Regenerative Retinal Laser and Light Therapies (RELITE): Proposal of a New Nomenclature, Categorization, and Trial Reporting Standard.

OBJECTIVES: Numerous laser and light therapies have been developed to induce regenerative processes in the choroid/retinal pigment epithelium (RPE)/photoreceptor complex, leaving the neuroretina undamaged. These therapies are applied to the macula for the treatment of various diseases, most prominently diabetic maculopathy, retinal vein occlusion, central serous chorioretinopathy, and age-related macular degeneration. However, the abundance of technologies, treatment patterns, and dosimetry protocols has made understanding these therapies and comparing different approaches increasingly complex and challenging. To address this, we propose a new nomenclature system with a clear categorization that will allow for better understanding and comparability between different laser and light modalities. We propose this nomenclature system as an open standard that may be adapted in future toward new technical developments or medical advancements. METHODS: A systematic literature review of reported macular laser and light therapies was conducted. A categorization into a standardized system was proposed and discussed among experts and professionals in the field. This paper does not aim to assess, compare, or evaluate the efficacy of different laser or dosimetry techniques or treatment patterns. RESULTS: The literature search yielded 194 papers describing laser techniques, 50 studies describing dosimetry, 272 studies with relevant clinical trials, and 82 reviews. Following the common therapeutic aim, we propose "regenerative retinal laser and light therapies (RELITE)" as the general header. We subdivided RELITE into four main categories that refer to the intended physical and biochemical effects of temperature increase (photothermal therapy, PTT), RPE regeneration (photomicrodisruption therapy, PMT), photochemical processes (photochemical therapy, PCT), and photobiomodulation (photobiomodulation therapy, PBT). Further, we categorized the different dosimetry approaches and treatment regimens. We propose the following nomenclature system that integrates the most important parameters to enable understanding and comparability: Pattern-Dosimetry-Exposure Time/Frequency, Duty Cycle/Irradiation Diameter/Wavelength-Subcategory-Category. CONCLUSION: Regenerative retinal laser and light therapies are widely used for different diseases and may become valuable in the future. A precise nomenclature system and strict reporting standards are needed to allow for a better understanding, reproduceable and comparable clinical trials, and overall acceptance. We defined categories for a systematic therapeutic goal-based nomenclature to facilitate future research in this field.

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.

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.

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.

[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.

Mechano- and thermosensitivity of injured muscle afferents 20 to 80 days after nerve injury.

Chronic injury of limb nerves leading to neuropathic pain affects deep somatic nerves. Here the functional properties of injured afferent fibers in the lateral gastrocnemius-soleus nerve were investigated 20 and 80 days after suturing the central stump of this muscle nerve to the distal stump of the sural nerve in anesthetized rats. Neurophysiological recordings were made from afferent axons identified in either the sciatic nerve (87 A-, 63 C-fibers) or the dorsal root L4/L5 (52 A-, 26 C-fibers) by electrical stimulation of the injured nerve. About 70% of the functionally identified A-fibers had regenerated into skin by 80 days after nerve suture; the remaining A-fibers could be activated only from the injured nerve. In contrast, 93% of the functionally identified C-fibers could only be activated from the injured sural nerve after 80 days. Nearly half of the injured A- (45%) and C-fibers (44%) exhibited ongoing and/or mechanically or thermally evoked activity. Because ~50% of the A- and C-fibers are somatomotor or sympathetic postganglionic axons, respectively, probably all injured muscle afferent A- and C-fibers developed ectopic activity. Ongoing activity was present in 17% of the A- and 46% of the C-fibers. Mechanosensitivity was present in most injured A- (99%) and C-fibers (85%), whereas thermosensitivity was more common in C-fibers (cold 46%, heat 47%) than in A-fibers (cold 18%, heat 12%). Practically all thermosensitive A-fibers and C-fibers were also mechanosensitive. Thus, unlike cutaneous axons, almost all A- and C-fibers afferents in injured muscle nerves demonstrate ectopic activity, even chronically after nerve injury. NEW & NOTEWORTHY After chronic injury of a muscle nerve, allowing the nerve fibers to regenerate to the target tissue, 1) most afferent A-fibers are mechanosensitive and regenerate to the target tissue; 2) ectopic ongoing activity, cold sensitivity, and heat sensitivity significantly decrease with time after injury in A-afferents; 3) most afferent C-fibers do not regenerate to the target tissue; and 4) injured C-afferents maintain the patterns of ectopic discharge properties they already show soon after nerve injury.

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.

Thinning of Inner Retinal Layers after Vitrectomy with Silicone Oil versus Gas Endotamponade in Eyes with Macula-Off Retinal Detachment.

PURPOSE: To evaluate retinal layer thickness with optical coherence tomography (OCT) in eyes with macula-off retinal detachment after silicone oil (SiO) or gas endotamponade. PROCEDURES: Cross-sectional study of 40 eyes with macula-off rhegmatogenous retinal detachment that underwent vitrectomy. 20 eyes received SiO tamponade and 20 matched eyes received gas. 33 healthy fellow eyes served as controls. Macular spectral domain OCT was performed with automated layer detection in the 5 inner subfields of the Early Treatment Diabetic Retinopathy Study (ETDRS) map. RESULTS: Comparing the SiO group with the gas group, the ganglion cell layer showed a significant thinning in all fields of the inner ring of the ETDRS map, the inner plexiform layer in the nasal, superior and temporal quadrants, and the outer plexiform layer in the nasal quadrant. CONCLUSIONS: Inner retinal layers in the fovea/parafovea were significantly thinner in the SiO group. Prospective studies are warranted to further elucidate possible retinal adverse effects of SiO tamponade.

Vision loss under silicone oil tamponade.

PURPOSE: We aimed to investigate frequency, time course and pathophysiology of vision loss in eyes with macula-on rhegmatogenous retinal detachment operated with vitrectomy and silicone oil tamponade. PATIENTS AND METHODS: Fifteen eyes of 15 patients who had been operated with 5,000 centistoke silicone oil between 2006 and 2014 were included in a retrospective case series. Examinations included logMAR best corrected visual acuity (BCVA), visual field testing (VF), spectral domain optical coherence tomography (OCT), electrophysiology, and fluorescein angiography. RESULTS: Vision loss was seen in eight (53 %) eyes of 15 patients with symptomatic central scotoma, which was confirmed by VF (5/6). Preoperative median BCVA of these patients was 0.15 (0.5 to 0), prior to oil removal 0.7 (1.0 to 0.5), and 6 weeks post oil removal 1.0 (1.5 to 0.2). BCVA recovered in five patients to a median of 0.15 (0.5 to 0.1), and it remained 1.0 in three (20 %) out of 15 eyes. OCT revealed significant thinning of the foveal and parafoveal combined nerve fiber, ganglion cell and inner plexiform layers in affected eyes (mean 58.3 µm +/-13, horizontal scan through fovea, 500 µm radius) compared to their healthy fellow eyes (mean 84.5 µm +/-12.3; p < 0.01, n = 6 patients, 12 eyes) and compared to eyes with no vision loss under silicone oil. CONCLUSIONS: We find persisting vision loss in three out of 15 patients treated for macula-on rhegmatogenous retinal detachment with silicone oil tamponade. Thinning of inner retinal layers possibly evoked by silicone oil tamponade might be a pathophysiological explanation for vision loss in these patients.

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.

In vitro comparison of human and murine trabecular meshwork cells: implications for glaucoma research.

The trabecular meshwork (TM) is crucial for regulating intraocular pressure (IOP), and its dysfunction significantly contributes to glaucoma, a leading cause of vision loss and blindness worldwide. Although rodents are commonly used as animal models in glaucoma research, the applicability of these findings to humans is limited due to the insufficient understanding of murine TM. This study aimed to compare primary human TM (hTM) and murine TM (mTM) cells in vitro to enhance the robustness and translatability of murine glaucoma models. In this in vitro study, we compared primary hTM and mTM cells under simulated physiological and pathological conditions by exposing both cell types to the glucocorticoid dexamethasone (DEX) and Transforming Growth Factor ß (TGFB2), both of which are critical in the pathogenesis of several ophthalmological diseases, including glaucoma. Phagocytic properties were assessed using microbeads. Cells were analyzed through immunocytochemistry (ICC) and Western blot (WB) to evaluate the expression of extracellular matrix (ECM) components, such as Fibronectin 1 (FN1) and Collagen IV (COL IV). Filamentous-Actin (F-Act) staining was used to analyze cross-linked actin network (CLAN) formation. Additionally, we evaluated cytoskeletal components, including Vimentin (VIM), Myocilin (MYOC), and Actin-alpha-2 (ACTA2). Our results demonstrated significant similarities between human and murine TM cells in basic morphology, phagocytic properties, and ECM and cytoskeletal component expression under both homeostatic and pathological conditions in vitro. Both human and murine TM cells exhibited epithelial-to-mesenchymal transition (EMT) after exposure to DEX or TGFB2, with comparable CLAN formation observed in both species. However, there were significant differences in FN1 and MYOC induction between human and murine TM cells. Additionally, MYOC expression in hTM cells depended on fibronectin coating. Our study suggests that murine glaucoma models are potentially translatable to human TM. The observed similarities in ECM and cytoskeletal component expression and the comparable EMT response and CLAN formation support the utility of murine models in glaucoma research. The differences in FN1 and MYOC expression between hTM and mTM warrant further investigation due to their potential impact on TM properties. Overall, this study provides valuable insights into the species-specific characteristics of TM and highlights opportunities to refine murine models for better relevance to human glaucoma.

Understanding the Ocular Hypertension Model in Mice Induced by Dexamethasone-21-Acetate - Implications for Glaucoma Research.

PURPOSE: This study aimed to assess the effectiveness of monocular and bilateral injections of Dexamethasone-21-acetate (Dex-21-Ac) into the murine fornix twice a week as a glucocorticoid-induced ocular hypertension model and investigated potential systemic side effects. METHODS: Dex-21-Ac was administered twice weekly in three groups: bilateral injections, monocular injections, and a control group receiving the vehicle solution bilateral. After 21 days, enucleated eyes were examined using immunocytochemistry (ICC), and organ histology was performed. RESULTS: All groups receiving Dex-21-Ac injections had a significant increase in intraocular pressure (IOP). Monocular injections also resulted in a significant increase in IOP in the fellow eye. The Dex-21-Ac-treated groups showed a bilateral increase in IOP of approximately 8 mmHg, accompanied by elevated expression of alpha smooth muscle actin and fibronectin in the anterior chamber angle. There were no significant changes in weight progression. Hepatic steatosis was observed in all Dex-21-Ac-treated animals, and some suffered from residual neuromuscular blockade under fentanyl anesthesia. CONCLUSION: Bilateral injections of Dex-21-Ac twice a week lead to a significant increase in daytime IOP and fibrotic changes in the trabecular meshwork. Unilateral application has a significant impact on the fellow eye. Local dexamethasone leads to notable systemic effects independent of changes in animal weight. Considering liver damage and associated influence on metabolization, hepatically eliminated injection anesthetics may lead to overdosing and are not recommended. They should be replaced by inhalation anesthesia.

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.

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.

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.

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.

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).