Microglial activation precedes and predominates over macrophage infiltration in transient focal cerebral ischemia: a study in green fluorescent protein transgenic bone marrow chimeric mice

Abstract

Resident microglia and hematogenous macrophages play crucial roles in the pathogenetic cascade following cerebral ischemia but may functionally differ regarding neuroprotective and cytotoxic properties. Distinction between these cells has not been possible due to a lack of discriminating cellular markers. We generated bone marrow chimeric mice by transplanting bone marrow from green fluorescent protein (GFP) transgenic mice into irradiated wild-type recipients. Transient focal cerebral ischemia was induced by transient middle cerebral artery occlusion (MCAO) for 30 min. Resident microglia and infiltrating macrophages were identified by immunohistochemistry and GFP fluorescence after 1–28 days. The first blood-derived cells infiltrating the infarct area were seen on Day 1 and identified as granulocytes. Hematogenous GFP⁺ macrophages were rarely observed on Day 2, reached peak numbers on Day 7, and decreased thereafter. In contrast, resident GFP⁻ microglial cells rapidly became activated already on Day 1 after MCAO. Even on Days 4 and 7, most macrophage-like cells remained GFP⁻, indicating their derivation from resident microglia. Hematogenous macrophages were able to acquire a ramified morphology indistinguishable from resident microglia while microglial cells could develop into a phagocytic phenotype indistinguishable from infiltrating macrophages. The vast majority of macrophages in the infarct area are derived from local microglia, revealing a remarkable predominance of local defense mechanisms over immune cells arriving from the blood. GFP bone marrow chimeric mice are a powerful tool to further differentiate the function of resident microglia and hematogenous macrophages following cerebral ischemia.

Introduction

Cerebral ischemia triggers a complex cellular response which includes both the activation of local glial cells and the recruitment of inflammatory cells from the blood Dirnagl et al 1999, Iadecola and Alexander 2001. Resident microglial cells and infiltrating hematogenous macrophages in particular play a pivotal 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). Examples of proinflammatory ischemia-induced cytokines include tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) Gregersen et al 2000, Boutin et al 2001 while transforming growth factor beta-1 (TGF β-1) may exert potent down-regulatory effects (Lehrmann et al., 1998). Experimental studies in rodents revealed an extremely rapid, progressive activation of microglial cells within hours after focal cerebral ischemia which is followed by a phagocytic response over several days Morioka et al 1993, Zhang et al 1994, Kato et al 1996, Lehrmann et al 1997. A microglial and macrophage response is also seen in human infarcts as revealed by both neuropathological investigations (Postler et al., 1997), as well as modern PET studies using the ligand PK11195 (Gerhard et al., 2000).

It is currently unknown whether local microglial cells and infiltrating macrophages act in a similar manner or exhibit distinct functional properties, since no cellular markers exist to date which can reliably differentiate between these two cell types. There is a considerable body of evidence suggesting that the inflammatory reaction following cerebral ischemia and the infiltration of inflammatory cells in particular are detrimental for the injured brain and contribute to infarct evolution. On that line, infarct size can be reduced by neutrophil depletion, inhibition or deletion of adhesion molecules, and inhibition of proinflammatory cytokines Dirnagl et al 1999, Iadecola and Alexander 2001. Investigations in MCP-1-deficient mice, a chemokine required for macrophage influx from the blood, revealed smaller infarcts underlining the destructive potential of infiltrating macrophages (Hughes et al., 2002). In contrast, studies in mice deficient in the microglial growth factor colony stimulating factor-1 (CSF-1) revealed a markedly attenuated microglial response to focal cerebral ischemia and an increase in infarct size, suggesting a protective role of microglial cells in ischemic stroke (Fedoroff et al., 1997). Activated microglial cells may, however, also express a plethora of cytotoxic cytokines and toxic radicals (Giulian et al., 1994), and sustained microglial activation may contribute to tissue destruction in neurodegenerative disorders including Alzheimer’s disease (Hanisch, 2002), possibly dependent on their specific state of activation (Streit, 2002).

To test the hypothesis that microglial cells and infiltrating macrophages act differently in ischemic stroke, their unequivocal selective identification would be required. Chimeric animals following bone marrow transplantation with cells carrying a differentiating genetic marker are potentially useful tools since hematogenous macrophages would carry that marker while resident, microglia-derived cells would not. However, while some studies suggested that microglial cells constitute a mostly stable resident cell population Hickey and Kimura 1988, Lassmann et al 1993, Bechmann et al 2001a, others identified bone marrow-derived ramified microglial cells Priller et al 2001, Nakano et al 2001 or other macrophage-like cells Eglitis et al 1999, Ono et al 1999 already within the uninjured brain, making a reliable differentiation from ischemia-attracted blood-derived macrophages impossible.

Technical issues related to irradiation and bone marrow preparation are likely to account for the difference since no hematogenous intraparenchymal microglial cells were found in one study not using bone marrow transplantation (Bechmann et al., 2001a). Here, we used the original protocol described by Hickey and colleagues (Hickey and Kimura, 1988) and generated chimeric mice by transplanting bone marrow from green fluorescent protein (GFP) transgenic mice (Okabe et al., 1997) into wild-type recipients. In these chimeric animals, all resident microglial cells remain GFP⁻ while hematogenous macrophages exhibit the green fluorescence of GFP.