Oxygen Distribution in the Rabbit Eye and Oxygen Consumption by the Lens

Abstract

purpose. Excessive exposure to oxygen has been proposed to be a risk factor for nuclear cataracts. For a better understanding of the metabolism of oxygen in the eye, oxygen distribution was mapped in the intraocular fluids, and the rate of oxygen consumption by the lens in rabbits breathing different levels of oxygen was calculated.

methods. Young albino rabbits were anesthetized, intubated, and exposed to normoxic, hypoxic, or hyperoxic conditions. The hemoglobin saturation of the blood was monitored with a pulse oximeter, and arterial oxygen levels were measured with a blood gas analyzer. A fiberoptic optical oxygen sensor (optode) was used to determine oxygen levels in different regions of the eye. Oxygen flux across the posterior of the lens was calculated from the measured oxygen gradients in the vitreous chamber.

results. Oxygen levels in the ocular fluids changed markedly when rabbits breathed air made hypoxic or hyperoxic. Oxygen levels were highest near the retinal vasculature, the iris vasculature, and the inner surface of the central cornea. Compared with nearby regions, oxygen levels were decreased in the aqueous humor closest to the pars plicata of the ciliary body and near the anterior chamber angle. Oxygen levels were generally lower closer to the lens. From the oxygen gradients in the vitreous body, oxygen consumption by the posterior half of the lens was calculated to be 0.2 to 0.4 μL/h under normoxic conditions. Oxygen consumption by the posterior of the lens increased in proportion to the amount of oxygen supplied.

conclusions. Intraocular oxygen is mostly derived from the retinal and iris vasculature and by diffusion across the cornea. Freshly secreted aqueous humor and the aqueous humor in the anterior chamber angle are relatively depleted of oxygen. The marked increase in oxygen consumption that occurs when the lens is exposed to increased oxygen is likely to result in the production of higher levels of reactive oxygen species and may provide a link between elevated oxygen levels and the risk of nuclear cataracts.

Aberrant distribution of oxygen is an important aspect of ocular diseases. In the retina, hypoxia contributes to the pathogenesis of diabetic retinopathy and other retinal ischemic diseases. ^{1 2} Retinal hypoxia leads to the excess production of angiogenic factors, like vascular endothelial growth factor (VEGF) and to the formation of abnormal vessels, which may grow out of the retina and into the vitreous body. These aberrant vessels often lead to blindness caused by hemorrhage or traction retinal detachment. ³ Increased leakage from the abnormal vasculature in diabetes also contributes to diabetic macular edema, a major cause of decreased visual acuity in this disease. ^{3 4} The importance of sufficient retinal oxygenation has focused attention on the mechanisms that regulate the retinal oxygen supply in normal, hypoxic, and hyperoxic conditions. ^{1 2 5 6 7}  

While maintaining adequate oxygen supply is essential for the retina, the opposite may be true of the lens. The oxygen level measured in the vitreous body of normal rabbit, feline, rat, and human eyes is low, between 8 and 20 mm Hg (∼1%–3% O₂). ^{8 9 10 11 12 13 14 15} Results in reported studies have led to the hypothesis that increased exposure of the lens to oxygen may contribute to nuclear cataract formation. ^{8 12 16 17 18 19} In accord with this hypothesis, vitrectomy leads to rapid-onset nuclear cataracts in humans and is associated with a substantial, long-term increase in oxygen levels near the lens. ^{8 12}  

In the present studies, we used an optical oxygen sensor (optode) to measure oxygen levels in the anterior, posterior, and vitreous chambers of rabbits breathing normal, higher or lower partial pressures of oxygen. These data show that intraocular oxygen is derived from several sources. Changes in the level of inspired oxygen leads to changes in the oxygen levels around the lens, which alters its rate of oxidative metabolism. 

Methods

Animals

Adult albino rabbits (2.5–4.5 kg; age, 2 –6 months) were obtained from Myrtle’s Rabbitry (Thompson Station, TN). All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and with the approval of the Animal Studies Committee of the Washington University School of Medicine. 

Anesthesia and Oxygen Delivery

To measure the partial pressure of oxygen in rabbit eyes under conditions that were similar to resting conditions, we first determined the hemoglobin saturation of the blood and the oxygen level in the center of the vitreous body of rabbits maintained in room air (∼21% O₂), hypoxic conditions (11% O₂), or hyperoxic conditions (60% O₂). Rabbits were kept in plastic enclosures that were gassed with room air or room air supplemented with the appropriate amount of nitrogen, or oxygen using an oxygen controller (Pro-Ox 110; Biospherix, Redfield, NY). After 2 hours, hemoglobin saturation was recorded and the animals were anesthetized, while still in the modified gas mixture, by intraperitoneal injection of ketamine and xylazine (5 and 35 mg/kg body weight, respectively). As soon as an animal was anesthetized, it was moved to room air, the fiberoptic optode was rapidly inserted through a small puncture wound made in the eye with a 23-gauge needle and positioned in the midvitreous cavity. Because these measurements were made within minutes of anesthesia, the oxygen levels that were recorded approximated the resting oxygen levels in the vitreous body under nonanesthetized conditions. In room air, over a period of 30 minutes after the initial measurements, oxygen levels slowly decreased when animals had been exposed to hyperoxic conditions or increased when animals had been exposed to hypoxic conditions (data not shown). 

For studies in which rabbits were maintained for longer periods under defined oxygen conditions, animals were first anesthetized with ketamine-xylazine, and then isoflurane was administered through a mask for 3 to 5 minutes before intubation. A 3-0 cuffed endotracheal tube was inserted and connected to an anesthesia machine (Narkomed 2A; North American Dräger, Louisville, KY). Anesthesia was then maintained with 2% isoflurane. During the oxygen measurements, hemoglobin saturation (SaO₂), heart rates, respiratory rates, CO₂ inspiration and expiration, and body temperature were monitored (model 1500; Nellcor, Pleasanton, CA). All animals were initially maintained in conditions that resembled normal breathing. They received 20% oxygen, and the rate of respiration was adjusted to keep the SaO₂ at the normal level of ∼97%. The partial pressure of oxygen in different regions of the eye was then measured. 

Two types of experiments were performed. In the first series, animals were equilibrated to 20% oxygen, and oxygen levels in the in several regions of the eye were measured. Then, while the same rate of respiration was maintained, the level of inspired oxygen was decreased or increased to 13% or 60%, respectively, for 1 hour, and a second set of measurements was made. In animals exposed to hyperoxia, the hemoglobin rapidly saturated. Under hypoxic conditions, the SaO₂ decreased to approximately 75%, which was the level measured in nonanesthetized rabbits maintained in 11% O₂. After measurements were made, the animals were killed by overdose of pentobarbital (100 mg/kg). 

Oxygen Measurements in Rabbit Eyes

An optical oxygen sensor and probe (Oxylab pO2 optode; Oxford Optronix, Oxford, UK) was used as previously described, to measure oxygen levels in several locations within the eye. ^{8 17} The diameter of the probe is 200 to 220 μm (∼25 gauge). Although this probe does not have the same spatial resolution as the microelectrodes used in some studies, ^{5 20} it has the advantage that it does not consume oxygen and is most sensitive at low oxygen levels. To measure oxygen partial pressures in rabbits under circumstances that were as close as possible to physiological, we avoided the use of drugs that might affect ocular blood flow. The pupils were not dilated. To avoid damaging the rabbit lens, which occupies a larger proportion of the globe than in the human eye, we used two sclerotomies. The first was 2.5 mm and the second 6.5 mm posterior to the corneal limbus. These locations are labeled A and B, respectively, in Figure 1 . The first sclerotomy was made with the tip of a 23-gauge needle at position A, the probe was inserted through the sclerotomy and gently moved into the posterior and anterior chambers. Because of the flexibility of the scleral wall in rabbits, the sclera and cortical vitreous sealed around the shaft of the probe, and there was no leakage of fluid or obvious loss of intraocular pressure. Measurements were first taken in the posterior chamber close to the pars plana of the ciliary body (Fig. 1 ; PC1), in the posterior chamber close to the pars plicata of the ciliary body (PC2), in the posterior chamber under the middle of the iris (PC3,) and in the posterior chamber at the edge of the pupil (PC4). The blue light from the tip of the optode was visible under the iris of the albino animals, assisting the positioning of the probe at locations PC3 and PC4. The probe was then positioned in the anterior chamber at the surface of the lens (AC lens), in the center of the anterior chamber (AC center), at the posterior surface of the cornea (AC cornea), and, finally at the anterior chamber angle (AC angle). The probe was then withdrawn to the posterior chamber, and a measurement was made at the equator of the lens (A LEQ). Oxygen measurements were recorded for 2 to 4 minutes at each position. To minimize the distortion of aqueous humor flow, the probe was moved from point to point, slowly and gently, under a surgical microscope. Oxygen readings were repeatable in the same animal by returning to the same point, signifying that the flow of aqueous humor and oxygen metabolism were not disturbed by the measurement procedure. When the oxygen measurements were completed in the anterior of the eye, the probe was removed. The wound site was sealed by a small plug of cortical vitreous, and no aqueous humor leaked out of the eye. A second, posterior sclerotomy was made 6.5 mm from the limbus and the probe was inserted. The partial pressure of oxygen was measured, in sequence, at the central posterior surface of lens (VC lens), in the center of the vitreous chamber (VC center), in the vitreous body just anterior to the central retina (VC retina), and near the equatorial retina on the opposite side of the eye (VC eq). The probe was then withdrawn to measure the oxygen level just posterior to the lens equator (B LEQ). Because bright light can alter the values obtained with the optode, only normal laboratory illumination was used during measurements. We confirmed that this amount of light did not alter the readings by checking oxygen levels with the lights on or off. In the low light that reached the interior of the eye, the blue excitation light at the tip of the probe permitted accurate positioning for each of the oxygen measurements. The mean and SEM of Po ₂ are reported for each location. IOP decreased by an average of 11% after the second sclerotomy and was at all times within the normal range for rabbits. 

Measuring Oxygen Consumption by the Lens

A second set of measurements was made to determine the effect of oxygen level on the rate of oxygen flux across the posterior half of the lens. In these studies, Po ₂ was measured at several locations near the surface of the retina, in the vitreous body midway between the retina and the lens, and near the posterior of the lens, while animals were breathing different concentrations of oxygen. Rabbits were anesthetized and intubated as described earlier, but the pupils were dilated with 10% phenylephrine to permit accurate positioning of the probe in the vitreous body and at the retinal surface. Eyelashes and fur around the eyelids were removed. After a pediatric lid speculum was inserted, a Landers vitrectomy ring was sutured at the corneal limbus with 6-0 silk to position a Machemer magnifying lens, allowing clear observation of the retina and vitreous chamber under the surgical microscope. Conjunctival tissue was removed from the nasal surface of the globe and the tip of a 23-gauge needle was used to make a sclerotomy 6.5 mm from the limbus. Rabbits were first equilibrated at 13%, 20%, or 30% inspired oxygen for 1 hour. The optode was inserted and detailed oxygen measurements were made at several locations at the surface of the retina close to the vascular supply and in avascular regions of the retina (see Fig. 6for locations). The probe was then moved to the midvitreous, and five measurements were taken at locations equidistant from the lens and retina. Finally, four measurements were made at equally spaced intervals along the posterior surface of the lens. Inspired oxygen was then increased to the next higher level (20%, 30%, or 40%, respectively) for 1 hour, and a second set of measurements was made in the same locations as the first. Repeat measures, made 2 hours after increasing the oxygen, yielded values that were indistinguishable from those at 1 hour, indicating that oxygen levels reached steady state within 1 hour. Each rabbit was used for only two sets of measurements. Arterial blood gases were monitored with a blood gas analyzer (Stat Profile; Nova Biomedical Co., Waltham, MA) after equilibration at each level of inspired oxygen (Table 1) . Measured intraocular Po ₂ was used to determine the oxygen gradient between the lens and retina. These gradients were used to estimate the oxygen consumption by the posterior of the lens. 

Calibration of the Optodes

The response profile of each optode to oxygen and temperature is calibrated by the manufacturer. A thermistor is incorporated into each probe, permitting the output of the probe to be adjusted for intraocular temperature during measurements. Each day before use, the response profile of the optode was entered into the detector (Oxylab) by means of a barcode reader. Before use, each optode was checked against water samples bubbled with 100% N₂ and 5% O₂, to confirm the accuracy of the probe in the range of oxygen levels that are typically encountered in the eye. Probes were rechecked after use, to assure the accuracy of the measurements. 

Estimation of Oxygen Consumption by the Posterior of the Lens

Oxygen flux across the posterior of the lens was estimated at different concentrations of inspired oxygen from the dimensions of the posterior of whole rabbit eyes that had been sectioned parallel to the optical axis), the oxygen gradients in the vitreous body and the diffusion constant of oxygen in water. These values were used to obtain the flux of oxygen (J) using equation (1) :  

\[J\ {=}\ {-}\mathrm{D}(dC/dX)\]

where D is the diffusion constant of oxygen in water, dC is the oxygen level gradient (in moles/cubic centimeter) and dX is the distance to the lens (in centimeters). We corrected J to account for the greater surface area of the retina compared with the lens by multiplying by the ratio of these areas. 

Measurement of Oxygen Level Gradients in the Vitreous Body

As described earlier, the oxygen partial pressures measured in several locations at the surface of the retina, in the middle of the vitreous body, and near the posterior surface of the lens at four different levels of inspired oxygen (Tables 2 3 and 4) . Near the retinal surface, the oxygen level varied greatly, depending on the proximity to the retinal vascular “streak” that lies in the horizontal plane of the globe (see Fig. 6 ). We accounted for the unequal distribution of oxygen on the retinal surface in two ways; In the first case, we averaged the oxygen level at all the points that were measured on the retina and used this mean value for the high end of the oxygen gradient. The average value at the posterior of the lens was used for the lower end of the gradient. Because it was not possible to obtain measures of Po ₂ along the 135° and 225° meridians at the surface of the retina, we used the values from the 45° and 315° meridians twice. We believe that averaging the Po ₂ at the surface of the retina was warranted, because, as shown in Figure 6 , the variation in retinal oxygen values diminishes greatly by the time the oxygen diffuses into the midvitreous, effectively “averaging” variations in retinal Po ₂. In a second approach, we calculated the oxygen gradient from the Po ₂ in the middle of the vitreous and at the posterior surface of the lens. 

Distance from the Retina to the Lens

Fresh rabbit eyes were immersed in PBS and carefully sectioned in a plane parallel to the optic axis and through the optic disc by manual dissection. The anterior and posterior chambers of the bisected eye were filled with glycerol to maintain their normal shape, the eye was photographed next to a ruler, and prints of the photographs were used to determine the dimensions of the eye. Before making measurements, we confirmed that the external dimensions of the bisected eye were the same as before dissection. The shortest distance from the inner surface of the retina to the posterior surface of the lens was measured at four equally spaced locations on either side of the optic disc. These positions were at the edge of the optic disc and at 1-disc-diameter intervals to a maximum of 3 disc diameters from the edge of the optic disc. For the first estimate of lens oxygen consumption, in which the mean of all retinal oxygen levels was used to calculate the oxygen gradient, we used the average of these eight distances to calculate the mean distance from the retina to the lens (5.8 mm). For the second set of calculations, in which the mean midvitreous oxygen level was used, we used one half of this value (2.9 mm). 

Surface Area of the Posterior of the Lens and the Inner Retina

The rabbit lens is ellipsoid. Therefore, we determined its perimeter (P) using equation (2) :  

\[P\ {=}\ {\pi}\sqrt{{[}2(a^{2}\ {+}\ b^{2}){]}}\]

in which a and b are the lengths of the major and minor axes. The perimeter was then multiplied by 2r to obtain the lens surface area. We divided the surface area by 2 to obtain the area of the posterior surface of the lens. For the perimeter of the retina, we used a string to trace, on a photograph of the bisected eye, the surface area of the retina that was exposed to the vitreous chamber. This length was then corrected for the magnification of the photograph and multiplied by 2r to obtain the surface area. The calculated surface area of the inner retina was more than twice that of the posterior of the lens (2.95 cm² vs. 1.1 cm²). Because oxygen diffuses from the inner surface of the retina across the posterior surface of the lens and there is more than twice as much retinal surface area as lens surface area, the calculated flux was adjusted for the difference in surface area by multiplying by the ratio of the surface areas (2.95/1.1 = 2.6). For measurements using the midvitreous-to-lens oxygen gradient, the surface area of the midvitreous was estimated by measuring the length of a line that passed through points midway between the retina and the lens. This perimeter was multiplied by 2r to obtain the area of this surface. The flux from the midvitreous to the lens was adjusted by multiplying the ratio of the area of the midvitreous surface (2.35 cm²) to the area of the posterior surface of the lens (2.35/1.1 = 2.1). 

Calculations

The Po ₂ gradients and the average distance from the lens to the retina were entered into equation (1)and corrected for differences in the surface areas of the lens and retina by using a spreadsheet (Excel; Microsoft, Redmond, WA), yielding values for J at different levels of inspired oxygen. Arterial blood saturation and mean oxygen levels at the surface of the retina were directly proportional to inspired oxygen (R ² = 0.98 for both). For convenience, oxygen flux was plotted against inspired oxygen. 

Results

The Effect of Anesthesia on Po ₂ in the Blood and Vitreous Body

In an initial series of experiments, rabbits were anesthetized with a combination of ketamine and xylazine, without mechanical ventilation. When the animals were in deep anesthesia, oxygen levels were measured in the posterior, anterior, and vitreous chambers. These measurements demonstrated standing gradients of oxygen in the vitreous and anterior chambers. However, during these studies, it was noted that the respiratory rate of the rabbits was significantly slower and shallower than normal. Subsequent tests, in which the optode was maintained in the center of the vitreous body, showed that oxygen levels gradually decreased after anesthesia (not shown). This suggested that ketamine-xylazine anesthesia, without the benefit of mechanical ventilation, depresses the respiratory drive, leading to a decline in the oxygen saturation of the blood. This could lead to incorrect estimates of intraocular oxygen levels in the eye. A pulse oximeter confirmed that SaO₂ began to decrease by 10% to 20% within minutes after ketamine-xylazine anesthesia (not shown). For this reason, all subsequent studies were conducted using animals maintained on isoflurane anesthesia and with mechanical ventilation. 

To obtain measurements of intraocular oxygen levels under baseline and hyperoxic conditions, SaO₂ was first stabilized at approximately 97% by regulating the respiratory rate of anesthetized rabbits breathing 20% oxygen. Baseline intraocular oxygen measurements were made and then the inspired gas was increased to 60% oxygen at the same rate of respiration. The SaO₂ rapidly saturated and oxygen levels in the intraocular fluids increased. By 60 minutes after increasing the level of oxygen, oxygen levels in the intraocular fluids stabilized and a second set of measurements was made. To obtain intraocular oxygen levels under baseline and hypoxic conditions, oxygen measurements were again made at 97% SaO₂ and then the inspired gas was lowered to 12% to 14% oxygen to maintain the SaO₂ between 75% and 80%—the SaO₂ found in rabbits maintained in an atmosphere of 11% oxygen. Oxygen levels in the intraocular fluids were measured 60 minutes after lowering the inspired oxygen. 

Oxygen Distribution in the Eyes of Rabbits Maintained at Normal SaO₂

Figure 2shows the mean partial pressure of oxygen in the intraocular fluids of rabbits maintained at SaO₂ of 97%. Inspection of the oxygen distribution in these eyes suggests that oxygen enters the eye by several routes. Oxygen levels were high at all locations around the periphery of the eye and decreased toward the lens. Therefore, oxygen is provided to the intraocular fluids by the vascular supply to the retina and iris and from the air by diffusion through the cornea. The standing oxygen gradients observed in the anterior chamber and the vitreous body confirmed the expectation that the rabbit lens is constantly consuming oxygen. 

Oxygen measurements were made along the path followed by the aqueous humor, from the ciliary body, between the lens and inner surface of the iris and into the anterior chamber. Oxygen levels were highest near the surface of the pars plana of the ciliary body (21 mm Hg) and beneath middle of the iris (23 mm Hg). Near the pars plicata of the ciliary body, oxygen levels were only 16 mm Hg. As the aqueous humor moved beyond the midpoint of the iris, oxygen levels decreased to below 20 mm Hg. These observations show that the aqueous humor entering the eye is relatively depleted of oxygen. Oxygen appears to enter the posterior chamber from the vasculature of the iris, where it is likely to be consumed by the lens epithelial cells. 

A gradient of increasing oxygen level was found in the anterior chamber from the anterior surface of the lens to the central corneal endothelium, suggesting that much of the oxygen in the middle of the anterior chamber comes from diffusion across the cornea. Compared with the levels found beneath the central cornea, the levels of oxygen were unexpectedly low near the anterior chamber angle. This observation suggests that oxygen transmission may be lower or consumption may be higher in this region. To be sure of these levels, measurements were made several times in each rabbit. Care was taken that the eye lids did not cover or approach the peripheral cornea during equilibration or when the measurements were being made, assuring that coverage by the eyelid did not cause a decrease in oxygen diffusion near the angle. 

The Po ₂ in the aqueous humor near the lens equator was less than half of that near the pars plana and pars plicata of the ciliary epithelium and much lower than the levels beneath the iris. Therefore, the cells in the germinative zone of the lens, near the lens equator, appear to be exposed to significantly lower levels of oxygen than the epithelial cells located in more anterior regions of the lens. 

Oxygen levels in the vitreous chamber were highest near the posterior retina and declined near the peripheral retina. In these initial measurements, no effort was made to account for the differences in oxygen distribution that occur due to the nonuniform distribution of vessels in the rabbit retina. A more detailed map of oxygen distribution at the surface of the retina is shown in studies described in the following section. A gradient of decreasing oxygen level was present from the surface of the retina to the posterior of the lens. 

Oxygen Distribution in the Eyes of Rabbits Breathing Increased or Decreased Oxygen

Breathing 60% oxygen for 60 minutes caused rapid saturation of blood hemoglobin and an increase in the oxygen levels inside the eye (Fig. 3) . The oxygen optode used in these studies was most sensitive to low levels of oxygen and could not reliably measure oxygen levels over 100 mm Hg. The oxygen levels in the eyes of rabbits exposed to 60% oxygen were, in most cases, greater than 100 mm Hg, precluding accurate quantification. Only around the lens (and away from the iris), the sites at which oxygen levels were lowest when animals breathed 20% oxygen, was quantification possible. At these locations, changing from breathing 20% to 60% oxygen caused the Po ₂ to increase by approximately 5 to more than 12 times the level in animals breathing 20% oxygen (compare Figs. 2and 3 ). 

The Po ₂ of the intraocular fluids of rabbits that were made hypoxic (SaO₂ 75% to 80%) were approximately half that in the eyes of normoxic animals, except in the aqueous humor at the inner surface of the central cornea (Fig. 4) . As in the normoxic animals, steep standing gradients of oxygen were highest near the tissues at the outer surfaces of the eye and declined toward the lens. The pattern of oxygen levels along the path followed by the aqueous humor also showed the same decreases near the surface of the pars plicata of the ciliary epithelium and beyond the midpoint of the iris that were seen in the eyes of animals breathing 20% oxygen. The oxygen levels in the aqueous humor near the anterior chamber angle were notably lower than in animals breathing 20% oxygen, even though the surface of the eye was exposed to room air. 

Figure 5summarizes the origins of oxygen in the intraocular fluids, as determined from the measurements of oxygen distribution under normoxic, hyperoxic, and hypoxic conditions. This diagram includes an estimate of the oxygen consumption by the anterior and posterior surfaces of the lens, which were derived from the known properties of lens cells and the consumption of oxygen by the posterior of the lens. 

Oxygen Flux across the Posterior of the Lens In Vivo

From the data in Figures 2 3and 4 , it was apparent that the gradient of oxygen from the surface of the retina to the posterior surface of the lens must reflect continuous consumption of oxygen by the lens. By measuring these gradients and the dimensions of the rabbit eye and using Fick’s Law and the diffusion constant of oxygen in water, it is possible to estimate the oxygen flux across the posterior half of the lens at different oxygen levels. 

Much of the rabbit retina is avascular. A narrow band of vessels arises from the optic disc, supplying a horizontal stripe of retinal tissue (Fig 6A) . From the initial measurements and knowledge of the vascular anatomy of the rabbit retina, it was expected that there would be large variations in Po ₂ over different regions of the retina, depending the proximity of the optode to the vasculature. We felt that accurate estimates of the oxygen gradients across the vitreous body might require detailed knowledge of the Po ₂ at the surface of the retina. Therefore, maps were made of oxygen level at many locations near the retinal surface (Fig. 6A) . Oxygen measurements were also obtained at five locations within the vitreous body, midway between the retina and the lens (Fig. 6B) . These measurements provided useful information about the Po ₂ at greater distance from the retina, where local fluctuations in oxygen tension at the retinal surface would be damped by diffusion. Po ₂ was also measured at four locations at the posterior surface of the lens (Fig. 6B) . The pupils of the rabbit eyes were also dilated for better visualization of the location of the probe in the vitreous chamber and at the retinal surface. The level of inspired oxygen was varied from 12% to 14% and 40%. The Po ₂ in different regions of the posterior of the eye obtained in animals breathing 20% oxygen are shown in Figs. 6Cand 6D . The SaO₂ and arterial Po ₂ of rabbits breathing different levels of oxygen are in Table 1 . Po ₂ at the retinal surface, midvitreous, and posterior of the lens at each oxygen level are in Tables 2 3 and 4 . 

When the average Po ₂ at the vitreal surface of the retina was used to calculate the oxygen level gradient, the calculated mean flux of oxygen across the posterior surface of the lens was 0.34 μL/h in normoxic conditions (see the Methods section). When the midvitreous-to-lens oxygen level gradient was used for this calculation, oxygen consumption by the posterior half of the lens was 0.21 μL/h. Despite the differences in these values, both methods for estimating the oxygen flux across the posterior of the lens demonstrated that oxygen consumption increased with an increase in the amount of oxygen supplied (Figs. 7) . Although the oxygen consumption calculated from the retina-to-lens gradient was best fit with an exponential (Fig. 7A) , a linear fit to the data was nearly as good (R ² = 0.76 vs. 0.71). Oxygen consumption calculated from the vitreous-to-lens gradient was best described by a linear relationship (Fig. 7B) . The slopes of the lines describing the fluxes of oxygen across the posterior of the lens at different oxygen levels were greater than 1, indicating that oxygen consumption increased more rapidly as oxygen levels increased. 

Discussion

This study describes the distribution of oxygen in the rabbit eye under normoxic, hyperoxic, and hypoxic conditions. In general, the values obtained for breathing 20% oxygen were similar to those reported previously in the vitreous body and anterior chamber of the living rabbit eye. ^{12 14 21} In the present work, we extended these studies by measuring oxygen distribution in eyes when animals were made hypoxic and hyperoxic and by mapping oxygen levels in the posterior chamber. These measurements suggest that oxygen primarily enters the ocular fluids from the retinal vasculature and the iris vasculature and by diffusion across the cornea. 

Because the ciliary body is well vascularized, we were surprised to find that oxygen levels were low in the aqueous humor near the pars plicata, the site of origin of the aqueous humor. Although the ciliary epithelium actively transports components of the aqueous humor, our measurements show that little oxygen enters the eye by this path. It seems likely that the abundant mitochondria in the ciliary folds use oxidative metabolism to power transepithelial transport, ²² thereby depleting oxygen from the secreted aqueous humor. As the aqueous humor flows beneath the iris, oxygen levels increase, presumably due to diffusion from the iris vasculature. 

Oxygen levels were substantially lower in the aqueous humor near the anterior chamber angle than beneath the central cornea. Hypoxia led to a decrease in the oxygen levels in the aqueous humor of the angle, but not in the aqueous humor adjacent to the central cornea. This was surprising, because the anatomy of the cornea in its central and peripheral regions is similar. These observations confirm that oxygen reaches the aqueous humor beneath the central cornea by diffusion from the surrounding air, as suggested previously. ^{21 23} By contrast, the oxygen in the aqueous humor in the angle appears to be derived primarily from the vasculature of the iris, ciliary body, and/or the limbus and not by diffusion across the peripheral cornea. Helbig et al. ²⁴ found levels of oxygen near the human angle that were higher than in the present study (45 mm Hg vs. 27 mm Hg). However, they did not compare oxygen levels near the angle to those beneath the central cornea. Therefore, it is not clear whether the relative difference in oxygen distribution between these regions, as detected in rabbits, is also present in humans. The lower oxygen levels near the anterior chamber angle could be relevant to the biology of the cells of the trabecular meshwork and the aqueous humor outflow pathway. 

Intraocular Oxygen Levels in Hyperoxia and Hypoxia

Breathing 60% oxygen caused the level of oxygen in most parts of the eye to exceed 100 mm Hg within 1 hour. Po ₂ at several locations around the lens remained measurable, however. In these locations, oxygen levels increased between 5- and 12-fold. It seems reasonable to ask how an approximate threefold increase in the level of inspired oxygen could lead to disproportionately large increases in Po ₂ near the surface of the lens. The answer to this question is likely to be related to the unusual way that the rabbit retina deals with exposure to increased oxygen. Under normal oxygen conditions in all species studied, oxygen consumption by the retina leads to a relatively low Po ₂ in the vitreous near the surface of the retina. In most of these animals (cat, rat, guinea pig, and monkey), increases in blood oxygen levels do not significantly alter preretinal (vitreous) oxygen levels. ^{10 11 15 25 26 27} However, in rabbits, small increases in inspired oxygen above normoxia lead to large relative increases in oxygen levels at the interface of the retina and vitreous. ^{5 20} Because the level of oxygen around the posterior of the lens is largely determined by the diffusion of oxygen from the preretinal vitreous and the consumption of oxygen by the lens, an increase in oxygen at the retinal surface results in an increase in oxygen tension around the posterior of the lens. Therefore, modest increases in inspired oxygen (∼3-fold) result in large relative increases in oxygen at the posterior of the lens (5- to 12-fold). 

At all levels of inspired oxygen, the level of oxygen near the lens equator was substantially lower than in more anterior regions of the posterior chamber. This means that the germinative zone of the lens epithelium is likely to be exposed to lower levels of oxygen than the central regions of the lens epithelium. We have observed that the rate of proliferation of germinative zone epithelial cells is suppressed by hypoxia in vivo (Shui, et al., manuscript in preparation). Therefore, the normally low level of oxygen near the lens equator may be essential in controlling lens growth. 

Consumption of Oxygen by the Lens

The level of oxygen in the intraocular fluids was always lower near the lens than near more peripheral tissues. We used the measured oxygen gradients in the vitreous body to estimate the rate of oxygen consumption by the posterior half of the lens in vivo. We did not attempt to calculate oxygen consumption by the anterior of the lens, because the geometry of the posterior and anterior chambers is complex, and estimates would be complicated by the flow of aqueous humor. Published rates of oxygen consumption by the rabbit lens in vitro vary greatly, ranging between 6.8 to 260 μL O₂/g wet weight/h (for review, see Refs. ²⁸ , ²⁹ ). This variability may be due to the relatively low rates of oxygen consumption by lenses, coupled with the insensitivity of some of the methods used to make these measurements and the different levels of ambient oxygen present during the measurement. ²⁸ It is also possible that some of this variability was due to damage to the lens that occurred during isolation and differences in the chemical composition between the test solutions and the intraocular fluids. These potential sources of error are avoided by estimating the consumption of oxygen by the lens in vivo. 

In normoxic conditions, the flux of oxygen across the posterior half of the lens, calculated using two methods of estimating the oxygen gradients, averaged 0.21 and 0.34 μL per hour. These seem to be reasonable levels, given that a whole adult rabbit lens consumes ∼1.8 μL O₂/h when measured in a well-stirred respirometer in 5% O₂. ²⁸ Compared with the levels of oxygen normally found around the lens in vivo, 5% oxygen (∼38 mm Hg) is actually quite high. Based on our observation that the rate of oxygen consumption increases with increasing oxygen exposure, measuring the oxygen consumption of the lens in 5% oxygen could overestimate the amount of oxygen that is normally consumed by the lens in situ. In addition, we only estimated oxygen consumption by the posterior of the lens. The epithelial cells in the anterior of the lens, because of their higher concentration of mitochondria, would be expected to consume more oxygen than would the posterior fiber cells. ^{30 31}  

Over most of the range of oxygen levels tested, the rate of oxygen consumption by the posterior of the rabbit lens increased in proportion to the level of inspired oxygen. This behavior is different from that in most tissues. For example, the rabbit retina consumes the same amount of oxygen under normoxic and hyperoxic conditions. ^{5 20} The unusual response of the lens to increased oxygen supply may be related to the fact that lens cells are normally hypoxic, whereas most vascularized tissues are usually provided with ample oxygen. Another normally avascular tissue, cartilage from the epiphyseal region of long bones, shows a proportional increase in oxygen consumption at low oxygen levels, but a hyperbolic (saturating) relationship between oxygen supplied and oxygen consumed at higher oxygen levels. ^{32 33} In the present studies, the oxygen flux across the posterior of the lens increased linearly up to the highest levels tested. We may have been able to saturate the response of the lens to oxygen, had we been able to reach higher oxygen levels. However, the range of the optodes used in our study precluded testing higher oxygen levels. Alternatively, the hyperbolic response of chondrocytes to increasing oxygen may reflect their need to reduce the rate of oxidative metabolism at high oxygen levels, since some chondrocytes will normally be close to the blood supply and exposed to higher levels of oxygen, while cells in the center of the tissue might be quite hypoxic. Fiber cells from adult lenses, due to their normally hypoxic environment and distance from the vascular supply, may not be able to limit their oxygen consumption in the face of higher levels of oxygen. 

It is well known that mitochondrial oxidative metabolism produces reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide. The production of ROS by isolated mitochondria increases as a linear function of oxygen tension. ^{34 35} Previous investigators have suggested that ROS contribute to cataract formation. ^{12 36 37 38 39 40} Several studies have suggested that exposure of the lens to elevated intraocular oxygen is a risk factor for nuclear cataracts. ^{8 12 16 17 18 19} The highest levels of intraocular oxygen measured in the present study were similar to those that are typically reached during vitrectomy ⁸ ; oxygen levels are likely to be even higher in patients treated with hyperbaric oxygen therapy. Both treatments are associated with the formation of nuclear cataracts. ^{8 18 19} Therefore, increased production of ROS may explain the association between exposure of the lens to higher levels of oxygen and increased risk of cataract formation. 

Our results show that the oxygen levels around the lens can be readily manipulated by altering the level of inspired oxygen. As described earlier, increasing the exposure of the lens to oxygen appears to increase the risk of cataract. Therefore, lowering the levels of oxygen around the lens might protect against nuclear cataract, perhaps by reducing the production of ROS. Based on the data obtained in this study, it should be possible to test this hypothesis in experimental animals by determining whether nuclear cataract formation is decreased in animals breathing lower levels of inspired oxygen. 

 Supported by the Department of Ophthalmology and Visual Sciences, an unrestricted grant from Research to Prevent Blindness, and National Eye Institute Grant EY04853 (DCB).

 Submitted for publication November 17, 2005; revised December 9, 2005; accepted February 10, 2006.

 Disclosure: Y.-B. Shui, None; J.-J. Fu, None; C. Garcia, None; L. K. Dattilo, None; R. Rajagopal, None; S. McMillan, None; G. Mak, None; N.M. Holekamp, None; A. Lewis, None; D.C. Beebe, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Map of locations at which oxygen measurements were recorded. Open symbols: data obtained through the sclerotomy at A; filled symbols: data obtained when the probe entered the eye through point B.

Table 1.

 

Average SaO₂ and Arterial Po ₂ in Rabbits Breathing Different Percentages of Inspired Oxygen

Inspired Oxygen (%)	SaO2	Arterial Po 2 (mmHg)	n
12–14	78 ± 1	37 ± 3	3
20	98 ± 0	89 ± 7	6
30	99 ± 0	125 ± 6	5
40	100 ± 0	196 ± 7	3

 Data are ± SEM.

Table 2.

 

Average Po ₂ at the Retinal Surface in Rabbits Breathing Different Percentages of Inspired Oxygen

Inspired Oxygen (%)	R0 1	R0 2	R0 3	R0 4	R45 1	R45 2	R45 3	R45 4	R90 1	R90 2	R90 3	R90 4	n
12–14	24 ± 3	19 ± 3	15 ± 3	8 ± 2	15 ± 1	7 ± 1	4 ± 1	3 ± 1	13 ± 2	6 ± 0	3 ± 0	3 ± 0	3
20	50 ± 2	39 ± 3	28 ± 4	19 ± 2	29 ± 2	17 ± 2	11 ± 2	10 ± 2	28 ± 4	14 ± 1	12 ± 2	10 ± 2	6
30	85 ± 4	71 ± 4	57 ± 1	48 ± 4	63 ± 4	50 ± 5	44 ± 7	38 ± 6	61 ± 5	47 ± 6	44 ± 6	41 ± 6	5
40	109 ± 6	84 ± 9	71 ± 7	64 ± 5	81 ± 9	71 ± 10	69 ± 9	58 ± 5	70 ± 10	67 ± 11	65 ± 9	59 ± 8	3
Inspired Oxygen (%)	R180 1	R180 2	R180 3	R180 4	R270 1	R270 2	R270 3	R270 4	R315 1	R315 2	R315 3	R315 4	n
12–14	25 ± 3	22 ± 3	15 ± 1	12 ± 1	14 ± 2	9 ± 1	5 ± 1	*	14 ± 3	11 ± 2	7 ± 1	4 ± 1	3
20	51 ± 1	44 ± 2	32 ± 3	21 ± 3	25 ± 3	19 ± 2	14 ± 3	*	26 ± 3	22 ± 2	16 ± 2	13 ± 2	6
30	89 ± 5	72 ± 4	58 ± 6	50 ± 6	54 ± 6	49 ± 6	39 ± 6	*	59 ± 8	52 ± 7	43 ± 5	40 ± 5	5
40	103 ± 13	90 ± 9	71 ± 6	62 ± 2	63 ± 7	52 ± 1	50 ± 1	*	70 ± 6	60 ± 4	56 ± 1	53 ± 2	3

 Refer to Figure 6for the location of the measurements on the retina. Data are mean mm Hg ± SEM. Asterisks in column R₂₇₀ 4 indicate that it was not possible to reach this location with the optode. Oxygen levels at position R₉₀ 4 were substituted for these values in calculating oxygen gradients.

Table 3.

 

Average Po ₂ in the Central Region of the Vitreous Body in Rabbits Breathing Different Percentages of Inspired Oxygen

Inspired Oxygen (%)	VC	V0	V90	V180	V270	n
12–14	6 ± 1	6 ± 1	5 ± 2	6 ± 1	6 ± 2	3
20	11 ± 2	12 ± 2	11 ± 2	11 ± 2	11 ± 2	6
30	34 ± 3	32 ± 5	31 ± 5	31 ± 5	31 ± 5	5
40	55 ± 8	56 ± 9	56 ± 10	56 ± 9	56 ± 9	3

 Data are mean mm Hg ± SEM.

Table 4.

 

Average Po ₂ at the Posterior Surface of the Lens in Rabbits Breathing Different Percentages of Inspired Oxygen

Inspired Oxygen (%)	LC	L1	L2	L3	n
12–14	3 ± 0	4 ± 0	3 ± 0	3 ± 0	3
20	5 ± 1	5 ± 1	5 ± 1	5 ± 1	6
30	22 ± 2	20 ± 3	23 ± 2	32 ± 1	5
40	35 ± 9	38 ± 9	38 ± 10	50 ± 7	3

 Data are mean mm Hg ± SEM.

Figure 2.

 

Mean oxygen levels (mm Hg) ± SEM in the eyes of rabbits breathing 20% oxygen at a respiration rate that resulted in normal hemoglobin saturation (∼97% SaO₂). Symbols are as in Figure 1 . n = 12.

Figure 3.

 

Mean oxygen levels in the eyes (mm Hg) ± SEM of rabbits breathing 60% oxygen at the same respiration rate as in Figure 2 . Symbols are as in Figure 1 . n = 6.

Figure 4.

 

Mean oxygen levels (mm Hg) ± SEM in the eyes of rabbits breathing 13% to 15% oxygen at the same respiration rate as in Figure 2 . n = 6.

Figure 5.

 

Paths of oxygen diffusion into the eye and consumption by the lens, as inferred from the oxygen distribution in Figures 2and 4and the known properties of the lens. Red arrows: oxygen entering the eye; orange arrows: consumption of oxygen by the lens; blue arrows: path of flow of the aqueous humor. Arrows originating from the retina represent average oxygen flux in the plane of the vascular streak; oxygen levels in the avascular regions of the retina would be lower.

Figure 6.

 

Measurements used to calculate oxygen flux across the posterior of the lens. (A) Locations at which Po ₂ was measured on the surface of the retina and (B) in the mid-vitreous and at the posterior surface of the lens. (C) Mean Po ₂ measured near the surface of the retina in a rabbit under normoxic conditions (20% inspired oxygen). (D) Mean Po ₂ in the midvitreous and at the posterior surface of the lens of a rabbit in normoxic conditions.

Figure 7.

 

Calculated oxygen flux across the posterior of the lens at different levels of inspired oxygen. (A) Oxygen gradients calculated from the mean oxygen levels at the surface of the retina and the posterior surface of the lens. (B) Oxygen gradients calculated from the mean oxygen levels in the center of the vitreous and the posterior surface of the lens.

The authors thank Bernard Becker for suggesting the approach used to measure oxygen levels in the posterior chamber; Michael Talcott, Director of Large Animal Surgery in the Division of Comparative Medicine, Washington University for many helpful suggestions; Stephen Cringle, for sharing data before publication and for helpful discussions of retinal oxygenation in the rabbit; and Steven Bassnett for constructive comments about the manuscript.