Inflammation of the intestine causes pain and altered motility , at least in part through effects on the enteric nervous system. While these changes may be reversed with healing , permanent damage may contribute to inflammatory bowel disease (IBD) and post-enteritis irritable bowel syndrome. Since little information exists , we induced colitis in male Sprague-Dawley rats with dinitrobenzene sulfonic acid and used immunocytochemistry to examine the number and distribution of enteric neurons at times up to 35 days later. Inflammation caused significant neuronal loss in the inflamed region by 24 hours , with only 49% of neurons remaining by days 4 to 6 and thereafter , when inflammation had subsided. Eosinophils were found within the myenteric plexus at only at the earliest time points , despite a general infiltration of neutrophils into the muscle wall. While the number of myenteric ganglia remained constant , there was significant decrease in the number of ganglia in the submucosal plexus. Despite reduced neuronal number and hyperplasia of smooth muscle , the density of axons among the smooth muscle cells remained unchanged during and after inflammation. Intracolonic application of the topical steroid budesonide caused a dose-dependent prevention of neuronal loss , suggesting that evaluation of anti-inflammatory therapy in inflammatory bowel disease should include quantitative assessment of neural components. (Am J Pathol 1999, 155:1051-1057)Inflammatory bowel disease (IBD) is a chronic idiopathic inflammation of the intestine that affects an increasing percentage of the population in Western society, with the highest incidence among the younger population, for whom there are no specific or effective treatments. The broad acting anti-inflammatory agents in wide use have serious side effects of immune suppression, loss of bone calcium, and growth retardation. Therefore, a strong need exists for increased information about the cellular basis of this disease, as well as new pharmacological tools and improved methods of use.Studies of IBD have relied heavily on immunological approaches, relating activation of immune cells to the periodic exacerbation and remissions of disease. However, a particular challenge lies in understanding the long-term or permanent changes present in the intestine in IBD, which are evident even in the periods of remission between acute episodes. Recently, attention has been paid to the other cell types in the intestine that may acquire the ability to participate in inflammation or that are previously unexpected targets of inflammatory change.1 This has shown that nonimmune cells can participate directly in inflammation, as well as pointing out the potential for long-term alterations in cell structure and function that may predispose to repeated episodes of inflammation. Thus, intestinal smooth muscle has been shown to have the potential to present antigen to activated T cells and may also be a source of cytokines that can directly affect neural function. 1,2Most, if not all, aspects of normal...

Inflammation of the rat jejunum with Trichinella spiralis causes altered smooth muscle contractility by day 6 postinfection (PI). We investigated the association of structural change in the smooth muscle layers with inflammation. By day 6 PI, smooth muscle area in cross sections of jejunum increased (P less than 0.05) in longitudinal (LM) and circular (CM) muscle layers. Nuclei counting in cross sections showed that cell number increased two- to threefold in CM and LM, and this increase was not reversed on day 23 PI. Estimation of cell size showed significant hypertrophy by day 6 PI in both muscle layers. [3H]thymidine autoradiography showed that the labeling index (LI) of jejunal LM and CM increased sharply on day 4 PI and peaked on day 6 PI (10- to 15-fold increase). The noninflamed ileum showed a smaller trophic response, with no significant change in area or nuclei number, the LI was increased only on day 6 PI in the ileal CM and was unchanged in LM. Thus extensive hyperplasia and hypertrophy of smooth muscle cells are associated with intestinal inflammation.

We investigated the involvement of nitric oxide in trinitrobenzene-sulfonic acid (TNB) colitis. Every 24 h after TNB, rats were orally dosed with NG-nitro-L-arginine methyl ester (L-NAME; 30 mg/kg), NG-nitro-D-arginine methyl ester (D-NAME), or water, and food intake, body weight, and plasma nitrite levels were measured. On day 6, colonic nitric oxide synthase and myeloperoxidase (MPO) activity, histology, intestinal muscle growth, NADPH-diaphorase, and myenteric nerve function were assessed. Food intake and body weight were reduced during the first 72 h of colitis. On day 6 post-TNB, a fourfold increase in mucosal nitric oxide synthase, a 30-fold increase in MPO, and a fivefold elevation in plasma nitrite were measured. Smooth muscle hyperplasia and hypertrophy in both colonic muscle layers, numerous diaphorase-positive macrophages in the myenteric plexus, and a suppression of myenteric nerve function were also observed. Unlike D-NAME, oral L-NAME reduced MPO and intestinal muscle hyperplasia by > 90%. Likewise, plasma nitrite and colonic nitric oxide synthase were reduced by > 70%. L-NAME completely prevented macrophage infiltration into the muscle. Conversely, it had no effect on anorexia or intestinal smooth muscle hypertrophy, nor did it affect suppressed myenteric nerve neurotransmitter release. These results demonstrate the selective transmural protective effects of L-NAME in the inflamed colon, implicating nitric oxide as a mediator.

Intestinal inflammation causes initial axonal degeneration and neuronal death but subsequent axon outgrowth from surviving neurons restores innervation density to the target smooth muscle cells. Elsewhere, the pro-inflammatory cytokines TNF␣ and IL-1␤ cause neurotoxicity, leading us to test their role in promoting enteric neuron death. In a rat coculture model, TNF␣ or IL-1␤ did not affect neuron number but did promote significant neurite outgrowth to twofold that of control by 48 h, while other cytokines (e.g., IL-4, TGF␤) were without effect. TNF␣ or IL-1␤ activated the NFB signaling pathway, and inhibition of NFB signaling blocked the stimulation of neurite growth. However, nuclear translocation of NFB in smooth muscle cells but not in adjacent neurons suggested a dominant role for smooth muscle cells. TNF␣ or IL-1␤ sharply increased both mRNA and protein for GDNF, while the neurotrophic effects of TNF␣ or IL-1␤ were blocked by the RET-receptor blocker vandetanib. Conditioned medium from cytokine-treated smooth muscle cells mimicked the neurotrophic effect, inferring that TNF␣ and IL-1␤ promote neurite growth through NFB-dependent induction of glial cell linederived neurotrophic factor (GDNF) expression in intestinal smooth muscle cells. In vivo, TNBS-colitis caused early nuclear translocation of NFB in smooth muscle cells. Conditioned medium from the intact smooth muscle of the inflamed colon caused a 2.5-fold increase in neurite number in cocultures, while Western blotting showed a substantial increase in GDNF protein. Pro-inflammatory cytokines promote neurite growth through upregulation of GDNF, a novel process that may facilitate re-innervation of smooth muscle cells and a return to homeostasis following initial damage.

The nervous and immune systems may communicate through the action of neurotransmitters on mast cells. We used patchclamp electrophysiology to assess the responses of rat peritoneal mast cells (PMC) to low levels of substance P (SP), which are likely to occur in situ. SP at 50 nM, or even 10,000 times reduced to 5 pM, triggered an outwardly rectified Cl- current (50 nM: 10 of 10 cells; 5 pM: 10 of 11 cells), although degranulation never occurred. Electrical responses were delayed (mean 102.6 s for 5 pM SP), appearing as brief current pulses. Reapplication of SP resulted in peak current augmentation (mean 15.3 pA before exposure to SP, 47.3 pA after 1st exposure, and 116.0 pA after 2nd exposure to 5 pM SP). Cells repetitively exposed to SP degranulated 5-15 min and > 25 min after the second exposure to 50 nM SP (10 of 10 cells) or 5 pM SP (5 of 9 cells), respectively. This effect was reduced by 10 microM 5-nitro-2-(3-phenylpropylamino)benzoic acid or when extracellular Ca2+ was removed, indicating a dependence on Cl- conductance and extracellular Ca2+. We propose that whole cell current oscillations in the absence of degranulation are the functional correlate of priming, a process that increases cellular responsiveness for the subsequent stimulation.

We examined the release of acetylcholine (ACh) from jejunal longitudinal muscle-myenteric plexus preparations in noninfected control rats and in rats infected 6, 23, or 40 days previously with Trichinella spiralis. ACh release was assessed by preincubating the tissue with [3H]choline and measuring the evoked release of tritium. The uptake of 3H was significantly less in tissue from T. spiralis-infected rats compared with control. In tissues from either infected or control animals, electrical field stimulation (30 V, 0.5 ms, 10 Hz for 1 min), or veratridine (6-30 microM) induced 3H release that was tetrodotoxin sensitive. Depolarization by KCl (25-75 mM) also caused 3H release, but this was only partially reduced by tetrodotoxin. Radiochromatographic analysis indicated evoked release of 3H to be almost entirely [3H]ACh. In rats infected 6 days previously with T. spiralis, [3H]ACh release induced by KCl, veratridine, and field stimulation were decreased at least 80%. The suppression of [3H]ACh release induced by veratridine or KCl was fully reversible after 40 days postinfection, but field-stimulated responses remained approximately 50% of control values. These results indicate that T. spiralis infection in the rat is accompanied by a reversible suppression of ACh release from the longitudinal muscle-myenteric plexus of the jejunum.

No abstract

Intestinal smooth muscle cells are normally quiescent, but in the widely studied model of trinitrobenzene sulfonic acid (TNBS)-induced colitis in the rat, the onset of inflammation causes proliferation that leads to increased cell number and an altered phenotype. The factors that drive this are unclear and were studied in primary cultures of circular smooth muscle cells (CSMC) from the rat colon. While platelet-derived growth factor (PDGF)-AA, fibroblast growth factor (FGF), and epidermal growth factor (EGF) were ineffective, PDGF-BB and insulin-like growth factor-1 (IGF-1) caused significant increase in [(3)H]thymidine incorporation, bromodeoxyuridine uptake, and increased CSMC number, with PDGF-BB (≥0.2 nM) substantially more effective than IGF-1. Surprisingly, CSMC lacked expression of PDGF receptor-β (PDGF-Rβ) upon isolation but by 4 days in vitro, CSMC gained expression of PDGF-Rβ as shown by quantitative PCR, Western blot analysis, and immunocytochemistry; these CSMC responded to PDGF-BB but not IGF-1. PDGF-BB caused PDGF-Rβ phosphorylation and mobilization from the surface membrane, leading to activation of both Akt and ERK signaling pathways, which were essential for subsequent proliferation. In contrast, PDGF-AA, FGF, EGF, and IGF-1 were ineffective. In vivo, control CSMC lacked expression of PDGF-Rβ. However, this changed rapidly with TNBS-colitis, and by day 2 when CSMC proliferation in vivo is maximal, freshly isolated CSMC showed on-going PDGF-Rβ phosphorylation that was further increased by exogenous PDGF-BB. This suggests that the onset of PDGF-Rβ expression is a key factor in CSMC growth in vitro and in vivo, where inflammation may damage intrinsic inhibitory mechanisms and thus lead to hyperplasia.