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Cryopreservation and transplantation of immature rat retina into adult rat retina.

A bank of freeze-stored donor retinas would free transplantation research from dependence on availability of fresh donor tissue. Donor retinas from E13, E16, E19 and E22 (P1) rat embryos were cryoprotected and stored in liquid nitrogen for up to 8 months. Cryopreserved and fresh donor retinas were grafted to adult rat retina. After 4 weeks survival, transplants were evaluated according to a scoring protocol for the criteria of size, viability, lamination and integration. All donor ages of fresh and cryopreserved retina resulted in successful transplants, with the exception of cryopreserved E13. Cryopreserved grafts were significantly less laminated than grafts of fresh tissue. The best lamination scores of cryopreserved transplants were achieved with donor age E16. Surviving transplants were found in the epiretinal and/or subretinal space. Subretinal transplants had higher viability scores than did epiretinal grafts; the difference was more pronounced with transplants of cryopreserved than with fresh tissue. Fresh subretinal transplants were also significantly better laminated than fresh epiretinal transplants. This study shows that (1) cryopreserved retinal donor tissue can successfully be transplanted to rat retina; and (2) the subretinal space appears to be more favorable than the epiretinal space for retinal transplants.

Animals↗

Photoreceptor and glial markers in human embryonic retina and in human embryonic retinal transplants to rat retina.

The purpose of this study was to compare the development of photoreceptor and glial cells in human embryonic retinal transplants with the development of normal human embryonic retina (13-20 weeks postconception). Human embryonic retinal cells (donor age 6-11 weeks postconception) were transplanted to the retinas of adult immunosuppressed rat hosts. Host animals were killed when the transplants were of 13-37 weeks total age (donor age+survival time after surgery). Immunohistochemistry was performed with antibodies specific for neuron-specific enolase (NSE), synaptophysin (SYN), cone-specific opsins, rhodopsin, rod alpha-transducin, S-antigen, vimentin, cellular retinaldehyde-binding protein (CRALBP) and glial fibrillary acidic protein (GFAP). With regards to photoreceptors, NSE and SYN immunoreactive cones were seen in transplants from 14-16 weeks of age, but cone opsin immunoreactivity was not seen until 25 weeks. Developing graft rods became S-antigen immunoreactive at 17-18 weeks. At 20 weeks, inner segments and some cell somas of graft rods stained faintly for alpha-transducin and rhodopsin. At 31 and 37 weeks, inner and outer rod segments were intensely labelled for the rod-specific antigens. The grafts exhibited areas of varying maturation with different staining intensities. Concerning the glial cells, vimentin immunoreactivity was seen in the earliest transplants studied (total age 14-16 weeks), but only in stages older than 19 weeks was the immunoreactivity of graft Müller cells comparable in intensity to those of the host retina. Host Müller cells were immunoreactive for GFAP near the lesion site at all times. At 20 weeks, some GFAP immunoreactive processes were seen inside the graft, apparently coming from the host retina. At 25 weeks, faintly stained Müller cells intrinsic to the graft were observed, indicating a gliosis within the graft. Graft Müller cells were first seen to express CRALBP immunoreactivity at 19-20 weeks and, at 25 weeks, intense immunoreactivity was seen in the transplant, mostly in regions near the host. In the transplants only the Müller cells were stained, whereas both Müller and retinal pigment epithelium cells were CRALBP immunoreactive in the host retina. The development of human embryonic retinal transplants appears to parallel approximately normal in utero development. Transplant cones, rods and Müller cells all express their cell-specific proteins. The photoreceptors develop both inner and outer segments and contain several essential proteins for processing light. The transplants can reach a degree of maturity comparable to newborn retina.

Animals↗

Uridine metabolism in the goldfish retina during optic nerve regeneration: whole retina studies.

Accumulation of radioactivity from [3H]uridine in incubations of whole goldfish retinas is increased in the ipsilateral retina during a period of regeneration that follows unilateral optic nerve crush. Brief incubations to investigate the nature of enhanced labeling of the acid-soluble fraction showed a peak uptake 4 days following crush, with a gradual decrease to control levels by 21 days following crush. That nucleoside uptake may not mediate the effect is supported by the observation that the rate of uptake of 5'-deoxyadenosine, a nonmetabolizable nucleoside analog, is the same in post-crush (PC) and normal (N) retinal incubations. Following brief incubations of PC and N retinas with [3H]uridine, there is enhanced labeling in PC retinas relative to N retinas of recovered UMP, UDP, UTP, and uridine nucleotide sugars, whereas recovery of labeled uridine itself is slightly decreased. The results suggest that the increased accumulation of radioactivity in PC retinas following incubation with uridine reflects an increase in the activities of retinal uridine kinase and uridine nucleotide kinases.

Animals↗

Co-transplantation of embryonic retina and retinal pigment epithelial cells to rabbit retina.

The retinal pigment epithelium (RPE) is important for normal development of the neural retina. We sought to investigate whether cografting RPE cells affected the differentiation and survival of retinal grafts. Pigmented embryonic day 16 (E16) rabbit retina was dissected either with or without attached RPE and injected into a lesion site in retinas of young adult rabbit hosts. Each host obtained a pure retina graft in one eye and a retina/RPE cograft in the other. Animals were sacrificed after 4, 8 and 12 weeks. After 4 weeks, grafts (1-2 mm in diameter) were seen in both experimental groups at the lesion site or in the subretinal space. However, 8 and 12 weeks after transplantation, the graft survival rate decreased. The grafts developed cell layers in folded sheets and many rosettes (a rosette consists of photoreceptors and cells of other retinal layers around a central lumen defined by an outer limiting membrane). Cografts of retina with RPE had areas of more distinct cell lamination than transplants of pure retina. Grafted RPE cells were organized in clusters of cells surrounded by extracellular matrix and often associated with blood vessels. If the extracellular matrix of RPE cell clusters was outside the rosettes close to inner retinal layers in the graft, transplant Müller cell endfeet developed an inner limiting membrane. Müller cell endfeet could also be observed in subretinal transplants attached to the denuded Bruch's membrane of the host. In 12-week grafts, when RPE cell clusters were inside rosettes, the surrounded photoreceptors survived better. No RPE effect could be seen if single RPE cells were dispersed among retinal donor cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Intact sheets of fetal retina transplanted to restore damaged rat retinas.

PURPOSE: The aim of this study was to establish a model for morphologic retinal reconstruction after destruction of photoreceptors. METHODS: Rat embryos were prelabeled by injection of bromodeoxyuridine (BrdU) into timed pregnant rats on 2 to 6 consecutive days. Pieces of fetal retinas (embryonic day [E] 17 to E22) were embedded in growth factor-reduced matrigel for protection and stored in medium on ice. With the use of a custom-mnade implantation tool, trimmed embedded pieces were placed into the subretinal space of albino rats whose photoreceptors had been damaged by continuous exposure to blue light for 3 to 4 days. RESULTS: Donor cells were unequivocally identified by the BrdU label. Approximately 25% of transplants in the subretinal space developed parallel layers, with photoreceptor outer segments facing the host pigment epithelium. Transplants developed rosettes if host pigment epithelium had been damaged, if trauma to the donor tissue occurred during preparation or transplantation, and if the donor tissue was misplaced into the choroid or into the epiretinal space on top of the host retina. If the surgery was performed more than 4 weeks after the light damage, continued degeneration of the host retina caused secondary pigment epithelium damage, and transplants did not develop parallel layers of photoreceptor outer segments. CONCLUSIONS: After transplantation to the subretinal space of a degenerated retina, gel-protected fetal retina can develop parallel layers and photoreceptor outer segments in contact with host pigment epithelium. Transplants can develop good fusion with the inner retina of a photoreceptor-deficient recipient.

Animals↗

Tunable retina encoders for retina implants: why and how.

Current research towards retina implants for partial restoration of vision in blind humans with retinal degenerative dysfunctions focuses on implant and stimulation experiments and technologies. In contrast, our approach takes the availability of an epiretinal multi-electrode neural interface for granted and studies the conditions for successful joint information processing of both retinal prosthesis and brain. Our proposed learning retina encoder (RE) includes information processing modules to simulate the complex mapping operation of parts of the 5-layered neural retina and to provide an iterative, perception-based dialog between RE and human subject. Alternative information processing technologies in the learning RE are being described, which allow an individual optimization of the RE mapping operation by means of iterative tuning with learning algorithms in a dialog between implant wearing subject and RE. The primate visual system is modeled by a retina module (RM) composed of spatio-temporal (ST) filters and a central visual system module (VM). RM performs a mapping 1 of an optical pattern P1 in the physical domain onto a retinal output vector R1(t) in a neural domain, whereas VM performs a mapping 2 of R1(t) in a neural domain onto a visual percept P2 in the perceptual domain. Retinal ganglion cell properties represent non-invertible ST filters in RE, which generate ambiguous output signals. VM generates visual percepts only if the corresponding R1(t) is properly encoded, contains sufficient information, and can be disambiguated. Based on the learning RE and the proposed visual system model, a novel retina encoder (RE*) is proposed, which considers both ambiguity removal and miniature eye movements during fixation. Our simulation results suggest that VM requires miniature eye movements under control of the visual system to retrieve unambiguous patterns P2 corresponding to P1. For retina implant applications, RE* can be tuned to generate optimal ganglion cell codes for epiretinal stimulation.

Action Potentials↗

Hemopexin in the human retina: protection of the retina against heme-mediated toxicity.

The existence of the blood-retinal barrier means that proteins that protect the retina from damage by reactive oxygen species must either be made locally or specifically transported across the barrier cells; however, such transepithelial transport does not seem to occur. Among the circulatory proteins that protect against iron-catalyzed production of free radicals are apo-transferrin, which binds ferric iron and has previously been shown to be made by cells of the neural retina (Davis and Hunt, 1993, J. Cell Physiol., 156:280-285), and the extracellular antioxidant, apo-hemopexin, which binds free heme (iron-protoporphyrin IX). Since hemorrhage and heme release can be important contributing factors in retinal disease, evidence of a hemopexin-based retinal protection system was sought. The human retina has been shown to contain apo-hemopexin which is probably synthesized locally since its mRNA can be detected in retinal tissue dissected from human donor eyes. It is likely that the retina contains a mechanism for the degradation of hemopexin-bound heme since the blood-retinal barrier also precludes the exit of heme-hemopexin from the retina. Retinal pigment epithelial cells have been found to bind and internalize heme-hemopexin in a temperature-dependent, saturable, and specific manner, analogous to the receptor-mediated endocytic system of hepatoma cells. Moreover, the binding of heme-hemopexin to the cells stimulates the expression of heme oxygenase-1, metallothionein-1, and ferritin.

Apoproteins↗

Human retina-specific amine oxidase: genomic structure of the gene (AOC2), alternatively spliced variant, and mRNA expression in retina.

Previously, we reported the isolation of cDNA for human retina-specific amine oxidase (RAO) and the expression of RAO exclusively in retina. Bacterial artificial chromosome clones containing the human RAO gene (AOC2) were mapped to human chromosome 17q21 (Imamura et al., 1997, Genomics 40: 277-283). Here, we report the complete genomic structure of the RAO gene, including 5' flanking sequence, and mRNA expression in retina. The human RAO gene spans 6 kb and is composed of four exons corresponding to the amino acid sequence 1-530, 530-598, 598-641, and 642-729 separated by three introns of 3000, 310, and 351 bp. Screening of a human retina cDNA library revealed the existence of an alternatively spliced cDNA variant with an additional 81 bp at the end of exon 2. The sizes of exons and the locations of exon/intron boundaries in the human RAO gene showed remarkable similarity to those of the human kidney diamine oxidase gene (AOC1). In situ hybridization revealed that mRNA coding for RAO is expressed preferentially in the ganglion cell layer of the mouse retina. We designed four sets of PCR primers to amplify four exons, which will be valuable for analyzing mutations in patients with ocular diseases affecting the retinal ganglion cell layer.

Alternative Splicing↗

Expression patterns of neurexin-1 and neuroligins in brain and retina of the chick embryo: Neuroligin-3 is absent in retina.

Neuroligins (NLs) constitute a family of cell-surface proteins that interact with neurexins (beta-Nxs), another class of neuronal cell-surface proteins, one of each class functioning together in synapse formation. The localization of the various neurexins and neuroligins, however, has not yet been clarified in chicken. Therefore, we studied the expression patterns of neurexin-1 (Nx-1) and neuroligin-1 and -3 during embryonic development of the chick retina and brain by reverse-transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). While neurexin-1 increased continuously in both brain and retina, the expression of both neuroligins was more variable. As shown by ISH, Nx-1 is expressed in the inner half retina along with differentiation of ganglion and amacrine cells. Transcripts of NL-1 were detected as early as day 4 and increased with the maturation of the different brain regions. In different brain regions, NL-1 showed a different time regulation. Remarkably, neuroligin-3 was entirely absent in retina. This study indicates that synaptogenetic processes in brain and retina use different molecular machineries, whereby the neuroligins might represent the more distinctly regulated part of the neurexin-neuroligin complexes. Noticeably, NL-3 does not seem to be involved in the making of retinal synapses.

Animals↗

Expression of substance P, neurokinin 1 receptors (NK1) and neurokinin 3 receptors in the developing mouse retina and in the retina of NK1 knockout mice.

To complete a series of studies on the expression of substance P and neurokinin receptors in mammalian retinas, we investigated the occurrence of these molecules in developing mouse retinas and in retinas of mice with genetic deletion of the neurokinin 1 receptor, the preferred substance P receptor. Using semi-quantitative reverse transcription-polymerase chain reaction, we measured detectable levels of the gamma isoform of preprotachykinin A (a substance P precursor) mRNA at postnatal day 4. Neurokinin 1 receptor and neurokinin 3 receptor mRNAs were also detected at postnatal day 4. While gamma preprotachykinin A and neurokinin 1 receptor mRNA levels significantly increased up to eye opening (postnatal day 11), neurokinin 3 receptor mRNA levels remained constant throughout development. Substance P, neurokinin 1 receptor and neurokinin 3 receptor immunoreactivities were present at postnatal day 5. Substance P was in amacrine cells, neurokinin 1 receptor in developing amacrine and bipolar cells and neurokinin 3 receptor in OFF-type cone bipolar cells. Interestingly, a transient increase in the density of neurokinin 1 receptor immunoreactive processes was observed at eye opening in lamina 3 of the inner plexiform layer, suggesting a role of substance P and neurokinin 1 receptor in this developmental phase. However, in neurokinin 1 receptor knockout retinas, besides a significant increase of the gamma preprotachykinin A mRNA levels, no major changes were detected: neurokinin 3 receptor mRNA levels as well as substance P and neurokinin 3 receptor immunostainings were similar to wild types. Together with previous studies, these observations indicate that there are major differences in neurokinin 1 receptor expression patterns among developing mammalian retinas. The observations in neurokinin 1 receptor knockout mice may not be applicable to rats or rabbits, and substance P and neurokinin 1 receptor may play different developmental roles in different species.

Aging↗

Does a tilted retina cause astigmatism? The ocular imagery and the retinoscopic reflex resulting from a tilted retina.

An astigmatic dial viewed by a tilted retina will have only one line in focus, simulating astigmatic blur. We compare and contrast this optical situation to actual astigmatism. Photographs were taken of an astigmatic dial blurred with a cylindrical lens, and also of the same astigmatic dial tilted, simulating tilted retinal imagery. A model eye for retinoscopy practice was provided with a retina tilted 30 degrees and was retinoscoped repeatedly with and without added confounding cylinder by five skilled retinoscopists. Photographs of the astigmatically blurred and tilted astigmatic dials were similar but not identical, as expected. The model eye with tilted retina showed no astigmatism by retinoscopy in 14/15 measurements and 0.25 D of astigmatism in one measurement. When confounding cylinder was present, the retinoscopic measurement was always within 0.25 D of the added cylinder. The ocular imagery resulting from a tilted retina can simulate astigmatic blur, but this is actually due to a type of curvature of field. Only a minute area of the tilted retina is viewed during retinoscopy, so the tilt has essentially no influence on the retinoscopic reflex.

Adult↗

Local differences in GABA release induced by excitatory amino acids during retina development: selective activation of NMDA receptors by aspartate in the inner retina.

Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the CNS. In the retina, it has been shown that glutamate and aspartate and their agonists kainate and NMDA promote the release of GABA. In the chick retina, at embryonic day 14 (E14), glutamate and kainate were able to induce the release of GABA from amacrine and horizontal cells as detected by GABA-immunoreactivity. NMDA also induced GABA release restricted to amacrine cell population and its projections to the inner plexiform layer (E14 and E18). Although aspartate reduced GABA immunoreactivity, specifically in amacrine cells of E18 retinas, it was not efficient to promote GABA release from retinas at E14. As observed in differentiated retinas, dopamine inhibited the GABA release promoted by NMDA and aspartate but not by kainate. Our data show that different retinal sites respond to distinct EAAs via different receptor systems.

Animals↗

Detached retina affects morphologic and biochemical changes in the retina adjacent to bullous retinal detachment in rabbits.

PURPOSE: Long-term results, more than 10 years after successful retinal detachment surgery, have shown gradually decreasing visual acuity in some cases. It is unclear if reduced functional recovery postoperatively is caused by anatomic changes or biochemical disorders. To determine the etiology of the reduced visual acuity, we cytochemically examined the changes in the cellular responses of the edges of retinal detachments. METHODS: We histochemically studied the glucose-6-phosphatase (G6P) and 5'-nucleotidase (5'-Nase) activity in the rabbit retina. Experimental rhegmatogenous retinal detachment was produced in a rabbit model after partial vitrectomy, followed by retinal tear formation. RESULTS: Although 5'-Nase activity gradually decreased during the period of detachment, activity was still detectable after 24 weeks. G6P activity increased in the region of the detached neural retina. Around the border of the detached retina, the decrease in 5'-Nase activity extended approximately 140 micrometers into the adjacent attached retina at 2 weeks after detachment and 270 micrometers at 24 weeks. CONCLUSIONS: These observations suggest that some anatomical and biochemical damages may occur in the retina adjacent to bullous retinal detachment and may explain the reduction in postoperative vision in some clinical cases.

5'-Nucleotidase↗

Outer-retina locus of increased flicker sensitivity of the peripheral retina.

We tested alternative hypotheses concerning the locus of enhanced flicker sensitivity observed in response to stimuli presented to the peripheral retina. The first hypothesis attributes increased temporal frequency sensitivity to ganglion cell and higher-order neural processing, whereas the second hypothesis states that the locus of these temporal effects is at the cone photoreceptors. To test these alternative hypotheses we measured retinal electrophysiological and psychophysical temporal modulation thresholds. We found that sensitivity for temporal frequencies < 30 Hz did not vary as a function of retinal location for either the focal electroretinogram (ERG) or the psychophysical measure. However, for both measures, sensitivity for temporal frequencies > or = 30 Hz was greater in the peripheral retina than in the central retina. In addition, critical flicker frequency for the central retina was linear as a function of retinal illuminance for both the psychophysical and the electrophysiological measures. For the peripheral retina the slopes of critical flicker frequency versus log illuminance functions were steeper than the central slopes for both threshold measures. Eccentrically measured focal ERG and psychophysical critical flicker frequency values showed a relative saturation, deviating from the linear slope above 3.5 log Td. The findings of similar focal ERG and psychophysical temporal sensitivity changes with eccentricity support an outer retinal origin of this phenomenon.

Adaptation, Ocular↗

Does 5HT play a neurotransmitter role in mammalian retina? Studies on uptake and potassium-stimulated release of 14C-5HT and 14C-GABA from bovine and rabbit retina slices.

Slices of the bovine and rabbit retina actively took up 14C-5HT from the incubation medium; the process was temperature- and Na+-dependent. Concentration-dependent inhibition of 14C-5HT uptake into retinal slices by fluoxetine and citalopram, as well as relative ineffectiveness of nomifensine and nisoxetine, indicate that the uptake process is specific. Exposition of the retina slices of both rabbit and cow to high K+ concentrations (25-70 mM) did not result in any change in the release of radioactivity (compared to basal outflow) when studied in a standard superfusion system. Under the same experimental conditions, 50 mM K+ stimulation of the bovine retina slices preloaded with 14C-GABA clearly enhanced outflow of 14C-radioactivity. Inability of K+ stimulation to increase the release of 14C-5HT from the bovine and rabbit retina slices does not support the idea that 5HT acts as a retinal neurotransmitter in the studied mammals. A possibility is discussed that in mammals 5HT, being actively and selectively taken up by some retinal elements, may serve as a precursor for a functionally active compounds, e.g. melatonin. In contrast to 5HT, K+ evoked 14C-GABA release from the bovine retina slices supports the contention that GABA is a neurotransmitter in some retinal cells.

Animals↗

Development of the rabbit retina. I. Size of eye and retina, and postnatal cell proliferation.

Measures of rabbit eyes and retinal wholemounts were used to evaluate the development of retinal area and shape. The retina is shown to have a horizontal axis about a third longer than the vertical axis just before birth, and to adopt an almost symmetrical shape during postnatal development to adulthood. In general, retinal thickness is shown to decrease after birth, but differently in particular retinal regions: the reduction is marked in the periphery, and less pronounced in the visual streak. As an exception, the myelinated region--after it becomes really myelinated, from 9 days p.p.--even increases in thickness. In all regions of the retina, the absolute and relative thickness of the nuclear layers decreases, whereas the relative thickness of plexiform and fibrous layers increases. Proliferation of cells within the rabbit retina was studied during the first three postnatal weeks. 3H-thymidine incorporation was used to demonstrate DNA synthesis autoradiographically in histological sections as well as in enzymatically isolated retinal cells. A first proliferation phase occurs in the neuroblastic cell layer and ceases shortly after birth in the retinal center, but lasts for about one week in the retinal periphery. We found, however, a few 3H-thymidine-labeled cells as late as in the third postnatal week. These late-labeled cells were found within the nerve fiber layer and in the inner plexiform layer. The latter cells were shown to express antigens detected by antibodies directed to the intermediate-sized filament protein vimentin, which are known to label Müller cells and neuroepithelial stem cells. This was confirmed in our preparation of enzymatically isolated cells; all cells with autoradiographically labeled nuclei revealed a characteristic elongated morphology typical for Müller radial glia (and also for early neuroepithelial stem cells). 3H-thymidine-labeled cells in the nerve fiber layer were most probably astrocytic. In analogy to the brain, we conclude that the mammalian retina undergoes a series of proliferation phases: first an early phase producing both neurons and glial cells, and then a late phase producing glial cells, e.g., in the nerve fiber layer. Most probably, the late phase within the inner nuclear layer is glial as well, i.e., consists of dividing Müller cells; it cannot be excluded, however, that there may remain some mitotically active stem cells.

Animals↗

[Changes of the phosphatides and their fatty acids in the retina and in the fasciculus opticus after retinal detachment: investigations of human and animal retinae (author's transl)].

In this study the effect of an experimentally provoked retinal detachment on the pattern of the phosphatides and fatty acids of the retina and the optic nerve of adult rabbits was investigated. The analysis was performed one month, and 4 months after the operation, and the values were related to the findings in control animals of the same age, and of the age of one day and 30 days respectively. In this way changes in the total lipide, in the phosphatides and in their fatty acids could be revealed, with a tendency towards the developmental stage of the 20th up to the 30th day of life. Between retina and optic nerve no difference was found neither temporally nor regarding the quantitative reaction. In the detached human retinae the same reactions can be proved as in the animal experiment. The relationship of these findings to the recovery of the operatively reatached human retina is discussed.

Adult↗

Neural cell adhesion molecule (NCAM) in adult vertebrate retinas: tissue localization and evidence against its role in retina-pigment epithelium adhesion.

The presence of neural cell adhesion molecule (NCAM) was examined in the neural retina, interphotoreceptor matrix (IPM), and retinal pigment epithelium (RPE) of adult bovine and frog eyes. Using polyclonal antibodies raised against adult isoforms of NCAM. Western blot analyses revealed the presence of NCAM in the neural retina, but not in the IPM or RPE of these species. As a control, Western blot analysis was used to demonstrate the presence of interphotoreceptor retinoid-binding protein (IRBP) in the IPM preparations. NCAM immunoreactivity was detected by light microscopic immunocytochemistry primarily in the plexiform layers and nerve fibre layer of the frog retina. Minor immunoreactivity was also detected in the inner and outer nuclear layers, but there was no detectable NCAM immunoreactivity in the IPM, outer segments, or RPE. These results indicate that NCAM is not a likely participant in the process of retina-RPE adhesion in the adult eye.

Animals↗