Oxidative Stress, Inflammatory, Angiogenic, and Apoptotic molecules in Proliferative Diabetic Retinopathy and Diabetic Macular Edema Patients.

The aim of this study is to evaluate molecules involved in oxidative stress (OS), inflammation, angiogenesis, and apoptosis, and discern which of these are more likely to be implicated in proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME) by investigating the correlation between them in the plasma (PLS) and vitreous body (VIT), as well as examining data obtained from ophthalmological examinations. Type 2 diabetic (T2DM) patients with PDR/DME (PDRG/DMEG; n = 112) and non-DM subjects as the surrogate controls (SCG n = 48) were selected according to the inclusion/exclusion criteria and programming for vitrectomy, either due to having PDR/DME or macular hole (MH)/epiretinal membrane (ERM)/rhegmatogenous retinal detachment. Blood samples were collected and processed to determine the glycemic profile, total cholesterol, and C reactive protein, as well as the malondialdehyde (MDA), 4-hydroxynonenal (4HNE), superoxide dismutase (SOD), and catalase (CAT) levels and total antioxidant capacity (TAC). In addition, interleukin 6 (IL6), vascular endothelial growth factor (VEGF), and caspase 3 (CAS3) were assayed. The VITs were collected and processed to measure the expression levels of all the abovementioned molecules. Statistical analyses were conducted using the R Core Team (2022) program, including group comparisons and correlation analyses. Compared with the SCG, our findings support the presence of molecules involved in OS, inflammation, angiogenesis, and apoptosis in the PLS and VIT samples from T2DM. In PLS from PDRG, there was a decrease in the antioxidant load (p < 0.001) and an increase in pro-angiogenic molecules (p < 0.001), but an increase in pro-oxidants (p < 0.001) and a decline in antioxidants (p < 0.001) intravitreally. In PLS from DMEG, pro-oxidants and pro-inflammatory molecules were augmented (p < 0.001) and the antioxidant capacity diminished (p < 0.001), but the pro-oxidants increased (p < 0.001) and antioxidants decreased (p < 0.001) intravitreally. Furthermore, we found a positive correlation between the PLS-CAT and the VIT-SOD levels (rho = 0.5; p < 0.01) in PDRG, and a negative correlation between the PSD-4HNE and the VIT-TAC levels (rho = 0.5; p < 0.01) in DMEG. Integrative data of retinal imaging variables showed a positive correlation between the central subfield foveal thickness (CSFT) and the VIT-SOD levels (rho = 0.5; p < 0.01), and a negative correlation between the CSFT and the VIT-4HNE levels (rho = 0.4; p < 0.01) in PDRG. In DMEG, the CSFT displayed a negative correlation with the VIT-CAT (rho = 0.5; p < 0.01). Exploring the relationship of the abovementioned potential biomarkers between PLS and VIT may help detecting early molecular changes in PDR/DME, which can be used to identify patients at high risk of progression, as well as to monitor therapeutic outcomes in the diabetic retina.

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.

Classifications for Proliferative Vitreoretinopathy (PVR): An Analysis of Their Use in Publications over the Last 15 Years.

Purpose. To evaluate the current and suitable use of current proliferative vitreoretinopathy (PVR) classifications in clinical publications related to treatment. Methods. A PubMed search was undertaken using the term "proliferative vitreoretinopathy therapy". Outcome parameters were the reported PVR classification and PVR grades. The way the classifications were used in comparison to the original description was analyzed. Classification errors were also included. It was also noted whether classifications were used for comparison before and after pharmacological or surgical treatment. Results. 138 papers were included. 35 of them (25.4%) presented no classification reference or did not use any one. 103 publications (74.6%) used a standardized classification. The updated Retina Society Classification, the first Retina Society Classification, and the Silicone Study Classification were cited in 56.3%, 33.9%, and 3.8% papers, respectively. Furthermore, 3 authors (2.9%) used modified-customized classifications and 4 (3.8%) classification errors were identified. When the updated Retina Society Classification was used, only 10.4% of authors used a full C grade description. Finally, only 2 authors reported PVR grade before and after treatment. Conclusions. Our findings suggest that current classifications are of limited value in clinical practice due to the inconsistent and limited use and that it may be of benefit to produce a revised classification.

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.

Proliferative vitreoretinopathy: A new concept of disease pathogenesis and practical consequences.

During the last four decades, proliferative vitreoretinopathy (PVR) has defied the efforts of many researchers to prevent its occurrence or development. Thus, PVR is still the major complication following retinal detachment (RD) surgery and a bottle-neck for advances in cell therapy that require intraocular surgery. In this review we tried to combine basic and clinical knowledge, as an example of translational research, providing new and practical information for clinicians. PVR was defined as the proliferation of cells after RD. This idea was used for classifying PVR and also for designing experimental models used for testing many drugs, none of which were successful in humans. We summarize current information regarding the pathogenic events that follow any RD because this information may be the key for understanding and treating the earliest stages of PVR. A major focus is made on the intraretinal changes derived mainly from retinal glial cell reactivity. These responses can lead to intraretinal PVR, an entity that has not been clearly recognized. Inflammation is one of the major components of PVR, and we describe new genetic biomarkers that have the potential to predict its development. New treatment approaches are analyzed, especially those directed towards neuroprotection, which can also be useful for preventing visual loss after any RD. We also summarize the results of different surgical techniques and clinical information that is oriented toward the identification of high risk patients. Finally, we provide some recommendations for future classification of PVR and for designing comparable protocols for testing new drugs or techniques.

Safety and Biocompatibility of a New High-Density Polyethylene-Based Spherical Integrated Porous Orbital Implant: An Experimental Study in Rabbits.

Purpose. To evaluate clinically and histologically the safety and biocompatibility of a new HDPE-based spherical porous orbital implants in rabbits. Methods. MEDPOR (Porex Surgical, Inc., Fairburn, GA, USA), OCULFIT I, and OCULFIT II (AJL Ophthalmic S.A., Vitoria, Spain) implants were implanted in eviscerated rabbis. Animals were randomly divided into 6 groups (n = 4 each) according to the 3 implant materials tested and 2 follow-up times of 90 or 180 days. Signs of regional pain and presence of eyelid swelling, conjunctival hyperemia, and amount of exudate were semiquantitatively evaluated. After animals sacrifice, the implants and surrounding ocular tissues were processed for histological staining and polarized light evaluation. Statistical study was performed by ANOVA and Kaplan-Meier analysis. Results. No statistically significant differences in regional pain, eyelid swelling, or conjunctival hyperemia were shown between implants and/or time points evaluated. However, amount of exudate differed, with OCULFIT I causing the smallest amount. No remarkable clinical complications were observed. Histological findings were similar in all three types of implants and agree with minor inflammatory response. Conclusions. OCULFIT ophthalmic tolerance and biocompatibility in rabbits were comparable to the clinically used MEDPOR. Clinical studies are needed to determine if OCULFIT is superior to the orbital implants commercially available.

Loss of Visual Acuity after Successful Surgery for Macula-On Rhegmatogenous Retinal Detachment in a Prospective Multicentre Study.

Purpose. To quantify the frequency of visual loss after successful retinal detachment (RD) surgery in macula-on patients in a multicentric, prospective series of RD. Methods. Clinical variables from consecutive macula-on RD patients were collected in a prospective multicentric study. Visual loss was defined as at least a reduction in one line in best corrected visual acuity (VA) with Snellen chart. The series were divided into 4 subgroups: (1) all macula-on eyes (n = 357); (2) macula-on patients with visual loss at the third month of follow-up (n = 53) which were further subdivided in (3) phakic eyes (n = 39); and (4) pseudophakic eyes (n = 14). Results. Fifty-three eyes (14.9%) had visual loss three months after surgery (n = 39 phakic eyes; n = 14 pseudophakic eyes). There were no statistically significant differences between them regarding their clinical characteristics. Pars plana vitrectomy (PPV) was used in 67.2% of cases, scleral buckle in 57.7%, and scleral explant in 11.9% (36.1% were combined procedures). Conclusions. Around 15% of macula-on RD eyes lose VA after successful surgery. Development of cataracts may be one cause in phakic eyes, but vision loss in pseudophakic eyes could have other explanations such as the effect of released factors produced by retinal ischemia on the macula area. Further investigations are necessary to elucidate this hypothesis.

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.

Hydatid disease of the liver: thirty years of surgical experience.

Hydatid disease of the liver is a relatively frequent disease. Although the natural history is almost completely known, several complications may occur. The aim of this study was to show that radical surgical resection of the hepatic hydatid cyst is a safe and very effective technique, based on our results after 30-year experience. A review of most significant studies was carried out. We retrospectively evaluated our surgical cases. From January 1973 to December 2003 we treated 216 patients, 98 males and 118 females. Survival was compared with the Kaplan-Meier test, using log-rank analysis to compare data. Differences with a p value less than 0.05 were considered significant. A total of 279 cysts were excised. We performed pericystectomy in 122 cases, 73 of which closed. We also performed 19 atypical resections, 10 segmentectomies, 20 lobectomies and 2 percutaneous treatments. In more than 90% of cases, preoperative data collection was completed by preoperative ultrasound. The cumulative morbidity was 13%. The recurrence rate amounted to 4.3% at 5 years and 7% at 10 years: of these, 6 occurred after non-radical surgery and 2 after total pericystectomy or liver resection (p < 0.001). Technical advances and accumulated experience permit safe treatment of hepatic hydatid cysts by radical resection, with an almost zero recurrence rate, making it the treatment of choice over partial resection. The utility of percutaneous treatment remains confined to limited indications, such as laparoscopy.