Diabetes Elevates Caspase-3 Enzyme Activity in the Rat Retina

Abstract

Abstract: : Purpose: Diabetes increases apoptosis in the neural retina of rats and humans (Barber et al, JCI, 102:783-791). The number of apoptotic cells in whole retinas was previously quantified by TUNEL labeling and by immunohistochemistry for the activated caspase-3 enzyme using CM-1, an antibody specific for the activated form of caspase-3. The purpose of this study was to quantify caspase-3 enzyme activity and determine more about the identity of the apoptotic cells. Methods: Diabetes was induced in male Sprague-Dawley rats by intravenous streptozotocin (STZ, 65 mg/kg). Apoptotic cells were labeled in flat-mount retinas by in situ terminal transferase dUTP nick end labeling (TUNEL) or by immunofluorescent histochemistry for activated caspase-3 using CM-1, a polyclonal antibody that only recognizes the cleaved form of caspase-3. Whole retinas were counter labeled for agrin, a marker of retinal vascular basement membrane. Caspase-3 enzyme activity was measured in whole retina lysate by Ac-DEVD-amc substrate cleavage. Duplicate incubations with the caspase-3 inhibitor Ac-DEVD-CHO were used to determine non-specific protease activity. Results: The numbers of TUNEL-positive and CM-1 immunoreactive cells were both significantly increased in STZ rat retinas compared to age-matched controls, as demonstrated previously. The majority of CM-1 immunoreactive cells did not colocalize with agrin immunoreactivity in whole-mount retinas. Caspase-3 enzyme activity was significantly elevated in STZ-diabetic rat retinas, compared to age-matched controls. Conclusions: Retinal apoptosis increases soon after the onset of STZ-diabetes and involves increased caspase-3 activity. The majority of cells containing active caspase-3 were not immediately associated with blood vessels. These data show that diabetes activates caspase-3 in the neural cells of the retina. This is a likely mechanism for neurodegeneration leading to loss of vision in diabetic retinopathy.

Keywords: diabetic retinopathy • apoptosis/cell death • degenerations/dystrophies