INTRAOCULAR EMULSION OF SILICONE OIL (ITEMS) GRADING SYSTEM: An Evidence-Based Expert-Led Consensus.

PURPOSE: To propose the InTraocular EMulsion of Silicone oil (ITEMS) grading system for the assessment of silicone oil (SiO) emulsion, applicable in a routine clinical setting and validated through an expert-led consensus procedure. METHODS: Seven experts on intraocular liquid tamponades, led by a facilitator, performed a literature review on the detection of SiO emulsion. Based on the proposed ideas, a questionnaire was developed and submitted to the experts on the methods to detect SiO emulsion and the items to grade. After 2 rounds of individual ranking using a 9-point scale and related discussion, the final grading system was developed including items that reached consensus (score ≥7 from ≥75% of members). RESULTS: The agreed ITEMS grading system includes the identification of SiO microbubbles and large SiO bubbles through slit-lamp biomicroscopy, gonioscopy, fundus examination under mydriasis, or ultra-wide-field fundus photography. Moreover, macular and disk optical coherence tomography are used to detect SiO-associated hyperreflective dots. CONCLUSION: An evidence-based expert-led consensus was conducted to develop grading system of SiO emulsion, allowing, for the first time, homogenous collection of data on SiO emulsion. This has the potential to improve the understanding of the role and clinical relevance of SiO emulsion, allowing comparisons between different studies.

Chemical compounds causing severe acute toxicity in heavy liquids used for intraocular surgery.

Perfluorocarbon liquids (PFCLs) have been considered safe for intraocular manipulation of the retina, but since 2013 many cases of acute eye toxicity cousing blindness have been reported in various countries when using various commercial PFCLs. All these PFCLs were CE marked (Conformité Européenne), which meant they had been subjected to evaluation complying with the International Organization for Standardization (ISO) guidelines. These dramatic events raised questions about the safety of PFCLs and the validity of some cytotoxicity tests performed under ISO guidelines. Samples from toxic batches were analyzed by gas chromatography-mass spectrometry combined with Raman and infrared spectrometry. Perfluorooctanoic acid, dodecafluoro-1-heptanol, ethylbenzene and tributyltin bromide were identified and evaluated by a direct contact cytotoxicity test using ARPE-19 cell line, patented by our group (EP 3467118 A1). Perfluorooctanoic acid at a concentration of >0.06 mM and tributyltin bromide at a concentration of ≥0.016 mM were shown to be toxic, whereas the concentration found in the toxic samples reached 0.48 mM, and 0.111 mM, respectively. These finding emphasized the idea that determination of partially fluorinated compounds are not enough to guarantee the safety of these medical devices.

Matrix Metalloproteinases in Age-Related Macular Degeneration (AMD).

Age-related macular degeneration (AMD) is a complex, multifactorial and progressive retinal disease affecting millions of people worldwide. In developed countries, it is the leading cause of vision loss and legal blindness among the elderly. Although the pathogenesis of AMD is still barely understood, recent studies have reported that disorders in the regulation of the extracellular matrix (ECM) play an important role in its etiopathogenesis. The dynamic metabolism of the ECM is closely regulated by matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs). The present review focuses on the crucial processes that occur at the level of the Bruch's membrane, with special emphasis on MMPs, TIMPs, and the polymorphisms associated with increased susceptibility to AMD development. A systematic literature search was performed, covering the years 1990-2020, using the following keywords: AMD, extracellular matrix, Bruch's membrane, MMPs, TIMPs, and MMPs polymorphisms in AMD. In both early and advanced AMD, the pathological dynamic changes of ECM structural components are caused by the dysfunction of specific regulators and by the influence of other regulatory systems connected with both genetic and environmental factors. Better insight into the pathological role of MMP/TIMP complexes may lead to the development of new strategies for AMD treatment and prevention.

Retinal Toxicity of Medical Devices Used during Vitreoretinal Surgery: A Critical Overview.

Retinal toxicity/biocompatibility of medical devices in direct contact with the retina is an important subject for clinicians and scientists. As these effects are not very frequent, there is also a relative lack of information for many clinicians. The past has taught us multiple times that there is a significant safety problem associated with severe loss of vision in affected patients. In this review, we want to classify medical products that are used in the back of the eye, describe recent examples of toxicity, critically reflect on the regulations that exist and suggest improvements that can be done to ensure patient safety without hindering innovation. METHODS: Critical review of the recent papers and personal experience of the authors in this issue. Medical devices used in the back of the eye and recent examples of toxicity are described, regulations that exist are critically reflected and improvements suggested that can ensure patient safety without hindering innovation. RESULTS: There is clear evidence of toxicity after intraocular surgery in any category. Some cytotoxic indirect methods have failed in detecting this toxicity. Some ISO rules do not seem appropriate. Postmarketing safety is missing. There is little data on this issue. CONCLUSIONS: The absence of a clear regulation of the production, purification and evaluation of the toxic effects of the medical devices supposes the possibility that products are not sufficiently safe to obtain the CE mark.

ACUTE RETINAL DAMAGE AFTER USING A TOXIC PERFLUORO-OCTANE FOR VITREO-RETINAL SURGERY.

PURPOSE: To describe a series of retinal acute toxicity cases with severe visual loss after intraocular use of a toxic perfluoro-octane (PFO). The clinical presentation is described, and the likely causes are analyzed. New biological methods for testing safety of intraocular medical devices are proposed. METHODS: Information regarding a series of eyes suffering acute severe events after intraocular use of a toxic PFO was analyzed. Four types of spectroscopy, nuclear magnetic resonance, and chromatography were used to identify the potential PFO contaminants. Cultures of human retinal pigment epithelial cells (ARPE-19) and porcine neuroretina were used to quantify the toxicity of the suspect PFO lots. RESULTS: Of 117 cases of intraocular toxicity, 96 were considered clearly related to the use of PFO. Fifty-three cases had no light perception, and 97 had no measurable visual acuity. Retinal necrosis (n = 38) and vascular occlusion (n = 33) were the most characteristic findings. Two hydroxyl compounds, perfluorooctanoic acid and dodecafluoro-1-heptanol, and benzene derivatives were identified as the suspected toxic agents. While existing toxicity testing failed, we proposed new tests that demonstrated clear toxicity. CONCLUSION: Protocols to determine cytotoxicity of intraocular medical devices should be revised to assure safety. Acute toxic events should be reported to health authorities and scientific media.

A novel coculture model of porcine central neuroretina explants and retinal pigment epithelium cells.

PURPOSE: To develop and standardize a novel organ culture model using porcine central neuroretina explants and RPE cells separated by a cell culture membrane. METHODS: RPE cells were isolated from porcine eyes, expanded, and seeded on the bottom of cell culture inserts. Neuroretina explants were obtained from the area centralis and cultured alone (controls) on cell culture membranes or supplemented with RPE cells in the same wells but physically separated by the culture membrane. Finally, cellular and tissue specimens were processed for phase contrast, cyto-/histological, and immunochemical evaluation. Neuroretina thickness was also determined. RESULTS: Compared to the neuroretinas cultured alone, the neuroretinas cocultured with RPE cells maintained better tissue structure and cellular organization, displayed better preservation of photoreceptors containing rhodopsin, lower levels of glial fibrillary acidic protein immunoexpression, and preservation of cellular retinaldehyde binding protein both markers of reactive gliosis. Neuroretina thickness was significantly greater in the cocultures. CONCLUSIONS: A coculture model of central porcine neuroretina and RPE cells was successfully developed and standardized. This model mimics a subretinal space and will be useful in studying interactions between the RPE and the neuroretina and to preclinically test potential therapies.

Triple-layered mixed co-culture model of RPE cells with neuroretina for evaluating the neuroprotective effects of adipose-MSCs.

Mesenchymal stem cell (MSC) therapy is promising for neuroprotection but there is no report of an appropriate in vitro model mimicking the situation of the in vivo retina that is able to test the effect of MSCs in suspension or encapsulated with/without a drug combination. This study aims to establish a viable mixed co-culture model having three layers: neuroretina explants (NRs), retinal pigment epithelium (RPE) cells and adipose tissue-derived MSCs (AT-MSCs) for evaluating adipose-MSC effects. AT-MSCs were grown on the lower surface of a transwell membrane and RPE cells were grown on the bottom of a culture plate as monocultures. A transwell membrane was inserted into a culture plate well. NR was placed as an organotypic culture on the upper surface of the transwell membrane. Thus, a triple-layered co-culture setup was constructed. In double-layered setups, NR were co-cultured with AT-MSCs or RPE cells. Optimum medium, experiment execution period and transwell membrane permeability (TMP) were determined. MSC effects on RPE cell proliferation and NR reactive gliosis were evaluated. Limitations were discussed. Our study shows that neurobasal A with DMEM (1:1) mixed medium was suitable for viability of all three layers. AT-MSC growth decreased TMP significantly, 30-60 % in 3- to 6-day periods. Spontaneous NR reactive gliosis limits the experiment execution period to 6 days. AT-MSCs maintained their undifferentiated nature and showed no or limited neuroprotective effects. In this study, we successfully assembled viable double- and triple-layered co-culture setups for AT-MSCs, RPE and NR, optimised conditions for their survival and explored setup Limitations.

External validation of existing formulas to predict the risk of developing proliferative vitreoretinopathy: the Retina 1 Project; report 5.

PURPOSE: To externally validate the accuracy of previously published formulas for predicting proliferative vitreoretinopathy development after retinal detachment surgery. METHODS: Clinical variables from consecutive retinal detachment patients (n = 1,047) were collected from the Retina 1 Project conducted in 17 Spanish and Portuguese centers. These data were used for external validation of four previously published formulas, F1 to F4. Receiver-operating characteristic curves were used to validate the quality of formulas, and measures of discrimination, precision, and calibration were calculated for each. Concordance among the formulas was determined by Cohen kappa index. RESULTS: The areas under the receiver-operating characteristic curves were as follows: F1, 0.5809; F2, 0.5398; F3, 0.5964; and F4, 0.4617. F1 had the highest accuracy, 74.21%. Almost 19% of proliferative vitreoretinopathy cases were correctly classified by F1 compared with 13%, 15%, and 10% for F2, F3, and F4, respectively. There was moderate concordance between F2 and F3 but little between the other formulas. CONCLUSION: After external validation, none of the formulas were accurate enough for routine clinical use. To increase its usefulness, other factors besides the clinical ones considered here should be incorporated into future formulas for predicting risk of developing proliferative vitreoretinopathy.

Mercury intoxication and ophthalmic involvement: An update review.

Human intoxication after mercury exposure is a rare condition that can cause severe damage to the central nervous, respiratory, cardiovascular, renal, gastrointestinal, skin, and visual systems and represents a major public health concern. Ophthalmic involvement includes impaired function of the extraocular muscles and the eyelids, as well as structural changes in the ocular surface, lens, retina, and optic nerve causing a potential irreversible damage to the visual system. Although, there are many pathways for poisoning depending on the mercury form, it has been suggested that tissue distribution does not differ in experimental animals when administered as mercury vapor, organic mercury, or inorganic mercury. Additionally, visual function alterations regarding central visual acuity, color discrimination, contrast sensitivity, visual field and electroretinogram responses have also been described widely. Nevertheless, there is still controversy about whether visual manifestations occur secondary to brain damage or as a direct affectation, and which ocular structure is primarily affected. Despite the use of some imaging techniques such as in vivo confocal microscopy of the cornea, optical coherence tomography (OCT) of the retina and optic nerve, and functional tests such as electroretinography has helped to solve in part this debate, further studies incorporating other imaging modalities such as autofluorescence, OCT angiography or adaptive optics retinal imaging are needed. This review aims to summarize the published structural and functional alterations found in the visual system of patients suffering from mercury intoxication.

Ala®sil chemical characterization and toxicity evaluation: an example of the need for the Medical Device Regulation 2017/745.

Introduction: Ala®sil infusion was on the market for clinical use under the Medical Devices Directive (MDD) 93/42/EEC as an irrigating solution based on polydimethylsiloxane (PDMS). The product was withdrawn in 2016, and to the best of our knowledge, it did not cause any health damage. Methods: A bibliographic review and experimental analysis were conducted to evaluate whether this CE-marked product could have been used in patients under the current Medical Device Regulation (MDR) 2017/745. Analytical results from gas chromatography-mass spectrometry (GC-MS) and matrixassisted laser desorption ionization (MALDI) were performed. Citotoxicity studies were also carried out. Results: Only one study related to Ala®sil clinical use was found, describing a pilot series of five patients. The authors rated the product as not helpful in three out of the five cases for internal searching of retinal breaks and in four out of the five cases for drainage of subretinal fluid. No other scientific papers or documentation was found regarding Ala®sil's safety. Nevertheless, the product was introduced in the market after achieving the CE marking. GC-MS and MALDI showed that the polymer has a low molecular weight of 1,000 g/mol. Several linear and cyclic low-molecular-weight components (LMWCs) were identified as impurities ranging from L3 to D8, with a molecular weight below 600 g/mol. The Ala®sil sample was found to be cytotoxic after 24 h of cell culture but non-cytotoxic after 72 h, probably due to the cellular regeneration capacity of an immortalized cell line. Tissular cytotoxicity revealed an increased apoptosis rate but without morphological modifications. Discussion: Although Ala®sil cannot be classified as cytotoxic, this substance appears to increase retinal cell death processes. This study supports the notion that the MDDwas not functioning adequately to ensure the safety of medical devices. However, the current MDR 2017/745 imposes stricter standards to prevent the commercialization of medical devices without high-quality preclinical and clinical information, as well as precise clinical verification for their use, information not available for Ala®sil infusion.

Quantification of Microvascular Lesions in the Central Retinal Field: Could It Predict the Severity of Diabetic Retinopathy?

Diabetic retinopathy (DR) is a neurodegenerative disease characterized by the presence of microcirculatory lesions. Among them, microaneurysms (MAs) are the first observable hallmark of early ophthalmological changes. The present work aims to study whether the quantification of MAs, hemorrhages (Hmas) and hard exudates (HEs) in the central retinal field could have a predictive value on DR severity. These retinal lesions were quantified in a single field NM-1 of 160 retinographies of diabetic patients from the IOBA's reading center. Samples included different disease severity levels and excluded proliferating forms: no DR (n = 30), mild non-proliferative (n = 30), moderate (n = 50) and severe (n = 50). Quantification of MAs, Hmas, and HEs revealed an increasing trend as DR severity progresses. Differences between severity levels were statistically significant, suggesting that the analysis of the central field provides valuable information on severity level and could be used as a clinical tool to assess DR grading in the eyecare routine. Even though further validation is needed, counting microvascular lesions in a single retinal field can be proposed as a rapid screening system to classify DR patients with different stages of severity according to the international classification.

Trypan Blue staining method for quenching the autofluorescence of RPE cells for improving protein expression analysis.

Retinal pigment epithelial (RPE) cells are currently in the "spotlight" of cell therapy approaches to some retinal diseases. The analysis of the expressed proteins of RPE primary cells is an essential step for many of these approaches. But the emission of autofluorescence by RPE cells produces higher background noise interference thereby creating an impediment to this analysis. Trypan Blue (TB), a routinely used counterstain, has the capacity to quench this autofluorescence, if it is used in optimized concentration. The results from the method developed in our study indicate that incubation of the cultured RPE cells with 20 µg/ml of TB after immunolabelling (post-treatment) as well as incubation of the retinal tissue specimens with same concentration before paraffin embedding, sectioning and immunolabelling (pre-treatment) can be applied to effectively quench the autofluorescence of RPE cells. Thus it can facilitate the evaluation of expressed cellular proteins in experimental as well as in pathological conditions, fulfilling the current requirement for developing a method which can serve to eliminate the autofluorescence of the cells, not only in cell cultures but also in tissues samples. This method should significantly increase the quality and value of RPE cell protein analysis, as well as other cell protein analysis performed by Flow cytometry (FC) and Immunohistochemistry (IHC) techniques.

Rho-Kinase Inhibitors for the Treatment of Refractory Diabetic Macular Oedema.

Diabetic macular oedema (DMO) is one of the leading causes of vision loss associated with diabetic retinopathy (DR). New insights in managing this condition have changed the paradigm in its treatment, with intravitreal injections of antivascular endothelial growth factor (anti-VEGF) having become the standard therapy for DMO worldwide. However, there is no single standard therapy for all patients DMO refractory to anti-VEGF treatment; thus, further investigation is still needed. The key obstacles in developing suitable therapeutics for refractory DMO lie in its complex pathophysiology; therefore, there is an opportunity for further improvements in the progress and applications of new drugs. Previous studies have indicated that Rho-associated kinase (Rho-kinase/ROCK) is an essential molecule in the pathogenesis of DMO. This is why the Rho/ROCK signalling pathway has been proposed as a possible target for new treatments. The present review focuses on the recent progress on the possible role of ROCK and its therapeutic potential in DMO. A systematic literature search was performed, covering the years 1991 to 2021, using the following keywords: "rho-Associated Kinas-es", "Diabetic Retinopathy", "Macular Edema", "Ripasudil", "Fasudil" and "Netarsudil". Better insight into the pathological role of Rho-kinase/ROCK may lead to the development of new strategies for refractory DMO treatment and prevention.

Intraocular toxicity caused by MEROCTANE perfluorocarbon liquid.

Serious intraocular toxicity cases have been reported worldwide after the use of different perfluorocarbon liquids. The current study reports for the first-time the clinical pictures of cases of acute intraocular toxicity caused by MEROCTANE, a perfluoro-octane commercialized by a Turkish company and distributed in many countries. A series of 18 cases from Chile and Spain was retrospectively analysed. To evaluate the impurity profile, a suspicious MEROCTANE sample (lot OCT.01.2013) was analysed by gas chromatography mass spectrometry and compared with a non-suspicious sample of the same commercial perfluoro-octane (lot OCT 722011). Cytotoxicity was tested following a direct-contact method, taking into consideration the high volatility and hydrophobicity of perfluoro-octane and following the ISO 10993 guideline. Cytotoxicity test showed clear cytotoxic effects of the analysed batch (less than 9% of cell viability). Moreover, chemical analysis demonstrated the presence of many contaminants, some highly toxic (acids and alcohols). Perfluorocarbon liquids are useful tools for intraocular surgery but companies and Agencies of Medical Devices must implement measures that guarantee the safety of these products based on both chemical and cytotoxicity analysis for every batch. Medical staff should be encouraged to report any suspected case to their respective National Agencies.

Histological Patterns of Epithelial Alterations in Keratoconus.

PURPOSE: The purpose of this study was to confirm the presence of specific patterns of epithelial response in corneal buttons from keratoconus patients. METHODS: This was a retrospective and descriptive study. 90 penetrating keratoplasty specimens obtained from patients diagnosed with keratoconus were evaluated using bright-field microscopy. Morphologically identifiable characteristics including epithelial cell density and epithelial thickness were analyzed on hematoxylin and eosin- (H&E-) and periodic acid of Schiff- (PAS-) stained slides. RESULTS: Three distinctive patterns of epithelial alteration of the central cornea were established. Pattern 3, in which the central epithelium was as thick as peripheral epithelium, was the commonest (44.4%), followed by the pattern 2, defined as central epithelium thinner than periphery epithelium (38.9%), and the uncommonest pattern was number 1, with central epithelium thicker than the periphery (16.7%). CONCLUSIONS: Three distinctive histologic patterns that could potentially have a diagnostic and prognostic value in keratoconus patients were found.

Evaluation of Potential Pain Biomarkers in Saliva and Pain Perception After Corneal Advanced Surface Ablation Surgery.

PURPOSE: To evaluate the evolution of a set of proposed pain biomarkers in the saliva of subjects following Advanced Surface Ablation (ASA), in order to determine their validity as objective pain measures. METHODS: A multicenter, prospective, and descriptive study was carried out to assess the variations between biomarkers and perceived pain. The Inclusion criteria were healthy subjects who underwent a bilateral, alcohol-assisted surface ablation with epithelial removal (ASA). Pain intensity before and after surgery was assessed by Visual Analog Scale (VAS) and the Numeric Pain Rating Scale (NPRS). Cortisol, sAA, sIgA, testosterone, and sTNFαRII were assayed at four-time points (V0, baseline; V1, pre-surgery; V2, 1 hr post-surgery, and V3, 72 hrs post-surgery). Comorbidities and Hospital Anxiety and Depression (HADS) questionnaires were administrated before and at 6 hrs after the surgery. All patients were treated with cold patches, topical steroids, topical cold antibiotics, and benzodiazepines after ASA surgery. A descriptive analysis of biomarkers and pain intensity evolution and the agreement between biomarkers and pain was performed. RESULTS: Concentration of sIgA and sTNFαRII post-surgery was significantly higher at each visit compared to baseline (p-value: 0.053, p-value: <0.001, respectively). Relations between VAS scale score and putative biomarker variations were not statistically significant except for the sIgA but only at visit 0 (p-value: 0.024). The HADS questionnaire showed anxiety scores between 0 and 7 in all patients before and at 6 hrs after surgery. CONCLUSION: In this study, sIgA and sTNFαRII are the two potential biomarkers that present correlation with the VAS and these salivary substances showed acceptable levels of reproducibility in healthy subjects.

Early prevention of diabetes microvascular complications in people with hyperglycaemia in Europe. ePREDICE randomized trial. Study protocol, recruitment and selected baseline data.

OBJECTIVES: To assess the effects of early management of hyperglycaemia with antidiabetic drugs plus lifestyle intervention compared with lifestyle alone, on microvascular function in adults with pre-diabetes. METHODS: Trial design: International, multicenter, randomised, partially double-blind, placebo-controlled, clinical trial. PARTICIPANTS: Males and females aged 45-74 years with IFG, IGT or IFG+IGT, recruited from primary care centres in Australia, Austria, Bulgaria, Greece, Kuwait, Poland, Serbia, Spain and Turkey. INTERVENTION: Participants were randomized to placebo; metformin 1.700 mg/day; linagliptin 5 mg/day or fixed-dose combination of linagliptin/metformin. All patients were enrolled in a lifestyle intervention program (diet and physical activity). Drug intervention will last 2 years. Primary Outcome: composite end-point of diabetic retinopathy estimated by the Early Treatment Diabetic Retinopathy Study Score, urinary albumin to creatinine ratio, and skin conductance in feet estimated by the sudomotor index. Secondary outcomes in a subsample include insulin sensitivity, beta-cell function, biomarkers of inflammation and fatty liver disease, quality of life, cognitive function, depressive symptoms and endothelial function. RESULTS: One thousand three hundred ninety one individuals with hyperglycaemia were assessed for eligibility, 424 excluded after screening, 967 allocated to placebo, metformin, linagliptin or to fixed-dose combination of metformin + linagliptin. A total of 809 people (91.1%) accepted and initiated the assigned treatment. Study sample after randomization was well balanced among the four groups. No statistical differences for the main risk factors analysed were observed between those accepting or rejecting treatment initiation. At baseline prevalence of diabetic retinopathy was 4.2%, severe neuropathy 5.3% and nephropathy 5.7%. CONCLUSIONS: ePREDICE is the first -randomized clinical trial with the aim to assess effects of different interventions (lifestyle and pharmacological) on microvascular function in people with pre-diabetes. The trial will provide novel data on lifestyle modification combined with glucose lowering drugs for the prevention of early microvascular complications and diabetes. REGISTRATION: - ClinicalTrials.Gov Identifier: NCT03222765 - EUDRACT Registry Number: 2013-000418-39.

Comparison between direct contact and extract exposure methods for PFO cytotoxicity evaluation.

A series of recent acute blindness cases following non-complicated retinal detachment surgery caused the release of several health alerts in Spain. The blindness was attributed to certain lots of perfluoro-octane (PFO; a volatile and transient medical device). Similar cases have been reported in other countries. This has raised questions regarding the validity of cytotoxicity test methods currently used to certify the safety of PFO lots. The tests were performed according to the International Organization for Standardization (ISO) norms, using the extract dilution method or the indirect contact method as applied to L929 cells, a line derived from mouse fibroblasts. The limitations of those methods have been resolved in this study by proposing a new cytotoxicity test method for volatile substances. The new method requires direct contact of the tested substance with cells that are similar to those exposed to the substance in the clinical setting. This approach includes a few new technical steps that are crucial for detecting cytotoxicity. Our new method detected toxic PFO lots that corresponded to the lots producing clinical blindness, which previous methods failed to detect. The study suggests applying this new method to avoid occurrence of such cases of blindness.