2022 Annual Meeting and Alumni Reunion
Category : Basic and Translational Research Candidate : Yilin Guan Poster #: B8
Ocular Surface Mast Cells Promote Nerve Damage and Trigeminal Ganglion Inflammation Following Corneal Injury
Yilin Guan , WonKyung Cho , Elsayed Elbasiony , Aastha Singh , Sharad Mittal , Sunil Chauhan
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 the 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 .