Cargando…

Liposomal clodronate selectively eliminates microglia from primary astrocyte cultures

BACKGROUND: There is increasing interest in astrocyte biology because astrocytes have been demonstrated to play prominent roles in physiological and pathological conditions of the central nervous system, including neuroinflammation. To understand astrocyte biology, primary astrocyte cultures are mos...

Descripción completa

Detalles Bibliográficos
Autores principales: Kumamaru, Hiromi, Saiwai, Hirokazu, Kobayakawa, Kazu, Kubota, Kensuke, van Rooijen, Nico, Inoue, Kazuhide, Iwamoto, Yukihide, Okada, Seiji
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3419615/
https://www.ncbi.nlm.nih.gov/pubmed/22651847
http://dx.doi.org/10.1186/1742-2094-9-116
_version_ 1782240743477215232
author Kumamaru, Hiromi
Saiwai, Hirokazu
Kobayakawa, Kazu
Kubota, Kensuke
van Rooijen, Nico
Inoue, Kazuhide
Iwamoto, Yukihide
Okada, Seiji
author_facet Kumamaru, Hiromi
Saiwai, Hirokazu
Kobayakawa, Kazu
Kubota, Kensuke
van Rooijen, Nico
Inoue, Kazuhide
Iwamoto, Yukihide
Okada, Seiji
author_sort Kumamaru, Hiromi
collection PubMed
description BACKGROUND: There is increasing interest in astrocyte biology because astrocytes have been demonstrated to play prominent roles in physiological and pathological conditions of the central nervous system, including neuroinflammation. To understand astrocyte biology, primary astrocyte cultures are most commonly used because of the direct accessibility of astrocytes in this system. However, this advantage can be hindered by microglial contamination. Although several authors have warned regarding microglial contamination in this system, complete microglial elimination has never been achieved. METHODS: The number and proliferative potential of contaminating microglia in primary astrocyte cultures were quantitatively assessed by immunocytologic and flow cytometric analyses. To examine the utility of clodronate for microglial elimination, primary astrocyte cultures or MG-5 cells were exposed to liposomal or free clodronate, and then immunocytologic, flow cytometric, and gene expression analyses were performed. The gene expression profiles of microglia-eliminated and microglia-contaminated cultures were compared after interleukin-6 (IL-6) stimulation. RESULTS: The percentage of contaminating microglia exceeded 15% and continued to increase because of their high proliferative activity in conventional primary astrocyte cultures. These contaminating microglia were selectively eliminated low concentration of liposomal clodronate. Although primary microglia and MG-5 cells were killed by both liposomal and free clodronate, free clodronate significantly affected the viability of astrocytes. In contrast, liposomal clodronate selectively eliminated microglia without affecting the viability, proliferation or activation of astrocytes. The efficacy of liposomal clodronate was much higher than that of previously reported methods used for decreasing microglial contamination. Furthermore, we observed rapid tumor necrosis factor-α and IL-1b gene induction in conventional primary astrocyte cultures after IL-6 stimulation, which was due to the activation of the Janus kinase/signal transducer and activator of the transcription pathway in contaminating microglia. CONCLUSIONS: Because contaminating microglia could result in erroneous data regarding the pro-inflammatory properties of astrocytes, astrocyte biology should be studied in the absence of microglial contamination. Our simple method will be widely applicable to experimental studies of astrocyte biology and provide clues for understanding the role of astrocytes in neural development, function and disease.
format Online
Article
Text
id pubmed-3419615
institution National Center for Biotechnology Information
language English
publishDate 2012
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-34196152012-08-16 Liposomal clodronate selectively eliminates microglia from primary astrocyte cultures Kumamaru, Hiromi Saiwai, Hirokazu Kobayakawa, Kazu Kubota, Kensuke van Rooijen, Nico Inoue, Kazuhide Iwamoto, Yukihide Okada, Seiji J Neuroinflammation Research BACKGROUND: There is increasing interest in astrocyte biology because astrocytes have been demonstrated to play prominent roles in physiological and pathological conditions of the central nervous system, including neuroinflammation. To understand astrocyte biology, primary astrocyte cultures are most commonly used because of the direct accessibility of astrocytes in this system. However, this advantage can be hindered by microglial contamination. Although several authors have warned regarding microglial contamination in this system, complete microglial elimination has never been achieved. METHODS: The number and proliferative potential of contaminating microglia in primary astrocyte cultures were quantitatively assessed by immunocytologic and flow cytometric analyses. To examine the utility of clodronate for microglial elimination, primary astrocyte cultures or MG-5 cells were exposed to liposomal or free clodronate, and then immunocytologic, flow cytometric, and gene expression analyses were performed. The gene expression profiles of microglia-eliminated and microglia-contaminated cultures were compared after interleukin-6 (IL-6) stimulation. RESULTS: The percentage of contaminating microglia exceeded 15% and continued to increase because of their high proliferative activity in conventional primary astrocyte cultures. These contaminating microglia were selectively eliminated low concentration of liposomal clodronate. Although primary microglia and MG-5 cells were killed by both liposomal and free clodronate, free clodronate significantly affected the viability of astrocytes. In contrast, liposomal clodronate selectively eliminated microglia without affecting the viability, proliferation or activation of astrocytes. The efficacy of liposomal clodronate was much higher than that of previously reported methods used for decreasing microglial contamination. Furthermore, we observed rapid tumor necrosis factor-α and IL-1b gene induction in conventional primary astrocyte cultures after IL-6 stimulation, which was due to the activation of the Janus kinase/signal transducer and activator of the transcription pathway in contaminating microglia. CONCLUSIONS: Because contaminating microglia could result in erroneous data regarding the pro-inflammatory properties of astrocytes, astrocyte biology should be studied in the absence of microglial contamination. Our simple method will be widely applicable to experimental studies of astrocyte biology and provide clues for understanding the role of astrocytes in neural development, function and disease. BioMed Central 2012-05-31 /pmc/articles/PMC3419615/ /pubmed/22651847 http://dx.doi.org/10.1186/1742-2094-9-116 Text en Copyright ©2012 Kumamaru et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research
Kumamaru, Hiromi
Saiwai, Hirokazu
Kobayakawa, Kazu
Kubota, Kensuke
van Rooijen, Nico
Inoue, Kazuhide
Iwamoto, Yukihide
Okada, Seiji
Liposomal clodronate selectively eliminates microglia from primary astrocyte cultures
title Liposomal clodronate selectively eliminates microglia from primary astrocyte cultures
title_full Liposomal clodronate selectively eliminates microglia from primary astrocyte cultures
title_fullStr Liposomal clodronate selectively eliminates microglia from primary astrocyte cultures
title_full_unstemmed Liposomal clodronate selectively eliminates microglia from primary astrocyte cultures
title_short Liposomal clodronate selectively eliminates microglia from primary astrocyte cultures
title_sort liposomal clodronate selectively eliminates microglia from primary astrocyte cultures
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3419615/
https://www.ncbi.nlm.nih.gov/pubmed/22651847
http://dx.doi.org/10.1186/1742-2094-9-116
work_keys_str_mv AT kumamaruhiromi liposomalclodronateselectivelyeliminatesmicrogliafromprimaryastrocytecultures
AT saiwaihirokazu liposomalclodronateselectivelyeliminatesmicrogliafromprimaryastrocytecultures
AT kobayakawakazu liposomalclodronateselectivelyeliminatesmicrogliafromprimaryastrocytecultures
AT kubotakensuke liposomalclodronateselectivelyeliminatesmicrogliafromprimaryastrocytecultures
AT vanrooijennico liposomalclodronateselectivelyeliminatesmicrogliafromprimaryastrocytecultures
AT inouekazuhide liposomalclodronateselectivelyeliminatesmicrogliafromprimaryastrocytecultures
AT iwamotoyukihide liposomalclodronateselectivelyeliminatesmicrogliafromprimaryastrocytecultures
AT okadaseiji liposomalclodronateselectivelyeliminatesmicrogliafromprimaryastrocytecultures