scholarly journals Efficient isolation of brain capillary from a single frozen mouse brain for protein expression analysis

2020 ◽  
pp. 0271678X2094144
Author(s):  
Seiryo Ogata ◽  
Shingo Ito ◽  
Takeshi Masuda ◽  
Sumio Ohtsuki
Keyword(s):  
Protein Expression ◽  
Mouse Brain ◽  
Expression Analysis ◽  
Brain Capillaries ◽  
Purification Step ◽  
Brain Capillary ◽  
Pathological Conditions ◽  
Protein Expressions ◽  
Isolation Methods ◽  
The Brain

Isolated brain capillaries are essential for analyzing the changes of protein expressions at the blood–brain barrier (BBB) under pathological conditions. The standard brain capillary isolation methods require the use of at least five mouse brains in order to obtain a sufficient amount and purity of brain capillaries. The purpose of this study was to establish a brain capillary isolation method from a single mouse brain for protein expression analysis. We successfully isolated brain capillaries from a single frozen mouse brain by using a bead homogenizer in the brain homogenization step and combination of cell strainers and glass beads in the purification step. Western blot and proteomic analysis showed that proteins expressed at the BBB in mouse brain capillaries isolated by the developed method were more enriched than those isolated from a pool of five mouse brains by the standard method. By using the developed method, we further verified the changes in expression of BBB proteins in Glut1-deficient mouse. The developed method is useful for the analysis of various mice models with low numbers and enables us to understand, in more detail, the physiology and pathology of BBB.

scholarly journals Altered ERK1/2 Signaling in the Brain of Learned Helpless Rats: Relevance in Vulnerability to Developing Stress-Induced Depression

2016 ◽  
Vol 2016 ◽  
pp. 1-18 ◽  
Author(s):  
Yogesh Dwivedi ◽  
Hui Zhang
Keyword(s):  
Protein Expression ◽  
Frontal Cortex ◽  
Learned Helplessness ◽  
Cellular Signaling ◽  
Structural Plasticity ◽  
Extracellular Signal Regulated Kinase ◽  
Catalytic Activities ◽  
Extracellular Signal ◽  
Protein Expressions ◽  
The Brain

Extracellular signal-regulated kinase 1/2- (ERK1/2-) mediated cellular signaling plays a major role in synaptic and structural plasticity. Although ERK1/2 signaling has been shown to be involved in stress and depression, whether vulnerability to develop depression is associated with abnormalities in ERK1/2 signaling is not clearly known. The present study examined ERK1/2 signaling in frontal cortex and hippocampus of rats that showed vulnerability (learned helplessness, (LH)) or resiliency (non-learned helplessness, (non-LH)) to developing stress-induced depression. In frontal cortex and hippocampus of LH rats, we found that mRNA and protein expressions of ERK1 and ERK2 were significantly reduced, which was associated with their reduced activation and phosphorylation in cytosolic and nuclear fractions, where ERK1 and ERK2 target their substrates. In addition, ERK1/2-mediated catalytic activities and phosphorylation of downstream substrates RSK1 (cytosolic and nuclear) and MSK1 (nuclear) were also lower in the frontal cortex and hippocampus of LH rats without any change in their mRNA or protein expression. None of these changes were evident in non-LH rats. Our study indicates that ERK1/2 signaling is differentially regulated in LH and non-LH rats and suggests that abnormalities in ERK1/2 signaling may be crucial in the vulnerability to developing depression.

Pathogenesis of Brain Edema and Hemorrhage: Role of the Brain Capillary

PEDIATRICS ◽  
1979 ◽  
Vol 64 (3) ◽  
pp. 357-360
Author(s):  
Gary W. Goldstein
Keyword(s):  
Endothelial Cells ◽  
Brain Edema ◽  
Tight Junctions ◽  
Interstitial Fluid ◽  
Water Soluble ◽  
Brain Capillaries ◽  
Reye's Syndrome ◽  
Anatomic Site ◽  
Brain Capillary ◽  
The Brain

It has recently been shown that the endothelial cells in brain capillaries are the anatomic site of the blood-brain barrier, and that these endothelial cells act to maintain a constant composition and volume of brain interstitial fluid.1-3 Defects in brain capillary function appear to play a role in the pathogenesis of brain edema and hemorrhage in a wide variety of diseases. Conditions as diverse as intraventricular hemorrhage of the premature, asphyxia neonatorum, lead poisoning, head injury, Reye's syndrome, osmolar coma, and the brain edema surrounding a tumor or abscess may all share the common feature of brain capillary failure. In this review, I will consider some recent advances in our understanding of the brain microvasculature that may explain their unusual susceptibility to injury. Brain capillaries have a number of important differences from capillaries in other organs. A schematic of a typical brain capillary is shown in the Figure. Unlike systemic capillaries, the endothelial cells in brain capillaries are joined together by tight junctions.3 These cellular junctions are present around the entire circumference of the capillary tube. The result is a continuous layer of endothelial cells that effectively separate the plasma from the interstitial fluid of the brain. The tight junctions are composed of a series of complex interdigitations that create a barrier so complete that water-soluble molecules and ions are unable to move into the brain between the endothelial cells. In other organs, the capillaries do not have tight junctions, and sugars, amino acids, ions, and drugs readily diffuse between endothelial cells into the interstitial fluid.

Filtration Coefficient of the Capillaries of the Brain

1958 ◽  
Vol 195 (2) ◽  
pp. 459-464 ◽  
Author(s):  
N. A. Coulter
Keyword(s):  
Filtration Rate ◽  
Filtration Coefficient ◽  
Secretory Process ◽  
Brain Capillaries ◽  
Brain Capillary ◽  
Average Value ◽  
Capillary Membranes ◽  
The Brain ◽  
Muscle Capillaries

The Monro-Kellie doctrine was used as the basis for determination of the filtration coefficient of the capillaries of the cat's brain. An average value of 10.3 x 10–8 ml/sec/cm H2O/cm2 was found. A linear relation was observed between filtration rate and filtration pressure. The fact that the filtration coefficient of brain capillaries is somewhat higher than that of muscle capillaries suggests that lipoid insoluble molecules actually penetrate the brain capillary membranes readily, but are pumped back by a secretory process.

scholarly journals An Atlas of the Quantitative Protein Expression of Anti-Epileptic-Drug Transporters, Metabolizing Enzymes and Tight Junctions at the Blood–Brain Barrier in Epileptic Patients

Pharmaceutics ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2122
Author(s):  
Risa Sato ◽  
Kotaro Ohmori ◽  
Mina Umetsu ◽  
Masaki Takao ◽  
Mitsutoshi Tano ◽  
...  
Keyword(s):  
Protein Expression ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Brain Capillaries ◽  
Metabolizing Enzymes ◽  
Expression Levels ◽  
Blood Brain ◽  
Anti Epileptic Drug ◽  
Epileptic Patients ◽  
The Brain

The purpose of the present study was to quantitatively elucidate the levels of protein expression of anti-epileptic-drug (AED) transporters, metabolizing enzymes and tight junction molecules at the blood–brain barrier (BBB) in the focal site of epilepsy patients using accurate SWATH (sequential window acquisition of all theoretical fragment ion spectra) proteomics. Brain capillaries were isolated from focal sites in six epilepsy patients and five normal brains; tryptic digests were produced and subjected to SWATH analysis. MDR1 and BCRP were significantly downregulated in the epilepsy group compared to the normal group. Out of 16 AED-metabolizing enzymes detected, the protein expression levels of GSTP1, GSTO1, CYP2E1, ALDH1A1, ALDH6A1, ALDH7A1, ALDH9A1 and ADH5 were significantly 2.13-, 6.23-, 2.16-, 2.80-, 1.73-, 1.67-, 2.47- and 2.23-fold greater in the brain capillaries of epileptic patients than those of normal brains, respectively. The protein expression levels of Claudin-5, ZO-1, Catenin alpha-1, beta-1 and delta-1 were significantly lower, 1.97-, 2.51-, 2.44-, 1.90- and 1.63-fold, in the brain capillaries of epileptic patients compared to those of normal brains, respectively. Consistent with these observations, leakage of blood proteins was also observed. These results provide for a better understanding of the therapeutic effect of AEDs and molecular mechanisms of AED resistance in epileptic patients.

scholarly journals Effect of Melatonin on Blood Pressure and Nitric Oxide Generation in Rats With Metabolic Syndrome

2016 ◽  
pp. S373-S380 ◽  
Author(s):  
J. KLIMENTOVA ◽  
M. CEBOVA ◽  
A. BARTA ◽  
Z. MATUSKOVA ◽  
S. VRANKOVA ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Blood Pressure ◽  
Metabolic Syndrome ◽  
Protein Expression ◽  
Brain Cortex ◽  
Brain Regions ◽  
Melatonin Treatment ◽  
Protein Expressions ◽  
Oxide Synthase ◽  
The Brain

Melatonin, a multitasking indolamine, seems to be involved in a variety of physiological and metabolic processes via both receptor-mediated and receptor-independent mechanisms. The aim of our study was to find out whether melatonin can affect blood pressure (BP), nitric oxide synthase (NOS) activity, eNOS and nNOS protein expressions in rats with metabolic syndrome (SHR/cp). Rats were divided into four groups: 6-week-old male WKY andSHR/cp and age-matched WKY and SHR/cp treated with melatonin (10 mg/kg/day) for 3 weeks. BP was measured by tail-cuff plethysmography. NOS activity, eNOS and nNOS protein expressions were determined in the heart, aorta, brain cortex and cerebellum. MT1 receptors were analyzed in the brain cortex and cerebellum. In SHR/cp rats, BP was decreased after melatonin treatment. In the same group, melatonin did not affect NOS activity and eNOS protein expression in the heart and aorta, while it increased both parameters in the brain cortex and cerebellum. Interestingly, melatonin elevated MT1 protein expression in the cerebellum. Neuronal NOS protein expression was not changed within the groups. In conclusion, increased NOS activity/eNOS upregulation in particular brain regions may contribute partially to BP decrease in SHR/cp rats after melatonin treatment. Participation of MT1 receptors in this melatonin action may be supposed.

scholarly journals Genotoxic and oxidative effect of duloxetine on mouse brain and liver tissues

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Isela Álvarez-González ◽  
Scarlett Camacho-Cantera ◽  
Patricia Gómez-González ◽  
Michael J. Rendón Barrón ◽  
José A. Morales-González ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Dna Damage ◽  
Mouse Brain ◽  
Control Level ◽  
Dna Oxidation ◽  
Methyl Methanesulfonate ◽  
Control Group ◽  
Oxidative Potential ◽  
The Brain ◽  
Oxidative Effect

AbstractWe evaluated the duloxetine DNA damaging capacity utilizing the comet assay applied to mouse brain and liver cells, as well as its DNA, lipid, protein, and nitric oxide oxidative potential in the same cells. A kinetic time/dose strategy showed the effect of 2, 20, and 200 mg/kg of the drug administered intraperitoneally once in comparison with a control and a methyl methanesulfonate group. Each parameter was evaluated at 3, 9, 15, and 21 h postadministration in five mice per group, except for the DNA oxidation that was examined only at 9 h postadministration. Results showed a significant DNA damage mainly at 9 h postexposure in both organs. In the brain, with 20 and 200 mg/kg we found 50 and 80% increase over the control group (p ≤ 0.05), in the liver, the increase of 2, 20, and 200 mg/kg of duloxetine was 50, 80, and 135% in comparison with the control level (p ≤ 0.05). DNA, lipid, protein and nitric oxide oxidation increase was also observed in both organs. Our data established the DNA damaging capacity of duloxetine even with a dose from the therapeutic range (2 mg/kg), and suggest that this effect can be related with its oxidative potential.

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