Confocal laser Doppler flowmeter measurements in a controlled flow environment in an isolated perfused eye

The aim of this study was to improve our ability to interpret and validate Heidelberg Retina Flowmeter (HRF) flow images by recording flow measurements from specific regions of the retinal vasculature by taking advantage of the ability to precisely regulate perfusion flow in an isolated eye preparation.The retinal vasculature in 16 isolated perfused pig eyes was perfused with a 50%/50% Krebs/RBC solution at known flow rates ranging from 0 to 300 μl min−1. At each flow rate, HRF images were obtained at a location approximately two disc diameters from the disc. After HRF image acquisition, the retinal vasculature was perfused with fluorescein isothiocyanate for fluorescence microscopy. Using the standard HRF software and a 10×10 pixel measurement window, flow rates were measured from a retinal artery, vein, arteriole, venule, and the retinal capillary bed and a capillary-free-zone. The relationship between HRF measured flow and perfusion flow in the different measurement locations was determined.At zero perfusion flow the measured HRF flow was consistently greater than zero (∼170 arbitrary units (AU)), and not significantly different at each measurement location except for the retinal vein, which had a significantly higher HRF flow value (∼230 AU). At higher perfusion flow rates the flow signal from the larger vascular elements (arteries and veins) increased rapidly thereafter to reach several thousand AU at a total perfusate flow of 50 μl min−1 and increased less rapidly at higher flow rates. In arterioles, the HRF flow was more linear over a broader range of perfusate flow rates but the peak flow signal was an order of magnitude smaller than that from the retinal artery. Both the linearity and magnitude of the flow signal in venules was less than that in arterioles. In capillary areas and in the capillary free zone, the HRF flow showed only a very weak relationship to perfusion flow when compared to the background noise.The choice of location for HRF flow analysis greatly influences the ability of the technique to measure changes in retinal blood flow. The major arteries and veins provide the strongest signal and greatest signal to noise ratio. However, the retinal arterioles produce an HRF signal that is more linear over a wider range of perfusate flow rates.