May 2007
Volume 48, Issue 5
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Retina  |   May 2007
Intraretinal Oxygen Distribution and Consumption during Retinal Artery Occlusion and Graded Hyperoxic Ventilation in the Rat
Author Affiliations
  • Dao-Yi Yu
    From the Centre for Ophthalmology and Visual Science, The University of Western Australia, Nedlands, Australia.
  • Stephen J. Cringle
    From the Centre for Ophthalmology and Visual Science, The University of Western Australia, Nedlands, Australia.
  • Paula K. Yu
    From the Centre for Ophthalmology and Visual Science, The University of Western Australia, Nedlands, Australia.
  • Er-Ning Su
    From the Centre for Ophthalmology and Visual Science, The University of Western Australia, Nedlands, Australia.
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 2290-2296. doi:10.1167/iovs.06-1197
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      Dao-Yi Yu, Stephen J. Cringle, Paula K. Yu, Er-Ning Su; Intraretinal Oxygen Distribution and Consumption during Retinal Artery Occlusion and Graded Hyperoxic Ventilation in the Rat. Invest. Ophthalmol. Vis. Sci. 2007;48(5):2290-2296. doi: 10.1167/iovs.06-1197.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To determine intraretinal oxygen distribution and consumption in a rat model of retinal artery occlusion during air breathing and stepwise systemic hyperoxia.

methods. Laser occlusion of the pair of retinal arteries feeding the area of retina under investigation was performed. Oxygen-sensitive microelectrodes were then used to measure oxygen tension as a function of depth through the retina. Breathing mixtures were manipulated to produce stepwise increments in systemic oxygen levels, and the measurement of intraretinal oxygen distribution was repeated. Oxygen distribution in the retina was analyzed by an established eight-layer mathematical model of retinal oxygen consumption.

results. Intraretinal oxygen distribution in the occluded area confirmed that the choroid was the only source of retinal oxygenation. Under air-breathing conditions, the oxygen supply from the choroid was sufficient to support the photoreceptor inner segments. Any remaining oxygen was consumed by the outer plexiform layer. Increases in inspired oxygen level reduced the extent of inner retinal anoxia. However, some degree of anoxia in the innermost retina was usually present.

conclusions. Occlusion of the retinal circulation renders most of the inner retina anoxic. Ventilation with 100% oxygen does not generally avoid some degree of intraretinal anoxia. With 100% oxygen ventilation, the oxygen consumption of the inner retina was more than four times that of the outer retina. A marked degree of heterogeneity in oxygen uptake of different retinal layers was evident. The dominant oxygen consumers were the inner segments of the photoreceptors, the outer plexiform layer, and the inner plexiform layer.

Oxygen is the most critical metabolite required for retinal function in humans. 1 The retina in humans and most mammals receives its oxygen from a dual blood supply. The choroid, lying immediately behind the retina, supports the outer retina, and the retinal circulation primarily supports the inner retina. The delicate balance between oxygen supply and consumption in the retina places the retina at particular risk for ischemic damage, and retinal ischemia is thought to play a major role in many retinal diseases. Understanding the metabolic requirements of different cell types in the retina can offer vital information about which cell types are most at risk from ischemic or hypoxic insult. The avascular nature of the outer retina, coupled with microelectrode-based measurements of intraretinal oxygen distribution, has allowed the high oxygen metabolism of the outer retina to be identified. 2 3 4 5 However, the presence of retinal vasculature in the inner retina normally precludes such an analysis, 3 and the oxygen needs of most layers within the inner retina of vascularized retinas remain unknown. This has led to the widely held, but mistaken, view that the oxygen requirements of the retina as a whole are dominated by the photoreceptors. Previous studies in the cat used an occluder probe to shut down the retinal circulation to allow the inner retina to be included in oxygen consumption analysis after intraretinal oxygen distribution was measured during breathing of 100% oxygen. 6 7 8 In the present study, we used laser occlusion of the retinal circulation in rats, combined with stepwise increases in oxygen delivery from the choroid, to allow the oxygen consumption demands of the inner retinal layers to be quantified using a mathematical model that reflected the potential for different oxygen consumption rates in different layers of the inner retina. We chose the rat as an animal model because of its increasing popularity in ophthalmic research and because of the similarities in retinal oxygen supply and consumption between rat and monkey eye, 3 4 9 thereby potentially providing useful insights into the behavior of the human retina. 
Materials and Methods
Animal Preparation
Adult male Sprague–Dawley rats were housed in a light/dark cycle of 12 hours light (50 lux) and 12 hours dark. They were fed standard laboratory rat chow with water ad libitum. On the day of the experiment, each rat was anesthetized with an intraperitoneal injection of 100 mg/kg 5-ethyl-5-(1′-methyl-propyl)-2-thiobarbiturate (Inactin, Byk Gulden, Konstantz, West Germany). Atropine sulfate (20 mg) was administered intramuscularly to minimize salivation. The trachea was cannulated for mechanical ventilation, the left internal jugular vein was cannulated for injection of Rose bengal, and the femoral artery was cannulated for continuous blood pressure monitoring. The rat was then mounted prone in a modified Stellar stereotaxic system (model 51400; Stoelting Co., Wood Dale, IL), and the head was fixed in position. The rat was artificially ventilated (model 683; Harvard rodent respirator; Harvard Apparatus, Holliston, MA) with a ventilation rate of 90 breaths/min and the tidal volume set to the manufacturer’s recommendations based on body weight. Rectal temperature was monitored and maintained at 37.5°C by a homeothermic blanket (Harvard Apparatus). All procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Laser-Induced Retinal Artery Occlusion and Ocular Surgery
The left eye was used for all experiments. The pupil was dilated with 1% tropicamide (Mydriacyl; Alcon, Frenchs Forest, NSW, Australia). An eye ring was sutured to the conjunctiva at the limbus and fixed to the stereotaxic framework. To allow insertion of the oxygen-sensitive microelectrode, a small incision was made in the superior nasal quadrant with a diamond knife, just posterior to the limbus. Damage to the larger choroidal vessels or the posterior lens capsule was avoided. A plano-concave contact lens was placed on the cornea to allow the vitreous and the fundus to be visualized under an operating microscope during all intraocular manipulations. After intravenous injection of 100 μL 1% Rose bengal (Minims; Smith and Nephew, Melbourne, VIC, Australia), laser occlusion of the pair of retinal arteries feeding the area of interest was performed using a 532 nm diode laser and delivery adaptor (OcuLight GL; Iridex Corp., Mountain View, CA) combined with an operation microscope (OPMI-6; Zeiss, Oberkochen, Germany). The laser spot size was set to 75 μm. Throughout the remainder of the experiment, occlusion was verified by a stationary or an empty blood column in the arteries that previously supplied the area under investigation. 
Intraretinal Oxygen Profiles
Microelectrode techniques were similar to those reported in our earlier studies in rats. 3 9 10 11 Whalen-type recessed oxygen-sensitive microelectrodes 12 were manufactured and calibrated in our own laboratory. The microelectrode entered the eye through the entry hole, which was also the locus of rotation of our microsurgical system, such that rotation of the positioning system pivoted about the entry point into the eye. 13 The small size of the electrode tip (1 μm), coupled with electrode beveling techniques and the high acceleration piezoelectric translation of the electrode, produced highly reproducible measurements of intraretinal oxygen distribution. Intraretinal oxygen profiles were measured in the inferior retina, approximately 2 to 3 disk diameters from the disk margin, in an area previously supplied by the occluded retinal arteries. The measurement site was therefore remote from the laser-treated area, minimizing the likelihood of direct laser damage to the retina or choroid in the measurement area. The electrode tip was placed at the surface of the chosen area of retina under observation. The electrode was then stepped through the retina (10-μm steps), under computer control, until a peak oxygen level within the choroid was reached. The measurement was repeated during stepwise withdrawal of the electrode. Although close agreement between the insertion and withdrawal profiles was routinely achieved, withdrawal profiles were used for data analysis because they tended to be less influenced by artifacts associated with mechanical stress on the electrode tip during penetration. Oxygen tension measured by the microelectrode and systemic conditions, such as arterial blood pressure and rectal temperature, were recorded continuously on an eight-channel chart recorder (LR8100; Yokogawa, Tokyo, Japan). Readings of each channel were also accessed by way of computer interface (GPIB-IEEE), and data were logged directly to a spreadsheet along with the relative position of the microelectrode. Typically, only one oxygen profile measurement was made at each ventilation condition. However, if a significant “noise” artifact occurred during measurement, the measurement was repeated before the next ventilation level. All experiments were performed under photopic conditions (55 μW at the cornea). 
Systemic Conditions
Ventilation mixtures were selected in increasing percentages of oxygen, in increments of 20%, from 20% to 100%. Intraretinal oxygen measurements were repeated under each ventilatory condition. Experiments usually lasted 8 hours, after which the rat was humanely killed with an overdose of anesthesia. 
Oxygen Consumption Model
An eight-layer model based on the Fick law of diffusion was used for the oxygen-consumption analysis. This model was developed with the highly layered structure of the retina in mind and has been used to quantify oxygen consumption in the rat under normoxic and hyperoxic conditions. 3 14 The present use of the model is more straightforward because only a single source of retinal oxygenation, the choroid, had to be considered. Under these conditions, we considered only the oxygen tension at the outer retinal boundary and the oxygen consumption rate of the specific retinal layers of interest. Based on previous knowledge, the oxygen consumption rates of some retinal layers (Q1, Q3, and Q5) were assumed to be zero. These corresponded to the outer segments of the photoreceptors, the outer nuclear layer, and the inner nuclear layer, respectively. The oxygen-consuming layers corresponded to the inner segments of the photoreceptors (Q2), the outer plexiform layer (Q4), the deeper portion of the inner plexiform layer (Q6), the more superficial region of the inner plexiform layer (Q7), and the superficial retina containing the ganglion cell/nerve fiber layer (Q8). The total oxygen consumption in any particular retinal layer is determined by the product of the local oxygen consumption rate (nL O2/min/cm3) and the thickness of the layer (cm), thereby giving oxygen consumption in terms of nL O2/min/cm2, as in previous applications of similar models. 3 4 11 15 16 17 Full justification for the principles involved in the derivation of a model applicable to an avascular (or occluded) retina are given elsewhere. 18  
Cytochrome Oxidase Staining
The retina was lightly fixed for 1 hour at 4°C and was immersed in increasing grades of sucrose-phosphate buffer (10%, 20%, and 30% sucrose in 0.1 M phosphate buffer) solution over 2 days until it sank. The tissue was then snap-frozen in optimal cutting temperature compound and cryosectioned at 10 μm. Sections were incubated at 37°C in the dark for 1 to 2 hours and checked every 30 minutes for dark brown reaction products. The incubation medium was made fresh immediately before incubation and consisted of 3,3′-diaminobenzidine (50 mg), 0.1 M phosphate buffer (pH 7.4; 90 mL); cytochrome c (type III, 15–30 mg; Sigma, St. Louis, MO), and sucrose (4 g). Incubation was stopped in 0.1 M phosphate buffer once clear differentiation between highly reactive and nonreactive portions could be discerned. Sections were dehydrated through increasing grades of alcohol and immersed in xylene before they were coverslipped in DPX. 
Statistical Analysis
All average values are stated as means ± SE. All statistical testing was performed using a statistics program (SigmaStat; Jandel Scientific Software, San Raphael, CA). Significant differences were determined using Student’s t-test or one-way ANOVA, with P < 0.05 accepted as significant, and regression curve fits were performed (SigmaPlot; Jandel Scientific Software). 
Results
Laser occlusion of the chosen retinal arteries was able to routinely stop blood flow to the area of retina under investigation. Stasis of blood in the arteries was verified throughout the experiment. Figure 1ashows a normal rat fundus before laser occlusion at the sites indicated by the dark arrows. The typical radial pattern of alternating arteries (a) and veins (v) is seen. After laser treatment (Fig. 1b) , the treated arteries were either cleared of blood or had segmented blood remaining with no visible flow (white arrows). A typical intraretinal oxygen profile in the occluded region is shown in Figure 2(filled circles). The best fit (solid line) of the data to the multilayer mathematical model of oxygen consumption is shown superimposed on the original data. Data were only fitted for the avascular portion of the profile, up to the level of Bruch membrane and the choriocapillaris. Also shown for easy comparison are the average data from a previous study of intraretinal oxygen distribution in air-breathing rats without retinal occlusion. 14 It became evident that after retinal occlusion, the choroid was the only source of retinal oxygenation, and that under these air breathing conditions retinal occlusion rendered much of the inner retina anoxic. Visual examination of the oxygen profile provides a guide to the local oxygen consumption because the intraretinal oxygen gradient changes most rapidly where the local oxygen consumption rate is highest. The first major change of gradient, at approximately 300-μm track distance, corresponds to the inner segments of the photoreceptors. Whatever oxygen flows through this region is consumed by the outer plexiform layer (approximately 230-μm track distance). 
Averaged oxygen profiles from occluded retinas (n = 10) are shown in Figure 3afor a range of increasing levels of inspired oxygen (20%–100% oxygen). With 20% oxygen ventilation, the choroidal oxygen supply was sufficient to avoid any anoxia in the outer retina, where the dominant oxygen-consuming layer was the inner segments of the photoreceptors. However, any oxygen diffusing across the outer retina was consumed by the outer plexiform layer, and anoxic regions were present in this and all the proximal retinal layers. Choroidal oxygen level rose with each increment in inspired oxygen level (P < 0.05). This increase in choroidal oxygen level increased the range of choroidally delivered oxygen to include more of the inner retina with each elevation in choroidal Po 2. Thus, as choroidal oxygen tension increased, the extent of inner retinal anoxia decreased. However, even with 100% oxygen ventilation, which produced an average choroidal Po 2 in excess of 300 mm Hg, the average oxygen tension in the innermost region of the inner retina was close to zero. The potential for different levels of oxygen consumption in specific retinal layers was indicated by the uneven distribution of cytochrome oxidase across the rat retina (Fig. 3b) . Relatively dense layers of cytochrome oxidase staining were seen in the inner segments of the photoreceptors, the outer plexiform layer, and the inner plexiform layer. 
Actual oxygen consumption rates of the individual retinal layers were calculated from mathematical analysis of the individual oxygen profiles in each case. Average values for oxygen consumption are shown in Figure 4for each level of inspired oxygen ventilation. With 20% oxygen ventilation, the oxygen consumption was dominated by the inner segments of the photoreceptors (Q2 = 142 ± 20 nL O2/min/cm2) and the outer plexiform layer (Q4 = 91.0 ± 9.7 nL O2/min/cm2), with the inner segments the significantly larger consumer (P < 0.05). With 40% oxygen ventilation, almost all the additional oxygen supplied by the choroid was consumed by the outer plexiform layer, which had an oxygen consumption rate not significantly different from that of the inner segments (P = 0.107). With 60% oxygen ventilation, the oxygen consumption rate of the outer plexiform layer increased further, but more oxygen was able to pass through the more proximal retina, and the oxygen uptake increased significantly (P < 0.05) in the deeper region of the inner plexiform layer (Q6) but not significantly (P = 0.125) in the more superficial region of the inner plexiform layer (Q7). With 80% oxygen ventilation, no further increase occurred in oxygen consumption in the outer plexiform layer (P = 0.467), but the oxygen consumption of the deeper region of the inner plexiform layer increased significantly (P < 0.05). With 100% oxygen ventilation, only the oxygen consumption of the innermost retina (Q8) increased significantly from the 80% oxygen ventilation level. Thus, the general pattern was for oxygen consumption in the inner retinal layers to increase as more oxygen became available from the choroid. In contrast, the oxygen uptake of the outer retina (Q2) showed no relationship to inspired oxygen level (P = 0.729). 
Figure 5shows the summed result of total inner and outer retinal oxygen consumption as a function of inspired oxygen level. Inner retinal oxygen consumption was highly dependent on oxygen ventilation level. With 20% oxygen ventilation, the outer retinal oxygen consumption (142 ± 20 nL O2/min/cm2) was significantly higher (P < 0.05) than the total inner retinal oxygen consumption (91.3 ± 9.5 nL O2/min/cm2). At 40% inspired oxygen, inner retinal oxygen consumption increased significantly (P < 0.001) and was not significantly different from that of outer retinal oxygen consumption (P = 0.065). At 60% and greater oxygen ventilation, total oxygen consumption of the inner retina was significantly greater that that of the outer retina (all P < 0.001). With 100% oxygen ventilation, the average inner retinal oxygen consumption (449 ± 40 nL O2/min/cm2) was more than four times that of the outer retina (95 ± 44 nL O2/min/cm2). 
Discussion
Oxygen requirements of the inner retina are less understood than those of the outer retinal layers, 4 reflecting the added complexities in studying inner retinal metabolism in vivo. However, understanding the oxygen requirements of the inner retina is important for elucidating the role of retinal hypoxia in diseases such as diabetic retinopathy, macular edema, retinopathy of prematurity, retinal vascular occlusion, and retinal detachment, in which therapeutic avenues for the improvement in inner retinal oxygen status are either in common clinical use or under investigation. Examples include laser therapy for the treatment of retinal neovascularization, 19 improved control of systemic oxygen levels to avoid retinopathy of prematurity in neonates, 20 and use of systemic hyperoxia in patients with diabetic macular edema 21 and retinal detachment. 22 In the outer retina, which is avascular, oxygen distribution can be analyzed to determine local oxygen consumption. 5 However, in the inner retina, the presence of the retinal vasculature precludes such simple analysis. Studying animals with naturally avascular retinas allows the inner retina to be included in the oxygen consumption analysis, but the extremely low oxygen uptake of the inner retina in these species 15 16 23 makes them of limited value for understanding the oxygen requirements of the inner retina in vascularized species. We already have indirect evidence that the inner retina in vascularized retinas has a need for high oxygen consumption that increases in hyperoxic conditions. We have previously demonstrated that the inner plexiform layer has a consistent minimum level of oxygen 9 and that this is maintained during systemic hyperoxia, 10 when oxygen delivery from the choroid increases dramatically. This behavior is very different from that in avascular retinas, in which inner retinal oxygen consumption is low, 15 23 and increases in choroidal oxygen level create equivalent increases in oxygen level in all layers of the inner retina. 16 24 Consequently, species with avascular retinas are not likely to provide a good model for inner retinal oxygen requirements in vascularized retinas. 
In vitro studies of retinal oxygen metabolism in the rat have previously used pharmacologic blockade of inner retinal function or models of dystrophic outer retinas to estimate the relative oxygen requirements of the inner and outer retina. 25 26 These studies demonstrate greater balance between inner and outer retinal oxygen requirements than found in the present study; however, intraretinal oxygen levels in those studies were unknown, and the presence of intraretinal anoxia limiting the extent of retinal oxygen consumption cannot be ruled out. 
In the present study, we have used laser-induced retinal arterial occlusion in the rat to eliminate the influence of the retinal circulation, and we examined intraretinal oxygen distribution and oxygen consumption in all retinal layers. In a preliminary study, we established the feasibility of shutting down the retinal vasculature by laser occlusion and increasing oxygen supply from the choroid to compensate for the loss of oxygen supply from the retinal circulation. 27 A previous study in cats treated the inner retina as a homogenous oxygen consumption zone. 8 We think that when some layers of the inner retina are anoxic, 24 28 or when heterogeneity in oxygen metabolism may be expected, a multilayer approach to inner retinal oxygen metabolism may be warranted. We have confirmed that after occlusion of the retinal circulation, the choroid is the only source of retinal oxygenation. In addition, we have confirmed that under air-breathing conditions, the choroid is able to supply all the oxygen needs of the inner segments of the photoreceptors but only a small proportion of the oxygen needs of the inner retina. In fact, only some of the oxygen needs of the outer plexiform layer are supported by the choroid during retinal occlusion under air-breathing conditions. The potentially high oxygen demand of the outer plexiform layer is further evident with moderate systemic hyperoxia (40% O2), where the oxygen uptake of the outer plexiform layer exceeds that of the outer retina. With still higher levels of systemic hyperoxia, the choroidal delivery of oxygen increases and oxygen penetrates greater distances into the inner retina. The oxygen uptake of the inner plexiform layer then becomes apparent. Layers Q6 and Q7 correspond to the deeper and more superficial regions of the inner plexiform layer, respectively. 14 In the present study, the maximum oxygen consumption rate of the innermost retinal layer containing the ganglion cells may be an underestimate because of the presence of some degree of anoxia in this region, even with 100% oxygen ventilation. We have no reason to believe the choroid was damaged by the laser treatment. The measurement site was remote from the laser treated area, and the choroidal Po 2 values in the choroid were not lower than we have previously reported in healthy rats without laser treatment. 10  
Thus, these findings from the occluded retina supported our earlier work suggesting that there are at least three zones of high oxygen requirements in the rat retina, namely the inner segments of the photoreceptors, the outer plexiform layer, and the inner plexiform layer. 27  
This result is consistent with our cytochrome oxidase staining of the normal rat retina (Fig. 3b) . The high oxygen demands of the plexiform layers probably reflect the metabolic requirements of synaptic activity. This finding may also be relevant to the brain, where significantly more complex architecture makes it more difficult to identify the metabolic requirements of different cell types. The major part of energy consumption in the retina and the brain serves to maintain and restore ionic gradients. 29 In diseased conditions, such as blood flow deficits, low ATP levels, ionic disruption and metabolic failure are severe, and cell death progresses within the ischemic territory. 30  
Another way of quantitatively addressing the oxygen requirements of some layers within the inner retina is offered by the rat retina, in which the inner plexiform layer has relatively few retinal capillaries and can be treated as an essentially avascular region sandwiched between capillary beds in the superficial retina and the outer plexiform layer. 3 11 14 Such an analysis of the intraretinal oxygen distribution in the in vivo rat has identified a high level of oxygen consumption in the inner plexiform layer 3 that increases significantly with hyperoxic ventilation. 14 However, the limitations imposed by the presence of capillaries in the outer plexiform layer and in the superficial retinal layer have previously precluded quantitative analysis of the oxygen requirements of these layers. Occlusion of the retinal circulation and increased oxygen supply from the choroid have the potential to allow the oxygen requirements of all retinal layers to be quantified. 
Previous studies in animal models of retinal ischemia have provided conflicting results with regard to the feasibility of supplying the oxygen requirements of the full thickness of the ischemic retina from the choroid. 31 32 33 34 35 36 37 This may point to the importance of duration of the ischemic insult, which is often not reported. Longer term insults may lead to loss of retinal function, reduction in oxygen uptake, and ability of choroidally delivered oxygen to support the full thickness of the ischemic retina. 
It is unlikely that the high oxygen requirement of the inner retina under hyperoxic conditions is an artifact resulting from the occlusion of retinal blood flow. In a normally perfused retina, a significant arteriovenous oxygen difference is observed, indicating oxygen delivery from the retinal circulation, even under hyperoxic ventilation conditions. Furthermore, in the intact retina, oxygen delivery from the choroid has been shown to increase almost fourfold during systemic hyperoxia, which, coupled with the stability of outer retinal oxygen consumption during systemic hyperoxia, suggests that inner retinal oxygen consumption increases dramatically during systemic hyperoxia in the normal retina. 14 Thus, the increase in inner retinal oxygen metabolism is not confined to a case in which extra oxygen from the choroid relieves inner retinal anoxia; rather, it is also apparent in the inner retina in which there is no anoxic insult. Anaerobic metabolism is known to exist in the rat inner retina. 38 When environmental oxygen levels are raised, a switch to more oxidative metabolism in the plexiform layers may be responsible for this effect. We have shown that the synaptic layers of the inner retina have a particularly high rate of oxygen metabolism, which may provide new insight regarding the vulnerability of the inner retina to ischemic or hypoxic insult. The use of hyperoxic ventilation in this acute model of arterial occlusion partially overcame the intraretinal anoxia seen in the inner retina, with successive increases in choroidal oxygen tension confining the anoxic zone to the more proximal retinal layers. However, even with 100% oxygen ventilation, complete relief from intraretinal anoxia could not be guaranteed. Under dark-adapted conditions, when the outer retina of the rat consumes approximately 50% more oxygen, 3 39 supporting the oxygen requirements of the full thickness of the occluded retina from the choroid would be even more difficult. 
 
Figure 1.
 
Fundus photographs of a normal rat retina (a) showing the radial distribution of retinal arteries (a) and veins (v) before the application of laser photocoagulation of the pair of retinal arteries feeding the area in which intraretinal oxygen profiles were subsequently measured. Chosen sites for laser occlusion are marked by the black arrowheads. After laser photocoagulation (b) the arteries have either disappeared or have a stationary blood column indicating occlusion of blood flow (white arrows).
Figure 1.
 
Fundus photographs of a normal rat retina (a) showing the radial distribution of retinal arteries (a) and veins (v) before the application of laser photocoagulation of the pair of retinal arteries feeding the area in which intraretinal oxygen profiles were subsequently measured. Chosen sites for laser occlusion are marked by the black arrowheads. After laser photocoagulation (b) the arteries have either disappeared or have a stationary blood column indicating occlusion of blood flow (white arrows).
Figure 2.
 
An example of an intraretinal oxygen measurement after laser occlusion of the retinal circulation (filled circles). The black line represents the best fit (R 2 = 0.998) of the oxygen profile to a mathematical model that determines the rate of oxygen consumption in particular retinal layers. Only data points to the outer retinal boundary (at 350-μm track length) are fitted to the model. Also shown for comparison (empty circles) is the average oxygen distribution in an earlier study in normal rats. 14 It is apparent that the laser occlusion of the retinal arteries has shut down any oxygen delivery from the retinal circulation. The only oxygen available to the retina now came from the choroid. Under the air-breathing conditions shown here, this was sufficient to supply oxygen to the inner segments of the photoreceptors (at approximately 300-μm track length), with the remaining oxygen supply consumed by the outer plexiform layer (at approximately 230 μm). The remainder of the inner retina was anoxic.
Figure 2.
 
An example of an intraretinal oxygen measurement after laser occlusion of the retinal circulation (filled circles). The black line represents the best fit (R 2 = 0.998) of the oxygen profile to a mathematical model that determines the rate of oxygen consumption in particular retinal layers. Only data points to the outer retinal boundary (at 350-μm track length) are fitted to the model. Also shown for comparison (empty circles) is the average oxygen distribution in an earlier study in normal rats. 14 It is apparent that the laser occlusion of the retinal arteries has shut down any oxygen delivery from the retinal circulation. The only oxygen available to the retina now came from the choroid. Under the air-breathing conditions shown here, this was sufficient to supply oxygen to the inner segments of the photoreceptors (at approximately 300-μm track length), with the remaining oxygen supply consumed by the outer plexiform layer (at approximately 230 μm). The remainder of the inner retina was anoxic.
Figure 3.
 
(a) Effect of increasing oxygen from the choroid to the retina. Average (n= 10) intraretinal oxygen distribution in occluded retinas is shown for different levels of inspired oxygen (20%, 40%, 60%, 80%, and 100% oxygen). Each increment in inspired oxygen level allows the choroid to deliver more oxygen to the inner retina; however, even with 100% oxygen ventilation, the average oxygen tension in the innermost retina is close to zero. (b) Uneven distribution of cytochrome oxidase across the rat retina. Relatively dense layers of cytochrome oxidase staining are seen in the inner segments of the photoreceptors, the outer plexiform layer, and the inner plexiform layer. A schematic of the starting position of each layer in the model is shown below the retinal section. Scale bar, 50 μm.
Figure 3.
 
(a) Effect of increasing oxygen from the choroid to the retina. Average (n= 10) intraretinal oxygen distribution in occluded retinas is shown for different levels of inspired oxygen (20%, 40%, 60%, 80%, and 100% oxygen). Each increment in inspired oxygen level allows the choroid to deliver more oxygen to the inner retina; however, even with 100% oxygen ventilation, the average oxygen tension in the innermost retina is close to zero. (b) Uneven distribution of cytochrome oxidase across the rat retina. Relatively dense layers of cytochrome oxidase staining are seen in the inner segments of the photoreceptors, the outer plexiform layer, and the inner plexiform layer. A schematic of the starting position of each layer in the model is shown below the retinal section. Scale bar, 50 μm.
Figure 4.
 
The result of applying the oxygen consumption model to each individual oxygen profile is shown in terms of oxygen consumption in each layer as a function of inspired oxygen percentage. As more oxygen was made available with hyperoxic ventilation, the oxygen consumption of the inner retina increased, and oxygen consumption was detected in the more proximal retinal layers.
Figure 4.
 
The result of applying the oxygen consumption model to each individual oxygen profile is shown in terms of oxygen consumption in each layer as a function of inspired oxygen percentage. As more oxygen was made available with hyperoxic ventilation, the oxygen consumption of the inner retina increased, and oxygen consumption was detected in the more proximal retinal layers.
Figure 5.
 
Average oxygen consumption of the inner and outer retina as a function of inspired oxygen level. With high levels of inspired oxygen, the oxygen consumption of the inner retina became many times that of the outer retina.
Figure 5.
 
Average oxygen consumption of the inner and outer retina as a function of inspired oxygen level. With high levels of inspired oxygen, the oxygen consumption of the inner retina became many times that of the outer retina.
The authors thank Dean Darcey for his expert technical assistance. 
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Figure 1.
 
Fundus photographs of a normal rat retina (a) showing the radial distribution of retinal arteries (a) and veins (v) before the application of laser photocoagulation of the pair of retinal arteries feeding the area in which intraretinal oxygen profiles were subsequently measured. Chosen sites for laser occlusion are marked by the black arrowheads. After laser photocoagulation (b) the arteries have either disappeared or have a stationary blood column indicating occlusion of blood flow (white arrows).
Figure 1.
 
Fundus photographs of a normal rat retina (a) showing the radial distribution of retinal arteries (a) and veins (v) before the application of laser photocoagulation of the pair of retinal arteries feeding the area in which intraretinal oxygen profiles were subsequently measured. Chosen sites for laser occlusion are marked by the black arrowheads. After laser photocoagulation (b) the arteries have either disappeared or have a stationary blood column indicating occlusion of blood flow (white arrows).
Figure 2.
 
An example of an intraretinal oxygen measurement after laser occlusion of the retinal circulation (filled circles). The black line represents the best fit (R 2 = 0.998) of the oxygen profile to a mathematical model that determines the rate of oxygen consumption in particular retinal layers. Only data points to the outer retinal boundary (at 350-μm track length) are fitted to the model. Also shown for comparison (empty circles) is the average oxygen distribution in an earlier study in normal rats. 14 It is apparent that the laser occlusion of the retinal arteries has shut down any oxygen delivery from the retinal circulation. The only oxygen available to the retina now came from the choroid. Under the air-breathing conditions shown here, this was sufficient to supply oxygen to the inner segments of the photoreceptors (at approximately 300-μm track length), with the remaining oxygen supply consumed by the outer plexiform layer (at approximately 230 μm). The remainder of the inner retina was anoxic.
Figure 2.
 
An example of an intraretinal oxygen measurement after laser occlusion of the retinal circulation (filled circles). The black line represents the best fit (R 2 = 0.998) of the oxygen profile to a mathematical model that determines the rate of oxygen consumption in particular retinal layers. Only data points to the outer retinal boundary (at 350-μm track length) are fitted to the model. Also shown for comparison (empty circles) is the average oxygen distribution in an earlier study in normal rats. 14 It is apparent that the laser occlusion of the retinal arteries has shut down any oxygen delivery from the retinal circulation. The only oxygen available to the retina now came from the choroid. Under the air-breathing conditions shown here, this was sufficient to supply oxygen to the inner segments of the photoreceptors (at approximately 300-μm track length), with the remaining oxygen supply consumed by the outer plexiform layer (at approximately 230 μm). The remainder of the inner retina was anoxic.
Figure 3.
 
(a) Effect of increasing oxygen from the choroid to the retina. Average (n= 10) intraretinal oxygen distribution in occluded retinas is shown for different levels of inspired oxygen (20%, 40%, 60%, 80%, and 100% oxygen). Each increment in inspired oxygen level allows the choroid to deliver more oxygen to the inner retina; however, even with 100% oxygen ventilation, the average oxygen tension in the innermost retina is close to zero. (b) Uneven distribution of cytochrome oxidase across the rat retina. Relatively dense layers of cytochrome oxidase staining are seen in the inner segments of the photoreceptors, the outer plexiform layer, and the inner plexiform layer. A schematic of the starting position of each layer in the model is shown below the retinal section. Scale bar, 50 μm.
Figure 3.
 
(a) Effect of increasing oxygen from the choroid to the retina. Average (n= 10) intraretinal oxygen distribution in occluded retinas is shown for different levels of inspired oxygen (20%, 40%, 60%, 80%, and 100% oxygen). Each increment in inspired oxygen level allows the choroid to deliver more oxygen to the inner retina; however, even with 100% oxygen ventilation, the average oxygen tension in the innermost retina is close to zero. (b) Uneven distribution of cytochrome oxidase across the rat retina. Relatively dense layers of cytochrome oxidase staining are seen in the inner segments of the photoreceptors, the outer plexiform layer, and the inner plexiform layer. A schematic of the starting position of each layer in the model is shown below the retinal section. Scale bar, 50 μm.
Figure 4.
 
The result of applying the oxygen consumption model to each individual oxygen profile is shown in terms of oxygen consumption in each layer as a function of inspired oxygen percentage. As more oxygen was made available with hyperoxic ventilation, the oxygen consumption of the inner retina increased, and oxygen consumption was detected in the more proximal retinal layers.
Figure 4.
 
The result of applying the oxygen consumption model to each individual oxygen profile is shown in terms of oxygen consumption in each layer as a function of inspired oxygen percentage. As more oxygen was made available with hyperoxic ventilation, the oxygen consumption of the inner retina increased, and oxygen consumption was detected in the more proximal retinal layers.
Figure 5.
 
Average oxygen consumption of the inner and outer retina as a function of inspired oxygen level. With high levels of inspired oxygen, the oxygen consumption of the inner retina became many times that of the outer retina.
Figure 5.
 
Average oxygen consumption of the inner and outer retina as a function of inspired oxygen level. With high levels of inspired oxygen, the oxygen consumption of the inner retina became many times that of the outer retina.
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