Modification of the Fluorescent Microsphere Impaction Method of Measuring Blood Flow in the Choriocapillaris

Abstract

Abstract: : Purpose: To modify the fluorescent microsphere impaction (FMI) method of measuring choriocapillaris blood flow (CBF) in order to permit rapid, direct visualization of the spheres in situ, thus allowing regional differences in CBF to be detected. Methods: In three pigmented rabbits, a 0.64 mm (outer diameter) vinyl tubing was inserted into the suprachoroidal space through a scleral incision. The tip of the tubing was placed in the posterior pole of the eye and the opposite end was connected to an osmotic minipump implanted subcutaneously. The pump was filled with endothelin–l (ET–1) and delivered a dose of 0.037 µg/hr. High speed indocyanine green (ICG) angiography and multifocal electroretinography were performed in vivo. 10 days following minipump implantation, the animals underwent left ventricular catherization via the right femoral artery. 5 million fluorescent microspheres (15 µm diameter) were injected over 25 seconds. The left femoral artery was also catherized and used for withdrawing a reference blood sample. The animals were sacrificed and the eyes enucleated, placed in 4% paraformaldehyde for 24 hrs at 4^o C, and stored in 0.1 M phosphate buffer at 4^o C. After removing the anterior segments of the eyes, the posterior poles were incubated in 0.25% potassium permanganate at 37^o C for 1 hr. They were then placed in 5% oxalic acid until clear, flattened with radial cuts, and pressed between two glass slides. The microspheres were then detected with an epifluorescence microscope. Results: All of the microspheres could be easily visualized. Little clumping was seen. Although the microspheres were fairly evenly distributed, patterns were evident. In the control eyes, a much higher concentration was noted in the region of the visual streak compared to the peripheral retina. In the eyes that had been administered ET–1, a paucity of microspheres was noted near the tubing tip in a pattern consistent with ICG changes suggesting reduced CBF in these areas. Conclusions: This modification of the FMI method of determining CBF is not only much more rapid than the traditional approach, but also permits direct visualization in relation to various features of the ocular anatomy and artifacts (such as the drug delivery tubing). Regional differences in CBF using this technique are clearly evident and can be quantified using standard image analysis software.

Keywords: blood supply • choroid • drug toxicity/drug effects