Abstract
Purpose :
To investigate the role of Substance P (SP) in a mouse model of corneal epithelial stem cell deficiency.
Methods :
Corneal epithelial stem cell deficiency was induced by means of total disepithelization in C57/BL6 (WT) and Substance P knock-out (Tac1-KO) mice. In vivo corneal biomicroscopy with fluorescein was performed for up to 2 weeks to quantify epithelial healing and corneal transparency. Upon animal sacrifice, the cornea was stained with Periodic Acid Schiff (PAS) to quantify goblet cells. We used immunostaining for cytokeratin 8 (CTK8) and ABCB5 to quantify conjunctival and epithelial stem cells. The expression of H2A histone family member X (H2AX) and phospho-P70S6 was measured in corneal epithelium of WT vs. Tac1-KO mice with western blot analysis.
Results :
Corneal wounds showed improved healing in Tac1-KO as opposed to WT mice (P<0.001). Corneal transparency was significantly higher in Tac1-KO mice (P<0.01).The number of goblet cells was reduced from 2.45±0.39 in WT to 0.77±0.52 in KO and infiltrating conjunctival cells declined from 18.44±1.85 to 6.11±2.98 in KO mice (P<0.04 and P<0.03, respectively). The number of ABCB5+ epithelial stem cells, instead, was higher in KO (19.08±0.46) vs. WT (16.61±0.44) mice (P<0.0001). Finally, the expression of H2AX and phospho-P70S6 was increased in WT vs. KO epithelium (P<0.0277 and P<0.0362, respectively).
Conclusions :
Our results suggest that Substance P expression is associated with worse clinical outcome in a pre-clinical model of corneal epithelial stem cell deficiency. Moreover, our data suggest that SP expression can induce stem cell exhaustion by means of DNA damage and m-TOR pathway hyper-activation. We anticipate that these findings can have clinical implications, because there are drugs in development that target Substance P and its receptor.
This is a 2020 ARVO Annual Meeting abstract.