1 Centre for Surgical Technologies, Catholic University of Leuven and 2 Department of Gynaecology and Obstetrics, University Hospital Gasthuisberg, Catholic University of Leuven, Belgium
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Abstract |
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Key words: adhesions/desiccation/insufflation pressure/intubation
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Introduction |
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Adhesions have been implicated as a cause of pelvic pain by their restriction in movement and distensibility of the pelvic organs (Kresch et al., 1984; Bahary and Gorodeski, 1987
; Peters, et al., 1992
). Adhesions are a major cause of infertility (Drake and Grunert, 1980
; DeCherney and Mezer, 1984
; Trimbos-Kemper et al., 1985
). These can be treated by surgery but recurrence remains problematic (Diamond et al., 1987
). Moreover, adhesive disease can produce a significant economic charge to community (Ray et al., 1993
).
Laparoscopic surgery has been claimed to be less adhesiogenic than laparotomy (Luciano et al., 1989). This, however, has never been demonstrated conclusively, at least partially because of ethical constraints in man, and because most efforts were devoted to show benefits of laparoscopic surgery, such as lower postoperative morbidity and pain. Animal models allow a better control of variables. The most commonly used animal models are rats (Filmar et al., 1987
; Evrard et al., 1996
) and rabbits (Doody et al., 1989
; Marana et al., 1994
). Rabbits are a good model and have been widely used for the study of induction and prevention of adhesion formation. The rabbit model moreover permits the performance of endoscopic procedures with conventional instruments. Using this model, it was shown (Ordonez et al., 1997
) that duration of pneumoperitoneum during endoscopic surgery was a co-factor in adhesion formation. This observation was confirmed in an endoscopic mouse model (Yesildaglar et al., 1999
), suggesting that either changes in pH or anoxaemia of the superficial mesothelial layers was the aetiological factor.
Since hypoxaemia of the superficial mesothelial layers of the peritoneum must increase with the pneumoperitoneum pressure, the effect of 5 and 20 mmHg insufflation pressure upon adhesion formation was evaluated to test this hypothesis.
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Materials and methods |
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Experiments
To evaluate the effect of intra-abdominal pressure during pneumoperitoneum upon adhesion formation, 5 and 20 mmHg insufflation pressure were used, being respectively the lowest pressure for adequate peritoneal distension, and the highest pressure which could reliably be used in rabbits without anaesthetic problems. Since this experiment was designed to test the hypothesis of mesothelial anoxaemia, and knowing that CO2 is readily absorbed and that the peritoneum has a high exchange capacity, a continuous flow of CO2 through the abdominal cavity was used to maintain a nearly 100% concentration of CO2 by removing continuously all oxygen which could have diffused from the circulation. Since some desiccation, a known co-factor of adhesion formation, could not be excluded, a 2x2 factorial design (Armitage and Berry, 1987) was used to evaluate the effect of insufflation pressure (5 and 20 mmHg) at two different flow rates (1 and 10 l/mm) together with the effect of flow rate. The effect of pressure was investigated at low and high flow rates since it was not possible to predict at which flow rate the effect of pressure would become most significant, and since this could be done without increasing the number of experiments simply by using a 2x2 factorial design. The four groups thus had an intra-abdominal pressure and a flow rate of 5 mmHg and 1 l/min (group 1, n = 5), of 5 mmHg and 10 l/min (group 2, n = 4), 20 mmHg and 1 l/min (group 3, n = 5) and 20 mmHg and 10 l/min (group 4, n = 4) respectively. The CO2 was warmed to 35 ± 1°C and humidified using the Thermoflator (Karl Storz, Brussels, Belgium) and a humidifier (Dräger®, Wemmel, Belgium).
Anaesthesia and intubation under endoscopic view
After premedication with 50 mg/kg i.m. ketamine (Ketalin; Apharmo, Arnhem, The Netherlands) and 0.3 mg/kg i.m. xylazin hydrochloride (Rompun, 2%; Bayer, Brussels, Belgium), anaesthesia was maintained with inhalation of 23% halothane (Fluothane®; Zeneca, Brussels, Belgium) using 1 l/min of oxygen. Pulse rate and oxygen saturation was monitored continuously using a pulse oximeter (Nellcor, Leuven, Belgium).
Because of the anatomy of the oral fissure and pharynx, orotracheal intubation of rabbits is difficult, with a high failure rate. Since intubation was necessary for these experiments using 20 mmHg of insufflation pressure, a new technique for endotracheal intubation of rabbits was developed. Approximately 5 min after the beginning of halothane anaesthesia, the animals were secured in a supine position on the operation table. A flexible salpingoscope (2.7 mm diameter; Karl Storz) in a straight, 20-cm-long metal tube with an external diameter of 3.2 mm, to obtain rigidity, was inserted in the endotracheal tube (3.5 mm internal diameter, Sheridan Catheter Corp., New York, NY, USA). The salpingoscope was connected to a conventional endoscopic surgery tower, which had a video-monitor and a cold light source. The tongue was grasped with a haemostatic clamp and pulled out and then the endotracheal tube was inserted through the oral cavity and oropharynx into the trachea under endoscopic vision. The whole procedure took less than a minute, was easy and safe.
Surgical procedure for induction of adhesions
The animals were placed in supine position and the abdomen was shaved and disinfected with polyvidone iodine (Iso-Betadine; Asta Medica, Brussels, Belgium). The surgical procedures were performed under strict aseptic conditions and no perioperative antibiotics were administered. A standard three-puncture laparoscopy was performed using conventional equipment. By open laparoscopy technique, a 10 mm trocar (Ethicon Endosurgery, Brussels, Belgium) was placed caudal to the sternum. For the pneumoperitoneum the Thermoflator was used. A 10 mm 0° scope connected to a single chip video camera and light source was used (Karl Storz). Under direct vision two 5 mm trocars were introduced in the left and right flank to allow the introduction of the necessary instruments. After the procedure, abdominal incisions were sutured with 30 Polyglactin (Vicryl, Ethicon Endosurgery).
In all animals, two opposing lesions were made on each side using either a CO2 laser (Sharplan, 1060, Brussels, Belgium) at 10 W in a continuous superpulse mode or a 5 mm endoscopic bipolar forceps (Ethicon Endosurgery) at 10 W. Randomly, 2 cm2 of the oviduct and of the ipsilateral pelvic side wall were superficially vaporized whereas on the other side, the same surface was coagulated using a bipolar forceps. Following this procedure, taking 56 min, the CO2 pneumoperitoneum was maintained for 30 min total time. Animals were assigned, on a daily basis using randomization tables with blocks of four animals. Each block of four animals was operated on during the same day. Thus only one animal in each treatment group was operated on in one day. These experiments were performed by one investigator (N.Y.) over a 10 day period.
Second-look laparoscopy and scoring system for assessment of adhesions
Seven days after the initial procedures, adhesions were scored during a second look laparoscopy under general anaesthesia. Adhesions were scored (Fiedler et al., 1996) as published before (Ordonez et al., 1997
). This scoring system took into account type (0 = no adhesions; 1 = filmy adhesions; 2 = firm adhesions; 3 = dense adhesions, require sharp dissection to be separated), tenacity (1 = easily fall apart; 2 = require traction; 3 = require sharp dissection) and extent of adhesions (1 to 4 points; 125%, 2650%, 5175%, 76100%) respectively. Total adhesion scores were the sum of type, tenacity and extent scores of lesions. All the procedures and second-look laparoscopies were videotaped, and subsequently scored blindly by two independent investigators in order to minimize inter-observer variability in adhesion scoring (Corson et al., 1995
).
The number of the animals was planned to be five in each group, but two rabbits (one rabbit from the second group and one rabbit from the fourth group) were excluded because of intraperitoneal infection observed during the second-look operations.
In-vitro experiments
Desiccation during high flow insufflation was evaluated in vitro as described previously (Yesildaglar et al., 1999). A 200 ml Falcon dish with two holes of 7 mm on each side, containing 20 ml of water, i.e. covering the entire bottom surface, was used to evaluate water loss/desiccation during insufflation at continuous flow rates of 1, 5, 10, 15 and 20 l/min with and without a humidifier (Dräger). The insufflation was carried out with a Thermoflator, which kept the insufflated CO2 at 35 ± 1°C. The dish was kept at exactly 36.5 ± 0.5°C using two heaters (Maquet Rastatt, Maquett, St-Pieters-Leeuw, Belgium; Ameda, Zug, Switzerland). During the experiments, the temperature of the dish was monitored continuously using a temperature probe (Hewlett Packard, Brussels, Belgium). In all experiments, observations were made in triplicate.
Statistics
Data were analysed by two-way analysis of variance (ANOVA; general linear models, GLM procedure) using the SAS system (SAS Release 6.12, 1998). All data are presented as means ± SD unless indicated otherwise. The advantage of a 2x2 factorial design, with five animals in each cell, was that to achieve the same statistical precision with a one-factor-at-a-time approach, twice as many observations would have been needed. The factorial design moreover had the advantage of permitting the detection of an effect of one factor at different levels of the other, i.e. it permitted the detection of an interaction between the two factors (Armitage and Berry, 1987). In these experiments, a difference in total adhesion score of >3 had a power of 89% given an SD of 2, and 8 and 10 animals in each group. The SAS GLM test also compensated for the unbalanced design.
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Results |
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Adhesion scores obtained in the four treatment groups are shown in Table I. By two-way ANOVA, total adhesion scores of laser lesions increased with flow rate (P = 0.0003) and insufflation pressure (P = 0.002). Total adhesion scores following a bipolar lesion increased with insufflation pressure (P = 0.02) but not with flow rate (P = 0.1). Sum of total adhesion scores of laser lesions and total adhesion scores of bipolar lesions increased with flow rate (P = 0.005) and with insufflation pressure (P = 0.004) (Figure 1
).
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In vitro, desiccation was linear with flow rate (Figure 2) being 1.61, 3.24, 4.83 and 6.53 g of water loss after insufflation for 30 min at 36.5 ± 0.5°C without humidification and at flow rates of 5, 10, 15 and 20 l/min respectively (Yesildaglar et al., 1999
). However, when the CO2 gas was humidified, water loss was much less and desiccation was exponential, with flow rates being 0.160 ± 0.018, 0.358 ± 0.015, 0.675 ± 0.021, 1.266 ± 0.027 and 2.153 ± 0.025 g of water after insufflation for 30 min at 1, 5, 10, 15 and 20 l/min flow rates for 30 min respectively (Figure 2
).
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Discussion |
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The increase of adhesion formation with the duration of CO2 pneumoperitoneum (Ordonez et al., 1997; Yesildaglar et al., 1999
) could be caused by changes in pH or by anoxaemia of the superficial mesothelial layers or both. These experiments were designed to investigate the effect of anoxaemia, assuming that higher insufflation pressures would increase the depth and extent of anoxaemia by mechanical compression of the capillary bed, together with passive diffusion of CO2. In order to maintain a near 100% concentration of CO2 in the peritoneal cavity, taking into account the high exchange capacity of the peritoneum, a continuous flow of CO2 was used, in order to remove any oxygen which could have diffused passively from the circulation. The result was that adhesions, following both a laser and a bipolar lesion, increased with the insufflation pressure, confirming the hypothesis that anoxaemia is a co-factor in adhesion formation, although an additional deleterious effect of changes in pH cannot be excluded.
The mechanism of adhesion formation as a consequence of anoxaemia remains unclear. Although it is logical to assume that the depth of anoxaemia increases with the insufflation pressure, the exact depth and extent of anoxaemia together with the consequences of the effects of CO2 pneumoperitoneum upon the peritoneal microcirculation (Taskin et al., 1998) can only be speculated. Little is known about the possible effects of anoxaemia in the mesothelium to induce angiogenic factors such as vascular endothelial growth factor (VEGF) (Wiczyk et al., 1998
) and upon the immune system, either directly upon the macrophages in the peritoneal fluid (Kopernik et al., 1998
) or by attraction of monocytes from the circulation (Zeyneloglu, 1998). Both factors, angiogenesis and the immune system, are candidates to mediate the observed effects upon adhesion formation.
Desiccation is a known co-factor of adhesion formation. Since it could not be ruled out that a longer duration of CO2 pneumoperitoneum would not cause some desiccation, the effects of anoxaemia and of desiccation were impossible to separate completely. Also the use of a humidifier could not guarantee the absence of desiccation. Therefore the effect of desiccation (i.e. flow rate) was introduced and evaluated in the model as a second factor using a 2x2 factorial design, permitting the simultaneous evaluation of the effects of pressure and of flow rate, together with the interaction between both factors. Since a higher flow rate probably resulted in more desiccation, the results suggest a direct effect of desiccation upon adhesion formation. In addition these experiments show that this effect cannot be prevented completely by using a humidifier, and that the effect increases with flow rate. From the in-vitro experiments, it can be postulated that in-vivo desiccation also must increase exponentially with flow rate since the efficacy of humidification decreases with increasing flow rate.
The relevance of desiccation at higher flow rates for surgery in the human is obvious, especially when using a high flow insufflator to remove smoke during CO2 laser surgery. Also the effect of insufflation pressure and superficial mesothelial anoxaemia could be important since operations of much longer duration than those in this study are performed. Moreover, the mechanisms involved in adhesion formation by CO2 pneumoperitoneum might be equalsly important for implantation and growth of malignant cells (Koster et al., 1996). Although the extrapolation of data from an animal model to the human should be done with great caution, the rabbit seems to be an appropriate model since reduction of adhesions by Interceed (Marana et al., 1997
) or by low dose aspirin (Muzii et al., 1998
) and the effect of thrombin application (Yarali et al., 1998
) are comparable with the effects observed in the human.
In conclusion, our data demonstrate that insufflation pressure is a co-factor in adhesion formation. These observations are consistent with the hypothesis that the increase in adhesion formation by CO2 pneumoperitoneum is caused by hypoxaemia of the superficial cell layers of peritoneum. Moreover, this study shows the importance of adequate humidification of CO2, especially at high flow rates.
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Acknowledgments |
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Notes |
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References |
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Bahary, C.M. and Gorodeski, I.G. (1987) The diagnostic value of laparoscopy in women with chronic pelvic pain. Ann. Surg., 53, 672674.
Corleta, O., Habazettl, H., Kreimeier, U. et al. (1992) Modified retrograde orotracheal intubation technique for airway access in rabbits. Eur. Surg. Res., 24, 129132[ISI][Medline]
Corson, S.L., Batser, F.R., Gocial, B., et al. (1995) Intra-observer and inter-observer variability in scoring in laparoscopic diagnosis of pelvic adhesions. Hum. Reprod., 10, 161164.[Abstract]
DeCherney, A.H. and Mezer, H.C. (1984) The nature of posttuboplasty pelvic adhesions as determined by early and late laparoscopy. Fertil. Steril., 41, 643646.[ISI][Medline]
Diamond, M.P., Daniell, J.F., Feste, J. et al. (1987) Adhesion reformation and de novo adhesion formation after reproductive pelvic surgery. Fertil. Steril., 47, 864866.[ISI][Medline]
Doody, K.J., Dunn, R.C. and Buttram, V.C.J. (1989) Recombinant tissue plasminogen activator reduces adhesion formation in a rabbit uterine horn model. Fertil. Steril., 51, 509512.[ISI][Medline]
Drake, T.S. and Grunert, G.M. (1980) The unsuspected pelvic factor in the infertility investigation. Fertil. Steril., 34, 2731.[ISI][Medline]
Ellis, H. (1982) Intestinal Obstruction. Appleton-Century-Crofts, New York.
Evrard, V.A.C., De Bellis, A., Boeckx, W. et al. (1996) Peritoneal healing after fibrin glue application: a comparative study in a rat model. Hum. Reprod., 12, 18771880.[Abstract]
Fiedler, E.P., Guzick, D.S., Guido, R. et al. (1996) Adhesion formation from release of dermoid contents in the peritoneal cavity and effect of copious lavage: a prospective, randomized, blinded, controlled study in a rabbit model. Fertil. Steril., 65, 852859.[ISI][Medline]
Filmar, S., Gomel, V. and McComb, P.F. (1987) Operative laparoscopy versus open abdominal surgery: A comparative study on postoperative adhesion formation in the rat model. Fertil. Steril., 48, 486489.[ISI][Medline]
Kopernik, G., Avinoach, E., Grossman, Y. et al. (1998) The effect of a high partial pressure of carbon dioxide environment on metabolism and immune functions of human peritoneal cellsrelevance to carbon dioxide pneumoperitoneum. Am. J. Obstet. Gynecol., 179, 15031510.[ISI][Medline]
Koster, S., Melchert, F. and Volz, J. (1996) Effect of CO2 pneumoperitoneum on intraperitoneal tumor growth in the animal model. Geburtshilfe Frauenheilkd., 56, 458461.[ISI][Medline]
Kresch, A.J., Seifer, D.B., Sachs, L.B. et al. (1984) Laparoscopy in 100 women with chronic pelvic pain. Obstet. Gynecol., 64, 672674.[Abstract]
Luciano, A.A., Maier, D.B., Koch, E.I. et al. (1989) A comparative study of postoperative adhesions following laser surgery by laparoscopy versus laparotomy in the rabbit model. Obstet. Gynecol., 74, 220224.[Abstract]
Marana, R., Luciano, A.A., Muzii, L. et al. (1994) Laparoscopy versus laparotomy for ovarian conservative surgery: A randomized trial in the rabbit model. Am. J. Obstet. Gynecol., 171, 861864.[ISI][Medline]
Marana, R., Catalano, G.F., Caruana, P. et al. (1997) Postoperative adhesion formation and reproductive outcome using Interceed after ovarian surgery: a randomized trial in the rabbit model. Hum. Reprod., 12, 19351938.[Abstract]
Menzies, D. and Ellis, H. (1990) Intestinal obstruction from adhesionshow big is the problem? Ann. R. Coll. Surg. Engl., 72, 6063.[ISI][Medline]
Muzii, L., Marana, R., Brunetti, L. et al. (1998) Postoperative adhesion prevention with low-dose aspirin: effect through the selective inhibition of thromboxane production. Hum. Reprod., 13, 14861489.[Abstract]
Ordonez, J.L., Dominguez, J., Evrard, V. et al. (1997) The effect of training and duration of surgery on adhesion formation in the rabbit model. Hum. Reprod., 12, 26542657.[Abstract]
Peters, A.A.W., Trimbos-Kemper, G.C.M., Admiral, C. et al. (1992) A randomized clinical trial on the benefit of adhesiolysis in patients with intraperitoneal adhesions and chronic pelvic pain. Br. J. Obstet. Gynaecol., 99, 5962.[ISI][Medline]
Ray, N.F., Larsen, Jr, J.W., Stillman, R.J. et al. (1993) Economic impact of hospitalizations for lower abdominal adhesiolysis in the United States in 1988. Surg. Gynecol. Obstet., 176, 271276.[ISI][Medline]
SAS Release 6.12. (1998) SAS Institute Inc., SAS Campus Drive, Cary, NC 27513, USA.
Taskin, O., Buhur, A., Birincioglu, M. et al. (1998) The effects of duration of CO2 insufflation and irrigation on peritoneal microcirculation assessed by free radical scavengers and total glutathion levels during operative laparoscopy. J. Am. Assoc. Gynecol. Laparosc., 5, 129133.[ISI][Medline]
Trimbos-Kemper, T.C., Trimbos, J.B. and van Hall, E.V. (1985) Adhesion formation after tubal surgery: results of the eighth day laparoscopy in 188 patients. Fertil. Steril., 43, 395400.[ISI][Medline]
Wiczyk, H.P., Grow, D.R., Adams, L.A. et al. (1998) Pelvic adhesions contain sex steroid receptors and produce angiogenesis growth factors. Fertil. Steril., 69, 511516.[ISI][Medline]
Yarali, H., Gomel, V., Uygur, D. et al. (1998) A comparative study of the effects of thrombin and electrodesiccation used for haemostasis on inflammation and adhesion formation. Hum. Reprod., 13, 14931495.[Abstract]
Yesildaglar, N., Ordonez, J.L., Laermans, I. et al. (1999) The mouse as a model to study adhesion formation following endoscopic surgery: a preliminary report. Hum. Reprod., 14, 5559.
Zeyneloglu, H.B., Senturk, L.M., Seli, E. et al. (1998) The role of monocyte chemotactic protein-1 in intraperitoneal adhesion formation. Hum. Reprod., 13, 11941199.[Abstract]
Submitted on April 15, 1999; accepted on December 7, 1999.