Study On Expression Of CHOP And Caspase-12 In Spinal Cord Injury Rat's

Background:Spinal cord injury(SCI)is a severe health problem worldwide,and it usually causes life-long disability for the patients.Normally,SCI is divided into three phases: the acute,the secondary and the chronic phases.The primary injury is the mechanical impact afflicted directly on the spine,while the secondary injury to the spinal cord is a progress involving a complex cascade of molecular events.The final outcome of Spinal cord injury not only depends on the primary injury,but also on the secondary injury.Secondary injury includes disturbances in ionic homeostasis,local edema,focal hemorrhage,excitotoxicity,presence free radicals and free fatty acids and activation of an inflammatory response Cell death in the form of apoptosis.Therefore,it is difficult to identify the specific mechanism of secondary injury.But with the improvement of modern experimental techniques and testing methods,the secondary pathology of SCI has been made great progress.Endoplasmic reticulum stress plays pivotal role in many diseases,such as:diabetes,Alzheimer's,Parkinson's disease,and so on.Endoplasmic reticulum stress-induced apoptosis were primarily activated by gene transcription CHOP/GADD153 and endoplasmic reticulum-specific cysteine protease caspase-12. CHOP/GADD153 is one of C/EBP a member in transcription factor family.CHOP expression was difficult to detect in the normal status,but significantly induced by endoplasmic reticulum stress,and involved in the regulation of the apoptosis-related cascade gene expression.Caspase-12 is one of the protein on membrane of the endoplasmic reticulum.it will be activated in the event of endoplasmic reticulum stress and trigger apoptosis.We suppose that the cell apoptosis is associated with the strong endoplasmic reticulum stress after SCI,and ultimately cause a variety of neuronal dysfunction,and we do not find any report in domestic.In this study,we try to observe the expression of CHOP and Caspasel2 in acute contusive spinal cord injury model in rats to further explore the mechanism of endoplasmic reticulum stress response in spinal cord injury.Objective:To observe the expression of CHOP and caspasel2 of spinal cord in acute contusive spinal cord injury model.To explore the endoplasmic reticulum stress in the cellular apoptosis after SCI.Methods:Adult male Sprague-Dawley rats aged four months were used.The rats weighing between 150 and 200g,provided by Animal Experimental Center of Zhejiang University.20 Sprague Dawley rats divided to two groups at random,10 rats' spinal cord contusive injuries were produced by using modified Allen's method(using a weight-drop device)after the T10 spinous process and the corresponding vertebral lamina were removed as experimental group.The rest 10 rats received only T10 laminectomies and didn't injury the spinal cord as Sham-operated control group.Spinal cord of two groups of rats were obtained after 12 hours.The BBB score, HR-dyed morphology and immunohistochemistry,Western-blot methods were used to determine the changes in motor function,pathological changes,and CHOP and Caspase-12 Expression Change after SCI.CHOP and Caspasel2 were dyed by immunohistochemistry to observe the number of the dyed cells by means of UTHSCSA Image Tools 3.0.The spinal cords from the same rats were tested for expression of CHOP and Caspasel2 by Western blot.Results:1.The rats were all paralyzed 12 hours after SCI.All of them had serious motor function,the BBB score range from 0 to 1.But the rats in Sham-operated control group had no motor function probrem,Means the success of SCI model.2.Histomorphology of spinal cord:In Sham-operated control group,cell nucleus is round and big,nucleolus clear.The pathological alterations were very apparent in the damaged spinal cord area in the experiment group.3.Immunohistochemistry of spinal cord:CHOP and caspase12 expression of spinal cord in experimental group were 168±12.5(optical density)and 6.3±1.2/mm~2 (positive cell count),which were significantly increased compare to Sham-operated control group with 120.4±5.6(optical density)and 2.9±0.5/mm~2(positive cell count) in,by means of UTHSCSA Image Tools(P＜0.01).4.Western Blotting:The relative absorbance ratio of CHOP and caspase12 in experimental group was 175.6±20.5%and 231.3±17.2%respectively,which were significantly higher than the Sham-operated control group tested by Western blotting (p＜0.01). Conclusions:1.The first domesticly found the expression of CHOP and Caspase-12 were both higher in spinal cord injury rat's model than the control group.2.It is further verified that the Increased ER stress responses are associated with the apoptotic neuronal loss after SCI.Moreover,developing novel pharmacological intervention targeting at inhibiting the expression of CHOP and Caspase-12 to prevent of ER stress-induced cell death represents a promising therapeutic strategy for apoptotic neuronal loss after SCI.