Hypothermia does not influence liver damage and function in a porcine polytrauma model

Abstract

BACKGROUND:

Previous studies revealed evidence that induced hypothermia attenuates ischemic organ injuries after severe trauma. In the present study, the effect of hypothermia on liver damage was investigated in a porcine long term model of multi-system injury, consisting of blunt chest trauma, penetrating abdominal trauma, musculoskeletal injury, and hemorrhagic shock

METHODS:

In 30 pigs, a standardized polytrauma including blunt chest trauma, penetrating abdominal trauma, musculoskeletal injury, and hemorrhagic shock of 45% of total blood volume was induced. Following trauma, hypothermia of 33 ${}^{\circ}$ C was induced for 12 h and intensive care treatment was evaluated for 48 h. As outcome parameters, we assessed liver function and serum transaminase levels as well as a histopathological analysis of tissue samples. A further 10 animals served as controls.

RESULTS:

Serum transaminase levels were increased at the end of the observation period following hypothermia without reaching statistical significance compared to normothermic groups. Liver function was preserved ( $p\leqslant$ 0.05) after the rewarming period in hypothermic animals but showed no difference at the end of the observation period. In H&E staining, cell death was slightly increased hypothermic animals and caspase-3 staining displayed tendency towards more apoptosis in hypothermic group as well.

CONCLUSIONS:

Induction of hypothermia could not significantly improve hepatic damage during the first 48 h following major trauma. Further studies focusing on multi-organ failure including a longer observation period are required to illuminate the impact of hypothermia on hepatic function in multiple trauma patients.

Keywords

Hypothermia porcine animal model trauma model liver laceration

1. Introduction

In multiple trauma patients, the liver is the most affected organ in the abdominal cavity [1, 2, 3, 4]. In addition to the direct injury, systemic hypotension as a consequence of bleeding and hemorrhagic shock may lead to decreased hepatic perfusion and consecutive impaired function. Ischemia to the liver leads to a loss of mitochondrial adenosine triphosphate (ATP) production resulting in cell swelling, membrane breakdown and oxidative stress, followed by disruption of microcirculation as well as inflammation causing further injury to the liver [5].

Induced hypothermia represents a novel therapeutic approach aiming at preserved organ function following multiple injuries [6] showing encouraging results [7, 8, 24]. However, to date, effects of hypothermia were mainly investigated in isolated hemorrhagic shock and small animal models [9, 10]. Despite numerous advantages, mild induced hypothermia is associated with various complications. Temperature-dependent impairment of enzyme activity as well as physiological activities like blood circulation, respiration, coagulation, renal and hepatic functions were described [11].

In the present study, the effect of hypothermia on liver damage was investigated in a porcine long term model of multi-system injury, consisting of blunt chest trauma, penetrating abdominal trauma, musculoskeletal injury, and haemorrhagic shock [12].

2. Methods

2.1 Animal experiments

This study was approved by local authorities (Ref. 22/2013; Regional board, Giessen, Germany), and the study was performed in compliance with the Helsinki convention for the use and care of animals. Report of this study is performed in agreement with the ARRIVE guidelines [13]. In the current study, data of 40 male pigs (Deutsche Landrasse) weighing in median 34.1 kg (29.7–44.8 kg) are described. Following the accepted principal of the “3 Rs” (restriction, refinement, reduction), we compared the data of hypothermic animals in this study to results of a normothermic cohort gained from an identical experimental setting (NT-T: $n=$ 15; sham: $n=$ 5) [12].

Animals were randomly assigned to one of 4 groups studied: Sham normothermia (Sn; $n=$ 5) Sham hypothermia (Sh; $n=$ 5) Trauma 45% blood loss, 90 min shock, normothermia (Tn; $n=$ 15) Trauma 45% blood loss, 90 min shock, hypothermia (Th; $n=$ 15)

All animals were intramuscularly injected with diazepam (1 mg/kg), ketamine (20 mg/kg) and atropine (0.5 mg). Anesthesia was induced and sustained with sufentanil and propofol during the entire study period of 48.5 hours. After orotracheally intubation pressure controlled ventilation with a tidal available ventilator (Draeger, Evita, Danvers, MA, USA) was performed. After adequate monitoring, a tracheotomy was performed. Respiratory rate was varied to achieve an endtidal CO ${}_{2}$ $<$ 7.3 kPa. Blood gas analyses (BGA, ABL700, Radiometer Copenhagen) were performed every 2 hours or if required. A balanced saline solution (Ringer-Acetate) was continuously infused at a rate of 2 ml/kg per hour. Under aseptic conditions an arterial catheter PiCCO-system (pulse contour cardiac output, picco plus, pulsion medical systems, Munich, Germany) was placed in the left femoral artery. A central venous line (3 lumen HD, Arrow, PA, USA) was inserted in the right jugular vein and a two-lumen hemodialysis line (two lumen HD, 14Fr, 15 cm, Arrow, PA, USA) was placed in the left femoral vein. For continuous measurement of urinary output a suprapubic urine catheter was placed.

All ventilation and hemodynamic parameters were monitored and recorded. Electrocardiogram, oxygen saturation, temperature and arterial pressure were continuously noted.

Figure 1.

Time schedule. Time flow in experimental setup of Group Th/ Tn; points of analysis of blood and urine samples are marked and differences in set up pointed out.

2.2 Trauma induction

The time schedule of the current study is depicted in Fig. 1. At baseline, the right hind leg was placed into the drop-weight device and 20 kg of plumb-cuboid was guided dropped from a height of 100 cm. Afterwards, a blunt chest trauma of the right thorax, (captive bolt stunner, direct contact on a panel 10 $\times$ 10 cm, upper layer of lead, lower layer of steel) was performed as described earlier [8], followed by an laparotomy and laceration of the liver (caudal lobe) induced by stabbing twice with a four edged scalpel (Fig. 2b). The consequent uncontrolled bleeding was addressed after 30 s with a tamponade.

Figure 2.

Experimental setup and macroscopic liver traumatisation. a) Experimental setup during hypothermia. b) Liver lacerations Grade II placed with four edged scalpel. c) Liver lacerations still bleeding after 24.5 h of observation period during second look.

Simultaneously, hemorrhage was induced by draining up to 45% of estimated total blood volume (TBV) in groups Tn and Th or until a mean arterial blood pressure (MAP) of 30 $\pm$ 5 mm/Hg was achieved and maintained for 90 min. Fluid resuscitation was performed with warmed lactated Ringer’s solution (four times hemorrhagic-volume/60 min.).

In hypothermia groups, two Gel Pads (Arctic sun 5000, Medivance, Louisville, USA) were placed at the flanks of animals (Fig. 2a) and programmed to cool animals down for 12 hours reaching a core temperature 33 ${}^{\circ}$ C followed by a 10 hour rewarming period (0.5 ${}^{\circ}$ C per hour) as previously described by Horst et al. [17]. Temperature was measured with an intraesophageal temperature sensor. After 24.5 h a second look surgery and repacking of the liver were performed.

During the whole observation period, standardized intensive care management (bedding, airway and volume management, temperature control) was performed. Complications were addressed according to current standards of emergency medicine and trauma surgery [14]. Typical emergency medications were intravenously applied as required for life-threatening events (i.e. seizure, tension pneumothorax, ventricular fibrillation, cardiac arrest). Sham animals underwent same procedure without traumatization. Methods and results of further analyses, gained from the same project, are explained in detail in separate publications [12, 15, 16, 17, 18].

2.3 Sample collection

At 6 time points, analyses of blood and urine samples were performed (Fig. 1):

1.
Baseline (before induction of trauma)
2.
After trauma
3.
After reperfusion
4.
At 14.5 h after trauma (following hypothermia)
5.
At 24.5 h after trauma (following rewarming)
6.
At 48.5 h after trauma

Samples were centrifuged (4 ${}^{\circ}$ C, 5 min, 4000 rpm, Centrifuge 5702R, Eppendorf, Hamburg, Germany), deep frozen and stored at $-$ 80 ${}^{\circ}$ C until further analyzed.

Euthanasia was followed by a standardized section. Tissue samples were taken from all livers.
2.4 Liver assessment

Liver function was monitored by elimination of indocyanine green (ICG) using the LiMON technology (Pulsion Medical Systems, Munich, Germany) [16]. Measurement was non-invasively performed at the ear or tongue. Analyses were performed at time points 1, 4, 5 and 6. The first measurement at the beginning of the experiment was defined as baseline value ( $=$ 100%). Values were described in percent of baseline value.

For assessment of liver damage, serum levels of AST, ALT and LDH were measured at time points described in Fig. 1 and histological evaluation was performed.

2.5 Histological evaluation

Liver tissue was fixed with 4% formalin for 7 days and afterwards embedded in paraffin. Sections of 4- $\mu$ m were stained with haematoxylin-eosin. A pathologist blinded to the study protocol reviewed stained samples with a light microscope and evaluated hepatic injury with the following semi-quantitative score to rate the degree of apparent cell death per hepatic lobule: 0: $<$ 5%, 1: $<$ 30%, 2: 30–60%, 3: $>$ 60%.

In addition, immunohistochemistry for cleaved caspase-3 (cleaved caspse-3, Asp175, rabbit polyclonal antibody; dilution 1:300; Cell Signalling Technology, Beverly, MA) using the ABC method (labelled biotin) was performed. Results were blindly assessed and semi-quantitatively analysed. Here, 10 randomized lobules were assessed by summarising the quantity (1–10 $=$ 10–100% of affected lobules) and the localisation of apoptosis in 10 related lobules.

2.6 Statistics

Data were collected using a Filemaker ${}^{\@setsize{\scriptsize}{9.5pt}{\viiipt}{\@viiipt}\textregistered}$ Database (FilemakerPro5.0, Filemaker Inc., Santa Clara, California, USA). Double entry with plausibility check was performed to improve data quality. Statistics were compiled using Excel ${}^{\@setsize{\scriptsize}{9.5pt}{\viiipt}{\@viiipt}\textregistered}$ (Microsoft 2010, Redmond, USA) and SPSS ${}^{\@setsize{\scriptsize}{9.5pt}{\viiipt}{\@viiipt}\textregistered}$ (PASW Statistics 22, Quarry Bay, Hong Kong). The Kolmogorov–Smirnov Test was used to test for normal distribution of data; homogeneity of variance was tested by the Levene Test.

Group differences were tested by the non-parametric Kruskal-Wallis Test. Individual groups were subjected to subsequent post-hoc analysis with the Mann-Witney-U Test; $p\leqslant$ 0.05 was considered statistically significant. Data are presented as mean $\pm$ standard deviation or as median and range.

Table 1
Baseline data

	Group Sn	Group Sh	Group Tn	Group Th	$p$
Number of animals	5	5	15	15
Weight (kg)	36.8 (SD $\pm$ 4.4)	35 (SD $\pm$ 2)	32.67 (SD $\pm$ 2.3)	35 (SD $\pm$ 3)
Mortality (%)	0	0	13 (N $=$ 2)	0	$p\geqslant$ 0.05
CPR (%)	0	0	26.67 (N $=$ 4)	7(N $=$ 1)	$p\geqslant$ 0.05
Chest tube (%)	0	0	40 (N $=$ 6)	27(N $=$ 4)	$p\geqslant$ 0.05

Sampled data is given as mean $\pm$ SD. Group differences were tested by the non-parametric Kruskal-Wallis test. Individual groups were subjected to subsequent post-hoc analysis with the Mann-Whitney U-test. $p<$ 0.05 was considered statistically significant. Sn $=$ Sham-normothermic, Sh $=$ Sham-hypothermic, Tn $=$ Trauma-normothermic, Th $=$ Trauma-hypothermic.

3. Results

3.1 Basic data

Basic data is shown in Table 1. In 6 normothermic trauma animals, insertion of a chest tube was required while 4 were inserted in hypothermic trauma animals. The incidence of required CPR was increased in the normothermic group as compared to the hypothermic group, (4 vs. 1) which was accompanied by a higher mortality in the normothermic group (2 vs. 0). Both parameters did not reach statistically significance. Liver trauma was performed without lethal complications. In all cases, a second-look operation after 24.5 h did not reveal relevant bleeding (Fig. 2c). In one animal in group Tn, liver lacerations strongly bled during the first hour after packing requiring suturing and repacking. Bleeding was diagnosed by sonography following an unexplained prolonged tachycardia after the reperfusion period. In control groups, in none of the animals an invasive procedure was required. Basic data has been partly published in previous publications of our study group [12, 15, 16, 17, 18] .

3.2 Serum transaminase analyses and LDH

Transaminases and LDH were measured at all 6 time points. AST levels were increasing during the observation period in trauma groups as compared to sham animals. Increased AST levels in the Th group were detected at later time points as compared to group Tn without reaching statistical significance (Fig. 3a). No differences in ALT levels were detected between both trauma groups (Fig. 3b).

Figure 3.

Serological parameters. Comparison between Trauma animals Th and Tn. a) AST, b) ALT, c) LDH levels in hypothermic and normothermic trauma group, data showed no statistical significant difference in nonparametric testing. Data is given as mean and SD, $p^{*}\geqslant$ 0.05. Points of analysis: immediately before the induction of trauma (1), after the trauma period (2), after reperfusion (3), at 14.5 h (4), 24.5 h (5), 48.5 h (6) and post trauma.

Severe liver impairment defined as at threefold increase of AST levels (standard value in German landrace 6–32 IU/l [20]) did not differ in sham groups. Hypothermia increased the number of animals with severe liver impairment ( $n=$ 9) as compared to normothermic animals ( $n=$ 6) 12 hours following trauma [12].

A threefold increase in ALT levels was shown in 4 animals of each trauma group at the end of investigation period (standard value in German landrace 8–31 IU/l [20]).

LDH levels as a marker for cell damage did not show significant differences between trauma groups (Fig. 3c).

3.3 Liver function

Complete data of LiMON analyses were available for 4 animals in both sham groups and 11 animals in both trauma groups, respectively. For a better comparability, the mean percentage of PDR decrease during the investigation period was calculated. The first measurement at the beginning of the investigation was defined as baseline value.

In the normothermic trauma group, a decreased liver function during the first 24 hours was detected. Following hypothermia (14.5 h), a significant decrease was observed as compared to normothermic animals (Fig. 4). Following the rewarming period (24.5 h), animals treated with hypothermia showed a significantly increased liver function as compared to the respective normothermic trauma animals. At the end of the observation period, no statistical differences were observed between both trauma groups.

Figure 4.

Plasma dilution rate of ICG. Measurements immediately before the induction of trauma (1) in deep hypothermia at 14.5 h (2), after rewarming at 24.5 h (3) and at the end of observation period 48.5 h (4). Data is given as percentage of initial value. ${}^{*}p\leqslant$ 0.05.

3.4 Histopathological changes

Cell death as well as vacuolisation of liver cells were detected as the major changes in H&E stained sections. Morphological changes were mostly localized in the central area around the central vein in Th (53%, N $=$ 15) and Tn (53%, N $=$ 15). A semi-quantitative score for cell death was assessed. Hypothermia in trauma animals was associated with a slight increase of animals displaying an overall cell death score $>$ 30% in non-traumatized (66 vs. 53%; Fig. 5b) as well as traumatized (66% vs. 62%; Fig. 5a) liver lobes. Vacuolisation showed to be the same amount in non-traumatized lobe, whereas normothermic trauma lobe displayed a higher rate of vacuolisation than hypothermic trauma lobe (Fig. 5d and c).

Figure 5.

Histological findings in peripheral and trauma lobe of the liver. a) Cell death $>$ 30% trauma lobe, b) Cell death $>$ 30% in non traumatized lobe, c) Vacuolization in trauma lobe, d) Vacuolization in non traumatized lobe. Data is given as percentage. Morphological findings are scored semi quantitatively.

Caspase-3 staining revealed positive signals in the margins of areas of cell death (Fig. 6b and d). The quantification of liver cell apoptosis showed a slightly pronounced signal in the pericentral area in hypothermic trauma animals (27% vs. 15%; Fig. 6c). Granulocyte infiltration occurred sparsely and did not differ between groups investigated.

Figure 6.

Histological staining. a) H&E staining showing pericentral accentuated damage of hepatocytes with -congestion and infiltration of granulocytes. b) Immunohistochemically work-up reveals positive caspase-3 labeling on the margin of the demarcated area of cell damage. c) Percentage of apoptosis in Th $=$ hypothermic and Tn $=$ normothermic trauma groups. d) High resolution of caspase-3 staining displays cytoplasmic dyeing of hepatocytes as expression of apoptotic pathway.

4. Discussion

For the current study, a porcine polytrauma model simulating the clinical situation regarding injury pattern and severity as well as intensive care treatment was used. In contrast to previous studies [4, 5, 25], hypothermia of 33 ${}^{\circ}$ C was applied for 12 hours following fluid resuscitation. Afterwards, a controlled rewarming (0.5 ${}^{\circ}$ C/h) for 10 hours was performed. Liver function was measured at different time points during the 48.5-hour study period. Liver damage was assessed by transaminase levels, metabolic function and histopathological findings.

The results of the current study showed that trauma and haemorrhage increased serum transaminase levels, displaying a pronounced, but not significant, increase at the end of the observation period in animals treated with hypothermia. Liver perfusion was preserved after 24 h in hypothermic trauma animals but did not show differences at the end of the observation period. Histopathological assessment showed no relevant difference in morphological liver cell damage after trauma in animals treated with hypothermia.

Liver injury represents an element of various experimental polytrauma models. Several small animal studies using either local or systemic hypothermia have shown protective effects regarding hepatic injury [21, 23, 24] Some porcine animal models confirmed these findings but are limited by short follow-up periods of about 5–30 minutes not simulating the clinical situation [24, 25]. Considering that liver dysfunction occurs at later time points an observation period beyond 7 hours is required [26, 27, 28, 29, 30].

Fröhlich et al. [25] analysed the effects of induced mild hypothermia of 34 ${}^{\circ}$ C for 3 h on the inflammation of the liver and kidney. In this study, hypothermia did not affect AST levels as compared to the respective normothermic trauma group after a study period of 15.5 h but granulocyte infiltration was decreased in the hypothermic trauma group. In general, these findings are confirmed with deeper hypothermia as well as an increased hypothermic period in the present study. The differences detected in the present study occurred at later time points which have not been investigated in the cited study [25]. In another porcine model of haemorrhage and resuscitation, decreased transaminase levels after 22 hours in animals treated with hypothermia (32 ${}^{\circ}$ C) were described [31]. In contrast to the current study, hypothermia was applied prior to haemorrhage and an autotransfusion was performed which may have influenced hepatic inflammatory response [32, 33].

In our study, a further increase of AST levels after 24 hours in trauma animals was observed which was not significantly affected by hypothermia treatment. This finding confirms results of a porcine model of haemorrhagic shock with a of observation period similar to the present study [34]. In this investigation, no effects of hypothermia (8 h, 33 ${}^{\circ}$ C) on transaminase levels 48 hours after a haemorrhage ( $\sim$ 40% total blood volume over 45 min) were observed. In contrast to our study, animals were applied to hypothermia directly after the haemorrhagic period without a period of resuscitation and stabilisation. Only 15 of 21 animals survived until the end of observation period, only 2 of them were in the normothermic group (39 ${}^{\circ}$ C). Due to a mortality rate of $>$ 50% in normothermic control group, interpretation of the data regarding differences in transaminase levels seems disputable.

Total blood flow and function of the liver were determined by ICG clearance which has been proven as a feasible method in swine [35, 36, 37]. Analyses showed a 50–60% decrease of liver function in trauma groups. Hypothermic trauma animals showed a decreased liver function during induced hypothermia but an improved function after beginning of the rewarming period as compared with normothermic trauma animals. This finding may be explained by a hypoperfusion of the liver during hypothermia [38] and a reactive hyperperfusion during the rewarming period which was not longer evident after 48.5 h suggesting a perfusion phenomenon rather than a reduced function of liver cells. This hypothesis is strengthened by another study showing a reduced perfusion of the liver during hypothermia [39]. Overall it can be concluded that metabolic effects of hypothermia may occur during the early rewarming period without being present 24 h post hypothermia. Long-time effects were not observed in the current study.

For further describing the trauma induced liver damage histological analyses were performed. The role of apoptosis in hepatic injury is not entirely understood. The mechanism of hypothermia induced organ protection has been attributed to a decrease in tissue metabolism and oxygen consumption [40, 41]. In ischemia-reperfusion (I/R) injury, anti-apoptotic therapy enhanced liver damage [42] and prevented inflammation as well as dysfunction [43]. In contrast, caspase-3 upregulation was associated as well with reduced proinflammatory cytokine expression [44].

In a previous study, hypothermia resulted in less histopathological findings as compared to normothermic trauma animals [25]. In contrast to these results, we found slightly increased cell damage in hypothermic trauma animals, containing simultaneously a higher amount of apoptotic changes. Positive labelling was mostly found around the central vein. This pattern is similar to the described distribution of liver damage in ischemic injuries in contrast to a rather periportal distribution pattern in toxic injury [45]. The observed focal distribution pattern of apoptosis in the non-traumatized liver lobe was described in a case report by Schmieg et al. [46]. They showed that a short period of hypotensive shock with rapid response to fluid resuscitation initiated the apoptotic program at non adjacent areas. As there was no significant difference in transaminase levels and metabolic outcome parameters in our study, the clinically relevance of these finding remains unclear.

As the present study, all animal models are limited due to inter-species differences. The induced liver trauma may have been too mild to induce primary liver failure in all animals. Furthermore, in a complex experimental multi system trauma model numerous factors may influence the outcome. Therefore, the impact of induced hypothermia may be attenuated. Animal’s individual inflammation response, infection, interventions (e.g. CPR, chest drainage), antiarrhythmic medication and sedation drugs may have further influenced outcome parameters.

5. Conclusion

In the current study the effect of induced hypothermia on liver function following multi system trauma was assessed. The induction of hypothermia of 33 ${}^{\circ}$ C for 12 h showed no clinically relevant significant protective effect after 48 h. Further studies focusing on multi-organ failure including a longer observation period are required to illuminate the impact of hypothermia on hepatic function in multiple trauma patients.

Footnotes

Acknowledgments

We thank J. Lippitz, K. Eisold and A. Gockel for making substantial contributions in practical acquisition of data. The data was partly presented at the ECTES 2015 Meeting in Amsterdam and at the DGOU 10/2015 Meeting in Berlin.

Conflict of interest

None to report.

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Hypothermia does not influence liver damage and function in a porcine polytrauma model

Abstract

BACKGROUND:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Methods

2.1 Animal experiments

2.5 Histological evaluation

2.6 Statistics

Table 1 Baseline data

3.1 Basic data

3.2 Serum transaminase analyses and LDH

5. Conclusion

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
Baseline data