A Novel Murine Model of Endothelial Keratoplasty

Abstract

Purpose: The purpose of this study was to establish a murine model of endothelial keratoplasty.

Methods: Endothelial keratoplasty (EK) was performed using C57BL/6 donor and BALB/c recipient mice. The central endothelium and Descemet membrane were removed from the recipient cornea, and a 1.5-mm posterior lamellar donor graft was made adherent to the recipient cornea with a small amount of viscoelastic. Mice were followed through slitlamp microscopy postoperatively, and OCT was used to assess the cornea and anterior chamber and measure central corneal thickness. Histology and immunohistochemistry were performed to confirm graft adherence and endothelial cell morphology.

Results: Successfully attached EK grafts were visualized in all transplanted animals. Histology and immunostaining confirmed proper graft orientation and adherence, as well as the presence of donor endothelium on transplanted grafts. We observed maximal corneal edema in all animals at day 1 postoperatively which gradually subsided. EK graft survival was 97% at 8 weeks.

Conclusions: In this study, we describe a novel murine model for EK which we anticipate will enable detailed investigation into the cellular and molecular mechanisms involved in EK pathobiology.

Fig 1.. Procurement of EK Donor Tissue.

(A) Mark the central cornea with 1.5mm trephine; (B) insert a 30G needle into the stroma, with the bevel pointing downwards, and create a lamellar pocket without entering the AC; (C) use Vannas scissors to enlarge the lamellar dissection; (D) separate the stroma by inserting a 30G needle through the trephine groove; (E) use 30G needle to enter AC then insert viscoelastic substance to form the AC; (F) using Vannas micro-scissors and Jeweler’s forceps, excise the donor tissue consisting of the remaining posterior stroma, Descemet’s membrane, and endothelium. AC, Anterior Chamber.

Fig 2.. Engraftment Technique.

(A) Mark the central cornea with a 2.0 mm trephine; (B) apply pressure to the eye using Jeweler’s forceps and penetrate the cornea using a 30G needle; (C) enter the anterior chamber and insert viscoelastic material; (D) using Jeweler’s forceps, bend a 30G needle; (E) insert the bent 30G needle and scratch the endothelial layer of the cornea; (F) scrape endothelial layer in a circular shape following the trephine mark; (G) insert EK donor tissue using Jeweler’s forceps; (H) adjust graft position; (I) insert small amount of viscoelastic material below the graft; (J) correct graft position using the tip of bent 30G needle; (K) close wound using an 11–0 nylon intrastromal suture; (L) confirm depth of the anterior chamber, integrity of iris, roundness of the pupil. EK, Endothelial Keratoplasty.

Fig 3.. Procurement of Donor Tissue.

(A) Mark the central cornea with a 1.5 mm diameter trephine. Apply enough pressure using Jeweler’s forceps to penetrate the deep stroma. Dashed line represents area of lamellar dissection; (B) insert a 30G needle through the groove created by the trephine and separate the stroma into an anterior and posterior portion. Dashed line represents area of lamellar dissection; (C) Excise the upper portion of the cornea using Vannas scissors and discard it. Enter the AC with a 30G needle. Dashed line represents posterior stroma and endothelium; (D) Using Vannas scissors and Jeweler’s forceps, excise the remaining lower part of the stroma, Descemet’s membrane, and the corneal endothelium. Dashed line represents tissue to be transplanted. AC, Anterior Chamber; EK, Endothelial Keratoplasty.

Fig 4.. Engraftment Technique.

(A) Insert bent 30G needle and scratch the endothelial layer. Arrow represents Descemet’s membrane being pulled by needle; (B) insert EK donor tissue using Jeweler’s forceps and adjust its position using a bent 30G needle. Arrows represent donor tissue entering anterior chamber; (C) Insert small amount of viscoelastic material below the graft. Dashed line represents graft; (D) close wound using an 11–0 nylon intrastromal suture. EK, Endothelial Keratoplasty.

Fig 5.. Assessment of Endothelial Keratoplasty.

(A) Representative slit-lamp photo and anterior segment OCT demonstrating proper orientation of the EK graft on day 1 post-transplant; (B) Representative slit lamp photo and anterior segment OCT demonstrating resolution of initial corneal edema and proper graft attachment by the end of follow-up (8 weeks post-transplantation). EK, Endothelial Keratoplasty. Arrow indicates EK graft border.

Fig 6.. Central Corneal Thickness.

Graft (red line), host (dashed blue line) and total (solid black line) corneal thickness measured over a period of 8 weeks. Data are presented as mean±SEM (N=8 mice).

Fig 7.. EK Graft Immunohistochemistry and Histology.

(A) ZO-1 immunostaining of grafted corneas 1 day post-operatively showed intact corneal endothelial cells in the center of the EK graft with corneal endothelial cell loss peripherally, as seen in this representative image (White arrows: donor graft border). (B) PAS staining at week 5 post-operatively showed complete graft attachment to the host corneal stroma (Box and Arrow: graft-host interface). EK, Endothelial Keratoplasty; PAS, Periodic-Acid-Schiff; ZO-1, Zonula Occludens-1.