C57BL/6 mice were purchased from Charles River (Sulzfeld, Germany). TLR-1 knock out (KO) mice were purchased from Oriental BioService, Inc (Tokyo, Japan) and TLR-2 KO mice (B6.129-Tlr2tm1Kir/J) were bought from the Jackson Laboratory (USA). All KO mice were on the C57BL/6 background. Mice were housed in a 12 h light-dark cycle and bred at Experimental Biomedicine (Gothenburg University, Gothenburg, Sweden) and were provided with a standard laboratory chow diet (B&K, Solna, Sweden) and drinking water ad libitum. All animal experiments were approved by the Ethical Committee of Gothenburg (No.277-07 and 374-09).
Induction of hypoxia-ischemia in neonatal mice
At postnatal day (PND) 9, mice were anesthetised with isoflurane (3.0% for induction and 1.0-1.5% for maintenance) in a mixture of nitrous oxide and oxygen (1:1). The left common carotid artery was ligated with prolene sutures (the whole procedure was less than 5 min). Mice were returned to the cage and allowed to recover for 1 h and then placed in an incubator circulated with a humidified gas mixture (10.00 ± 0.01% oxygen in nitrogen) at 36°C for 50 min. After hypoxia, the pups were returned to their dam until sacrifice. Control animals received no surgery and were not subjected to HI. Two wild type and one TLR-1 KO mice died during the HI procedure.
Tissue collection and processing
For mRNA expression analysis, wild type pups were deeply anesthetized and intracardially perfused with saline at 30 min (n = 5), 6 h (n = 5) and 24 h (n = 5) after HI. Brains were rapidly dissected out, divided into ipsi- and contralateral hemispheres, snap frozen and then stored at -80°C until analysis. Brain tissue was homogenised with Qiasol lysis reagent homogeniser (Qiagen, Solna, Sweden) and total RNA was extracted using RNeasy Lipid Tissue Mini Kit (Qiagen, Solna, Sweden) according to the manufacturer's instructions. RNA was measured in a spectrophotometer at 260-nm absorbance.
For immunohistochemical analysis, animals were deeply anesthetised and intracardially perfused with saline and 5% buffered formaldehyde (Histofix; Histolab, Sweden) at 30 min, 6 h, 24 h and 5 days after HI. Brains were rapidly removed and immersion fixed in 5% formaldehyde for 24 h. Brains were then kept in a 30% sucrose solution until they were cut or put through dehydration with graded ethanol and xylene and embedded in paraffin. Coronal sectioning (25 μm/section) was performed on a sliding microtome (Leica SM2000R, Leica Microsystems, Sweden), and sections were stored in tissue cryoprotectant solution (25% ethylene glycol, 25% glycerol and 0.1 M phosphate buffer) at -20°C. For detection of infarction volume paraffin embedded tissue was used. Paraffin embedded brains were cut coronally (10 μm/section) on a rotating microtome (Leica RM2165, Leica Microsystems, Sweden).
TLR signalling pathway RT2-PCR-Profiler PCR Array
cDNA-synthesis was performed by using the RT2 First Strand Kit (SABiosciences, Frederick, MD, USA) following the manufacturer's instructions. The mouse TLR signalling pathway RT2-PCR-Profiler PCR Array (SABiosciences, Frederick, MD, USA) was carried out according to manufacturer's instructions using the LightCycler 480 system (Roche, Bromma, Sweden). The raw data obtained from the Lightcycler 480 software was uploaded into GEarray Analyzer software (SABiosciences, Frederick, MD, USA) for analysis.
Reverse transcription-quantitative PCR
To confirm the RT2-PCR-Profiler PCR Array results, TLR-1 and -2 mRNA expressions were determined by reverse transcription-quantitative PCR (RT-qPCR). Superscript RNase H- reverse transcriptase kit (Invitrogen, CA, USA) random hexamer primers and dNTP (Roche Molecular Biochemicals, IN, USA) were used to synthesise first strand cDNA as previously described . Each PCR (20 μl) contained 2 μl cDNA diluted 1:8, 10 μl Quanti Fast SYBR Green PCR Master Mix (Qiagen, Sweden) and 2 μl PCR primer. The following primers were used: TLR-1 QuantiTech Primer Assay (QT00157430), TLR-2 QuantiTech Primer Assay (QT00129752) and Hprt-1 QuantiTech Primer Assay (QT00166768), all from Qiagen, Sweden. The amplification protocol comprised an initial 5 min denaturation at 95°C, followed by 40 cycles of denaturation for 10 sec at 95°C and annealing/extension for 30 sec at 60°C on a LightCycler 480 (Roche, Sweden). Melting curve analysis was performed to ensure that only one PCR product was obtained. For quantification and for estimation amplification efficiency, a standard curve was generated using increasing concentrations of cDNA. The amplification transcripts were quantified with the relative standard curve and normalised against the reference gene hypoxanthine guanine phosphoribosyltransferase-1(Hprt-1).
Immunohistochemical staining was performed on free-floating 25 μm sections pretreated with 0.6% H2O2 in Tris-buffered saline (TBS; 0.15 M NaCl and 0.1 M Tris-HCl, pH 7.5) for 30 min to block endogenous peroxidase activity. Nonspecific binding was blocked for 30 min in blocking solution (3% goat serum and 0.1% Triton-X 100 in TBS). After rinsing, sections were incubated with primary antibody (TLR-1, IMG-5012, 1:500, TLR-2, IMG-526, 1:100, Imgenex, CA, USA) in blocking solution at 4°C for 48 h. The tissue sections were then incubated for 1 h with biotinylated goat anti-rabbit IgG secondary antibody (1:500, Vector Laboratories, CA, USA) in blocking solution and then rinsed in TBS. Visualisation was performed using Vectastain ABC Elite (Vector Laboratories, CA, USA) with 0.5 mg/ml 3, 3'-diaminobenzidine (DAB) enhanced with 0,01% H2O2 and 0,04% NiCl (all from Sigma-Aldrich, Sweden). Sections were analysed on an Olympus BX60 fluorescence microscope equipped with an Olympus DP50 cooled digital camera.
For paraffin sections, antigen recovery was performed by boiling the sections in 10 mM sodium citrate buffer (pH 6.0) for 10 min. Nonspecific binding was blocked for 30 min in blocking solution (1% horse serum, 3% bovine serum albumin, 0.1% NaN3 in phosphate buffered saline (PBS)). Sections were incubated in primary antibody against microtubule-associated protein-2 (MAP-2; clone HM-2, 1:1000; Sigma-Aldrich) at 4°C overnight, followed by biotinylated horse anti-mouse secondary anti body (1:250, Vector Laboratories) for 60 min at room temperature. Visualization was performed using Vectastain ABC Elite with 0.5 mg/ml 3,3--diaminobenzidine enhanced with 15 mg/ml ammonium nickel sulfate, 2 mg/ml β-D-glucose, 0.4 mg/ml ammonium chloride, and 0.01 mg/ml β-glucose oxidase (all from Sigma-Aldrich).
To identify the cell type specific localisation of TLRs, multi-immunofluorescence staining was performed. Non-specific binding was blocked for 30 min in blocking solution (3% donkey serum and 0.1% Triton-X 100 in TBS). TLR antibodies were then incubated simultaneously with antibodies against specific markers for neurons (NeuN 1:1000, MAB377, or alexa 488 conjugated NeuN 1:1000, MAB377X, Chemicon International, USA, and HuC/D 1:500, A21271, Molecular Probes, Netherlands), oligodendrocytes (Olig2 1:1000, AF2418, R&D systems, UK), microglia (Iba-1 1:1000, ab5076, Abcam, UK), and astrocytes (GFAP 1:1000, PCK-591P, Covance, USA) diluted in blocking solution for 48 h at 4°C. Samples stained for HuC/D were subjected to sodium citrate antigen retrieval (10 mM NaCi, pH6 for 30 min at 80°C) prior to blocking. Immunoreactivity was visualised via appropriate combinations of the following secondary antibodies: donkey anti-chicken DyLight 549 (1:1000, Jackson ImmunoResearch, USA), donkey anti-mouse Alexa 488, donkey anti rabbit Alexa 594, donkey anti-goat Alexa 647, and donkey anti-rabbit Alexa 488 (1:1000, all from Molecular probes, Netherlands). Multi-channel confocal images were captured with a Leica TCS SP2 confocal system (Leica, Heidelberg, Germany) with channel settings appropriate to the fluorophores present. Sequentially scanned, grey scale Z-stacks were pseudocolored and processed in ImageJ (version 1.42u; National Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij) before final processing in Adobe Photoshop (version 11.0.2; Adobe Systems Inc., San Jose, CA).
Brain infarct evaluation
For brain infarct evaluation, coronal paraffin sections throughout the hippocampus were used. Every 40th section was stained for MAP-2 and analyzed under a Nikon Optiphot-2 microscope equipped with an Olympus DP50 cooled digital camera. Images were captured and processed using Micro Image version 4.0 (Olympus). Infarct area was assessed as the MAP-2 negative area in the ipsilateral hemisphere and total infarct volume was calculated according to the Cavalieri Principle using the following formula: V = ∑A × P × T, where V = total volume, ∑A = the sum of areas measured, P = the inverse of the sections sampling fraction, and T = the section thickness.
Data analysis and Statistics
For normalisation of gene expression on the RT2-PCR-Profiler PCR Array, five housekeeping genes, β-glucuronidase, Hprt-1, heat shock protein 90 kDa alpha, glyceraldehyde-3-phosphate dehydrogenase, and β-actin, were used. The cycle threshold (CT) was determined for each sample and normalised to the average CT of the five housekeeping genes. A comparative CT method was used to calculate relative gene expression. Data are represented as fold change relative to control. The p-value was calculated using a student's t-test (two-tailed, equal variance).
Brain infarct data were analyzed with one way ANOVA followed by Dunnett's Multiple Comparison Test to compare total infarct volume between genotypes or infarct area at each brain level between groups (WT, TLR-1 KO and TLR-2 KO). Data are presented as mean ± SD and significance was set at p ≤ 0.05. All statistical analyses were performed using GraphPad Prism 4.0 (GraphPad Software).