Changes of Ocular Surface and the Inflammatory Response in a Rabbit Model of Short-Term Exposure Keratopathy.

PURPOSE: To evaluate the ocular surface change and the inflammatory response in a rabbit model of short-term exposure keratopathy. METHODS: Short term exposure keratopathy by continuous eyelid opening was induced in New Zealand white rabbits for up to 4 hours. Ultrasound pachymetry was used to detect central total corneal thickness. In vivo confocal microscopy and impression cytology were performed to evaluate the morphology of ocular surface epithelium and the infiltration of inflammatory cells. Immunohistochemistry for macrophage,neutrophil, CD4(+) T cells, and CD8(+) T cells were performed to classify the inflammatory cells. Scanning electron microscopy(SEM) was performed to detect ocular surface change.The concentrations of IL-8, IL-17, Line and TNF-αwere analyzed by multiplex immunobead assay. TUNEL staining was performed to detect cellular apoptosis. RESULTS: Significant decrease ofcentral total cornealthickness were found within the first 5 minutes and remained stable thereafter, while there were no changes of corneal epithelial thickness.No significant change of corneal, limbal and conjunctival epithelial morphology was found by in vivo confocal microscopy except the time dependent increase of superficial cellular defects in the central cornea. Impression cytology also demonstrated time dependent increase of sloughing superficial cells of the central cornea. Aggregations ofinflammatory cells were found at 1 hour in the limbal epithelium, 2 hours in the perilimbal conjunctival epithelium, and 3 hours in the peripheral corneal epithelium.In eyes receiving exposure for 4 hours, the infiltration of the inflammatory cells can still be detected at 8 hours after closing eyes.Immunohistochemical study demonstrated the cells to be macrophages, neutrophils, CD4-T cells and CD-8 T cells.SEM demonstrated time-depending increase of intercellular border and sloughing of superficial epithelial cells in corneal surface. Time dependent increase of IL-8, IL-17 and TNF-α in tear was found.TUNEL staining revealed some apoptotic cells in the corneal epithelium and superficial stroma at 3 hours after exposure. CONCLUSIONS: Short term exposure keratopathy can cause significant changes to the ocular surface and inflammatory response. Decrease of central total corneal thickness, aggregation of inflammatory cells, and cornea epithelial cell and superficial keratocyte apoptosis were found no less than 4 hours following the insult.

[Cross-linking in an artificial human cornea via induction of tissue transglutaminases]. / Vernetzung einer humanen künstlichen Hornhaut durch Induktion von Tissue-Transglutaminasen.

PURPOSE: In recent years many three-dimensional cornea models have been developed. However, they show poor collagen stability in the stroma. Transglutaminases (Tgases) are calcium-dependent proteins which play an important role in cross-linking of the corneal stroma. The purpose of this study was to find out whether it is possible to induce in vitro cross-linking of the stroma in an artificial hemicornea model with the help of Tgases. MATERIALS AND METHODS: For the construction of the hemicornea, human SV40 adenovector corneal epithelial cells (HCE) and human SV40 adenovector corneal keratocytes (HCK) were cultivated. Confluent HCK cells were treated for 24 h with transforming growth factor beta (TGFb) 1, 2 and 3 at different concentrations as well as with other growth factors and the treated cells were compared to untreated cultivated cells. The quantification of the expression of the Tgases by HCKs was examined with the use of real time PCR, Western blot imaging and immunochemistry. RESULTS: All concentrations of TGFbs used resulted in a significant increase of Tgase-mRNA, Tgase protein level and Tgase activity. The Tgases remained unaffected after treatment with other growth factors in comparison to untreated control cells. Treatment of the hemicornea with TGFb2 showed a very strong contraction and haze in comparison to the untreated hemicornea. CONCLUSION: It has been shown for the first time that TGFb induces a strong expression of Tgases in HCK cells. This effect caused an undesired contraction and haze of the human hemicornea model. Further research is necessary in order to find out whether the induction of Tgases in the HCK cells can be regulated without losing stability of the constructed hemicornea.