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The ability to accurately and precisely determine the perfusion of specific ocular tissues is fundamental to understanding the pathophysiology of eye disease. This requires blood flow measurements that are non-invasive, safe, robust, accurate, and reproducible. Despite much research; however, a number of issues regarding the reproducibility and validity of Heidelberg retinal flowmetry (HRF) measurements remain. Chauhan et al. (86), using modified optics in order to image the rat retina, were unable to visualize retinal capillaries in five adult Sprague-Dawley rats. They extend the meaning of their findings to use of the commercial instrument in humans suggesting the analysis of retinal capillaries is limited. In our experience, we have compared HRF images of retinal capillaries with fluorescein angiograms, and found good agreement between the two imaging techniques in humans. The author's use of the 2X2, 4X4 and 10X10 pixel measurement windows (averaged flow values) also brings into question the validity of their conclusions. HRF data are not normally distributed and cannot be represented as a single mean value. Our in-vitro model investigations1 and validation studies2-4 have shown many factors need to be considered when imaging the retina with HRF. The HRF allows reproducible measurements of low capillary perfusion areas when a pixel by pixel analysis is used by trained graders.4
Foremost, the present findings suggest that the HRF is difficult or impossible to use with a rodent animal model. The projection of a 300-micrometer focal plane in a rat eye, with the modified optics, likely includes all layers from vitreous to choroid contributing to background noise levels. Good focus is also vital for reliable HRF data, and the authors failed to obtain a well focused imaged as evidenced by the non-visualized retinal capillaries. Further research regarding ocular blood flow technologies, including the HRF and the various analysis techniques utilized, and cautious interpretation, will continue to shed light on the accuracy and limitations of ocular perfusion measurements.