Method for selective quantification of immune and inflammatory cells in the cornea using flow cytometry
The cornea serves as a protective surface against the environment (i.e., allergens, pollutants, desiccation and microorganisms) and promotes vision, made possible by corneal transparency. This protocol describes corneal preparation for flow cytometry to assess cells localized in the cornea. Our model details the process, from determining how many corneas are needed in the experiment to corneal excision to digestion and staining of the cornea cells. The simplicity of the model allows for systematic analysis of different corneal mechanisms of immunity, inflammation, angiogenesis and wound healing. In corneal transplantation, residential immune and inflammatory cells are key to the mechanisms that underlie angiogenesis, opacity, and graft rejection. In addition, this model can also elucidate cellular mechanisms mediating corneal graft outcomes and wound healing. Lastly, this model can be used to analyze the efficacy of new medications such as instillation and subconjunctival injections and assess the potential of therapeutic molecules to enhance graft survival and wound healing in vivo.
1. Niederkorn JY. Cornea: Window to Ocular Immunology. Curr Immunol Rev. 2011;7(3):328-35.
2. Gipson IK. The ocular surface: the challenge to enable and protect vision: the Friedenwald lecture. Invest Ophthalmol Vis Sci. 2007;48(10):4390; 1-8.
3. Inomata T, Mashaghi A, Di Zazzo A, Lee SM, Chiang H, Dana R. Kinetics of Angiogenic Responses in Corneal Transplantation. Cornea. 2017;36(4):491-6.
4. Hua J, Jin Y, Chen Y, Inomata T, Lee H, Chauhan SK, et al. The resolvin D1 analogue controls maturation of dendritic cells and suppresses alloimmunity in corneal transplantation. Invest Ophthalmol Vis Sci. 2014;55(9):5944-51.
5. Stevenson W, Chauhan SK, Dana R. Dry eye disease: an immune-mediated ocular surface disorder. Arch Ophthalmol. 2012;130(1):90-100.
6. Inomata T, Hua J, Nakao T, Shiang T, Chiang H, Amouzegar A, et al. Corneal Tissue From Dry Eye Donors Leads to Enhanced Graft Rejection. Cornea. 2017.
7. Chen Y, Chauhan SK, Shao C, Omoto M, Inomata T, Dana R. IFN-gamma-Expressing Th17 Cells Are Required for Development of Severe Ocular Surface Autoimmunity. Journal of immunology. 2017;199(3):1163-9.
8. Ljubimov AV, Saghizadeh M. Progress in corneal wound healing. Prog Retin Eye Res. 2015;49:17-45.
9. Inomata T, Hua J, Di Zazzo A, Dana R. Impaired Function of Peripherally Induced Regulatory T Cells in Hosts at High Risk of Graft Rejection. Sci Rep. 2016;6:39924.
10. Abud TB, Amparo F, Saboo US, Di Zazzo A, Dohlman TH, Ciolino JB, et al. A Clinical Trial Comparing the Safety and Efficacy of Topical Tacrolimus versus Methylprednisolone in Ocular Graft-versus-Host Disease. Ophthalmology. 2016;123(7):1449-57.
11. Dastjerdi MH, Sadrai Z, Saban DR, Zhang Q, Dana R. Corneal penetration of topical and subconjunctival bevacizumab. Invest Ophthalmol Vis Sci. 2011;52(12):8718-23.
12. Price RD, Berry MG, Navsaria HA. Hyaluronic acid: the scientific and clinical evidence. J Plast Reconstr Aesthet Surg. 2007;60(10):1110-9.
13. Dua HS, Gomes JA, Singh A. Corneal epithelial wound healing. Br J Ophthalmol. 1994;78(5):401-8.
14. Itoi M, Kim O, Kimura T, Kanai A, Momose T, Kanki K, et al. Effect of sodium hyaluronate ophthalmic solution on peripheral staining of rigid contact lens wearers. CLAO J. 1995;21(4):261-4.
15. Gupta D, Illingworth C. Treatments for corneal neovascularization: a review. Cornea. 2011;30(8):927-38.
16. Utine CA, Stern M, Akpek EK. Clinical review: topical ophthalmic use of cyclosporin A. Ocul Immunol Inflamm. 2010;18(5):352-61.
17. Chang JH, Garg NK, Lunde E, Han KY, Jain S, Azar DT. Corneal neovascularization: an anti-VEGF therapy review. Surv Ophthalmol. 2012;57(5):415-29.
18. Philipp W, Speicher L, Humpel C. Expression of vascular endothelial growth factor and its receptors in inflamed and vascularized human corneas. Invest Ophthalmol Vis Sci. 2000;41(9):2514-22.
19. Emami-Naeini P, Dohlman TH, Omoto M, Hattori T, Chen Y, Lee HS, et al. Soluble vascular endothelial growth factor receptor-3 suppresses allosensitization and promotes corneal allograft survival. Graefes Arch Clin Exp Ophthalmol. 2014;252(11):1755-62.
20. Di Zazzo A, Tahvildari M, Subbarayal B, Yin J, Dohlman TH, Inomata T, et al. Proangiogenic Function of T Cells in Corneal Transplantation. Transplantation. 2017;101(4):778-85.
21. Chauhan SK, Dohlman TH, Dana R. Corneal Lymphatics: Role in Ocular Inflammation as Inducer and Responder of Adaptive Immunity. J Clin Cell Immunol. 2014;5.
22. Hua J, Stevenson W, Dohlman TH, Inomata T, Tahvildari M, Calcagno N, et al. Graft Site Microenvironment Determines Dendritic Cell Trafficking Through the CCR7-CCL19/21 Axis. Invest Ophthalmol Vis Sci. 2016;57(3):1457-67.
23. Tahvildari M, Omoto M, Chen Y, Emami-Naeini P, Inomata T, Dohlman TH, et al. In Vivo Expansion of Regulatory T Cells by Low-Dose Interleukin-2 Treatment Increases Allograft Survival in Corneal Transplantation. Transplantation. 2016;100(3):525-32.
24. Yamagami S, Dana MR, Tsuru T. Draining lymph nodes play an essential role in alloimmunity generated in response to high-risk corneal transplantation. Cornea. 2002;21(4):405-9.
25. Afsaneh Amouzegar SKC, Reza Dana. Alloimmunity and tolerance in corneal transplantation. Journal of immunology. 2016;In press.
26. Crnej A, Omoto M, Dohlman TH, Dohlman CH, Dana R. Corneal inflammation after miniature keratoprosthesis implantation. Invest Ophthalmol Vis Sci. 2014;56(1):185-9.
27. Inomata T, Mashaghi A, Di Zazzo A, R. D. Ocular surgical models for immune and angiogenic responses. J Biol Methods. 2015;2(3):e27.