bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiological activation of liver x receptor provides protection against ocular inflammation in uveitic glaucoma
Authors: Choi Jin A. , Lee Ji Young , Ahn Jin‐Hyun , Skiba Nikolai , Rao Vasantha
Published: January 2025

Aims/Purpose: In the pathogenesis of uveitic glaucoma, virus‐induced trabeculitis is considered as a significant cause, markedly by a sudden increase in intraocular pressure (IOP) and relatively mild inflammation in the anterior chamber. In the previous proteome analyses of the aqueous humor derived from the CMV uveitic glaucoma patients using IPA analyses, we observed that the Liver X receptor (LXR) pathway is among the most prominently activated canonical pathways. Thus, we explored the role of LXR pathway in the etiology of glaucoma in association with ocular inflammation.

Methods: Regulation of expression by external cues, and the distribution and secretion of LXR‐ABCA1 by human trabecular meshwork (TM) primary cell cultures and aqueous humor derived from patients with uveitic glaucoma. The effects of LXR agonist were evaluated in the context of LPS‐mediated inflammation in human TM and in a rat model of endotoxin‐induced uveitis.

Results: LXRα/β and ABCA1 were confirmed to be distributed throughout the conventional aqueous humor outflow pathway of the human eye. LXRα/β and ABCA1 were significantly increased in human TM cells in response to CMV infection and LPS. LXR agonists inhibited LPS induction of IL‐1, IL‐6, MCP‐1, and ATX. In both LXRα/LXRβ knockdown cells, LXR agonist induced suppression was reversed, suggesting that such suppression is LXRα or LXRβ receptor mediated. In rat model, LXR agonist significantly reduced infiltrating cells and decreased the expression of proinflammatory cytokines, including TNF‐α, IL‐1, IL‐6, MCP‐1, NFkB2, and TGF‐β1 in iris and retina.

Conclusions: In the conventional outflow tract, LXR‐ABCA molecules were physiologically distributed, showing a robust induction in response to inflammatory insult. Moreover, the activation of LXR exhibited a potent anti‐inflammatory effect, suggesting its physiological protective role in the glaucoma associated with ocular inflammation.
Clinical and genetic profile of patients with leber's congenital amaurosis
Authors: Sahli Esra , Çavdarlı Büşranur , Kızıltunç Pınar Bingöl , Atilla Huban
Published: January 2025

Aims/Purpose: To report clinical examination findings and responsible genetic variants in a cohort of patients with a prediagnosis of Leber's congenital amaurosis (LCA).

Methods: The children with severely decreased visual acuity (20/200 or less), oculo‐digital sign, roving eye movements, high hyperopia, and normal or LCA‐specific fundus appearance at presentation were included in the study. Mutations were revealed using whole exome sequencing to the proband in a family, and Sanger DNA sequencing of the specific mutation to the other affected family members.

Results: Forty‐three patients with a prediagnosis of LCA were included in the study. After excluding 2 patients with Senior‐Loken syndrome (IQCB1 gene mutation), 1 patient with Alström syndrome (ALMS1 gene mutation), and 2 patients without a mutation in a disease‐associated gene, the most common mutated gene was GUCY2D. It was responsible for the disease in 8 patients (21.05%). This was followed by CEP290 gene mutation in 5 patients (13.1%), but the mutation was heterozygous in 1 patient. AIPL1 gene mutation was identified in 4 patients (9.5%). The following mutations were found in the LCA5 gene in 5 patients (13.1%) including two siblings, RPGRIP gene in 3 patients (7.8%) including two siblings, RDH12 gene in 3 siblings (7.8%), RP2 gene in 2 siblings (5.2%), RPE65 gene in 1 patient and SPATA7 gene in 1 patient. Heterozygous mutation in the ABCA4 gene was defined in 3 children and hemizygous mutation in the CACNA1F gene was defined in 1 child with LCA presentation. Children with CEP290 mutation had the worst visual functions, with none having light fixation at baseline examination and only 1 patient having a visual acuity of 20/400, which can be measured at the age of 5 years. Visual functions of patients with mutations in the AIPL1 gene at baseline and follow‐up were limited to light following at 20 cm.

Conclusions: Unlike genes previously reported in the literature for different races, the most frequently mutated gene in this Turkish cohort is GUCY2D.
From leigh syndrome to isolated optic neuropathy: A new piece to the mitochondrial complex I genetic puzzle
Authors: Caporali Leonardo , Fiorini Claudio , Jurkute Neringa , Torraco Alessandra , Yu‐Wai‐Man Patrick , Carelli Valerio , Carrozzo Rosalba , Arno Gavin
Published: January 2025

Aims/Purpose: The genetic landscape of inherited optic neuropathies is continuously expanding and includes numerous nuclear DNA‐encoded genes or loci1, encompassing dominant, recessive, and X‐linked inheritance patterns. Pathogenic variants in respiratory Complex I (CI) genes, typically are associated to severe early onset phenotypes, but have occasionally identified also in syndromic optic atrophy. Therefore, we investigated CI genes to identify pathogenic variants in patients with optic atrophy who previously tested negative for optic atrophy panel screenings.

Methods: We conducted whole exome, clinical exome or whole genome sequencing on all available patients. Those who were found to carry pathogenic variants underwent additional clinical evaluations, including MRI and neurological examinations.

Results: We identified pathogenic or likely pathogenic variants in 24 patients from 19 families, with biallelic or hemizygous variants in 10 different genes coding for subunits or assembly factors of CI. These variants span all major types of peptides involved in the structure of CI, including 3 core subunits (NDUFS7, NDUFV1, NDUFV2), 3 accessory subunits (NDUFA1, NDUFA10, NDUFB11), and 4 assembly factors (NDUFAF2, NDUFAF3, NDUFAF4, NDUFAF8). Notably, defects in core CI subunits lead to isolated optic atrophy, defects in accessory CI subunits resulted in an optic atrophy plus phenotype, and defects in assembly CI factors lead to a spectrum ranging from isolated to syndromic optic atrophy. In some cases, the disease presentation closely resembled LHON, further expanding the list of genes that may cause recessive LHON‐like phenotype.

Conclusions: Pathogenic variants in nuclear CI‐related genes, typically linked to Leigh syndrome, are also common causes of isolated or syndromic optic atrophy. This discovery further broadens the genetic landscape of inherited optic atrophy and LHON.

References: 1Fiorini C et al. The Italian reappraisal of the most frequent genetic defects in hereditary optic neuropathies and the global top 10. Brain. 2023;146(9):e67‐e70. doi:10.1093/brain/awad080
Efficacy and safety of lenadogene nolparvovec gene therapy for leber hereditary optic neuropathy in the real‐life setting
Authors: Yu‐Wai‐Man Patrick , Vignal‐Clermont Catherine , Carelli Valerio , La Morgia Chiara , Moster Mark , Sergott Robert , Donahue Sean , Dollfus Hélène , Klopstock Thomas , Priglinger Claudia , Hage Rabih , Smirnov Vasily , Cochard Catherine , Rougier Marie‐Benedicte , Tournaire‐Marques Emilie , Lebranchu Pierre , Froment Caroline , Pollet‐Villard Frederic , Laville Marie‐Alice , Prospero‐Ponce Claudia , Walter Scott D. , Munier Francis , Zoppe Pauline , Taiel Magali , Sahel José‐Alain
Published: January 2025

Aims/Purpose: Early access programs enable patients with Leber hereditary optic neuropathy (LHON) due to the m.11778G>A MT‐ND4 mutation to receive lenadogene nolparvovec, a gene therapy that has not yet received marketing authorization.

Methods: Lenadogene nolparvovec was provided based on unsolicited requests and its use was authorized by local regulations. Patients received lenadogene nolparvovec in 4 countries (France, Italy, the United Kingdom [UK], and the United States of America [USA]) as a unilateral or bilateral intravitreal injection at 9x1010 viral genomes/eye.

Results: Lenadogene nolparvovec was administered to 63 patients with MT‐ND4 LHON, mainly in France (56%) and the USA (29%); 42 (67%) patients received bilateral injections. At the time of the first injection, mean age (standard deviation [SD]) was 33.7 (16.6) years and mean (SD) duration of disease was 11.4 (9.6) months. Most (84%) patients were treated with idebenone at the same time as or after gene therapy injection. At one year, mean change in best‐corrected visual acuity (BCVA) from nadir was ‐0.42 (0.54) LogMAR (+21 ETDRS letters equivalent) (n = 53 patients). An improvement of at least 0.3 LogMAR from nadir was observed in 61% of patients. Lenadogene nolparvovec safety was favorable and comparable to that of phase 3 clinical trials.

Conclusions: Injection of lenadogene nolparvovec in the real‐life setting was associated with a clinically meaningful improvement in BCVA from nadir and a favorable safety profile similar to that observed in phase 3 clinical trials.
OCT‐ Angiography evaluation of the autonomic influence on the retinal microvasculature in the peripapillary region of optic nerve head
Authors: Marques‐Neves Carlos , Nunes Beatriz , Rocha Isabel , Rosa Michele , Santos Mariana , Lima‐Cabrita Afonso , Sousa David
Published: January 2025

Aims/Purpose: The autonomic nervous system (ANS) is crucial for regulating blood circulation, including ocular circulation, which is vital for vision. Diseases like glaucoma, age‐related macular degeneration, diabetes, and hypertension can disrupt this regulation, impacting retinal blood flow (RBF). Understanding the effects of ANS on RBF could aid in developing targeted and personalized treatments for these conditions.

Objective: This study aims to investigate ANS influence on peripapillary vessel density (PPVD) using optical coherence tomography angiography (OCTA) in healthy individuals.

Methods: OCTA scans of the right eye were performed in 12 healthy individuals at 3 different time points: at the beginning of the study (B), after parasympathetic stimulation‐deep breathing (PSY) and after sympathetic stimulation‐handgrip (SY). Vessel density was measured within a specific circular crown analysis was conducted within a circular crown with an (inner/outer) radius of (0.6/1.19) mm area, using the Erlangen Angio Tool (EA Tool). ANS function was assessed through beat to beat recordings of blood pressure (BP) and ECG signals (Task Force Monitor) immediately before and after each ANS provocation using the Hilbert‐Huang transform as stated in Laranjo et al 2017. Heart rate (HR), cardiac output (CO) and vascular resistance (TPR) were also computed. Statistical analysis was performed using R software and a p < 0.05 was significant.

Results: Changes in the optic nerve head (ONH) circuit were observed during (PSY) increasing 14.09% and (SY) increasing 11.20 % in both ANS provocative manoeuvres (p < 0.05). Mean BP 79.15 (B) mmHg(69.09/83.43), HR (B) 73.9 bp(74.51/76.91), TPR (B) 2448.42 dyne.s.m2.m−5 (1947.0/2393.75) and CO(B) 2,71 l.mn−1.m−2 (2.98/2.85) changed with ANS stimuli (PSY/SY), respectively, in line with the changes in PPVD. A strong linear correlation was observed between the ONH perfusion index and the reciprocal of mean TPR index under both provocative conditions TPR−1versus PPVD 0.5466/0.4771 (PSY/SY) respectively.

Conclusions: ANS input significantly affects ONH blood flow and can be measured with OCTA, reflecting adaptive responses. Understanding ANS effects on PPVD is crucial for developing precise treatments for diseases affecting ANS and ocular conditions.
Myopia macular atrophy in the two‐continent population‐based study
Authors: Panda‐Jonas Songhomitra , Mukharram Bikbov , Gyulli M. , Wang Yaxing , Xu Jie , Jonas Rahul , Jonas Jost
Published: January 2025

Aims/Purpose: To examine myopic macular atrophy (myopic macular degeneration (MMD) stage‐4) and myopic patchy atrophies (MMD‐stage‐3) in three ethnically different cohorts recruited in a population‐based manner.

Methods: The population‐based Ural Eye and Medical Study (UEMS) and the Beijing Eye Study (BES) included individuals aged 40+ years, and the Ural Very Old Study (UVOS) examined individuals aged 85+ years.

Results: Among 5794 UEMS participants, 19 eyes had MMD‐stage‐4, with 17 (89%) eyes showing a foveal BMD; two eyes could not fully be explored. All 19 eyes showed localized SRPs. Among 21 eyes with MMD‐stage‐3, BMD and SRP prevalence was 9/21 (44%) and 7/21 (33%), respectively. Among 930 UVOS participants, 17 eyes had MMD‐stage‐4, with 16 (94%) eyes showing foveal BMDs and SRPs; one eye could not clearly be assessed. Among 18 eyes with MMD‐stage‐3, BMD and SRP prevalence was 3/18 (17%) and 2/18 (11%), respectively. Among 3468 BES participants, 8 eyes had MMD‐stage‐4, with all eyes showing foveal BMDs and SRPs. Among 14 eyes with MMD‐stage‐3, BMD and SRP prevalence was 10/14 (71%) and 7/21 (33%), respectively. In all three study cohorts, BMD and SRP prevalence was higher (P < 0.001) in MMD‐stage‐4 than MMD‐stage‐3. In MMD‐stage‐3, 12 (55%) of 22 eyes with BMDs showed SRPs, while 12 (86%) of 14 eyes with SRP showed BMDs. SRP prevalence correlated with BMD prevalence (OR:78.3;95%CI:16.1,382).

Conclusions: Independent of the ethnic background, all assessable eyes with myopic macular atrophy showed foveal BMDs associated with SRPs, while patchy atrophies could be differentiated into eyes with BMDs and SRPs and eyes without BMDs and without SRPs. The results suggest that SRP as sequel of myopic choroidal neovascularization is a complication of BMDs, and that patchy atrophies may develop due to a primary BMD with secondary RPE defect, or due to an expansion defect in the RPE without concomitant underlying BMD.
Gene editing in ipscs derived from patients affected by inherited retinal dystrophies exhibits rescue of the disease‐associated phenotype
Authors: Siles Laura , Ruiz‐Nogales Sheila , Méndez‐Vendrell Pilar , Pomares Esther
Published: January 2025

Aims/Purpose: Inherited retinal dystrophies (IRD) are rare disorders triggering blindness and other ocular affectations that currently have no effective treatment. Gene editing allows the precise and permanent correction of pathogenic variants in the DNA of the patient itself. By using two different gene editing technologies, we aimed to repair pluripotent stem cell lines (iPSCs) derived from IRD patients.

Methods: We used CRISPR/Cas9 and TALEN methodologies for the correction of mutations in iPSCs from seven patients affected by IRD like Retinitis Pigmentosa or Stargardt disease. Mutant and corrected isogenic cell clones were differentiated into retinal cell models (photoreceptors, RPE cells and retinal organoids) to analyze the potential rescue of the disease‐associated phenotype.

Results: We efficiently corrected all the iPS cell lines and without detected off‐target effects. In the majority of cases, at least 50% of the clones exhibit correction without undesired genomic aberrations. The best gene editing results were in the small insertion/deletion or homozygous mutations, reaching the 80% of efficiency. In addition, the design of the sgRNA guides decisively influence gene editing outcomes, and, in case of heterozygous carriers, the specific recognition of the mutant allele only. Also, we achieved successful gene editing by using the patient's own wild‐type allele and not the exogenous ssODN template. Finally, corrected clones differentiated into several retinal cell models exhibit the rescue of the phenotype associated to the disease.

Conclusions: Gene editing represents a promising tool for the treatment of IRD. TALENs were less effective than CRISPR/Cas9, but they could be useful in cases where CRISPR not due to genomic concerns. Altogether, these data significantly contribute and encourage the study and application of gene editing as a therapeutic strategy for IRD, and suggest an unexpected application of CRISPR‐mediated correction of pathogenic variants in ssODN‐free assays.
Genetically modified mesenchymal stem cells with cxcr4 and tsg‐6 as future cell therapy for the ocular surface: in vitro study of the cell migration and the regenerative capacity in an inflammation model
Authors: Figueroa‐Haro Jessica Nataly , de la Parte Esther González , Marceñido Beatriz , Vázquez Carmen García , de la Rosa Sara Galindo , Paniagua Marina López , Calonge Margarita , López‐Miguel Alberto , Miguel Teresa Nieto , de la Mata Sampedro Ana
Published: January 2025

Aims/Purpose: Regeneration of the corneal epithelium, mediated by limbal epithelial stem cells, is essential for the maintenance of corneal transparency. Loss or dysfunction of these cells or their niche, known as limbal stem cell deficiency (LSCD), causes corneal inflammation, ulceration, neovascularization, chronic pain, and conjunctivalization, leading to corneal blindness. Mesenchymal stem cells (MSCs) are a safe and effective treatment for LSCD, however, there is controversy regarding their migration potential. The aim of this work is to enhance the migration and regenerative capacity of adipose tissue‐derived MSCs (AT‐MSCs) by overexpressing the TNF‐stimulated gene 6 (TSG‐6) and the chemokine receptor CXCR4, while maintaining their phenotype.

Methods: AT‐MSCs were transduced with TSG‐6 and CXCR4 using lentiviral vectors. The immunophenotype of the transduced AT‐MSCs was analyzed evaluating the expression of CD14, CD19, CD34, CD45, HLA‐DR, CD73, CD90 and CD105. Moreover, the migration capacity and the regenerative potential of the modified AT‐MSCs were analyzed using a Transwell cell migration assay in the presence or absence of the pro‐inflammatory cytokine TNFα, and a wound healing assay in human corneal epithelial cells (HCE).

Results: TSG‐6 and CXCR4‐modified AT‐MSCs expressed the MSCs surface markers, maintaining the immunophenotype. Both cells, CXCR4 or TSG‐6‐modified, showed significantly higher migration than control AT‐MSCs in standar conditions. Under inflammatory conditions, a significant increase in the migratory capacity of TSG‐6‐modified AT‐MSCs was observed compared to the control and the CXCR4‐modified ones. The HCE wound healing was faster in the presence of TSG‐6‐modified AT‐MSCs.

Conclusions: TSG‐6 and CXCR4‐modified AT‐MSCs maintain the MSCs immunophenotype and improve their migration capacity under standar conditions. Transduction of AT‐MSCs with TSG‐6 enhances both migratory and regenerative capacity of AT‐MSCs under inflammatory conditions.
Surgical detachment of irido‐ and lenticulocorneal adhesions in peters anomaly
Authors: Kotola Elsa‐Leea , Turunen Joni A. , Lindahl Päivi , Krootila Kari , Majander Anna
Published: January 2025

Aims/Purpose: To report anatomical and visual acuity outcome of surgical detachment of iridocorneal or lenticulocorneal adhesions in pediatric patients with Peters anomaly.

Methods: An observational study of pediatric patients with Peters anomaly treated with surgical detachment of iridocorneal or lenticulocorneal adhesions between 2011 and 2024 at the Helsinki University Hospital. Ocular and extraocular diagnoses, age, sex, best corrected visual acuity (BCVA), intraocular pressure, ocular surgeries, surgical complications, family history, genetics and anterior segment optical coherence tomography data were collected from the medical records.

Results: Fourteen eyes from nine patients underwent the surgery. The median age at the time of surgery was 14 months (range 5‐56) and the median follow‐up period was 156 months (range 6‐166). Of the treated eyes, 12 had iridocorneal adhesions, while two eyes also had lenticulocorneal adhesions. No surgical complications were reported. Anterior segment anatomy and corneal clarity were stable or improved during the follow‐up. Among the fourteen eyes, three eyes achieved a BCVA better than 0.3 (Snellen decimal), while none had a BCVA worse than 0.05. BCVA was not available for three treated eyes due to the patients' young age. The following additional ocular conditions were recorded: microphthalmia, cataract, glaucoma, retinal degeneration, nystagmus, corectopia and strabismus. Four eyes underwent further ocular surgeries, such as cataract surgery with intraocular lens implantation, anterior vitrectomy, pupilloplasty, and temporal iridectomy. Five patients had systemic findings. Pathogenic variants in the PITX3 and RARB genes were detected in three patients and one patient, respectively.

Conclusions: This study demonstrates that surgical detachment of irido‐ and even lenticulocorneal adhesions in Peters anomaly is safe and results stable anatomical and visual acuity outcome over the period of critical visual development.
Extraction and quantification of human tear proteins sampled by schirmer test strips. A validated and standardized method
Authors: Porru Emanuele , Ciavarella Carmen , Comito Rossana , Astolfi Gloria , Violante Francesco , Versura Piera
Published: January 2025

Aims/Purpose: The identification of biomarkers in tears help in diagnosis and management of ocular diseases, and beyond [1]. We aimed to develop and validate a method for extracting and quantifying proteins from human tears collected using Schirmer test strips, which are typically discarded after clinical use.

Methods: Tears from 18 healthy volunteers were collected by aspiration with a micropipette and by paper strips after use for Schirmer test (5 minutes at closed eyes). Aspirated tears were centrifuged and stored at ‐80°C until processing. Tears from strips were eluted using a developed extraction buffer (ABC 100mM, Chaps X‐100 0,25%), and both samples were analyzed using the Agilent 2100 Bioanalyzer. The conversion of the wetted portion (mm) into volume tears (μL) was accurately assessed. The method's accuracy, precision, and sensitivity were estimated, and it was further validated using mass spectrometry.

Results: The elution buffer enabled high recovery values for proteins such as albumin, lysozyme, zinc‐alpha2‐glycoprotein (ZAG), lipocalin A, transferrin, and lactoferrin, as compared to the results from the aspirated tears. The method demonstrated high accuracy (bias%<10%), precision (CV%<10%), and sensitivity, with a matrix effect of less than 5% for the studied proteins. The recovery rates were greater than 90% for lysozyme and albumin, and greater than 80% for ZAG, lipocalin, transferrin, and lactoferrin. Validation using mass spectrometry confirmed the reliability of protein identifications and quantifications.

Conclusions: The developed protocol is simple, cost‐effective, and achieves significant protein recovery, making it suitable for routine clinical analyses in the clinical setting. The method's validation through mass spectrometry highlights its robustness and applicability for identifying and quantifying tear proteins.

References

Rentka, Aniko, et al. "Evaluation of commonly used tear sampling methods and their relevance in subsequent biochemical analysis." Annals of clinical biochemistry 54.5 (2017): 521‐529.
Engineering multicellular organoid for corneal niche modelling and regenerative therapies
Authors: Gulzar Ayesha , Goktan Isilay , Yalcin Esra , Sahin Afsun , Kizilel Seda
Published: January 2025

Purpose: The corneal niche is composed of various cell types, including limbal stem cells (LSCs), corneal epithelial cells (CECs), and neuronal cells. LSCs are pivotal for corneal epithelial regeneration, CECs maintain the corneal transparency, and neuronal cells contribute to sensory and trophic functions. Despite advances in corneal research, the interplay among these cell types within the corneal niche remains insufficiently understood. This study aims to engineer organoids combining LSCs, CECs, and neuronal cells to create a robust model for studying the corneal niche and to explore their transplantation potential.

Methods: LSCs, CECs, and SHSY‐5Y neuronal cells were separately seeded in their growth mediums. After reaching confluency, cells were trypsinized and co‐cultured to form multicellular organoids using the hanging drop technique [1]. Various parameters including cell number, cell‐cell ratio and incubation time were optimized for organoid formation. The organoids were characterized for their cellular composition, structural integrity, and viability using immunohistochemical staining and confocal microscopy. Functional assays, including cell proliferation and differentiation markers, were performed to assess their regenerative potential.

Results: To determine the optimal conditions for organoid formation, we experimented with different ratios of LSCs, CECs, and SHSY‐5Y cells, specifically 1:1:1, 2:2:1, 4:2:1, and 8:2:1. The results showed that the engineered organoids formed under all four ratios exhibited a well‐organized structure with high viability for a week. Functional assays including Ki67 and CD44 revealed active proliferation and differentiation, indicating the regenerative potential of the spheroids.

Conclusions: This study underscores the potential use of tri‐coculture organoid as a promising therapeutic strategy for limbal stem cells deficiency and a valuable tool for investigating the complex cellular interactions within the corneal environment.

References

M. Birgul Akolpoglu, Yasemin Inceoglu, Seda Kizilel, An all‐aqueous approach for physical immobilization of PEG‐lipid microgels on organoid surfaces, Colloids and Surfaces B: Biointerfaces, Volume 186, 2020, 110708, ISSN 0927‐7765, https://doi.org/10.1016/j.colsurfb.2019.110708.
Morphometric histological description of fuchs endothelial dystrophy guttae
Authors: Jonas Rahul , Howaldt Antonia , Matthaei Mario , Schlereth Simona , Bachmann Björn , Cursiefen Claus
Published: January 2025

Aims: Morphometric histological description of Fuchs corneal endothelial dystrophy guttae as well as anterior banded and posterior layer of Descemet's membrane

Methods: After routine DMEK procedures, 47 consecutive explants were taken for histopathological examination as part of clinical routine from 38 patients. 6μm thick sections were cut and stained with PAS and HE. The morphometric analysis was done using a Leica DM‐2500 microscope. Guttae were counted in a line measuring 400μm randomly selected in the middle of the tissue. The largest guttae with the best cross section was used to measure height and width at the base, the middle and the top. The anterior banded layer (ABL) and the posterior (banded and non‐banded as one) layer (PL) were measured below that gutta. The endothelial cells closest to that gutta were measured to get the height and width. Measurements were done using Discus Software (Ingenieursbüro Hilgen). Statistical analysis was done using linear regression on SPSS (Version 29.0 IBM).

Results: The mean amount of guttae in the 400μm section was 11.76 ± 4.8. The mean thickness of ABL was 2.92 ± 0.96μm and of PL 8.59 ± 2.7μm. The guttae measured in height 8.68 ± 2.99μm, in width (base) 14.55 ± 4.22μm, in width (middle) 13.13 ± 4.54μm and in width (top) 14.72 ± 4.69μm. The endothelial cells measured in height 3.28 ± 0.98μm and in width 8.02 ± 2.8μm. The width (middle) of the guttae correlated with the width (base) (p < 0.001), width (top) (p < 0.001) and the width of the endothelial cells (p = 0.041). The PL correlated with the height of the guttae (p = 0.028), which also correlated with the height of the endothelial cells (p = 0.044). The width (top) correlated, additionally to the width (middle), with the width of the endothelial cells (p = 0.031). The PL did not correlate with the age of the patients. Most of the correlations kept their statistical significance after Bonferroni correction.

Conclusions: The thicker the PL was, the higher were the guttae (r2 = 0,14). The wider the width (base) was, the wider the width(top) (r2 = 0,209) and width (middle) (r2 =0,455) was. Considering the observed variability in corneal guttae shape and dimensions even at the same corneal location at, there might be differences in the pathophysiological processes warranting further analysis.
Quality control in corneal eye banking: impact of preservation time on graft endothelium
Authors: Cabanas João Castro , Cardoso Daniel Ferreira , Martins Pedro Moreira , Soares Ricardo Machado , Ribeiro Ana Clara , Arêde Carlos , Sequeira Joaquim
Published: January 2025

Aims/Purpose: To evaluate the impact of preservation duration on the endothelial quality of corneal grafts and to identify the most effective parameters for quality control of these grafts.

Methods: Retrospective analysis of specular microscopy data from corneal grafts collected by the Cornea and Amniotic Membrane Department of Unidade Local de Saúde Gaia e Espinho between 2021 and 2024. According to department protocol, corneal grafts were stored at 4°C in Optisol‐GS (Bausch and Lomb, Rochester, USA) solution, and specular microscopy was performed at 24 hours (D0), day 5 (D5), and day 10 (D10) post‐harvest. Only grafts with at least two specular microscopy exams (D0‐D5) before transplantation were considered. Data collected included donor age, endothelial cell density (ECD), hexagonality (HEX), and coefficient of variation (VC) at all time points. Data was compared across all time points using repeated‐measures ANOVA adjusted for confounders, with a significance level set at 0.05.

Results: Of 93 corneal grafts, 58 met the inclusion criteria, with 12 having specular microscopy analysis at D10. The mean donor age was 61.7 ± 12.9 years. Mean ECD was 2846 ± 434 cells/mm2 at D0, 3025 ± 535 cells/mm2 at D5, and 2880 ± 440 cells/mm2 at D10. Mean HEX was 58.7 ± 11.7% at D0, 51.1 ± 10.5% at D5, and 49.6 ± 12.3% at D10. Mean VC was 34.0 ± 6.7% at D0, 37.9 ± 6.2% at D5, and 38.5 ± 8.7% at D10. Variations in HEX and VC between D0 and D5 were statistically significant (p<0.001), while no significant difference was found between D5 and D10 (p = 0.915 and p = 0.274, respectively). These differences were independent of graft donor age (p>0.05).

Conclusions: In this study, preservation time affects the endothelial quality as represented by the decrease of cell HEX and increased VC, especially in the first days of preservation (D0‐D5) while this effect is more attenuated between D5 and D10. Moreover, these two parameters might be more reliable markers of endothelial cell quality in corneal grafts during preservation compared to ECD as this measure is more prone to exam variation as seen in our results.
In‐situ printing of gelma‐based hydrogels for cornea regeneration
Authors: Goodarzi Hamid , Om Boda , Griffith May , Boutopoulos Christos
Published: January 2025

Aims/Purpose: Corneal perforation is a medical emergency that can lead to blindness. Treatment options encompass cyanoacrylate or fibrin glue, both linked to side effects, including cytotoxicity. Gelatin methacryloyl‐based biomaterials offer an alternative to these adhesives [1, 2]. Achieving optical clarity and smooth surface integrity is crucial for the restoration of vision in treated corneal wounds.

Methods: Currently, the delivery of corneal biomaterials utilizes standard syringe systems, which lack the accuracy to reconstruct the cornea's shape. Therefore, the use of a laser bioprinting technology, developed in the Boutopoulos lab [3], for achieving precise in‐situ corneal wound repair was examined. We used a photocrosslinkable ink comprising Gelatin Methacryloyl (GelMa), hydroxyethyl acrylate (HEA), and Lithium phenyl‐2,4,6‐trimethylbenzoylphosphinate (LAP) as a photoinitiator.

Results: Printability was optimized by generating nanoliter‐volume individual droplets using 230 μJ laser energy, a flow rate 20 microliter per minute, and a temperature of 37°C. Rheology, optical characterization, and bursting pressure measurements assessed its potential to seal corneal perforations. Results indicated a hydrogel storage modulus of 1.31 ± 0.31 KPa for printed LiQD cornea and a bursting pressure of 38 ± 6 mmHg when used to seal full thickness cornea perforation in cadaveric pig eyes. Optical clarity akin to the native cornea was observed (%light transmission: 93.12 ± 1.02 printed vs 92.61 ± 1.50). The OCT results showed that the LIST technique could potentially fill the corneal wounds and reconstruct the natural curvature of the wounded cornea.

Conclusions: In conclusion, a precise printing system for delivering adhesive corneal regenerative biomaterials to wounds was developed and characterized.

References

1. Sharifi, S., et al., 2021. 6(11): p. 3947‐3961.

2. Barroso, I.A., et al., 2022. 9(2).

3. Ebrahimi Orimi, H., et al., Sci Rep, 2020. 10(1): p. 9730.
Do estrogens receptor agonists have a neuroprotective effect on mouse rgc after optic nerve axotomy?
Authors: Rodríguez‐Ramírez Kristy T. , Galindo‐Romero Caridad , Vidal‐Sanz Manuel , Agudo‐Barriuso Marta
Published: January 2025

Aims/Purpose: Estrogens are known to have neuroprotective effects in the central nervous system1,2 (CNS) and only this steroid hormone has the capability of rescue neuronal cell death caused by an injury. Estrogen receptors (ERs) are expressed in both sexes and are distributed throughout the CNS, while the ER‐β is the most predominant in the brain.

The aim of this study is:

1. To compare the course of death of RGCs in female and male mice after optic nerve axotomy during the fast (up to 9 days) and slow (from 9 day onwards) phases of RGC loss.

2. To analyse the expression of ER‐α and ER‐β in intact retinas.

3. To study the neuroprotective effect of ER agonism in the two phases of RGC loss.

Methods: Optic nerve crush (ONC) was performed on the left eye of pigmented male and female mice, and the course of retinal ganglion cell3 (RGC) loss analysed up to 21 days after the injury. In intact retinas of both sexes the number of RGCs expressing the estrogen receptors alpha (ER‐α) and beta (ER‐β) was studied. To test the neuroprotective potential of both ERs, ER‐β or ER‐α agonists1,2 were injected into the vitreous just after ONC and 9 days later, and the retinas analysed at 21 days.

Results: The time course of axotomy‐induced RGC death differs between male and female mice. In males, axotomy‐induced RGC loss is significant after 3 days, whereas in females it is delayed until 5 days. In both sexes, ERs are expressed by more than 70% of RGCs. ER‐β agonism neuroprotects axotomised RGCs at 21 days in both sexes: RGC survival in ER‐β treated animals is 19% higher in males and 17% higher in females than in vehicle treated animals.

Conclusions: The loss of RGCs following axotomy is significant at an earlier in males than in females. RGCs from both sexes express both ERs. Intravitreal administration of ER‐β agonist delays axotomy‐induced RGCs loss during the second, slow phase of death.

References

1. Chakrabarti M., et al. Estrogen receptor agonists for attenuation of neuroinflammation and neurodegeneration. Brain Res Bull. 2014; 109:22‐31.

2. Das A., et al. Estrogen receptor agonists and estrogen attenuate TNF‐α‐induced apoptosis in VSC4.1 motoneurons. J Endocrinol. 2011; 208(2):171‐82.

3. Galindo‐Romero C., et al. Axotomy‐induced retinal ganglion cell death in adult mice: quantitative and topographic time course analyses. Exp Eye Res. 2011; 92(5):377‐87.
Clinical and morphological parallels in the assessment of the lamina cribrosa of sclera as a risk factor and potential target in promotion for survival of optic nerve axons
Authors: Huseva Yuliya
Published: January 2025

Purpose: To study the lamina cribrosa (LC) of sclera in people of different age.

Methods: Longitudinal sections of trepanned fragments of 31 enucleated eyeballs of deceased donors aged from 27 to 81 years stained by Masson trichrome were studied histologically and immunohistochemically with an antibody to glial fibrillary acidic protein (GFAP). The results were compared with optical coherence tomography (OCT) of the optic disc (OD) of 32 eyes of patients aged 34–74 years. Four age groups were identified. The optic nerve (ON) prelaminar and retrolaminar parts and LC of sclera were assessed. The data was statistically approved, the Pearson, Spearman correlation coefficients, the Kruskal‐Wallis and Mann‐Whitney tests were calculated.

Results: Three patterns of LC were identified based on connective and glial tissue ratio: mixed predominated in all age groups; elastic was more common until 44 years (45.5%); collagenous was typical for group III over the age of 60 (40%) and after 75 years (50%). The perivascular proliferation of collagen fibres was mostly after 75 years (group IV), correlated with deformation of LC anterior surface resulting in a decrease in LC elasticity in groups III and IV, increased frequency of axonal deviation in group III (40%) and IV (50%) and a ‘slowing down’ myelination of ON axons (35.5%). The age‐related tendency to LC superficial location, disorganisation of its structure and deformation of its anterior surface in all groups was revealed. The prelaminar zone showed a decreased number of fibroblast processes in groups II and III compared to group I. All ON zones demonstrated immunoreactivity to GFAP, predominantly in the prelaminar determining its susceptibility to ischaemia. Distribution and morphology of GFAP‐positive cells demonstrated that the structure of astrocytes decreased with age.

Conclusions: Age‐related morphological features of the LC beyond 60 years are considered to be risk factors for ischaemia and blockade of axonal transport. The effect of age on LC should be taken into account when predicting the results of treatment of optic neuropathies. The variability of cells in LC opens up new prospects for developing protection and regeneration of ON, in particular, influencing the process of LC remodelling.
Generation of an isogenic control from an induced pluripotent stem cell line of a patient with dominant optic atrophy harbouring the genetic variant C.1024 A>G (P.K342E) in the OPA1 gene
Authors: Jabardo Raquel González , López Marta García , Anda Natalia Robles , Vicente Lydia Jimenez , Monreal Helena Dorado , de Arriba Pablo Rueda , Gallardo Maria Esther
Published: January 2025

Aims/Purpose: Dominant Optic Atrophy (DOA) is an inherited optic neuropathy commonly caused by OPA1 gene mutations, affecting mitochondrial function. The main symptom of patients is vision loss, but the syndromic form of DOA, DOA‐plus, presents with additional manifestations such as deafness and myopathy, among others. The visual impairment occurring in DOA is caused by the degeneration of Retina Ganglion Cells (RGCs), specialized neurons that convey visual information from the retina to the brain. Currently, there is a lack of an effective treatment for DOA patients, which highlights the importance of developing a proper experimental model. Induced Pluripotent Stem Cells (iPSCs) technology could be a promising approach to generate a model of DOA, useful not only for studying mechanisms of disease and therapeutic strategies, but also to confirm if a genetic variant is pathogenic. Our final aim is to determine if a genetic variant in OPA1 gene is pathogenic in a patient with DOA‐plus by the generation of an isogenic human RGC model.

Methods: An iPSC line, OPA1.15, carrying the variant NM_015560.3: c.1024 A>G (p.K342E) in OPA1 gene, obtained from a patient with DOA‐plus was created. Subsequently, a battery of assays was performed to confirm the pluripotency and good quality of the OPA1.15 iPSC line. In this work, as a first step for the creation of the RGC model, the generation of an isogenic control from OPA1.15 with the CRISPR/Cas9 system has been developed.

Results: A bona fide iPSC line from a patient with DOA‐plus has been created. Using this line, an isogenic control was generated with the genome editing tool CRISPR/Cas9.

Conclusions: The iPSC line OPA1.15 and its matched isogenic control will be very useful to generate a RGC model to delve into the pathogenicity of this variant.
Optic nerve damage model: A preliminary study to determine injury duration and degree of gliosis
Authors: Kaleli Humeyra Nur , Kesim Cem , Hasanreisoğlu Murat
Published: January 2025

Aims/Purpose: The aim of this study was to investigate the effects of varying durations of optic nerve compression on retinal function and morphology in an adult DA/HanR rat with optic nerve crush model.

Methods: In the study, adult DA/HanR rats (4‐8 mo) were divided into 5 experimental groups. Lateral canthotomy and lateral bulbar conjunctiva incision were performed to reach the area. The optic nerve was held 2‐3 mm posterior to the globe with the help of calibrated forceps and compressed for four different periods of time: 10 seconds, 15 seconds, 30 seconds and 2 minutes. During the injury, retinal fundus observation of rats was checked with a 90‐diopter fundus lens. To evaluate changes in vision after optic nerve damage, in vivo electrophysiological tests (VEP, EEG and ERG) with daylight stimulator, a light intensity of 500 μW/cm2, was studied. Changes in optic nerve and retina morphology after injury were evaluated by hematoxylin‐eosin (H&E), TUNEL assay, immunofluorescence staining of gliosis and retina cell markers.

Results: It was observed that the response to light was reduced in both the retina (ERG) and visual cortex (VEP‐EEG) in the damaged eyes compared to the control group. Retina cell survival showed decrease in injured groups by TUNEL assay. In immunofluorescence staining, an increase in the severity of gliosis with activation of the microglia and Müller cells in the retina and optic nerve depending on the duration of damage was shown by the expressions of GFAP, Glutamine Synthetase and Vimentin. It was observed that the expression of the neural marker NeuN and the photoreceptor marker Rhodopsin decreased upon injury.

Conclusions: Our study provided preliminary results to further optic nerve regeneration studies to select optimum time points for improve axonal healing in retina and optic nerve injuries.

Funding Information: This work is supported by the Scientific and Technological Research Council of Turkey (TÜBİTAK) under Grant ARDEB‐121S762
Comparison of synchrotron‐based ftir spectra in lens epithelial‐ and idiopathic epiretinal membrane‐ cells
Authors: Josifovska Natasha , Ducic Tanja , Andjelic Sofija , Petrovski Goran
Published: January 2025

Purpose: To determine the synchrotron radiation‐based FTIR microspectroscopy spectrum of lens capsules (LCs) from cataract surgery and that of idiopathic epiretinal membranes (iERMs) towards finding a possible causal relationship between lens fragments falling onto the posterior segment during phacoemulsification procedure and development of iERMs.

Methods: LCs from cataract surgery and iERMs were collected from 5 patients. FTIR measurements were performed at the MIRAS beamline of the ALBA Synchrotron, Barcelona, Spain, and the complete bio‐macromolecular spectral information at cellular level (proteins, lipids, and nucleic acids) in the lens epithelium of LCs from cataract surgery and iERMs were evaluated and compared.

Results: The FTIR spectra revealed several bands corresponding to the vibration of various groups associated with proteins, lipids, and nucleic acids, demonstrating the complex biochemical composition of the LCs and iERMs. Overall, the spectra of the LCs and iERMs were very similar, showing slight differences in the spectral region of the proteins, particularly in the Amide I, then in the carbonyl groups, and phosphate groups of DNA. Additionally, the lipid region showed differences in the spectral ratio between the LCs and iERMs, characterized by CH2 and CH3 bands.

Conclusions: The FTIR spectra of lens epithelial cells on LCs and cells within the iERMs appeared similar, with slight differences in some spectral regions. It is possible that the lens fragments falling onto the retina during cataract surgery could contribute to the pathogenesis of iERMs.
Artificial intelligence‐powered glaucoma screening: Demographic and clinical comparison between screening attendees and non‐attendees
Authors: Lobo Vasco , Lima‐Cabrita Afonso , Monteiro Bernardo , Whitfield Rafael , Theriaga Sofia , Marques Rodrigo , Ourique Matilde , Barão Rafael , Pazos Marta , Stalmans Ingeborg , Abegão‐Pinto Luís
Published: January 2025

Aims/Purpose: Investigate demographic and socio‐economic differences between individuals who accepted and declined participation in a glaucoma screening program in Sete Rios Health Center, Lisbon, and identify factors potentially responsible for decrease in screening adherence.

Methods: Subset analysis of ongoing prospective study (Clinicaltrial.gov: NCT05875090). Diabetic and healthy patients between 55 and 65 years were invited to an AI‐powered glaucoma screening by both telephone and letter. Demographic, socio‐economic and clinical data were collected through both surveys to the patients and through medical records.

Results: 545 subjects (out of 690) were reachable by telephone. 80.0% of those (n = 436) accepted scheduling and roughly 4 out of 5 (72.5%, n = 316) attended the screening visit. Additional 27 people attended screening having received the letter only. Telephone contact revealed overall effectiveness of 45.7% (overall effectiveness of 47.8%). Both screening attendees (S group, n = 343) and non‐attendees (NS group, n = 357) showed homogeneity in age, sex and family history of glaucoma. NS group had a higher percentage of people with more than 12 years of education compared to the S group (42.0% vs 30.5%, p = 0.036). Notably, managers were the professional group which exhibited the highest avoidance rate (47.7%), whereas conversely armed forces occupations showed no refusals. The prevalence of diabetes was significantly higher in the NS group (49.3% vs 32.2%, p = 0.004). Most non‐participants (50.6%) cited prior ophthalmology consultation as their reason for their refusal. Only 15.6% actively refused the screening.

Conclusions: These findings shed light on potential barriers to participation and underscore the importance of tailored approaches to engage diverse populations in preventive eye care initiatives.
Preoperative fluorometholone in glaucoma surgery: 1 year clinical and anatomical outcomes with in vivo confocal microscopy (IVCM)
Authors: Tomaselli Davide , Monsellato Gianluca , Calzi Gaia Li , Fantaguzzi Francesca , Lizzio Umberto , Sacchi Matteo , Nucci Paolo
Published: January 2025

Aims/Purpose: To report preliminary data on the preoperative application of topical Fluorometholone in patients requiring glaucoma surgery due to primary open‐angle glaucoma (POAG), evaluating clinical and anatomical outcomes with in vivo confocal microscopy (IVCM) at 1 year follow‐up.

Methods: We conducted a prospective study involving 54 eyes with POAG requiring glaucoma surgery: Trabeculectomy or Minimally Invasive Bleb Surgery (MIBS) (XEN® and PreserFlo®). The sample was randomized: the treatment group (25 eyes) received Fluorometholone 1mg/ml QID for 1 month pre‐surgery, while the control group (29 eyes) did not. IVCM of the conjunctiva was performed at baseline, after 1 month of treatment, and 1 year post‐surgery. Preoperative parameters included goblet cell density (GCD) and dendritic cell density (DCD) as inflammatory markers, while postoperative parameters were density and mean area of intraepithelial cysts (MMD and MMA) as indices of bleb function. Pre and post‐surgery stromal meshwork reflectivity (SMR) served as an indirect marker of stromal collagen.

Results: Preoperative Fluorometholone led to significantly decreased in DCD (p < 0.001) compared to baseline and in DCD (p < 0.001) and SMR (p = 0.014) compared to the control group. At 1 year, there were no significant differences between the two groups in mean IOP, medications, needling procedures, and failure rates. IVCM analysis showed increased MMD in the treatment group compared to the control group (p = 0.028). In the separate analysis, the MIBS treatment subgroup group had lower IOP (p = 0.046) and less medication use (p = 0.032), but this was not observed in the trabeculectomy treatment subgroup. MMD increased in both the MIBS (p = 0.037) and trabeculectomy (p = 0.024) treatment subgroups.

Conclusions: Preoperative Fluorometholone reduced ocular inflammation, improving bleb function at 1 year as detected by IVCM. However, its impact on overall bleb functionality remains unclear, with a clinically detectable improvement only in the MIBS group. Larger studies with longer follow‐up are necessary to fully understand the implications of preoperative Fluorometholone on long‐term surgical outcomes across different types of glaucoma surgeries.