Prevention of adhesions during laparoscopic surgery in mice with crystalloids

November 2002, Vol. 9, No. 4 The Journal of the American Association of Gynecologic Laparoscopists Prevention of Adhesions with Crystalloids
during Laparoscopic Surgery in Mice

Osama Ali Elkelani, M.D., Carlos Roger Molinas, M.D., Ospan Mynbaev, M.D., Ph.D., and Philippe Robert Koninckx, M.D., Ph.D.
Study Objective. To evaluate the effect of saline and Ringer's lactate solutions in preventing adhesions during lapa-
roscopic surgery in mice.
Design. Prospective, randomized trial (Canadian Task Force classification I).
Setting. Academic research center.
Subjects. Ninety-two female Naval Medical Research Institute mice.
Intervention. Adhesions were induced laparoscopically by opposing bipolar lesions in the uterine horns and pelvic
sidewalls, and saline or Ringer's lactate solution was added at different times during the procedure.
Measurements and Main Results. Adhesions were scored quantitatively and qualitatively for extent, type, and tenac-
ity after 7 days under microscopic vision during laparotomy. After 45 minutes of pneumoperitoneum, neither solu-
tion reduced adhesion formation, but when added immediately after surgery they did (p = 0.002). Coagulation
was not significantly different with addition of either solution immediately after coagulation. In the third experi-
ment the presence of fluid during pneumoperitoneum decreased adhesion formation (p = 0.0001) but Ringer's
lactate was more effective than saline (p = 0.0005).
Conclusion. Crystalloids reduced CO2 pneumoperitoneum-enhanced adhesion formation in a laparoscopic mouse

model, but Ringer's lactate solution was more effective than saline. (J Am Assoc Gynecol Laparosc 9(4):447–452, 2002)
Postoperative adhesion formation is an important as rats,6 rabbits,7,8 and monkeys.9 After laparoscopic clinical problem. Good surgical technique, that is, surgery these materials were effective in humans but atraumatic and bloodless surgery, and prevention of not in animal models.4 Separation of injured sur- desiccation are emphasized as preventing adhesions.
faces by hydrofloatation with viscous substances such Mechanical barriers and hydrofloatation with fluids as dextran reduced adhesions after laparotomy in help reduce adhesion formation in humans and ani- humans10 and in rats,11 and after laparoscopy in mals. After laparotomy both Interceed and Gore-Tex humans.10,12 Hydrofloatation with crystalloids such as were effective in humans1–5 and in animal models such saline or Ringer's lactate solution in humans has a From the Center for Surgical Technologies (all authors), and Department of Obstetrics and Gynaecology, University Hospital Gasthuisberg, KatholiekeUniversiteit Leuven (Drs. Elkelani, Molinas, and Koninckx), Leuven, Belgium.
Supported in part by grants from Fonds Wetenschappelijk Onderzoek, Brussels, Belgium, and Onderzoeks Toelagen Katholieke Universiteit Leuven,Leuven, Belgium.
Address reprint requests to Osama Ali Elkekani, M.D., Centre for Surgical Technologies, Minderbroederstraat 17, 3000 Leuven, Belgium; fax32 16 344 238.
Accepted for publication April 25, 2002.
Prevention of Adhesions in Mice with Crystalloids Elkelani et al controversial effect,13,14 but was effective after lapa- Pneumoperitoneum was maintained with an insuf- rotomy in rats15–17 and rabbits.18 No data are available flator (Thermoflator Plus, Karl Storz) delivering car- regarding crystalloids after laparoscopy.
bon dioxide (CO2) heated to 37° C and humidified with vapor (Drager, Lubeck, Germany). Since most insuf- Materials and Methods
flators have intermittent delivery of gas, a water valve with free escape of gas was used to provide contin- We investigated the effect of saline and Ringer's uous flow and constant pressure. An elastic balloon lactate solutions on adhesion formation in a mouse was placed next to the water valve to eliminate virtu- model of CO2 pneumoperitoneum-enhanced adhe- ally all pressure changes in the animal's tiny abdom- sion formation.19 The study was approved by the insti- inal cavity. Since peritoneum has a high exchange tutional review animal care committee.
capacity, continuous removal of oxygen that could diffuse from the circulation is required to maintain the predefined concentration of gas inside the abdominal Ninety-two Naval Medical Research Institute cavity. Therefore, a 26-gauge needle was inserted next mice (age 10–12 wks, weight 25–35 g) were kept under to the endoscope to obtain continuous flow of some standard laboratory conditions (temperature 20–25° C, 10 ml/minute of gas through the animal.
relative humidity 40–70%, 14 hrs light, 10 hrs dark).
After establishing pneumoperitoneum, two 14- They were fed a standard laboratory diet (Hope Farm, gauge catheters were inserted under direct laparo- Woerden, The Netherlands) and had free access to scopic guidance for working instruments in both right water and food before and after the laparoscopic and left flanks. With a 1.5-mm grasper the bicornuate uterus was individualized by removing surrounding fat tissue and grasped in the midline. Using the bicap for- Anesthesia and Endotracheal Intubation
ceps (Circon ACMI, Stanford, CT) with a 1.6-mm After intramuscular anesthesia with pentobarbital ball electrode, a linear bipolar coagulation lesion of 0.06 mg/g, the abdomen was shaved and the animal 10 mm was created in the antimesenteric border of was secured to the table in supine position. For intu- both uterine horns for 3 to 5 seconds and in ipsilateral bation, the animal was placed with the neck under a pelvic sidewalls.
light source; the tongue was grasped with a hemostatic The procedure took 3 to 4 minutes. Pneumoperi- clamp and pulled out, and vocal cords were visualized toneum was maintained for 45 minutes. In groups I by transillumination. A 20-gauge catheter with a blunt to III, macroscopic lesions were made with 10 W in guidewire was inserted into the trachea. After remov- standard coagulation mode (Autocon 350, Karl Storz), ing the wire, the mouse was ventilated with room air whereas in group IV (under fluid) 30 W had to be used using a mechanical respirator (Rodent Ventilator, Har- for 3 to 5 seconds. These settings were obtained in pre- vard Apparatus, Hollison, MA) with 1.5 ml tidal vol- vious pilot experiments. At the end of surgery the ume and 85 strokes/minute.
incisions were sutured with 6-0 polyglycolic acid in single layers.
Induction of Adhesions
Laparoscopic surgery was performed under asep- tic conditions as described elsewhere.19 After disin- After 7 days laparotomy was performed to eval- fection, a 3.5-mm abdominal incision was made caudal uate adhesions under an operative microscope. The to the xiphoid process. A 2.1-mm zero-degree endo- whole abdominal cavity was visualized through xypho- scope with an outer sheath for protection and insuf- pubic midline and bilateral subcostal incisions. After flation of 3.3 mm (Karl Storz, Tuttlingen, Germany), evaluating ports sites and viscera for de novo adhe- connected to a single-chip videocamera and light sions, fatty tissue surrounding the uterus was care- source, was introduced into the abdominal cavity. The fully removed. The lengths of visceral and parietal endoscope was secured in a holder and the incision lesions and adhesions were measured. Adhesions, was sutured around the endoscope with 6-0 polygly- when present, were lysed to evaluate their type and colic acid suture (Dexon II; Davis+Geck, Gosport, tenacity. They were scored by the surgeon and an UK) to avoid gas leakage.
independent observer in random order, and codes of November 2002, Vol. 9, No. 4 The Journal of the American Association of Gynecologic Laparoscopists the intervention were broken only at the end of the one of the laparoscopic ports. Ringer's lactate was left experiment. The qualitative evaluation assessed extent in situ (group III) or evacuated (group IV). Two, three, (0 = no adhesions, 1 = 1–25%, 2 = 26–50%, 3 = and three mice died after surgery in the first, second, 51–75%, 4 = 76–100%), type (0 = no adhesions, 1 = and third experiments, respectively.
filmy, 2 = dense, 3 = capillaries present), tenacity (0 = no adhesions, 1 = essentially fall apart, 2 = require traction, 3 = require sharp dissection), and totals of these (extent + type + tenacity).
Statistical analysis was performed with the SAS For quantitative evaluation the proportion of adhe- system (SAS Institute, Cary, NC) using a nonpara- sions was calculated by measuring the length of metric two-way analysis of variance (Proc GLM), the line covered by adhesions and the length of the both giving similar results. Only significances obtained lesions. Calculation was made according to the for- by regression analysis are given. Since the design of mula: adhesions (%) = (sum of the length of the experiments I and II was identical, and since both individual attachments/length of the lesion) ´ 100.
were randomized, the data of these experiments were Adhesions in visceral and parietal peritoneum were analyzed together to evaluate differences among the evaluated separately. The animal was sacrificed imme- four groups. During this analysis, results were cor- diately after evaluation.
rected for eventual differences between saline (exper- iment I) and Ringer's lactate (experiment II) as follows: adhesion scores are influenced simultaneously by the All experiments were performed using block ran- experimental group and by type of fluid.
domization by day. Therefore, a block of animals, Since data were obtained in two experiments and consisting of one animal of each group, was always thus were obviously nonrandomized, differences operated on the same day, avoiding day-to-day between fluids were disregarded. Therefore, a third experiment was performed using a 2 ´ 2 factorial The first experiment evaluated the effect of adding design to evaluate and ascertain differences between saline at the end of pneumoperitoneum, immediately solutions and to confirm results of the first two exper- after surgery or before surgery in 20 animals. In group iments. In the analysis, parietal and uterine and right I, no fluid was added; in group II, 20 ml of NaCl 0.9% and left sides of adhesions were averaged and differ- (normal saline) was added at the end of the procedure; ences were not taken into account.
in group III, 20 ml of normal saline was added imme- Means and standard errors are indicated unless diately after coagulation; and in group IV, 20 ml of normal saline was added before induction of pneumo- peritoneum, and coagulation was performed under The design of the second experiment was the Addition of saline or Ringer's lactate after 45 same, but Ringer's lactate was used instead of saline minutes of pneumoperitoneum (group II) did not reduce adhesion formation (Figure 1). Addition of The third experiment in 32 animals was designed either solution immediately after surgery decreased to evaluate simultaneously (factorial 2 ´ 2 design) adhesion formation (group III) expressed as the total differences between saline and Ringer's lactate and the adhesions (p = 0.002) or as quantitative adhesion score effect of fluid during coagulation. In the first two (p = 0.007). Coagulation under saline or Ringer's lac- experiments a different power setting had to be used tate (group IV) was not significantly different with for normal and underwater coagulation to obtain sim- addition of the solutions immediately after coagula- ilar macroscopic lesions. Differences in tissue dam- tion (group III). Similar observations were seen for age thus could not be ruled out with certainty. To extent, type, and tenacity of adhesions. Scores for avoid differences caused by depth of coagulation, all extent in group I were 2.5 ± 0.4 and 2.1 ± 0.2 for saline coagulations were performed under fluid, which was and Ringer's lactate, respectively; in group II, 2.6 ± either left or drained. Saline was left in the peritoneal 0.3 and 1.7 ± 0.2, respectively; in group III, 1.8 ± 0.3 cavity (group I) or evacuated at the end of coagula- and 1.2 ± 0.2, respectively (p = 0.003 vs group I, p = tion (group II) by inserting a 14-gauge catheter through 0.02 vs group II); and in group IV, 1.5 ± 0.3 and 0.9 Prevention of Adhesions in Mice with Crystalloids Elkelani et al Quantitative score Qualitative score roportion of A 20 FIGURE 1. Quantitative and total adhesion scores in mice 7 days after induction of peritoneal adhe-
sions by bipolar lesions and 45-minute pneumoperitoneum (group I). Saline (dark bars) or Ringer's lac-
tate solution (light bars) 20 ml was added at the end of pneumoperitoneum, immediately after or before
coagulation, respectively. Mean ± SEM together with significances (p vs group I *
£0.02, ** £0.002; p
vs group II #
£0.02, ## £0.002) between groups are shown.
± 0.2, respectively (p = 0.0003 vs group I, p = 0.001 In the third experiment (Figure 2) both the pres- vs group II).
ence of fluid during pneumoperitoneum and type of Scores for type were group I, 2 ± 0.1 and 2.3 ± fluid (Proc Logistic) strongly affected adhesion for- 0.2 for saline and Ringer's lactate, respectively; group mation as assessed by quantitative evaluation (p = II, 2.1 ± 0.4 and 1.9± 0.2, respectively; group III, 1.6 0.0001 and 0.005 for presence and type of fluid, ± 0.3 and 1.6 ± 0.3, respectively (p = 0.003 vs group respectively), total adhesions score (p = 0.0001 and I, p = 0.02 vs group II); and in group IV, 1.6 ± 0.2 and 0.0005, respectively), extent (p = 0.0001 and 0.003, 1.1± 0.2, respectively (p = 0.002 vs group I, p = 0.008 respectively), type (p = 0.0001 and 0.003, respec- vs group II). Scores for tenacity were group I, 2.1 ± tively), and tenacity (p = 0.0001 and 0.01, respec- 0.1and 2.2 ± 0.2 for saline and Ringer's lactate, respec- tively) of adhesions. The same factors were important tively; group II, 2.0 ± 0.3 and 1.8 ± 0.1, respectively; for total parietal (p = 0.005 and 0.0001, respectively) group III, 1.8 ± 0.3 and 1.1 ± 0.2, respectively (p = and total visceral (p = 0.007 and 0.0001, respectively) 0.003 vs group I, p = 0.02 vs group II); and group IV, adhesion scores. For experimental groups I, II, III, and 1.6 ± 0.1 and 0.9 ± 0.2, respectively (p = 0.0002 vs IV, scores for extent were 1.4 ± 0.2, 2.6 ± 0.2, 0.8 ± group I, p = 0.0008 vs group II).
0.1, and 2.0 ± 0.1, respectively; scores for type were 70 Fluid left vs. Fluid removed P=0.0001 Fluid left vs. Fluid removed P=0.0001 Saline vs. Ringer P=0.005 Saline vs. Ringer P= 0.0005 FIGURE 2. Quantitative total adhesion score in mice 7 days after induction of peritoneal adhesions.
Bipolar lesions were created under saline (dark bars) or Ringer's lactate solution (light bars) at lapa-
roscopy. Fluid was left or evacuated at the end of creating lesion. Mean ± SEM are indicated.

November 2002, Vol. 9, No. 4 The Journal of the American Association of Gynecologic Laparoscopists 1.4 ± 0.3, 2.4 ± 0.1, 0.8 ± 0.1, and 1.9 ± 0.2, respec- thermal injury after coagulation under saline or Ringer's tively; and for tenacity 1.3 ± 0.3, 2.4 ± 0.2, 0.7 ± 0.7, lactate cannot be ruled out, this is highly unlikely and and 2.0 ± 0.2, respectively.
we suggest that Ringer's lactate will more effectively protect cells from hypoxia-induced cell damage. The importance of this for preventing adhesions is that solutions that better support cell viability, such as Our data failed to confirm that saline or Ringer's those used during cell culture (phosphate-buffered lactate solution prevents primary adhesions (group I saline), could be superior.
vs group II) when instilled at the end of surgery. This The advantage of the 2 ´ 2 factorial design is that is in contradiction with data in rats for Ringer's lac- to achieve the same statistical precision as with a one- tate15,17,20 and saline.16 In humans, however, a review at-a-time approach, twice as many observations would and meta-analysis could not confirm the effective- have been required. This increase in power by facto- ness of either solution. The discrepancy between our rial design is valid only when the effects of the two fac- data and previous animal reports could be explained tors are additive; that is, when no interaction between by different models. In all previous reports fluid was factors is present. The possibility to detect an inter- instilled after laparotomy. We used a laparoscopic action, a different effect of one factor at different levels model. This also is the first report in mice, which of the other factor, can also be considered an advan- could respond slightly differently from other animals.
tage of the design, since this effect could otherwise This mouse model was validated in previous experi- easily be missed. When the number of observations ments,19–22 in which CO2 pneumoperitoneum was a is low, one should be aware that a positive interaction cofactor in adhesion formation. Duration of pneumo- (with subsequent reduction of power to demonstrate peritoneum therefore was standard at 45 minutes.
the effect of the two factors) can be missed, espe- Our data clearly show that the two crystalloids are cially when between-subject variability is high.24 ineffective in reducing CO2 pneumoperitoneum- These data extend our previous observation on enhanced adhesion formation. This could be extremely CO2 pneumoperitoneum as a cofactor in adhesion important for future investigations on adhesion pre- formation. Prevention of adhesions is fully consistent vention. It indeed suggests that mechanisms involved with the concept of superficial mesothelial hypoxia as in adhesion formation might not be identical after the driving mechanism. Absence of an effect when laparotomy and laparoscopy. Data for laparotomy instilled after pneumoperitoneum suggests that the therefore should not be extrapolated to laparoscopy mechanisms involved after laparoscopic surgery might without evidence.
be at least partly different from those after laparotomy.
Both crystalloids prevented CO2 pneumoperi- These concepts require further experiments to clar- toneum-enhanced adhesions if instilled immediately ify the exact mechanisms. If substantiated, however, after the lesion was created. During that time the this will be clinically extremely important for adhe- lesions were covered by fluid. This observation is sion prevention since results after laparotomy can- fully consistent with our hypothesis that CO2 pneumo- not be extrapolated to laparoscopy. In addition, new peritoneum induces hypoxia in superficial mesothe- approaches to adhesion prevention after laparoscopic lial layers.19 Partial pressure of oxygen in superficial surgery could be developed.
mesothelial layers will be decreased during CO2 pneu- moperitoneum, and this obviously will be less if peri- toneum is covered by layers of fluid. The importance of this observation is that any substance covering 1. No authors listed: Prevention of postsurgical adhesions peritoneum during laparoscopic surgery has the poten- by INTERCEED(TC7), an absorbable adhesion bar- tial of reducing adhesion formation, at least those rier: A prospective randomized multicenter clinical enhanced by CO2 pneumoperitoneum.
Ringer's lactate was superior to saline in this 2. Sekiba K: Use of Interceed (TC7) absorbable adhesion study, but it is unlikely that pH and buffering capac- barrier to reduce postoperative adhesion reformation in ity could explain this difference. Indeed we clearly infertility and endometriosis surgery. The obstetrics and showed that the effects of helium and CO2 pneumo- gynecology adhesion prevention committee.
peritoneum were identical.23 Although differences in Prevention of Adhesions in Mice with Crystalloids Elkelani et al 3. No authors listed: Prophylaxis of pelvic sidewall adhe- 14. Olaf GJ, Naether OG: Significant reduction of adnexal sions with Gore-Tex surgical membrane: A multicenter adhesions following laparoscopic electrocautry of the ovarian surface (LEOS) by lavage and artificial ascitis.
Gynaecol Endosc 4:17–19, 1995 4. Farquhar C, Vandekerckhove P, Watson A, et al: Barrier agents for preventing adhesions after surgery for sub- 15. Canbaz MA, Ustun C, Kocak I, et al: The comparison fertility. Cochrane Database Syst Rev (2):CD000475, of gonadotropin-releasing hormone agonist therapy and intraperitoneal Ringer's lactate solution in preventionof postoperative adhesion formation in rat models 5. Crain J, Curole D, Hill G, et al: Laparoscopic implant of Gore-Tex surgical membrane. J Am Assoc Gynecol 16. Bhatia DS, Allen JE: The prevention of experimentally Laparosc 2(4):417–420, 1995 induced postoperative adhesions.
6. Bleichrodt RP, Simmermacher RK, van der Lei B , et al: Expanded polytetrafluoroethylene patch versus 17. Pagidas K, Tulandi T: Effects of Ringer's lactate, Inter- polypropylene mesh for the repair of contaminated ceed (TC7) and Gore-Tex surgical membrane on post- defects of the abdominal wall 7. Diamond MP, Cunningham T, Linsky CB, et al: Inter- 18. Sahakian V, Rogers RG, Halme J, et al: Effects of car- ceed (TC7) as an adjuvant for adhesion reduction: Ani- bon dioxide-saturated normal saline and Ringer's lac- tate on postsurgical adhesion formation in the rabbit.
8. Marana R, Catalano GF, Caruana P, et al: Postoperative 19. Molinas CR, Mynbaev O, Pauwels A, et al: Peritoneal adhesion formation and reproductive outcome using mesothelial hypoxia during pneumoperitoneum is a Interceed after ovarian surgery: A randomized trial in cofactor in adhesion formation in a laparoscopic mouse 9. Grow DR, Seltman HJ, Coddington CC, et al: The 20. Ustun C, Yanik FF, Koçak MA, et al: Effects of Ringer's reduction of postoperative adhesions by two different lactate, medroxyprogesterone acetate, gonadotropin- barrier methods versus control in cynomolgus mon- releasing hormone analogue and its dilutent on the pre- keys: A prospective, randomized, crossover stud vention of postsurgical adhesion formation in rat model.
10. Puchalski A: The influence of cumulative dexametha- 21. Yesildaglar N, Ordonez JL, Laermans I, et al: The mouse sone, promethazine and dextran 70 used as protection as a model to study adhesion formation following endo- against intraperitoneal adhesions on selected parame- scopic surgery: A preliminary report.
ters of humoral immunity in women operated on for infertility. Ann Acad Med Stetin 44:115–136, 1998 22. Haney AF, Doty E: Expanded-polytetrafluoroethylene 11. Nagelschmidt M, Saad S: Influence of polyethylene but not oxidized regenerated cellulose prevents adhe- glycol 4000 and dextran 70 on adhesion formation in sion formation and reformation in a mouse uterine horn model of surgical injury 12. Krinsky AH, Haseltine FP, DeCherney A: Peritoneal 23. Molinas CR, Koninckx PR: Hypoxaemia induced by fluid accumulation with dextran 70 instilled at time of CO2 or helium pneumoperitoneum is a co-factor in adhesion formation in rabbits.
13. Wiseman DM, Trout JR, Diamond MP: The rates of adhesion development and the effects of crystalloid 24. Armitage P, Berry G: Factorial designs. Statistical Meth- solutions on adhesion development in pelvic surgery.
ods in Medical Methods in Medical Research. Oxford, Blackwell Scientific Publications, 1987, pp 227–239


Dental Care for the Patient with Bipolar Disorder • David B. Clark, BSc, DDS, MSc (Oral Path), MRCDC • Chronic mental illness and its treatment carry inherent risks for significant oral diseases. Given the shift in treatmentregimens from the traditional institutionally based approach to more community-focused alternatives, generaldental practitioners can expect to see and be asked to treat patients with various forms of psychiatric disorders. Onesuch group consists of patients with bipolar disorder (including type I bipolar disorder or manic-depressive disor-der). The purpose of this paper is to acquaint the dental practitioner with the psychopathological features of bipo-lar disorder and to highlight the oral health findings and dental management considerations for these patients.Bipolar disorder is considered one of the most treatable forms of psychiatric illness once it has been diagnosedcorrectly. Through a combination of pharmacotherapy, psychotherapy and life-adjustment skills counselling, thesepatients are better able to understand and cope with the underlying mood swings that typify the condition and inturn to interact more positively and progressively within society as a whole. Both the disease itself and its variouspharmacologic management modalities exact a range of oral complications and side effects, with caries, peri-odontal disease and xerostomia being encountered most frequently. It is hoped that after reading this article thegeneral dental practitioner will feel more confident about providing dental care for patients with bipolar disorderand in turn to become a vital participant in the reintegration of these patients into society.

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