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, any cell damage caused by disease or trauma stimulates the remaining endothelial cells to enlarge and migrate, to cover resulting defects and to maintain corneal functionality⁵. However, if the CEC density falls below a critical threshold, decompensation of the endothelium leads to an edema, resulting in blurred vision and discomfort or even severe pain⁴. Despite the availability of drugs to relieve symptoms, currently the only definitive treatment in these cases is corneal transplantation, which can be performed in the form of a full-thickness graft or a lamellar endothelial transplantation. The latter procedure is available as Descemet's membrane endothelial keratoplasty (DMEK) as well as Descemet's stripping automated endothelial keratoplasty (DSAEK)⁶. However, the protection of remaining CEC and enhancing their survival could be an alternative target, which needs an adequate disease model to test potential therapeutic drugs.

Current CEC loss disease models focus on the destruction of the endothelium through the injection of toxic agents (e.g., benzalkonium chloride) into the anterior chamber or by mechanical abrasion of the cells using an invasive descemetorhexis technique^7,8. While these models are well established, disadvantages such as general inflammatory response and imprecise collateral damage exist. Therefore, these models are more likely to represent final stages of the disease, when the above-mentioned surgical options are inevitable.

With advances in cellular treatment strategies such as stem cells and gene therapy, the application of these cellular therapies could be useful in early stages of CEC loss⁹. Subsequently, we need a model that represents these earlier stages of the disease more adequately. In this regard, cell culture models have improved over the last decade but are still limited in their validity, as cells in vitro cannot come close to replicating the complex interactions that occur between the different cell types within the cornea¹⁰. Therefore, ex vivo and in vivo disease models are still in high demand and improving the existing ones is of utmost interest.

Noninvasive, intraocular surgery by photodisruption using a neodymium:YAG (Nd:YAG) laser has become a routine procedure for ophthalmologists worldwide since its introduction in the late 1970s¹¹. Photodisruption relies on nonlinear light absorption leading to the formation of plasma, generation of acoustic shock waves, and creation of cavitation bubbles, whenever the application site is located in a liquid environment¹². In general, these processes contribute to the intended effect of precise tissue cutting. However, they can also be the source of unnecessary collateral damage limiting the local confinement of laser surgery¹³.

The prediction of resulting mechanical effects has significantly improved through characterization of the shock wave propagation and cavitation course. It is our goal to target CEC with as little damage to surrounding tissue as possible to provide a noninvasive, laser-assisted experimental disease model for the early stages of CEC loss. For this purpose, it is necessary to determine the optimal pulse energies and positions of the focal spots of the laser.

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 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.

2. Preparation for histology

1. Prepare Sorensen’s buffer with a pH of 7.4 containing 19.6 mL of 133 mM KH₂PO₄ and 80.4 mL of 133 mM Na₂HPO₄.
2. Remove the medium from the cornea-containing well and fixate the tissue for 20 min at room temperature (RT) using methanol-free paraformaldehyde (4%) in Sorensen’s buffer.
3. Place tissue in 20% sucrose in PBS until tissue sinks (1 h) and then in 30% sucrose in PBS overnight at RT. Take care to avoid contact with bubbles and the air surface interface. Embed tissue in optimal cutting temperature (OCT) compound and store at -80 °C.
4. Cut sections 10 μm thick using a cryostat at -27 °C.
   NOTE: A camel hairbrush is useful to help guide the emerging section over the knife blade.
5. Transfer the section to a microscope slide by touching the slide to the tissue within 1 min of cutting it to avoid freeze-drying of the tissue. Store the slides at -80 °C.

3. Hematoxylin and eosin (H&E) staining

1. Air dry sections for several minutes to remove moisture.
2. Stain with filtered 0.1% Mayers hematoxylin for 10 min in a 50 mL tube.
3. In a Coplin jar, rinse in cool running ddH₂O for 5 min and dip in 0.5% eosin 10x.
4. Dip in ddH₂O until the eosin stops streaking and then dip in 50% (10x) as well as 70% (10x) EtOH.
5. Equilibrate in 95% EtOH (30 s) and 100% EtOH (60 s) before dipping in xylene several times.
6. Finally mount and coverslip the specimen before taking images using a light microscope.

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...

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

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