Anaesthesia and set-up
The study was approved by the Ethical Committee for Animal Research at Lund University, Sweden (application no. M180-10). The animals were treated in accordance with the guidelines of the National Institutes of Health for Care and Use of Laboratory Animals. Male adult Sprague-Dawley rats were used, weighing 337 ± 26 g (mean ± SD). Anaesthesia was induced using a covered glass container with a continuous supply of 5% isoflurane in air (Forene® 100%; Abbot Scandinavia AB, Solna, Sweden), in which the animals were placed. After induction, the animals were removed from the container, and anaesthesia was maintained with 1.5% to 1.8% isoflurane in air using a mask, while tracheostomy was performed. Thereafter, the animals were connected to a ventilator (Ugo Basile; Biological Research Apparatus, Comerio, Italy) and ventilated in a volume-controlled mode with a positive end expiratory pressure of 4 cm H2O. End-tidal PCO2 was continuously monitored (Capstar-100; CWE, Ardmore, PA, USA). Anaesthesia was maintained with 1.5% to 1.8% isoflurane in air throughout the experiment. Body temperature, measured rectally, was kept at 37.1°C to 37.3°C using a feedback-controlled heating pad. The left femoral artery was cannulated to monitor arterial blood pressure and to obtain blood samples for analysis of electrolytes, haematocrit, lactate, arterial blood gases (I-STAT; Abbot Point of Care Inc, Abbot Park, IL, USA) and plasma volumes. The left femoral vein was cannulated and used for infusions, and kept open with a continuous infusion of saline at 0.2 μL/min. The right internal jugular vein was cannulated and used for injection of 125I-albumin for plasma volume measurements. At the end of the experiments, the animals were sacrificed with an intravenous injection of potassium chloride.
Experimental procedure
A well-established rat model of severe sepsis was used [16, 18]. A longitudinal midline skin incision in the abdominal wall with diathermia was performed, followed by laparotomy by incision along the linea alba. After ligation just below the ileocaecal valve, an incision of 1 cm in length was made in the caecum, allowing leakage of faeces into the abdominal cavity, thereby inducing sepsis/systemic inflammatory response syndrome (SIRS). The abdominal wall and the skin were then closed with clips. There was no bleeding during the experiment.
Plasma volume
Plasma volume (PV) was determined with a reliable and established technique, shown to produce reproducible and reliable results [16, 19–21]. As described previously [16], PV was determined by measuring the radioactivity in 100 μL of plasma taken 5 min after an intravenous injection of human 125I-albumin (0.5 mL) with a known amount of activity. The increase in radioactivity was calculated by subtracting the activity in a blood sample taken just before the injection from that taken 5 min after the injection, thereby adjusting for any remaining radioactivity from previous measurements. To calculate the amount of radioactivity given, the remaining activity in the emptied vial, syringe and needle used was measured and subtracted from the total activity in the prepared dose. Sources of error are small with the technique used. Free iodine was measured regularly following precipitation with 10% trichloroacetic acid and was found to be less than 2.0% in the prepared samples. Radioactivity was measured with a gamma counter (Wizard 1480; LKB-Wallac, Turku, Finland).
Experimental protocol
In this study, we evaluated the effect of intravenous vitamin C on plasma volume in the early stage of sepsis in the rat. The septic rats were divided into three groups: a bolus + infusion group (the B + I group, n = 9), a bolus group (the B group, n = 9) and a sham group (the S group, n = 9). Animals that did not show a decrease in PV 3 h after the preparation were considered to be non-septic and were excluded from the study. These animals and animals that died before the end of the experiment were replaced with new animals.
After cannulation and surgical preparation, the animals were left undisturbed for 3 h, a time period previously shown to be sufficient for systemic inflammation and plasma leakage to develop [16]. Three hours after surgical preparation, the treatment was initiated. The B + I group received an intravenous injection of ascorbic acid (2,3-didehydro-l-threo-hexono-1,4-lactone, Askorbinsyra 100 mg/ml, APL, Stockholm, Sweden) of 66 mg/kg, followed by an infusion of 33 mg/kg/h during the rest of the experiment (Figure 1). In previous studies, this dose regime has been shown to be effective in decreasing microvascular permeability after burns in the rat [9, 10]. The B group received a single intravenous bolus injection of 200 mg/kg of ascorbic acid (Figure 1), previously shown to be effective in septic mice [4, 6]. The S group received no treatment, as it was meant to represent a non-treatment situation.
Plasma volumes were measured at baseline, at 3 h after the end of surgical preparation and at the end of the experiment another 3 h later. Blood samples for measurement of arterial pH, PCO2, PO2, lactate, haematocrit, sodium and potassium were taken at the same time points.
Urine was collected in a glass vial placed at the external meatus of the urethra throughout the whole experiment, and the bladder was emptied by external compression at the end of the experiment.
Statistical analysis
Statistical analyses were performed with GraphPad Prism software version 5.0c for Mac OS X (GraphPad Software, San Diego, CA, USA). Physiological data and plasma volumes were compared using two-way ANOVA for repeated measures followed by Bonferroni post hoc test and unpaired two-tailed Student's t test. Urine productions were compared using unpaired two-tailed Student's t test. Differences were considered significant when p < 0.05. To achieve a statistical power of 90% with a difference in PV (PV3 − PV2) between groups of 4 mL/kg, the calculated sample size for each group was 9. All data were normally distributed. The results are presented as mean ± SD.