Experimental model
This protocol received veterinary authority approval (Authorization notification No. 01008.02 from the French Ministry of Higher Education and Research).
We designed a randomized, blind, controlled, experimental prospective study using male Wistar rats (weight: 250–350 g) to compare 0.9% NaCl (Cl [154 mmol/L]) and PL (Cl [98 mmol/L]) used as resuscitation fluids in rats with severe sepsis induced by cecal ligature and puncture.
Surgical preparation
Induction of anesthesia was performed with isoflurane (Piramal Healthcare Inc., Mumbai, India), followed by an intraperitoneal injection of thiopental (Rotex Medica Inc., Trittau, Germany) and ketamine (Renaudin Inc., Paris, France). After additional local anesthesia on the surgical sites (described below), rats were placed on a heating support. Then, they were tracheotomized, ventilated and perfused with a central vein catheter inserted via the left femoral vein. Mean arterial pressure was continuously monitored by a left femoral arterial catheter. The left renal and carotid arteries were isolated, and transit time ultrasound Doppler probes (Transonic System Inc., Ithaca, USA) were placed in contact with them. The bladder was cannulated. Finally, a cecal ligature and puncture (CLP) was performed to induce sepsis. After this surgical preparation was completed (T0, end of preparation), the rats were randomized to 0.9% NaCl or balanced crystalloid (PL) groups.
Blinding and randomization methods
Before the start of the experiment, 0.9% NaCl and PL were sampled in 40 unmarked kits (20 of each fluid) containing 100 mL of 0.9% NaCl or PL and 10 mL of inulin diluted in 0.9% NaCl or PL (1 mg/mL). Kits were numbered randomly and supplied to the investigator who was blinded for the group assignment.
Sepsis induction and monitoring
After sepsis induction by CLP, depending on the randomization arm, the rats were hydrated with 1 mL/H of 0.9% NaCl or PL. Mean arterial pressure was monitored every 10 min until arterial pressure dropped below 90 mm Hg (T1, resuscitation baseline, T0 to T1 = supervision). The rats were then considered hypotensive, and depending on their randomization arm, continuous infusion was increased to 3 mL/H (base infusion). In addition, animals received additional fluid challenges targeted to maintain a mean arterial pressure at 90 mm Hg. The rats were monitored for 200 min from the T1 (T2, end of resuscitation, T1 to T2 = resuscitation) (Fig. 1).
Data collection
Heart rate, mean arterial blood pressure, pressure in the inferior vena cava considered as a surrogate of central venous pressure, left carotid and left renal arteries blood flow, core temperature and urine output were recorded every 10 min.
Doppler probes on left renal and left carotid arteries allowed a continuous monitoring of blood flows (displayed as a numeric mean value in milliliter per minute). The quality of signal was assessed by the adequate pulsatility and the absence of short-term lability (the lack of these criteria led to probe adjustments). The absence of contralateral stenosis, dilatation or renal malformation was controlled after animal killing.
Renal microcirculation was evaluated by measuring velocity in cortical capillaries with Side Dark Field Camera movies (Micro Vision Medical Inc., Amsterdam, the Netherlands). Videos were sampled at T1, T1 + 90 min and T1 + 180 min. Analyses were performed off-line with AVA software (Micro Vision Medical Inc., Amsterdam, the Netherlands). Twenty capillaries (5 capillaries per quadrant) per video were analyzed, and velocities were determined on, at least, three blood cells, as previously described [18].
Renal function was assessed by measuring inulin and creatinine clearance. We used FTIC-labeled inulin (Sigma Inc., Paris, France) diluted in the 3-mL/H base infusion of 0.9% NaCl or PL (depending on the randomization arm), with a 1-mg/mL concentration. Clearance was calculated at the end of the experiment according to the formulae: U × V/P (U for urine concentration, V for urine volume/time and P for plasma concentration). We used for inulin blood concentration measurement blood sampled at the end of resuscitation and for inulin urine concentration measurement urine sampled from the last hour of resuscitation. The urine volume corresponded to the volume sampled during the last hour of resuscitation. Rats of similar age and weight were supposed to have similar and normal baseline renal function. To reduce blood sampling volume before a severe surgical stress and to assess RIFLE score, we measured baseline blood creatinine in ten training test rats and considered the mean creatinine value for the RIFLE calculation.
Urine was collected at T0 and T2 to measure the concentration of chloride, sodium, potassium, blood urea nitrogen (BUN) and creatinine. Arterial blood was sampled at T2 to measure chloride, sodium, potassium, BUN, creatinine, hematocrit, proteins, pH, phosphate, calcium, bicarbonates, PaO2 and PaCO2.
Data analysis
Quantitative data are expressed as the mean and standard deviation. A nonparametric Mann–Whitney test was used for intergroup comparisons. For repeated measurements, we used a mixed linear regression model [19]. All statistics were calculated with R software 3.2.2 (free software developed by R Core Team (2008) for the R Foundation for Statistical Computing, Vienna, Austria) and Nlme R package version 3.1-122 (free software developed by Pinheiro J, Bates D, DebRoy S, Sarkar D and R Core Team (2015) for the R Foundation for Statistical Computing, Vienna, Austria). All tests were bilateral, and a p value of <0.05 was considered statistically significant.