Rat Brain Samples Research Articles

Evaluation of H2S-producing enzymes in cerebrospinal fluid and its relationship with interleukin-6 and neurologic deficits in subarachnoid hemorrhage

Background and purposeRecent studies have suggested that H2S may be involved in the pathophysiology of subarachnoid hemorrhage (SAH). Endogenous H2S is mainly formed by cystathionine β-synthase (CBS), d-amino-acid oxidase (DAO), and 3-mercaptopyruvate sulfotransferase (3-MST) from the substrate cysteine in the central nervous system. In this study, we assessed the expression of CBS, 3-MST, and DAO in cerebrospinal fluid (CSF) from patients with SAH and rats and the expression in the rat brain. MethodsCSF samples were collected within 48 h of aneurysm rupture in SAH patients. The CBS, DAO and 3-MST levels in CSF were measured using Western blot analyses, and correlations with the inflammatory parameter Interleukin-6 (IL-6) were assessed. Six months after SAH, the clinical outcomes were assessed. ResultsIn human CSF samples, the CBS and DAO protein levels were detected and increased after SAH. However, 3-MST was not detected in the control group CSF but increased after SAH. Strong correlations were observed between the increasing levels of CBS, DAO, and 3-MST and IL-6 2 days after SAH. Furthermore, high CBS, 3-MST and DAO levels in the CSF samples were correlated with poor outcomes at 6 months after SAH onset. We also found that the expression of CBS, DAO and 3-MST in the rat CSF and brain (parietal cortex and hippocampus) increased following SAH. We detected strong correlations between the increases in CBS, 3-MST and IL-6 in the rat CSF and brain samples. ConclusionsThese results indicate that the upregulated expression of CBS, DAO and 3-MST after SAH was closely associated with the inflammatory response and neurological deficits after SAH.

Tin disulfide nanorod-graphene-β-cyclodextrin nanocomposites for sensing dopamine in rat brains and human blood serum.

In the present work describes a facile synthesis of tin disulfide (SnS2) nanorods decorated graphene-β-cyclodextrin (SnS2/GR-β-CD) nanocomposite for robust and novel dopamine (DA) electrochemical biosensor applications. The DA biosensor was fabricated using the glassy carbon electrode (GCE) modified with SnS2/GR-β-CD nanocomposite. The sonochemical and hydrothermal methods have been used for the synthesis of SnS2/GR-β-CD. Different physicochemical methods were used to confirm the formation of the GR-β-CD, SnS2, and SnS2/GR-β-CD nanocomposite. The cyclicvoltammetric cathodic current response of DA was 5 folds higher than those observed at bare, β-CD, SnS2-β-CD, and GR-β-CD modified GCEs. Under optimised conditions, the biosensor's DPV response current is linear to DA from the concentration of 0.01-150.76 μM. The detection limit of the biosensor was 4 nM. The SnS2/GR-β-CD biosensor shows an excellent selectivity towards DA in the presence of common interfering species, including ascorbic acid and uric acid. Also, the as-prepared nanocomposite-modified electrode exhibited satisfactory long-term stability, sensitivity (2.49 μAμM-1 cm-2) along with reusability for detection of DA. The fabricated SnS2/GR-β-CD biosensor was successfully used for the detection of DA in the rat brain and human blood serum samples.

Inhibitory influence of agmatine in ethanol withdrawal-induced depression in rats: Behavioral and neurochemical evidence.

Although ethanol withdrawal depression is one of the prominent reasons for ethanol consumption reinstatement and ethanol dependence, its neurochemical basis is not clearly understood. The present study investigated the role of the agmatinergic system in ethanol withdrawal-induced depression using the forced swim test (FST) in rats. Chronic exposure of animals to ethanol for 21 days and its abrupt withdrawal produced depression-like behavior, as evidenced by increased immobility time in the FST, compared to the pair-fed control animals. The ethanol withdrawal-induced depression was significantly attenuated by agmatine (20-40 μg/rat, i.c.v. [intracerebroventricularly]), moxonidine (50 μg/rat, i.c.v.), 2-BFI (20 μg/rat, i.c.v.), L-arginine (80 μg/rat, i.c.v.), amino-guanidine (25 μg/rat, i.c.v.), and arcaine (50 μg/rat, i.c.v.) by their once-daily administration during the withdrawal phase (Days 21, 22, and 23). The antidepressant effect of agmatine in ethanol-withdrawn rats was potentiated by the imidazoline receptor I1 agonist moxonidine (25 μg/rat, i.c.v.) and the imidazoline receptor I2 agonist, 2-BFI (10 μg/rat, i.c.v.) at their sub-effective doses. On the other hand, it was completely blocked by the imidazoline receptor I1 antagonist, efaroxan (10 μg/rat, i.c.v.) and the imidazoline receptor I2 antagonist, idazoxan (4 μg/rat, i.c.v.). In addition, agmatine levels were significantly reduced in brain samples of ethanol-withdrawn rats as compared to the pair-fed control animals. In conclusion, the present study suggests the importance of the endogenous agmatinergic system and the imidazoline receptors system in ethanol withdrawal-induced depression. The data project agmatine as a potential therapeutic target for the alcohol withdrawal-induced depression.

Optimised lyophilisation-based method for different biomolecule single-extractions from the same rat brain sample: Suitability for RNA and protein expression analyses after ischemic stroke

BackgroundOptimisation of tissue processing procedures in preclinical studies reduces the number of animals used and allows integrated multilevel study in the same sample. Multiple extraction of different biomolecules from the same sample has several limitations. New methodUsing brain samples from rats subjected to ischemic stroke, we combined lyophilisation of flash-frozen tissue, mechanical pulverisation and cryopreservation in a method to optimise tissue handling and preservation for independent RNA or protein single-extract methods, and subsequent RT-qPCR or Western blot analyses. ResultsLyophilisation resulted in 70% tissue weight loss. RNA (OD260/280∼1.8) and protein yields were similar in non-ischemic and ischemic brain samples, subjected to either flash freezing (FF) or flash freezing followed by lyophilisation (FF + Lyo). RNA transcription of reference genes (Actb and Rn18s), expression of housekeeping proteins (β-actin and α-tubulin), and mRNA overexpression of stroke-regulated genes (Nos2, Mmp9 and Tnfa) was similar in FF and FF + Lyo samples. Comparison with existing method(s)Contrary to high heat stress of baking method in a drying oven, lyophilisation maintains the integrity of dried samples for subsequent extractions and analyses. Sample lyophilisation allows different manual representative extractions/analyses from the same rat, it is much cheaper than using commercial kits, and shows higher yields that multiple manual or kit-based extractions. ConclusionsThe lyophilisation-based method for different biomolecule single-extractions from tissue powder aliquots, representing the same rat brain sample, is sample saving, contributes to the reduction principle in animal research, and allows coordinated analysis for accurate correlations between the transcriptome and proteome in stroke and other neuroscience research.

Determination of Ochratoxin-A in the brain microdialysates and plasma of awake, freely moving rats using ultra high performance liquid chromatography fluorescence detection method

In this study, a simple, efficient and rapid Ultra High Performance Liquid Chromatography method with fluorescence detection (UHPLC-FLD) has been developed and validated for the determination of Ochratoxin-A (OTA) in rat brain microdialysates and plasma samples. Six adult male wistar rats were used in the study and a single dose (5 mg/kg b.w.) of OTA was given by intraperitoneal (i.p.) injection. Rat blood and microdialysate samples were collected simultaneously after i.p. injection in awake freely moving rats, over a twelve-hour period. An UHPLC analysis was performed on a Zorbax Eclipse Plus C8 (150 mm × 3.0 mm ID × 1.8 μm particles) column with a mobile phase of acetonitrile:water:phosphoric acid (50:50:0.1, v/v) using a flow rate of 0.6 mL/min. The fluorescence detector was set at 330 nm excitation and 460 nm emission wavelengths. Diflunisal (DIF) was used as an internal standard (IS). OTA and IS were separated within 5 min under these conditions. The method was validated in terms of linearity, precision, accuracy, limit of detection, limit of quantification, and stability. Calibration curves obtained with spiked biological matrices show good linearity with high correlation coefficients. The intra- and inter-day assay variability was <5% for the OTA. The limit of detection and the limit of quantification values were found to be 0.490 ng/mL and 1.48 ng/mL for plasma; 0.0900 ng/mL and 0.270 ng/mL for microdialysate samples, respectively. This method was successfully applied for the monitoring of OTA levels in the rat brain and plasma samples.

Trim47 is a critical regulator of cerebral ischemia-reperfusion injury through regulating apoptosis and inflammation

Cerebral ischemia is a leading cause of death and long-term disability in the world. Tripartite motif-47 (Trim47), a member of the TRIM family proteins, has been reported to be involved in apoptosis and inflammation in various types of diseases. Nevertheless, the underlying molecular mechanism of Trim47 in cerebral ischemia/reperfusion (I/R) injury remains unclear. This study aimed to explore the role of Trim47 in cerebral I/R injury and the potential underlying mechanisms. The results indicated that Trim47 expression was markedly induced in rats after stroke onset. By the use of genetic approaches, we indicated that Trim47 knockdown significantly reduced the infarct size, mitigated the neurological deficits scores and decreased brain water contents in rats with cerebral I/R injury induced by middle cerebral artery occlusion (MCAO). In addition, Trim47 knockdown-alleviated cerebral I/R was correlated with the suppression of apoptosis through inhibiting Caspase-3 cleavage. Furthermore, reducing Trim47 apparently decreased the release of pro-inflammatory factors, including interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS), in brain samples of MCAO rats, which was partly by the blockage of nuclear factor-kappa B (NF-κB) signaling. However, Trim47 over-expression markedly accelerated cerebral ischemia injury through promoting apoptosis and inflammation. The suppressive effects of Trim47 knockdown on cerebral I/R were verified in human neuron-like cells stimulated by oxygen and glucose deprivation (OGD). Thus, this study demonstrated a new mechanism for the effect of Trim47 on cerebral I/R injury, and targeting Trim47 might provide feasible therapies for stroke treatment.

Pharmacokinetics, excretion and metabolites analysis of DL0410, a dual‑acting cholinesterase inhibitor and histamine‑3 receptor antagonist.

DL0410, a dual-action cholinesterase inhibitor and histamine-3 receptor antagonist with a novel structural scaffold, may be a potential candidate for the treatment of Alzheimer's disease (AD). To the best of the authors' knowledge, this is the first study to demonstrate a reliable method for the measurement of DL0410 in rat plasma, brain, bile, urine and feces samples, and identification of its primary metabolites. The pharmacokinetic properties of DL0410 were analyzed by liquid chromatography-mass spectrometry at oral doses of 25, 50 and 100 mg/kg and intravenous dose of 5 mg/kg. The investigation of the excretion and metabolism of DL0410 was determined following liquid-liquid extraction for biliary, urinary and fecal samples. Finally, the cytochrome (CY)P450 isoforms involved in the production of DL0410 metabolites with recombinant human cytochrome P450 enzymes were characterized. The results suggested that DL0410 was not well absorbed; however, was distributed to the entorhinal cortex and hippocampus of the brain. A total of two common metabolites of the reduction of DL0140 in the bile, urine and feces were identified and CYP2D6 was involved in this reaction. The pharmacokinetic results of DL0410 provided information for the illustration of its pharmacodynamic properties, mechanism of action and promoted its continued evaluation as a therapeutic agent for AD treatment.

Rational design and facile synthesis of binary metal sulfides VS2-SnS2 hybrid with functionalized multiwalled carbon nanotube for the selective detection of neurotransmitter dopamine

In this work, we report a sensitive and selective electrochemical sensor for the detection of dopamine (DA) neurotransmitter based on VS2-SnS2/f-MWCNT hybrids. Herein, the binary metal sulfide (VS2-SnS2) was synthesized via single step hydrothermal route and hybrids with f-MWCNT via the ultrasonication process. The as-prepared VS2-SnS2/f-MWCNT hybrids were characterized through the FESEM, EDX and elemental mapping, TEM, XPS, Raman and XRD techniques. The electrochemical performance and catalytic activity of the modified electrodes were probed using electrochemical impedance spectra (EIS), cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Interestingly, DPV results exhibits an appreciable linear range from 0.025 to 1017 μM and LOD of 0.008 μM. The selectivity study was performed to prove the high selectivity of the VS2-SnS2/f-MWCNT hybrids modified electrode. Furthermore, the practical applicability of the DA sensor was scrutinized in human serum sample and rat brain sample.

An integrated strategy for ascertaining quality marker of Schisandra chinensis (Turcz.) Baill based on correlation analysis between depression-related monoaminergic metabolites and chemical components profiling

Traditional Chinese Medicines (TCMs) have been widely used in orient countries for thousands of years, while their inconsistent quality and therapy issues have become increasingly serious as a result of the absence of effective methods for quality control. Therefore, it is necessary to develop a novel and specific evaluation system for TCMs’ quality involved with not only composition but also bioactivity. In this study, we used Schisandra chinensis (Turcz.) Baill as an example and developed a novel integrated approach involved with various chemical analysis and data processing methods to explore its quality marker (Q-marker) underlying its anti-depressive effects. First, six bioactive lignans were identified and semi-quantified in rat brain samples via high resolution mass spectrometry. Then, the bioinformation analysis showed that all the six bioactive components could modulate various diseases relative to noradrenergic, dopaminergic and serotonergic pathways. Thus, the monoaminergic metabolites contained in these three pathways were selected to screen potential biomarkers of depression treated by S. chinensis based on target metabolomics using a rapid HPLC-MS/MS method. Finally, the correlation analysis between the six components and potential biomarkers was employed to uncover the Q-markers of S. chinensis. It is suggested that schisandrol A, schisandrin A, schisandrin C and gomisin N could be determined as Q-markers for S. chinensis. Thus, the integrated approach describing here for discovering Q-markers was expected to offer an alternative quality assessment strategy of herbal medicines for the first time.

Liquid Chromatography Analysis of Clozapine in Rat Brain Tissue, Using its Molecularly Imprinted Polymer after Administration of Toxic Dose of Drug and Lipid Emulsion Therapy

Background:Molecularly imprinted polymers (MIPs) are synthetic polymers that have a selective site for a given analyte, or a group of structurally related compounds, that make them ideal polymers to be used in separation processes.Objective:An optimized molecularly imprinted polymer was selected and applied for selective extraction and analysis of clozapine in rat brain tissue.Methods:A molecularly imprinted solid-phase extraction (MISPE) method was developed for preconcentration and cleanup of clozapine in rat brain samples before HPLC-UV analysis. The extraction and analytical process was calibrated in the range of 0.025-100 ppm. Clozapine recovery in this MISPE process was calculated between 99.40 and 102.96%. The limit of detection (LOD) and the limit of quantification (LOQ) of the assay were 0.003 and 0.025 ppm, respectively. Intra-day precision values for clozapine concentrations of 0.125 and 0.025 ppm were 5.30 and 3.55%, whereas inter-day precision values of these concentrations were 9.23 and 6.15%, respectively. In this study, the effect of lipid emulsion infusion in reducing the brain concentration of drug was also evaluated.Results:The data indicated that calibrated method was successfully applied for the analysis of clozapine in the real rat brain samples after administration of a toxic dose to animal. Finally, the efficacy of lipid emulsion therapy in reducing the brain tissue concentration of clozapine after toxic administration of drug was determined.Conclusion:The proposed MISPE method could be applied in the extraction and preconcentration before HPLC-UV analysis of clozapine in rat brain tissue.

Abstract TP139: MicroRNAs Expression Patterns of Hemorrhagic Transformation After Mechanical Reperfusion for Acute Ischemic Stroke in Rat

Background: Severe hemorrhagic transformation (HT) after mechanical thrombectomy and intravenous thrombolysis predicts poor clinical outcome in acute ischemic stroke. The specific roles of microRNAs in the pathogenesis of HT, however, are still unclear. Thus, the aim of this study is to investigate miRNA expression profiles in the rat ischemic brain with HT after mechanical reperfusion. Methods: A model of reperfusion-induced HT was established in rats with hyperglycemic challenge and 5h middle cerebral artery occlusion (MCAO) followed by 19h reperfusion. Infarct volume, brain water content, HT, neurological score, and blood-brain barrier damage were determined at 24h after cerebral ischemia. The miRNA expression profiling of rat brain samples in the MCAO group and those in the sham-operated group was performed using the miRCURYLNA Array. The differentially expressed miRNAs were further validated by qRT-PCR. Then, after predicting their target genes from top-ranked miRNAs, the KEGG and GO analyses were further used to predict the associated significant cell signaling pathways and functions. Results: Reperfusion with 5h of MCAO induced HT in all hyperglycemic rats, and most of them were the parenchymal hematoma. The results from the miRNA array revealed that 57 miRNAs were significantly altered 24h after reperfusion in the ischemic brain with HT. The expressions of these 57 miRNAs were further verified using qRT-PCR, and 33 top-ranked miRNAs were confirmed. Among them, 30 miRNAs were significantly downregulated and 3 were significantly upregulated in rats with HT. The top-listed downregulated miRNAs included miR-383-5p, miR-335, miR-376a-5p, miR-212-5p, miR-219a, miR-126a-5p. Three miRNAs were significantly upregulated, including miR-125b, miR-195, miR-214. The most enriched pathways are associated with the Hippo signaling pathway which regulates cell survival and death. Twenty significantly regulated miRNAs involve into the Hippo signaling pathway, including miR-383-5p. Conclusions: Alteration of the miRNAs in the ischemic brain with HT may serve as biomarkers for HT after mechanical reperfusion in acute ischemic stroke. The role of reperfusion-inducible miR-383-5p and Hippo signaling pathway in the HT requires further investigation.

Sexual dimorphism in brain transcriptomes of Amami spiny rats (Tokudaia osimensis): a rodent species where males lack the Y chromosome

BackgroundBrain sexual differentiation is sculpted by precise coordination of steroid hormones during development. Programming of several brain regions in males depends upon aromatase conversion of testosterone to estrogen. However, it is not clear the direct contribution that Y chromosome associated genes, especially sex-determining region Y (Sry), might exert on brain sexual differentiation in therian mammals. Two species of spiny rats: Amami spiny rat (Tokudaia osimensis) and Tokunoshima spiny rat (T. tokunoshimensis) lack a Y chromosome/Sry, and these individuals possess an XO chromosome system in both sexes. Both Tokudaia species are highly endangered. To assess the neural transcriptome profile in male and female Amami spiny rats, RNA was isolated from brain samples of adult male and female spiny rats that had died accidentally and used for RNAseq analyses.ResultsRNAseq analyses confirmed that several genes and individual transcripts were differentially expressed between males and females. In males, seminal vesicle secretory protein 5 (Svs5) and cytochrome P450 1B1 (Cyp1b1) genes were significantly elevated compared to females, whereas serine (or cysteine) peptidase inhibitor, clade A, member 3 N (Serpina3n) was upregulated in females. Many individual transcripts elevated in males included those encoding for zinc finger proteins, e.g. zinc finger protein X-linked (Zfx).ConclusionsThis method successfully identified several genes and transcripts that showed expression differences in the brain of adult male and female Amami spiny rat. The functional significance of these findings, especially differential expression of transcripts encoding zinc finger proteins, in this unusual rodent species remains to be determined.

Metabolomic analysis of the brain and blood from rats exposed to high-dose chlorpyrifos

Chlorpyrifos is an organophosphate pesticide used to kill pests such as insects and worms. Wide use of chlorpyrifos has led to serious safety concerns worldwide. Research on the mechanism of action of chlorpyrifos poisoning is continuing. We investigated changes in the small-molecular metabolites in the brain and blood of rats upon exposure to chlorpyrifos at an acute-poisoning dose. Rats were given twice the lowest dose of chlorpyrifos that is lethal for 100% of exposed animals (2 × LD100) and then killed after 2 h. After treatment, gas chromatography-mass spectrometry was used to analyze the metabolomic changes in the brain and blood samples of rats. An increase in blood levels of creatinine and uric acid were noted, along with a decrease in levels of various amino acids. These changes suggested that chlorpyrifos exposure may damage kidney function and cause disorders in amino-acid metabolism of rats. Decreased concentrations of gamma-aminobutyric acid and niacinamide in the brain and increased concentrations of 3-hydroxybutyric acid in rats with acute poisoning by chlorpyrifos were observed, which may suggest oxidative damage in the body.

Optimized liquid chromatography-tandem mass spectrometry for Otaplimastat quantification in rat plasma and brain tissue.

An optimized liquid chromatography-tandem mass spectrometry method for simple and sensitive quantification of Otaplimastat in rat plasma and brain tissue was developed and validated. Protein precipitation with acetonitrile was selected for sample preparation method based on recovery and matrix effect. The chromatographic separation of the sample was performed on a reverse-phase AQ column with an isocratic mobile phase consisting of 10 mM ammonium acetate (pH 4.0) and acetonitrile (50:50, v/v). The analyte was quantified by multiple reaction monitoring with a Waters Quattro micro™ API mass spectrometer. The lower limits of quantification were 20 ng/mL in plasma and 2 ng/g in brain, with the relative standard deviation % of 7.6 and 8.0% for plasma and brain samples, respectively. Acceptable intra-day and inter-day precisions and accuracies were obtained. Otaplimastat was sufficiently stable under all relevant analytical conditions, including a temperature of 4°C for 24 hr, room temperature 20°C for 24 hr, -80°C for 10 days and three freeze-thaw cycles (each at -80°C for 24 hr), for rat plasma and brain tissue. The validated method was successfully used to measure Otaplimastat concentrations in rat plasma and brain samples.

Validated HPLC-MS/MS method for quantification of ethylmethylhydroxypyridine succinate in rat brain and its application to a pharmacokinetic study

2-ethyl-6-methyl-3-hydroxypyridine (EMHP) succinate is the original antioxidant and antihypoxic drug commonly prescribed in Russia. The objective of this study was to develop a rapid, simple and sensitive high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) method for EMHP quantitation in rat brain tissue with the use of a bead beating homogenizer. The comparison between two approaches to brain tissue preparation was performed, when spiking the blank brain tissue with EMHP reference standard and internal standard (IS) before and after homogenization step. Chromatographic separation was achieved using Zorbax Eclipse Plus C18 column (1.8 μm, 2.1 × 50 mm) and elution was performed with the mobile phase, consisting of 10 mM of ammonium formate aqueous solution with 0.1% formic acid as solvent A and 0.1% formic acid in methanol as solvent B [44%(А):56%(В), v/v]. Flow rate was 0.4 mL/min and the total run time for each sample analysis was 2.0 min. EMHP and amantadine, IS of this study, were analyzed in positive ionization mode. Ion transitions of m/z 138.0 → 123.0 for EMHP and m/z 152.0 → 135.0 for amantadine were selected in multiple reaction monitoring mode. The developed method for EMHP determination in rat brain samples was validated for selectivity, linearity, accuracy, precision, matrix effects, and stability. The lower and upper limits of quantification were determined to be 1 and 1500 ng/g, respectively. The developed and validated HPLC-MS/MS method was successfully applied to determine EMHP concentrations in rat brain tissue following the intraperitoneal administration at a dose of 3.4 mg/kg.

Untargeted rat brain metabolomics after oral administration of a single high dose of cannabidiol

Cannabidiol (CBD), for long time considered as a minor cannabinoid of Cannabis sativa, has recently gained much attention due to its antioxidant, anti-inflammatory, analgesic and anticonvulsant properties. A liquid chromatography coupled to mass spectrometry based method was developed for the quantitative determination of CBD and other cannabinoids (Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC and 11-nor-9-carboxy-THC) in rat brain samples after oral administration of a single high dose (50 mg/kg) of CBD. The main challenge of the present work was to study CBD pharmacokinetics in rat cortex: the identification of its metabolites and pharmacodynamics through the study of variations in endogenous compounds’ concentrations following CBD administration. An untargeted metabolomics approach revealed the formation of some CBD metabolites that are not commonly found in other body tissues or fluids. Lastly, the changes in some endogenous compounds’ concentrations were correlated with some of the pharmacological properties of this cannabinoid.

Development and validation of rapid and sensitive LC methods with PDA and fluorescence detection for determination of piribedil in rat plasma and brain tissues and their pharmacokinetic application.

Simple, selective and sensitive high-performance liquid chromatographic (HPLC) bioanalytical methods using fluorescence (FL) and photodiode array (PDA) detectors were developed and validated for determination of piribedil (PBD), an anti-Parkinson's drug, in rat plasma and brain samples, with telmisartan as internal standard (IS). Protein precipitation technique was used to extract PBD from both biological matrices. Chromatographic separation was achieved on a Phenomenex Kinetex C18 end-capped column (250 × 4.6 mm, 5 μm), with 38:62 v/v acetonitrile and ammonium acetate buffer (pH 5.0) as mobile phase at 1.0 mL/min flow rate. Linear response in the concentration ranges 5-300 and 150-3000 ng/mL in plasma, and 15-900 and 450-9000 ng/g in brain tissue were achieved in FL and PDA detectors, respectively. The chromatograms were extracted at 239 nm in case of PDA detection and at excitation wavelength of 239 nm and emission wavelength of 385 nm in case of FL detection. FL detection was found to be more sensitive compared with PDA detection. The developed methods were successfully employed in determining the plasma time course, brain distribution and the pharmacokinetic parameters of PBD following intravenous bolus administration of the drug in male Wistar rats.

Effect of montelukast in experimental model of Parkinson’s disease

Despite the availability of many drugs offering symptomatic relief in Parkinson’s disease, there are no drugs available offering neuroprotective effect. Hence, it was decided to evaluate the neuroprotective effect of montelukast, an anti-inflammatory drug, in rotenone induced model of Parkinson’s disease in rats. Forty eight male wistar rats were randomly divided into three groups. Group 1: Vehicle control, Group 2: Montelukast 5 mg/kg, Group 3: Montelukast 10 mg/kg. All the groups received rotenone 2.5 mg/kg intraperitoneally for 10 days as a disease inducing agent. The study drug montelukast was administered to respective groups orally from day 11 to day 24. On day 25, 24 h after 14 days of study drug administration, the rats were subjected to open field test, rota rod test and catalepsy test. Brain samples of rats from each group were collected for Malondialdehyde(MDA), Glutathione(GSH) and TNFα analysis. In the open field test both the doses of montelukast showed significant increase in the locomotor activity and also decreased the immobility time compared to vehicle (p < 0.05). In rotarod test, montelukast 5 mg/kg and 10 mg/kg showed significant increase in the time to fall, compared to vehicle (p < 0.05). In catalepsy test, both doses of montelukast significantly decreased the retraction time compared to vehicle(p < 0.05). The brain MDA levels were decreased and GSH levels were found to be higher in the two montelukast groups compared to vehicle (p < 0.05). TNFα levels too were decreased significantly on montelukast administration. Montelukast showed potential neuroprotective effect by virtue of its anti-oxidant and anti-inflammatory actions.

A Simple and Highly Sensitive Quantitation of Eicosanoids in Biological Samples Using Nano-flow Liquid Chromatography/Mass Spectrometry.

A simple sample preparation method for eicosanoid was developed by the combination of deproteinization and nanoLC-ESI-MS/MS. Eicosanoids are a group of bioactive lipid mediators, present in trace amounts in the body. Therefore, an analytical method for eicosanoids requires superior sensitivity. The method described in this report, which takes advantage of the highly sensitive power of nanoLC-ESI-MS/MS, enabled a simplification of the sample-preparation process. Eicosanoid extraction was performed just by homogenization in methanol with subsequent phospholipid removal, and then the liquid phase was directly subjected to nanoLC-ESI-MS/MS analysis without a condensation process. The quantitation range achieved 0.01 - 100 ng/mL for thromboxane B2, and 0.05 - 100 ng/mL for prostaglandin E2, prostaglandin D2, prostaglandin F2, leukotriene B4, 6-keto prostaglandin F1α and 11-dehydro thromboxane B2. Rat brain sample analyses demonstrated the feasibility of the quantification of those seven eicosanoids from biological samples.

A-to-I RNA editing in the rat brain is age-dependent, region-specific and sensitive to environmental stress across generations

BackgroundAdenosine-to-inosine (A-to-I) RNA editing is an epigenetic modification catalyzed by adenosine deaminases acting on RNA (ADARs), and is especially prevalent in the brain. We used the highly accurate microfluidics-based multiplex PCR sequencing (mmPCR-seq) technique to assess the effects of development and environmental stress on A-to-I editing at 146 pre-selected, conserved sites in the rat prefrontal cortex and amygdala. Furthermore, we asked whether changes in editing can be observed in offspring of stress-exposed rats. In parallel, we assessed changes in ADARs expression levels.ResultsIn agreement with previous studies, we found editing to be generally higher in adult compared to neonatal rat brain. At birth, editing was generally lower in prefrontal cortex than in amygdala. Stress affected editing at the serotonin receptor 2c (Htr2c), and editing at this site was significantly altered in offspring of rats exposed to prereproductive stress across two generations. Stress-induced changes in Htr2c editing measured with mmPCR-seq were comparable to changes measured with Sanger and Illumina sequencing. Developmental and stress-induced changes in Adar and Adarb1 mRNA expression were observed but did not correlate with editing changes.ConclusionsOur findings indicate that mmPCR-seq can accurately detect A-to-I RNA editing in rat brain samples, and confirm previous accounts of a developmental increase in RNA editing rates. Our findings also point to stress in adolescence as an environmental factor that alters RNA editing patterns several generations forward, joining a growing body of literature describing the transgenerational effects of stress.