Keywords: Desferrioxamin, vitamin E, rat, sepsis, peritonitis, survival rates

Aim: The aim of the study was to determine the effects of vitamin E and the iron chelating agent desferrioxamin (Dfx), supplemented by clindamycin and gentamycin therapy, on peritonitis caused by caecal ligation of a puncture wound in an experimental model. Materials and Methods: One hundred and twenty Spraque Dawley rats were divided into eight groups. Three groups were used as controls; intraperitoneal (ip), subcutaneous (sc) and ip and sc, respectively. Group 4 was treated with Dfx, Group 5 with vitamin E and Group 6 with antibiotics. Group 7 was treated with vitamin E in combination with antibiotics, and Group 8 with a combination of antibiotics and Dfx. The rats were studied for 14 days following treatment, and survivors then humanely dispatched. Post-mortem examination was undertaken on all the rats studied. Results: In the control groups, mortality at 14 days was 66%. Rats treated with antibiotics alone (Group 5) had a mortality rate of 40%. Those treated with a combination of antibiotics and vitamin E (Group 7), however, had a mortality rate of only 14%, and those treated with antibiotics and Dfx had a mortality rate of only 7%. Conclusion: This study suggests that treatment of peritonitis in rats with a combination of Dfx and antibiotics has a significant beneficial effect on survival, in comparison with treatment with antibiotics alone

This experimental study with its title and responsible research member has been reviewed by the Ethics Committee of Haseki Research and Educational State Hospital. We would like to confirm the approval for the study was designed and performed under the conditions fitting to the Helsinki Experimental Animal Rights Agreement and to all other ethical rules of experimental study.

J.R.Coll.Edinb., 47, October 2002, 700-704 

INTRODUCTION

Peritonitis is a major cause of death in surgical intensive care units. This mortality is high, despite the use of broad spectrum antibiotics, aggressive surgical intervention and careful monitoring. Patients at higher risk of infection may benefit from the newer regimens of antibiotic management or compounds that might enhance the effect of the antibiotics.

Oxygen-free radicals are generated in large amounts during endotoxic shock and septic peritonitis.^1,2 Free oxygen radicals are postulated to play a role in the organ dysfunction associated with septic peritonitis, and possibly participate in the pathogenesis of experimental endotoxaemia.³ The use of free oxygen radical scavengers, in addition to antibiotics, may improve survival in abdominal sepsis.^4,5

Vitamin E is a widely used antioxidant. It controls excessive accumulation of peroxides by the inhibition of lipid peroxidation, neutralizing and reducing free radicals; a process called the ‘free radical scavenger effect’.^6,7 Hydroxyl radicals, which are more toxic than peroxide and superoxide, are formed mainly by Haber-Weiss and Fenton reactions. These reactions may be arrested by blocking the iron during the reaction.⁸ Desferrioxamin (Dfx) is an ironchelating agent and prevents free oxygen radical formation by inhibiting the catalyzing role of iron in the Fenton reaction. Desferrioxamin is a powerful inhibitor of hydroxyl production, lipid peroxidation and cell damage.⁹

This experimental study was undertaken to assess the effect of vitamin E and Dfx, in combination with antibiotics, in an animal model of peritonitis.

MATERIALS AND METHODS

One hundred and twenty male Spraque Dawley rats, weighing between 180 and 220 g, were obtained from Charles Rivers Breeding Laboratories, Germany. All rats were housed in regular rat cages, on a 12 hour light, 12 hour dark cycle and in a temperature-controlled room. All animals were fed a standard laboratory chow diet and drinking water ad libitium.

Animals were divided into eight groups, each consisting of 15 rats. All were subjected to caecal ligation and puncture, as described by Wichterman et al (1980).¹⁰ Four rats died during the anaesthesia in groups 4, 5, 7 and 8, and were excluded from the study. The groups and treatments are listed in Table 1.

All rats underwent a general anesthetic with an injection of Ketamine intraperitoneally (IP) (10ml/100gr body weight).

Table 1: The groups, treatments, survival data and statistical comparison (Log Rank Test) of the groups in terms of survival

Each received a 4-6 mm lower midline incision. The caecum was filled with faeces by milking the stools back from the descending colon. The wound was then ligated with a 3-0 silk ligature in a manner that did not obstruct the ileocecal valve. The antimesenteric caecal surface was punctured twice with an 18-gauge needle below the ligature. The bowel was then placed back into the peritoneal cavity, and the abdomen closed in two layers. The animals were allowed food following the surgical procedure.

Treatment with antibiotics (Clindamycin and Gentamycin) and/or Dfx or vitamin E commenced just before the midline incisions were made. Group 1 to 3 were used as untreated controls. Group 1 received 1ml of normal saline ip, Group 2, 0.5ml subcutaneously (sc), and Group 3 both forms of saline. Group 4 received 80 mg/kg Dfx (Desferal^®, Ciba-Geigy, Istanbul) sc. Group 5 received 1000 mg/kg ip vitamin E (a-Tocopherol, Sigma Chemie, Germany). Group 6 received 25mg/kg Clindamycin phosphate ip (Cleocin^®, Eczacibasi, Istanbul) and 2,5mg/kg Gentamycin sulphate (Garamycin^®, Eczacibasi, Istanbul). Group 7 received both 1000 mg/kg ip vitamin E and 25mg/kg ip Clindamycin phosphate and 2,5mg/ kg Gentamycin sulphate (a combination of the treatment of rats in Groups 5 and 6). Group 8 received 80 mg/kg Dfx sc, 25mg/ kg ip of Clindamycin phosphate and 2,5mg/kg Gentamycin sulphate (a combination of the treatments received by Group 4 and 6. The daily doses were given with a single injection in all the groups.

All rats were followed-up for survival for 14 days. Rats surviving 14 days were dispatched by cervical dislocation. Post-mortem examination was undertaken on all rats.

Data are expressed as mean and percentage of survival for each group and the groups were compared. Statistical evaluations were conducted with the Statistical Package for Social Sciences (SPSS; Chicago, Illinois, USA). The Log Rank test was used for statistical evaluation and the Kaplan-Meier method was used for statistical curves. Significance was determined as p<0.05 with 95 % confidence interval.

RESULTS

Table 1 shows the groups, treatments, and survival rates of the groups. All the control groups had a 33% survival rate by the 14th day. The survival rate of the rats in the experimental groups varied from 50 to 96%. Group 8 (antibiotics plus Dfx) had a 96% survival rate on day 14 (Figure 1).

There were statistically significant differences between the control groups and the rats which received antibiotics alone (p<0.05), antibiotics and vitamin E (p<0.01) and antibiotics with Dfx (p<0.001) groups. The difference between the antibiotics alone and antibiotics and vitamin E groups was not significant. Group 8 (Dfx and antibiotics) showed significant improvement in survival (p<0.05), when compared with the group treated with antibiotics alone (Table 1; Figure 1).

Post-mortem examination showed diffuse peritonitis and intraabdominal abscess formation in all the animals in Groups 1 to 5. Localized abscesses near the caecal perforation site were detected in 11 rats in Group 6, 9 rats in Group 7, and 10 rats in Group 8. The rest of the rats in Group 6, 7 and 8 had diffuse peritonitis and abscess formation. Post-mortem histologic evaluation of the livers of all the rats showed no pathological difference between the various groups.

DISCUSSION

Septic peritonitis continues to be a major problem in surgery and remains the major cause of death in trauma patients who survive the initial resuscitation period. The standard treatment - wide debridement of necrotic tissue, drainage of abscesses, and broad spectrum antibiotic coverage of suspected bacterial infections - remains the cornerstone of therapy, but improved treatment is necessary to increase host response. The possible role of oxygen-free radicals contributing to the cycle of peroxidation of plasma membranes and cell damage is well established.¹¹ Both lipid peroxidation and hydroxyl production would be stimulated by the release of low molecular weight iron complexes from the injured cell.¹² Recent studies suggest that haemoglobulin can act as a biological Fenton reagent to enhance the production of reactive oxygen species, and can impair antimicrobial defense of the peritoneal cavity.^13,14 Yoo et al (1999) noted the decreased oxygen radical activity and viability of phagocytes in the presence of iron in the peritoneal cavity, and concluded that the lethal toxicity of haemoglobin in bacterial peritonitis was assocciated with a Fenton-type reaction. In Yoo’s study, Fenton inhibitors (desferrioxamin) removed most of the cytotoxic activity of the iron.¹⁵ It has been demonstrated that treatment with an antibiotic in combination with 2,3- dihydroxybenzoic acid (an iron chelating agent and free radical scavenger) improves survival in a septic rat model.¹⁶ In the present study, rats in the groups receiving antibiotics had a better survival rate than the rats in the control groups. Our study has also demonstrated the beneficial effect of Dfx as a supplement to antibiotics, in the survival of peritonitis (Figure 1). However, this experiment does not provide an understanding of the mechanism by which Dfx favorably alters survival. It may be that the radical scavenging effect of iron chelating agent Dfx influences the survival rate. It is also known that iron is an essential ingredient for bacterial growth and multiplication.¹⁷ Without iron, cells are unable to proceed the G1 phase to the S-phase of the cell cycle.^18,19 Dfx may also exert a bacteriostatic effect on bacteria in the peritoneal cavity by competing with microbial siderophores for the available extracellular iron. We suggest that further research is required to determine the mechanisms of action of Dfx in sepsis.

Alpha-tocopherol is the most important lipid soluble, chainbreaking antioxidant, and plays an important role in preventing lipid peroxidation in septic peritonitis.²⁰ Although vitamin E as a supplement to antibiotics in the treatment of sepsis led to a significant improvement (p<0.001) in survival, that improvement was not significantly superior to that of the group treated with antibiotics alone in our experimental model.

Vitamin E, in combination with antibiotics, also improved survival rates in our study. However, it is not possible to conclude from our results that the positive effect of vitamin E supplementing antibiotics is actually significant. The debate continues regarding the effect and mechanism of action of vitamin E in the treatment of sepsis. Several investigators have demonstrated improved survival in experimental models of murine endotoxaemia with alpha-tocopherol.^4,21,22 Siguno et al (1999) demonstrated a decrease in hepatocyte membrane lipid peroxidation, improved cell energy charge, and increased survival in alpha-tocopherol treated animals exposed to endotoxin.²¹

Figure 1: Conversion of survival in the groups receiving antibiotics with or without Dfx over a defined (15 day) time course.

Konukoglu et al (1987), suggested that an oxidant/antioxidant imbalance is involved in animal peritonitis, and that the use of alpha-tocopherol is effective in decreasing oxidative stress of tissues during peritonitis.²³ Improved survival rates have been reported as a result of pre-treatment with alphatocopherol, and by treatment with vitamin E analogues in an experimental model with abdominal sepsis.⁴ Castillo et al (1991) suggested that the use of free radical scavengers alone is not associated with an increase in survival in sepsis: in their study, however, the combination of free radical scavengers with certain antibiotics led to improved results.¹¹

Although post-mortem histopathological evaluation of the livers did not indicate any significant pathological difference between the groups in our study, the difference in survival between the groups treated with antibiotics and vitamin E, when compared with those treated with Dfx and antibiotics, may have been influenced by the side effects associated with the high dosages of drugs.

The results of our study suggest that the use of vitamin E or Dfx alone is not associated with an increase in survival when used to treat septic peritonitis in rats. Vitamin E in combination with antibiotics did not have a significant effect on survival rates when compared with the survival rate of rats treated with antibiotics alone. However, treatment with a combination of Dfx with antibiotics resulted in a survival rate significantly higher than that for rats treated with antibiotics alone. More effort in this field is needed to clarify the mechanism of action of Dfx in sepsis.

REFERENCES

1. Zhang H, Slutsky AS, Vincent JL. Oxygen free radicals in ARDS, septic shock and organ dysfunction. Intensive Care Med 2000; 26(4): 474-476
2. Rotstein OD. Oxidants and antioxidant therapy. Crit Care Clin 2001; 17 (1): 239-247
3. Lehmann C, Egerer K, Georgiew A, Weber M, Grune T, Kox WJ. Inhibition of tumor necrosis factor-alpha release in rat experimental endotoxemia by treatment with the 21-aminosteroid U-74389G. Crit Care Med 1999; 27(6): 1164-1167
4. Powell RJ, Machiýedo GW, Rush BF, Dikdtan GS. Effect of oxygen-free radical scavengers in survival in sepsis. Am Surg 1991; 57: 86-888
5. Fujimura N, Sumita S, Narimatsu E. Alteration in diaphragmatic contractility during septic peritonitis in rats: Effect of polyethylene glycol-absorbed superoxide dismutase. Crit Care Med 2000; 28(7): 2406-2414
6. Wang X, Quinn PJ. Vitamin E and its function in membranes. Prog Lipid Res 1999; 38(4): 309-336
7. Chaudiere J, Ferrari-Iliou R. Intracellular antioxidants: from chemical to biochemical mechanisms. Food Chem Toxicol 1999; 37(9-10): 949-962
8. Farber JL. Mechanisms of cell injury by activated oxygen species. Environ Health Perspect 1994; 102(S 10): 17-24
9. Libra S, Pagano D, Curella G, Litrico V, Cancelliere M, Audibert D, La Terra S, Gruttadauria S, Pappalardo A, D’Alessandro M. Experimental research on the use of deferoxamine in the prevention of renal damage from acute ischemia. Ann Ital Chir 1999; 70(4): 569-573
10. Wichterman KA, Baue AE, Chaudry IH. Sepsis and septic shock: a review of laboratory models and proposal. J Surg Res 1980; 29:189-201
11. Castillo M, Toledo-Pereyra LH, Gutierrez R, Prough D, Shapiro E. Peritonitis after cecal perforation. An experimental model to study the therapeutic role of antibiotics associated with allopurinol and catalase. Am Surg 1991; 57: 313-316
12. Halliwell B. Oxidants and human disease: Some new concepts. FASEB J 1987; 1(5): 358-364
13. Huffman LJ, Miles PR, Shi X, Bowman L. Hemoglobin potentiates the production of reactive oxygen species by alveolar macrophages. Exp Lung Res 2000; 26(3): 203-217
14. Kim YM. Counterprotective effect of erithrocytes in experimental bacterial peritonitis is due to scavenging of nitric oxide and reactive oxygen intermediates. Infect Immun 1996; 64(8): 3074-3080
15. Yoo YM, Kim KM, Kim SS, Han JA, Lea HZ, Kim YM. Hemoglobin toxicity in experimental bacterial peritonitis is due to production of reactive oxygen species-Clin Diagn Lab Immunol 1999; 6(6): 938-945
16. Halliwell B, Gutteridge JMC. Free radicals and human disease: the importance of free radicals and catalytic metal ions in human disease. Mol Aspect Med 1985; 8: 135-153
17. Bullen J, Griffiths E, Rogers H, Ward G. Sepsis: The critical role of iron. Microbes Infect 2000; 2 (4): 409-415
18. Simonart T, Degraef C, Andrei G, Mosselmans R, Hermans P, Van Vooren JP, Noel JC, Boelaert JR, Snoeck R, Heenen M. Iron chelators inhibit the growth and induce the apoptosis of Kaposi’s sarcoma cells and of their putative endothelial precursors. J Invest Dermatol 2000; 115(5): 893-900
19. Rakba N, Loyer P, Gilot D, Delcros JG, Glaise D, Baret P, Pierre JL, Brissot P, Lescoat G. Antiproliferative and apoptotic effects of O-Trensox, a new synthetic iron chelator, on differentiated human hepatoma cell lines. Carcinogenesis 2000; 21(5): 943-951
20. Vlasov AP, Tarasova TV, Sudakova GI, Ashirov RS, Kil’diushov AN, Dubovskaia TN, Rubtsov OI, Lazareva OA. Effect of antioxidants on endotoxicosis in experimental peritonitis. Eksp Klin Farmakol 2000;63(6): 58-61
21. Pekkanen T, Lindberg P, Sankari S. The effect of pretreatment with vitamin E on the effects of endotoxin in the rat. Acta Pharmacol Toxicol 1983; 53: 64-69
22. Sugino K, Dohi K, Yamada K, Kaawasaki T. The role of lipid peroxidation in the endotoxin-induced hepatic damage and the protective effect of antioxidants. Surgery 1987; 101: 746-752
23. Konukoglu D, Iynem H, Ziylan E. Antioxidant status in experimental periotonitis: Effects of alpha tocopherol and taurolin. Pharmacol Res 1999; 39(3): 247-251

Copyright: 21 May 2002

Correspondence: G. Soybir, Tesvikiye Mah. Husrev Gerede Cad. Lavkan Apt. No:80 D:1 Nisantasi 80200 Istanbul- Turkey