Simultaneous HPLC–MS–MS quantification of 8-iso-PGF2α and 8,12-iso-iPF2α in CSF and brain tissue samples with on-line cleanup

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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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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On-line Flow Cytometry for Real-time Surgical Guidance

Neurosurgery ◽

10.1227/01.neu.0000134470.13971.0c ◽

2004 ◽

Vol 55 (3) ◽

pp. 551-561 ◽

Cited By ~ 7

Author(s):

Ali H. Mesiwala ◽

Louis D. Scampavia ◽

Peter S. Rabinovitch ◽

Jaromir Ruzicka ◽

Robert C. Rostomily

Keyword(s):

Decision Making ◽

Real Time ◽

Dna Content ◽

Time Frame ◽

Tissue Samples ◽

Surgical Decision ◽

Surgical Guidance ◽

On Line ◽

Surgical Decision Making ◽

Line Analysis

Abstract OBJECTIVE: This study tests the feasibility of using on-line analysis of tissue during surgical resection of brain tumors to provide biologically relevant information in a clinically relevant time frame to augment surgical decision making. For the purposes of establishing feasibility, we used measurement of deoxyribonucleic acid (DNA) content as the end point for analysis. METHODS: We investigated the feasibility of interfacing an ultrasonic aspiration (USA) system with a flow cytometer (FC) capable of analyzing DNA content (DNA-FC). The sampling system design, tissue preparation requirements, and time requirements for each step of the on-line analysis system were determined using fresh beef brain tissue samples. We also compared DNA-FC measurements in 28 nonneoplastic human brain samples with DNA-FC measurements in specimens of 11 glioma patients obtained from central tumor regions and surgical margins after macroscopically gross total tumor removal to estimate the potential for analysis of a biological marker to influence surgical decision making. RESULTS: With minimal modification, modern FC systems are fully capable of real-time, intraoperative analysis of USA specimens. The total time required for on-line analysis of USA specimens varies between 36 and 63 seconds; this time includes delivery from the tip of the USA to complete analysis of the specimen. Approximately 60% of this time is required for equilibration of the DNA stain. When compared with values for nonneoplastic human brain samples, 50% of samples (10 of 20) from macroscopically normal glioma surgical margins contained DNA-FC abnormalities potentially indicating residual tumor. CONCLUSION: With an interface of existing technologies, DNA content of brain tissue samples can be analyzed in a meaningful time frame that has the potential to provide real-time information for surgical guidance. The identification of DNA content abnormalities in macroscopically normal tumor resection margins by DNA-FC supports the practical potential for on-line analysis of a tumor marker to guide surgical resections. The development of such a device would provide neurosurgeons with an objective method for intraoperative analysis of a clinically relevant biological parameter that can be measured in real time.

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A Validated UPLC–MS-MS Assay for the Rapid Determination of Lorcaserin in Plasma and Brain Tissue Samples

Journal of Analytical Toxicology ◽

10.1093/jat/bkv126 ◽

2015 ◽

Vol 40 (2) ◽

pp. 133-139 ◽

Cited By ~ 4

Author(s):

Amal A. Bajrai ◽

Essam Ezzeldin ◽

Khalid A. Al-Rashood ◽

Mohammad Raish ◽

Muzaffar Iqbal

Keyword(s):

Brain Tissue ◽

Rapid Determination ◽

Tissue Samples

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

Medical academic journal ◽

10.17816/maj70770 ◽

2021 ◽

Vol 21 (2) ◽

pp. 63-73

Author(s):

Valeria A. Razenkova ◽

Dmitrii E. Korzhevskii

Keyword(s):

Brain Tissue ◽

Glutamate Decarboxylase ◽

Brain Structures ◽

Gabaergic Neurons ◽

Laboratory Animals ◽

Tissue Samples ◽

Tissue Sections ◽

Background Staining ◽

The Central Nervous System ◽

Rat Forebrain

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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Glutathione S-Transferases and Cytochrome P450 Enzyme Expression in Patients with Intracranial Tumors: Preliminary Report of 55 Patients

Medical Principles and Practice ◽

10.1159/000494496 ◽

2018 ◽

Vol 28 (1) ◽

pp. 56-62

Author(s):

Cahit Kural ◽

Arzu Kaya Kocdogan ◽

Gulcin Güler Şimşek ◽

Serpil Oğuztüzün ◽

Pınar Kaygın ◽

...

Keyword(s):

Cytochrome P450 ◽

Brain Tissue ◽

Pituitary Adenomas ◽

Normal Brain ◽

Cytochrome P450 Enzyme ◽

Intracranial Tumors ◽

Glutathione S Transferase ◽

Normal Brain Tissue ◽

Tissue Samples ◽

P450 Enzyme

Objective: Intracranial tumors are one of the most frightening and difficult-to-treat tumor types. In addition to surgery, protocols such as chemotherapy and radiotherapy also take place in the treatment. Glutathione S-transferase (GST) and cytochrome P450 (CYP) enzymes are prominent drug-metabolizing enzymes in the human body. The aim of this study is to show the expression of GSTP1, GSTM1, CYP1A1, and CYP1B1 in different types of brain tumors and compare our results with those in the literature. Subjects and Methods: The expression of GSTP1, GSTM1, CYP1A1, and CYP1B1 was analyzed using immunostaining in 55 patients with intracranial tumors in 2016–2017. For GST and CYP expression in normal brain tissue, samples of a portion of surrounding normal brain tissue as well as a matched far neighbor of tumor tissue were used. The demographic features of the patients were documented and the expression results compared. Results: The mean age of the patients was 46.72 years; 29 patients were female and 26 were male. Fifty-seven specimens were obtained from 55 patients. Among them, meningioma was diagnosed in 12, metastases in 12, glioblastoma in 9, and pituitary adenoma in 5. The highest GSTP1, GSTM1, and CYP1A1 expressions were observed in pituitary adenomas. The lowest GSTP1 expression was detected in glioblastomas and the lowest CYP1B1 expression in pituitary adenomas. Conclusion: GSTP1 and CYP expression is increased in intracranial tumors. These results should be confirmed with a larger series and different enzyme subtypes.

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Scatterometry Measurements With Scattered Light Imaging Enable New Insights Into the Nerve Fiber Architecture of the Brain

Frontiers in Neuroanatomy ◽

10.3389/fnana.2021.767223 ◽

2021 ◽

Vol 15 ◽

Author(s):

Miriam Menzel ◽

Marouan Ritzkowski ◽

Jan A. Reuter ◽

David Gräßel ◽

Katrin Amunts ◽

...

Keyword(s):

Human Brain ◽

Nerve Fiber ◽

Brain Tissue ◽

Scattered Light ◽

Polar Angle ◽

Nerve Fibers ◽

Fiber Architecture ◽

Whole Brain ◽

Tissue Samples ◽

The Brain

The correct reconstruction of individual (crossing) nerve fibers is a prerequisite when constructing a detailed network model of the brain. The recently developed technique Scattered Light Imaging (SLI) allows the reconstruction of crossing nerve fiber pathways in whole brain tissue samples with micrometer resolution: the individual fiber orientations are determined by illuminating unstained histological brain sections from different directions, measuring the transmitted scattered light under normal incidence, and studying the light intensity profiles of each pixel in the resulting image series. So far, SLI measurements were performed with a fixed polar angle of illumination and a small number of illumination directions, providing only an estimate of the nerve fiber directions and limited information about the underlying tissue structure. Here, we use a display with individually controllable light-emitting diodes to measure the full distribution of scattered light behind the sample (scattering pattern) for each image pixel at once, enabling scatterometry measurements of whole brain tissue samples. We compare our results to coherent Fourier scatterometry (raster-scanning the sample with a non-focused laser beam) and previous SLI measurements with fixed polar angle of illumination, using sections from a vervet monkey brain and human optic tracts. Finally, we present SLI scatterometry measurements of a human brain section with 3 μm in-plane resolution, demonstrating that the technique is a powerful approach to gain new insights into the nerve fiber architecture of the human brain.

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MicroRNA Analysis of Human Stroke Brain Tissue Resected during Decompressive Craniectomy/Stroke-Ectomy Surgery

Genes ◽

10.3390/genes12121860 ◽

2021 ◽

Vol 12 (12) ◽

pp. 1860

Author(s):

Andrew P. Carlson ◽

William McKay ◽

Jeremy S. Edwards ◽

Radha Swaminathan ◽

Karen S. SantaCruz ◽

...

Keyword(s):

Signaling Pathways ◽

Brain Tissue ◽

Stroke Patient ◽

Mirna Expression ◽

Strong Association ◽

Bioinformatic Analysis ◽

Experimental Models ◽

Aberrant Expression ◽

Tissue Samples ◽

Human Stroke

Background: Signaling pathways mediated by microRNAs (miRNAs) have been identified as one of the mechanisms that regulate stroke progression and recovery. Recent investigations using stroke patient blood and cerebrospinal fluid (CSF) demonstrated disease-specific alterations in miRNA expression. In this study, for the first time, we investigated miRNA expression signatures in freshly removed human stroke brain tissue. Methods: Human brain samples were obtained during craniectomy and brain tissue resection in severe stroke patients with life-threatening brain swelling. The tissue samples were subjected to histopathological and immunofluorescence microscopy evaluation, next generation miRNA sequencing (NGS), and bioinformatic analysis. Results: miRNA NGS analysis detected 34 miRNAs with significantly aberrant expression in stroke tissue, as compared to non-stroke samples. Of these miRNAs, 19 were previously identified in stroke patient blood and CSF, while dysregulation of 15 miRNAs was newly detected in this study. miRNA direct target gene analysis and bioinformatics approach demonstrated a strong association of the identified miRNAs with stroke-related biological processes and signaling pathways. Conclusions: Dysregulated miRNAs detected in our study could be regarded as potential candidates for biomarkers and/or targets for therapeutic intervention. The results described herein further our understanding of the molecular basis of stroke and provide valuable information for the future functional studies in the experimental models of stroke.

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Intraoperative Detection of Pediatric Brain Tumor Margins Using Raman Spectroscopy.

Neurosurgery ◽

10.1093/neuros/nyz310_132 ◽

2019 ◽

Vol 66 (Supplement_1) ◽

Cited By ~ 1

Author(s):

Rashad Jabarkheel ◽

Jonathon J Parker ◽

Chi-Sing Ho ◽

Travis Shaffer ◽

Sanjiv Gambhir ◽

...

Keyword(s):

Machine Learning ◽

Raman Spectroscopy ◽

Brain Tumor ◽

Brain Tumors ◽

Brain Tissue ◽

Pediatric Brain Tumor ◽

Tissue Samples ◽

Tumor Margins ◽

Pediatric Brain ◽

Tumor Debulking

Abstract INTRODUCTION Surgical resection is a mainstay of treatment in patients with brain tumors both for tissue diagnosis and for tumor debulking. While maximal resection of tumors is desired, neurosurgeons can be limited by the challenge of differentiating normal brain from tumor using only microscopic visualization and tactile feedback. Additionally, intraoperative decision-making regarding how aggressively to pursue a gross total resection frequently relies on pathologic preliminary diagnosis using frozen sections which are both time consuming and fallible. Here, we investigate the potential for Raman spectroscopy (RS) to rapidly detect pediatric brain tumor margins and classify brain tissue samples equivalent to histopathology. METHODS Using a first-of-its-kind rapid acquisition RS device we intraoperatively imaged fresh ex vivo pediatric brain tissue samples (2-3 mm × 2-3 mm × 2-3 mm) at the Lucille Packard Children's Hospital. All imaged samples received standard final histopathological analysis, as RS is a nondestructive imaging technique. We curated a labeled dataset of 575 + unique Raman spectra gathered from 160 + brain samples resulting from 23 pediatric patients who underwent brain tissue resection as part of tumor debulking or epilepsy surgery (normal controls). RESULTS To our knowledge we have created the largest labeled Raman spectra dataset of pediatric brain tumors. We are developing an end-to-end machine learning model that can predict final histopathology diagnosis within minutes from Raman spectral data. Our preliminary principle component analyses suggest that RS can be used to classify various brain tumors similar to “frozen” histopathology and can differentiate normal from malignant brain tissue in the context of low-grade glioma resections. CONCLUSION Our work suggests that machine learning approaches can be used to harness the material identification properties of RS for classifying brain tumors and detecting their margins.

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Evaluation of an Immunochromatographic Assay as a Canine Rabies Surveillance Tool in Goa, India

Viruses ◽

10.3390/v11070649 ◽

2019 ◽

Vol 11 (7) ◽

pp. 649 ◽

Cited By ~ 3

Author(s):

Yale ◽

Gibson ◽

Mani ◽

P.K. ◽

Costa ◽

...

Keyword(s):

Brain Tissue ◽

Low Cost ◽

Antibody Test ◽

Immunochromatographic Assay ◽

Human Rabies ◽

Post Mortem ◽

Laboratory Equipment ◽

Tissue Samples ◽

Test Kit ◽

The Government

Rabies is a fatal zoonotic disease transmitted by the bite of a rabid animal. More than 95% of the human rabies cases in India are attributed to exposure to rabid dogs. This study evaluated the utility of a lateral flow immunochromatographic assay (LFA) (Anigen Rapid Rabies Ag Test Kit, Bionote, Hwaseong-si, Korea) for rapid post mortem diagnosis of rabies in dogs. Brain tissue was collected from 202 animals that were screened through the Government of Goa rabies surveillance system. The brain tissue samples were obtained from 188 dogs, nine cats, three bovines, one jackal and one monkey. In addition, 10 dogs that died due to trauma from road accidents were included as negative controls for the study. The diagnostic performance of LFA was evaluated using results from direct fluorescence antibody test (dFT); the current gold standard post mortem test for rabies infection. Three samples were removed from the analysis as they were autolysed and not fit for testing by dFT. Of the 209 samples tested, 117 tested positive by LFA and 92 tested negative, while 121 tested positive by dFT and 88 tested negative. Estimates of LFA sensitivity and specificity were 0.96 (95% CI 0.91–0.99) and 0.99 (95% CI 0.94–1.00), respectively. The LFA is a simple and low-cost assay that aids in the rapid diagnosis of rabies in the field without the need for expensive laboratory equipment or technical expertise. This study found that Bionote LFA has potential as a screening tool in rabies endemic countries.

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