A spontaneous animal model of intestinal dysmotility evoked by inflammatory nitrergic dysfunction

Abstract

Background and aims: Recent reports indicate the presence of low grade inflammation in functional gastrointestinal disorders (FGID), in these cases often called "post-inflammatory" FGIDs. However, suitable animal models to study these disorders are not available. The Biobreeding (BB) rat consists of a diabetes-resistant (BBDR) and a diabetes-prone (BBDP) strain. In the diabetes-prone strain, 40-60% of the animals develop diabetes and concomitant nitrergic dysfunction. Our aim was to investigate the occurrence of intestinal inflammation, nitrergic dysfunction and intestinal dysmotility in non-diabetic animals.

Methods: Jejunal inflammation (MPO assay, Hematoxylin&Eosin staining and inducible nitric oxide synthase (iNOS) mRNA expression), in vitro jejunal motility (video analysis) and myenteric neuronal numbers (immunohistochemistry) were assessed in control, normoglycaemic BBDP and diabetic BBDP rats. To study the impact of iNOS inhibition on these parameters, normoglycaemic BBDP rats were treated with aminoguanidine.

Results: Compared to control, significant polymorphonuclear (PMN) cell infiltration, enhanced MPO activity, increased iNOS mRNA expression and a decreased ratio of nNOS to Hu-C/D positive neurons were observed in both normoglycaemic and diabetic BBDP rats. Aminoguanidine treatment decreased PMN infiltration, iNOS mRNA expression and MPO activity. Moreover, it restored the ratio of nNOS to Hu-C/D positive nerves in the myenteric plexus and decreased the abnormal jejunal elongation and dilation observed in normoglycaemic BBDP rats.

Conclusions: Aminoguanidine treatment counteracts the inflammation-induced nitrergic dysfunction and prevents dysmotility, both of which are independent of hyperglycaemia in BB rats. Nitrergic dysfunction may contribute to the pathophysiology of "low-grade inflammatory" FGIDs. Normoglycaemic BBDP rats may be considered a suitable animal model to study the pathogenesis of FGIDs.

Figure 1. Intestinal inflammation in BB rats.

Representative images of the myenteric ganglia and the muscular layer of the jejunum as stained by H&E (A). Intra-ganglionic infiltration of PMN cells (arrows) and thicker muscular layers were observed in both inflamed and diabetic rats. All scale bars: 50 µm. Numbers of PMN cells per 50 myenteric ganglia (B) in both inflamed and diabetic rats increased, compared to control rats (**p<0.01, *p<0.05, respectively). Sum of circular and longitudinal muscular layer thickness (C) in both inflamed and diabetic rats increased, compared to control rats (**p<0.01, ***p<0.001, respectively by One-way ANOVA followed by Dunnett post hoc test). Jejunal MPO activity (D) in both inflamed rats and diabetic rats increased, compared to control rats (**p<0.01, *p<0.05,). Jejunal iNOS mRNA expression (E) in both inflamed and diabetic rats increased, compared to control rats (**p<0.01). If not mentioned, p values were obtained by Kruskal-Wallis test followed by Steel post hoc test.

Figure 2. Changes of myenteric neurons in BB rats.

Representative images of immunohistochemical stainings of myenteric ganglia by anti-nNOS antibody (blue), anti-Hu-C/D antibody (green) and anti-neurofilament 200 antibody (red) in BB rats (A). Arrows indicate nNOS positive neuronal cells. Scale bar: 50 µm. Hu-C/D positive cells (C) in both inflamed rats and diabetic rats did not change significantly. However, the ratio of nNOS positive cells to Hu-C/D positive cells (B) in both inflamed and diabetic rats decreased, compared to control rats (***p<0.001, ***p<0.001, respectively by One-way ANOVA followed by Dunnett post hoc test). Both nNOSα (D) and nNOSβ (E) mRNA expression in inflamed rats and diabetic rats increased, compared to control rats (**p<0.01). If not mentioned, p values were obtained by Kruskal-Wallis test followed by Steel post hoc test.

Figure 3. Dysmotility in BB rats.

Macroscopic abdominal findings in BB rats (A). Dilated intestines were observed in both inflamed rats and diabetic rats. Representative images of organ bath video analysis and spatiotemporal maps depicting the diameter of the intestinal segment upon an oral 3 cm H₂O stimulus (A). The short-lived aborally propagating contractions that are clearly observed in control gut are impaired in both inflamed and diabetic animals. Dilated and elongated intestines were observed in both inflamed and diabetic rats. The jejunal diameter (B) in oral 0 and 3 cm H₂O stimulus of both inflamed and diabetic rats were increased compared to control rats (***p<0.001). Changes in jejunal length (C) under 3 cm H₂O stimulus in inflamed rats were increased compared to control rats (***p<0.001). In diabetic rats, this increase was not statistically significant (p = 0.14). Both p values were obtained by Kruskal-Wallis test followed by Steel post hoc test

Figure 4. Mast cell infiltration in myenteric plexus of inflamed rats.

In the myenteric plexus of control rats, no toluidine blue positive cells were found (left). In the myenteric plexus of inflamed rats, toluidine blue positive mast cells were found (right). Scale bar: 100 µm. SM: Sub-mucosal layer, CM: Circular muscle layer, MP: Myenteric plexus LM: Longitudinal muscle layer.

Figure 5. Effect of AG on intestinal inflammation in inflamed rats.

Representative images of the myenteric ganglia and the muscular layer by H&E stainings in inflamed rats with or without AG treatment (A). All scale bars: 50 µm. AG treatment prevented infiltration of PMN cells in myenteric ganglia (arrows). AG treatment decreased numbers of PMN cells per 50 myenteric ganglia (B) (*p<0.05). However, AG treatment had no effect on the muscular layer thickness (C) (by Student's t-test). AG treatment dercreased jejunal MPO activity (D) (*p<0.05) and jejunal iNOS mRNA expression (E), however, not statistically significant (p = 0.11). If not mentioned, p values were obtained by Mann-Whitney U test.

Figure 6. Effect of AG on myenteric neurons in inflamed rats.

Representative images of immunohistochemical stainings of myenteric ganglia by anti-nNOS antibody (blue), anti-Hu-C/D antibody (green) and anti-neurofilament 200 antibody (red) in inflamed rats with or without AG treatment (A). Arrows indicate nNOS positive neuronal cells. Scale bar: 50 µm. AG had no effect on Hu-C/D positive cells (C) (p = 0.35 by Student's t-test). However, AG treatment increased the ratio of nNOS positive cells to Hu-C/D positive cells (B). (*p<0.05 by Student's t-test). Enhanced nNOSα mRNA expression (D) (*p<0.05) and enhanced nNOSβ mRNA expression (E) are decreased by AG treatment, however, not statistically significant (p = 0.14). If not mentioned, p values were obtained by Mann-Whitney U test.

Figure 7. Effect of AG on dysmotility in inflamed rats.

Representative images of organ bath video analysis and spatiotemporal maps depicting the diameter of the intestinal segment under oral 3₂O stimulus in inflamed rats with or without AG treatment (A). AG treatment partially reinstalled the short-lived propagating contractions that were prominent in control. AG treatment reduced jejunal diameters (B) in inflamed rats (***p<0.001 by Student's t-test) and also abolished the changes in jejunal length (C) in inflamed rats (*p<0.05 by Mann-Whitney U test).