| ObjectiveOn the basis of previous studies,we intend to establish a rat model of spinal cord injury by means of heavy object impact method,detect the bladder compliance of the rat model of spinal cord injury with urodynamics,screen out the bladder model of low compliance,observe the effect of electroacupuncture on the bladder compliance of the rat model of low compliance,discuss the mechanism of electroacupuncture regulating the bladder compliance of low compliance neurogenic bladder based on TRPV1 channel,and demonstrate the specificity of acupoint effect.The research is also to provide experimental basis for the current acupuncture and acupoint theory of traditional Chinese medicine,and to provide necessary theoretical basis for the clinical treatment of low compliance neurogenic bladder.Method80 adult female SD rats of SPF rank were randomly divided into two groups:sham operation group and neurogenic bladder model group after spinal cord injury(10 rats and 70 rats respectively).After 12 weeks of feeding,60 low compliant bladder models were selected from the neurogenic bladder model group after spinal cord injury.They were randomly divided into 10 rats in low compliant bladder models group,10 rats in TRPV1 activators group,10 rats in TRPV1 inhibitors,10 rats in EA Guanyuan points group,10 rats in EA non acupoints group,and 10 rats in EA Guanyuan points+TRPV1 inhibitors group.in the sham operation group,only the intact dura mater was exposed surgically,and the incision muscles and skin were sutured layer by layer,after the whole width of spinal cord was exposed.In the model group:only make model,no treatment.In TRPV1 activator group:intravesical instillation of TRPV1 activator capsaicin.In the TRPV1 inhibitor group:capsazepine,a TRPV1 inhibitor,was infused into the bladder.In the group of electroacupuncture Guanyuan point:electroacupuncture Guanyuan point treatment.Lh202h type Korean instrument was adopted,and dilatational wave was selected with frequency 2Hz/15Hz and intensity ImA.In the group of electroacupuncture non meridians and non acupoints:the acupuncture point was located at 1 cm beside the level of left side of Guanyuan point,and the needling method is to stab outward 15° horizontally.In the electroacupuncture at Guanyuan point+TRPV1 inhibitor group:after 30min of intravesical instillation of TRPV1 inhibitor capsazepine,electroacupuncture at Guanyuan point was given.The intervention time of each intervention group was 30 minutes,once a day for 14 days.At the end of the intervention,the indexes of urodynamic bladder compliance in each group were detected first,then the bladder materials were taken and fixed in turn to detect the indexes.After 14 days of intervention,for each group,the bladder compliance of rats was measured by urodynamic method.The verhoeff van Gieson staining method was used to detect the expression and proportion of collagen and elastic fibers in detrusor of rat bladder.The ultrastructures of detrusor of bladder in rats were examined by electron microscopy method and the relative gray levels of mitochondria of detrusor cells in each group were calculated.The method of detrusor muscle strip experiment in vitro was used to detect the frequency of detrusor muscle and the minimum contractile tension in rat bladder.The Immunohistochemistry was used to detection of a-SMA expression in detrusor muscle of rat bladder.The immunofluorescence double labeling was used to detect the co expression and relative expression of icc-lc cell receptor c-kit and receptor p2x5 in detrusor of rat bladder.Western blot was used to detect the protein expression level of TRPV1 and P2X5 in rat bladder.The detection of TRPV1 and P2X5 gene expression in rat bladder by RT-PCR.The statistical analysis was carried out after the completion of index test.Result1.The effect of electroacupuncture at Guanyuan point on urodynamic bladder compliance in model rats:The model group was lower than the group of sham operation(P<0.01);The group of TRPV1 activator was markedly higher than the model group(P<0.01);The electroacupuncture at Guanyuan point group was significantly higher than the model group and the electroacupuncture at non meridian and non acupoint group(P<0.01);There was no significant difference between the electroacupuncture at non meridian non acupoint group and the model group(P>0.05);There was no statistical difference between the TRPV1 inhibitor group and the electroacupuncture at Guanyuan+TRPV 1 inhibitor group(P>0.05).2.Effect of electroacupuncture at Guanyuan point on the viscoelasticity of detrusor in model rats:The proportion of collagen fiber in model group was higher than that in sham operation group,and the proportion of elastic fiber was lower than that in sham operation group(P<0.01);The proportion of collagen fiber in TRPV 1 activator group was lower than that in model group,and the proportion of elastic fiber was higher than that in model control group(P<0.01);The proportion of collagen fiber in TRPV1 inhibitor group was higher than that in model group,and the proportion of elastic fiber was lower than that in model group(P<0.01).The percentage of collagen fiber in the group of electroacupuncture Guanyuan was markedly lower than that in the model group,and the percentage of elastic fiber was significantly higher than that in the model control group(P<0.01);The percentage of collagen fiber and elastic fiber in the electroacupuncture at non meridian and non acupoint group were slightly different than that in the model control group,but there was no statistical significance(P>0.05);The percentage of collagen fiber in electroacupuncture Guanyuan group was significantly lower than that in the electroacupuncture at non meridian and non acupoint group(P<0.01),and the percentage of elastic fiber was higher than that in the electroacupuncture at non meridian and non acupoint group(P<0.01);There was no significant difference in the percentage of collagen fiber and elastic fiber between TRPV1 inhibitor+electroacupuncture Guanyuan group and TRPV1 inhibitor group(P>0.05).3.Effect of electroacupuncture at Guanyuan point on the ultrastructure of detrusor in model rats:It was observed under 12000 times electron microscope that the size and shape of detrusor cells in the sham operated group were basically the same and arranged in parallel.The intercellular connections were mostly intermediate connections and a small number of desmosomes and hemidesmosomes.There was no obvious collagen fiber between cells.There was no edema,vacuolation and calcification in various organelles such as linear bodies and rough endoplasmic reticulum.However,in the model group,TRPV1 inhibitor group,electroacupuncture at non meridian non acupoint group,electroacupuncture at Guanyuan point+TRPV1 inhibitor group,the detrusor of bladder had a lot of mitochondrial edema,some of which were vacuolated,mitochondrial cristae disappeared,calcified,rough endoplasmic reticulum expanded,with edema and local vacuolation,collagen fibers increased in large numbers,with different directions and disordered arrangement.In the electroacupuncture Guanyuan group and TRPV 1 activator group,a small amount of mitochondrial edema,vacuolation,disappearance of mitochondrial cristae,slight expansion of rough endoplasmic reticulum with edema and local vacuolation,a small amount of collagen fibers with relatively consistent direction and clear arrangement were observed.As for the relative gray value of detrusor mitochondria,the model group was higher than the sham operation group(P<0.01);The TRPV1 activator group was significantly lower than the model group(P<0.01);The TRPV1 inhibitor group was significantly higher than the model group(P<0.01);The electroacupuncture Guanyuan group was significantly lower than the model group(P<0.01);The electroacupuncture at non meridian and non acupoint group was a little different from the group of model,but there was no statistical significance(P>0.05);The electroacupuncture Guanyuan group was significantly lower than the electroacupuncture at non meridian and non acupoint group.There was no prominent difference between the two groups(P>0.05).There was no significant difference between TRPV1 inhibitor+electroacupuncture Guanyuan group and TRPV1 inhibitor group(P>0.05).4.Effect of electroacupuncture at Guanyuan point on detrusor contractility in model rats:The minimum contractile tension of model group was higher than that of sham operation group,and the contractile frequency was lower than that of sham operation group(P<0.01);The minimum contractile tension of TRPV1 activator group was significantly lower than that of model group,and the contractive frequency was significantly higher than that of model group(P<0.01);The minimum contractive tension of TRPV1 inhibitor group was significantly higher than that of model group,and the contractive frequency was significantly lower than that of model group(P<0.01).The minimum contractile tension of electroacupuncture Guanyuan group was significantly lower than that of model group,and the contractile frequency was significantly higher than that of model group(P<0.01);The minimum contractile tension and contractile frequency of the electroacupuncture at non meridian non acupoint group were slightly different from that of model group,but there was no statistical significance(P>0.05);the minimum contractile tension of electroacupuncture Guanyuan group was significantly lower than that of the electroacupuncture at non meridian non acupoint group(P<0.01),and the contractile frequency was significantly higher than that of electroacupuncture at non meridian non acupoint group(P<0.01).The minimum contractile tension of TRPV1 inhibitor+electroacupuncture Guanyuan group was lower than that of TRPV1 inhibitor group,and the contractile frequency of TRPV1 inhibitor+electroacupuncture Guanyuan group was higher than that of TRPV1 inhibitor group,but the difference was not statistically significant(P>0.05).As for the expression of ?-SMA in detrusor of rats,the model group was lower than that of sham operation group(P<0.01);The TRPV1 activator group was significantly higher than that of model group(P<0.01);The TRPV1 inhibitor group was significantly lower than that of model group(P<0.01);The electroacupuncture at Guanyuan point group was significantly higher than that of model group and the electroacupuncture at non meridian non acupoint group(P<0.01);There was no prominent difference between the electroacupuncture at non meridian non acupoint group and model group(P>0.05);The electroacupuncture Guanyuan+TRPV1 inhibitor group was higher than that of TRPV1 inhibitor group,but the difference was not statistically significant(P>0.05).5.The effect of electroacupuncture at Guanyuan point on the function of ICC pacemaker cells of bladder in model rats:There existed co expression of c-kit and P2X5 receptors in ICC cells under the fluorescence microscope.As for the positive expression rate of icc-lc cells and P2X5 receptor in detrusor muscle of rats,the model group was lower than the sham operation group(P<0.01);The TRPV1 activator group was significantly higher than the model group(P<0.01);The TRPV1 inhibitor group was significantly lower than the model group(P<0.01);The electroacupuncture at Guanyuan point group was significantly higher than the model group and the electroacupuncture at non meridian non acupoint group(P<0.01);There was no prominent diversity between the electroacupuncture at non meridian non acupoint group and the model group(P>0.05).There was no prominent diversity between the TRPV1 inhibitor group and the electroacupuncture Guanyuan+TRPV1 inhibitor group(P>0.05).6.Effects of electroacupuncture at Guanyuan point on tprvl channel related factors in model rats:As for the gray ratio of TRPV1 and P2X5 protein in bladder of rats.The grey ratio of TRPV1 protein in bladder in the model group was prominently lower than that in the sham operation group(P<0.01);The gray ratio of TRPV1 protein in the TRPV1 activator group was higher than that in the model group(P<0.01);The gray ratio of TRPV1 protein in the TRPV1 inhibitor group was lower than that in the model group(P<0.01);The gray ratio of TRPV 1 protein in the electroacupuncture Guanyuan point group was higher than that in the model group(P<0.01).The grey ratio of TRPV1 protein in bladder of rats in Guanyuan point group was higher than that in the electroacupuncture at non meridian non acupoint group(P<0.01);The grey ratio of TRPV 1 protein in bladder of rats in the electroacupuncture at non meridian non acupoint group was higher than that in model group,however,the diversity was not statistically significant(P>0.05);The grey ratio of TRPV1 protein in bladder in the group of TRPV1 inhibitor+electroacupuncture at Guanyuan point was lower than that in the group of TRPV1 inhibitor(P>0.05).The gray ratio of P2X5 protein in the model group was prominently lower than that in the sham operation group(P<0.01);The gray ratio of P2X5 protein in the TRPV1 activator group was higher than that in the model group(P<0.01);The gray ratio of P2X5 protein in the TRPV1 inhibitor group was lower than that in the model group(P<0.01);The gray ratio of P2X5 protein in the electroacupuncture Guanyuan point group was higher than that in the model group(P<0.0 1).The gray ratio of P2X5 protein of bladder in the electroacupuncture Guanyuan point group was higher than that in the electroacupuncture at non meridian non acupoint group(P<0.01);The gray ratio of P2X5 protein in the group of the electroacupuncture at non meridian non acupoint was prominently higher than that in the group of model,but the diversity was not statistically significant(P>0.05);The gray ratio of P2X5 protein in the TRPV 1 inhibitor+electroacupuncture Guanyuan point group was lower than that in the TRPV1 inhibitor group,however the diversity was not statistically significant(P>0.05)?As for TRPV1 and P2X5 gene level of bladder in rats:The mRNA amplification of TRPV 1 of bladder in the model group was lower than that in the sham operation group(P<0.01);The mRNA amplification of TRPV1 in the TRPV1 activator group was higher than that in the model group(P<0.01);The mRNA amplification of TRPV 1 in the TRPV 1 inhibitor group was lower than that in the model group(P<0.01);The mRNA amplification of TRPV1 in the electroacupuncture at Guanyuan point group was higher than that in the model group(P<0.01).The mRNA amplification times of TRPV1 of bladder in electroacupuncture Guanyuan point group were higher than that in electroacupuncture at non meridian non acupoint group(P<0.01);The mRNA amplification times of TRPV 1 in the electroacupuncture at non meridian non acupoint group were lower than that in model group(P>0.05);The mRNA amplification times of TRPV1 of bladder in electroacupuncture Guanyuan group were prominent lower than that in TRPV1 inhibitor group,however there was no significant difference(P>0.05);There was no significant diversity between the TRPV1 inhibitor+electroacupuncture Guanyuan point group and the TRPV1 inhibitor group(P>0.05).The mRNA amplification of detrusor P2X5 in model group was prominent lower than that in sham operation group(P<0.01);The mRNA amplification of bladder P2X5 in TRPV1 activator group was higher than that in model group(P<0.01);The mRNA amplification of bladder P2X5 in TRPV1 inhibitor group was lower than that in model group(P<0.01);The mRNA amplification of bladder P2X5 in electroacupuncture Guanyuan point group was higher than that in model group(P<0.01).The mRNA amplification of P2X5 in bladder of rats in Guanyuan point group was higher than that in the electroacupuncture at non meridian non acupoint group(P<0.01);The mRNA amplification of P2X5 in the electroacupuncture at non meridian non acupoint group was lower than that in model group,but there was no significant difference(P>0.05);The mRNA amplification of P2X5 in TRPV 1 inhibitor+electroacupuncture Guanyuan point group was prominent lower than that in TRPV1 inhibitor group,however there was no significant difference(P>0.05).Conclusion1.The viscoelasticity,cell ultrastructure,contractility,number of ICC pacemaker cells,TRPV1 channel and other factors of bladder detrusor involved in the pathophysiological process of low compliance bladder after spinal cord injury.2.TRPV 1 channel activator can improve the mechanical sensory function of bladder,improve the function of ICC pacemaker cells,improve the function of ICC pacemaker cells,improve the function of ICC pacemaker cells,improve the viscoelasticity and contractility of detrusor,improve pathological state of cells ultrastructural and detrusor kinetic energ,and improve the compliance of bladder detrusor.The effect of TRPV1 inhibitor is opposite.3.Electroacupuncture at Guanyuan point has the similar effect as TRPV1 channel activator.Electroacupuncture at Guanyuan point can activate TRPV1 channel,upregulate the protein and gene expression level of TRPV1 and ICC cell receptor P2X5,increase ICC expression,stimulate the biological effect of ICC cell,and increase ?-SMA and other factors expression,so as to improve urodynamic bladder compliance.However,the electroacupuncture at non meridian non acupoint has no such effect.Acupoint effect has specificity.4.ICC pacemaker cell function may be the key bridge between bladder sensory afferent and detrusor contraction.Electroacupuncture at Guanyuan point may improve the mechanical sensory function of bladder,promote the function of ICC pacemaker cells,strengthen the signal relationship between the mechanical sensory afferent and the contractile movement of bladder detrusor,improve the contractility and the kinetic energy of detrusor,improve the ultrastructural and pathological state of detrusor cells and viscoelasticity and other physiological characteristics of detrusor,and further improve the compliance of bladder in rats with low compliance bladder after spinal cord injury by regulating TRPV1 channel. |
PDF Full Text Request