1. Corneal Inflammation After Penetrating Keratoplasty and Miniature Keratoprosthesis Implantation Alja Crnej, Masahiro Omoto, Thomas H. Dohlman, Claes H. Dohlman, and Reza Dana Massachusetts Eye and Ear Infirmary and Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA No financial interests to disclose. This work was supported by the Boston-KPro research fund and the National Institute of Health (grant R01-EY 12963).

2. Background Boston Keratoprosthesis (B-KPro), an artificial cornea, is well- established alternative to re-grafting and high-risk transplantation. Glaucoma, other optic neuropathies, epiretinal membrane, macular edema, and retinal detachment are still threats to good long-term visual outcomes after B-KPro surgery and it has been hypothesized that chronic postoperative corneal inflammation may be a contributing factor to these complications. Boston Keratoprosthesis

3. We have established a novel murine model of KPro implantation (m-KPro) for evaluating host responses after KPro surgery. Background Design and dimensions of the m-KPro.A mouse with implanted m-KPro.

4. Purpose To compare corneal inflammation after: 1)Syngeneic* PK** 2)Allogeneic PK 3)Syngeneic m-KPro*** implantation 4)Allogeneic m-KPro implantation in mice. *Syngeneic = autograft **PK = penetrating keratoplasty ***m-KPro = miniature Keratoprosthesis

5. Methods BALB/C (syngeneic) or C57BL/6 (allogeneic) corneas were transplanted onto BALB/C host beds as part of PK or m-KPro implantation. Corneal inflammation was assessed by determining the frequencies of CD45 + leukocytes, CD4 + T cells, CD11b + cells, and Gr-1 + granulocytes/monocytes by flow cytometry at 2, 4, and 8 weeks post-transplantation. In addition, expression levels of the pro-inflammatory cytokines Tumor necrosis factor alpha (TNFα) and interleukin 1 beta (IL-1β) were analyzed using Real-Time qPCR at 8 weeks post- transplantation.

6. Results Quantification Of Graft-infiltrating Immune Cells B) CD4 + CD45+ CD11b+ CD4+ Gr1+ N – naïve SP – syngeneic PK SK – syngeneic m-KPro AP – allogeneic PK AK – allogeneic m-KPro Corneal grafts plus host corneal beds were collected and analyzed for the presence of CD45+, CD4+, CD11b+ and Gr1+ cells using flow cytometry. n=3 eyes/group; ***p<0.001, **p<0.005, *p<0.05. Data from one experiment of two is shown.

7. Results Quantification Of Pro-inflammatory Cytokines In The Graft N – naïve SP – syngeneic PK SK – syngeneic m-KPro AP – allogeneic PK AK – allogeneic m-KPro Corneal grafts plus host corneal beds were collected 8 weeks after the procedure and analyzed for the expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β by Real-Time qPCR. *p<0.01. Data from one representative experiment of two is shown. TNFα IL-1β

8. Conclusion Although the m-KPro device results in the inflammatory response in the cornea, allogenicity (of the carrier tissue) is also a significant contributor to inflammation. This suggests using the patient’s own cornea as a carrier for the B-KPro, if possible. When an autograft carrier is not an option, -methods to reduce the allogenicity (such as decellularization), or -usage of inhibitors of inflammatory cytokines, should be considered.

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