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.

Reliability and reproducibility of a rodent model of choroidal neovascularization based on the subretinal injection of polyethylene glycol.

Purpose: To evaluate the reliability and reproducibility of a rodent choroidal neovascularization (CNV) model by subretinal injection of polyethylene glycol (PEG). Methods: C57BL/6 mice were injected subretinally with 2 µl PBS (Gibco, Invitrogen, Paisley, UK; n=14) or PEG (1 mg; n=18). Animals were sacrificed at either 0, 5, 14 or 21 days. Eyes were embedded in paraffin wax and serial sections were stained with haematoxylin and eosin or Fontana-Masson or immunostained for cytokeratin 8/18, isolectin B4 (IB4), vascular endothelial growth factor (VEGF) and von Willebrand factor (vWF). Results: Both the PBS and PEG groups had retinal degeneration and retinal pigment epithelium (RPE)/choroid modifications at 5 and 14 days. Pigment clumps and cell vacuolization at the RPE/choroid were identified as melanin-containing RPE cells. In PEG-injected eyes, CK8/18-positive cellular elements were present at the subretinal space, IB4 immunoreactivity was significantly increased and choroidal vessels appeared diffusely thickened. However, neither VEGF nor vWF (angiogenesis/neovascularization markers) were detected in either group. At 21 days, the retina/choroid of PBS-injected animals was normal in appearance, while retina/choroid changes remained in some PEG-injected mice. Conclusions: Subretinal injection of PEG induced retina/choroid degenerative modifications that mimic the initial steps of human CNV. However, ocular changes were heterogeneous among animals from PBS and PEG groups and did not follow a consistent pattern while most PBS-injected animals showed similar degenerative changes. Abnormal growth of new vessels originating from the choroidal vasculature was not observed. Therefore, we consider that this model does not consistently reproduce CNV and that researchers should choose other rodent models of CNV to avoid misinterpreting their results.

Mesenchymal stem cells provide paracrine neuroprotective resources that delay degeneration of co-cultured organotypic neuroretinal cultures.

Through the paracrine effects of stem cells, including the secretion of neurotrophic, immunomodulatory, and anti-apoptotic factors, cell-based therapies offer a new all-encompassing approach to treatment of neurodegenerative diseases. In this study, we used physically separated co-cultures of porcine neuroretina (NR) and human mesenchymal stem cells (MSC) to evaluate the MSC paracrine neuroprotective effects on NR degeneration. NR explants were obtained from porcine eyes and cultured alone or co-cultured with commercially available MSCs from Valladolid (MSCV; Citospin S.L.; Valladolid, Spain), currently used for several approved treatments. Cultures were maintained for 72 h. MSC surface markers were evaluated before and after co-culture with NRs. Culture supernatants were collected and the concentration of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and glial-derived neurotrophic factor (GDNF) were determined by enzyme-linked immunosorbent assays. NR sections were stained by haematoxylin/eosin or immunostained for terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), glial fibrillary acidic protein, ß-tubulin III, and neuronal nuclei marker. NR morphology, morphometry, nuclei count, apoptosis rate, retinal ganglion cells, and glial cell activation were evaluated. Treatment effects were statistically analysed by parametric or non-parametric tests. The MSCs retained stem cell surface markers after co-culture with NR. BDNF and CNTF concentrations in NR-MSCV co-cultures were higher than other experimental conditions at 72 h (p < 0.05), but no GDNF was detected. NR general morphology, total thickness, and cell counts were broadly preserved in co-cultures, and the apoptosis rate determined by TUNEL assay was lower than for NR monocultures (all p < 0.05). Co-cultures with MSCV also protected retinal ganglion cells from degenerative changes and reduced reactive gliosis (both p < 0.05). In this in vitro model of spontaneous NR degeneration, the presence of co-cultured MSCs retarded neuroglial degeneration. This effect was associated with elevated concentrations of the neurotrophic factors BDNF and CNTF. Our data suggest that the paracrine secretion of these, and possibly other molecules, are a potential resource for the treatment of several neuroretinal diseases.

Biocompatibility of intravitreal injection of human mesenchymal stem cells in immunocompetent rabbits.

PURPOSE: To evaluate the feasibility, safety, and biocompatibility of intravitreal injection of human mesenchymal stem cells (MSCs) in immunocompetent pigmented rabbits. MATERIALS AND METHODS: Thirty-two pigmented rabbits (24 females, 8 males; Chinchilla-New Zealand White) were divided into 8 groups of 4 animals. Commercially prepared human MSCs were injected (0.05 ml) into the post-lens vitreous of the right eyes. Groups 1 and 4 received isotonic medium (Ringer lactate-based), groups 2, 5, 7, and 8 received a low dose of 15 × 106 cells/ml. Groups 3 and 6 received a high dose of 30 × 106 cells/ml. Clinical signs were evaluated and scored before MSCs injection and weekly for 2 or 6 weeks. Animals were sacrificed at 2 or 6 weeks after injection. Eyes, liver, spleen, and gonads were assessed by histology and by fluorescent in situ hybridization to evaluate survival and extraocular migration of MSCs. RESULTS: There were no relevant clinical findings between control and MSC-injected rabbit eyes at any time point. There were also no relevant histological findings between control and MSC-injected rabbits related to ocular, liver, spleen, or gonad tissues modifications. MSCs survived intravitreally for at least 2 weeks after injection. Extraocular migration of MSCs was not detected. CONCLUSIONS: MSCs are safe and well-tolerated when administered intravitreally at a dose of 15 × 106 cells/ml in pigmented rabbits. These findings enable future research to explore the intravitreal use of commercially prepared allogenic human MSCs in clinical trials of retinal diseases.

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.

Clinical exome analysis and targeted gene repair of the c.1354dupT variant in iPSC lines from patients with PROM1-related retinopathies exhibiting diverse phenotypes.

BACKGROUND: Inherited retinal dystrophies (IRD) are one of the main causes of incurable blindness worldwide. IRD are caused by mutations in genes that encode essential proteins for the retina, leading to photoreceptor degeneration and loss of visual function. IRD generates an enormous global financial burden due to the lack of understanding of a significant part of its pathophysiology, molecular diagnosis, and the near absence of non-palliative treatment options. Patient-derived induced pluripotent stem cells (iPSC) for IRD seem to be an excellent option for addressing these questions, serving as exceptional tools for in-depth studies of IRD pathophysiology and testing new therapeutic approaches. METHODS: From a cohort of 8 patients with PROM1-related IRD, we identified 3 patients carrying the same variant (c.1354dupT) but expressing three different IRD phenotypes: Cone and rod dystrophy (CORD), Retinitis pigmentosa (RP), and Stargardt disease type 4 (STGD4). These three target patients, along with one healthy relative from each, underwent comprehensive ophthalmic examinations and their genetic panel study was expanded through clinical exome sequencing (CES). Subsequently, non-integrative patient-derived iPSC were generated and fully characterized. Correction of the c.1354dupT mutation was performed using CRISPR/Cas9, and the genetic restoration of the PROM1 gene was confirmed through flow cytometry and western blotting in the patient-derived iPSC lines. RESULTS: CES revealed that 2 target patients with the c.1354dupT mutation presented monoallelic variants in genes associated with the complement system or photoreceptor differentiation and peroxisome biogenesis disorders, respectively. The pluripotency and functionality of the patient-derived iPSC lines were confirmed, and the correction of the target mutation fully restored the capability of encoding Prominin-1 (CD133) in the genetically repaired patient-derived iPSC lines. CONCLUSIONS: The c.1354dupT mutation in the PROM1 gene is associated to three distinct AR phenotypes of IRD. This pleotropic effect might be related to the influence of monoallelic variants in other genes associated with retinal dystrophies. However, further evidence needs to be provided. Future experiments should include gene-edited patient-derived iPSC due to its potential as disease modelling tools to elucidate this matter in question.

Evaluation of the neuroprotective efficacy of the gramine derivative ITH12657 against NMDA-induced excitotoxicity in the rat retina.

Purpose: The aim of this study was to investigate, the neuroprotective effects of a new Gramine derivative named: ITH12657, in a model of retinal excitotoxicity induced by intravitreal injection of NMDA. Methods: Adult Sprague Dawley rats received an intravitreal injection of 100 mM NMDA in their left eye and were treated daily with subcutaneous injections of ITH12657 or vehicle. The best dose-response, therapeutic window study, and optimal treatment duration of ITH12657 were studied. Based on the best survival of Brn3a + RGCs obtained from the above-mentioned studies, the protective effects of ITH12657 were studied in vivo (retinal thickness and full-field Electroretinography), and ex vivo by quantifying the surviving population of Brn3a + RGCs, αRGCs and their subtypes α-ONsRGCs, α-ONtRGCs, and α-OFFRGCs. Results: Administration of 10 mg/kg ITH12657, starting 12 h before NMDA injection and dispensed for 3 days, resulted in the best significant protection of Brn3a + RGCs against NMDA-induced excitotoxicity. In vivo, ITH12657-treated rats showed significant preservation of retinal thickness and functional protection against NMDA-induced retinal excitotoxicity. Ex vivo results showed that ITH12657 afforded a significant protection against NMDA-induced excitotoxicity for the populations of Brn3a + RGC, αRGC, and αONs-RGC, but not for the population of αOFF-RGC, while the population of α-ONtRGC was fully resistant to NMDA-induced excitotoxicity. Conclusion: Subcutaneous administration of ITH12657 at 10 mg/kg, initiated 12 h before NMDA-induced retinal injury and continued for 3 days, resulted in the best protection of Brn3a + RGCs, αRGC, and αONs-RGC against excitotoxicity-induced RGC death. The population of αOFF-RGCs was extremely sensitive while α-ONtRGCs were fully resistant to NMDA-induced excitotoxicity.

Does PLEX® Elite 9000 OCT Identify and Characterize Most Posterior Pole Lesions in Highly Myopic Patients?

High myopia (HM) is defined as an axial length (AL) ≥ 26 mm that may result in various pathologies that constitute pathologic myopia (PM). The PLEX® Elite 9000 (Carl Zeiss AC, Jena, Germany) is a new swept-source optical coherence tomography (SS-OCT) underdevelopment that allows wider, deeper and more detailed posterior-segment visualization; it can acquire ultra-wide OCT angiography (OCTA) or new ultra-wide high-density scans in one image. We assessed the technology's ability to identify/characterize/quantify staphylomas and posterior pole lesions or image biomarkers in highly myopic Spanish patients and estimate the technology's potential to detect macular pathology. The instrument acquired 6 × 6 OCTA, 12 × 12 or 6 × 6 OCT cubes, and at least two high-definition spotlight single scans. A hundred consecutive patients (179 eyes; age, 51.4 ± 16.8 years; AL, 28.8 ± 2.33 mm) were recruited in one center for this prospective observational study. Six eyes were excluded because images were not acquired. The most common alterations were perforating scleral vessels (88.8%), classifiable staphyloma (68.7%), vascular folds (43%), extrafoveal retinoschisis (24%), dome-shaped macula (15.6%), and more uncommonly, scleral dehiscence (4.46%), intrachoroidal cavitation (3.35%), and macular pit (2.2%). The retinal thickness of these patients decreased, and the foveal avascular zone increased in the superficial plexus compared with normal eyes. SS-OCT is a novel potent tool that can detect most main posterior pole complications in PM and may provide us with a better understanding of the associated pathologies; some pathologies were identifiable only with this new kind of equipment, such as perforating scleral vessels, which seem to be the most common finding and not so frequently related to choroidal neovascularization, as previously reported.

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.

Safety, biocompatibility, and potential functionality of a new accommodative intraocular lens: An experimental study in rabbits.

This study aims to evaluate the safety, biocompatibility, and functionality of a new accommodative intraocular lens (IOL) (LUZ, patent PCT/ES2016/070,813) after implantation in rabbit eyes. LUZ (Study) and EyeCee® plus a capsular ring (Control) were implanted in rabbits (n = 8 each) after phacoemulsification. Intraoperative follow-up, long-term clinical follow-up, and functional IOL studies were carried out periodically for up to 180 days. A macroscopic examination of the eyeballs to reveal abnormalities and determine the implant centering and a microscopic examination to semi-quantify cell and tissue response were performed. Statistical analysis of the collected data was finally achieved. During follow-up, no significant changes in the general condition nor the clinical evaluation were observed between both groups. However, Study IOL remained centered throughout the study and did not present severe complications as observed in the Control group. Functional studies did not reveal significant differences between both materials. Study showed better centering, fewer adhesions, and maintenance of an opening capsular bag compared to the Control. Local biological effects caused by Study implantation are minimal and comparable to the Control. Therefore, LUZ showed no clinical signs or histological response of adverse reaction to the implanted material, according to UNE-EN ISO 11979-5 and 10993-6. Functionality must be confirmed in another animal species with greater lens accommodation capacity than the rabbit. LUZ keeps the capsular bag open, favoring its centering and avoiding fibrosis and adherence to the bag; this allows potential accommodation of this IOL and theoretically enables the patient to focus dynamically.

Time course modifications in organotypic culture of human neuroretina.

The purpose of this study was to characterize organ culture of human neuroretina and to establish survival and early degeneration patterns of neural and glial cells. Sixteen neuroretina explants were prepared from 2 postmortem eyes of 2 individuals. Four explants were used as fresh retina controls, and 12 were evaluated at 3, 6, and 9 days of culture. Neuroretina explants (5 × 5 mm) were cultured in Transwell(®) dishes with the photoreceptor layer facing the supporting membrane. Culture medium (Neurobasal A-based) was maintained in contact with the membrane beneath the explant. Cryostat and ultrathin sections were prepared for immunohistochemistry and electron microscopy. Neuroretinal modifications were evaluated after toluidine blue staining and after immunostaining for neuronal and glial cell markers. Ultrastructural changes were analyzed by electron microscopy. From 0 to 9 days in culture, there was progressive retinal degeneration, including early pyknosis of photoreceptor nuclei, cellular vacuolization in the ganglion cell layer, decrease of both plexiform layer thicknesses, disruption and truncation of photoreceptor outer segments (OS), and marked reduction in the number of nuclei at both nuclear layers where the cells were less densely packed. At 3 days there was swelling of cone OS with impairment of pedicles, loss of axons and dendrites of horizontal and rod bipolar cells that stained for calbindin (CB) and protein kinase C (PKC-α), respectively. After 9 days, horizontal cells were pyknotic and without terminal tips. There were similar degenerative processes in the outer plexiform layer for rod bipolar cells and loss of axon terminal lateral varicosities in the inner plexiform layer. Glial fibrillary acidic protein (GFAP) staining did not reveal a dramatic increase of gliosis in Müller cells. However, some Müller cells were CB immunoreactive at 6 days of culture. Over 9 days of culture, human neuroretina explants underwent morphological changes in photoreceptors, particularly the OS and axon terminals, and in postsynaptic horizontal and bipolar cells. These early changes, not previously described in cultured human samples, reproduce some celullar modifications after retinal damage. Thus, this model may be suitable to evaluate therapeutic agents during retinal degeneration processes.

Factors influencing mesenchymal stromal cells in in vitro cellular models to study retinal pigment epithelial cell rescue.

Mesenchymal stromal cells (MSC) stop or slow retinal pigment epithelium (RPE) and neuroretina (NR) degeneration by paracrine activity in oxidative stress-induced retinal degenerative diseases. However, it is mandatory to develop adequate in vitro models that allow testing new treatment strategies against oxidative stress before performing in vivo studies. The viable double- and triple-layered setups are composed of separate layers of NR, MSC, and RPE (NR-MSC-RPE, NR-RPE, MSC-RPE) partially mimic in vivo retinal conditions. In this study, the paracrine neuroprotective effect of each setup's microenvironment on hydrogen peroxide (H2O2)-stressed was compared with unstressed RPE cells. RPE cell proliferation viability was assessed on day 1, 3, and 6 using Alamar Blue® (10%), MTT (10%) and a cell viability/cytotoxicity assay kit followed by data analysis. The results showed that RPE cells, highly viable (> 90%) in mixed medium of DMEM and neurobasal A (1:1), lost 50% viability on exposure to 400 µM of H2O2 (P < 0.05). The unexposed groups differed significantly from exposed groups for RPE cell growth (RPE and [Formula: see text]RPE (P < 0.0001), NR-MSC-RPE, and NR-MSC-[Formula: see text]RPE (P < 0.05), NR-RPE and NR-[Formula: see text]RPE (P < 0.01), and MSC-RPE and MSC-[Formula: see text]RPE (P < 0.01). NR-[Formula: see text]RPE and NR-RPE supported RPE cell proliferation viability better than other setups (P < 0.01) and RPE cells proliferated 0.49-fold more in NR-MSC-[Formula: see text]RPE than NR-MSC-RPE. Thus, NR and MSC presence improved significantly each setup's microenvironment for cell rescue, nevertheless, each setup also showed limitations for its use as an in vitro study tool. Health of microenvironment of such setups depends on many factors including cell-secreted trophic factors.

Retinal Neuroprotective Effect of Mesenchymal Stem Cells Secretome Through Modulation of Oxidative Stress, Autophagy, and Programmed Cell Death.

Purpose: Degenerative mechanisms of retinal neurodegenerative diseases (RND) share common cellular and molecular signalization pathways. Curative treatment does not exist and cell-based therapy, through the paracrine properties of mesenchymal stem cells (MSC), is a potential unspecific treatment for RND. This study aimed to evaluate the neuroprotective capability of human bone marrow (bm) MSC secretome and its potential to modulate retinal responses to neurodegeneration. Methods: An in vitro model of spontaneous retinal neurodegeneration was used to compare three days of monocultured neuroretina (NR), NR cocultured with bmMSC, and NR cultured with bmMSC secretome. We evaluated retinal morphology markers (Lectin peanut agglutinin, rhodopsin, protein kinase C α isoform, neuronal-specific nuclear protein, glial fibrillary acidic protein, TdT-mediated dUTP nick-end labeling, and vimentin) and proteins involved in apoptosis (apoptosis-inductor factor, caspase-3), necroptosis (MLKL), and autophagy (p62). Besides, we analyzed the relative mRNA expression through qPCR of genes involved in apoptosis (BAX, BCL2, CASP3, CASP8, CASP9), necroptosis (MLKL, RIPK1, RIPK3), autophagy (ATG7, BCLIN1, LC3B, mTOR, SQSTM1), oxidative stress (COX2, CYBA, CYBB, GPX6, SOD1, TXN2, TXNRD1) and inflammation (IL1, IL6, IL10, TGFb1, TNFa). Results: The bmMSC secretome preserves retinal morphology, limits pro-apoptotic- and pro-necroptotic-related gene and protein expression, modulates autophagy-related genes and proteins, and stimulates the activation of antioxidant-associated genes. Conclusions: The neuroprotective ability of the bmMSC secretome is associated with activation of antioxidant machinery, modulation of autophagy, and inhibition of apoptosis and necroptosis during retinal degeneration. The neuroprotective effect of bmMSC secretomes in the presence/absence of MSC looks similar. Our current results reinforce the hypothesis that the human bmMSC secretome slows retinal neurodegeneration and may be a therapeutic option for treating RND.

Programmed Cell Death and Autophagy in an in vitro Model of Spontaneous Neuroretinal Degeneration.

Retinal neurodegenerative diseases are the leading causes of visual impairment and irreversible blindness worldwide. Although the retinal response to injury remains closely similar between different retinal neurodegenerative diseases, available therapeutic alternatives are only palliative, too expensive, or very specific, such as gene therapy. In that sense, the development of broad-spectrum neuroprotective therapies seems to be an excellent option. In this regard, it is essential to identify molecular targets involved in retinal degeneration, such as cell death mechanisms. Apoptosis has been considered as the primary cell death mechanism during retinal degeneration; however, recent studies have demonstrated that the only use of anti-apoptotic drugs is not enough to confer good neuroprotection in terms of cell viability and preservation. For that reason, the interrelationship that exists between apoptosis and other cell death mechanisms needs to be characterized deeply to design future therapeutic options that simultaneously block the main cell death pathways. In that sense, the study aimed to characterize the programmed cell death (in terms of apoptosis and necroptosis) and autophagy response and modulation in retinal neurodegenerative diseases, using an in vitro model of spontaneous retinal neurodegeneration. For that purpose, we measured the mRNA relative expression through qPCR of a selected pool of genes involved in apoptosis (BAX, BCL2, CASP3, CASP8, and CASP9), necroptosis (MLKL, RIPK1, and RIPK3), and autophagy (ATG7, BCLIN1, LC3B, mTOR, and SQSTM1); besides, the immunoexpression of their encoding proteins (Casp3, MLKL, RIPK1, LC3B, and p62) were analyzed using immunohistochemistry. Our results showed an increase of pro-apoptotic and pro-necroptotic related genes and proteins during in vitro retinal neurodegeneration. Besides, we describe for the first time the modulation between programmed cell death mechanisms and autophagy in an in vitro retinal neurodegeneration model. This study reinforces the idea that cell death mechanisms are closely interconnected and provides new information about molecular signaling and autophagy along the retinal degeneration process.

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.

The parameters of the porcine eyeball.

BACKGROUND: The eye of the domestic pig (Sus scrofa domestica) is an ex vivo animal model often used in vision sciences research (retina studies, glaucoma, cataracts, etc.). However, only a few papers have compiled pig eye anatomical descriptions. The purpose of this paper is to describe pig and human eye anatomical parameters to help investigators in their choice of animal model depending on their study objective. METHODS: A wide search of current medical literature was performed (English language) using PubMed. Anteroposterior axial length and corneal radius, astigmatism, vertical and horizontal diameter, and pachymetry (slit-scan and ultrasound) were measured in five enucleated pig eyes of animals 6 to 8 months old. RESULTS: Horizontal corneal diameter was 14.31 ± 0.25 mm (CI 95% 14.03 mm-14.59 mm), vertical diameter was 12.00 ± 0 mm, anteroposterior length was 23.9 ± 0.08 mm (CI 95% 23.01 mm-29.99 mm), central corneal ultrasound pachymetry was 877.6 ± 13.58 µm (CI 95% 865.70 µm-889.50 µm) and slit-scan pachymetry was 906.2 ± 15.30 µm (CI 95% 892.78 µm-919.61 µm). Automatic keratometry (main meridians) was 41.19 ± 1.76D and 38.83 ± 2.89D (CI 95% 40.53D-41.81D and 37.76D-39.89D respectively) with an astigmatism of 2.36 ± 1.70D (CI 95% 1.72D-3.00D), and manual keratometry was 41.05 ± 0.54D and 39.30 ± 1.15D (CI 95% 40.57D-41.52D and 38.29D-40.31D respectively) with an astigmatism of 1.75 ± 1.31D (CI 95% 0.60D-2.90D). CONCLUSION: This paper describes the anatomy of the pig eyeball for easy use and interpretation by researchers who are considering their choice of animal model in vision sciences research.

Ex Vivo Model of Spontaneous Neuroretinal Degeneration for Evaluating Stem Cells' Paracrine Properties.

Ex vivo neuroretina cultures closely resemble in vivo conditions, retaining the complex neuroretina cells dynamics, connections, and functionality, under controlled conditions. Therefore, these models have allowed advancing in the knowledge of retinal physiology and pathobiology over the years. Furthermore, the ex vivo neuroretina models represent an adequate tool for evaluating stem cell therapies over neuroretinal degeneration processes.Here, we describe a physically separated co-culture of neuroretina explants with stem cells to evaluate the effect of stem cells paracrine properties on spontaneous neuroretinal degeneration.

Ex-Vivo Method to Quantifiably Evaluate the Staining Effectiveness of Anterior Lens Capsule Dyes.

Purpose: High frequency of cataracts and the requirements of new European Union regulations for medical devices require the availability of preclinical models to adequately evaluate anterior lens capsule dyes before their use in patients. Herein, we describe an ex vivo method to quantifiably evaluate the macroscopic and microscopic staining effectiveness of anterior lens capsule dyes using porcine eyes. Methods: Commercially available trypan blue-based products or physiological saline solution (negative control) was injected into porcine eyes. Anterior pole and lens (after extraction) were macroscopically photographed, and the images were quantitatively analyzed. Lenses were histologically processed, and the staining intensity microscopically was semiquantified. Results: Macroscopic evaluation of the anterior pole revealed bluish staining of the anterior capsule; however, this coloring cannot be macroscopically discerned after lens extraction. Quantitative image analyses showed significant (P < 0.01) staining of the lens capsule compared to the negative control, but not significant (P > 0.05) between the products tested. Quantitative analysis of dying on lens images could not be performed. Microscopic semiquantification of the capsule staining intensity allows us to appreciate differences between products. Conclusions: The described method is a quick and useful tool for macroscopic evaluation by surgeons to choose an anterior capsule staining for use during everyday surgeries, and a more specific microscopic evaluation also allows us to determine the effectiveness and usefulness of these products. Translational Relevance: This method satisfies preclinical effectiveness evaluations required by European Union regulations and complements the safety and toxicity evaluations that new products must guarantee before they enter the market and are used in clinical practice.