Experimental models of glaucoma filtration surgery.

Glaucoma filtration surgery plays an important role in achieving intraocular pressure (IOP) reduction in patients who have high IOP despite maximum medical therapy. Preclinical experimental models of glaucoma filtration surgery contribute a great deal to our knowledge of the wound healing processes that predispose to scarring and may lead to poor outcomes. However, this research needs to be interpreted in the light of the specific study design, animal model and methods used. We review the existing literature addressing various models of experimental glaucoma filtration surgery, discuss the considerations in assessing these models and describe future steps in evaluating potential therapeutics and bleb characteristics that could impact translational research in this field.

Nox4 Facilitates TGFß1-Induced Fibrotic Response in Human Tenon's Fibroblasts and Promotes Wound Collagen Accumulation in Murine Model of Glaucoma Filtration Surgery.

Collagen accumulation in sub-conjunctival tissue at the surgical wound is one of the major complications associated with glaucoma filtration surgery (GFS). This process often leads to unwanted fibrotic scar formation at the lesion site and dysfunction of tissues. Previously, we demonstrated that NADPH oxidase 4 (Nox4) is implicated in transforming growth factor-beta (TGFß)-induced collagen production in ocular fibroblasts and scarring responses in a mouse model of corneal injury. Here, we propose that Nox4 is an important facilitator of TGFß-induced responses. We tested this hypothesis in human Tenon's fibroblasts (HTF) and also assessed a role of Nox4 in an experimental mouse model of GFS. TGFß1 induced Nox4 mRNA expression but downregulated Nox5 in HTF. Targeting Nox4 gene expression with an adenovirus carrying a Nox4 small interfering RNA (siRNA) (Ad-Nox4i) or removal of hydrogen peroxide (H2O2) with EUK-134 (25 µM) in HTFs significantly reduced TGFß1-induced Nox4 expression, H2O2 production, and collagen synthesis (p < 0.05, n = 3-6). SIS3 (5 µM) that prevents Smad3 phosphorylation is found to suppress TGFß1-induced collagen production in HTFs. Furthermore, Ad-Nox4i and EUK-134 both abolished TGFß1-stimulated proliferation of HTFs. We also compared collagen deposition at the wound arising from GFS between wildtype (WT) and Nox4 knockout (KO) mice. Both collagen deposition and fibrovascularization at the wound were significantly decreased in Nox4 KO mice at 14 days after GFS. Our results provide comprehensive evidence that Nox4 is an important mediator for TGFß1-induced responses in HTFs and collagen deposition in surgical wound following GFS in mice. As such, pharmacological inhibition of Nox4 would be a viable therapeutic strategy for the control of scarring after glaucoma surgery.

Prevalence and associations of non-retinopathy ocular conditions among older Australians with self-reported diabetes: The National Eye Health Survey.

AIM: To determine the prevalence and associations of non-retinopathy ocular conditions among older Australian adults with diabetes. METHODS: Multistage random-cluster sampling was used to select 3098 non-indigenous Australians aged 50y or older (46.4% male) and 1738 indigenous Australians aged 40y or older (41.1% male) from all levels of geographic remoteness in Australia. Participants underwent a standardised questionnaire to ascertain diabetes history, and a clinical examination to identify eye disease. We determined the prevalence of uncorrected refractive error, visually significant cataract, cataract surgery, age-related macular degeneration, glaucoma, ocular hypertension, retinal vein occlusion and epiretinal membrane among those with and without self-reported diabetes. RESULTS: Participants with self-reported diabetes had a higher prevalence of cataract surgery than those without diabetes (28.8% vs 16.9%, OR 1.78, 95%CI: 1.35-2.34 among non-indigenous Australians, and 11.3% vs 5.2%, OR 1.62, 95%CI: 1.22-2.14 among indigenous Australians). Diabetic retinopathy (DR) increased the odds of cataract surgery among self-reported diabetic indigenous and non-indigenous Australians (OR 1.89, P=0.004 and OR 2.33, P<0.001 respectively). Having diabetes for ≥20y and having vision-threatening DR increased the odds of cataract surgery among indigenous Australians with diabetes (OR 3.73, P=0.001 and 7.58, P<0.001, respectively). CONCLUSION: Most non-retinopathy ocular conditions are not associated with self-reported diabetes. However, to account for Australia's worsening diabetes epidemic, interventions to reduce the impact of diabetes-related blindness should include increased cataract surgery services.

Effects of age on retinal macrophage responses to acute elevation of intraocular pressure.

There is accumulating evidence that aging shifts the central nervous system milieu towards a proinflammatory state, with increased reactivity of microglia in the aging eye and brain having been implicated in the development of age-related neurodegenerative conditions. Indeed, alterations to microglial morphology and function have been recognized as a part of normal aging. Here, we sought to assess the effects of age on the retinal microglial and macrophage response to acute intraocular pressure (IOP) elevation. Further, we performed experiments whereby bone marrow from young or middle-aged mice was used to reconstitute the bone marrow of whole-body irradiated 12 month old mice. Bone marrow chimeric mice then underwent cannulation and IOP elevation 8 weeks after whole-body irradiation and bone marrow transplantation in order to determine whether the age of bone marrow alters the macrophage response to retinal injury. Our data show retinal macrophage reactivity and microglial morphological changes were enhanced in older mice when compared to younger mice in response to injury. When IOP elevation was performed after whole-body irradiation and bone marrow rescue, we noted subretinal macrophage accumulation and glial reactivity was reduced compared to non-irradiated mice that had also undergone IOP elevation. This effect was evident in both groups of chimeric mice that had received either young or middle-aged bone marrow, suggesting irradiation itself may alter the macrophage and glial response to injury rather than the age of bone marrow.

Selective laser trabeculoplasty versus topical medication as initial glaucoma treatment: the glaucoma initial treatment study randomised clinical trial.

BACKGROUND/AIMS: To determine if selective laser trabeculoplasty (SLT) is superior to topical medication as a first-line treatment for glaucoma on quality of life (QoL) and clinical outcomes. METHODS: In this international, longitudinal, multisite randomised controlled trial, treatment naïve mild-to-moderate primary open angle or exfoliation glaucoma patients were randomised 1:1 to SLT or topical medication. Glaucoma-specific QoL (primary outcome) was measured using the Glaucoma Outcomes Assessment Tool (GOAT; 342 items, 12 domains). Secondary outcomes included rate of successful intraocular pressure (IOP) reduction (>25% reduction from baseline) and presence of ocular surface disease including conjunctival hyperaemia and eyelid erythema. Our intention-to-treat analysis was performed at months 12 and 24. RESULTS: Of 167 enrolled patients, 83 and 84 were randomised to SLT and topical medication, respectively; and 145 (n=75 SLT, n=70 medication) completed 24-month follow-up. While both treatment arms achieved significant within-group gains in GOAT outcomes at both endpoints, SLT patients reported a greater between-group improvement in 'social well-being' compared with medication patients (mean±SE=0.28±0.13; p=0.034) at 24 months. At month 24, the rate of successful IOP reduction was 18.6% (95% CI 3.0% to 34.3%, p=0.022) higher (absolute difference) in the medication compared with SLT group. More individuals in the medication group had conjunctival hyperaemia and eyelid erythema compared with SLT at 24 months. CONCLUSION: Overall, we did not find evidence that SLT was superior to medication in improving glaucoma-specific QoL. While we found superior IOP reduction in the medication arm, eyelid erythema and conjunctival hyperaemia were more prevalent in these patients compared with the SLT group. TRIAL REGISTRATION: ACTRN12611000720910.

Bleb-associated endophthalmitis: Proportion, presentation, management and outcomes in Victoria, Australia.

IMPORTANCE: Bleb-associated endophthalmitis is a potentially vision-threatening complication of trabeculectomy. With improvements in surgical technique and changing patterns of intraoperative antimetabolite use, a re-evaluation of the incidence of bleb-associated endophthalmitis is warranted. BACKGROUND: To investigate changes in the incidence, presentation, management and outcomes of bleb-associated endophthalmitis between 1997 and 2015 in Victoria, Australia. DESIGN: A retrospective cohort analysis. PARTICIPANTS: Consecutive cases of bleb-associated endophthalmitis managed at the Royal Victorian Eye and Ear Hospital (RVEEH) between 1997 and 2015. METHODS: Medical record review of consecutive cases of bleb-associated endophthalmitis and statistical analysis were performed. MAIN OUTCOME MEASURES: Visual acuity, including loss of light perception, intraocular pressure, and need for further surgery. RESULTS: Sixty-seven eyes with bleb-associated endophthalmitis (BAE) were identified. Of these, 41 had trabeculectomy performed in Victoria during the study period, over which time 11 129 trabeculectomies were performed. The proportion of BAE was stable over time (0.4%). The mean age at presentation was 73.7 ± 12.1 years old and the majority of patients were Caucasian (79.1%). The mean duration between glaucoma filtration surgery and the development of bleb-associated endophthalmitis was 3 years (Interquartile Range = 0.4-6.0 years). The cultures were positive in 71.6% of cases. Approximately 1 in 8 patients required enucleation. The final visual acuity was poor with a Snellen Visual Acuity (VA) of 6/60 or worse in two-thirds of patients. CONCLUSIONS AND RELEVANCE: Bleb-associated endophthalmitis is an uncommon complication following glaucoma filtration surgery. The proportion has remained stable over time. Visual outcomes remain poor.

Amyloid Precursor Protein Mediates Neuronal Protection from Rotenone Toxicity.

Mitochondrial complex I dysfunction is the most common respiratory chain defect in human disorders and a hotspot for neurodegenerative diseases. Amyloid precursor protein (APP) and its non-amyloidogenic processing products, in particular soluble APP α (sAPPα), have been shown to provide neuroprotection in models of neuronal injury; however, APP-mediated protection from acute mitochondrial injury has not been previously reported. Here, we use the plant-derived pesticide rotenone, a potent complex I-specific mitochondrial inhibitor, to discover neuroprotective effects of APP and sAPPα in vitro, in neuronal cell lines over-expressing APP, and in vivo, in a retinal neuronal rotenone toxicity mouse model. Our results show that APP over-expression is protective against rotenone toxicity in neurons via sAPPα through an autocrine/paracrine mechanism that involves the Pi3K/Akt pro-survival pathway. APP-/- mice exhibit greater susceptibility to retinal rotenone toxicity, while intravitreal delivery of sAPPα reduces inner retinal neuronal death in wild-type mice following rotenone challenge. We also show a significant decrease in human retinal expression of APP with age. These findings provide insights into the therapeutic potential of non-amyloidogenic processing of APP in complex I-related neurodegeneration.

Prevalence of glaucoma in the Australian National Eye Health Survey.

AIM: To estimate the prevalence of glaucoma in Australia. METHODS: This was a population-based study of 3098 non-Indigenous Australians (50-98 years) and 1738 Indigenous Australians (40-92 years) stratified by remoteness. Each participant underwent a standard examination that included visual field assessment, tonometry and non-mydriatic fundus photography. Two fellowship-trained glaucoma specialists independently assessed relevant case notes (past ocular history, best-corrected visual acuity, frequency doubling technology visual fields, Van Herick grade, intraocular pressure and optic disc-centred photographs) and assigned a diagnosis ranked on a scale of certainty: none, possible, probable or definite glaucoma. RESULTS: A total of 4792 (99.1%, 3062 non-Indigenous and 1730 Indigenous) participants had retinal photographs in at least one eye that were gradable for glaucoma. The weighted prevalence of glaucoma (definite) in non-Indigenous Australians and Indigenous Australians was 1.5% (95% CI 1.0 to 2.2) and 0.6% (95% CI 0.4 to 1.1), respectively. When definite and probable cases of glaucoma were combined, rates were 3.4% (95% CI 2.7 to 4.3) among non-Indigenous and 1.6% (95% CI 1.1 to 2.3) in Indigenous Australians. Only 52.4% of non-Indigenous Australians and 28.0% of Indigenous Australians with glaucoma self-reported a known history of glaucoma. CONCLUSION: We estimate that 198 923 non-Indigenous Australians aged 50 years and over and 2139 Indigenous Australians aged 40 years and over have glaucoma. Given the high rates of undiagnosed glaucoma coupled with a significant ageing of the Australian population, improvements in case detection and access to low vision rehabilitation services may be required to cope with the growing burden of glaucoma.

Amyloid precursor protein drives down-regulation of mitochondrial oxidative phosphorylation independent of amyloid beta.

Amyloid precursor protein (APP) and its extracellular domain, soluble APP alpha (sAPPα) play important physiological and neuroprotective roles. However, rare forms of familial Alzheimer's disease are associated with mutations in APP that increase toxic amyloidogenic cleavage of APP and produce amyloid beta (Aß) at the expense of sAPPα and other non-amyloidogenic fragments. Although mitochondrial dysfunction has become an established hallmark of neurotoxicity, the link between Aß and mitochondrial function is unclear. In this study we investigated the effects of increased levels of neuronal APP or Aß on mitochondrial metabolism and gene expression, in human SH-SY5Y neuroblastoma cells. Increased non-amyloidogenic processing of APP, but not Aß, profoundly decreased respiration and enhanced glycolysis, while mitochondrial DNA (mtDNA) transcripts were decreased, without detrimental effects to cell growth. These effects cannot be ascribed to Aß toxicity, since higher levels of endogenous Aß in our models do not cause oxidative phosphorylation (OXPHOS) perturbations. Similarly, chemical inhibition of ß-secretase decreased mitochondrial respiration, suggesting that non-amyloidogenic processing of APP may be responsible for mitochondrial changes. Our results have two important implications, the need for caution in the interpretation of mitochondrial perturbations in models where APP is overexpressed, and a potential role of sAPPα or other non-amyloid APP fragments as acute modulators of mitochondrial metabolism.

Transforming Growth Factor ß1-Induced NADPH Oxidase-4 Expression and Fibrotic Response in Conjunctival Fibroblasts.

Purpose: Fibrotic scarring after ocular surgeries and chemical burn injuries can impede clarity of the cornea and cause vision impairment. Transforming growth factor ß (TGFß) signaling pathway is known to mediate fibrotic scarring, and NADPH oxidase-derived reactive oxygen species has been shown to be an effector molecule that facilitates TGFß1-mediated responses. The present study explores the expression profile and functional importance of NADPH oxidase (Nox) in conjunctival fibroblasts. In addition, the effect of curcumin on the TGFß1-induced NADPH oxidase expression and collagen synthesis was also investigated. Methods: The mRNA expression of Nox isoforms in rabbit conjunctival fibroblasts was measured by real-time PCR. The production of hydrogen peroxide (H2O2) and total collagen by these cells was measured by Amplex Red assay and Picro-Sirius red assay, respectively. Nox4 was knocked down by adenovirus-mediated siRNA targeting Nox4 (Adv-Nox4i). Results: We describe for the first time that conjunctival fibroblasts express mRNA encoding for Nox2, Nox3, Nox4, and Nox5. TGFß1 was found to induce Nox4 mRNA expression and total collagen release by these cells (P < 0.05; n = 4), and both responses are blocked by Smad3 inhibitor SIS3. Suppressing Nox4 gene transcription with Adv-Nox4i completely attenuated TGFß1-stimulated H2O2 release and collagen production by conjunctival fibroblasts (P < 0.05; n = 4-6). Similarly, curcumin also inhibited TGFß1-induced Smad3 phosphorylation, Nox4-derived H2O2 production, and total collagen synthesis by conjunctival fibroblasts (P < 0.05; n = 4-6). Conclusions: The present study suggests that TGFß1-mediated production of collagen by conjunctival fibroblasts involves Nox4-derived H2O2 pathway and this effect of Nox4 is abrogated by curcumin. This mechanism might be exploited to prevent fibrotic scarring after surgeries and chemical burn injuries in the eye.

Surgical outcomes of trabeculectomy and glaucoma drainage implant for uveitic glaucoma and relationship with uveitis activity.

IMPORTANCE: This study provides ophthalmologists who manage uveitic glaucoma with important information on factors that can affect the success of surgical management of this challenging disease. BACKGROUND: This study examines surgical outcomes of trabeculectomy and glaucoma device implant (GDI) surgery for uveitic glaucoma, in particular the effect of uveitis activity on surgical outcomes. DESIGN: Retrospective chart review at a tertiary institution. SAMPLES: Eighty-two cases with uveitic glaucoma (54 trabeculectomies and 28 (GDI) surgeries) performed between 1 December 2006 and 30 November 2014. METHODS: Associations of factors with surgical outcomes were examined using univariate and multivariate analysis. MAIN OUTCOME MEASURES: Surgical outcomes as defined in Guidelines from World Glaucoma Association. RESULTS: Average follow up was 26.4 ± 21.5 months. Overall qualified success rate of the trabeculectomies was not statistically different from GDI, being 67% and 75%, respectively (P = 0.60). Primary and secondary GDI operations showed similar success rates. The most common postoperative complication was hypotony (~30%). Active uveitis at the time of operation was higher in trabeculectomy compared with GDI group (35% vs. 14%). Active uveitis at the time of surgery did not significantly increase risk of failure for trabeculectomies. Recurrence of uveitis was significantly associated with surgical failure in trabeculectomy group (odds ratio 4.8, P = 0.02) but not in GDI group. CONCLUSIONS AND RELEVANCE: Surgical success rate of GDI was not significantly different from trabeculectomy for uveitic glaucoma in this study. Regular monitoring, early and prolonged intensive treatment of ocular inflammation is important for surgical success particularly following trabeculectomy.

Porosity of Bleb Capsule declines rapidly with Fluid Challenge.

INTRODUCTION: The porosity of the fibrous capsule around a glaucoma drainage device (GDD) may be the most important functional attribute. The factors that determine capsular porosity are not well understood. Failed GDD surgeries are usually associated with thick impervious capsules and components of aqueous have been implicated in this process. PURPOSE: In this study, we interrogated the effect of passage of nonaqueous fluid on capsular porosity in mature (but aqueous naïve) blebs in a previously reported GDD model (the "Center for Eye Research Australia Implant"). MATERIALS AND METHODS: The study was performed at two centers using 17 New Zealand White (NZW) rabbits. An experimental GDD was implanted into the subconjunctival space but without connection to the anterior chamber. After 28 days, balanced salt solution (BSS) was passed through the implant for 30 to 40 minutes at 12 mm Hg. Capsular porosity was measured as flow (µL/min) at a constant pressure. Porosity of the capsule was retested at 3 and 6 days. RESULTS: There was a marked reduction in capsular porosity within 3 days of exposure to BSS (fluid challenge). Even though the baseline porosity was significantly different in the two groups (3.00 ± 0.5 µL/min and 29.67 ± 12.12 µL/min, p < 0.001), the effect of passage of BSS was similar. Capsular porosity fell by approximately 80% in both groups from baseline after single BSS challenge. Capsular thickness was significantly less in Advanced Eye Center (AEC) rabbits at baseline. There was no change in the capsular thickness before and after single fluid challenge. CONCLUSION: Passage of BSS at physiological pressures for under 40 minutes caused marked reduction in the porosity of the fibrous capsule within 3 days. This was not associated with any significant thickening of the fibrous capsule within this time frame. HOW TO CITE THIS ARTICLE: Pandav SS, Ross CM, Thattaruthody F, Nada R, Singh N, Gautam N, Beirne S, Wallace GG, Sherwood MB, Crowston JG, Coote M. Porosity of Bleb Capsule declines rapidly with Fluid Challenge. J Curr Glaucoma Pract 2016;10(3):91-96.

A Comparison of Perimetric Results from a Tablet Perimeter and Humphrey Field Analyzer in Glaucoma Patients.

PURPOSE: To determine the correlation between the perimetric outcomes from perimetry software Melbourne Rapid Fields (MRF) run on an Apple iPad tablet and those from the Humphrey Field Analyzer (HFA). METHODS: The MRF software was designed with features including variable fixation and fast thresholding using Bayes logic. Here, we report a cross-sectional study on 90 eyes from 90 participants: 12 had normal optic nerves and 78 had glaucoma with various degrees of visual field loss (41 mild and 37 moderate-severe). Exclusion criteria were patients with worse than 20/40 vision or recent intraocular surgery. The visual field outcomes of MRF were compared against those returned from the HFA 24-2 SITA standard. Participants were tested twice on the MRF to establish test-retest repeatability. RESULTS: The test durations were shorter on MRF than HFA (5.7 ± 0.1 vs. 6.3 ± 0.1 minutes, P < 0.001). MRF showed a high level of concordance in its outcomes with HFA (intraclass coefficient [ICC] = 0.93 for mean defect [MD] and 0.86 for pattern deviation [PD]) although the MRF tended to give a less negative MD (1.4 dB bias) compared with the HFA. MRF also showed levels of test-retest reliability comparable to HFA (ICC = 0.93 for MD and 0.89 for PD, 95% limits of agreement -4.5 to 4.3 dB). CONCLUSION: The perimetry results from the MRF have a strong correlation to the HFA outcomes. MRF also has test-retest reliability comparable to HFA. TRANSLATIONAL RELEVANCE: Portable tablet perimetry may allow accurate assessment of visual field when standard perimetry machines are unavailable or unsuitable.

Primary open-angle glaucoma.

Glaucoma is an optic neuropathy that is characterized by the progressive degeneration of the optic nerve, leading to visual impairment. Glaucoma is the main cause of irreversible blindness worldwide, but typically remains asymptomatic until very severe. Open-angle glaucoma comprises the majority of cases in the United States and western Europe, of which, primary open-angle glaucoma (POAG) is the most common type. By contrast, in China and other Asian countries, angle-closure glaucoma is highly prevalent. These two types of glaucoma are characterized based on the anatomic configuration of the aqueous humour outflow pathway. The pathophysiology of POAG is not well understood, but it is an optic neuropathy that is thought to be associated with intraocular pressure (IOP)-related damage to the optic nerve head and resultant loss of retinal ganglion cells (RGCs). POAG is generally diagnosed during routine eye examination, which includes fundoscopic evaluation and visual field assessment (using perimetry). An increase in IOP, measured by tonometry, is not essential for diagnosis. Management of POAG includes topical drug therapies and surgery to reduce IOP, although new therapies targeting neuroprotection of RGCs and axonal regeneration are under development.

AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo.

PURPOSE: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has recently been adapted to enable efficient editing of the mammalian genome, opening novel avenues for therapeutic intervention of inherited diseases. In seeking to disrupt yellow fluorescent protein (YFP) in a Thy1-YFP transgenic mouse, we assessed the feasibility of utilizing the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for gene modification of retinal cells in vivo. METHODS: Single guide RNA (sgRNA) plasmids were designed to target YFP, and after in vitro validation, selected guides were cloned into a dual AAV system. One AAV2 construct was used to deliver Streptococcus pyogenes Cas9 (SpCas9), and the other delivered sgRNA against YFP or LacZ (control) in the presence of mCherry. Five weeks after intravitreal injection, retinal function was determined using electroretinography, and CRISPR/Cas-mediated gene modifications were quantified in retinal flat mounts. RESULTS: Adeno-associated virus 2-mediated in vivo delivery of SpCas9 with sgRNA targeting YFP significantly reduced the number of YFP fluorescent cells of the inner retina of our transgenic mouse model. Overall, we found an 84.0% (95% confidence interval [CI]: 81.8-86.9) reduction of YFP-positive cells in YFP-sgRNA-infected retinal cells compared to eyes treated with LacZ-sgRNA. Electroretinography profiling found no significant alteration in retinal function following AAV2-mediated delivery of CRISPR/Cas components compared to contralateral untreated eyes. CONCLUSIONS: Thy1-YFP transgenic mice were used as a rapid quantifiable means to assess the efficacy of CRISPR/Cas-based retinal gene modification in vivo. We demonstrate that genomic modification of cells in the adult retina can be readily achieved by viral-mediated delivery of CRISPR/Cas.

Glaucoma laser and surgical procedure rates in Australia.

AIM: To determine trends in the number of glaucoma laser and surgical procedures performed in Australia between 1994 and 2014. METHODS: Medicare claims were analysed to determine the number of glaucoma-related procedures reimbursed in Australia between 1994 and 2014. Glaucoma procedures were identified by Medicare Benefits Schedule item number and analysed by age range, gender, state, month and year. RESULTS: Laser trabeculoplasty rates declined 60% between 1994 and 2003 before increasing a dramatic 353% between 2003 and 2014. Laser iridotomies increased 281% over the study period while cyclodestructive procedures increased 207%. The number of primary filtering operations for glaucoma fell 68% from a peak in 1996 to a low in 2006 and then remained stable. However, the number of filtering operations in eyes where a previous filtering operation had been performed increased 27%. There was a marked increase in glaucoma drainage device insertion, increasing 234% over the study period. CONCLUSIONS: There has been a substantial increase in laser trabeculoplasty procedures in Australia, following a decline between 1994 and 2003. Primary filtering operations for glaucoma have declined in number while glaucoma drainage devices are playing an increasingly prominent role in the surgical management of glaucoma.

Effects of ranibizumab on TGF-ß1 and TGF-ß2 production by human Tenon's fibroblasts: An in vitro study.

PURPOSE: Inhibiting exaggerated wound healing responses, which are primarily mediated by human Tenon's fibroblast (HTF) migration and proliferation, has become the major determining factor for a successful trabeculectomy. Antivascular endothelial growth factor (anti-VEGF) has showed promising results as a potential antifibrotic candidate for use concurrently in trabeculectomy. Preliminary cohort studies have revealed improved bleb morphology following trabeculectomy augmented with ranibizumab. However, the effects on HTFs remain unclear. This study was conducted to understand the effects of ranibizumab on transforming growth factor (TGF)-ß1 and transforming growth factor (TGF)-ß2 expression by HTFs. METHODS: The effect of ranibizumab on HTF proliferation and cell viability was determined using 3-(4,5-dimethylthiazone-2-yl)-2,5-diphenyl tetrazolium (MTT) assay. Ranibizumab at concentrations ranging from 0.01 to 0.5 mg/ml were administered for 24, 48, and 72 h in serum and serum-free conditions. Supernatants and cell lysates from samples were assessed for TGF-ß1 and TGF-ß2 mRNA and protein levels using quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: At 48 h, 0.5 mg/ml of ranibizumab significantly induced cell death under serum-free culture conditions (p<0.05). Ranibizumab caused a significant reduction in TGF-ß1 mRNA, but not for TGF-ß2. However, the total protein production of TGF-ß1 and TGF-ß2 was unaffected by this anti-VEGF treatment. CONCLUSIONS: Exposure of HTFs to an intravitreal dose of ranibizumab significantly suppresses cell viability in vitro; however, the application seemed unable to affect the ultimate production of TGF-ß. Therefore, we highlighted ranibizumab as a potential antiscarring agent that acts via a different mechanism when used synergistically with another antifibrotic agent. Understanding the mechanism of actions of ranibizumab offers an additional view of a possible new rational therapeutic strategy.

Measurement of Systemic Mitochondrial Function in Advanced Primary Open-Angle Glaucoma and Leber Hereditary Optic Neuropathy.

Primary Open Angle Glaucoma (POAG) is a common neurodegenerative disease characterized by the selective and gradual loss of retinal ganglion cells (RGCs). Aging and increased intraocular pressure (IOP) are glaucoma risk factors; nevertheless patients deteriorate at all levels of IOP, implying other causative factors. Recent evidence presents mitochondrial oxidative phosphorylation (OXPHOS) complex-I impairments in POAG. Leber Hereditary Optic Neuropathy (LHON) patients suffer specific and rapid loss of RGCs, predominantly in young adult males, due to complex-I mutations in the mitochondrial genome. This study directly compares the degree of OXPHOS impairment in POAG and LHON patients, testing the hypothesis that the milder clinical disease in POAG is due to a milder complex-I impairment. To assess overall mitochondrial capacity, cells can be forced to produce ATP primarily from mitochondrial OXPHOS by switching the media carbon source to galactose. Under these conditions POAG lymphoblasts grew 1.47 times slower than controls, whilst LHON lymphoblasts demonstrated a greater degree of growth impairment (2.35 times slower). Complex-I enzyme specific activity was reduced by 18% in POAG lymphoblasts and by 29% in LHON lymphoblasts. We also assessed complex-I ATP synthesis, which was 19% decreased in POAG patients and 17% decreased in LHON patients. This study demonstrates both POAG and LHON lymphoblasts have impaired complex-I, and in the majority of aspects the functional defects in POAG were milder than LHON, which could reflect the milder disease development of POAG. This new evidence places POAG in the spectrum of mitochondrial optic neuropathies and raises the possibility for new therapeutic targets aimed at improving mitochondrial function.

Determination of bleb capsule porosity with an experimental glaucoma drainage device and measurement system.

IMPORTANCE: Control of intraocular pressure after implantation of a glaucoma drainage device (GDD) depends on the porosity of the capsule that forms around the plate of the GDD. OBJECTIVE: To compare capsular porosity after insertion of 2 different GDDs using a novel implant and measurement system. DESIGN, SETTING, AND SUBJECTS: We performed an experimental interventional study at an eye research facility in a tertiary eye care center. Testing was performed on 22 adult New Zealand white rabbits that received the experimental GDD or an existing GDD. INTERVENTIONS: A new experimental GDD, the Center for Eye Research Australia (CERA) implant, was created using computer-aided design and a 3-dimensional printer. The CERA GDDs were implanted in the eyes of rabbits randomized into 1 of the following 3 groups: with no connection to the anterior chamber (n = 7), with connection to the anterior chamber for 1 week (n = 5), and with connection to the anterior chamber for 4 weeks (n = 5). In a control group (n = 5), a pediatric GDD was implanted without connection to the anterior chamber. We measured the capsular porosity using a pressure-gated picoliter pump at a driving pressure of 12 mm Hg. The animals were killed humanely for histologic study. MAIN OUTCOMES AND MEASURES: Porosity of the fibrous capsule around the implant. RESULTS: We found no difference in mean (SEM) capsular porosity between the CERA (3.39 [0.76; 95% CI, 1.43-5.48] µL/min) and pediatric (4.52 [0.52; 95% CI, 3.19-5.95] µL/min) GDDs (P = .28, unpaired t test) at 4 weeks without aqueous exposure. Mean (SEM) capsular porosity of CERA GDDs connected to the anterior chamber at 1 week was 2.46 (0.36; 95% CI, 1.55-3.44) µL/min but decreased to 0.67 (0.07; 95% CI, 0.49-0.86) µL/min at 4 weeks (P = .001, unpaired t test). CONCLUSIONS AND RELEVANCE: Our experimental method permits direct measurement of capsular porosity of an in situ GDD. In a comparison between an experimental (CERA) and an existing GDD, no differences were identified in capsular porosity or histologic reaction between the implants. These results suggest that the CERA GDD model can be used to test key components of glaucoma surgery and implant design.

Role of adenosine receptors in resveratrol-induced intraocular pressure lowering in rats with steroid-induced ocular hypertension.

BACKGROUND: Steroid-induced ocular hypertension is currently treated in the same way as primary open-angle glaucoma. However, the treatment is often suboptimal and is associated with adverse effects. We evaluated the oculohypotensive effects of topical trans-resveratrol in rats with steroid-induced ocular hypertension and involvement of adenosine receptors (AR) in intraocular pressure (IOP) lowering effect of trans-resveratrol. METHODS: The oculohypotensive effect of unilateral single-drop application of various concentrations of trans-resveratrol was first studied in oculonormotensive rats. Concentration with maximum effect was similarly studied in rats with steroid-induced ocular hypertension. Involvement of AR was studied by observing the alterations of IOP in response to trans-resveratrol after pretreating animals with AR subtype-specific antagonists. Additionally, we used computational methods, including 3D modelling, 3D structure generation and protein-ligand interaction, to determine the AR-trans-resveratrol interaction. RESULTS: All concentrations of trans-resveratrol produced significant IOP reduction in normotensive rat eyes. Maximum mean IOP reduction of 15.1% was achieved with trans-resveratrol 0.2%. In oculohypertensive rats, trans-resveratrol 0.2% produced peak IOP reduction of 25.2%. Pretreatment with A1 antagonist abolished the oculohypotensive effect of trans-resveratrol. Pretreatment with A3 and A2A AR antagonists produced significant IOP reduction in both treated and control eyes, which was further augmented by trans-resveratrol application in treated eyes. Computational studies showed that trans-resveratrol has highest affinity for A2B and A1, followed by A2A and A3 AR. CONCLUSION: Topically applied trans-resveratrol reduces IOP in rats with steroid-induced ocular hypertension. Trans-resveratrol-induced oculohypotension involves its agonistic activity at the A1 AR.