Introduction: Oxygen delivery, or utilization, is a function of retinal blood flow and blood oxygen saturation. The retinal pigment epithelium (RPE), in particular, has been shown to have the highest levels of metabolic activity within the human body. Oxygen delivery is therefore of extreme importance to the maintenance of the health and integrity of the retina. Animal models presuppose that the oxygen tension in the retina is highest in the innermost layers at the level of the choriocapillaris, less in the photoreceptors and further decreases throughout the outer retinal structures. The choroid provides by far the largest component of the oxygen for consumption by the photoreceptors. A lack of oxygen stores in the inner retina therefore makes a constant supply crucial for its normal functioning. Blood flow dysfunction and subsequent hypoxia are both a feature in the pathogenesis of several major ocular diseases such as retinopathy of prematurity (ROP), age-related macular degeneration (ARMD), diabetic retinopathy (DR) and glaucoma. The development of methods to measure retinal blood flow and blood oxygen saturation is crucial to improve understanding of the patho-physiology of major ocular diseases. Purpose: The aims of this work were, firstly, to determine the least variable (range ± standard deviation) wavelength combination (610/548, 600/569 and 605/586) and subsequent ODR with the prototype HRC device. Secondly, using the ODR with the lowest measurement variability, we sought to quantify retinal blood SO2 in arterioles and venules and investigate the relationship between retinal blood SO2 and total retinal blood flow (TRBF) in response to stepwise changes in PETO2 in healthy participants. Retinal blood SO2 and TRBF were assessed using the IRIS HRC (Photon etc. Inc. Montreal, Canada) and the RTvue Doppler Fourier Domain OCT (Optovue Inc, Freemont, CA) instruments, respectively. Methods: Ten healthy participants between the ages of 23 and 37, with an average age of 28.3 years were evaluated in two descriptive cross-sectional studies. Two gas provocation protocols; hyperoxia (end-tidal oxygen; PETO2 of 100, 200, 300, 400mmHg) and hypoxia (PETO2 of 100, 80, 60, 50mmHg) were administered in a fixed sequential order. In each phase of gas provocation (via modulation of PETO2), retinal blood SO2 and TRBF measurements were acquired with the HRC and Doppler FD-OCT. The precise and repeated control of the partial end tidal pressures of oxygen (PETO2) and carbon dioxide (PETCO2) over the pre-determined phase duration, irrespective of the individuals’ respiratory rate, was made possible with the RespirAct (Thornhill Research Inc., Toronto, Canada); a sequential re-breathing gas delivery Results: In arterioles, the group range (±SD) of ODR values for baseline measurements (PETO2 of 100mmHg) was 0.169±0.061 for the 605/586 wavelength combination, 0.371±0.099 for the 600/569 wavelength combination and 0.340±0.104 for the 610/548 wavelength combination. In venules, the group range (±SD) of ODR values was 0.600±0.198 for the 605/586 wavelength combination, 0.569±0.169 for the 600/569 wavelength combination and 0.819±0.274 for the 610/548 wavelength combination. With the 605/586 combination at baseline 1 and 2 in arterioles, the group range (±SD) of ODR values was 0.607 ± 0.224 and 0.619 ± 0.158, respectively (p = 0.370), while in venules the group range (±SD) of ODR at baseline 1 and 2 was 0.289±0.750 and 0.284 ± 0.729, respectively (p = 0.714). For the 600/569 combination at baseline 1 and 2 in arterioles, the group range (±SD) of ODR values was 0.747±0.350 and 0.761±0.391, respectively (p = 0.424) while in venules the group range (±SD) of ODR at baseline 1 and 2 was 0.329±0.675 and 0.366±0.659, respectively (p = 0.372). For the 610/548 combination at baseline 1 and 2 in arterioles, the group range (±SD) of ODR values was 0.604±0.263 and 0.685±0.450, respectively (p = 0.056) while in venules, the group range (±SD) of ODR at baseline 1 and 2 was 0.292±0.746 and 0.285±1.009, respectively (p = 0.131). There was no statistical difference found between baseline ODR values (baseline 1 and 2) across all three wavelength combinations in both arterioles and venules. The mean retinal blood SO2 value at baseline in arterioles for 4 participants was 95.19% ± 31.04% and venules was 53.89% ± 17.24% (p = 0.115). There was a negative linear relationship between group retinal blood SO2 and TRBF values in the 10 participants studied, although the results of any of the 10 individuals did not show evidence of such a relationship using the described methodology. The Pearson’s correlation coefficient (r) between TRBF and SaO2 was r = -0.354 and p = 0.001 and between TRBF and SvO2 was r = - 0.295, p = 0.008 Conclusion: Of the three wavelength combinations investigated (605/586, 600/569 and 610/548), the 605/586 combination was shown to have the overall least variability. It would be unwise at this stage to adopt this wavelength combination for clinical usage, however, since it is presupposed that the 605/586 combination is also the most reliable combination to detect change in retinal blood SO2 i.e. lower variability of the 605/586 combination may be irrelevant if this combination proves to be insensitive to change in retinal blood SO2. The absolute mean ± SD retinal blood SO2 in the arterioles (SaO2) was 95.19% ± 31.04% and in the venules (SvO2) was 53.89% ± 17.24%. These values fell within the range expected and described in the literature. The magnitude of the difference between the SaO2 and SvO2 was also consistent with the literature. These findings were all appropriate for a low flow, high oxygen exchange vascular network typical of the inner retinal vascular system. Using group rather than individual data, TRBF was found in this study to relate inversely with SaO2 (r = -0.354 and p = 0.001) and SvO2 (r = – 0.295 and p=0.008), respectively. This relationship between TRBF and SaO2 and SvO2, was as expected based upon data derived primarily from animal models. This study is ground-breaking and unique, in that, it is the first study to concomitantly measure both retinal blood SO2 and TRBF in human participants. Individual data showed extensive variability and noise, thus limiting the strength of the association between TRBF and SaO2 and SvO2..

Introduction: Oxygen delivery, or utilization, is a function of retinal blood flow and blood oxygen saturation. The retinal pigment epithelium (RPE), in particular, has been shown to have the highest levels of metabolic activity within the human body. Oxygen delivery is therefore of extreme importance to the maintenance of the health and integrity of the retina. Animal models presuppose that the oxygen tension in the retina is highest in the innermost layers at the level of the choriocapillaris, less in the photoreceptors and further decreases throughout the outer retinal structures. The choroid provides by far the largest component of the oxygen for consumption by the photoreceptors. A lack of oxygen stores in the inner retina therefore makes a constant supply crucial for its normal functioning. Blood flow dysfunction and subsequent hypoxia are both a feature in the pathogenesis of several major ocular diseases such as retinopathy of prematurity (ROP), age-related macular degeneration (ARMD), diabetic retinopathy (DR) and glaucoma. The development of methods to measure retinal blood flow and blood oxygen saturation is crucial to improve understanding of the patho-physiology of major ocular diseases. Purpose: The aims of this work were, firstly, to determine the least variable (range ± standard deviation) wavelength combination (610/548, 600/569 and 605/586) and subsequent ODR with the prototype HRC device. Secondly, using the ODR with the lowest measurement variability, we sought to quantify retinal blood SO2 in arterioles and venules and investigate the relationship between retinal blood SO2 and total retinal blood flow (TRBF) in response to stepwise changes in PETO2 in healthy participants. Retinal blood SO2 and TRBF were assessed using the IRIS HRC (Photon etc. Inc. Montreal, Canada) and the RTvue Doppler Fourier Domain OCT (Optovue Inc, Freemont, CA) instruments, respectively. Methods: Ten healthy participants between the ages of 23 and 37, with an average age of 28.3 years were evaluated in two descriptive cross-sectional studies. Two gas provocation protocols; hyperoxia (end-tidal oxygen; PETO2 of 100, 200, 300, 400mmHg) and hypoxia (PETO2 of 100, 80, 60, 50mmHg) were administered in a fixed sequential order. In each phase of gas provocation (via modulation of PETO2), retinal blood SO2 and TRBF measurements were acquired with the HRC and Doppler FD-OCT. The precise and repeated control of the partial end tidal pressures of oxygen (PETO2) and carbon dioxide (PETCO2) over the pre-determined phase duration, irrespective of the individuals’ respiratory rate, was made possible with the RespirAct (Thornhill Research Inc., Toronto, Canada); a sequential re-breathing gas delivery Results: In arterioles, the group range (±SD) of ODR values for baseline measurements (PETO2 of 100mmHg) was 0.169±0.061 for the 605/586 wavelength combination, 0.371±0.099 for the 600/569 wavelength combination and 0.340±0.104 for the 610/548 wavelength combination. In venules, the group range (±SD) of ODR values was 0.600±0.198 for the 605/586 wavelength combination, 0.569±0.169 for the 600/569 wavelength combination and 0.819±0.274 for the 610/548 wavelength combination. With the 605/586 combination at baseline 1 and 2 in arterioles, the group range (±SD) of ODR values was 0.607 ± 0.224 and 0.619 ± 0.158, respectively (p = 0.370), while in venules the group range (±SD) of ODR at baseline 1 and 2 was 0.289±0.750 and 0.284 ± 0.729, respectively (p = 0.714). For the 600/569 combination at baseline 1 and 2 in arterioles, the group range (±SD) of ODR values was 0.747±0.350 and 0.761±0.391, respectively (p = 0.424) while in venules the group range (±SD) of ODR at baseline 1 and 2 was 0.329±0.675 and 0.366±0.659, respectively (p = 0.372). For the 610/548 combination at baseline 1 and 2 in arterioles, the group range (±SD) of ODR values was 0.604±0.263 and 0.685±0.450, respectively (p = 0.056) while in venules, the group range (±SD) of ODR at baseline 1 and 2 was 0.292±0.746 and 0.285±1.009, respectively (p = 0.131). There was no statistical difference found between baseline ODR values (baseline 1 and 2) across all three wavelength combinations in both arterioles and venules. The mean retinal blood SO2 value at baseline in arterioles for 4 participants was 95.19% ± 31.04% and venules was 53.89% ± 17.24% (p = 0.115). There was a negative linear relationship between group retinal blood SO2 and TRBF values in the 10 participants studied, although the results of any of the 10 individuals did not show evidence of such a relationship using the described methodology. The Pearson’s correlation coefficient (r) between TRBF and SaO2 was r = -0.354 and p = 0.001 and between TRBF and SvO2 was r = - 0.295, p = 0.008 Conclusion: Of the three wavelength combinations investigated (605/586, 600/569 and 610/548), the 605/586 combination was shown to have the overall least variability. It would be unwise at this stage to adopt this wavelength combination for clinical usage, however, since it is presupposed that the 605/586 combination is also the most reliable combination to detect change in retinal blood SO2 i.e. lower variability of the 605/586 combination may be irrelevant if this combination proves to be insensitive to change in retinal blood SO2. The absolute mean ± SD retinal blood SO2 in the arterioles (SaO2) was 95.19% ± 31.04% and in the venules (SvO2) was 53.89% ± 17.24%. These values fell within the range expected and described in the literature. The magnitude of the difference between the SaO2 and SvO2 was also consistent with the literature. These findings were all appropriate for a low flow, high oxygen exchange vascular network typical of the inner retinal vascular system. Using group rather than individual data, TRBF was found in this study to relate inversely with SaO2 (r = -0.354 and p = 0.001) and SvO2 (r = – 0.295 and p=0.008), respectively. This relationship between TRBF and SaO2 and SvO2, was as expected based upon data derived primarily from animal models. This study is ground-breaking and unique, in that, it is the first study to concomitantly measure both retinal blood SO2 and TRBF in human participants. Individual data showed extensive variability and noise, thus limiting the strength of the association between TRBF and SaO2 and SvO2..