Nanosecond laser therapy reverses pathologic and molecular changes in age-related macular degeneration without retinal damage.

Age-related macular degeneration (AMD) is a leading cause of vision loss, characterized by drusen deposits and thickened Bruch's membrane (BM). This study details the capacity of nanosecond laser treatment to reduce drusen and thin BM while maintaining retinal structure. Fifty patients with AMD had a single nanosecond laser treatment session and after 2 yr, change in drusen area was compared with an untreated cohort of patients. The retinal effect of the laser was determined in human and mouse eyes using immunohistochemistry and compared with untreated eyes. In a mouse with thickened BM (ApoEnull), the effect of laser treatment was quantified using electron microscopy and quantitative PCR. In patients with AMD, nanosecond laser treatment reduced drusen load at 2 yr. Retinal structure was not compromised in human and mouse retina after laser treatment, with only a discrete retinal pigment epithelium (RPE) injury, and limited mononuclear cell response observed. BM was thinned in the ApoEnull mouse 3 mo after treatment (ApoEnull treated 683 ± 38 nm, ApoEnull untreated 890 ± 60 nm, C57Bl6J 606 ± 43 nm), with the expression of matrix metalloproteinase-2 and -3 increased (>260%). Nanosecond laser resolved drusen independent of retinal damage and improved BM structure, suggesting this treatment has the potential to reduce AMD progression.

A single EFEMP1 mutation associated with both Malattia Leventinese and Doyne honeycomb retinal dystrophy.

Malattia Leventinese (ML) and Doyne honeycomb retinal dystrophy (DHRD) refer to two autosomal dominant diseases characterized by yellow-white deposits known as drusen that accumulate beneath the retinal pigment epithelium (RPE). Both loci were mapped to chromosome 2p16-21 (refs 5,6) and this genetic interval has been subsequently narrowed. The importance of these diseases is due in large part to their close phenotypic similarity to age-related macular degeneration (AMD), a disorder with a strong genetic component that accounts for approximately 50% of registered blindness in the Western world. Just as in ML and DHRD, the early hallmark of AMD is the presence of drusen. Here we use a combination of positional and candidate gene methods to identify a single non-conservative mutation (Arg345Trp) in the gene EFEMP1 (for EGF-containing fibrillin-like extracellular matrix protein 1) in all families studied. This change was not present in 477 control individuals or in 494 patients with age-related macular degeneration. Identification of this mutation may aid in the development of an animal model for drusen, as well as in the identification of other genes involved in human macular degeneration.

Role of genetic factors in lower- and higher-order aberrations--the genes in myopia twin study.

AIMS: We intended to investigate the relative genetic contribution in wavefront aberrations using a sub-group of twins recruited in the Genes in Myopia twin study, and subsequently provide direction for future studies into the aetiology of mono-chromatic aberrations. To our knowledge, the Genes in Myopia twin study is the first study to explore the role of genetic factors in both lower- and higher-order aberrations in a Caucasian population. METHODS: Each individual completed a general questionnaire and underwent a comprehensive eye examination. Higher-order wavefront aberrations were calculated with Zernike coefficients up to the fourth order. RESULTS: A total of 46 twin pairs with a mean age of 65.3 years were included in the analysis. Monozygotic intra-pair correlations were significantly higher compared to those in dizygotic twin pairs for defocus aberrations (p < 0.05). A trend for a genetic component was identified for higher-order aberrations. CONCLUSION: Genetic studies into refraction typically explore the genetic effects of lower-order aberrations such as myopia and hypermetropia; however, there is little to no research into the genetic basis of higher-order aberrations. The Genes in Myopia twin study indicates a potential genetic role for higher-order aberrations and provides useful insights into the aetiology of refractive error.

Age-related macular degeneration and mortality: the Melbourne Collaborative Cohort Study.

AimsTo assess associations between features of age-related macular degeneration (AMD) and mortality.MethodsA total of 21 129 participants from the Melbourne Collaborative Cohort Study aged 47-85 years (60% female) were assessed for AMD (2003-2007). Mortality data to December 31, 2012 were obtained through linkage with the National Death Index. Associations were assessed using Cox regression, adjusting for age, sex, smoking, region of birth, education, physical activity, diet and alcohol.ResultsLate AMD was identified in 122 (0.6%) participants, including those with choroidal neovascularisation (n=55, 0.3%), geographic atrophy (n=87, 0.4%) and reticular pseudodrusen (n=87, 0.4%). After a median follow-up period of 8.1 years, 1669 (8%) participants had died, including those from cardiovascular diseases (386), tobacco-related cancers (179), and neurodegenerative disease (157). There was evidence of an increased rate of all-cause mortality for those with choroidal neovascularisation (Hazard Ratio (HR) 1.71 95% CI 1.06-2.76) and geographic atrophy (HR 1.46 95% CI 0.99-2.16). Choroidal neovascularisation was also associated with an increased rate of cardiovascular mortality (HR 3.16 95% CI 1.62-6.15) and geographic atrophy was associated with an increased rate of death from tobacco-related cancer (HR 2.86 95% CI 1.15-7.09). Weak evidence was also present for an association between choroidal neovascularisation and death from neurodegenerative disease (HR 2.49 95% CI 0.79-7.85). Neither reticular pseudodrusen nor the earlier stages of AMD were associated with mortality.ConclusionsLate AMD is associated with an increased rate of all-cause mortality. Choroidal neovascularisation and geographic atrophy were associated with death from cardiovascular disease and tobacco-related cancer, respectively.

A comparison of corneal, pancreas, and skin grafts in mice. A study of the determinants of tissue immunogenicity.

A model of murine heterotopic allogeneic transplantation was used to study the rejection characteristics of three tissues--adult cornea, fetal pancreas, and fetal skin--for attributes that might explain their variation in rejection rates and help define the determinants of graft immunogenicity. Under identical conditions, tissues were transplanted to the renal subcapsular space and their base-line rejection rates compared. The expression of MHC class I and II and intercellular adhesion molecule-1 (ICAM-1), was determined for each tissue, as was their ability to produce interleukin-6, IL-3, interferon-gamma, and granulocyte-macrophage colony-stimulating factor in vitro. These studies were performed under basal conditions and after stimulation with concanavalin A-stimulated spleen cell supernatant (CAS) or INF gamma. Corneal grafts had a slow rejection rate compared with pancreas and skin. While all three tissues had low basal expression of MHC class II, both fetal skin and pancreas, but not adult cornea, were able to increase this under our experimental conditions. Pancreas and skin produced IL-6 under basal conditions and could be stimulated to increase production 2-3-fold but the cornea did not basally produce IL-6 and showed minimal upregulation. We postulate that delayed corneal rejection, compared with pancreas and skin, results from two compounding deficiencies: the relative lack of class II MHC-positive APC and the inability to overcome this deficiency by upregulating class II expression and producing accessory molecules for antigen presentation.

Urocanic acid as an immunosuppressant in allotransplantation in mice.

Urocanic acid (UCA) was investigated for its activity as an immunosuppressive agent in murine heterotopic allotransplantation. Mice grafted with cornea, pancreas, and skin under the renal capsule were given either intravenous, subcutaneous, or topical cis-UCA in the peritransplant period. Grafts were performed across complete and minor MHC barriers. Peritransplant immunosuppression with cis-UCA prolonged survival of these grafts, but no route of administration was clearly superior to another. Survival of cornea was prolonged more than survival of skin or pancreas in all MHC disparate combinations. cis-UCA was also used to assess its effect on stimulator cells, derived from mice that had received UCA, in mixed lymphocyte responses. cis-UCA adversely affected the ability of stimulator cells to provoke T cell proliferation in an allogeneic MLR. These findings suggest that cis-UCA may be a potentially useful immunosuppressive agent.

Laser treatment in subjects with high-risk clinical features of age-related macular degeneration. Posterior pole appearance and retinal function.

OBJECTIVES: To verify that a few laser lesions in the posterior pole can cause drusen to resolve in patients with age-related macular degeneration, and to document central retinal sensitivity as drusen resolve. DESIGN: In a pilot study, 12 patients considered to be at high risk for sight-threatening complications from age-related macular degeneration were treated with 12 argon laser lesions in the posterior pole, with review for 12 to 24 months. RESULTS: Choroidal neovascularization developed in 1 patient 8 months after treatment, with consequent loss of central vision. In 9 of the remaining 11 patients, high-risk characteristics of drusen were reduced. Four patients had retinal pigment epithelial depigmentation, and all maintained 20/40 visual acuity at 12 months. One patient lost 3 lines of vision due to geographic atrophy after 12 months. Scotopic retinal threshold was elevated before treatment in 8 patients, compared with an age-matched comparison group. Of these, 4 patients underwent retesting 3 to 6 months after treatment, and all had improved thresholds, but only 1 patient sustained the improvement at 12 months. At 12 months, 3 of the 8 patients showed an improvement in their mean retinal threshold. Of those in whom the mean retinal threshold worsened, the mean elevation in threshold was not more than 0.6 log units. CONCLUSIONS: A few laser lesions in the posterior pole leads to resolution of drusen. There does not appear to be an increased risk for choroidal neovascularization. Retinal threshold measurements show no indication of geographic atrophy at 1 year, but cannot be excluded as a late outcome. Laser treatment may reduce the risk for profound sight-threatening lesions in age-related macular degeneration.

Variation of codons 1961 and 2177 of the Stargardt disease gene is not associated with age-related macular degeneration.

OBJECTIVES: To investigate the role of 2 specific alleles of the Stargardt disease gene (ABCA4) in the pathogenesis of age-related macular degeneration (AMD). Secondary objectives were to investigate differences in frequency of the G1961E allele in selected ethnic groups as well as to examine the segregation of both G1961E and D2177N alleles in 5 multiplex families with AMD. METHODS: Five hundred forty-four patients with AMD and 689 controls were ascertained from 3 continents. Blood samples from 62 normal individuals of Somalian ancestry were also obtained. Participants were screened for the presence of these ABCA4 alleles with a combination of restriction digestion and single-strand conformation polymorphism analysis of polymerase chain reaction amplification products. Detected alleles were confirmed by DNA sequencing. The number of subjects exhibiting the G1961E or D2177N variants were compared between AMD and control groups using a 2-tailed Fisher exact test. RESULTS: There was no significant difference (P >.1) in the frequency of the G1961E and D2177N alleles in patients with AMD (2.2%) vs controls (1.0%). In contrast, there was a significant difference (P< .001) in the frequency of the G1961E alleles between normal individuals of Somali ancestry (11.3%) and normal individuals from other populations (0.4%). There was no evidence of cosegregation of these alleles and the AMD phenotype in the 5 multiplex families with AMD examined. These two ABCA4 alleles were slightly more frequent in patients with AMD with choroidal neovascularization (2.7%) than those without this complication (2.5%). CONCLUSIONS: Somali ancestry is more than 100 times more strongly associated with presence of the G1961E allele than the AMD phenotype. This study did not find any statistically significant evidence for involvement of the G1961E or D2177N alleles of the ABCA4 gene in AMD. CLINICAL RELEVANCE: The ABCA4 gene is definitively involved in the pathogenesis of Stargardt disease and some cases of photoreceptor degeneration. However, it does not seem to be involved in a statistically significant fraction of AMD cases.

Prophylactic laser treatment to fellow eyes of unilateral retinal pigment epithelial tears.

PURPOSE: To evaluate prophylactic laser treatment of the macula in reducing the risk of visual loss in the fellow eye of patients with a retinal pigment epithelial tear caused by age-related macular degeneration in the first eye. METHODS: In a prospective study, 12 patients with a retinal pigment epithelial tear in one eye caused by age-related macular degeneration and drusen in the fellow eye received prophylactic laser treatment of the retina in their fellow eyes and were followed up for 2 years or more after prophylactic treatment. RESULTS: In 12 fellow eyes that received prophylactic laser treatment, a reduction in best-corrected visual acuity to 20/80 or worse occurred in one (8%) of 12 eyes in the first year and two (18%) of the remaining 11 eyes in the second year after treatment. The cumulative risk of visual loss in the treated fellow eye was 25% in 2 years. CONCLUSIONS: In historical control subjects in a natural history study of patients with retinal pigment epithelial tear in one eye, central visual loss occurred in 16 (37%) of 43 eyes in the first year and in seven (30%) of 23 eyes in the second year for a cumulative loss of 59% in the first 2 years. Compared with these historical control subjects, our findings suggest that visual loss in the fellow eyes of patients with a retinal pigment epithelial tear in the first eye is reduced by prophylactic low intensity laser photocoagulation of the macula.

Fluorescein angiographic abnormalities after prophylactic macular photocoagulation for high-risk age-related maculopathy.

PURPOSE: Initial studies suggest that drusen associated with age-related maculopathy resolve in response to laser photocoagulation; there are conflicting reports regarding whether this treatment might prevent neovascular complications and blindness. The goal of the Drusen Laser Study is to maintain good visual acuity in eyes at the highest risk for neovascular complications of age-related maculopathy. In this report, we alert the ophthalmic community to possible laser-induced complications in patients treated within the context of this clinical trial. METHODS: A double-masked, randomized, controlled clinical trial of prophylactic macular photocoagulation for high-risk age-related maculopathy is in progress. Patients randomly assigned to treatment received a ring-type distribution of 12 light spots of argon laser photocoagulation. Drusen were treated directly only if they were present at the protocol treatment locations. Fluorescein angiography was performed in all patients at yearly review, and at nonprotocol visits if symptoms or clinical examination were suggestive of choroidal neovascularization. RESULTS: Fluorescein angiographic abnormalities suggestive of choroidal neovascularization have been seen in treated eyes only: one patient in the pilot study and six patients in the Drusen Laser Study. No fluorescein angiographic abnormalities were seen in eyes of control subjects. CONCLUSIONS: Laser photocoagulation in high-risk age-related maculopathy may induce choroidal neovascularization and, therefore, is not recommended outside the context of a randomized, controlled clinical trial.

Influence of laser photocoagulation on choroidal capillary cytoarchitecture.

AIM: To identify if laser photocoagulation induces morphological changes specifically related to the choroidal capillary endothelial processes that protrude into Bruch's membrane. METHODS: Two human eyes and one adult macaque monkey eye received retinal laser photocoagulation that was just suprathreshold, before enucleation or exenteration. They were examined by electron microscopy to determine the length of the endothelial processes emanating from the choroidal capillaries in the region around the laser burn. One human and two monkey untreated eyes were used for comparison. RESULTS: In human eyes, there was no increase in the number of processes 15 hours after laser treatment but at 5 days the processes were more numerous and longer within 400-500 microm of the burn than in the untreated half of the same eye. The processes were longer 9 days after photocoagulation in the monkey, when compared with untreated monkeys, and some breached the elastic lamina, a phenomenon not seen in the untreated eyes. Qualitative differences were also noted in the endothelial cell processes following photocoagulation. Neovascularisation was not observed. CONCLUSIONS: Protrusion of choroidal endothelial cell processes into Bruch's membrane is a normal anatomical feature but the number, length, and morphology of the processes change following mild photocoagulation. It is plausible that these processes may play a part in the clearance of debris from Bruch's membrane, and represent an early stage of angiogenesis. If the latter is true prophylactic laser photocoagulation at just suprathreshold levels may carry a risk of inducing choroidal neovascularisation.

Generating mouse models of retinal disease using ENU mutagenesis.

We used the chemical mutagen, N-ethyl-N-nitrosourea, to induce random point mutations in the germline of the mouse strain C57BL/6 in order to generate models of retinal diseases. 1163 mutagenised first generation mice produced using this approach were examined for eye abnormalities. Approximately one-third (412) presented with some form of ocular abnormality. Most changes were unilateral and confined to the anterior segment of the eye. Less than 10% (44) of identified changes affected the posterior segment of the eye. 21 mice with varying ocular abnormalities, including 17 with retinal changes, were bred to produce second generation mice to confirm genetic inheritance. Genetic inheritance was confirmed in several of these lines including three with retinal changes.

Heritability of the spatial distribution and peak density of macular pigment: a classical twin study.

PURPOSE: To elucidate the heritability of peak density and spatial width of macular pigment (MP) using a Classical Twin Study. METHODS: Fundus autofluorescence images were obtained at 488 nm from 86 subjects or 43 twin pairs (21 monozygotic (MZ) and 22 dizygotic (DZ)) (27 male, 59 female) aged from 55 to 76 years (mean 62.2 ± 5.3 years). The relative topographic distribution of MP was measured using a grey scale of intensity (0-255 units) in a 7° eccentricity around the fovea. Relative peak MP density (rPMPD) and relative spatial distribution of MP (rSDMP) were used as the main outcome measure in the statistical analysis. RESULTS: A significantly higher correlation was found within MZ pairs as compared with that within DZ pairs for rPMPD, (r=0.99, 95% confidence interval (95% CI) 0.93 to 1.00) and 0.22, 95% CI -0.34 to 0.71), respectively, suggesting strong heritability of this trait. When rSDMP was compared, there was no significant difference between the correlations within MZ pairs (r=0.48, 95% CI -0.02 to 0.83) and DZ pairs (r=0.63, 95% CI 0.32 to 0.83), thus rSDMP is unlikely to have a considerable heritable component. In addition, there was no difference between any MP parameter when normal maculae were compared with early age-related macular degeneration (AMD) (rPMPD 0.36 vs 0.34, t=1.18 P=0.243, rSDMP 1.75 vs 1.75, t=0.028 P=0.977). CONCLUSIONS: rPMPD is a strongly heritable trait whereas rSDMP has minimal genetic influence and a greater influence by environmental factors. The presence of macular changes associated with early AMD did not appear to influence any of these pigment parameters.

Retinal vascular changes following intravitreal ranibizumab injections for neovascular AMD over a 1-year period.

PURPOSE: To assess retinal vascular calibre changes in eyes with neovascular age-related macular degeneration (AMD), treated with intravitreal anti-vascular endothelial growth factor agents, over a 1-year period and compare any such changes to untreated fellow eyes. METHODS: Treatment naïve patients with neovascular AMD received three consecutive intravitreal injections of ranibizumab, followed by a pro re nata dosing regimen up to 1 year, with the aim of maintaining a 'fluid-free' macula. Retinal arteriolar and venular calibre was measured from digital fundus photographs at baseline and at three monthly intervals to 1 year, and summarised as central retinal artery equivalent (CRAE) and central retinal venular equivalent (CRVE), respectively. RESULTS: A total of 53 injected eyes and 41 fellow, non-injected eyes were analysed. At baseline, there were no differences in retinal vascular calibre between injected and non-injected eyes (mean CRAE (SD) 144.93 (14.07) vs 145.74 (13.10) µm, P=0.80 and mean CRVE (SD) 216.23 (25.93) vs 219.91 (22.82) µm, P=0.53). Over a 12-month period, retinal venular calibre dilatation occurred in injected eyes (mean CRVE change +5.71 (14.71) µm, P=0.007), with no change in retinal arterioles, +0.69 (14.71) µm, P=0.68. In non-injected eyes, arteriolar narrowing occurred as a whole, mean CRAE change -4.20 (7.00) µm, P=0.001, over 12 months, with a trend for narrowing in venules, -2.16 (11.56) µm, P=0.28. In injected eyes, after controlling for covariates, the changes in CRVE over 12 months mirrored improvements in macular thickness, -0.06 (-0.005, -0.11) µm, P=0.04, and visual acuity, +9.66 (-0.30, +19.32) µm, P=0.06. CONCLUSION: Intravitreal ranibizumab significantly dilated retinal venules after a 1-year period.

Best's macular dystrophy in Australia: phenotypic profile and identification of novel BEST1 mutations.

PURPOSE: (1) To evaluate the spectrum of BEST1 mutations within Australian Best Disease or vitelliform macular dystrophy (VMD) pedigrees, including any novel mutations; (2) to analyse the range of clinical presentations of this cohort; (3) to determine any possible genotype-phenotype correlations and (4) to compare clinical data of patients with phenotypic VMD, both with and without a BEST1 mutation. PATIENTS AND METHODS: Patients with suspected VMD were referred to clinical centres for ophthalmological assessment and genetic screening. When a mutation was identified in a proband, further family members were invited for clinical and genetic screening. RESULTS: We identified 42 patients with one of 13 BEST1 mutations. Seven mutations were novel. There were a further 14 probands in whom a BEST1 mutation was not identified. Median visual acuity in both VMD (mutation positive) and clinical VMD (no BEST1 mutation identified) groups reached driving standards (6/12 or better). CONCLUSION: We did not identify any firm genotype-phenotype correlations in our Australian VMD pedigrees, in which there was a spectrum of BEST1 mutations and marked variation in clinical presentation. Genetic screening remains the gold standard for VMD diagnosis. Patients should be counselled that visual acuity might remain at or above driving standards in at least one eye even in the presence of a BEST1 mutation.

The dot-and-fleck retinopathy of X linked Alport syndrome is independent of complement factor H (CFH) gene polymorphisms.

BACKGROUND AND AIMS: X linked Alport syndrome is characterised by renal failure, hearing loss, lenticonus, and a central and peripheral dot-and-fleck retinopathy. complement factor H (CFH) gene variants are strongly associated with retinal drusen in macular degeneration and mesangiocapillary glomerulonephritis, and this study examines their role in the development of the Alport retinopathy. METHODS: Twenty-three males and 27 females from 27 unrelated families were examined and their DNA tested for the CFH risk allele (1277 T>C, h1, Y402H) and protective haplotypes (h2 and h4) using a MALDI-TOF-based method. RESULTS: The prevalence of the CFH risk allele was not increased in males with a central or peripheral retinopathy. Three of the nine (33%) with the central retinopathy had at least one copy of the risk allele, and five of the 14 (36%) without the retinopathy did (NS, OR 0.900, CI 0.154 to 5.259). Four of the 12 (33%) with either retinopathy had the risk allele, and two of the six (33%) with none did (NS OR 1.0, CI 0.125 to 7.996). CONCLUSION: The pathogenesis of the retinal dots and flecks in Alport syndrome is independent of CFH-dependent mechanisms and, like other clinical features, may depend on the nature of the underlying COL4A5 mutations.