All animal experiments were performed in strict accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and approved by the local Animal Care and Use Committee of the ‘Regierungspräsidium’ Giessen, Hessen, Germany.
Adult male Sprague Dawley rats (290 to 320 g) were purchased from Harlan Laboratories (Harlan, Rossdorf, Germany). They were housed five to a cage with food and water available ad libitum and were maintained on a 12-hour light/dark cycle (lights on at 07:00). For the experiments, rats were initially anesthetized with isoflurane, tracheotomized, paralyzed with pancuronium bromide (0.2 mg/kg/h) and mechanically ventilated (Harvard Rodent Ventilator; Harvard Apparatus, South Natick, MA, USA). The right femoral artery and vein were cannulated for blood pressure recording, blood sampling, and drug administration. Rectal body temperature was maintained at 37°C using a feedback-controlled heating pad.
Isoflurane anesthesia was discontinued and replaced by an intravenous application of α-chloralose (80 mg/kg; Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) for a period of 60 minutes to washout isoflurane. α-chloralose anesthesia was continued for the duration of the experiment. Supplementary doses of chloralose (30 mg/kg) were given every hour. During chloralose anesthesia, the animals were ventilated with a 1:1 mixture of nitrogen and oxygen. Arterial blood gas analyses and pH were measured repeatedly at least every 30 minutes (blood gas analyzer model Rapidlab 348; Bayer Vital GmbH, Fernwald, Germany). Glucose and lactate levels were measured simultaneously (Glukometer Elite XL, Bayer Vital GmbH, Fernwald, Germany; Lactate pro, Arkray Inc. European Office, Düsseldorf, Germany), while glucose concentrations were maintained at
60 mg/dl. A moderate volume therapy of 1.2 ml/hour of 0.9% NaCl was given to replace renal and perspirative fluid losses.
Neurovascular coupling measurements
The head of each animal was fixed in a stereotaxic frame without perforating the tympanum. The apex of the skull was exposed, and the bone over the left parietal cortex was thinned with a saline-cooled drill to allow transcranial laser Doppler flowmetry (LDF). The laser probe (BRL-100; Harvard Apparatus, South Natick, MA, USA) was placed 3.5 mm lateral and 1 mm rostral to the bregma in accordance with the coordinates of the somatosensory cortex, a location that corresponds closely to the region of maximal hemodynamic response during contralateral forepaw stimulation. The laser Doppler signal and the systemic mean arterial blood pressure were recorded continuously and processed on a personal computer running data acquisition software (Neurodyn; HSE, March-Hugstetten, Germany). Since the laser Doppler measures flow changes rather than absolute values, resting LDF signals had arbitrary units. However, evoked signal changes were used to assess flow changes, which are given in percentage changes from the baseline.
Brain electrical activity was recorded monopolarily with an active calomel electrode placed 0.5 mm behind the laser probe and an indifferent calomel electrode placed on the nasal bone. Signals were recorded and amplified (BPA Module 675; HSE, March-Hugstetten, Germany) and somatosensory evoked potentials (SEP) were averaged using the Neurodyn acquisition software (HSE, March-Hugstetten, Germany).
Somatosensory stimulation was achieved with electrical pulses applied by small needle electrodes inserted under the skin of the right forepaw (PSM Module 676; HSE, March-Hugstetten, Germany). The pulses consisted of rectangular bipolar pulses of 1.5 mA and a pulse width of 0.3 ms. The pulse repetition frequency was set at 2 Hz and stimulations were carried out for 30 seconds. Stimulation with 1.5 mA ensured that pain fibers were unaffected, thereby preventing changes in blood pressure. A rest of 30 seconds followed each stimulation train while activation rest cycles were repeated 10 times in order to increase the signal to noise ratio.
Flow velocity responses were averaged and calculated relative to the resting phase, which was set to zero. The evoked flow velocity responses (EFVR) were calculated from the averaged relative flow velocity signals under conditions of stimulation.
Evoked potential amplitudes were calculated from the N2-P1 amplitude differences and the latency between the beginning of stimulation and the occurrence of the P1 peak was examined.
Vagus nerve stimulation
The nervus vagus of each side was exposed at the cervical level lateral of the trachea running in parallel with the common carotid artery. Both vagus nerves were carefully dissected from their artery in each rat. The right vagus nerve mainly innervates the thoracic organs such as the lung and heart, whereas the left trunk mainly innervates the intestinal organs and the plexus mesentericus. For vagus nerve stimulation, the left nerve was cut. The distal part of the nerve was then fixed in a special nerve stimulation clamp (HSE, March-Hugstetten, Germany) to allow electrical stimulation of the distal trunk of the vagus nerve. To exclude aberrant innervations of the intestinal organs from the right nervus vagus, this nerve was also cut. Electrical pulses of 2 mA were applied during a 0.3 ms pulse width, and 2 Hz repetition frequency for a period of 10 minutes [15
]. Stimulation blocks were repeated every 45 minutes, starting before the application of endotoxin and continuing until the end of experimentation (4.5 hours). Additional experiments were performed to measure neurovascular coupling (Figure ).
Illustration of the preparation period and experimental protocol together with time points of vagal interaction or neurofunctional measurements.
In random order, 60 rats received 5 mg/kg body weight intravenous lipopolysaccharide (LPS, from Escherichia coli
, O111:B4; Sigma-Aldrich Chemie GmbH, Germany) or 0.5 ml of 0.9% NaCl. LPS was dissolved in 0.5 ml of 0.9% NaCl and slowly administered for 5 minutes. Endotoxinemic and sham groups were further divided into three subgroups. In one subgroup, bilateral cervical vagotomy (VGX) was performed without stimulation of the nerve (LPS group: VGX LPS; nonseptic group: VGX). In a second group, the distal trunk of the left vagus nerve was stimulated after bilateral cervical vagotomy (LPS group: VGX LPS
STIM; sham group: VGX
STIM). In a third group, the operation of the vagus nerve was stopped before vagotomy and the vagus nerve was not stimulated (LPS group: SHAM LPS; nonseptic group: SHAM). Each experimental group consisted of 10 animals. Experiments were performed up to 4.5 hours after LPS/vehicle administration since previous studies have shown that blood pressure levels remain above the lower limit of the cerebral autoregulation during this time period [7
], thus excluding secondary ischemic brain damage due to a decline in systemic blood pressure. Plasma samples were obtained before the rats were killed. Brains were removed and stored at −80°C.
Biochemical determination of the effects of VNS or VGX on inflammatory parameters
The following experiments were carried out on the endotoxinemic groups (SHAM LPS, VGX LPS, VGX LPS
STIM) and the non-treated control group (SHAM).
Rat cortex tissues obtained from three animals from each group were lysed in RIPA buffer containing Na3VO4, protease inhibitors, and PMSF. The lysates were resolved with SDS-PAGE (10% acrylamide) and transferred onto nitrocellulose membranes, blocked for one hour in 5% non-fat milk at room temperature and probed with the appropriate primary antibody overnight at 4°C at the following dilutions: β-actin (Abcam plc, Cambridge, United Kingdom; ab6276,1:3000), iNOS (Santa Cruz Biotechnology, Heidelberg, Germany; iNOS (M-19) sc-650,1:800), HIF-2α (Novus Biologicals, Cambridge, United Kingdom; NB100-132,1:500). After overnight incubation in primary antibody, membranes were washed with wash buffer and incubated with the respective horseradish peroxidase-conjugated polyclonal secondary antibodies for one hour at room temperature (RT). After washing, the blots were developed with an enhanced chemiluminescence (ECL) kit (GE Healthcare Europe GmbH, Freiburg, Germany) followed by film exposure. The reported optical densities are the means and SEMs of three separate experiments.
RNA isolation, real-time PCR (RT-PCR), and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from frozen rat cortex tissue using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocols. Each group consisted of n
6 rats. cDNA synthesis was performed with the ImProm-II™ Reverse Transcription System (Promega, Madison, WI, USA) by incubating 0.8 μg of RNA according to the manufacturer’s instructions. qRT-PCR was performed with 2 μl cDNA. The oligonucleotide primer pairs (rat origin) were as follows: rat porphobilinogen deaminase (PBGD) 5′-CAAGGTTTTCAGCATCGCTACCA-3′ (forward) and 5′-ATGTCCGGTAACGGCGGC-3′ (reverse); rat inducible nitric oxide synthase (iNOS) 5′-CAGCCACCTTGGTGAGGGGA-3′ (forward) and 5′- TCCAGGGGCAAGCCATGTCT-3′ (reverse); rat Bcl-2-associated X protein (BAX) 5′-CAAGAAGCTGAGCGAGTGTCT -3′ (forward) and 5′-CAATCATCCTCTGCAGCTCCATATT-3′ (reverse).
qRT-PCR was carried out in a Stratagene Mx3000P™ qPCR system (Stratagene, La Jolla, CA, USA) and data were analyzed with accompanying software. The cycling protocol was as follows: polymerase activation, 95°C for 10 minutes; 40 cycles with denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds and extension at 72°C for 30 seconds. To ensure that a single, specific product was generated, dissociation curves were evaluated. Relative expression levels were calculated as ΔCt values by normalizing Ct values of target genes to Ct values of PBGD as previously described. For every gene we performed three independent measurements. All data reported are the means and SEM of three separate experiments.
Following the experiments, blood samples were drawn into tubes containing heparin sodium 2500 (Ratiopharm, Ulm, Germany). They were immediately centrifuged and separated, and the plasma stored at −80°C until analysis. Cytokine determinations for IFN-γ, TNF-α, IL-6, IL-10 concentrations in plasma samples were performed with ELISA sets (BD Bioscience, Heidelberg, Germany) for each LPS group (n
6) as well as for the SHAM group.
Measurement of NO concentration in plasma samples
NO metabolite (nitrite and nitrate) concentrations were determined for each of 10 samples in the sepsis groups and in the SHAM group using NOA Sievers 280 (FMI GmbH, Seeheim, Germany) according to the manufacturer’s instructions. Briefly, plasma samples were drawn 4.5 hours after LPS injection into heparin-containing tubes, immediately centrifuged, and stored at −20°C until measurements were taken. NO reaction products in samples were reduced by vanadium chloride. Resulting gaseous NO was detected by NOA Sievers 280, which was connected to a computer for data transfer and analysis by NOAWIN 32 software (DeMeTec, Langgöns, Germany).
Statistical evaluation of physiological data
Statistical analyses were performed with a two-way ANOVA with the factors within and between groups to assess differences between the groups. In cases of significance, a Scheffe’s post hoc test was applied. For intergroup comparisons the statistical analyses were performed against baseline measurements. If the assumptions of normal distribution and equality of variances could not be assured in the statistic F-test, a nonparametric paired-sign or Kolmogorov-Smirnov test was conducted (Statview, SAS, Cary, NC, USA). The significance level was set to P