1 Centre for Surgical Technologies (CHT) and 2 Department of Obstetrics and Gynaecology, University Hospital Gasthuisberg, K. U. Leuven, Minderbroederstraat 17, B-3000, Leuven, Belgium
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Abstract |
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Key words: adhesions/endoscopy/mouse
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Introduction |
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Laparoscopic surgery is claimed to be less adhesiogenic than laparotomy in humans, since it is less traumatic and more precise, with a lower postoperative morbidity. In experimental studies, the incidence of postoperative adhesions after laparotomy or laparoscopy is controversial. Filmar et al. (1987) concluded that adhesions following laparotomy and laparoscopy were not different. In 1989, Luciano et al. reported more postoperative adhesions after laparotomy than after laparoscopy in a rabbit model. According to the results of studies carried out by Marana et al. (1994) and Jorgensen et al. (1994), there was no significant difference between the adhesions after laparotomy and laparoscopy. Chen et al. (1998) reported that for para-aortic lymphadenectomy, the transperitoneal laparoscopic approach induced fewer adhesions than laparotomy (Chen et al., 1998).
CO2 is generally used for the pneumoperitoneum during laparoscopy, since its solubility in water is an important safety factor. CO2 pneumoperitoneum, however, causes peritoneal acidosis, which is most severe at the site directly exposed to CO2 (Volz et al., 1997). Moreover, during CO2 pneumoperitoneum especially with high flow rates, desiccation could occur which is a known co-factor in peritoneal injury and adhesiogenesis (Ryan et al., 1973
; Holmdahl et al., 1997
). Duration of CO2 pneumoperitoneum in endoscopic surgery increased adhesion formation in rabbits (Ordonez et al., 1997
). In this study, we wanted to confirm and extend these observations in the mouse model.
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Materials and methods |
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The study was approved by the Institutional Animal Care Committee of K. U. Leuven.
Experiments
Preliminary studies were necessary to establish a suitable anaesthesia for mice and the intra-abdominal pressures to permit endoscopic procedures up to 2 h. Since anaesthesia of long duration permitting endoscopic surgery, was difficult with i.m. and s.c. anaesthetics, inhalational anaesthesia was required and halothane (Fluothane, Zeneca, Destelbergen, Belgium) with mask was used. In the preliminary study for insufflation pressure, the mortality rate was evaluated with insufflation pressures of 2.5, 5, 7.5, 10 and 15 cm of water for 10 min (n = 100) and subsequently with an insufflation pressure of 2.5 cm of water for 15, 30, 60, 90 and 120 min, respectively (n = 100). To evaluate adhesion formation, six monopolar coagulation lesions using 5 W (Autocon, Karl Storz
, Belgium) for 2 s, were inflicted endoscopically to all animals using a specifically designed ball electrode. Three 1 mm diameter lesions were made on the uterus (one on each horn and one on the midline) and three identical lesions in the opposing mid-lower abdominal wall. These lesions were at 1 mm distance from each other. This procedure took 34 min. To evaluate the effect of CO2 pneumoperitoneum upon adhesion formation, four groups were studied (n = 60). In group I (control group) pneumoperitoneum was not prolonged after the surgical procedure and thus consisted of a pneumoperitoneum of 34 min (n = 15). In all other groups the pneumoperitoneum was prolonged for 60 min with non-humidified CO2. In group II (n = 15), there was no flow through the abdominal cavities of the animals whereas in group III (n = 15), a continuous low flow rate of ~1 ml/min per mouse and in group IV (n = 15), a continuous high flow rate of ~10 ml/min per mouse was maintained. Assuming a weight of 3040 g and a pneumoperitoneum volume of 23 ml in mice and a weight of 5070 kg and of a pneumoperitoneum volume of 34 l in humans, a flow rate of 1 ml/min in mice is estimated to be comparable to 11.5 l/min in humans.
After 1 week, mice were killed and immediately afterwards a laparotomy was performed. Adhesions were scored blindly by two researchers under an operative microscope (Zeiss, Brussels, Belgium) using the registration numbers of the animals. Type of adhesions was scored as 0 (no adhesions), 1 (filmy adhesions), 2 (firm adhesions) or 3 (vascular firm or dense adhesions). Tenacity of adhesions was scored as 1 (adhesions which essentially fall apart), 2 (adhesions which require traction) or 3 (adhesions which require sharp dissection). Maximum total adhesion score (type + tenacity) thus was 6.
Surgical procedures
After preanaesthesia with 100 mg/kg of ketamine i.m. (Ketalin® Apharmo, Arnhem, The Netherlands) and 16 mg/kg of xylazine s.c. (Rompun® 2%, Bayer, Brussels, Belgium), anaesthesia was maintained with inhalational Halothane (Fluothane, Zeneca) at 0.51% using 1 l/min of O2. The animals were secured to the table in supine position. A 5 mm abdominal skin incision on the midline 1 cm below the sternum, was made. The Veress needle together with a miniscope (Storz Miniscope, a 1.2 mm 0° fibroscope in a Veress needle, Karl Storz®) was introduced into the peritoneal cavity. The pneumoperitoneum was initiated and maintained with a conventional CO2 insufflator (Thermoflator, Karl Storz
) connected to a water valve (Koninckx and Vandermeersch, 1991
), using non-humidified CO2. This water valve was used, since it accurately permits insufflation pressures as low as 2.5 cm of water (1.9 mmHg). It also prevents extreme pressures due to the intermittent insufflation by the thermoflator. An elastic balloon was placed next to the water valve to eliminate virtually all pressure changes. Endoscopy was performed using a camera (Telecam, CCU, Karl Storz®) attached to the miniscope. Because of the small size of a mouse, the miniscope was held by the surgeon who was sitting and facing the monitor with his elbows stabilized. A secondary operating port was placed by the surgeon using his free hand in the left lower quadrant of the abdomen using a 14-gauge angiocatheter (Insyte
, Vialon
, Becton Dickinson, Madrid, Spain) under direct laparoscopic vision. The needle was withdrawn and a monopolar home made ball electrode which was 10 cm long and 1.5 mm in diameter, was inserted through the angiocatheter. The animal was tilted to a 45° Trendelenburg position to explore the lower abdominal cavity and six lesions (5 W, 2 s) were applied. After this, the miniscope was withdrawn and replaced by a 2 mm introducer (MiniSite Introducers
, Auto Suture, Mechelen, Belgium) through which pneumoperitoneum was maintained. The angiocatheter in the lower abdomen, was closed in group II with no flow and left open in group IV to maintain a high flow rate of ~10 ml/min per mouse. The lumen of the angiocatheter was reduced in group III to have a low flow rate of ~1 ml/min per mouse by inserting the needle of a 24-gauge angiocatheter through the angiocatheter in the lower abdomen.
Pneumoperitoneum can be maintained in 10 mice simultaneously using a multi-channelled-set-up (Figure 1). The pressure and flow rate settings of the insufflator, were 15 mmHg and 1 l/min respectively. Since the water valve limits the insufflation pressure to 2.5 cm of water, the insufflation pressure is identical in each mouse and virtually independent of the pressure setting of the insufflator in this multi-channelled-set-up because of the identical tubes with an internal diameter of 7 mm. In this set-up, the insufflation pressure depends only on the height of the water column in the water valve, since excess CO2 escapes freely.
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Statistical analysis
Data were analysed using analysis of variance (ANOVA). Paired group comparisons were performed using the Tukey-HSD multiple range test (GraphPad, Prism(TM), San Diego, CA, USA). To estimate the accuracy of desiccation experiments, the SD of the whole experiment was calculated from all duplicate experiments using: SD = square root of the sum of the differences between the two measurements performed for each point, divided by the number of measurements. All data from the mice experiments are reported as means ± SD.
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Results |
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In vitro, desiccation was linear with time and flow rate, being 1.28, 2.41, 3.50, 4.49, 5.48 and 6.44 g of water loss following 10, 20, 30, 40, 50 and 60 min of insufflation at 10 l/min flow rate respectively and being 1.61, 3.24, 4.83 and 6.53 g of water loss after insufflation at 5, 10, 15 and 20 l/min flow rates for 30 min (Figure 2a,b).
The total adhesion scores were 0.9 ± 0.8 (n = 15) in control group with some 5 min of pneumoperitoneum (Figure 3). The adhesion scores increased to 2.4 ± 0.8 (n = 15) (P < 0.001 versus control group) in group II with 60 min of pneumoperitoneum without flow and to 2.6 ± 1.3 (n = 15) (P < 0.001 versus control group) in group III with 60 min of pneumoperitoneum using continuous low flow (P < 0.001 versus control group). The total adhesion score increased further to 4.3 ± 0.9 (n = 15) (P < 0.001 versus control group, P < 0.001 versus group II and III) in group IV with 60 min of pneumoperitoneum using continuous high flow. There was no mortality in this study.
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Discussion |
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The in-vitro desiccation set-up showed that at 37°C desiccation is linear with time and flow rate, at least within the limits of the experiment. The amount of desiccation which was 0.674 g/( l/min flow rate)/h in vitro, can only give a rough estimation of the in-vivo situation. Indeed, surface area, temperature and transudation of the peritoneum are crucial factors, which will ultimately determine the drying of the peritoneum.
Many scoring systems have been used for postoperative adhesions. Generally extent, type, tenacity and inflammation were scored macroscopically and/or microscopically (Rijhwani et al., 1995; Evrard et al., 1996
; Wallwiener et al., 1998
). We used the same scoring as in rabbits (Ordonez et al., 1997
), which is a scoring system essentially comparable to the scoring system of Fiedler et al. (1996) and Boyers et al. (1988). Because of the small dimensions of mice, adhesions have to be scored by laparotomy, preferentially using magnification such as an operating microscope. Inflammation was not scored since histological evaluation was not used in this study. Extent was not scored either, because in virtually all animals >75% of the lesion sites were covered with adhesions probably due to small lesion sites.
Our data clearly demonstrate that the duration of pneumoperitoneum using non-humidified CO2, is an important co-factor in adhesion formation in the mouse model and also confirm the results of the study which showed that shorter operation times reduced adhesion formation in the rabbit model (Ordonez et al., 1997). Pneumoperitoneum alone for 60 min without any flow through the abdomens of the mice, increased adhesion formation. This increase was probably mediated by acidosis (Volz et al., 1997
), hypoxaemia and/or desiccation. Desiccation was probably minimal, since there was no flow and since desiccation is flow related at least in vitro. Desiccation does increase adhesion formation, which was clearly apparent with higher flow rates through the abdomens of the mice, whereas a low flow rate had an effect similar to CO2 alone without flow. Although the in-vitro observations can not be extrapolated to the situation in vivo, it is clear that high flow rates cause more desiccation being linear with time. For this reason, proper humidification of CO2, especially when used with a high flow insufflator, seems to be mandatory. The mouse model permitting surgery in 10 mice seems suited to investigate the exact correlation between flow rate, duration of surgery, degree of humidification, desiccation of the peritoneal lining and adhesion formation. Since most biological effects are not linear, this knowledge could be important to delineate the conditions when endoscopic surgery with high flow insufflation becomes dangerous for adhesion formation.
A mouse model for the studies of adhesion formation following endoscopic procedures, has some advantages. Firstly, the mouse is a known model in adhesion formation after a laparotomy. Secondly, this model permits the anaesthesia and maintenance of pneumoperitoneum even in 10 mice simultaneously, which is an obvious advantage to investigate the effect of variables such as the duration of CO2 pneumoperitoneum. Thirdly, since implantation of human cells from endometriosis and malignant tumours is possible in some immunodeficient mice strains such as Nude and SCID (severe combined immunodeficiency) mice, this model opens new possibilities.
In conclusion, the mouse model can be used to study adhesion formation following endoscopic procedures. The duration of CO2 pneumoperitoneum is a co-factor in adhesion formation.
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Acknowledgments |
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Notes |
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3 To whom correspondence should be addressed
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References |
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Submitted on July 27, 1998; accepted on October 16, 1998.