Dietary prevention of cataracts in the pink-eyed RCS rat

The American Institute of Nutrition purified ingredient diet (AIN-76) prevented occurrence of mature cataracts associated with hereditary retinal degeneration in pink-eyed, tan-hooded Royal College of Surgeons (RCS) rats. Rats fed a natural ingredient open formula NIH diet or a closed formula commercial diet had a cataract incidence of 27-29% by 3 to 12 months of age. In contrast, only 1 of 50 rats fed the AIN diet developed a mature cataract in one year. When the NIH diet and the commercial diet were pelleted with 25% of ground sunflower kernels, rats fed these diets had a delay in onset and a reduced incidence of mature cataracts to 18% and 5%, respectively. No mature cataracts occurred in rats fed the AIN diet supplemented with 25% sunflower kernels. All diets were fed to the parental generation as well as the progeny (experimental group). The rats were reared at a low level of illumination (1-3 footcandles inside the cage) to minimize effects of light. Prevention of mature cataracts by the AIN purified diet suggests that diets permitting cataracts to occur may have a constituent at a concentration innocuous for normal rats but beyond the homeostatic control of the RCS rat. Posterior subcapsular cataracts of RCS rats are a model for cataracts associated with human hereditary retinal degenerations, such as retinitis pigmentosa and gyrate atrophy. Manipulation of dietary ingredients allowed by the use of the AIN diet may permit identification of nutrients, nutrient interactions of toxic factors involved in cataractogenesis and its prevention.     

The rat pink-eyed dilution (p) mutation: An identical intragenic deletion in pink-eye dilute-coat strains and several Wistar-derived albino strains

We identified the rat pink-eyed dilution (p) and pink eye Mishima (p^m) mutations. The p^m mutation, which was isolated from a wild rat caught in Mishima Japan in 1961 and is carried in the NIG-III strain, is a splice donor site mutation in intron 5. The p mutation, which was first described in 1914 and is carried in several p/p rats including the RCS and BDV strains, is an intragenic deletion including exons 17 and 18. In addition to RCS and BDV strains, several albino strains, KHR, KMI and WNA, all descendants of albino stock of the Wistar Institute, are homozygous for the p allele. Analyses revealed that the colored p strains and the Wistar-derived albino p strains had the same marker haplotype spanning approximately 4 Mb around the P locus. This indicates that these p strains share a common ancestor and the p allele did not arise independently via recurrent mutations. The historical relationship among the p strains suggests that the p deletion had been maintained in stock heterogeneous for the C and P loci and then was inherited independently by the ancestor of the Wistar albino stock and the ancestor of the pink-eyed agouti rats in Europe.     

Persistent induction of somatic reversions of the pink-eyed unstable mutation in F1 mice born to fathers irradiated at the spermatozoa stage

Untargeted mutation and delayed mutation are features of radiation-induced genomic instability and have been studied extensively in tissue culture cells. The mouse pink-eyed unstable (p(un)) mutation is due to an intragenic duplication of the pink-eyed dilution locus and frequently reverts back to the wild type in germ cells as well as in somatic cells. The reversion event can be detected in the retinal pigment epithelium as a cluster of pigmented cells (eye spot). We have investigated the reversion p(um) in F1 mice born to irradiated males. Spermatogonia-stage irradiation did not affect the frequency of the reversion in F1 mice. However, 6 Gy irradiation at the spermatozoa stage resulted in an approximately twofold increase in the number of eye spots in the retinal pigment epithelium of F1 mice. Somatic reversion occurred for the paternally derived p(un) alleles. In addition, the reversion also occurred for the maternally derived, unirradiated p(un) alleles at a frequency equal to that for the paternally derived allele. Detailed analyses of the number of pigmented cells per eye spot indicated that the frequency of reversion was persistently elevated during the proliferation cycle of the cells in the retinal pigment epithelium when the male parents were irradiated at the spermatozoa stage. The present study demonstrates the presence of a long-lasting memory of DNA damage and the persistent up-regulation of recombinogenic activity in the retinal pigment epithelium of the developing fetus.     

The interaction of the coloboma and aphakia mutant genes in mice

The eye development has been studied in the 12-day-old, 14-day-old embryos and in neonates of Cm/+ ak/ak genotype. The gene coloboma (Cm) in heterozygous state causes a typical coloboma of the iris and the gene aphakia (ak) blocks the lens development in the homozygotes. It has been shown that in Cm/+ ak/ak mice the eyes go through mainly the same abnormal development as that in +/+ ak/ak animals. In mice of both genotypes the lens morphogenesis blocking at the vesicle stage and the retinal fold in the dorsal half of the eye develops. However, the ventral retinal fold which is characteristic for the +/+ ak/ak mice does not form in the Cm/+ ak/ak animals that is the result of the interaction of Cm and ak genes in the eye morphogenesis. The Cm gene suppressing the growth of the retina ventral half inhibits the formation of its fold in Cm/+ ak/ak embryos. As a result of the gene interaction a certain normalization of the eye development compared to the +/+ ak/ak mice is observed in the Cm/+ ak/ak animals. The obtained data show that the Cm gene expresses in the cell clones of the retina ventral half.     

Developmental expression of nerve growth factor in the eye of rats affected by inherited retinopathy: correlative aspects with retinal structural degeneration

We have previously reported that exogenous administration of nerve growth factor (NGF) in C3H/HeJ mouse strain affected by retinitis pigmentosa (RP) delayed retinal degeneration, suggesting that NGF may be implicated in retinal development. Whether NGF is present in the developing eye was not investigated. To address this question we have used Royal College of Surgeons (RCS) rats, characterised by photoreceptor loss during postnatal life. The results of these studies showed that while the thickness of the outer nuclear layer (ONL) of RCS is comparable to controls, while the amount of NGF expressed in the eye of this mutant rat is significant lower, as compared to control eye. This observation suggests that the lower presence of NGF in the eye of RCS rats during early postnatal life might be one critical key factor implicated in RP. The results of these studies will be presented and discussed.     

Disrupted retinal development in the embryonic belly spot and tail mutant mouse

The Belly spot and tail (Bst) semidominant mutation, mapped to mouse Chromosome 16, leads to developmental defects of the eye, skeleton, and coat pigmentation. In the eye, the mutant phenotype is characterized by the presence of retinal colobomas, a paucity of retinal ganglion cells, and axon misrouting. The severity of defects in the Bst/+ retina is variable among individuals and is often asymmetric. In order to determine the role of the Bst locus during retinal morphogenesis, we searched for the earliest observable defects in the developing eye. We examined the retinas of Bst/+ and +/+ littermates from embryonic day 9.5 (E9.5) through E13.5 and measured retinal size, cell density, cell death, mitotic index, and cell birth index. We have found that development of the Bst/+ retina is notably dilatory by as early as E10.5. The affected retinas are smaller than their wildtype counterparts, and optic fissure fusion is delayed. In the mutant, there is a marked lag in the exit of retinal cells from the mitotic cycle, even though there are no observable differences in the rate of cellular proliferation or cell death between the two groups. We hypothesize that Bst regulates retinal cell differentiation and that variability of structural defects in the mutant, such as those affecting optic fissure fusion, is a reflection of the extent of developmental delay brought about by the Bst mutation.     

Safety and Efficacy of Human Wharton's Jelly-Derived Mesenchymal Stem Cells Therapy for Retinal Degeneration

Purpose

To investigate the safety and efficacy of subretinal injection of human Wharton’s Jelly-derived mesenchymal stem cells (hWJ-MSCs) on retinal structure and function in Royal College of Surgeons (RCS) rats.

Methods

RCS rats were divided into 2 groups: hWJ-MSCs treated group (n = 8) and placebo control group (n = 8). In the treatment group, hWJ-MSCs from healthy donors were injected into the subretinal space in one eye of each rat at day 21. Control group received saline injection of the same volume. Additional 3 animals were injected with nanogold-labelled stem cells for in vivo tracking of cells localisation using a micro-computed tomography (microCT). Retinal function was assessed by electroretinography (ERG) 3 days before the injection and repeated at days 15, 30 and 70 after the injection. Eyes were collected at day 70 for histology, cellular and molecular studies.

Results

No retinal tumor formation was detected by histology during the study period. MicroCT scans showed that hWJ-MSCs stayed localised in the eye with no systemic migration. Transmission electron microscopy showed that nanogold-labelled cells were located within the subretinal space. Histology showed preservation of the outer nuclear layer (ONL) in the treated group but not in the control group. However, there were no significant differences in the ERG responses between the groups. Confocal microscopy showed evidence of hWJ-MSCs expressing markers for photoreceptor, Müller cells and bipolar cells.

Conclusions

Subretinal injection of hWJ-MSCs delay the loss of the ONL in RCS rats. hWJ-MSCs appears to be safe and has potential to differentiate into retinal-like cells. The potential of this cell-based therapy for the treatment of retinal dystrophies warrants further studies.     

Ocular distribution of 70-kDa heat-shock protein in rats with normal and dystrophic retinas

Summary Stress proteins are thought to play an important role in cellular development and in survival mechanisms. We compared the immunolocalization of the 70-kDa stress protein (SP70) in the ocular tissue of the normal Sprague-Dawley (SD) rat with that in the Royal College of Surgeons (RCS) rat with retinal dystrophy. SP70 was present in the maturing ocular tissues of both rat strains. However, once retinal degeneration began in the RCS rat, the retinal pigment epithelium and photoreceptor cells showed increased immunostaining for SP70 over that observed in age-matched SD rats. In late stages of retinal degeneration, immunostaining for SP70 was considerably reduced in the RCS retina, whereas normal distribution of immunostaining for SP70 in the SD retina was preserved, albeit decreased, through postnatal day 180. The optic nerve, ciliary body, and corneal epithelium were also influenced by the dystrophic disease condition, although the pattern of changes in SP70 immunostaining differed for each tissue. These results suggest that the genetic defect in the RCS rat produces a state of metabolic stress in all ocular tissues as the degeneration progresses, but that the subsequent rise in ocular SP70 is insufficient to prevent progression of the disease.     

Mapping of genes for cytochromes P-450b, P-450e, P-450g, and P-450h in the rat. Demonstration of two gene clusters with P450-b,e localized on chromosome 1

Inbred ACI, WF, and RCS rats having characteristic markers for albino (c), hemoglobin beta-chain (Hbb), and pink-eyed dilution (p) on chromosome 1 and expressing variants for hepatic cytochromes P-450b, P-450e, P-450g, and P-450h were used in genetic mapping studies for these hemoproteins. The results of WF X (ACI X WF)F1 and RCS X (WF X RCS)F1 backcrosses revealed the existence of two gene clusters designated the P450-b,e and P450-g,h loci. The linkage map P450-b,e-p-c-Hbb on rat chromosome 1 was demonstrated and found to be congruent with Coh(P450-b,e)-p-c-Hbb on mouse chromosome 7. P450-g,h is not linked with P450-b,e and the other markers tested on rat chromosome 1. It appears that close genetic linkage, rather than common functional/regulatory properties, typify members of cytochrome P-450 families/subfamilies.     

RCS大鼠和Wistar大鼠视网膜酸性磷酸酶活性的动态观察

本实验观察了不同年龄组ＲＣＳ大鼠和Ｗｉｓｔａｒ大鼠视网膜中酸性磷酸酶的动态变化及其与ＲＰＥ细胞消化功能的关系。运用偶氮偶联法显示１２ｄ、２１ｄ、２ｍ的ＲＣＳ大鼠和７ｄ、２ｍ的Ｗｉｓｔａｒ大鼠视网膜中的酸性磷酸酶;通过图像分析仪测定ＲＰＥ细胞层和光感受器外节部分的酸性磷酸酶含量,并进行统计学分析。结果：酸性磷酸酶阳性反应呈暗红色,主要位于ＲＰＥ细胞层,视网膜外核层、内核层,节细胞层亦有少量阳性反应颗粒。２ｍ的ＲＣＳ大鼠视细胞内、外节的酸性磷酸酶含量则明显高于其它组（Ｐ＜０．０１）,其余结构的酸性酶各组间无显著性差异（Ｐ＞０．０５）。结论：ＲＣＳ大鼠和Ｗｉｓｔａｒ大鼠的视网膜色素上皮细胞可能具有相同的消化功能。     

The localization of glutathione peroxidase in the photoreceptor cells and the retinal pigment epithelial cells of Wistar and Royal College of Surgeons dystrophic rats

INTRODUCTION: If degenerating photoreceptor outer segments not phagocytized by RPE cells in the retina of Royal College Surgeons (RCS) rats were to undergo peroxidation, the distribution of glutathione peroxidase (GSH-PO) in the mitochondria or cytoplasm of the retina might be altered. We evaluated the immunocytochemical localization of GSH-PO to identify subcellular organelles in sections of the retinas of RCS rats. METHODS: Immunoblot analysis confirmed the presence of GSH-PO molecules in the retinas of RCS and Wistar rats aged 3 weeks. Sections were reacted with the F(ab) fragment of anti-rat alphaGSH-PO and then examined by laser scanning microscopy (LSM) and transmission electron microscopy (TEM). RESULTS: The size of the GSH-PO molecule in the retina was about 21 KD in the mitochondria and 23 KD in the cytosol in both strains of rats. LSM revealed fluorescent granules in the photoreceptor inner segments of the Wistar rats, and immunohistochemical TEM revealed GSH-PO in the mitochondria of their photoreceptor inner segments and retinal pigment epithelial (RPE) cells. In the RCS rats, the degenerating photoreceptor outer segments were clearly seen to be positive for anti-GSH-PO by conventional light microscopy (CLM). However, the photoreceptor inner segments of the RCS rats were negative for staining with anti-GSH-PO by LSM, and no GSH-PO could be detected in the mitochondria of the photoreceptor inner segments or RPE cells by immuno-TEM. CONCLUSION: Degeneration of the photoreceptor outer segments induced mitochondrial damage in the photoreceptor inner segments, and as a result GSH-PO shifted from the photoreceptor inner segments to the degenerating outer segments.     

An extracellular retinol-binding glycoprotein in the eyes of mutant rats with retinal dystrophy: development, localization, and biosynthesis

Interstitial retinol-binding protein (IRBP) is a soluble glycoprotein in the interphotoreceptor matrix of bovine, human, monkey, and rat eyes. It may transport retinol between the retinal pigment epithelium and the neural retina. In light-reared Royal College of Surgeons (RCS) and RCS retinal dystrophy gene (rdy)+ rats, the amount of IRBP in the interphotoreceptor matrix increased in corresponding proportion to the amount of total rhodopsin through postnatal day 22 (P22). In the RCS-rdy+ rats, the amount increased slightly after P23. However, in the RCS rats there was a rapid fall in the quantity of IRBP as the photoreceptors degenerated between P23 and P29. No IRBP was detected by immunocytochemistry in rats at P28. The amount of rhodopsin fell more slowly. Although retinas from young RCS and RCS-rdy+ rats were able to synthesize and secrete IRBP, this ability was lost in retinas from older RCS rats (P51, P88) but not their congenic controls. The photoreceptor cells have degenerated at these ages in the RCS animals, and may therefore be the retinal cells responsible for IRBP synthesis. The putative function of IRBP in the extracellular transport of retinoids during the visual cycle is consistent with a defect in retinol transport in the RCS rat reported by others.     

Systemic Administration of Erythropoietin Inhibits Retinopathy in RCS Rats

Objective

Royal College of Surgeons (RCS) rats develop vasculopathy as photoreceptors degenerate. The aim of this study was to examine the effect of erythropoietin (EPO) on retinopathy in RCS rats.

Methods

Fluorescein angiography was used to monitor retinal vascular changes over time. Changes in retinal glia and vasculature were studied by immunostaining. To study the effects of EPO on retinal pathology, EPO (5000 IU/kg) was injected intraperitoneally in 14 week old normal and RCS rats twice a week for 4 weeks. Changes in the retinal vasculature, glia and microglia, photoreceptor apoptosis, differential expression of p75 neurotrophin receptor (p75^NTR), pro-neurotrophin 3 (pro-NT3), tumour necrosis factor-α (TNFα), pigment epithelium derived factor (PEDF) and vascular endothelial growth factor-A (VEGF-A), the production of CD34⁺ cells and mobilization of CD34⁺/VEGF-R2⁺ cells as well as recruitment of CD34⁺ cells into the retina were examined after EPO treatment.

Results

RCS rats developed progressive capillary dropout and subretinal neovascularization which were accompanied by retinal gliosis. Systemic administration of EPO stabilized the retinal vasculature and inhibited the development of focal vascular lesions. Further studies showed that EPO modulated retinal gliosis, attenuated photoreceptor apoptosis and p75^NTR and pro-NT3 upregulation, promoted the infiltration of ramified microglia and stimulated VEGF-A expression but had little effect on TNFα and PEDF expression. EPO stimulated the production of red and white blood cells and CD34⁺ cells along with effective mobilization of CD34⁺/VEGF-R2⁺ cells. Immunofluorescence study demonstrated that EPO enhanced the recruitment of CD34⁺ cells into the retina.

Conclusions

Our results suggest that EPO has therapeutic potentials in treatment of neuronal and vascular pathology in retinal disease. The protective effects of EPO on photoreceptors and the retinal vasculature may involve multiple mechanisms including regulation of retinal glia and microglia, inhibition of p75^NTR-pro-NT3 signaling together with stimulation of production and mobilization of bone marrow derived cells.     

Benzo(a)pyrene and X-rays induce reversions of the pink-eyed unstable mutation in the retinal pigment epithelium of mice

The pink-eyed unstable (p(un)) mutation is the result of a 70kb tandem duplication within the murine p gene. Homologous deletion/recombination of the locus to wild-type occurs spontaneously in embryos and results in pigmented spots in the fur and eye that persist for life. Such deletion events are also inducible by a variety of DNA damaging agents, as we have observed previously with the fur spot assay. Here, we describe the use of the retinal pigment epithelium (RPE) of the eye to detect reversion events induced with two differently acting agents. Benzo(a)pyrene (B(a)P) induces a high frequency, and X-ray exposure a more modest increase, of p(un) reversion in both the fur and the eye. The eye-spot assay requires fewer mice for significant results than the fur spot assay. Previous work had elucidated the cell proliferation pattern in the RPE and a position effect variegation phenotype in the pattern of p(un) reversions, which we have confirmed. Acute exposure to B(a)P or X-rays resulted in an increased frequency of reversion events. The majority of the spontaneous reversions lie toward the periphery of the RPE whereas induced events are found more centrally, closer to the optic nerve head. The induced distribution corresponds to the major sites of cell proliferation in the RPE at the time of exposure, and further advocates the proposal that dividing cells are at highest risk to develop deletions.     

PHOTORECEPTOR-PIGMENT EPITHELIAL CELL RELATIONSHIPS IN RATS WITH INHERITED RETINAL DEGENERATION : Radioautographic and Electron Microscope Evidence for a Dual Source of Extra Lamellar Material

Protein synthesis and displacement in photoreceptor and pigment epithelial cells of inbred normal (Fisher) and mutant (RCS) rats with inherited retinal degeneration has been studied by light and electron microscope radioautography. Groups of animals 14, 15, 17, 19, 27, 35, and 50 days of age were injected with amino acids-H³ and killed at subsequent time intervals. In normal rats, radioactive protein synthesized in the rod inner segments was incorporated into outer segment saccules and displaced outward; the total renewal time of outer segments at all ages was approximately 9 days. In RCS photoreceptors, outer segment displacement was slowed from the normal rate before day 17 and at all subsequent stages. Most of the newly synthesized protein appeared to migrate only into the basal third of the outer segments. Labeling of pigment epithelial cells in RCS rats was always heavier than in controls. Labeled protein was displaced as early as 1 hr postinjection from pigment epithelial cell somas into the apical processes, and by 2 hr postinjection was located in the adjacent lamellar whorls characteristic of the mutant rat retina. After 1 day, radioactivity was present in the 14, 15, 17, and 19 day series of RCS rats in the apical third of the outer segment layer (occupied mainly by extra lamellar material) while there were few silver grains in the middle third of the layer (occupied mainly by distal parts of outer segments). The RCS pigment epithelial cells thus have an unusual synthetic role and appear to be a source of the extra lamellar material. Electron microscope examination revealed that many intact pigment epithelial cell processes were incorporated into the large whorls of extra lamellae. In addition, many disorganized outer segment saccules were observed in continuity with longer membranous lamellae and large lamellar whorls. The extra lamellar material therefore appears to be derived from both rod outer segments and pigment epithelial cells.     

The mouse pink-eyed dilution allele of the P-gene greatly inhibits eumelanin but not pheomelanin synthesis

The mouse pink-eyed dilution (p) locus is known to control eumelanin synthesis, melanosome morphology, and tyrosinase activity in melanocytes. However, it has not been fully determined whether the mutant allele, p affects pheomelanin synthesis. Effects of the p allele on eumelanin and phemelanin synthesis were investigated by chemical analysis of dorsal hairs of 5-week-old mice obtained from the F(2) generations (black, pink-eyed black, recessive yellow, pink-eyed recessive yellow, agouti, and pink-eyed agouti) between C57BL/10JHir (B10)-congenic pink-eyed black mice (B10-p/p) and recessive yellow (B10-Mc1r(e)/Mc1r(e)) or agouti (B10-A/A) mice. The eumelanin content was dramatically (>20-fold) decreased in pink-eyed black and pink-eyed agouti mice, whereas the pheomelanin content did not decrease in pink-eyed black, pink-eyed recessive yellow, or pink-eyed agouti mice compared to the corresponding P/- mice. These results suggest that the pink-eyed dilution allele greatly inhibits eumelanin synthesis, but not pheomelanin synthesis.     

Retinal Gangliosides in RCS Mutant Rats

Abstract: The distribution of retinal gangliosides was studied in normal and mutant rats with retinal dystrophy at 30 and 180 days of age. The loss of photoreceptor cells in the retinal dystrophic RCS rats was not associated with a significant reduction in the relative distribution of any of the major retinal gangliosides. The loss of photoreceptors, however, caused a marked increase in total retinal ganglioside concentration. These findings suggest that photoreceptor cells contain a low concentration of gangliosides and that no major retinal ganglioside is localized or concentrated in these cells. The cellular localization and function of the most abundant retinal ganglioside, G_D3, is discussed.     

Culture of rat retinal pigment epithelium.

A method of preparing monolayer cultures of retinal pigment epithelium from normal pigmented neonatal rats is described. Critical features include incubating the eyes in balanced salt solution and treating with trypsin before dissecting the eyes. The tissue also has been culured from RCS rats with inherited retinal degeneration. Since the pigment epithelium has been shown to be the primary site of action of the gene for retinal dystrophy in the RCS rat, the method should be usefull in studying the defect(s) associated with this mutation.     

Pink-eyed dilution, a coat color mutation in the Japanese field vole (Microtus montebelli)

Inheritance of dilute coat color with pink eye in the Japanese field vole (Microtus montebelli) was investigated by mating of the dilute mutant with a normal agouti vole and a white vole. As the results, it was cleared that an autosomal recessive gene p is responsible for the pink-eyed dilution in M. montebelli.     

Increased phagocytosis of outer segments in the presence of serum by cultured normal, but not dystrophic, rat retinal pigment epithelium

Cultured normal rat retinal pigment epithelium (PE) ingested six times more rod outer segments in the presence of 20% fetal bovine serum than in serum-free medium. PE cultured from Royal College of Surgeons (RCS) rats with hereditary retinal dystrophy, known to have a defect in vivo in the phagocytosis of shed outer segment tips, ingested amounts of outer segments comparable to normal PE in serum-free medium but did not show an increase in the presence of serum. In both strains of rat PE phagocytosis of latex spheres was similar in the absence of serum and was six-fold higher in the presence of serum, showing that the RCS phagocytic deficiency for outer segments in vitro is not due to a general defect in the phagocytic capacity of the cell. Increased phagocytosis of outer segments by normal PE was observed in the presence of the high molecular weight fraction of ultrafiltered serum but was not seen with serum that was heated at 93 degrees C or precipitated with 5% trichloroacetic acid. Bovine serum albumin had no effect on phagocytosis. These results are consistent with the idea that the phagocytosis of outer segments by cultured normal rat PE, but not by cultured RCS rat PE, is increased in the presence of a specific protein or other macromolecular component of fetal bovine serum.