| Objective: To establish the model of moderate-severe acute thoracic cord injury with clip compression in rats,through observing behavior and histomorphology changes whthin 12 weeks and detecting the related indexes of inflammatory pathway within 1 week after spinal cord injury in rats,to explore the effect of every-other-day fasting(EODF)on the motor function recovery after acute thoracic cord injury with clip compression in rats,and to preliminarily discusses the effect of EODF on inflammation response mechanism after moderate-severe spinal cord injury.Providing theoretical foundation and experimental evidence for the application of spinal cord injury with EODF.Methods: The study was conducted in two experiments.(1)In experiment one,SPF female adult SD rats in experiment one were randomly divided into EODF group(model+EODF),AL group(model+ad libitum)and Sham group(Sham-operated+ad libitum).The models of T10 spinal cord injury were established in rats with medical temporary cerebral aneurysm clip.EODF Group was started immediately fasting for 24 h after operation,then ad libitum for 24 h,again fasting for 24 h,repeated until the experiment completed,free drinking water in the whole process.AL and Sham groups were given a normal diet immediately and free drinking after operation.At pre-operation and post-operation 2d,1w,2w,4w,6w,8w,10 w,12w,inclined plane test were calculated in each group.Then put the rats to death at 12 w.After paraffin sectioning of 1cm long injured spinal cord tissue with the changes of histological structure was observed by Hematoxylin-eosin(HE)staining.(2)In experiment two,rats were randomly divided into EODF group,AL group and Sham group,and 30 rats were in every group.Interventions and models were identical with experiment one.The rats were euthanized at 6h,12 h,1d,3d and 7d after operation,then tested injured spinal cord segments.There was IKK? kinase activity kit used to dissect the kinase activity.The content of NF-?B p65 was tested by Western blotting.ELISA was used to detect the level of IL-1?,IL-10 in serum.Preliminary probing the effect of EODF on inflammation response mechanism after ASCI in rats.Results:1.The effect of EODF on the motor function and spinal cord tissue structure after SCI with clip compression in rats(1)Body weight Body weight of rats pre-operation in three groups have no significant difference(P>0.05).The body weight of rats in EODF and AL group decreased after operation,then gradually increased,which were less than Sham group(P<0.05).Compared with the Sham group at 12 w after operation,body weight of rats in EODF group was reduced by 19% and body weight of rats in AL group was reduced by 7%.(2)Inclined plane test The angles of inclined plane test of rats in three groups were found no statistically significant differences(P>0.05).In Sham group angles of various time points have no significant difference(P>0.05).In EODF and AL groups the hind limbs of the rats were completely paralyzed after operation,and the angles of inclined plane test at each time point after operation were less than those of Sham group(P< 0.05).In EODF and AL groups at 1w after operation,the angles of inclined plane test have no obvious change,then the angles of inclined plane test showed an increasing trend.At 4w?6w?8w?10w?12w postoperatively,the angles of inclined plate test were higher than those of Sham group,the difference was statistically significant(P<0.05).(2)HE staining results: In Sham group the structure of the spinal cord were integrity,the boundary between white and gray matter were clear,neurons were rich which structure was complete,a large number of Nissl bodies may be seen in cytoplasm.In AL group and EODF group the boundary between white and gray matter were unclear,different degrees of necrosis appeared in the damaged region,Nissl bodies in residual neurons decreased or disappeared,accompanied by varying degrees of infiltration of lymphocyte and microglia,nerve fiber were edema which arrangement were disordered.The pathological results in EODF group were significantly improved compared with those in AL group.2.The effect of EODF on inflammation mechanism after moderate-severe acute spinal cord injury(1)The specific kits detect IKK? kinase activity: In Sham group at each time point the results through pairwise comparison were not statistically significant(P>0.05).At 6h,12 h and 1d,EODF group and AL group with a significant rise compared with Sham group(P<0.05).In EODF group IKK? kinase activity showed a similar trend to AL group,which rising first and falling later,at 12 h after operation to peak,at 3d later returned to the level of Sham group,significant difference between two groups appeared only at 12h(P<0.05).(2)Western blot test the expression of NF-?B p65 : In Sham group there was no significant difference between two time points(P>0.05).At 6h,12 h and 1d,in EODF and AL groups the expression of NF-?B p65 were more than Sham group,the difference were statistically significant(P<0.05).EODF group curved trend with rising first and falling later,and was similar to AL group.EODF group reached a peak at 12 h,then started to decline at 1d,while AL group arrived a peak at 12 h,then went down,and two groups returned to sham-operation levels at 3d(P>0.05).Only at 12 h and 1d there was a significant difference between EODF group and AL group(P<0.05).(3)ELISA detect IL-1? in serum: In Sham group there were no significant difference in serum IL-1? level between five time points(P>0.05).Compared with Sham group at 6h,12 h,1d after operation,the level of IL-1? in serum in EODF group and AL group were significantly higher(P<0.05).EODF group and AL group presented rising first and falling later,at 12 h to a peak,then dwon,and at 7d back to the control level,there were no statistical significance between two groups at any time point(P>0.05).(4)ELISA detect IL-10 in serum: In Sham group and AL group there was no significant difference between any two time points(P>0.05).Compared to Sham group.The level of IL-10 in serum in AL and EODF groups have increased significantly at every time point(P<0.05),and the contrast of IL-10 in serum in EODF group rised gradually over time.At 1d,3d,7d after operation the content of IL-10 in serum in EODF group were more than AL group,however,at each time point there were no statistical significance between two groups(P>0.05).Conclusions:1.Long-term EODF treatment may promote behavioral changes in rats after moderate-severe thoracic spinal cord injury with clip compression,and reduce the degree of pathological damage of the injured spinal cord tissue,thereby it is confirmed that EODF have obvious effect on rats of moderate-severe thoracic spinal cord injury model with clip compression.2.EODF treatment for 1 week has no significant improvement in inflammation response after moderate-severe acute spinal cord injury. |
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