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Cited by 216Traumatic Brain Injury and Neurovascular DisturbancesNeuroinflammation and Neurodegeneration MechanismsTraumatic Brain Injury Research

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Temporal Changes in Cell Marker Expression and Cellular Infiltration in a Controlled Cortical Impact Model in Adult Male C57BL/6 Mice

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Jin, Xuemei, Ishii, Hiroshi, Bai, Zhongbin, Itokazu, Takahide, Yamashita, Toshihide·2012·PLoS ONE
DOI 10.1371/journal.pone.0041892PMC PMC3404031
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Abstract

BACKGROUND: Traumatic injury to the central nervous system (CNS) triggers a robust inflammatory response that leads to axonal damage and secondary degeneration of spared tissue. In contrast, some immune responses have neuroprotective effects. However, detailed information regarding the dynamics of immune responses after traumatic CNS injury is still unavailable. METHODS: In the present study, changes in the immune cells present in the injured brain, spleen, and cervical lymph nodes (CLNs), which are draining lymphatic organs from the CNS, were analyzed after controlled cortical impact (CCI) by flow cytometry and immunohistochemistry. RESULTS: The number of neutrophils and macrophages that infiltrated the injured brain immediately increased 1 d post-injury and declined rapidly thereafter. In the injured brain, resident microglia showed a bimodal increase during the first week and in the chronic phase (≥3 weeks) after injury. Increase in the Iba-1(+) microglia/macrophages was observed around the injured site. Morphologic analysis showed that Iba-1(+) cells were round at 1 week, whereas those at 3 weeks were more ramified. Furthermore, CD86(+)/CD11b(+) M1-like microglia increased at 4 weeks after CCI, whereas CD206(+)/CD11b(+) M2-like microglia increased at 1 week. These results suggest that different subsets of microglia increased in the acute and chronic phases after CCI. Dendritic cells and T cells increased transiently within 1 week in the injured brain. In the CLNs and the spleen, T cells showed dynamic changes after CCI. In particular, the alteration in the number of T cells in the CLNs showed a similar pattern, with a 1-week delay, to that of microglia in the injured brain. CONCLUSION: The data from this study provide useful information on the dynamics of immune cells in CNS injuries.

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Figures

Figure 1 Analysis of neutrophils, macrophages, and microglia in the injured brain. A: Dot plots of isolated immune cells in the brain, gated for live cell analysis. B: Representative cytometry data for neutrophils (CD45+/Gr-1+ cells) in the injured brain at the indicated days after CCI. C: Representative cytometry data for macrophages (CD45high/CD11b+ cells) and microglia (CD45low/CD11b+ cells) in the injured brain at the indicated days after CCI. D: Graph illustrating quantitative data for accumulated neutrophils, macrophages, and microglia in the injured brain up to 28 d after CCI. All the values are presented in terms of mean ± standard error of mean (n = 3–6). Macrophages/neutrophils: ** p<0.01 at 1, 3, and 14 dpi compared with no injury; microglia: ** p<0.01 at 14 and 28 dpi compared with no injury, * p<0.05 at 14 dpi compared with 1 dpi.
Figure 1 Analysis of neutrophils, macrophages, and microglia in the injured brain. A: Dot plots of isolated immune cells in the brain, gated for live cell analysis. B: Representative cytometry data for neutrophils (CD45+/Gr-1+ cells) in the injured brain at the indicated days after CCI. C: Representative cytometry data for macrophages (CD45high/CD11b+ cells) and microglia (CD45low/CD11b+ cells) in the injured brain at the indicated days after CCI. D: Graph illustrating quantitative data for accumulated neutrophils, macrophages, and microglia in the injured brain up to 28 d after CCI. All the values are presented in terms of mean ± standard error of mean (n = 3–6). Macrophages/neutrophils: ** p<0.01 at 1, 3, and 14 dpi compared with no injury; microglia: ** p<0.01 at 14 and 28 dpi compared with no injury, * p<0.05 at 14 dpi compared with 1 dpi.CC BY · PubMed Central
Figure 2 Analysis of T cells and dendritic cells in the injured brain. A: Dot plots of isolated immune cells in the brain, gated for live cell analysis. B: Representative cytometry data for T cells (CD45+/CD3+ cells) in the injured brain at the indicated days after CCI. C: Representative cytometry data for CD45+/CD11c+ dendritic cells (DCs) in the injured brain after CCI. D: Graph illustrating quantitative data for accumulated T cells and DCs in the injured brain after CCI. n = 3–6 for each experiment. T cells: ** p<0.01 at 1, 3, and 7 dpi compared with no injury, * p<0.05 at 28 dpi compared with no injury; DCs: ** p<0.01 at 3 and 7 dpi compared with no injury.
Figure 2 Analysis of T cells and dendritic cells in the injured brain. A: Dot plots of isolated immune cells in the brain, gated for live cell analysis. B: Representative cytometry data for T cells (CD45+/CD3+ cells) in the injured brain at the indicated days after CCI. C: Representative cytometry data for CD45+/CD11c+ dendritic cells (DCs) in the injured brain after CCI. D: Graph illustrating quantitative data for accumulated T cells and DCs in the injured brain after CCI. n = 3–6 for each experiment. T cells: ** p<0.01 at 1, 3, and 7 dpi compared with no injury, * p<0.05 at 28 dpi compared with no injury; DCs: ** p<0.01 at 3 and 7 dpi compared with no injury.CC BY · PubMed Central
Figure 3 T cells and DCs in the cervical lymph nodes (CLNs) after CCI. A: Representative cytometry data for T cells in the CLNs at the indicated days after CCI. B: Graph illustrating quantitative data for T cells in the CLNs after CCI. C: Representative cytometry data for DCs in the CLNs at the indicated days after CCI. D: Graph illustrating quantitative data for DCs in the CLNs after CCI. (B, D) n = 3–4 for each experiment. T cells: * p<0.05 at 28 dpi compared with 1 dpi; DCs: ** p<0.01 at 3 dpi compared with no injury, * p<0.05 at 7, 14, and 21 dpi compared with no injury.
Figure 3 T cells and DCs in the cervical lymph nodes (CLNs) after CCI. A: Representative cytometry data for T cells in the CLNs at the indicated days after CCI. B: Graph illustrating quantitative data for T cells in the CLNs after CCI. C: Representative cytometry data for DCs in the CLNs at the indicated days after CCI. D: Graph illustrating quantitative data for DCs in the CLNs after CCI. (B, D) n = 3–4 for each experiment. T cells: * p<0.05 at 28 dpi compared with 1 dpi; DCs: ** p<0.01 at 3 dpi compared with no injury, * p<0.05 at 7, 14, and 21 dpi compared with no injury.CC BY · PubMed Central
Figure 4 Neutrophils and macrophages in the CLNs after CCI. A: Dot plots of immune cells in the CLNs for live cell analysis. B: Representative cytometry data for neutrophils in the CLNs at the indicated days after CCI. C: Representative cytometry data for CD45+/CD11b+ macrophages in the CLNs at the indicated days after CCI. D: Graph illustrating quantitative data for neutrophils and macrophages in the CLNs after CCI. n = 3–4 for each experiment. Neutrophils: ** p<0.01 at 1 and 14 dpi compared with no injury; macrophages: ** p<0.01 at 7, 14, and 21 dpi compared with 1 dpi.
Figure 4 Neutrophils and macrophages in the CLNs after CCI. A: Dot plots of immune cells in the CLNs for live cell analysis. B: Representative cytometry data for neutrophils in the CLNs at the indicated days after CCI. C: Representative cytometry data for CD45+/CD11b+ macrophages in the CLNs at the indicated days after CCI. D: Graph illustrating quantitative data for neutrophils and macrophages in the CLNs after CCI. n = 3–4 for each experiment. Neutrophils: ** p<0.01 at 1 and 14 dpi compared with no injury; macrophages: ** p<0.01 at 7, 14, and 21 dpi compared with 1 dpi.CC BY · PubMed Central
Figure 5 T cells and DCs in the spleen after CCI. A: Representative cytometry data for CD45+/CD3+ T cells in the spleen at the indicated days after CCI. B: Graph illustrating quantitative data for T cells in the spleen after CCI. C: Representative cytometry data for DCs in the spleen at the indicated days after CCI. D: Graph illustrating quantitative data for DCs in the spleen after CCI. (B, D) n = 3–7 for each experiment. T cells: * p<0.05 at 14 and 21 dpi compared with no injury, * p<0.05 at 3 dpi compared with 1 dpi, ** p<0.01 at 14 and 21 dpi compared with 1 dpi; DCs: ** p<0.01 at 7 dpi compared with no injury, * p<0.05 at 28 dpi compared with 7 dpi.
Figure 5 T cells and DCs in the spleen after CCI. A: Representative cytometry data for CD45+/CD3+ T cells in the spleen at the indicated days after CCI. B: Graph illustrating quantitative data for T cells in the spleen after CCI. C: Representative cytometry data for DCs in the spleen at the indicated days after CCI. D: Graph illustrating quantitative data for DCs in the spleen after CCI. (B, D) n = 3–7 for each experiment. T cells: * p<0.05 at 14 and 21 dpi compared with no injury, * p<0.05 at 3 dpi compared with 1 dpi, ** p<0.01 at 14 and 21 dpi compared with 1 dpi; DCs: ** p<0.01 at 7 dpi compared with no injury, * p<0.05 at 28 dpi compared with 7 dpi.CC BY · PubMed Central
Figure 6 Neutrophils and macrophages in the spleen after CCI. A: Dot plots of immune cells in the spleen for live cell analysis. B: Representative cytometry data for neutrophils in the spleen at the indicated days after CCI. C: Representative cytometry data for macrophages in the spleen at the indicated days after CCI. D: Graph illustrating quantitative data for neutrophils and macrophages in the CLNs after CCI. n = 3–5 for each experiment. Neutrophils: ** p<0.01 at 28 dpi compared with no injury.
Figure 6 Neutrophils and macrophages in the spleen after CCI. A: Dot plots of immune cells in the spleen for live cell analysis. B: Representative cytometry data for neutrophils in the spleen at the indicated days after CCI. C: Representative cytometry data for macrophages in the spleen at the indicated days after CCI. D: Graph illustrating quantitative data for neutrophils and macrophages in the CLNs after CCI. n = 3–5 for each experiment. Neutrophils: ** p<0.01 at 28 dpi compared with no injury.CC BY · PubMed Central
Figure 7 Bimodal increase in the Iba-1-positive cells in the brain after CCI. (A–E) Brain sections immunostained for Iba-1 at the indicated days after CCI. The red signals show Iba-1-positive microglia/macrophages in sham-operated mouse brains (A) and in brains after CCI at 1 dpi (B), 7 dpi (C), 14 dpi (D), and 21 dpi (E). The small image on the right lower side of each large image is a magnified view of the square indicated. Scale bars: large image, 600 µm; small image 100 µm. (F) The graph shows the number of microglia/macrophages at the indicated days after CCI. Microglia/macrophages observed around the injured side significantly increased at 7 dpi and 21 dpi. Sham, sham-operated mice. n = 4 for each group. ** p<0.01 at 7 and 21 dpi compared with sham controls.
Figure 7 Bimodal increase in the Iba-1-positive cells in the brain after CCI. (A–E) Brain sections immunostained for Iba-1 at the indicated days after CCI. The red signals show Iba-1-positive microglia/macrophages in sham-operated mouse brains (A) and in brains after CCI at 1 dpi (B), 7 dpi (C), 14 dpi (D), and 21 dpi (E). The small image on the right lower side of each large image is a magnified view of the square indicated. Scale bars: large image, 600 µm; small image 100 µm. (F) The graph shows the number of microglia/macrophages at the indicated days after CCI. Microglia/macrophages observed around the injured side significantly increased at 7 dpi and 21 dpi. Sham, sham-operated mice. n = 4 for each group. ** p<0.01 at 7 and 21 dpi compared with sham controls.CC BY · PubMed Central
Figure 8 Changes in specific subsets of microglia in the injured brain. A: Representative cytometry data showing the purity of microglia isolated. Of the isolated cells, 91% were microglia. B: Representative cytometry data for CD86+/CD11b+ M1-like microglia in the brain at the indicated days after CCI. C: Representative cytometry data for CD206+/CD11b+ M2-like microglia in the brain at the indicated days after CCI. D: Graph illustrating quantitative data for CD86+/CD11b+ cells in the brain after CCI. CD86+/CD11b+ cells were higher in number than in the intact brain at 28 dpi. E: Graph illustrating quantitative data for CD206+/CD11b+ cells in the brain after CCI. CD206+/CD11b+ cells were higher in number at 7 dpi than in the intact brain, and decreased thereafter until 28 dpi. n = 3–5 for each group. * p<0.05 compared with no injury.
Figure 8 Changes in specific subsets of microglia in the injured brain. A: Representative cytometry data showing the purity of microglia isolated. Of the isolated cells, 91% were microglia. B: Representative cytometry data for CD86+/CD11b+ M1-like microglia in the brain at the indicated days after CCI. C: Representative cytometry data for CD206+/CD11b+ M2-like microglia in the brain at the indicated days after CCI. D: Graph illustrating quantitative data for CD86+/CD11b+ cells in the brain after CCI. CD86+/CD11b+ cells were higher in number than in the intact brain at 28 dpi. E: Graph illustrating quantitative data for CD206+/CD11b+ cells in the brain after CCI. CD206+/CD11b+ cells were higher in number at 7 dpi than in the intact brain, and decreased thereafter until 28 dpi. n = 3–5 for each group. * p<0.05 compared with no injury.CC BY · PubMed Central

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Metadata

Authors
Jin, X., Ishii, H., Bai, Z., Itokazu, T., & Yamashita, T.
Affiliations
  • Japan Science and Technology Agency (JP)
  • Yunnan Agricultural University (CN)
Corresponding author
Yamashita, Toshihide
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2012-07-24
Volume
7
Issue
7
Pages
e41892
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0041892
PMC
PMC3404031
OpenAlex
W2025828188
Cited by
211
References
48
Citations by year
2026: 7 · 2025: 9 · 2024: 13 · 2023: 13 · 2022: 20 · 2021: 22 · 2020: 23 · 2019: 20 · 2018: 13 · 2017: 19 · 2016: 9 · 2015: 17 · 2014: 10 · 2013: 14 · 2012: 1
FWCI
8.78
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Japan Science and Technology Agency (CREST)
  • Core Research for Evolutional Science and Technology
Topics
  • Health Sciences › Medicine › Clinical Neurology › Traumatic Brain Injury and Neurovascular Disturbances
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Clinical Neurology › Traumatic Brain Injury Research
Keywords
controlled cortical impact, traumatic brain injury, microglia, macrophages, M1/M2 microglial polarization, dendritic cells, T cells, neutrophils, cervical lymph nodes, spleen, immune cell infiltration, neuroinflammation, Iba1, CD86, CD206
MeSH terms
Aging, Animals, Calcium-Binding Proteins, Cerebral Cortex, Dendritic Cells, Disease Models, Animal, Immunohistochemistry, Lymph Nodes, Macrophages, Male, Mice, Inbred C57BL, Microfilament Proteins, Neutrophils, Spleen, T-Lymphocytes, Time Factors, Biomarkers, Microglia, Mice

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