Determining the Efficacy of Crosslinking Stiffening Agents in Rat Sclera

Abstract

Purpose : Scleral stiffening has been proposed as a possible therapeutic treatment for glaucoma and myopia. Several studies have characterized the effects of various biocompatible stiffening agents on rabbit, porcine, and human sclerae with uniaxial and inflation testing. However, the majority of in vivo studies use rodent models of glaucoma and myopia where these agents have not been tested. Thus, we have experimentally determined the mechanical effects of various stiffening agents (methylglyoxal, MG; glyceraldehyde, GC; and genipin, GP) on the posterior rat sclera and identified the concentrations necessary to obtain an approximate doubling in scleral stiffness, as reported in previous studies.

Methods : 78 eyes were enucleated from male retired breeder (9-12 months old) Brown Norway rats. Each eye was partially incubated overnight (Fig. 1A) in one of the three stiffening agents at various concentrations. After incubation, inflation testing with mechanical characterization was performed on eyes at three IOPs (5, 15, and 30 mmHg). Strain throughout the posterior sclera was determined using digital image correlation and the strain in each region (control or experimental, Fig. 1B) was used to calculate relative stiffness.

Results : Dose-dependent stiffening effects were observed for all agents (Fig. 2A-C) and relative stiffness ranged from 12% for MG to 1.3x10³% for GP. Concentrations of each agent resulting in nearly doubled scleral stiffness were 7 mM for MG, 30 mM for GC, and 1 mM for GP (Fig. 2D).

Conclusions : We have determined the efficacy of stiffening the posterior rat sclera using three different stiffening agents: MG, GC, and GP. Additionally, we have identified the optimal concentrations of each agent to increase stiffness from natural levels by approximately 100%. These data provide a foundation for further study on scleral stiffening’s possible therapeutic effects in glaucoma and myopia.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

Fig. 1: (A) Overnight partial incubation method. Control region was misted with PBS to prevent drying. (B) Posterior sclera with control and experimental regions outlined.

 

Fig. 2: Dose-response curves at 15 mmHg for MG (A), GC (B), and GP (C), and their concentrations that produced approximately 100% relative stiffening (dashed line; D). Data shown as mean ± SD.