Intercellular gap junctions in the developing retina and pigment epithelium of the chick

Summary Gap junctions are found in the pigment epithelium, between retina and pigment epithelium and in the retina of 5–14 day chick embryos, they are identified using block staining and extracellular tracer techniques. In the pigment epithelium gap junctions are found between cell bodies and interdigitating processes and many change their position during development. Gap junctions between retina and pigment epithelium are only made by undiferentiated retinal ventricular cells and may provide intercytoplasmic pathways important for photoreceptor differentiation. Retinal gap junctions are found in an outer zone next to the pigment epithelium and inner zone near the vitreous, they are only seen between ventricular cells but may provide pathways for ganglion cell specification. The role of gap junctions in the generation of retinal neurons is discussed.     

TOPORS, implicated in retinal degeneration, is a cilia-centrosomal protein

We recently reported that mutations in the widely expressed nuclear protein TOPORS (topoisomerase I-binding arginine/serine rich) are associated with autosomal dominant retinal degeneration. However, the precise localization and a functional role of TOPORS in the retina remain unknown. Here, we demonstrate that TOPORS is a novel component of the photoreceptor sensory cilium, which is a modified primary cilium involved with polarized trafficking of proteins. In photoreceptors, TOPORS localizes primarily to the basal bodies of connecting cilium and in the centrosomes of cultured cells. Morpholino-mediated silencing of topors in zebrafish embryos demonstrates in another species a comparable retinal problem as seen in humans, resulting in defective retinal development and failure to form outer segments. These defects can be rescued by mRNA encoding human TOPORS. Taken together, our data suggest that TOPORS may play a key role in regulating primary cilia-dependent photoreceptor development and function. Additionally, it is well known that mutations in other ciliary proteins cause retinal degeneration, which may explain why mutations in TOPORS result in the same phenotype.     

Cone loss is delayed relative to rod loss during induced retinal degeneration in the diurnal cone-rich rodent Arvicanthis ansorgei

Cone photoreceptor breakdown underlies functional vision loss in many blinding diseases. Cone loss is often secondary to that of rods, but little experimental data are available on the relationship between the two populations. Because of its high cone numbers, we used the diurnal rodent Arvicanthis ansorgei to explore changes in rod and cone survival and function during chemically-induced retinal degeneration. Adult animals received intraperitoneal injections of N-methyl-N-nitrosourea (MNU), and changes in retinal fundus appearance, histology, phenotype, apoptosis (TUNEL staining) and functionality (scotopic and photopic electroretinography) were monitored as a function of post-treatment time and retinal topography. Relative to control animals injected with vehicle only, MNU-injected animals showed time-, region- and population-specific changes as measured by morphological and immunochemical criteria. Histological (gradual thinning of photoreceptor layer) and phenotypical (reduced immunostaining of rhodopsin and rod transducin, and mid wavelength cone opsin and cone arrestin) modifications were first observed in superior central retina at 11 days post-injection. These degenerative changes spread into the superior peripheral and inferior hemisphere during the following 10 days. Rod loss preceded that of cones as visualized by differential immunolabelling and presence of apoptotic cells in rod but not cone cells. By 3 months post-injection, degeneration of the photoreceptor layer was complete in the superior hemisphere, but only partial in the inferior hemisphere. Despite the persistence of cone photoreceptors, scotopic and photopic electroretinography performed at 90 days post-treatment showed that both rod and cone function were severely compromised. In conclusion, MNU-induced retinal degeneration in Arvicanthis follows a predictable spatial and temporal pattern allowing clear separation of rod- and cone-specific pathogenic mechanisms. Compared to other rodents in which MNU has been used, Arvicanthis ansorgei demonstrates pronounced resistance to photoreceptor cell loss.     

Eye Histology and Ganglion Cell Topography of Northern Elephant Seals (Mirounga angustirostris)

Northern elephant seals are one of the deepest diving marine mammals. As northern elephant seals often reach the bathypelagic zone, it is usually assumed that their eyes possess evolutionary adaptations that provide better ability to see in dim or scotopic environments. The purpose of this study was to carefully describe anatomical and histological traits of the eye that may improve light sensitivity. Northern elephant seals have large, somewhat elliptical eyes, with equatorial and anteroposterior diameters of 5.03 and 4.4 cm, respectively. The cornea is large in diameter and the lens is completely spherical. The iris has pronounced constrictor and dilator muscles, whereas the ciliary muscle is notably less developed. The tapetum lucidum is more prominent than in other pinnipeds, making up about 63% of retinal thickness in the posterior aspect of the globe. Within the retina, the pigmented epithelium lacks pigment except for the region close to the ora serrata. Parts of the photoreceptor and outer nuclear layers are folded. Although the photoreceptor layer is composed predominantly of rods, cone photoreceptors were also observed. Cells within the retinal ganglion cell layer are arranged in a single level. Ganglion cells reach their maximum density (～1,300 cells per mm²) dorsal to the optic disc, whereas the periphery of the retina is sparsely populated (<100 cells per mm²). All above mentioned features are consistent with the predicted evolutionary adaptations to the photic environment of the bathypelagic zone. Anat Rec, 299:798–805, 2016. © 2016 Wiley Periodicals, Inc.     

Retinal degeneration induced in adult mice by a single intraperitoneal injection of N-ethyl-N-nitrosourea

Seven-week-old female BALB/c mice received a single intraperitoneal injection of N-ethyl-N-nitrosourea (ENU) (50, 100, 200, 400, or 600 mg/kg), and retinal damage was evaluated after 7 days. Sequential morphological features of the retina and retinal apoptosis, as determined by the TUNEL assay, were analyzed 6, 12, 24, and 72 hr and 7 days after treatment with 600 mg/kg of ENU. Moreover, older mice (25 to 34 weeks of age) received an intraperitoneal injection of 600 mg/kg ENU and were sacrificed 7 days later. All animals were necropsied, and both eyes were examined histopathologically. Two of the 5 mice that received 600 mg/kg ENU died during the experimental period. Histopathologically, all mice that received 600 mg/kg of ENU experienced retinal degeneration characterized by the loss of photoreceptor cells (disappearance of the outer nuclear layer and photoreceptor layer) in both the central and peripheral retina within 7 days. One of 5 mice treated with 400 mg/kg ENU exhibited retinal damage that was restricted to the central retina. Older mice treated with 600 mg/kg ENU exhibited retinal damage that was similar to the retinal damage in younger mice. In the 600 mg/kg ENU-treated mice, TUNEL-positive photoreceptor cells peaked 72 hr after ENU treatment. Retinal thickness and the photoreceptor cell ratio in the central and peripheral retina were significantly decreased, and the retinal damage ratio was significantly increased 7 days after treatment. In conclusion, ENU induces retinal degeneration in adult mice that is characterized by photoreceptor cell apoptosis.     

Histochemical localization of zinc and copper in rat ocular tissues

Zinc and copper were histochemically localized in rat ocular tissues. Zinc was demonstrated by the dithizone method, and copper by the rubeanic acid and rhodanine methods. The retinal photoreceptor's outer segment showed the presence of zinc, but no zinc reaction was seen in other parts of the retina or other ocular tissues, even in rats fed excess zinc. It seems that photoreceptor cells contain the highest concentration of zinc. Copper was seen in the corneal epithelium and endothelium, iris, ciliary body, lens epithelium, retinal outer nuclear layer, photoreceptor inner and outer segments, retinal pigment epithelium, choroid, sclera, and optic nerve. These trace elements seems to play roles as components of some metalloenzymes and may have other functions till now unknown.     

Rhodopsin和S-opsin在MNU诱导的大鼠视网膜损伤过程中的表达变化

目的:观察在N-甲基-N亚硝酸脲(MNU)诱导的大鼠视网膜外核层细胞损伤过程中视紫质(rhodopsin)和蓝光视蛋白(blue-sensitive opsin/S-opsin)在视网膜中的表达变化,分析其与MNU诱导的视网膜损伤的关系。方法:将30只SPF级50 d龄雌性SD大鼠随机分为正常对照组和MNU模型1、3、7、10 d组,每组各6只大鼠。模型组以腹腔注射MNU(40 mg/kg)建立MNU模型;正常对照组大鼠腹腔注射生理盐水(5 mL/kg)。右眼冰冻切片行苏木素-伊红(HE)染色判断视网膜损伤程度。通过逆转录-聚合酶链反应(RT-PCR)和免疫荧光检测各组视网膜中rhodopsin和S-opsin的mRNA及蛋白表达情况。结果:病理检测确定造模结果与以往实验一致,各组病理分级之间的差异显著(2=16.838,P0.01)。RT-PCR检测结果表明,与正常对照组相比,各MNU模型组rhodopsin和S-opsin的mRNA表达水平随MNU作用时间增加而逐渐降低且差异均显著(rhodopsin 1 d组P0.05;S-opsin 1 d组P0.01;3、7、10 d组rhodopsin和S-opsin均P0.01)。免疫荧光检测结果显示,在正常大鼠视网膜rhodopsin主要在光感受器细胞外段表达,MNU作用后rhodopsin主要在外核层表达,少量在内核层表达,并随MNU作用时间增加而逐渐表达降低。S-opsin在正常视网膜各层均有表达,在各模型组随MNU作用时间增加S-opsin的表达逐渐降低,在外核层和光感受器细胞内、外段尤为明显。结论:MNU诱导的视网膜外核层细胞损伤可降低rhodopsin和S-opsin的表达,并与MNU选择性引起光感受器细胞丧失有关。     

Growth factor-responsive progenitors in the postnatal mammalian retina.

It is thought that the adult mammalian retina lacks the regenerative capacity of fish and amphibians retina because it does not harbor a progenitor population. However, recent observations suggest that another derivative of the optic neuroepithelium, the ciliary body, contains a mitotically quiescent population of neural progenitors that proliferate in the presence of growth factors and demonstrate properties of neural stem cells. Examination of the hypothesis that similar mitotically quiescent and growth factor-responsive progenitors may exist in the postnatal retina revealed a population of cells located in the periphery of the retina that displayed proliferative responsiveness to growth factors and possessed potential to support neurogenesis. Given their marginal position and neural properties and potential, these cells may represent a residual population of retinal progenitors, analogous to those found in the ciliary marginal zone of fish and amphibians. Their progressive decrease in proliferative potential and number in postnatal stages suggests a temporal decline in regulatory signaling that supports their maintenance during retinal neurogenesis.     

Retinal degeneration in experimental Creutzfeldt-Jakob disease

Mice with experimental Creutzfeldt-Jakob disease (CJD) develop a progressive retinal degeneration after a prolonged incubation period. Sections of the eyes stained with hematoxylin and eosin revealed pathologic changes in the optic nerve and a marked degeneration of photoreceptor cell inner and outer segment areas. Both peripheral and central retina, normally 10 cells thick, were reduced to one photoreceptor cell or less in thickness. Ultrastructural analysis revealed total loss of outer segment and most inner segment elements. Only Müller cell microvilli and macrophages remained in the subretinal space. Macrophages were also visible in the remnant photoreceptor cell layer. The inner nuclear layer and pigment epithelial cell layers appeared normal. Müller cell hypertrophy was evident but was not accompanied by spongiform vacuolation. Several of the degenerative changes of the eye in mice with experimental Creutzfeldt-Jakob disease differ from those observed for scrapie in rodents. The pathologic similarities between the retinal degenerations occurring in mice with experimental Creutzfeldt-Jakob disease and those found in some forms of human retinal degeneration are provocative. These similarities raise the question whether or not other retinal degenerative diseases might be caused by infectious agents such as prions or slow viruses.     

The Leber congenital amaurosis gene product AIPL1 is localized exclusively in rod photoreceptors of the adult human retina

Leber congenital amaurosis (LCA) is the most severe inherited retinal dystrophy resulting in markedly impaired vision or blindness at birth. LCA is characterized by an extinguished electroretinogram in infancy, which is thought to be indicative of an early and severe impairment of both the rod and cone photoreceptors in the human retina. Recently, the aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) gene was identified as the fourth causative gene of LCA. AIPL1 encodes a 384 amino acid protein of unknown function. We have generated a polyclonal antibody against a peptide from a unique region within the primate AIPL1 protein, which detects a protein of approximately 43 kDa in human retinal extracts. A screen of human tissues and immortalized cell lines with this antibody reveals AIPL1 to be specific to human retina and cell lines of retinal origin (Y79 retinoblastoma cells). Within the retina, AIPL1 was detected only in the rod photoreceptor cells of the peripheral and central human retina. The AIPL1 staining pattern extended within the rod photoreceptor cells from the inner segments, through the rod nuclei to the rod photoreceptor synaptic spherules in the outer plexiform layer. AIPL1 was not detected in the cone photoreceptors of peripheral or central human retina. This study is the first to suggest that AIPL1 performs a function essential to the maintenance of rod photoreceptor function.     

Expression of developmentally defined retinal phenotypes in the histogenesis of retinoblastoma.

Retinoblastoma, the most common intraocular tumor of childhood, is a malignant neoplasm that arises during retinal development. The embryonal cell target for neoplastic transformation is not yet clearly defined. To better understand the histogenetic potential of this tumor, the expression of photoreceptor and glial cell-associated proteins were examined in 22 primary retinoblastomas. Interphotoreceptor retinol-binding protein (IRBP), cone and rod opsins were selected as the photoreceptor specific proteins due to their different temporal patterns of expression during normal retinal development. Neoplastic Müller cell differentiation, and non-neoplastic reactive astrocytes were identified using cellular retinaldehyde binding-protein (CRAlBP), and glial fibrillary acidic protein (GFAP), respectively. Photoreceptor proteins were present in 16 cases and showed different cellular patterns of expression. IRBP and cone opsin were usually abundant. Although rod opsin was clearly identified in eight tumors, its expression was more restricted than either IRBP or cone opsin. This differential pattern of expression, opposite to the normal pattern of photoreceptor gene expression in the adult retina, corresponded to a marked decrease in mRNA for rod opsin. Cone opsin and IRBP colocalized in fleurettes demonstrating that neoplastic human cone cells are capable of IRBP synthesis. Müller cell differentiation was present in 12 of the 16 cases in which photoreceptor proteins were detected. In contrast, GFAP was only present in reactive, stromal astrocytes associated with blood vessels. Our data suggest that the retinoblastoma has the histogenetic potential of the immature neural retinal epithelium which can give rise to both photoreceptor and Müller cell lineages. The differential expression of cone and rod phenotypes in retinoblastoma is consistent with the "default" mechanism of cone cell differentiation.     

Increased expression of ciliary neurotrophic factor receptor alpha mRNA in the ischemic rat retina

Using in situ hybridization, we investigated the expression of ciliary neurotrophic factor receptor ((CNTFRalpha) mRNA in the rat retina rendered ischemic by elevation of the intraocular pressure (IOP). The IOP was increased to 120 mmHg and maintained for 60 min. The rats were sacrificed on the day of reperfusion (DRP) 1, 3, 7, 14, and 28. In the normal retina, the signal for CNTFRalpha mRNA was present in retinal cells in the inner nuclear layer (INL) and in the ganglion cell layer (GCL). On DRP 1, numerous cells in the INL and GCL showed a CNTFRalpha mRNA signal. From DRP 3 onwards, CNTFRalpha mRNA appeared in photoreceptor cells located in the outer part of the outer nuclear layer. The signal in these cells increased up to DRP 14 and then decreased at DRP 28. Our findings suggest that cells expressing CNTFRalpha mRNA may resist the degenerative processes induced by ischemic insult in the rat retina.     

Experimental retinal detachment causes widespread and multilayered degeneration in rabbit retina

Retinal detachment remains one of the most frequent causes of visual impairment in humans, even after ophthalmoscopically successful retinal reattachment. This study was aimed at monitoring (ultra-) structural alterations of retinae of rabbits after experimental detachment. A surgical procedure was used to produce local retinal detachments in rabbit eyes similar to the typical lesions in human patients. At various periods after detachment, the detached retinal area as well as neighbouring attached regions were studied by light and electron microscopy. In addition to the well-known degeneration of photoreceptor cells in the detached retina, the following progressive alterations were observed, (i) in both the detached and the attached regions, an incomplete but severe loss of ganglion cell axons occurs; (ii) there is considerable ganglion cell death, particularly in the detached area; (iii) even in the attached retina distant from the detachment, small adherent groups of photoreceptor cells degenerate; (iv) these photoreceptor cells degenerate in an atypical sequence, with severely destructed somata and inner segments but well-maintained outer segments; and (v) the severe loss of retinal neurons is not accompanied by any significant loss of Müller (glial) cells. It is noteworthy that the described progressive (and probably irreparable) retinal destructions occur also in the attached retina, and may account for visual impairment in strikingly large areas of the visual field, even after retinal reattachment.     

Experimental retinal detachment causes widespread and multilayered degeneration in rabbit retina

Retinal detachment remains one of the most frequent causes of visual impairment in humans, even after ophthalmoscopically successful retinal reattachment. This study was aimed at monitoring (ultra-) structural alterations of retinae of rabbits after experimental detachment. A surgical procedure was used to produce local retinal detachments in rabbit eyes similar to the typical lesions in human patients. At various periods after detachment, the detached retinal area as well as neighbouring attached regions were studied by light and electron microscopy. In addition to the well-known degeneration of photoreceptor cells in the detached retina, the following progressive alterations were observed, (i) in both the detached and the attached regions, an incomplete but severe loss of ganglion cell axons occurs; (ii) there is considerable ganglion cell death, particularly in the detached area; (iii) even in the attached retina distant from the detachment, small adherent groups of photoreceptor cells degenerate; (iv) these photoreceptor cells degenerate in an atypical sequence, with severely destructed somata and inner segments but well-maintained outer segments; and (v) the severe loss of retinal neurons is not accompanied by any significant loss of Müller (glial) cells. It is noteworthy that the described progressive (and probably irreparable) retinal destructions occur also in the attached retina, and may account for visual impairment in strikingly large areas of the visual field, even after retinal reattachment.     

Prenatal development of the retina in a diurnal primate (Macaca mulatta)

Observations were made on the pre- and postnatal development of the photoreceptors in the central region of the retina in the diurnal rhesus monkey. Histologic findings on the level of development of the retina at birth revealed a macula with the characteristic adult foveal depression. Observations on the ultrastructural development of the receptor inner and outer segments revealed that the photoreceptors were well differentiated even at birth. An evaluation of the prenatal morphogenesis of the photoreceptors indicated that the initial differentiation may take place as early as 100–125 days after conception. At 100 days after conception, the pigment epithelium consisted of a single layer of cuboidal cells with cytoplasmic content of smooth endoplasmic reticulum, but was only sparsely populated with mitochondria and rough endoplasmic reticulum. The photoreceptor inner segments contained vesiculation in the supranuclear region, while the incipient outer segments consisted of outbudding ciliary processes. By 125 days postconception, the pigment epithelium contained such cytoplasmic specializations as increases in rough endoplasmic reticulum and mitochondria, as well as greater organelle complexity. In the photoreceptor outer segments there were arrays of closed bimembranous discs, with vesiculation at the apical tip in the inner segments. A comparison of the pre- and postnatal ultrastructural changes in the choroid and the interstitial zone comprising the photoreceptor outer segments and the cell processes of the pigment epithelium suggested that the changes in these areas represent a critical aspect in the maturation of the retina.     

Dendrimer-based targeted intravitreal therapy for sustained attenuation of neuroinflammation in retinal degeneration

Retinal neuroinflammation, mediated by activated microglia, plays a key role in the pathogenesis of photoreceptor and retinal pigment epithelial cell loss in age-related macular degeneration and retinitis pigmentosa. Targeted drug therapy for attenuation of neuroinflammation in the retina was explored using hydroxyl-terminated polyamidoamine (PAMAM) dendrimer-drug conjugate nanodevices. We show that, upon intravitreal administration, PAMAM dendrimers selectively localize within activated outer retinal microglia in two rat models of retinal degeneration, but not in the retina of healthy controls. This pathology-dependent biodistribution was exploited for drug delivery, by covalently conjugating fluocinolone acetonide to the dendrimer. The conjugate released the drug in a sustained manner over 90 days. In vivo efficacy was assessed using the Royal College of Surgeons (RCS) rat retinal degeneration model over a four-week period when peak retinal degeneration occurs. One intravitreal injection of 1 μg of FA conjugated to 7 μg of the dendrimer was able to arrest retinal degeneration, preserve photoreceptor outer nuclear cell counts, and attenuate activated microglia, for an entire month. These studies suggest that PAMAM dendrimers (with no targeting ligands) have an intrinsic ability to selectively localize in activated microglia, and can deliver drugs inside these cells for a sustained period for the treatment of retinal neuroinflammation.     

The distribution of intercellular gap junctions in the developing retina and pigment epithelium of Xenopus laevis

Summary The distribution of gap junctions in the developing retina and pigment epithelium of Xenopus has been examined from optic vesicle to photoreceptor outer segment stages. In the retina and pigment epithelium walls of the optic vesicle gap junctions are found between ventricular cells in the apical contact zone and close to the outside of the wall; a neural tube type of distribution. Optic cup formation brings the retina and pigment epithelium into contact and gap junctions form between them. After the stage of retinal specification gap junctions, previously more numerous dorsally, are more often found in the ventral retina and pigment epithelium. They continue to be found in the retinal centre between ventricular cell processes in the apical contact zone and near the vitreal basement lamella above the ganglion cell layer until stage 35. Gap junctions are occasionally made by ganglion cells and their axons, providing possible pathways initiating cell differentiation. After stage 35 gap junctions seem to be confined to the peripheral rim of the eye cup where new cells are formed throughout larval life. The observed distribution suggests that gap junctions may provide intercytoplasmic pathways for retinal specification intrinsic to the retina and pigment epithelium.     

Distribution of three retinal proteins in developing octopus photoreceptors

Summary The expression of proteins unique to plasma membrane domains of developing photoreceptors is used as a marker for retinal differentiation in vertebrates. Invertebrate photoreceptors are also compartmentalized, but little information is available on the development of these compartments or the expression of retinal proteins specific to these cellular regions. Using routine electron microscopy techniques, we have made observations on the formation of photoreceptor organelles, including myeloid bodies and rhabdomeres, in embryonic octopus eyes from an early stage in development through hatching. Immunocytochemical experiments on the embryos demonstrate a timed expression of three retinal proteins during development, and the early separation of the octopus photoreceptor plasma membrane into distinct domains. Using polyclonal antibodies for opsin, retinochrome and retinal binding protein we have shown that opsin appears first and is confined to the distal end of the photoreceptor that will eventually differentiate into rhabdomeres. This membrane domain is separated from the proximal/inner segment plasma membrane by a septate junction. Retinochrome is expressed later when the myeloid bodies appear in the inner segments, and retinal binding protein is apparently not synthesized until sometime after hatching. These results suggest that, in the cephalopod retina, protein components of the retinoid cycling apparatus appear in a specific developmental sequence during the differentiation of this tissue.