There are a number of important limitations to our study. Certain physiological parameters, including , , blood glucose, and pH were not measured or controlled, in large part because measurement of these parameters requires direct arterial cannulation, which complicates the procedure, particularly in longitudinal studies. Lacking knowledge of these parameters means that it is possible that at times we were imaging in the hyperoxic/hypoxic and/or hypercapniac/hypocapniac regimes. Other parameters, such as blood pressure, were measured using techniques known to be less accurate than invasive methods (e.g., we measured blood pressure using a blood pressure cuff instead of via direct arterial cannulation). Furthermore, in our study, only rats anesthetized with ketamine–xylazine–vecuronium were ventilated, while ventilation could be used in conjunction with any of the tested anesthetic protocols. Because isoflurane is known to be a potent respiratory depressant, potentially inducing hypercapnia during spontaneous breathing, and because previous studies have shown that hypercapnia is associated with increased ocular blood flow due to vasodilation, the higher blood flows observed in rats anesthetized with isoflurane may be partially due to hypercapnia, which could be controlled in part by ventilation. While these limitations in monitoring and physiological control prevent us from ascribing the observed blood flow differences to the tested anesthetic agents, per se, they do not affect the goal of evaluating the effects of the different anesthetic protocols, taken as a whole. Moreover, a secondary aim of this study was to investigate anesthetic protocols commonly used in optical imaging of the small animal eye. This is especially true when our study is considered in the context of optical imaging studies of small animal retinal blood flow, where the above parameters are rarely measured or controlled. Another limitation of this study is that we did not test different drug dosages or consider all of the various methods of delivery. While such data would be informative, the number of animals needed to perform this analysis serves as a high barrier. Furthermore decreased dosages are likely to exacerbate ocular motion, making optical imaging difficult. Other limitations of the study include (1) that the protocols were evaluated using criteria that emphasized measurement precision rather than accuracy, which could lead to incorrect conclusions being reached when the results of this study are extended to disease models, and (2) that there was an absence of blood flow measurements from unanesthetized rats, which would have served as a baseline against with which the tested anesthetic protocols could be compared. Both of these limitations could be addressed in future studies by performing blood flow measurements using the microsphere method; this method has the distinct advantage that it can be performed in the absence of anesthesia. Despite these limitations, we believe that this study makes a useful contribution to the current functional blood flow imaging literature.