Stimulation of Corneal Nociceptors Results in Pro-inflammatory Molecular and Cellular Responses

Abstract

Purpose : We have recently shown immediate changes in neuropeptides levels following CO₂ stimulation of corneal nociceptors. The peripheral nervous system, in part through neuropeptides, is able to alter the balance towards a pro- or anti-inflammatory environment by influencing the activity of resident antigen presenting cells (APCs) such as dendritic cells (DCs), modulating their chemotaxis, maturation and T-cell stimulatory capacity. We therefore hypothesized that CO₂-induced nociceptor stimulation will result in alterations in downstream inflammatory cytokines and adhesion molecules, consistent with, and resulting in pain-induced neurogenic inflammation.

Methods : CO₂ gas was applied to the central cornea of C57BL/6 and BALB/c mice in a series of 3 pulses every hour over 4 hours, and collected 1 hour following the final burst. Corneas were then excised, and gene and protein analysis for pro-inflammatory cytokines and adhesion markers, as well as whole-mount staining for CD11b (leukocyte marker), CD11c (DCs) and MHC class II (antigen presenting cells), was performed. Naïve un-stimulated and air-stimulated corneas served as controls.

Results : Long-term stimulation of corneal nociceptors resulted in increased expression of the pro-inflammatory neuropeptides substance P (7-fold, p<0.001) and CGRP (3-fold, p<0.05), as well as the anti-inflammatory neuropeptide urocortin (70 fold, p<0.001) in C57BL/6 mice. IL-1β was up-regulated (3-fold, p<0.05), and whilst IL-6 expression increased, it was not significant (p=0.12) compared to controls. Increased expression of adhesion molecules P- and E-Selectin (p=0.059 and p<0.05, respectively) as well as elevated ICAM-1 expression (p<0.05) was found. Analysis of BALB/c mice revealed a similar increase in inflammatory cytokines (p=0.06 and p<0.01 for IL-1β and IL-6, respectively) and adhesion markers (p=0.053 and p<0.05 for E-Selectin and ICAM, respectively). Fractalkine (CX3CL1) mRNA levels increased (p<0.05) following CO₂ stimulation. Finally, nociceptor stimulation resulted in alterations in resident APC density and maturation levels.

Conclusions : Our study demonstrates downstream changes in inflammatory cytokines, chemokines and adhesion markers, ultimately leading to alterations in resident APC populations following nociceptor activation. To our knowledge, this is the first report to reveal direct neurogenic effect of pain-induced corneal nociceptor activation.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.