Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 May 24;11(11):1723.
doi: 10.3390/cells11111723.

Different Methods for Evaluating Microglial Activation Using Anti-Ionized Calcium-Binding Adaptor Protein-1 Immunohistochemistry in the Cuprizone Model

Mariela Wittekindt  1 , Hannes Kaddatz  1 , Sarah Joost  1 , Anna Staffeld  1 , Yamen Bitar  1 , Markus Kipp  1 , Linda Frintrop  1
Affiliations

Different Methods for Evaluating Microglial Activation Using Anti-Ionized Calcium-Binding Adaptor Protein-1 Immunohistochemistry in the Cuprizone Model

Mariela Wittekindt et al. Cells. .

Abstract

Microglia play an important role in the pathology of various central nervous system disorders, including multiple sclerosis (MS). While different methods exist to evaluate the extent of microglia activation, comparative studies investigating the sensitivity of these methods are missing for most models. In this study, we systematically evaluated which of the three commonly used histological methods (id est, quantification of microglia density, densitometrically evaluated staining intensity, or cellular morphology based on the determination of a ramification index, all measured in anti-ionized calcium-binding adaptor protein-1 (IBA1) immunohistochemical stains) is the most sensitive method to detect subtle changes in the microglia activation status in the context of MS. To this end, we used the toxin-induced cuprizone model which allows the experimental induction of a highly reproducible demyelination in several central nervous system regions, paralleled by early microglia activation. In this study, we showed that after 3 weeks of cuprizone intoxication, all methods reveal a significant microglia activation in the white matter corpus callosum. In contrast, in the affected neocortical grey matter, the evaluation of anti-IBA1 cell morphologies was the most sensitive method to detect subtle changes of microglial activation. The results of this study provide a useful guide for future immunohistochemical evaluations in the cuprizone and other neurodegenerative models.

Keywords: IBA1; cuprizone model; microglia; multiple sclerosis.

PubMed Disclaimer

Conflict of interest statement

The authors have no relevant financial or non-financial interests to disclose.

Figures

Figure 1
Figure 1
Regions of interest at brain level region 315 (Bregma −2.3; Nissl staining). (A) The medial corpus callosum (mcc) was defined as the corpus callosum area between a perpendicular line dropped from the interhemispheric fissure and a perpendicular line dropped from the peak of the cingulum (cg). (B) The medial retrosplenial cortex was defined as the cortical areas above the mcc with a perpendicular line dropped from the brain surface and the peak of the cingulum as lateral boundary. (C) The entire hippocampus was defined as a third region of interest (ROI). (D) The ramification index was calculated from the ratio of the cell area (Ac) and the projection area (Ap). (E) Ap is the area of a polygonal object that is defined by the cells’ most prominent projections. A resting microglia cell displays long and thin ramified processes with a relatively high Ap (encircled in yellow) and a relatively small Ac (encircled in blue). (F) An activated microglia cell is characterized by a hypertrophic cell body with retracted processes resulting in a ramification index value close to 1.
Figure 2
Figure 2
Microglia activation in the medial corpus callosum of cuprizone-intoxicated mice (n = 5 per group). C57BL/6 mice were intoxicated with cuprizone for 1, 3, or 5 weeks (1 w Cup; 3 w Cup; 5 w Cup). Controls (Ctrl) were fed with normal chow throughout the experiment. (A) Representative anti-IBA1 staining of Ctrl, 1, 3, and 5 w Cup mice. Insets depict representative IBA1+ cells in the corpus callosum (panels beneath). (B) IBA1+ cell numbers were manually counted by two independent evaluators (MW and YB) blinded to the treatment groups. The microglia cell numbers gradually increased during the course of cuprizone intoxication. (C) Anti-IBA1+ staining intensities evaluated by densitometrical analysis also increased during the course of cuprizone intoxication. Densitometrically measured IBA1+ staining intensities were increased in 3 and 5 w Cup mice compared to Ctrl mice. (D) Ramification indices of randomly selected microglia cells in the corpus callosum were determined. After 3 and 5 w of cuprizone intoxication, the ramification indices were increased compared to Ctrl mice. Kruskal–Wallis test followed by Dunn’s multiple comparisons test were used to test for significant differences between cuprizone-intoxicated and Ctrl groups. Ac: cell area. Ap: projection area. ** p ≤ 0.01, *** p ≤ 0.001.
Figure 3
Figure 3
Microglia activation in the medial retrosplenial cortex of cuprizone-intoxicated mice (n = 5 per group). (A) Representative anti-IBA1 staining of control (Ctrl), 1, 3, and 5 w Cup mice. Insets depict representative IBA1+ cells in the cortex. (B) IBA1+ cell numbers were manually counted by two independent evaluators (MW and AS) blinded to the treatment groups. The densities of IBA1+ microglia cells were significantly increased after 5 w of cuprizone intoxication. (C) Densitometrically measured IBA1+ staining intensities were increased in 5 w Cup mice compared to Ctrl mice. (D) The ramification indices of microglia cells in the retrosplenial cortex gradually increased during the course of cuprizone intoxication. Kruskal–Wallis test followed by Dunn’s multiple comparisons test were used to test for significant differences between Ctrl and all cuprizone-intoxicated groups. Ac: cell area. Ap: projection area. * p ≤ 0.05, ** p ≤ 0.01.
Figure 4
Figure 4
Microglia activation in the hippocampus of cuprizone-intoxicated mice (n = 5 per group). (A) Representative anti-IBA1 staining of control (Ctrl), 1, 3, and 5 w Cup mice. Insets depict representative IBA1+ cells in the hippocampus. (B) IBA1+ cell numbers were manually counted by two independent evaluators (MW and YB) blinded to the treatment groups. IBA1+ cell densities were significantly increased after 3 and 5 w of cuprizone intoxication. (C) Densitometrically measured IBA1+ staining intensities demonstrated a significant increase during the course of cuprizone intoxication. (D) The ramification indices of microglia in the hippocampus were increased after 3 w of cuprizone intoxication. Kruskal–Wallis test followed by Dunn’s multiple comparisons test were used to test for significant differences between Ctrl and all cuprizone-intoxicated groups. Ac: cell area. Ap: projection area. * p ≤ 0.05, ** p ≤ 0.01.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ginhoux F., Greter M., Leboeuf M., Nandi S., See P., Gokhan S., Mehler M.F., Conway S.J., Ng L.G., Stanley E.R., et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science. 2010;330:841–845. doi: 10.1126/science.1194637. - DOI - PMC - PubMed
    1. Menassa D.A., Gomez-Nicola D. Microglial Dynamics During Human Brain Development. Front. Immunol. 2018;9:1014. doi: 10.3389/fimmu.2018.01014. - DOI - PMC - PubMed
    1. Safaiyan S., Kannaiyan N., Snaidero N., Brioschi S., Biber K., Yona S., Edinger A.L., Jung S., Rossner M.J., Simons M. Age-related myelin degradation burdens the clearance function of microglia during aging. Nat. Neurosci. 2016;19:995–998. doi: 10.1038/nn.4325. - DOI - PMC - PubMed
    1. Wake H., Moorhouse A.J., Jinno S., Kohsaka S., Nabekura J. Resting microglia directly monitor the functional state of synapses in vivo and determine the fate of ischemic terminals. J. Neurosci. 2009;29:3974–3980. doi: 10.1523/JNEUROSCI.4363-08.2009. - DOI - PMC - PubMed
    1. Akiyoshi R., Wake H., Kato D., Horiuchi H., Ono R., Ikegami A., Haruwaka K., Omori T., Tachibana Y., Moorhouse A.J., et al. Microglia Enhance Synapse Activity to Promote Local Network Synchronization. eNeuro. 2018;5 doi: 10.1523/ENEURO.0088-18.2018. - DOI - PMC - PubMed

Publication types

Grants and funding

This work was supported by the Deutsche Forschungsgemeinschaft (KI 1469/8-1; MK).