Studies into endothelial cell migration in corneal models: towards application of ROCK inhibitor in treatment

A clear cornea comprising the front surface of the eye is essential for normal vision. In part, a single layer of corneal endothelial cells located on the inner surface of the cornea helps regulate corneal transparency. In corneal endothelial dysfunctions such as Fuchs’ endothelial corneal dystrophy (FECD), however, deteriorating endothelial cells lead to corneal cloudiness and a progressive loss of vision. FECD is currently treated via corneal replacement surgeries, which generally are successful but are associated with some potentially problematic issues, including donor shortage and graft rejections. Thus, there is a pressing need for new, less-invasive medical treatments for corneal endothelial dysfunction and the resultant loss of vision. Recently, there has been a growing interest in selective inhibitors of a Rho-associated kinase (ROCK) as agents that can help dysfunctional endothelial cells recover. The research described in this thesis has as its main focus an investigation of new treatment options for corneal endothelial diseases such as FECD. As a baseline, corneal endothelial development is studied because some biological processes in corneal healing have been reported to recapitulate those in corneal embryogenesis. Following this, an assessment is made of the human corneal endothelium and the morphologic changes that occur in FECD. Next, to judge potential new therapeutic approaches to treat vision loss caused by corneal endothelial dysfunction, experiments to assess the potential of transcoreal freezing were conducted. These identified the optimal use of a newly designed cryoprobe and its application for transcorneal freezing to reproducibly damage corneal endothelial cells that line the inner aspect of the cornea. Ingress into the cornea of dyes and medicinal agents was also tested. The concept of this approach is to eliminate the diseased corneal endothelial cells, prior to the medicinal encouragement of more peripheral corneal endothelial cells to regenerate the ablated area. Current research in many laboratories indicates that inhibitors of the Rho kinase pathway within cells lead to them becoming more fibroblast-like in their phenotype, accelerating the migration and inhibit cell death encouraging the endothelial wound healing. This study suggests that a less invasive transcorneal freezing using a 3.4 mm-diameter cryoprobe can be used for reproducible and targeted endothelial cell destruction to be followed by selective ROCK inhibitor application to treat corneal endothelial pathologies such as FECD and potentially other corneal endothelial dysfunctions

Topical delivery of a Rho-kinase inhibitor to the cornea via mucoadhesive film

The application of inhibitors of the Rho kinase pathway (ROCK inhibitors) to the surface of the eye in the form of eyedrops has beneficial effects which aid the recovery of diseased or injured endothelial cells that line the inner surface of the cornea. The aim of this study was to test the plausibility of delivering a selective ROCK inhibitor, Y-27,632, to the cornea using a thin polymeric film. Mucoadhesive polymeric thin films were prepared incorporating Y-27,632 and diffusional release into PBS was determined. Topical ocular delivery from the applied film was investigated using freshly excised porcine eyes and eyedrops of equivalent concentration acted as comparators; after 24 h the formulations were removed and the corneas extracted. Drug-loaded thin polymeric films, with high clarity and pliability were produced. ROCK inhibitor Y-27,632 was weakly retained within the film, with release attaining equilibrium after 1 h. This in turn facilitated its rapid ocular delivery, and an approximately three-fold greater penetration of Y-27,632 into cryoprobe-treated corneas was observed from the thin film (p < 0.01) compared to eyedrop. These findings support the further development of ROCK inhibitor delivery to the cornea via release from thin mucoadhesive films to treat vision loss cause by corneal endothelial dysfunction

Enzymatic resistance of corneas crosslinked using riboflavin in conjunction with low energy, high energy, and pulsed UVA irradiation modes

Purpose: To investigate the effect of various riboflavin/ultraviolet light (UVA) crosslinking (CXL) protocols on corneal enzymatic resistance. Methods: A total of 66 enucleated porcine eyes, with the corneal epithelium removed, were divided into 6 groups. Group 1 remained untreated. Groups 2 to 6 received riboflavin/dextran for 30 minutes. Group 3 underwent standard CXL (SCXL) with 3 mW/cm2 UVA for 30 minutes (total energy dose 5.4 J/cm2). Groups 4 and 5 underwent high intensity CXL (HCXL) using 30 mW/cm2 UVA for 3 minutes (5.4 J/cm2) and 30 mW/cm2 for 4 minutes (7.2 J/cm2), respectively. Group 6 was exposed to 8 minutes of 30 mW/cm2 UVA in a 10-second on/10-second off pulsed-radiation mode (p-HCXL; 7.2 J/cm2). A central 8-mm disk from each cornea was submerged in pepsin digest solution at 23°C and measured daily. After 13 days, the dry weight was recorded from 5 samples in each group. Results: The CXL-treated corneas took longer to digest than nonirradiated corneas (P < 0.0001). Differences in digestion time also were observed between CXL groups, such that, HCXL (5.4 J/cm2) < SCXL (5.4 J/cm2) < HCXL (7.2 J/cm2) < p-HCXL (7.2 J/cm2; P < 0.0001). The dry weight of the SCXL (5.4 J/cm2) group was higher than the HCXL (5.4 and 7.2 J/cm2; P < 0.001) and p-HCXL 7.2 J/cm2 (P <0.05) groups. No difference was detected between the HCXL and p-HCXL 7.2 J/cm2 groups. Conclusions: The intensity and distribution of the crosslinks formed within the cornea vary with different UVA protocols. The precise location and amount of crosslinking needed to prevent disease progression is unknown

A Surgical Cryoprobe for Targeted Transcorneal Freezing and Endothelial Cell Removal

PURPOSE: To examine the effects of transcorneal freezing using a new cryoprobe designed for corneal endothelial surgery. METHODS: A freezing console employing nitrous oxide as a cryogen was used to cool a series of different cryoprobe tip designs made of silver for high thermal conductivity. In vitro studies were conducted on 426 porcine corneas, followed by preliminary in vivo investigations on three rabbit corneas. RESULTS: The corneal epithelium was destroyed by transcorneal freezing, as expected; however, the epithelial basement membrane remained intact. Reproducible endothelial damage was optimally achieved using a 3.4 mm diameter cryoprobe with a concave tip profile. Stromal edema was seen in the pre-Descemet's area 24 hrs postfreeze injury, but this had been resolved by 10 days postfreeze. A normal collagen fibril structure was seen 1 month postfreeze, concurrent with endothelial cell repopulation. CONCLUSIONS: Transcorneal freezing induces transient posterior stromal edema and some residual deep stromal haze but leaves the epithelial basement membrane intact, which is likely to be important for corneal re-epithelialization. Localized destruction of the endothelial monolayer was achieved in a consistent manner with a 3.4 mm diameter/concave profile cryoprobe and represents a potentially useful approach to remove dysfunctional corneal endothelial cells from corneas with endothelial dysfunction