Identification of proteins associated with clinical and pathological features of proliferative diabetic retinopathy in vitreous and fibrovascular membranes.

PURPOSE: To identify the protein profiles in vitreous associated with retinal fibrosis, angiogenesis, and neurite formation in epiretinal fibrovascular membranes (FVMs) in patients with proliferative diabetic retinopathy (PDR). METHODS: Vitreous samples of 5 non-diabetic control patients with vitreous debris and 7 patients with PDR membranes were screened for 507 preselected proteins using the semi-quantitative RayBio® L-series 507 antibody array. From this array, 60 proteins were selected for a custom quantitative antibody array (Raybiotech, Human Quantibody® array), analyzing 7 control patients, 8 PDR patients with FVMs, and 5 PDR patients without FVMs. Additionally, mRNA levels of proteins of interest were measured in 10 PDR membranes and 11 idiopathic membranes and in retinal tissues and cells to identify possible sources of protein production. RESULTS: Of the 507 proteins screened, 21 were found to be significantly elevated in PDR patients, including neurogenic and angiogenic factors such as neuregulin 1 (NRG1), nerve growth factor receptor (NGFR), placental growth factor (PlGF) and platelet derived growth factor (PDGF). Angiopoietin-2 (Ang2) concentrations were strongly correlated to the degree of fibrosis and the presence of FVMs in patients with PDR. Protein correlation analysis showed PDGF to be extensively co-regulated with other proteins, including thrombospondin-1 and Ang2. mRNA levels of glial-derived and brain/derived neurotrophic factor (GDNF and BDNF) were elevated in PDR membranes. These results were validated in a second study of 52 vitreous samples of 32 PDR patients and 20 control patients. CONCLUSIONS: This exploratory study reveals protein networks that potentially contribute to neurite outgrowth, angiogenesis and fibrosis in the formation of fibrovascular membranes in PDR. We identified a possible role of Ang2 in fibrosis and the formation of FVMs, and of the neurotrophic factors NRG1, PDGF and GDNF in neurite growth that occurs in all FVMs in PDR.

Optical coherence tomography evaluation of patients with macula-off retinal detachment after different postoperative posturing: a randomized pilot study.

PURPOSE: To assess the presence of outer and inner retinal folds (RFs) and drop-out of the ellipsoid zone (EZ) occurring after surgical repair of macula-off rhegmatogenous retinal detachment (RRD) with different postoperative posture and preoperative use of adjuvant perfluorocarbon liquid (PFCO). METHODS: In this prospective study, 56 eyes of 56 consecutive patients affected by RRD were subjected to 23- or 25-gauge pars plana vitrectomy (PPV). The patients were randomized in four groups (14 prone 5 hr without PFCO, 14 supine 5 hr without PFCO, 14 prone 5 hr with PFCO and 14 supine 5 hr with PFCO) and followed up with spectral domain optical coherence tomography (SD-OCT). RESULTS: Spectral domain optical coherence tomography (SD-OCT) was recorded before surgery, at days 30 and 90 to detect the presence of outer RFs, inner RFs and drop-out of EZ and to follow their variation over time. No statistical significance was found in our groups for outer RFs, inner RFs, drop-out of EZ formation and evolution. The postoperative best-corrected visual acuity (BCVA) improved in all groups (mean preoperative BCVA 1.47 logMar ± 0.19, mean postoperative BCVA 0.27 logMar ± 0.11, p < 0.01), without statistical variations between the four groups in BCVA after surgery. CONCLUSION: The use of adjuvant and variation in postoperative position did not change the risk of presenting outer RFs, inner RFs and drop-out of EZ after RRD.

Incidence of Cataract Surgery after Vitrectomy for Vitreous Opacities.

PURPOSE: A computational model of vitreous oxygen consumption and transport predicts that limited vitrectomy will result in lower retrolental oxygen levels than extensive vitrectomy, and that higher retrolental oxygen would promote cataractogenesis. This study compared the incidence and timing of cataract surgery after limited versus extensive vitrectomy for vitreous opacities. METHODS: Ninety-six phakic eyes in 75 patients (aged 55±14 years) underwent limited 25 G vitrectomy with preservation of 3 to 4 mm of retrolental vitreous and without surgical posterior vitreous detachment induction. Of these 96 eyes, 48 eyes in 37 patients (aged 56±14 years) had a minimum of 24 months' follow-up and were compared with 23 eyes from 18 patients (aged 63±8 years) who underwent extensive vitrectomy. RESULTS: Limited vitrectomy patients were older than extensive vitrectomy patients (P < 0.015), yet only 17 of 96 eyes (18%) required cataract surgery after limited vitrectomy (mean follow-up = 20±17 months). In eyes with a minimum follow-up of 24 months, 17 of 48 eyes (35%; age = 53-81 years) with limited vitrectomy required cataract surgery, versus 20 of 23 eyes (87%; age = 50-75 years) with extensive vitrectomy (P < 0.0001). Just before cataract surgery, visual acuity was comparable in each group (0.47±0.18 in the limited vitrectomy group vs. 0.54±0.30 in the extensive vitrectomy group; P = 0.23). Cataract surgery occurred an average of 12.4±5.1 months after limited vitrectomy, compared with 7.3±3.9 months after extensive vitrectomy (P < 0.002). CONCLUSIONS: The incidence of cataract surgery was lower after limited vitrectomy, which had a longer interval until cataract surgery compared with extensive vitrectomy. These findings are consistent with the computational model of oxygen consumption and transport and suggest clinical strategies to mitigate postvitrectomy cataractogenesis.

Mobile ultra-clean unidirectional airflow screen reduces air contamination in a simulated setting for intra-vitreal injection.

The aim of this study is to determine whether the use of a mobile ultra-clean laminar airflow screen reduces the air-borne particle counts in the setting of a simulated procedure of an intra-vitreal injection. A mobile ultra-clean unidirectional airflow (UDF) screen was tested in a simulated procedure for intra-vitreal injections in a treatment room without mechanical ventilation. One UDF was passed over the instrument tray and the surgical area. The concentration of particles was measured in the background, over the instrument table, and next to the ocular area. The degree of protection was calculated at the instrument table and at the surgical site. Use of the UDF mobile screen reduced the mean particle concentration (particles > 0.3 microns) on the instrument table by a factor of at least 100.000 (p < 0.05), and over the patient's eye by at least a factor of 436 (p < 0.05), which in clinical practice translates into significantly reduced air contamination. Mobile UDF screen reduces the mean particle concentration substantially. The mobile UDF screen may therefore allow for a safer procedural environment for ambulatory care procedures such as intra-vitreal injections in treatment rooms.

Rhegmatogenous retinal detachment following intravitreal ocriplasmin.

PURPOSE: To describe the characteristics and outcomes of patients presenting with rhegmatogenous retinal detachment (RRD) after ocriplasmin (OCP) injection. METHODS: Retrospective, multi-centre, observational case series with case note review. RESULTS: Eight patients with symptomatic vitreomacular traction (six with concomitant macular hole) were diagnosed with RRD after a median of 16 days (range 3-131 days) post-OCP injection. Presentation was within 3 weeks of the OCP injection in six of the cases. Five patients presented with symptoms post-OCP, and three were diagnosed asymptomatically on planned visits. Seven cases were phakic, one had high myopia (>8 dioptres), and two cases had lattice degeneration. Following RRD surgery, hole closure was achieved in 5/6 MH cases. The final median BCVA at 7 months was 20/80 (range 20/40-20/1200) similar to the baseline BCVA 20/80, with four patients gaining ≥1 line of vision compared to baseline but three losing ≥3 lines. CONCLUSIONS: RRD is a non-negligible risk associated with intravitreal OCP, and it should be used with caution in eyes with high myopia and peripheral retinal pathology predisposing to RRD. Detailed peripheral retinal examination is recommended pre- and postoperatively at all visits. Patients should be advised to seek attention if symptoms recur after initial presentation.

Vitreous TIMP-1 levels associate with neovascularization and TGF-ß2 levels but not with fibrosis in the clinical course of proliferative diabetic retinopathy.

In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and CCN2 (connective tissue growth factor; CTGF) cause blindness by neovascularization and subsequent fibrosis. This angio-fibrotic switch is associated with a shift in the balance between vitreous levels of CCN2 and VEGF in the eye. Here, we investigated the possible involvement of other important mediators of fibrosis, tissue inhibitor of metalloproteinases (TIMP)-1 and transforming growth factor (TGF)-ß2, and of the matrix metalloproteinases (MMP)-2 and MMP-9, in the natural course of PDR. TIMP-1, activated TGF-ß2, CCN2 and VEGF levels were measured by ELISA in 78 vitreous samples of patients with PDR (n = 28), diabetic patients without PDR (n = 24), and patients with the diabetes-unrelated retinal conditions macular hole (n = 10) or macular pucker (n = 16), and were related to MMP-2 and MMP-9 activity on zymograms and to clinical data, including degree of intra-ocular neovascularization and fibrosis. TIMP-1, CCN2 and VEGF levels, but not activated TGF-ß2 levels, were significantly increased in the vitreous of diabetic patients, with the highest levels in PDR patients. CCN2 and the CCN2/VEGF ratio were the strongest predictors of degree of fibrosis. In diabetic patients with or without PDR, activated TGF-ß2 levels correlated with TIMP-1 levels, whereas in PDR patients, TIMP-1 levels, MMP-2 and proMMP-9 were associated with degree of neovascularization, like VEGF levels, but not with fibrosis. We confirm here our previous findings that retinal fibrosis in PDR patients is significantly correlated with vitreous CCN2 levels and the CCN2/VEGF ratio. In contrast, TIMP-1, MMP-2 and MMP-9 appear to have a role in the angiogenic phase rather than in the fibrotic phase of PDR.

Evolution of outer retinal folds occurring after vitrectomy for retinal detachment repair.

PURPOSE: To assess the evolution of outer retinal folds (ORFs) occurring after repair of rhegmatogenous retinal detachment (RRD) using spectral domain-optical coherence tomography (sd-OCT) and fundus autofluorescence (FAF), and to discuss their pathogenesis. METHODS: Twenty patients were operated on with 25-gauge pars plana vitrectomy and 20% sulfur hexafluoride gas injection for primary macula-off RRD repair and were followed prospectively. Sd-OCT and FAF images were recorded at 1, 3 and 6 months postoperatively. RESULTS: ORFs appeared on sd-OCT as hyperreflective lesions consisting of folded inner segment/outer segment of photoreceptors band and external limiting membrane band. Corresponding lines of increased or decreased autofluorescence were observed on FAF. Over the follow-up, the thick hypoautofluorescent lines progressively evolved to thick hyperautofluorescent lines and to thin hyperautofluorescent lines and eventually disappeared. Concomitantly, OCT scans revealed that the corresponding hyperreflective lesions decreased in number, height, and size. In six cases FAF assessment at month 6 was precluded by cataract development. CONCLUSIONS: ORFS tend to resolve spontaneously within a few months from operation leaving no or subtle abnormalities at the level of the outer retinal layers. OCT is superior to FAF to follow the evolution of orfs in phakic eyes. The following factors might be involved in ORFS pathogenesis: structural changes occurring in the detached retina, residual pockets of subretinal fluid after retinal reattachment, intravitreal gas, unintentional retinal translocation, and intraoperative or perioperative hypotony.

A shift in the balance of vascular endothelial growth factor and connective tissue growth factor by bevacizumab causes the angiofibrotic switch in proliferative diabetic retinopathy.

INTRODUCTION: In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) may cause blindness by neovascularisation followed by fibrosis of the retina. It has previously been shown that a shift in the balance between levels of CTGF and VEGF in the eye is associated with this angiofibrotic switch. This study investigated whether anti-VEGF agents induce accelerated fibrosis in patients with PDR, as predicted by this model. METHODS: CTGF and VEGF levels were measured by ELISA in 52 vitreous samples of PDR patients, of which 24 patients had received intravitreal bevacizumab 1 week to 3 months before vitrectomy, and were correlated with the degree of vitreoretinal fibrosis as determined clinically and intra-operatively. RESULTS: CTGF correlated positively, and VEGF correlated negatively with the degree of fibrosis. The CTGF/VEGF ratio was the strongest predictor of fibrosis. Clinically, increased fibrosis was observed after intravitreal bevacizumab. CONCLUSIONS: These results confirm that the CTGF/VEGF ratio is a strong predictor of vitreoretinal fibrosis in PDR, and show that intravitreal anti-VEGF treatment causes increased fibrosis in PDR patients. These findings provide strong support for the model that the balance of CTGF and VEGF determines the angiofibrotic switch, and identify CTGF as a possible therapeutic target in the clinical management of PDR.

Early simultaneous fundus autofluorescence and optical coherence tomography features after pars plana vitrectomy for primary rhegmatogenous retinal detachment.

PURPOSE: To describe fundus autofluorescence and optical coherence tomography (OCT) features of the macula after pars plana vitrectomy for rhegmatogenous retinal detachment. METHODS: Thirty-three eyes of 33 consecutive patients with repaired rhegmatogenous retinal detachment with or without the involvement of the macula were prospectively investigated with simultaneous fundus autofluorescence and OCT imaging using the Spectralis HRA+OCT (Heidelberg Engineering, Heidelberg, Germany) within a few weeks after the operation. RESULTS: Fundus autofluorescence imaging of the macula showed lines of increased and decreased autofluorescence in 19 cases (57.6%). On OCT, these lines corresponded to the following abnormalities: outer retinal folds, inner retinal folds, and skip reflectivity abnormalities of the photoreceptor inner segment/outer segment band. Other OCT findings, not related to abnormal lines on fundus autofluorescence, consisted of disruption of photoreceptor inner segment/outer segment band and collection of intraretinal or subretinal fluid. The presence of outer retinal folds significantly related to metamorphopsia but did not relate to poor postoperative visual acuity. CONCLUSION: Partial-thickness retinal folds occur commonly after vitrectomy for rhegmatogenous retinal detachment repair and may represent an important anatomical substrate for postoperative metamorphopsia. Fundus autofluorescence and OCT are both sensitive techniques for the detection of these abnormalities.

Safety of vitrectomy for floaters.

PURPOSE: To assess the risks of vitrectomy for the removal of primary and secondary vitreous opacities. DESIGN: Retrospective, nonrandomized, interventional case series. METHODS: We reviewed the results of 116 consecutive cases of vitrectomy for vitreous floaters. Eighty-six cases were primary and 30 cases were secondary floaters. Main outcome measures were the incidence of iatrogenic retinal breaks and postoperative rhegmatogenous retinal detachments. RESULTS: We found iatrogenic retinal breaks in 16.4% of operations. There was no statistically significant difference in risk between cases of primary and secondary floaters. Intraoperative posterior vitreous detachment induction was found to increase significantly the risk of breaks. Retinal detachment occurred in 3 cases (2.5%), all after operations for primary floaters. One case of complicated retinal detachment ended with a low visual acuity of hand movements. Cataract occurred in 50% of phakic cases. Transient postoperative hypotony was found after 5.2% of our operations, and transient postoperative high intraocular pressure was encountered in 7.8%. An intraoperative choroidal hemorrhage occurred in 1 case, which resolved spontaneously. The mean visual acuity improved from 0.20 to 0.13 logarithm of the minimal angle of resolution units. CONCLUSIONS: The risk profile of vitrectomy for floaters is comparable with that of vitrectomy for other elective indications. Retinal breaks are a common finding during surgery and treatment of these breaks is crucial for the prevention of postoperative retinal detachment. Patients considering surgery for floaters should be informed specifically about the risks involved.

Evidence that neurites in human epiretinal membranes express melanopsin, calretinin, rod opsin and neurofilament protein.

AIMS: We have previously identified neurofilament-protein-containing neurites in human epiretinal membranes (ERMs). The aim of this study was to further characterise these neurites by examining the expression of additional specific proteins in human ERMs and to correlate this expression with various retinal disease conditions. METHODS: Epiretinal membranes originating from 43 patients with proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR) or with no known pathology (idiopathic epiretinal membrane; iERM) were removed during vitrectomy at varying durations after diagnosis and immediately placed in fixative. The membranes were labelled immunohistochemically with different combinations of antibodies to the proteins melanopsin, calretinin and neurofilament (to identify subclasses of ganglion cells), rod opsin (to identify rod photoreceptors), synaptophysin and synaptic vesicle glycoprotein 2A (SV2) (identifies synaptic vesicles) and vimentin (identifies glial cells). RESULTS: Anti-melanopsin-, anti-calretinin-, anti-neurofilament- and anti-rod-opsin-labelled neurites were routinely observed in the epiretinal membranes. Their presence did not appear to correlate with a specific disease condition or duration of the membrane. Generally neurites were observed in regions of glial cells. CONCLUSIONS: Based on the expression of selected markers for neurites, we show neurite processes in human ERMs of various aetiologies originating from rod photoreceptors and different populations of retinal ganglion cells, although there was no obvious correlation with specific disease condition. In addition, synaptophysin and SV2 labelling was observed associated with all types of neurites, indicating the presence of at least one component necessary for synaptic transmission. Our data suggest that the adult human retina retains a significant capacity for neuronal remodelling under various disease conditions.

Enhanced internal search for iatrogenic retinal breaks in 20-gauge macular surgery.

PURPOSE: To evaluate the incidence and characteristics of iatrogenic retinal breaks in 20-gauge macular surgery with an intensified search strategy. DESIGN: Retrospective, non-comparative interventional case series. PARTICIPANTS: 218 consecutive operations in 209 patients who underwent 20-gauge vitrectomy vitrectomy for idiopathic macular pucker or idiopathic macular hole. METHODS: Retrospective review of patient records undergoing 20-gauge vitrectomy with intensified peripheral search for retinal defects. MAIN OUTCOME MEASURES: Incidence of breaks related to the sclerotomies, the incidence of breaks occurring elsewhere, the incidence of lesions suspicious for traction, the location of identified breaks and intraoperative induction of posterior vitreous detachment. RESULTS: Retinal breaks occurred in 24.3% of operations. In 17.4% breaks were related to the sclerotomies and in 9.6% of breaks were found elsewhere. In 6.4% of eyes, only lesions suspicious for traction were detected. Retinal detachment occurred in 1.8% of cases. The occurrence of breaks was significantly related to induction of PVD. CONCLUSIONS: With intensified intraoperative search, a much higher incidence of retinal breaks was found than previously reported in the literature. Despite the high incidence of breaks, the incidence of postoperative rhegmatogenous retinal detachment was low. These findings support the rationale that intensive intraoperative search for iatrogenic breaks is crucial for the prevention of postoperative retinal detachments in macular surgery.

Heavy trypan blue staining of epiretinal membranes: an alternative to infracyanine green.

BACKGROUND: By using dyes, it is easier to identify the extent of an epiretinal membrane (ERM) or the inner limiting membrane (ILM) during surgery. Trypan blue (TB) stains ERM and ILM weakly, but with less apparent toxicity than other intraocular dyes. Its main drawback in vitreoretinal surgery is the requirement of an air-fluid exchange (AFX) before its use. AIM: To propose a modified form of TB denser than water, thus obviating the need for an AFX. DESIGN: A prospective, consecutive trial with heavy trypan blue in vitreoretinal surgery. METHODS: A consecutive group of patients with ERMs was recruited prospectively. Patients were operated on using conventional methods. Heavy TB was prepared by mixing glucose 10% with Membrane blue (Dorc, Zuidland, The Netherlands) isovolumetrically. Patients were preoperatively and postoperatively assessed at 3 and 6 months (vision and ocular coherence tomography (OCT)). Ease of surgery was also assessed. RESULTS: 29 eyes were included in the study. Reapplication of dye was necessary in 25% of the cases, leading to improved contrast further facilitating the peeling process. In no case was an AFX necessary to obtain sufficient staining. All patients with ERM had an improvement in vision (from median 0.30 to 0.55) and macular volume and foveal thickness (from median 450 to 238 mm) on OCT. No retinal detachment or other complications developed as a result of surgery. CONCLUSION: Heavy TB can be delivered efficiently to the retinal surface without an AFX. Staining was sufficient to allow a safe and efficient peeling of ERM. Repeat applications were easily performed. Its use was associated with vision improvement and decreased in foveal thickness, and the absence of adverse events in this small case series.

Identification of ganglion cell neurites in human subretinal and epiretinal membranes.

AIM: To determine whether neural elements are present in subretinal and epiretinal proliferative vitreoretinopathy (PVR) membranes as well as in diabetic, fibrovascular membranes removed from patients during vitrectomy surgery. METHODS: Human subretinal and epiretinal membranes of varying durations were immunolabelled with different combinations of antibodies to glial fibrillary acidic protein, vimentin, neurofilament protein and laminin. RESULTS: Anti-neurofilament-labelled neurites from presumptive ganglion cells were frequently found in epiretinal membranes and occasionally found in subretinal membranes. In addition, the neurites were only observed in regions that also contained glial processes. CONCLUSIONS: These data demonstrate that neuronal processes are commonly found in human peri-retinal cellular membranes similar to that demonstrated in animal models. These data also suggest that glial cells growing out of the neural retina form a permissive substrate for neurite growth and thus may hold clues to factors that support this growth.