ODR, which correlates inversely to hemoglobin oxygen saturation, can be measured in the choroidal vasculature in lightly pigmented individuals with a spectrophotometric oximeter. Even though calculation of oxygen saturation from choroidal ODR has not been attempted, a low ODR is consistent with high oxygen saturation and high saturation in the choroid is in agreement with earlier studies on oxygenation in animals.
20,21 Shahidi et al.
20 and Shakoor et al.
21 used noninvasive phosphorescence imaging system to measure pO
2 in rat eyes and measured higher pO
2 in the choroid than in retinal arterioles under normal and increased oxygen breathing conditions; this agrees with our findings in the human eye.
While measuring the choroidal vessel ODR is new in this field, measurements on the retinal arterioles and venules is not new and our measurements on the retinal vessels are also in good agreement with previous studies made using an automated image analysis technique based on dual-wavelength oximetry similar to our technique.
7,22
Alm and Bill
2 found that the arteriovenous difference in oxygen content in the cat choroid is only 3%. This agrees with our experience that choroidal arterioles and venules are difficult to distinguish with spectrophotometric oximetry and that choroidal vortex veins only have slightly higher ODR (indication of lower oxygen saturation) than the other choroidal vessels (
Fig. 3).
The Oxymap T1 oximeter (Oxymap ehf., Reykjavik, Iceland) has been shown to be sensitive to changes in oxygen saturation in retinal vessels and to give repeatable and reliable results when measuring hemoglobin oxygen saturation and vessel diameter.
8,9,18,19 This is the first time it has been applied to choroidal vessels. By using the standard Oxymap T1 retinal calibration for the choroidal ODR the calculated hemoglobin oxygen saturation is 107 ± 12% (mean ± SD,
n = 16) for the choroidal vessels, 106 ± 13% for the vortex veins (mean ± SD,
n = 12), 94 ± 5% for retinal arterioles (mean ± SD,
n = 16), and 59 ± 9% (mean ± SD,
n = 16) for retinal venules. This calibration is obviously not appropriate for the vessels within the choroid, but the oxygen saturation for the retinal vessels compares well to our previous results on retinal oxygen saturation.
8,9 Subjects for this study were selected because their choroidal vasculature was visible (due to light pigmentation) and measureable. That does not seem to affect the results on oxygen saturation for the retinal arterioles and venules. Inhalation of 100% oxygen (hyperoxia,
n = 6) lowered the ODR levels for all measured vessel types, both choroidal and retinal, which corresponds to an increase in oxygen saturation. By using the standard retinal calibration, the increase in hemoglobin percentage was found to be 4% for choroidal vessels, 2% for retinal arterioles, and 26% for the retinal venules. This demonstrates that with hyperoxia the oximeter is sensitive to changes in oxygen saturation for both the choroidal and retinal vessels. (Vortex veins were not visible on images taken for the hyperoxia part of the study.)
We measured only individuals with the most visible choroidal vessels, which included only 16 individuals from a group of 148 healthy individuals. Of these, only six were available for the hyperoxia experiment. The small sample sizes may make the parametric statistical tests used vulnerable to deviations of the population from normal distribution. We therefore recalculated all P values using the Wilcoxon signed rank test. This did not change the conclusions of the study, although the difference between normoxia and hyperoxia in choroidal vessels became borderline statistically significant (P = 0.063), and the same was true for the comparison of retinal arterioles and choroidal vessels during hyperoxia (P = 0.059).
The repeatability of retinal oxygen measurements with the oximeter has been determined previously.
18 The standard deviation of repeated measurements was 1.0% for arterioles and 1.4% for venules. Repeatability was not tested in the same way in this study but can nonetheless be estimated. We calculated the standard deviation between measurements of the left and right eye in the same individual. This is displayed in ODR values in the results. If the ODR values are transformed into saturation (with standard retinal calibration), the standard deviation between measurements of the left and right eye in the same individual was 7% (
Fig. 5). Although the study was not designed to estimate repeatability, these values indicate that the variability is greater for the choroidal measurements than it is for retinal measurements.
The optical properties of the intravascular tissue in the choroid may play a role in these measurements. The ODR was lower in the choroidal vessels than in the retinal arterioles, potentially indicating higher oxygen saturation in the choroid; however, it is also observed that the OD of choroidal vessels is reduced at both 570 and 600 nm, which can affect the ODR and oxygen saturation conversion. This is compatible with scattering of light within the choroid reducing the contrast of choroidal vessels as follows. The OD of retinal vessels is measured against a bright background dominated by scattering from interstitial tissue. Choroidal blood vessels are embedded within this interstitial tissue and components of this tissue lying between blood vessels and the retinal pigment epithelium backscatter incident illumination, which reduces contrast of choroidal vessels; that is, it reduces OD. The magnitude of the scattering from interstitial tissue is approximately equal at both 570 and 600 nm but causes a proportionately greater reduction in the OD for the lower OD measurements at 600 nm. In consequence, there is a reduction in the ODR for choroidal vessels. If the reduction in ODR due to scattering is neglected, this would imply higher oxygenation levels and suggests nonrealistic oxygen saturation in excess of 100% in choroidal vessels. It is probable that the different illumination of the vessel by the surrounding choroidal tissue also has an effect, though this is expected to be less significant.
Different optical properties result from the fact that retinal vessels and choroidal vessels lie in different tissues at different depths. The result is that the standard calibration, which has been used for retinal vessels to transform ODR into oxygen saturation, is not appropriate for choroidal vessels. However, the lowering of ODR in the choroidal vessels with hyperoxia demonstrates that the oximeter is sensitive to changes in oxygen saturation in choroidal vessels as well as in retinal vessels.
It is furthermore observed that for some of the imaged retinas, the OD of choroidal vessels imaged at 600 nm is negative; that is, vessels appear brighter than the surrounding choroidal tissue, and this leads to the negative ODRs shown in
Figure 3. Study of these images suggests that this brightness is associated with diffuse structure in the scattering interstitial tissue that correlates with the vessel structure and that there is insufficient contrast to detect an OD due to the vessel. This may be because the vessels are located more deeply within the choroidal tissue than the vessels for which positive ODs can be measured. In these cases the ODR for these vessels is effectively zero.
The physical optics leading to the observed ODR of choroidal vessels is inherently different from that underpinning oximetry of retinal vessels. The determination of choroidal vessel oxygenation will require some modification and refinement to the physical optics model established for retinal vessel oximetry, and this is the subject of ongoing investigation. It is clear, however, from these results that it is nevertheless possible to detect changes in choroidal vessel oxygenation associated with changes in inspired oxygen.