Retardation of Photoreceptor Degeneration in the Detached Retina of rd1 Mouse

Abstract

purpose. To study the neuroprotective effect of experimental retinal detachment (RD) on photoreceptor degeneration in rd1 mice.

methods. RD was produced in the eyes of rd1 mice at postnatal day (P) 9. These eyes were collected and compared to controls without RD. The effects of RD on retinal degeneration were evaluated by histochemical staining of nuclei in the outer nuclear layer (ONL), rod and cone photoreceptors, and retinal vessels at P30 in retinal sections and flatmounts. Apoptotic photoreceptors were detected by TdT-mediated dUTP nick-end labeling (TUNEL) at P15. Mice with or without RD were also reared in darkness and evaluated immunohistochemically at P30.

results. The numbers of rhodopsin-positive (rod), peanut agglutinin-positive (cone), and diamino-2-phenyl-indol-stained (rod-plus-cone) cells in the ONL were increased by 2.0-fold, 1.3-fold, and 1.2-fold, respectively, in the rd1 eyes with RD compared to those without RD at P30. In the detached retina, the cone photoreceptor inner/outer segment structures and the deep retinal vessels surrounding the inner nuclear layer and the ONL, but not the ganglion cell layer, were preserved. At P15, TUNEL-positive cell numbers in the ONL were significantly reduced in the eyes with RD. Light exposure had no effect on photoreceptor degeneration in the eyes with or without RD.

conclusions. RD mediates the preservation of cone and rod photoreceptors in the ONL and surrounding vascular structures by reducing the rate of apoptosis of photoreceptors in rd1 mice. Light deprivation does not appear to be one of the mechanisms of photoreceptor protection in the detached retinas in these mice.

Retinitis pigmentosa (RP) is a group of inherited human eye diseases that affects approximately 1 in 4000 persons. The disease is clinically characterized by the progressive loss of rod photoreceptors followed by cone photoreceptor degeneration and vessel attenuation. ¹ In the early stages of the disease, affected patients show evidence of rod photoreceptor dysfunction/degeneration, such as night blindness or constriction of the visual field. Later stages of the disease are characterized by additional symptoms related to cone photoreceptor dysfunction/degeneration, which eventually results in loss of central vision. ^{1 2} To date, genetic defects in more than 45 genes have been identified as causes of this disease ³ (http://www.sph.uth.tmc.edu/RetNet/home.htm). In particular, recessive defects in the gene encoding the β-subunit of cyclic GMP phosphodiesterase have been identified as causes not only of RP in humans ^{4 5} but also of retinal degeneration in mice widely used for the eye research (rd1 mice). ^{6 7}  

In rd1 mice, rapid rod photoreceptor degeneration begins at approximately postnatal day (P)10, with most of the cells degenerated by P30. ^{8 9} Consequently, the remaining photoreceptors at this age are mostly cones. ⁸ These cone photoreceptors are distributed unevenly across the retina at P30, and their concentration decreases first in the central and far peripheral retina, leaving a ring of cells in the midperipheral region. ¹⁰ Retinal vessels begin to degenerate at approximately the second week, and by P30 vessel lengths are reduced by approximately 50%. ¹¹  

We have been searching for factors that may delay the process of photoreceptor degeneration by protecting them from apoptosis or promoting their regeneration in rd1 mice. Vascular endothelial growth factor (VEGF), a known angiogenic factor, has been shown to stimulate one of its ligands, VEGFR2/Flk1, expressed in retinal progenitor cells to promote their proliferation at the peripheral retina in these mice. ¹² During the course of the VEGF study, we found that intravitreal injection of VEGF in rd1 mice often induced retinal detachment (RD) from the underlying retinal pigment epithelium (RPE), the mechanism of which was unknown. Interestingly, among the eyes injected with VEGF, those with RD clearly and consistently had more photoreceptors than those without. This observation was unexpected because RD is a blinding disease known to cause slow photoreceptor apoptosis in humans ^{13 14 15} and mice. ^{16 17 18} Although the precise molecular pathology of retinal degeneration in RD is still unclear, photoreceptor apoptosis is mediated through pathways dependent or independent of caspases. ^{13 14 15 16 18 19 20 21} Moreover, such photoreceptor apoptosis is partially promoted by the recruitment of macrophages into the detached retina through monocyte chemoattractant protein 1, expressed by Müller glial cells. ¹⁸ On the other hand, it has been reported that the progression of photoreceptor loss in rd1 mice was delayed by removing the retina from the RPE and keeping it in culture, ^{22 23 24} whereas similar tissue culture of retinas from wild-type mice resulted in fewer photoreceptor layers. ^{22 24} Survival factors in the medium have been postulated as among the mechanisms for the observed photoreceptor preservation in retinal culture from rd1 mice. ²³  

In this study, we defined the influence of RD on the rd1 retina by statistical analysis of its effect on the numbers of surviving and apoptotic photoreceptors. We showed that experimentally induced RD preserved photoreceptors and the surrounding retinal vasculatures by reducing apoptosis of photoreceptors in rd1 mice. 

Methods

Animals

All experimental procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Guidelines for the Use of Experimental Animals at Nagoya University School of Medicine. We used mice of the C3H/HeJ strain (Clea, Tokyo, Japan) homozygous for the rd1 mutation as an RP model. ^{6 7 9} Mice were reared under a 12-hour light/12-hour dark cycle unless otherwise indicated. 

Experimental Retinal Detachment

RD was experimentally generated in P9 rd1 mice using a technique based on one previously reported ¹⁹ (Fig. 1) . In brief, the pupils were dilated with 0.5% tropicamide/0.5% phenylephrine. After perforation of the nasal globe at the equator using a 30-gauge needle with its tip slightly bent, the needle tip was slowly advanced through the vitreous cavity and into the temporal retina. The retina was mechanically detached from the underlying RPE by gentle pulling away of the needle, with its bent tip stuck in or under the retina (Fig. 1A) . Meanwhile, during the procedure, a small detachment occurred spontaneously at the nasal retina adjacent to the perforation site. The needle was released from the detached temporal retina and carefully retracted into the nasal subretinal space. There the nasal retina was gently pushed away from the RPE several times with the bent end of the needle to enlarge the RD, and the entire retina detached from the RPE. This appeared to be important for maintaining RD in rd1 mice until P30 because focal RD induced by subretinal saline injection at P9 disappeared by P15. Non-RD eyes underwent a similar procedure except that perforation was performed roughly 0.5 to 1 mm posterior to the corneal limbus (Fig. 1B) , thereby creating a retinal break at the perforation site without inducing RD (Fig. 1C) . Eyes from mice with no injury also served as controls (untouched eyes). We conducted fundus examination on each eye under a surgical microscope and analyzed histologic sections to confirm the presence or absence of RD before using the eye for experimental purposes. Eyes with incidental cataracts were excluded from the study. 

Histochemical Analyses

Histochemical analyses were performed on eyes enucleated at P30, as described previously ¹² with some modifications. To evaluate histologic sections, the eyes were enucleated after cauterization of the superior cornea for orientation. The cornea and lens were removed from the eyes, which were fixed with 4% paraformaldehyde (PFA) for 2 hours, cryoprotected with 30% sucrose at 4°C overnight, and processed through the ventral to dorsal meridian into sections 10 μm thick. These sections were permeabilized with 0.01% Triton X-100/PBS (PBST) for 5 minutes, blocked with 5% goat serum/PBS for 1 hour, and incubated with a first antibody for 1 hour and a second antibody for 1 hour. For analysis of retinal flatmount specimens, eyecups were fixed with 4% PFA and the entire retina was dissected from the RPE and incised radially for each sample. For evaluation of the vascular structures, samples were permeabilized with 0.5% PBST for 3 hours, blocked in 5% goat serum/PBS for 1.5 hours, and incubated with a primary antibody for 12 hours and with a secondary antibody for 6 hours. For analysis of cone photoreceptor inner/outer segments, the flatmounts were permeabilized with 0.5% PBST for 3 hours and incubated in fluorescent dye–conjugated peanut agglutinin (PNA; Alexa 488,1:100; Molecular Probes, Eugene, OR) for 6 hours. 

Specimens were incubated with first antibodies against rhodopsin (1:1500; Chemicon, Temecula, CA), M-cone opsin (1:1000; Chemicon), or collagen IV (1:500; Neomarkers, Vancouver, WA) followed by staining with fluorescent dye–conjugated antibody (Alexa 488, 1:1500; Molecular Probes) and diamino-2-phenyl-indol (DAPI; 1:1000, Molecular Probes). The same concentrations of antibodies were applied to sections and flatmount specimens. 

TUNEL Staining

To detect apoptotic cells in the retina, the eye sections from P15 rd1 mice were treated with an in situ cell detection kit (Roche, Indianapolis, IN) using the TUNEL technique according to the manufacturer’s instructions. 

Rearing Mice in Darkness

To study the effects of light deprivation on photoreceptor degeneration, rd1 mice were reared in darkness immediately after the induction of RD or control injury at P9. The eyes were enucleated at P30 and processed into sections as described. 

Statistical Analyses

In 600-μm wide portions of the central retina and at equal distances from the anterior margin of the optic nerve and the posterior margin of the ciliary body, DAPI-positive nuclei, rhodopsin-positive cells, or TUNEL-positive cells were evaluated from histologic sections of the outer nuclear layer (ONL). DAPI-positive nuclei and rhodopsin-positive cells in the ONL were counted from a single image, whereas TUNEL-positive cells in the ONL were counted from three independent images and averaged. Numbers of DAPI-positive nuclei or rhodopsin-positive cells were normalized by retinal thickness (average of three independent measurements) measured within the same image to reduce the effect of the tangential sectioning of the retina. The average retinal thickness from untouched P30 rd1 mice (n = 11) was used as a reference. 

Numbers of collagen IV-positive retinal vessel lumens in three major layers in which the vascular networks were most highly concentrated ²⁵ —the nerve fiber layer/ganglion cell layer (NFL/GCL), the inner plexiform layer (IPL), and the inner nuclear layer/outer plexiform layer (INL/OPL)—were determined from three independent images of retinal sections and averaged. 

Numbers of PNA-positive cone photoreceptor inner/outer segments within a 320 μm × 320 μm square located 1 mm dorsal and ventral to the optic nerve were determined and averaged from images obtained from flatmounts. All experimental data were evaluated by an operator masked to the treatment conditions (RD, non-RD, or untouched). Differences were examined using the unpaired Student t-test, and P < 0.05 was considered significant. 

Results

Photoreceptor Preservation in the rd1 Mouse Eyes with RD

At P30, rd1 mice lose most of their photoreceptors. ^{8 9} As a consequence, the ONL is composed of approximately one layer of photoreceptors and almost no detectable inner/outer segment structures. ^{8 9} After experimental RD was produced in rd1 mice at P9, the eyes were enucleated at P30, and DAPI- and rhodopsin-positive cells in the ONL were counted in histologic sections. We observed increased numbers of DAPI-positive cells (1.22-fold; Fig. 2E ) in the ONL, representing the sum of rod and cone photoreceptors in the eyes with RD (Figs. 2A 2B)compared to those without RD (Figs. 2C 2D) . Significant differences were detected only in the dorsal retina (1.29-fold), whereas differences in the ventral retina did not reach statistical significance (P = 0.09) when they were evaluated separately (Fig. 2E) . Similarly, rhodopsin-positive rod photoreceptors, constituting a minor portion of the residual photoreceptors at P30, ^{8 9} were also more frequently identified (2.09-fold; Fig. 2F ) in the ONL of eyes with RD (Figs. 2A 2B)than in those without RD (Figs. 2C 2D) . We found significant differences when the dorsal (2.12-fold) and ventral (2.06-fold) retinas were evaluated separately (Fig. 2F) . No significant difference was detected in the numbers of DAPI-positive cells (P = 0.29) or rhodopsin-positive cells (P = 0.07) in the ONL between the untouched and non-RD eyes of P30 rd1 mice (Figs. 2E 2F) . 

The number of cone photoreceptors was determined by counting the PNA-positive cone inner/outer segments (Fig. 3A)in retinal flatmounts, which was increased by 1.34-fold in eyes with (P = 4.9 × 10⁻⁴) compared to those without RD (Fig. 3B) . In addition, more prominent cone photoreceptor inner/outer segment structures positive for M-cone opsin were frequently seen in the eyes with RD (Fig. 3C) , whereas such structures were less obvious and rare in those without RD (Fig. 3D) . However, gross abnormalities were observed in the alignment and structure of M-cone photoreceptor outer segments in the eyes with RD compared to those of wild-type mice (Supplementary Fig. S1). 

Preservation of Retinal Vascular Structures in the rd1 Mouse Eyes with RD

In the rodent eyes, vascular networks are mainly identified around the GCL and at the interface anterior to the INL and ONL to nourish adjacent retinal neurons. With the progress of photoreceptor degeneration, secondary attenuation of retinal vessels, probably correlated with the degree of photoreceptor loss, is observed in rd1 mice. ^{9 11 25} To further study the neuroprotective effect of RD on the rd1 retina, we evaluated its effect on retinal vessels by immunohistochemical analyses of flatmount specimens and histologic sections of the retina with and without RD using anti-collagen IV antibody as a vascular marker. Collagen IV is mainly expressed in the basement membrane of retinal vessels. ²⁶ Obvious preservation of vascular structures, particularly those of minor vessels in the deeper vascular plexus, was detected in flatmount specimens and retinal sections in the eyes with RD (Figs. 4A 4C)compared to those without RD (Figs. 4B 4D) . To statistically quantify remaining retinal vessels, we divided the images obtained from retinal sections into three compartments—NFL/GCL, IPL, and INL/OPL, ²⁵ and separately counted the number of collagen IV-positive vascular lumens (Fig. 4E) . The number of identified collagen IV-positive retinal vascular lumens was significantly larger in the eyes with than in the eyes without RD in the IPL (2.09-fold) and the INL/OPL (8.12-fold) but not in the NFL/GCL. 

Reduced Apoptosis in the rd1 Mouse Retina with RD

To compare the number of apoptotic photoreceptors in the retinas of rd1 mice with and without RD, TUNEL staining was performed on retinal sections (Fig. 5) . Because only rare TUNEL-positive cells were detected at P30, when most of the photoreceptors had already degenerated, the eyes were evaluated at P15, when the retina still contained numerous photoreceptors, a few of which were apoptotic cells. The number of TUNEL-positive cells in the ONL was significantly (31%) smaller in the eyes with than in the eyes without RD at P15. 

Effects of Light Deprivation on Photoreceptor Degeneration and RD in rd1 Mice with and without RD

We found that the reduction of apoptosis could account for the preservation of photoreceptors in the rd1 mouse eyes with RD. However, the precise mechanism of the decrease in photoreceptor apoptosis in the detached retina is unclear. Light exposure is associated with the development or exacerbation of photoreceptor degeneration in rodents ^{27 28 29 30} and light deprivation with protection of photoreceptors in those with retinal degeneration. ^{31 32} We postulated that reduced phototransduction in the rd1 eyes with RD may confer neuroprotective effects similar to those of light deprivation in other rodent models of hereditary retinal degeneration. ^{31 32} To test this hypothesis, we evaluated the relationship between light exposure and RD on photoreceptor degeneration in rd1 mice. 

First, the numbers of DAPI- and rhodopsin-positive photoreceptors in the ONL were compared between rd1 mice with and without RD. Both groups were reared in darkness immediately after the induction of RD or control injury at P9. Similar to the results in rd1 mice reared under a regular light-dark cycle (Fig. 2) , the number of DAPI-positive cells in the ONL at P30 was significantly increased (1.23-fold) in the eyes with RD compared to those without RD (Fig. 6A) . We also found significant differences when the dorsal (1.28-fold) and ventral (1.17-fold) retinas were evaluated separately (Fig. 6A) . Similarly, the numbers of rhodopsin-positive cells in the ONL were significantly increased (1.99-fold) in the eyes with RD compared to those without RD. Significant differences were also detected when the dorsal (2.27-fold) and ventral (1.77-fold) retinas were evaluated separately (Fig. 6B) . The degrees of differences were comparable to those in which rd1 mice were reared under a regular light-dark cycle. 

Second, the numbers of DAPI- and rhodopsin-positive photoreceptors in the ONL were compared in rd1 mice without RD reared in darkness and under a regular light-dark cycle. Among the eyes without RD, we observed no significant difference in the number of DAPI-positive cells in the ONL between mice reared in darkness and those reared under a regular light-dark cycle (Fig. 6C) . This was also true when the dorsal and ventral retinas were evaluated separately. Similarly, we observed no significant difference in the number of rhodopsin-positive cells in the ONL in mice reared in darkness and in those reared under a regular light-dark cycle; this applied as well to the dorsal and ventral retinas when evaluated separately (Fig. 6D) . 

Discussion

We found significantly more rod and cone photoreceptors in detached than in nondetached rd1 retinas. This neuroprotective effect of RD on photoreceptors was likely mediated by reduced apoptosis. We also found preservation of the inner/outer segment structures of M-cone photoreceptors and retinal vascular plexus, both of which are likely to be secondary effects. ^{8 9 10 11 25}  

Rod photoreceptors are primarily affected because of defects in the gene Pde6b encoding the rod photoreceptor-specific phosphodiesterase in rd1 mice. ^{6 7} It is reasonable then to assume that the induction of RD preferentially protects diseased rod photoreceptors from apoptosis. This was supported by the observation that the number of rhodopsin-positive rod photoreceptors was increased by approximately 2-fold compared with the nondetached retina, whereas DAPI-positive nuclei in the ONL, representing the sum of rod and cone photoreceptors, was increased by only approximately1.2-fold at P30. Given that more than 90% of photoreceptors in the ONL are cone photoreceptors at this stage in the rd1 retina, ⁸ these results suggested that cone photoreceptors are also preserved, though to a lesser degree. This finding was further supported by the quantification of the PNA-positive inner/outer segments of cone photoreceptors in retinal flatmounts, which were increased by approximately 1.3-fold in the detached retinas. 

Although we found that the neuroprotective effect of RD on photoreceptor degeneration was probably mediated by the reduction of apoptosis, the precise mechanism of the neuroprotective effect remains unclear. One of the most feasible factors contributing to the rescue effects is the widely known upregulation of neurotrophic factors or cytokines induced by ocular injuries in rodents ^{33 34 35 36 37} and in humans. ^{38 39 40} However, unlike other forms of trauma known to be neuroprotective against retinal degeneration, such as puncture ^{34 36 41 42} or lens injury, ^{43 44 45} RD is directly toxic to photoreceptors and induces their apoptosis in wild-type mice. ^{16 17 18} Therefore, the degree to which upregulated cytokines or neurotrophic factors counteract photoreceptor degeneration is unclear. At the same time, it is possible that important factors, in addition to the injury itself, may account for the observed paradoxical neurotrophic effect of RD on photoreceptors in rd1 mice. 

A pathway of photoreceptor apoptosis dependent on phototransduction has been described in mice with retinal degeneration. ^{33 46} Moreover, light deprivation has been reported to protect retinal neurons in other rodent models of retinal degeneration. ^{31 32} Because the defective gene in rd1 mice, Pde6b, encodes an essential component of the phototransduction cascade, we initially postulated that the reduction of aberrant visual transduction in rd1 eyes with RD may protect photoreceptors from apoptosis. However, further experiments revealed that this hypothesis does not apply to RD-induced neuroprotection in rd1 mice. Other factors—such as separation of the retina from the RPE, which is involved in phagocytosis of the subretinal debris, ^{47 48} and reduction of excessive oxidative supply from the choroidal circulation, which results in reduced oxidative stress ⁴⁹ —are under evaluation. 

Analyses of the collagen IV-positive vascular structures showed significant preservation of the small caliber vessels in the deeper retina but not the larger vessels around the GCL. The greatest effect was observed in the deepest vascular network at the interface between the INL and the severely attenuated OPL, which showed an increase of approximately 8.2-fold in the number of vascular lumens in the eyes compared to those without RD. The anatomic proximity of these vessels and the adjacent photoreceptors indicate that preservation of these two retinal components is related, which is consistent with the hypothesis that vascular structures are preserved secondarily to the protection of photoreceptors from apoptosis. The greater amount of blood needed in the deeper retinal layers because of the increased number of remaining photoreceptors may account for the preservation of deep vascular plexus. 

Our finding that RD, a known blinding disease that results in photoreceptor degeneration and severely reduced visual transduction, could have a protective effect against photoreceptor apoptosis is unique and unexpected, though the underlying mechanisms are still unclear. This study also indicates the need for careful evaluation of data involving subretinal injection of neurotrophic substances or cells accompanied by the presence of RD in rodents with retinal degeneration, particularly rd1 mice. 

 Supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (B16390497, B18390466, C19592013) and a Grant-in Aid from the Ministry of Health, Labor, and Welfare of Japan, Tokyo, Japan.

 Submitted for publication June 14, 2007; revised August 21 and October 23, 2007; accepted December 14, 2007.

 Disclosure: H. Kaneko, None; K.M. Nishiguchi, None; M. Nakamura, None; S. Kachi, None; H. Terasaki, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Creating RD in rd1 mice eyes. (A) After perforating the nasal globe, the needle was advanced through the vitreous cavity and into the temporal retina. The retina was detached from the underlying RPE by pulling away the needle, with its bent tip stuck in or under the retina. Then the needle tip was retracted into the nasal subretinal space, where it was used to push the nasal retina away from the RPE to enlarge RD. (B) The distance from the corneal limbus to the retinal margin was 120 ± 13.6 μm (n = 6). The retina, roughly 0.5 to 1 mm posterior to the corneal limbus, was perforated with a needle while avoiding the ciliary body in the control non-RD eyes. (C) The retinal flatmount of P30 rd1 eyes without RD showing an example of a perforation site. CB, ciliary body. Scale bar, 200 μm.

Figure 2.

 

Photoreceptors are preserved in the retinas of rd1 mice with RD. (A–D) Increased numbers of rhodopsin-positive rod photoreceptors (green; arrowhead) were found in the rd1 eyes with (A, B) and without (C, D) RD at P30. (B) and (D) are magnified views of (A) and (C), respectively. RD had a lesser neuroprotective effect on DAPI-positive (blue) cells, primarily photoreceptors, in the ONL. (E, F) Numbers of DAPI- or rhodopsin-positive cells in the ONL of the eyes with and without RD and of eyes untouched were quantified (mean ± SEM). The number of DAPI-positive cells in the ONL was increased by 1.2-fold in the eyes with RD (P = 3.0 × 10⁻³) compared to those without RD (E). Significant differences were observed when the dorsal retinas were evaluated (P = 3.3 × 10⁻³; E). Similarly, the number of rhodopsin-positive rod photoreceptors was larger in the eyes with RD (P = 3.3 × 10⁻⁴) than in eyes without RD (F). Significant differences were observed when the dorsal and ventral retinas were evaluated separately (P = 7.9 × 10⁻³ and 1.3 × 10⁻², respectively; F). We detected no significant difference in the numbers of DAPI-positive cells or rhodopsin-positive cells in the ONL between the untouched and non-RD eyes from P30 rd1 mice. RD or control ocular injury was induced at P9 in these mice. Scale bars: (A, C) 100 μm; (B, D) 10 μm. *P < 0.05.

Figure 3.

 

Cone photoreceptors are preserved in the retina of rd1 mouse with RD. (A) PNA-positive cone photoreceptor inner/outer segments (green) in flatmounts from P30 rd1 mice. (B) Significantly larger numbers of PNA-positive cone photoreceptors were observed (1.3-fold) in eyes with RD (P = 4.9 × 10⁻⁴) than in those without RD. (C, D) Structures of the M-cone opsin-positive inner/outer segments (green) were relatively preserved, though abnormal (see Supplementary Fig. S1 showing wild-type retina for comparison), in the P30 rd1 eyes with RD (C) compared to those without RD (D). RD or control ocular injury was induced at P9 in these mice. Scale bar, 10 μm (M-cone, M-cone opsin).

Figure 4.

 

Deep retinal vascular plexus is preserved in the retinas of rd1 mice with RD. (A, B) The collagen IV-positive (green) small caliber vessels are preferentially preserved in the flatmounted retinas from P30 rd1 mice with RD (A) compared to those without RD (B). (C–E) Histologic sections from the eyes with RD (C) showed larger numbers of collagen IV-positive (green) vascular lumens in the IPL (P = 3.2 × 10⁻⁴; filled arrowhead) and INL/OPL (P = 7.3 × 10⁻⁴; arrow) in the deep retina compared to those without RD (D), whereas no significant difference (P = 0.08) was observed in superficially located vessels in the NFL/GCL (open arrowhead; E). RD or control ocular injury was induced at P9 in these mice. Scale bar, 50 μm. *P < 0.05.

Figure 5.

 

TUNEL-positive apoptotic photoreceptors were decreased in the rd1 eyes with RD at P15. Quantification of TUNEL-positive cells in the ONL showed reduced photoreceptor apoptosis in the eyes with (P = 0.036) compared to those without RD at P15. RD or control ocular injury was induced at P9. *P < 0.05.

Figure 6.

 

Light deprivation had no effect on photoreceptor degeneration in rd1 eyes with or without RD. (A, B) Increased numbers of DAPI- (A) and rhodopsin-positive (B) photoreceptors in the ONL were identified and compared in eyes with and eyes without RD (P = 8.5 × 10⁻³ and P = 0.02, respectively) in rd1 mice reared in darkness. These results were comparable to those of a similar experiment conducted in rd1 mice reared under a regular light-dark cycle (Fig. 2) . Significant differences were found when the dorsal and ventral retinas were evaluated separately with DAPI (P = 0.01 and P = 0.02, respectively; A) or rhodopsin (P = 0.03 and P = 0.04, respectively; B) staining. (C, D) No significant differences (P = 0.21 and P = 0.88, respectively) in the number of DAPI- (C) or rhodopsin-positive (D) photoreceptors were detected between rd1 mice without RD reared in darkness and under a regular light-dark cycle. No significant differences were found when the dorsal and ventral retinas were evaluated separately with DAPI (P = 0.27 and P = 0.52, respectively; C) or rhodopsin (P = 0.97 and P = 0.78, respectively; D) staining. *P < 0.05.

Supplementary Materials

Supplementary Figure S1 - (PDF)