Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Summary

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet’s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

Abstract

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Introduction

Transparency of the cornea is essential for the transmission of light to the retina and its photoreceptors¹. In this regard, a relative state of dehydration is critical to keep the collagen fibers within the corneal stroma correctly aligned. This homeostasis is maintained by corneal endothelial cells (CEC) located on the Descemet’s membrane (DM)². The endothelium is the innermost corneal layer. It has an important barrier and pump function, which is crucial for corneal transparency³. In contrast to the epithelium, the endothelium is not able to self-renew⁴. Therefore,....

Protocol

All procedures involving animal tissue follow the guidelines of the local Animal Care and Ethics Committee.

1. Preparation of organ culture and laser treatment

1. Obtain freshly enucleated porcine eyes from the local abattoir. Keep them cool (4 °C) in Dulbecco's modified Eagle medium (DMEM) with high glucose, supplemented with L-glutamine, sodium pyruvate, penicillin/streptomycin (1%), and porcine serum (10%), henceforth referred to in this article as full medium.
2. Remove extracellular tissues with scissors and soak the eyes in 5% povidone-iodine ophthalmic solution for 5 min before placing them in sterilized phos....

Results

Using the procedure presented here, we treated eyes with a Nd:YAG laser, evaluating different pulse energies (1.0−4.6 mJ) and positions of focal points (distance from the posterior surface of the cornea: 0.0−0.2 mm) to find the optimal parameters. Multiple replicates (n = 3) were evaluated for each constellation of the laser parameters (12 x 21).

In addition to the above-mentioned protocol, specimen was analyzed with a two-photon microscope before fixation and H&E staining. The two-photon .......

Discussion

The results of this pilot study indicate that a Nd:YAG laser can be used to selectively ablate corneal endothelial cells when appropriate parameters for energy dose and focus point position are chosen.

As the endothelial function is important for corneal transparency and safeguarding the cornea from stromal edema, models of endothelial dysfunction play an important role in the development of anti-edematous drugs or surgical procedures. There are several established in vitro models for mimickin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BARRON VACUUM TREPHINE	Katena	K20-2058
Cryostat	Leica	CM 3050S
Dulbecco’s Modified Eagle’s Medium - high glucose	PAA	E-15009
Eye holder	Self	N/A
Inverted Microscope	Leica	DMI 6000 B
KH2PO4	Merck	529568
Na2HPO4	Merck	1065860500
Nd:YAG laser	Zeiss Meditec	visuLAS YAG II plus
OCT Tissue Tek	Sakura Finetechnical	4583
Penicillin-Streptomycin	Sigma-Aldrich	P4333
Phosphate Buffered Saline (PBS)	Gibco	10010056
Porcine serum	Sigma-Aldrich	12736C
Spectral-domain optical coherence tomograph	Heidelberg Engineering	Spectralis
Tissue culture plate 12-well	Sarstedt	833921
Two-Photon Microscope	JenLab	DermaInspect
Viscoelastic	OmniVision	Methocel