(Investigative Ophthalmology and Visual Science. 2001;42:842-849.)
© 2001
by The Association for Research in Vision and Ophthalmology, Inc.
Extended Photoreceptor Viability by Light Stress in the RCS Rats but not in the Opsin P23H Mutant Rats
Izhak Nir1,
Joseph M. Harrison2,
Changdong Liu3 and
Rong Wen3
1 From the Departments of Pharmacology and
2 Ophthalmology, The University of Texas Health Science Center at San Antonio; and the
3 Department of Ophthalmology, University of Pennsylvania, School of Medicine, Philadelphia.
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Abstract
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PURPOSE. To determine the effect of light stress on retinal function and long-term
photoreceptor viability in Royal College of Surgeons (RCS) rats and the
applicability of the light treatment to the opsin P23H mutant rats.
METHODS. RCS rats at postnatal day (P)23 were illuminated with 120 foot-candles
(fc) white light for 10 hours. Photoreceptor survival and basic
fibroblast growth factor (bFGF) expression were measured at P60 and
P83. Retinal function was evaluated by electroretinography. Opsin P23H
transgenic rats were treated with light at P28 and analyzed at P70 for
photoreceptor viability, ultrastructure, and bFGF expression.
RESULTS. Light-treated RCS rats at P60 had four to five rows of nuclei versus
one to two rows in untreated littermates. The average amplitude of the
ERG b-wave was 28 µV in treated rats, compared with 6 µV in
untreated littermates. By P83 there was still significant preservation
of the ONL in treated rats. Immunoblot analysis showed a high
expression of bFGF in the treated retinas even 2 months after
treatment. Illumination of P23H rats at P28 with 120 fc white light for
10 hours caused substantial photoreceptor cell death, although bFGF
expression was upregulated. Lowered illumination dosages continued to
cause photoreceptor damage until levels were reached that neither
caused damage nor enhanced survival.
CONCLUSIONS. Although light stress promotes photoreceptor survival and function in
the RCS rat, it elicits death signals in the P23H rats that may not be
overcome by survival-promoting factors. Therefore, use of light stress
to promote photoreceptor survival should be considered with regard to
sensitivity of the mutation to light damage.
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Introduction
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Exposure of the retina to intense visible light elicits a
series of reactions leading to apoptotic photoreceptor cell
death.1
2
The severity of the damage is determined by the
light’s intensity, length of exposure, and
wavelength.3
4
5
Recently, it has been recognized that in
addition to its damaging effect, exposure to intense light also evokes
a response that protects photoreceptors. By using a preconditioning
paradigm, it has been shown that exposure to intense light for a short
period protects photoreceptors from subsequent exposure to damaging
light.6
Because exposure to light stimulates the
expression of basic fibroblast growth factor (bFGF) in the
retina,7
8
the protection induced by light could be
attributed to elevated levels of bFGF which are measured in the
light-stressed retinas.6
Direct involvement of bFGF in
protection of photoreceptors was demonstrated in studies in which
intraocular injections of bFGF reduced photoreceptor cell death due to
excessive light.9
10
11
In a previous study, we showed that light treatment enhances
photoreceptor survival in dystrophic retinas of Royal College of
Surgeons (RCS) rats. Illumination with 12 hours of bright light at
postnatal day (P)23 resulted in the retention of numerous photoreceptor
nuclei at P42. Elevated expression of bFGF was measured in the treated
rats.12
These findings extend the light–rescue paradigm
to the retinal degeneration that is caused by a genetic mutation. In
the present study, we explored the functional significance of enhanced
survival in RCS rats, evaluated by electroretinogram (ERG), and the
long-term effect of light treatment on photoreceptor survival. To
evaluate the applicability of light treatment to a different animal
model of retinal dystrophy, the transgenic P23H rat was investigated.
The RCS rat is characterized by reduced capacity of the pigment
epithelium to phagocytose shed rod outer segment tips. The phagocytic
malfunction results in accumulation of membranous debris in the
subretinal space and subsequently to apoptotic photoreceptor cell death
and blindness.13
14
15
The mutation in the RCS rat was
recently localized to a receptor tyrosine kinase mertk
gene.16
The P23H transgenic rats carry a point mutation in
the opsin gene in which histidine in position 23 is replaced by
proline.17
18
19
The mutation leads to progressive loss of
photoreceptors and to blindness, possibly due to misrouting of
transduction proteins or defective disc membrane
morphogenesis.20
21
In humans, the P23H mutation is the
most common mutation found in patients with autosomal dominant
retinitis pigmentosa.22
Mutant rats were treated with light at a postnatal age before onset of
measurable degenerative cell death. Photoreceptor survival, bFGF
expression, and retinal function were evaluated. Although light
treatment significantly enhanced photoreceptor survival and function in
RCS rats, light stress negatively affected photoreceptors in the P23H
retinas. Therefore, the potential use of light therapy to extend
photoreceptor survival in dystrophic retinas should be selective.
Appropriate animal models for the specific genotype should be evaluated
to determine the consequences of the light treatment.
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Methods
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All animal procedures were performed in accordance with the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research.
Animals
Tan-hooded, pink-eyed RCS rats23
and transgenic
P23H rats18
(line 3, kindly provided by Matthew M. LaVail,
University of California, San Francisco) were raised at the local
animal facility. The rats were maintained under a diurnal cycle of 12
hours dark and 12 hours light with an illumination intensity of 3
foot-candles (fc; 1 fc = 10.76 lux). Normal Sprague–Dawley rats
(Harlan, Indianapolis, IN) were kept under the same illumination
conditions for at least 2 weeks before experimentation.
Light Stress
Bright white light was produced by two fluorescent lamps, 40 W
each (Cool White; General Electric, Fairfield, CT).23
Illumination intensities were determined by the distance of the lamps
from the cage floor.12
In some experiments, the light was
filtered through a green sheet of Plexiglas (number 2092; DuPont,
Wilmington, DE) to produce transmitted light with a peak at 520
nm.3
Temperature inside the cage was maintained at 22°C
to 24°C. Untreated littermates were kept under a low cyclic light
level (3 fc) and used as control subjects.
Morphology and Nuclei Measurements
Eyes were fixed for 3 hours in 4% formaldehyde and 2%
glutaraldehyde (in 0.1 M phosphate buffer; pH 7.0). After they were
maintained for 30 minutes in fixative, the eyes were bisected along the
vertical meridian. Tissue was postfixed with 1%
OsO4 and embedded in Araldite.12
Sections of appropriate thickness were used for structural analysis
under light and electron microscopy.
Photoreceptor survival was determined by quantitative assessments of
photoreceptor nuclei in the outer nuclear layer (ONL). Measurements
were performed on thick (1-µm) sections cut along the vertical
meridian, extending from the optic nerve head to the ora
serrata.23
The width of the ONL was measured at 100-µm
intervals along the posterior–periphery axis, in both the superior and
inferior hemispheres. In some experiments, the number of photoreceptor
nuclei was counted in 100-µm segments of the retina from the optic
nerve head to the ora serrata. All measurements were performed using an
eyepiece graticule.
bFGF Immunoblot Analysis
Retinas were dissected, snap-frozen in liquid nitrogen, and
stored at -80°C. Protein preparation and immunoblot analysis were
performed as previously described.6
Briefly, total protein
was obtained by homogenizing retinas in a lysis buffer. The tissue
homogenate was centrifuged, the supernatant collected, and the protein
concentration determined. Total proteins (100 µg) of each sample were
size fractionated on sodium dodecyl sulfate (SDS)–polyacrylamide gels.
Blots were probed with anti-bFGF antibodies (Santa Cruz Biotechnology,
Santa Cruz, CA). Signals were recorded on film and scanned on a
densitometer (Molecular Dynamics, Sunnyvale, CA). Quantitative analyses
of the digitized data were performed by computer with image analysis
software (ImageQuant; Molecular Dynamics). To ensure that
the signals were recorded below saturation of the film, a blot was
recorded on film through a series of exposures with durations ranging
from 2 to 20 minutes. Two such records of different exposure duration
were analyzed quantitatively. The consistency of the results indicated
that the film was not saturated.
Electroretinogram
Twelve-hour dark-adapted rats were prepared under dim red
illumination. The rats were anesthetized with intraperitoneal ketamine
(75 mg/kg) and xylazine (10 mg/kg). Pupils were dilated with 1%
tropicamide. The corneas were kept moist with 2.5% hydroxypropyl
methylcellulose. The rats were placed prone on a catalytic heating pad
(Deltaphase Isothermal Pad; Braintree Scientific, Braintree, MA). ERGs
were recorded with an active carbon fiber electrode on the cornea, an
indifferent gold ring electrode on the tongue, and a ground platinum
needle subcutaneous electrode near the tail. The rat eye was centered
in the aperture of the Ganzfeld (model 2503; LKC Technologies,
Gaithersburg, MD). Bioelectric signals were amplified with a gain of
10,000 and filtered from 1 to 1000 Hz (model P5 amplifier; Grass,
Quincy, MA). The amplified signals were digitized, recorded, and
averaged by a digital oscilloscope (model 4094 B; Nicolet, Madison,
WI). The signals were digitized at 10,000 samples/sec with 12-bit
analog-to-digital resolution in a trace of 0.396 seconds’ duration.
The flash stimulus source (model PS22; Grass) that evoked the ERG was
seen by diffuse reflection off the interior surface of the Ganzfeld.
Flash luminance was 9 scotopic candelas (cd)/sec2
(model 1700 with SED033 detector and ZCIE scotopic filter;
International Light, Newburyport, MA). The rats were dark adapted a
further 10 minutes after preparation. Two ERGs were recorded with a
minimum interval of 10 seconds. The b-wave amplitude was measured from
the preceding trough to the peak of the b-wave.
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Results
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Photoreceptor Survival and ERG Measurements in Light-Treated RCS
Rats
Animals were treated with a single dose of 120 fc light for 10
hours at P23 and analyzed at P60, an age by which major cell loss
occurred in untreated RCS rats. A significant increase in photoreceptor
survival was noted in the 3-mm length of retina from the optic nerve
head to the midperiphery. In the far periphery, a region approximately
1 mm from the ora serrata, protection by light treatment was very
limited. The most consistent levels of ONL width were measured at a
distance of approximately 1 to 2 mm from the optic nerve head. The
results of quantitative analysis of the ONL width in this region are
presented in Figure 1 . The average ONL width of light-treated retinas in this area was
16.73 ± 1.13 µm (n = 10), whereas in the
untreated retinas the same area measured 2.04 ± 0.3 µm (n
= 7), an eightfold difference. In normal Sprague–Dawley rats at
P56 to P66, the ONL width in the posterior retinas measured 41.7 ± 1.4 µm (n = 4). Thus, approximately 40% of the normal
ONL width is preserved in the light-treated RCS retina in the region 1
to 2 mm away from the optic nerve head. Examination of serial sections
of treated retinas revealed that the ONL survival extended to all
quadrants of the retina.
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Figure 1. Width of photoreceptor ONL in RCS rats at P60. The data are from six
rats that were illuminated with 120 fc for 10 hours at P23
(left) and four untreated control littermates
(right). Each double column depicts the superior and
inferior hemispheres of one retina. Measurements were made in the
posterior retina along the vertical meridian, in an area that extends 1
to 2 mm from the optic nerve head toward the equator. The value is an
average of 10 measurements, 100 µm apart.
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As shown in Figure 2A
, the light-treated rats at P60 had a relatively well-preserved ERG
b-wave with prominent oscillatory potentials on the ascending slope of
the b-wave. The ERG of the untreated littermates was typical of
dystrophic RCS rats and exhibited a barely detectable b-wave with no
oscillatory potentials (Fig. 2B)
. The within-animal variability of the
ERG data was very low, as illustrated by the superimposed repeated ERGs
of the light-treated and untreated rats (Figs. 2A
2B)
. Data from eight
light-treated and seven untreated RCS rats are illustrated in Figure 2C
. There were larger b-wave amplitudes in all treated rats than in
untreated littermates. The average b-wave amplitude in treated retinas
was 28 µV versus 6 µV in untreated retinas. The mean difference in
b-wave amplitudes in the two groups was significant by a two-tailed
t-test with a Welch correction for the difference in
variability (t = 4.62, P < 0.01).
Considerable variability was measured in b-wave amplitude among the
eight light-treated RCS rats, possibly reflecting different levels of
photoreceptor preservation as revealed by measurements of ONL width
(Fig. 2C)
. For comparison purposes, b-wave amplitude in Sprague–Dawley
retinas under the same stimulus conditions was 625 µV.
Long-Term Photoreceptor Survival in Light-Treated RCS Rats
The end point of photoreceptor degeneration in the retina of the
pink-eyed RCS rat occurs between P80 and P90.24
We
investigated the long-term effect of light treatment in RCS rats at
P83, 2 months after exposure to a single dose of intense light. This
selected experimental period of P23 to P83 also correlates with the
period used in a study in which application of exogenous bFGF was found
to protect photoreceptors in the RCS rat.11
By P83, the
ONL in the retinas of untreated littermates was completely absent in
some areas or reduced to a few pyknotic nuclei in a single row in other
areas (Fig. 3A
). The retinas of animals treated with light, however, contained areas
that had a considerable number of normal photoreceptor nuclei. The
surviving nuclei were unevenly distributed along the posterior
periphery axis of the retina. Whereas some sites were devoid of nuclei,
others had two to four rows of nuclei (Fig. 3B)
. Ultrastructural
analysis of surviving nuclei in light-treated retinas revealed normal
morphologic characteristics (Fig. 3C)
. Because of the great variation
in the number of surviving photoreceptor nuclei at various sites in the
retina, measurements of ONL width at given intervals produced great
variation in values. To reliably reflect the extent of increase in
photoreceptor nuclei survival, the number of nuclei along a 100-µm
segment of the retina was counted in light-treated and untreated
retinas (Fig. 4)
. The analysis was performed at a distance of 1 to 2 mm from the optic
nerve head, which corresponds to the region measured at P60 in Figure 1 . For comparison, the number of nuclei in light-treated and untreated
retinas at P60 are included (Fig. 4)
. Substantial variations in number
of nuclei between different retinas and between the two hemispheres of
the same retina were observed. However, the number of remaining nuclei
in the treated retinas was much larger than in the untreated control
retinas.
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Figure 3. Microscopic analysis of light-treated and untreated RCS retina at P83.
Treated rats were illuminated with 120 fc for 10 hours at P23.
(A) Light microscopy of the posterior retina in untreated
RCS rats showing a single row of pyknotic photoreceptor nuclei
(arrows). (B) Light microscopy of light-treated
retinas, showing several rows of photoreceptor nuclei in the ONL and a
layer of remaining inner segments (arrow). (C)
Ultrastructure of an area in the posterior retina of light-treated
rats. Most of the photoreceptor nuclei in the selected field had normal
morphology (N), a few were pyknotic (P). A narrow rod inner segment
layer (RIS) and outer segment (ROS) debris were seen. Magnification,
(A, B) x500; (C) x3040.
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Figure 4. The number of photoreceptor nuclei in light-treated
and untreated control RCS retinas at P80 through P90.
Treated rats were illuminated with 120 fc for 10 hours at P23 and
analyzed from P80 through P90. For comparison, the number of nuclei in
retinas of RCS rats that were similarly treated at P23 and killed at
P60 and the number in untreated control retinas is shown. Each double
column depicts superior and inferior hemispheres of one retina.
Measurements were made in the posterior retina along the
vertical meridian, in an area 1 to 2 mm from the optic nerve head
toward the equator. The number of nuclei in 100-µm segments of the
retina was counted. The value for each hemisphere is an
average of 10 sequential measurements that cover a 1000-µm-long area
of retina.
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For further quantitative assessment, the total number of nuclei was
counted along the full length of a 1-µm-thick section, which included
the complete length of the retina from the optic nerve head to the ora
serrata, a distance of approximately 4 mm. Superior and inferior
hemispheres along the vertical meridian were analyzed in 14 eyes of
treated rats. Four eyes of untreated littermates were used as control
retinas. In treated retinas, 665.6 ± 45 (mean ± SD; n
= 14) nuclei per section were counted, whereas in untreated
retinas the number was 186.1 ± 35.3 (n = 4). As noted,
all the remaining nuclei in untreated retinas were highly pyknotic.
Thus, by P83 there were 3.5 times more photoreceptor nuclei in retinas
treated with a single dose of intense light 2 months earlier at P23.
The ERG of two treated RCS rats at P83 showed b-wave amplitudes of 9.6
and 9.3 µV, whereas an untreated control RCS rat at P83 had a b-wave
amplitude of 2.2 µV. Thus, the survival of photoreceptor nuclei with
normal morphology in light-treated RCS rats at P83 was reflected by the
functional measurements.
bFGF Protein Expression in Light-Treated RCS Retinas at P60 and P83
For analysis of bFGF protein expression, retinas were collected at
P60 and P83 from RCS rats that had been treated with a single exposure
of 120 fc light for 10 hours at P23. Retinas of untreated littermates
served as control specimens. Immunoblot analyses revealed a significant
increase in bFGF protein in the treated retinas at both P60 and P83. At
P60 (Fig. 5A
), there was a 40% increase in the 18-kDa bFGF. A higher increase of
102% in the sum of the two high-molecular-weight forms (24 and 22.5
kDa) of bFGF was also seen. By P83, there was still a significant
upregulation of bFGF (Fig. 5B)
. Light-treated retinas had a 42%
increase in the 18-kDa bFGF protein and a 50% increase in the sum of
the two high-molecular-weight forms of bFGF.
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Figure 5. Expression of bFGF at P60 and P83 in RCS retinas treated with bright
light (120 fc) for 10 hours at P23. The amount of bFGF proteins was
determined by immunoblot analysis. Retinas were collected at P60 and
P83 from animals that had been treated with a single dose of 10 hours
of bright light at P23. Each lane represents a sample of 100 µg total
protein from two retinas of the same animal. Proteins of bFGF were
detected as three distinct bands of 24, 22.5, and 18 kDa.
(A) P60: Data for each band were averaged from
lanes 1 through 4 (untreated control
retinas) and lanes 5 through 8
(light-treated retinas). The amount of the 18-kDa form of bFGF protein
was 40% higher in the light-treated retinas (n = 4,
P < 0.001; Student’s t-test for all
statistical analyses). The sums of the 24- and 22.5-kDa forms of the
bFGF proteins were 102% higher in the light-treated retinas (n
= 4, P < 0.01). (B) P83: Data for each
band were averaged from lanes 1 through 3
(untreated control retinas) and lanes 4 through
6 (light-treated). The amount of the 18-kDa form of bFGF
protein was 42% higher in the light-treated retinas (n = 3;
P < 0.01). The sum of the 24- and 22.5-kDa forms of
the bFGF proteins were 50% higher in the light-treated retinas
(n = 3, P < 0.01).
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Photoreceptor Viability in Light-Treated Transgenic P23H Rats
In P23H (line 3) rats, the ONL is reduced to four to seven rows of
nuclei by P60.18
In preliminary studies, we did not find
measurable photoreceptor cell loss before P30. Therefore, to evaluate
the effect of light stress, P23H rats were treated at P28 and analyzed
at P70. In the first set of experiments, the same protocol as was
applied to RCS rats was used with P23H rats. Animals were illuminated
for 10 hours with 120 fc of bright light. Unlike the results in RCS
rats, illumination of P23H rats at P28 with bright light for 10 hours
caused substantial photoreceptor cell loss in all regions of the retina
at P70, when compared with untreated rats. In Figure 6
, the ONL width in control (Fig. 6A)
and light-treated (Fig. 6B)
P23H
rats at P70 is depicted. The posterior region of the superior retina
where the light damage was more pronounced is shown. Whereas the ONL in
untreated retina contained five to six rows of nuclei, it was reduced
to three to four in the light-treated retinas, indicating
light-mediated photoreceptor cell death. Ultrastructural analysis of
light-treated retinas and untreated P23H retina is shown in Figures 6C
and 6D
. In the untreated retina at P70, intact outer and inner segments
were observed. In the light-treated retina, shortened malformed outer
segments and inner segments were seen. Only four rows of photoreceptor
nuclei remained in the observed site.
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Figure 6. Light and electron microscopy of P23H retinas at P70. Sites at the
posterior region of the superior hemisphere are shown. Light
microscopy: (A) Untreated control retina showing five to six
rows of densely stained photoreceptor nuclei in the ONL. (B)
Retina treated with 120 fc for 10 hours at P28 shows that the ONL was
reduced to three to four rows of nuclei. Electron microscopy:
(C) Untreated control retina. Intact rod outer segments
(ROS) and rod inner segments (RIS) are seen. PE, pigment
epithelium. (D) Light-treated retina. Short, damaged
RIS and ROS and were seen. Four rows of remaining photoreceptor nuclei
(N) were observed. Magnification, (A, B) x690;
(C) x6620; (D) x4750.
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In view of the considerable light damage caused by 10 hours’
illumination at 120 fc, subsequent experiments with lower levels of
illumination were performed. However, considerable reduction in
illumination levels and duration continued to produce light-damage in
the P23H retina. Quantitative analysis of ONL width after different
light treatments is presented in Figure 7
. ONL width in untreated normal Sprague–Dawley rats at P55 through P65
and in untreated P23H rats at P70 was compared with that in
light-treated P23H rats at P70. A 33% reduction in ONL width in the
untreated P23H rats, compared with normal Sprague–Dawley rats, is a
measure of the degenerative photoreceptor cell loss in the P23H rat
retina. A 34.5% reduction in ONL width in P23H rats treated with 120
fc for 10 hours (at P28) is a measure of light-mediated photoreceptor
cell death (in addition to degenerative cell death). Lowering the
illumination dosage to 50 fc for 8 hours produced a 19% reduction in
ONL width in comparison with untreated P23H retinas. Further reduction
of illumination levels to 50 fc for 4 hours and use of a green filter
to eliminate possible damaging short wavelength did not prevent light
damage. Finally, reduction of light dosage to 0.5 to 1 hour at 120 fc
had no effect on the width of ONL at P70, when compared with the ONL
width of untreated littermates (data not shown). Thus, unlike the RCS
rats, the P23H rats are highly susceptible to light damage and, under a
wide range of illumination dosages, light rescue could not be obtained.
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Figure 7. Measurements of width of photoreceptor ONL at P70 in P23H rats. Retinas
of untreated normal Sprague–Dawley (SD) rats at P56 through P66 and
dystrophic P23H rats were compared with those of light-treated P23H
rats. Measurements were made in the posterior retina along the vertical
meridian, in an area extending 0.1 to 2 mm from the optic nerve head
toward the equator. Each column depicts the average ONL width in the
superior and inferior hemispheres of one retina. ONL width in untreated
P23H rat (27.8 ± 0.6 µm, n = 8) was
significantly lower than in normal untreated SD rats (41.7 ± 1.4
µm, n = 4). Illumination of P23H for 10 hours at 120
fc resulted in significant reduction in ONL width (18.2 ± 0.2
µm, n = 8), and reduction continued at illumination of
50 fc for 8 hours (22.5 ± 1.8 µm, n = 6).
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bFGF Expression in Light-Treated P23H Retina
Because light stress in the RCS rat resulted in elevated bFGF
expression that could be a cause of the observed photoreceptor rescue
in this mutant, immunoblot analysis of bFGF was performed on
illuminated P23H rat retinas to determine whether a similar response
would be observed. P23H rats were treated with a single dose of 10
hours of bright light at P28. The rats were then returned to cyclic
light, and retinas were collected 2.5 days or 5 days after treatment.
Retinas of untreated littermates served as a control (Fig. 8)
. At 2.5 days after treatment, a 1.4-fold increase in the two
high-molecular-weight forms (24 and 22.5 kDa) of bFGF protein was
observed. A still larger 2.3-fold increase in bFGF protein was measured
5 days after treatment. Therefore, light treatment produced
considerable upregulation of bFGF in the P23H retina.
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Figure 8. Expression of bFGF in P23H retinas treated with bright light (120 fc
for 10 hours) at P28. Retinas were collected at 2.5 days and 5 days
after illumination. Each lane represents a sample of 100
µg total protein from two retinas of the same animal. Proteins of
bFGF were detected as three distinct bands of 24, 22.5, and 18 kDa.
Lanes 1 through 3: untreated control
littermates; lanes 4 to 6: 2.5 days after
treatment; and lanes 5 through 6: 5 days
after treatment. A 1.4-fold increase in bFGF protein levels occurred at
2.5 days after light treatment in the two high-molecular-weight forms
(24 and 22.5 kDa; n = 3, P < 0.001)
and a 2.3-fold increase at 5 days after treatment (n =
2, P < 0.001). There was little change in the
18-kDa form of bFGF after light treatment.
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Discussion
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Evidence collected in this study reveals a surprisingly
long-lasting effect of the light-induced protective response in the RCS
retina. A single exposure to intense light at P23 extended the survival
of numerous photoreceptor nuclei to P60. By P83 the number of nuclei in
light-treated retinas was still 3.5-fold higher than in untreated
control retinas. The dose of light exposure used in the present work
(120 fc for 10 hours) has been found to cause minimal damage to normal
photoreceptors, together with maximal protection from subsequent light
damage.6
Although it has been reported that the RCS rat is
more sensitive to light damage than normal animals,25
26
it is apparent that the extent of damage caused by 10 hours’ exposure
to 120 fc light at P23 is not significant and that the net outcome is a
long-term increase in photoreceptor survival.
The increased survival of photoreceptors in the light-treated RCS
retina was clearly reflected in enhanced retinal function as measured
by the ERG. There was a large and significant difference in the ERG
b-wave amplitudes of the light-treated RCS retinas and the untreated
littermates. The b-wave amplitudes of the light-treated rats ranged
from a factor of 1.7 to a factor of 11 times greater than those of the
untreated rats. The barely detectable ERG observed in the present study
in untreated RCS rats at P60 is in agreement with previous reports of
reduced ERGs in RCS rats of similar age.25
27
The larger
b-wave amplitudes in the light-treated rats are consistent with the
presence of a larger number of functioning photoreceptors in the
treated retinas. Previous studies in which b-wave amplitude was
extensively used to measure retinal function in light damage and
retinal degeneration models demonstrated that the b-wave amplitude is
related to the number of remaining photoreceptors.28
29
A major increase in bFGF expression observed in light-stressed RCS
retina is in agreement with previous studies in which light stress
resulted in upregulation of bFGF in normal6
7
8
and RCS
retinas.12
Evidence that bFGF is a trophic factor for
photoreceptors comes from experiments in which intraocular injection of
purified bFGF protected photoreceptors in inherited and induced retinal
degeneration9
10
11
as well as in age-related photoreceptor
degeneration.30
Adenovirally expressed bFGF was shown to
protect photoreceptors in RCS rats.31
In the present study
we were surprised to see that upregulation of bFGF protein after a
brief exposure of RCS rats to intense light at P23 lasted for up to 2
months. That the long-lasting protection of photoreceptors is
accompanied by an increase in bFGF protein may provide additional
evidence that bFGF is involved in light-induced photoreceptor
protection.
In the P23H rats, unlike the RCS rats, light stress did not result in
photoreceptor rescue but in increased cell death. It has been proposed
that exposure to intense light induces two opposing processes in the
retina: a degenerating process that kills photoreceptors (light damage)
and a protective response that enhances photoreceptor
survival.6
Whereas light damage was minimal in the RCS
rats after illumination for 10 hours at 120 fc, the same treatment
caused significant photoreceptor loss in P23H rats. Additional
experiments revealed that photoreceptors in the P23H rats were highly
susceptible to light damage, because cell loss was also measured at
much reduced light dosages. Thus, in the light-treated P23H rats, any
possible protection would have been masked by the excessive light
damage.
The increased susceptibility of the P23H rat to light damage over that
seen in the RCS retina may be due to differences in the nature and site
of the mutations. Although in the RCS rat photoreceptor degeneration is
secondary to a receptor kinase mutation in the pigment
epithelium,16
cell death in the opsin P23H rat is a
consequence of a primary mutation in a photoreceptor-specific gene. The
high susceptibility of the P23H rats to light damage is consistent with
previous reports of similar findings in transgenic P23H
mice.32
33
The abnormal dislocation of opsin, transducin,
and phosphodiesterase to the inner segment and outer plexiform plasma
membrane20
may increase photoreceptor vulnerability to
insults. Susceptibility to light damage in opsin P23H mutants may also
be a result of the prolonged life of phototransduction
intermediates.33
Because light treatment resulted in significant upregulation of bFGF in
the P23H retina, it is apparent that the P23H retina is capable of
responding to light stress in a manner similar to that seen in the RCS
retina. It is noteworthy, however, that the 1.4-fold upregulation of
bFGF in the P23H rat at 2.5 days after light treatment is lower than
the 5-fold increase measured in RCS rats at 2.5 days after similar
light treatment.12
Of note, a recent study reported that
subretinal transplantation of normal neuroretinal cells prolongs
survival of photoreceptors in RCS rats but not in P23H rats. It was
proposed that higher levels of trophic factors may be necessary to
promote rescue in the P23H retina.34
Thus, it is possible
that light stress in the P23H rat does not produce sufficient levels of
trophic factors to overcome intense death signals that are a result of
both light damage and the mutation.
Our results do not rule out alternative explanations. For example, the
expression and distribution of FGF receptors are known to be altered
under pathologic conditions,35
36
which would certainly
alter bFGF signaling in affected cells. Recently, an FGF soluble
receptor (SR1) was found to inhibit bFGF activity during retinal
degeneration.37
38
An increase of SR1 expression could
also counterbalance the bFGF increase.
In conclusion, the use of light to recruit endogenous survival factors
may circumvent potential harmful side effects, such as cataracts and
angiogenesis, which were observed in RCS retinas after treatment with
exogenous bFGF.10
39
However, in view of the negative
results that were obtained with the light-treated P23H rats, it is
necessary that the extent of sensitivity to light damage and levels of
upregulation of trophic responses first be determined. Mutations with
low sensitivity to light damage can then be considered for noninvasive
light therapy.
|
Acknowledgements
|
|---|
The authors thank Annemarie Brown for excellent technical
support.
|
Footnotes
|
|---|
Supported by Grants RO1-EY10286 (IN) and RO1-EY12727 (RW) from the National Institutes of Health; an unrestricted departmental research grant from Research to Prevent Blindness (JMH); and a grant from The Foundation Fighting Blindness (RW). RW is the recipient of a Career Development Award from Research to Prevent Blindness.
Submitted for publication July 20, 2000; revised October 2, 2000; accepted November 3, 2000.
Commercial relationships policy: N.
Corresponding author: Rong Wen, Department of Ophthalmology, University of Pennsylvania School of Medicine, D-603 Richards Building, Philadelphia, PA 19104. rwen{at}mail.med.upenn.edu
|
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Top
Abstract
Introduction
) had an ONL width of 25.6 ± 1.87 µm (n
= 10), whereas an eye with a lower b-wave (