Investigative Ophthalmology & Visual Science, Vol 34, 3320-3328, Copyright © 1993 by Association for Research in Vision and Ophthalmology

ARTICLES AND REPORTS

Expression of collagen I, smooth muscle alpha-actin, and vimentin during the healing of alkali-burned and lacerated corneas

M Ishizaki, G Zhu, T Haseba, SS Shafer and WW Kao
Department of Ophthalmology, University of Cincinnati, OH 45267-0527.

PURPOSES. Alkali-burned corneas can seldom heal properly to restore corneal transparency. To provide a better understanding of this devastating corneal injury, we compared the expression of collagen I, smooth muscle alpha-actin (alpha-SMA), and vimentin in lacerated and alkali-burned rabbit corneas. METHODS. A radiolabeled cDNA probe of alpha 1(I) chain was used in slot-blot hybridization to determine the levels of alpha 1(I) mRNA in alkali-burned corneas. In situ hybridization was used to identify the cell types that express the alpha 1(I) chain. Antibodies against collagen I, alpha-SMA, and vimentin were used in immunohistochemical studies to determine the tissue distribution of collagen I and to identify cells expressing alpha-SMA and vimentin. RESULTS. The levels of alpha 1(I) mRNA in alkali-burned corneas increased steadily after the alkali burn and reached a plateau within 2 weeks. One day after alkali burn, specific in situ hybridization signals were detected in stromal cells immediately surrounding the edge of the corneal injury. As the healing proceeded, the fibroblastic cells migrated into the injured stroma, and they showed positive reactions by in situ hybridization and by immunostaining with anti-collagen I probes. In alkali-burned corneas, retrocorneal membranes were formed 1 week after injury. This fibrillar membrane was stained by anti-collagen I antibody, and the fibroblastic cells in the membrane were hybridized by the 3H-labeled alpha 1(I) cDNA probe. No retrocorneal membrane was formed in the lacerated corneas, even after the injured corneas were allowed to heal for 3 weeks. The epithelial cells in the epithelial plug of lacerated corneas were positive by in situ hybridization, whereas the epithelial cells in the regenerated epithelium of alkali-burned cornea was not. Antibodies against alpha-SMA reacted with the migrating fibroblastic cells but did not react with epithelial cells or endothelial cells in the injured corneas. Anti-vimentin antibody reacted with fibroblastic cells, endothelial cells, and keratocytes in normal and injured corneas, and with the basal epithelial cells of injured corneas. CONCLUSIONS. During wound healing, the keratocytes that migrate to injured stroma transform into myofibroblasts. These myofibroblasts express high levels of alpha 1(I) mRNA, alpha-SMA, and vimentin. The healing of alkali-burned corneas differ from that of lacerated corneas in that the retrocorneal membranes are formed in the former but not in the latter. In addition, the epithelial cells of alkali-burned corneas lack alpha 1(I) mRNA, whereas it is found in the epithelium of lacerated corneas. These differences may result from the persistence of inflammatory cells in the alkali-burned corneas.

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