Exacerbation of retinal degeneration in the absence of alpha crystallins in an in vivo model of chemically induced hypoxia

This study evaluated the role of crystallins in retinal degeneration induced by chemical hypoxia. Wild-type, αA-crystallin (−/−), and αB-crystallin (−/−) mice received intravitreal injection of 12 nmol (low dose), 33 nmol (intermediate dose) or 60 nmol (high dose) cobalt chloride (CoCl2). Hematoxylin and eosin and TdT-mediated dUTP nick-end labeling (TUNEL) stains were performed after 24 h, 96 h, and 1 week post-injection, while immunofluorescent stains for αA- and αB-crystallin were performed 1 week post-injection. The in vitro effects of CoCl2 on αB-crystallin expression in ARPE-19 cells were determined by real time RT–PCR, Western blot, and confocal microscopy and studies evaluating subcellular distribution of αB-crystallin in the mitochondria and cytosol were also performed. Histologic studies revealed progressive retinal degeneration with CoCl2 injection in wild-type mice. Retinas of CoCl2 injected mice showed transient increased expression of HIF-1α which was maximal 24 h after injection. Intermediate-dose CoCl2 injection was associated with increased retinal immunofluorescence for both αA- and αB-crystallin; however, after high-dose injection, increased retinal degeneration was associated with decreased levels of crystallin expression. Injection of CoCl2 at either intermediate or high dose in αA-crystallin (−/−) and αB-crystallin (−/−) mice resulted in much more severe retinal degeneration compared to wild-type eyes. A decrease in ARPE-19 total and cytosolic αB-crystallin expression with increasing CoCl2 treatment and an increase in mitochondrial αB-crystallin were found. We conclude that lack of α-crystallins accentuates retinal degeneration in chemically induced hypoxia in vivo.