Study of Intrinsic Bladder Activity by Optical Imaging

描述(申请人提供)：在人类中，逼尿肌过度活动存在于婴儿中，通常在成年人中消失，并经常因出口阻塞而再次出现。同样，在大鼠中，自发的起搏器活动负责固有的平滑肌收缩，在新生膀胱中存在，在成年膀胱中不存在，并在膀胱出口梗阻后重新出现。已经有许多尝试使用膀胱测压、细胞外、细胞内和膜片钳记录技术来表征膀胱起搏器活动和逼尿肌过度活动。然而，起搏器活动是起源于有限的特定部位还是随机发生，兴奋如何在平滑肌内传播以及逼尿肌过度活动的机制仍不清楚。 我们已经发展了光学成像技术来映射逼尿肌动作电位和钙离子通量。我们的光电二极管阵列系统允许我们以高达4000帧/S的速度同时记录来自膀胱壁256个部位的动作电位和钙瞬变。我们建议应用我们的方法来研究新生和成年大鼠膀胱自发活动的起源(S)和传播机制，神经调节的参与，以及部分出口梗阻后逼尿肌过度活动的机制。 大鼠的膀胱将被切除，在尿道处插管，并用适当的荧光染料染色/装入。完整的膀胱将被放置在温度调节的浴缸中，连接到压力传感器，并从腹侧、背侧或外侧浆膜表面成像。或者，膀胱会被打开并以薄片的形式安装在滑动支架上，这样我们就可以施加均匀的拉伸，测量发展的张力，并从整个管腔表面获取光学信号。我们将记录自发活动，以及通过刺激主要骨盆神经节或直接双极刺激浆膜或腔表面而诱发的活动。 在使用新生儿膀胱的初步研究中，起搏器活动起源于膀胱顶附近的一个单一部位，并引发大幅度(15-30 cm H20)收缩。相反，在正常成人膀胱中，自发活动同时起源于多个部位，且幅度较低(2-5 cm H20)。然而，在梗阻的肥厚的成人膀胱中，局灶性起搏器活动重新出现，引发大规模的协调收缩。同时阻断乙酰胆碱、三磷酸腺苷、P物质、一氧化氮以及α和β肾上腺素能激动剂的作用可以改变但不能完全阻断新生儿、成人或梗阻膀胱的自发活动。然而，缝隙连接阻滞剂(10 mU/M 18β-甘草次酸)可逆地抑制新生儿和阻塞膀胱的自发活动，但不能消除正常成人膀胱的自发活动。这表明，缝隙连接对于局灶性启动的大规模自发活动是必要的，这种自发活动以协调的方式传播到整个新生儿的膀胱，这种连接在发育过程中丢失，并在梗阻后重新出现。因此，我们的具体目标是：i.)2.研究出生后膀胱固有活动的调节；研究自发性逼尿肌活动的神经化学控制；目的：确定部分梗阻膀胱逼尿肌过度活动的原因。

DESCRIPTION (provided by applicant): In humans, detrusor overactivity is present in infants, usually disappears in adults, and frequently reappears with outlet obstruction. Similarly, in rats, spontaneous pacemaker activity, which is responsible for intrinsic smooth muscle contraction, is present in neonatal bladders, absent in adult bladders and re-emerges following bladder outlet obstruction. There have been numerous attempts to characterize bladder pacemaker activity and detrusor overactivity using cystometry, extracellular, intracellular and patch clamp recording techniques. However, whether pacemaker activity originates from restricted specialized sites or occurs randomly, how excitation propagates within the smooth muscle and the mechanism of detrusor over activity remain unclear. We have developed optical imaging techniques to map detrusor action potentials and Ca 2+ fluxes. Our photodiode array system allows us to simultaneously record, at up to 4000 frames/s, action potentials and Ca 2+ transients from 256 sites in the bladder wall. We propose to apply our methodology to investigate the origin(s) and mechanism of propagation of spontaneous activity in the bladders of neonate and adult rats, the involvement of neural modulation, and the mechanism of detrusor over activity following partial outlet obstruction. Rat bladders will be excised, cannulated at the urethra and stained/loaded with the appropriate fluorescent dyes. Intact bladders will be placed in a temperature-regulated bath, connected to a pressure transducer, and imaged from the ventral, dorsal or lateral serosal surface. Alternatively, bladders will be opened and mounted as sheets on a sliding rack that allows us to apply uniform stretch, measure developed tension and acquire optical signals from the entire luminal surface. We will record spontaneous activity, and activity evoked through stimulation of the major pelvic ganglion or direct bipolar stimulation of the serosal or luminal surfaces. In preliminary studies using neonatal bladders, pacemaker activity originated at a single site near the bladder dome and initiated large amplitude (15-30 cm H20) contractions. By contrast, in normal adult bladders, spontaneous activity originated simultaneously at multiple sites and was of low amplitude (2-5 cm H20). In obstructed hypertrophied adult bladders however, focal pacemaker activity re-emerged initiating large amplitude coordinated contractions. Simultaneous blockade of the effects of acetylcholine, ATP, substance P, nitric oxide and alpha- and beta-adrenergic agonists altered but did not completely block spontaneous activity in neonatal, adult or obstructed bladders. However, gap junction blockade (10 mu/M 18beta-glycyrrhetinic acid) reversibly abolished spontaneous activity in neonatal and obstructed bladders, but not normal adult bladders. This suggests that gap junction connectivity is necessary for focally initiated large amplitude spontaneous activity that spreads in a coordinated manner throughout the neonate's bladder, and that this connectivity is lost during development and re-emerges following obstruction. Accordingly, our specific aims are: I.) To investigate the postnatal modulation of intrinsic bladder activity; II.) To study the neural-chemical control of spontaneous detrusor activity; and III.) To determine the origin of detrusor overactivity in partially obstructed bladders.