Mild hypothermia in rat with acute myocardial ischaemia‐reperfusion injury complicating severe sepsis

Abstract Myocardial ischemia‐reperfusion injury (MIRI) with concurrent severe sepsis has led to substantial mortality. Mild hypothermia (MHT) has been proved to have a therapeutic effect in either MIRI or severe sepsis, which suggests it might be beneficial for MIRI complicating severe sepsis. In this study, Sprague‐Dawley rats with MIRI complicating severe sepsis were allotted in either MHT (33 ± 0.5°C) group or normothermia (NT, 37 ± 0.5°C) group; as control, rats receiving sham surgery and normal saline were kept at NT. After 2h of temperature maintenance, blood and heart tissue were acquired for detections. Lactate dehydrogenase (LDH) and MB isoenzyme of creatine kinase (CK‐MB) in blood, triphenyl tetrazolium chloride and Evans blue staining, hematoxylin and eosin staining for myocardium were employed to detect myocardial damage. Tumor necrosis factor (TNF)‐α and caspase‐3 was performed by immunohistochemistry to exam myocardial inflammation and apoptosis. Detection of NADPH oxidase (NOX) 2 was for myocardial oxidative stress. In MHT group, systolic blood pressure was improved significantly compared with NT group. Myocardial infarct size, morphological change, LDH and CK‐MB levels were attenuated compared to NT group. Moreover, less expressions of TNF‐α, caspase‐3 and NOX2 in MHT group were presented compared with NT group. MHT showed cardioprotection by improving cardiac dysfunction, reducing myocardial infarct size and attenuating myocardial injury, inflammation, apoptosis and oxidative stress.

that causes tissue hypoxia, sepsis-induced organ damage might be associated with impaired oxygen utilization, also referred to as 'cytopathic hypoxia'. 4 Thus, the heart may suffer from both insufficient oxygen supply and impaired oxygen utilization in patients with MIRI complicating severe sepsis, which is a conundrum for clinicians.
Mild hypothermia (MHT) has been recognized beneficial in either MIRI or sepsis. It decreases myocardial infarct size, prevents cardiac apoptosis, improves cardiac function and alleviates myocardial morphological change. 5,6 Albeit the popular recognition that MHT weakens body immune system, whether hypothermia is protective in infection is yet disputed, for evidence has shown MHT attenuates inflammation and oxidative stress in animals with severe sepsis. 3,7,8 However, due to the intricate mechanisms of the co-existence of MIRI and severe sepsis, none contributions have been found to study treatment on MIRI complicating severe sepsis.
Herein, based on the presupposition of the salubrious effects of MHT, we presumed MHT is effective in treating MIRI with severe sepsis and firstly applied MHT into the disease model of Sprague-Dawley (SD) rat with MIRI combining severe sepsis and substantiated the beneficial effects of MHT through improving the systemic and cellular symptoms. MHT can be a potential treatment for MIRI complicating severe sepsis, which lies a foundation of more profound studies in the future.

| Animals and experimental protocol
The experimental protocol was approved by the local ethics committee, and the study was in consistent with guidelines in the Guide for the Care and Use of Laboratory Animals. Rats were purchased from Vital River Laboratory Animal Technology Co., Ltd.
A total of 15 SD rats (male, weight: 350-450 g, n = 5) were randomly assigned into MIRI with severe sepsis in normothermia (NT, 37 ± 0.5°C) or MHT (model, 33 ± 0.5°C) group; for control, rats received sham surgery and normal saline (NS) were in NT ( Figure 1A). MIRI was achieved, and the related electrocardiogram (ECG) and haemodynamic values were recorded according to the method in previous studies. 9,10 Lipopolysaccharide (LPS, 15 mg/kg, ip, Escherichia coli, O111:B4, Sigma-Aldrich) was injected when reperfusion commenced while control group received a tantamount dosage of NS. During the surgery, body temperatures of the rats were maintained within a range of 37 ± 0.5°C.
Mild hypothermia was introduced immediately after the injection of LPS. Cooling was maintained for whole 2 hours at 33 ± 0.5°C, and the target temperature reached within 30 minutes. NT group received the same procedure. After 2 hours, heart and blood samples were acquired. The heart was used to detect infarct size, morphological change, inflammation, apoptosis and oxidization. The blood was used to test levels of lactate dehydrogenase (LDH) and MB isoenzyme of creatine kinase (CK-MB) according to the preceding study. 11

| Triphenyl tetrazolium chloride (TTC) and Evans blue staining
Procedures have been described previously. 9 The ratio was infarcted areas to the overall region ×100%.

| Haematoxylin and eosin (HE) staining
Procedures have been described previously. 11 Myocardial injury was based on the infiltration of inflammatory cells and myocardial morphological change.

| Immunohistochemistry
Procedures have been described previously. 11 The sections were incubated with anti-TNFα (1:200, ab220210, Abcam) and anticaspase-3 (pro-caspase-3, 1:200, ab184787, Abcam) antibodies. In each group, 5 heart tissues were used for immunohistochemistry detection. At least 3 random sights were analysed in each tissue. TNFα and caspase-3 positive cells were quantified by Image J (Version 1.52q, National Institutes of Health) and determined by the ratio of positive cells to overall cells.

| Statistical analysis
Student's t test was used to compare difference between two groups, and one-way ANOVA was for comparison among more than two groups. Differences were mean ± SD and considered statistically significant at P < .05.

| RE SULTS AND D ISCUSS I ON
The classic animal models of MIRI have been used in numerous experiments throughout a long-time span from 1980s to today. 9,[12][13][14] Thanks to endotoxin that usually generates severe sepsis, lipopolysaccharide (LPS)-induced sepsis in animals is widely recognized. 15 In this study, to mimic clinical condition of MIRI complicating with severe sepsis, we combined MIRI and LPS-induced sepsis in rats.
Myocardial impairment of the rats caused by MIRI and severe sepsis was achieved conclusively through the change in haemodynamics, the patent myocardial infarct size, morphological change as well as the incremental expressions of TNFα, caspase-3 and NOX2.

| Mild hypothermia improved systolic and diastolic function
Haemodynamics was monitored for cardiac function. Table 1 summarized haemodynamic parameters of all experimental rats.
Baselines of heart rate (HR), min dp/dt, diastolic pressure (DBP) and reperfusion were compared to the baselines and among groups. Left ventricular (LV) systolic function was assessed by max dp/dt, and diastolic function was assessed by min dp/dt. Values remained at a stable range at all stages in control group. Compared with baselines, in NT and MHT group, all values diminished during ischaemic status; at the 2 hours of reperfusion, max dp/dt, min dp/dt, SBP, DBP and MAP elevated in both NT group and MHT group. All values showed no significances compared among groups. However, tendency shows values but HR in MHT groups improved more significantly compared with NT group (P > .05). HR decreased in MHT group compared with NT group (P > .05) at the 2 hours of reperfusion, suggesting an improved myocardial contractility.
Whether MHT improves cardiac function remains a question.
Although some previous investigations found no significant change in haemodynamics, or even an impaired cardiac diastolic function, it is affirmed MHT leads to stronger myocardial contractility. 16,17 Our study proved MHT improves myocardial contractility and presented better systolic and diastolic function in haemodynamics, though without statistical difference. This can correlate to scant samples or insufficient duration of MHT. In the study of Huang and colleagues, 10 MHT improved both LV systolic and diastolic function (P < .001).
In their study, MHT was being monitored for 4 hours, which was longer than 2 hours of ours. As for samples, 6 rats were used in each group in theirs while 5 were in ours. However, an animal study in vivo showed MHT did not exert positive inotropic effect. 18 Thus, MHT on cardiac functional change of MIRI complicating severe sepsis needs more exploration.

| Mild hypothermia reduced infarct size and myocardial damages
Mild hypothermia can reduce myocardial infarct size. 6  TA B L E 1 Haemodynamic parameters: heart rate, cardiac systolic function, diastolic function and blood pressure (n = 5, mean ± SD)

| Mild hypothermia palliated myocardial inflammation, apoptosis and oxidative stress
Proinflammatory cytokines, which relate to inflammation and cell apoptosis, can be released to the damaged myocardial area rapidly after the cardio being attacked. Inflammation was assessed by TNFα (Figure 2A). Higher TNFα expressed in NT and MHT group than control (P < .05 vs P < .05). Moreover, TNFα expressed decidedly less in MHT group than in NT group (P < .05). MHT can inhibit inflammatory response in either MIRI or sepsis in other experimental studies, which declines inflammatory cytokines and preserve organ functions. 18,19 In our study, TNFα was shown less in the MHT group, suggesting the anti-inflammatory response effect of MHT in MIRI with sepsis.
Pro-caspase-3 is an inactive form of caspase-3 and is a sign of caspase-3 activation. Studies show pro-caspase-3 can protect cell from apoptosis. 20,21 In this study, apoptosis was assessed by procaspase-3 ( Figure 2B). Though not strong, caspase-3 was presented higher expression in NT group than control (P < .05). The expression in MHT was also observed higher compared to control (P < .05).
Accordingly, caspase-3 was expressed less in MHT group than in NT group (P < .05). Thus, the results illustrated MHT also has an effect of anti-myocardial apoptosis.
Superoxide dismutase (SOD) and malondialdehyde (MDA) are major oxidative products. Cheng et al 2 found hypothermia decreases SOD and MDA and shows a cardioprotective effect against MIRI.
Additionally, MHT reduces organ SOD and MDA in sepsis. 8 NOX2 is proved to be a target in cardiac oxidative stress reaction, which generates reactive oxygen species (ROS), such as SOD and MDA, and elevated ROS leads to oxidative stress. 22 Therefore, elevation of NOX2 can reveal existence of oxidative stress reaction. NOX2 expression was performed in WB analysis ( Figure 2C).

| CON CLUS ION
Both MIRI and sepsis can injure heart by generating myocardial inflammation, apoptosis and oxidative stress reaction in the host, leading to cardiac structural and functional damage. In our study, MHT showed a cardioprotective effect from aspects of haemodynamic change, myocardial infarct size, myocardial injury, inflammation, apoptosis and oxidative stress in rats with MIRI combining severe sepsis.

| LI M ITATI O N S
The LPS-induced sepsis differs from sepsis caused by microorganisms clinically. Other treatments and rewarming process were not incorporated in this study. Moreover, interspecies difference may limit the transfer from our results to mankind and sensibility of each rat to MHT was distinct.

ACK N OWLED G EM ENTS
The authors all thank JAT Biodiscovery Co., Ltd. for the assistance with the study.

CO N FLI C T O F I NTE R E S T S
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.