Retina
Assessing Sodium Iodate–Induced Outer Retinal Changes in Rats Using Confocal Scanning Laser Ophthalmoscopy and Optical Coherence Tomography Yaping Yang,1,2 Tsz Kin Ng,1 Cong Ye,1 Yolanda W. Y. Yip,1 Kasin Law,1 Sun-On Chan,3 and Chi Pui Pang1 1Department
of Ophthalmology and Visual Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China 2 Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, China 3 School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China
Correspondence: Chi Pui Pang, Department of Ophthalmology and Visual Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong Eye Hospital, 147K Argyle Street, Kowloon, Hong Kong SAR, China; [email protected]. S-OC and CPP contributed equally to the work presented here and should therefore be regarded as equivalent authors. Submitted: May 24, 2013 Accepted: February 1, 2014 Citation: Yang Y, Ng TK, Ye C, et al. Assessing sodium iodate–induced outer retinal changes in rats using confocal scanning laser ophthalmoscopy and optical coherence tomography. Invest Ophthalmol Vis Sci. 2014;55:1696–1705. DOI:10.1167/ iovs.13-12477
PURPOSE. Sodium iodate induces RPE atrophy and photoreceptor degeneration, as seen in the pathogenesis of many retinal diseases. We investigated a new approach of analyzing retinal images using confocal scanning laser ophthalmoscopy (cSLO) that allows longitudinal assessment of sodium iodate–induced lesions in the retina of living rats. METHODS. A single dose of sodium iodate (25–75 mg/kg) was given intravenously to adult Sprague-Dawley rats. Control animals were given normal saline or sodium iodide. The retina was examined by cSLO and optical coherence tomography (OCT) in living rats, which were then killed for histologic assessments. RESULTS. Confocal scanning laser ophthalmoscopy revealed the appearance of dark patchy blots in planar images of the retina 7 days after intravenous injection of sodium iodate (25–75 mg/kg). This finding coincided with the observations of degenerative changes in the outer retinal layers in OCT images and in histology of the retina. Further analyses showed a concomitant localization of degenerative profiles in histologic preparations of this retina, suggesting that the blots corresponded to the deteriorating photopigments and outer nuclear layer (ONL). In histologic sections, these degenerative profiles appeared as irregular folds or rosettes in the ONL. Quantitative analyses showed that the changes in blot number were dose dependent, which again coincided with results showing a dose-dependent lesion in the photopigment layer and ONL in histologic sections of the retina. CONCLUSIONS. Sodium iodate–induced degenerative changes can be assessed quantitatively and reliably by in vivo retinal imaging using cSLO in adult rats, allowing efficient evaluation of lesions in a large area of retina in longitudinal studies. Keywords: in vivo, retinal degeneration, histology
etinal pigment epithelium and photoreceptor degeneration is typically found in retinal diseases, including early-stage AMD and retinitis pigmentosa,1,2 and can lead to severe visual loss and eventual blindness. Sodium iodate is a retinotoxin known to selectively damage the RPE by oxidative stress, resulting in photoreceptor apoptosis.3 Systemic administration of different doses of sodium iodate has been used in mice to evaluate the effect on visual function,4,5 retinal morphology,3 and other functional features.6 Sodium iodate–induced retinal degeneration in rats was used as an animal model to explore the capability of stem cells to differentiate into RPE and photoreceptors after transplantation into the subretinal space7,8 and to investigate the protective effect of hepatocyte growth factor against retinal degeneration.9 Sodium iodate–induced retinal degeneration has also been reported in rabbits10–13 and sheep.14 Traditional assessments of sodium iodate–induced retinal degeneration are based on electrophysiology and histologic examination. Electrophysiology allows longitudinal monitoring of the retinal function but not structural changes of the retina.
While histologic examination provides clear visualization of individual layers of the retina, many animals are often needed for longitudinal study because they have to be killed at specific intervals. In vivo imaging of the retina is thus the preferred approach to investigate the structural changes of the retina in live animals. Spectral-domain optical coherence tomography (OCT) has been developed as a noninvasive technique for in vivo crosssectional imaging of the retina.15 This technique has been reported for examination of real-time longitudinal changes in the retina of mutant mice with photoreceptor degeneration16,17 and in mice with intravitreal injection of N-methyl-D-aspartate.18 Spectral-domain OCT also revealed degenerative changes in the outer retinal layers in transgenic rabbits19 and in rats with retinal lesions induced by sodium iodate.20 However, while live information on the site of lesions within a confined area of the retina can be provided, spectral-domain OCT is limited in deciphering whether a lesion is widespread throughout different areas of the retina and to quantify the damage across different retinal quadrants. We therefore attempted a novel in
Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783
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cSLO Imaging of Rat Retina vivo method using confocal scanning laser ophthalmoscopy (cSLO) that allows efficient quantitative evaluation of damage in different quadrants of the retina for assessments of lesions induced by sodium iodate in the adult rat.
METHODS Animal Ethics Statement All rats were treated according to guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The experimental protocol in this study was approved by the Animal Experimentation Ethics Committee of The Chinese University of Hong Kong. Adult SpragueDawley rats, weighing 200 to 250 g and aged 8 weeks, were obtained from the Laboratory Animal Service Center of The Chinese University of Hong Kong. The animals were housed in standard conditions and maintained at 22 6 18C, 40% 6 10% humidity, and a cycle of 12 hours of dark and 12 hours of light. Standard rodent chow and water were provided ad libitum. For each experimental group, at least three rats were used and included in the analyses.
IOVS j March 2014 j Vol. 55 j No. 3 j 1697 scanning the rat eye by altering configurations at the software level. The zero-point length of the reference arm was adjusted from the default setting of 20,480 lm to 20,060 lm. Thus, infrared retinal fundus photographs and OCT images could be simultaneously captured on the exact retinal focus, which ensures the high quality of OCT imaging in the retina. In each retina, four different square regions (each from the superotemporal, inferotemporal, inferonasal, and superonasal quadrants) around the optic nerve head were scanned separately without the widefield lens by the volume scan protocol, which consists of 19 evenly distributed B-scans (1024 A-scans for each B-scan) covering a 208 3 158 area of the retina.23–25 Nine images for each B-scan at the same retinal location were captured and averaged. The OCT system registers the cSLO and OCT images in the retina simultaneously. All retinas were examined before (baseline) and after sodium iodate injection (days 1, 4, 7, and 14). Prior to imaging, the rat was anesthetized and immediately placed on a custom-made platform. The head was fixed in position for imaging. Pupils were dilated by topical 1% tropicamide. No contact lens was required. During imaging, 1 drop of sterile saline was applied to the cornea every 2 minutes to maintain media clarity.
Sodium Iodate Application Sodium iodate (Sigma-Aldrich Corp., St. Louis, MO) was dissolved in sterile normal saline as a 4% stock solution (wt/ vol). The rats were anesthetized by intraperitoneal injection of ketamine (35 mg/kg, Ketaset; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (5 mg/kg, TranquiVed; Vedco, Inc., St. Joseph, MO). A single intravenous or intraperitoneal injection of sodium iodate was given. For intravenous injection, the rats were divided into four groups; each received a single injection of 25, 40, 50, or 75 mg/kg sodium iodate. The control animals were injected with the same volume of normal saline or 75 mg/ kg sodium iodide; the latter was administered at an ionic strength comparable to that of the highest dose of sodium iodate used in this study. For intraperitoneal injection, only two doses were tested (50 and 75 mg/kg) in order to compare the effect on retina with that in rats injected intravenously. After the injection, the rats were returned to the colony and kept under standard conditions.
Retinal Examination by cSLO and OCT We used cSLO and spectral-domain OCT (SPECTRALIS HRA þ OCT system; Heidelberg Engineering GmbH, Dossenheim, Germany) for in vivo imaging utilizing an adaptive device specific for imaging rat eye. The cSLO infrared reflectance was recorded using a light source with 820-nm wavelength to provide planar visualization of the retina. The scan rate of the cSLO was 16 frames per second. A 558 widefield noncontact lens (Heidelberg Engineering GmbH) was added to the camera in order to capture high-quality images in a wider view of the fundus. Eye tracking (a retinal recognition technology enabling the exact same retinal location to be scanned and ‘‘locked on’’) was activated during imaging. Fifteen images at the same retinal location at the same focal depth were captured and averaged automatically by the built-in software to increase the signal-to-noise ratio and simultaneously displayed on a computer screen.21,22 These images were used for subsequent counting of degenerating blots in optical sections of the retina. The OCT system used a superluminescent diode light source with a center wavelength of 870 lm. The OCT parameters were modified to adapt OCT imaging in rats according to the technical advice from the manufacturer (Heidelberg Engineering GmbH). The length of the reference arm was adjusted to match the length of the sample arm
Histologic Assessment of the Retina At postinjection day 14, the rats were killed with an overdose of pentobarbital sodium (20% wt/vol) and perfused with PBS, followed by 4% paraformaldehyde in PBS at pH 7.4. Eyes were removed and further fixed in 10% formalin before paraffin embedding. Five-micrometer sections of the eye were obtained at the pupil–optic nerve position. The sections were stained with hematoxylin-eosin and imaged using a light microscope (DMRB; Leica, Wetzlar, Germany) connected to a SPOT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI).
Evaluation of Retinal Lesions For the paraffin sections, morphologic analyses were performed within a 1000-lm2 area in the superotemporal quadrant of the retina 300 lm from the optic nerve head using ImageJ (version 1.46e; National Institutes of Health, Bethesda, MD). Measurements included thickness of inner/ outer segments (IS/OS) of photoreceptors, outer nuclear layer (ONL), inner nuclear layer (INL), and inner plexiform layer (IPL). The INL cell densities were also counted in these regions. To correlate the changes in histologic sections with the OCT images, the changes in ONL thickness, which were readily identified, were measured in OCT images captured at corresponding regions of the retina using ImageJ (National Institutes of Health). In vivo images were captured by cSLO at baseline, day 7, and day 14 after sodium iodate injection. In each retina, four different square regions (each 400 3 400 lm) with clear visualization of the blots that correspond to the location of lesions were analyzed. The images were exported to a computer for generating montages of the central retina (Fig. 1) and for counting dark blots using Photoshop (11.0; Adobe Systems, Incorporated, San Jose, CA). The blots in the central retina in each eye were counted manually before and after the injection by a colleague blinded to the experiment. Mann-Whitney U test was used to compare the means between different experimental groups and the controls. Data were expressed as means (SDs). All analyses were performed using PASW Statistics 18 (SPSS Science, Chicago, IL), and differences were considered significant at P < 0.05.
cSLO Imaging of Rat Retina
IOVS j March 2014 j Vol. 55 j No. 3 j 1698 Cross-sectional images of the retina were also taken from these animals using spectral-domain OCT. Images from day 0 animals showed a typical laminated pattern of the retina (Fig. 2A), which was observed also in retinas 4 days after sodium iodate injection (Fig. 2B). Structural changes were first detected at day 7, which were characterized by the appearance of dome-shaped hyperreflective areas in outer layers of the retina, corresponding to the ONL and IS/OS of photoreceptors (Fig. 2C). These abnormalities were more severe and abundant 14 days after injection (Fig. 2D), representing lesions induced by sodium iodate that were known to confine preferentially to the outer retinal layers.6,20 Such abnormalities were not observed in control animals injected with saline (Fig. 2E) or sodium iodide (Fig. 2F). Further analyses of images collected by cSLO (n ¼ 3) and their corresponding cross-sectional OCT images showed good correlation spatially of the lesions in the planar image of the retina with degenerative regions in crosssectional images (Figs. 3A, 3B). Moreover, the analyses of the width of the blots in cSLO strongly correlated positively with the width of degenerative profiles in the ONL revealed in OCT images (Fig. 3C), indicating that degenerative changes in the outer retinal layers are visualized readily in cSLO images of the retina.
Dose Effect of Sodium Iodate
FIGURE 1. Sodium iodate–induced retinal lesions under infrared cSLO. Montages of retinal images are generated by cSLO. (A–D) Typical changes in the retina of a rat injected intravenously with 40 mg/kg sodium iodate. Dark patchy dots or blots (white arrows) were detected in all retinal quadrants 7 and 14 days after injection. (E, F) Retina in control animals 14 days after injection of saline or 75 mg/kg sodium iodide. OD, optic disc. Superior is up and temporal to the right in these images. Scale bar: 200 lm.
RESULTS cSLO and OCT in the Rat Retina Retinal lesions were induced in adult rats with intravenous injection of 40 mg/kg sodium iodate. The retina was examined using cSLO before the injection (day 0) (n ¼ 12). In these retinal images, typical appearances of the retinal vessels and optic nerve head were readily recognizable. No obvious change was visible in the retina of the animals 4 days after injection (Fig. 1B). However, obvious changes started to appear at day 7 and were characterized by the presence of many small dark dotted profiles in the retina (Fig. 1C). These dark profiles or blots were not observed in any optical section through the whole thickness of the retina at day 0 and day 4. The changes were more obvious in the retina when examined at day 14 (Fig. 1D). In most rats, such dark blots were found throughout the retina, whereas in others the distribution was limited to certain regions (see Fig. 10). In control animals that had been injected either with saline (n ¼ 6) (Fig. 1E) or 75 mg/kg sodium iodide (n ¼ 3) (Fig. 1F), a chemical with ionic strength similar to that of sodium iodate, no such changes were observed.
We examined the change in numbers of dark blots in response to different doses of sodium iodate using cSLO. Quantitative analyses showed a dose-dependent increase in the number of blots at 7 and 14 days after intravenous sodium iodate injection. The response was progressive in animals injected intravenously with 25 or 40 mg/kg sodium iodate and was more acute in those that received higher doses (Fig. 4). Moreover, animals with injection of 50 mg/kg sodium iodate administered intraperitoneally showed a milder response than those with a similar dose given intravenously (mean [SD], 666.5 [33.2]–fold at day 7 vs. 2.1 [42.6]–fold at day 14; P < 0.05).
Histopathology of Sodium Iodate–Induced Lesions in the Rat Retina To further investigate relationships of the structural changes in cSLO and OCT images, we examined the histologic changes in paraffin sections of the retina after intravenous injection of 40 mg/kg sodium iodate. Morphologic analyses showed no detectable change in cellular arrangement 1 day (n ¼ 3) and 4 days (n ¼ 3) after injection (Figs. 5B, 5C). The retinal layers were readily identified as in the normal controls (n ¼ 3) (Fig. 5A). Obvious lesions were found at day 7 (n ¼ 3), which appeared as massive disruption of the photopigment layer IS/ OS and focal reduction of photoreceptor cells in the ONL (Fig. 5D), producing irregular profiles of folding (or rosettes) in the outer retinal layers. The inner retinal layers, including the ganglion cell layer, IPL, and INL, were relatively spared. Similar abnormalities were observed in the retina 14 days after injection (n ¼ 3) but with a further increase in folding and a reduction in retinal thickness (Fig. 5E). In another experiment, we investigated the morphologic changes in retinal sections 14 days after intravenous injection of different doses of sodium iodate. Sodium iodate at 25 mg/kg (n ¼ 3), the lowest dose examined, generated obvious degeneration in the outer retinal layers and a reduction in retinal thickness compared with the controls injected with saline (n ¼ 3) (Figs. 6A, 6B). Higher doses (40 or 50 mg/kg) produced further degenerative changes in the retina. At 75 mg/ kg (n ¼ 6), the highest dose tested, there was massive disruption of all retinal layers (Fig. 6E).
cSLO Imaging of Rat Retina
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FIGURE 2. Sodium iodate–induced retinal lesions under spectral-domain OCT. (A–D) Cross-sectional OCT images of the retina in adult rats treated intravenously with 40 mg/kg sodium iodate showing the appearance of retinal layers in OCT images from a representative experimental animal. (C, D) Obvious degenerative profiles (white arrows) in the photopigment layer and ONL were first observed at day 7 and became more prominent at day 14. They were indicated by the appearance of a thin line with hyporeflectivity within the photoreceptor IS/OS interface, in addition to patches of degeneration in the INL that reflected the pattern of lesions seen in histologic sections. (E, F) This damage was not observed in control retinas 14 days after injection of saline (E) or sodium iodide (F). Scale bar: 200 lm.
Quantitative analyses of morphologic changes in these histologic sections showed a significant dose-dependent reduction in thickness of the photoreceptor layer IS/OS and ONL and in the number of rows of cells in the ONL in animals with intravenous injection of sodium iodate (25–75 mg/kg) at 7 and 14 days after injection compared with the saline control (Fig. 7). However, in animals injected intraperitoneally with sodium iodate, a significant reduction was observed only at 75 mg/kg and not at 50 mg/kg, indicating that intravenous injection is more effective in generating degeneration in the outer retinal layers. The analyses of IPL and INL revealed less severe damage compared with the saline control, particularly at day 7 (Fig. 8). The OCT images revealed a similar trend of changes in the ONL (Fig. 9A). While 25 mg/kg sodium iodate caused a mild reduction in thickness of the ONL 14 days after injection, injection of 40 and 75 mg/kg generated significant thinning (P < 0.05) both at days 7 and 14 compared with the saline control. Further analyses showed clearly that the reduction in ONL thickness compared with the corresponding saline control was similar between histologic sections and OCT images (P > 0.05) (Figs. 9B, 9C). These findings indicated that sodium iodate imposed selective effects on cellular structures
in the outer retina. The morphologic changes observed in histologic preparations were reflected in OCT analyses. The effects of sodium iodate on other organs were determined in these animals. Sodium iodate at 25 mg/kg (n ¼ 3) or 40 mg/kg (n ¼ 6) administered intravenously did not produce any obvious damage in kidney and liver (Figs. 10B, 10D) compared with the respective controls that had received saline injection (n ¼ 6; Figs. 10A, 10C). Obvious damages in cellular structures were observed at higher doses (50 or 75 mg/ kg; n ¼ 6) in both kidney and liver. Moreover, while all animals with 40 mg/kg injection showed lesions in the retina, only approximately 50% of rats with 25 mg/kg injection had retinal lesions, indicating that 40 mg/kg is the maximal dose that generates toxicity consistently to the retina but not to other organs.
Correlation of cSLO Images With Histology of the Retina To further investigate the anatomical basis of lesions in cSLO images, we selected six animals injected with 25 mg/kg sodium iodate that produced a restricted distribution of dark blots in live images of the retina. These retinas were later fixed with the
cSLO Imaging of Rat Retina
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FIGURE 3. Dark blots in cSLO images registered to degenerative profiles in OCT images. The cSLO and OCT images were collected from the superotemporal quadrant of a retina 7 days after injection of 40 mg/kg sodium iodate. The OCT captured the cSLO and OCT images from the retina simultaneously. (A) Confocal scanning laser ophthalmoscopy images depicting scan lines (white lines) that cut across regions without blots (lines 1 and 2) and regions that contain blots (white solid arrows) (lines 3–6). Hyperreflectivity was also observed in the blood vessels (white empty arrow). (B) Cross-sectional OCT images correspond to the six locations in A. Note the location of a blood vessel on the surface (black empty arrow in panel 1) and degenerative profiles (solid white arrows) in the ONL (panels 3–6) that registered to the blots in the cSLO image of this retina. A thin line with hyporeflectivity within the photoreceptor IS/OS interface and patches of degeneration in the INL (similar to Figs. 2C, 2D) were observed, more obvious within the lesion territory (panels 4 and 5) and less obvious along the periphery of the lesion (panels 1–3). Scale bars: 50 lm (A) and 100 lm (B). (C) The width of the blots in cSLO is strongly correlated with the width of dots in the ONL of the retina as revealed by OCT (P < 0.01, Spearman correlation output), collected simultaneously from the scanning of four retinas.
FIGURE 4. Dose-dependent effect of sodium iodate on the blot number in the retina. The number of blots in cSLO images increased with increasing dose of sodium iodate. Comparison of the number at day 14 versus at day 7 showed significant increases in animals injected intravenously with 25 or 40 mg/kg of drug compared with those injected with 75 mg/kg, the maximum dose tested. *P < 0.05, Mann-Whitney U test. Data are the mean (SD).
cSLO Imaging of Rat Retina
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FIGURE 5. Temporal changes in a retinal lesion after sodium iodate injection. Shown is a paraffin section of a rat retina stained with hematoxylineosin. (A) Saline control observed at 14 days. (B–E) On different days after injection of 40 mg/kg sodium iodate. The retinal layers are clearly depicted in the control retina and in retinas 1 and 4 days after sodium iodate injection. Disruption of the outer retinal layers (arrows) was detected at day 7, and the lesions became more severe at day 14. Note the progressive increase in folding in the outer retinal layers and thinning out of retinal thickness at the end of the examination period. GCL, ganglion cell layer; OPL, outer plexiform layer. Scale bar: 100 lm.
orientation marked, paraffin embedded, and sectioned at the level that cut across the blot-rich regions. We found a correlation of the blot-rich regions with retinal regions that showed extensive damage in the outer retinal layers. In one example, the blots were confined to the inferonasal quadrant
of the retina as revealed by cSLO. In histologic sections of this retina cutting across similar regions, lesions were observed in only nasal and not temporal hemiretina (Figs. 11A, 11B). In another example, the blots were found in regions surrounding the optic disc (Fig. 11C). Sections across this retina slightly
FIGURE 6. Dose effect of a sodium iodate–induced retinal lesion. Representative micrographs show responses of the retina to different doses of sodium iodate at day 14. (A) Saline control. (B–E) Sodium iodate at 25 to 75 mg/kg. Note the progressive changes of lesions that spread from the photopigment layer at low dose to the ONL and INL at higher doses. GCL, ganglion cell layer; OPL, outer plexiform layer. Scale bar: 50 lm.
cSLO Imaging of Rat Retina
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FIGURE 7. Quantitative analyses of the outer retinal layer damages after sodium iodate insults. (A) Dose-dependent reduction in thickness of the photoreceptor IS/OS in a histologic section of retina (n ¼ 3 in each group). Intravenous injection gave a more prominent effect than intraperitoneal injection at comparable doses of sodium iodate. (B) Similar changes were observed in the ONL. (C) Counting the number of rows of photoreceptor nuclei in the ONL confirmed the dose-dependent response. *P < 0.05, **P < 0.01 compared with the saline control (Mann-Whitney U test). Each plot indicates the mean (SD).
FIGURE 8. Quantitative analyses of changes in the inner retinal layers after sodium iodate injection. (A) Reduction in thickness of the IPL was only obvious at day 14, and again intravenous injection showed a more substantial effect than intraperitoneal injection at comparable doses of drug. (B) Cell density counts showed a dose-dependent reduction in the INL, which was particularly obvious at day 14. (C) Similar findings were obtained from analyses of the thickness of INL. Legends of the plots are the same as those of Figure 7. *P < 0.05, **P < 0.01 compared with the saline control (Mann-Whitney U test).
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cSLO Imaging of Rat Retina
FIGURE 9. Quantitative analyses of ONL thickness in OCT images. (A) Intravenous injection of sodium iodate at 40 or 75 mg/kg produced a significant reduction in thickness of the ONL in OCT images of the retina at days 7 and 14 (*P < 0.05, Mann-Whitney U test) compared with the saline control. Such reduction was not obvious in animals injected with 25 mg/kg sodium iodate. (B, C) The percentage of ONL thickness reduction caused by sodium iodate compared with the saline control was similar between histologic sections (hematoxylin-eosin) and OCT images. No significant difference was observed at all doses tested and on days 7 and 14 after sodium iodate injection (P > 0.05, Mann-Whitney U test) (n ¼ 6 in each plot). Each plot indicates the mean (SD).
inferior to the optic disc revealed obvious lesions in the central but not peripheral retina (Fig. 11D). These findings showed that dark blots revealed by cSLO corresponded to regions with degeneration of the outer retinal layers.
DISCUSSION
FIGURE 10. Histologic assessment of liver and kidney in sodium iodate–treated rats. (A, B) Hematoxylin-eosin staining of a paraffin section of kidney in a control rat (saline injected) and in a rat treated intravenously with 40 mg/kg sodium iodate 14 days after injection. (C, D) Hematoxylin-eosin–stained sections of liver in control and sodium iodate–injected rats.
In this study, we investigated in vivo imaging of sodium iodate– induced retinal lesions in adult rats. The major findings were as follows: (1) using infrared cSLO, degenerative profiles in the outer retinal layers could be recognized as dark patchy dots or blots in planar images of the retina; (2) these profiles appeared at the time when lesions were observed in the photoreceptor IS/OS and ONL as revealed by OCT imaging; (3) the distribution of these degenerative profiles correlated well with the lesion in the outer retinal layers in histologic sections; and (4) these degenerative profiles showed a dose-dependent change in response to sodium iodate insult. Results from this study indicate that infrared cSLO can be used reliably to assess retinal lesions induced by sodium iodate in living adult rats. Previous in vivo imaging studies16–18 reported the use of spectral-domain OCT as a tool to evaluate degenerative changes in the retina. It provides high-resolution cross-sectional views of the retina and has been used widely in the assessment of retinal lesions in experimental and mutant rodents. It has also been found useful and reliable in the assessment of longitudinal changes in macular thickness and optic fiber layer thickness in human retina.24–26 In the present study, we found that the appearance of dark blots in the cSLO
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cSLO Imaging of Rat Retina
FIGURE 11. Lesions in cSLO images indicated the location of outer retinal degeneration in histologic sections. (A) Confocal scanning laser ophthalmoscopy revealed in this rat a localized sodium iodate–induced lesion that was confined to the inferonasal quadrant of the retina. (B) Sectioning of this retina at the level indicated in A (broken line) showed concomitant degeneration in the outer retinal layers (black arrows) observed only in the nasal and not the temporal retina in hematoxylin-eosin preparation. (C) In another rat, dark blots were observed in central regions around the optic disc (white arrows). (D) Histologic section at the level indicated by the broken line in C showed restricted damage (black arrows) in the central retina. Scale bar: 500 lm.
retinal images in live rats corresponded to degenerative profiles in the photopigment layers and ONL of the retina after they were killed. Distributions of the blots restricted to certain areas in the live retina showed good correlation with lesion sites in the corresponding histologic sections. Moreover, these lesions were detected 7 days after injection of sodium iodate, coinciding with the time when obvious degenerative changes were observed in OCT images and histologic preparations. We therefore concluded that the lesions observed under infrared cSLO indicated degenerative profiles in the outer retinal layers. Moreover, changes in the number of lesions were dependent on the dose of sodium iodate. This phenomenon was consistent with morphometric analyses of the outer retinal layers in histologic sections. These dose-dependent responses suggested that cSLO can be used as a reliable tool to evaluate real-time longitudinal damage to the retina with the outer retinal layer lesions. However, it remains to be determined whether cSLO can be applied to degenerative changes in the inner retinal layers and in other animal species. The appearance of degenerative profiles in the form of folding or rosettes as observed in our findings has been reported in histologic preparations of the retina of SpragueDawley rats after injection of 40 mg/kg sodium iodate9 and in albino mice with experimentally induced retinal detachment.26 However, a similar pattern of degeneration was not reported by Hariri et al.,20,27 who utilized ultrahigh-resolution OCT to study degenerative changes after treatment with 40 mg/kg sodium iodate in Long-Evans rats. They observed a progressive destruction of cellular structure in the outer retinal layers, which seemed to initiate at the interphase between the IS/OS
of photoreceptors and the pigment epithelium as early as 1 hour after injection, producing a general and even thinning of the retina. The reported differences in the pattern of degeneration could be due to a difference in the subspecies of animals used. Pigmented rats were used in the studies by Hariri et al., while the study by Ohtaka et al.9 and the present study used rats lacking ocular pigment. How the folding and rosettes are formed in the retina is largely unknown. A study26 in mice showed the development of folds and rosettes in regions of retinal detachment, generated probably by an elongation of outer segments of some photoreceptors that resulted in rearrangement and misalignment of the ONL layer. It remains to be determined whether similar changes occur in photoreceptor cells in the rat after sodium iodate injection. In this study, we also showed that sodium iodate generated severe lesions on the photoreceptor IS/OS and ONL and milder changes in the IPL and INL. These pathologic changes were similar to those observed in early-onset AMD.28 Moreover, we showed from the dose-dependent assays that 40 mg/kg was the optimal concentration generating consistent retinal lesions in the outer retinal layers without obvious adverse effects to other organs, indicating that intravenous administration of drug at this dose can be used as a reliable model for experimental study of ocular degeneration diseases that involve the outer retinal layers such as AMD and retinitis pigmentosa.
Acknowledgments The authors thank Waiying Li, Shaoying Tan, Yongjie Qin, Kai-On Chu, and Benjamin Fuk Loi Li for technical assistance with the
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cSLO Imaging of Rat Retina OCT experiments and histologic preparations, as well as Christopher Leung for helpful comments on the manuscript. Supported in part by a block grant from the University Grants Committee Hong Kong, a General Research Fund (Project No. CUHK461612), and a seed grant from Lui Che Woo Institute of Innovative Medicine (Project No. 8303107). Disclosure: Y. Yang, None; T.K. Ng, None; C. Ye, None; Y.W.Y. Yip, None; K. Law, None; S.-O. Chan, None; C.P. Pang, None
15. 16.
17.
References 1. Berson EL. Retinitis pigmentosa: The Friedenwald Lecture. Invest Ophthalmol Vis Sci. 1993;34:1659–1676. 2. de Jong PT. Age-related macular degeneration. N Engl J Med. 2006;355:1474–1485. 3. Kiuchi K, Yoshizawa K, Shikata N, Moriguchi K, Tsubura A. Morphologic characteristics of retinal degeneration induced by sodium iodate in mice. Curr Eye Res. 2002;25:373–379. 4. Enzmann V, Row BW, Yamauchi Y, et al. Behavioral and anatomical abnormalities in a sodium iodate–induced model of retinal pigment epithelium degeneration. Exp Eye Res. 2006; 82:441–448. 5. Franco LM, Zulliger R, Wolf-Schnurrbusch UE, et al. Decreased visual function after patchy loss of retinal pigment epithelium induced by low-dose sodium iodate. Invest Ophthalmol Vis Sci. 2009;50:4004–4010. 6. Machalinska A, Lubinski W, Klos P, et al. Sodium iodate selectively injures the posterior pole of the retina in a dosedependent manner: morphological and electrophysiological study. Neurochem Res. 2010;35:1819–1827. 7. Enzmann V, Howard RM, Yamauchi Y, Whittemore SR, Kaplan HJ. Enhanced induction of RPE lineage markers in pluripotent neural stem cells engrafted into the adult rat subretinal space. Invest Ophthalmol Vis Sci. 2003;44:5417–5422. 8. Gong L, Wu Q, Song B, Lu B, Zhang Y. Differentiation of rat mesenchymal stem cells transplanted into the subretinal space of sodium iodate–injected rats. Clin Exp Ophthalmol. 2008; 36:666–671. 9. Ohtaka K, Machida S, Ohzeki T, et al. Protective effect of hepatocyte growth factor against degeneration of the retinal pigment epithelium and photoreceptor in sodium iodate– injected rats. Curr Eye Res. 2006;31:347–355. 10. Grignolo A, Orzalesi N, Calabria GA. Studies on the fine structure and the rhodopsin cycle of the rabbit retina in experimental degeneration induced by sodium iodate. Exp Eye Res. 1966;5:86–97. 11. Suyama T. Electron microscopic study on experimental retinal degeneration induced by sodium iodate injection. Yonago Acta Med. 1967;11:222–231. 12. Flage T. Changes in the juxtapapillary retinal pigment epithelium following intravenous injection of sodium iodate: a light and electron microscopic study using horseradish peroxidase as a tracer. Acta Ophthalmol. 1983;61:20–28. 13. Yoon YH, Marmor MF. Retinal pigment epithelium adhesion to Bruch’s membrane is weakened by hemicholinium-3 and sodium iodate. Ophthalmol Res. 1993;25:386–392. 14. Nilsson SE, Knave B, Persson HE. Changes in ultrastructure and function of the sheep pigment epithelium and retina induced by sodium iodate, I: the ultrastructure of the normal
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
pigment epithelium of the sheep. Acta Ophthalmol. 1977;55: 994–1006. Huang D, Swanson EA, Lin CP, et al. Optical coherence tomography. Science. 1991;254:1178–1181. Ruggeri M, Wehbe H, Jiao S, et al. In vivo three-dimensional high-resolution imaging of rodent retina with spectral-domain optical coherence tomography. Invest Ophthalmol Vis Sci. 2007;48:1808–1814. Fischer MD, Huber G, Beck SC, et al. Noninvasive, in vivo assessment of mouse retinal structure using optical coherence tomography. PloS One [serial online]. 2009;4:e7507. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759518. Accessed February 14, 2014. Nakano M, Otsuka F, Virmani R. Letter by Nakano et al regarding article, ‘‘Optical Coherence Tomographic Analysis of In-Stent Neoatherosclerosis After Drug-Eluting Stent Implantation.’’ Circulation [serial online]. 2011;124:e954. Available at: http://circ.ahajournals.org/content/124/25/e954.full. Accessed February 14, 2014. Muraoka Y, Ikeda HO, Nakano N, et al. Real-time imaging of rabbit retina with retinal degeneration by using spectral-domain optical coherence tomography. PloS One [serial online]. 2012;7: e36135. Available at: http://www.ncbi.nlm.nih.gov/pmc/ articles/PMC3338600. Accessed February 14, 2014. Hariri S, Moayed AA, Choh V, Bizheva K. In vivo assessment of thickness and reflectivity in a rat outer retinal degeneration model with ultrahigh resolution optical coherence tomography. Invest Ophthalmol Vis Sci. 2012;53:1982–1989. Liu S, Li ZW, Weinreb RN, et al. Tracking retinal microgliosis in models of retinal ganglion cell damage. Invest Ophthalmol Vis Sci. 2012;53:6254–6262. Li ZW, Liu S, Weinreb RN, et al. Tracking dendritic shrinkage of retinal ganglion cells after acute elevation of intraocular pressure. Invest Ophthalmol Vis Sci. 2011;52:7205–7212. Guo L, Normando EM, Nizari S, Lara D, Cordeiro MF. Tracking longitudinal retinal changes in experimental ocular hypertension using the cSLO and spectral domain–OCT. Invest Ophthalmol Vis Sci. 2010;51:6504–6513. Leung CK, Cheung CY, Lin D, Pang CP, Lam DS, Weinreb RN. Longitudinal variability of optic disc and retinal nerve fiber layer measurements. Invest Ophthalmol Vis Sci. 2008;49: 4886–4892. Leung CK, Cheung CY, Weinreb RN, et al. Comparison of macular thickness measurements between time domain and spectral domain optical coherence tomography. Invest Ophthalmol Vis Sci. 2008;49:4893–4897. Secondi R, Kong K, Blonska AM, Staurenghi G, Sparrow JR. Fundus autofluorescence findings in a mouse model of retinal detachment. Invest Ophthalmol Vis Sci. 2012;53:5190–5197. Hariri S, Tam MC, Lee D, Hileeto D, Moayed AA, Bizheva K. Noninvasive imaging of the early effect of sodium iodate toxicity in a rat model of outer retina degeneration with spectral domain optical coherence tomography. J Biomed Optics [serial online]. 2013;18:26017. Available at: http://biomedicaloptics. spiedigitallibrary.org/article.aspx?articleid¼1569950. Accessed February 14, 2014. Fleckenstein M, Charbel Issa P, Helb HM, et al. High-resolution spectral domain–OCT imaging in geographic atrophy associated with age-related macular degeneration. Invest Ophthalmol Vis Sci. 2008;49:4137–4144.
Assessing Sodium Iodate–Induced Outer Retinal Changes in Rats Using Confocal Scanning Laser Ophthalmoscopy and Optical Coherence Tomography Yaping Yang,1,2 Tsz Kin Ng,1 Cong Ye,1 Yolanda W. Y. Yip,1 Kasin Law,1 Sun-On Chan,3 and Chi Pui Pang1 1Department
of Ophthalmology and Visual Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China 2 Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, China 3 School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China
Correspondence: Chi Pui Pang, Department of Ophthalmology and Visual Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong Eye Hospital, 147K Argyle Street, Kowloon, Hong Kong SAR, China; [email protected]. S-OC and CPP contributed equally to the work presented here and should therefore be regarded as equivalent authors. Submitted: May 24, 2013 Accepted: February 1, 2014 Citation: Yang Y, Ng TK, Ye C, et al. Assessing sodium iodate–induced outer retinal changes in rats using confocal scanning laser ophthalmoscopy and optical coherence tomography. Invest Ophthalmol Vis Sci. 2014;55:1696–1705. DOI:10.1167/ iovs.13-12477
PURPOSE. Sodium iodate induces RPE atrophy and photoreceptor degeneration, as seen in the pathogenesis of many retinal diseases. We investigated a new approach of analyzing retinal images using confocal scanning laser ophthalmoscopy (cSLO) that allows longitudinal assessment of sodium iodate–induced lesions in the retina of living rats. METHODS. A single dose of sodium iodate (25–75 mg/kg) was given intravenously to adult Sprague-Dawley rats. Control animals were given normal saline or sodium iodide. The retina was examined by cSLO and optical coherence tomography (OCT) in living rats, which were then killed for histologic assessments. RESULTS. Confocal scanning laser ophthalmoscopy revealed the appearance of dark patchy blots in planar images of the retina 7 days after intravenous injection of sodium iodate (25–75 mg/kg). This finding coincided with the observations of degenerative changes in the outer retinal layers in OCT images and in histology of the retina. Further analyses showed a concomitant localization of degenerative profiles in histologic preparations of this retina, suggesting that the blots corresponded to the deteriorating photopigments and outer nuclear layer (ONL). In histologic sections, these degenerative profiles appeared as irregular folds or rosettes in the ONL. Quantitative analyses showed that the changes in blot number were dose dependent, which again coincided with results showing a dose-dependent lesion in the photopigment layer and ONL in histologic sections of the retina. CONCLUSIONS. Sodium iodate–induced degenerative changes can be assessed quantitatively and reliably by in vivo retinal imaging using cSLO in adult rats, allowing efficient evaluation of lesions in a large area of retina in longitudinal studies. Keywords: in vivo, retinal degeneration, histology
etinal pigment epithelium and photoreceptor degeneration is typically found in retinal diseases, including early-stage AMD and retinitis pigmentosa,1,2 and can lead to severe visual loss and eventual blindness. Sodium iodate is a retinotoxin known to selectively damage the RPE by oxidative stress, resulting in photoreceptor apoptosis.3 Systemic administration of different doses of sodium iodate has been used in mice to evaluate the effect on visual function,4,5 retinal morphology,3 and other functional features.6 Sodium iodate–induced retinal degeneration in rats was used as an animal model to explore the capability of stem cells to differentiate into RPE and photoreceptors after transplantation into the subretinal space7,8 and to investigate the protective effect of hepatocyte growth factor against retinal degeneration.9 Sodium iodate–induced retinal degeneration has also been reported in rabbits10–13 and sheep.14 Traditional assessments of sodium iodate–induced retinal degeneration are based on electrophysiology and histologic examination. Electrophysiology allows longitudinal monitoring of the retinal function but not structural changes of the retina.
While histologic examination provides clear visualization of individual layers of the retina, many animals are often needed for longitudinal study because they have to be killed at specific intervals. In vivo imaging of the retina is thus the preferred approach to investigate the structural changes of the retina in live animals. Spectral-domain optical coherence tomography (OCT) has been developed as a noninvasive technique for in vivo crosssectional imaging of the retina.15 This technique has been reported for examination of real-time longitudinal changes in the retina of mutant mice with photoreceptor degeneration16,17 and in mice with intravitreal injection of N-methyl-D-aspartate.18 Spectral-domain OCT also revealed degenerative changes in the outer retinal layers in transgenic rabbits19 and in rats with retinal lesions induced by sodium iodate.20 However, while live information on the site of lesions within a confined area of the retina can be provided, spectral-domain OCT is limited in deciphering whether a lesion is widespread throughout different areas of the retina and to quantify the damage across different retinal quadrants. We therefore attempted a novel in
Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783
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cSLO Imaging of Rat Retina vivo method using confocal scanning laser ophthalmoscopy (cSLO) that allows efficient quantitative evaluation of damage in different quadrants of the retina for assessments of lesions induced by sodium iodate in the adult rat.
METHODS Animal Ethics Statement All rats were treated according to guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The experimental protocol in this study was approved by the Animal Experimentation Ethics Committee of The Chinese University of Hong Kong. Adult SpragueDawley rats, weighing 200 to 250 g and aged 8 weeks, were obtained from the Laboratory Animal Service Center of The Chinese University of Hong Kong. The animals were housed in standard conditions and maintained at 22 6 18C, 40% 6 10% humidity, and a cycle of 12 hours of dark and 12 hours of light. Standard rodent chow and water were provided ad libitum. For each experimental group, at least three rats were used and included in the analyses.
IOVS j March 2014 j Vol. 55 j No. 3 j 1697 scanning the rat eye by altering configurations at the software level. The zero-point length of the reference arm was adjusted from the default setting of 20,480 lm to 20,060 lm. Thus, infrared retinal fundus photographs and OCT images could be simultaneously captured on the exact retinal focus, which ensures the high quality of OCT imaging in the retina. In each retina, four different square regions (each from the superotemporal, inferotemporal, inferonasal, and superonasal quadrants) around the optic nerve head were scanned separately without the widefield lens by the volume scan protocol, which consists of 19 evenly distributed B-scans (1024 A-scans for each B-scan) covering a 208 3 158 area of the retina.23–25 Nine images for each B-scan at the same retinal location were captured and averaged. The OCT system registers the cSLO and OCT images in the retina simultaneously. All retinas were examined before (baseline) and after sodium iodate injection (days 1, 4, 7, and 14). Prior to imaging, the rat was anesthetized and immediately placed on a custom-made platform. The head was fixed in position for imaging. Pupils were dilated by topical 1% tropicamide. No contact lens was required. During imaging, 1 drop of sterile saline was applied to the cornea every 2 minutes to maintain media clarity.
Sodium Iodate Application Sodium iodate (Sigma-Aldrich Corp., St. Louis, MO) was dissolved in sterile normal saline as a 4% stock solution (wt/ vol). The rats were anesthetized by intraperitoneal injection of ketamine (35 mg/kg, Ketaset; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (5 mg/kg, TranquiVed; Vedco, Inc., St. Joseph, MO). A single intravenous or intraperitoneal injection of sodium iodate was given. For intravenous injection, the rats were divided into four groups; each received a single injection of 25, 40, 50, or 75 mg/kg sodium iodate. The control animals were injected with the same volume of normal saline or 75 mg/ kg sodium iodide; the latter was administered at an ionic strength comparable to that of the highest dose of sodium iodate used in this study. For intraperitoneal injection, only two doses were tested (50 and 75 mg/kg) in order to compare the effect on retina with that in rats injected intravenously. After the injection, the rats were returned to the colony and kept under standard conditions.
Retinal Examination by cSLO and OCT We used cSLO and spectral-domain OCT (SPECTRALIS HRA þ OCT system; Heidelberg Engineering GmbH, Dossenheim, Germany) for in vivo imaging utilizing an adaptive device specific for imaging rat eye. The cSLO infrared reflectance was recorded using a light source with 820-nm wavelength to provide planar visualization of the retina. The scan rate of the cSLO was 16 frames per second. A 558 widefield noncontact lens (Heidelberg Engineering GmbH) was added to the camera in order to capture high-quality images in a wider view of the fundus. Eye tracking (a retinal recognition technology enabling the exact same retinal location to be scanned and ‘‘locked on’’) was activated during imaging. Fifteen images at the same retinal location at the same focal depth were captured and averaged automatically by the built-in software to increase the signal-to-noise ratio and simultaneously displayed on a computer screen.21,22 These images were used for subsequent counting of degenerating blots in optical sections of the retina. The OCT system used a superluminescent diode light source with a center wavelength of 870 lm. The OCT parameters were modified to adapt OCT imaging in rats according to the technical advice from the manufacturer (Heidelberg Engineering GmbH). The length of the reference arm was adjusted to match the length of the sample arm
Histologic Assessment of the Retina At postinjection day 14, the rats were killed with an overdose of pentobarbital sodium (20% wt/vol) and perfused with PBS, followed by 4% paraformaldehyde in PBS at pH 7.4. Eyes were removed and further fixed in 10% formalin before paraffin embedding. Five-micrometer sections of the eye were obtained at the pupil–optic nerve position. The sections were stained with hematoxylin-eosin and imaged using a light microscope (DMRB; Leica, Wetzlar, Germany) connected to a SPOT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI).
Evaluation of Retinal Lesions For the paraffin sections, morphologic analyses were performed within a 1000-lm2 area in the superotemporal quadrant of the retina 300 lm from the optic nerve head using ImageJ (version 1.46e; National Institutes of Health, Bethesda, MD). Measurements included thickness of inner/ outer segments (IS/OS) of photoreceptors, outer nuclear layer (ONL), inner nuclear layer (INL), and inner plexiform layer (IPL). The INL cell densities were also counted in these regions. To correlate the changes in histologic sections with the OCT images, the changes in ONL thickness, which were readily identified, were measured in OCT images captured at corresponding regions of the retina using ImageJ (National Institutes of Health). In vivo images were captured by cSLO at baseline, day 7, and day 14 after sodium iodate injection. In each retina, four different square regions (each 400 3 400 lm) with clear visualization of the blots that correspond to the location of lesions were analyzed. The images were exported to a computer for generating montages of the central retina (Fig. 1) and for counting dark blots using Photoshop (11.0; Adobe Systems, Incorporated, San Jose, CA). The blots in the central retina in each eye were counted manually before and after the injection by a colleague blinded to the experiment. Mann-Whitney U test was used to compare the means between different experimental groups and the controls. Data were expressed as means (SDs). All analyses were performed using PASW Statistics 18 (SPSS Science, Chicago, IL), and differences were considered significant at P < 0.05.
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IOVS j March 2014 j Vol. 55 j No. 3 j 1698 Cross-sectional images of the retina were also taken from these animals using spectral-domain OCT. Images from day 0 animals showed a typical laminated pattern of the retina (Fig. 2A), which was observed also in retinas 4 days after sodium iodate injection (Fig. 2B). Structural changes were first detected at day 7, which were characterized by the appearance of dome-shaped hyperreflective areas in outer layers of the retina, corresponding to the ONL and IS/OS of photoreceptors (Fig. 2C). These abnormalities were more severe and abundant 14 days after injection (Fig. 2D), representing lesions induced by sodium iodate that were known to confine preferentially to the outer retinal layers.6,20 Such abnormalities were not observed in control animals injected with saline (Fig. 2E) or sodium iodide (Fig. 2F). Further analyses of images collected by cSLO (n ¼ 3) and their corresponding cross-sectional OCT images showed good correlation spatially of the lesions in the planar image of the retina with degenerative regions in crosssectional images (Figs. 3A, 3B). Moreover, the analyses of the width of the blots in cSLO strongly correlated positively with the width of degenerative profiles in the ONL revealed in OCT images (Fig. 3C), indicating that degenerative changes in the outer retinal layers are visualized readily in cSLO images of the retina.
Dose Effect of Sodium Iodate
FIGURE 1. Sodium iodate–induced retinal lesions under infrared cSLO. Montages of retinal images are generated by cSLO. (A–D) Typical changes in the retina of a rat injected intravenously with 40 mg/kg sodium iodate. Dark patchy dots or blots (white arrows) were detected in all retinal quadrants 7 and 14 days after injection. (E, F) Retina in control animals 14 days after injection of saline or 75 mg/kg sodium iodide. OD, optic disc. Superior is up and temporal to the right in these images. Scale bar: 200 lm.
RESULTS cSLO and OCT in the Rat Retina Retinal lesions were induced in adult rats with intravenous injection of 40 mg/kg sodium iodate. The retina was examined using cSLO before the injection (day 0) (n ¼ 12). In these retinal images, typical appearances of the retinal vessels and optic nerve head were readily recognizable. No obvious change was visible in the retina of the animals 4 days after injection (Fig. 1B). However, obvious changes started to appear at day 7 and were characterized by the presence of many small dark dotted profiles in the retina (Fig. 1C). These dark profiles or blots were not observed in any optical section through the whole thickness of the retina at day 0 and day 4. The changes were more obvious in the retina when examined at day 14 (Fig. 1D). In most rats, such dark blots were found throughout the retina, whereas in others the distribution was limited to certain regions (see Fig. 10). In control animals that had been injected either with saline (n ¼ 6) (Fig. 1E) or 75 mg/kg sodium iodide (n ¼ 3) (Fig. 1F), a chemical with ionic strength similar to that of sodium iodate, no such changes were observed.
We examined the change in numbers of dark blots in response to different doses of sodium iodate using cSLO. Quantitative analyses showed a dose-dependent increase in the number of blots at 7 and 14 days after intravenous sodium iodate injection. The response was progressive in animals injected intravenously with 25 or 40 mg/kg sodium iodate and was more acute in those that received higher doses (Fig. 4). Moreover, animals with injection of 50 mg/kg sodium iodate administered intraperitoneally showed a milder response than those with a similar dose given intravenously (mean [SD], 666.5 [33.2]–fold at day 7 vs. 2.1 [42.6]–fold at day 14; P < 0.05).
Histopathology of Sodium Iodate–Induced Lesions in the Rat Retina To further investigate relationships of the structural changes in cSLO and OCT images, we examined the histologic changes in paraffin sections of the retina after intravenous injection of 40 mg/kg sodium iodate. Morphologic analyses showed no detectable change in cellular arrangement 1 day (n ¼ 3) and 4 days (n ¼ 3) after injection (Figs. 5B, 5C). The retinal layers were readily identified as in the normal controls (n ¼ 3) (Fig. 5A). Obvious lesions were found at day 7 (n ¼ 3), which appeared as massive disruption of the photopigment layer IS/ OS and focal reduction of photoreceptor cells in the ONL (Fig. 5D), producing irregular profiles of folding (or rosettes) in the outer retinal layers. The inner retinal layers, including the ganglion cell layer, IPL, and INL, were relatively spared. Similar abnormalities were observed in the retina 14 days after injection (n ¼ 3) but with a further increase in folding and a reduction in retinal thickness (Fig. 5E). In another experiment, we investigated the morphologic changes in retinal sections 14 days after intravenous injection of different doses of sodium iodate. Sodium iodate at 25 mg/kg (n ¼ 3), the lowest dose examined, generated obvious degeneration in the outer retinal layers and a reduction in retinal thickness compared with the controls injected with saline (n ¼ 3) (Figs. 6A, 6B). Higher doses (40 or 50 mg/kg) produced further degenerative changes in the retina. At 75 mg/ kg (n ¼ 6), the highest dose tested, there was massive disruption of all retinal layers (Fig. 6E).
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FIGURE 2. Sodium iodate–induced retinal lesions under spectral-domain OCT. (A–D) Cross-sectional OCT images of the retina in adult rats treated intravenously with 40 mg/kg sodium iodate showing the appearance of retinal layers in OCT images from a representative experimental animal. (C, D) Obvious degenerative profiles (white arrows) in the photopigment layer and ONL were first observed at day 7 and became more prominent at day 14. They were indicated by the appearance of a thin line with hyporeflectivity within the photoreceptor IS/OS interface, in addition to patches of degeneration in the INL that reflected the pattern of lesions seen in histologic sections. (E, F) This damage was not observed in control retinas 14 days after injection of saline (E) or sodium iodide (F). Scale bar: 200 lm.
Quantitative analyses of morphologic changes in these histologic sections showed a significant dose-dependent reduction in thickness of the photoreceptor layer IS/OS and ONL and in the number of rows of cells in the ONL in animals with intravenous injection of sodium iodate (25–75 mg/kg) at 7 and 14 days after injection compared with the saline control (Fig. 7). However, in animals injected intraperitoneally with sodium iodate, a significant reduction was observed only at 75 mg/kg and not at 50 mg/kg, indicating that intravenous injection is more effective in generating degeneration in the outer retinal layers. The analyses of IPL and INL revealed less severe damage compared with the saline control, particularly at day 7 (Fig. 8). The OCT images revealed a similar trend of changes in the ONL (Fig. 9A). While 25 mg/kg sodium iodate caused a mild reduction in thickness of the ONL 14 days after injection, injection of 40 and 75 mg/kg generated significant thinning (P < 0.05) both at days 7 and 14 compared with the saline control. Further analyses showed clearly that the reduction in ONL thickness compared with the corresponding saline control was similar between histologic sections and OCT images (P > 0.05) (Figs. 9B, 9C). These findings indicated that sodium iodate imposed selective effects on cellular structures
in the outer retina. The morphologic changes observed in histologic preparations were reflected in OCT analyses. The effects of sodium iodate on other organs were determined in these animals. Sodium iodate at 25 mg/kg (n ¼ 3) or 40 mg/kg (n ¼ 6) administered intravenously did not produce any obvious damage in kidney and liver (Figs. 10B, 10D) compared with the respective controls that had received saline injection (n ¼ 6; Figs. 10A, 10C). Obvious damages in cellular structures were observed at higher doses (50 or 75 mg/ kg; n ¼ 6) in both kidney and liver. Moreover, while all animals with 40 mg/kg injection showed lesions in the retina, only approximately 50% of rats with 25 mg/kg injection had retinal lesions, indicating that 40 mg/kg is the maximal dose that generates toxicity consistently to the retina but not to other organs.
Correlation of cSLO Images With Histology of the Retina To further investigate the anatomical basis of lesions in cSLO images, we selected six animals injected with 25 mg/kg sodium iodate that produced a restricted distribution of dark blots in live images of the retina. These retinas were later fixed with the
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FIGURE 3. Dark blots in cSLO images registered to degenerative profiles in OCT images. The cSLO and OCT images were collected from the superotemporal quadrant of a retina 7 days after injection of 40 mg/kg sodium iodate. The OCT captured the cSLO and OCT images from the retina simultaneously. (A) Confocal scanning laser ophthalmoscopy images depicting scan lines (white lines) that cut across regions without blots (lines 1 and 2) and regions that contain blots (white solid arrows) (lines 3–6). Hyperreflectivity was also observed in the blood vessels (white empty arrow). (B) Cross-sectional OCT images correspond to the six locations in A. Note the location of a blood vessel on the surface (black empty arrow in panel 1) and degenerative profiles (solid white arrows) in the ONL (panels 3–6) that registered to the blots in the cSLO image of this retina. A thin line with hyporeflectivity within the photoreceptor IS/OS interface and patches of degeneration in the INL (similar to Figs. 2C, 2D) were observed, more obvious within the lesion territory (panels 4 and 5) and less obvious along the periphery of the lesion (panels 1–3). Scale bars: 50 lm (A) and 100 lm (B). (C) The width of the blots in cSLO is strongly correlated with the width of dots in the ONL of the retina as revealed by OCT (P < 0.01, Spearman correlation output), collected simultaneously from the scanning of four retinas.
FIGURE 4. Dose-dependent effect of sodium iodate on the blot number in the retina. The number of blots in cSLO images increased with increasing dose of sodium iodate. Comparison of the number at day 14 versus at day 7 showed significant increases in animals injected intravenously with 25 or 40 mg/kg of drug compared with those injected with 75 mg/kg, the maximum dose tested. *P < 0.05, Mann-Whitney U test. Data are the mean (SD).
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FIGURE 5. Temporal changes in a retinal lesion after sodium iodate injection. Shown is a paraffin section of a rat retina stained with hematoxylineosin. (A) Saline control observed at 14 days. (B–E) On different days after injection of 40 mg/kg sodium iodate. The retinal layers are clearly depicted in the control retina and in retinas 1 and 4 days after sodium iodate injection. Disruption of the outer retinal layers (arrows) was detected at day 7, and the lesions became more severe at day 14. Note the progressive increase in folding in the outer retinal layers and thinning out of retinal thickness at the end of the examination period. GCL, ganglion cell layer; OPL, outer plexiform layer. Scale bar: 100 lm.
orientation marked, paraffin embedded, and sectioned at the level that cut across the blot-rich regions. We found a correlation of the blot-rich regions with retinal regions that showed extensive damage in the outer retinal layers. In one example, the blots were confined to the inferonasal quadrant
of the retina as revealed by cSLO. In histologic sections of this retina cutting across similar regions, lesions were observed in only nasal and not temporal hemiretina (Figs. 11A, 11B). In another example, the blots were found in regions surrounding the optic disc (Fig. 11C). Sections across this retina slightly
FIGURE 6. Dose effect of a sodium iodate–induced retinal lesion. Representative micrographs show responses of the retina to different doses of sodium iodate at day 14. (A) Saline control. (B–E) Sodium iodate at 25 to 75 mg/kg. Note the progressive changes of lesions that spread from the photopigment layer at low dose to the ONL and INL at higher doses. GCL, ganglion cell layer; OPL, outer plexiform layer. Scale bar: 50 lm.
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FIGURE 7. Quantitative analyses of the outer retinal layer damages after sodium iodate insults. (A) Dose-dependent reduction in thickness of the photoreceptor IS/OS in a histologic section of retina (n ¼ 3 in each group). Intravenous injection gave a more prominent effect than intraperitoneal injection at comparable doses of sodium iodate. (B) Similar changes were observed in the ONL. (C) Counting the number of rows of photoreceptor nuclei in the ONL confirmed the dose-dependent response. *P < 0.05, **P < 0.01 compared with the saline control (Mann-Whitney U test). Each plot indicates the mean (SD).
FIGURE 8. Quantitative analyses of changes in the inner retinal layers after sodium iodate injection. (A) Reduction in thickness of the IPL was only obvious at day 14, and again intravenous injection showed a more substantial effect than intraperitoneal injection at comparable doses of drug. (B) Cell density counts showed a dose-dependent reduction in the INL, which was particularly obvious at day 14. (C) Similar findings were obtained from analyses of the thickness of INL. Legends of the plots are the same as those of Figure 7. *P < 0.05, **P < 0.01 compared with the saline control (Mann-Whitney U test).
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FIGURE 9. Quantitative analyses of ONL thickness in OCT images. (A) Intravenous injection of sodium iodate at 40 or 75 mg/kg produced a significant reduction in thickness of the ONL in OCT images of the retina at days 7 and 14 (*P < 0.05, Mann-Whitney U test) compared with the saline control. Such reduction was not obvious in animals injected with 25 mg/kg sodium iodate. (B, C) The percentage of ONL thickness reduction caused by sodium iodate compared with the saline control was similar between histologic sections (hematoxylin-eosin) and OCT images. No significant difference was observed at all doses tested and on days 7 and 14 after sodium iodate injection (P > 0.05, Mann-Whitney U test) (n ¼ 6 in each plot). Each plot indicates the mean (SD).
inferior to the optic disc revealed obvious lesions in the central but not peripheral retina (Fig. 11D). These findings showed that dark blots revealed by cSLO corresponded to regions with degeneration of the outer retinal layers.
DISCUSSION
FIGURE 10. Histologic assessment of liver and kidney in sodium iodate–treated rats. (A, B) Hematoxylin-eosin staining of a paraffin section of kidney in a control rat (saline injected) and in a rat treated intravenously with 40 mg/kg sodium iodate 14 days after injection. (C, D) Hematoxylin-eosin–stained sections of liver in control and sodium iodate–injected rats.
In this study, we investigated in vivo imaging of sodium iodate– induced retinal lesions in adult rats. The major findings were as follows: (1) using infrared cSLO, degenerative profiles in the outer retinal layers could be recognized as dark patchy dots or blots in planar images of the retina; (2) these profiles appeared at the time when lesions were observed in the photoreceptor IS/OS and ONL as revealed by OCT imaging; (3) the distribution of these degenerative profiles correlated well with the lesion in the outer retinal layers in histologic sections; and (4) these degenerative profiles showed a dose-dependent change in response to sodium iodate insult. Results from this study indicate that infrared cSLO can be used reliably to assess retinal lesions induced by sodium iodate in living adult rats. Previous in vivo imaging studies16–18 reported the use of spectral-domain OCT as a tool to evaluate degenerative changes in the retina. It provides high-resolution cross-sectional views of the retina and has been used widely in the assessment of retinal lesions in experimental and mutant rodents. It has also been found useful and reliable in the assessment of longitudinal changes in macular thickness and optic fiber layer thickness in human retina.24–26 In the present study, we found that the appearance of dark blots in the cSLO
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cSLO Imaging of Rat Retina
FIGURE 11. Lesions in cSLO images indicated the location of outer retinal degeneration in histologic sections. (A) Confocal scanning laser ophthalmoscopy revealed in this rat a localized sodium iodate–induced lesion that was confined to the inferonasal quadrant of the retina. (B) Sectioning of this retina at the level indicated in A (broken line) showed concomitant degeneration in the outer retinal layers (black arrows) observed only in the nasal and not the temporal retina in hematoxylin-eosin preparation. (C) In another rat, dark blots were observed in central regions around the optic disc (white arrows). (D) Histologic section at the level indicated by the broken line in C showed restricted damage (black arrows) in the central retina. Scale bar: 500 lm.
retinal images in live rats corresponded to degenerative profiles in the photopigment layers and ONL of the retina after they were killed. Distributions of the blots restricted to certain areas in the live retina showed good correlation with lesion sites in the corresponding histologic sections. Moreover, these lesions were detected 7 days after injection of sodium iodate, coinciding with the time when obvious degenerative changes were observed in OCT images and histologic preparations. We therefore concluded that the lesions observed under infrared cSLO indicated degenerative profiles in the outer retinal layers. Moreover, changes in the number of lesions were dependent on the dose of sodium iodate. This phenomenon was consistent with morphometric analyses of the outer retinal layers in histologic sections. These dose-dependent responses suggested that cSLO can be used as a reliable tool to evaluate real-time longitudinal damage to the retina with the outer retinal layer lesions. However, it remains to be determined whether cSLO can be applied to degenerative changes in the inner retinal layers and in other animal species. The appearance of degenerative profiles in the form of folding or rosettes as observed in our findings has been reported in histologic preparations of the retina of SpragueDawley rats after injection of 40 mg/kg sodium iodate9 and in albino mice with experimentally induced retinal detachment.26 However, a similar pattern of degeneration was not reported by Hariri et al.,20,27 who utilized ultrahigh-resolution OCT to study degenerative changes after treatment with 40 mg/kg sodium iodate in Long-Evans rats. They observed a progressive destruction of cellular structure in the outer retinal layers, which seemed to initiate at the interphase between the IS/OS
of photoreceptors and the pigment epithelium as early as 1 hour after injection, producing a general and even thinning of the retina. The reported differences in the pattern of degeneration could be due to a difference in the subspecies of animals used. Pigmented rats were used in the studies by Hariri et al., while the study by Ohtaka et al.9 and the present study used rats lacking ocular pigment. How the folding and rosettes are formed in the retina is largely unknown. A study26 in mice showed the development of folds and rosettes in regions of retinal detachment, generated probably by an elongation of outer segments of some photoreceptors that resulted in rearrangement and misalignment of the ONL layer. It remains to be determined whether similar changes occur in photoreceptor cells in the rat after sodium iodate injection. In this study, we also showed that sodium iodate generated severe lesions on the photoreceptor IS/OS and ONL and milder changes in the IPL and INL. These pathologic changes were similar to those observed in early-onset AMD.28 Moreover, we showed from the dose-dependent assays that 40 mg/kg was the optimal concentration generating consistent retinal lesions in the outer retinal layers without obvious adverse effects to other organs, indicating that intravenous administration of drug at this dose can be used as a reliable model for experimental study of ocular degeneration diseases that involve the outer retinal layers such as AMD and retinitis pigmentosa.
Acknowledgments The authors thank Waiying Li, Shaoying Tan, Yongjie Qin, Kai-On Chu, and Benjamin Fuk Loi Li for technical assistance with the
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cSLO Imaging of Rat Retina OCT experiments and histologic preparations, as well as Christopher Leung for helpful comments on the manuscript. Supported in part by a block grant from the University Grants Committee Hong Kong, a General Research Fund (Project No. CUHK461612), and a seed grant from Lui Che Woo Institute of Innovative Medicine (Project No. 8303107). Disclosure: Y. Yang, None; T.K. Ng, None; C. Ye, None; Y.W.Y. Yip, None; K. Law, None; S.-O. Chan, None; C.P. Pang, None
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References 1. Berson EL. Retinitis pigmentosa: The Friedenwald Lecture. Invest Ophthalmol Vis Sci. 1993;34:1659–1676. 2. de Jong PT. Age-related macular degeneration. N Engl J Med. 2006;355:1474–1485. 3. Kiuchi K, Yoshizawa K, Shikata N, Moriguchi K, Tsubura A. Morphologic characteristics of retinal degeneration induced by sodium iodate in mice. Curr Eye Res. 2002;25:373–379. 4. Enzmann V, Row BW, Yamauchi Y, et al. Behavioral and anatomical abnormalities in a sodium iodate–induced model of retinal pigment epithelium degeneration. Exp Eye Res. 2006; 82:441–448. 5. Franco LM, Zulliger R, Wolf-Schnurrbusch UE, et al. Decreased visual function after patchy loss of retinal pigment epithelium induced by low-dose sodium iodate. Invest Ophthalmol Vis Sci. 2009;50:4004–4010. 6. Machalinska A, Lubinski W, Klos P, et al. Sodium iodate selectively injures the posterior pole of the retina in a dosedependent manner: morphological and electrophysiological study. Neurochem Res. 2010;35:1819–1827. 7. Enzmann V, Howard RM, Yamauchi Y, Whittemore SR, Kaplan HJ. Enhanced induction of RPE lineage markers in pluripotent neural stem cells engrafted into the adult rat subretinal space. Invest Ophthalmol Vis Sci. 2003;44:5417–5422. 8. Gong L, Wu Q, Song B, Lu B, Zhang Y. Differentiation of rat mesenchymal stem cells transplanted into the subretinal space of sodium iodate–injected rats. Clin Exp Ophthalmol. 2008; 36:666–671. 9. Ohtaka K, Machida S, Ohzeki T, et al. Protective effect of hepatocyte growth factor against degeneration of the retinal pigment epithelium and photoreceptor in sodium iodate– injected rats. Curr Eye Res. 2006;31:347–355. 10. Grignolo A, Orzalesi N, Calabria GA. Studies on the fine structure and the rhodopsin cycle of the rabbit retina in experimental degeneration induced by sodium iodate. Exp Eye Res. 1966;5:86–97. 11. Suyama T. Electron microscopic study on experimental retinal degeneration induced by sodium iodate injection. Yonago Acta Med. 1967;11:222–231. 12. Flage T. Changes in the juxtapapillary retinal pigment epithelium following intravenous injection of sodium iodate: a light and electron microscopic study using horseradish peroxidase as a tracer. Acta Ophthalmol. 1983;61:20–28. 13. Yoon YH, Marmor MF. Retinal pigment epithelium adhesion to Bruch’s membrane is weakened by hemicholinium-3 and sodium iodate. Ophthalmol Res. 1993;25:386–392. 14. Nilsson SE, Knave B, Persson HE. Changes in ultrastructure and function of the sheep pigment epithelium and retina induced by sodium iodate, I: the ultrastructure of the normal
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
pigment epithelium of the sheep. Acta Ophthalmol. 1977;55: 994–1006. Huang D, Swanson EA, Lin CP, et al. Optical coherence tomography. Science. 1991;254:1178–1181. Ruggeri M, Wehbe H, Jiao S, et al. In vivo three-dimensional high-resolution imaging of rodent retina with spectral-domain optical coherence tomography. Invest Ophthalmol Vis Sci. 2007;48:1808–1814. Fischer MD, Huber G, Beck SC, et al. Noninvasive, in vivo assessment of mouse retinal structure using optical coherence tomography. PloS One [serial online]. 2009;4:e7507. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759518. Accessed February 14, 2014. Nakano M, Otsuka F, Virmani R. Letter by Nakano et al regarding article, ‘‘Optical Coherence Tomographic Analysis of In-Stent Neoatherosclerosis After Drug-Eluting Stent Implantation.’’ Circulation [serial online]. 2011;124:e954. Available at: http://circ.ahajournals.org/content/124/25/e954.full. Accessed February 14, 2014. Muraoka Y, Ikeda HO, Nakano N, et al. Real-time imaging of rabbit retina with retinal degeneration by using spectral-domain optical coherence tomography. PloS One [serial online]. 2012;7: e36135. Available at: http://www.ncbi.nlm.nih.gov/pmc/ articles/PMC3338600. Accessed February 14, 2014. Hariri S, Moayed AA, Choh V, Bizheva K. In vivo assessment of thickness and reflectivity in a rat outer retinal degeneration model with ultrahigh resolution optical coherence tomography. Invest Ophthalmol Vis Sci. 2012;53:1982–1989. Liu S, Li ZW, Weinreb RN, et al. Tracking retinal microgliosis in models of retinal ganglion cell damage. Invest Ophthalmol Vis Sci. 2012;53:6254–6262. Li ZW, Liu S, Weinreb RN, et al. Tracking dendritic shrinkage of retinal ganglion cells after acute elevation of intraocular pressure. Invest Ophthalmol Vis Sci. 2011;52:7205–7212. Guo L, Normando EM, Nizari S, Lara D, Cordeiro MF. Tracking longitudinal retinal changes in experimental ocular hypertension using the cSLO and spectral domain–OCT. Invest Ophthalmol Vis Sci. 2010;51:6504–6513. Leung CK, Cheung CY, Lin D, Pang CP, Lam DS, Weinreb RN. Longitudinal variability of optic disc and retinal nerve fiber layer measurements. Invest Ophthalmol Vis Sci. 2008;49: 4886–4892. Leung CK, Cheung CY, Weinreb RN, et al. Comparison of macular thickness measurements between time domain and spectral domain optical coherence tomography. Invest Ophthalmol Vis Sci. 2008;49:4893–4897. Secondi R, Kong K, Blonska AM, Staurenghi G, Sparrow JR. Fundus autofluorescence findings in a mouse model of retinal detachment. Invest Ophthalmol Vis Sci. 2012;53:5190–5197. Hariri S, Tam MC, Lee D, Hileeto D, Moayed AA, Bizheva K. Noninvasive imaging of the early effect of sodium iodate toxicity in a rat model of outer retina degeneration with spectral domain optical coherence tomography. J Biomed Optics [serial online]. 2013;18:26017. Available at: http://biomedicaloptics. spiedigitallibrary.org/article.aspx?articleid¼1569950. Accessed February 14, 2014. Fleckenstein M, Charbel Issa P, Helb HM, et al. High-resolution spectral domain–OCT imaging in geographic atrophy associated with age-related macular degeneration. Invest Ophthalmol Vis Sci. 2008;49:4137–4144.
