Laser-assisted Selective Ablation of Porcine Corneal Endothelial Cells: A Technique to Induce Focused Porcine Corneal Endothelial Injury for Ex Vivo Studies
Abstract
Source: Holzhey, A. et al., Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss. J. Vis. Exp. (2020)
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
- 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.
- 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.
- 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).
- 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). - Use a magnification of 12x and deflect the illumination to visualize the individual corneal layers.
- Set the pulse energy (e.g., 1.6 mJ) and focus point (e.g., 0.16 mm) for selective ablation of endothelial cells.
- Place a clear cornea paracentesis close to the limbus and inject viscoelastic (Table of Materials) to stabilize the anterior chamber.
- Excise the laser-treated central cornea using an 8 mm trephine.
- 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).
Divulgaciones
The authors have nothing to disclose.
Materials
| 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 |
