All procedures with animals were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the institutional animal care and use committee (Animal Welfare Assurance no. A3307–01). Male Sprague-Dawley rats, weighing 250 to 300 g at the beginning of the study, were used. Diabetes was induced by intravenous injection of streptozotocin (STZ, 50 mg/kg, dissolved in 0.1 M sodium citrate buffer [pH 4.5]). Two groups of diabetic rats received either simvastatin (5 mg/kg/d, SC) or no treatment. Based on body surface area, this dose is 27.8 mg/m
2/d, which falls within the dose range used in humans (i.e., 20–80 mg/d or 10.8–43.2 mg/m
2/d). Age-matched control rats received only the vehicle. Rats were considered diabetic if their blood glucose was greater than 350 mg/dL. After 4 weeks, the animals were killed, and their blood was collected for analysis of blood glucose, cholesterol, and triglyceride levels. The results of these blood chemistry analyses, which have been reported by us previously,
19 showed that simvastatin treatment of the diabetic animals significantly lowered the total cholesterol levels ([asterisks refer to *
P < 0.05 versus control, **
P < 0.05 versus diabetic] control = 78 mg/dL, diabetic 121 mg/dL*, diabetic + statin = 81 mg/dL**) and triglyceride levels (control = 53 mg/dL, diabetic = 391 mg/dL*, diabetic+statin = 135 mg/dL**). Blood glucose levels were not significantly altered by statin treatment (control = 106 mg/dL, diabetic = 506 mg/dL*, diabetic + statin = 486 mg/dL*). These results are consistent with those of previous studies showing that statin treatment blocks diabetes-induced increases in cholesterol and triglycerides in the STZ diabetic rat model.
20 21
Retinas from different subgroups of animals were prepared for biochemical and morphologic analysis. For the biochemical studies, the animals were killed by decapitation, and the retinas were removed, snap frozen in liquid nitrogen, and stored at −80°C. Other groups were prepared for analysis of BRB function, immunofluorescence analysis, or dihydroethidine imaging, as explained later.