Effect of Leptin Treatment on Small Intestinal Damage Induced by Hypoxia Reoxygenation in Newborn Rats

Abstract

Objective: To evaluate whether leptin has an protective effect on small intestinal damage induced by hypoxia reoxygenation in newborn rats. Materials and Methods: There were three equal Groups each consisted of 10 newborn rats. Group 1; control, Group 2; hypoxia-reoxygenation, Group 3; leptin-hypoxia-reoxygenation. The rats of Group 3 were pretreated with leptin (20 microgram/kg) one hour before experiments then rat pubs in Group 2 and Group 3 were stressed twice daily with asphyxia followed by cold (+4º C for 10 min). Breathing 100% CO2 for ten minutes in a chamber followed 10 minutes 100% O2 breathing was the asphyxia model. This protocol was repeated for the following two days and the rat pups were decapitated on the third day. The entire gastrointestinal tract was removed. A 3-cm section of distal ileum from each animal was taken. Histopathological damage and epidermal growth factor receptors (EGFR) are evaluated. For biochemical evaluation, tissue malondialdehyde (MDA) and nitric oxide (NO) levels were measured. Results: In Group 2 and Group 3 macroscopic intestinal injuries were observed. Histopathological grading and immunohistochemical EGFR evaluation showed similar damage in the Group 2 and the Group 3. In these Groups tissue MDA levels were also in the same ranges but NO levels were different each other with the higher levels of Group 2. Group 3 had same NO levels with control Group. Conclusions: Leptin pretreatment has no protective effect on hypoxia reoxygenation induced intestinal injury in newborn rats although it changes NO levels in the injured intestine.

Keywords

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Leptin Tedavisi Yeni Doğan Rat İncebarsağında Hipoksi/Reoksijenasyon Hasarına Karşı Koruyucu mudur?

Öz

Amaç: Hipoksi reoksijenasyonun neden olduğu yenidoğan rat incebarsak hasarına leptin'in etkinliğini arastırmak. Gereç ve Yöntem: Doğum sonrası rastgele seçilmiş 30 adet yavru rat kendi aralarında üç eşit gruba bölünerek deneysel gruplar oluşturuldu. Grup 1 kontrol, Grup 2 hipoksi-reoksijenasyon , Grup 3 leptin hipoksi-reoksijenasyon grubu olarak belirlendi. Grup 3 deki yavru ratlara deneysel işlemden 1 saat önce ciltaltı enjeksiyonla 20 mikrogram/kg leptin verildi. Bu gruplardaki yavrular bir cam kavanoz içerisine koyuldular. Kavanoz içerisindeki hava aspiratör yardımı boşaltılıp tekrar karbon dioksit ile dolduruldu. Yavruların 10 dk bu ortamda solumaları sağlandı ve ardından kavanoz oksijen ile dolduruldu. Böylece reoksijenasyon yapılmış oldu. Onuncu dakikadan sonra yavrular tekrar dışarı alındı ve 10 dakika soğuğa maruz bırakıldı. Bu işlem birkez daha baştan sona tekrarlandı. Uygulanan bu işlemler serisi 2. ve 3. günlerde de tekrarlandı. Üçüncü günü yavrular dekapite edildi ve gasrointestinal sistemleri çıkarıldı. Histopatolojik ve biyokimyasal inceleme için terminal ileumdan 3 cm uzunluğunda ince barsak dokusu alındı. Histopatolojik incelemede ileum hasarı bakıldı ve immunhistokimyasal boyama ile epidermel büyüme faktörü reseptorleri (EBFR) araştırıldı. Kalan barsak dokularında malondialdehit (MDA), ve nitrik oksit (NO) düzeyleri ölçüldü. Bulgular: Grup 2 ve grup 3 de tipik NEK bulguları gözlendi. Histopatolojik incelemelerde gruplar farksızdı (P>.05) Yine grup 2 ve grup 3 de MDA düzeyleri farksızdı (p>.05) Ancak bu grupların NO düzeyleri birbirinden farklıydı . NO Grup 2 de yüksek ve Grup 3 de kontrol grubu ile aynıydı (P>.05) Sonuç: Leptin'in NO düzeylerini değiştirmesine rağmen yeni doğan ratlarda hipoksi reoksijenasyonun neden olduğu barsak hasarını önleyici etkisi yoktur.

Anahtar Kelimeler

• Leptin, hipoksi/reoksijenasyon, nekrotizan enterokolit

References

1. Neu J. Necrotizing enterocolitis: the search for a unifying pathogenic theory leading to prevention. Pediatr Clin North Am 1996; 43: 409-432.
2. Morecroft JA, Spitz L, Hamilton PA, Holmes SJ. Necrotizing enterocolitis--multisystem organ failure of the newborn ? Acta Paediatr Suppl 1994; 396: 21-23.
3. Wilson R, Kanto WP Jr, McCarthy BJ, Feldman RA. Age at onset of necrotizing enterocolitis. Risk factors in small infants. Am J Dis Child 1982; 136: 814-16.
4. Kliegman RM. Models of the pathogenesis of necrotizing enterocolitis. J Pediatr 1990; 117: 2-5.
5. Go LL, Albanese CT, Watkins SC, Simmons RL, Rowe MI. Breast milk protects the neonate from bacterial translocation. J Pediatr Surg 1994; 29: 1059-1063; discussion 1063-1064.
6. Scheifele DW. Role of bacterial toxins in neonatal necrotizing enterocolitis. J Pediatr 1990; 117:44-46.
7. Albanese CT, Rowe MI. Necrotizing enterocolitis. Semin Pediatr Surg 1995; 4: 200-206.
8. Kazez A, Küçükaydın N, Küçükaydın M et al. A model of hypoxia-induced necrotizing enterocolitis: the role of distension. J Pediatr Surg. 1997; 32: 1466-1469.

9. Caplan MS, Sun XM, Hseuh W, Hageman JR. Role of platelet activating factor and tumor necrosis factor-alpha in neonatal necrotizing enterocolitis. J Pediatr 1990; 116: 960-964.
10. Caplan MS, Sun XM, Hsueh W. Hypoxia causes ischemic bowel necrosis in rats: the role of platelet-activating factor (PAF- acether). Gastroenterology 1990 ; 99 : 979-986
11. Caplan MS, Sun XM, Hsueh W. Hypoxia, PAF, and necrotizing enterocolitis. Lipids 1991; 26: 1340-1343.
12. Furukawa M, Lee EL, Johnston JM. Platelet-activating factor- induced ischemic bowel necrosis: the effect of platelet-activating factor acetylhydrolase. Pediatr Res 1993; 34: 237-241.
13. Topalian SL, Ziegler MM. Necrotizing enterocolitis: a review of animal models. J Surg Res 1984; 37:320-336.
14. Saugstad OD. Hypoxanthine as an indicator of hypoxia: its role in health and disease through free radical production. Pediatr Res 1988; 23:143-150.
15. Hebra A, Brown MF, McGeehin K, Broussard D, Ross AJ 3rd. The effects of ischemia and reperfusion on intestinal motility. J Pediatr Surg 1993; 28: 362-365; discussion 365-366.
16. Szabo JS, Stonestreet BS, Oh W. Effects of hypoxemia on gastrointestinal blood flow and gastric emptying in the newborn piglet. Pediatr Res 1985; 9: 466-471.
17. Bielefeldt K, Conklin JL. Intestinal motility during hypoxia and reoxygenation in vitro. Dig Dis Sci 1997; 42: 878-884.
18. Kowalewski K, Zajac S, Kologej A. Effect of ischemic anoxia on electrical and mechanical activity of the totally isolated porcine stomach. Eur Surg Res 1976; 8: 12-25.
19. Hayashi S, Gleason WA, McFee AS, Park MK. Effects of pH alterations and hypoxia on isolated human intestine. Scand J Gastroenterol 1986; 21: 9-15.
20. Hill RA, Margetic S, Pegg GG, Gazzola C. Leptin: its pharmacokinetics and tissue distribution. Int J Obes Relat Metab Disord 1998; 22: 765-770.
21. Wolinski J, Biernat M, Guilloteau P, Westrom BR, Zabielski R. Exogenous leptin controls the development of the small intestine in neonatal piglets. J Endocrinol 2003; 177: 215-222.
22. Alavi K, Schwartz MZ, Prasad R, O'connor D, Funanage V. Leptin: a new growth factor for the small intestine. J Pediatr Surg 2002; 37: 327-330.
23. Gaige S, Abysique A, Bouvier M. Effects of leptin on cat intestinal motility. J Physiol 2003; 546: 267-277.
24. Pearson PY, O'Connor DM, Schwartz MZ. Novel effect of leptin on small intestine adaptation. J Surg Res 2001; 97: 192-195.
25. Saleri R, Giustina A, Tamanini C, et al. Leptin stimulates growth hormone secretion via a direct pituitary effect combined with a decreased somatostatin tone in a median eminence-pituitary perifusion study. Neuroendocrinology 2004; 79: 221-228.
26. Green LC, Wagner DA, Glogowski J et al. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem 1982; 126:131-138.
27. Crissinger KD, Granger DN. Mucosal injury induced by ischemia and reperfusion in the piglet intestine: influences of age and feeding. Gastroenterology 1989; 97: 920-926.
28. Santulli TV, Schullinger JN, Heird WC et al. Acute necrotizing enterocolitis in infancy: a review of 64 cases. Pediatrics 1975; 55: 376-387.
29. Nowicki P. Intestinal ischemia and necrotizing enterocolitis. J Pediatr 1990; 117: 14-19.
30. Nadler EP, Dickinson E, Knisely A, et al. Expression of inducible nitric oxide synthase and interleukin-12 in experimental necrotizing enterocolitis. J Surg Res 2000; 92: 71-77.
31. Zhang BH, Yu HG, Sheng ZX, Luo HS, Yu JP. The therapeutic effect of recombinant human trefoil factor 3 on hypoxia-induced necrotizing enterocolitis in immature rat. Regul Pept 2003; 15: 53-60.
32. Okur H, Kucukaydin M, Kose K, et al: Hypoxia-induced necrotizing enterocolitis in the immature rat: the role of lipid peroxidation and management by vitamin E. J Pediatr Surg 1995; 30: 1416-1419.
33. Kabul Tarihi:09.03.2005