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Reperfusion Injury in Rabbits
Effects of L-Nitro-Arginine Methyl Ester, an Inhibitor of Nitric Oxide Biosynthesis, on Intestinal Ischemia/Reperfusion Injury in Rabbits M.O. Taha, R. Miranda-Ferreira, A.L. Fagundes, D.J. Fagundes, R.S. Simões, J.M. Santos, P.D.F. Souza, I.S. Oliveira Júnior, A. Marchini, I.T. Gomes, H.P. Monteiro, L.O. Mendonça, and A. Caricati-Neto ABSTRACT To study whether treatment with L-nitro-arginine methyl ester (L-NAME), an inhibitor of nitric oxide biosynthesis, attenuates intestinal dysfunction caused by ischemia (I) and/or reperfusion (R), rabbits were treated with L-NAME (15 mg · kg⫺1, intervenously) or saline olution (SS) prior to I (60 minutes) induced by occlusion of superior mesenteric artery and/or R (120 minutes). After I or I/R, isolated jejunal segments (2 cm) were mounted in an organ bath to study nerve-mediated contractions stimulated by electrical pulses or KCI using a digital recording system. Thin jejunal slices were stained (hematoxylin and eosin) for analysis by optical microscopy. Compared with a sham group, the jejunal contractions were similar in the I/R ⫹ L-NAME, but reduced in I ⫹ SS, I/R ⫹ SS, and I ⫹ L-NAME groups. The jejunal enteric nerves were damaged in the I ⫹ SS, I/R ⫹ SS, and I ⫹ L-NAME cohorts, but not among the I/R ⫹ L-NAME cohort. These results suggested that L-NAME attenuated intestinal dysfunction caused by R but not by I. SCHEMIA (I)-REPERFUSION (R) intestinal injury is a significant problem in numerous situations, such as abdominal aortic aneurysm surgery, small bowel transplantation, cardiopulmonary bypass, strangulated herniae, and neonatal necrotizing enterocolitis.1 These situations cause cellular lesions and death due to oxygen and nutrient deprivation.1 Blood R after I also contributes to produce cellular lesions and death due mainly to lipid peroxidation of cell membranes resulting from accumulation of free oxygen radicals and other cytotoxic substances.2,3 Decreased contractile function of the mucosal barrier is associated with intestinal I and R2,3 due to severely compromised motor and secretory functions.1–3 Intestinal motility is regulated by several excitatory and inhibitory transmitters released from enteric nerves, including acetylcholine, 5= triphosphate adenosine, nitric oxide (NO), and neuropeptides.4 The actions of these transmitters are highly dependent on the integrity of the enteric nerves.5 However, intestinal motor activity is reduced by I/R due mainly to a loss of structural and functional integrity of enteric nerves.5,6 Several drugs been proposed to attenuate or prevent the cellular dysfunctions caused by I and by R,7,8 including drugs that interfere with NO production.9 –14 NO is a endogenous mediator released from various cell types, including endothelial, immune, and neural cells, producing the physiological responses of vasodilatation, neurotransmis-
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© 2010 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 42, 457– 460 (2010)
sion, and inflammation.4,15 Cells produce NO by means of the action of NO synthase on L-arginine.15 Some studies have suggested that NO attenuates the cellular lesions caused by I and R due to inhibition of lipid peroxidation via a lipophilic free radical effect. 9 –15 In the present work, we studied the effects of treatment with L-nitro-arginine methyl ester (L-NAME), an inhibitor of NO biosynthesis, on the motility and histology of rabbit jejunal undergoing intestinal I and R. MATERIALS AND METHODS Male New Zeland rabbits (2.5 to 3.0 kg) that were anesthetized (ketamine 60 mg · kg⫺1 and xylazine 40 mg · kg⫺1, intravenously) underwent occlusion of the superior mesenteric artery using metallic clip for 60 minutes (I) followed by blood recirculation (R) for 120 minutes following clip removal. Twelve rabbits underwent only I (I group); 12, I plus R (I/R group); and six, a sham group. From the Departments of Pharmacoloy, Biochemistry, Morphology and Surgery of the Federal University of São Paulo (UNIFESP) and Federal University of Great Dourados (UFGD), Brazil. Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil. Address reprint request to Dr Murched Omar Taha, Department of Surgery, UNIFESP/EPM; Rua Botucatu, 740, CEP 04023-900, São Paulo-SP, Brazil. E-mail: [email protected] 0041-1345/10/$–see front matter doi:10.1016/j.transproceed.2010.01.037 457
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TAHA, MIRANDA-FERREIRA, FAGUNDES ET AL
Fig 1. Typical records of nervemediated contractions induced by electrical field stimulation (5 and 30 Hz) or KCl (100 mmol/L) in rabbits jejunum treated with L-nitro-arginine methyl ester (LNAME) or saline solution 0.9% (SS) and submitted to intestinal ischemia (I) or I/reperfusion. *,#,@Statistically different of I ⫹ SS (P ⬍ .05, n ⫽ 6). ⵜ, ●, Statistically different of I/R ⫹ SS (P ⬍ .05, n ⫽ 6). Among the I group, six rabbits were treated with saline solution 0.9% (SS) and six with L-NAME (15 mg · kg⫺1), injected into the femoral vein 5 minutes prior to I. In the I/R group, six rabbits were treated with SS and six with L-NAME (15 mg · kg⫺1) injected into the femoral vein 5 minutes before I/R and 5 minutes before R and then 55 minutes after R. After I or I/R, the rabbits were sacrificed and jejunal segments (2 cm) isolated, washed, cleared of surrounding tissues, and mounted under 1 g tension at 37°C in an organ bath containing 10 mL of aerated nutrient solution (composition in mmol/L): NaCl 138, KCl 5.7, CaCl2 1.8, NaH2PO4, 0.36, NaHCO3 15, and dextrose 5.5, pH 7.4). We studied nerve-mediated contractions induced by electrical field stimulation (EFS) or by the depolarizing agent KCl (100 mmol/L) using a digital recording system.6 – 8 EFS (5 and 30 Hz, 1-ms duration, 60 V) was performed by platinum electrodes connected to an electrical stimulator S88 (Grass, Astro-Med Inc, West Warwick, RI, USA).6 – 8 Responses to EFS and KCl were recorded by force-displacement transducers connected via a bridge amplifier to an analog/digital recording system (AD Instruments, Colorado Springs, Colo, USA).6–8 Contrac-
tile response data were evaluated by one-way analysis of variance and Student t test.6–8 We also performed histological analyses by staining pieces of jejunal tissue mounted in paraffin, cut in thin slices, and stained with hematoxylin and eosin for optical microscopy.7,8 Table 1. Values of Amplitude of Nerve-Mediated Contractions (Expressed in Grams of Tension) Induced by EFS (5 and 30 Hz) or KCl (100 mmol/L) in Jejunum of Rabbits Treated with L-NAME or SS and Submitted to Intestinal I or I/R Groups
5 Hz
30 Hz
KCl
I ⫹ SS I ⫹ L-NAME I/R ⫹ SS I/R ⫹ L-NAME
0.98 ⫾ 0.09 0.44 ⫾ 0.03* 1.31 ⫾ 0.20 2.15 ⫾ 0.20*
1.89 ⫾ 0.09 1.02 ⫾ 0.11* 2.05 ⫾ 0.15 2.97 ⫾ 0.25*
2.21 ⫾ 0.23 1.05 ⫾ 0.10* 2.08 ⫾ 0.30 3.20 ⫾ 0.31*
Data corresponding to mean ⫾ standard error of the mean (n ⫽ 6). EFS, electrical field stimulation; L-NAME, L-nitro-arginine methyl ester; SS, saline solution 0.9%; I, ischemia; R, reperfusion. *Statistically different of SS (P ⬍ .05).
L-NAME AND INTESTINAL ISCHEMIA-REPERFUSION INJURY
459
Fig 2. Histological aspects of rabbit jejunum treated with saline solution 0.9% (SS) or L-nitroarginine methyl ester (L-NAME) and submitted to intestinal ischemia and/or reperfusion. The images show the longitudinal muscle (LM), circular muscle (CM), epithelial layer (E), and enteric nerves (black arrows; hernatoxylin and eosin method, 400⫻).
RESULTS
Figure 1 shows that EFS (5 and 30 Hz) and KCl produced contractile responses in the jejunal segments of all groups: sham, I ⫹ SS, I ⫹ L-NAME, I/R ⫹ SS, I/R ⫹ L-NAME. Compared with the sham group the amplitude of the contractions was similar in the I/R ⫹ L-NAME group, but reduced in I ⫹ SS, IR ⫹ SS, and I ⫹ L-NAME cohorts, (Fig 1, Table 1). In contrast, the contractions were smaller in the I ⫹ L-NAME group compared with I ⫹ SS (Fig 1, Table 1). Histological analysis showed a loss of structural integrity of enteric nerves in the jejunum of I ⫹ SS, I/R ⫹ SS, and I ⫹ L-NAME, but not in the I/R ⫹ L-NAME groups (Fig 2).
Luo et al reported that L-NAME reduces the I/R injury by inhibiting endogenous NO.17 Kurose et al showed that L-NAME causes the formation of platelet-leukocyte aggregates. They reported that decreased NO production produced an increase in aggregate formation.11 It is possible that these molecular mechanisms may be involved in the protective effects of L-NAME against cellular lesions caused by R, but not by I. In conclusion, treatment with L-NAME attenuated the motor and neural dysfunctions of rabbit small bowel caused by R, but not by I.
REFERENCES DISCUSSION
The present work showed that the amount of and the motility stimulated by transmitters released by enteric nerves were significantly reduced among rabbit jejunum undergoing intestinal I or I/R. However, the dysfunctions in the R, but not in the 1, cohorts were reduced or absent among rabbits treated with L-NAME; suggesting that LNAME, an inhibitor of NO biosynthesis, attenuated or prevented motor and neural dysfunctions. Deleterious effects of R on the intestine and its exacerbation of ischemic events have been strongly suggested by previous work.16 The mechanisms responsible for the injury caused by R include formation of free oxygen radicals with consequent oxidative stress. In fact, the mechanism of injury is not fully understood; there are other factors involved such as activation of phospholipase A2 and alterations of calcium flux. Mason et al reported increased free oxygen radicals after a decrease in tissue NO levels.13 Endogenous NO can inhibit or delay the injury caused by free oxygen radicals in the early periods of I/R.13 However, the functions of NO are still not fully understood, as NO reacts with superoxide anion to form peroxynitrite anion (ONOO2).13
1. Barie PS: Schemes against ischemia; solutions for reperfusion (injury)? Crit Care Med 27:684, 1999 2. Horton JW, Walker PB: Oxygen radicals, lipid peroxidation, and permeability changes after intestinal ischemia and reperfusion. J Appl Physiol 74:1515, 1993 3. Schoenberg MH, Beger HG: Reperfusion injury after intestinal ischemia. Crit Care Med 21:1376, 1993 4. Furness JB: Types of neurons in the enteric nervous system. J Auton Nerv Syst 81:87, 2000 5. Bauer AJ: Transplantation-induced injuries of the intestinal muscularis and its innervation: from preservation to chronic rejection. Transplant Proc 28:2539, 1996 6. Taha MO, Fraga MM, Bandeira CF, et al: Effect of preservation conditions on autonomic transmission in rat small bowel. Transplant Proc 34:1021, 2002 7. Taha MO, Fraga MM, Fagundes DJ, et al: Effect of allopurinol on autonomic dysfunction in rat jejunal segments exposed to cold ischemic preservation for transplantation. Transplant Proc 36:293, 2004 8. Taha MO, Fraga MM, Fagundes DJ, et al: Ascorbic acid prevents autonomic dysfunction in rat jejunal submitted to cold ischemic preservation for transplantation. Transplant Proc 36:289, 2004 9. Van Ye TM, Roza AM, Pieper GM, et al: Inhibition of intestinal lipid peroxidation does not minimize morphologic damage. J Surg Res 55:553, 1993
460 10. Chan KL, Zhang XH, Fung PC, et al: Role of nitric oxide in intestinal ischemia-reperfusion injury studied using electron paramagnetic resonance. Br J Surg 86:1427, 1999 11. Kurose I, Wolf R, Grisham MB, et al: Modulation of ischemia/reperfusion-induced microvascular dysfunction by nitric oxide. Circ Res 74:376, 1994 12. Payne D, Kubes P: Nitric oxide donors reduce the rise in reperfusion-induced intestinal mucosal permeability. Am J Physiol 265(1 Pt 1):G189, 1993 13. Mason RB, Pluta RM, Walbridge S, et al: Production of reactive oxygen species after reperfusion in vitro and in vivo: protective effect of nitric oxide. J Neurosurg 93:99, 2000
TAHA, MIRANDA-FERREIRA, FAGUNDES ET AL 14. Chen JC, Chen HM, Shyr MH, et al: Selective inhibition of inducible nitric oxide in ischemia-reperfusion of rat small intestine. J Formos Med Assoc 99:213, 2000 15. Nijkamp FP, Folkerts G: Nitric oxide: inhibitor and modulator. Clin Exp Allergy 27:347, 1997 16. Sies H, de Groot H: Role of reactive oxygen species in cell toxicity. Toxicol Lett 64-65:547, 1992 17. Luo CC, Chen HM, Chiu CH, et al: Effect of N (G)-nitroL-arginine methyl ester on intestinal permeability following intestinal ischemia reperfusion injury in a rat model. Biol Neonate 80:60, 2001
I
© 2010 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 42, 457– 460 (2010)
sion, and inflammation.4,15 Cells produce NO by means of the action of NO synthase on L-arginine.15 Some studies have suggested that NO attenuates the cellular lesions caused by I and R due to inhibition of lipid peroxidation via a lipophilic free radical effect. 9 –15 In the present work, we studied the effects of treatment with L-nitro-arginine methyl ester (L-NAME), an inhibitor of NO biosynthesis, on the motility and histology of rabbit jejunal undergoing intestinal I and R. MATERIALS AND METHODS Male New Zeland rabbits (2.5 to 3.0 kg) that were anesthetized (ketamine 60 mg · kg⫺1 and xylazine 40 mg · kg⫺1, intravenously) underwent occlusion of the superior mesenteric artery using metallic clip for 60 minutes (I) followed by blood recirculation (R) for 120 minutes following clip removal. Twelve rabbits underwent only I (I group); 12, I plus R (I/R group); and six, a sham group. From the Departments of Pharmacoloy, Biochemistry, Morphology and Surgery of the Federal University of São Paulo (UNIFESP) and Federal University of Great Dourados (UFGD), Brazil. Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil. Address reprint request to Dr Murched Omar Taha, Department of Surgery, UNIFESP/EPM; Rua Botucatu, 740, CEP 04023-900, São Paulo-SP, Brazil. E-mail: [email protected] 0041-1345/10/$–see front matter doi:10.1016/j.transproceed.2010.01.037 457
458
TAHA, MIRANDA-FERREIRA, FAGUNDES ET AL
Fig 1. Typical records of nervemediated contractions induced by electrical field stimulation (5 and 30 Hz) or KCl (100 mmol/L) in rabbits jejunum treated with L-nitro-arginine methyl ester (LNAME) or saline solution 0.9% (SS) and submitted to intestinal ischemia (I) or I/reperfusion. *,#,@Statistically different of I ⫹ SS (P ⬍ .05, n ⫽ 6). ⵜ, ●, Statistically different of I/R ⫹ SS (P ⬍ .05, n ⫽ 6). Among the I group, six rabbits were treated with saline solution 0.9% (SS) and six with L-NAME (15 mg · kg⫺1), injected into the femoral vein 5 minutes prior to I. In the I/R group, six rabbits were treated with SS and six with L-NAME (15 mg · kg⫺1) injected into the femoral vein 5 minutes before I/R and 5 minutes before R and then 55 minutes after R. After I or I/R, the rabbits were sacrificed and jejunal segments (2 cm) isolated, washed, cleared of surrounding tissues, and mounted under 1 g tension at 37°C in an organ bath containing 10 mL of aerated nutrient solution (composition in mmol/L): NaCl 138, KCl 5.7, CaCl2 1.8, NaH2PO4, 0.36, NaHCO3 15, and dextrose 5.5, pH 7.4). We studied nerve-mediated contractions induced by electrical field stimulation (EFS) or by the depolarizing agent KCl (100 mmol/L) using a digital recording system.6 – 8 EFS (5 and 30 Hz, 1-ms duration, 60 V) was performed by platinum electrodes connected to an electrical stimulator S88 (Grass, Astro-Med Inc, West Warwick, RI, USA).6 – 8 Responses to EFS and KCl were recorded by force-displacement transducers connected via a bridge amplifier to an analog/digital recording system (AD Instruments, Colorado Springs, Colo, USA).6–8 Contrac-
tile response data were evaluated by one-way analysis of variance and Student t test.6–8 We also performed histological analyses by staining pieces of jejunal tissue mounted in paraffin, cut in thin slices, and stained with hematoxylin and eosin for optical microscopy.7,8 Table 1. Values of Amplitude of Nerve-Mediated Contractions (Expressed in Grams of Tension) Induced by EFS (5 and 30 Hz) or KCl (100 mmol/L) in Jejunum of Rabbits Treated with L-NAME or SS and Submitted to Intestinal I or I/R Groups
5 Hz
30 Hz
KCl
I ⫹ SS I ⫹ L-NAME I/R ⫹ SS I/R ⫹ L-NAME
0.98 ⫾ 0.09 0.44 ⫾ 0.03* 1.31 ⫾ 0.20 2.15 ⫾ 0.20*
1.89 ⫾ 0.09 1.02 ⫾ 0.11* 2.05 ⫾ 0.15 2.97 ⫾ 0.25*
2.21 ⫾ 0.23 1.05 ⫾ 0.10* 2.08 ⫾ 0.30 3.20 ⫾ 0.31*
Data corresponding to mean ⫾ standard error of the mean (n ⫽ 6). EFS, electrical field stimulation; L-NAME, L-nitro-arginine methyl ester; SS, saline solution 0.9%; I, ischemia; R, reperfusion. *Statistically different of SS (P ⬍ .05).
L-NAME AND INTESTINAL ISCHEMIA-REPERFUSION INJURY
459
Fig 2. Histological aspects of rabbit jejunum treated with saline solution 0.9% (SS) or L-nitroarginine methyl ester (L-NAME) and submitted to intestinal ischemia and/or reperfusion. The images show the longitudinal muscle (LM), circular muscle (CM), epithelial layer (E), and enteric nerves (black arrows; hernatoxylin and eosin method, 400⫻).
RESULTS
Figure 1 shows that EFS (5 and 30 Hz) and KCl produced contractile responses in the jejunal segments of all groups: sham, I ⫹ SS, I ⫹ L-NAME, I/R ⫹ SS, I/R ⫹ L-NAME. Compared with the sham group the amplitude of the contractions was similar in the I/R ⫹ L-NAME group, but reduced in I ⫹ SS, IR ⫹ SS, and I ⫹ L-NAME cohorts, (Fig 1, Table 1). In contrast, the contractions were smaller in the I ⫹ L-NAME group compared with I ⫹ SS (Fig 1, Table 1). Histological analysis showed a loss of structural integrity of enteric nerves in the jejunum of I ⫹ SS, I/R ⫹ SS, and I ⫹ L-NAME, but not in the I/R ⫹ L-NAME groups (Fig 2).
Luo et al reported that L-NAME reduces the I/R injury by inhibiting endogenous NO.17 Kurose et al showed that L-NAME causes the formation of platelet-leukocyte aggregates. They reported that decreased NO production produced an increase in aggregate formation.11 It is possible that these molecular mechanisms may be involved in the protective effects of L-NAME against cellular lesions caused by R, but not by I. In conclusion, treatment with L-NAME attenuated the motor and neural dysfunctions of rabbit small bowel caused by R, but not by I.
REFERENCES DISCUSSION
The present work showed that the amount of and the motility stimulated by transmitters released by enteric nerves were significantly reduced among rabbit jejunum undergoing intestinal I or I/R. However, the dysfunctions in the R, but not in the 1, cohorts were reduced or absent among rabbits treated with L-NAME; suggesting that LNAME, an inhibitor of NO biosynthesis, attenuated or prevented motor and neural dysfunctions. Deleterious effects of R on the intestine and its exacerbation of ischemic events have been strongly suggested by previous work.16 The mechanisms responsible for the injury caused by R include formation of free oxygen radicals with consequent oxidative stress. In fact, the mechanism of injury is not fully understood; there are other factors involved such as activation of phospholipase A2 and alterations of calcium flux. Mason et al reported increased free oxygen radicals after a decrease in tissue NO levels.13 Endogenous NO can inhibit or delay the injury caused by free oxygen radicals in the early periods of I/R.13 However, the functions of NO are still not fully understood, as NO reacts with superoxide anion to form peroxynitrite anion (ONOO2).13
1. Barie PS: Schemes against ischemia; solutions for reperfusion (injury)? Crit Care Med 27:684, 1999 2. Horton JW, Walker PB: Oxygen radicals, lipid peroxidation, and permeability changes after intestinal ischemia and reperfusion. J Appl Physiol 74:1515, 1993 3. Schoenberg MH, Beger HG: Reperfusion injury after intestinal ischemia. Crit Care Med 21:1376, 1993 4. Furness JB: Types of neurons in the enteric nervous system. J Auton Nerv Syst 81:87, 2000 5. Bauer AJ: Transplantation-induced injuries of the intestinal muscularis and its innervation: from preservation to chronic rejection. Transplant Proc 28:2539, 1996 6. Taha MO, Fraga MM, Bandeira CF, et al: Effect of preservation conditions on autonomic transmission in rat small bowel. Transplant Proc 34:1021, 2002 7. Taha MO, Fraga MM, Fagundes DJ, et al: Effect of allopurinol on autonomic dysfunction in rat jejunal segments exposed to cold ischemic preservation for transplantation. Transplant Proc 36:293, 2004 8. Taha MO, Fraga MM, Fagundes DJ, et al: Ascorbic acid prevents autonomic dysfunction in rat jejunal submitted to cold ischemic preservation for transplantation. Transplant Proc 36:289, 2004 9. Van Ye TM, Roza AM, Pieper GM, et al: Inhibition of intestinal lipid peroxidation does not minimize morphologic damage. J Surg Res 55:553, 1993
460 10. Chan KL, Zhang XH, Fung PC, et al: Role of nitric oxide in intestinal ischemia-reperfusion injury studied using electron paramagnetic resonance. Br J Surg 86:1427, 1999 11. Kurose I, Wolf R, Grisham MB, et al: Modulation of ischemia/reperfusion-induced microvascular dysfunction by nitric oxide. Circ Res 74:376, 1994 12. Payne D, Kubes P: Nitric oxide donors reduce the rise in reperfusion-induced intestinal mucosal permeability. Am J Physiol 265(1 Pt 1):G189, 1993 13. Mason RB, Pluta RM, Walbridge S, et al: Production of reactive oxygen species after reperfusion in vitro and in vivo: protective effect of nitric oxide. J Neurosurg 93:99, 2000
TAHA, MIRANDA-FERREIRA, FAGUNDES ET AL 14. Chen JC, Chen HM, Shyr MH, et al: Selective inhibition of inducible nitric oxide in ischemia-reperfusion of rat small intestine. J Formos Med Assoc 99:213, 2000 15. Nijkamp FP, Folkerts G: Nitric oxide: inhibitor and modulator. Clin Exp Allergy 27:347, 1997 16. Sies H, de Groot H: Role of reactive oxygen species in cell toxicity. Toxicol Lett 64-65:547, 1992 17. Luo CC, Chen HM, Chiu CH, et al: Effect of N (G)-nitroL-arginine methyl ester on intestinal permeability following intestinal ischemia reperfusion injury in a rat model. Biol Neonate 80:60, 2001