B.E. MALYUGIN, E.A. MALYUTINA, Kh.D. TONAEVA, S.A. BORZENOK, D.S. OSTROVSKIY

The S. Fyodorov Eye Microsurgery Federal State Institution, 59a Beskudnikovskiy Blvd, Moscow, Russian Federation, 127486

Malyugin B.E. — D.Sc. (medicine), Professor, tel. (495) 488-85-11, e-mail: malyugin@mntk.ru, ORCID ID: 0000-0001-5666-3493

Malyutina E.A. — post-graduate student, e-mail: malyutinaekaterinamd@gmail.com, ORCID ID: 0000-0003-0270-0847

Borzenok S.A. — D.Sc. (medicine), Professor, tel. (495) 488-85-52, e-mail: mdborzenok@yandex.ru, ORCID ID: 0000-0001-9160-6240

Tonaeva Kh.D. — PhD (medicine), e-mail: dr.tonaeva@gmail.com, ORCID ID: 0000-0002-9034-0660

Ostrovskiy D.S. — post-graduate student, laboratory assistant, e-mail: dmitriy.ostrovskiy@gmail.com, ORCID ID: 0000-0002-2817-7102

Objective: experimental modeling of central descemetorhexis and human cornea endothelial cells migration and phenotype study in experiment ex vivo with imminohystochemical analyses.

Materials and methods: Three donors of 31 y.o. (endothelial cells density 3115±64 kl/mm2), 54 y.o. (ECD 2930±86 kl/mm2), 69 y.o. (ECD 2865±112 kl/mm2). Central circular descemethorhexis (d=4.00 mm) was provided using micro forceps. The cornea was divided into 4 segments with intact endothelial zone and plane (without endotheliocytes) zone. Each sample was cultivated during 14 days in preservative culture DMEM/ F12 with addition of fetal calf serum. The medium was changed once in 7 days. Then the material was fixated in formaldehyde (10%). The staining for particular markers was made: α-SMA, ki67, lumikan and Na/K АTFase. The examination of samples was made on laser-scanning confocal microscope Olympus FV10i.

Results: By the 14^th day of experiment, there was no active α-SMA expression in descemetorhexis zone. Single cells with ki67 expression were detected. Active lumican detection was in defect zones of all samples. Also in descemethorhexis zone, the Na/K АTFase expression was detected.

Conclusion: The results show complete or almost complete restoration of the defect by the 14^th day of the experiment. The large zone filled with new cells was detected in a young patient with maximal ECD in the center. The obtained data testify that phenotype and functional activity of endotheliocytes were preserved, which substantiates the reliability of the method of central descemethorhexis in treating patients with primary endothelial-epithelial corneal Fuks dystrophy.

Key words: human corneal endothelium, migration, cultivation, immunocytological analyses, limikan, α-SMA, ki67, Na/K АTFase.

(For citation: Malyugin B.E., Malyutina E.A., Borzenok S.A., Tonaeva Kh.L., Borzenok S.A., Tonaeva Kh.D., Ostrovskiy D.S. Study of human cornea endothelial cells migration into iatrogenic defect zone in experiment ex vivo. Practical Medicine. 2018)

REFERENCES

1. Katikireddy K.R., Schmedt T., Price M.O., Price F.W., Jurkunas U.V. Exictence of neural crest-derived progenitor cells in normal and Fuchs endothelial dystrophy corneal endothelium. Am J Pathol., 2016 Oct;186(10):2736-50.
2. Joyce N.C., Meklir B., Joyce S.J., Zieske J.D. Cell cycle protein expression and proliferative status in human corneal cells. Invest Ophthalmol Vis Sci., 1996, Mar;37(4):645-55.
3. Joyce N.C. Proliferative capacity of the corneal endothelium. Prog Retin Eye Res., 2003 May;22(3):359-89.
4. Bourne W.M., Nelson L.R., Hodge D.O. Central corneal endothelial cell changes over a ten-year period. Invest Ophthalmol Vis Sci., 1997, Mar;38(3):779-82.
5. Oie Y., Watanabe S., Nishida K. Evaluation of visual quality in patients with Fuchs endothelial corneal dystrophy. Cornea, 2016, Nov;35 Suppl 1:S55-S58. Review.
6. Murphy C., Alvarado J., Juster R., Maglio M. Prenatal and postnatal cellularity of the human corneal endothelium: a quantitative histologic study. Invest Ophthalmol Vis Sci., 1984;25(3):312-322.
7. Okumura N., Kakutani K., Inoue R. et al. Generation and feasibility assessment of a new vehicle for cell-based therapy for treating corneal endothelial dysfunction. PLoS One, 2016;11(6): e0158427.
8. Malyugin B.E., Izmaylova S.B., Malyutina E.A., Antonova O.P., Gelyastanov A.M. Clinical and functional results of surgical treatment of cataract and primary endothelial dystrophy of Fuchs’ cornea by single-stage phacoemulsification and central descemethorexis. Vestnik oftal’mologii, 2017, no. 6, pp. 16–22 (in Russ.).
9. Koizumi N., Okumura N., Kinoshita S. Development of new therapeutic modalities for corneal endothelial disease focused on the proliferation of corneal endothelial cells using animal models. Exp Eye Res., 2012;95(1):60-67.
10. Rizwan M., Peh G.S., Adnan K. et al. In vitro topographical model of Fuchs dystrophy for evaluation of corneal endothelial cell monolayer formation. Adv Hea.
11. Peh G.S., Chng Z., Ang H.P. et al. Propagation of human corneal endothelial cells: a novel dual media approach. Cell Transplant., 2015;24(2):287-304.
12. Peh G.S., Adnan K., George B.L. et al. The effects of Rho-associated kinase inhibitor Y-27632 on primary human corneal endothelial cells propagated using a dual media approach. Sci Rep., 2015;5:9167.
13. Maksimov A.A. Limfotsit kak obshchaya stvolovaya kletka razlichnykh elementov krovi v embrional’nom razvitii i postfetal’noy zhizni mlekopitayushchikh. Folia Haematologica, 8.1909, 125-134 (in Russ.).
14. Ng K.W., Abraham M.C., Leong D.TW, Morris C., Schantz J-T. Primary culture of specific cell types and the establishment of cell lines. Animal cell culture: essential methods. [Internet]. John Wiley & Sons, Ltd; 2011:205-230. doi:10.1002/9780470669815.ch7.
15. Ahmad S. Concise review: limbal stem cell deficiency, dysfunction, and distress. Stem Cells Trans Med., 2012;1(2):110-115. doi:10.5966/sctm.2011-0037.
16. Kasparova E.A., Subbot A.M., Kalinina D.B. Proliferative potential of the posterior epithelium of the human cornea. Vestnik oftal’mologii. 2013;129(3):82-88 (in Russ.).
17. Sabater A.L., Guarnieri A., Espana E.M., Li W., Prósper F., Moreno-Montañés J. Strategies of human corneal endothelial tissue regeneration. Regen Med., 2013;8(2):183-195. doi:10.2217/rme.13.11.
18. Mimura T., Yamagami S., Yokoo S., Usui T., Tanaka K., Hattori S., Irie S., Miyata K., Araie M., Amano S. Cultured human corneal endothelial cell transplantation with a collagen sheet in a rabbit model. Invest Ophthalmol Vis Sci., 2004;45(9):2992-2997. doi:10.1167/iovs.03-1174.
19. Bayyoud T., Thaler S., Hofmann J., Maurus C., Spitzer M.S., Bartz-Schmidt K-U., Szurman P., Yoeruek E. Decellularized bovine corneal posterior lamellae as carrier matrix for cultivated human corneal endothelial cells. Curr Eye Res., 2012;37(3):179-186. doi:10.3109/02713683.2011.644382.
20. Teichmann J., Valtink M., Nitschke M., Gramm S., Funk RHW., Engelmann K., Werner C. Tissue engineering of the corneal endothelium: a review of carrier materials. J Funct Biomater., 2013;4(4):178-208. doi:10.3390/jfb4040178.
21. Parikumar P., Haraguchi K., Ohbayashi A., Senthilkumar R., Abraham S.JK. Successful transplantation of in vitro expanded human cadaver corneal endothelial precursor cells on to a cadaver bovine’s eye using a nanocomposite gel sheet. Curr Eye Res. 2013;39(5):522-526. doi:10.3109/02713683.2013.838633.
22. Koizumi N., Okumura N., Kinoshita S. Development of new therapeutic modalities for corneal endothelial disease focused on the proliferation of corneal endothelial cells using animal models. Exp Eye Res. 2012;95(1):60- 67. doi:10.1016/j.exer.2011.10.014.
23. Patel S.V., Bachman L.A., Hann C.R., Bahler C.K., Fautsch M.P. Human corneal endothelial cell transplantation in a human ex vivo model. Invest Ophthalmol Vis Sci., 2009;50(5):2123-2131. doi:10.1167/iovs.08-2653.
24. Mimura T., Yamagami S., Usui T., Ishii Y., Ono K, Yokoo S., Funatsu H., Araie M., Amano S. Long-term outcome of iron-endocytosing cultured corneal endothelial cell transplantation with magnetic attraction. Exp Eye Res., 2005;80(2):149-157. doi:10.1016/j.exer.2004.08.021.
25. Mimura T., Yokoo S., Araie M., Amano S., Yamagami S. Treatment of rabbit bullous keratopathy with precursors derived from cultured human corneal endothelium. Invest Ophthalmol Vis Sci., 2005;46(10):3637-3644. doi:10.1167/iovs.05-0462.
26. Fan T., Ma X., Zhao J., Wen Q., Hu X., Yu H., Shi W. Transplantation of tissueengineered human corneal endothelium in cat models. Mol Vis., 2013;19:400- 407.
27. Shao C., Chen J., Chen P., Zhu M., Yao Q., Gu P., Fu Y., Fan X. Targeted transplantation of human umbilical cord blood endothelial progenitor cells with immunomagnetic nanoparticles to repair corneal endothelium defect. Stem Cells Dev., 2015;24(6):756-767. doi:10.1089/scd.2014.0255
28. Shao C., Chen J., Chen P., Zhu M., Yao Q., Gu P., Fu Y., Fan X. Targeted transplantation of human umbilical cord blood endothelial progenitor cells with immunomagnetic nanoparticles to repair corneal endothelium defect. Stem Cells Dev., 2015;24(6):756-767. doi:10.1089/scd.2014.0255.
29. Koizumi N., Okumura N., Ueno M., Kinoshita S. New therapeutic modality for corneal endothelial disease using rho-associated kinase inhibitor eye drops. Cornea, 2014;33 Suppl 11:S25-S31. doi:10.1097/ICO.0000000000000240.
30. Okumura N., Kinoshita S., Koizumi N. Cell-based approach for treatment of corneal endothelial dysfunction. Cornea, 2014; 33, Suppl 11:S37-S41. doi:10.1097/ICO.0000000000000229.
31. Malyugin B.E., Borzenok S.A., Kolokol’tsova T.D., Komakh Yu.A., Zheltonozhko A.A., Popov I.A., Saburina I.N., Repin V.S., Kosheleva N.V., Zurina I.M., Davydova L.I., Bogush V.G., Agapov I.I. Development of a bio-engineering design of an artificial cornea on the basis of a film matrix from spirodiainakultivirovanny cells of a limbal zone of an eyeball. Oftal’mokhirurgiya, 2013;4:89-97. 97 (in Russ.).
32. Okumura N., Okazaki Y., Inoue R. et al. Effect of the Rho-associated kinase inhibitor eye drop (ripasudil) on corneal endothelial wound healing. Invest Ophthalmol Vis Sci., 2016;57(3):1284-1292.
33. Okumura N., Inoue R., Okazaki Y. et al. Effect of the Rho kinase inhibitor Y-27632 on corneal endothelial wound healing. Invest Ophthalmol Vis Sci., 2015;56(10):6067-6074.
34. Bostan C., Thériault M., Forget K.J. et al. In vivo functionality of a corneal endothelium transplanted by cell-injection therapy in a feline model. Invest Ophthalmol Vis Sci., 2016;57(4):1620-1634.
35. Okumura N., Koizumi N., Kay E.P. et al. The ROCK inhibitor eye drop accelerates corneal endothelium wound healing. Invest Ophthalmol Vis Sci., 2013;54 (4):2493-2502.
36. Koizumi N., Okumura N., Ueno M., Kinoshita S. New therapeutic modality for corneal endothelial disease using Rho-associated kinase inhibitor eye drops. Cornea, 2014;33(suppl 11):S25-S31.
37. Okumura N., Sakamoto Y., Fujii K. et al. Rho kinase inhibitor enables cell-based therapy for corneal endothelial dysfunction. Sci Rep., 2016;6: 26113.
38. Iovieno A., Neri A., Soldani A.M., Adani C., Fontana L. Descemetorhexis without graft placement for the treatment of Fuchs endothelial dystrophy: preliminary results and review of the literature. Cornea, 2017;36(6):637-641.
39. Moloney G., Petsoglou C., Ball M. et al. Descemetorhexis without grafting for Fuchs endothelial dystrophy-supplementation with topical ripasudil. Cornea, 2017;36(6):642-648.
40. Arbelaez J.G., Price M.O., Price F.W. Jr. Long-term follow-up and complications of stripping descemet membrane without placement of graft in eyes with Fuchs endothelial dystrophy. Cornea, 2014;33(12): 1295-1299.
41. Bleyen I., Saelens I.E., van Dooren B.T., van Rij G. Spontaneous corneal clearing after Descemet’s stripping. Ophthalmology, 2013;120(1):215.