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Original Article

A novel method for evaluating microglial

activation using ionized calcium‑binding adaptor

protein‑1 staining: cell body to cell size ratio

Iris Bertha Hovens , Csaba Nyakas , Regien Geertruida Schoemaker 1,3
1
1,2
1 Department of Molecular Neurobiology, University of Groningen, 9747 AG, Groningen, The Netherlands.
2 Department of Brain Physiology Research Unit, Semmelweis University, H-1085, Budapest, Hungary.
3 Department of Cardiology, University of Groningen, 9747 AG, Groningen, The Netherlands.

ABSTRA CT
Aim: The aim was to validate a newly developed methodology of semi‑automatic image analysis to analyze microglial morphology as
marker for microglial activation in ionized calcium‑binding adaptor protein‑1 (IBA‑1) stained brain sections. Methods: The novel method was
compared to currently used analysis methods, visual characterization of activation stage and optical density measurement, in brain sections
of young and aged rats that had undergone surgery or remained naïve. Results: The cell body to cell size ratio of microglia was strongly
correlated to the visual characterization activation stage. In addition, we observed specific surgery and age‑related changes in cell body
size, size of the dendritic processes and cell body to cell size ratio. Conclusion: The novel analysis method provides a sensitive marker
for microglial activation in the rat brain, which is quick and easy to perform and provides additional information about microglial morphology.
Key words: Image analysis, immunohistochemistry, ionized calcium-binding adaptor protein-1, microglia, neuroinflammation

INTRODUCTION activation provides a defense against injury and
infection, chronic or excessive activation is considered
Microglia, the primary immune cells in the central to be detrimental and has been implicated in many
nervous system, are highly plastic cells. Under resting neurodegenerative and psychiatric disorders. [2,5,7]
[1]
conditions, microglia have a ramified morphology,
characterized by small cell bodies and numerous Ionized calcium-binding adaptor protein-1 (IBA-1)
long branching processes. The ramified microglia is a 17-kDa actin-binding protein that is specifically
[2]
continuously scan the environment for danger signals and constitutively expressed in all microglia. [8,9] It is
associated with pathogens or injury. [2,3] When a danger widely employed as an immunohistochemical marker
signal is detected microglia undergo a rapid change for both ramified and activated microglia. [9,10] IBA-1
in morphology and function, a process that has been is shown to have a function in the actin-crosslinking
termed activation. Microglial activation is classically involved in membrane ruffling of microglia. [11] Since
[4]
described as a graded process, in which the processes membrane ruffling is essential for the morphological
retract and thicken, cell body size increases and the changes from quiescent ramified microglia to activated
cell starts excreting cytokines and radical species. [1,4,5] amoeboid microglia, microglial activation is associated
Eventually, the microglia may become amoeboid cells with increased IBA-1 expression. [8,11] Densitometry
capable of phagocytosis. More recently, it has become can, therefore, be utilized for measuring microglial
[5]
apparent that depending on the signal detected and local activation, especially when microglia is strongly
environment microglia can undergo various changes activated and/or microglia number is substantially
in morphology and function. [2,6] Although microglial increased. However, morphological changes of
microglia can occur without significantly affecting the
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optical density (OD) in IBA-1 stained brain areas and
Quick Response Code: densitometry does not provide any specific information
Website:
www.nnjournal.net on the nature of the morphological changes.

DOI: Alternatively, the stadia of microglial activation
10.4103/2347-8659.139719 described by Kreutzberg are often used visually
[4]
to determine the activation stage of all individual

Corresponding Author: Iris Bertha Hovens, Department of Molecular Neurobiology, University of Groningen, Nijenborgh 7,
9747 AG, Groningen, The Netherlands. E‑mail: i.b.hovens@rug.nl

82 Neuroimmunol Neuroinflammation | Volume 1 | Issue 2 | September 2014