bluuebunny/crossref_metadata_2025 huggingface dataset:
Interplay between autophagy and mitochondrial metabolism in retinal pigment epithelium cells obtained by differentiation of induced pluripotent stem cells generated from wet AMD patients
Authors: Blasiak Janusz , Ali Koskela , Tomi Tuomainen , Heli Skottman , Pasi Tavi , Kai Kaarniranta
Published: December 2022

Purpose: Age‐related macular degeneration (AMD) presents a limited potential of experimental studies on the target tissue from AMD patients. Therefore, it is important to have a reliable experimental model to study AMD pathogenesis. We differentiated induced pluripotent stem cells (PSCs) derived from individuals suffering from wet AMD into retinal pigment epithelium (RPE) cells (iPSCs‐RPE). Impaired autophagy and mitochondrial metabolism are recognized factors of AMD pathogenesis, but the underlined mechanism is not fully known. The aim of this study was to assess mitochondrial metabolism in dependence on autophagy in iPSCs‐RPE derived from wet AMD patients and non‐AMD controls.

Methods: Bafilomycin a1 (Baf1) and MG‐132 were used as autophagy inhibitor and inducer, respectively. Expression of autophagy markers were evaluated by RT‐PCR and western blotting. The Seahorse FX test was used to evaluate mitochondrial metabolism with carbonyl cyanide‐p trifluoromethoxyphenylhydrazone (FCCP) to uncouple oxidative respiration.

Results: We observed differences between the expression of the autophagy‐related genes MAP1LC3B (Microtubule Associated Protein 1 Light Chain 3 Beta) and SQSTM1 (Sequestosome 1) in RPE cells derived from wet AMD and non‐AMD controls. In addition, differences in the expression of the HSP701A (Heat Shock Protein Family A (Hsp70) Member 1A, chaperone) gene were also observed. Autophagy differentially affected mitochondrial metabolism as evaluated by basal and maximal respiration, ATP production, spare capacity and proton link.

Conclusions: Autophagy differentially influences mitochondrial metabolism in iPSCs‐RPE derived from wet AMD and non‐AMD eyes.

Acknowledgement: This work was supported by National Science Centre, Poland grant number 2017/27/B/NZ3/00872.
Role of optical coherence tomography in diagnosis of Alzheimer's disease
Authors: Castro‐Roger Luisa , Mallen Victor , Palomar Elisa Viladés , Ciordia Beatriz Cordón , Altabás María José Vicente , Tello Alvaro , Rodrigo Maria Jesus , Perié Manuel Subías , Campo Lorena Arias , Munuera Inés , Garcia‐Martin Elena
Published: December 2022

Purpose: To evaluate changes in visual function and anatomical structures in the neuroretina of patients diagnosed with Alzheimer's disease (AD) in comparison with healthy controls.

Methods: A total of 25 eyes of 13 AD patients and 80 eyes of 40 healthy subjects were analysed. Best corrected visual acuity (BCVA) was assessed in different contrast situations (100%, 2.5%, 1.25%) with ETDRS chart (Early Treatment Diabetic Retinopathy Study). Also, retinal nerve fibre layer (RNFL) and ganglion cell layer (GCL) thickness were analysed using Spectralis and Triton optical coherence tomography (OCT), globally and by sectors, both in peripapillary and macular area.

Results: A statistically significant decrease was found in BCVA in AD patients in comparison with controls (p < 0.001), specifically in 100% and 1.25% contrast charts. Regarding structural analysis, a significant reduction in retinal thickness was found in AD patients in comparison with controls. Global thickness was found to be significantly thinner in peripapillary retina in Triton OCT (p < 0.001). In a similar way, macular GCL, both in Triton and Spectralis OCT, showed a significant thinning (p < 0.001) in global and sectorial analysis. RNFL showed a significant tendency towards thinning, but specifically in infero‐temporal sector of macular area in Spectralis OCT (p < 0.001).

Conclusions: The neurodegeneration that AD produces to the brain affects the retina in a parallel way, and that can be objectified by measuring BCVA parameters and thickness measurements in OCT. Compared to healthy controls, AD has a significant thinning in several neuroretinal layers, which can be considered a potential diagnostic tool considering that OCT is a quick, reproducible, non‐invasive test.
Acute glaucoma secondary to posterior scleritis uveal effusion syndrome
Authors: Abellán Maria Victoria Navarro , Perea Juan Antonio Sánchez , Menargues Manuel Almarcha , López Carmen MIquel , Velázquez Elena Rubio , Molina Celia Gómez , Medina José Javier García
Published: December 2022

Purpose: To describe an observational case of closed‐angle glaucoma secondary to uveal effusion syndrome after a first episode of posterior scleritis.

Methods: Morales Meseguer University Hospital. Ophthalmology service. Uveitis section.

Results: A 76‐year‐old woman presented to the emergency department with a red, painful right eye (RO) and decreased visual acuity (VA). Initial VA was light perception and intraocular pressure was 40 mmHg, presenting a narrowed chamber and corticonuclear cataract. The fundus was not assessable due to media opacity. Optical coherence tomography showed choroidal folds in the left eye, and the OD was unapproachable. B‐mode ocular ultrasound showed scleral thickening of 2 mm in both eyes and a superior temporal hematic choroidal detachment in the left eye. She was diagnosed with acute glaucoma secondary to uveal effusion after the first outbreak of posterior scleritis. In the emergency department she was treated with intravenous mannitol, acetazolamide orally and hypotensive eye drops. Acute angle‐closure glaucoma and choroidal detachment remitted under systemic steroid treatment, cycloplegic eye drops and topical hypotensive agents. After screening for pathologies associated with posterior scleritis, idiopathic origin of the condition was concluded.

Conclusions: Patients with posterior scleritis may develop uveal effusion syndrome, which can lead to secondary angle‐closure glaucoma. A screening for pathologies associated with posterior scleritis (Wegener's, rheumatoid arthritis, herpes zoster, tumours, porphyria, post‐surgical or pharmacological causes) should be performed, although most cases are idiopathic. Treatment differs from glaucoma due to pupillary block, being necessary the use of systemic corticosteroids.
SMAD2/3 activation and myofibroblast conversion of corneal fibroblasts is fully dependent on the activation of MELK by the noncanonical TGFβ1 pathway
Authors: Wolosin Jose , Wang Zheng , Bernstein Audrey
Published: December 2022

Purpose: To identify the role of noncanonical responses to TGFβ on the early canonical response and the subsequent fibroblasts into myofibroblasts conversion (FMC).

Methods: Human corneal fibroblasts were seeded in a serum‐free medium for 48 h. TGFβ1 (TGFβ‐2 n/ml) was added and cultures were lysed 1–15 min later. Kinase inhibitors were added 10 min beforehand. Identical amounts of soluble protein were then used for Western blot, to measure the level of expression of p.SMAD2 (S465/467), p.SMAD3(S423/425), p.TAK1 (S412), p.JNK1/2 (JNK;T183/185), p.p38 (T180/182), p.MELK(T167/171), α‐SMA, and GAPDH. MELK esiRNA was electroporated 48 h prior to the addition of TGFβ. FMC was assessed by α‐SMA immunohistology and morphology.

Results: In the corneal fibroblasts, the TGFβ‐induced SMAD2/3 phosphorylation (canonical response), displayed a 6–8 min latency period that was followed by rapid, p.SMAD2/3 accumulation to peak response within 10 min. Overt de novo expression of α‐SMA and morphological FMC was observed after 48 h. Inhibition of MELK, a leucine zipper kinase, by OTSS167 or MELK8A, or its downregulation by esiRNA, blocked both responses. In contrast to the delayed canonical response, phosphorylation of TAK1 (noncanonical response), reached its peak within 1 min, as did JNK and p38, TAK1 downstream targets in the mitogen‐activated kinase cascade. The temporal difference was exploited to examine the relationship between the two responses. Inhibition of TAK1 by TAKinib or 5Z‐7‐oxozeaenol blocked the phosphorylation of JNK, p38, MELK, and SMAD2/3 and the FMC. In turn, inhibition of JNK by SP600125 blocked the phosphorylation of MELK and SMAD2/3; p38 inhibition had no effect. MELK inhibition did not inhibit the phosphorylation of either TAK1 or JNK. Finally, phosphorylation of JNK and MELK via TNFα or LPS, did not induce p.SMAD2/3 or blocked the effect of TGFβ on SMAD. These results are consistent with the event sequence, TGFβ → p.TAK → p.JNK1/2 → p.MELK + TGFβ → p.SMAD2/3.

Conclusions: SMAD2/3 activation is fully dependent on TAK1 activity, challenging the notion of separated canonical ‐ noncanonical responses. MELK, a previously unknown component of the response to TGFβ, plays a central role in SMAD phosphorylation. The molecular interaction between the TGFβ receptor, MELK, and SMAD2/3 remains to be elucidated.
Swept‐source optical coherence tomography angiography in toxoplasmic retinochoroiditis
Authors: Doukh Meryem , Ksiaa Imen , Amor Hager Ben , Abroug Nesrine , Jelliti Bechir , Khochteli Sana , Khairallah Moncef
Published: December 2022

Purpose: To describe optical coherence tomography angiography (OCTA) findings in eyes with active and scarred toxoplasmic retinochoroiditis.

Methods: OCTA scans in active (25 eyes) and scarred (17 eyes) retinochoroiditis were retrospectively reviewed at Fattouma Bourguiba University Hospital, Monastir, Tunisia.

Results: In active lesions, OCTA findings included a non‐detectable flow signal area in retinal vascular plexuses and choriocapillaris in all 25 eyes (100%), an abnormal intraretinal vascular process in two eyes (8%), and an associated area of retinal flow deficit secondary to branch retinal artery occlusion in one eye (4%). In scarred lesions, OCTA findings included a flow deficit area in retinal vascular plexuses and choriocapillaris in all 17 eyes (100%), a visibility of larger deeper choroidal vessels at the level of choriocapillaris in nine eyes (53%), and a well‐defined intraretinal vascular network in one eye (5.9%). Peripapillary scars were associated on OCTA with wedge‐shaped loss of radial peripapillary capillaries with corresponding localized retinal nerve fibre layer defect and visual field loss.

Conclusions: OCTA allows to non‐invasively detect retinal and choroidal vascular changes in active and scarred toxoplasmic retinochoroiditis.

References

1. Perez AL, Lozada RA, Emanuelli A, Oliver AL. Optical coherence tomography angiography findings in macular toxoplasma retinochoroiditis: A case report. Am J Ophthalmol Case Rep. 2020;19:100764.

2. Atas F, Kaya M, Toprak T, Akbulut Yagci B, Selver A, Saatci AO. Measurement of the active toxoplasma retinochoroiditis lesion size during the disease course with swept‐source optical coherence tomography angiography: A retrospective image analysis. Int Ophthalmol. 2021;41(12):4127–4135.
Effect of photobiomodulation in suppression of oxidative stress on retinal pigment epithelium
Authors: Lee Yeojin , Kim Jongmin , Won Jae Yon
Published: December 2022

Purpose: As the world undergoes aging, the number of age‐related diseases has increased. One of them is disease related to retinal pigment epithelium (RPE) degeneration, such as age‐related macular degeneration, causing vision loss without physical damage in the ocular system. It is the leading cause of blindness, with no cure. Although the exact pathogenesis is still unknown, the research shows that oxidative stress is one of the risk factors. Various molecules have been reported as anti‐oxidative materials; however, the disease has not yet been conquered. Here, we would like to introduce photobiomodulation (PBM). PBM is a non‐invasive treatment based on red and near‐infrared light and has been used to cure various diseases by regulating cellular functions.

Methods: The BM‐ECM was prepared and the RPE cell line was cultured. The hypoxia condition was induced via cobalt chloride treatment. The barrier function was evaluated via ZO‐1, a marker of tight junction, expression, and Transepithelial Electrical Resistance (TEER) measurement. The clearance function was evaluated via MERTK expression and digestion of polystyrene beads. The visual function of RPE was examined via expression of RPE65, the enzyme for phototransduction. The oxidative stress was compared via ROS generation.

Results: In this study, we confirm the antioxidant effect of PBM in retinal pigment epithelium via an RPE model with hypoxia. The function of RPE is protected by PBM against damage from hypoxia. Furthermore, we observed the protective mechanism of PBM by its suppression effect on reactive oxygen species generation.

Conclusions: These results indicate that PBM shows great potential to cure RPE degeneration to help patients with blindness.
Assessment of compliance with current driving legislation in Spanish patients with visual disturbances
Authors: Franco Pedro Esteban Quílez , de Imperial Ollero Juan Antonio Miralles , Alacid Alberto López , Navarro Inmaculada Sellés
Published: December 2022

Purpose: To determine whether patients who do not fulfil some of the requirements of current Spanish driving regulation but still have their driving licence have stopped driving. Furthermore, to assess the extent to which ophthalmologists know such legislation and whether they counsel their patients accordingly.

Methods: Two surveys were conducted. For the first one, we contacted 133 patients who did not fulfil either current visual acuity (VA) or visual field requirements. We asked them to answer 10 questions about their driving habits and their subjective perception of visual quality and road safety. For the second one, we contacted 137 ophthalmologists and presented them with 12 questions: 6 of them inquired about current legislation requirements and the rest of them looked at their opinion about the importance of vision in driving matters.

Results: 65% of patients (95% CI: [57%–74%]) kept driving despite not fulfilling minimal legal requirements. Continuation of driving was associated with having better VA (p < 0.005), better subjective perception of visual quality (p < 0.0000001), having had a previous conversation with their ophthalmologist about driving (p < 0.05; OR = 3.50) and not being pressured by their families for driving cessation (p < 0.05, OR = 2.67). Regression models showed that subjective perception of visual quality was not a good predictor of objective VA neither among non‐drivers (R2 = 0.228) nor drivers (R2 = 0.072). Most ophthalmologists (72.3%) correctly answered no more than 3 out of 6 questions about the requirements of current legislation. Only 7 (5.1%) had received specific training about these matters. Most of them (97.1%) agreed that sufficient visual quality is of great importance for driving safely.

Conclusions: Most drivers who keep their driving licence despite falling below the minimum legal requirements to drive continue driving. Current evaluations for licence renewal could be insufficient. Family pressure seems to be important in driving cessation. Ophthalmologists play a role in patients' counsel about driving, but their knowledge seems to be limited. New training initiatives could be beneficial.
Are choroidal vascularity parameters influenced by age, hypertension and visual acuity in pachychoroid spectrum diseases?
Authors: Abid Yosra , Sassi Hela , Ouederni Meriem , Nouri Slim , Chaabouni Karim , Cheour Monia
Published: December 2022

Purpose: To identify correlations of subfoveal choroidal thickness (SFCT) and choroidal vascularity index (CVI) with age and best corrected visual acuity in eyes with pachychoroid spectrum diseases (PSD) and their association to systemic hypertension using a retrospective study design.

Methods: Our study included 82 eyes of 45 patients with PSD: 15 eyes had uncomplicated pachychoroid (UCP), 27 had pachychoroid pigment epitheliopathy (PPE), 32 had central serous chorioretinopathy (CSC) and 8 had pachychoroid neovasculopathy (PNV). All patients underwent DRI TRITON Swept‐Source Optical Coherence Tomography (SS‐OCT). A 1500 mm width subfoveal choroidal area was defined in SS‐OCT B‐scans and divided into luminal area (LA) and stromal area (SA) by the imageJ binarization technique. CVI was defined as the ratio of LA to the total subfoveal choroidal area (TCA).

Results: The mean SFCT was 393, 23 ± 95, 65 μm and it was correlated negatively with age (p = 0.003, R = −0.326). We found a negative correlation of TCA and LA with age (p = 0.043, r = −0.227, p = 0.04, r = −0.230). Mean choroidal vascularity index was 64.45 ± 2, 74% with no correlation with age (p = 0.169, R = −0.155). No parameter among SFCT, CVI, TCA, LA and SA had a significant correlation with BCVA or spherical equivalent. Patients with essential hypertension were found to have significantly thinner SFCT when compared with healthy controls (374, 49 ± 83 141 μm vs. 423, 98 ± 130 466 μm, p = 0.046). CVI was not affected by a history of systemic hypertension (64.05 ± 1.97 vs. 64.83 ± 3.3, p = 0.207). The total choroidal area, luminal area, and stromal area, as well as the CVI correlated with SFCT (p < 0.001, r = 0.817, p < 0.001, r = 0.821, p < 0.001, r = 0.750 and p < 0.001, r = 0.371 respectively).

Conclusions: SFCT and CVI are dynamic parameters that are affected in patients with pachychoroid diseases. Unlike CVI, SFCT is also affected by age, ocular and systemic factors like hypertension. CVI may be a more specific biomarker for PSD.
Administration of minocycline reduces microglia and Caspase‐3 activation but does not mitigate retinal ganglion cell loss after ocular hypertension in mice
Authors: Valiente‐Soriano Francisco Javier , Sánchez‐Migallón María Cielo , Di Pierdomenico Johnny , Ortega Alejandro Gallego , García‐Ayuso Diego , Vidal‐Sanz Manuel , Agudo‐Barriuso Marta
Published: December 2022

Purpose: To study the effect of minocycline administration on the evolution of retinal ganglion cell (RGC) loss, the number of microglial cells and the activation of the apoptotic signal Caspase‐3 after ocular hypertension (OHT) in mice.

Methods: In left eyes of Swiss albino mice, OHT was induced by diode laser photocoagulation of the limbal and episcleral veins and the intraocular pressure (IOP) evolution was monitored with a TonoLab®. Mice received daily intraperitoneal injections of minocycline hydrochloride (45 mg/kg) diluted in saline starting the day before OHT induction until the day of sacrifice (4 or 15 days, n = 12–14 per group/time point). Control groups received saline injections. Retinas were divided into two groups. Retinas of the first group were double immunodetected against Brn3a and active Caspase‐3 to identify surviving (Brn3a+RGCs) and apoptotic (a‐Casp3+RGCs) RGCs. In the second group, RGCs were retrogradely traced with Fluorogold 7 days before OHT induction and retinas were immunodetected against Iba‐1. Total Brn3a+RGCs, a‐Casp3+RGCs, Iba‐1+cells and FG+microglial cells in the RGC layer were manually or automatically quantified, and the distributions were analysed by topographical maps.

Results: IOP was significantly elevated 24 hours after OHT induction (33.2 ± 6.1 mmHg) and returned to baseline at 1 week (17.3 ± 5.3 mmHg). This evolution was not altered by minocycline administration. Although minocycline treatment was not successful in protecting Brn3a+RGCs at 4 (40 878 ± 4860 vs 43 526 ± 2714) nor at 15 (16 161 ± 12 650 vs 12 919 ± 6085) days after OHT, the numbers of a‐Casp3+RGCs, Iba‐1+cells and FG+microglial cells were significantly lower in the minocycline‐treated groups at both 4 (58.6%, 46.8% and 24.5%, respectively) and 15 (33.6%, 54.2% and 37.3%, respectively) days compared to the saline‐treated groups (100%).

Conclusions: Minocycline treatment decreases the number of microglial cells and caspase‐3 activation but does not reduce RGC loss after OHT.
Report of two cases of subconjunctival haemorrhage as first sign of post‐pregnancy acquired haemophilia a (AHA)
Authors: Fruschelli Mario , Marini Davide , Luciani Diego , Caroni Federico , Puccetti Luca
Published: December 2022

Purpose: To show that subconjunctival haemorrhage may be the first sign of post‐pregnancy acquired A haemophilia (AHA), a potentially fatal bleeding disorder.

Methods: Case reports.

Results: Two thirty‐year‐old caucasic women at first pregnancy presented slight pain without visual loss 2 weeks after uncomplicated spontaneous full‐term delivery. The only clinical findings were a subconjunctival bleeding for both and a small haematoma in the first case. The clinical history was negative for spontaneous bleeding also in infantile age and at previous surgery and no bleeding tendency was observed in the main relatives. The first patient was lost on follow‐up and admitted to urgent haemathologic evaluation due to large multiple cutaneous hematomas, a massive deep muscle haematoma, asthenia and dyspnea. In the second case quick routine coagulation labs were performed immediately after the subconjunctival bleeding. In both cases PTT ratio was increased. An extensive study of blood coagulation suggested the presence of a circulating inhibitor against factor VIII [1,2], confirming the diagnosis of AHA. First case was treated with a bypassing agent, however the eradication was obtained only with immunosuppressive therapy. Second case was initially placed in close follow‐up, since the post‐pregnancy form is often self‐limiting, however an immunosuppressive therapy was set after clinical progression due to the appearance of spontaneous hematoma.

Conclusions: AHA is a relevant acquired bleeding disorder that can be fatal if not quickly recognized and treated even when the initial clinical signs are minor [3], how it can happen in post‐pregancy mainly in the first month [4]. Since it may present as spontaneous subconjunctival haemorrhage, routine hemathological and coagulation labs should be performed to exclude this serious condition or once identified, allow for proper clinical management.
Osmoprotective and bioprotective effect of threalose 3% under desiccating conditions: An in vitro study
Authors: del Papa Melina Sol , Passerini María Silvia , Berra Alejandro
Published: December 2022

Purpose: To study the osmoprotective and bioprotective effect of trehalose 3% during desiccation conditions.

Methods: Normal human conjunctival epithelial cells (IOBA‐NHC) were exposed to: 1) culture media DMEM‐F12 (Control); 2) Carboxymethylcellulose 0.5% + glycerin 0.9% + PEG 400 0.25% (Vehicle); 3) Vehicle + trehalose 3% (Trehalose). Cultures were treated for 1 h and then exposed to desiccating conditions (25°C and 36–38% relative humidity). Metabolic activity by MTT assay and cell viability by Triptan Blue assay was evaluated. To evaluate water retention capacity, a gravimetric test was carried out by soaking a filter paper with 5 ml of each solution under evaluation and recording its weight over time. The data were analysed by analysis of variance (ANOVA) with a post hoc Tukey multiple comparisons test.

Results: MTT assay: During the first 30 min under desiccating conditions there were no differences between groups (Control: 0.586 ± 0.012; Vehicle: 0.575 ± 0.278; Trehalose: 0.523 ± 0.035). After 45 min of exposure metabolic activity sharply decreased in control group (0.111 ± 0.007) and vehicle group (0.107 ± 0.027), while trehalose group maintained metabolic activity values (0.560 ± 0.008; p < 0.001).

Triptan Blue assay: The cell viability of the trehalose group was maintained throughout the time (10 min: 62.7 ± 2.5; 15 min: 58.7 ± 8.1; 20 min: 62.3 ± 10.8; 30 min:41.7 ± 8.5%) and was statistically superior to the control and vehicle groups at all evaluated times (p < 0.01).

Water retention capacity: Water content of papers soaked with control and vehicle solution was almost completely evaporated (less than 20% of water retention) at 10 min on desiccating conditions, while trehalose group show a significant delay of water evaporation of 5 and 7 min regarding the vehicle and control group, respectively.

Conclusions: The addition of trehalose 3% prolonged cell viability and extended water retention time in conditions of high environmental dryness. This study confirms the bioprotective and osmoprotective role of trehalose evaluated in previous investigations and provides scientific evidence of its properties, representing an additional benefit in the treatment of dry eye.
Gene editing strategy for development of an inherited retinal dystrophy model based on iPSC‐derived photoreceptors with mutation in the PROM1 gene
Authors: Fernandez‐Bueno Ivan , Puertas‐Neyra Kevin , Hernández‐Rodríguez Leticia , Gobelli Dino , Simarro Maria , Usategui‐Martín Ricardo , Coco Rosa , de la Fuente Miguel Angel
Published: December 2022

Purpose: The PROM1 gene encodes the protein Prominin‐1 which plays a critical role in the morphogenesis of photoreceptors outer segments. Mutations in the PROM1 gene are related to Inherited Retinal Dystrophy (IRD) phenotypes. The c.1354dupT mutation in the PROM1 gene causes a premature stop codon. The aim of the study was to define the more accurate gene editing strategy for development of an IRD model based on iPSC‐derived photoreceptors.

Methods: Firstly, the most accurate CRISPR/Cas9 machinery was determined by the web tool for genome editing CRISPOR and the DNAStar Lasergene software. gRNAs were selected, accordingly to their proximity to the mutation position and their specificity and efficiency scores. Then, silent mutations were added to the repair oligonucleotides (Alt‐R HDR Donor Oligos), by using the web tool MolBiotools, to generate a restriction site for the Ssp1enzyme. Afterwards, to confirm the in silico information, in vitro studies were performed. The U2OS cell line and the control iPSC line ([FiPS] Ctrl1‐Ep6F‐5) were transfected with each selected gRNA, their respective repair oligonucleotide, and the endonuclease Cas9‐protein, by using the Neon Transfection System. Finally, a digestion with Ssp1 were performed to specifically detect the edited genomes.

Results: Several gRNAs were selected in silico. In vitro studies showed higher genome editing efficiency in two of them (40RE and 47RE). Finally, the gRNA guide 40RE and its respective repair oligonucleotide were selected.

Conclusions: In‐silico and in‐vitro studies concluded that the gRNA guide 40RE was the most accurate for development of an in vitro IRD model based on iPSC‐derived photoreceptors with a mutation in the PROM1 gene. Gene editing strategy designed in silico should be validated in vitro before transfecting the target cells.
Activation of Nrf2‐mediated protective pathways by nature‐inspired hybrids improves proteostasis in retinal pigment epithelial cells
Authors: Koskela Ali , Manai Federico , Basagni Filippo , Liukkonen Mikko , Rosini Michela , Govoni Stefano , Monte Massimo Dal , Smedowski Adrian , Kaarniranta Kai , Amadio Marialaura
Published: December 2022

References: Antioxidant systems are essential in many elderly diseases, including age‐related macular degeneration (AMD). Oxidative stress, inflammation, protein degradation impairment, are well‐described in AMD retinal pigment epithelial (RPE) cells. Nrf2 (NF‐E2‐related factor 2), master regulator of antioxidant defence, is linked to AMD, autophagy, inflammation. We previously showed that nature‐inspired hybrids (NIH1‐3) induce Nrf2‐mediated protection against AMD‐related pro‐oxidant stimuli in RPE cells.

Purpose: In human RPE cells we aimed to further investigate NIH1‐3 for capability to modulate Nrf2 pathway and provide cytoprotection against stress caused by dysfunction of protein homeostasis.

Methods: ARPE‐19 cells were treated with 5 μM NIH 1–3, NIH4 (the derivative unable to activate Nrf2) or vehicle (dimethyl sulfoxide; control) for a minimum of 3 h. Proteasome inhibitor MG‐132 (5 μM) and autophagy inhibitor Bafilomycin‐A1 (50 nM) were added after 24 h NIH pre‐treatment. Levels of Nrf2, its target HO‐1 and SQSTM1/p62, the autophagy flux marker LC3‐II, were analysed by Western blotting, whilst IL‐8 release by ELISA, respectively. Cell survival was also assessed.

Results: NIH1‐3 activate Nrf2‐pathway and increase HO‐1 and SQSTM1/p62 expression, in turn exerting beneficial effects on intracellular redox balance, without modification of the autophagy flux. NIH1‐3 treatments predispose ARPE‐19 cells to a better response to the following exposure to proteasome and autophagy inhibitors, as revealed by the higher cell survival and decreased secretion of the pro‐inflammatory IL‐8 compared to NIH‐untreated cells. Interestingly, also NIH4 compound, through an Nrf2‐independent pathway, exerts pro‐survival and anti‐inflammatory effects, although to a less extent than NIH1‐3.

Conclusions: Our findings suggest that all NIHs are interesting for their cytoprotective properties and confirm Nrf2 as a valuable pharmacological target in contexts characterized by oxidative stress.
Ocular biometry in adults from a population‐based study in Germany
Authors: Michael Ralph , Wirkner Kerstin , Engel Christoph , Loeffler Markus , Kirsten Toralf , Rauscher Franziska
Published: December 2022

Purpose: To obtain normal ocular biometry data in healthy European subjects.

Methods: As part of the large population‐based LIFE‐Adult study (Leipzig Research Centre for Civilization Diseases), randomly selected participants from Leipzig, Germany were evaluated with the ZEISS IOL‐Master 700 (Carl Zeiss Meditec, Jena, Germany). Age range was 25–85 years, with 50% of participants above 70 years by study design and mean age of 64 years. Axial length (AL), central corneal thickness (CCT), aqueous depth (AQD), lens thickness (LT) and corneal radii (R1 & R2) were assessed in 1365 right eyes (two mandatory repeated measurements, with quality check passed).

Results: Mean AL was 23.53 mm and mean CCT was 558 micrometres with no significant change with age. AQD decreased from 2.61 mm at 50 years to 2.44 mm at 80 years. LT increased from 4.36 mm at 50 years to 4.50 mm at 80 years. LT was 4.69 mm in eyes with AL of 22 mm and 4.38 in eyes with AL of 25 mm. Mean R1 was 7.81 mm and mean R2 7.66 mm with no significant change with age.

Conclusions: Above 25 years of age, we found no difference of axial length or corneal thickness with age. The lens was found continuously thicker with age and aqueous depth decreased with age. Short eyes had thicker lenses compared to long eyes; probably an adaptation to maintain a sharp retinal image.
Orbital abscess surgery in a patient with odontogenic pansinusitis
Authors: Carsi Marta Comes , Tepper Sergio Maugard , García Maria Amparo Lanuza , Duch Samper Antonio M.
Published: December 2022

Purpose: In orbital abscesses with sinus origin, the most frequent treatment is a endoscopic sinus surgery (ENSS), if there is a subperiosteal abscess that may compromise the vision, an external drainage should be performed, depending on the location of the abscess. We present an orbital abscess of odontogenic cause that required an orbital approach.

Methods: We present an orbital abscess in a 44‐year‐old man with no relevant medical or ophthalmological history, who, after complicated dental extraction, suffered a left orbital cellulitis.

Surgical technique: Ethmoidectomy was performed by endoscopic sinus surgery (ENSS), without resolution of the infection, requiring drainage of the abscess through an external orbital superomedial approach.

Results: This abscess was located between the middle and superior rectus muscles. A superomedial incision was made, dissecting up to the periosteum and incising the periorbita, observing the exit of purulent material. A drainage was left in place. At the same time, ENSS was performed with exeresis of the lamina papyracea. The patient evolved torpidly, with productive drainage. Computed tomography (CT) shows two collections adjacent to the drainage and the thickening of the lateral rectus muscle. Reoperation was performed, draining the abscesses visualized and washing the area with hydrogen peroxide and metronidazole. New drainage was placed. The evolution was favourable.

Conclusions: In orbital abscesses, antibiotic therapy is usually not sufficient, and surgical intervention is indicated. There are various types of procedures and surgical approaches for the drainage of orbital abscesses, the most common is ENSS, but in complex abscesses an external approach may be necessary. Imaging tests facilitate the location of the abscess, serving as a guide for surgery.
Repurposing dimethyl fumarate? In vitro evidence of Nrf2‐pathway activation and cytoprotective effects in human retinal endothelial cells
Authors: Amadio Marialaura , Manai Federico
Published: December 2022

References: Nrf2 (NF‐E2‐related factor 2) pathway is a major defensive system orchestrating cellular response to pro‐oxidant and pro‐inflammatory stress and regulating the expression of many cytoprotective genes. Pharmacologic activation of Nrf2 has emerged as an attractive strategy for many pathologic conditions. The best‐known Nrf2 activator is dimethyl fumarate (DMF), currently used for the treatment of relapsing–remitting multiple sclerosis and psoriasis. However, literature evidence in animal models of eye diseases reports that DMF displays beneficial effects also at ocular level, suggesting its potential therapeutic use also in retinal pathologies.

Purpose: The aim of this study was to evaluate in vitro whether DMF activates Nrf2‐pathway and exerts beneficial effects in human retinal endothelial cells, and to test its possible cytoprotection against a diabetic retinopathy‐related insult.

Methods: Human Retinal Endothelial Cells (HREC) were exposed to increasing concentrations of DMF and tested for viability and Nrf2‐pathway activation. Expression of Nrf2 and its target HO‐1 (Heme Oxygenase‐1) coding an antioxidant and detoxifying enzyme relevant for vascular endothelium, were evaluated by Western blotting and real‐time quantitative PCR. Intracellular levels of reactive oxygen species (ROS) were investigated after DMF treatment through flow cytometry. DMF‐mediated pro‐survival effects were also tested in HREC under hyper‐glucose conditions.

Results: In basal conditions, DMF is optimally tolerated and activates Nrf2/HO‐1 pathway, decreasing intracellular ROS levels in HREC. DMF protects HREC under high glucose conditions.

Conclusions: Together with the most recent evidence of DMF effects at ocular level, our findings open a new perspective for the repositioning of DMF in eye pathologies involving alterations of the endothelium and, more extensively, for ocular diseases characterized by oxidative stress and inflammation.
A CYP1B1 associated irido‐trabeculodysgenesis
Authors: Gupta Shikha , Panigrahi Arnav , Mahalingam Karthikeyan , Singh Abhishek , Gupta Viney
Published: December 2022

Purpose: CYP1B1 mutations are commonly associated with primary congenital glaucoma (PCG). PCG is due to an isolated trabeculodysgenesis and classically does not involve iris dysgenesis. We describe a cohort of atypical PCG phenotype showing iris atrophy.

Methods: In this study, exome sequencing was carried out in five patients with neonatal onset congenital glaucoma who also had features of iris atrophy that were detected once the children were old enough for slit lamp examination.

Results: The five patients identified had presented with bilateral neonatal onset glaucoma with iris abnormalities like iris atrophy, corectopia and polycoria detected when they were older. All patients had undergone bilateral trabecutectomy with ab externo trabeculotomy during early childhood. The mean age of these children at the time of this study was 14.4 years (range: 8–25 years). The iris atrophy was characteristically peripheral and circumferential. However, none of these patients had evidence of a posterior embryotoxon or peripheral anterior synechiae in the angle (PAS), typical of Axenfeld Rieger Syndrome (ARS). Whole exome sequencing identified biallelic CYP1B1 mutations in all of these five patients as pathogenic variants.

Conclusion: This variant of neonatal onset PCG associated with biallelic CYP1B1 mutations may have associated iridodysgenesis and may resemble the ARS phenotype.
Biological processes and clinical variables affecting trabeculectomy surgery outcome
Authors: Nättinen Janika , Aapola Ulla , Nukareddy Praveena , Gielen Fabian , Vaajanen Anu , Uusitalo Hannu
Published: December 2022

Purpose: To establish what preoperative clinical factors and tear protein levels influence the trabeculectomy outcome.

Methods: In this prospective study, we followed 80 glaucomatous eyes for 5 years after trabeculectomy. Preoperatively, tear fluid samples and clinical data were collected from all patients. Postoperative statuses, i.e., qualified success (QS) in case of 5‐fluorouracil (5FU), needling or restart of glaucoma medication and failure (F) in case of reoperation, were recorded. The patients' postoperative statuses were then compared against the preoperative tear proteomics and clinical variables with Wilcoxon rank sum test. Proteomic pathway analyses were performed using Ingenuity Pathway Analysis (IPA) software.

Results: Within 1 year of the surgery, 15 (19%) QS + F patients were detected. Patients with early failure were younger than those with a successful early outcome (64.7 ± 9.5 vs 70.1 ± 7.6 years, respectively, p = 0.04) and according to pathway analysis results, had increased activation of immune responses prior to surgery (all p‐values <0.05). Overall, 24 (31%) QS + F cases occurred during the 5‐year follow‐up. Pathway analysis revealed that these patients had increased levels of apoptosis prior to surgery (activation z‐score = 2.2, p‐value = 0.02).

Conclusions: These results further emphasize the importance of observing the preoperative state of the ocular surface. The tear proteomic profiles reveal biological functions that are associated with the trabeculectomy outcome.
Development of a biocompatible allogenic extra cellular matrix (MEC) for gradual dispensation of active substances
Authors: Barnouin Laurence , Grossetete Florine
Published: December 2022

Purpose: TBF aims to design an allogeneic extra cellular matrix that can be loaded with a large range of active substances: antibiotic, anti‐inflammatory or biological components and able to provide a gradual dispensation of these substances.

Methods: TBF developed an amniotic membrane of umbilical cord (AM‐UC). The product is a moulded foam or fibres to be reconstituted in dry eyes or gel. The product is virally inactivated, freeze‐dried and sterilized. Products are safe and biocompatible. The testing product consists in a moulded small strip that can be inserted behind the lower eyelid. These products contain hyaluronic acid, growth factors and proteoglycans. They have been impregnated after gamma irradiation with a known quantity of gentamycin, dexamethasone or extracellular micro‐vesicles extracted from mesenchymal stem cells cultures. Impregnated products have been placed in an extraction medium, which has been removed and replaced at defined times. Actives substances have then been quantified in the different extracts according to appropriate methods. Gentamycin has been quantified according to the Method A from the Eur. Ph., dexamethasone by an ELISA detection kit then supported by HPLC‐MS/MS method (after a bioreactor study) and exosomes using an ELISA detection kit based on CD63 protein binding.

Results: For gentamycin and dexamethasone, quantifications have shown that the products enable a progressive release during the first 6 h of extraction of 40%–60% of the active substance with still extraction after 24 h. Then a threshold is reached, and the release is stabilized over the remaining time of extraction until reaching 55%–70% of extraction. Exosomes are constantly released in extraction medium; all observation points are similar.

Conclusions: The results obtained tend to show that the product developed can be impregnated with different type of active substances and allow a gradual dispensation. It could be used in ophthalmological pathologies in order to increase contact duration between the eye and the active substance.
Analysis of glaucoma genes in Finnish patients with juvenile open‐angle glaucoma
Authors: Liuska Perttu , Tadji Abdesallam , Repo Pauliina , Hiltunen Juho , Backlund Michael , Järvinen Reetta‐Stiina , Ojanen Eeva , Majander Anna , Kivelä Tero , Harju Mika , Turunen Joni
Published: December 2022

Purpose: Juvenile open‐angle glaucoma (JOAG) is a rare type of glaucoma diagnosed between the age of 4 and 40 years. Predisposition to JOAG is often inherited as a Mendelian autosomal dominant pattern. Pathogenic myocilin (MYOC) variants are reported in 8%–36% of patients but rarer variants in other genes could also predispose to JOAG. We investigated germline variants in known glaucoma genes in Finnish patients with JOAG.

Methods: Finnish patients with JOAG treated between 2010 and 2018 at the Department of Ophthalmology, Helsinki University Hospital, Finland, were enrolled in the study. We sequenced all exonic regions and flanking splice sites of MYOC for six patients and one healthy relative using Sanger sequencing. In 55 patients, we performed exome sequencing to investigate variants also in 26 other glaucoma genes.

Results: A total of 61 patients with JOAG from 58 pedigrees, and one healthy relative, were included. The mean [SD; range] age at diagnosis was 31.9 [7.5; 11–39]. Eight subjects (13.8%) had a normal‐tension disease, while 50 individuals (86.2%) had a disease with elevated intraocular pressure. Five probands had probable pathogenic variants in MYOC: c.1102C > T p.(Gln368Ter), c.1109C > T p.(Pro370Leu), c.1130C > T p.(Thr377Met), c.1132G > A p.(Asp378Asn), and c.1456C > T p.(Leu486Phe). Four of these patients had a family history of dominantly inherited JOAG. One patient and his mother with JOAG had a novel loss‐of‐function variant in the FOXC1 gene, c.366G > A p.(Trp122Ter). A patient with sporadic JOAG had a homozygous likely pathogenic variant in the LTBP2 gene, c.3938G > A p.(Cys1313Tyr). The frequency of MYOC variants was 8.6% (5/58). The genetic variants explained 12.1% (7 out of 58; 95% CI, 6.0%–22.8%) of JOAG in our cohort.

Conclusions: The prevalence of pathogenic variants in known glaucoma genes is low in Finnish patients with JOAG. Due to the specific population history of Finland, it might be possible to identify novel glaucoma genes in the future.
Nerve regeneration and corneal wound healing by ophthalmic formulations based on sodium hyaluronate, taurine, vitamin B6 and vitamin B12
Authors: Bucolo Claudio , Maugeri Grazia , Giunta Salvatore , Romano Giovanni Luca
Published: December 2022

Purpose: To evaluate the pharmacological profile of ocular formulations based on cross‐linked sodium hyaluronate (CL‐SH), taurine (Tau), Vit B6 and Vit B12 using in vitro and in vivo paradigms.

Methods: Rabbit corneal epithelial cells (SIRC) were used to assess wound healing and ROS formation by scratch assay and oxidative stress (0.3 mM H2O2; 30 min), respectively w/ or w/o ocular formulations exposure. In vivo studies were carried out on albino rabbits to evaluate corneal nerve regeneration and corneal wound healing with or without treatment with six different formulations. Animals were anaesthetised, the corneal epithelium was removed, and formulations were topically administered (30 μl/eye; 3 times/day for 6 days). Slit‐lamp observation was carried out at the following day: T0, T2 and T5. After 6 days the animals were killed, and corneas were collected to evaluate corneal re‐innervation by immunohistochemistry of selective neuronal marker β‐III tubulin.

Results: Formulations containing the concentrations 0.16% or 0.32% of CL‐SH, Tau, Vit B6 and B12 accelerated corneal wound healing. Cells exposed to H2O2 led to significant (p < 0.001) increase of ROS concentration that was significantly (p < 0.001) counteract by formulations containing CL‐SH (0.32%) and Tau w/ or w/o Vit B6 and B12. The extent of re‐innervation, in terms of β‐III tubulin staining, was 5‐fold greater (p < 0.01) in the eye of rabbits treated with formulation containing 0.32% CL‐SH, Tau, Vit B6 and Vit B12 (RenerviX®) compared with the control group (no treatment). Furthermore, re‐innervation with RenerviX® group was significantly greater (p < 0.01) also compared with the groups treated with the formulation containing 0.32% CL‐SH and Tau w/o vitamins and with the formulation containing 0.5% linear SH, Tau, and Vit B12.

Conclusions: Among all formulations tested, the new ophthalmic gel RenerviX® was able to contrast oxidative stress, to accelerate corneal re‐epithelization and to promote nerve regeneration.
Ambra1 haploinsufficiency results in metabolic alterations and exacerbates age‐associated retinal degeneration
Authors: Villarejo‐Zori Beatriz , Ramírez‐Pardo Ignacio , Jiménez‐Loygorri Juan Ignacio , Sierra‐Filardi Elena , Alonso‐Gil Sandra , Gómez‐Sintes Raquel , Boya Patricia
Published: December 2022

Purpose: Retina is a highly complex and metabolically active tissue that sense the light. Persistent light‐induced stimuli lead to constant turnover of the damaged cellular structures by proteostatic mechanisms such as autophagy. During aging there is a gradual decrease of autophagy which compromises retinal homeostasis and visual function. AMBRA1 (autophagy and beclin‐1 regulator 1) is a key protein involved in the initiation phase of autophagy pathway. To further determine the importance of autophagy along aging, we will use Ambra1+/gt heterozygous mice, mimicking the decrease of autophagy during the aging process.

Methods: We have used young (3 months), middle‐aged (12 months) and old (25 months) Ambra1+/+ and Ambra1+/gt mice for this study. Visual function was determined by electroretinographic recordings (ERGs) along aging. Eyes were enucleated for histological porpoises or retinas were isolated for biochemical procedures. Retinal morphology and cellular components were assessed by immunofluorescence techniques on retinal cryosections or flatmounts. mRNA and protein lysates were obtained from isolated retinas. Metabolomic analyses were conducted using mass spectrometry and bioinformatic analysis was performed.

Results: Ambra1+/gt retinas display a reduced autophagic flux at young without changes in the retinal integrity and function. However, aged Ambra1+/gt animals show an exacerbated loss of visual function also evidenced by morphological changes as increased gliosis, reduced nuclear density and cone number. Interestingly, we observed increased bipolar cell protrusions that might indicate the loss of synapsis between retinal layers. Partial loss of autophagy results in increased damaged mitochondria evidenced by a reduction in the mitochondrial membrane potential (MMP) in young age, higher oxidative stress at middle‐aged and accumulation of mitochondrial mass in old Ambra1+/gt retinas. Finally, by metabolomics, we demonstrate marked metabolic alterations suggestive of defective oxidative metabolism in Ambra1+/gt animals at 1 year of age.

Conclusions: Reduced autophagic response in Ambra1 haploinsufficient retinas leads to an exacerbated age‐associated declines in retinal function, metabolic alterations and accumulation of damaged mitochondria.