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PARADOXICAL EFFECTS OF INFLAMMATION ON ENTERIC NEURONS
S Venkataramana, MG Blennerhassett
GIDRU, Department of Physiology, Queen’s University, Kingston, Ontario
BACKGROUND: Inflammatory bowel diseases (IBD) cause structural and functional damage, and animal models show this to include major remodeling of the enteric nervous system. In the TNBS-induced model of colitis, a 30-50% loss of neurons was observed in the initial stages of inflammation followed by dramatic axon proliferation, involving a 5-fold increase in axons. The onset of inflammation involves the activation of resident immune cells as well as the rapid influx of infiltrating immune cells, resulting in the release of both proinflammatory and potentially damaging factors. We hypothesized that the neurotoxic levels of nitric oxide (NO) from the enzyme iNOS cause the early loss of neurons early in colitis. This was tested in vitro using a co-culture model of myenteric neurons and smooth muscle cells from the rat intestine.
METHODS AND RESULTS: NO concentrations in the inflamed intestine were determined using the Griess reaction. Similar concentrations were produced in vitro by addition of the chemical donors SNAP or SNP with verification of NO levels, and assayed for neuronal toxicity. Neuron number was determined after immunocytochemistry for the pan neuronal marker HuD, and axons were visualized with an anti-SNAP-25. NO (40-80 µM) selectively decreased neuron survival by 15-20% (±3.9). To mimic the inflammatory conditions in vitro, peritoneal immune cells were activated by prior application of LPS (10 µg/ml) and placed above neuronal co-cultures using a transwell system. This induced iNOS expression in the immune cells as indicated by iNOS-specific NADPH diaphorase staining, and the release of NO (60-80 µM per 106 cells). Despite these NO levels, neuronal survival was unimpaired (119% ±11.3). Unexpectedly, axon outgrowth was strongly potentiated (228% ±18%).
CONCLUSIONS: We have duplicated in vitro several key early events in colitis, showing both an NO-mediated initial loss of neurons and immune cell-mediated axon proliferation. However, the NO levels that exerted neurotoxic effects on the co-cultures when derived from chemical NO-donors were ineffective when present in combination with other products of activated immune cells. Since the net outcome was highly conducive to axon growth, it appears that immune cells can exert neurotrophic effects on enteric neurons despite the potential for neurotoxic effects. Overall, this study highlights the paradoxical roles played by immune cells in intestinal inflammation.
Funded by the CIHR training grant, NSERC and the CCFC.