Joseph M. Fuentes

General anesthesia delays the inflammatory response and increases survival for mice with endotoxic shock.

ABSTRACT Anesthesia is an indispensable component of any operative procedure. In this study, we demonstrate that continuous isoflurane anesthesia for 1 h after a lethal dose (20 mg/kg of body weight) of Escherichia coli lipopolysaccharide (LPS) results in a significant increase in survival of C57BL/6J (B6) mice in comparison with survival of nonanesthetized mice. Protection by anesthesia correlates with a delay in plasma LPS circulation, resulting in a delayed inflammatory response, particularly DNA binding activity of NF-κB and serum levels of tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-10. Disparate classes of anesthetic agents produce the same effects on the inflammatory response, which is also independent of the inbred mouse strain used. These results suggest that anesthesia has an important impact on the outcome from endotoxemia. Moreover, the immunomodulatory effects of anesthetics should be considered when interpreting data from experimental animal models.

Carbon dioxide pneumoperitoneum prevents mortality from sepsis

BackgroundCarbon dioxide (CO2) pneumoperitoneum has been shown to attenuate the inflammatory response after laparoscopy. This study tested the hypothesis that abdominal insufflation with CO2 improves survival in an animal model of sepsis and investigated the associated mechanism.MethodsThe effect of CO2, helium, and air pneumoperitoneum on mortality was studied by inducing sepsis in 143 rats via intravenous injection of lipopolysaccharide (LPS). To test the protective effect of CO2 in the setting of a laparotomy, an additional 65 animals were subjected to CO2 pneumoperitoneum, helium pneumoperitoneum, or the control condition after laparotomy and intraperitoneal LPS injection. The mechanism of CO2 protection was investigated in another 84 animals. Statistical significance was determined via Kaplan– Meier analysis for survival and analysis of variance (ANOVA) for serum cytokines.Results Among rats with LPS-induced sepsis, CO2 pneumoperitoneum increased survival to 78%, as compared with using helium pneumoperitoneum (52%; p < 0.05), air pneumoperitoneum (55%; p = 0.09), anesthesia control (50%; p < 0.05), and LPS-only control (42%; p < 0.01). Carbon dioxide insufflation also significantly increased survival over the control condition (85% vs 25%; p < 0.05) among laparotomized septic animals, whereas helium insufflation did not (65% survival). Carbon dioxide insufflation increased plasma interleukin-10 (IL-10) levels by 35% compared with helium pneumoperitoneum (p < 0.05), and by 34% compared with anesthesia control (p < 0.05) 90 min after LPS stimulation. Carbon dioxide pneumoperitoneum resulted in a threefold reduction in tumor necrosis factor-α (TNF-α) compared with helium pneumoperitoneum (p < 0.05), and a sixfold reduction with anesthesia control (p < 0.001).ConclusionAbdominal insufflation with CO2, but not helium or air, significantly reduces mortality among animals with LPS-induced sepsis. Furthermore, CO2 pneumoperitoneum rescues animals from abdominal sepsis after a laparotomy. Because IL-10 is known to downregulate TNF-α, the increase in IL-10 and the decrease in TNF-α found among the CO2-insufflated animals in our study provide evidence for a mechanism whereby CO2 pneumoperitoneum reduces mortality via IL-10-mediated downregulation of TNF-α.

Abdominal insufflation with CO2 causes peritoneal acidosis independent of systemic pH

We have shown that the inflammation-attenuating effects of CO2 pneumoperitoneum during laparoscopy are not due to changes in systemic pH. However, acidification of peritoneal macrophages in an in vitro CO2 environment has been shown to reduce LPS-mediated cytokine release. We tested the hypothesis that the peritoneum is locally acidotic during abdominal insufflation with CO2deven when systemic pH is corrected. Rats (n = 20) were anesthetized and randomized into two groups: continued spontaneous ventilation (SV) or intubation and mechanical ventilation (MV). All animals were then subjected to abdominal insufflation with CO2. Mean arterial pH among SV rats decreased significantly from baseline after 15 and 30 minutes of CO2 pneumoperitoneum (7.329→7.210→7.191, P < 0.05), while arterial pH among MV rats remained relatively constant (7.388→7.245→7.316, P = NS). In contrast, peritoneal pH dropped significantly from baseline and remained low for both groups during CO2 abdominal insufflation (SV 6.74 → 6.41 → 6.40, P < 0.05; MV 6.94 → 6.45 → 6.45, P < 0.05). In a second experiment, rats (n = 10) were randomized to receive abdominal insufflation with either CO2 or helium. Abdominal insufflation with helium did not significantly affect peritoneal pH (7.10 → 7.02 →6.95, P = NS), and the decrease in pH among CO2-insufflated animals was significant compared with helium-insufflated animals (P < 0.05). Peritoneal pH returned to baseline levels in all groups within 15 minutes of desufflation in both experiments. A significant local peritoneal acidosis occurs during laparoscopy which is specifically attributable to the use of CO2 and which is independent of systemic pH. These data provide additional evidence that localized peritoneal acidosis is central to the mechanism of CO2-mediated attenuation of the inflammatory response following laparoscopic surgery.

CO2 Abdominal Insufflation Pretreatment Increases Survival After a Lipopolysaccharide-Contaminated Laparotomy

Carbon dioxide (CO2)-pneumoperitoneum is known to favorably modify the systemic immune response during laparoscopic surgery. The presented studies were designed to determine whether treating animals with CO2 abdominal insufflation before undergoing a lipopolysaccharide (LPS)-contaminated laparotomy would serve as “shock prophylaxis” and thus improve survival and attenuate cytokine production. Rats were randomized into five groups: CO2-pneumoperitoneum, helium-pneumoperitoneum, anesthesia control, laparotomy/LPS control, and LPS only control. Animals in the first four groups all received a laparotomy and a lethal dose of LPS. Immediately preceding their laparotomy, animals in the pneumoperitoneum groups received a 30-minute pretreatment of abdominal insufflation with either CO2 or helium. The anesthesia control group received a 30-minute pretreatment of isoflurane. Animal mortality was then recorded during the ensuing 72 hours. Subsequently, a similar protocol was repeated for measurements of cytokines. CO2-pneumoperitoneum increased survival at 48 hours compared with LPS control (P<.05), and decreased interleukin-6 plasma levels at 2 hours (P<.05). Abdominal insufflation with CO2 before the performance of a laparotomy contaminated with endotoxin increases survival and attenuates interleukin-6. The beneficial immune-modulating effects of CO2-pneumoperitoneum endure after abdominal insufflation. CO2-pneumoperitoneum pretreatment may improve outcomes among patients undergoing gastrointestinal surgery who are at high risk for abdominal fecal contamination.

PROTECTION FROM LETHAL ENDOTOXIC SHOCK AFTER TESTOSTERONE DEPLETION IS LINKED TO CHROMOSOME X

Men are considered more susceptible to sepsis after severe injury than are women, which has been attributed to a suppressing effect of male sex steroids on the inflammatory response. Moreover, the effect of sex steroids on the inflammatory process depends on the genetic background. The present study examined the genetic contribution to survival after endotoxic shock in mice depleted of testosterone by surgical castration. Six-week-old male mice, from strains A/J, AKR/J, C57BL/6J (B6), BALBc/J, DBA/2J, and C3H/HeN, were castrated (CX) or nonoperated (NoOp). Two weeks after surgery, mice were injected intraperitoneally with Escherichia coli lipopolysaccharide (15 mg/kg) and the frequency of mortality was monitored. CX A/J mice showed a significantly higher survival rate than NoOp mice, but this protective effect was not observed in the other strains. Administration of 5-α-dihydrotestosterone to CX A/J mice reverted the protection by CX. The protective effect of CX was also observed in crosses of female A/J and male B6 (AXB), but not female B6 and male A/J (BXA), suggesting that protection is linked to the A/J X chromosome. This possibility was corroborated by using consomic mice containing A/J chromosome X and the remaining chromosomes from B6. These results suggest that testosterone is a negative factor in the recovery from endotoxic shock, depending on the genetic background.

Atropine treatment modifies LPS-induced inflammatory response and increases survival

Abstract.Objective:To explore the effect that Atropine, a competitive antagonist for the muscarinic acetylcholine receptor (mAChR), has on the response to LPS.Subjects:Eight-week-old, male, B6 mice.Treatment:Mice were treated with Atropine prior to, or after LPS challenge.Methods:Survival was monitored and analyzed via Kaplan-Meier analysis using the log-rank test. The effects of atropine on the inflammatory response (TNF-α, IL-6 and IL-10) were monitored at various time intervals following LPS injection in mice that were treated and not treated with atropine.Results:Atropine administration prior to LPS induction of the inflammatory response resulted in reduced TNF-α and elevated IL-10 plasma levels without affecting the production of IL-6. This reduction in TNF-α levels was independent of the increase in IL-10 production. Atropine pretreatment improved the rate of survival from endotoxic shock in mice. The improved survival of mice after endotoxic shock could still be observed when atropine was administered several hours after LPS injection.Conclusion:The administration of atropine after injury may have a beneficial clinical effect.

Laparoscopic surgery and the parasympathetic nervous system

BackgroundLaparoscopic surgery preserves the immune system and has anti-inflammatory properties. CO2 pneumoperitoneum attenuates lipopolysaccharide (LPS)-induced cytokine production and increases survival. We tested the hypothesis that CO2 pneumoperitoneum mediates its immunomodulatory properties via stimulation of the cholinergic pathway.MethodsIn the first experiment, rats (n = 68) received atropine 1 mg/kg or saline injection 10 min prior to LPS injection and were randomization into four 30-min treatment subgroups: LPS only control, anesthesia control, CO2 pneumoperitoneum, and helium pneumoperitoneum. In a second experiment, rats (n = 40) received atropine 2 mg/kg or saline 10 min prior to randomization into the same four subgroups described previously. In a third experiment, rats (n = 96) received atropine 2 mg/kg or saline 10 min prior to randomization into eight 30-min treatment subgroups followed by LPS injection: LPS only control; anesthesia control; and CO2 or helium pneumoperitoneum at 4, 8, and 12 mmHg. In a fourth experiment, rats (n = 58) were subjected to bilateral subdiaphragmatic truncal vagotomy or sham operation. Two weeks postoperatively, animals were randomized into four 30-min treatment subgroups followed by LPS injection: LPS only control, anesthesia control, CO2 pneumoperitoneum, and helium pneumoperitoneum. Blood samples were collected from all animals 1.5 h after LPS injection, and cytokine levels were determined by enzyme-linked immunosorbent assay.ResultsSerum tumor necrosis factor-α (TNF-α) levels were consistently suppressed among the saline–CO2 pneumoperitoneum groups compared to saline–LPS only control groups (p < 0.05 for all four experiments). All chemically vagotomized animals had significantly reduced TNF-α levels compared to their saline-treated counterparts (p < 0.05 for all), except among the CO2 pneumoperitoneum-treated animals. Increasing insufflation pressure with helium eliminated differences (p < 0.05) in TNF-α production between saline- and atropine-treated groups but had no effect among CO2 pneumoperitoneum-treated animals. Finally, vagotomy (whether chemical or surgical) independently decreased LPS-stimulated TNF-α production in all four experiments.ConclusionCO2 pneumoperitoneum modulates the immune system independent of the vagus nerve and the cholinergic pathway.