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On-line version ISSN 1806-907X
Rev. Bras. Anestesiol. vol.58 no.5 Campinas Sept./Oct. 2008
Effects of peritoneal lavage with bupivacaine on survival of mice with fecal peritonitis*
Efecto del lavado peritoneal con bupivacaína en la sobrevida de ratones con peritonitis fecal
Marcos Célio Brocco, TSAI; Danilo Nagib Salomão PauloII; João Florêncio de Abreu BaptistaIII; Antônio Roberto Carraretto, TSAIV; Thiago Antunes FerrariV; Thiago Caetano V. de AzevedoV; Alcino Lázaro da SilvaVI
Adjunto IV de Anestesiologia da UFES; Mestrando em Cirurgia da Universidade
Federal de Minas Gerais (UFMG)
IIProfessor Titular de Cirurgia da EMESCAM
IIIProfessor Adjunto IV de Anestesiologia da UFES, Mestre em Cirurgia pela UFMG; Anestesiologista do Hospital Universitário Cassiano Antônio de Moraes Vitória, ES
IVProfessor de Anestesiologia da UFES, Mestre em Anestesiologia pela Faculdade de Medicina de Botucatu UNESP; Responsável CET Integrado HUCAM-HAFPES
VEstudante do 10º período do Curso de Medicina da EMESCAM
VIProfessor Emérito de Cirurgia da Faculdade de Medicina da UFMG
OBJECTIVES: Based on the knowledge of the anti-inflammatory and anti-bacterial
actions of local anesthetics (LA), the objective of this study was to determine
the effects of peritoneal lavage with bupivacaine on survival of mice with fecal
METHODS: Forty-eight Wistar mice, weighing between 300 and 330 g (311.45 ± 9.67 g), undergoing laparotomy 6 hours after induction of peritonitis were randomly divided in 4 groups: 1 - Control, without treatment (n = 12); 2 - Drying of the abdominal cavity (n = 12); 3 - Lavage with 3 mL NS and posterior drying of the abdominal cavity (n = 12); and 4 - Lavage with 8 mg.kg-1 (± 0.5 mL) of 0.5% bupivacaine added to 2.5 mL of NS followed by drying out of the abdominal cavity (n = 12). Animals that died underwent necropsy and the time of death was recorded. Surviving animals were killed on the 11th postoperative day and underwent necropsy.
RESULTS: Group 1 presented a 100% mortality rate in 52 hours, 100% mortality rate in Group 2 in 126 hours, and Group 3 presented a 50% mortality rate in 50 hours. Animals in Group 4 survived. Survival on the 11th day was greater in groups 3 and 4 than in Groups 1 and 2 (p < 0.001) and greater in Group 4 than in Group 3 (p < 0.01).
CONCLUSIONS: Peritoneal lavage with a solution of bupivacaine diluted in NS was effective in preventing death for 11 days in 100% of animals with fecal peritonitis.
Key Words: ANESTHETIC, Local: bupivacaine; ANIMALS: mice; COMPLICATIONS, Infection: peritonitis.
Y OBJETIVOS: Basados en los conocimientos sobre la acción antiinflamatoria
y antibacteriana de los anestésicos locales (AL), el estudio tuvo el
objetivo de verificar el efecto del lavado peritoneal con solución de
bupivacaína en la sobrevida de ratones con peritonitis fecal por heces
MÉTODO: Se usaron 48 ratones de la raza Wistar, con peso entre 300 g y 330 g (311,45 ± 9,67), sometidos a la laparotomía 6 horas después de la inducción de peritonitis, distribuidos aleatoriamente en 4 grupos: 1 - Control, ningún tratamiento (n = 12); 2 - Secado de la cavidad abdominal (n = 12); 3 - Lavado de la cavidad abdominal con 3 mL de solución fisiológica a 0,9% y secado (n = 12); 4 - Lavado de la cavidad abdominal con 8 mg.kg-1 (± 0,5 mL) de bupivacaína 0,5%, adicionada a 2,5 mL de solución fisiológica a 0,9% y secado (n = 12). Los animales que murieron fueron llevados a necropsia y el horario del óbito se anotó. Los animales sobrevivientes se sacrificaron al 11º día del postoperatorio y se realizó la necropsia.
RESULTADOS: Hubo un 100% de mortalidad en los animales del Grupo 1 en 52 horas, 100% en los animales del Grupo 2, en 126 horas y un 50% en los animales del Grupo 3 en 50 horas. Los animales del Grupo 4 sobrevivieron. La sobrevida, al 11° día del postoperatorio, fue mayor en los grupos 3 y 4 con relación a los grupos 1 y 2 (p < 0,001) y mayor en los Grupo 4 con relación al Grupo 3 (p < 0,01).
CONCLUSIONES: El lavado peritoneal con solución de bupivacaína diluida en solución fisiológica fue eficaz para evitar el óbito, por 11 días, en un 100% de los animales con peritonitis fecal.
Despite advances in the treatment of peritonitis, its mortality rate has not decreased in the last two decades 1. The mortality rate increases when multiple organ and system failure are present. This dysfunction, although its pathogeny has not been elucidated, seems to be secondary to a complex inflammatory complex. The septic response is associated with the release of anti-inflammatory and proinflammatory cytokines 12-4 with subsequent activation of leukocytes, and complement and coagulation cascades 5, besides the production of antibodies and bacterial destruction by polymorphonuclear cells 6. Mediators, such as TNF-alpha, interleukins (IL-1 beta, IL-6, and IL-8), and nitric oxide (NO) have a fundamental role in sepsis, and inflammatory mediators are present concomitantly, modulating the effects and release of inflammatory mediators 7.
Local anesthetics have shown to be effective in modulating the inflammatory cascade during ischemia and reperfusion of the heart 8,9, lungs 1-,11, and liver 2,13. They have an anti-inflammatory action in several types of cells, including monocytes, macrophages, and neutrophils. Ropivacaine reduced the pulmonary inflammatory response caused by lipopolysaccharides in mice 15. Lidocaine, at 1%, and 0.5% bupivacaine demonstrated to be effective on preventing peritonitis induced by 0.1 M of hydrochloric acid when compared with NS 16. The mortality of mice with septic peritonitis induced according to an experimental model 17 treated with the subcutaneous administration of 5% and 10% lidocaine and 1% and 2% bupivacaine with an infusion pump was reduced and protected against hepatic and renal dysfunctions, by attenuating the hyper-inflammatory response 18. Besides, some anesthetics have a bactericidal action against some bacteria in vitro 19-20. Based on those studies, it was questioned whether the application of intra-peritoneal local anesthetic could interfere with survival of animal with peritonitis. Thus, the objective of the present study was to determine the effects of the infusion of bupivacaine on the abdominal cavity of mice with induced fecal peritonitis.
This study was approved by the Ethics on Research Committee of the Faculdade de Medicina da Universidade Federal de Minas Gerais (UFMG), according to protocol number 144/06 (COEP-CETEA).
Forty-eight Wistar mice, weighing 300 g to 330 g (311.45 ± 9.67), provided by the Biotherium of the Escola Superior de Ciências da Santa Casa de Misericórdia de Vitória, ES (EMESCAM) were used.
Animals were anesthetized with 12.5 mg.kg-1 of S(+)-ketamine hydrochloride, the left inferior quadrant of the abdomen was punctured with a 16G catheter, and the animals were weighed on an electronic scale with a 1 g sensitivity. Peritonitis was induced by the intrabadominal injection of 5 mL.kg-1 of a suspension of 2 g of feces recently defecated diluted in 17 mL of NS and filtered through a 4 × 4 bandage to allow its free flow through the needle. Six hours after induction of peritonitis, mice were anesthetized with a mixture of 2.5 mg.kg-1 of xylazine chloride with 25 mg.kg-1 of S(+)-ketamine hydrochloride, and underwent median laparotomy approximately 2 cm long, assessment of the cavity, collection of 0.5 mL of secretion for bacterioscopy, and culture and sensitivity. At this moment, animals were randomly distributed in four groups (Table I).
Group 3 received 3 mL of NS; and Group 4, 8 mg.kg-1 of 0.5% bupivacaine (± 0.5 mL) mixed with 2.5 mL of NS, for a total volume of 3 mL.
In groups 3 and 4, NS, with or without anesthetic, remained in the abdominal cavity for three minutes. During this period, the solution was manually distributed among the abdominal organs, to allow greater contact with the peritoneum. Afterwards, the peritoneal fluid was dried out with gauze pads to remove as much as possible of the solution. The abdominal wall was sutured in two planes using continuous running suture with 4.0 nylon monofilament. The muscular-aponeurotic plane was sutured followed by the skin. In the postoperative period, all animals were hydrated with a single subcutaneous dose of 10 mL of NS every 24 hours for two days. Subcutaneous nalbufine chlorhydrate, 0.1 mg.kg-1 every 8 hours for two days, was used for analgesia.
Animals that died underwent necropsies and the time of death was recorded. Surviving animals were killed on the 11th postoperative day with the administration of 50 mg.kg-1 of sodium pentobarbital in the abdominal cavity. At this time, the abdominal cavity, possible adhesions, and foci of infection were examined, and material was collected for bacteriological studies. Adhesions were classified in six degrees 21 (Tale II).
Mean survival, number of survivors in each group, and survival curve were analyzed. For such, non-parametric studies were used to compare length of survival (Student t test for independent samples), number of survivors (Fisher's exact test), and the survival by the Kaplan Meier curve with the Log Rank test. A p < 0.05 was considered significant.
Laparotomy performed 6 hours after abdominal puncture and injection of fresh feces showed edema, hyperemia among the intestinal loops, and the presence of purulent secretion in the abdominal cavity.
Bacteria isolated from the peritoneal fluid at the time of laparotomy included: Proteus mirabilis, Klebsiela pneumoniae, Enterococcus faecalis, Escherichia coli, Micrococcus, Proteus penneri, Enterococcus gallinarum, Staphylococcus sciuri, Bacillus species, Staphylococcus epidermidis, and Aerococcus viridans. Chart I shows the sensitivity of those bacteria to antibiotics.
Mice that survive the postoperative period were dynamic, ingested liquid meals, and the exam of the abdominal cavity showed 2nd and 3rd degree adhesions. Mice that died presented apathy, erected hairs, dark circles around their eyes, tachycardia, and anorexia. Exam of the abdominal cavity revealed a small amount of purulent secretion and loose adhesions between the intestinal loops, classified as 0 and 1st degree adhesions (Table I).
Animal survival was more frequent in Group 4 than in Group 3 (p < 0.01) and than in Groups 2 and 1 (p < 0.001) (Table III).
The survival curve demonstrated 100% mortality in Group 1 in 52 hours, 100% mortality in Group 2 in 126 hours, and 50% mortality in Group 3 in 50 hours. (The log-Rank test demonstrated a significant increase in the survival curve in Group 4 when compared with Group 3 (p < 0.01) and when compared with groups 2 and 1 (p < 0.001), and between Groups 3 and 1 (p < 0.001); there were no statistically significant differences between Groups 1 and 2 (Figure 1).
In the present study, lavage of the peritoneal cavity with bupivacaine solution avoided the death of animals subjected to autologous fecal peritonitis. A similar result was observed when the peritoneal cavity was bathed in antibiotics to treat Escherichia coli peritonitis 22. This did not happen when the peritoneal cavity with peritonitis was not treated (control Group), or even when it was dried out (Group 2) and dried out and flushed with NS (Group 3). Group 3 had a greater survival rate than the control group. This shows that lavage was beneficial in this model of peritonitis. Although it is the subject of controversy, peritoneal lavage in peritonitis is used by a large number of surgeons 23,24. Peritoneal lavage with NS in mice with peritonitis resulted in decreased mortality when compared with just cleaning the abdominal cavity with gauze pads. In group 4, the addition of bupivacaine to NS was effective in the treatment of peritonitis. Basically, local anesthetics behave as anti-inflammatories. Continuous subcutaneous infusion of 5% and 10% lidocaine and 1% and 2% bupivacaine with a pump reduced the mortality of mice with peritonitis 18. It has been shown that local anesthetics are effective modulators of the inflammatory cascade in ischemia and reperfusion of the heart 8,9, lungs 10, and liver 12, and they have an anti-inflammatory action in different types of cells, including monocytes, macrophages, and neutrophils 14. Ropivacaine attenuated the pulmonary inflammatory response to lipopolysaccharides in mice 15. Lidocaine at 1% and 0.5% bupivacaine were also capable to prevent peritonitis caused by 0.1 M of hydrochloric acid 16. Activation of the coagulation cascade has been associated with the development of multiple organ failure, with a poor prognosis in septic patients. This is probably due to disseminated intravascular coagulation that compromises the blood flow vital for organ survival, resulting in organic failure and death 25. In the present study, this was not demonstrated in animals treated with local anesthetic solution because they all survived.
Besides the anti-inflammatory action, some local anesthetics are bactericidal against some bacteria in vitro 19,20. In this study, the sensitivity of the bacteria isolated from the abdominal cavity was not done because in a pilot study it was not possible to reach a conclusion regarding the bacteriostatic and bactericidal actions of those drugs. Therefore, besides the probable anti-inflammatory actions of local anesthetics, one should consider that drying out of the abdominal cavity of animals in Group 4 is also a treatment for peritonitis because it removes bacteria and toxins.
In surviving animals, their abdominal cavities showed the presence of more severe adhesions than in animals that died. Adhesions work by isolating septic processes and protect the organism against bacteremia. The inhibition of adhesion formation is accompanied by increased mortality secondary to the generalized intra-abdominal septic process 26.
Animals in the control group and groups 2 and 3 that died presented, in the immediate postoperative period, manifestations of sepsis, such as: tachypnea, anorexia, apathy, erection of their fur, and dark circles around their eyes, similar to the report of Guilgen 27. Animals that survived were active and fed themselves. In a pilot study, the authors observed that mice that survived until the 10th day did not die as a consequence of peritonitis. For this reason, we used the 10-day period for the macroscopic study of the abdominal cavity (post-mortem) and as a parameter to for the statistical analysis of animal survival. Considering that there were no statistically significant differences among the weights of the animals in all four groups, animals were all from the same lineage, and the same peritonitis technique was used, one can compare the survival among the different groups.
The dose of local anesthetic used was minimal if one considers that the mean lethal dose (LD50) of intraperitoneal bupivacaine is 57.7 to 58.7 mg.kg-1 28. In the peritoneal lavage performed after first drying out the abdominal cavity, the local anesthetic was spread manually to guarantee the greatest contact possible of the drug with the abdominal organs. The anesthetic solution remained for three minutes in the abdominal cavity to allow enough time for its action. The second drying out of the abdominal cavity was done with smooth movements, avoiding the complete removal of the solution. This method was satisfactory since there were no deaths in Group 4. Other studies can be done to evaluate other local anesthetics in other models of peritonitis, associated with other therapeutic resources or not. New studies will also be able to be conducted to determine the effects of each local anesthetic on bacteria that cause peritonitis, on organ function, and on the inflammatory reaction produced before and after the use of those drugs. In face of peritonitis, surgeons have shown concerns with the degree of compromise of mesothelial cells (peritoneum) and the real value of peritoneal lavage.
Peritoneal lavage with bupivacaine diluted in NS was effective in avoiding death in animals with induced autologous peritonitis.
We would like to acknowledge the Clinical and Experimental Research Supporting Department of the Instituto de Desenvolviment Sustentável (Instituto Solidário do Espírito Santo) for its financial support.
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Correspondence to: Submitted em 16
de março de 2008 *
Received from Escola de Medicina da Santa Casa de Misericórdia de Vitória
(EMESCAM) e Universidade Federal do Espírito Santo (UFES), Vitória,
Dr. Marcos Célio Brocco
Rua Pedro Luis Zanandréa, 55 - Mata da Praia
29065-610 Vitória, ES
Accepted para publicação em 23 de junho de 2008
Submitted em 16
de março de 2008
* Received from Escola de Medicina da Santa Casa de Misericórdia de Vitória (EMESCAM) e Universidade Federal do Espírito Santo (UFES), Vitória, ES