Aflibercept monotherapy versus aflibercept with targeted retinal laser to peripheral retinal ischemia for diabetic macular oedema (LADAMO).

OBJECTIVE: We tested the hypothesis that targeted retinal laser photocoagulation (TPRP) to peripheral retinal ischaemia reduces the overall burden of aflibercept injections when treating diabetic macular oedema (DMO) over a 24-month period. METHODS: Prospective, double-masked, multicentre, randomised controlled trial in Australia comparing aflibercept monotherapy, following a treat-and-extend protocol, or combination therapy of aflibercept and TPRP for DMO. The aflibercept monotherapy group received placebo laser. The primary outcome measure was the mean number of intravitreal aflibercept injections for each group at 24 months. Secondary outcome included: mean change in central macular thickness (CMT) and vision at trial completion, the proportion of eyes whose DMO resolved and the mean injection treatment interval. Ocular and systemic adverse events were recorded. RESULTS: We enrolled 48 eyes of 47 patients; 27 eyes were randomised to combination therapy (aflibercept and TPRP) and 21 to aflibercept monotherapy. Thirty-two eyes (67%) completed the 2-year study. The number of intravitreal treatments given were similar for combination therapy (10.5 (SD 5.8) and monotherapy (11.8 (SD5.6)) (P = 0.44). The mean visual improvement (+4.0 (-1.8, 9.8) and +7.8 (2.6, 12.9) letters, P = 0.32), mean decrease in CMT (-154 (-222,-87) µm and -152 (-218,-86) µm, P = 0.96), proportion of eyes with CMT < 300 µm (48% and 67%; P = 0.50) and safety outcomes were similar in both the combination and monotherapy treatment groups (respectively). CONCLUSIONS: Laser to areas of ischaemic peripheral retina does not reduce the burden of intravitreal aflibercept injections when treating diabetic macular oedema.

Optogenetic approaches to vision restoration.

Inherited retinal disease (IRD) affects about 1 in 3000 to 1 in 5000 individuals and is now believed to be the most common cause of blindness registration in developed countries. Until recently, the management of such conditions had been exclusively supportive. However, advances in molecular biology and medical engineering have now seen the rise of a variety of approaches to restore vision in patients with IRDs. Optogenetic approaches are primarily aimed at rendering secondary and tertiary neurons of the retina light-sensitive in order to replace degenerate or dysfunctional photoreceptors. Such approaches are attractive because they provide a "causative gene-independent" strategy, which may prove suitable for a variety of patients with IRD. We discuss theoretical and practical considerations in the selection of optogenetic molecules, vectors, surgical approaches and review previous trials of optogenetics for vision restoration. Optogenetic approaches to vision restoration have yielded promising results in pre-clinical trials and a phase I/II clinical trial is currently underway (ClinicalTrials.gov NCT02556736). Despite the significant inroads made in recent years, the ideal optogenetic molecule, vector and surgical approach have yet to be established.

Glucocorticoids: structure, signaling and molecular mechanisms in the treatment of diabetic retinopathy and diabetic macular edema.

Diabetic retinopathy (DR) is one of the leading causes of blindness in the working population worldwide. Vascular leakage, angiogenesis and neuronal degeneration are key features of DR. Current effective interventions for DR include treatment of systemic risk factors such as elevated blood glucose, blood pressure and dyslipidemia. Ocular treatments include vascular endothelial growth factor A (VEGF-A) inhibitors, laser photocoagulation and surgery. While anti-VEGF therapy has become as first-line treatment for diabetic macular edema (DME) that causes reduced vision, intravitreal glucocorticoids also have been shown to be efficacious in this situation. It has been reported that all the major pathological processes of DR are susceptible to glucocorticoid treatment. The effects of glucocorticoids on vascular leakage and angiogenesis may be mediated through their well established anti-inflammatory role. Alternatively, glucocorticoids may affect other mechanisms known to be activated in DR. Potential mechanisms for the anti-inflammatory effects of glucocorticoids include blockage of cytokine production and inhibition of leukocyte adhesion induced by VEGF-A. Glucocorticoids decrease the expression of VEGF-A directly, and increase the production, or decrease phosphorylation, of tight junction-associated proteins. Glucocorticoids have also been shown to be neuroprotective, in contrast to VEGF-A inhibitors which animal studies suggest may be neurotoxic. This review outlines the biological properties of synthetic glucocorticoids, with particular emphasis on the potential beneficial effect of combining glucocorticoids with anti-VEGF treatment for DME and DR.

Triamcinolone acetonide inhibits p38MAPK activation and neuronal apoptosis in early diabetic retinopathy.

OBJECTIVE: Intravitreal glucocorticoids and anti-vascular endothelial growth factor (VEGF) therapies are novel strategies for the treatment of advanced diabetic retinopathy, a condition with inflammatory and neuropathic elements. In contrast with anti-VEGF therapy, glucocorticoids may also exert neuroprotective effects. How glucocorticoids protect retinal neurons is unknown. The aims of the study are to investigate the anti-apoptotic actions of glucocorticoids on diabetic retinal neurons, and characterize the signalling pathways involved. RESEARCH DESIGN AND METHODS: The regulation of gene expression of the four p38 mitogen-activated protein kinase (MAPK) isoforms (α, ß, δ and γ) and the glucocorticoid receptor (GR) in the retinas was evaluated using quantitative RT-PCR, Western blot and immunohistochemistry. Phosphorylation of all isoforms p38MAPK (Thr180/Tyr182) and GR (S-211) was further evaluated. Apoptosis was confirmed by immunolocalization of active CASPASE-3 and the subsequent cleavage of poly (ADP-ribose) polymerase (PARP) following intravitreal injection of triamcinolone acetonide (IVTA), in an early diabetic rat model (26 days after induction of diabetes. RESULTS: IVTA significantly down-regulated mRNA expression of Caspase 3. Activation of CASPASE-3, the subsequent cleavage of PARP-1 and phosphorylation of p38MAPK induced by diabetes were attenuated by IVTA treatment, concomitantly with activation by phosphorylation of the glucocorticoid receptor (GR S-211). CONCLUSIONS: IVTA activates the GR and exerts neural protective effects on retinal neurons. Inhibition of the p38MAPK pathway and activation of GR play a critical anti-apoptotic role in retinal neurons of diabetes following IVTA treatment. Both the anti-inflammatory and anti-apoptotic effects of glucocorticoids may be mediated through inhibition of the p38MAPK pathway in diabetic retinopathy.

Diabetes impairs mobilization of mouse bone marrow-derived Lin(-)/VEGF-R2(+) progenitor cells.

Endothelial progenitor cells circulating in the peripheral blood (PB) contribute to vascular repair. This study aimed to evaluate the potential of a 'cocktail' consisting of erythropoietin, granulocyte colony-stimulating factor and tetrahydrobiopterin to mobilize hematopoietic lineage negative/vascular endothelial growth factor receptor 2 positive (Lin(-)/VEGF-R2(+)) cells from the bone marrow (BM) to PB in non-diabetic and diabetic mice. Diabetes was induced in mice by intraperitoneal injection of streptozotocin. Diabetic mice were studied after 16weeks of hyperglycemia. Half the mice in each group (non-diabetic and diabetic) received daily intraperitoneal injections of the cocktail for 6 consecutive days while the other half received vehicle buffer. Mobilization of Lin(-)/VEGF-R2(+) cells, which were expanded in MCP301 medium, was evaluated after isolating them from BM and PB and their phenotypic and morphological properties were studied. We found that 16weeks of diabetes affected neither the total number of BM mononucleated cells nor the number of Lin(-)/VEGF-R2(+) cells in BM compared with non-diabetic controls. In non-diabetic mice, cocktail treatment resulted in a significant decrease in BM Lin(-)/VEGF-R2(+) cells, paralleled by a significant increase of these cells in PB. Such changes in the number of Lin(-)/VEGF-R2(+) cells in BM and PB after the cocktail treatment were less marked in diabetic mice. In vitro studies of BM Lin(-)/VEGF-R2(+) cells from diabetic and non-diabetic mice did not reveal any differences in either phenotypes or colony forming potential. These findings indicate that diabetes impairs the mobilization of Lin(-)/VEGF-R2(+) cells from BM to PB. Impaired mobilization of BM Lin(-)/VEGF-R2(+) cells soon after the onset of diabetes may contribute to complications such as diabetic retinopathy.

Bruch's membrane and choroidal macrophages in early and advanced age-related macular degeneration.

AIM: To determine the sub-macular Bruch's membrane (BrM) macrophage count and the choroidal and BrM macrophage immunophenotype in normal eyes and in eyes with early and advanced age-related macular degeneration (AMD). METHODS: BrM macrophages were counted in 125 human eyes (normal, normal aged, early AMD and geographical atrophy), and CD68 and inducible nitric oxide synthase (iNOS) immunohistochemistry was performed on 16 human eyes (normal, normal aged, early AMD, geographical atrophy and disciform scarring). All eyes were examined clinically ante mortem. Results were correlated with histopathological features, including basal laminar deposit and membranous debris, and with clinical fundus appearance. RESULTS: CD68(+) macrophages were found in the choroid of normal human eyes, and did not express iNOS. Expression of iNOS by choroidal macrophages (as well as endothelial cells and pericytes) was associated with: (1) recruitment of macrophages to BrM in early AMD eyes with soft drusen or thick continuous basal laminar deposit, corresponding to clinically detectable soft drusen or pigment changes; and (2) active disciform scarring. iNOS expression was absent in BrM macrophages, suggesting immunomodulatory differences between the choroid and BrM. The highest BrM macrophage counts were found in eyes with subclinical choroidal neovascularisation. CONCLUSION: The presence of extracellular deposits (soft drusen and thick continuous basal laminar deposit) is associated with macrophage recruitment to BrM and alteration in the immunophenotype of resident choroidal macrophages.

Conditioned medium from mixed retinal pigmented epithelium and Müller cell cultures reduces in vitro permeability of retinal vascular endothelial cells.

AIM: To investigate the in vitro effect of laser photocoagulation on blood-retinal barrier permeability. METHODS: Retinal capillary endothelial cells were exposed to supernatants from long term co-cultured cells that were argon laser treated. Endothelial cell permeability was analysed by (1) measurement of transendothelial electrical resistance and (2) equilibration of [(3)H] inulin and [(14)C] albumin across the cell monolayer. RESULTS: Laser photocoagulation of various retinal cells and control ECV304 cells in the lower chamber did not appreciably improve permeability of the endothelial cell monolayer compared with that of unlasered cells. However, medium that was conditioned by mixed retinal pigmented epithelium and Müller cells significantly reduced both inulin (43.2% (SD 6.5%) equilibration in mixed cultures v 59.8% (SD 7.0%) control cells, p<0.05) and albumin (15.1% (SD 3.8%) v 31.1% (SD 6.7%), p<0.05) permeability of the endothelial cell monolayers. A fourfold increase in transendothelial electrical resistance was also seen. CONCLUSIONS: These results are consistent with the hypothesis that interaction of Müller cells with retinal pigmented epithelium induced by laser treatment results in secretion of soluble factor(s), which reduces permeability of retinal vascular endothelium. Identification of these factor(s) may have implications for the clinical treatment of macular oedema secondary to diabetic retinopathy and other diseases.

Pseudo-endophthalmitis after intravitreal injection of triamcinolone.

AIMS: To describe an unusual endophthalmitis-like reaction after an intravitreal injection of triamcinolone acetonide in four patients. METHODS: Retrospective case series. RESULTS: Four patients are reported with an endophthalmitis-like reaction following an intravitreal injection of triamcinolone acetonide. There was a dense vitreous haze with severe reduction of fundus view in all cases. One case was treated as an infectious endophthalmitis but the vitreous tap showed no evidence of an endophthalmitis and no bacterial or fungal growth in culture. In all four cases, the vitreous haze cleared without specific treatment. The anterior chamber remained quiet in all cases but one, which was examined 30 minutes after the injection, and there was no periorbital inflammation or pain. CONCLUSION: Pseudo-endophthalmitis after an intravitreal injection of triamcinolone acetonide seems to be a distinct clinical entity that may resolve without specific treatment.

Immunological and aetiological aspects of macular degeneration.

Aetiological and immunological aspects of AMD, a leading cause of blindness in Western countries, have been reviewed. Developmental studies suggest that anatomical features unique to the fovea result in a critical relationship between metabolic demand and blood supply at the macula, which is maintained throughout life. Recent studies show a sufficient degree of consistency in the link between smoking and both dry and wet AMD to regard it as causative. Dry AMD is considered to be the natural endstage of the disease; epidemiological and morphological studies point to choroidal vascular atrophy as the causative event and it is suggested that signals associated with acute vascular compromise lead to the development of subretinal neovascularisation. The relationship between sub-pigment epithelial deposits, including basal laminar deposit, and the pathogenesis of AMD is examined. Much of the literature is consistent with a choroidal origin for the constituents of drusen. The blood-retinal barrier preserves the physiological environment of the neural retina and limits inflammatory responses. The factors, including cytokines, adhesion molecules and the presence of resident immunocompetent cells (microglia), which determine the immune status of the retina are considered. Historical descriptions of the involvement of inflammatory cells are provided, evidence implicating inflammation in the pathogenesis of AMD involving macrophages, giant cells and microglia has been derived from observations of human and animal subretinal neovascular lesions. The role of humoral factors such as anti-retinal autoantibodies and acute phase proteins together with clinical observations has been surveyed. Taken together these data demonstrate the involvement of both cellular and humoral immunity in the pathogenesis of AMD. It remains to be determined to what degree the influence of immunity is causative or contributory in both wet and dry AMD, however, the use of anti-inflammatory agents to ameliorate the condition further indicates the existence of an inflammatory component.

Triamcinolone acetonide modulates permeability and intercellular adhesion molecule-1 (ICAM-1) expression of the ECV304 cell line: implications for macular degeneration.

Whilst animal studies and a pilot clinical trial suggest that intravitreal triamcinolone acetonide (TA) may be useful in the treatment of age-related macular degeneration (AMD), its mode of action remains to be fully elucidated. The present study has investigated the capacity of TA to modulate the expression of adhesion molecules and permeability using a human epithelial cell line (ECV304) as a model of the outer blood-retinal barrier (BRB). The influence of TA on the expression of ICAM-1 and MHC-I was studied on resting and phorbol myristate acetate (PMA)- or interferon-gamma (IFN-gamma)- and/or tumour necrosis factor-alpha (TNF-alpha)-activated cells using flow cytometry and immunocytochemistry. Additionally, ECV304 cells were grown to confluence in uncoated Transwell chambers; transepithelial resistance (TER) across resting and PMA-activated cells was monitored. TA significantly decreased the paracellular permeability of ECV304 cells and down-regulated ICAM-1 expression, consistent with immunocytochemical observations. PMA-induced permeability changes were dose-dependent and TA decreased permeability of both resting and PMA-activated monolayers. MHC-I expression by ECV304 cells however, was not significantly affected by TA treatment. The modulation of TER and ICAM-1 expression in vitro correlate with clinical observations, suggesting re-establishment of the BRB and down-regulation of inflammatory markers are the principal effects of intravitreal TA in vivo. The results further indicate that TA has the potential to influence cellular permeability, including the barrier function of the retinal pigment epithelium (RPE) in AMD-affected retinae.

A randomized phase II trial of interferon-alpha2b versus 5-fluorouracil after trabeculectomy.

PURPOSE: The aim of the present study was to investigate the safety and potential efficacy of subconjunctival interferon-alpha2b (IFN-alpha), either alone or in combination with 5-fluorouracil (5-FU), in reducing the risk of failure of glaucoma surgery METHODS: A prospective, masked randomized phase II study was undertaken in which patients received three subconjunctival injections per week for 3-4 weeks postoperatively. Three treatments were compared: (i) IFN-alpha (1 x 10(6)IU per dose); (ii) 5-FU (5 mg per dose); and (iii) alternating IFN-alpha and 5-FU (BOTH). The primary outcome measures were: (i) rate of successful control of intra-ocular pressure without further surgery; and (ii) the incidence of side effects. RESULTS: Fifty-seven patients undergoing glaucoma surgery with an increased risk of failure were evaluated, including 23 patients (40%) undergoing trabeculectomy combined with extracapsular cataract extraction as well as other conventional high-risk groups. With 53 patients (93%) completing 2 years follow up,there was no significant difference in success rates among the three groups. Intra-ocular pressure was controlled without further surgery in 79% of patients (95% confidence interval (CI): 61, 97%) receiving IFN-alpha, in 89% of patients (76, 100%) receiving 5-FU and in 89% of patients (76, 100% receiving BOTH. Side effects were similar among the three groups. CONCLUSIONS: These results are consistent with a beneficial effect of IFN-alpha2b given either alone or in combination with 5-FU after glaucoma filtering surgery. However, the lack of a clear and substantial benefit over conventional anti-fibrotic therapy does not support the further clinical evaluation of these treatments.

Regulators of vascular permeability: potential sites for intervention in the treatment of macular edema.

Rather than being a non-specific reaction to a noxious stimulus, breakdown of the capillary blood-retinal barrier causing macular edema appears to be dependent on a number of active processes which may be open to pharmacological manipulation. Extracellular influences which may affect barrier function include serum and neighboring cell types, which act though cytokines, such as vascular endothelial growth factor and transforming growth factor-beta, and other factors. A number of intracellular pathways acting on the cytoskeleton and components of the intercellular junctional complexes have been identified which mediate agonist-induced leak of the vascular endothelium. The further elucidation of these processes may be useful in the development of better treatments for breakdown of the inner blood-retinal barrier.

Exudative macular degeneration and intravitreal triamcinolone: 18 month follow up.

PURPOSE: To evaluate the safety and efficacy of intravitreal triamcinolone after 18 months of follow up in patients with age-related macular degeneration and subfoveal or juxtafoveal choroidal neovascularization considered unsuitable for laser photocoagulation. METHODS: Thirty eyes of 28 patients, referred from general eye clinics as well as the private clinic of one of the authors to a hospital-based retinal out-patient clinic, were treated with an intravitreal injection of triamcinolone (4 mg). The primary outcome measure was the proportion of eyes with loss of six or more lines on a Bailey-Lovie Chart. The incidence of adverse events associated with treatment was also observed. RESULTS: Of the 20 eyes with initial visual acuity (VA) of 6/60 or better, the vision was maintained (+/-1 Bailey-Lovie lines) in 11 eyes (55%), while six eyes (30%) suffered severe visual loss (six or more lines). The VA improved by five to six lines in three of 10 eyes with initial vision of 3/60 or worse. Three of four eyes receiving a second injection suffered either progressive cataract or elevated intra-ocular pressure (IOP) requiring cataract surgery and/or filtering surgery. One of 26 eyes (3%) receiving a single injection showed progression of cataract and elevation of IOP within 6 weeks of treatment and required anti-glaucoma medication for 6 weeks. Progression of nuclear sclerosis 8-12 months after treatment was observed in six of 26 eyes (23%) receiving a single injection. CONCLUSIONS: The results of the present study suggest that a single intravitreal injection of 4 mg triamcinolone is reasonably well tolerated by the human eye. The rate of development of severe visual loss was less than reported for historical controls. Because the results are preliminary and uncontrolled, the treatment should not be used routinely until its benefit to patients is established by a prospective, randomized controlled study.

Effect of high glucose on permeability of retinal capillary endothelium in vitro.

PURPOSE: To study the effect of high glucose on the permeability of bovine retinal capillary endothelial cell (BRCEC) monolayers. METHODS: The paracellular permeability of second-passage BRCEC cultured on millipore filters in two chamber transwell inserts was assayed by measuring the peak trans-monolayer electrical resistance and percent equilibration of 14C-inulin 48 hours after it had been added to the luminal chamber. RESULTS: High glucose increased the paracellular permeability of BRCEC monolayers independently of its hypertonic action (5 mM glucose: 154.2 +/- 21.2 and 19.5 +/- 2.4; 30 mM glucose: 134.2 +/- 5.1 [P = 0.01] and 23.5 +/- 2.1 [P = 0.01]; 5 mM glucose + 25 mM mannitol: 168.7 +/- 13.7 ohm.cm2 [P = 0.04] and 19.3% +/- 1.2% 48-hour equilibration of inulin [P = 0.008]). In a separate series of experiments, the authors were unable to show that either aminoguanidine or ponalrestat prevented the effect of high glucose on permeability (30 mM glucose 95.1 +/- 16.7 and 45.4 +/- 5.6; 5 mM glucose: 122.9 +/- 14.2 [P = 0.02] and 36.6 +/- 5.6 [P = 0.001]; 30 mM glucose + aminoguanidine 87.9 +/- 17.5 [P = 0.04] and 75.3 +/- 14.9 [P = 0.6]; 30 mM glucose + ponalrestat 79.9 +/- 12.7 ohm.cm2 [P = 0.1] and 48.2 +/- 2.5% 48-hour equilibration of inulin [P = 0.15]). Ponalrestat did not abrogate the effect of high glucose despite its ability to reduce a high glucose-induced increase in BRCEC intracellular sorbitol levels. CONCLUSIONS: The data are consistent with a role for increased paracellular permeability in breakdown of the blood-retinal barrier in diabetic retinopathy, which appears to be independent of both nonenzymatic glycosylation and the polyol pathway.

Topical interferon alpha 2b for corneal haze after excimer laser photorefractive keratectomy. The Melbourne Excimer Laser Group.

PURPOSE: To determine whether topical interferon alpha 2b (IFN-alpha) prevents corneal haze after excimer laser photorefractive keratectomy (PRK). SETTING: Tertiary referral ophthalmic hospital. METHOD: A prospective, double-blind, placebo-controlled, randomized study of 31 patients was undertaken. After surgery in a single institution, patients received a drop of either a placebo or IFN-alpha (5 x 10(6) IU/ml) four times daily for 4 weeks. The main outcome measures were corneal haze, refraction, and visual acuity. RESULTS: The major side effect of interferon alpha treatment was a significant delay in epithelial healing by a mean of 2 days. The means of the average post-treatment clinical scores for haze in all patients up to 12 months after surgery were 0.46 +/- 0.25 for the IFN-alpha group and 0.64 +/- 0.43 for the placebo group (P = .20). Of patients with a correction of greater than 5.00 diopters (D), the IFN-alpha group had significantly less haze over the course of the study (0.39 +/- 0.23 versus 0.98 +/- 0.50; P = .03). After 12 months, the mean absolute spherical equivalent in the two groups was not significantly different (1.02 +/- 1.13 D versus 1.44 +/- 2.64 D). There was a tendency toward better uncorrected visual acuity in the INF-alpha group (P < .10, Kolmogorov-Smirnov). CONCLUSION: Topical IFN-alpha may merit further investigation as a treatment to reduce corneal haze after excimer laser PRK for corrections greater than 5.00 D.

Electrical resistance and macromolecular permeability of retinal capillary endothelial cells in vitro.

This study was undertaken to examine the ability of retinal capillary endothelial cells to retain blood-retinal barrier properties in vitro. Second passage bovine retinal capillary endothelial cells were grown to confluence on polycarbonate filters in two chamber systems coated with laminin, fibronectin and type IV collagen. The electrical resistances, permeability of 3H-insulin and expression of blood-brain barrier related enzymes by retinal cells was observed and compared with bovine aortic endothelial cells and bovine fibroblasts. The electrical resistance of retinal cells rose over the first week of culture, peaking after 5-9 days in culture. In eleven separate experiments (n = 5 for each experiment) the average peak resistance of retinal endothelial cells ranged from 89.3-186.6 with a mean average of 129.0 ohm.cm2. In one of these experiments, the peak electrical resistance of retinal cells was 149.0 +/- 10.3 compared with 34.8 +/- 6.8 for aortic cells and 37.8 +/- 3.8 ohm.cm2 for fibroblasts. The permeability coefficients of inulin were: retinal cells 0.17 +/- 0.09, aortic cells 3.47 +/- 1.58 (p = 0.015), fibroblasts 3.93 +/- 0.78 (p = 0.002) x 10(-6) cm/sec. Retinal cells expressed significantly higher activities of gamma-glutamyl transpeptidase and alkaline phosphatase than the other cell types. Treatment of the monolayers with the calcium ionophore, A23187, resulted in a reversible increase in permeability as has been described for peripheral vascular endothelium. We conclude that BRCEC retain at least some of their specialised barrier properties in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)