Regular ArticlePredominant phagocytic activity of resident microglia over hematogenous macrophages following transient focal cerebral ischemia: An investigation using green fluorescent protein transgenic bone marrow chimeric mice
Introduction
After cerebral ischemia, complex pathophysiological events are involved in infarct development over time and space. Excitotoxicity, periinfarct depolarization, inflammation and programmed cell death are the predominant mechanisms regulating neuronal survival and development of tissue damage and final infarct size after vessel occlusion (Dirnagl et al., 1999). The inflammatory response after focal cerebral ischemia is characterized by an extremely rapid activation of microglia/macrophages within hours (Morioka et al., 1993, Kato et al., 1996, Lehrmann et al., 1997, Zhang et al., 1994). Resident microglial cells and infiltrating hematogenous macrophages play an important role during the pathogenetic cascade following cerebral ischemia since they express a plethora of growth factors, chemokines and regulatory cytokines as well as free radicals and other toxic mediators (Raivich et al., 1999) which are involved in secondary infarct expansion (Del Zoppo et al., 2000, Hallenbeck, 2002). Further, microglial cells are essential as scavenger cells in tissue repair and are of functional importance since insufficient removal of cell debris has been identified as one of the major causes for regeneration failure (Stoll et al., 2004). Phagocytosis is stimulated by specific epitopes on phagocytic targets and requires activation of downstream signaling cascades that lead to the rearrangement of the actin cytoskeleton and incorporation of the cell debris (Koenigsknecht and Landreth, 2004). A large number of actin-regulatory proteins are responsible for the formation of multiform actin assemblies and macrophages have been reported to contain various actin-binding proteins (Ohsawa et al., 2004). However, little is known about factors that regulate microglial phagocytosis (Mitrasinovic et al., 2003). Traditionally, hematogenic macrophages are considered to be responsible for phagocytosis (Perry et al., 1987). But once activated, resident microglia and hematogenous macrophages are not distinguishable by morphological criteria due to a lack of discriminating cellular markers (Kreutzberg, 1996). Using GFP transgenic bone marrow chimeric mice, we were enabled to conduct a definitive distinction between these two cell types. Recently, we found a rapid activation of resident microglial cells and a remarkable delay of infiltration and unexpected small number of hematogenous macrophages after transient focal cerebral ischemia (Schilling et al., 2003). These findings evoke the hypothesis that microglial cells and infiltrating macrophages act differently in phagocytosis after ischemic stroke. In order to answer this question and to quantify the proportion of phagocytosis performed by hematogenous macrophages or activated microglial cells, we examined GFP transgenic bone marrow chimeric mice after transient focal cerebral ischemia using the methyl methacrylate embedding technique for immunohistochemical analysis of multiple antigens in semithin serial sections (Mueller et al., 2000).
Section snippets
Production of bone marrow chimeric mice
The animal experiments were approved by the local governmental authorities. Male C57BL/6J-mice (20–30 g) were obtained from Charles-River (Sulzfeld, Germany). GFP-transgenic mice (C57BL/6J-GFP) were generously donated by Dr. Masaru Okabe, Osaka, Japan (Okabe et al., 1997). Bone marrow chimeric mice were created as described previously (Mueller et al., 2001). In brief, 6–8 weeks old male C57BL/6J-recipients (20–30 g) were sublethally irradiated with 7 Gy in a cobalt source. Male donor animals
Results
The evolution of ischemic damage after transient MCAO for 30 min reproducibly followed a profile of unilateral infarction within the lateral caudate putamen with little variation between different animals. Cerebral blood flow measurements confirmed a drop of cerebral blood flow below 15% after placement of the intraarterial thread, followed by adequate reperfusion following thread withdrawal. As mentioned above, only chimeric mice with nearly complete chimerism were used containing a minimum of
Discussion
Traditionally, hematogenic macrophages are thought to be the primary cell type for phagocytosing of cellular debris after cerebral ischemia and to promote scar formation (Mabuchi et al., 2000, Ito et al., 2001). In this study, we were able to dissect in detail the proportion of identified activated resident microglial cells phagocytosing neuronal cell debris after transient focal cerebral ischemia on the one hand and infiltrating hematogenous macrophages on the other. Until now, no quantitative
Acknowledgments
We thank Antje Stöber and Karin Wacker for excellent technical assistance, Dr. M. Okabe, Osaka, Japan, for his generous gift of GFP-transgenic mice, and Dr. Y. Imai, Tokyo, Japan, for providing us with Iba-1 antibody. This study was supported by a grant from the Interdisciplinary Center of Clinical Research Münster (IZKF Project No. G5).
References (29)
- et al.
Pathobiology of ischemic stroke: an integrated view
Trends Neurosci.
(1999) - et al.
Hematopoietic origin of microglial and perivascular cells in brain
Exp. Neurol.
(2004) - et al.
Progressive expression of immunomolecules on activated microglia and invading leukocytes following focal cerebral ischemia in the rat
Brain Res.
(1996) Microglia: a sensor for pathological events in the CNS
Trends Neurosci.
(1996)- et al.
Macrophage colony stimulating factor promotes phagocytosis by murine microglia
Neurosci. Lett.
(2003) - et al.
Co-localization of multiple antigens and specific DNA—a novel method using methyl methacrylate-embedded semithin serial sections and catalyzed reporter deposition
Am. J. Pathol.
(2000) - et al.
Rapid response of identified resident endoneurial macrophages to nerve injury
Am. J. Pathol.
(2001) - et al.
‘Green mice’ as a source of ubiquitous green cells
FEBS Lett.
(1997) - et al.
Neuroglial activation repertoire in the injured brain: graded response, molecular mechanisms and cues to physiological function
Brain Res. Rev.
(1999) - et al.
Microglial activation precedes and predominates over macrophage infiltration in transient focal cerebral ischemia: a study in green fluorescent protein transgenic bone marrow chimeric mice
Exp. Neurol.
(2003)