Functional Role And Involved Mechanisms Of Prokineticin 2 In Regulating Bladder Function And Sensation

Part I:The establishment of a novel animal model for urodynamic study in rats under restrained or freely-moving conditionObjective This study aimed to develop a novel animal model for urodynamic examination in conscious rats and collect basal urodynamic parameters of normal rat. In addition, the potential implact of restraint on rat bladder activities would also be addressed.Materials and Methods Twenty four female Sprague-Dawley rats were used in the current study. Intravesical catheter implantation was conducted on all the animals. On the eighth day after catheter implantation, all the rats undergone urodynamic test. They were firstly placed in a dorsal-access plastic restrainer and subsequently transferred to a filter paper-coated capacious transparent cage where they could move freely. For each rat, at least 6 voiding cycles were recorded. All the technique issues, such as tube twisting and catheter disconnection, occurring during the urodynamic examination were also recorded. Subsequently, the rats were sacrificed by cervical dislocation and the bladders were harvested for HE staining. The potential statistical difference of the urodynamic values and incidences of technological problems between the two groups was compared by paired two tail t-test and chi-square test respectively.Results Our present findings demonstrated that urodynamic investigation through a pre-implanted catheter is a feasible and repeatable approach for studying bladder functions in restrained waking rats. The current model enabled the researchers to successfully acquire stable and reliable urodynamic data of rats maintained in either restraining cages or capacious cabinets where they were able to move freely. Fewer technique problems need to be addressed in urodynamic examination in restrained rats than in freely-moving ones, and the use of experimental animals probably would be greatly reduced in the restrained condition. In addition, our data shows that there was no significant difference between the urodynamic parameters collected from restrained or freely-moving rats, which suggested that restrained urodynamic investigation is ideal for bladder function evaluation in rats. Histological examination of the bladder slides showed no apparent inflammation signs such as edema, erosion and leukocyte infiltration.Conclusions Intravesical catheter implantation followed with appropriate recovery period does not result in observable inflammation in the bladder wall. It is putatively a suitable way for the establishment of channel used for conscious bladder pressure recording in rats. Conscious cystometry in rats placed in a restraining cage as described above appears to be a reliable and practical method for evaluating detrusor activity and bladder function. Complemented with appropriate managements, restraint has no apparent influences on the urodynamic parameters of rat, which instead would reduce the use of experiment animal. This novel model can be widely used to investigate bladder dysfunction related to various disorders such as overactive bladder and interstitial cystitis as well to assess new therapeutic agents and/or strategies for these diseases.Part ?:PK2/PKR1 signaling regulates bladder function and sensation in rats with cyclophosphamide-induced cystitisObjective This study was designed to explore the expression and functional role of prokineticin family, a novel bioactive system with multiple pro-inflammatory activities, in modulating bladder activity and sensation in rats with cyclophosphamide (CYP)-induced cystitis.Materials and Methods Female Sprague-Dawley rats were treated with CYP to induce cystitis with different durations (4h,48h, and 8d). The severity of inflammation in the bladders was determined by general observation, histological evaluation and IL-1? mRNA detection. The expression of PKs (Prokineticin 1 & Prokineticin 2) and prokineticin receptors (PKRs) in normal and cystitis bladders were examined at several levels by IHC, real-time qPCR, western blot as and ELISA. Combining a nonselective prokineticin receptor antagonist (PKRA), physiological and pathological activities of Prokineticin 2 (PK2) in regulating bladder function and related visceral and somatic pain sensation were evaluated in normal rats as well as rats with cystitis through conscious cystometry, pain behavioral scoring and von-frey filaments.Results PK2 and Prokineticin receptor 1 (PKR1) were detected in all the rat bladders at both mRNA and protein levels. By contrast, Prokineticin 1 (PK1) and Prokineticin receptor 2 (PKR2) were not detected. CYP treatment dynamically increased the expression of PK2 in bladders with cystitis regardless of duration. Immunohistochemistry staining localized PKR1 primarily in the urothelium and demonstrated its up-regulation following CYP administration. Intravesical perfusion of PKRA showed no effect on micturition reflex activity and bladder sensation in control rats. In rats treated with CYP (48 h), PKRs inhibition with PKRA significantly increased the voiding volume and voiding interval without effect on maximum bladder pressure, while ameliorating visceral hyperalgesia.Conclusions PK2/PKR1 signaling pathway contributes to the modulation of inflammation-mediated voiding dysfunction, spontaneous visceral pain as well related somatic hyperalgesia. Local blockade of PKRs may be a novel and promising therapeutic strategy for the clinical management of inflammation-related bladder diseases.Part III:PK2 stimulates ATP release from the urothelium and regulates bladder function.Objective:This study tried to elucidate the potential mechanisms how PK2 regulates bladder function and sensation upon activation of PKR1 on the urothelial cells and further address the modulatory role of PK2/PKR1 signaling in bladder activity under physiological condition.Materials and Methods:Normal SD rats were randomly divided to two groups, control and experimental groups. On the eighth day after intravesical catheter implantation, experimental rats received PK2 intravesical perfusion and controls were treated with same volume of saline. Urodynamical examination and pain scoring were conducted on all the rats to determine the effect of PK2 on bladder function and sensation. In addition, fresh bladders were harvested from additional normal rats and incubated in Krebs solution. After equilibrium, the baseline levels of ATP and ACh release from the bladder wall were examined. Then half of the bladders were administrated with PK2 peptide solution, and then filled and distended with saline per 30 minutes. The left ones served as control and received saline intravesical instillation, and then were filled using the same protocol. The concentration of ATP and ACh on both sides of the bladder wall were measured by special kits at 30 min and 60 min after treatment.Results:Physiologically, PK2 (10 nM) intravesical perfusion changed the bladder function but did not cause bladder related spontaneous visceral pain. It increased voiding frequency and non-voiding contractions, decreased voiding volume and voiding interval, while did not affect maximum bladder pressure, basal bladder pressure, voiding threshold pressure as well. ATP and ACh were continuously detected on both mucosal and serosal side of the bladder wall. PK2 treatment remarkably promoted the secretion of ATP on both sides of the urothelium, however, the ACh production was not changed by PK2 administration. In contrast, intravesical irrigation with saline in control rats showed no effect on the release of both ATP and ACh from the urothelium on both sides.Conclusion:PK2 intravesical perfusion result in bladder dysfunction but do not evoke bladder related spontaneous pain. The urothelium bilaterally and continuously secret ATP and ACh under physiological conditions. PK2 is capable to improve ATP, but not ACh, release from the urothelium in normal bladders.