Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

Summary

A way to gain new insights into the complexity of the brain inflammatory response is presented. We describe immunofluorescence-based protocols followed by three-dimensional confocal analysis to investigate the pattern of co-expression of microglia/macrophage phenotype markers in a mouse model of focal ischemia.

Abstract

After brain stroke microglia/macrophages (M/M) undergo rapid activation with dramatic morphological and phenotypic changes that include expression of novel surface antigens and production of mediators that build up and maintain the inflammatory response. Emerging evidence indicates that M/M are highly plastic cells that can assume classic pro-inflammatory (M1) or alternative anti-inflammatory (M2) activation after acute brain injury. However a complete characterization of M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is still missing.

Immunofluorescence protocols specifically staining relevant markers of M/M activation can be performed in the ischemic brain. Here we present immunofluorescence-based protocols followed by three-dimensional confocal analysis as a powerful approach to investigate the pattern of localization and co-expression of M/M phenotype markers such as CD11b, CD68, Ym1, in mouse model of focal ischemia induced by permanent occlusion of the middle cerebral artery (pMCAO). Two-dimensional analysis of the stained area reveals that each marker is associated to a defined M/M morphology and has a given localization in the ischemic lesion. Patterns of M/M phenotype marker co-expression can be assessed by three-dimensional confocal imaging in the ischemic area. Images can be acquired over a defined volume (10 μm z-axis and a 0.23 μm step size, corresponding to a 180 x 135 x 10 μm volume) with a sequential scanning mode to minimize bleed-through effects and avoid wavelength overlapping. Images are then processed to obtain three-dimensional renderings by means of Imaris software. Solid view of three dimensional renderings allows the definition of marker expression in clusters of cells. We show that M/M have the ability to differentiate towards a multitude of phenotypes, depending on the location in the lesion site and time after injury.

Introduction

After acute brain injury, microglia are rapidly activated and undergo dramatic morphological and phenotypic changes^1-3. This intrinsic response is associated to recruitment of blood-born macrophages which migrate into the injured brain parenchyma^4,5. The role of microglia and macrophages which are antigenically not distinguishable (henceforth referred to as M/M) in brain injury is still debated. An increasing number of studies indicate that, similarly to what described for peripheral macrophages, microglia and brain recruited macrophages can assume different phenotypes whose extremes correspond to classic pro-inflammatory toxic (M1) or anti-inflammatory protective (M2) phenotype. The different activation states, including secretion of pro- or anti-inflammatory factors, release of neurotrophic molecules and lysosomal activity are characterized by a specific pattern of phenotypic markers, whose expression depends on the temporal evolution of the surrounding environment. The characterization of these M/M phenotypes in the injured brain is still scanty. We used a well-established murine model of pMCAo to analyze M/M expression and evolution after stroke. Immunofluorescence based protocols presented here aim at getting insight into the appearance of specific M/M phenotype markers, their localization and cellular co-expression in the ischemic area. We investigated a few molecules associated to different activation state or phenotype, namely CD11b, a surface marker expressed by leukocytes and a widely used marker of M/M activation/recruitment^6-8, CD68 a marker of lysosomes^6,7 and Ym1 a secretory protein expressed by alternatively activated (M2) macrophages and associated to recovery and function restoration^9-10.

When two markers are expressed by the same cell, but in different subcellular compartments, colocalization alone may not be much informative. In this case, analysis of coexpression can be performed by using single plane view and by three-dimensional renderings. We here describe a protocol to obtain a thorough three-dimensional analysis of marker coexpression.

Protocol

1. Immunofluorescence The following protocol is performed on coronal brain cryosections obtained from transcardially perfused mice (20 ml of PBS, 0.1 mol/liter, pH 7.4, followed by 50 ml of chilled paraformaldehyde 4% in PBS). After perfusion, brains are carefully removed and transferred to 30% sucrose in PBS at 4 °C overnight for cryoprotection. The brains are then rapidly frozen by immersion in isopentane at – 45 °C for 3 min before being sealed into vials and stored at -70 °C u…

Representative Results

An example of the results obtained when labeling protocols and confocal acquisitions are carried out into the ischemic region is illustrated in Figures 1A and 1B. A two dimensional view of acquired images shows that at twenty-four hours after ischemia (A), the lysosomal marker CD68 (green) is expressed in hypertrophic ameboid CD11b cells (red) present in the ischemic core. In the border zone (B) CD11b positive cells display round cell bodies and ramified…

Discussion

We present here immunofluorescence-based protocols followed by three-dimensional confocal analysis as a powerful approach to investigate localization and co-expression of M/M phenotype markers into the ischemic area (for a more detailed analysis see ref ⁶). This method combines specific staining of relevant marker of M/M activation with three-dimensional confocal imaging. The fine tuning of antibodies, serum and fluorconjugated working dilutions allows optimal signal to noise ratio of the investigated m…

Materials

			Materials
Rat Anti-mouse CD11b	Kindly provided by Dr. A. Doni, Istituto Clinico Humanitas, Milan, Italy
Rat Anti-mouse CD68	AbD Serotec	MCA 1957
Rabbit Anti-mouse Ym1	Stem Cell Technologies	1404
Hoechst 33342	Life technologies	H21492
Mouse Anti-Neural Nuclei (NeuN)	CHEMICON	MAB377
Biotinilated Goat Anti-Rat antibody	Jackson Immuno Research	112-065-143
TSA Cyanine 5 System	Perkin Elmer	NEL705A001KT
Prolong Gold	Invitrogen	P36930
Anti-rat alexa 546	Invitrogen	A-11081
Anti mouse Alexa 488	Invitrogen	A-21121
Anti-rabbit Alexa 594	Invitrogen	A-11037
Normal Goat Serum	Vectors Laboratories	S-1000
Tritin X-100	Sigma	T8787
Phosphate Buffered Saline	Sigma	P4417-100
			Equipment
Cryostat CM1850	Leica
Olympus IX81 confocal microscope	Olympus
AnalySIS software	Olympus
Imaris software 5.0	Bitplane
Photoshop cs2	Adobe Systems
Software packages GraphPad Prism version 4.0	GraphPad Software Inc.