Effect Of Mechanical Stretch On Excitability Of Bladder ICC And Its Contribution To Detrusor Overactivity

Background and Objective: Bladder function regulation of urine storage and emptying involves complicated mechanisms. Many researches indicated that besides neural modulation, myogenic factors in bladder take part in the process of detrusor contractions. Detrusor strips show spontaneous contractions under static mechanical stretch, which are independent with neural regulation. Many studies suggested some myogenic changes lead to bladder dysfunction, such as overactive bladder. Since then, lots of theories about bladder myogenic factors have been presented to explain bladder function regulation such as secretion of urothelium, gap junctions and some special cell population. However, the exact mechanisms of those myogenic factors are not fully understood. In recent years, researchers found some special interstitial cells in bladder and believe those cells are important myogenic factors that take part in bladder modulation. Those interstitial cells have the same receptor tyrosine kinase c-kit molecular marker as interstitial cells of Cajal (ICC) in gastrointestinal tract and some scientist named those cells as bladder ICC. Previous researches of bladder ICC about their potential pacemaker function and relationship with nerve brought new understandings and recognitions of physiology and pathophysiology underlining bladder function regulation.Interstitial cells of Cajal in gut have potential pace-making property. They have spontaneous excitability and generate periodic"slow waves"to aid local coordination to sense excitatory and inhibitory neural inputs and spread those signal to the adjacent myocytes and they can sense mechanical stimulations and integrate gut motility. McCloskey firstly identified c-kit positive interstitial cells in guinea pig bladder in 2002. They also widely distribute in urinary tract, including renal pelvic, ureter, bladder, urethral and even in cavernous body. Those cells share the same molecular marker with gut ICC, but their morphological properties and potential functions may not assemble with their analogue in gastrointestinal tract. The 5th International Symposium on Interstitial Cells of Cajal (Ireland, 2007) recommended that all such cells in the urinary tract be classified as ICC and we adopted this nomenclature in the present paper. ICC in bladder have close connection with detrusor myocytes and show intimate relationship with nerve fibers. Those specific characters indicate that bladder ICC may play a similar role as gut ICC. Although there are little direct evidence of ICC in bladder is a pacemaker, ICC networking is a potential candidate to modulate bladder sensation and coordinate autonomous activities in bladder wall.Several studies have demonstrated that ICC in gut can respond the mechanical stimulation and convert this stimulus into electrical signals and consequently modulate intestinal peristalsis. Since ICC has also been identified in the urinary bladder for years, and they were presumed to relate with spontaneous phasic activity of the detrusor muscle and maintaining local tension in storage phase, we speculate those functions may associate with mechanical stretch stimulation. However, the correlation between bladder ICC and mechanical stretch has not been defined yet.In our previous studies, c-kit positive ICC in guinea pig bladder have been demonstrated to have spontaneous depolarization potentials(If), and have close relationship with detrusors spontaneous contractions; ICC in bladder have different distributive density; bladder trigone have higher density of ICC and smooth muscle strips derived from that region of bladder have higher spontaneous contraction frequency. We also found that ICC can sense and respond to neurotransmitters such as ATP, carbacol, et al. The effects of Glevic on rat pelvic nerve afferent discharge evoked by ATP indicated that bladder ICC play very important role in bladder sensation and neurotransmission. Since the potential pacemaker and neurotransmmition function have beem demonstrated in bladder ICC, we speculated that bladder ICC have similar functions of mechanical sensitivity and take part in the the regulation of detrusor contractions. Moreover, the special function of bladder ICC in mechanical sensitivities may contribute to the normal bladder regulation and mechanisms underline bladder dysfunction.In summary, bladder detrusor have spontaneous excitability and present periodical contraction under static mechanical stretch. Whether bladder ICC is the origin of the spontaneous excitability? Whether bladder ICC have mechanical sensitivity and whether the changes of ICC in structure and function contribute to bladder dysfunction? Answering those questions and exploring bladder ICC provide a new approach to further understand of bladder regulation and pathogenesis of bladder dysfunction.In this study, we tried to investigate the variation of morphology, distribution and excitability of bladder ICC in chronical bladder obstructive animal models in vivo and detected the mechanical sensitiveity of cultured ICC and its preliminary pharmacological properties in vitro.Our studies were carried out as following steps: first, we constructed partial bladder outlet obstruction (PBOO) models and investigated the morphological and quantitative changes of bladder ICC in PBOO bladders to elucidate the adaptive variation of bladder ICC underline long-term mechanical stretch; Second, at the base of the results of the first part, we detected the spontaneous [Ca2+]i transient properties of ICC after PBOO in vitro to explore the possible mechanisms underline detrusor overactivity (DO); Third, ICC were cultured in vitro in the static uniaxial mechanical stretch device and applied step-wise static stretch on cultured ICC. Stretch induced [Ca2+]i transient properties were detected. We also investigated the preliminary mechanisms of those [Ca2+]i transient with calcium influx and intracellular calcium stores inhibitors application.Materials and methods:1. Female guinea pigs of 2～3 months old were used in the study. Proximal urethra was partial ligated to produce the models of partial bladder outlet obstruction (PBOO), sham operated animals were set as control. Intravesical pressure was measured 6 weeks later. Animals were divided into DO group, DS group and control group based on the urodynamic results. Immunohistochemical studies under confocal microscopy were performed in whole mounted preparations and cryosections to identified the morphological and distributional properties of c-kit positive ICC in bladders;2. The quantitative evaluation of c-kit positive cells in bladders of each group was performed with FCM analysis as well as manually counting of c-kit positive ICC under confocal microscopy;3. Static mechanical stretch device was constructed with StageFlexer silicone membrane and polyethylene toothcombs ect. Mechanical stretch was also obtained from hypotonic distention; those two methods were compared in the effeciency of stretch and cell morphology;4. Urinary bladder tissues were dispersed with colleagenase and cultured in vitro and c-kit positive ICC were identified with immunofluorescent staining;5. Spontaneous [Ca2+]i transient properties of ICC were identified with living cell calcium indicator Fluo-4AM.6. After 3-4 days cultured on the mechanical stretch silicone mambranes, step-wise mechanical stretch was applied to the membrane and the cells cultured onto it, meanwhile, [Ca2+]i transient induced by mechanical stretch (SICT) of ICC were detected under confocal microscopy.7. The calcium transient properties of cultured cells under mechanical stretch were also been measured after exposed respectively to ruthenium red (a ryanodine sensitive intracellular Ca2+ stores inhibitor), 2-APB (an IP3 sensitive intracellular Ca2+ stores inhibitor) and replacing extracellular solution by no calcium HBS to investigate the composition of SICT in cultured ICC.Results:1. C-kit positive ICC mainly located in suburothelium and intermuscular layers. In control and DS bladders, we found those intramuscular c-kit positive cells were discrete and did not contact with each other. Stellate-shaped c-kit positive cells in DO bladders presented many cytoplasmic branches emanating from them. Those lateral branches connected with each other to construct a reticular network between smooth muscle bundles2. C-kit positive cells in DO bladders were significant increased comparing to the DS and control bladders.3. Static mechanical stretch device for in vitro cultured cells was successfully constructed. Mechanical stretch could be applied approximate 10-30% step-wisely and extension was subjected uniformly to the silicone membrane, and the cells cultured on it. Hypotonic distension could also expand the cell volume to obtain mechanical stretch in vitro. However, the nonuniform swelling and extracellular environment changing underline hypotonic restrict its application.4. Successfully cultured bladder ICC in vitro. Those fusiform cells with dendritic branches were identified as ICC with c-kit immunocytochemistry experiment.5. Freshly isolated ICC from DO bladders showed spontaneous calcium waves with higher frequency and lower amplitude comparing to those from DS and control bladder.6. Strech-induced [Ca2+]i transient could be detected in cultured bladder ICC when 20-30% extension was applied to the cultured cells via lengthening the silicone membrane. The similar calcium augment were also detected in the cultured smooth muscle cells with longer responding time comparing to ICC. SICT generated in ICC could transfer to adjacent smooth muscle cells throough cell membrane connection.7. The amplitude of SICT was significantly reduced when removing the extracellular Ca2+ or exposed to ruthenium red . 2-APB nearly abolished the augment of [Ca2+]i response to mechanical stretch application.Conclusion:1. Long-term obstruction following PBOO caused morphological changes and increasing quantity of ICC, which may play a role in the pathogenesis of DO. Freshly dispersed ICC from DO bladders showed spontaneous calcium waves with high frequency and lower amplitude comparing to those from DS and control bladder, which indicated that ICC have different excitability in DO bladders.2. Cultured bladder ICC showed mechanical sensitivity and presented enhanced [Ca2+]i augment under stretch load. ICC were more sensitive than SMC to mechanical stretch and the calicium waves could transfer from ICC to adjacent SMC. Mechanosensitive role of ICC provide a novel mechanism underling myogenic contraction.3. IP3 sensitive Ca2+ stores release compose the major part of initiation of [Ca2+]i augment in responding to mechanical stretch.