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To investigate oxygen tension (PO2) changes in the retinal and choroidal vasculatures in response to visual stimulation by light flicker.
A previously developed optical section phosphorescence imaging system was used to measure PO2 separately in the retinal veins, arteries, and capillaries and in the choroid before and during light flicker. Imaging was performed in rats during light flicker at frequencies between 0 and 14 Hz. Light flicker–induced changes in the chorioretinal vasculature PO2 and arteriovenous PO2 differences were determined. Retinal arterial and venous PO2 were measured along blood vessels as a function of the distance from the optic nerve head.
Retinal arterial PO2 and arteriovenous PO2 differences increased with increasing light flicker at frequencies up to 10 Hz, after which no further increase was observed. Significant increases in retinal arterial PO2 (P = 0.009; n = 10) and in retinal capillary PO2 (P = 0.04, n = 10) were measured in response to light flicker at 10 Hz. Retinal arteriovenous PO2 differences during light flicker were significantly greater than differences before light flicker (P = 0.01; n = 10). Retinal arterial PO2 decreased significantly with increased distance from the optic nerve head (P ≤ 0.004), whereas retinal venous PO2 remained relatively unchanged (P ≥ 0.4).
Measurement of changes in the chorioretinal vasculature PO2 can potentially advance the understanding of oxygen dynamics in challenged physiological states and in animal models of human retinal diseases.
Oxygen consumption is a major factor in retinal metabolism. Oxygen is delivered to the retinal tissue by the choroidal and retinal circulations. The supply of oxygen is adapted according to the metabolic activity of the retinal tissue. Visual stimulation by light flicker increases the metabolic demand of the inner retina.1–5 Therefore, changes in the PO2 of the chorioretinal vascular beds are likely to be induced by light flicker, particularly in the retinal circulation, which supplies the inner retina. Light flicker–induced changes in the intravascular PO2 have been reported based on the blood oxygen level–dependent signals on magnetic resonance imaging, which only provide a combined measurement in all the chorioretinal vasculatures.6 Although optic nerve head PO2 increases from light flicker with phosphorescence imaging,7 PO2 changes in the retinal arteries, veins, capillaries, and the choroid have not been investigated.
Increased retinal blood vessel diameter and blood flow in response to light flicker, under normal physiological conditions, have been demonstrated,8–10 and reduced retinal hemodynamic response to flicker has been reported in patients with hypertension, glaucoma, and diabetes.11–13 According to the Fick principle, retinal tissue oxygen consumption equals the product of retinal blood flow and arteriovenous oxygen content difference.14 Therefore, measuring retinal intravascular PO2 separately in the artery and in the vein, coupled with the knowledge of blood flow, may provide a better means for assessing retinal oxygen consumption than blood flow measurement alone.
In the retina, a measurable efflux of oxygen from the arterial vasculature has been reported.15,16 Moreover a reduction in the arterial PO2 gradient along the vessel caused by increased blood flow has been demonstrated in isolated small arterial preparations.17 Therefore, arterial PO2 changes resulting from altered blood flow must be considered in interpreting the retinal intravascular PO2 changes in response to light flicker.
We have previously developed a technique for measuring PO2 separately in the retinal and choroidal vasculatures.18–20 The purpose of the present study was to measure PO2 changes in the chorioretinal vasculatures and to determine the retinal arteriovenous PO2 difference in response to light flicker. Given that arterial PO2 is influenced by oxygen diffusion,15,17 variations in retinal arterial PO2 along blood vessels in the absence of visual stimulation were also investigated.
Seventeen male Long Evans pigmented rats (each weighing 450–650 g) were used for this study. The animals were treated in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were anesthetized using ketamine (85 mg/kg intraperitoneally) and xylazine (3.5 mg/kg intraperitoneally). Anesthesia was maintained by intraperitoneal infusion of ketamine and xylazine at the rate of 0.5 mg/kg per minute and 0.02 mg/kg per minute, respectively. The pupils were dilated with 2.5% phenylephrine and 1% tropicamide. A molecular probe (Pd-porphyrin; Frontier Scientific, Logan, UT) was dissolved (12 mg/mL) in bovine serum albumin solution (60 mg/mL) and physiological saline buffered to a pH of 7.4, and injected intravenously (20 mg/kg) through a tongue vein. Before imaging, 1% hydroxypropyl methylcellulose was applied to the cornea, and a glass coverslip was placed on the cornea to eliminate its refractive power and to prevent corneal dehydration.
We have previously developed an optical section phosphorescence imaging system for quantitative measurement of PO2 in the chorioretinal vasculatures.20 Briefly, a laser beam (λ = 532 nm) was projected at an oblique angle on the retina after intravenous injection of porphyrin (an oxygen-sensitive molecular probe), and phosphorescence emission was imaged. The incident laser beam was not coaxial with the viewing axis; hence, choroidal and retinal vasculatures appeared laterally displaced according to their depth location on the phosphorescence optical section image. Because of the quenching of the probe phosphorescence emission by the surrounding oxygen, PO2 is related to phosphorescence lifetime according to the Stern-Volmer expression: PO2 = (τ0 / τ)/[1 + (κQ)(τ0)], where PO2 (mm Hg) is the oxygen tension, τ (μsec) is the phosphorescence lifetime, κQ (1/mm Hg μsec) is the quenching constant for the triplet-state phosphorescence probe, and τ0 is the lifetime in zero oxygen environment. Phosphorescence lifetime was measured with a frequency-domain approach by varying the phase relationship between the modulated excitation source and the sensitivity of the camera detector, as previously described.20,21
For visual stimulation, the illumination light from a slit lamp biomicroscope was used as the source for light flicker. A filter that transmitted light at a wavelength of 568 ± 5 nm (Edmund Industrial Optics, Barrington NJ) was placed in the slit lamp illumination housing to eliminate overlap with the phosphorescence emission and thus to permit imaging during light flicker. The light power was 110 µW at the pupil. A shutter was attached to a solenoid and placed in front of the illumination housing of the slit lamp biomicroscope to alter the flicker frequency. The solenoid was externally driven by a square wave generator (Tenma, Springboro, OH) to vary the flicker frequency between 2 and 14 Hz.
Each rat was placed in front of the imaging instrument. Laser power was adjusted to 100 µW, which is safe for viewing according to the American National Standard Institute for Safety Standards.22 Imaging was performed at single locations within 1 disk diameter from the edge of the optic nerve head, before and during light flicker at a frequency of 10 Hz (n = 10) and at varying frequencies (n = 3). Three sets of phase-delayed phosphorescence optical section images were acquired before light flicker was initiated, and three sets of images were acquired at the same location during light flicker, after 2 minutes of visual stimulation. In the absence of visual stimulation, imaging was performed at 20 spatially consecutive locations along a retinal artery and vein, spanning a distance of 2 disk diameters, beginning from a location near the edge of the optic nerve head (n = 4). Images were acquired at 6-second intervals, permitting resolution of physiological events that occurred within a factor of this time scale.
The set of phase-delayed phosphorescence optical section images was analyzed by relating the phosphorescence intensity and the experimentally varied phase delay between the modulated laser and camera sensitivity.20 Measurement of PO2 was based on the parameters that described the best-fit sinusoidal curve to the data points.21 The mean and SD of PO2 in the retinal arteries, veins, and capillaries and in the choroid were calculated. Analysis of variance (ANOVA; repeated measures) was performed to determine the effect of flicker frequency on retinal arterial and venous PO2 measurements. Paired Student t test was performed to compare measurements obtained before and during light flicker. Flicker-induced change in PO2 was determined by the following expression: (PO2during flicker − PO2before flicker) × 100/PO2before flicker. Linear regression analysis was performed to determine the significance of the association between retinal arterial (or venous) PO2 measurements and the distance from the optic nerve head. Statistical significance was accepted at P < 0.05.
The optimum light flicker frequency for altering PO2 in the retinal vasculatures was determined based on data obtained in three rats during light flicker at frequencies of 2, 6, 10, and 14 Hz. The relationship between retinal arterial (and venous) PO2 and light flicker frequency is depicted in Figure 1. Retinal arterial PO2 increased with increasing flicker frequencies up to 10 Hz, after which no further increase in PO2 was observed (P = 0.004). Retinal venous PO2 remained relatively constant at all frequencies (P = 0.5).
Before light flicker, the average PO2 readings in the retinal veins, arteries, and capillaries and in the choroid were 31 ± 5, 41 ± 5, 39 ± 5, and 55 ± 7 mm Hg (mean ± SD; n = 10), respectively. During light flicker at 10 Hz, the average PO2 readings in the retinal veins, arteries, and capillaries and in the choroid were 32 ± 5, 46 ± 8, 41 ± 7, and 54 ± 7 mmHg (n = 10), respectively. A significant increase in the retinal arterial PO2 (P = 0.009; n = 10) and the retinal capillary PO2 (P = 0.04; n = 10) was measured in response to light flicker. Retinal venous and choroidal PO2 did not vary significantly during light flicker (P > 0.05; n = 10). The percentage PO2 changes in the retinal veins, arteries, capillaries and in the choroid are shown in Figure 2. Flicker-induced PO2 changes were greatest in the retinal artery (13%) and capillaries (5%).
Retinal arteriovenous PO2 difference was measured within 1 disk diameter from the edge of the optic nerve head before and during light flicker, as shown in Figure 3. The retinal arteriovenous PO2 difference during flicker (14 ± 6 mm Hg) was significantly greater than the difference before flicker (9 ± 2 mm Hg; P = 0.01; n = 10).
Retinal arterial (and venous) PO2 was measured at 20 locations along the retinal arteries and veins over a distance spanning 2 disk diameters (approximately 600 µm).23 Results obtained in one rat are shown in Figure 4. Arterial PO2 decreased linearly with increased distance (slope, −0.04; y-intercept, 63), whereas venous PO2 remained relatively unchanged (slope, −0.004; y-intercept, 33). The significance of the association between PO2 in the retinal vasculature and distance from the optic nerve head was determined (Table 1). Retinal arterial PO2 decreased significantly with increased distance from the optic nerve head (P ≤ 0.004; n = 20), but retinal venous PO2 did not vary significantly with increased distance from the optic nerve head (P ≥ 0.4; n = 20).
In the present study, retinal arterial PO2 and capillary PO2 were significantly greater during light flicker than before flicker. No significant change in retinal venous PO2 or choroidal PO2 was observed. The arteriovenous PO2 difference measured before light flicker was consistent with previously reported values in rats21 and increased significantly in response to light flicker. Animals in the present study were allowed to breathe spontaneously under anesthesia and, hence, were likely to be hypoxemic, as reported in our previous studies.20 Although this might have altered the magnitude of the compensatory response, our results showed that the rats were capable of substantial retinal vascular compensation in response to light flicker.
Retinal arterial PO2 increased with each successive increment in flicker frequency until it reached a maximum at 10 Hz. No further increase was observed at the subsequent flicker frequency of 14 Hz. In mice24 and in rats (Qian H, personal communication, February 2006), the electrophysiological response to variable temporal frequencies of flicker stimulation has been shown to be maximal at a frequency similar to that at which the retinal arterial PO2 change was greatest. The corresponding flicker frequency at which maximal electrophysiological and intravascular PO2 responses have been observed in rats may be indicative of a relationship between retinal oxygenation and neuronal activity.
In the optic nerve head, a decrease in the optic nerve head tissue PO2 concurrent with an increase in blood flow has been demonstrated with flicker stimulation.25,26 Contrary to our results, venous PO2 measured by phosphorescence imaging was reported to increase at the optic nerve head during flicker.7 The change in the venous PO2 may be different because the optic nerve head veins receive blood from the retina, choroid, and the optic nerve head microvasculature and because oxygen consumption in response to flicker may differ between the retina and the optic nerve head tissues.
In the retina, a measurable efflux of oxygen from the pre-capillary vasculature has been demonstrated.15 The occurrence of diffusion of oxygen from the retinal arteries is further substantiated by the presence of periarterial capillary-free zones.27 Studies in isolated arterial preparations have shown that increased blood flow in small arteries and in arterioles results in a decrease in the efflux of oxygen per unit of blood volume and, consequently, a smaller longitudinal oxygen tension gradient. 17 In the present study, a decrease in the intravascular PO2 along the length of the retinal arteries, from proximal to distal, was measured, substantiating the presence of oxygen loss from the retinal arterial lumen. The presence of an increase in retinal blood flow in response to flicker stimulation8–10 and the flow-dependent gradient in the PO2 along the artery apparently explain the finding of increased retinal arterial PO2 in the present study.
Retinal tissue oxygen consumption is related to the product of the blood flow and the arteriovenous oxygen content difference.14 The observation of increased arteriovenous PO2 difference coupled with the previously reported increase in blood flow8–10 implies increased inner retinal oxygen consumption during visual stimulation by light flicker. Moreover, the finding that the increased arteriovenous PO2 difference in the present study was caused by a lack of change in the retinal venous PO2 and an increase in the arterial oxygen PO2 indicates that the effect of increased flow on retinal arterial PO2 is dominant in the regulation of oxygen supply to the inner retinal tissue during light flicker. Furthermore, this finding suggests at least a partial match between the increased oxygen consumption and supply. Retinal vascular PO2, as with the vascular PO2 of other tissue, depends on the balance between the amount of oxygen delivered and the amount consumed by tissue. During light flicker, inner retinal metabolic rates are increased,1,2 which causes reductions in retinal venous PO2. However, the vasodilatory response to light flicker increases retinal blood flow,8,25,28 resulting in increased retinal arterial PO2 that counteracts the effects of increased oxygen consumption on the retinal venous PO2. The arteriovenous PO2 difference may vary depending on the distance from the optic nerve head because the volume of measured tissue that is consuming oxygen changes. Therefore, the findings of the present study may be limited to locations 1 disk diameter from the optic nerve head edge.
Similar to findings of a previous study that reported no change in choroidal blood flow with flicker,28 the choroidal PO2 remained relatively unchanged during light flicker, supporting the selective effect of the light flicker on the inner retinal metabolic activity. This finding corroborates previous studies that have reported light flicker–induced changes in the electrophysiological response4,5 and glucose uptake3 of the inner retina, along with a minimal change in the outer retina.
In summary, increased retinal arterial and capillary PO2 caused by light flicker were demonstrated in this study. Measuring changes in the chorioretinal vasculature PO2 may advance the understanding of oxygen dynamics in challenged physiological states and in animal models of human retinal diseases.
Supported by the National Eye Institute Grants EY14917 (MS) and EY1792 (UIC) and by an unrestricted grant from Research to Prevent Blindness (UIC).
Disclosure: A. Shakoor, None; N.P. Blair, None; M. Mori, None; M. Shahidi, None