Elsevier

Experimental Neurology

Volume 196, Issue 2, December 2005, Pages 290-297
Experimental Neurology

Regular Article
Predominant phagocytic activity of resident microglia over hematogenous macrophages following transient focal cerebral ischemia: An investigation using green fluorescent protein transgenic bone marrow chimeric mice

https://doi.org/10.1016/j.expneurol.2005.08.004Get rights and content

Abstract

Activated microglia and hematogenous macrophages are known to be involved in infarct development after cerebral ischemia. Traditionally, hematogenic macrophages are thought to be the primary cells to remove the ischemic cell debris. However, phagocytosis is a well known property also of activated microglia. Due to a lack of discriminating cellular markers, the cellular origin of phagocytes and the temporal course of phagocytosis by these two cell types are largely unknown. In this study, we used green fluorescent protein (GFP) transgenic bone marrow chimeric mice and semithin serial sections after methyl methacrylate embedding of the brains to dissect in detail the proportion of identified activated resident microglial cells and infiltrating hematogenous macrophages in phagocytosing neuronal cell debris after 30 min of transient focal cerebral ischemia. Already at day one after reperfusion, we found a rapid decrease of neurons in the ischemic tissue reaching minimum numbers at day seven. Resident GFP-negative microglial cells rapidly became activated at day one and started to phagocytose neuronal material. By contrast, hematogenous macrophages incorporating neuronal cell debris were observed in the ischemic area not earlier than on day four. Quantitative analysis showed maximum numbers of phagocytes of local origin within 2 days and of blood-borne macrophages on day four. The majority of phagocytes in the infarct area were derived from local microglia, preceding and predominating over phagocytes of hematogenous origin. This recruitment reveals a remarkable predominance of local defense mechanisms for tissue clearance over immune cells arriving from the blood after ischemic damage.

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).

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