Meiotic recombination analysis in female ducks (Anas platyrhynchos).

Meiotic recombination in female ducks was directly studied by immunolocalization of MLH1 protein, a mismatch repair protein of mature recombination nodules. In total, 6820 crossovers were scored along the autosomal synaptonemal complexes in 122 meiotic nuclei. From this analysis we predict that the female map length of the duck is 2845 cM, with a genome wide recombination rate of 2 cM/Mb. MLH1-focus mapping along the six largest bivalents shows regional variations of recombination frequencies that can be linked to differences in chromosome morphology. From this MLH1 mapping it can be inferred that distally located markers will appear more separated in genetic maps than physically equidistant markers located near the centromeres on bivalents 1 and 2. Instead, markers at interstitial positions on the acrocentric bivalents 3-6 will appear more tightly linked than expected on the basis of their physical distance because recombination is comparatively lower at the mid region of these chromosomes. The present results provide useful information to complement linkage mapping in ducks and extend previous knowledge about the variation of recombination rates among domestic Galloanserae.

Chromosomal axis formation and meiotic progression in chicken oocytes: a quantitative analysis.

The assembly and disassembly of the synaptonemal complexes (SCs) correlate with the progression of meiotic prophase I. Using immunostaining of the cohesin component SMC3, which is present in the axial elements of the SC, we characterized the synaptic process in chicken oocytes and quantified the frequency of the different prophase stages at hatching and at 3 different ages after hatching. The analysis provides detailed quantitative data regarding the meiotic stages in the chicken ovary showing that the maximum amount of pachytene oocytes is found around hatching and that oocytes reach the diplotene stage 5 days after entering into meiosis. We confirmed the asynchrony of the meiotic development in the female chicken gonad showing that the ovary has a composite population of cells at different stages from day 17 before hatching and for several days after hatching. The significance of these results is discussed in relationship to functional experimental procedures that involve avian oocytes.

Age-related accumulation of 3-nitrotyrosine and nitro-A2E in human Bruch's membrane.

Age-related macular degeneration (AMD) is a disease leading to severe visual loss and legal blindness in the elderly population. The pathophysiology of AMD is complex and may include genetic predispositions, accumulation of lipofuscin and drusen, local inflammation and neovascularization. Recently four independent research groups have identified a commonly inherited variant (Y402H) of the complement factor H gene in the genome from different groups of AMD patients. The Y402H variant of CFH significantly increases the risk of AMD and links the genetics of the disease with inflammation. During inflammation there is activation of inducible nitric oxide synthase and release of nitric oxide, which in principal could lead to non-enzymatic nitration within extracellular deposits and/or intrinsic extracellular matrix protein components of human Bruch's membrane. We have identified two biomarkers for non-enzymatic nitration in aged human Bruch's membrane, indicative of inflammation, that include 3-nitrotyrosine identified in Bruch's membrane preparations and nitrated A2E from the lipid soluble extract of the Bruch's membrane preparation. Approximately 30-40 times more A2E is observed in samples of the organic soluble extract of lipofuscin compared to the extract of Bruch's membrane. It is of interest to note that although A2E is a major constituent of RPE lipofuscin, nitrated A2E could not be detected in RPE extracts. We show here that nitro-A2E is a specific biomarker of nitrosative stress in Bruch's membrane and its concentration correlates directly with tissue age.

Vanadate, tungstate and molybdate activate rod outer segment phosphodiesterase in the dark.

Anionic activation of rod outer segment phosphodiesterase by vanadate, molybdate and tungstate is demonstrated. Comparisons are made to adenylate cyclase, which is known to be activated by vanadate and molybdate but not by tungstate. In view of the differences in anionic activation between these two important enzymatic regulators of intracellular cyclic nucleotide metabolism, it is possible that tungstate can be used as a selective probe for the effects of phosphodiesterase activity in photoreceptors and other cells. The known electrophysiological stimulation of Limulus photoreceptors by these anions is also interpreted in light of our results. If anionic production of quantum bumps in Limulus photoreceptors is mediated by changes in cyclic nucleotides, then the electrophysiological response of Limulus photoreceptors to tungstate may indicate a role for phosphodiesterase rather than adenylate cyclase in mediating light-induced cyclic nucleotide alterations in this cell.

Retinopathy in African Americans and whites with insulin-dependent diabetes mellitus.

BACKGROUND: The development and progression of diabetic retinopathy in African Americans with insulin-dependent diabetes mellitus is not known. METHODS: Two hundred subjects with insulin-dependent diabetes mellitus with duration of diabetes 16 years or less at first visit were studied; 58 were African Americans and 142 were whites. All had gradable stereoscopic color fundus photographs (seven standard fields) from at least two visits (mean time between first and second visit was 4.1 years). Subjects with hemoglobinopathy or proliferative retinopathy or subjects who had evidence of treatment for proliferative retinopathy at first visit were excluded. Masked grading of photographs was conducted using the modified Airlie House classification scheme. RESULTS: African Americans were older, heavier, had higher systolic blood pressure (all P < .05), and marginally higher hemoglobin A1 (HbA1) values (P = .06) than the whites at first visit. African Americans had a lower rate of two steps or more progression from preexistent retinopathy (19%) than whites (43%). Progression to proliferative retinopathy or treatment was similar by race. Multivariate analysis predicting development oe progression of retinopathy, while controlling for length of follow-up, found higher HbA1 (odds ratio [OR] = 2.15), longer duration of insulin-dependent diabetes mellitus (OR = 1.69), higher serum creatinine concentration (OR = 1.59), and white race (OR = 2.62) to be independent risk factors. CONCLUSIONS: These data suggest a previously unsuspected reduction in the adjusted risk for development and progression of retinopathy in African Americans. The reason for this apparently reduced risk are not known.

Repopulation of different layers of host human Bruch's membrane by retinal pigment epithelial cell grafts.

PURPOSE: To determine the morphology of human retinal pigment epithelium (RPE) after reattachment to different ultrastructural layers of human Bruch's membrane (BM). METHODS: Bruch's membrane explants were prepared from eyes of 23 human donors (age range, 11-89 years). The basal lamina of the RPE, inner collagenous layer, and elastin layer were removed sequentially by mechanical and enzymatic techniques. First-passage cells of human RPE (15,000 cells/6 mm explant) from three donors (ages, 52, 64, and 80 years) were plated onto different layers of human BM, and the explants were examined by scanning and transmission electron microscopy up to 21 days later. RESULTS: RPE flattened and extended footplates 6 hours after plating onto basal lamina. Cells remained round 6 and 24 hours after plating onto the inner collagenous, elastin, or outer collagenous layer. The RPE cells became confluent 14 days after plating onto basal lamina but did not become confluent up to 21 days after plating onto the inner collagenous or elastin layer. Sparse round cells were observed 21 days after plating onto deeper layers, suggesting extensive loss of RPE. CONCLUSIONS: The morphology and subsequent behavior of the RPE reattached to BM depends on the anatomic layer of BM available for cell reattachment. The results suggest that the ability of transplanted RPE to repopulate BM in age-related macular degeneration and other disorders may depend on the layer of BM available to serve as a substrate for cell reattachment.

Reattachment of cultured human retinal pigment epithelium to extracellular matrix and human Bruch's membrane.

PURPOSE: To determine the mechanism of reattachment of harvested human retinal pigment epithelium (RPE) to RPE-derived extracellular matrix and Bruch's membrane. METHODS: Confluent first-to third-passage human RPE were harvested from tissue culture and plated onto RPE-derived extracellular matrix or human Bruch's membrane exoplants denuded of cells by treatment with 0.02 N ammonium hydroxide. The authors measured RPE reattachment to uncoated surfaces or surfaces precoated with extracellular matrix proteins (fibronectin, laminin, vitronectin, or type IV collagen), antibodies to extracellular matrix-proteins, or the synthetic peptide RGDS (arginine-glycine-aspartate-serine). Some RPE were pretreated with anti-beta 1 integrin antibodies before plating onto either substrate. RESULTS: Coating the surface of either RPE-derived extracellular matrix or Bruch's membrane with fibronectin, laminin, vitronectin, or type IV collagen increased the RPE attachment rate. Exposing RPE to anti-beta 1 integrin antibodies or RGDS or precoating the surface with antibodies to fibronectin, laminin, vitronectin, or type IV collagen decreased the RPE attachment rate to both surfaces. The RPE attachment rate to Bruch's membrane was lower when the exoplants were harvested from the macula of older (age, 70 to 90 years) versus younger (age, 30 to 40 years) persons (52.4 +/- 3.6% versus 64.3 +/- 3.5%, respectively; P < 0.05). CONCLUSIONS: The attachment of cultured human RPE cells to human Bruch's membrane or to RPE-derived extracellular matrix is mediated by an interaction between the beta 1-subunit of integrin on the RPE surface and ligands in the extracellular matrix that include laminin, fibronectin, vitronectin, and type IV collagen. The lower rate of RPE reattachment to the macula from older human cadaveric eyes may have implications for studies aimed at RPE transplantation in elderly persons.

Fate of human retinal pigment epithelial cells seeded onto layers of human Bruch's membrane.

PURPOSE: To determine the fate of human retinal pigment epithelial (RPE) cells seeded onto different layers of human Bruch's membrane (BM). METHODS: Bruch's membrane explants were prepared from 16 human cadaver eyes (7 eyes age <50 years; 9 eyes >50 years) by removing native RPE cells with ammonium hydroxide to expose the RPE cell basal lamina (BL). The inner collagenous layer (ICL) and elastin layer (EL) were exposed by removing apical layers sequentially by mechanical and enzymatic means. Synchronized first passage human RPE cells (15,000 cells/(6-mm-diameter explant) were plated onto each layer of human BM. The RPE cell reattachment and apoptosis rates at 24 hours, proliferation rates and mitotic index 24 hours after growth stimulation, and the ability of RPE cells to repopulate the explant surface were determined on each layer. RESULTS: RPE cell reattachment was highest on BL but decreased on deeper layers of BM. The apoptosis rate of attached cells increased as deeper layers of BM were exposed. The proliferation rate and mitotic index of the grafted cells were higher on BL than on deeper layers. RPE cells plated onto BL repopulated the explant surface within 14 +/- 3 days, whereas cells plated onto the ICL and EL eventually died and never reached confluence. CONCLUSIONS: The fate of RPE cells seeded onto BM depends on the ultrastructural layer of BM available for reattachment. These findings suggest that the ability of transplanted RPE cells to repopulate bare BM will depend on the layer of BM available for RPE cell reattachment.

Fluorescence light microscopy of F-actin in retinal rods and glial cells.

The actin cytoskeleton of rod photoreceptors and glial cells in toad retina has been directly viewed using fluorescence microscopy of cells labeled with a potent phallotoxin that specifically binds to F-actin. The three-dimensional organization of this cytoskeletal protein consists of actin filaments, which course through the inner segment and end at the tips of the calycal processes surrounding the base of the outer segment. A transverse layer of actin staining is also observed at the base of rod outer segments in the region where new discs are formed. At the level of the external limiting membrane, evidence has been found for rings of actin within the glial cells that surround the photoreceptors. These actin rings form a structural meshwork in which photoreceptor cells are embedded.

Reattachment rate of human retinal pigment epithelium to layers of human Bruch's membrane.

OBJECTIVES: To determine the reattachment rate of human retinal pigment epithelium (RPE) to different layers of human Bruch's membrane (BM). METHODS: Explants of BM were prepared from 10 human cadaver eyes by removing native RPE. The RPE basal lamina, inner collagenous layer, elastin layer, and outer collagenous layer were exposed by sequentially removing each apical layer by mechanical or enzymatic means. First-passage human RPE was plated onto the surface and the RPE reattachment rate to each layer of BM was determined. RESULTS: Retinal pigment epithelial cell reattachment was highest to the inner aspects of BM and decreased as deeper layers of BM were exposed (ie, reattachment rate to basal lamina was higher than to the inner collagenous layer, which was higher than to the elastin layer, which was higher than to the outer collagenous layer). The reattachment rate to the inner collagenous layer, elastin layer, and outer collagenous layer harvested from elderly donors (age >60 years) was less than the reattachment rate to corresponding layers harvested from younger (age <50 years) donors. CONCLUSIONS: Retinal pigment epithelial cell reattachment depends on the anatomical layer of BM present in the host tissue. Age-related changes in BM may interfere with RPE reattachment. Our observations may have implications for understanding the pathogenesis of age-related macular degeneration and its potential treatment with RPE transplantation techniques.

Spontaneous resolution of retinal detachment occurring after macular hole surgery.

OBJECTIVE: To document the spontaneous resolution of retinal detachment developing after macular hole surgery. METHODS: We identified all patients who developed a postoperative retinal detachment after undergoing macular hole surgery at Washington University School of Medicine, St Louis, Mo; the surgery was performed by one of us (L.V.D.P. or H.J.K.) between 1991 and 1996. RESULTS: Six of 73 eyes developed a postoperative retinal detachment; the retinal detachment was inferior in all cases. Two eyes that had inferior retinal breaks underwent further surgery to repair the retinal detachment. Retinal breaks could not be identified in the other 4 eyes; the retinal detachment resolved without further surgery in all 4 of these eyes. CONCLUSION: The recognition that retinal detachment occurring after macular hole surgery can resolve without additional surgery may result in the avoidance of further surgical intervention in some eyes.

Morphology of pig retinal pigment epithelium maintained in organ culture.

Exoplants of porcine retinal pigment epithelium (RPE) attached to Bruch's membrane, choroid, and sclera were maintained in organ culture for up to four weeks. Four-millimeter round buttons of eye wall that contained RPE, choroid, and sclera were trephined from freshly enucleated pig eyes and incubated at 37 degrees C in Eagle's minimum essential medium with 10% fetal calf serum. The RPE cells remained as a monolayer for at least four weeks in organ culture, and individual RPE cells became taller and dome shaped. The RPE cells retained several prominent ultrastructural features, including apical microvilli, intracellular melanosomes and mitochondria, and intercellular tight junctions. Since the cellular substratum can exert important influences on cell behavior, the ability to maintain RPE cells attached to Bruch's membrane provides a new in vitro tool for studying the metabolic activity of this tissue and its response to external stimuli, including laser photocoagulation.

Human photoreceptor transplantation in retinitis pigmentosa. A safety study.

OBJECTIVE: To establish the technical feasibility and safety of photoreceptor transplantation in retinitis pigmentosa. METHODS: A sheet of human photoreceptor cells was harvested from 2 human cadaveric eyes with a vibratome and transplanted into the subretinal spaces of 2 patients with advanced retinitis pigmentosa and visual acuity of no light perception by means of submacular surgery techniques. Preoperative and postoperative electrophysiologic testing, fundus photography, fluorescein angiography, and scanning laser ophthalmoscopy were performed. RESULTS: Twelve months after photoreceptor transplantation, the visual acuity of each patient remained no light perception. The temporal edge of the retinotomy in 1 patient was folded but was not associated with a retinal detachment. The patients were not immunosuppressed, and there was no evidence of rejection of the allogeneic transplant. Cystoid macular edema, uveitis, and macular pucker were not observed. CONCLUSION: A sheet of adult human photoreceptor cells can be harvested from human cadaveric eyes and safely transplanted to the subretinal spaces of patients with retinitis pigmentosa without systemic immunosuppression.

Response of pig retinal pigment epithelium to laser photocoagulation in organ culture.

Laser photocoagulation was applied in vitro to organ culture exoplants of porcine retinal pigment epithelium (RPE) attached to Bruch's membrane. Four-millimeter-round buttons of eye wall containing RPE, choroid, and sclera were treated with 25 spots from the argon blue-green laser using 300 mW of power, a 500-micron spot size, and 0.1-s duration. Laser photocoagulation disrupts individual RPE cells acutely and lifts damaged RPE cells from Bruch's membrane. Treated areas become covered with irregular mounds of RPE cells within seven days. The acute damage and subsequent repair of the RPE in organ culture mimic the response of the RPE following laser photocoagulation in vivo. Thus, the morphologic response of the RPE to laser photocoagulation is an intrinsic property of this tissue that does not depend on the presence of the overlying retina.

Débridement of the pig retinal pigment epithelium in vivo.

OBJECTIVE: To study the morphologic effects of surgical débridement of the retinal pigment epithelium (RPE) in an animal model. METHODS: A pars plana vitrectomy was performed in the domestic pig, and a neurosensory retinal detachment was created by injecting the calcium-chelating agent edetic acid (commonly referred to as ethylenediaminetetraacetic acid or EDTA) into the subretinal space through a retinotomy. Twenty minutes later, the RPE was débrided by gently brushing Bruch's membrane with a soft-tip silicone catheter. Dissociated RPE was aspirated from the subretinal space, and the retina was reattached with a fluid-gas exchange. RESULTS: Light microscopic analysis confirmed that Bruch's membrane was devoid of native RPE and the choriocapillaris was morphologically intact immediately after débridement. Photoreceptor outer segments were disrupted and foreshortened immediately after RPE débridement. One to 4 weeks later, a layer of hypopigmented RPE covered most of the previously débrided areas of Bruch's membrane. The choriocapillaris was intact in areas of Bruch's membrane that were repopulated by hypopigmented RPE, and remained intact 12 weeks after débridement. Some regions of Bruch's membrane near the retinotomy remained devoid of RPE for more than 4 weeks after débridement. The choriocapillaris was atrophic and there was extensive disruption of the outer retinal layers in these areas. CONCLUSIONS: The RPE healed in most areas after surgical débridement of the RPE in the experimental animal. Atrophy of the choriocapillaris was present in areas of poor RPE healing near the retinotomy.

Retinal pigment epithelial repopulation in monkeys after submacular surgery.

BACKGROUND: Transplantation of retinal pigment epithelium may be a treatment for retinal diseases, such as age-related macular degeneration and hereditary macular degeneration. Before transplantation studies are undertaken, questions concerning repopulation of retinal pigment epithelial cells in situ and photoreceptor repair after submacular surgery need to be addressed. METHODS: We removed the retinal pigment epithelium from Bruch's membrane in the macaque monkey in the macula and outside the vascular arcades. This model allowed the study of in situ retinal pigment epithelium regrowth and photoreceptor repair for 9 months following débridement. RESULTS: Fluorescein angiography revealed a window defect in the area of denuded retinal pigment epithelium. Histologic studies revealed repopulated nonpigmented retinal pigment epithelial cells in the denuded areas in both the early and late periods. At 9 months, the repopulated retinal pigment epithelium was associated with repaired, normal-appearing photoreceptor outer segments. Retinal pigment epithelium regrowth was observed only if Bruch's membrane was intact. CONCLUSIONS: Repopulation of retinal pigment epithelium in the adult primate can occur rapidly and can support the repair of damaged photoreceptors following submacular surgery.

Submacular surgery for subfoveal choroidal neovascular membranes in patients with presumed ocular histoplasmosis.

OBJECTIVE: To determine the visual results, recurrence rates, and postoperative complications of surgical removal of subfoveal choroidal neovascularization (CNV) in patients with the presumed ocular histoplasmosis syndrome. DESIGN: A consecutive surgical series of 63 eyes of 62 patients with subfoveal CNV and the presumed ocular histoplasmosis syndrome with longer than 6 months of follow-up. SETTING: Tertiary care university medical center. METHODS: Patients underwent surgical removal of subfoveal CNV using vitreoretinal surgical techniques. The anatomical and functional results of surgery were analyzed. RESULTS: The median age of the patients was 42 years (range, 16-68 years), and the median follow-up time was 24 months (range, 6-48 months). Visual acuity improved by 2 or more Snellen lines in 22 (35%) of the 63 eyes, was unchanged in 28 (44%) of the eyes, and worsened in 13 (21%) of the eyes. Eleven (17%) of the 63 eyes improved to a visual acuity of 20/50 or better. Eyes with an initial visual acuity of 20/200 or worse had a better prognosis for improved vision (ie, 26 [41%] of the eyes) than those with an initial visual acuity of 20/100 or better (ie, 5 [8%] of the eyes). Recurrence of the subfoveal CNV occurred in 24 (38%) of the 63 eyes and was more common in those eyes that received preoperative laser photocoagulation (ie, 15 [47%] of the eyes). The median time to recurrence was 5 months after surgery. Post-operative complications included macular striae in 4 (6%) of the 63 eyes, rhegmatogenous retinal detachment in 2 (3%) of the eyes, retinal tear in 1 (1.6%) of the eyes, and progression of cataract in 19 (30%) of the eyes. CONCLUSIONS: Surgical excision of subfoveal CNV may be an effective therapeutic modality in patients with the presumed ocular histoplasmosis syndrome that offers the possibility of improving central vision in many patients. Factors possibly associated with a favorable visual prognosis include younger patient age and the absence of previous laser photocoagulation.

Treatment choice and quality of life in patients with choroidal melanoma.

OBJECTIVE: To determine if quality of life differs between patients with choroidal melanoma treated with enucleation and those treated with radiation therapy. MATERIALS AND METHODS: Patients treated for choroidal melanoma at 5 Midwest centers were asked to participate. There were 65 participants treated with enucleation and 82 treated with radiation therapy. Quality of life was assessed using the Medical Outcome Study Short Form 36 and the National Eye Institute Visual Function Questionnaire and by the Time-Tradeoff interview method. RESULTS: The average length of follow-up was 4.9 years for the group treated with radiation therapy and 6.3 years for the group treated with enucleation (P = .05). After adjusting for age, sex, years of follow-up, and the number of chronic conditions, there were few differences in any of the quality-of-life measures by treatment status. Participants in the group treated with radiation therapy were more likely to have higher (better) scores on the Vitality and Mental Component subscales of the Medical Outcome Study Short Form 36 than participants treated with enucleation. There were no differences on the National Eye Institute Visual Function Questionnaire or the Time-Tradeoff measures of quality of life. CONCLUSION: Choice of treatment for choroidal melanoma does not seem to be associated with large differences in quality of life in long-term follow-up.

Drusen in age-related macular degeneration: pathogenesis, natural course, and laser photocoagulation-induced regression.

Drusen are subretinal pigment epithelial deposits that are characteristic of but not uniquely associated with age-related macular degeneration (AMD). Age-related macular degeneration is associated with two types of drusen that have different clinical appearances and different prognoses. Hard drusen appear as small, punctate, yellow nodules and can precede the development of atrophic AMD. Areolar atrophy of the retinal pigment epithelium (RPE), choriocapillaris, and outer retina develop as the drusen disappear, but drusen can regress without evidence of atrophy. Soft drusen appear as large (usually larger than 63 microm in diameter), pale yellow or grayish-white, dome-shaped elevations that can resemble localized serous RPE detachments. They tend to precede the development of clinically evident RPE detachments and choroidal neovascularization. Drusen characteristics correlated with progression to exudative maculopathy include drusen number (five or more), drusen size (larger than 63 microm in diameter), and confluence of drusen. Focal hyperpigmentation in the macula and systemic hypertension also are associated with an increased risk of developing choroidal new vessels (CNVs). Large drusen are usually a sign of diffuse thickening of Bruch's membrane with basal linear deposit, a vesicular material that probably arises from the RPE, constitutes a diffusion barrier to water-soluble constituents in the plasma, results in lipidization of Bruch's membrane, and creates a potential cleavage plane between the RPE basement membrane and the inner collagenous layer of Bruch's membrane through which CNVs can grow. Disappearance of drusen spontaneously and in areas adjacent to laser photocoagulation scars was first noted by Gass (Gass JD: Arch Ophthalmol 90:206-217, 1973; Trans Am Acad Ophthalmol Otolaryngol 75:580-608, 1971). Subsequent reports have confirmed these observations. Photocoagulation-induced drusen regression might prevent patients with drusen from developing exudative maculopathy. The mechanism for spontaneous drusen regression probably involves RPE atrophy. The mechanism for photocoagulation-induced drusen regression is unknown. If photocoagulation-induced drusen regression is anatomically similar to atrophy-associated drusen regression, then the former will be associated with dissolution of basal linear deposit and a residuum of basal laminar deposit. Sarks and coworkers (Sarks JP, Sarks SH, Killingsworth MC: Eye 11:515-522, 1997) proposed that this in turn will eliminate the potential cleavage plane between the RPE basement membrane and inner collagenous layer of Bruch's membrane through which CNVs grow, thus retarding the growth of CNVs.