Methods
Anesthesia and surgical preparation
After a fasting period of 4 hours, mice were anesthetized with 2% isoflurane in air
followed by an intraperitoneal (i.p.) injection of ketamine, midazolam and atropine. To ensure
adequate fluid resuscitation and perioperative antibiotic treatment, animals received a
subcutaneous (s.c.) injection of Lactated Ringer´s solution containing glucose, ceftriaxon, and
clindamycine. A laparotomy was performed, and the cecum was ligated. A single puncture
using a 18-gauge needle was performed, and the cecum was lightly squeezed to expel a small
amount of feces from the puncture site to ensure a full-thickness perforation. The abdomen
was closed thereafter. Postoperatively, water and food were provided ad libitum. After a
period of 8 hours, all mice received a second s.c. injection of fluids and antibiotics together
with buprenorphine to provide adequate postoperative analgesia. 15 hours post-CLP animals
were anesthetized by a second i.p. injection and placed on the procedure bench equipped with
heating pads and lamps to maintain body temperature between 36.5-37.5 °C. Thereafter, they
were tracheostomized to allow for endotracheal intubation and mechanical ventilation.
Normoventilation was controlled by monitoring the endexpiratory CO2 concentration. A
central venous line was placed in the right jugular vein, and anesthesia was maintained by
continuous i.v. ketamine and fentanyl, the infusion rates being identical in the three
experimental groups [1,18]. Since we had previously shown that CLP-challenged wild-type
mice died from refractory hypotension with fluid resuscitation alone, continuous intravenous
colloids and norepinephrine were administered simultaneously using a fixed catecholamine
concentration (0.033 µg·mL-1 hetastarch) [1]. This approach allowed a sustained 50 – 100%
increase in cardiac output (CO) at a mean arterial pressure (MAP) of that seen in shamoperated animals [1]. Practically, in septic animals the infusion rate of this solute was titrated
to maintain the MAP > 65 mmHg throughout the entire observation period and thereby to
achieve normotensive and hyperdynamic hemodynamics as indicated by a sustained increase
in CO. Normoglycemia was maintained by continuous i.v. glucose (2 mg·g-1·h-1) which
comprised 50% non-radioactive labelled 1,2,3,4,5,6,-13C6-glucose (Campro-Scientific GmbH
(ISOTEC), Berlin, Germany).
A 1.4-F catheter with pressure and conductance sensors (SPR 864, Millar Instruments,
Houston, TX, USA) was introduced into the heart via the right carotid artery. The
conductance catheter was interfaced with a pressure-volume analogue signal for the
assessment of systemic hemodynamics and cardiac output (CO) in mice as described
previously [18]. Thereafter,
perivascular flow probes were placed around the superior
mesenteric artery (SMA) and the portal vein (PV) to obtain regional blood flow data using a
multi-channel
ultrasonic transit-time flowmetry. Microvascular perfusion, capillary
hemoglobin concentration ([µHb]) and oxygen saturation (Hb-O2) were determined
simultaneously using combined laser Doppler flowmetry and remission spectroscopy
technique as described in detail previously [1]. Hemodynamic parameters recorded over 6
hours were mean arterial pressure (MAP), heart rate (HR), cardiac output (CO), SMA and PV
as well as the microvascular parameters.
Estimation of hepatic glucose production rate
Hepatic glucose production was estimated from the levels of intrahepatic glucose isotope
enrichment. Katz et al. previously showed that plasma and liver tissue isotope labelling values
are close to equilibrium [56, 57]. In fact, in separate sham-operated and CLP mice
simultaneous measurement of the isotope enrichment in liver tissue specimen and in
consecutive blood samples taken from the tail-tip yielded virtually identical results, and the
blood isotope enrichment did not show any time dependent variation (77.2, 77.4, 77.2 %
tracer/tracee ratio at 2, 4 and 6 hours after the start of the glucose isotope infusion,
rescpectively). These findings imply stationary conditions for the isotope labelling levels both
in the intravascular and the intrahepatic compartment. Consequently, based on these results
established tracer dilution concepts [23, 24] were applied on levels of the intrahepatic
glucose isotope enrichment to estimate the rate of hepatic glucose production. For this
purpose, glucose was isolated from the liver tissue samples and converted to a penta(trifluoro-acetyl) derivate (wildtype controls, n=12; iNOS-/- n=8, wildtype+GW274150, n=8).
Under positive chemical ionisation molecular fragments were analysed for the mass range m/z
319 – 326 using a gas chromatography-mass spectrometry system (Agilent 6890GC/5973MS,
Palo Alto, CA). The mass distribution derived from these ions was separated using standard
deconvolution approaches (Lee W-NP, Byerley LO, Bergner EA, Edmond J (1991) Mass
isotopomer analysis: theoretical and practical considerations. BiolMass Spect 1991, 20:451458) into a component arising from unlabelled and labelled recycling glucose, which mainly
contributes to the ions at m/z 320 and m/z 321 arising from the infused tracer, and a
component arising from the infused tracer. The level of the latter was used to estimate glucose
production.