March 2013
Volume 54, Issue 3
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Retina  |   March 2013
Xenopus laevis Tadpoles Can Regenerate Neural Retina Lost after Physical Excision but Cannot Regenerate Photoreceptors Lost through Targeted Ablation
Author Notes
  • From the Department of Ophthalmology and Visual Sciences and Centre for Macular Research, University of British Columbia, Vancouver, British Columbia, Canada. 
  • Current affiliation: *Department of Optometry and Vision Science, University of Auckland, Auckland, New Zealand. 
  • Corresponding author: Orson L. Moritz, Department of Ophthalmology and Visual Sciences and Centre for Macular Research, UBC/VGH Eye Care Centre, 2550 Willow Street, Vancouver, BC, Canada V5Z 3N9; [email protected]
Investigative Ophthalmology & Visual Science March 2013, Vol.54, 1859-1867. doi:https://doi.org/10.1167/iovs.12-10953
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      Damian C. Lee, Lisa M. Hamm, Orson L. Moritz; Xenopus laevis Tadpoles Can Regenerate Neural Retina Lost after Physical Excision but Cannot Regenerate Photoreceptors Lost through Targeted Ablation. Invest. Ophthalmol. Vis. Sci. 2013;54(3):1859-1867. https://doi.org/10.1167/iovs.12-10953.

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

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Abstract

Purpose.: To determine whether the Xenopus laevis retina is capable of regenerating photoreceptor cells lost through apoptotic cell death in an inducible transgenic X. laevis model of retinitis pigmentosa (RP).

Methods.: Acute rod photoreceptor apoptosis was induced in transgenic X. laevis expressing drug-inducible caspase 9. We subsequently monitored the ability of the retina to regenerate lost photoreceptors in the absence of drug, and in combination with physical injury or ectopic supplementation of basic fibroblast growth factor (FGF2).

Results.: Direct activation of caspase 9 in rod photoreceptors resulted in the initiation of apoptosis and complete removal of rod photoreceptors within 4 days. Photoreceptors lost by apoptosis were not replaced over a 4-week recovery time frame. In contrast, physical disruption of rod-ablated retina was repaired by the end of a 3-week time frame, but did not result in rod photoreceptor regeneration other than at the site of injury. Furthermore, ectopic supplementation of FGF2 did not stimulate regeneration of photoreceptors lost by apoptosis. However, FGF2 supplementation increased the rate of regeneration of retina (including rod photoreceptors) in eyes from which retinal tissue was surgically removed.

Conclusions.: In the X. laevis retina, rod photoreceptors that undergo drug-induced caspase-9–mediated apoptosis are permanently lost and do not regenerate. In contrast, the neural retina (including rod photoreceptors) can regenerate in injured or retinectomized eyes, and this regeneration is promoted by supplementation with FGF2. However, FGF2 does not promote regeneration of rod photoreceptors that are selectively lost by apoptosis.

Introduction
Retinal degenerative diseases are a major cause of blindness in industrialized/developed countries. 1 One form of inherited retinal degeneration, retinitis pigmentosa (RP), is caused by the progressive degeneration of rod photoreceptors in the outer retina. RP patients experience tunnel vision and night blindness as their rods degenerate and die. Eventually, their cone photoreceptors die as well, resulting in loss of central vision and ultimately, irreversible blindness. 2  
There is currently no cure for RP. In humans and most mammalian vertebrates, the loss of neurons, including retinal neurons, is permanent. However, some nonmammalian vertebrates are capable of repairing and restoring retinal neurons lost through injury. The retina of the African clawed frog, Xenopus laevis , has the capacity to regenerate after traumatic physical injury. Previous studies demonstrated regeneration of the eye and neural retina after physical removal of sections of the orbit and/or retinectomy. 38 This ability to regenerate retinal tissue makes X. laevis an interesting model for the process and mechanisms of neural retina repair and regeneration. 9  
A transgenic X. laevis model of retinal degeneration in which rod photoreceptor apoptosis is directly drug inducible was previously developed and characterized. 10 These iCaspase 9 (iCasp9) transgenic animals express a drug-activatable form of caspase 9 in the rod photoreceptors in which procaspase 9 is fused to FK506-binding domains. Caspase 9 is an initiator caspase that, once activated, directly activates the caspase cascade, resulting in apoptosis. Dimerization and subsequent activation of procaspase-9 chimeric protein is driven by the binding of AP20187 (a small molecule based on a dimer of the drug FK506) to the FK506-binding domains. Hamm et al. 10 previously showed that chronic AP20187 administration selectively killed rods in the retinas of these animals. 
Previous findings from animal models of RP indicate that, despite heterogeneity of underlying causes, apoptosis of the rod photoreceptor is the final common end point. 1114 The completely inducible nature of retinal degeneration in the iCasp9 model presented us with a unique opportunity to examine the potential for regeneration of rod photoreceptors. This question is of interest for two reasons: (1) we have developed a number of transgenic X. laevis models of RP, and it is important to know whether X. laevis photoreceptors may regenerate in these animals in long-term studies, and (2) understanding the regenerative responses of the X. laevis retina may allow us to propose methods for initiating regeneration in human retina from either endogenous retinal cells or stem cell transplants. 
Methods
Transgenic X. laevis Generation and Rearing
Transgenic X. laevis tadpoles expressing iCasp9 in rod photoreceptors were generated from founder transgenic frogs originally described by Hamm et al. 10 Breeding was induced by injection of 700 units of human chorionic gonadotropin into the dorsal lymph sack of the female frog. Tadpoles were reared in an 18°C incubator on a 12-hour dark and 12-hour light (1700 lux) cycle as previously described. 10 Rod photoreceptor death was induced in the iCasp9 tadpoles by the addition of 10 μM AP20187 (Clontech, Mountain View, CA) to the tadpole rearing medium as previously described. 10 Green fluorescent protein (GFP)-labeled rod photoreceptors were generated by mating with transgenic frogs expressing enhanced green fluorescent protein (eGFP) under control of the X. laevis opsin promoter.15–17  
Tadpole Microsurgery
Tadpoles were anesthetized by immersion in tadpole-rearing medium containing 0.01% tricaine methanesulfonate (Sigma-Aldrich, St. Louis, MO) for 1 to 2 minutes. The anesthetized tadpoles were transferred to a well that was fashioned from modeling clay and covered with tissue paper soaked in Ringer's solution to prevent desiccation during the course of microsurgery. To create retinal injury, an incision was made on the dorsal part of a tadpole between the eyes to allow a glass-pulled pipette access to the posterior of the eye. Using the glass-pulled pipette, a hole was punched through the posterior of the eye into the retina and the circular plug of choroid, retinal pigment epithelium, and neural retina were removed. To retinectomize tadpole eyes, an incision was first made to the cornea using a 30-gauge hypodermic needle. A Dumont #5 fine forceps was used to remove the lens through the pupil through the incision. Buffer (0.1x Marc's Modified Ringer's [MMR]) was gently blown into the eye through a glass-pulled pipette to detach and flush out the neural retina. To perform a lentectomy and subsequent bead implantation, an incision was made in the cornea and the lens was pulled out through the pupil with Dumont #5 fine forceps. A heparin-coated acrylic bead (Sigma-Aldrich) presoaked in either MMR buffer or basic fibroblast growth factor (FGF2) was inserted through the same opening and placed into the space left by removal of the lens. All microsurgeries were performed when tadpoles were at least Nieuwkoop and Faber (NF) stage 47/48 for optimal recovery and survival rate. 
Histology and Immunohistochemistry
Anesthetized tadpoles were euthanized and fixed overnight with 4% paraformaldehyde in 0.1M sodium phosphate buffer (pH 7.4). The fixed eyes were dissected and infiltrated with 20% sucrose/0.1M sodium phosphate buffer (pH 7.4). The cryoprotected eyes were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA) and frozen before cyrosectioning into 12-μm serial sections. For histological processing, the sections were washed in deionized water to remove the O.C.T compound before staining with PROTOCOL Hema 3 manual stain kit (Fisher Scientific, Hampton, NH). For immunohistochemistry processing, sections were washed in PBS to remove the O.C.T compound before blocking with 1% goat serum in PBS containing 0.1% Triton X-100 for 1 hour at room temperature. The blocked sections were incubated overnight with the primary antibody diluted in dilution buffer (0.1% Triton X-100, 0.1% goat serum in PBS). The sections were then washed three times for 10 minutes each in PBS before incubating in secondary antibody diluted in dilution buffer for 4 hours at room temperature. Retinal sections were counterstained with wheat germ agglutinin (WGA) conjugated to Alexa Fluor 488 or 555, diluted 1:200 (Invitrogen, Carlsbad, CA) and Hoescht 33342, diluted 1:1000 (Sigma-Aldrich) to label rod outer segments and nuclei, respectively. The stained sections were washed three times for 10 minutes each in PBS before mounting with Mowiol 4-88 (Calbiochem-EMD Millipore, Billerica, MA). Confocal images were acquired using Zeiss 510 Meta laser scanning confocal microscope (Carl Zeiss, Thornwood, NY). Representative images are shown in the figures. 
EdU Proliferation Assay
Proliferating cells were labeled by the detection of EdU (5-ethynyl-2′-deoxyuridine) incorporation; 4 μL of 2.5 mM EdU was injected intra-abdominally into anesthetized tadpoles. At the indicated time points, tadpoles were euthanized and prepared for cryosectioning as described above. EdU incorporation was detected according to the Click-iT EdU Alexa Fluor 555 Imaging Kit recommendations (Invitrogen). 
Primary Antibodies
Primary antibodies were B630N antirod opsin diluted 1:20 (courtesy of W. Clay Smith); PC-10 anti-PCNA diluted 1:500 (Sigma-Aldrich); anticalbindin diluted 1:250 (Calbiochem); Cy3- or Cy5-conjugated antimouse and antirabbit secondary antibodies diluted 1:1000 (Jackson ImmunoResearch, West Grove, PA). 
All procedures were performed in accordance to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Results
The X. laevis Retina Does Not Regenerate Rod Photoreceptors Ablated by Targeted Apoptosis
The iCasp9 transgenic X. laevis expresses a drug-activatable form of caspase 9 in rod photoreceptors. Administration of the otherwise innocuous drug AP20187 to iCasp9 transgenic tadpoles induces rapid death of the rod photoreceptors. 10 To label rod photoreceptors in vivo, green fluorescent protein (GFP) was coexpressed in the rod photoreceptors under the control of the X. laevis rod opsin promoter. 15  
To induce rod photoreceptor apoptosis, we treated NF stage 47/48 (equivalent to 14 days postfertilization) iCasp9/GFP double transgenic tadpoles with AP20187 for 4 days. To test the inherent regenerative capacity of X. laevis retina, drug treatment was withdrawn and tadpoles were allowed to recover for 4 weeks in normal rearing conditions. Retinal cryosections were prepared, immunolabeled, and examined by confocal microscopy. We confirmed that administration of AP20187 induced rapid rod cell death, with ablation of a majority of rod photoreceptors after the 4 days of drug treatment (Fig. 1A). After the 4-week recovery period, the rod outer segment (ROS) layer remained absent in the central retina of the drug treated iCasp9 tadpoles (Fig. 1C). There was no detectable GFP signal in the photoreceptor layer, namely, the outer nuclear layer (ONL), inner segment (IS), and outer segment (OS), indicating the absence of rod photoreceptors in the central retina. Only cone photoreceptors remained in the ONL of the central retina, as evidenced by immunolabeling with cone-specific calbindin antibodies (Fig. 1C'). However, rod photoreceptors were observed adjacent to the ciliary marginal zone (CMZ) at the periphery of the growing tadpole retina, indicating that acute drug treatment did not prevent the generation of new rod photoreceptors. The CMZ is a zone of retinal stem cells at the anterior margin of the X. laevis retina that facilitates the addition of new retina as the eye grows. 18 In contrast, there was no evidence of new rod cells repopulating sections of the retina where rods were lost due to drug-induced apoptosis. In the untreated retinas, rod photoreceptors could be detected by GFP in the photoreceptor layer (Figs. 1B, 1D). This methodology is capable of detecting rod photoreceptors even if their morphology is significantly altered. 16 These observations indicate that rods lost by caspase 9–mediated apoptosis do not regenerate after the death-inducing signal is removed. Furthermore, new rods from the expanding CMZ do not migrate along the retina to repopulate the ablated region. Interestingly, we found that the morphology of the cone photoreceptors differed between rod-ablated retinas and untreated retinas (Figs. 1C', 1D'), with the overall length of the cones in the rod-ablated retinas being shorter in length. We previously reported a similar result in drug-treated adult frogs. 10  
Figure 1
 
Tadpoles do not regenerate rod photoreceptors ablated by targeted apoptosis. (A) Administration of the drug AP20187 rapidly induces apoptosis in iCasp9-expressing rod photoreceptors of 2-week old tadpoles. Four days after drug administration, rods are completely ablated from the retina. Note the absence of GFP signal (green) in the central retina. Inset shows higher magnification of the boxed region in the central retina. The retinal cell layers labeled are ganglion cell layer (GCL), inner nuclear layer (INL), ONL, and OS. (B) Representative image of age-matched untreated iCasp9 retinas. (C) Drug-treated iCasp9 tadpole retina after 4-week recovery period. Inset shows higher magnification of the rodless central retina. (C') Anticalbindin immunolabeling showing that only cone photoreceptors remain in ONL of central retina. (D, D') Untreated iCasp9 tadpole retina at 6 weeks after fertilization. Note the ONL is populated with both GFP-positive rods ([D] inset, green) and cones ([D'], red). All images shown are representative confocal micrographs of retinal cryosections. (AD) Sections were stained with wheat germ agglutinin (red) to label membranes and Hoechst 33342 (blue) to label nuclei. (C', D') Anticalbindin colabeling to visualize cone photoreceptors. Scale bars: 100 μm (A, C); 10 μm (inset panels, [C']).
Figure 1
 
Tadpoles do not regenerate rod photoreceptors ablated by targeted apoptosis. (A) Administration of the drug AP20187 rapidly induces apoptosis in iCasp9-expressing rod photoreceptors of 2-week old tadpoles. Four days after drug administration, rods are completely ablated from the retina. Note the absence of GFP signal (green) in the central retina. Inset shows higher magnification of the boxed region in the central retina. The retinal cell layers labeled are ganglion cell layer (GCL), inner nuclear layer (INL), ONL, and OS. (B) Representative image of age-matched untreated iCasp9 retinas. (C) Drug-treated iCasp9 tadpole retina after 4-week recovery period. Inset shows higher magnification of the rodless central retina. (C') Anticalbindin immunolabeling showing that only cone photoreceptors remain in ONL of central retina. (D, D') Untreated iCasp9 tadpole retina at 6 weeks after fertilization. Note the ONL is populated with both GFP-positive rods ([D] inset, green) and cones ([D'], red). All images shown are representative confocal micrographs of retinal cryosections. (AD) Sections were stained with wheat germ agglutinin (red) to label membranes and Hoechst 33342 (blue) to label nuclei. (C', D') Anticalbindin colabeling to visualize cone photoreceptors. Scale bars: 100 μm (A, C); 10 μm (inset panels, [C']).
FGF2 Does Not Stimulate Regeneration of Rods Lost by Targeted Cell Death
Although our experiments involving acute induction of apoptosis showed that X. laevis tadpoles do not spontaneously regenerate rod photoreceptors ablated by targeted cell death, it is well known that X. laevis can regenerate retina after physical trauma, resections, and retinectomy. 35,7,19 When we performed rectinectomies on NF stage 47/48 wild-type tadpoles, removing the neural retina and lens but leaving intact the RPE and choroid, we confirmed that X. laevis tadpoles are capable of regenerating neural retina over a 3-week recovery period (Fig. 2A). However, the extent of regeneration was variable. We categorized the degrees of regeneration as “extensive” when the amount of retina present was at least 50% as much as the control eye; “intermediate” when regeneration had clearly occurred but was incomplete, and “none” when no retinal cells were observed. Of the 16 animals tested and examined, 44% of the retinectomized eyes showed extensive regeneration of neural retina, while 19% of the eyes had no significant retina, with the remaining 37% of the eyes showing an intermediate level of regeneration (Fig. 2B, left chart). Interestingly, this regenerative capacity was negatively modulated by the implantation of a heparin-coated acrylic bead into the retinectomized space (Fig. 2B, right chart). We found that inhibition by implantation was counteracted by presoaking the bead in FGF2 (Fig. 2B, right chart). 
Figure 2
 
Retina can regenerate after retinectomy. (A) Histological sections of X. laevis tadpole eyes 1 day after (Day+1) and 21 days after (Day+21) retinectomy. Retinectomies were performed on NF stage 47/48 tadpoles (equivalent to 14 days postfertilization [dpf]); 21 days after retinectomy, most eyes spontaneously regenerated retina. However, the extent of regeneration varied. Representative images shown. Scale bar: 100 μm. (B) Percentage of retinectomized eyes that regenerate retina after 21 days without intervention (left) or with heparin-coated bead implants (right). Heparin-coated beads were implanted in the eye immediately after retinectomy and allowed to recover for 21 days. Implantation of a bead negatively affected the regenerative capacity after retinectomy (+blank bead). However, presoaking the beads in FGF2 (+FGF2 bead) significantly reversed the inhibition (*P = 0.019, chi-squared test). Chart legend shows representative images of the varying extents of regeneration.
Figure 2
 
Retina can regenerate after retinectomy. (A) Histological sections of X. laevis tadpole eyes 1 day after (Day+1) and 21 days after (Day+21) retinectomy. Retinectomies were performed on NF stage 47/48 tadpoles (equivalent to 14 days postfertilization [dpf]); 21 days after retinectomy, most eyes spontaneously regenerated retina. However, the extent of regeneration varied. Representative images shown. Scale bar: 100 μm. (B) Percentage of retinectomized eyes that regenerate retina after 21 days without intervention (left) or with heparin-coated bead implants (right). Heparin-coated beads were implanted in the eye immediately after retinectomy and allowed to recover for 21 days. Implantation of a bead negatively affected the regenerative capacity after retinectomy (+blank bead). However, presoaking the beads in FGF2 (+FGF2 bead) significantly reversed the inhibition (*P = 0.019, chi-squared test). Chart legend shows representative images of the varying extents of regeneration.
It has been previously reported that FGF2 can promote the transdifferentiation of RPE cells into different neural retinal cells types. 2022 We wanted to determine whether supplementation with FGF2 could stimulate regeneration of rods lost by induced apoptosis. Using the rod-ablated tadpole system, a lentectomy was performed and an FGF2-soaked bead was placed into the space left by removal of the lens. A blank bead (soaked in 0.1X MMR buffer) was implanted in the control animals. The operated tadpoles were allowed to recover for 3 weeks under normal rearing conditions. After the recovery period, the animals were prepared for histological analysis of the eyes. Although we observed spontaneous regeneration of lens, no regeneration of ablated rods was observed in FGF2-treated or control eyes (Fig. 3). Our data indicate that while ectopic supplementation of FGF2 can influence regeneration of retina lost due to excision, it has no influence on the regeneration of rod photoreceptors ablated by apoptosis. 
Figure 3
 
FGF2 does not stimulate retina to replace rods lost by induced apoptosis. Rod ablated iCasp9 eyes 21 days after bead implantation. iCasp9 tadpoles (14 dpf, NF stage 47/48) were treated with AP20187 to induce rod apoptosis. After a 4-day incubation period to allow for complete rod ablation, the lens was replaced with a buffer-soaked bead (+blank bead) or an FGF2-soaked bead (+FGF2-soaked bead) and subsequently allowed to recover for 21 days. Retinal sections were stained with wheat germ agglutinin (green) to label membranes and Hoechst 33342 (blue) to label nuclei. Scale bar: 100 μm.
Figure 3
 
FGF2 does not stimulate retina to replace rods lost by induced apoptosis. Rod ablated iCasp9 eyes 21 days after bead implantation. iCasp9 tadpoles (14 dpf, NF stage 47/48) were treated with AP20187 to induce rod apoptosis. After a 4-day incubation period to allow for complete rod ablation, the lens was replaced with a buffer-soaked bead (+blank bead) or an FGF2-soaked bead (+FGF2-soaked bead) and subsequently allowed to recover for 21 days. Retinal sections were stained with wheat germ agglutinin (green) to label membranes and Hoechst 33342 (blue) to label nuclei. Scale bar: 100 μm.
De Novo Regeneration of Neural Retina
Previous studies have shown that the X. laevis tadpole can repair and regenerate resections of up to two-thirds of the eye. 3,19 We confirmed this previously observed ability to repair physical trauma by performing a punch biopsy to the eye of NF stage 47/48 tadpoles and allowing the tadpoles to recover from the injury. Our punch biopsy removed a transverse section of the eye, including the choroid, RPE, and retina (Figs. 4A, 4A'). After allowing the tadpole to recover for 3 weeks, we found that the lesion was repaired to an extent that it was difficult to distinguish the site of injury from the unaffected rest of the retina (Figs. 4B, 4B'). The RPE of the injured eye was intact with good lamination of the neural retinal cell layers. 
Figure 4
 
Physical injuries to the eye and retina are spontaneously repaired. Retinal injuries were created in NF stage 47/48 (14 dpf) tadpoles by punching a hole through the posterior of the eye into the retina and removing the circular plug of choroid, RPE, and neural retina. (A, A') Representative image of tadpole eyes 1 day after punch injury. (B, B') Section of eye from tadpole 21 days after injury. Arrowheads indicate site of injury. (A, B) Light micrographs of retinal cryosections stained with PROTOCOL Hema 3 stain. (A', B') Confocal micrographs of retinal cryosections labeled with wheat germ agglutinin (green) and Hoechst 33342 (blue). Scale bar: 100 μm.
Figure 4
 
Physical injuries to the eye and retina are spontaneously repaired. Retinal injuries were created in NF stage 47/48 (14 dpf) tadpoles by punching a hole through the posterior of the eye into the retina and removing the circular plug of choroid, RPE, and neural retina. (A, A') Representative image of tadpole eyes 1 day after punch injury. (B, B') Section of eye from tadpole 21 days after injury. Arrowheads indicate site of injury. (A, B) Light micrographs of retinal cryosections stained with PROTOCOL Hema 3 stain. (A', B') Confocal micrographs of retinal cryosections labeled with wheat germ agglutinin (green) and Hoechst 33342 (blue). Scale bar: 100 μm.
From our observations that punch biopsies to the tadpole eye are robustly repaired, we wanted to determine if injury and subsequent repair can stimulate regeneration of rod photoreceptors ablated by apoptosis. To test this hypothesis, we performed the punch biopsy on rod-ablated tadpole retinas. The contralateral eye was left intact as a control. After a 3-week recovery period, the animals were euthanized, fixed whole, and cyrosections were prepared for histological analysis. As expected, we found that the injury was repaired after the 3-week period (Fig. 5A). Although we did not find robust regeneration of rods previously ablated by apoptosis, we did observe rod photoreceptors present at the site of injury and repair, as evidenced by the presence of rod outer segments (Fig. 5A, red bracket). These cells were confirmed to be rhodopsin-positive rod photoreceptors by antibody staining (Figs. 5B, 5B', upper panels). The site of repair was also found to be immunoreactive to proliferating cell nuclear antigen (PCNA), a nuclear protein expressed in S-phase mitotic cells, indicative that proliferation was occurring (Figs. 5B, 5B', lower panels). In addition, these results indicate that the drug AP20187, in and of itself, does not prevent regeneration. 
Figure 5
 
De novo rod photoreceptors are present at site of injury repair. iCasp9 tadpoles (14 dpf, NF stage 47/48) were treated with AP20187 for 4 days to induce rod apoptosis. Subsequently one eye was subjected to punch injury while the contralateral eye was left intact. (A) Rod-ablated iCasp9 eyes 21 days after punch injury. Upper panels: injured eye; lower panels: intact eye. Arrows indicate site of injury. Red brackets highlight regenerated rod outer segments that coincide with site of injury. (B) Immunolabeling with rhodopsin antibodies confirmed that rod outer segments were at site of injury repair (upper panels). Site of repair was also PCNA immunoreactive (lower panels). (B') Higher magnification of respective boxed regions. Asterisks indicate a nonspecific crossreaction of anti-PCNA label in rod inner segments. All sections were colabeled with wheat germ agglutinin (green) and Hoechst 33342 (blue). Scale bars: 100 μm.
Figure 5
 
De novo rod photoreceptors are present at site of injury repair. iCasp9 tadpoles (14 dpf, NF stage 47/48) were treated with AP20187 for 4 days to induce rod apoptosis. Subsequently one eye was subjected to punch injury while the contralateral eye was left intact. (A) Rod-ablated iCasp9 eyes 21 days after punch injury. Upper panels: injured eye; lower panels: intact eye. Arrows indicate site of injury. Red brackets highlight regenerated rod outer segments that coincide with site of injury. (B) Immunolabeling with rhodopsin antibodies confirmed that rod outer segments were at site of injury repair (upper panels). Site of repair was also PCNA immunoreactive (lower panels). (B') Higher magnification of respective boxed regions. Asterisks indicate a nonspecific crossreaction of anti-PCNA label in rod inner segments. All sections were colabeled with wheat germ agglutinin (green) and Hoechst 33342 (blue). Scale bars: 100 μm.
To confirm that the repair at the site of injury is facilitated by the proliferation of new cells, we used EdU to label mitotic cells. EdU is a thymidine analogue that is incorporated into the DNA of replicating cells during S-phase. We performed the punch injury on rod-ablated tadpoles and allowed the animals to recover over 3 weeks as above. Over the course of recovery, we injected the animals once with EdU at 3, 6, or 15 days postinjury, followed by a short incubation period (8 hours) or for an extended incubation period (3–9 days). After the incubation period, the tadpoles were euthanized and fixed whole. Cryosections were prepared for EdU detection and histochemical analysis (Fig. 6). At 3 days postinjury, after an 8-hour incubation, EdU-positive cells were detected at the periphery of the retina corresponding to the CMZ, in both the injured and intact eyes (Fig. 6A, upper panels). After a 3-day incubation period, we observed that EdU-positive cells at the periphery of the retina had migrated away from the CMZ, while some EdU positive cells were detected at the site of injury in cell layers corresponding to the RPE and choroid (Fig. 6A, lower panels). At 6 days postinjury, more EdU-positive cells were observed at the site of injury near the choroid; this was more obvious after a longer incubation (Fig. 6B). At 15 days postinjury, we observed a similar EdU pattern to day 3–labeled retinas (Fig. 6C). In all of the contralateral uninjured eyes, no EdU-positive cells were detected in the central retina (Figs. 6A', 6B', 6C'). These single-pulse EdU incorporation data indicate that injury repair elicits proliferation in the proximity of the site of injury. 
Figure 6
 
Actively proliferating cells coincide with location of retinal injury and repair. iCasp9 tadpoles (14 dpf, NF stage 47/48) were treated with AP20187 for 4 days to induce rod apoptosis. Rod-ablated iCasp9 tadpoles were injected with EdU on day 3 (A, A'), day 6 (B, B'), or day 15 (C, C') after retinal injury. EdU incorporation was followed for a short period (8 hours) or for an extended period (3–9 days). At the end of the incubation period, tadpoles were killed and retinas stained for EdU detection (red) and co-stained with WGA (green). In some instances, the lens was lost during sectioning and histological processing. Double daggers indicate nonspecific labeling of lens. Arrowheads indicate region corresponding to CMZ of retina.
Figure 6
 
Actively proliferating cells coincide with location of retinal injury and repair. iCasp9 tadpoles (14 dpf, NF stage 47/48) were treated with AP20187 for 4 days to induce rod apoptosis. Rod-ablated iCasp9 tadpoles were injected with EdU on day 3 (A, A'), day 6 (B, B'), or day 15 (C, C') after retinal injury. EdU incorporation was followed for a short period (8 hours) or for an extended period (3–9 days). At the end of the incubation period, tadpoles were killed and retinas stained for EdU detection (red) and co-stained with WGA (green). In some instances, the lens was lost during sectioning and histological processing. Double daggers indicate nonspecific labeling of lens. Arrowheads indicate region corresponding to CMZ of retina.
The single-pulse EdU experiment provided a temporal profile of the proliferating cells over the 3-week recovery period. To provide a spatial profile of proliferating cells, we used multiple pulses of EdU labeling. Tadpoles were treated as above in the single-pulse experiment, but injected with EdU consecutively on days 3, 6, and 15 postinjury. Animals were killed and fixed on day 22. Cryosections were prepared for EdU detection and histochemical analysis (Fig. 7). We found four distinct clusters of EdU-positive cells in the uninjured eyes (Fig. 7A). The bands of EdU-labeled cells were consistent with two pulses of EdU incorporation in the CMZ at the periphery of the retina. It is likely that the pulses on days 3 and 6 were too close to be separated, resulting in a single cluster of label (Fig. 7A, brackets). In contrast, there were five distinct bands or clusters of EdU-positive cells in the injured contralateral eyes (Fig. 7B). In addition to the four bands at the periphery of the retina, there was a fifth band in the central retina, corresponding to site of injury/repair (Figs. 7B, 7B', asterisk). Also contrasting from the uninjured eyes, the band of EdU cells farthest away from periphery was broader and more dispersed in the injured eyes (Fig. 7B, brackets). The data from the single-pulse EdU and multiple-pulse EdU labeling taken together indicate that there is active cellular proliferation at the site of injury repair. Additionally, there is greater proliferation at the CMZ in the injured eye. 
Figure 7
 
Retinal injury and repair induces proliferation of retinal cells. Rod-ablated iCasp9 tadpoles were injected consecutively with EdU on days 3, 6, and 15 postinjury. Tadpoles were killed on day 22 after injury and retinas stained for EdU detection (red) and counterstained with WGA (green). (A) Stained section of uninjured eye. Brackets indicate clusters of EdU-positive cells corresponding to injections from days 3 and 6. (B) Stained section of contralateral injured eye. Brackets indicate clusters of EdU-positive cells corresponding to injections from days 3 and 6. Asterisks indicate a fifth cluster of EdU-positive cells corresponding to the site of injury and repair. (B') Higher magnification of boxed region showing regenerated rhodopsin immunoreactive (red) rod photoreceptors.
Figure 7
 
Retinal injury and repair induces proliferation of retinal cells. Rod-ablated iCasp9 tadpoles were injected consecutively with EdU on days 3, 6, and 15 postinjury. Tadpoles were killed on day 22 after injury and retinas stained for EdU detection (red) and counterstained with WGA (green). (A) Stained section of uninjured eye. Brackets indicate clusters of EdU-positive cells corresponding to injections from days 3 and 6. (B) Stained section of contralateral injured eye. Brackets indicate clusters of EdU-positive cells corresponding to injections from days 3 and 6. Asterisks indicate a fifth cluster of EdU-positive cells corresponding to the site of injury and repair. (B') Higher magnification of boxed region showing regenerated rhodopsin immunoreactive (red) rod photoreceptors.
Discussion
Our data demonstrates that the X. laevis tadpole is capable of regenerating de novo neural retina to repair and replace injured and lost retina. We observed this regenerative capacity after resection of the eye, after retinectomy and retinal injury. Our observations support previously described reports of retinal regeneration in X. laevis larvae. 3,4,7,8,19,23,24 Furthermore, we confirmed that administration of FGF2 can influence the rate of retinal regeneration in retinectomized eyes. 
We showed that retinal injury is repaired by the proliferation cells at the site of injury as well as from increased proliferation arising from the CMZ. Our EdU labeling experiments showed the temporal profile of proliferating cells after punch injury. The most proliferation and regenerative activity at a site of injury occurs between 3 and 15 days postinjury, which was also observed by other groups. 24 In response to injury, we observed two distinct sites of proliferation, at both the CMZ and the site of injury. Thus, the response to injury is not entirely local. This “injury-induced neurogenesis” was also previously observed in Rana pipiens larvae, in which the proportion of new cells generated was comparable to the proportion of retinal cells ablated by injury. 25  
In contrast, we found that rod photoreceptors ablated by direct activation of apoptosis are not regenerated. While neurogenesis, including the addition of new photoreceptors, continued at the periphery of the retina subsequent to the insult (consistent with the growth of the eye), there was no regeneration of ablated rod photoreceptors, and the central retina of drug-treated tadpoles remained rodless. Based on these results, it is unlikely that regeneration of rods is a complicating factor in our previously reported transgenic X. laevis models of RP. 2630  
We attempted to induce regeneration of the retina by two means: administration of FGF2, and simultaneous injury of the eye. We successfully induced regeneration of rods in a region that would otherwise remain rodless via injury. However, this regeneration was limited to the site of injury, despite the fact that we observed nonlocalized effects on proliferation, that is, increased proliferation was also induced at the CMZ (Fig. 7B). Interestingly, FGF2 did not have an effect on regenerating ablated rods, although we observed a positive effect on neural retina regeneration after retinectomy. 
Our results present an intriguing question: given that the X. laevis retina can repair gross structural damage, why does it fail to repair the more modest injury of ablation of rod photoreceptors? The most likely reason is the mechanism of injury: targeted cellular ablation by direct activation of apoptosis versus physical damage/removal of large sections of retina lamellae. Apoptosis is an active programmed cell death mechanism where apoptotic cells are fragmented into membrane-bound vesicles (apoptotic bodies). These apoptotic bodies are subsequently cleared by neighboring cells and/or macrophages in a process that avoids eliciting inflammatory responses. In contrast, with physical resection and retinectomy, normal morphology is dramatically altered. Additionally, there is epithelium tearing, extracellular matrix disruption and cell debris release. This trauma may constitute a regeneration signal, or may invoke a regeneration signal, leading to a reestablishment of appropriate morphology. It is possible that the damage caused by apoptosis is not gross enough to elicit a regenerative response, and that the greater or more traumatic the destruction, the more effective the regenerative response. Indeed, Kuriyama et al. 21 found that alteration of tissue interactions, such as detachment of RPE cells from the choroid, is required for RPE transdifferentiation. Furthermore, in a rod-ablated retina, additional traumatic injury and subsequent repair do not stimulate regeneration of rods lost by apoptosis except at the site of trauma, although other remote effects are seen (compensatory proliferation at the CMZ). Thus, regeneration of rods would appear to require not only a regenerative signal, but also a traumatic cue that identifies the missing rods as a morphological abnormality. 
Interestingly, a recent report by Choi et al. 31 using a similar paradigm suggested that regeneration of lost rod photoreceptors in X. laevis retina does occur. Choi et al. 31 reported that after rod ablation initiated by drug-induced DNA damage, tadpole rods at least partially regenerated. It is not clear what produced the differences between our studies. One possibility is that the mechanism of cell death in the model from Choi et al. 31 is sufficiently different (i.e., not caspase 9–mediated apoptosis) that the retina is able to recognize this insult as a traumatic injury, resulting in regeneration. A second possibility is that in the study by Choi et al., 31 a significant number of rods were still present at the end point of drug treatment, albeit dysmorphic. This would be similar to our recent report of morphologically abnormal rods persisting for long periods in a transgenic X. laevis Pro23His rhodopsin model of RP. 16 This possibility is avoided in our current study by identification of rod photoreceptors via GFP expression. 
Although our data in this current study cannot unambiguously identify the source of progenitor cells during regeneration, it is likely that they originate from the RPE. We found proliferating cells at the site of injury at the interface of the choroid and RPE (Figs. 6, 7). Previous studies also suggest that the RPE is required for retinal regeneration after retinectomy. 6,23 Additionally, we and others 24 observed that the RPE is first to be repaired after resection, followed by relamination of retinal layers. It is thought that the RPE cells undergo de-differentiation to become retinal precursor cells. FGF2 has also been reported to be required for RPE transdifferentiation in vitro and in vivo; without supplementation in vitro, or the use of blocking antibodies in vivo, RPE-mediated retinal regeneration is inhibited. 21,23  
Our data support the hypothesis that retinal regeneration in frogs occurs only after physical trauma. In support, accounts of retinal regeneration in lower vertebrates (other than fish) have been described only after surgical and/or physical injury. These include the urodeles amphibians. Interestingly, it is also widely accepted that amphibians regenerate retina by RPE transdifferentiation. In contrast, zebrafish (which have been reported to regenerate photoreceptors lost by apoptosis) regenerate retina by either Müller transdifferentiation or inner nuclear layer precursor cells. 32 Rod photoreceptor regeneration is also observed in fish in which photoreceptors have been ablated by intense light exposure. Currently, we do not have a similar paradigm available to use for X. laevis , but it would be interesting to determine whether this is due to differences in regenerative capacity of fish and frog retina, or differences in the cell death mechanisms in the respective models. 
Rod photoreceptor apoptosis is the unifying first event in animal models of RP regardless of the genetic cause, 1114 and is likely the first event in the pathology of human RP. Like the X. laevis retina, mammalian retinas are unable to regenerate rods lost due to apoptosis. However, the inherent regenerative capacity of the X. laevis retina suggests that it should be possible to manipulate the X. laevis retina into entering a regenerative state. To accomplish this, we require a better understanding of the signaling pathways involved. Furthermore, if these signaling pathways are evolutionarily conserved, this knowledge may suggest therapeutic interventions that could be useful in treatment of human RP. Future comparative studies may provide insight to the mechanisms regulating retinal neurogenesis. Similarly, regeneration of rods, features of the rod precursor cells, and pathways involved in regeneration would be of specific interest to those investigating stem cell or transplant treatments for RP. Our studies may suggest therapeutic approaches for increasing the regenerative potential of the retina. 
Acknowledgments
The authors thank Jenny Wong for technical assistance. 
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Footnotes
 Supported by grants from the Foundation Fighting Blindness of Canada.
Footnotes
 Disclosure: D.C. Lee, None; L.M. Hamm, None; O.L. Moritz, None
Figure 1
 
Tadpoles do not regenerate rod photoreceptors ablated by targeted apoptosis. (A) Administration of the drug AP20187 rapidly induces apoptosis in iCasp9-expressing rod photoreceptors of 2-week old tadpoles. Four days after drug administration, rods are completely ablated from the retina. Note the absence of GFP signal (green) in the central retina. Inset shows higher magnification of the boxed region in the central retina. The retinal cell layers labeled are ganglion cell layer (GCL), inner nuclear layer (INL), ONL, and OS. (B) Representative image of age-matched untreated iCasp9 retinas. (C) Drug-treated iCasp9 tadpole retina after 4-week recovery period. Inset shows higher magnification of the rodless central retina. (C') Anticalbindin immunolabeling showing that only cone photoreceptors remain in ONL of central retina. (D, D') Untreated iCasp9 tadpole retina at 6 weeks after fertilization. Note the ONL is populated with both GFP-positive rods ([D] inset, green) and cones ([D'], red). All images shown are representative confocal micrographs of retinal cryosections. (AD) Sections were stained with wheat germ agglutinin (red) to label membranes and Hoechst 33342 (blue) to label nuclei. (C', D') Anticalbindin colabeling to visualize cone photoreceptors. Scale bars: 100 μm (A, C); 10 μm (inset panels, [C']).
Figure 1
 
Tadpoles do not regenerate rod photoreceptors ablated by targeted apoptosis. (A) Administration of the drug AP20187 rapidly induces apoptosis in iCasp9-expressing rod photoreceptors of 2-week old tadpoles. Four days after drug administration, rods are completely ablated from the retina. Note the absence of GFP signal (green) in the central retina. Inset shows higher magnification of the boxed region in the central retina. The retinal cell layers labeled are ganglion cell layer (GCL), inner nuclear layer (INL), ONL, and OS. (B) Representative image of age-matched untreated iCasp9 retinas. (C) Drug-treated iCasp9 tadpole retina after 4-week recovery period. Inset shows higher magnification of the rodless central retina. (C') Anticalbindin immunolabeling showing that only cone photoreceptors remain in ONL of central retina. (D, D') Untreated iCasp9 tadpole retina at 6 weeks after fertilization. Note the ONL is populated with both GFP-positive rods ([D] inset, green) and cones ([D'], red). All images shown are representative confocal micrographs of retinal cryosections. (AD) Sections were stained with wheat germ agglutinin (red) to label membranes and Hoechst 33342 (blue) to label nuclei. (C', D') Anticalbindin colabeling to visualize cone photoreceptors. Scale bars: 100 μm (A, C); 10 μm (inset panels, [C']).
Figure 2
 
Retina can regenerate after retinectomy. (A) Histological sections of X. laevis tadpole eyes 1 day after (Day+1) and 21 days after (Day+21) retinectomy. Retinectomies were performed on NF stage 47/48 tadpoles (equivalent to 14 days postfertilization [dpf]); 21 days after retinectomy, most eyes spontaneously regenerated retina. However, the extent of regeneration varied. Representative images shown. Scale bar: 100 μm. (B) Percentage of retinectomized eyes that regenerate retina after 21 days without intervention (left) or with heparin-coated bead implants (right). Heparin-coated beads were implanted in the eye immediately after retinectomy and allowed to recover for 21 days. Implantation of a bead negatively affected the regenerative capacity after retinectomy (+blank bead). However, presoaking the beads in FGF2 (+FGF2 bead) significantly reversed the inhibition (*P = 0.019, chi-squared test). Chart legend shows representative images of the varying extents of regeneration.
Figure 2
 
Retina can regenerate after retinectomy. (A) Histological sections of X. laevis tadpole eyes 1 day after (Day+1) and 21 days after (Day+21) retinectomy. Retinectomies were performed on NF stage 47/48 tadpoles (equivalent to 14 days postfertilization [dpf]); 21 days after retinectomy, most eyes spontaneously regenerated retina. However, the extent of regeneration varied. Representative images shown. Scale bar: 100 μm. (B) Percentage of retinectomized eyes that regenerate retina after 21 days without intervention (left) or with heparin-coated bead implants (right). Heparin-coated beads were implanted in the eye immediately after retinectomy and allowed to recover for 21 days. Implantation of a bead negatively affected the regenerative capacity after retinectomy (+blank bead). However, presoaking the beads in FGF2 (+FGF2 bead) significantly reversed the inhibition (*P = 0.019, chi-squared test). Chart legend shows representative images of the varying extents of regeneration.
Figure 3
 
FGF2 does not stimulate retina to replace rods lost by induced apoptosis. Rod ablated iCasp9 eyes 21 days after bead implantation. iCasp9 tadpoles (14 dpf, NF stage 47/48) were treated with AP20187 to induce rod apoptosis. After a 4-day incubation period to allow for complete rod ablation, the lens was replaced with a buffer-soaked bead (+blank bead) or an FGF2-soaked bead (+FGF2-soaked bead) and subsequently allowed to recover for 21 days. Retinal sections were stained with wheat germ agglutinin (green) to label membranes and Hoechst 33342 (blue) to label nuclei. Scale bar: 100 μm.
Figure 3
 
FGF2 does not stimulate retina to replace rods lost by induced apoptosis. Rod ablated iCasp9 eyes 21 days after bead implantation. iCasp9 tadpoles (14 dpf, NF stage 47/48) were treated with AP20187 to induce rod apoptosis. After a 4-day incubation period to allow for complete rod ablation, the lens was replaced with a buffer-soaked bead (+blank bead) or an FGF2-soaked bead (+FGF2-soaked bead) and subsequently allowed to recover for 21 days. Retinal sections were stained with wheat germ agglutinin (green) to label membranes and Hoechst 33342 (blue) to label nuclei. Scale bar: 100 μm.
Figure 4
 
Physical injuries to the eye and retina are spontaneously repaired. Retinal injuries were created in NF stage 47/48 (14 dpf) tadpoles by punching a hole through the posterior of the eye into the retina and removing the circular plug of choroid, RPE, and neural retina. (A, A') Representative image of tadpole eyes 1 day after punch injury. (B, B') Section of eye from tadpole 21 days after injury. Arrowheads indicate site of injury. (A, B) Light micrographs of retinal cryosections stained with PROTOCOL Hema 3 stain. (A', B') Confocal micrographs of retinal cryosections labeled with wheat germ agglutinin (green) and Hoechst 33342 (blue). Scale bar: 100 μm.
Figure 4
 
Physical injuries to the eye and retina are spontaneously repaired. Retinal injuries were created in NF stage 47/48 (14 dpf) tadpoles by punching a hole through the posterior of the eye into the retina and removing the circular plug of choroid, RPE, and neural retina. (A, A') Representative image of tadpole eyes 1 day after punch injury. (B, B') Section of eye from tadpole 21 days after injury. Arrowheads indicate site of injury. (A, B) Light micrographs of retinal cryosections stained with PROTOCOL Hema 3 stain. (A', B') Confocal micrographs of retinal cryosections labeled with wheat germ agglutinin (green) and Hoechst 33342 (blue). Scale bar: 100 μm.
Figure 5
 
De novo rod photoreceptors are present at site of injury repair. iCasp9 tadpoles (14 dpf, NF stage 47/48) were treated with AP20187 for 4 days to induce rod apoptosis. Subsequently one eye was subjected to punch injury while the contralateral eye was left intact. (A) Rod-ablated iCasp9 eyes 21 days after punch injury. Upper panels: injured eye; lower panels: intact eye. Arrows indicate site of injury. Red brackets highlight regenerated rod outer segments that coincide with site of injury. (B) Immunolabeling with rhodopsin antibodies confirmed that rod outer segments were at site of injury repair (upper panels). Site of repair was also PCNA immunoreactive (lower panels). (B') Higher magnification of respective boxed regions. Asterisks indicate a nonspecific crossreaction of anti-PCNA label in rod inner segments. All sections were colabeled with wheat germ agglutinin (green) and Hoechst 33342 (blue). Scale bars: 100 μm.
Figure 5
 
De novo rod photoreceptors are present at site of injury repair. iCasp9 tadpoles (14 dpf, NF stage 47/48) were treated with AP20187 for 4 days to induce rod apoptosis. Subsequently one eye was subjected to punch injury while the contralateral eye was left intact. (A) Rod-ablated iCasp9 eyes 21 days after punch injury. Upper panels: injured eye; lower panels: intact eye. Arrows indicate site of injury. Red brackets highlight regenerated rod outer segments that coincide with site of injury. (B) Immunolabeling with rhodopsin antibodies confirmed that rod outer segments were at site of injury repair (upper panels). Site of repair was also PCNA immunoreactive (lower panels). (B') Higher magnification of respective boxed regions. Asterisks indicate a nonspecific crossreaction of anti-PCNA label in rod inner segments. All sections were colabeled with wheat germ agglutinin (green) and Hoechst 33342 (blue). Scale bars: 100 μm.
Figure 6
 
Actively proliferating cells coincide with location of retinal injury and repair. iCasp9 tadpoles (14 dpf, NF stage 47/48) were treated with AP20187 for 4 days to induce rod apoptosis. Rod-ablated iCasp9 tadpoles were injected with EdU on day 3 (A, A'), day 6 (B, B'), or day 15 (C, C') after retinal injury. EdU incorporation was followed for a short period (8 hours) or for an extended period (3–9 days). At the end of the incubation period, tadpoles were killed and retinas stained for EdU detection (red) and co-stained with WGA (green). In some instances, the lens was lost during sectioning and histological processing. Double daggers indicate nonspecific labeling of lens. Arrowheads indicate region corresponding to CMZ of retina.
Figure 6
 
Actively proliferating cells coincide with location of retinal injury and repair. iCasp9 tadpoles (14 dpf, NF stage 47/48) were treated with AP20187 for 4 days to induce rod apoptosis. Rod-ablated iCasp9 tadpoles were injected with EdU on day 3 (A, A'), day 6 (B, B'), or day 15 (C, C') after retinal injury. EdU incorporation was followed for a short period (8 hours) or for an extended period (3–9 days). At the end of the incubation period, tadpoles were killed and retinas stained for EdU detection (red) and co-stained with WGA (green). In some instances, the lens was lost during sectioning and histological processing. Double daggers indicate nonspecific labeling of lens. Arrowheads indicate region corresponding to CMZ of retina.
Figure 7
 
Retinal injury and repair induces proliferation of retinal cells. Rod-ablated iCasp9 tadpoles were injected consecutively with EdU on days 3, 6, and 15 postinjury. Tadpoles were killed on day 22 after injury and retinas stained for EdU detection (red) and counterstained with WGA (green). (A) Stained section of uninjured eye. Brackets indicate clusters of EdU-positive cells corresponding to injections from days 3 and 6. (B) Stained section of contralateral injured eye. Brackets indicate clusters of EdU-positive cells corresponding to injections from days 3 and 6. Asterisks indicate a fifth cluster of EdU-positive cells corresponding to the site of injury and repair. (B') Higher magnification of boxed region showing regenerated rhodopsin immunoreactive (red) rod photoreceptors.
Figure 7
 
Retinal injury and repair induces proliferation of retinal cells. Rod-ablated iCasp9 tadpoles were injected consecutively with EdU on days 3, 6, and 15 postinjury. Tadpoles were killed on day 22 after injury and retinas stained for EdU detection (red) and counterstained with WGA (green). (A) Stained section of uninjured eye. Brackets indicate clusters of EdU-positive cells corresponding to injections from days 3 and 6. (B) Stained section of contralateral injured eye. Brackets indicate clusters of EdU-positive cells corresponding to injections from days 3 and 6. Asterisks indicate a fifth cluster of EdU-positive cells corresponding to the site of injury and repair. (B') Higher magnification of boxed region showing regenerated rhodopsin immunoreactive (red) rod photoreceptors.
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