Intravitreal acetazolamide implant for pseudophakic cystoid macular edema.

BACKGROUND: Pseudophakic cystoid macular edema (PCME) is the most common cause of visual acuity deterioration after uncomplicated cataract surgery. There is no consensus regarding how to manage recurrent or refractory cases. REPORT: A 54-year-old woman complained of decreased vision and central metamorphopsia in the right eye (OD) 3 months after uneventful cataract surgery. Visual acuity was 0.3 logMAR (20/40) OD and 0.1 logMAR (20/25) OS. Reduced macular brightness was seen OD on funduscopy associated with increased macular thickness on optical coherence tomography (OCT). Pseudophakic cystoid macular edema (PCME) was diagnosed, and treatment with oral acetazolamide was tried without success. The patient underwent a single intravitreal injection of an acetazolamide implant (260 µg) OD as off-label treatment. Four weeks following the injection, she reported complete resolution of her metamorphopsia and visual loss OD. Four months later, her visual acuity was 0.0 logMAR (20/20) in OD and 0.1 logMAR (20/25) in OS. The patient reported no discomfort after the injection procedure. Laboratory and ophthalmologic tests did not identify any adverse effects of the medication. CONCLUSION: We show that PCME refractory to conventional treatment improved after intravitreal acetazolamide implant injection. Further investigation is warranted to confirm these preliminary findings.

Assessment of the safety of intravitreal injection of metoprolol tartrate in rabbits.

PURPOSE: To verify the safety of different doses of intravitreal metoprolol tartrate (MT) after intravitreal injection in rabbit eyes. METHODS: Animals were randomly assigned into 2 groups: group I received 50 µg of MT and group II 100 µg of MT. A volume of 0.05 mL of the drug solution was administered through an intravitreal injection, while the control eyes received an equal volume of saline solution. Safety was assessed by clinical observation, electroretinography (ERG) and histological evaluation. RESULTS: No evidence of clinical toxicity was observed. ERG waveforms from the MT treated eyes were similar to those recorded from the control eyes in dark-adapted state, amplitude and the implicit time are similar between the groups in light-adapted state, and their retinas had no signs of toxicity by histological evaluation 7 days after intravitreal injection. CONCLUSIONS: The intravitreal use of metoprolol at 50 and 100 µg dosages does not cause short-term retinal toxicity in rabbits.

Sirolimus-Loaded Intravitreal Implant for Effective Treatment of Experimental Uveitis.

Non-infectious uveitis, an ocular inflammatory condition that affects the iris, ciliary body, choroid, and adjacent tissues (retina, optic nerve, and vitreous), is an important cause of blindness worldwide. Sirolimus (SRL), a potent immunomodulatory drug, has shown promising results in the treatment of inflammatory ocular diseases. Despite this therapeutic potential, its clinical use is a major challenge due to low bioavailability and poor solubility. Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable polymer commonly used for ophthalmic drug delivery due to its suitable characteristics such as biocompatibility, good mechanical properties, and improvement of the pharmacokinetic profile of the drug. In the present study, we investigated the effects of SRL-PLGA implant on experimental autoimmune uveitis in rabbits. Clinical and histopathological examinations were performed, followed by assessment of protein levels and determination of myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) activity in the aqueous humor/vitreous. As a result, treated eyes had decreased average inflammatory scores, protein significant decreases in treated eyes, assessed after 35 days. Histopathological examination showed less severe intraocular inflammation and decreased tissue damage in treated eyes. According to these results, the SRL-PLGA implant evaluated in this study was apparently safe, reducing inflammation in treated eyes, with an extended effect possibly associated with prolonged release of SRL in the posterior segment of the eye. Therefore, intravitreal SRL-PLGA implant could be a promising alternative for treatment of non-infectious uveitis.

Central nervous system (CNS) tumor cell heterogeneity contributes to differential platinum-based response in an in vitro 2D and 3D cell culture approach.

One of the models that best explains the cellular heterogeneity observed in central nervous system (CNS) tumors is the presence of cancer stem cells (CSCs). CSCs can originate from differentiated adult cells that return to an undifferentiated stage through the mechanism known as epithelial-mesenchymal transition (EMT). In this paper, we evaluated cellular and molecular heterogeneity and the participation of the epithelial-mesenchymal transition (EMT) in glioblastoma (U-87 MG and LN-18) and neuroblastoma (KELLY and IMR-32) cell lines cultured in monolayer (2D) and neurosphere (CSC enrichment- 3D) models. For this, after treatment with cisplatin, we studied different cell subpopulations by immunophenotyping using neural stem cell/progenitor markers (ALDH, CD24, CD56, and CD133), mesenchymal stem cell markers (CD73, CD90, CD105, and CD146) and hematopoietic markers (CD14, CD19, CD34, CD45, and HLA-DR) and mRNA expression profiles of genes related to EMT, such as ZEB1, TWIST1, TGFB1, STAT3, and lncRNA HOTAIR. In addition, we evaluated the growth capacity of residual cells when treated with cisplatin using the chorioallantoic membrane (CAM) model to study disease relapse. After treatment with cisplatin, we found that the expression of STAT3 and TGFB1 genes markedly increased in the neurosphere of the IMR-32 cell line, and TWIST1 was upregulated in the neurosphere of LN-18. Only the nontreated monolayer of LN-18, KELLY, and IMR-32 amplified the lncRNA HOTAIR. The IMR-32 cell line exhibited an enrichment of CD24+/ALDH+ and this cell subset decreased after cisplatin treatment. We observed the loss of CD146+/CD73+ cell subpopulations in U-87 MG monolayer and neurosphere models, after cisplatin treatment, while in LN-18 monolayer cisplatin-treated cells, CD73+/CD90+ cell subpopulations increased. Neuroblastoma cell lines showed CD14+/HLA-DR- cell subpopulations representative of myeloid-derived suppressor cells (MDSCs). Tumors generated from residual cells, after exposure to cisplatin, grafted on CAM showed patterns of organization different from those of the controls. Thus, our findings strongly supported the idea that definitions of tumor phenotypic characteristics may help to establish better therapeutic strategies for the development of new drug targets.

Toxicity and in vivo release profile of sirolimus from implants into the vitreous of rabbits' eyes.

PURPOSE: To assess the in vivo release profile and the retinal toxicity of a poly (lactic-co-glycolic acid) (PLGA) sustained-release sirolimus (SRL) intravitreal implant in normal rabbit eyes. METHODS: PLGA intravitreal implants containing or not SRL were prepared, and the viability of ARPE-19 and hES-RPE human retinal cell lines was examined after 24 and 72 h of exposure to implants. New Zealand rabbits were randomly divided into two groups that received intravitreal implants containing or not SRL. At each time point (1-8 weeks), four animals from the SRL group were euthanized, the vitreous was collected, and drug concentration was calculated. Clinical evaluation of the eyes was performed weekly for 8 weeks after administration. Electroretinography (ERG) was recorded in other eight animals, four for each group, at baseline and at 24 h, 1, 4, 6, and 8 weeks after the injection. ERG was carried out using scotopic and photopic protocols. The safety of the implants was assessed using statistical analysis of the ERG parameters (a and b waves, a and b implicit time, B/A ratio, oscillatory potential, and Naka-Rushton analysis) comparing the functional integrity of the retina between the PLGA and SRL-PLGA groups. After the last electrophysiological assessment, the rabbits were euthanized and retinal histopathology was realized. RESULTS: After 24 and 72 h of incubation with PLGA or SRL-PLGA implants, ARPE-19 and hES-RPE cells showed viability over 70%. The maximum concentration of SRL (199.8 ng/mL) released from the device occurred within 4 weeks. No toxic effects of the implants or increase in the intraocular pressure was observed through clinical evaluation of the eye. ERG responses showed no significant difference between the eyes that received PLGA or SRL-PLGA implants at baseline and throughout the 8 weeks of follow-up. No remarkable difference in retinal histopathology was detected in rabbit eyes treated with PLGA or SRL-PLGA implants. CONCLUSIONS: Intravitreal PLGA or SRL-PLGA implants caused no significant reduction in cell viability and showed no evident toxic effect on the function or structure of the retina of the animals. SRL was released from PLGA implant after application in the vitreous of rabbits during 8 weeks.

Biodegradable core-shell electrospun nanofibers containing bevacizumab to treat age-related macular degeneration.

Age-related macular degeneration (AMD) is a degenerative ocular disease that affects the central retina. It is considered the main cause of blindness and loss of vision worldwide. Angiogenic factors are associated with AMD, which has led to the use of antiangiogenic drugs, such as bevacizumab, to treat the disease using frequent intravitreal injections. In the present study, biodegradable core shell nanofibers containing bevacizumab were prepared by the coaxial electrospinning technique. It is thought that the shell could control the release of the drug, while the core would protect and store the drug. Poly(caprolactone) (PCL) and gelatin were used to form the shell of the nanofibers, while poly(vinyl alcohol) (PVA) and bevacizumab comprised the core. The nanofibers were characterized using microscopy techniques, thermal analysis, and FTIR. The results showed that core-shell nanofibers were produced as designed. Bevacizumab activity was evaluated using a chicken embryo chorioallantoic membrane (CAM) assay. An enzyme-linked immunosorbent assay was used to quantify the amount of the drug released from the different nanofibers in vitro. The toxicity of the nanofibers was evaluated in human retinal pigment epithelial (ARPE) cells. The CAM results demonstrated that bevacizumab maintained its antiangiogenic activity when incorporated into the nanofibers. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests revealed that the nanofibers showed no cellular toxicity, even in the presence of bevacizumab. The core-shell structure of the nanofibers reduced the release rate of bevacizumab compared with PVA nanofibers. The bevacizumab-loaded biodegradable nanofibers presented interesting properties that would potentially constitute an alternative therapy to intravitreal injections to treat AMD.

Etoposide-Loaded Poly(Lactic-co-Glycolic Acid) Intravitreal Implants: In Vitro and In Vivo Evaluation.

Etoposide-loaded poly(lactic-co-glycolic acid) implants were developed for intravitreal application. Implants were prepared by a solvent-casting method and characterized in terms of content uniformity, morphology, drug-polymer interaction, stability, and sterility. In vitro drug release was investigated and the implant degradation was monitored by the percent of mass loss. Implants were inserted into the vitreous cavity of rabbits' eye and the in vivo etoposide release profile was determined. Clinical examination and the Hen Egg Test-Chorioallantoic Membrane (HET-CAM) method were performed to evaluate the implant tolerance. The original chemical structure of the etoposide was preserved after incorporation in the polymeric matrix, which the drug was dispersed uniformly. In vitro, implants promoted sustained release of the drug and approximately 57% of the etoposide was released in 50 days. In vivo, devices released approximately 63% of the loaded drug in 42 days. Ophthalmic examination and HET-CAM assay revealed no evidence of toxic effects of implants. These results tend to show that etoposide-loaded implants could be potentially useful as an intraocular etoposide delivery system in the future.

Ocular safety of Intravitreal Clindamycin Hydrochloride Released by PLGA Implants.

BACKGROUND: Drug ocular toxicity is a field that requires attention. Clindamycin has been injected intravitreally to treat ocular toxoplasmosis, the most common cause of eye posterior segment infection worldwide. However, little is known about the toxicity of clindamycin to ocular tissues. We have previously showed non intraocular toxicity in rabbit eyes of poly(lactic-co-glycolic acid) (PLGA) implants containing clindamycin hydrochloride (CLH) using only clinical macroscotopic observation. In this study, we investigated the in vivo biocompatibility of CLH-PLGA implants at microscotopic, cellular and molecular levels. METHODS: Morphology of ARPE-19 and MIO-M1 human retinal cell lines was examined after 72 h exposure to CLH-PLGA implant. Drug delivery system was also implanted in the vitreous of rat eyes, retinal morphology was evaluated in vivo and ex vivo. Morphology of photoreceptors and inflammation was assessed using immunofluorescence and real-time PCR. RESULTS: After 72 h incubation with CLH-PLGA implant, ARPE-19 and MIO-M1 cells preserved the actin filament network and cell morphology. Rat retinas displayed normal lamination structure at 30 days after CLH-PLGA implantation. There was no apoptotic cell and no loss in neuron cells. Cones and rods maintained their normal structure. Microglia/macrophages remained inactive. CLH-PLGA implantation did not induce gene expression of cytokines (IL-1ß, TNF-α, IL-6), VEGF, and iNOS at day 30. CONCLUSION: These results demonstrated the safety of the implant and highlight this device as a therapeutic alternative for the treatment of ocular toxoplasmosis.

Montmorillonite clay based polyurethane nanocomposite as substrate for retinal pigment epithelial cell growth.

The subretinal transplantation of retinal pigment epithelial cells (RPE cells) grown on polymeric supports may have interest in retinal diseases affecting RPE cells. In this study, montmorillonite based polyurethane nanocomposite (PU-NC) was investigated as substrate for human RPE cell growth (ARPE-19 cells). The ARPE-19 cells were seeded on the PU-NC, and cell viability, proliferation and differentiation were investigated. The results indicated that ARPE-19 cells attached, proliferated onto the PU-NC, and expressed occludin. The in vivo ocular biocompatibility of the PU-NC was assessed by using the HET-CAM; and through its implantation under the retina. The direct application of the nanocomposite onto the CAM did not compromise the vascular tissue in the CAM surface, suggesting no ocular irritancy of the PU-NC film. The nanocomposite did not elicit any inflammatory response when implanted into the eye of rats. The PU-NC may have potential application as a substrate for RPE cell transplantation.

Development and evaluation of sustained-release etoposide-loaded poly(ε-caprolactone) implants.

Poly(ε-caprolactone) implants containing etoposide, an important chemotherapeutic agent and topoisomerase II inhibitor, were fabricated by a melt method and characterized in terms of content uniformity, morphology, drug physical state, and sterility. In vitro and in vivo drug release from the implants was also evaluated. The cytotoxic activity of implants against HeLa cells was studied. The short-term tolerance of the implants was investigated after subcutaneous implantation in mice. The original chemical structure of etoposide was preserved after incorporation into the polymeric matrix, in which the drug was dispersed uniformly. Etoposide was present in crystalline form in the polymeric implant. In vitro release study showed prolonged and controlled release of etoposide, which showed cytotoxicity activity against HeLa cells. After implantation, good correlation between in vitro and in vivo drug release was found. The implants demonstrated good short-term tolerance in mice. These results tend to show that etoposide-loaded implants could be potentially applied as a local etoposide delivery system.

Efficacy and Safety Evaluation of Mometasone Furoate in Treating Ocular Inflammation.

Mometasone furoate (MF) is a medium-potency synthetic glucocorticosteroid with anti-inflammatory, antipruritic, and vasoconstrictive properties. However, its role in the treatment of ocular inflammation has not yet been explored. This work investigated the anti-inflammatory activity of MF in ocular tissues. First, the in vivo safety of the intravitreal (IVT) injection of MF (80, 160, and 240 µg) was evaluated via clinical examination (including the assessment of intraocular pressure), electroretinography (ERG), and histopathology. Second, MF was tested in an experimental model of bacillus Calmette-Guérin (BCG)-induced uveitis in Wistar rats. Intraocular inflammation was then evaluated via a slit-lamp and fundus examination, ERG, histopathology, and the quantification of pro-inflammatory markers. Intravitreal MF showed no toxicity in all the investigated doses, with 160 µg leading to attenuated disease progression and improvement in clinical, morphological, and functional parameters. There was a significant reduction in the levels of inflammatory markers (myeloperoxidase, interleukins 6 and 1ß, CXCL-1, and tumor necrosis factor-alpha) when compared to the levels in untreated animals. Therefore, MF should be further investigated as a promising drug for the treatment of ocular inflammation.

Clinical outcomes of intravitreal treatment for ocular toxoplasmosis: systematic review and meta-analysis.

BACKGROUND: Ocular toxoplasmosis is the leading cause of infectious posterior uveitis worldwide, accounting for 30-50% of all cases in immunocompetent patients. Conventional treatment is associated with adverse effects and does not prevent recurrence. Intravitreal drug administration can improve disease outcomes and reduce side effects. Herein, we conducted a systematic review and meta-analysis on the efficacy of intravitreal injections for treating ocular toxoplasmosis. METHODS: The systematic search was conducted using PubMed, SciELO, and Google Scholar with the descriptors "ocular toxoplasmosis" AND "intravitreal". We analyzed studies that met the inclusion criteria, i.e., experimental cases in patients treated intravitreally for ocular toxoplasmosis. Considering the systematic review, we focused on the number of intravitreal injections, the therapeutic drug class, and the presence of preexisting conditions. To assess the efficacy of intravitreal injections, a meta-analysis was performed using visual acuity, side effects, disease recurrence, and inflammatory responses as variables. RESULTS: Intravitreal injection-induced side effects were rarely observed (0.49% [0.00, 1.51%] ). The use of antiparasitic and anti-inflammatory drugs afforded improved visual acuity (99.81% [98.60, 100.00%]) and marked effectiveness in treating ocular toxoplasmosis. CONCLUSIONS: Intravitreal injections may facilitate the successful treatment of ocular toxoplasmosis. However, clinicians should carefully evaluate the presence of preexisting conditions for ocular toxoplasmosis or previous diseases, as these can impact the decision to administer intravitreal injections.

Nanofiber-coated implants: Development and safety after intravitreal application in rabbits.

Intravitreal injections are the preferred choice for drug administration to the posterior segment of the eye. However, the required frequent injections may cause complications to the patient and low adherence to the treatment. Intravitreal implants are able to maintain therapeutic levels for a long period. Biodegradable nanofibers can modulate drug release and allow the incorporation of fragile bioactive drugs. Age-related macular degeneration is one of the world major causes of blindness and irreversible vision loss. It involves the interaction between VEGF and inflammatory cells. In this work we developed nanofiber-coated intravitreal implants containing dexamethasone and bevacizumab for simultaneously delivery of these drugs. The implant was successfully prepared and the efficiency of the coating process was confirmed by scanning electron microscopy. Around 68% of dexamethasone was released in 35 days and 88% of bevacizumab in 48hs. The formulation presented activity in the reduction of vessels and was safe to the retina. It was not observed any clinical or histopathological change, neither alteration in retina function or thickness by electroretinogram and optical coherence tomography during 28 days. The nanofiber-coated implants of dexamethasone and bevacizumab may be considered as a new delivery system that can be effective for the treatment of AMD.

Markers to sensibility and relapse on IMR-32 neuroblastoma cell line cultured in monolayer (2D) and neurosphere (3D) models cisplatin-treated.

The complexity of different components of tumor stroma poses huge challenges for therapies targeting the neuroblastoma (NB) microenvironment. The present study aimed to evaluate platinum-based response in IMR-32 neuroblastoma cell line cultured in monolayer (2D) and neurosphere (3D) models. For this, we evaluated mRNA expression of heat shock proteins HSPA1A, HSPB1, TRAP1, HSPA1AL, HSPD1, and DNA damage repair gene ERCC1. After treatment, residual cells were grafted on CAM (chicken chorioallantoic membrane) to evaluate the growth capability and histological paraffin sections were made to assess Ki-67 and HER-2 proteins by immunofluorescence. Our results showed that cisplatin induces mRNA downregulation of Heat Shock Proteins and ERCC1 in IMR-32 cells cultured in 2D or 3D models. In addition, the cisplatin-treatment approach increased HER-2 expression in residual IMR-32 cells grafted on the CAM. Therefore, these insights provide many advances in neuroendocrine tumor biology and knowledge about cisplatin-response in neuroblastoma.

Influence of SARS-CoV-2 inactivation by different chemical reagents on the humoral response evaluated in a murine model.

Viral inactivation for antibody induction purposes, among other applications, should ensure biosafety, completely avoiding the risk of infectivity, and preserving viral immunogenicity. ß-propiolactone (BPL) is one of the most used reagents for viral inactivation, despite its high toxicity and recent difficulties related to importation, experienced in Brazil during the SARS-CoV-2 pandemic. In this context, the main objectives of this work were to test different inactivation procedures for SARS-CoV-2 and to evaluate the induction of neutralizing antibodies in mice immunized with antigenic preparations obtained after viral treatment with formaldehyde (FDE), glutaraldehyde (GDE), peroxide hydrogen (H2O2), as well as with viral proteins extract (VPE), in parallel with BPL. Verification of viral inactivation was performed by subsequent incubations of the inactivated virus in Vero cells, followed by cytopathic effect and lysis plaques observation, as well as by quantification of RNA load using reverse transcription-quantitative real time polymerase chain reaction. Once viral inactivation was confirmed, cell culture supernatants were concentrated and purified. In addition, an aliquot inactivated by BPL was also subjected to viral protein extraction (VPE). The different antigens were prepared using a previously developed microemulsion as adjuvant, and were administered in a four-dose immunization protocol. Antibody production was comparatively evaluated by ELISA and Plaque Reduction Neutralization Tests (PRNT). All immunogens evaluated showed some level of IgG anti-SARS-CoV-2 antibodies in the ELISA assay, with the highest levels presented by the group immunized with FDE-inactivated viral antigen. In the PRNT results, except for VPE-antigen, all other immunogens evaluated induced some level of neutralizing anti-SARS-CoV-2 antibodies, and the FDE-antigen stood out again with the most expressive values. Taken together, the present work shows that FDE can be an efficient and affordable alternative to BPL for the production of inactivated SARS-CoV-2 viral antigen.

Low-dose melittin is safe for intravitreal administration and ameliorates inflammation in an experimental model of uveitis.

Uveitis is a group of sight-threatening ocular inflammatory disorders, whose mainstay of therapy is associated with severe adverse events, prompting the investigation of alternative treatments. The peptide melittin (MEL) is the major component of Apis mellifera bee venom and presents anti-inflammatory and antiangiogenic activities, with possible application in ophthalmology. This work aims to investigate the potential of intravitreal MEL in the treatment of ocular diseases involving inflammatory processes, especially uveitis. Safety of MEL was assessed in retinal cells, chick embryo chorioallantoic membranes, and rats. MEL at concentrations safe for intravitreal administration showed an antiangiogenic activity in the chorioallantoic membrane model comparable to bevacizumab, used as positive control. A protective anti-inflammatory effect in retinal cells stimulated with lipopolysaccharide (LPS) was also observed, without toxic effects. Finally, rats with bacille Calmette-Guerin- (BCG) induced uveitis treated with intravitreal MEL showed attenuated disease progression and improvement of clinical, morphological, and functional parameters, in addition to decreased levels of proinflammatory mediators in the posterior segment of the eye. These effects were comparable to the response observed with corticosteroid treatment. Therefore, MEL presents adequate safety profile for intraocular administration and has therapeutic potential as an anti-inflammatory and antiangiogenic agent for ocular diseases.

Sodium butyrate-loaded nanoparticles coated with chitosan for the treatment of neovascularization in age-related macular degeneration: Ocular biocompatibility and antiangiogenic activity.

Sodium butyrate-loaded nanoparticles coated chitosan (NaBu-loaded nanoparticles/CS) were developed to treat the choroidal neovascularization in wet age-related macular degeneration (AMD). The nanoparticles were produced by double emulsification and solvent evaporation technique, optimized by experimental statistical design, characterized by analytical methods, investigated in terms of in vitro and in vivo ocular biocompatibility, and evaluated as an antiangiogenic system in vivo. The NaBu-loaded nanoparticles/CS were 311.1 ± 3.1 nm in diameter with a 0.208 ± 0.007 polydispersity index; had a +56.3 ± 2.6 mV zeta potential; showed a 92.3 % NaBu encapsulation efficiency; and sustained the drug release over 35 days. The NaBu-loaded nanoparticles/CS showed no toxicity to human retinal pigment epithelium cells (ARPE-19 cells); was not irritant to the chorioallantoic membrane (CAM); did not interfere in the integrity of the retinal layers of rat's eyes, as detected by the Optical Coherence Tomography and histopathology; and inhibited the angiogenesis in CAM assay. The NaBu-loaded nanoparticles/CS could be a therapeutic alternative to limit the neovascularization in AMD.

Intravitreal ketamine promotes neuroprotection in rat eyes after experimental ischemia.

Retinal ischemia, one of the most common cause of visual loss, is associated with blood flow inadequacy and subsequent tissue injury. In this setting, some treatments that can counteract glutamate increase, arouse interest in ischemic pathogenesis. Ketamine, a potent N-methyl-d-aspartate (NMDA) receptor antagonist, provides a neuroprotective pathway via decreasing the excitotoxicity triggered by excess glutamatergic. Thus, the goal of this study was to evaluate the safety of intravitreal use of ketamine and their potential protective effects on retinal cells in retinal ischemia/reperfusion model. Initially, ketamine toxicity was evaluated by cytotoxicity assay and Hen's egg chorioallantoic membrane (HET-CAM) method. Afterward, some ketamine concentrations were tested in rat's eyes to verify the safety of the intravitreal use. To investigate the neuroprotective effect on retinal, a single intravitreal injection of ketamine in concentrations of 0.059 mmol.L-1 and 0.118 mmol.L-1 was performed one day before the retinal injury by ischemia/reperfusion model. After 7 and 15 days, the retina activity was evaluated by electroretinogram (ERG) records and, lastly, by morphological analyzes. Cytotoxicity assay reveals that the maximum ketamine concentration that could reach retinal pigmented epithelium cells is 0.353 mmol.L-1. HET-CAM assay showed that concentrations above 0.237 mmol.L-1 are irritants to the eye. Thus, Ketamine in concentrations of 0.0237 mmol.L-1, 0.118 mmol.L-1, and 0.059 mmol.L-1 were selected for in vivo toxicity test. ERG records reveal a tendency of b-wave amplitude to decrease as the luminous intensity increased, in the group receiving ketamine at 0.237 mmol.L-1. Therefore, ketamine in concentrations at 0.059 mmol.L-1 and 0.118 mmol.L-1 were chosen for the following tests. In the ischemia retinal degeneration model, pretreatment with ketamine was capable to promote a recovery of retinal electrophysiological function minimizing the ischemic effects. In histological analysis, the groups that received intravitreal ketamine showed a number of retinal cells significantly higher than the vehicle group. In TUNEL assay a reduction on TUNEL-positive cells was observed in all the layers for both concentrations which allow to affirm that ketamine contributes to reducing cell death in the retina. Transmission electron microscopy (TEM) reaffirms this finding. Ketamine intravitreal pretreatment showed reduced ultrastructural changes. Our findings demonstrate that ketamine is safe for intravitreal use in doses up to 0.118 mmol.L-1. They seem to be particularly efficient to protect the retina from ischemic injury.

Ischemia-induced retinal injury is attenuated by Neurovespina, a peptide from the venom of the social wasp Polybia occidentalis.

Neurovespina is a synthetic peptide modified from Occidentalin-1202, a nine amino acid residue peptide isolated from the venom of the social wasp Polybia occidentalis. Previous studies showed that this peptide has a neuroprotective effect on the central nervous system, but its action on the eye has not been explored. So, the objective of this work was to investigate the neuroprotective effect of Neurovespina on the retina and its angiogenic potential in the chicken chorioallantoic membrane (CAM). Retinal ischemia was induced in rats by acute elevation of intraocular pressure (IOP). Electroretinography (ERG) measurements, histopathological and immunohistochemical analysis, and transmission electronic microscopy (TEM) records were performed to check the neuroprotection effect of Neurovespina in the retina of the animals. The angiogenic activity of the peptide was investigated by CAM assay. The results showed that Neurovespina was able to reduce the effects induced by ischemic injury, preventing the reduction of a- and b-waves in the scotopic ERG. Histopathological and immunohistochemistry assays showed that Neurovespina, mainly at 60 µg/ml, protected all layers of the retina. The CAM assay revealed that the peptide promoted the reduction of CAM vessels. So, Neurovespina was able to protect retinal cells from ischemic insult and has an antiangiogenic effect, which can be considered as a promising neuroprotective agent for intravitreal application.

Phenotypic changes on central nervous system (CNS) tumor cell lines cultured in vitro 2D and 3D models and treated with cisplatin.

Despite aggressive therapy, most patients with brain tumors present disease relapse due to the cellular and molecular nature of these tumors. One of the models that best explains the heterogeneity observed in CNS tumors is the presence of cancer stem cells (CSCs). In this paper, we evaluated platinum-based response in brain tumor U-87 MG, LN-18, and KELLY cell lines cultured in monolayer (2D) and neurosphere (CSC enrichment- 3D) models. We evaluated mRNA expression of heat shock proteins (HSPA1A, HSPB1, HSPA1AL, TRAP1, and HSPD1), and DNA repair gene ERCC1. Changes in cell cycle and glycosylation profile were assessed by flow cytometry. After treatment with cisplatin, we found that the mRNA expression of HSPs markedly increased in the U-87 MG and LN-18 neurosphere cells. In KELLY monolayer cells, cisplatin induced upregulation of all genes. In KELLY neurosphere cells, only the HSPA1A, HSPB1, TRAP1, and HSPD1 genes were upregulated. The proportion of cells in the G0/G1 phase was significantly higher in U-87 MG neurosphere cisplatin-treated cells. A trend towards a greater proportion of cells in the S phase of U-87 MG monolayer cisplatin-treated cells was also observed. On the other hand, a significant decrease in the number of cells in the S phase and an increase in G2/M was observed in LN-18 monolayer cisplatin-treated cells. Glycosylation analysis using lectins showed a higher surface binding for PNA in the U-87 MG treated monolayer and a lower binding for Concanavalin A in the treated neurospheres. The binding of Isolectin GS-IB4, GSII, and SBA in KELLY monolayer cisplatin-treated cells was lower whereas the proportion of cells labeled with Concanavalin A was higher. In the KELLY neurosphere cisplatin-treated cells, the binding of Concanavalin A was lower than nontreated cells. Thus, our findings strongly supported the idea that definitions of phenotypic characteristics may help to establish better therapeutic strategies for brain tumors.