Burn injury activates an inflammatory cascade, which plays a major role in the development of subsequent immune dysfunction, sepsis, and multiple organ failure (1,2). One of the accepted models used to explain this sequence of events has been termed the “two-hit” phenomenon. According to this theory, major trauma (such as burn injury) increases the production of proinflammatory mediators, mainly from macrophages. This, in turn, compromises the immune system of the host, making the host vulnerable to a second hit (i.e., infection), leading to multiple organ failure and, ultimately, to death (2). Therefore, down-regulation of the production and release of proinflammatory mediators may help prevent post-burn immune dysfunction.
Ketamine, a commonly used anesthetic drug, decreases the release of proinflammatory mediators (3–5). In a previous study, we demonstrated a significant beneficial effect of ketamine on survival after Escherichia coli (E. coli)-induced sepsis in rats (6). This effect was attributed to the antiinflammatory activity of ketamine, as evidenced by lower serum interleukin (IL)-6 and tumor necrosis factor (TNF)-α concentrations in the treated animals compared with controls. The present study was conducted to test the effect of ketamine on survival and on IL-6 concentrations in rats sustaining either burn injury alone or burn injury followed by sepsis.
Three hypotheses were examined in this study. First, based on a previous report that ketamine decreased IL-6 and improved survival after sepsis, and recognizing that sepsis and burn injury share the common characteristic of activating inflammation, we hypothesized that ketamine would decrease IL-6 and improve survival after burn injury alone (6). Second, based on our expectation that ketamine would decrease IL-6 and improve survival after burn injury, we hypothesized that giving ketamine after burn injury, but before sepsis, in a model of burn injury + sepsis would decrease post-sepsis IL-6 production and improve survival. Third, based on an expected greater inflammatory response after sepsis than after burn injury, we hypothesized that giving ketamine at the time of sepsis in a model of burn injury + sepsis would cause a larger decrease of post-sepsis IL-6 production and improvement in survival as compared with giving ketamine after burn injury but before sepsis.
The protocol was approved by the Committee for the Ethical Care and Use of Laboratory Animals of the Ben-Gurion University of the Negev. The protocol included a provision that rats exhibiting evidence of distress (such as restlessness or aggressive behavior) be euthanized. Rats were acclimated to the laboratory for 2 weeks before beginning the study and had free access to water and food at all times. We used 176 male Sprague-Dawley rats (weighing 200 to 250 g). The animals were assigned to 1 of 8 groups (Table 1). The groups were defined by experimental condition (burn injury or burn injury followed by sepsis), treatment (ketamine or saline [controls]), and timing of treatment (1 h post-burn injury or at the onset of sepsis [24 h after burn in the no-sepsis groups]). In the ketamine groups, the dose of ketamine (Ketalar; Parke Davis, Hampshire, UK) was 10 mg/kg given IP. After each calculated dose of ketamine was drawn up into a 1 mL syringe, saline solution was added to the ketamine dose so that a standardized total injective volume of 1 mL was administered to each rat. In the saline group, the volume of saline given IP was 1 mL. In a previous study we found that this dose of ketamine had no detectable bactericidal effect (6). In addition, a previous in vitro study reported that clinically recommended doses of ketamine produced minor effects on E. coli elimination and that larger concentrations prolonged E. coli elimination; thus it is unlikely that the dose of ketamine used in the present study affected bacterial clearance (7).
Because of expected increased mortality after burn injury + sepsis as compared with burn injury only, 76 rats were assigned to the burn only groups (Groups 1–4) and 100 rats were assigned to the burn injury + sepsis groups (Groups 5–8). Rats failing to survive the first 60 min after burn injury were excluded from the study (5 rats in Groups 1–4 and 6 rats in Groups 5–8). The remaining 71 rats in groups 1–4 received either ketamine or saline at either 1 or 24 h after burn injury. The remaining 94 rats in Groups 5–8 received IP inoculation of E. coli bacteria 24 h after the burn injury and either ketamine or saline either at 1 h after burn injury or at the onset of sepsis (E. coli inoculation). All eight groups were then further subdivided into subgroups for determination of outcome measures. In the assessment of IL-6 subgroups (n = 5 per group), blood was withdrawn from the tail vessel for determination of serum concentration of IL-6 at 6 h after burn injury in Groups 1–2, 30 h after burn injury in Groups 3–4, and at 6 h after sepsis (E. coli inoculation) (30 h after burn injury) in Groups 5–8. A n = 5 was considered satisfactory for IL-6 determinations based on a one-sided sample size determination (power analysis), assuming sem = 0.25, σ (sd) of 0.56, power of 0.85, significance of 0.05, and a meaningful μo − μ1 (difference between means; in this case increase of IL-6) of 120% (i.e., more than 2 sd). With n = 5 the actual threshold for detection of significant difference for μo − μ1 was 0.72 (i.e., this study could have detected a μo − μ1 as small as 0.72 even though we sought to detect a μo − μ1 of 1.2 or more). In the assessment of survival subgroups (n ∼ 12–13 per group in the burn injury-only groups [1–4] and ∼18–19 per group in the burn injury + sepsis groups [5–8]), rats were observed from the time they sustained the burn injury to 1 wk after inoculation. Behavior, appearance and mortality were recorded every 24 h.
Rats were anesthetized with inhaled halothane 4%. The hair of the torso was removed from a rectangular area using an electrical trimmer. Rats were subjected to a 30% body surface area full-thickness burn injury by an electrical radiator generating a temperature of 4000°C and placed 1–2 cm from the skin for 20 s. In preliminary studies, histopathologic examination of the burned area indicated that this injury model produces a reproducible, full-thickness skin burn, generally with no direct injury to underlying internal organs. The hemodynamic effect of edema formation and serum exudation at the burn site was minimized by administration of 5 mL of normal saline IP immediately after burn injury. A preliminary experiment showed that the mortality rate of this reproducible model was 13% at 24 h. Causes of mortality were not investigated but presumably include shock, thrombosis, hemorrhage, and excessive release of humoral substances. Animals received the experimental treatment (ketamine or saline) after recovery from anesthesia.
Sepsis was induced by IP inoculation with E. coli. A description of the use of this technique to model clinical sepsis and a discussion of the clinical relevance and reproducibility of this technique in comparison with other models of sepsis have been published (6,8). E. coli (HB-109) were cultured in 5 L of Lennox broth (Pronadisa, La Forja, Spain) medium. Bacteria were harvested at their logarithmic growth phase, resuspended in freezing medium (phosphate-buffered saline + 30% glycerol), divided into small aliquots, and frozen at −800°C. Each bacterial batch was assayed for colony-forming units (CFU) and for its lethal dose. In preliminary experiments in rats, we found that the survival rate after inoculation with E. coli 0.2 × 109 CFU of the batch used in this trial was 30%. The bacterial freezing medium (up to 3 mL) has no effect on mortality or cytokine levels, as determined previously. The bacterial suspension was thawed to room temperature 15 min before injection.
We selected IL-6 as a good marker of proinflammatory activity because it reaches peak concentrations and remains stable longer (7–10 days) than other proinflammatory cytokines such as TNF-α and IL-1 (9). Blood samples were centrifuged at 1500 rpm for 10 min. Serum fractions were analyzed with a commercial rat IL-6 ELISA kit (Biosource International, Camarillo, CA). Assays were performed in duplicate. If serum concentrations exceeded kit standards, they were diluted in assay buffer and assayed again to ensure accurate results.
This study was designed with the intent to compare the primary measures (survival and IL-6) of each study group to its control (i.e., within burn Groups 1–4 and within burn + sepsis Groups 5–8). The only exceptions were the comparison of 7-day survival and 30-h IL-6 concentrations in burn injury controls with those in burn injury + sepsis controls (to test our expectation that burn injury + sepsis represented a more severe injury than burn alone). Survival rates of ketamine-treated and control rats were compared using a one-tailed log rank test (for comparison of survival curves). The means of IL-6 concentrations were compared using a one-way analysis of variance followed by Bonferroni-corrected, one-tailed, unpaired Student's t-test, as indicated. The results are presented as mean ± sem. A P value < 0.05 was considered significant.
Burn Injury Only
The 7-day survival rate in rats given ketamine at 1 h (Group 1, 86.7%) or 24 h (Group 3, 69.2%) was not significantly different from that in the corresponding saline-treated groups (Group 2, 75%, P = 0.48, Fig. 1; Group 4, 81.8%, P = 0.43, Fig. 2). There was no difference between the survival rates of the two saline-treated groups (Groups 2 and 4, P = 0.71).
The serum IL-6 concentrations of control animals collected at 30 h after burn injury were increased compared with those collected at 6 h after the injury (545.8 ± 47.1 pg/mL versus 430.0 ± 36.7 pg/mL; P = 0.04). Ketamine given 1 h after burn injury caused a significant decrease of IL-6 concentrations (106.5 ± 3.4 pg/mL; P < 0.0001 versus 430.0 ± 36.71 pg/mL). In contrast, ketamine given 24 h after burn injury failed to decrease IL-6 concentrations significantly (486.3 ± 72.6 pg/mL versus 545.8 ± 47.1 pg/mL; P = 0.25).
Burn Followed By Sepsis
As expected, survival rates were significantly lower for saline-treated rats sustaining burn injury + sepsis compared to rats that had burn injury only (10.3% versus 78.3%; P < 0.0001). The survival rates for animals that were subjected to burn injury + sepsis were numerically higher in the ketamine-treated groups. The group given ketamine 1 h after burn injury (Group 5) had a survival rate six-fold better than that of the corresponding saline-treated group (Group 6) (42.1% versus 7.1%). However, this difference did not reach statistical significance (P = 0.14) (Fig. 3). The group given ketamine immediately after the E. coli inoculation (i.e., 24 h after burn injury) (Group 7) demonstrated a better survival rate than the corresponding saline-treated group (Group 8) (46.1% versus 13.3%. P = 0.008) (Fig. 4). No statistical difference was found between the 2 control groups (Groups 6 and 8; P = 0.97).
At 30 h, saline-treated groups receiving burn injury + sepsis demonstrated a larger increase in IL-6 concentrations compared with animals that sustained only burn injury (332,300 ± 32,300 pg/mL and 398,000 ± 116,600 pg/mL versus 545.8 ± 47.1 pg/mL, P < 0.0001). Ketamine given 1 h after the burn injury (23 h before sepsis) decreased the IL-6 concentrations measured 6 h after the inoculation of E. coli; however, this decline was not significant (117,300 ± 41,380 pg/mL versus 398,000 ± 116,600 pg/mL, P = 0.069). Ketamine given immediately after E. coli inoculation (i.e., 24 h after burn injury) significantly attenuated the increase of IL-6 (72,640 ± 40,990 pg/mL versus 332,300 ± 32,300 pg/mL, P = 0.008).
The present results support some of the elements of our three hypotheses but not others. Specifically, the results support a portion of our first hypothesis, i.e., that ketamine would decrease IL-6 production after burn injury alone, but not the remainder of our first hypothesis, i.e., that ketamine would improve survival after burn injury alone. In this regard, the timing of ketamine administration was critical, as treatment at 1 hour after burn injury was effective whereas treatment at 24 hours after burn injury was not. The results did not support our second hypothesis, i.e., that ketamine given after burn injury but before sepsis would decrease post-sepsis IL-6 production and improve survival in a model of burn injury + sepsis. Lastly, the results did support our third hypothesis, i.e., that ketamine given at the time of sepsis in a model of burn injury + sepsis would decrease post-sepsis IL-6 production and improve survival. As was the case with burn injury alone, the timing of ketamine administration was critical in burn injury + sepsis. Ketamine affected post-sepsis measures when given at the time of the greater inflammatory stimulus (sepsis) but did not affect post-sepsis measures when given 1 h after burn injury (23 hours before sepsis).
Our finding that ketamine given 1 h after burn injury alone did not improve survival was unexpected, considering a report that ketamine reduced mortality in severely burned rats (10). The apparent inconsistency between our results and those of the previous study may relate to differences in the severity of burn injury and dose of ketamine. In the previous study, the burn caused 80% mortality without ketamine treatment; whereas, in our study, the burn caused 15%–20% mortality. In the previous study, the dose of ketamine was 90 mg/kg, considerably larger than the dose used in our study, 10 mg/kg. The combined results from our study and the previous one suggest that a reduction of burn-induced mortality can be demonstrated when a large dose of ketamine is given in a model of severe burn injury but not when a smaller dose of ketamine is given after a less severe burn injury.
Inspection of Figure 1B may convey the impression that giving ketamine 24 hours after burn injury increased mortality despite statistical analysis indicating no significant difference between the group receiving ketamine and the group receiving saline. With statistical “power” of 0.85, there is a 3 in 20 chance that ketamine did worsen post-burn injury mortality and our finding of no difference between saline and ketamine represents a “false negative.” Ketamine could worsen outcome if there was some delayed effect (i.e., at 24 hours) of burn injury altering the anticipated cardiovascular and respiratory effects of ketamine (stimulation and minor depression/bronchodilation, respectively) to unanticipated effects (either excessive stimulation or severe depression, and severe depression/bronchoconstriction, respectively).
The finding that ketamine suppresses increase of IL-6 and/or improves survival after endotoxin administration is well established. Ketamine suppressed IL-6 after lipopolysaccharide (LPS) administration to isolated cells (3–5) and after E. coli inoculation in rats (11). Ketamine improved survival when given during inoculation with E. coli (12,13) and Pseudomonas aeruginosa (14). However, the present study is the first to report attenuation of the IL-6 surge by ketamine in burn injury.
The therapeutic significance of altering the concentration of IL-6, or any other cytokine, is uncertain. In major burns, there is an abundant release of proinflammatory mediators that contribute to macrophage hyperstimulation. The stimulated macrophage, which is necessary in the acute phase of burns, becomes an inhibitory macrophage after several days. Although it still produces IL-1 and IL-6, it also changes its co-signaling, such that lymphocytes respond differently. The stimulated macrophage also initiates the production of IL-10, which enhances the Th2 cascade and is by itself a global immunosuppressant. The end result of these processes is depression of the global immune proliferative and cytokine response (15).
Burn Injury and Sepsis
Burn injuries often lead to complications, including infection, sepsis, multiple organ failure, and, ultimately, death. During the past decades, the role of the immune system in modulating post-burn organ failure has received greater attention. It is generally accepted that an initial injury such as burn trauma alters immune function such that a secondary bacterial insult increases morbidity and mortality over that observed with either insult individually. Investigations have implicated the gut as a main source of the infection that occurs after burn injury (16). The treatment of both conditions, burn injury and sepsis, is still dependent largely on supportive hemodynamic and respiratory management. A specific treatment that would significantly improve the outcome of these lethal conditions has yet to be found.
It has been shown that there are differences between the two pathophysiologic states, sepsis and thermal injury, in signal transduction and cytokine expression (17,18). The large variability in inflammatory mediator expression between the two conditions may explain their different responses to ketamine treatment. In the past decade, researchers have identified the suppressors of cytokine signaling (SOCS) family of proteins, which act as intracellular inhibitors of several cytokine signal transduction pathways. Ogle et al. (19) showed that thermal injury causes increases in SOCS3 within 4 hours after a burn, reaching a maximum at 24 hours after injury. Levels continue to be elevated for up to 10 days after injury. They concluded that SOCS3 may regulate the balance between immunosuppression and inflammation after thermal injury (19). Our results indicate less activation of inflammatory processes in burn injury than in infection, as reflected by smaller IL-6 concentrations. Severe injury can initiate an exaggerated systemic inflammatory response and multiple organ failure if a subsequent immune stimulus, i.e., a second hit, occurs. Results from our model are consistent with this theory, as reflected by the worse outcome and the significantly larger IL-6 concentrations of animals that sustained burn injury and infection compared with those that sustained only burn injury. Ketamine treatment in the case of animals that sustained the two hits resulted in blunting of the highly exaggerated inflammatory response, which was ultimately beneficial to these subjects. Opposing responses of different inflammatory conditions to specific treatment have been documented. Kinoshita et al. (20) described how IL-8 therapy that had a beneficial effect on survival of burned mice with severe bacterial infection caused higher mortality rates in the immunocompromised hosts with mild infection.
Timing and Dose
This study emphasizes again the importance of the timing of ketamine treatment. Our finding that there was better survival and smaller IL-6 concentrations when ketamine was administered close to the insult, either burn injury or inoculation, is consistent with our previous results that there was a trend towards better survival with administration of ketamine 5 min after E. coli inoculation as compared to 2 hours after inoculation when signs of sepsis were overt (6). Other authors also reported that ketamine pretreatment more effectively suppressed increases of IL-6 and TNF-α after E. coli inoculation than did ketamine posttreatment (11). It is of potential clinical importance that the improved survival of infected animals was achieved despite the fact that the cumulative dose of ketamine in the present study was less than that used previously (10 mg/kg versus 50 mg/kg, respectively) and the insult the animals sustained was more serious (6).
In addition to attenuating endotoxin-induced increase of IL-6, a second major means by which ketamine may reduce endotoxin-induced tissue injury is via its effects on nitric oxide (NO). Inducible NO synthase (iNOS) has been implicated as a mediator of endotoxin-induced tissue injury (21). In a rat model in which serum and gastric fluid and mucosa were sampled, ketamine was reported to attenuate LPS–induced upregulation of iNOS mRNA and protein during endotoxemia (22). Ketamine also attenuated LPS-induced iNOS protein immunoreactivity/expression in the stomach, duodenum, jejunum, ileum, colon, liver, kidney, and spleen (21). These suppressive mechanisms occur not only by pretranslational inhibition of NOS expression but also by a posttranslational decrease in NOS activity (23). In the absence of endotoxemia, ketamine was reported to increase the density of reduced neuronal NOS-positive hippocampal interneurons (24).
Ketamine induces anesthetic and analgesic activities by blocking the N-methyl-d-aspartate receptor in the central nervous system and by activation of the NO-cyclic GMP pathway at the supraspinal, but not the spinal, level (4,25). Newer data indicate that in addition to these activities, ketamine has potent antiinflammatory effects. Although the anesthetic and analgesic activities of ketamine involve N-methyl-d-aspartate receptor antagonism, the mechanism of ketamine's antiinflammatory action was uncertain until recently. Recent work from our laboratory (8) suggests that adenosine is involved in the antiinflammatory effects of ketamine. We demonstrated that agonists of adenosine-2A (A2A) receptors produced ketamine-like effects on leukocyte recruitment and on TNF-α and IL-6 concentrations. In addition, administration of an A2A antagonist blocked the inhibitory effects of ketamine on the inflammatory process.
The present study is limited by measurement of a single proinflammatory marker and no measurement of antiinflammatory cytokines or elements of the NO pathway. As above, outcome from processes in which inflammation plays a major role is influenced by proinflammatory and antiinflammatory cytokines and elements of the NO pathway. Including determination of these mediators in future studies of burn injury and burn injury + sepsis would help clarify their relative importance to outcomes.
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© 2006 International Anesthesia Research Society
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