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Progressive Change of Optical Coherence Tomography Scans in Retinal Degeneration Slow Mice
Naoichi Horio, MD;
Syu Kachi, MD;
Kenji Hori, MD;
Yoko Okamoto, MD;
Etsuko Yamamoto, MD;
Hiroko Terasaki, MD;
Yozo Miyake, MD
Arch Ophthalmol. 2001;119:1329-1332.
ABSTRACT
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Objective To study whether optical coherence tomography (OCT) scans correlate
retinal histologic findings with the progression of retinal degeneration in
retinal degeneration slow (rds) mice.
Methods Sensory retinal thickness (SRT) and outer retinal thickness (ORT), representing
photoreceptor cell layer, in temporal retina at a distance 1 to 2 disc diameters
from the optic disc were measured using scan profile in OCT from 6 healthy
mice (16 weeks old) and 2-week-old (n = 6), 6-week-old (n = 4), and 60-week-old
(n = 2) rds mice. Histologic sections were obtained
from Epon-embedded retinas from the corresponding location.
Results Cross-sectional OCT images correlated to the corresponding histologic
sections in each mouse. Both SRT and ORT of 2-week-old rds mice (150 ± 4 µm and 28 ± 4 µm, respectively)
lacking photoreceptor outer segments were already shorter than those of healthy
mice (174 ± 5 µm and 37 ± 6 µm, respectively) (P<.001). In 6-week-old mice, microscopic findings revealed
a decreased number of nuclei in the outer nuclear layer, and SRT and ORT (136
± 2 µm and 20 ± 1 µm, respectively) were shorter
than those of 2-week-old rds mice (P<.001). The SRT of 60-week-old rds mice
without a photoreceptor layer was remarkably reduced (120 ± 7 µm),
and no ORT could be measured.
Conclusion Our findings suggest a possible relationship between SRT and ORT, as
measured by OCT, and histologic change in retinal degenerative diseases.
Clinical Relevance The quantitative analysis obtained by OCT scans may have potential to
detect progressive change in degenerative retina and may be used in studying
human retinal degeneration.
INTRODUCTION
OPTICAL COHERENCE tomography (OCT) provides a noninvasive, high-resolution,
cross-sectional image analog to ultrasound imaging.1-2
The resolution of OCT is reportedly 10 µm with the use of low-coherence
interferometry. Optical coherence tomography has been used as a diagnostic
method in studying several retinal diseases,2-4
because it provides quantitative information for longitudinally tracking small
changes in tissue structure and the development of disease processes. Recently,
OCT has been used to study degenerative retina in humans5-6
and animals.7-9
These studies have described a thinner retina in eyes with retinal degeneration
compared with healthy subjects. Optical coherence tomography may prove a valuable
noninvasive technique in the study of human retinal degeneration. However,
only a few studies have compared OCT scans with histologic sections, and the
potential of OCT to detect progression in degenerative retina is unclear.
In retinal degeneration slow (rds) mice, the
outer segments of photoreceptors fail to develop and are followed by slow
degeneration.10-12
The rds mice have been studied because they presumably
make a good model for some forms of hereditary retinal degeneration occurring
in humans.13-15
To study the relationship between changes of OCT image and histologic
progress of retinal structure in degenerative retina, we used OCT scan profiles
in healthy and rds mice.
MATERIALS AND METHODS
ANIMALS
Six adult (16-week-old) mice (Balb/c-57) served as normal controls,
and six 2-week-old, four 6-week-old, and two 60-week-old rds mice were used in this study. All animals were maintained in accordance
with the Association for Research in Vision and Ophthalmology Statement for
the Use of Animals in Ophthalmic and Vision Research, and institutional approval
was obtained. The mice were anesthetized with an intraperitoneal injection
of 15 µL/g of isotonic sodium chloride solution containing ketamine
hydrochloride (1 mg/mL), xylazine hydrochloride (0.4 mg/mL), and urethane
(40 mg/mL). The pupils were dilated with 0.5% phenylephrine hydrochloride
and 0.5% tropicamide. Mice were placed on a heating pad throughout the experimental
session.
OCT IMAGING
Cross-sectional imaging of the retina was performed using an OCT instrument
(Humphrey Instruments, San Leandro, Calif), which has been described previously.1-2 In brief, low-coherence light (center
wavelength, 850 nm) from a superluminescent diode source is guided into a
fiberoptic Michelson interferometer and is divided at a fiber coupler into
reference and sample paths. The reflected light from the reference mirror
and the eye is recombined in the coupler. The 2 light pulses produce an interference
signal at the detector only when the reference arm distance matches the length
of a reflective path through the eye to within the source coherence length,
which predicts a longitudinal resolution of 10 µm full width at half-maximum
in the retina. This longitudinal resolution is unaffected by ocular aberrations
or limited pupil diameter.
A single 1-dimensional, longitudinal profile of optical reflectivity
vs distance into the tissue is created by translating the reference arm mirror
and measuring the magnitude of the interference signal at the detector. A
2-dimensional, cross-sectional image is created by obtaining multiple, longitudinal
reflectivity profile (LRP) and is displayed using a pseudocolor scale while
rapidly scanning the probe beam through tissue. The fundus view with a visible
light source placed coincident with the probe beam can be obtained by an infrared
sensitive video camera.
Anesthetized mice were aligned with the OCT so that the optic disc of
their right eye was visible. The OCT images were oriented horizontally, and
each scan was recorded from the nasal edge of the optic disc, across the center
of the optic disc, to the temporal retina. The lateral extent of scans was
2.8 mm.
HISTOLOGY
Eyecups were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde,
refixed with 1% osmium tetroxide, dehydrated, and embedded in epoxylysin.
Semithin sections (0.5-0.7 µm) were mounted on silanized glass slides,
stained with toluidine blue, and observed with conventional light microscopy.
QUANTITATIVE ANALYSIS
Sensory retinal thickness (SRT) and outer retinal thickness (ORT) were
measured in temporal retina at a distance 1 to 2 disc diameters from the temporal
margin of the optic disc. The disc diameter was determined from the distance
between the edges of the red reflective layer, which delineates the posterior
boundary of the retina and terminates at the margin of the optic disc. This
layer probably corresponds to the retinal pigment epithelium (RPE) and choriocapillaris
(Figure 1).8
Another highly reflective red layer in pseudocolor scale delineating the surface
of the retina in OCT image may correspond to retinal nerve fiber layer (NFL),
including the internal limiting membrane (ILM).7-8
Measurement of SRT and ORT was performed manually using LRP in a scan profile
program of OCT (Figure 2).16-17 For the SRT measurement, cursors
were placed at the steepest portion of each rising slope produced at the ILM
and RPE. The ORT was measured as the distance between the descending slope
anterior to the RPE and the anterior border of the RPE. This relative low
reflectivity layer anterior to the RPE may correspond to the photoreceptor
layer.7-9 Retinal
thickness of each eye represented the average of 10 measurements at different
regions in the right eye of each mouse.
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Figure 1. Optical coherence tomogram of
a section of the optic disc (A) and its corresponding histologic section (B)
in a healthy mouse. Logarithm of reflectivity is mapped to a pseudocolor scale.
Retinal layers are labeled as follows: nerve fiber layer (NFL), ganglion cell
layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer
plexiform layer (OPL), outer nuclear layer (ONL), inner segments of photoreceptors
(IS), outer segments of photoreceptors (OS), retinal pigment epithelium (RPE),
and choriocapillaris (CC). Calibration bar is 100 µm.
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Figure 2. Representative longitudinal reflectivity
profile. Measurement cursors were placed at the surface of nerve fiber layer
and retinal pigment epithelium to measure sensory retinal thickness (SRT).
Outer retinal thickness (ORT) was measured as the distance between the descending
slope anterior to the RPE and the anterior border of the retinal pigment epithelium
and that was assumed to be the length of photoreceptor cells.
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STATISTICAL ANALYSIS
The analyses were performed using the StatView (Abacus Concepts Inc,
Berkeley, Calif) statistical analysis package. The SRT and ORT in each group
were compared using the 1-way factorial analysis of variance and Fisher protected
least significant difference for post hoc test. Values of P<.05 were considered significantly different. All data were expressed
as mean ± SD.
RESULTS
Cross-sectional OCT images were obtained, suggesting a close relationship
with the corresponding histologic sections in normal and rds mice (Figure 3). Highly
reflective red layers that delineate the surface and the posterior boundary
of the retina and corresponding to NFL and RPE with choriocapillaris, respectively,
were clearly observed in all mice. A low reflective layer, located anterior
to the RPE and corresponding to the photoreceptor layer, was also observed
clearly in healthy mice, but it progressively decreased as the rds mice aged. The 60-week-old mice showed no photoreceptor layer.
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Figure 3. Optical coherence tomographic
scans and the corresponding histologic sections (A) and longitudinal reflectivity
profiles (B) in each group of healthy and 2-, 6-, and 60-week-old retinal
degeneration slow (rds) mice. As the retinal degeneration progresses, the
sensory retinal thickness and the outer retinal thickness decrease. Calibration
bar is 50 µm. Arrowheads indicate the steepest portion of each rising
slope produced at the internal limiting membrane (upper arrowheads) and retinal
pigment epithelium (lower arrowheads). Arrows indicate the descending slope,
representing the inner surface of the photoreceptor layer.
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The SRT and ORT of healthy mice measured by OCT scan profile were 174
± 5 µm and 37 ± 6 µm, respectively (Figure 4). The SRTs of 2-week-old and 6-week-old rds mice were 150 ± 4 µm and 136 ± 2 µm,
respectively, and the ORTs of each group were 28 ± 4 µm and 20
± 1 µm, respectively. The SRT and ORT of 2-week-old rds mice were statistically shorter than those of normal mice (P<.001). Moreover, SRT and ORT of 6-week-old mice were
statistically shorter than those of 2-week-old mice (P<.001).
Histologic findings revealed the progress of retinal degeneration in 6-week-old
mice, which had a decreased number of nuclei in the outer nuclear layer compared
with 2-week-old mice. The photoreceptor layer of 60-week-old rds mice was almost gone, and SRT was 120 ± 7 µm. The
SRT of 60-week-old rds mice was shorter than that
of any other group of mice (P<.001). No relative
low reflectivity layer was found in a cross-sectional OCT image in 60-week-old rds mice.
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Figure 4. Sensory retinal thickness (SRT)
and outer retinal thickness (ORT) in healthy and 2-, 6-, and 60-week-old retinal
degeneration slow (rds) mice. Data are expressed as mean ± SD. Significant
differences were found among each group (P<.001).
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COMMENT
We demonstrated that SRT and ORT measured by OCT scan profile decreased
with progression of retinal degeneration in rds mice
and the cross-sectional OCT image corresponded with histologic findings. Previous
reports8-9 in animal models compared
normal and degenerative retina. Those studies defined the exact relationships
between the cross-sectional image and the microanatomy of the retina; however,
the potential to detect microanatomical change in degenerative retina using
OCT is still unknown. Our findings indicated that the quantitative analysis
using OCT scan profile has potential to detect progressive change in retinal
degenerative diseases.
The murine rds allele is a semidominant null
allele that causes abnormal development of photoreceptors, followed by their
slow degeneration.10-12,18
The wild-type sequence at the rds locus encodes a
photoreceptor disc membrane protein named peripherin/RDS.19
The rds mice have been studied because it is thought
that they represent a model for some forms of hereditary retinal degeneration
occurring in humans.13-15
In rds mice, the outer segments of photoreceptor
cells fail to develop, and a progressive loss of photoreceptor cells occurs
throughout life. In the present study, the cross-sectional image demonstrated
the change of outer retinal structure was consistent with histologic features.
Several techniques have reportedly measured retinal thickness.2, 8, 17 Optical coherence tomography
has original software for automatically measuring SRT, and it has been widely
used in clinical study.20-21 Baumann
et al17 have described the technique using
a manually assisted method of computer software. In their study, measurement
cursors were placed at the steepest portion of each rising slope produced
at the ILM and RPE when the observer visualized the representative LRP. In
our study, this technique was used to measure SRT, and SRT was measured clearly
because the reflectivity from ILM and RPE could be obtained as a sharply rising
slope. In contrast, the technique to measure ORT has not been established,
and the other cursor was placed at the steepest portion of the descending
slope of the outer nuclear layer to measure ORT in this study. Although this
technique of measuring ORT should be evaluated in detail, our data showing
the reduction of the outer retina in rds mice indicated
that this technique may be effective in measuring ORT.
In summary, our findings suggested a possible relationship between SRT
and ORT and histologic change in retinal degenerative diseases. Although the
quantitative analysis obtained with OCT scans should be followed by functional
tests,5-6 OCT has the potential
to detect microanatomical change of degenerative retina and may be useful
for noninvasive assessment of human retinal degeneration.
AUTHOR INFORMATION
Accepted for publication February 12, 2001.
This study was supported by Grant-in-Aid for Scientific Research (B2-11470363)
from the Ministry of Education, Science, Sports and Culture, Tokyo, Japan.
Corresponding author and reprints: Naoichi Horio, MD, Department
of Ophthalmology, Nagoya University School of Medicine, 65 Tsuruma-cho, Showa-ku,
Nagoya 466-0065, Japan (e-mail: naoichi{at}med.nagoya-u.ac.jp).
From the Department of Ophthalmology, Nagoya University School of Medicine,
Nagoya, Japan.
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