The Study Of Unstable Contraction Mechanism In The Overactive Bladder

Chapter One TShtued Eyl oefc tUrnospthaybslieo lCoognictrala catnido nB Mioemcehcahnainsimc ainl The overactive BladderBackground & Objective:The determination of overactive bladder (OAB) may be made of urinay urgency or the unstable contraction during filling cystometrogram (CMG) in urodynamical study. Clinically, the hallmark of OAB diagnosis is urgency even without urodynamical study. Firstly, the sensation of urgency in OAB indicates there are afferent signals from the bladder to the central nervous system (CNS) through the pelvic nerve. Secondly, during the unstable contraction, the phasic contractile activity might involve efferent signals from parasympathetic nerve which was also contained in the pelvic nerve. Given local surroundings of the bladder can evoke unstable contraction which even was absolutely independent of the signals via the pelvic nerve, nevertheless, the afferent fiber of the bladder could be activated with bladder pressure increase during unstable contraction. Thus, these afferent signals are high correlative with the symptom of urgency.However, the urinary urgency is merely subjective evidence. It has not been cleared what the exactly afferent and efferent signals are among the pelvic nerve as well as what relevant reflexes involve when the unstable contraction occurs in OAB. Therefor, in this chapter study, on the basis of an OAB rat model which was induced by bladder outlet obstruction (BOO), we aim to determinate whether efferent signals in the pelvic nerve participate in the unstable contraction in OAB. In the other side, we also aim to determinate if there are afferent signals in the pelvic during the unstable contraction in OAB. The urgency would have an objective support if afferent signals'activity has been determinate in the pelvic nerve during the unstable contraction in OAB. Thus, it indicates that there is a neurogenic basis in the unstable contaction in OAB, and the determinated afferent signals are high relevant to the symptom of urgency.Materals & Methods:1. Besides the suprapubic surgical pathway (obstruction of proximal urethra) and the perineal pathway (obstruction of distal urethra) to BOO, a new surgerical method, transobturator-vaginal pathway (obstruction of muddle urethra) was performed in this study to induce OAB rats after these BOO surgical procedures.2. 6 -8 weeks after the BOO surgery, OAB rat model was determinated by filling cystometry if there were unstable contractions (over 15 cmH2O) according to the conventional standard in the ICS terminology. Quantitive comparsions of the filling cystometry results between OAB rats and normal rats were determinated.3. OAB rats were studied under urethane anesthesia, during filling CMG (6ml/h; model A) or isovolumetric CMG (bladder neck ligation, volume 0.8 ml; model B). After microsurgerical preparation (exposure of the pelvic nerve, the external urinary sphincter (EUS), and the motor branch of the pudendal nerve), an electrophysiological multi-channel simultaneous recording system was set for the recordings of pelvic afferent fiber potentials (crush central end of the pelvic nerve) or pelvic efferent fiber potentials (crush peripheral end of the pelvic nerve) as well as the pudendal nerve motor beanch potentials, EUS electromyogram (EMG) and abdominal muscle EMG. The results of these simultaneous recordings during the constable contraction in OAB rats were quantitive analyzed in potentials of frequency and amplitude to determinate effects of the unstable contraction in OAB rats.4. To test the effect of the unstable contraction in OAB rats after the decentralization of the CNS, during filling CMG (model A) or isovolumetric CMG (model B), OAB rats were studied the changes of the unstable contraction after the transection of the spinal cord (T8 level) or some nerves which are relevant to the bladder function. The transected nerves included the pelvic nerve, the sympathetic trunk, and the pudendal nerve. Before and after transections, the quantitive comparsions of the unstable contraction in OAB rats were determinated.5. To test effects of the spontaneous contraction in isolated OAB detursor strips after substantial deinnervation by a cocktail which contains mixed nervous receptors to the relevant nerves in the bladder. OAB detruor strips were harversted from the bladder and incubated in a physiological saline solution (PSS;37°C). Both electrical stimulation trial and isometric tension trial were studied before and after administration of the cocktail. Quantitive comparisons of the spontaneous contraction and the stimulation-evoked contraction were characterized before and after administration of the cocktail.Results:1. The rate of OAB rats by the new procedure was significantly different to the rate of OAB rats both by the suprapubic pathway and by the peripheral pathway in filling cystometry. The urodynamical results were no significant difference bwteen the three surgey methods in determinated OAB rats, and significantly different between OAB rats and normal rats.2. During filling CMG (model A), according to different changes of pelvic afferent fiber signals, pelvic efferent fiber signals, the relevant nerves and muscles responses, the unstable contraction in OAB rats was determinaetd as two kinds of the unstable contractions: the small pressure of the unstable contraction (S-Poab) and the big pressure of the unstable contraction (B-Poab). For the B-Poab, there were significant differences in the potentials of amplitudes (μV) between before B-Poab and B-Poab in the recordings of both pelvic affernt fiber and pelvic efferent fiber. It also showed significant differences in the potentials of amplitudes (μV) and frequency (Hz) between before B-Poab and B-Poab during the recordings of the motor branch of the pudendal nerve, EUS EMG, and abdominal muscle EMG. All these differences have not been determinated in the unstable contraction of S-Poab.3. During isovolumetic CMG (model B), compared to model A, the bladder baseline pressure (Pb) significantly increased. There were voiding-like contraction cycles in the CMG. Similarly, the unstable contractions of S-Poab and B-Poab were also determinated according to the above standard in model A. For the B-Poab, there were significantly differences between before B-Poab and B-Poab in the potentials of amplitudes (μV) both in pelvic affernt fiber and in pelvic efferent fiber. It showed significant differences in the potentials of amplitudes (μV) and frequency (Hz) between before B-Poab and B-Poab during the recordings of the motor branch of pudendal nerve, EUS EMG, and abdominal muscle EMG. However, for the S-Poab, all comparsions of before S-Poab and S-Poab in these electrophysiologcal recordings have not shown any significant difference.4. Effects of the unstable contraction in OAB rats after T8 transection: During filling CMG model A, Both the filling-voiding cycle and the unstable contraction of B-Poab were eliminated by such spinal cord transection. There was a significant increase in the Pb. The S-Poab was still good with a little increase in pressure amplitude without significant change. During isovolumteic CMG model B, the voiding-like contraction cycle did not recur after the transection. The B-Poab had also could not restore. Similarly, a significant increase in the Pb and a little increase in the S-Poab were determinated.5. Effects of bladder relevant nerves transection by the sequence of the pelvic nerve, the sympathetic trunk, and the pudendal nerve: During filling CMG model A, the filling-voiding cycle was eliminated. The Pb was a significant increase. No B-Poab could be determinated. There was no signifiannt change in the S-Poab. The Pb significantly dropped after transection of the pudendal nerve. During CMG model B, the voiding-like contraction and the B-Poab cound not be determinated after these transections. No signifiannt changes happened in the S-Poab.6. The effect of the spontaneous contraction and the stimulation-evoked contraction in isolated OAB detrusor strips by the Cocktail. 1 The stimulation-evoked contraction was eliminated with the administration of the Cocktail, and restored with washing-out of the Cocktail. It indicated that the Cocktail can eliminate the local nervous effect in detursor strip. 2 The spontaneous contraction did not weaken after the presence of the Cocktail. Moreover, the amplitude of the spontaneous contraction was showed a significant increase after the presence of the Cocktail.Conliusion:1. There are two kinds of mechanisms of the unstable contraction in OAB rats, the B-Poab and the S-Poab. The former has signals of both pelvic afferent fiber and pelvic affernt fiber which indicates there is a nervous objective basis of urgency. Moreover, the B-Poab can trigger some relevant reflexes. The S-Poab doesn't dependent on above nervous mechanism. It indicates the unstable contraction in OAB has also a detrusor mechanism.2. After the transection of the T8 spinal cord or bladder relevant nerves, the unstable contraction of B-Poab was eliminated. But the S-Poab maintained the above transections. It indicated some of the unstable contractions are independent of the nervous system.3. The excitation of the spontanenous contraction in OAB isolated detrusor strip has its myogentic basis. It might indicate some unstable contractions in OAB were induced by this prerequisite detrusor mechanism on excitation of the spontenous contraction.Chapter Two A Pilot Study of Expressions and Regulative Effects of Ryanodine Receptor Calcium Channel in The Overactive BladderBackground & Objective:A variety of changes occur during overactive bladder (OAB). The above study, along with the other studies, suggests that there is a myogenic detrusor basis excitability and contractile overactivity in OAB rat. Ryanodine receptors (RyR) in the sarcoplasmic reticulum act as an important modulator of excitation and contraction in detrusor smooth muscle, contributing transient localized Ca²⁺ release events (Ca²⁺ sparks) associated with activation of Ca²⁺-activated K+ channels (Kca) and negative feedback regulation of voltage-dependent Ca²⁺ channels (VDCC), and thus regulation corresponding in the excitation of membrane and the spontaneous contractility, which presumably has a high relevant to the spontaneous contractility and exactation overactivity in OAB detrusor. In this study, we aim to determinate if the alterations in RyR mRNA & protein expression, and if the change of RyR regulative function to the excitation of membrane and the spontaneous contractility. Therefor, it would be determinated if the changed RyR in OAB detrusor is responsible for increased spontaneous contractility which is the prerequisite of detrusor mechanism in the unstable contraction.Materals & Methods:1. RT-PCR test was investigated for determinating the sub-type of RyR in OAB detrusor. Semiquantitative RT-PCR test and western blot analysis determined RyR expression respectively in the level of mRNA & protein in OAB and normal detrusor preparations.2. Small detrusor strips were dissected from fresh bladders, and isometric tension was recorded from OAB detrusors and normal detrusors. Under the incubation of PSS-Cocktail (37°C), the spontaneous contractions were recorded and compared by adding various agents which including VDCC blocker (nimodipine), RyR agonist (caffeine), or RyR blocker (ryanodine) in the spontaneous contraction test of both OAB detrusor strips and normal detrusor strips. Under the effects of these agents, quantitive comparisons of the freqnency (Hz) and amplitude (mN) in the spontaneous contraction were analyzed.Results:1.â‘ The sub-type of RyR2 was determinated in OAB rat detrusor by RT-PCR testing. There is no sub-type difference in beteen OAB detrusor and normal detrusor.â‘¡Compared to normal preparations, semiquantitative RT-PCR test and western blot analysis showed that RyR expression of mRNA as well as protein significantly decreased in OAB detursor preparations.2.â‘ With the RyR blocker (ryanodine), the frequency of the spontaneous contractility significantly increased in normal bladder strips, while ryanodine failed to affect the frequency and amplitude of the spontaneous contractions in OAB detursor strips.â‘¡The administration of Caffeine inhibited the frequency and amplitude of the spontaneous contractile activity in the detursor strips preparations of both OAB and control.â‘¢The administration of L-type Ca²⁺ channel antagonist (nimodipine), the contractile activity was almost abolished in both OAB and control except for little activity below countable aplitude in OAB detrusor strips.Conliusion:This study results provide the first characterization of a loss of regulation of spontaneous contractile activity by RyR in OAB detrusor associated with a significant decrease in RyR expression. RyR in normal detrusor act as negative feedback regulators of spontaneous contractile activity, presumably by releasing Ca²⁺ that activates Kca channels to decrease contractility. This cross talk mechanism might be weakened in OAB smooth muscle cell, which results in spontaneous contractile overactivity.