Study On The Effects Of Body-weight-support Treadmill Training On Sperm Quality Of Male Rats With Spinal Cord Injury

Background: Spinal cord injury(SCI)affects patients' physical,psychological and social health.SCI mostly occurs in young men at the peak of fertility,and 90% of them have reproductive dysfunction,and the decline of sperm quality is one of the important reasons.SCI male patients have unique semen characteristics,that is,sperm concentration is normal,sperm motility and viability are decreased,and sperm morphology is abnormal.However,the exact time point when sperm quality began to decline,whether it will continue to decline with time in chronic phase and the reasons for the characteristic changes are not completely clear.Exercise training is an irreplaceable means of rehabilitation for SCI patients.Studies have shown that exercise training can reduce the levels of proinflammatory cytokines and oxidative markers in semen of non-SCI male infertility patients,enhance the antioxidant defense system,and improve the semen quality and sperm DNA integrity.However,the effects of exercise training on sperm quality of SCI male patients have not been studied.Does exercise training also have improving effect on sperm quality of SCI male patients? What are the effects of different intensity exercise training? The solution of these problems can help the clinical rehabilitation nursing staffs to understand the physiological changes of SCI patients more comprehensively,and to clarify the time-varying characteristics and influencing factors of sperm quality.To further understand the effects of exercise training on rehabilitation of SCI male patients.It is of great clinical significance for rehabilitation nursing staffs to carry out comprehensive evaluation for SCI male patients with reproductive needs,select targeted intervention measures and better formulate rehabilitation strategies,and promote the comprehensive rehabilitation of patients.Sperm generation and maturation involve many organs and tissues,and the process is complicated.Due to the limitation of materials and other factors,this study chose male rats as the research objects,and made an in-depth discussion on the sperm quality changes and influencing factors caused by SCI and exercise training after SCI.Objectives: To establish a standardized SCI rat models,and clarify the time-varying characteristics of sperm quality of male rats after SCI and analyze its influencing factors.On this basis,the effects of different intensity body-weight-support Treadmill Training(BWSTT)on sperm quality of SCI male rats were studied.Methods: 1.Study on the method of establishing rat models To design a novel laminectomy auxiliary device(LAD)and evaluate its application effects.24 rats were randomly divided into N group(normal),CONV group(conventional laminectomy)and LAD group(LAD laminectomy).The application effects of LAD was evaluated from the aspects of incision size,success rate,operation time,body weight,motor function(BBB score,RRMPS gait analysis),spinal nerve function(tce MEPs),and oxidative stress(SOD activity and MDA content in serum measured by ELISA).2.Effects evaluation of LAD lamina removal on sperm quality of rats 12 rats were randomly divided into N group(normal)and LAD group(LAD laminectomy).1 week after operation,the sperm quality of epididymis was measured by blood cell counting plate.Serum FSH,LH and T levels,SOD activity and MDA content in testis homogenate,and IL-1?,IL-6 and TNF-? concentrations in seminal plasma were measured by ELISA.HE staining was used to observe testicular tissue structure.TUNEL staining was used to observe the apoptosis of spermatogenic cells in testis.3.Time-varying characteristics and influencing factors of sperm quality of SCI rats 60 rats were randomly divided into Sham group(only do laminectomy without damaging the spinal cord)and SCI group(damage the spinal cord).Six rats in each group were killed respectively at 1,2,3,4 and 5 weeks after SCI.The measurement indexes and methods were the same as "Method 2".4.Effects of different intensity BWSTT on sperm quality of SCI rats 30 rats were randomly divided into Sham group(only do laminectomy without damaging the spinal cord),Sedentary group(injure the spinal cord but do not exercise),BWSTT-A group(low intensity,7cm/s),BWSTT-B group(moderate intensity,15cm/s),BWSTT-C group(high intensity,21cm/s).BWSTT started on the 14 th day after SCI and lasted for 3 weeks.The measurement indexes and methods were the same as "Method 2".In addition,BBB score of hind limb function and Gn RH protein expression in hypothalamus of rats were increased(immunohistochemical staining).Results: 1.Study on the method of establishing rat models(1)General situation: Compared with CONV group,LAD group had smaller incision(1.3cm vs.2.2cm),shorter operation time(4.82±0.67 min vs.14.64±1.62min)and higher success rate(100% vs.88.89%).(2)Weight: 1 week after operation,there was no significant difference in weight gain between CONV group and LAD group,but they were lower than that of N group(both P<0.01).There was no significant difference in weight gain among the three groups at 2 and 3 weeks after operation.(3)Hindlimb motor function: After awakening from anesthesia,compared with N group,the step height in CONV group decreased significantly(left: P<0.01,right: P<0.05),but there was no significant change in LAD group;There was no significant difference in step length among the three groups.1 week after operation,there was no significant difference in step height and step length among the three groups.(4)Spinal nerve conduction function: 1 day and 1 week after operation,there was no significant difference in latency and amplitude of spinal nerve conduction among the three groups.(5)Serum SOD activity and MDA content: 1 week after operation,compared with N group,SOD activity in CONV group and LAD group decreased significantly(both P<0.01);MDA content in CONV group increased significantly(P<0.05),there was no significant change in LAD group.2 and 3 weeks after operation,there was no significant difference in SOD activity and MDA content among the three groups.2.Effects evaluation of LAD lamina removal on sperm quality of rats(1)Sperm quality: There was no significant difference in count,viability,rapid forward movement and abnormal morphology between LAD group and N group.(2)HPT axis function: There was no significant difference in serum LH,FSH and T levels between LAD group and N group.(3)Testicular tissue structure and spermatogenic cell apoptosis: In LAD group and N group,the seminiferous tubules in testicular tissue were complete in structure and the cells were arranged orderly.There was no significant difference in apoptosis index(AI)of spermatogenic cells.(4)Oxidative damage of testis and inflammation of seminal plasma: SOD activity,MDA content of testis;IL-1?,IL-6,TNF-? concentration of seminal plasma between LAD group and N group had no significant difference.3.Time-varying characteristics and influencing factors of sperm quality of SCI rats(1)Sperm quality: Sperm count did not change significantly after SCI.Viability and rapid forward movement rate began to decrease 1 week after injury,and decreased to the lowest level 2 weeks(both P<0.01).The incidence of abnormal sperm morphology did not change significantly at 1 week after injury,but increased significantly at 2 weeks(P<0.01).After 3 weeks of injury and later,sperm parameters improved slightly and remained stable,but they were worse than those in Sham group.(2)HPT axis function: Serum T level decreased significantly at 1 week(P<0.05)and 2 weeks(P<0.01)after SCI,and returned to normal at 3 weeks,then remained stable.Serum LH and FSH levels had no significant changes.(3)Testicular tissue structure and spermatogenic cell apoptosis: 1 week after SCI,the seminiferous tubule structure of some testicular tissues was destroyed,and the AI of spermatogenic cells increased(P<0.01).The change was most significant 2 weeks(P<0.001)after SCI and improved slightly after 3 weeks and remained stable,but they were worse than those in Sham group(P<0.01).(4)Oxidative damage of testis and inflammation of seminal plasma: SOD activity of testis decreased significantly,and MDA content increased significantlyafter SCI.The concentrations of IL-1?,IL-6 and TNF-? in seminal plasma increased significantly.These changes were the most obvious 1 week after injury,and then improved slightly and remained stable,but there were statistical differences compared with Sham group.4.Effects of different intensity BWSTT on sperm quality of SCI rats(1)BBB score of hind limbs: BWSTT-A was higher than Sedentary(P<0.05);BWSTT-B and BWSTT-C were higher than BWSTT-A(both P<0.05).(2)Sperm quality: Compared with Sedentary group,there was no significant change in sperm count,viability and rapid forward movement rate in BWSTT-A group,but the incidence of abnormal morphology increased significantly(P<0.05);BWST-B group and BWST-C group had no significant change in count and viability,while the rate of rapid forward movement decreased significantly(both P<0.05)and the incidence of abnormal morphology increased significantly(both P<0.001).The abnormal morphology of sperm in BWST-C group was higher than that in BWSTT-A group(P<0.05).(3)HPT axis function: Compared with Sedentary group,there was no significant change in serum LH,FSH,T levels and hypothalamic Gn RH protein expression in BWSTT-A group;serum FSH,T levels and hypothalamic Gn RH protein expression were significantly decreased in BWSTT-B and BWSTT-C groups(all P<0.05);serum LH level in BWSTT-C group also decreased significantly(P<0.05).Compared with BWSTT-A group,serum T level in BWSTT-B group decreased significantly(P<0.05);serum FSH and T levels and hypothalamic Gn RH protein expression in BWSTT-C group were significantly decreased(all P<0.05).(4)Testicular tissue structure and spermatogenic cell apoptosis: The structure of seminiferous tubules in BWSTT-A group was slightly worse than that in Sedentary group.In BWSTT-B and BWSTT-C groups,the testicular tissue structure was obviously broken,and the sperm in the seminiferous tubule decreased obviously.The AI of spermatogenic cells in BWSTT-A group was higher than that in Sedentary group(P<0.001);Group BWSTT-B and BWSTT-C were higher than group BWSTT-A(both P<0.001). (5)Oxidative damage of testis and inflammation of seminal plasma: Compared with Sedentary group,SOD activity and MDA content of testis in BWST-A group had no significant changes;In BWST-B and BWST-C groups,SOD activity decreased significantly(both P<0.05)and MDA content increased significantly(both P<0.01);MDA content in BWST-B group and BWST-C group was higher than that in BWST-A group(both P<0.05).Compared with Sedentary group,the concentrations of IL-1?,IL-6 and TNF-? in seminal plasma of BWSTT-A group had no significant changes;The TNF-? concentration in BWSTT-B and BWSTT-C groups increased significantly(both P<0.05).There was no significant difference in the concentrations of IL-1?,IL-6 and TNF-? in seminal plasma among BWSTT-A,BWSTT-B and BWSTT-C groups.Conclusions: 1.In this study,a novel laminectomy auxiliary device(LAD)was designed,which is more suitable for laminectomy in SCI modeling process than conventional laminectomy method.It is also suitable for lamina removal of SCI rat models for sperm quality research.2.The time-varying characteristics of sperm quality of SCI rats are as follows: the sperm quality began to decrease 1 week after injury,then decreased to the lowest level in 2 weeks,and improved slightly and remained stable in chronic stage,but it was always lower than the normal level.The parameters are characterized by normal count,decreased viability and fast forward movement rate,and abnormal morphology.3.After SCI,the function of HPT axis was temporarily inhibited,which may be involved in the decline of sperm quality in early stage.The persistent oxidative damage in testicular tissue after SCI is an important reason for abnormal spermatogenesis in testis.Inflammation of seminal plasma persists after SCI,which is also an important reason for poor sperm quality.4.BWSTT promotes the rehabilitation of hind limb motor function of rats,and the higher the intensity,the better the rehabilitation effect.However,BWSTT aggravated the decline of sperm quality of SCI rats,and the higher the intensity,the more obvious the decline was. 5.BWSTT can inhibit the function of HPT axis,enhance the oxidative damage of testicular tissue,seriously damage the microenvironment of spermatogenesis and maturation;BWSTT can also increase the production of inflammatory cytokines in seminal plasma,which plays an important role in the decline of sperm quality induced by exercise.