Method for the simultaneous isolation of primary astrocytes and microglia from the neonatal rats cerebral cortex.

AMP-Activated Protein Kinase Signaling Protects Oligodendrocytes that Restore Central Nervous System Functions in an Experimental Autoimmune Encephalomyelitis Model

Dye exclusion tests are used to determine the number of live and dead cells. Theseassays are based on the principle that intact plasma membranes in live cells excludespecific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB)exclusion assay has limitations. The dye can be incorporated by live cells after ashort exposure time, and personal reliability, related to the expertise of theanalyst, can affect the results. We propose an alternative assay for evaluating cellviability that combines the TB exclusion test and the high sensitivity of the flowcytometry technique. Previous studies have demonstrated the ability of TB to emitfluorescence when complexed with proteins. According to our results, TB/bovine serumalbumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which isdetectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v)was defined as the optimum concentration for distinguishing unstained living cellsfrom fluorescent dead cells, and fluorescence emission was stable for 30 min aftercell treatment. Although previous studies have shown that TB promotes greenfluorescence quenching, TB at 0.002% did not interfere with green fluorescence inhuman live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonalantibody. We observed a high correlation between the percentage of propidiumiodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similardouble-stained cell profiles in flow cytometry dot-plot graphs. Taken together, theresults indicate that a TB exclusion assay by flow cytometry can be employed as analternative tool for quick and reliable cell viability analysis.

Objective: Assessment of anticancer activity of the plant cell suspension cultures of Ocimum sanctum by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Trypan blue dye exclusion assay against A549 (human lung cancer cell line). Materials and Methods: In vitro anticancer activity of ethanol, acetone, and aqueous leaf extracts of O. sanctum was evaluated on A549 cancerous cell line by MTT assay and Trypan blue dye exclusion assay. MTT assay is based on the capacity of mitochondrial enzymes of viable cells to reduce the yellow soluble salt MTT to purple-blue insoluble formazan precipitate which is then quantified spectrophotometrically at 570 nm. Trypan blue assay is based on staining of cells. Cells are then counted using hemocytometer under the microscope, non-viable cells were stained blue and viable cells remain unstained. Results: The aqueous leaf extract of O. sanctum has not shown any anticancer activity. However, potent anticancer activity was shown by the acetone and ethanol leaf extracts of O. sanctum on A549 (human lung cancer cell line). Conclusions: The medicinal plant, i.e., O. sanctum was studied by in vitro evaluation methods, i.e., MTT assay and Trypan blue exclusion assay. The acetone and ethanol leaf extract of O. sanctum have shown potent anticancer activity on A549 cancerous cell line.

Exposure to brominated flame retardant PBDE-99 affects cytoskeletal protein expression in the neonatal mouse cerebral cortex

The rise in antifungal-resistant strains of Candida albicans indicates an urgent need for alternative treatments. This study investigated the potential of six homeopathic medications as affordable therapies for drug-resistant fungal infections by assessing their antifungal activities. This study aimed to investigate the antifungal activity of homeopathic medicines using microdilution assays, biofilm inhibition assays, and trypan blue exclusion assays. Assays for broth microdilution, followed by trypan blue exclusion and biofilm formation assays, were used to verify the antifungal properties of Mercurius solubilis (6C, 12C, 30C, 200C), Kreosotum (6C, 12C, 30C, 200C), Curcuma longa (200C), Sepia officinalis (6C, 12C, 30C, 200C), Mezereum (6C, 30C), and Myristica sebifera (12C, 30C, 200C). The assays were performed using 91% ethanol and fluconazole as negative and positive controls, respectively. This study was repeated thrice to verify the accuracy and reliability. Compared to the negative control and control, Kreosotum 12C (0.024 ± 0.007) had the most significant effect in microdilution and demonstrated the highest inhibition in the biofilm experiment (74.46%), whereas Sepia officinalis 30C demonstrated a substantial value in the trypan blue exclusion assay (89.41%). This study concluded that six homoeopathic medicines have antifungal activities against C. albicans.

What is the central question of the study? Does neonatal stress, in the form of neonatal maternal separation, influence the maturation of microglial density, morphology and neuronal signalling in medullary regions regulating cardiorespiratory function in rat pups? What is the main finding and its importance? Using Iba-1 immunohistochemistry, we show that neonatal maternal separation augments microglial density and the proportion of cells with an amoeboid morphology in the medulla. Although the current understanding of the effect of early life stress on medullary development is relatively limited, these data show that within this area, microglia are affected by neonatal stress. Microglia could therefore be important effectors in cardiorespiratory disorders resulting from maternal separation. Neonatal stress has wide-ranging consequences for the developing brain, including the medullary cardiorespiratory network. In rat pups, the reflexive cardiorespiratory inhibition triggered by the presence of liquids near the larynx is augmented by neonatal maternal separation (NMS), especially in males. Sex-specific enhancement of synaptic connectivity by NMS might explain this cardiorespiratory dysfunction. Microglia influence the formation, maturation, activity and elimination of developing synapses, but their role in the wiring of medullary networks is unknown. Owing to their sensitivity to sex hormones and stress hormones, microglial dysfunction could contribute to the abnormal cardiorespiratory phenotype observed in NMS pups. Here, we first used ionized calcium-binding adapter molecule-1(Iba-1) immunolabelling to compare the density and morphology of microglia in the medulla of male versus female rat pups (14-15days old) that were either undisturbed or subjected to NMS (3hday-1 ; postnatal days3-12). Neonatal maternal separation augmented the density of Iba-1+ cells (caudal region of the NTS), increased the size of the soma and reduced the arborization area (especially in the dorsal motor nucleus of the vagus). Sex-based differences were not observed. Given that the actions of microglia are regulated by neuronal fractalkine (CX3 CL1 ), we then used western blot analysis to compare the expression of CX3 CL1 and its microglial receptor (CX3 CR1 ) in medullary homogenates from control and NMS pups. Although CX3 CR1 expression was 59% greater in males versus females, NMS had no effect on CX3 CL1 /CX3 CR1 signalling. Given that an amoeboid morphology reflects an immature phenotype in developing microglia, NMS could interfere with synaptic pruning via a different mechanism.

A prolonged exposure to ketamine triggers significant neurodegeneration and long-term neurocognitive deficits in the developing brain. Monosialotetrahexosylganglioside (GM1) can limit the neuronal damage from necrosis and apoptosis in neurodegenerative conditions. We aimed to assess whether GM1 can prevent ketamine-induced developmental neurotoxicity. Postnatal day 7 (P7) rat pups received 5 doses of intraperitoneal ketamine (20 mg/kg per dose) at 90-minute intervals for 6 hours. Cognitive functions, determined by using Morris water maze (MWM) including escape latency (at P32-36) and platform crossing (at P37), were compared among the ketamine-exposed pups treated with or without exogenous GM1 (30 mg/kg; n = 12/group). The effect of GM1 on apoptosis in hippocampus was determined by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining and activated caspase 3 measurement. The hippocampal expression of brain-derived neurotrophic factor (BDNF), along with the phosphorylation of protein kinase B (AKT) and extracellular signal-related kinases 1 and 2 (ERK1/2), was detected by western blotting (n = 6/group). Anti-BDNF antibody (2 μg per rat) administered before GM1 treatment was applied to determine the neuroprotective mechanisms of GM1. The rats receiving ketamine exposure experinced cognitive impairment in MWM test compared to the control rats, indicated by prolonged escape latency at P34 (P = .006), P35 (P = .002), and P36 (P = .005). However, in GM1-pretreated rats, ketamine exposure did not induce prolonged escape latency. The exogenous GM1 increased the platform-crossing times at P37 (3.00 ± 2.22 times vs 5.40 ± 1.53 times, mean ± standard deviation; P = .041) and reduced the hippocampal TUNEL-positive cells and cleaved-caspase 3 expression in ketamine-exposed young rats. Ketamine decreased BDNF expression and phosphorylation of AKT and ERK in the hippocampus, whereas exogenous GM1 blocked these ketamine-caused effects. However, for the ketamine-exposed rat pups receiving exogenous GM1, compared to immunoglobulin Y (IgY) isotype control, the BDNF-neutralizing antibody treatment counteracted the exogenous GM1-induced improvement of the escape latency at P36 (41.32 ± 12.37 seconds vs 25.14 ± 8.97 seconds, mean ± standard deviation; P = .036), platform-crossing times at P37 (2.16 ± 1.12 times vs 3.92 ± 1.97 times, mean ± standard deviation; P < .036), apoptotic activity, as well as AKT and ERK1/2 phosphorylation in the hippocampus of ketamine-challenged young rats. Our data suggest that the exogenous GM1 acts on BDNF signaling pathway to ameliorate the cognitive impairment and hippocampal apoptosis induced by ketamine in young rats. Our study may indicate a potential use of GM1 in preventing the cognitive deficits induced by ketamine in the young per se.

Glutamate is a representative excitatory neurotransmitter. However, excessive glutamate exposure causes neuronal cell damage by generating neuronal excitotoxicity. Excitotoxicity in neonates caused by glutamate treatment induces neurological deficits in adults. The 14–3-3 family proteins are conserved proteins that are expressed ubiquitously in a variety of tissues. These proteins contribute to cellular processes, including signal transduction, protein synthesis, and cell cycle control. We proposed that glutamate induces neuronal cell damage by regulating 14–3-3 protein expression in newborn animals. In this study, we investigated the histopathological changes and 14–3-3 proteins expressions as a result of glutamate exposure in the neonatal cerebral cortex. Rat pups at post-natal day 7 were intraperitoneally administrated with vehicle or glutamate (10 mg/kg). Animals were sacrificed 4 h after treatment, and brain tissues were fixed for histological study. Cerebral cortices were isolated and frozen for proteomic study. We observed serious histopathological damages including shrunken dendrites and atypical neurons in glutamate-treated cerebral cortices. In addition, we identified that 14–3-3 family proteins decreased in glutamate-exposed cerebral cortices using a proteomic approach. Moreover, Western blot analysis provided results that glutamate treatment in neonates decreased 14–3-3 family proteins expressions, including the β/α, ζ/δ, γ, ε, τ, and η isoforms. 14–3-3 proteins are involved in signal transduction, metabolism, and anti-apoptotic functions. Thus, our findings suggest that glutamate induces neonatal neuronal cell damage by modulating 14–3-3 protein expression.

The iron chelator deferasirox (DFX) prevents complications related to transfusional iron overload in several haematological disorders characterized by marrow failure. It is also able to induce haematological responses in a percentage of treated patients, particularly in those affected by myelodysplastic syndromes. The underlying mechanisms responsible for this feature, however, are still poorly understood. In this study, we investigated the effect of DFX-treatment in human haematopoietic/progenitor stem cells, focussing on its impact on the redox balance, which proved to control the interplay between stemness maintenance, self-renewal and differentiation priming. Here we show, for the first time, that DFX treatment induces a significant diphenyleneiodonium-sensitive reactive oxygen species (ROS) production that leads to the activation of POU5F1 (OCT4), SOX2 and SOX17 gene expression, relevant in reprogramming processes, and the reduction of the haematopoietic regulatory proteins CTNNB1 (β-Catenin) and BMI1. These DFX-mediated events were accompanied by decreased CD34 expression, increased mitochondrial mass and up-regulation of the erythropoietic marker CD71 (TFRC) and were compound-specific, dissimilar to deferoxamine. Our findings would suggest a novel mechanism by which DFX, probably independently on its iron-chelating property but through ROS signalling activation, may influence key factors involved in self-renewal/differentiation of haematopoietic stem cells.

Corticosteroid responses following hypoxic preconditioning provide neuroprotection against subsequent hypoxic-ischemic brain injury in the newborn rats

Long-Term Cryopreservation of Peripheral Blood Stem Cell Harvest Using Low Concentration (4.35%) Dimethyl Sulfoxide with Methyl Cellulose and Uncontrolled Rate Freezing at -80 °C: An Effective Option in Resource-Limited Settings

Sex differences in microglia morphology and function across the lifespan are mediated by the early hormone environment.

Post-thaw cell viability assessment is very important in cryopreservation because it is the main assessment method used to optimize cryopreservation protocols for each cell type; hence, having standardized accurate, quick, and reliable assays for post-thaw cell viability measurements is of utmost importance. The trypan blue exclusion assay and nucleic-acid-binding fluorescence-based assays are two different methods for cell viability assessment. Both assays identify cells with damaged membranes by whether they let a compound enter the cell. In this study, these two assays are compared in the context of cryopreservation and the impacts of important cryopreservation parameters on the differences in measurements are investigated. H9c2 myoblasts were cryopreserved with different freezing protocols. Cell membrane integrities were measured immediately after thaw as well as after cryoprotectant removal by a hemocytometer-based trypan blue dye exclusion assay and a dual fluorometric SYTO 13/GelRed assay; and the results were compared. This study quantifies how (i) the absence or presence of different cryoprotectants, (ii) different cell–cryoprotectant incubation conditions, and (iii) the presence or removal of cryoprotectants after thaw affect the differences between these two viability assays.

Objective To determine the role of metformin in regulating intracellular redox status and cell growth in prostate cancer cells Background : Prostate cancer is the second leading cause of cancer-related death among males in the United States. Despite several advances in treatment options, there are several underlying challenges that affect prostate cancer therapy, including patient age, cancer resistance and undesirable side effects. Moreover, current treatment options focus on two or more cellular targets, such as, androgen synthesis, DNA repair enzymes and immune cells. Such cellular targets bypass the underlying supportive metabolic and molecular phenotypes that contribute to a cancer cell's ability to evade apoptosis, proliferate, and develop chemoresistance. Over the last decade, both preclinical and clinical studies have indicated the potential of metformin in prostate cancer therapy. Notably, these extensive studies demonstrated metformin's pleiotropic effects on several molecular and metabolic pathways such as androgen signaling, cell cycle and cellular bioenergetics, including its unique ability to activate the energy sensor 5 AMP activated kinase (AMPK). We previously demonstrated that metformin alters mitochondrial function in LNCaP prostate cancer cells resulting in elevated glycolysis. In light of this, we hypothesized that metformin induced oxidative stress, modulates prostate cancer cell survival, and is involved in its antiproliferative effects via the transsulfuration pathway. Methods : Using Seahorse XP extracellular analysis, we measured LNCaP prostate cancer cellular bioenergetics oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in the presence of metformin and SBI0206965 (an AMPK inhibitor). RT-PCR, Liquid Chromatography/ Mass Spectrophotometry (LC/MS), Trypan Blue Exclusion and MTT assays were used to assess the effect of metformin on intracellular redox status and prostate cancer survival. Additionally, we also measured transsulfuration-related enzymatic activities using an ELISA Assay. Results Metformin altered prostate cancer cellular bioenergetics resulting in decreased OCR and increased ECAR, reflecting oxidative phosphorylation and glycolytic activity, respectively. LC/MS analysis of metformin-treated cells showed a significant reduction in GSH/GSSG ratio and metabolite levels associated with the transsulfuration pathway such as glutathione (GSH) and cystathionine (CYS). These low levels of GSH and CYS were consistent with an increase in the gene expression of transsulfuration related enzymes, cystathionine beta synthase (CBS) and cystathionine gamma-lyase (CSE). Additionally, metformin increased the gene expression of the glutathione synthesis-related enzyme γ-glutamyl cysteine ligase (GCL), with no observable change in glutathione synthetase (GSS) gene expression. Notably, MTT and trypan blue exclusion assays indicated that metformin significantly decreased cell viability and had an anti-proliferative effect on LNCaP prostate cancer cells. Conclusion :Metformin induced oxidative stress modulates transsulfuration and glutathione synthesis pathways and exerts antiproliferative effects in LNCaP prostate cancer cells.

Study aims were to evaluate the safety and efficacy of the Food and Drug Administration-approved drug Vorinostat [suberoylanilide hydroxamic acid (SAHA)] in the treatment of canine corneal fibrosis using an in vitro model. Healthy donor canine corneas were collected and used to generate primary canine corneal fibroblasts (CCFs) by growing cultures in minimal essential medium supplemented with 10% fetal bovine serum. Canine corneal myofibroblasts, used as a model for corneal fibrosis, were produced by growing CCF cultures in serum-free medium containing transforming growth factor β1 (1 ng/mL). Trypan blue exclusion assays were used to determine the optimal SAHA dose for this in vitro model. Four hour after culturing with TGFβ1, CCF cultures were treated with 0.06% SAHA for 5 min (group 1) and for 24 h (group 2), representing single and multiple dose treatment regimes, respectively. Cultures were then further incubated in the presence of TGFβ1 (1 ng/μL) under serum-free conditions until they reached 70% confluence. Trypan blue exclusion, immunocytochemistry, and TUNEL assays were used to evaluate the cytotoxicity of SAHA. Real-time PCR, western blot analysis, and immunocytochemistry were used to determine the efficacy of SAHA to inhibit canine corneal myofibroblast formation. Topical SAHA application in both treatment groups successfully decreased α-smooth muscle actin expression when compared to the TGFβ1 only treatment group (P < 0.05). Tested SAHA did not affect CCF phenotype or cellular viability and did not cause significant cell death. Suberoylanilide hydroxamic acid safely and effectively inhibits TGFβ1-induced CCFs transformation to myofibroblast in vitro.