Rat Brain Tissue Extracts Research Articles

Induction of Tyrosine Hydroxylase Gene Expression in Embryonal Carcinoma Stem Cells Using a Natural Tissue-Specific Inducer.

A large number of studies have focused on the generation of dopaminergic neurons from pluripotent cells. Differentiation of stem cells into distinct cell types is influenced by tissue-specific microenvironment. Since, central nervous system undergoes further development during postnatal life, in the present study neonatal rat brain tissue extract (NRBE) was applied to direct the differentiation of embryonal carcinoma stem cell line, P19 into dopaminergic (DA) phenotypes. Additionally, a neuroprotective drug, deprenyl was used alone or in combination with the extract. Results from morphological, immunofluorescence, and qPCR analyses showed that during a period of one to three weeks, a large percentage of stem cells were differentiated into neural cells. The results also indicated the greater effect of NRBE on the differentiation of the cells into tyrosine hydroxylase-expressing cells. MS analysis of NRBE showed the enrichment of gene ontology terms related to cell differentiation and neurogenesis. Network analysis of the studied genes and some DA markers resulted in the suggestion of potential regulatory candidates such as AVP, ACHE, LHFPL5, and DLK1 genes. In conclusion, NRBE as a natural native inducer was apparently able to simulate the brain microenvironment and support neural differentiation of P19 cells.

The effects of grafted mesenchymal stem cells labeled with iron oxide or cobalt-zinc-iron nanoparticles on the biological macromolecules of rat brain tissue extracts

IntroductionRat mesenchymal stem cells (rMSCs) labeled with 1) poly-l-lysine-coated superparamagnetic iron oxide nanoparticles or 2) silica-coated cobalt-zinc-iron nanoparticles were implanted into the left brain hemisphere of rats, to assess their effects on the levels of oxidative damage to biological macromolecules in brain tissue.MethodsControls were implanted with unlabeled rMSCs. Animals were sacrificed 24 hours or 4 weeks after the treatment, and the implantation site along with the surrounding tissue was isolated from the brain. At the same intervals, parallel groups of animals were scanned in vivo by magnetic resonance imaging (MRI). The comet assay with enzymes of excision DNA repair (endonuclease III and formamidopyrimidine-DNA glycosylase) was used to analyze breaks and oxidative damage to DNA in the brain tissue. Oxidative damage to proteins and lipids was determined by measuring the levels of carbonyl groups and 15-F2t-isoprostane (enzyme-linked immunosorbent assay). MRI displayed implants of labeled cells as extensive hypointense areas in the brain tissue. In histological sections, the expression of glial fibrillary acidic protein and CD68 was analyzed to detect astrogliosis and inflammatory response.ResultsBoth contrast labels caused a similar response in the T2-weighted magnetic resonance (MR) image and the signal was clearly visible within 4 weeks after implantation of rMSCs. No increase of oxidative damage to DNA, lipids, or proteins over the control values was detected in any sample of brain tissue from the treated animals. Also, immunohistochemistry did not indicate any serious tissue impairment around the graft.ConclusionBoth tested types of nanoparticles appear to be prospective and safe labels for tracking the transplanted cells by MR.

BDNF expression is up-regulated by progesterone in human umbilical cord mesenchymal stem cells

Objective: To investigate whether promotion of neuronal differentiation of human umbilical cord mesenchymal stem cells (HUMSCs) by progesterone (PROG) involves changes in brain-derived neurotrophic factor (BDNF) levels.Methods: We used rat brain tissue extracts to mimic the brain microenvironment. Quantitative sandwich enzyme-linked immunosorbent assay was performed to measure levels of BDNF in cultured medium with or without PROG.Results: Progesterone increased levels of BDNF in HUMSCs.Conclusion: Progesterone enhancement of brain-derived neurotrophic factor levels may be involved in PROG activated-pathways to promote neuronal differentiation of HUMSCs.

Evaluation of Hadamard Transform Atmospheric Pressure Ion Mobility Time-of-Flight Mass Spectrometry for Complex Mixture Analysis

Ion mobility mass spectrometry (IMMS) has gained popularity in the analysis of complex mixtures such as those encountered in metabolomics and proteomics. However, the challenge that exists in conventional pulsed IMMS is its inherent low duty cycle. The first application of Hadamard transform (HT)-type signal coupled with atmospheric pressure IMMS to complex mixtures is presented. Performance of the prototype was assessed by the analysis of metabolite standard mixture. With 200 times increased IMS duty cycle in HT mode compared with conventional pulsed mode, the limit of detection (LOD) was decreased by ∼10 times. Evaluation for application to complex mixtures was achieved using the NIST Standard Reference Material 1950 Metabolites in Human Plasma. Approximately 180 metabolite ions were detected within 1 min with an IMS resolving power (Rp) of ∼100. Rapid chromatographic separation prior to IMMS analysis was also demonstrated for improving the response of metabolite ions in rat brain tissue extract.

Effect of rat brain tissue extracts on osteoblast proliferation and differentiation

The reason for enhanced fracture healing in traumatic brain injury patients is not clearly understood. It is possible that factors inherent in the brain passing through the blood-brain barrier to the peripheral circulation, or a disruption of central nervous system (CNS) control of the sympathetic nervous system (SNS), stimulates the process of fracture healing. In this study, we assessed proliferation [using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay] and differentiation [using alkaline phosphatase (ALP)] in rat osteoblasts incubated with gray matter or other tissue extracts with and without the addition of an α- or β-adrenergic receptor blocker (phentolamine or propranolol). Gray matter extract from normal brain caused a dose-dependent increase in osteoblast proliferation and differentiation. Serum from normal rats enhanced differentiation but not proliferation. Alpha-receptor blockade had no effect on proliferation or differentiation. Beta-receptor blockade caused a partial, but statistically significant, decrease in gray matter stimulation of osteoblast differentiation. The results of this study indicate that gray matter extract from normal brain increases osteoblast proliferation and differentiation and that β receptors may be involved in differentiation under these conditions.

Radiosynthesis and in vivo evaluation of 1-[ 18F]fluoroelacridar as a positron emission tomography tracer for P-glycoprotein and breast cancer resistance protein

Aim of this study was to label the potent dual P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) inhibitor elacridar ( 1) with 18F to provide a positron emission tomography (PET) radiotracer to visualize Pgp and BCRP. A series of new 1- and 2-halogen- and nitro-substituted derivatives of 1 ( 4a– e) was synthesized as precursor molecules and reference compounds for radiolabelling and shown to display comparable in vitro potency to 1 in increasing rhodamine 123 accumulation in a cell line overexpressing human Pgp (MDCKII-MDR1). 1-[ 18F]fluoroelacridar ([ 18F] 4b) was synthesized in a decay-corrected radiochemical yield of 1.7 ± 0.9% by a 1-step no-carrier added nucleophilic aromatic 18F-substitution of 1-nitro precursor 4c. Small-animal PET imaging of [ 18F] 4b was performed in naïve rats, before and after administration of unlabelled 1 (5 mg/kg, n = 3), as well as in wild-type and Mdr1a/b (−/−) Bcrp1 (−/−) mice ( n = 3). In PET experiments in rats, administration of unlabelled 1 increased brain activity uptake by a factor of 9.5 ( p = 0.0002, 2-tailed Student’s t-test), whereas blood activity levels remained unchanged. In Mdr1a/b (−/−) Bcrp1 (−/−) mice, the mean brain-to-blood ratio of activity at 60 min after tracer injection was 7.6 times higher as compared to wild-type animals ( p = 0.0002). HPLC analysis of rat brain tissue extracts collected at 40 min after injection of [ 18F] 4b revealed that 93 ± 7% of total radioactivity in brain was in the form of unchanged [ 18F] 4b. In conclusion, the in vivo behavior of [ 18F] 4b was found to be similar to previously described [ 11C] 1 suggesting transport of [ 18F] 4b by Pgp and/or BCRP at the rodent BBB. However, low radiochemical yields and a significant degree of in vivo defluorination will limit the utility of [ 18F] 4b as a PET tracer.

Development of mesoporous TiO 2 microspheres with high specific surface area for selective enrichment of phosphopeptides by mass spectrometric analysis

In this study, mesoporous TiO 2 microspheres were synthesized by simple hydrothermal reaction, and successfully developed for phosphopeptides enrichment from both standard protein digestion and real biological sample such as rat brain tissue extract. The mesoporous TiO 2 microspheres (the diameter size of about 1.0 μm) obtained by simple hydrothermal method were found to have a specific surface area of 84.98 m 2/g, which is much larger than smooth TiO 2 microspheres with same size. The surface area of mesoporous TiO 2 microspheres is almost two times of commercial TiO 2 nanoparticle (a diameter of 90 nm). Using standard proteins digestion and real biological samples, the superior selectivity and capacity of mesoporous TiO 2 microspheres for the enrichment of phosphorylated peptides than that of commercial TiO 2 nanoparticles and TiO 2 microspheres was also observed. It has been demonstrated that mesoporous TiO 2 microspheres have powerful potential for selective enrichment of phosphorylated peptides. Moreover, the preparation of the mesoporous TiO 2 microspheres obtained by the hydrothermal reaction is easy, simple and low-cost. These mesoporous TiO 2 microspheres with the ability of large scale synthesis can widely be applied for phosphorylated proteomic research.

Quantitative analysis of phenibut in rat brain tissue extracts by liquid chromatography–tandem mass spectrometry

Phenibut (3-phenyl-4-aminobutyric acid) is a gamma-aminobutyric acid mimetic drug, which is used clinically as a mood elevator and tranquilizer. In the present work, a rapid, selective and sensitive liquid chromatography-tandem mass spectrometry method for quantification of phenibut in biological matrices has been developed. The method is based on protein precipitation with acidic acetonitrile followed by isocratic chromatographic separation using acetonitrile-formic acid (0.1% in water; 8:92, v/v) mobile phase on a reversed-phase column. Detection of the analyte was performed by electrospray ionization mass spectrometry in multiple reaction monitoring mode with the precursor-to-product ion transition m/z 180.3 --> m/z 117.2. The calibration curve was linear over the concentration range 50-2000 ng/mL. The lower limit of quantification for phenibut in rat brain extracts was 50 ng/mL. Acceptable precision and accuracy were obtained over the whole concentration range. The validated method was successfully applied in a pharmacological study to analyze phenibut concentration in rat brain tissue extract samples.

201 Stage-dependent activation of cell cycle and apoptosis mechanisms in the right ventricle by pressure overload

induction and development of cardiac hypertrophy (CH). In this study we investigated, whether calcineurin might be involved in the development of the left/right ventricle differences in NE-induced CH. Female Sprague-Dawley rats were treated with continuous NaCl-infusion (controls, C) or NE-infusion (0.1 mg/kg.h i.v.) for 12 and 96 hours. The effect of NE-treatment was documented by changes in the LV and RV function and in the change in the LV weight to body weight (LVW/BW) and RV weight to body weight (RVW/BW) ratios. Western blots were done with calcineurin-A (Cn-A) antibody (Stressgene Biotechnologies). We used the rat brain tissue extract as positive control for calcineurin antibody. After 12h of NE-treatment the RV systolic pressure (C 28.7±4.1, NE 65.9±2.7 mm Hg), heart rate (C 381±10.5, NE 490±12.7 beats/min), RV dP/dt max (C 1927±462.2, NE 5380±508 mm Hg/s) as well as LV dP/dt max (C 9276±1192, NE 18405±1538 mm Hg/s) were significantly increased (all p < 0.01). The LVW/BW ratio, but not the RVW/BW ratio was significantly increased in NE-treated rats (p < 0.05). Also the mRNA for atrial natriuretic factor (ANP), a marker of hypertrophy, was significantly increased in the LV only as shown previously by Barth et al. (J Mol Cell Cardiol 32, 2000, 273-284). At this time point neither in the LV, nor in the RV was the Cn-A protein significantly changed after NEtreatment. After 96h of NE-treatment heart function remained elevated to a similar extent as after 12h. The LVW/BW, but not the RVW/BW ratio was significantly increased (from 2.19±0.07 to 2.5±0.05, p < 0.01). There was a significant increase in the Cn-A protein in the LV only (factor of change for LV 1.49±0.26, p < 0.05). This increase was also significantly different from the change in the RV (factor of change 1.01 ± 0.15; p < 0.05). Thus, calcineurin seems to be differently expressed in the left and right ventricle in the NE-induced hypertrophy and may play a role in the signalling for ventricle-specific cardiac hypertrophy.

Immunodiagnosis of the primary brain tumor (glioma) by the endogenous lectin

Background: A sugar-binding protein, lectin, was identified in the cystic fluid of brain tumor (glioma). Methods: The protein was purified by high performance liquid chromatography. Results: The molecular weight of the lectin was 29 kDa. The activity of the purified protein was inhibited strongly by p-nitro-β- d-galactose and asialofetuin. The lectin activity was not Ca 2+- or Mg 2+-dependent. The antiserum of this pure lectin specifically cross-reacted in the cerebrospinal fluid (CSF) of glioma tumor patients. The same antibody had no reaction with CSF of other neurological patients, normal rat brain tissue extract, oncofetal antigens such as alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), plant lectins, and other body fluids like plural and ascites. Conclusions: This antilectin antibody may be useful in the evaluation of patients with primary brain tumor (glioma).

Zymographic measurement of gelatinase activity in brain tissue after detergent extraction and affinity-support purification

Several methods have been developed for the measurement of gelatinase activity from various tissues using detergent extraction. Gelatin-affinity chromatography has been employed for the large-scale purification of gelatinases from conditioned medium obtained from cultured cells. The objective of this paper was to develop a rapid method whereby gelatinase activity could be extracted from regional brain tissues without tedious, intervening purification steps. After Triton X-100 extraction and gelatin-Sepharose 4B purification of rat brain tissue extracts, two major activities were observed on gelatin zymograms. These were identified as gelatinase A and B using co-migration with astrocyte-derived enzymes and inhibition of activity by tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). The non-ionic detergents, Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) were equally effective in extracting activities from brain tissue. Little difference in recovery was observed among 0.1, 1 and 10% concentrations of Triton X-100. The method developed here was capable of recovering gelatinase activities from rat brain tissue over a 4–10-fold range using gelatin zymography for the measurement of activity. It is possible that this method may be modified for the measurement of gelatinases in tissues such as biopsy samples of gliomas or astrocytomas or other cancers where gelatinases are thought to play a role in tumor invasion and/or metastasis.

Biochemical characterization of related microtubule proteins in Drosophila melanogaster and adult rat brain

We describe the biochemical characteristics of three proteins isolated from Drosophila embryos and the rat brain. We refer to these proteins as DMAPs (Drosophila microtubule- associated proteins) since they were identified by monoclonal antibodies generated against microtubule protein (MTP) purified from Drosophila melanogaster embryos. DMAP-45 is a 45 kDa protein that binds microtubules in an ATP dependent manner. Preliminary biochemical evidence suggests that DMAP-45 may be an actin-related protein. DMAP-55 is a 55 kDa protein and based on its molecular weight and isoelectric point, may be a novel isoform of tubulin. DMAP-66 is a 66 kDa protein that binds strongly to microtubules in vitro and has multiple isoforms. Analyses of proteins in rat brain tissue extracts and purified rat brain MTP identified proteins of similar molecular weight and isoelectric points and are designated DMAP-45R, -55R and -66R. The presence of proteins with common biochemical properties in these widely divergent animal species suggests that they are related proteins.

Measurement of nitrite and nitrate levels in biological samples by capillary electrophoresis.

Nitrite is one of the products of NO-synthase in biological media. It is slowly oxidized in animals to nitrate. We developed a simple and rapid method to determine simultaneously nitrite and nitrate in biological samples. Capillary ion electrophoresis with direct UV detection at 214 nm was used employing a carrier electrolyte consisting of 10 mM sodium sulfate and an electroosmotic flow modifier. The detection limit in ultrafiltrates of plasma, urine and brain tissue extracts was 25 ng/ml for both compounds. Nitrate levels in human plasma and urine were in the microgram/ml range. Nitrite could not be detected. Rat brain tissue extracts contained detectable amounts of nitrite and nitrate.

Cysteinyl-leukotriene production during limbic seizures triggered by kainic acid

In rats kainic acid-induced seizures were accompanied by time-dependent cerebral cysteinyl-leukotriene (LT) and prostaglandin (PG) F 2α formation. Cysteinyl-LT were identified in the rat brain tissue extracts by their immunoreactive properties and their retention times upon reversed phase HPLC profiling. In perfused blood-free brain tissue contents of LTC 4-like material were significantly elevated in cortex, hippocampus, midbrain and hypothalamus at 3 h after kainic acid injection. PGF 2α tissue contents were significantly elevated in all brain areas studied with very large amounts in the hippocampus and smaller amounts in the cortex. The cyclooxygenase inhibitor indomethacin significantly inhibited formation of PGF 2α in whole brain tissue while leaving unaffected the production of cysteinyl-LT. A dose of indomethacin which nearly completely inhibited cyclooxygenase activity as monitored by cerebral PGF 2α contents also tended to aggravate behavioral changes and significantly increased the mortality. Phenidone, a lipoxygenase inhibitor, significantly and dose-dependently inhibited formation of cysteinyl-LT but did not significantly affect PGF 2α formation. Seizure activity tended to be attenuated by a higher dose of this compound. Dexamethasone which supposedly inhibits phospholipase A 2 activity by induction of lipocortins, did not significantly reduce either cysteinyl-LT or PGF 2α biosynthesis. Flunarizine, trifluoperazine and diazepines protected a certain percentage of animals from kainic acid-induced seizures. In rats in which seizures occurred in spite of pretreatment with these compounds, the eicosanoid formation was not inhibited but in the case of flunarizine was even found to be somewhat enhanced. Thus, systemic application of the excitatory amino acid and neurotoxin, kainic acid, results in cerebral cysteinyl-LT production which might be of pathological significance for the edemogenic and neurotoxic actions of the compound.

Tissue-specific inhibition of embryonic neural cell proliferation by rat brain extract.

The effect of rat brain tissue extract on the proliferative activity of chicken embryonic neural tube and other tissues was studied. Only tissue-specific inhibitory action was found to be similar to substances of the chalone group.