Abstract
Purpose :
We have shown that mast cells are key immune cells in initiating and promoting ocular surface inflammation. Here we investigated the role of mast cells in neuroinflammation and pain sensitization during ocular inflammation.
Methods :
Corneal injury was induced by mechanical removal of the epithelium and anterior stroma in C57BL/6 mice. To visualize mast cells and damaged nerves, corneas were harvested 6h post-injury and stained with β-tubulin III (corneal nerves), and avidin (mast cells) for immunofluorescence analysis. TGs were harvested post-injury and lysates or single-cell suspensions were prepared to measure levels of mast cell-specific tryptase and frequencies of CD45+ inflammatory cells via ELISA and FACS, respectively. To assess direct interaction between mast cells and corneal nerves, primary TGs were co-cultured with mast cells ± mast cell inhibitor cromolyn (100 μM) for 24h. β-tubulin III-stained TGs were assessed under fluorescent microscope and neurite length was quantified using ImageJ. TGs harvested from co-cultures were assessed for the expression of nerve activation marker Substance P (SubP). To assess the in vivo effect of mast cell activation, injured corneas were treated with 2% cromolyn and TG inflammation and hyperalgesia were measured.
Results :
Immunofluorescence micrographs showed that ocular surface mast cells juxtapose damaged corneal nerves. Corneal injury resulted in significant upregulation of tryptase levels (p=0.003) and frequencies of CD45+ cells (p=0.01) in the TG, compared to naive controls. In vitro assay demonstrated a 50% decrease in neurite length of TGs co-cultured with mast cells compared to TGs cultured alone (p<0.0001), which was suppressed when treated with cromolyn (p<0.0001). Co-culturing of TGs with mast cells resulted in a 4-fold upregulation in SubP (p<0.0001), compared to TGs cultured alone or co-cultures treated with cromolyn. Moreover, pharmacological inhibition of ocular surface mast cell activation reduced infiltration of mast cells to damaged corneal nerves and resulted in a significant decline in tryptase levels (p<0.01) and CD45+ infiltration in the TG (p=0.03), compared to PBS control. Cromolyn treatment prevented injury-induced hyperalgesia, as shown by decreased eye wipes (p=0.005).
Conclusions :
Ocular surface mast cells interact with corneal nerves to augment nerve damage and inflammation in the trigeminal ganglion to induce hyperalgesia.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.