Neurogenic mechanisms in bladder and bowel ageing.

The prevalence of both urinary and faecal incontinence, and also chronic constipation, increases with ageing and these conditions have a major impact on the quality of life of the elderly. Management of bladder and bowel dysfunction in the elderly is currently far from ideal and also carries a significant financial burden. Understanding how these changes occur is thus a major priority in biogerontology. The functions of the bladder and terminal bowel are regulated by complex neuronal networks. In particular neurons of the spinal cord and peripheral ganglia play a key role in regulating micturition and defaecation reflexes as well as promoting continence. In this review we discuss the evidence for ageing-induced neuronal dysfunction that might predispose to neurogenic forms of incontinence in the elderly.

The TNF-α antagonist etanercept reverses age-related decreases in colonic SERT expression and faecal output in mice.

Treatment for chronic constipation in older people is challenging and the condition has a major impact on quality of life. A lack of understanding about the causes of this condition has hampered the development of effective treatments. 5-HT is an important pro-kinetic agent in the colon. We examined whether alterations in colonic 5-HT signalling underlie age-related changes in faecal output in mice and whether these changes were due to an increase in TNF-α. Components of the 5-HT signalling system (5-HT, 5-HIAA, SERT) and TNF-α expression were examined in the distal colon of 3, 12, 18 and 24-month old mice and faecal output and water content monitored under control conditions and following the administration of etanercept (TNF-α inhibitor; 1 mg Kg-1). Faecal output and water content were reduced in aged animals. Age increased mucosal 5-HT availability and TNF-α expression and decreased mucosal SERT expression and 5-HIAA. Etanercept treatment of old mice reversed these changes, suggesting that age-related changes in TNFα expression are an important regulator of mucosal 5-HT signalling and pellet output and water content in old mice. These data point to "anti-TNFα" drugs as potential treatments for age-related chronic constipation.

Age-associated changes in distribution of the P2X2 receptor in the major pelvic ganglion of the male rat.

P2X purinoceptors constitute a group of ligand-gated ion channels one of which, the P2X2 receptor has previously been described in neurons within autonomic ganglia, including the major pelvic ganglion (MPG). Earlier work strongly suggests that age-associated attrition of sympathetic but not parasympathetic MPG neurons occurs but there have been no investigations of age-related changes in P2X2 receptor expression in autonomic ganglia or to determine whether the receptor is localised in one or both of the two MPG neuronal subpopulations. In the current study, immunocytochemistry was employed to label cells expressing the P2X2 receptor in the MPG from young and aged male Wistar rats. By combining P2X2 receptor immunocytochemistry with the immunolocalisation of tyrosine hydroxylase (TH), the numbers of sympathetic (TH+) and parasympathetic (TH-) neurons expressing the P2X2 receptor were determined. In young rats P2X2 receptor expression was found in 93.08+/-3.2% of TH- (parasympathetic) neurons. In aged rats a similar analysis revealed no significant difference in the number of TH- neurons expressing the P2X2 receptor. In contrast a significant increase in the number of TH+ sympathetic neurons expressing P2X2 was observed in the MPG of aged rats (10.70+/-2.26%) in comparison to the young group (2.38+/-0.78%). Age-related changes in the numbers of small intensely fluorescent (SIF) cells which were highly P2X2 positive were also quantified, revealing a small reduction in number with age. This study has demonstrated the preferential localisation of P2X2 receptors to parasympathetic MPG neurons and suggests that purinergic transmission in the pelvic organs maybe largely unaffected by ageing.

Changes in the substance P-containing innervation of the lumbosacral spinal cord in male Wistar rats as a consequence of ageing.

Quantitative image analysis was used to determine age-related changes in the substance P-containing innervation of autonomic and somatic nuclei in the lumbosacral spinal cord, which are associated with the control of micturition and sexual reflexes. In the upper lumbar segments (L1-L2), significant declines in the distribution density of substance P-containing processes were observed in the dorsal grey commissure, the intermediolateral cell column and the ventral horn. More caudally, at levels corresponding to L5 through S1, significant reductions were seen in the dorsal grey commissure and within the sacral parasympathetic nucleus. In contrast to these observations, the substance P-immunoreactive innervation of the dorsolateral nucleus remained robust in aged animals and was not significantly different from young adults. It is possible that these distinct age-related patterns of change in substance P-containing innervation, are reflected in the urinary/sexual dysfunction's in aged animals.

Biogenic amine and neuropeptide inputs to identified pelvic floor motoneurons that also express SRC-1.

In the rat, the neurochemical phenotypes of neurons that are presynaptic to motoneurons innervating the levator ani are poorly defined. In this study, motoneurons within the spinal nucleus of the bulbospongiosus (SNB) were revealed, using retrograde labelling, following injection of cholera toxin B subunit into the levator ani muscle. Different classes of neuron making substantial inputs onto these labelled neurons were revealed by using immunocytochemistry for dopamine beta hydroxylase, serotonin and substance P. Appositions (sites of presumptive synapses) between immunoreactive terminals and both the somata and dendrites of labelled SNB motoneurons were commonly seen suggesting that substance P, noradrenaline and serotonin are likely to exert a significant influence on the activity of perineal motoneurons and thus on sexual reflexes. Additionally, steroid receptor coactivator-1 was found to be present in the nuclei of 96% of SNB neurons retrogradely labelled from the levator ani. This suggests that practically all of the neurons that innervate the levator ani are likely to be modulated by circulating steroid hormones.

SRC-1 localisation in lumbosacral spinal cord of male and female Wistar rats.

Nuclear receptor co-activators play an important role in enhancing transcriptional activity of steroid hormone receptors, however there is currently little information concerning their distribution within the spinal cord. In this study, the distribution of steroid receptor co-activator-1 (SRC-1) was examined with immunocytochemistry, in the lumbosacral cord of Wistar rats of both sexes. In all rats, regardless of sex, SRC-1 was predominant in neurons of the superficial laminae of the dorsal horn and within motorneurons of lamina IX. Sexually dimorphic nuclei exhibited robust SRC-1 immunoreactivity in young rats, including orchidectomised animals, but this appeared to decline in aged rats. Dorsal horn labelling appeared similarly reduced suggesting a possible age-related down-regulation of the transcription mediated by steroid receptors in some spinal neurons.

Age-associated changes in the monoaminergic innervation of rat lumbosacral spinal cord.

The effects of ageing on the innervation patterns of lumbosacral spinal nuclei involved in controlling lower urinary tract functions, including micturition, were studied using immunohistochemistry for serotonin (5-HT) and tyrosine hydroxylase (TH) in male Wistar rats of 3 and 24 months. Quantitative image analysis revealed significant age-associated declines in the innervation of most regions including the intermediolateral cell nucleus, sacral parasympathetic nucleus, dorsal grey commissure and in the ventral horn including the dorsolateral nucleus which in the rat is one of the component nuclei homologous to Onuf's nucleus in man. Notable exceptions to this generalised decline were observed in the 5-HT innervation of the sacral parasympathetic nucleus, which was maintained, and in the region of the dorsolateral motor nucleus where TH-like immunoreactivity did not significantly decline. These results suggest that the changes in micturition characteristics observed in aged rats may in part be a consequence of the alterations in, and decline of, aminergic inputs to both autonomic and somatic spinal nuclei associated with bladder function.

The effects of ageing and of DSP-4 administration on the micturition characteristics of male Wistar rats.

This study sought to determine the effects of ageing on the in vivo micturition characteristics of male Wistar rats and to assess whether they might be replicated in young rats by using the neurotoxin DSP-4 to lesion locus coeruleus-derived noradrenergic pathways projecting to spinal cord nuclei controlling micturition. Significant age-related changes in micturition patterns were observed. There was a loss of a diurnal rhythm in micturition patterns and a large increase in voided volume, maximal between 21 and 24 months, which was paralleled by an increased water intake. DSP-4 lesions neither altered micturition patterns nor water intake in the young adult rat. DSP-4 induced changes in the pattern of tyrosine hydroxylase-like immunoreactivity (TH-LI), most notably almost complete depletion of TH-LI in the dorsolateral nucleus and retention of TH-LI in lumbosacral autonomic preganglionic nuclei, did not mimic the changes in the pattern of TH-LI seen in aged rats.

Differential susceptibility to ageing of rat preganglionic neurones projecting to the major pelvic ganglion and of their afferent inputs.

We have analysed age-related changes in the morphology of preganglionic neurones in the lumbosacral spinal cord, labelled following injection of retrograde tracers into the major pelvic ganglion of young adult and aged male rats. We have also examined changes in neurotransmitter-characterised spinal afferent inputs to these neurones, or to the nuclei in which they lie, using light and electron microscope immunohistochemistry. In previous investigations of the major pelvic ganglion, the sympathetic, but not parasympathetic, postganglionic neurones were seen to exhibit age-related changes and the same pattern is seen in the preganglionic neurones. This included an apparent reduction in the numbers of sympathetic preganglionic neurones, and a reduction in the length of their dendrites and the complexity of their branches. Ultrastructural immunohistochemical studies described here reveal significant reductions in the area of synaptic contact made by glutamate-immunoreactive boutons onto the dendrites of sympathetic (but not parasympathetic) preganglionic neurones, while contacts from boutons immunoreactive for glycine or gamma-aminobutyric acid (GABA) were unchanged. There is also a reduction in synaptic contacts received by sympathetic somata from boutons immunoreactive for none of these amino acids. Serotonin-immunoreactive terminals are closely associated with preganglionic autonomic neurones, and these are reduced in number in sympathetic, but not parasympathetic, spinal nuclei of aged rats. However, serial section electron microscopy has so far failed to demonstrate conventional synaptic contacts between serotonergic terminals and the dendrites or somata of the preganglionic autonomic neurones. In young animals, axon terminals immunoreactive for thyrotropin-releasing hormone (TRH) are abundant in all spinal laminae including area X, but in aged animals, such terminals are significantly reduced in number in regions containing preganglionic sympathetic, but not parasympathetic, neurones. These results indicate that the sympathetic preganglionic neuron populations that project to the major pelvic ganglion, and the spinal inputs they receive, show a number of degenerative changes in aged rats which are not seen parasympathetic preganglionic neuronal populations.

Impaired colonic motility and reduction in tachykinin signalling in the aged mouse.

Ageing is associated with an increased incidence of constipation in humans. The contribution that the ageing process makes to this condition is unclear. The aim of this study was to determine the effects of age on faecal output and colonic motility in male C57BL/6J mice and to determine the role that altered tachykinin signalling plays in this process. Total faecal output recorded over a 24h period decreased with age due to a reduction in the number of pellets produced and their water content. These changes occurred in the absence of any significant change in food and water intake. There was an increase in the amount of faecal matter stored in the isolated colon with age which caused a proportional increase in colonic length. Analysis of colonic motility using an artificial pellet demonstrated that pellets moved in a stepwise fashion through the colon. There was an age-related increase in pellet transit time due to decreases in the step distance, velocity, and frequency of stepwise movements. These changes were reversed using the neurokinin 2 (NK2) receptor agonist neurokinin A. Addition of the NK2receptor antagonist GR159897 significantly increased transit time in the young animals by decreasing step distance, velocity and frequency, but was without effect in the aged colon. In summary, the ageing C57BL/6J mouse shows an impaired motility phenotype. These effects appear, at least in part, to be due to an attenuation of tachykinin signalling via NK2 receptors.

Nuclear expression of PG-21, SRC-1, and pCREB in regions of the lumbosacral spinal cord involved in pelvic innervation in young adult and aged rats.

In rats, ageing results in dysfunctional patterns of micturition and diminished sexual reflexes that may reflect degenerative changes within spinal circuitry. In both sexes the dorsal lateral nucleus and the spinal nucleus of the bulbospongiosus, which lie in the L5-S1 spinal segments, contain motor neurons that innervate perineal muscles, and the external anal and urethral sphincters. Neurons in the sacral parasympathetic nucleus of these segments provide autonomic control of the bladder, cervix and penis and other lower urinary tract structures. Interneurons in the dorsal gray commissure and dorsal horn have also been implicated in lower urinary tract function. This study investigates the cellular localisation of PG-21 androgen receptors, steroid receptor co-activator one (SRC-1) and the phosphorylated form of c-AMP response element binding protein (pCREB) within these spinal nuclei. These are components of signalling pathways that mediate cellular responses to steroid hormones and neurotrophins. Nuclear expression of PG-21 androgen receptors, SRC-1 and pCREB in young and aged rats was quantified using immunohistochemistry. There was a reduction in the number of spinal neurons expressing these molecules in the aged males while in aged females, SRC-1 and pCREB expression was largely unchanged. This suggests that the observed age-related changes may be linked to declining testosterone levels. Acute testosterone therapy restored expression of PG-21 androgen receptor in aged and orchidectomised male rats, however levels of re-expression varied within different nuclei suggesting a more prolonged period of hormone replacement may be required for full restoration.