Eighteen New-Zealand rabbits (weight 2.5 ± 0.1 kg), supplied by the Reproduction Laboratory of the University of Cadiz, were maintained at a controlled temperature (23°C) in individual cages on a 12-h light/dark cycle with free access to food and water up to the time of experimental procedures. Animals were allowed to acclimatize to the laboratory for one week before the beginning of the experiments. All methods and protocols in this investigation were reviewed and approved (project 06-04-15-230) by the Ethical Committee for Animal Experimentation of the School of Medicine of the University of Cadiz (license 07–9604) and the Dirección General de la Producción Agrícola y Ganadera of the Junta de Andalucía. Animal care and use procedures conformed to national and European Union regulations and guidelines (Spanish Royal Decree 53/2013 and EU Directive 2010/63/EU).
Anesthesia and instrumentation. Animals were premedicated with an intramuscular dose of xylazine (10 mg·kg−1) and ketamine (40 mg·kg−1). Then they were tracheotomized and their lungs mechanically ventilated in volume-controlled mode (Servo 900c, Siemens-Elema, Solna, Sweden), with 8 ml·kg-1 of tidal volume, positive end-expiratory pressure of 0 cmH2O, an inspiration to expiration ratio of 1:2, inspired oxygen fraction of 0.6, and a respiratory rate of 35-40 breaths·min-1 adjusted to maintain an end-tidal CO2 between 35–45 mmHg. The right internal jugular vein was catheterized for continuous sedation with ketamine (10–40 mg·kg-1·h-1) and midazolam (1–3 mg·kg-1·h-1). The muscular blockade was maintained with a rocuronium bromide infusion (0.6–1.2 mg·kg-1·h-1)[14]. Adequacy of anesthesia before neuromuscular blockade and through the experiment was evaluated by the absence of any significant blood pressure and/or heart rate change in response to an external noxious stimulus. A Ringers lactate solution (6 ml·kg-1·h-1) was administered as a maintenance fluid therapy. Temperature was continuously monitored by a rectal probe and maintained between 38−40º using a heating pad. A 22G sterile polyethylene catheter was introduced into the right femoral artery and connected to a pressure transducer (TruWave, Edwards Lifesciences LLC, Irvine, CA, USA). This was zeroed against atmospheric pressure, and optimal damping of the arterial waveform was checked by a square wave test. The left femoral vein was used to administer vasoactive drugs and fluid bolus.
Hemodynamic monitoring. A pediatric esophageal Doppler probe (KDP72; CardioQ Combi, Deltex Medical, Chichester, UK) was introduced into the esophagus until the best outline and maximal peak velocity of aortic blood waveform was obtained. Cardiac output (CO) was calculated using the minute distance of aortic blood flow, which represents the distance traveled by a column of blood in 1 min and is calculated by the Doppler system as the product of HR and the velocity-time integral of the aortic flow waveform. The arterial pressure signal was transferred from the multi-parametric monitor (S/5, Datex-Ohmeda, Helsinki, Finland) to the Doppler system and automatically synchronized with the aortic blood flow waveform for pressure-flow analysis.
Evaluation of arterial load. A 3-element Windkessel model was used for characterizing the arterial system[15], consisting of systemic vascular resistance [SVR=mean arterial pressure (MAP) / CO]; arterial compliance (Cart= stroke volume / arterial pulse pressure)[16] and characteristic impedance (Zc). Zc represents the arterial input impedance in the absence of arterial wave reflections[17]. Assuming that arterial reflections are negligible during early systole[17, 18], Zc was estimated as the slope of the early ejection pressure-flow relationship, using the ratio between the maximum of the first derivate of pressure and flow averaged during one minute [19, 20].
The effective arterial elastance (Ea) was used as a lumped parameter of arterial load accounting for both mean and pulsatile components[21]:
[Please see the supplementary files section to view the equation.]
Where RT is the total mean vascular resistance (SVR + Zc), ts and td are systolic and diastolic periods, respectively, and τ the diastolic time constant (τ = SVR × Cart) [21].
PPV, SVV, and dynamic arterial elastance. PPV was calculated as the percentage changes in arterial pulse pressure during a ventilatory cycle as [(PPmax – PPmin) / (PPmax + PPmin) /2] × 100, where PPmax and PPmin represent the maximal and minimal arterial pulse pressure, respectively. The calculation of SVV was performed similarly. SVmax and SVmin were calculated by integrating the systolic component of aortic blood flow waveform, whereas PPV was derived from the femoral arterial pressure waveform. PPV and SVV values were simultaneously analyzed and averaged during 1-min using a custom Excel macro (Microsoft Corporation, Redmond, WA, USA) (Figure 1). Eadyn was calculated as PPV/SVV [22].
Left ventricular energetics. Left ventricular energetics were analyzed from the instantaneous pressure and flow recordings. Aortic blood flow and femoral arterial pressure waveforms were recorded simultaneously during at least 20 seconds at 180 Hz and ensemble-averaged, foot-to-foot aligned using the maximum of the second derivative, and interpolated to the duration of the cardiac cycle to provide a representative waveform for analysis. An illustrative example of the signal processing is shown in Figure 2. The contribution of kinetic energy was considered negligible and not included in the analysis[18].
The left ventricular (LV) total power (Ẇtot) transferred to the systemic circulation was calculated as the time-averaged integral of the instantaneous product of blood pressure (P) by flow (Q) during the whole cardiac period (T):
[Please see the supplementary files section to view the equation.]
The product of mean pressure by mean flow, or steady power (Ẇstd), corresponds to the energy maintaining forward blood flow and represents the fraction of Ẇtot useful for organ perfusion [18, 23, 24].
[Please see the supplementary files section to view the equation.]
The oscillatory power (Ẇosc) refers to the energy lost in pulsatile phenomena due to cardiac contractions:
[Please see the supplementary files section to view the equation.]
Cardiovascular efficiency. The oscillatory power fraction (%Osc) represents the portion of Ẇtot wasted in oscillatory power and quantifies the efficiency with which the external mechanical power was transferred into useful work from the LV to the arterial system [23-25]. Therefore, the lower the %Osc, the more efficiently LV external work is delivered to the arterial system and converted into useful work for creating blood flow. %Osc has been used as a measure of the optimization of ventriculo-arterial coupling[23, 24].
[Please see the supplementary files section to view the equation.]
We also calculated the LV power necessary for generating one unit cardiac output for a given arterial load, as the energy efficiency ratio (EER) [25, 26]:
[Please see the supplementary files section to view the equation.]
Therefore, the lower the EER, the lower the energy required for generating blood flow for a given LV afterload.
Tissue O2 saturation. Tissue oxygen saturation (StO2) was used as a surrogate for microcirculatory perfusion. StO2 was continuously assessed by using near-infrared spectroscopy (NIRS) with the INVOS 5100C monitor (Covidien, Boulder, CO, USA), as previously described[27]. A neonatal self-adhesive sensor was placed over a shaved and cleaned skin of the lateral area of the hind leg (biceps femoris muscle), contralateral to the femoral venous access. The sensor was held on place with adhesive tape and covered with an opaque wrapper to avoid ambient light interference. Measurements were obtained every 4 secs, and 1-min averaged for analysis.
We also considered the ratio between LV mechanical work and StO2 as a measure of the work required for the cardiovascular system for given tissue perfusion, which represents an index of the performance of the coupling between the central hemodynamics and the microcirculation.
[Please see the supplementary files section to view the equation.]
So, the lower the Mmi, the lower the LV mechanical work required for sustaining tissue perfusion.
Experimental protocol. After completion of the surgical procedures, animals were allowed to stabilize MAP and HR (variation <5%) at least for 10 minutes. After that, they were assigned using a computer-generated random sequence to three groups (6 animals each): a sham-operated group (SHAM), a septic group (EDX), and a septic group with hemodynamic resuscitation (EDX-R). In septic animals, a purified lipopolysaccharide (LPS) prepared from Escherichia coli serotype 055:B5 (Sigma Chemical, St. Louis, MO) was intravenously infused over 10 mins through the femoral vein (1 mg·kg-1 diluted in normal saline for a total volume of 8 ml, and flushed by 2 ml of normal saline to ensure a complete delivery). The dose and rate of LPS administration were previously established on a pilot experiment with 8 animals, in which the dose was varied from 1 to 2 mg·kg-1. SHAM animals received an equivalent amount of normal saline. Three hours after LPS infusion, animals in the EDX-R group received a fluid bolus of 20 ml·kg-1 for 10 min. A norepinephrine infusion started at 0.25 mcg·kg-1·min-1 was started after fluid administration if MAP was below the baseline level. Norepinephrine was increased by 0.10 mcg·kg-1·min-1 every 3 mins until reaching a MAP value similar to the baseline level (±5% deviation). Hemodynamic measurements, aortic blood flow, and arterial pressure waveforms were recorded a least during 1 min at baseline and every hour up to 4 hours after LPS or placebo administration. In EDX-R animals, measurements after fluid bolus (post-infusion) and norepinephrine infusion (which corresponds to 4 hours after LPS infusion) were also obtained. After completion of the study protocol, animals were euthanized using a lethal dose of intravenous potassium chloride under deep anesthesia. Animal death was confirmed by verification of the absence of blood flow and arterial pressure tracings.
Statistical analysis. Data are expressed as the mean ± standard deviation or median (25th to 75th interquartile). The normality of data was checked by the Shapiro Wilk test. Differences between groups at baseline were performed using a one-way analysis of variance (ANOVA), and differences over time were assessed by two-way mixed ANOVA for repeated measurements. The Greenhouse–Geisser correction was used when the Mauchly test detected violation of sphericity. Whenever a significant interaction was found, pairwise comparison between groups was performed using a one-way ANOVA with the Tukey-Kramer test. Mixed-effect regression analyses were used to evaluate the impact of sepsis on the relationship between Eadyn (the dependent variable) and arterial load (Ea, and its determinants: SVR, C, and Zc), cardiac function variables (heart rate and maximal acceleration of aortic blood flow, as an index LV performance[28]); cardiac energetics (Ẇtot and its components: Ẇstd and Ẇosc) and variables of cardiovascular efficiency (%Osc and EER). The effects of sepsis on Eadyn were evaluated in the EDX group and on animals in the EDX-R group before the hemodynamic resuscitation. Models were constructed using individual animals and experimental groups as subjects for random factors, and experimental stages as repeated measurements with a heterogeneous first-order autoregressive covariance structure. Model selection based on the corrected Akaike’s Information Criteria, in which lower scores indicate superior fit [29]. Model parameters were estimated via the restricted maximum likelihood method, and the estimated fixed effect of each parameter quantified by using estimated value and standard error (SE). A P value < 0.05 was considered statistically significant. All statistical analyses were two-tailed and performed using MedCalc Statistical Software version 19.1 (MedCalc Software bvba, Ostend, Belgium; https://www.medcalc.org; 2016).