Autonomic and circulatory alterations persist despite adequate resuscitation in a 5-day sepsis swine experiment

Abstract

Autonomic and vascular failures are common phenotypes of sepsis, typically characterized by tachycardia despite corrected hypotension/hypovolemia, vasopressor resistance, increased arterial stiffness and decreased peripheral vascular resistance. In a 5-day swine experiment of polymicrobial sepsis we aimed at characterizing arterial properties and autonomic mechanisms responsible for cardiovascular homeostasis regulation, with the final goal to verify whether the resuscitation therapy in agreement with standard guidelines was successful in restoring a physiological condition of hemodynamic profile, cardiovascular interactions and autonomic control. Twenty pigs were randomized to polymicrobial sepsis and protocol-based resuscitation or to prolonged mechanical ventilation and sedation without sepsis. The animals were studied at baseline, after sepsis development, and every 24 h during the 3-days resuscitation period. Beat-to-beat carotid blood pressure (BP), carotid blood flow, and central venous pressure were continuously recorded. The two-element Windkessel model was adopted to study carotid arterial compliance, systemic vascular resistance and characteristic time constant τ. Effective arterial elastance was calculated as a simple estimate of total arterial load. Cardiac baroreflex sensitivity (BRS) and low frequency (LF) spectral power of diastolic BP were computed to assess autonomic activity. Sepsis induced significant vascular and autonomic alterations, manifested as increased arterial stiffness, decreased vascular resistance and τ constant, reduced BRS and LF power, higher arterial afterload and elevated heart rate in septic pigs compared to sham animals. This compromised condition was persistent until the end of the experiment, despite achievement of recommended resuscitation goals by administered vasopressors and fluids. Vascular and autonomic alterations persist 3 days after goal-directed resuscitation in a clinically relevant sepsis model. We hypothesize that the addition of these variables to standard clinical markers may better profile patients' response to treatment and this could drive a more tailored therapy which could have a potential impact on long-term outcomes.

Introduction

The autonomic nervous system (ANS) plays a central role in maintaining cardiovascular homeostasis both in physiological and pathological conditions; there is, indeed, a large body of research works reporting a failure of the cardiovascular autonomic control mechanisms during sepsis and septic shock^1,2,3,4,5. The initial compensatory increase of sympathetic outflow to the heart and peripheral vessels has been shown to become detrimental to the successful correction of hypovolemia and hypotension if prolonged^6,7,8. A sustained sympathetic overstimulation is a common phenotype in septic patients, proved also by the high concentration of circulating catecholamines, and it has been found significantly associated to a higher risk of mortality and morbidity^9,10,11,12. This condition of adrenergic stress is not routinely targeted during sepsis resuscitation, although it may be a crucial determinant of many complications secondary from sepsis, such as septic cardiomyopathy, vasopressor resistance, immunomodulation and generally organ dysfunctions^13,14,15. In particular, the vascular endothelium regulates the vascular tone, haemostasis and/or vascular permeability, and arterial vascular functions have been reported to be severely impaired during sepsis¹⁶; recent direct and indirect evidence suggests that sympathetic overactivity and vascular dysfunction may be closely and causally related to each other in a very complex fashion¹⁷. For example, sympatho-excitatory manoeuvres have been shown to induce endothelial dysfunction and they typically lead to increased stiffness of large arteries^18,19; on the other side, stiffer arteries may alter the baroreflex function, thus interfering with the sympathetic control of smooth muscle vascular tone²⁰. Furthermore, the α1-adrenergic receptors, mainly located in the peripheral vessels, may undergo the desensitization process, a feedback mechanism to protect the receptor against overstimulation, and this further contributes to the failure of autonomic modulation of vascular tone and a less effectiveness of vasopressor therapy²¹. Finally, experimental evidence indicates that the sympathetic ANS is critically influenced, both at central and peripheral level, by the most relevant factors regulating vascular functions, such as nitric oxide (NO), reactive oxygen species (ROS), and endothelin, that are all known to be involved in the dysregulated inflammatory response to sepsis and septic shock¹⁶.

Previous studies of our group have proposed several indices able to highlight the cardiovascular autonomic dysfunction in different experimental populations during septic shock resuscitation, and we hypothesize that the introduction of these indices to standard therapy targets, such as mean arterial pressure, lactate, and oxygen saturation as suggested by the international SSC guidelines, could be helpful to better evaluate the therapy effectiveness in restoring circulatory characteristics and cardiovascular regulatory mechanisms and to implement an improved resuscitation strategy^22,23. However, the limited period of observation after the full resuscitation has not permitted to obtain conclusive results, but only an indication about the short-term effects of resuscitation, i.e. about 5/6 h after the resuscitation procedure.

The aim of this work is to characterize the arterial properties and the autonomic cardiovascular response in a long-term swine experiment of polymicrobial sepsis, in order to investigate whether the standard resuscitation therapy was successful in restoring a physiological condition of hemodynamic profile, cardiovascular interactions and autonomic control. Moreover, the analyses exploited the long-time window after resuscitation (72 h) to disentangle and explore possible differences in the early and late response to the therapy.

Methods

Animal population, anaesthesia and ventilatory setup

The animal experiment was carried out in collaboration with the Inselspital University Hospital of Bern, Switzerland, and was performed in accordance with the EU Directive 2010/63/EU for animal experiments and the ARRIVE guidelines for animal research, and with the approval of the Animal Care Committee of the Canton of Bern (BE103/16).

Twenty mechanically ventilated and sedated domestic pigs (weight: 39.8 2.7 kg; male/female: 1:1) were used for the experiment. Ketamine (20 mg/kg) and xylazine (2 mg/kg) were intramuscularly administered for animal sedation. General anaesthesia was induced by intravenous infusion of midazolam (0.5–1.0 mg/kg) and atropine (0.02 mg/kg) and maintained with propofol (4–10 mg/kg/h) and fentanyl (3–20 g/kg/h), standard drugs for sedation and analgesia also in critically ill patients. Additional bolus of propofol (20–40 mg) and fentanyl (50–200 g) were administered if necessary, according to sedation depth evaluated hourly by noise pinching and presence of spontaneous movements. After tracheal intubation, the pigs underwent mechanical ventilation in volume-controlled mode, initially with FI_O2 at 30%, PEEP at 5 cmH₂O, tidal volume at 8 mL/kg, and respiratory rate at 20 breaths/min and later adjusted with the goal to keep pH between 7.35 and 7.45 and Pa_CO2 between 35 and 45 mmHg. Gastric output was measured by a gastric probe inserted into the stomach and used also for the later enteral nutrition infusion. During surgical preparation, ringer lactate (RL) solution was infused at 3 mL/kg/h, and measurable blood loss was 1:1 replaced with RL. Rocuronium was administrated (30–50 mg as bolus) only during instrumentation if necessary for better exposure (10 of 20 animals, 5 in sepsis and 5 in sham). After surgeries, 10 mL/kg RL was infused as bolus to all animals to prevent hypovolemia.

Surgical preparation and data acquisition

The animal was placed in a supine position for instrumentation, a midline neck incision was performed to allow placement of an arterial catheter (5F, Cordis AVANTI, Fremont, CA, USA) in the left carotid artery, a pulmonary artery catheter (8F, Edwards Lifesciences, Irvine, USA) via left external jugular vein, a venous catheter in the left internal jugular vein (5F, Cordis AVANTI, Fremont, CA, USA) for volume infusion, a triple-lumen catheter (7F, Arrow international, Inc, PA, USA) in the right internal jugular vein, and a flow probe (Transonic Systems Inc., Ithaca, NY, USA) around the right carotid artery. Thereafter, a midline laparotomy was performed for placement of a catheter into the urinary bladder for drainage and measurement of urine output. Also, a large-bore drainage tube was placed intraperitoneally for induction of fecal peritonitis.

All pressures were measured and displayed on a multi-modular monitor (S/5 Critical Care Monitor; Datex-Ohmeda, GE Healthcare, Helsinki, Finland). Continuous cardiac output (L/min, by thermodilution) was measured with a Vigilance monitor (Baxter Healthcare Corporation, Edwards Critical Care Division, Irvine, CA), and O₂ saturations were measured continuously with a Vigilance monitor or intermittently with a blood gas analyzer (Radiometer, Copenhagen, Denmark). Blood flows were measured with ultrasonic transit time perivascular flowmeters using double-channel TS 140 420 flowmeters (Transonic Systems Inc., Ithaca, New York, USA). The signals of all the pressures and blood flows were recorded at 100 Hz by a data acquisition system (Soleasy™; National Instruments Corp., Austin, Tx, USA). More details can be found in²⁴.

Experimental protocol

Instrumentation was followed by a one hour stabilization phase, during which no manipulation on the animal was allowed, thereafter baseline blood samples were taken (time point T1). Subsequently pigs were randomized to fecal peritonitis (SS n = 10)/sham (SH n = 10) and to groups with/without abdominal negative pressure therapy, which was the objective of a separate study with no influence on systemic and splanchnic hemodynamics nor abdominal organ function (all p > 0.05, not published). Sepsis was induced by peritoneal instillation of 2 g/kg of autologous feces dissolved in 250 mL warmed glucose 5% solution. After 8 h of observation without resuscitation, another set of blood samples was collected (T2). During stabilization and observation periods, RL infusion rate was reduced to 1.5 mL/kg/h. Before peritonitis induction, to avoid hypovolemia, a bolus of 150 mL of RL was infused to all animals and repeated until there was no fluid response (i.e. increase in cardiac output or stroke volume ≥ 10% after 10 min of fluid bolus). Then, the protocol-based resuscitation was started and continued for approximately 76 h (resuscitation period, RP), including fluid infusion, vasopressor support, electrolyte maintenance, antibiotic therapy, and reduction of body temperature. During the resuscitation period, a basal infusion with RL solution, glucose solution (G50%) and enteral nutrition (36 h after RP) was maintained at 3.0 ml/kg/h. Additional fluid boluses were given to treat hypovolemia. In particular, after 150 ml bolus of RL was given, if cardiac output or stroke volume increased by more than 10%, the fluid response was considered positive, and a repeated fluid bolus was given. This was repeated until the bolus resulted in a negative fluid response. If correction of hypovolemia was not sufficient to keep the mean arterial pressure above 65 mmHg, a continuous infusion of noradrenaline was started to keep the blood pressure at this level, as suggest in the 2013 sepsis guidelines²⁵, which were the only available at the time this experimental protocol was designed.

If the mixed venous oxygen saturation (SvO₂) was ≤ 50%, dobutamine administration was started at a dose of 5 mg/h. This dose was increased by the same amount every 30 min until the SvO₂ was 50% or higher or until a maximal dose of 20 mg/h was reached. The sepsis guidelines from 2013 suggested dobutamine infusion in the presence of myocardial dysfunction or ongoing signs of hypoperfusion despite achieving adequate intravascular volume and adequate mean arterial pressure²⁵. As a surrogate of low cardiac output, we used mixed venous oxygen saturation which represents the body's oxygen reserve by taking into account not only cardiac output, but rather systemic oxygen delivery in relation to oxygen consumption.

Blood glucose was maintained between 3.5 and 7.0 mmol/L by G50% or insulin infusion. Respiratory rate and minute ventilation were adjusted in order to maintain pH between 7.35 and 7.45. Arterial oxygen partial pressure was kept between 100 and 150 mmHg and oxygen saturation above 90%. PEEP and FiO2 were adjusted accordingly and tracheal suctioning as well as pulmonary recruitment manoeuvres were performed regularly. As in human abdominal sepsis, piperacillin-tazobactam (2.25 g/8 h iv; Sandoz Pharmaceuticals, Rotkreuz, Switzerland) was used as an antibiotic. Liquemin 10000 IU/24 h was infused IV to prevent deep vein thrombosis. If core temperature exceeded 39.5 °C, the animal was cooled by fan, lowering of room temperature, alcohol spray and ice bags. Further blood samples were taken just before RP + 24 h (T3), RP + 48 h (T4) and two of hours after RP + 72 h (T5). At the end of the experiment animals were euthanized by bolus infusion of 40 mmol of potassium chloride. Further details can be found in²⁶.

Hemodynamic analyses

All signals were continuously recorded for the entire duration of the experiment. The time points used for blood analyses were considered as reference to identify and select stationary portion of signals (5–10 min) for successive analyses. The carotid arterial blood pressure (ABP) and blood flow (CBF) were analysed and the onsets of each heart pulse were identified using available algorithms^27,28. The following beat-to-beat time series were derived from ABP signals: systolic arterial pressure (SAP), diastolic arterial pressure (DAP), mean arterial pressure (MAP), pulse pressure (PP), and heart period (HP), defined as the time difference between consecutive onsets (surrogate for RR-intervals). Beat-to-beat mean CBF was also computed. Continuous cardiac output (CCO) and central venous pressure (CVP) were averaged on each HP time interval to get beat-to-beat series. Beat-to-beat values of carotid stroke volume (carotid SV) was assessed from CBF by integrating it over each heart cycle by means of the trapezoid method; beat-to-beat values of global stroke volume (global SV) were computed as $CCO/HR$. Finally, mean values of all time series were computed for each phase of the experiment.

Autonomic cardiovascular indices

ANS cardiovascular control was evaluated by estimating the power spectral density of DAP component and the cardiac baroreflex sensitivity (BRS). The time series of DAP values were resampled at 2 Hz to obtain evenly spaced signals, successively were detrended using a 10th order polynomial function, and then normalized by dividing the detrended signal by its standard deviation in order to have zero-mean oscillations and unitary SD. The stationarity of the resulting time series was assessed by the Kwiatkowski-Phillips-Schmidt-Shin (KPSS) test. Power spectra were estimated via autoregressive spectral analysis. Spectral indices were computed as low-frequency power (LF, 0.04–0.15 Hz), total power (TP), which is the total area under the spectrum, and the normalized LF power, i.e. LF/(TP-VLF)%, where VLF is the very low frequency power [0–0.04 Hz].

The bivariate closed-loop model method²⁹ was adopted to estimate the cardiac BRS on the time series of SAP and HP which were resampled at 2 Hz. We decided to restrict the analysis of BRS only to the LF component of the feedback gain, since this component gives indications on the sympathetic activity³⁰. The autonomic cardiovascular indices were computed over 2-min windows with 50% overlap and then averaged.

Windkessel vascular indices

The arterial tree was modelled as a two-element Windkessel model, and several indices were derived to characterize its properties. The Windkessel time constant $\tau$ is defined as $\tau =TPR*AC$, where TPR is the total peripheral resistance and AC is the total arterial compliance, it represents the exponential decay of ABP pulse from systolic peak to end of diastole³¹. The method proposed by Mukkamala and colleagues³² estimates the exponential decay over pretty large windows (6 min) so to avoid cumulative effects of the wave reflections, which are attenuated in the central ABP waveforms only. More specifically, the ABP response to a single, solitary cardiac contraction is estimated from the ABP waveform. Then, the Windkessel time constant is measured by fitting an exponential to the tail end of this estimated response once the faster wave reflections have vanished. We adopted this method for a robust estimate of $\tau .$

Arterial compliance is defined as the change in arterial blood volume due to a given change in arterial blood pressure³³; we thus computed the beat-to-beat carotid arterial compliance (cAC) as $carotid SV/PP$ according to the definition. For clarity sake the CBF was measured in the right carotid while carotid ABP in the left carotid, we reasonably assumed that the changes and values were similar.

We calculated the vascular resistance, previously named TPR, with two different approaches based on the Windkessel model³¹. We named systemic vascular resistance (SVR) the estimate obtained with the global CCO, and carotid vascular resistance (carotid VR) the estimate obtained with the CO computed from the carotid SV (CO = SV*HP). Both vascular resistance indices were then normalized by the animal weight:

$$SVR = \frac{MAP - CVP}{{CCO}}/weight$$

(1)

$$carotid{ }VR = \frac{MAP - CVP}{{carotid{ }CO}}/weight$$

(2)

The indices of vascular resistance and cAC were computed on a beat-to-beat basis and then averaged at each time point. The index $\tau$ was computed over 6-min 50% overlapping time window and then averaged to get a single value at each time point.

Effective arterial elastance

Effective arterial elastance (Ea) is considered a simple estimate of total arterial load. Monge Garcia et al.³⁴ proposed an estimate of Ea from a blood pressure measurement recorded in a peripheral vascular district; they proved that, given a reliable SV measurement, the ratio MAP/SV provides a robust Ea surrogate over a wide range of hemodynamic conditions and is interchangeably in any peripheral artery. In this case MAP is proposed as a surrogate for the left ventricular end-systolic pressure (LV ESP). We similarly estimated Ea by using carotid artery pressure and carotid stroke volume according to Eq. (3). Carotid is a direct branch of aorta so we can suppose that this measure can provide reliable information of heart-vascular coupling and total cardiac afterload.

$$Ea = MAP/carotid{ }SV$$

(3)

Ea was computed on a beat-to-beat basis and then averaged at each time point.

Therapy and laboratory data

The overall dosages of fluids and vasopressors (VP) were calculated by summing up the dosage from the start of the experiment to each time point. The only vasopressor administered was noradrenaline (100 μg/mL and 200 μg/mL), and the fluids used were NaCl 0.9%, ringer lactate and glucose solution (G50%). Urine output UO (mL) was retrieved as the cumulative amount from the start of the experiment until each time point. Fluid balance (mL) was computed as the difference between the amount of intravenous fluids given and UO at each time point. The amount of sedative drugs (propofol and fentanyl) was computed as cumulative dosage at each time point. VP, fluids, UO, fluid balance and sedation amount were normalized to the animal's weight. We also considered the following parameters: mixed venous oxygen saturation (SvO2, %), hematocrit (Htc, L/L), hemoglobin (Hb, g/L), platelets (Plat, 10⁹/L), leukocytes (WBC, 10⁹/L), creatinine (Creat, µmol/L), prothrombin time (PT, sec), bilirubin (µmol/L), ammonia (µmol/L), and band neutrophils (N%, %). Finally, high sensitive cardiac troponin I (hs-cTnI, ng/L) was measured from plasma samples using a specific ELISA kit (CTNI-9-US, Life Diagnostics) to have an indirect measure of cardiac stress.

Statistical analysis

Wilcoxon rank-sum test was adopted to verify significant differences in the indices between the two groups at each time point. Friedman test was adopted to find differences within each group among time points across multiple tests attempts. In case Friedman test p-value < 0.05, we used then the Wilcoxon signed-rank test to assess significant changes among pairs of time points within each group of animals. For the post-hoc multiple comparisons we adopted the Tukey's honestly significant difference correction for p-value significance. Significance was considered with a p-value < 0.05.

Results

Four pigs out of 20 were excluded from the original population since the quality of collected signals was not guaranteed for all the time points and the artefact-free segments were too short due to technical reasons. The resulting dataset was composed by 9 pigs in the septic group and 7 in the sham group.

The measurement of CCO at time point T2 was not available for the majority of the septic animals due to technical reasons, i.e. unavailable acquisition due to hyperthermia during sepsis development, therefore CCO and derived indices (i.e. global SV, Ea, SVR) at T2 were excluded from the statistical analyses.

Clinical parameters and hemodynamic indices

Table 1 and Fig. 1 report the laboratory, hemodynamic and therapy data of septic and sham pigs at all time points. At baseline, all animals present a similar condition in terms of both hemodynamic profile and laboratory variables. After 6–8 h from inoculation of feces, SS animals showed clear signs of sepsis (time point T2 in Fig. 1 and Table 1); a marked increase in band neutrophils percentage confirmed the inflammation; the inflammatory response resulted in an increased endothelial permeability and capillary leakage represented by a significant increase in hematocrit and hemoglobin values at T2 (Fig. 1 and Table 1); moreover, these indices were significantly higher in SS with respect to SH group. Tissue hypoperfusion is suggested by the significant reduction in blood pressure and SV, together with an increase in serum creatinine and ammonia. Lactate concentration was significantly higher in septic animals than in controls, but remained below 2 mmol/L (Fig. 1 and Table 1). However, due to species differences with humans, normal lactate levels are usually observed in swine septic shock³⁵ despite developing all the criteria for a septic shock and tissular hypoperfusion³⁶. According to the surviving sepsis campaign guidelines from 2013²⁵, which were the only available at the time this protocol was designed, severe sepsis was defined as sepsis plus sepsis-induced organ dysfunction or tissue hypoperfusion; our porcine sepsis phenotype is therefore compatible with this definition. Moreover, the hypotensive state together with the systemic inflammation triggered a tachycardic condition in SS pigs, that was characterized by a significantly higher HR compared to SH at T2 (Fig. 1 and Table 1). Unfortunately, CCO and global SV were not available at T2; however, CBF was significantly decreased in SS pigs, together with the computed carotid SV, hinting a global decrease in systemic CO (Fig. 1 and Table 1). Finally, the significantly higher values of creatinine, bilirubin and ammonia in SS pigs compared to SH, may indicate an initial hepatic and renal dysfunction (Table 1).

Table 1 Medians (25–75th percentiles) values of the laboratory analyses, hemodynamic measures, and clinical and therapy data at each time point for the two groups (septic pigs SS n = 9, sham pigs SH n = 7, if not stated otherwise in the table).

Full size table

The main targets of hemodynamic resuscitation, according to international guidelines, is to restore a MAP ≥ 65 mmHg by means of fluids and vasopressors therapy and to maintain a sufficient blood oxygenation for organ perfusion. The resuscitation protocol was effective in restoring and keeping MAP in the target range and to significantly increase SvO2 which was maintained above 60% in almost all animals (Fig. 1, Table 1). However, we found that the tachycardic response did not resolve in the 24 h after resuscitation and, although the HR decreased, SS group maintained significantly higher values than SH group for the next three days until the end of the experiment (Fig. 1). Besides, at T4 and T5, SS group showed a significant increase in the values of CO with respect to baseline, and the values were significantly higher with respect to SH group (Fig. 1). SV was instead lower in SS pigs with respect to SH (Fig. 1). This condition of persistent tachycardia can be a source of cardiac stress, as documented by the significantly increased concentration of cardiac troponin I in the arterial plasma of septic animals with respect to sham until the end of the experiment (Fig. 2).

Figure 3 shows the distribution of vasopressor dosage and fluids input normalized by pig's weight, both computed as cumulative values from the start of the experiment until each time point. As expected, SS pigs received significantly higher dosages of VP; moreover, the fluid balance resulted higher in SS group, due to the decreased urine output (significant at T5, Table 1) and the slightly higher amount of fluids administered.

Cardiovascular autonomic control

Sepsis induced a significant drop in BRS (time point T2, Table 2), hinting a failure of autonomic control of the cardiovascular system. To note, this impaired condition seems not resolved after resuscitation as SS group maintained significantly lower values of BRS with respect to SH group until the end of the experiment, and the BRS values in septic pigs were slightly lower than baseline condition even at T5, i.e. after 3 days of treatment. Moreover, at T2, SS group showed a reduction in LF power of DAP series with respect to baseline values (Table 2). DAP can be seen as a surrogate of systemic vascular resistance, therefore, this result might indicate a decreased sympathetic outflow to the vasculature, probably associated to the vasodilation triggered by sepsis. Noteworthy, the dosage of sedative drugs was similar between septic and sham animals (Table 1), despite a larger range of fentanyl in septic animals (electronic supplement Fig. S1); this allows to exclude sedation as important factor affecting the different autonomic response in the two groups of animals.

Table 2 Medians (25–75th percentiles) values of cardiac baroreflex sensitivity and power spectral indices of diastolic arterial pressure time series computed at each time point for the two groups (septic pigs SS n = 9, sham pigs SH n = 7).

Full size table

Vascular alterations

The values of the Windkessel time constant τ and cAC were reduced after sepsis development in SS group and remained lower than those of sham group until the end of the experiment (Fig. 4). Higher Ea values were found in SS group compared to SH group during the entire resuscitation phase, meaning an elevated afterload (Fig. 4). Finally, the values of carotid VR were significantly higher in SS group compared to SH group at T2 (Table 3). In the next three days of resuscitation, instead, SS group was characterized by a lower SVR (Fig. 4); in particular, the values of SVR were significantly lower in SS group with respect to SH group at T4 and T5 and to baseline values. Figure 5 shows the relationship between vascular properties and vasopressor therapy in terms of SVR and $\tau$ values together with VP dosage normalized by each pig's weight. As expected, SS pigs received significantly higher dosages of VP, although they exhibited relatively low SVR compared to sham, which, on the contrary, displayed higher values of SVR and Windkessel time constant $\tau$, and most of them received no vasopressor. Only two sham pigs received noradrenaline, one developed systemic inflammatory response syndrome (SIRS) during the experiment, the other went into respiratory failure at day 4. From these results septic pigs may be considered less responsive to VP therapy in terms of vascular resistance, and this condition may be related to a modification of arterial tree characteristics and mechanical properties induced by sepsis; in these terms the therapy was ineffective in restoring the basal condition.

Table 3 Medians (25–75th percentiles) values of carotid vascular resistance computed at each time point for the two groups (septic pigs SS n = 9, sham pigs SH n = 7, if not stated otherwise in the table).

Full size table

Discussion

In this study we evaluated the effect of sepsis development and protocolled resuscitation on autonomic mechanisms for cardiovascular control and arterial vascular properties during a 5-day swine experiment. Our results generally confirmed previous studies^22,37, highlighting a persisting autonomic failure and vascular disarray despite successful resuscitation, as defined by the hemodynamic and clinical targets suggested by the international guidelines (e.g. MAP > 65 mmHg)³⁸.

As expected, sepsis induced a severe inflammation, hypoperfusion, hypotension, hypovolemia and tachycardia, as shown in Fig. 1 and Table 1. The resuscitation manoeuvres, including antibiotics, were able to restore MAP > 65 mmHg and SvO2 > 60%, to reduce inflammation, and to increase the circulating volume as the CO values increased. While these changes can be considered signs of therapy effectiveness, we observed a persistent tachycardia in SS pigs until the end of the experiment, i.e. after three days of therapy administration. Tachycardia, typically triggered by sepsis, is the main compensatory mechanism to maintain a sufficient CO despite reduced SV either due to reduction of preload or to diastolic and/or systolic dysfunction. However, even after adequate fluid resuscitation to correct for hypovolemia, septic patients often show an elevated HR, and this condition has been demonstrated to be associated to higher mortality, incidence of septic cardiomyopathy and cardiac dysfunctions^9,13,39. Indeed, the raised cardiac troponin I observed in septic pigs (Fig. 2) indicate an elevated stress of the cardiomyocytes, which can further worsen and lead to severe dysfunctions if long lasting⁴⁰. For this reason, several preclinical and clinical studies have investigated new drugs able to attenuate this persistent tachycardia in order to improve cardiac functionality and the clinical outcome^41,42. Despite a lack of guidelines recommending lowering heart rate, very promising results have been achieved by administering cardioselective β1-blocker drugs, such as esmolol, in large randomized clinical trials⁴³. Recent studies have hypothesized an anti-inflammatory action exerted by esmolol, decreasing lactate and proinflammatory cytokines concentrations, but also improving vascular responsiveness to vasopressors^44,45.

Acute inflammation and sepsis are known to impair endothelial functions and to induce a stiffening of large compliant vessels, such as the aorta¹⁶. Our results confirmed this evidence as the values of the carotid compliance decreased after sepsis development (cAC T2, Fig. 4); similarly, the values of arterial time constant τ were significantly reduced. The present work demonstrates that hemodynamic resuscitation was able to restore neither τ nor carotid compliance when compared to sham animals after 3 days of therapy (T5). Moreover, SVR was also found to be significantly reduced in SS pigs with respect to SH after 48 h and 72 h of resuscitation (T4 and T5, respectively), despite increasing dosage of noradrenaline (Fig. 3). These vascular alterations can be observed from the graph of carotid ABP waveform (Fig. 6). The ABP morphological characteristics in a sham pig at T5 are similar to baseline (T1), on the contrary, the ABP waveform in a septic pig is completely different from baseline and SH pig, in particular, a much steeper decay of the diastolic phase, consistently with the lower values of τ and cAC previously described. In a previous study of our group²², we have reported a similar decreasing trend of τ and total arterial compliance in a septic shock swine population undergoing hemodynamic resuscitation with fluids and noradrenaline; in that case, the observation window was very short, about 5–6 h after therapy, and the insult was more severe (MAP reached values < 50 mmHg). All together these results point out that the vascular alterations occurring in the early phase of sepsis development characterize not only the first hours of therapy administration, when recovery from hypotension is of primary importance and massive fluids and vasopressors infusion are crucial for survival, but they persist also after days, when hemodynamic stabilization of the patient has been achieved. Long-term post-ICU disabilities are known to occur commonly in sepsis survivors, leading to a higher risk for future cardiovascular adverse events or hospital readmission⁴⁶. Our results support the hypothesis that the persisting arterial stiffening plays a major role in this process, and a timely intervention to limit this phenomenon could positively impact the long-term outcome of septic patients. For example, in a previous experimental study²³ we observed an improvement in compliance and τ values when septic shock pigs were treated with the adjunction of esmolol in comparison to septic pigs treated with fluids and noradrenaline only. The potential anti-inflammatory effect of esmolol together with its adrenergic antagonist effect could explain the improvement of vascular properties, as the proposed indices showed.

The increase in arterial stiffness may also help to explain the higher cardiac afterload in SS pigs compared to SH, measured from the effective elastance Ea index. In the original publication of Sunagawa et al.⁴⁷, Ea was proposed as a steady-state arterial parameter that expresses all the extracardiac forces opposing to v...

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