Fig 1.
Schematic representation of the timeline for corneal sensory nerve suppression and neuropeptide treatment.
Mice received subconjunctival injections (SCI) of 0.5% bupivacaine (BUP; 5 μL) using a Hamilton syringe, followed by topical (TOP) application (5 μL) to the central cornea every other day for 15 days to achieve sustained sensory nerve suppression (black line). Control mice received equivalent volumes of phosphate-buffered saline (PBS) via both routes. In a separate experimental group, neuropeptides CGRP (10.5 μM) or SP (15 μM) were administered subconjunctivally 24 hours before the final bupivacaine treatment (day 14) and again one hour prior to the final bupivacaine injection on day 15 (red line). All assessments, including tear production, fluorescein staining, corneal nerve density, corneal and tear fluid neuropeptide levels, and bacterial adhesion, were conducted four hours after the last bupivacaine injection on day 15.
Fig 2.
A. Representative confocal microscopy images of corneal nerves labeled with β-tubulin III (green) in WT mice treated with 0.5% bupivacaine (BUP) via subconjunctival injection (SCI) alone or in combination with topical (TOP) application, as well as PBS-treated controls receiving similar subconjunctival and topical applications. Corneal tissues were processed on day 15 post-injection. Scale bar = 70 µm. B. Quantification of corneal nerve density using a grading scale (0–10) assessed by a blinded researcher in WT mice across different treatment groups.
A significant reduction in corneal nerve density was observed in mice treated with 0.5% bupivacaine (subconjunctival alone or in combination with topical application) compared to PBS-treated controls. Data are presented as mean ± SD. ** P < 0.01, **** P < 0.0001 (One-way ANOVA with Tukey’s multiple comparisons test).
Fig 3.
Tear production was quantified using the SMTube assay, revealing a significant reduction in tear secretion in mice treated with 0.5% bupivacaine (BUP), either via subconjunctival injection (SCI) alone or in combination with topical (TOP) application, compared to PBS-treated controls.
The SMTube assay was performed four hours after the final BUP treatment on day 15 to assess aqueous tear volume. Data are presented as mean ± SD. * P < 0.05, **** P < 0.0001 (One-way ANOVA with Tukey’s multiple comparisons test).
Fig 4.
ELISA analysis revealed a significant decrease in SP (A & B) and CGRP (C & D) protein levels in the corneas and tear fluid of mice treated with 0.5% bupivacaine (BUP) every alternate day for 15 days (administered via subconjunctival injection (SCI) alone or in combination with topical (TOP) application) compared to PBS-treated controls.
Protein levels were assessed on day 15 four hours post-injection and data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 [One-way ANOVA with Tukey’s multiple comparison test].
Fig 5.
Quantification of bacterial adhesion showed a significant increase in adhered S. aureus (A), S. epidermidis (B), and P. aeruginosa (C) on blotted corneas of mice treated with 0.5% bupivacaine (BUP), either subconjunctival injection (SCI) alone or in combination with topical (TOP) application, compared to PBS-treated controls.
The level of bacterial adhesion was assessed on Day 15 post-injection and data are presented as mean ± SD of Log CFU bacterial adhesion. ** P < 0.01, **** P < 0.0001, and ns = not significant (One-way ANOVA with Tukey’s multiple comparisons test).
Fig 6.
Quantification of bacterial adhesion revealed a significant decrease in the adhesion of S. aureus, S. epidermidis, and P. aeruginosa on blotted corneas of mice treated with 0.5% bupivacaine (BUP) combined with topical (TOP) application, following subconjunctival injection (SCI) of CGRP, 24 hours before the final BUP treatment (day 14) and again one hour prior to the final bupivacaine injection on day 15.
The impact of neuropeptides on bacterial adhesion was assessed on day 15 and data are presented as mean ± SD of Log CFU bacterial adhesion. * P < 0.05, ** P < 0.01, **** P < 0.0001 (One-way ANOVA with Tukey’s multiple comparisons test).
Fig 7.
ELISA results showing SP (A & B) and CGRP (C & D) protein levels in mice treated with SP or CGRP via subconjunctival injection (SCI) and topical (TOP) application, 24 hours before the final bupivacaine (BUP) treatment (day 14) and again one hour prior to the final bupivacaine injection on day 15, compared to PBS-treated controls.
Both SP and CGRP were significantly increased in corneas and tear fluid on day 15 post treatment. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001, and ns = not significant (One-way ANOVA with Tukey’s multiple comparisons test).