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B Khoobehi; Freeze Frame Video Technique to Measure Volumetric Blood Flow of the Retina in Diabetic Rats . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3318.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To quantify volumetric blood flow in the rat retina using freeze frame video of fluorescent erythrocytes and to compare blood flow in diabetic and normal rats. Methods: Diabetes was induced in 12 rats with the injection of streptozotocin (65 mg/kg); 12 normal rats served as controls. Fourteen days later, volumetric retinal blood flow was measured in each animal using the scanning laser ophthalmoscope (SLO). Blood (0.1 ml) was drawn from the tail vein and transferred to a test tube. Then, 5 ml of mouse plasma buffer was added, the suspension was centrifuged, and plasma and excess buffer aspirated. Leukocytes and erythrocytes were separated. Erythrocytes were labeled with 10 ml of a membrane stain (2 mg D-307 in 1 ml ethanol) and injected back into the tail vein. The eyes were dilated, the animal was positioned in front of the SLO, and the number of cells passing through the fundus was determined by the freeze frame technique. The erythrocytes were excited by the SLO He-Ne laser, and fundus images were recorded on digital tape in 10-second segments. A 633-nm filter was positioned in front of the SLO detector to eliminate incident He-Ne laser wavelengths. Data from each 10-second block (30 frame/sec) were stored on digital tape and transferred to a PC for analysis. Erythrocytes were counted in every frame, and cells seen in previous frames were subtracted. Simultaneously with the SLO imaging, a blood sample was drawn and used to determine the concentration of cells/ml. A measure of absolute volumetric blood flow in the entire retinal vessels was generated based on the total calculated number of labeled erythrocytes passing through the retina in a specific amount of time combined with the concentration of cells in the blood sample. Results: Total volumetric blood flow was 33.6 ± nl/sec (N=12) for normal rats and 27.5 ± 4.8 nl/sec for diabetic rats (N = 12) (P < 0.05). Thus, this techniques demonstrates significantly reduced volumetric blood flow in the diabetic rat retina. Conclusion: This technique allows for comparison of the hemodynamics of the normal retina with that of retinas in disease states such as diabetic retinopathy. Volumetric blood flow obtained from this technique is direct. With this technique, not only is it possible to assess absolute volumetric blood flow of the entire retina in the field of view, but also regional volumetric blood flow can be obtained by mapping the entire retina and dividing it into sectors.
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