From Intestinal Permeability to Dysmotility: The Biobreeding Rat as a Model for Functional Gastrointestinal Disorders

<div><p>Background</p><p>Impaired intestinal barrier function, low-grade inflammation and altered neuronal control are reported in functional gastrointestinal disorders. However, the sequence of and causal relation between these events is unclear, necessitating a spontaneous animal model. The aim of this study was to describe the natural history of intestinal permeability, mucosal and neuromuscular inflammation and nitrergic motor neuron function during the lifetime of the BioBreeding (BB) rat.</p><p>Methods</p><p>Normoglycemic BB-diabetes prone (DP) and control rats were sacrificed at different ages and jejunum was harvested to characterize intestinal permeability, inflammation and neuromuscular function.</p><p>Results</p><p>Both structural and functional evidence of increased intestinal permeability was found in young BB-DP rats from the age of 50 days. In older animals, starting in the mucosa from 70 days and in half of the animals also in the muscularis propria from 110 days, an inflammatory reaction, characterized by an influx of polymorphonuclear cells and higher myeloperoxidase activity, was observed. Finally, in animals older than 110 days, coinciding with a myenteric ganglionitis, a loss of nitrergic neurons and motor function was demonstrated.</p><p>Conclusion</p><p>In the BB-rat, mucosal inflammatory cell infiltration is preceded by intestinal barrier dysfunction and followed by myenteric ganglionitis and loss of nitrergic function. This sequence supports a primary role for impaired barrier function and provides an insightful model for the pathogenesis of functional gastrointestinal disorders.</p></div

Early increased intestinal permeability.

<p>(A) TEER was lower in BB-DP (white circles) rats compared to controls (black circles) starting from 50 days, indicating increased permeability. (B) Increased cumulative passage of FITC-Dx20 at 70, 90 and 110 days, but only the effect at 90 and 110 days remained significant after correction for multiple testing. Data are presented as mean ± SEM. *P<0.05; **P<0.01; ***P<0.001 after correction for multiple testing. BB-DP: normoglycemic Diabetes-Prone BioBreeding; FITC-Dx20: Fluorescein-isothiocyanate labeled dextrans of 20 kiloDalton; TEER: transepithelial-electrical resistance.</p

Molecular mechanisms of impaired intestinal barrier function.

<p>(A) Lower gene expression of CLDN1 was found at 30, 50, 70 and 110 days and increased expression of CLDN2 at 50, 70 and 110 days in BB-DP rats (white bars) compared to controls (black bars). (B-D) Semi-quantitative immunofluorescence confirmed decreased protein expression of CLDN1 (B, C) and increased expression of CLDN2 (B, D) at 50 days. (scale bar = 10 µm). (E) Western Blot analysis at 50 days shows decreased CLDN1 expression and a tendency towards increased CLDN2 expression (P = 0.07) in BB-DP rats (white bars) compared to controls (black bars). Liver tissue was used as a positive control. Data are presented as mean ± SEM. *P<0.05; **P<0.01. BB-DP: normoglycemic Diabetes-Prone BioBreeding; CLDN1: Claudin 1; CLDN2: Claudin 2; VINC: Vinculin.</p

Intestinal inflammation.

<p>(A) From 70 days of age, the mucosal MPO-activity was increased in the BB-DP rats (white circles) vs. controls (black circles), however statistical significance was lost in animals older than 110 days. (B) The number of PMN cells per HPF (0.233 mm²) in the lamina propria was elevated starting from 70 days. Jejunal histology (H&E staining) at 50 (C) and 160 (D) days (scale bar = 100 µm). (E) In rats older than 110 days, MPO-activity was also elevated in the muscularis propria, but reached significance only at the age of 160 days. (F) Quantification of the number of intra-ganglionic PMN cells in the myenteric plexus in BB-DP (white circles) vs. control (black circles) rats. Intra- and periganglionic PMN cells in BB-DP rats are indicated by black arrows (Scale bar = 25 µm). Data are presented as mean ± SEM. *P<0.05; **P<0.01; ***P<0.001. BB-DP: normoglycemic Diabetes-Prone BioBreeding; HPF: High-Power Field; MPO: Myeloperoxidase; PMN: Polymorphonuclear.</p

Gene expression analysis of inflammatory markers in BB rats of 50–70 days expressed as fold-changes compared to the control group.

<p>Data are expressed as median (interquartile range). (n = 8 animals per group at 50 days and n = 7 animals per group at 70 days).</p><p>BB-DP: normoglycemic Diabetes-Prone BioBreeding Rat; IFN: Interferon; IL: Interleukin; iNOS: inducible isoform of Nitric Oxide Synthase; TNF: Tumor-Necrosis Factor.</p><p>Gene expression analysis of inflammatory markers in BB rats of 50–70 days expressed as fold-changes compared to the control group.</p