Organ blood flow and O2 transport during hypothermia (27°C) and rewarming in a pig model
Funding information:
This work was generously supported by grants from The Norwegian Research Council (Petromax2) and Ministry of Foreign Affairs (Barents 2020).
Edited by: Jeremy Ward
Abstract
New Findings
What is the central question of this study?
Absence of hypothermia-induced cardiac arrest is a strong predictor for a favourable outcome after rewarming. Nevertheless, detailed knowledge of preferences in organ blood flow during rewarming with spontaneous circulation is largely unknown.
What is the main finding and its importance?
In a porcine model of accidental hypothermia, we find, despite a significantly reduced cardiac output during rewarming, normal blood flow and O2 supply in vital organs owing to patency of adequate physiological compensatory responses. In critical care medicine, active rewarming must aim at supporting the spontaneous circulation and maintaining spontaneous autonomous vascular control.
The absence of hypothermia-induced cardiac arrest is one of the strongest predictors for a favourable outcome after rewarming from accidental hypothermia. We studied temperature-dependent changes in organ blood flow and O2 delivery (
) in a porcine model with spontaneous circulation during 3 h of hypothermia at 27°C followed by rewarming. Anaesthetized pigs (n = 16, weighing 20–29 kg) were randomly assigned to one of two groups: (i) hypothermia/rewarming (n = 10), immersion cooled to 27°C and maintained for 3 h before being rewarmed by pleural lavage; and (ii) time-matched normothermic (38°C) control animals (n = 6), immersed for 6.5 h, the last 2 h with pleural lavage. Regional blood flow was measured using a neutron-labelled microsphere technique. Simultaneous measurements of 
and O2 consumption (
) were made. During hypothermia, there was a reduction in organ blood flow, 
and 
. After rewarming, there was a 40% reduction in stroke volume and cardiac output, causing a global reduction in 
; nevertheless, blood flow to the brain, heart, stomach and small intestine returned to prehypothermic values. Blood flow in the liver and kidneys was significantly reduced. Cerebral 
and 
returned to control values. After hypothermia and rewarming there is a significant lowering of 
owing to heart failure. However, compensatory mechanisms preserve O2 transport, blood flow and 
in most organs. Nevertheless, these results indicate that hypothermia-induced heart failure requires therapeutic intervention.
1 INTRODUCTION
Accidental hypothermia is a clinical term known as involuntary lowering of body (core) temperature to <35°C. Over the past three decades, encouraging case reports and surveys have reported increased survival with favourable neurological outcome in patients rewarmed from accidental hypothermia (Boue et al., 2014; Gilbert, Busund, Skagseth, Nilsen, & Solbø, 2000; Mark et al., 2012; Meyer, Pelurson, Khabiri, Siegenthaler, & Walpoth, 2014; Walpoth et al., 1997; Wanscher et al., 2012). This notion is substantiated by the fact that over the same time span overall mortality of accidental hypothermia patients has decreased from 52–80% (Maclean & Emslie-Smith, 1977; Murray & Hall, 1998) in previous reports to the present 28–35% (Megarbane, Axler, Chary, Pompier, & Brivet, 2000; Roeggla, Roeggla, Wagner, & Hoedl, 1994; van der Ploeg, Goslings, Walpoth, & Bierens, 2010; Vassal et al., 2001).
This favourable outcome is closely linked to accidental hypothermia in patients with maintained spontaneous circulation during rescue and rewarming but is in contrast to the outcome in hypothermic patients with cardiac arrest. Hypothermic patients with spontaneous circulation are rewarmed using a wide diversity of methods, which can be divided into non-invasive (warmed blankets, hot air mattress and warmed illumination) or invasive techniques [warmed fluid infusion, warmed stomach and bladder irrigation, pleural lavage (PL; as used in the present study) and cardiopulmonary bypass (CPB)] (van der Ploeg et al., 2010). However, in some cases rewarming of accidental hypothermia patients with preserved spontaneous circulation may not be successful. The reasons for this are mainly uncontrolled factors taking place during the preceding hypothermic insult and the extent of circulatory dysfunction, which can range from modestly reduced cardiac output (CO) to complete circulatory collapse (Polderman, 2009) (‘rewarming shock’).
In comparison, the survival rate of accidental hypothermia patients with hypothermia-induced cardiac arrest, often taking place at core temperatures below 30–28°C during rescue, is much lower. The current state-of-the-art method to rewarm these hypothermia patients in cardiac arrest is to apply CPB. This technique has been routinely used over the last 60 years to cool and rewarm patients for cardiac surgery. An extensive volume of cardiovascular research work, both clinical and preclinical, conducted over the past 60 years has been a prerequisite for safe use of CPB during cardiac surgery. Results from these studies are, however, not directly applicable for accidental hypothermia patients rewarmed with CPB. Differences between cardiac surgery patients and accidental hypothermia patients include the duration of the hypothermic insult, circulatory function and the patency of O2 transport, factors that might also warrant different approaches for the use of CPB for rewarming. This view may find support in a recent survey from a Norwegian university hospital reporting no change in survival rate over the last 30 years when using CPB to rewarm accidental hypothermia patients (Svendsen, Grong, Andersen, & Husby, 2017).
Existing preclinical information on haemodynamic function, organ blood flow and O2 transport during hypothermia and rewarming is conflicting. Some report that rewarming after acute (Blair, Montgomery, & Swan, 1956) or prolonged (Steen, Milde, & Michenfelder, 1980) hypothermia might be unsuccessful owing to inadequate tissue perfusion, whereas others report maintained tissue oxygenation during cooling to 29°C (Anzai, Turner, Gibson, & Neely, 1978; Gutierrez, Warley, & Dantzker, 1986; Morray & Pavlin, 1990). Despite differences in experimental protocols, one may obtain the impression that larger animal models tolerate hypothermia/rewarming better (Anzai et al., 1978; Filseth, How, Kondratiev, Gamst, & Tveita, 2010) than smaller animals (Kondratiev, Flemming, Myhre, Sovershaev, & Tveita, 2006; Tveita, Mortensen, Hevrøy, Refsum, & Ytrehus, 1994; Tveita, Skandfer, Refsum, & Ytrehus, 1996a). We therefore used a pig model of experimental hypothermia and rewarming (Filseth et al., 2010). Essential factors for survival during hypothermia and rewarming are O2 transport and regional blood flow (Kondratiev et al., 2006; Tveita et al., 1996b), and therefore it appears essential to explore and learn ‘physiological strategies’ of O2 transport from euthermic organisms successfully rewarmed with maintained spontaneous circulation.
The goal of the present study was to examine the changes in organ blood flow and O2 delivery (
) in a porcine model with spontaneous circulation during hypothermia and rewarming. To simulate the representative duration for evacuation and transport of a hypothermic patient (Gilbert et al., 2000; Mark et al., 2012; Wanscher et al., 2012), a 3 h period of hypothermia was chosen. Cooling to 27°C core temperature was selected because this is close to the lower limit to maintain spontaneous circulation in humans. For rewarming, the model was instrumented for pleural lavage, an invasive method increasingly applied for rewarming in clinical practice.
2 METHODS
2.1 Ethical approval
The Norwegian Food Safety Authority approved the study (reference no. 14/56323). Sixteen castrated male juvenile pigs (20–29 kg) from NOROC stock were used. On arrival, the animals acclimated for 2–5 days before experiments were conducted. The animals were fed twice daily, had free access to water at all times, and received humane care in accordance with the Norwegian Animal Welfare Act.
2.2 Anaesthesia and instrumentation
After an overnight fast, anaesthesia was administered by an i.m. bolus of ketamine hydrochloride 20 mg kg−1 (Ketalar, Pfizer Norge AS, Oslo, Norway), midazolam 30 mg (B. Braun Melsungen AG, Germany) and atropine 1.0 mg (Takeda AS, Asker, Norway). After transfer to the experimental laboratory, an ear vein catheter was inserted, and a bolus injection of fentanyl (10 μg kg−1) (Fentanyl-Hameln, Hameln Pharma plus Gmbh, Hameln, Germany) and pentobarbital sodium (10 mg kg−1) (Ås produksjonslab, Ås, Norway) was given. After tracheostomy, a continuous infusion of fentanyl (20 μg kg−1 h−1), midazolam (0.3 mg kg−1 h−1) and pentobarbital sodium (4 mg kg−1 h−1) along with Ringer acetate (9 ml kg−1 h−1) in the right external jugular vein was started and maintained throughout the experiment. No neuromuscular blockers were used at any time. During experiments in normothermic and hypothermic animals, the level of surgical anaesthesia was assessed frequently by applying a pinching stimulus to the septum of the nose. Animals were ventilated without positive end-expiratory pressure (Siemens Servo 900D, Solna, Sweden). The fraction of inspired O2 was adjusted to maintain arterial 
>10 kPa, and alveolar ventilation adjusted to keep arterial 
between 4.5 and 6 kPa uncorrected for temperature. Arterial blood gases were analysed (ABL800 FLEX; Radiometer Medical, Copenhagen, Denmark) to confirm adequate ventilation. After termination of the experiment, animals were killed by an i.v. lethal dose of pentobarbital and 20 ml potassium chloride, 1 mmol ml−1.
Left ventricular pressure recordings and microsphere injections were made by introducing a 6 F fluid-filled pigtail catheter (Cordis Corporation, Miami, FL, USA) into the right common carotid artery through a 10 F Super Arrowflex (Arrow International Inc., Reading, PA, USA) introducer. Pulmonary artery pressure (PAP), central venous pressure (CVP), blood core temperature measurements and determination of mixed venous and venous blood gases were enabled by introducing a 7 F Swan-Ganz thermodilution catheter (Edwards Lifesciences LLC, Irvine, CA, USA) into the pulmonary trunk via the right external jugular vein. A single dose of 5000 IU heparin was given after placement of the thermodilution catheter. The tip of another 7 F Swan-Ganz thermodilution catheter was positioned in the aortic arch via the left femoral artery for arterial blood gas analysis, mean arterial pressure (MAP) recordings and collection of the reference blood sample for the microsphere technique. An 18-gauge central venous catheter (Arrow International Inc.) was introduced cranially into the left external jugular vein and advanced to the jugular bulb for blood sampling. A 14 F urinary bladder catheter was introduced via a lower abdominal incision for continuous monitoring of urinary output.
2.3 Experimental protocol
After instrumentation and 30 min stabilization, baseline haemodynamic recordings were obtained. Animals were divided into the following two groups: (i) hypothermia group (n = 10), immersion cooled to 27°C (1.5 h), kept at 27°C with spontaneous circulation for 3 h before rewarming (3 h) to 38°C using pleural lavage (2 h); and (ii) a time-matched normothermia control group (n = 6), kept at 38°C for 6.5 h, the first 1.5 h by immersion, the last 2 h by pleural lavage. Evaluation of haemodynamic variables, assessment of global and cerebral O2 transport, and determination of regional blood flow by means of stable isotope-labelled microspheres [BioPhysics Assay Laboratory (BioPAL), Inc., Worcester, MA, USA], were performed at baseline (38°C), during cooling at 32 and 27°C, hourly during 3 h stable hypothermia (27°C), and during rewarming at 32 and 38°C. Blood flow was measured in the brain, heart, liver, kidneys, stomach and small intestine.
2.4 Cooling and rewarming
Animals were cooled by circulating cold water (5°C) in ice slush, leaving two-thirds of the dependent animal submerged in a tarpaulin tub mounted on top of the operating table. The head was placed on a cushion out of the water and not covered with ice slush. Blood core temperature was monitored by a thermistor on the 7 F thermodilution catheter (Edwards Lifesciences). After core temperature was reduced to 28°C, the cooling exposure was terminated by draining the tub of cold water, after which core temperature subsequently dropped to 27°C. To prevent further reduction in core temperature, careful warming was performed by repositioning the operation lamp closer to the animal. For rewarming, two PVC tubes were placed in the left pleural space, one 16 F in the mid-clavicular line in the second intercostal space, the other (24 F) in the mid-axillar line in the sixth intercostal space. Via tubing, the upper (inlet) pleural tube was connected to a roller pump circulating water at 40–42°C from the reservoir, whereas drainage was obtained via the outlet tube by gravity. Care was taken to keep flow rate <500 ml min−1 to avoid a potential tension hydrothorax, as described by Barr, Halvorsen, and Donovan (1988), with close attention to the water temperature and water level in the reservoir. Time-matched normothermia control animals were kept immersed in warm water (38–40°C) to maintain core body temperature at 38°C for 6.5 h. Pleural lavage was performed during the last 2 h in the same manner as in the hypothermia group.
2.5 Data sampling
Each data sampling lasted 10–15 min and was carried out in the following order: (i) sampling of heart rate (HR), MAP, CVP and left ventricular pressures (LVP); (ii) recording of core temperature, diuresis and respirator settings; (iii) simultaneous blood sampling from catheters placed in the femoral artery, pulmonary artery and right and left jugular veins for arterial, mixed venous, venous and jugular bulb for blood gas measurements (not corrected for temperature); (iv) injection of stable isotope-labelled microspheres into the left ventricle, and simultaneous collection of a reference blood sample from the aortic arch. Data series were collected at baseline, during cooling at 32 and 27°C, hourly during 3 h stable hypothermia, and during rewarming at 32 and 38°C in the hypothermia group. In the normothermia control group, data series were collected at baseline, at 45 and 90 min after baseline, and thereafter hourly.
2.6 Calculation of haemodynamic variables, global and cerebral O2 transport and O2 extraction rate
Cardiac output was calculated using the following formula: CO (in litres per minute) = 
× D/d/1000, where 
is sample rate of the reference blood sample in millilitres per minute, D is total activity of injected microspheres, and d activity of the microspheres in the reference blood sample in counts per minute. Stroke volume (SV) was calculated as follows: SV (in millilitres) = CO/HR × 1000. Total peripheral resistance (TPR) was calculated as follows: TPR ( in dynes seconds per centimetre raised to the fifth power) = (MAP − CVP) × 80/CO. Oxygen content (in millilitres per 100 ml) values (arterial, mixed venous and jugular bulb) were calculated according to the formula: 
× Hb × (1.34 × 10−2), where 
is blood O2 saturation as a percentage and Hb is the haemoglobin value in millilitres per decilitre measured in the corresponding blood samples. Global O2 delivery (
) was calculated as the product of CO and arterial O2 content per kilogram of body weight (in millilitres per minute per kilogram). Global O2 consumption (
) was calculated as the product of CO and the difference between arterial and mixed venous O2 content per kilogram of body weight (in millilitres per minute per kilogram). Cerebral 
was calculated as the product of cerebral blood flow (in millilitres per 100 g brain tissue) and arterial O2 content (in millilitres per minute per 100 g). Cerebral 
was calculated as the product of cerebral blood flow in millilitres per 100 g brain tissue and the difference between arterial and jugular bulb O2 content (in millilitres per minute per 100 g). Cerebral blood flow values were calculated from pooled data of left and right brain blood flow (regional blood flow estimation technique described below). Global and cerebral O2 extraction rate (ER) was calculated as the ratio of corresponding 
to 
values.
2.7 Stable isotope-labelled microsphere technique for measuring regional blood flow
Stable isotope-labelled microspheres were used for measuring regional organ blood flow (Reinhardt, Dalhberg, Tries, Marcel, & Leppo, 2001). This is a refined method for measuring regional blood flow with the use of neutron-activated microspheres. At every data sampling time point an injection of ∼106 microspheres, of different specificity, was given through a fluid-filled pigtail catheter placed in the left ventricle. At the same time, a reference blood sample was drawn from the aortic arch using a withdrawal pump set at a fixed sample rate (5 ml min−1) for 2 min (New Era Pump Systems, Inc., Farmingdale, NY, USA). Each reference blood sample, with the predetermined type of microsphere in accordance with the data collection point, was collected in 20 ml sample vials. The syringe was rinsed with ‘sansSaLine’ medium (BioPAL, Inc.) to secure removal of microspheres attached to the wall. Reference blood samples were centrifuged twice with ‘sansSaLine’ to remove sodium and chloride together with the supernatant to improve the signal-to-noise ratio of the sample. After the end of the experiment, animals were killed and organ tissue samples were taken from the same locations in all animals and rinsed with ‘sansSaLine’ to remove surface blood and other potential contaminants. Both reference blood samples and organ tissue samples were dried in an oven (70°C overnight). After processing, reference blood samples and tissue samples were analysed at the BioPAL laboratory for specific activity. Tissue samples for evaluation of regional blood flow were taken from the heart, brain, kidneys, liver, stomach and small intestine. Determination of regional tissue blood flow (expressed in millilitres per minute per gram) was calculated using the following equation: 
, where 
is blood flow in millilitres per minute, TisCPM is the number of radioactive counts in the tissue sample in counts per minute, 
is the reference flow rate in millilitres per minute, RefCPM is the number of radioactive counts in the reference blood sample in counts per minute, and g is weight of the tissue sample in grams.
2.8 Statistical analyses
Statistical analyses were performed using SigmaPlot statistical software version 12 [Systat Software Inc. (SSI), Richmond, CA, USA]. Intragroup comparisons were performed by one-way repeated-measures ANOVA. If significant differences were found, Dunnett's post hoc test was used to compare values within group versus baseline, and intragroup values during ongoing thoracic lavage versus pre-lavage values. The level of significance was set at P ≤ 0.05. Data are presented as means ± SD.
3 RESULTS
All animals survived the experiments. Cooling to 27°C lasted 80 ± 15 min, and rewarming lasted 167 ± 16 min. Neuromuscular blocking agents were not used in these experiments and, therefore, visible shivering could be detected. Shivering occurred during cooling in every animal but disappeared after an i.v. bolus of fentanyl. Shivering was not observed during rewarming.
3.1 Normothermic control animals
According to the protocol, six animals were kept at 38°C for 6.5 h and served as normothermic controls. During the last 2 h, to simulate pleural rewarming in the hypothermia group, continuous pleural lavage with warm water was given under core temperature control.
3.1.1 Haemodynamics (Figure 1a–f)
During the first 4.5 h, before pleural lavage was activated, all haemodynamic variables recorded remained unaltered (P > 0.05). After activation of the pleural lavage, simultaneous reductions (P < 0.05) in SV and CO and a significant (P < 0.05) increase in CVP from 4.8 ± 1.9 to 7.5 1.5 mmHg (data not tabulated) took place when compared with its baseline valve at 0 h. In more detail, at the end of experiment (6.5 h), SV was reduced by 43% and, despite signs of simultaneous activation of compensatory mechanisms (increase in HR, MAP and TPR), CO remained reduced by 25% at 6.5 h. Left ventricular systolic pressure (LVSP) remained unchanged (P > 0.05) during 6.5 h at 38°C (data not tabulated). However, if we compared the same variables during pleural lavage using 4.5 h as a new baseline instead of 0 h, we found a significant (P < 0.05) increase in MAP and TPR, whereas SV, CO and HR remained unaltered (P > 0.05). At the end of protocol (6.5 h), the only changes from 4.5 h were a significant (P < 0.05) reduction in SV and an increase (P < 0.05) in HR.
3.1.2 Global and cerebral O2 transport, 
, plasma lactate, mixed venous O2 saturation (
) and O2 ER (Figures 2 and 3)
Throughout the experiment, global and cerebral 
and 
all remained unaltered (P > 0.05) as were plasma lactate and 
. Global and cerebral O2 ER both touched the upper critical limit (0.7) during the period with pleural lavage.
), O2 consumption (
) and O2 extraction ratio (ER). (a, c) Time-matched normothermia (38°C) control group; n = 6 animals. (b, d) Variables during cooling, for 3 h at 27°C, and rewarming; n = 10 animals. Values are means ± SD. *P < 0.05 compared with 380°C in the normothermia control group or the prehypothermic value in the hypothermia and rewarming group. #P < 0.05 compared with 384.5h°C in the normothermia control group or 273h°C in the hypothermia and rewarming group
). (a) Time-matched normothermia (38°C) control group; n = 6 animals. (b) Variables during cooling, for 3 h at 27°C, and rewarming; n = 10 animals. Values are means ± SD. *P < 0.05 compared with 380°C in the normothermia control group or the prehypothermic value in the hypothermia and rewarming group. #P < 0.05 compared with 384.5h°C in the normothermia control group or 273h°C in the hypothermia and rewarming group
3.1.3 Regional blood flow (data not tabulated)
After rewarming, organ blood flow in brain, kidneys, heart, liver, stomach and small intestine all remained unchanged (P > 0.05). However, there was a significant (P < 0.05) reduction in blood flow to the right and left kidney that occurred during the last 2 h at 38°C.
3.2 Cooling to 27°C and 3 h stable hypothermia
3.2.1 Haemodynamics (Figure 1a–f)
Compared with their prehypothermic control values, an almost linear reduction in HR, MAP, CO and SV occurred during immersion cooling. Already at 32°C, CO was significantly (P < 0.05) reduced, whereas HR, MAP, LVSP (data not tabulated), CVP (data not tabulated) and SV were significantly reduced (P < 0.05) first at 27°C. An apparent linear increase in TPR took place during cooling, but the increase was significant (P < 0.05) only after 2 h at 27°C (Figure 1a–f). Except for CVP, which returned to within its normothermic control value after 1 h at 27°C and remained unchanged (P > 0.05) for the rest of the experiment, all the other haemodynamic variables remained stable at altered levels for the rest of the 27°C hypothermia period except for during short episodes with nodal rhythm and various ventricular arrhythmias occurring in six out of 10 animals but eliminated by i.v. injections of lidocaine hydrochloride or amiodarone. In addition, in three out of 10 animals ventricular fibrillations occurred during stable hypothermia but were converted to sinus rhythm by single or repeated DC discharges at 150 J.
3.2.2 Global and cerebral 
and 
, ER, plasma lactate and 
(Figures 2 and 3)
During cooling to 27°C, global 
and 
decreased linearly and were reduced (P < 0.05) by 56 and 43%, respectively, at 27°C and remained statistically significantly reduced (P < 0.05) at this level throughout the remaining stable hypothermia period. Changes in cerebral 
and 
were almost identical to the changes in global 
and 
. Calculated ER remained statistically unchanged during cooling but was significantly (P < 0.05) increased after 2 h stable hypothermia in both brain and global circulation and touched its critical level (0.6–0.7) at this time point. A simultaneous significant reduction (P < 0.05) in 
was measured after 2 h at 27°C, but the plasma lactate concentration remained unchanged (P > 0.05).
3.2.3 Regional blood flow (Figure 4a–d)
At 27°C during cooling, blood flow in heart, brain, kidneys and liver was significantly (P < 0.05) reduced and remained reduced at this low level throughout the stable hypothermia period (27°C). Blood flow in small intestine was reduced (P < 0.05) after 1 h at 27°C and remained reduced during the next 2 h at this temperature. In the stomach, however, blood flow remained unchanged (P > 0.05) throughout cooling and stable hypothermia.
3.3 Rewarming after 3 h at 27°C
3.3.1 Haemodynamics (Figure 1a–f)
During rewarming, CO and SV remained significantly (P < 0.05) reduced, at the same level as at 27°C, with both ending up being reduced by 40% after rewarming. In contrast, MAP, LVSP, CVP and HR returned to within their prehypothermic baseline values after rewarming to 38°C. During and after rewarming, TPR remained significantly (P < 0.05) increased when compared with its prehypothermic control value.
3.3.2 Global and cerebral 
and 
, ER, plasma lactate and 
(Figures 2 and 3)
In parallel with CO and SV, 
remained significantly (P < 0.05) reduced throughout rewarming to 38°C, whereas 
increased linearly and returned to within its prehypothermic control level at the end of rewarming. Global ER, which was below the critical value (0.6–0.7) during hypothermia and rewarming, was significantly (P < 0.05) increased after rewarming and ended up between 0.6 and 0.7 simultaneous with a significant (P < 0.05) reduction in 
. However, brain ER remained below this critical level after rewarming.
3.3.3 Regional blood flow (Figure 4a–d)
After rewarming, the blood flows to the heart, brain and small intestine returned to their individual prehypothermic control levels, whereas blood flow in the right and left kidneys and liver remained significantly (P < 0.05) reduced. Unlike flow to all other organs monitored, stomach blood flow remained unaltered (P > 0.05) throughout cooling, stable hypothermia and rewarming.
4 DISCUSSION
The results of the present study using an intact porcine model of accidental hypothermia and rewarming with maintained spontaneous circulation showed that blood flow in essential organs, such as the brain and heart, returned to their prehypothermic values after rewarming. In addition, owing to physiological compensatory mechanisms for O2 extraction, spontaneous circulation provided adequate global and cerebral 
during hypothermia and rewarming to support tissue 
despite simultaneous alterations in haemodynamic function.
Only a limited number of accidental hypothermia patient case reports have been published, but fortunately, national and international hypothermia registries have been established (Walpoth et al., 2017). However, these registries are relatively new, the annual number of patients added is relatively low, the mechanisms for cooling are heterogeneous, and it will therefore take time before essential new information can be extracted. Therefore, the new information needed must be collected from preclinical animal studies. Accordingly, the results of the present study conducted using a pig model are important to help guide clinical practice. The pig shares a number of anatomical and physiological characteristics with humans, especially with respect to the cardiovascular system (Swindle, Makin, Herron, Clubb, & Frazier, 2012).
Numerous preclinical studies have reported that hypothermia and rewarming induce an impairment of haemodynamic function, which may create therapeutic challenges, especially during rewarming. Multiple pathophysiological mechanisms have been suggested (Chen & Chien, 1977; Hammersborg et al., 2005; Han, Tveita, Prakash, & Sieck, 2010; Kondratiev, Wold, Aasum, & Tveita, 2008), and most of these describe cardiac mechanical dysfunction after rewarming, with a reduction of circulating blood volume, a gradual increase of blood viscosity and an increase of vascular tone. The severity of these changes in haemodynamic function appears to depend on the extent and duration of hypothermic exposure (Kondratiev et al., 2008). In the present study, MAP was restored after rewarming, although TPR remained unchanged compared with 27°C throughout the rewarming period. The mechanisms for the restored MAP reflect the significant increase in CO and HR during the rewarming from 27°C. However, when compared with baseline prehypothermic values, the significant reduction of SV and CO without any compensatory increase in HR after hypothermia and rewarming indicate the presence of a decompensated hypothermia-induced cardiac dysfunction. These results are in contrast to data provided by Filseth et al. (2010) using the same model. These investigators reported moderately diminished SV and changes in indices of left ventricular contractility indicating isolated left ventricular systolic cardiac dysfunction. However, they found that CO returned to prehypothermic control levels owing to an apparent compensatory increase in HR after rewarming following 1 h at 25°C. The results of the present study are more similar to the findings of Blair et al. (1956), using a dog model. Numerous previous studies performed on rats by our research group (Kondratiev et al., 2006; Tveita et al., 1996a) showed similar circulatory dysfunction after rewarming from 2–5 h hypothermia.
Normally, 
exceeds tissue 
, set by its metabolic activity, and 
is largely independent of delivery. The O2 extraction ratio (
) is normally in the range of excess O2 supply (Schumacker & Cain, 1987), but in cases with limited available O2 or increased demand this ratio increases and approaches a critical value at which tissue O2 consumption is limited and depends on O2 supply (Schumacker, Rowland, Saltz, Nelson, & Wood, 1987). This critical O2 extraction ratio is reported to be 0.6–0.7 in normothermic organisms (Leach & Treacher, 1992), but the critical value appears to be narrowed (∼0.65) after decreasing core temperature in experimental animals (Schumacker et al., 1987). Thus, if compared with normothermia, cooling makes tissues apparently more dependent on O2 supply to cover tissue O2 demand because of a decreased efficacy of O2 extraction. This decrease in efficacy of O2 extraction during hypothermia might be caused by a hypothermia-induced increase in vascular resistance (Schumacker et al., 1987), which was also observed in the present study. Such an increase in vascular resistance might interfere with local metabolic feedback to arterioles regulating the distribution of blood flow. The presence of latent physiological compensatory mechanisms at low core temperatures is also supported by other experimental results. First, global 
was reduced during hypothermia owing to a Q10 effect (Schumacker et al., 1987) and returned to prehypothermic values after rewarming. This finding also receives support in data from our previous studies (Kondratiev et al., 2006; Tveita et al., 1996a, b), indicating that mechanisms responsible for O2 extraction to the tissues are also functioning at low core temperatures. Second, the O2 extraction ratio never exceeded the critical level (0.6–0.7) during hypothermia but approached this level first after rewarming, when SV and CO were both reduced by 40%. This level of O2 extraction is below the critical O2 delivery point and indicates preserved use-dependent O2 transport (Leach & Treacher, 1992; Schumacker & Cain, 1987). The presence of a patent O2 delivery also finds support in our plasma lactate and 
data. Apart from ending up being significantly reduced to a critically low level after rewarming, 
remained largely stable and within the physiological range during hypothermia and rewarming in concert with unaltered plasma lactate concentrations.
During hypothermia, blood flow was reduced in all organs that were evaluated. However, despite a 40% reduction in SV and CO after rewarming, blood flow returned to prehypothermic control levels in all organs, except for the liver and kidneys. Blood flow in the brain was determined from four different areas, and the pattern of blood flow changes was uniform throughout hypothermia and rewarming. The results of the present study are generally in agreement with those reported by Anzai et al. (1978), who found total restitution of regional blood flow after rewarming from a short (30 min) exposure to hypothermia in dogs. Therefore, our results indicate that there are physiological compensatory responses to a low CO in severe hypothermia and rewarming similar to the well-recognized compensatory mechanisms taking place in other critical conditions accompanied by reduced CO (Carter, Tompkins, Yarmush, Walker, & Burke, 1988; Seyde, McGowan, Lund, Duling, & Longnecker, 1985). Accordingly, cardiovascular homeostasis appears to be preserved after rewarming from 3 h of hypothermia. In contrast, the blood flow distribution found in pigs in the present study is different from that which we reported in rats, where, after 5 h at 15°C, blood flow in essential organs was not restored after rewarming (Tveita et al., 1996b). Again, this indicates that the duration and level of hypothermic exposure are essential factors for survival during hypothermia and after rewarming.
Unexpected observations were made in the present study with respect to changes in haemodynamic function over time during PL. Most prominent was the significant reduction in SV and CO in the normothermia control animals that took place during the last 2 h of the experiment, with PL. This finding is in contrast to previous reports from our laboratory, using pigs (Filseth et al., 2010, 2012) and other intact animal models, in which we documented haemodynamic stability during continuous anaesthesia with controlled ventilation for hours at normothermia. However, after comparing the same variables with those measured immediately before activating PL (4.5 h), instead of at the start of experiment, only SV was reduced at the end of experiment. Compared with previous experiments, the only methodological change in the present study was the surgical placement of two pleural catheters for introducing and draining warm water for central rewarming. This procedure was performed in accordance with techniques described in previous publications (Barr et al., 1988; Brunette, Sterner, Robinson, & Ruiz, 1987; Otto & Metzler, 1988). These publications all documented a return to the precooling MAP level after rewarming with PL, similar to what we found after rewarming in the present study (Otto & Metzler, 1988). However, out of these three publications only Otto & Metzler (1988) reported CO in addition to MAP, although they questioned the validity of their CO measurement owing to the use of the thermodilution technique. After having performed methodological adjustments, these authors estimated a 48.5% restitution of CO after rewarming. We measured a significant reduction in CO after 2 h with PL in both our experimental groups. The reduction in CO measured after rewarming can be attributed to hypothermia-induced cardiac dysfunction, as already discussed, but the reduction in CO in the normothermic control group was unexpected and difficult to explain other than being attributable to the activation of PL. We measured CO using two independent methods, thermodilution and microsphere injections, and both showed similar reductions in CO. Pleural rewarming is being used increasingly in clinical practice for rewarming accidental hypothermia patients, both with preserved spontaneous circulation and in cardiac arrest (Kjaergaard & Bach, 2006; Plaisier, 2005; Turtiainen, Halonen, Syväoja, & Hakala, 2014; Winegard, 1997). The only major complication reported so far when applying this technique is an increase in intrathoracic hydrostatic pressure if pleural adhesions are present, which may result in accidental catheter occlusion. The present results, however, are in support of the notion that in hypothermic patients, often suffering from hypothermia-induced cardiac dysfunction, the use of PL for rewarming must be considered, and therefore used with caution.
4.1 Conclusions
In conclusion, the present study shows that after rewarming, despite the existence of a significantly reduced CO, blood flow distribution and O2 availability are preserved in vital organs owing to the presence of adequate physiological compensatory responses. When using CPB for rewarming accidental hypothermia patients in cardiac arrest, detailed insight into these physiological compensatory responses can be useful when applying complex therapeutic approaches.
AUTHOR CONTRIBUTIONS
Conception and design: S.V., T.T., G.C.S. and T.K. Completion of experiments and collection of data: S.V., R.M., J.H.N., T.S. and T.K. Data analysis and interpretation: T.T., G.C.S., T.K., S.V., R.M., J.H.N. and T.S. Drafting the manuscript for intellectual content: S.V., T.T. and G.C.S. Revision of the manuscript: S.V., R.M., J.H.N., T.S., T.K., G.C.S. and T.T. All authors approved the final version of the manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.
COMPETING INTERESTS
None declared.
