Expression and significance of caspase-3 after spinal cord injury in rats:Selection of proper time window of intervention for secondary spinal cord injury / 中国组织工程研究

ABSTRACT

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 prop e r 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, 54SD 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.rats were divided into the control and injury group. Laminectomy was only done on Ts and T9 in the control and the injury group was subdivided into 9 subgroups, in which, the materials were collected on the 4th and 8th hours and on the 1st, 2rd, 3rd, 7th 14th and 21st days successively, 6 rats in each one. After abdominal anesthesia with 30 g/L pentobarbitol sodium,sternal cord on T8 andT9 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 mn×5.0 mm for 5 minutes. After injury, artificial bladder urination was done 3 times at 10:00, 16:00and 22:00 successively everyday till the bladder reflex was established.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, immunohistocheistry 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 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 neuronal apoptosis.pase-3 expression after spinal cord injury in rats of each group.RESULTS: Six rats were maintained in each group and included in result 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 with immunohistochemistry in rats of each group: Very few caspase-3 expressions (2.1±0.5) presented in neurons of spinal cord in normal rat. In 8hoursafter spinal cord injury, caspase-3 expression of positive neurons was increased remarkably (89.2±10.5) and up to the peak (189.6±12.7) in 3 days. Caspase-3 expression of positive cell and apoptotic cell appeared alexpression 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).In the control and 4 hours group, stained cell was seen occasionally and positive cell appeared 8 hours later, mainly localized in gray matter. Afterwards, 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 14th day and 21st 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.