982 Prolonged IL-1β Exposure of Jejunal Myenteric Neurons Alters Their Intracellular CA 2+ Response to Oral Electrical Tract Stimulation and Serotonin Application

Abstract

Background: Infection as well as inflammatory diseases of the gut result in profound changes of intestinal function. One of the pro-inflammatory cytokines, interleukin-1β (IL-1β) has been attributed excitatory and neuromodulatory roles on both central andmyenteric neurons. Here we aimed to study the response ofmyenteric neurons to different stimuli after incubation with IL-1β enriched medium.Material and methods: Longitudinal muscle myenteric plexus preparations (LMMP) of the guinea pig (GP) jejunum were incubated for 24h in medium with or without human recombinant IL-1β, followed by loading with the calcium indicator Fluo-4. Confocal Ca2+ imaging was used to visualize activation of neurons, which were identified by an application of 75 mM K+-Krebs solution. In a first series of experiments, the response to a 30 s application of acetylcholine (Ach), serotonin (5-HT), substance P (SP) and a 15 s application of ATP (all 10-6M) was tested on LMMP's from 5 GP. In a next series (5 GP), internodal strands in the LMMP's were activated by electrical tract stimulation (ETS). All experiments were performed in the presence of nicardipine 10-6 M to reduce tissue movement. Images were analyzed using a custom made macro in Igor Pro. After normalization, the percentage of activated neurons per ganglionwas compared using unpaired t-tests. Immunohistochemistry for nNOS, HuD, calbindin and calretinin was performed on the incubated LMMPs. Data are given as mean±SEM. Results: 1/ In response to 5-HT, Ca2+transients were observed in twice as many neurons after incubation with IL-1β 10-9 M (1.00±0.24 vs 2.08±0.32), while no difference was observed in the Ach, SP or ATP responses. The effect on 5-HT responses was absent at an IL-1β concentration of 10-12 M. 2/ Ca2+transients were observed in significantly more neurons per ganglion after oral and lateral ETS following incubation with IL-1β 10-10 M (resp 1.52±0.20 and 1.35±0.11) as compared to 10-9 M (resp 0.84±0.12 and 0.74±0.13) or 10-12 M (resp 0.89±0.20 and 0.66±0.12), all p<0.03. No difference was observed after aboral ETS. Neuronal coding was not influenced by prior incubation with IL-1β 10-9 M. Conclusion: IL-1β differentially modulates the neuronal response to ETS and neurotransmitter application in the myenteric plexus of guinea pigs and could be directly implicated in the observed changes in intestinal secretomotor function during inflammation.