Nadroparine blunts lipopolysaccharide-ınduced hypothermia and behavioral depression in mice

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Journal of Investigative Surgery

ISSN: 0894-1939 (Print) 1521-0553 (Online) Journal homepage: http://www.tandfonline.com/loi/iivs20

Nadroparine Blunts Lipopolysaccharide-Induced

Hypothermia and Behavioral Depression in Mice

Cihangir Akyol, Erpulat Özis, Atíl Çakmak, Eyüp S. Akarsu & M. Ayhan Kuzu

To cite this article: Cihangir Akyol, Erpulat Özis, Atíl Çakmak, Eyüp S. Akarsu & M. Ayhan Kuzu (2008) Nadroparine Blunts Lipopolysaccharide-Induced Hypothermia and Behavioral Depression in Mice, Journal of Investigative Surgery, 21:6, 311-317, DOI: 10.1080/08941930802348253

To link to this article: https://doi.org/10.1080/08941930802348253

Published online: 09 Jul 2009.

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Journal of Investigative Surgery, 21:311–317, 2008 Copyrightc Informa Healthcare USA, Inc.

ISSN: 0894-1939 print / 1521-0553 online DOI: 10.1080/08941930802348253

ORIGINAL ARTICLE

Nadroparine Blunts

Lipopolysaccharide-Induced Hypothermia

and Behavioral Depression in Mice

Cihangir Akyol, MD Specialist

Department of General Surgery, School of Medicine, Ankara University, Ankara

Erpulat ¨Ozis, MD Specialist

Department of General Surgery, School of Medicine, Ufuk University, Ankara

Atıl C¸akmak, MD Specialist

Ey ¨up S. Akarsu, MD Prof.

Department of Pharmacology and Clinical Pharmacology, School of Medicine, Ankara University, Ankara

M. Ayhan Kuzu, MD Prof.

ABSTRACT Introduction: Despite the use of appropriate antimicrobial ther-apy and intensive care support, sepsis remains a major cause of morbidity and mortality in surgical clinics. Low-molecular weight heparin treatment may re-duce mortality and end-organ failure in sepsis. The purpose of this study was to compare the effects of low-molecular weight heparins such as nadroparine, enoxaparine, and dalteparine on lipopolysaccharide-induced acute phase re-action in mice. Methods: Lipopolysaccharide was injected intraperitoneally to produce a systemic inflammatory response and septic shock-like effects in adult male BALB/c mice. Mices were treated with low-molecular weight heparins (nadroparine, enoxaparine, dalteparine) and unfractioned heparin in different doses and times. Rectal temperature and spontaneous locomotor activity of the mice were evaluated. Results: Lipopolysaccharide (1 mg/kg, intraperitoneal) produced a hypothermia that occurred 20 minutes after injection. Nadroparine pretreatment (23.75 U/kg, sc) 2 hours before lipopolysaccharide challenge, but not synchronous injection, inhibited the hypothermic response. Pretreatment with equivalent doses of enoxaparine or dalteparine had no effect on the hy-pothermia. The high dose of lipopolysaccharide (60 mg/kg, intraperitoneal) caused more profound hypothermia and also inhibited spontaneous locomo-tor activity 24 hours after injection. Synchronous nadroparine administration partially attenuated the hypothermia and significantly abolished the depres-sion of spontaneous locomotor activity. Concludepres-sions: The results suggest that some low-molecular weight heparins such as nadroparine might be beneficial in high-risk surgical patients because of their potential anti-inflammatory action, in addition to their efficiency in preventing thrombo-embolic complications.

KEYWORDS hypothermia, lipopolysaccharide, low-molecular weight heparins, nadro-parine, sepsis, systemic inflammationbehavioral depression

INTRODUCTION

D

espite the use of appropriate antimicrobial therapy and intensive care support, sepsis remains a major cause of morbidity and mor-tality in surgical clinics. Following septic insult, a wide variety of

Received March 6, 2008; accepted June 4, 2008.

Adress correspondence to Dr. M. Ayhan Kuzu, Ibni Sina Hastanesi Genel Cerrahi Ek bina K4, Samanpazari 06100, Ankara, Turkey. E-mail: ayhankuzu@gmail.com

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proinflammatory cytokines are released and devastating consecutive physiopathological events are activated. It has been documented that the release of macrophage-derived proinflammatory cytokines such as interleukin-1 or tumour necrosis factor-α is a critical step for the pathophysiology of sepsis. These pleiotropic cytokines then activate a multisystem response, of which some components may be harmful for the host. For instance, cytokine-mediated activation of the coagulation cas-cade results in progressive inhibition of fibrinolysis and impaired fibrin dissolution. These changes lead to mi-crovascular thrombosis that ultimately causes multisys-tem organ damage [1, 2].

Lipopolysaccharide (LPS) is now well-documented as a potent microbial toxin that plays a crucial role in the pathogenesis of sepsis [3]. Systemic injection of LPS into animals causes a multisystem response, which is collectively known as the acute phase reac-tion. Depression of spontaneous locomotor activity and anorexia are some of the behavioral components of this reaction. Body temperature changes such as hy-pothermia or fever also accompany this response [4, 5]. These behavioral alterations may have an adaptive value against the immunological challenge. The inten-sity of this reaction changes depending primarily on the dose of LPS. Sublethal doses of LPS produce a septic shock-like effect that is prominent with more profound hypothermia and excessive release of proinflammatory cytokines [6].

Experimental studies have shown that both hep-arin and low-molecular weight hephep-arin (LMWH) reduce mortality and decrease end-organ failure following sep-tic challenge in primates due to their anti-inflammatory actions [7, 8]. There are significant variations among the LMWH preparations produced by different manu-facturers, but little is known regarding their preventive value in either systemic inflammation or sepsis [9].

The aim of this study was to assess the effects of LMWHs such as nadroparine, enoxaparine, and dalteparine on LPS-induced acute phase reaction in mice.

MATERIAL AND METHODS

Animals: Adult male BALB/c mice (weighing 26–40 g) were used. They were kept in standard polyethylene cages in groups of 6 and fed ad libitum with rat-chow and water. Mice were housed in an air-conditioned an-imal facility at an ambient temperature of 21 ± 1◦C

and a 12-hour light/dark cycle (lights on at 7.00 am). Each mouse was used only once for the experiments. All the animals were handled in accordance with the guidelines for the care and use of laboratory animals (NIH, publication no: 86–23, revised, 1985). All an-imal experiments described herein were approved by Ankara University Medical School Ethics Committee (2003; 33–76).

Body temperature measurement: Rectal temperature (Trectal) of the mice was measured by computer controlled-thermometer (Iso-Thermex, model 256, Columbus Instruments, Ohio, USA). The animals were gently handled, taken out of their cages every 20 min-utes and the rectal probe (TX-9) was inserted 2 cm into the rectum and left for about 20 seconds before readings were taken.

Behavioral analysis: The spontaneous locomotor ac-tivity (SLMA) of the mice was evaluated in a 40× 40 cm Plexiglas box during a 5-minute period using a com-puterised behavioral analysis system (Flex Field, Pho-tobeam Activity System, San Diego Instruments, CA, USA). The SLMA parameters were horizontal activity and the number of rearings. All the behavioral experi-ments were carried out during the light period.

Compounds and drugs: Phenol-extracted LPS of E. Coli serotype 055:B5 (Sigma, Lot 69H 4046) was used. LPS was aliquoted under aseptic conditions and kept at −20◦C until use. For injection, LPS was further diluted with sterile pyrogen-free physiological saline solution. Nadroparine calcium (0.1 ml = 950 anti-Xa IU; Fraxiparine, Sanofi-Synthelabo, France), enoxa-parin sodium (0.1 ml = 1000 anti-Xa IU; Clexane, Aventis, France), dalteparine sodium (0.1 ml = 1250 anti-Xa IU; Fragmin, Pharmacia, Germany), and hep-arin sodium (unfractionated hephep-arin, 0.1 ml= 500 IU; Nevparin, Mustafa Nevzat, Turkey) were used for the study. All the drugs were diluted with sterile pyrogen-free physiological saline solution.

LPS doses: In order to produce a systemic inflam-matory response in mice, LPS was injected 1 mg/kg, intraperitoneally (ip) (low dose). We did not observe any significant behavioral depression to implicate a sep-tic shock-like effect. Thus, we decided to evaluate dose response relationships in a small group of mice to de-termine the sublethal dose of LPS in our experimental conditions. Eighty percent of mice died after 120 mg/kg ip injection of LPS; thus we selected 60 mg/kg ip (high dose) LPS as a sublethal dose to simulate septic shock-like conditions.

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Experimental protocol: The experiments were started at 9:00 am. Three or four control Trectal values were recorded before LPS and/or drug treatment and the av-erage of those was regarded as the basal Trectal for each mouse. Thereafter, Trectal was recorded for the indi-cated time periods (see protocol) and expressed as a difference from the basal Trectal (T). LPS injection was carried out between 11.00 and 11.30 am in order to minimize the influence of the circadian rhythm. All the experiments were carried out in an air-conditioned laboratory, maintained at 24–26◦C ambient tempera-ture. The injection volume was 0.1 ml/10 g of body weight and the same volume of saline was injected for the control experiments.

After determination of the basal Trectal of each mouse two different protocols were followed:

Low-dose LPS (1 mg/kg, ip) protocol: LPS was injected and Trectal was recorded for 300 minutes after injec-tion. Nadroparine at various doses (11.875, 23.75, 47.5, 95.0, and 190.0 U/kg, sc) was injected 2 hours be-fore LPS challenge (pretreatment). The effects of other LMWHs (enoxaparine 11.90 and 23.8 U/kg, sc or dal-teparine 12.01 and 24.03 U/kg, sc) and unfraction-ated heparine (500 IU/kg, sc) pretreatments were also tested.

Based on the finding that 23.75 U/kg sc nadroparine had the most effect on LPS-induced changes, this was chosen as a test dose and used for the other experiments. Thus, the effect of a single test-dose of nadroparine (23.75 U/kg, sc) was evaluated when synchronously in-jected with LPS.

High-dose LPS (60 mg/kg, ip) protocol: LPS was in-jected and Trectal was recorded for 300 minutes at 20 minutes intervals. The test dose of nadroparine (23.75 U/kg, sc) was administered either 2 hour be-fore (pretreatment) or synchronously with LPS. In an-other group of mice, a test dose of nadroparine was injected 5 hours after LPS treatment. Then, the Trectal and SLMA of all mice were evaluated 24 hours after LPS injection.

Statistical Analysis:T values were expressed as mean ± standard errors of the mean. Multiple comparisons of the Trectal curves were made by analysis of variance (one-way ANOVA) with Newman-Keuls post hoc test. Comparisons of the two independent groups were made by Student’s t test or the Mann-Whitney U test (be-havioral data). The α value was corrected by using the Bonferroni procedure for multiple comparisons. Signif-icance was noted when p< .05.

RESULTS

Effects of LMWHs on low dose LPS (1 mg/kg, ip)-induced hypothermia: The Trectal of the mice treated with saline decreased slightly in comparison to the basal values during the observation period (300 minutes). LPS pro-duced a significant hypothermic response that appar-ently occurred after 20 minutes and reached a nadir (−2.73 ± 0.36◦C) ∼ 100 minutes after injection. The hypothermia was sustained throughout the experiment (F [5,84]: 34.1, p < .0001). Nadroparine pretreatment (23.75 U/kg, sc) 2 hours before LPS challenge almost completely inhibited the LPS-induced hypothermic re-sponse. A higher dose of nadroparine pretreatment (47.5 U/kg, sc) partially reversed the hypothermia (Figure 1). Nadroparine pretreatment at doses of 11.875, 95.0, or 195.0 U/kg sc did not change the course of LPS-induced hypothermia (data not shown). On the other hand, the antihypothermic effect of nadroparine (23.75 U/kg, sc) was not observed when injected synchronously with LPS. Nadroparine treatment alone did not produce any significant Trectal change in comparison with saline-treated mice (Figure 1).

Pretreatment with an equivalent dose of enoxaparine (23.8 U/kg, sc) or dalteparine (24.03 U/kg, sc), in terms of anti-Xa activity with nadroparine, had no effect on LPS-induced hypothermia (F [2,42]: 6.41; p< .28) (Fig-ure 2). A lower dose of each compound (11.9 U/kg and 12.01 U/kg, sc; respectively) also had no effect on the hypothermia. Similarly, unfractionated sodium heparine pretreatment did not prevent the LPS-induced hypothermic response (data not shown).

Effects of nadroparine on high dose LPS (60 mg/kg, sc)-induced hypothermia: High dose LPS caused a pro-found hypothermic response that reached a nadir (−4.24 ± 0.29◦C) ∼40 minutes after injection. The mice remained hypothermic throughout the obser-vation period (300 minutes) (F [3,42]: 64.6, p < .0001). Nadroparine pretreatment (23.75 U/kg, sc) did not abolish the LPS-induced response. However, syn-chronous nadroparine administration with LPS signif-icantly attenuated the first hour of the hypothermia (Figure 3).

Effects of nadroparine on high dose LPS-induced SLMA depression and hypothermia that occurred 24 hours after injec-tion: High dose LPS dramatically inhibited the parame-ters of SLMA, namely the counts of horizontal activity and rearings, 24 hours after injection. The Trectal was also significantly lower than in saline-treated control

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FIGURE 1 The effects of nadroparine injection either as pretreatment or synchronous on low dose of LPS-induced hypothermic re-sponce. Each point represents the mean± SEM of the specified observations.

∗_p_{< .05 compared to saline + saline-injected group. The statistically significant portion of the curves are indicated as:}

saline + LPS;

nadroparine 23.75 U/kg (pretreatment) + LPS; nadroparine 47.5 U/kg (pretreatment) + LPS; nadroparine 23.75 U/kg + LPS (synchronous).

mice at this time (Table 1, Treatment B). Nadroparine pretreatment (23.75 U/kg, sc) potentiated the LPS-induced inhibition of SMLA and hypothermia (Table 1, Treatment C). However, synchronous nadroparine treatment significantly inhibited the depressive effect of LPS on the horizontal component of SLMA. Further-more, LPS-induced hypothermia and the reduction in

rearing were also attenuated (Table 1, Treatment D). The beneficial effect of nadroparine was partially observed when injected 5 hours after LPS treatment. Thus, the hypothermia and inhibition of the rearing component of SLMA were still prominent, but the reduction in horizontal activity was completely reversed (Table 1, Treatment E).

FIGURE 2 The effects of enoxaparine or dalteparine pretreatment on low dose of LPS-induced hypothermic responce. Each point represents the mean± SEM of the specified observations.

saline + LPS; enoxaparine + LPS; dalteparine + LPS.

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FIGURE 3 The effects of nadroparine injection either as pretreatment or synchronous on high dose of LPS-induced hypothermic responce. Each point represents the mean_{± SEM of the specified observations.}

saline + LPS;

nadroparine 23.75 U/kg (pretreatment) + LPS; nadroparine(pretreatment) + LPS.

DISCUSSION

Severe sepsis is the tenth most common cause of death in the United States [10]. Twenty percent of pa-tients who develop severe sepsis are surgical papa-tients, and severe sepsis is a major cause of both preoperative and postoperative morbidity and mortality. The bur-den of disease associated with severe sepsis is stagger-ing. Total hospital costs for the care of severe sepsis in the United States in 1995 were estimated at $16.7 billion [11]. Intra- and postoperative factors that put patients at increased risk for sepsis include the presence of a pronounced systemic inflammatory response and a greater degree of early postoperative organ dysfunction

[12]. Thus, preventive approaches have a crucial value in clinical practice.

Previous studies have reported that macrophage-derived proinflammatory cytokines such as interleukin-1 or tumour necrosis factor-α can modulate the endothelial surface, making it procoagulant and impair-ing fibrinolysis. These lead to inappropriate intravas-cular coagulation and multiple organ damage [1–6]. Although a variety of mechanisms may be involved, the generation of thrombin, factor Xa and the tissue fac-tor VIIa complex play an essential role, expanding the acute inflammatory response via the activation of spe-cific receptors on endothelial cells. Expression of adhe-sion molecules and platelet-activating factor facilitates

TABLE 1 Effects of differential timing of nadroparine administration on LPS-induced SLMA depression and hypothermia that occurred 24 hours after injection

SLMA (counts/5 min)

Treatment

Delta

Trectal (◦C) horizontal act. rearings

A Salin+ salin 0.17± 0.12 37± 6 14± 2

B Salin+ LPS −5.8 ± 1.07∗ 17± 3∗ 2± 0.3∗

C Nadroparine+ LPS (pretreatment) −7.9 ± 0.87∗# 5± 2∗# 0∗

D Nadroparine+ LPS (synchronous) −2.5 ± 0.4∗# 27± 4# ₇_{± 0.8}∗#

E LPS+ nadroparine (5 hours after LPS) −4.5 ± 0.79∗ 25± 5# ₅_{± 2}∗ ∗_p_{< .05 compared to saline control.}

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leucocyte-endothelium interactions, which play a cen-tral role in the host inflammatory response [1–6, 13– 16]. Beside their anticoagulant activity, LMWHs, which have an antifactor Xa activity, are expected to reduce this acute inflammatory response by blocking this in-teraction during sepsis and therefore have the potential to be useful drugs.

To assess the hypothesis that the use of low dose or low-molecular weight heparin would improve sur-vival in sepsis, Davidson et al. [17] searched the liter-ature published between 1991 and 2001. They found that either low dose or low-molecular weight heparin administration significantly improved survival. Other investigators also supported this conclusion, reporting that heparin derivatives are effective in the prevention of endotoxin-induced coagulation disorders, reducing mortality and moreover decreasing end-organ damage [7, 8]. However, little is known about whether differ-ences among the various LMWHs, the timing of admin-istration, and the efficacy of LMWHs against varying intensities of septic insult do exist.

The results of the present study demonstrate that nadroparine, a LMWH, might have a protective effect against the LPS-induced systemic inflammatory response in mice. It appears that this is a non-dose-dependent effect and that other LMWHs, such as enoxaparine or dalteparine, do not show such an effect, at least in the dose range studied. There are substantial variations among the LMWH preparations produced by different manufacturers[18, 19]. Thus, it is not possible to standardise the dose of each LMWH on a molecular weight basis. For this reason, the dose-response relationships of the compounds were constructed in terms of their anti-Xa activity. This variable effect on low dose LPS-induced hypother-mia indicates that the antihypothermic activity of nadroparine may not be related to its anticoagulant effect. It is conceivable that some molecular structural differences may account for the possible beneficial effects of LMWHs on the systemic inflammatory response. The other critical point is that the antihy-pothermic activity of nadroparine is a time-dependent response, i.e., nadroparine elicits an effect when injected as a pretreatment but loses its activity when injected synchronously with LPS. This implies that nadroparine may not be able to suppress the systemic inflammatory process once the process is initiated.

Our results also suggest that nadroparine might be able to attenuate some (presumably septic shock-like)

changes due to high doses of LPS. In contrast, nadroparine has an effect if injected synchronously with or after LPS administration. Interestingly, nadroparine may potentiate the high dose LPS-induced changes if injected as a pretreatment. This peculiar finding may underline the fact that LPS-induced changes such as hypothermia may have different pathophysiological mechanisms depending on the dose of LPS.

This clear-cut pharmacological activity profile of nadroparine deserves some possible explanations for the possible mechanisms of actions. Systemic LPS injection triggers the release of proinflammatory cy-tokines into the peripheral blood circulation. These cir-culated cytokines pleiotrophically affect various organ systems including the brain. Thus, some changes due to LPS treatment, such as hypothermia, are mediated by central neuronal mechanisms [20]. It is unlikely that nadroparine interferes with the central mechanisms to attenuate the hypothermic response, since it is a large molecule that cannot pass through the blood-brain bar-rier. Alternatively, nadroparine may abolish the release of various proinflammatory cytokines such as TNF-α in the periphery [21–27].

In conclusion, the results of this study reveal that the use of LMWHs such as nadroparine might be beneficial in high-risk surgical patients because of their potential anti-inflammatory action, in addition to their efficiency in preventing thrombo-embolic complications. Further studies are underway to elucidate the possible mecha-nisms of action.

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