Abstract
Purpose :
Nerve damage following injury has been associated with impaired wound healing. We have previously shown that corneal injury leads to increased activation of ocular surface mast cells. Here, we investigated whether mast cells interact with corneal nerve and contribute to nerve degeneration and neuroinflammation.
Methods :
Corneal injury was induced by mechanical removal of the epithelium (3 mm) and one-third of the anterior stroma in C57BL/6 mice using an Algebrush II. To evaluate the proximity of mast cells and damaged nerves, corneas were harvested 6 hours post-injury and stained with β-tubulin III (corneal nerves), and avidin (mast cells), for immunohistochemistry (IHC) analysis. Trigeminal ganglions (TGs) were harvested post-injury and lysates were prepared to measure levels of tryptase (mast cell activation marker), CD11b and Substance P (SubP), using colorimetric assay and PCR analysis. To assess direct interaction between mast cells and inflamed corneal nerves, primary TGs were co-cultured with bone marrow-derived mast cells for 24h. Brightfield images were captured, and neurite length was quantified using ImageJ software. TGs harvested from co-cultures were assessed for expression of nerve activation marker SubP and CGRP. To assess the in vivo effect of mast cell activation on nerve damage, injured corneas were treated with mast cell inhibitor cromolyn (2% in PBS) and hyperactivation of nerves were measured using eye wipe test.
Results :
IHC analysis demonstrated that ocular surface mast cells infiltrated into the injured cornea in close proximity to the damaged corneal nerves. Corneal injury resulted in a significant activation of TG mast cells and inflammation, as indicated by increased levels of tryptase (p=0.003), CD11b (p<0.001) and SubP (p=0.009). Co-culturing of TGs with mast cells resulted in an approximate 13-fold upregulation in CGRP (p<0.001), and ~1.5-fold increase in SubP (p=0.008), compared to TGs cultured alone. Moreover, mast cells resulted in significant neuronal degeneration, as indicated by 50% decrease in neurite length compared to control TG cultures (p<0.001). Pharmacological inhibition of ocular surface mast cell activation resulted in a significant decline in TG inflammation and hyperalgesia, as shown by decreased eye wipes (p=0.005).
Conclusions :
Ocular surface mast cells exacerbate nerve degeneration and promote inflammation in the trigeminal ganglion.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.