Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa.

Ribozymes, catalytic RNA molecules that cleave a complementary mRNA sequence, have potential as therapeutics for dominantly inherited disease. Twelve percent of American patients with the blinding disease autosomal dominant retinitis pigmentosa (ADRP) carry a substitution of histidine for proline at codon 23 (P23H) in their rhodopsin gene, resulting in photoreceptor cell death from the synthesis of the abnormal gene product. Ribozymes can discriminate and catalyze the in vitro destruction of P23H mutant mRNAs from a transgenic rat model of ADRP. Here, we demonstrate that in vivo expression of either a hammerhead or hairpin ribozyme in this rat model considerably slows the rate of photoreceptor degeneration for at least three months. Catalytically inactive control ribozymes had less effect on the retinal degeneration. Intracellular production of ribozymes in photoreceptors was achieved by transduction with a recombinant adeno-associated virus (rAAV) incorporating a rod opsin promoter. Ribozyme-directed cleavage of mutant mRNAs, therefore, may be an effective therapy for ADRP and also may be applicable to other inherited diseases.

Light-evoked changes in the interphotoreceptor matrix.

The normal function of vertebrate photoreceptor cells depends on multiple interactions and transfer of substances between the photoreceptors and the retinal pigment epithelium (RPE), but the mechanisms of these interactions are poorly understood. Many are thought to be mediated by the interphotoreceptor matrix (IPM), a complex extracellular matrix that surrounds the photoreceptors and lies between them and the RPE. Histochemical, immunocytochemical, and lectin probes for several IPM constituents revealed that components of the IPM in the rat undergo a major shift in distribution or molecular conformation after the transition between light and dark. In the light, various IPM constituents concentrated in bands at the apical and basal regions of the outer segment zone; in the dark, they distributed much more uniformly throughout the zone. The change in IPM distribution was triggered by the light-dark transition; it was not a circadian event, and it was not driven by a systemic factor. The light-evoked change in IPM distribution may facilitate the transfer of substances between the photoreceptors and the RPE.

Retinal degeneration in the nervous mutant mouse. I. Light microscopic cytopathology and changes in the interphotoreceptor matrix.

Nervous is an autosomal recessive mutation in mice (gene symbol, nr) that produces a progressive cerebellar and retinal degeneration. We have examined various cytopathological features of the photoreceptor degeneration by light microscopy. An increase in the number of pyknotic photoreceptor nuclei in the outer nuclear layer (ONL) is first seen at postnatal day (P) 11. Between P13 and P19 there is a rapid loss of photoreceptors, with the ONL about 60% the thickness of littermate controls at P19. Between P19 and 2.5 months of age, photoreceptor cell loss is minimal, and there is a relatively slow loss of these cells between 3 and 7.5 months of age. At 7.5 months, the ONL consists of single row of nuclei, most of which are lost over the ensuing months, although a few photoreceptor nuclei persist at 17 months of age and older. Both rods and cones are lost at comparable rates for the first 2 months of life, but rods are somewhat preferentially lost at later ages. A very slight central-to-peripheral gradient of photoreceptor degeneration exists in the nr/nr retina, but no superior-inferior hemispheric differences are evident. The rate, spatiotemporal gradient, and hemispheric similarity in photoreceptor degeneration are the same in albino nr/nr mice reared either in cyclic light or in the dark, and in pigmented nr/nr mice. Autoradiographic analysis of rod outer segment renewal shows that outer segment membranes are synthesized in nervous homozygotes. Rhythmic outer segment disc shedding and phagocytosis by the retinal pigment epithelium occur at approximately normal rates in nr/nr mice. Histochemical and immunocytochemical study of the interphotoreceptor matrix (IPM) reveals the exclusion of stainable IPM from the outer segment zone by lamellar whorls of outer segment membrane, accumulation of stainable IPM in the basal region of the outer segment zone, and the absence of an intense band of stainable IPM at the apical surface of the retinal pigment epithelium. These changes in the IPM are similar to those seen in the Royal College of Surgeons rat. However, comparison of cytopathological changes in these two mutants reveal that the IPM defect probably is not the primary cause of photoreceptor cell death in nr/nr mice, and that similar phenotypic appearance does not necessarily signify similar pathological processes.

Immunogold localization of chondroitin 6-sulfate in the interphotoreceptor matrix of normal and RCS rats.

Localization of chondroitin 6-sulfate (6S) in the interphotoreceptor matrix (IPM) of both normal and RCS rats with inherited retinal dystrophy has been carried out using light and electron microscopic immunogold cytochemistry. In the normal rat, 6S antibody labeling was found in highest concentration at the apical surface of the retinal pigment epithelium (RPE) and between adjacent photoreceptors near their basal inner segment-outer segment junction. In the apical zone, label was localized in the IPM and toward the distal portions of the RPE apical processes. In the basal zone, label was found in the IPM and near the outer plasma membranes of inner and outer segments. Interstitial labeling, between the shafts of outer segments, occurred at much lower concentration than in the apical and basal zones. In all zones, the extent of labeling appeared to be space-dependent; it was most abundant where extracellular spaces were large, and little was present where adjacent cell membranes were contiguous. In the dystrophic rat, apical zone labeling of the RPE apical processes was minimal; instead, the relatively small amount of label that was present was localized primarily in the IPM and within encapsulated spaces of membranous whorls of debris. Label was most concentrated in the basal zone, primarily associated with the IPM within abundant interphotoreceptor spaces, and near the plasma membranes of disorganized inner and outer segments. In isolated eyecups and neural retinas, some labeling persisted after extensive buffer rinses which suggests that chondroitin 6-sulfate is a somewhat insoluble component of the IPM in the rat retina.

Rod- and cone-associated interphotoreceptor matrix in the rat retina. Differences in light-evoked distributional changes.

Several of the components of the interphotoreceptor matrix (IPM) in the rat have recently been shown to undergo a light-evoked shift in distribution or molecular conformation. In the light, the IPM appears concentrated at the apical surface of the retinal pigment epithelium (RPE) and in the basal outer segment region at the inner and outer segment junction, with relatively little present in the intervening interstitial zone adjacent to the outer segments. By contrast, the IPM in the dark is distributed almost uniformly across the outer segment layer. In the present study, the authors explored whether specialized domains of IPM known as the cone matrix sheaths undergo light-induced changes similar to those previously shown for the IPM as a whole. Fluorescence lectin histochemistry was used on retinal sections of light- and dark-adapted rats with lectins that show selective or preferential binding to cone matrix sheaths, peanut agglutinin (PNA), and Ricinus communis agglutinin (RCA-1). Lectin binding to cone matrix sheaths was the same in both lighting conditions, unlike the rod-associated IPM. These experiments and others using pretreatment of sections with neuraminidase suggested different roles of RCA-1 and PNA-binding components of the IPM in different photoreceptor-RPE cell interactions, including the transfer of substances between the two cell types and retinal adhesion.

The interphotoreceptor matrix in rats with inherited retinal dystrophy.

The retinas of Royal College of Surgeons (RCS) rats with inherited retinal dystrophy and of genetic control RCS-rdy+ rats have been examined histochemically to determine whether the stainable interphotoreceptor matrix (IPM) is abnormal in dystrophic retinas. The mucosubstances that are stained with Alcian blue, Metachromatically stained with toluidine blue, or reacted with colloidal iron appear in normal retinas beginning on postnatal day 12 as an intense band of stain at the apical surface of the pigment epithelium and with less intense staining between the outer segments throughout the rest of the outer segment zone. In RCS retinas the distribution of stainable IPM differs from that in normal retinas beginning on day 12. At this time, there is a failure of the intense band of IPM staining to form completely at the apical surface of the pigment epithelium. As whorls of outer segment membranes accumulate due to the phagocytosis defect in RCS pigment epithelial cells, IPM staining almost disappears along the pigment epithelial cell surface and in the debris zone. In addition, the basal outer segment region stains much more heavily in RCS retinas than in normal retinas, a feature that presumably represents an abnormal accumulation of IPM in this region of mutant retinas. Since the abnormal distribution of stainable IPM is evident 6 to 8 days before the first pyknotic photoreceptor cell nuclei are seen, it may play a role in photoreceptor cell death in the RCS rat. Furthermore, since the difference in IPM distribution between mutant and normal retinas is first evident on the same day that disc shedding and phagocytosis begin in the normally developing retina, the abnormal IPM distribution in RCS rats may also be related to the phagocytosis defect in the mutant pigment epithelial cells.

Increased susceptibility to constant light in nr and pcd mice with inherited retinal degenerations.

PURPOSE: To determine whether the degenerating photoreceptors in nervous (nr/nr) and Purkinje cell degeneration (pcd/pcd) mutant mice are more susceptible to the damaging effects of constant light than those in age-matched normal mice. METHODS: Beginning at two ages for each mutant, albino nr/nr and pcd/pcd mice were placed into constant fluorescent light at an illuminance of 115 foot-candles to 130 foot-candles for a period of 1 week. Age-matched (usually littermate) normal (+/-) mice were exposed at the same time. The degree of photoreceptor cell loss was quantified histologically by obtaining a mean outer nuclear layer thickness for each animal. The light-exposed mice were compared with age-matched mutant and normal mice that were maintained in cyclic light. RESULTS: The homozygous mutants at each age showed a significantly greater loss of photoreceptor cells caused by constant light exposure than did the normal +/- mice in the same period of light exposure. The nr/nr and pcd/pcd mutants lost two to three times the number of photoreceptor cells than did the +/- mice during the constant light exposure. CONCLUSIONS: It has long been thought that excessive light may be harmful to patients with inherited or age-related photoreceptor degenerations. The present data add to other experimental evidence suggesting that photoreceptors already undergoing inherited or other forms of degeneration may be particularly susceptible to the damaging effects of excessive light.

Increased susceptibility to light damage in an arrestin knockout mouse model of Oguchi disease (stationary night blindness)

PURPOSE: To determine whether constitutive signal flow arising from defective rhodopsin shut-off causes photoreceptor cell death in arrestin knockout mice. METHODS: The retinas of cyclic-light-reared, pigmented arrestin knockout mice and wild-type littermate control mice were examined histologically for photoreceptor cell loss from 100 days to 1 year of age. In separate experiments, to determine whether constant light would accelerate the degeneration in arrestin knockout mice, these animals and wild-type control mice were exposed for 1, 2, or 3 weeks to fluorescent light at an intensity of 115 to 150 fc. The degree of photoreceptor cell loss was quantified histologically by obtaining a mean outer nuclear layer thickness for each animal. RESULTS: In arrestin knockout mice maintained in cyclic light, photoreceptor loss was evident at 100 days of age, and it became progressively more severe, with less than 50% of photoreceptors surviving at 1 year of age. The photoreceptor degeneration appeared to be caused by light, because when these mice were reared in the dark, the retinal structure was indistinguishable from normal. When exposed to constant light, the retinas of wild-type pigmented mice showed no light-induced damage, regardless of exposure duration. By contrast, the retinas of arrestin knockout mice showed rapid degeneration in constant light, with a loss of 30% of photoreceptors after 1 week of exposure and greater than 60% after 3 weeks of exposure. CONCLUSIONS: The results indicate that constitutive signal flow due to arrestin knockout leads to photoreceptor degeneration. Excessive light accelerates the cell death process in pigmented arrestin knockout mice. Human patients with naturally occurring mutations that lead to nonfunctional arrestin and rhodopsin kinase have Oguchi disease, a form of stationary night blindness. The present findings suggest that such patients may be at greater risk of the damaging effects of light than those with other forms of retinal degeneration, and they provide an impetus to restrict excessive light exposure as a protective measure in patients with constitutive signal flow in phototransduction.

Protection of mouse photoreceptors by survival factors in retinal degenerations.

PURPOSE: To examine the protective effect of a number of survival factors on degenerating photoreceptors in mutant mice with naturally occurring inherited retinal degenerations, including retinal degeneration (rd/rd), retinal degeneration slow (rds/rds), nervous (nr/nr), and Purkinje cell degeneration (pcd/pcd), in three different forms of mutant rhodopsin transgenic mice and in light damage in albino mice. METHODS: Various survival factors were injected intravitreally into one eye of mice at or soon after the beginning of photoreceptor degeneration, with the opposite eye serving as the control, and the eyes were examined histologically at later ages. The survival factors included brain-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin-4, ciliary neurotrophic factor (CNTF), Axokine (a mutein of CNTF), leukemia inhibitory factor, basic fibroblast growth factor, and nerve growth factor and insulin-like growth factor II, either alone or in various combinations. RESULTS: Photoreceptor degeneration was slowed in rd/rd and nr/nr mutant mice and in Q344ter mutant rhodopsin mice by certain forms of CNTF; the degeneration in Q344ter mice was slowed by Axokine and by leukemia inhibitory factor; and the degeneration in a few nr/nr mice was slowed by BDNF. The other agents were ineffective in these mice, and none of the agents were effective in the other mutants and other mutant rhodopsin transgenic mice. However, light damage experiments that compared agent effectiveness in albino mice versus rats suggested a significant delivery problem with the very small mouse eye, thereby making the interpretation of negative findings equivocal in mutant mice. Basic fibroblast growth factor failed to protect the mouse retina from the damaging effects of constant light, whereas it showed a strong protective effect in the rat, indicating an important species difference. CONCLUSIONS: The slowing of degeneration in the rd/rd and Q344ter mutant mice demonstrated that intraocularly injected survival factors can protect photoreceptors from degenerating in animal models with the same or similar genetic defects as those in human inherited retinal degenerations.

Human fatality due to ingestion of the crab Demania reynaudii that contained a palytoxin-like toxin.

Clinical accounts of a human fatality resulting from ingestion of the crab Demania reynaudii are documented. The causative toxin was suggested to be palytoxin on the basis of dose-death time relationships and chromatographic properties.

Lectin binding of the interphotoreceptor matrix during retinal development in normal and RCS rats.

The retinas of both normal and Royal College of Surgeons (RCS) rats with inherited retinal dystrophy have been examined using lectin histochemistry to determine the developmental and degenerative changes of the glycoconjugates in the interphotoreceptor matrix (IPM) between postnatal day (P) 10 and P25, when the adult lectin binding patterns are seen in normal rats. Wheat germ agglutinin (WGA; recognizing sialic acid and/or N-acetyl-D-glucosamine) bound to the apical surface of the retinal pigment epithelium (RPE) sparsely at P10 and prominently at P12 in both strains. In both strains at P14, WGA also stained the basal outer segment zone at the inner segment-outer segment junction. Between P14 and P16 in both strains, there was a dramatic increase in the binding of the interstitial region, the space alongside the outer segments and between the apical and basal outer segment zones. The binding pattern of WGA in normal rats remained basically unchanged from P16 to P25, although the intensity of binding was increased somewhat. Ricinus communis agglutinin-1 (RCA-1; specific for galactosyl residues) bound to the outer segment zone prominently and diffusely with increasing intensity with age at P10, P12 and P14 in both strains. At P16 and older, the intense binding of the interstitial zone was dramatically reduced and the RCA-1 bound primarily to the inner and outer segment junctional region, with weak binding to the apical surface of the RPE in both strains. At P25, the binding of the inner and outer segment junctional region was even more restricted, limited to punctate sites in this zone in normal rats and almost missing in RCS rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Continuous exposure to bright light upregulates bFGF and CNTF expression in the rat retina.

PURPOSE: To examine mRNA expression of neurotrophic factors in the retina after exposure to bright light. METHODS: Male adult Sprague-Dawley rats were exposed to light of 115-130 ft-c. Retinas were collected after 1, 2, 4 or 7 days of exposure. Northern blot analysis was performed to determine mRNA levels for the following factors and their receptors: basic fibroblast growth factor (bFGF), ciliary neurotrophic factor (CNTF), acidic fibroblast growth factor (aFGF), brain-derived neurotrophic factor (BDNF), insulin-like growth factor 1 (IGF-1) and glial fibrillary acidic protein (GFAP). Expression of bFGF, CNTF and GFAP was localized by in situ hybridization. RESULTS: Exposure to light of 115-130 ft-c resulted in a substantial increase in bFGF and CNTF expression that persisted during the entire 7-day period of exposure. The peak expression of bFGF was almost 9-fold at day 2. The increase in CNTF mRNA reached a maximum of 6-fold at day 4. A small increase (50%) in IGF-1 mRNA was also seen at day 4. Among the receptors, an elevation of 3-fold in FGF receptor 1 (FGFR-1) was observed at day 2. There was also a small increase (70%) in IGF-1 receptor (IGF-1R) at day 2. In addition, the expression of GFAP showed a rapid elevation of about 8-fold by day 1 and 9-fold by day 2, and 18-fold by day 4. There was, however, no significant alteration in the expression of aFGF and BDNF. In situ hybridizations showed that the elevation of bFGF, CNTF and GFAP occurred across the entire retina with especially prominent increases over specific layers for each gene. CONCLUSIONS: Continuous exposure to bright light upregulates bFGF, CNTF, FGFR-1 and GFAP expression in the rat retina. The pattern of induced expression closely resembles that induced by mechanical injury, implying a common underlying mechanism.

[Aging effects on the light response of the interphotoreceptor matrix as revealed by binding of Ricinus communis agglutinin-1].

This study intended to explore whether the light response of the interphotoreceptor matrix (IPM) is affected by aging. The binding pattern of fluorescence-labeled Ricinus communis agglutinin-1 (RCA) to IPM was examined histochemically in 2 month-old and 1.5 year-old rats under light- and dark-adapted conditions. Two month-old animals showed obvious light-evoked changes in the rod associated IPM: the photoreceptor inner segment zone showed a greater fluorescence than the outer segment zone in the light, whereas the staining-intensity of the former was less than that of the latter in the dark. On the other hand, 1.5 year-old rats did not show such light-evoked IPM responses as in 2 month-old animals: no light-dark differences were found in RCA-1 binding. The scarce, linear, preferential binding of RCA-1 to the cone-associated IPM was the same in both lighting conditions independent of the age.

[Detection of dual phase of light response of interphotoreceptor matrix].

The postnatal development of light-evoked changes in the interphotoreceptor matrix (IPM), a complex of the extracellular matrix that surrounds the photoreceptors and lies between them and the retinal pigment epithelium, was studied by use of a histochemical probe colloidal iron in rats at various postnatal days of age. In the dark, IPM constituents distributed uniformly throughout the outer segment zone and in the apical region of the inner segment zone; this dark pattern of IPM distribution was commonly observed in animals ranging from postnatal day 12 to one year. In the light, little changes were observed at postnatal day 12, followed by varying light-evoked changes in the IPM with increasing ages. At postnatal day 14, the IPM constituents showed a distribution towards the basal region of the inner segment zone along with the dark pattern. At postnatal day 16, they concentrated in bands at the apical and basal region of the outer segment zone, and also in the inner segment zone with expansion from the apical to basal region with increasing time after light exposure. At age one year, the light-adapted IPM constituents were distributed in a manner similar to that on postnatal day 14. The present results confirm the light-evoked changes in the IPM as revealed by colloidal iron-recognized IPM components and provide evidence for postnatal development of the light-evoked changes in the IPM components, whereby the changes appeared earlier and were preserved longer in the basal region of the inner segment.

[Light response of the interphotoreceptor matrix in inherited degenerative retina].

The light-evoked distributional changes of the interphotoreceptor matrix (IPM) in mice with three types of inherited retinal degeneration were examined by histochemistry using fluorescence isocyanate-labeled wheat germ agglutinin. In mice with nervous and Purkinje cell degeneration, the light response of the IPM was still somewhat preserved during the early stage of photoreceptor degeneration, whereas it became extinct when the outer segments (OS) became moderately or markedly shortened. In mice with slow retinal degeneration mice without development of OS, the light response of the IPM was absent throughout the developmental stages. These findings suggest that the presence of normal OS is necessary for the light response of the IPM to occur.

[Lectin binding in the interphotoreceptor matrix in neuraminidase-induced retinal detachment].

Binding sites of fluorescence isocyanate-labeled lectins, peanut agglutinin (PNA) and Ricinus communis agglutinin-1 (RCA-1), were studied in the interphotoreceptor matrix (IPM) of adult rat retinas that were pretreated with an intravitreous injection of neuroaminidase. The localization of binding sites to the subretinal IPM differed significantly between the two lectins. PNA bound to the subretinal IPM components in a cord-like fashion that formed a bridge between the apical photoreceptor outer segments and the surface of the RPE. On the other hand, RCA-1 showed homogeneously prominent binding to the subretinal space IPM, the intensity of which appeared to inversely correlate with the extent of neuraminidase-induced retinal separation. The results suggest that the IPM may consist of mucin-type glycoconjugates as recognized by PNA and serum-type ones as bound by RCA-1, and that these IPM components may play different roles in the maintenance and organization of photoreceptor-RPE complex, e.g. the mucin-type for the retinal adhesion and the serum-type for the transport of metabolites.

[Effects of fixation and light conditions on distribution of interphotoreceptor matrix].

By using albino, adult rats, the effects of different light conditions during enucleation and fixation on the staining of the interphotoreceptor matrix (IPM) with colloidal iron were examined. When the eyes were enucleated in the dark or under a fluorescent lamp, followed by immersion-fixing in the dark, the IPM around the photoreceptor outer segments (OS) and apical inner segments (IS) was uniformly stained. When the eyes were enucleated in the light, and were immersion-fixed in the light, the staining pattern of the IPM was dependent on the light conditions during the fixation. By increasing the intensity and the exposure-time of the light, the intensity of the interstitial IPM-staining around the OS decreased except for the apical- and basal-regions, whereas that of the IPM staining around the IS increased from the basal- to the apical-region. When the rat was perfused with the fixative in the light, the diffuse IPM staining around the IS was especially remarkable. In cases in which it is only possible to apply immersion-fixation, it is necessary to remove the light effects during the fixation in order to obtain consistent results with IPM-histochemistry. For this purpose, it may be effective to cover the bottle for fixation with aluminum foil immediately after the enucleation.

[Classification of light-evoked distribution of interphotoreceptor matrix].

Distributional patterns of the interphotoreceptor matrix (IPM) under several different light conditions were examined in adult rats using colloidal iron staining. In the dark, the IPM distributed uniformly throughout photoreceptor outer segments including apical halves of inner segments. After exposure to light for 1 min, the colloidal iron-bound IPM concentrated in bands at apical and basal regions of outer segments and at apical inner segments. After exposure to light for 5-30 min, the IPM distributed throughout inner segments, in addition to the distribution at apical and basal outer segments. In excessive light, diffuse IPM staining was observed throughout the photoreceptor layer. These findings suggest that the light response of the IPM around outer segments precedes that around basal inner segments. It is also remarkable that the excessive light-induced distributional pattern of the IPM is similar to patterns in inherited retinal dystrophy and light-induced retinal degenerations.