CO₂-Induced Corneal Nociceptor Stimulation Reveals Neuropeptide Changes in a Novel Murine Pain Model

Abstract

Purpose: The cornea is highly innervated with sensory nociceptors, the stimulation of which result in the sensation of pain and defensive reflex actions, such as blinking and tear secretion. Following stimulation, nociceptors also release neuropeptides which in turn can affect cell proliferation and migration, vasodilation and permeability of vessels, and promote wound healing after injury. To our knowledge, there have been no studies examining neuropeptide expression alterations in the cornea following induction of pain and in the absence of tissue injury.

Methods: A modified version of the Belmonte CO₂ esthesiometer was developed for murine use to investigate nerve stimulation in the absence of injury. In short, one or three series of pulses of 100% CO₂ gas, triplicates of 5 seconds (3x5 seconds) duration each, were applied to the central cornea of mice with 10 minute intervals between stimuli. Blink was induced manually every 2 minutes during the procedure to avoid dryness. Corneas were excised 10 minutes following final CO₂ application and processed for neuropeptide gene quantification by real-time PCR. Neuropeptides analyzed include substance P (SP), nerve growth factor (NGF), urocortin and secretoneurin (SN). Naïve unstimulated corneas served as controls.

Results: CO₂ administration to the central cornea successfully stimulated polymodal nociceptors of the murine cornea where a blinking response indicative of pain was observed in all mice. Substance P mRNA expression significantly increased in both the one and three CO₂ pulse groups (2.1 and 4.1 fold change respectively, p<0.05) compared to non-stimulated control corneas with SP expression increasing with the number of pulses; this increase was not significant (p=0.07). Further, NGF expression increased (1.7 fold, p<0.01), whereas urocortin and secretoneurin expression levels decreased following CO₂ stimulation as compared to non-stimulated control corneas (0.5 fold change, p<0.01 and 0.4 fold change, p<0.05, respectively). Fluorescein staining of the cornea revealed these changes were not due to epithelial damage or dry eye.

Conclusions: Our study demonstrates immediate changes in corneal neuropeptides including increase in SP and NGF expression levels after CO₂ induced pain. This murine model may serve as the basis for future studies into understanding the pathophysiology of peripheral pain.