Long-term in vitro monitoring of AAV-transduction efficiencies in real-time with Hoechst 33342.

Adeno-associated viral transduction allows the introduction of nucleic fragments into cells and is widely used to modulate gene expressions in vitro and in vivo. It enables the study of genetic functions and disease mechanisms and, more recently, serves as a tool for gene repair. To achieve optimal transduction performance for a given cell type, selecting an appropriate serotype and the number of virus particles per cell, also known as the multiplicity of infection, is critical. Fluorescent proteins are one of the common reporter genes to visualize successfully transduced cells and assess transduction efficiencies. Traditional methods of measuring fluorescence-positive cells are endpoint analysis by flow cytometry or manual counting with a fluorescence microscope. However, the flow cytometry analysis does not allow further measurement in a test run, and manual counting by microscopy is time-consuming. Here, we present a method that repeatedly evaluates transduction efficiencies by adding the DNA-stain Hoechst 33342 during the transduction process combined with a microscope or live-cell imager and microplate image analysis software. The method achieves fast, high-throughput, reproducible, and real-time post-transduction analysis and allows for optimizing transduction parameters and screening for a proper approach.

A simple dissection method for the isolation of mouse trabecular meshwork cells.

PURPOSE: The outflow pathway, especially trabecular meshwork (TM), plays an essential role in glaucoma, and the availability of TM cells is crucial for in vitro research. So far, the isolation of TM cells from mice has been anything but manageable due to the small size of the eye. Direct isolation using a stereomicroscope and forceps requires a high grade of dexterity. Indirect isolation is based on the phagocytic properties of TM cells and involves injecting magnetic microspheres into the anterior chamber of live mice followed by isolation. Therefore, a simpler, less expensive, and nonexperimental strategy for isolating mouse TM cells would be desirable. METHODS: After enucleation, the eyes were cut in half anterior-to-posteriorly. The lens and posterior segment were removed. Iris and the attached ciliary body were gently pulled backward and disconnected from the remaining tissue to expose the TM. By incising through the cornea anteriorly and posteriorly of the TM, the cornea/TM stripe could be isolated. The cornea/TM stripe was cultured with the pigmented side down in a 6-well. The outgrowing pigmented cells were analyzed by immunocytochemistry and mRNA expression for previously described TM cell markers. The phagocytic properties of the cells were additionally confirmed using fluorescent microspheres. RESULTS: Pigmented phagocytic cells were the first to grow out of the cornea/TM strips after approximately 4-7 days. Cells were positive for Collagen IV, Fibronectin1, Vimentin, and Actin alpha 2 and could phagocytize fluorescent microbeads. Cross-linked actin networks were visible after 9 days of exposure to TGFB2 (transforming growth factor-beta 2). Additionally, treatment with 500 nM Dexamethasone for one week increased myocilin expression, as previously reported for TM cells. In addition, we proved that this method can also be used in albino mice, which lack pigmentation of the trabecular meshwork. CONCLUSIONS: The isolated cells show phagocytic properties and specific expression of markers reported in TM cells. Therefore, our dissection-based method is inexpensive and reproducible for isolating TM cells in mice.

Investigation of OCTA Biomarkers in Fabry Disease: A Long Term Follow-Up of Macular Vessel Area Density and Foveal Avascular Zone Metrics.

INTRODUCTION: Retinal microvasculature is known to be altered in patients with Fabry disease (FD). We aimed to investigate the long-term changes in macular microvasculature and explore a reliable retinal biomarker for treatment monitoring in FD. METHODS: Prospective study of 26 eyes with FD followed up to 48 months (mean 24, range 8-48). OCT angiography (OCTA) images (2.9 × 2.9 mm) were obtained using Heidelberg Spectralis II at baseline and follow-up. Macular vessel area density (VAD, %) was measured in three layers: superficial vascular plexus (SVP), intermediate capillary plexus (ICP) and deep capillary plexus (DCP) in three peri-macular circular sectors (c1, c2, c3). Additionally, foveal avascular zone (FAZ) area (mm2) and horizontal and vertical diameters (µm) were assessed. RESULTS: VAD decreased over time in SVP, ICP (in sectors c2 and c3) and DCP (all sectors) (p < 0.04). VAD reduction was predominantly seen in treated FD patients. FAZ and horizontal diameters increased at follow-up in FD patients compared to baseline (p ≤ 0.025). Correlation analysis showed a moderate to strong negative correlation between VAD of SVP and DCP in the innermost circle and FAZ in treated patients (r = - 0.6; p < 0.0001). CONCLUSIONS: This is the first long-term follow-up OCTA study in FD to our knowledge. A decrease in VAD, pronounced in the peripheral circle and deeper layers, as well as an enlargement of the FAZ could be observed over time. These changes reflect the vascular remodelling during the course of the disease. Interestingly, the reduction of VAD was more pronounced in treated patients. This could be a result of enzyme replacement therapy and could be potentially used as a reliable biomarker for monitoring the treatment of the disease. A baseline examination of VAD and FAZ before treatment initiation is meaningful. Larger studies are needed to establish the use of VAD and FAZ as biomarkers for treatment monitoring.

Long-Term Multimodal Imaging Analysis of Selective Retina Therapy Laser Lesions.

This study evaluates the long-term effects of selective retina therapy (SRT) on the retinal pigment epithelium (RPE) and neuroretina in patients with central serous chorioretinopathy. SRT was performed on 36 patients using a Nd:YLF-Laser at 527 nm (R:GEN®, Lutronic, Goyang-Si, Republic of Korea). A total of 994 titration spots were examined using up to three years' multimodal imaging. Leakage in fluorescein angiography (FA) was observed after SRT in 523 lesions and resolved after one month. SRT lesions were not visible clinically, but appeared as brightly reflective areas in infrared and multicolor images. Normal morphology was observed in optical coherence tomography (OCT) immediately after SRT. After one month, thickening of the RPE and interdigitation zone changes were seen and disappeared after 539 ± 308 days. No RPE atrophies occurred during the observation period. Decreased fundus autofluorescence (FAF) was mostly observed directly after SRT followed by increased FAF at one month, which faded over time. A significant decrease in the number of visible lesions in the FA and FAF was observed within the three-year follow-up. OCT findings are consistent with animal studies showing SRT-related defect closure by hypertrophy and migration of neighboring cells without RPE atrophy or photoreceptor damage. This suggests that SRT is a safe treatment option for macular diseases and does not lead to retinal atrophy.

MiR-302d inhibits TGFB-induced EMT and promotes MET in primary human RPE cells.

PURPOSE: Transforming growth factor-beta (TGFB)-mediated epithelial-mesenchymal transition (EMT) plays a crucial role in the pathogenesis of retinal fibrosis, which is one of the leading causes of impaired vision. Current approaches to treating retinal fibrosis focus, among other things, on inhibiting the TGFB signaling pathway. Transient expression of microRNAs (miRNAs) is one way to inhibit the TGFB pathway post-transcriptionally. Our previous study identified the miRNA miR-302d as a regulator of multiple TGFB-related genes in ARPE-19 cells. To further explore its effect on primary cells, the effect of miR-302d on TGFB-induced EMT in primary human retinal pigment epithelium (hRPE) was investigated in vitro. METHODS: hRPE cells were extracted from patients receiving enucleation. Transfection of hRPE cells with miR-302d was performed before or after TGFB1 stimulation. Live-cell imaging, immunocytochemistry staining, Western blot, and ELISA assays were utilized to identify the alterations of cellular morphology and EMT-related factors expressions in hRPE cells. RESULTS: hRPE cells underwent EMT by TGFB1 exposure. The transfection of miR-302d inhibited the transition with decreased production of mesenchymal markers and increased epithelial factors. Meanwhile, the phosphorylation of SMAD2 activated by TGFB1 was suppressed. Moreover, miR-302d expression promoted TGFB1-induced fibroblast-like hRPE cells to revert towards an epithelial stage. As confirmed by ELISA, miR-302d reduced TGFB receptor 2 (TGFBR2) and vascular endothelial growth factor A (VEGFA) levels 48 hours after transfection. CONCLUSIONS: The protective effect of miR-302d might be a promising approach for ameliorating retinal fibrosis and neovascularization. MiR-302d suppresses TGFB-induced EMT in hRPE cells via downregulation of TGFBR2, even reversing the process. Furthermore, miR-302d reduces the constitutive secretion of VEGFA from hRPE cells.

Changes in Intraocular Pressure following Narcosis With Medetomidine, Midazolam, and Fentanyl in Association With Initial Intraocular Pressure in Mice.

PURPOSE: This article describes the development of decreased intraocular pressure (IOP) under general anesthesia with medetomidine, midazolam, and fentanyl in mice with normal and elevated IOP. METHODS: IOP was measured using the iCare Tonolab rebound tonometer. Twelve 3-4 months-old male and female C57BL/6J mice were randomized to a control group with physiological IOP and a high IOP group with experimentally induced ocular hypertension using tarsal injections of dexamethasone-21-acetate. For anesthesia, medetomidine and midazolam were used, subgroups additionally received fentanyl. IOP was measured every 2.5 min for 30 min. RESULTS: Control group differed with 14.89 mmHg (SEM: 0.58) significantly (p = 0.0002) from the high IOP group with initial 20.44 mmHg (SEM: 0.75). All groups showed a significant (p < 0.05) decrease in IOP under general anesthesia. There was no significant difference in IOP development and decrease between the group additionally receiving fentanyl and the group without fentanyl. The decrease in IOP was highly dependent on the initial value, with the high IOP group showing a greater decrease. After 10 min, no significant difference in IOP could be detected between the high IOP and control group. CONCLUSIONS: In mice, general anesthesia with medetomidine and midazolam leads to a declining IOP over time. Adding fentanyl to the anesthesia did not alter these effects. The decline is time-dependent and IOP-dependent.

Self-Examination Low-Cost Full-Field Optical Coherence Tomography (SELFF-OCT) for neovascular age-related macular degeneration: a cross-sectional diagnostic accuracy study.

OBJECTIVES: Self-Examination Low-Cost Full-Field Optical Coherence Tomography (SELFF-OCT) is a novel OCT technology that was specifically designed for home monitoring of neovascular age-related macular degeneration (AMD). First clinical findings have been reported before. This trial investigates an improved prototype for patients with AMD and focusses on device operability and diagnostic accuracy compared with established spectral-domain OCT (SD-OCT). DESIGN: Prospective single-arm diagnostic accuracy study. SETTING: Tertiary care centre (University Eye Clinic). PARTICIPANTS: 46 patients with age-related macular degeneration. INTERVENTIONS: Patients received short training in device handling and then performed multiple self-scans with the SELFF-OCT according to a predefined protocol. Additionally, all eyes were examined with standard SD-OCT, performed by medical personnel. All images were graded by at least 2 masked investigators in a reading centre. PRIMARY OUTCOME MEASURE: Rate of successful self-measurements. SECONDARY OUTCOME MEASURES: Sensitivity and specificity of SELFF-OCT versus SD-OCT for different biomarkers and necessity for antivascular endothelial growth factor (anti-VEGF) treatment. RESULTS: In 86% of all examined eyes, OCT self-acquisition resulted in interpretable retinal OCT volume scans. In these patients, the sensitivity for detection of anti-VEGF treatment necessity was 0.94 (95% CI 0.79 to 0.99) and specificity 0.95 (95% CI 0.82 to 0.99). CONCLUSIONS: SELFF-OCT was used successfully for retinal self-examination in most patients, and it could become a valuable tool for retinal home monitoring in the future. Improvements are in progress to reduce device size and to improve handling, image quality and success rates. TRIAL REGISTRATION NUMBER: DRKS00013755, CIV-17-12-022384.

Algorithms for optoacoustically controlled selective retina therapy (SRT).

OBJECTIVES: Selective Retina Therapy (SRT) uses microbubble formation (MBF) to target retinal pigment epithelium (RPE) cells selectively while sparing the neural retina and the choroid. Intra- and inter-individual variations of RPE pigmentation makes frequent radiant exposure adaption necessary. Since selective RPE cell disintegration is ophthalmoscopically non-visible, MBF detection techniques are useful to control adequate radiant exposures. It was the purpose of this study to evaluate optoacoustically based MBF detection algorithms. METHODS: Fifteen patients suffering from central serous chorioretinopathy and diabetic macula edema were treated with a SRT laser using a wavelength of 527 nm, a pulse duration of 1.7 µs and a pulse energy ramp (15 pulses, 100 Hz repetition rate). An ultrasonic transducer for MBF detection was embedded in a contact lens. RPE damage was verified with fluorescence angiography. RESULTS: An algorithm to detect MBF as an indicator for RPE cell damage was evaluated. Overall, 4646 irradiations were used for algorithm optimization and testing. The tested algorithms were superior to a baseline model. A sensitivity/specificity pair of 0.96/1 was achieved. The few false algorithmic decisions were caused by unevaluable signals. CONCLUSIONS: The algorithm can be used for guidance or automatization of microbubble related treatments like SRT or selective laser trabeculoplasty (SLT).

Selective retina therapy and thermal stimulation of the retina: different regenerative properties - implications for AMD therapy.

BACKGROUND: Selective Retina Therapy (SRT), a photodisruptive micropulsed laser modality that selectively destroys RPE cells followed by regeneration, and Thermal Stimulation of the Retina (TSR), a stimulative photothermal continuous wave laser modality that leads to an instant sublethal temperature increase in RPE cells, have shown therapeutic effects on Age-related Macular Degeneration (AMD) in mice. We investigate the differences between both laser modalities concerning RPE regeneration. METHODS: For PCR array, 6 eyes of murine AMD models, apolipoprotein E and nuclear factor erythroid-derived 2- like 2 knock out mice respectively, were treated by neuroretina-sparing TSR or SRT. Untreated litter mates were controls. Eyes were enucleated either 1 or 7 days after laser treatment. For morphological analysis, porcine RPE/choroid organ cultures underwent the same laser treatment and were examined by calcein vitality staining 1 h and 1, 3 or 5 days after irradiation. RESULTS: TSR did not induce the expression of cell-mediators connected to cell death. SRT induced necrosis associated cytokines as well as inflammation 1 but not 7 days after treatment. Morphologically, 1 h after TSR, there was no cell damage. One and 3 days after TSR, dense chromatin and cell destruction of single cells was seen. Five days after TSR, there were signs of migration and proliferation. In contrast, 1 h after SRT a defined necrotic area within the laser spot was seen. This lesion was closed over days by migration and proliferation of adjacent cells. CONCLUSIONS: SRT induces RPE cell death, followed by regeneration within a few days. It is accompanied by necrosis induced inflammation, RPE proliferation and migration. TSR does not induce immediate RPE cell death; however, migration and mitosis can be seen a few days after laser irradiation, not accompanied by necrosis-associated inflammation. Both might be a therapeutic option for the treatment of AMD.

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.

Self-examination low-cost full-field OCT (SELFF-OCT) for patients with various macular diseases.

PURPOSE: The treatment guidelines for many macular diseases rely on frequent monitoring with optical coherence tomography (OCT). However, the burden of frequent disease control leads to low therapy adherence in real life. OCT home monitoring would address this issue but requires an inexpensive and self-operable device. With self-examination low-cost full-field OCT (SELFF-OCT), our group has introduced a novel technology that may fulfill both requirements. In this pilot study, we report the initial experiences with a clinical prototype. METHODS: Fifty-one patients with different macular diseases were recruited in a cross-sectional study. The most common diseases were age-related macular degeneration (AMD; 39/51), diabetic macular edema (DME; 6/51), and retinal vein occlusion (RVO; 3/51). Patients received a short training in device usage and then performed multiple self-scans with the SELFF-OCT device. For comparison, scans with a standard clinical spectral domain (SD-)OCT were taken. RESULTS: After a brief training, 77% of the patients were able to successfully acquire images that were clinically gradable. No significant influence on success could be found for age (p = 0.08) or BCVA (p = 0.97). Relevant disease biomarkers in the most common retinal diseases could be detected. CONCLUSIONS: SELFF-OCT was used successfully for retinal self-examination and in the future could be used for retinal home monitoring. Future improvements in technology are expected to improve success rates and image quality. TRIAL REGISTRATION: The Trial was registered in the German Trial Register under the number DRKS00013755 on 14.03.2018.

Emulsified silicone oil is taken up by and induces pro-inflammatory response in primary retinal microglia.

PURPOSE: Silicone oil is used as endotamponade in combination with vitrectomy. Thinning of retinal layers and loss of retinal cells under silicone oil use have been found. Here, we investigate the influence of silicone oil on primary microglia cells. METHODS: Primary microglia cells were prepared from the porcine retina. Microglia identity was assessed with Iba1 staining. Silicone oil was emulsified by sonification. Cell morphology and silicone oil uptake were evaluated by light microscopy after Coomassie blue staining. Cytokine secretion was evaluated with ELISA. Toxicity of silicone oil on microglia and toxic effect of silicone oil-treated microglia on neuronal cell line PC12 were evaluated by MTT or WST assay, respectively. RESULTS: Microglia took up silicone oil droplets after 72 h of incubation. Silicone oil induced no toxicity but increased the metabolism in microglial cells. In addition, the secretion of IL-6 and IL-8, but not of IL-1ß or TNF-α, was induced. Silicone oil-treated microglia did not exert any neurotoxic effect on differentiated PC12 cells but induced an increase in metabolism. CONCLUSION: Emulsified silicone oil changes the activity level of microglia and induces the secretion of IL-6 and IL-8. Neurotoxicity is not induced. Further experiments are required to investigate the long-term effect of silicone oil on microglia and their consequent effect on neuronal cells.

CRB1rd8 mutation influences the age-related macular degeneration phenotype of NRF2 knockout mice and favors choroidal neovascularization.

PURPOSE: We examined the influence of retinal degeneration 8 (rd8) mutation of crumbs homolog 1 (CRB1) gene on age-related macular degeneration (AMD) phenotype in nuclear factor E2-related factor 2 knock out (NRF2-/-) mouse model. METHODS: CRB1rd8 mutation genotype was determined by polymerase chain reaction from tail clips in 73 NRF2-/- mice originating from C57BL/6J background on mixed C57BL/6J and C57BL/6N ancestry. The clinical grade of AMD-like fundus alterations was determined by funduscopy, optical coherence tomography (OCT) and fluorescein angiography (FLA) at the age of 9 or 12 months. RESULTS: Twelve NRF2-/- mice were wildtype CRB1+/+, 61 NRF2-/- were homozygous CRB1rd8/rd8. NRF2-/-CRB1rd8/rd8 mice had a significantly higher probability to show an advanced grade (grade 4 and 5) of AMD-like fundus alterations known to appear in NRF2-/- mice. Choroidal neovascularization (CNV) was only detected in NRF2-/-CRB1rd8/rd8 homozygous mice. CONCLUSIONS: Homozygous CRB1rd8/rd8 mutation is common in commercial vendor mice strains of C57BL/6J origin if partly on C57BL/6N ancestry. The mutation has an influence on the extent of AMD-like retinal alterations in NRF2-/- mice and favors CNV formation.

Modulation of inflammatory processes by thermal stimulating and RPE regenerative laser therapies in age related macular degeneration mouse models.

PURPOSE: Inflammatory processes play a major role within the multifactorial pathogenesis of age-related macular degeneration (AMD). Neuroretina sparing laser therapies, thermal stimulation of the retina (TSR) and selective retina therapy (SRT), are known to reduce AMD-like pathology in vitro and in vivo. We investigated the effect of TSR and SRT on inflammatory processes in AMD mouse models. METHODS: One randomized eye of 8 months old apolipoprotein (Apo)E and 9 months old nuclear factor (erythroid-derived 2) -like 2 (NRF2) knock out mice were treated by TSR (10 ms, 532 nm, 50 µm2 spot size, mean 4.5 W, ~200 spots) or SRT (~1.4 µs pulses, 532 nm, 50 µm spot size, 100 Hz over 300 ms, mean 2.5 µJ per pulse, ~200 spots). Fellow eyes, untreated knock out mice and wild-type BL/6J mice acted as controls. All mice were examined funduscopically and by optical coherence tomography (OCT) at the day of laser treatment. Mice were euthanized and enucleated either 1 day or 7 days after laser treatment and examined by gene expression analysis of 84 inflammatory genes. RESULTS: The inflammatory gene expression profile of both knock out models compared to healthy BL/6J mice suggests a regulation of pro- and anti-inflammatory processes especially concerning T-cell activity and immune cell recruitment. TSR resulted in downregulation of several pro-inflammatory cell-mediators both in ApoE -/- and NRF2-/- mice compared to treatment naïve litter mates one day after treatment. In contrast, SRT induced pro-inflammatory cell-mediators connected with necrosis one day after treatment as expected following laser-induced selective RPE cell death. Seven days after laser treatment, both findings were reversed. CONCLUSIONS: Both TSR and SRT influence inflammatory processes in AMD mouse models. However, they act conversely. TSR leads to anti-inflammatory processes shortly after laser therapy and induces immune-cell recruitment one week after treatment. SRT leads to a quick inflammatory response to laser induced RPE necrotic processes. One week after SRT inflammation is inhibited. It remains unclear, if and to what extent this might play a role in a therapeutic or preventive approach of both laser modalities on AMD pathology.

[Effect of Selective Retina Therapy (SRT) on the Inflammatory Microenvironment of the Subretinal Space]. / Einfluss der Selektiven Retinatherapie (SRT) auf inflammatorische Zellmediatoren des subretinalen Raums.

PURPOSE: To investigate the effect of Selective Retina Therapy (SRT) on inflammatory key factors such as complement factor-C3 (CC3), tumor growth factor-beta2 (TGF-ß2), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). MATERIALS AND METHODS: Porcine RPE-Bruch's membrane-choroid explants were irradiated with two SRT laser systems, SRTYLF and SRTYAG (Nd : YLF laser, wave length 527 nm, pulse duration 1.7 µs and Nd : YAG laser, wave length 532 nm, pulse duration 2.4 - 3 µs). Laser irradiation was performed on a spot size of 200 × 200 µm, 30 pulses, with a repetition rate of 100 Hz, and a radiant exposure of 140 (threshold RPE death) and 180 mJ/cm2 per pulse (above threshold RPE death). Explants were cultivated in modified Ussing chambers and culture viability was assessed by calcein-AM cell staining. Secretion of inflammatory factors was analyzed by ELISA. Protein expression of tissue explants was assessed by Western blot. RESULTS: Regeneration of RPE was observed after 4 days. One day after SRT with 140 mJ/cm2 per pulse the secretion of basal CC3 decreased in ELISA. Following 180 mJ/cm2 radiant exposure, the level of IFN-γ decreased at day 4. CONCLUSION: SRT does not induce the release of the pro-inflammatory factors analyzed in this in-vitro study.

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.

Selective retina therapy: toward an optically controlled automatic dosing.

Selective retina therapy (SRT) targets the retinal pigment epithelium (RPE) with pulsed laser irradiation by inducing microbubble formation (MBF) at the intracellular melanin granula, which leads to selective cell disruption. The following wound healing process rejuvenates the chorio-retinal junction. Pulse energy thresholds for selective RPE effects vary intra- and interindividually. We present the evaluation of an algorithm that processes backscattered treatment light to detect MBF as an indicator of RPE cell damage since these RPE lesions are invisible during treatment. Eleven patients with central serous chorioretinopathy and four with diabetic macula edema were treated with a SRT system, which uses a wavelength of 527 nm, a repetition rate of 100 Hz, and a pulse duration of 1.7 µs. Fifteen laser pulses with stepwise increasing pulse energy were applied per treatment spot. Overall, 4626 pulses were used for algorithm parameter optimization and testing. Sensitivity and specificity were the metrics maximized through an automatic optimization process. Data were verified by fluorescein angiography. A sensitivity of 1 and a specificity of 0.93 were achieved. The method introduced in this paper can be used for guidance or automatization of microbubble-related treatments like SRT or selective laser trabeculoplasty.

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.