The microcirculatory failure could not weaken the increase of systematic oxygen extraction rate in septic shock: An observational study in canine models

Abstract

OBJECT: This study aimed at exploring what level of the microcirculation alteration could weaken the decrease of ScvO₂ (or the increase of O₂ER) and further result in an abnormally elevated ScvO₂.

METHODS: Beagles were randomly assigned into control (n = 5) and shock group (n = 5). The canines in shock group were intravenously injected with live E. coli (3.5×108 cfu/kg), and the ones in control group were injected with sterile saline. The experiment continued to the animals’ death or for a maximum of 24 hours. Hemodynamic parameters, blood gas and inflammatory cytokines level were collected. Microcirculatory parameters were assessed with Sidestream Dark Field (SDF) imaging. The correlation between the microcirculation and oxygen metabolism or inflammatory cytokine, meanwhile the correlation between the oxygen metabolism and inflammatory cytokine was assessed.

RESULTS:E. coli infusion induced hypodynamic shock. The correlation between microcirculation and oxygen metabolism or inflammatory cytokine, and The correlation between the oxygen metabolism and inflammatory cytokine (O₂ER vs. MFI: r = –0.700, P < 0.01; O₂ER vs. PVD: r = –0.677, P < 0.01; O₂ER vs. PPV: r = –0.538, P < 0.01; MFI vs. IL-6: r = –0.780, P < 0.01; PPV vs. IL-6: r = –0.621, P < 0.01; MFI vs. TNF-α: r = –0.636, P < 0.01; PPV vs. TNF-α: r = –0.561, P < 0.01) were observed.

CONCLUSIONS: The increase of O₂ER cannot be weakened by the microcirculatory failure.

Keywords

Oxygen consumption rate microcirculation septic shock inflammatory cytokines canine

1 Introduction

Severe sepsis and septic shock are the major causes of intensive care unit (ICU) patients’ high mortality [12]. In these episodes, the imbalance between oxygen delivery (DO₂) and demand of tissues are considered as an important influencing factor to sepsis/septic shock [2]. As the result of overwhelmingly systemic inflammatory response, the increase of systemic oxygen consumption (VO₂) accompanied with insufficiency DO₂ may result in the increase of oxygen extraction rate (O₂ER), tissue hypoxia (hyperlactacidemia) and multiple organs dysfunction syndromes (MODS). Rivers et al. demonstrated that early goal-directed therapy (EGDT) resulted in mortality reduction of absolute [26]. Mixed venous oxygen saturation (SvO₂) is an important targeted endpoint of the EGDT protocol, which is often used as a marker to reflect the balance between DO₂ and VO₂. Central venous oxygen saturation (ScvO₂) is an easily obtained parameter via the central venous catheter which is already in most critically patients, and it has a good correlation with SvO₂ [25]. Thus ScvO₂ has become the substitution of SvO₂ in clinical practise; in addition, this method has already been recommended by practice guidelines and medical professional organizations [1, 7]. These EGDT-based guidelines aim to correct the low ScvO₂ level (an abnormally elevated O₂ER) which may indicate a decrease in oxygen delivery or an increase in oxygen consumption, or both.

In the clinical arena, it has been reported that abnormally elevated ScvO₂ levels represented hypoxia on the cellular level [10]. Jennifer V. Pope et al. found that the high initial ScvO₂ was associated with a worse outcome [21]. They assumed that high ScvO₂ might partially due to the microcirculatory alterations in sepsis which was arisen from impaired vascular autoregulatory mechanisms and functional shunt of oxygen. Indeed, the microcirculatory alterations played a crucial role in the pathogenesis and the development of sepsis/septic shock. It had been confirmed that the persistent deterioration of the microcirculation was one of the independent risk factors for the prognosis of the critical patients [4, 31]. The experimental studies had demonstrated that the decreased tissue capillary density and the increased heterogeneity of perfusion in sepsis led to a functional shunting of oxygen [18]. All of these changes might bring about the shunting of blood, and hinder the circulation of oxygen to the metabolically active tissues via microcirculation, and make more high oxygen saturation blood flow back to thevenous.

For well understanding the pathophysiology in this circumstance, it is important to identify the effect of altered microcirculation on the ScvO₂ or systematic O₂ER in sepsis/septic shock. As there were few studies specially aimed at the correlation between the microcirculation and ScvO₂ or systematic O₂ER, we did the present experiment to explore to what extent the altered microcirculation could slow down the increase of oxygen availability and consumption, because which may led to an abnormally elevated ScvO₂ or decrease of O₂ER_. We also want to know whether there is a certain degree of correlation between the microcirculatory status and systematic metabolic parameters, such as O₂ER and lactate etc., and also whether there is a correlation between the inflammatory cytokines and parameters of microcirculation or oxygen metabolism.

2 Materials and methods

2.1 Experiment animals

Purebred Beagle dogs were purchased from the Laboratory Animal Center of Dalian Medical University used in this study with the animal qualification number SCXX (Liao) 2008–0002. The experimental protocol conformed to the Guide for the Care and Use of Laboratory Animals as promulgated by the Council of the American Physiologic Society (8th Edition, 2011). The following procedures have been approved by the Ethical Committee for Animal Research of China Medical University (The protocol number: L2011001). All of the animals were transferred to the Experimental Animal Department of China Medical University and fed with standard dog food for a minimum of one-week observation. Artificial environment had been set in animal housing rooms (every 12 hours in the alternation of day and night, room temperature 22±1°C, relative humidity 30% ∼60%).

2.2 Bacterial preparation

The pathogen (E. coli, serotype O79) used in this study was isolated from a blood culture of clinical septic shock patient (this strain of E. coli had been investigated elsewhere [34]). Before each experiment the bacteria was inoculated to the culture dish, incubated at 37°C over 16 hours then withdrew for grinding and dissolved in sterile saline. Spectrophotometry was applied to measure McBurney turbidity (Mc fold) of the bacterial suspension each time, then the concentration was calculated. According to the bacteria amount of 3.5×10⁸ cfu/kg per dog, sterile saline was used to mix the suspension into 20 mldissolution.

2.3 Surgical operation

The dogs were fed nothing but water for 24 hours before the experiment began. Anesthesia was induced by intramuscular injection of pentobarbital (25 mg/kg). The continuous intravenous anesthesia was maintained through intravenous infusion of pentobarbital (5-6 mg/kg/h) combined with fentanyl (0.3-0.4 μg/kg/hour) after right external jugular venous catheterization. Then arterial catheter (14F) for pulse indicator continuous cardiac output (PiCCO; Piccoplus Pc8100, PULSION Medical System AG, Munich, Germany) was intubated into the right femoral through surgical dissection, in order to measure hemodynamic parameters and collect blood samples. Meanwhile to perform orotracheal intubation (7.5-mm tracheal tubes) for mechanical ventilation (Servo ventilator 900 C, Siemens-Elema; Ventilation setting: Pressure control, Pi 12–16 cmH₂O, RR 12–14/min, FiO₂:25–40%) as the method of maintaining the arterial oxygen partial pressure (PaO₂)≥80 mmHg, partial pressure of carbon dioxide (PaCO₂) 28 35 mmHg. The stomach was emptied with an orogastric tube. A heating pad was used to keep the core body temperature at 36.5±1.0°C.

Paramedian incision was performed for cystostomy at the vertex point of the bladder. The urethral catheter for adult (14F) was indwelled and fixed onto the abdominal wall.

Later on, a proximal-loop jejunostomy was performed 15–20 cm from the Treitz’s ligament and the orificium fistulae was fixed at the right lateral abdomen for microcirculation parameters collection by the Sidestream Dark Field (SDF) imaging (Microscan; Video Microscope System, Amsterdam, The Netherlands). The initiating terminal of posterior jejunum was sutured completely and put back into the abdominal cavity. The jejunostomy was covered with moisturized gauze of warm saline solution by local application.

2.4 Experiment protocol

The canines were randomly assigned into 2 groups: a) control group and b) shock group. In both groups the steady hemodynamic status was maintained for 2–3 hours after surgical operation. Then in the shock group, bacterial suspension was injected into the canine superior venous for 2 hours via central venous catheter. Meanwhile in the control group, 20 ml 0.9% saline was injected as placebo. T-0 h was designated as the time point on which the injection of E. coli or saline started. When mean arterial pressure (MAP) decreased by 20% compared to the baseline the septic shock was considered completed [11]. The experiment was last until animal death or 24 hours of most. Animals which lived through 24 hours were considered survivors yet subsequently were euthanized with overdose potassiumchloride.

2.5 Hemodynamic parameters

PiCCO was applied for measuring heart rate (HR), mean arterial pressure (MAP) and cardiac index (CI) were recorded at 0 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 18 h and 24 h (indicated as T-0 to T-24 h).

2.6 Blood sampling

Blood sample was collected with heparinized 1 ml syringe. Arterial PH, partial pressure of oxygen (PaO2), base excess (BE), arterial lactate acid concentration (Lac) and systemic central venous oxygen saturation (ScvO₂) were measured by blood gas analyzer (GEM premier 3000 Model 5700 Instrumentation Laboratory Co. USA) at 0 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 18 h and 24 h. Another 1.8 ml anti-coagulated with EDTA was for measuring the biomarkers of inflammation [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α)] at 0 h, 1 h, 2 h, 3 h, 6 h, 12 h, 18 h and 24 h. Blood samples were centrifuged at 3000×g for 30 min, then plasma was aliquoted in polypropylene tubes and stored at –70°C. The concentration of plasma IL-6, TNF-α were determined by the enzyme-linked immunosorbent assay kits (R&D Systems, US) according to the instruction for use.

2.7 Calculation of oxygen metabolism

DO₂, VO₂ and O₂ER were calculated as follow:

DO₂ = CO× (1.34×Hb×SaO₂ + 0.0031×PaO₂)

VO₂ = CO×[1.34×Hb×(SaO₂–ScvO₂) + 0.0031×PcvO₂)

O₂ER = (DO₂–VO₂)/DO₂

2.8 Microcirculation measurements

Consistent with the principle of the recommendations [19], the microcirculation of the small intestinal villous were investigated through the jejunostomy by insertion of SDF, which was objective to the depth of 5 to 7 cm from the edge of the stoma and slight angulation with disposable sterile cap at 5 different sites each time. The microcirculation parameters of the jejunum villus were recorded at 0 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 18 h, and 24 h. 20 seconds long videos were acquired, which were then stored under a random number for the later blinded off-line analysis using software AVA 3.0 (automated vascular analysis; Academic Medical Center, University of Amsterdam, Netherlands). The proportion of perfused vessels (PPV), the perfused vessel density (PVD), microvascular flow index (MFI), and the total vessel density (TVD) were evaluated according to the methods described by De Backer D et al. [6]. 37°C warm saline was used to wash the secretion of intestinal lumens surface before each observation.

2.9 Statistical analysis

All parameters were expressed as the mean±SD. The data analysis was performed using one-way analysis of variance (ANOVA) for a single time point comparison. Statistical comparison of data over time and between groups was conducted by a two-way analysis of variance for repeated measurements with time and treatments as factors followed by Dunnett’s multiple comparisons. The correlation between the microcirculation and oxygen metabolism or inflammatory cytokine, and the correlation between the oxygen metabolism and inflammatory cytokine were assessed with the Spearman rho. The data were analyzed using SPSS 20.0 software for Windows (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered to be the statistical difference.

3 Result

3.1 Changes of systematic hemodynamic, oxygen metabolism and inflammatory cytokine

Ten Beagle dogs were randomly assigned into shock group (n = 5) and control group (n = 5), and survival time of septic shock dogs was 12.4±1.6 hours. The dogs in control group all survived over the 24 hours. Therefore in the septic shock group, T12 represented the moment just before the heartbeat stopped. All the animals in the shock group developed a hypodynamic state which characterized by the low cardiac index, increased systemic vascular resistance, and decreased mean arterial pressure. At least one episode of systemic arterial hypotension happened within the first 2 hours (54±45 minutes) after starting the infusion of E. coli, which was recorded in all sepsis cases. Accompanied by the collapse of systematic circulation, oxygen metabolism disorder had been observed, including significantly increased DO₂ and VO₂ after E. coli injection. In our study, contrary to the gradually increased ScvO₂ in the control group, the ScvO₂ in the septic shock group irreversibly declined until the animals’ death without any relief.

Based on the calculation, the O₂ER in the septic shock group rose progressively until the dogs died even though the DO₂ decreased at 12 h. IL-6 level increased sharply at 1 h, and gradually increased from 2 h to 12 h, reaching around 3 times of the baseline. The serum concentration of TNF-α did not increase at 1 h but began to increase at 2 h (Table 1).

3.2 Deterioration of the jejunum villus microcirculation

The jejunum villus microcirculation showed irreversible deterioration along with the development of septic shock, as well as the continued reduction of the PPV and PVD after the infusion of E. coli. And the deterioration accelerated from 3 h to 6 h, and then obvious perfusion in jejunum villus could hardly be found until the animals died. Meanwhile MFI showed a linear decrease until the animals died. Both of these parameters in shock group had significant differences comparing to the control group (P < 0.01). TVD showed no significant change (Fig. 1).

3.3 Correlation between microcirculation and oxygen metabolism or inflammatory cytokine, and the correlation between oxygen metabolism and inflammatory cytokine

Lactate correlated negatively with microcirculatory parameters (Lac vs. MFI: r = –0.658, P < 0.01; Lac vs. PVD: r = –0.684, P < 0.01; Lac vs. PPV: r = –0.592, P < 0.01) (Fig. 2A–C). We found that ScvO₂ had no correlation with microcirculatory parameters (P > 0.05), but the O₂ER was increased while both the convective and diffusive oxygen transport (MFI and PPV) broken down. O₂ER negatively correlated to microcirculatory parameters (O₂ER vs. MFI: r = –0.700, P < 0.01; O₂ER vs. PVD: r = –0.677, P < 0.01; O₂ER vs. PPV: r = –0.538, P < 0.01) (Fig. 2D–F).

Then we tested the correlation between the inflammatory cytokine and the O₂ER or microcirculatory parameters. O₂ER correlated positively with inflammatory cytokine (O₂ER vs. IL-6: r = 0.627, P < 0.01; O₂ER vs. TNF-α: r = 0.565, P < 0.01) (Fig. 3A, D). The microcirculatory parameters correlated negatively with inflammatory cytokine (MFI vs. IL-6: r = –0.780, P < 0.01; PPV vs. IL-6: r = –0.621, P < 0.01; MFI vs. TNF-α: r = –0.636, P < 0.01; PPV vs. TNF-α: r = –0.561, P < 0.01) (Fig. 3B, C, E, F).

4 Discussion

In the present study, we reproduced the microcirculatory deterioration during septic shock and confirmed that the ScvO₂ declined irreversibly and the O₂ER increased continuously in our hypodynamic septic shock model set on healthy young dogs. The most interesting finding was, we observed that the microcirculatory deterioration did not blunt the decrease of ScvO₂ and the O₂ER was increasing throughout the septic shock, until the animals died. Meanwhile, we found that the increased O₂ER and microcirculatory deterioration significantly correlated to systematic proinflammatory cytokine. Although we observed a negative correlation between the microcirculatory status and O₂ER, we would like to believe there is a parallel relationship between microcirculatory status and O₂ER rather than causal relationship.

We set up this kind of models with young healthy dogs because we would like to minimize the impact of mitochondria on the ScvO₂ or O₂ER. The function and quantity insufficient of mitochondria could be found in the aged patients with other comorbidities [20]. Moreover, the immune systems of elderly people are less effective than earlier in life, so-called immunosenescence [23]. Thus we use the young healthy dogs so that we could evaluate the relatively independent effect of microcirculation on the ScvO₂ or O₂ER.

Our study showed that intravenous injection of lethal dose of E. coli induced a hypodynamic state of septic shock of which symptoms are low CI, MAP and hyperlactatemia. Hypodynamic sepsis could also be induced by gram negative bacteria in different kind of animals in other studies [17, 22]. However, in our models we did not administer any interference such as antibiotic, fluid resuscitation or vasopressor, etc., as these interferences may impact the status of microcirculation and O₂ER. Another reason for this protocol was that the purpose of our study was to explore the basic mechanism of sepsis/septic shock and to simulate the clinical patients who were just admitted into the emergency room or intensive care unit (ICU) without any interference for septic shock. Our models matched the hemodynamic changes perfectly and reproduced many features of severe sepsis in human, including hypotension, hyperlactatemia, as well as a concomitantly significant deterioration of microcirculation.

Alterations in microcirculation had been observed in a variety of diseases including cardiogenic shock [14, 15], labour pain [13] and intestinal ischemia/reperfusion [36]. In sepsis/septic shock, the pivotal role of microcirculation had been confirmed in clinical studies [4 , 32] and animal experiments [9 , 35]. According to these studies, using the application of SDF technology we observed the similar deterioration of jejunum villus microcirculation without any reversion during the development of septic shock. Consequently the decreased perfusion vascular density and blood flow velocity might further impair O₂ availability in sepsis [8]. This vicious circle resulted in irreversible organ impairment and even death in experiment animals. The observed microcirculation alterations in our models may be associated with microcirculation hypoperfusion, increased local metabolism, cytokine activation, thrombosis and other factors [5]. Especially the inflammatory cytokine such as IL-6 and TNF-α which significantly impacted the alterations in microcirculation (Fig. 3). This correlation suggested that the severity of microcirculatory alternation was closely related to the inflammatory activation. In addition, the observed microcirculatory deterioration of jejunum villus in our study exactly represented microcirculatory alternation in the small vessel in the gut, and the sluggish or stopped blood flow in these vessels might indicate more arteriovenous shunt in some larger vessels of the gastrointestinal tract or other organs, which might lead to more blood with higher oxygen saturation return to the center venous. Thus the microcirculation alterations we observed in the models are eligible for our purpose.

In our study, we also observed that the inflammatory cytokines increased during the septic shock development, which can result in cardiac depression and peripheral hypoperfusion [24]. The continuous growth of serum concentration of IL-6 and TNF-α was different from that transiently increasing induced by lipopolysaccharide (LPS) [29]. It might be due to the persistent destruction of the E. coli. The correlation between the inflammatory cytokine and the O₂ER or microcirculatory parameters might remind us that we could attribute both the oxygen metabolism disorder and microcirculatory dysfunction/failure to the excessive proinflammatory reaction.

O₂ER is the quotient of VO₂ divided by DO_2, thus it is determined by the changes of those two determinants. In the context of septic shock, the increase of VO₂ is caused by the systematic inflammation. Also, it was believed that the oxygen is consumed by leukocytes to generate oxidant radicals for killing bacteria [33]. Meanwhile, the dependent correlation between VO₂ and DO₂ may disappear [28]. Indeed in our study, the VO₂ of the animals in septic shock group increased after the infusion of E. coli, and maintained a high level until the animals died. Although at 12 h VO₂ and DO₂ were both decreased, we did not find any relationship between VO₂ and DO₂, which addressed that relatively high level oxygen consumption existing through the whole study. Combining with the high heart rate and serum concentration of IL-6 and TNF-α, we would like to attribute the increase of O₂ER to the enhanced metabolism and inflammatory reaction.

In clinical tests, both high and low ScvO₂, in another word, both low and high O₂ER could be found in patients who have sepsis/septic shock [21]. For the patients with increased ScvO₂ (decreased O₂ER), some authors considered that it was partially due to the microcirculatory failure. However, in our study, we did not find that the microcirculatory failure blunt the decrease of O₂ER. Conversely, we observed a negative correlation between the microcirculatory status and O₂ER, which means along with the development of microcirculatory dysfunction or failure, the systematic O₂ER increased gradually. Other study demonstrated that the correlation between the increased O₂ER and microcirculatory dysfunction/failure in the skeletal muscle but not the whole body [8]. Some authors might believe that the decreased O₂ER or increased ScvO₂ may be due to increased DO₂ which surpassed the tissue oxygen demand caused by excessive resuscitation, which was also associated with worse outcome observed in the clinical trial [21]. But it is hard to explain how the patient who was just admitted into the emergency room or intensive care unit without any interference of resuscitation could get an increased DO₂. Furthermore, we could image that both patients who with low or high O₂ER may have similar microcirculatory dysfunction/failure throughout the sepsis/septic shock from previous clinical studies [4 , 27]. However, why the similar microcirculatory dysfunction/failure lead to different O₂ER and clinical outcomes? Therefore, we would like to assume that the microcirculatory dysfunction/failure takes a very little part of the reasons of the ScvO₂ increase which was observed in the clinical arena, and we speculated that the decreased O₂ER or increased ScvO₂ may be due prior to: 1) mitochondria dysfunction; 2) inflammatory hyporesponsiveness to infection.

Some authors may doubt about that in our study, the O₂ER may be further increased without the influence from microcirculation. We cannot deny this, yet we couldn’t verify this neither. Because there is no widely accepted interventions can effectively improve the microcirculation in septic shock, we could not compare the changes of O₂ER with and without presence of microcirculation disorder. Moreover, according to the current evidences, we can’t rule out some protective significance of the microcirculatory disorder in the process of septic shock. Thus we don’t know whether the critically ill patients can be benefit from such simple elimination of the microcirculation disorder.

There were certain limitations of this study, including the differences in human and canine physiology, restricting the transformation of the obtained results into clinical practice. Our models were set up on the healthy young dogs, which could not mimic the variety of clinical patients with different ages and pre-existed underlying comorbidities. However, these purified models might help us to understand the mechanism underlying sepsis/septic shock.

5 Conclusion

In this study, a parallel relationship between microcirculatory alteration and O₂ER in sepsis/septic shock canine models is observed. This finding revealed that the increase of O₂ER cannot be weakened by the microcirculatory failure. Maybe both the oxygen metabolic disorder and microcirculatory dysfunction/failure are induced by excessive inflammatory reaction.

Footnotes

Acknowledgments

Special thanks to Dr. Yunzhuo Chu (Department of Laboratory Medicine, the First Hospital of China Medical University) for the clinical biochemical analysis and provision of live bacterial strain.

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