Effectiveness and safety of VISULAS® green selective laser trabeculoplasty: a prospective, interventional multicenter clinical investigation.

PURPOSE: To evaluate the effectiveness and safety of Selective Laser Trabeculoplasty (SLT) with the SLT mode of the VISULAS® green laser in patients with primary open-angle glaucoma (POAG). METHODS: This prospective, interventional multicenter clinical investigation included patients with POAG who either needed a treatment escalation because the individual intraocular pressure (IOP) target was not met or treatment initiation and had an IOP ≥ 17 mmHg at baseline in the study eye. The study was conducted in five research centers across Germany. Approximately 100 laser applications were delivered to 360° of the trabecular meshwork. Glaucoma medications were not modified during the 3-month follow-up to allow evaluation of the sole effect of VISULAS® green with SLT. Efficacy outcomes were postoperatively absolute and relative IOP changes at 1 and 3 months. Safety outcomes analyzed the rate of intra- and postoperative adverse events. RESULTS: Thirty-four eyes of 34 POAG patients were included. The overall mean number of preoperative glaucoma medications was 2.2 ± 1.4 in 29 treated eyes, 5 eyes were treatment naïve. Mean baseline IOP (mmHg) was 21.0 ± 2.69 and was reduced by - 3.53 ± 3.34 [95% CI - 4.61; - 2.45] and - 3.59 ± 3.41 [95% CI - 4.64; - 2.53] at the 1- and 3-month follow-up, respectively (p < 0.0001), with 48.5% of cases achieving a ≥ 20% IOP reduction at 3 months [95% CI = 30.8%; 66.5%]. The mean relative IOP reduction was - 16.4% and - 16.3% at 1 and 3 months, respectively (p < 0.0001). Potentially device- or procedure-related adverse events were mild to moderate and included 3 postoperative IOP-spikes and 6 reports regarding eye pain and discomfort. All were resolved without sequelae. CONCLUSIONS: SLT performed with the VISULAS® green laser achieved clinically significant additional IOP reductions in medically treated as well as in treatment naïve eyes with POAG and there were no relevant safety issues. The results are comparable to other reported SLT studies.

Spatial and temporal immunoreaction of nestin, CD44, collagen IX and GFAP in human retinal Müller cells in the developing fetal eye.

The purpose of this study was to investigate Müller cells during the fetal development of the human eye. Müller cells in eyes of 39 human fetuses (11-38 weeks of gestation, WOG) and 6 infants (5 died of abusive head trauma, AHT, aged 1-9 months) were immunohistochemically stained and investigated for spatial and temporal immunoreaction of nestin, CD44, collagen IX and GFAP, which are stem cell markers or markers of intermediate filaments, respectively, in one of the hitherto largest cohorts of fetal eyes. Müller cells could be detected immunohistochemically as early as 12 WOG by immunohistochemical staining with nestin. Nestin was more strongly expressed in Müller cells of the peripheral retina and a centroperipheral gradient of immunoreaction over time was observed. CD44 was predominantly expressed in fetal eyes of the late second and early third trimester between (23 and 27 WOG) and significantly stronger in the infant eyes. Collagen IX labeling in the central retina was significantly stronger than in more peripheral sectors and increased with fetal age. GFAP staining in Müller cells was seen in the eye of a fetus of 38 WOG who died postnatally and in the infant eyes with and without history of AHT. Additionally, GFAP staining was present in the astrocytes of fetal and infant eyes. All examined markers were expressed by Müller cells at different developmental stages highlighting the plasticity of Müller cells during the development of the human eye. GFAP should be cautiously used as a marker for AHT as it was also expressed in fetal astrocytes and Müller cells in eyes without history of AHT.

Response of Retinal Pigment Epithelium (RPE)-Choroid Explants to Thermal Stimulation Therapy of the RPE (TSR).

BACKGROUND AND OBJECTIVES: The thermal stimulation therapy of the retinal pigment epithelium (TSR) is a sublethal laser technique for thermal stimulation of the retinal pigment epithelium (RPE)-Bruch's membrane (BrM)-complex. The aim of this study was to investigate the influence of TSR on the release of age-related macular degeneration (AMD)-relevant cell mediators. STUDY DESIGN/MATERIALS AND METHODS: Porcine RPE-BrM-choroid explants were irradiated with a 532 nm continuous wave laser using different spot sizes (100-300 µm, duration 100 milliseconds, 15-100 mW). Cell death was investigated by calcein staining. Explants were treated with grids of sublethal spots and cultivated in modified Ussing chambers. The effect on matrix metalloproteinase-2 (MMP-2) and -9 was investigated by zymography and quantitative reverse transcription polymerase chain reaction. Secretion of vascular endothelial growth factor (VEGF), pigment epithelium derived factor (PEDF), and transforming growth factor-ß (TGF-ß) was analyzed by enzyme-linked immunosorbent assay and expression of HSP70 was examined by western blot. Integrity of the RPE/BrM-complex was analyzed by scanning electron microscopy. RESULTS: Laser powers of 15 mW (100 µm) and 45 mW (300 µm) did not induce RPE cell death. The integrity of the RPE/BrM-complex was not impaired after TSR. After TSR with 300 µm spot size, we observed a significant increase of active MMP-2 in the basal compartments. The content of PEDF significantly increased in treated explants in both compartments with 100 and 300 µm spot sizes. VEGF and TGF-ß secretion was not triggered by TSR. CONCLUSIONS: TSR represents a possible RPE stimulating treatment for dry AMD. TSR increases the basal release of active MMP-2, which might reverse age-related thickening of BrM. VEGF secretion was not triggered by TSR while anti-angiogenic PEDF was increased, indicating an induction of an anti-angiogenic and neuroprotective environment. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Retinal volume change is a reliable OCT biomarker for disease activity in neovascular AMD.

PURPOSE: Current algorithms for automated computer interpretation of optical coherence tomography (OCT) imaging of patients suffering from neovascular age-related macular degeneration (AMD) mostly rely on fluid detection. However, fluid detection itself and correct interpretation of the fluid currently limits diagnostic accuracy. We therefore performed a detailed analysis of the requirements that would have to be met for fluid detection approaches. We further investigated if monitoring retinal volume would be a viable alternative to detect disease activity. METHODS: Retrospective analysis and manual grading of 764 OCT volume scans of 44 patients with exudative AMD treated with intravitreal anti-VEGF injections at a pro-re-nata (PRN) treatment regimen for at least 24 months. RESULTS: Detection of subretinal fluid (SRF) or intraretinal fluid (IRF) alone is not sufficient for disease detection. A combination of SRF and IRF can detect disease activity with a sensitivity of 98.6% and a specificity of 82%. With further characterization of IRF into exudative and degenerative cysts, specificity can be increased to 100%. However, correct characterization is currently not achieved by published fluid detection approaches. Change of macular retinal volume (MRV) can depict disease activity with sensitivity of 88.4% and specificity of 89.6%. Combination with the detection of SRF can further improve diagnostic accuracy to a specificity of 93.3% and sensitivity of 93.9% without relying on IRF or IRF characterization. CONCLUSION: Fluid detection without further characterization is not sufficient for AMD monitoring. Either further distinction between exudative and degenerative cysts is necessary, or other activity markers have to be taken into account. MRV offers good potential to fill this diagnostic gap and might become an important monitoring marker.

Intravitreal injection of anti-Interleukin (IL)-6 antibody attenuates experimental autoimmune uveitis in mice.

PURPOSE: To evaluate the effect of an intravitreally applied anti-IL-6 antibody for the treatment of experimental autoimmune uveitis (EAU). METHODS: EAU was induced in female B10.RIII mice by Inter-Photoreceptor-Binding-Protein (IRBP) in complete Freund's adjuvant, boosted by Pertussis toxin. Single blinded intravitreal injections of anti-IL-6 antibody were applied 5-7days as well as 8-10days (3day interval) after EAU induction into the randomized treatment eye and phosphate buffered saline (PBS) into the fellow control eye. Clinical and fluorescein angiography scoring (6 EAU grades) was done at each injection day and at enucleation day 14. Enucleated eyes were either scored histologically (6 EAU grades) or examined by ELISA for levels of IL-6, IL-17 and IL-6 soluble Receptor (sIL-6R). RESULTS: Uveitis developed in all 12 mice. Clinical uveitis score was significantly reduced (p=0.035) in treated eyes (median 2.0, range 0-4.0, n=12) compared to the fellow control eyes (median 3.0, range 1.0-4.0, n=12). Angiography scores were reduced in 9/12 treated eyes and histological scores in 3/4 treated eyes compared to the fellow control eyes. Cytokine levels were determined in 8 mice, of which 4 responded to anti-IL-6 treatment and 4 did not respond. All mice responding to treatment had a significant reduction of IL-6 (p<0.01) and IL-17 (p=0.01) levels in treated eyes compared to the fellow control eyes. This difference was not seen in non-responding mice. CONCLUSIONS: Intravitreal anti-IL-6 treatment significantly attenuates experimental autoimmune uveitis in mice. EAU activity correlates with ocular IL-6 and IL-17 levels.

Variability of panretinal photocoagulation lesions across physicians and patients. Quantification of diameter and intensity variation.

BACKGROUND: Photocoagulation lesion intensity relies on the judgement of retinal blanching. Lesions turn out variable due to observer-dependent judgement and time dependency of blanching. We investigated lesion variability per patient and per physician in clinical routine treatments. METHODS: In this observational clinical trial, different physicians performed panretinal photocoagulation for diabetic retinopathy. Study eyes received 20-30 study lesions at 20 ms (three physicians, nine eyes) and 200 ms (four physicians, 12 eyes) irradiation time (532 nm continuous wave photocoagulator, 300 µm spot size). Lesions were imaged after 1 hour with photography and optical coherence tomography (OCT). We measured lesion diameters in fundus and OCT images, and graded intensities according to a previously published six-step classifier. RESULTS: 200-ms lesions were larger and more severe (568, 474-625 µm [median, IQR], predominantly class 6) than 20-ms lesions (397, 347-459 µm, predominantly classes 3-4). The impact of laser power was small compared to other factors. Lesion intensities and diameters in fundus and OCT images varied significantly between patients and between physicians. Median photographic lesion diameters varied by up to a factor of 1.61 (20 ms) or 1.5 (200 ms) respectively. CONCLUSIONS: In this study, the treated area of retina varied by up to a factor of 1.612 = 2.59 for a given spot number. As clinical efficacy depends on the treated area, which is a function of lesion number by area per lesion, our results implicate poor control of the overall treatment effect if treatments are administered according to lesion number or spacing alone. Better ways of laser effect control should be sought.

Calibration of histological retina specimens after fixation in Margo's solution and paraffin embedding to in-vivo dimensions, using photography and optical coherence tomography.

BACKGROUND: The extent of retinal tissue deformation by histological processing needs to be separately measured for every workup protocol. This work presents a simple approach for its quantitative assessment, and shows lateral and axial scaling factors for a common protocol. We calibrated histological measurements by in-vivo photographic and optical coherence tomographic (OCT) measurements, using retinal photocoagulation lesions as calibration markers. METHODS: We evaluated four rabbit eyes that were examined histologically after fixation in Margo's solution (1 % paraformaldehyde:1.25 % glutaraldehyde), isopropanol dehydration, paraffin embedding and hematoxylin and eosin staining. Distances between 51 pairs of laser lesions were compared in photographs and on histological slides. Retinal thickness measurements were performed at 15 anatomically defined sites in these eyes, and related to anatomically matched OCT thickness measurements of six different rabbit eyes. RESULTS: We found that the ratio of histological over photographic lesion distances was 1.17 (95 % CI 1.13-1.22), indicating 17 % lateral retinal stretching or expansion by the processing. Thickness measurements in histology were 65.6 % of the in-vivo thickness as measured in OCT, indicating 1/3 axial tissue compression or shrinkage. CONCLUSIONS: We provide an analysis of retinal tissue deformation after fixation in Margo's solution and paraffin embedding. In spite of protocol optimization for reduced tissue deformation, the workup caused 1/3 axial compression/shrinkage and 17 % lateral elongation, which was unexpected. We show a simple way how to calibrate retina specimens by fundus photography and OCT, two methods that are readily available to most ophthalmologists. Our findings underline the necessity to calibrate specimens prior to morphometry.

Photocoagulation in rabbits: optical coherence tomographic lesion classification, wound healing reaction, and retinal temperatures.

BACKGROUND AND OBJECTIVE: The rabbit is the most common animal model to study retinal photocoagulation lesions. We present a classification of retinal lesions from rabbits, that is based on optical coherence tomographic (OCT) findings, temperature data, and OCT-follow-up data over 3 months. MATERIALS AND METHODS: Four hundred eighty-six photocoagulation lesions (modified Zeiss Visulas® 532 nm CW laser, lesion diameter 133 µm, exposure duration 200 milliseconds or variable, power variable) were analyzed from six eyes of three chinchilla gray rabbits. During the irradiation of each lesion, we used an optoacoustics-based method to measure the retinal temperature profile. Two hours, 1 week, 1 month, and 3 months after the treatment, we obtained fundus color and OCT (Spectralis®) images of each lesion. We classified the lesions according to their OCT morphology and correlated the findings to ophthalmoscopic and OCT lesion diameters, and temperatures. RESULTS: Besides an undetectable lesion class 0, we discerned subthreshold lesions that were invisible on the fundus but detectable in OCT (classes 1 and 2), very mild lesions that were partly visible on the fundus (class 3), and 3 classes of suprathreshold lesions. OCT greatest linear diameters (GLDs) were larger than ophthalmoscopic lesion diameters, both increased for increasing classes, and GLDs decreased over 3 months within each class. Mean peak end temperatures for 200 milliseconds lesions ranged from 61°C in class 2 to 80°C in class 6. CONCLUSION: The seven step rabbit lesion classifier is distinct from a previously published human lesion classifier. Threshold lesions are generated at comparable temperatures in rabbits and humans, while more intense lesions are created at lower temperatures in rabbits. The OCT lesion classifier could replace routine histology in some studies, and the presented data may be used to estimate lesion end temperatures from OCT images.

Deferoxamine mesylate is toxic for retinal pigment epithelium cells in vitro, and its toxicity is mediated by p38.

Deferoxamine mesylate is clinically used as a chelating agent but might induce retinopathy. To evaluate its effect on the retinal pigment epithelium (RPE), porcine RPE cells were stimulated with deferoxamine. Cell death was assessed with trypan blue exclusion assay. To investigate the pathway of cell death, the mitogen-activated protein kinases (MAPKs) Erk, JNK, and p38 were inhibited with U0126, SP600125, and SB203580, respectively. Their activity was determined by Western blot. Deferoxamine induces significant cell death in RPE cells, accompanied by phosphorylation of p38 and Erk. Inhibition of p38 attenuates cell death. In conclusion, deferoxamine is directly toxic on RPE cells, its toxicity depending on p38.

Selective Retina Therapy Reduces Bruch's Membrane Thickness and Retinal Pigment Epithelium Pathology in Age-Related Macular Degeneration Mouse Models.

PURPOSE: To investigate the effect of selective retina therapy (SRT) on age-related macular degeneration (AMD)-like alterations of retinal pigment epithelium (RPE) and Bruch's membrane (BrM) in AMD mouse models as therapeutic approach for the treatment of dry AMD. METHODS: In B6.129P2-Apoetm1Unc /J (ApoE-/-) and B6.129X1-Nfe2I2 tm1Ywk /J (NRF2-/-), one randomized eye of each mouse in groups of 15 mice was treated by SRT (532 nm, 300 ms, ∼1.4-µs pulse, 100 Hz, 50-µm spot), the fellow eye and healthy C57BL/6J mice served as controls. Clinical examinations were obtained at treatment day and 1 month later, followed by enucleation to analyze BrM thickness and ultrastructural RPE morphology. RESULTS: Nearly all ApoE-/- and NRF2-/- mice showed AMD-like retinal alterations. BrM thickness was increased in both mouse models, RPE had vacuoles within the cell body and shortened apical microvilli. SRT neither affected neuroretinal anatomy nor function. BrM thickness as well as AMD-like ultrastructural alterations of the RPE were significantly reduced in laser-treated eyes compared with fellow control and untreated control eyes. CONCLUSIONS: SRT reduces BrM thickness and AMD-like RPE alterations in AMD mouse models without damage to structural or functional properties of neuroretina. It may be a prophylactic or therapeutic option for dry AMD. TRANSLATIONAL RELEVANCE: SRT shows therapeutic effectivity in murine AMD models and might therefore become an option for the treatment of dry AMD.

Thermal Stimulation of the Retina Reduces Bruch's Membrane Thickness in Age Related Macular Degeneration Mouse Models.

PURPOSE: To investigate the effect of thermal stimulation of the retina (TS-R) on Bruch's membrane (BrM) thickness in age-related macular degeneration (AMD) mouse models as a novel concept for the prophylaxis and treatment of dry AMD. METHODS: Two knockout AMD mouse models, B6.129P2-Apoetm1Unc/J (ApoE-/-) and B6.129X1-Nfe2I2tm1Ywk/J (NRF2-/-), were chosen. One randomized eye of each mouse in four different groups (two of different age, two of different genotype) of five mice was treated by TS-R (532 nm, 10-ms duration, 50-µm spot size), the fellow eye served as control. Laser power was titrated to barely visible laser burns, then reduced by 70% to guarantee for thermal elevation without damage to the neuroretina, then applied uniformly to the murine retina. Fundus, optical coherence tomography (OCT), and fluorescein angiography (FLA) images were obtained at the day of treatment and 1 month after treatment. Eyes were enucleated thereafter to analyze BrM thickness by transmission electron microscopy (TEM) in a standardized blinded manner. RESULTS: Fundus images revealed that all ApoE-/- and NRF2-/- mice had AMD associated retinal alterations. BrM thickness was increased in untreated controls of both mouse models. Subvisible TS-R laser spots were not detectable by fundus imaging, OCT, or FLA 2 hours or 1 month after laser treatment. TEM revealed a significant reduction of BrM thickness in laser-treated eyes of all four groups compared to their fellow control eyes. CONCLUSIONS: TS-R reduces BrM thickness in AMD mouse models ApoE-/- and NRF2-/- without damage to the neuroretina. It may become a prophylactic or even therapeutic treatment option for dry AMD. TRANSLATIONAL RELEVANCE: TS-R may become a prophylactic or even therapeutic treatment option for dry AMD.

Release of Different Cell Mediators During Retinal Pigment Epithelium Regeneration Following Selective Retina Therapy.

Purpose: To investigate the effect of selective retina therapy (SRT) on the release of AMD-relevant cell mediators, such as matrix metalloproteinases (MMPs), VEGF, and pigment epithelium derived factor (PEDF) using different laser spot sizes and densities. Methods: Porcine RPE-choroid explants were treated with a pulsed 532 nm Nd:YAG laser using (1) large spot sizes, (2) small spot sizes with a high-density (hd) treatment, and (3) small spot sizes with a low-density (ld) treatment. Explants were cultivated in modified Ussing chambers. RPE regeneration and RPE cell death were investigated by calcein-AM staining and immunofluorescence. The MMP release was examined via zymography and immunofluorescence. VEGF and PEDF secretion was analyzed by ELISA. Results: During pigment epithelium regeneration (PER), mitosis and RPE cell migration were observed. Four days after SRT (large spot size) the content of active MMP2 increased significantly (P < 0.01). Hd treatment with small spot sizes resulted also in an increase of active MMP2 (P < 0.05). In immunofluorescence explants showed a localized expression of MMP2 within the healing lesions after irradiation. The PEDF level increased significantly (P = 0.01) after SRT with large spot sizes. VEGF secretion decreased significantly (P < 0.05) following SRT with large spot sizes and with hd treatment of small spot sizes. Conclusions: SRT induces a cytokine profile, which may improve the flux across Bruch's membrane, slows down progression of early AMD by RPE regeneration, and inhibits the formation of choroidal neovascularization. The cytokine release depends on the size and density of applied laser spots.

Power-controlled temperature guided retinal laser therapy.

Laser photocoagulation has been a treatment method for retinal diseases for decades. Recently, studies have demonstrated therapeutic benefits for subvisible effects. A treatment mode based on an automatic feedback algorithm to reliably generate subvisible and visible irradiations within a constant irradiation time is introduced. The method uses a site-individual adaptation of the laser power by monitoring the retinal temperature rise during the treatment using optoacoustics. This provides feedback to adjust the therapy laser power during the irradiation. The technique was demonstrated on rabbits in vivo using a 532-nm continuous wave Nd:YAG laser. The temperature measurement was performed with 523-nm Q-switched Nd:YLF laser pulses with 75-ns pulse duration at 1-kHz repetition rate. The beam diameter on the fundus was 200 µm for both lasers, respectively. The aim temperatures ranged from 50°C to 75°C in 11 eyes of 7 rabbits. The results showed ophthalmoscopically invisible effects below 55°C with therapy laser powers over a wide range. The standard deviation for the measured temperatures ranged from 2.1°C for an aim temperature of 50°C to 4.7°C for 75°C. The ED50 temperature value for ophthalmoscopically visible lesions in rabbits was determined as 65.3°C. The introduced method can be used for retinal irradiations with adjustable temperature elevations.

Lesion strength control by automatic temperature guided retinal photocoagulation.

Laser photocoagulation is an established treatment for a variety of retinal diseases. However, when using the same irradiation parameter, the size and strength of the lesions are unpredictable due to unknown inter- and intraindividual optical properties of the fundus layers. The aim of this work is to investigate a feedback system to generate desired lesions of preselectable strengths by automatically controlling the irradiation time. Optoacoustics were used for retinal temperature monitoring. A 532-nm continuous wave Nd:YAG laser was used for photocoagulation. A 75-ns/523-nm Q-switched Nd:YLF laser simultaneously excited temperature-dependent pressure transients, which were detected at the cornea by an ultrasonic transducer embedded in a contact lens. The temperature data were analyzed during the irradiation by a LabVIEW routine. The treatment laser was switched off automatically when the required lesion strength was achieved. Five different feedback control algorithms for different lesion sizes were developed and tested on rabbits in vivo. With a laser spot diameter of 133???m, five different lesion types with ophthalmoscopically visible diameters ranging mostly between 100 and 200???m, and different appearances were achieved by automatic exposure time control. The automatically controlled lesions were widely independent of the treatment laser power and the retinal pigmentation.

Long-term, therapy-related visual outcome of 49 cases with retinal arterial macroaneurysm: a case series and literature review.

PURPOSE: Retinal arterial macroaneurysms (RAMAs) are acquired dilations of branches of the central retinal artery. Treatment depends on vision-limiting complications. We compare the long-term visual acuity (VA) in three groups according to treatment. METHODS: 49 charts of patients with RAMA were reviewed. 16 remained untreated, 15 received photocoagulation and 18 vitrectomy. Patients underwent full ophthalmological examinations and up-to-date imaging. We evaluated chosen therapy, complications and final VA at the last visit. RESULTS: 65% of the cohort was female, aged 75±11 years (mean±SD). Follow-up was 34±23 months. These parameters did not differ significantly between the three groups. In the observed group, initial VA was 0.48 (mean log MAR) vs 0.35 at the final visit, in the photocoagulation group 0.55 vs 0.59, and in the vitrectomy group 1.8 vs 0.77. VA was significantly worse at enrolment in the vitrectomy group, while all other VA differences were not significant. CONCLUSIONS: The overall visual prognosis of RAMA was good, even after macular complications. VA remained unchanged in the observed and the laser groups and was comparable in all groups after 3 years. Based on an individual treatment decision, all therapies were effective and efficient. If subfoveal haemorrhage caused a macular hole, the VA outcome was limited.

Oxidative Stress Induces Biphasic ERK1/2 Activation in the RPE with Distinct Effects on Cell Survival at Early and Late Activation.

PURPOSE: Oxidative stress is considered a major factor in the deterioration of retinal pigment epithelium (RPE) cells in dry age-related macular degeneration (AMD). The MAPK ERK1/2 can be activated by oxidative stress, may exert both pro- and anti-apoptotic functions, and has recently been proposed as a major factor in RPE degeneration in atrophic changes. Nrf2 is a master regulator of oxidative stress defense and ERK1/2 is an upstream activator of Nrf2. In this study, we investigate the participation of ERK1/2 in oxidative stress pathways in connection with Nrf2. METHODS: Nrf2 knock-out and wild-type primary RPE cells were prepared from mouse eyes. Oxidative stress was induced by different concentrations of t-butylhydroperoxide. Mitogen-activated protein kinases (MAPKs) were blocked by commercially available inhibitors (SB203580, U0126, SP600125). Cell viability was determined by MTT assay. ERK1/2 expression and activation were assessed by Western blotting. RESULTS: Oxidative stress induced concentration dependent cell death, which occurred at lower concentrations in Nrf2 knock-out RPE. Western blot analysis displayed a biphasic activation of ERK1/2 in murine wild-type RPE and the inhibition of late, but not early activation of ERK1/2 exerted protection in wild-type murine RPE cells. The biphasic activation of ERK1/2 is lost in Nrf2 knock-out mice, and inhibition of ERK1/2 was generally protective. The inhibition of MAPK JNK or p38 exerted no protection, irrespective of Nrf2. CONCLUSION: RPE cells display a biphasic activation of ERK1/2 after oxidative insult, of which the late activation is pro-apoptotic. The biphasic activation is lost in Nrf2 knock-outs, suggesting that early ERK1/2 activation may be connected to Nrf2 signaling. In addition, ERK1/2 activation in Nrf2 knock-outs mediates oxidative stress-induced cell death.

Temperature-Controlled Retinal Photocoagulation Reliably Generates Uniform Subvisible, Mild, or Moderate Lesions.

PURPOSE: Conventional retinal photocoagulation produces irregular lesions and does not allow reliable control of ophthalmoscopically invisible lesions. We applied automatically controlled retinal photocoagulation, which allows to apply uniform lesions without titration, and aimed at five different predictable lesion intensities in a study on rabbit eyes. METHODS: A conventional 532-nm photocoagulation laser was used in combination with a pulsed probe laser. They facilitated real-time fundus temperature measurements and automatic exposure time control for different predefined time/temperature dependent characteristics (TTC). We applied 225 control lesions (exposure time 200 ms) and 794 TTC lesions (5 intensities, exposure times 7-800 ms) in six rabbit eyes with variable laser power (20-66.4 mW). Starting after 2 hours, we examined fundus color and optical coherence tomographic (OCT) images over 3 months and classified lesion morphologies according to a seven-stage OCT classifier. RESULTS: Visibility rates in funduscopy (OCT) after 2 hours were 17% (68%) for TTC intensity group 1, 38% (90%) for TTC group 2 and greater than 94% (>98%) for all consecutive groups. TTC groups 1 through 4 correlated to increasing morphological lesion intensities and increasing median funduscopic and OCT diameters. Group 5 lesions were as large as, but more intense than group 4 lesions. CONCLUSIONS: Automatic, temperature controlled photocoagulation allows to apply predictable subvisible, mild, or moderate lesions without manual power titration. TRANSLATIONAL RELEVANCE: The technique will facilitate standardized, automatically controlled low and early treatment of diabetic retinopathy study (ETDRS) intensity photocoagulation independently of the treating physician, the treated eye and lesion location.

Comprehensive detection, grading, and growth behavior evaluation of subthreshold and low intensity photocoagulation lesions by optical coherence tomographic and infrared image analysis.

PURPOSE: To correlate the long-term clinical effect of photocoagulation lesions after 6 months, as measured by their retinal damage size, to exposure parameters. We used optical coherence tomographic (OCT)-based lesion classes in order to detect and assess clinically invisible and mild lesions. METHODS: In this prospective study, 488 photocoagulation lesions were imaged in 20 patients. We varied irradiation diameters (100/300 µm), exposure-times (20-200 ms), and power. Intensities were classified in OCT images after one hour, and we evaluated OCT and infrared (IR) images over six months after exposure. RESULTS: For six consecutive OCT-based lesion classes, the following parameters increased with the class: ophthalmoscopic, OCT and IR visibility rate, fundus and OCT diameter, and IR area, but not irradiation power. OCT diameters correlated with exposure-time, irradiation diameter, and OCT class. OCT classes discriminated the largest bandwidth of OCT diameters. CONCLUSION: OCT classes represent objective and valid endpoints of photocoagulation intensity even for "subthreshold" intensities. They are suitable to calculate the treated retinal area. As the area is critical for treatment efficacy, OCT classes are useful to define treatment intensity, calculate necessary lesion numbers, and universally categorize lesions in clinical studies.

Toll-like receptor 3 activation in retinal pigment epithelium cells - Mitogen-activated protein kinase pathways of cell death and vascular endothelial growth factor secretion.

PURPOSE: Toll-like receptor 3 (TLR3) is a receptor of the innate immune system, recognizing double-stranded RNA. TLR3 can lead to cytokine release or apoptosis and has recently been associated with the development of geographical atrophy via cytotoxic effects on the retinal pigment epithelium (RPE). The current study was conducted to elucidate the underlying pathways of TLR3 effects in the RPE. METHODS: TLR3 activation via polyinosinic acid/polycytidylic acid (Poly I:C) was investigated in primary porcine RPE cells, focussing on cell death and vascular endothelial growth factor (VEGF) secretion. Primary cells were stimulated with different concentrations of Poly I:C. Cell death was investigated in trypan blue exclusion assay and cell death detection ELISA. VEGF and IFN-ß secretion were also detected in ELISA. As Mitogen-activated protein kinases (MAPK) play an important part in TLR3-mediated signal transduction, we investigated the influence of JNK, ERK1/2 and p38 on cell death and VEGF secretion, using commercially available inhibitors. RESULTS: Activation of TLR3 by Poly I:C induced concentration-dependent cell death, partly mediated by JNK. ERK1/2 was activated and exerted some protection. Furthermore, higher concentrations of Poly I:C increased VEGF secretion after 4 and 24 hr, which was independent of MAPK. CONCLUSION: The induction of cell death in RPE cells by TLR3 activation confirms possible involvement of TLR3 activation in GA. As cell death is partly mediated by JNK, more studies should be conducted investigating the role of JNK in RPE cell death to evaluate whether its inhibition might be a new therapeutic opportunity for the treatment of geographical atrophy. Additionally, effects on VEGF secretion can be found.