1. Introduction

The function of retinal tissue can be adversely affected by inadequate delivery and/or consumption of oxygen. Derangements in retinal oxygenation are thought to contribute significantly to the development of vision threatening eye diseases, including retinal vascular occlusion, diabetic retinopathy, retinopathy of prematurity, and glaucoma [1–4]. However, thus far, fundamental mechanisms that implicate oxygen in the development of retinal pathologies and impairment of retinal function are not completely understood. This is in part because measurement of oxygen consumption of the inner retina in vivo has been challenging.

The oxygen-sensitive microelectrode technique has provided direct measurements of inner retinal oxygen tension (PO₂) and oxygen consumption in the rat [5], though the assumption of one dimensional oxygen diffusion gradients from the retinal capillaries for estimation of oxygen consumption may not be valid. To circumvent the complicated three-dimensional oxygen diffusion gradients, inner retinal oxygen consumption has been estimated by occlusion of the retinal vascular supply and inspiration of 100% oxygen [6–8]. However, these measurements may not accurately reflect oxygen consumption under a normal physiological condition. Other methods have assessed inner retinal oxygen consumption in an indirect manner. Magnetic resonance imaging has provided measurements of retinal oxygenation in response to hyperoxic inhalation challenge, though with limited depth resolution for isolating the inner retinal response [9,10]. Cell labeling and microsphere imaging methods in animals [11–14] and Doppler velocimetry techniques in humans [15–18] have measured retinal blood flow, providing only limited information about the retinal oxygen metabolic function. Combined blood flow and oxygen saturation (SO₂) measurements for estimating metabolic rate of oxygen (MO₂) have been reported in human retina [19], and more recently in human brain and mouse ear [20,21]. We report an optical imaging technique for assessing inner retinal MO₂ of rat by combined measurements of retinal blood flow and vascular PO₂.

2. Methods

2.1. Metabolic Rate of Oxygen

Inner retinal MO₂ can be calculated from retinal blood flow and vascular oxygen content measurements, according to Fick’s principle [22]:

where F_vi is the blood flow in the i ^th of n veins, O_2a is the oxygen (O₂) content of arterial blood, and O_2vi is the O₂ content of blood in the i ^th vein. It was assumed O_2a is the same in all retinal arteries, and the total arterial and venous blood flows are identical. Blood flow was measured in veins because they are less affected by pulsation and have larger diameters as compared to arteries. Inner retinal MO₂ was measured both globally and locally for assessing the function of the entire retinal tissue (perfused by all retinal capillaries) and a region of retinal tissue near the optic disc (perfused by capillaries draining into several adjacent smaller veins (venules)), respectively. Global inner retinal MO₂ was derived by measuring PO₂ in all major retinal arteries and veins around the optic disc and F in all major retinal veins. Local inner retinal MO₂ was determined based on measurements of PO₂ in the major feeding artery, coupled with PO₂ and F measured in smaller draining venules.

2.2. Imaging

Vascular PO₂ was measured using our optical section phosphorescence lifetime imaging system [23,24], which combines principles of optical sectioning and phosphorescence lifetime imaging. Due to an angle between the incident laser light and imaging path, phosphorescence from structures in different retinal depths were imaged. Phosphorescence imaging was performed in 10 anesthetized Long Evans rats following intravenous injection of an oxygen-sensitive molecular probe (albumin bound Pd-porphine). Since phosphorescence emission was quenched by oxygen, its lifetime was a measure of PO₂. Phosphorescence lifetime was determined using a frequency-domain approach that involved incrementally varying the phase delay between modulated laser light and camera sensitivity. Lifetime was converted to PO₂ according to the Stern-Volmer expression: PO₂ = 1/(κ_Q τ) - 1/(κ_Q τ₀), where τ is the phosphorescence lifetime, κ_Q is the quenching constant for the triplet-state phosphorescence probe, and τ₀ is the lifetime in a zero oxygen environment.

For global inner retinal MO₂ calculation, PO₂ was measured in major retinal arteries and veins around the optic nerve head from cross-sectional phosphorescence images, according to our previously published methodology [24]. Three repeated PO₂ measurements were averaged in each major retinal artery and vein. For local inner retinal MO₂ calculation, an enface PO₂ map of a retinal area either temporal or nasal to the optic nerve head was generated by a laser scan, as described in our previous publication [23]. Global and local MO₂ measurements were obtained in 2 separate groups of 5 rats. The O₂ content of blood was determined as the sum of oxygen bound to hemoglobin and dissolved in blood [25]. The amount of oxygen bound to hemoglobin was calculated as the product of SO₂, the hemoglobin concentration of 14 g/dl (based on a measured mean of 14.4 ± 0.6 g/dl in 7 rats), and the oxygen carrying capacity of 1.39 mlO₂/g [26]. SO₂ was calculated from the measured PO₂ and blood pH values using the hemoglobin dissociation curve [27]. The amount of dissolved oxygen in blood was calculated as the product of oxygen solubility in blood (0.003 mlO₂/dl*mmHg) and PO₂ measurements._.

Blood velocity was measured by imaging intravascular motion of fluorescent microspheres [28], within 20 minutes of phosphorescence imaging. The method involved intravenous injection of 2 micron fluorescent spheres, while the retina was imaged using a prototype slitlamp biomicroscope. The slitlamp was equipped with a 488 nm diode laser for illumination and a 560 ± 60 nm emission filter placed in front of a charge-coupled device camera, which had high sensitivity and image acquisition rate. The movement of microspheres in the retinal vasculature was captured by a sequence of images acquired at a frame rate of 60 or 108 Hz. A higher acquisition rate was preferred for measuring higher blood velocity in the major retinal veins. Images were analyzed to measure the displacement of microspheres over time and derive blood velocity measurements. Blood velocity was determined based on measured velocities of 4 to 47 microspheres in major retinal veins and 1 to 10 microspheres in retinal venules. The number of microspheres visualized depended on the duration of image acquisition, and the concentration and volume of the injected microspheres, which were progressively optimized as the technique was developed. Blood vessel diameter was measured from red free retinal images for veins and from fluorescein angiograms for venules, which provided better contrast for visualization of the smaller blood vessels. Diameters were measured based on the full width at half maximum of an intensity profile of a line perpendicular to the centerline of the blood vessel, similar to our previous publication [29]. Blood vessel diameter was derived by averaging 3 to 38 measurements in major retinal veins and 2 to 19 measurements in retinal venules. The number of measurements was dependent on the length of the centerline that could be identified from the image of the blood vessel. F was calculated by the following equation: F = blood velocity*π*diameter²/4.

3. Results

3.1. Global Inner Retina MO₂

Figure 1a shows an example of a retinal image acquired in one rat, with major retinal arteries and veins labeled. An example of a retinal vein with edges outlined based on multiple diameter measurements is shown. The locations of a single microsphere traversing a major retinal vein as visualized over 8 consecutive images is depicted in Fig. 1b. A cross-sectional vascular PO₂ map overlaid on the retinal image is shown in Fig. 1c. Major retinal arteries and veins were differentiated based on the direction of F and level of PO₂.

 

Fig. 1 (a) Retinal image with major arteries and veins labeled (a and v); the outlined edges of a vein (v3) were identified by multiple diameter measurements; (b) Red crosses overlaid on the retinal image indicate the positions of a microsphere traversing a vein (v3), visualized over 8 consecutive images; (c) A cross-sectional vascular PO₂ map (rectangle) overlaid on the retinal image, depicting values in veins (v1, v2, v3) and arteries (a2, a3). Color bar displays PO₂ in mmHg.

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Measured blood vessel diameter, velocity, PO₂, and calculated F and O₂ content of blood in the major retinal veins of a rat (shown in Fig. 1) are listed in Table 1 . Since retinal arterial PO₂ is assumed to be constant, an average PO₂ over all major retinal arteries was calculated. Following Eq. (1), global inner retina MO₂ was calculated to be 639 nlO₂/min. Global inner retinal MO₂ in rats was on average 499 ± 117 nlO₂/min (N = 5).

Table 1. Vessel diameter, blood velocity, and blood flow in major retinal veins; PO₂ and O₂ content in major retinal veins and averaged over major retinal arteries (mean ± SD) in one rat

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For global MO₂ measurements, the standard deviations (SD) of repeated vessel diameter, blood velocity, and PO₂ measurements in the major retinal blood vessels were on average 3 microns, 6 mm/s, and 2 mmHg (N = 5), respectively. Using principles of propagation of error [30], the SD of MO₂ was estimated to be approximately 46 nlO₂/min, indicating changes of 90 nlO₂/min can be detected with 95% confidence.

3.2. Local Inner Retina MO₂

Figure 2a shows an example of a fluorescein angiogram acquired in one rat, with selected retinal blood vessels numbered and labeled. The positions of a microsphere traversing a venule and a major retinal vein, overlaid on a retinal image, are shown in Fig. 2b. In Fig. 2c, an enface PO₂ map of a segmented major retinal artery, vein, and venules displays values in pseudo color.

 

Fig. 2 (a) Fluorescein angiogram with draining retinal venules labeled; edges of a venule (v1) are outlined; (b) Red crosses overlaid on the retinal image indicate the positions of a microsphere traversing a venule (v1) and major vein, visualized over 8 consecutive images; (c) An enface PO₂ map depicting values in a segmented major retinal artery, vein, and venules. Color bar displays PO₂ in mmHg.

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Measured blood vessel diameter, velocity and calculated F in the draining venules connected to a major vein of one rat (shown in Fig. 2) are listed in Table 2 . Measured PO₂ and O₂ content of blood in the major retinal artery and venules of the same rat are also listed in Table 2. PO₂ in the artery supplying the retinal area was assumed to be constant and the same as in the branching arterioles, since the branching arterioles are directly supplied by the major artery. It is not valid to use PO₂ in the major vein to calculate the local inner retinal MO₂ because the vein has substantial oxygen contributions from peripheral retinal tissue. Local inner retinal MO₂ was calculated according to Eq. (1) to be 4.5 nlO₂/min. The range of local inner retinal MO₂ measurements was from 3.6 to 11.4 nlO₂/min (5.1 ± 3.6 nlO₂/min; N = 5). Local inner retinal MO₂ measurements vary among rats depending on the volume of the retinal tissue drained by the selected venules.

Table 2. Vessel diameter, blood velocity, and flow in retinal venules; PO₂ and O₂ content in retinal venules and a major retinal artery in one rat

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4. Discussion

The feasibility of our methodology to measure quantitatively retinal blood flow and oxygen tension and to determine inner retinal metabolic rate of oxygen in rats was demonstrated. Our measurements in major retinal veins were in agreement with previously reported blood velocity [11,31] and vessel diameter [32] measurements in rats. Since blood velocity was determined by averaging velocities of multiple microspheres in each blood vessel, it is likely a reasonable estimate of the mean of the distribution of blood velocities that occur due to laminar flow. PO₂ measurements in retinal arteries and veins were consistent with values obtained with microelectrodes near the surface of retinal blood vessels in rats [33]. For comparison to previously published studies of retinal oxygen consumption, global inner retina MO₂ measurements obtained in the present study were divided by the estimated mass of inner retinal tissue in rat [34], yielding an average inner retinal oxygen consumption of 7.1 mlO₂/min*100g. This value is somewhat higher than inner retinal oxygen consumption measured by determining arteriovenous oxygen differences and blood flow in separate groups of pigs (4.6 and 3.8 mlO₂/min*100g) [35,36]. It is also higher than inner retinal oxygen consumption measurements obtained by oxygen microelectrodes for only a selected part of inner retina of rat, converted to the same units (1.1 mlO₂/min*100g) [5], and with retinal artery occlusion and 100% oxygen inspiration in cat and rat (3.7 and 2.7 mlO₂/min*100g) [7,8]. Differences in estimated parameters, such as the mass of inner retinal tissue, may in part account for variations of oxygen consumption values reported by various techniques.

In future studies, our method will allow investigation of the relationships among retinal blood flow, vascular oxygen levels, and oxygen consumption under normal and challenged physiological conditions. For example, by examining the same retinal tissue with and without light flicker, alterations in global and local inner retinal MO₂ measurements can be evaluated. Such studies will improve understanding of neurovascular coupling under a normal physiological condition, as well as assessment of abnormalities due to pathological states. Overall, quantitative measurements of inner retinal MO₂ have the potential to advance knowledge of retinal oxygen dynamics in health and disease.