Protocol
The study was carried out in accordance with the principles for the care and use of animals in research established by the Guide for the Care and Use of Laboratory Animals (NIH Guide) 11, Resolution 008430 of 1993 issued by the Colombian Ministry of Health, and Law 84 of 1989 issued by the Congress of Colombia, the "Estatuto Nacional de Protección de Animales" (National Statute for the Protection of Animals). Additionally, this research was conducted according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines 12.
Preparation of animals
This study was approved by the Bioethics Committee of the Faculty of Veterinary Medicine of Universidad Nacional de Colombia (CB-FMVZ-UN-031-19). The study was conducted at the simulation laboratory of Instituto de Simulacion Medica (INSIMED) from January to December 2019. Ten female pigs (Yorkshire 40 ± 1.0 kg; ± 4 months old) were premedicated using tiletamine-zolazepam (Zoletil®, Virbac Colombia) at 4.4 mg/kg doses through intramuscular injection. Then, each pig was cannulated through the marginal ear vein, and anesthesia was induced through the inhalation of isoflurane at a 1.5 minimum alveolar concentration (MAC) using an anesthesia mask. Once under general anesthesia, the pig was intubated and mechanically ventilated with a tidal volume of 10 ml/kg and a respiratory rate of 20 breaths/minute. For the maintenance of general anesthesia, isoflurane at doses of 1.5 MAC was used. Additionally, in all animals, the internal jugular vein and the femoral artery were cannulated using a central venous catheter (ARROW® CV-17702) and an artery catheter (PiCCO® PV2015L20-A). The quality of blood pressure signals was tested using a rapid washout test. All measurements were performed with the animal in a supine position, considering the phlebostatic axis as the zero reference.
Animals were placed on a stationary operating table and thermoregulated using medical blanket warmers, keeping their body temperature at a minimum level of 38°C. During the development of the model, intravenous fluid was administered using normal saline solution (SS) at an infusion rate of 3 ml/kg/hour.
Measurements
A monitor with the pulse contour cardiac output system (TFT Mindray BeneVision N22 Patient Monitor) was used. CO was calculated as the average of three thermodilution boluses (20 ml of < 8°C SS through the jugular venous catheter). SV was calculated as CO/heart rate (HR). Systemic vascular resistance (SVR) and PPV were automatically calculated by the PiCCO® system. Finally, MSFP was estimated using the method of Parkin 13: MSFP = 0.96 (CVP) + 0.04 (MAP) + 0.5 (CO).
Experimental protocol
After hemodynamic stabilization (variation in MAP < 10% for at least 10 minutes), animals were assigned to the control group (3 animals) or the endotoxin group (7 animals).
Animals in the endotoxin group received a continuous infusion of endotoxin (LPS E. Coli 055: B5, Sigma, St. Louis, MO) through the central venous catheter at an infusion rate of 7 µg/kg/hour that was increased every 10 minutes (7, 14, and 20 µg/kg/hour) until reaching 20 µg/kg/hour 14. The infusion of endotoxin ended when an MAP < 50 mm Hg for at least 10 minutes was achieved. Pigs in the control group were not administered endotoxin. Time zero (T0) was defined in the endotoxin group and the control group as the time immediately after hemodynamic stabilization.
When an MAP < 50 mm Hg was reached in the endotoxin group (TH0), a fluid load was administered at a dose of 20 ml/kg for 20 to 30 minutes through the central venous catheter. In the control group, a similar fluid load was administered at 3 hours of observation (TH0). This time was selected in the control group because it was the median time required to reach an MAP < 50 mm Hg for at least 10 minutes during the pre-experimental standardization phase of the endotoxin models. Then, a noradrenaline infusion at a dose of 0.05 mcg/kg/minute was started after the fluid load, and the infusion rate was increased 0.05 mcg/kg/minute every 5 minutes until an MAP of 65 mmHg was reached. Noradrenaline was not administered in the control group. After completing the protocol, all animals were euthanized by a certified veterinarian with a bolus of pentobarbital + diphenylhydantoin at 100 mg/kg (Euthanex®), in accordance with the international criteria for this procedure established by the American Veterinary Medical Association (AVMA).
Fluid challenges
A total of 6 fluid challenges were performed in each group. The first three fluid challenges were performed every hour starting at T0 (T0, T1, and T2). Afterward, three fluid challenges were performed every hour starting at TH0 (TH0, TH1, and TH2). Hemodynamic measurements were performed each time. This approach allowed us to assure that both groups had the same amount of fluid administered and that the variables could be compared.
All fluid challenges consisted of 4 ml/kg SS IV infused for 5 minutes through a central venous catheter. This is a standardized approach to perform a fluid challenge in humans 15. Animals in which a fluid challenge induced an increase in CO > 10% were defined as fluid responders 6. CO was measured by transpulmonary thermodilution in all cases. The study protocol is depicted in Fig. 1.
Statistical analysis
Data are presented using medians and interquartile ranges. Receiver operating characteristic (ROC) curve analysis was conducted for each fluid challenge to assess changes in the PPV operative performance over time in the endotoxin group. The cutoff was calculated as the maximum (sum (sensitivity + specificity)).
Two-way analysis of variance (ANOVA) was conducted to assess differences between both groups. Additionally, one-way ANOVA was performed to determine changes in the variables over time in the endotoxin group. Post hoc analysis was carried out using the Bonferroni test.
A linear mixed model was performed to determine the associations between PPV and SVR, MAP, SV, baseline PPV, MSFP, and the endotoxin or the control group. Time was used as the random effect, the variables in the model were assessed by the restricted maximum likelihood (REML), and the contribution on each variable was quantified using the estimated value and its standard deviation. Models were compared using the Akaike information criterion (AIC), the Bayesian information criterion (BIC), and the REML. The models with the lowest AIC and BIC were considered the best models.
Data were analyzed using R statistical software. A p value < 0.05 was considered statistically significant.