April 2015
Volume 56, Issue 4
Free
Retina  |   April 2015
In Vivo Imaging of Subretinal Bleb-Induced Outer Retinal Degeneration in the Rabbit
Author Affiliations & Notes
  • Hammurabi Bartuma
    Department of Clinical Neuroscience, Section for Ophthalmology and Vision, St. Erik Eye Hospital, Karolinska Institutet, Stockholm, Sweden
  • Sandra Petrus-Reurer
    Department of Clinical Neuroscience, Section for Ophthalmology and Vision, St. Erik Eye Hospital, Karolinska Institutet, Stockholm, Sweden
  • Monica Aronsson
    Department of Clinical Neuroscience, Section for Ophthalmology and Vision, St. Erik Eye Hospital, Karolinska Institutet, Stockholm, Sweden
  • Sofie Westman
    Department of Clinical Neuroscience, Section for Ophthalmology and Vision, St. Erik Eye Hospital, Karolinska Institutet, Stockholm, Sweden
  • Helder André
    Department of Clinical Neuroscience, Section for Ophthalmology and Vision, St. Erik Eye Hospital, Karolinska Institutet, Stockholm, Sweden
  • Anders Kvanta
    Department of Clinical Neuroscience, Section for Ophthalmology and Vision, St. Erik Eye Hospital, Karolinska Institutet, Stockholm, Sweden
  • Correspondence: Anders Kvanta, St. Erik Eye Hospital and Karolinska Institutet, Polhemsgatan 50, SE11282 Stockholm, Sweden; anders.kvanta@ki.se
Investigative Ophthalmology & Visual Science April 2015, Vol.56, 2423-2430. doi:10.1167/iovs.14-16208
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      Hammurabi Bartuma, Sandra Petrus-Reurer, Monica Aronsson, Sofie Westman, Helder André, Anders Kvanta; In Vivo Imaging of Subretinal Bleb-Induced Outer Retinal Degeneration in the Rabbit. Invest. Ophthalmol. Vis. Sci. 2015;56(4):2423-2430. doi: 10.1167/iovs.14-16208.

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

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Abstract

Purpose.: To analyze the morphologic effects of subretinal blebs in rabbits using real-time imaging by spectral-domain optical coherence tomography (SD-OCT), infrared-confocal scanning laser ophthalmoscopy (IR-cSLO), and blue-light fundus autofluorescence (BAF).

Methods.: Subretinal blebs of PBS or balanced salt solution (BSS) were induced in albino or pigmented rabbits using a transvitreal pars plana technique. Spectral-domain optical coherence tomography, IR-cSLO, and BAF were done at multiple intervals for up to 12 weeks after subretinal bleb injection. The morphologic effects were compared with histologic analysis on hematoxylin-eosin–stained sections of the neurosensory retina and on flat-mounts of phalloidin-labeled RPE.

Results.: Scans of SD-OCT of the normal rabbit posterior segment revealed 11 bands including six layers of the photoreceptors. Subretinal blebs of PBS or BSS caused acute swelling of the neurosensory retina followed by gradual atrophy. Outer retinal thickness was significantly reduced with pronounced degeneration of all the photoreceptor OCT layers. En face IR-cSLO showed a hyperreflective area corresponding to the progressive photoreceptor degeneration, whereas BAF revealed both hyper- and hypofluorescent changes in the RPE layer. The in vivo results were confirmed by histology and on subretinal flatmounts demonstrating extensive photoreceptor loss and disruption of the RPE mosaic.

Conclusions.: Subretinal blebs induce pronounced photoreceptor degeneration and RPE changes in the rabbit as demonstrated by in vivo imaging using SD-OCT, IR-cSLO, and BAF.

Noninvasive techniques including spectral-domain optical coherence tomography (SD-OCT), infrared-confocal scanning laser ophthalmoscopy (IR-cSLO), and blue-light fundus autofluorescence (BAF) have revolutionized clinical imaging of the posterior pole of the eye. Spectral-domain optical coherence tomography produces a real-time cross-sectional image of the neurosensory retina, RPE, and choroid by using the interference between the reference optical path and the path reflected back from the eye.1 Infrared-confocal scanning laser ophthalmoscopy instead uses an 820-nm light source to create an en face fundus reflectance image of the outer retina and RPE, whereas BAF uses a 488-nm blue laser excitation to capture the in vivo autofluorescence (AF) emitted by fluorophores in the RPE.2,3 When combined with IR-cSLO or BAF, the cross-sectional SD-OCT scan can be precisely positioned further. Together, these multimodel methods thus provide detailed frontal and sagittal segmentation of the neurosensory retina and RPE.4 For example, in patients with geographic atrophy (GA), the end-stage form of dry AMD, these methods have come into clinical use as diagnostic and prognostic tools.5 Geographic atrophy is typically detected as an area of increased IR-cSLO reflectance (i.e., a bright signal) and decreased BAF (i.e., a dark signal), whereas the SD-OCT scans show loss of the outer neurosensory retina layers and RPE. 
Due to their potential to provide detailed morphologic information noninvasively, these methods have been used recently also in animal models. In rodents, OCT has been used to delineate the neurosensory retina using either specialized or modified clinical devices.6,7 However, information on individual layers, in particular, of the outer retina, has been limited. Using a modified method for averaging the OCT scans, Berger and coworkers colleagues8 were able to obtain excellent delineation of the outer retinal layers. The rabbit, with its near–human-sized eye, is well suited for SD-OCT imaging as demonstrated previously.912 Several studies have analyzed BAF in both rodents and lagomorph models of retinal degeneration.1315 In the mouse, an increased BAF signal has been attributed to both photoreceptor rosette formation and presence of RPE lipofuscin. Imaging of IR-cSLO has gained less attention in animal models. However, successful monitoring of sodium iodate–induced retinal degeneration by IR-cSLO was recently described in pigmented rats.7 
Artificial separation of the neurosensory retina from the RPE allows drug delivery to the subretinal space and has recently been used for gene and cell administration in patients.16,17 However, despite using isotonic solutions for creating the subretinal bleb, there is strong evidence of injection-induced damage to the outer retina.18,19 In the rabbit, it has been demonstrated that the force applied to release the neurosensory retinal adhesion is sufficient to create ultrastructural alterations to the photoreceptors and the RPE.20 To optimize treatment outcome of current and future clinical studies, it is therefore essential to minimize these injection-induced effects and to establish relevant in vivo models. 
In the present study, we analyze the effects of subretinal isotonic saline injections on the outer retina and RPE in rabbits using SD-OCT, IR-cSLO, and BAF and compare the in vivo results to histology. 
Materials and Methods
Animals
After approval by the Northern Stockholm Animal Experimental Ethics Committee, 14 New Zealand white albino rabbits and two pigmented Dutch belted rabbits (provided by Lidköpings Rabbit Farm, Lidköping, Sweden) aged 5 months, weighing 2.5 to 4.0 kg, were used in this study. All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Subretinal Injection
Animals were put under general anesthesia by intramuscular administration of 35 mg/kg ketamine (Ketaminol, 100 mg/mL; Intervet International BV, Boxmeer, The Netherlands) and 5 mg/kg xylazine (Rompun vet. 20 mg/mL; Bayer Animal Health, Leverkusen, Germany), and the pupils were dilated with a mix of 0.75% cyclopentolate/2.5% phenylephrine (APL, Stockholm, Sweden). Microsurgeries were performed on both eyes using a three-port, 25-G transvitreal pars plana technique (Alcon Accurus; Alcon Nordic, Copenhagen, Denmark). Phosphate-buffered saline (Life Technologies Europe, Stockholm, Sweden) or balanced salt solution (BSS; Alcon Nordic) was drawn into a 1-mL syringe connected to an extension tube and a 38-G cannula (PolyTip; MedOne Surgical, Inc., Sarasota, FL, USA). We inserted 25-G trocars 1 mm from the limbus, and an infusion cannula was connected to the lower temporal trocar. Without infusion or prior vitrectomy, the cannula was inserted through the upper temporal trocar, and the tip of the cannula allowed to slowly penetrate the retina just below the optic nerve head (Supplementary Fig. S1A). After proper tip positioning, ascertained by a focal whitening of the retina, 50 μL PBS (or BSS) was injected slowly subretinally, forming a uniform bleb that was clearly visible under the operating microscope. Care was taken to maintain the tip within the bleb during the injection to minimize reflux. After instrument removal, light pressure was applied to the self-sealing sutureless sclerotomies. No postsurgical topical steroids or antibiotics were given. On postsurgical follow-up, none of the eyes showed signs of extra- or intraocular infection or inflammation. 
In Vivo Retinal and Subretinal Imaging
Anesthetized rabbits were placed in an adjustable mount. A commercial SD-OCT device (Spectralis HRA+OCT; Heidelberg Engineering, Heidelberg, Germany) with the accompanying software (Heidelberg Eye Explorer version 1.9.10.0; Heidelberg Engineering) was used to obtain cross-sectional b-scans of treated animals. The device (Heidelberg Engineering) has a real-time motion tracking system that minimizes eye motion artifacts. At least three scans were obtained with simultaneous IR-cSLO reflectance reference images representing the upper, central, and lower portion of the injected area. The best overall image quality was obtained when the OCT setting was on high-speed acquisition with at least 50 averaged automatic real-time images. En face fundus images were obtained by cSLO blue, green, infrared, and multicolor (i.e., multiple simultaneous laser colors) laser reflectance, respectively. These modalities have a higher contrast level compared with conventional fundus camera photography. In addition, corresponding BAF images were captured using a blue-light laser (Heidelberg Engineering) with an excitation wavelength of 488 nM and a barrier filter of 500 nM. One human specimen was used for comparison with albino rabbit SD-OCT scans. This was an SD-OCT scan of a 51-year-old healthy male as part of an observational study approved by the Local Ethics Committee in Stockholm, Sweden. 
To measure retinal thickness (RT) and outer retinal thickness (ORT), SD-OCT images were imported into the ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). The thickness of untreated and treated retina was measured 500 μm from either side of the temporal transition zone of the bleb area. All measurements were made on a horizontal OCT scan through the temporal center of the bleb, approximately 6 mm below the margin of the optic nerve head (Supplementary Fig. S1A). Retinal thickness and ORT were defined as the distance from the inner retinal surface to the RPE or from the inner nuclear layer (INL) to the RPE, respectively. Distances were normalized to the 200-μm scale bar of the original image. Ten consecutive measurements on three independent OCT scans from nine PBS-injected eyes were acquired. The relative IR-cSLO reflectance of subretinal blebs was estimated by using the histogram tool of the ImageJ software. The average pixel brightness was obtained by inserting a standardized circle (1-mm diameter) in the lower-right quadrant of the image immediately outside or inside of the injection area, respectively. The brightness ratio was then used as relative measure of bleb reflectance. 
Histology
Immediately after euthanasia by intravenous injection of 100 mg/kg pentobarbital (Allfatal vet. 100 mg/mL; Omnidea, Stockholm, Sweden), the eyes were enucleated, and the bleb injection area marked with green tissue-marking dye (TMD; Histolab Products, Gothenburg, Sweden). An intravitreal injection of 100 μL fixing solution (FS) consisting of 4% buffered formaldehyde (Solvenco, Rosersberg, Sweden) was made before fixation in FS for 24 to 48 hours and embedding in paraffin. Serial sections of 4-μm made through the TMD-labeled area were stained with hematoxylin and eosin (HE). Images were captured with a camera phone (iPhone 4S; Apple, Inc., Cupertino, CA, USA) mounted to a bright-field microscope (Zeiss Axioskop 40; Carl Zeiss, Jena, Germany). 
Phalloidin Flatmounts
Immediately after euthanasia, the eyes were enucleated, and the injection area marked with TMD. After removal of the anterior segment and vitreous, the posterior pole including the marked area was excised with a scalpel. After careful dissection of the neurosensory retina with a blunt spatula, the RPE–choroid–scleral flat-mounts were fixed in 1.5 mL FS overnight at 4°C. Postfixing, the tissue was extensively washed with PBS and stained with Hoechst 33258 (5 g/L in PBS) and rhodamine-phalloidin (300 IU/mL in methanol; both reagents from Sigma-Aldrich Corp., St. Louis, MO, USA). Staining (both dyes at 1:1000) was performed in PBS containing 0.1% Triton X-100 (Sigma-Aldrich Corp.) for 1 hour at room temperature under slow agitation. Subsequently, samples were extensively washed with PBS and flat-mounted in between two microscope slides with fluorescent mounting medium (Dako, Carpinteria, CA, USA). Deconvoluted images of the RPE layer were obtained with a fluorescence microscope (Axioskop 2 plus using the AxioVision software, Carl Zeiss, Jena, Germany). 
Results
cSLO Reflectance and SD-OCT of the Rabbit Retina
We initially found that subretinal injections of PBS in albino rabbits caused a well-demarked hyperreflective circular area on cSLO that was detectable even after the bleb had resolved. This area was notably not visible by conventional fundus ophthalmoscopy. The bleb area appeared brightest by IR-cSLO reflectance compared with blue or green reflectance, suggesting structural changes in the outer retinal layers. We next tested if cross-sectional SD-OCT scans could provide further morphologic information. To address this, we first analyzed the posterior segment of normal albino and pigmented rabbits. Retinal scans comprised 11 hyper- or hyporeflective bands of varying width and intensity from the innermost ganglion cell layer (GCL) to the outermost RPE/Bruch's layer (Fig. 1A). The six layers of the photoreceptors were also delineated although sometimes the interdigitation zone (IZ) was difficult to separate from the RPE/Bruch's. Comparison of albino and pigmented rabbit SD-OCT scans did not reveal any apparent differences in layer reflection or thickness (Fig. 1B). 
Figure 1
 
Spectral-domain OCT of the normal albino, pigmented rabbit, and human retina. Cross-sectional horizontal SD-OCT b-scans of the normal albino rabbit (A1) and human (A2) retina demonstrate 11 distinct hyper- and hyporeflective layers. The left margin of the human image is 1 mm from the temporal side of the foveal center. Comparison between SD-OCT scans of pigmented (B1) and albino (B2) rabbits reveals no apparent difference in retinal delineation. The underlying choriocapillaris (*) is diffusely defined and often difficult to separate from the RPE/Bruch's layer (arrowhead). IPL, inner plexiform layer; MZ, myoid zone; EZ, ellipsoid zone; IZ, interdigitation zone; BM, Bruch's membrane. Scale bars: 100 μm.
Figure 1
 
Spectral-domain OCT of the normal albino, pigmented rabbit, and human retina. Cross-sectional horizontal SD-OCT b-scans of the normal albino rabbit (A1) and human (A2) retina demonstrate 11 distinct hyper- and hyporeflective layers. The left margin of the human image is 1 mm from the temporal side of the foveal center. Comparison between SD-OCT scans of pigmented (B1) and albino (B2) rabbits reveals no apparent difference in retinal delineation. The underlying choriocapillaris (*) is diffusely defined and often difficult to separate from the RPE/Bruch's layer (arrowhead). IPL, inner plexiform layer; MZ, myoid zone; EZ, ellipsoid zone; IZ, interdigitation zone; BM, Bruch's membrane. Scale bars: 100 μm.
Effect of Subretinal Injection on the Neurosensory Retina
To analyze the morphologic effects of subretinal injections in the rabbit retina, a series of imaging methodologies were acquired through time. Immediately after injection, the subretinal bleb was clearly detectable by en face multicolor or IR-cSLO fundus reflectance, as shown by pooling of subretinal fluid (Fig. 2A). Spectral-domain optical coherence tomography confirmed acute separation of the neurosensory retina at the RPE layer that resolved 1 day post injection (Fig. 2B). In the injection area, the outer retina was diffusely hyperreflective with lost layering extending from the outer plexiform layer (OPL) to the RPE between days 1 and 2 (Figs. 2B, 2C). On multicolor- and IR-cSLO, the injection bleb was detected as a sharply demarked circular area with a hyporeflective margin and a hyperreflective center. At day 5, outer retinal degeneration became evident with loss of the ellipsoid zone (EZ) and outer segment (OS) layers and an enhanced outer limiting membrane (OLM; Fig. 2D). By day 14, further degeneration was observable with loss of the OLM and most of the outer nuclear layer (ONL; Fig. 2E), an appearance that persisted to day 28 (Fig. 2F). From days 0 to 28, the gradual loss of the outer retinal layers was paralleled by increased IR-cSLO reflection (Fig. 3). Thickness comparisons between PBS- and nontreated areas were done 28 days after injection. Since eccentricity may have a significant effect on retinal thickness in the rabbit, we first analyzed the vertical and horizontal thickness in the bleb area.21,22 In accordance with previous studies, we could confirm that vertical thickness decreased toward the periphery, whereas horizontal thickness was constant (Supplementary Figs. S1B, S1C). The mean neurosensory retinal thickness in the studied area was 134 μm from the GCL to the RPE and 157 μm from the GCL to the choriocapillaris, respectively. The retinal thickness was reduced by 37% (P < 0.05), whereas the ORT was reduced by 68% (P < 0.05; Fig. 4). Similar results were obtained with subretinal injection of BSS, as well as in pigmented rabbits (data not shown). 
Figure 2
 
Subretinal injection–induced outer retinal degeneration. Multicolor- and IR-cSLO images demonstrate a progressive hyperreflective area (AF) corresponding to the subretinal bleb 1 hour to 28 days after injection. Simultaneous SD-OCT scans reveal rapid clearance of subretinal fluid with subsequent outer retinal swelling followed by gradual loss of the photoreceptor layers. Higher magnification SD-OCT images of the bleb transition zone are shown to the right. The scan plane is shown (green arrow), and the borders of the bleb are marked (arrowhead) on the cSLO and SD-OCT images, respectively. MC-cSLO, multicolor-confocal scanning laser ophthalmoscopy.
Figure 2
 
Subretinal injection–induced outer retinal degeneration. Multicolor- and IR-cSLO images demonstrate a progressive hyperreflective area (AF) corresponding to the subretinal bleb 1 hour to 28 days after injection. Simultaneous SD-OCT scans reveal rapid clearance of subretinal fluid with subsequent outer retinal swelling followed by gradual loss of the photoreceptor layers. Higher magnification SD-OCT images of the bleb transition zone are shown to the right. The scan plane is shown (green arrow), and the borders of the bleb are marked (arrowhead) on the cSLO and SD-OCT images, respectively. MC-cSLO, multicolor-confocal scanning laser ophthalmoscopy.
Figure 3
 
Time-course of subretinal injection–induced outer retinal changes. The time-dependent relative increase in IR-cSLO reflectance (black line) and relative decrease of ORT thickness (gray line), when compared to the untreated control retina as depicted in Figure 2.
Figure 3
 
Time-course of subretinal injection–induced outer retinal changes. The time-dependent relative increase in IR-cSLO reflectance (black line) and relative decrease of ORT thickness (gray line), when compared to the untreated control retina as depicted in Figure 2.
Figure 4
 
Subretinal injection decreases total retinal and outer retinal thickness. The total RT and ORT were measured on SD-OCT scans of albino rabbits at 28 days after injection of PBS. Measurements of RT or ORT were done from the inner retinal surface to the RPE/BM (black arrow) and from the INL to the RPE/BM (white arrow), respectively. Subretinal injection caused a significant decrease in RT and ORT (*P < 0.05, as determined by two-tailed Student's t-test).
Figure 4
 
Subretinal injection decreases total retinal and outer retinal thickness. The total RT and ORT were measured on SD-OCT scans of albino rabbits at 28 days after injection of PBS. Measurements of RT or ORT were done from the inner retinal surface to the RPE/BM (black arrow) and from the INL to the RPE/BM (white arrow), respectively. Subretinal injection caused a significant decrease in RT and ORT (*P < 0.05, as determined by two-tailed Student's t-test).
The morphologic effects captured by SD-OCT were next compared to histologic analysis in the same eyes. Scans of SD-OCT and HE staining 4 and 12 weeks post PBS injection showed the same overall degenerative changes to the outer retina, whereas the inner retina appeared normal (Fig. 5). On histology, a distinct degenerative transition zone was found at the border of the bleb. The normal ONL of the rabbit comprises approximately five nuclei layers that were gradually reduced to a single layer in the area of the subretinal bleb. The outer segments showed corresponding changes with gradual thinning in the transition zone to an almost complete loss inside the bleb. 
Figure 5
 
Subretinal injection causes loss of photoreceptor nuclei and outer segments. Cross-sectional IR-cSLO and SD-OCT images of albino retina 4 (A1, A2) and 12 weeks (B1, B2) after subretinal injection are shown. Corresponding HE-stained histologic sections demonstrate a transition zone at the border of the bleb where the normal ONL of the rabbit comprising approximately five layers is gradually reduced to a single layer in the area of the subretinal bleb. The OS becomes thinner in the transition zone and is almost completely lost inside the bleb area. The RPE, choroid, and sclera were lost during tissue processing and are not shown. ONL, outer nuclear layer; OS, outer segments. Scale bars: 100 μm.
Figure 5
 
Subretinal injection causes loss of photoreceptor nuclei and outer segments. Cross-sectional IR-cSLO and SD-OCT images of albino retina 4 (A1, A2) and 12 weeks (B1, B2) after subretinal injection are shown. Corresponding HE-stained histologic sections demonstrate a transition zone at the border of the bleb where the normal ONL of the rabbit comprising approximately five layers is gradually reduced to a single layer in the area of the subretinal bleb. The OS becomes thinner in the transition zone and is almost completely lost inside the bleb area. The RPE, choroid, and sclera were lost during tissue processing and are not shown. ONL, outer nuclear layer; OS, outer segments. Scale bars: 100 μm.
Effects of Subretinal Injection on Subretinal Layers
We subsequently investigated if subretinal injection caused morphologic effects also in the subretinal layers, in particular, the RPE. By SD-OCT, it was challenging to identify any clear changes in the RPE/Bruch's layer although some alternations in the degree of hyperreflection were sporadically observed. We therefore analyzed BAF, which mainly captures the RPE, in both pigmented and albino rabbits. In the normal rabbit retina, BAF was very low and frequently undetectable (Fig. 6A). However, 4 weeks after subretinal injection, the bleb area of albino rabbits was covered with hyper- and hypofluorescent salt- and pepper-like dots as shown in Figure 6B. In some eyes, larger hypofluorescent areas were also identified that had no corresponding appearance on IR-cSLO, suggesting an effect at the RPE level (Fig. 6C). On SD-OCT, the photoreceptor layer overlying these hypofluorescent areas showed the same degree of thinning as in areas with salt- and pepper-like changes (data not shown). Blue-light fundus autofluorescence on pigmented rabbits provided results similar to albino animals (data not shown). To further support the conclusion that the observed BAF changes were localized in the RPE, phalloidin-stained RPE flatmounts were analyzed. In PBS-injected areas, the normal hexagonal RPE morphology was disturbed with prominent variability in the size and shape of the RPE cells (Fig. 7). 
Figure 6
 
Subretinal injection–induced changes in the RPE. We used BAF to visualize the RPE of albino rabbits before (A1, A2) and 4 weeks after subretinal injection (B1, B2, C1, C2). Subretinal injection caused widespread changes in the RPE presented as hyper- and hypofluorescent dots (B1, B2) and occasionally as larger confluent hypofluorescent areas ([C1, C2] arrowheads). Infrared-confocal scanning laser ophthalmoscopy of the same areas did not show corresponding changes. Scale bar: 1 mm.
Figure 6
 
Subretinal injection–induced changes in the RPE. We used BAF to visualize the RPE of albino rabbits before (A1, A2) and 4 weeks after subretinal injection (B1, B2, C1, C2). Subretinal injection caused widespread changes in the RPE presented as hyper- and hypofluorescent dots (B1, B2) and occasionally as larger confluent hypofluorescent areas ([C1, C2] arrowheads). Infrared-confocal scanning laser ophthalmoscopy of the same areas did not show corresponding changes. Scale bar: 1 mm.
Figure 7
 
Subretinal injection disturbs the normal RPE morphology. Normal (A) and PBS-injected (B) flatmounts demonstrate marked alterations in the hexagonal RPE mosaic within the subretinal bleb 4 weeks after treatment as depicted by phalloidin staining. Scale bar: 50 μm.
Figure 7
 
Subretinal injection disturbs the normal RPE morphology. Normal (A) and PBS-injected (B) flatmounts demonstrate marked alterations in the hexagonal RPE mosaic within the subretinal bleb 4 weeks after treatment as depicted by phalloidin staining. Scale bar: 50 μm.
Discussion
In the present study, we show that subretinal injection of isotonic saline solutions causes significant and sustained outer retinal degeneration and morphologic RPE alterations in the rabbit as demonstrated by in vivo imaging and histology. 
Infrared-confocal scanning laser ophthalmoscopy, SD-OCT, and BAF were used for in vivo imaging of bleb-induced outer retinal damage. On SD-OCT, the progressive changes in the neurosensory retina were clearly observable. Clearance of the subretinal fluid was rapid and never persisted 1 day after injection. Outer retina swelling followed and was then replaced by apparent structural damage, first observed as a loss of the EZ by day 5. These observations are somewhat similar to those reported after subretinal BSS injection in cynomolgus macaque.19 In contrast, we found that the degeneration progressed further involving all six photoreceptor layers by day 14 with corresponding loss of ONL nuclei also seen in the histologic analysis. From this time point, the degeneration remained stable for up to 12 weeks. There was good overall agreement between the SD-OCT and IR-cSLO, both showing time-dependent thinning and hyperreflection. The increasing IR-cSLO hyperreflection most likely reflects the gradual loss of the photoreceptor layer as this method mainly captures changes in the outer retina and RPE.2,3 This is supported by a previous study on sodium iodate–induced outer retinal degeneration in albino rats where IR-cSLO showed hyporeflective dots, representing ONL folding and rosette formation, surrounded by hyperreflective areas corresponding to ONL thinning.7 
Since the albino RPE did not appear to reflect light, IR-cSLO was not useful for identifying changes at the RPE level. However, by BAF, a characteristic salt- and pepper-like pattern was observed in the bleb area that most likely originated in the RPE. The cause of this pattern is not clear, but it is well known that pathologic RPE undergoes different stages of increased BAF.23 We therefore propose that these injection-induced BAF changes represent damaged and metabolically altered RPE, a conclusion that was further supported by the results on phalloidin-labeled flat-mounts. In some cases, we found larger confluent areas of decreased BAF that were not detected by either IR-cSLO or SD-OCT. These areas of RPE loss were located in proximity to the injection site, thus probably being mechanically induced. An alternative explanation, albeit less likely, is that such areas represent end-stage RPE degeneration. 
The finding that subretinal bleb formation by isotonic saline solutions caused damage to the outer retinal layers and RPE is in overall accordance with previous in vivo studies. In a short-term ultrastructural study, subretinal injection of BSS in rabbits was shown to cause fragmentation of photoreceptor outer segments and disruption of the RPE within 3 minutes after injection.20 In cynomolgus macaque, a similar treatment led to milder ultrastructural changes in the photoreceptor/RPE interphase that persisted for up to 3 months.19 In a recent retrospective study in patients operated for retinal detachment, it was found that the outer retinal becomes significantly thinner after successful surgery and that the time until surgery is correlated with the extent of the degeneration.24 Compared to these results, the changes observed in our study were apparently much more pronounced and long-lasting and included significant loss of the photoreceptor layer. The force of the retinal–RPE adhesion has been well characterized and is known to be higher in primates than in rabbits.25 Indeed, the rabbit retina possesses several unique anatomic features not present in the humans or nonhuman primates.26 The retinal vessels are merangiotic, and the myelinated nerve fibers radiate horizontally from the optic nerve forming the medullary ray. In addition, the macula is replaced by a horizontal visual streak below the optic nerve head. These differences may potentially affect retinal adhesiveness in the rabbit. Furthermore, adhesiveness is influenced by metabolic factors, and pretreatment of the vitreous cavity with low Ca2+ and Mg2+ solutions reduces retinal adhesion.20,27 In the present study, we performed subretinal injections without prior vitrectomy or vitreous pretreatment and used standard PBS or BSS solutions. It is likely that together these factors contributed to maintain a strong retinal adhesion that in turn lead to the damage observed after bleb induction. At the molecular level, retinal adhesion is complex and not fully understood. It presumably involves apical surface receptors on the RPE and ligands in the interphotoreceptor matrix.28 Recently, αvβ5 integrin, exclusively located on the apical part of the RPE, was the first receptor to be implicated as demonstrated in mice lacking αvβ5 integrin showing reduced RPE–photoreceptor OS adherence.29 The pronounced loss of photoreceptors demonstrated by our data suggests that acute disruption of such receptor–ligand interactions may not be recovered, thus potentially leading to irreversible damage. 
Toxicity of PBS and BSS to the rabbit retina is one possible explanation of the observed outer retinal damage. Phosphate-buffered saline has the same osmolarity, and the ion concentration matches those of the human body and is extensively used as a nontoxic isotonic solution in cell culture studies. Balanced salt solution is a physiologic salt solution routinely used for irrigation during intraocular ocular surgery. Although not directly addressed in the present study, it is unlikely that these commonly used and generally nontoxic solutions should be toxic to the rabbit retina. The volume injected subretinally is another factor that may affect the extent of the damage. In the present study, we injected a volume of 50 μL of PBS or BSS, similar to the volume (100 μL) previously used in cynomolgus macaque,19 possibly resulting in less pronounced damage compared to larger volumes. In this respect, it is noteworthy that current gene and cell therapy trials use subretinal volumes of 100 to 150 μL.16,17 
As pointed out, a well-defined hyperreflective area by IR-cSLO with thinning or loss of the photoreceptor layer on SD-OCT and loss of RPE on BAF are all hallmarks of GA.4 Current animal models used to mimic GA fail to capture many of these alterations. For instance, the commonly used RCS rat has impaired RPE phagocytosis of OS leading to photoreceptor degeneration but with intact RPE.30 Another common model is induced RPE atrophy and outer retinal degeneration by systemic sodium iodate.31 However, the changes are panretinal and histologically distinctly different from those observed in GA.7 In the present study, we show that subretinal injection of PBS, or BSS, causes photoreceptor and RPE changes that in many ways are similar to clinical GA including a well-defined hyperreflective and sometimes hypofluorescent area with markedly thinned photoreceptor layers. The potential use of injection-induced retinal degeneration in the rabbit as an in vivo model of GA thus merits further study. Although a direct comparison with the clinical situation should be addressed with caution, our results nevertheless suggest that injection-associated retinal damage has to be accounted for when considering subretinal administration as a treatment route. This is particularly important since many of the conditions considered for subretinal treatment, including hereditary retinal dystrophies and GA, are associated with photoreceptor damage.16,17 
In summary, we show that subretinal blebs induce pronounced photoreceptor degeneration and RPE changes in the rabbit as demonstrated by in vivo imaging using SD-OCT, IR-cSLO, and BAF. The rabbit model in combination with these noninvasive real-time techniques could be ideally suited for studying models of retinal degeneration and for optimizing and monitoring new treatments targeting the subretinal space. 
Acknowledgments
Supported by grants from the Karolinska Institutet, Crown Princess Margareta's Foundation for the Visually Impaired, Edwin Jordan Foundation for Ophthalmological Research, the Swedish Eye Foundation, and the Cronqvist Foundation. 
Disclosure: H. Bartuma, None; S. Petrus-Reurer, None; M. Aronsson, None; S. Westman, None; H. André, None; A. Kvanta, None 
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Figure 1
 
Spectral-domain OCT of the normal albino, pigmented rabbit, and human retina. Cross-sectional horizontal SD-OCT b-scans of the normal albino rabbit (A1) and human (A2) retina demonstrate 11 distinct hyper- and hyporeflective layers. The left margin of the human image is 1 mm from the temporal side of the foveal center. Comparison between SD-OCT scans of pigmented (B1) and albino (B2) rabbits reveals no apparent difference in retinal delineation. The underlying choriocapillaris (*) is diffusely defined and often difficult to separate from the RPE/Bruch's layer (arrowhead). IPL, inner plexiform layer; MZ, myoid zone; EZ, ellipsoid zone; IZ, interdigitation zone; BM, Bruch's membrane. Scale bars: 100 μm.
Figure 1
 
Spectral-domain OCT of the normal albino, pigmented rabbit, and human retina. Cross-sectional horizontal SD-OCT b-scans of the normal albino rabbit (A1) and human (A2) retina demonstrate 11 distinct hyper- and hyporeflective layers. The left margin of the human image is 1 mm from the temporal side of the foveal center. Comparison between SD-OCT scans of pigmented (B1) and albino (B2) rabbits reveals no apparent difference in retinal delineation. The underlying choriocapillaris (*) is diffusely defined and often difficult to separate from the RPE/Bruch's layer (arrowhead). IPL, inner plexiform layer; MZ, myoid zone; EZ, ellipsoid zone; IZ, interdigitation zone; BM, Bruch's membrane. Scale bars: 100 μm.
Figure 2
 
Subretinal injection–induced outer retinal degeneration. Multicolor- and IR-cSLO images demonstrate a progressive hyperreflective area (AF) corresponding to the subretinal bleb 1 hour to 28 days after injection. Simultaneous SD-OCT scans reveal rapid clearance of subretinal fluid with subsequent outer retinal swelling followed by gradual loss of the photoreceptor layers. Higher magnification SD-OCT images of the bleb transition zone are shown to the right. The scan plane is shown (green arrow), and the borders of the bleb are marked (arrowhead) on the cSLO and SD-OCT images, respectively. MC-cSLO, multicolor-confocal scanning laser ophthalmoscopy.
Figure 2
 
Subretinal injection–induced outer retinal degeneration. Multicolor- and IR-cSLO images demonstrate a progressive hyperreflective area (AF) corresponding to the subretinal bleb 1 hour to 28 days after injection. Simultaneous SD-OCT scans reveal rapid clearance of subretinal fluid with subsequent outer retinal swelling followed by gradual loss of the photoreceptor layers. Higher magnification SD-OCT images of the bleb transition zone are shown to the right. The scan plane is shown (green arrow), and the borders of the bleb are marked (arrowhead) on the cSLO and SD-OCT images, respectively. MC-cSLO, multicolor-confocal scanning laser ophthalmoscopy.
Figure 3
 
Time-course of subretinal injection–induced outer retinal changes. The time-dependent relative increase in IR-cSLO reflectance (black line) and relative decrease of ORT thickness (gray line), when compared to the untreated control retina as depicted in Figure 2.
Figure 3
 
Time-course of subretinal injection–induced outer retinal changes. The time-dependent relative increase in IR-cSLO reflectance (black line) and relative decrease of ORT thickness (gray line), when compared to the untreated control retina as depicted in Figure 2.
Figure 4
 
Subretinal injection decreases total retinal and outer retinal thickness. The total RT and ORT were measured on SD-OCT scans of albino rabbits at 28 days after injection of PBS. Measurements of RT or ORT were done from the inner retinal surface to the RPE/BM (black arrow) and from the INL to the RPE/BM (white arrow), respectively. Subretinal injection caused a significant decrease in RT and ORT (*P < 0.05, as determined by two-tailed Student's t-test).
Figure 4
 
Subretinal injection decreases total retinal and outer retinal thickness. The total RT and ORT were measured on SD-OCT scans of albino rabbits at 28 days after injection of PBS. Measurements of RT or ORT were done from the inner retinal surface to the RPE/BM (black arrow) and from the INL to the RPE/BM (white arrow), respectively. Subretinal injection caused a significant decrease in RT and ORT (*P < 0.05, as determined by two-tailed Student's t-test).
Figure 5
 
Subretinal injection causes loss of photoreceptor nuclei and outer segments. Cross-sectional IR-cSLO and SD-OCT images of albino retina 4 (A1, A2) and 12 weeks (B1, B2) after subretinal injection are shown. Corresponding HE-stained histologic sections demonstrate a transition zone at the border of the bleb where the normal ONL of the rabbit comprising approximately five layers is gradually reduced to a single layer in the area of the subretinal bleb. The OS becomes thinner in the transition zone and is almost completely lost inside the bleb area. The RPE, choroid, and sclera were lost during tissue processing and are not shown. ONL, outer nuclear layer; OS, outer segments. Scale bars: 100 μm.
Figure 5
 
Subretinal injection causes loss of photoreceptor nuclei and outer segments. Cross-sectional IR-cSLO and SD-OCT images of albino retina 4 (A1, A2) and 12 weeks (B1, B2) after subretinal injection are shown. Corresponding HE-stained histologic sections demonstrate a transition zone at the border of the bleb where the normal ONL of the rabbit comprising approximately five layers is gradually reduced to a single layer in the area of the subretinal bleb. The OS becomes thinner in the transition zone and is almost completely lost inside the bleb area. The RPE, choroid, and sclera were lost during tissue processing and are not shown. ONL, outer nuclear layer; OS, outer segments. Scale bars: 100 μm.
Figure 6
 
Subretinal injection–induced changes in the RPE. We used BAF to visualize the RPE of albino rabbits before (A1, A2) and 4 weeks after subretinal injection (B1, B2, C1, C2). Subretinal injection caused widespread changes in the RPE presented as hyper- and hypofluorescent dots (B1, B2) and occasionally as larger confluent hypofluorescent areas ([C1, C2] arrowheads). Infrared-confocal scanning laser ophthalmoscopy of the same areas did not show corresponding changes. Scale bar: 1 mm.
Figure 6
 
Subretinal injection–induced changes in the RPE. We used BAF to visualize the RPE of albino rabbits before (A1, A2) and 4 weeks after subretinal injection (B1, B2, C1, C2). Subretinal injection caused widespread changes in the RPE presented as hyper- and hypofluorescent dots (B1, B2) and occasionally as larger confluent hypofluorescent areas ([C1, C2] arrowheads). Infrared-confocal scanning laser ophthalmoscopy of the same areas did not show corresponding changes. Scale bar: 1 mm.
Figure 7
 
Subretinal injection disturbs the normal RPE morphology. Normal (A) and PBS-injected (B) flatmounts demonstrate marked alterations in the hexagonal RPE mosaic within the subretinal bleb 4 weeks after treatment as depicted by phalloidin staining. Scale bar: 50 μm.
Figure 7
 
Subretinal injection disturbs the normal RPE morphology. Normal (A) and PBS-injected (B) flatmounts demonstrate marked alterations in the hexagonal RPE mosaic within the subretinal bleb 4 weeks after treatment as depicted by phalloidin staining. Scale bar: 50 μm.
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