G-CSF administration to adult mice stimulates the proliferation of microglia but does not modify the outcome of ischemic injury

doi:10.1016/j.nbd.2010.11.013

Neurobiology of Disease

Volume 41, Issue 3, March 2011, Pages 640-649

https://doi.org/10.1016/j.nbd.2010.11.013 Get rights and content

Abstract

Recent evidence suggests that adult bone marrow stem cells reduce tissue damage and promote repair following CNS ischemic injury. Since granulocyte-colony stimulating factor (G-CSF) mobilizes hematopoietic stem cells to the circulating compartment, here we tested whether administration of this drug modifies the outcome of a permanent occlusion of the middle cerebral artery in adult mice. To elucidate the behavior and fate of blood-borne cells in the ischemic brain, we produced chimeric animals, in which hematopoietic derivatives are genetically tagged. G-CSF administration enhances the proliferation of microglia in the uninjured CNS but has no effect on the amount of hematopoietic cells that infiltrate the ischemic tissue and on the size of the lesion. The blood-borne elements acquire different mesodermal identities but fail to adopt neural phenotypes, even though they occasionally fuse with Purkinje neurons. These results indicate that G-CSF treatment does not exert a significant beneficial effect on the ischemic injury.

Research Highlights

► Granulocyte-colony Stimulating Factor (G-CSF) mobilizes hematopoietic stem cells to circulating blood. ► We asked whether G-CSF exerts beneficial effects on CNS ischemic injury. ► In the intact mouse CNS, G-CSF stimulates proliferation of microglia. ► Following ischemic injury, G-CSF. • Does not enhance infiltration of blood-borne cells. • Does not modify the fate of infiltrating cells. • Does not reduce the extent of the ischemic injury. ► Albeit effective on the CNS, G-CSF does not modify the outcome of ischemic injury.

Introduction

Granulocyte-colony stimulating factor (G-CSF) is a hematopoietic growth factor that stimulates proliferation and specification of neutrophil precursors (Morrison et al., 1997, Wright et al., 2001) and induces their large-scale mobilization from bone marrow (BM) to the peripheral blood (Levesque et al., 2003, Zou et al., 2004, Katayama et al., 2006). Because of these properties, G-CSF has been widely used in clinical practice to treat neutropenia induced by chemotherapy, or in the case of BM transplantation (Welte et al., 1996). Recent studies highlighted the efficacy of G-CSF in animal models of cerebral ischemia, reporting significant reduction of infarct size and improved functional recovery (Minnerup et al., 2008, Sevimli et al., 2009, Zhang et al., 2009). The beneficial effects of G-CSF have been attributed to different concurrent mechanisms, including anti-apoptotic activity (Schäbitz et al., 2003, Schneider et al., 2005), immunomodulation (von Aulock et al., 2004), stimulation of neurogenesis (Schneider et al., 2005, Kawada et al., 2006, Diederich et al., 2009), and angiogenesis (Lee et al., 2005). These phenomena have been related to direct effects of G-CSF on neural cells or to enhanced colonization of ischemic regions by hematopoietic stem cells (HSCs) (Schäbitz and Schneider, 2007). The latter cells may produce and release molecules endowed with neuroprotective or neurotrophic activity (Schäbitz and Schneider, 2007, Hokari et al., 2009) but could also contribute directly to nervous tissue repair by adopting local phenotypes (Brazelton et al., 2000, Mezey et al., 2000) or fusing with damaged neurons (Magrassi et al., 2007).

The beneficial effects of G-CSF have been consistently observed following transient cerebral ischemia. Nevertheless, a dose-dependent expansion of cortical atrophy, associated with severe functional defects, has been reported following G-CSF administration in a model of permanent ligation of the middle cerebral artery (MCA) (Taguchi et al., 2007). These negative outcomes have been attributed to an exaggerated inflammatory response, consequent to the enhanced G-CSF-induced infiltration of the infarct region by hematopoietic cells.

In spite of such contrasting reports, and considering their relevance for a potential use of G-CSF in the clinical treatment of stroke, little is known about the behavior and fate of blood-borne cells that colonize the ischemic brain following G-CSF stimulation and the consequent effects on ischemic injury. To address these issues, here we evaluated the effect of G-CSF application following permanent occlusion of the medial cerebral artery (MCAo). In this context, to relate the effects of G-CSF administration to the mobilization of hematopoietic elements and to the extension of ischemic brain damage, we prepared chimeric mice in which blood cells had been tagged by enhanced Green Fluorescent Protein (eGFP) expression. Analysis of these animals was aimed at monitoring the G-CSF-induced infiltration of the ischemic brain and at identifying the phenotypes acquired by the blood-borne elements. Our results show a moderate but consistent activity of G-CSF on the uninjured brain but fail to disclose significant effects on the extent of the injury, on the amount of blood cells that colonize the ischemic region, and on their phenotypic differentiation.

Section snippets

Animals and surgical procedures

Adult CD1 or C57BL/6 mice (body weight 20–30 g, age 3–6 months; Harlan, San Pietro al Natisone, Italy) were used for the study. All experimental procedures on living animals were performed according to the European Communities Council Directive of 1986 (86/609/EEC), the National Institutes of Health guidelines, and the Italian law for care and use of experimental animals (DL116/92) and were approved by the Italian Ministry of Health and the Bioethical Committee of the University of Turin. All

G-CSF administration stimulates cell proliferation in the uninjured CNS

The main goal of our work was to assess the effects of G-CSF administration on the fate of BM-derived cells after ischemia and, hence, on the evolution of injury itself. Since G-CSF can cross the blood-brain barrier (Zhao et al., 2007a), we first examined intact animals to determine the direct effects of the substance on the CNS, and to set basal conditions to be compared with ischemic injury. Uninjured mice received injections of G-CSF or vehicle plus BrdU for 4 days and were killed 7 days after

Discussion

Starting from recent evidence suggesting that BM-derived stem cells promote repair and recovery following ischemic brain injury, we asked whether pharmacological mobilization of these cells can help to ameliorate the outcome of permanent MCAo, a condition that reproduces the features of human ischemic stroke (Stroke Therapy Academic Industry Roundtable, 1999, Mohr et al., 2004). Our results show that, although G-CSF stimulates mitotic activity of microglia in the intact brain, it has no clear

Acknowledgments

We thank Francesco Frassoni for initial help in generating the hemopoietic chimeras. F.R. was supported by grants from Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MIUR-PRIN 2007 prog. no. 2007F7AJYJ), Regione Piemonte (Project A14/05; Ricerca Sanitaria Finalizzata, 2008, 2009), Fondazione Cavaliere del Lavoro Mario Magnetto of Turin. L.M. was supported by grants from MIUR (PRIN 20074MW29N-004 2007), Ministero della Salute (RC cod. 08016098/07).

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G-CSF administration to adult mice stimulates the proliferation of microglia but does not modify the outcome of ischemic injury

Abstract

Research Highlights

Introduction

Section snippets

Animals and surgical procedures

G-CSF administration stimulates cell proliferation in the uninjured CNS

Discussion

Acknowledgments

Brain Res. Rev.

Cytokine

Biochem. Biophys. Res. Commun.

Cell

Brain Res.

Neurosci. Res.

FEBS Lett.

Resuscitation

Trends Pharmacol. Sci.

Neuroscience

Exp. Neurol.

Eur. J. Pharmacol.

Prog. Neurobiol.

Blood

Blood

Arch. Med. Res.

Blood

Exp. Neurol.

Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes

Nature

Granulocyte-colony stimulating factor delays PWI/DWI mismatch evolution and reduces final infarct volume in permanent-suture and embolic focal cerebral ischemia models in the rat

Stroke

From marrow to brain: expression of neuronal phenotypes in adult mice

Science

Prominin: a story of cholesterol, plasma membrane protrusion and human pathology

Traffic

In vivo over-expression of interleukin-10 increases resistance to focal brain ischemia in mice

J. Neurochem.

The role of granulocyte-colony stimulating factor (G-CSF) in the healthy brain: a characterization of G-CSF-deficient mice

J. Neurosci.

Targets for vascular protection after acute ischemic stroke

Stroke