| IntroductionOveractive bladder(OAB)symptoms are characterized by urinary urgency,frequency,and nocturia with or without incontinence,and it does not contain same symptoms due to the urinary infection or other certainly pathologic change.It affects the patient’ s life quality and creates high pressure on them both economically and psychologically.The pathology and etiology of OAB are still uncertain.Currently,antimuscarinic drugs and behavioral therapies are the first-line pharmacotherapy for OAB,but have a limited efficacy with significant adverse effect such as dry,constipation,dizziness.Neuromodulation is working by stimulating peripheral nerves.If pharmacotherapy fails,neuromodulation is one of the alternative treatment options for OAB.Sacral neuromodulation has been approved by the Food and Drug Administration(FDA)to treat OAB for more than a decade.FDA also approved TNS as one of the effective therapy for OAB,Both AUA and EAU guidelines recommend neuromodulation as the way to treat the patients whom pharmacotherapy fails to work on.And also PNS had effect on urinary incontinence and OAB.However,their mechanisms,location and neurotransmitters involved in neuromodulation are still uncertain.The storage and voiding of the bladder are mediated by the co-working of the bladder and urethral muscle.And it is mediated by the brain,pon,spinal cord and peripheral nerve ganglion.It is well known that reflex bladder activity is controlled by two distinct neural pathways:one is AS afferents enabled a spinobulbospinal micturition reflex pathway and another one is C-fiber afferents enabled a spinal micturition reflex pathway.Currently,many neurotransmitters involved in mediating bladder activity by simulating peripheral nerves.Our previous studies in cat revealed that spinal GABAA receptors play an important role in pudendal neuromodulation of bladder overactivity,whereas opioid and glycine receptors have no or a minor role.In contrast,we showed that opioid receptors have an essential role in tibial neuromodulation of bladder overactivity in the cat,and application of naloxone(an opioid receptor antagonist)to the surface of pons removed tibial inhibition of irritation-induced bladder overactivity,and spinal cord transection at the T9-T10 level eliminated tibial but not pudendal inhibition,indicating that tibial neuromodulation probably acts at supraspinal sites.CB receptors are G protein-coupled receptors,which include type 1(CB1)and type 2(CB2)receptors.CB1 are involved in CNS while CB2 in immune system.Previous animal studies have indicated that both CB1 and CB2 receptors are involved in micturition control,and cannabinoid and opioid mechanisms have interactions in the central nervous system.Because afferent axons passing through the pudendal enter the spinal cord through the sacral S1-S2 dorsal roots,and afferent axons passing through the tibial nerve enter the spinal cord through the lumbar L7.The interactions between cannabinoid and opioid mechanisms in the central nervous system are well known.Our previous study have shown that the bladder could be irritated by dilute(0.5%)acetic acid,in order to enable the nociceptive bladder C-fiber afferents and induce bladder overactivity.And,sacral neuromodulation,tibial nerve stimulation and pudendal nerve stimulation have been proved to be effective on OAB.Therefore,this study consists of two parts:(1)To examine the effects of a GABAA receptor antagonist(picrotoxin),a glycine receptor antagonist(strychnine),and an opioid receptor antagonist(naloxone)on the modulation of bladder overactivity elicited by electrical stimulation of the S1 or S2 sacral dorsal roots.(2)After induce bladder overactivity of cats,electrical stimulation was applied to tibial or pudendal nerve to inhibit the bladder overactivity.Then,AM 251(a selective CB1 receptor antagonist)was administrated intravenously to determine the involvement of CB1 receptors in the two types of neuromodulation.AM251 was also administered intrathecally to determine the central site of actionPart 1:Contribution of GABAA,Glycine,and Opioid Receptors to Sacral Neuromodulation of Bladder Overactivity in CatsObjectiveTo examine the role of GABAA,glycine,and opioid receptors in sacral neuromodulation induced inhibition of bladder overactivity enabled by intravesical infusion of 0.5%acetic acid(AA).Method1.Surgical procedureA total of twenty cats was used in this study.After the animals were anesthetized by isoflurane(2-5%in oxygen),we performed a tracheotomy and put into a tube in order to hold the trachea open.A catheter was inserted into the right carotid artery in order to monitor the systemic blood pressure.Left and right cephalic veins were putted in catheter for intravenous administration of drugs and fluid.Through an abdominal incision,the ureters were isolated,tied,and cut for external drainage.A double lumen catheter was inserted through the urethra into the bladder.One lumen was connected to apump to slowly infuse saline or 0.5%AA in saline.The other lumen was attached to apressure transducer to measure bladder pressure.The spinal cord and cauda equina were exposed via a dorsal laminectomy.The spinal dura was cut,and the S1 and S2 dorsal roots on the right side were separated for electrical stimulation.A bipolar stainless steel hook electrode was used during the experiment to stimulate individual S1/S2 dorsal roots by delivering electrical pulses that were generated by an electricalstimulator.In five cats,a small catheter(PE10)was inserted rostrally underneath the dura to position the catheter tip between S1 and S2 spinal cord for intrathecal administration of picrotoxin ornaloxone.After the sugery,we switched to a-chloralose anesthesia(initial 65 mg/kg followed by slow i.v.infusion at 2mg/kg per hour)during data collection.Pancuronium(initial 0.1 mg/kg followed by slow i.v.infusion at 0.1 mg/kg per hour)was also given during data collection to prevent striated muscle contractions and movement of the animal.The skin and muscle layers were closed by sutures after the surgery.2.Stimulation parametersBased on our previous study in cats,stimulation(5 Hz,0.2 millisecond)of S1 or S2 dorsal roots at motor threshold intensity for inducing reflex twitching of the anal sphincter or toe was used in this study to inhibit reflex bladder activity.The motor threshold was determined before administering pancuronium.3.Stimulation protocol and drug administrationAt the beginning of each experiment,multiple cystometrograms(CMGs)were performed by slowly infusing the bladder with saline to determine the bladder capacity that was defined as the bladder volume threshold to induce a bladder contraction of large amplitude(>30 cm H20)and long duration(>20 seconds).Then 0.5%AA was infused into the bladder to irritate the bladder and induce bladder overactivity.Once the control bladder capacity stabilized during repeated AA CMGs,the inhibitory effect of sacral dorsal root stimulation was determined by additional four AA CMGs:1)control CMG without stimulation;2)CMG during S1 dorsal root stimulation;3)CMG during S2 dorsal root stimulation;and 4)control CMG again to examine any post-stimulation effect.Then the animals were separated into three groups.In the first group(N=9 cats),cumulative doses(0.01,0.03,0.1,0.3,and 1.0 mg/kg,i.v.)of picrotoxinwere given.In the second group(N=6 cats),strychninewas administered in cumulative doses(0.001,0.003,0.01,0.03,0.1,and 0.3 mg/kg,i.v.)followed by picrotoxin(0.3 mg,i.v.).In the third group(N=5 cats),a single dose(0.4 mg in 0.2 mL saline,i.t.)of picrotoxin was given,which was followed by a single dose(0.3 mg in0.1mLsaline,i.t.)of naloxone.After administering each dose of drug,the four CMGs(control,S1 stimulation,S2 stimulation,control)were repeated to determine the drug effects.A 10-minute waiting period for each i.v.dose of picrotoxin or strychnine and a 5-minute period for i.t.picrotoxin or naloxone were used to allow time for the drugs to take effect.A waiting period of 2-3 minutes was also used between CMGs to allow the bladder reflex to recover.4.Data Analysis:The bladder capacity was measured from each CMG and normalized to the capacity measured during the first control CMG in different test groups.Repeated measurements in the same animal under the same conditions were averaged.The normalized data from different animals were presented as mean±S.E.Statistical significance(P<0.05)was determined by a paired Student t test or analysis of variance followed by Bonferroni multiple comparisons.Results1.Inhibition of Bladder Overactivity by S1 or S2 Dorsal Root Stimulation.AA irritation induced bladder overactivity and significantly(P<0.01)reduced bladder capacity to59.5±4.8%of saline control capacity(N=20 cats).S1 or S2 dorsal root stimulation at threshold intensity for inducing reflex twitching of anal sphincter or toe inhibited bladder overactivity and significantly(P<0.01)increased bladder capacity to 105.3±9.0%and 134.8±8.9%of saline control,respectively.After the stimulation,AA control capacityreturned to pre-stimulation level,indicating that there was no post-stimulation effect.2.Effect of i.v.Picrotoxin on Sacral Inhibition of Bladder Overactivity.Picrotoxin(i.v.)slightly increasedthe pre-stimulation bladder capacity at 0.3-1.0 mg/kg doses,but the increase was notstatistically significant(P<0.05,N=9 cats).The0.3 mg/kg dose of picrotoxin blocked(P<0.05)the increase inbladder capacity elicited by S1 dorsal root stimulation,but notthe increase elicited by S2 dorsal root stimulation.Picrotoxin at 1 mg/kg blocked(P<0.05)the increase inbladder capacity induced by either S1 or S2 dorsal rootstimulation.After the stimulation,the bladdercapacity returned to pre-stimulation level at every dose ofpicrotoxin,that is,no post-stimulation effect.3.Combined Effect of i.v.Strychnine and Picrotoxin on Sacral Inhibition of Bladder Overactivity.Strychnine at 0.03-0.3 mg/kg(i.v.)significantly(P<0.05)increased the pre-stimulation bladder capacity without affecting the increase in bladder capacity caused by S1 or S2 dorsal root stimulation(N=6 cats).Following strychnine treatment,picrotoxin(0.3 mg/kg,i.v.)further significantly(P<0.05)increased the pre-stimulation bladder capacity and blocked the inhibition induced by S1 or S2 dorsal root stimulation(N=6 cats),whereas the same dose of picrotoxin without strychnine pretreatment only blocked the inhibition induced by S1 but not S2 dorsal root stimulation.There was no post-stimulation effect at any dose of the drugs.4.Effect of i.t.Picrotoxin and Naloxone on SacralInhibition of Bladder Overactivity.Picrotoxin(0.4 mg,i.t.)did not significantly change the pre-stimulation bladdercapacity and had no effect on the capacity increase induced by either S1 or S2 dorsal root stimulation(N=5 cats).However,after sacral dorsal root stimulation,the post-stimulation bladder capacity was significantly(P<0.05)increased(about 100%),that is,the stimulation induced a significantpost-stimulation inhibitory effect.Following picrotoxin treatment,naloxone(0.3 mg,i.t.)significantly(P<0.05)reduced the pre-stimulation bladder capacity and removed the post-stimulation inhibition induced by sacral dorsal root stimulation.However,even after i.t.administration of both picrotoxin and naloxone,S1 or S2 dorsal root stimulation still significantly(P<0.05)increased bladder capacity during stimulation.ConclusionIn this study,the effects of selective receptor antagonists administered alone or in combination revealed that GABA,glycine,and opioids contribute in varying ways to sacral neuromodulation of bladder overactivity in anesthetized cats.GABA acting on GABAA receptors at supraspinal sites plays a major role in sacral neuromodulation,whereas glycine seems to have a minor role to facilitate the GABAergic inhibition.In contrast,spinal opioid mechanisms have an unusual function.They do not contribute to the increase in bladder capacity elicited during stimulation of the S1 or S2 dorsal roots,but do contribute to the post-stimulation increase in capacity that is unmasked by blocking GABAA receptors in the spinal cord with i.t.administration of picrotoxin.Part 2:Role of cannabinoid receptor type 1 in tibial and pudendal neuromodulation of bladder overactivity in catsObjectiveTo determine the role of cannabinoid type 1(CB1)receptors in tibial and pudendal neuromodulation of bladder overactivity induced by intravesical infusion of 0.5%acetic acid(AA)in cats.Methods1.Surgical procedureA total of seventeen cats was used in this study.After the animals were anesthetized by isoflurane(2-5%in oxygen),a tracheotomy was performed and a tube was inserted to keep the airway patent.A catheter was inserted into right carotid artery to monitor systemic blood pressure.Left cephalic veins were catheterized for intravenous administration of drugs and fluid.Through an abdominal incision,the ureters were isolated,tied,and cut for external drainage.A double lumen catheter was inserted through the urethra into the bladder.One lumen was connected to a pump to slowly infuse saline or 0.5%AA in saline.The other lumen was attached to a pressure transducer to measure bladder pressure.The tibial nerve was exposed in the left leg above the ankle,and a tripolar cuff electrode was implanted for stimulation.The right pudendal nerve was dissected via a 3-to 4-cm incision in the sciatic notch lateral to the tail for implantation of another tripolar cuff electrode.The tibial and pudendal nerve electrodes were connected to different output channels of an electrical stimulator via constant voltage stimulus isolators.In 5 cats,a small incision was made to remove the L3 spinal process and expose the spinal cord.Then,the spinal dura was pierced and a fine catheter(PE 10)was inserted caudally underneath the dura to position the catheter tip at the level of L7 spinal cord for intrathecal administration of AM251.After the surgery,the animals were switched to α-chloralose anesthesia(initial 65 mg/kg i.v.and supplemented as needed)during data collection.The skin andmuscle layers were closed by sutures.2.Stimulation parametersUniphasic rectangular pulses(5-Hz frequency,0.2-m pulse width)were used to stimulate the tibial or pudendal nerve via the cuff electrode.The intensity threshold(T)for inducing observable toe movement or anal sphincter twitch was determined by gradually increasing the stimulation intensity.Based on our previous studies,intensities of 2T or 4T were used in this study to suppress bladder overactivity.3.Stimulation protocol and drug administration At the beginning of each experiment,multiple cystometrograms(CMGs)were performed by slowly infusing the bladder with saline to determine the bladder capacity that was defined as the bladder volume threshold to induce a bladder contraction of large amplitude(>30cm H20)and long duration(>20 s).Then,AA was infused into the bladder to irritate the bladder,activate nociceptive C-fiber afferent nerves,and induce bladder overactivity.Once the control bladder capacity stabilized during repeated AA CMGs,the inhibitory effect of tibial nerve stimulation(TNS)was determined by additional four AA CMGs 1):control CMG without TNS;2)CMG during 2T TNS;3);CMG during 4T TNS;and 4);control CMG again to examine any post-stimulation effect.Then,the animals were seperated into three experimental groups.In the first group(n = 5 cats),cumulative doses(0.01,0.03,0.1,0.3,and 1.0 mg/kg i.v.)of AM251 were given.Since AM251 was dissolved in 30%Cremophor in the second group(n=5 cats)the vehicle solution(30%Cremophor in saline)was administered in increasing volumes corresponding to the volumes of cumulative doses of AM251(0.01,0.03,0.1,0.3,and 1.0 mg/kg i.v.).In the third group(n=5cats,3 cats from the vehicle control group),a single dose(0.03 mg it)of AM 251 was given.After administering each dose of drug/vehicle,the four CMGs(control,2T TNS,4T TNS,control)were repeated to determine the drug/vehicle effects.A 10-min waiting period after each intravenous dose of AM251/vehicle and a 5-min waiting period after intrathecal AM251 were used for the drug/vehicle to take effect.Waiting period of 2-3 min was also used between CMGs for the bladder reflex to recover.The same experimental protocol used to test TNS was also used to test the effect of cumulative doses(0.01,0.03,0.1,0.3,and 1.0 mg/kg.i.v.)of AM251 on pudendal nerve stimulation(PNS)in the fourth group of five cats.4.Data Analysis:Bladder capacity was measured during each CMG and normalized to the capacity measured during the first control CMG in different experimental groups.Repeated measurements(2-3 CMGs)of control bladder capacity in the same animal under the same conditions(saline or AA)were averaged.The normalized data from different animals are presented as mean±SE.Statistical significance(P<0.05)was determined by repeated-measures ANOVA followed by Dunnett’s(one-way)or Bonferroni’s(two-way)multiple comparison.Results1.TNS inhibition of bladder overactivity induced by AA irritation.AA irritation induced bladder overactivity that significantly(P<0.01)reduced bladder capacity to 36.6±4.8%of saline control capacity(n=12 cats).TNS at 2T or 4T intensity inhibited the bladder overactivity and significantly(P<0.01)increased bladder capacity to 69.2±9.7%and 79.5±7.2%of saline control,respectively.After the stimulation,AA control capacity returned to pre-stimulation level,indicating that there was no post-stimulation effect.2.Effect of AM251 on TNS or PNS inhibition of bladder overactivity.AM251(intravenous)dose dependently reduced TNS inhibition of bladder overactivity without changing the pre-stimulation bladder capacity during repeated AA CMGs.The increase in bladder capacity induced by TNS was significantly(P<0.05)reduced by AM251 at 0.03 and 0.1 mg/kg for 2T TNS and 4T TNS,respectively.The vehicle(30%Cremophor in saline)at volumes corresponding to those used to administer different intravenous doses of AM215 did not have a significant effect on TNS inhibition or pre-stimulation bladder capacity during repeated AA CMGs,AM251(0.03 mg it)had no effect on TNS inhibition or pre-stimulation bladder capacity during AA CMGs,indicating that the site of action of the intravenous doses of drug is not within the lumbar L7 spinal cord.AM251 at 0.01-1 mg/kg i.v.had no effect on PNS inhibition of bladder overactivity or pre-stimulation bladder capacity during repeated AA CMGs.No post-stimulation effect was observed after any dose of drug/vehicle treatment.ConclusionThis study revealed that CB1 receptors at supraspinal sites play a critical role in TNS but not PNS inhibition of bladder overactivity induce by AA irritation of the bladder in cats. |
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