Development of the Retina in the Porcine Fetus A Light Microscopic Study

Morphogenesis of the porcine retina was studied using light microscopy from 4 weeks of gestation until birth (18 to 310 mm crown-rump length), and compared with the adult stage (6 months). Tissue samples were examined from the posterior and peripheral parts of the retina. At 18 mm the retina consists of an inner marginal layer and an outer layer of neuroblastic cells. At 18-40 mm the latter layer is divided into an inner and an outer neuroblastic layer by the transient layer of Chievitz. Subsequently, the development of the different retinal layers begins at the inner retinal border and moves progressively outwards; it also spreads from the posterior to the peripheral part of the neural retina. Many cells of the inner neuroblastic layer are prospective ganglionic cells which migrate inwards, thus forming the ganglion cell layer and the inner plexiform layer at 90 mm. At 120 mm, primitive horizontal cells appear within the outer neuroblastic layer. Separation of this layer into the inner nuclear, outer plexiform and outer nuclear layers is first evident at 180 mm. At this stage all retinal layers are present, except the layer of the photoreceptor cells which is not widespread until at 220 mm. The inner and outer segments of the photoreceptor cells lengthen considerably during the last month of gestation. During the late fetal stage the nerve fiber layer, the inner and outer plexiform layers and the layer of rods and cones all continue to increase in thickness. Concurrently, the ganglion cell layer and the inner and outer nuclear layers have reached their maximal thickness and become thinner. After the total thickness of the neural retina amounts to approximately 180 microns at two to three weeks before birth, it then thins to approximately 160 microns in the adult stage.     

An immunohistochemical study of protein kinase C in the bovine retina

The expression of protein kinase C (PKC) was studied in the bovine retina by immunohistochemical analysis. Western blot analysis showed that PKC isoforms, including alpha, betaI, delta and theta, were detected in the bovine retina. By immunohistochemistry, both PKC alpha and betaI were expressed in all retinal layers, with an intense localization of both PKC alpha and betaI detected in bipolar cells in the inner nuclear cell layer and in some glial cells in ganglion cell layers. The immunoreactivity of both PKC delta and theta was quite weak in the retinal layers, compared with that of PKC alpha and betaI. These findings suggest that both conventional and novel PKCs are differentially expressed in the bovine retina.     

Canine tissue-specific expression of multiple small leucine rich proteoglycans

Small leucine rich proteoglycans (SLRPs) are important constituents of extracellular matrix (ECM) and contribute to the production, organization and remodelling of collagen and elastin through complex biological systems. The relative expression and distribution of SLRPs in a variety of different mammalian tissues is poorly characterized. The aim of this study was to map the expression of seven SLRPs (biglycan, versican, prolargin, fibromodulin, osteoglycin, decorin and lumican) in seven tissues (bone, cartilage, cruciate ligament, skin, ventricular myocardium, mitral valve and cornea) in young adult dogs using a combination of quantitative real-time PCR, immunohistochemistry and protein immunoblotting. Clear and consistent patterns of SLRP expression and distribution were identified for the seven tissues examined, with the greatest SLRP expression in cartilage, skin, cornea and mitral valve, and the least expression in myocardium. In general, lumican and prolargin had the greatest expression across the seven tissues whilst osteoglycin was the least abundantly expressed SLRP. These data provide a SLRP profile for different canine tissues which can inform future studies of SLRP expression in development and disease.     

In vivo imaging reveals novel aspects of retinal disease progression in Rs1h(-/Y) mice but no therapeutic effect of carbonic anhydrase inhibition

Purpose X-linked juvenile retinoschisis (XLRS) is the most common juvenile maculopathy in men and is caused by mutations in the gene encoding retinoschisin (RS1). Evidence in the literature on the therapeutic effect of carboanhydrase inhibitors (CAIs) to treat schisis formation in the retina has remained equivocal. Here, we evaluate the effect of the CAI dorzolamide on the structural and functional disease progression in the mouse model for XLRS (Rs1h(-/y) ). Methods Rs1h ( -/y ) mice were treated unilaterally with dorzolamide eye drops (Trusopt(?) 20?mg/mL) every 12?h for 2?weeks starting on postnatal day 14 (n?=?27). Changes of retinal structure were monitored by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography 12?h, 14?days, 4?weeks, 2?months, and 6?months after completion of the treatment. Results Schisis formation (peak at 3?months) preceded photoreceptor degeneration and hyper-fluorescence (peak at 7?months). Structural pathology was most severe in the superior hemi-retina with previously unreported hyper-fluorescent lesions. Quantitative analysis showed no significant differences regarding the inner or outer retinal thickness of the treated vs. untreated eyes 12?h after the completion of treatment (IRT(12?h) =?-1.29?±?1.89?μm; ORT(12?h) =?0.61?±?2.08?μm; mean?±?95%CI) or at any later time point. Conclusion Time line analysis after short-term treatment with CAI failed to show short-, intermediate-, or long-term evidence of structural improvement in Rs1h(-/y) mice. Schisis formation in the inner retina peaked at the age of 3?months and was followed by photoreceptor degeneration predominantly in the superior hemi-retina. Previously unreported hyper-fluorescent lesions co-register with structural retinal pathologies.     

Abnormal prion accumulation associated with retinal pathology in experimentally inoculated scrapie-affected sheep

The purpose of this study was to characterize the patterns of PrP(Sc) immunoreactivity in the retinae of scrapie-affected sheep and to determine the extent of retinal pathology as indicated by glial fibrillary acidic protein immunoreactivity (GFAP-IR) of Müller glia. Sections from the retina of 13 experimentally inoculated scrapie-affected and 2 negative control sheep were examined with immunohistochemical staining for PrP(Sc), GFAP, and PrP(Sc)/GFAP double staining. GFAP-IR of Müller glia is suggestive of retinal pathology in the absence of morphologic abnormality detected by light microscopy. Sheep with the least amount of PrP(Sc) in the retina have multifocal punctate aggregates of prion staining in the outer half of the inner plexiform layer and rarely in the outer plexiform layer. In these retinae, GFAP-IR is not localized with prion accumulation, but rather is present in moderate numbers of Müller glia throughout the sections of retina examined. The majority of sheep with retinal accumulation of PrP(Sc) have intense, diffuse PrP(Sc) staining in both plexiform layers, with immunoreactivity in the cytoplasm of multiple ganglion cells and lesser amounts in the optic fiber layer and between nuclei in nuclear layers. This intense PrP(Sc) immunoreactivity is associated with diffuse, intense GFAP-IR that extends from the inner limiting membrane to the outer limiting membrane. This is the first report of a prion disease in a natural host that describes the accumulation of PrP(Sc) in retina associated with retinal pathology in the absence of overt morphologic changes indicative of retinal degeneration.     

Pathogenetic studies of infection of the bovine fetus with bovine viral diarrhea virus. II. Ocular lesions.

Twenty-three susceptible pregnant heifers were inoculated with bovine viral diarrhea virus at 150 +/- 1 days of gestation. Seven additional heifers were inoculated between 65 and 115 days of gestation. Acute ocular lesions were seen in fetuses taken 17-21 days after inoculation of the dams at 150 days. By the fourth week, the acute lesions were beginning to resolve, and in newborn animals focal to total retinal atrophy was seen. The acute lesions were characterized by a mild to moderate retinitis that resulted in various degrees of destruction of the different layers, mononuclear cuffing of inner retinal vessels, proliferation of pigment epithelium, and choroiditis. Residually there was an absence of cellular elements in the atrophied areas of the retina, frequently a loss of layering and various numbers of pigment-containing cells. Moderately severe acute inflammation was seen in the retina of the fetus taken at 22 days after inoculation of its dam at 95 days. Ocular lesions did not occur in the other fetuses taken from heifers inoculated at earlier stages of gestation.     

Angiostatin and integrin alphavbeta3 in the feline, bovine, canine, equine, porcine and murine retina and cornea

PURPOSE: Angiogenesis is tightly controlled in the ocular tissues of domestic animals but its mechanisms are not fully understood. This is largely because of insufficient data on the expression of molecules that impact angiogenesis. Because angiostatin and one of its receptors integrin alphavbeta3 inhibit and promote angiogenesis, respectively, we hypothesized that the normal retina and cornea of domestic animals would express angiostatin but not integrin alphavbeta3. PROCEDURE: Normal eyes of the cat, cow, dog, horse, pig and rat were evaluated for angiostatin and integrin alphavbeta3 by light and electron immunocytochemistry and estern blots. RESULTS: Angiostatin was detected in the corneal epithelium of the cat, dog, horse, pig and rat, but was not found in cow corneal epithelium. Angiostatin was localized in the nerve fiber layer, ganglion cell layer, inner and outer plexiform layers, and the photoreceptor layer of the cat, cow, dog and rat. Horse and pig retinas showed additional staining in the matrix of the inner nuclear layer. Immunogold electron microscopy further confirmed angiostatin in cat retina. Western blots showed angiostatin in corneal and retinal homogenates. Integrin alphavbeta3 was absent in cornea and retina of all the species studied. CONCLUSION: These data show that angiostatin, an inhibitor of angiogenesis, is present while integrin alphavbeta3, which promotes angiogenesis, is absent in normal cornea and retina of the domestic animals in this study with the exception being angiostatin absence in cow corneal epithelium. Therefore, angiostatin may contribute to the anti-angiogenic environment in the normal domestic animal eye while its absence in the cow may contribute to greater propensity for corneal vascularization. Because integrin alphavbeta3 is one of the receptors for angiostatin, its absence may prevent angiostatin from killing normal retinal and corneal cells.     

Sarcocystis neurona retinochoroiditis in a sea otter (Enhydra lutris kenyoni)

Sarcocystis neurona is an important cause of fatal disease in sea otters in the USA. Encephalitis is the predominant lesion and parasites are confined to the central nervous system and muscles. Here we report retinochoroiditis in a sea otter (Enhydra lutris kenyoni) found dead on Copalis Beach, WA, USA. Salient lesions were confined to the brain and eye. Multifocal nonsuppurative meningoencephalitis was present in the cerebrum and cerebellum associated with S. neurona schizonts. The retina of one eye had a focus of inflammation that contained numerous S. neurona schizonts and merozoites. The focus extended from the retinal pigment epithelium inward through all layers of the retina, but inflammation was most concentrated at the inner surface of the tapetum and the outer retina. The inner and outer nuclear layers of the retina were disorganized and irregular at the site of inflammation. There was severe congestion and mild hemorrhage in the choroid, and mild hemorrhage into the vitreous body. Immunohistochemistry with S. neurona-specific polyclonal rabbit antibodies stained schizonts and merozoites. To our knowledge this is the first report of S. neurona-associated retinochoroiditis in any naturally infected animal.     

Immunohistochemical study of caveolin-1 and -2 in the rat retina

The expression of caveolin-1 and -2 in the retina was examined; Western blot analysis showed that both were present. Immunohistochemistry indicated that caveolin-1 was expressed in the majority of retinal layers, including the ganglion cell layer, inner plexiform layer, outer plexiform layer, and in the vascular endothelial cells of the retina. Caveolin-2 was primarily immunostained in the vessels, but in a few other elements as well. This is the first demonstration of caveolin differential expression in the retina of rats, and suggests that caveolin plays an important role in signal transduction in glial cells and neuronal cells.     

Ultrastructural ocular lesions of 6-aminonicotinamide toxicosis in rabbits

6-Aminonicotinamide, given by intraperitoneal injection to male and female Dutch belted rabbits, produced swelling and vacuolation of ciliary and iridal epithelium plus vacuolation of the retinal pigment epithelial and outer plexiform layers of the retina. By transmission electron microscopy, inner and outer ciliary epithelial cells and inner iridal epithelial cells contained numerous coalescing, membrane-bound vacuoles of the cytocavitary network. These vacuoles were viewed as numerous interconnecting, intracytoplasmic cavities in scanning electron micrographs. Swelling of vacuolated epithelial cells and the presence of fibrin and proteinaceous fluid in the ciliary stroma resulted in thickening of the anterior ciliary processes with the formation of surface alterations detectable by scanning electron microscopy. In transmission electron micrographs the vacuoles in the retinal pigment epithelium were large, electron-lucent spaces and the vacuoles in the outer plexiform layer of the retina appeared to be intracytoplasmic spaces in axons of photoreceptor cells. Distention of cytocavitary structures has been reported in glial cells of animals given 6-aminonicotinamide and this change was apparently due to alterations in ion and water movement across cellular membranes that resulted in intracellular edema.     

Number and density of retinal photoreceptor cells with emphasis on oil droplet distribution in the Mallard Duck (Anas platyrhynchos var. domesticus)

This study was intended to determine the number and regional distribution of photoreceptor cells and different colored oil droplets in the retina of the Mallard Duck (Anas platyrhynchos var. domesticus). To estimate the number and density of photoreceptor cells, adult ducks were killed and both eyes were enucleated under deep anesthesia to prepare Nissl‐stained retinal whole‐mount samples. Different colored oil droplets were counted from color microphotographs of the freshly prepared retina. The mean number of retinal photoreceptors was approximately 6 308 828 ± 521 927, with a peak density of 33 573/mm² in the central retina. The density was similar in the nasal, temporal, ventral and dorsal areas of the retina. Five types of oil droplets were identified on the basis of color: red, orange, greenish‐yellow, yellow and clear. The mean density of oil droplets was highest in the central retina (17 639/mm²) and gradually declined towards the nasal, temporal, ventral and dorsal areas. The size of oil droplets gradually increased with retinal eccentricity and varied even within an area. The greenish‐yellow oil droplets were most abundant across the retina. Taken together, these results demonstrate the differential retinal distribution of photoreceptor cells and oil droplets in duck retina. We conclude that the area of high photoreceptor cell density, which is matched by high neuron densities of the ganglion cell layer, corresponds to the site of acute vision in duck retina.     

Expression of KA1 kainate receptor subunit in the substantia gelatinosa of the trigeminal subnucleus caudalis in mice

The KA1 kainate receptor (KAR) subunit in the substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) has been implicated in the processing of nociceptive information from the orofacial region. This study compared the expression of the KA1 KAR subunit in the SG of the Vc in juvenile, prepubescent and adult mice. RT-PCR, Western blot and immunohistochemistry analyses were used to examine the expression level in SG area. The expression levels of the KA1 KAR subunit mRNA and protein were higher in juvenile mice than in prepubescent or adult mice. Quantitative data revealed that the KA1 KAR subunit mRNA and protein were expressed at levels approximately two and three times higher, respectively, in juvenile mice than in adult mice. A similar expression pattern of the KA1 KAR subunit was observed in an immunohistochemical study that showed higher expression in the juvenile (59%) than those of adult (35%) mice. These results show that the KA1 KAR subunits are expressed in the SG of the Vc in mice and that the expression level of the KA1 KAR subunit decreases gradually with postnatal development. These findings suggest that age-dependent KA1 KAR subunit expression can be a potential mechanism of age-dependent pain perception.     

Quantitative Histomorphology of Liver Growth in Sheep at Prenatal and Postnatal Stages

This study was carried to determine quantitative histomorphologically on the development of the liver of sheep in prenatal and postnatal stages, and to prove the relationship between functional and structural differentiation of liver. There were more blood cells than hepatocytes, and haemotopoieisis was the primary function of the liver in the first half of gestation. As observed in the fetal stages bile ducts and Kiernan areas are formed from the 12th week. The distance between the two adjacent central veins was 401.2 ? 20.8 micron in the fetuses and 629.77 ? 34.7 micron in the lambs, while rising in the adult to 740 + 14.35 micron. This increase was directly proportional to age. The average diameter of sheep hepatocyte and nuclei, and the ratio between the diameters of nuclei and their hepatic cells were compared according to the prenatal and postnatal stages, and the difference between these stages was found to be statistically significant (P < 0.05). This difference was sourced from adult sheep. The number of hepatocytes per unit area were 107.48 ? 6.63, 133.6 ? 7.01, 100.84 ? 6.63 in the fetus, lamb and adult liver of sheep, respectively, and the differentia that earned statistical importance was sourced from the young stages. The number of ductus biliferi was 2.75 ? 0.47 in the fetuses, however, this had risen to 5.8 ? 0.6 and 6.8 ? 0.37 respectively in the lambs and adult sheep. The portai lobule areas rose according to the age and were 0.17796 ? 0.00086 mm² and 2.022650 ? 0.0097 mm² respectively in the lambs and adult sheep and the differentia between young and adult sheep was statistically important (P < 0.05).     

Treatment of serous retinal detachments associated with optic disk pits in dogs

Serous retinal detachment, associated with optic disk pit, was diagnosed in 28 eyes of 24 dogs. Xenon arc photocoagulation was used in treatment of the detachment. Of 24 dogs, 21 were Collies. In 23 eyes, retinal detachments affected temporal and/or inferior portions of the retina. In 5 eyes, detachments were predominantly superior and/or nasal. A single photocoagulation treatment resulted in reattachments in 25 eyes. Of the 3 remaining detachments, 2 eyes improved with additional photocoagulation, and 1 eye, which was not treated further, had a complete retinal detachment.     

N -ethyl- N -nitrosourea induces retinal photoreceptor damage in adult rats

Seven-week-old male Lewis rats received a single intraperitoneal injection of N-ethyl-N-nitrosourea (ENU) (100, 200, 400 or 600 mg/kg), and retinal damage was evaluated 7 days after the treatment. Sequential morphological features of the retina and retinal DNA damage, as determined by a TUNEL assay and phospho-histone H2A.X (γ-H2AX), were analyzed 3, 6, 12, 24 and 72 hr, 7 days, and/or 30 days after 400 mg/kg ENU treatment. Activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) was analyzed immunohistochemically by poly (ADP-ribose) (PAR) expression in response to DNA damage of the retina. All rats that received ≥ 400 mg/kg of ENU developed retinal degeneration characterized by the loss of photoreceptor cells in both the central and peripheral retina within 7 days. In the 400 mg/kg ENU-treated rats, TUNEL-positive signals were only located in the photoreceptor cells and peaked 24 hr after ENU treatment. The γ-H2AX signals in inner retinal cells appeared at 24 hr and peaked at 72 hr after ENU treatment, and the PAR signals selectively located in the photoreceptor cell nuclei appeared at 12 hr and peaked at 24 hr after ENU treatment. However, degeneration was restricted to photoreceptor cells, and no degenerative changes in inner retinal cells were seen at any time points. 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 ENU treatment. In conclusion, ENU induced retinal degeneration in adult rats that was characterized by photoreceptor cell apoptosis through PARP activity.     

The central retinal artery and regression of the hyaloid artery in perinatal cattle

The arterial supply to the retina and lens of 10 fetal, 10 neonatal and four adult Zavot-bred cattle of both sexes was studied macroscopically and by stereoscopic microscopy by means of vascular perfusion with latex, giving special emphasis on the hyaloid artery. The central retinal artery ramified in four major retinal arterioles, which formed a compact network throughout the retina (holangiotic or euangiotic pattern). The hyaloid artery was patent in all fetal stages and extended through the vitreous cavity of the eye to the caudal surface of the capsule of the lens. Atrophy of the hyaloid artery began immediately after birth and was completed on day 17 after parturition. No remnant of the hyaloid artery in the vitreous cavity was observed in the adult cattle examined at stereoscopic microscopic level.     

Chronic ocular lesions associated with bi-directional microbeam radiation therapy in an experimental rat study for therapy of C6 and F98 gliomas

Objectives  To describe the chronic ocular lesions associated with microbeam radiation therapy (MRT) in an experimental rat study.
Procedures  MRT was administered bi-directionally with a skin entry dose of 350 Gy. During laterally directed irradiation, the beam entered the head on the right with the center of the beam array 3 mm posterior to the center of the right eye. During irradiation in anterior-posterior direction, the right eye was almost completely in the path of the beam array. Twelve months after MRT ophthalmic examinations were completed on 37 treated (MRT+) and 16 control (MRT–) rats. Electroretinography (ERG) was completed in two MRT+ and one MRT– rat. Histopathology was performed on eyes of 16 MRT+ and 9 MRT– rats, and retinal and choroidal thicknesses were measured.
Results  Biomicroscopic and indirect ophthalmoscopic examinations revealed fundus pallor and retinal vascular attenuation in 33 of 37 right and 2 of 37 left eyes of MRT+ rats. Cataracts were present in the right eyes of 12 of 37 MRT+ rats. ERG amplitudes were reduced in the eyes of MRT+ rats. Light microscopy revealed retinal lesions ranging in severity with loss of outer to inner retinal cell layers, in 16 of 16 right and of 8 of 16 left eyes MRT+ rats. The mean right retinal thickness of MRT+ rats was reduced.
Conclusions  Eyes within the field treated with MRT at a dose of 350 Gy develop retinal degeneration and occasionally, cataract.     

Ocular lesions of 6-aminonicotinamide toxicosis in rabbits

6-Aminonicotinamide, an antimetabolite of nicotinamide, given by intraperitoneal injection produced diarrhea, ascending paresis/paralysis, death, and bilateral ocular alterations in both sexes of New Zealand white and Dutch belted rabbits. Ocular vascular lesions consisted of iridal congestion with iridal hemorrhage and associated acute iritis and aqueous flare in some rabbits. Cytoplasmic swelling, vacuolation, and loss of staining affinity that represented hydropic change were present in both layers of ciliary epithelium and the inner layer of iridal epithelium. Cells in the outer layer of the iridal processes and the ciliary ridge, were most severely affected. Vacuoles were also present in the retinal pigment epithelium and scattered throughout the outer plexiform layer of the retina with a few in the inner and outer nuclear layers. Ocular alterations were prevented by simultaneous administration of nicotinamide and their development appeared related to nicotinamide deficiency. No ocular alterations were caused by nicotinamide administration alone.     

Cataracts and optic nerve hypoplasia in turkey poults

Newly hatched commercial turkey poults culled because of grossly visible cataracts were studied. A total of 43 affected and 23 unaffected control poults at various ages were necropsied, and the ocular changes in affected poults were compared with those of aged-matched controls. Affected poults had consistent cataracts coupled with a marked depletion in retinal inner plexiform, ganglion cell, and optic nerve fiber layers, with a resultant reduction in the size of the optic nerves. Lesions were seen in 1-day-old poults. Lens changes included microphakia and lens fiber degeneration throughout the lens, with nuclear liquefaction. The depletion in the numbers of retinal ganglion cells did not appear to progress over several weeks time. The ganglion cell depletion was not uniform within the retina. The cause for these ocular changes is unknown.     

Topography of ganglion cells in the retina of the duck (Anas platyrhynchos var. domesticus)

The purpose of this study was to define ganglion cell density, size and topography in the retina of the mallard duck. After killing adult mallard ducks (Anas platyrhynchos var. domesticus), their eyes were removed using pentobarbital (30 mg/kg). The retinas were isolated, whole mount specimens were prepared by staining with 0.1% cresyl violet and then fixing the tissues for study. The retinal ganglion cells were counted, mapped and measured. The mean total number of ganglion cells was estimated at approximately 1.7 × 10⁶ and the retinal area centralis had the highest ganglion cell density with 15 820 cells/mm². The number of ganglion cell bodies was highest in the temporal area, followed by the nasal, dorsal and ventral areas. Ganglion cell size ranged from 56 to 406 μm². A population of small ganglion cells persisted into the central area just above the optic disc and the largest soma area was in the ventral zone of the retina. This localization of ganglion cells suggests that the quality of vision is not equal in all the areas of the duck retina and the central part may have the highest vision quality as a function of the retinal ganglion cells.