Ian L. McAllister

Correlation of Histologic and Clinical Images to Determine the Diagnostic Value of Fluorescein Angiography for Studying Retinal Capillary Detail

PURPOSE To delineate morphometric and quantitative features of the capillary image derived from high-resolution fundus fluorescein angiography (FFA) and consequently determine the diagnostic value of FFA for studying the retinal capillary circulation. METHODS Retinal capillary images obtained from healthy young subjects using high-resolution FFA were compared with confocal scanning laser microscopic capillary images derived from the retinas of age-matched human donors. Confocal microscopic images were acquired from retinal flatmount tissue after central retinal artery cannulation, perfusion fixation, and antibody labeling. Capillary images from equivalent retinal regions were morphologically and quantitatively analyzed in both groups. RESULTS Ten human subjects (mean age, 27.4 years) were used for FFA studies, and five cadaveric eyes (mean donor age, 26.5 years) were used for histologic studies. In histologic specimens the density of the superficial capillary network was significantly greater than that of the deep capillary network. Despite use of a healthy young population, only 30% of high-resolution FFA studies provided clear capillary images. The configuration of the capillary network in FFA images was comparable to the superficial capillary network in confocal microscope images; however, the density of the capillary network in FFA images was consistently lower than that of histologic images. CONCLUSIONS FFA provides incomplete morphologic information about the superficial capillary network and even less information about the deep capillary network. Caution should, therefore, be exercised when using FFA data to extrapolate information about microvascular histopathologic processes. The usefulness of newer technology for studying retinal capillary detail should be investigated.

A Randomized Clinical Trial of Intravitreal Bevacizumab versus Intravitreal Dexamethasone for Diabetic Macular Edema: The BEVORDEX Study

OBJECTIVE To report the 12-month results of the first head-to-head comparison of a dexamethasone implant (Ozurdex; Allergan, Inc., Irvine, CA) versus bevacizumab (Avastin; Genentech, South San Francisco, CA) for center-involving diabetic macular edema (DME). DESIGN Phase 2, prospective, multicenter, randomized, single-masked clinical trial (clinicaltrials.gov identifier NCT01298076). PARTICIPANTS We enrolled 88 eyes of 61 patients with center-involving DME. METHODS Forty-two eyes were randomized to receive bevacizumab every 4 weeks and 46 eyes were randomized to receive a dexamethasone implant every 16 weeks, both pro re nata. Results were analyzed using linear regression with generalized estimation equation methods to account for between-eye correlation. MAIN OUTCOME MEASURES The primary outcome was the proportion of eyes that improved vision by 10 logarithm of minimum angle of resolution letters. Secondary outcomes included mean change in best-corrected visual acuity (BCVA), change in central macular thickness (CMT), injection frequency, and adverse events. Patient-reported outcomes were measured using the Impact of Vision Impairment (IVI) questionnaire. RESULTS Improvement in BCVA of 10 or more letters was found in 17 of 42 eyes (40%) treated with bevacizumab compared with 19 of 46 dexamethasone implant-treated eyes (41%; P = 0.83). None of the 42 bevacizumab eyes lost 10 letters or more, whereas 5 of 46 (11%) dexamethasone implant eyes did, mostly because of cataract. Mean CMT decreased by 122 μm for bevacizumab eyes and by 187 μm for dexamethasone implant eyes (P = 0.015). Bevacizumab-treated eyes received a mean of 8.6 injections compared with 2.7 injections for dexamethasone implant eyes. Significant improvement in IVI scores occurred for both treatment groups. CONCLUSIONS Dexamethasone implant achieved similar rates of visual acuity improvement compared with bevacizumab for DME, with superior anatomic outcomes and fewer injections. Both treatments were associated with improvement in visual quality-of-life scores. However, more dexamethasone implant-treated eyes lost vision, mainly because of cataract.

Quantitative confocal imaging of the retinal microvasculature in the human retina.

PURPOSE We investigated quantitatively the distribution of blood vessels in different neural layers of the human retina. METHODS A total of 16 human donor eyes was perfusion-fixed and labeled for endothelial f-actin. Retinal eccentricity located 3 mm superior to the optic disk was studied using confocal scanning laser microscopy. Immunohistochemical methods applied to whole-mount and transverse sections were used to colocalize capillary networks with neuronal elements. Capillary morphometry, diameter, and density measurements were compared among networks. RESULTS Four different capillary networks were identified and quantified in the following regions: Nerve fiber layer (NFL), retinal ganglion cell (RGC) layer, border of the inner plexiform layer (IPL) and superficial boundary of the inner nuclear layer (INL), and boundary of the deep INL and outer plexiform layer. The innermost and outermost capillary networks demonstrated a laminar configuration, while IPL and deep INL networks displayed a complex three-dimensional configuration. Capillary diameter in RGC and IPL networks were significantly less than in other networks. Capillary density was greatest in the RGC network (26.74%), and was significantly greater than in the NFL (13.69%), IPL (11.28%), and deep INL (16.12%) networks. CONCLUSIONS The unique metabolic demands of neuronal sub-compartments may influence the morphometric features of regional capillary networks. Differences in capillary diameter and density between networks may have important correlations with neuronal function in the human retina. These findings may be important for understanding pathogenic mechanisms in retinal vascular disease.

The distribution of angioarchitectural changes within the vicinity of the arteriovenous crossing in branch retinal vein occlusion.

OBJECTIVE Branch retinal vein occlusions (BRVOs) are known to occur most commonly in the vicinity of arteriovenous (A/V) crossings. The authors aimed to identify types of venous wall abnormalities in BRVO and document their position in relation to the A/V crossing. DESIGN A retrospective review of the color photographs and fluorescein angiograms from the most recent 110 patients with first- or second-order BRVO was performed. MAIN OUTCOME MEASURES The films were examined for the presence of angioarchitectural changes of specified type within one-quarter disk diameter of the A/V crossing involved in the BRVO. The specific changes noted were fluorescein leakage, presumed thrombi, and flow abnormalities, which were recorded along with their position in relation to the A/V crossing. RESULTS Of the 110 patients diagnosed with BRVO, 59 had photography of satisfactory quality. Forty-one (70%) of these 59 patients had venous lesions, of which significantly more (chi-square -5.74, P < 0.02) were downstream (56%) than upstream (12%) from the A/V crossing. Thirty-two percent were upstream and downstream. Of the hemodynamic changes seen, 49% had late venous phase leakage of fluorescein, 85% had abnormal flow, and 7% had presumed thrombi. All thrombi seen were downstream. CONCLUSIONS Venous lesions in the vicinity of the A/V crossing commonly are seen in BRVO, most of which occur downstream. This suggests that the venous narrowing at the crossing may induce downstream hemodynamic changes predisposing to endothelial damage and thrombus generation.

Quantitative morphometry of perifoveal capillary networks in the human retina.

PURPOSE To quantify the distribution and morphometric characteristics of capillary networks in the human perifovea. To determine correlations between the location of neuronal subcellular compartments and the morphometric features of regional capillary networks in the layered retina. METHODS The perifoveal region, located 2 mm nasal to the fovea, was studied in 17 human donor eyes. Novel micropipette technology was used to cannulate the central retinal artery and label the retinal microcirculation using a phalloidin perfusate. γ-synuclein, Goα, and parvalbumin antibodies were also used to co-localize the nerve fiber layer (NFL), retinal ganglion cell layer (RGCL), inner plexiform layer (IPL), and inner nuclear layer (INL). Confocal scanning laser microscopy was used for capillary imaging. Capillary diameter, capillary density, and capillary loop area measurements were compared between networks. RESULTS Four capillary networks were identified in the following retinal layers: (1) NFL, (2) RGCL and superficial portion of IPL, (3) deep portion of IPL and superficial portion of INL, and (4) deep portion of INL. Laminar configurations were present in NFL and deep INL networks. Remaining networks demonstrated three-dimensional configurations. Capillary density was greatest in the networks serving the IPL. Capillary loop area was smallest in the two innermost networks. There was no difference in capillary diameter between networks. CONCLUSIONS Capillary networks in the human perifovea are morphometrically heterogeneous. Morphometric features of regional capillary networks in the layered retina may serve a critical role in supporting neuronal homeostasis. Improved knowledge of these features may be important for understanding pathogenic mechanisms underlying retinal vascular diseases.

Intravitreal Aflibercept for Macular Edema Secondary to Central Retinal Vein Occlusion: 18-Month Results of the Phase 3 GALILEO Study

PURPOSE To evaluate intravitreal aflibercept for treatment of macular edema secondary to central retinal vein occlusion (CRVO). DESIGN Randomized, double-masked, phase 3 study. METHODS A total of 177 patients with macular edema secondary to CRVO were randomized to receive 2 mg intravitreal aflibercept (n = 106) or sham (n = 71) every 4 weeks for 20 weeks. From weeks 24 to 48, patients were monitored every 4 weeks; the former group received intravitreal aflibercept as needed (PRN), and the sham group received sham. From weeks 52 to 76, patients were monitored every 8 weeks, and both groups received intravitreal aflibercept PRN. The primary endpoint (proportion of patients who gained ≥15 letters) was at week 24. This study reports exploratory outcomes at week 76. RESULTS The proportion of patients who gained ≥15 letters in the intravitreal aflibercept and sham groups was 60.2% vs 22.1% at week 24 (patients discontinued before week 24 were considered nonresponders; P < .0001), 60.2% vs 32.4% at week 52 (last observation carried forward, P < .001), and 57.3% vs 29.4% at week 76 (last observation carried forward; P < .001). Mean μm change from baseline central retinal thickness was -448.6 vs -169.3 at week 24 (P < .0001), -423.5 vs -219.3 at week 52 (P < .0001), and -389.4 vs -306.4 at week 76 (P = .1122). Over 76 weeks, the most common ocular serious adverse event in the intravitreal aflibercept group was macular edema (3.8%). CONCLUSIONS The visual and anatomic improvements seen after fixed, monthly dosing at week 24 were largely maintained when treatment intervals were extended. Patients with macular edema following CRVO benefited from early treatment with intravitreal aflibercept.

Effect of triamcinolone acetonide on vascular endothelial growth factor and occludin levels in branch retinal vein occlusion.

PURPOSE To investigate the molecular mechanism by which triamcinolone acetonide (TA) may reduce edema in a porcine model of branch retinal vein occlusion (BRVO). DESIGN Animal study. METHOD After baseline ophthalmoscopic examination and fundus photography, a BRVO was created photothrombotically in each eye of 6 pigs, using argon green photocoagulation and intravenous Rose Bengal. Following this, the left eye was injected intravitreally with 4 mg/0.1 ml TA. After 11 weeks, the eyes were re-examined. Fluorescein angiography, in addition to ophthalmoscopy and fundus photography, was performed. Following sacrifice of the animals, the eyes were enucleated and processed. The distribution of vascular endothelial growth factor (VEGF), occludin, and glial fibrillary acidic protein (GFAP) were localized by immunofluorescence cytochemistry on 10 microm frozen retinal sections of TA-treated and untreated eyes. RESULTS Retinal VEGF levels were significantly lower in the TA-treated eyes as compared with the untreated eye (P = .002). Conversely occludin levels were significantly higher in the treated eye (P = .026). There was also a significant reduction in GFAP immunoreactivity in the Muller cells of the treated eyes (P = .015) with no statistical significance in the astrocytes (P = .065). CONCLUSION Intravitreal TA down regulates VEGF, which may prevent a decrease in occludin and also inhibits an increase in GFAP expression in Muller cells. These events may contribute to a reduction in the blood retinal barrier breakdown that occurs in BRVO and promote resolution of the associated retinal edema.

Microstructure and Network Organization of the Microvasculature in the Human Macula

PURPOSE To characterize the topography and cellular structure of the macular microvasculature using a recently developed technique of arterial cannulation, perfusion, fixation, and staining of human donor eyes. METHODS Sixteen human donor eyes were used. The central retinal artery was cannulated and perfused with Ringers, then fixative, membrane permeabilizing, and selected labeling solutions. The eyes were immersion fixed, and the retina was flat mounted for confocal microscopy. The macular area, including the foveola, fovea, and parafovea, was sampled. The intracellular cytoskeleton of vascular endothelial and smooth muscle cells was studied in different orders of arterioles and venules and in the capillaries. To evaluate the degree of asymmetry within vascular networks, the distribution of generation numbers and the Horton-Strahler approach to vessel naming were compared. RESULTS The distribution of the microvascular network in the macular region was complex but followed a general theme. The parafoveal region was supplied by dense vasculature with approximately nine closely arranged pairs of arterioles and venules. Each arteriole had abundant branches and a high degree of asymmetry (∼10 generations and 3.5 orders within 1.2-mm length). Only a few arterioles (average ∼2.9) supplied the terminal capillary ring. Very long spindle endothelial cells were seen in the superficial and deep capillaries. Significant heterogeneity of distribution and shape of the endothelial and smooth muscle cells was evident in different orders of the macular vasculature. CONCLUSIONS The authors have demonstrated for the first time the cellular structure and topographic features of the macular microvasculature in human donor eyes.

The Structural Relationship between the Microvasculature, Neurons, and Glia in the Human Retina

PURPOSE To develop a new technique for detailed study of the spatial distribution of retinal and choroidal microvasculature and their relationship to neurons and glial cells at the cellular level in human cadaveric eyes. METHODS Twenty-six human donor eyes were used. Wherever possible, the central retinal artery and a branch of the posterior ciliary artery were individually cannulated and perfused with oxygenated Ringers solution with 0.5% bovine serum albumin. The perfusion pressure was continuously monitored. Once residual blood was washed out, the perfusate solutions were switched to fixative, membrane-permeabilizing solution and selected labeling solutions. The eyes were then immersion fixed and the retina and choroid flat-mounted for immunolabeling and confocal imaging before cryosectioning. The microstructures of vascular, glial, and neuronal cells in the retina and the stroma in the choroid were studied. RESULTS The retinal microvasculature was fully perfused and stained by cannulation of the central retinal artery. Regional distribution of choroidal vasculature perfusion was dependent on the specific feeder artery cannulated. The detailed spatial relationship between endothelial cells, glial cells, and neurons at the cellular and subcellular levels was identified with confocal microscopy and immunohistochemical labeling of retinal sections. In the choroid, endothelial cells were clearly identifiable down to the level of the intracellular cytoarchitecture of the choriocapillaris, along with their relationship to Bruchs membrane and the feeding and drainage vessels. CONCLUSIONS A microperfusion fixation and staining technique has been developed that allows studies of the structural relationships of vascular, glial, and neuronal elements at the cellular level in human donor eyes.

Two-year outcomes of "treat and extend" intravitreal therapy for neovascular age-related macular degeneration.

PURPOSE To report 24-month outcomes of anti-vascular endothelial growth factor (VEGF) therapy for treatment-naïve eyes with neovascular age-related macular degeneration (nAMD) using a treat and extend treatment regimen in routine clinical practice. DESIGN Database observational study. PARTICIPANTS We included treatment-naïve eyes receiving predominantly ranibizumab for nAMD in routine clinical practice treated using a treat and extend regimen that were tracked in the Fight Retinal Blindness observational registry. METHODS A cohort of eyes treated by practitioners using exclusively a treat and extend regimen was extracted from the Fight Retinal Blindness observational registry. MAIN OUTCOME MEASURES Change in visual acuity (VA) over 2 years and number of injections and visits. RESULTS Data from 1198 eyes from 1011 patients receiving anti-VEGF therapy using a treat and extend regimen for treatment-naïve nAMD between January 2007 and December 2012 and with 24-month follow-up were included in the analysis. Mean VA increased by +5.3 logarithm of the minimum angle of resolution letters from 56.5 letters (20/80+1) at initial visit to 61.8 (20/60+2) letters at 24 months. Mean VA gains improved and number of injections increased with successive years from +2.7 letters for eyes commencing in 2007 after a mean of 9.7 injections in 2 years, to +7.8 letters for eyes commencing in 2012 after a mean of 14.2 injections over 2 years. The proportion of eyes with VA >20/40 increased from 27% when starting treatment to 45% after 24 months; the proportion with vision of <20/200 remained unchanged (13% initial, 11% at 24 months). Of the included eyes, 90.5% avoided a vision loss of ≥15 letters. There was an overall mean of 13.0 injections over the 24 months, 7.5 injections in the first year and 5.5 in the second year, with a mean of 14.8 clinic visits. CONCLUSIONS These data indicate that eyes managed in routine clinical practice with a treat and extend regimen can achieve good visual outcomes while decreasing the burden of treatments and clinic visits.