Trigeminal ganglion neurons directly modulate leukocyte migration via chemotactic mediators

Abstract

Purpose : An association between corneal nerves and resident leukocytes during steady state and their dissociation following injury has previously been shown. We hypothesize that corneal nerves may directly modulate leukocyte migration via chemotactic mediators. Thus, our aim was to assess the migration of leukocytes and characterize alterations in chemotactic neuropeptide and chemokine expression within the trigeminal ganglion (TG), following corneal nociceptor stimulation.

Methods : Expression of select chemotactic neuropeptides/chemokines in the TG was assessed by RT-qPCR. TG neurons were co-cultured with plasmacytoid dendritic cells (pDCs) in a modified Boyden chamber, using transwell inserts with 3-mm pore size for the cell migration assay. To investigate the effect of nociceptor stimulation in the absence of injury, CO₂ was applied to the central cornea of adult C57BL/6 mice in a series of 3 pulses every hour over 4 hours, using a modified Belmonte CO₂ esthesiometer. TGs were excised at 30 minutes or 24 hours following final CO₂ application and RNA isolated for RT-qPCR analysis for candidate molecules.

Results : RT-qPCR results indicated that TG neurons directly express various chemotactic neuropeptides and chemokines, including CGRP, Substance P, ADM, and CXCL12. Higher density of TG neurons co-cultured in the modified Boyden chamber (10⁴ and 10⁵ cells) had higher percentage of pDCs migrating through the transwell membrane after 24 hours (1.67±0.72% vs. 6.67±1.44%) and 48 hours incubation period (2.08±0.72% vs 9.58±2.60%) (p<0.05) compared to no migration in the absence of neurons. Following corneal nociceptor stimulation with CO₂, the cornea had elevated inflammatory cytokine IL-1β expression (4-fold; p<0.05). In the TG, there was significant 12.83-fold increase in CGRP (p<0.05), 4.35-fold increase in Substance P (p<0.05), 0.11-fold decrease in NGF (p<0.05), 0.02-fold decrease in Urocortin (p<0.05) and no significant change in ADM and CXCL12 expression compared to naïve controls.

Conclusions : Our study demonstrates that TG neurons express chemotactic molecules and have a direct chemotactic effect on leukocytes, and that changes in expression levels of pro-inflammatory and anti-inflammatory neuropeptides in the TG following CO₂-induced pain may further mediate leukocyte chemotaxis.

This is a 2021 ARVO Annual Meeting abstract.