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.

Duration of action of intravitreal ranibizumab and bevacizumab in exudative AMD eyes based on macular volume measurements.

BACKGROUND/AIMS: Sequential macular volume and central foveal point thickness (CFPT) measurements on optical coherence tomography (OCT) were used to determine the efficacy and duration of action of ranibizumab versus bevacizumab in wet age-related macular degeneration (AMD). METHODS: Retrospective chart review of patients who received their first treatment of intravitreal ranibizumab or bevacizumab for exudative AMD. 316 patients (202 ranibizumab;114 bevacizumab) who received 823 injections (313 ranibizumab;510 bevacizumab) were identified. 74 patients had pre- and post-treatment OCTs performed to determine CFPT and macular volume changes. RESULTS: Ranibizumab caused a significant reduction in CFPT (278 (SD 84) before treatment vs 227 (80) microm after treatment; p = 0.001) and macular volume (7.22 (0.96) vs 6.69 (0.74) mm(3); p = 0.002). Intravitreal bevacizumab caused a similar reduction in CFPT (288 (94) vs 220 (55) microm; p = 0.008) and macular volume (7.36 (1.08) vs 6.50 (0.42) mm(3); p<0.001). The mean duration of action was 74.0 (19.1) days for ranibizumab compared with 101.8 (16.6) days for bevacizumab (p = 0.036; t test). The ratio of the relative duration of action of bevacizumab versus ranibizumab was 1.40 (0.19). CONCLUSIONS: Both drugs are equally effective at reducing CFPT or macular volume. Bevacizumab appears to take longer to achieve the minimum macular volume, and its effects take longer to wear off, suggesting it can be given less often.

Epiretinal membrane removal in diabetic eyes: comparison of viscodissection with conventional methods of membrane peeling.

AIMS: To compare conventional methods of epiretinal membrane peeling with viscodissection. METHODS: 154 eyes with proliferative diabetic retinopathy (PDR) that underwent pars plana vitrectomy with membrane dissection (89 traditional, 65 viscodissection) were studied retrospectively. Incidence of retinal breaks (RBs), length of time under anaesthesia, postoperative intraocular pressure, retinal reattachment rate, and final visual acuity (VA) were measured. RESULTS: To compare cases of similar complexity, a "complexity score" was defined. The average complexity score for cases done with and without viscodissection was 4.7 and 3.2, respectively. The mean frequency of RBs in eyes undergoing viscodissection was 0.43 (SD 0.5) v 0.14 (0.35) RBs/eye without viscodissection. In complex cases, the frequency of posterior/peripheral RBs was 0.31 (0.47)/0.13 (0.34) RBs/eye, respectively, with viscodissection v 0.12 (0.33)/0.23 (0.43) RBs/eye without viscodissection. None of these differences were statistically significant. The average preoperative/postoperative VA (logMAR) in the viscodissection cohort was 1.7/1.3 (range 0.3 to >1.9/0.1 to >1.9) v 1.4/1 (range 0.48 to >1.9/0.1 to >1.9) in the non-viscodissection cohort, among eyes with 6 months of follow up. Anaesthesia duration was significantly shorter for cases done without viscodissection (p=0.03), but cases done with viscodissection were significantly more complex than cases done without viscodissection (p<0.0001). CONCLUSION: Viscodissection appears to be a safe and effective alternative technique in eyes with PDR. Owing to the retrospective nature of the study, additional studies are warranted.

Vitrectomy with silicone oil infusion in severe diabetic retinopathy.

AIMS: To determine the results of pars plana vitrectomy (PPV) and silicone oil infusion (SOI) in severe proliferative diabetic retinopathy (PDR). METHODS: The records of 23 eyes (21 patients: 12 males, nine females) with PDR who had undergone PPV and SOI were reviewed retrospectively. RESULTS: Average follow up was 5.4 months (range 1-25). Surgical indications were tractional retinal detachment (TRD) (17.4%), traction-rhegmatogenous retinal detachment (TRRD) (8.7%), TRD with vitreous haemorrhage (VH) (48%), TRD with neovascular glaucoma (NVG) (8.6%), TRD with fibrinoid syndrome (FS) (17.3%). With one operation, the retinal reattachment rate was 17/23 (74%). Among these 23 eyes, 11 (48%) had previously failed vitrectomy, and the retina was attached in 8/11 (73%) with a single procedure. With additional surgery employing PPV and SOI, the final reattachment rate was 20/23 (87%). The only cases with intraocular pressure <5 mm Hg had retinal detachment. Postoperative visual acuity (VA) improved in 10 eyes (44%), was unchanged in three (12%), and decreased in 10 eyes (44%). CONCLUSION: SO tamponade is useful in severely diseased eyes with PDR, even in the presence of rubeosis iridis (RI) and NVG, FS, or in cases with previously failed vitrectomy, especially in the presence of RI.

Use of viscodissection and silicone oil in vitrectomy for severe diabetic retinopathy.

Improvement in surgical techniques has led to improved anatomic and functional success rates following surgery for severe complications of proliferative diabetic retinopathy (PDR). We compared the anatomic and functional outcomes of surgery in a non-randomized, consecutive case series of patients with severe PDR. We found that viscodissection using Healon provides outcomes comparable to conventional pick and scissors dissection. We also found that adjunctive use of silicone oil can salvage selected cases with particularly severe manifestations of PDR (e.g., the fibrinoid syndrome). With proper selection of patients and techniques, the anatomic success rate can exceed 80% even in the most severe cases. The goal of this paper is to show the applicability of using viscodissection and silicone oil infusion during vitrectomy in eyes with severe PDR.

Pharmacokinetics help optimizing mycophenolate mofetil dosing in kidney transplant patients.

BACKGROUND: Mycophenolic acid (MPA), the active metabolite of mycophenolate mofetil (MMF), is now routinely used as immunosuppressant in solid organ transplantation in a fixed daily dose regimen (2 g/d) in association with cyclosporine (CsA) and steroids. However, no correlation has been shown between fixed MMF dose and clinical outcome. METHODS: Here we examined the possibility of optimizing MMF dosing by drug pharmacokinetic monitoring in 46 stable kidney transplant recipients. MPA plasma concentration profiles were measured by a reverse-phase high-performance liquid chromatography method 6-9 months after transplantation and related with routine laboratory analysis tests. Since MPA is extensively bound to serum albumin and only the free fraction is pharmacologically active, in a subgroup of 23 patients free plasma MPA was also determined. RESULTS: Despite a comparable MMF dose, a large interindividual variability in both MPA area under the curve (AUC) from 0 to 12 h (range 10.1-99.8 microg/mL. h) and in trough levels (range 0.24-7.04 microg/mL) was found. Patients with AUC >40 microg/mL. h showed a better (p<0.05) renal function than patients with lower AUC (creatinine clearance 85.7+/-23.2 versus 64.5+/-17.5 mL/min), despite no difference in CsA dose, CsA AUC and blood CsA trough level. The percentage of free plasma MPA but not total MPA correlated with the red blood cell and leukocyte count. CONCLUSIONS: Therapeutic MMF drug monitoring might contribute to a better management of kidney transplant recipient with the goal of optimizing drug dosing and limiting the risk of MMF-related toxicity.

Retinal pigment epithelial cell transplantation after subfoveal membranectomy in age-related macular degeneration: clinicopathologic correlation.

PURPOSE: To report the histopathology after retinal pigment epithelial cell transplantation and subfoveal membranectomy in age-related macular degeneration. METHODS: An 85-year-old white woman with bilateral choroidal neovascularization underwent subfoveal membranectomy combined with transplantation of a sheet of human adult retinal pigment epithelium (retinal pigment epithelium) under the foveal center in the right eye. The patient was immunosuppressed postoperatively with prednisone, cyclosporine, and azathioprine. The patient died from congestive heart failure 114 days after surgery. RESULTS: A patch of hyperpigmentation was visible at the transplant site under the foveola after surgery. Mound-like clusters of individual round, large densely pigmented cells were present in the subretinal space and outer retina in this area. There was loss of the photoreceptor outer segments and native retinal pigment epithelium in the center of the transplant bed, with disruption of the outer nuclear layer predominantly over regions of multilayered pigmented cells. Cystic spaces were present in the inner and outer retina. A residual intra-Bruchs membrane component of the original choroidal neovascular complex was present under the transplant site. CONCLUSIONS: The transplant site contained clusters of round, pigmented cells that did not form a uniform monolayer in most areas. The morphology at the transplant site is consistent with the lack of visual improvement seen after surgery in this patient.

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.

TGF beta secretion modulates the density-dependent growth of pig retinal pigment epithelium in vitro.

BACKGROUND: We aimed to identify the cytokine(s) responsible for the density-dependent growth regulation of pig retinal pigment epithelium (RPE) in vitro. METHODS: Confluent monolayers of primary pig RPE were established on bovine corneal endothelial extracellular matrix-coated tissue culture well inserts wrapped with dialysis membranes with different molecular weight cutoffs (0.5-50 kDa). These confluent RPE monolayers were then cocultured with first passage porcine RPE plated at a density of 1 cell/mm2, so that the newly plated RPE was bathed with different molecular weight fractions of the confluent cell media. Growth rates of the newly plated RPE were determined 72 h after plating and the molecular weight fraction of the confluent cell medium that inhibits the RPE proliferation was determined. First passage pig RPE (1 cell/mm2) were cocultured with confluent monolayers of primary pig RPE on inserts in the presence of different amounts of TGF-beta neutralizing antibody (0.1-100 microg/ml). Growth rates of the newly plated RPE were calculated 72 h after plating to determine the antibody concentration that would maximize the growth rate of the newly plated RPE in the presence of an adjacent confluent RPE monolayer. RESULTS: The growth rate of the newly plated RPE decreased when RPE were bathed with the 10- to 25-kDa fractions of medium from an adjacent confluent RPE monolayer. This growth inhibition reached statistical significance with the 25- to 50-kDa fractions (p < 0.05), and was abolished by adding pan-specific neutralizing antibody against TGF-beta (0.1-5 microg/ml). Blocking greater amounts of TGF-beta in the medium with higher doses of antibody (>10 microg/ml) also inhibited the growth of the newly plated RPE, in the presence or absence of a neighboring confluent cell layer. CONCLUSION: The TGF-beta family of cytokines mediates the density-dependent growth suppression of RPE in vitro. Neutralizing the effect of these cytokines by adding anti-TGF-beta antibodies can result in more rapid growth of the RPE in vivo.

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.

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.

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.

Minimum number of adult human retinal pigment epithelial cells required to establish a confluent monolayer in vitro.

PURPOSE: To determine the minimum number of cells required to establish a confluent monolayer of retinal pigment epithelium (RPE) with an epitheloid morphology in vitro. METHODS: Primary or passaged human RPE were harvested by trypsinization from 6 donors and plated onto bovine corneal endothelium extracellular matrix-coated tissue culture plastic in 96-well plates. Plating densities ranged from 1 to 66,000 viable cells/well (0.03-2062 viable cells/mm2) for primary cells or 1 to 100,000 viable cells/well (0.03-3112 viable cells/mm2) for passaged cells. The time required to reach confluence was determined by monitoring the cultures daily until they reached confluence. Mean cell area and circularity index at confluence was calculated to determine the effect of different plating densities on final RPE morphology. RESULTS: Primary RPE plated at densities above 10 viable cells/mm2 (320 cells/well) and passaged RPE plated above 2 viable cells/mm2 (64 cells/well) reached confluence on every occasion. There was a negative correlation between the plating density and time required to reach confluence. Plating densities above 3 viable cells/mm2 (96 cells/well) and 50 viable cells/mm2 (1600 cells/well) yielded smaller, rounder cells at confluence for primary and passaged RPE, respectively. CONCLUSIONS: As few as 96 primary RPE cells and 1600 passaged RPE are required to obtain a confluent, 6mm (4-disc diameter) patch of RPE in vitro. This suggests that autologous RPE grafts can be prepared with high efficiency for subsequent transplantation into the subretinal space in vivo.

Surgical removal of extensive peripapillary choroidal neovascularization associated with presumed ocular histoplasmosis syndrome.

OBJECTIVE: This study aimed to report the visual outcome of surgical removal of extensive peripapillary choroidal neovascularization (CNV) due to presumed ocular histoplasmosis syndrome (POHS). DESIGN: Retrospective review of the records of all patients seen at the Barnes Retina Institute who underwent surgical removal of extensive peripapillary CNV associated with POHS and who had at least 12 months of follow-up. PARTICIPANTS: Seventeen consecutive eyes (in 14 patients) undergoing surgical removal of extensive peripapillary CNV associated with POHS were studied. INTERVENTION: Pars plana vitrectomy and surgical removal of CNV were performed. MAIN OUTCOME MEASUREMENTS: Best-corrected Snellen visual acuity, funduscopic examination, and intravenous fluorescein angiography were obtained before surgery and at regular intervals after surgery. RESULTS: In 14 of 17 eyes, the peripapillary CNV was subfoveal, and in 3 eyes, it was extrafoveal. All three eyes with extrafoveal CNV were not eligible for laser treatment according to Macular Photocoagulation Study guidelines because treatment would have spared less than 1.5 contiguous clock-hours of retina temporal to the optic disc. Follow-up ranged from 17 to 57 months, with a median of 32 months. In eyes with subfoveal CNV, best-corrected preoperative Snellen visual acuity ranged from 20/25 to counting fingers at 2 feet with a median of 20/200, and best-corrected final Snellen visual acuity ranged from 20/25 to 20/200 with a median of 20/40. In 7 (50%) of 14 eyes, a final Snellen acuity of 20/40 or better was achieved, and in all cases except 1, visual acuity improved or did not change with surgery. In the three eyes with extrafoveal CNV, best-corrected preoperative Snellen visual acuity ranged from 20/20 to 20/400 with a median of 20/200, and best-corrected final Snellen visual acuity was 20/20 in all cases. In addition, visual acuity improved with surgery. CONCLUSIONS: The data from this small retrospective study suggest that surgical removal may provide visual benefit in selected cases of extensive peripapillary CNV due to POHS.