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UP Andley, J-H Xi; Reduced Survival of Lens Epithelial Cells in the alphaA-crystallin Knockout Mouse . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1920.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have shown that cultured lens epithelial cells derived from αA-crystallin knockout mice grow at a ≷50% slower rate and have a higher apoptotic index than wild type cells. To investigate whether αA-crystallin affects lens epithelial cell growth and apoptosis in vivo, the effect of expression of αA-crystallin on newly synthesized DNA in proliferating cells was studied. Methods: Seven day-old 129Sv (wild type) and αA knockout mice (a kind gift from Dr. Eric Wawrousek, NEI) were injected with 5-bromo-2-deoxyuridine (BrdU) to label newly synthesized DNA in proliferating cells. Whole mounts of capsule-epithelial explants were made at various times after the BrdU pulse (1, 3, 8, 24 and 48 hours and 5 days). The number of BrdU labeled cells was detected by immunofluorescence and confocal microscopy. The total number of cells was determined using the DNA binding dye TOTO-1. TUNEL labeling was used to detect apoptotic cells in the whole mounts. Lens size was determined by staining sagittal sections with hematoxylin and eosin. Results: The size of the αA-crystallin knockout lens was 50% smaller than the wild type at 1, 2, and 7 days of age. The total number of nuclei was 33% lower in the knockout lens than the wild type. One hour after BrdU injection, the labeling index in the central region of the whole mount of both wild type and αA knockout lens epithelium was the same (∼0.04). Some of the cells in the S phase were heavily labeled with BrdU and other cells were labeled lightly. The first labeled mitoses were detected ∼3 hours after the BrdU pulse, indicating that the length of the G2 phase in the wild type lens is about 3 hours. Twenty-four hours after BrdU injection, the labeling index in the wild type lens had doubled due to mitosis. The number of labeled cell pairs was 50% less in the αA knockout lens suggesting that some of the cells failed to divide or that the daughter cells died during or soon after mitosis. The number of BrdU-labeled cell pairs was also lower in the knockout than the wild type lens epithelium 48 hours after labeling. Five days after BrdU injection, the number of BrdU-labeled cells per lens decreased dramatically. TUNEL labeling was sparse (1 per lens) in wild type, and 6 per lens in the αA-knockout. Conclusions: αA-crystallin expression appears to protect against cell death in vivo. The smaller size of the αA-crystallin knockout lens appears to be due to a decrease in the net production of epithelial cells. Support: NIH Grants EY05681, EY02687 and RPB.
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