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Supplemental Digital Content is Available in the Text.PRIVENT study report No. 3—Prescreening data—Objective tyndallometry (laser flare photometry) is independently influenced by age, sex, lens status, and the presence and the extent of rhegmatogenous retinal detachment.

Supplemental Digital Content is Available in the Text.In this study, a chronic vision-threatening phenomenon characterized by silicone oil clouding without intraocular inflammation or silicone oil emulsification is reported. This discoloration of silicone oil may disturb vision and prevent proper fundus examination; however, it seems to be a nontoxic phenomenon without serious long-term consequences.

PurposeTo determine differences in postoperative pupil diameter in eyes that undergo pars plana vitrectomy (PPV) for rhegmatogenous retinal detachment (RRD) with endolaser retinopexy (ELR), comparing 360° vs focal ELR.MethodsPatients with uncomplicated RRD who underwent PPV were prospectively analysed regarding the postoperative pupil diameter difference (PDD) between the affected eye and the partner eye. Group 1 underwent 360° ELR and group 2 received focal ELR. Postoperative vision and complications, including redetachment rate, macular oedema and epiretinal membrane formation, were also compared.ResultsA total of 72 patients, 42 in group 1 and 30 in group 2, were analysed. PDD, as observed at 6 weeks, was significantly greater than the preoperative values in both groups 1 and 2. It increased by a mean of 1±1.11 mm in group 1 and by 0.5±0.78 in group 2. This initial increase in PDD receded over time, but remained statistically significant in both groups, even at 6 months. The top 20% of patients with the largest PDD change comprised 13 out of 15 eyes from group 1, which was a statistically significant overrepresentation (p=0.0435).ConclusionsModerate pupillotonia was induced post-ELR in vitrectomy and correlated to the extent of ELR. The pupillotonia effect of ELR was significantly less marked in pseudophakic eyes.

AimsTo determine long-term safety and efficacy outcomes of a subretinal gene therapy for CNGA3-associated achromatopsia. We present data from an open-label, nonrandomised controlled trial (NCT02610582).MethodsDetails of the study design have been previously described. Briefly, nine patients were treated in three escalating dose groups with subretinal AAV8.CNGA3 gene therapy between November 2015 and October 2016. After the first year, patients were seen on a yearly basis. Safety assessment constituted the primary endpoint. On a secondary level, multiple functional tests were carried out to determine efficacy of the therapy.ResultsNo adverse or serious adverse events deemed related to the study drug occurred after year 1. Safety of the therapy, as the primary endpoint of this trial, can, therefore, be confirmed. The functional benefits that were noted in the treated eye at year 1 were persistent throughout the following visits at years 2 and 3. While functional improvement in the treated eye reached statistical significance for some secondary endpoints, for most endpoints, this was not the case when the treated eye was compared with the untreated fellow eye.ConclusionThe results demonstrate a very good safety profile of the therapy even at the highest dose administered. The small sample size limits the statistical power of efficacy analyses. However, trial results inform on the most promising design and endpoints for future clinical trials. Such trials have to determine whether treatment of younger patients results in greater functional gains by avoiding amblyopia as a potential limiting factor.

Glaucoma is the second leading cause of irreversible blindness worldwide. Among others, elevated intraocular pressure (IOP) is one of the hallmarks of the disease. Antiglaucoma drugs such as brimonidine can lower the IOP but their adherence to the ocular surface is low, leading to a low drug uptake. This results in a frequent dropping regime causing low compliance by the patients. Lipid DNA nanoparticles (NPs) have the intrinsic ability to bind to the ocular surface and can be loaded with different drugs. Here, we report DNA NPs functionalized for loading of brimonidine through specific aptamers and via hydrophobic interactions with double stranded micelles. Both NP systems exhibited improved affinity toward the cornea and retained release of the drug as compared to controls both in vitro and in vivo. Both NP types were able to lower the IOP in living animals significantly more than pristine brimonidine. Importantly, the brimonidine-loaded NPs showed no toxicity and improved efficacy and hence should improve compliance. In conclusion, this drug-delivery system offers high chances of an improved treatment for glaucoma and thus preserving vision in the aging population.

IMPORTANCE: Achromatopsia linked to variations in the CNGA3 gene is associated with day blindness, poor visual acuity, photophobia, and involuntary eye movements owing to lack of cone photoreceptor function. No treatment is currently available. OBJECTIVE: To assess safety and vision outcomes of supplemental gene therapy with adeno-associated virus (AAV) encoding CNGA3 (AAV8.CNGA3) in patients with CNGA3-linked achromatopsia. DESIGN, SETTING, AND PARTICIPANTS: This open-label, exploratory nonrandomized controlled trial tested safety and vision outcomes of gene therapy vector AAV8.CNGA3 administered by subretinal injection at a single center. Nine patients (3 per dose group) with a clinical diagnosis of achromatopsia and confirmed biallelic disease-linked variants in CNGA3 were enrolled between November 5, 2015, and September 22, 2016. Data analysis was performed from June 6, 2017, to March 12, 2018. INTERVENTION: Patients received a single unilateral injection of 1.0 × 1010, 5.0 × 1010, or 1.0 × 1011 total vector genomes of AAV8.CNGA3 and were followed up for a period of 12 months (November 11, 2015, to October 10, 2017). MAIN OUTCOMES AND MEASURES: Safety as the primary end point was assessed by clinical examination of ocular inflammation. Systemic safety was assessed by vital signs, routine clinical chemistry testing, and full and differential blood cell counts. Secondary outcomes were change in visual function from baseline in terms of spatial and temporal resolution and chromatic, luminance, and contrast sensitivity throughout a period of 12 months after treatment. RESULTS: Nine patients (mean [SD] age, 39.6 [11.9] years; age range, 24-59 years; 8 [89%] male) were included in the study. Baseline visual acuity letter score (approximate Snellen equivalent) ranged from 34 (20/200) to 49 (20/100), whereas baseline contrast sensitivity log scores ranged from 0.1 to 0.9. All 9 patients underwent surgery and subretinal injection of AAV8.CNGA3 without complications. No substantial safety problems were observed during the 12-month follow-up period. Despite the congenital deprivation of cone photoreceptor–mediated vision in achromatopsia, all 9 treated eyes demonstrated some level of improvement in secondary end points regarding cone function, including mean change in visual acuity of 2.9 letters (95% CI, 1.65-4.13; P = .006, 2-sided t test paired samples). Contrast sensitivity improved by a mean of 0.33 log (95% CI, 0.14-0.51 log; P = .003, 2-sided t test paired samples). CONCLUSIONS AND RELEVANCE: Subretinal gene therapy with AAV8.CNGA3 was not associated with substantial safety problems and was associated with cone photoreceptor activation in adult patients, as reflected by visual acuity and contrast sensitivity gains. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02610582

IMPORTANCE: Choroideremia (CHM) is a rare, degenerative, genetic retinal disorder resulting from mutation of the CHM gene, leading to an absence of functional ras-associated binding escort protein 1 (REP1). There is currently no approved treatment for CHM. OBJECTIVE: To assess the safety and efficacy of retinal gene therapy with an adeno-associated virus vector (AAV2) designed to deliver a functional version of the CHM gene (AAV2-REP1) for treatment of patients with choroideremia. DESIGN, SETTING, AND PARTICIPANTS: Tübingen Choroideremia Gene Therapy (THOR) was a single-center, phase 2, open-label randomized clinical trial. Data were collected from January 11, 2016, to February 26, 2018. Twenty-four–month data are reported for 6 men with a molecularly confirmed diagnosis of CHM. Intention-to-treat analysis was used. INTERVENTIONS: Patients received AAV2-REP1 by a single, 0.1-mL subretinal injection of 1011 genome particles during vitrectomy into 1 eye randomly assigned to receive treatment. MAIN OUTCOMES AND MEASURES: Primary end point was change in best-corrected visual acuity (BCVA) on the Early Treatment Diabetic Retinopathy Study chart from baseline to month 24 in the treated eye vs the control eye. Secondary end points included microperimetry variables, change in fundus autofluorescence, and spectral-domain optical coherence tomographic evaluations from baseline to month 24 in the treated eye vs the control eye. RESULTS: On enrollment, the mean (SD) age of the 6 men included in the study was 54.9 (4.1) years. The mean (SD) BCVA score was 60.3 (13.4) (approximately 20/63 Snellen equivalent) in the study eyes and 69.3 (20.6) (approximately 20/40 Snellen equivalent) in the control eyes. At 24 months, the BCVA change was 3.7 (7.5) in the treated eyes and 0.0 (5.1) in the control eyes (difference, 3.7; 95% CI, −7.2 to 14.5; P = .43). Mean change in retinal sensitivity was 10.3 (5.5) dB in the treated eyes and 9.7 (4.9) dB in the control eyes (difference, 0.6; 95% CI, −10.2 to 11.4; P = .74). A total of 28 adverse events were reported; all were consistent with the surgical procedure (eg, conjunctival hyperemia, foreign body sensation), and none were regarded as severe. CONCLUSIONS AND RELEVANCE: Among 6 participants, gene therapy with AAV2-REP1 was associated with maintenance or improvement of visual acuity, although no significant difference was found from control eyes. All safety issues were associated with the surgical procedure and none were judged severe. Continued investigations could more precisely define the efficacy and safety of gene therapy with AAV2-REP1 in CHM. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02671539

Ex vivoorgan cultures represent unique research models, as they combine the advantages of cell cultures with those of animal models. Being able to mimic in vivosituations through the use of organ cultures provides an excellent opportunity to investigate cellular processes, molecular pathways and cell–cell interactions, as well as structural and synaptic organisation. Human and animal organ cultures are now well established and comprise sensitive, easy-to-manipulate experimental systems that raise minimal ethical concerns. The eye, in particular, is a very complex organ that is not easy to reproduce in vitro. However, a lot of research has been dedicated to the development of suitable ocular organ cultures. This review covers the various ex vivoretinal organ culture systems available for use in ophthalmology research and compares them with commonly used animal models. In particular, bovine and porcine retinal organ culture systems are described, because the size, anatomy, physiology and vessel morphology of bovine and porcine eyes are similar to the human eye in an undisputed way, thus making them good models. In addition, these animals are widely used by the food industry and the eyes are considered surplus material. A short overview of murine, rat, rabbit, cat, canine and simian retinal organ cultures is also provided.

Background/aimsThe aim of this study was to investigate a change in visual acuity and contrast sensitivity (CS) during high altitude exposure in healthy subjects due to the effects of hypobaric hypoxia. This study is related to the Tu¨bingen High Altitude Ophthalmology study.MethodsVisual acuity and Weber CS were tested monocularly using the Freiburger Visual Acuity and Contrast Test under standardised conditions in 14 healthy subjects at high altitude at the Capanna Margherita (4559 m, Italy) and compared with baseline measurements in Tu¨bingen (341 m, Germany). Intraindividual differences between baseline and follow-up examinations were calculated by multivariate analysis of variance for repeated measures. Clinical parameters of peripheral oxygen saturation (SpO2) and heart rate (HR) as well as scores for acute mountain sickness (AMS) were correlated to psychophysical tests by Pearson’s correlation coefficient.ResultsA significant decrease in CS with a mean effect size of −0.13 logCS was found for Weber CS (day 1=−0.16±0.22, p=0.01; day 2=−0.10±0.2, p=0.049; day 3=−0.12±0.19, p=0.03) at high altitude compared with baseline. Visual acuity remained unchanged. Decreased CS correlated with SpO2(r=0.53, p=0.046) but not with HR (r=− 0.16, p=0.59) and occurred irrespective of AMS at high altitude.ConclusionHigh altitude exposure leads to decreased CS. Changes occur independent of AMS. This finding is of clinical importance to trekkers and mountaineers exposed to high altitude as visual processing in particular under mesopic conditions at dusk and dawn is altered. Furthermore, it provides novel insight into hypoxia related changes in CS function.

AimTo evaluate and correlate mean light sensitivity thresholds (MLST) in patients with congenital stationary night-blindness (CSNB) in comparison with healthy subjects using microperimetry (MP1).MethodsEleven patients with CSNB and 13 healthy subjects were compared. In all subjects, static threshold perimetry was performed using MP1 evaluating the central 6 mm of the retina. This central retinal area was divided into three rings through using the ETDRS grid algorithm with an innermost (1 mm), inner (3 mm) and outer ring (6 mm). The MLSTs were acquired in nine sectors of the ETDRS grid. A comparison of MLST was performed between both groups using a t-test (significance level p<0.005).ResultsA significant reduction of MLST in the fovea (innermost ring, 1 mm) was observed for patients with CSNB (7.2±3.90 dB) in comparison to healthy subjects (19.7±0.75; p<0.0001). Similarly, comparison of MLST in all other sectors (superior/inferior/temporal and nasal) within the inner and outer ring revealed a statistically significant reduction in patients with CSNB compared with healthy subjects (p<0.001).ConclusionsExamination of macular retinal sensitivity intensity using MP1 revealed for the first time a significant reduction of MLST within the central 6 mm of the retina in patients with CSNB compared with healthy subjects. This finding supports MP1 as an additional diagnostic tool when examining patients with retinal dysfunctions such as CSNB.

Background/aimsThe study aims to investigate changes in the optic nerve sheath diameter (ONSD) at high altitude and to assess correlation to optic disc oedema (ODE) and acute mountain sickness (AMS). This investigation is part of the Tu¨bingen High Altitude Ophthalmology study.MethodsFourteen volunteers ascended to 4559 m for 4 days before returning to low altitude. Ultrasonography of ONSD, quantification of optic disc parameters using a scanning laser ophthalmoscope and fluorescein angiography were performed at 341 m and at high altitude. Pearson’s coefficient was used to correlate changes in ONSD with the optic disc and AMS. Assessment of AMS was performed using the Lake Louise (LL) and AMS-cerebral (AMS-C) scores of the Environmental Symptom Questionnaire-III. All volunteers were clinically monitored for heart rate (HR) and oxygen saturation (SpO2).ResultsThe mean ONSD at high altitude (4.6±0.3 mm, p<0.05) was significantly increased compared with baseline (3.8±0.4 mm) and remained enlarged throughout high-altitude exposure. This change in ONSD did not correlate with AMS (AMS-C, r=0.26, p=0.37; LL, r=0.21, p=0.48) and high-altitude headache (r=0.54, p=0.046), or clinical parameters of SpO2(r=0.11, p=0.72) and HR (r=0.22, p=0.44). Increased ONSD did not correlate with altered key stereometric parameters of the optic disc describing ODE at high altitude (r<0.1, p>0.5).ConclusionHigh-altitude exposure leads to marked oedema formation of the optic nerve independent of AMS. Increased ONSD and ODE reflect hypoxia-driven oedema formation of the optic nerve at high altitude, providing important pathophysiological insight into high-altitude illness development and for future research.

Ocular gene therapy has evolved rapidly into the clinical realm due to promising pre-clinical proof-of-concept studies, recognition of the high unmet medical need of blinding disorders, and the excellent safety profile of the most commonly used vector system, the adeno-associated virus (AAV). With several trials exposing subjects to AAV, investigators independently report about cases with clinically evident inflammation in treated eyes despite the concept of ocular immune privilege. Here, we provide a detailed analysis of innate and adaptive immune response to clinical-grade AAV8 in non-human primates and compare this to preliminary clinical data from a retinal gene therapy trial for CNGA3-based achromatopsia (ClinicalTrials.gov: 02610582).

BACKGROUND AND OBJECTIVE:The aim of this study was to assess changes in retinal structure and thickness after subretinal implantation of the Retina Implant Alpha IMS (Retina Implant AG, Reutlingen, Germany).PATIENTS AND METHODS:Spectral-domain optical coherence tomography (SD-OCT) imaging was performed to assess the structure and thickness of the retina anterior to the microphotodiode array preoperatively, within 6 weeks and 6 months ± 1 month after implantation. Thickness measurements were performed using the distance tool of the built-in software. Three thickness measurements were performed in each of the four quadrants of the retina on the microchip within 6 weeks and 6 months ± 1 month after implantation.RESULTS:The mean ± standard deviation change in retinal thickness from within 6 weeks to 6 months ± 1 month after implantation in all four quadrants combined was 24 μm ± 68 μm. None of the tested variables (location, time, or their interaction) had a statistically significant effect on the mean retinal thickness (The mean ± standard deviation change in retinal thickness from within 6 weeks to 6 months ± 1 month after implantation in all four quadrants combined was 24 μm ± 68 μm. None of the tested variables (location, time, or their interaction) had a statistically significant effect on the mean retinal thickness (P= .961, P= .131, and P= .182, respectively; n = 19).CONCLUSION:The authors report on qualitative and quantitative findings in retinal structure in 27 patients after subretinal implantation of the Retina Implant Alpha IMS using OCT technology. No significant changes of retinal thickness could be observed in a period of 6 months after surgery. With more patients receiving subretinal implants and with advanced OCT technology, the data set will be extended to study possible changes in retinal structure in finer detail.[[Ophthalmic Surg Lasers Imaging Retina. 2017;48:993–999.]

Oxidative stress is a key player in many ophthalmic diseases. However, the role of oxidative stress in most degenerative processes is not yet known. Therefore, accurate and practical models are required to efficiently screen for therapeutics. Porcine eyes are closely related to the human eye, and can be obtained from the abattoir as a by-product of the food industry. Therefore, they offer excellent opportunities for the development of culture models with which to pre-screen potential therapies, while reducing the use of laboratory animals. To induce oxidative stress, organotypic cultures of porcine retina were treated with different doses of hydrogen peroxide (H2O2; 100, 300 and 500µM) for three hours. On days 3 and 8, the retinas were conserved for histological and Western blotting analyses and for evaluation of gene expression, which determined the number of retinal ganglion cells (RGCs), the activation state of glial cells, and the expression levels of several oxidative stress markers. H2O2treatment led to a reduction in the number of RGCs and to an increase in apoptotic RGCs. In addition, a dose-dependent increase of microglia and an elevation of CD11bexpression was observed. On day 3, a reduction of IL-1ß, and an increase of iNOS, as well as of HSP70mRNA were found. On day 8, an increase in TNF-aand IL-1ßmRNA expression was detected. In conclusion, this ex vivomodel offers an opportunity to study the molecular mechanisms underlying certain eye disorders and to test new therapeutic approaches to diminish the effects of oxidative stress.

Supplemental Digital Content is Available in the Text.

Vitreoretinal diffuse large B-cell lymphoma is a rare disorder, occurring as primary ocular disease or as secondary involvement by primary central nervous system lymphoma. It is usually diagnosed by cytologic, immunocytochemical, and molecular examination of vitreous aspirates. However, distinguishing vitreoretinal diffuse large B-cell lymphoma from uveitis remains difficult, and clonality analysis may be either unsuccessful or misleading. Diffuse large B-cell lymphoma arising in immune-privileged sites (eg, the central nervous system) shows a high frequency of MYD88 mutations. Therefore, we retrospectively assessed the frequency of MYD88 mutations in vitreoretinal lymphoma (VRL) and their diagnostic potential in 75 vitrectomy samples of 69 patients, and validated our results in a separate cohort (n = 21). MYD88 mutations were identified in 20 of 29 (69%) clinically, histologically, and molecularly confirmed VRL, including 6 cases of the test cohort initially diagnosed as reactive (3/6) or suspicious (3/6) for lymphoma. MYD88 mutations, especially L265P, are very frequent in VRL and their detection significantly improves the diagnostic yield of vitrectomy specimens.

Vitreoretinal diffuse large B-cell lymphoma is a rare disorder, occurring as primary ocular disease or as secondary involvement by primary central nervous system lymphoma. It is usually diagnosed by cytologic, immunocytochemical, and molecular examination of vitreous aspirates. However, distinguishing vitreoretinal diffuse large B-cell lymphoma from uveitis remains difficult, and clonality analysis may be either unsuccessful or misleading. Diffuse large B-cell lymphoma arising in immune-privileged sites (eg, the central nervous system) shows a high frequency of MYD88mutations. Therefore, we retrospectively assessed the frequency of MYD88mutations in vitreoretinal lymphoma (VRL) and their diagnostic potential in 75 vitrectomy samples of 69 patients, and validated our results in a separate cohort (n = 21). MYD88mutations were identified in 20 of 29 (69%) clinically, histologically, and molecularly confirmed VRL, including 6 cases of the test cohort initially diagnosed as reactive (3/6) or suspicious (3/6) for lymphoma. MYD88mutations, especially L265P, are very frequent in VRL and their detection significantly improves the diagnostic yield of vitrectomy specimens.

The aim of this study was to evaluate clinical outcomes of Descemet membrane endothelial keratoplasty (DMEK) in vitrectomized eyes.

Purpose To compare the outcomes after switching between bevacizumab and ranibizumab therapy due to poor treatment effect in neovascular age-related macular degeneration (AMD).Methods This is a retrospective review of patients with neovascular AMD with first treatment using intravitreal bevacizumab (group 1) or ranibizumab (group 2) who switched to the other drug due to poor treatment effect. Primary outcome measures were change in mean best-corrected visual acuity (BCVA) and mean central retinal thickness (CRT) at 1 year and last visit.Results Eighty-seven eyes met the inclusion criteria. In group 1 (43 eyes), the mean BCVA decreased from 20/94 to 20/100 at 1 year after being switched (p = 0.573) and to 20/150 (p = 0.015) at final visit (mean 29.2 months, range 12-53). In group 2 (44 eyes), mean BCVA decreased from 20/72 to 20/90 (p = 0.401) and 20/100 (p = 0.081) at 1 year after switch and at final visit (mean 20.1 months, range 10-40), respectively. The mean CRT at switch, 1 year after switch, and at final visit were 344.4 ± 140 µm (mean ± SD), 286.26 ± 155 µm (p = 0.019), and 290.58 ± 196 µm (p = 0.009) in group 1 and 329.36 ± 144 µm, 302.0 ± 179 µm (p = 0.215), and 309.5 ± 220 µm (p = 0.154) in group 2, respectively.Conclusions The mean BCVA decreased over time in both groups; however, nearly 30% of the eyes in each group showed vision improvement after switching. Mean CRT decreased in both groups, which was more pronounced after being switched from bevacizumab to ranibizumab. In neovascular AMD, a switch between ranibizumab and bevacizumab can be considered as a further therapy option if poor treatment effect is seen with the initial therapy.

The effects of a glutathione-containing intraocular irrigation solution, BSS Plus®, on retinal function and on the survival of ganglion cells in whole-mount retinal explants were studied. Evidence is provided that the perfused ex vivobovine retina can serve as an alternative to in vivoanimal testing. Isolated bovine retinas were prepared and perfused with an oxygen-saturated standard irrigation solution, and an electroretinogram was recorded to assess retinal function. After stable b-waves were detected, the isolated retinas were perfused with BSS Plus for 45 minutes. To investigate the effects of BSS Plus on photoreceptor function, 1mM aspartate was added to the irrigation solution in order to obtain a-waves, and the ERG trace was monitored for 75 minutes. For histological analysis, isolated whole retinal mounts were stored for 24 hours at 4°C, in the dark. The percentages of cell death in the retinal ganglion cell layer and in the outer and inner nuclear layers were estimated by using an ethidium homodimer-1 stain and the TUNEL assay. General swelling of the retina was examined with high-resolution optical coherence tomography. During perfusion with BSS Plus, no significant changes in a-wave and b-wave amplitudes were recorded. Retinas stored for 24 hours in BSS Plus showed a statistically significant smaller percentage (52.6%, standard deviation [SD] = 16.1%) of cell death in the retinal ganglion cell layer compared to the control group (69.6%, SD = 3.9, p= 0.0031). BSS Plus did not seem to affect short-term retinal function, and had a beneficial effect on the survival of retinal ganglion cells. This method for analysing the isolated perfused retina represents a valuable alternative for testing substances for their retinal biocompatibility and toxicity.

OBJECTIVE To evaluate the early effects of triamcinolone acetonide (TA) on inflammation, proliferation, and vascular endothelial growth factor (VEGF) in human choroidal neovascularization (CNV). METHODS Retrospective review of an interventional case series of 29 patients who underwent macular translocation. Fourteen CNV membranes without previous therapy (control CNV group) and 4 CNV membranes excised 3 days after photodynamic therapy (PDT CNV group) comprised the control groups. Eleven patients were treated with intravitreal TA (TA CNV group; n = 5) or PDT and TA combined (PDT+TA CNV group; n = 6) 3 to 9 days preoperatively. The CNV membranes were stained for cytokeratin 18, CD34, VEGF, intercellular adhesion molecule-1 (ICAM-1), E-selectin, CD68, CD45, Ki-67, and Thy-1. RESULTS Treatment with TA and PDT+TA resulted in increased immunostaining of ICAM-1 in endothelial cells and the stroma and a higher percentage of Thy-1 expression than controls. The density of macrophages was significantly increased in PDT+TA CNV membranes. Leukocyte density and proliferative activity were lower in TA and PDT+TA CNV membranes. The total VEGF score was significantly increased in TA and PDT+TA CNV membranes compared with the control CNV membranes. Evidence of VEGF in the retinal pigment epithelium of PDT+TA CNV membranes was stronger than in control CNV membranes. CONCLUSIONS Triamcinolone acetonide has no inhibitory effect on macrophage infiltration or ICAM-1, Thy-1, or VEGF expression in CNV membranes in the early term. The clinical benefits of TA are probably not based on pure antiinflammatory or VEGF-suppressing mechanisms.Arch Ophthalmol. 2009;127(3):275-281--

OBJECTIVE To evaluate the effect of bevacizumab (Avastin; Genentech, Inc, South San Francisco, California) on inflammation and proliferation in human choroidal neovascularization (CNV) secondary to age-related macular degeneration. METHODS Retrospective review of interventional series of 38 patients who underwent choroidal neaovascular membrane (CNVM) extraction. Twenty-four patients received intravitreal bevacizumab 1 to 154 days preoperatively (bevacizumab CNV group). Fourteen patients received no preoperative therapy (control CNV group). The CNVM were stained for cytokeratin 18, CD68, CD45, intercellular adhesion molecule (ICAM)–1, E-selectin, Ki-67, Thy-1, and endostatin. RESULTS No significant difference was detected in ICAM-1 and E-selectin expression between groups. The density of leukocytes in the bevacizumab CNV group (median, 271.61 cells/mm2) was higher than in the control CNV group (median, 116.87 cells/mm2; P = .07), but without significance. Density of macrophages (median, 4661.95 cells/mm2), proliferative activity (median, 160.19 cells/mm2), and percentage of Thy-1–expressing vessels (median, 100%) were significantly higher in the bevacizumab CNV group than in the control CNV group (median, 882.66 cells/mm2, P &lt; .001; median, 34.34 cells/mm2, P &lt; .001; and median, 80%, P &lt; .001, respectively). Endostatin immunoreactivity was considerably stronger in the retina pigment epithelium (RPE)–Bruch membrane complex (median, 3; range, 2-3; P &lt; .001), and stroma (median, 3; range, 1-3; P &lt; .001) of the bevacizumab CNV group than control CNV group (median, 1.5; range, 0-3 and median, 1; range, 0-3, respectively). CONCLUSIONS Unexpectedly, CNVM from patients treated by bevacizumab are characterized by significantly high inflammatory and proliferative activity and enhanced endostatin expression. These characteristics need to be considered when protocols for combination therapies are established.Arch Ophthalmol. 2008;126(6):782-790--

OBJECTIVE To assess long-term functional and morphological changes after macular translocation in patients with exudative age-related macular degeneration. METHODS Evaluation of a noncomparative cohort study of 90 patients with a follow-up of 14 to 79 months (mean, 38.2 months). RESULTS Visual acuity increased by 3 or more lines in 15 patients, remained stable in 35 patients, and deteriorated in 40 patients at final examination. Pigment epithelium atrophy extending to the new fovea was detected in 44 patients; in 25 patients this new atrophy was associated with loss of visual function. CONCLUSIONS Long-term follow-up of macular translocation with 360° retinotomy showed stabilization or improvement in half of the patients. Progressive atrophy of the pigment epithelium represented one major limiting factor of the beneficial effect of the treatment. Macular translocation may be an option for cases of exudative age-related macular degeneration that are not eligible for or do not respond to alternative treatments.Arch Ophthalmol. 2007;125(10):1367-1372--

OBJECTIVE To examine the impact of photodynamic therapy (PDT) on angiogenesis in human choroidal neovascular membranes with respect to vascular endothelial growth factor (VEGF) expression, proliferation, and vascularization. METHODS Retrospective review of an interventional case series of 50 patients (50 eyes) who underwent removal of choroidal neovascular membranes. Choroidal neovascularization was secondary to age-related macular degeneration. Twenty patients were treated with PDT 3 to 655 days before surgery. Choroidal neovascular membranes were stained for CD34, CD105, Ki-67, cytokeratin 18, and VEGF. Thirty choroidal neovascular membranes secondary to age-related macular degeneration without previous treatment were used as controls. RESULTS Specimens without pretreatment disclosed varying degrees of vascularization, proliferative activity, and VEGF expression by different cells. Specimens treated with PDT 3 days earlier showed mostly occluded vessels, damaged endothelial cells, and low proliferative activity. In contrast, specimens excised at later time points after PDT were highly vascularized and proliferating. This chronology was associated with an impressive VEGF immunoreactivity unique to retinal pigment epithelial cells shortly after PDT that also shifted to other cells at later time points. CONCLUSIONS Photodynamic therapy induces selective vascular damage in choroidal neovascular membranes. The effectiveness and selectivity of this treatment, however, seem to be jeopardized by a rebound effect initiated by enhanced VEGF expression in retinal pigment epithelial cells.Arch Ophthalmol.2006;124:815-823 --

AbstractPurpose: The controlled orientation of subretinal or intraretinal delivery of delicate tissue sheets is a critical problem in retinal transplantation research. Methods: A surgical device (explant injector) was designed and built for the controlled delivery of tissue explants into the subretinal or intraretinal space via a vitrectomy approach. The technical feasibility was demonstrated in enucleated porcine eyes. Results: Using negative pressure, the injector allows for the fixation of an explant, such as a monolayer of cells cultured on a collagen membrane, on the carrier platform. The sheet can then be placed safely without loss of orientation in the subretinal space. Conclusions: With the aid of the explant injector, it is possible to deliver an explant with the proper orientation to a precise location in the subretinal space.Copyright © 2006 S. Karger AG, Basel

Monoamine receptors are found throughout the body. Reports about the presence of monoamine receptors in the human cornea are inconsistent.Immunohistochemistry, immunofluorescence and immunoblotting were used to localize monoamine receptor sites on human corneal epithelium and endothelium.Antibodies to alpha-1, beta-1 and beta-2 adrenergic receptors and to D1-like and 5HT-7 receptors were bound in corneal epithelium. Antibodies to alpha-1, alpha-2A, beta-1 and beta-2 adrenergic receptors and to 5HT-7 receptors were bound in corneal endothelium.Our data demonstrate the presence of several monoamine receptors in the human cornea. These receptors may play a role in the regulation of fluid transport or corneal homeostasis.

AbstractPurpose: To analyze chloroquine-induced morphological changes in the retinal pigment epithelium (RPE) and Bruch’s membrane (BM). Methods: Retina-choroid complexes of chloroquine-treated Long-Evans rats were analyzed by electron microscopy. Results: Intercellular spaces between the RPE cells and BM were enlarged. Residual material from phagosomes was released into these enlarged spaces. Debris accumulated within BM and encircled choriocapillaris endothelial cells. Conclusion: There is a release of undegraded phagocytic material (rod outer segments) into the extracellular space between BM and RPE cells, following inhibition of lysosomal degradation. Electron-dense deposits in BM and choriocapillaris may lead to reduced oxygen and nutrition flow.Copyright © 2006 S. Karger AG, Basel

AbstractPurpose: To analyze and compare the angiographic features of retinal vascular anomalous complex (RVAC) in patients with a vascularized pigment epithelial detachment (PED) secondary to age-related macular degeneration. Methods: Retrospective evaluation of fluorescein and indocyanine green angiographies of 180 patients with occult choroidal neovascularization and PED. Results: On indocyanine green angiography (ICGA), RVAC demonstrated as a focal hyperfluorescence in connection with retinal choroidal anastomosis was identified in 12 of the 180 eyes (6.7%) with a vascularized PED. In comparison, fluorescein angiography revealed RVAC in 6 of the 12 eyes. Associated clinical and angiographic features were intraretinal hemorrhage in all 12 eyes, cystoid macular edema in 6 eyes and preretinal hemorrhage in 4 eyes. Conclusions: RVAC is better delineated on ICGA than on fluorescein angiography. In clinically suspected cases with a vascularized PED in connection with intraretinal hemorrhage and/or cystoid macular edema, ICGA should be performed to enhance management considerations.Copyright © 2005 S. Karger AG, Basel

Abstract

Background To report long-term functional and morphological outcome of verteporfin photodynamic therapy (PDT) for classic extrafoveal choroidal neovascularisation (CNV) secondary to pathologic myopia. Methods This retrospective case series included three eyes of three consecutive patients with classic extrafoveal CNV secondary to pathologic myopia, who underwent PDT at a tertiary retinal referral centre. Change in visual acuity and fluorescein leakage was the main outcome criteria. Results The patients were followed up an average of 36 months (33–40 months). In all eyes increase of visual acuity (one eye 1 ETDRS line, two eyes 2 ETDRS line) and no fluorescein leakage of the CNV were seen. Conclusions PDT can achieve long-term improvement of the visual acuity and morphological stability in selected cases with classic extrafoveal CNV in pathologic myopia. Large scale randomised studies are warranted for assessment of the benefit of PDT in such eyes.

Background Following multiple promising investigations into restoration of vision in degenerative retinal disease by implantation of a sub- or epiretinal prosthesis, the step to clinical use in humans is impending. In this study we intended to establish optical coherence tomography (OCT) and fluorescein angiography (FA) first in research animals for noninvasive assessment of the condition of the posterior pole of eyes after intraocular implant surgery. Methods Three adult cats that had undergone subretinal implant surgery were evaluated by OCT and FA between 1 and 470 days postoperatively. Eight adult cats served as control. In addition histology was performed. Results In all three cats OCT demonstrated stable positioning of the implants in the subretinal space during the complete examination period. Transient retinal edema was found in the early postoperative period but decreased during follow-up. The retina over the implants was well attached at all times in cats 1 and 2; however, in cat 3 localized retinal detachment was demonstrated. FA showed intact retinal vasculature over the subretinal implant in high detail without interference from choroidal background fluorescence. Conclusions OCT and FA have been fruitfully applied to cats to assess the morphological and circulatory conditions of the neuroretina and of its interface with the subretinal implant. The techniques may therefore provide a tool for objective, noninvasive in vivo evaluation of eyes that have undergone subretinal implant surgery, both in research animals and in humans.

Purpose:Successful engraftment of retinal pigment epithelial cells (RPE) to treat RPE-related retinopathy will depend, at least in part, on controlling the immune response. In order to understand this process we evaluated the fate of RPE xenografts in the subretinal space, anterior chamber, and subcutis of nonimmunosuppressed Royal College of Surgeons rats.

The Royal College of Surgeons (RCS) rat is an animal model for retinal degeneration such as the age-related macular degeneration. The RCS rat undergoes a progressive retinal degeneration during the early postnatal period. A potential treatment to prevent this retinal degeneration is the transplantation into the subretinal space of cells that would replace functions of the degenerating retinal pigment epithelium (RPE) cells or may form neurotrophic factors. In this study we have investigated the potential of subretinally transplanted embryonic stem cells to prevent the genetically determined photoreceptor cell degeneration in the RCS rat. Embryonic stem cells from the inner cell mass of the mouse blastocyst were allowed to differentiate to neural precursor cells in vitro and were then transplanted into the subretinal space of 20-day-old RCS rats. Transplanted and sham-operated rats were sacrificed 2 months following cell transplantation. The eyes were enucleated and photoreceptor degeneration was quantified by analyzing and determining the thickness of the outer nuclear layer by light and electron microscopy. In the eyes transplanted with embryonic cells up to 8 rows of photoreceptor cell nuclei were observed, whereas in nontreated control eyes the outer nuclear layer had degenerated completely. Transplantation of embryonic stem cells appears to delay photoreceptor cell degeneration in RCS rats.

Abstract. Background: In patients with more complex rhegmatogenous retinal detachments (RRD) not complicated by proliferative vitreoretinopathy (PVR), the most appropriate operating method is controversial, and different surgeons use different techniques. The Scleral Buckling Versus Primary Vitrectomy in Rhegmatogenous Retinal Detachments Study (SPR Study) is designed to compare primary vitrectomy and scleral buckling techniques in these patients. Methods: The SPR Study is a multicentre, randomised, controlled clinical trial stratified by lens status. Patients with RRD which is not complicated by PVR grade B or C and which cannot be treated with a single meridional sponge are randomised to either scleral buckling or pars plana vitrectomy as first surgical intervention. Four hundred consecutive patients are to be recruited per subtrial (phakic and aphakic/pseudophakic patients), and followed up for 1 year. The primary endpoint (functional outcome) is the change in visual acuity. Secondary endpoints (anatomical outcome) include postoperative PVR, retinal reattachment and the number of reoperations necessary to achieve retinal reattachment. Twenty-seven institutions (49 surgeons) in six European countries have been recruited for participation in the study. Conclusion: The SPR Study is the first randomised prospective clinical trial to compare scleral buckling and primary vitrectomy in patients with RRD. The results of this study should enable vitreoretinal surgeons to improve the surgical therapy of patients with the more complicated manifestations of RRD.

Uveal melanoma is the most common primary ocular cancer among adults and patients with distant metastases seldom survive longer than a year. Melanomas of the eye have the advantage of growing in the special environment of an immune privileged site and it has long been shown, that the special immunosuppressive properties of the intraocular microenvironment are strongly mediated by cytokines, especially transforming growth factor‐beta (TGF‐beta). Here, we sought to investigate the presence of TGF‐β in surgically removed uveal melanoma specimens using immunohistochemical methods to verify possible autocrine mechanisms. Immunocytochemistry for pan‐TGF‐β and TGF‐β2was performed on 13 melanoma specimens using an alkaline phosphatase labeling procedure. Melanocytic origin of the tumors was confirmed by HMB‐45 staining. All tissue samples exhibited positive staining using either pan‐TGF‐β or TGF‐β2antibody regardless of cell‐type, size of the tumor, or tumor location. The intensity of staining did not vary significantly within a given tumor. All tumors stained positive against the HMB‐45 antibody. Many cytokines have been found to act on melanoma tumors. The presence of the TGF‐β2isoform in all specimens points to progressive tumor‐growth as has been shown for melanomas of the skin. Based on our immunohistochemical findings and the immunosuppressive properties of TGF‐β, we suppose that ocular melanomas should be able to create their own immunosuppressive environment even in the uvea, which might be a non‐privileged site. Microsc. Res. Tech. 52:396–400, 2001. © 2001 Wiley‐Liss, Inc.

Abstract: · Background. The assessment of the cup of the optic disc in follow-up of glaucomatous optic nerve heads depends on the variability of the cup area over time. We examined the variability of topographic measurements depending on scan focus settings and evaluated the long-term fluctuations of the normalised rim/disc area ratio quotient of normal subjects for 1 year. · Methods. Part 1. Evaluation of the influence of scan focus and corneal curvature on the number of pixels per millimetre in one emmetropic volunteer. The scan focus was varied using contact lenses of different refraction. Part 2. HRT examinations of the optic nerve head of five eyes of five volunteers were repeatedly recorded during a 12-month period. The contour line from the first mean topographic image was transferred into the following seven mean topographic images of each subject. · Results. Part 1. The size of pixels depends on the adjusted scan focus for mean corneal curvature radius between 6 and 9 mm. However, after image to image scaling the size of pixels is nearly independent to the scan focus. Part 2. We found the highest variation of normalised rim/disc area ratio for the location of the temporal horizontal sectors, followed by the temporal inferior and the temporal superior sectors. Variation was smallest in the nasal sectors. Mean fluctuation of the normalised rim/disc area ratio quotient of the 95th percentile was 0.09 following image to image scaling compared to 0.16 when using individual image scaling. · Conclusion. Scan focus variation during HRT follow-up examinations can influence optic disc parameters, especially when image to image scaling has not been performed. A confidence area of an individual optic disc could now be calculated. These confidence bands could help to distinguish more easily between progress of glaucomatous optic nerve head damage and physiological intertopographic variation of optic cup measurements. A decrease of more than 43% in the fluctuations of the normalised rim/disc area ratio could be achieved using image to image scaling techniques. For that reason follow-up of glaucomatous optic nerve heads should only be performed using this software application.

Transplantation of autologous iris pigment epithelium (IPE) into the subretinal space has been suggested as one approach for the treatment of age-related macular degeneration. Autologous rabbit IPE cells were transplanted to the subretinal space to define the technique of transplantation and examine the survival of the transplanted cells.

Abstract: · Background: To investigate in iris pigment epithelium (IPE) the expression of mRNA for proteins involved in retinol metabolism we used a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. · Methods: RNA was prepared from freshly isolated bovine IPE and retinal pigment epithelium (RPE) cells and reverse transcribed. The expression of mRNA for cellular retinaldehyde binding protein (CRALBP), p63 (RPE63), the presumed retinal pigment epithelial membrane receptor for retinoids, and 11-cis-dehydrogenase (11cisRDH ) was determined by RT-PCR using specific primers. Semi-quantitative expression data were obtained by using a series of fivefold dilution of each cDNA with a fixed number of PCR cycles. · Results: Bovine IPE and RPE cells express mRNA for CRALBP, 11cisRDH, and RPE63. The mRNA expression for CRALBP and 11cisRDH is high and equal in both cell types. However, RPE63 mRNA expression in IPE cells is relatively low compared with the expression in RPE cells. · Conclusions: The presence of mRNA for CRALBP, RPE63, and 11cisRDH suggests that IPE cells may be able to metabolize retinol.

The mechanism of the toxicity of light on the retina remains unclear despite a large number of investigations. The purpose of this study is to identify and localize the ultrastructural changes and the site of the earliest damage after intense light exposure. Nine adult Syrian golden hamsters (Mesocricetus auratus) have been maintained under constant illumination with a high-pressure mercury lamp (HQJ R 80 W Deluxe, Osram, Berlin, light intensity 1000 lx) for 12 h, followed by an additional 3 h in the dark. Light damage is assessed by light and electron microscopy. Morphological evaluation reveals focal damage to the retinal pigment epithelial (RPE) cells in close proximity to less-affected RPE cells and normal photoreceptors. Collagen fibers in Bruch's membrane lose their parallel orientation. Occasionally, fusion of cell membranes of neighboring rod outer segments (ROS) is also observed. Continuous, 12 h exposure of hamsters to intense light results in initial focal damage to some RPE cells, such that severely damaged RPE cells are found adjacent to intact RPE cells. Only slight damage to the photoreceptors is evident, suggesting that the sequence of the pathological changes resulting from light begins with damage to the RPE cells and associated Bruch's membrane.

• Background: The assessment of the cup of the optic disc depends, among other criteria, on the disc area. A small cup in a small optic disc can indicate an advanced glaucomatous lesion, while on the other hand a large cup in a large optic disc can be normal. Therefore, a cumulative normalised rim/disc area ratio curve could help to distinguish between glaucomatous and normal optic cups. The aim of our study was to calculate and to evaluate such a cumulative normalised rim/disc area ratio curve. • Methods: Heidelberg Retina Tomograph examinations of the optic nerve head of 100 randomly selected eyes of 100 normal subjects were evaluated. We calculated the disc area-adjusted normalised rim/disc area ratio in sectors of 10°. The 95th, 90th and 50th percentiles of each of the 36 sectors were displayed in descending order. • Results: In relation to the normal percentile curves, it is possible to display an individual normalised rim/disc area ratio curve. We obtained such curves for a normal optic disc, optic nerve heads with moderate and advanced lesions and a small optic disc with glaucomatous damage. • Conclusion: We present a new display mode for the results of Heidelberg Retina Tomograph optic nerve head examination, which may be helpful for easy and reliable assessment of the local, diffuse and combined components of glaucomatous optic nerve head damage depending on optic disc size.

• Purpose: The purpose of this study was to evaluate recovery data for the b-wave of the electroretinogram (ERG) elicited using multiple flash stimulation with increasing stimulus intervals in normal controls and in patients with macular diseases. The results will describe effects of age and macular disease and define indexes characterizing the recovery process. • Methods: Scotopic Ganzfeld flash ERGS were elicited using interstimulus intervals of 140, 280, and 560 ms. Relative b-waves were calculated as the ratio b140 ms/ b560 ms and b280 ms/b560 ms, respectively. Responses obtained in 134 eyes of 134 normal controls served as reference data. Fifty-four patients with different macular diseases were also examined and their data compared to the reference data. • Results: Relative b-wave amplitudes correlated with interstimulus interval and with flash luminance, but not with age. All patients had a normal ERG when recorded following the standard of clinical electroretinography. A sigmoidal model was suggested, providing three indexes characterizing the b-wave recovery process. Relative b-waves and recovery indexes varied in age-related macular degeneration, central serous retinopathy, vitelliform macular degeneration, Stargardt's disease, and pattern dystrophy. • Conclusion: The triple flash ERG reflects energy-yielding and -utilizing mechanisms. It proved to be more sensitive in detecting functional lesions in macular diseases than the standardized explorating procedure. The b-wave recovery model allows differentiation between two independent mechanisms contributing to the b-wave recovery process. One or more of the three characterizing indexes are affected in different macular diseases.

The targeted induction of apoptosis is a novel therapeutic approach to control the unlimited growth of proliferating cells. Since massive proliferation of cells at the vitreoretinal interface is a key feature of proliferative vitreoretinal disorders, we sought to identify apoptosis in epiretinal membranes from patients with proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR) and macular pucker. Further, we evaluated the possible induction of apoptosis of retinal pigment epithelial (RPE) cells by transforming growth factor-β(TGF-β).