Reagents
Ac2-26 (Ac-AMVSEFLKQAWFIENEEQEYVQTVK) was purchased from Bankpeptide Limited (Heifei, China). The peptide was dissolved in phosphate-buffered saline (PBS) buffer. The mice were injected intraperitoneally with Ac2-26 (1 mg/kg body weight) 2, 8 and 24 hours after the infection.
Animals:
Fpr1
−/−,
Fpr2−/− and wildtype (WT) mice were kept at the Central Animal Care Facility of the RWTH Aachen University.
Fpr1−/− mice were a kind gift from Dr Philip Murphy of the National Institute for Allergy and Infectious Diseases, NIH, Bethesda, MD (
21). The
Fpr2−/− mice were generated as described previously (
22). Both mouse strains were maintained at a C57BL/6 background. The wild-type (WT) mice were back-crossed on the C57BL/6J background for at least five generations. The resulting WT were used as control mice.
Induction of experimental pneumococcal meningitis
All
in vivo experiments were approved by the Animal Care Committee of the University Hospital of Aachen and by the District Government in Recklinghausen, North Rhine-Westphalia, Germany (84-02.04.2015.A157) and performed according to international ARRIVE guidelines on the use of laboratory mice. The mouse model of pneumococcal meningitis used in this study has been previously described (
10,
23,
24). In brief, 8 week old WT,
Fpr1−/− and
Fpr2−/− mice were anaesthetized with ketamine (100 mg/kg body weight) and xylazine (20 mg/kg body weight) and infected with the
Streptococcus pneumoniae D39 (type 2) strain (10
4 colony-forming units/ml). The bacteria were injected directly through the skull into the subarachnoid space. Control mice received a sterile saline solution. Infected mice developed clinical signs of infection within the first 24 h (see as well Fig.
1). For histology, gene expression studies and blood analyses, mice were sacrificed within 28 to 30 hours post infection (each group n ≥ 5). Unless stated otherwise, three independent experiments were performed. By the end of the experiments, the mice were euthanized and perfused with either 4% formalin or 0.9% NaCl for immunohistochemistry or gene expression studies, respectively. Brain hemispheres were divided in the sagittal plane and used for each application. The bacterial burden was analyzed in the spleen and cerebellum as well as in each mouse’s blood sample as described by our group (
25).
Immunohistochemistry:
For immunohistochemistry, sections were rehydrated and, if necessary, antigens were unmasked with Tris/EDTA buffer (pH 9.0) or citrate (pH 6.0) heating as previously described (
8,
23). The sections were washed in PBS and incubated overnight at 4⁰C with the following primary antibodies diluted in blocking solution (serum of the species in which the secondary antibody was produced): Anti-GFAP (1:75.000; RPCA-GFAP, EnCor, Gainesville, FL, USA) or anti-IBA1 (1:10.000; 019-19741, Wako, Neuss, Germany). On the following day, the slides were incubated with biotinylated secondary antibodies) (1:50; BA-1000; Biozol, Eching, Germany) for 1 h, and, after another washing step in PBS, incubated with a peroxidase-coupled avidin-biotin complex (ABC kit; Vector Laboratories, Peterborough, UK). Finally, the sections were treated with 3,3’-diaminobenzidine (DAKO, Hamburg, Germany) as a peroxidase substrate. After visualization of the antigen-antibody complexes, the slides were counterstained with hematoxylin and covered with DePeX (Serva, Heidelberg Germany).
Stained and processed sections were digitalized using the BZ-9000 microscope from Keyence (Keyence, Neu-Isenburg, Germany). The densities of immunolabeled cells are expressed as cells per square millimetre within the respective region of interest (ROI). The activity of naphthol AS-D chloroacetate esterase (CAE) was used to visualize neutrophil granulocytes (91-C Kit; Sigma-Aldrich, Munich, Germany) according to the manufacturer’s instructions (8, 23).
Meningeal inflammation score:
The extent of granulocyte infiltration was used as a surrogate marker for meningeal inflammation. Granulocyte density was determined 30 h post-infection, in the following ROIs: frontal interhemispheric region, the hippocampal fissure (both sides), three randomly chosen superficial meningeal regions over the entire convexities, and within the third ventricle. For each region, granulocyte density was evaluated in one field of view (20 x objective), according to the following score: no granulocytes: 0; < 10 granulocytes: 1; 10 to 50 granulocytes: 2; and > 50 granulocytes: 3. The sum of the individual regions can therefore reach a maximum value of 21 (
24,
26).
RNA isolation and realtime RT-PCR analyses:
Following brain perfusion with 0.9% NaCl and brain dissection, RNA was prepared using peqGold Trifast reagent (Peqlab, Erlangen, Germany) as previously described (
23). For cDNA synthesis, moloney murine leukemia virus (MMLV) reverse transcriptase (Fermentas, Burlington, Canada) and random hexamer primers (Invitrogen, Darmstadt, Germany) were used. Gene expression levels were then determined using SYBR green (Biotool, Houston, USA) real-time PCR and a StepOne Plus apparatus (Applied Biosystems, Darmstadt, Germany). The ΔΔCt method was used for the relative quantification of gene expression levels. Ribosomal Protein L13a (
Rpl13a) was used as internal reference gene. To determine
Gfap expression levels, the QuantiTect Primer Assay from Qiagen (QT00101143, QT00156471; Qiagen, Hilden, Germany) was used. Primers used to measure
Rpl13a (
8), Tumor necrosis factor-α (
Tnf-α)(
24), Chemokine (C-C motif) ligand 3 (
Ccl3) (
27) and C-X-C motif chemokine (
Cxcl10) (
28) were produced by Eurofins MWG Operon (Ebersberg, Germany). The sequences and used annealing temperatures were listed in the cited publications. To exclude contamination of the reagents with either RNA or DNA, appropriate negative controls were performed. Melting curve analysis was used to determine reaction specificity whereas amplification efficiency was determined with the LinRegPCR software package (version 12.7) (
23).
Statistical analysis:
All data are given as the arithmetic means ± SEM. Differences between groups were statistically tested using the software package GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA). The D’Agostino and Pearson test was applied to test for Gaussian distribution of the data. Bacterial load (CFU/ml) was statistically compared with the non-parametric Mann-Whitney U-test. Real-time RT-PCR data were determined as duplicates. The four groups were statistically compared using the Two-way ANOVA followed by Bonferroni’s multiple comparison test. The definite statistical procedure applied for the different analyses is provided in the text and figure legends. p-values ≤ 0.05 were considered statistically significant. The following symbols are used to indicate the level of significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, ns = not significant.