scholarly journals Visualizing subcellular structures in neurons with expansion microscopy

2020 ◽  
Author(s):  
Logan A. Campbell ◽  
Katy E. Pannoni ◽  
Niesha A. Savory ◽  
Dinesh Lal ◽  
Shannon Farris
Keyword(s):  
Golgi Apparatus ◽  
Brain Tissue ◽  
Mouse Brain ◽  
Biological Samples ◽  
Proteinase K ◽  
Fluorescence Signal ◽  
Expansion Process ◽  
Physical Separation ◽  
Subcellular Organelles ◽  
The Brain

ABSTRACTProtein expansion microscopy (proExM) is a powerful technique that crosslinks proteins to a swellable hydrogel to physically expand and optically clear biological samples. The resulting increased resolution (~70 nm) and physical separation of labeled proteins make it an attractive tool for studying the localization of subcellular organelles in densely packed tissues, such as the brain. However, the digestion and expansion process greatly reduces fluorescence signals making it necessary to optimize ExM conditions per sample for specific end goals. Here we describe a proExM workflow optimized for resolving subcellular organelles (mitochondria and the Golgi apparatus) and reporter-labeled spines in fixed mouse brain tissue. By directly comparing proExM staining and digestion protocols, we found that immunostaining before proExM and using a Proteinase K based digestion for 8 hours consistently resulted in the best fluorescence signal to resolve subcellular organelles while maintaining sufficient reporter labeling to visualize spines and trace individual neurons. With these methods, we more accurately quantified mitochondria size and number and better visualized Golgi ultrastructure in reconstructed CA2 neurons of the hippocampus.

scholarly journals Full-Scale Label-Free Surface-Enhanced Raman Scattering Analysis of Mouse Brain Using a Black Phosphorus-Based Two-Dimensional Nanoprobe

2019 ◽  
Vol 9 (3) ◽  
pp. 398 ◽  
Author(s):  
Tiejun Guo ◽  
Fangsheng Ding ◽  
Dongling Li ◽  
Wen Zhang ◽  
Liren Cao ◽  
...  
Keyword(s):  
Raman Scattering ◽  
Brain Tissue ◽  
Mouse Brain ◽  
Au Nanoparticles ◽  
Full Scale ◽  
Label Free ◽  
Surface Enhanced ◽  
Surface Enhanced Raman ◽  
Enhanced Raman Scattering ◽  
The Brain

The brain takes the vital role in human physiological and psychological activities. The precise understanding of the structure of the brain can supply the material basis for the psychological behavior and cognitive ability of human beings. In this study, a fast molecular fingerprint analysis of mouse brain tissue was performed using surface-enhanced Raman scattering (SERS) spectroscopy. A nanohybrid consisting of flake-like black phosphorus (BP) and Au nanoparticles (BP-AuNSs) served as the novel SERS substrate for the spectral analysis of brain tissue. BP-AuNSs exhibited outstanding SERS activity compared to the traditional citrate-stabilized Au nanoparticles, which could be largely ascribed to the plentiful hot spots formed in the BP nanosheet. Rapid, full-scale and label-free SERS imaging of mouse brain tissue was then realized with a scanning speed of 56 ms per pixel. Fine textures and clear contour were observed in the SERS images of brain tissue, which could be well in accordance with the classical histological analysis; however, it could avoid the disadvantages in the processing procedure of tissue section. Additionally, the SERS spectra illustrated plentiful biochemical fingerprint of brain tissue, which indicated the molecular composition of various encephalic regions. The SERS difference spectrum of the left versus right hemisphere revealed the biochemical difference between the two hemispheres, which helped to uncover the psychological and cognitive models of the left and right hemispheres.

scholarly journals Voltage-sensitive dye delivery through the blood brain barrier using adenosine receptor agonist Regadenoson

2018 ◽  
Author(s):  
Rebecca W. Pak ◽  
Jeeun Kang ◽  
Heather Valentine ◽  
Leslie M. Loew ◽  
Daniel L.J. Thorek ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Blood Brain Barrier ◽  
Near Infrared ◽  
Brain Activity ◽  
Brain Barrier ◽  
Fluorescence Signal ◽  
Voltage Sensitive Dye ◽  
Blood Brain ◽  
Adenosine Receptor Agonist ◽  
The Brain

AbstractOptical imaging of brain activity has mostly employed genetically manipulated mice, which cannot be translated to clinical human usage. Observation of brain activity directly is challenging due to difficulty in delivering dyes and other agents through the blood brain barrier (BBB). Using fluorescence imaging, we have demonstrated the feasibility of delivering the near-infrared voltage-sensitive dye (VSD) IR-780 perchlorate to the brain tissue through pharmacological techniques, via an adenosine agonist (Regadenoson). Comparison of VSD fluorescence of mouse brains without and with Regadenoson showed significantly increased residence time of the fluorescence signal in the latter case, indicative of VSD diffusion into the brain tissue. Dose and timing of Regadenoson were varied to optimize BBB permeability for VSD delivery.

scholarly journals Direct Visualization of Mouse Brain Oxygen Distribution by Electron Paramagnetic Resonance Imaging: Application to Focal Cerebral Ischemia

2009 ◽  
Vol 29 (10) ◽  
pp. 1695-1703 ◽  
Author(s):  
Jiangang Shen ◽  
Rohit Sood ◽  
John Weaver ◽  
Graham S Timmins ◽  
Aaron Schnell ◽  
...  
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Brain Tissue ◽  
Mouse Brain ◽  
Focal Cerebral Ischemia ◽  
Imaging Method ◽  
Resonance Imaging ◽  
Paramagnetic Resonance ◽  
Electron Paramagnetic Resonance Imaging ◽  
Electron Paramagnetic ◽  
The Brain

Electron paramagnetic resonance imaging (EPRI) is a new modality for visualizing O2 distribution in tissues, such as the brain after stroke or after administration of drugs of abuse. We have recently shown that 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl [1] is a pro-imaging agent that can cross the blood–brain barrier. After hydrolysis by esterases, the anion of 3-carboxy-2,2,5,5-tetramethyl-1-tetramethyl-1-pyrrolidinyloxyl [2] is trapped in brain tissue. In this study, we investigated the feasibility of using this to map the changes of O2 concentration in mouse brain after focal ischemia. The decrease in tissue O2 concentration in the ischemic region of mouse brain was clearly visualized by EPRI. The hypoxic zone mapped by EPRI was spatially well correlated with the infarction area in the brain imaged by diffusion-weighted magnetic resonance imaging (MRI). Finally, we observed a decrease in the size of the hypoxic region when the mouse breathed higher levels of O2. This finding suggests that EPRI with specifically designed nitroxides is a promising imaging modality for visualizing O2 distribution in brain tissue, especially in an ischemic brain. We believe that this imaging method can be used for monitoring the effects of therapeutic intervention aimed at enhancing brain O2 supply, which is crucial in minimizing brain injury after stroke.

Simulation of Mouse Brain Tissue Under Controlled Cortical Impact

2018 ◽  
Author(s):  
Haifeng Zhao ◽  
Changxin Lai ◽  
Ke Wang ◽  
Suhao Qiu ◽  
Tianyao Wang ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Mouse Brain ◽  
Impact Angle ◽  
Von Mises Stress ◽  
Fe Model ◽  
Controlled Cortical Impact ◽  
Cortical Impact ◽  
Von Mises ◽  
The Impact ◽  
The Brain

Traumatic brain injury is one of the leading causes of injury and death in both developed and developing countries. Animal models are important preclinical tools for injury level studies. In this study, a finite element (FE) model of mouse brain was constructed to investigate the biomechanical responses of brain tissue during a controlled cortical impact (CCI). Impact of the brain tissue was simulated with varying impact speeds and angles. Computational results indicated that the viscoelastic properties of the brain tissue and the impact angle could greatly influence the injury responses. Comparison with the experimental observation showed that energy based stress parameters such as the von Mises stress has the potential to be descriptive of the injury levels.

scholarly journals Viscoelastic mapping of mouse brain tissue: relation to structure and age

2020 ◽  
Author(s):  
Nelda Antonovaite ◽  
Lianne A. Hulshof ◽  
Elly M. Hol ◽  
Wytse J. Wadman ◽  
Davide Iannuzzi
Keyword(s):  
Mechanical Properties ◽  
Brain Tissue ◽  
Mouse Brain ◽  
Brain Slices ◽  
Relative Area ◽  
Linear Regression Models ◽  
Brain Functioning ◽  
Mechanical Factors ◽  
The Relationship ◽  
The Brain

AbstractThere is growing evidence that mechanical factors affect brain functioning. However, brain components responsible for regulating the physiological mechanical environment and causing mechanical alterations during maturation are not completely understood. To determine the relationship between structure and stiffness of the brain tissue, we performed high resolution viscoelastic mapping by dynamic indentation of hippocampus and cerebellum of juvenile brain, and quantified relative area covered by immunohistochemical staining of NeuN (neurons), GFAP (astrocytes), Hoechst (nuclei), MBP (myelin), NN18 (axons) of juvenile and adult mouse brain slices. When compared the mechanical properties of juvenile mouse brain slices with previously obtained data on adult slices, the latter was ~ 20-150% stiffer, which correlates with an increase in the relative area covered by astrocytes. Heterogeneity within the slice, in terms of storage modulus, correlates negatively with the relative area of nuclei and neurons, as well as myelin and axons, while the relative area of astrocytes correlates positively. Several linear regression models are suggested to predict the mechanical properties of the brain tissue based on immunohistochemical stainings.

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

2019 ◽  
Vol 15 (3) ◽  
pp. 251-257
Author(s):  
Bahareh Sadat Yousefsani ◽  
Seyed Ahmad Mohajeri ◽  
Mohammad Moshiri ◽  
Hossein Hosseinzadeh
Keyword(s):  
Rat Brain ◽  
Brain Tissue ◽  
Molecularly Imprinted Polymer ◽  
Lipid Emulsion ◽  
Limit Of Detection ◽  
Imprinted Polymer ◽  
Toxic Dose ◽  
Molecularly Imprinted ◽  
The Brain ◽  
Rat Brain Tissue

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.

scholarly journals Safety profile of the transcription factor EB (TFEB)-based gene therapy through intracranial injection in mice

2020 ◽  
Vol 11 (1) ◽  
pp. 241-250
Author(s):  
Zhenyu Li ◽  
Guangqian Ding ◽  
Yudi Wang ◽  
Zelong Zheng ◽  
Jianping Lv
Keyword(s):  
Gene Therapy ◽  
Transcription Factor ◽  
Neurodegenerative Diseases ◽  
Brain Tissue ◽  
Inflammatory Responses ◽  
Tissue Samples ◽  
Protein Levels ◽  
Virus Serotype ◽  
Transcription Factor Eb ◽  
The Brain

AbstractTranscription factor EB (TFEB)-based gene therapy is a promising therapeutic strategy in treating neurodegenerative diseases by promoting autophagy/lysosome-mediated degradation and clearance of misfolded proteins that contribute to the pathogenesis of these diseases. However, recent findings have shown that TFEB has proinflammatory properties, raising the safety concerns about its clinical application. To investigate whether TFEB induces significant inflammatory responses in the brain, male C57BL/6 mice were injected with phosphate-buffered saline (PBS), adeno-associated virus serotype 8 (AAV8) vectors overexpressing mouse TFEB (pAAV8-CMV-mTFEB), or AAV8 vectors expressing green fluorescent proteins (GFPs) in the barrel cortex. The brain tissue samples were collected at 2 months after injection. Western blotting and immunofluorescence staining showed that mTFEB protein levels were significantly increased in the brain tissue samples of mice injected with mTFEB-overexpressing vectors compared with those injected with PBS or GFP-overexpressing vectors. pAAV8-CMV-mTFEB injection resulted in significant elevations in the mRNA and protein levels of lysosomal biogenesis indicators in the brain tissue samples. No significant changes were observed in the expressions of GFAP, Iba1, and proinflammation mediators in the pAAV8-CMV-mTFEB-injected brain compared with those in the control groups. Collectively, our results suggest that AAV8 successfully mediates mTFEB overexpression in the mouse brain without inducing apparent local inflammation, supporting the safety of TFEB-based gene therapy in treating neurodegenerative diseases.

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.

scholarly journals High-fat diet-induced obesity and impairment of brain neurotransmitter pool

2020 ◽  
Vol 11 (1) ◽  
pp. 147-160
Author(s):  
Ranyah Shaker M. Labban ◽  
Hanan Alfawaz ◽  
Ahmed T. Almnaizel ◽  
Wail M. Hassan ◽  
Ramesa Shafi Bhat ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Brain Tissue ◽  
High Fat Diet ◽  
Density Lipoprotein ◽  
Obese Group ◽  
Control Group ◽  
High Fat ◽  
Brain Neurotransmitter ◽  
The Brain ◽  
Lean Group

AbstractObesity and the brain are linked since the brain can control the weight of the body through its neurotransmitters. The aim of the present study was to investigate the effect of high-fat diet (HFD)-induced obesity on brain functioning through the measurement of brain glutamate, dopamine, and serotonin metabolic pools. In the present study, two groups of rats served as subjects. Group 1 was fed a normal diet and named as the lean group. Group 2 was fed an HFD for 4 weeks and named as the obese group. Markers of oxidative stress (malondialdehyde, glutathione, glutathione-s-transferase, and vitamin C), inflammatory cytokines (interleukin [IL]-6 and IL-12), and leptin along with a lipid profile (cholesterol, triglycerides, high-density lipoprotein, and low-density lipoprotein levels) were measured in the serum. Neurotransmitters dopamine, serotonin, and glutamate were measured in brain tissue. Fecal samples were collected for observing changes in gut flora. In brain tissue, significantly high levels of dopamine and glutamate as well as significantly low levels of serotonin were found in the obese group compared to those in the lean group (P > 0.001) and were discussed in relation to the biochemical profile in the serum. It was also noted that the HFD affected bacterial gut composition in comparison to the control group with gram-positive cocci dominance in the control group compared to obese. The results of the present study confirm that obesity is linked to inflammation, oxidative stress, dyslipidemic processes, and altered brain neurotransmitter levels that can cause obesity-related neuropsychiatric complications.

scholarly journals Regulation of Protein Synthesis in Developing Mouse Brain Tissue

1970 ◽  
Vol 245 (6) ◽  
pp. 1388-1393
Author(s):  
Michael P. Lerner ◽  
Terry C. Johnson
Keyword(s):  
Protein Synthesis ◽  
Brain Tissue ◽  
Mouse Brain ◽  
Developing Mouse Brain
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