Mo1848 Characterization of Dorsal Horn Neurons Activated by Noxious Colorectal Distension in Health and Chronic Visceral Hypersensitivity

Abstract

Background: Little is known about the spinal cord dorsal horn (DH) neurons activated by colonic mechanical stimuli. Even less is known about how chronic hypersensitivity of colonic nociceptors effects DH neuron activity. Methods: Experiments were performed in healthy mice (N=4) and in a TNBS model of post-inflammatory chronic visceral hypersensitivity (CVH; N=4). TNBS (130μL/ml) was administered by colorectal enema and after 28 days mice underwent 80 mmHg of colorectal distension (CRD). By this time inflammation is resolved and the peripheral endings of colonic nociceptors are mechanically hypersensitive1. Following CRD, mice were perfused fixed, spinal cord T10-L1 removed and processed for phosphorylated MAP kinase ERK 1/2 (pERK) immunohistochemistry to identify DH neurons activated by CRD. Spinal sections were co-labeled for calcitonin gene related peptide (CGRP), isolectin B4 (IB4), calbindin, GABA and NMDA receptor subunit 2B (NMDAr2B). The average number of pERK-immunoreactive (IR) neurons from 6-10 spinal sections was compared between healthy and post-inflamed mice using Two-way ANOVA with Bonferroni's posttests whilst unpaired student t-tests determined differences in co-labelling. Results: Significantly more DH neurons were pERK-IR following CRD in CVH mice compared to healthy mice (P<0.0001), specifically in T12-T13 (P<0.05) and T13-L1 (P<0.001). In healthy mice, pERK-IR neurons were located in CGRP-IR laminae I (LI), but not in the IB4-IR substantia gelatinosa. 37±3% of pERK-IR neurons were calbindin-IR, which accounted for 3±0.8% of all calbindin-IR DH neurons, classified as projection neurons in LI and a subtype of excitatory interneuron in LI-LII. All pERK/calbindin-IR neurons were located in LI and NMDAr2B-IR. 3.8±2% of pERK-IR neurons were GABA-IR, which accounted for 0.9±0.6% of all GABAIR DH inhibitory interneurons. In CVH mice, pERK-IR neurons were located in CGRP-IR LI and ventral to the IB4-IR substantia gelatinosa. A smaller proportion of pERK-IR neurons were calbindin-IR (14±5%, P=0.07), which was a consequence of more pERK-IR neurons not containing calbindin-immunoreactivity and the number of pERK/calbindin-IR neurons unchanged. Significantly more pERK-IR neurons were GABA-IR in CVH mice (13.45±3%, P<0.05), with pERK-IR neurons accounting for a greater number of GABA-IR neurons (8±1%, P<0.05). Conclusion: We identified DH neurons responsive to colonic mechanical stimulation. We also show that the chronic hypersensitivity of colonic afferent endings following inflammation leads to a significant increase in the number of DHneurons responsive to noxious colonic stimulation. These data also indicate a change in the types of DH neurons activated by colonic mechanical stimuli post-inflammation. Such DH changes may correlate with increased pain perception associated with CVH. 1) Hughes & Brierley, et al. Gut. 2009.