Selective enzymatic lysis of the corneal stroma as a method to prepare a transplant for endothelial keratoplasty

Abstract:

Endothelial keratoplasty (EK) is a targeted surgery for the treatment of diseases associated with insufficient function of endothelial corneal cells. Despite the variety of available techniques for EK, the development of new methods of transplant preparation remains relevant, primarily due to the limited predictability of preparation of thin transplants, the use of which allows achieving the greatest functional result of treatment. To overcome this problem, the authors propose a method of selective enzymatic lysis of the corneal stroma to prepare a transplant for the EK.

Purpose: To study the possibility of using enzymatic lysis for controlled, predictable dissolution of the corneal stroma.

Materials and methods: Three experimental studies were carried out sequentially:

1. Dissolution of isolated corneal stroma with an enzyme solution of collagenase type I at concentrations of 125 and 500 U/ml for 1 hour at 37 ° C. The results were monitored by examining the samples in an sugical microscope.
2. Application of collagenase type I solution during a 2-stage exposure with a total duration of 180 minutes on the corneoscleral disc of an animal with mechanically removed surface layers of the corneal stroma. The thickness of the residual stroma was controlled using an optical coherence tomography (OCT).
3. Application of the collagenase type I enzyme solution on the animal's corneoscleral disc, with previously removed surface layers using a femtosecond laser, with a pre-calculated incubation time of 91 minutes. Monitoring of treatment results using OCT and histological examination (hematoxylin-eosin).
   
   In experiments 2 and 3, an artificial anterior chamber was used, it was filled with a Borzenka-Moroz conservation medium.

Results:

Experiment 1: When the tissue was exposed to a low concentration enzyme, the tissue was partially lysed. In a solution with a high concentration of the proteolytic enzyme, the tissue dissolved completely.

Experiment 2: We obtained a gradual decrease in the residual thickness of the stroma in the center from 925 microns of the initial thickness to 197 microns of the final thickness. At the same time, in some places, the only one hyperreflective layer remaining intact was determined by OCT.

Experiment 3: As a result of exposure, we obtained a uniform dissolution of the corneal layers up to 44 microns of residual thickness without the formation of local defects and perforations. Histological staining confirmed the findings detected by OCT scanning, and also it allowed us to visualize the intact Descemet membrane and endothelial cells attached to it.

Conclusion: The results of pilot experiments on the use of enzyme solutions to dissolve the corneal stroma open the way for further research, which in the future may expand the arsenal of tools available to the ophthalmologist for the treatment of corneal diseases and that will increase the effectiveness of EK.

The use of selective enzymatic lysis of the corneal stroma may become a new approach for preparation of a transplant for EK, however, the effectiveness and safety of such a procedure must be confirmed in further studies.