The mesenteric circulation is highly specialized, supplying the metabolic needs of the gut and other organs; blood flow is altered in response to mechanical stimulation of food in the lumen17 and as part of inflammatory responses.18 Mesenteric veins form the largest capacitance bed in man and control of venous tone are important in the effective maintenance of blood pressure,19 particularly when posture changes. The greater nerve density in human mesenteric veins compared with arteries shown in this study may reflect this role.
Sympathetic perivascular nerves were previously thought to contain only NA, but are now recognized as utilizing cotransmitters, notably ATP and NPY.10 The neuronal markers for this study were selected on the basis of neurotransmitters commonly identified in the four classes of perivascular nerves,20 TH and NPY for sympathetic nerves, SP and CGRP for sensory-motor nerves, and NO and VIP for intrinsic nerves projecting from the enteric nervous system (ENS). 5-HT has previously been identified in human mesenteric perivascular nerves, probably after being taken up by sympathetic nerves.14 ATP is a cotransmitter in most, if not all of these nerve types, but there is no immunohistochemical method for its localization, and detecting its presence relies on functional studies. Noradrenaline and ATP are the major sympathetic vasoconstrictors, acting via postjunctional α1-adrenoceptors and P2X1 receptors.10 Although in some blood vessels NPY acts as a vasoconstrictor, in many vessels NPY acts as a neuromodulator, postjunctionally enhancing the contraction produced by other transmitters and/or acting prejunctionally to reduce transmitter release.10 Perivascular nerves, in concert with signals from the endothelium, regulate local blood flow.1
Expression of immunomarkers
In arteries and veins from IBD patients, there was an increase in overall nerve density measured as the TFA for PGP9.5, compared with controls. While increased immunoreactivity implies an increased number of fibres, it may also be indicative of increased branching or increased immunoreactivity within fibres. The majority of human mesenteric perivascular nerves were of sympathetic origin. All vessels had a plexus of TH- and NPY-containing nerves. Neuropeptide Y is also found in subpopulations of neurons in the ENS21 and in some parasympathetic nerves.22 There was a greater nerve density for NPY and NA in arteries and veins from patients with IBD.
A moderately dense plexus of nerves containing VIP was identified in arteries and veins. The VIP is a cotransmitter with acetylcholine and other neuropeptides in some parasympathetic nerves.22 However, the role of parasympathetic innervation of the human mesenteric circulation has been questioned23 and, in mesenteric vessels of the pig, VIP-immunoreactive nerves are derived from the ENS.24 In IBD vessels, VIP-immunoreactive nerves accounted for 30 and 25% of the total innervation (as shown by PGP9.5 staining) of arteries and veins, respectively, although this was not significantly different from control vessels. It has been suggested that VIP is involved in suppression of chronic inflammatory responses of the gut.25 Studies on changes in VIP innervation in the bowel wall have been contradictory26,27 and they may only occur close to the site of mucosal inflammation.28
Fewer than 5% of nerve fibres in control vessels were SP- and CGRP-immunoreactive probably indicating extrinsic sensory-motor nerves.2,29 Both are potent vasodilators and also interact with mast cells and may have a role in immune functions in inflamed bowel25,30 and in the protection of the mucosa.31 However, some of these nerves may represent projections of intrinsic neurons of the ENS, which have been shown to contain CGRP and SP in humans.32 There were no significant differences in the SP-immunoreactive perivascular nerve density between IBD and control arteries. In control veins, CGRP was absent, but present in all veins from IBD patients, suggesting a change in the role of the sensory motor innervation in IBD.
5-Hydroxytryptamine is often described as a ‘false neurotransmitter’ in sympathetic nerves.33 It is not synthesized in the nerve varicosities, but is taken up from extracellular sources after release from platelets and stored in vesicles for subsequent release, although a subpopulation of enteric neurons contain 5-HT as a principal transmitter.34 Vasoconstrictor nerves containing 5-HT have been identified in human mesenteric vessels.14 In spite of the low density of innervation in healthy vessels, there was a marked increase (three to four times) in the number of 5-HT-immunoreactive nerves in IBD, indicating greater release of 5-HT from platelets and perhaps enterochromaffin cells in this pathological condition. The number of 5-HT-containing mucosal enterochromaffin and mast cells was increased in inflamed bowel.35 Release of 5-HT could contribute to vasospasm and disturbances in blood flow, characteristic of the inflammatory response. This may indicate an underlying abnormality in 5-HT release or metabolism in the gut of IBD patients.36 Nitric oxide synthase-positive nerves were not detected in this study. This is consistent with a previous study on human infant mesenteric vessels.21
These neurochemical changes that we have identified in mesenteric vessels in IBD are similar to those identified in the myenteric plexus of ileum from patients with CD, where an increase in the number of nerves with TH-, NPY- and 5-HT-immunoreactivity was observed.37 Similarly, in a study comparing biopsies of normal and UC bowel, the number of sympathetic nerves, the mean diameter and the number of varicosities were seen to be increased in inflamed bowel.38 However, a loss of sympathetic nerve fibres from the mucosa and submucosa has been reported in IBD patients39 and in a mouse model of colitis.40 It was suggested that loss of sympathetic fibres might be more apparent in ‘hot’ inflammatory areas in the mucosa and submucosa. It has also been speculated that loss of sympathetic fibres is due to a repulsion of the fibres from the inflammatory area,41 which could lead to the concertinaed appearance of nerve fibres seen in this study. Ultrastructural studies in CD identified axonal proliferation accompanied by necrosis, both in the inflamed region of the bowel and the resection margins.42 In our study of mesenteric vessels in IBD, nerve proliferation was identified but there was no evidence of axonal degeneration. Nerve proliferation appears to be a widespread feature in chronic gut inflammation.43 The increase in sympathetic innervation would be likely to contribute to the vasospasm, non-synchronized bowel contraction and oedema observed in IBD. Whether nerve proliferation is the result of the inflammatory processes, perhaps by initiating increase in sympathetic nerve activity, or to an unknown environmental factor, has yet to be determined.
It should be noted that the IBD patients in this study were receiving corticosteroids. This may have an effect on the innervation, as intracolonic application of the corticosteroid, budesonide, to rats in which colitis had been induced, resulted in a dose-dependent prevention of nerve loss.44
Contractions to NA in IBD artery and vein were reduced, although not significantly, compared to controls. However, contractions to ATP were significantly decreased in both IBD artery and vein, suggesting that the purinergic component of sympathetic cotransmission may be selectively altered in the inflamed state. In a recent study of a mouse model of colitis, ATP did not induce vasoconstriction of submucosal arerioles,45 further supporting this view. In contrast to veins, arteries from control and IBD subjects did not respond to NPY. The contractile effects of NPY on healthy human mesenteric veins have been described previously.46 Significant decreases in the contractile responses of human mesenteric arteries to phenylephrine in CD have been described.47
The alteration in contractility of veins is likely to be of clinical importance. Localized venous contraction would contribute to engorgement and oedema of the gut, which is a prominent feature of CD. Furthermore, non-invasive tests of autonomic nervous system function have identified systemic abnormalities in UC and CD. Changes in contractility of splanchnic veins would be reflected in tests such as these.48 Abnormalities in blood flow in mesenteric arteries have been identified using Doppler sonography.49 Differential blood flow in the layers of the bowel wall may divert blood away from the mucosa, leading to paradoxical ischaemia. Increased flow in the face of contraction of the postcapillary resistance vessels may also contribute to vascular engorgement and oedema. It has been suggested that ischaemia plays a part in the pathogenesis of IBD.7 Experimental evidence in mice indicates that microcirculatory disturbances precede histological abnormalities.8
A further factor that may be of importance is ATP, which is released from both nerves and non-neuronal cells, as it has many actions in the inflammatory process, including mast cell degranulation, leucocyte adhesion to the endothelium, production of prostaglandins and inflammatory cytokines and potentiation of the oxidative burst. The response to ATP is determined by the receptor subtypes present in the tissues, which have been found to be altered in chronically inflamed tissues45,50, as well as alterations in ATP degradation.45
Abnormalities in the perivascular innervation of human mesenteric vessels have been demonstrated in the present study. The perivascular nerves are one arm of the dual control system for controlling blood flow,1 and changes in this system in patients with IBD may be important in its pathogenesis.