microglia in Rat Traumatic Brain Injury

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Lesional Accumulation of CD163 + Macrophages/microglia in Rat Traumatic Brain Injury Zhiren Zhanga, b,⁎, Zhi-Yuan Zhangb , Yuzhang Wua , Hermann J. Schluesenerb a

Institute of Immunology, Third Military Medical University of PLA, Gaotanyan Main Street 30, 400038, Chongqing, People's Republic of China Institute of Brain Research, University of Tuebingen, Calwer Str. 3, D-72076 Tuebingen, Germany

b

A R T I C LE I N FO

AB S T R A C T

Article history:

A robust neuroinflammation, contributing to the development of secondary injury, is a common

Accepted 20 April 2012

histopathological feature of traumatic brain injury (TBI). Characterization of leukocytic

Available online 27 April 2012

subpopulations contributing to the early infiltration of the damaged tissue might aid in further understanding of lesion development. Reactive macrophages/microglia can exert protective or

Keywords:

damaging effects in TBI. CD163 is considered a marker of M2 (alternatively activated)

Traumatic brain injury

macrophages. Therefore we investigated the accumulation of CD163+ macrophages/microglia

CD163

in the brain of TBI rats. TBI was induced in rats using an open skull weight-drop contusion model

Macrophages

and the accumulation of CD163+ cells was analyzed by immunohistochemistry. In normal rat

Heme oxygenase-1

brains, CD163 was expressed by meningeal, choroid plexus and perivascular macrophages. Significant parenchymal CD163+ cell accumulation was observed two days post TBI and continuously increased in the investigated survival time. The accumulated CD163+ cells were mainly distributed to the lesional areas and exhibited macrophage phenotypes with amoeboid morphologic characteristics but not activated microglial phenotypes with hypertrophic morphology and thick processes. Double-labeling experiments showed that most CD163+ cells co-expressed heme oxygenase-1 (HO-1). In addition, in vitro incubating of macrophage RAW264.7 cells or primary peritoneal macrophages with hemoglobin– haptoglobin (Hb-Hp) complex suppressed LPS-induced inflammatory macrophages phenotype and induced CD163 and HO-1 upregulation, indicating that CD163+ macrophages/microglia in TBI might have antiinflammatory effects. And further study is necessary to identify functions of these cells in TBI. © 2012 Elsevier B.V. All rights reserved.

1.

Introduction

In TBI, the primary injury is due to the effects of biomechanical impact but a pronounced secondary injury due to endogenous pathological processes manifests itself over a period of hours to days and months. Among the complex cellular, biochemical and pathophysiological processes, a robust neuroinflammation, which involves the activation of glia and neurons as well as

cerebral accumulation of leukocytes, occurs after TBI and contributes to the development of the secondary injury. This inflammatory response post TBI has various consequences on outcome, depending on timing and severity of inflammation (Lenzlinger et al., 2001; Morganti-Kossmann et al., 2001; Whitney et al., 2009; Ziebell and Morganti-Kossmann, 2010). Microglia/macrophage activation/infiltration is a well-known response following TBI. Activated microglia/macrophages secrete

⁎ Corresponding author at: Institute of Immunology, Third Military Medical University of PLA, Gaotanyan Main Street 30, 400038, Chongqing, People's Republic of China. Fax: +86 23 68752779. E-mail address: [email protected] (Z. Zhang). 0006-8993/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2012.04.038

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a variety of proinflammatory cytokines that are crucial to the development of secondary injury (Kreutzberg, 1996; Perry et al., 2010). In the central nervous system (CNS) injury, activated microglia/macrophages have been reported to remove inhibitory tissue debris and secrete growth promoting factors resulting in regeneration (Glezer et al., 2007; Napoli and Neumann, 2009; Whitney et al., 2009). On the other hand, uncontrolled activation of microglia/macrophages can cause deleterious effects to CNS tissue (Dheen et al., 2007; Stoll and Jander, 1999). Activated microglia/macrophages transform their morphology, up-regulate certain membrane proteins and express a panel of cytokines which can contribute to inflammation, loss of tissue and glial scar formation (Kreutzberg, 1996; Perry et al., 2010; Schwartz, 2003). These reactive microglia/ macrophages are composed of different subpopulations, which may be derived from resident microglia or blood-derived macrophages, express different membrane proteins, and secrete diverse inflammatory molecules (Colton, 2009; Ladeby et al., 2005; Schwartz, 2003). The reason for the high heterogeneity of activated microglia/macrophages is unknown but different subpopulations have been speculated to have different functions at a given time (Ladeby et al., 2005; Schwartz, 2003). The functional heterogeneity of reactive macrophages/ microglia in TBI could be due to the existence of different forms of macrophages. Recruited macrophages can be subclassified into two extremely distinct subsets, categorized as either classically activated (M1) or alternatively activated (M2). M1 macrophages induced by lipopolysaccharide (LPS) or inflammatory cytokines, like interferon-γ (IFN-γ), are characterized by high expression of interleukin-12 (IL-12), IL-23, tumour necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), etc., and are mainly considered to cause autoimmune tissue damage or host defencse to infection. M2 macrophages induced by IL4, IL13, IL10 or transforming growth factor-β (TGFβ), highly express anti-inflammatory molecules, such as IL-10 and TGF-β,extracellular matrix molecules, like fibronectin, and scavenger receptors, which underlie their roles in antiinflammation and tissue repair (Fairweather and Cihakova, 2009; Gordon, 2003; Olefsky and Glass, 2010). CD163 is a member of the scavenger receptor cysteine-rich family class B and expressed mainly on monocytes and macrophages (Onofre et al., 2009; Van Gorp et al., 2010). CD163 functions as an endocytic receptor for Hb–Hp complexes and as such is proposed to mediate the clearance of free Hb from the circulation (Moestrup and Moller, 2004; Nielsen and Moestrup, 2009). CD163 expression is induced by IL-10, IL-6, and glucocorticoid, but is downregulated by LPS and IFNγ (Buechler et al., 2000; Van Gorp et al., 2010). Interestingly, CD163 is considered as a marker of alternatively activated or anti-inflammatory macrophages (Abraham and Drummond, 2006; Komohara et al., 2006). CD163+ macrophages are found during the late downregulatory phase of acute inflammation (Zwadlo et al., 1987) and in chronic inflammation (Topoll et al., 1989). In normal brain, CD163 is restricted to perivascular macrophages (Fabriek et al., 2005). Furthermore, the up-regulation of CD163 by perivascular macrophages in brains was observed under several CNS disorders, such as viral encephalitis and multiple sclerosis (MS) (Fabriek et al., 2005; Kim et al., 2006). While CD163 is not expressed on parenchymal microglial cells in normal brain, accumulation of parenchymal CD163+

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macrophages/microglia was seen in human immunodeficiency virus and simian immunodeficiency virus encephalitis (Borda et al., 2008; Roberts et al., 2004), astrocytoma (Komohara et al., 2008). Devic's disease (Satoh et al., 2008) and multiple sclerosis brains (Fabriek et al., 2005). It has been reported that CD163 mediates both Hb-Hp complex and Hb clearance, and induces activation of the enzyme heme oxygenase-1 (HO-1) in macrophages providing an important anti-inflammatory and cytoprotective activity (Schaer et al., 2006a, 2006b). Given the intracranial hemorrhage occurs often after TBI (Chang et al., 2005) and the deposition of Hb following vascular disorders contributes to tissue damage it is important to know the expression and functions of CD163 following TBI. Therefore, in the present study, we have investigated the spatio-temporal accumulation of CD163+ cells following open-skull weight-drop-induced TBI in rat brains.

2.

Results

Open skull weight drop injury generates a reproducible local lesion in the ipsilateral cortex. The development of the lesion was first analysed by HE staining and has been described previously (Zhang et al., 2006). In brief, selective neuronal loss and necrotic loci condensed at the edge of impact site were observed 12 hours post injury. Significant leukocyte infiltration and hemorrhage was already seen at Day 1. At Day 4 post TBI, a lesioned cavity together with its surrounding perilesional tissue loss was formed under the impact areas. At the lesional areas, most tissue was lost, no neurons remained and leukocyte infiltration together with hemorrhage appeared. In the perilesional areas, selective neuron loss with leukocyte infiltration was observed. In the subcortical white matter, no morphological changes were seen.

2.1. CD163 expression pattern in rat brains following experimental TBI The expression of CD163 in normal and TBI brains was studied using immunohistochemistry. No immunoreactivity (IR) was detected for the negative control without primary antibody (data not shown). In normal rat brain, immunohistochemistry revealed CD163 expression by perivascular (Fig. 1A), meningeal (Fig. 1A) and choroid plexus macrophages (Fig. 1B), but not in the parenchyma. In perivascular spaces, corresponding to VirchowRobin-like spaces, which represent the infiltrative route for blood-borne leukocytes, CD163+ macrophages had the appearance of flattened and elongated cells, and were located outside the vascular basement membrane (Fig. 1A). The CD163+ meningeal macrophages showed elongated morphologic characteristics and choroid plexus macrophages showed amoeboid morphologic characteristics (Fig. 1B). CD163 IR was seen in lesional and perilesional areas which represent the regions of ongoing secondary injury where accumulation of CD163+ cells was quantified (Fig. 2). A relatively strong increase of CD163+ cells in lesioned areas was observed from 18-h post TBI (3.3±0.5 per HPF, Figs. 1C and 2). Accumulation of CD163+ cells increased over time. A significant accumulation of CD163+ cells began on Day 2 post injury (19.0±0.3 per HPF, P<0.05, Figs. 1D and 2) when compared with normal control

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Fig. 1 – Accumulation of CD163+ cells in TBI. (A–B): In normal brains, CD163 was expressed by perivascular macrophages (A), meningeal macrophages (A) and choroid plexus macrophages (B). Arrow indicated the perivascular macrophage and arrow head indicated the meningeal macrophage. (C–F): Accumulation of CD163+ macrophages in the lesioned regions at 18 hours (C), 2 days (D), 3 days (E) and 4 days (F) after TBI. (G): Micrographs show that, in TBI brains, CD163+ cells were predominantly identified as macrophages with amoeboid morphologic characteristics. Some lipid-laden, foamy-appearing CD163+ macrophages were seen (arrow indicated). (H): CD163+ perivascular macrophages in lesioned rats were often found in parenchymal areas. Scale bar is 50 μm for A-F and 25 μm for E–F.

brains. And the maximal accumulation of CD163+ cells (49.5±5.6 per HPF, P<0.01; Figs. 1F and 2) was observed on Day 4 in our study. However, the accumulation of CD163+ cells following TBI may continue longer. And the magnitude of CD163 expression on cells was comparable among different time points in our study. In TBI brains, CD163+ cells were mainly identified as macrophages or fully activated microglia with amoeboid morphologic characteristics (Fig. 1G). Phagocytic macrophages,

with characteristic multivacuolated cytoplasm and large round eccentric nuclei, constituted another population of CD163+ cells (Fig. 1G, arrow indicated). CD163+ perivascular macrophages in lesional parenchymal were often seen (Fig. 1H), but in contrast CD163+ cells with ramified microglial phenotypes with hypertrophic morphology and thick elongated processes were rare. We further characterized CD163+ cells by double-staining with antibodies against activated microglia/macrophages (ED1)

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in Fig. 3, most CD163+ cells co-expressed CD68, but only part of ED1+ cells co-expressed CD163 (Fig. 3A–C). Specifically, ED1+ macrophages/fully activated microglia localized to the lesional areas, co-expressed CD163, however, ED1+ microglia localized to the perilesional areas, which were characterized with hypertrophic morphology and thick elongated processes, were CD163 negative, suggesting that CD163 was mainly expressed on macrophages or fully activated microglia in TBI. Thereafter the co-expression of CD163 with HO-1 was analysed by double-staining. As shown in Fig. 3D, post TBI all CD163+ macrophages co-expressed HO-1 and almost all HO-1+ cell co-expressed CD163, indicating that CD163+ macrophages might be activated by Hb-Hp complex to express HO-1 in TBI. Fig. 2 – Time course of parenchymal CD163+ macrophage accumulation in TBI. The numbers of parenchymal CD163+ cells of every rat brain coronal section were counted in 8 HPFs. In each field, only positive cells with the nucleus at the focal plane were counted. Results were given as arithmetic means of positive cells per HPF and standard errors of means (SEM). Statistical analysis was performed by one-way ANOVA followed by Dunnett´s Multiple Comparison test. *: P < 0.05, **: P < 0.01 compared to normal control.

using brain sections from days 4 post TBI. ED1 stains CD68, a lysosomal membrane protein, which is mainly found in phagocytosing macrophages and reactive microglia. As shown

2.2.

Effects of Hb-Hp complex on macrophage function

Because CD163+ macrophages co-expressed HO-1 and hemorrhage was observed post TBI, it's reasonable to assume that CD163+ macrophages might be activated by Hb-Hp complex. Therefore, the effects of Hb-Hp complex on macrophages were investigated in vitro using murine macrophage cell line RAW 264.7 and primary rat peritoneal macrophages. We investigated the effects of Hb-Hp complex on CD163 and HO-1 expression in macrophages. As shown in Fig. 4, mRNA levels of CD163 and HO-1 were greatly induced following incubation with Hb-Hp complex for 24 h compared to saline control. Subsequently, the effects of Hb-Hp complex on inflammatory macrophages were analyzed. Inflammatory

Fig. 3 – CD163 double labelling in brain sections from day 4 post TBI. (A): Most CD163+ macrophages (brown) co-expressed CD68 (blue) but only part of ED1+ cells (blue) co-expressed CD163 (brown). The boxed area indicated the regions that were further observed under high-power magnification shown in B and C. (B): ED1+ macrophages (blue) localized to the lesion areas, which were characterized by amoeboid morphology, co-expressed CD163 (brown). (C): However, ED1+ microglia (blue) localized to the perilesional areas, which were characterized with hypertrophic morphology and thick elongated processes, were CD163-. (D): Almost all CD163+ macrophages (brown) co-expressed HO-1 (blue). Scale bar is 100 μm for A and 25 μm for B–C.

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Fig. 4 – Hb–Hp complex increased CD163 and HO-1 mRNA in macrophages in vitro. Murine macrophage cell line RAW 264.7 cells (A) or primary rat peritoneal macrophages (B) were grown in complete RPMI 1640 media containing penicillin, streptomycin and 10% fetal calf serum at 37 °C and 5% CO2. 105 cells were seeded into 12-well cell culture plates and cultured overnight. Then cells were incubated with Hb-Hp complex for 24 hours, total RNA was extracted and mRNA levels of CD163 and HO-1 were quantified by real-time PCR. Results were calculated as levels of target mRNAs relative to those of the housekeeping gene β-actin (three samples from each group were analyzed). *: p < 0.05, **: p < 0.01 compared to their respective control.

macrophage phenotype was induced by LPS (1 μg/mL) for 24 h. Following LPS treatment, mRNA expression of TNF-α, iNOS, IL-6, and CCL-3 was greatly induced (Fig. 5) compared to medium control, indicating an inflammatory phenotype. To observe Hb-Hp complex effects, macrophages were incubated with LPS and Hb–Hp complex for 24 h and then mRNA levels of certain cytokines were measured. As shown in Fig. 5, Hb–Hp complex significantly attenuated mRNA expression of TNF-α, iNOS, IL12p35 and CCL-3, but increased mRNA expression of IL-10, suggesting a suppression of inflammatory macrophage phenotype. Furthermore, cell viability of LPS and Hb-Hp complex treated cells was analyzed by MTT assay and no significant differences were seen among these groups (data not shown). These data suggests that Hb-Hp complex could suppress inflammatory macrophage phenotype.

3.

Discussion

We have analyzed early accumulation of CD163+ macrophages in rat brains up to 96 h after TBI induced by weight-drop

contusion. Significant CD163+ cell accumulation was observed 2 days post TBI and increased steadily. The CD163+ cells were mainly distributed to lesioned regions and showed an amoeboid but not ramified morphology. Further, double-labeling experiments showed that most CD163+ macrophages coexpressed HO-1. In addition, in vitro experiment with macrophage RAW264.7 cells and primary rat peritoneal macrophages revealed that Hb-Hp complex could induce CD163 and HO-1 expression, and suppress LPS-induced inflammatory macrophages phenotype. After TBI, the accumulation of reactive microglia/macrophages is an early response and the injury-activated microglia/ macrophages consist of functionally distinct cell populations (Colton, 2009; Ladeby et al., 2005; Lenzlinger et al., 2001). CD163 is expressed by cells of the monocyte/macrophage lineage and can be induced by IL-10, IL-6, and glucocorticoids, whereas the proinflammatory LPS and IFN-γ down-regulate its expression (Buechler et al., 2000). The best studied function of CD163 is binding and clearing of Hb–Hp complexes, and this process can exert anti-inflammatory effects via the release of IL-10 and heme metabolites (Kristiansen et al., 2001; Schaer et al., 2006a; Van Gorp et al., 2010). Recently CD163 has been proposed as receptor for TNF-like weak inducer of apoptosis (TWEAK). Upon binding to CD163-expressing macrophages, TWEAK is internalized and degraded (Bover et al., 2007; Moreno et al., 2009). Because CD163 is up-regulated by anti-inflammatory molecules, such as IL-10 and glucocorticoids, and exerts antiinflammatory effects, expression of CD163 may regulate the anti-inflammatory nature of macrophages and is thought to be a useful marker for alternatively activated M2 macrophages (Buechler et al., 2000; Komohara et al., 2006). In normal brain, expression of CD163 is restricted to perivascular and meningeal macrophages but not to parenchymal microglia, which is in accordance with our observations here (Fabriek et al., 2005). The up-regulation of CD163 by perivascular macrophages and parenchymal microglia in brains has been reported from several CNS disorders, such as viral encephalitis (Kim et al., 2006), MS (Fabriek et al., 2005), astrocytoma (Komohara et al., 2008) and Devic's disease (Satoh et al., 2008). Here we further analyzed the spatiotemporal accumulation of CD163+ macrophages in brains of TBI rat. The significant accumulation of CD163+ macrophages was seen on Day 2 post injury, mainly localized to the lesioned areas and showed macrophage or fully activated microglia morphology in TBI. Interestingly, most CD163+ macrophages co-expressed HO-1, which is an inducible and rate-limiting enzyme that catalyzes the degradation of heme to generate carbon monoxide, biliverdin and free iron (Shibahara et al., 2002). Induction of HO-1 protects against the cytotoxicity of oxidative stress and apoptotic cell death and has major immunomodulatory and anti-inflammatory effects (Jazwa and Cuadrado, 2010; Paine et al., 2010). Following TBI, upregulation of HO-1 in macrophages/ microglia was observed in human and rat TBI and was suggestted to exert a protective role against secondary insults including oxidative stress (Beschorner et al., 2000; Fukuda et al., 1996). Therefore, CD163+ macrophages in TBI brain exhibited an anti-inflammatory and possibly neuroprotective phenotype. It has been reported that hemoglobin can induce HO-1 expression through CD163 mediated endocytosis (Abraham and Drummond, 2006). In our study, hemorrage was observed

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Fig. 5 – Hb–Hp complex suppressed inflammatory macrophage phenotype in vitro. Murine macrophage cell line RAW 264.7 cells (A) or primary rat peritoneal macrophages (B) were grown in complete RPMI 1640 media containing penicillin, streptomycin and 10% fetal calf serum at 37 °C and 5% CO2. 105 cells were seeded into 12-well cell culture plates and cultured overnight, cells were stimulated by LPS with or without Hb–Hp complex for 24 h and then total RNA from cultured cells was prepared and mRNA levels of TNF-α, iNOS, IL12p35, CCL-3, IL-6 and IL-10 was measured by real-time PCR. Results were calculated as levels of target mRNAs relative to those of the housekeeping gene β-actin (three samples from each group were analyzed). *: p < 0.05, **: p < 0.01 compared to their respective LPS group.

following TBI and in vitro investigation showed that Hb–Hp complex greatly increased HO-1 mRNA level, indicating that the expression of HO-1 in CD163+ macrophages might be induced by hemoglobin post TBI. In TBI brains, our data first revealed that CD163 expression was up-regulated in perivascular and parenchymal macrophages. While potential inducers of CD163 up-regulation are yet unknown in TBI, our data together with others’ showed that up-regulation of CD163 in macrophages could be induced by hemoglobin (Ugocsai et al., 2006). As hemorrhage is common after TBI, hemoglobin might contribute to CD163 induction in TBI. A pathophysiological role of CD163 might relate to binding to hemoglobin and TWEAK and therefore CD163 might be important for their clearance (Bover et al., 2007; Moestrup and Moller, 2004). Intracranial hemorrhage occurs often after TBI (Chang et al., 2005). The removal of hemoglobin from red blood cells is partly dependent on the CD163 (Schaer et al., 2006b) and this clearing process can exert anti-inflammatory effects via stimulation of release of IL-10 and heme metabolites (Kristiansen et al., 2001; Van Gorp et al., 2010). TWEAK is a member of the tumor necrosis factor superfamily which function via binding to fibroblast growth factor-inducible 14 (Fn14) receptor (Zheng and Burkly, 2008). TWEAK signaling is associated with disruption of the blood–brain barrier and the activation of NF-kappaB in the

CNS to release proinflammatory cytokines and matrix metalloproteinases (Polavarapu et al., 2005; Zhang et al., 2007). While the expression of TWEAK following TBI hasn't been reported recent studies have indicated an increase in TWEAK and Fn14 expression following cerebral ischemia, a disorder with similar pathological process with TBI (Inta et al., 2008). Therefore, CD163+ macrophages in brain might function in removing hemoglobin and TWEAK to decrease inflammation and tissue damage in TBI. In addition, it is known that CD163 is expressed in anti-inflammatory or alternatively activated macrophages (Komohara et al., 2006). In our study, CD163+ macrophages were HO-1 positive as well, suggesting that CD163+ macrophages may endocytose Hb-Hp complex. Furthermore, our in vitro experiment showed that Hb-Hp complex turned classically activated inflammatory macrophages into anti-inflammatory macrophages, which highly expressed the anti-inflammatory cytokine IL-10. So, in TBI brain, CD163+ macrophages might exert anti-inflammation and tissue protection functions. Infiltration and activation of macrophages are well-known following TBI. These reactive macrophages are composed of different subpopulations and have different or even adverse functions. While our investigations here clearly demonstrated an accumulation of CD163+ macrophages following TBI in rats, the origin and functions of these cells are still unclear. Further

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studies using specific techniques, like clodronate liposomes to deplete CD163+ cells or conditional gene knockout to reduce CD163 expression in macrophages, would be helpful to confirm the anti-inflammatory and neuroprotective effects of these cells following TBI (Galea et al., 2008). In summary, we observed a lesional accumalation of CD163+ macrophages, which were HO-1 positive, in parenchyma of TBI rat brains. Furthermore, Hb–Hp complex induced CD163 and HO-1 upregulation in macrophages and turned LPS-induced inflammatory macrophages into an anti-inflammatory phenotype in vitro. Our study suggested that CD163+ macrophages might have an anti-inflammation and tissue protection role in TBI and probably could be a suitable target for the development of immunotherapeutic, anti-inflammatory therapies.

4.

Experimental procedures

4.1.

Rat brain tissue library

Brain libraries of normal and TBI rats have been described previously (Zhang et al., 2006). In brief, Lewis rats (8–9 weeks of age, 350–400 g, Elevage Janvier, Le Genest-St-Isle, France) were housed with equal daily periods of light and dark and free access to food and water. All procedures were performed in accordance with the published International Health Guidelines under a protocol approved by the Administration District Official Committee. The number of rats used and their suffering were minimized. TBI was induced in anesthetized rats using an open skull weight-drop contusion model. Rats were randomly grouped, anesthetized with Ketamine (120 mg/kg)/Rompun (8 mg/kg) and underwent craniotomy, in which a circular region of the skull (3.0 mm diameter, centered 2.3 mm caudal and 2.3 mm lateral to bregma) was removed over the right somatosensory cortex. A weight-drop device was placed over the dura and adjusted to stop an impact transducer (foot plate) at a depth of 2.5 mm below the dura. Then, a 20 g weight was dropped from 15 cm above the dura, through a guiding tube onto the foot plate. Body temperature was maintained using an overhead heating lamp during surgery. After injury, the scalp was closed tightly. TBI rats survived without further treatment and were euthanasied at different times (6 h, 12 h, 18 h, 24 h, 48 h and 96 h; 3–5 rats per time point). For euthanasie, rats (26 TBI rats, 5 normal control rats) were deeply anesthetized with Ketamine (120 mg/kg)/Rompun (8 mg/kg) and perfused intracardially with 4 °C 4% paraformaldehyde (PFA) in PBS. Brains were quickly removed and post-fixed in 4% paraformaldehyde overnight at 4 °C. Fixed rat whole brains were put in rodent brain matrices (coronal) and were sliced to get the cortical coronal blocks containing the contusion regions. Such cortical coronal blocks were embedded in paraffin, serially sectioned (3 μm) through the center of the traumatized area and mounted on silan-covered slides. The contused areas were numbered and during the following immunostaining the same antibody was applied to sections with the same number. 4.2.

Immunohistochemistry

After dewaxing, brain sections were boiled (in an 850 W microwave oven) for 15 min in citrate buffer (2.1 g citric acid

monohyrate/L, pH 6) (Carl Roth, Karlsruhe, Germany). Endogenous peroxidase was inhibited by 1% H2O2 in pure methanol (Merck, Darmstadt, Germany) for 15 min. Sections were incubated with 10% normal pig serum (Biochrom, Berlin, Germany) to block non-specific binding of immunoglobulins and then with the mouse monoclonal antibody against CD163 (Serotec, Oxford, Great Britain; dilution 1:100) overnight at 4 °C. Antibody binding to tissue sections was visualized with a biotinylated rabbit anti-mouse IgG F(ab)2 antibody fragment (1:400; DAKO, Hamburg, Germany). Subsequently, sections were incubated with a horseradish peroxidase-conjugated Streptavidin complex for 30 min (1:100; DAKO, Hamburg, Germany), followed by development with diaminobenzidine (DAB) substrate (Fluka, Neu-Ulm, Germany). Finally, sections were counterstained with Maier's Hemalaun. As negative controls, the primary antibodies were omitted. After immunostaining, brain sections of each time point were examined by light microscopy. The numbers of CD163+ cells were counted in 8 non-overlapping high-power fields (HPFs, x400 magnification) for each section. The HPFs were selected to have a maximum of positive cells. In each field studied, only positive cells with the nucleus at the focal plane were counted. Results were given as arithmetic means of CD163+ cells per HPF and standard errors of means (SEM). 4.3.

Double staining

In double staining experiments, brain sections were immunolabeled as described above. Then they were once more irradiated in a microwave for 15 min in citrate buffer and were incubated with 10% normal pig serum (Biochrom, Berlin, Germany). Subsequently the sections were incubated with the appropriate second primary monoclonal antibodies for 1 h at room temperature. The monoclonal antibody ED1 (1:100; Serotec, Oxford, UK) and the polyclonal antibody HO-1 (HSP 32, Usbio, 1:100; Swampscott, Massachusatts, USA) were used. Consecutively, visualization was achieved by adding secondary antibody (biotinylated rabbit anti-mouse IgG or swine anti-rabbit F(ab’)2) at a dilution of 1:400 in TBS-BSA for 30 min and then alkaline phophatase-conjugated Avidin complex diluted 1:100 in Tris-BSA for another 30 min. Finally immunostaining was developed with Fast Blue BB salt chromogensubstrate solution, but by omission of counterstaining with hemalaun. 4.4.

Preparation of Hb–Hp complex

Hb-Hp complex was prepared as described (Borda et al., 2008). Briefly, blood from normal mice was collected in two vacutainer tubes, one containing sodium heparin and the other containing silica clot activator and polymer gel to obtain serum. The red blood cells were separated from the plasma and rinsed twice with isotonic PBS. The packed red blood cells were lysed mechanically by adding 2-mm glass beads and vigorously mixing the tube for 5 to 10 minutes on a vortex. Disruption of the red blood cells was confirmed by microscopic examination. The blood lysate was centrifuged at 15,000 rpm in a microcentrifuge and the supernatant (soluble Hb) was separated from the particulate pellet. The soluble Hb was mixed at a 1:10 ratio with serum from the same animal. The mixture was left at room

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temperature for 10 minutes to allow binding of Hp to the free Hb (Hb-Hp complex formation). 4.5.

Rat peritoneal macrophages

Peritoneal macrophages were collected from 8 to 10-week-old Lewis rats by peritoneal lavage with 10 ml of phosphate-buffered saline for several times, centrifuged at 1,000 rpm for 10 min, resuspended in RPMI1640 media supplemented 10% fetal bovine serum, and incubated on the 24-well plate for 2 h at 37 °C and 5% CO2 to remove non-adherent cells. The remanent cells were harvested and the RNA was prepared for RT-PCR assay. 4.6.

In vitro cell culture

The mouse leukaemic monocyte macrophage cell line RAW 264.7 was utilized to determine effects of myelin loading on macrophages in vitro. RAW 264.7 cells were grown in complete RPMI 1640 media (Gibco, Grand Island, NY) containing penicillin (100 U/mL), streptomycin (100 U/mL) and 10% fetal calf serum at 37 °C and 5% CO2. 105 cells were seeded into each well of 12-well cell culture plates and cultured overnight. Afterwards cells were stimulated with the LPS (1 μg/mL) together with either the Hb–Hp complex as described above (1:100 diluted) or the same volume of saline for 24 h. Thereafter, cells were harvested and centrifuged, and total RNA from cultured cells was prepared using the RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacture's instruction. 1 μg RNA was reverse transcribed into cDNA using QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). The resulting cDNA was used to measure quantitatively the expression of genes using SYBR green qPCR master mix according to the manufacturer's protocol (BioRad). Real-time measurements of gene expression were performed with an iCycler thermocycler system and iQ5 optical system software (BioRad). Results were calculated as levels of target mRNAs relative to those of the housekeeping gene β-actin (three samples from each group were analyzed). Primers used to measure gene expression are: β-actin (sense, TGG AAT CCT GTG GCA TCC ATG AAA; antisense, TAA AAC GCA GCT CAG TAA CAG TCC G), TNF- α (sense, AAC TAG TGG TGC CAG CCG AT; antisense, CTT CAC AGA GCA ATG ACT CC), IL-1β (sense, TGC TGA TGT ACC AGT TGG GG; antisense, CTC CAT GAG CTT TGT ACA AG), iNOS (sense, CAG CTG GGC TGT ACA AAC CTT; antisense, CAT TGG AAG TGA AGC GTT TCG), IL-6 (sense, ACA ACC ACG GCC TTC CCT ACT T; antisense, CAC GAT TTC CCA GAG AAC ATG TG), IL-10 (sense, TCA TTC ATG GCC TTG TAG ACA C; antisense, AGC TGG ACA ACA TAC TGC TAA C), HO-1 (sense, CAC GCA TAT ACC CGC TAC CT; antisense, CCA GAG TGT TCA TTC GAG A) and CD163 (sense, AGC ATG GAA GCG GTC TCT GTG ATT; antisense, AGC TGA CTC ATT CCC ACG ACA AGA). 4.7.

MTT assay

Cell viability of treated RAW 264.7 macrophages was detected by MTT assay. Briefly, cells were treated as described above. Following stimulation, cells were washed with PBS and MTT solution (5 mg/mL, Sigma-Aldrich Chemie GmbH, Munich, Germany) was added to each well. After 4 h of incubation at

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37 °C and 5% CO2, cells were washed again with PBS and then DMSO was added to each well to thoroughly dissolve the formazan. Thereafter, optical density of each well was read at 560 nm and background at 670 nm was substracted. 4.8.

Statistics

Statistical analysis was performed by one-way ANOVA followed by Dunnett's multiple comparison tests or non-parametric t test (Graph Pad Prism 4.0 software). For all statistical analyses, significance levels were set at P < 0.05.

Acknowledgments This research was partly supported by Natural Science Foundation Project of CQ CSTC (contract No. 2010BB5025) and National Nature Science Foundation of China (No: 81070954). Author disclosure statement No competing financial interests exist.

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