Intraretinal Oxygen Levels before and after Photoreceptor Loss in the RCS Rat

Abstract

purpose. To measure the intraretinal oxygen environment at different stages in the Royal College of Surgeons (RCS) rat model of retinal degeneration to determine whether changes in oxygen level are an important aspect of the disease.

methods. Oxygen-sensitive microelectrodes were used to measure oxygen tension as a function of depth through the retina of anesthetized, mechanically ventilated RCS rats at ages ranging from postnatal day (P)20 to P104. The oxygen profiles were correlated with histologic observations of the cellular changes within the dystrophic retinas and compared with those in RCS-rdy ⁺ control animals and published values in normal mature rats.

results. Although the youngest rats studied exhibited some differences in intraretinal oxygen distribution compared with mature animals, the distribution in dystrophic RCS rats at P20 was not significantly different from that in age-matched control subjects. However, the intraretinal oxygen distribution in dystrophic RCS rats was clearly affected after approximately P30, reflecting a loss of photoreceptor oxygen consumption consistent with histologic observations. In contrast, oxygen uptake by the inner retina was still evident long after the loss of photoreceptors was essentially complete.

conclusions. There was no significant tissue hypoxia during photoreceptor degeneration in the dystrophic RCS rat. The changes in intraretinal oxygen distribution are consistent with the loss of outer retinal oxygen uptake but the preservation of inner retinal oxygen metabolism.

Retinitis pigmentosa (RP) refers to a heterogeneous group of inherited human diseases that cause primary degeneration of rod and cone photoreceptors. ¹ The Royal College of Surgeons (RCS) rat is commonly used as an animal model of photoreceptor degeneration of relevance to RP. ^{2 3} There are several lines of evidence implying a role for oxygen in the RCS rat model of retinal degeneration. LaVail and Battelle ⁴ noted a significant sparing of photoreceptors at the extreme periphery of the retina and around the optic disc, and increased oxygen availability in these regions due to the diffusion of oxygen around the edges of the photoreceptor array has been put forward as an explanation for this phenomena. ⁵ A similar explanation was proposed for the sparing of photoreceptors around the edges of photocoagulation lesions in dystrophic RCS rats. ⁶ Manipulation of environmental oxygen conditions at key stages in the dystrophic RCS rat has reportedly been able to modulate the rate of photoreceptor cell death. Systemic hyperoxia has been shown to slow down and systemic hypoxia to accelerate the rate of cell death in the dystrophic RCS rat model. ⁷ Similar findings have been reported in normal animals that undergo a more controlled process of cell death during maturation of the retina. ^{5 7}  

Despite these compelling arguments for a possible role for oxygen in the photoreceptor degeneration in the dystrophic RCS rat, there has, to date, been no direct measurement of retinal oxygen levels at any stage of the disease. The present study therefore set out to characterize for the first time the intraretinal oxygen distribution at different stages in the RCS rat model of photoreceptor degeneration. 

Methods

General

Thirty-two pink-eyed, dystrophic RCS rats ³ and 14 RCS-rdy ⁺ congenic control rats were used for this study. The animals were bred in our own animal facilities from colonies originally sourced from Matthew LaVail. All procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The rats were anesthetized with an intraperitoneal injection of 5-ethyl-5- (1′-methyl-propyl)-2-thiobarbiturate (Inactin; Byk Gulden, Konstantz, Germany), 100 mg/kg and atropine sulfate (20 μg). Artificial ventilation was performed with tidal volume determined by body weight. Whenever possible, the femoral artery was cannulated for continuous arterial blood pressure (106.6 ± 2.9 mm Hg, n = 33) and blood gas monitoring (Po ₂ = 79.4 ± 3.0 mm Hg, Pco ₂ = 35.9 ± 1.57 mm Hg, [pH 7.40 ± 0.02], n = 32). Rectal temperature was maintained between 36.5°C and 37.5°C using a homeothermic blanket. The rat was positioned prone in a modified stereotaxic instrument (Stellar, Pompano Beach, FL) with the head clamped to the stereotaxic frame and the eye immobilized by suturing to an eye ring. The pupil was dilated with 1% tropicamide (Mydriacyl; Alcon, Fort Worth, TX). 

Oxygen Measurements

Recessed oxygen-sensitive microelectrodes (1 μm tip) were manufactured and calibrated in our own laboratory. The microelectrode was inserted into the rat eye through a small hole posterior to the limbus. A high-quality stereoscopic view of the fundus and the electrode was achieved by an operating microscope and a plano concave corneal contact lens. This enabled the observer to determine the relationship between the retina and the electrode tip and provided a clear view of both the retinal and choroidal vasculature. The retinal locations used for data collection were in the inferior retina, two or three disc diameters from the optic disc margin. Intraretinal Po ₂ profiles were measured in 10-μm steps from the retinal surface through to the deep choroid and measurements repeated during microelectrode withdrawal. Experience has shown that clean penetrations of the retina are best accomplished by using a freshly made and sharpened electrode in each experiment. ^{8 9} No correction was made for the slightly nonperpendicular angle of the electrode track through the retina. Measurements were typically performed in light-adapted conditions, but both light- and dark-adapted measurements were performed in P20 dystrophic RCS rats and RCS-rdy ⁺ control animals to determine the magnitude of any dark adaptation effects on outer retinal oxygen tension. All statistical testing was performed using the statistics program SigmaStat (Jandel Scientific, San Raphael, CA). A nonparametric, Mann–Whitney rank sum test was used to compare oxygen levels in the RCS and RCS-rdy ⁺ groups, with a significance acceptance level of P < 0.05 (one tailed). All data are expressed as mean ± SE, with error bars on graphs showing the SE. 

Histology

At the conclusion of each experiment, both eyes were enucleated and used for histologic studies to characterize the retinal structure, both for comparison with the oxygen measurements and for assessment of morphologic changes. The eyes were placed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 24 hours. Full-thickness pieces of retina and choroid were dissected and placed in 10% sucrose for 1 to 2 days. The specimens were then postfixed in osmium tetroxide, dehydrated in graded ethanols, and embedded in epoxy resin. Sections (2μ m) were cut at selected locations in the region of the microelectrode studies and stained with toluidine blue for light microscopy. 

Results

The dystrophic RCS rat data were collected into five groups corresponding to increasing postnatal age, and therefore severity of retinal degeneration (P20, P25–P30, P30–P35, P35–P50, and P50–P104). There were at least six animals in each group. Representative oxygen profiles are presented for each group, along with retinal histology from that animal. The nature of the oxygen distribution across the retina reflects the sources of oxygen from the choroidal and retinal vascular systems and oxygen consumption within the predominantly avascular retinal layers in the inner retina, such as the inner plexiform layer, and within the completely avascular layers of the outer retina. Regions that contain a significant net oxygen source, or sink, have the most dramatic changes in oxygen gradient. Figure 1A shows the intraretinal oxygen distribution in a dystrophic RCS rat at P20. The profile exhibited characteristics similar to that reported for normal mature rats, ^{8 9 10} except that the oxygen level in the superficial retina and at the level of the deep capillary layer were somewhat higher. The high value in the superficial retina may have been associated with the presence of a patent hyaloid arterial system in the vitreous of such young animals, but the prominence of the oxygen peak in the deep capillary layer indicated increased oxygen delivery from this vascular bed. The three sources of oxygen correspond to the superficial retinal capillary layer, the deep capillary layer, and the choroid. The minimum oxygen level in the inner plexiform layer between the superficial and deep capillary layers ⁹ and a region of high oxygen uptake in the outer retina were consistent with normal mature rats. The retinal histology from this animal (Fig. 1B) is shown together with labels of specific retinal layers to allow comparison with later stages of the disease. At P20 the retina showed a mild degree of deterioration of the outer segments and an accumulation of amorphous debris between the outer segments and the retinal pigment epithelium (RPE). There was evidence of deterioration of the inner segments of the photoreceptors, but other cell layers appeared largely unchanged. 

At P29 in the dystrophic RCS rat (Fig. 2A ), the oxygen level in the superficial retina had reduced, but the regions of high oxygen uptake in the inner and outer retina were still evident. The histology (Fig. 2B) showed some thinning of the outer nuclear layer, and both the outer and inner segments of the photoreceptors were less distinct. There was an increased amount of debris and vacuole formation. 

At P32 in the dystrophic RCS rat (Fig. 3A ), the level of oxygen uptake in the outer retina was clearly reduced, but the minimum oxygen level in the inner retina persisted. Histologically (Fig. 3B) , the outer nuclear layer was continuously thinned. The outer segments were indistinct because of the amorphous debris accumulation, and the remaining inner segments were markedly deteriorated. Thinning of the outer and inner plexiform layer with vacuole formation was noted. The deep capillaries were still evident. 

At P41 in the dystrophic RCS rat (Fig. 4A ), the oxygen uptake in the outer retina was dramatically reduced, but the oxygen utilization by the inner retina was still evident. Oxygen delivery from the choroid and the superficial capillaries was evident, but a contribution from the deep capillary layer could not be seen. Histologically (Fig. 4B) , thinning of the outer nuclear layer was more clearly evident, and only half the original thickness remained. The outer and inner segments were impossible to distinguish among the amorphous debris accumulation. The outer plexiform layer was thinned and hard to define in some areas. The nerve fiber layer, ganglion cell layer, and inner plexiform layer were also thinned. The deep capillaries were no longer evident. 

A very similar oxygen distribution was evident at P62 in the dystrophic RCS rat (Fig. 5A ). Histologically, the degeneration of the photoreceptors was essentially complete (Fig. 5B) , the outer nuclear layer was almost totally absent, and the space between the inner nuclear layer and the RPE was filled with amorphous debris accumulation and a few scattered nuclei. The outer plexiform layer was also absent. The inner nuclear layer was well preserved, but there was relative thinning of the inner plexiform layer and nerve fiber layer. 

The changes in intraretinal oxygen distribution at different stages of the RCS model of retinal degeneration can best be illustrated by superimposition of the mean data for each group (Fig. 6) . A total of 103 oxygen profiles were included, with at least 15 from each age group (2 or 3 profiles for each animal). To accommodate the marked degree of retinal thinning as the degeneration progressed, the data were aligned with respect to the most consistent feature across all ages, the inner retinal minimum oxygen tension. Figure 6 demonstrates the intraretinal oxygen distribution before, during, and after the loss of oxygen uptake by the photoreceptors. Thus, at all ages the intraretinal oxygen distribution in the dystrophic RCS rats exhibited a minimum oxygen tension between the superficial and deep capillary layers of the retinal circulation, reflecting a significant oxygen uptake by the inner plexiform layer. ⁹ In the outer retina, before approximately P30 there was a high level of oxygen uptake in the region of the inner segments of the photoreceptors, which was more pronounced at P20 than that seen in normal mature rats. ⁹ However, at later stages in the dystrophic RCS model, the oxygen distribution in the outer retina changed markedly, and the oxygen uptake by the photoreceptors was massively reduced. 

The mean values for oxygen tension in the choroid in the dystrophic RCS rats at all ages was not significantly different from that previously reported for normal mature rats under equivalent ventilation conditions. ⁹ The lowest oxygen tensions seen within the inner retina in the dystrophic RCS rats was also not different from that in normal mature animals. ⁹ In the superficial retina in the P20 dystrophic RCS group, the oxygen level (28.2 ± 0.6 mm Hg) was significantly higher than in normal mature rats. This, we believe, was a reflection of the oxygen contribution of the hyaloid artery system, which is still patent in such young animals. Measurements of oxygen tension in the vicinity of the hyaloid vessels in the youngest RCS rats confirmed that they were indeed a source of vitreal oxygenation. 

In comparison to mature rats, the influence of the deep capillary layer and the trough in the outer retina was more evident in the younger (P20) dystrophic RCS rats. ^{8 9} However, the minimum oxygen level in the outer retina was not significantly lower than in mature rats. This suggests that much of the additional oxygen contribution from the deep capillary layer was consumed within the outer retina. To examine this further, we performed a series of measurements in age-matched RCS-rdy ⁺ control rats. A typical profile is shown in Figure 7 for a P20 RCS-rdy ⁺ control animal, together with a retinal section from that animal. The average data from all P20 RCS-rdy ⁺ control animals tested (n= 6) are shown in Figure 8 , along with the mean data for the P20 dystrophic RCS rats (n= 6). The notable oxygen contribution from the deep capillary layer and the outer retinal trough were also evident in the RCS-rdy ⁺ control group at P20. There was no significant difference in oxygen level at any retinal location between the dystrophic RCS and RCS-rdy ⁺ groups. The intraretinal oxygen distribution in more mature RCS-rdy ⁺ control rats is shown in Figure 9 for the P40 to P50 age group (n = 8). The intraretinal oxygen distribution was indistinguishable from that seen in mature Sprague–Dawley rats. ^{8 9}  

The effect of dark adaptation was investigated in a subgroup of P20 dystrophic RCS rats (n = 5) and their age-matched control animals (n = 5). Figure 10A shows a pair of intraretinal oxygen measurements under light-adapted and dark-adapted conditions in a dystrophic RCS rat. The equivalent data for an RCS-rdy ⁺ control rat at P20 are shown in Figure 10B . Dark adaptation caused a reduction in the oxygen tension in the region of the outer retinal minimum in both groups. The magnitude of the effect was not significantly different between the dystrophic RCS and RCS-rdy ⁺ control groups: 6.1 ± 0.5 (n = 5) mm Hg, and 5.1 ± 0.4 (n= 5) mm Hg, respectively. The minimum oxygen level in the outer retina under dark-adapted conditions did not reach zero in either group, reducing to mean values of 5.3 ± 0.6 mm Hg and 7.7 ± 1.3 mm Hg in dystrophic RCS and RCS-rdy ⁺ control groups at P20, and these minimum values were not significantly different. 

Discussion

This study provides the first quantitative data for oxygen distribution within the retina of the dystrophic RCS rat. The changes in oxygen distribution correlated well with the loss of photoreceptors that is characteristic of this model of retinal degeneration. The loss of oxygen uptake by the photoreceptors coincided with the loss of distinction of the photoreceptor inner segments in histologic sections. In contrast, the oxygen distribution across the inner retina was much less disturbed, even at the late stages of the disease, long after the photoreceptor loss was essentially complete. This finding is consistent with the in vitro work of Graymore ¹¹ who found a significant level of oxygen uptake in isolated retinas from RCS rats with retinal dystrophy. The maintenance of inner retinal oxygen metabolism in the dystrophic RCS rat is in stark contrast to the findings from an alternative model of retinal degeneration. In the urethane-induced retinopathy model in rats, the oxygen distribution across the inner retina was markedly affected, becoming uniform, which indicates a loss of oxygen uptake in the inner retina. ¹² Histologic examination of the inner retina in these two models does not reveal any overt changes to retinal structure that could account for the very different outcomes in oxygen metabolism. ¹² Presumably, the different behavior of the inner retina is related to the different mechanisms responsible for the retinal degeneration in each case. The substantial maintenance of inner retinal oxygen uptake, even after the complete loss of signal input from the photoreceptors, is intriguing and provides further evidence that the inner retina may remain viable for considerable periods after the degeneration of the outer retina. 

In the present study, we found no evidence for decreased oxygen levels in either the retinal or choroidal vascular beds in dystrophic RCS rats. Neither was there a significant decrease in oxygen levels within the retina in the dystrophic RCS rats when compared with age matched controls or published values for mature rats. It had been hypothesized that the build-up of cell debris in the outer retina could constitute a diffusion barrier for oxygen diffusing from the choroid ⁷ and result in intraretinal hypoxia. Although our measurements cannot quantify the oxygen diffusion properties of the outer retinal layers, the absence of overt intraretinal hypoxia suggests that oxygen supply is not compromised at P20. Previous observations that supplemental oxygen exposure during a critical period (P16–P22) was able to slow down the rate of photoreceptor cell degeneration ⁵ cannot apparently be explained in terms of a simple relief from intraretinal hypoxia at the level of the photoreceptors. ⁷ However, we cannot rule out the possibility that hypoxic events before P20 somehow triggered the later photoreceptor cell loss. 

The suggestion that outer retinal oxygen uptake may be increased at P20 in both dystrophic RCS and RCS-rdy ⁺ control rats requires further investigation. A quantitative study of oxygen consumption in specific retinal layers at even earlier stages may indicate which regions are most at risk from oxygen stress during the maturation of the juvenile rat retina. Isolated retina studies have confirmed that there is a dramatic increase in total retinal oxygen consumption between P10 and P20 in control and dystrophic RCS rats. ¹¹ It is possible that an inability of the dystrophic RCS rat retina to cope with such fluctuations in energy demand may play a vital role in the subsequent disease. Such an analysis must await technological improvements to allow the measurement of the intraretinal oxygen environment in even younger animals to be accomplished. 

A similar study in normal rats would also be of interest in confirming the role that changes in oxygen supply and consumption is thought to have in the normal development processes within the retina. ¹³ Aside from the surgical difficulty in conducting work of this sort on such young animals, there are also differences in the intraocular environment when compared with more mature animals. ⁸ The hyaloid system and associated membranes are more evident in younger animals, making intraocular manipulation of microelectrodes considerably more difficult. 

Many other factors, apart from oxygen metabolism, have also been proposed as important to the disease in the dystrophic RCS. ^{1 2 14} A recent study has reported that a short period of light stress at P23 has a sustained protective effect on the photoreceptors in the dystrophic RCS rat model. ¹⁵ This protective effect was attributed to a sustained increase in basic fibroblast growth factor expression and not to any alterations in oxygen supply. ¹⁵ Winkler et al. ¹⁶ have previously demonstrated that the rat retina is remarkably resistant to hypoxic insult and have demonstrated the ability of the rat outer retina to switch to glycolytic pathways under hypoxic conditions. The interaction between these metabolic pathways provides other avenues by which manipulation of oxygen supply could have effects on retinal metabolism far more complex than the simple relief of tissue hypoxia. 

Dark adaptation in the rat did not result in the development of anoxic regions in the outer retina, a finding that is consistent with a much smaller light–dark effect in the rat ¹⁶ when compared with that reported for the cat. ¹⁷  

We have demonstrated that a consequence of the photoreceptor depletion in the dystrophic RCS rats is that oxygen levels are increased in some regions of the remaining outer retina. Other groups have proposed that oxygen toxicity may play an important role in photoreceptor degeneration. ^{18 19} Our studies were not able to assess the cause and effect of the oxygen changes that we observed in the dystrophic RCS animals. 

An intriguing aspect of the intraretinal oxygen distribution in the later stages of dystrophic RCS disease is the absence of any apparent oxygen contribution from the deep capillary layer. Our histologic observations suggest a reduction in the number of capillaries in the deep capillary layer, a point that has been confirmed quantitatively elsewhere. ¹³ It is not clear whether such changes are a consequence of photoreceptor loss or a factor contributing to the disease. 

In summary, our measurements do not support the case that a simple hypoxic insult is responsible for the pathologic changes seen in the dystrophic RCS rat. The changes in intraretinal oxygen level that are seen during the time course of photoreceptor cell depletion are consistent with a reduction in outer retinal oxygen uptake and a relative maintenance of inner retinal oxygen metabolism. 

 Supported by the National Health and Medical Research Council of Australia, and by Retina Australia.

 Submitted for publication November 2, 1999; revised March 17 and July 13, 2000; accepted July 19, 2000.

 Commercial relationships policy: N.

 

Figure 1.

 

(A) Intraretinal oxygen distribution in a dystrophic RCS rat at P20, along with a retinal section (B) from the same animal. The oxygen tension is shown as a function of electrode track distance from the surface of the retina. The retinal layers are labeled in the adjacent bar. NF/GC, nerve fiber and ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments of the photoreceptors; OS, outer segments; RPE/BM, retinal pigment epithelium and Bruch’s membrane (abbreviations apply to Figs. 1 2 3 4 5 6 7 ). Long arrows: Signs of the early stages of debris accumulation; short arrows: location of the deep capillaries. Magnification, ×344.

Figure 2.

 

Intraretinal oxygen distribution (A) in a dystrophic RCS rat at postnatal day 29, along with a retinal section (B) from the same animal. Long arrows: Debris accumulation; short arrows: location of the deep capillaries. Magnification, ×344.

Figure 3.

 

Intraretinal oxygen distribution (A) in a dystrophic RCS rat at postnatal day 32, along with a retinal section (B) from the same animal. The deep capillaries were still visible (short arrows). Magnification, ×344.

Figure 4.

 

Intraretinal oxygen distribution (A) in a dystrophic RCS rat at P41, along with a retinal section (B) from the same animal. The outer plexiform layer was missing in localized areas (thin arrows), and the deep capillaries were no longer apparent. Magnification, ×344.

Figure 5.

 

Intraretinal oxygen distribution (A) in a dystrophic RCS rat at P62, along with a retinal section (B) from the same animal. Magnification, ×344.

Figure 6.

 

Average intraretinal oxygen distribution in dystrophic RCS rats at different age groups. To accommodate the changes in retinal thickness, the oxygen profiles were aligned in relation to the inner retinal minimum, which was preserved at all stages of the disease. The loss of oxygen uptake in the outer retina in the older animals and an overall thinning of the retina were evident.

Figure 7.

 

Intraretinal oxygen distribution (A) in an RCS-rdy control rat at postnatal day 20, along with a retinal section (B) from the same animal. Magnification,× 344.

Figure 8.

 

Average intraretinal oxygen distribution in dystrophic RCS rats and RCS-rdy ⁺ control rats at P20. There was no significant difference in oxygen level at any retinal location between the two groups.

Figure 9.

 

Average intraretinal oxygen distribution in RCS-rdy control rats at P40 through P50. The intraretinal oxygen distribution was indistinguishable from that previously reported in mature rats. ⁹

Figure 10.

 

Sequential oxygen profiles in the light- and dark-adapted state in P20 dystrophic RCS rats (A) and RCS-rdy ⁺ control rats (B). Anoxic regions within the retina were not present in either group during dark adaptation, and the magnitude of the dark-induced reduction in the outer retinal oxygen minimum was not significantly different.

The authors thank Dean Darcey for expert technical assistance.