scholarly journals Quantitative evidence of suppressed TMEM119 microglial immunohistochemistry in fatal morphine intoxications

2021 ◽  
Author(s):  
Simone Bohnert ◽  
Kosmas Georgiades ◽  
Camelia-Maria Monoranu ◽  
Michael Bohnert ◽  
Andreas Büttner ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Specific Marker ◽  
Phagocytic Function ◽  
Human Brain Tissue ◽  
Tissue Samples ◽  
Quantitative Evidence ◽  
Single Substance ◽  
Diagnostic Potential ◽  
Drug Type ◽  
Cell Densities

AbstractThe aim of this pilot study was to investigate the diagnostic potential of TMEM119 as a useful microglia-specific marker in combination with immunostainings for phagocytic function and infiltrating capacity of monocytes in cases of lethal monosubstance intoxications by morphine (MOR), methamphetamine (METH), and of ethanol-associated death (ETH) respectively. Human brain tissue samples were obtained from forensic autopsies of cases with single substance abuse (MOR, n = 8; ETH, n = 10; METH, n = 9) and then compared to a cohort of cardiovascular fatalities as controls (n = 9). Brain tissue samples of cortex, white matter, and hippocampus were collected and stained immunohistochemically with antibodies against TMEM119, CD68KiM1P, and CCR2. We could document the lowest density of TMEM119-positive cells in MOR deaths with highly significant differences to the control densities in all three regions investigated. In ETH and METH deaths, the expression of TMEM119 was comparable to cell densities in controls. The results indicate that the immunoreaction in brain tissue is different in these groups depending on the drug type used for abuse.

scholarly journals TMEM119 as a specific marker of microglia reaction in traumatic brain injury in postmortem examination

2020 ◽  
Vol 134 (6) ◽  
pp. 2167-2176
Author(s):  
Simone Bohnert ◽  
Anja Seiffert ◽  
Stefanie Trella ◽  
Michael Bohnert ◽  
Luitpold Distel ◽  
...  
Keyword(s):  
Traumatic Brain Injury ◽  
Brain Injury ◽  
Survival Time ◽  
Brain Tissue ◽  
Causes Of Death ◽  
Cell Number ◽  
Specific Marker ◽  
Human Brain Tissue ◽  
Tissue Samples ◽  
Forensic Assessments

Abstract The aim of the present study was a refined analysis of neuroinflammation including TMEM119 as a useful microglia-specific marker in forensic assessments of traumatic causes of death, e.g., traumatic brain injury (TBI). Human brain tissue samples were obtained from autopsies and divided into cases with lethal TBI (n = 25) and subdivided into three groups according to their trauma survival time and compared with an age-, gender-, and postmortem interval-matched cohort of sudden cardiovascular fatalities as controls (n = 23). Brain tissue samples next to cortex contusions and surrounding white matter as well as samples of the ipsilateral uninjured brain stem and cerebellum were collected and stained immunohistochemically with antibodies against TMEM119, CD206, and CCR2. We could document the highest number of TMEM119-positive cells in acute TBI death with highly significant differences to the control numbers. CCR2-positive monocytes showed a significantly higher cell count in the cortex samples of TBI cases than in the controls with an increasing number of immunopositive cells over time. The number of CD206-positive M2 microglial cells increased survival time-dependent. After 3 days of survival, the cell number increased significantly in all four regions investigated compared with controls. In sum, we validate a specific and robustly expressed as well as fast reacting microglia marker, TMEM119, which distinguishes microglia from resident and infiltrating macrophages and thus offers a great potential for the estimation of the minimum survival time after TBI.

Enrichment of single neurons and defined brain regions from human brain tissue samples for subsequent proteome analysis

2015 ◽  
Vol 122 (7) ◽  
pp. 993-1005 ◽  
Author(s):  
Mariana Molina ◽  
Simone Steinbach ◽  
Young Mok Park ◽  
Su Yeong Yun ◽  
Ana Tereza Di Lorenzo Alho ◽  
...  
Keyword(s):  
Human Brain ◽  
Brain Tissue ◽  
Proteome Analysis ◽  
Brain Regions ◽  
Single Neurons ◽  
Human Brain Tissue ◽  
Tissue Samples

scholarly journals Hybridization-based in situ sequencing (HybISS) for spatially resolved transcriptomics in human and mouse brain tissue

2020 ◽  
Vol 48 (19) ◽  
pp. e112-e112 ◽  
Author(s):  
Daniel Gyllborg ◽  
Christoffer Mattsson Langseth ◽  
Xiaoyan Qian ◽  
Eunkyoung Choi ◽  
Sergio Marco Salas ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Rolling Circle Amplification ◽  
Cell Types ◽  
Probe Design ◽  
Sequencing Data ◽  
Human Brain Tissue ◽  
Tissue Samples ◽  
Rolling Circle ◽  
Spatial Detection

Abstract Visualization of the transcriptome in situ has proven to be a valuable tool in exploring single-cell RNA-sequencing data, providing an additional spatial dimension to investigate multiplexed gene expression, cell types, disease architecture or even data driven discoveries. In situ sequencing (ISS) method based on padlock probes and rolling circle amplification has been used to spatially resolve gene transcripts in tissue sections of various origins. Here, we describe the next iteration of ISS, HybISS, hybridization-based in situ sequencing. Modifications in probe design allows for a new barcoding system via sequence-by-hybridization chemistry for improved spatial detection of RNA transcripts. Due to the amplification of probes, amplicons can be visualized with standard epifluorescence microscopes for high-throughput efficiency and the new sequencing chemistry removes limitations bound by sequence-by-ligation chemistry of ISS. HybISS design allows for increased flexibility and multiplexing, increased signal-to-noise, all without compromising throughput efficiency of imaging large fields of view. Moreover, the current protocol is demonstrated to work on human brain tissue samples, a source that has proven to be difficult to work with image-based spatial analysis techniques. Overall, HybISS technology works as a targeted amplification detection method for improved spatial transcriptomic visualization, and importantly, with an ease of implementation.

scholarly journals Improved Reproducibility of Protein Detection in Neuromelanin-containing Human Substantia Nigra Tissue enriched by Laser Microdissection

2020 ◽  
Author(s):  
Steffen Kösters ◽  
Mandy Rettel ◽  
Britta Eggers ◽  
Peter Riederer ◽  
Manfred Gerlach ◽  
...  
Keyword(s):  
Substantia Nigra ◽  
Brain Tissue ◽  
Protein Identification ◽  
Laser Microdissection ◽  
Protein Detection ◽  
Protein Quantification ◽  
Human Brain Tissue ◽  
Limited Sample ◽  
Tissue Samples ◽  
Proteomics Research

Abstract Background: Processing of post-mortem human brain tissue samples like Neuromelanin-containing substantia nigra is a challenging task in proteomics research in the context of neurodegenerative diseases such as Parkinson’s disease, due to the availability of only limited sample amounts. Therefore, a highly efficient lysis and digestion approach is needed to guarantee high reproducibility and a good quantification performance. We conducted a systematic study in order to compare qualitatively and quantitatively six different sample preparation protocols for substantia nigra brain tissue collected by laser microdissection.Results: LC-MS/MS analysis revealed that the combination of formic acid and in-solution digestion with trypsin leads to an enhanced peptide and protein identification as well as a robust and reliable protein quantification with a very good reproducibility.Conclusion: We recommend this protocol for future studies of human substantia nigra tissue as well as NM granules in the context of neurodegenerative disorders like Parkinson syndrome.

scholarly journals Improved Reproducibility of Protein Detection in Neuromelanin-containing Human Substantia Nigra Tissue enriched by Laser Microdissection

2020 ◽  
Author(s):  
Steffen Kösters ◽  
Mandy Rettel ◽  
Britta Eggers ◽  
Peter Riederer ◽  
Manfred Gerlach ◽  
...  
Keyword(s):  
Substantia Nigra ◽  
Brain Tissue ◽  
Protein Identification ◽  
Laser Microdissection ◽  
Protein Detection ◽  
Protein Quantification ◽  
Human Brain Tissue ◽  
Limited Sample ◽  
Tissue Samples ◽  
Proteomics Research

Abstract Background: Processing of post-mortem human brain tissue samples like Neuromelanin-containing substantia nigra is a challenging task in proteomics research in the context of neurodegenerative diseases such as Parkinson’s disease, due to the availability of only limited sample amounts. Therefore, a highly efficient lysis and digestion approach is needed to guarantee high reproducibility and a good quantification performance. We conducted a systematic study in order to compare qualitatively and quantitatively six different sample preparation protocols for substantia nigra brain tissue collected by laser microdissection.Results: LC-MS/MS analysis revealed that the combination of formic acid and in-solution digestion with trypsin leads to an enhanced peptide and protein identification as well as a robust and reliable protein quantification with a very good reproducibility.Conclusion: We recommend this protocol for future studies of human substantia nigra tissue as well as NM granules in the context of neurodegenerative disorders like Parkinson syndrome.

Shear Properties of Human Brain Tissue

1997 ◽  
Vol 119 (4) ◽  
pp. 423-432 ◽  
Author(s):  
B. R. Donnelly ◽  
J. Medige
Keyword(s):  
Shear Stress ◽  
Human Brain ◽  
Brain Tissue ◽  
Reaction Force ◽  
Single Pulse ◽  
High Rate ◽  
Human Brain Tissue ◽  
Tissue Samples ◽  
Fresh Cadaver ◽  
Constant Rate

The objective of this study was to determine a relationship between shear stress and strain for human brain tissue by performing transient, single-pulse, high-rate, shear displacement tests. A constant velocity, parallel plate shear test device was designed and fabricated. This equipment generated constant rate shear strains in cylindrical tissue samples mounted between the shear plates. The transverse reaction force at the upper end of the sample was measured during the event with a sensitive quartz piezoelectric force transducer, thus obtaining the force associated with the displacement versus time ramp. Shear tests were performed on 125 tissue samples taken from twelve fresh cadaver brain specimens. The average true shear stress and finite strain were calculated. A nonlinear, viscoelastic, standard solid model was fit to the constant rate test data and the material constants were determined.

scholarly journals Hybridization-based In Situ Sequencing (HybISS): spatial transcriptomic detection in human and mouse brain tissue

2020 ◽  
Author(s):  
Daniel Gyllborg ◽  
Christoffer Mattsson Langseth ◽  
Xiaoyan Qian ◽  
Sergio Marco Salas ◽  
Markus M. Hilscher ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Rolling Circle Amplification ◽  
Probe Design ◽  
Sequencing Data ◽  
Human Brain Tissue ◽  
Tissue Samples ◽  
Rolling Circle ◽  
Spatially Resolved ◽  
Spatial Detection

Visualization of the transcriptome in situ has proven to be a valuable tool in exploring single-cell RNA-sequencing data, providing an additional dimension to investigate spatial cell typing and cell atlases, disease architecture or even data driven discoveries. The field of spatially resolved transcriptomic technologies is emerging as a vital tool to profile gene-expression, continuously pushing current methods to accommodate larger gene panels and larger areas without compromising throughput efficiency. Here, we describe a new version of the in situ sequencing (ISS) method based on padlock probes and rolling circle amplification. Modifications in probe design allows for a new barcoding system via sequence-by-hybridization chemistry for improved spatial detection of RNA transcripts. Due to the amplification of probes, amplicons can be visualized with standard epifluorescence microscopes with high-throughput efficiency and the new sequencing chemistry removes limitations bound by sequence-by-ligation chemistry of ISS. Here we present hybridization-based in situ sequencing (HybISS) that allows for increased flexibility and multiplexing, increased signal-to-noise, all without compromising throughput efficiency of imaging large fields of view. Moreover, the current protocol is demonstrated to work on human brain tissue samples, a source that has proven to be difficult to work with image-based spatial analysis techniques. Overall, HybISS technology works as a target amplification detection method for improved spatial transcriptomic visualization, and importantly, with an ease of implementation.

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 RBIO-06. IMPLEMENTATION OF HUMAN INDUCED PLURIPOTENT STEM CELL DERIVED CEREBRAL ORGANOIDS TO MODEL NORMAL TISSUE RADIORESPONSE

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii193-ii193
Author(s):  
Lawrence Bronk ◽  
Sanjay Singh ◽  
Riya Thomas ◽  
Luke Parkitny ◽  
Mirjana Maletic-Savatic ◽  
...  
Keyword(s):  
Stem Cell ◽  
Human Brain ◽  
Brain Tissue ◽  
Human Tissue ◽  
Model Systems ◽  
Human Brain Tissue ◽  
Post Irradiation ◽  
Mature Neurons ◽  
Induced Pluripotent ◽  
Effects Of Radiation

Abstract Treatment-related sequelae following cranial irradiation have life changing impacts for patients and their caregivers. Characterization of the basic response of human brain tissue to irradiation has been difficult due to a lack of preclinical models. The direct study of human brain tissue in vitro is becoming possible due to advances in stem cell biology, neuroscience, and tissue engineering with the development of organoids as novel model systems which enable experimentation with human tissue models. We sought to establish a cerebral organoid (CO) model to study the radioresponse of normal human brain tissue. COs were grown using human induced pluripotent stem cells and a modified Lancaster protocol. Compositional analysis during development of the COs showed expected populations of neurons and glia. We confirmed a population of microglia-like cells within the model positive for the makers Iba1 and CD68. After 2-months of maturation, COs were irradiated to 0, 10, and 20 Gy using a Shepard Mark-II Cs-137 irradiator and returned to culture. Subsets of COs were prepared for immunostaining at 30- and 70-days post-irradiation. To examine the effect of irradiation on the neural stem cell (NSC) population, sections were stained for SOX2 and Ki-67 expression denoting NSCs and proliferation respectively. Slides were imaged and scored using the CellProfiler software package. The percentage of proliferating NSCs 30-days post-irradiation was found to be significantly reduced for irradiated COs (5.7% (P=0.007) and 3.4% (P=0.001) for 10 and 20 Gy respectively) compared to control (12.7%). The reduction in the proliferating NSC population subsequently translated to a reduced population of NeuN-labeled mature neurons 70 days post-irradiation. The loss of proliferating NSCs and subsequent reduction in mature neurons demonstrates the long-term effects of radiation. Our initial results indicate COs will be a valuable model to study the effects of radiation therapy on normal and diseased human tissue.

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