Long-Term Porcine Retina Explants as an Alternative to In Vivo Experimentation.

Purpose: The porcine retina represents an optimal model system to study treatment approaches for inherited retinal dystrophies owing to close anatomical similarities to the human retina, including a cone enriched visual streak. The aim of this work was to establish a protocol to keep explants in culture for up to 28 days with good morphological preservation. Methods: Two to four retina explants per eye were obtained from the central part of the retina and transferred onto a membrane insert with the photoreceptors facing down. Different medium compositions using Neurobasal-A medium containing 100 or 450 mg/dL glucose and combinations of fetal calf serum, B-27 with or without insulin and N-2 were tested. We developed a tissue quality score with robust markers for different retinal cell types (protein kinase C alpha, peanut agglutinin and 4',6-diamidino-2-phenylindol). Results: Retinae were kept until 28 days with only little degradation. The best results were attained using Neurobasal-A medium containing 100 mg/dL glucose supplemented with B-27 containing insulin and N-2. For an easy preparation process, it is necessary to minimize transport time and keep the eyes on ice until dissected. Heat-mediated decontamination by the butcher has to be avoided. Conclusions: Using a standardized protocol, porcine retina explants represent an easy to handle intermediate model between in vitro and in vivo experimentation. This model system is robustly reproducible and contributes to the implementation of the 3R principle to minimize animal experimentation. Translational Relevance: This model can be used to test future therapeutic approaches for inherited retinal dystrophies.

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.

Rapid and Reliable Quantification of Prime Editing Targeting Within the Porcine ABCA4 Gene Using a BRET-Based Sensor.

Stargardt disease (STGD) leads to blindness in children and young adults. So far, no curative therapy is available and gene augmentation therapies have not yet advanced to the clinics, in part, due to the limited packaging capacity of adeno-associated viruses used to transfer genes into photoreceptor cells. Prime editing offers a new perspective to treat mutations on the genomic level. A nicking variant of Cas9 fused to a reverse transcriptase complex with an elongated guideRNA force intracellular mismatch repair to correct the targeted mutation even in postmitotic cells such as photoreceptors in the eye. Using a custom-made bioluminescence resonance energy transfer (BRET)-based editing sensor in HEK293 cells, we tested 27 different prime editing guide RNAs (pegRNAs) and additional 4 nicking guide RNAs (ngRNAs) with regard to their efficiency to induce sequences changes in exon 43 of the porcine ATP binding cassette subfamily A member 4 (ABCA4) gene that eliminate a mutagenic adenine frameshift insertion, which has been associated with STGD in humans. We identified nine working pegRNAs, and in combination with ngRNAs, we achieved a correction rate of up to ≈92% measured with the BRET-based reporter system. Our data prove the high efficiency of prime editors to correct mutations and highlight the importance of optimal ngRNA design, thus offering a promising editing tool to correct ABCA4 mutations in the disease context.

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.

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.

Characterization of Double-Strand Break Repair Protein Ku80 Location Within the Murine Retina.

Purpose: To characterize the spatial distribution of the DNA-double strand break-repair protein Ku80 in the murine retina. Even though robust data exist on the complexity of DNA repair mechanisms in dividing cells in vitro, almost nothing is known about it in post-mitotic neurons or photoreceptors (PRs). This knowledge is an important prerequisite for in vivo therapeutic approaches by genome editing in retina and PRs. Recently, it was shown that mouse rod PRs are incapable of repairing double-strand breaks induced by radiation. Material and Methods: Retinae from wild-type, rd10, and RPGR-KI mouse lines were obtained and stained with antibodies against Ku80, and cellular markers CtBP2, beta-Dystropglycan, Lamin B, and peanut agglutinin. Organotypic explant cultures were generated and maintained for up to 10 days. Laser microdissection was performed to obtain photoreceptor nuclei, and Ku80 expression was compared to whole retina by real-time PCR (RT-PCR). Results: Strong Ku80 immunoreactivity was observed in rod but not cone photoreceptor terminals localized in the outer plexiform layer of the retina in all three mouse lines. During retinal explant culture, we observed that Ku80-positive globules translocate into the heterochromatin region of nuclei in the outer nuclear layer (ONL). By quantitative PCR, we showed upregulation of relative Ku80 expression in the ONL during wild-type retinal explant culture. Discussion: The unexpected localization of Ku80 to murine rod terminals indicates another tissue-specific modification to the canonical DNA repair mechanisms and warrants further investigation.

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.

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.

Retinal tissue develops an inflammatory reaction to tobacco smoke and electronic cigarette vapor in mice.

Cigarette smoke has been identified as a major risk factor for the development of age-related macular degeneration (AMD). As an alternative to conventional cigarettes (C-cigarette), electronic cigarettes (E-cigarette) have been globally promoted and are currently widely used. The increasing usage of E-cigarettes raises concerns with regard to short- (2 weeks), medium- (3 months), and long- (8 months) term consequences related to retinal tissue. In this report, a controlled study in mouse models was conducted to probe the comprehensive effects of E-cigarette vapor on retina, retinal pigmented epithelium (RPE), and choroidal tissues by (1) comparing the effects of C-cigarette smoke and E-cigarette vapor on retina separately and (2) determining the effects of E-cigarette vapor on the RPE and analyzing the changes with regard to inflammatory (IL-1ß, TNFα, iNOS) and angiogenic (VEGF, PEDF) mediators in retina/RPE/choroid by ELISA assays. The data showed that C-cigarette smoke exposure promoted an inflammatory reaction in the retina in vivo. Mice exposed to E-cigarette (nicotine-free) vapor developed inflammatory and angiogenic reactions more pronounced in RPE and choroid as compared to retinal tissue, while nicotine-containing E-cigarette vapor caused even a more serious reaction. Both inflammatory and pro-angiogenic reactions increased with the extension of exposure time. These results demonstrate that exposure to C-cigarette smoke is harmful to the retina. Likewise, the exposure to E-cigarette vapor (with or without nicotine) increases the occurrence and progression of inflammatory and angiogenic stimuli in the retina, which might also be related to the onset of wet AMD in humans. KEY MESSAGES: C-cigarette smoke exposure promotes an inflammatory reaction in the retina in vivo. Mice exposed to E-cigarette (nicotine-free) vapor develop inflammatory and angiogenic reactions more pronounced in RPE and choroid compared to retinal tissue, while nicotine-containing E-cigarette vapor causes even a more serious reaction. Both inflammatory and pro-angiogenic reactions increase with the extension of E-cigarette vapor exposure time.

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.

The Major Ciliary Isoforms of RPGR Build Different Interaction Complexes with INPP5E and RPGRIP1L.

X-linked retinitis pigmentosa (XLRP) is frequently caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. A complex splicing process acts on the RPGR gene resulting in three major isoforms: RPGRex1-19, RPGRORF15 and RPGRskip14/15. We characterized the widely expressed, alternatively spliced transcript RPGRskip14/15 lacking exons 14 and 15. Using the CRISPR/eSpCas9 system, we generated HEK293T cell lines exclusively expressing the RPGRskip14/15 transcript from the endogenous RPGR gene. RPGRex1-19 and RPGRORF15 were knocked out. Immunocytochemistry demonstrated that the RPGRskip14/15 protein localizes along primary cilia, resembling the expression pattern of RPGRex1-19. The number of cilia-carrying cells was not affected by the absence of the RPGRex1-19 and RPGRORF15 isoforms. Co-immunoprecipitation assays demonstrated that both RPGRex1-19 and RPGRskip14/15 interact with PDE6D, further supporting that RPGRskip14/15 is associated with the protein networks along the primary cilium. Interestingly, interaction complexes with INPP5E or RPGRIP1L were only detectable with isoform RPGRex1-19, but not with RPGRskip14/15, demonstrating distinct functional properties of the major RPGR isoforms in spite of their similar subcellular localization. Our findings lead to the conclusion that protein binding sites within RPGR are mediated through alternative splicing. A tissue-specific expression ratio between RPGRskip14/15 and RPGRex1-19 seems required to regulate the ciliary concentration of RPGR interaction partners.

Novel Needle for Intravitreal Drug Delivery: Comparative Study of Needle Tip Aspirates, Injection Stream and Penetration Forces.

PURPOSE: To study the efficacy of a novel needle for intravitreal injection (IVI) in comparison to the conventional needle under experimental conditions. METHODS: The newly designed 30-gauge (G) needle (NDN) (EP 18158 542.3, patent pending) with occluded outer orifice and a side port for drug delivery was compared to the conventional standard hypodermic 30 G needle for IVI (SHN). An animal study to obtain needle tip aspirates was performed on 10 albino rat eyes. During IVIs, cellular content, which was cut by the needle tip, was aspirated. Cellular material was studied in regard to cell types and their quantity. The injection stream was studied using trypan blue dye in vitro and pig cadaver eyes. The penetration force was tested on polyurethane Testing Foil Strips PU 04 (Melab, Leonberg, Germany) by applying a velocity of 100 mm/min. The results were analyzed using descriptive statistics, correlation matrices and t-test methods with p<0.05 as statistically significant. RESULTS: Cytological analysis of the needle aspirates showed the presence of cellular content in each case. The amount of conjunctival, ciliary body epithelial cells and granulated basophilic protein sediments (sign of cellular damage) in the case of the NDN tips was significantly lower compared to the SHN. The average penetration force of the NDN was 0.791 N, and in the case of the SHN was 0.566 N. The injection stream study revealed a difference in the initial injection phase between the two needle types, although the diffuse filling of the vitreous area which surrounded the needle tip appeared to be similar. DISCUSSION: The NDN demonstrated superior performance with regard to a significantly reduced number of cells being captured by the needle tip. Delivery of the injected fluid into the vitreous cavity was comparable. In order to investigate superior properties of the NDN needle design, further studies with improved prototypes would be necessary.

Trifocal diffractive intraocular lens implantation in patients after previous corneal refractive laser surgery for myopia.

BACKGROUND: With the difficulties in IOL power calculation and the potential side effects occurring postoperatively, multifocal IOL implantation after previous corneal refractive surgery are rarely reported especially for the trifocal IOL. Herein we report the clinical observation of trifocal IOL implantation in patients with previous myopia excimer laser correction. In this study, a multi-formula average method was performed for the IOLs power calculation to improve the accuracy. Visual and refractive outcomes were analyzed, and the subjective quality of patients' life was evaluated by questionnaires survey. METHODS: This retrospective case series included patients with previous myopia excimer laser correction who underwent femtosecond laser assisted phacoemulsification and trifocal IOL (AT LISA tri 839 MP) implantation. Follow-up was done at 1-day, 1-month and 3-month to assess the visual outcomes. Outcome measures were uncorrected distance, intermediate and near visual acuity (UDVA, UIVA, UNVA), manifest refraction, defocus curve, and subjective quality of vision. RESULTS: Twenty-one Eyes from sixteen patients (14 eyes with previous laser in situ keratomileusis and 7 eyes with previous photorefractive keratectomy) were included. Mean postoperative spherical equivalent (SE) at 3-month was - 0.56 D ± 0.49 SD, wherein, 10 eyes (47.6%) were within ±0.50 D of the desired emmetropia and 19 eyes (90.5%) were within ±1.0 D. Mean monocular UDVA, UIVA and UNVA (logMAR) at last visit were 0.02 ± 0.07, 0.10 ± 0.10, and 0.15 ± 0.11 respectively. Three patients (19%) reported halos and glare in postoperative 3 months, two of them needed to use spectacles to improve the intermediate visual acuity. Fifteen patients (94%) reported a satisfaction score of ≥3.5 out of 4.0, without any difficulty in daily activity. Thirteen patients (81%) did not need spectacles at all distances, while the other 3 patients (19%) used spectacles for near-distance related visual activity. Mean composite score of the VF-14 questionnaire was 95.00 ± 7.29 out of 100. CONCLUSIONS: Trifocal IOL implantation after myopia excimer laser correction could restore good distance, intermediate visual acuity and acceptable near visual acuity, and provide accurate refractive outcomes as well as high spectacles independence rate.

Creation of different bioluminescence resonance energy transfer based biosensors with high affinity to VEGF.

In age-related macular degeneration (AMD) or diabetic retinopathy (DR), hypoxia and inflammatory processes lead to an upregulation of the vascular endothelial growth factor (VEGF) expression and thereby to pathological neovascularisation with incorrectly formed vessels prone to damage, thus increasing the vascular permeability and the risk of bleeding and oedema in the retina. State of the art treatment is the repeated intraocular injection of anti-VEGF molecules. For developing improved individualized treatment approaches, a minimally invasive, repeatable method for in vivo quantification of VEGF in the eye is necessary. Therefore, we designed single molecule eBRET2 VEGF biosensors by directly fusing a Renilla luciferase mutant (Rluc8) N-terminal and a green fluorescent protein (GFP2) C-terminal to a VEGF binding domain. In total, 10 different VEGF biosensors (Re01- Re10) were generated based on either single domains or full length of VEGF receptor 1 or 2 extracellular regions as VEGF binding domains. Full length expression of the biosensors in HEK293-T cells was verified via Western Blot employing an anti-Rluc8-IgG. Expression of alternative splice variants was eliminated through the deletion of the donor splice site by introduction of a silent point mutation. In all ten biosensors the energy transfer from the Rluc8 to the GFP2 occurs and generates a measurable eBRET2 ratio. Four biosensors show a relevant change of the BRET ratio (ΔBR) after VEGF binding. Furthermore, each biosensor shows a unique detection range for VEGF quantification and especially Re06 and Re07 have a high sensitivity in the range of in vivo VEGF concentrations in the eye, previously measured by invasive methods. In conclusion, we generated several eBRET2 biosensors that are suitable for VEGF quantification in vitro and could identify two eBRET2 biosensors, which may be suitable for non-invasive in vivo VEGF quantification with an implantable device.

Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in vivo Genome Editing.

Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases. Genome editing represents a promising emerging field in the treatment of monogenic disorders, as it aims to correct disease-causing mutations within the genome. Genome editing relies on highly specific endonucleases and the capacity of the cells to repair double-strand breaks (DSBs). As DSB pathways are cell-cycle dependent, their activity in postmitotic retinal neurons, with a focus on photoreceptors, needs to be assessed in order to develop therapeutic in vivo genome editing. Three DSB-repair pathways are found in mammalian cells: Non-homologous end joining (NHEJ); microhomology-mediated end joining (MMEJ); and homology-directed repair (HDR). While NHEJ can be used to knock out mutant alleles in dominant disorders, HDR and MMEJ are better suited for precise genome editing, or for replacing entire mutation hotspots in genomic regions. Here, we analyzed transcriptomic in vivo and in vitro data and revealed that HDR is indeed downregulated in postmitotic neurons, whereas MMEJ and NHEJ are active. Using single-cell RNA sequencing analysis, we characterized the dynamics of DSB repair pathways in the transition from dividing cells to postmitotic retinal cells. Time-course bulk RNA-seq data confirmed DSB repair gene expression in both in vivo and in vitro samples. Transcriptomic DSB repair pathway profiles are very similar in adult human, macaque, and mouse retinas, but not in ground squirrel retinas. Moreover, human-induced pluripotent stem-cell-derived neurons and retinal organoids can serve as well suited in vitro testbeds for developing genomic engineering approaches in photoreceptors. Our study provides additional support for designing precise in vivo genome-editing approaches via MMEJ, which is active in mature photoreceptors.

[Fluorescence Angiography-assisted Management of Recurrences in Aggressive Posterior Retinopathy of Prematurity (APROP) after Intravitreal Monotherapy with 0.312 mg Bevacizumab]. / Fluoreszenzangiografieassistiertes Management von Rezidiven bei aggressiver posteriorer Frühgeborenenretinopathie (APROP) nach intravitrealer Monotherapie mit 0,312 mg Bevacizumab.

BACKGROUND: In cases of aggressive posterior retinopathy of prematurity (APROP), recurrences can occur after intravitreal injection of bevacizumab (IVB), in spite of successful treatment of the acute stage. Therefore, long-term examinations in extremely premature patients are needed. We defined recurrences as a relapse of plus disease and leakage (with or without proliferation) at the vascularisation border, but also anterior and posterior to it. METHODS: RetCam wide-field colour images and fluorescein angiography were performed before the first IVB (0.312 mg bevacizumab in 0.025 ml per eye), before each further therapy, i.e. additional intravitreal injection, laser- or cryocoagulation or pars-plana vitrectomy, and at the end of the therapy. We analysed the images of 18 eyes with APROP of 9 extreme premature patients treated between 08/2007 and 12/2017 (GA 21 - 27 weeks, BW 430 - 890 g). RESULTS: Long-term therapeutic success was achieved in only 4 eyes/2 children (22%) with one single injection. In 2 eyes/2 children (11%), a second and third injection was given within 2 weeks because of an insufficient therapeutic effect. Up to 3 injections together with laser coagulation were needed in 12 eyes/6 children (67%), in order to achieve complete resolution of ROP activity. In 6 eyes/2 children (33%), resolution of leakage at the original vascularisation border was achieved only with further laser coagulation. In one single eye, retinal detachment occurred after unsuccessful retinal surgery. Before IVB, fluorescein angiography disclosed leakage due to proliferation in most of the patients (12 eyes/6 children). In recurrences after IVB, a posterior shift of the leakage site was found (14 eyes/4 children), whereas after laser photocoagulation proliferative changes were also detected anterior to the vascularisation border (5 eyes/3 children). Treatment was indicated based on angiographic findings in 14 eyes/4 children where wide-field colour images did not show plus disease or proliferation. CONCLUSIONS: Intravitreal injection of 0.312 mg bevacizumab has been shown to be an effective therapy for the acute stage of APROP. Long-term success required consequent monitoring and treatment of APROP recurrences. Fluorescein angiography was particularly useful to detect recurrences that were not evident in wide-field colour images.

Fundus-Controlled Dark Adaptometry in Young Children Without and With Spontaneously Regressed Retinopathy of Prematurity.

PURPOSE: We correlate dark adaptation course with foveal morphologic alterations in preterm and term-born children using a modified fundus-controlled perimeter and spectral domain-optical coherence tomography (SD-OCT) imaging. METHODS: We performed fundus-controlled chromatic dark adaptometry in premature children aged 6 to 13 years without retinopathy of prematurity (no-ROP; n = 61) and with spontaneously regressed ROP (sr-ROP, n = 29), and in 11 age-matched term-born children. The degree of macular developmental arrest (MDA), defined as a disproportion of the outer nuclear layer to inner retinal layers in the fovea (ONL+/IRL-ratio), was analyzed with the DiOCTA tool in SD-OCT scans. RESULTS: Children with MDA showed a flatter dark adaptation course progression with a significant rod-mediated sensitivity recovery delay (0.0113 vs. 0.0253 dB/s; P < 0.001). Preterm-born children with regular foveal morphology reached the final rod-mediated dark-adapted threshold at 12 minutes after bleach at 18.8 dB, compared to after 18.7 minutes at 17.6 dB in children with MDA (no significant difference in final threshold; P = 0.773). The cone-mediated dark adaptation progression showed a significant lower final threshold in children with MDA (6.0 vs. 8.1 dB; P = 0.004). CONCLUSIONS: Changes in dark adaptation were seen in the presence of MDA observed in premature children in the no-ROP and sr-ROP groups. MDA in former premature children is associated with functional deficits of cone and rod photoreceptor visual pathways. TRANSLATIONAL RELEVANCE: Morphologic alterations in the central retina of premature children, evident in SD-OCT, are associated with long-term functional deficits in the rod and cone pathways, particularly evident in the rod dark adaptation course measured at 12° eccentricity. This indicates a more widespread retinal functional pathology not limited to the fovea, but occurring together with foveal alterations best defined as MDA.

Optimizing Measurement of Vascular Endothelial Growth Factor in Small Blood Samples of Premature Infants.

To establish a method that allows for the reliable assessment of vascular endothelial growth factor (VEGF-A) concentrations in very small blood samples of preterm infants. Systemic VEGF measurements are important in view of the most appropriate Anti-VEGF drug to be used for the treatment of acute retinopathy of prematurity (ROP). Cord blood samples from preterm (n = 6) infants, blood samples from preterm infants with treatment requiring ROP (n = 12), and blood samples from healthy adults (n = 10) were collected. Serum, citrate plasma, and serum from recalcified citrate blood were obtained. Levels of VEGF-A and platelet factor-4 (PF-4) were quantified by ELISA or AlphaLISA immunoassay. VEGF-A levels could be detected by both assays, with the AlphaLISA generating slightly lower levels in healthy adults, but not in cord blood of preterm infants. In plasma samples, VEGF levels ranged from non detectable to 181 pg/ml. PF-4 concentrations were between 0.16-3.88 µg/ml. Values of VEGF-A and PF-4 in serum and recalcified serum were significantly higher compared to plasma through the release of these cytokines after platelet activation. In plasma samples of infants with ROP, VEGF-A could always be detected and its values ranged from 19.50 to 245.91 pg/ml and PF-4 concentrations were between 0.1 and 3.3 µg/ml. Using the AlphaLISA kit, we were able to detect VEGF in small sample volumes (5 µl plasma or serum/well) in premature infants with treatment requiring ROP and to monitor platelet activation by PF-4 detection. Minimal blood probe volumes reduce phlebotomy losses avoiding the risk of iatrogenic anemia, thus allowing close monitoring of the cytokine levels in these very fragile infants.