Visualisation of GABAergic neurons and synapses in the rat brain using immunohistochemistry for two forms of glutamate decarboxylase

BACKGROUND: Taking into account the importance of GABAergic brain system research and also the opportunity to achieve specific and accurate results in laboratory studies using immunohistochemical approaches, it seems important to have a reliable method of visualization GABA-synthesizing cells, their projections and synapses, for the morphofunctional analysis of GABAergic system both in normal conditions and in the experimental pathology. AIM: The aim of the study was to visualize analyze GABAergic neurons and synapses within rats brain using three different antibody types against glutamate decarboxylase and to identify the optimal conditions for reaction performing. MATERIALS AND METHODS: The study was performed on paraffin brain tissue sections of 5 adult Wistar rats. Immunohistochemical reactions using three antibody types against glutamate decarboxylase isoform 67 (GAD67) and glutamate decarboxylase isoform 65 (GAD65) were performed. Additional controls on C57/Bl6 mice and Chinchilla rabbits brain samples were also carried out. RESULTS: Antibodies used in the research made it possible to achieve high quality of GABAergic structures visualizing without increasing background staining. At the same time different antibody types are distinct in their efficacy to perform immunohistochemistry reaction on laboratory animal brain tissue samples. By performing additional controls, we discovered that there is necessary to adsorb secondary reagents immunoglobulins in order to eliminate nonspecific staining. It was found that GAD67 and GAD65 distribution in rat forebrain structures is different. It was stated that GAD67 immunohistochemistry most completely reveals GABAergic brain structures compared to GAD65 immunhistochemistry. The possibility of determining morphological features of GABAergic neurons and synaptic terminals, as well as performing quantitative analysis, was demonstrated. CONCLUSIONS: The approach proposed makes it possible to specifically visualize GABAergic structures of the central nervous system of different laboratory animals. This could be useful both in fundamental studies and in pathology research.

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Localization of Classical Swine Fever Virus from Chronically Infected Pigs by In Situ Hybridization and Immunohistochemistry

Veterinary Pathology ◽

10.1354/vp.40-1-107 ◽

2003 ◽

Vol 40 (1) ◽

pp. 107-113 ◽

Cited By ~ 14

Author(s):

C. Choi ◽

C. Chae

Keyword(s):

In Situ Hybridization ◽

Nucleic Acid ◽

Mononuclear Cells ◽

Classical Swine Fever ◽

Lymphoid Tissues ◽

Hybridization Technique ◽

Tissue Sections ◽

Background Staining ◽

The Central Nervous System

Classical swine fever (CSF) virus (CSFV) nucleic acid and antigen were detected in 15 pigs with naturally occurring chronic CSF by in situ hybridization and immunohistochemistry. The most consistent and prominent microscopic lesions were perivascular mononuclear cell infiltration and gliosis in the central nervous system of pigs with chronic CSF. Positive cells typically exhibited a dark brown (in situ hybridization) or red (immunohistochemistry) reaction product in the cytoplasm without background staining. A positive signal for both in situ hybridization and immunohistochemistry was detected in mononuclear cells and lymphocytes of lymphoid tissues. Viral nucleic acid was detected in some tissue sections in the absence of viral antigen. The in situ hybridization technique developed in this study was useful for the detection of CSFV RNA in tissues taken from chronically infected pigs and may be a valuable technique for studying the pathogenesis of chronic CSFV infection.

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Targeted UHPLC-ESI-MS/MS Analysis of Selected Neurotransmitters, Tryptophan and Its Metabolite Kynurenine in Tau Transgenic Rat Brain Tissue: A Pivotal Study

Separations ◽

10.3390/separations9010016 ◽

2022 ◽

Vol 9 (1) ◽

pp. 16

Author(s):

Juraj Piestansky ◽

Andrea Forgacsova ◽

Dominika Olesova ◽

Jaroslav Galba ◽

Peter Mikus ◽

...

Keyword(s):

Brain Tissue ◽

Aspartic Acid ◽

High Performance ◽

Aminobutyric Acid ◽

Transgenic Rat ◽

Elevated Concentration ◽

Tissue Samples ◽

Validation Parameters ◽

On Line ◽

The Central Nervous System

Neurotransmitters (NT) are widely distributed in the central nervous system. These molecules are important for many physiological processes and the function of the immune system. Imbalance of NT are linked to numerous neurological disorders and diseases, including tauopathies. Here, a targeted approach based on on-line combination of ultra-high performance liquid chromatography with tandem mass spectrometry was validated and applied to the quantitative analysis of nine NT (acetylcholine, choline, aspartic acid, asparagine, glutamic acid, glutamine, pyroglutamate, γ-aminobutyric acid, N-acetyl-L-aspartic acid), tryptophan and its metabolite kynurenine in brain tissue samples of a rat model for tauopathy. The applied analytical method was characterized by excellent validation parameters for all analytes, such as limits of detection in the range of 0.01–1.70 µg/mL, regression coefficients of the calibration curves ≥ 0.9946, intra-day and inter-day precision expressed as coefficient of variation in the range of 0.6–11.9% and 0.6–14.4%, and accuracy in the range of 87.6–107.1% and 87.2–119.6%. Our analytical approach led to the identification of increased levels of choline and γ-aminobutyric acid in pons, and elevated concentration levels of pyroglutamate in medulla oblongata. These findings indicate that NT could play a valuable role in the study and clarification of neuroinflammation and neurodegenerative diseases.

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Discovery of intracellular 2,8 dihydroxyadenine crystals in bovine lymphatic and hepatic cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010012881x ◽

1987 ◽

Vol 45 ◽

pp. 906-907

Author(s):

W. E. Rigsby ◽

D. M. Hinton ◽

V. J. Hurst ◽

P. C. McCaskey

Keyword(s):

Polarized Light ◽

Paraffin Sections ◽

Tissue Samples ◽

Whole Cells ◽

Tissue Sections ◽

Hepatic Cells ◽

Slaughter House ◽

Mammalian Tissues ◽

Carbon Coated ◽

Cellular Components

Crystalline intracellular inclusions are rarely seen in mammalian tissues and are often difficult to positively identify. Lymph node and liver tissue samples were obtained from two cows which had been rejected at the slaughter house due to the abnormal appearance of these organs in the animals. The samples were fixed in formaldehyde and some of the fixed material was embedded in paraffin. Examination of the paraffin sections with polarized light microscopy revealed the presence of numerous crystals in both hepatic and lymph tissue sections. Tissue sections were then deparaffinized in xylene, mounted, carbon coated, and examined in a Phillips 505T SEM equipped with a Tracor Northern X-ray Energy Dispersive Spectroscopy (EDS) system. Crystals were obscured by cellular components and membranes so that EDS spectra were only obtainable from whole cells. Tissue samples which had been fixed but not paraffin-embedded were dehydrated, embedded in Spurrs plastic, and sectioned.

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P1-377: BIIB092 DOES NOT INTERFERE WITH RADIOLABELED MK6240 BINDING TO NEUROFIBRILLARY TANGLES: RESULTS FROM IN VITRO COMPETITION BINDING STUDY IN ALZHEIMER'S BRAIN TISSUE SECTIONS

Alzheimer s & Dementia ◽

10.1016/j.jalz.2019.06.932 ◽

2019 ◽

Vol 15 ◽

pp. P399-P399

Author(s):

Sara Girmay ◽

Roser Farre Garros ◽

Abha Yadav ◽

Richelle Sopko ◽

Heike Hering ◽

...

Keyword(s):

Brain Tissue ◽

Neurofibrillary Tangles ◽

Binding Study ◽

Tissue Sections ◽

Competition Binding

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

Translational Neuroscience ◽

10.1515/tnsci-2020-0132 ◽

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.

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Rapid brain structure and tumour margin detection on whole frozen tissue sections by fast multiphotometric mid-infrared scanning

Scientific Reports ◽

10.1038/s41598-021-90777-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tim Kümmel ◽

Björn van Marwick ◽

Miriam Rittel ◽

Carina Ramallo Guevara ◽

Felix Wühler ◽

...

Keyword(s):

Imaging System ◽

Frozen Section ◽

Computational Effort ◽

Primary Hepatocellular Carcinoma ◽

Measuring System ◽

Sufficient Information ◽

Tissue Samples ◽

Mid Infrared ◽

Tissue Sections ◽

Frozen Tissue

AbstractFrozen section analysis is a frequently used method for examination of tissue samples, especially for tumour detection. In the majority of cases, the aim is to identify characteristic tissue morphologies or tumour margins. Depending on the type of tissue, a high number of misdiagnoses are associated with this process. In this work, a fast spectroscopic measurement device and workflow was developed that significantly improves the speed of whole frozen tissue section analyses and provides sufficient information to visualize tissue structures and tumour margins, dependent on their lipid and protein molecular vibrations. That optical and non-destructive method is based on selected wavenumbers in the mid-infrared (MIR) range. We present a measuring system that substantially outperforms a commercially available Fourier Transform Infrared (FT-IR) Imaging system, since it enables acquisition of reduced spectral information at a scan field of 1 cm2 in 3 s, with a spatial resolution of 20 µm. This allows fast visualization of segmented structure areas with little computational effort. For the first time, this multiphotometric MIR system is applied to biomedical tissue sections. We are referencing our novel MIR scanner on cryopreserved murine sagittal and coronal brain sections, especially focusing on the hippocampus, and show its usability for rapid identification of primary hepatocellular carcinoma (HCC) in mouse liver.

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In Vivo and In Vitro Toxicodynamic Analyses of New Quinolone-and Nonsteroidal Anti-Inflammatory Drug-Induced Effects on the Central Nervous System

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.5.1091 ◽

1999 ◽

Vol 43 (5) ◽

pp. 1091-1097 ◽

Cited By ~ 7

Author(s):

Hideki Kita ◽

Hirotami Matsuo ◽

Hitomi Takanaga ◽

Junichi Kawakami ◽

Koujirou Yamamoto ◽

...

Keyword(s):

Brain Tissue ◽

Threshold Concentration ◽

Current Response ◽

Drug Induced ◽

Inhibition Ratio ◽

New Quinolones ◽

The Central Nervous System ◽

Anti Inflammatory

ABSTRACT We investigated the correlation between an in vivo isobologram based on the concentrations of new quinolones (NQs) in brain tissue and the administration of nonsteroidal anti-inflammatory drugs (NSAIDs) for the occurrence of convulsions in mice and an in vitro isobologram based on the concentrations of both drugs for changes in the γ-aminobutyric acid (GABA)-induced current response in Xenopus oocytes injected with mRNA from mouse brains in the presence of NQs and/or NSAIDs. After the administration of enoxacin (ENX) in the presence or absence of felbinac (FLB), ketoprofen (KTP), or flurbiprofen (FRP), a synergistic effect was observed in the isobologram based on the threshold concentration in brain tissue between mice with convulsions and those without convulsions. The three NSAIDs did not affect the pharmacokinetic behavior of ENX in the brain. However, the ENX-induced inhibition of the GABA response in the GABAA receptor expressed in Xenopus oocytes was enhanced in the presence of the three NSAIDs. The inhibition ratio profiles of the GABA responses for both drugs were analyzed with a newly developed toxicodynamic model. The inhibitory profiles for ENX in the presence of NSAIDs followed the order KTP (1.2 μM) > FRP (0.3 μM) > FLB (0.2 μM). These were 50- to 280-fold smaller than those observed in the absence of NSAIDs. The inhibition ratio (0.01 to 0.02) of the GABAA receptor in the presence of both drugs was well-fitted to the isobologram based on threshold concentrations of both drugs in brain tissue between mice with convulsions and those without convulsions, despite the presence of NSAIDs. In mice with convulsions, the inhibitory profiles of the threshold concentrations of both drugs in brain tissue of mice with convulsions and those without convulsions can be predicted quantitatively by using in vitro GABA response data and toxicodynamic model.

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