缺血性脑损伤神经细胞凋亡及其相关基因的表达

缺血性脑损伤后神经元的死亡包括凋亡和坏死,近年来,缺血性脑损伤的研究中主要集中在缺血后的细胞凋亡过程。细胞凋亡又称程序性细胞死亡,是指在某些生理或病理刺激诱导下,机体为维护内环境的稳定,通过多种调控因子相互激活和表达,激活DNA内切酶,从而使细胞正常死亡的过程。细胞凋亡主要发生在缺血周围区,是一种涉及多种基因及其蛋白相互作用的自主死亡的过程。因此,

新生大鼠缺氧缺血性脑损伤神经细胞凋亡及卡配因抑制剂-3对其的影响

卡配因(Calpain)是一种Ca2+依赖性中性蛋白酶,广泛存在于包括脑组织在内的各种组织中,静息状态下Calpain主要以无活性的酶原形式存在,当胞内Ca2+浓度增高时被激活,并参与脑缺血等多种疾病的发生.近年来研究表明,脑缺血时神经细胞的凋亡也与Calpain有关,而Calpain抑制剂可显著减少凋亡的发生,减轻脑损伤.目前Calpain抑制剂对新生儿缺氧缺血性脑损伤(hypoxic ischemic brain damage,HIBD)神经元凋亡的影响尚未见报道.

亚低温对大鼠脑缺血再灌注损伤后HSP_(70) mRNA表达及损伤神经细胞凋亡的影响

目的 探讨亚低温对大鼠脑缺血再灌注损伤后HSP70 mRNA、HSP70 (热休克蛋白70 )表达及损伤神经细胞凋亡的影响。方法 采用大鼠局灶性脑缺血再灌注损伤模型 ,大脑中动脉阻塞 2小时 ,再灌注损伤 10小时 ,用逆转录聚合酶链反应 (RT PCR)技术、免疫组织化学法和原位缺口末端标记 (TUNEL)法分别检测假手术组、对照组和亚低温组HSP70 mRNA、HSP70 表达水平和凋亡细胞百分率。结果 亚低温组HSP70 mRNA、HSP70 表达水平较对照组显著升高 (P <0 .0 5 ) ,而凋亡细胞百分率明显低于对照组 (P <0 .0 5 )。结论 亚低温上调大鼠脑缺血再灌注损伤后HSP70 mRNA、HSP70 表达水平可能与其抗损伤神经细胞凋亡作用有关。

Expression of NF-κB in Schwann cells and its effect on motor neuron apoptosis in spinal cord following sciatic nerves injury in rats

Objective：To explore the expression of nuclear factor-kappa B （NF-kB） in Schwann cells （SCs） and its effect on motor neuron apoptosis in spinal cord following sciatic nerves injury in adult rats. Methods： Thirty-six adult Sprague-Dawley （SD） rats were divided randomly into normal control group （n=6）, and sciatic nerves crushing group （n= 30）, and the later was further equally randomized into 5 subgroups： 1, 3, 7, 14, and 21 d post-injury groups. The expression of NF-kB of normal and injured nerves were examined by immunohistochemistry staining, and the apoptosis of motor neurons in spinal cord of lumbar 4 to lumbar 6 （L4-L6） was investigated by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling （TUNEL） assay. Both were qua.ntitated by image analysis. Results： In crushing group, except 21 d post-injury group, the expression of NF-kB was markedly higher than that in the normal control group （P〈0.05, P〈0. 01）. At 1 d after sciatic nerves crushing, the expression of NF-kB was obviously up-regulated, reached peak at 3 d, and recovered at 21 d. The same trend was observed in the time-course on motor neuron apoptosis after sciatic nerves injury. Correlation analyses revealed that motor neuron apoptosis was significantly and positively correlated with the expression of NF-kB following sciatic nerves injury （r= 0. 976 0, P〈0. 01）. Conclusion： After injury of sciatic nerves, the presence and up-regulation of NF-kB in SCs may be involved in motor neuron apoptosis in L4-L6 spinal cord.

Role of Caspase 3 in neuronal apoptosis after acute brain injury

Objective: To analyze the role of Caspase 3 in neuronal apoptosis after acute brain injury. Methods: Experiments were carried out with rat diffuse brain trauma model. The neuronal DNA injury in cortex and hippocampus was observed by TUNEL stain. The mRNA and protein expressions and enzyme activation of Caspase 3 were observed by Northern blot, in situ hybridization, immunohistochemistry stain and Western blot, respectively. Special Caspase 3 enzyme inhibitor was used to observe the therapeutic effect.Results: TUNEL positive neurons appeared 2 hours after severe trauma, peaked at 1 day and lasted for 7 days. Northern blot showed that the Caspase 3 mRNA expression was increased and peaked at 1 day, about twice higher than the control. In the area of cortex and hippocampus, positive mRNA staining neurons appeared most distinct on one day. With the antibody for Caspase 3 P20 subunit, the active Caspase 3 expression peaked at 1 3 days. The electrophoresis band of PARP degradation would be seen by Western blot. Caspase 3 enzyme inhibitor could reduce apoptotic neuronal death without any effect on Caspase 3 P20 subunit expression. Conclusions: After brain trauma, Caspase 3 mRNA and protein expressions and enzyme activation are enhanced in combination with neuronal apoptosis. Special Caspase 3 enzyme inhibitor can apparently decrease the neuronal apoptosis.

Human neuronal apoptosis secondary to traumatic brain injury and the regulative role of apoptosis-related genes

Objective: To observe human neuronal apoptosis secondary to traumatic brain injury, and to elucidate its regulative mechanism and the change of expression of apoptosis-related genes. Methods: Specimens of brain were collected from cases of traumatic brain injury in humans. The histological and cellular morphology was examined by light and electron microscopy. The extent of DNA injury to cortical neurons was detected by using TUNEL. By in situ hybridisation and immunohistochemistry the mRNA changes and protein expression of Bcl-2, Bax, p53, and caspase 3 p20 subunit were observed. Results: Apoptotic neurons appeared following traumatic brain injury, peaked at 24 hours and lasted for 7 days. In normal brain tissue activated caspase 3 was rare, but a short time after trauma it became activated. The activity peaked at 20-28 hours and remained higher than normal for 5-7 days. There was no expression of Bcl-2 mRNA and Bcl-2 protein in normal brain tissue but 8 hours after injury their expression became evident and then increased, peaked at 2-3 days and remained higher than normal for 5-7 days. The primary expression of Bax-mRNA and Bax protein was high in normal brain tissue. At 20-28 hours they increased and remained high for 2-3 days; on the 7th days they returned to a normal level. In normal brain tissue, p53mRNA and P53 were minimally expressed. Increased expression was detected at the 8th hour, and decreased at 20-28 hours but still remained higher than normal on the 5th day. Conclusions: Following traumatic injury to the human brain, apoptotic neurons appear around the focus of trauma. The mRNA and protein expression of Bcl-2, Bax and p53 and the activity of caspase 3 enzyme are increased.

Spinal cord decompression reduces rat neural cell apoptosis secondary to spinal cord injury

Objective： To determine whether spinal cord decompression plays a role in neural cell apoptosis after spinal cord injury. Study design： We used an animal model of compressive spinal cord injury with incomplete paraparesis to evaluate neural cell apoptosis after decompression. Apoptosis and cellular damage were assessed by staining with terminal deoxynucleotidyl transferase （TdT）-mediated deoxyuridine triphosphate nick-end labelling （TUNEL） and immunostaining for caspase-3, Bcl-2 and Bax. Methods： Experiments were conducted in male Sprague-Dawley rats （n-78） weighing 300-400 g. The spinal cord was compressed posteriorly at T10 level using a custom-made screw for 6 h, 24 h or continuously, followed by decompression by removal of the screw. The rats were sacrificed on Day I or 3 or in Week 1 or 4 post-decompression. The spinal cord was removed en bloc and examined at lesion site, rostral site and caudal site （7.5 mm away from the lesion）. Results： The numbers of TUNEL-positive cells were significantly lower at the site of decompression on Day 1, and also at the rostral and caudal sites between Day 3 and Week 4 post-decompression, compared with the persistently compressed group. The numbers of cells between Day 1 and Week 4 were immunoreactive to caspase-3 and B-cell lymphoma-2 （Bcl-2）-associated X-protein （Bax）, but not to Bcl-2, correlated with those of TUNEL-positive cells. Conclusion： Our results suggest that decompression reduces neural cell apoptosis following spinal cord injury.