| BackgroundBladder dysfunction will cause dysuria,such as frequent urination,dysuria,urgency,urinary incontinence,urinary retention and other uncomfortable symptoms.Acupuncture has a certain regulatory effect on bladder dysfunction diseases,and there are a large number of clinical literature reports.Therefore,it is important to actively explore the mechanism of acupuncture regulating bladder function and provide experimental evidence for clinical acupuncture treatment of bladder dysfunction diseases.The normal realization of bladder function requires the coordination of the bladder detrusor and the urethra,both of which are innervated by the sacral parasympathetic nerve(pelvic nerve),sympathetic nerve(hypogastric nerve)and somatic nerve(pudendal nerve).Among them,the pelvic nerve can contract the detrusor and relax the urethral sphincter to cause urination,the hypogastric nerve can relax the detrusor and contract the internal urethral sphincter to store urine,and the pudendal nerve mainly innervates the extern al urethral sphincter.However,currently there are few systematic studies on the regulation of physiological bladder function by acupuncture.Most of the studies selected Ciliao/Zhongliao,Sanyinjiao,Pangguangshu,Shenshu,etc.on rats with different pathological models and observed the bladder urodynamic index to explore acupuncture effect using electrophysiological methods.A few of studies simultaneously recorded the activities of the bladder detrusor and the afferent/efferent nerves of the pelvic nerve to explain the mechanism of acupuncture effect.In addition,the acupuncture effect is closely related to the physiological and pathological states of the bladder.Previous studies have used rats with different pathological models as experimental subjects,but under physiological conditions(isovolumic bladder and isotonic bladder)there are few studies.In recent years,the research on the nature of acupoint believes that acupoints are dynamic and sensitized body surface reaction sites(reaction points),and the location of acupuncture points is a regular summary of this reaction point.Based on the above background information,this study first observed the distribution characteristics of reaction points of the body surface in the acute cystitis model rat to determine the study acupoint,then explored the segmental relationship of the research acupoint and the bladder.To further design acupoints of different segments at study acupoint as control,and use electrophysiological methods to observe the effects of acupuncture acupoints at different segments on urodynamics,urethral sphincter,hypogastric nerve and pelvic nerves to explore its regulatory effects and corresponding neural mechanisms in the isovolumic bladder and isotonic bladder.ObjectiveBy observing the distribution characteristics of reaction points of the body surface in the acute cystitis model rats to determine the research acupoint,then explore the segmental relationship between the study acupoint and the bladder.To further design acupoints of different segments at study acupoint as control,and use electrophysiological methods to observe the effects of acupuncture acupoints at different segments on urodynamics,urethral sphincter,hypogastric and pelvic nerves to explore its regulatory effects and corresponding neural mechanisms in the isovolumic bladder and isotonic bladder.MethodsThe first part of the study mainly observed the distribution of Evans Blue(EB)exudation points under acute cystitis to determine the research acupoint.9 SD female rats were divided into two groups,control group(n=3)and acute cystitis group(n=6).0.3 mL 1.5%hydrogen peroxide was injected into the bladder via the urethral to induce acute cystitis and was drained from the bladder after 30 min by pressing the lower abdomen.All rats were injected with 5%EB for 2 hours,then the hair of the body surface was removed and the rats were perfused via the heart by saline.EB exudation points were observed by natural light and fluorescent labeling organic active imaging system.The second part of the study explored the relationship between the research acupoint and the bladder wall in the dorsal root ganglia(DRG).5 normal female rats were injected with CTB-488 in the research acupoint and CTB-594 in the bladder wall.After 4 days,the L5-S2 DRG were harvested to observe the labeled sensory neurons to reveal the relationship of nerve segment between the study acupoint and the bladder.The third part is to study the effect and corresponding mechanism of different acupoint stimulation on the function of the bladder in different states using electrophysiological methods.30 normal female rats were anesthetized and catheterized via the urethra and infused with 0.9%NaCl solution until the bladder produced stable rhythmic contraction,called isovolumic bladder.Then,the electromyogrphy(EMG)of the urethral sphincter,discharges of hypogastric nerve and pelvic nerves were recorded.Design non-noxious stimuli(brush),noxious stimuli(pinch and acupuncture)to randomly stimulate the research acupoints Huiyin,Yinlingquan at the adjacent segment of the research acupoint,Hegu at the distal different segment of the research acupoint,then observed their effects on the rat's urodynamics,activities of the urethral sphincter,hypogastric nerve and pelvic nerve to explore the mechanism and regulating effect of different acupoint stiimulation in isovolumic bladder.In the fourth part of the study,40 normal female rats were catheterized through the bladder dome and continuously perfused with 0.9%NaCl solution.When the intravesical pressure reached the threshold pressure,the bladder expanded,produced micturition contraction and became an isotonic bladder.At the same time,EMG of the urethral sphincter,discharges of the hypogastric and pelvic nerves were recorded.Acupuncture randomly stimulated Huiyin,Yinlingquan and Hegu for 1min to explore the regulating effect and mechanism of acupuncture at different acupoints on isotonic bladder.Further changes in the urodynamics of the bladder were observed after one side of the pelvic nerve was cut to reveal the role of the pelvic nerves in acupuncture regulating bladder efficacy.Results1.The distribution of Evans Blue(EB)exudation points under acute cystitis is mainly distributed in the pubic,urethra,and the perinium surrounding the urethra.Huiyin(CV1)was determined as the research acupoint considering convenience of the anatomical position and electrical signal recording,etc.2.After injecting micro doses of the cholera toxin subunits CTB-488 and CTB-594 into CV1 and the bladder,it was observed that the sensory neurons related to CV1 and the bladder appeared in the dorsal root ganglions of the L5-S2 and L6-S2,respectively.L6 and S1 DRGs and their tissue sections were the mostly distributed ones,labeled with the tracers.Most of the neurons labeled by CTB-594 appeared in S1 DRG.3.In rats with isovolumic bladder,brush stimulation of CV1,Yinlingquan(SP9),Hegu(LI4)had no effect on urodynamics,the activities of urethral sphincter,HGN and PN(P>0.05).Pinch at CV1,SP9,LI4 prolonged contraction intervals,increased contraction duration(P<0.01).When pinch prolonged contraction intervals,pinch prolonged ed the rhythmic burst discharges of urethral sphincter,hypogastric nerve and pelvic nerve accompanied with bladder contraction.Pinch at CV1 reduced the maximum contraction pressure(MCP)(P<0.05);LI4 had no effect on them(P>0.05).The effect of pinch at CV1 to prolong contraction intervals was better than that of SP9(P<0.05)and LI4(P<0.01).Acupuncture at CV1 and SP9 could prolong contraction intervals,reduce MCP,increase contraction duration(P<0.01).When acupuncture prolonged contraction intervals,it prolonged the rhythmic burst discharges of urethral sphincter,hypogastric and pelvic nerves.While LI4 had no effect on them(P>0.05),the effect of acupuncture at CV1 to prolong contraction intervals was better than that of SP9(P<0.05)and LI4(P<0.01).The effect of acupuncture at the CV1 to prolong the contraction intervals was better than the effect of brush(P<0.01)and pinch(P<0.05).4.In rats with isotonic bladder,acupuncture at CV1 and SP9 could prolong the urination intervals,increase the threshold pressure,MCP and contraction duration(P<0.01),while LI4 had no effect on them(P>0.05).When acupuncture prolonged urination intervals,it prolonged the burst discharges of urethral sphincter,hypogastric and pelvic nerves.The effect of acupuncture at CV1 to increase threshold pressure and MCP was better than that of SP9(P<0.05)and LI4(P<0.01).After cutting one side of the pelvic nerve,the urination interval was shortened(P<0.05)and MCP was decreased(P<0.05),while the urination threshold pressure,basal pressure and contraction duration did not change(P>0.05).Acupuncture at CV1 could prolong the urination intervals and increase the threshold pressure,MCP and basal pressure(P<0.05)after cutting the pelvic nerve of one side,while acupuncture at SP9 had no effect on urinary dynamics.There was no significant difference in the effect of the acupuncture at CV1 on regulating the urinary dynamics pre and post cutting one side pelvic nerve(P>0.05).The effect of acupuncture at SP9 to increase threshold pressure and MCP before cutting one side pelvic nerve was better than after cutting one side pelvic nerve(P<0.05).Conclusion1.The sensory neurons that innervate CV1 and bladder had a segmental connection,mainly in L6-S1 DRG.This may be the neural pathway for acupuncture at CV1 to regulate bladder function.It could provide morphological basis for the clinical application of CV1 in treating urinary system diseases.2.In isovolumic bladder and isotonic bladder,the effects of acupuncture,pinch at CV1 and SP9 on urodynamics and urethral sphincter may be related to prolong the activities of pelvic and hypogastric nerves.These effects are all segmental effect,and the CV1 at the same segment of the bladder had the best effect.3.It is observed that the acupuncture effect was better than pinch and brush stimulation,suggesting that different tissue structures of the body are stimulated to produce different effects.Acupuncture acted on both the skin and muscle layers,and its effect is more obvious.4.In isotonic bladder,after cutting one side of the pelvic nerve,the effect of acupuncture at CV1 was still present,while the effect of acupuncture at SP9 disappeared,suggesting that acupuncture effects play a role by the pelvic nerve. |
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