Measurement of Retinal Blood Flow Using Fluorescently Labeled Red Blood Cells

Tess E Kornfield,Eric A Newman

doi:10.1523/eneuro.0005-15.2015

Abstract

Blood flow is a useful indicator of the metabolic state of the retina. However, accurate measurement of retinal blood flow is difficult to achieve in practice. Most existing optical techniques used for measuring blood flow require complex assumptions and calculations. We describe here a simple and direct method for calculating absolute blood flow in vessels of all sizes in the rat retina. The method relies on ultrafast confocal line scans to track the passage of fluorescently labeled red blood cells (fRBCs). The accuracy of the blood flow measurements was verified by (1) comparing blood flow calculated independently using either flux or velocity combined with diameter measurements, (2) measuring total retinal blood flow in arterioles and venules, (3) measuring blood flow at vessel branch points, and (4) measuring changes in blood flow in response to hyperoxic and hypercapnic challenge. Confocal line scans oriented parallel and diagonal to vessels were used to compute fRBC velocity and to examine velocity profiles across the width of vessels. We demonstrate that these methods provide accurate measures of absolute blood flow and velocity in retinal vessels of all sizes.

Highlights

Blood flow is closely tied to the metabolic state of cells in the CNS (Attwell et al, 2010) and is disrupted in manyCNS disorders including stroke and Alzheimer’s disease (Girouard and Iadecola, 2006)
Confocal line scans oriented parallel and diagonal to vessels were used to compute fluorescently labeled red blood cells (fRBCs) velocity and to examine velocity profiles across the width of vessels. We demonstrate that these methods provide accurate measures of absolute blood flow and velocity in retinal vessels of all sizes
We have measured blood flow in rat retinal blood vessels by introducing fluorescently labeled red blood cells into the vasculature (Fig. 1). fRBC flux was measured with perpendicularly oriented confocal line scans in vessels ranging from primary arterioles and venules to capillaries by counting individual fRBCs as they passed through a vessel (Fig. 1A, yellow line, B,C)

Summary

Introduction

Blood flow is closely tied to the metabolic state of cells in the CNS (Attwell et al, 2010) and is disrupted in manyCNS disorders including stroke and Alzheimer’s disease (Girouard and Iadecola, 2006). Blood flow is closely tied to the metabolic state of cells in the CNS (Attwell et al, 2010) and is disrupted in many. Acknowledgements: The authors thank Michael Burian for his invaluable technical assistance; and Kyle Biesecker, Michael Burian, Joanna Kur, and Anja Srienc for their helpful discussions and for reviewing the manuscript.

Methods

Results

Discussion

Conclusion