| Chapter 1The expression of autophagy in the process of neurogenic bladder in different stages after spinal cord injury in ratsObjectiveTo investigate the expression of autophagy in different stages of neurogenic bladder after spinal cord injury in rats.MethodsThe total of 36 wistar rats were divided into the spinal cord injury group and the sham-operated group,6 experimental animals were sacrificed and sampled from each group at 1 d,4 d and 14d after surgery.BBB scale,residual urine volume,and urinary bladder function score was measured at each time point.The expression of LC3 and P62 was detected using western blot analysis,immunofluorescence analysis and RT-PCR analysis.ResultsWe found that the hindlimbs of the rats of the SCI group lost locomotor functions after surgery and were dragged behind.Starting at 1d,the locomotor functions of the rats in SCI group was gradually recovered.The urinary bladder function was damaged after surgery,but was recovered gradually.Western blot analysis showed that the LC3-II/actin was higher in the SCI group compared with the control group.The expression of LC3-II was reached the peak at 4d in the SCI group.Immunofluorescence analysis showed that the expression of LC3Ⅱ proteins,increased in the SCI group than in the control group at 1d,4d and 14d.In addition,P62 was founded at 1d,decreased at 4d,and lasted at 14d.RT-PCR analysis showed that LC3mRNA was significantly increased in the rats of the SCI group compared to the rats of the control group at 1d,4d(P<0.01),and 14d(P<0.05)after surgery.It was revealed that the expressing level of LC3mRNA was higher at 4d compared to 1d,and that was less at 14d than 4d.And it was no significant difference of P62mRNA level of the bladder tissues in the SCI group compared to the bladder in the control group at 1d after surgery.However,it was found that the P62mRNA level was significantly decreased at 4d in the SCI group compared to the control group.And at 14d,P62mRNA level of the bladder tissues was less in the SCI group than the ones in the control group,but it was showed that the expression level of P62mRNA was higher at 14d than 4d.ConclusionAutophagy is activated in the recovery of the bladder after spinal cord injury,and sustained over a period time.Autophagy may play an important role in the process of neurogenic bladder and may represent one of the mechanisms of bladder self-repair.Chapter 2The effects of 3-MA on neurogenic bladder after spinal cord injury in rats by inhibiting autophagyObjectiveTo observe the effect of the neurogenic bladder after spinal cord injury in rats and the expression of autophagy in the urinary muscle cells by the intervention of 3-MA of autophagy inhibitor.MethodsThe total of 27 wistar rats were randomly divided into sham-operated group,the control group,and the 3-MA injection group.All groups were subjected to kinematic scoring,residual urine volume and urinary bladder function score at 1 d,4 d and 14d after surgery.Three experimental animals were sacrificed and sampled from each group at every time point.The expression of LC3 and P62 was detected using western blot analysis,immunofluorescence analysis and RT-PCR analysis.ResultsThe hindlimbs of the rats of the control and 3-MA group lost locomotor functions after surgery and were dragged behind.The BBB scores were lower in the 3-MA groups compared to the control group(P<0.05).The residual urine volume of control group and 3-MA group reduced gradually from 1d to 14d after surgery,especially among 3d to 9d.However,the residual urine volumes were significantly increased in the3-MA groups compared to the control group(P<0.05).The urinary bladder function scores were lower in the 3-MA group compared to the control group at 1d,9d,12d,14d(P<0.05),4d,6d(P<0.01).Western blot analysis showed that the LC3-Ⅱ/actin was decreased in the 3-MA group compared with the control group(P<0.05).Immunofluorescence staining results showed that the positive red dots,which represent the expression of LC3Ⅱ proteins,decreased in the 3-MA group than in the control group at 1d,4d and 14d.In addition,P62 was founded increased in the 3-MA group than in the control group at 4d and 14d.The RT-PCR analysis results showed that LC3mRNA was significantly decreased in the rats of the 3-MA group compared to the rats of the control group at 1d,4d(P<0.01),and 14d(P<0.05)after surgery;at 4d and 14d,P62mRNA level was higher in the 3-MA group than in the control group(P<0.05).Conclusion3-MA inhibited the expression of autophagy,and prevented the recovery of bladder function and motor function of lower extremity.And further confirmed that autophagy in the development of neurogenic bladder after spinal cord injury may play a role in the development of bladder detrusor,and may be involved in self-repair.Chapter 3The effect of rapamycin on neurogenic bladder after spinal cord injury in ratsObjectiveTo observe the effect of the neurogenic bladder after spinal cord injury in rats by the intervention of rapamycin.MethodThe total of 15 wistar rats were randomly divided into sham-operated group,the control group,and the rapamycin injection group.All groups were subjected to kinematic scoring,residual urine volume and urinary bladder function score at 1d,3d,5d,7d,9d,11d and 14d after surgery.ResultsThe hindlimbs of the rats of the control and rapamycin group lost locomotor functions after surgery and were dragged behind.Starting at 1d,the locomotor functions of the rats in control and rapamycin group was gradually recovered.The BBB scores were higher in the rapamycin groups compared to the control group at 3d,5d,9d(P<0.05)and 11d,14d(P<0.01)after surgery.The residual urine volumes were significantly decreased in the rapamycin groups compared to the control group(P<0.05)at 7d,9d,11d,14d after surgery.The urinary bladder function scores were no difference at 1d,3d,5d,7d after surgery between the rapamycin group and control group,and higher in the rapamycin group compared to the control group at 9d,11d,14d(P<0.05).ConclusionIntraperitoneal injection of rapamycin promoted the recovery of hindlimbs locomotor function and bladder function of rats after spinal cord injury.Autophagy is expressed in neurogenic bladder after spinal cord injury,and may promote the function of bladder function recovery. |
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