strain: C57Bl/6J gender: male age: 12-14 weeks sepsis: Sham 3 days meropenem: s.c. 3 days enrofloxacin: no tissue: mouse whole brain
Experimental sepsis was induced by the standardized and established PCI model as described previously . In brief, PCI mice received an intraperitoneal injection of human fecal slurry (diluted 1:4 in saline solution, 3 µl/g B.W.) with a 21-gauge cannula. Control mice were injected with 100µl of 0.9 M saline solution. Mice were closely monitored and the Clinical Severity Score (CSS) was assessed as described previously. To avoid premature death due to acute severe peritonitis, mice were systemically treated with antibiotics. PCI mice received the first subcutaneous (s.c.) injection of the beta-lactam antibiotic meropenem (650µl, 1mg/ml) when the CSS reached a point value of 3. To increase long-time survival after PCI, meropenem was s.c. injected b.i.d. for 7 days and one time per day at the following three days. Thereafter, enrofloxacin (Baytril 2,5%, Bayer AG, Germany) was added to the sugared drinking water (saccharose solution 2,5%) in a final concentration of 2 mg/ml until the end of the experiment. Both, PCI and control animals received equal amounts of antibiotic treatment
All animal experiments were approved by the state authorities of Thuringia (UKJ-02-085-14) and were performed in accordance with animal welfare and the ARRIVE guidelines for reporting animal research. All efforts were made to minimize animal suffering and to reduce the number of animals used. Adult male C57BL6/J mice were kept in a 12 h light-/dark cycle in standard cages with free access to food and water.
Total cellular RNA from murine brain biopsies were isolated according to standard totalRNA extraction procedures using QIAzol lysis reagent and RNeasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturer instructions.
Label incorporation metho: Hybridized arrays were stained with 1 µg/µl Alexa Fluor 555 Streptavidin (Invitrogen, Karlsruhe, Germany), washed dried and scanned immediately on a Illumina iScan array Reader. Label used: Alexa Fluor 555 Streptavidin (Invitrogen, Karlsruhe, German).
250ng of total RNA of each sample were reversely transcribed and amplified using the TargetAmp-Nano Labeling Kit for Illumina Expression BeadChips (Epicentre, Biozym, Hessisch Oldendorf, Germany) a Biorad MJ Thermal Cycler (Biorad, München, Germany). All cRNA samples were quantified with a NanoDrop spectrophotometer (Nanodrop-Technologies) before proceeding to sample hybridization. Samples were prepared for hybridization according to the Illumina WGGEX Direct Hybridization Assay Guide (Illumina, San Diego, USA). Hybridization is performed according to the Illumina WGGEX Direct Hybridization Assay Guide (Illumina). Quantity of target used: 0.75 µg cRNA of each sample was hybridized on a Mouse-Ref-8 v2.0 Expression BeadChip (Illumina, San Diego, USA/Eindhoven, The Netherlands) containing 25,697 probes for murine genome-wide expression analysis and several control probes for sample- preparation, hybridisation, washing and staining quality Hybridization time: 20 h Volume: 15 µl Temperature: 58° C
Data processing was performed using Genome Studio Software V2011.1 and R Package “lumi”. Raw data was subjected to average normalization (RSN, log2 signal transformation), quality control and background correction. Bead types with detection values p < 0.01 in at least one sample (sham 3 d, sham 10 w, PCI 3 d, or PCI 10 w) were called ‘present’ and analyzed. Hierarchical clustering identified sample 9298740075_G as an outlier and was removed from further processing. Differentially expressed genes (DEG) with LFC=log2FC >1 for PCI vs. Sham at same time point for False Discovery Rate adjustment (FDR-adj.) were filtered. False Discovery Rate was adjusted at p-value <0.05 using the Welch-modified t-test.