Segmental distribution and peptide content of primary afferent neurons innervating the urogenital organs and colon of male rats.

Many visceral afferent neurons contain peptides, which have been proposed as histochemical markers for nerve pathways of particular targets or as transmitter candidates. The former possibility was investigated in the present study. Primary afferent neurons which project to the urinary bladder, distal colon or penis of rats, and the colon of cats were labelled with retrogradely transported fluorescent dyes (Fast Blue, True Blue, or Fluoro Gold). One to six weeks after dye injection into the organs, lumbosacral dorsal root ganglia were removed, treated with colchicine, and processed for immunohistochemical identification of five peptides. Dye-labelled neurons were distributed in an organ-specific manner in the lower lumbosacral ganglia, where colon afferent neurons were almost exclusively found in S1 ganglia, penis neurons primarily in L6, and bladder neurons at both levels. Substance P- (SP), calcitonin gene-related peptide-(CGRP), vasoactive intestinal peptide- (VIP), enkephalin- (ENK), and somatostatin- (SOM) immunoreactivity (IR) were detected in neurons in all lumbosacral ganglia but only some of these peptides were present in a large percentage of labelled neurons. The numbers of peptide-containing neurons innervating each organ were CGRP greater than SP greater than VIP greater than ENK greater than SOM; however some differences were observed in the relative proportions of these neuronal populations between upper lumbar and lower lumbosacral ganglia and between different organs. The major difference seen at the upper lumbar level was amongst the SP-IR neurons, which were common (25-30%) amongst bladder and colon afferent neurons but absent in penis neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasoactive intestinal polypeptide elicits a discharge in chronically decentralized sympathetic ganglia

Vasoactive intestinal polypeptide (VIP, 5-50 micrograms) injected intraarterially to the chronically decentralized cat superior cervical ganglion elicited a prolonged (2-5 min) postganglionic discharge which was resistant to cholinergic blocking agents but was blocked by [Leu5]enkephalin and GABA (10-200 micrograms i.a.). VIP did not elicit a discharge in acutely decentralized ganglia. These findings indicate that VIP has direct excitatory effects on ganglion cells and that these excitatory effects are enhanced following degeneration of the preganglionic nerve terminals.     

An electrophysiological study of the sacral parasympathetic pathway to the colon of the cat.

1. Electrophysiological techniques were used to study the sacral parasympathetic pathway to the colon of the cat. 2. Electrical stimulation of the sacral ventral roots or the pelvic nerve elicited contractions of the colon and firing in nerve filaments on the serosal surface of the colon. Both responses were markedly reduced by the administration of ganglionic blocking agents. It is concluded that sacral preganglionic fibres to the colon make synaptic contacts with extramural ganglion cells. These cells were identified histologically in small ganglia on the serosal surface of the distal colon and rectum. 3. Transmission in extramural colonic ganglia was cholinergic and mediated by nicotinic receptors. Colonic ganglia did not exhibit large recruiting responses during repetitive (1-4 c/s) preganglionic nerve stimulation or an adrenergic inhibitory mechanism, both of which have been identified in bladder parasympathetic ganglia. It is concluded that colonic ganglia unlike bladder function primarily as simple relay stations and have little potential for modulating the neral activity arising in the central nervus system. 4. The preganglionic input to colonic ganglia was mediated by C fibres with maximal conduction velocities ranging from 0-5 to 1-4 m/sec. Bladder ganglia, on the other hand, received a preganglionic input composed of B fibres with maximal conduction velocities ranging from 8 to 10 m/sec. The possible physiological significance of different types of preganglionic fibres in the sacral outflow is discussed.     

An excitatory action of 5-Hydroxytryptamine on sympathetic preganglionic neurones

Summary Preganglionic neurones in the fourth thoracic segment of anaesthetized cats fired spontaneously at a rate of about 2/sec. The effects of micro-electrophoretic administration of 5-hydroxytryptamine, noradrenaline, acetylcholine and DL-homocysteic acid on this spontaneous firing were determined. Many cells were excited by 5-hydroxytryptamine (5HT) and DL-homocysteic acid (DLH), a few were depressed by noradrenaline (NAdr), but acetylcholine (ACh) was inactive. The data are consistent with the view that 5HT and NAdr may function as excitatory and inhibitory transmitters which are released from the terminals of descending pathways in the spinal cord.National Science Foundation Postdoctoral Fellow, Riker Fellow.     

Autologous primary muscle-derived cells transfer into the lower urinary tract

The goal of these experiments was to establish the basic methodology for future clinical applications of muscle-derived cells (MDC) tissue engineering and gene transfer for the treatment of urological dysfunction. Primary MDC isolated via preplating techniques from adult female SD rats were transduced with retrovirus encoding the expression of beta-galactosidase reporter gene. The MDC were injected into the right and left lateral walls of the bladder and proximal urethra of the autologous animals (n = 6) with a 10 microl Hamilton micro syringe. The amount of injected MDC ranged from 1 to 2 x 10(6) cells. The injected tissue was harvested after 7, 14, and 28 days, sectioned and examined histologically for beta-galactosidase and immunohistochemically for fast myosin heavy chain specific to skeletal muscle. The tissues were also stained for anti-CD4 and anti-CD8 antibodies to assess for cellular immune reaction. We have detected a large number of autologous MDC expressing beta-galactosidase and positively stained for fast myosin heavy chain in the bladder and urethral wall. Many injected myoblasts and myotubes were also seen in the bladder and urethral wall at each time point. Staining of lymphocytes with anti-CD4 and anti-CD8 antibodies was negative after MDC injection at each time point. We have demonstrated the long-term survival of autologous MDC and MDC mediated gene transfer into the bladder and urethral wall. Autologous MDC and MDC mediated gene transfer may be a promising treatment to augment bladder and urethral sphincter function.     

Effect of intravesical nitric oxide therapy on cyclophosphamide-induced cystitis

PURPOSE: This study was conducted to examine effects of nitric oxide (NO) donors on bladder hyperactivity induced by cyclophosphamide (CYP)-induced cystitis. MATERIALS AND METHODS: Female Sprague-Dawley rats received a single intraperitoneal injection of CYP (100 mg./kg.), and then their micturition pattern including mean micturition volume and the number of micturitions during 24 hours was recorded in a metabolic cage before and after CYP treatment. Forty-eight hours after CYP injection, bladder function under urethane anesthesia was evaluated by cystometry with continuous saline infusion (0.04 ml. per minute) or under isovolumetric conditions (0.8 ml. bladder volume). NO donors, S-nitroso-N-acetyl-penicillamine (SNAP, 2 mM) or sodium nitroprusside (SNP, 1 mM), and an NO synthase (NOS) inhibitor, N-nitro-L-arginine methyl ester (L-NAME, 20 mM) were administered intravesically. Direct action of SNAP on bladder afferent neurons was also tested in a patch-clamp recording study. RESULTS: The number of micturitions significantly increased during the first 24 hours after CYP injection (19.0 +/- 0.88 versus 92.1 +/- 16.3 micturitions/24 hours, mean +/- SE, n = 25) (p <0.001). There was no significant difference in total micturition volume before (12.3 +/- 1.0 ml./24 hours) and after CYP treatment (15.6 +/- 1.5 ml./24 hours). During continuous infusion cystometry, intercontraction interval (ICI) was smaller in CYP-injected rats than in control rats. In CYP-injected animals, NO donors increased the ICI, but did not change the amplitude of bladder contractions. Continuous intravesical infusion of the NOS inhibitor did not alter the cystometric parameters. During cystometry under isovolumetric conditions, contraction frequency was decreased after NO donor administration. NO donors did not influence bladder activity in control rats. In patch clamp recordings, when SNAP (500 microM) was directly applied to dissociated afferent neurons innervating the urinary bladder, high-voltage-activated Ca2+ channel currents were suppressed by approximately 30%. CONCLUSIONS: Intravesical NO donors can suppress CYP-induced bladder hyperactivity. We hypothesize that the effect of NO donors is not due to smooth muscle relaxation, but rather due to an inhibitory effect on bladder afferent pathways that was manifested by an increase in intercontraction interval without changes in contraction amplitude. NO donors may be considered as a possible treatment of CYP-induced and other types of bladder inflammation.