Expression of neuropeptides and nitric oxide synthase in neurones innervating the inflamed rat urinary bladder

Abstract

Micturition reflexes become hyperexcitable with the development of a cystitis. In the present study the question is addressed, whether alterations in the expression of neuropeptides and nitric oxide synthase (NOS) in the neuronal pathways to the bladder may be involved in the hyperexcitability. Primary sensory neurones in the dorsal root ganglia (DRG) L1, L2, L6 and S1 as well as postganglionic efferent neurones in the major pelvic ganglia (MPG) that innervate the rat urinary bladder were labeled with retrogradely transported Fast Blue (FB). Immunocytochemical techniques were used to determine alterations in the expression of calcitonin gene-related peptide (CGRP), substance P (SP), galanin (GAL) and NOS in these neurones following mustard oil-induced inflammation of the urinary bladder. Instillation of 2.5% mustard oil into the bladder led to a massive leukocyte infiltration of the vesical tissues, partial damage of the mucosal layer and a marked hyperreflexia of the detrusor muscle. 48 h after induction of the cystitis the proportion of FB-labeled bladder afferent neurones that expressed CGRP and SP were significantly increased in both the rostral lumbar DRGs (L1, L2) and the lumbosacral DRGs (L6, S1) (CGRP, +15–38%; SP, +47–158%) as compared to control animals. However, there was a differential effect of the inflammation on the expression of GAL and NOS in bladder afferents at the two segmental levels examined. Significant alterations in the number of FB-labeled afferents exhibiting GAL immunoreactivity were mainly restricted to the lumbosacral DRGs L6 (+169%) and S1 (+60%). On the contrary, the proportion of NOS-immunoreactive bladder afferents significantly increased only in the rostral lumbar DRGs L1 (+144%) and L2 (+193%), while the level of NOS-expression was unaffected at the lumbosacral levels. Inflammation furthermore induced a significant increase (+275%) in the number of FB-labeled neurones in the MPGs that exhibited NOS immunoreactivity. These results indicate that an upregulation of CGRP-, SP-, GAL- and NOS-synthesis in sensory and efferent neurones is involved in the response to an acute cystitis. Because of the differences in the segmental pattern and degree of upregulation of these substances in bladder afferents that project to the rostral lumbar and lumbosacral spinal cord a different regulation of the sympathetic and parasympathetic efferent outflow to the urinary bladder is suggested. The involvement of CGRP, SP, GAL and NOS in the modulation of both excitatory and inhibitory mechanisms that control the cystitis-induced detrusor hyperreflexia is discussed.

Introduction

Several important changes of urinary bladder function and sensation accompany an acute urinary bladder inflammation in humans. The neuronal and mechanical events associated with bladder filling and micturition become hypersensitive and progressively painful. This affects normal bladder capacity and frequency of micturition. In animals, experimental inflammation of the urinary bladder has been induced by instillation of irritant chemicals such as mustard oil, turpentine oil or capsaicin into the bladder 1, 16, 17, 18, 19, 25, 33, 34. The inflammatory vesical response is characterized by oedema, plasma protein extravasation from the vasculature, leukocyte infiltration of the bladder tissue and a marked and prolonged hyperactivity of the detrusor muscle. Since the bladder contractions following application of mustard oil are absent in denervated bladders, a neurogenic mechanism for the induction of an unstable and hyperreflexic detrusor muscle was postulated [34]. It was suggested that detrusor hyperexcitability was mainly due to alterations in the afferent innervation of the bladder. Indeed, previously unresponsive primary afferent neurones in the pelvic nerves were shown to change their receptive properties and develop ongoing activity following the induction of a cystitis 16, 17, 18. However, the rat urinary bladder receives a dual afferent innervation that originates from sensory neurones located in lumbosacral dorsal root ganglia (L5–S1) and thoracolumbar dorsal root ganglia (T11–L3) and whose axons travel in the pelvic and hypogastric nerves, respectively. Yet, little attention has been paid so far to the latter population of afferent neurones. Furthermore, changes in the efferent innervation of the bladder could not be excluded.

Whether neuropeptides are involved in the mediation of bladder hyperactivity following inflammation has been a matter of discussion for many years [29]. Yet, little information is available about alterations in the expression of neuropeptides in the bladder reflex pathway in this type of functional disorder. Neuropeptide-containing, capsaicin-sensitive primary afferent neurones innervating the bladder may be a part of the spinal, vesicovesical excitatory reflex-loop mediating detrusor hyperreflexia 13, 19, 29. Capsaicin-sensitive afferents, expressing neuropeptides like tachykinins and calcitonin gene-related peptide (CGRP), serve not only a sensory but also a local effector function, due to a release of peptides from their peripheral endings upon stimulation. Substance P (SP) and CGRP released from these nerves following induction of a cystitis may have pro-inflammatory effects in the periphery [15]and may play a role as neurotransmitters and/or neuromodulators in bladder ganglia and in the spinal cord.

Galanin (GAL) has been reported to be present in numerous nerve fibres in the urinary bladder of man and rat [5]. GAL was shown to influence the activity of vesical smooth muscles 11, 47including the rat urinary bladder and modulate neural transmission in autonomic ganglia 46, 56and at neuromuscular junctions [30]. However, the origin and distribution of the galaninergic innervation of the bladder is still an unsettled question.

In recent years, there is accumulating evidence indicating that nitric oxide synthase (NOS), the enzyme which forms the gaseous NO in various tissues including the nervous system is of high significance in this context. Histochemical 49, 51and pharmacological studies 22, 40have suggested an involvement of NO in visceral sensory pathways in pathological states. NO may further play an important role as a non-adrenergic, non-cholinergic (NANC) neurotransmitter or modulator in the efferent innervation to the urinary bladder 39, 48. However, the functional significance of the nitrergic innervation of the detrusor muscle remained uncertain.

In the present study, we investigated the expression of SP, CGRP, GAL and NOS in neurones of the dorsal root ganglia and major pelvic ganglia innervating the rat urinary bladder. Moreover, a comparison was made between tissue from normal animals and rats with a mustard oil-induced cystitis. The question was addressed, whether the reported electrophysiological changes that occur after induction of bladder inflammation are paralleled by neuroplastic changes in the chemical coding of these neurones. Thus indirect evidence for an involvement of the examined substances in the modulation of hyperexcitability of bladder reflexes was obtained.