Structural, neuronal, and functional adaptive changes in atrophic rat ileum

Abstract

Background: Inactivity of the gut leads to atrophic changes of which little is known.

Aims: To investigate structural, neuronal, and functional changes occurring in bypassed rat ileum.

Methods: Morphometry was used to characterise the atrophic changes. The numbers of enteric neurones, their expression of neurotransmitters, and the presence of interstitial cells of Cajal were studied using immunocytochemistry and in situ hybridisation. Motor activity was studied in vitro.

Results: Adaptive changes in bypassed ileum include atrophy and remodelling of the gut wall. The total numbers of submucous and myenteric neurones per unit length increased one and four weeks after bypass but were identical to sham operated intestine 10 weeks after bypass. Neurones expressing vasoactive intestinal peptide, neuropeptide Y, or pituitary adenylate cyclase activating peptide decreased gradually in number in bypassed ileum. Nitric oxide synthase expressing neurones were increased, particularly in the myenteric ganglia. No change in the frequency and distribution of interstitial cells of Cajal was noted. The contractile response elicited by electrical stimulation of sham operated ileum consisted of a fast cholinergic twitch followed by a slower non-adrenergic, non-cholinergic contraction. In the bypassed ileum an identical biphasic contraction was elicited; however, the entire response was non-adrenergic, non-cholinergic. The relaxatory response to electrical stimulation in sham operated ileum was nitric oxide mediated; after bypass it was non-nitrergic.

Conclusions: Notable atrophic changes were seen in the rat ileum after bypass. The enteric nervous system reacted with neuronal cell death and plasticity in terms of release and expression of neurotransmitters.

Figure 1

Schematic outline of the surgical procedures used.

Figure 2

Measurements on the circumference (A) and thickness of the whole wall, mucosa, submucosa, and muscle layers (B) in rat ileum, sham operated and bypassed for one, four, or 10 weeks. (C) Total numbers of submucous and myenteric nerve cell bodies (as estimated by EtBr staining) per visual field in longitudinally cut sections from sham operated and bypassed rats. Mean (SEM). *p<0.05, **p<0.01, ***p<0.001 compared with sham operated.

Figure 3

Cryostat sections from rat, sham operated (A and D), one week (B and E), and 10 weeks (C and F) bypassed ileum, immunostained for VIP (A-C) and nNOS (D-F). VIP immunoreactive nerve fibres are numerous in mucosa/submucosa, muscular layers, and enteric ganglia at all time points studied. nNOS immunoreactive nerve fibres are restricted to the muscle layers and myenteric ganglia. The number of nNOS immunoreactive cell bodies within myenteric ganglia (indicated by arrows) is increased in bypassed ileum, particularly 10 weeks postoperatively (F). Original magnification × 130.

Figure 4

Cryostat sections from rat, sham operated (A and E), one week (B and F), four weeks (C and G), and 10 weeks (D and H) bypassed ileum autoradiographically labelled for VIP mRNA (A-D) and galanin mRNA (E-H). VIP mRNA containing submucous neurones (indicated by arrows) are intensely labelled in sham operated ileum (A) but only weakly labelled in the bypassed ileum (B-D). The labelling of galanin mRNA containing enteric neurones is equally intense in both sham operated (E) and bypassed (F-H) ileum. Original magnification × 130.

Figure 5

Numbers of CGRP, galanin, GRP, NPY, PACAP, SP, VIP, and nNOS immunoreactive, NADPH diaphorase positive (A and C); and CGRP mRNA, galanin mRNA, NPY mRNA, VIP mRNA, and nNOS mRNA expressing (B and D) neuronal cell bodies in submucous (A and B) and myenteric (C and D) ganglia from sham operated and bypassed (1-10 weeks) rat ileum. The numbers of positive cell bodies are expressed as the percentage of the total number of cell bodies, established by EtBr staining. Mean (SEM). *p<0.05, **p<0.01, ***p<0.001 compared with sham operated; p values refer to one, two, or three bars, as indicated.

Figure 6

Cryostat sections from rat: (A) sham operated, (B) one week, and (C) 10 weeks bypassed ileum, showing the presence of ICC using antiserum against c-kit receptor. Numerous ICC are found within the deep muscular plexus and at the border of longitudinal and circular muscle in both sham operated and bypassed ileum. Original magnification × 180.

Figure 7

Tracings of electrically induced responses of rat sham operated, one week, and four weeks bypassed ileum. Stimulation (20 Hz, 4 V, 400 mA) was maintained for five seconds (indicated by vertical bars). The contractile response elicited by electrical stimulation of sham operated ileum consists of a fast cholinergic twitch followed by a slower non-adrenergic, non-cholinergic (NANC) mediated contraction; in the bypassed ileum an identical biphasic contraction is elicited; however, the entire response was NANC mediated—that is, not blocked by atropine. After precontraction by PGF_2α and in the presence of atropine a relaxation followed by a contraction is elicited by EFS. The EFS induced relaxatory response in sham operated ileum is abolished by L-NAME and thus, NO mediated. After bypass the relaxation was unaffected by pretreatment with L-NAME.