Gastrointestinal dysfunction in mice with a targeted mutation in the gene encoding vasoactive intestinal polypeptide: a model for the study of intestinal ileus and Hirschsprung's disease

Abstract

In 1970, Drs. Said and Mutt isolated a novel peptide from porcine intestinal extracts with powerful vasoactive properties, and named it vasoactive intestinal peptide (VIP). Since then, the biological actions of VIP in the gut as well as its signal transduction pathways have been extensively studied. A variety of in vitro and in vivo studies have indicated that VIP, expressed in intrinsic non-adrenergic non-cholinergic (NANC) neurons, is a potent regulator of gastrointestinal (GI) motility, water absorption and ion flux, mucus secretion and immune homeostasis. These VIP actions are believed to be mediated mainly by interactions with highly expressed VPAC(1) receptors and the production of nitric oxide (NO). Furthermore, VIP has been implicated in numerous physiopathological conditions affecting the human gut, including pancreatic endocrine tumors secreting VIP (VIPomas), insulin-dependent diabetes, Hirschsprung's disease, and inflammatory bowel syndromes such as Crohn's disease and ulcerative colitis. To further understand the physiological roles of VIP on the GI tract, we have begun to analyze the anatomical and physiological phenotype of C57BL/6 mice lacking the VIP gene. Herein, we demonstrate that the overall intestinal morphology and light microscopic structure is significantly altered in VIP(-/-) mice. Macroscopically there is an overall increase in weight, and decrease in length of the bowel compared to wild type (WT) controls. Microscopically, the phenotype was characterized by thickening of smooth muscle layers, increased villi length, and higher abundance of goblet cells. Alcian blue staining indicated that the latter cells were deficient in mucus secretion in VIP(-/-) mice. The differences became more pronounced from the duodenum to the distal jejunum or ileum of the small bowel but, became much less apparent or absent in the colon with the exception of mucus secretion defects. Further examination of the small intestine revealed larger axonal trunks and unusual unstained patches in myenteric plexus. Physiologically, the VIP(-/-) mice showed an impairment in intestinal transit. Moreover, unlike WT C57BL/6 mice, a significant percentage of VIP(-/-) mice died in the first postnatal year with overt stenosis of the gut.

Figure 1

A: Low magnification picture of the GI tract of representative WT, VIP^+/−, and VIP^−/− mice. Of interest, total GI length and appendix size are reduced when compared to their dimensions in the tract from a representative age-matching control animal B. High magnification photomicrographs showing some of the anatomical details reported in Table III. Left panel shows individual villi: arrows point to enterocytes (EC) and mucous-secreting (goblet) cells (GC); VW- and EL-labeled brackets denote villus width and enterocyte length, respectively. Right panel is a high power view showing the muscularis propria (MP), consisting of the outer (longitudinal) cell layer (OCL) and the inner (circular) cell layer (ICL). Arrow points to a myenteric plexus (MyP), which lies between the two muscle layers of the muscularis propria.

Figure 2

Microscopic structure of the proximal jejunum in WT and VIP^−/− mice. Small bowel sections were stained using standard H&E staining. Pictures at low (x2.5, x10, upper panel) and higher magnification (x20, x40; mid and lower panels, respectively) revealed an overall increase in thickness of muscularis mucosa in VIP KO (B, D and F) vs. controls (A, C and E), as well as higher numbers of mucus-forming cells (G vs. E).

Figure 3

Microscopic structure of the colon in WT and VIP^−/− mice. Gut sections were stained using standard H&E staining.

Figure 4

Alcian blue staining to visualize mucus deposits on sections in duodenum (A–D) and distal colon (E–H) from WT (left panel) and VIP^−/− mice (right panels). As expected from H&E staining shown in figures 2 & 3, Alcian blue staining revealed a significant difference in mucus-positive cells in the small intestine, but no change in colon. However, sections from VIP^−/− showed a dramatic reduction in the amount of mucus released in the lumen in both small (A, C vs B, D), and large (E, G vs F, H) bowels.

Figure 5

Microscopic structure of myenteric plexus in duodenum and distal colon of WT and VIP^−/− mice. H&E (A, B, G and H) and S100β (C–D & I–J) and pan-neurofilaments (NF) (E, F) stainings are shown in WT and VIP KO mice (left and right panels, respectively). Structural differences were observed in duodenum of VIP deficient mice when compared to WT controls. Plexuses from KO mice showed enlarged unstained patches (A vs B) that were non-immunoreactive with S100β or NF antibodies used to specifically reveal Schwann cells and axons (C vs. D & E vs. F, respectively). However, immunofluorescence emitted by the pan-NF antibody coupled with green fluorescent dye-labeled conjugates (E vs. F) revealed larger area signals within the external plexus of the KO mice than WT controls, suggesting the presence of bigger axons within the myenteric plexus of VIP-deficient mice. Conversely, no obvious differences in plexus structures of the large bowel (G–J) were using these techniques (pan-NF not shown).

Figure 6

Peristaltic activity in WT vs. VIP^−/− mice. A: migration of charcoal-stained bolus along the GI tract 30 min after gavage. B: Distribution of fluorescein-labeled dextran along the gastro-intestinal tract 60 minutes post administration in WT controls vs. VIP^−/− mice. Procedures are described in methods. Data are presented as mean ± SEM (n=3).