Study On The Effects Of Body-weight-support Treadmill Training On Depressive Behaviors Of Rats With Spinal Cord Injury

Background:Spinal cord injury（SCI）caused severe motor and sensory dysfunction in the limbs below the injured segment,which greatly affected the physical and mental health of patients.The incidence of early depressive symptoms in SCI patients was as high as63.9%.If they could not be accurately screened and identified early,depressive symptoms could not only gradually develop into depression,but also hindered the physical recovery process.A literature review found that the current law of changes in early depressive symptoms（depressive behaviors）and related evaluation indicators in individuals with SCI was still unclear,and relevant research was urgently needed to explore.A large number of studies have shown that exercise training was an irreplaceable means of rehabilitation for patients with SCI,and it also significantly prevented and reduced the depressive symptoms of patients with SCI.However,as an important element of exercise training strategy,exercise intensity affected the antidepressant effect,there was currently a lack of relevant research.This study was based on the most commonly used exercise method to promote the recovery of motor function after SCI—body-weight-support treadmill training（BWSTT）.Starting from another key element of exercise training strategy—exercise intensity,SCI male rats were used as the research object to clarify the early depressive behaviors of SCI,serological and molecular biological indicators of time changes and compare the effects of different intensities of BWSTT on the depressive behaviors of rats after SCI,in order to explore the exercise intensity that achieved the optimal rehabilitation of motor function and prevention and alleviation of depressive behaviors at the same time.Because this study needed to explore molecular biological indicators related to depressive behaviors,and involved the limitations of tissue materials and other factors,it was necessary to use animal experiments to evaluate the depressive symptoms of rats through depressive behaviors related measurement indicators.Objectives:To clarify the changing trend of early depressive behaviors,serological and molecular biological indicators in rats with SCI and analyze the underlying mechanism of the changing trend of early depressive behaviors in SCI;On this basis,to determine the start time of BWSTT and compare the effects of different intensities of BWSTT on depressive behaviors in rats after SCI and clarify that the best intensity of BWSTT promoted motor function for rehabilitation and prevention and alleviation of depressive behaviors.Methods:1.Study on the changing trend of early depressive behaviors in rats with spinal cord injury（1）Experimental animal grouping method:72 male SD rats were randomly divided into Normal group,Sham group（only laminectomy,no spinal cord injury）and SCI group.（2）Motor function,depressive behaviors,serological and molecular biological index measurement methods:the three groups of rats of hind limb motor function（BBB score）was measured at 7^th day,14^th day,21^st day,28^th day,and 35^th day after SCI;depressive behaviors were respectively 2^nd to 3^rd day before SCI,5^th to 6^th day,11^th to12^th day,18^th to 19^th day,25^th to 26^th day,32^nd to 34^th after SCI.HPA axis function was measured at 7^th day,21^st day,and 35^th day after SCI（ELISA method to measure serum corticosterone;Western blot analysis of hippocampal protein GR expression level）,hippocampal neurotrophic factor（Western blot analysis of hippocampal protein BDNF and Trk B expression levels）,hippocampal neurogenesis（Immunohistochemical staining analysis of hippocampus protein DCX expression level）.2.Study on the effects of body-weight-support treadmill training at different intensities on depressive behaviors of rats with spinal cord injury（1）Grouping method of experimental animals:40 male SD rats were randomly divided into five groups:Sham group（only received laminectomy,no spinal cord injury,no BWSTT）;Sedentary group（SCI,no BWSTT）;BWSTT-A group（SCI,low intensity:7cm/s）;BWSTT-B group（SCI,moderate intensity:15cm/s）;BWSTT-C group（SCI,high intensity:21cm/s）.（2）BWSTT implementation method:BWSTT was implemented with the aid of the Rodent Robotic Motor Performance System（RRMPS）.BWSTT exercise started in the 14^th day after SCI,5 days a week,once a day,15 minutes each time（without rest）,and body-weight-support was 80%for 3 weeks.（3）The method of measuring the antidepressant effect of different intensities of BWSTT:the measuring method of motor function,depressive behaviors,and serological and molecular biological indicators were the same as"Method 1".Results:1.Study on the changing trend of early depressive behaviors in rats with spinal cord injury（1）Changes in hindlimb motor function of rats in each experimental group at different time pointsBBB score:the BBB score of rats recovered quickly within 21 days after injury,and the plateau began to enter the 21^st day.During the experiment,the BBB score of the SCI group was significantly lower than that of the Normal group and Sham group(F_5.162,54.202=115.583,P<0.001).（2）Changes in depressive behaviors of rats in each experimental group at different time pointsSucrose preference:at the 6^th day after injury,the SCI group was significantly lower than the Normal group（P<0.001）and the Sham group（P<0.05）,and the Sham group was lower than the Normal group（P<0.05）.After the 12^th day,the sucrose preference in the SCI group increased slightly,but both were significantly lower than the Normal group（P<0.001）and the Sham group（P<0.001）;the Sham group began to increase until the 26^th day after SCI reached the normal level.Time in the central area:at the 5^th day after injury,time in central area in the SCI group（P<0.001）and Sham group（P<0.05）was significantly less than that of the Normal group,and the SCI group was significantly less than that of the Sham group（P<0.05）.After injury,the time in the central area of the SCI group continued to drop to the 18^th day,and then stabilized,but it was still significantly lower than the Normal group（P<0.001）and the Sham group（P<0.001）.At the 11^th day after the injury,the Sham group returned to normal levels.Social time:at the 5^th day after injury,the social time of the SCI group（P<0.01）and the Sham group（P<0.05）were significantly less than that of the Normal group,and the SCI group was significantly less than that of the Sham group（P<0.01）.After the injury,the social time of the SCI group continued to decline to the 18^th day,and then stabilized,but it was still significantly lower than the Normal group（P<0.01）and the Sham group（P<0.01）.At the 11^th day after the injury,the Sham group returned to normal levels.Immobility time:at the 34 day after injury,the immobility time of rats in the SCI group was significantly higher than that of the Normal group（P<0.01）and the Sham group（P<0.05）.（3）Correlation between BBB score and depressive behaviors:at the 7^th day,14^thday,21^st day,28^th day and 35^th day after injury,there was no correlation between the BBB score of the SCI group and the time in the central area and social time.（4）Changes in HPA axis function of rats in each experimental group at different time pointsSerum corticosterone:at the 7^th day after injury,the levels of serum corticosterone in the SCI group（P<0.01）and Sham group（P<0.01）were significantly higher than those in the Normal group.At the 21^st day after the injury,the serum corticosterone concentration of the SCI group decreased temporarily,but it was still significantly higher than that of the Normal group（P<0.01）.The concentration of serum corticosterone in the Sham group returned to normal 21^th day after injury.Correlation analysis between serum corticosterone and depressive behaviors:at the 7^th day after injury（P<0.001）,21^st day（P<0.01）and 35^th day（P<0.01）,the concentration of serum corticosterone and sucrose preference in the SCI group were compared strong negative correlation.There was a strong positive correlation between serum corticosterone concentration and immobility time in the SCI group（P<0.01）.At the 7^th day after injury,there was a strong negative correlation between serum corticosterone concentration and sucrose preference in Sham group（P<0.01）.Hippocampal protein GR:at 7^th,21^st and 35^th day after injury,the expression level of hippocampal protein GR in the SCI group was significantly lower than that of the Normal group（P<0.01）and the Sham group（P<0.01）.At the 7^th day after injury,the expression level of hippocampal protein GR in the Sham group（P<0.01）was significantly lower than that in the Normal group（P<0.01）,and then increased,and there was no difference from the Normal group.（5）Changes of hippocampal neurotrophic factors in each experimental group at different time pointsAt 7^th,21^st and 35^th day after injury,the expression levels of hippocampal proteins BDNF and Trk B in the SCI group were significantly lower than those in the Normal group（all P<0.01）and the Sham group（all P<0.01）.During the experiment,the expression levels of hippocampal proteins BDNF and Trk B in the Sham group were not statistically different from those in the Normal group.（6）Changes in hippocampal nerves in each experimental group at different time pointsAt the 7^th day after injury,there was no significant difference in the expression level of hippocampal protein DCX in the SCI group compared with the Normal group and the Sham group.At 21^st day and 35^th day after injury,the expression of hippocampal protein DCX in the SCI group was significantly lower than that of the Normal group（P<0.01）and the Sham group（P<0.01）.During the experiment,the expression level of hippocampal protein DCX in the Sham group was not significantly different from that in the Normal group.2.Study on the effects of body-weight-support treadmill training at different intensities on depressive behaviors of rats with spinal cord injury（1）Comparison of motor function of hind limbs of rats in each experimental groupAfter the end of BWSTT,the BBB score of the BWSTT-A group was slightly higher than that of the Sedentary group,but the difference was not statistically significant.The BBB scores of the BWSTT-B group（P<0.01）and the BWSTT-C group（P<0.001）were significantly higher than those of the BWSTT-A group.（2）Comparison of depressive behaviors of rats in each experimental groupSucrose preference:The sucrose preference of the BWSTT-A group was slightly higher than that of the Sedentary group,but the difference was not statistically significant.The BWSTT-C group（P<0.001）and the BWSTT-B group（P<0.01）were significantly higher than the BWSTT-A group.Time in the central area:Time in the central area of the BWSTT-A group was slightly higher than that of the Sedentary group,but the difference was not statistically significant.The time in the central area of the BWSTT-C group（P<0.05）and the BWSTT-B group（P<0.05）were basically the same,and both were significantly higher than the BWSTT-A group.Social time:The social time of rats in the BWSTT-A group was slightly higher than that in the Sedentary group,but the difference was not statistically significant.The BWSTT-C group（P<0.001）and the BWSTT-B group（P<0.01）were significantly higher than the BWSTT-A group.Immobility time:The immobility time of rats in the BWSTT-A group was slightly lower than that in the Sedentary group,but the difference was not statistically significant.The BWSTT-C group（P<0.01）and the BWSTT-B group（P<0.01）were significantly lower than those in the BWSTT-A group.（3）Comparison of HPA axis function of rats in each experimental groupSerum corticosterone:The concentration of serum corticosterone in the BWSTT-A group was slightly higher than that in the Sedentary group,but the difference was not statistically significant.The BWSTT-C group（P<0.05）and the BWSTT-B group（P<0.05）were significantly lower than those in the BWSTT-A group.Hippocampal protein GR:The level of BWSTT-A group was higher than that of Sedentary group,but the difference was not statistically significant.The expression levels of hippocampal protein GR in the BWSTT-C group（P<0.001）and BWSTT-B group（P<0.001）were significantly higher than those in the BWSTT-A group.（4）Comparison of expression levels of hippocampal neurotrophic factors in rats in each experimental groupHippocampal protein BDNF:The level of BWSTT-A group was slightly lower than that of Sedentary group（P>0.05）.The BWSTT-C group was significantly higher than the BWSTT-B group（P<0.05）.The expression levels of hippocampal protein BDNF in the BWSTT-C group（P<0.001）and BWSTT-B group（P<0.001）were significantly higher than those in the BWSTT-A group.Hippocampal protein Trk B:The level of BWSTT-A group was slightly lower than that of Sedentary group（P>0.05）.The expression level of hippocampal protein Trk B in the BWSTT-C group increased significantly,which was significantly higher than that in the BWSTT-A group（P<0.001）and the BWSTT-B group（P<0.001）.（5）Comparison of hippocampal neurogenesis expression of rats in each experimental groupThe level of BWSTT-A group was slightly higher than that of Sedentary group（P>0.05）.The BWSTT-C group was significantly higher than the BWSTT-B group（P<0.05）.The expression levels of hippocampal protein DCX in the BWSTT-C group（P<0.001）and BWSTT-B group（P<0.01）were significantly higher than those in the BWSTT-A group.Conclusions:1.In the early stage of SCI,the rats showed obvious and lasting depressive behaviors,and the clearer changing trend.2.Serum corticosterone was an important objective evaluation index that reflected the changes of early depressive behaviors in rats with SCI.HPA axis hyperfunction might be the neuroendocrine mechanism of early depressive behaviors changes in rats with SCI.The persistently low expression of hippocampal neurotrophic factors and the suppression of hippocampal neurogenesis were the underlying mechanisms for the deterioration of early depressive behaviors in rats with SCI.3.Moderate and high-intensity BWSTT effectively promoted the rehabilitation of hindlimb motor function in SCI rats.Moderate and high-intensity BWSTT prevented and alleviated depressive behaviors in rats with SCI by improving HPA axis function,increasing the expression level of hippocampal neurotrophic factors,and promoting hippocampal neurogenesis,especially the high-intensity effect was better.The high-intensity BWSTT was based on the conditions of this study and was also conducive to the recovery of the motor function of rats after SCI and the best intensity to prevent and reduce depressive behaviors.