VEGF-A165a and angiopoietin-2 differently affect the barrier formed by retinal endothelial cells.

Exposure to VEGF-A165a over several days leads to a persistent dysfunction of the very tight barrier formed by immortalized endothelial cells of the bovine retina (iBREC). Elevated permeability of the barrier is indicated by low cell index values determined by electric cell-substrate impedance measurements, by lower amounts of claudin-1, and by disruption of the homogenous and continuous staining of vascular endothelial cadherin at the plasma membrane. Because of findings that suggest modulation of VEGF-A's detrimental effects on the inner blood-retina barrier by the angiogenic growth factor angiopoietin-2, we investigated in more detail in vitro whether this growth factor indeed changes the stability of the barrier formed by retinal endothelial cells or modulates effects of VEGF-A. In view of the clinical relevance of anti-VEGF therapy, we also studied whether blocking VEGF-A-driven signaling is sufficient to prevent barrier dysfunction induced by a combination of both growth factors. Although angiopoietin-2 stimulated proliferation of iBREC, the formed barrier was not weakened at a concentration of 3 nM: Cell index values remained high and expression or subcellular localization of claudin-1 and vascular endothelial cadherin, respectively, were not affected. Angiopoietin-2 enhanced the changes induced by VEGF-A165a and this was more pronounced at lower concentrations of VEGF-A165a. Specific inhibition of the VEGF receptors with tivozanib as well as interfering with binding of VEGF-A to its receptors with bevacizumab prevented the detrimental effects of the growth factors; dual binding of angiopoietin-2 and VEGF-A by faricimab was marginally more efficient. Uptake of extracellular angiopoietin-2 by iBREC can be efficiently prevented by addition of faricimab which is also internalized by the cells. Exposure of the cells to faricimab over several days stabilized their barrier, confirming that inhibition of VEGF-A signaling is not harmful to this cell type. Taken together, our results confirm the dominant role of VEGF-A165a in processes resulting in increased permeability of retinal endothelial cells in which angiopoietin-2 might play a minor modulating role.

Laser Treatment of Central Serous Chorioretinopathy - An Update.

Laser treatment has been a mainstay for management of central serous chorioretinopathy for a few decades. Different types of lasers have been used and non-damaging retinal laser is the most recent option. The aim of this review is to provide an update on this form of treatment, based on the research published during last 5 years, in comparison with earlier studies published. A MEDLINE database search was performed with a combination of the following terms: central serous chorioretinopathy and laser photocoagulation or subthreshold laser or subthreshold micropulse laser or nanosecond laser or microsecond laser or end-point management or photodynamic therapy. Results were analyzed separately for each modality of laser treatment. Reports published in recent years confirm findings of previous research and do not distinguish treatments of this clinical entity. Among all analyzed laser options, photodynamic therapy provides the fastest and most prominent morphological improvements, including subretinal fluid resorption and reduction of choroidal thickness. This modality is also associated with fewer recurrences than with other treatments. Subthreshold micropulse laser allows the physician to maintain and, in selected cases, improve the patient's vision. Conventional photocoagulation is still effective, especially with the introduction of navigated laser systems. Despite the availability of variable laser treatment options, long-term functional improvements in chronic cases are minor for each modality. Long-lasting central serous chorioretinopathy cases with significantly altered retinal morphology do not usually present with functional improvement, despite satisfactory morphological outcomes. Early initiation of treatment has the potential to prevent visual loss and to improve the patient's quality of life.

Comparison of vascular risk profile and clinical outcomes among patients with central (branch) retinal artery occlusion versus amaurosis fugax.

BACKGROUND: Retinal artery occlusions lead to sudden, painless vision loss, affecting millions globally. Despite their significance, treatment strategies remain unestablished, contrasting with acute ischemic stroke (AIS), where IVT has proven efficacy. Similar to AIS, retinal artery occlusions demand urgent evaluation and treatment, reflecting the principle "time is retina". Even for patients with transient monocular vision loss, also known as amaurosis fugax (AF), pertinent guidelines meanwhile recommend immediate emergency assessment in a specialized facility. However, data on the clinical benefit and comparability with persistent occlusions are missing. This study aimed to compare the results of a comprehensive stroke-workup among patients with persistent retinal artery occlusions (RAO), including both central retinal (CRAO) and branch retinal artery occlusion (BRAO) and those with AF. METHODS: Conducted at the University Hospital Giessen, Germany, this exploratory cross-sectional study enrolled patients with transient or permanent unilateral vision loss of non-arteritic origin. The primary outcome were differences between the two groups RAO and AF with regard to cardiovascular risk profiles and comorbidities, vascular and pharmacological interventions and clinical neurological and ophthalmological outcomes. Secondary outcome was a sub-group analysis of patients receiving IVT. RESULTS: Out of 166 patients assessed, 76 with RAO and 40 with AF met the inclusion criteria. Both groups exhibited comparable age, gender distribution, and cardiovascular risk profiles. Notably, RAO patients did not show significantly more severe vascular comorbidities than AF patients. However, AF patients received vascular interventions more frequently. Pharmacological intervention rates were similar across groups. RAO patients had slightly worse neurological outcomes, and IVT did not yield favorable ophthalmological outcomes within any observed patients. CONCLUSION: The study found similar vascular burden and risk factors in patients with RAO and AF, with implications for clinical workflows. IVT for RAO may only be effective in very early treatment windows. This emphasizes the need for public awareness and collaborative protocols between ophthalmologists and neurologists to improve outcomes.

Feasibility of Direct Vitrectomy-Sparing Subretinal Injection for Gene Delivery in Large Animals.

PURPOSE: To assess the safety and feasibility of direct vitrectomy-sparing subretinal injection for gene delivery in a large animal model. METHODS: The experimental Libechov minipigs were used for subretinal delivery of a plasmid DNA vector (pS/MAR-CMV-copGFP) with cytomegalovirus (CMV) promoter, green fluorescent protein (GFP) reporter (copGFP) and a scaffold/matrix attachment region (S/MAR) sequence. The eyes were randomized to subretinal injection of the vector following pars plana vitrectomy (control group) or a direct injection without prior vitrectomy surgery (experimental group). Intra- and post-operative observations up to 30 days after surgery were compared. RESULTS: Six eyes of three mini-pigs underwent surgery for delivery into the subretinal space. Two eyes in the control group were operated with a classical approach (lens-sparing vitrectomy and posterior hyaloid detachment). The other four eyes in the experimental group were injected directly with a subretinal cannula without vitrectomy surgery. No adverse events, such as endophthalmitis, retinal detachment and intraocular pressure elevation were observed post-operatively. The eyes in the experimental group had both shorter surgical time and recovery while achieving the same surgical goal. CONCLUSIONS: This pilot study demonstrates that successful subretinal delivery of gene therapy vectors is achievable using a direct injection without prior vitrectomy surgery.

Clinical significance of signal shadowing during intraoperative optical coherence tomography-assisted vitreoretinal surgery.

This study aimed to analyze the clinical significance of signal shadowing during intraoperative optical coherence tomography (iOCT)-assisted vitreoretinal surgery caused by vitreoretinal instruments, tissue dyes, and vitreous substitutes, and to objectively quantify its impact on iOCT imaging. This is a retrospective observational study of postoperative image analysis from one hundred seventeen (117) patients who underwent iOCT-assisted vitrectomy. The image data were divided into three groups: vitreoretinal instruments, tissue dyes, and vitreous substitutes. The data was then processed using graphic software to measure the grade of picture quality distortion and compared to paired image controls without clinically perceptive interference, then analyzed statistically. The intraocular portion of all studied vitreoretinal instruments caused a high average gray level interference compared to controls ranging from 32 to 68% reduction, obscuring the area of interest significantly. The tips of the instruments produced low-grade shadowing, allowing the underlying tissue to be distinguished. The analyzed dyes demonstrated a wide interference range: ICG (- 75.12%), and triamcinolone (- 26.13%) showed dose-dependent high shadowing, while VITREODYNE™ (49.3%) and brilliant blue G (14.06%) exhibited no perceived distortions whilst increasing average gray levels. All analyzed vitreous substitutes (air, SF6, C3F8, PFCL, and silicone oil) showed an insignificant shadowing effect on iOCT. Certain dyes and vitreous substitutes produce a negligible shadowing effect compared to controls and other dyes, providing an advantage during real-time iOCT imaging. All analyzed vitreoretinal instruments showed a significant interference that should prompt the development of new imaging techniques or the implementation of materials with low-grade interference to overcome a clinically relevant shadowing effect on iOCT, maximizing the technology's visual accuracy and surgical diagnostic aid proficiency.

Progress in Stem Cells-Based Replacement Therapy for Retinal Pigment Epithelium: In Vitro Differentiation to In Vivo Delivery.

Retinal pigment epithelium (RPE) is a critical cell monolayer forming the blood-retina-barrier (BRB) and a permeable bridge between the choriocapillaris and the retina. RPE is also crucial in maintaining photoreceptor function and for completing the visual cycle. Loss of the RPE is associated with the development of degenerative diseases like age-related macular degeneration (AMD). To treat diseases like AMD, pluripotent stem cell-derived RPE (pRPE) has been recently explored extensively as a regenerative module. pRPE like other ectodermal tissues requires specific lineage differentiation and long-term in vitro culturing for maturation. Therefore, understanding the differentiation process of RPE could be useful for stem cell-based RPE derivation. Developing pRPE-based transplants and delivering them into the subretinal space is another aspect that has garnered interest in the last decade. In this review, we discuss the basic strategies currently employed for stem cell-based RPE derivation, their delivery, and recent clinical studies related to pRPE transplantation in patients. We have also discussed a few limitations with in vitro RPE culture and potential solutions to overcome such problems which can be helpful in developing functional RPE tissue.

Macular hole Delphi consensus statement (MHOST).

PURPOSE: To derive a Delphi method-based consensus for the surgical management of Full Thickness Macular Hole (FTMH) and Lamellar Macular Hole (LMH). METHODS: 37 expert VR surgeons from 21 mainly European countries participated in Delphi method-based questionnaire for diagnosis and treatment of FTMHs and LMHs. RESULTS: A total of 36 items were rated in round 1 by 37 participants, of which 10 items achieved consensus: intraoperative verification of PVD; clinical superiority of OCT-based FTMH classification; practical ineffectiveness of ocriplasmin; circular 360° ILM peeling for small macular holes; use of regular surgical technique for the size of the hole in concomitant retinal detachment; performing complete vitrectomy; SF6 gas as preferred tamponade; cataract surgery if crystalline lens is mildly/moderately opaque; removal of both ILM and LHEP in LMH surgery. In round 2, 18 items with moderate consensus (45-70% agreement) in round 1 were rated by 35 participants. Final consensus was reached in 35% of questions related to both diagnosis and surgical procedures. CONCLUSIONS: This Delphi study provides valuable information about the consensus/disagreement on different scenarios encountered during FTMH and LMH management as a guide tosurgical decision-making. High rate of disagreement and/or variable approaches still exist for treating such relatively common conditions.

Biomacromolecular Profile in Human Primary Retinal Pigment Epithelial Cells-A Study of Oxidative Stress and Autophagy by Synchrotron-Based FTIR Microspectroscopy.

Synchrotron radiation-based Fourier Transform Infrared (SR-FTIR) microspectroscopy is a non-destructive and chemically sensitive technique for the rapid detection of changes in the different components of the cell's biomacromolecular profile. Reactive oxygen species and oxidative stress may cause damage to the DNA, RNA, and proteins in the retinal pigment epithelium (RPE), which can further lead to age-related macular degeneration (AMD) and visual loss in the elderly. In this study, human primary RPEs (hRPEs) were used to study AMD pathogenesis by using an established in vitro cellular model of the disease. Autophagy-a mechanism of intracellular degradation, which is altered during AMD, was studied in the hRPEs by using the autophagy inducer rapamycin and treated with the autophagy inhibitor bafilomycin A1. In addition, oxidative stress was induced by the hydrogen peroxide (H2O2) treatment of hRPEs. By using SR-FTIR microspectroscopy and multivariate analyses, the changes in the phosphate groups of nucleic acids, Amide I and II of the proteins, the carbonyl groups, and the lipid status in the hRPEs showed a significantly different pattern under oxidative stress/autophagy induction and inhibition. This biomolecular fingerprint can be evaluated in future drug discovery studies affecting autophagy and oxidative stress in AMD.

Retinal Pigment Epithelium Cell Development: Extrapolating Basic Biology to Stem Cell Research.

The retinal pigment epithelium (RPE) forms an important cellular monolayer, which contributes to the normal physiology of the eye. Damage to the RPE leads to the development of degenerative diseases, such as age-related macular degeneration (AMD). Apart from acting as a physical barrier between the retina and choroidal blood vessels, the RPE is crucial in maintaining photoreceptor (PR) and visual functions. Current clinical intervention to treat early stages of AMD includes stem cell-derived RPE transplantation, which is still in its early stages of evolution. Therefore, it becomes essential to derive RPEs which are functional and exhibit features as observed in native human RPE cells. The conventional strategy is to use the knowledge obtained from developmental studies using various animal models and stem cell-based exploratory studies to understand RPE biogenies and developmental trajectory. This article emphasises such studies and aims to present a comprehensive understanding of the basic biology, including the genetics and molecular pathways of RPE development. It encompasses basic developmental biology and stem cell-based developmental studies to uncover RPE differentiation. Knowledge of the in utero developmental cues provides an inclusive methodology required for deriving RPEs using stem cells.

A simple twist technique for lens-sparing one-handed peripheral vitrectomy in phakic patients: a learning approach for junior surgeons.

The evolution of vitrectomy has led to improved suturless techniques and minimally invasive surgery. Nevertheless, the procedure requires great bimanual dexterity and poses risk for lens touch, especially in the hands of less experienced junior surgeons. We hereby present a twist technique which allows for one-handed (right or left) peripheral vitrectomy without the need for one or several hand-switches with the vitreous cutter and avoids lens touch. The technique can be used as a learning approach for junior vitreoretinal surgeons.

Intraoperative Optical Coherence Tomography in the Management of Macular Holes: State of the Art and Future Perspectives.

BACKGROUND: Non-invasive diagnostic technologies in ophthalmology have substantially transformed contemporary clinical practice. Intraoperative optical coherence tomography (iOCT) systems have recently been used for various surgical interventions, including the treatment of full-thickness macular holes (FTMHs). MATERIALS AND METHODS: We conducted a systematic review on the use of iOCT and its possible benefits in the management of FTMHs, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines (PRISMA). The level of evidence according to the Oxford Centre for Evidence-Based Medicine (OCEM) 2011 guidelines, and the quality of evidence according to the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system, were assessed for all included articles. RESULTS: 1131 articles were initially extracted, out of which 694 articles were obtained after duplicates were removed and their abstracts screened. A total of 65 articles was included for full-text review. Finally, 17 articles remained that fulfilled the inclusion criteria. CONCLUSIONS: Even though there is just a small number of studies with solid results, the use of iOCT in FTMH surgery may be a helpful tool for both novice and experienced surgeons planning and managing difficult cases. Additionally, it could be used for teaching reasons and for exploring novel surgical techniques.

Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care.

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial (RPE) layer, lead to a progressive disorganization of the retinal anatomy and the deterioration of visual function. The substitution of damaged RPE cells (RPEs) with in vitro cultured RPE cells using a subretinal cell carrier has shown potential for re-establishing the anatomical structure of the outer retinal layers and is, therefore, being further studied. Here, we present the principles of a surgical technique that allows for the effective subretinal transplantation of a cell carrier with cultivated RPEs into minipigs. The surgeries were performed under general anesthesia and included a standard lens-sparing three-port pars plana vitrectomy (PPV), subretinal application of a balanced salt solution (BSS), a 2.7 mm retinotomy, implantation of a nanofibrous cell carrier into the subretinal space through an additional 3.0 mm sclerotomy, fluid-air exchange (FAX), silicone oil tamponade, and closure of all the sclerotomies. This surgical approach was used in 29 surgeries (18 animals) over the past 8 years with a success rate of 93.1%. Anatomic verification of the surgical placement was carried out using in vivo fundus imaging (fundus photography and optical coherence tomography). The recommended surgical steps for the subretinal implantation of RPEs on a carrier in minipig eyes can be used in future preclinical studies using large-eye animal models.

Internal Limiting Membrane Peeling and Gas Tamponade For Full-Thickness Macular Holes of Different Etiology - Is It Still Relevant?

Background: Despite the abundance of novel surgical approaches proposed for full thickness macular hole (FTMH) treatment, the choice of the optimal technique remains debatable Vitrectomy with «classic¼ internal limiting membrane peeling and gas tamponade remains the standard of FTMH surgery in many cases, but there are still very limited recent publications on the outcomes of such surgery. Purpose: To investigate the anatomical and functional result and to analyze the significance of outcome-related risk factors of the classic 25-gauge pars plana vitrectomy (PPV) with ILM peeling and gas tamponade (GT) for treatment of FTMH of different etiology. Patients and methods: Thirty-eight eyes of thirty-seven patients with FTMH who underwent 25-gauge PPV, ILM peeling and GT were recruited for this retrospective, consecutive, interventional study. Four eyes with persistent holes underwent a re-operation. Outcome-related factors were discussed. Results: The primary closure rate was 89.5% (34/38). All eyes that underwent the repeated surgery (4 cases) obtained final closure. A hole size of >500 µm has a statistically significant effect on the primary macular hole closure (F = 0.048; φ = 0.38; p ˂ 0.05). In the general group (N = 38), the duration of symptoms directly correlated with age (ρ = 0.34; p = 0.04), size of the hole (ρ = 0.66; p ˂ 0.001) and BCVA before surgery (ρ = 0.59; p ˂ 0.001), after 1 month (ρ = 0.36; p = 0.03), and after 3 months (ρ = 0.35; p = 0.03). Preoperative BCVA was better in initially closed cases (Group 1) (U = 26.0; p = 0.05). In the Group 2 with primary unclosed holes, 75% of the eyes (3/4) had an axial length (AL) >26 mm, while in Group 1 such eyes were 12.5 times less (2/34) 5.9% (F = 0.004; φ = 0.63; р ˂ 0.01). The ELM recovery rate at 3 months was 92% (35/38 eyes) and the restoration of EZ at 3 months was 47% (18/38 eyes). Best-corrected visual acuity of all individuals improved significantly from 0.72 ± 0.35 (logMAR) (Me = 0.7; IQR: 0.5-0.8) to 0.25±0.14 (logMAR) (Me = 0.2; IQR: 0.2 - 0.3) at 1 month and 0.17 ± 0.13 (logMAR) (Me = 0.2; IQR: 0.1 - 0.2) at 3 months after surgery (P = 0.0001). Conclusion: 25G PPV with ILM and GT for FTMH of different etiology provide satisfactory morphologic and functional outcomes. Elongated AL, large diameter of MH and long duration of symptoms are the risk factors for initial closure. Proper second surgery can obtain satisfactory outcomes for persistent holes.

Mitochondrial Dysfunction in a High Intraocular Pressure-Induced Retinal Ischemia Minipig Model.

Purpose: Retinal ischemia (RI) and progressive neuronal death are sight-threatening conditions. Mitochondrial (mt) dysfunction and fusion/fission processes have been suggested to play a role in the pathophysiology of RI. This study focuses on changes in the mt parameters of the neuroretina, retinal pigment epithelium (RPE) and choroid in a porcine high intraocular pressure (IOP)-induced RI minipig model. Methods: In one eye, an acute IOP elevation was induced in minipigs and compared to the other control eye. Activity and amount of respiratory chain complexes (RCC) were analyzed by spectrophotometry and Western blot, respectively. The coenzyme Q10 (CoQ10) content was measured using HPLC, and the ultrastructure of the mt was studied via transmission electron microscopy. The expression of selected mt-pathway genes was determined by RT-PCR. Results: At a functional level, increased RCC I activity and decreased total CoQ10 content were found in RPE cells. At a protein level, CORE2, a subunit of RCC III, and DRP1, was significantly decreased in the neuroretina. Drp1 and Opa1, protein-encoding genes responsible for mt quality control, were decreased in most of the samples from the RPE and neuroretina. Conclusions: The eyes of the minipig can be considered a potential RI model to study mt dysfunction in this disease. Strategies targeting mt protection may provide a promising way to delay the acute damage and onset of RI.

Outcomes from the Retrospective Multicenter Cross-Sectional Study on Lamellar Macular Hole Surgery.

Purpose: To analyze the functional and anatomical parameters of lamellar macular hole (LMH) surgery with internal limiting membrane peeling and determine which surgical technique provides the best visual outcome. Methods: This is a retrospective multicenter cross-sectional study on patients who underwent pars plana vitrectomy (PPV) for LMH with or without combined phaco-vitrectomy, as well as gas-, air- or BSS-tamponade. Pre- and postoperative examination included best corrected visual acuity (BCVA) measurements for functional comparison and optical coherence tomography (OCT) scans to determine the contributing anatomical parameters. Results: A total of 66 consecutive patients were included (age: 71.79 ± 8.52 years), of which 47 (71.2%) were diagnosed as tractional type LMH, and 19 patients (28.8%) as degenerative type. An epiretinal membrane (ERM) was present in 63 of the patients (95.5%), LMH-associated epiretinal proliferation (LHEP) was present in 19 patients (28.8%), and 16 patients (24.2%) had concomitant ERM and LHEP. In the group of tractional LMH, the mean central foveal thickness (CFT) was 81.1% thicker (P < 0.05) than in the degenerative group. Thirty-one patients (47.0%) underwent a combined phaco-vitrectomy procedure, while the rest underwent 23G, 25G or 27G PPV. Seventeen of the 66 patients received gas-tamponade (25.7%)-either SF6 or C3F8, 26 received air-tamponade (39.4%), while the remaining 23 patients received balanced salt solution (BSS)-tamponade (34.9%) during vitrectomy. The total BCVA showed significant improvement postoperatively (p < 0.001) and accordingly in the following groups: tractional LMH type (p < 0.001), degenerative type (p < 0.001), simple PPV (p < 0.001), phaco-vitrectomy (p < 0.001), BSS injection (p < 0.01), gas-tamponade (p < 0.05). None of the patients included in the study developed a full thickness macular hole postoperatively. Conclusion: PPV provided a high success rate and functional improvement for treating LMH for both tractional and degenerative types, as well as combined phaco-vitrectomy treatment when cataract was present.

Subretinal Implantation of Human Primary RPE Cells Cultured on Nanofibrous Membranes in Minipigs.

PURPOSE: The development of primary human retinal pigmented epithelium (hRPE) for clinical transplantation purposes on biodegradable scaffolds is indispensable. We hereby report the results of the subretinal implantation of hRPE cells on nanofibrous membranes in minipigs. METHODS: The hRPEs were collected from human cadaver donor eyes and cultivated on ultrathin nanofibrous carriers prepared via the electrospinning of poly(L-lactide-co-DL-lactide) (PDLLA). "Libechov" minipigs (12-36 months old) were used in the study, supported by preoperative tacrolimus immunosuppressive therapy. The subretinal implantation of the hRPE-nanofibrous carrier was conducted using general anesthesia via a custom-made injector during standard three-port 23-gauge vitrectomy, followed by silicone oil endotamponade. The observational period lasted 1, 2, 6 and 8 weeks, and included in vivo optical coherence tomography (OCT) of the retina, as well as post mortem immunohistochemistry using the following antibodies: HNAA and STEM121 (human cell markers); Bestrophin and CRALBP (hRPE cell markers); peanut agglutining (PNA) (cone photoreceptor marker); PKCα (rod bipolar marker); Vimentin, GFAP (macroglial markers); and Iba1 (microglial marker). RESULTS: The hRPEs assumed cobblestone morphology, persistent pigmentation and measurable trans-epithelial electrical resistance on the nanofibrous PDLLA carrier. The surgical delivery of the implants in the subretinal space of the immunosuppressed minipigs was successfully achieved and monitored by fundus imaging and OCT. The implanted hRPEs were positive for HNAA and STEM121 and were located between the minipig's neuroretina and RPE layers at week 2 post-implantation, which was gradually attenuated until week 8. The neuroretina over the implants showed rosette or hypertrophic reaction at week 6. The implanted cells expressed the typical RPE marker bestrophin throughout the whole observation period, and a gradual diminishing of the CRALBP expression in the area of implantation at week 8 post-implantation was observed. The transplanted hRPEs appeared not to form a confluent layer and were less capable of keeping the inner and outer retinal segments intact. The cone photoreceptors adjacent to the implant scaffold were unchanged initially, but underwent a gradual change in structure after hRPE implantation; the retina above and below the implant appeared relatively healthy. The glial reaction of the transplanted and host retina showed Vimentin and GFAP positivity from week 1 onward. Microglial activation appeared in the retinal area of the transplant early after the surgery, which seemed to move into the transplant area over time. CONCLUSIONS: The differentiated hRPEs can serve as an alternative cell source for RPE replacement in animal studies. These cells can be cultivated on nanofibrous PDLLA and implanted subretinally into minipigs using standard 23-gauge vitrectomy and implantation injector. The hRPE-laden scaffolds demonstrated relatively good incorporation into the host retina over an eight-week observation period, with some indication of a gliotic scar formation, and a likely neuroinflammatory response in the transplanted area despite the use of immunosuppression.

Advantages of nanofibrous membranes for culturing of primary RPE cells compared to commercial scaffolds.

PURPOSE: Dysfunction of the retinal pigment epithelium (RPE) causes numerous forms of retinal degeneration. RPE replacement is a modern option to save vision. We aimed to test the results of transplanting cultured RPEs on biocompatible membranes. METHODS: We cultivated porcine primary RPE cells isolated from cadaver eyes from the slaughterhouse on two types of membranes: commercial polyester scaffolds Transwell (Corning Inc., Kenneburg, ME, USA) with 0.4 µm pore size and prepared Poly (L-lactide-co-DL-lactide) (PDLLA) nanofibrous membranes with an average pore size of 0.4 µm. RESULTS: Five types of assays were used for the analysis: immunocytochemistry (ICC), phagocytosis assay, Western blotting, real-time qPCR (RT-qPCR) and electron microscopy. RT-qPCR demonstrated that RPEs cultured on nanofibrous membranes have higher expressions of BEST1 (bestrophin 1), RLBP1 (retinaldehyde-binding protein 1), RPE65 (retinal pigment epithelium-specific 65 kDa protein), PAX6 (transcription factor PAX6), SOX9 (transcription factor SOX9), DCT (dopachrome tautomerase) and MITF (microphthalmia-associated transcription factor). ICC of the RPEs cultured on nanofibrous membranes showed more intensive staining of markers such as BEST1, MCT1 (monocarboxylate transporter 1), Na+ /K+ ATPase, RPE65 and acetylated tubulin in comparison with commercial ones. Additionally, the absence of α-SMA proved the stability of the RPE polarization state and the absence of epithelial-to-mesenchymal transition. RPE possessed high phagocytic activity. Electron microscopy of both membranes confirmed a confluent layer of RPE cells and their genuine morphological structure, which was comparable to native RPEs. CONCLUSIONS: Retinal pigment epitheliums cultured on polylactide nanofibrous membranes improved the final quality of the cell product by having better maturation and long-term survival of the RPE monolayer compared to those cultured on commercial polyester scaffolds. PDLLA-cultured RPEs are a plausible source for the replacement of non-functioning RPEs during cell therapy.

A Bioluminescence Resonance Energy Transfer-Based Reporter System: Characterization and Applications.

Genome editing strategies and DNA repair research need powerful analytical tools. We generated a bioluminescence resonance energy transfer (BRET)-based reporter for the quantification of indel frequencies induced by DNA repair. The BRET reporter, expressed as a single molecule, consists of a mutated Renilla reniformis luciferase domain and a GFP2 domain separated by a shuttle-cloning box for the integration of any given endonuclease target sequence. The luciferase activity acts both as energy donor and as the internal standard, while the loss of GFP2 fluorescence acts as a reporter for the out-of-frame sequence alterations that result from the DNA repair via the non-homologous end joining/microhomology-mediated end joining DNA repair pathways of the endonuclease-mediated DNA double-strand break. This results in a decrease of the fluorescence/luminescence ratio. Employing this reporter in different experimental scenarios, using different cell lines and diseases targeted, we quantified the influence of both protein knockdown of DNA repair pathways as well as guide RNA mismatches on CRISPR-mediated nuclease activity and subsequent repair based on mutagenic repair on the reporter. In conclusion, we demonstrated this BRET-based reporter to be a robust and sensitive analytical tool for assessment of variety of different genome editing-based approaches.

Combination of Inverted ILM Flap Technique and Subretinal Fluid Application Technique for Treatment of Chronic, Persistent and Large Macular Holes.

INTRODUCTION: The choice of surgical treatment for chronic, persistent and large full-thickness macular holes (FTMH) continues to be undefined and challenging, as some of these cases remain refractory to the treatment. We report the efficacy of combination of inverted internal limiting membrane flap technique (IILMFT) and subretinal application of the fluid (SR fluid application) technique for treatment of refractory FTMHs. METHODS: Nine patients (nine eyes) were enrolled into this retrospective non-randomized exploratory consecutive case series study. All patients were diagnosed with chronic, persistent or large FTMH and were treated with a combination of IILMFT and SR fluid application technique. The following outcome parameters were analysed during 1- and 6-month follow-up visits: anatomical FTMH closure rate on spectral domain optical coherence tomography (SD-OCT), best-corrected visual acuity (BCVA), degree of postoperative retinal displacement. RESULTS: The mean preoperative diameter of FTMH was 542.0 µm (range 154-1930 µm). Final closure of FTMH was achieved in nine of nine cases (100%). In one case a second operation was required because of postoperative rhegmatogenous retinal detachment. The mean BCVA after the FTMH closure increased from 1.0 logMAR (0.7-1.3) to 0.4 logMAR (0.2-0.8 logMAR) (W = 2.67; p = 0.008). A positive correlation was revealed between preoperative BCVA and axial length (ρ = 0.67, p = 0.048), between preoperative BCVA and duration of the symptoms (ρ = 0.818, p = 0.007), as well as between postoperative BCVA at 1-month follow-up and BCVA at 6-month follow-up (ρ = 0.821, p = 0.007). CONCLUSION: Combination of IILMFT with SR fluid application technique for refractory FTMH surgery appears to be effective and safe. Improvement of anatomical and visual outcomes after the single surgery benefits from and is ensured by the advantages of both novel surgical approaches.