General animal preparation
Adult male Sprague Dawley rats (weighing 290 to 350 g) were purchased from Charles River (Sulzfeld, Germany). They were maintained on a 12-h light/dark cycle and housed four to a cage with food and water available ad libitum. Initially, rats were anesthetized with 1.5 to 3 % isoflurane, tracheotomized, paralyzed with pancuronium bromide (0.2 mg/kg/h; Inresa Arzneimittel GmbH, Freiburg, Germany) and mechanically ventilated (Harvard Rodent Ventilator; Harvard Apparatus, South Natick, MA, USA) with a 1:1 mixture of nitrogen and oxygen. For recording of blood pressure, blood sampling, and drug administration, the right femoral artery and vein were cannulated. Isoflurane anesthesia was then replaced by an intravenous application of an α-chloralose bolus (60 mg/kg; Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and maintained by a continuous administration of α-chloralose (40 mg/kg/h). A washout period of 60 min was maintained before the neurophysiological measurements were started. Arterial blood gas analysis and pH (blood gas analyzer model Rapidlab 348; Bayer Vital GmbH, Fernwald, Germany) as well as glucose (Glukometer Elite XL, Bayer Vital GmbH, Fernwald, Germany) and lactate (Arkray Inc. European Office, Düsseldorf, Germany) were measured at least every 60 min. To replace renal and perspirative fluid losses, a moderate volume therapy of 1.2 ml/h of 0.9 % NaCl was provided. Additionally, glucose concentrations in the blood were maintained at >60 mg/dl and body core temperature was maintained at 37 °C using a heating pad.
All animal experiments were conducted in strict accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and approved by the local ethics committee (ethics approval number GI 20/18–81/2011).
Neurovascular coupling measurement
The head of the animals was placed in a stereotaxic frame and the apex of the skull was exposed. To allow transcranial laser-Doppler flowmetry (LDF), the bone over the left parietal cortex was thinned with a saline-cooled drill (Dremel Moto-flex, Dremel Europe, Breda, The Netherlands) . In accordance with the coordinates of the somatosensory cortex, the laser probe (BRL-100, Harvard Apparatus, Holliston, MA, USA) was placed 3.5 mm lateral and 1 mm rostral to bregma; this corresponds to the region of maximal hemodynamic response during contralateral forepaw stimulation [16–18]. For the continuous recording and processing of the laser-Doppler signal and mean arterial blood pressure, a data acquisition software (Neurodyn, HSE, March-Hugstetten, Germany) was used.
Electric brain activity was measured monopolarily with one active calomel electrode about 0.5 mm behind the laser probe and one indifferent calomel electrode located on the nasal bone. Signals were recorded and amplified (BPA Module 675, HSE, March-Hugstetten, Germany) and in the following somatosensory evoked potentials (SEP) were averaged (Neurodyn acquisition software, HSE, March-Hugstetten, Germany). To calculate the SEP amplitudes, the difference between N2 and P1 was used.
To achieve somatosensory activation, rectangular bipolar pulses of 1.5 mA, 0.3 ms, and 2 Hz were applied by small needle electrodes placed under the skin of the right forepaw (PSM Module 676; HSE, March-Hugstetten, Germany). Stimulation with 1.5 mA ensured that there were no pain-associated changes in systemic blood pressure [16–18]. Electric stimulation was carried out for 30 s, followed by 30 s of non-stimulation. These activation-rest cycles were performed ten times to increase the signal to noise ratio.
Whereas the Doppler does not measure absolute values, signal changes closely correlate with flow changes [16, 17]. Therefore, flow velocity responses were averaged and the relative responses were calculated in relation to the resting phase, which was set to zero. The evoked flow velocity responses (EFVR) were then calculated from the averaged relative flow velocity signals under conditions of stimulation .
Vagus nerve stimulation
Both vagus nerves were exposed at the cervical level and carefully dissected from the common carotid artery in each rat. Animals received either no further manipulation of the vagus nerve (sham surgery (SHAM) and SHAM + LPS), a bilateral vagotomy (LPS + VGX), or a bilateral vagotomy and a stimulation of the distal trunk of the left nervus vagus (LPS + VGX + VNS) with a special nerve stimulation clamp (HSE, March-Hugstetten, Germany). For electrical stimulation, pulses of 2 mA, 0.3 ms, and 2 Hz were applied 15 min before administration of LPS and during the time course of the experiment and were only interrupted during the neurophysiological measurements .
Rats were assigned to the abovementioned groups in random order with 15 rats per group. Animals received 5 mg/kg body weight intravenous LPS (LPS from Escherichia coli, O111:B4; Sigma-Aldrich Chemie GmbH, Germany) resolved in 0.9 % NaCl or 0.5 ml of 0.9 % NaCl. LPS was administered slowly for 5 min. Experiments ended 4.5 h after LPS/vehicle administration, plasma samples were obtained, and brains were removed and stored at −80 °C. The reason for limiting the study to 4.5 h after LPS application was that the blood-brain barrier and the macrocirculation were still in the normal range .
Coronal 20 μm brain sections at the level of the subfornical organ (SFO, bregma −0.8 to −1.6 mm) were cut using a cryostat (2800 Grigocut E, Reichert-Jung, Nußloch, Germany). Brain sections were then thaw-mounted on poly-l-lysin-coated glass-slides and stored at −55 °C for immunohistochemistry. Additionally, brain sections between the vascular organ of the lamina terminalis and the median eminence (bregma 0.6 to −2.8) were stacked on glass-slides, and the hypothalamus was dissected. The cortex and the hypothalamus were divided into two pieces each (left and right hemisphere) and separately stored at −55 °C for polymerase chain reaction (PCR) analysis. Brain structures were identified using “The rat brain in stereotaxic coordinates” .
Frozen brain sections were briefly air-dried at room temperature and fixed in 2 % paraformaldehyde (Merck, Darmstadt, Germany) diluted in phosphate-buffered saline (PBS) for 10 min. The sections were then washed three times in PBS and incubated in a blocking solution containing PBS, 10 % normal donkey serum (NDS, Biozol, Eching, Germany), and 0.1 % Triton X-100 (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) for 1 h at room temperature. Subsequently, sections were incubated with the primary antibodies diluted in the blocking solution (rabbit-anti-myeloperoxidase (MPO), dilution 1:400, A 0398, Dako GmbH, Hamburg, Germany; goat-anti-intercellular adhesion molecule (ICAM)-1, dilution 1:1000, AF583, R&D Systems Inc, Minneapolis, MN, USA; goat-anti-nuclear factor interleukin 6 (NF-IL6), dilution 1:500, Sc-150-G, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 20–22 h at 4 °C. For the double labeling of neutrophil granulocytes and ICAM-1, the two antibodies were mixed in one solution. After washing three times in PBS, the sections were incubated for 2 h at room temperature with the secondary antibodies diluted in blocking solution (Alexa488-conjugated anti-rabbit IgG, dilution 1:500, AZA21206, MoBiTec GmbH, Göttingen, Germany; Cy3-conjugated anti-goat, dilution 1:600, 705-165-147, Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA). Following another three washing steps, cell nuclei were counterstained with 4.6-diamidino-2-phenylindole (DAPI, dilution 1:8000 in PBS, Mobitec GmbH, Göttingen, Germany) for 10 min. Finally, sections were washed three times in PBS, cover slipped using Citifluor (Citifluor Ltd., London, UK) and stored at 4 °C until analysis.
Specificity of the used antibodies has previously been tested . Moreover, control experiments by substitution of the primary antibodies with non-immunized animals IgG were performed.
A light/fluorescent Olympus BX50 microscope (Olympus Optical, Hamburg, Germany) and a black and white Spot Insight camera (Diagnostic Instruments, Visitron Systems, Puchheim, Germany) were used to acquire images from the stained sections. For each staining and time point, microphotographs were taken with the same exposure times using MetaMorph 18.104.22.168 software (Molecular Devices Inc., Downingtown, PA, USA). For NF-IL6 and the colocalization between neutrophil granulocytes and ICAM-1, the images were combined to RGB color images with help of the MetaMorph 5.05 software. All images were optimized for brightness and contrast using Adobe Photoshop 6.0 to the exact same extend within one analysis to preserve comparability (Adobe Systems Incorporated, San Jose, CA, USA).
For the quantification of nuclear NF-IL6 immunoreactivity, the DAPI-stained nuclei were labeled with “1” and counted on microphotographs with a ×100 magnification. Subsequently, NF-IL6-positive nuclei were labeled with “2”, counted, and given as percentage of all stained nuclei.
For the quantification of neutrophil granulocytes and ICAM-1, Fiji (Fiji Is Just ImageJ) software 2014 (open source software based on ImageJ modified by BioVoxxel, Mutterstadt, Germany) was used. After a median filter was applied to improve image noise, a threshold was set and the watershed function was used. Then, all particles over 20 pixels were automatically counted. For the number of neutrophil granulocytes, the number of particles on microphotographs with a ×5 and ×10 magnification and for the ICAM-1 immunoreactivity the percentage of the stained area on microphotographs with a ×20 magnification was used as readout.
The colocalization of neutrophil granulocytes and ICAM-1 was determined similar to the nuclear NF-IL6 immunoreactivity with a labeling of all neutrophil granulocytes and those that were colocalized with ICAM-1 on microphotographs with a ×10 magnification. The number of colocalized neutrophil granulocytes is given as percentage of all neutrophil granulocytes.
All images were processed in the same way, to guarantee comparability.
For all quantifications, three sections of all animals and five animals of each group were used. The mean of the three sections was taken for calculating the mean of each group.
Brain maps for overviews were modified from the digital version of “The rat brain in stereotaxic coordinates”  using CorelDraw 9 (Corel Corporation, Ottawa, Canada).
Total ribonucleic acid (RNA) of the collected frozen cortex and hypothalamic sections (approximately 40 to 50 mg tissue) was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Reverse transcription of 1 μg total RNA was performed using 50 U murine leukemia virus reverse transcriptase, 50 μM random hexamers, and 10 mM deoxynucleoside triphosphates (dNTP) mix (Applied Biosystems, Foster City, CA, USA) in a total reaction volume of 20 μl. Afterwards, quantitative real-time PCR was performed in duplicate with preoptimized primer/probe mixture (TaqMan Gene Expression Assay, Applied Biosystems, Foster City, CA, USA) and TaqMan universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The following cycling protocol was used: polymerase activation, 50 °C for 2 min, denaturation, 95 °C for 10 min, and 35 to 44 cycles of 15 s denaturation at 95 °C followed by 1 min of annealing and elongation at 60 °C.
The cDNA quantities were normalized by measurement of a housekeeping gene as a reference. Out of the six tested genes (Double-dye (Hydrolysis) probe geNorm 6 gene kit, ge-DD-6, Primer Design Ltd, Southampton UK), β actin (4352340E, Applied Biosystems, Foster City, CA, USA) was chosen as the best housekeeping gene using DataAssist 3.01 (Thermo Fisher Scientific Inc., Waltham, MA, USA) and NormFinder version 4 (Department of Molecular Medicine (MOMA) Aarhus University Hospital, Aarhus, Denmark).
Using the ΔΔCT method, sample values were calculated as x-fold difference from a control sample (SHAM, value determined as 1) within the same experiment. Samples from the same five animals of each group that were used for immunohistochemistry were also used for PCR analysis.
The following gene expression assays from Applied Biosystems were used: ICAM-1 Rn00564227_m1, NF-IL6 Rn00824635_s1, IkBα Rn01473658_g1, SOCS3 Rn00585674_s1, IL-1β Rn00580432_m1, IL-6 Rn01410330_m1, IL-10 Rn99999012_m1, IL-17 Rn01757168, TNFα Rn99999017_m1, Elane (neutrophil elastase) Rn01535456_g1, CD168 Rn01495634, CXCL1 Rn00578225_m1, COX2 Rn00568225_m1, and mPGES1 Rn00572047ml.
Blood samples were drawn into tubes with heparin sodium 2500 (Ratiopharm, Ulm, Germany) at the end of the experiment. They were immediately centrifuged and separated, and plasma was stored at −80 °C. Tumor necrosis factor (TNF)α (Rat TNF ELISA Kit, 560479, BD Bioscience, San Diego, CA, USA), interleukin (IL)-6 (Rat IL-6 ELISA Kit, 550319, BD Bioscience, San Diego, CA, USA), IL-10 (Rat IL-10 ELISA Kit, 555134, BD Bioscience, San Diego, CA, USA), and leptin (Rat Leptin ELISA, EZRL-83K, Merck KgaA Millipore, Darmstadt, Germany) plasma levels were measured using enzyme-linked immunosorbent assays according to the manufacturer’s protocol.
The detection limits for the assays were 31.3 pg TNFα/ml, 78 pg IL-6/ml, 15.6 pg IL-10/ml, and 0.2 ng leptin/ml, respectively. For TNFα, IL-6, and IL-10 measurements, 8 samples of the SHAM group and 13 samples of all septic groups were used, and for leptin, 5 samples of the SHAM group and 8 samples of all septic groups were used.
Statistical analyses were performed using a one-way analysis of variance. In cases of significance, a Fisher post hoc test was applied (Statview, SAS, Cary, NA, USA). The SEP and EFVR data were analyzed separately for each time point. The significance level was set to P < 0.05. All data are presented as means ± standard error of the mean (SEM) or as means ± standard deviation (SD), respectively.