Exfoliation syndrome: association with systemic diseases-the Maccabi glaucoma study.

PURPOSE: To investigate the relationship between exfoliation syndrome (XFS) and systemic diseases. METHODS: A population-based, retrospective study with control group was conducted using the electronic medical database of Maccabi Health Services, the second largest Health Maintenance Organization (HMO) in Israel. Study population included Maccabi members from January 2003 to April 2016. Cases consisted of patients diagnosed with XFS regardless of glaucoma. The control group included Maccabi members without XFS, matched on age, sex, and ancestry, that were examined by an ophthalmologist within the last year. MAIN OUTCOME MEASURES: Associations between XFS and systemic diseases. RESULTS: We identified 16,388 patients with XFS, in whom 40.3% (n = 6613) had glaucoma. The control group included 14,015 patients. Mean age was 78.3 ± 8.9 years and 76.2 ± 8.5 years for the XFS and control group, respectively. In unconditional logistic regression analyses, after adjusting for age, sex, and ancestry, XFS was significantly associated with risk of cardiovascular diseases including hypertension (OR 1.07, 95% CI 1.01-1.13, p = 0.02), myocardial infarction (OR 1.21, 95% CI 1.17-1.31, p < 0.0001), and congestive heart failure (OR 1.70, 95% CI 1.55-1.88, p < 0.0001) as well as higher risk for high creatinine (OR 1.28, 95% CI 1.2-1.37, p < 0.0001). Diabetes mellitus and body mass index were inversely associated with XFS (OR 0.70, 95% CI 0.67-0.73, p < 0.0001 and OR 0.88, 95% CI 0.84-0.93, p < 0.0001, respectively). Overall cancer diagnoses were more common in the XFS group (OR 1.05, 95% CI 1.0-1.1, p = 0.05). XFS was associated with more hospitalizations (mean 5 ± 5.3 hospitalizations in the XFS group and 3.3 ± 4.0 in the controls, p < 0.0001). CONCLUSION: XFS is significantly associated with cardiovascular systemic diseases (in a population living in Israel and predominantly born in Russia).

Association of clusterin (CLU) variants and exfoliation syndrome: An analysis in two Caucasian studies and a meta-analysis.

Exfoliation syndrome (XFS) is an important risk factor for glaucoma (XFG) worldwide. LOXL1 variants are highly associated with XFS in most populations; however, the high frequency of risk alleles in normal individuals and the reversal of risk alleles in different ethnic populations suggest that other factors contribute to XFS pathogenesis. Clusterin (CLU) is an extracellular matrix chaperone that prevents protein aggregation and is highly expressed in ocular tissues affected by XFS. Studies examining common CLU variants for association with XFS have been inconsistent. The purpose of this study was to evaluate CLU variants for association with XFS in two independent datasets from the United States (222 cases and 344 controls) and Israel (92 cases and 102 controls). Seven tag SNPs that captured >95% of alleles at r(2) greater than 0.8 across the CLU genomic region were genotyped using TaqMan assays. Genotypes for an additional SNP, rs2279590, were imputed using phased haplotypes of HapMap reference CEU samples. Of the 8 CLU SNPs selected for the study, none were significantly associated with XFS in either case-control group (age and sex adjusted P > 0.14 and 0.36, respectively, in the US and Israeli datasets), or when they were meta-analyzed together (age and sex adjusted P > 0.13). Haplotype analysis using all 8 SNPs or only the promoter region SNPs also did not show significant associations of CLU with XFS in the combined US and Israeli dataset (P > 0.28). Meta-analysis of the data from this study and previous studies in Caucasian populations (1184 cases and 978 controls) resulted in statistically significant association of rs2279590 with XFS (summary OR = 1.18, 95% CI: 1.03-1.33, P = 0.01). Significant association between rs2279590 and XFS was also found in Indian populations (summary OR = 0.76, 95% CI: 0.61-0.96; P = 0.02); however, significant heterogeneity between the Caucasian and Indian populations possibly due to reversal of the risk allele precluded an overall meta-analysis for rs2279590 (Q = 0.001, I(2) = 91%). No significant association was identified for rs3087554 in either Caucasian populations (summary OR = 0.90, 95% CI: 0.77-1.05, P = 0.17) or Indian populations (summary OR = 0.89, 95% CI: 0.72-1.10, P = 0.28), or in both populations combined (1705 cases and 3713 controls; summary OR = 0.90, 95% CI: 0.79-1.01, P = 0.08). Significant heterogeneity precluded the addition of the Japanese data to the meta-analysis for rs3087554 (Q = 0.006, I(2) = 87%). Our results suggest that common CLU variants may contribute to modest XFS risk but even larger datasets are required to confirm these findings.

Transplantation of human bone marrow mesenchymal stem cells as a thin subretinal layer ameliorates retinal degeneration in a rat model of retinal dystrophy.

Vision incapacitation and blindness associated with retinal degeneration affect millions of people worldwide. Cell based therapy and specifically transplantation of human adult bone marrow-derived stem cells (hBM-MSCs) present possible treatment strategy. Subretinal transplantation of human or rat BM-MSCs was shown previously to improve retinal function in Royal College Surgeons (RCS) rats. In those studies cells were transplanted via a transscleral-transchoroidal approach, creating a localized subretinal bleb. Limited number of cells could be injected and photoreceptor rescue was restricted to areas in proximity to the injection site. Here we describe a new surgical method for subretinal transplantation that facilitates uniform distribution of transplanted cells as a thin layer along most of the subretinal space. We assessed the therapeutic effect of hBM-MSCs on RCS rats when transplanted either subretinally or intravitreally. We also examined whether a second transplantation can prolong the therapeutic effect. A cell suspension of 2.5 × 10(6) cells in 5 µl was injected subretinally or intravitreally in RCS rats at 28 days postnatal. In the subretinal group, hBM-MSCs were transplanted posterior to the limbus in the superotemporal part of the eye through a longitudinal triangular scleral tunnel reaching the choroid. In the intravitreal group, the cells were injected into the superotemporal part of the vitreous cavity. In cross sections of subretinally transplanted eyes, removed 2 h following transplantation, hBM-MSCs were distributed as a near-homogenous thin layer along most of the subretinal space. In some animals the cells were also detected in the choroid. In the intravitreal injection group, hBM-MSCs were clustered in the vitreous cavity. Transplanted cells could be detected up to 2 weeks after transplantation but not at later time points. Retinal function and structure were assessed by electroretinogram (ERG) and histology analysis, respectively. Six weeks post transplantation, the mean maximal scotopic ERG b-wave amplitude response recorded in RCS control eyes was 1.2 µV. By contrast, in transplanted eyes mean responses of 56.4 µV and 66.2 µV were recorded in the intravitreally and subretinally transplanted eyes, respectively. In the subretinal group, retinal function was significantly higher in transplanted compared with control eyes up to 20 weeks following transplantation. By contrast, in the intravitreal group, rescue of retinal function persisted only up to 12 weeks following transplantation. Histological analysis revealed that 8 weeks following subretinal transplantation, the retinas of control eyes were dystrophic, with outer nuclear layer (ONL) containing a single cell layer. An extensive photoreceptor rescue was demonstrated in transplanted eyes at this time point, with 3-4 cell layers in the ONL along the entire retina. A second subretinal transplantation at 70 days postnatal did not enhance or prolong the therapeutic effect of hBM-MSCs. No immunosuppressants were used and long-term safety analysis demonstrated no gross or microscopic adverse effects. Taken together our findings suggest that transplantation of hBM-MSCs as a thin subretinal layer enhances the therapeutic effect and the safety of cell transplantation.

Minocycline upregulates pro-survival genes and downregulates pro-apoptotic genes in experimental glaucoma.

PURPOSE: Minocycline, a second-generation tetracycline with anti-inflammatory and anti-apoptotic properties, was reported to be neuroprotective in experimental glaucoma and optic nerve transection as well as in other neurodegenerative diseases. The purpose of this study was to investigate the mechanism underlying that neuroprotective effect in murine glaucoma. METHODS: Elevated intraocular pressure was induced in 159 rats by the translimbal photocoagulation laser model. Minocycline 22 mg/kg or saline was injected intraperitoneally starting 3 days before the induction of glaucoma, and continued daily until the animals were sacrificed. The effect of minocycline on gene expression was evaluated using a quantitative polymerase chain reaction (PCR) array for apoptosis. The involvement of selected pro-apoptotic, pro-survival, and inflammatory genes was further analyzed by quantitative real-time PCR at multiple time points. Immunohistochemistry was used to study the effect of minocycline on microglial activation and to localize Bcl-2 changes. RESULTS: Minocycline significantly increased the anti-apoptotic gene Bcl-2 expression at day 8 and day 14 after the induction of glaucoma (p = 0.04 and p = 0.03 respectively), and decreased IL-18 expression in the retina at day 14 and day 30 (p = 0.04 and p < 0.001 respectively). PCR arrays suggested that additional genes were affected by minocycline, including Tp53bp2, TRAF4, osteoprotegerin, caspase 1 and 4, and members of the tumor necrosis factor superfamily. Additionally, minocycline decreased the amount of activated microglia in glaucomatous eyes. CONCLUSIONS: These results suggest that minocycline upregulates pro-survival genes and downregulates apoptotic genes, thus shifting the balance toward the anti-apoptotic side in experimental glaucoma.

Minocycline mechanism of neuroprotection involves the Bcl-2 gene family in optic nerve transection.

The second-generation tetracycline, minocycline, has been shown to exhibit neuroprotective therapeutic benefits in many neurodegenerative diseases including experimental glaucoma and optic nerve transection (ONT). This study investigated the mechanism underlying minocycline neuroprotection in a model of ONT. ONT was applied unilaterally in 36 Wistar rat eyes. The rats were randomly divided into a minocycline (22 mg/kg/d) treatment group and a saline treatment group (control). Treatment (minocycline or saline) was given by intraperitoneal injections initiated 3 d before ONT and continued daily until the end of the experiment. The involvement of pro-apoptotic, pro-survival and inflammatory pathways was analyzed by quantitative Real-Time Polymerase Chain Reaction at 4 h and 3 d after the transection in both treatment groups. The involvement of Bcl-2 protein was evaluated by immunohistochemistry. We found that Minocycline significantly increased the expression of the antiapoptotic gene bcl-2 4 h after transection (n = 8, p = 0.008) and decreased the expression of Bax at the same time point (n = 8, p = 0.03). Tumor Necrosis Factor α (TNFα), Inhibitor of Apoptosis Protein (IAP1) and Gadd45α were significantly upregulated in the retinas of eyes with ONTs compared to control (n = 10 for each gene, p = 0.02, p = 0.03, p = 0.04, respectively) but this effect was unaffected by minocycline. This study further support that the mechanism underlying minocycline neuroprotection involves the Bcl-2 gene family, suggesting that minocycline has antiapoptotic properties that support its value as a promising neuroprotective drug.

Increase in retinal ganglion cells' susceptibility to elevated intraocular pressure and impairment of their endogenous neuroprotective mechanism by age.

PURPOSE: To investigate age-associated changes in retinal ganglion cell (RGC) response to elevated intraocular pressure (IOP), and to explore the mechanism underlying these changes. Specifically, the effect of aging on inhibitor of apoptosis (IAP) gene family expression was investigated in glaucomatous eyes. METHODS: IOP was induced unilaterally in 82 Wistar rats using the translimbal photocoagulation laser model. IOP was measured using a TonoLab tonometer. RGC survival was evaluated in 3-, 6-, 13-, and 18-month-old animals. Changes in the RNA profiles of young (3-month-old) and old glaucomatous retinas were examined by PCR array for apoptosis; changes in selected genes were validated by real-time PCR; and changes in selected proteins were localized by immunohistochemistry. RESULTS: There were no significant IOP differences between the age groups. However, there was a natural significant loss of RGCs with aging and this was more prevalent in glaucomatous eyes. The number of RGCs in glaucomatous eyes decreased from 669±123 RGC/mm² at 3 months to 486±114 RGC/mm² at 6 months and 189±46.5 RGC/mm² at 18 months (n=4-8, p=0.048, analysis of variance). The PCR array revealed different changes in proapoptotic and prosurvival genes between young and old eyes. The two important prosurvival genes, IAP-1 and X-linked IAP (XIAP), acted in opposite directions in 3-month-old and 15-month-old rats, and were significantly decreased in aged glaucomatous retinas, while their expression increased significantly in young glaucomatous eyes. P53 levels did not vary between young glaucomatous and normal fellow eyes, but were reduced with age. B-cell leukemia/lymphoma 2 (Bcl-2) family members and tumor necrosis factor (TNF)-α expression were unaffected by age. Immunohistochemistry results suggested that the sources of changes in IAP-1 protein expression are RGCs and glial cells, and that most XIAP secretion comes from RGCs. CONCLUSIONS: Decreased IAP-1 and XIAP gene expression in aged eyes may predispose RGCs to increased vulnerability to glaucomatous damage. These findings suggest that aging impairs the endogenous neuroprotective mechanism of RGCs evoked by elevated IOP.

Comparison between axonal and retinal ganglion cell gene expression in various optic nerve injuries including glaucoma.

PURPOSE: The pathogenesis of retinal ganglion cell loss in glaucoma remains incompletely understood. Current evidence suggests that the optic nerve (ON) head and axons are the main site of injury in glaucoma. This study compares changes in prosurvival and proapoptotic gene expression in ONs with those in retinas in three models of ocular injury, specifically ON transection (ONTX), N-methyl-D-aspartate (NMDA) retinal toxicity, and experimental glaucoma. METHODS: Rats (n=240) were divided into three models (ONTX, NMDA retinal toxicity, and experimental glaucoma). The experimental model was induced unilaterally and the contralateral eye served as control. Rats were sacrificed at 4-5 different time points specific for each model. ONs and retinas were isolated for real-time PCR investigation of expression of selected genes. Immunohistochemistry localized changes in inhibitor of apoptosis (IAP)-1 and X-linked IAP (XIAP) proteins in retinas and ONs. Colocalization was measured using Imaris colocalization software (three-dimensional analysis). RESULTS: The earliest changes in gene expression occurred in ONs in the ONTX model and in retinas in the NMDA model, as expected. However, some gene changes occurred first in ONs, while others occurred first in retinas in the glaucoma model. The expression patterns of the prosurvival genes IAP-1 and XIAP differed between retinas and ONs of glaucomatous eyes: Both were upregulated in the retinas, but XIAP was downregulated in the ONs, while IAP-1 stayed unchanged. Colocalization of IAP-1, XIAP, glial fibrillary acidic protein, and Thymus cell antigen-1 (Thy-1) suggested that IAP-1 was colocalized mostly with Thy-1 and XIAP with glial fibrillary acidic protein in the ONs. Members of the B-cell lymphoma 2 (BCL-2) family were similarly involved in the ONs and retinas of glaucomatous, transected, and NMDA-injected eyes. The expression of the prosurvival genes, Bcl-2 and Bcl-xl, decreased significantly in both the ONs and retinas of injured eyes. The proapoptotic genes, BCL2-associated X protein (BAX) and Bcl-2-associated death promoter (BAD), were significantly upregulated in both injured retinas and ONs. CONCLUSIONS: The overexpression of XIAP and IAP-1 genes in the retinas was not associated with similar changes in the ONs of glaucomatous eyes. The lack of activation of these prosurvival genes in the ONs may explain the increased vulnerability of ONs to elevated intraocular pressure.

Functional and structural evaluation of lamotrigine treatment in rat models of acute and chronic ocular hypertension.

Voltage gated sodium channels (Nav), are proposed mediators of neuronal damage in ischemic and excitotoxicity disease models. We evaluated the neuroprotective effects of lamotrigine, a Nav blocker, in the acute and chronic rat ocular hypertension models. Additionally, expression of the main Nav subtypes in the optic nerve (ON) was assessed to test whether their upregulation plays a role in the pathogenesis of ocular hypertension induced optic neuropathy. Unilateral intraocular pressure (IOP) elevation was induced for 60 min (80 mmHg) and 14-21 days (670-859 mmHg*day) in the acute and chronic models, respectively. Lamotrigine was administered at dosages of 10 mg/kg twice daily and 12.5 mg/kg once daily in the acute (n = 9) and chronic (n = 11) trials, respectively. Treatment began 2 days prior to IOP elevation until sacrifice. Outer and inner retinal function was evaluated with dark- and light-adapted flash electroretinography and pattern electroretinography, respectively, 6 and 14 days post acute IOP elevation and 13, 28 and 48 days post chronic IOP elevation. Retinal ganglion cell and axon densities and inflammatory reaction were evaluated through Fluorogold, Bielschowsky's silver impregnation and ED1 labeling respectively. Immunohistochemistry for Nav1.1, 1.2 and 1.6 was performed in ONs of untreated rats 7 and 15 days post IOP elevation in the acute model and after 7, 28 and 50 days in the chronic model. In the acute model, no differences were found in the a-wave amplitudes between lamotrigine-treated and vehicle-treated rats. B-wave amplitudes decreased by 40-66% in both treatment groups 6 days post IOP elevation, with no significant difference between groups (p = 0.38). However, a partial recovery of b-wave amplitudes was found in lamotrigine-treated rats between day 6 and day 14 post procedure (p < 0.05). No differences were found in any other parameter tested in this model. Similarly, lamotrigine treatment did not result in any beneficial effect in structural parameters of the chronic model. Functional evaluation of this model was inconclusive due to super-normal values in the hypertensive eyes. Up-regulation of Nav1.1 and 1.2 expression was found in both models, beginning by day 7; an increase of the former continued in a time-dependent manner in the chronic model. Nav1.6 labeling was inconclusive. In conclusion we found lamotrigine treatment to be mostly ineffective in both acute and chronic ocular hypertension models.

Peripapillary and fovea avascular zone optical coherence tomography angiography parameters in exfoliation glaucoma versus primary open-angle glaucoma versus healthy eyes.

Purpose: To examine the differences in the peripapillary vascular parameters and foveal-avascular-zone (FAZ) vascularity parameters between primary open-angle-glaucoma (POAG) patients versus exfoliation-glaucoma (XFG) patients versus healthy subjects. Methods: This is cross-sectional study and a comparative clinical study. POAG and XFG patients and healthy subjects underwent a comprehensive ophthalmic examination, including visual field optical coherence tomography (OCT) and OCT angiography (OCTA) of the optic disc and FAZ. Differences in peripapillary vessel density (VD), perfusion density (PD), and FAZ area and circularity were examined between all groups, as well as correlations between clinical parameters and vascularity parameters for each glaucoma group. Results: A total of 109 subjects (one eye for each patient) were analyzed, including 45 with POAG, 30 with XFG, and 34 controls. The average peripapillary VDs were the lowest among the XFG patients and the highest among the controls (P < 0.05, ANOVA). The average peripapillary PD of the central ring was the lowest in the XFG group and the highest in the control group (P = 0.02, ANOVA). A significant negative correlation was found between the average peripapillary VDs and PDs of the inner ring and full ring and disease severity of the POAG patients. There was a significant positive correlation between the average peripapillary PDs of the central rings and full ring and the central macular thickness of the XFG patients (P < 0.01 and P < 0.04, respectively, Pearson correlation). Conclusion: The peripapillary vascular parameters of the POAG and XFG patients were lower compared to those of normal participants. A correlation between clinical characteristics of POAG and XFG patients and PD was found. This may hint to a vascular mechanism in glaucoma either primary or secondary to intra-ocular pressure/OAG damage.

Similarities and differences between primary and secondary degeneration of the optic nerve and the effect of minocycline.

PURPOSE: To investigate the mechanism of secondary degeneration of the optic nerve, and to evaluate the neuroprotective effect of minocycline in this process. METHODS: A partial transection model that morphologically separates primary and secondary degeneration was applied unilaterally in 152 Wistar rat eyes. The involvement of pro-apoptotic, pro-survival and inflammatory pathways was analyzed by quantitative real-time PCR and immunohistochemistry at multiple time points. The neuroprotective effect of daily intraperitoneal injections of minocycline 22 mg/kg/day was evaluated at 7, 11 and 21 days post-injury. Retrograde labeling of retinal ganglion cells (RGCs) with fluorogold was via the superior colliculus, and surviving RGCs were counted using retinal whole mounts. RESULTS: Both primary and secondary degeneration led to a significant up-regulation of the pro-apoptotic genes, GADD45α, ei24 and CDK2, and the pro-survival gene, IAP-1. These processes differed, however, in their reaction to minocycline. Minocycline protected RGC death from secondary degeneration at 11 days (6 ± 8% loss compared to 37 ± 7% in the saline-treated group, n = 15, P = 0.012), and at 21 days (42 ± 7% versus 64 ± 7% respectively, n = 15, P = 0.06) after partial transection. In contrast, its effect on primary degeneration was not significant. CONCLUSIONS: While the genetic profile supported similarities between primary and secondary degeneration of the optic nerve, the specific effect of minocycline on secondary degeneration revealed a potential difference between the two. The mechanism underlying secondary degeneration, and its role in optic neuropathies such as glaucoma, awaits further studies.

Mechanism of retinal ganglion cells death in secondary degeneration of the optic nerve.

In central nervous system injury, the secondary degeneration process is known to play a major role in determining the final extent of impairment. Here, we investigated the mechanism of retinal ganglion cell (RGC) death in secondary degeneration of the optic nerve using a unique model that allows morphological separation between primary and secondary degeneration. A partial transection model was applied unilaterally in 110 Wistar rat eyes. The rate of apoptosis was evaluated in primary and secondary degeneration over a period of 6 months using the Hoechst staining technique. The involvement of caspase 3 and members of the Bcl-2 family (Bax, Bad, Bcl-2 and Bcl-xl) was evaluated at multiple time points for 6 months after the injury by immunohistochemistry and RT-PCR. We found that in secondary degeneration of the optic nerve, RGCs died by apoptosis from day 3-6 months following the injury, peaking at 3 months (16.3% +/- 2.5% apoptotic cells, p < 0.01). Both primary and secondary degeneration of the optic nerve resulted in caspase 3 activation, which was longer and more intense in the former. Similarly, both primary and secondary degeneration led to significant (p < 0.05) downregulation of the pro-survival genes Bcl-2 and Bcl-x-L and up-regulation of the pro-apoptotic genes Bax and Bad (p < 0.05), with a suggested delay in secondary degeneration. Thus, secondary degeneration of the optic nerve leads to RGC apoptosis over long periods in a similar mechanism as in primary degeneration.

Mesopic foveal contrast sensitivity is impaired in diabetic patients without retinopathy.

BACKGROUND: Contrast sensitivity (CS) has been studied extensively to determine its effectiveness as a test for diagnosing early and advanced diabetic retinopathy. Various techniques have been adopted to measure CS, and most of them reported a significant difference between diabetic and normal eyes. Our purpose is to demonstrate differences in foveal CS between diabetic patients without retinopathy and healthy subjects under mesopic and photopic conditions, using a simple, rapid computerized test. METHODS: Seventeen eyes of nine patients with type 2 diabetes without diabetic retinopathy were included. Fourteen eyes of seven non-diabetic patients served as controls. All the patients underwent a careful ophthalmologic examination, including ETDRS chart visual acuity, color photographs, and optical coherence tomography (OCT). Patients with any ocular disease were excluded. All eyes had a visual acuity of 20/25 or better, a normal eye examination and optical coherence tomography (OCT). Photopic and mesopic contrast sensitivity was tested using a computerized psychophysical static method involving four forced-choice procedures. The targets were Gabor patches with spatial frequencies of 3-12 cycles per degree (cpd). The mesopic testing was conducted in a completely darkened room; the monitor was covered with a neutral density filter, allowing luminance of only 0.9 cd/m(2). RESULTS: The average age was similar: 59.1 ± 5.3 years in the diabetic group vs 61.4 ± 3.2 years in the control group. The average duration of diabetes was 16 years (range 6-26). The average visual acuity was 0.04 ± 0.01 logMAR and 0.01 ± 0.01 logMAR in the diabetic and control groups respectively. Photopic foveal CS was similar in both groups. Significantly lower CS was found in diabetic patients under mesopic conditions at a spatial frequency of 3 (p < 0.008). At higher spatial frequencies, the mesopic contrast sensitivity was very low in both groups and without a significant difference. CONCLUSIONS: Mesopic foveal CS is impaired in diabetic patients despite good visual acuity, a normal fundus examination and normal OCT. Early central visual function impairment may occur in diabetic patients before the appearance of retinopathy.

Constitutive expression of HIF-1alpha and HIF-2alpha in bone marrow stromal cells differentially promotes their proangiogenic properties.

Bone marrow stromal cells (BMSCs) contain progenitors capable of participating in postnatal angiogenesis. Hypoxia-inducible factors (HIFs) mediate endothelial activation by driving the expression of multiple angiogenic factors. We explored the potential of HIF-1alpha and HIF-2alpha modification in BMSCs, as a tool to improve cell-based angiogenic therapy. BMSCs were retrovirally transduced to express stable forms of HIF-1alpha and HIF-2alpha. HIF-1alpha and, to a greater extent, HIF-2alpha overexpression promoted differentiation of BMSCs to the endothelial lineage, evident by CD31 and Tie-2 expression and improved adhesive properties. Whereas chemotaxis toward stromal-derived factor 1 was higher in both HIF-alpha-expressing BMSCs, enhanced migration toward vascular endothelial growth factor was found only following overexpression of HIF-2alpha, supported by a robust expression of its receptor, Flk-1. HIF-alpha expression was associated with upregulation of angiogenic proteins and improved tube formation. Cytokine arrays of endothelial cells stimulated by medium collected from HIF-alpha-expressing BMSCs revealed further angiogenic activation and improved adhesive capacity. Eventually, delivery of HIF-2alpha-transduced BMSCs induced a more robust angiogenic response, compared with sham-transduced or HIF-1alpha-transduced BMSCs in the corneal micropocket angiogenesis model. Our results support the use of HIF-alpha genes, particularly HIF-2alpha, to augment the efficacy of future cell-based therapy. Disclosure of potential conflicts of interest is found at the end of this article.

Overuse and Underuse of Visual Field Testing Over 15 Years.

PRéCIS:: A 15-year analysis of 198,843 visual field (VF) tests revealed a growing trend for their performance for nonglaucoma indications. Adherence to glaucoma management guidelines was suboptimal. Guidelines for referral to VF assessments should be established. PURPOSE: The purpose of this study was to identify trends in VF assessments over 15 years among patients with and without suspected or confirmed glaucoma, in a large healthcare maintenance organization. METHODS: This was a population-based retrospective cohort study, conducted by means of electronic medical database analyses. STUDY POPULATION: Maccabi Healthcare Services is an healthcare maintenance organization that insures 2 million members constituting 25% of the population. All members who underwent at least 1 VF test between January 2000 and December 2014 were included. In addition, all members with glaucoma or suspected glaucoma diagnosis or who were prescribed with antiglaucoma medications were evaluated. MAIN OUTCOME MEASURES: VF performance rates. RESULTS: A total of 93,617 Maccabi Healthcare Services members underwent 198,843 VF tests; of whom 47.9% involved patients without any glaucoma-related conditions. There was a growing trend over time toward more of those members to undergo VF tests and, by 2014, non-glaucoma-related members comprised 74.0% of new VF assessments. In contrast, 32.3% of glaucoma-related patients did not perform even 1 VF test throughout the entire study period. Although over 2 years (25.95±6.33 mo) passed between the first glaucoma-related diagnosis and first VF test, once a patient underwent the first VF test, an average once-a-year VF follow-up (0.95±0.37 annual tests) began. CONCLUSION: There is a growing trend for VF tests being apparently overused for indications other than glaucoma. Concurrently, adherence to glaucoma management guidelines on VF tests is suboptimal, leading to discernible underuse. Guidelines for VF assessments in nonglaucoma patients should be established. Adherence to existing glaucoma management guidelines should be improved.

Microarchitecture of Schlemm Canal Before and After Cataract Extraction Surgery.

PRECIS: Schlemm canal (SC) expands after cataract extraction (CE), both in the area and in volume by 25% as was measured using enhanced-depth imaging optical coherent tomography (EDI-OCT) in patients before and 1 week after CE. PURPOSE: This study aims to characterize the structural and volume changes on the microstructure of SC in patients before and after uneventful phacoemulsification CE by using EDI-OCT. MATERIALS AND METHODS: Forty-one serial horizontal EDI-OCT B-scans (interval between B-scans, 69 µm) were obtained in the nasal corneoscleral limbus before and 1 week after CE. The structure of aqueous channels, conjunctival blood vessels and iris anatomy in each scan were used as landmarks to select for overlapping scans taken before and following CE. The SC cross-section area was measured in each of the selected scans and SC volume was determined following a 3-dimensional reconstruction. RESULTS: Eleven eyes (6 females and 5 males) were imaged successfully before and after CE. Mean age was 70.54±11.38 years. The mean axial length was 23.10±0.87 mm. After CE, the mean best-corrected visual acuity in logMAR improved from 0.4±0.13 to 0.2±0.13 (P=0.028). There was no significant change in the mean intraocular pressure before and after CE (15.09±1.33 to 15.0±2.16 mm Hg; P=0.39). The mean SC cross-section area increased by 25%, from 4744±376 to 5941±1048 µm (P<0.001). SC volume in the analyzed region increased by 25% from 6,641,473±585,954 to 8,317,909±1,328,809 µm (P<0.001). CONCLUSION: CE expands SC dimensions in healthy eyes. EDI-OCT imaging of SC may prove useful in the evaluation of the SC dimensions in vivo before and after CE.

Drug modification of angiogenesis in a rat cornea model.

PURPOSE: To evaluate the influence of some widely used antiglaucoma agents on angiogenesis in a novel rat cornea model. METHODS: Angiogenesis was induced in 32 rats by slow-release polymer pellets containing basic fibroblast growth factor (bFGF) placed in a corneal micropocket. Angiogenesis was later measured and compared in groups of rats given one of four antiglaucoma drug therapies and one control group. The drugs were commercially available preparations of prostaglandins, beta-blockers, alpha-2 agonists, and carbonic anhydrase inhibitors given for 7 days in a manner similar to that used in humans. Growth was measured by calculating the maximum linear vessel growth divided by pellet-limbus distance. RESULTS: Biomicroscopic observation disclosed that all tested animals showed an induction of neovascular reactions in their corneal stroma. The growth index results for the control, latanoprost, dorzolamide, brimonidine, and timolol malate groups were 1.65 +/- 0.16, 1.98 +/- 0.18, 1.85 +/- 0.19, 2.03 +/- 0.38, and 1.65 +/- 0.14, respectively, confirming the hypothesis that topically delivered antiglaucoma drugs modify the normal angiogenic response. Of them, the prostaglandins showed the most prominent angiogenic stimulatory effect (P = 0.03). CONCLUSIONS: This modified micropocket assay of corneal angiogenesis in rats demonstrated the stimulatory effect of several widely used topically delivered antiglaucoma medications on the angiogenic process. The results indicate that the selection of drugs for treating different ophthalmic diseases should take into account their influence on angiogenic processes.

Intraocular pressure elevation within the first 24 hours after cataract surgery in patients with glaucoma or exfoliation syndrome.

OBJECTIVE: To investigate whether eyes with glaucoma or exfoliation syndrome without glaucoma are prone to exhibit intraocular pressure (IOP) elevation shortly after cataract surgery and, if so, whether timolol maleate 0.5% reduces these spikes. DESIGN: Prospective randomized double-masked clinical trial. PARTICIPANTS: One hundred twenty-two patients with normal eyes, medically well-controlled glaucoma, or exfoliation syndrome who underwent uneventful phacoemulsification cataract extraction. METHODS: Patients were randomly assigned to an immediately postoperative drop of either timolol maleate 0.5% or no treatment. Intraocular pressure was measured preoperatively and 4, 8, and 24 hours and 1 week later. MAIN OUTCOME MEASURES: Intraocular pressure measurements. RESULTS: The changes in postoperative IOP over time differed significantly between glaucoma, exfoliation syndrome, and normal (P = 0.005). Intraocular pressure was significantly lower in the normal group (n = 25) than in both the glaucoma (n = 18) and exfoliation syndrome (n = 19) groups (P<0.001). With 1 drop of prophylactic timolol maleate 0.5% at completion of surgery, the normal group (n = 25) again had IOP significantly lower than those of the glaucoma (n = 15) and exfoliation syndrome (n = 20) groups (P<0.001). Treatment with timolol maleate 0.5% significantly changed postoperative IOP over time in the glaucomatous eyes (P = 0.003), but it made no difference in the exfoliation syndrome (P = 0.4) or normal (P = 0.5) eyes. Intraocular pressure > 25 mmHg did not occur among normal eyes. Intraocular pressure > 25 mmHg and > 30 mmHg occurred in 10 (55%) and 5 (28%) glaucoma patients, respectively, and 5 (27%) and 2 (11%) exfoliation syndrome patients, respectively. Timolol maleate 0.5% eliminated IOP spikes > 30 mmHg and reduced the frequency of IOP > 25 mmHg in both groups to 14% in the glaucoma group and 5% in the exfoliation syndrome group. Most IOP elevation occurred at 4 hours postoperatively. The mean IOP was <20 mmHg in all groups 1 day postoperatively. CONCLUSIONS: Medically well-controlled glaucoma patients and patients with exfoliation syndrome may experience IOP elevation shortly after cataract surgery. Instillation of timolol maleate 0.5% at the end of the procedure in this series eliminated IOP > 30 mmHg, but IOP elevation below that level can still occur.

LOXL1 Polymorphisms: Genetic Biomarkers that Presage Environmental Determinants of Exfoliation Syndrome.

An agnostic high throughput search of the genome revealed a robust association between LOXL1 genetic polymorphisms and exfoliation syndrome (XFS), a discovery that likely would not have been possible with candidate or family-based gene search strategies. While questions remain regarding how LOXL1 gene variants contribute to XFS pathogenesis, it is clear that the frequencies of disease-related alleles do not track with the varying disease burden throughout the world, prompting a search for environmental risk factors. A geo-medicine approach revealed that disease load seemed to increase as a function of the distance from the equator. The exact reason for this extraequatorial disease distribution pattern remains unclear, but a greater amount of time spent outdoors is a robust risk factor for XFS, suggesting climatic factors such as ocular solar exposure and colder ambient temperature may be involved in disease pathogenesis. Prospective studies have also implicated higher coffee consumption and lower dietary folate intake in association with incident XFS. The discovery of environmental risk factors for XFS suggests that preventive measures may help to reduce ocular morbidity from XFS.

A Role for Clusterin in Exfoliation Syndrome and Exfoliation Glaucoma?

The multifunctional protein clusterin (CLU) is a secreted glycoprotein ubiquitously expressed throughout the body, including in the eye. Its primary function is to act as an extracellular molecular chaperone, preventing the precipitation and aggregation of misfolded extracellular proteins. Clusterin is commonly identified at fluid-tissue interfaces, and has been identified in most body fluids. It is a component of exfoliation material, and CLU mRNA is reduced in eyes with exfoliation syndrome compared with controls. SNPs located in the CLU genomic region have been associated with Alzheimer disease (AD) at the genome-wide level and several CLU SNPs located in an apparent regulatory region have been nominally associated with XFS/XFG in Caucasians with European ancestry and in south Indians. Interestingly, clusterin associates with altered elastic fibers in human photoaged skin and prevents UV-induced elastin aggregation in vitro. In light of the known geographic risk factors for XFS/XFG, which could include UV light, investigations of CLU-geographic interactions could be of interest. Future studies investigating rare CLU variation and other complex interactions including gene-gene interactions in XFS/XFG cases and controls may also be fruitful. Although CLU has been considered as a therapeutic target in AD, cancer and dry eye, a role for clusterin in XFS/XFG needs to be better defined before therapeutic approaches involving CLU can be entertained.