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J. Chodosh, J. Rajaiya, I. S. Barequet, X. Zhou, T. Suzuki, M. S. Gilmore; The Corneal Facsimile: A Three Dimensional Model of Corneal Inflammation. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2018.
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Evidence from existing studies suggests that corneal stromal inflammation associated with ocular adenovirus infection occurs in response to the chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), produced by infected corneal cells. However, in vitro infections performed on two dimensional cell monolayers cannot adequately reproduce conditions in vivo. For example, studies done on cell monolayers do not take into account the possible contribution of extracellular matrix, which has been shown to play an important role in corneal inflammation. Hence, we developed a novel 3 dimensional corneal replica (cornea facsimile), to more closely mimic the human corneal stroma in adenovirus infection.
Cornea facsimiles were built in transwell plates using a neutralized solution containing PBS, DMEM, collagen type I, and second passage primary keratocytes, and then coated on their upper surface with Matrigel. Chemical signaling inhibitors were applied several hours before infection with human adenovirus type 19. A lymphocyte migration assay was performed using total lymphocytes isolated from human peripheral blood. Facsimiles were immunostained with antibodies to IL-8 and myloperoxidase (MPO) and evaluated under confocal microscopy. Supernatants were analyzed for IL-8 and MCP-1 expression using ELISA.
In adenovirus infected corneal facsimiles, we observed increased chemokine expression with increasing multiplicity of infection. Lymphocytes migrated into the sub-Matrigel stroma in adenovirus but not in mock infected facsimiles. Interestingly, we observed multifocal infiltration by leukocytes in a sub-Matrigel location, mimicking the subepithelial infiltrates seen clinically in adenovirus keratitis. Inhibitors of p38 MAPK and Src, when applied prior to infection, reduced subsequent leukocyte infiltration.
The corneal facsimile model of adenovirus keratitis closely mimics the human cornea after adenovirus infection, and shows substantial promise as a general model of tissue inflammation. The corneal facsimile may permit complex and relatively high throughput studies of viral or microbial pathogenesis not feasible with other methods.
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