Advanced glycation end products in age-related macular degeneration

OBJECTIVE: To investigate the localization of N epsilon-(carboxymethyl)lysine (CML), a component and major immunologic epitope of advanced glycation end products, in aged eyes and choroidal neovascular membranes (CNVMs) surgically excised from eyes with age-related macular degeneration. METHODS: Immunohistochemistry for CML was performed using 8 snap-frozen, surgically excised CNVMs. Twelve eyes from patients aged 69 to 82 years and 2 donor eyes, 1 each from a 23-week-old fetus and 21-year-old patient, without age-related macular degeneration or diabetic retinopathy were also examined. To determine if retinal pigment epithelial cells in CNVMs accumulate advanced glycation end products, cytokeratin and CML were stained in paired serial sections. RESULTS: Soft, macular drusen and/or basal laminar and basal linear deposits were observed in 8 of 12 aged eyes. Each case showed CML accumulation, while overlying retinal pigment epithelial cells showed no accumulation in all 12 eyes. In CNVMs, however, retinal pigment epithelial cells showed CML accumulation in their cytoplasm. CONCLUSION: The additional accumulation of advanced glycation end products in soft, macular drusen and/or retinal pigment epithelial cells may play a role in the pathogenesis of CNVM formation in age-related macular degeneration. CLINICAL RELEVANCE: Recently, advanced glycation end products have been found to play a role both in aging changes and neovascularization. Localization of advanced glycation end products in the above-mentioned tissue may lead to a better understanding of the pathogenesis of age-related macular degeneration.     

Mosaicism in sporadic neurofibromatosis 2 patients

More than half of neurofibromatosis 2 (NF2) patients represent de novo mutations which could have occurred at either pre-zygotic or post-zygotic stages. A post-zygotic mutation can result in mosaicism. In four sporadic NF2 patients, we found NF2 mutations in only a portion of corresponding leukocytes. In two other sporadic patients, no mutations were found in leukocytes but constitutional NF2 mutations were suggested by identical mutations in different tumors from each patient. We screened leukocyte DNA from a total of 16 inherited and 91 sporadic NF2 patients, and found NF2 mutations in 13 (81%) of the former and in 46 (51%) of the latter cases. The 30% difference in the rate of detection of mutations ( P = 0.051) might be partially explained by mosaicism in a portion of sporadic NF2 patients who carry the mutations in such a fashion that their leukocytes are unaffected. Among sporadic cases, we found mutations more frequently in patients with severe phenotypes (59%) than in patients with mild phenotypes (23%) (difference of 36%, P = 0.007). Mosaicism might be more common in the latter patient group since small populations of mutation-bearing cells can in some cases result in mild phenotypes and can also lead to difficulties in identifying mutations. No mutations were found in eight patients suspected of having NF2. Mosaicism with an extremely small population of affected cells may explain the incomplete phenotypes in some of these patients and the lack of mutations in their leukocytes. These findings suggest that mosaicism is relatively common in NF2 and may have important implications for diagnosis, prognosis and genetic counseling.     

Nailfold capillary microscopy in patients with antiphospholipid syndrome

In this paper we tried to define the capillaroscopic pattern of anti phospholipid syndrome able to differentiate between the primary (PAPS) and the systemic lupus erythematosus-associated form (SLE-APS) and to be a predictive marker of thrombotic manifestations. Eight PAPS and five SLE-APS patients were studied. In each patient the evaluation was based on anti cardiolipin antibody levels, nailfold capillaroscopy, retinal fluorangiography and transcranial doppler sonography. Statistical analysis has been performed using chi 2 analysis. Morphological alterations of capillary loops, venular visibility and sludging of blood were often observed in both groups. While we found in higher prevalence a variability of capillary loop length in PAPS patients, the SLE-APL group significantly differed for the presence of microhaemorrhages (p < 0.001). When we evaluated the clinical history, a marked microcirculatory damage was related with the occurrence of thrombotic manifestations in the PAPS patients. Anti cardiolipin antibody levels, retinal fluorangiography and transcranial doppler sonography did not correlate with clinical history in either group. In conclusion, nailfold capillaroscopy can be usefully employed in the differentiation between primary and SLE-associated anti phospholipid syndrome, and it can help to identify the patients at higher risk of thrombotic disease.     

Germ-line mutations in the neurofibromatosis 2 gene: correlations with disease severity and retinal abnormalities

Neurofibromatosis 2 (NF2) features bilateral vestibular schwannomas, other benign neural tumors, and cataracts. Patients in some families develop many tumors at an early age and have rapid clinical progression, whereas in other families, patients may not have symptoms until much later and vestibular schwannomas may be the only tumors. The NF2 gene has been cloned from chromosome 22q; most identified germ-line mutations result in a truncated protein and severe NF2. To look for additional mutations and clinical correlations, we used SSCP analysis to screen DNA from 32 unrelated patients. We identified 20 different mutations in 21 patients (66%): 10 nonsense mutations, 2 frameshifts, 7 splice-site mutations, and 1 large in-frame deletion. Clinical information on 47 patients from the 21 families included ages at onset and at diagnosis, numbers of meningiomas, spinal and skin tumors, and presence of cataracts and retinal abnormalities. We compared clinical findings in patients with nonsense or frameshift mutations to those with splice-site mutations. When each patient was considered as an independent random event, the two groups differed (P < or = .05) for nearly every variable. Patients with nonsense or frameshift mutations were younger at onset and at diagnosis and had a higher frequency and mean number of tumors, supporting the correlation between nonsense and frameshift mutations and severe NF2. When each family was considered as an independent random event, statistically significant differences between the two groups were observed only for mean ages at onset and at diagnosis. A larger data set is needed to resolve these discrepancies. We observed retinal hamartomas and/or epiretinal membranes in nine patients from five families with four different nonsense mutations. This finding, which may represent a new genotype-phenotype correlation, merits further study.     

Ageing and degeneration in the macular region: a clinico-pathological study

Clinical and pathological examination was performed on 378 eyes from 216 patients aged 43 to 97 years. This series represented eyes in which the fundi were normal or showed various manifestations of senile macular degeneration. The eyes were divided into six groups according to the histological appearance of a linear deposit at the base of the retinal pigment cells. Groups I and II were considered to represent normal ageing, Groups III and IV the progressive development of senile macular degeneration and Groups V and VI the end-results. Group I showed no basal linear deposit. Thickening and hyalinization of Bruch's membrane was noted as early as the fifth decade. Group II showed patchy development of the basal linear deposit in relation to thickened or basophilic segments of Bruch's membrane, or over intercapillary hyalinization extending to the level of the outer surface of the choriocapillaris. Almost all eyes in these two groups retained a normal fundus appearance but visual acuity declined with age even in the absence of other causes. In Group III the basal deposit formed a thin continuous layer associated with moderate degeneration of the retinal pigment epithelium. More than half the eyes had developed a clinical disturbance of pigmentation and in most vision was reduced. Group IV was characterized by thickening of the deposit and more pronounced disturbance of the pigment epithelium. Clinically most eyes showed coarse pigmentary changes and vision was in the order of 6/24. 14-3 per cent of eyes in this group showed early neovascularization from the choroid. In Group V the pigment epithelium disappeared to produce circumscribed areas of depigmentation. The basal linear deposit could be traced throughout the depigmented area in most eyes. Thin fibrovascular sheets were found beneath the pigment epithelium in 41-7 per cent of eyes. Group VI represented disciform degeneration. The basal linear deposit could often be demonstrated as a disrupted hyalinized layer incorporated into the scar. Disciform degeneration was an alternative end-result to geographical atrophy. In each group the clinical and histological findings may be modified by the presence of drusen or by atrophy of the choroid. The basal linear deposit consisted of banded fibres embedded in granular material lying between the plasma infoldings and the basement membrane of the retinal pigment epithelium. This deposit seems to be a manifestation of gradual failure of the pigment epithelium and proved to be the most suitable criterion by which to study the natural history of senile macular degeneration.     

Objective evaluation of endoscopy skills during training

Chromosome region 15q is thought to contain one or more genes that are important for melanin pigment synthesis in the hair, skin, and eyes. Hypopigmentation has been identified in the Prader-Willi (PWS) and Angelman (AS) syndromes. We have examined 6 individuals with AS to further characterize the pigment pattern in this condition. The age of the 5 girls and one boy ranged from 2.4 to 7.0 years. None had obvious albinism. Hair color ranged from light blond to brown. Skin was type I in 3 and type II in 3. Eye changes included nystagmus in 2, strabismus in 4, and reduced retinal pigment in 5. The mean hairbulb tyrosinase activity was 0.37 +/- 0.44 pmol/hb/120 min for the individuals with AS, with a range of 0.00 to 1.13 (normal brown control 1.49 +/- 0.79, normal blond control 1.50 +/- 0.85). Electron microscopic examination of hairbulb melanocytes showed normal melanosome and melanocyte architecture and number, but reduced melanin formation, with many stage II and III premelanosomes but few stage IV fully melanized melanosomes. Hypopigmentation characterized by light skin, reduced retinal pigment, low hairbulb tyrosinase activity, and incomplete melanization of melanosomes is part of the phenotype of AS, and is similar to that found in PWS.     

Botulinum toxin A, adjunctive therapy for refractory headaches associated with pericranial muscle tension

A set of neurofibromatosis type 1 (NF1) patients was screened for large NF1 gene deletions by comparing patient and parent genotypes at 10 intragenic polymorphic loci. Of 67 patient/parent sets (47 new mutation patients and 20 familial cases), five (7.5%) showed loss of heterozygosity (LOH), indicative of NF1 gene deletion. These five patients did not have severe NF1 manifestations, mental retardation, or dysmorphic features, in contrast to previous reports of large NF1 deletions. All five deletions were de novo and occurred on the maternal chromosome. However, two patients showed partial LOH, consistent with somatic mosaicism for the deletion, suggesting that mosaicism may be more frequent in NF1 than previously recognised (and may have bearing on clinical severity). We suggest that large NF1 deletions (1) are not always associated with unusual clinical features, (2) tend to occur more frequently on maternal alleles, and (3) are an important mechanism for constitutional and somatic mutations in NF1 patients.     

The S1(1A1)-S0(1A1) Electronic Transition of Jet-Cooled o-Difluorobenzene

Retinal pigment epithelium transplantation has been proposed as adjunctive treatment for age-related macular degeneration following surgical excision of choroidal neovascular membranes. The goal of this study was to develop a model to evaluate retinal pigment epithelium transplantation onto human Bruch's membrane in vitro. We investigated the ability of cultured fetal human retinal pigment epithelium to colonize human cadaver Bruch's membrane, determined the incubation time needed to form a monolayer and to exhibit apical microvilli and tight junctions, and assessed the production of basement membrane. Freshly enucleated (less than 48 hours old) human eyes were cut through the pars plana, and the anterior segment, vitreous, and retina were removed. The native retinal pigment epithelium was debrided with a surgical sponge. Bruch's membrane and choroid at the macula were trephined with a 7.0 mm diameter trephine and then incubated with 1/2 ml of Dulbecco's modified Eagle's medium +15% fetal calf serum+basic fibroblast growth factor (1 ng ml-1), and fetal human retinal pigment epithelium at a concentration of 242,000 cells ml-1. Specimens were incubated for 1, 4, 6, 8, 12, or 24 hours. The specimens were fixed in half strength Karnovsky's fixative, processed, and analysed with scanning and transmission electron microscopy. The retinal pigment epithelium covered the debrided macular specimens to different degrees at different incubation times. After 1 hour, the cells started to attach and flatten (median percent coverage: 78%). The extent of Bruch's membrane coverage by fetal retinal pigment epithelium varied greatly between specimens. After 4-6 hours, the cells covered the entire debrided surface in a monolayer (median percent coverage: 97.2% at 4 hours, 99.8% at 6 hours). Tight junctions were observed, and the cells had few apical microvilli. The lateral cell borders were obliquely oriented with respect to Bruch's membrane, and the nuclei were elongated, exhibited prominent nucleoli, and were oriented parallel to Bruch's membrane. After 6-8 hours, cells started to become hexagonal (median percent coverage at 8 hours: 99.97%). Cells attached to the inner collagenous layer tended to be flatter than cells attached to residual native basement membrane. At 12 and 24 hours, expression of hexagonal shape, tight junctions, and apical microvilli were observed more frequently (median percent coverage: 99.87% at 12 and 100% at 24 hours). No newly formed basement membrane was observed at these time points. In separate experiments comparing attachment in the presence and absence of native RPE basement membrane, the presence of native retinal pigment epithelial basement membrane promoted the early attachment of the cells and more rapid expression of normal morphology. This in vitro system provides a reproducible way to study the adherence of retinal pigment epithelium to normal and diseased human Bruch's membrane.     

Rod outer segment renewal in the retinae of deep-sea fish

Outer segment renewal involves the synthesis of disc material in the photoreceptor inner segments, the shedding of the tips of the photoreceptor outer segments, and their phagocytosis by the retinal pigment epithelial cells. It has been suggested that in the retinae of deep-sea fish no renewal of outer segments may take place. In order to assess outer segment renewal in deep-sea fish retinae we counted (i) periciliary vesicles in rod inner segments as a parameter for disc-synthesis activity and (ii) phagosomes in retinal pigment epithelial cells as a parameter of shedding and phagocytosis in 12 species of deep-sea fish with multibank or single bank retinae. We also measured the lengths of rod outer segments in order to evaluate the balance between synthesis and phagocytotic activity. In four of these species (Synaphobranchus kaupi, Nematonurus armatus, Coryphaenoides guentheri and Halosauropsis macrochir) we further recorded size-related changes of these parameters and their relation to the position of a given rod within the banks in the retina. The number of periciliary vesicles was highest in inner segments of the most vitread bank and in the periphery of the retina. Phagosomes were most abundant in retinal pigment epithelial cells of the central retina. Long rod outer segments were most frequently recorded in the peripheral retina indicating that in this region new synthesis may outbalance shedding. Vitread rod outer segments were only slightly longer than sclerad ones. Larger animals had shorter rod outer segments than small ones. We present evidence that rod outer segment renewal takes place in the retina of all deep-sea fish. Vitread rods may be more active in this respect than sclerad ones.     

Long-term follow-up of severe central serous chorioretinopathy using indocyanine green angiography

BACKGROUND: The severe types of central serous chorioretinopathy (CSC) have a chronic nature, suggesting that a pathological process persists subclinically. Indocyanine green (ICG) angiography recently revealed intrachoroidal dye leakage and its static nature in CSC. As the intrachoroidal dye leakage was suspected to be relevant to the disease process, the long-term persistence of intrachoroidal ICG leakage was examined in four patients of the severe types of CSC. METHODS: ICG angiography was performed periodically over more than three years in three patients and two years in one patient. One patient had CSC with bullous retinal detachment, and the other three had chronic CSC or diffuse retinal pigment epitheliopathy. RESULTS: Intrachoroidal ICG leakage persisted in all the patients. However, a change in location of persistent intrachoroidal leakage or disappearance of intrachoroidal leakage regardless of no progression of retinal pigment epithelial alteration was noted in one eye of two patients. CONCLUSIONS: Pathology causing intrachoroidal ICG leakage persisted subclinically for a long period. However, location and extent of the intrachoroidal leakage could change during a long-term follow-up period.     

Retinal degeneration induced by N-methyl-N-nitrosourea in Syrian golden hamsters

BACKGROUND: The sequential retinal changes in Syrian golden hamsters induced by N-methyl-N-nitrosourea (MNU) have not been studied. METHODS: Female hamsters received a single intraperitoneal injection of 90 mg/kg MNU at 50 days of age, and the retina was examined light and electron microscopically, immunohistochemically and by the TdT-mediated dUTP-digoxigenin nick end labeling (TUNEL) method until 20 weeks after the treatment. RESULTS: The retinal changes were as follows: (1) Photoreceptor apoptosis occurred 1 day after the treatment and resulted in photoreceptor loss at day 7. During the degeneration, Müller cell proliferation was conspicuous at day 5. (2) After the photoreceptor cell loss, migration of the pigment epithelial cells in all layers of the retina which were in contact with blood vessels occurred. Due to the Müller cell proliferation, gliosis was prominent at the later stage. CONCLUSIONS: The MNU injection caused photoreceptor apoptosis followed by pigment epithelial cell migration around the blood vessels, accompanied by gliosis. The primary event and the course of this disease closely resemble those of retinitis pigmentosa in humans.     

What''s in a name? The 22q11.2 deletion

PURPOSE: To document that lacunar, atrophic lesions of the retinal pigment epithelium, previously reported as a complication of treatment, can result from the natural course of retinopathy of prematurity. METHODS: We reviewed photographs of patients diagnosed with retinopathy of prematurity at the Casey Eye Institute between 1979 and 1996. Lacunar atrophic lesions of the retinal pigment epithelium were correlated with the clinical records of the affected patients. RESULT: Three untreated eyes of three patients with retinopathy of prematurity had lacunar atrophic lesions of the retinal pigment epithelium. CONCLUSIONS: The spectrum of findings associated with untreated retinopathy of prematurity includes lacunar, atrophic lesions of the retinal pigment epithelium. These lesions are distinct from scars secondary to treatment and are possibly linked to macular dragging and exudative or serous retinal detachment.     

The effect of high-dose methylprednisolone on laser-induced retinal injury in primates: an electron microscopic study

BACKGROUND: Previously we reported an ameliorative effect of high-dose methylprednisolone in laser injury to monkey retinas. The ultrastructural modification by methyl-prednisolone has not been examined. METHODS: Cynomolgus monkeys were given severe (grade III) retinal laser burns and treated with an intravenous megadose of methylprednisolone. Pathologic features of the retinal lesions with or without methylprednisolone treatment were evaluated by light and electron microscopy. RESULTS: Ultrastructurally, the treated lesions showed rapid recanalization of choriocapillaris; proliferation of retinal pigment epithelium to replace the necrotic and damaged cells, resulting in rapid re-establishment of blood retinal barrier; mild macrophagic activity; and rapid reformation of the outer limiting membrane by Mueller cells. CONCLUSION: A high dose of methylprednisolone affected the responses of the choriocapillaris, retinal pigment epithelium, photoreceptor cells and Mueller cells to laser injury, showing an overall beneficial effect. These modifications might be ascribed to methylprednisolone's anti-inflammatory action, protection of the microcirculation and anti-lipid peroxidation effect.     

A technique of minilaparotomy-assisted vaginal hysterectomy

PURPOSE: To report a patient with acute retinal pigment epitheliitis examined less than 24 hours after onset of symptoms. METHOD: One day after the onset of blurred vision in her left eye, a 33-year-old woman had a best-corrected visual acuity of LE, 20/60 -2. The left eye had classic uniform golden-colored nodules in a honeycomb pattern in the foveal retinal pigment epithelium. Intravenous fundus fluorescein angiography disclosed staining of the foveal pigment epithelium. RESULTS: One month after initial examination, visual acuity was LE, 20/20, and fine subfoveal pigmentary clumping was present. CONCLUSION: The pigmentary maculopathy of acute retinal pigment epitheliitis may be nonspecific, resulting from more than one type of primary foveal inflammation.     

Intraocular silicone oil tamponade. A clinico-pathologic study of 36 enucleated eyes

BACKGROUND: Previous histological studies have shown that intraocular silicone oil induces irreversible changes in ocular tissues, especially the retina. The purpose of this study was to analyze, in a larger group of enucleated eyes, changes in intraocular tissue after silicone oil injection, dependent on intraocular pressure, how long the oil was in the eye, and the viscosity of intraocular silicone oil. PATIENTS AND METHODS: We did histological examinations on 36 enucleated globes with intraocular silicone oil after vitreoretinal surgery and compared them with 68 enucleated globes treated with buckle and encircling band using immunohistochemistry (n = 5) and electron microscopy (n = 7). For statistical evaluation we used the chi(2) test and analysis of variance. RESULTS: After silicone oil injection we observed a more pronounced reduction in corneal endothelial cells (58%), more frequent closed chamber angle (86%), atrophy of the ciliary body (80%) (P < 0.05), proliferative vitreoretinopathy (89%), and glaucomatous atrophy of the optic nerve (56%) (P < 0.01). The retinae showed independent of the use of silicone oil a loss of inner and outer segments of photoreceptors and of ganglion cells and thinning and rareficaton of all other retinal layers. Globes with silicone oil revealed vacuoles both free and incorporated by macrophages in all layers of the retina. Similar vacuoles were seen in the optic nerve, choroid, retinal pigment epithelium, ciliary body, iris, chamber angle and the corneal endothelium. Silicone oil vacuoles were seen in the retina and optic nerve by 1 month after surgery in two eyes with high intraocular pressure (42 mmHg). Six of eight eyes with normal intraocular pressure levels showed retinal vacuoles, 3 of them after 2 months. Vacuoles in the optic nerve were found in eight of nine eyes with intraocular instillation of 1000 mPa silicone oil. There was no clinicohistopathological correlation between the presence of vacuoles in the retina or optic nerve and the duration and viscosity of intraocular silicone oil. CONCLUSIONS: This study suggests that vacuoles in eyes with silicone oil instillation can be found in the retina after 4 weeks. The period of intraocular silicone oil should be limited to 3-6 months.     

NF2 gene in neurofibromatosis type 2 patients

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder that predisposes to nervous system tumors. The schwannomin (also termed merlin) protein encoded by the NF2 gene shows a close relationship to the family of cytoskeleton-to-membrane proteins linkers ERM (ezrin-radixin-moesin proteins). Even though penetrance of the disease is >95% and no genetic heterogeneity has been described, point mutations in the NF2 gene have been observed in only 34-66% of the screened NF2 patients, depending on the series. In order to generate tools that would enable an exhaustive alteration screening for the NF2 gene, we have deduced its entire genomic sequence. This knowledge has provided the delineation of a mutation screening strategy which, when applied to a series of 19 NF2 patients, has revealed a high recurrence of large deletions in the gene and has raised the efficiency of mutation detection in NF2 patients to 84% of the cases in this series. The remaining three patients who express two functional NF2 alleles are all sporadic cases, an observation compatible with the presence of mosaicism for NF2 mutation.     

Antibiotic susceptibility among aerobic gram-negative bacilli in intensive care units in 5 European countries. French and Portuguese ICU Study Groups

BACKGROUND: Many successful pigment epithelium transplantation studies involving pink-eyed Royal College of Surgeons (RCS) dystrophic rats showed highly pigmented transplanted cells forming a double layer with slightly pigmented cells, attached to Bruch's membrane. Since it is not clear whether transplanted pigmented cells can displace retinal pigment epithelial (RPE) host cells from Bruch's membrane, we suggested that RPE cells of RCS dystrophic rats can phagocytize melanin granules, possibly derived from perished transplanted cells. METHODS: In a series of three experiments, RPE cells of nine pink-eyed, 2 1/2-month-old RCS dystrophic rats were isolated by trypsinization and mechanical dissection and cultivated in Dulbecco's modified Eagles' medium. These cells were then fed with melanin granules, isolated from bovine RPE cells, double-trypsinized after phagocytosis and viewed by light and electron microscopy. We also transplanted iris pigment epithelial (IPE) cells of 20-day-old Long-Evans rats into the subretinal space of pink-eyed RCS dystrophic rats of the same age, shown in light-microscopic photography after 42 days. RESULTS: Living RPE cells were heavily pigmented after feeding with isolated melanin granules in all three experiments as viewed by light microscopy. In addition, we identified melanin granules phagocytized by dystrophic RPE cells in electron microscopy. After transplantation of pigmented IPE cells into the subretinal space of pink-eyed RCS dystrophic rats' eyes, a layer of slightly pigmented cells was seen on Bruch's membrane below the transplanted IPE cells, shown in light microscopy. CONCLUSION: We have shown by phagocytosis assay that dystrophic RPE cells can take up melanin granules in vitro. Our results assume that pigmented cells in transplantation studies, found as a monolayer, attached to Bruch's membrane, cannot automatically be identified as transplanted cells. Instead, the possibility of perished transplanted cells serving as melanin donors for RPE host cells must be taken into consideration.