Iron deposition in the tumor cells was observed in 8/15 (53%) angiomatous meningioma cases, 2/6 (33%) microcystic meningiomas and 2/20 (10%) meningothelial meningiomas, which included clustered microvessels,

but not in fibrous, atypical or anaplastic meningiomas (P = 0.001). Cytoplasmic CD71 expression was largely negative in angiomatous meningioma cases, but positive in meningothelial and high-grade meningiomas, suggesting that the transferrin-dependent iron transporter was involved in iron uptake in meningiomas. Nuclear expression of 8-OHdG was observed in ≥50% of the tumor cells in all 15 cases of angiomatous meningioma and was associated with the presence of regressive histopathological findings, such as hyalinized vessels and cystic changes. In addition, the fraction of iron-containing tumor cells was correlated to those expressing 8-OHdG (P = 0.005). Our finding indicates BMS-907351 research buy that cytoplasmic iron deposition in tumor cells is characteristic of highly vascularized benign meningiomas and related to increased oxidative DNA damage markers. “
“It has been reported that bisphenol A (BPA), a widespread xenoestrogen employed in the production of polycarbonate plastics, affects brain development in both humans and rodents. VX-770 price In the present study

employing mice, we examined the effects of exposure to BPA (500 μg/kg/day) during fetal and lactational periods on the development of the locus coeruleus (LC) at the

age of embryonic day 18 (E18), postnatal 3 weeks (P3W), P8W and P16W. The number of tyrosine hydroxylase-immunoreactive cells (TH-IR cells) in females exposed to BPA was decreased, Resveratrol compared with the control females at P3W. At P8W, the number of TH-IR cells in females exposed to BPA was significantly decreased, compared with the control females, whereas the number of TH-IR cells in males exposed to BPA was significantly increased, compared with the control males, which resulted in reversed transient sexual differences in the numbers of TH-IR cells observed in the controls at P8W. However, no significant changes were demonstrated at E18 or P16W. Next, we examined the density of the fibers containing norepinephrine transporter (NET) in the anterior cingulate cortex (ACC) and prefrontal cortex, at P3W, P8W and P16W, because NET would be beneficial in identifying the targets of the LC noradrenergic neurons. There were no significant differences shown in the density of the NET-positive fibers, between the control and the groups exposed to BPA. These results suggested that BPA might disrupt the development of physiological sexual differences in the LC-noradrenergic system in mice, although further studies are necessary to clarify the underlying mechanisms.

2 Infection caused by Candida sp. was confirmed by positive culture of the blood or device lead or on the basis of consistent histopathological studies. The appropriate management of persons with PPM/ICD infections has been described by Sohail et al. [7] and the current approach to patients with CRMD Candida infections was recently defined by Pappas et al. [15]. From publications spanning a 40-year period (1969–2009), we documented 15 patients, including our current case, with well-defined CRMD-associated Candida endocarditis (12 PPM, 3 ICD; Table 1). All were men with a mean age of 65.1 years (range = 38–87 years). Use of device prior to infection was documented for 13 patients and varied widely

from <1 month to 16 years. Manipulation of the CRMD within 3 months of infection (generator change) occurred in two patients. Infection symptoms were defined for 13 selleck screening library patients and fever was present in 10. All patients had lead vegetations and vegetation size ranged from 0.5 to 7 cm. Four patients experienced a major fungal embolus

to a pulmonary artery with C. albicans recovered from three of these and C. parapsilosis from one. Microbiology results revealed C. albicans (seven patients), C. parapsilosis (four patients), Candida tropicalis (two patients) and Candida glabrata (two patients). Included Daporinad in vivo in these results are one patient with both C. albicans and C. glabrata16 and one case where both C. albicans and Staphylococcus epidermidis were isolated.17 In one case, blood cultures were negative but histopathology at the time of autopsy was consistent with CRMD Candida endocarditis.18 Antimicrobial interventions varied with five patients receiving an amphotericin B formulation alone, two received amphotericin B with 5-flucytosine, four received fluconazole alone, therapy was undefined for two patients, one patient received

only antibacterial therapy18 and one patient received an echinocandin agent (caspofungin) Ketotifen followed by fluconazole and posaconazole.19 Twelve patients underwent CRMD explantation as part of the management of Candida endocarditis (five thoracotomy, three percutaneous extractions, four methods undefined), one patient refused surgical intervention, one was felt not to be a candidate for explantation and one expired without intervention. Eight of the 15 patients (53%) died whilst receiving treatment for infection. Amongst the 10 patients who received clearly documented anti-fungal therapy and also underwent CRMD explantation, there were two deaths (20%) that could be attributed to the Candida endocarditis. The number of hospitalisations associated with CRMD infections increased substantially in the United States during a 7-year period (1996–2003) when a 49% rise in CRMD implantations occurred.1 Increase in infection occurred in both PPM and ICD populations, and the complication increased the risk of in-hospital death by over twofold.

Further studies are needed to investigate the reasons for false positives. We found that the sensitivity of MgEDTA–CAZ is higher than that of MgEDTA–IPM. This makes CAZ preferable to IPM as a substrate in DDSTs. However, one IMP-1-producing A. baumannii and two NDM-1-producing Enterobacteriaceae were positive when IPM was used, but negative when CAZ was used. Kim et al. have reported that, because these organisms have other CAZ resistant mechanisms such as ESBL and AmpC β-lactamase production, DDSTs using CAZ have difficulty detecting MBL-producing Acinetobacter [21]. Therefore, DDSTs using Mg-EDTA should use both IPM and CAZ disks as substrates

in order to further reduce false negative results. False positive results reportedly also occur with MBL phenotypic methods using EDTA and IPM. It is believed that such false positive results are attributable to increasing membrane permeability Omipalisib cell line caused by chelating agents [24, 25] and the anti-bacterial activity of EDTA [19, 24, 25]. DDSTs using Mg-EDTA yielded no false positive results among 25 non-MBL producers. The disk content SP600125 ic50 of Mg-EDTA was 10 mg, this concentration being higher than that of the EDTA was used in previous reports. Because false positive results were confirmed for P. aeruginosa and Acinetobacter spp. by the Etest MBL and combined disk test, DDST using Mg-EDTA should be evaluated for specificity using non-MBL-producing P. aeruginosa or Acinetobacter

spp. In conclusion, this is the first report to evaluate several metal-EDTA complexes as inhibitors of MBL. Use of Mg-EDTA in DDSTs is the most useful Y-27632 2HCl phenotypic method for detecting MBL producers, including NDM-1 producing strains, in clinical laboratories. Because we tested only two NDM-1 producers by the Mg-EDTA DDST method, other NDM-1 producers should be confirmed by subsequent studies in actual clinical practice.

M. Fujisaki and S. Sadamoto are employees of Eiken Chemical. “
“CD1d-restricted invariant natural killer T (iNKT) cells bear characteristics of innate and adaptive lymphocytes, which allow them to bridge the two halves of the immune response and play roles in many disease settings. Recent work has characterized precisely how their activation is initiated and regulated. Novel antigens from important pathogens have been identified, as has an abundant self-antigen, β-glucopyranosylcaramide, capable of mediating an iNKT-cell response. Studies of the iNKT T-cell receptor (TCR)–antigen–CD1d complex show how docking between CD1d–antigen and iNKT TCR is highly conserved, and how small sequence differences in the TCR establish intrinsic variation in iNKT TCR affinity. The sequence of the TCR CDR3β loop determines iNKT TCR affinity for ligand–CD1d, independent of ligand identity. CD1d ligands can promote T helper type 1 (Th1) or Th2 biased cytokine responses, depending on the composition of their lipid tails.

Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized check details integrin β1 to lysosomal compartments with

a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking. “
“The transcobalamin II (TCN2)

MK-8669 776C>G polymorphism has been reported to be a genetic risk factor for idiopathic recurrent

spontaneous abortion (RSA). However, the sample size in previous studies was small, and other TCN2 polymorphisms have not been studied. Moreover, the TCN2 67A>G and 776C>G polymorphisms, and the transcobalamin II receptor (TCblR/CD320) Casein kinase 1 1104C>T polymorphism, have demonstrated associations with immune responses. Three hundred and seventy-eight RSA patients who had at least two consecutive spontaneous abortions were enrolled. Two hundred and seven control subjects were collected from a convenience sample. Polymerase chain reaction and restriction fragment length polymorphism analysis were performed to identify the TCN2 67A>G and 776C>G polymorphisms, and the TCblR 1104C>T polymorphism. RSA patients showed significantly different frequencies of the TCN2 67AG+GG genotypes compared with control subjects. The TCN2 67G allele is a possible risk factor for idiopathic RSA. “
“Infection with murine gammaherpesvirus 68 has become an accepted model for studying the virus/host interactions with regard to gammaherpesvirus infections. Previous studies using gene-deficient mice have revealed that neither IFNγ nor perforin is essential in controlling the outcome of infection or the virus load during chronic infection in C57BL/6 mice. However, pronounced multiorgan fibrosis and splenic atrophy are observed in mice lacking IFNγ or the IFNγ receptor.

Our results were inconsistent with data described by Tajik et al. [28] showing that compound KIR/HLA genotype had no major impact on limiting M. tuberculosis infection. The different results can be explained as follows: KIR-HLA interactions may transmit inhibitory and activating signals with different strengths. The way by which M. tuberculosis is processed may depend on the properties of the associated HLA alleles. The binding and presentation depend mainly on the polymorphic HLA alleles present in the infected population. Thus, the diversity in the genetic locus is a major part for finding an association of HLA

alleles Akt inhibitor with disease development. Collectively, the above-mentioned results suggested that various factors could be of importance for the development of PTB, such as gene polymorphisms, polymorphisms between ethnicity and

geographical location and so on. Despite the small number of subjects included in this study, we are able to demonstrate the significant effects of KIRs and HLA-Cw genes on the susceptibility to PTB infection. These basic mechanisms will be of help in designing treatment strategies. Nevertheless, additional genetic and functional studies will be necessary to clarify the involvement of the mechanism in PTB infection. This manuscript was supported by the Shandong Provincial Scientific and Technological Development Projects Foundation (2009GG10002014) to Z. M. Lu and Chinese National Natural Science Foundation (grant 30371304) to Y. R. Zhao. “
“Medical Corporation Katsurakai Hirao Hospital, BTK inhibitor 6-28 Hyobu, Kashihara, Nara 634-0076, Japan Milk fat globule-EGF factor 8 (MFG-E8) promotes the phagocytosis of apoptotic cells by serving as a bridging molecule between apoptotic cells and phagocytes. Many apoptotic cells are left unengulfed in the germinal centers of the spleen of MFG-E8−/− mice, which develop a human systemic lupus erythematosus (SLE)-like autoimmune disease. Here, we analyzed the MFG-E8 gene in human SLE patients, and found in two out of 322 female

patients a heterozygous intronic Epothilone B (EPO906, Patupilone) mutation, which caused a cryptic exon from intron 6 to be included in the transcript. The cryptic exon contained a premature termination codon, generating a C-terminally truncated MFG-E8 protein. The mutant MFG-E8 was aberrantly glycosylated and sialylated, but bound to phosphatidylserine and enhanced the phagocytosis of apoptotic cells. When intravenously injected into mice, the mutant MFG-E8 was sustained longer in the blood circulation than wild-type MFG-E8. Repeated administrations of the mutant MFG-E8 protein induced the production of autoantibodies, such as anti-cardiolipin and anti-nuclear antibodies, at a lower dose than that required for the wild-type protein. These results suggested that the intronic mutation in the human MFG-E8 gene can lead to the development of SLE.

The spectra were normalized to the total ion current intensity in the m/z range over 2000–50,000 to modulate peak dimension. The peaks ranging between m/z 0 and m/z 2000 were eliminated

from analysis to avoid the interference of adducts, artefacts of the energy-absorbing molecules and other possible chemical contaminants. Biomarker Wizard Version 3.1 (BMW; Ciphergen) was used to identify corresponding peaks in each spectrum (peak clusters). The settings for autodetect peaks to cluster were as follows: signal-to-noise ratio was 5 and minimum peak threshold was 0% for the first pass; for cluster completion, cluster mass window was 0.3%, and signal-to-noise ratio for the second pass was 2. Also, BMW helps pick out differently expressed Panobinostat molecular weight peaks by evaluating the differences of peak intensities between groups by non-parametric Kruskal–Wallis test and Mann–Whitney test [23, 24]. Peak intensities were considered statistically significantly different at P-values below 0.05. Construction of classification tree model.  Construction of the Daporinad price classification tree model was based on a platform of bioinformatic, Biomarker Patterns Software Version 5.0 (BPS; Ciphergen), which was developed basing on the Classification

and Regression Trees decision tree system [25]. The BPS helps build a binary decision tree algorithm with the peak information of the training set, and that algorithm assigns each sample into one of the two nodes according to some rules established by the intensity of certain peaks [16–19]. Staurosporine order The data of differently expressed peaks generated by BMW between active TB and non-TB group were used in this proceeding. The BPS generated some classification tree models and evaluated the

error cost (represented as ‘relative cost’ in BPS) for each one. Of those models, the one with the lowest error cost was the best, and then this resulting model was applied to the data of the test set for evaluating the efficiency of classification. The spectra of 178 serum samples were detected by MALDI-TOF MS combined with WCX magnetic beads. This combination was particularly effective in resolving low molecular weight proteins and peptides, as shown in Fig. 1. Peak of m/z 48 was found differently expressed between active TB group and non-TB group (Table 2). Reproducibility was evaluated by performing an 8-spot assay (intra-assay), a 20-chip assay (interassay) and a 20-day assay with a mixed serum sample (age and sex matched, two patients with TB and two health volunteers), and the coefficient of variations for peak intensity of spectra were 1.7%, 1.9% and 13.3%, respectively. These data derived from averaging values for nine of the highest amplitude peaks as follows: m/z 4309, 4963, 5344, 7772, 7846, 8058, 8608, 9413 and 16105.

The empty vector was used to generate CAL-1-EV cells. Lentiviral particles were produced in 293T cells by calcium phosphate transfection. Spin transduction of CAL-1 cells with 8 μg/mL Polybrene was performed at 1800 TSA HDAC purchase rpm for 90 min. GFP-positive CAL-1 cells were sorted under low-pressure conditions on the

FACSAria. For RNA interference, CAL-1 cells were transfected with 75 nM siRNA directed against NAB2 (siRNA ID: s9248; Ambion/Applied Biosystems) or the Silencer Selected Negative Control siRNA #1; Ambion/Applied Biosystems) together with 25 nM siGLO Transfection Indicator (Dharmacon) with transfection reagent DharmaFECT 4 (Dharmacon) according to the manufacturer’s protocol. Transfection efficiency was determined by flow cytometry (Supporting Information Fig. 3A), and silencing was confirmed at protein levels by western

blot (Supporting Information Fig. 3B). A total of 105 primary human pDCs were stimulated with 12.5 μg/mL CpG A (Invivogen) or left untreated for 4 h or overnight in complete medium in a 96-well plate for RT-PCR and flow cytometry or western blot analysis, respectively. CAL-1 cells (7 × 105) were seeded overnight in a 24-well plate in 2 mL medium. A total of 1.1 mL medium was replaced with 100 μL FBS-free RPMI medium containing 12.5 μg/mL CpG B or Ctrl CpG B, 5 μg/mL Imiquimod (Invivogen), or 100–200 ng/mL IFN-β (PBL Medical Laboratories) to prevent FBS-mediated NAB2 induction ABT-263 ic50 ([14], data not shown). A total of 50 μM SB203580, 2.5 μM BAY11–7082, 5 μM PI-103 (Tocris Bioscience), 200 mM Rapamycin (Calbiochem) or DMSO alone, or 0.1 μg/mL B18R (eBioscience) were added to cells 30 min prior to CpG stimulation. After stimulation, supernatant was harvested for cytokine analysis and cells were washed once with PBS before further analysis. pDC cell sorting was performed with anti-CD45RA-FITC (BD Biosciences) and anti-CD123-PE (Miltenyi Biotec). Cell surface staining was performed Phloretin with Anti-CD40-PE (Beckman Coulter) or isotype control

IgG1-PE (BD Biosciences), and anti-TRAIL (2E5; Enzo Life Sciences), or control mouse IgG1 (BD Biosciences), followed by anti-mouse IgG1-Biotin (Enzo Life Sciences) and Steptavidin-allophycocyanin (BD Pharmingen). Dead cell exclusion was performed with propidium iodide. Intracellular IRF-7 staining was performed by fixation and permeabilization with Cytofix/cytoperm Solution (BD Biosciences) and PBS containing 0.5% saponin and 2% FCS, followed by staining with IRF-7 (H-246; Santa Cruz Biotechnologies) or isotype control (Imgenex) and anti-rabbit IgG Alexa 568 (Invitrogen). Flow cytometry was performed with FACS Calibur or LSRII (BD Biosciences). Analysis was performed with FlowJo software (Tristar).

Among them, IL-6 plays a pivotal and not redundant role acting on the survival and on the Ig secretion capacity of B cells [23]. Thus, to characterize the mechanisms underlying Selleck Daporinad the differential responsiveness

of B cells from MS patients to TLR7 and TLR9 stimulation, we measured by ELISA the production of IL-6 in PBMCs isolated from 10 HDs and from 15 MS patients before and after IFN-β therapy (Fig. 3A). PBMCs were treated for 24 h with the TLR7 and TLR9 ligands, 3M001 and CpG, respectively, and cytokine release was quantified. In line with the data obtained for Ig production, HD PBMCs secreted a robust level of IL-6 following TLR7 triggering that was significantly higher than the production seen in PBMCs of therapy-free MS patients. The lower cytokine release observed in MS patients was almost completely restored following IFN-β administration. TLR9 stimulation induced low amounts of this cytokine in HDs and the level of production was not differently modulated in MS individuals

before and after IFN-β therapy. In the same way, another key component of the milieu responsible for B-cell proliferation and differentiation into plasma cells is the B-cell-activating factor of the TNF family (BAFF), whose mRNA expression was found to be comparable between PBMCs of HDs and untreated MS patients but strongly induced upon IFN-β therapy (Fig. 3B). Accordingly, similar levels of BAFF were present in the sera Cabozantinib price of HDs and MS patients and were induced in response to IFN-β therapy (Fig. 3C), confirming previous data from Krumbholz et al. [24]. All together these results show that in MS patients, the lower humoral immune response upon TLR7 triggering is replenished by IFN-β treatment likely Temsirolimus through the release of factors, such as IL-6 and BAFF, that mediate B-cell differentiation

into Ig-secreting cells. Having found that in MS patients monocytes display an impaired expression of the TLR7 gene (Fig. 2E), we hypothesized that this cell type might have a role in the defective TLR7-induced Ab response of MS patients through the release of cytokines involved in B-cell differentiation and activation, such as IL-6 and BAFF [25-27]. To test our hypothesis, we depleted PBMCs from 4 IFN-β-treated MS patients of monocytes and cultured total or monocyte-depleted PBMCs with the TLR7- or TLR9-specific ligands. While the poor induction of IL-6 was not affected by the depletion of monocytes upon TLR9 stimulation, a strong dependence on monocytes was observed for IL-6 release in response to TLR7 (Fig. 4A). In a similar manner, BAFF mRNA, strongly expressed in freshly drawn total PBMCs upon IFN-β therapy, displayed a clear reduction in the level of expression in IFN-β-treated monocyte-depleted PBMCs (Fig. 4B).

However, there is marked regional variation in the uptake of home haemodialysis (HD) and peritoneal dialysis (PD) suggesting further scope for the expansion of these modalities. Methods:  Between 1 April and 5 August 2009, Australian nephrologists were invited to complete an online survey. Seventy-six questions were Selleckchem SAHA HDAC asked covering characteristics of the dialysis units, responders’ experience, adequacy of facilities and support structures, attitudes to the use of home HD and PD and issues impeding the increased uptake of home dialysis. Results:  Completed surveys were received and analysed from 71 respondents; 27 from Heads of Units (35% response rate) and 44 (16%) from other nephrologists. There was strong agreement

that HD with long hours was advantageous

and that this was most easily accomplished in the home. PD was not considered to be an inferior therapy. A ‘PD first’ policy existed in 34% of Renal Units. The most commonly reported impediments to expanding home dialysis services were financial disadvantage for home HD patients, and lack of physical infrastructure for training, support and education. Areas of concern for expanding home dialysis programmes included psychiatry support, access to respite care and home visits, and lack of support from medical administration and government. The majority of nephrologists would recommend home dialysis to more patients if these impediments could be overcome. Conclusion:  This survey identified support from nephrologists for the expansion of home dialysis in Australia and highlighted important barriers to improving access to these therapies. “
“Aim:  KU-57788 order Autophagy is a cellular process of degradation of damaged cytoplasmic components and regulates cell death or proliferation. Unilateral

ureteral obstruction (UUO) is a model of progressive renal fibrosis in the obstructed kidney. And UUO is followed by compensatory cellular proliferation in the contralateral kidney. We investigate the role of autophagy Wnt inhibitor in the obstructed kidney and contralateral kidney after UUO. Methods:  To obtain the evidence and the patterns of autophagy during UUO, the rats were sacrificed 3, 7 and 14 days after UUO. To examine the efficacy of the autophagy inhibitors, 3-methyladenine (3-MA), the rats were treated daily with intraperitoneal injection of 3-MA (30 mg/kg per day) for 7 days. Results:  After UUO, autophagy was induced in the obstructed kidney in a time-dependent manner. Inhibition of autophagy by 3-MA enhanced tubular cell apoptosis and tubulointerstitial fibrosis in the obstructed kidney after UUO. In the contralateral kidney, autophagy was also induced and prolonged during UUO. Inhibition of autophagy by 3-MA increased the protein expression of proliferating cell nuclear antigen significantly in the contralateral kidney after UUO. The Akt-mammalian target of rapamycin (mTOR) signalling pathway was involved in the induction of autophagy after UUO in both kidneys.

In addition, our Treg depletion experiment shows that Regorafenib clinical trial the reduced number of Treg alone is sufficient to explain the aggravated EAE course. Therefore, additional functional defects of the Treg appear to be unlikely but can, on the other

hand, not totally be excluded. Taken together, our results point toward a crucial involvement for LFA-1 in Treg homeostasis and highlight the importance of Treg in limiting EAE. Future study needs to determine how Treg generation depends on the presence of LFA-1. LFA-1-deficient mice 24 were obtained from the Jackson Laboratories and were backcrossed to C57BL/6 for 13 generations. We further crossed them with C57BL/6 WT mice and used littermates of LFA-1+/−inter-se matings for the experiments. Animal handling and experiments were conducted according to the German animal protection laws and approved by the responsible governmental authority. For EAE induction, 6- to 10-wk-old mice were anaesthetized with ketamine (94 mg/kg body weight) and xylazine (6.25 mg/kg) and immunized subcutaneously at two sites of the back close to inguinal lymph nodes with 200 μg MOG35–55 in CFA (EAE Induction Kit™, MOG35–55/CFA Emulsion PTX (3.75×), Hooke Laboratories). VX-809 supplier Directly after immunization, mice received a first dose of 400 ng pertussis toxin

i.p. followed by a second injection the day after. After 1 wk, mice were scored daily for clinical signs according to the following scale: 0, no obvious changes in motor functions; 1, limp tail; 2, limp tail and weakness of hind legs; 3, limp tail and complete paralysis of hind legs; 4, limp tail, complete hind leg and partial front leg paralysis; and 5, complete hind and complete front leg paralysis. 8 days prior induction of EAE mice were treated with 500 μg anti-CD25 (clone PC61.5) i.p. The Ab preparation was controlled to contain less than 0.1 ng endotoxin/mg of protein

by limulus amoebocyte lysate assay. Mice were perfused under deep anaesthesia through the left cardiac ventricle with PBS OSBPL9 followed by 4% paraformaldehyde. Brain and spinal cord were removed, post-fixed in paraformaldehyde over night, and embedded in paraffin. Briefly, 5-μm thick sections were stained for haematoxylin-eosin, Luxol Fast Blue/periodic acid-Schiff, and Bielschowsky’s silver impregnation. Immunohistochemistry was performed with an avidin–biotin technique. For immunohistochemistry, sections were deparaffinised and intrinsic peroxidase activity was blocked by incubation with 5% H2O2 in PBS for 20 min. Nonspecific Ab binding was inhibited with 10% FCS in PBS for 25 min. Macrophages/microglial cells were detected using an anti-Mac-3 Ab (BD Biosciences) with biotinylated anti-mouse Ig (GE Healthcare) as secondary reagent.