Mechanism Of Sacral Neuromodulation To Alleviate Spinal Cord Injury-induced Detrusor Overactivity

Background and objective:Spinal cord injury(SCI)is a serious disease with complex mechanisms and high morbidity and mortality,which brings huge burden to the economy and society.According to statistics,almost all SCI patients eventually developed neurogenic lower urinary tract dysfunction,which manifested as detrusor overactivity(DO),detrusor-sphincter dyssynergia(DSD)and detrusor acontractility.If not managed properly,these symptoms will cause continuously aggravated urinary retention,urinary incontinence,and serious upper urinary tract damage,which will greatly threaten the life quality and life expectancy of SCI patients.Therefore,relieving bladder dysfunction is essential to protect the upper urinary tract and improve the prognosis of SCI patients.Sacral neuromodulation(SNM)has been used to treat overactive bladder,urge urinary incontinence,non-obstructive urinary retention and defecation dysfunction.However,the application of neurogenic lower urinary tract dysfunction is still controversial.Previous studies have shown that early SNM alleviated DO caused by SCI.However,due to the small number of related studies,the role and mechanism of SNM in neurogenic lower urinary tract dysfunction after SCI have not yet been fully clarified,which needs further research and confirmation.Prostaglandin E2(PGE2)is a major metabolite produced by the oxidation of arachidonic acid catalyzed by cyclooxygenase,which has been found to be closely related to bladder dysfunction.PGE2 plays an important role in the regulation of bladder function by acting on a group of G-protein-coupled receptors.In the bladder,there are four main types of PGE2 receptors,namely E-type prostaglandin receptors 1,2,3 and 4(EP1,EP2,EP3 and EP4).Activation of different types of EP receptors will generate different effect on bladder function.Disorder of PGE2 pathway was found in patients with overactive bladder(OAB)and animal models of DO.Studies have shown increased level of PGE2 released from bladder smooth muscle of SCI rats,suggesting PGE2 signaling may play an important role in the development of DO after SCI.Intervention of the pathologically disordered PGE2 signaling pathways may be a potential mechanism for SNM therapy to alleviate detrusor overactivity,but no relevant research has been reported.Based on those above,our study used rats with complete spinal cord injury at T9-T10 segment as SCI model to explore:(1)the therapeutic effect of SNM on detrusor overactivity in SCI rats;(2)Whether the regulation of PGE2 pathway would be a potential mechanism for SNM to alleviate detrusor overactivity in SCI rats;(3)Whether intervention of PGE2 pathway would alleviate detrusor overactivity after SCI.Through the exploration of the above contents,this study proved the therapeutic effect of SNM in alleviating detrusor overactivity in SCI rats,revealed the potential mechanism of SNM,and provided a new target for the treatment of detrusor overactivity.Methods:1.After establishing SCI rat model,SNM treatment(20 Hz、100μs、0.1-1 V、30 s train duration、80 s training cycle)was performed in early phase of these SCI rats.Body weight,bladder weight,bladder weight/body weight were collected and calculated.H&E and Masson staining were used to evaluate the histological changes of bladder after SNM treatment.Cystometry was used to examine the urodynamic changes of rats after treatment of SNM to evaluate the therapeutic effect of SNM on detrusor overactivity in SCI rats.2.ELISA was used to evaluate the expression of PGE2 in bladders of SCI rats after SNM treatment.Then,expression of EP receptors in bladder after SNM treatment was evaluated by qRT-PCR,Western blot and immunohistochemistry.The effect of the changed components of PGE2 signaling on bladder function were then verified.After screening out the changes of PGE2 signaling after SNM treatment,cystometry was used to verify that SNM alleviates SCIinduced detrusor overactivity by intervening the related changes in PGE2 signaling.3.Overactivation of C-fibers was one of the main pathological mechanisms of bladder detrusor overactivity after SCI.This study aims to explore whether SNM can interfere with the expression of PGE2 signaling pathway by inhibiting C-fibers.By using heavy dose of capsaicin subcutaneously(125mg/kg,divided into 3 injections,25,50,50mg/kg,interval of 12 hours,respectively),desensitization of C-fibers was performed to inhibit the activity of C-fibers in SCI rats.Cystometry was performed to evaluate the alleviation of DO after capsaicin desensitization.ELISA,qRT-PCR,Western blot were used to examine PGE2 signaling to explore whether capsaicin desensitization would cause changes in PGE2 signaling.Cystometry was then used to verify that capsaicin desensitization alleviates SCI-induced detrusor overactivity by intervening the related changes in PGE2 signaling.4.To determine the therapeutic effect of inhibition of EP3 on DO in SCI rats,cystometry was used to examine the changes of bladder function on SCI rats after tail vein injection of different concentration(0.1mg/kg,1.Omg/kg)of EP3 receptor antagonist L-826266.5.To determine the effect of EP3 on bladder function under physiological condition,cystometry was performed to examine the changes of bladder function on sham rats after tail vein injection of different concentration(0.1mg/kg,1.Omg/kg)of EP3 receptor agonist Sulprostone or EP3 receptor antagonist L-826266.Results:1.No significant changes were observed on body weight,bladder weight,body weight/bladder weight ratio after SNM treatment comparing to the control group rats(28 days post SCI surgery).Cystometry results showed that SNM significantly increased intercontration interval,bladder capacity,while the non-voiding contraction(NVC)frequency was significantly decreased.No significant changes in the maximum pressure,baseline pressure,postvoid residual,and NVC amplitude.These results indicated that SNM relieved the DO after SCI.2.The expression of EP3 rather than PGE2 or other EP receptors,was significantly decreased after SNM treatment.EP3 receptor agonist was administered to SNM treatment group rats and control group rats(28 days post SCI surgery).Cystometry results showed that administration of EP3 receptor agonist significantly decreased intercontraction interval and bladder capacity,while NVC frequency was also increased in the control group rats.In SNM treatment group rats,no significant changes were observed in these parameters after administration of EP3 receptor agonist.These results suggested that SNM relieved EP3 receptor agonist-induced DO in SCI rats.3.Comparing to the control group,inhibition of C-fibers using high-dose capsaicin desensitization in SCI rats(28 days post SCI surgery)showed increased intercontraction interval,bladder capacity and reduced NVC frequency in cystometry tests,suggesting inhibition of C-fibers alleviated DO in SCI rats.Comparing to the control group,the expression of EP3 rather than PGE2 or other EP receptors was also decreased after inhibition of C-fibers via capsaicin desensitization.Administration of EP3 receptor agonist significantly reduced intercontraction interval,bladder capacity and increased NVC frequency in control group,while no significant changes in these parameters were observed in capsaicin-desensitized SCI rats,suggesting that inhibition of C-fibers by capsaicin desensitization can alleviate EP3 receptor agonist-induced DO in SCI rats.4.Tail vein injection of different concentration of EP3 receptor antagonist L-826266 significantly increased intercontraction interval,bladder capacity and decreased NVC frequency.No significant changes were observed on maximum pressure,basal pressure,postvoid residual,NVF amplitude after administration of EP3 receptor antagonist.These results indicated that inhibition of EP3 alleviated DO in SCI rats(28 days post SCI surgery).5.After administration of EP3 receptor agonist to sham rats,cystometry showed decreased intercontraction interval and bladder capacity while no significant changes in maximum pressure,basal pressure,postvoid residual,and NVC amplitude.NVC frequency was significantly increased after injection of 1.Omg/kg EP3 receptor agonist.In rats treated with EP3 receptor antagonist,1.Omg/kg L-826266 significantly increased the intercontraction interval and bladder capacity.No significant changes were observed in maximum pressure,basal pressure,postvoid residual,NVC amplitude,and NVC frequency.Conclusion:SNM may alleviate detrusor overactivity in SCI rats by interfering with the expression of EP3 receptors in the bladder.Inhibition of EP3 receptors may be a potential treatment for relieving detrusor overactivity secondary to SCI.