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Objective To investigate the role of JAK2/STAT3 signaling pathway in regulating the expression and nuclear-cytoplasm translocation of high mobility group box 1 (HMGB1) in early brain injury (EBI) after subarachnoid hemorrhage (SAH).Methods Ninety SD rats were divided into a sham group (15 rats),an SAH group (altogether 45 rats,with 15 ones for each time point of 6 h,1 d,and 3 d),an SAH+AG490 (JAK2/STAT3 inhibitor) group (15 rats) and an SAH+dimethyl sulfoxide (DMSO)group (15 rats).The SAH models in the later 3 groups were established by endovascular perforation;the blood vessels were not perforated in the sham group but the other operations were the same as in the SAH groups.(1) Western blotting was used to detect the expression of HMGB1 and phosphorylated JAK2/STAT3 (p-JAK2/p-STAT3) in the 4 groups (at different time points in the SAH group) and compared the expression changes between the 4 groups after AG490 intervention.(2)Immunofluorescence confocal microscopy was used to detect HMGB1 nuclear translocation in the 4 groups.(3) TUNEL staining was used to detect apoptosis in the 4 groups.(4) Brain water contents and neurobehavioral scores in the 4 groups were measured.Results (1) Western blotting showed that the expression levels ofp-JAK2 and p-STAT3 were significantly increased at 6 h,1 d,and 3 d after SAH,and there were significant differences between the sham group and the SAH group (P＜0.05).HMGB1 total protein,cytoplasmic HMGB1 and nucleus HMGB1 also increased significantly at different time points after SAH,and statistically significant differences existed between the sham group and the SAH group (P＜0.05).The expression levels ofp-JAK2/p-STAT3,HMGB1 and cytoplasm and nucleus HMGB1 in the SAH+AG490 group were significantly lower than in the SAH group and SAH+DMSO group(P＜0.05).(2) The immunofluorescence staining showed that HMGB1 staining was positive in the SAH group while the positive staining of HMGB1 was present mainly in the nucleus but not in the cytoplasm in the sham and SAH+AG490 groups,suggesting that AG490 might inhibit the nucleus-cytoplasm transposition of HMGB1.(3) Compared with the SAH and SAH+DMSO groups,the TUNEL staining positive cells in the SAH+AG490 group were significantly decreased (P＜0.05).(4) Compared with the SAH and SAH+DMSO groups,the brain water contents in the SAH+AG490 group decreased significantly and the neurobehavioral scores increased significantly (P＜0.05).Conclusions JAK2/STAT3 signaling pathway is involved in the pathological process of early brain injury after SAH,and its mechanism may be related to the regulation of HMGB1 expression and nuclear-cytoplasm transposition.The regulation of JAK2/STAT3 may contribute to the neuroprotection dependent of HMGB 1.
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Objective To investigate the role of AMP-activated protein kinase (AMPK) signal path in cell autophagy activation at early brain injury in rats after subarachnoid hemorrhage (SAH).Methods Adult male SD rats (weighting 300-350 g) were divided into five groups (n=12):sham-operated group,SAH group,and SAH+AICAR group,SAH+Compound C group and SAH+vehicle group.SAH models in the later four groups were established by endovascular perforation technique,and rats in the later three groups were performed left intracerebroventricular injection of AMPK agonist AICAR,AMPK inhibitor Compound C or normal saline 30 min before modeling;animals were subsequently sacrificed at 24 h after modeling.Immunohistochemical method was used to detect the phosphorylated mammalian target of rapamycin (p-mTOR) expression.Expressions of cortex autophagy related proteins LC3,AMPK and phosphorylated AMPK (p-AMPK) were observed by Western blotting.Loeffler's method was used to evaluate the neurologic behavior scores.Results As compared with those in the sham-operated group,the p-AMPK level,p-mTOR expression level and LC3Ⅱ/LC3Ⅰ ratio were significantly increased,while the behavioral deficit scores were significantly lower in the SAH group,with statistical differences (P＜0.05);the p-mTOR mainly expressed at cortex surrounding the hemorrhage areas,and integration areas of deep cortex and brain white matter.As compared with the sham-operated group and SAH+vehicle group,SAH+AICAR group had significantly increased p-AMPK level,decreased p-mTOR expression level,increased LC3Ⅱ/LC3Ⅰ ratio,and decreased behavioral deficit scores (P＜0.05);as compared with the sham-operated group and SAH+vehicle group,SAH+Compound C group had significantly decreased p-AMPK level,decreased LC3Ⅱ/LC3Ⅰ ratio,and decreased behavioral deficit scores (P＜0.05).Conclusion AMPK is involved in the process ofautophagy activation after SAH through regulating mTOR,and the regulation of AMPK may contribute to neuroprotection related to autophagy.
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Objective To investigate the mechanism of unbalanced expressions of endothelin receptors (ETA/ETB )in cerebral vasospasm (CVS)after subarachnoid hemorrhage (SAH).Methods The rat CVS models were established by injecting autologous blood into the cisterna magna the second time.Basilar artery morphology was observed under light microscope and immunofluorescence staining was conducted to dynamically detect ETA/ETB receptor expression.Results The cross-sectional area of the basilar artery in the SAH model group decreased at 2 d to 3 d,and then gradually returned to normal.ETA receptor expression in endothelial cells of the basilar artery increased at 2 d after SAH,peaked at 3 d and remained increased till 14 d.ETB receptor expression increased significantly in endothelial cells at 3 d,peaked at 7 d and remained the same level till 14 d.Conclusion The results suggest that ETA/ETB receptors play an important role in cerebral vasospasm after SAH.The specific expression differences of ETB receptor subtypes in the brain vascular layers need further study.
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Objective To study the dynamic expression and distribution of high mobility group box 1 (HMGB-1)in diffuse axonal injury (DAI)in rats and to clarify its involvement in the inflammatory reaction after DAI in rats,in order to provide new targets for the clinical treatment of DAI.Methods A DAI model was established using a coronal rotation device and evaluated by HE,Glees-Marsland silver staining,and Mallory phosphotungstic acid hematoxylin staining.Immunohistochemistry,Western blot and RT-PCR were used to detect the expression and distribution of HMGB-1 in the cortex of DAI rats at 6 h,1 d,3 d and 7 d.And TUNEL was used to examine the apoptosis of neurons in DAI rats.Results Immunohistochemical results showed that at 6 h and 1 d after DAI,the number of HMGB-1-positive cells decreased,but at 3 and 7 d it began to increase.Western blot also showed that during the early stage after DAI (6 h and 1 d),the level of HMGB-1 protein in the cortex was significantly lower than that in the control group,but at the late stage (3 and 7 d)after DAI it significantly increased compared with that in the control group until 7 d.RT-PCR showed that at 6 h after DAI there was no significant increase in the level of HMGB-1mRNA,but at 1 d there was a slight increase compared with the control group;at 3 and 7 d,it showed an obvious significance.TUNEL staining indicated that the significant neuronal apoptosis appeared as early as 6 h after DAI,and reached the peak at 3 d;it started to decrease at 7 d but still remained at a relatively high level.Conclusion The dynamic expression and distribution of HMGB-1 showed significant changes with the time course after DAI in rats.They decreased at the early stage but increased at the late stage.At the early stage, HMGB-1 is mainly passively released by the necrotic neurons,and at the late stage it may be actively secreted by the active inflammatory cells.HMGB-1 may mediate the post-DAI neural cell apoptosis by inducing the inflammatory reaction.
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Cerebral vasospasm is one of the most severe complications of subarachnoid hemorrhage. Its incidence is as high as 30-90%. It often causes severe regional cerebral ischemia or delayed ischemic brain damage, even resulting in cerebral infarction, and it thus becomes the primary cause of mortality and disability. In recent years, with the development of studies, people have realized that the oxyhemoglobin, inflammatory reaction, accumulation of vasoconstrictive substances, apoptosis, blood hypercoagulability and blood vessel cell proliferation play important roles in the development of cerebral vasospasm. Although its mechanism remains unclear, better effects have been achieved by using related treatment methods according to the available pathogenesis.
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Objective To design a new experimental facility to induce rat diffuse axonal injury model. Methods Rat diffuse axonal injury was induced by a new experimental facility,which was developed to let the rat head spin 90 degree at the moment to cause shearing injury.Vital sign and behavior of rat were measured simultaneously.The rats were sacrificed at 2 h,6 h,12 h,24 h,36 h,72 h and 10 d after injury,respectively,and the brain tissues were removed to prepare paraffin section.Then silver staining and HE staining were conducted to investigate changes of axonal fibers. Results There were unconsciousness,respiratory rhythm disturbance and hyporeflexia of pupil light reflex immediately after injury,and reactiveness decrease and activity retardation still existed even after resuming consciousness.At anatomical scene,subarachnoid hemorrhage or cerebroventricular haemorrhage were widespread.At an early stage,there were swelling,collapse,and axonal retraction ball formation at cortico-medulla junction,callosum,brainstem,and cerebellar white matter under microscope.But at the later stage,gitter cell proliferation and nest-like aggregation were major pathophysiological changes at focal brain tissue. Conclusion The new experimental facility is suit able to be used to induce rat diffuse axonal injury,since it is convenient,controlable,and precise.
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Objective To discuss the ultrastructural pathological characteristics and dynamic changes of brain vessel after subarachnoid hemorrhage(SAH),and the mechanism of these changes in delayed cerebral vasospasm.Methods SAH model was made by infusing blood twice into the cistern magna of Japanese rabbits.The animals were divided randomly into SAH group,saline group,puncture group and blank group,at 1 h,3 d,5 d,7 d and 10 d after the first infusion the animals were perfused and basilar artery was harvested.Ultrastructural changes were observed under light microscope and electron microscope.Results Under the light microscope,the vessel wall became thick,the vessel cavity became narrow,the endothelia cells became swollen,vacuoles could be found in the chromatin,inner elastic membrane became reductus and broke.Under the electron microscope,the close connection between the endothelial cells disappeared,the membrane of the cells fell off,and the mitochondria became swollen,vacuoles could be seen,the chromatin became concentrated,heterochromatin could be seen,smooth muscle became deformed,chromatin became uneven, myofilament had derangement and fragmentation and dissolved,vacuolus could be seen in the kytoplasm,mitochondrion became swollen.The structural change of basilar artery under the light microscope got similar to that under the electron microscope;slight change was observed right after 1 h of SAH,significant change was observed at 3 d,and most obvious change was observed between 5 d and 7 d.Conclusion Ultrastructural changes were observed in the basilar artery after SAH,and significant dynamic changes were observed in the progress.The damage of endothelia cells may be the important factors which cause delayed cerebral vasospasm.
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Objective To discuss the changes of the S100B protein concentration in serum and cerebral spinal fluid(CSF) after subarachnoid hemorrhage(SAH) in rabbit model and their significance.Methods Rabbit SAH model was induced by the cisterna magna puncture and injection two times of autogeneic blood into the cisterna magna.The animals were divided randomly into SAH group,saline group,puncture group and blank group.The serum and CSF were taken in blank group after 3 days' breeding.At 1 h,3 d,5 d,7 d and 10 d after the first infusion,the serum and CSF of the other groups were taken.ELISA method was used to detect S100B protein concentration in serum and CSF.The result data was analyzed by software SPSS13.0.Results S100B protein concentration in serum and CSF of SAH group was much higher than that in the other three groups(P=0).S100B protein concentration in serum ascended from 1 h after SAH,reached the peak at 3～5 d after SAH,and then descended slowly.S100B protein concentration in CSF ascended from 1 h after SAH,then slightly descended,ascended and reached the peak at 5～7 d after SAH,and then descended slowly.S100B protein concentration in serum and CSF of saline group was higher than that in puncture group and blank group from 1 h after model establishment(P