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General introductionAetiology, incidence and clinical significance Intraperitoneal adhesions may be classified, according to the aetiology, in congenital or acquired, which can be postinflammatory or postoperative. Some 10% of patients without previous surgery have adhesions (9% postinflammatory and 1% congenital). In contrast, 94% of patients with at least one previous surgery have adhesions (92% postoperative, 1% postinflammatory and 1% congenital) QUOTE "(1)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\03(1)\00\03\00-D:\5CProgram Files\5CReference Manager 9\5CDatabase\03\00\041561\13Ellis 1997 1561 /id\00\13\00 (1). Intraperitoneal adhesions are clinically important because of the associated complaints and complications. They are a major cause of intestinal obstruction, chronic pelvic pain and female infertility. In addition, adhesions can cause major difficulties at the time of reoperation, increasing the difficulty to access to the abdomen or to the operation site, the complication rates, the anaesthesia and operation time, the use of surgical materials and the need of blood transfusion. For all of these reasons, adhesions have a huge economical impact for patients and health care systems QUOTE "(2)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\03(2)\00\03\00-D:\5CProgram Files\5CReference Manager 9\5CDatabase\03\00\044732\13Ellis 2000 4732 /id\00\13\00 (2). PathophysiologyPeritoneal injury initiates an inflammatory reaction that increases all components of the peritoneal fluid such as the concentration of fibrinogen and the cellular population. Fibrinogen is transformed into fibrin and immediately deposited on the wound surface. Polymorphonuclears (PMN), macrophages, fibroblasts and mesothelial cells migrate, proliferate and/or differentiate. During the first 2 postoperative days, a large number of PMN enter and, in the absent of infection, depart within 3-4 days. Macrophages increase in number and change their functions, becoming the most important component of the leukocyte population after day 5. Mesothelial cells migrate, form islands and proliferate throughout the injured area. These cells release plasminogen system components, arachidonic acid metabolites, oxygen-derived free radicals, cytokines and growth factors, which play a role at different stages of the process of peritoneal healing and adhesion formation. The deposited fibrin can be degraded by plasmin, which is transformed from plasminogen by plasminogen activators, i.e. tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). This action can be counteracted by plasminogen activator inhibitors (PAI), i.e. PAI-1, PAI-2, PAI-3 and protease nexin 1, and in lesser extent by plasmin inhibitors, i.e. α2-macroglobulin, α2-antiplasmin and α1-antitrypsin. If fibrin is completely lysed normal peritoneal healing will occur, but if it is not, fibrin serves as a scaffold for fibroblasts and capillaries, resulting in extracellular matrix deposition and angiogenesis, leading to adhesion formation QUOTE "(3)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\03(3)\00\03\00-D:\5CProgram Files\5CReference Manager 9\5CDatabase\03\00\041560\16DiZerega 1997 1560 /id\00\16\00 (3). The process of angiogenesis, which is meditated in a large extent by hypoxia, hypoxia inducible factors (HIFs) and angiogenic factors such as vascular endothelial growth factor (VEGF), has been however poorly addressed.Laparoscopic surgery and adhesion formationIt has been claimed that laparoscopy is less adhesiogenic than laparotomy but this is controversial. To fully understand the differences it is necessary to differentiate between adhesion formation (adhesions formed at operative sites), de novo adhesion formation (adhesions formed at non-operative sites) and adhesion reformation (adhesions formed after the lysis of previous adhesions). Animal and human studies indicate, in comparison with laparotomy, that laparoscopy induces less adhesion formation, but with controversial reports, that laparoscopy induces less de novo adhesion formation, and that both laparoscopy and laparotomy induce similar adhesion reformation QUOTE "(4)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\03(4)\00\03\00-D:\5CProgram Files\5CReference Manager 9\5CDatabase\03\00\044733\1EPouly & Seak-San 2000 4733 /id\00\1E\00 (4). Laparoscopy induces less surgical trauma because of gentle tissue handling, meticulous hemostasis, constant irrigation, the use of microsurgical instruments and the more precise operative field, which may reduce the risk of adhesion formation. This assumption, however, implies an adequate surgical technique, which can only be achieved with an appropriate training. Since laparoscopy is rapidly replacing laparotomy and since it requires specific skills a variety of in vitro and in vivo training models have been developed to obtain better quality of surgery and shorter operation times. Laparoscopic surgery, considered a minimal access surgery, only becomes a minimal invasive surgery, with the advantage of inducing less surgical trauma, when the criteria of adequate training are fulfilled. Laparoscopy is performed in an especial gas environment, the pneumoperitoneum. CO2 is the most common gas used for pneumoperitoneum because of safety reasons, i.e. its high solubility in water and its high exchange capacity in lungs. CO2 pneumoperitoneum, however, induces adverse effects such as hypercarbia and acidosis QUOTE "(5)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\03(5)\00\03\00-D:\5CProgram Files\5CReference Manager 9\5CDatabase\03\00\03590&Neuberger, Andrus, et al. 1996 590 /id\00&\00 (5), and hypothermia and desiccation QUOTE "(6)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\03(6)\00\03\00-D:\5CProgram Files\5CReference Manager 9\5CDatabase\03\00\044734\11Ott 2000 4734 /id\00\11\00 (6). It alters the peritoneal fluid QUOTE "(7)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\03(7)\00\03\00-D:\5CProgram Files\5CReference Manager 9\5CDatabase\03\00\03283\10Ott 2001 283 /id\00\10\00 (7), the morphology of the mesothelial cells QUOTE "(8)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\03(8)\00\03\00-D:\5CProgram Files\5CReference Manager 9\5CDatabase\03\00\03103!Volz, Koster, et al. 1999 103 /id\00!\00 (8), and the microcirculation QUOTE "(9)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\03(9)\00\03\00-D:\5CProgram Files\5CReference Manager 9\5CDatabase\03\00\0221!Taskin, Buhur, et al. 1998 21 /id\00!\00 (9), which can be deleterious inducing adhesion formation. Indeed, it was recently demonstrated that adhesion formation increases with the duration of the pneumoperitoneum and the insufflation pressure QUOTE "(10-12)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(10-12)\00\07\00-D:\5CProgram Files\5CReference Manager 9\5CDatabase\03\00\03183'Ordonez, Dominguez, et al. 1997 183 /id\00'\00 (10-12) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\00-D:\5CProgram Files\5CReference Manager 9\5CDatabase\03\00\03168#Yesildaglar & Koninckx 2000 168 /id\00#\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\00-D:\5CProgram Files\5CReference Manager 9\5CDatabase\03\00\03368)Yesildaglar, Ordonez, et al. 1999 368 /id\00)\00 .Adhesion preventionLess surgical trauma during surgical procedures will obviously diminish adhesion formation, but this alone will not prevent adhesions. A variety of approaches have been developed to prevent adhesions such as peritoneal instillates (crystalloids, dextran, hyaluronic acid, cross-linked hyaluronic acid, icodextrin), barriers (oxidized regenerated cellulose, hyaluronic acid-carboxymethylcellulose, expanded polytetrafluoroethylene, polyetilenglycol), drugs (corticosteroids, non-steroidal antiinflammatory drugs, fibrinolytic agents, anticoagulants, antibiotics, hormones) and other adjuvant (calcium channel blockers, cytostatics, motility promoters, modulators of macrophage function, cytokines and growth factors). Some of these adjuvant, however, are difficult to use, have side effects or show controversial results and no one have been proven to be universally effective QUOTE "(13;14)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(13;14)\00\07\00-D:\5CProgram Files\5CReference Manager 9\5CDatabase\03\00\044735\16DiZerega 2000 4735 /id\00\16\00 (13;14) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\00-D:\5CProgram Files\5CReference Manager 9\5CDatabase\03\00\044736\15Wiseman 2000 4736 /id\00\15\00 . Aim of the studyTo develop a training model for laparoscopic surgery and to evaluate its effect in the quality and duration of laparoscopic surgery.To evaluate the effect of the pneumoperitoneum on adhesion formation in the rabbit model.To develop a laparoscopic mouse model and to evaluate the effect of the pneumoperitoneum on adhesion formation in this model.To evaluate the role of the plasminogen system, VEGF family and HIFs in adhesion formation after laparoscopic surgery. To evaluate the effects of the pneumoperitoneum, under different conditions such as duration, insufflation pressure, insufflation gas, on blood gases. To evaluate the association between pneumoperitoneum-enhanced adhesion formation and pneumoperitoneum-induced changes in blood gases and the effects of the assisted ventilation.To evaluate the effects of adhesion prevention adjuvant specifically after laparoscopic surgery.General material and methodsAnimals and anaesthesiaThe study was performed in adult, female, New Zealand, white rabbits weighing 2.7-3.3 kg and in wild-type or transgenic, adult, female, mice of different strains (NMRI, Swiss, C57Bl/6J and 129SvJ) weighing 30-40 g. Rabbits were anaesthetized with inhalational halothane, intubated with a 3.5-mm tube and mechanically ventilated. Mice were anaesthetized with IM pentobarbital, intubated with a 20-ga tube and mechanically ventilated. Laparoscopy A midline incision (15-mm in rabbits, 3.5-mm in mice) was performed caudal to the xyphoides. In rabbits, a 10-mm trocar was introduced into the abdominal cavity for insufflation and for a 10-mm endoscope. In mice, a 2-mm endoscope with a 3.3-mm external sheath for insufflation was introduced into the abdominal cavity. To avoid any gas leakage, the incision was closed gas-tight around the trocar or the endoscope. For the pneumoperitoneum, heated and humidified gas was insufflated using the Thermoflator Plus (Karl Storz, Germany), which allows the mixture of CO2 with up to 12% of oxygen. A water valve was used to ascertain a continuous insufflation pressure. Since the peritoneum has a large surface and high exchange capacity, some oxygen could diffuse from the circulation to the abdominal cavity. To ascertain continuously the desired gas concentration in the abdominal cavity, the gas was continuously replaced by inserting a 26-gauge needle through the abdominal wall. Duration, insufflation gas and insufflation pressure were determined according to the experimental design. Induction of adhesionsAfter the establishment of the pneumoperitoneum, two 5-mm trocars (in rabbits) or 14-ga catheters (in mice) were inserted under laparoscopic vision in both right and left flank for the working instruments. Standardised lesions were performed in the antimesenteric border of right and left uterine horns and in right and left pelvic sidewalls. In rabbits, 2-cm2 lesions were performed with bipolar or CO2 laser coagulation. In mice, 10-mm linear lesions were performed with monopolar or bipolar coagulation. Scoring of adhesionsAdhesions were blindly scored after 7 days, during laparoscopy in rabbits and under microscopic vision during laparotomy in mice, using a qualitative (for rabbits and mice) and a quantitative (for mice) scoring system. The qualitative scoring system assess the following characteristics: extent (0: no adhesions; 1: 1-25%; 2: 26-50%; 3: 51-75%; 4: 76-100% of the injured surface involved, respectively), type (0: no adhesions; 1: filmy; 2: dense; 3: capillaries present), tenacity (0: no adhesions; 1: essentially fall apart; 2: require traction; 3: require sharp dissection) and total (extent + type + tenacity) QUOTE "(15)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(15)\00\04\00-D:\5CProgram Files\5CReference Manager 9\5CDatabase\03\00\03181!Leach, Burns, et al. 1998 181 /id\00!\00 (15). The quantitative scoring system measures the proportion of the lesions covered by adhesions using the following formula: adhesions (%) = (sum of the length of the individual attachments/length of the lesion) x 100 QUOTE "(16)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(16)\00\04\00-D:\5CProgram Files\5CReference Manager 9\5CDatabase\03\00\041992*Holmdahl, al Jabreen, et al. 1994 1992 /id\00*\00 (16). StatisticsStatistical analyses were performed with the SAS System according to the requirements of each experiment. Individual comparisons were performed with t-test or Wilcoxon, multiple comparisons with logistic regression or ANOVA and correlation analysis with Pearson or Spearman for parametric and non-parametric variables, respectively. Factorial design was used when needed. The rabbit model: a novel training model for laparoscopic surgery AimTo develop a standard animal training model for laparoscopic surgery and to evaluate its feasibility and reproducibility.Material and MethodsThe study was performed in 210 rabbits. A pilot study was carried out (n=10) in order to evaluate the feasibility of reproducing surgical tasks needed in human surgeries. Several exercises were performed including nephrectomy, hysterectomy, dissection of major vessels, enterolysis, models for salpingostomy, neosalpingostomy and cystectomy, stitching and knot-tying, hysteroscopy and transvaginal hydrolaparoscopy. Nephrectomy was chosen as a “gold standard” since it permits and objective quantification. This exercise includes the dissection of renal vessels, ureter and kidney. Firstly, both renal artery and vein are dissected individually from the hilus up to the aorta, ligated and cut. Secondly, the ureter is dissected from the hilus up to its intersection with the ovarian vessels, since this is an obvious anatomical landmark. Finally, the kidney is dissected until its completed isolation, without surrounding fat, from the renal fossa. Each nephrectomy is individually scored for duration and quality of surgery. Quality of surgery was determined by subtracting accidents (0-6) from quality of dissection (0-9). Accident scores were determined by adding bleeding (0-3) and lesion of adjacent organs (0-3). Quality of dissection was determined by adding quality of renal vessel (0-3), ureter (0-3) and kidney (0-3) dissection. Once the model was established, the learning curve was evaluated in 10 experienced and 10 inexperienced surgeons (200 rabbits, 20 nephrectomies/surgeon). ResultsDuration of surgery decreased whereas quality of surgery increased with training, assessed by the consecutive number of nephrectomies performed. These effects were observed for both inexperienced and experienced surgeons. For the inexperienced surgeons duration of surgery decreased from some 60 min in the first interventions to some 10 min in the last interventions, whereas the quality of surgery scores increased from 4 to 9. For the experienced surgeons duration of surgery decreased from some 20 min in the first interventions to some 10 min in the last interventions, whereas the quality of surgery scores increased from 6 to 9. Effect of the diameter of the endoscope and of surgeon training on the duration and quality of laparoscopic surgery in a rabbit model(J Am Assoc Gynecol Laparosc 1999;6:447-452)AimTo evaluate the effect of the diameter of the endoscope and training on the duration and quality of laparoscopic surgery.Material and MethodsThe study was performed in 60 rabbits. Laparoscopic nephrectomies were performed and duration and quality of surgery were scored as described. Sixty consecutive procedures were performed by an experienced (series 1) and by an inexperienced (series 2) surgeon using 2-, 4-, 5- or 10-mm endoscopes. ResultsFor the inexperienced surgeon, duration of surgery and bleeding decreased whereas quality of dissection increased with larger endoscopes and with training, assessed by the number of nephrectomies, demonstrating the learning curve. For the experienced surgeon, only a slight effect of training on duration of surgery was observed. The experienced surgeon achieved shorter operation times and better qualities at the beginning of the training and with smaller endoscopes, but the differences decreased with the progressive gain in experience during consecutive nephrectomies and with larger endoscopes. Hypoxia induced by co2 or helium pneumoperitoneum is a cofactor in adhesion formation in rabbits (Partially published in Hum Reprod 2000;15:1758-1763)AimTo evaluate the effect of the duration of the pneumoperitoneum and the insufflation gas on adhesion formation.Material and MethodsThe study was performed in 68 rabbits. In experiment 1 (4 groups, 5 rabbits/group), pure CO2 pneumoperitoneum was maintained for 10, 20, 30 or 60 min. In experiment 2 (6 groups, 8 rabbits/group), pure CO2 or helium pneumoperitoneum was maintained for 10 or 45 min, and CO2 or helium pneumoperitoneum with 4% of oxygen was maintained for 45 min. The mixture of gases was achieved using two insufflators connected to a mixing chamber. ResultsIn rabbits with 10, 20, 30 or 60 min of pure CO2 pneumoperitoneum, adhesion formation increased with the duration of the CO2 pneumoperitoneum. In rabbits with 10 or 45 min of pure CO2 or helium pneumoperitoneum, no differences in adhesion formation were observed between both insufflation gases, whereas adhesion formation increased with the duration of the pneumoperitoneum.In rabbits with 45 min of CO2 or helium pneumoperitoneum with 0% or 4% of oxygen, no differences in adhesion formation were observed between CO2 and helium pneumoperitoneum, whereas adhesion formation decreased with the adhesion of oxygen.Peritoneal mesothelial hypoxia during pneumoperitoneum is a cofactor in adhesion formation in a laparoscopic mouse modelIntroductionThe CO2 pneumoperitoneum is a cofactor in adhesion formation in rabbits, as was demonstrated in chapter 6. Indeed, adhesions increased with the duration of the pneumoperitoneum and with the insufflation pressure and decreased with the addition of 4% of oxygen. Similar effects were observed with CO2 and helium pneumoperitoneum without differences between both insufflation gases. In the mouse model was also demonstrated that adhesion formation increased with the duration of the pneumoperitoneum, but using an insufflation pressure of 2.5 cm H2O only (23). All these data together lead to the hypothesis that mesothelial hypoxia, due to the compression of the capillary flow in the superficial peritoneal layers during pneumoperitoneum, plays a role in adhesion formation. In order to investigate in detail the intrinsic mechanisms involved, a model suitable for laparoscopies at high insufflation pressures and for the evaluation of cytokines and growth factors expression is required. Therefore, this study was performed to develop a laparoscopic mouse model at high insufflation pressures, to confirm the known effects of pneumoperitoneum, and to investigate more specifically the effect of the addition from 0.5% up to 12% of oxygen to CO2 pneumoperitoneum on adhesion formation.Material and MethodsAnimalsThe study was performed in 130 female NMRI mice, 6-8 months old weighing 45-55 g. LaparoscopyLaparoscopy was performed as described in detail in chapter 2. For the pneumoperitoneum, a mixture of CO2 or helium with different concentrations of oxygen was used. The insufflation gas, the insufflation pressure and the duration of the pneumoperitoneum were determined according to the experimental design.Induction of intraperitoneal adhesions Adhesions were induced by standardized monopolar lesions (Autocon 350, Karl Storz, Tüttlingen, Germany) as described in detail in chapter 2. Scoring of adhesions Adhesions were scored during laparotomy after 7 or 28 days as described in chapter 2. Experimental designAll experiments were performed using block randomization by days. In the first experiment (n=35), survival rate was evaluated after one hour of pneumoperitoneum with insufflation pressures of 5, 10, 15, 20, 25 and 30 cm H2O, respectively (n=5 per group). Besides, survival rate was evaluated maintaining the pneumoperitoneum for three hours at 15 cm H2O (n=5).In the second experiment (n=35), a 2X2 factorial design was used to evaluate the effect of the duration of the pneumoperitoneum (10 and 60 min) and the insufflation pressure (5 and 15 cm H2O) on adhesion formation. Pneumoperitoneum at 5 cm H2O was maintained for 10 min (n=7) and 60 min (n=7). Similarly, pneumoperitoneum at 15 cm H2O was maintained for 10 min (n=7) and 60 min (n=7). In an additional group (n=7), the pneumoperitoneum was maintained for 60 min at 15 cm H2O and adhesions were scored after 28 days. In the third experiment (n=60), a 2X2 factorial design was used to evaluate the effect of the insufflation gas (CO2 and helium) and the addition of oxygen (0 and 3%) on adhesion formation. CO2 pneumoperitoneum was used with 0% (n=5) and with 3% (n=5) of oxygen. Similarly, helium pneumoperitoneum was used with 0% (n=5) and with 3% (n=5) of oxygen. The effect of adding other concentrations of oxygen, i.e. 0.5%, 1%, 1.5%, 2%, 2.5%, 6%, 9% and 12%, to CO2 pneumoperitoneum was also specifically assessed (n=5 per group) maintaining the pneumoperitoneum for 60 minutes at 15 cm H2O.StatisticsStatistical analyses were performed with the SAS System using logistic regression (Proc logistic) for comparing several variables and Wilcoxon test for individual comparisons. ResultsIntubation was a safe and quick procedure taking less than one min without any single injury or mortality. Insufflation pressures of 5, 10, 15, and 20 H2O for one hour did not caused mortality, whereas insufflation pressures of both 25 and 30 cm H2O caused a 20% of mortality during the surgery. All animals survived when the pneumoperitoneum was maintained for three hours at 15 cm H2O. All mice surviving the surgery survived after one week indicating that both intubation and laparoscopy did not have long-term complications in terms of mortality.In adhesion formation experiments, none of the mice showed signs of intestinal obstruction or other serious complications. Adhesions consistently formed to the injured parietal and visceral sites attaching pelvic fat. Two mice presented filmy adhesions of the omentum to the laparoscopic ports and no de novo adhesions were found. In the second experiment one mouse was excluded because the lesion completely destroyed the uterine horn and consequently six instead of seven animals were evaluated in one group (pneumoperitoneum for 60 min at 15 cm H2O, scored after 28 days). The proportion of adhesions were 38 5% after seven days and 40 6% after 28 days, respectively (P=NS). Extent, type, tenacity and total adhesions scores were 1.8 0.2, 1.8 0.1, 1.8 0.1, and 5.4 0.4 after seven days and 1.8 0.3, 2.0 0.2, 2.0 0.3, and 5.8 0.7 after 28 days, respectively (P=NS). In mice with 10 or 60 min of CO2 pneumoperitoneum at 5 or 15 H2O, which were analysed simultaneously (4 groups, 2 variables, proc logistic), adhesion formation increased with the duration of the pneumoperitoneum (proportion: P=0.001; extent: P=0.004; type: P=0.001; total: P=0.002) and the insufflation pressure (proportion: P=0.005; extent: P=0.03; tenacity: P=0.01; total: P=0.02). When groups were individually compared (Wilcoxon), some differences only reached significance. In mice with 60 min of pneumoperitoneum, adhesion formation increased with the insufflation pressure (proportion: P=0.004; extent: P=0.03). In mice with 15 cm H2O of insufflation pressure, adhesion formation increased with the duration of the pneumoperitoneum (proportion: P=0.002; extent: P=0.005; type: P=0.002; total: P=0.005) (Figure 1 and Table I). Figure 1: Effect of the duration of the CO2 pneumoperitoneum and the insufflation pressure on adhesion formation.Quantitative scoringQualitative scoringTable I: Effect of the duration of the CO2 pneumoperitoneum and the insufflation pressure on adhesion formation. Duration (min)Pressure (cm H2O)Adhesion scoresExtentTypeTenacity1050.8 0.21.1 0.40.9 0.26051.1 0.21.4 0.31.2 0.310150.9 0.11.0 0.11.4 0.260151.8 0.21.8 0.11.8 0.1In mice with CO2 or helium pneumoperitoneum with 0% or 3% of oxygen, which were analysed simultaneously (4 groups, 2 variables, proc logistic), adhesion formation decreased with the addition of 3% of oxygen (proportion: P=0.0001; extent: P=0.0003; type: P=0.0003; tenacity: (P=0.0002); total: P=0.0001) and was higher with helium pneumoperitoneum (extent: P=0.05; type: P=0.01; tenacity: P=0.004; total: P=0.008). When groups were individually compared (Wilcoxon), adhesion formation also decreased with the addition of 3% of oxygen in mice with CO2 pneumoperitoneum (proportion: P=0.004; extent: P=0.005; type: P=0.01; tenacity: P=0.004; total: P=0.003) and in mice with helium pneumoperitoneum (proportion: P=0.01; extent: P=0.01; type: P=0.01; tenacity: P=0.02; total: P=0.01). The differences between CO2 and helium pneumoperitoneum were observed with 0% of oxygen only (type: P=0.02; tenacity: P=0.04; total: P=0.02) (Figure 2 and Table II). Figure 2: Effect of the insufflation gas and the addition of 3% of oxygen on adhesion formation.Quantitative scoringQualitative scoringTable II: Effect of the duration of the CO2 pneumoperitoneum and the insufflation pressure on adhesion formation.Insufflation gasConcentration of oxygen (%)Adhesion scoresExtentTypeTenacityCO201.8 0.21.8 0.11.8 0.1CO230.7 0.20.9 0.30.7 0.2Helium02.1 0.22.5 0.22.4 0.2Helium31.1 0.21.3 0.21.4 0.2In comparison with mice with pure CO2 pneumoperitoneum, adhesion formation also decreased with the addition of 2% (proportion: P=0.003; extent: P=0.01; type: P=0.01; total: P=0.02), 2.5% (proportion: P=0.01), 6% (proportion: P=0.03; extent: P=0.05), 9% (proportion: P=0.05; extent: P=0.04; type: P=0.01; tenacity: P=0.01; total: P=0.005), and 12% (type: P=0.05; total: P=0.04) of oxygen. No differences were found between these different groups (Figure 3, Table 3).Figure 3: Effect of the addition of different concentrations of oxygen to the CO2 pneumoperitoneum on adhesion formation.Quantitative scoringQualitative scoringTable III: Effect of the addition of different concentrations of oxygen to the CO2 pneumoperitoneum on adhesion formation.Insufflation gasConcentration of oxygen (%)Adhesion scoresExtentTypeTenacityCO201.8 0.21.8 0.11.8 0.10.51.7 0.41.4 0.31.5 0.311.6 0.21.3 0.31.5 0.31.51.4 0.21.9 0.22.0 0.020.7 0.30.9 0.31.3 0.42.51.0 0.41.4 0.41.4 0.430.7 0.20.9 0.30.7 0.260.9 0.41.3 0.41.2 0.491.1 1.21.1 0.11.0 0.0121.3 0.21.2 0.21.3 0.2DiscussionThe mouse model is well suited to investigate adhesion formation after laparoscopic surgery. Firstly, the mouse is a well-established model for the study of intraabdominal adhesion formation after laparotomy (26). Secondly, the mouse model is one of the best-developed animal models with many specific assays available. In addition, the availability of nude, severe combined immunodeficient (SCID) and knockout mice permit detailed investigations. Therefore it is not surprising that laparoscopic surgery in mice was recently developed to study postoperative tumor growth (27), port-site metastases (28), immune response (29), gastrointestinal transit (30) and morphologic alterations of the mesothelium (31). These procedures were, however, all done in non-intubated mice with a low insufflation pressure. In addition, a high mortality was reported with insufflation pressures higher than 5 cm H2O in non-intubated mice, making surgical procedures virtually impossible (23).Our model with intubated mice is, to the best of our knowledge, the first model permitting insufflation pressures up to 20 cm H2O (15 mm of Hg), i.e. comparable to human surgery, and surgical procedures. This model permits 1-3 hours of surgery without anaesthetic mortality. This is extremely important to investigate the effect of laparoscopy on adhesion formation since we know from rabbit experiments that adhesions increase with insufflation pressure (22). It should be stressed that in this mouse model humidification will be close to 100% because of low flow rates (22, 23). In addition, using the Thermoflator Plus, insufflation can be done with gas heated at 37 C because of the distant heating device close to the animal, whereas this set up permits to use a variable oxygen tension between 0% and 12%. A water valve and an elastic balloon were introduced to prevent any overpressure, which is critical in mice.Adhesions evaluated after 7 and 28 days were almost identical confirming previous observations that adhesion formation did not vary significantly after seven days (24). Evaluation after seven days is advantageous for research purposes because it permits to induce adhesions during one week and to evaluate them the following week. Results from the first experiment can be obtained before beginning the second experiment.Our data are consistent with the following hypothesis: CO2 or helium pneumoperitoneum, especially at higher insufflation pressures, will compress the capillary flow in the superficial peritoneal layers inducing local ischemia, whereas diffusion of pure CO2 or helium from the abdominal cavity to the blood stream will create a gradient of hypoxia, the superficial layers being exposed to virtually 100% CO2 or helium without oxygen. This hypothesis is supported by the observation that adhesions increase with duration and pressure of pneumoperitoneum and decrease by the addition of oxygen, 3% having a maximal effect and 1.5% a half-maximal effect.Our hypothesis of mesothelial hypoxia as a driving mechanism in adhesion formation is consistent with the intracellular partial pressure of oxygen (PO2), being 5 to 40 mm Hg (average 23 mm Hg), in contrast with the arterial, interstitial and venous PO2, which are 95, 40, and 40 mm Hg, respectively (32). Compression of the capillary flow in the superficial peritoneal layers by the pneumoperitoneum will reduce the PO2 in the mesothelial cells. A PO2 bellow 5 mm Hg, which causes hypoxic changes, thus could be reached. A mixture of 3% of oxygen in 97% of CO2, insufflated at 15 cm H2O ( 12 mm Hg), results in a PO2 of 23 mm Hg (3% of 760 mm Hg atmospheric pressure + 12 mm Hg insufflation pressure). This PO2 is obviously sufficient to increase the mesothelial PO2 to normal levels thus abolishing any local hypoxia.Hypoxia is the main stimulus of VEGF (35) and a few studies have already reported its role in adhesion formation. Indeed, a polyclonal rabbit antibody to VEGF limited adhesions after laparotomy in mice (36) and VEGF was found in peritoneal adhesions by inmunohistochemistry (37), enzyme-linked immunosorbent assay (38) and reverse transcriptase-polymerize chain reaction (39). Other effects of hypoxia cannot be ruled out. Prior reports indicate that hypoxic conditions (2% of oxygen) in cultured mesothelial cells up-regulate the expression of TGF-1 and 2 (40) and TIMPs, possibly through a TGF-1-dependent mechanism (41). TGF- up-regulates PAI-1 and down-regulates tPA, decreasing plasmin and thus inhibiting the lysis of fibrin increasing adhesion formation (42). TGF- decreases the expression of MMPs and increases the expression of TIMPs and therefore decreases matrix degradation increasing adhesions (43). Further studies are required to clarify the intrinsic mechanism whereby mesothelial hypoxia induces adhesions and the laparoscopic mouse model is well suited for these investigations.The similarity between CO2 and helium, which is chemically, pharmacologically and physiologically inert and does not produce hypercarbia and acidosis (44, 45), showed at least that these two effects -hypercarbia and acidosis- were less pronounced in adhesion formation, thus pointing to the hypoxia hypothesis. It is unclear, however, why helium pneumoperitoneum induced slighter more adhesions than CO2 in mice, an observation that was not made in rabbits in chapter 6. We only can speculate that hypoxia with a less soluble gas was more pronounced, whereas the lower interanimal variability of the mouse model permitted to detect this. This observation is another argument that hypoxia, rather than pH changes, is the driving mechanism whereby CO2 pneumoperitoneum induces adhesion formation.In conclusion, these experiments demonstrate the feasibility of the laparoscopic mouse model at high insufflation pressures, confirm the effect of the pneumoperitoneum on adhesion formation and are consistent with the hypothesis of mesothelial hypoxia as a driving mechanism.Role of the plasminogen system in basal adhesion formation and in CO2 pneumoperitoneum-enhanced adhesion formation following laparoscopic surgery in miceIntroductionThe plasminogen system is a complex system comprising the serine protease plasmin, the zymogen plasminogen, plasminogen activators (tPA and uPA), plasminogen activator inhibitors (PAI-1, PAI-2, PAI-3 and protease nexin 1) and plasmin inhibitors (α2-macroglobulin, α2-antiplasmin and α1-antitrypsin) QUOTE "(20;20;21;21-23)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\10(20;20;21;21-23)\00\10\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044197\14Collen 1999 4197 /id\00\14\00 (20-23) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044196\1DCollen & Lijnen 1995 4196 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044196\1DCollen & Lijnen 1995 4196 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044197\14Collen 1999 4197 /id\00\14\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044245\1DLijnen & Collen 1995 4245 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044193\16Chandler 1996 4193 /id\00\16\00 . Plasminogen is abundant in virtually all tissues and is converted to plasmin by tPA and uPA, both being equally efficient in degradation of fibrin clots in blood but not in tissues, where tPA seems to have a higher efficacy QUOTE "(24;25;25;25;26;26;26;27;27;27)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\1F(24;25;25;25;26;26;26;27;27;27)\00\1F\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044360\1FSprengers & Kluft 1987 4360 /id\00\1F\00 (24-27) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044355(Runge, Quertermous, et al. 1989 4355 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044354$Pannell, Black, et al. 1988 4354 /id\00$\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044351\1CLu, Wu, et al. 1992 4351 /id\00\1C\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044355(Runge, Quertermous, et al. 1989 4355 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044354$Pannell, Black, et al. 1988 4354 /id\00$\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044351\1CLu, Wu, et al. 1992 4351 /id\00\1C\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044355(Runge, Quertermous, et al. 1989 4355 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044354$Pannell, Black, et al. 1988 4354 /id\00$\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044351\1CLu, Wu, et al. 1992 4351 /id\00\1C\00 . The activity of tPA and uPA is modulated mainly by the glycoproteins PAI-1 and PAI-2 through the formation of inactive complexes, PAI-1 having a stronger inhibitory action than PAI-2. The other PAIs and the plasmin inhibitors are considerably less active QUOTE "(28;29;29;29;30;30;30;31;31;31)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\1F(28;29;29;29;30;30;30;31;31;31)\00\1F\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044357*van Mourik, Lawrence, et al. 1984 4357 /id\00*\00 (28-31) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044356%Kopitar, Rozman, et al. 1985 4356 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044358 Baker, Low, et al. 1980 4358 /id\00 \00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044359&Stump, Thienpont, et al. 1986 4359 /id\00&\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044356%Kopitar, Rozman, et al. 1985 4356 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044358 Baker, Low, et al. 1980 4358 /id\00 \00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044359&Stump, Thienpont, et al. 1986 4359 /id\00&\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044356%Kopitar, Rozman, et al. 1985 4356 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044358 Baker, Low, et al. 1980 4358 /id\00 \00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044359&Stump, Thienpont, et al. 1986 4359 /id\00&\00 . The plasminogen system is crucial in the processes of fibrin deposition/degradation and adhesion formation. After complete fibrinolysis, little or no adhesions will be formed whereas insufficient fibrinolysis will provide a scaffold for migrating cells, i.e. fibroblasts and capillaries, resulting in extra-cellular matrix (ECM) deposition and angiogenesis, leading to adhesion formation QUOTE "(1;2;2;3;3;3)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\0D(1;2;2;3;3;3)\00\0D\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041560\16DiZerega 1997 1560 /id\00\16\00 (1-3) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041558\16Holmdahl 1997 1558 /id\00\16\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041183!Holmdahl & Ivarsson 1999 1183 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041558\16Holmdahl 1997 1558 /id\00\16\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041183!Holmdahl & Ivarsson 1999 1183 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041183!Holmdahl & Ivarsson 1999 1183 /id\00!\00 . This is based on a series of studies demonstrating more adhesions with decreased fibrinolysis QUOTE "(4)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\03(4)\00\03\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041334)Ivarsson, Bergstrom, et al. 1998 1334 /id\00)\00 (4) and less adhesions with increased fibrinolysis, i.e. following the administration of plasminogen activator inhibitor-1 (PAI-1) antibodies QUOTE "(5)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\03(5)\00\03\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041026%Falk, Bjorquist, et al. 2001 1026 /id\00%\00 (5) or of recombinant tissue-type plasminogen activator (tPA) QUOTE "(6;7;7;7;7;8;8;8;8;9;9;9;9;10;10;10;10;11;11;11;11;12;12;12;12)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00?(6;7;7;7;7;8;8;8;8;9;9;9;9;10;10;10;10;11;11;11;11;12;12;12;12)\00?\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041860%Vipond, Whawell, et al. 1994 1860 /id\00%\00 (6-12) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042516!Doody, Dunn, et al. 1989 2516 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042310\1DMenzies & Ellis 1991 2310 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042493\1DMenzies & Ellis 1989 2493 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042359!Dorr, Vemer, et al. 1990 2359 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042314%Orita, Fukasawa, et al. 1991 2314 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044485%Hill-West, Dunn, et al. 1995 4485 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042516!Doody, Dunn, et al. 1989 2516 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042310\1DMenzies & Ellis 1991 2310 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042359!Dorr, Vemer, et al. 1990 2359 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042493\1DMenzies & Ellis 1989 2493 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042314%Orita, Fukasawa, et al. 1991 2314 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044485%Hill-West, Dunn, et al. 1995 4485 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042516!Doody, Dunn, et al. 1989 2516 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042310\1DMenzies & Ellis 1991 2310 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042493\1DMenzies & Ellis 1989 2493 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042359!Dorr, Vemer, et al. 1990 2359 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042314%Orita, Fukasawa, et al. 1991 2314 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044485%Hill-West, Dunn, et al. 1995 4485 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042516!Doody, Dunn, et al. 1989 2516 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042310\1DMenzies & Ellis 1991 2310 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042359!Dorr, Vemer, et al. 1990 2359 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042493\1DMenzies & Ellis 1989 2493 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042314%Orita, Fukasawa, et al. 1991 2314 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044485%Hill-West, Dunn, et al. 1995 4485 /id\00%\00 .Besides its role in fibrin degradation, the plasminogen system has a direct role in other processes of tissue repair, such as activation of pro-enzymes of the matrix metalloprotease (MMP) family QUOTE "(13)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(13)\00\04\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044349&Murphy, Atkinson, et al. 1992 4349 /id\00&\00 (13), ECM degradation QUOTE "(14)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(14)\00\04\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044346"Wong, Cortez, et al. 1992 4346 /id\00"\00 (14), activation of pro-urokinase-type plasminogen activator (uPA) QUOTE "(15)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(15)\00\04\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044488$Petersen, Lund, et al. 1988 4488 /id\00$\00 (15), liberation and activation of growth factors QUOTE "(16;17;17)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\0A(16;17;17)\00\0A\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044347\1ESaksela & Rifkin 1990 4347 /id\00\1E\00 (16-17) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044348\1BSato & Rifkin 1989 4348 /id\00\1B\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044348\1BSato & Rifkin 1989 4348 /id\00\1B\00 , angiogenesis QUOTE "(18)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(18)\00\04\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044489#Devy, Blacher, et al. 2002 4489 /id\00#\00 (18) and cellular migration QUOTE "(19)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(19)\00\04\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044044&Carmeliet, Moons, et al. 1997 4044 /id\00&\00 (19). It is unclear whether the reported effects on adhesion formation only involve fibrinolysis or also some of the other processes. QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044245\1DLijnen & Collen 1995 4245 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044193\16Chandler 1996 4193 /id\00\16\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044356%Kopitar, Rozman, et al. 1985 4356 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044358 Baker, Low, et al. 1980 4358 /id\00 \00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044359&Stump, Thienpont, et al. 1986 4359 /id\00&\00 The overall peritoneal fibrinolytic activity is crucial in the pathogenesis of adhesion formation. tPA, uPA, PAI-1 and PAI-2 have been widely explored but not yet PAI-3, protease nexin 1 and plasmin inhibitors. The peritoneal fibrinolytic activity decreases after different types of peritoneal trauma such as suturing, retractors, foreign bodies and infection QUOTE "(2;2;2;32;32;33;33)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\13(2;2;2;32;32;33;33)\00\13\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041646(Holmdahl, Eriksson, et al. 1996 1646 /id\00(\00 (2, 32, 33) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041558\16Holmdahl 1997 1558 /id\00\16\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044502(Holmdahl, Eriksson, et al. 1998 4502 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041558\16Holmdahl 1997 1558 /id\00\16\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044502(Holmdahl, Eriksson, et al. 1998 4502 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041646(Holmdahl, Eriksson, et al. 1996 1646 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041558\16Holmdahl 1997 1558 /id\00\16\00 . This is modulated by a variety of pro-inflammatory cytokines released after a peritoneal injury, such as tumor necrosis factor-α (TNF-α), interleukin 1 and 6 and transforming growth factor- (TGF-), which decrease the fibrinolytic activity of human mesothelial cells in vitro QUOTE "(2;32;34;35;35;35;35;36;36;36;36;37;37;37;37)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00-(2;32;34;35;35;35;35;36;36;36;36;37;37;37;37)\00-\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044512$Badia, Whawell, et al. 1996 4512 /id\00$\00 (34-37) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044515,Whawell, Scott-Coombes, et al. 1994 4515 /id\00,\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044501(Ivarsson, Holmdahl, et al. 1998 4501 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041438&Tietze, Elbrecht, et al. 1998 1438 /id\00&\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044515,Whawell, Scott-Coombes, et al. 1994 4515 /id\00,\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044501(Ivarsson, Holmdahl, et al. 1998 4501 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041438&Tietze, Elbrecht, et al. 1998 1438 /id\00&\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044515,Whawell, Scott-Coombes, et al. 1994 4515 /id\00,\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044501(Ivarsson, Holmdahl, et al. 1998 4501 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041438&Tietze, Elbrecht, et al. 1998 1438 /id\00&\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041646(Holmdahl, Eriksson, et al. 1996 1646 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041558\16Holmdahl 1997 1558 /id\00\16\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044515,Whawell, Scott-Coombes, et al. 1994 4515 /id\00,\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044501(Ivarsson, Holmdahl, et al. 1998 4501 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041438&Tietze, Elbrecht, et al. 1998 1438 /id\00&\00 . Postoperative adhesion formation in vivo has been investigated mainly following open surgery. We recently demonstrated in rabbits and mice that the pneumoperitoneum is a cofactor in adhesion formation following laparoscopic surgery. We suggested mesothelial hypoxia as the driving mechanism for this pneumoperitoneum-enhanced adhesion formation since adhesions increase with the duration of pneumoperitoneum and the insufflation pressure and decrease with the addition of oxygen to both CO2 and helium pneumoperitoneum QUOTE "(38;39;39;39;40;40;40;41;41;41)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\1F(38;39;39;39;40;40;40;41;41;41)\00\1F\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03183'Ordonez, Dominguez, et al. 1997 183 /id\00'\00 (38-41) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03168#Yesildaglar & Koninckx 2000 168 /id\00#\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03164\1FMolinas & Koninckx 2000 164 /id\00\1F\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043673&Molinas, Mynbaev, et al. 2001 3673 /id\00&\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03168#Yesildaglar & Koninckx 2000 168 /id\00#\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03164\1FMolinas & Koninckx 2000 164 /id\00\1F\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043673&Molinas, Mynbaev, et al. 2001 3673 /id\00&\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03168#Yesildaglar & Koninckx 2000 168 /id\00#\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03164\1FMolinas & Koninckx 2000 164 /id\00\1F\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043673&Molinas, Mynbaev, et al. 2001 3673 /id\00&\00 . This has lead to the concept of basal adhesions (adhesions following a peritoneal lesion only) and pneumoperitoneum-enhanced adhesions (adhesions following a peritoneal lesion with the additional effect of the pneumoperitoneum).The importance of trauma with tissue necrosis and the inflammatory reaction and repair mechanism, involving local cellular hypoxia, has been recognized in adhesion formation QUOTE "(42;43;43;44;44;45;45;46;46)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\1C(42;43;43;44;44;45;45;46;46)\00\1C\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042927\13Ellis 1982 2927 /id\00\13\00 (42-46) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043797\15Raftery 1973 3797 /id\00\15\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\0425190Tsimoyiannis, Tsimoyiannis, et al. 1989 2519 /id\000\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043681$Wiseman, Huang, et al. 1994 3681 /id\00$\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043684'Wiseman, Gottlick, et al. 1992 3684 /id\00'\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043797\15Raftery 1973 3797 /id\00\15\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\0425190Tsimoyiannis, Tsimoyiannis, et al. 1989 2519 /id\000\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043681$Wiseman, Huang, et al. 1994 3681 /id\00$\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043684'Wiseman, Gottlick, et al. 1992 3684 /id\00'\00 . Furthermore, hypoxia modulate the expression of several molecules involved in different stages of adhesion formation, such as PAI-1, tPA, TGF-, MMPs and tissue inhibitors of metalloproteinases (TIMPs) QUOTE "(47-56)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(47-56)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043475!Saed, Zhang, et al. 1999 3475 /id\00!\00 (47-56) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043476!Saed, Zhang, et al. 2000 3476 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03992 Saed, Zhang, et al. 2001 992 /id\00 \00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043474!Saed, Zhang, et al. 2000 3474 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044698\1CSaed & Diamond 2002 4698 /id\00\1C\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044699\1CSaed & Diamond 2002 4699 /id\00\1C\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044529+Uchiyama, Kurabayashi, et al. 2000 4529 /id\00+\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044117"Pinsky, Liao, et al. 1998 4117 /id\00"\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044519&Fink, Kazlauskas, et al. 2002 4519 /id\00&\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041559\15Chegini 1997 1559 /id\00\15\00 . QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03992 Saed, Zhang, et al. 2001 992 /id\00 \00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043474!Saed, Zhang, et al. 2000 3474 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044698\1CSaed & Diamond 2002 4698 /id\00\1C\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044699\1CSaed & Diamond 2002 4699 /id\00\1C\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044529+Uchiyama, Kurabayashi, et al. 2000 4529 /id\00+\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044117"Pinsky, Liao, et al. 1998 4117 /id\00"\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044519&Fink, Kazlauskas, et al. 2002 4519 /id\00&\00 This study was performed to evaluate the role of PAI-1, uPA and tPA in basal and in pneumoperitoneum-enhanced postoperative adhesion formation using a laparoscopic mouse model. The choice of PAI-1, uPA and tPA was dictated by the availability of knockout mice for genes encoding for these factors. Material and MethodsAnimalsThe study was performed in 70 female, 10 to 12 week old mice that weighed 30 to 40 g. For the first experiment, twenty 87.5% C57Bl/6J - 12.5% 129SvJ wild-type and transgenic mice, deficient for the gene encoding for PAI-1 (PAI-1+/+ and PAI-1-/-) were used. For the second experiment, thirty 75% C57Bl/6J - 25% 129SvJ wild-type and transgenic mice, deficient for the genes encoding for uPA (uPA+/+ and uPA-/-) and tPA (tPA+/+ and tPA-/-) were used. All wild-type and transgenic mice were obtained from the Center for Transgene Technology and Gene Therapy of the Katholieke Universiteit Leuven (KUL). The uPA, tPA and PAI-1 knockout mice were generated as described QUOTE "(57;58;58;59;59)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\10(57;58;58;59;59)\00\10\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044032)Carmeliet, Kieckens, et al. 1993 4032 /id\00)\00 (57-59) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044031(Carmeliet, Stassen, et al. 1993 4031 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044034+Carmeliet, Schoonjans, et al. 1994 4034 /id\00+\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044031(Carmeliet, Stassen, et al. 1993 4031 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044034+Carmeliet, Schoonjans, et al. 1994 4034 /id\00+\00 . QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044034+Carmeliet, Schoonjans, et al. 1994 4034 /id\00+\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044031(Carmeliet, Stassen, et al. 1993 4031 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044034+Carmeliet, Schoonjans, et al. 1994 4034 /id\00+\00 For the third experiment, 20 NMRI mice were used. Anaesthesia Animals were anaesthetised, intubated with a 22-ga catheter (Insyte-W, Vialon, Becton Dickinson, Madrid, Spain) and mechanically ventilated with room air (tidal volume 500 ?l, 85 strokes/minute, Rodent Ventilator, Harvard Apparatus, Holliston, MA, USA) as described.Laparoscopy Laparoscopy was performed as described in detail previously. For the pneumoperitoneum, 100% CO2 at 20 cm H2O was used and maintained for different time periods according to the experimental design. Induction of intraperitoneal adhesionsAdhesions were induced as described in detail previously by standardized monopolar and bipolar lesions (Autocon 350, Karl Storz, Tüttlingen, Germany). Scoring of adhesions Adhesions were scored after seven days as described previously. Tissue sampling, protein extraction and PAI-1, tPA and total proteins assaysThe abdomen was opened at different time periods before or after pneumoperitoneum exposure as described above, and biopsies were taken rapidly from the pelvic sidewall within the first four minutes. The samples were rinsed with ice cold phosphate buffered saline (PBS), frozen immediately in liquid nitrogen and stored at –80°C. Tissues were homogenized in 500 ?l of PBS containing 1% triton X-100 diluted, 0.1% sodium dodecylsulfate, 0.5% sodium deoxicholate, 0.2% sodium azide, and a cocktail of protease inhibitors (Complete, Roche Diagnostics GmbH, Mannhein, Germany). Following centrifugation (8500 g, 4°C, 10 minutes), the supernatants were assayed for total proteins, PAI-1 and tPA concentration. Tissue protein concentration was measured with a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantification of total proteins. Bovine serum albumin (BSA) was used as a standard (BCA Protein Assay Kit, Pierce, Rockford, UK). PAI-1 and tPA concentration were measured as previously described QUOTE "(62;63;63)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\0A(62;63;63)\00\0A\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044518)Kawasaki, Dewerchin, et al. 2000 4518 /id\00)\00 (62-63) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044556*Declerck, Verstreken, et al. 1995 4556 /id\00*\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044556*Declerck, Verstreken, et al. 1995 4556 /id\00*\00 using a home-made ELISA assay. All samples were assayed at four serial dilutions and the results (pg PAI-1/mg protein or pg tPA/mg protein) are expressed as means SE. Experimental designAll experiments were performed using block randomisation by day. In the first experiment (n=20), basal adhesions and pneumoperitoneum-enhanced adhesions were assessed in PAI-1+/+ mice (n=5 and n=5, respectively) and PAI-1-/- mice (n=5 and n=5, respectively).In the second experiment (n=30), basal adhesions and pneumoperitoneum-enhanced adhesions were assessed in uPA/tPA+/+ mice (n=5 and n=5, respectively), uPA-/- mice (n=5 and n=5, respectively) and tPA-/- mice (n=5 and n=5, respectively). In the third experiment (n=20), the effect of 60 minutes CO2 pneumoperitoneum exposure, without any other peritoneal lesion, on the time course expression of PAI-1 and tPA was evaluated in the abdominal wall. Samples were collected before (n=5) and at different time points after 60 minutes of pneumoperitoneum, i.e. immediately at the end (n=5) and after 3 (n=5) and 6 hours (n=5).StatisticsStatistical analysis was performed with the SAS System (SAS institute, Cary, NC, USA) using a non-parametric test (Kruskal-Wallis) to compare individual groups and Spearman correlation to evaluate association. All data are presented as the mean SE.ResultsAll animals survived the surgical procedures and, in adhesion formation experiments, all of them were available for adhesion scoring after seven days. Adhesions formed between the injured visceral site and the pelvic fat or between the injured parietal site and the pelvic fat. No adhesions were observed at the site of the laparoscopic ports or at other sites. Monopolar lesions systematically induced more adhesions than bipolar lesions. The proportion of adhesions for monopolar and bipolar lesions were, respectively, as follows. In PAI-1 wild-type mice, 16 3% and 4 2% for basal adhesions and 25 4% and 14 3% for pneumoperitoneum-enhanced adhesions. In PAI-1-/- mice, 6 4% and 3 3% for basal adhesions and 8 3% and 2 2% for pneumoperitoneum-enhanced adhesions. In uPA/tPA wild-type mice, 8 5% and 4 3% for basal adhesions and 18 5% and 15 3% for pneumoperitoneum-enhanced adhesions. In uPA-/- mice, 12 4% and 12 6% for basal adhesions and 18 2% and 8 3% for pneumoperitoneum-enhanced adhesions. In tPA-/- mice, 29 2% and 21 7% for basal adhesions and 24 7% and 19 3% for pneumoperitoneum-enhanced adhesions. Similar data were obtained for the extent, type, tenacity and total adhesion scores (data not shown). In order to maximize statistical significance, only the means of both lesions are used for further analysis.In PAI-1 wild-type mice, pneumoperitoneum enhanced adhesion formation (proportion: P=0.02; extent: P=0.01; type: P=0.01; tenacity: P=0.01; total: P=0.01). In comparison with PAI-1 wild-type mice, basal adhesions were lower in PAI-1-/- mice (proportion: P=0.03; extent: P=0.02; type: P=0.01; tenacity: P=0.02; total: P=0.01). In PAI-1-/- mice, pneumoperitoneum did not enhance adhesions. Therefore, in comparison with PAI-1 wild-type mice, pneumoperitoneum-enhanced adhesions were obviously even lower in PAI-1-/- mice (proportion: P=0.01; extent: P=0.01; type: P=0.01; tenacity: P=0.01; total: P=0.01) (Figure 1, Table I). Similar effects were observed for monopolar and bipolar lesions analysed individually. Figure 1: Basal adhesions (□) and pneumoperitoneum-enhanced adhesions (■) in PAI-1+/+ mice and PAI-1-/- mice Quantitative scoringQualitative scoringTable I: Basal adhesions and pneumoperitoneum-enhanced adhesions in PAI-1+/+ mice and PAI-1-/- miceGenotypeAdhesionsAdhesion scoresExtentTypeTenacityPAI-1+/+Basal0.6 0.10.8 0.10.9 0.1Pneumoperitoneum-enhanced 1.3 0.1a 1.6 0.1a 1.7 0.1aPAI-1-/-Basal 0.2 0.1b 0.2 0.1b 0.3 0.1bPneumoperitoneum-enhanced 0.3 0.1b 0.3 0.1b 0.4 0.2bIn uPA/tPA wild-type mice, pneumoperitoneum enhanced adhesion formation (proportion: P=0.03; extent: P=0.02; type: P=NS; tenacity: P=0.02; total: P=0.03). In comparison with uPA/tPA wild-type mice, basal adhesions were higher in both uPA-/- mice (P=NS) and tPA-/-mice (proportion: P=0.02; extent: P=0.02; type: P=0.03; tenacity: P=0.03; total: P=0.02). In both uPA-/- and tPA-/- mice, pneumoperitoneum did not enhance adhesions. In comparison with uPA/tPA wild-type mice, pneumoperitoneum-enhanced adhesions were similar in both uPA-/- and tPA-/- mice. tPA-/- mice developed more basal adhesions (extent: P=0.04) and more pneumoperitoneum-enhanced adhesions (type: P=0.05; tenacity: P=0.03) than uPA-/- mice (Figure 2, Table II). Similar effects were observed for monopolar and bipolar lesions analyzed individually.Figure 2: Basal adhesions (□) and pneumoperitoneum-enhanced adhesions (■) in uPA/tPA+/+ mice, uPA-/- mice and tPA-/- Quantitative scoringQualitative scoringTable II: Basal adhesions and pneumoperitoneum-enhanced adhesions in PAI-1+/+ mice and PAI-1-/- miceGenotypeAdhesionsAdhesion scoresExtentTypeTenacityuPA/tPA+/+Basal0.4 0.10.5 0.20.5 0.2Pneumoperitoneum-enhanced 0.9 0.1a0.9 0.1 1.1 0.1auPA-/-Basal0.6 0.10.9 0.20.8 0.1Pneumoperitoneum-enhanced0.8 0.10.8 0.10.8 0.1tPA-/-Basal 1.3 0.2b 1.9 0.4b 1.8 0.4bPneumoperitoneum-enhanced1.2 0.21.5 0.31.7 0.3The protein concentration of PAI-1 in the abdominal wall increased (Spearman correlation) for at least 6 hours after 1 hour of CO2 pneumoperitoneum exposure (P<0.001). By Kruskal-Wallis these increases in PAI-1 concentration were significant after 1 hour (P=0.02), 3 hours (P=0.02) and 6 hours (P=0.01) (Figure 3). The protein concentration of tPA did not change significantly over time after 1 hour of CO2 pneumoperitoneum exposure (Spearman correlation: P=NS; Kruskal-Wallis in comparison with control group: P=NS) (Figure 3). DiscussionTo the best of our knowledge this is the first report using PAI-1, uPA and tPA knockout mice for the study of adhesion formation in a laparoscopic model. The effects of these components of the plasminogen system on basal adhesions were as expected, confirming previous studies QUOTE "(2-4;4;5;5;6;6;7;7;7;8;8;8;9;9;9;10;10;10;11;11;11;12;12;12)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00<(2-4;4;5;5;6;6;7;7;7;8;8;8;9;9;9;10;10;10;11;11;11;12;12;12)\00<\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041558\16Holmdahl 1997 1558 /id\00\16\00 (2-12) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041334)Ivarsson, Bergstrom, et al. 1998 1334 /id\00)\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041026%Falk, Bjorquist, et al. 2001 1026 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041860%Vipond, Whawell, et al. 1994 1860 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042516!Doody, Dunn, et al. 1989 2516 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042310\1DMenzies & Ellis 1991 2310 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042493\1DMenzies & Ellis 1989 2493 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042359!Dorr, Vemer, et al. 1990 2359 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042314%Orita, Fukasawa, et al. 1991 2314 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044485%Hill-West, Dunn, et al. 1995 4485 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041334)Ivarsson, Bergstrom, et al. 1998 1334 /id\00)\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041026%Falk, Bjorquist, et al. 2001 1026 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041860%Vipond, Whawell, et al. 1994 1860 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042516!Doody, Dunn, et al. 1989 2516 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042310\1DMenzies & Ellis 1991 2310 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042493\1DMenzies & Ellis 1989 2493 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042359!Dorr, Vemer, et al. 1990 2359 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042314%Orita, Fukasawa, et al. 1991 2314 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044485%Hill-West, Dunn, et al. 1995 4485 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041183!Holmdahl & Ivarsson 1999 1183 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042516!Doody, Dunn, et al. 1989 2516 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042310\1DMenzies & Ellis 1991 2310 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042359!Dorr, Vemer, et al. 1990 2359 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042493\1DMenzies & Ellis 1989 2493 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042314%Orita, Fukasawa, et al. 1991 2314 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044485%Hill-West, Dunn, et al. 1995 4485 /id\00%\00 . Indeed, in comparison with wild-type mice, PAI-1 knockout mice developed less adhesions whereas both uPA and tPA knockout mice developed more adhesions. This effect was expected since the lack of uPA/tPA reduces plasmin activation and fibrin degradation, thus leading to adhesion formation, whereas the lack of PAI-1 reduces the inactivation of uPA/tPA, increasing plasmin levels and fibrin degradation, thus reducing adhesion formation. The less important increase in adhesion formation in uPA than in tPA knockout mice is also consistent with the effects of recombinant uPA and tPA in animal models. Indeed, while tPA administration clearly reduced adhesion formation, the results of uPA administration were controversial QUOTE "(6;7;7;7;8;8;9;9;9;10;10;11;11;11;12;12;12;64;64;64)" ADDIN REFMAN ?\11\05‘\19\01\00\00\004(6;7;7;7;8;8;9;9;9;10;10;11;11;11;12;12;12;64;64;64)\004\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041860%Vipond, Whawell, et al. 1994 1860 /id\00%\00 (6-12, 64) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042516!Doody, Dunn, et al. 1989 2516 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042310\1DMenzies & Ellis 1991 2310 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042493\1DMenzies & Ellis 1989 2493 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042359!Dorr, Vemer, et al. 1990 2359 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042314%Orita, Fukasawa, et al. 1991 2314 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044485%Hill-West, Dunn, et al. 1995 4485 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042786(Rivkind, Lieberman, et al. 1985 2786 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042516!Doody, Dunn, et al. 1989 2516 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042310\1DMenzies & Ellis 1991 2310 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042493\1DMenzies & Ellis 1989 2493 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042359!Dorr, Vemer, et al. 1990 2359 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042314%Orita, Fukasawa, et al. 1991 2314 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044485%Hill-West, Dunn, et al. 1995 4485 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042786(Rivkind, Lieberman, et al. 1985 2786 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042516!Doody, Dunn, et al. 1989 2516 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042493\1DMenzies & Ellis 1989 2493 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042314%Orita, Fukasawa, et al. 1991 2314 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042786(Rivkind, Lieberman, et al. 1985 2786 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044485%Hill-West, Dunn, et al. 1995 4485 /id\00%\00 . These differences between uPA and tPA could be explained by the fact that whereas uPA activity requires the specific binding of plasminogen to fibrin, tPA activity is greatly enhanced by fibrin, since its binding to its specific receptor exposes a strong plasminogen-binding site on the surface of the fibrin molecule QUOTE "(26;26;26;65;65;65;66;66;66;67)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\1F(26;26;26;65;65;65;66;66;66;67)\00\1F\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044362%Norrman, Wallen, et al. 1985 4362 /id\00%\00 (26, 65-67) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044354$Pannell, Black, et al. 1988 4354 /id\00$\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044361$Collen, Lijnen, et al. 1989 4361 /id\00$\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044362%Norrman, Wallen, et al. 1985 4362 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044361$Collen, Lijnen, et al. 1989 4361 /id\00$\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044354$Pannell, Black, et al. 1988 4354 /id\00$\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044363%Ichinose, Takio, et al. 1986 4363 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044362%Norrman, Wallen, et al. 1985 4362 /id\00%\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044354$Pannell, Black, et al. 1988 4354 /id\00$\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044361$Collen, Lijnen, et al. 1989 4361 /id\00$\00 .This study also confirmed our previous finding that the pneumoperitoneum is a cofactor in adhesion formation QUOTE "(38-41)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(38-41)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03183'Ordonez, Dominguez, et al. 1997 183 /id\00'\00 (38-41) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03168#Yesildaglar & Koninckx 2000 168 /id\00#\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03164\1FMolinas & Koninckx 2000 164 /id\00\1F\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043673&Molinas, Mynbaev, et al. 2001 3673 /id\00&\00 . QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03164\1FMolinas & Koninckx 2000 164 /id\00\1F\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043673&Molinas, Mynbaev, et al. 2001 3673 /id\00&\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03183'Ordonez, Dominguez, et al. 1997 183 /id\00'\00 Pneumoperitoneum-enhanced adhesions were observed in all wild-type mice. Absence of pneumoperitoneum-enhanced adhesions in PAI-1, uPA and tPA knockout mice can be explained by postulating that pneumoperitoneum-enhanced adhesion formation observed in wild-type mice was due, at least in part, to an up regulation of PAI-1. In PAI-1 knockout mice up-regulation of PAI-1 and thus pneumoperitoneum-enhanced adhesions would obviously be impossible. Mice lacking either uPA or tPA already have increased basal adhesions. Up-regulation of PAI-1 will not inhibit the non-existing uPA/tPA activity and will thus not further increase adhesion formation. CO2 pneumoperitoneum up regulation of PAI-1 was confirmed by the ELISA assay of the abdominal wall. This is moreover consistent with the reported up regulation of PAI-1 by hypoxia QUOTE "(53;54;54;54;55;55;55)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\16(53;54;54;54;55;55;55)\00\16\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044529+Uchiyama, Kurabayashi, et al. 2000 4529 /id\00+\00 (53-55) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044117"Pinsky, Liao, et al. 1998 4117 /id\00"\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044519&Fink, Kazlauskas, et al. 2002 4519 /id\00&\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044117"Pinsky, Liao, et al. 1998 4117 /id\00"\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044519&Fink, Kazlauskas, et al. 2002 4519 /id\00&\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044117"Pinsky, Liao, et al. 1998 4117 /id\00"\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044519&Fink, Kazlauskas, et al. 2002 4519 /id\00&\00 , since the CO2 pneumoperitoneum-enhanced adhesion formation is probably mediated by mesothelial hypoxia as described QUOTE "(41)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(41)\00\04\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043673&Molinas, Mynbaev, et al. 2001 3673 /id\00&\00 (41). It is unclear at present whether the pneumoperitoneum-induced mesothelial damage causes an inflammatory reaction, since this equally could up regulates PAI-1. Indeed, PAI-1 is regulated by many factors, such as thrombin, endotoxin, interleukine-1, TNF-, TGF-, trauma and infection, a common denominator or these factors being the inflammatory reaction QUOTE "(68-70)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(68-70)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041210$Brosens, Campo, et al. 1999 1210 /id\00$\00 (68-70) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041165#Gordts, Campo, et al. 2000 1165 /id\00#\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041350#Gordts, Campo, et al. 1998 1350 /id\00#\00 . The tissue injury locally up regulates PAI-1 and down regulates tPA QUOTE "(33;71;72)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\0A(33;71;72)\00\0A\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044502(Holmdahl, Eriksson, et al. 1998 4502 /id\00(\00 (33, 71-73) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042514.Thompson, Paterson-Brown, et al. 1989 2514 /id\00.\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044514,Scott-Coombes, Whawell, et al. 1995 4514 /id\00,\00 , probably in response to local hypoxia, inflammation or both. The CO2 pneumoperitoneum up regulates PAI-1 and, although we failed to confirm in this study, down regulates tPA QUOTE "(73)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(73)\00\04\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044721,Nagelschmidt, Gerbecks, et al. 2001 4721 /id\00,\00 (74), probably in response to hypoxia, inflammation or both in the whole peritoneal mesothelium. It therefore remains unclear whether the mechanisms of PAI-1 up regulation following tissue injury (hypoxia and inflammation) or following pneumoperitoneum (hypoxia followed by inflammation) are identical or not. QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042514.Thompson, Paterson-Brown, et al. 1989 2514 /id\00.\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041792,Scott-Coombes, Whawell, et al. 1995 1792 /id\00,\00 To fully understand the mechanisms involved more detailed investigation are required in the normal and in the damaged peritoneum, i.e. by studying the effects of the duration of pneumoperitoneum, the insufflation pressure and the addition of oxygen. In conclusion, our data confirm the role of PAI-1, uPA and tPA in basal adhesions and the role of the pneumoperitoneum as a cofactor in adhesion formation. Our findings in knockout mice and in the ELISA assay indicate PAI-1 up-regulation as a mechanism for pneumoperitoneum-enhanced adhesion formation. This is consistent with our concept that CO2 pneumoperitoneum causes mesothelial hypoxia and with the up regulation of PAI-1 through hypoxia in the peritoneal injured areas.Role of vascular endothelial growth factor and placental growth factor in basal adhesion formation and in CO2 pneumoperitoneum-enhanced adhesion formation following laparoscopic surgery in miceIntroductionThe speed of fibrin degradation following peritoneal injury is a critical process in peritoneal repair and adhesion formation. If the local fibrinolysis is insufficient, the remaining fibrin serves as a scaffold for fibroblast growth, extra cellular matrix (ECM) deposition and capillary growth, leading to adhesion formation. The role of fibrin and other components of the plasminogen system, fibroblasts and ECM in adhesion formation is well known QUOTE "(1;2;2-6)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\09(1;2;2-6)\00\09\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041558\16Holmdahl 1997 1558 /id\00\16\00 (1-6) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041183!Holmdahl & Ivarsson 1999 1183 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041334)Ivarsson, Bergstrom, et al. 1998 1334 /id\00)\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03979&Holmdahl, Kotseos, et al. 2001 979 /id\00&\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043808&Chegini, Kotseos, et al. 2001 3808 /id\00&\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043809&Chegini, Kotseos, et al. 2001 3809 /id\00&\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041183!Holmdahl & Ivarsson 1999 1183 /id\00!\00 . The role of angiogenesis, however, has barely been explored neither after laparotomy nor after laparoscopy.Angiogenesis, the formation of new blood vessels extending from existing vessels, occurs when the distance from the nearest capillary exceeds an efficient diffusion range maintaining adequate supply of oxygen and nutrients to cells. Angiogenesis is regulated mainly by cellular hypoxia through the modulation of angiogenic factors expression, such as the up-regulation of VEGF-A QUOTE "(7-10)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\06(7-10)\00\06\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044365\15Folkman 2000 4365 /id\00\15\00 (7-10) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044056\17Carmeliet 1999 4056 /id\00\17\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044064 Carmeliet & Collen 2000 4064 /id\00 \00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044054 Carmeliet & Collen 1998 4054 /id\00 \00 . A role for angiogenesis and hypoxia in adhesion formation is suspected for several reasons. Firstly, tissue necrosis and the inflammatory reaction following trauma involves cellular hypoxia that have been recognized as important in adhesion formation QUOTE "(11-13)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(11-13)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042927\13Ellis 1982 2927 /id\00\13\00 (11-13) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043797\15Raftery 1973 3797 /id\00\15\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\0425190Tsimoyiannis, Tsimoyiannis, et al. 1989 2519 /id\000\00 . Secondly, hypoxia modulates the expression of several molecules involved in different stages of adhesion formation, such as PAI-1, tPA, TGF-, MMPs and TIMPs QUOTE "(14-17)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(14-17)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043475!Saed, Zhang, et al. 1999 3475 /id\00!\00 (14- QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043476!Saed, Zhang, et al. 2000 3476 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03992 Saed, Zhang, et al. 2001 992 /id\00 \00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043474!Saed, Zhang, et al. 2000 3474 /id\00!\00 QUOTE "(18-23)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(18-23)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044698\1CSaed & Diamond 2002 4698 /id\00\1C\00 23) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044699\1CSaed & Diamond 2002 4699 /id\00\1C\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044529+Uchiyama, Kurabayashi, et al. 2000 4529 /id\00+\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044117"Pinsky, Liao, et al. 1998 4117 /id\00"\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044519&Fink, Kazlauskas, et al. 2002 4519 /id\00&\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041559\15Chegini 1997 1559 /id\00\15\00 . Thirdly, the CO2 pneumoperitoneum is a cofactor in adhesion formation after laparoscopic surgery and s QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03168#Yesildaglar & Koninckx 2000 168 /id\00#\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03164\1FMolinas & Koninckx 2000 164 /id\00\1F\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043673&Molinas, Mynbaev, et al. 2001 3673 /id\00&\00 ince adhesions increase in a time- and pressure-dependent manner and decrease with the addition of oxygen, mesothelial hypoxia has been suggested as the driving mechanism QUOTE "(24-27)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(24-27)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03183'Ordonez, Dominguez, et al. 1997 183 /id\00'\00 (24-27). It is unknown, however, whether the mechanism of adhesion formation following a peritoneal lesion only (“basal adhesions”) and of the increased adhesion formation following a peritoneal lesion with the additional effect of the pneumoperitoneum (“pneumoperitoneum-enhanced adhesions”) are different.Vascular endothelial growth factor (VEGF) is a family of angiogenic factors including VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PlGF). VEGF-A is transcribed from a single gene and processed into four isoforms in humans (VEGF-A121, VEGF-A165, VEGF-A189 and VEGF-A206) and three isoforms in mice (VEGF-A120, VEGF-A164 and VEGF-A188) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044416\15Ferrara 1996 4416 /id\00\15\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044381\15Ferrara 2000 4381 /id\00\15\00 . VEGF-B is transcribed from a single gene and processed into two isoforms in humans and in mice (VEGF-B167 and VEGF-B186). PlGF is also transcribed from a single gene and processed into three isoforms in humans (PlGF-1, PlGF-2 and PlGF-3) and one isoform in mice (PlGF-2) QUOTE "(28-41)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(28-41)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043489$Neufeld, Cohen, et al. 1999 3489 /id\00$\00 (28-41) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044416\15Ferrara 1996 4416 /id\00\15\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044381\15Ferrara 2000 4381 /id\00\15\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043610)Maglione, Guerriero, et al. 1991 3610 /id\00)\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043611)Maglione, Guerriero, et al. 1993 3611 /id\00)\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043541\1DCao, Ji, et al. 1997 3541 /id\00\1D\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043628'Persico, Vincenti, et al. 1999 3628 /id\00'\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043559$DiPalma, Tucci, et al. 1996 3559 /id\00$\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044708'Olofsson, Jeltsch, et al. 1999 4708 /id\00'\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044704+Olofsson, Korpelainen, et al. 1998 4704 /id\00+\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043624(Olofsson, Pajusola, et al. 1996 3624 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044712(Olofsson, Pajusola, et al. 1996 4712 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044710'Joukov, Kaipainen, et al. 1997 4710 /id\00'\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044714\14Clauss 2000 4714 /id\00\14\00 .VEGF-A, VEGF-B and PlGF are dimeric glycoproteins that bind to two tyrosine kinase receptors: VEGFR-1 (VEGF-A, VEGF-B and PlGF) and VEGFR-2 (VEGF-A) QUOTE "(28-30;41)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\0A(28-30;41)\00\0A\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043489$Neufeld, Cohen, et al. 1999 3489 /id\00$\00 (28-30, 41) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044416\15Ferrara 1996 4416 /id\00\15\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044381\15Ferrara 2000 4381 /id\00\15\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044714\14Clauss 2000 4714 /id\00\14\00 . Heterodimerization of VEGF-A with VEGF-B or PlGF controls the bioavailability of VEGF-A, modulating its mitogenic, chemotactic and vascular permeability-inducing properties QUOTE "(36-45)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(36-45)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043560$DiSalvo, Bayne, et al. 1995 3560 /id\00$\00 (36-45) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043534+Birkenhager, Schneppe, et al. 1996 3534 /id\00+\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043627 Park, Chen, et al. 1994 3627 /id\00 \00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043539!Cao, Linden, et al. 1996 3539 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044708'Olofsson, Jeltsch, et al. 1999 4708 /id\00'\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044704+Olofsson, Korpelainen, et al. 1998 4704 /id\00+\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043624(Olofsson, Pajusola, et al. 1996 3624 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044712(Olofsson, Pajusola, et al. 1996 4712 /id\00(\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044710'Joukov, Kaipainen, et al. 1997 4710 /id\00'\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044714\14Clauss 2000 4714 /id\00\14\00 . In contrast with the marked up-regulation of VEGF-A by hypoxia, the up-regulation of VEGF-B and PlGF by hypoxia is controversial QUOTE "(46-49)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(46-49)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043503%Simpson, Murphy, et al. 1999 3503 /id\00%\00 (46-49) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043659)Viglietto, Maglione, et al. 1995 3659 /id\00)\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043525!Ahmed, Dunk, et al. 2000 3525 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043576%Green, Lichtlen, et al. 2001 3576 /id\00%\00 . The formation of VEGF-A/VEGF-B and of VEGF-A/PlGF heterodimers, however, is clearly modulated by hypoxia QUOTE "(45;50)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(45;50)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043539!Cao, Linden, et al. 1996 3539 /id\00!\00 (45, 50) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043540\1FCao, Chen, et al. 1996 3540 /id\00\1F\00 . These experiments were designed to investigate the role of the angiogenic factors VEGF-A, VEGF-B and PlGF in both “basal adhesions” and “CO2 pneumoperitoneum-enhanced adhesions” in a laparoscopic mouse model using transgenic mice and monoclonal antibodies.Material and MethodsAnimalsThe study was performed in 110 female, 10-12 weeks old mice weighing 30-40 g. For the first study twenty 75% Swiss - 25% 129SvJ wild-type mice (VEGF-A+/+) and transgenic mice deficient for VEGF-A120 and for VEGF-A188 and over expressing exclusively VEGF-A164 (VEGF-A164/164) were used. For the second study twenty 100% C57Bl/6J wild-type mice (VEGF-B+/+) and transgenic mice deficient for VEGF-B (VEGF-B-/-) were used. For the third study twenty 50% Swiss - 50% 129SvJ wild-type mice (PlGF+/+) and transgenic mice deficient for PlGF (PlGF-/-) were used. For the fourth study fifty 100% Swiss wild-type mice were used. VEGF-A and PlGF wild-type mice and transgenic mice were obtained from the Centre for Transgene Technology and Gene Therapy of the Katholieke Universiteit Leuven (KUL) and VEGF-B wild-type mice and transgenic mice from the Ludwig Institute for Cancer Research, Stockholm Branch, Sweden. The transgenic mice were generated as described QUOTE "(51-54)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(51-54)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044059#Carmeliet, Ng, et al. 1999 4059 /id\00#\00 (51-54) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044700"Stalmans, Ng, et al. 2002 4700 /id\00"\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043492&Carmeliet, Moons, et al. 2001 3492 /id\00&\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044702%Aase, von Euler, et al. 2001 4702 /id\00%\00 . No major phenotypic differences were observed between wild-type and transgenic mice of the same strain. Little differences, however, have been reported such as an atrial conduction abnormality in VEGF-B-/- mice characterized by a prolonged PQ interval in the electrocardiogram, which did not generate arrhythmia or any other malfunction of the heart (54) and a subtle remodelling defect of retinal vessels in PlGF-/- mice (53). Anaesthesia Animals were anaesthetised, intubated with a 22-ga catheter (Insyte-W, Vialon, Becton Dickinson, Madrid, Spain) and mechanically ventilated with room air (tidal volume 500 ?l, 85 strokes/minute, Rodent Ventilator, Harvard Apparatus, Holliston, MA, USA) as described.LaparoscopyLaparoscopy was performed as described in detail previously. For the pneumoperitoneum, 100% CO2 at 20 cm H2O was used and maintained for different time periods according to the experimental design.Induction of intraperitoneal adhesions Adhesions were induced as described in detail previously by standardized monopolar and bipolar lesions (Autocon 350, Karl Storz, Tüttlingen, Germany). PlGF antibodies preparation and administrationMouse monoclonal antibodies were raised against murine PlGF-2 (R&D Systems, Abingdon, U.K.) using PlGF-/- mice and methods as described QUOTE "(55)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(55)\00\04\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044073)Declerck, Carmeliet, et al. 1995 4073 /id\00)\00 (55). Purified clones were screened for their ability to inhibit the binding of PlGF to VEGFR-1/Flt-1 in a homemade ELISA assay as described QUOTE "(53)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(53)\00\04\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043492&Carmeliet, Moons, et al. 2001 3492 /id\00&\00 (53). The antibodies varied from complete neutralization of the binding of PlGF to VEGFR-1/Flt-1 to no neutralization. In this in vivo study of adhesion formation, we used 1 clone that does not neutralize (PLGE1G11: PlGF antibody A), 2 clones that completely neutralizes (PL17A10F12: PlGF antibody B and PL5D11D4: PlGF antibody C) and 1 clone that partially neutralizes (PLGH12G5: PlGF antibody D) the binding of PlGF to VEGFR-1/Flt-1. Animals received 4 intraperitoneal doses of 20?g/g of either mouse IgG (Sigma Chemical Company, St. Louis, MO, USA) or 1 of the 4 PlGF antibodies (A, B, C or D) diluted in 200 ?l of saline. The first dose was administrated on day 0 at the beginning of the surgery and under direct laparoscopic vision, whereas the subsequent doses were injected on days 2, 4 and 6 following surgery. Scoring of adhesions Adhesions were scored after seven days as described previously. Experimental designAll experiments were performed using block randomization by days. In the first experiment (n=20), basal adhesions and pneumoperitoneum-enhanced adhesions were assessed in VEGF-A+/+ mice (n=5 and n=5, respectively) and VEGF-A164/164 mice (n=5 and n=5, respectively).In the second experiment (n=20), basal adhesions and pneumoperitoneum-enhanced adhesions were assessed in VEGF-B+/+ mice (n=5 and n=5, respectively) and VEGF-B-/- mice (n=5 and n=5, respectively).In the third experiment (n=20), basal adhesions and pneumoperitoneum-enhanced adhesions were assessed in PlGF+/+ mice (n=5 and n=5, respectively) and PlGF-/- mice (n=5 and n=5, respectively).In the fourth experiment (n=50), basal adhesions and pneumoperitoneum-enhanced adhesions were assessed in mice treated with mouse IgG (n=5 and n=5, respectively) or with PlGF antibodies A (n=5 and n=5, respectively), B (n=5 and n=5, respectively), C (n=5 and n=5, respectively) or D (n=5 and n=5, respectively). StatisticsStatistical analysis was performed with the SAS System (SAS institute, Cary, NC, USA) using the non-parametric Kruskal-Wallis test to compare individual groups. All data are presented as the mean SE.ResultsAll animals survived the surgical procedures and all of them were available for adhesion scoring after seven days. Adhesions only formed between the injured visceral site and the pelvic fat or between the injured parietal site and the pelvic fat. No adhesions were observed at the site of the laparoscopic ports or at other sites. In all experiments monopolar lesions systematically induced more adhesions than bipolar lesions (data not shown) for both basal and pneumoperitoneum-enhanced adhesion formation and the effect of the pneumoperitoneum. The effect of the pneumoperitoneum, however, was equally observed with both monopolar and bipolar lesions separately analyzed. In order to maximize statistical significance, only the means of both lesions are discussed in this manuscript.In VEGF-A wild-type mice, pneumoperitoneum enhanced adhesion formation (proportion: P=0.03; extent: P=0.04; total: P=0.05). In comparison with VEGF-A wild-type mice, basal adhesions were higher in VEGF-A164/164 mice (proportion: P=0.01; extent: P=0.02; type: P=0.02; tenacity: P=0.02; total: P=0.01). In VEGF-A164/164 mice, pneumoperitoneum did not enhance adhesion formation. In comparison with VEGF-A wild-type mice, pneumoperitoneum-enhanced adhesions were higher in VEGF-A164/164 mice (proportion: P=0.05; extent: P=0.02; tenacity: P=0.04; total: P=0.05) (Figure 1, Table I). Figure 1: Basal adhesions (□) and pneumoperitoneum-enhanced adhesions (■) in VEGF-A+/+ mice and VEGF-A164/164 mice Quantitative scoringQualitative scoringTable I: Basal adhesions and pneumoperitoneum-enhanced adhesions in VEGF-A+/+ mice and VEGF-A164/164 miceGenotypeAdhesionsAdhesion scoresExtentTypeTenacityVEGF-A+/+Basal0.6 0.30.6 0.30.5 0.3Pneumoperitoneum-enhanced 1.6 0.2a1.6 0.21.4 0.2VEGF-A164/164Basal 2.0 0.2b 2.0 0.2b 1.9 0.2bPneumoperitoneum-enhanced 2.8 0.4b2.1 0.3 2.3 0.2bIn VEGF-B wild-type mice, pneumoperitoneum enhanced adhesion formation (proportion: P=0.02; type: P=0.04; total: P=0.05). In comparison with VEGF-B wild-type mice, basal adhesions were similar in VEGF-B-/- mice (P=NS). In VEGF-B-/- mice, pneumoperitoneum did not enhance adhesion formation. Therefore, in comparison with VEGF-B wild-type mice, pneumoperitoneum-enhanced adhesions were obviously lower in VEGF-B-/- mice (proportion: P=0.05; type: P=0.03; total: P=0.05) (Figure 2, Table II). Figure 2: Basal adhesions (□) and pneumoperitoneum-enhanced adhesions (■) in VEGF-B+/+ mice and VEGF-B-/- mice Quantitative scoringQualitative scoringTable II: Basal adhesions and pneumoperitoneum-enhanced adhesions in VEGF-B+/+ mice and VEGF-B-/- miceGenotypeAdhesionsAdhesion scoresExtentTypeTenacityVEGF-B+/+Basal0.4 0.20.5 0.30.6 0.3Pneumoperitoneum-enhanced0.7 0.2 1.1 0.2a0.9 0.2VEGF-B-/-Basal0.5 0.20.7 0.20.7 0.2Pneumoperitoneum-enhanced0.4 0.2 0.6 0.3b0.5 0.2In PlGF wild-type mice, pneumoperitoneum enhanced adhesion formation (proportion: P=0.01; extent: P=0.01; type: P=0.01; tenacity: P=0.02; total: P=0.01). In comparison with PlGF wild-type mice, basal adhesions were slightly lower (P=NS) in PlGF-/- mice. In PlGF-/- mice, pneumoperitoneum did not enhance adhesion formation. Therefore, in comparison with PlGF wild-type mice, pneumoperitoneum-enhanced adhesions were obviously lower in PlGF-/- mice (proportion: P=0.01; extent: P=0.01; type: P=0.01; tenacity: P=0.01; total: P=0.01) (Figure 3, Table III). Figure 3: Basal adhesions (□) and pneumoperitoneum-enhanced adhesions (■) in PlGF +/+ mice and PlGF -/- mice Quantitative scoringQualitative scoringTable III: Basal adhesions and pneumoperitoneum-enhanced adhesions in PlGF +/+ mice and PlGF -/- miceGenotypeAdhesionsAdhesion scoresExtentTypeTenacityPlGF +/+Basal0.4 0.10.4 0.10.5 0.2Pneumoperitoneum-enhanced 1.3 0.2a 1.3 0.2a 1.2 0.1aPlGF -/-Basal0.3 0.10.4 0.10.5 0.2Pneumoperitoneum-enhanced 0.2 0.1b 0.3 0.1b 0.3 0.1bIn the experiment with PlGF antibodies two control groups were used, i.e. mice treated with IgG and mice treated with the non-neutralizing PlGF antibody A, and therefore all other groups were compared with both control groups. In mice treated with IgG, pneumoperitoneum enhanced adhesion formation (proportion: P=0.01; extent: P=0.01; total: P=0.03). In comparison with mice treated with IgG, basal adhesions were similar in mice treated with the PlGF antibody A (P=NS). In mice treated with PlGF antibody A, pneumoperitoneum enhanced adhesion formation (proportion: P=0.02; extent: P=0.04; tenacity: P=0.02; total: P=0.05). In comparison with mice treated with IgG, pneumoperitoneum-enhanced adhesions were similar in mice treated with PlGF antibody A (P=NS) (Figure 4, Table IV). In mice treated with the neutralizing PlGF antibody B, basal adhesions were lower than in mice treated with IgG or with PlGF antibody A (proportion: P=0.03, P=NS; type: P=0.02, P=NS; tenacity: P=0.05, P=NS; total: P=0.04, P=NS, respectively). In mice treated with PlGF antibody B, pneumoperitoneum did not enhance adhesion formation. Therefore, in these mice pneumoperitoneum-enhanced adhesions were lower than in mice treated with IgG or with PlGF antibody A (proportion: P=0.01, P=0.02; extent: P=0.01, P=0.02; tenacity: P=0.04, P=NS; total: P=0.02, P=0.05, respectively) (Figure 4, Table IV). Figure 4: Basal adhesions (□) and pneumoperitoneum-enhanced adhesions (■) in mice treated with IgG or with PlGF mice antibodies with different neutralizing capacity Quantitative scoringQualitative scoringTable IV: Basal adhesions and pneumoperitoneum-enhanced adhesions in mice treated with IgG or with PlGF mice antibodies with different neutralizing capacityGenotypeAdhesionsAdhesion scoresExtentTypeTenacityIgG Basal1.1 0.11.4 0.11.5 0.2Pneumoperitoneum-enhanced 2.2 0.2a1.8 0.22.0 0.1Ab A Basal1.0 0.21.0 0.31.1 0.2Pneumoperitoneum-enhanced 1.9 0.3a1.8 0.1 1.9 0.2aAb B Basal0.7 0.1 0.7 0.2b 0.8 0.2bPneumoperitoneum-enhanced 0.7 0.2bc0.9 0.4 1.0 0.3bAb C Basal 0.3 0.2b 0.3 0.2b 0.3 0.2bPneumoperitoneum-enhanced 0.5 0.1bc 0.5 0.1bc 0.8 0.1bcAb D Basal0.9 0.21.0 0.21.1 0.2Pneumoperitoneum-enhanced1.6 0.31.5 0.21.6 0.2In mice treated with the neutralizing PlGF antibody C, basal adhesions were lower than in mice treated with IgG or with PlGF antibody A (proportion: P=0.01, P=0.03; extent: P=0.03, P=NS; type: P=0.01, P=NS; tenacity: P=0.01, P=0.03; total: P=0.01, P=NS, respectively). In mice treated with PlGF antibody C, pneumoperitoneum did not enhance adhesion formation. Therefore, in these mice pneumoperitoneum-enhanced adhesions were lower than in mice treated with IgG or with PlGF antibody A (proportion: P=0.01, P=0.02; extent: P=0.01, P=0.01; type: P=0.01, P=0.01; tenacity: P=0.01, P=0.01; total: P=0.01, P=0.01, respectively) (Figure 4, Table IV). In mice treated with the semi neutralizing PlGF antibody D, basal adhesions were similar than in mice treated with IgG (P=NS) or with PlGF antibody A (P=NS). In mice treated with PlGF antibody D, pneumoperitoneum enhanced adhesion formation but not significantly. In these mice, pneumoperitoneum-enhanced adhesions were similar than in mice treated with IgG (P=NS) or with PlGF antibody A (P=NS) (Figure 4, Table IV).Comparing the effects of the neutralizing PlGF antibodies B and C with the effect of the semi neutralizing antibody D, the following differences were significant: basal adhesions were higher in mice treated with PlGF antibody D than in mice treated with PlGF antibody C (proportion: P=0.01) and pneumoperitoneum-enhanced adhesions were higher in mice treated with PlGF antibody D than in mice treated with PlGF antibody B and C (proportion: P=0.05, P=0.02; extent: P=NS, P=0.01; type: P=NS, P=0.01; tenacity: P=NS, P=0.02; total: P=NS, P=0.02, respectively). These data are consistent with PlGF antibody D having a partial neutralizing effect. The effects of antibodies B and C were comparable (Figure 4, Table IV). DiscussionThis study confirms our previous finding that the pneumoperitoneum is a cofactor in adhesion formation QUOTE "(24-27)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(24-27)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03183'Ordonez, Dominguez, et al. 1997 183 /id\00'\00 (24-27) since pneumoperitoneum-enhanced adhesions were observed in all wild-type mice used as control animals QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03168#Yesildaglar & Koninckx 2000 168 /id\00#\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03164\1FMolinas & Koninckx 2000 164 /id\00\1F\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043673&Molinas, Mynbaev, et al. 2001 3673 /id\00&\00 . To the best of our knowledge this is the first study demonstrating directly a role of VEGF-A, VEGF-B and PlGF in postoperative adhesion formation. Our results can be explained by postulating that pneumoperitoneum enhances adhesion formation, at least in part, through an up-regulation of VEGF-A164, VEGF-B and PlGF. This is consistent with the reported up-regulation of VEGF-A and VEGF-A/PlGF heterodimers by hypoxia QUOTE "(21;22;28-30;45;50)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\13(21;22;28-30;45;50)\00\13\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043489$Neufeld, Cohen, et al. 1999 3489 /id\00$\00 (21, 22, 28-30, 45, 50) and confirms the hypothesis that QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044416\15Ferrara 1996 4416 /id\00\15\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044381\15Ferrara 2000 4381 /id\00\15\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043539!Cao, Linden, et al. 1996 3539 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043540\1FCao, Chen, et al. 1996 3540 /id\00\1F\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044117"Pinsky, Liao, et al. 1998 4117 /id\00"\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044519&Fink, Kazlauskas, et al. 2002 4519 /id\00&\00 CO2 pneumoperitoneum-enhanced adhesion formation is mediated by mesothelial hypoxia QUOTE "(27)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(27)\00\04\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043673&Molinas, Mynbaev, et al. 2001 3673 /id\00&\00 (27). In VEGF-A164/164 mice, basal adhesions were higher than in wild-type mice, demonstrating a direct role of VEGF-A164 in basal adhesion formation. This was not unexpected since VEGF-A was found in peritoneal adhesions by immunohistochemistry QUOTE "(58)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(58)\00\04\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041425"Wiczyk, Grow, et al. 1998 1425 /id\00"\00 (58) and by reverse transcriptase polymerase chain reaction QUOTE "(59)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(59)\00\04\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041103"Rout, Oommen, et al. 2000 1103 /id\00"\00 (59). Moreover, a reduction in adhesion formation following open surgery was reported in mice after the administration of polyclonal antibodies against VEGF-A QUOTE "(60)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(60)\00\04\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041648%Saltzman, Olson, et al. 1996 1648 /id\00%\00 (60). The slight but not significant increased in adhesion formation following 60 minutes of CO2 pneumoperitoneum in VEGF-A164/164 mice does not rule out an up-regulation of VEGF-A164 by the CO2 pneumoperitoneum, since the effect of VEGF-A on adhesion formation could already be near maximal in mice over expressing this VEGF-A isoform. In VEGF-B-/- mice, basal adhesions were comparable with those in wild-type mice, suggesting that VEGF-B has no major role in basal adhesion formation. In these VEGF-B-/- mice, adhesion formation did not increase following 60 minutes of CO2 pneumoperitoneum, demonstrating that the mechanism of CO2 pneumoperitoneum-enhanced adhesions involves VEGF-B, which obviously cannot be up-regulated in these mice. In PlGF-/- mice, basal adhesions were identical or possibly slightly lower than in wild-type mice, suggesting that PlGF does not play a major role in basal adhesion formation. It should be recognized, however, that the design of the study and the number of animals involved do not permit to detect minor changes in basal adhesions. Adhesion formation clearly did not increase following 60 minutes of CO2 pneumoperitoneum, demonstrating that the mechanism of CO2 pneumoperitoneum-enhanced adhesions involves PlGF. This was confirmed by the absence of pneumoperitoneum-enhanced adhesions in mice treated with PlGF neutralizing antibodies. We recently evaluated the role of the plasminogen system, i.e. PAI-1, uPA and tPA, in adhesion formation in transgenic mice using the same model (61), confirming the major role of these factors in basal adhesions. The up-regulation of PAI-1 by the CO2 pneumoperitoneum was shown to be involved in pneumoperitoneum-enhanced adhesions. To fully understand the exact importance of the plasminogen system and of the VEGF family on basal adhesions and pneumoperitoneum-enhanced adhesions more detailed investigations in the normal and in the damaged peritoneum are obviously required, e.g. by studying the effects of the duration of pneumoperitoneum, the insufflation pressure and the addition of oxygen. The available data, however, show clearly that the mechanisms involved in basal adhesions and in pneumoperitoneum enhanced adhesions are, at least partially, different. In conclusion, our data confirm the role of the CO2 pneumoperitoneum as a cofactor in adhesion formation and demonstrate a direct role of VEGF-A in basal adhesions and of VEGF-B and PlGF in pneumoperitoneum-enhanced adhesions, suggesting VEGF-A164, VEGF-B and PlGF up-regulation as a mechanism for this pneumoperitoneum-enhanced adhesion formation. This is fully consistent with the up-regulation of these angiogenic factors by hypoxia and with the concept that CO2 pneumoperitoneum causes mesothelial hypoxia. These observations open new insights in the pathophysiology of adhesion formation. Since the mechanisms involved in basal adhesions and in pneumoperitoneum-enhanced adhesions are, at least partially, different, new methods for adhesion prevention following laparoscopic surgery could be developed.Role of hypoxia inducible factors 1 and 2 in basal adhesion formation and in CO2 pneumoperitoneum-enhanced adhesion formation following laparoscopic surgery in miceIntroductionSurgical peritoneal trauma initiates an inflammatory reaction determining fibrin deposition on the peritoneal injured surface and migration, proliferation and differentiation of several cell types that modulate the subsequent peritoneal healing. The local fibrinolytic activity is critical because fibrin, if not completely degraded, will provide a scaffold for fibroblasts growth, extracellular matrix (ECM) deposition and angiogenesis, leading to adhesion formation. The roles of fibrin and other members of the plasminogen system, fibroblasts and ECM in adhesion formation are well known QUOTE "(1-6)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\05(1-6)\00\05\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041560\16DiZerega 1997 1560 /id\00\16\00 (1-6) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041558\16Holmdahl 1997 1558 /id\00\16\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03992 Saed, Zhang, et al. 2001 992 /id\00 \00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043475!Saed, Zhang, et al. 1999 3475 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043476!Saed, Zhang, et al. 2000 3476 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043808&Chegini, Kotseos, et al. 2001 3808 /id\00&\00 . The role of angiogenesis and angiogenic factors, however, remains largely unknown. Angiogenesis, the formation of new blood vessels extending from existing vessels, occurs when the distance between cells and the nearest capillary exceeds an efficient diffusion range to maintain an adequate supply of oxygen and nutrients. Angiogenesis is highly regulated by cellular hypoxia QUOTE "(7-12)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\06(7-12)\00\06\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044369!Folkman & Klagsbrun 1987 4369 /id\00!\00 (7-12) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044368\15Folkman 1995 4368 /id\00\15\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044365\15Folkman 2000 4365 /id\00\15\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044056\17Carmeliet 1999 4056 /id\00\17\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044064 Carmeliet & Collen 2000 4064 /id\00 \00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044054 Carmeliet & Collen 1998 4054 /id\00 \00 . The importance of hypoxia in adhesion formation has been postulated for several reasons besides for its role in angiogenesis. Firstly, surgical trauma induces tissue necrosis and an inflammatory reaction involving local hypoxia as was recognized in adhesion formation QUOTE "(13-17)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(13-17)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042927\13Ellis 1982 2927 /id\00\13\00 (13-17) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043797\15Raftery 1973 3797 /id\00\15\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\0425190Tsimoyiannis, Tsimoyiannis, et al. 1989 2519 /id\000\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043681$Wiseman, Huang, et al. 1994 3681 /id\00$\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043684'Wiseman, Gottlick, et al. 1992 3684 /id\00'\00 . Secondly, hypoxia modulates the expression of several molecules involved in different stages of adhesion formation, such as plasminogen activator inhibitor-1 (PAI-1), tissue type plasminogen activator (tPA), transforming growth factor- (TGF-), matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) QUOTE "(3-5;18-24)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\0B(3-5;18-24)\00\0B\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044529+Uchiyama, Kurabayashi, et al. 2000 4529 /id\00+\00 (3-5, 18-24) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044117"Pinsky, Liao, et al. 1998 4117 /id\00"\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044519&Fink, Kazlauskas, et al. 2002 4519 /id\00&\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044698\1CSaed & Diamond 2002 4698 /id\00\1C\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044699\1CSaed & Diamond 2002 4699 /id\00\1C\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043475!Saed, Zhang, et al. 1999 3475 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043474!Saed, Zhang, et al. 2000 3474 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043476!Saed, Zhang, et al. 2000 3476 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03992 Saed, Zhang, et al. 2001 992 /id\00 \00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041559\15Chegini 1997 1559 /id\00\15\00 . Thirdly, we recently demonstrated that the pneumoperitoneum during laparoscopic surgery is a cofactor enhancing adhesion formation QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03164\1FMolinas & Koninckx 2000 164 /id\00\1F\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043673&Molinas, Mynbaev, et al. 2001 3673 /id\00&\00 . Since adhesions increase with the duration of pneumoperitoneum and with the insufflation pressure and decrease with the addition of oxygen and since similar effects were found with CO2 and helium pneumoperitoneum, mesothelial hypoxia has been postulated as the driving mechanism QUOTE "(25-28)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(25-28)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03183'Ordonez, Dominguez, et al. 1997 183 /id\00'\00 (25-28) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03168#Yesildaglar & Koninckx 2000 168 /id\00#\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03164\1FMolinas & Koninckx 2000 164 /id\00\1F\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043673&Molinas, Mynbaev, et al. 2001 3673 /id\00&\00 . It is unknown whether the mechanisms of adhesion formation following a peritoneal lesion only (“basal adhesions”) and of the increased adhesion formation following a peritoneal lesion with the additional effect of the pneumoperitoneum (“pneumoperitoneum-enhanced adhesions”) are similar or different.The hypoxic response is not restricted to specific specialized cell types and a general similar mechanism might act in a variety of cell types. Most mammalian cells can respond to alterations in oxygen levels by increasing or decreasing the expression of specific genes QUOTE "(29;30)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(29;30)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044588\15Semenza 1999 4588 /id\00\15\00 (29, 30) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044651!Gleadle & Ratcliffe 1998 4651 /id\00!\00 . The hypoxic regulation of many of these genes, such as vascular endothelial growth factor (VEGF), takes place at both transcriptional and posttranscriptional levels. The transcriptional regulation is mediated by transcription factors known as hypoxia inducible factors (HIFs) QUOTE "(31-33)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(31-33)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044578\15Semenza 2000 4578 /id\00\15\00 (31-33) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044596\15Semenza 1998 4596 /id\00\15\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044583\15Semenza 1999 4583 /id\00\15\00 .Hypoxia inducible factors are nuclear proteins that bind to hypoxia response elements (HRE) in the promoter or enhancer regions of hypoxia inducible genes, activating gene transcription in response to reduced cellular oxygen concentration QUOTE "(30)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(30)\00\04\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044651!Gleadle & Ratcliffe 1998 4651 /id\00!\00 (30). Hypoxia inducible factors are members of the basic helix-loop-helix (bHLH) periodic (Per) aryl hydrocarbon receptor nuclear translocator (ARNT) single-minded (Sim) (PAS) domain protein family. Several proteins have been identified in this bHLH-PAS family that belong to the α class or the β class. Each member of the α class is able to heterodimerize with a member of the β class to form a stable activation complex. Whereas β class members are constitutively expressed in a ubiquitous or a tissue-specific way, α class members are often inducible by environmental stimuli such as light or hypoxia QUOTE "(34;35)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(34;35)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044662#Gu, Hogenesch, et al. 2000 4662 /id\00#\00 (34, 35) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044685\1DTaylor & Zhulin 1999 4685 /id\00\1D\00 . Hypoxia inducible factor-1, the first HIF identified, is a heterodimer composed of HIF-1α and HIF-1β subunits QUOTE "(36-38)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(36-38)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044633\1CSemenza & Wang 1992 4633 /id\00\1C\00 (36-38) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044622!Wang, Jiang, et al. 1995 4622 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044624\1CWang & Semenza 1995 4624 /id\00\1C\00 . Hypoxia inducible factor-2 is a heterodimer composed of HIF-2α and HIF-1β subunits QUOTE "(39)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(39)\00\04\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044697\1FEma, Taya, et al. 1997 4697 /id\00\1F\00 (39). HIF-1α and HIF-2α, the specific hypoxia-regulated subunits, are structurally very similar and share the same heterodimerization partner. Therefore, both HIF-1 and HIF-2 have a high similarity in structure and regulatory domains and are able to bind to the same HRE of target genes. This study was performed to evaluate the role of HIF-1α and HIF-2α in both “basal adhesions” and “CO2 pneumoperitoneum-enhanced adhesions” in a laparoscopic mouse model using transgenic mice partially deficient for the genes encoding for these factors.Material and MethodsAnimalsThe study was performed in 40 female, 10-12 weeks old mice weighing 30-40 g. For the first experiment, twenty 50% Swiss - 50% 129SvJ wild-type mice (HIF-1α+/+) and transgenic mice partially deficient for the gene encoding for HIF-1α (HIF-1α+/-) were used. For the second experiment, twenty 87.5% Swiss – 12.5% 129SvJ wild-type mice (HIF-2α+/+) and transgenic mice partially deficient for the gene encoding for HIF-2α (HIF-2α+/-) were used. All wild-type and transgenic mice were obtained from the Center for Transgene Technology and Gene Therapy of the Katholieke Universiteit Leuven (KUL). The transgenic mice were generated as described QUOTE "(40;41)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(40;41)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044052$Carmeliet, Dor, et al. 1998 4052 /id\00$\00 (40, 41) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044717'Brusselmans, Bono, et al. 2001 4717 /id\00'\00 . No phenotypic differences were observed between wild-type and transgenic mice of the same strain. It has been reported, however, that HIF-1α+/- mice exposed to chronic hypoxia present significantly delayed development of polycythemia, right ventricular hypertrophy, pulmonary hypertension, and pulmonary vascular remodeling and significantly greater weight loss than HIF-1α+/+ mice (42).Anesthesia Animals were anaesthetised, intubated with a 22-ga catheter (Insyte-W, Vialon, Becton Dickinson, Madrid, Spain) and mechanically ventilated with room air (tidal volume 500 ?l, 85 strokes/minute, Rodent Ventilator, Harvard Apparatus, Holliston, MA, USA) as described.LaparoscopyLaparoscopy was performed as described in detail previously. For the pneumoperitoneum, 100% CO2 at 20 cm H2O was used and maintained for different time periods according to the experimental design.Induction of intraperitoneal adhesions Adhesions were induced as described in detail previously by standardized monopolar and bipolar lesions (Autocon 350, Karl Storz, Tüttlingen, Germany). Scoring of adhesions Adhesions were scored after seven days as described previously. Experimental designAll experiments were performed using block randomization by days. In the first experiment (n=20), basal adhesions and pneumoperitoneum-enhanced adhesions were assessed in HIF-1α+/+ mice (n=5 and n=5, respectively) and HIF-1α+/- mice (n=5 and n=5, respectively).In the second experiment (n=20), basal adhesions and pneumoperitoneum-enhanced adhesions were assessed in HIF-2α+/+ mice (n=5 and n=5, respectively) and HIF-2α+/- mice (n=5 and n=5, respectively).StatisticsStatistical analysis was performed with the SAS System (SAS institute, Cary, NC, USA) using the Kruskal-Wallis test to compare individual groups. All data are presented as the mean SE.ResultsAll animals survived the surgical procedures and were available for adhesion scoring after seven days. Adhesions formed between the injured visceral site and the pelvic fat and/or between the injured parietal site and the pelvic fat. No adhesions were observed at the site of the laparoscopic ports or at other sites. Monopolar lesions systematically induced more adhesions than bipolar lesions. The proportion of adhesions for monopolar and bipolar lesions were, respectively, as follows. In HIF-1α wild-type mice, 13 3% and 6 3% for basal adhesions and 23 4% and 21 5% for pneumoperitoneum-enhanced adhesions. In HIF-1α -/- mice, 7 4% and 1 1% for basal adhesions and 7 7% and 4 3% for pneumoperitoneum-enhanced adhesions. In HIF-2α wild-type mice, 17 4% and 13 7% for basal adhesions and 41 4% and 35 5% for pneumoperitoneum-enhanced adhesions. In HIF-2α -/- mice, 15 5% and 2 2% for basal adhesions and 20 7% and 7 5% for pneumoperitoneum-enhanced adhesions. Similar effect was observed for the extent, type, tenacity and total adhesion scores (Tables I and II). In order to maximize statistical significance, only the means of both lesions are used for further analysis.In HIF-1α wild-type mice, pneumoperitoneum enhanced adhesion formation (proportion: P=0.01; extent: P=0.01). In comparison with HIF-1α wild-type mice, basal adhesions were lower in HIF-1α+/- mice (extent: P=0.03; type: P=0.04; total: P=0.05). In HIF-1α+/- mice, pneumoperitoneum did not enhance adhesions. Therefore, in comparison with HIF-1α wild-type mice, pneumoperitoneum-enhanced adhesions were obviously lower in HIF-1α+/- mice (proportion: P=0.04; extent: P=0.02; type: P=0.02; tenacity: P=0.01; total: P=0.01) (Figure 2, Table I).In HIF-2α wild-type mice, pneumoperitoneum enhanced adhesion formation (proportion: P=0.02; type: P=0.01; total: P=0.03). In comparison with HIF-2α wild-type mice, basal adhesions were similar in HIF-2α+/- mice. In HIF-2α+/- mice, pneumoperitoneum did not enhance adhesions. Therefore, in comparison with HIF-2α wild-type mice, pneumoperitoneum-enhanced adhesions were obviously lower in HIF-2α+/- mice (proportion: P=0.01; extent: P=0.01; type: P=0.01; tenacity: P=0.01; total: P=0.01) (Figure 3, Table II).DiscussionThis study confirms that CO2 pneumoperitoneum is a cofactor in adhesion formation since pneumoperitoneum-enhanced adhesions were observed in all wild-type mice. QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03164\1FMolinas & Koninckx 2000 164 /id\00\1F\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043673&Molinas, Mynbaev, et al. 2001 3673 /id\00&\00 To the best of our knowledge this is the first study demonstrating the role of HIFs in postoperative adhesion formation. In mice partially deficient in HIF-1α or HIF-2α, adhesion formation did not increase following 60 minutes of CO2 pneumoperitoneum, demonstrating that the mechanism of CO2 pneumoperitoneum-enhanced adhesions involves HIF-1α and HIF-2α, which obviously cannot be up-regulated in these mice. In mice partially deficient in HIF-1α, but not in HIF-2α, basal adhesions were lower than in wild-type animals suggesting that HIF-1α, but not HIF-2α, also has a role in basal adhesion formation. Our results can be explained by postulating that CO2 pneumoperitoneum enhances adhesion formation, at least in part, through an up-regulation of HIF-1α and HIF-2α. Since HIF-1α and HIF-2α are expressed in response to hypoxia, this study confirms that CO2 pneumoperitoneum-enhanced adhesion formation is mediated by mesothelial hypoxia as suggested QUOTE "(27)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(27)\00\04\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043673&Molinas, Mynbaev, et al. 2001 3673 /id\00&\00 (27). This is also consistent with the reported role of hypoxia in adhesion formation QUOTE "(3-5;13-24)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\0B(3-5;13-24)\00\0B\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\042927\13Ellis 1982 2927 /id\00\13\00 (3-5, 13-24) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043797\15Raftery 1973 3797 /id\00\15\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\0425190Tsimoyiannis, Tsimoyiannis, et al. 1989 2519 /id\000\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043681$Wiseman, Huang, et al. 1994 3681 /id\00$\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043684'Wiseman, Gottlick, et al. 1992 3684 /id\00'\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044529+Uchiyama, Kurabayashi, et al. 2000 4529 /id\00+\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044117"Pinsky, Liao, et al. 1998 4117 /id\00"\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044519&Fink, Kazlauskas, et al. 2002 4519 /id\00&\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044698\1CSaed & Diamond 2002 4698 /id\00\1C\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044699\1CSaed & Diamond 2002 4699 /id\00\1C\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043475!Saed, Zhang, et al. 1999 3475 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043474!Saed, Zhang, et al. 2000 3474 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043476!Saed, Zhang, et al. 2000 3476 /id\00!\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\03992 Saed, Zhang, et al. 2001 992 /id\00 \00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\041559\15Chegini 1997 1559 /id\00\15\00 . This hypothesis is moreover supported by the similarity in partial oxygen tensions regulating HIF-1 expression and adhesion prevention following laparoscopic surgery. Indeed, HIF-1 levels increase exponentially with lower intracellular oxygen tensions (evaluated in human cervical carcinoma HeLa S3 cells in culture), with a half maximal expression of HIF-1 around 1.5% - 2% of oxygen and a maximal response at 0.5% of oxygen QUOTE "(44)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(44)\00\04\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043491$Jiang, Semenza, et al. 1996 3491 /id\00$\00 (45). This is consistent with our observation that adhesion formation decreases by the addition of oxygen to the CO2 pneumoperitoneum with a half maximal effect around 1.5% - 2% of oxygen and a maximal effect from 3% of oxygen onwards QUOTE "(27)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\04(27)\00\04\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\043673&Molinas, Mynbaev, et al. 2001 3673 /id\00&\00 (27). We recently evaluated the role of vascular endothelial growth factor (VEGF), i.e. VEGF-A, VEGF-B and placental growth factor (PlGF) (Molinas et al, submitted) and of the plasminogen system, i.e. PAI-1, uPA, tPA, (46) in adhesion formation in transgenic mice using the same model, demonstrating that pneumoperitoneum-enhanced adhesions involve up-regulation of PAI-1, VEGF-A, VEGF-B and PlGF. These findings and the role of HIFs are consistent with the hypothesis of mesothelial hypoxia as the driving mechanism of pneumoperitoneum enhanced adhesions. Indeed, it is known that VEGF-A expression is up-regulated by hypoxia through HIF-1α QUOTE "(31-33)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(31-33)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044578\15Semenza 2000 4578 /id\00\15\00 (31-33) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044596\15Semenza 1998 4596 /id\00\15\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044583\15Semenza 1999 4583 /id\00\15\00 . Similarly, PAI-1 is known to be up-regulated by hypoxia and recently a HRE capable to bind HIF-1 was described in the gene encoding for this protein QUOTE "(18-20)" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\07(18-20)\00\07\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044529+Uchiyama, Kurabayashi, et al. 2000 4529 /id\00+\00 (18-20) QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044117"Pinsky, Liao, et al. 1998 4117 /id\00"\00 QUOTE "" ADDIN REFMAN ?\11\05‘\19\01\00\00\00\00\01\00\002C:\5CProgram Files\5CReference Manager 9\5CAll data base\03\00\044519&Fink, Kazlauskas, et al. 2002 4519 /id\00&\00 . It is too early for a comprehensive model integrating the relative importance of HIFs, of the VEGF family and of the plasminogen system on basal adhesions and on pneumoperitoneum-enhanced adhesions. This model has to integrate the effects on normal and damaged peritoneum together with the effects of the duration of pneumoperitoneum, the insufflation pressure and the addition of oxygen. The available data, however, derived from experiments with transgenic mice for PAI-1, VEGF-A, VEGF-B and PlGF and now for HIF-1α and HIF-2α, show clearly that the mechanisms involved in basal adhesions and in pneumoperitoneum-enhanced adhesions are, at least partially, different. In conclusion, our data confirm the role of the CO2 pneumoperitoneum as a cofactor in adhesion formation and demonstrate a role of HIF-1α in basal adhesions and of both HIF-1α and HIF-2α in pneumoperitoneum-enhanced adhesions, suggesting HIF-1α and HIF-2α up-regulation as a mechanism for this pneumoperitoneum-enhanced adhesion formation. This is fully consistent with the up-regulation of these factors by hypoxia and with the concept that CO2 pneumoperitoneum causes mesothelial hypoxia and confirms the role of hypoxia in adhesion formation. These observations open new insights in the pathophysiology of adhesion formation. Since the mechanisms involved in basal adhesions and in pneumoperitoneum-enhanced adhesions are, at least partially, different, new methods for adhesion prevention following laparoscopic surgery could be developed.Pathogenesis of CO2 pneumoperitoneum induced metabolic hypoxia in a rabbit model(J Am Assoc Gynecol Laparosc 2002;9:306-314)AimTo evaluate the effect of CO2 pneumoperitoneum on blood gases, acid-base balance and oxygen homeostasis. Material and MethodsThe study was performed in 28 rabbits. CO2 pneumoperitoneum was maintained for 2 h at 10 mm Hg in animals with spontaneous breathing or superficial ventilation and at 6 or 10 mm Hg in animals with optimal ventilation. Animals without pneumoperitoneum were used as controls, i.e. spontaneously breathing, superficially or optimally ventilated (7 groups, 4 rabbits/group). Since no differences were observed between control groups, they were grouped for further analysis. Arterial blood samples were taken every 30 min from the ear artery and analysed for pH, partial pressures of oxygen (pO2) and CO2 (pCO2), oxygen saturation (sO2), oxyhemoglobin (O2Hb), reduced hemoglobin (RHb), oxygen tension at half-saturation (p50), total concentration of oxygen (tO2), CO2 (tCO2), lactate, bicarbonate, standard bicarbonate (SBC), actual base excess (ABE) and standard base excess (SBE). ResultsIn control groups no major changes were observed. Rabbits with CO2 pneumoperitoneum and spontaneous breathing or superficial ventilation developed respiratory acidosis that leaded to metabolic acidosis and finally to metabolic hypoxia. This was evidenced by the increase in pCO2, bicarbonate, lactate, p50 and RHb and by the decrease in pH, ABE, SBE, SBC, sO2 and O2Hb. In animals optimally ventilated, these changes were less pronounced, being minimal at 6 mm Hg of insufflation pressure.Reduction of CO2 pneumoperitoneum-induced metabolic hypoxia by the addition of small amounts of O2 to the CO2 in a rabbit ventilated model(Hum Reprod 2002;17:1623-1629)AimTo evaluate the effect of CO2 pneumoperitoneum with the addition of oxygen on blood gases, acid-base balance and oxygen homeostasis. Material and MethodsThe study was performed in 21 rabbits. In series A (3 groups, 4 rabbits/group), rabbits superficially ventilated without pneumoperitoneum or with CO2 pneumoperitoneum with 0% or 6% of oxygen at 10 mm Hg were used. In series B (3 groups, 3 animals/group), rabbits optimally ventilated without pneumoperitoneum or with CO2 pneumoperitoneum with 0% or 6% of oxygen at 6 mm Hg were used. The procedures took 210 min (series A) or 120 min (series B) and blood samples were taken and analysed every 30 min (series A) or 15 min (series B) as described previously.ResultsIn control groups no major changes were observed. In animals with CO2 pneumoperitoneum, an increase in pCO2, bicarbonate, lactate, p50 and RHb was observed, together with a decrease in pH, ABE, SBE, SBC, sO2 and O2Hb. These changes were more pronounced in superficially ventilated animals with 10 mm Hg of insufflation pressure (series A). The addition of 6% of oxygen reduced significantly these changes. Prevention of adhesions with crystalloids during laparoscopic surgery in mice(J Am Assoc Gynecol Laparosc 2002;9:447-452)AimTo evaluate the effect of saline and Ringer’s lactate solutions in preventing adhesion formation after laparoscopic surgery.Material and MethodsThe study was performed in 92 NMRI mice. In experiment 1, CO2 pneumoperitoneum was maintained for 45 min and saline (series 1) or Ringer’s lactate (series 2) was added at the end of the pneumoperitoneum, immediately after or before bipolar coagulation. No solutions were used as control groups (series 1: 4 groups, 5 mice/group; series 2: 4 groups, 10 mice/group). In experiment 2 (4 groups, 8 mice/group), saline or Ringer’s lactate was added at the beginning of the procedure and was left of drained at the end of the coagulations.ResultsIn comparison with 45 min of pneumoperitoneum only, i.e. without any additional fluid, the addition of saline or Ringer’s lactate at the end of the pneumoperitoneum did not reduce adhesion formation, whereas both solutions added immediately before or after coagulation decreased adhesion formation, without differences between both approaches, i.e. before or after coagulation. Adhesion formation was lower with the use Ringer’s lactate and when any of both solutions were maintained in the abdominal cavity during the whole pneumoperitoneum. Perspectives for completion of the workThe data obtained so far indicate that CO2 pneumoperitoneum increases adhesion formation in a time- and pressure-dependent manner, which is counteracted by the addition of 3% of oxygen, and that it upregulates HIF-1, PAI-1, VEGF-A, VEGF-B and PlGF, strongly suggesting mesothelial hypoxia as the driving mechanism. For completion of this thesis we would like to investigate [1] the relation between this pneumoperitoneum-induced mesothelial hypoxia and pneumoperitoneum-induced acidosis, [2] the effect of adding a high concentration of oxygen to the CO2 pneumoperitoneum (mesothelial hyperoxia) on adhesion formation, and [3] the effects of some adhesion prevention adjuvants after laparoscopic surgery in a hypoxic or a hyperoxic environment.Pneumoperitoneum-enhanced adhesion formation and pneumoperitoneum-induced acidosis: the effects of assisted ventilation.CO2 pneumoperitoneum induces acidosis and enhances adhesion formation, being the former highly regulated by assisted ventilation. Therefore, we will evaluate the effect of assisted ventilation on adhesion formation and the association between pneumoperitoneum-enhanced adhesion formation and pneumoperitoneum-induced acidosis. In mice ventilated with different ventilation patterns, pneumoperitoneum will be maintained for different time periods and at different insufflation pressures. Adhesions will be induced and scored as described and blood gases will be analysed. Time needed for the experimental work and for the analysis of the results: 4 weeks. The effect of the addition of higher concentration of oxygen to the CO2 pneumoperitoneum (mesothelial hyperoxia)It was already demonstrated the beneficial effect of adding small amounts of oxygen to the CO2 pneumoperitoneum for reducing both adhesion formation and acidosis. Our data indicate, however, that adding higher concentrations could be again deleterious. Therefore, we will specifically evaluate the addition of up to 12% of oxygen to the CO2 pneumoperitoneum on adhesion formation, since this is the maximum concentration that can be obtained with the Thermoflator Plus.In mice, CO2 pneumoperitoneum with 0%, 3% or 12% of oxygen will be maintained for different times and at different insufflation pressures and adhesions will be induced and scored as described. Time needed for the experimental work and for the analysis of the results: 4 weeks. Prevention of adhesions after laparoscopic surgerySurgical lesions performed during laparotomy (air environment) could have a different evolution than surgical lesions performed during laparoscopy (CO2 pneumoperitoneum environment), leading eventually in differences in adhesion formation. Therefore, adhesion prevention adjuvant need to be specifically evaluated in each condition. It was already demonstrated that crystalloids reduce adhesion formation when they are used during laparoscopic surgery immediately after performing the surgical trauma, avoiding the effects of pure CO2 pneumoperitoneum. We will evaluate the effects of other adhesion prevention adjuvant during laparoscopic surgery with the standard pure CO2 pneumoperitoneum (mesothelial hypoxia) or with the addition of 3% (mesothelial normoxia) or 12% of oxygen (mesothelial hyperoxia). Time needed for the experimental work and for the analysis of the results: 8 weeks.General DiscussionReferences ................
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