BACKGROUND： Numerous studies have shown that transient ischemic preconditioning induces cerebral ischemic tolerance. However, the underlying mechanisms of endogenous protection following ischemic preconditioning rema...BACKGROUND： Numerous studies have shown that transient ischemic preconditioning induces cerebral ischemic tolerance. However, the underlying mechanisms of endogenous protection following ischemic preconditioning remain unclear. OBJECTIVE： To dynamically measure erythropoietin and hypoxia-inducible factor-1α （HIF-1α） mRNA and protein expression at various times following preconditioning, and to investigate effects of erythropoietin and HIF-1α on cerebral ischemic tolerance in a model of focal ischemia/reperfusion established using the twice suture method. DESIGN, TIME AND SETTING： The randomized, controlled study was performed at the Institute of Anatomy, Medical College, Qingdao University, China from March 2006 to March 2007. MATERIALS： Rabbit anti-rat HIF-1α monoclonal antibody and biotinylated goat anti-rabbit IgG （Boster, China）, rabbit anti-rat erythropoietin monoclonal antibody （Santa Cruz Biotechnology, USA）, and one-step RT-PCR kit （Qiagen, Germany） were used in this study. METHODS： A total of 99 healthy, male, Wistar rats were randomly assigned to three groups： sham surgery （n = 9）, non-ischemic preconditioning （n = 45）, and ischemic preconditioning （n = 45）. In the ischemic preconditioning group, rat models of pre-ischemia-reperfusion-ischemia-reperfusion were established by occluding the left middle cerebral artery using the twice suture method. In the non-ischemic preconditioning group, pre-ischemia was replaced by sham surgery. Subsequently, the ischemic preconditioning and non-ischemic preconditioning groups were equally divided into five subgroups according to time of first reperfusion, including 1-, 3-, 7-, 14-, and 21-day subgroups. The sham surgery group received the sham surgery twice. MAIN OUTCOME MEASURES： HIF-la and erythropoietin protein expression was measured in the cerebral cortex, corpus striatum, and hippocampus of the ischemic hemisphere. HIF-1α and erythropoietin mRNA expression were determined in the frontal and parietal cortex of the ischemic hemisphere. RESULTS： （1） Intergroup comparison： compared with the non-ischemic preconditioning group, HIF-1α protein expression significantly increased in the rat cerebral cortex, corpus striatum, and hippocampus in the ischemic hemisphere at 1,3, and 7 days following reperfusion in the ischemic preconditioning group （P 〈 0.05 or P 〈 0.01）. Erythropoietin protein expression significantly increased in the cerebral cortex, corpus striatum, and hippocampus, as well as HIF-1α and erythropoietin mRNA expression in the frontal and parietal cortex in the ischemic hemisphere, at 3 and 7 days following reperfusion in the ischemic preconditioning group （P 〈 0.05）. （2） Temporal expression： HIF-1α protein expression in the rat cerebral cortex, corpus striatum, and hippocampus, as well as HIF-la mRNA expression in the frontal and parietal cortex, in the ischemic hemisphere increased at 3 days, and gradually decreased from 7 days following reperfusion in the ischemic preconditioning group. Temporal erythropoietin protein and mRNA expression was consistent with HIF-1α protein expression. （3） Correlation： erythropoietin mRNA expression positively correlated with HIF-1α mRNA expression （r= 0.737, P 〈 0.01）. CONCLUSION： Ischemic preconditioning induced cerebral ischemic tolerance. Pre-ischemiainduced increase in endogenous HIF-1αexpression, as well as its target gene erythropoietin, participated in the formation of cerebral ischemic tolerance.展开更多
Previous studies of integrin αvβ3 have focused on ischemic brain damage, although the role of integrin αvβ3 in ischemic preconditioning （IP） has rarely been reported. The present study analyzed the effects of IP...Previous studies of integrin αvβ3 have focused on ischemic brain damage, although the role of integrin αvβ3 in ischemic preconditioning （IP） has rarely been reported. The present study analyzed the effects of IP on integrin αvβ3 mRNA expression following cerebral ischemia through the use of hematoxylin-eosin staining and real-time quantitative polymerase chain reaction techniques. Integrin avid3 mRNA expression in the ischemia group peaked at 24 hours after ischemia-reperfusion. In the IP ＋ ischemia group, integrin αvβ3 mRNA expression increased after 24 hours, but remained significantly less than the ischemia group, and expression continued to increase until 7 days after ischemiaJreperfusion. These results demonstrate that IP effectively attenuated upregulation of integrin αvβ3 mRNA expression at 24 hours after ischemia.展开更多
The astrocyte is a critical regulator of neuronal survival after ischemic brain injury. Electroacupuncture may be an effective therapy for cerebral ischemia, as electroacupuncture frequency can affect the structural i...The astrocyte is a critical regulator of neuronal survival after ischemic brain injury. Electroacupuncture may be an effective therapy for cerebral ischemia, as electroacupuncture frequency can affect the structural integdty of astrocytes. In this study, a rat model of middle cerebral artery occlusion established using the modified thread embolism method was treated with electroacupuncture of the bilateral Quchi （Llll） and Zusanfi （ST36） at 15, 30, and 100 Hz frequencies. Behavioral testing, immunohistochemistry and electron microscopy were used to explore the effect of these electroacupuncture frequencies used on maintaining the structural integrity of ischemic brain tissue. Compared with the model and 100 Hz electroacupuncture groups the 15 and 30 Hz electroacupuncture groups displayed decreased neurological deficit scores, as evaluated by the ＂Longa＂ method, significantly increased glial fibrillary acidic protein expression, and alleviated ultrastructural damage of astrocytes at the edge of the infarct. Our experimental findings indicate that 15 and 30 Hz electroacupuncture intervention can favorably maintain the structural integrity of astrocytes and play a protective role in cerebral ischemic injury. Astrocyte structural integrity may be the mechanism underlying acupuncture production of ischemic tolerance展开更多
Hypoxia-inducible factor 1 （HIF-1） can lead to the adaptative reaction of body for hypoxia and ischemia. HIF-1 plays an important role in the response of ischemia-hypoxia. At present, there has been no overall repor...Hypoxia-inducible factor 1 （HIF-1） can lead to the adaptative reaction of body for hypoxia and ischemia. HIF-1 plays an important role in the response of ischemia-hypoxia. At present, there has been no overall report on the significance for the expression of HIF-1 following experimental cerebral ischemia. OBJECTIVE： To observe the expression of HIF-1 after middle cerebral artery occlusion （MCAO） by immunohistochemical method. DESIGN： Completely randomly grouped controlled animal experiment. SETTING： Second Hospital, Xi＇an Jiaotong University. MATERIALS： Thirty-six Sprague-Dawley healthy male rats, with body mass of 250 - 330 g, were used in this study. Thirty-six rats were randomized into 3 groups： preischemia group, sham-operation group and control group, with 12 rats in each. METHODS： This study was carried out in the clinical laboratory, People＇s Hospital of Ningjin County of Shandong Province from March 2006 to January 2007. Rats in the pre-ischemia group were created into preischemia models by two embolisms twice. Three days after ischemic preconditioning, middle cerebral artery （MCA） was occluded for 2 hours with the same method. After being perfused for 22 hours, the rats were euthanized. In the sham-operation group, rats were not given the treatment of preischemia. In the first operation, only common carotid artery （CCA） and its crotch were exposed in the first operation, and MCA was not blocked by inserting embolism. At postoperative 3 days, rats were euthanized after being subjected to MCAO for 2 hours and reperfusion 22 hours by the same procedure as that in the preischemia group. As for each rat in the control group, only CCA and its crotch were exposed, and no any other treatment was carried out on them. MAIN OUTCOME MEASURES： Brain tissue of each rat was performed immunohistochemical staining at reperfusion 22 hours after preischemia, HIF-1 expression and brain infarct volume were detected. RESULTS： Thirty-six Sprague-Dawley rats were involved in the experiment. During the experiment, 8 rats dropped out, and another 8 rats were supplemented. The infarct volume of rats in the preischemia group was significantly smaller than that in the sham-operation group （t=3.22, P 〈 0.01 ） . HIF-1 expression was not found in the control group, but many HIF-I positive cells were found in the other two groups. Absorbance in the preischemia group was significantly higher than that in the sham-operation group （t=4.31, P 〈 0.01）. CONCLUSION： Slight ischemia caused preconditioning can increase HIF-1 content, and it is one of protective mechanisms for nerve cells.展开更多
In this study, we hypothesized that an increase in integrin αβand its co-activator vascular endothelial growth factor play important neuroprotective roles in ischemic injury. We performed ischemic preconditioning wi...In this study, we hypothesized that an increase in integrin αβand its co-activator vascular endothelial growth factor play important neuroprotective roles in ischemic injury. We performed ischemic preconditioning with bilateral common carotid artery occlusion for 5 minutes in C57BL/6J mice. This was followed by ischemic injury with bilateral common carotid artery occlusion for 30 minutes. The time interval between ischemic preconditioning and lethal ischemia was 48 hours. Histopathological analysis showed that ischemic preconditioning substantially diminished damage to neurons in the hippocampus 7 days after ischemia. Evans Blue dye assay showed that ischemic preconditioning reduced damage to the blood-brain barrier 24 hours after ischemia. This demonstrates the neuroprotective effect of ischemic preconditioning. Western blot assay revealed a significant reduction in protein levels of integrin αβ, vascular endothelial growth factor and its receptor in mice given ischemic preconditioning compared with mice not given ischemic preconditioning 24 hours after ischemia. These findings suggest that the neuroprotective effect of ischemic preconditioning is associated with lower integrin αβand vascular endothelial growth factor levels in the brain following ischemia.展开更多
基金the Scientific and Technological Development Program of Qingdao City, No.05-1-NS-73
文摘BACKGROUND： Numerous studies have shown that transient ischemic preconditioning induces cerebral ischemic tolerance. However, the underlying mechanisms of endogenous protection following ischemic preconditioning remain unclear. OBJECTIVE： To dynamically measure erythropoietin and hypoxia-inducible factor-1α （HIF-1α） mRNA and protein expression at various times following preconditioning, and to investigate effects of erythropoietin and HIF-1α on cerebral ischemic tolerance in a model of focal ischemia/reperfusion established using the twice suture method. DESIGN, TIME AND SETTING： The randomized, controlled study was performed at the Institute of Anatomy, Medical College, Qingdao University, China from March 2006 to March 2007. MATERIALS： Rabbit anti-rat HIF-1α monoclonal antibody and biotinylated goat anti-rabbit IgG （Boster, China）, rabbit anti-rat erythropoietin monoclonal antibody （Santa Cruz Biotechnology, USA）, and one-step RT-PCR kit （Qiagen, Germany） were used in this study. METHODS： A total of 99 healthy, male, Wistar rats were randomly assigned to three groups： sham surgery （n = 9）, non-ischemic preconditioning （n = 45）, and ischemic preconditioning （n = 45）. In the ischemic preconditioning group, rat models of pre-ischemia-reperfusion-ischemia-reperfusion were established by occluding the left middle cerebral artery using the twice suture method. In the non-ischemic preconditioning group, pre-ischemia was replaced by sham surgery. Subsequently, the ischemic preconditioning and non-ischemic preconditioning groups were equally divided into five subgroups according to time of first reperfusion, including 1-, 3-, 7-, 14-, and 21-day subgroups. The sham surgery group received the sham surgery twice. MAIN OUTCOME MEASURES： HIF-la and erythropoietin protein expression was measured in the cerebral cortex, corpus striatum, and hippocampus of the ischemic hemisphere. HIF-1α and erythropoietin mRNA expression were determined in the frontal and parietal cortex of the ischemic hemisphere. RESULTS： （1） Intergroup comparison： compared with the non-ischemic preconditioning group, HIF-1α protein expression significantly increased in the rat cerebral cortex, corpus striatum, and hippocampus in the ischemic hemisphere at 1,3, and 7 days following reperfusion in the ischemic preconditioning group （P 〈 0.05 or P 〈 0.01）. Erythropoietin protein expression significantly increased in the cerebral cortex, corpus striatum, and hippocampus, as well as HIF-1α and erythropoietin mRNA expression in the frontal and parietal cortex in the ischemic hemisphere, at 3 and 7 days following reperfusion in the ischemic preconditioning group （P 〈 0.05）. （2） Temporal expression： HIF-1α protein expression in the rat cerebral cortex, corpus striatum, and hippocampus, as well as HIF-la mRNA expression in the frontal and parietal cortex, in the ischemic hemisphere increased at 3 days, and gradually decreased from 7 days following reperfusion in the ischemic preconditioning group. Temporal erythropoietin protein and mRNA expression was consistent with HIF-1α protein expression. （3） Correlation： erythropoietin mRNA expression positively correlated with HIF-1α mRNA expression （r= 0.737, P 〈 0.01）. CONCLUSION： Ischemic preconditioning induced cerebral ischemic tolerance. Pre-ischemiainduced increase in endogenous HIF-1αexpression, as well as its target gene erythropoietin, participated in the formation of cerebral ischemic tolerance.
基金the National Natural Science Foundation of China,No. 30870849,81071068the Science and Technology Planning Project of Guangdong Province,No. 2009B030801101
文摘Previous studies of integrin αvβ3 have focused on ischemic brain damage, although the role of integrin αvβ3 in ischemic preconditioning （IP） has rarely been reported. The present study analyzed the effects of IP on integrin αvβ3 mRNA expression following cerebral ischemia through the use of hematoxylin-eosin staining and real-time quantitative polymerase chain reaction techniques. Integrin avid3 mRNA expression in the ischemia group peaked at 24 hours after ischemia-reperfusion. In the IP ＋ ischemia group, integrin αvβ3 mRNA expression increased after 24 hours, but remained significantly less than the ischemia group, and expression continued to increase until 7 days after ischemiaJreperfusion. These results demonstrate that IP effectively attenuated upregulation of integrin αvβ3 mRNA expression at 24 hours after ischemia.
基金supported by a grant from the Guangxi Administration of Traditional Chinese Medicine,No.GZZC0962
文摘The astrocyte is a critical regulator of neuronal survival after ischemic brain injury. Electroacupuncture may be an effective therapy for cerebral ischemia, as electroacupuncture frequency can affect the structural integdty of astrocytes. In this study, a rat model of middle cerebral artery occlusion established using the modified thread embolism method was treated with electroacupuncture of the bilateral Quchi （Llll） and Zusanfi （ST36） at 15, 30, and 100 Hz frequencies. Behavioral testing, immunohistochemistry and electron microscopy were used to explore the effect of these electroacupuncture frequencies used on maintaining the structural integrity of ischemic brain tissue. Compared with the model and 100 Hz electroacupuncture groups the 15 and 30 Hz electroacupuncture groups displayed decreased neurological deficit scores, as evaluated by the ＂Longa＂ method, significantly increased glial fibrillary acidic protein expression, and alleviated ultrastructural damage of astrocytes at the edge of the infarct. Our experimental findings indicate that 15 and 30 Hz electroacupuncture intervention can favorably maintain the structural integrity of astrocytes and play a protective role in cerebral ischemic injury. Astrocyte structural integrity may be the mechanism underlying acupuncture production of ischemic tolerance
文摘Hypoxia-inducible factor 1 （HIF-1） can lead to the adaptative reaction of body for hypoxia and ischemia. HIF-1 plays an important role in the response of ischemia-hypoxia. At present, there has been no overall report on the significance for the expression of HIF-1 following experimental cerebral ischemia. OBJECTIVE： To observe the expression of HIF-1 after middle cerebral artery occlusion （MCAO） by immunohistochemical method. DESIGN： Completely randomly grouped controlled animal experiment. SETTING： Second Hospital, Xi＇an Jiaotong University. MATERIALS： Thirty-six Sprague-Dawley healthy male rats, with body mass of 250 - 330 g, were used in this study. Thirty-six rats were randomized into 3 groups： preischemia group, sham-operation group and control group, with 12 rats in each. METHODS： This study was carried out in the clinical laboratory, People＇s Hospital of Ningjin County of Shandong Province from March 2006 to January 2007. Rats in the pre-ischemia group were created into preischemia models by two embolisms twice. Three days after ischemic preconditioning, middle cerebral artery （MCA） was occluded for 2 hours with the same method. After being perfused for 22 hours, the rats were euthanized. In the sham-operation group, rats were not given the treatment of preischemia. In the first operation, only common carotid artery （CCA） and its crotch were exposed in the first operation, and MCA was not blocked by inserting embolism. At postoperative 3 days, rats were euthanized after being subjected to MCAO for 2 hours and reperfusion 22 hours by the same procedure as that in the preischemia group. As for each rat in the control group, only CCA and its crotch were exposed, and no any other treatment was carried out on them. MAIN OUTCOME MEASURES： Brain tissue of each rat was performed immunohistochemical staining at reperfusion 22 hours after preischemia, HIF-1 expression and brain infarct volume were detected. RESULTS： Thirty-six Sprague-Dawley rats were involved in the experiment. During the experiment, 8 rats dropped out, and another 8 rats were supplemented. The infarct volume of rats in the preischemia group was significantly smaller than that in the sham-operation group （t=3.22, P 〈 0.01 ） . HIF-1 expression was not found in the control group, but many HIF-I positive cells were found in the other two groups. Absorbance in the preischemia group was significantly higher than that in the sham-operation group （t=4.31, P 〈 0.01）. CONCLUSION： Slight ischemia caused preconditioning can increase HIF-1 content, and it is one of protective mechanisms for nerve cells.
基金supported by grants from the National Natural Science Foundation of China,No.81071068the Israel Science Foundation-the National Natural Science Foundation of China(Joint Program),No.813111290the Natural Science Foundation of Guangdong Province in China,No.2014A030313172
文摘In this study, we hypothesized that an increase in integrin αβand its co-activator vascular endothelial growth factor play important neuroprotective roles in ischemic injury. We performed ischemic preconditioning with bilateral common carotid artery occlusion for 5 minutes in C57BL/6J mice. This was followed by ischemic injury with bilateral common carotid artery occlusion for 30 minutes. The time interval between ischemic preconditioning and lethal ischemia was 48 hours. Histopathological analysis showed that ischemic preconditioning substantially diminished damage to neurons in the hippocampus 7 days after ischemia. Evans Blue dye assay showed that ischemic preconditioning reduced damage to the blood-brain barrier 24 hours after ischemia. This demonstrates the neuroprotective effect of ischemic preconditioning. Western blot assay revealed a significant reduction in protein levels of integrin αβ, vascular endothelial growth factor and its receptor in mice given ischemic preconditioning compared with mice not given ischemic preconditioning 24 hours after ischemia. These findings suggest that the neuroprotective effect of ischemic preconditioning is associated with lower integrin αβand vascular endothelial growth factor levels in the brain following ischemia.
