Localisation of muscarinic receptor M1-M3 immunoreactivity in human colon

Physiological, pharmacological, radioligand binding and in-situ hybridisation studies show human intestine has M1, M2 and M3 muscarinic receptor subtypes, but the precise localisation of receptor subtypes is not known due to lack of specific antibodies for immunhistological studies. This study used new antibodies, fluorescence immunohistochemistry and confocal microscopy to determine the precise cellular location of cholinergic muscarinic receptor subtypes M1-M3 in human colon. Methods: Mid transverse colon removed from children with familial adenomatous polyposis was fixed, frozen, sectioned and incubated with M1, M2 or M3 antisera followed by fluorescent secondary antibodies. Sections were double labelled with antibodies against synaptophysin to display nerve varicosities and cKit to identify interstitial cells of Cajal. Images were collected on a Leica LSM confocal microscope to allow the subcellular location of receptors to be defined. Preabsorption with the immunising peptide and western blotting were used to confirm antibody specificity. Colon was also snap frozen, RNA extracted and RT-PCR performed to confirm expression of all receptor subtypes. Results: In paediatric human colon, mRNA was abundant for all 3 receptors (M1-M3). M1-immunoreactivity (-IR) was present in myenteric and submucosal neuron cell bodies and in endothelial cells of submucosal blood vessels. M2-IR was present on the surface of smooth muscle cells in both muscle layers and in varicosities on nerve fibres in myenteric ganglia and in the circular muscle. Strong M2-IR was present on endothelial cells in submucosal and serosal blood vessels. M3-IR was present on smooth muscle cells in both muscle layers, at lower abundance than M2. M3-IR was also present in some myenteric neuron cell bodies. Immunoreactivity was absent following preabsorption with immunising peptide. Western blotting confirmed the antibodies bound to proteins of the expected size. Conclusion: Consistent with pharmacological studies M2-IR and M3-IR were present on muscle with M2 more abundant thanM3. Nerves contained all 3 receptors with different distributions. M1-IR was present in cell bodies of many myenteric and submucosal neurons suggesting thatM1 receptors mediate cholinergic neuro-neuronal transmission in human colon. M3 was present in only a small subset of myenteric neurons. M2-IR was located presynaptically on nerve fibres suggesting that M2 receptors act as autoreceptors regulating acetylcholine release. M1-IR and M2-IR were present on endothelial cells in blood vessels suggesting they mediate cholinergic regulated vaso-activity.A. M. Harrington, J. M. Hutson and B. R. Southwel

Predictors of the outcome of percutaneous nerve evaluation for faecal incontinence

Not available

Recto-anal motility responses in the Göttingen minipig to selective stimulation of the ventral sacral nerve roots

Introduction: Following spinal cord injury control of normal bowel activities is lost, leading to constipation associated with episodic faecal impaction and overflow faecal incontinence. Rectal evacuation might improve if the rectum could be activated by selective stimulation of the ventral sacral nerve roots (VSNR) without activation of the external anal sphincter (EAS). <p></p>Aim: The aim was to investigate whether selective activation of the rectum, without activation of the EAS, can be obtained by selective stimulation of the VSNR (S2 -S3 ). <p></p>Methods and material: The recto-anal motility response to electrical stimulation was investigated in 7 alpha-1-chloralose anaesthetised pigs. Stimulation was accomplished through cuff-electrodes placed on the intradural VSNR. Anal pressure and rectal motility were recorded by manometry and impedance planimetry, respectively. <p></p>Results: Unilateral stimulation of the S2 (n=4) or S3 (n=3) roots with trains of short duration pulses at low as wel as high amplitude resulted in a contraction of the EAS. Using trains of long duration pulses, anodal blocking was achieved in all pigs resulting in an anal pressure response, which was reduced more than 80% compared to stimulation without blocking. In 3 pigs rectal responses were obtained. <p></p>Conclusion: The results of this study demonstrate that selective activation of the rectum, with reduced activation of the EAS is feasible when stimulating the VSNR

Anorectal motility responses to selective stimulation of the ventral sacral nerve roots in an experimental model

Background: Control of defaecation and continence may be lost in patients with spinal cord injury. Electrical stimulation of sacral nerve roots to promote defaecation simultaneously activates both the rectum and the external anal sphincter (EAS), and may actually obstruct defaecation. The aim of this study was to investigate whether the EAS could be blocked selectively by selective stimulation of the ventral sacral nerve roots, and whether activation of the rectum without activation of the EAS could be obtained by stimulation of the ventral sacral nerve roots. Methods: Selective electrical stimulation was performed using anodal blocking, a tripolar cuff electrode and monophasic rectangular current pulses applied to the sacral nerve roots in nine Göttingen minipigs. Results: Simultaneous responses in the rectum and the anal canal were observed in five animals, whereas only anal responses were noted in four. Variations in cross‐sectional area and an increase in rectal pressure seemed to facilitate defaecation. Without blocking, the increase in anal canal pressure was 16–45 cmH2O. With blocking, this increase was abolished in seven and reduced to 3–6 cmH2O in two animals. Conclusion: Selective activation of the rectum is possible using an anodal block of somatic motor fibres. This technique holds promise in further development of electro‐defaecation

Recto-anal motility response in the Göttingen minipig by selective stimulation of the ventral sacral nerve roots

Electrical stimulation of sacral roots for electrodefecation results in simultaneous activation of the rectum and the external anal sphincter. The sphincter contraction hinders evacuation of the rectum. In this study anodal blocking has been used to reduce the activation of the external anal sphincter. Using a tripolar cuff electrode and monophasic rectangular current pulses in 7 acute minipigs experiments the pressure response in the anal canal was reduced more than 80 % in all animals compared to stimulation without blocking. The result confirms previous results from the bladder, in other species, in that selective small fibre activation can be obtained using an anodal block