Short-Wavelength Light-Blocking Filters and Oral Melatonin Administration in Patients With Retinitis Pigmentosa: Protocol for a Randomized Controlled Trial.

BACKGROUND: The medical community is beginning to recognize that retinitis pigmentosa (RP), due to its disabling progression, eventually leads to a reduction in the patient´s quality of life, a direct economic impact, and an increase in the burden on the health care system. There is no curative treatment for the origin of the disease, and most of the current interventions fail in reducing the associated negative psychological states, such as anxiety and depression, which lead to increased variability of vision and pose a continuous threat to the patient's independence. OBJECTIVE: The aim of this study is to assess the effect of oral melatonin (OM) administration alone and combined with short-wavelength light (SWL)-blocking filters on patients with RP and test their effectiveness in improving the level of stress and sleep problems in many of these patients. METHODS: We have developed a low-cost therapy protocol for patients with RP with sleep disorders and negative psychological stress. Patients will be randomized to receive a combined intervention with SWL-blocking filters and OM, SWL-blocking filters alone, or OM alone. There will also be a nonintervention arm as a control group. This study will be conducted across 2 retinal units in patients with RP with sleep disorders and high perceived stress and anxiety score reports. Patients will be assessed in the preintervention period, weekly during the 4 weeks of intervention, and then at 6 months postintervention. The primary outcomes are the differences in changes from baseline to postintervention in hormone release (α-amylase, cortisol, and melatonin) and sleep quality, as measured with the visual analog scale. Secondary outcome measures include clinical macular changes, as measured with optical coherence tomography and optical coherence tomography angiography; retinal function, as measured using the visual field and best-corrected visual acuity; sleep data collected from personal wearables; and several patient-reported variables, such as self-recorded sleep diaries, quality of life, perceived stress, and functional status. RESULTS: This project is still a study protocol and has not yet started. Bibliographic research for information for its justification began in 2020, and this working group is currently seeking start-up funding. As soon as we have the necessary means, we will proceed with the registration and organization prior to the preliminary phase. CONCLUSIONS: In this feasibility randomized clinical controlled trial, we will compare the effects of SWL blocking alone, administration of OM alone, and a combined intervention with both in patients with RP. We present this study so that it may be replicated and incorporated into future studies at other institutions, as well as applied to additional inherited retinal dystrophies. The goal of presenting this protocol is to aid recent efforts in reducing the impact of sleeping disorders and other psychological disorders on the quality of life in patients with RP and recovering their self-autonomy. In addition, the results of this study will represent a significant step toward developing a novel low-cost therapy for patients with RP and validating a novel therapeutic target. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/49196.

Deep Learning Application to Detect Glaucoma with a Mixed Training Approach: Public Database and Expert-Labeled Glaucoma Population.

INTRODUCTION: Artificial intelligence has real potential for early identification of ocular diseases such as glaucoma. An important challenge is the requirement for large databases properly selected, which are not easily obtained. We used a relatively original strategy: a glaucoma recognition algorithm trained with fundus images from public databases and then tested and retrained with a carefully selected patient database. METHODS: The study's supervised deep learning method was an adapted version of the ResNet-50 architecture previously trained from 10,658 optic head images (glaucomatous or non-glaucomatous) from seven public databases. A total of 1,158 new images labeled by experts from 616 patients were added. The images were categorized after clinical examination including visual fields in 304 (26%) control images or those with ocular hypertension and 347 (30%) images with early, 290 (25%) with moderate, and 217 (19%) with advanced glaucoma. The initial algorithm was tested using 30% of the selected glaucoma database and then re-trained with 70% of this database and tested again. RESULTS: The results in the initial sample showed an area under the curve (AUC) of 76% for all images, and 66% for early, 82% for moderate, and 84% for advanced glaucoma. After retraining the algorithm, the respective AUC results were 82%, 72%, 89%, and 91%. CONCLUSION: Using combined data from public databases and data selected and labeled by experts facilitated improvement of the system's precision and identified interesting possibilities for obtaining tools for automatic screening of glaucomatous eyes more affordably.

Towards the international interoperability of clinical research networks for rare diseases: recommendations from the IRDiRC Task Force.

BACKGROUND: Many patients with rare diseases are still lacking a timely diagnosis and approved therapies for their condition despite the tremendous efforts of the research community, biopharmaceutical, medical device industries, and patient support groups. The development of clinical research networks for rare diseases offers a tremendous opportunity for patients and multi-disciplinary teams to collaborate, share expertise, gain better understanding on specific rare diseases, and accelerate clinical research and innovation. Clinical Research Networks have been developed at a national or continental level, but global collaborative efforts to connect them are still lacking. The International Rare Diseases Research Consortium set a Task Force on Clinical Research Networks for Rare Diseases with the objective to analyse the structure and attributes of these networks and to identify the barriers and needs preventing their international collaboration. The Task Force created a survey and sent it to pre-identified clinical research networks located worldwide. RESULTS: A total of 34 responses were received. The survey analysis demonstrated that clinical research networks are diverse in their membership composition and emphasize community partnerships including patient groups, health care providers and researchers. The sustainability of the networks is mostly supported by public funding. Activities and research carried out at the networks span the research continuum from basic to clinical to translational research studies. Key elements and infrastructures conducive to collaboration are well adopted by the networks, but barriers to international interoperability are clearly identified. These hurdles can be grouped into five categories: funding limitation; lack of harmonization in regulatory and contracting process; need for common tools and data standards; need for a governance framework and coordination structures; and lack of awareness and robust interactions between networks. CONCLUSIONS: Through this analysis, the Task Force identified key elements that should support both developing and established clinical research networks for rare diseases in implementing the appropriate structures to achieve international interoperability worldwide. A global roadmap of actions and a specific research agenda, as suggested by this group, provides a platform to identify common goals between these networks.

Reduction in the Risk of Peripheral Neuropathy and Lower Decrease in Kidney Function with Metformin, Linagliptin or Their Fixed-Dose Combination Compared to Placebo in Prediabetes: A Randomized Controlled Trial.

OBJECTIVE: To compare the effect of glucose-lowering drugs on peripheral nerve and kidney function in prediabetes. METHODS: Multicenter, randomized, placebo-controlled trial in 658 adults with prediabetes treated for 1 year with metformin, linagliptin, their combination or placebo. Endpoints are small fiber peripheral neuropathy (SFPN) risk estimated by foot electrochemical skin conductance (FESC < 70 µSiemens) and estimated glomerular filtration rate (eGFR). RESULTS: Compared to the placebo, the proportion of SFPN was reduced by 25.1% (95% CI:16.3-33.9) with metformin alone, by 17.3% (95% CI 7.4-27.2) with linagliptin alone, and by 19.5% (95% CI 10.1-29.0) with the combination linagliptin/metformin (p < 0.0001 for all comparisons). eGFR remained +3.3 mL/min (95% CI: 0.38-6.22) higher with the combination linagliptin/metformin than with the placebo (p = 0.03). Fasting plasma glucose (FPG) decreased more with metformin monotherapy -0.3 mmol/L (95%CI: -0.48; 0.12, p = 0.0009) and with the combination metformin/linagliptin -0.2 mmol/L (95% CI: -0.37; -0.03) than with the placebo (p = 0.0219). Body weight (BW) decreased by -2.0 kg (95% CI: -5.65; -1.65, p = 0.0006) with metformin monotherapy, and by -1.9 kg (95% CI: -3.02; -0.97) with the combination metformin/linagliptin as compared to the placebo (p = 0.0002). CONCLUSIONS: in people with prediabetes, a 1 year treatment with metformin and linagliptin, combined or in monotherapy, was associated with a lower risk of SFPN, and with a lower decrease in eGFR, than treatment with placebo.

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.

Acute intraocular toxicity caused by perfluorocarbon liquids: safety control systems of medical devices.

BACKGROUND: Acute retinal toxicity has been demonstrated to be associated with the intraoperative use of perfluorocarbon liquids (PFCLs), especially perfluorooctane (PFO). Recently, several cases of PFO-associated blindness have been reported in Spain, Holland, France, Italy, the Middle East, and South America. METHODS: As a result, a new ISO guideline (ISO 16672:2020) was drafted, discussed, approved, and released in 2019. This recent ISO16672:2020 guideline recommends performing direct cytotoxicity tests as an option along with chemical analysis to measure PFCL quality (purity and safety). RESULTS: In this review paper, it has been emphasized why an appropriate biological test, specifically direct exposure of PFCL to live cells, for measuring cytotoxicity must be performed with each PFCL batch along with chemical analysis. CONCLUSIONS: The paper intends to compile all available information to discuss possible approaches for avoiding adverse clinical cases in future.

Complications associated with the use of silicone oil in vitreoretinal surgery: A systemic review and meta-analysis.

Silicone oil (SO) still represents the main choice for long-term intraocular tamponade in complicated vitreoretinal surgery. This review compared the complications associated with the use of SO and other vitreous substitutes after pars plana vitrectomy in patients with different underlying diseases. Meta-analysis was conducted in accordance with PRISMA guidelines. We retrieved randomized clinical trials (RCTs), retrospective case-control and cohort studies evaluating the risk of using SO, published between 1994 and 2020, conducting a computer-based search of the following databases: PubMed, Web of Science, Scopus and Embase. Primary outcome was the rate of complications such as intraocular hypertension, retinal re-detachment, unexpected vision loss or hypotony. Secondary outcome was to compare the rate of adverse events of different SO viscosities, especially emulsification. Forty-three articles were included. There were significant differences in intraocular hypertension (p = 0.0002, OR = 1.66; 95% CI = 1.27-2.18) and the rate of retinal re-detachment (p < 0.0009, OR = 0.65; 95% CI = 0.50-0.64) between SO and other agents, including placebo. However, there were no differences in other complication rates. Silicone oil (SO)-emulsification rate was non-significantly higher in low than high SO viscosity, and results from other complications were comparable in both groups. The high quality of most of the studies included in this study is noteworthy, which provides some certainty to the conclusions. Among them is the high variability of the SO residence time. The fact that ocular hypertension and not hypotension is related to SO use. A clear relationship is not found for the so-called unexplained vision loss, which affects a significant percentage of eyes. Re-detachment cases are less if SO is used and that surprisingly there does not seem to be a relationship in the percentage of emulsification between the low- and high-viscosity silicones. All these data warrant more standardized prospective studies.

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.

Cell Replacement Therapy for Retinal and Optic Nerve Diseases: Cell Sources, Clinical Trials and Challenges.

The aim of this review was to provide an update on the potential of cell therapies to restore or replace damaged and/or lost cells in retinal degenerative and optic nerve diseases, describing the available cell sources and the challenges involved in such treatments when these techniques are applied in real clinical practice. Sources include human fetal retinal stem cells, allogenic cadaveric human cells, adult hippocampal neural stem cells, human CNS stem cells, ciliary pigmented epithelial cells, limbal stem cells, retinal progenitor cells (RPCs), human pluripotent stem cells (PSCs) (including both human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs)) and mesenchymal stem cells (MSCs). Of these, RPCs, PSCs and MSCs have already entered early-stage clinical trials since they can all differentiate into RPE, photoreceptors or ganglion cells, and have demonstrated safety, while showing some indicators of efficacy. Stem/progenitor cell therapies for retinal diseases still have some drawbacks, such as the inhibition of proliferation and/or differentiation in vitro (with the exception of RPE) and the limited long-term survival and functioning of grafts in vivo. Some other issues remain to be solved concerning the clinical translation of cell-based therapy, including (1) the ability to enrich for specific retinal subtypes; (2) cell survival; (3) cell delivery, which may need to incorporate a scaffold to induce correct cell polarization, which increases the size of the retinotomy in surgery and, therefore, the chance of severe complications; (4) the need to induce a localized retinal detachment to perform the subretinal placement of the transplanted cell; (5) the evaluation of the risk of tumor formation caused by the undifferentiated stem cells and prolific progenitor cells. Despite these challenges, stem/progenitor cells represent the most promising strategy for retinal and optic nerve disease treatment in the near future, and therapeutics assisted by gene techniques, neuroprotective compounds and artificial devices can be applied to fulfil clinical needs.

OCT Variability Prevents Their Use as Robust Biomarkers in Multiple Sclerosis.

OBJECTIVE: To evaluate the agreement between the peripapillary retinal nerve fiber layer (pRNFL) and foveal thickness (FT) measurements among three different spectral domain-optical coherence tomography (SD-OCT) instruments in a sample of multiple sclerosis (MS) patients and a healthy age-matched control group. METHODS: An observational cross-sectional study with three groups: healthy subjects and MS patients w/w a previous clinical diagnosis of optic neuritis (ON) was conducted. The pRNFL and FT were measured using three different SD-OCT instruments (OCT PRIMUS 200 and OCT CIRRUS 500 SD-OCT [Carl Zeiss Meditec] and OCT 3D 2000 [Topcon]). RESULTS: Twenty eyes from 10 healthy subjects matched in age with MS patients without a previous history of eye disease and 62 MS eyes from 31 MS patients (29 eyes without history of ON and 33 eyes with history of ON) were enrolled. Healthy subjects and MS patients without ON did not show differences between the pRNFL and FT thickness (P>0.99) with any of the instruments. However, MS eyes with a previous episode of ON showed thinner pRNFL and FT (P<0.01). PRIMUS and CIRRUS OCT showed better agreement of the pRNLF and FT in both healthy and MS eyes. However, 3D OCT showed less agreement in the pRNFL measurement with CIRRUS in both healthy and MS eyes. INTERPRETATION: Although OCT is a valuable technology to improve MS patient assessment, differences between devices must be taken into account. It is necessary to create an international group that standardizes the measurement conditions and above all that provides reference bases for normal subjects.

Impact of contact lens wear on epithelial alterations in keratoconus

PURPOSE: The purpose of this study was to characterize the central epithelial thickness (CET) of penetrating keratoplasty corneal specimens obtained from patients with keratoconus (KC) and correlate the histological patterns with their clinical history. METHODS: Ex vivo histological imaging was performed to measure CET and total corneal thickness (TCT) in 56 patients with KC. Microscopic slides from penetrating keratoplasty corneal specimens, stained with hematoxylin and eosin were evaluated using bright field microscopy. CET and TCT were measured, and morphological features were studied. Clinical history regarding duration of KC prior to surgery and length of and tolerance to contact lens wear were compared and analyzed. RESULTS: The microscopic slides of all patients available for follow up (n = 48) were analyzed and CET and TCT were measured. The histological evaluation revealed 3 distinctive epithelial patterns. Pattern 1 with central hypertrophic and hydropic changes (n = 19) measured 70.89 ± 25.88 Mum in CET and 308.63 ± 100.74 Mum in TCT; Pattern 2 (n = 14) had not changed, similar to normal epithelium CET and TCT measuring 36.5 ± 7.02 Mum and 260.14 ± 87.93 Mum respectively. Pattern 3 (n = 15) demonstrated thinner central epithelium characterized by atrophy and focal hydropic changes measuring 19.93 ± 4.60 Mum and 268.00 ± 79.39 Mum in CET and TCT respectively (all p < 0.0001). The presence of Pattern 2 characterized by similar to normal CET was correlated with the duration of the condition (R = 0.600, p = 0.030). There was a significant difference in the length of CL wear comparing those with patterns 1 and 2 versus 3 (least no. of CL years) (p = 0.05 and p = 0.33 respectivelly). CONCLUSIONS: Patients with advanced disease have various central corneal epithelial changes detected with histology. Although each central epithelial pattern type was distinctive comparing the 3 patterns, there was no correlation with years of CL wear but only with the duration of the condition

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Impact of contact lens wear on epithelial alterations in keratoconus.

PURPOSE: The purpose of this study was to characterize the central epithelial thickness (CET) of penetrating keratoplasty corneal specimens obtained from patients with keratoconus (KC) and correlate the histological patterns with their clinical history. METHODS: Ex vivo histological imaging was performed to measure CET and total corneal thickness (TCT) in 56 patients with KC. Microscopic slides from penetrating keratoplasty corneal specimens, stained with hematoxylin and eosin were evaluated using bright field microscopy. CET and TCT were measured, and morphological features were studied. Clinical history regarding duration of KC prior to surgery and length of and tolerance to contact lens wear were compared and analyzed. RESULTS: The microscopic slides of all patients available for follow up (n=48) were analyzed and CET and TCT were measured. The histological evaluation revealed 3 distinctive epithelial patterns. Pattern 1 with central hypertrophic and hydropic changes (n=19) measured 70.89±25.88µm in CET and 308.63±100.74µm in TCT; Pattern 2 (n=14) had not changed, similar to normal epithelium CET and TCT measuring 36.5±7.02µm and 260.14±87.93µm respectively. Pattern 3 (n=15) demonstrated thinner central epithelium characterized by atrophy and focal hydropic changes measuring 19.93±4.60µm and 268.00±79.39µm in CET and TCT respectively (all p<0.0001). The presence of Pattern 2 characterized by similar to normal CET was correlated with the duration of the condition (R=0.600, p=0.030). There was a significant difference in the length of CL wear comparing those with patterns 1 and 2 versus 3 (least no. of CL years) (p=0.05 and p=0.33 respectivelly). CONCLUSIONS: Patients with advanced disease have various central corneal epithelial changes detected with histology. Although each central epithelial pattern type was distinctive comparing the 3 patterns, there was no correlation with years of CL wear but only with the duration of the condition.

Mesenchymal Stem Cell Secretome Enhancement by Nicotinamide and Vasoactive Intestinal Peptide: A New Therapeutic Approach for Retinal Degenerative Diseases.

Mesenchymal stem cells (MSC) secrete neuroprotective molecules that may be useful as an alternative to cell transplantation itself. Our purpose was to develop different pharmaceutical compositions based on conditioned medium (CM) of adipose MSC (aMSC) stimulated by and/or combined with nicotinamide (NIC), vasoactive intestinal peptide (VIP), or both factors; and to evaluate in vitro their proliferative and neuroprotective potential. Nine pharmaceutical compositions were developed from 3 experimental approaches: (1) unstimulated aMSC-CM collected and combined with NIC, VIP, or both factors (NIC+VIP), referred to as the aMSC-CM combined composition; (2) aMSC-CM collected just after stimulation with the mentioned factors and containing them, referred to as the aMSC-CM stimulated-combined composition; and (3) aMSC-CM previously stimulated with the factors, referred to as the aMSC stimulated composition. The potential of the pharmaceutical compositions to increase cell proliferation under oxidative stress and neuroprotection were evaluated in vitro by using a subacute oxidative stress model of retinal pigment epithelium cells (line ARPE-19) and spontaneous degenerative neuroretina model. Results showed that oxidatively stressed ARPE-19 cells exposed to aMSC-CM stimulated and stimulated-combined with NIC or NIC+VIP tended to have better recovery from the oxidative stress status. Neuroretinal explants cultured with aMSC-CM stimulated-combined with NIC+VIP had better preservation of the neuroretinal morphology, mainly photoreceptors, and a lower degree of glial cell activation. In conclusion, aMSC-CM stimulated-combined with NIC+VIP contributed to improving the proliferative and neuroprotective properties of the aMSC secretome. Further studies are necessary to evaluate higher concentrations of the drugs and to characterize specifically the aMSC-secreted factors related to neuroprotection. However, this study supports the possibility of improving the potential of new effective pharmaceutical compositions based on the secretome of MSC plus exogenous factors or drugs without the need to inject cells into the eye, which can be very useful in retinal pathologies.

Human Mesenchymal Stem Cell Secretome Exhibits a Neuroprotective Effect over In Vitro Retinal Photoreceptor Degeneration.

Retinal photoreceptor degeneration occurs frequently in several neurodegenerative retinal diseases such as age-related macular degeneration, retinitis pigmentosa, or genetic retinal diseases related to the photoreceptors. Despite the impact on daily life and the social and economic consequences, there is no cure for these diseases. Considering this, cell-based therapy may be an optimal therapeutic option. This study evaluated the neuroprotective in vitro potential of a secretome of human bone marrow mesenchymal stem cells (MSCs) for retinal photoreceptors in vitro. We analyzed the photoreceptor morphologic changes and the paracrine factors secreted by human bone marrow MSCs in a physically separated co-culture with degenerated neuroretinas, using organotypic neuroretinal cultures. The results showed that the secretome of human bone marrow MSCs had a neuroprotective effect over the neuroretinal general organization and neuropreserved the photoreceptors from degeneration probably by secretion of neuroprotective proteins. The study of the expression of 1,000 proteins showed increased paracrine factors secreted by MSCs that could be crucial in the neuroprotective effect of the stem cell secretome over in vitro retinal degeneration. The current results reinforce the hypothesis that the paracrine effect of the human bone marrow MSCs may slow photoreceptor neurodegeneration and be a therapeutic option in retinal photoreceptor degenerative diseases.

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.