Abstract
Abstract: :
Purpose : Lens organ culture has been extensively used to study cataract induction and prevention. Cultured rat lenses remain transparent and viable for a week or longer. This study compares aspects of the biology of lenses after a week in culture with freshly extracted ones. Methods : Lenses from Sprague-Dawley rats (~4weeks old) were cultured in modified TC-199 medium under standard conditions. Some lenses were fixed in glutaraldehyde for histological analysis or in formalin for preparation of paraffin sections for immunohistochemistry. S-35 methionine incorporation , SDS-PAGE and autoradiography were used to analyze protein synthesis patterns . Cell proliferation was assessed by BrdU incorporation and proteomic analysis was by 2-D electrophoresis and MALDI mass spectrometry. Results : Although remaining transparent and metabolically active, the cultured lenses did not increase in weight. In vivo during the same 7 day period lenses increase in weight by about 20%. Protein synthesis in the cultured lenses decreases with time in culture, but after 7 days is still about 2/3 of that in freshly extracted lenses. It appears that crystallin synthesis is decreased while no general decrease is apparent in the non-crystallin proteins. The synthesis of a few particular non-crystallin proteins is affected by culture as determined by autoradiography of 1D and 2-D SDS-PAGE gels. MALDI mass spectrometry is being used to attempt to identify these species. Preliminary results from BrdU immunohistochemistry indicate significant proliferation of epithelial cells. The histology of the bow region of the cultured lenses is abnormal. Conclusion : The data at present suggest that while epithelial cells are proliferating they are not differentiating and elongating into fiber cells normally. Studies are continuing to confirm this and to determine its molecular basis.
Keywords: cataract • crystallins • immunohistochemistry