Development of Peritoneal Adhesions in Macrophage Depleted Mice

doi:10.1016/j.jss.2005.08.026

Journal of Surgical Research

Volume 131, Issue 2, April 2006, Pages 296-301

https://doi.org/10.1016/j.jss.2005.08.026 Get rights and content

Background

We present a new mouse model for the study of peritoneal adhesions using macrophage Fasinduced apoptosis (Mafia) transgenic mice expressing a Fas-FKBP construct under control of the murine c-fms promoter. Mafia mice allow systemic macrophage depletion by dimerization of Fas with a synthetic dimerizer, AP20187. Results demonstrate that macrophage depletion in Mafia mice induces peritoneal adhesion formation when the peritoneal cavity is also exposed to an irritant. The Mafia mouse model presents a reproducible, non-surgical approach for research in adhesion formation and prevention.

Materials and methods

Mafia mice were treated with AP20187 using an intravenous (i.v.) or intraperitoneal (i.p.) injection. Control groups included mock-treated Mafia mice and both AP20187 and mock-treated wild type mice. Seven days after treatment, mice were observed for the presence of adhesions.

Results

After i.p. injection with AP20187, 76% of Mafia mice developed adhesions whereas none of the mock-treated Mafia or wild-type mice developed adhesions, and only one AP20187-treated wild-type mouse (5.8%) developed a mild adhesion. Mafia mice treated with AP20187 i.v. exhibited macrophage depletion not significantly different than i.p. treated mice, but did not develop adhesions. In contrast, Mafia mice treated with AP20187 i.v. developed adhesions when diluent was also injected into the peritoneal cavity, whereas i.p diluent alone had no effect.

Conclusion

Macrophage depletion, combined with a peritoneal irritant, results in peritoneal adhesion formation in transgenic Mafia mice. Macrophages appear to play a protective role in the development and/or repair of peritoneal adhesions.

Introduction

The formation of peritoneal adhesions is a common complication of abdominal surgery, and can lead to abdominal pain, intestinal obstruction, and infertility. Peritoneal adhesions cause more than 60% of all small bowel obstructions, perhaps the most severe consequence of adhesions [1, 2]. A recent study in England involving 138 patients found that 29% of intestinal obstructions require surgery [3]. Another study performed in the United States reported that adhesions resulted in over 300,000 hospitalizations and $1.3 billion in hospitalization and surgical expenditures in 1994 [4]. Infection and surgical trauma are leading causes of peritoneal adhesions. Over 90% of patients who have undergone a prior laparotomy have adhesions at their second operation [5]. It is thought that even slight injury to the mesothelial cell layer lining the peritoneal cavity results in the release of a fibrous exudate from blood vessels [6]. This exudate would normally be degraded by the process of fibrinolysis. However, during adhesion formation, fibrinolytic activity has been shown to be reduced [7, 8, 9].

Many cells have been implicated in the process of adhesiogenesis, including neutrophils [10, 11], lymphocytes [12], mast cells [13, 14], and macrophages [15]. Macrophages are the predominant immune cell type within the peritoneum, and histological data have shown macrophages to be present early in adhesion development [16]. Macrophages express urokinase and tissue plasminogen activators (uPA and tPA) and plasminogen activator inhibitors (PAI-1 and PAI-2), as well as other cytokines that regulate fibrinolysis and inflammation. Ar’Rajab and colleagues have shown that adoptive transfer of inflammatory macrophages can reduce the incidence of adhesion formation in a surgical model in rabbits [17]. Post-surgical macrophages often have a reduction in fibrinolytic activity, which may allow the persistence of adhesions [9, 17]. Fibrinogen is released after peritoneal trauma and may affect the expression pattern of peritoneal macrophages, thereby reducing macrophage fibrinolytic activity [18, 19].

We have developed a new transgenic mouse model using macrophage Fas-induced apoptosis (Mafia) transgenic mice to study the role of macrophages in peritoneal adhesion formation. The transgene in Mafia mice utilizes the murine c-fms promoter for monocyte/macrophage specific expression of enhanced green fluorescent protein (EGFP) as well as a drug-inducible suicide gene capable of initiating Fas-mediated apoptosis when cross-linked with the synthetic dimerizer, AP20187 [20]. Mafia transgenic mice can be depleted selectively of macrophages by treatment with AP20187. Macrophage depletion is systemic and inducible at any age.

In this study, we observed the development of adhesions in approximately 76% of macrophage-depleted Mafia mice when AP20187 was given by an intraperitoneal route. Histological analysis indicated that these adhesions were consistent with those previously described in surgically induced adhesion experiments [21]. The Mafia mouse model represents a reproducible, non-surgical model to study the induction and repair of peritoneal adhesions and the role of macrophages in the regulation of adhesion formation.

Section snippets

Mice

All wild-type C57Bl/6J mice were purchased from Charles River Laboratories (Boston, MA). Mafia mice were offspring from homozygous Mafia breeders at the University of Kentucky. The development and characterization of the Mafia transgenic mouse has been described previously [20]. Mafia mice have been catalogued with the Jackson Laboratory as strain C57BL/6J-Tg(Csf1r-GFP, NGFR/FKBP12)2Bck/J. Animals were maintained by the Division of Laboratory Animal Resources at the University of Kentucky

Effects of Macrophage Depletion on Adhesion Formation

The development of peritoneal adhesions in Mafia mice was initially described by us in correlation with systemic macrophage depletion [20], where approximately 80% of Mafia mice developed adhesions within 7 days after macrophage depletion via i.p. injection of AP20187. To confirm the apparent correlation between macrophage depletion and adhesion formation in Mafia mice [20], adhesion formation in AP20187-treated Mafia mice was compared to control groups including diluent-treated Mafia and

Discussion

Adhesion formation in patients recovering from peritoneal trauma or surgery remains a prevalent and costly clinical problem and much more research in animal models is necessary to develop appropriate therapeutic approaches. Several animal models have been used over the years to study adhesion development and these models typically involve laparotomy and physical abrasion of the mesothelial linings in the peritoneum. In a comparative study of laparoscopic induction of adhesion formation in mice,

Acknowledgments

AP20187 was kindly provided by Ariad Pharmaceuticals.

References (37)

N.F. Ray et al.
Abdominal adhesiolysisInpatient care and expenditures in the United States in 1994
J. Am. Coll. Surg
(1998)
M. Fukasawa et al.
Production of protease inhibitors by postsurgical macrophages
J. Surg. Res
(1989)
A. Ar’Rajab et al.
The role of neutrophils in peritoneal adhesion formation
J. Surg. Res
(1996)
A.F. Haney
Identification of macrophages at the site of peritoneal injuryEvidence supporting a direct role for peritoneal macrophages in healing injured peritoneum
Fertil. Steril
(2000)
A. Ar’Rajab et al.
Enhancement of peritoneal macrophages reduces postoperative peritoneal adhesion formation
J. Surg. Res
(1995)
F.M. Szaba et al.
Roles for thrombin and fibrin(ogen) in cytokine/chemokine production and macrophage adhesion in vivo
Blood
(2002)
G.-D. Chen et al.
The results of laparoscopic adhesiolysis for intractable urinary frequency
Clin. Urol
(1997)
P. Martin et al.
Wound healing in the PU. 1 null mouseTissue repair is not dependent on inflammatory cells
Curr. Biol
(2003)
A. Lichanska et al.
Differentiation of the mononuclear phagocyte system during mouse embryogenesisThe role of transcription factor PU. 1
Blood
(1999)
K. Anderson et al.
Neutrophils deficient in PU.1 do not terminally differentiate or become functionally competent
Blood
(1998)

J.A.O. Kossi et al.

Surgical workload and cost of postoperative adhesion-related intestinal obstructionImportance of previous surgery

World J. Surg

(2004)

H. Ellis

The clinical significance of adhesionsFocus on intestinal obstruction

Eur. J. Surg

(1997)

J. Kossi et al.

Population-based study of the surgical workload and economic impact of bowel obstruction caused by postoperative adhesions

Br. J. Surg

(2003)

D. Menzies et al.

Intestinal-obstruction from adhesionsHow big is the problem

Ann. R. Coll. Surg. Engl

(1990)

L. Holmdahl

The role of fibrinolysis in adhesion formation

Eur. J. Surg

(1997)

H. Sulaiman et al.

Role of plasminogen activators in peritoneal adhesion formation

Biochem. Soc. Trans

(2002)

M.L. Ivarsson et al.

Characterization and fibrinolytic properties of mesothelial cells isolated from peritoneal lavage

Scand. J. Clin. Lab. Invest

(1998)

B. Vural et al.

The role of neutrophils in the formation of peritoneal adhesions

Hum. Reprod

(1999)

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¹: This work was supported by the following NIH grants: HL69459 and HL57399.

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Wound healing/plastic surgeryDevelopment of Peritoneal Adhesions in Macrophage Depleted Mice1

Background

Materials and methods

Results

Conclusion

Introduction

Section snippets

Mice

Effects of Macrophage Depletion on Adhesion Formation

Discussion

Acknowledgments

J. Am. Coll. Surg

J. Surg. Res

J. Surg. Res

Fertil. Steril

J. Surg. Res

Blood

Clin. Urol

Curr. Biol

Blood

Blood

Surgical workload and cost of postoperative adhesion-related intestinal obstructionImportance of previous surgery

World J. Surg

The clinical significance of adhesionsFocus on intestinal obstruction

Eur. J. Surg

Population-based study of the surgical workload and economic impact of bowel obstruction caused by postoperative adhesions

Br. J. Surg

Intestinal-obstruction from adhesionsHow big is the problem

Ann. R. Coll. Surg. Engl

The role of fibrinolysis in adhesion formation

Eur. J. Surg

Role of plasminogen activators in peritoneal adhesion formation

Biochem. Soc. Trans

Characterization and fibrinolytic properties of mesothelial cells isolated from peritoneal lavage

Scand. J. Clin. Lab. Invest

The role of neutrophils in the formation of peritoneal adhesions

Hum. Reprod

Wound healing/plastic surgery
Development of Peritoneal Adhesions in Macrophage Depleted Mice1