53 male Wistar rats (Charles River, Sulzfeld, Germany) weighing 250 – 300 g were housed in groups under standard conditions at a temperature of 22°C (± 1°C) and a 12 hour light-dark cycle – with free access to standard food (Altromin, Soest, Germany) and tap water. Animal care and handling were performed according to the Declaration of Helsinki and approved by local ethical committees (approval number 50.203.2 BN 33,34/00).
Rats were randomized and received either 10 mg/kg of LPS (0127:B8, E. coli; Sigma, München, Germany) dissolved in 1 ml sodium chloride (0.9%) intraperitoneally (i. p.) or the vehicle alone. 24 hours after induction of sepsis, animals were anaesthetized with a combination of ketamine (80 mg/kg) and xylazine (10 mg/kg). The trachea was cannulated to facilitate respiration and rectal temperature was maintained at 37°C using a heating blanket. The femoral artery was exposed and catheterized with a polyethylene tube connected to a pressure transducer for continuous recording of arterial blood pressure and heart rate (Harvard Apparatus, March-Hugstetten, Germany). The head was fixed in a stereotactic frame and four stainless steel skull screws were placed epidurally, two electrodes per parietal bone at bregma coordinates 0 mm and -6.5 mm, 4 mm from the midline. A reference electrode was placed on the anterior midline over the frontal sinus. All electrodes were connected through insulated wire with a 2-channel amplifier (Harvard Apparatus, March-Hugstetten, Germany). Electrical brain activity was amplified (× 10 000 – 20 000), digitized and transferred to a PC for storage and further analysis. EEG was recorded for 15 – 20 min periods.
After EEG-recording, the screws were removed, and a 5 × 3 mm large cranial window was drilled (thinning of the scull until translucency, leaving the dura mater intact), centered 4 mm lateral and 4 mm caudal to the bregma. CBF was assessed using a laser flow blood perfusion monitor (PeriFlux 5000, Perimed, Stockholm, Sweden) with a 1.0 mm diameter laser Doppler probe (wavelength 780 μm; probe 407, Perimed, Stockholm Sweden). Local CBF was measured sequentially at 32 (8 × 4) parietocortical sites 300 μm apart, just above the dura over the exposed hemisphere using a micromanipulator as previously published . Data were sampled, on average, for 8 s at each site. Intraarterial pressure curve and CBF signals were transferred to a PC (Haemodyn, Harvard Apparatus, March-Hugstetten, Germany). Signals were sampled at 500 Hz with a 12 bit resolution. All experiments were carried out in accordance with the animal welfare guidelines and laws of the Federal Republic of Germany and were approved by the local ethics committee.
Small animal positron emission tomography (microPET)
microPET was performed on a 32-module quadHIDAC scanner (Oxford Positron Systems, Weston-on-the-Green, UK) dedicated to small animal imaging. The scanner has an effective resolution of 0.7 mm (FWHM) in the transaxial and axial directions when using an iterative resolution recovery reconstruction algorithm .
Before the scan, a tail vein catheter was inserted under short-term isofluorane anesthesia. The conscious rat was afterwards kept in a restraining device. 40 MBq of 18F-Fluordeoxyglucose (FDG) in 800 μl 0.9% saline were injected via the tail vein catheter. Following a 60 min interval, animals were again anaesthetized using isofluorane and placed in the PET scanner on a heating pad to maintain normal body temperature. List mode PET data were acquired for 15 minutes and subsequently reconstructed into a single image volume with a voxel size of 0.8 × 0.8 × 0.8 mm3.
Rats were perfused transcardially using heparanized saline and brains were subsequently dissected. Serial sagittal sections were cut (10 μm) from cryo-conserved preserved hemispheres (Leica Cryostat CM 3050S), embedded in tissue freezing medium (Leica Microsystems #0201-08926, Nussloch, Germany) and mounted (Microscope Slides #K0123b, Engelbrecht, Germany). After drying for 30 minutes at room temperature, for fixation slides were incubated in 4% paraformaldehyde (Roti Histofix 4% #P087.4, Roth, Karlsruhe, Germany) for 20 minutes. Blocking of non-specific binding was achieved by one hour incubation in 5% normal goat serum (Linaris #S-1000, Wertheim, Germany). Between the steps, slides were rinsed three times for five minutes in PBST. Immunostaining was performed overnight by incubation at 4°C with the following primary antibodies: 1.) polyclonal antibody rabbit-anti-mouse GFAP (1:1000 in 2% normal goat serum in PBST; DAKO Z0334, Glostrup, Denmark). 2.) monoclonal antibody mouse-anti-rat CD68/ED1 (1:100 in 2% normal goat serum in PBST; Serotec MCA341G, Düsseldorf, Germany). 3.) monoclonal rabbit anti mouse NeuN (1:250 in 2% normal donkey serum in PBS; Clone A60, Chemicon, Temecula, CA). Afterwards slides were incubated with Alexa Fluor 594-labeled secondary antibodies for one hour (1:400 in PBST; Invitrogen #A11037 & #A11020 Karlsruhe, Germany). For co-staining with Hoechst Dye 33342 (10 μg/ml; Fluka/Sigma-Aldrich #14533, Steinheim, Germany) an incubation time of two minutes was set. Again, slides were rinsed with PBST between the steps. Finally, the slides were covered in Mowiol 4–88 (Calbiochem/VWR #475904, Darmstadt, Germany) and stored at -20°C in the dark until microscopy was performed.
Real time PCR
RNA from brain hemispheres were extracted using Trizol (Life Technologies Invitrogen, Karlsruhe, Germany) using an Ultra Turrax (IKA Labortechnik, Staufen, Germany). Total RNA was quantified photometrically and reverse transcribed using the RevertAid First Strand cDNA Synthesis kit (Fermentas, St. Leon-Rot, Germany) according to the manufacturer's instructions. Real time qPCR was performed using the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, USA). Power SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, USA) was used for PCR amplification and real time detection of PCR products. 1 μl of the RT product corresponding to 40 ng of total RNA, 0.2 μM of each primer and 10 μl of the master mix were mixed and run under the following conditions: 95°C for 10 min and 40 cycles of 95°C for 15 s and 60°C for 1 min. Amplification specificity was checked using a melting curve analysis after PCR. mRNA expression was normalized to GAPDH. Primers used were: GAPDH forward ACG ACA GTC CAT GCC ATC AC and reverse TCC ACC ACC CTG TTG CTG TA, IL-1β forward GCT ACC TAT GTC TTG CCC GTG GAG and revers GTC CCG ACC ATT GCT GTT TCC TA, IL-4 forward GGA TGT AACGAC AGC CCT C and revers GAC ACC TCT ACA GAG TTT CC, IL-6 forward CTT GGG ACT GAT GTT GTT GA and revers CTC TGA ATG ACT CTG GCT TTG, IL-10 forward CCT GCT CTT ACT GGC TGG AG and reverse CTG CAG TAA GGA ATC TGT CAG, TNF-α forward AAA ACT CGA GTG ACA AGC CC and reverse GGT TGA CCT CAG CGC TGA GC, TGF-β forward TGC GCC TGC AGA GAT TCA AG and reverse TCT CTG TGG AGC TGA AGC AG, MCP-1 forward CTG TTG TTC ACA GTT GCT GC and revers CTG ATC TCA CTT GGT TCT GG and iNOS forward CCA GAG CAG TAC AAG CTC AC and revers CCA CAA CTC GCT CCA AGA TC.
Electrophysiological recordings were analyzed using a moving window analysis with a window length of 16.384 s (8192 data points). Prior to analysis signals were scanned for movement and recording artifacts using automated artifact detection. Windows containing constant signals for more than 100 consecutive points or signal jumps exceeding 10 standard deviations of a window's amplitude distribution were discarded.
Arterial pressure signals were decomposed into their instantaneous frequency and envelope signals using the Hilbert transform  in order to extract heart rate as well as systolic and diastolic arterial pressure. The shock index (SI) was determined from the ratio of the heart rate to systolic blood pressure. Doppler flow signals were averaged over time. EEG signals were subjected to spectral analysis to extract signal power in different frequency bands using conventional band definitions (delta 0.5–4 Hz, theta 4–8 Hz, alpha 8–13 Hz, beta 13–20 Hz, gamma 20–40 Hz). In order to quantify electroencephalographic signs of septic encephalopathy in terms of slowing of oscillatory brain activity, we determined the main EEG frequency from the maximum of the power spectrum.
All electrophysiological variables extracted in the moving window analysis were averaged over time (i.e., over different windows) for each animal. Results from different groups (septic animals vs. control animals) were compared using a nonparametric test (two-sided Mann-Whitney test for independent samples). In addition, to address the question whether electroencephalographic signs of encephalopathy exhibit dependence on hemodynamic sepsis parameters, we tested for significant correlation using a one-sided non-parametric correlation test (Spearman's rho).
For analyses of FDG-PET the individual volume data sets of all ten PET scans were coregistered and an averaged data set of the control group was calculated on pixel-by-pixel basis using the MPI-Tool (Advanced Tomo Vision, Kerpen, Germany). On this average image regions of interest (ROI) encompassing neocortex, caudate nucleus, thalamus and hippocampus were defined. ROIs were projected onto the individual data sets of all animals in both study groups to assess regional FDG uptake. Cerebral glucose uptake was finally quantified as the count ratio of individual ROIs and a reference ROI placed in the cerebellum in each animal.
For immunofluorescence quantification, 5 randomly chosen areas from 10 parallel sections per animal were analyzed using an Olympus BX61 microscope (Olympus, Hamburg, Germany) at 10× magnification. Evaluation was performed by determining the stained area using Cell^P software (Olympus Soft Imaging Solutions, Münster, Germany). Statistical analysis was performed using the Prism 4 Software, (GraphPad, San Diego, CA).