Ca~（2+）/cbl-b/AKT通路在脑缺血预处理保护机制中的作用研究
摘要
在中国,缺血性脑血管病的发病率排名世界第一,比美国高出一倍。脑卒中已经升为中国第一位死因,死亡率高于欧美国家的4-5倍。我国存活的脑卒中患者中,约有3/4不同程度地丧失了劳动能力,其中重度致残者约占40%,存在偏瘫、失语、智能低下、生活不能自理等残疾,严重影响患者的生存质量。急性脑卒中的治疗目标为限制缺血的严重性及减少缺血持续的时间,以保存缺血半暗带的低灌注区的面积,缺血瀑布理论为急性期的干预提供了治疗靶点。但急性期的治疗干预效果有限,要根本解决脑血管病造成的脑损伤,就要从脑细胞对缺血应激损伤的适应机制上干预治疗。
在本研究中,采用了大脑中动脉栓塞法制作了SD大鼠脑缺血预处理(cereb- ral ischemic preconditioning,CIPC)模型,并给予Ca~(2+)调节剂的干预,观测了各组大鼠海马神经元内Ca~(2+)浓度的变化,检测各组大鼠海马神经元的凋亡情况。评价了神经功能缺损评分及脑组织的梗死体积;应用Western blotting检测Ca~(2+)信号传导通路上的神经钙调磷酸酶(calcineurin,CaN)、B系淋巴瘤-b(Casitas B-cell lineage lymphoma -b,cbl-b)及磷酸化蛋白激酶B(phosphorylated proteion kinasep B,p-AKT)的蛋白表达,RT-PCR检测cbl-b的转录。以进一步探讨Ca~(2+)信号传导通路在CIPC中的作用机制,及脑神经保护的机制。
目的: 1.SD大鼠实验模型制备采用的是局灶性大脑中动脉栓塞法,在本实验中探讨
了实验方法的改进,以提高实验的成功率。
2.采用TUNEL凋亡检测法检测各组大鼠海马神经元的凋亡情况,以明确经CIPC及Ca~(2+)调节剂干预后神经元凋亡的情况。
3.应用激光扫描共聚焦显微镜检测各组大鼠海马神经元内Ca~(2+)浓度变化情况,已明确CIPC及Ca~(2+)调节剂干预后神经元内Ca~(2+)的浓度变化。
4.评价各组大鼠神经功能缺损评分及脑梗死体积,以明确CIPC及各种Ca~(2+)调节剂对大鼠神经功能缺损及脑梗死体积的影响。
5.应用Western Blot技术检测海马神经元内凋亡相关蛋白CaN、cbl-b及AKT、p-AKT的蛋白表达情况,进一步探讨凋亡相关蛋白在CIPC中的表达变化情况及如何调整神经元的存亡。
6.采用RT-PCR技术检测海马神经元内cbl-b的转录变化情况,进一步探讨CIPC对神经元的凋亡的干预是否是从转录水平参与调控的。
方法:
第一部分:大鼠大脑中动脉栓塞(the occlusion of cerebral middle artery, MCAO)模型制备的改进
参考Koizumi线栓方法及焦卓敏局灶缺血模型的方法,做出以下几个方面的调整:1)采用颈部正中偏右侧的手术切口,2)根据大鼠的体重为200-250g,选择的线栓直径为0.26mm,3)应用统一购买的成品线栓,制备CIPC模型,脑缺血模型及假手术模型,并依据是否出现Hornor征、bederson神经功能缺损评分及TTC染色判定模型制备是否成功。
第二部分:各组大鼠海马神经元内游离Ca~(2+)及凋亡神经元的检测
1.试验动物分组雄性SD大鼠60只,随机分为假手术组、脑缺血组、CIPC组、尼莫地平+CIPC组(尼莫地平组)组、MK801+CIPC组(MK801组),共5组。
2.采用寡核苷酸末端脱氧核糖转移酶(TdT)介导的dUTP缺口末端标记法(TUNEL)检测各组大鼠右侧海马凋亡阳性神经元。
3.各组大鼠在预定时间断头取脑后,经分离海马神经元、荧光标记、激光扫描共聚焦显微镜检测,计算出不同组大鼠的海马神经元内游离Ca~(2+)平均荧光像素值,进行海马神经元游离Ca~(2+)的检测。
第三部分:各组大鼠海马神经元内CaN、cbl-b、AKT及p-AKT的蛋白表达
1.雄性健康SD大鼠108只,随机分为假手术组、脑缺血组、CIPC组、尼莫地平+CIPC组(尼莫地平组)组、MK801+CIPC组(MK801组)、环孢素A+CIPC组(环孢素A组)组共6组,每组18只。
2.采用bederson法评价各组大鼠的神经功能缺损情况及应用TTC染色进行脑梗死体积比较。
3.各组大鼠在预定时间点断头取脑,采用RT-PCR及Western Blot检测cbl-b的转录及CaN、cbl-b、AKT及p-AKT的蛋白表达。
结果:
第一部分
1.各组大鼠神经功能缺损评分假手术组神经功能缺损评分均为0分。脑缺血组神经功能缺损评分最高,与脑缺血组比较,CIPC组评分降低明显(P<0.05)。
2.各组大鼠脑梗死体积假手术组基本无脑梗死病灶。脑缺血组脑梗死体积最大,CIPC组梗死体积减小明显(P<0.05)。
第二部分
1.神经元内的游离Ca~(2+)的变化情况缺血组及MK801组大鼠的海马神经元相对荧光像素值最高,两组间差异无统计学意义(P>0.05);与脑缺血组比较,CIPC组的相对像素值明显减低(P<0.05);与CIPC组比较,尼莫地平组的相对像素值降低(P<0.05)。
2.神经元的凋亡情况假手术组神经元凋亡最少;缺血组及MK801组凋亡的神经元数最多,两组间差异无统计学意义(P>0.05);CIPC组的神经元凋亡数明显减少,与缺血组比较差异有统计学意义(P<0.05);尼莫地平组的海马神经元凋亡较CIPC组明显减少,差别有显著的统计学意义(P<0.01)。
第三部分
1.神经功能缺损评分脑缺血组及MK801组神经功能缺损评分最高,与脑缺血组比较, CIPC组、尼莫地平组及环孢素A组评分降低明显,差异有统计学意义(P<0.05,P<0.01);尼莫地平组与环孢素A组评分最低,与CIPC组比较,差异均有统计学意义(P<0.05);但环孢素A组与尼莫地平组比较,差异无统计学意义(P>0.05)。
2.脑梗死体积比较脑缺血组及MK801组脑梗死体积最大,CIPC组、尼莫地平组及环孢素A组梗死体积减小明显,与脑缺血组比较,差异有统计学意义(P<0.05,P<0.01);尼莫地平组与环孢素A组梗死体积最小,与CIPC组比较,差异有统计学意义(P<0.01),但环孢素A组与尼莫地平组比较,差异无统计学意义(P>0.05)。
3.CaN、cbl-b、AKT及p-AKT的蛋白表达假手术组大鼠CaN、cbl-b蛋白表达极少,与假手术组比较,其他各组大鼠CaN、cbl-b蛋白表达明显升高,差异有统计学意义(P<0.01)。脑缺血组及MK801组大鼠CaN、cbl-b蛋白表达最高,与脑缺血组比较,CIPC组明显降低,差异有统计学意义(P<0.05)。与CIPC组比较,尼莫地平组及环孢素A组降低明显,差异有统计学意义(P<0.05,P<0.01)。但尼莫地平组与环孢素A组比较,差异没有统计学意义(P>0.05)。在各组中,AKT的蛋白表达没有差异。脑缺血组的p-AKT的蛋白表达最低,MK801组与脑缺血组比较,差异没有统计学意义(P>0.05)。CIPC组、尼莫地平组及环孢素A组的p-AKT蛋白表达增高,与脑缺血组比较,差异有统计学意义(P<0.05,P<0.01)。尼莫地平组及环孢素A组的p-AKT蛋白表达明显升高,与CIPC组比较,差异有统计学意义(P<0.05,P<0.01)。但尼莫地平组与环孢素A组比较,差异没有统计学意义(P>0.05)。
4.cbl-b mRNA转录的变化脑缺血组及MK801组均增加了cbl-b的mRNA水平的转录,两组间差异无统计学意义(P>0.05);在脑缺血前给予亚致死量的CIPC,降低了cbl-b的转录,较脑缺血组,差异有统计学意义(P <0.05);尼莫地平组及环孢素A组更加降低了cbl-b在mRNA水平的表达,与CIPC组比较,差异有统计学意义(P<0.05);但与尼莫地平组比较,没有转录水平的差异(P>0.05)。
结论:
第一部分
1.采用颈部正中偏右侧手术切口,线栓直径调整为0.26mm,统一购买线栓明显缩短了手术的时间,提高了线栓介入的成功率,减小了大鼠损伤率,提高了MCAO模型制备成功率。
第二部分
1.CIPC可适量增加神经元内Ca~(2+)浓度,减少海马神经元的凋亡,产生神经保护作用。
2.CIPC前给予MK801,抵消了CIPC的保护作用,最终使海马神经元内Ca~(2+)浓度升高,增加了海马神经元的凋亡。
3.CIPC前给予尼莫地平可明显降低神经元内Ca~(2+)浓度,减轻海马神经元的凋亡,对神经保护有叠加作用。
第三部分
1.持续性脑缺血时,细胞内的过量Ca~(2+)作用于钙调蛋白,增加了CaN的表达,在核内促进了cbl-b基因的转录,增加了cbl-b的表达,增强的cbl-b泛素化了PI3K的P85亚基,使PI3K的含量降低,减少了AKT的磷酸化表达,促进了凋亡的发生。
2.CIPC抑制了这一过程,减少了CaN及cbl-b的表达,增加了p-AKT的表达,减少神经元的凋亡。
3.尼莫地平通过降低神经元内Ca~(2+)浓度,环孢素A抑制了CaN的活性,进一步减少CaN及cbl-b的表达,增加p-AKT的表达。
4.MK801抵消了CIPC的神经保护作用,使其在脑缺血时达到了钙超载,增加CaN及cbl-b的表达,抑制p-AKT的表达,促进了细胞的凋亡。
There is the highest incidence of ischemic cerebrovascular disease in china. Stroke has been the first cause of death. The death rate of stroke is higher 4 or 5 multiples than that in the united states. The number of disability to work amount to threes of forth in all strokers in our country,which may be hemiplegia, aphasia, intelligence is low, the life cannot provide for oneself, etc .The object of treatment of stroke in the acute period limit the seriousness of ischemia and reduce the duration of the ischemia. But the effect of the intervention in the acute period is dissatisfied. In order to less the injury of brain tissue, we should intervene the adaptation mechanism of ischemic stress damage. Cerebral ischemic preconditioning (CIPC) is a phenomenon in which the brain protects itself against lethal injury by adapting to sub-lethal insults. CIPC can decrease neuron injury and promote neuron survival.
We established rat models with the technique of the occlusion of middle cerebral artery , The concentration of Ca~(2+) in the hippocampal neurons of the each group were detected by relative fluorescent value(using the Fluo-3/AM as the fluorescent indicator) under the confocal laser scanning microscope. The apoptotic neurons was detected by TUNEL apoptotic detection assay. We discussed expression changes in CaN, cbl-b and p-AKT after CIPC or pretreatment with a Ca~(2+)-regulator,such as nimodipine, MK801 or cyclosporine A before CIPC. We also discussed a possible transduction pathway of Ca~(2+).
Objective:
1.We established rat models with the technique of the occlusion of middle cerebral artery, and we improved the operation method, so as to raise the success rateof rat models in our study.
2.The neurons apoptosis was detected by TUNEL apoptosis assay, so as to understand the effect of CIPC and Ca~(2+)-regulator on neurons apoptosis.
3.The concentration of Ca~(2+) in the hippocampal neurons of the each group were detected by relative fluorescent value(using the Fluo-3/AM as the fluorescent indicator) under the confocal laser scanning microscope, so as to understand the effect of CIPC and Ca~(2+)-regulator on the concentration of Ca~(2+) in the hippocampal neurons.
4.The neurological deficit scores and volume of the cerebral infarction were assessed, so as to understand the effect of CIPC and Ca~(2+)-regulator .
5.The expression of CaN, cbl-b and p-AKT by Western blotting assay were detected, so we can know the change of apoptotic protein in CIPC and pretreatment of Ca~(2+)-regulator.
6.The transcription of cbl-b in hippocampus neurons by RT-PCR was detected ,we can understand weather cbl-b regulate apoptosis from the transcription level of cbl-b.
Methods:
Part one:The improvement of MCAO rat model According to the technique of Koizumi and jiao zuo min, we established the rat models and changed the following :1) the partial right by the neck of incision;2)the diameter of the fish line is 0.26mm,according to the weight of rat;3)we used the purchased fish lines. The rat model of CIPC,sham and ischemic groups were established. We judged the successful model with Horner syndrome, bederson neurologic deficits score and TTC staining method.
Part two: The detection of apoptosis neurons and the relative fluorescent value of intracellular free calcium in hippocampus neurons in each group.
1.The sham,cerebral ischemia,CIPC,nimodipine-pretreated,MK801- pretreated models were established . The apoptosis of the hippocampus neurons was detected in each group with TUNEL apoptosis detection kit.
2.The brain tissue in all groups were removed at the scheduled time, then the hippocampus neurons were separated, the fluorescence was marked, and the intensity of fluorescence was detected by relative fluorescent value under the confocal laser scanning microscope.
Part three:Effect of CIPC and intervention of Ca~(2+)-regulated factors on CaN, cbl-b and p-AKT expression in neurons.
We established rat models including sham, ischemia, CIPC, nimodipine, MK801 and cyclosporine A. The neurological deficit scores were processed. The right hippocampus was removed and stained with TTC, and the volume of cerebral infarction was calculated. The expressions of CaN, cbl-b and p-AKT at the protein level were examined by Western blotting, and the transcription of cbl-b by RT-PCR, respectively.
Results:
Part one:The judgement of the success of rats model
1.The neurological deficit scores in three groups: The scores in the sham group were 0. There were the highest scores in the ischemia group, the scores in CIPC group were lower than those in the ischemia group(P<0.05).
2.The volume of the cerebral infarction in three groups: The volume of the cerebral infarction in the sham group were 0. There were the largest volume in the ischemia group, the volumes of the cerebral infarction in CIPC group were less than those in the ischemia group(P<0.05).
Part two:
1.Apoptotic neurons: Apoptotic neurons were most in the ischemia and MK801 groups, and there was no difference between these two groups(P>0.05);Apoptotic neurons in the CIPC groups were less than those in the ischemia group(P<0.05);and were much less in the nimodipine group than those in the CIPC group(P<0.01 ).
2.The relative fluorescent values of free calcium in hippocampus neurons: The relative fluorescent values of free calcium in hippocampus neurons were highest in the ischemia and MK801 groups, and there was no difference between these two groups(P>0.05);The relative fluorescent values in the CIPC groups were lower than those in the ischemia group(P<0.05);and were much lower in the nimodipine group than those in the CIPC group(P<0.05 ).
Part three:
1.The neurological deficit scores: the neurological deficit scores were the highest in the ischemia and MK801 groups, there were no difference between the two groups(P>0.05); this factors in CIPC group were lower than those in the ischemia group(P<0.05); and much lower in the nimodipine and cyclosporine A group than those in the CIPC group (P<0.05), but no significant difference existed between the nimodipine and cyclosporine A groups(P>0.05).
2.The volume of the cerebral infarction: The volumes of the cerebral infarction were the largest in the ischemia and MK801 groups, there were no difference between the two groups(P>0.05); this factor in CIPC group was smaller than that in the ischemia group(P<0.05); and much smaller in the nimodipine and cyclosporine A group than that in the CIPC group (P<0.01), but no significant difference existed between the nimodipine and cyclosporine A groups(P>0.05).
3.The protein expression of CaN, cbl-b and p-AKT: The protein expression of CaN, cbl-b were high in the ischemia and MK801 groups, there were no difference between the two groups(P>0.05); these factors in CIPC group were lower than those in the ischemia group(P<0.05); and much lower in the nimodipine and cyclosporine A group than those in the CIPC group (the expression of CaN in nimodipine group P<0.01, others were P<0.05), but no significant difference existed between the nimodipine and cyclosporine A groups(P>0.05). The expression of AKT in all groups was alike, the expression of p-AKT was the lowest in the ischemia and MK801 groups, and there was no difference between the two groups (P>0.05), This factor was higher in CIPC group than that in the ischemia group (P<0.05); it was the highest in the nimodipine and cyclosporine A groups among these groups (the nimodipine group P<0.01, the cyclosporine A group P<0.05), no significant difference existed between the nimodipine and cyclosporine A groups (P>0.05).
4.The transcription of cbl-b:The transcription of cbl-b were the highest in the ischemia and MK801 groups, there were no difference between the two groups(P>0.05); this factor in CIPC group were lower than that in the ischemia group(P<0.05); and much lower in the nimodipine and cyclosporine A group than that in the CIPC group (P<0.05), but no significant difference existed between the nimodipine and cyclosporine A groups(P>0.05).
Conclusions
1.The improvement of the operation in rat models brings about the following advantages:shorten operation time, increasing embolism rate, decreasing rats damage ratio,and improving the success rate of MCAO models .
2.CIPC can moderately increase the free calcium in the neurons, decreasing the apoptosis of the hippocampus neurons, and protecting the neurons from ischemic damage, inducing the cerebral ischemic tolerance;MK801 can increase free calcium in the neurons, raising the apoptotic neurons in the hippocampus, blocking neuroprotection. Nimodipine can reduce the concentration of the free calcium in the hippocampus neurons, decreasing the apoptosis of the neurons, increasing neuroprotection.
3.During the continuous ischemic period, calcium overload increases the expression of CaN by the activation of calmodulin, then promoting the transcription and the protein expression of cbl-b in the neuron nucleus. Cbl-b reduces the content of PI3K, decreasing the phosphorylated expression of AKT through ubiquitinating the subunit p85 of PI3K, ultimately activating apoptosis. CIPC inhibits above process and reduces the expression of CaN and cbl-b, and increases the expression of p-AKT, thereby inhibiting apoptosis in neurons. Nimodipine reduce the concentration of free calcium in the neurons, and cyclosporine A inhibit the activity of CaN, which can reduce the expression of CaN and cbl-b, and increase the expression of p-AKT,leading to neuroprotection. MK801 counteracts the effect of CIPC by getting to calcium overload during the ischemic period, and increasing the expression of CaN and cbl-b as the ischemic process, then inhibiting the the phosphorylated expression of AKT ,promoting the apoptosis of neurons.
在本研究中,采用了大脑中动脉栓塞法制作了SD大鼠脑缺血预处理(cereb- ral ischemic preconditioning,CIPC)模型,并给予Ca~(2+)调节剂的干预,观测了各组大鼠海马神经元内Ca~(2+)浓度的变化,检测各组大鼠海马神经元的凋亡情况。评价了神经功能缺损评分及脑组织的梗死体积;应用Western blotting检测Ca~(2+)信号传导通路上的神经钙调磷酸酶(calcineurin,CaN)、B系淋巴瘤-b(Casitas B-cell lineage lymphoma -b,cbl-b)及磷酸化蛋白激酶B(phosphorylated proteion kinasep B,p-AKT)的蛋白表达,RT-PCR检测cbl-b的转录。以进一步探讨Ca~(2+)信号传导通路在CIPC中的作用机制,及脑神经保护的机制。
目的: 1.SD大鼠实验模型制备采用的是局灶性大脑中动脉栓塞法,在本实验中探讨
了实验方法的改进,以提高实验的成功率。
2.采用TUNEL凋亡检测法检测各组大鼠海马神经元的凋亡情况,以明确经CIPC及Ca~(2+)调节剂干预后神经元凋亡的情况。
3.应用激光扫描共聚焦显微镜检测各组大鼠海马神经元内Ca~(2+)浓度变化情况,已明确CIPC及Ca~(2+)调节剂干预后神经元内Ca~(2+)的浓度变化。
4.评价各组大鼠神经功能缺损评分及脑梗死体积,以明确CIPC及各种Ca~(2+)调节剂对大鼠神经功能缺损及脑梗死体积的影响。
5.应用Western Blot技术检测海马神经元内凋亡相关蛋白CaN、cbl-b及AKT、p-AKT的蛋白表达情况,进一步探讨凋亡相关蛋白在CIPC中的表达变化情况及如何调整神经元的存亡。
6.采用RT-PCR技术检测海马神经元内cbl-b的转录变化情况,进一步探讨CIPC对神经元的凋亡的干预是否是从转录水平参与调控的。
方法:
第一部分:大鼠大脑中动脉栓塞(the occlusion of cerebral middle artery, MCAO)模型制备的改进
参考Koizumi线栓方法及焦卓敏局灶缺血模型的方法,做出以下几个方面的调整:1)采用颈部正中偏右侧的手术切口,2)根据大鼠的体重为200-250g,选择的线栓直径为0.26mm,3)应用统一购买的成品线栓,制备CIPC模型,脑缺血模型及假手术模型,并依据是否出现Hornor征、bederson神经功能缺损评分及TTC染色判定模型制备是否成功。
第二部分:各组大鼠海马神经元内游离Ca~(2+)及凋亡神经元的检测
1.试验动物分组雄性SD大鼠60只,随机分为假手术组、脑缺血组、CIPC组、尼莫地平+CIPC组(尼莫地平组)组、MK801+CIPC组(MK801组),共5组。
2.采用寡核苷酸末端脱氧核糖转移酶(TdT)介导的dUTP缺口末端标记法(TUNEL)检测各组大鼠右侧海马凋亡阳性神经元。
3.各组大鼠在预定时间断头取脑后,经分离海马神经元、荧光标记、激光扫描共聚焦显微镜检测,计算出不同组大鼠的海马神经元内游离Ca~(2+)平均荧光像素值,进行海马神经元游离Ca~(2+)的检测。
第三部分:各组大鼠海马神经元内CaN、cbl-b、AKT及p-AKT的蛋白表达
1.雄性健康SD大鼠108只,随机分为假手术组、脑缺血组、CIPC组、尼莫地平+CIPC组(尼莫地平组)组、MK801+CIPC组(MK801组)、环孢素A+CIPC组(环孢素A组)组共6组,每组18只。
2.采用bederson法评价各组大鼠的神经功能缺损情况及应用TTC染色进行脑梗死体积比较。
3.各组大鼠在预定时间点断头取脑,采用RT-PCR及Western Blot检测cbl-b的转录及CaN、cbl-b、AKT及p-AKT的蛋白表达。
结果:
第一部分
1.各组大鼠神经功能缺损评分假手术组神经功能缺损评分均为0分。脑缺血组神经功能缺损评分最高,与脑缺血组比较,CIPC组评分降低明显(P<0.05)。
2.各组大鼠脑梗死体积假手术组基本无脑梗死病灶。脑缺血组脑梗死体积最大,CIPC组梗死体积减小明显(P<0.05)。
第二部分
1.神经元内的游离Ca~(2+)的变化情况缺血组及MK801组大鼠的海马神经元相对荧光像素值最高,两组间差异无统计学意义(P>0.05);与脑缺血组比较,CIPC组的相对像素值明显减低(P<0.05);与CIPC组比较,尼莫地平组的相对像素值降低(P<0.05)。
2.神经元的凋亡情况假手术组神经元凋亡最少;缺血组及MK801组凋亡的神经元数最多,两组间差异无统计学意义(P>0.05);CIPC组的神经元凋亡数明显减少,与缺血组比较差异有统计学意义(P<0.05);尼莫地平组的海马神经元凋亡较CIPC组明显减少,差别有显著的统计学意义(P<0.01)。
第三部分
1.神经功能缺损评分脑缺血组及MK801组神经功能缺损评分最高,与脑缺血组比较, CIPC组、尼莫地平组及环孢素A组评分降低明显,差异有统计学意义(P<0.05,P<0.01);尼莫地平组与环孢素A组评分最低,与CIPC组比较,差异均有统计学意义(P<0.05);但环孢素A组与尼莫地平组比较,差异无统计学意义(P>0.05)。
2.脑梗死体积比较脑缺血组及MK801组脑梗死体积最大,CIPC组、尼莫地平组及环孢素A组梗死体积减小明显,与脑缺血组比较,差异有统计学意义(P<0.05,P<0.01);尼莫地平组与环孢素A组梗死体积最小,与CIPC组比较,差异有统计学意义(P<0.01),但环孢素A组与尼莫地平组比较,差异无统计学意义(P>0.05)。
3.CaN、cbl-b、AKT及p-AKT的蛋白表达假手术组大鼠CaN、cbl-b蛋白表达极少,与假手术组比较,其他各组大鼠CaN、cbl-b蛋白表达明显升高,差异有统计学意义(P<0.01)。脑缺血组及MK801组大鼠CaN、cbl-b蛋白表达最高,与脑缺血组比较,CIPC组明显降低,差异有统计学意义(P<0.05)。与CIPC组比较,尼莫地平组及环孢素A组降低明显,差异有统计学意义(P<0.05,P<0.01)。但尼莫地平组与环孢素A组比较,差异没有统计学意义(P>0.05)。在各组中,AKT的蛋白表达没有差异。脑缺血组的p-AKT的蛋白表达最低,MK801组与脑缺血组比较,差异没有统计学意义(P>0.05)。CIPC组、尼莫地平组及环孢素A组的p-AKT蛋白表达增高,与脑缺血组比较,差异有统计学意义(P<0.05,P<0.01)。尼莫地平组及环孢素A组的p-AKT蛋白表达明显升高,与CIPC组比较,差异有统计学意义(P<0.05,P<0.01)。但尼莫地平组与环孢素A组比较,差异没有统计学意义(P>0.05)。
4.cbl-b mRNA转录的变化脑缺血组及MK801组均增加了cbl-b的mRNA水平的转录,两组间差异无统计学意义(P>0.05);在脑缺血前给予亚致死量的CIPC,降低了cbl-b的转录,较脑缺血组,差异有统计学意义(P <0.05);尼莫地平组及环孢素A组更加降低了cbl-b在mRNA水平的表达,与CIPC组比较,差异有统计学意义(P<0.05);但与尼莫地平组比较,没有转录水平的差异(P>0.05)。
结论:
第一部分
1.采用颈部正中偏右侧手术切口,线栓直径调整为0.26mm,统一购买线栓明显缩短了手术的时间,提高了线栓介入的成功率,减小了大鼠损伤率,提高了MCAO模型制备成功率。
第二部分
1.CIPC可适量增加神经元内Ca~(2+)浓度,减少海马神经元的凋亡,产生神经保护作用。
2.CIPC前给予MK801,抵消了CIPC的保护作用,最终使海马神经元内Ca~(2+)浓度升高,增加了海马神经元的凋亡。
3.CIPC前给予尼莫地平可明显降低神经元内Ca~(2+)浓度,减轻海马神经元的凋亡,对神经保护有叠加作用。
第三部分
1.持续性脑缺血时,细胞内的过量Ca~(2+)作用于钙调蛋白,增加了CaN的表达,在核内促进了cbl-b基因的转录,增加了cbl-b的表达,增强的cbl-b泛素化了PI3K的P85亚基,使PI3K的含量降低,减少了AKT的磷酸化表达,促进了凋亡的发生。
2.CIPC抑制了这一过程,减少了CaN及cbl-b的表达,增加了p-AKT的表达,减少神经元的凋亡。
3.尼莫地平通过降低神经元内Ca~(2+)浓度,环孢素A抑制了CaN的活性,进一步减少CaN及cbl-b的表达,增加p-AKT的表达。
4.MK801抵消了CIPC的神经保护作用,使其在脑缺血时达到了钙超载,增加CaN及cbl-b的表达,抑制p-AKT的表达,促进了细胞的凋亡。
There is the highest incidence of ischemic cerebrovascular disease in china. Stroke has been the first cause of death. The death rate of stroke is higher 4 or 5 multiples than that in the united states. The number of disability to work amount to threes of forth in all strokers in our country,which may be hemiplegia, aphasia, intelligence is low, the life cannot provide for oneself, etc .The object of treatment of stroke in the acute period limit the seriousness of ischemia and reduce the duration of the ischemia. But the effect of the intervention in the acute period is dissatisfied. In order to less the injury of brain tissue, we should intervene the adaptation mechanism of ischemic stress damage. Cerebral ischemic preconditioning (CIPC) is a phenomenon in which the brain protects itself against lethal injury by adapting to sub-lethal insults. CIPC can decrease neuron injury and promote neuron survival.
We established rat models with the technique of the occlusion of middle cerebral artery , The concentration of Ca~(2+) in the hippocampal neurons of the each group were detected by relative fluorescent value(using the Fluo-3/AM as the fluorescent indicator) under the confocal laser scanning microscope. The apoptotic neurons was detected by TUNEL apoptotic detection assay. We discussed expression changes in CaN, cbl-b and p-AKT after CIPC or pretreatment with a Ca~(2+)-regulator,such as nimodipine, MK801 or cyclosporine A before CIPC. We also discussed a possible transduction pathway of Ca~(2+).
Objective:
1.We established rat models with the technique of the occlusion of middle cerebral artery, and we improved the operation method, so as to raise the success rateof rat models in our study.
2.The neurons apoptosis was detected by TUNEL apoptosis assay, so as to understand the effect of CIPC and Ca~(2+)-regulator on neurons apoptosis.
3.The concentration of Ca~(2+) in the hippocampal neurons of the each group were detected by relative fluorescent value(using the Fluo-3/AM as the fluorescent indicator) under the confocal laser scanning microscope, so as to understand the effect of CIPC and Ca~(2+)-regulator on the concentration of Ca~(2+) in the hippocampal neurons.
4.The neurological deficit scores and volume of the cerebral infarction were assessed, so as to understand the effect of CIPC and Ca~(2+)-regulator .
5.The expression of CaN, cbl-b and p-AKT by Western blotting assay were detected, so we can know the change of apoptotic protein in CIPC and pretreatment of Ca~(2+)-regulator.
6.The transcription of cbl-b in hippocampus neurons by RT-PCR was detected ,we can understand weather cbl-b regulate apoptosis from the transcription level of cbl-b.
Methods:
Part one:The improvement of MCAO rat model According to the technique of Koizumi and jiao zuo min, we established the rat models and changed the following :1) the partial right by the neck of incision;2)the diameter of the fish line is 0.26mm,according to the weight of rat;3)we used the purchased fish lines. The rat model of CIPC,sham and ischemic groups were established. We judged the successful model with Horner syndrome, bederson neurologic deficits score and TTC staining method.
Part two: The detection of apoptosis neurons and the relative fluorescent value of intracellular free calcium in hippocampus neurons in each group.
1.The sham,cerebral ischemia,CIPC,nimodipine-pretreated,MK801- pretreated models were established . The apoptosis of the hippocampus neurons was detected in each group with TUNEL apoptosis detection kit.
2.The brain tissue in all groups were removed at the scheduled time, then the hippocampus neurons were separated, the fluorescence was marked, and the intensity of fluorescence was detected by relative fluorescent value under the confocal laser scanning microscope.
Part three:Effect of CIPC and intervention of Ca~(2+)-regulated factors on CaN, cbl-b and p-AKT expression in neurons.
We established rat models including sham, ischemia, CIPC, nimodipine, MK801 and cyclosporine A. The neurological deficit scores were processed. The right hippocampus was removed and stained with TTC, and the volume of cerebral infarction was calculated. The expressions of CaN, cbl-b and p-AKT at the protein level were examined by Western blotting, and the transcription of cbl-b by RT-PCR, respectively.
Results:
Part one:The judgement of the success of rats model
1.The neurological deficit scores in three groups: The scores in the sham group were 0. There were the highest scores in the ischemia group, the scores in CIPC group were lower than those in the ischemia group(P<0.05).
2.The volume of the cerebral infarction in three groups: The volume of the cerebral infarction in the sham group were 0. There were the largest volume in the ischemia group, the volumes of the cerebral infarction in CIPC group were less than those in the ischemia group(P<0.05).
Part two:
1.Apoptotic neurons: Apoptotic neurons were most in the ischemia and MK801 groups, and there was no difference between these two groups(P>0.05);Apoptotic neurons in the CIPC groups were less than those in the ischemia group(P<0.05);and were much less in the nimodipine group than those in the CIPC group(P<0.01 ).
2.The relative fluorescent values of free calcium in hippocampus neurons: The relative fluorescent values of free calcium in hippocampus neurons were highest in the ischemia and MK801 groups, and there was no difference between these two groups(P>0.05);The relative fluorescent values in the CIPC groups were lower than those in the ischemia group(P<0.05);and were much lower in the nimodipine group than those in the CIPC group(P<0.05 ).
Part three:
1.The neurological deficit scores: the neurological deficit scores were the highest in the ischemia and MK801 groups, there were no difference between the two groups(P>0.05); this factors in CIPC group were lower than those in the ischemia group(P<0.05); and much lower in the nimodipine and cyclosporine A group than those in the CIPC group (P<0.05), but no significant difference existed between the nimodipine and cyclosporine A groups(P>0.05).
2.The volume of the cerebral infarction: The volumes of the cerebral infarction were the largest in the ischemia and MK801 groups, there were no difference between the two groups(P>0.05); this factor in CIPC group was smaller than that in the ischemia group(P<0.05); and much smaller in the nimodipine and cyclosporine A group than that in the CIPC group (P<0.01), but no significant difference existed between the nimodipine and cyclosporine A groups(P>0.05).
3.The protein expression of CaN, cbl-b and p-AKT: The protein expression of CaN, cbl-b were high in the ischemia and MK801 groups, there were no difference between the two groups(P>0.05); these factors in CIPC group were lower than those in the ischemia group(P<0.05); and much lower in the nimodipine and cyclosporine A group than those in the CIPC group (the expression of CaN in nimodipine group P<0.01, others were P<0.05), but no significant difference existed between the nimodipine and cyclosporine A groups(P>0.05). The expression of AKT in all groups was alike, the expression of p-AKT was the lowest in the ischemia and MK801 groups, and there was no difference between the two groups (P>0.05), This factor was higher in CIPC group than that in the ischemia group (P<0.05); it was the highest in the nimodipine and cyclosporine A groups among these groups (the nimodipine group P<0.01, the cyclosporine A group P<0.05), no significant difference existed between the nimodipine and cyclosporine A groups (P>0.05).
4.The transcription of cbl-b:The transcription of cbl-b were the highest in the ischemia and MK801 groups, there were no difference between the two groups(P>0.05); this factor in CIPC group were lower than that in the ischemia group(P<0.05); and much lower in the nimodipine and cyclosporine A group than that in the CIPC group (P<0.05), but no significant difference existed between the nimodipine and cyclosporine A groups(P>0.05).
Conclusions
1.The improvement of the operation in rat models brings about the following advantages:shorten operation time, increasing embolism rate, decreasing rats damage ratio,and improving the success rate of MCAO models .
2.CIPC can moderately increase the free calcium in the neurons, decreasing the apoptosis of the hippocampus neurons, and protecting the neurons from ischemic damage, inducing the cerebral ischemic tolerance;MK801 can increase free calcium in the neurons, raising the apoptotic neurons in the hippocampus, blocking neuroprotection. Nimodipine can reduce the concentration of the free calcium in the hippocampus neurons, decreasing the apoptosis of the neurons, increasing neuroprotection.
3.During the continuous ischemic period, calcium overload increases the expression of CaN by the activation of calmodulin, then promoting the transcription and the protein expression of cbl-b in the neuron nucleus. Cbl-b reduces the content of PI3K, decreasing the phosphorylated expression of AKT through ubiquitinating the subunit p85 of PI3K, ultimately activating apoptosis. CIPC inhibits above process and reduces the expression of CaN and cbl-b, and increases the expression of p-AKT, thereby inhibiting apoptosis in neurons. Nimodipine reduce the concentration of free calcium in the neurons, and cyclosporine A inhibit the activity of CaN, which can reduce the expression of CaN and cbl-b, and increase the expression of p-AKT,leading to neuroprotection. MK801 counteracts the effect of CIPC by getting to calcium overload during the ischemic period, and increasing the expression of CaN and cbl-b as the ischemic process, then inhibiting the the phosphorylated expression of AKT ,promoting the apoptosis of neurons.
引文
1.刘亢丁,苏志强,饶明俐,等.实验性局灶性脑缺血再灌注动物模型的改进及评价中风与神经疾病杂志1997;14(2):87-89
2.马常升,马文领,戴维国,等.插线法制备大鼠局灶性脑缺血再灌注模型的研究解剖学杂志1999;22(3):209
3.徐佳,葛林宝,徐明曙,等.大鼠栓线法MACO模型中插入深度的研究上海实验动物科学2002;22(4):209-212
4.诸晓凡,董家政,吴军,等.颈内动脉线拴与环扎建立局灶性脑缺血再灌注模型中风与神经疾病杂志2000;17(3):152-154
5.Longa Z, Weinstein PR, Carson S, et al. Reversible middle cerebral artery:without craniotomy in rats Stroke 1989l;20:84-91
6.Koizumi J, Yoshid Y, Nakazawa T, et al. Experimental studies of ischemic brain edema, a new experiment model of cerebral embolism in rats in which recirculation can be introduced in the ischemia area Stroke 1986;8:1-3
7.王伟.严格控制试验条件建立标准化脑缺血动物模型中华神经科杂志1998;31(5):261
8.Bederson JB, Pitts LH, Tsuji M, et al. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination Stroke 1986; 17(3): 472-476
9.Dahl N, Balfour W. Prolonged anoxic survival due to anoxia pre-exposure: brain ATP, lactate, and pyruvate Am J Physiol 1964; 207:452-456
10.寞万臣,王任直,张波,等.大鼠局灶性脑缺血模型制作方法的改进中国脑血管病杂志2004;1(2):81-84
11.Xing BZ, Chen H, Zhang M. Ischemic postconditioning inhibits apoptosis after focal cerebral ischemia/reperfusion injury in the rat Stroke 2008; 39:2362-2369
12.闫明,王晶晶,乔欣,等. Cdk5与大鼠脑缺血再灌注损伤时细胞凋亡的关系中国实验动物学报2008;16(2):117-121
13.贾杰,胡永善,吴毅,等.跑台运动训练促进脑梗死大鼠外源性移植的神经干细胞迁移分化的实验研究中国康复医学杂志2008;23(7):594-597
1.Blckler PE, Fahlman CS. Moderate increases in intracellular calcium activate neuroprotective signals in hippocampal neurons Neuroscience 2004;127:673–683
2.Raval AP, Dave KR, Daria MR,et al. PKC is required for the induction of tolerance by ischemic and NMDA-mediated preconditioning in the organotypic hippocampal slice Neuroscience 2003;23(2):384–391
3.Koizumi J, Yoshida Y, Nakazawa T, et al. Experimental studies of ischemic brain edema: a new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area Stroke 1986;8:1-8
4.焦卓敏,赵瑞波,毕彦忠,等.大鼠脑局灶性缺血对再次缺血的影响中风与神经疾病杂志1999;16(3):138-140
5.黄勇华,张微微,郝小淑,等.钙离子与鼠脑出血继发性损伤的关系及尼莫地平的保护作用中华老年心脑血管病杂志2000;6(2):200-203
6.Mabuchi T, Kitagawa K, Kuwabara K, et al. Phosphorylation of cAMP response element-binding protein in hippocampal neurons as a protective response after exposure to glutamate in vitro and ischemia in vivo Neuroscience 2001;21(23):9204–9213
7.许建新,周跃飞,赵洪洋.创伤性脑损伤后脑神经细胞内Ca2+变化试验研究中华神经外科疾病研究杂志2009;8(1):73-74
8.Miller.RJ.Calcium signaling in neurons. Trends.Neurosci,1988,11:415- 441.
9.PE blckler, CS fahlman. Moderate increases in intracellular calcium activate neuroprotective signals in hippocampal neurons Neuroscience 2004; 127: 673–683
10.金惠铭,王建枝.病理生理学第6版231-233
11.Wong.MC, Haley EC. Jr. Calcium antagonists: stroke therapy coming of age Stroke 1990;21(3): 494-451
12.Lipton P. Regulation of glycogen in the dentate gyrus of the in vitro guinea pig hippocampus: effect of combined deprivation of glucose and oxygen J Neurosci Methods 1988;28:147–154
13.Mitani A, Yanase H, Sakai K, Wake Y, Kataoka K. Origin of intracellular Ca2+ elevation induced by in vitro ischemia-like condition in hippocampal slices Brain Res 1993;601:103–110
14.Mitani A, Yanase H, Namba S, Shudo M, Kataoka K. In vitro ischemia-induced intracellular Ca2+ elevation in cerebellar slices: a comparative study with the values found in hippocampal slices Acta Neuropathol,1995;89:2–7
15.Hoehner PJ,Blanck TJ, Roy R, Rosenthal RE, Fiskum G. Alteration of voltage-dependent calcium channels in canine brain during global ischemia and reperfusion J Cereb Blood Flow Metab 1992;12:418–424
16.Friedman JE, Haddad GG. Major differences in [Ca2+]i response to anoxiabetween neonatal and adult rat CA1 neurons: role of Ca2+ and Na+ J Neurosci 1993;13:63–72
17.Grishin AA, Gee CE, Gerber U, et al. Differential calcium-dependent modulation of NMDA currents in CA1 and CA3 hippocampal pyramidal cells Neuroscience 2004;24(2):350–355
18.Philip E, Bickler, Xinhua Z, Christian S. Fahlman. Isoflurane preconditions hippocampal neurons against oxygen–glucose deprivation role of intracellular Ca2+ and mitogen-activated protein kinase signaling anesthesiology 2005;103:532–539
19.姜长斌,丁聪,尹琳.脑缺血预处理大鼠脑IL-6和Ngb的表达及尤瑞克林对其表达的干预中国神经免疫学和神经病学杂志2009;01:46-50
20.尹琳,李芳,金萍,闵连秋.脑缺血预处理对再次脑缺血大鼠神经功能、脑含水量及脑组织中Bcl-xl、P53蛋白表达的影响中国临床康复2005(1):111-113
21.Lin CH, Chen PS, Gean PW. Glutamate preconditioning prevents neuronal death induced by combined oxygen-glucose deprivation in cultured cortical neurons Eur J Pharmacol 2008;589(3): 85-93
22. Zhang Y, Lipton PJ. Cytosolic Ca2+ changes during in vitro ischemia in rat hippocampal slices: major roles for glutamate and Na+-dependent Ca2+ release from mitochondria Neuroscience 1999;19:3307–3315
23.Wang RM, Yang F, Zhang YX. Preconditioning-induced activation of ERK5 is dependent on moderate Ca2+ influx via NMDA receptors and contributes to ischemic tolerance in the hippocampal CA1 region of rats Life Sci 2006; 4;79(19):1839-1846
24.QuanGuang Zhang, RuiMin Wang, Dong Han, LiCai Yang ,Jie Li,Darrell W. Brann.Preconditioning neuroprotection in global cerebral ischemia involves NMDA receptor-mediated ERK-JNK3 crosstalk Neurosc Res 2009;63(3):205-212
25.Miao B, Yin XH, Pei DS, et al. Neuroprotective effects of preconditioning ischemia on ischemic brain Injury through down-regulating activation of JNK1/2 via N-Methyl-D-aspartate receptor-mediated Akt1 activation Biol Chem 2005;80:21693–21699
26.Yin XH, Zhang QG, Miao B, et al. Neuroprotective effects of preconditioning ischaemia on ischaemic brain injury through inhibition of mixed-lineage kinase 3 via NMDA receptor-mediated Akt1 activation Neurochem 2005;93:1021–1029
27.吕少萍,管勇.尼莫通对脑缺血再灌注细胞凋亡相关基因表达影响青岛大学医学院学报2005;41(2):167-168
28.Wen JY, Chen ZW. Protective effect of pharmacological preconditioning of total flavones of abelmoschl manihot on cerebral ischemic reperfusion injury in rats Am J Chin Med 2007;35(4): 653-661
29.Liu C, Zhou R, Sun S.Nimodipine modulates Bcl-2 and Bax mRNA expression after cerebral ischemia Huazhong Univ Sci Technolog Med Sci 2004;24(2):170-172
30.苏静姜长斌.大鼠脑缺血预处理后线粒体通透性转换及细胞色素C水平的变化及意义中国优秀硕士学位论文全文数据库医药卫生神经病学2009:1-44
31.陈道文,周永,赵薛旭,李作汉,狄晴.大鼠脑缺血预处理再灌注模型Bcl-2和Bax的表达临床神经病学杂志2006;01:52-53
32.王晶余,徐忠信,康治臣,韩雪梅,邬英全,钱佳利.局灶脑缺血预处理缺血耐受与Bcl-xl、Bax表达的研究中国实验诊断学2005;01:40-42
33.刁尧,陈春梅,李雪娜,李亚明,高杰,蔡奎,赵恂,于成国.NF-κB、Bcl-2和Bax蛋白在大鼠脑缺血预处理脑组织中的表达及意义解剖科学进展2008;03:41-44
1.Gidday, JM. Cerebral preconditioning and ischaemic tolerance. Nat. Rev. Neurosci.2006,7(6): 437–448
2.Shamloo.M, Rytter. A, Wieloch.T. Activation of the extracellular signal-regulated protein kinase cascade in the hippocampal CA1 region in a rat model of global cerebral ischemic preconditioning Neuroscience 1999;93(1):81–88
3.Gonzalez-Zulueta.M, Feldman.AB, Klesse.LJ, Kalb.RG, Dillman. JF, Parada. LF, Dawson.TM, Dawson.VL. Requirement for nitric oxide activation of p21(ras)/extracellular regulated kinase in neuronal ischemic preconditioning Proc. Natl Acad. Sci 2000; 97(1):436–441
4.Kurkinen. K, Busto.R, Goldsteins. G, Koistinaho. J, Pérez-Pinzón. MA. Isoform-specific membrane translocation of protein kinase C after ischemic preconditioning Neurochem Res 2001;26(10):1139–1144
5.Miao.B, Yin.X., Pei.DS, Zhang.QG, Zhang.GY.Neuroprotective effects of preconditioning ischemia on ischemic brain injury through down-regulating activation of JNK1/2 via N-methyl -D-aspartate receptor-mediated Akt1 activation Biol Chem 2005 ;280(23):21693–21699
6.Chen.J, Graham.SH, Zhu.RL, Simon.RP. Stress proteins and tolerance to focal cerebral ischemia Cereb Blood Flow Metab 1996;16(4):566–577
7.Sónia.C, Correia.Cristina. C, Susana.C, Renato. X, Santos,Maria, Catarina. RO, George.P, Zhu.XW, Smith.MA, Moreira. Mitochondrial preconditioning: a potential neuroprotective strategy Front Aging Neurosci2010; 26(2):138
8.Stevens.SL, Ciesielski.TM, Marsh. BJ, Yang.T, Homen. DS.,Boule.JL, Lessov.NS, Simon.RP, Stenzel-Poore.MP. Toll-like receptor 9: a new target of ischemic preconditioning in the brain Cereb Blood Flow Metab 2008;28(5):1040- 1047
9.Goto S, Matsukado Y, Mihara Y, Inoue N, Miyamoto E.The distribution of calcineurin in rat brain by light and electron microscopic immunohistochemistry and enzyme-immunoassay Brain Res 1986;397(1):161-172
10.Subramaniam Jayanthi,Xiaolin Deng , Bruce Ladenheim. Calcineurin/NFAT-induced up-regulation of the Fas ligand/Fas death pathway is involved in methamphetam -ine-induced neuronal apoptosis PNAS 2005;102(3):868-873
11.Tan Dong Ping,Liu Qi Ying,Koshiya N,Gu H, Alkon D.Enhancement of long-term memory retention and short-term synaptic plasticity in cbl-b null mice Neuroscience 2006;103(13): 5125–5130
12.黄勇华,张微微,郝小淑. Ca2+与鼠脑出血继发性损伤的关系及尼莫地平的保护作用中华老年心脑血管病杂志2000;6(2): 200-203
13.Mabuchi T, Kitagawa K, Kuwabara K, Takasawa K, Ohtsuki T, Xia Z, Storm D, Yanagihara T, Hori M, Matsumoto M. Phosphorylation of cAMP response element-binding protein in hippocampal neurons as a protective response after exposure to glutamate in vitro and ischemia in vivo Neuroscience 2001;21(23):9204–9213
14.Yamaguchi1 T,Miyata1 K, Shibasaki F, et al. Effect of cyclosporine A on immediately gene in rat global ischemia and its neuroprotection Pharmacol Sci 2006;100:73– 81
15.Bederson JB, Pitts LH, Tsuji M, Isshiki A, Uchino H. Rat middle cerebral cnfery occlusion: evalution of the model and development of a neurologic examination Stroke 1986; 17(3): 472-476
16.Dirnagl.U,Simon.RP,Hallenbeck.JM.Ischemic tolerance and endogenous neuroprotection Trends neurosci 2003 May;26(5):248-54
17.Kapinya KJ. Ischemic tolerance in the brain Acta Physiol Hung 2005;92:67-92
18.Dirnagl U, Becker K, Meisel A. Preconditioning and tolerance against cerebral ischaemia: from experimental strategies to clinical use Lancet Neurol 2009;8:398-412
19.邓珊,秦超.缺血预处理与后处理的脑保护作用医学综述2008;(14):377-379
20.Blckler PE,Fahlman CS. Moderate increases in intracellular calcium activate neuroprotective signals in hippocampal neuron Neuroscience 2004;127:673– 668
21.朱心红,杨建明,高天明.脑心通对大鼠脑缺血海马CA1区神经元损害的保护作用第一军医大学学报2004;24(10):1123-1125
22.Jian-Feng GAO ,Jian-Sheng LI ,You-Long ZHOU ,Ke LIU .Effects of naomaitong recipe on expressions of nuclear factor-κB and nitric-oxide synthases in brain tissue after focal cerebral ischemia reperfusion in aged rats Zhong Xi Yi Jie He Xue Bao/J Chin Integr Med 2006;4(5): 530-534
23.Antonio Pisani, Paolo Calabresi, Alessandro Tozzi, Vincenza D'Angelo, Giorgio Bernardi.L-Type Ca2+ channel blockers attenuate electrical changes and Ca2+ rise induced by oxygen/glucose deprivation in cortical neurons Stroke 1998;29:196- 202
24.JoséM. Roda, Fernando Carceller, Exuperio Díez-Tejedor, Carlos Avenda?o.Reduction of Infarct size by intra-arterial nimodipine administered at reperfusion in a rat model of partially reversible brain focal ischemia Stroke 1995;26:1888-1892
25.Wen JY,Chen ZW. Protective effect of pharmacological preconditioning of total flavones of abelmoschlmanihot on cerebral ischemic reperfusion injury in rats Am J Chin Med 2007;35 (4):653-661
26.Liu C, Zhou R, Sun S.Nimodipine modulates Bcl-2 and Bax mRNA expression after cerebralischemia Huazhong Univ Sci Technolog Med Sci 2004;24(2):170-172
27.Steiner JP,Dawson TM,Fotuhi M,et al.Snyder High brain densities of the immunophilin FKBP colocalized with calcineurin Nature 1992;358:584-587
28.Bond A, Lodge D, Hicks CA, Ward MA, O'Neill MJ.NMDA receptor antagonism, but not AMPA receptor antagonism attenuates induced ischaemic tolerance in the gerbil hippocampus Eur J Pharmacol 1999 ;10;380(3):91-99
29.Lin CH, Chen PS, Gean PW. Glutamate preconditioning prevents neuronal death induced by combined oxygen-glucose deprivation in cultured cortical neurons Eur J Pharmacol 2008; 589(1-3):85-93
30.Takuma Mabuchi, Kazuo Kitagawa, Keisuke Kuwabara, Kenichiro Takasawa, Toshiho Ohtsuki,Zhengui Xia, Daniel Storm, Takehiko Yanagihara, Masatsugu Hori, Masayasu Matsumoto. Phosphorylation of cAMP Response Element-Binding Protein in Hippocampal Neurons as a Protective Response after Exposure to Glutamate In Vitro and Ischemia In Vivo The Journal of Neuroscience 2001;21(23):9204–9213
31.Fix AS, Horn JW, Wightman KA, et al. Neuronal vacuolization and necrosis induced by the noncompetitive N-methyl-D-aspartate (NMDA) antagonist MK801 (dizocilpine maleate): a light and electron microscopic evaluation of the rat retrosplenial cortex Exp Neurol 1993 ;123(2):204-215
32.Lipton SA. Failures and successes of NMDA receptor antagonists:molecular basis for the use of open-channel blockers like memantine in the treatment of acute and chronic neurologic insults NeuroRx 2004 ;1(1):101-110.
33.Wang RM, Yang F, Zhang YX.Preconditioning-induced activation of ERK5 is dependent on moderate Ca2+ influx via NMDA receptors and contributes to ischemic tolerance in the hippocampal CA1 region of rats Life Sci 2006 ;79(19):1839-1846
34.Okamura H, Aramburu J, García-Rodríguez C,et al. Concerted dephosphorylation of the transcription factor NFAT1 induces a conformational switch that regulates transcriptional activity mol cell 2000;6(3):539-550
35.Aramburu J, Rao A. Klee C. B.Calcineurin: from structure to function Curr Top Cell Regul 2000; 36, 237–295
36.Klee CB, Ren H , Wang X.Regulation of the Calmodulin stimulated Protein phosphatase, Calcineurin Biol Chem 1998;273, 13367–13370
37.Wang HG, Pathan N, Ethell IM, et al. Ca2+-induced apoptosis through calcineurin dephosphorylation of BADScience 1999;284:339–343
38.Norifumi Shioda, Shigeki Moriguchi, Yasufumi Shirasaki, Kohji Fukunaga. Generation ofconstitutively active calcineurin by calpain contributes to delayed neuronal death following mouse brain ischemia Neurochem 2006; 98:310–320
39.ZHANG Yingjun , MEI Heshan , WANG Chuan , WANG Yongli, ZHANG Yongjian. Involvement of nuclear factor of activated T-cells ( NFATc) in calc- ineurin mediated ischemic brain damage in vivo Acta Pharmaceutica Sinica 2005; 40 (4) : 299– 305
40.Akio Asai, Jian-hua Qiu, Yoshitaka Narita, Shunji Chi, Nobuhito Saito, Nobusada Shinoura, Hirofumi Hamada, Yoshiyuki Kuchino, Takaaki Kirino. High levelcalcineurin activity predisposes neuronal cells to apoptosis Biol Chem 1999; 274( 48):34450-34458
41.Yan Shou, Li Li Krishnan Prabhakaran, Joseph L. Borowitz Gary E. Isom,Calcineurin-mediated Bad translocation regulates cyanide-induced neuronal apoptosis Biochem 2004;379:805-813
42.Shibasaki F, Kondo E, Akagi T McKeon F. Suppression of signaling through transcription factor NFAT by interactions between calcineurin and Bcl-2 Nature (London)1997; 386: 728-731
43.Shitashige M, Tou M, Yano T, Shibata M, Matsuo Y and Shibasaki F. Dissociation of Bax from a Bcl-2/Bax heterodimer triggered by phosphorylation of serine 70 of Bcl-2 Biochem 2001;130:741-748
44.Andrew A, Sproul, Zhiheng Xu, Michael Wilhelm, Stephen Gire, Lloyd A. Greene.Cbl negatively regulates JNK activation and cell death Cell Res 2009; 19(8): 950–961
45.Widmann C, Gibson S, Johnson GL. Caspase-dependent cleavage of signaling proteins during apoptosis A turn-off mechanism for anti-apoptotic signals Biol Chem 1998;273(12):7141-7147
46.ZR Zhou, HC Gao,CJ Zhou, YG Chang, J Hong, AX Song, DH Lin, HY Hu.Differential ubiquitin binding of the UBA domainsfrom human c-Cbl and Cbl-b: NMR structural and biochemical insights Protein Science 2008;17:1805–1814
47.DY Fang,YC Liu.Proteolysis-independent regulation of PI3K by Cbl-b?mediated ubiquitination in T cell Nat Immun 2001; 2: 870 - 875
48.Hashiguchi A, Yano S, Morioka M, Hamada J, Ushio Y, Takeuchi Y, Fukunaga K. Upregulation of endothelial nitric oxide synthase via phosphatidylinositol 3-kinase pathway contributes to ischemic tolerance in the CA1 subfield of gerbil hippocampus Cereb Blood Flow Metab 2004; 24(3):271-279
49.Yano S, Morioka M, Fukunaga K, Kawano T, Hara T, Kai Y, Hamada J, Miyamoto E, Ushio Y.Activation of Akt/protein kinase B contributes to induction of ischemic tolerance in the CA1 subfield of gerbil hippocampus Cereb Blood Flow Metab 2001;21(4):351-360
50.Nakajima T, Iwabuchi S, Miyazaki H, Okuma Y, Kuwabara M, Nomura Y, Kawahara K. Preconditioning prevents ischemia-induced neuronal death through persistent Akt activation in the penumbra region of the rat brain Vet Med Sci 2004 ;66(5):521-527
51.Johnnides, Hickey RW,Jun Chen, Wei Yin,et al. Preconditioning suppresses Inflammation in neonatal hypoxic ischemia via Akt activation Stroke 2007;38:1017-1024
52.Hillion JA, Li YiXin, Maric D,et al. Involvement of Akt in preconditioning-induced tolerance to ischemia in PC12 cells Cereb Blood Flow Metab 2006 ;26(10): 1323–1331
53.Burgering BM, Coffer PJ.Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction Nature 1995;17:599–602
54.Franke T, Yang S, Chan T, Datta K, Kazlauskas A, Morrison D ,Kaplan D Tsichlis P. The protein kinase encoded by the Akt protooncogene is a target of the PDGF–activated phosphatidylinositol 3-kinase Cell 1995; 81: 727–736
55.XiaoHui Yin, QuanGuang Zhang, Bei Miao, Guang-Yi Zhang. Neuroprotective effects of reconditioning ischaemia on ischaemic brain injury through inhibition of mixed-lineage kinase 3 via NMDA receptor-mediated Akt1 activation Neurochem 2005;93: 1021–1029
56.Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu L S,Anderson MJ, Arden KC, Blenis J and Greenberg ME. Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor Cell 1999;96:857–868
57.Wiggins AK, Shen PJ , Gundlach AL. Neuronal-NOS adaptor protein expression after spreading depression: implications for NO production and ischemic tolerance Neurochem 2003; 87:1368–1380
1.Dahl NA, Balfour WM. Prolonged anoxic survival due to anoxia pre-exposure:brain ATP, lactate, and pyruvate Am J Physiol 1964;207:452-456
2.Yao H , Sugimori H , Fukda K, et al . Photot hrombotic middle cerebral artery occlusion and reperfusion laser system in spontaneously hypertensive rats Stroke 2003;34(11):2716 -2721
3.Traystman RJ . Animal models of focal and global cerebral ischemia ILAR 2003; 44(2):85-95
4.龚彪,李长清,黄剑.SD大鼠线栓法局灶性脑缺血/再灌注模型的改进重庆医学2006;35(4):313 -315
5.Jensen HA, Loukogeorgakis S, Yannopoulos F, Rimpil?inen E, Petzold A,Tuominen H, Lepola P, Macallister RJ, Deanfield JE, M?kel? T, Alestalo K, Kiviluoma K, Anttila V, Tsang V, Juvonen T. Remote ischemic preconditioning protects the brain against injury after hypothermic circulatory arrest Circulation 2011 Feb2;123(7):714-721
6.Kapinya KJ, Lowl D, Futterer C, Maurer M, Waschke KF, Isaev NK,et al. Tolerance against ischemic neuronal injury can be induced by volatile anesthetics and is inducible NO synthase dependent Stroke 2002;33:1889-1898
7.Weih M, Bergk A, Isaev NK, Ruscher K, Megow D, Riepe M, et al.Induction of ischemic tolerance in rat cortical neurons by 3-nitropropionic acid: chemical preconditioning Neurosci Lett 1999;272:207-210
8.Hausenloy DJ, Yellon DM. Remote ischaemic preconditioning: underlying mechanisms and clinical application Cardiovasc Res 2008;79:377-386
9.Dave KR, Saul I, Prado R, Busto R, Perez-Pinzon MA. Remote organ ischemic preconditioning protect brain from ischemic damage following asphyxial cardiac arrest Neurosci Lett 2006;404:170-175
10.Ali ZA, Callaghan CJ, Lim E, Ali AA, Nouraei SA, Akthar AM, et al. Remote ischemic preconditioning reduces myocardial and renal injury after elective abdominal aortic aneurysm repair: a randomized controlled trial Circulation 2007;116:98-105
11.Bitsch A, KleneW,Murtada L, et al. A longitudinal p rospective study of soluble adhesion molecules in acute stroke Stroke 1998; 29(10) : 21-29
12.E-selectin nasal spray to prevent stroke recurrence accessed Feb 13, 2009
13.Endres M, Gertz K, Lindauer U, Katchanov J, Schultze J, Schrock H,et al. Mechanisms of stroke protection by physical activity Ann Neurol 2003;54:582-590
14.Vlasov TD, Korzhevskii DE, Polyakova EA. Ischemic preconditioning of the rat brain as a method of endothelial protection from ischemic/repercussion injury Neurosci Behav Physiol2005;35:567-572
15.Zhu Y, Zhang Y, Ojwang BA, Brantley MA, Gidday JM. Long-term tolerance to retinal ischemia by repetitive hypoxic preconditioning: role of HIF-1alpha and heme oxygenase-1 Investig Ophthalmol Visual Sci 2007;48:1735–1743
16.Gidday JM. Cerebral preconditioning and ischaemic tolerance Nat Rev Neurosci 2006;7:437-448
17.Kapinya KJ. Ischemic tolerance in the brain Acta Physiol Hung 2005;92:67-92
18.Dirnagl U, Becker K, Meisel A. Preconditioning and tolerance against cerebral ischaemia: from experimental strategies to clinical use Lancet Neurol 2009;8:398-412
19.Tauskela JS, Morley P. On the role of Ca2+ in cerebral ischemic preconditioning Cell Calcium 2004;36:313-322
20.Raval AP, Dave KR, Mochly-Rosen D, Sick TJ, Perez-Pinzon MA. Epsilon PKC is required for the induction of tolerance by ischemic and NMDA mediated preconditioning in the organotypic hippocampal slice Neurosci 2003; 23:384-391
21.Blckler PE, Fahlman CS.Moderate increases in intracellular calcium activate neuroprotective signals in hippocampal neurons Neuroscience 2004;127:673–683
22.Rui-Min Wang , Fang Yang, Yu-Xin Zhang. Preconditioning-induced activation of ERK5 is dependent on moderate Ca2+influx via NMDA receptors and contributes to ischemic tolerance in the hippocampal CA1 region of rats Life Sciences 2006;79:1839–1846
23.Tauskela JS, Comas T, Hewitt K, Monette R, Paris J, Hogan M, Morley P Cross-tolerance to otherwise lethal N-methyl-D-aspartate and oxygenglucose deprivation in preconditioned cortical cultures Neuroscience 2001; 107:571-584
24.Wang RM, Yang F, Zhang YX.Preconditioning-induced activation of ERK5 is dependent on moderate Ca2+ influx via NMDA receptors and contributes to ischemic tolerance in the hippocampal CA1 region of rats Life Sci 2006; 79:1839-1846
25.Mabuchi T, Kitagawa K, Kuwabara K,et al.Phosphorylation of cAMP response element-binding protein in hippocampal neurons as a protective response after exposure to glutamate in vitro and ischemia in vivo .Neuroscience 2001;21(23):9204–9213
26.Zhang Y, Lipton PJ. Cytosolic Ca2+ changes during in vitro ischemia in rat hippocampal slices: major roles for glutamate and Na+-dependent Ca2+ release from mitochondri Neuroscience 1999;19:3307–3315
27.Grishin AA, Gee CE, Gerber U, et al. Differential calcium-dependent modulation of NMDA currents in CA1 and CA3 hippocampal pyramidal cells Neuroscience 2004;24(2):350–355
28.Lin CH, Chen PS, Gean PW. Glutamate preconditioning prevents neuronal death induced bycombined oxygen-glucose deprivation in cultured cortical neurons Eur J Pharmacol 2008;589(1-3):85-93
29.Miao B, Yin XH, Pei DS, Zhang QG, Zhang GY. Neuroprotective effects of preconditioning ischemia on ischemic brain injury through downregulating activation of JNK1/2 via N-methyl-D-aspartate receptormediated Akt1 activation J Biol Chem 2005; 280:21693-21699
30.Zhang Y, Park TS, Gidday JM.Hypoxic preconditioning protects human brain endothelium from ischemic apoptosis by Akt-dependent surviving activation Am J Physiol Heart Circ Physiol 2007;292:2573-2581
31.Yuan Y, Guo Q, Ye Z, Pingping X, Wang N, Song Z.Ischemic postconditioning protects brain from ischemia/reperfusion injury by attenuating endoplasmic reticulum stress-induced apoptosis through PI3K-Akt pathway Brain Res 2011;7,1367:85-93
32.Miyawaki T, Mashiko T, Ofengeim D, Flannery RJ, Noh KM, Fujisawa S, Bonanni L, Bennett MV, Zukin RS, Jonas EA.Ischemic preconditioning blocks BAD translocation, Bcl-xL cleavage, and large channel activity in mitochondria of postischemic hippocampal neurons Proc Natl Acad Sci 2008; 25;105(12):4892-4897
33.Chung H, Seo S, Moon M,ParkS.Phosphatidylinositol-3-kinase/Akt/glycogen synthase kinase-3 beta and ERK1/2 pathways mediate protective effects of acylated and unacylated ghrelin against oxygen-glucose deprivation-induced apoptosis in primary rat cortical neuronal cells Endocrinol 2008;198(3):511-521
34.Pong K. Ischaemic preconditioning: therapeutic implications for stroke? Expert Opin Ther Targets 2004 Apr;8(2):125-139
35.Kirino T.Ischemic tolerance Cereb Blood Flow Metab 2002; 22:1283-1296
36.Okamura H, Aramburu J, García-Rodríguez C,et al.Concerted dephosphorylation of the transcription factor NFAT1 induces a conformational switch that regulates transcriptional activity mol.cell 2000;6:539-550
37.Kim MG, Jo DG, Hong GS,et al.Calpain-d ependent cleavage of cain cabin1 activates calcineurin to mediate calcium-triggered cell death PNAS 2002 Jul 23;99(15):9870-9875
38.Katsura KI, Kurihara J, Kato H, Katayama Y.Ischemic pre-conditioning affects the subcellular distribution of protein kinase C and calcium/calmodulin-dependent protein kinase II in the gerbil hippocampal CA1 neurons Neurol Res 2001;23(7):751-754
39.Fix AS, Horn JW, Wightman KA, et al. Neuronal vacuolization and necrosis induced by the noncompetitive N-methyl-D-aspartate (NMDA) antagonist MK_801 (dizocilpine maleate): a light and electron microscopic evaluation of the rat retrosplenial cortex Exp Neurol 1993;123:204-215
40.Tan DP,Liu QY, Koshiva N, et al. Enhancement of long-term memory retention and short-term synaptic plasticity in cbl-b null mice Proc Natl Acad Sci USA 2006; 103 (13) : 5125 - 5130
41.SaffordM, Collins S,LutzMA, et al. Egr-2 and Egr-3 are negative regulators of T cell activation Nat Immunol 2005; 6 (5) : 472 - 480
42.BergholdtB, Taxvig C, Eising S, et al. CBL-B variants in type 1 diabetes and their genetic interaction with CTLA4 Leukoc Biol 2005; 77 (4): 579 - 585
43.Krawczyk CM, Jones RG, Atfield A, et al. Differential control of CD28-regulated in vivo immunity by the E3 ligase Cbl-b Immunol 2005; 174 (3) : 1472 - 1478
44.Qu X,Miah SM, Hatani T, et al. Selective inhibition of fcep silon Imediated mast cell activation by a truncated variant of Cbl-b related to the rat model of type 1 diabetes mellitus Biochem 2005;137(6):711-720
45.Tran T, Hoffmann S,Wiesehan K, et al. Insights into human Lck SH3 domain binding specificity: different binding modes of artificial and native ligands Biochemistry 2005;44(45):15042-15052
46.Jozic D, Cardenes N,Deribe YL, et al. Cbl promotes clustering of endocytic adaptor proteins Nat Struct Mol Biol 2005; 12 (11) : 972- 979
47.Horne WC, Sanjay A,Bruzzaniti A, et al. The role of Src kinase and Cbl proteins in the regulation of osteoclast differentiation and function Immunol Rev 2005; 208 (1): 106 - 125
48.Wohlfert EA, Gorelik L,Mittler R, et al. Cutting edge: deficiency in the E3 ubiquitin ligase Cbl-b results in a multifunctional defect in T cell TGF-beta sensitivity in vitro and in vivo Immunol 2006;176(3):1316-1320
49.Suzue N, Nikawa T, Onishi Y, et al. Ubiquitin ligase Cbl-b downregulates bone formation through suppression of IGF-I signaling in osteoblasts during denervation Bone Miner Res 2006;21(5):722 -734
50.Babu S, Blauvelt CP, Kumaraswami V, et al. Regulatory networks induced by live parasites impair both Th1 and Th2 pathways in patent lymphatic filariasis: imp lications for parasite persistence Immunol 2006;176(5):3248-3256
51.Qu X, Sada K, Kyo S, et al. Negative regulation of Fcep silonR I-mediated mast cell activation by a ubiquitin-protein ligase Cbl-b Blood 2004;103(5):1779-1786
52.Wohlfert EA, Callahan MK, Clark RB. Resistance to CD4+CD25 +regulatory T cells and TGF-beta in Cbl-b-/-mice Immunol 2004;173(2):1059-1065
53.Leng Q, Bentwich Z, Borkow G. Increased TGF-beta, Cbl-b and CTLA-4 levels and immunosuppression in association with chronic immune activation Int Immunol 2006;18(5):637-644
54.Andrew A. Sproul, Zhiheng Xu, Michael Wilhelm, Stephen Gire, Lloyd A. Greene.Cblnegatively regulates JNKactivation and cel death Cell Res 2009; 19(8):950–961
55.Widmann C, Gibson S, Johnson GL. Caspase-dependent cleavage of signaling proteins during apoptosis.A turn-off mechanism for anti-apoptotic signals Biol Chem 1998,20;273(12):7141-7147
56.ZiRen Z, HongChang G,ChenJie Z, YongGang C, Jing H, AiXin S, DongHai L,HongYu H.Differential ubiquitin binding of the UBA domainsfrom human c-Cbl and Cbl-b:NMR structural and biochemical insights Protein Science 2008; 17:1805–1814
57.Stenzel-Poore MP, Stevens SL, Xiong Z, et al. Effect of ischaemic preconditioning on genomic response to cerebral ischaemia: similarity to neuroprotective strategies in hibernation and hypoxiatolerant states Lancet 2003;362:1028–1037
58.Yildirim F, Gertz K, Kronenberg G, et al. Inhibition of histone deacetylation protects wildtype but not gelsolin-deficient mice from ischemic brain injury Exp Neurol 2008;210:531–542
59.Langley B, Gensert JM, Beal MF, Ratan RR. Remodeling chromatin and stress resistance in the central nervous system: histone deacetylase inhibitors as novel and broadly effective neuroprotective agents Curr Drug Targets CNS Neurol Disord 2005;4:41–50
2.马常升,马文领,戴维国,等.插线法制备大鼠局灶性脑缺血再灌注模型的研究解剖学杂志1999;22(3):209
3.徐佳,葛林宝,徐明曙,等.大鼠栓线法MACO模型中插入深度的研究上海实验动物科学2002;22(4):209-212
4.诸晓凡,董家政,吴军,等.颈内动脉线拴与环扎建立局灶性脑缺血再灌注模型中风与神经疾病杂志2000;17(3):152-154
5.Longa Z, Weinstein PR, Carson S, et al. Reversible middle cerebral artery:without craniotomy in rats Stroke 1989l;20:84-91
6.Koizumi J, Yoshid Y, Nakazawa T, et al. Experimental studies of ischemic brain edema, a new experiment model of cerebral embolism in rats in which recirculation can be introduced in the ischemia area Stroke 1986;8:1-3
7.王伟.严格控制试验条件建立标准化脑缺血动物模型中华神经科杂志1998;31(5):261
8.Bederson JB, Pitts LH, Tsuji M, et al. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination Stroke 1986; 17(3): 472-476
9.Dahl N, Balfour W. Prolonged anoxic survival due to anoxia pre-exposure: brain ATP, lactate, and pyruvate Am J Physiol 1964; 207:452-456
10.寞万臣,王任直,张波,等.大鼠局灶性脑缺血模型制作方法的改进中国脑血管病杂志2004;1(2):81-84
11.Xing BZ, Chen H, Zhang M. Ischemic postconditioning inhibits apoptosis after focal cerebral ischemia/reperfusion injury in the rat Stroke 2008; 39:2362-2369
12.闫明,王晶晶,乔欣,等. Cdk5与大鼠脑缺血再灌注损伤时细胞凋亡的关系中国实验动物学报2008;16(2):117-121
13.贾杰,胡永善,吴毅,等.跑台运动训练促进脑梗死大鼠外源性移植的神经干细胞迁移分化的实验研究中国康复医学杂志2008;23(7):594-597
1.Blckler PE, Fahlman CS. Moderate increases in intracellular calcium activate neuroprotective signals in hippocampal neurons Neuroscience 2004;127:673–683
2.Raval AP, Dave KR, Daria MR,et al. PKC is required for the induction of tolerance by ischemic and NMDA-mediated preconditioning in the organotypic hippocampal slice Neuroscience 2003;23(2):384–391
3.Koizumi J, Yoshida Y, Nakazawa T, et al. Experimental studies of ischemic brain edema: a new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area Stroke 1986;8:1-8
4.焦卓敏,赵瑞波,毕彦忠,等.大鼠脑局灶性缺血对再次缺血的影响中风与神经疾病杂志1999;16(3):138-140
5.黄勇华,张微微,郝小淑,等.钙离子与鼠脑出血继发性损伤的关系及尼莫地平的保护作用中华老年心脑血管病杂志2000;6(2):200-203
6.Mabuchi T, Kitagawa K, Kuwabara K, et al. Phosphorylation of cAMP response element-binding protein in hippocampal neurons as a protective response after exposure to glutamate in vitro and ischemia in vivo Neuroscience 2001;21(23):9204–9213
7.许建新,周跃飞,赵洪洋.创伤性脑损伤后脑神经细胞内Ca2+变化试验研究中华神经外科疾病研究杂志2009;8(1):73-74
8.Miller.RJ.Calcium signaling in neurons. Trends.Neurosci,1988,11:415- 441.
9.PE blckler, CS fahlman. Moderate increases in intracellular calcium activate neuroprotective signals in hippocampal neurons Neuroscience 2004; 127: 673–683
10.金惠铭,王建枝.病理生理学第6版231-233
11.Wong.MC, Haley EC. Jr. Calcium antagonists: stroke therapy coming of age Stroke 1990;21(3): 494-451
12.Lipton P. Regulation of glycogen in the dentate gyrus of the in vitro guinea pig hippocampus: effect of combined deprivation of glucose and oxygen J Neurosci Methods 1988;28:147–154
13.Mitani A, Yanase H, Sakai K, Wake Y, Kataoka K. Origin of intracellular Ca2+ elevation induced by in vitro ischemia-like condition in hippocampal slices Brain Res 1993;601:103–110
14.Mitani A, Yanase H, Namba S, Shudo M, Kataoka K. In vitro ischemia-induced intracellular Ca2+ elevation in cerebellar slices: a comparative study with the values found in hippocampal slices Acta Neuropathol,1995;89:2–7
15.Hoehner PJ,Blanck TJ, Roy R, Rosenthal RE, Fiskum G. Alteration of voltage-dependent calcium channels in canine brain during global ischemia and reperfusion J Cereb Blood Flow Metab 1992;12:418–424
16.Friedman JE, Haddad GG. Major differences in [Ca2+]i response to anoxiabetween neonatal and adult rat CA1 neurons: role of Ca2+ and Na+ J Neurosci 1993;13:63–72
17.Grishin AA, Gee CE, Gerber U, et al. Differential calcium-dependent modulation of NMDA currents in CA1 and CA3 hippocampal pyramidal cells Neuroscience 2004;24(2):350–355
18.Philip E, Bickler, Xinhua Z, Christian S. Fahlman. Isoflurane preconditions hippocampal neurons against oxygen–glucose deprivation role of intracellular Ca2+ and mitogen-activated protein kinase signaling anesthesiology 2005;103:532–539
19.姜长斌,丁聪,尹琳.脑缺血预处理大鼠脑IL-6和Ngb的表达及尤瑞克林对其表达的干预中国神经免疫学和神经病学杂志2009;01:46-50
20.尹琳,李芳,金萍,闵连秋.脑缺血预处理对再次脑缺血大鼠神经功能、脑含水量及脑组织中Bcl-xl、P53蛋白表达的影响中国临床康复2005(1):111-113
21.Lin CH, Chen PS, Gean PW. Glutamate preconditioning prevents neuronal death induced by combined oxygen-glucose deprivation in cultured cortical neurons Eur J Pharmacol 2008;589(3): 85-93
22. Zhang Y, Lipton PJ. Cytosolic Ca2+ changes during in vitro ischemia in rat hippocampal slices: major roles for glutamate and Na+-dependent Ca2+ release from mitochondria Neuroscience 1999;19:3307–3315
23.Wang RM, Yang F, Zhang YX. Preconditioning-induced activation of ERK5 is dependent on moderate Ca2+ influx via NMDA receptors and contributes to ischemic tolerance in the hippocampal CA1 region of rats Life Sci 2006; 4;79(19):1839-1846
24.QuanGuang Zhang, RuiMin Wang, Dong Han, LiCai Yang ,Jie Li,Darrell W. Brann.Preconditioning neuroprotection in global cerebral ischemia involves NMDA receptor-mediated ERK-JNK3 crosstalk Neurosc Res 2009;63(3):205-212
25.Miao B, Yin XH, Pei DS, et al. Neuroprotective effects of preconditioning ischemia on ischemic brain Injury through down-regulating activation of JNK1/2 via N-Methyl-D-aspartate receptor-mediated Akt1 activation Biol Chem 2005;80:21693–21699
26.Yin XH, Zhang QG, Miao B, et al. Neuroprotective effects of preconditioning ischaemia on ischaemic brain injury through inhibition of mixed-lineage kinase 3 via NMDA receptor-mediated Akt1 activation Neurochem 2005;93:1021–1029
27.吕少萍,管勇.尼莫通对脑缺血再灌注细胞凋亡相关基因表达影响青岛大学医学院学报2005;41(2):167-168
28.Wen JY, Chen ZW. Protective effect of pharmacological preconditioning of total flavones of abelmoschl manihot on cerebral ischemic reperfusion injury in rats Am J Chin Med 2007;35(4): 653-661
29.Liu C, Zhou R, Sun S.Nimodipine modulates Bcl-2 and Bax mRNA expression after cerebral ischemia Huazhong Univ Sci Technolog Med Sci 2004;24(2):170-172
30.苏静姜长斌.大鼠脑缺血预处理后线粒体通透性转换及细胞色素C水平的变化及意义中国优秀硕士学位论文全文数据库医药卫生神经病学2009:1-44
31.陈道文,周永,赵薛旭,李作汉,狄晴.大鼠脑缺血预处理再灌注模型Bcl-2和Bax的表达临床神经病学杂志2006;01:52-53
32.王晶余,徐忠信,康治臣,韩雪梅,邬英全,钱佳利.局灶脑缺血预处理缺血耐受与Bcl-xl、Bax表达的研究中国实验诊断学2005;01:40-42
33.刁尧,陈春梅,李雪娜,李亚明,高杰,蔡奎,赵恂,于成国.NF-κB、Bcl-2和Bax蛋白在大鼠脑缺血预处理脑组织中的表达及意义解剖科学进展2008;03:41-44
1.Gidday, JM. Cerebral preconditioning and ischaemic tolerance. Nat. Rev. Neurosci.2006,7(6): 437–448
2.Shamloo.M, Rytter. A, Wieloch.T. Activation of the extracellular signal-regulated protein kinase cascade in the hippocampal CA1 region in a rat model of global cerebral ischemic preconditioning Neuroscience 1999;93(1):81–88
3.Gonzalez-Zulueta.M, Feldman.AB, Klesse.LJ, Kalb.RG, Dillman. JF, Parada. LF, Dawson.TM, Dawson.VL. Requirement for nitric oxide activation of p21(ras)/extracellular regulated kinase in neuronal ischemic preconditioning Proc. Natl Acad. Sci 2000; 97(1):436–441
4.Kurkinen. K, Busto.R, Goldsteins. G, Koistinaho. J, Pérez-Pinzón. MA. Isoform-specific membrane translocation of protein kinase C after ischemic preconditioning Neurochem Res 2001;26(10):1139–1144
5.Miao.B, Yin.X., Pei.DS, Zhang.QG, Zhang.GY.Neuroprotective effects of preconditioning ischemia on ischemic brain injury through down-regulating activation of JNK1/2 via N-methyl -D-aspartate receptor-mediated Akt1 activation Biol Chem 2005 ;280(23):21693–21699
6.Chen.J, Graham.SH, Zhu.RL, Simon.RP. Stress proteins and tolerance to focal cerebral ischemia Cereb Blood Flow Metab 1996;16(4):566–577
7.Sónia.C, Correia.Cristina. C, Susana.C, Renato. X, Santos,Maria, Catarina. RO, George.P, Zhu.XW, Smith.MA, Moreira. Mitochondrial preconditioning: a potential neuroprotective strategy Front Aging Neurosci2010; 26(2):138
8.Stevens.SL, Ciesielski.TM, Marsh. BJ, Yang.T, Homen. DS.,Boule.JL, Lessov.NS, Simon.RP, Stenzel-Poore.MP. Toll-like receptor 9: a new target of ischemic preconditioning in the brain Cereb Blood Flow Metab 2008;28(5):1040- 1047
9.Goto S, Matsukado Y, Mihara Y, Inoue N, Miyamoto E.The distribution of calcineurin in rat brain by light and electron microscopic immunohistochemistry and enzyme-immunoassay Brain Res 1986;397(1):161-172
10.Subramaniam Jayanthi,Xiaolin Deng , Bruce Ladenheim. Calcineurin/NFAT-induced up-regulation of the Fas ligand/Fas death pathway is involved in methamphetam -ine-induced neuronal apoptosis PNAS 2005;102(3):868-873
11.Tan Dong Ping,Liu Qi Ying,Koshiya N,Gu H, Alkon D.Enhancement of long-term memory retention and short-term synaptic plasticity in cbl-b null mice Neuroscience 2006;103(13): 5125–5130
12.黄勇华,张微微,郝小淑. Ca2+与鼠脑出血继发性损伤的关系及尼莫地平的保护作用中华老年心脑血管病杂志2000;6(2): 200-203
13.Mabuchi T, Kitagawa K, Kuwabara K, Takasawa K, Ohtsuki T, Xia Z, Storm D, Yanagihara T, Hori M, Matsumoto M. Phosphorylation of cAMP response element-binding protein in hippocampal neurons as a protective response after exposure to glutamate in vitro and ischemia in vivo Neuroscience 2001;21(23):9204–9213
14.Yamaguchi1 T,Miyata1 K, Shibasaki F, et al. Effect of cyclosporine A on immediately gene in rat global ischemia and its neuroprotection Pharmacol Sci 2006;100:73– 81
15.Bederson JB, Pitts LH, Tsuji M, Isshiki A, Uchino H. Rat middle cerebral cnfery occlusion: evalution of the model and development of a neurologic examination Stroke 1986; 17(3): 472-476
16.Dirnagl.U,Simon.RP,Hallenbeck.JM.Ischemic tolerance and endogenous neuroprotection Trends neurosci 2003 May;26(5):248-54
17.Kapinya KJ. Ischemic tolerance in the brain Acta Physiol Hung 2005;92:67-92
18.Dirnagl U, Becker K, Meisel A. Preconditioning and tolerance against cerebral ischaemia: from experimental strategies to clinical use Lancet Neurol 2009;8:398-412
19.邓珊,秦超.缺血预处理与后处理的脑保护作用医学综述2008;(14):377-379
20.Blckler PE,Fahlman CS. Moderate increases in intracellular calcium activate neuroprotective signals in hippocampal neuron Neuroscience 2004;127:673– 668
21.朱心红,杨建明,高天明.脑心通对大鼠脑缺血海马CA1区神经元损害的保护作用第一军医大学学报2004;24(10):1123-1125
22.Jian-Feng GAO ,Jian-Sheng LI ,You-Long ZHOU ,Ke LIU .Effects of naomaitong recipe on expressions of nuclear factor-κB and nitric-oxide synthases in brain tissue after focal cerebral ischemia reperfusion in aged rats Zhong Xi Yi Jie He Xue Bao/J Chin Integr Med 2006;4(5): 530-534
23.Antonio Pisani, Paolo Calabresi, Alessandro Tozzi, Vincenza D'Angelo, Giorgio Bernardi.L-Type Ca2+ channel blockers attenuate electrical changes and Ca2+ rise induced by oxygen/glucose deprivation in cortical neurons Stroke 1998;29:196- 202
24.JoséM. Roda, Fernando Carceller, Exuperio Díez-Tejedor, Carlos Avenda?o.Reduction of Infarct size by intra-arterial nimodipine administered at reperfusion in a rat model of partially reversible brain focal ischemia Stroke 1995;26:1888-1892
25.Wen JY,Chen ZW. Protective effect of pharmacological preconditioning of total flavones of abelmoschlmanihot on cerebral ischemic reperfusion injury in rats Am J Chin Med 2007;35 (4):653-661
26.Liu C, Zhou R, Sun S.Nimodipine modulates Bcl-2 and Bax mRNA expression after cerebralischemia Huazhong Univ Sci Technolog Med Sci 2004;24(2):170-172
27.Steiner JP,Dawson TM,Fotuhi M,et al.Snyder High brain densities of the immunophilin FKBP colocalized with calcineurin Nature 1992;358:584-587
28.Bond A, Lodge D, Hicks CA, Ward MA, O'Neill MJ.NMDA receptor antagonism, but not AMPA receptor antagonism attenuates induced ischaemic tolerance in the gerbil hippocampus Eur J Pharmacol 1999 ;10;380(3):91-99
29.Lin CH, Chen PS, Gean PW. Glutamate preconditioning prevents neuronal death induced by combined oxygen-glucose deprivation in cultured cortical neurons Eur J Pharmacol 2008; 589(1-3):85-93
30.Takuma Mabuchi, Kazuo Kitagawa, Keisuke Kuwabara, Kenichiro Takasawa, Toshiho Ohtsuki,Zhengui Xia, Daniel Storm, Takehiko Yanagihara, Masatsugu Hori, Masayasu Matsumoto. Phosphorylation of cAMP Response Element-Binding Protein in Hippocampal Neurons as a Protective Response after Exposure to Glutamate In Vitro and Ischemia In Vivo The Journal of Neuroscience 2001;21(23):9204–9213
31.Fix AS, Horn JW, Wightman KA, et al. Neuronal vacuolization and necrosis induced by the noncompetitive N-methyl-D-aspartate (NMDA) antagonist MK801 (dizocilpine maleate): a light and electron microscopic evaluation of the rat retrosplenial cortex Exp Neurol 1993 ;123(2):204-215
32.Lipton SA. Failures and successes of NMDA receptor antagonists:molecular basis for the use of open-channel blockers like memantine in the treatment of acute and chronic neurologic insults NeuroRx 2004 ;1(1):101-110.
33.Wang RM, Yang F, Zhang YX.Preconditioning-induced activation of ERK5 is dependent on moderate Ca2+ influx via NMDA receptors and contributes to ischemic tolerance in the hippocampal CA1 region of rats Life Sci 2006 ;79(19):1839-1846
34.Okamura H, Aramburu J, García-Rodríguez C,et al. Concerted dephosphorylation of the transcription factor NFAT1 induces a conformational switch that regulates transcriptional activity mol cell 2000;6(3):539-550
35.Aramburu J, Rao A. Klee C. B.Calcineurin: from structure to function Curr Top Cell Regul 2000; 36, 237–295
36.Klee CB, Ren H , Wang X.Regulation of the Calmodulin stimulated Protein phosphatase, Calcineurin Biol Chem 1998;273, 13367–13370
37.Wang HG, Pathan N, Ethell IM, et al. Ca2+-induced apoptosis through calcineurin dephosphorylation of BADScience 1999;284:339–343
38.Norifumi Shioda, Shigeki Moriguchi, Yasufumi Shirasaki, Kohji Fukunaga. Generation ofconstitutively active calcineurin by calpain contributes to delayed neuronal death following mouse brain ischemia Neurochem 2006; 98:310–320
39.ZHANG Yingjun , MEI Heshan , WANG Chuan , WANG Yongli, ZHANG Yongjian. Involvement of nuclear factor of activated T-cells ( NFATc) in calc- ineurin mediated ischemic brain damage in vivo Acta Pharmaceutica Sinica 2005; 40 (4) : 299– 305
40.Akio Asai, Jian-hua Qiu, Yoshitaka Narita, Shunji Chi, Nobuhito Saito, Nobusada Shinoura, Hirofumi Hamada, Yoshiyuki Kuchino, Takaaki Kirino. High levelcalcineurin activity predisposes neuronal cells to apoptosis Biol Chem 1999; 274( 48):34450-34458
41.Yan Shou, Li Li Krishnan Prabhakaran, Joseph L. Borowitz Gary E. Isom,Calcineurin-mediated Bad translocation regulates cyanide-induced neuronal apoptosis Biochem 2004;379:805-813
42.Shibasaki F, Kondo E, Akagi T McKeon F. Suppression of signaling through transcription factor NFAT by interactions between calcineurin and Bcl-2 Nature (London)1997; 386: 728-731
43.Shitashige M, Tou M, Yano T, Shibata M, Matsuo Y and Shibasaki F. Dissociation of Bax from a Bcl-2/Bax heterodimer triggered by phosphorylation of serine 70 of Bcl-2 Biochem 2001;130:741-748
44.Andrew A, Sproul, Zhiheng Xu, Michael Wilhelm, Stephen Gire, Lloyd A. Greene.Cbl negatively regulates JNK activation and cell death Cell Res 2009; 19(8): 950–961
45.Widmann C, Gibson S, Johnson GL. Caspase-dependent cleavage of signaling proteins during apoptosis A turn-off mechanism for anti-apoptotic signals Biol Chem 1998;273(12):7141-7147
46.ZR Zhou, HC Gao,CJ Zhou, YG Chang, J Hong, AX Song, DH Lin, HY Hu.Differential ubiquitin binding of the UBA domainsfrom human c-Cbl and Cbl-b: NMR structural and biochemical insights Protein Science 2008;17:1805–1814
47.DY Fang,YC Liu.Proteolysis-independent regulation of PI3K by Cbl-b?mediated ubiquitination in T cell Nat Immun 2001; 2: 870 - 875
48.Hashiguchi A, Yano S, Morioka M, Hamada J, Ushio Y, Takeuchi Y, Fukunaga K. Upregulation of endothelial nitric oxide synthase via phosphatidylinositol 3-kinase pathway contributes to ischemic tolerance in the CA1 subfield of gerbil hippocampus Cereb Blood Flow Metab 2004; 24(3):271-279
49.Yano S, Morioka M, Fukunaga K, Kawano T, Hara T, Kai Y, Hamada J, Miyamoto E, Ushio Y.Activation of Akt/protein kinase B contributes to induction of ischemic tolerance in the CA1 subfield of gerbil hippocampus Cereb Blood Flow Metab 2001;21(4):351-360
50.Nakajima T, Iwabuchi S, Miyazaki H, Okuma Y, Kuwabara M, Nomura Y, Kawahara K. Preconditioning prevents ischemia-induced neuronal death through persistent Akt activation in the penumbra region of the rat brain Vet Med Sci 2004 ;66(5):521-527
51.Johnnides, Hickey RW,Jun Chen, Wei Yin,et al. Preconditioning suppresses Inflammation in neonatal hypoxic ischemia via Akt activation Stroke 2007;38:1017-1024
52.Hillion JA, Li YiXin, Maric D,et al. Involvement of Akt in preconditioning-induced tolerance to ischemia in PC12 cells Cereb Blood Flow Metab 2006 ;26(10): 1323–1331
53.Burgering BM, Coffer PJ.Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction Nature 1995;17:599–602
54.Franke T, Yang S, Chan T, Datta K, Kazlauskas A, Morrison D ,Kaplan D Tsichlis P. The protein kinase encoded by the Akt protooncogene is a target of the PDGF–activated phosphatidylinositol 3-kinase Cell 1995; 81: 727–736
55.XiaoHui Yin, QuanGuang Zhang, Bei Miao, Guang-Yi Zhang. Neuroprotective effects of reconditioning ischaemia on ischaemic brain injury through inhibition of mixed-lineage kinase 3 via NMDA receptor-mediated Akt1 activation Neurochem 2005;93: 1021–1029
56.Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu L S,Anderson MJ, Arden KC, Blenis J and Greenberg ME. Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor Cell 1999;96:857–868
57.Wiggins AK, Shen PJ , Gundlach AL. Neuronal-NOS adaptor protein expression after spreading depression: implications for NO production and ischemic tolerance Neurochem 2003; 87:1368–1380
1.Dahl NA, Balfour WM. Prolonged anoxic survival due to anoxia pre-exposure:brain ATP, lactate, and pyruvate Am J Physiol 1964;207:452-456
2.Yao H , Sugimori H , Fukda K, et al . Photot hrombotic middle cerebral artery occlusion and reperfusion laser system in spontaneously hypertensive rats Stroke 2003;34(11):2716 -2721
3.Traystman RJ . Animal models of focal and global cerebral ischemia ILAR 2003; 44(2):85-95
4.龚彪,李长清,黄剑.SD大鼠线栓法局灶性脑缺血/再灌注模型的改进重庆医学2006;35(4):313 -315
5.Jensen HA, Loukogeorgakis S, Yannopoulos F, Rimpil?inen E, Petzold A,Tuominen H, Lepola P, Macallister RJ, Deanfield JE, M?kel? T, Alestalo K, Kiviluoma K, Anttila V, Tsang V, Juvonen T. Remote ischemic preconditioning protects the brain against injury after hypothermic circulatory arrest Circulation 2011 Feb2;123(7):714-721
6.Kapinya KJ, Lowl D, Futterer C, Maurer M, Waschke KF, Isaev NK,et al. Tolerance against ischemic neuronal injury can be induced by volatile anesthetics and is inducible NO synthase dependent Stroke 2002;33:1889-1898
7.Weih M, Bergk A, Isaev NK, Ruscher K, Megow D, Riepe M, et al.Induction of ischemic tolerance in rat cortical neurons by 3-nitropropionic acid: chemical preconditioning Neurosci Lett 1999;272:207-210
8.Hausenloy DJ, Yellon DM. Remote ischaemic preconditioning: underlying mechanisms and clinical application Cardiovasc Res 2008;79:377-386
9.Dave KR, Saul I, Prado R, Busto R, Perez-Pinzon MA. Remote organ ischemic preconditioning protect brain from ischemic damage following asphyxial cardiac arrest Neurosci Lett 2006;404:170-175
10.Ali ZA, Callaghan CJ, Lim E, Ali AA, Nouraei SA, Akthar AM, et al. Remote ischemic preconditioning reduces myocardial and renal injury after elective abdominal aortic aneurysm repair: a randomized controlled trial Circulation 2007;116:98-105
11.Bitsch A, KleneW,Murtada L, et al. A longitudinal p rospective study of soluble adhesion molecules in acute stroke Stroke 1998; 29(10) : 21-29
12.E-selectin nasal spray to prevent stroke recurrence accessed Feb 13, 2009
13.Endres M, Gertz K, Lindauer U, Katchanov J, Schultze J, Schrock H,et al. Mechanisms of stroke protection by physical activity Ann Neurol 2003;54:582-590
14.Vlasov TD, Korzhevskii DE, Polyakova EA. Ischemic preconditioning of the rat brain as a method of endothelial protection from ischemic/repercussion injury Neurosci Behav Physiol2005;35:567-572
15.Zhu Y, Zhang Y, Ojwang BA, Brantley MA, Gidday JM. Long-term tolerance to retinal ischemia by repetitive hypoxic preconditioning: role of HIF-1alpha and heme oxygenase-1 Investig Ophthalmol Visual Sci 2007;48:1735–1743
16.Gidday JM. Cerebral preconditioning and ischaemic tolerance Nat Rev Neurosci 2006;7:437-448
17.Kapinya KJ. Ischemic tolerance in the brain Acta Physiol Hung 2005;92:67-92
18.Dirnagl U, Becker K, Meisel A. Preconditioning and tolerance against cerebral ischaemia: from experimental strategies to clinical use Lancet Neurol 2009;8:398-412
19.Tauskela JS, Morley P. On the role of Ca2+ in cerebral ischemic preconditioning Cell Calcium 2004;36:313-322
20.Raval AP, Dave KR, Mochly-Rosen D, Sick TJ, Perez-Pinzon MA. Epsilon PKC is required for the induction of tolerance by ischemic and NMDA mediated preconditioning in the organotypic hippocampal slice Neurosci 2003; 23:384-391
21.Blckler PE, Fahlman CS.Moderate increases in intracellular calcium activate neuroprotective signals in hippocampal neurons Neuroscience 2004;127:673–683
22.Rui-Min Wang , Fang Yang, Yu-Xin Zhang. Preconditioning-induced activation of ERK5 is dependent on moderate Ca2+influx via NMDA receptors and contributes to ischemic tolerance in the hippocampal CA1 region of rats Life Sciences 2006;79:1839–1846
23.Tauskela JS, Comas T, Hewitt K, Monette R, Paris J, Hogan M, Morley P Cross-tolerance to otherwise lethal N-methyl-D-aspartate and oxygenglucose deprivation in preconditioned cortical cultures Neuroscience 2001; 107:571-584
24.Wang RM, Yang F, Zhang YX.Preconditioning-induced activation of ERK5 is dependent on moderate Ca2+ influx via NMDA receptors and contributes to ischemic tolerance in the hippocampal CA1 region of rats Life Sci 2006; 79:1839-1846
25.Mabuchi T, Kitagawa K, Kuwabara K,et al.Phosphorylation of cAMP response element-binding protein in hippocampal neurons as a protective response after exposure to glutamate in vitro and ischemia in vivo .Neuroscience 2001;21(23):9204–9213
26.Zhang Y, Lipton PJ. Cytosolic Ca2+ changes during in vitro ischemia in rat hippocampal slices: major roles for glutamate and Na+-dependent Ca2+ release from mitochondri Neuroscience 1999;19:3307–3315
27.Grishin AA, Gee CE, Gerber U, et al. Differential calcium-dependent modulation of NMDA currents in CA1 and CA3 hippocampal pyramidal cells Neuroscience 2004;24(2):350–355
28.Lin CH, Chen PS, Gean PW. Glutamate preconditioning prevents neuronal death induced bycombined oxygen-glucose deprivation in cultured cortical neurons Eur J Pharmacol 2008;589(1-3):85-93
29.Miao B, Yin XH, Pei DS, Zhang QG, Zhang GY. Neuroprotective effects of preconditioning ischemia on ischemic brain injury through downregulating activation of JNK1/2 via N-methyl-D-aspartate receptormediated Akt1 activation J Biol Chem 2005; 280:21693-21699
30.Zhang Y, Park TS, Gidday JM.Hypoxic preconditioning protects human brain endothelium from ischemic apoptosis by Akt-dependent surviving activation Am J Physiol Heart Circ Physiol 2007;292:2573-2581
31.Yuan Y, Guo Q, Ye Z, Pingping X, Wang N, Song Z.Ischemic postconditioning protects brain from ischemia/reperfusion injury by attenuating endoplasmic reticulum stress-induced apoptosis through PI3K-Akt pathway Brain Res 2011;7,1367:85-93
32.Miyawaki T, Mashiko T, Ofengeim D, Flannery RJ, Noh KM, Fujisawa S, Bonanni L, Bennett MV, Zukin RS, Jonas EA.Ischemic preconditioning blocks BAD translocation, Bcl-xL cleavage, and large channel activity in mitochondria of postischemic hippocampal neurons Proc Natl Acad Sci 2008; 25;105(12):4892-4897
33.Chung H, Seo S, Moon M,ParkS.Phosphatidylinositol-3-kinase/Akt/glycogen synthase kinase-3 beta and ERK1/2 pathways mediate protective effects of acylated and unacylated ghrelin against oxygen-glucose deprivation-induced apoptosis in primary rat cortical neuronal cells Endocrinol 2008;198(3):511-521
34.Pong K. Ischaemic preconditioning: therapeutic implications for stroke? Expert Opin Ther Targets 2004 Apr;8(2):125-139
35.Kirino T.Ischemic tolerance Cereb Blood Flow Metab 2002; 22:1283-1296
36.Okamura H, Aramburu J, García-Rodríguez C,et al.Concerted dephosphorylation of the transcription factor NFAT1 induces a conformational switch that regulates transcriptional activity mol.cell 2000;6:539-550
37.Kim MG, Jo DG, Hong GS,et al.Calpain-d ependent cleavage of cain cabin1 activates calcineurin to mediate calcium-triggered cell death PNAS 2002 Jul 23;99(15):9870-9875
38.Katsura KI, Kurihara J, Kato H, Katayama Y.Ischemic pre-conditioning affects the subcellular distribution of protein kinase C and calcium/calmodulin-dependent protein kinase II in the gerbil hippocampal CA1 neurons Neurol Res 2001;23(7):751-754
39.Fix AS, Horn JW, Wightman KA, et al. Neuronal vacuolization and necrosis induced by the noncompetitive N-methyl-D-aspartate (NMDA) antagonist MK_801 (dizocilpine maleate): a light and electron microscopic evaluation of the rat retrosplenial cortex Exp Neurol 1993;123:204-215
40.Tan DP,Liu QY, Koshiva N, et al. Enhancement of long-term memory retention and short-term synaptic plasticity in cbl-b null mice Proc Natl Acad Sci USA 2006; 103 (13) : 5125 - 5130
41.SaffordM, Collins S,LutzMA, et al. Egr-2 and Egr-3 are negative regulators of T cell activation Nat Immunol 2005; 6 (5) : 472 - 480
42.BergholdtB, Taxvig C, Eising S, et al. CBL-B variants in type 1 diabetes and their genetic interaction with CTLA4 Leukoc Biol 2005; 77 (4): 579 - 585
43.Krawczyk CM, Jones RG, Atfield A, et al. Differential control of CD28-regulated in vivo immunity by the E3 ligase Cbl-b Immunol 2005; 174 (3) : 1472 - 1478
44.Qu X,Miah SM, Hatani T, et al. Selective inhibition of fcep silon Imediated mast cell activation by a truncated variant of Cbl-b related to the rat model of type 1 diabetes mellitus Biochem 2005;137(6):711-720
45.Tran T, Hoffmann S,Wiesehan K, et al. Insights into human Lck SH3 domain binding specificity: different binding modes of artificial and native ligands Biochemistry 2005;44(45):15042-15052
46.Jozic D, Cardenes N,Deribe YL, et al. Cbl promotes clustering of endocytic adaptor proteins Nat Struct Mol Biol 2005; 12 (11) : 972- 979
47.Horne WC, Sanjay A,Bruzzaniti A, et al. The role of Src kinase and Cbl proteins in the regulation of osteoclast differentiation and function Immunol Rev 2005; 208 (1): 106 - 125
48.Wohlfert EA, Gorelik L,Mittler R, et al. Cutting edge: deficiency in the E3 ubiquitin ligase Cbl-b results in a multifunctional defect in T cell TGF-beta sensitivity in vitro and in vivo Immunol 2006;176(3):1316-1320
49.Suzue N, Nikawa T, Onishi Y, et al. Ubiquitin ligase Cbl-b downregulates bone formation through suppression of IGF-I signaling in osteoblasts during denervation Bone Miner Res 2006;21(5):722 -734
50.Babu S, Blauvelt CP, Kumaraswami V, et al. Regulatory networks induced by live parasites impair both Th1 and Th2 pathways in patent lymphatic filariasis: imp lications for parasite persistence Immunol 2006;176(5):3248-3256
51.Qu X, Sada K, Kyo S, et al. Negative regulation of Fcep silonR I-mediated mast cell activation by a ubiquitin-protein ligase Cbl-b Blood 2004;103(5):1779-1786
52.Wohlfert EA, Callahan MK, Clark RB. Resistance to CD4+CD25 +regulatory T cells and TGF-beta in Cbl-b-/-mice Immunol 2004;173(2):1059-1065
53.Leng Q, Bentwich Z, Borkow G. Increased TGF-beta, Cbl-b and CTLA-4 levels and immunosuppression in association with chronic immune activation Int Immunol 2006;18(5):637-644
54.Andrew A. Sproul, Zhiheng Xu, Michael Wilhelm, Stephen Gire, Lloyd A. Greene.Cblnegatively regulates JNKactivation and cel death Cell Res 2009; 19(8):950–961
55.Widmann C, Gibson S, Johnson GL. Caspase-dependent cleavage of signaling proteins during apoptosis.A turn-off mechanism for anti-apoptotic signals Biol Chem 1998,20;273(12):7141-7147
56.ZiRen Z, HongChang G,ChenJie Z, YongGang C, Jing H, AiXin S, DongHai L,HongYu H.Differential ubiquitin binding of the UBA domainsfrom human c-Cbl and Cbl-b:NMR structural and biochemical insights Protein Science 2008; 17:1805–1814
57.Stenzel-Poore MP, Stevens SL, Xiong Z, et al. Effect of ischaemic preconditioning on genomic response to cerebral ischaemia: similarity to neuroprotective strategies in hibernation and hypoxiatolerant states Lancet 2003;362:1028–1037
58.Yildirim F, Gertz K, Kronenberg G, et al. Inhibition of histone deacetylation protects wildtype but not gelsolin-deficient mice from ischemic brain injury Exp Neurol 2008;210:531–542
59.Langley B, Gensert JM, Beal MF, Ratan RR. Remodeling chromatin and stress resistance in the central nervous system: histone deacetylase inhibitors as novel and broadly effective neuroprotective agents Curr Drug Targets CNS Neurol Disord 2005;4:41–50
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