Design and validation of a custom-made system to measure transepithelial electrical impedance in human corneas preserved in active storage machine.

Corneal epithelial barrier represents one of the major limitations to ocular drug delivery and can be explored non-invasively through the evaluation of its electrical properties. Human corneas stored in active storage machine (ASM) could represent an interesting physiological model to explore transcorneal drug penetration. We designed a new system adapted to human corneas preserved in ASM to explore corneal epithelial barrier function ex-vivo. A bipolar set-up including Ag/AgCl electrodes adaptors to fit the corneal ASM and a dedicated software was designed and tested on freshly excised porcine corneas (n = 59) and human corneas stored 14 days in ASM (n = 6). Porcine corneas presented significant and proportional decrease in corneal impedance in response to increasing-size epithelial ulcerations and acute exposure to benzalkonium chloride (BAC) 0.01 and 0.05%. Human corneas stored 14 days in ASM presented a significant increase in corneal impedance associated with the restoration of a multi-layer epithelium and an enhanced expression of tight junctions markers zonula occludens 1, claudin 1 and occludin. These results support the relevance of the developed approach to pursue the exploration and development of human corneas stored in ASM as a physiological pharmacological model.

P03-A138†Comparison of specular and light transmission microscopy for endothelial control of corneas stored in the active storage machine.

PURPOSE: The Active Storage Machine (ASM), designed by Sincler (a company of group Laboratoires Théa) for eyebanks, will be used for long term donor corneas preservation at 31°C before transplantation. In this device, the endothelial cell density (ECD) counting is expected to be performed non-invasively throughout the storage, thus without changing the storage medium nor handling the cornea. To meet these constraints, specular microscopy (SM), also used for cold storage was selected, instead of the standard light transmission microscopy (LTM-NaCl) used in eye banks storing corneas in organ culture at 31-34°C. The purpose of this study is to compare both imaging methods for ECD measurement of corneas preserved in ASM. METHODS: Five human corneas from body donation to Science were preserved in a prototype ASM with 35mmHg in the endothelial chamber, 2.5µl/min of Corneamax® (Eurobio, France) at 31°C for 5 days. The endothelium of the cornea was imaged through the ASM window using the CellChek® D+ SM (Konan Medical, California, United-States) equipped with an add on device at customized stage. The cornea was then immediately removed from the ASM and prepared for standard endothelial assessment (dilation of the intercellular spaces using 0.9% NaCl and light transmission imaging). Finally, the endothelium was stained with alizarine red and trypan blue and observed again with the same microscope, to determine ECD using the referenced method up to now. For each cornea and each observation method, 5 images were acquired: 1 central and 4 paracentral. The SM images were counted with the Konan software. The LTM-NaCl and 'Alizarin Red' counts were performed with a dedicated plugin of ImageJ after microscope calibration. RESULTS: The means ± SD of the ECD calculated for SM, LTM-NaCl and 'Alizarine' images were respectively of 2314 ± 537, 2243 ± 506 and 2354 ± 543 cells/mm2. There was no significant difference between the 3 methods (ANOVA one-way, p-value = 0.1066). The percentage error was -1.7% +/- 3.3% for SM and -4.7 +/- 4.0% for light transmission microscopy. CONCLUSION: Quality control of the endothelium of corneas stored in ASM can be performed non-invasively with a standard eye bank SM. The ECD measured by SM does not differ from that measured by the conventional microscopy technique used until now in organoculture.

Repeat corneal transplantation: indication, surgical technique, and early graft failures trends in France from 2004 to 2019.

PURPOSE: To investigate the repeat corneal transplantation trend in France from 2004 to 2019. METHODS: Review of the prospectively compiled French Biomedicine Agency electronic database containing all corneal transplantation records from 2004 to 2019. The surgical technique, demographic characteristics, diagnosis, and previous graft data were retrieved and analyzed using the Cochran-Armitage trend test. RESULTS: A total of 66,584 corneal transplantations were performed, 51,260 of which were first grafts and 15,324 (23%) were regrafts. For regrafts, 77% were penetrating keratoplasties (PK) and 19.6% were lamellar keratoplasties (LK). Age, hypertonia, glaucoma, trauma, lens surgery, immune disorders, diameter > 8.5 mm, and neovessels in > 2 quadrants were associated with a higher rate of repeat keratoplasty. Keratoconus, secondary endothelial dystrophy, and Fuchs' dystrophy were the principal indications for regrafting. When a previous graft failed, it occurred earlier for patients with LK (4.6 years, median = 2, SD = 7.54) than PK (8.48 years, median = 5, SD = 9.51). Failure within a year was the reason why 28.3% of the LK regrafts and 12.5% of PK regrafts were performed, while for failure within two years these values were 49.9% and 27.8%, respectively. Graft survival decreased with the number of repeat keratoplasty, being more pronounced after a second LK regraft and after a first PK regraft. CONCLUSION: The number of LK regrafts increased continuously, and 1/3 were performed for failure within the year. This rate increased until 2015, after which it stabilized until 2019, probably due to the better mastery of the technique.

Tissue engineered endothelial keratoplasty in rabbit: tips and tricks.

PURPOSE: To report a detailed surgical procedure of tissue engineered endothelial keratoplasty (TEEK) in a rabbit model and its postoperative evaluation. METHODS: TEEKs were prepared 7 days before transplantation by seeding human or rabbit corneal endothelial cells on either femtosecond laser-cut ultrathin human stromal lamellae (fs-UTSL) or femtosecond laser-cut human anterior lens capsule (fs-HALC). Thirty transplantations were performed on aphakic eyes. Recombinant tissue plasminogen activator (rTPA) was used throughout the surgery. The native endothelium was removed by full-surface scraping and central descemetorhexis. The transplantation was performed as a human Descemet's membrane endothelial keratoplasty. Controls included Descemetorhexis only and transplantation of carrier alone. Postoperative follow-up was performed by slit lamp and optical coherence tomography, followed by histology. RESULTS: Controls remained oedematous. No fibrin occurred during surgery. All but three TEEKs adhered immediately. One/6 fs-UTSL and 9/16 fs-HALC cleared perfectly (p = 0.161). All failures could be explained by at least one of the following causes intraoperative bleeding, vitreous prolapsus, early partial detachment, postoperative irido corneal synechiea/angle closure. Presumed immune rejection was observed in three rabbits only after 4 weeks. Immunostaining with anti-human CD166 allowed to perfectly differentiate human cells from rabbit cells. In successful TEEK at 3 or 4 weeks, human cells formed a normal endothelium and started migrating outside the carrier. CONCLUSION: Though the transplantation of a TEEK in rabbits is a complex model with many causes of failure, established procedure including use of rTPA allows reliable preclinical study. In addition, we suggest that fs-HALC might be a potential carrier for TEEK.