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Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ
Published on: June 5, 2018
The in situ morphology of microglia is highly sensitive to the mode of tissue fixation
Bogdan Cătălin1, Laura Stopper2, Tudor-Adrian Bălşeanu3
1Department of Molecular Physiology, CIPMM (Center for Integrative Physiology and Molecular Medicine), Building 48, University of Saarland, Homburg, Germany; Department of Functional Sciences, University of Medicine and Pharmacy of Craiova, Romania.
Abstract:
Microglia are known as the most motile cells in the central nervous system (CNS). It was shown in vivo that they permanently scan their direct microenvironment and react to pathological conditions within minutes. Many studies of brain pathologies use fixed brain tissue to investigate cellular changes. Unfortunately, due to technical reasons, the time span between the induction of the fixation procedure (start of the perfusion) and the finally-fixed tissue lasts several minutes, giving time to microglia to start reacting to the ischemic conditions due to perfusion start. Here, we investigated the microglial changes generated by the fixation itself in TgH(CX3CR1-EGFP) mice with fluorescent labelled microglia using confocal laser scanning microscopy (CLSM) of fixed brain tissue as well as two-photon laser scanning microscopy (2P-LSM) during the perfusion of a living animal. We revealed the impact of fixation and buffer parameters on cell morphology. The largest morphological differences compared to physiological in vivo branch arborization were observed when the directly dissected brain was immersed in paraformaldehyde fixation solution overnight, without prior fixative perfusion of the animal. But even perfusion with a fixative, followed by post-fixation leads to small changes in microglial process length and number and could not be prevented when compared to physiological in vivo microglia morphology acquired using in vivo 2P-LSM. Interestingly, perfusion with different buffers either oxygenated artificial cerebrospinal fluid or phosphate buffered saline prior to perfusion-fixation showed minor microglia changes in arborization and/or number of processes. Fixation methods influence microglia morphology. Therefore, to define microglia activation states immunohistochemical stainings or genetic labelling of the cells have to be included in addition to morphological analysis.
Insights
Brain fixation methods significantly alter microglia morphology, affecting their appearance compared to their in vivo state. Researchers must consider these changes when analyzing microglia activation states in fixed tissues.
Area of Science:
- Neuroscience
- Cell Biology
- Immunology
Background:
- Microglia, the immune cells of the central nervous system (CNS), are highly motile and rapidly respond to stimuli.
- Standard methods for studying brain pathologies involve tissue fixation, but the fixation process itself can induce changes in microglia morphology.
- The time delay between initiating fixation and achieving fully fixed tissue allows microglia to react to the procedure, potentially confounding results.
Purpose of the Study:
- To investigate the morphological changes in microglia induced by different fixation protocols and buffer conditions.
- To compare in vivo microglia morphology with morphology in fixed tissue using advanced microscopy techniques.
- To determine the impact of fixation parameters on microglia morphology and identify potential confounds in pathological studies.
Main Methods:
- Utilized TgH(CX3CR1-EGFP) mice with fluorescently labeled microglia.
- Employed confocal laser scanning microscopy (CLSM) for fixed brain tissue.
- Used two-photon laser scanning microscopy (2P-LSM) for in vivo imaging during perfusion.
- Compared morphology under various fixation conditions, including immersion and perfusion, with different buffer solutions (oxygenated artificial cerebrospinal fluid, phosphate-buffered saline).
Main Results:
- The most significant morphological alterations were observed when brain tissue was directly immersed in paraformaldehyde without prior perfusion.
- Even with perfusion-fixation, microglia process length and number showed minor changes compared to in vivo morphology.
- Different buffer solutions (oxygenated artificial cerebrospinal fluid vs. phosphate-buffered saline) prior to fixation resulted in minimal differences in microglia morphology.
- Fixation methods demonstrably influence microglia morphology, deviating from their physiological in vivo state.
Conclusions:
- Microglia morphology is significantly influenced by the fixation methods used in tissue preparation.
- Standard fixation procedures can introduce artifacts that alter the appearance of microglia, potentially mimicking or masking activation states.
- Accurate assessment of microglia activation requires complementary methods like immunohistochemical staining or genetic labeling alongside morphological analysis to account for fixation-induced changes.
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