Simultaneous flow cytometric characterization of multiple cell types and metabolic states in the rat brain after repeated mild traumatic brain injury.

Animals Brain Brain Concussion Flow Cytometry Microglia Neurons Rats
Flow cytometry Neurotrauma Oxidative stress Rat brain Repeated mild traumatic brain injury

Journal

Journal of neuroscience methods
ISSN: 1872-678X
Titre abrégé: J Neurosci Methods
Pays: Netherlands
ID NLM: 7905558

Informations de publication

Date de publication:
15 07 2021
Historique:
received: 20 02 2021
revised: 11 05 2021
accepted: 11 05 2021
pubmed: 19 5 2021
medline: 1 7 2021
entrez: 18 5 2021
Statut: ppublish

Résumé

Cellular responses at the sub-acute phase of mild traumatic brain injury (mTBI), and their contribution to ongoing damage, are unclear, complex and require simultaneous assessment of multiple cells to elucidate. An 11-colour flow cytometry method for analysing brain cells was evaluated in a weight-drop rat model of repeated mTBI. Animals received sham, one, two or three mTBI delivered at 24 h intervals (n = 6/group). Cerebrum homogenates were prepared 11 days after first mTBI, in two cohorts of n = 3/group to enable same-day staining of fresh tissue. Percentages of neurons, astrocytes, microglia, mature oligodendrocytes and NeuN + CC1+ cells, neutrophils, macrophages and non-myeloid leukocytes, and their immunoreactivity for cell damage indicators (inducible nitric oxide synthase; iNOS, proliferating cell nuclear antigen; PCNA, 8-Oxo-2'-deoxyguanosine; 8OHDG and 4-hydroxynonenal; HNE), were assessed. Median fluorescence intensity (MFI) of iNOS in activated microglia increased following two, but not one or three, mTBI (p = 0.04). However, there were differences between processing cohorts in terms of percentages and MFI of some PCNA+, iNOS+, 8OHDG + and HNE + cell populations. Previous applications of flow cytometry for rat brain analysis were typically limited to three or four markers. This method uses 11 markers to identify nine cell populations and evaluate their immunoreactivity to four metabolic indicators of cell damage. Flow cytometry can be useful for discerning injury-related changes in multiple rat brain cells. However, markers sensitive to subtle changes in experimental conditions must be identified in pilot experiments and subsequently analysed in the same tissue-processing cohort.

Sections du résumé

BACKGROUND
Cellular responses at the sub-acute phase of mild traumatic brain injury (mTBI), and their contribution to ongoing damage, are unclear, complex and require simultaneous assessment of multiple cells to elucidate.
NEW METHOD
An 11-colour flow cytometry method for analysing brain cells was evaluated in a weight-drop rat model of repeated mTBI. Animals received sham, one, two or three mTBI delivered at 24 h intervals (n = 6/group). Cerebrum homogenates were prepared 11 days after first mTBI, in two cohorts of n = 3/group to enable same-day staining of fresh tissue. Percentages of neurons, astrocytes, microglia, mature oligodendrocytes and NeuN + CC1+ cells, neutrophils, macrophages and non-myeloid leukocytes, and their immunoreactivity for cell damage indicators (inducible nitric oxide synthase; iNOS, proliferating cell nuclear antigen; PCNA, 8-Oxo-2'-deoxyguanosine; 8OHDG and 4-hydroxynonenal; HNE), were assessed.
RESULTS
Median fluorescence intensity (MFI) of iNOS in activated microglia increased following two, but not one or three, mTBI (p = 0.04). However, there were differences between processing cohorts in terms of percentages and MFI of some PCNA+, iNOS+, 8OHDG + and HNE + cell populations.
COMPARISON WITH EXISTING METHODS
Previous applications of flow cytometry for rat brain analysis were typically limited to three or four markers. This method uses 11 markers to identify nine cell populations and evaluate their immunoreactivity to four metabolic indicators of cell damage.
CONCLUSIONS
Flow cytometry can be useful for discerning injury-related changes in multiple rat brain cells. However, markers sensitive to subtle changes in experimental conditions must be identified in pilot experiments and subsequently analysed in the same tissue-processing cohort.

Identifiants

DOI: 10.1016/j.jneumeth.2021.109223 PMID: 34004202
pubmed: 34004202
pii: S0165-0270(21)00158-8
doi: 10.1016/j.jneumeth.2021.109223
pii:
doi:

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Pagination

109223

Informations de copyright

Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.

Auteurs

Chidozie C Anyaegbu (CC)

Curtin Health Innovation Research Institute, Curtin University, Belmont, Western Australia, Australia. Electronic address: chidozie.anyaegbu@curtin.edu.au.

Yilin Mao (Y)

Curtin Health Innovation Research Institute, Curtin University, Belmont, Western Australia, Australia.

Terry McGonigle (T)

Curtin Health Innovation Research Institute, Curtin University, Belmont, Western Australia, Australia.

Sushmitha Raja (S)

Curtin Health Innovation Research Institute, Curtin University, Belmont, Western Australia, Australia.

Thomas Clarke (T)

Curtin Health Innovation Research Institute, Curtin University, Belmont, Western Australia, Australia.

Anna M B Black (AMB)

Curtin Health Innovation Research Institute, Curtin University, Belmont, Western Australia, Australia.

Tanya Solomon (T)

Curtin Health Innovation Research Institute, Curtin University, Belmont, Western Australia, Australia.

Kathy Fuller (K)

Division of Cancer Biology, School of Biomedical Sciences, The University of Western Australia, 35 Stirling Hwy, Nedlands 6009 Western Australia, Australia.

Melinda Fitzgerald (M)

Curtin Health Innovation Research Institute, Curtin University, Belmont, Western Australia, Australia; Perron Institute for Neurological and Translational Science, Sarich Neuroscience Research Institute Building, 8 Verdun St, Nedlands 6009 Western Australia, Australia.

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Classifications MeSH

questionsmedicales.fr › Eucaryotes › Animaux › Simultaneous flow cytometric characterization...
questionsmedicales.fr › Système nerveux › Système nerveux central › Encéphale › Simultaneous flow cytometric characterization...
questionsmedicales.fr › Plaies et blessures › Plaies non pénétrantes › Traumatismes crâniens fermés › Commotion de l'encéphale › Simultaneous flow cytometric characterization...
questionsmedicales.fr › Techniques d'investigation › Techniques cytologiques › Cytophotométrie › Cytométrie en flux › Simultaneous flow cytometric characterization...
questionsmedicales.fr › Cellules › Névroglie › Microglie › Simultaneous flow cytometric characterization...
questionsmedicales.fr › Cellules › Neurones › Simultaneous flow cytometric characterization...
questionsmedicales.fr › Eucaryotes › Animaux › Chordés › Vertébrés › Mammifères › Eutheria › Rodentia › Muridae › Murinae › Rats › Simultaneous flow cytometric characterization...