Brain Tissue Homogenates Research Articles

N-Acetylcysteine Inhibits Kynurenine Aminotransferase II

The tryptophan metabolite kynurenic acid (KYNA) may play an important role in normal and abnormal cognitive processes, most likely by interfering with α7 nicotinic and NMDA receptor function. KYNA is formed from its immediate precursor kynurenine either by non-enzymatic oxidation or through irreversible transamination by kynurenine aminotransferases. In the mammalian brain, kynurenine aminotransferase II (KAT II) is the principal enzyme responsible for the neosynthesis of rapidly mobilizable KYNA, and therefore constitutes an attractive target for pro-cognitive interventions. N-acetylcysteine (NAC), a brain-penetrant drug with pro-cognitive efficacy in humans, has been proposed to exert its actions by increasing the levels of the anti-oxidant glutathione (GSH) in the brain. We report here that NAC, but not GSH, inhibits KAT II activity in brain tissue homogenates from rats and humans with IC50 values in the high micromolar to low millimolar range. With similar potency, the drug interfered with the de novo formation of KYNA in rat brain slices, and NAC was a competitive inhibitor of recombinant human KAT II (Ki: 450 μM). Furthermore, GSH failed to S-glutathionylate recombinant human KAT II treated with the dithiocarbamate drug disulfiram. Shown by microdialysis in the prefrontal cortex of rats treated with kynurenine (50 mg/kg, i.p.), peripheral administration of NAC (500 mg/kg, i.p., 120 and 60 min before the application of kynurenine) reduced KYNA neosynthesis by ∼50%. Together, these results suggest that NAC exerts its neurobiological effects at least in part by reducing cerebral KYNA formation via KAT II inhibition.

Cobalt accumulation and iron-regulatory protein profile expression in immature mouse brain after perinatal exposure to cobalt chloride

Developing brain is very sensitive to the influence of environmental factors during gestation and the neonatal period. The aim of the study is to assess cobalt and iron accumulation in the brain as well as changes in the expression of iron-regulatory proteins transferrin receptor 1, hepcidin, and ferroportin in suckling mice. Perinatal exposure to cobalt chloride increased significantly cobalt content in brain tissue homogenates of 18-day-old (d18) and 25-day-old (d25) mice inducing alterations in brain iron homeostasis. Higher degree of transferrin receptor 1 expression was demonstrated in cobalt chloride-exposed mice with no substantial changes between d18 and d25 mice. A weak ferroportin expression was found in 18-day-old control and cobalt-treated mouse brain. Cobalt exposure of d25 mice resulted in increased ferroportin expression in brain compared to the untreated age-matched control group. Hepcidin level in cobalt-exposed groups was decreased in d18 mice and slightly increased in d25 mice. The obtained data contribute for the better understanding of metal toxicity impact on iron homeostasis in the developing brain with further possible implications in neurodegeneration.

Cigarette smoke extract triggers neoplastic change in lungs and impairs locomotor activity through wnt3a-β-catenin signaling in aged COPD rodent model

Background Chronic cigarette smoking primes immense decline in lung functions and retardation of motor functions with increase in age. This raise the question of whether age status overwhelm the susceptibility to smoking induced lung inflammatory diseases and neuro-motor dysfunctions. Methods To study the hypothesis 11–12 month old aged wistar rats (n = 6) were administered cigarette smoke extract (CSE) through intraperitoneal route (0.5 ml/rat) twice a week for 2 months. Respiratory lung functions were measured through whole body plethysmography. Lung histopathological evaluation and neuronal degeneration were observed by using H&E, picrosirius red and nissl staining respectively. Motor function tests were done through panel of neuro-behavioral tests and protein expressions were performed in lung and brain tissue homogenates through western blotting. Results Sub-chronic CSE exposure worsened the lung functions including decreased tidal volume (p < 0.05), peak inspiratory flow (p < 0.05) and enhanced pause (p < 0.05). Grossly, solid neoplastic lesions were visible on the supra-lateral surface of the lungs of the CSE treated animals. Histopathological examination revealed immune cell infiltration, dominated with macrophages and alveolar type II cells stained positive for PCNA. Increased expression of BAX, PCNA, Wnt-3a, p-β-catenin (p < 0.05) was seen in the lungs of CSE treated aged animals. Elevated expression of inflammatory markers including NF-ϏB, TNF-α, TNF-R1, p-AKT was found in CSE treated lung tissues. Moreover, our result showed increased MCP-1, VEGF and IL-6 levels in BALF and plasma (p < 0.01) which might lead to neo-vascularization and excessive cell proliferation in lungs of CSE induced rats. Sub-chronic cigarette smoke exposure retarded the motor activity with suppression of D1 and D2 receptor expression in brain tissues. Brain tissue revealed the abundance of hyperchromatic and pyknotic nuclei suggesting neuronal degeneration. Conclusion So in conclusion, chronic cigarette smoking in old age creates susceptibility to fast onset of lung inflammatory diseases and neuro-motor retardation than their nonsmoker counterparts.

Sensitive UPLC–MS/MS Method for Oxycodone Quantification in Serum and Brain Tissue Homogenates: Application to an Interaction Study in Rats

Objectives: An ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) assay has been developed and validated for the quantification of oxycodone in rat serum and brain tissue homogenates. Methods: A simple extraction method using methyl-t-butyl ether was used for sample extraction with oxycodone–D3 as an internal standard (IS). A Symmetry® C18 column (100 mm x 4.6 mm, 5 µm) connected to a Phenomenex Luna® guard column (4 x 3 mm, 5 µm) was used for chromatographic separation. The mobile phase was composed of a mixture of acetonitrile and 0.1 % formic acid in water (pH = 2.7) (15:85, v/v). The flow rate was 0.4 mL/min, the total run time was 5 min, and the injection volume was 10 µL. The column and autosampler temperatures were maintained at 60 °C and 25 °C, respectively. Results: The calibration curve for oxycodone was linear over the range of 10–2000 ng/mL. Oxycodone extraction recovery from rat serum and brain samples ranged from 90.78–105.85 % and 98.42–104.38 % with relative standard deviation (RSD) values of 0.94–3.87 % and 1.04–2.82 %, respectively. The inter-day accuracy values ranged from 87.67–104.83 %, while the intra-day accuracy values ranged from 86.95–105.67 %. Conclusion: This method can be used for the quantification of oxycodone in samples obtained from preclinical animal studies and has great promise for applications in the quantification of oxycodone in human biological matrices.

PET Detection of Cerebral Necrosis Using an Infarct-Avid Agent 2-Deoxy-2-[18F]Fluoro-D-Glucaric Acid (FGA) in a Mouse Model of the Brain Stroke.

Ischemic stroke is a leading cause of disability worldwide. The volume of necrotic core in affected tissue plays a major role in selecting stroke patients for thrombolytic therapy or endovascular thrombectomy. In this study, we investigated a recently reported positron emission tomography (PET) agent 2-deoxy-2-[18F]fluoro-D-glucaric acid (FGA) to determine necrotic core in a model of transient middle cerebral artery occlusion (t-MCAO) in mice. The radiopharmaceutical, FGA, was synthesized by controlled, rapid, and quantitative oxidation of clinical doses of 2-deoxy-2-[18F]fluoro-D-glucose (FDG) in a one-step reaction using a premade kit. Brain stroke was induced in the left cerebral hemisphere of CD-1 mice by occluding the middle cerebral artery for 1h, and then allowing reperfusion by removing the occlusion. One day post-ictus, perfusion single-photon emission tomography (SPECT) was performed with 99mTc-lableled hexamethylpropyleneamine oxime (HMPAO), followed by PET acquisition with FGA. Plasma and brain tissue homogenates were assayed for markers of inflammation and neurotrophins. The kit-based synthesis was able to convert up to 2.2GBq of FDG into FGA within 5min. PET images showed 375% more accumulation of FGA in the ipsilateral hemisphere than in the contralateral hemisphere. SPECT images showed that the ipsilateral HMPAO accumulation was reduced to 55% of normal levels; there was a significant negative correlation between the ipsilateral accumulation of FGA and HMAPO (p < 0.05). FGA accumulation in stroke also correlated with IL-6 levels in the ipsilateral hemisphere. There was no change in IL-6 or TNFα in the plasma of stroke mice. Accumulation of FGA correlated well with the perfusion defect and inflammatory injury. As a PET agent, FGA has potential to image infarcted core in the brain stroke injury with high sensitivity, resolution, and specificity.

Alzheimer's amyloid-β and tau protein accumulation is associated with decreased expression of the LDL receptor-associated protein in human brain tissue.

IntroductionOne of the major neuropathological features of Alzheimer's disease (AD) is the accumulation of amyloid‐β (Aβ) protein in the brain. Evidence suggests that the low‐density lipoprotein receptor‐associated protein (RAP) binds strongly to Aβ and enhances its cellular uptake and that decreased RAP expression correlates with increased Aβ production in animal models of AD.MethodsThe current study examined whether RAP levels change in AD human brain tissue and whether they are related to the amount of AD pathology. RAP and NeuN levels were determined by Western blot, while low‐density lipoprotein receptor‐related protein 1 (LRP1), tau and Aβ levels were determined by ELISA in the temporal cortex of 17 AD and 16 control cases.ResultsAn increase in total Aβ and insoluble and soluble tau protein was observed in AD brain tissue. In contrast, RAP levels were significantly decreased in AD brain tissue compared to controls. Correlation analysis revealed that levels of RAP correlated with both total Aβ and soluble and insoluble tau levels. Neither LRP1 nor NeuN levels were significantly altered in AD brain tissue homogenates and did not correlate with Aβ or tau protein levels.ConclusionReduction in RAP may contribute to the accumulation and aggregation of Aβ in the AD brain.

Effect of Natural Vs. Synthetic α-tocopherol on Neurogenesis-Related Genes in Cerebella of Juvenile α-tocopherol Transfer Protein-Null Mice

Abstract Objectives Vitamin E (α-tocopherol, α-T) restriction during brain development alters the expression of neurogenesis-related genes in cerebella of juvenile α-tocopherol transfer protein-null (Ttpa−/−) mice. Synthetic α-T (SYN), compared to natural α-T (NAT), downregulates cerebellar myelin genes in adolescent Ttpa−/− mice. We studied how early-life exposure to SYN or NAT affects the expression of neurogenesis-related genes in juvenile Ttpa−/− mice. Methods Male and female Ttpa+/+ and Ttpa−/− mice were nursed by Ttpa+/−dams fed AIN-93G-based diets containing either SYN (∼816 mg α-T/kg diet) or NAT (∼600 mg α-T/kg diet). Homogenized brain tissues from 21 day old weanlings (n = 9/group) were used to measure total α-T concentrations via HPLC-PDA. The expression of genes critical for brain development (Rora, Shh), myelination (Plp1, Cntnap1, Mbp, Mobp, Nr1h3), and synaptic function (Cplx1, Necab1, Prkcg) were measured in the cerebellum via real-time qPCR. Results α-T concentrations were significantly lower in brains of Ttpa−/− mice (17.7 nmol/g) compared to Ttpa+/+ mice (37.5 nmol/g) (P &amp;lt; 0.001). Exposure to SYN vs. NAT resulted in similar total α-T brain levels within each genotype (Ttpa−/−: 19.8 vs. 15.6 nmol/g; Ttpa+/+: 42.5 vs. 32.6 nmol/g). Consistent with previous studies, Necab1 was significantly downregulated in Ttpa−/− mice (P &amp;lt; 0.05). The other selected neurogenesis-related genes were similarly expressed between all groups, regardless of genotype or dietary α-T source. Conclusions Brain α-T concentrations at weaning depended on the presence of Ttpa. α-T source did not modulate the selected neurogenesis genes, possibly because the natural and synthetic α-T diets each provided sufficient total α-T during development. Funding Sources Abbott Nutrition through the Center for Nutrition, Learning, and Memory (CNLM), Division of Nutritional Sciences Vision 20/20 Grant Program, and Division of Nutritional Sciences Margin of Excellence Research Program (all through the University of Illinois at Urbana-Champaign). KMR was supported by the AFRI NIFA Predoctoral Fellowships Grant Program (2019–67,011-29,514) from the U.S. Department of Agriculture, National Institute of Food and Agriculture.

Single-nucleus transcriptomics of the prefrontal cortex in major depressive disorder implicates oligodendrocyte precursor cells and excitatory neurons.

Major depressive disorder (MDD) has an enormous impact on global disease burden, affecting millions of people worldwide and ranking as a leading cause of disability for almost three decades. Past molecular studies of MDD employed bulk homogenates of postmortem brain tissue, which obscures gene expression changes within individual cell types. Here we used single-nucleus transcriptomics to examine ~80,000 nuclei from the dorsolateral prefrontal cortex of male individuals with MDD (n = 17) and of healthy controls (n = 17). We identified 26 cellular clusters, and over 60% of these showed differential gene expression between groups. We found that the greatest dysregulation occurred in deep layer excitatory neurons and immature oligodendrocyte precursor cells (OPCs), and these contributed almost half (47%) of all changes in gene expression. These results highlight the importance of dissecting cell-type-specific contributions to the disease and offer opportunities to identify new avenues of research and novel targets for treatment.

In vitro Studies on Antioxidant Potential of Aqueous Extract of Bridelia ferruginea Stem Bark in Brain and Liver of Wistar-albino Rats using Sodium Nitroprusside as Pro-oxidant

Thiobarbituric acid reactive species (TBARS) assay was used to study the antioxidant potential of Bridelia ferruginea, a widely used medicinal plant in Nigeria, sub-tropical Africa and parts of Asia. The aqueous extract of Bridelia ferruginea stem bark showed inhibition against the formation of TBARS induced by the pro-oxidant, sodium nitroprusside, in the liver and brain tissue homogenates of the locally bred male and female albino-Wistar rats. The inhibition of TBARS is an indication of the antioxidant potential of the plant extract. The extent of antioxidant potential depends on concentrations, showing varying degrees of inhibition with different concentrations. It showed a 54.16% inhibition in the liver and 60.65% in the brain, both at a concentration of 0.33 mg/ml, with IC50 values of 3.00 ± 1.58 mg/ml and 2.99 ± 1.59 mg/ml for the liver and brain homogenates respectively. The results suggest the effectiveness of the aqueous extract of Bridelia ferruginea stem bark in reversing the effect of lipid peroxidation that may result from sodium nitroprusside overload.

Effect of Thymoquinone against Aluminum Chloride-Induced Alzheimer-Like Model in Rats: A Neurophysiological and Behavioral Study

Abstract Background: Alzheimer Disease (AD) is a progressive neurodegenerative disorder characterized by oxidative stress, neuro-inflammation and synaptic dysfunction, which caused in part by abnormal aggregation of senile plaques and neu-rofibrillary tangles outside and inside the cerebral neurons, respectively. Thymoquinone (TQ) is the major bioactive ingredient of the volatile oil of Nigella sativa seed and it possess anti-inflammatory and antioxidant effects in some neuronal diseases. Aim of Study: To evaluate the neuroprotective potential of TQ on the oxidative stress status of the brain in aluminum chloride (AlCl3)-induced AD in rats. Material and Methods: Thirty adult male Sprague Dawley albino rats were used in this study. They were randomly divided into 3 groups. Group 1 (control group). Group 2 (AD group): Supplemented orally with AlCl3 (17mg/kg/day) for 4 weeks. Group 3 (TQ/AD group) supplemented concomitantly with oral TQ (10mg/kg/day) and AlCl3 (17mg/kg/day) for 4 weeks. At the end of the experiment, spatial working memory was assessed using the Y-maze spontaneous alternation test. Then, serum levels of Malondialdehyde (MDA) and Glutath-ione Peroxidase Enzyme (GPX) were assessed. Then, the rats were sacrificed and the brain tissue homogenate content of tau protein, AR and acetyle choline were determined. Results: In AD group, spatial working memory was im-paired as indicated by a significant decline of Spontaneous Alteration Performance (SAP%) at Y-maze behavior test. Also, AR, tau protein, acetyle choline in brain and serum MDA were significantly increased with a significant decrease of serum GPX. Co-administration of TQ with AlCl3 in TQ/AD group, significantly decreased AR, tau protein, acetyle choline in brain and serum MDA and increased (SAP%) and serum GPX. Conclusion: TQ could mitigate the neurodegenerative markers and oxidative stress indices encountered in AD, presumably via its antioxidant and anti-inflammatory effects.This may implement TQ as an adjuvant alternative medical strategy in ameliorating this devastating disease

Selective Fluorogenic β-Glucocerebrosidase Substrates for Convenient Analysis of Enzyme Activity in Cell and Tissue Homogenates.

Within mammals, there are often several functionally related glycoside hydrolases, which makes monitoring their activities problematic. This problem is particularly acute for the enzyme β-glucocerebrosidase (GCase), the malfunction of which is a key driver of Gaucher's disease (GD) and a major risk factor for Parkinson's disease (PD). Humans harbor two other functionally related β-glucosidases known as GBA2 and GBA3, and the currently used fluorogenic substrates are not selective, which has driven the use of complicated subtractive assays involving the use of detergents and inhibitors. Here we describe the preparation of fluorogenic substrates based on the widely used nonselective substrate resorufin β-d-glucopyranoside. Using recombinant enzymes, we show that these substrates are highly selective for GCase. We also demonstrate their value through the analysis of GCase activity in brain tissue homogenates from transgenic mice expressing mutant human GCase and patient fibroblasts expressing mutant GCase. This approach simplifies the analysis of cell and tissue homogenates and should facilitate the analysis of clinical and laboratory tissues and samples.

Mass Spectrometry-Based Rapid Quantitative Bioanalysis of Flibanserin: Pharmacokinetic and Brain Tissue Distribution Study in Female Rats.

Flibanserin (FLB) is the first United States Food and Drug Administration (USFDA) approved serotonin modulator recently marketed to treat acquired generalized women hypoactive sexual desire disorder. The scope of this study was to develop and validate a sensitive, selective and reliable ultra-performance liquid chromatography-mass spectroscopy/mass spectroscopy-based quantification method for FLB in rat plasma as well as brain tissue samples. The method includes a simple liquid-liquid sample extraction procedure. FLB was subjected to chromatographic separation using a poroshell C18 column with the mobile phase comprising a mixture of acetonitrile (ACN), 10mM ammonium acetate and acetic acid (90:10:0.1, v/v/v). Detection and quantification of FLB after positive electrospray ionization were carried out in selective ion monitoring mode. The fragment ions (m/z) of FLB (parent ion: 391.1741) and IS (parent ion: 448.1550) were monitored at 161.0704 and 285.0917, respectively. A linear response of FLB was observed over a concentration range of 2.5-600ng/mL in plasma and 5-500ng/mL in brain tissue homogenate. The intra- and inter-day precision and accuracy of the method met the acceptable limits specified in the USFDA bioanalytical method validation guideline. The analyte was found to be stable in benchtop, freeze-thaw, auto-injector and dry extract stability studies. The developed method was used to quantitate FLB in the plasma and brain tissue of a single-dose oral pharmacokinetic and brain tissue distribution study in female rats. Maximum FLB concentration in plasma and brain was achieved within an hour; however, the total amount of the drug that reached the brain was significantly less than in plasma. Rate of elimination of FLB from brain was also faster resulting in a lesser half-life in brain compared to the plasma.

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals. Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-β and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

Neuroprotective effect of Egyptian Hyphaene thebaica fruit extract against Alzheimer’s disease in an animal model

Background/aim Alzheimer’s disease (AD) is a chronic neurodegenerative disease. The AD incidence is rising and is becoming a heavy economic burden on society and patients’ families. The present study was directed to investigate the protective and therapeutic effects of Hyphaene thebaica fruit extract (HTE) on AD in an animal model. Materials and methods A total of 90 adult albino male rats were involved in this study and were classified into six groups (15 each). Two negative control groups were used: one did not take any supplementation (G1) and a second negative control group in which normal rats were supplemented with HTE (G2). AD induction was performed by orally injecting the animals with 50 mg AlCl3/kg body weight for 6 weeks. Four groups took AlCl3. First group served as a positive control (G3). Two groups were used to investigate the effect of the orally injected HTE on AD rats either as a therapeutic (G4) or preventive (G5) agent. The last group was treated with the reference drug donepezil (G6). After treatment with HTE for 4 weeks, biochemical analyses of acetylcholinesterase activity, lipid peroxidation (malondialdehyde), and reduced glutathione were done in the brain tissue homogenate. Lipid profile was determined in sera. Expression level assessments of genes encoding microtubule-associated protein tau and amyloid precursor protein were carried out as well as DNA adducts were measured in brain tissues. Histopathological investigations of brain tissues were also performed. Results The results showed that brain samples of AD group exhibited significant changes in biochemical, molecular, and histopathological parameters. The samples of the groups treated with HTE showed improvements in the studied parameters. The treatment of induced AD group with HTE showed better results than supplementing the extract after occurrence of the signs of Alzheimer. Conclusion It was concluded that HTE acts as a promising candidate natural product to ameliorate and protect brain injury in AD-induced rats.

Involvement of N- and C-terminal region of recombinant cervid prion protein in its reactivity to CWD and atypical BSE prions in real-time quaking-induced conversion reaction in the presence of high concentrations of tissue homogenates

ABSTRACT The real-time quaking-induced conversion (RT-QuIC) reaction is a sensitive and specific method for detecting prions. However, inhibitory factors present in tissue homogenates can easily interfere with this reaction. To identify the RT-QuIC condition under which low levels of chronic wasting disease (CWD) and bovine spongiform encephalopathy (BSE) prions can be detected in the presence of high concentrations of brain tissue homogenates, reactivities of various recombinant prion proteins (rPrPs) were tested. Among the tested rPrPs, recombinant cervid PrP (rCerPrP) showed a unique reactivity: the reactivity of rCerPrP to CWD and atypical BSE prions was not highly affected by high concentrations of normal brain homogenates. The unique reactivity of rCerPrP disappeared when the N-terminal region (aa 25–93) was truncated. Replacement of aa 23–149 of mouse (Mo) PrP with the corresponding region of CerPrP partially restored the unique reactivity of rCerPrP in RT-QuIC. Replacement of the extreme C-terminal region of MoPrP aa 219–231 to the corresponding region of CerPrP partially conferred the unique reactivity of rCerPrP to rMoPrP, suggesting the involvement of both N- and C-terminal regions. Additionally, rCerN–Mo–CerCPrP, a chimeric PrP comprising CerPrP aa 25–153, MoPrP aa 150–218, and CerPrP aa 223–233, showed an additive effect of the N- and C-terminal regions. These results provide a mechanistic implication for detecting CWD and atypical BSE prions using rCerPrP and are useful for further improvements of RT-QuIC.

Protein aggregate formation permits millennium-old brain preservation.

Human proteins have not been reported to survive in free nature, at ambient temperature, for long periods. Particularly, the human brain rapidly dissolves after death due to auto-proteolysis and putrefaction. The here presented discovery of 2600-year-old brain proteins from a radiocarbon dated human brain provides new evidence for extraordinary long-term stability of non-amyloid protein aggregates. Immunoelectron microscopy confirmed the preservation of neurocytoarchitecture in the ancient brain, which appeared shrunken and compact compared to a modern brain. Resolution of intermediate filaments (IFs) from protein aggregates took 2-12 months. Immunoassays on micro-dissected brain tissue homogenates revealed the preservation of the known protein topography for grey and white matter for type III (glial fibrillary acidic protein, GFAP) and IV (neurofilaments, Nfs) IFs. Mass spectrometry data could be matched to a number of peptide sequences, notably for GFAP and Nfs. Preserved immunogenicity of the prehistoric human brain proteins was demonstrated by antibody generation (GFAP, Nfs, myelin basic protein). Unlike brain proteins, DNA was of poor quality preventing reliable sequencing. These long-term data from a unique ancient human brain demonstrate that aggregate formation permits for the preservation of brain proteins for millennia.

Evaluation of Neuroprotective and In-vitro Anti-oxidant Activity Isolated Hexane and Ethyl Acetate Fraction from Methanolic Extract of Biophytum reinwardtii

Nature is the best source of complementary and alternative medicine. The plant Biophytum reinwardtii has been used traditionally in pain, inflammatory and oxidative stress related disorders. In this consequence, fraction of methanolic extract of Biophytum reinwardtii was selected to explore the ability of this plant to enhance cognitive function, brain antioxidant enzymes and anti-acetyl cholinesterase activity which can be used for the treatment of oxidative stress related disorders like Alzheimer’s disease (AD). The purpose of this study was to investigate the neuroprotective effect of HEMBR on learning and memory impairment in scopolamine-induced rats of dementia and oxidative stress. Treatment with HEMBR (i.e., 50 and 100 mg/kg b.w.) was investigated in scopolamine-treated Swiss albino male rats for 7 days and its neuroprotective effects were examined using Elevated Plus Maze (EPM) test, Passive Avoidance (PA) test and, Morris Water Maze (MWM) test as well as level of antioxidant enzymes such as catalase (CAT), reduced glutathione (GSH) and acetylcholinesterase (AChE) activity in rat brain tissue homogenates. The present study demonstrates that HEMBR showed the neuroprotective effect by improving cognitive functions and reduces oxidative stress by increasing the level of brain antioxidant enzymes as well as decreasing acetylcholinesterase activity. Therefore, this plant extract faction can be used for enhancing learning, memory, antioxidant potentiality and anti-acetylcholinesterase activity in neurodegenerative disorders like AD.

Neurodynamic Influence of Hydrocortisone on Rhinencephalic and Telencephalic Brain Areas

The neurodynamic effect of graded hydrocortisone treatment on rhinencephalic and telencephalic brain regions was studied in an experimental animal design that sampled isolated hippocampal, basal nucleic and frontal cortical brain regions of male wistar rats. Four test groups, ii to v were administered 2.5 mg, 5 mg, 7.5 mg and 10 mg respectively. The study period lasted for 6 weeks. Results were statistically analyzed and considered significantly different at a confidence interval of 95%. There was a progressive decline in olfactory response as dose of hydrocortisone treatment was increased. There was a significant dose-dependent decrease in assayed frontal cortical acetylcholine and hippocampal glutamate in brain tissue homogenates. Similar change was observed in brain tissue calcium, magnesium and sodium. For the behavioral, histological and biochemical tests conducted in this study, 6 weeks hydrocortisone treatment showed adverse manifestations from 5 mg, which was more obvious from 7.5 mg. The outcome of this study revealed a possible dose-dependent adverse effect of hydrocortisone on specific brain regions responsible for learning, memory, olfaction and psychosocial behavior.

Alpha technology: A powerful tool to detect mouse brain intracellular signaling events

BackgroundPhosphorylation by protein kinases is a fundamental molecular process involved in the regulation of signaling activities in living organisms. Understanding this complex network of phosphorylation, especially phosphoproteins, is a necessary step for grasping the basis of cellular pathophysiology. Studying brain intracellular signaling is a particularly complex task due to the heterogeneous complex nature of the brain tissue, which consists of many embedded structures. New methodOvercoming this degree of complexity requires a technology with a high throughput and economical in the amount of biological material used, so that a large number of signaling pathways may be analyzed in a large number of samples. We have turned to Alpha (Amplified Luminescent Proximity Homogeneous Assay) technology. Comparison with existing methodWestern blot is certainly the most commonly used method to measure the phosphorylation state of proteins. Even though Western blot is an accurate and reliable method for analyzing modifications of proteins, it is a time-consuming and large amounts of samples are required. Those two parameters are critical when the goal of the research is to comprehend multi-signaling proteic events so as to analyze several targets from small brain areas. ResultHere we demonstrate that Alpha technology is particularly suitable for studying brain signaling pathways by allowing rapid, sensitive, reproducible and semi-quantitative detection of phosphoproteins from individual mouse brain tissue homogenates and from cell fractionation and synaptosomal preparations of mouse hippocampus. ConclusionAlpha technology represents a major experimental step forward in unraveling the brain phosphoprotein-related molecular mechanisms involved in brain-related disorders.

Spectrofluorimetric Method for Determination of Letrozole: Analytical Applications to Brain Tissue Samples and Alkaline Degradation Kinetic Study

A simple and sensitive spectroflourimetric method has been proposed for the determination of the antitumor agent letrozole in tablets, spiked human plasma, and rat brain tissue homogenates. Our method involves measuring the native fluorescence of letrozole at 590 nm upon excitation at 239 nm as indicated upon scanning its three-dimensional spectrum. Various experimental parameters were intensively studied and the method was validated as per ICH guidelines. The calibration curve was linear over the concentration range 5–160 ng/mL, with limit of detection 1.36 ng/mL. It was successfully applied to the analysis of letrozole in Femara® tablets with mean recovery 99.35 ± 1.49% and was further applied to study the alkaline degradation kinetics of letrozole. The pseudo first-order rate constant and half-life were calculated. Moreover, successful application of our proposed procedure was carried out on spiked human plasma and rat brain tissue samples. Linear ranges were found to be 5–30 and 10–130 ng/mL, with detection limits 1.25 and 1.71 ng/mL for plasma and brain samples, respectively. Thanks to the method's simplicity, selectivity, and high sensitivity, it can be used for routine analysis in quality control laboratories and for further clinical investigations involving letrozole.