Birch, David John; (2001) The perivascular innervation of human mesenteric blood vessels in health and inflammatory bowel disease. Doctoral thesis (M.D), UCL (University College London).

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Abstract

Vascular abnormalities and ischaemia have been implicated in the pathophysiology of Crohn's disease and ulcerative colitis. Perivascular nerves and the endothelium form the dual control mechanism for local blood flow. This study investigates the structure and function of the perivascular nerve plexus to identify changes in IBD which may contribute to the disease process. Immunohistochemical techniques using computerised quantitative image analysis were used to assess the nerve density and structure of the nerve plexus in resected colonic specimens. Noradrenaline (NA), neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and 5-hydroxytryptamine (5-HT) were identified in the perivascular nerves. The neuronal marker, protein gene product 9.5 (PGP), was used to determine the total number of nerves. In the control arteries, the number of nerves containing NPY, NA and VIP, in proportion to those with PGP-like immunoreactivity (LI), was 100%, 60% and 30% respectively, compared to 100%, 85% and 30% in the veins. 5-HT nerves were sparse and SP and CGRP were identified in less than 5% of the nerves of the control vessels; CGRP-LI was absent in the control veins. In IBD there was an increase in the number of nerve fibres in both the arteries and veins (control vs IBD arteries; 6.94 ± 1.22 vs 19.83 ± 1.52, p=0.005; veins; 9.31 ± 1.06 vs 14.39 ± 0.59, p=0.01; mean PGP total fluorescent area ± 95% CI; Student's t-test). This increase was mainly of NA and NPY-containing sympathetic nerves and was accompanied by the appearance of a less organised nerve plexus. The relative proportion of nerve types was similar to the control vessels, although 5-HT-LI was identified more consistently and CGRP-LI was found in the veins. The response of the vessels to vasoactive substances and to transmural electrical field stimulation (EFS) was examined. NA and adenosine triphosphate (ATP), cotransmitters with NPY in sympathetic nerves, produced concentration-dependent contractions of the arteries and the veins. The strength of the contraction to NA was greater in the arteries than the veins (control arteries vs veins; 124.3 ± 19.3 vs 64.1 ± l9.0, p=0.0008; % maximal contraction to KCl (Emax); mean ± 95% CI; Student's t-test). NPY produced dose-dependent contraction in the veins, but not in the arteries. In control veins, but not arteries, the presence of a sub-threshold dose of NPY increased the sensitivity to NA, and increased the force of contraction. There was no difference in the maximal contraction to NA of the IBD vessels compared with controls, but there was a right-shift of the concentration-response curves of the IBD arteries to ATP, indicating decreased sensitivity (3.22 ± 0.15 V5 2.59 ± 0.45, p=0.021; [-log] producing 25% of the maximal contraction of the vessel to KCl 120mM (p[A]₂₅); mean ± 95%CI; Student's t-test). The modulatory effects of NPY on the contractions of the veins to NA were not seen. Transmural EFS of perivascular nerves produced contractions of the veins but not of the arteries which was abolished by tetrodotoxin (1μM). The neurogenic contractions were partially blocked by the α₁-adrenoceptor antagonist prazosin (1μM), P₂-purinoceptor antagonism with suramin (10μM) or desensitisation of P₂ᵪ-purinoceptors with α, β-methylene adenosine triphosphate (1μM). In the presence of both α₁-adrenoceptor and purinoceptor blockade any small residual contraction was abolished by the addition of the α₂-adrenoceptor antagonist, yohimbine. In IBD, there was a significant enhancement of the contractile response (control vs IBD veins; 10.2 ± 4.2 vs 33.0 ± 14.35, p=0.038; % maximal contraction to 120mM KCl; mean ± 95% Cl; Student's t-test). The proportion of this response to α₁-adrenoceptor stimulation was less than in the control vessels. After P₂-purinoceptor antagonism, there was often a large residual contraction which was abolished by yohimbine, but in some, it was abolished only by tetrodotoxin. Electron microscope studies on control vessels identified differences between the arteries and veins, including wide separation of nerves and vascular smooth muscle with interposed collagen and fibroblasts, which may explain the lack of responsiveness to nerve stimulation. The increase in nerve density in IBD arteries was confirmed. This study defines changes in the structure and function of human mesenteric perivascular nerves in IBD. These changes in the mechanism of blood flow control may contribute to the ischaemia hypothesised as a factor in the pathogenesis of IBD.

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