Fine structure of the retinal pigment epithelium, Bruch's membrane and choriocapillaris in the horse

The fine structure of the retinal pigment epithelium (RPE), Bruch's membrane and choriocapillaris was investigated by light and transmission electron microscopy in both the tapetal and non-tapetal fundus of the horse eye. In all locations, the RPE consisted of a single layer of low cuboidal cells. The epithelial cells were joined laterally by apically located tight junctions. These cells displayed numerous basal infoldings and abundant thin apical processes which enclosed the rod outer segments. The epithelial cell nuclei were large and located basally. Within the epithelial cells, smooth endoplasmic reticulum was very abundant, while rough endoplasmic reticulum was scarce, polysomes and mitochondria, which often display a ring-shaped structure, were abundant. Melanosomes were abundant in the non-tapetal area but absent in the tapetal area. Bruch's membrane was pentalaminate throughout the retina. The endothelium of the choriocapillaris was heavily fenestrated.     

Alginate as a new biomaterial for the growth of porcine retinal pigment epithelium

Objective Determine the effect of a 3‐dimensional alginate matrix on the growth and differentiation of cells isolated from porcine retinal pigment epithelium (RPE). Procedures Porcine RPE cells were harvested from enucleated eyecups, isolated by differential gravity sedimentation and cultured in either alginate alone (Group 1) or on plastic tissue culture plates followed by alginate (Group 2). Group 1 cells were cultured in alginate to evaluate the efficacy of the matrix as a culture medium. Group 2 cells were initially cultured on plastic to induce dedifferentiation. The cells were then harvested, suspended in alginate beads, and incubated for a second culture period to determine if the induced dedifferentiation was reversible. Results The number of Group 1 cells was significantly greater (P ≤ 0.01) at the end of the culture period. The amount of pigment and cell morphology of Group 1 cells at the end of the culture period was similar to that seen at initial cell isolation. The initial culture of Group 2 cells on plastic showed characteristic features of dedifferentiation marked by the loss of pigment and alterations in microscopic appearance. Secondary culture of dedifferentiated Group 2 cells in alginate beads resulted in a return to pigmentation and characteristic morphology for a majority of the cultured cells. Conclusions Porcine RPE cells can be propagated in alginate culture with a significant increase in cell numbers while maintaining normal morphology. Under the conditions described in the present study, the dedifferentiation of porcine RPE induced by standard in vitro culture methods is reversible.     

Study of retinal microvessels in a Rhesus monkey model of chronic high intraocular pressure

Objective  To investigate the changes in retinal vessels, especially macular capillaries, under high IOP using the Rhesus monkey high IOP model.
Methods  The trabecular meshwork of the adult Rhesus monkey was cauterized by laser to induce increased IOPs with different degrees of damage. The eyeballs were enucleated, and the optic nerves were stained with toluidine blue in semithick slices. Part of the retina was observed under electron microscope, and the rest was stained by the ADPase method. The damage levels of the optic nerve were evaluated by axon count, and the pathological appearance of the macular capillaries were observed.
Results  Five mildly damaged eyes, three moderately damaged eyes and three severely damaged eyes were evaluated. Dense and intact perifoveal vascular rings were observed in all the eyes. The vessels' area percentages, as well as area, perimeter and diameter of the foveal avascular zones, were measured, and no statistically differences were found among different groups ( P -values were 0.269, 0.500, 0.951, and 0.555 separately). The ultra structures of the normal capillaries showed regular tubes and intact basement membranes, while lipoid substances in capillary tubes, swollen mitochondria in endothelial cell bodies, and uneven basement membranes were found in the high IOP-damaged eyes.
Conclusions  Compared with normal eyes, no obvious differences were found in macular microvessels and foveal avascular zones in the Rhesus monkey model of high IOP. However, presence of swollen mitochondria in endothelial cells and lipoid substances in capillary tubes might suggest that high IOP could damage the capillary endothelial cells.     

The cytoskeletal intermediate filaments of canine retinal pigment epithelial cells in vivo and in vitro

The cytoskeletal intermediate filament characteristics of normal, freshly isolated and subcultured canine retinal pigment epithelial ( ) cells were studied using immunocytochemistry and immunoblotting techniques. Commercially available primary antibodies recognising a broad range of cytokeratins and vimentin were selected. Cytokeratin reactivity was a constant feature of all canine cells. The main cytokeratins expressed by cultured RPE cells included 8, 18 and 19. This finding is consistent with the published findings of work carried out in other mammalian species including man. Freshly isolated cells stained positively with broad-spectrum antibodies to cytokeratins but generally did not stain with antibodies specific to cytokeratins 18 or 19 and did not stain with antibodies to vimentin, or stained only very weakly. After a short time in culture however, cells demonstrated intense positive staining for vimentin. This study demonstrated that cytokeratin immunoreactivity (in conjunction with vimentin immunoreactivity in vitro) is a useful and consistent marker for canine RPE cells.     

Characteristics of structures and lesions of the eye in laboratory animals used in toxicity studies

Histopathology of the eye is an essential part of ocular toxicity evaluation. There are structural variations of the eye among several laboratory animals commonly used in toxicity studies, and many cases of ocular lesions in these animals are related to anatomical and physiological characteristics of the eye. Since albino rats have no melanin in the eye, findings of the fundus can be observed clearly by ophthalmoscopy. Retinal atrophy is observed as a hyper-reflective lesion in the fundus and is usually observed as degeneration of the retina in histopathology. Albino rats are sensitive to light, and light-induced retinal degeneration is commonly observed because there is no melanin in the eye. Therefore, it is important to differentiate the causes of retinal degeneration because the lesion occurs spontaneously and is induced by several drugs or by lighting. In dogs, the tapetum lucidum, a multilayered reflective tissue of the choroid, is one of unique structures of the eye. Since tapetal cells contain reflecting crystals in which a high level of zinc has been demonstrated chemically, drug-induced tapetum degeneration is possibly related to zinc chelation. The eye of the monkey has a macula similar to that of humans. The macula consists only of cones with a high density, and light falls directly on the macula that plays an important role in visual acuity. Macular degeneration occurring in monkeys resembles histopathologically that of humans. Hence, the eye of the monkey is a suitable model to investigate macular degeneration and to assess drug-induced macular lesions.     

Fine structure of the retinal pigment epithelium, Bruch's membrane and choriocapillaris in the camel

The morphology of the retinal pigment epithelium (RPE) and closely associated Bruch's membrane and choriocapillaris was investigated by light and transmission electron microscopy in the camel (Camelus dromedarius). The study showed that RPE is composed of a single layer of hexanocuboidal cells that were joined laterally by a series of apically located tight junctions. In addition, adjacent from internal side of cell membrane at the level of tight junctions, an undefined structure which resembled the myofibrillar organization of skeletal muscles in appearance was located. These cells displayed numerous short basal infoldings and abundant thin apical processes which enclosed the rod outer segments. The epithelial cell nuclei were large, vesicular and eccentrically located. Within the epithelial cells, smooth endoplasmic reticulum was very abundant, while rough endoplasmic reticulum was present only in small amounts. Polysomes were also numerous and the mitochondria often displayed a ring-shaped structure. Lipofuscin granules were plentiful in all locations. Bruch's membrane (complexus basalis) was typically pentalaminate throughout the retina. The endothelium of the choriocapillaris facing Bruch's membrane was extremely thin and heavily fenestrated. These fenestrations displayed typical single-layered diaphragm as noted in most species.     

Fine structure of the retinal pigment epithelium, Bruch's membrane and choriocapillaris in the ostrich (Struthio camelus)

The fine structure of the retinal pigment epithelium (RPE), Bruch's membrane and choriocapillaris in the ostrich (Struthio camelus) was investigated by using light microscopy and transmission electron microscopy. In this species, the RPE consisted of a single layer of low columnar cells. The epithelial cells were joined laterally by two type junctions, zonulae occludentes and zonulae adherentes located in the midregion of the cells. These cells displayed numerous deep basal infoldings and thick extensive apical processes, which enclosed the outer segments of the rods. The epithelial cell nuclei were large, vesicular and located basally within the epithelial cells. Smooth endoplasmic reticulum was very abundant, while rough endoplasmic reticulum was scarce. Mitochondria of various shapes were abundant basally while polysomes were plentiful and widespread. In the light-adapted state melanosomes were located in the apical region and in apical processes of the epithelial cells. Myeloid bodies were large, numerous and often showed ribosomes on their outer surface. Bruch's membrane (complexus basalis) was typical pentalaminate throughout the retina, as noted in the majority of other vertebrates. The endothelium of the choriocapillaris facing Bruch's membrane was extremely thin but only moderately fenestrated. Some of the fenestrations displayed a double-layered diaphragm while the majority showed the more typical single-layered diaphragm noted in most species.     

Ocular albinism and hypopigmentation defects in Slc24a5-/- mice

As part of a high-throughput mutagenesis and phenotyping process designed to discover novel drug targets, we generated and characterized mice with a targeted mutation in Slc24a5, a gene encoding a putative cation exchanger. Upon macroscopic examination, Slc24a5-/- mice were viable, fertile, and indistinguishable by coat color from their heterozygous and wild-type litter mates. Ophthalmoscopic examination revealed diffuse retinal hypopigmentation, and a histologic examination of the eye confirmed the presence of moderate-to-marked hypopigmentation of the retinal pigmented epithelium (RPE), ciliary body, and iris pigment epithelium (IPE). Hypopigmentation was most severe in the anterior layer cells of the IPE, where melanosomes were smaller, paler, and more indistinct than those of the anterior stroma and posterior IPE. The pigment granules of the posterior IPE appeared to be nearly as dark as those in stromal melanocytes; however, both cell layers were thinner and paler than corresponding layers in wild-type mice. Ultrastructural analysis of the RPE, IPE, and ciliary body pigmented cells confirmed that mutation of Slc24a5 results in marked hypopigmentation of melanosomes in optic cup-derived pigmented neuroepithelium in the eyes. Milder reductions in melanosome size and pigmentation were noted in neural crest-derived melanocytes. The severe hypopigmentation of neuroepithelium-derived cells in the eyes resulted in a novel form of ocular albinism in Slc24a5-/- mice. Our findings suggest that SLC24A5 may be a candidate gene for some forms of ocular albinism and for the BEY1/EYCL2 locus previously associated with central brown eye color in humans.     

Neuronal ceroid lipofuscinosis: clinical and morphologic findings in nine affected Polish Owczarek Nizinny (PON) dogs

OBJECTIVE: The aim of this study was to characterize the clinical and morphologic features of neuronal ceroid lipofuscinosis (NCL) in the Polish Owczarek Nizinny (PON) breed of dog. ANIMALS: Nine Swedish PON dogs of both sexes were included in the study. PROCEDURE: All dogs underwent a detailed clinical evaluation, with emphasis on ophthalmic exams. Histopathology and electron microscopy were performed on the eyes, brain and various internal organs. Immunohistochemical staining for detection of sphingolipid activator proteins (SAPs) and mitochondrial ATP synthase (SCMAS) was performed on the eyes and brain. RESULTS: The dogs showed behavioral abnormalities, motor disturbances and visual impairment or blindness. Pupillary responses were abnormal while fundus changes varied from normal to severe retinal atrophy. Electroretinography (ERG) showed variable changes, from slight alterations in the process of dark adaptation to severely reduced or nonrecordable ERG a- and b-wave amplitudes. Histopathology revealed intracytoplasmic storage bodies within neurons of the brain and in retinal cells, especially the retinal pigment epithelium (RPE). Round to oval granular type of inclusion bodies, known as granular osmiophilic dense deposits (GRODS), were found in neuronal cells in the brain and in the retina. Immunohistochemistry identified the storage material in the brain and retina as consisting of SAPs. CONCLUSION: The presently described NCL disease in PON dogs shows similarities to previously recorded cases in the Miniature Schnauzer. The closest human equivalent to this disease is infantile NCL (CLN1), in which the major stored proteins are SAPs and the ultrastructure of the inclusion bodies of neuronal cells is granular.     

Differences in expression of retinal pigment epithelium mRNA between normal canines

A reference database of differences in mRNA expression in normal healthy canine retinal pigment epithelium (RPE) has been established. This database identifies non-informative differences in mRNA expression that can be used in screening canine RPE for mutations associated with clinical effects on vision. Complementary DNA (cDNA) pools were prepared from mRNA harvested from RPE, amplified by PCR, and used in a subtractive hybridization protocol (representational differential analysis) to identify differences in RPE mRNA expression between canines. The effect of relatedness of the test canines on the frequency of occurrence of differences was evaluated by using 2 unrelated canines for comparison with 2 female sibling canines of blue heeler/bull terrier lineage. Differentially expressed cDNA species were cloned, sequenced, and identified by comparison to public database entries. The most frequently observed differentially expressed sequence from the unrelated canine comparison was cDNA with 21 base pairs (bp) identical to the human epithelial membrane protein 1 gene (present in 8 of 20 clones). Different clones from the same-sex sibling RPE contained repetitions of several short sequence motifs including the human epithelial membrane protein 1 (4 of 25 clones). Other prevalent differences between sibling RPE included sequences similar to a chicken genetic marker sequence motif (5 of 25), and 6 clones with homology to porcine major histocompatibility loci. In addition to identifying several repetitively occurring, noninformative, differentially expressed RPE mRNA species, the findings confirm that fewer differences occurred between siblings, highlighting the importance of using closely related subjects in representational difference analysis studies.     

Retinal Pigment Epithelial Fine Structure in the Red-Tailed Hawk (Buto jamaicensis)

As part of a comparative morphological study, the fine structure of the retinal epithelium (RPE), choriocapillaris and Bruch's membrane (complexus basalis) has been studied by electron microscopy in the red-tailed hawk (Buteo jamaicensis). In this species the RPE consists of a single layer of low cuboidal cells which display numerous basal (scleral) infoldings and extensive apical (vitreal) processes which interdigitate with photoreceptor outer segments. These epithelial cells are joined laterally by a series of basally located tight junctions. Internally SER is the most abundant cell organelle while only small amounts of RER are present. Polysomes are however abundant as are mitochondria. The RPE cell nucleus is large and vesicular. Melanosomes are mainly located in the apical processes of the RPE cells in light-adaptation. Myeloid bodies are large and numerous in light-adaptation and often show ribosomes on their outer border. Bruch's membrane (complexus basalis) shows the typical pentalaminate structure noted in most vertebrates but with only a poorly defined central elastic layer. The endothelium of the choriocapillaris is very thin facing the RPE but is only moderately fenestrated. The choriocapillaris in this species is unusual however in that many of the fenestrae show a double-layered diaphragm.     

Retinal Epithelial Fine Structure in the Domestic Cat (Felis catus)

The morphology of the retinal epithelium (RPE) and closely associated choriocapillaris and Bruch's membrane (complexus basalis) has been investigated in the eye of the domestic cat (felis catus) by light and electron microscopy. The RPE consists of a single layer of cuboidal cells joined laterally by apically-located junctional complexes. Basally (sclerally) these cells display numerous infoldings while apically (vitreally) two distinct types of processes enclose photoreceptor outer segments. Internally the large vesicular nucleus is centrally located. Smooth endoplasmic reticulum, mitochondria, polysomes and lysosome-like bodies are abundant. Rough endoplasmic reticulum while present is not plentiful. Over the tapetum lucidum, melanosomes are absent from the RPE cells while in non-tapetal locations melanosomes are large and plentiful. The endothelium of the choriocapillaris is highly fenestrated and these capillaries are deeply indented into the epithelial layer over the tapetum. Bruch's membrane (complexus basalis) is non-tapetal regions is the typical pentalaminate structure noted for most mammals. Over the tapetum however it is reduced to a trilaminate structure and when associated with the indented capillary profiles is further reduced to a single thickened basal lamina.     

Ocular manifestations of equine motor neuron disease

The characteristics of the ocular manifestations of equine motor neuron disease (EMND) are described. Forty-two horses with histories, clinical signs and necropsies compatible with EMND were the subjects of this study. Ophthalmoscopic lesions that varied in severity were found in 40 of 42 horses and appeared as a distinct pigmented reticulated pattern at the tapetal-nontapetal junction or throughout the fundus, depending upon severity. The pattern colours ranged from yellow brown to black. Areas of hyperreflectivity formed mosaic patterns in the tapetal fundus. ERG B-wave amplitudes were usually at least 50% reduced and many animals showed extinguished amplitudes. None of the horses had apparent visual impairment. Histopathologically, all 42 horses had retinal pigment epithelial (RPE) congestion with ceroid-lipofuscin. Retinal degeneration was variable even within the eyes. Thin layer chromatography (TLC) analysis of the RPE and neural retina identified both green and orange emitting fluorescent compounds not found in normal horses. All unsupplemented horses had plasma vitamin E levels <1.0 microg/ml. The potential significance of this report is the pathognomonic role the ocular manifestations exhibit in helping to diagnose equine motor neuron disease.     

Retinal Pigment Epithelial Fine Structure in the American Crow (Corvus brachyrhynchos)

The fine structure of the retinal epithelial (RPE) region has been investigated by light and electron microscopy in the American crow (Corvus brachyrhynchos). In this species the RPE consists of a single layer of cuboidal cells which display numerous deep basal (scleral) infoldings and plentiful apical (vitreai) microvillar processes which surround photoreceptor inner and outer segments. The RPE cells are joined laterally by a series of tight junctions (VERHOEFF'S membrane) located in the mid to basal region. Within the epithelial cells, smooth endoplasmic reticulum is very abundant while rough endoplasmic reticulum (RER) is scarce. Mitochondria of various shapes are abundant basally while polysomes are plentiful and widespread. In the light-adapted state RPE nuclei are large and vesicular and basally located while the melanosomes of these cells are predominantly located with the apical processes indicating photomechanical movements. Myeloid bodies are large and numerous and often have ribosomes on their outer surface. Bruch's membrane (complexus basalis) is typical of avian species in that it is pentalaminate and the lamina densa is displaced near the cho-riocapillaris. The endothelium of the choriocapillaris is thin facing Bruch's membrane but is only moderately fenestrated. Some of these fenestrations display a double-layered diaphragm while the majority show the more typical single-layered diaphragm noted in most species.     

Retinal Pigment Epithelial Fine Structure in the Great Blue Heron (Ardea herodias)

The fine structure of the retinal epithelium (RPE), choriocapillaris and Bruch's membrane (complexus basalis) has been studied by light and electron microscopy in the great blue heron (Ardea herodias). In this species the RPE consists of a single layer of cuboidal cells which display numerous basal (scleral) infoldings and plentiful apical (vitreal) processes which surround photoreceptor outer segments. These epithelial cells are joined laterally by a series of tight junctions located in the mid to basal region. Within the epithelial cells, smooth endoplasmic reticulum is very abundant while rough ER is not. Mitochondria (some of which are ring-shaped) and polysomes are abundant. In light-adaptation the RPE nuclei are large vesicular and basally located while the melanosomes of these cells are almost exclusively located within the apical processes. Myeloid bodies are large and numerous and often show ribosomes on their outer surface. Bruch's membrane (complexus basalis) shows the typical pentalaminate structure noted in the majority of vertebrates except teleosts. The choriocapillary endothelium is very thin facing Bruch's membrane but is only moderately fenestrated. The majority of these fenestrations show a single-layered diaphragm but double-layered diaphragms are also noted.     

Viral vectors for targeting the canine retina: a review

Clinical trials are currently underway using gene therapy to treat retinal disease such as Leber congenital amaurosis (LCA). Viral vectors that have been utilized to target retinal cells include adenoviruses, lentiviruses, and recombinant adeno-associated viruses (rAAV). Of the three classes, rAAV vectors show the greatest promise for retinal gene therapy. Recent developments in virus technology such as the development of hybrid and capsid mutant rAAV vectors mean that specific retinal cells can be targeted and faster stronger transgene expression is now possible compared to that achieved with the first generation of vectors. Gene therapy trials in dogs have been very important in the development of therapy for RPE65 LCA which is currently in phase I/II clinical trials in humans. Recent successes in using gene therapy to treat canine achromatopsia, X-linked progressive retinal atrophy (PRA) and the more severe rapid degenerations such as rod-cone dysplasia type 3 may lead also to the translation to human clinical trials. Dogs have played and continue to play an important role as animal models for proof-of-concept studies of retinal gene therapy. As modifications and improvements in gene therapy protocols are made from experience gathered from human clinical trials perhaps gene therapy for the treatment of canine clinical patients will become available to veterinary ophthalmologists.     

Retinal Photoreceptor Fine Structure in the Great Blue Heron (Ardea herodias)

The morphology of the retinal photoreceptors of the great blue heron (Ardea herodias) has been investigated by light and electron microscopy. They consist of rods, single cones and double (unequal) cones present in a ratio of about 2:1:1 respectively. The rods are slender elongated cells with outer segments that reach to the retinal epithelial (RPE) cells and are surrounded by pigment-rich apical processes of the RPE cells in the light-adapted state. The rod inner segment displays an ellipsoid of mitochondria, an hyperboloid of glycogen, much rough ER, numerous polysomes, Golgi zones and autophagic vacuoles. The rod nucleus is located deep in the outer nuclear layer and the rod synaptic pedicle displays both invaginated and superficial synaptic sites. Single cones display a slightly tapered outer segment, a large electron lucent oil droplet and an ellipsoid of mitochondria in the apex of the inner segment. Double cones consist of a long thin chief member which shows an electron dense oil droplet and a shorter, stouter accessory cone with no oil droplet but a paraboloid of glycogen below the ellipsoid. As in the single cone, polysomes, RER and Golgi zones are present in the myoid region of both members of the double cone. All photoreceptor types have a connecting cilium joining inner and outer segments. Near the external limiting membrane, the chief and accessory cones show intercellular junctions. All cone photoreceptors are relatively small in diameter and hence tightly packed. While rods are felt to undergo retinomotor movements, cones are felt to move minimally or not at all. Both single and double cones display several invaginated (ribbon) synapses as well as numerous superficial (conventional) synaptic sites.     

IMPG1 gene variation in rhesus macular drusen

Drusen is a hallmark of human age-related maculopathy. Rhesus macaques (Macaca mulatta) represent a natural model of age-related maculopathy with drusen. We have already mapped the macular drusen susceptibility locus in rhesus macaques to the homolog of human chromosome 6q14-15 and shown that a particular IMPG1 gene SNP haplotype was apparently associated with drusen formation in the rhesus macaques maintained by the Caribbean Primate Research Center (CPRC), Puerto Rico, USA. The aim of the present study was to verify this finding in the macaques kept at the German Primate Research Center (DPZ), Germany. The study group comprised 64 animals (34 affected, 30 unaffected). These monkeys were genotyped for all known variations in the IMPG1 gene and haplotype analysis was performed. A total absence of the previously identified risk haplotype of the IMPG1 gene, and a much lower drusen prevalence in comparison to the CPRC group, was observed in the DPZ samples. This prompted a re-analysis of the original disease association in the CPRC, which revealed that the implied risk haplotype was in fact a sequencing artifact. Taken together, the data highlight that additional factors, other than IMPG1 variation, must play a role in drusen pathogenesis in rhesus macaques.     

Retinal degeneration in the dog and cat

Retinal degenerations in the dog and cat are an important cause of blindness in these species. Particularly in the dog, many retinal degenerations, collectively called progressive retinal atrophy, seen in clinical practice are inherited. The clinical signs, electrophysiological findings, pathology, and underlying biochemical defects in the retina vary from breed to breed. Specific categories of inherited retinal degeneration are now recognized, and classified into early onset photoreceptor dysplasias, late-onset retinal degenerations, or retinal degenerations secondary to primary RPE dystrophy. As new inherited retinal degenerations are reported in different breeds they can generally be assigned to one these categories. Other causes of retinal degeneration include nutritional deficiencies, glaucoma, inflammation, ischemia, and toxins. Idiopathic retinal degeneration occurs in the dog with some frequency.