Investigation On The Mechanism Of Electroacupuncture(EA) At CILIAO (BL32) To Overactive Bladder (OAB) Inhibition

Background:Overactive Bladder (OAB) is a syndrome characterized by urinary urgency accompanied byurinary frequency and nocturia with or without urge incontinence. Detrusor overactivity, detrusorinstability and other types urethra-bladder dysfunction can be presented in Urodynamicexaminations. The pathogenesis of OAB is still unclear, but unstable detrusor contractions may bethe main pathologic and pathophysiologic changes of OAB. M receptor is distributed in the bladderdetrusor. Acetylcholine secreted by the excited efferent nerve of bladder，which acts on M-receptorselicits detrusor contraction. But current therapy with anticholinergics for the overactive bladder isonly partially effective. Therefore，many scholars are making an effort to afferent mechanism inOAB.The afferent innervation of the urinary bladder consists of myelinated (Aδ) and unmyelinated(C-fiber) axons. Recently, the effect of C-fiber in OAB has attracted more attention. It was reportedthat afferent C-fibers overactivity was the most important pathological mechanism of detrusorhyperreflexia. Fos protein shows a increased express in the spinal cord after irritation of the lowerurinary tract. The overexpression of c-fos gene in the lumbar-sacral spinal cord micturition centercould be used as a positive index of afferent C-fibers overactivity.To investigate the etiology and to evaluate the effect of different treatment for OAB, lots ofOAB animal models have been established. Cyclophosphamide(CYP)-induced OAB models havebeen applied extensively with advantages of simple operation, controllable and easy to observe thechanges of voiding function. There were three types of cyclophosphamide-induced OAB models,including acute, subacute and chronic. Subacute models were less applied.OAB is a chronic disease, so chronic OAB rats may be more appropriate model in experiment. ifthe objective of the experiment is not to investigate other mechanism of OAB but to observe thechanges of urodynamic and neuroelectrophysiology during micturating, both acute and chronic OABmodels are desirable. It was not reported that whether the urodynamic changes between acute OABmodel and chronic OAB model which have the similar pathophysiologic changes to OAB weresignificantly different.Electro-acupuncture(EA) with little side effect has a significant long-term effect on overactivebladder better than the improvement offered by pharmacologic therapy and physical or behavioral therapy. The animal experiments showed that: electro-acupuncture at CiLiao (BL32) decreasedmicturition frequence, increased capacity of bladder and inhibited detrusor spontaneous contractionin OAB rats. There was high evidence suggesting the strongest effect where the nerves enter thesacral segment1-2close to sacral spinal cord micturition center after electro-acupuncture at CiLiaoenter. Howeve, whether the effect of electro-acupuncture at CiLiao (BL32) engendered throughstimulating afferent innervation of the urinary bladder especially activating afferent C-fibers is stillnot clear.Object:This study compared the urodynamics parameter in acute and chronic OAB rats which inducedby cyclophosphamide to select the the most convenient animal models Through the analysis ofurodynamic parameter changes before and after electro-acupuncture at CiLiao (BL32) in OAB ratsdesensitized by capsaicin, We aim to investigate the mechanism of bladder afferent nerve withelectro-acupuncture for treatment of OAB. To overcome these limitations, a single-fiber method wasdeveloped for recording afferent pressure-related activity of C-fibers and Aδ fibers of the pelvicnerve during the whole bladder filling phase, further to study afferent nerve mechanism ofoveractive bladder. This research focuses on observing the expression of c-fos gene in OAB ratsinduced by CYP at the lumbar-sacral spinal cord micturition center after pretreatment withelectroacupunctue at CiLiao to probe into the possible molecular mechanism,which will provide thetheory basis for treatment of OAB with electroacupunctue at CiLiao point.Method and Result:This paper consists of four parts. The first part is to investigate the influence of acute andchronic models of Cyclophosphamide (CYP)-induced cystitis on urinary bladder motor activity. Atotal of30female Sprague-Dawley rats weighing180-220g were divided into, control group (n=10),acute model group (n=10) and chronic model group (n=10), acute model group were induced bysingle injection of CYP (200mg/kg, ip) and chronic model group (n=10) were induced by by fourinjections of CYP in14days (75mg/kg, ip). Control rats received saline injections. Cyctometry wascarried out including intercontraction interval (ICI), basal pressure, threshold pressure, andmicturition pressure in each group. Intercontraction interval decreased significantly in acute andchronic model group（P<0.05）and basal pressure increased compared with control group (P<0.01),there was no differences between the model group (P>0.05）. There were no differences in thresholdpressure and micturition pressure among these3groups (P>0.05).The second part is to investigate the urodynamic effects of electroacupuncture (EA) at CiLiao(BL32) on cyclophosphamide-induced overactive bladder, with particular focus on themechanism of bladder afferent nerve. A total of24female Sprague-Dawley rats were divided into overactive bladder (OAB) group, capsaicin group, and control group. Before electro-acupuncturecystometry including inter-contraction interval (ICI), basal pressure, threshold pressure, andmicturition pressure was carried out. Cystometry was repeated after electroacupuncture atCiLiao(BL32). Inter-contraction interval was decreased significantly in OAB group compared withcontrol group (p<0.05) and was increased in the capsaicin group compared with OAB group(p<0.01). After electroacupuncture, the inter-contraction interval was increased in OAB rats andthere were no differences in capsaicin group and control group. Basal pressure was increased inmodel group (p<0.01) compared with control group and was reduced afterelectroacupuncture(p<0.05). Basal pressure was increased after electroacupunture in control group(p<0.01) and was decreased in the capsaicin group (p<0.01) compared with OAB group. There wereno differences before and after electroacupunture in capsaicin group(p>0.05).The third part is to evaluate the influence of electroacupuncture on single fiber pelvic afferent nervein the anaesthetized female rats with cyclophosphamide-induced overactive bladder(OAB). Thirtysix adult female Sprague-Dawley (SD) rats were randomly divided into OAB group20rats andcontrol group16rats. Fifty six single bladder afferent fibers were isolated. The fibers were dividedin Aδ or C-fibers dependent on conduction velocities (CV). The effect of repeat bladder filling wasstudied on the mechanosensitive properties of these units. Unitary afferent activity was analyzed inOAB group rats before and after electroacupuncture. The inter-contraction interval decreased andbasal pressure increased in OAB group (p<0.01). Micturition pressure was not significantly changedbetween the2groups. In OAB group rats32single unit afferent fibers were isolated, including14Aδ and18C-fibers with a mean CV of5.32±3.16m per second and1.28±0.28m per second,respectively. In control rats24single unit afferent fibers were isolated, including8Aδ and16C-fibers with a CV of6.82±3.16m per second and1.45±0.18m per second, respectively. In OABgroup rats,20min-60min after electroacupuncture, the afferent spike rate in C-fibers showed atendency to a significant decrease compared with the spike rate before treatment forpressure-rerelated activity(P<0.05). There no differences in Aδ fibers in the afferent spike rate,before and after electroacupuncture.The fourth part is to observe the expression of c-fos gene in the lumbar-sacral spinal cordmicturition center（L6） before and after Electroacupuncture（EA） at CILIAO(BL32) and to probeinto the possible molecular mechanism of EA on overactive bladder （OAB）. Thirty-seven adultfemale Sprague-Dawley rats weighing180to220g were randomly divided into control group (n=5),OAB group (n=16) and EA+OAB (n=16)group. Total32rats in OAB group and EA+OAB groupwere received intraperitoneal injections of cyclophosphamide (CYP,200mg/kg) to elicit acuteoveractive bladder. Control rats received equal amount of normal saline. Electroacupuncture preconditioning at CiLiao (BL32) was started5days before CYP induction in EA+OAB grouponce daily for5days. A cystometrogram was performed to confirm the development of overactivebladder. L6spinal cord segment was removed in all groups and an immunocytochemical methodwas applied to stain fos-protein that was encoded by the c-fos gene, then counting fos-proteinpositive neurons. The inter-contraction interval decreased and basal pressure increased in OABgroup compared with control group（P<0.01）. Inter-contraction interval increased and basal pressuredecreased significantly compared to OAB group （P<0.05）. Micturition pressure was notsignificantly changed among the3groups. The numbers of fos-protein positive neurons in the L6spinal cord segment in OAB group was increased significantly compared with control group, after5days of EA preconditioning, the fos-protein positive neuron numbers were reduced significantlycompared with OAB group (p=0.032) and was very close to the baseline (control group). There wasno significant difference in the number of fos-protein positive neurons between the control groupand the EA group (p=0.92).Conclusions:1. Current results prove that both acute and chronic models of cyclophosphamide(CYP)-induced cystitis on urinary bladder motor activity were equally credible for cystometricevaluation.2. Electro-acupuncture at CiLiao could contribute to improve cyclophsphamide–inducedoveractive bladder through inhibition of capsaicin-sensitive C-fiber activation.3. There was an increased afferent activity in C-fibers in CYP-induced OAB, and decreasedafferent activity in C-fibers after electroacupuncture. The results of this study suggested that themechanism of acupuncture on bladder inhibition was related to act on nociceptive afferent-mediatedC-fibers.4. Electroacupuncture at CiLiao acupoint significantly inhibits overactive bladder and reducesc-fos gene expression in rats with CYP induced OAB. Inhibition of afferent C-fiber activity in thespinal reflex pathway may be one of the underlying mechanisms of EA.