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Despite an aging confined population, the current state of organ transplantation in carceral systems is largely unknown. This scoping review aimed to assess the literature on organ transplantation in populations experiencing incarceration. The review used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for a scoping review. Included references were published between January 2000 and January 2022 in PubMed, Cumulative Index to Nursing and Allied Health Literature via EBSCO, EMBASE.com, PsycInfo via EBSCO, Sociological Abstracts via ProQuest, and Scopus. Two reviewers conducted title and abstract screening, full-text review, and data extraction in order to generate common themes. The initial search yielded 3,225 studies, and 2,129 references underwent screening. Seventy studies underwent full-text review, and 10 met inclusion criteria. These studies revealed heterogeneous perspectives and policies by providers and transplant centers regarding transplant consideration of individuals with incarceration history or current involvement. Two studies on a kidney transplant program for patients experiencing incarceration showed transplant as a sustainable and potentially superior option for people who are incarcerated, as compared with chronic hemodialysis. Literature on transplantation for populations experiencing incarceration is sparse. More research is required to understand the demand for transplants and the ethical implications of the heterogeneous perspectives and policies on practice patterns.

Aberrant growth of blood vessels in the choroid layer of the eye, termed choroidal neovascularization (CNV), is the pathological hallmark of exudative age-related macular degeneration (AMD), causing irreversible blindness among the elderly. Co-localization of proangiogenic factors and hypoxia inducible factors (HIF) in neovascular membranes from AMD eyes suggests the role of hypoxia in pathogenesis of CNV. In order to utilize hypoxic conditions in RPE for therapeutic purposes, we developed an optimized hypoxia regulated, RPE cell-specific gene therapy to inhibit choroidal neovascularization. An adeno-associated virus (AAV2) vector comprising a RPE-specific promoter and HIF-1 response elements (HRE) was designed to regulate production of human endostatin (a powerful angiostatic protein) in RPE. The vector was tested in a mouse model of laser-induced CNV using subretinal delivery. Spectral domain optical coherence tomography (SD-OCT) images from live mice and confocal images from lectin stained RPE flat mount sections demonstrated reduction in CNV areas by 80% compared to untreated eyes. Quantitative real-time polymerase chain reaction (qPCR) confirmed exogenous endostatin mRNA expression from the regulated vector that was significantly elevated 3, 7, and 14 days following laser treatment, but its expression was completely shut off after 45 days. Thus, RPE-specific, hypoxia-regulated delivery of anti-angiogenic proteins could be a valuable therapeutic approach to treat neovascular AMD at the time and in the ocular space where it arises., Key Points: An optimized gene therapy vector targeting hypoxia and tissue-specific expression has been designed. The inhibitory role of gene therapy vector was tested in a mouse model of laser-induced CNV. An 80% reduction in choroidal neovascularization was achieved by the optimized vector. The expression of endostatin was limited to retinal pigment epithelium and regulated by hypoxia.

Purpose: Müller cells, the major glial cell in the retina, play a significant role in retinal neovascularization in response to tissue hypoxia. We previously designed and tested a vector using a hypoxia-responsive domain and a glial fibrillary acidic protein (GFAP) promoter to drive green fluorescent protein (GFP) expression in Müller cells in the murine model of oxygen-induced retinopathy (OIR). This study compares the efficacy of regulated and unregulated Müller cell delivery of endostatin in preventing neovascularization in the OIR model., Methods: Endostatin cDNA was cloned into plasmids with hypoxia-regulated GFAP or unregulated GFAP promoters, and packaged into self-complementary adeno-associated virus serotype 2 vectors (scAAV2). Before placement in hyperoxia on postnatal day (P)7, mice were given intravitreal injections of regulated or unregulated scAAV2, capsid, or PBS. Five days after return to room air, on P17, neovascular and avascular areas, as well as expression of the transgene and vascular endothelial growth factor (VEGF), were compared in OIR animals treated with a vector, capsid, or PBS., Results: The hypoxia-regulated, glial-specific, vector-expressing endostatin reduced neovascularization by 93% and reduced the central vaso-obliteration area by 90%, matching the results with the unregulated GFAP-Endo vector. Retinas treated with the regulated endostatin vector expressed substantial amounts of endostatin protein, and significantly reduced VEGF protein. Endostatin production from the regulated vector was undetectable in retinas with undamaged vasculature., Conclusions: These findings suggest that the hypoxia-regulated, glial cell-specific vector expressing endostatin may be useful for treatment of neovascularization in proliferative diabetic retinopathy., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: Retinal Müller cells span the retina and secrete several trophic factors and represent the functional link between blood vessels and neurons, making them attractive targets for gene therapy. Therefore, a hypoxia-regulated, retinal glial cell-specific vector was constructed and tested for its response to hypoxia., Methods: A hybrid promoter containing domains of human glial fibrillary acidic protein (GFAP) and several hypoxia-responsive and aerobically silenced elements (HRSE) was incorporated separately into plasmid vectors for generation of self-complementary adeno-associated virus. Müller cells trasfected with plasmids or virus were compared with other cell lines using standard, Methods: The mouse model of oxygen-induced retinopathy (OIR) was used to analyze retinas from mice exposed to high oxygen or room air to evaluate the induction of the regulated promoter., Results: The regulated promoter was silenced under aerobic conditions in comparison with unregulated promoter in Müller cells. Hypoxia induced a 12-fold and 16-fold increase in promoter activity in primary Müller cells and human Müller cell lines, respectively. In the OIR model, intravitreal injection of the regulated promoter at postnatal day 7 (P7) resulted in high levels of green fluorescent protein expression only in retinal Müller cells at P17. GFP expression was absent in retinas of mice only exposed to room air. In vivo studies confirm normoxia silencing, hypoxic induction, and cell specificity of the regulated promoter in the mouse retina., Conclusions: This hypoxia-regulated, retinal glial cell-specific AAV vector provides a platform for gene therapy within regions of retinal hypoxia which are found in diabetic retinopathy and age-related macular degeneration.

Purpose: Metallothioneins (MTs) in the brain and retina are believed to bind metals and reduce free radicals, thereby protecting neurons from oxidative damage. This study was undertaken to investigate whether retinal photoreceptor (PR) cells lacking MTs are more susceptible to hyperbaric oxygen (HBO)-induced cell death in vivo., Methods: Wild-type (WT) and MT-knockout (MT-KO) mice lacking metallothionein (MT)-1 and MT-2 were exposed to three atmospheres of 100% oxygen for 3 hours, 3 times per week for 1, 3, or 5 weeks. The control animals were not exposed. Histologic analysis of PR viability was performed by counting rows of nuclei in the outer nuclear layer (ONL). Ultrastructure studies verified PR damage., Results: HBO exposure produced a major loss of PR cells in the central retinas of WT and MT-KO mice, with no effect on the peripheral retina even at the longest (5 weeks) exposures. The degree of PR damage and cell death increased with duration of HBO exposure. One week of HBO exposure was insufficient to cause PR death, but tissue damage was observed in the inner and outer segments. At 3 weeks, the rows of PR nuclei in the central retina were significantly reduced by 38% in WT and 28% in MT-KO animals. At 5 weeks, PR loss was identical in WT (34%) and MT-KO (34%) animals and was comparable to that in WT at 3 weeks., Conclusions: The data suggest that MT-1 and -2 alone are not sufficient for protecting PRs against HBO-induced cell death. The selective degeneration of central PRs may provide clues to mechanisms of oxidative damage in retinal disease.

Purpose: To develop an hypoxia-regulated retinal pigment epithelium (RPE)-specific adeno-associated virus (AAV) gene transfer platform that exploits hypoxia as a physiologic trigger for an early antiangiogenic treatment strategy directed at arresting neovascularization in the eye., Methods: Tissue-specific and hypoxia-regulated expression vectors were constructed with tandem combinations of hypoxia responsive elements and aerobically silenced elements (HRSE) that together induce gene expression in hypoxia and suppress it in normoxia. For RPE-specific expression, the HRSE and a (6x) HRE (hypoxia responsive element) oligomer were ligated upstream of the Rpe65 promoter in a pGL3 firefly luciferase plasmid (pGL3-HRSE-6xHRE-Rpe65). The cell specificity of this novel hybrid promoter was tested in human RPE (ARPE-19), human glioblastoma, rat C6 glioma, mouse hippocampal neurons, and human embryonic kidney cell lines. Expression of all cell types in normoxia, and following 40 h hypoxia, was analyzed by dual luciferase assay. After confirmation of its tissue-specificity and hypoxia-inducibility, the hybrid promoter construct was integrated into a replication-deficient AAV delivery system and tested for cell-selectivity and hypoxia-inducible green fluorescent protein (GFP) reporter expression., Results: The HRSE-6xHRE-Rpe65 promoter was highly selective for RPE cells, strongly induced in hypoxia, and similar in activation strength to the cytomegalovirus (CMV) promoter. The AAV.HRSE.6xHRE.Rpe65 vector induced robust GFP expression in hypoxic ARPE-19 cells, but elicited no GFP expression in hypoxia in other cell types or in normoxic ARPE-19 cells., Conclusions: The hypoxia-regulated, aerobically-silenced RPE-targeting vector forms a platform for focal autoregulated delivery of antiangiogenic agents in hypoxic regions of the RPE. Such autoinitiated therapy provides a means for early intervention in choroidal neovascularization, when it is most sensitive to inhibition.

Purpose: High oxygen consumption and cyclical changes related to dark-adaptation are characteristic of the outer retina. Oxygenation changes may contribute to the selective vulnerability of the retina in retinitis pigmentosa (RP) patients, especially for those forms involving genes with global cellular functions. Genes coding for components of the U4/U6.U5 tri small nuclear ribonucleoprotein (tri-snRNP) complex of the spliceosome stand out, because mutations in four genes cause RP, i.e., RP9 (PAP1), RP11 (PRPF31), RP13 (PRPF8), and RP18 (PRPF3), while there is no degeneration outside the retina despite global expression of these genes. With the assumption that variable oxygenation plays a role in RP forms related to pre-mRNA splicing and the retina and brain are similar, we searched a data collection of ischemia-hypoxia regulated genes of the brain for oxygen regulated genes of the U4/U6.U5 tri-snRNP complex., Methods: A database of ischemia-hypoxia response (IHR) genes in the brain was generated from gene expression profiling studies [n=24]. Public databases (NCBI) were searched for RP genes with global function that are expressed in the brain. From the IHR gene list, we extracted genes that were directly related to retinal degeneration through a listed mutation (OMIM, Retnet, RISN). The database was then examined for indirect links to RP forms affecting the U4/U6.U5 tri-snRNP complex by searching for IHR genes contributing to this complex. Potential expression of matched genes in the retina was ascertained using NEIBank. Immunohistochemistry was used to localize a selected protein of the U4/U6.U5 tri-snRNP complex in cynomolgus monkey and human retina specimens., Results: The approach identified genes that cause retinal degeneration (CNGB1, SEMA4A, RRG4) or developmental changes (SOX2) when mutated. One IHR gene, Pim1, is the immediate binding partner for PAP1 (RP9). Three IHR genes linked the U4/U6.U5 tri-snRNP complex to regulation by oxygenation: PRPF4; SART1, also known as 110 kDa SR-related protein of the U4/U6.U5 tri-snRNP or as hypoxia associated factor (HAF); and LSM8, U6 snRNA-associated Sm-like protein. The 110 kDa SR-related protein was localized in all retinal cells including photoreceptors., Conclusions: Regulation by changes in oxygenation within the U4/U6.U5 tri-snRP complex could be particularly important for photoreceptors where oxygen consumption follows a circadian rhythm. If the U4/U6.U5 tri-snRP complex is already impaired by mutations in any of the four genes causing RP, it may be unable to follow properly the physiological demands of oxygenation which are mediated by the four hypoxia-regulated proteins emerging in this study. Selective vulnerability may involve complex combinations of widely expressed genes, specific cellular functions and local energy availability.

Purpose: To compare the transduction efficiency of a lentiviral vector in the retina of normal mice and retinal degenerative (rd) mice following subretinal injection at various postnatal ages., Methods: Subretinal injections of lentiviral vector (pHR-CMV-GFP, 107IU/ml) were performed in normal (C57/6J) and rd mice on postnatal days P1 to P7 using a trans-scleral method and on days P14-P35 by a trans-corneal method. One to six weeks later the eyes were prepared for histological analysis. GFP positive cells were identified in retinal sections and retinal whole mounts to determine the overall extent and distribution of lentiviral transduction., Results: Expression of GFP was observed adjacent to the injection site starting about 1 week after injection in both normal and rd mice and lasted 6 weeks (the longest period examined). In normal mice, GFP expression continued to increase and peaked around 2-3 weeks after injection with expression varying from approximately one quarter to the entire retina. GFP expression peaked earlier in rd mice injected from P1 to P7 compared to normal mice. Lenti-GFP expression decreased rapidly in rd mice older than P15. This was attributed to a period of intensive photoreceptor (PR) degeneration characteristic to this mutant. Retinal GFP expression was virtually absent in eyes injected after P14 in both normal and rd mice. Histological sections from P3 injected eyes showed GFP expression 9 days post-injection in both retinal pigment epithelium (RPE) and photoreceptor (PR) cells. GFP expression in RPE cells was stronger than that in PR cells. Both rods and cones expressed the lenti-GFP. GFP expression was limited to the RPE of normal mice if injections were performed at P14 or later. In rd mice, GFP expression in RPE was observed one week after injection at P1; GFP+-PR and -RPE cells were first detected 9 days after injection at P1, and 7 days after injection at P3-P7; RPE cells and occasional Muller cells around the injection site were GFP+ when the injection was performed at P14 or later., Conclusions: Lentiviral-mediated GFP transduction of RPE was efficient and sustained at all ages examined in both the normal and rd mouse. Trans-scleral, subretinal injection of lenti-GFP during the first postnatal week produced age-dependent transduction of PR cells in both mouse strains. Lenti-GFP expression was absent in both mouse strains if injections occurred after P14. There was a dramatic decrease in the transduction efficiency in rd mouse retinas corresponding to the degeneration of PR cells. However, the early stages of retinal degeneration in rd mice appeared to increase the transduction efficiency of PR cells. These data suggest that both age and degree of PR degeneration are important parameters to consider when designing gene therapy experiments or protocols.

Naturally occurring mutations of the beta subunit of the cyclic guanosine monophosphate (cGMP) phosphodiesterase (beta-PDE) gene in rod photoreceptors of mice and dogs are similar to one of the inherited retinal degenerations termed retinitis pigmentosa in humans. Defects in the rod beta-PDE gene leading to photoreceptor cell degeneration in retinal degenerative (rd) mice can be corrected by transfer of a wild type beta-PDE gene. However, the rapid photoreceptor degeneration in this mutant makes the study of gene therapy difficult. Since the retinal degeneration is slowed in vitro, we have employed retinal explants from rd mice to study factors influencing viral transduction. Retinal explants provide a rapid, efficient method to compare the transduction efficiency of adenoviral vector-mediated reporter gene delivery at different ages in normal and rd mice. Retinal explants from postnatal day (P)2 to P28 control (C57BL/6J) and P2-P42 rd mice were exposed for 20 hr to 2.5 x 10(8) plaque forming units (pfu) ml(-1) of adenoviral vector with a beta-galactosidase (Lac Z) reporter gene (Ad-CMV-Lac Z). After incubation in vector-free media for an additional 3 days, the explants were fixed and histochemically stained for beta-galactosidase to reveal Lac Z gene expression. The explants were also embedded and sectioned for light microscopic observation. Transduction efficiency was higher in rd mice than in controls on all postnatal days examined. In normal retinal explants, expression of the Lac Z gene increased from P2 to a peak around P7-P8, then decreased at subsequent ages; little transduction could be found after P17. In rd mice transduction efficiency of Ad-CMV-Lac Z increased from P2 to P7, decreased by P10 and increased again after P10. The most dramatic increase in the transduction efficiency occurred in the rd retina between P10 and P15 when Lac Z was intensely expressed throughout the retina. Microscopic examination of retinal sections revealed the types and distribution of Lac Z-positive cells responsible for the deep blue staining in the retinal whole mount. In normal and rd mice, Lac Z-positive cells were located throughout the retina. However, larger numbers of Lac Z-positive cells were present at all ages examined in retinal explants from rd mice compared to normal mice. These data indicate a difference in transduction efficiency between normal and rd mice, especially after P12, and suggest efficient adenovirus-mediated gene transfer is more attainable in developing or degenerating retina. Thus, transduction efficiency in rd mice depends on the relationship between development, maturation and the degenerative state of the photoreceptor cells.

Mouse retinal photoreceptor cell generation and morphogenesis take place in a well-characterized temporal sequence. Both rod and cone photoreceptor differentiation and synaptogenesis occur postnatally, but the relative timing of these events has been difficult to document due to the paucity of cell-specific markers. We have found that antibodies to neuron-specific enolase (NSE) preferentially label a subpopulation of photoreceptors in the outer nuclear layer (ONL) of the mouse retina in addition to labeling ganglion, amacrine, bipolar, and horizontal cells within the inner layers of the retina. The appearance of NSE immunoreactivity in the different classes of retinal neurons during development showed a close temporal relationship to the onset of expression of the synaptic vesicle-associated protein SV2 and clearly preceded the sequential development of synaptic connections in both inner and outer synaptic layers. The NSE-immunoreactive photoreceptors were identified as cones by dual labeling of their inner segments with the lectin peanut agglutinin or by colabeling with antisera to cone photopigments. Axonal extensions of NSE-labeled cone cells were shown to interact with those of differentiating horizontal cells as early as postnatal day 3 (P3). Colocalization of NSE with SV2 indicated that cone cells began to make synaptic contacts with horizontal cell processes several days prior to the development of rod synaptic terminals. Between P4 and P11, cone photoreceptor cell nuclei were observed to be scattered at various levels throughout the ONL and thus appeared to have become displaced from their previous position directly beneath the outer limiting membrane (OLM). By P12, the cone nuclei had migrated sclerad once again and were now observed to be neatly aligned adjacent to the OLM. In the rd mouse mutant, this migratory process was delayed, so that, at P12, positioning of the cone cell nuclei within the ONL was still quite irregular. Thus, we have identified a late migratory phase for cone photoreceptors during the second week after birth that correlates with the timing of maturation of the rod synaptic terminals just prior to eye opening. The types of cues used by maturing cone cells for their eventual sclerad location remain to be elucidated.

Purpose: A retroviral marker was used to label daughter cells arising from individual neuroblasts in the rd mouse retina, in order to investigate the hypothesis that a clonal relationship exists among degenerating photoreceptor cells., Methods: On the day of birth, a single injection of retrovirus with a lac Z (beta-galactosidase) reporter construct was injected into the retina in the vicinity of the subretinal space. Descendants of single neuroblasts were identified histochemically by examining the retinas at P14 (postnatal day 14). Light and electron microscopic studies were used to identify the retrovirally-induced marker beta-galactosidase using Bluo-gal dye. Double-labeling of degenerating cone cells was accomplished by taking 100 microns vibratome sections of retrovirally-injected eyes and using either FITC-PNA or HRP-PNA to visualize clusters of degenerating cones as well as Bluo-gal labeled clones of photoreceptor cells on the same tissue section., Results: A relatively large number of clones of primarily photoreceptor cells were observed in the peripheral retinas of both normal and rd mice. In a few cases in the rd, photoreceptor cells in a given clone consisted of both PNA- and Bluo-gal-labeled cells as well as of only Bluo-gal-labeled cells., Conclusion: These results suggest that during the period of intense cell death in the rd retina, a single dying photoreceptor cell can be surrounded by photoreceptors (either rods or cones) from the same clone that appear morphologically normal without evidence of degeneration.

Selective degeneration of rod photoreceptor cells in the retinal degenerative (rd) mouse prior to their complete maturation is thought to result from elevated cyclic guanosine monophosphate (cGMP) levels owing to the inherited defect in cGMP-phosphodiesterase. To investigate potential signaling pathways which might lead to apoptotic death of photoreceptors in the rd retina, the expression of immediate-early genes (IEG) of the activating protein-1 transcription factor (AP-1) family was examined. Increasing numbers of apoptotic photoreceptor nuclei were observed in the outer nuclear layer of the rd mouse beginning at postnatal day (P) 10. The peak incidence of apoptotic cells was observed at P13; by P16, almost the entire population of photoreceptors had been lost. Although c-Fos-like immunoreactivity was absent in photoreceptors of normal retinas, we observed that commencing at around P10, increasing numbers of rod photoreceptors in the rd retina exhibited nuclear staining for c-Fos protein. While no change in the distribution patterns of other members of the AP-1 family (c-Jun, JunB, and JunD) was observed in photoreceptors, Müller cell nuclei were transiently immunoreactive for c-Jun on P11. The incidence of c-Fos-positive photoreceptors peaked sharply at P12, 1 day earlier than the peak in apoptosis. Furthermore, the population of c-Fos-positive photoreceptors was distinct from apoptotic photoreceptors exhibiting chromatin condensation. The aberrant expression of c-Fos protein in rod photoreceptors immediately prior to their death in the rd mouse raises the possibility that c-Fos may be directly or indirectly involved in triggering the apoptotic cascade. Furthermore, the additional finding of c-Jun induction in Müller glia suggests that the IEG response to photoreceptor degeneration involves both intra- and intercellular signal transduction pathways.

Purpose: To compare retinal light damage in rats with either normal or reduced levels of rod outer segment (ROS) docosahexaenoic acid., Methods: Weanling male albino rats were maintained in a weak cyclic light environment and fed either a nonpurified control diet or a purified diet deficient in the linolenic acid precursor of docosahexaenoic acid (DHA). Half the rats on the deficient diet were given linseed oil, containing more than 50 mol% linolenic acid, once a week to maintain ROS DHA at near normal levels. Diets and linseed oil supplementation were continued for 7 to 12 weeks. To replenish DHA in their ROS, some 10-week-old rats on the deficient diet were given linseed oil three times a week for up to 3 additional weeks. Groups of animals were killed at various times for ROS fatty acid determinations or were exposed to intense green light using intermittent or hyperthermic light treatments. The extent of retinal light damage was determined biochemically by rhodopsin or photoreceptor cell DNA measurements 2 weeks after exposure and morphologically by light and electron microscopy at various times after light treatment., Results: Rats maintained for 7 to 12 weeks on the linolenic acid-deficient diet had significantly lower levels of DHA and significantly higher levels of n-6 docosapentaenoic acid (22:5n-6) in their ROS than deficient-diet animals supplemented once a week with linseed oil or those fed the nonpurified control diet. As determined by rhodopsin levels and photoreceptor cell DNA measurements, deficient diet rats exhibited protection against retinal damage from either intermittent or hyperthermic light exposure. However, the unsaturated fatty acid content of ROS from all three dietary groups was the same and greater than 60 mol%. In 10 week-old deficient-diet rats given linseed oil three times a week, ROS DHA was unchanged for the first 10 days, whereas 22:5n-6 levels declined by 50%. After 3 weeks of treatment with linseed oil, ROS DHA and 22:5n-6 were nearly the same as in rats supplemented with linseed oil from weaning. The time course of susceptibility to retinal light damage, however, was different. Hyperthermic light damage in rats given linseed oil for only 2 days was the same as for rats always fed the deficient diet. Six days after the start of linseed oil treatment, retinal light damage was the same as in rats given the linseed oil supplement from weaning. Morphologic alterations in ROS of linseed oil-supplemented rats immediately after intermittent light exposure were more extensive than in either the deficient-diet animals or those fed the control diet. The deficient-diet rats also exhibited better preservation of photoreceptor cell nuclei and structure 2 weeks after exposure., Conclusions: Rats fed a diet deficient in the linolenic acid precursor of DHA are protected against experimental retinal light damage. The relationship between retinal light damage and ROS lipids does not depend on the total unsaturated fatty acid content of ROS; the damage appears to be related to the relative levels of DHA and 22:5n-6.

Purpose: Viral-mediated gene transfer to retina, as well as to other tissues, is evolving rapidly. We have evaluated the potential of a retroviral vector with an internal opsin promoter fragment to direct gene expression to retinal photoreceptor cells., Methods: Two recombinant retroviral vectors were prepared; in each Vector, a 1.4 kb fragment of the mouse opsin promoter was placed downstream from the neoR gene in the Moloney murine leukemia virus-based vector G1Na. The opsin promoter fragment was linked either to the cDNA for mouse rod photoreceptor phosphodiesterase (PDE) beta-subunit or to the bacterial lacZ reporter gene. These vectors were tested for their ability to direct gene expression after transduction of 3T3 and Y79 cells, or of dissociated retinal cell cultures or retinal explants from neonatal mice., Results: As expected, PDE beta-subunit and beta-galactosidase mRNAs were expressed only at low levels in 3T3 fibroblasts and Y79 retinoblastoma cells. Northern blot analysis indicated that expression was derived from the viral long terminal repeat (LTR) promoter. Infection of primary retinal cell cultures or explants from neonatal mice with BAG retrovirus, in which beta-galactosidase is driven by the viral LTR, resulted in expression in many cell types, while the opsin-lacZ vector mediated the expression of the lacZ reporter gene specifically in photoreceptor cells., Conclusions: The internal opsin promoter fragment appears capable of selectively directing gene expression to photoreceptor cells after retroviral-mediated gene transfer. These findings serve as a basis for future studies using the opsin promoter-beta PDE retroviral vector to rescue photoreceptor cells in the rd mutant mouse, in which the beta-PDE gene is mutated resulting in degeneration of photoreceptor cells during the early postnatal period.

Detailed analyses of neuronal and astrocyte cell numbers in the ganglion cell layer (GCL) of whole-mounted peripheral retinas from 16 Alzheimer's disease (AD) and 11 control eyes (11 and 9 cases, respectively) demonstrate extensive neuronal loss throughout the entire retina in AD as compared to control eyes. The observed neuronal loss is most pronounced in the superior and inferior quadrants, ranging between 40 and 49% throughout the midperipheral regions, and reaching 50-59% in the far peripheral inferior retina, while the overall neuronal loss throughout the entire retina amounts to 36.4% (p < 0.004). Although the 16% increase in astrocyte numbers is not significant, the ratio of astrocytes to neurons is significantly higher (82%; p < 0.0008) in AD as compared to normal retina (0.238 +/- 0.070 vs. 0.131 +/- 0.042). These results are strengthened by the close agreement (within +/- 15% of respective means) found between fellow eyes. Analysis of glial fibrillary acidic protein immunoreactivity (GFAP-ir) in sections of retinas from an additional 12 AD and 19 control cases show increased GFAP-ir with more extensive labeling of astrocytes in the GCL as well as increased labeling of Müller cell end-feet and radial processes in AD as compared to control retinas. The extensive loss of neurons documented in these retinas, accompanied by an increased astrocyte/neuron ratio, provides further support for the substantial involvement of the retina in AD.

Morphometric analysis of the numbers of neurons in the ganglion cell layer (GCL) of the central retina (fovea/foveola/parafoveal retina) in eyes from 9 Alzheimer's disease (AD) and 11 age-matched control cases revealed an overall decrease of 25% in total numbers of neurons in AD as compared with control eyes. Detailed analyses of GCL neurons at various eccentricities from the foveola showed that the greatest decrease in neuronal density (43% decrease) occurred in the central 0-0.5 mm (foveal region), while at 0.5-1 mm and at 1-1.5 mm eccentricities, neuronal loss amounted to 24 and 26%, respectively. The temporal region of the central retina appeared most severely affected, with up to 52% decrease in neuronal density near the foveola (central 0-0.5 mm eccentricity). There was close agreement between fellow eyes analyzed separately for three AD and three control cases. Analysis of neuronal sizes showed that all sizes of neurons were similarly affected in AD. In the GCL of control retinas, neurons decreased with age (coefficient of correlation = -0.67), while in AD retinas no such relationship was evident. Since in the central 0-2 mm region of the retina 97% of neurons in the GCL are ganglion cells (while the remaining 3% consist of displaced amacrine cells), these results demonstrate extensive ganglion cell loss in the central retina in AD.

Somatostatin (SRIF) is a neuroactive peptide that is distributed throughout the nervous system, including the retina. This peptide has been localized to populations of amacrine cells in a variety of vertebrate species. In the rabbit retina, SRIF immunoreactivity is present in a sparse population of medium to large neurons (13.72 microns in diameter, or 147.84 mu 2) in the ganglion cell layer and in a small number of neurons in the inner nuclear layer. These cells display a preferential distribution to the inferior retina, with the highest density near the ventral and ventrolateral retinal margins (11.33 cells/mm2). SRIF-immunoreactive cells have two to five primary processes that arborize in the proximal inner plexiform layer (IPL). These give rise to a plexus of finer processes in the distal IPL. Occasional immunoreactive processes are also present in the outer plexiform layer. In the IPL, these laminar networks are present in all retinal regions. In addition, SRIF-immunoreactive cells often have a fine-caliber axonlike process that eminates from the soma or perisomal region. These processes travel for great distances across the retina in either the nerve fiber layer or in the distal IPL but are never seen to enter the optic nerve head. In addition, the number of SRIF-immunoreactive somata remains unchanged following transection of the optic nerve. Taken together, these data indicate that SRIF-immunoreactive neurons of the rabbit retina are displaced amacrine cells. Furthermore, the sparse distribution of SRIF-immunoreactive somata, the wide-ranging, asymmetric arborization of their cellular processes, and previous pharmacological studies suggest that these neurons mediate a broad modulatory role in retinal function.

Müller cells have been proposed to play an important role in photoreceptor cell development during the final stages of retinal maturation. The effect of disrupting Müller cells during mouse retinal development was investigated using the specific glial cell toxin, DL-alpha-aminoadipic acid (AAA). By giving multiple systemic injections over several days, impairment of Müller cell function was maintained during the period of photoreceptor migration and differentiation. Following three consecutive days of AAA treatment [commencing on post-natal (P) day 3, 5, 7 or 9, and examined at P8-P14], clumps of photoreceptor nuclei were displaced through the inner segments, lying immediately beneath the retinal pigment epithelium (RPE). Apart from the scalloped appearance of the outer retina, the overall lamination pattern of the retina was relatively well preserved. Even when AAA treatment commenced as early as P3, several days prior to the formation of the outer nuclear layer, the majority of photoreceptors migrated to their correct position and formed inner and outer segments. Therefore, the signals for photoreceptor migration are either provided by the Müller cells prior to P3, or, alternatively, are derived from different intrinsic or extrinsic cues. Disruption of Müller cell function was evidenced by decreased glutamine synthetase activity as well as by increased glial fibrillary acidic protein (GFAP) and decreased cellular retinaldehyde-binding protein (CRALBP) immunoreactivity. Immunocytochemistry with an antibody to CD44, which labels the microvilli of Müller cells at the outer limiting membrane, coupled with electron microscopic analysis, demonstrated that the zonulae adherentes between Müller cells and photoreceptors were either irregular or absent in areas adjacent to displaced clumps of photoreceptors. Thus AAA treatment of early post-natal mice results in localized disruption of the contacts between Müller cells and photoreceptors. These pathologic changes persist into adulthood since at P28, while short stretches of photoreceptors appeared relatively normal with fully developed outer segments, periodic clumps of displaced photoreceptor nuclei were still present adjacent to the RPE. In conclusion, Müller cell processes at the outer limiting membrane appear to play a critical role in providing a barrier to aberrant photoreceptor migration into the subretinal space.

Purpose: To study the time course of visual cell damage resulting from hyperthermic light exposure and the possible involvement of rod outer segment (ROS) lipids in the process., Methods: Rats were acclimated in darkness for 2 hours in a hyperthermic chamber to elevate core body temperature and then exposed to intense green light for up to 4 hours during hyperthermia. After light exposure, the animals were either sacrificed immediately for biochemical or morphologic analysis of retinal light damage or returned to darkness for up to 2 weeks at ambient temperature before analysis. Rod outer segment lipid profiles were characterized, and visual cell loss was determined by rhodopsin and visual cell DNA measurements. Morphology was performed at the light and electron microscopic level., Results: Retinal damage resulting from hyperthermic light exposure was found to be temperature, time, and light intensity dependent. At an elevated environmental temperature of 34.5 degrees, 50% visual cell loss was found after 1.5 hours of 1100 lux light exposure; the same degree of visual cell loss occurred after only 1 hour when rats were maintained at 37 degrees C. At ambient temperatures, 4 hours of light exposure had no effect on visual cell loss. Irrespective of environmental temperature, when rats were maintained in darkness no visual cell loss occurred. Whereas docosahexaenoic acid (22:6) was unchanged in the purest fraction of ROS isolated immediately after light treatment, a 5 mol% loss of the polyunsaturated fatty acid was found in ROS isolated 2 or 24 hours after light exposure. Rod outer segment lipid composition was largely unaffected by hyperthermic light exposure, but the density of some ROS increased. Morphologically, the ROS appeared to be nearly normal immediately after hyperthermic light exposure and structurally more abnormal 2 and 24 hours later. The retinal pigment epithelium exhibited damage immediately after exposure, which also increased 2 and 24 hours later., Conclusions: Hyperthermia in rats dramatically accelerates retinal light damage compared with light exposure under euthermic conditions. Over loss of ROS 22:6 does not occur during hyperthermic light exposure, but it is apparent during the 24-hour period after light treatment. This suggests that the disappearance of 22:6 from ROS occurs in tandem with the process of visual cell death resulting from retinal light damage.

A rhodopsin-related protein is preferentially expressed at high levels in retinal pigment epithelium (RPE) and in Müller cells. The putative RPE-retinal G protein-coupled receptor (RGR) was localized in light-adapted bovine retina by means of electron microscopic immunocytochemistry. In the RPE, the protein was localized to a widespread intracellular compartment. Except for the region adjacent to the basal surface, the RPE cytoplasm was labeled throughout the cell including the apical surface. In Müller cells also RGR was found in the intracellular compartment, especially in the cytoplasm in the region of the Müller cell endfeet and proximal cell processes. Subcellular fractionation studies of bovine RPE and neural retina indicated that RGR is a membrane-bound protein. The intracellular localization of RGR is a unique variation in the subcellular distribution of seven-transmembrane-domain receptors and suggests an unconventional role for RGR in the signal transduction process.

Peanut agglutinin, a lectin with high binding affinity for galactose-galactosamine disaccharide, was used to monitor changes in the photoreceptor cell layer of mice with inherited retinal degeneration. Mice homozygous for the retinal degeneration (rd) gene exhibit a rapid loss of rod photoreceptor cells in the first postnatal month. Previous studies have shown that aggregates of peanut agglutinin-binding cells are observed in the outer nuclear layer in the retinal degenerative mouse at between postnatal days 10 and 18, a period during which massive photoreceptor degeneration occurs in this mutant. This study was performed to determine whether these peanut agglutinin-positive cell clusters represent degenerating photoreceptor cells or, alternatively, macrophages that have migrated into the photoreceptor cell layer. Electron microscopic cytochemistry, using horseradish-peroxidase-conjugated peanut agglutinin, was used to trace cellular processes of peanut-agglutinin-stained cell clusters. Additionally, macrophage-specific antibodies were employed to determine whether macrophages were present in the clusters. The cell clusters did not react with macrophage-specific antibodies. However, processes of cells in peanut-agglutinin-bound clusters could be traced by electron microscopic serial sections to both the outer limiting membrane and the outer synaptic layer. These results provide strong evidence that peanut-agglutinin-bound cells seen during this stage of degeneration in the rd mouse are degenerating photoreceptor cells. Since peanut agglutinin has been shown to bind preferentially to cone, but not to rod, photoreceptor cells, the results also suggest that the clusters may be aggregates of degenerating cones.

In dark-reared albino rats, exposure to 2 or 3 hr of intense light interrupted by 2 hr dark periods resulted in extensive degeneration of photoreceptor cells and degeneration of the retinal pigment epithelium (RPE). Ascorbate (ie, vitamin C) administration prior to light exposure protected photoreceptors and the RPE from light damage. In the present study, ascorbate-treated and untreated rats were exposed to various cycles of intermittent light. Immediately after this light exposure, phagosome frequency in the RPE was morphologically evaluated in comparable 50 microns sections. In untreated rats, exposure to 2 or 3 hr of intermittent light resulted in a five- to sixfold increase in phagosome density compared to unexposed controls. In contrast, no increase in phagosome density was observed in ascorbate-treated rats. In these animals, under all lighting regimens, phagosome levels remained essentially identical to those in rats not exposed to light. After a single nondamaging light exposure, phagosome density remained at the level of dark controls in ascorbate-treated and untreated rats. These results indicate that phagosome frequency may serve as an index for light damage and that the protective effect of ascorbate may be linked to its capacity to prevent rod outer segment shedding and phagocytosis under intense light conditions.

The Purkinje cell degeneration (pcd) mutant mouse rapidly loses cerebellar Purkinje cells and about 50% of its retinal photoreceptor cells at between 3 and 5 weeks of age, and thereafter slowly loses the remaining photoreceptor cells during the first year of life. An ultrastructural study of the developing photoreceptor cells of the pcd/pcd retina was undertaken using both transmission and scanning electron microscopy to characterize further the previously reported retinal vesicles associated with this mutation. Transmission electron microscopy (TEM) revealed an abundance of 'bead-like' vesicles or excrescences in the extracellular matrix surrounding the inner segment region at post-natal day (P) 25. The vesicles are membrane bound, amorphous in appearance and vary in size from 125 to 370 nm. Scanning electron microscopy suggests that the vesicles seen with TEM are actually spherules formed from outpocketing and pinching off of the plasma membrane in the mitochondria-rich region of the rod inner segment. At P25, the spherules are concentrated in the interphotoreceptor space at the level of rod inner segments; at P40, however, they are displaced from their origin and appear mostly at the level of rod outer segments and in the subretinal space.

The protective effect of dimethylthiourea (DMTU) against retinal light damage was determined in albino rats reared in darkness or in weak cyclic light. Rats maintained under these conditions were treated with DMTU at different concentrations and dosing schedules and then exposed for various times to intense visible light, either intermittently (1 hr light and 2 hr dark) or continuously. The extent of retinal light damage was determined 2 weeks after light exposure by comparing rhodopsin levels in experimental rats with those in unexposed control animals. To determine the effect of DMTU on rod outer segment (ROS) membrane fatty acids, ROS were isolated immediately after intermittent light exposure, and fatty acid compositions were measured. The time course for DMTU uptake and its distribution in serum, retina, and the retinal pigment epithelium (RPE)/choroid complex was determined in other rats not exposed to intense light. After intraperitoneal injection of the drug (500 mg/kg body weight), DMTU appeared rapidly in the serum, retina, and the RPE and choroid. In the ocular tissues, it was distributed 70-80% in the retina and 20-30% in the RPE and choroid. This antioxidant appears to have a long half-life because it was present in these same tissues 72 hr after a second intraperitoneal injection. For rats reared in the weak cyclic light environment, DMTU (two injections) provided complete protection against rhodopsin loss after intense light exposures of up to 16 hr. Only 15% rhodopsin loss was found in cyclic-light DMTU-treated rats after 24 hr of intermittent or continuous light. For rats reared in darkness, DMTU treatment resulted in a rhodopsin loss of less than 20% after 8-16 hr of continuous light and approximately 40% after similar exposure to intermittent light. Irrespective of the type of light exposure, rhodopsin loss in the dark-reared DMTU-treated rats was nearly identical to that found in uninjected cyclic light-reared animals. In rats from both light-rearing environments, DMTU treatment prevented the light-induced loss of docosahexaenoic acid from ROS membranes. As measured by rhodopsin levels 2 weeks later, DMTU was most effective when given as two doses administered 24 hr before and just before intense light exposure. As a single dose given during continuous light exposure, DMTU protected cyclic light-reared rats for at least 4 hr after the start of exposure but was ineffective in dark-reared animals if injected 1 hr after the start of light. It was also ineffective in both types of rats when given after light exposure.(ABSTRACT TRUNCATED AT 400 WORDS)

Perfluorocarbon liquids (PFCL) are fully fluorinated, synthetic, transparent compounds with a high specific gravity. These compounds are being increasingly used as an intraoperative tool for repair of complicated retinal detachments. A known complication of their use, however, is liquid entering the subretinal space via a retinal break. The purpose of this study was to evaluate the effects of two of these liquids when placed subretinally in the rabbit eye. Vitrectomy, retinotomy, and subretinal injection of 0.03 cc of either perfluoroctane, perfluorotributylamine, or balanced salt solution (control eyes) were performed on 36 rabbit eyes. Animals were monitored clinically by indirect ophthalmoscopy and fundus photography for up to 21 days. After the 21-day observation period, electroretinograms (ERG) were taken before the rabbits were killed. Histopathologic studies were done at 3 hours, 24 hours, 3 days, 7 days, 14 days, and 21 days after injection. Three eyes demonstrated tearing of the retinotomy site due to downward migration of the PFCL droplet. Results of the ERGs were normal in all animals tested. Phagocytosis of PFCL droplets by the retinal pigment epithelium (RPE) was observed in 1 eye 3 hours after injection. Three of the eyes that received PFCL injections and all of the control eyes demonstrated moderate intracellular edema of both inner and outer nuclear layers as early as 24 hours after injection. In one eye injected with PFCL, these changes progressed to swelling and cystic formation of the inner nuclear layer and mild degeneration of the outer photoreceptor segments 3 days after injection. It was assumed that these effects occurred on a mechanical basis and were not related to PFCL toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent reports (Hinton et al. 1986; Blanks et al. 1989) document the involvement of the retina in the constellation of neurodegenerative changes present in Alzheimer's disease (AD). These studies demonstrate the degeneration of large numbers of optic nerve axons and loss of retinal ganglion cells (RGCs) in patients with AD, but the quantitative changes in the retina of patients with AD compared with age-matched controls have not been examined. An important question is whether the lesion affects the macula, the area of highest visual acuity and the region of the greatest density of cone photoreceptor cells and RGCs. Additionally, it is unknown if patients with AD have a uniform thinning of cells in the ganglion cell layer (GCL) or if there is a differential loss of the medium- to large-sized cells, as suggested earlier (Bassi et al. 1987) and documented histopathologically in some areas of the central nervous system of patients with AD (Kemper 1984). If patients with AD were to show a differential loss of large versus small RGCs with characteristic differences in density, distribution, central projections, and physiologic properties (see review by Rowe and Stone 1977), then a loss of the visual functions normally ascribed to these classes of mammalian RGCs might be expected. This quantitative study of the retinal lesions in the macula of patients with AD provides important data on the progression of the disease and may eventually be the basis for diagnostic procedures for assessing the severity of AD.

Although lectins have been used to study surface oligosaccharides of photoreceptor cells in intact retinas and dissociated retinal cells, the specificity of lectin binding to cones versus rods in a variety of species has not been examined closely. The authors previously found that application of fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA), a lectin with high affinity for galactose-galactosamine disaccharide residues, to cryostat sections of unfixed mouse retina results in staining that is confined to synaptic regions and a subpopulation of photoreceptor cells. To further investigate the possibility that PNA binding is specific for cone photoreceptors, the authors extended their studies to include the duplex retinas of fish, rabbit, monkey, and human in addition to the cone-dominant retina of the chick. These studies show that PNA binding is specific for cone inner and outer segments and also is likely to be associated with the large synaptic pedicles of cone photoreceptor cells. In addition, the authors compared PNA binding with that of Ricinus communis agglutinin I (RCA), another lectin that preferentially binds terminal D-galactose moieties. While RCA does bind to cones in the species examined, it also binds to a lesser extent to rod photoreceptor inner segments. The pattern of binding of RCA in other regions of the retina differs markedly from that of PNA. Significantly, RCA serves as a specific marker for retinal vasculature in the human, monkey, and mouse. These results suggest that certain PNA-binding macromolecules may be important in defining the molecular and cellular specificity of cone photoreceptor cells and that PNA may provide a means for the isolation of cones and cone-specific molecules. RCA may prove to be of value in monitoring vascular changes associated with normal development and pathologic conditions.

Light microscopic autoradiography was used to study the pattern of glycoprotein labeling following intravitreal injection of 3H-fucose in the developing mouse retina. Autoradiograms from three postnatal age groups (7-day, 12-day, and adult) were examined. Distinct labeling patterns were observed in all three age groups which followed the general scheme of incorporation into cell bodies followed by localization in the synaptic layers. Thus, 1 to 2 hr after injection, the label was present in all layers but concentrated within the cell bodies of amacrine, ganglion, and horizontal cells in P7 and P12 animals and amacrine and ganglion cells in the adult animals. In all age groups, the synaptic layers showed increased incorporation compared to nuclear layers and a greater retention of glycoproteins. The major differences noted during development were that the turnover rate of 3H-fucose was faster in 7-day animals than in the P12 or adult animals.

Incorporation of [3H]-cytidine into messenger RNAs (polyadenylated RNAs) was enhanced in light in isolated intact photoreceptor cells of the rat. The increase in polyadenylated (PolyA+)RNAs appeared selective relative to other photoreceptor RNAs since incorporation of [3H]-cytidine into this fraction was up to 10-fold higher while labeling of total cellular RNAs was only two- to three-fold higher in light compared with dark. The photoreceptor cells were isolated in vivo through destruction of inner retina neurons with injections of combinations of neurotoxic substances during early postnatal development. The photoreceptor cells attained normal adult morphology and function: the alpha-wave of the electroretinogram, as well as the thickness of the outer nuclear layer and the length of photoreceptor inner and outer segments, were found to be within the normal range at 4 and 10 weeks of age. In addition to RNA synthesis, such photoreceptor cell preparations when incubated in vitro demonstrated a capacity for regulating light-dependent sodium fluxes comparable to that within the intact retina. The potential usefulness of this model for exploring the molecular biology of photoreceptor cells is discussed.

The use of acrylamide as an embedding medium for vertebrate retinal tissue and its applicability to lectin and antibody-based cytochemical studies is described. The acrylamide technique has numerous advantages over those using fresh-frozen or paraffin embedded material. The morphological integrity of retinal tissue prepared in acrylamide is equivalent to that obtainable with paraffin and superior to that of fresh-frozen material. In addition, this technique alleviates problems often encountered with the thermal and chemical treatments required in the paraffin method. The acrylamide technique allows the localization of lectin and antibody-binding sites essentially unaltered by the fixation and embedding protocol, as in frozen sections, while maintaining tissue morphology similar to that of paraffin-embedded tissue. It is hoped that this approach will be useful to other workers in vision research employing lectin, antibody or other cytochemical approaches to the study of cellular structure and function.

The internal limiting membrane (ILM) and cortical vitreous of the rabbit and primate were studied with transmission electron microscopy following staining with the cationic dye, Alcian blue GX. An unusual feature of the cortical vitreal collagen fibril was its displacement from the ILM: it did not insert into the lamina densa. The separation between vitreal collagen and the ILM was especially noticeable in the rabbit eye, which possessed an extremely strong vitreal retinal attachment in the posterior fundus. The lack of fibril insertion was observed in rabbit tissue that had been fixed by quick freezing on a helium-cooled copper block. The similarity in the appearance of tissue fixed by glutaraldehyde, glutaraldehyde supplemented with Alcian blue, or by quick freezing suggested that the lack of collagen fibril insertion into the ILM was an accurate representation of the relationship between collagen and the ILM. It was found that these two animal species had radically different ILM's; the rabbit ILM was a thin basement membrane throughout all areas of the posterior fundus, whereas the ILM of the cynomolgus monkey was a thick basement membrane in the peripapillary region and a thin basement membrane in the region of the fovea centralis. The topographic variations in the primate ILM thickness and appearance followed the pattern observed in human eyes. Like man, the thickening of the cynomolgus ILM in the posterior fundus was age related. The similarity between the cynomolgus and human ILM suggests that this animal would be more suitable than the rabbit for studying age-related changes or alterations in the strength of vitreal attachment following trauma.

Glycoconjugates, including glycolipids, glycoproteins, and proteoglycans, are present in the plasma membrane of photoreceptor cells and in the interphotoreceptor matrix surrounding photoreceptor cell ellipsoids and outer segments. Although the precise function of these molecules is unknown, they may be important in mediating photoreceptor-pigment epithelial cell interactions, outer segment membrane assembly, and/or disc shedding. Lectins, affinity ligands for defined carbohydrate sequences, have proven particularly useful in studying the glycoconjugate composition of the interphotoreceptor matrix. The peanut lectin selectively binds to domains of the interphotoreceptor matrix surrounding cone ("cone matrix sheaths"), but not rod inner and outer segments. This is evidence for the existence of chemical and structural heterogeneity within the interphotoreceptor matrix. The studies described herein utilized ultrastructural pre-embedding histochemical labeling to assess whether, in addition to the surrounding interphotoreceptor matrix, peanut lectin binding is associated directly with that plasma membrane of cone inner and outer segments. This study confirms that ferritin-conjugated peanut agglutinin binds to cone matrix sheaths, and, in addition, provides ultrastructural evidence for the presence of binding to the plasma membrane surrounding cone inner and outer segments. The data suggest that cone membrane-associated peanut agglutinin-binding molecules may differ from those located within cone matrix sheaths.

Neuronal degeneration is one of the hallmarks of Alzheimer disease (AD). Given the paucity of molecular markers available for the identification of neuronal subtypes, the specificity of neuronal loss within the cerebral cortex has been difficult to evaluate. With a panel of four monoclonal antibodies (mAbs) applied to central nervous system tissues from AD patients, we have immunocytochemically identified a population of vulnerable cortical neurons; a subpopulation of pyramidal neurons is recognized by mABs 3F12 and 44.1 in the hippocampus and neocortex, and clusters of multipolar neurons in the entorhinal cortex reactive with mAb 44.1 show selective degeneration. Closely adjacent stellate-like neurons in these regions, identified by mAB 6A2, show striking preservation in AD. The neurons recognized by mAbs 3F12 and 44.1, to the best of our knowledge, do not comprise a single known neurotransmitter system. mAb 3A4 identifies a phosphorylated antigen that is undetectable in normal brain but accumulates early in the course of AD in somas of vulnerable neurons. Antigen 3A4 is distinct from material reactive with thioflavin S or antibody generated against paired helical filaments. Initially, antigen 3A4 is localized to neurons in the entorhinal cortex and subiculum, later in the association neocortex, and, ultimately in cases of long duration, in primary sensory cortical regions. mAb 3F12 recognizes multiple bands on immunoblots of homogenates of normal and AD cortical tissues, whereas mAb 3A4 does not bind to immunoblots containing neurofilament proteins or brain homogenates from AD patients. Ultrastructurally, antigen 3A4 is localized to paired helical filaments. Using these mAbs, further molecular characterization of the affected cortical neurons is now possible.

Light microscopic autoradiography with 3H-thymidine was used as a probe for DNA synthesis on the retinas of the guinea pig, rabbit, and monkey. Relatively heavy labeling was found in the ganglion cell and inner nuclear layers as well as in the photoreceptor inner segments in the guinea pig and monkey. Electron microscopic autoradiography demonstrated that in the ganglion cells and photoreceptor inner segments in the monkey, 89% of silver grains, representing thymidine uptake, were on or near the mitochondria.

The retinal interphotoreceptor matrix (IPM) occupies the space between the neural retina and the retinal pigmented epithelium (RPE), two neuroectoderm-derived epithelia. While the IPM appears to be a major route by which photoreceptor cells receive vital metabolic factors, relatively little is known concerning its structure and function. The studies reported here describe the presence of specialized domains of the IPM that ensheath cone, but not rod, inner and outer segments in pig, monkey, and human retinae. These cone extracellular matrix sheaths are chemically and structurally distinct from the remainder of the IPM as revealed by their specific binding of the lectin peanut agglutinin (PNA) and their structural stability during physical dissociation of the retina. Biochemical studies suggest that the PNA-binding components of the cone matrix sheaths are trypsin-sensitive glycoproteins. These structures may play a role in establishing a specialized microenvironment for cone photoreceptors, maintaining proper orientation of cone outer segments, and/or facilitating cone-RPE interactions.

A battery of eight lectins with different carbohydrate specificities was used to study changes in glycoconjugate expression during cell differentiation in the mouse retina. The lectins tested included concanavalin A (Con A), wheat germ agglutinin (WGA), soybean agglutinin (SBA), peanut agglutinin (PNA), Ulex europaeus agglutinin (UEA), Ricinus communis agglutinin I (RCA), Dolichos biflorus agglutinin (DBA), and Limulus polyphemus agglutinin (LPA). Unfixed frozen sections of adult and early postnatal mouse retina were treated with fluorescein isothiocyanate-conjugated lectins and examined by fluorescence microscopy. The results showed selective lectin binding in both cellular and synaptic retinal layers of the adult mouse and throughout postnatal development. In general, an increase in intensity of fluorescent lectin staining during retinal development was observed for Con A, WGA, DBA, LPA, RCA, and PNA. This suggests an increase in the expression or accessibility of carbohydrate moieties during development. SBA and UEA showed little to no binding to adult or neonatal retina. Retinal vasculature was intensely stained by RCA, both during development and in the adult. All lectins binding to adult or neonatal retinal layers showed some degree of reactivity with the inner segment region of photoreceptor cells. However, only Con A, PNA and WGA bound to photoreceptor outer segments, suggesting significant differences in the glycosylated components of inner and outer segment membranes. PNA bound specifically to a subpopulation of photoreceptor cells and to discrete regions within the outer synaptic layer. The pattern of PNA binding suggests that this lectin binds preferentially to cone photoreceptor inner and outer segments and cone synaptic pedicles rather than to rod photoreceptor cells. This marked specificity of PNA binding suggests that it may provide a basis for the physical separation of cone and rod photoreceptor cells.

We have observed that the lectin Ricinus communis agglutinin I (RCA), which binds to terminal galactose moieties, serves as a marker for vasculature in the mouse retina. The binding of fluorescein-isothiocyanate-conjugated RCA was used to study the development of retinal vasculature in normal mice and in rd (retinal degeneration) mutant mice, which exhibit a massive loss of photoreceptor cells during the first month of life. In the normal mouse, retinal capillaries develop in an ordered manner and are concentrated in three major zones between the inner limiting membrane and the outer plexiform layer. In the rd mouse, the vessels appear to form normally but begin to degenerate by the end of the second postnatal week. By the end of the fourth postnatal week there is approximately a 35% reduction in the total number of vascular profiles in the rd retina compared to normal littermate controls. This reduction in vascularity is temporally associated with the photoreceptor degeneration.

Posterior vitreous detachment (PVD) and epiretinal membranes occur in a number of vitreoretinal diseases. We have developed an experimental model in which we can provide the morphologic correlation of these dynamic processes. The method provides the opportunity to study epiretinal membrane formation with the scanning electron microscope (SEM); with SEM, some epiretinal membranes that could not be readily detected either clinically or by routine light microscopy can now be identified and studied in detail. We performed an experimental posterior penetrating injury with injection of autologous whole blood or blood and lens material into the vitreous. Five eyes with posterior vitreous detachment but no retinal detachment were selected for SEM. A reduction in the cortical vitreous filaments and the presence of epiretinal membranes was apparent with SEM. In most areas the epiretinal membranes were separated from the internal limiting membrane by a narrow cleft; however, limited attachment sites between the epiretinal membranes and retina were observed in areas overlying retinal blood vessels. In two eyes we observed microscopic retinal folds beneath the membranes, demonstrating a possible morphologic correlation between epiretinal cellular contraction and traction on the retina.

Alzheimer's disease is a dementing disorder of unknown cause in which there is degeneration of neuronal subpopulations in the central nervous system. In postmortem studies, we found widespread axonal degeneration in the optic nerves of 8 of 10 patients with Alzheimer's disease. The retinas of four of the patients were also examined histologically, and three had a reduction in the number of ganglion cells and in the thickness of the nerve-fiber layer. There was no retinal neurofibrillary degeneration or amyloid angiopathy, which are typically seen in the brains of patients with Alzheimer's disease. The changes we observed in the patients with Alzheimer's disease were clearly distinguishable from the findings in 10 age-matched controls and represent a sensory-system degeneration that occurs in Alzheimer's disease. Study of the retina in patients with this disease may be helpful diagnostically, and isolation of the affected ganglion cells may facilitate molecular analysis of the disorder.

The cerebellar ataxia in Purkinje cell degeneration (pcd) mutant mice results from the rapid loss of Purkinje cells between 3 and 5 weeks after birth. The loss of photoreceptors in these mutants begins about the same time but proceeds slowly, with most photoreceptors being lost by 1 year of age. In this study the retinas of pcd/pcd mice and their littermate controls from the age of 10 postnatal days to 15 months were analyzed by electron microscopy. The first signs of photoreceptor cell degeneration are apparent in the region of photoreceptor inner segments as early as postnatal day 13, and more prominently at day 18. During this time, the degeneration is characterized by a large number of vesicles, ranging in diameter from 150 to 350 nm, which are located in the extracellular space adjacent to the photoreceptor inner segments. Analysis of serial sections shows that most of these membrane-bound degeneration profiles are tubular in shape and some are continuous with the cell membrane of the inner segment. Therefore, these "profiles" are thought to arise from tubular outpocketings of the inner segments which cleave off to form isolated membrane-bound profiles. This represents a new and unusual form of photoreceptor degeneration. While the most obvious abnormality in the retina is degeneration of photoreceptor cells, Müller cells also appear to be affected, with swollen apical processes often seen coursing through the outer nuclear layer.

A new experimental model of subretinal cellular proliferation, based on injection of autologous vitreous into the subretinal space of rabbits, was studied by light and electron microscopy. As early as five days after injection, proliferation of retinal pigment epithelial (RPE) and retinal glial cells was observed in the subretinal space. These morphologically distinct proliferating cells were sometimes joined by junctional complexes. Morphologically, the proliferating RPE cells resembled either RPE cells or fibroblasts. Some proliferating RPE cells also retained their epithelial characteristics (ie, basement membranes and cell junctions), while others were partially dedifferentiated and showed some embryonic features. New formation of melanin could be identified within the proliferated RPE cells, which could account, in part, for the hyperpigmentation at the site of the bleb caused by the injection of vitreous. The results demonstrated that injection of autologous vitreous into the subretinal space can lead to subretinal proliferation of retinal glial and RPE cells in the rabbit.

The effect of intraocular liquid silicone on the electroretinogram (ERG) and on retinal morphology was studied in rabbits. After vitrectomy, liquid silicone (1000 centistokes) or balanced salt solution (BSS) was injected into the eyes. The intraocular silicone was well tolerated clinically in all eyes that were followed over a period of 3 days to 6 months. ERG responses were equivalent in operated control and silicone-injected eyes. Light and electron microscopy showed slight but comparable changes in both operated control and silicone-injected eyes that were consistent with either fixation artifact and/or surgical trauma. The results suggest that exposure to silicone for up to 6 months does not have a toxic effect on the rabbit retina.

Peanut agglutinin (PNA), a lectin with affinity for galactose-galactosamine disaccharides, has been employed to monitor alterations in carbohydrate expression in retinal degenerative (rd) mice. Mice homozygous for the rd gene exhibit a rapid loss of rod photoreceptor cells in the first postnatal month; outer segment degeneration is first detected between postnatal days (P) 12-P14 and most rod photoreceptors degenerate by P30. In the rd mouse, PNA-binding cells are observed in the outer nuclear layer (ONL) between P10 and P18, a time corresponding to the intense phase of photoreceptor cell death (LaVail et al., 1982). At no time are PNA-positive cells identified within the ONL of control retinas. PNA-binding cells typically occur in groups and binding of the lectin appears to be restricted to granular elements of the cytoplasm. This report represents the first documentation of changes in the expression and/or accessibility of glycoconjugates in the degenerating ONL of the rd retina. Further analysis of the relationship between regional distribution of PNA-binding cells in the ONL and degenerating photoreceptors of the rd may lead to a better understanding of the mechanism of cell death induced by this mutation.