investigations have been performed to study the role of AMPK in subarachnoid hemorrhage (SAH)-induced early brain injury (EBI). This study was undertaken to investigate the time course of AMPK activation in the early stage of SAH and to evaluate the influence of AICAR (which is known to mimic AMP and activates AMPK) and compound C (a commonly used AMPK inhibitor) on EBI in rats following SAH. METHODS: Adult male rats were divided into six groups: control, sham, SAH, SAH + vehicle, SAH + AICAR and SAH + compound C. SAHs were induced by a modified endovascular perforation method. Immunohistochemistry, real-time PCR and Western blot were used to detect the spatial and dynamic expression of AMPK after SAH. Cortical apoptosis and the expressions of apoptosis-related proteins such as FOXO3a (forkhead box, class O, 3a) and Bim (Bcl-2-interacting mediator of cell death) were detected after different drug interventions. RESULTS: We found SAH induced prolonged activation of AMPK. Treatment with AICAR markedly induced overactivation of AMPK and upregulation of FOXO3a and Bim. AICAR also significantly exacerbated cerebral apoptosis and neurological impairment following SAH. On the other hand, pre-administration of compound C attenuated EBI in this SAH model by modulating cerebral apoptosis by inhibiting FOXO3a and Bim. CONCLUSIONS: Our findings suggest that the AMPK pathway may play an important role in SAH-induced neuronal apoptosis, and the use of AMPK inhibitors can provide neuroprotection in EBI after SAH.

Receptor-associated protein promotes t-PA expression, reduces PAI-1 expression and improves neurorecovery after acute ischemic stroke.

Receptor-associated protein (RAP) is a receptor antagonist that inhibits ligand interactions with the receptors that belong to the low density lipoprotein receptor gene family. The low-density lipoprotein receptor-related protein 1 (LRP1) has a crucial role in regulating tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) expression. Furthermore, the functional balance of these two proteins is directly associated with the initiation and development of cerebral ischemic stroke. In the present study, the effect of RAP post-treatment was investigated in a rat autologous thromboembolic model. The expression and activity of t-PA and PAI-1 were detected and the neurological function was tested. The results suggest that post-treatment with RAP is able to improve neurorecovery after ischemic stroke by decreasing vascular damage and regulating t-PA and PAI-1 expressions. Post-treatment with RAP promotes t-PA expression, suppresses PAI-1 expression, significantly improves functional outcomes and decreases the amount of TUNEL-positive cells. RAP-treated rats show lower intracranial hemoglobin levels and a smaller ischemic zone. In conclusion, post-treatment with RAP regulates t-PA and PAI-1 expressions and thereby contributes to the improvement of functional outcomes after cerebral ischemia. Our findings strongly suggest that RAP may be of value in neurorecovery after stroke.

The rapid lipopolysaccharide-induced release of matrix metalloproteinases 9 is suppressed by simvastatin.

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.

Etanercept alleviates early brain injury following experimental subarachnoid hemorrhage and the possible role of tumor necrosis factor-α and c-Jun N-terminal kinase pathway.

Cerebral inflammation plays a crucial role in early brain injury (EBI) after subarachnoid hemorrhage (SAH). This study investigated the effects of c-Jun N-terminal kinase (JNK) inhibitor SP600125, acetylcholine (Ach), etanercept, and anti-TNF-α on cellular apoptosis in the cerebral cortex and the hippocampus, in order to establish the role of JNK and TNF-α in EBI. The SAH model was established using an endovascular puncture protocol. The reliability of the EBI model was determined by phosphorylated-Bad (pBad) immunohistochemistry. Neurological scores were recorded and western blot was used to detect the expression of JNK and TNF-α, and TUNEL assay was used to mark apoptotic cells. The results showed that pBad positive cells were evenly distributed in the cerebral cortex at different time points. The highest expression of pBad was reached 1 day after SAH, and pJNK and TNF-α reached their peak expression at 2 days after SAH. SP600125, Ach, and etanercept significantly decreased the level of pJNK and TNF-α in the cerebral cortex and the hippocampus. In addition, SP600125 and etanercept reduced cellular apoptosis in the cerebral cortex and the hippocampus and significantly improved neurological scores at 2 days after SAH potentially via inhibition of the JNK-TNF-α pathway. Ach reduced cellular apoptosis only in the cerebral cortex. It is possible that JNK induces TNF-α expression, which in turn enhances JNK expression in EBI after SAH, leading to increased apoptosis in the cerebral cortex and the hippocampus. Thus, our results indicate that that etanercept may be a potential therapeutic agent to alleviate EBI.

Suppression of STIM1 in the early stage after global ischemia attenuates the injury of delayed neuronal death by inhibiting store-operated calcium entry-induced apoptosis in rats.

Ca²âº overload is considered to be the most important ion imbalance in the neuronal injury. Store-operated Ca²âº entry has been suggested to be a significant mechanism of excessive Ca²âº influx in many cells. The role of store-operated Ca²âº entry in neuronal ischemic injury has yet to be elucidated. The aim of this study was to assess the role of store-operated calcium channel (SOCC) proteins involved with calcium overload in the induction of delayed neuronal death after global ischemia in rats. A transient RNA interference model of global ischemia in rats was established to determine the role of SOCC-induced Ca²âº overload in delayed neuronal death. We found that STIM1 and ORAI1 expression in the hippocampus increased continuously after global ischemia and peaked on day 4. These data were consistent with an increase in the intracellular calcium concentration. Using Stim1 siRNA to suppress SOCC activity in the early stage of ischemia significantly inhibited STIM1 and ORAI1 expression and decreased the intracellular calcium concentration in neurons. In addition, the neurological function of rats improved after the Stim1 siRNA injection. High expression of STIM1 and ORAI may be the source of excessive calcium influx after ischemic damage. Blocking of this SOCC-induced calcium influx could lead to an improved neuronal survival. These data suggest that calcium influx through SOCC is another nonexcitotoxicity mechanism of ischemic neuronal death.

Role of hydrogen sulfide in secondary neuronal injury.

In acute neuronal insult events, such as stroke, traumatic brain injury, and spinal cord injury, pathological processes of secondary neuronal injury play a key role in the severity of insult and clinical prognosis. Along with nitric oxide (NO) and carbon monoxide (CO), hydrogen sulfide (H2S) is regarded as the third gasotransmitter and endogenous neuromodulator and plays multiple roles in the central nervous system under physiological and pathological states, especially in secondary neuronal injury. The endogenous level of H2S in the brain is significantly higher than that in peripheral tissues, and is mainly formed by cystathionine ß-synthase (CBS) in astrocytes and released in response to neuronal excitation. The mechanism of secondary neuronal injury exacerbating the damage caused by the initial insult includes microcirculation failure, glutamate-mediated excitotoxicity, oxidative stress, inflammatory responses, neuronal apoptosis and calcium overload. H2S dilates cerebral vessels by activating smooth muscle cell plasma membrane ATP-sensitive K channels (KATP channels). This modification occurs on specific cysteine residues of the KATP channel proteins which are S-sulfhydrated. H2S counteracts glutamate-mediated excitotoxicity by inducing astrocytes to intake more glutamate from the extracellular space and thus increasing glutathione in neurons. In addition, H2S protects neurons from secondary neuronal injury by functioning as an anti-oxidant, anti-inflammatory and anti-apoptotic mediator. However, there are still some reports suggest that H2S elevates neuronal Ca(2+) concentration and may contribute to the formation of calcium overload in secondary neuronal injury. H2S also elicits calcium waves in primary cultures of astrocytes and may mediate signals between neurons and glia. Consequently, further exploration of the molecular mechanisms of H2S in secondary neuronal injury will provide important insights into its potential therapeutic uses for the treatment of acute neuronal insult events.

The roles of MMP-9/TIMP-1 in cerebral edema following experimental acute cerebral infarction in rats.

Matrix metalloproteinases 9 (MMP-9) and its endogenous inhibitor, tissue inhibitor of metalloproteinases 1 (TIMP-1), regulate homeostasis and turnover of the extra cellular matrix (ECM). They play important roles in acute cerebral infarction (ACI). The contributions of MMP-9 and TIMP-1 to the early stages of ACI are not completely understood. This study investigates the time course of MMP-9 and TIMP-1 and their relations to edema after ACI in rats. Serum concentrations of MMP-9 and TIMP-1 protein were measured using ELISA and mRNA level were measured using real-time PCR. Brain samples were harvested and the brain water content (BWC) was measured. Results revealed that MMP-9 concentration increased fast during the first 12 h after ACI, while after 12 h the increase was much slower. The MMP-9 protein concentration was elevated earlier than the mRNA level. BWC increased starting at 6 h after ACI to reach a peak at 12 h and decreased back to normal levels at 72 h. Both the MMP-9 protein and its mRNA were positively correlated with BWC, however no correlation was found between TIMP-1 levels and BWC. The MMP-9/TIMP-1 protein ratio was more closely correlated with BWC than the MMP-9 concentration. These results indicate that brain edema induced by ACI is associated with increased MMP-9 levels and MMP-9/TIMP-1 ratio in serum.