Laser-assisted Selective Ablation of Porcine Corneal Endothelial Cells: A Technique to Induce Focused Porcine Corneal Endothelial Injury for Ex Vivo Studies

Overview

This video demonstrates a non-invasive laser-assisted procedure to selectively damage the corneal endothelial cells in the porcine eye. The developed ex vivo model of endothelial cell damage can be used to study diseases of the corneal endothelium.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

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 phosphate-buffered saline (PBS) until use.
3. Screen eyes for major anterior segment pathologies, such as corneal scarring, edema, and other opacities with a spectral-domain optical coherence tomography device (Table of Materials).
4. Position the eyes in front of a slit-lamp unit equipped with a Nd:YAG laser (Table of Materials), which has a wavelength of 1,064 nm and a focal spot diameter of 10 µm in air.        
   NOTE: For optimal positioning a 3D-printed holding apparatus is used, which was designed to hold the eye firm, without putting too much pressure on it (Figure 1).
5. Use a magnification of 12x and deflect the illumination to visualize the individual corneal layers.
6. Set the pulse energy (e.g., 1.6 mJ) and focus point (e.g., 0.16 mm) for selective ablation of endothelial cells.
7. Place a clear cornea paracentesis close to the limbus and inject viscoelastic (Table of Materials) to stabilize the anterior chamber.
8. Excise the laser-treated central cornea using an 8 mm trephine.
9. Place the excised cornea in a well of a 12-well plate with the endothelial site facing upwards and incubate the specimen in 3 mL of full medium at 37 °C for up to 3 days.
   NOTE: Potential cytoprotective agents can be added to the medium during this step.

Representative Results

Figure 1: Experimental setup. (A) Eyes were fixed in a partially 3D-printed holding apparatus, which allowed precise alignment with respect to the laser beam. (B) Before laser treatment, the tissue was evaluated with an anterior segment optical coherence tomography device to check for major anterior segment pathologies. Positions of focal laser points are indicated with exemplary for 0.0 mm (black asterisk), 0.1 mm (red asterisk), and 0.2 mm (blue asterisk). 

Materials

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
Viscoelastic	OmniVision	Methocel