Introduction

Endotoxemia is a major cause of sepsis. Circulating endotoxins induce the activation of complement and the release of proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β [1]. The proinflammatory cytokines, particularly TNF-α, IL-1β, and IL-6, can in turn trigger secondary inflammatory cascades, including the production of cytokines, lipid mediators, and reactive oxygen species [2, 3]. The proinflammatory cytokines and nitric oxide induced by endotoxin can decrease systemic vascular resistance, resulting in profound hypotension [4, 5]. The combination of hypotension and microvascular occlusion results in tissue ischemia and ultimately leads to multiple organ failure [6].

Our previous study demonstrated that a new cytokine adsorbent, CTR, effectively adsorbed small to middle-sized proteins, such as cytokines, in vitro and that direct hemoperfusion using a CTR column (CTR treatment) reduced mortality and inhibited inflammatory responses in endotoxemic rats [7]. Moreover, our other study showed that CTR treatment 2 h after endotoxin injection had beneficial effects on mortality and cytokine responses in rats [8]. However, we did not determine whether CTR treatment provided different beneficial effects when given at different doses after endotoxin injection. The current study was undertaken to clarify the dose-related effects of CTR treatment on mortality and inflammatory responses in rats with endotoxin-induced shock.

Materials and methods

Forty-eight male Wistar rats, weighing 382 ± 14 g (mean ± SD), were used in this study. The experimental protocol was approved by the Animal Care Committee of our institute, and the care and handling of the animals were in accord with the National Institute of Health's guidelines.

CTR, a cytokine adsorbent, developed by Kaneka Corporation (Osaka, Japan) is composed of porous cellulose beads to which a hydrophobic organic compound with a hexadecyl alkyl chain has been covalently bound to the surface as a ligand.

General procedure

The animal preparation method was reported previously [9, 10]. Briefly, after an intraperitoneal injection of pentobarbital sodium (30 mg/kg), ventilation was performed through a tracheotomy. The femoral artery was cannulated to monitor the blood pressure and to draw blood samples. Lactated Ringer's solution containing a muscle relaxant (pancuronium bromide, 0.02 mg/ml) and pentobarbital sodium (0.5 mg/ml) was infused continuously at a rate of 10 ml/kg/h through the femoral vein cannula. The rats were connected to a pressure-controlled ventilator (Servo 900B, Siemens-Elema, Solna, Sweden) delivering 100% oxygen at a frequency of 30 breaths/min. After this procedure, the animals were allowed to rest for more than 30 min to stabilize. Baseline HR and systolic arterial pressure (SAP) reading were then performed.

After baseline measurements, all rats were injected intravenously with Escherichia coli lipopolysaccharide derived from E. coli 0111:B4 (Difco Laboratories, Detroit, MI, USA) at a rate of 15 mg/kg over 2 min. Then rats were randomly allocated to one of the following four groups (n = 12 per group).

Control column group (group C)

Beginning 15 min after endotoxin injection, direct hemoperfusion alone using a control column without CTR was performed for 120 min. The volume of the column was 0.5 ml.

Quarter-dose treatment group (group Q)

Beginning 15 min after endotoxin injection, CTR treatment was performed for 120 min. The volume of the column was 0.75 ml and it contained 0.25 ml of CTR.

Half-dose treatment group (group H)

The CTR treatment was performed as in group Q. The volume of the column was 1.0 ml and it contained 0.5 ml of CTR.

Full-dose treatment group (group F)

The CTR treatment was performed as in group Q. The volume of the column was 1.5 ml and it contained 1.0 ml of CTR.

For all groups, the blood circuit volume was 1.0 ml, the blood volume into the column was 0.5 ml and the blood flow rate was 1.5 ml/min. Low-dose heparin was administered as an anticoagulant at a rate of 200 units/kg/h.

The rectal body temperature was maintained at between 36 and 38°C with the aid of a heating pad. Arterial blood samples were drawn 2, 4, 6, and 8 h after endotoxin injection for the measurement of pH, PaCO2, and PaO2 levels. Additionally, arterial blood samples were drawn for the measurement of plasma cytokine concentrations 2 and 4 h after endotoxin injection. All cytokine (TNF-α and IL-6) concentrations were measured using ELISA kits (BioSource, Camarillo, CA, USA). A total amount of 4.75 ml of blood was drawn from each animal over 8 h. Mortality was observed for up to 8 h after the endotoxin injection.

Statistical analysis

Data are presented as the mean ± SD. Differences between groups at baseline were analyzed using an one-way analysis of variance (ANOVA), followed by a post hoc test (Dunnett's method). Differences between groups were analyzed using a two-way ANOVA with repeated measures, followed by a post hoc test (Bonferroni's method). The mortality rates were compared using the Kaplan–Meier and the Mantel–Cox methods. Statistical significance was defined as p< 0.05. All statistical analyses were performed using StatView (Version 5.0, Abacus Concepts, Berkeley, CA, USA).

Results

Hemodynamics and mortality rate

No significant differences in baseline HR or SAP were noted among the groups (Fig. 1). Four hours after endotoxin injection, the SAP in group C was reduced, but those in the CTR treatment groups were not. The mortality rates at 8 h after endotoxin injection were 92%, 58%, 42% and 17% for group C, group Q, group H and group F, respectively (Fig. 2). The mortality rates for the CTR treatment groups were significantly lower than the rate in group C. The mortality rate of group F was also significantly lower than that of group Q.

Fig. 1
figure 1

The systolic arterial pressure (top) and heart rate (bottom) at baseline and after endotoxin injection (mean ± SD). Open squares, control column group; filled squares, quarter-dose treatment group; open circles, half-dose treatment group; filled circles, full-dose treatment group. * p < 0.05 versus baseline value (within-group); # p < 0.05 versus control column group

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Fig. 2
figure 2

Survival curves for control column, quarter-dose treatment, half-dose treatment, and full-dose treatment groups. * and #, p< 0.05

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Plasma cytokine concentrations

All baseline values were similar for the four groups (Fig. 3). Endotoxin injection increased the TNF-α and IL-6 concentrations in all the groups, but the TNF-α concentrations were significantly lower in the group H and group F than in the group C. The IL-6 concentrations were significantly lower in the CTR treatment groups than in group C.

Fig. 3
figure 3

Changes of plasma TNF-α (top) and IL-6 (bottom) at baseline and after endotoxin injection (mean ± SD). Open squares, control column group; filled squares, quarter-dose treatment group; open circles, half-dose treatment group; filled circles, full-dose treatment group. * p< 0.05 versus baseline value (within-group); # p< 0.05 versus control column group

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Blood gases

No significant differences in baseline pHa were noted among the groups. The pHa values in groups H and F were significantly higher than in group C at 8 h after endotoxin injection. The PaCO2 and PaO2 values did not differ significantly among the four groups at any point during the experimental period.

Discussion

Endotoxemia in the absence of CTR treatment produced hypotension, acidosis, and an increase in plasma cytokine concentrations, leading to a high mortality rate. In contrast, CTR treatment dose-dependently decreased the mortality rate and inhibited inflammatory responses in our rat endotoxic shock model.

Our previous study showed that CTR columns are useful for the selective adsorption of sepsis-related proteins and that CTR treatment immediately after the exposure to endotoxemia reduced mortality and inhibited inflammatory responses in rats [7]. Moreover, we showed that CTR treatment 2 h after endotoxin injection had beneficial effects on mortality and inflammatory responses in rats [8]. However, we did not evaluate whether CTR treatment provided different beneficial effects when given at different doses after endotoxin injection. There have been no studies to show the dose-related effects of CTR on mortality and inflammatory responses during endotoxic shock both in vitro and in vivo. The present study demonstrated that CTR treatment dose-dependently decreased the mortality rate and inhibited inflammatory responses in vivo. These findings suggest that a large CTR dose may have more far-reaching effects during sepsis and septic shock.

The dose-dependent effect of CTR treatment on the production of inflammatory cytokines after endotoxin injection is interesting. Circulating endotoxin induces the release of cytokines such as TNF and IL-6, which can lead to hypotension and metabolic acidosis [2, 3, 4, 5, 6]. The present study found that CTR treatment dose-dependently inhibited increases in the plasma concentrations of TNF and IL-6 induced by endotoxemia in vivo. Preliminarily, we used a very large dose of CTR (2.0 ml) in our model. However, we stopped CTR treatment because of hypotension caused by the large column volume. These findings suggest that a large CTR dose may have greater effects during sepsis and septic shock but that the CTR treatment may cause hypotension. Further investigation of this point is needed.

The present study did not evaluate changes in plasma endotoxin concentration after the endotoxin injection. The ability of CTR to effectively adsorb endotoxin in vitro was also uncertain. Therefore, whether CTR can effectively adsorb endotoxins remains uncertain. The present study involved injection of a large dose of endotoxin. Buras et al. reported that endotoxemic models may not necessarilyreflect clinical septic situations [11]. Moreover, questions remain as to whether CTR treatment is effective against different models of sepsis, such as the direct infusion of bacteria or cecal rupture, and whether CTR treatment is effective in different animal models, such as pig or monkey. Further investigation of these points is also needed.

In summary, the present study showed that CTR treatment dose-dependently decreased the mortality rate and inhibited inflammatory responses in our rat model for endotoxic shock. These findings suggest that CTR treatment may be effective against sepsis and septic shock.