大鼠脊髓损伤后半胱氨酸天冬氨酸蛋白酶3的表达及意义 继发性脊髓损伤适宜干预时间窗的选择(英文)

Expression and significance of caspase-3 after spinal cord injury in rats:Selection of proper time window of intervention for secondary spinal cord injury

背景:半胱氨酸天冬氨酸蛋白酶家族被认为是细胞凋亡的执行因子,脊髓损伤后可发生神经细胞凋亡。目的:观察大鼠脊髓损伤后半胱氨酸天冬氨酸蛋白酶3的表达变化,探讨其与神经细胞凋亡发生的关系,为判定减轻继发性脊髓损伤的适宜干预时间窗提供依据。设计:自身对照、相互对照动物实验。单位:华中科技大学同济医学院附属同济医院创伤外科、骨科。材料:实验于2001-09/12在华中科技大学同济医学院附属同济医院骨科实验室完成。54只SD大鼠,雌雄不限,体质量220～250g,由华中科技大学同济医学院动物实验中心提供。方法:①动物模型建立及分组:将大鼠分为对照组和损伤组,对照组仅做T8,T9椎板切除术,损伤组于4、8h和1,2,3,7,14和21d取材共9组,每组6只。以30g/L戊巴比妥钠腹腔麻醉后,按Nystrom法暴露T8,T9节段胸髓,将50g重物通过2.2mm×5.0mm弧型光滑金属垫片压迫该段脊髓后正中部5min,损伤后每天10:00,16:00和22:003次人工膀胱排尿,直至建立反射性膀胱排空。②取材及切片准备:在脊髓损伤后各时间点完整取出脊髓全长,将每组4只损伤段脊髓组织块,长约8mm,石蜡包埋,连续切片,分别行苏木素伊红染色、免疫组化及原位末端脱氧核苷酸转移酶介导的脱氧三磷酸尿苷缺口末端标记法(TdT-mediateddUTP-biotinnickendlabeling,TUNEL)标记;每组2只在冰上处死,将损伤脊髓中心组织置入液氮罐中保存备用。③检测指标:以免疫组化法检测半胱氨酸天冬氨酸蛋白酶3的表达,以逆转录-聚合酶链反应半定量测定半胱氨酸天冬氨酸蛋白酶3mRNA的表达变化,以TUNEL法检测神经细胞的凋亡水平,并以直线相关分析半胱氨酸天冬氨酸蛋白酶3的表达和神经细胞凋亡的相关性。主要观察指标:①光镜下各组大鼠脊髓损伤观察。②免疫组化结果。③各组大鼠脊髓损伤后半胱氨酸天冬氨酸蛋白酶3的表达变化。④各组大鼠脊髓损伤后神经细胞凋亡情况及与半胱氨酸天冬氨酸蛋白酶3的表达的相关性。结果:纳入结果分析的各组动物数均为6只。①光镜下各组大鼠脊髓损伤观察结果:损伤段1h有广泛出血;4～8h脊髓结构开始破坏,大量的神经元死亡;24h脊髓破坏严重;7～21d损伤范围确定,脊髓内有空洞形成。②免疫组化检测的各组大鼠半胱氨酸天冬氨酸蛋白酶3表达的结果:正常大鼠脊髓神经细胞中很少有半胱氨酸天冬氨酸蛋白酶3表达(2.1±0.5);脊髓损伤后8h,半胱氨酸天冬氨酸蛋白酶3表达阳性的神经细胞明显增加(89.2±10.5),3d达到高峰(189.6±12.7);半胱氨酸天冬氨酸蛋白酶3表达的阳性细胞与凋亡细胞出现的时限相似,呈正相关(r=0.941)。③逆转录-聚合酶链反应检测的各组大鼠半胱氨酸天冬氨酸蛋白酶3表达的结果:半胱氨酸天冬氨酸蛋白酶3mRNA4h开始增高(0.442±0.024),48h达到高峰(0.634±0.028),7d后恢复正常(0.351±0.013),早于凋亡出现的时期,与神经细胞凋亡水平呈正相关(r=0.622)。④TUNEL标记及计数的各组大鼠半胱氨酸天冬氨酸蛋白酶3表达的结果:对照组及4h组仅见偶染细胞,8h后开始出现阳性细胞,主要位于灰质中;此后阳性细胞逐渐增多,3d达到高峰,7d灰质中凋亡染色的阳性细胞逐渐减少,主要在周围白质中,14和21d可见少量的阳性细胞。结论:在正常的脊髓组织半胱氨酸天冬氨酸蛋白酶3是以活性很低的酶原形式存在;大鼠脊髓损伤后半胱氨酸天冬氨酸蛋白酶3表达增强,8h开始大量表达,24～48h达到高峰,与TUNEL所检测的阳性凋亡细胞在时间上相重叠,从半胱氨酸天冬氨酸蛋白酶3阳性细胞先后出现的区域上看,与脊髓损伤阳性凋亡细胞一致,可见半胱氨酸天冬氨酸蛋白酶3参与了脊髓损伤细胞凋亡的调节。从本实验可以看出,从脊髓损伤后到半胱氨酸天冬氨酸蛋白酶3活化这段时间是脊髓损伤干预细胞凋亡、减轻继发性脊髓损伤治疗时间窗,应用基因干预或特异性半胱氨酸天冬氨酸蛋白酶抑制剂宜在48h内应用。

BACKGROUND： Caspase family is viewed as the executive factor of cell apoptosis. Neuronal apoptosis happens probably after spinal cord injury. OBJECTIVE： To observe the changes in caspase-3 expression after spinal cord injury in rats so as to probe into the relationship between it and neuronal apoptosis and provide the evidence on the proper time window of intervention on alleviating secondary spinal cord injury. DESIGN： Self-control and mutual-control were designed in animal experiment. SETTING： Department of Traumatic Surgery and Department of Orthopedics of Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology. MATERIALS： The experiment was performed in Experiment Room of Tongji Hospital affiliated to Tongji Medical College of Huazhong University of Science and Technology from January to December 2001, in which, 54 SD rats were employed, of either sex, mass weighted varied from 220 to 250 g and provided from Animal Experimental Center of Tongji Medical College of Huazhong University of Science and Technology. METHODS： ① Establishment of animal model and group division： The rats were divided into the control and injury group. Laminectomy was only done on T8 and T9 in the control and the injury group was subdivided into 9 subgroups, in which, the materials were collected on the 4^th and 8^th hours and on the 1^st, 2^rd, 3^rd, 7^th, 14^th and 21^st days successively, 6 rats in each one. After abdominal anesthesia with 30 g/L pentobarbitol sodium, sternal cord on T8 and T9 segments were exposed with Nystrom method and 50 g weight compressed the front middle region of the spinal cord of such segments with arch smooth metal pad 2.2 mm×5.0 mm for 5 minutes. After injury, artificial bladder urination was done 3 times at 10：00, 16：00 and 22：00 successively everyday till the bladder reflex was established. ② Selection of materials and preparation of slices： The complete spinal cord was collected at various time spots after spinal cord injury. 4 pieces of spinal cord tissue masses from each group, about 8mm in length, were embedded with paraffin and sectioned continuously. Afterwards, HE staining, immunohistocbeistry and TUNEL （TdT-mediated dUTP-biotin nick end labeling） were performed successively. Two rats were sacrificed on ice in each group and central tissue of injured spinal cord was placed in liquid nitrogen jar to preserve. ③ Determination of indexes： Caspase-3 expression was assayed with immunohistochemistry method, neuronal apoptosis was assayed with TUNEL method and linear correlation was used to analyze the correlativity between caspase-3 expression and neu- ronal apoptosis. MAIN OUTCOME MEASURES： ① Observation of spinal cord injury in rats of each group under optic microscope. ② Results of immunohistochemistry. ③ Changes in caspase-3 expression after spinal cord injury in rats of each group. ④ Correlativity between neuronal apoptosis and caspase-3 expression after spinal cord injury in rats of each group. RESULTS： Six rats were maintained in each group and included in result analysis. ① Observation of spinal cord injury in rats of each group under optic microscope： Extensive hemorrhage appeared in 1 hour in injured segment. In 4 to 8 hours, spinal structure began destructive and a large amount of neuronal death appeared. In 24 hours, the destruction of spinal cord became severe and in 7 to 21days, the range of injury was defined and cavitation appearedl in spinal cord. ② Caspase-3 expression assayed with immunohistechemistry in rats of each group： Very few easpase-3 expressions （2.1±0.5） presented in neurons of spinal cord in normal rat. In 8 hours after spinal cord injury, caspase-3 expression of positive neurons was increased remarkably （89.2：1：10.5） and up to the peak （189.6：1：12.7） in 3 days. Caspase-3 expression of positive cell and apoptotic cell appeared almost at same time, indicating positive correlation （r=0.941）. ③ Caspase-3 expression assayed with transcription-polymerase chain reaction （RT-PCR） in rats of each group： Caspese-3 mRNA （0.442±0.024） began increased in 4h, was up to the peak （0.634±0.028） in 48 hours and was restored to be normal （0.351±0.013） in 7 days, which appeared early than apoptosis, indicating positive correlation with the level of neuronal apoptosis （r=0.622）. ④ Caspase-3 expression with TUNEL and counting in rats of every group： In the control and 4 hours group, stained cell was seen occasionally and positive cell appeared 8 hours later, mainly localized in gray matter. Mterwards, positive cell was increased and up to the peak in 3 days. In 7 days, positive cell of apoptosis and staining was decreased gradually in gray matter, mainly around the white matter. Little amount positive cells appeared on the 14^th day and 21^st day. CONCLUSION： In normal spinal cord tissue, caspase-3 existed in form of zymogen with very low activity. Caspase-3 is enhanced in expression after spinal cord injury in rats, expresses in large amount in 8 hours and is up to the peak in 24 to 48 hours, which is overlapped in time with positive apoptotic cell assayed with TUNEL and concerning to the localization, it is in conformity with positive apoptotic cell of spinal cord injury compared with positive cell of caspase-3. It is indicated that caspase-3 is involved in regulation of cell apoptosis after spinal cord injury. It is seen in this experiment that the time from spinal cord injury to the activation of caspase-3 is the time window of treatment for cell apoptosis intervened by spinal cord and alleviating secondary spinal cord injury. It is suggested that genetic intervention or specific caspase-3 inhibitor should be applied in 48 hours.