The Effect Of End-to-side Neurorrhaphy And FK1706 On The Recovery Of Bladder Morphology And Function In Rats With Neurogenic Bladder

Background and objectiveNeurogenic bladder(NB)is a neurogenic bladder urethral dysfunction abbreviation,caused by the nerve itself lesions or trauma,surgery,etc.The clinical manifestations of NB are characterized by dysfunction of the bladder detrusor muscle and/or urethral sphincter resulting in abnormal urine storage and voiding.NB patients may eventually die due to urinary tract infection and renal failure.NB is common in clinical and the pathology and pathogenesis of NB is not completely clear,resulting in treatment difficulties.In the past decades,many methods have been used to treat NB.These methods can improve urine storage and urination to some extent,but cannot fundamentally restore the innervation of the bladder,so the clinical effect is not good.Sacral nerve root electrostimulation,developed by Brindley,is the only effective clinical treatment for NB that restores as well voiding as storage,but it is only applicable in complete spinal cord injury and is only successful in experienced,highly specialized hands.In 2001,Xiao et al and Chang and Havton reported that neurorrhaphy is an effective approach,a conclusion echoed by Lin et al in 2011.On the contrary,Rasmussen et al reported no improvement in lower urinary tract function in patients with SCI by neurorrhaphy in 2015.Because neurological anastomosis is controversial for the treatment of NB,it is necessary to study whether neurological anastomosis can treat NB from animal experiments.In 2012,Gao established a NB model through cutting off spinal nerves,and then performed nerve end-to-side anastomosis.The neurological end-to-side anastomosis was proved effectively in treatment of the NB model by retrograde nerve tracing and electrical stimulation.However,it cannot fully prove the effect of nerve end-to-end anastomosis on the recovery of bladder function in NB rats,because there is no urodynamic examination,and urodynamic examination is the gold standard for the diagnosis of NB.Therefore,we need to investigate the effect of nerve end-to-side anastomosis on the recovery of bladder function and morphology of NB rats through urodynamics and morphological examination.An important factor that leads to poor neurological anastomosis is that the growth rate of nerve is too slow.The time it takes for a target organ from denervation to reinnervation has a major impact on the recovery of target organ function.Nerve growth factor(NGF)and other members of the neurotrophin family are critical for the survival and differentiation of neurons within the peripheral and central nervous systems.But inconvenient pharmacokinetics(necessity of injection and poor brain permeability),as well as non-specific effects and toxicity,have limited the clinical usefulness of systemic application of neurotrophic factors.Compounds that enhance neurotrophin signaling and overcome these other barriers might show greater therapeutic potential.Tacrolimus(FK506)is a macrolide immunosuppressant widely used in allogeneic organ transplantation.Domestic and foreign scholars found that FK506 not only has the role of immunosuppression,but also has neuroprotection and promotion of nerve regeneration in clinical applications.However,immunosuppression is unavoidable as a side effect when performing autonomic anastomosis.FK1706 is a non-immunosuppressive derivative of FK506 that has weaker immunosuppressive effects than FK506.FK1706 not only promotes axonal regeneration but also has no immunosuppressive effect and has no significant immunosuppressive effect at doses that exert neuroprotective effects.At present,there is no other report on the application of FK1706 in NB in addition to the preliminary research report of our team.Therefore,it is necessary to establish NB rat model to study the effect of FK1706 on nerve regeneration and and to further improve the bladder function and morphology of NB rats by promoting nerve regeneration is very necessary.The mechanism by which FK1706 promotes axonal regeneration is still unclear.FK506 activity is mediated by binding to members of the FK506-binding proteins(FKBPs),creating a complex that binds to and inhibits the calcineurin phosphatase activity.Calcineurin can inhibit the expression and activity of growth-associated protein 43(GAP43),which is the main component of neuronal growth cone phosphoprotein.Calcineurin is one of the most active proteins in the growth cone during peripheral nerve regeneration and plays an important role in the regeneration of peripheral nerves.Therefore,FK1706 promotes the growth of nerve by promoting the expression and activity of GAP43 by inhibiting calcineurin.Another hypothesis is that FK1706 promotes neurogenesis independent of FKBP12 but binds heat shock protein 90(Hsp90)and promotes the dissociation of heat shock protein 90(Hsp90)from the steroid receptor complex(FKBP52/Hsp90/p23/SR).Dissociated Hsp90 enhances the effect of NGF on promoting nerve regeneration by regulating the Ras/Raf/MEK/ERK signaling pathway.Gold found that immunophilin ligands can promote nerve regeneration in primary knockout of FKBP12 mouse hippocampal cells,indicating that FKBP12 is not necessary for immunophilin ligands to promote nerve regeneration.Gold believe that immunophilin FK506-binding protein 52(not FK506-binding protein 12)mediates the neurotrophic action of immunophilin ligands.This neurite potentiation could be blocked by an anti-FKBP-52 antibody with a dose-dependency,which is suggested that FKBP52 is necessary for immunophilin ligands to promote nerve regeneration.However,it remains unclear how FK1706 promotes nerve regeneration after binding to FKBP52.Therefore,the main purposes of this study include(1)To establish the NB model of lumbosacral spinal nerve transection and anastomosis in adult SD rats,and investigate the effect of neural anastomosis on the morphological and functional recovery of bladder by urodynamic studies and bladder morphometry.(2)To establish the NB model of L6 and L4 spinal nerve anastomosis in adult SD rats,and investigate the effect of FK1706 on nerve regeneration and the effect of FK1706 on bladder function and morphology in NB rats by promoting nerve regeneration after anastomosis.(3)To preliminary investigate the mechanism of FK1706 on axon growth by intervening SH-SY5Y cells with FK1706,NGF and geldanamycin.Part One The establishment of NB model in adult SD rats and study of the bladder function and morphology after end-to-side neruorrhaphyMaterials and Methods1.Fifty male SD(Sprague-Dawley)rats[mean weight(260?300g)]were randomly divided into end-to-side nerve coaptation group(ECG,n=20),no nerve coaptation group(NCG,n= 20),and control group(CG,n=10).In the ECG,the left ventral root(VR)and dorsal root(DR)of L6 and S1 were transected,and the distal stump of L6VR was sutured to the lateral face of L4VR.In the NCG,the left VR and DR of L6 and S1 were transected,but coaptation was not performed.In the CG,no operation was performed.2.Four months later,the rats were anesthetized again,the pelvic ganglia were exposed,and 4%FluoroGold(FG)was injected into the pelvic ganglia using a microinjector.Seven days later,cystometry and electrical stimulation were performed.Bladder was weighed,then the bladder tissue was placed in 10%neutral formalin,embedded in paraffin,sliced,and stained with hematoxylin and eosin(HE)and VG(Van Gieson,VG).The whole body was perfused through the heart with 10%neutral formalin,and then the L3-S1 spinal cord tissue was taken and frozen sections were performed.Record and compare the maximum cystometric capacity(MCC),postvoid residual volume(PVR),bladder compliance(BC),maximal detrusor pressure during urination,area of bladder fibrosis and fluorescent labeled cells in spinal cord tissue of rats in each group.3.Data are presented as mean ± standard deviation(SD).Statistically significant differences among the three groups were evaluated using one-way analysis of variance in SPSS 17(IBM,Armonk,NY).P<0.05 was considered statistically significant.Results1.In the ECG,FG-labeled neurons were observed in the left ventral horn of L4 but not in the left intermediolateral area of L6 and S1.In the NCG,no FG-labeled neurons were seen in L3-S1.In the CG,FG-labeled neurons were mainly located in the left intermediolateral area of L6 and S1,but not in the left ventral horn of L4.2.Maximum cystometric capacity(MCC),postvoid residual volume(PVR),and bladder compliance(BC)were less in the ECG than in the NCG but higher than in the CG.There was no significant difference in maximum detrusor voiding pressure(Pdet.void.max)between the ECG and CG;however,both were greater than in the NCG When the left L6VR was stimulated in the CG,there was a significant increase in intravesical pressure(IVP).The increased maximum pressure was 35.8±3.3mmHg.In the NCG,no change in IVP occurred after stimulation.When we stimulated the left L4VR proximal to the coaptation in the ECG,IVP increased(18.1±2.2mmHg).The differences in increased mean maximum IVP were statistically significant.3.The mean bladder weights of the NCG and ECG rats were significantly heavier than CG rats.Interestingly,the mean bladder weights of the ECG rats were lighter than NCG rats.H&E-stained CG bladder sections showed normal epithelial and smooth muscle layers in SCG rats.In contrast,the smooth muscle layer was irregular and had distinctive edema in the NCG and ECG.Interestingly,the degree of irregularity and edema was lower in the ECG than in the NCG.VG staining revealed significantly different size of the fibrotic area in the CG,NCG,and ECG bladders.Part Two The study of FK1706 on nerve regeneration and bladdermorphology and function recovery in rats with end-to-sideneurorrhaphy.Materials and Methods1.Fifty male SD(Sprague-Dawley)rats[mean weight(260?300g)]were randomly divided into FK1706 and end-to-side nerve coaptation group(FK1706+ECG,n=20),end-to-side nerve coaptation group(ECG,n= 20),and surgical control group(SCG,n=10).In the FK1706+ECG,the left ventral root(VR)and dorsal root(DR)of L6 and S1 were transected,and the distal stump of L6VR was sutured to the lateral face of L4VR,then subcutaneous injection of FK1706 solution was given for 8 weeks.The ECG rats were operated in the same manner as the FK1706 + ECG rats but use 30%DMSO instead of FK1706.In the SCG,the left DR of L6 and VR and DR of S1 were transected,continuous subcutaneous injection of 30%DMSO for 8 weeks postoperative.2.Four months later,nerve regeneration,bladder function and morphology were evaluated by FluoroGold(FG)retrograde tract tracing,cystometry,electrical stimulation,histology assays,nerve fiber count,western blotting and qPCR.3.Data are presented as mean ± standard deviation(SD).Statistically significant differences among the three groups were evaluated using one-way analysis of variance in SPSS 17(IBM,Armonk,NY).P<0.05was considered statistically significant.Results1.Maximum cystometric capacity(MCC),postvoid residual volume(PVR),and bladder compliance(BC)were less in the FK1706+ECG than in the ECG but higher than in the SCG.There was no significant difference in Pdet.void.max among the three groups.When the left L6VR was stimulated in the SCG,there was a significant increase in intravesical pressure(IVP).The mean increased maximum pressure was 34.6±3.8mmHg.When we stimulated the left L4VR proximal to the coaptation in the ECG and FK1706+ECG,IVP increased.The mean increased maximum pressure were 17.8±3.1mmHg and 22.3±2.7mmHg.The mean maximum pressure was greatest in the SCG rats,intermediate in the FK1706+ECG rats,and lowest in the ECG rats.2.The mean bladder weights of the FK1706+ECG and ECG rats were significantly heavier than SCG rats.Interestingly,the mean bladder weights of the FK1706+ECG rats was lighter than ECG rats.H&E-stained CG bladder sections showed normal epithelial and smooth muscle layers in SCG rats.In contrast,the smooth muscle layer was irregular and had distinctive edema in the FK1706+ECG and ECG.Interestingly,the degree of irregularity and edema was lower in the FK1706+ECG than in the ECG.VG staining revealed significantly different size of the fibrotic area in the SCG,ECG,and FK1706+ECG bladders.3.The average number of myelinated axons of the preganglionic PPN was 590.4± 55.6 in FK1706 + ECG rats,392.6 ± 38.8 in ECG rats,and 717.5 ± 29.1 in SCG rats.There are significant differences between the three groups.4.The semi quantitative western blotting results shows that the relative quantity of S100?,GAP43 and ?? tubulin in FK1706+ECG and ECG rats are higher than SCG rats.The relative level of S100?,GAP43 and ?? tubulin in FK1706+ECG rats are the highest,ECG take the second place,while the SCG are the lowest.5.Real-time PCR showed that the level of the three mRNA among the three groups were significantly different.The level of S100? in FK1706 + ECG rats was 2.1 folds of SCG rats,the value of S100? in ECG rats was 1.8 folds of SCG rats.The level of GAP43 in FK1706 + ECG rats was 2.2 folds of SCG rats,the value of GAP43 in ECG rats was 1.5 folds of SCG rats.The level of ??tubulin in FK1706 +ECG rats was 2.3 folds of SCG rats,the level of ??tubulin in ECG rats was 1.9 folds of SCG rats.Part Three The preliminary experiment study of FK1706 enhances thefunction of NGF to promote peripheral nerve axon regeneration andmechanism of action.Materials and Methods1.SH-SY5Y neuroblastoma cells were cultured and made into single cell suspension,the cell density was adjusted to 1 × 105/ml,100?ln of cell suspension was added to each well and spread evenly on a 96-well plate.The cells were divided into control group,NGF(lOng/ml)group,FK1706(lnM)+ NGF group,FK1706 + NGF+ geldanamycin(lOng/ml)group.Three replicates were treated in each group.After culturing for 24h,the p-Raf-1 protein were analyzed by Western blot.Five replicates were treated in each group.After culturing for 96h,the cells were photographed,and the neurite length of the cells were measured.Each repeated three fields.2.Data are presented as mean ± standard deviation(SD).Statistically significant differences among the three groups were evaluated using one-way analysis of variance in SPSS 17(IBM,Armonk,NY).P<0.05was considered statistically significant.Results1.In order to observe the effect of FK1706 on SH-SY5Y neuroblastoma cells and preliminary study of its mechanism of action,the cells were grouped and treated differently.The results showed that the length of neurite outgrowth in NGF group was significantly longer than that in control group,and the length of neurite outgrowth in FK1706 + NGF group was significantly longer than that in NGF group,the difference was statistically significant.In the FK1706 + NGF + Geldanamycin group,the neurite length was shorter than that in the FK1706 + NGF group,with significant difference.2.The relative expression of p-Raf-1 protein in NGF group was significantly higher than that in control group,and the relative expression of p-Raf-1 protein in FK1706 + NGF group was significantly higher than that in NGF group,the difference was statistically significant.In the FK1706 + NGF + Geldanamycin group,the relative expression of p-Raf-1 protein was lower than that in the FK1706+ NGF group,with significant difference.Part four Conclusions1.The transection of L6 and S1 spinal nerves can establish a rat model of neurogenic bladder.2.The new neural reflex pathway can be established between the autonomic nerve of L6VR and somatic nerve of L4VR by end-to-side neurorrhaphy in rats,and the new neural reflex pathway can reinnervate the bladder.3.The bladder function and morphology can be partially improved by L6VR and L4VR end-to-side neurorrhaphy in rats,which is caused by the transection of lumbosacral spinal nerve.4.FK1706 can promote nerve regeneration in neurogenic rats after L6VR and L4VR end-to-side neurorrhaphy.5.FK1706 can improve bladder morphology and function in rats by promoting nerve regeneration after L6VR and L4VR end-to-side neurorrhaphy.6.FK1706 can enhances NGF-induced neurite outgrowth,and it is related to the interaction of dissociated Hsp90 and Raf-1.The dissociation of Hsp90 is achieved after the combination of FK1706 and FKBP52.