March 2004
Volume 45, Issue 3
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Retina  |   March 2004
Caspase-3 in Postnatal Retinal Development and Degeneration
Author Affiliations
  • Caroline J. Zeiss
    From the Section of Comparative Medicine, Yale School of Medicine, New Haven, Connecticut.
  • Jason Neal
    From the Section of Comparative Medicine, Yale School of Medicine, New Haven, Connecticut.
  • Elizabeth A. Johnson
    From the Section of Comparative Medicine, Yale School of Medicine, New Haven, Connecticut.
Investigative Ophthalmology & Visual Science March 2004, Vol.45, 964-970. doi:https://doi.org/10.1167/iovs.03-0439
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      Caroline J. Zeiss, Jason Neal, Elizabeth A. Johnson; Caspase-3 in Postnatal Retinal Development and Degeneration. Invest. Ophthalmol. Vis. Sci. 2004;45(3):964-970. https://doi.org/10.1167/iovs.03-0439.

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

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Abstract

purpose. The primary purpose of this study was to evaluate the impact of caspase-3 ablation on photoreceptor degeneration in the rd-1 mouse. Concurrently, the role of caspase-3 in postnatal retinal development was evaluated. Caspase-3 is an important effector caspase that mediates many of the terminal proteolytic events of apoptosis. Its activation has been demonstrated in rodent models of photoreceptor degeneration and its ablation results in exencephaly and neonatal death.

methods. Retinal morphometry was performed at the light microscopic level in caspase-3 mutant mice from PN0 through PN23, and in rd-1/caspase-3 double mutant mice at PN14, -16, and -18. This was supplemented by terminal dUTP transferase nick end labeling (TUNEL) and immunohistochemical staining for activated caspase-3, rhodopsin, factor VII–related antigen and proliferating cell nuclear antigen (PCNA).

results. Caspase-3–deficient animals display marginal microphthalmia, peripapillary retinal dysplasia, delayed regression of vitreal vasculature, and retarded apoptotic kinetics of the inner nuclear layer. Ablation of caspase-3 provided transient photoreceptor protection in rd-1, but TUNEL-positive rod death proceeded, despite the absence of caspase-3 activation.

conclusions. In vivo, caspase-3 is not critical for rod photoreceptor development, nor does it play a significant role in mediating pathologic rod death. Peripapillary dysplastic lesions suggest that there is delayed fusion of the optic fissure, and inner nuclear layer abnormalities indicate a cell-specific dependency on the mitochondria-caspase axis during development. The temporal nature of apoptotic retardation in the absence of caspase-3 implies the presence of caspase-independent mechanisms of developmental and pathologic cell death.

In the retina, caspase-3 is activated in disorders resulting in photoreceptor degeneration. Activation of caspase-3 has been noted in the rd-1 mouse, 1 2 in the rhodopsin mutant rat, 3 in chemically induced models of retinal degeneration, 4 and in photoreceptor degeneration due to exposure to blue light. 5 There is also in vivo evidence that supports the role of caspase-3 in retinal development. Caspase-3 knockout mice display cerebral overgrowth, retinal hyperplasia and a high rate of perinatal death. 6 This suggests that apoptotic mechanisms used during development may be subsequently used to kill postmitotic cells exposed to a pathologic stimulus. 
Despite the importance of caspases, their role in promoting photoreceptor degeneration is unclear. Although many papers describe activation of caspases accompanying retinal degeneration, 1 2 3 4 5 these data do not establish a clear cause and effect relationship between caspase activation and photoreceptor death. Furthermore, in retinal light damage, there is evidence that caspase activation does not occur, 7 and attempts to prevent photoreceptor death in rd-1 mouse by pancaspase inhibitors are marginally successful. 8 Increasing evidence suggests that cell death can be mediated by coexistent caspase-dependent and caspase-independent mechanisms. 9 The relative proportions of these two broad mechanisms is likely to be highly cell and context specific. The phenotypes of mice lacking individual caspase genes reveal distinct temporal and tissue-specific roles of individual apoptotic effectors in animal development. 10 Genetic or pharmacologic elimination of caspase activation fails to prevent cell death, but often changes its morphology from an apoptotic to a necrotic one. 9 11 12 These data suggest that caspase-dependent and caspase-independent mechanisms can coexist and that inhibition of the former can result in cell death due to compensation by the latter. 
The primary purpose of this study was to explore the impact of caspase-3 ablation on photoreceptor death in the rd-1 mouse. Concurrently, the role of caspase-3 in normal postnatal retinal development was examined. These data are used to discuss the possible relationship of apoptotic mechanisms used to drive development and subsequent degeneration of individual retinal cell layers. The rd-1 gene defect results in massive rod death in the first month of life. 13 Caspase-3–deficient mice infrequently survive beyond 1 month of age, making it impossible to study the in vivo effects of caspase-3 ablation in models of slower retinal degeneration. The combined use of the caspase-3 knockout mouse and the rd-1 mouse provide a unique opportunity to explore the role of caspase-3 in both a developmental and neurodegenerative context. 
Methods
Animals
All experiments were performed in accordance with the ARVO Statement for use of Animals in Ophthalmic and Vision Research. All animals were husbanded in accordance with the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care. For developmental studies, heterozygous caspase-3 mutant mice (a gift from Richard Flavell) maintained on a mixed C57BL/6J-129sv strain were bred to generate homozygous mutant mice and wild-type (WT) control littermates. For the study of the effect of caspase-3 genotype on the rd-1 phenotype, mice were generated as follows. F1 heterozygotes were generated by breeding caspase+/− mice on a mixed C57BL/6J-129sv background to C3H/FeJ mice homozygous for the rd-1 mutation. F1 animals were bred back to C3H/FeJ parental stock, maintaining the caspase-3 locus in the heterozygous state for three to four generations. Then, the progeny of these matings (approximately 60%–70% C3H/FeJ derived), heterozygous at the caspase-3 locus and homozygous mutant at the rd-1 locus, were bred to generate animals for analysis. Pups from these matings resembled those of C57BL/6J-129sv caspase-3+/− matings. The expected Mendelian ratios were obtained in each litter at birth. Caspase-3−/− pups were usually identifiable by a slightly domed cranium, and a runtish appearance. These pups would generally live no longer than 4 to 5 weeks, irrespective of whether they were on a C57BL/6J-129sv or C57BL/6J-129sv/C3H background. Animals were genotyped by PCR using previously described protocols for caspase-3 6 and rd-1 (http://www.jax.org; provided in the public domain by Jackson Laboratories, Bar Harbor, ME). Two to four animals per genotype were examined at each time point. 
Tissues
For the developmental series, entire litters were killed by CO2 anesthesia and decapitation at postnatal days (PN)0, -2, -4, -5, -13, -16, -18, and -23. The rd-1/caspase-3 mutants were examined only at PN14, -16, and -18. Entire heads were placed in Bouin’s fixative for 2 days, and sectioned at 5 μm after paraffin embedding. Eyes were sectioned sagittally so that the entire eye, from the ora serrata to the optic nerve, was visible. 
Morphometric Analysis
Sections were examined with a light microscope (Axioplan; Carl Zeiss Meditec, Dublin, CA) and digital imaging camera (Axiocam; Carl Zeiss Meditec). Morphometric analysis was performed with the accompanying software (Axiovision 3.1; Carl Zeiss Meditec). The width of individual retinal layers was calculated at the same location in each retina, 200 μm from the optic nerve and 200 μm from the ora serrata. Width of the optic nerve was measured in sagittal sections. Dying cells in the inner nuclear layer were quantitated by counting the number of pyknotic nuclear profiles in an entire retinal section. To evaluate the numbers of cells in the ganglion cell layer, all neurons in this layer in a 5000-μm2 rectangle adjacent to the optic nerve and a 5000-μm2 rectangle adjacent to the ora serrata were counted. The size of the entire globe was evaluated by taking two measurements across the entire globe at right angles to each other, from the optic nerve to the cornea, and across the eye through the lens. 
TUNEL Labeling
Photoreceptor death in all animals was detected by terminal deoxynucleotide transferase-mediated nick end labeling (TUNEL; Roche Diagnostics, Nutley, NJ), according to the manufacturer’s instructions. 
Immunohistochemistry
Bouin’s-fixed paraffin-embedded sections were examined for immunohistochemical expression of rhodopsin (RET-P1; mouse anti-human rhodopsin monoclonal primary antibody; a gift from Colin Barnstable), Factor-VII–related antigen (DakoUSA, Carpenteria, CA; polyclonal rabbit anti-human Factor VII–related antigen, catalog no. N1505), PCNA (polyclonal rabbit anti-human PCNA, catalog no. U7032; DakoUSA, Carpinteria, CA), and the cleaved subunit (p17) of caspase-3 (polyclonal rabbit anti-human caspase-3, catalog no. AF835; R&D Systems, Minneapolis, MN). Commercial antibodies were used according to the manufacturer’s instructions. RET-P1 was used at a concentration of 1:25, and visualized with a biotinylated goat anti-mouse secondary antibody (concentration 1:400). All reactions were visualized with diaminobenzidine (DAB). All secondary reagents were obtained from Vector Laboratories (Burlingame, CA). 
Results
Effect of Absence of Caspase-3
Retinal dysplasia was limited to a 100- to 200-μm region encircling the optic nerve. In its least-severe manifestation, dysplasia consisted only of the absence of the retinal pigment epithelium (Fig. 1B , arrows). More commonly, however, the opposing neuroretina, was whorled and folded, although it retained normal retinal layering (Figs. 1C 1E) . This finding was noted in animals of all ages. By 1 month of age, peripapillary lesions had shrunken, and although residual dysplasia was noted, lesions were relatively inconspicuous. In some cases, neuroretinal tissue was present in the choroid, most commonly around the optic nerve (Fig. 1F) , or within the intraretinal portion of the optic nerve (Fig. 1G) , often accompanied by melanocytes. Cells within ectopic neuroretina had features of differentiated retinal neurons. Neurons with nuclei characteristic of differentiated rods (two to three large clumps of heterochromatin) were most easily recognized (Fig. 1G) but cells with larger, euchromatic nuclei characteristic of inner retinal neurons could also be identified. In many cases, retinal neurons were arranged in laminar fashion reminiscent of inner and outer nuclear layering present in the normal retina. 
The width and morphology of the extraretinal portion of the optic nerve in caspase-3−/− mice was comparable to that of WT mice, with the exception that in caspase-3−/− mice there was often mislocalization of retinal neurons along the sheath of the optic nerve (Fig. 1I , star). This finding was never noted in WT animals. 
Caspase-3−/− eyes were consistently 85% to 90% smaller than heterozygous or WT counterparts at all ages examined (Fig. 2B) . Temporary intraocular vascular abnormalities were also present. Like posterior retinal dysplasia, these varied in severity between individual caspase-3−/− mice. In the most severe cases, regression of the vasa hyaloidea propria (Fig. 2B , arrow), tunica vasculosa lentis and inner retinal vasculature was slightly delayed. 
The pupillary membrane appeared unaffected. The vascular discrepancy between caspase-3−/− and WT animals was most noticeable at PN13. By PN18, vitreal and retinal vasculature was comparable between both genotypes, although focal thickening of the nerve fiber layer by vascular profiles was still present (Figs. 3D 3F ; stars). 
There were no significant differences in cell number or apoptotic kinetics within the ganglion cell layer, or in the width of the nerve fiber and plexiform layers between caspase-3−/− and WT mice at any age. Apoptotic cells were identified by their pyknotic nuclei at the light microscopic level and by positive TUNEL staining. Apart from peripapillary dysplasia, retinal morphology was indistinguishable between WT and caspase-3−/− retinas at PN0 (Figs. 3A 3B) . As in WT mice, apoptotic nuclei were noted in the ganglion cell layer of caspase-3−/− mice in the first week after birth (Fig. 3B , arrows). Abnormal apoptotic kinetics were limited to the inner nuclear layer and were first noted at PN5. Compared with WT mice, in which the thickness of inner and outer nuclear layers was equivalent from PN5 onward (Fig. 3C) , in caspase-3−/− mice, the inner nuclear layer at PN5 was slightly thickened and nuclei in the outermost region exhibited heterochromia reminiscent of rod nuclear morphology (Fig. 3D) . This phenomenon was still present, but was less noticeable by PN13 and resolved at later time points (Figs. 3F 4A) . In WT retina at PN5, pyknotic TUNEL-positive nuclei were present in the inner nuclear layer (Fig. 3C) . The number of apoptotic nuclei decreased markedly over the next week, and by PN18, only isolated dying cells were noted (Fig. 3E 4A) . In contrast, the inner nuclear layer of caspase-3−/− retina contained fewer apoptotic nuclei than WT retina at PN5 (Figs. 3D 4A) . However, the number of apoptotic nuclei increased over the next week and then declined gradually over the next 2 weeks (Fig. 4A) . By PN16–18, a moderate number of apoptotic nuclei were still present in the inner nuclear layer (Figs. 3F 6F) . These were absent by PN23 (data not shown). Therefore, the kinetics of inner nuclear layer apoptosis in the caspase-3−/− mouse replicated that of WT, but was delayed by 7 to 10 days. Retinal morphology in caspase-3 heterozygous mice was indistinguishable from that of WT mice. 
Immunohistochemical staining for the cleaved portion (p17 subunit) of caspase-3 in WT mice was present in the ganglion cell layer (Fig. 5A) , in the neuroblastic layer of mice younger than 5 days (Fig. 5C) , and in the inner nuclear layer (Fig. 5E) , but was rarely seen in the outer nuclear layer. Similar staining was absent in all retinal layers of the caspase-3−/− animals, despite the presence of dying cells (Figs. 3B 3D 3F 5B 5D 5F) . PCNA labeling failed to reveal any differences in the distribution of PCNA-positive neuroblasts from PN0 through PN18 (data not shown). Rhodopsin-positive photoreceptor cell bodies were identified from PN5 onward and were limited primarily to the outer nuclear layer in both caspase-3−/− and WT mice. They were also seen in dysplastic peripapillary retinal tissue located in the choroid (data not shown). Although individual rhodopsin-positive cells were noted in the inner nuclear layer of caspase-3−/− mice, these were also present in WT littermates (Fig. 5G 5H)
Effect of Caspase-3 Ablation in rd-1 Mice
Caspase-3−/− rd-1 double mutant mice were examined to assess the effects of caspase-3 ablation on the progression of photoreceptor degeneration. Developmental abnormalities (retarded inner nuclear layer apoptosis, and peripapillary dysplasia) were identical irrespective of strain background (C57BL/6J-129sv or mixed C57BL/6J-129sv/C3H/FeJ). From PN14 through PN18, the rd-1 mutation resulted in loss of 60% to 90% of the rod population in mice that were WT at the caspase-3 locus. Cells immunopositive for activated caspase-3 were present in the ONL, but in far fewer numbers than those that were TUNEL positive. In rd-1 mice lacking caspase-3 (Fig. 6C 6D) , the outer nuclear layer was slightly thicker than in rd-1/caspase-3+/+ animals (Fig. 6A 6B) . More neurons with typical rod nuclear morphology were present in rd-1 mice lacking caspase-3 (Figs. 6A 6C ; vertical gray arrow), and inner segments were more evident. Nuclear morphology of dying cells (dense, pyknotic nuclei) was unchanged whether caspase-3 was present or not (Figs. 6A 6C ; thin black arrow). Preservation of the outer nuclear layer in caspase-3−/− rd-1 mice was most obvious in the central retina (data not shown). Nevertheless, photoreceptor death by apoptosis continued in rd-1 mice, despite elimination of caspase-3 activity (Figs. 6D 6F) . These results indicate that in the absence of caspase-3, the rd-1 mutation still kills photoreceptors in the central-to-peripheral gradient typical of the disease; however, cell death occurs at a slightly slower rate. The peripapillary retinal dysplasia and persistent inner nuclear layer apoptosis (Fig. 6D) typifying caspase-3 deficiency were also present in the double-mutant animals. In both INL and ONL, TUNEL-staining identified fragmented DNA in the pyknotic nuclei of dying cells. 
Discussion
The peripapillary dysplasia seen in caspase-3–deficient retinas is typical of those disorders resulting from delayed fusion of the posterior portion of the optic fissure. 14 Peripapillary absence of perioptic retinal pigment epithelium results in folding and whorling of the opposing retina, because normal development of the neuroretina requires contact with retinal pigment epithelium. 15 This lesion was originally reported as retinal hyperplasia. 6  
Choroidal localization of differentiated neuroretina is limited to the region surrounding the optic nerve and can be present from birth. Regression of posterior vascular network in caspase-3−/− eyes is temporarily delayed by approximately 7 to 10 days, but eventually occurs resulting in normal retinal vascularization. These findings suggest localized delay of developmental mechanisms directing closure of the posterior fetal fissure, resulting in retinal dysplasia and microphthalmia which persist into adulthood. Delayed vitreal vascular regression resolves completely. In the mouse, many TUNEL-positive cells are present at the lips of the embryonic fissure on embryonic days 10, 11, and 12. 16 Very few are present in the corresponding area on embryonic day 13 after the complete closure of the embryonic fissure, indicating a transient apoptotic requirement for formation and closure of the optic fissure. A temporal delay in apoptosis resulting from caspase-3 deficiency could result in delayed closure of the optic fissure to produce a posterior coloboma. 
Microphthalmia and posterior coloboma occur in other murine mutants with delayed closure of the optic fissure. These include microphthalmia (mi), 17 ocular retardation (or), 18 the mouse homologue of hairy and enhancer of split homologue (HES-1), 19 and the belly spot and tail heterozygote (Bst). 20 All these mutants have more severe and pervasive abnormalities of neuroretinal development than the caspase-3 mutant mouse. Mutation of the retinal homeobox gene Chx10 in the or mouse impedes proliferation of retinal precursors resulting in a thin hypocellular retina and optic nerve aplasia, 18 and mutant alleles of the Mitf transcription factor (mi) 17 result in neural transdifferentiation of the retinal pigment epithelium. In HES-1–null mutant mice, retinal progenitor differentiation is accelerated, resulting in premature generation of rod and horizontal cells to form rosettelike structures and eventual loss of all bipolar cells. 19 Lens and cornea development is also disturbed. The autosomal semidominant mutation Bst results in ganglion cell loss and optic nerve hypoplasia, 20 features not apparent in the caspase-3−/− mutant. 
In humans, abnormal closure of the posterior fetal fissure results in a complex of colobomatous disorders including optic nerve pit and morning glory syndrome. 14 They are characterized by folding of the perioptic retina and posterior coloboma involving retinal pigment epithelium, sclera, or choroid. Subretinal neovascularization can be associated with retinochoroidal colobomas, 21 22 suggesting that vascular penetration of the choroid will occur if the subretinal space is unprotected by intact RPE. This the most likely cause of abnormal localization of the neuroretina in the choroidal stroma in caspase-3−/− mice, but has not been described in humans. 
Several mouse mutants have both molecular and histopathologic relevance to the inner nuclear layer abnormalities displayed by the caspase-3−/− mutant. The group of animals whose phenotype most closely resembles that of the caspase-3−/− mouse are those with genetic alterations in the Bcl-2 superfamily of pro- and antiapoptotic proteins. 23 24 25 These proteins localize to the mitochondria, and interact stoichiometrically to promote both pro- and antiapoptotic downstream events. They function upstream of the effector caspases. Deficiency of the proapoptotic protein Bax results in reduced apoptosis of ganglion cells and inner retinal neurons, resulting in a thicker inner retina (inner nuclear layer, inner plexiform layer, and ganglion cell layer) and resultant thickened optic nerve. 23 These changes are reminiscent of those seen in mice overexpressing Bcl-2, 24 an antiapoptotic protein. In this model, reduced developmental apoptosis results in retention of a greater number of rod bipolar cells, horizontal cells, dopaminergic amacrine cells, and ganglion cells (but not cholinergic amacrine cells) indicating that these populations are protected by Bcl-2 overexpression. In Bax−/− Bak−/− double-mutant mice, similar thickening of inner nuclear and ganglion cell layers is noted. In addition, the hyaloid vascular system also exhibits incomplete regression. 25 In addition, rod photoreceptor cells, which normally die during development during their migration through the inner nuclear layer, survive in Bax/Bak double-mutant mice. 25 This phenomenon does not occur in caspase-3−/− retinas. In none of the animals with alterations in Bcl-2, Bax, or Bak are the peripapillary dysplastic lesions that characterize caspase-3 deficiency noted. 
These results suggest highly cell-specific roles for individual apoptotic effectors in retinal development. In contrast to the Bax/Bak/Bcl-2 group of mutants, in which reduced neuronal apoptosis results in persistent thickening of both inner retinal layers, caspase-3−/− mice display normal morphology of the optic nerve, ganglion cell layer, and nerve fiber layers. Furthermore, abnormalities of the inner nuclear layer are temporary. Developmental apoptosis of the inner nuclear layer is retarded by approximately 2 weeks, but still occurs so that the retina assumes normal architecture. This implies that caspase-3 is only one of several downstream molecules that mediate the proapoptotic effects of Bax and Bak. Caspase-3 appears to be completely dispensable in the ganglion cell layer and partially redundant in the inner nuclear layer. The persistence of TUNEL-positive cells in the inner retina of caspase-3−/− mice implies that apoptotic mechanisms, as measured by this indicator of DNA fragmentation, are still intact. This implies that apoptosis is delayed by caspase-3 deficiency, but that alternate mechanisms prevail to allow resumption of normal development. The identity of those inner nuclear neurons in which apoptosis is delayed in caspase-3−/− mice is unknown. They may represent the spectrum of neurons in which death is reduced in Bax mutants, or they may be rod precursors (which are postmitotic but do not yet express rhodopsin) destined to migrate from the inner nuclear layer to the outer nuclear layer after formation of the outer plexiform layer at PN5. The latter possibility may explain the increased thickness of the inner nuclear layer compared with the outer nuclear layer at PN5 and why this difference resolves over time. 
Rods that normally die as they migrate through the inner nuclear layer still do so in the absence of either Bax or caspase-3, but survive when both Bak and Bax are eliminated. 23 25 On the contrary, inner retinal neurons, require Bax 23 and to a lesser extent, caspase-3 for developmental apoptosis. These data indicate that developmental rod death, although dependent on the mitochondria–caspase axis, requires fewer of its constituent components than inner retinal neurons. This is supported by evidence indicating that cell-specific caspase expression occurs in retinal development 26 and in retinas exposed to ischemia–reperfusion injury. 27 28 Alternatively, the preferential effects of caspase-3 deletion on the inner nuclear layer may simply reflect different degrees of developmental cell death in rod versus inner retinal neurons. Young 29 describes loss of rod precursors during their migration from inner to outer retinal layers after development of the outer plexiform layer and sporadic rod death in the outer nuclear layer after this stage. In a study in developing chick retina, 30 rod apoptosis is minimal compared with apoptotic death of developing neurons in the inner nuclear and ganglion cell layers. 
Activation of caspase-3 is reported in photoreceptors of the rd-1 mouse 1 2 and was also demonstrated in this study. Both presence 5 and absence 7 of caspase-3 activation are described in light-induced retinal degeneration. In vitro, the functional importance of caspase-3 is demonstrated by the ability of the caspase-3 inhibitor DEVD-fmk to prevent photoreceptor death. 31 However, in this study, we demonstrate that genetic elimination of caspase-3 provides minimal and transient photoreceptor protection in rd-1. This is consistent with results of pharmacologic inhibition of caspase-3 in rd-1. 8  
Therefore, it appears that different neuronal subpopulations in the developing and degenerating retina use different and distinct proapoptotic pathways. Bax deficiency also fails to protect photoreceptors in rd-1 mice, 23 despite (like caspase-3 deficiency) its impact on inner retinal development. The failure of Bax and caspase-3 deficiency to protect rods in rd-1 indicates that these two components of the mitochondria-dependent apoptotic pathway are not necessary to mediate the effects of a degenerative stimulus. Donovan and Cotter 7 have demonstrated that retinal maturity is associated with reduced Apaf-1 levels and reduced caspase activation through the mitochondrial pathway. These data suggest that mechanisms not used during development of a particular cell type are also not available during its subsequent degeneration. Nevertheless, if transgenic expression of mitochondria-associated antiapoptotic proteins such as Bcl-2 and BAG-1 are directed to photoreceptors using an opsin promotor, retardation of photoreceptor degeneration can be achieved. 32 33 These data suggest that components of the mitochondrial axis are involved in photoreceptor death and that the neither caspase-3 nor Bax ablation completely blocks the mitochondrial axis. 
Compensatory activation of caspases-6 and -7 in the absence of caspase-3 34 is unlikely to play a significant role in rd-1/caspase-3−/− mice, because pancaspase inhibition fails to protect photoreceptors in rd-1. 35 Alternate caspase-independent mechanisms of photoreceptor death are likely to contribute to retinal degeneration in rd-1 mice. Photoreceptor loss in rd-1 is accompanied by marked intracellular influx of Ca2+. 36 Elevated cytoplasmic (and consequently mitochondrial) Ca2+ accumulation results in mitochondrial permeabilization. 37 Uncoupling of mitochondrial oxidative phosphorylation is an inevitable consequence of loss of mitochondrial membrane potential. Release of cytochrome c disrupts electron transport, resulting in a decrease in production of ATP and generation of reactive oxygen species, both of which can contribute to cell death in the absence of caspases. In addition, as the apoptosome is dependent on ATP for its proapoptotic activity, progressive energy depletion would result in a greater proportion of cells being committed to necrotic, rather than apoptotic, death. 38 As TUNEL staining can be seen in both apoptotic and necrotic nuclei, it is possible that photoreceptor death in rd-1 has a necrotic component. Apoptosis-inducing factor is released after increased mitochondrial permeability and can induce caspase-independent cell death with apoptotic morphology. 39 Additional cell death pathways can occur through activation of noncaspase proteases such as cathepsin B and calpain. 37 40 Light-induced rod death can occur through calpain activation 7 and can be prevented by the calcium channel blocker d-cis-diltiazem. However, in three of four reports of studies in which this agent was used in animal models of inherited retinal degeneration, it failed to prevent photoreceptor death. 40 41 42 43 44  
In conclusion, we have demonstrated that despite evidence of caspase-3 activation in numerous models of retinal degeneration, this mechanism is not required for rod death in one in vivo model, the rd-1 mouse. Similarly, elimination of caspase-3 also fails to alter development of the outer nuclear and ganglion cell layers and only transiently retards development of the inner nuclear layer. The relative immunity of rods compared with inner retinal neurons to the developmental aberrations induced by caspase-3 deficiency may be a reflection of differential apoptotic kinetics in these cell populations. Alternatively, it supports the notion that both developmental and pathologic cell death pathways are cell-type–specific and temporally restricted and can occur through caspase-dependent and caspase-independent means. 
 
Figure 1.
 
Posterior retinal morphology in WT (A, D, H) and caspase-3−/− (B, C, EG, I) mice. (AC) WT (A) and caspase-3−/− (B, C) mice at PN0. In caspase-3−/− mice, retinal pigment epithelium was absent in a 100- to 200-μm region encircling the optic nerve (compare arrows in A and B). In more severe cases, the posterior retina was folded and elevated at the optic nerve head (C). (D) WT mouse, PN18 showed a normal posterior retina. (EG) Caspase-3−/− mice, PN18. Posterior absence of the retinal pigment epithelium was accompanied by folding of adjacent neuroretina, with thickening and distortion of the intraretinal portion of the optic nerve (E). In some cases, the neuroretina penetrated the choroid (F, vertical arrows), usually in the region of the optic nerve (F, star). The intraretinal portion of the optic nerve was often thickened with dysplastic neurons, which in this case, displayed a nuclear phenotype characteristic of rod photoreceptor cells (G, horizontal arrow). (H) WT mouse, PN16. Optic nerve head. (I) Caspase-3−/− mouse, PN16. The extraretinal optic nerve contained retinal neurons within its sheath (star), but was otherwise comparable to its WT counterpart (H). Bar: 100 μm (AE); 50 μm (F); 15 μm (G); 20 μm (H, I).
Figure 1.
 
Posterior retinal morphology in WT (A, D, H) and caspase-3−/− (B, C, EG, I) mice. (AC) WT (A) and caspase-3−/− (B, C) mice at PN0. In caspase-3−/− mice, retinal pigment epithelium was absent in a 100- to 200-μm region encircling the optic nerve (compare arrows in A and B). In more severe cases, the posterior retina was folded and elevated at the optic nerve head (C). (D) WT mouse, PN18 showed a normal posterior retina. (EG) Caspase-3−/− mice, PN18. Posterior absence of the retinal pigment epithelium was accompanied by folding of adjacent neuroretina, with thickening and distortion of the intraretinal portion of the optic nerve (E). In some cases, the neuroretina penetrated the choroid (F, vertical arrows), usually in the region of the optic nerve (F, star). The intraretinal portion of the optic nerve was often thickened with dysplastic neurons, which in this case, displayed a nuclear phenotype characteristic of rod photoreceptor cells (G, horizontal arrow). (H) WT mouse, PN16. Optic nerve head. (I) Caspase-3−/− mouse, PN16. The extraretinal optic nerve contained retinal neurons within its sheath (star), but was otherwise comparable to its WT counterpart (H). Bar: 100 μm (AE); 50 μm (F); 15 μm (G); 20 μm (H, I).
Figure 2.
 
Ocular morphology in WT and caspase-3−/− mice at PN13. (A) WT mouse, PN13. Hyaloid vasculature was inconspicuous. (B) Caspase-3−/− mouse at PN13. Persistent hyaloid vasculature was present (arrow). Subtle micro-ophthalmia was also evident.
Figure 2.
 
Ocular morphology in WT and caspase-3−/− mice at PN13. (A) WT mouse, PN13. Hyaloid vasculature was inconspicuous. (B) Caspase-3−/− mouse at PN13. Persistent hyaloid vasculature was present (arrow). Subtle micro-ophthalmia was also evident.
Figure 3.
 
Retinal morphology in WT (A, C, E) and caspase-3−/− mice (B, D, F) at PN0, -5, and -16. Retinal morphology at birth was comparable in WT (A) and caspase-3 (B) littermates. Pyknotic dying cells were noted in the GCL of both WT (not shown) and caspase-3−/− mutant (B, arrows). WT mice at PN5 (C) displayed numerous pyknotic nuclei in the inner nuclear layer (arrows). In contrast, the caspase-3–deficient retina at PN5 had few dying neurons in the inner nuclear layer (D, arrow). In addition, the inner nuclear layer was slightly thicker and the outer nuclear layer slightly thinner than in the age-matched WT counterpart. Vasculature of the nerve fiber layer was more prominent in the caspase-3−/− retina (D, star). Width of the inner and outer nuclear layers was comparable in WT (E) and caspase-3−/− mice (F) by PN18. However, in the caspase-3 mutant at PN18, apoptotic nuclei persisted in the inner nuclear layer (F, arrows) and multifocal thickened vasculature persisted in the nerve fiber layer (F, star). Bar, 15 μm.
Figure 3.
 
Retinal morphology in WT (A, C, E) and caspase-3−/− mice (B, D, F) at PN0, -5, and -16. Retinal morphology at birth was comparable in WT (A) and caspase-3 (B) littermates. Pyknotic dying cells were noted in the GCL of both WT (not shown) and caspase-3−/− mutant (B, arrows). WT mice at PN5 (C) displayed numerous pyknotic nuclei in the inner nuclear layer (arrows). In contrast, the caspase-3–deficient retina at PN5 had few dying neurons in the inner nuclear layer (D, arrow). In addition, the inner nuclear layer was slightly thicker and the outer nuclear layer slightly thinner than in the age-matched WT counterpart. Vasculature of the nerve fiber layer was more prominent in the caspase-3−/− retina (D, star). Width of the inner and outer nuclear layers was comparable in WT (E) and caspase-3−/− mice (F) by PN18. However, in the caspase-3 mutant at PN18, apoptotic nuclei persisted in the inner nuclear layer (F, arrows) and multifocal thickened vasculature persisted in the nerve fiber layer (F, star). Bar, 15 μm.
Figure 4.
 
Apoptosis and development of the inner nuclear layer in WT and caspase-3−/− mice. Data were compiled from measurements of six to eight eyes of each genotype at PN5, -13, and -16 and three to six eyes at PN18. (A) Apoptotic kinetics of the inner nuclear layer in WT and caspase-3−/− mice from PN5 through PN18. In WT retina at PN5, pyknotic nuclei were prominent in the inner nuclear layer. The number of apoptotic nuclei decreased markedly over the next week, and by PN18, only isolated dying cells were noted. In contrast, the inner nuclear layer of caspase-3−/− retina contained fewer apoptotic nuclei than WT retina at PN5. However, the number of apoptotic nuclei increased over the next week, and then declined gradually over the next 2 weeks. (B) Width of the inner nuclear layer in WT and caspase-3−/− mice from PN5 through PN18. In contrast, in the caspase-3 mutant animal, INL width was thicker at PN5, but approximated normal width with maturity.
Figure 4.
 
Apoptosis and development of the inner nuclear layer in WT and caspase-3−/− mice. Data were compiled from measurements of six to eight eyes of each genotype at PN5, -13, and -16 and three to six eyes at PN18. (A) Apoptotic kinetics of the inner nuclear layer in WT and caspase-3−/− mice from PN5 through PN18. In WT retina at PN5, pyknotic nuclei were prominent in the inner nuclear layer. The number of apoptotic nuclei decreased markedly over the next week, and by PN18, only isolated dying cells were noted. In contrast, the inner nuclear layer of caspase-3−/− retina contained fewer apoptotic nuclei than WT retina at PN5. However, the number of apoptotic nuclei increased over the next week, and then declined gradually over the next 2 weeks. (B) Width of the inner nuclear layer in WT and caspase-3−/− mice from PN5 through PN18. In contrast, in the caspase-3 mutant animal, INL width was thicker at PN5, but approximated normal width with maturity.
Figure 5.
 
Retinas from WT (A, C, E, G) and caspase-3−/− (B, D, F, H) mice immunostained for the p17 subunit of caspase-3 (AF) and rhodopsin (G, H). (A, B) PN0. Active caspase-3 immunopositivity was noted in the ganglion cell layer in WT (A) but not in caspase-3−/− retinas (B). (C, D) PN4: neuroblasts of WT retina were immunopositive for caspase-3 (C, arrows). No staining was noted in caspase-3 retina (D). (E, F) PN13: inner nuclear layer neurons displayed caspase-3 immunoreactivity in WT mice (E, arrows), but not in caspase-3 mice, despite the presence of dying cells (F, arrows). (G, H) PN16: rhodopsin immunostaining. Occasional ectopic rods were noted in the INL of both WT (G) and caspase-3−/− (H) retinas. Bar: (AF) 10 μm; (G, H) 15 μm.
Figure 5.
 
Retinas from WT (A, C, E, G) and caspase-3−/− (B, D, F, H) mice immunostained for the p17 subunit of caspase-3 (AF) and rhodopsin (G, H). (A, B) PN0. Active caspase-3 immunopositivity was noted in the ganglion cell layer in WT (A) but not in caspase-3−/− retinas (B). (C, D) PN4: neuroblasts of WT retina were immunopositive for caspase-3 (C, arrows). No staining was noted in caspase-3 retina (D). (E, F) PN13: inner nuclear layer neurons displayed caspase-3 immunoreactivity in WT mice (E, arrows), but not in caspase-3 mice, despite the presence of dying cells (F, arrows). (G, H) PN16: rhodopsin immunostaining. Occasional ectopic rods were noted in the INL of both WT (G) and caspase-3−/− (H) retinas. Bar: (AF) 10 μm; (G, H) 15 μm.
Figure 6.
 
Retinal morphology, TUNEL staining, and immunohistochemistry for activated caspase-3 at PN18 in rd-1 mice with (A, B, E, G) and without (C, D, F, H) caspase-3. (AD) Retinal morphology: in rd-1 mice lacking caspase-3 (C, D), the outer nuclear layer was slightly thicker than in rd-1/caspase-3+/+ animals (A, B). Morphology of pyknotic nuclei was comparable between both genotypes (A, C, thin black arrow); however, more cells with typical rod photoreceptor nuclear features were present in rd-1 mice lacking caspase-3 (A, C, vertical gray arrow). (E, F) TUNEL staining: positive TUNEL staining was present in rd-1, whether caspase-3 was present (E) or not (F). The persistent inner nuclear layer apoptosis typifying caspase-3 deficiency was also present in the double-mutant animals (D, F, arrows). (G, H) Rod death in rd-1 mice with an intact caspase-3 gene was accompanied by caspase-3 activation (G, arrows). (H) No staining was noted in rd-1 mice with caspase-3. Bar: (A, C) 5 μm; (B, D, EH) 15 μm.
Figure 6.
 
Retinal morphology, TUNEL staining, and immunohistochemistry for activated caspase-3 at PN18 in rd-1 mice with (A, B, E, G) and without (C, D, F, H) caspase-3. (AD) Retinal morphology: in rd-1 mice lacking caspase-3 (C, D), the outer nuclear layer was slightly thicker than in rd-1/caspase-3+/+ animals (A, B). Morphology of pyknotic nuclei was comparable between both genotypes (A, C, thin black arrow); however, more cells with typical rod photoreceptor nuclear features were present in rd-1 mice lacking caspase-3 (A, C, vertical gray arrow). (E, F) TUNEL staining: positive TUNEL staining was present in rd-1, whether caspase-3 was present (E) or not (F). The persistent inner nuclear layer apoptosis typifying caspase-3 deficiency was also present in the double-mutant animals (D, F, arrows). (G, H) Rod death in rd-1 mice with an intact caspase-3 gene was accompanied by caspase-3 activation (G, arrows). (H) No staining was noted in rd-1 mice with caspase-3. Bar: (A, C) 5 μm; (B, D, EH) 15 μm.
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Figure 1.
 
Posterior retinal morphology in WT (A, D, H) and caspase-3−/− (B, C, EG, I) mice. (AC) WT (A) and caspase-3−/− (B, C) mice at PN0. In caspase-3−/− mice, retinal pigment epithelium was absent in a 100- to 200-μm region encircling the optic nerve (compare arrows in A and B). In more severe cases, the posterior retina was folded and elevated at the optic nerve head (C). (D) WT mouse, PN18 showed a normal posterior retina. (EG) Caspase-3−/− mice, PN18. Posterior absence of the retinal pigment epithelium was accompanied by folding of adjacent neuroretina, with thickening and distortion of the intraretinal portion of the optic nerve (E). In some cases, the neuroretina penetrated the choroid (F, vertical arrows), usually in the region of the optic nerve (F, star). The intraretinal portion of the optic nerve was often thickened with dysplastic neurons, which in this case, displayed a nuclear phenotype characteristic of rod photoreceptor cells (G, horizontal arrow). (H) WT mouse, PN16. Optic nerve head. (I) Caspase-3−/− mouse, PN16. The extraretinal optic nerve contained retinal neurons within its sheath (star), but was otherwise comparable to its WT counterpart (H). Bar: 100 μm (AE); 50 μm (F); 15 μm (G); 20 μm (H, I).
Figure 1.
 
Posterior retinal morphology in WT (A, D, H) and caspase-3−/− (B, C, EG, I) mice. (AC) WT (A) and caspase-3−/− (B, C) mice at PN0. In caspase-3−/− mice, retinal pigment epithelium was absent in a 100- to 200-μm region encircling the optic nerve (compare arrows in A and B). In more severe cases, the posterior retina was folded and elevated at the optic nerve head (C). (D) WT mouse, PN18 showed a normal posterior retina. (EG) Caspase-3−/− mice, PN18. Posterior absence of the retinal pigment epithelium was accompanied by folding of adjacent neuroretina, with thickening and distortion of the intraretinal portion of the optic nerve (E). In some cases, the neuroretina penetrated the choroid (F, vertical arrows), usually in the region of the optic nerve (F, star). The intraretinal portion of the optic nerve was often thickened with dysplastic neurons, which in this case, displayed a nuclear phenotype characteristic of rod photoreceptor cells (G, horizontal arrow). (H) WT mouse, PN16. Optic nerve head. (I) Caspase-3−/− mouse, PN16. The extraretinal optic nerve contained retinal neurons within its sheath (star), but was otherwise comparable to its WT counterpart (H). Bar: 100 μm (AE); 50 μm (F); 15 μm (G); 20 μm (H, I).
Figure 2.
 
Ocular morphology in WT and caspase-3−/− mice at PN13. (A) WT mouse, PN13. Hyaloid vasculature was inconspicuous. (B) Caspase-3−/− mouse at PN13. Persistent hyaloid vasculature was present (arrow). Subtle micro-ophthalmia was also evident.
Figure 2.
 
Ocular morphology in WT and caspase-3−/− mice at PN13. (A) WT mouse, PN13. Hyaloid vasculature was inconspicuous. (B) Caspase-3−/− mouse at PN13. Persistent hyaloid vasculature was present (arrow). Subtle micro-ophthalmia was also evident.
Figure 3.
 
Retinal morphology in WT (A, C, E) and caspase-3−/− mice (B, D, F) at PN0, -5, and -16. Retinal morphology at birth was comparable in WT (A) and caspase-3 (B) littermates. Pyknotic dying cells were noted in the GCL of both WT (not shown) and caspase-3−/− mutant (B, arrows). WT mice at PN5 (C) displayed numerous pyknotic nuclei in the inner nuclear layer (arrows). In contrast, the caspase-3–deficient retina at PN5 had few dying neurons in the inner nuclear layer (D, arrow). In addition, the inner nuclear layer was slightly thicker and the outer nuclear layer slightly thinner than in the age-matched WT counterpart. Vasculature of the nerve fiber layer was more prominent in the caspase-3−/− retina (D, star). Width of the inner and outer nuclear layers was comparable in WT (E) and caspase-3−/− mice (F) by PN18. However, in the caspase-3 mutant at PN18, apoptotic nuclei persisted in the inner nuclear layer (F, arrows) and multifocal thickened vasculature persisted in the nerve fiber layer (F, star). Bar, 15 μm.
Figure 3.
 
Retinal morphology in WT (A, C, E) and caspase-3−/− mice (B, D, F) at PN0, -5, and -16. Retinal morphology at birth was comparable in WT (A) and caspase-3 (B) littermates. Pyknotic dying cells were noted in the GCL of both WT (not shown) and caspase-3−/− mutant (B, arrows). WT mice at PN5 (C) displayed numerous pyknotic nuclei in the inner nuclear layer (arrows). In contrast, the caspase-3–deficient retina at PN5 had few dying neurons in the inner nuclear layer (D, arrow). In addition, the inner nuclear layer was slightly thicker and the outer nuclear layer slightly thinner than in the age-matched WT counterpart. Vasculature of the nerve fiber layer was more prominent in the caspase-3−/− retina (D, star). Width of the inner and outer nuclear layers was comparable in WT (E) and caspase-3−/− mice (F) by PN18. However, in the caspase-3 mutant at PN18, apoptotic nuclei persisted in the inner nuclear layer (F, arrows) and multifocal thickened vasculature persisted in the nerve fiber layer (F, star). Bar, 15 μm.
Figure 4.
 
Apoptosis and development of the inner nuclear layer in WT and caspase-3−/− mice. Data were compiled from measurements of six to eight eyes of each genotype at PN5, -13, and -16 and three to six eyes at PN18. (A) Apoptotic kinetics of the inner nuclear layer in WT and caspase-3−/− mice from PN5 through PN18. In WT retina at PN5, pyknotic nuclei were prominent in the inner nuclear layer. The number of apoptotic nuclei decreased markedly over the next week, and by PN18, only isolated dying cells were noted. In contrast, the inner nuclear layer of caspase-3−/− retina contained fewer apoptotic nuclei than WT retina at PN5. However, the number of apoptotic nuclei increased over the next week, and then declined gradually over the next 2 weeks. (B) Width of the inner nuclear layer in WT and caspase-3−/− mice from PN5 through PN18. In contrast, in the caspase-3 mutant animal, INL width was thicker at PN5, but approximated normal width with maturity.
Figure 4.
 
Apoptosis and development of the inner nuclear layer in WT and caspase-3−/− mice. Data were compiled from measurements of six to eight eyes of each genotype at PN5, -13, and -16 and three to six eyes at PN18. (A) Apoptotic kinetics of the inner nuclear layer in WT and caspase-3−/− mice from PN5 through PN18. In WT retina at PN5, pyknotic nuclei were prominent in the inner nuclear layer. The number of apoptotic nuclei decreased markedly over the next week, and by PN18, only isolated dying cells were noted. In contrast, the inner nuclear layer of caspase-3−/− retina contained fewer apoptotic nuclei than WT retina at PN5. However, the number of apoptotic nuclei increased over the next week, and then declined gradually over the next 2 weeks. (B) Width of the inner nuclear layer in WT and caspase-3−/− mice from PN5 through PN18. In contrast, in the caspase-3 mutant animal, INL width was thicker at PN5, but approximated normal width with maturity.
Figure 5.
 
Retinas from WT (A, C, E, G) and caspase-3−/− (B, D, F, H) mice immunostained for the p17 subunit of caspase-3 (AF) and rhodopsin (G, H). (A, B) PN0. Active caspase-3 immunopositivity was noted in the ganglion cell layer in WT (A) but not in caspase-3−/− retinas (B). (C, D) PN4: neuroblasts of WT retina were immunopositive for caspase-3 (C, arrows). No staining was noted in caspase-3 retina (D). (E, F) PN13: inner nuclear layer neurons displayed caspase-3 immunoreactivity in WT mice (E, arrows), but not in caspase-3 mice, despite the presence of dying cells (F, arrows). (G, H) PN16: rhodopsin immunostaining. Occasional ectopic rods were noted in the INL of both WT (G) and caspase-3−/− (H) retinas. Bar: (AF) 10 μm; (G, H) 15 μm.
Figure 5.
 
Retinas from WT (A, C, E, G) and caspase-3−/− (B, D, F, H) mice immunostained for the p17 subunit of caspase-3 (AF) and rhodopsin (G, H). (A, B) PN0. Active caspase-3 immunopositivity was noted in the ganglion cell layer in WT (A) but not in caspase-3−/− retinas (B). (C, D) PN4: neuroblasts of WT retina were immunopositive for caspase-3 (C, arrows). No staining was noted in caspase-3 retina (D). (E, F) PN13: inner nuclear layer neurons displayed caspase-3 immunoreactivity in WT mice (E, arrows), but not in caspase-3 mice, despite the presence of dying cells (F, arrows). (G, H) PN16: rhodopsin immunostaining. Occasional ectopic rods were noted in the INL of both WT (G) and caspase-3−/− (H) retinas. Bar: (AF) 10 μm; (G, H) 15 μm.
Figure 6.
 
Retinal morphology, TUNEL staining, and immunohistochemistry for activated caspase-3 at PN18 in rd-1 mice with (A, B, E, G) and without (C, D, F, H) caspase-3. (AD) Retinal morphology: in rd-1 mice lacking caspase-3 (C, D), the outer nuclear layer was slightly thicker than in rd-1/caspase-3+/+ animals (A, B). Morphology of pyknotic nuclei was comparable between both genotypes (A, C, thin black arrow); however, more cells with typical rod photoreceptor nuclear features were present in rd-1 mice lacking caspase-3 (A, C, vertical gray arrow). (E, F) TUNEL staining: positive TUNEL staining was present in rd-1, whether caspase-3 was present (E) or not (F). The persistent inner nuclear layer apoptosis typifying caspase-3 deficiency was also present in the double-mutant animals (D, F, arrows). (G, H) Rod death in rd-1 mice with an intact caspase-3 gene was accompanied by caspase-3 activation (G, arrows). (H) No staining was noted in rd-1 mice with caspase-3. Bar: (A, C) 5 μm; (B, D, EH) 15 μm.
Figure 6.
 
Retinal morphology, TUNEL staining, and immunohistochemistry for activated caspase-3 at PN18 in rd-1 mice with (A, B, E, G) and without (C, D, F, H) caspase-3. (AD) Retinal morphology: in rd-1 mice lacking caspase-3 (C, D), the outer nuclear layer was slightly thicker than in rd-1/caspase-3+/+ animals (A, B). Morphology of pyknotic nuclei was comparable between both genotypes (A, C, thin black arrow); however, more cells with typical rod photoreceptor nuclear features were present in rd-1 mice lacking caspase-3 (A, C, vertical gray arrow). (E, F) TUNEL staining: positive TUNEL staining was present in rd-1, whether caspase-3 was present (E) or not (F). The persistent inner nuclear layer apoptosis typifying caspase-3 deficiency was also present in the double-mutant animals (D, F, arrows). (G, H) Rod death in rd-1 mice with an intact caspase-3 gene was accompanied by caspase-3 activation (G, arrows). (H) No staining was noted in rd-1 mice with caspase-3. Bar: (A, C) 5 μm; (B, D, EH) 15 μm.
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