World Journal of Surgery

, Volume 30, Issue 1, pp 127–133

Effects of Peritoneal Lavage with Scolicidal Agents on Survival and Adhesion Formation in Rats

Authors

    • Department of General SurgeryCumhuriyet University, School of Medicine
  • Isinsu Kuzu
    • Department of PathologyAnkara University, School of Medicine
  • Kaan Karayalcin
    • Department of General SurgeryAnkara University, School of Medicine
Article

DOI: 10.1007/s00268-005-7960-4

Cite this article as:
Topcu, O., Kuzu, I. & Karayalcin, K. World J. Surg. (2006) 30: 127. doi:10.1007/s00268-005-7960-4

Abstract

Intraoperative irrigation of the peritoneal cavity with scolicidal agents is frequently recommended when dealing with traumatic or spontaneous rupture of hydatid cysts. The present experimental study was designed to examine the influence of various scolicidal agents on adhesion formation and survival. A total of 149 rats were randomly allocated to nine groups. Peritoneal lavage through a median laparotomy was performed with the following scolicidal agents. Group 1 (0.9% saline: controls), group 2 (20% hypertonic saline), group 3 (0.04% chlorhexidine gluconate), group 4 (3% hydrogen peroxide), group 5 (0.5% silver nitrate), group 6 (1% polyvinylpyrrolidone-iodine, or PVP-I ), group 7 (5% PVP-I), group 8 (0.5% cetrimide/0.05% chlorhexidine), and group 9 (10% PVP-I). The surviving animals were sacrificed on postoperative day 15. Adhesion formation was macroscopically graded by the Nair criteria. The severity of adhesion formation was evaluated microscopically using the fibrosing scoring criteria and the strain test. Group 9 (10% PVP-I) was excluded from the adhesion evaluation because all of the rats died in this group. The mortality rate was significantly higher in groups 5 and 7 than in groups 1, 2, 3, 4, 6, and 8. Adhesion scores were significantly lower in groups 1, 2, 3, and 4 than in groups 5, 6, 7, and 8. The lowest adhesion score was found in group 3 and the highest in the group 7. These results indicate that 0.04% chlorhexidine gluconate, the most potent scolicidal agent in vitro and in vivo, was associated with the lowest adhesion formation and mortality among various scolicidal agents in this experimental study.

Postoperative peritoneal adhesions represent a major surgical problem. They are the main cause (80–90%) of intestinal obstruction.1,2 Adhesions are also a significant cause of morbidity among women of reproductive age3. Adhesions create various medical problems, including infertility and chronic pelvic pain along with increased health expenses.3,4 The total cost of all hospitalizations in 1994 during which adhesiolysis was performed was estimated to be $1.33 billion in the United States. More than three-fourths (84%) of the expenditures were related to procedures in the digestive system ($1.11 billion). Adhesiolysis procedures associated with the female reproductive system, pregnancy, and hepatobiliary system and pancreas accounted for $166.9 million, $25.4 million, and $23.2 million, respectively.4

Surgical intervention is still the preferred method of treatment, but it carries the risk of intraoperative spillage of scolices.5,6 Although injecting a scolicidal solution into the hydatid cyst and preserving the operative field with sponges soaked in scolicidal agents have been used to avoid dissemination of the parasite during surgery, intraperitoneal hydatidosis (IPH) remains the most important complication of hydatid cyst surgery. IPH can occur after spontaneous, traumatic, or iatrogenic rupture of, or leakage from, the cyst.7 Peritoneal lavage with scolicidal agents plus chemotherapy have been suggested as a therapeutic approach to peritoneal dissemination that occurs prior to the surgical intervention or during the operation.8

Because adhesion is an important problem associated with surgery, it is crucial clinically that the agents used in hydatid cysts and IPH should not increase adhesion formation. Therefore, the aim of the present experimental study was to determine the effects of scolicidal agents on adhesion formation and survival rates following peritoneal lavage.

METHODS

The present study was approved by the local ethics committee of Ankara University and was performed according to the guidelines for the care and use of laboratory animals. Randomly selected 3-month-old male Wistar albino rats (n = 149) weighing 250 ± 50 g were obtained from the Experimental Animal Laboratory at Ankara University School of Medicine. During the study period, all animals were fed rat chow ad libitum, given tap water, and kept at room temperature (18°–20°C) in separate cages. All manipulations described below were performed with strict compliance to antiseptic principles.

The rats were subjected to a 2 cm long median laparotomy with a scalpel under ether anesthesia. Peritoneal lavage was performed with the following scolicidal agents: group 1 (0.9% saline: controls, n = 15); group 2 [20% hypertonic saline (HS), n = 15]; group 3 [0.04% chlorhexidine gluconate (Chx-Glu), n = 15]; group 4 [3% hydrogen peroxide (HP, H2O2), n = 15]; group 5 [0.5% silver nitrate (SN), n = 15]; group 6 [1% polyvinylpyrrolidone-iodine (PVP-I ), n = 17]; group 7 (5% PVP-I, n = 20); group 8 [0.5% cetrimde/0.05% chlorhexidine (Cet-Chx), n = 17]; and group 9 (10% PVP-I, n = 20). Each of the nine solutions (in 5 ml aliquots) was injected into the peritoneal cavity of each rat. After 10 minutes, a 3 ml sample of the excess fluid in the lower part of the abdominal cavity was gently aspirated with an injector to facilitate closure of the laparotomy incision with a continuous subcuticular 4/0 polyglactin suture.

Fifteen days later, the surviving rats were killed (in a humanitarian way, by cervical dislocation) under ether anesthesia, and a second laparotomy was performed with a wide inverted-U incision.

Adhesion formation was macroscopically graded according to the Nair criteria9 as follows: grade 0, complete absence of adhesions; grade 1, single band of adhesions between viscera or from viscera to abdominal wall; grade 2, two bands, either between viscera or from viscera to abdominal wall; grade 3, more than two bands, between viscera, or viscera to abdominal wall, or the intestines forming a mass without being adherent to the abdominal wall; grade 4, viscera directly adherent to abdominal wall, irrespective of the number and extent of the adhesive bands.

The severity of the adhesion formation was evaluated microscopically by the amount of fibrosis and scored: 0, no fibrosis; 1, fibrosis with thin collagen bundles; 2, tissue with wider and less vascularized collagen fibrosis; 3, tissue with thick collagen bundles.10

Masson trichrome histochemistry was carried out on each sample to examine the collagen bundles. The severity of adhesion formation was also measured by the strain test. Adhesions, including both viscera and abdominal wall, were removed and the strain test performed. The strain test required use of a straining apparatus, which moved steadily at the vertical axis, connected to a sensitive sensor (Hema, Ankara, Turkey), which had a mobile tip and a fixed tip. The “pick hold” circuit of the straining apparatus ensured that the measurement remained constant on the scale during the time of breakdown.

All data were analyzed using the χ2, Kruskal-Wallis variance analysis, and Mann-Whitney U-test. SPSS computer program software (version 9.0; SAS Institute, Cary, NC, USA) was used. A value of P< 0.05 was accepted as significant.

RESULTS

Evaluation of the Survival and Mortality Rates

All deaths occurred during the first 24 hours of the postoperative period. All animals in group 9 (10% PVP-I) died within the first 2 hours of the experiment, demonstrating the highest mortality rate of all the groups (P < 0.001). The comparison of mortality rates between group 5 (0.5% SN) and group 7 (5% PVP-I) showed no difference (P > 0.05). On the other hand, mortality rates for group 5 (0.5% SN) and group 7 (5% PVP-I) were found to be significantly higher (P < 0.001) than those for the other groups (groups 1, 2, 3, 4, 6, 8) (Table 1). In addition, there were no significant difference (P > 0.05) between the mortality rates of groups 1, 2, 3, 4, 6, and 8.
Table 1

Survival and mortality rates for groups 1 to 9

Group

Survivors (no.)

Dead (no.)

Total (no.)

1 (control)

15

0

15

2 (20% HS)

14

1

15

3 (0.04% Chx-Glu)

15

0

15

4 (3% HP)

15

0

15

5 (0.5% SN)

10

5

15

6 (1% PVP-I)

16

1

17

7 (5% PVP-I)

10

10

20

8 (0.5% Cet-0.05% Chx)

16

1

17

9 (10% PVP-I)

0

20

20

HS: hypertonic saline; Chx-Glu: chlorhexidine gluconate; SN: silver nitrate; PVP-I: polyvinylpyrrolidone-iodine; Cet: cetrimide.

Evaluation of Adhesion Formation

Because all of the animals died within the first perioperative and postoperative 2 hours in group 9, they could not be evaluated.

Macroscopic Evaluation of the Adhesion Formation via the Nair Criteria

Table 2 shows the adhesion number and extent, and Table 3 shows the severity of the adhesions (mean ± standard deviation) in the groups. A comparison of the adhesion rates in groups revealed that there was no difference between groups 1, 2, 3, and 4 (P > 0.05) or between groups 5, 6, 7, and 8 (P > 0.05). Adhesion formation in groups 1, 2, 3, and 4 was found to be lower (P < 0.05) than that observed in groups 5, 6, 7, and 8. The most numerous adhesion formations were found in group 7 (5% PVP-I), and the lowest adhesion formation rate was observed in group 3 (0.04% Chx-Glu).
Table 2

Number and extent of adhesions for groups 1 to 8 (described by Nair et al.9

Group

Total no.

Grade 0

Grade 1

Grade 2

Grade 3

Grade 4

1 (control)

15

5

8

2

2 (20% HS)

14

6

8

3 (Chx-Glu)

15

9

6

4 (3% HP)

15

5

4

3

2

1

5 (0.5% SN)

10

1

2

2

2

3

6 (1% PVP-I)

16

3

7

6

7 (5% PVP-I)

10

1

1

6

2

8 (Cet-Chx)

16

9

7

Grade 0: complete absence of adhesions.

Table 3

Mean adhesion scores for groups 1 to 8

Group

Mean ± SD

Median

Range

1 (control)

0.80 ± 0.68

1.00

0–2

2 (20% HS)

0.57 ± 0.51

1.00

0–1

3 (0.04% Chx-Glu)

0.40 ± 0.50

0

0–1

4 (3% HP)

1.33 ± 1.29

1.00

0–4

5 (0.5% SN)

2.40 ± 1.43

2.50

0–4

6 (1% PVP-I)

2.18 ± 0.75

2.00

1–3

7 (5% PVP-I)

2.90 ± 0.88

3.00

1–4

8 (Cet-Chx)

2.44 ± 0.51

2.00

2–3

Histopathologic Evaluation of the Severity of Adhesion Formation via the Fibrosis Score Criteria

Fibrosis scores are given in Table 4, and their averages are in Table 5. The mean fibrosis scores in groups 1, 2, 6, and 7 were similar (P > 0.05), as was seen for groups 3, 4, 5, and 8 (P > 0.05). In contrast, the mean fibrosis scores were lower (P < 0.05) in groups 3, 4, 5, and 8 than in groups 1, 2, 6, and 7. The highest average was observed in group 7 (5% PVP-I) and the lowest in group 5 (0.5% SN).
Table 4

Fibrosis scores for groups 1 to 8

Group

Total no.

Adhesions (no.)

Score 0

Score 1

Score 2

Score 3

1 (control)

15

10

2

4

4

2 (20% HS)

14

8

6

2

3 (Chx-Glu)

15

6

2

4

4 (3% HP)

15

10

4

4

2

5 (0.5% SN)

10

9

6

3

6 (1% PVP-I)

16

16

2

7

7

7 (5% PVP-I)

10

10

4

6

8 (Cet-Chx)

16

16

4

9

3

Score 0: no fibrosis.

Table 5

Mean fibrosis scores for groups 1 to 8

Group

X ± SD

Median

Range

1 (control)

1.30 ± 0.67

1.00

0–2

2 (20% HS)

1.25 ± 0.46

1.00

1–2

3 (0.04% Chx-Glu)

0.60 ± 0.54

1.00

0–1

4 (3% HP)

0.80 ± 0.78

1.00

0–2

5 (0.5% SN)

0.37 ± 0.74

0.00

0–2

6 (1% PVP-I)

1.31 ± 0.70

1.00

0–2

7 (5% PVP-I)

1.70 ± 0.67

2.00

1–2

8 (Cet-Chx)

0.87 ± 0.61

1.00

0–2

Measurement of the Severity of Adhesion Formation via the Strain Test

The mean values of the strain test are shown in Table 6. The mean values of the straining test in groups 1, 2, 3, and 4 were similar (P > 0.05), as were those in groups 5, 6, 7, and 8 (P > 0.05). The mean values for groups 1, 2, 3, and 4 were significantly lower (P < 0.05) than those in groups 5, 6, 7, and 8. The highest mean value was in group 7 (5% PVP-I) and the lowest in group 3 (0.04% Chx-Glu).
Table 6

Mean strain test results for groups 1 to 8

Groups

Mean ± SD

Median

Range

1 (Control)

1.82 ± 1.40

2.50

1.70–2.65

2 (20% HS)

1.42 ± 1.28

2.50

1.32–2.55

3 (0.04% Chx-Glu)

0.88 ± 0.20

0.90

0.68––1.13

4 (3% HP)

2.12 ± 1.75

2.50

1.95–2.60

5 (0.5% SN)

5.05 ± 2.91

5.35

4.80–5.50

6 (1% PVP-I)

3.82 ± 0.94

3.95

3.70–4.10

7 (5% PVP-I)

5.24 ± 1.91

5.30

4.90–5.60

8 (Cet-Chx)

3.16 ± 0.39

3.20

2.90–3.30

DISCUSSION

The effects of scolicidal agents on intraperitoneal adhesion formation were determined in the current study. The most extensive and severe adhesion formation occurred in the 5% PVP-I group according to the macroscopic evaluation (Nair criteria), the histopathologic evaluation (fibrosis score criteria), and the strain test (severity of adhesion formation). The lowest number of adhesions and the least severe adhesion formation were in the 0.04% Chx-Glu group according to the strain test and by macroscopic evaluation. In addition, the least severe adhesion formation was in the 0.5% SN group according to the histopatholgic evaluation.

The incidence of intraperitoneal adhesions ranges from 67% to 93% after general surgical abdominal operations11 and up to 97% after open gynecologic pelvic procedures. Adverse sequelae associated with postsurgical abdominal adhesions include bowel obstruction, difficult reoperative surgery, chronic pain, and infertility in women. The treatment of these conditions significantly increases health care costs.4,12

Hydatid disease continues to be an important endemic problem in many parts of the world including Turkey. The mainstay of treatment for hydatid disease is surgery. The surgical approach depends on the location, size, and complications of the cyst. Although inactivation of the scolex with a scolicidal agent prior to opening or removing a cyst is strongly recommended, spillage of the cyst contents is far too common despite all the technical precautions taken. This is the major cause of recurrence, which is seen in approximately 10% (8.5–22.0%) of postoperative cases.7,13 Operative spillage can sometimes also lead to secondary disseminated IPH, a disaster leading to persistent disease, which is difficult to treat, often requiring multiple operations with high complication and fatality rates.7,1315 Peritoneal lavage with scolicidal agents plus chemotherapy prior to the surgical intervention or during the operation is suggested as a therapeutic approach to any peritoneal dissemination that might occur.8

To date, several scolicidal agents were used for this purpose. Scolicidal agents used during hydatid cyst surgery are not safe for treating IPH and secondary hydatidosis because of their associated side effects. In fact, limited numbers of agents to treat experimental peritoneal hydatidosis have been studied.1618

It was reported that 1% PVP-I was more potent than 20% hypertonic saline in the treatment of IPH.16 It has also been suggested that application of low-dose iodine (1%) may be necessary to avoid the toxicity caused by high concentrations of iodine.16 Besim et al. showed that 1% PVP-I was not effective as a scolicidal agent at 5 and 10 minutes in vitro.19 Furthermore, it was reported that irrigation of the serosal surface with PVP-I caused renal shutdown, sterile peritonitis, sclerosing serositis, and constructive pericarditis.20 Landa Garcia et al. tested four scolicidal agents (10% H2O2, 10% PVP-I, praziquantel, 10% hypertonic saline) in experimental peritoneal hydatidosis. They reported that 10% H2O2 and 10% PVP-I were much more potent than the other agents.17 Data on the effects of PVP-I on adhesion formation have been controversial. Mazuji et al.,21 Badowski and Daciews,22 Janik et al.,23 and Gilmore and Reid24 reported that PVP-I decreased adhesion formation, whereas Van Westreenen et al.,25 Roberts et al.,26 and Mund-Hoym and Roberg27 reported increased adhesion after PVP-I use.

In the present study, three concentrations of PVP-I (1%, 5%, 10%) were used. Because 10% PVP-I is highly toxic, all rats in the group died within the first 2 hours of the experiment. Moreover, 5% PVP-I was found to cause the most extensive and severe adhesions, and 1% PVP-I increased adhesion formation. The higher mortality rates seen in the 5% and 10% PVP-I groups might have been due to iodine toxicity, which was used in high concentrations.

Even though 10% H2O2 was noted to be a powerful scolicidal agent in vitro by Meymerian et al.,28 it may not have any effect in vivo because of its instability [29, 30]. Fatal air embolism and anaphylactic shock have also been reported with 10% H2O2.31,32 Injection of H2O2 into cysts could cause bursting, spillage, and tears.

Hypertonic saline is one of the most common scolicidal agents in the world. The effectiveness of hypertonic saline at various concentrations and exposure times has been studied in vitro and in vivo by Kayaalp et al., who looked at whether hypertonic saline [20%, 30%] could be used to irrigate the abdomen when there was a free intraperitoneal perforation of the hydatid disease.33 They found that 20% and 30% hypertonic saline concentrations were effective for only a short period of time (6 and 3 minutes, respectively). Hypertonic saline can cause acute hypernatremia, leading to convulsions, intracranial bleeding, necrosis, and myelinolysis; whereas adding it to the cyst cavity without evacuation might cause dilution of the hypertonic saline. Therefore, it has been postulated that hypertonic saline should not be used as a scolicidal agent for the treatment of intraperitoneal disease or hydatid cysts.33 Findings of the present study revealed no serious mortality associated with 20% hypertonic saline, which was also found to cause the second least amount of adhesion formation among the scolicidal agents tested.

Besim et al. reported that cetrimide-chlorhexidine was the most potent scolicidal agent in vitro.19 The results of Sonisik et al. further supported the potent scolicidal effect of cetrimide-chlorhexidine in a clinical study.34 Because of the toxicity and side effects of cetrimide (e.g., methemoglobinemia, increasing metabolic acidosis, convulsions, coma), it has limited use clinically.35,36 Gilchrist reported three cases of sterile chemical peritonitis with massive adhesions after hydatid liver surgery, which was attributed to peritoneal washout with 0.5% cetrimide.37 Karayalcin et al. reported a seriously increased mortality rate [60%] following experimental peritoneal lavage with Cet-Chx.38 Their results showed that although Cet-Chx did not increase the incidence of adhesions, the use of 0.5% Cet/0.05% Chx solution as a peritoneal washout solution could be dangerous because of its associated toxicity. In contrast to their findings, the present study found no serious increase in the mortality rate after the use of 0.5% Cet/0.05% Chx, although adhesion formation was found to be increased.

Chlorhexidine gluconate (Chx-Glu) is an antiseptic agent with many attractive features for local therapy of intraabdominal infection. It has a broad spectrum of activity against gram-positive and gram-negative bacteria and fungi.39 Chx-Glu also has a low toxicity profile at therapeutic concentrations of 0.05% in animal models of peritonitis40,41 and in the clinical setting [42]. Bondar et al. determined that lavage with 0.05% Chx-Glu was effective and nontoxic in patients with an intraabdominal infection.43 Chx-Glu at a concentration of 0.04% was found to be the most potent scolicidal agent that acted in a short period of time (5 minutes) in vitro and was 100% effective in vivo for IPH [18]; thus Chx-Glu 0.04% could be used safely for treating IPH and hydatid cysts.

Van Westreenen et al.25 reported increased adhesion formation in a standard peritoneal ischemia model in rats after peritoneal lavage with certain solutions (1% PVP-I, 0.02% Chx-Glu, 0.9% saline) compared with a control group (no lavage). Maleckas et al. reported that the peritoneal adhesion score was significantly higher after lavage with 0.05% Chx-Glu solution than with saline in both gastric and fecal peritonitis.44 In the present study, Chx-Glu 0.04% was found to cause the least adhesion formation. In addition, although the histopathologic findings did not support those findings, 0.04% Chx-Glu demonstrated the lowest adhesion severity according to the strain test. Furthermore, the findings of this study did not agree with the findings of the Van Westreenen et al. and Maleckas et al. studies;25,44 however, the present study did not use an adhesion model such as cecal abrasion, peritonitis, or ischemia.

CONCLUSIONS

These results indicate that 0.04% chlorhexidine gluconate, the most potent scolicidal agent in vitro and in vivo, is associated with the lowest rate of adhesion formation and mortality among various scolicidal agents tested in this experimental study. Therefore, it can be used in the treatment of hydatid cysts and IPH.

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© Société Internationale de Chirurgie 2005