Note the different scales for offset between brain and retina. Data shown for capillaries only, arteries and veins were similarly altered Figure S3. We have directly compared in vivo cortical intraparenchymal and inner retinal vessels using video fluorescein angiography Hui et al. As retinal and cortical intraparenchymal vessels are at different distances from the heart, with different sized feeder vessels and thus resistance , it is not surprising that cortical vessels showed faster fluorescein filling half-rise.
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The timing of cortical filling however, was similar to previous quantification using contrast-enhanced imaging where Lucifer Yellow dye took on average 5. In addition, the cortical surface vessels imaged were significantly larger than the inner retinal arteries brain: Consistent with faster filling, cortical vessels also showed faster fluorescence decay. It is of interest that the difference in half-rise and half-fall for arteries and veins was greater in the retina compared with cortical vessels.
Again this would be consistent with a bolus of contrast agent entering and leaving a larger cortical vessel faster than a smaller retinal vessel. Thus, within the small cranial window through which imaging was undertaken, fluorescein filling in a given artery may not drain into any of the visible veins.
Furthermore, a cortical vein collects blood from a wider network of venules, many of which may not be visible through the limited window, which can also contribute to faster contrast dye filling. A higher residual fluorescence or offset was also seen in the brain. This is not surprising given the greater depth of cortical tissue and lack of pigmentation, meaning that surface as well as sub-surface vasculature contribute to the measured fluorescence Prager et al. Furthermore, the density of capillaries is higher in the cortex though not homogenously so compared to the retina Cavaglia et al.
The inner retinal vessels have only two capillary beds, in the retinal nerve fiber layer and inner plexiform layer Paques et al. As more retinas were imaged compared to brains, this may have led to less variability in the retinal cohort. Inherent differences in blood vessel type, diameter, and number between each cranial window preparation is likely responsible for greater variability noted in the brain data.
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The retinal and cortical vasculature were similarly affected by systemic blood-neural barrier disruption, with an elevated relative offset, which is consistent with fluorescein leakage. There was little change in the rate of fluorescein filling Figure 5E. It is important to note that systemic blood pressure can also affect the offset. Given the small increase in blood pressure at 6 h Table 2 and the slightly lower blood pressure at 24 h, it is unlikely that differences in blood pressure account for the observed elevation of offset and rightward shift of the offset histogram Figure 5C post-DOC delivery.
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The elevated offset however, is consistent with previous work showing that DOC directly applied to the surface of the brain resulted in increased residual fluorescence with normal filling Prager et al. There is a clear indication for a more gradual build-up of DOC related injury in the brain compared to the retina.
One explanation for this difference may be because of the heterogeneous vascular beds included in the brain imaging. In particular, as a craniotomy was not performed, the dura and pial vasculature could have contributed to the observed leakage. Notably, two populations of pial vessels have been described: those with similar tight junctions to the cortical vasculature and those with gaps between endothelial cell membranes and thus a more permeable BBB Allt and Lawrenson, Pial vessels are also thought to have less astrocyte coverage than intraparenchymal cortical vessels Bundgaard, ; Stewart and Hayakawa, ; Cassella et al.
Given the above, one might expect that a substantial contribution from pial vessels would have the effect of increasing leakage in the brain which occurred at 24 h but not at 6 h post-DOC. We compared the fluorescence profiles of the pial and cortical vessels in our cohort by analysing distinct regions of interest Figure S5. The pial vasculature could be differentiated from cortical surface vessels as they reside above the cortical vasculature and the camera gave sufficient depth of focus to discern the difference.
Given this, it is unlikely that leakage from pial vessels accounts for the earlier leakage in the retina compared with the brain. However, pial leakage may make a later contribution to the higher offset seen later at 24 h.
The earlier leakage of fluorescein in the retina compared with cortical vessels following DOC administration suggests that the BRB is sensitive to systemic disruption of tight junctions. As the imaged retinal blood vessels were generally smaller, preferential earlier leakage is possibly seen in the retina simply due to smaller blood vessels being more susceptible to DOC-induced injury. However, it is also worth noting that we have only assessed leakage at 6 and 24 h, thus how much earlier leakage appears in retinal compared with intraparenchymal vessels remains to be identified.
Nevertheless, this shares some parallels with previous studies showing that retinal vascular abnormalities can be indicative of systemic diseases such as diabetes Wessels et al. Here we build on this idea to show that imaging the retinal vasculature may be a sensitive but simpler means to quantify injury to the cortical vasculature. It is worth considering that the elevated offset may have arisen from damage to the vasculature during surgery to create the cranial window. Whilst the skull thinning process is less invasive than a craniotomy, it can nevertheless cause some trauma to the surface vasculature Grutzendler et al.
Care was taken during surgery to avoid this, including frequent breaks and the provision of a constant stream of cooled saline to minimize friction and heat damage. Despite these precautions, some damage may have occurred during the surgical procedure. However, one might have expected that this type of trauma would manifest in relatively increased leakage at the 6 h time point which was not observed Figure 5G.
Our study has a number of limitations.
First, the compromised barriers were probed using fluorescein sodium salt MW Comparison of a range of fluorescent tracers with different molecular structures and sizes will further define the extent to which the BRB can be used as a surrogate for the BBB. Second, our imaging has been focused on surface cortical vessels, thus care must be taken in extrapolating similarities in retinal vessels to brain vessels other than those superficial on the brain surface. Although, these vessels share many similarities with deeper cerebral vessels in structure and in the way they are affected by bile salts Greenwood et al.
Another difference has been that two vascular beds have been compared in two separate cohorts of animals. Whilst it would be ideal to image the retina and brain of the same animal simultaneously to allow intra-animal comparisons and remove confounds such as different blood pressures, it was not logistically feasible using our camera system. Fortunately the average blood pressures for the retinal and cortical imaging groups were not significantly different, minimizing possible confounds.
Lastly, only male rats were used in this study to remove sex as a potential confound. This is due to inherent differences between male and female physiology, such that they may respond differently to the same pharmacological insult. However, this possibility requires further exploration to ascertain the exact differences that can occur. Care must be taken to rule out retinal conditions using additional signs and symptoms to avoid confounding this interpretation.
Overall, there are some clear advantages in imaging the retinal vasculature compared to the cortical vessels. First there is no need for any preparatory procedures i. Second, there is lower intra-animal variability for retinal vessels, as there is more homogeneity between the number and diameters of retinal arteries and veins between eyes.
Inter-animal variability is also lower as the vasculature is organized in a similar, spoke-like pattern, with two major inner retinal layers, with only minor variations Figure 1B. In contrast, there is more variability in blood vessel type and size observed in the intraparenchymal vessels Figure 1A. This is evident in the histograms Figure 2G where multiple peaks can be seen in the brain data, likely reflecting the different flow dynamics in differently sized blood vessels.
Third, retinal pigment epithelium limits fluorescence from the choroid, thus improving signal-to-noise characteristics for inner retinal vasculature. This allows for robust calculations of circulation times between retinal arteries and veins Bursell et al. In conclusion, we have developed a method to quantitatively compare fluorescein angiography dynamics in the retina and superficial cortical vessels. We show that systemic disruption of tight junctions results in vascular leakage in both tissues. Interestingly retinal leakage was detectable earlier than in the cortical vessels, thus suggesting that the eye may be a sensitive marker for pharmacological barrier disruption of the CNS.
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