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1 From the Neuroscience Research Institute and the 3 Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara; and the 2 Department of Ophthalmology, Jikei University School of Medicine, Tokyo, Japan.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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METHODS. Retinal detachments were created in the right eyes of ground squirrels and the animals immediately placed in normoxic (room air) or hyperoxic (70% oxygen) conditions for 3 days. The retinas were sampled from different regions and investigated morphologically or immunocytochemically by light or confocal microscopy. Agarose embedded sections were immunostained with antibody probes to cytochrome oxidase, synaptophysin, medium-to-long wavelength–sensitive (M/L) cone opsin, rod opsin, excitatory amino acid transporter 1 (EAAT1), glutamate synthetase (GS), cellular retinaldehyde-binding protein (CRALBP), and peanut agglutinin (PNA) lectin. Retinal wholemounts were labeled with PNA and antibodies to short (S)-wavelength–sensitive cone opsin and rod opsin. Cell death was examined using a TUNEL assay on agarose sections or using toluidine blue staining on semithin sections.
RESULTS. The percentage of dying cells relative to the total nuclei in the photoreceptor layer was significantly reduced, and the total number of nuclei was greater in hyperoxic animals. Triple labeling using TUNEL, anti-M/L cone opsin and anti-rod opsin showed that hyperoxia had a remarkable effect both on the reduction of cone cell death and the maintenance of the overall structure of cone photoreceptors. Analysis of the retinal wholemounts demonstrated the preservation of PNA, S-cone, and rod opsin antibody labeling in the detachments maintained in hyperoxic conditions. Although the disruption of cytochrome oxidase and synaptophysin was seen in normoxic animals, there was minimal disruption in hyperoxic animals. Labeling with anti-EAAT1, anti-GS, and anti-CRALBP was increased in the Müller cells of normoxic animals with detachments, but was decreased in the hyperoxic animals.
CONCLUSIONS. Hyperoxia prevents the degeneration of both rods and cones in retinas heavily dominated by cones and mitigates the effect of detachment on Müller cell reactivity. The current results suggest that the rescue of cones is not secondary to that of rods.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Müller cells play various roles in maintaining the retinal environment. It has been suggested that they interact with photoreceptors and RPE cells in both glutamate cycling and the visual cycle.9 10 11 12 13 14 However, specific interactions between Müller cells and cone photoreceptors in vivo remain mostly unknown. Indeed, there are striking differences in the reactivity of Müller cells in the cone-dominated ground squirrel and rod-dominated cat retinas.15 Müller cells in the ground squirrel do not proliferate, upregulate glial fibrillary acidic protein (GFAP) expression, or lose their expression of various soluble proteins, as they do in the cat retina in response to detachment.
In the present study, hyperoxia had a protective effect on cone degeneration after retinal detachment in the cone-dominated California ground squirrel (Spermophilus beecheyi). This effect was dramatic, even in a region of high detachment. Moreover, we provide evidence that hyperoxia mitigates the active responses of Müller cells during detachment in this model, even though these responses are very different from those in rod-dominated retinas.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Retinal Detachments
Retinal detachments were created in the right eyes of ground
squirrels (n = 6 normoxia; n = 5 hyperoxia),
as described previously.15
16
Briefly, ground squirrels
were anesthetized with an injection of a mixture of ketamine
hydrochloride (18 mg/ml) and xylazine hydrochloride (1.2 mg/ml). A
scleral incision was made 2 mm from the limbus at the 10 o’clock
position. Using a fundus contact lens, a glass micropipette (100-µm
approximate tip diameter) attached to a micromanipulator was inserted
through the scleral incision and advanced while its progress was viewed
with an operating microscope. A solution of 0.25% sodium hyaluronate
(Healon; Pharmacia, Piscataway, NJ) in balanced salt solution (Alcon,
Fort Worth, TX) was injected slowly through the pipette to maintain a
constant detachment height. By this procedure, a single medium-sized
detachment was produced in the inferior retina. The scleral and
conjunctival incision sites were closed with 8-0 nylon suture. The
height of the detachment was noted at this time, through the surgical
microscope, and at the time of the animal’s death by observation
through a dissecting microscope.
Oxygen Exposure
Animals were placed in standard cages which in turn were
put inside a box made of clear plastic, built to function as an oxygen
chamber. The level of oxygen in the box was set and maintained by a
computer-controlled feedback device (Oxycycler; Reming Bioinstruments,
Redfield, NY). Ground squirrels were exposed to 70% oxygen
immediately after the surgery and remained in the chamber for 3 days.
The animals were provided food and water as usual, and ambient
illumination was kept on a 12-hour/12-hour light–dark cycle. In
the descriptions of our experiments, "hyperoxia" refers to
70% oxygen, as described previously.1
2
17
Tissue Preparation
Three days after detachment surgery, the animals were killed
with an overdose of sodium pentobarbitone (120 ng/kg), and the eyes
were enucleated and immersion fixed for 10 minutes in 4%
paraformaldehyde in sodium cacodylate buffer (0.1 N; pH 7.4). The
cornea and lens were then removed and the eyecup was cut in half. One
half of the tissue was stored in the fixative solution. From this
sample, small areas of retina were excised and embedded in
low-melting-point agarose for immunocytochemical analysis by confocal
microscopy. The other half was immersion fixed in 1% glutaraldehyde
and 1% paraformaldehyde in sodium phosphate buffer (0.086 M; pH 7.3)
overnight at 4°C for high-resolution transmitted light or
ultrastructural analysis. This tissue was then fixed in
phosphate-buffered osmium tetroxide (2%) for 1 hour and embedded in
Spurr resin.
Sampling Area
Previous immunocytochemical labeling experiments have provided
maps of the density distribution of cones and rods in the ground
squirrel retina.18
The density for the cones reaches a
plateau 2 mm inferior to the elongated optic nerve head, defining a
horizontally oriented visual streak (VS). Rod density is lowest in the
VS, where they comprise less than 5% of the local photoreceptor
population, and increases conspicuously in the ventral retina, where
the rods achieve 30% of the population.
In this series, to examine the effect of detachment height as well as areas with differences in rod population densities, three different regions within the detached retinas were chosen as sample areas. Figure 1 shows examples of these sampled areas in a photograph of a detached retina. Sampled areas A and C were approximately 1 to 2 mm from the superior and inferior detachment edges, respectively, and area B was from the center of the detachment. The length of detached retina averaged 6.95 ± 1.05 mm (range, 5.60–8.34). The sampled area and the size of the detachment were measured on computer (Image Tool software; University of Texas Health Science Center, San Antonio, TX). Similar sampling areas were used for the retinas embedded in resin.
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The primary antibodies used in this study were a mouse monoclonal antibody to rod opsin (Rho4D2, provided by Robert Molday, University of British Columbia, Vancouver; 1:50), two rabbit polyclonal antisera to short wavelength-sensitive (S) and medium/long wavelength-sensitive (M/L) cone opsins (JH455 and JH492 provided by Jeremy Nathans, Johns Hopkins Medical School, Baltimore, MD; both 1:1000), a mouse monoclonal antibody to cytochrome oxidase (1 µg/ml; Molecular Probes, Eugene, OR), a rabbit polyclonal antibody to synaptophysin (1:100; Dako, Carpinteria, CA), a rabbit polyclonal antiserum to cellular retinaldehyde binding protein (CRALBP, 1:400; provided by John Saari, University of Washington, Seattle, WA), a rabbit polyclonal antibody to glutamine synthetase (GS, 1:600; provided by Paul Linser, University of Florida, St. Augustine, FL), and a goat polyclonal antibody to excitatory amino acid transporter 1 (EAAT1, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA). In addition, staining with biotinylated peanut agglutinin (PNA) lectin (400 µg/ml; Vector Laboratories, Burlingame, CA) was used on some sections.
Retinal Wholemount Immunocytochemistry
Retinal wholemount immunocytochemistry was performed according
to the following protocol. Retinas were removed and rinsed three times
in PBS. They were incubated with blocking solution for 6 hours and
incubated in biotinylated PNA, anti-rod opsin and -S opsin (all at
1:10,000) for 3 days. After the incubation, they were washed in PBTA
and then incubated in a mixture of streptavidin-Cy3 and anti-mouse and
-rabbit IgG conjugated to the fluorochromes Cy5 and Cy2 respectively.
The retinal wholemounts were rinsed in PBTA and mounted with the
photoreceptor side up.
TUNEL Assay
To detect dying (apoptotic) cells, we performed terminal
deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin
nick-end labeling (TUNEL), as described previously.17
19
The agarose-embedded sections were used for this series. After the
sections were rinsed three times in PBS, they were immersed in 70%
alcohol for 30 minutes, followed by a wash in double-distilled water.
They were then incubated in 1% citrate and 1% Triton in PBS at 4°C
for 4 minutes and, after washing, were placed in terminal
deoxynucleotidyl transferase (TdT) buffer (Roche Molecular
Biochemicals) at room temperature for 30 minutes, followed by reaction
with TdT enzyme and 2 µM biotinylated deoxyuridine triphosphate
(dUTP; Roche Molecular Biochemicals) at 37°C for 120 minutes. The
sections were then washed in SSC (150 mM sodium chloride and 15 mM
sodium citrate, pH 7.4) for 15 minutes. After a rinse in PBS,
avidin-Cy2 or -Cy3 (1:200) was added for 3 hours, at which time the
sections were examined with a confocal microscope. For double or triple
labeling, the sections were incubated with primary antibodies overnight
after the TUNEL procedure and, after rinsing, were reacted with
appropriate fluorescent secondary antibody.
TUNEL-positive cells, which were regarded as dying cells, were counted in four areas of each section from three different eyes. The percentage of dying photoreceptor cells was expressed as the number of TUNEL-positive cells divided by the total number of nuclei in the outer nuclear layer (ONL). The nuclear counts were represented by the number of cells per millimeter of retinal length.
Light Microscopic Analysis
For light microscopy, three different eyes were embedded in
resin, divided into four regions extending from superior to inferior
retina, sectioned at 1 µm, and stained with toluidine blue. The cells
with dark nuclei were counted as representative of dying
photoreceptors. In this study, the identity of the tissue was masked
from the observer.
Statistical Analysis
All data are presented as means ± SD. The cell counts for
the study of photoreceptor cell death were analyzed by one-way ANOVA
and Student’s t-test. P < 0.05 was
considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Quantitation of the Effects of Hyperoxia
Figure 3
reflects how hyperoxia effects the relative percentage of dying cells
and the actual number of nuclei in the ONL, as obtained from cell
counts in semithin resin sections. In normoxic detached retinas, the
percentage of dying cells was highest in area B. There was no
significant difference between areas A and C. The percentage of dying
photoreceptors in the oxygen-treated eyes was significantly decreased
compared with that in untreated eyes (P < 0.01). In
contrast, the number of nuclei in the ONL was significantly greater in
the hyperoxic detached retinas (P < 0.01), where the
effects were dramatic in all areas.
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Anti-Cytochrome Oxidase and Anti-Synaptophysin.
In normal retina, immunocytochemical labeling of the outer retina with
the antibody to cytochrome oxidase occurred mainly in the IS of
photoreceptor cells (Fig. 5A)
. At 3 days after detachment, the IS were disrupted and their labeling
was faint (Fig. 5B)
. In contrast, the oxygen-treated eyes showed less
of a reduction of labeling with this antibody (Fig. 5C)
, even though
the IS morphology was far from normal.
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The Effects of Detachment Height: Triple-Labeling Studies.
To further examine the correlation between detachment height, TUNEL and
the expression patterns of various proteins, we performed
triple-labeling experiments with TUNEL, anti-cytochrome oxidase, and
anti-synaptophysin (Figs. 5D
5E
5F
5G)
as well as TUNEL, anti-rod, and
anti-M/L cone opsins (Fig. 6)
. In these studies the transition zone from attached to detached
retinas (Figs. 5D
5E
6A
5B)
was also included and comparisons made
between normoxic and hyperoxic conditions. In both experiments the
transition to a higher detachment showed a more intense TUNEL signal
(blue in Fig. 5
; red in Fig. 6
) and an attenuation of staining by the
other antibodies in the photoreceptor layer. A comparison of normoxic
(Figs. 5D 6A)
and hyperoxic (Figs. 5E
6B)
retina showed the same
trend but an overall attenuation of the response. The same effects are
shown at higher magnification in Figures 5F
and 5G
and 6C
and 6D . One
striking observation we made throughout was that cells double labeled
with TUNEL and the antibodies to rod opsin were rarely observed,
although the rods showed other characteristic signs of photoreceptor
degeneration.15
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Anti-CRALBP and PNA.
Immunolabeling with the antibody to CRALBP occurred throughout the
Müller cell cytoplasm and the RPE cytoplasm in normoxic attached
retina (Fig. 8G)
. After detachment for 3 days, the signal was increased
in the Müller cell processes, especially noticeable where these
filled in the ONL as photoreceptor cells were lost (Fig. 8H)
. In
hyperoxic detached retina, there was more PNA labeling of cone matrix
and less labeling with the anti-CRALBP antibody (Fig. 8I)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we compared the use of the TUNEL assay on agarose sections and toluidine blue staining of pyknotic nuclei in resin sections as ways of evaluating cell death. As another measure of cell death, we counted the remaining total nuclei in the ONL on both types of sections. The use of these different quantitative methods provided us with confidence in our ability to evaluate the effects of hyperoxia on cell death and survival in the ground squirrel retina. Three days’ treatment in hyperoxia had marked effects on reducing cell death as assessed by TUNEL, or morphology, or simply by the size of the remaining population of photoreceptor cells. The efficacy of this treatment was dramatic in all regions, regardless of height of the detachment and rod distribution. It may be somewhat surprising that the oxygen supplementation was useful in rescuing cells in the highest detached region, because recent modeling analysis predicted that hyperoxia could rescue photoreceptors during small and low detachments.7 8 Certainly, we found the best evidence for rescue in the shallower detached areas (Figs. 5 6) but saw some effect even in the region of highest detachment. These results from the ground squirrel detachment model may differ from the predicted modeling outcome for several reasons. First, as Linsenmeier and Padnick-Silver8 point out, convection in subretinal fluid during eye movements may enhance the effects. Second, we do not rule out the possibility that some flattening of detached retina may occur during an episode of detachment. Third, oxygen supply from the retinal circulation may supplement the demand during the detachment.7 8 24 Finally, the effects on photoreceptors may be indirect, mediated through other retinal cells such as Müller cells, which border both the subretinal space and the retinal circulation. Although retinal and choroidal circulation and their respective contribution to the ONL have not been studied in ground squirrels, taken together, the present findings suggest that supplemental oxygen reaches the photoreceptors of the detached retinas generating a positive effect in the 3-day detachments.
Our combined immunocytochemical and TUNEL data in this model suggest that most rods do not show apoptosis in the 3-day detachments; indeed, rods in this species may be more resistant to the effects of detachment than the cones. The result indicates that most of the dying photoreceptors we observed were cones, and that oxygen supplementation had a marked effect on reducing cone cell death. These data would seem to indicate that oxygen deprivation may be one of the major factors influencing the survival of cone photoreceptors after detachment.
Although some previous studies suggest it,25 26 27 28 we have shown for the first time quantitative evidence that photoreceptor cell death has a positive relationship with the height of the detachment. Therefore the present study provides specific evidence that cone cell death is related to retinal elevation during an episode of detachment.
Based on our morphologic, immunocytochemical, and wholemount data, oxygen supplementation appears to better maintain many aspects of photoreceptor cells in a cone-dominant retina, just as it does in the rod-dominated feline retina.2 This result suggests that oxygen supplementation may improve cone metabolism through one or more effects and, as in the case with cell death, there is also a positive relationship with the height of the detachment. Cytochrome oxidase is a key enzyme involved in adenosine triphosphate (ATP) generation within the mitochondria and is an indicator of neuronal oxidative capacity.29 30 The synaptic vesicles found in the synaptic terminals of photoreceptors each contain approximately 10,000 glutamate molecules.31 32 Thus, our results may provide further evidence that the major effect of detachment is to induce hypoxia in the photoreceptor layer resulting in disturbance of mitochondrial ATP production which could lead to cone metabolic death and may also result in abnormal glutamate cycling. The data suggest that hyperoxia could generate its dramatic effect on cone survival by maintaining ATP production in the mitochondria and/or maintaining functional capacity in physiological processes such as glutamate cycling.
Recently, we reported a distinct and dramatic difference in the reactivity of Müller cells in the rod-dominant feline retina compared with the cone-dominant squirrel retina.15 By comparison with cat Müller cells, those in ground squirrel retina appear to react in reverse with respect to proliferation and the expression of GS, CRALBP, carbonic anhydrase C, and intermediate filament proteins after detachment.33 In the current study, we focused on EAAT1 and GS, because of their important roles in glutamate cycling in the Müller cells. EAAT1 acts to remove glutamate from the synaptic cleft,34 and GS is the primary enzyme for glutamate metabolism,34 converting glutamate to glutamine in Müller cells. It has been suggested that the level of GS in mature Müller cells may be determined by cell interactions at the OPL and ONL.35 Thus, EAAT1 and GS may be jointly related to glutamatergic neurotransmission and cycling.34 Our results showing that EAAT1 and GS immunoreactivity were upregulated in normoxic detached retina but not in the hyperoxic detached retina, may suggest that the Müller cells have an active response to changes in retinal glutamate levels that occur as a result of detachment. A previous report indicated that RPE cells, photoreceptor cells, and bipolar cells also possess a glutamate transporter to take up glutamate from the extracellular space.36 37 38 39 Thus, in normal outer retina, there is a well-organized glutamate uptake system to eliminate excess glutamate. The increase in EAAT1 and GS that appeared to occur in the ground squirrel Müller cells may indicate an acceleration of glutamate cycling in them in normoxic detached retinas, perhaps induced by changes in glutamate balance in the outer retina. The suppression in hyperoxic detached retinas may result from the maintenance of that system in the surviving photoreceptors. It is interesting that in the feline retina there are also apparent shifts in glutamate balance after detachment40 but in that case, there is a dramatic decrease in the GS content of Müller cells. It is significant that in both animal models the effectiveness of oxygen is to return GS toward normal levels of expression.
There is increasing evidence that Müller cells are linked to the visual cycle through CRALBP,41 42 a retinoid-binding protein, found both in Müller cells and RPE. It has been suggested that cones may regenerate their visual pigment through Müller cells in the absence of RPE.10 This suggests that CRALBP upregulation in the Müller cells may help maintain cone metabolism through the regeneration of cone visual pigments. The improved overall cone survival and presumed metabolic capacity due to hyperoxia would explain the absence of CRALBP upregulation in the oxygen treated eyes.
In conclusion, we have shown clearly that oxygen supplementation has a protective effect on cone degeneration during retinal detachment in a second species, one whose retina is cone dominated. It reduces cell death and improves the overall structural integrity and probably the metabolic state of the photoreceptor cells. This in turn appears to be accompanied by a metabolic change in glutamate cycling and perhaps the visual cycle between the photoreceptors and the Müller cells. We believe that this study strengthens the case for hyperoxia as a candidate for improving the ability of the cone photoreceptors to recover in reattachment. In addition, this study suggests that there may be special metabolic interactions, which are related to visual function, between cone photoreceptors and Müller cells in the macula. Hyperoxia may also be an important adjunct to surgical procedure that involves the production of a macular detachment, such as macular translocation in the treatment of AMD.43 44 45 46
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Acknowledgements |
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Footnotes |
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Submitted for publication July 3, 2001; revised August 4, 2001; accepted September 5, 2001.
Commercial relationships policy: N.
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.
Corresponding author: Steven K. Fisher, Neuroscience Research Institute, Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA 93106-5060. fisher{at}lifesci.ucsb.edu
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) of
the detachment to high detachment. The increase of TUNEL positivity and
the reduction of anti-cytochrome oxidase and anti-synaptophysin
staining showed a positive relationship with the height of the
detachment in normoxic detached retina (D). The
hyperoxia-treated retina showed a decrease of TUNEL positivity and
minimal reduction of anti-cytochrome oxidase and anti-synaptophysin
labeling (E). Higher magnification better showed the
respective differences of labeling (F, G) using
the triple-labeling protocol. (
