Our study is potentially confounded by factors including smoking status and use of vasoconstrictors like phenylephrine for pupillary dilation. Other limitations include the number of participants and the method of vascular stimulation. However, the effect sizes we observed are very large and significant. Our findings are also consistent and explained by trends reported by the previous studies.
18,23,39 Although we did not assess end-tidal CO
2 during the gas inhalation, the method used in the study has been very well characterized by Yezhuvath et al.,
25 from whom we adopted the procedure. For example, Yezhuvath et al.
25 found that after 60 seconds of CO
2 nonbreathing, the end-tidal CO
2 significantly increased by 28% while the arterial oxygen saturation only decreased by 0.1%. Heart rate and breathing rate were unchanged.
25,45 In addition, Yezhuvath and colleagues
25 reported that 1 minute of 5% CO
2 gas breathing (which is much more comfortable for human participants) yielded similar cerebrovascular reactivity as 4 minutes. Given the essentially identical approach we use, we do not expect the physiologic changes induced by the gas breathing in our study to significantly differ from that already reported in the literature.
25,45 Furthermore, this gas inhalation method has been validated across hundreds of participants and is being used in a multicenter study as a lead candidate for a biomarker of cerebrovascular reactivity.
18,23,26,27 While this gas provocation approach has proven effective, it may benefit from additional systemic evaluations such as PaCO
2, PaO
2, and pH. In the unlikely case that any of our healthy human volunteers (with no known respiratory conditions) were underventilating for some reason, our results would be an underestimate of the true effect of the gas stimuli. Additional studies comparing the blood flow response measured with OCTA and systemic blood oxygen levels in both healthy participants and those with vascular disease will be helpful. Other methods of vascular stimulation, including light–dark adaptation and flickering light, can also complement our understanding of the retinal vascular physiology in future studies.
20,46–48