Article, Critical Care

Accompanying mild hypothermia significantly improved the prognosis of septic mice than artificial mild hypothermia

a b s t r a c t

Background: Some patients with sepsis are found with accompanying Mild hypothermia (ACMH); however, the effects of ACMH on the patients with sepsis are poorly understood.

Objective: To compare the impacts of ACMH and artificial mild hypothermia (ATMH) on mortality, systemic in-

flammatory reactions, and organ functions in mice with sepsis. Methods: Septic mouse models were induced and divided into ACMH, un-hypothermia, keep normothermia, and ATMH groups, according to the anal temperature and the thermic intervention strategy. The mortality rate, serum levels of tumor necrosis factor ? (TNF-?), interferon ? (IFN-?), and interleukin (IL)-4 and liver and renal functions of the mice in each group were recorded. Liver, lung, and renal tissues of the mice were stained and examined under optic microscope.

Results: The mortality rate in the ACMH group was the lowest among all the sepsis groups. Increased serum levels of TNF-?, IFN-?, and IL-4 and impairments of the liver and renal functions were found in the septic mice. The serum levels of TNF-?, IFN-?, and IL-4 were significantly lower and the liver and renal functions of ACMH group were not impaired significantly as compared with other sepsis groups. Pathological examinations of the lung, liver, and renal tissues showed that the ACMH group were with the lowest pathological score among all the sepsis groups.

Conclusion: Accompanying mild hypothermia and ATMH could both reduce mortalities in mice with sepsis, and ACMH could reduce mortality even lower, and more alleviate systemic inflammatory responses and the damages in lung, kidney, and other organs were lighter.

(C) 2015

  1. Introduction

Hypothermia, a protective treatment method for acute brain dam- age that has been considered as the only effective method in the 20th century, has now been popularly accepted in clinical practices [1-5]. Hy- pothermia therapy could reduce the bleeding and edema of blood vessels, prevent the infiltration of neutrophils, reduce the release of excitatory neurotransmitter, lower the intracellular calcium accumulation, prevent the generation of oxygen free radicals, decrease the expression of cyto- kines, and reduce the cellular apoptosis [6]. Studies in recent years have demonstrated that a mild hypothermia therapy could significantly reduce the mortality rate of sepsis [7-11] from different aspects including proin- flammatory and anti-inflammatory factors [7], cell apoptosis [6], and functions of autonomic nerves and different organs [8-11].

Sepsis is a systemic inflammatory response syndrome caused by an infection. The pathophysiologic processes of sepsis involved multiple cytokines and inflammatory factors. Despite the improvements of novel anti-inflammatory treatments and Organ function-supporting

* Corresponding author. Department of Emergency, Clinical College of Pediatrics, Anhui Medical University, Anhui Provincial Children’s Hospital, Hefei 230051, China. Tel.: +86 551 62237328; fax: +86 551 62237007.

E-mail addresses: [email protected] (X. Li), [email protected] (Q. Xie).

techniques, sepsis is still one of the most important causes of death for the critically ill patients [12-15]. Interestingly, we found clinically that some septic patients would also appear the status of accompanying hy- pothermia, among whom some might exhibit the status of accompany- ing mild hypothermia (ACMH), but there still existed controversies about whether keeping normothermia should be performed toward this kind of accompanying hypothermia [16,17]. Few studies have in- vestigated if the strategy is appropriate, the changes of organ functions of the patients under normothermia or hypothermia, the effects of ac- companying hypothermia on the patients with sepsis, and the differ- ences of the efficacies as comparing with artificial mild hypothermia (ATMH). The aim of this study were examined to investigate whether the sepsis accompanied mild hypothermia could result in protective ef- fects on the body and the possible mechanisms involved; in addition, which one between ACMH and ATMH could better improve the progno- sis of the mice was also investigated.

  1. Materials and methods

Endotoxin plays a very important role in the pathogenesis of sepsis caused by the Gram-negative bacterial infections; in addition, lipopoly- saccharide (LPS) could also induce hypothermic reactions in rodents [18,19]. Hence, in the present study, intraperitoneal injection of LPS

http://dx.doi.org/10.1016/j.ajem.2015.08.003

0735-6757/(C) 2015

was performed to induce sepsis in BALB/c mice [20-23], and then the mice were divided into different groups, namely, ACMH, un- hypothermia (UH), keep normothermia (KN), and ATMH groups, ac- cording to the anal temperature and the thermic intervention strategy [8-11]. The mortality rate in each group was evaluated; the levels of tumor necrosis factor ? (TNF-?), interferon ? (IFN-?), and interleukin (IL)-4 were measured; and the changes in the renal and liver functions, as well as the pathological changes in the liver, lung, and renal tissues, were examined.

Animals, reagents, and equipment

Specific Pathogen Free grade BALB/c mice (6-8 weeks old) were pur- chased from the Laboratory Animal Centre of Anhui Medical University, Anhui Province, China (scxk [Wan] 2011-002]. The present study was approved by the Ethics Committee of Anhui Medical University (No. LLSC2013028), China, and the usage of the animals and the procedures were performed in accordance with the guidelines issued by the Ethics Committee of Anhui Medical University. The TNF-?, IFN-?, and IL-4 levels of the mice were measured using enzyme-linked immunosorbent assay kits (R&D, Minneapolis, MN). The LPS (Escherichia coli 055:B5) was purchased from Sigma-Aldrich (St Louis, MO). Sodium chloride in- jection (0.9%) was purchased from Limin pharmaceutical Co, Ltd. (Jinan, China). All other reagents were of analytical grade and were prepared by the investigators. Automatic biochemical analyzer (C16000; Abbott, Tochigi, Japan), mild hypothermia therapy apparatus (YYT-1 ice blanket machine; Chinese People’s Liberation Army 6904 Factory, Taiyuan, China), and newborn’s incubator (YP-920 medical infant incubator; Daiwei, Ningbo, China) were kindly provided by the Anhui Province Children’s Hospital; TH212 mice thermometer (Haichuang High-Tech Science and Technology Co, LTD, Beijing, China) was provided by the Comprehensive Laboratory of Anhui Medical University, China.

Study design and regulation and management of the anal temperature of the mice

All mice had free access to drinking water and food. The mice were acclimated for 1 week at a Room temperature of 22?C to 24?C, humidity of 40% to 60%, and 12-hour light/12-hour dark cycle. The study consisted of 2 parts: the first part was designed to observe the mortality rate of the mice, and the second part was designed to measure the included pa- rameters. Different mice were used in the first and the second parts of the study.

In the first part of the study, BALB/c mice (n = 160) were divided into 2 groups, namely, sepsis group (n = 145) and normal control (NC) group (n = 15). Intraperitoneal injection of LPS (10 mg/kg) was administered for all the mice in the sepsis group, and then the anal tem- perature of the mice was measured 1 hour later. The mice were further divided into accompanying hypothermia group (anal temperature

<= 36?C) and nonhypothermia group (anal temperature N 36?C). In the accompanying hypothermia group, mice with the anal temperature lower than 34?C were excluded, and the mice with the anal temperature of 34?C to 36?C were included, which were further randomly divided into ACMH group and KN group. No thermic intervention was per- formed for the mice in the ACMH group, whereas the mice in the KN group were placed in the incubator to maintain the anal temperature at 36.0?C to 37.5?C. The mice in the nonhypothermia group were further randomly divided into UH group and ATMH group. No thermic inter- vention was performed for the mice in the UH group, whereas the mice in the ATMH group were placed in the ice blanket to maintain the anal temperature at 34?C to 36?C. The temperature of the mice in the ATMH and KN groups was tweaked to change 0.5?C/h to 1?C/h. In- traperitoneal injection of same volume of normal saline was adminis- tered for the mice in the NC group. For the mice in all the groups, appropriate food and clean water were provided. Thermic intervention lasted for 5 continuous hours. The mental state, reactions to

stimulations, mobility, hair, bilateral eyes, stool, and mortality rate within 24 hours after the injection of LPS were recorded.

The processes in the second part of the study were identical to the processes in the first part. Eighteen mice from each group were selected for the examinations.

Collection and examination of the blood samples

The time at the end of the LPS injection was recorded as 0 hour. Six mice each were selected from every group at 6, 12, and 24 hours, respec- tively. The mice were anesthetized with ether, and then the eyeballs of the mice were removed rapidly to collect 1.0 mL of blood. The mice were then euthanized by cervical dislocation, whereas the collected blood was processed to separate serum. The serum levels of TNF-?, IFN-?, and IL-4 were measured using enzyme-linked immunosorbent assay kits, accord- ing to the manufacturer’s instructions. The levels of alanine aminotransfer- ase (ALT), aspartate aminotransferase , creatinine (CREA), Blood urea nitrogen , and lactate were measured by the automatic biochemical analyzer (Abbott Laboratories; provided by the Laboratory Department of the Anhui Province Children’s Hospital, China).

Hematoxylin and eosin staining and scoring of the lung, liver, and renal tissues

Left lung, right lobe of the liver, and the right kidney of the mice were chosen at a time point of 24 hours. The tissues were fixed with 40 g/L of paraformaldehyde, and then rinsed, dehydrated, and sliced. The slices were stained with hematoxylin and eosin, and then the pathological ex- amination was performed with an optic microscope. Four slices were se- lected for each mouse for the double-blinded evaluation, and the mean scores were calculated. acute lung injury scoring was performed by a board-certified pathologist masked to treatment assignment and was classified into 4 categories based on the severity of alveolar congestion and hemorrhage, infiltration of neutrophils in the air spaces or vessel walls, and the thickness of the alveolar wall/hyaline membrane formation [9]. The severity of each category was graded from 0 (minimal) to 4 (maximal), and the total score was calculated by summing the individual scores. The score of each animal was calculated as the mean of 4 sections. Acute liver injury scoring was measured with the following morpho- logic criteria [24]: spotty necrosis, capsular inflammation, portal inflam- mation, ballooning degeneration, and steatosis. Spotty necrosis was graded and scored as follows: 0, none; 1, 1 focus or less per x10 objec- tive; 2, 2 to 4 foci per x10 objective; 3, 5 to 10 foci per x10 objective; and 4, more than 10 foci per x 10 objective. Capsular inflammation was graded and scored in each 10 x area, after magnification, for the presence of capsular inflammation as follows: 0, none; 1, capsular in- flammation in a 1 x 10 magnification area; 2, capsular inflammation in a 2 x 10 magnification area; and 3, capsular inflammation in a 3 x 10 magnification area. Portal inflammation was scored as follows: 0, none; 1, mild, some, or all portal areas; 2, moderate, some, or all portal areas; and 3, marked, all portal areas. Ballooning degeneration was scored as follows: 0, none; 1, ballooning degeneration in one third of the hepatic lobule; 2, ballooning degeneration in two-thirds of the he- patic lobule; and 3, ballooning degeneration in all parts of the hepatic lobule. Steatosis was scored as follows: 0, none; 1, b 30% of hepatocytes contain fat; 2, 30%-70% of hepatocytes contain fat; and 3, N 70% of hepato- cytes contain fat. The liver injury severity score ranged from 0 (none) to 16 (severe).Acute kidney injury scoring was measured with the following morphologic criteria [25]: kidney damage was scored by grading glomeru- lar, tubular, and interstitial changes. Glomerular damage (sclerotic changes such as matrix expansion, the narrowing or disappearance of Bowman space, the adhesion of the capillary tuft to Bowman capsule, the capillary collapse, and the thickening of the glomerular basement membrane) was evaluated as follows: 0, absent; 1, b 25% of glomeruli affected; 2, 25%-50% glomeruli affected; and 3, N 50% of glomeruli affected. The grading for tubular changes (intracellular vacuolization) was scaled as follows: 0, absent; 1, b 25% of tubules injured; 2, 25% to 50% of tubules injured; and

3, N 50% of tubules injured. The presence of interstitial inflammation was judged as follows: 0, absent; 1, mild; 2, moderate; and 3, severe.

Statistical analyses

All quantitative data were described as mean +- SD. Normal- ity test and homogeneity test of variances were performed for

all data, and then 1-way analysis of variance was used to com- pare the differences among the groups; least significant differ- ence method was used for pairwise comparisons when statistical significance was found by the 1-way analysis of vari- ance. Comparisons between 2 independent groups were per- formed by independent t test. A P value less than .05 was considered statistically significant.

Image of Fig. 1

Fig. 1. A, Survival rate of the mice. Significant difference in the survival rate was found between the ACMH group and other sepsis groups (#P b .01), whereas no significant difference was found between the KN group and the ATMH group (P N .05). B, The anal temperature of the mice in the NC group maintained between 36.0?C and 37.5?C; the anal temperature of the mice in the ACMH group was basically between 34?C and 36.5?C at 1 to 6 hours after the LPS injection, whereas the anal temperature of the mice in the UH group was generally between 36.8?C and 39.9?C. C, The way the groups were divided is expressed in the form of graphs.

  1. Results
    1. Change of the anal temperature of the mice in the nonthermic interven- tion group and the mortality rate in different groups

Substantially decreased mobile activity, with refuse to eat, piloerection, slow responses, secretions from both eyes, and mucous stool were noted in septic mice at 0.5 hours after the intraperitoneal in- jection of LPS; the anal temperature of the mice was also changed grad- ually. The mice were divided into different groups according to the anal temperature at 1 hour after the LPS injection, which were as follows: the mice with the anal temperature basically between 34?C and 36.5?C at 1 to 6 hours after the LPS injection were categorized as the ACMH group; the mice with the anal temperature between 36.0?C and 37.5?C were categorized as the UH group; and the other mice were with the anal temperature between 36.8?C and 39.9?C at 1 to 6 hours after the LPS in- jection. The anal temperature of the mice in the ACMH and UH groups gradually returned to normal range after 6 hours after the injection. Six (6/55; 10.9%), 18 (18/53; 34.0%), 9 (9/13; 69.2%), and 6 (6/15;

40%) mice from the ACMH, KN, UH, and ATMH groups died at 1 to 12 hours after the LPS injection, respectively, and most of them died be- tween 3 and 9 hours after the injection. Substantial gastrointestinal swelling was found for the dead mice. The anal temperature of the mice in the NC group maintained between 36.0?C and 37.5?C, whereas no abnormality or death was noted (Fig. 1).

Changes in the serum levels of IFN-?, TNF-?, and IL-4

The IFN-? level of the septic mice increased gradually after the injec- tion of LPS and peaked at 6 hours after the injection. The IFN-? level was highest in the UH group and lowest in the ACMH group. The difference in the IFN-? level was significant between the sepsis groups and the NC group (P b .01); in addition, the IFN-? level in the ACMH group was not statistically different compared with ATMH group (P N .05); however, statistically significant difference was found with the IFN-? levels in the other groups (P b .01) at 6 hours after the LPS injection. The differ- ence in the IFN-? level at 12 hours after the injection was only signifi- cantly different between the UH and NC groups (P b .05), and no significant difference was found between each of the sepsis group and the NC group at 24 hours after the injection (P N .05; Table 1).

The TNF-? level in the sepsis groups increased gradually after the in- jection of LPS and peaked at 6 hours after the injection. The TNF-? level was highest in the UH group and lowest in the ACMH group. The differ- ence in the TNF-? level was significant between the sepsis groups and the NC group (P b .05 for ACMH group vs NC group, and P b .01 for the other sepsis groups vs NC group); in addition, the TNF-? level in the ACMH group was also statistically significant as compared with the level in the KN and ATMH groups (P b .05; Table 2) at 6 hours after the LPS injection.

The IL-4 level of the mice in the sepsis group also increased gradually after the injection of LPS and peaked at 12 hours after the injection. The IL-4 level was highest in the UH group and lowest in the ACMH group. The IL-4 levels in the sepsis groups and the NC group were statistically

Table 2

Changes of the serum TNF-? level in the mice (means +- SD; n = 6) Group Changes of TNF-? level with time (pg/mL)

6 h

12 h

24 h

NC

121.67 +- 20.45

132.17 +- 18.19

127.33 +- 23.86

ACMH

187.50 +- 51.32?

148.50 +- 26.74

124.00 +- 24.55

KN

262.17 +- 75.40??,???

179.67 +- 32.26?

129.17 +- 24.08

UH

288.00 +- 65.39??,+

194.50 +- 39.94??,???

140.17 +- 32.52

ATMH

252.00 +- 37.88??,???

161.83 +- 33.64

121.67 +- 29.73

* P b .05, compared with the NC group.

?? P b .01, compared with the NC group.

??? P b .05, compared with the ACMH group.

+ P b .01, compared with the ACMH group.

significant at both 6 and 12 hours after the injection (P b .05); however, no significant difference was found between the ACMH and ATMH groups (P N .05; Table 3).

Changes in the liver and renal functions

Comparing with the NC group, the serum levels of ALT and AST in- creased significantly in the sepsis groups. The levels of ALT and AST in each group peaked at 6 hours after the LPS injection and then decreased gradually thereafter; however, the levels did not return to normal range at 24 hours after the injection. The ALT and AST levels were highest in the UH group and lowest in the ACMH group. The level of ALT was not significantly different between the ACMH group and the NC group at 24 hours after the LPS injection (P N .05), whereas the ALT level was sig- nificantly different between the other sepsis groups and the NC group at all the time points measured (Fig. 2A). In terms of AST level, significant difference was found between the ACMH and KN groups and the NC group only at 24 hours after the injection, whereas the levels of the other sepsis groups at each time point as well as the ACMH group at other time points and the NC group were significantly different (Fig. 2B). Similarly, CREA levels also significantly increased in the sepsis groups compared with the NC group. The CREA level peaked at 6 hours after the LPS injection in each group and then decreased gradually; however, the level did not return to normal range at 24 hours after the injection (Fig. 2C). The CREA level was highest in the UH group and lowest in the ACMH group at 6 hours after the LPS injection. No sig- nificant difference in the serum BUN and lactate level was found among these groups (Fig. 2D, E).

Pathological scores of the lung, liver, and kidney of the mice in the sepsis group

The pathological scores of the liver, lung, and kidney of the mice in the sepsis group are shown in Fig. 3. The pathological score of acute lung damage in the ACMH group (1.67 +- 0.82) was significantly lower than that in the KN group (3.00 +- 0.63, P b .01), UH group (4.33 +- 0.75, P b .01), and ATMH group (2.83 +- 0.68, P b .05); in addi-

tion, the score was also significantly lower in the ATMH group than in the UH group (2.83 +- 0.68 vs 4.33 +- 0.75, P b .01). The pathological

Table 1

Changes of the serum IFN-? level in the mice (means +- SD; n = 6) Group Changes of IFN-? level with time (pg/mL)

6 h

12 h

24 h

6 h

12 h

24 h

NC

215.33 +- 31.22

210.17 +- 17.44

221.33 +- 16.91

NC

392.33 +- 37.67

380.50 +- 54.29

404.50 +- 48.92

ACMH

310.83 +- 69.10??

229.00 +- 46.56

213.17 +- 24.71

ACMH

458.50 +- 48.62?

517.00 +- 73.90??

367.00 +- 60.41

KN

416.17 +- 44.87??,???

251.83 +- 66.99

206.50 +- 25.37

KN

470.67 +- 54.28?

536.50 +- 74.30??

398.83 +- 66.43

UH

452.00 +- 71.15??,???

274.17 +- 52.83?

209.33 +- 33.90

UH

537.00 +- 66.04??,???

628.83 +- 86.97??,???

440.50 +- 83.88

ATMH

372.50 +- 56.78??

216.50 +- 37.39

227.17 +- 24.08

ATMH

511.00 +- 59.27??

580.00 +- 82.58??

421.00 +- 74.03

Table 3

Changes of the serum IL-4 level in the mice (means +- SD; n = 6) Group Changes of IL-4 level with time (pg/mL)

  • P b .05, compared with the NC group.

?? P b .01, compared with the NC group.

??? P b .01, compared with the ACMH group.

  • P b .05, compared with the NC group.

?? P b .01, compared with the NC group.

??? P b .05, compared with the ACMH group.

Image of Fig. 2

Fig. 2. Comparison between the sepsis groups and NC group (*P b .05, #P b .01); comparison between the sepsis groups and the ACMH group (?P b .05, ?P b .01). A, Significant difference was found in the serum ALT between the ATMH group and the UH group (P b .05). B and C, No significant difference was found in the serum AST and IL-4 levels between the ATMH group and the UH group (P N .05). D and E, No significant difference was found in the serum BUN and lactate levels among the different groups (P N .05).

score of acute liver damage was significantly lower in the ACMH group (3.25 +- 0.69) than in the other sepsis groups (P b .01), and the score was also significantly lower in the ATMH group than in the UH group (6.42 +- 1.60 vs 8.25 +- 0.69, P b .05). The pathological score of acute Renal damage was significantly lower in the ACMH group (2.92 +- 0.74) than in the UH group (5.50 +- 0.71, P b .01), KN group (4.08 +- 0.86, P b .05), and ATMH

group (3.92 +- 0.74, P b .05); in addition, the score was also significantly lower in the ATMH group than in the UH group (3.92 +- 0.74 vs 5.50 +- 0.71, P b .01).

  1. Discussion

In clinical practice, body temperature variation has been considered as one of the most important diagnostic criteria for several diseases. The

temperature increase and decrease are, in certain degree, protective re- actions of the body. However, the increase or decrease of the body tem- perature should be preserved in an appropriate range. Too high or Low Temperature will result in detrimental effects on the body. Previous studies have shown that hypothermia is an independent predictor of the 28-day mortality rate of patients with sepsis [26]. However, in the study by Elaine et al [27], it was proved that the naturally occurred mild hypothermia could benefit more for the rats with systemic inflam- mation than hyperthermia. In the present study, it was found that re- storing the ACMH to normal temperature in the septic mice resulted in significantly increased mortality rate of the mice, and the difference was statistically significant. It is suggesting that the ACMH is beneficial for the prognosis of the septic mice, and the aggressive thermic inter- vention could be detrimental. Numerous studies have shown that

A

B

Lung

NC

ACMH KN

UH

ATMH

Fig. 3. A, Pathological scores of the lung, liver, and renal damages in the sepsis groups. Significant difference was found between the ACMH group and the other sepsis groups (*P b .05, #P b .01) as well as between the ATMH group and the UH group (?P b .05, ?P b .01). B-D, Images of the lung tissues (B), liver tissues (C), and renal tissues (D) in the NC and sepsis groups (x400).

mild hypothermic intervention could significantly reduce the mortality rate of the animals with sepsis [7-11,28,29]. Our study also found that ATMH could reduce the mortality rate of septic mice, but the reduction degree of mortality in septic mice was not as significant as that reduced by ACMH. Hypothermia therapy is a process that passively tunes the body temperature to under the normal “set-point,” which could imme- diately induce the reactions that increase the heat production and de- crease the heat dissipation, and thus reduce the effects of hypothermia therapy, Hence, in the present study, the poor effects of hypothermia therapy on the nonhypothermic septic mice could be caused by the facts that the positive effects of the hypothermia therapy were some neutralized by the negative effects of passive down-regulation of tem- perature set-point, which were in accordance with previous findings by Gordon [30]. The pathological damages of the lung and liver in the ACMH group were the lowest among the sepsis groups, which were in accordance with previous findings that hypothermia therapy could re- duce the liver and lung damages of septic rats [9].

During the immune hyperfunction phase in early sepsis, the secretion of proinflammatory factors is greatly up-regulated [31]. Several proinflam- matory factors including TNF-? and IFN-? could be secreted by multiple

immune cells at the early stage of sepsis [32]. In the present study, blood was collected from the mice at 6 hours after the LPS injection, and then the serum levels of TNF-? and IFN-? were measured. The results showed that the serum levels of TNF-? and IFN-? were significantly lower in the ACMH group than in the KN group and UH group; however, only the dif- ference in IFN-? level was statistically significant between the ATMH and UH groups. Further studies are needed to verify whether ACMH could bet- ter down-regulate the levels of proinflammatory factors than ATMH. In the study by Karalle et al [10], it was shown that the TNF-? level was not sig- nificantly different between the mild hypothermia group and the normal temperature group; however, in that study, the septic rat models were in- duced by cecal ligation and perforation, and the blood was collected 4 hours after the procedures, which were different from the present study. In the present study, only 1 anti-inflammatory factor, namely IL-4, was in- vestigated, which limited the significance of the present study. The present study compared the IL-4 levels among the different sepsis groups, and the significant difference was found only between the ACMH group and the UH group. The next experiment could be designed to observe more inflam- matory markers, such as procalcitonin, to much more fully understand the impacts of ACMH on septic body.

Previous studies have shown that about 33.6% of the patients with sepsis could develop organ failure [33], among which acute renal failure and renal damages are the most common critical complications for pa- tients with sepsis or septic shock [34]. The mortality rate of sepsis ac- companied with acute renal failure is up to 70%, which is greatly higher than the mortality rate in acute renal failure patients (45%) [35]. Liver is the major organ involved in the process of detoxification, mainly for intestinal endotoxins. Previous studies have already demon- strated that LPS could cause acute liver damages [36], whereas abnor- mal liver functions are associated with worse outcomes [37]. Several studies have shown that the mild hypothermia therapy could effectively alleviate the acute damages on the heart, liver, and lungs of septic ani- mals [8,9], as well as the liver damages in mice with fulminant hepatitis [38]. Hence, in the present study, serum levels of ALT, AST, CREA, and BUN were selected to evaluate the liver and renal functions of the septic mice. The results of the present study showed that the ALT, AST, and CREA levels in the ACMH group and the ATMH group were significantly lower than those in the KN group and the UH group; in addition, the levels were lower in the ACMH group than in the ATMH group.

The Serum lactate level could reflect the severity of tissue hypoxia. In the present study, no significant difference in the lactate level was found among the sepsis groups, which was in accordance with the findings of the study by Corrigan et al [39], which showed that hypometabolism and hypothermia might not necessarily cause hypoxia in the cases with endotoxic shock. However, the present study did not measure the lactate level in the same mice at different time points but measured the levels in different mice that killed at different time points, which also limited the significance of the present study. It is believed that the Lactate clearance could better reflect the severity of tissue hypoxia [40]. Currently, the aim of common treatment for patients with sepsis and ACMH is to bring back the body temperature to normal range with the use of heavy blankets or by increasing the environmental temperature via air conditioner. The findings of the present study showed that the state of ACMH is beneficial for the recovery of septic mice. However, only very few studies (especially clinical studies) have investigated the effects of ACMH on patients with sepsis, and a lot of questions in- cluding how to maintain the state of ACMH in patients with sepsis, the time of maintaining the state, the Possible complications, and how to treat the possible side effects are to be elucidated. The clinical applica- tion of ACMH in treating patients with sepsis should be further investi-

gated with multicenter clinical trials with larger sample sizes.

In summary, our experiments suggested that ACMH had significant organ-protective effects. The state of ACMH of the patients with sepsis should be considered, and appropriate management of the temperature should be performed to improve the clinical efficacies in treating pa- tients with sepsis and ACMH.

Conflict of interest

All authors have no conflict of interest regarding this manuscript.

Acknowledgments

This study was supported by Science and Technology Research Pro- jects of Anhui Province, China (No. 1301042213).

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