Peripheral Neuronal Mechanisms Of Reduced Sensory Function Of Aged Bladder

Background and ObjectiveAging is a global phenomenon.The incidence of lower urinary tract symptoms(LUTS)caused by bladder dysfunction are increasing with aging both in men and women.Urologists should pay much attention to the diagnosis and treatment of age associated bladder diseases and their pathogenesis.Activation of bladder primary sensory afferents is the first step of the urination reflex.The enhancement of sensory function is the main manifestation of overactive bladder(OAB).Underactive bladder(UAB),another type of bladder dysfunction that is contrary to OAB has received much attention in recent years.UAB caused solely by aging but without specific reasons is called senile idiopathic UAB.Studies have found that senile idiopathic UAB could be caused by the reduction of sensory afferent activity and thus the inactivation of the detrusor muscle,but rather than the decrease of the detrusor muscle contractility.One clinical study published in the European Journal of Urology also found that the decrease in bladder sensory function is an important factor in the development of UAB.Experts have reached a consensus:bladder sensory function especially volume sensory disorders is an important cause of idiopathic UAB in the aged people.However,up to now there are few studies in the literature to study the mechanisms underlying the decreased bladder sensory function especially the peripheral neuronal mechanisms in the aged bladder.At present,many studies have agreed that a variety of TRP channels such as TRPV4 and TRPV1 on the sensory terminals are the main channels involved in bladder sensation.At the same time,the active and passive electrophysiological characteristics of sensory neurons are also key factors that determine the generation and conduction of bladder sensory signals.Although the TRP channels on the bladder primary sensory neurons and its electrophysiological properties have been studied elsewhere,there is still no relevant reports about their roles in age-related decline of bladder sensory function.Because old male patients with LUTS usually have bladder outlet obstruction result from prostate hyperplasia,whereas,old women usually do not have this factor.Therefore,we chose woman and female rats to study the age-related changes in bladder sensory function.Based on the above background,this study was aimed to mainly explore:(1)whether there are age associated changes in bladder sensory function and bladder contraction function in female patients;(2)the role of bladder primary sensory neurons and urothelium in aging associated changes of bladder sensory function in female rats,particularly focused on TRPV1 and TRPV4 channels and electrical properties of bladder sensory neurons;(3)the cellular mechanisms of mechanical stimulation evoked intracellular Ca2+increase in urothelial cells and its age-related changes.Part Ⅰ Age-related decline in female bladder sensory functionPurpose:To observe whether there are age-related changes in the bladder sensory function and bladder contractility by analyzing the urodynamic data from female patients.Methods:1.Women with mild urinary incontinence and uterine prolapse who came to our hospital for urodynamic testing were collected in our study.Finally,two hundred and thirty-five subjects were enrolled for analysis according to the exclusion and inclusion criteria.2.For each patient,free urine flow was measured,and urine was collected for ATP measurement with luciferin-luciferase bioluminescence assay.Volume-pressure during storage stage and pressure-flow during urination were measured.3.According to the principle of Matbias age division,patients were divided into two groups:young group(30-50 years old)and old group(50-70 years old).The bladder sensory function and contractile function of the two groups were compared.Results:1.Comparison of free urine flow:The maximum urine flow rate(Qmax)and average urine flow rate(Qace)were significantly higher in the young group than the old group(p<0.001).There was no significant difference in voiding volume and the duration of voiding(p>0.05).2.Comparison of sensory function during storage period:The bladder volume and the pressure at the first sensation(FS),first desire to urinate(FD)and strong desire to urinate(SD)are higher in the old group than the young group(p<0.001).3.Comparison of detrusor contraction function:There are no difference in maximum urine flow rate(Qmax),detrusor pressure at maximum flow rate(Pdet@Qmax)and maximum detrusor pressure(Pdetmax)between the two groups(p>0.05).4.Comparison of urinary ATP concentration and the correlation analysis of urinary ATP with bladder volume threshold:The urinary ATP concentration of the young group is higher than old group(p<0.001).The urinary ATP levels are negatively correlated with the volume threshold either in the young group(r=-0.706,p<0.001)or the old group(r=-0.733,p<0.001).Conclusion:The volume threshold and pressure threshold increase with aging suggesting a reduction in bladder sensory function,but the detrusor contraction function does not change with aging.These results suggest that decreased bladder sensory function may play an important role in the occurrence of LUTS in aged women,and imply that the neurophysiological mechanisms involving in generation of bladder sensation may be altered in aged women.Part Ⅱ Peripheral neuronal mechanisms of the decreased bladder sensory function in aged female ratsPurpose:To explore the underling peripheral neuronal mechanisms for the reduced sensory function in old female rats by examining age associated changes in bladder sensory function,changes in the excitability of primary sensory neurons,and changes in expression of TRP channels(TRPV1,TRPV4)in sensory neurons or in the bladder urothelium.Methods:Female Sprague-Dawley(SD)rats were divided into young group(2-3 months)and old group(15-18 months).1.The blood estrogen levels were compared between the two groups.Vaginal smears were performed to determine whether the reproductive cycle is still present in old rats.2.Metabolic cage experiments were done to record the urination behavior in awake rats.3.In cystometry(CMG)experiments,the pressure threshold and volume threshold for voiding initiation,maximum bladder pressure during voiding,bladder capacity and residual urine volume were recorded.The changes of the above parameters in response to intravesical infusion of TRPV1 agonist(capsaicin)and TRPV4 agonist(GSK)were observed.4.The bladder muscle strip experiments were performed in vitro to record the tension changes in response to the M receptor agonist(carbachol),capsaicin and GSK.5.Immunohistochemistry experiments were done to observe the protein expression level of TRPV4 in urothelium.In cultured urothelial cells,calcium imaging recording was performed to record GSK induced intracellular Ca2+ increase.6.Bladder primary sensory neurons were labeled with DiI 10-14 days before cell culture.DiI labeled DRG neurons were isolated and cultured for whole-cell patch clamp recording.The inward current evoked by the TRPV1 agonist capsaicin was recorded.7.The active and passive electrophysiological characteristics of bladder sensory neurons were recorded with current clamp model.The voltage threshold,the current threshold(Rheobase)for action potential generation,and the responses to the sup-threshold were used to measure excitability.Results:1.The blood estradiol of young rats is higher than old rats(p<0.05).The vaginal cytology showed that young rats have regular and periodic changes,while most of the old rats showed diestrus.2.Metabolic cage experiments showed that the urine output,24-hour water intake and 24-hour urination volume of old rats are significantly higher than young rats(p<0.01),but the frequency of 24-hour urination was significantly lower than young rats(p<0.05).3.CMG recording showed that the voiding interval(IVI),volume threshold(Vthreshold),pressure threshold(Pthresh)for voiding and bladder compliance in old rats was significantly higher than young rats(p<0.01).In both young and old rats,intravesical application of capsaicin or GSK can shorten IVI,decrease the Pthresh,increase maximum bladder pressure(Pmax)and basal bladder pressure(Pb).However,the changes of the above parameters in old rats were significantly smaller than those in young rats(p<0.01).4.Isolated bladder strip experiments in vitro showed that the contraction amplitude and the area under the contraction curve(AUC)of the muscle strips evoked by carbachol are significantly lower in young rats than in old rats(p<0.01).However,the amplitude and the AUC of electrical field stimulation,GSK and capsaicin evoked contractions were significantly lower in old rats than young rats(p<0.01).5.Immunohistochemistry experiments showed that the TRPV4 channel expression level in urothelium was significantly lower in old rats than young rats(p<0.05).Calcium imaging recording showed that GSK induced intracellular Ca2+ increase was significantly lower in old rats than young rats(p<0.001).6.Voltage clamp recording showed that the amplitude of capsaicin induced inward current was significantly lower in old rats than young rats(p<0.05).7.Current clamp recording showed that two types of firing pattern are present in bladder sensory neurons:tonic and phasic.Compared with phasic neurons,tonic neurons have a small cell diameter(p<0.05),a slower AP rise rate(p<0.05),a longer AP duration(p<0.05),a higher voltage threshold and rheobase for AP generation(p<0.05).8.There was no difference in membrane capacitance(Cm),resting potential(RMP),AP duration,AP overshoot,voltage threshold and rheobase required for AP generation between the two groups of rats(p>0.05).9.Phasic neurons in old rats have a longer AP duration(p<0.05),a slower AP increase rate(p<0.05),a higher voltage threshold(p<0.05)and a higher rheobase for AP generation(p<0.05)than young rats suggesting a higher excitability in old rats.Conclusion:The functional expression of TRPV1 channels in bladder sensory neurons in old female rats is reduced;the excitability of phasic neurons is reduced;the expression of TRPV4 channels in the urothelium is reduced.The above changes may contribute to the reduction of bladder sensory function in aged rats.The elucidation of these mechanisms helps to explain the pathogenesis of senile idiopathic DU/UAB.The TRP channels,especially the TRPV4 channel,may be a key pharmaceutical target for DU/UAB treatment in the future.The age-related decline in the excitability of the bladder phasic neurons provides a theoretical basis for neuron subtype-based treatment of idiopathic DU/UAB.Part Ⅲ The mechanisms underlying intracellular Ca2+ increase induced by mechanical stimulation in urothelial cells and its age-related changesPurpose:To explore the mechanisms of mechanical stimulation induced intracellular Ca2+increase in urothelial cells and its age-related changes using molecular biology,Ca2+imaging and pharmacology approaches.Methods:1.The urothelial cells were cultured in vitro and mechanical stimulation was given to the urothelial cells by forming an air liquid-interface(ALI).2.Calcium imaging was performed to record the increase of intracellular Ca2+([Ca2+]i)induced by ALI,and to examine the ion channels or receptors on the cell membrane or endoplasmic reticulum(ER)involved in the increase of[Ca2+]i and ATP release with a variety of channel or receptor’s blockers.3.ATP concentration in perfusate after ALI stimulation was measured using a luciferin-luciferase bioluminescence assay.ALI induced[Ca2+]i increase and ATP release were compared between young and old rats.4.Western Blot was done to compare the expression of TRPV4 and Pannexin-1 channel in the urothelium between young and old rats.Results:1.ALI can induce[Ca2+]i increase and ATP release in cultured urothelial cells(p>0.05),and these responses were repeatable.GdCl3,an non-specific mechanical sensitive channel(MSC)blocker,significantly reduced ALI induced[Ca2+]i increase(p<0.05).2.ENaC channel antagonist-amiloride,Piezo channel blocker-GsMTX4 and connexin channel blocker-carbenoxolone failed to block ALI-induced[Ca2+]i increase(p>0.05),while TRP channel non-specific blocker-RR,TRPV4-specific antagonist-HC0674 and Pannexin-1 channel peptide blocker-10panx can block the ALI induced[Ca2+]i increase(p<0.001).Meanwhile,10panx can reduce ALI induced ATP release(p<0.001).3.Zero calcium HBSS solution,endoplasmic reticulum Ca2+-ATPase pump blocker-TG,IP3 receptor antagonist-Xest and ryanodine receptor antagonist-ryanodine(100μM)all reduced ALI induced[Ca2+]i increase(P<0.001).Apyrase and non-selective purine receptor blocker-PPADS can significantly inhibit ALI induced[Ca2+]i increase(p<0.001).5.Immunofluorescence showed that TRPV4 and Pannexin-1 channels are highly expressed in the urothelium both from young and old rats.Western Blot quantitative analysis showed that the expression of TRPV4 and Pannexin-1 channels in the urothelium of old rats was significantly lower than young rats(p<0.05).The amplitude of ALI induced[Ca2+]i increase and the amount of ATP release in old rats were significantly lower than young rats(p<0.001).Conclusion:Our results suggest that Ca2+ release from intracellular Ca2+ store via IP3 or ryanodine receptors play dominate role than Ca2+ influx via membrane bound TRPV4 and Pannexin-1 channels in urothelial mechanical response.ATP release further enhance[Ca2+]i increase by acting P2X and P2Y receptors via autocrine or paracrine mechanisms.