Effects of Hypothermia on Mortality and Inflammatory Responses to Endotoxin-Induced Shock in Rats

We studied the effects of hypothermia on mortality rate, concentrationsof tumor necrosis factor alpha and interleukin 6 in plasma,and the end products of nitric oxide (NO) in endotoxemia. Itwas found that moderate and mild hypothermia improved the mortalityrate and attenuated cytokine responses and the elevation ofthe end products of NO after endotoxin injection and that thesebeneficial effects were similar for moderate and mild hypothermia.

INTRODUCTION

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Introduction

Endotoxin shock, a common problem in patients with endotoxemiathat often resists even intensive medical treatment, is characterizedby profound hypotension, progressive metabolic acidosis, anddysfunction of multiple organs. Although its pathophysiologyis not well defined, cytokines and nitric oxide (NO) are consideredimportant in mediating the cardiovascular disturbance (10, 11,17). Circulating endotoxin increases the production and releaseof tumor necrosis factor alpha (TNF- ${alpha}$ ) and interleukin 6 (IL-6),which are dilators of resistance vessels in skeletal muscle(5). When macrophages are activated by endotoxin or cytokines,the inducible NO synthase gene is expressed. This, in turn,leads to the production of NO synthase, which, once activated,continuously releases large amounts of NO, with a vasodilatingeffect (13, 14). Enhanced production of NO thus contributesimportantly to cardiovascular dysfunction.

Several investigators have documented that hypothermia has anorgan-protective effect in controlled hemorrhagic shock andprovides myocardial protection (6, 18). Moreover, several studieshave shown that hypothermia delays the induction of proinflammatorycytokines in vitro (7). At the same time, however, several studieshave reported that the mortality rate of hypothermic patientswith sepsis is twice that of febrile septic patients (2, 4,9). We studied the effects of hypothermia on mortality rate,concentrations of TNF- ${alpha}$ and IL-6 in plasma, and the end productsof NO in endotoxin-exposed rats.

MATERIALS AND METHODS

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Materials and Methods

Animal preparation. All experimental procedures were approved by the Animal CareCommittee of Kanazawa University School of Medicine and werein accordance with the National Institutes of Health guidelinesfor animal use. Male Wistar rats (n = 36) weighing 375 ±15 g (mean ± standard deviation [SD]) were anesthetizedwith an intraperitoneal injection of pentobarbital sodium (30mg/kg of body weight). Ventilation was performed through a tracheotomy.The femoral artery was cannulated to monitor blood pressureand to draw blood samples. Lactated Ringer's solution containinga muscle relaxant (0.02 mg of pancuronium bromide/ml) and pentobarbitalsodium (0.5 mg/ml) was infused continuously at a rate of 10ml · kg^-1 · h^-1 through a femoral vein cannulabecause animals anesthetized with this dose of pentobarbitalalone did not demonstrate escape behavior. The heart rate (HR)was recorded from lead II of the electrocardiogram. The ratswere connected to a pressure-controlled ventilator (Servo 900B;Siemens-Elema, Solna, Sweden) delivering 100% oxygen at a frequencyof 30 breaths/min with an inspiratory/expiratory ratio of 1:1.The animals were then rested for at least 15 min to allow hemodynamicparameters to be established, and baseline readings of HR andsystolic arterial pressure (SAP) were taken.

Study protocols. After baseline measurements were taken, the animals were allocatedrandomly to one of three groups, all of which received endotoxin(n = 12 per group).

Normothermia group. Endotoxin shock was induced by a bolus injection of endotoxin(15 mg/kg of body weight). We used lipopolysaccharide preparedfrom Escherichia coli (O111:B4; Difco Laboratories, Detroit,Mich.) as the endotoxin. Rectal body temperatures of this normothermiagroup were maintained between 36 and 38°C with the aid ofa heating pad.

Mild hypothermia group. Endotoxin shock was induced as for the normothermia group. Immediatelyafter the injection of endotoxin, the animals were cooled externallyto maintain rectal body temperatures between 34 and 35°C.

Moderate hypothermia group. Endotoxin shock was induced as for the normothermia group. Immediatelyafter the injection of endotoxin, the animals were cooled externallyto maintain rectal body temperatures between 30 and 31°C.

Arterial blood samples (1.0 ml) were drawn for the measurementof the baseline for cytokine concentrations in plasma 2 and5 h after the endotoxin injection. Another set of samples (0.5ml) was drawn for the measurement of the end products of NO6 h after the endotoxin injection. The total amount of blooddrawn from each animal was 4.0 ml over 6 h.

Sample analysis. The blood used for determination of cytokine concentrationswas centrifuged for 10 min at 3,000 x g at 4°C. Plasma wasthen decanted and stored at -70°C until analysis. Cytokine(TNF- ${alpha}$ and IL-6) concentrations were measured by an enzyme-linkedimmunosorbent assay (BioSource, Camarillo, Calif.). The lowerlimits of detection were 4.5 pg of TNF- ${alpha}$ and 7.0 pg of IL-6/ml.NO-related compounds were measured by a chemiluminescent assayusing a nitric oxide analyzer (NOA 280; Sievers Instruments,Boulder, Colo.). Data are reported as nitrite plus nitrate concentrations(NOx) in µmol/liter of plasma.

Statistical analysis. Data are presented as the means ± SDs. Differences betweenthe groups at baseline were analyzed with the unpaired Student'st test and Mann-Whitney U test. Hemodynamic and cytokine changesduring the study were analyzed by means of two-way analysisof variance with repeated measures followed by a post hoc test(Bonferroni's correction). Comparisons of mortality rates amongthe groups were made by the Kaplan Meier and Mantel-Cox methods.Statistical analyses were performed using the StatView application(Macintosh version 5.0; Abacus Concepts, Berkeley, Calif.).Statistical significance was defined as a P of < 0.05.

RESULTS

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Results

Hemodynamics and mortality rate. No significant differences were noted in baseline HR or SAPamong the groups (Fig. 1). Injections of endotoxin reduced SAPin the normothermia group but not in the mild hypothermia andmoderate hypothermia groups. Mortality rates 6 h after endotoxininjection were 75, 16, and 8% for the normothermia, mild hypothermia,and moderate hypothermia groups, respectively (Fig. 2). Themortality rate for the normothermia group was thus significantlyhigher than that for the other groups (P < 0.001).

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FIG. 1. HR and SAP at baseline and after injection of endotoxin (means ± SDs). An asterisk (*) indicates a P of < 0.05 that SAP will decrease versus that for the normothermia group. bpm, beats per min.

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FIG. 2. Survival curves for the normothermia, mild hypothermia, and moderate hypothermia groups.

Plasma cytokine concentrations. All baseline values for the three groups were similar (Fig.3). Endotoxin injection increased the TNF- ${alpha}$ concentrations inall the groups, but concentrations remained significantly lowerin the mild hypothermia and moderate hypothermia groups thanin the normothermia group 2 h after injection. Concentrationsof IL-6 in plasma became elevated in all the groups. Significantlylower concentrations were seen in the moderate hypothermia groupthan in the normothermia group (P < 0.01).

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FIG. 3. Changes in concentrations of TNF- ${alpha}$ and IL-6 in plasma from baseline after endotoxin injection (means ± SDs). An asterisk (*) indicates a P of < 0.05 that TNF- ${alpha}$ and IL-6 concentrations will increase versus those for the normothermia group.

Concentrations of NOx in plasma. All baseline values were similar for the three groups (Fig.4). Concentrations of NOx in plasma significantly increasedin all the groups 6 h after endotoxin injection, but the concentrationswere lower in the mild hypothermia and moderate hypothermiagroups than in the normothermia group (P < 0.05).

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FIG. 4. Changes in concentrations of NOx in plasma from baseline to 4 h after endotoxin injection. An asterisk (*) indicates a P of < 0.05 that NOx concentrations will decrease versus those for the normothermia group.

DISCUSSION

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Discussion

Endotoxin produced hypotension as well as increased cytokineand NOx concentrations in plasma in rats. Moreover, the mortalityrate 6 h after endotoxin injection was very high. With moderateand mild hypothermia, however, these endotoxin-induced changeswere attenuated. In addition, the mortality rates for moderate-and mild-hypothermic rats were significantly lower than thosefor normothermic rats. These inhibitory effects of moderateand mild hypothermia were the most important findings of ourstudy.

Several investigators have shown that hypothermia has beneficialeffects on endotoxemia, and their clinical studies have demonstratedthat hypothermia improves outcomes for patients in comas afterresuscitation (3) and that moderate hypothermia during cardiopulmonarybypass surgery reduces myocardial cell damage and myocardialcell death (18). On the other hand, several studies have foundthat hypothermia with endotoxemia has a deteriorating effecton hemodynamics and survival rate. Marik and Zaloga observedthat hypothermic patients in septic shock had a higher mortalityrate with a higher incidence of organ dysfunction than febrileseptic shock patients (9), while other studies have reportedthat the mortality rate of hypothermic patients with sepsiswas twice that of febrile septic patients (2, 4). Thus, it isinconclusive whether hypothermia is beneficial or not, althoughour study demonstrated that moderate and mild hypothermia improvedthe survival rate during endotoxemia in rats.

Of particular interest are the anti-inflammatory effects ofhypothermia on endotoxemia observed in our study. Several investigatorshave examined the relationship between cytokine responses andhypothermia. Kimura et al. (7) showed that moderate hypothermiadelayed the induction of proinflammatory cytokines in humanperipheral blood mononuclear cells in vitro, while several animaland clinical studies reported that hypothermia attenuated theelevation of circulating concentrations of IL-6 (1). In contrast,Lee et al. (8) showed that hypothermia induced an anti-inflammatory-T-cell-cytokineprofile in vitro, and several other reports showed that concentrationsof TNF- ${alpha}$ and IL-6 in plasma were not significantly differentin hypothermic and normothermic septic patients (2, 9). Thestudy presented here showed that moderate and mild hypothermiaattenuated the elevation of TNF- ${alpha}$ and IL-6 concentrations afterendotoxin injection. NOx is known to increase in endotoxemia,causing vasodilation and myocardial toxicity (13, 14). However,there are few reports on the relationship between NO and hypothermiain endotoxemia. Our study showed that, during normothermia,concentrations of NOx in plasma became remarkably elevated butthat the elevation of NOx concentrations in plasma is attenuatedduring moderate and mild hypothermia. These findings suggestthat these anti-inflammatory effects of hypothermia may be thereason for the improvement in mortality rates.

We compared the effects of moderate and mild hypothermia onendotoxemia in rats. Qing et al. (12) showed that moderate hypothermia,in contrast to normothermia and deep hypothermia, produced anti-inflammatoryeffects, including reduction of leukocyte mobilization and inhibitionof TNF- ${alpha}$ production during cardiac surgery. However, there arefew other reports on different degrees of hypothermia. We wereable to show that both moderate and mild hypothermia improvedmortality rates and attenuated endotoxin-induced changes, includingattenuation of TNF- ${alpha}$ , IL-6, and NOx production.

Two important and related questions remain unanswered. One iswhether hypothermia has similar beneficial effects when inducedat different times after endotoxin injection and whether thedose of endotoxin used in the present study is clinically feasible.With regard to endotoxin, previous studies found that the sameendotoxin dose as used in the present study caused endotoxin-inducedshock at 4 h and a high mortality rate (60 to 80%) at 6 h (15,16). This dose was obviously high, but we used this dose inthe present study in order to evaluate the mortality rate. Moreover,another question, that of species, remains. We used rats asthe endotoxin shock model, but the responses to endotoxin inthis model may be different from those in humans and other animals.Further studies need to focus on these questions.

In summary, our study showed that moderate and mild hypothermiaimproved mortality rates and attenuated cytokine responses andNOx elevation in rats receiving a bolus injection of endotoxin.Our findings also indicated that these beneficial effects weresimilar for moderate and mild hypothermia.

FOOTNOTES

* Corresponding author. Mailing address: Department of Emergency and Critical Care Medicine, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8641, Japan. Phone: 81-76-265-2826. Fax: 81-76-234-0973. E-mail: taniyan{at}med.kanazawa-u.ac.jp.

REFERENCES

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