Mortality
In total, there was a 42% mortality (8 died out of 19) during the observation period in rats treated with HBO
2, whereas we saw 50% mortality (9 died out of 18) in rats treated with NBO
2 group and 61% mortality in the control group (11 died out of 18). Treatment with 2.5 bar p
abs HBO
2 did not alter the survival significantly compared with NBO
2 treatment at any hours (24 h,
p = 0.13, 48 h,
p = 0.63 and 72 h,
p = 0.63). Additionally, we found no statistically significant difference in cumulative survival between the HBO
2 group and the NBO
2 group (
p = 0.59, log-rank test), between the HBO
2 group and the control group (
p = 0.24, log-rank test) or between the NBO
2 group and the control group (
p = 0.59, log-rank test) (Fig.
2).
Cytokines
Tables
2,
3 and
4 show the differences in the median cytokine concentration at hour 0, 24, 48 and 72. The difference between HBO
2 and both NBO
2 and control groups was calculated for all three cytokines. We found a statistically significant difference between HBO
2 (489.4 pg/mL; IQR 332.7–650.1) and control /228.6 pg/mL; IQR 40.4–313.2) according to the anti-inflammatory IL-10 at hour 48, however this was not the case for the other time points (hour 0,
p = 0.82; hour 24,
p = 0.94; hour 48,
p = 0.01; hour 72,
p = 0.60). We found no statistically significant difference between HBO
2 and control at any time point according to the pro-inflammatory IL-6 (hour 0,
p = 0.06; hour 24,
p = 0.85; hour 48,
p = 0.97; hour 72,
p = 0.86) and TNF-α (hour 0,
p = 0.48; hour 24,
p = 0.85; hour 48,
p = 0.36; hour 72,
p = 0.72). Neither did we find any statistically significant difference between HBO
2 and NBO
2 at any time point according to the pro-inflammatory IL-6 (hour 0,
p = 0.87; hour 24,
p = 0.98; hour 48,
p = 0.91; hour 72,
p = 0.94) and TNF-α (hour 0,
p = 0.99; hour 24,
p = 0.88; hour 48,
p = 0.97; hour 72,
p = 0.94). This was also the case for the anti-inflammatory IL-10 (hour 0,
p = 0.66; hour 24,
p = 0.31; hour 48,
p = 0.13; hour 72,
p = 0.37).
Table 2
Differences in median TNF-α concentration
0 | 25.7 (0.4–102.9) | 11.9 (0.4–188.7) | 57.2 (0.4–114.9) |
24 | 59.4 (0.4–138.5) | 33.5 (0.4–136.3) | 64.0 (0.4–157.8) |
48 | 9.5 (0.4–74.0) | 0.4 (0.4–93.2) | 47.0 (1.2–137.6) |
72 | 55.8 (0.4–122.4) | 62.1 (0.4–125.5) | 5.1 (0.4–131.8) |
Table 3
Differences in median IL-6 concentration
0 | 2.8 (2.8–30.5) | 2.8 (2.8–153.2) | 33.4 (2.8–70.0) |
24 | 161.5 (14.6–296.5) | 197.9 (2.8–278.6) | 144.3 (53.4–322.9) |
48 | 2.8 (2.8–77.9) | 2.8 (2.8–101.3) | 2.8 (2.8–95.5) |
72 | 5.5 (2.8–134.9) | 2.8 (2.8–224.7) | 2.8 (2.8–182.6) |
Table 4
Differences in median IL-10 concentration
0 | 64.7 (0.1–148.5) | 0.1 (0.1–278.8) | 79.4 (0.1–155.9) |
24 | 591.0 (336.2–969.1) | 405.8 (296.6–579.9) | 517.2 (289.6–1138) |
48 | 489.4 (332.7–650.1)* | 337.8 (203.6–453.4) | 228.6 (40.4–313.2)* |
72 | 250.9 (99.4–330.5) | 72.5 (0.1–538.5) | 169.5 (585.8–0.1) |
We assessed the concentration of cytokines on hour 24 on all animals alive longer than 24 h after CLP. We grouped them in survivors (alive until 72 h after CLP) and non-survivors (died between 24 and 72 h after CLP). We found non-survivors to have a significant higher IL-6 concentration at 24 h compared to survivors (non-survivors IL-6302.3 pg/mL (102.8–581.6) vs. survivors IL-6 = 107.9 pg/mL (20.5–252.2), (p = 0.04)). There was no statistically difference in the concentration of TNF-α and IL-10 between survivors and non-survivors (non-survivors TNF-α = 59.37 pg/mL (0.37–179.8) vs. survivors TNF-α 37.52 pg/mL (0.37–122.6), p = 0.31; non-survivors IL-10 = 1050 pg/mL (274.1–5318) vs. survivors IL-10 = 450.2 pg/mL (342.2–627.4), p = 0.13).
Discussion
In this study, we evaluated delayed intervention of hyperbaric oxygen as a treatment for sepsis. We found no difference in the concentrations of IL-6, TNF-α and IL-10 between the groups treated with hyperbaric or normobaric oxygen during the first 72 h of sepsis. However, we found a significantly higher concentration of IL-10 at hour 48 in the HBO2 group compared with the control group, but not at any other time points. Moreover, we found a significantly higher concentration of IL-6 at hour 24 in non-survivors compared to survivors. Lastly, we found no difference in survival rates between all the treatment groups.
In previous reports, the experimental animals were either terminated or anesthetized at varied time points in order to collect samples and to obtain knowledge of cytokine concentrations at different time points [
8,
19,
25,
26]. This weakens longitudinal conclusions and results in a larger amount of animal sacrifice. In the present study, each rat was monitored with intermittent blood sampling which allows evaluation of the changes in cytokine concentrations on a daily basis and to follow each rat individually. In addition, we were able to reduce the number of rats by 75% and still obtain the same number of samples, which gives this study an enormous ethical advantage. Moreover, it is a strength that we have combined the gold standard of sepsis induction models with intermittent blood sampling in the individual rat making the model applicable to clinical practice with respect to the study of different interventions, alone or in combination [
20].
The present study uses only clinical signs and blood cultures to ensure that the animals are septic, since earlier studies have shown that CLP imitates sepsis according to vascular derangement, alterations in the metabolic state and with clinical signs showing disease [
15,
20,
27]. In this study we found that 63% of the blood cultures were positive (Table
1) with
E. coli, E. Faecalis and
E. cloacae as the most frequent bacteria isolated, corresponding to peritonitis from fecal matter. A post mortem laparotomy was performed in all animals, disclosing a necrotic cecum tightly adherent to close-lying anatomical structures, including signs of peritonitis with excessive cloudy intraperitoneal fluid combined with a foul smell. The information from the clinical symptoms and signs in combination with the positive blood cultures and the laparotomy findings suggest that all rats were septic.
Sepsis is a critical illness currently treated with fluids and antibiotics. Turnbull et al. has demonstrated that antibiotics can improve the outcome in murine sepsis lowering overall mortality, but if a concentration of 14,000 pg/mL of IL-6 is reached, the animals are destined to die despite antibiotic treatment [
28]. High plasma concentrations of IL-6 in humans are also associated with the development of septic shock and is a predictor of mortality [
5,
29‐
31]. The current experiment confirms that IL-6 was a predictor of death with a significantly higher concentration of IL-6 in non-surviving animals at 24 h indicating a state of septic shock (Table
5). As shown by Singleton KD and Wischmeyer PE, CLP distance and size of needle puncture determines mortality. Performing a 30% CLP with a 16G double-puncture as in the current experiment should give a 90% mortality after 72 to 96 h [
19]. Accordingly, the severity of the model presented here, in combination with the high expression of IL-6 at 24 h measured before oxygen intervention, indicates this study applies to a severe state of sepsis.
Table 5
Difference between survivors and non-survivors in median cytokine levels at hour 24
TNF-α | 59,37 (0.37–179.8) | 37.52 (0.37–122.6) | 0.31 |
IL-10 | 1050 (274.1–5318) | 450.2 (342.2–627.4) | 0.13 |
IL-6 | 302.3 (102.8–581.6) | 107.9 (20.5–252.2) | 0.04* |
It has been suggested that HBO
2 might exert its effect through production of the anti-inflammatory IL-10, which acts by lowering the pro-inflammatory IL-6 and thereby reducing mortality [
5,
7,
18,
29,
30]. IL-10 has furthermore been demonstrated to play a critical step in the progression to a lethal state of sepsis and that endogenous production of IL-10 delays the onset of mortality in CLP induced sepsis [
18]. In the present study, we found a significantly higher concentration of IL-10 at hour 48 in the HBO
2 group compared with control. This finding is in accordance to previous studies showing that HBO
2 treatment might boost the production of IL-10 [
7]. In this study, we found no statistical difference in the mortality between the HBO
2 group and the control group, hence the statistically higher concentration of IL-10 in the HBO
2 group at 48 h did not influence survival. Interestingly, we found no significant difference between HBO
2 and NBO
2 in the concentrations of IL-6, TNF-α and IL-10 at any hours. Overall, the concentration of all three measured cytokines decreased after the first 24 h. This is probably due to the most severely ill animals dying within the first 24 h of the study. The cytokine value on hour 24 and forth may therefore represent less ill animals, explaining the lower values of both pro- and anti-inflammatory cytokines as measured. As later described, the cytokine concentrations on Day 0 fluctuated greatly and may have contributed to the lack of statistically significant results in the measured cytokines.
Buras et al. examined the effect of HBO
2 treatment according to survival in septic mice and found that two daily treatments at 2.5 atm with a 12-h interval and a total of eight treatments were required to reduce mortality [
7]. They also found that NBO
2 had no effect in terms of survival. In the present study, HBO
2 was delayed by 24 h after CLP-induction of sepsis, whereas Buras et al. administered two HBO
2 sessions within the first 24 h [
7]. Undoubtedly, the 24 h delay of the HBO
2 treatment were fatal to overall survival in this CLP model, although the treatment pressure was chosen with reference in Buras et al.’s studies [
7]. The delay of treatment was chosen to study the rats in a circulatory hypodynamic state of sepsis, which follows the initial hyperdynamic state and subsequently progresses into a state of severe sepsis [
32]. Experimentally, early intervention with hyperbaric oxygen has been shown to ameliorate sepsis. In most studies, however, the treatment is given shortly after induction of sepsis before the signs of manifest sepsis appear [
7,
14]. In clinical practice, however, we cannot treat sepsis before the symptoms and signs appear. Moreover, logistic challenges and patient transfer between hospitals and other procedures can delay hyperbaric oxygen treatment. Therefore, the chosen delay of the hyperbaric treatment in this report reflects clinical practice. However, the delay also constitute a limitation since many animals die before the first intervention.
There are some other limitations of the present study. Firstly, no sample size calculation was performed prior to the experiment, which increases the risk of a type II error due to a potential insufficient number of animals included in each group. However, others studies have investigated sepsis and outcomes in animals using similar sample size, which is why we chose the number of animals in the current study [
7,
15,
18]. Secondly, the cytokine concentrations on Day 0 varied greatly (see Tables
2,
3 and
4 for concentrations). It is well known in animal models to see a stress response from anesthesia, which can vary between the animals, however in the current study it varied greater than we expected. It is important to outline, that the baseline sample was taken prior to CLP hence no infection was present yet. The fluctuating concentrations on Day 0 may have contributed to the lack of statistically significant results in cytokines concentrations between groups. Thirdly, although rats were randomized by groups a difference occurred in rat weight at the time of inclusion (Table
1). Minor changes in weight (< 10%) induced by high fat diet has earlier been shown to be associated with increased oxidative stress and inflammation causing increased rat mortality in CLP models of sepsis [
33,
34]. However, our rats were all fed standard chow so a worsening effect of the weight is not to be expected. The weight differences could be related to differences in age at the time of randomization, but all rats were provided similar acclimatization of 1½ week and previous reports have failed to find any correlation between age, weight and final outcome in similar CLP models [
35]. Finally, as the Kaplan-Mayer curves show (Fig.
2) most of the mortality occurred during the first 24 h and therefore before the various treatment modalities were commenced. This means that NBO
2 and HBO
2 therapies were studied in a small number of rats, hence the size of the surviving group may have been too small to demonstrate a possible beneficial effect. Measurements of other biomarkers e.g. nuclear protein high mobility group box protein 1 (HMGB1) has emerged in sepsis, and we cannot exclude other biomarkers to link to survival in our model [
36].