February 2010
Volume 51, Issue 2
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Retina  |   February 2010
Fourier Domain Optical Coherence Tomography as a Noninvasive Means for In Vivo Detection of Retinal Degeneration in Xenopus laevis Tadpoles
Author Affiliations & Notes
  • Damian C. Lee
    From the Department of Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, British Columbia, Canada; and
  • Jing Xu
    the School of Engineering Science, Simon Fraser University, Burnaby, British Columbia, Canada.
  • Marinko V. Sarunic
    the School of Engineering Science, Simon Fraser University, Burnaby, British Columbia, Canada.
  • Orson L. Moritz
    From the Department of Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, British Columbia, Canada; and
  • Corresponding author: Orson L. Moritz, Department of Ophthalmology and Visual Sciences, University of British Columbia, Room 330, 2550 Willow Street, Vancouver, BC, V5Z 3N9, Canada; olmoritz@interchange.ubc.ca
Investigative Ophthalmology & Visual Science February 2010, Vol.51, 1066-1070. doi:10.1167/iovs.09-4260
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      Damian C. Lee, Jing Xu, Marinko V. Sarunic, Orson L. Moritz; Fourier Domain Optical Coherence Tomography as a Noninvasive Means for In Vivo Detection of Retinal Degeneration in Xenopus laevis Tadpoles. Invest. Ophthalmol. Vis. Sci. 2010;51(2):1066-1070. doi: 10.1167/iovs.09-4260.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To determine the efficacy of Fourier domain optical coherence tomography (FD-OCT) as a noninvasive, nonlethal method for detecting in vivo, pathologic signs of retinal degeneration in Xenopus laevis larvae.

Methods.: A prototype OCT system using FD detection customized for tadpole imaging was used to noninvasively obtain retinal scans in two different transgenic X. laevis models of retinal degeneration. FD-OCT retinal scans were compared with laser scanning confocal micrographs of histologic sections of the same eye. Retinal thickness was measured in the histologic micrographs and compared with in vivo measurements acquired with FD-OCT.

Results.: In vivo retinal images of X. laevis tadpoles were obtained that visualized the major retinal layers. FD-OCT successfully detected the ablation of rod outer segments (OS) in degenerating tadpole eyes. Measurements from FD-OCT and histology showed a decrease in retinal thickness in transgenic mutant tadpoles relative to the wild-type control. The accumulation of phagosomes from dying rod OS was also visualized in the retinal pigment epithelium (RPE) in a degenerating tadpole retina.

Conclusions.: This report demonstrates that FD-OCT is a viable technique for screening, diagnosing, and monitoring retinal degeneration in X. laevis tadpoles in vivo.

Transgenic animals are crucial to the study of cellular pathways and molecular mechanisms underlying retinal degenerative disease, and Xenopus laevis, a frog species commonly used as a laboratory animal, is highly amenable to the development of animal models of retinal degeneration. The X. laevis retina has a rod/cone photoreceptor ratio similar to that of humans (unlike the rod-dominated retina of rodents), and the large size of the principle rod photoreceptors makes them ideal for biochemical, morphologic, and electrophysiological studies. 1 Compared with the generation of transgenic mice, the Kroll and Amaya 2 method of transgenesis produces large numbers of transgenic X. laevis in a single day, and rapid retinal development allows the analysis of the larval retina within 1 week after fertilization. 3 As a research subject, X. laevis has advantages over other common laboratory animals, as they have high numbers of offspring, low maintenance requirements, and a very long lifespan. However, at present, detailed assessment of X. laevis retinal morphology and substructure can only be achieved by histologic examination after the animals are euthanatized. Because of this, researchers' ability to evaluate the presence or progression of retinal disease in a given animal is limited. To identify animals with retinal disease and perform longitudinal studies for monitoring retinal disease progression in individual animals, a noninvasive in vivo assessment of the retina without killing the animal is required. 
Optical coherence tomography (OCT) is an optical imaging technique for acquiring cross-sectional images of biological tissue noninvasively based on optical reflectivity. 4 OCT has become an integral diagnostic tool for clinical ophthalmology and is rapidly being adopted for retinal research in animal models. OCT has been used to noninvasively obtain cross-sectional images of retina from chickens, pigs and rodents in vivo. 57 This technique allows the investigation of the time course of disease progression in individual animals, dramatically reducing the number of animals required for a longitudinal study. Since an in vivo image is acquired, OCT also overcomes the problems of artifacts from fixation and tissue processing in histology. 
Fourier domain (FD) detection with OCT (FD-OCT) imaging modality has been extensively used to study retinal degeneration in rodents. 811 In this report, we show that FD-OCT can be used to visualize retinal layers in X. laevis larvae (tadpoles) in vivo. Using FD-OCT, we were able to noninvasively image loss of the rod outer segments (OS) in two different transgenic X. laevis models of retinal degeneration and visualize the accumulation of phagosomes from dying rod OS by the retinal pigment epithelium (RPE). This report demonstrates that FD-OCT is a viable system for screening, diagnosing, and monitoring retinal degeneration in X. laevis tadpoles. 
Materials and Methods
Transgenic X. laevis Generation and Rearing
Transgenic X. laevis tadpoles expressing either bovine P23H rhodopsin or inducible-caspase 9 (iCasp9) in the rod photoreceptors were bred from transgenic frogs carrying the respective transgenes under the control of the X. laevis opsin promoter. Breeding was induced by injection of 700 units of human chorionic gonadotropin into the dorsal lymph sack of the female frog. Transgenic X. laevis expressing bovine rhoP23H were originally described in Tam and Moritz, 12 and transgenic X. laevis expressing iCasp9 were originally described in Hamm et al. 13 Tadpoles were reared in an 18°C incubator on a 12-hour dark and 12-hour light (1700 lux) cycle. Rod photoreceptor death was induced in the iCasp9 tadpoles by the addition of 10 nM AP20187 (Ariad Pharmaceuticals, Cambridge, MA) to their rearing medium as previously described. 13  
OCT
The FD-OCT system used in this study was a prototype that was customized for tadpole imaging (Fig. 1). In OCT, the axial resolution is independent of the optics used to deliver light to the sample and is determined only by the spectral bandwidth of the light source. The low-coherence light source used (Superlum, Moscow, Russia) had a central wavelength of 826 nm and a full-width-at-half-maximum bandwidth of 72 nm, corresponding to an axial resolution of 4 μm in tissue. The sample arm optics were redesigned for imaging the tadpole retina. In contrast to human retinal imaging which uses a collimated beam, a converging beam was incident on the tadpole cornea. Backscattered light was collected through sample arm optics and delivery fiber, and combined with a reference beam by using a 70/30 fiber coupler. The interfering sample and reference beams were directed to a custom-built spectrometer with software controlled integration time. B-scan images consisting of 512 lines were acquired at a line rate of 20 kHz. 
Figure 1.
 
FD-OCT system.
Figure 1.
 
FD-OCT system.
The OCT sample arm was constructed from bulk optic achromatic lenses (Thorlabs, Newton, NJ). A 4× beam expander was placed after the galvanometer scanning mirrors, followed by a lens to focus the light onto the sample. The sample arm was designed for a calculated depth of focus of ∼70 μm (defined as twice the Rayleigh range) and corresponding to a 1/e2 lateral spot diameter of ∼6 μm (in air). The long depth of focus of the interrogating beam approximately matched the thickness of the tadpole retina, permitting the visualization and measurement of all the retinal layers in vivo. The optical power incident on the eye was adjusted below 750 μW, which is in accordance with the ANSI recommended limit for ocular exposure in humans. 
Before OCT imaging, tadpoles were anesthetized by immersion in tadpole rearing medium (Ringer's solution) containing 0.01% tricaine methanesulfonate (Sigma-Aldrich, St. Louis, MO) for 1 to 2 minutes. The anesthetized tadpoles were transferred to a stage soaked in Ringer's solution to prevent desiccation during the course of OCT imaging. High-speed screening was facilitated by FD detection in the OCT; imaging was performed quickly, with alignment and acquisition requiring, nominally, 5 minutes per specimen. Volumetric images were acquired by raster scanning the OCT beam across the eye, and reconstructions were generated (Amira; Visage Imaging, San Diego, CA). The rapid image acquisition rate also permitted the acquisition of multiple frames at single position. The two-dimensional B-scans presented in this article were generated by averaging 40 frames to reduce speckle. No further postprocessing was performed. All procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Immunohistochemistry and Microscopy
Anesthetized tadpoles were euthanatized and fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). The fixed eyes were dissected and infiltrated with 20% sucrose/0.1 M sodium phosphate buffer (pH 7.4). The cryoprotected eyes were embedded in OCT compound (Tissue-Tek; Sakura Finetek, Torrance, CA) and frozen for cryosectioning. Twelve-micrometer serial sections were cut and labeled with Alexa Fluor 488–conjugated wheat germ agglutinin (WGA; Molecular Probes-Invitrogen, Eugene, OR) and Hoechst 33342 (Sigma-Aldrich) to label rod outer segments and nuclei, respectively. Images were acquired with a laser scanning confocal microscope (510 Meta; Carl Zeiss Meditec, Dublin, CA). 
Results
FD-OCT Retinal Scans of X. laevis Tadpoles
FD-OCT was used to image wild-type X. laevis tadpole retinas at various stages of development, from Nieuwkoop-Faber developmental stage 46/47 to stage 55/56. 14 Tadpoles were first anesthetized before they were placed on a moistened pad on the OCT stage for scanning. We found that an experienced user could locate the tadpole retina and acquire retinal scans in as little as 5 minutes. The FD-OCT setup described in the Materials and Methods section was used to acquire cross-sectional images of the X. laevis tadpole eye (Fig. 2). Although the light was focused on the retina, the cornea and iris were also easily identifiable in the FD-OCT images, allowing for reconstruction of the gross anatomy of a healthy tadpole eye. The source of contrast in OCT is Fresnel reflection from tissue boundaries as well as the relative amount of backscattered light between different tissues. In the retina, the plexiform layers and the RPE backscatter strongly and appear white, whereas the nuclear layers appear darker. These differences in scattering intensity between layers give the retina a banded appearance in OCT images, permitting visual delineation and measurement. 
Figure 2.
 
FD-OCT images of a normal tadpole eye. (A) 2-D cross-sectional view and (B) 3-D volumetric view of the tadpole eye. Tissues that had high optical reflectivity (e.g., iris and retinal pigment epithelium) appear bright in the images, whereas tissues that backscattered less (e.g., lens) appear dark. Scale bars, 100 μm.
Figure 2.
 
FD-OCT images of a normal tadpole eye. (A) 2-D cross-sectional view and (B) 3-D volumetric view of the tadpole eye. Tissues that had high optical reflectivity (e.g., iris and retinal pigment epithelium) appear bright in the images, whereas tissues that backscattered less (e.g., lens) appear dark. Scale bars, 100 μm.
The optics in the FD-OCT sample arm were customized to a shorter depth of focus than that used for rodent imaging because of the comparatively thinner retina of the tadpole. OCT provided B-scans of the tadpole retina showing the distinct banding pattern of the retinal layers (Fig. 3A). Subsequent to imaging, the tadpoles were euthanatized while still under anesthesia and fixed with 4% paraformaldehyde to prepare histologic sections of the corresponding scanned eyes. We identified and assigned the individual retinal layers by visual inspection and comparing cross-sectional FD-OCT images with fluorescence labeled confocal micrographs of the histologic sections. We were able to identify the inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), rod outer segment (OS), and retinal pigment epithelium (RPE) in the FD-OCT images. However, the ganglion cell layer (GCL) and the inner segments (IS) were not easily identifiable from the FD-OCT retinal scans. 
Figure 3.
 
Comparison of tadpole retina images from histology and FD-OCT. (A) Representative images of laser scanning confocal micrographs of cryosections (left) and in vivo FD-OCT scans (right) of healthy wild-type retina (top) and degenerating transgenic retina expressing rhoP23H (bottom). Cryosections were stained with Alexa Fluor 488 conjugated–wheat germ agglutinin (green) and Hoechst 33342 (blue) to label photoreceptor outer segments and nuclei, respectively. IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium. Scale bar, 20 μm. (B) Relative thickness of wild-type and transgenic P23H retinas as measured from histologic sections (left) and FD-OCT (right) (n > 7, mean ± SD). From both measurements, the relative thickness of the mutant retina is significantly thinner than the wild-type control (*P < 0.001, t-test).
Figure 3.
 
Comparison of tadpole retina images from histology and FD-OCT. (A) Representative images of laser scanning confocal micrographs of cryosections (left) and in vivo FD-OCT scans (right) of healthy wild-type retina (top) and degenerating transgenic retina expressing rhoP23H (bottom). Cryosections were stained with Alexa Fluor 488 conjugated–wheat germ agglutinin (green) and Hoechst 33342 (blue) to label photoreceptor outer segments and nuclei, respectively. IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium. Scale bar, 20 μm. (B) Relative thickness of wild-type and transgenic P23H retinas as measured from histologic sections (left) and FD-OCT (right) (n > 7, mean ± SD). From both measurements, the relative thickness of the mutant retina is significantly thinner than the wild-type control (*P < 0.001, t-test).
In both clinical and research settings, OCT can be used as a diagnostic tool to image in vivo morphologic changes in degenerating retinas. To assess the ability of FD-OCT to detect differences between normal and degenerating tadpole retinas, we compared FD-OCT retinal scans from transgenic P23H mutant rhodopsin tadpoles and retinal scans from wild-type tadpoles. The X. laevis P23H model has been used to model rod photoreceptor degeneration and is well characterized. 12,15 The mutant rhodopsin protein causes light-mediated rod cell death, which is easily identifiable in histologic sections from the loss of rod OS (Fig. 3A, lower panels). FD-OCT images from the matching mutant retina showed a corresponding decrease in retinal thickness and loss of OS layer. Table 1 shows the thickness of the INL and OS as measured from confocal micrographs of histologic sections and FD-OCT retinal scans. The OS layer in the P23H mutant retina was significantly thinner (P < 0.001, by t-test) than the wild-type retina in both measurements. We also measured the relative thickness of the retina (the INL+ ONL+OS thickness normalized to the thickness of the entire retina) from both histology and OCT. The relative thickness of the mutant (degenerated) retina was significantly less (P < 0.001, by t-test) than that of the control wild-type retina in both the histologic sections and FD-OCT scans (Fig. 3B). The percentage of decrease measured from both histology and OCT was 13% ± 1%. These measurements indicate that our interpretation and assignments of the retinal layers in OCT images are consistent and that FD-OCT is capable of detecting the ablation of rod OS in the degenerating P23H mutant tadpole retina. Of interest, we found that the relative thickness of the ONL with respect to the INL in FD-OCT images was larger than that observed in histologic sections. This apparent discrepancy may be due to artifacts from histology or OCT (see the Discussion section). 
Table 1.
 
Thicknesses of INL and OS Measured by Histology and OCT
Table 1.
 
Thicknesses of INL and OS Measured by Histology and OCT
Histology OCT
INL OS INL OS
Wild-type 25.3 ± 3.5 24.9 ± 4.4 20.10 ± 2.3 15.3 ± 1.3
P23H mutant 25.9 ± 2.6 6.7 ± 2.3* 19.85 ± 2.3 5.7 ± 2.4*
In Vivo Imaging of Progressive Retinal Degeneration in X. laevis Larvae with FD-OCT
As FD-OCT is noninvasive, it should be possible to visualize and monitor, in vivo, the time course of disease progression in individual animals. To assess the utility of FD-OCT for imaging progressive retinal degeneration in a given tadpole, we used a drug-inducible X. laevis model of retinal degeneration that allows rapid induction of rod cell death by direct activation of apoptosis (programmed cell death). 13 These transgenic X. laevis express a drug-activatable form of caspase-9 (iCasp-9) in the rod photoreceptors. Caspase-9 is an initiator caspase that, once activated, triggers the caspase cascade, resulting in apoptosis. Administration of the drug AP20187 to these transgenic tadpoles induces rapid death of the rod photoreceptors, with ablation of most of the rods by day 5 afterward. 13 We used FD-OCT to monitor the retina of stage 47/48 tadpoles before administering the drug and at various time points over 4 days after drug administration. At each time point, a subset of tadpoles were euthanatized for histology. Figure 4 shows representative images of iCasp-9 tadpole retinas at three time points: immediately before, 2 days after, and 4 days after administration of the drug. Before exposure to the drug, the INL, ONL, OS and RPE can be observed from FD-OCT, indicative of a healthy retina. At the intermediate time point (2 days) after drug exposure, histology showed that the majority of rods were dysmorphic and rod OS bodies were seen within the RPE, indicating rod cell death and clearing of dead rods by the RPE. We observed an analogous disruption in the OS layer in the FD-OCT images and a corresponding disruption in the RPE layer as well (Fig. 4, lower panels). The disruption in the RPE layer in OCT images indicates a change in reflectivity and backscatter in the RPE, consistent with the accumulation of phagosomes in the RPE. At day 4 after drug administration, histology indicated that most of the dead rods were cleared, and relatively few phagosomes remained in the RPE. Similarly, the OS layer could not be identified in FD-OCT retinal scans of 4 day postdrug tadpoles, and the RPE appeared as a featureless single layer. These data demonstrate that FD-OCT is able to image intermediate rod degeneration as well as phagosomes in the RPE in vivo. 
Figure 4.
 
In vivo imaging of the progression of retinal degeneration. Representative images of X. laevis tadpole undergoing induced rod photoreceptor degeneration are shown. Rapid rod photoreceptor death was induced by the drug AP20187 in transgenic iCasp9 tadpoles. Laser scanning confocal micrographs (left) and FD-OCT images (right) show progressive retinal degeneration over a period of 4 days after induction of cell death. On day 2 after treatment, the disruption in the RPE from OCT indicates a change in reflectivity and backscatter, consistent with the accumulation of phagosomes in the RPE (red asterisk). By day 4 after drug, the rod OS was completely ablated (red brackets). Scale bar, 20 μm.
Figure 4.
 
In vivo imaging of the progression of retinal degeneration. Representative images of X. laevis tadpole undergoing induced rod photoreceptor degeneration are shown. Rapid rod photoreceptor death was induced by the drug AP20187 in transgenic iCasp9 tadpoles. Laser scanning confocal micrographs (left) and FD-OCT images (right) show progressive retinal degeneration over a period of 4 days after induction of cell death. On day 2 after treatment, the disruption in the RPE from OCT indicates a change in reflectivity and backscatter, consistent with the accumulation of phagosomes in the RPE (red asterisk). By day 4 after drug, the rod OS was completely ablated (red brackets). Scale bar, 20 μm.
Discussion
We have demonstrated for the first time that FD-OCT can be applied to image X. laevis tadpole retina in vivo. This noninvasive technique was used to obtain retinal scans from tadpoles as early as Nieuwkoop-Faber developmental stage 46/47. Despite the small size of tadpole eyes (∼1.5mm), we were able to acquire details regarding retinal layer profiles and thickness. By comparing the FD-OCT images alongside fluorescence-labeled histologic sections, we confirmed that the same anatomic structures were observed with OCT and with conventional histology. We performed a longitudinal study of actively degenerating retina and were able to discern abnormalities in the tadpole retina in vivo, specifically the progressive loss of rod outer segments and the accumulation of phagosomes in the RPE. 
Transgenic X. laevis is an ideal vertebrate system for retinal research. Transgenic tadpoles are easily generated and their fast retinal development (retina is fully developed in 2 weeks) means that FD-OCT can be used to obtain retinal scans from transgenic tadpoles as early as 2 weeks after transgenesis. Our data demonstrate that we now have the ability to track and follow the time course of retinal disease progression in individual transgenic animals. Our scanned tadpoles recovered from the short anesthesia and we were able to subject individual tadpoles to multiple scans over the course of several days with no observable effect on developmental health. This method allowed us to use FD-OCT to monitor the retinal health of the same animal over a prolonged period (e.g., the course of development of the tadpole). 
Primary transgenic X. laevis larvae can be reared to adulthood, and subsequently bred to give large numbers of transgenic offspring with very consistent phenotypes. 12 However, X. laevis require many months (8–12) to reach sexual maturity. As expression levels are often low and can vary dramatically between animals, the development of strains of X. laevis can necessitate rearing a large number of animals to sexual maturity and subsequent lengthy breeding and screening procedures to identify animals with offspring that have the desired retinal phenotype. With FD-OCT, it is possible to identify primary transgenic X. laevis larvae with retinal degeneration and thus dramatically limit the number of animals that must be housed, bred, and screened. 
One concern was the ability to directly correlate histology sections with OCT scans. Taking measurements of the retinal layers, we found that the relative thickness of the ONL with respect to the INL was consistently larger in FD-OCT images than that measured from confocal micrographs of histologic sections. We found that the differences in the thickness of the INL relative to the ONL in OCT versus histology was also apparent in studies on rodent retina. 811 In addition, artifacts from the processing steps of histology, such as shrinkage of the tissue during fixation, cannot be ruled out. However, we found that OCT measurements of retinal layers showed less variation compared to measurements of histologic sections. Taking into consideration the potential artifacts from routine histologic processing such as tissue deformations and variations in sectioning levels and angles, it is not unexpected that measurements from OCT are more consistent. 
One inherent limitation of OCT is its maximum resolution. Although FD-OCT can resolve the major retinal layers (OPL, INL, ONL, OS, and RPE), histology is still necessary for single-cell resolution and analysis. Furthermore, histologic analysis has the advantage of the ability to immuno- and chemically label cells, thereby unambiguously identifying various cell types and consequently, retinal layers. However, compared with the labor-intensive process of histology, the speed at which OCT retinal scans can be acquired noninvasively implies that FD-OCT may be used to rapidly screen a large number of tadpoles, which is a practical limitation of histology. The strength in FD-OCT is its ability to image retinal morphology noninvasively and without killing the animal, to monitor changes in a degenerating retina in vivo. FD-OCT is an important new addition to the existing techniques of biochemistry, immunohistochemistry, and electroretinography used to study retinal disease in the X. laevis model. 
Footnotes
 Supported by grants from the Canadian Institutes for Health Research (CIHR) and Foundation Fighting Blindness (Canada; FFB-Ca; OLM). OLM is a CIHR New Investigator and an FFB-Ca W. K. Stell Scholar. MVS is a MSFHR (Michael Smith Foundation for Health Research) Scholar and was supported by the NSERC (Natural Sciences and Engineering Research Council) and CIHR collaborative health research and team grants.
Footnotes
 Disclosure: D.C. Lee, Ariad Pharmaceuticals (F); J. Xu, None; M.V. Sarunic, None; O.L. Moritz, Ariad Pharmaceuticals (F)
References
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Figure 1.
 
FD-OCT system.
Figure 1.
 
FD-OCT system.
Figure 2.
 
FD-OCT images of a normal tadpole eye. (A) 2-D cross-sectional view and (B) 3-D volumetric view of the tadpole eye. Tissues that had high optical reflectivity (e.g., iris and retinal pigment epithelium) appear bright in the images, whereas tissues that backscattered less (e.g., lens) appear dark. Scale bars, 100 μm.
Figure 2.
 
FD-OCT images of a normal tadpole eye. (A) 2-D cross-sectional view and (B) 3-D volumetric view of the tadpole eye. Tissues that had high optical reflectivity (e.g., iris and retinal pigment epithelium) appear bright in the images, whereas tissues that backscattered less (e.g., lens) appear dark. Scale bars, 100 μm.
Figure 3.
 
Comparison of tadpole retina images from histology and FD-OCT. (A) Representative images of laser scanning confocal micrographs of cryosections (left) and in vivo FD-OCT scans (right) of healthy wild-type retina (top) and degenerating transgenic retina expressing rhoP23H (bottom). Cryosections were stained with Alexa Fluor 488 conjugated–wheat germ agglutinin (green) and Hoechst 33342 (blue) to label photoreceptor outer segments and nuclei, respectively. IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium. Scale bar, 20 μm. (B) Relative thickness of wild-type and transgenic P23H retinas as measured from histologic sections (left) and FD-OCT (right) (n > 7, mean ± SD). From both measurements, the relative thickness of the mutant retina is significantly thinner than the wild-type control (*P < 0.001, t-test).
Figure 3.
 
Comparison of tadpole retina images from histology and FD-OCT. (A) Representative images of laser scanning confocal micrographs of cryosections (left) and in vivo FD-OCT scans (right) of healthy wild-type retina (top) and degenerating transgenic retina expressing rhoP23H (bottom). Cryosections were stained with Alexa Fluor 488 conjugated–wheat germ agglutinin (green) and Hoechst 33342 (blue) to label photoreceptor outer segments and nuclei, respectively. IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium. Scale bar, 20 μm. (B) Relative thickness of wild-type and transgenic P23H retinas as measured from histologic sections (left) and FD-OCT (right) (n > 7, mean ± SD). From both measurements, the relative thickness of the mutant retina is significantly thinner than the wild-type control (*P < 0.001, t-test).
Figure 4.
 
In vivo imaging of the progression of retinal degeneration. Representative images of X. laevis tadpole undergoing induced rod photoreceptor degeneration are shown. Rapid rod photoreceptor death was induced by the drug AP20187 in transgenic iCasp9 tadpoles. Laser scanning confocal micrographs (left) and FD-OCT images (right) show progressive retinal degeneration over a period of 4 days after induction of cell death. On day 2 after treatment, the disruption in the RPE from OCT indicates a change in reflectivity and backscatter, consistent with the accumulation of phagosomes in the RPE (red asterisk). By day 4 after drug, the rod OS was completely ablated (red brackets). Scale bar, 20 μm.
Figure 4.
 
In vivo imaging of the progression of retinal degeneration. Representative images of X. laevis tadpole undergoing induced rod photoreceptor degeneration are shown. Rapid rod photoreceptor death was induced by the drug AP20187 in transgenic iCasp9 tadpoles. Laser scanning confocal micrographs (left) and FD-OCT images (right) show progressive retinal degeneration over a period of 4 days after induction of cell death. On day 2 after treatment, the disruption in the RPE from OCT indicates a change in reflectivity and backscatter, consistent with the accumulation of phagosomes in the RPE (red asterisk). By day 4 after drug, the rod OS was completely ablated (red brackets). Scale bar, 20 μm.
Table 1.
 
Thicknesses of INL and OS Measured by Histology and OCT
Table 1.
 
Thicknesses of INL and OS Measured by Histology and OCT
Histology OCT
INL OS INL OS
Wild-type 25.3 ± 3.5 24.9 ± 4.4 20.10 ± 2.3 15.3 ± 1.3
P23H mutant 25.9 ± 2.6 6.7 ± 2.3* 19.85 ± 2.3 5.7 ± 2.4*
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