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Original Article |

Increased Systemic Inflammation After Laparotomy vs Laparoscopy in an Animal Model of Peritonitis FREE

C. A. Jacobi, MD; J. Ordemann, MD; H. U. Zieren, PhD; H. D. Volk, Prof; A. Bauhofer, PhD; E. Halle, PhD; J. M. Müller, Prof
[+] Author Affiliations

From the Department of Surgery (Drs Jacobi, Ordemann, Zieren, and M[[uuml]]ller), the Institute of Medical Immunology (Dr Volk), and the Institute of Microbiology (Dr Halle), University of Berlin, Berlin, Germany; and the Institute of Theoretical Surgery (Dr Bauhofer), University of Marburg, Marburg, Germany.


Arch Surg. 1998;133(3):258-262. doi:10.1001/archsurg.133.3.258.
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Published online

Objective  To study the influence of laparotomy and laparoscopy on local and systemic inflammation in a rat model of peritonitis.

Design  Bacteremia, peripheral leukocyte subpopulations, tumor necrosis factor α (TNF-α) plasma levels, and ex vivo secretion of peripheral blood mononuclear cells were investigated after laparotomy and laparoscopy in a prospective randomized experimental study.

Setting  Surgical department of a university hospital.

Animals  60 male inbred Wistar rats.

Interventions  Standardized fecal inoculum was injected intraperitoneally and rats underwent laparotomy (n=20), laparoscopy (n=20), or no further manipulation (control group, n=20). Blood samples were obtained during the perioperative course to determine bacteremia, leukocytic subpopulations, TNF-α plasma levels, and ex vivo secretion. The number of intraperitoneal abscesses was determined in each animal after 1 week.

Main Outcome Measure  The hypothesis of the experiment was that laparoscopy with carbon dioxide leads to an increase of local and systemic inflammation in comparison with the laparotomy and control groups.

Results  One hour after intervention, bacteremia was significantly higher in the laparotomy and laparoscopy groups compared with the control group (P=.01). Fecal inoculum caused significant monocytopenia and lymphocytopenia in all groups within 1 hour after intervention (P<.05), with complete recovery on day 2 only in the laparoscopy and control groups. Laparotomy caused a significant increase in TNF-α plasma levels and decrease of ex vivo production of TNF-α compared with the other 2 groups (P<.05).

Conclusions  Laparotomy and laparoscopy increased the incidence of bacteremia and systemic inflammation in this peritonitis model. The inflammatory response was significantly higher in the laparotomy group compared with the laparoscopy group.

Figures in this Article

LAPAROSCOPIC SURGERY is increasingly used for intra-abdominal diseases complicated by inflammatory processes and peritonitis. Successful treatment of appendicitis, perforated peptic ulcer, and diverticulitis has been reported with low morbidity.15 However, a theoretical concern is that elevated intra-abdominal pressure may promote bacteremia and systemic inflammatory response during laparoscopic surgery. Data regarding the effects of pneumoperitoneum on physiological changes and systemic inflammation during sepsis are scarce and controversial.6,7 Furthermore, only early effects of pneumoperitoneum during peritonitis have been evaluated and the differences between conventional and laparoscopic surgery have not been compared in all studies.

Therefore, the influence of laparotomy and laparoscopy on bacteremia, peripheral leukocytic subpopulations, proinflammatory tumor necrosis factor α (TNF-α) plasma levels, and ex vivo secretion of peripheral blood mononuclear cells (PBMCs) was studied in a rat model.

EXPERIMENTAL COURSE

Sixty male inbred 2-month-old Wistar rats (Charles River, Sulzfeld, Germany) were acclimated to a climate- and light cycle–controlled environment for at least 7 days prior to investigations. The animals were allowed standard laboratory food and water ad libitum. All animals were initially anesthetized by intraperitoneal injection of pentobarbital (60 mg/kg) under sterile conditions. A standardized fecal inoculum (1×0.5 mL/kg) was applied intraperitoneally under sterile conditions in all animals. The preparation has been previously described and has been shown to produce a nonfatal bacterial peritonitis after intraperitoneal instillation in rats.8

The rats were randomized into 3 different groups. In the first group (n=20), laparotomy was performed and the abdomen was closed after 30 minutes. In the second group (n=20), laparoscopy was performed with insufflation of carbon dioxide at a pressure of 8 mm Hg over a period of 30 minutes. The control group (n=20) underwent no further manipulation after stool injection. Blood samples were obtained from the femoral artery and placed into sterile heparinized vials (pyrogen-free) 2 days before, 1 hour after, and 2 and 7 days after fecal inoculation. Leukocyte subpopulations were determined with a differential blood smear and plasma was separated from blood samples by centrifugation at 3000g at 4°C for 10 minutes to determine TNF-α plasma levels using commercially available enzyme immunoassays (Laboserv, Gießen, Germany). Additionally, 50 µL of blood from each sample was used to measure the ex vivo TNF-α secretion by PBMCs. Microbiological analysis of the blood was performed before, 1 hour after, and 7 days after intervention. All animals underwent laparotomy on day 7 to determine the number of intraperitoneal abscess formations. All studies were performed under protocols approved by the local committees of animal use and care.

TNF-α PLASMA LEVELS AND TNF-α EX VIVO SECRETION BY PBMCs

Plasma was separated from blood samples by centrifugation at 3000g at 4°C for 10 minutes immediately after withdrawal. Tumor necrosis factor α serum levels were determined by using commercially available enzyme immunoassays (Laboserv). Additionally, 50 µL of blood from each sample was diluted with 200 µL of fetal calf serum–supplemented (2% vol/vol) RPMI 1640 (Biochrom, Berlin, Germany) and incubated for 4 hours (37°C, 5% carbon dioxide) with 100 ng/mL of endotoxin Escherichia coli 0127:B8 (Sigma, Deisenhofen, Germany) to assess the ex vivo TNF-α secretion of PBMCs. Cell-free supernatants were harvested by centrifugation at 3000g at 4°C for 10 minutes. All samples were stored in 2-mL pyrogen-free polypropylene screw-cap tubes (Sarstedt, Numbrecht-Rommelsdorf, Germany) at −85°C until final analysis.

MICROBIOLOGICAL ANALYSIS

The microorganisms were grown on chocolate agar (tryptic soy agar supplemented with 10% defibrinated sheep blood, heated for 10 minutes to 80°C), blood agar (Columbia agar supplemented with 5% defibrinated sheep blood), Endo agar, and Sabouraud agar in both an aerobic and anaerobic atmosphere. The phenotypical identification of all strains was carried out by testing the carbohydrate fermentation reactions or by using commercially available enzyme activity and fermentation tests (API, Bio Mérieux, Nürtingen, Germany).

STATISTICS

Data are given as mean±SD. Data between groups were compared using the Kruskal-Wallis test for continuous data and the Fisher exact test for categorical data, if appropriate. P<.05 was considered significant.

MICROBIOLOGICAL ANALYSIS

The number of blood cultures positive for organisms was increased in the laparotomy and laparoscopy group as compared with the control group (P=.01) 1 hour after stool application (Table 1). There was no difference in bacteremia between the groups 1 week after fecal inoculum and surgical intervention.

Table Graphic Jump LocationTable 1. Number of Positive Blood Cultures After 1 Hour and 7 Days

Microbiological analysis of blood cultures detected 12 different bacteria species after laparotomy, 7 species after laparoscopy, and 7 species in the control group (Table 2).

Table Graphic Jump LocationTable 2. Bacterial Species in Blood Cultures 1 Hour and 7 Days After Endotoxin Application*
LEUKOCYTE SUBPOPULATIONS

Anesthesia and fecal inoculum caused a significant leukocytopenia 1 hour after intervention in all groups (Figure 1). Leukocyte counts recovered on day 2 in the laparoscopy and control groups (P=.02), while they did not recover in the laparotomy group during the entire postoperative course. In contrast to the laparotomy group, laparoscopy and control groups showed a decrease of leukocyte counts in the late postoperative course (P=.01). Decrease and recovery of the leukocyte count was mainly caused by a change in the number of lymphocytic and monocytic cells (Figure 2 and Figure 3). Decrease of monocytes and lymphocytes was significantly higher after laparotomy compared with laparoscopy and in the control group 1 hour after intervention. Neutrophil granulocyte counts increased in the postoperative course (Figure 4). While rats in the laparoscopic and laparotomy groups showed a significant increase 1 hour after fecal inoculum and surgery, fecal inoculum alone caused only a slight increase of neutrophil granulocytes on day 2, with complete recovery after 1 week. Early granulocytosis after laparoscopy or laparotomy was followed by complete recovery on day 2 and a second increase on day 7.

Place holder to copy figure label and caption
Figure 1.

Perioperative leukocyte counts in laparotomy, laparoscopy, and control groups (n=20 in each group; mean±SD). Asterisks indicate P<.05, laparotomy group vs laparoscopy and control groups.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Perioperative lymphocyte counts in laparotomy, laparoscopy, and control groups (n=20 in each group; mean±SD). Asterisks indicate P<.05, laparotomy group vs laparoscopy and control groups.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

Perioperative monocyte counts in laparotomy, laparoscopy, and control groups (n=20 in each group; mean±SD). Asterisks indicate P<.05, laparotomy group vs laparoscopy and control groups.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Perioperative neutrophil granulocyte counts in laparotomy, laparoscopy, and control groups (n=20 in each group; mean±SD). Asterisk indicates P<.05, laparotomy and laparoscopy groups vs control group.

Graphic Jump Location
TNF-α PLASMA LEVELS AND TNF-α EX VIVO SECRETION BY PBMCs

Plasma levels of TNF-α sharply increased 1 hour after fecal inoculum and intervention in all groups (Figure 5). Levels were significantly higher after laparotomy compared with laparoscopy and the control group at this time. There were no group differences in the plasma levels of TNF-α on days 2 and 7.

Place holder to copy figure label and caption
Figure 5.

Mean tumor necrosis factor α (TNF-α) levels in blood plasma after laparotomy and laparoscopy and in the control group (n=20 in each group; mean±SD). Asterisk indicates P<.05, laparotomy group vs the laparoscopy and control groups.

Graphic Jump Location

One hour after intervention, the TNF-α ex vivo production of PBMCs decreased significantly in all groups, with significantly lower levels after laparotomy compared with laparoscopy and in the control group (Figure 6). On postoperative days 2 and 7 there was no significant difference among the 3 groups.

Place holder to copy figure label and caption
Figure 6.

Mean tumor necrosis factor α (TNF-α) ex vivo secretion of peripheral blood mononuclear cells after laparotomy and laparoscopy and in the control group (n=20 in each group; mean±SD). Asterisk indicates P<.05, laparotomy group vs laparoscopy and control groups.

Graphic Jump Location
DEVELOPMENT OF INTRAPERITONEAL ABSCESSES

Intraperitoneal abscess formation was detected in all 20 rats after laparotomy, in 15 of 20 rats after laparoscopy, and in 18 of 20 rats in the control group 1 week after inoculation. The mean number of abscesses in each animal was significantly higher (P<.05) after laparotomy (10±6.2) compared with laparoscopy (6±5.1) and the control group (5±4.8).

Laparoscopic techniques are being increasingly used in surgical conditions complicated by diffuse or localized peritonitis. It has been demonstrated that laparoscopic is superior to conventional open appendectomy in terms of postoperative complications and recovery in prospective randomized trials.1,3,4 Further, laparoscopic management of perforated peptic ulcers has been reported to be simple and followed by a short recovery time.5 In rabbits, Gurtner et al6 did not find an increase of bacteremia or endotoxemia after pneumoperitoneum of 12 mm Hg. In comparison with these results, experimental studies in rats showed a significant increase of extent and severity of peritonitis and bacteremia after laparoscopy and ulcus perforation compared with the control group.9 Unfortunately, a comparison between laparoscopy and laparotomy was not performed in this experiment. In our study, laparoscopy was superior to laparotomy in terms of postoperative systemic inflammation as well as intraperitoneal abscess formation.

The nonfatal peritonitis produced in the rats resembles the clinical situation8 and the spectrum of bacterial species (E coli, Enterococcus faecalis, Staphylococcus aureus ) found in blood cultures of the rats were similar to what is commonly detected in patients with abdominal sepsis.10,11

The number of blood cultures positive for organisms was significantly higher after laparoscopy as compared with the control group 1 hour after intervention. It has been demonstrated that elevated intra-abdominal pressure leads to an increase of the patency of lymphatic openings through which intra-abdominal fluids are removed from the peritoneal cavity.12 This may explain the difference of bacteremia between these 2 groups. However, aerobic bacteria were not found after 1 hour in the laparoscopy or control groups. Since carbon dioxide is bacteriostatic on aerobic bacteria,13 this may explain why aerobic bacteria were only detected in blood cultures after laparotomy at this time.

Although there was a higher incidence of bacteremia after laparoscopy, systemic inflammation was only slightly increased. While the neutrophil granulocyte count was significantly higher 1 hour after fecal inoculum after laparoscopy compared with the control group, there was no difference between the other leukocytic subpopulations between these groups during the postoperative course. Plasma levels of TNF-α and ex vivo secretion of PBMCs showed almost parallel changes in the postoperative course, indicating that rats had similar systemic inflammation in both groups. It seems that laparoscopy does not significantly promote systemic inflammation compared with controls, which is also confirmed by the number of intraperitoneal abscess formations showing no difference between the 2 groups.

In contrast to laparoscopy, laparotomy caused a significant increase of systemic inflammation in the early postoperative course. While leukocyte counts recovered on day 2 in the laparoscopy and control groups, they did not recover after laparotomy and the postoperative decrease of monocytes and lymphocytes was significantly higher after laparotomy. In parallel to the higher incidence of bacteremia, TNF-α plasma levels showed significantly higher values and TNF-α secretion by PBMCs was mostly decreased in the laparotomy group 1 hour after intervention. It may be that abdominal incision causes greater tissue trauma, leading to an increase of proinflammatory cytokines and enhanced postoperative systemic inflammation. In patients undergoing colectomy, systemic inflammation was significantly higher after an open approach than a laparoscopic procedure, which is due to less trauma in the laparoscopic group.14 This hypothesis is also supported by the histopathological findings in this study. Intraperitoneal abscess formation was detected in all 20 rats after laparotomy and the mean number of abscesses in each animal was significantly higher in this group as compared with the laparoscopy and control groups. Early enhanced postoperative systemic inflammation may cause lower transient immunologic defense after laparotomy, leading to enhanced sepsis in the rats.

Corresponding author: C. A. Jacobi, MD, Department of Surgery, Charité, Humboldt-University of Berlin, Schumann Straße 20/21, D-10098 Berlin, Germany.

Attwood  SEHill  ADMurphy  PGThorton  JStephens  RB A prospective randomized trial of laparoscopic vs open appendectomy. Surgery. 1992;112497- 501
Geis  WPKim  HC Use of laparoscopy in the diagnosis and treatment of patients with surgical abdominal sepsis. Surg Endosc. 1995;9178- 182
Hansen  JBSmithers  BMSchache  DWall  DRMiller  BJMenzies  BL Laparoscopic vs open appendectomy: a prospective randomized trial. World J Surg. 1996;2017- 20
Tate  JDawson  JWChung  SCLau  WYLi  AK Laparoscopic vs open appendectomy: prospective randomised trial. Lancet. 1993;342633- 637
Urbano  DRossi  MDe Simone  PBerloco  PAlfani  DCortesini  R Alternative laparoscopic management of perforated peptic ulcers. Surg Endosc. 1994;81208- 1211
Gurtner  GCRobertson  CSChung  SCSLing  TKWIp  SMLi  AKC Effect of carbon dioxide pneumoperitoneum on bacteraemia and endotoxaemia in an animal model of peritonitis. Br J Surg. 1995;82844- 848
Eleftheriadis  EKotzampassi  KPapanotas  KHeliadis  NSarris  K Gut ischemia, oxidative stress, and bacterial translocation in elevated abdominal pressure in rats. World J Surg. 1996;2011- 16
Lorenz  WReimund  KPWeitzel  F  et al.  Granulocyte colony–stimulating factor prophylaxis before operation protects against lethal consequences of postoperative peritonitis. Surgery. 1994;116925- 934
Blöchle  CEmmermann  AAchilles  ETreu  HZornig  CBroelsch  CE Effect of a pneumoperitoneum on the extent and severity of peritonitis induced by gastric ulcer perforation in the rat. Surg Endosc. 1995;9898- 901
Walker  APKrepel  CJGohr  CMEdmiston  CE Microflora of abdominal sepsis by locus of infection. J Clin Microbiol. 1994;32557- 558
Zubkov  MNMenshikov  DDGugutsidze  ENChegin  VMVasina  TA Microbiologic diagnosis of mixed anaerobic and aerobic surgical infections. Antibiot Khimioter. 1995;4046- 50
Tsilibary  ECWissig  SL Lymphatic absorption from the peritoneal cavity: regulation of patency of mesothelial stomata. Microvasc Res. 1983;2522- 39
Gill  CODelacy  KM Growth of Escherichia coli and Salmonella typhimurium on high-pH beef packed under vacuum or carbon dioxide. Int J Food Microbiol. 1991;1321- 30
Harmon  GDSenagore  AJKilbride  MJWarzynski  MJ Interleukin 6 response to laparoscopic and open colectomy. Dis Colon Rectum. 1994;37754- 759

Figures

Place holder to copy figure label and caption
Figure 1.

Perioperative leukocyte counts in laparotomy, laparoscopy, and control groups (n=20 in each group; mean±SD). Asterisks indicate P<.05, laparotomy group vs laparoscopy and control groups.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Perioperative lymphocyte counts in laparotomy, laparoscopy, and control groups (n=20 in each group; mean±SD). Asterisks indicate P<.05, laparotomy group vs laparoscopy and control groups.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

Perioperative monocyte counts in laparotomy, laparoscopy, and control groups (n=20 in each group; mean±SD). Asterisks indicate P<.05, laparotomy group vs laparoscopy and control groups.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Perioperative neutrophil granulocyte counts in laparotomy, laparoscopy, and control groups (n=20 in each group; mean±SD). Asterisk indicates P<.05, laparotomy and laparoscopy groups vs control group.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 5.

Mean tumor necrosis factor α (TNF-α) levels in blood plasma after laparotomy and laparoscopy and in the control group (n=20 in each group; mean±SD). Asterisk indicates P<.05, laparotomy group vs the laparoscopy and control groups.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 6.

Mean tumor necrosis factor α (TNF-α) ex vivo secretion of peripheral blood mononuclear cells after laparotomy and laparoscopy and in the control group (n=20 in each group; mean±SD). Asterisk indicates P<.05, laparotomy group vs laparoscopy and control groups.

Graphic Jump Location

Tables

Table Graphic Jump LocationTable 1. Number of Positive Blood Cultures After 1 Hour and 7 Days
Table Graphic Jump LocationTable 2. Bacterial Species in Blood Cultures 1 Hour and 7 Days After Endotoxin Application*

References

Attwood  SEHill  ADMurphy  PGThorton  JStephens  RB A prospective randomized trial of laparoscopic vs open appendectomy. Surgery. 1992;112497- 501
Geis  WPKim  HC Use of laparoscopy in the diagnosis and treatment of patients with surgical abdominal sepsis. Surg Endosc. 1995;9178- 182
Hansen  JBSmithers  BMSchache  DWall  DRMiller  BJMenzies  BL Laparoscopic vs open appendectomy: a prospective randomized trial. World J Surg. 1996;2017- 20
Tate  JDawson  JWChung  SCLau  WYLi  AK Laparoscopic vs open appendectomy: prospective randomised trial. Lancet. 1993;342633- 637
Urbano  DRossi  MDe Simone  PBerloco  PAlfani  DCortesini  R Alternative laparoscopic management of perforated peptic ulcers. Surg Endosc. 1994;81208- 1211
Gurtner  GCRobertson  CSChung  SCSLing  TKWIp  SMLi  AKC Effect of carbon dioxide pneumoperitoneum on bacteraemia and endotoxaemia in an animal model of peritonitis. Br J Surg. 1995;82844- 848
Eleftheriadis  EKotzampassi  KPapanotas  KHeliadis  NSarris  K Gut ischemia, oxidative stress, and bacterial translocation in elevated abdominal pressure in rats. World J Surg. 1996;2011- 16
Lorenz  WReimund  KPWeitzel  F  et al.  Granulocyte colony–stimulating factor prophylaxis before operation protects against lethal consequences of postoperative peritonitis. Surgery. 1994;116925- 934
Blöchle  CEmmermann  AAchilles  ETreu  HZornig  CBroelsch  CE Effect of a pneumoperitoneum on the extent and severity of peritonitis induced by gastric ulcer perforation in the rat. Surg Endosc. 1995;9898- 901
Walker  APKrepel  CJGohr  CMEdmiston  CE Microflora of abdominal sepsis by locus of infection. J Clin Microbiol. 1994;32557- 558
Zubkov  MNMenshikov  DDGugutsidze  ENChegin  VMVasina  TA Microbiologic diagnosis of mixed anaerobic and aerobic surgical infections. Antibiot Khimioter. 1995;4046- 50
Tsilibary  ECWissig  SL Lymphatic absorption from the peritoneal cavity: regulation of patency of mesothelial stomata. Microvasc Res. 1983;2522- 39
Gill  CODelacy  KM Growth of Escherichia coli and Salmonella typhimurium on high-pH beef packed under vacuum or carbon dioxide. Int J Food Microbiol. 1991;1321- 30
Harmon  GDSenagore  AJKilbride  MJWarzynski  MJ Interleukin 6 response to laparoscopic and open colectomy. Dis Colon Rectum. 1994;37754- 759

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