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H. Gong, U.V. Jurkunas, A. Plaas, E. Frank, A. Grodzinsky; A novel role for keratocytes in corneal stromal swelling . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3828.
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Purpose: It has been assumed that corneal stromal edema, when unaccompanied by inflammation, is solely the result of epithelium or endothelial compromise. Biochemical studies have shown that, during corneal edema, reduction in stromal glycosaminoglycan (GAG) content occurs in both vitro and in vivo. This study was to determine whether the stromal keratocytes play a role in controlling corneal swelling by regulating stromal GAG content. Methods: : Fifty 6 mm rabbit corneal buttons were cultured in Medium 199, either free or embedded in 1% agarose for 14 days. Amounts of keratan sulfate (KS)–proteoglycan (PG) and chondroitin/ dermatan sulfate (CS/DS)–PG in the corneal stroma, argarose and culture medium were analyzed on days 1, 7 and 14 by fluorophore–assisted carbohydrate electrophoresis and Western blot. The corneal swelling pressure and its relationship with stromal GAG content was evaluated. Morphological changes were examined by light microscopy. Changes in sulfated PGs were visualized by electron microscopy using cuprolinic blue (CB) staining. Results: : At days 1 and 7, both the agarose–embedded and free–floating corneal buttons exhibited stroma swelling compared to fresh corneas, but the amount swelling was much greater in the free–floating than in the agarose–embedded samples. By day 7 there was a 25% loss of KS–PGs from the stroma in argarose–embedded corneas. The loss of KS–PGs increased to 60% by day 14. CS/DS content was also decreased (25%). Importantly, the lost PGs were not recovered in either the agarose or the culture medium. By electron microscopy, in day 7 specimens, many focal losses of PG–CB complexes were evident around keratocytes compared to fresh corneas. The keratocytes were increased in size and contained many large vacuoles with multiple, large PG–CB complexes. By day 14, the amount of stromal PG–CB complexes was decreased and keratocyte size was increased even more. Many large vacuoles were still present in the cytoplasm, but fewer PG–CB complexes were found within them. Decreasing the GAG content in the corneal stroma reduced the swelling pressure. By contrast, in the day 7 free–swelling corneas, few keratocytes remained in the stroma. In the absence of keratocytes, the GAG content in the corneal stroma was not decreased during swelling. Conclusions: Keratocytes play an active role in reducing corneal edema by increasing endocytosis of stromal GAGs, thus reducing the osmotic pressure and swelling in the corneal stroma.
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