- Email: [email protected]
Role of Adenosine on Intestinal Ischemia-Reperfusion Injury in Rabbits
Role of Adenosine on Intestinal Ischemia-Reperfusion Injury in Rabbits M.O. Taha, R. Miranda-Ferreira, R.S. Simões, M.S. Abrão, I.S. Oliveira-Junior, H.P. Monteiro, J.M. Santos, P.H. Rodrigues, J.V. Rodrigues, A.E.M. Alves, E.C. Nascimento, T.L.B. Silva, W.M. Zeviani, and A. Caricati-Neto ABSTRACT To study if the treatment with adenosine (ADO), an agonist of adenosine receptors, attenuates intestinal dysfunction caused by ischemia (I) and reperfusion (R), we treated rabbits with ADO (15 mg · kg⫺1, intravenously) or saline solution (SS) to I (60 minutes) before occlusion of superior mesenteric artery and/or R (120 min). 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 KCl using a digital recording system. Thin jejunal slices were stained (hematoxylin and eosin) for analysis by optical microscopy. Compared to the sham group, the jejunal contractions were similar in I ⫹ ADO, but reduced in I ⫹ SS, I/R ⫹ SS, and I/R ⫹ ADO groups. We concluded that the jejunal enteric nerves were damaged in I ⫹ SS, I/R ⫹ SS, and I/R ⫹ ADO, but not in I ⫹ ADO group. These results suggested that ADO attenuated intestinal dysfunction due to I, but not to R. NTESTINAL ISCHEMIA (I) AND REPERFUSION (R) injury contributes to the morbidity and mortality of critical illness.1 In the mammalian intestine, I results from local (mechanical vascular obstruction) or systemic (hypovolemia, hypotension, hypoxia, sepsis) factors, which can produce cellular lesions and death due to oxygen and nutrient deprivation.1 Blood R after I also contributes to cellular lesions and death due mainly to lipid peroxidation of cell membranes due to accumulation of free oxygen radicals and other cytotoxic substances.2,3 The cellular dysfunctions caused by I and R severely compromised motor and secretory functions of the intestines.1–3 Intestinal motility is regulated by several excitatory and inhibitory transmitters released from enteric nerves, including acetylcholine, neuropeptides, purines (adenosine triphosphate [ATP]), nitric oxide, and others.4 The actions of these transmitters are highly dependent on the integrity of enteric nerves.5 However, intestinal motor activity is reduced by I/R due mainly to a loss structural and functional integrity of enteric nerves.5,6 Several drugs been proposed to attenuate or prevent the cellular dysfunctions caused by I and R,7,8 including drugs that interfere with adenosine receptors (AR).9 –13 In mammalians, the degradation of ATP produces adenosine (ADO),9,10 which is involved in several physiological process, including inflammation and platelet aggregation.9,10 The actions of ADO are mediated by four distinct subtypes of AR: A1, A2A, A2B, and A3.9,10 Stressed or
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injured tissues release endogenous ADO, which blocks potentially destructive inflammatory cascades, decreasing activation of platelets, leukocytes, and endothelial cells.9,10 We sought to investigate whether ADO attenuated or prevented intestinal I/R injury, by analyzing the effects of ADO treatment on the motility and histology of jejunal segments of rabbits undergoing I/R. MATERIALS AND METHODS Male New Zealand rabbits (2.5–3.0 kg) were anesthetized (ketamine 60 mg · kg⫺1 and xylazine 40 mg · kg⫺1, intravenously) and submitted to occlusion of superior mesenteric artery using a metallic clip for 60 minutes (I) followed by blood recirculation (R) for 120 minutes following removal of the clip. Twelve rabbits underwent only I (I group) and 12, I and R (I/R group). We also included a sham group (n ⫽ 6). In the I group, six rabbits were treated with saline solution 0.9% (SS) and six with ADO (15 mg · kg⫺1) injected into the femoral vein From the Departments of Pharmacoloy, Biochemistry, Morphology and Surgery of the Federal University of São Paulo (UNIFESP) and Federal University of Great Dourados (UFGD), São Paulo, Brazil. Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil. Address reprint requests to Dr Murched Omar Taha, Department of Surgery, UNIFESP/EPM, Rua Botucatu, 740, CEP 04023-900, São Paulo-SP, Brazil. E-mail: [email protected] © 2010 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 42, 454 – 456 (2010)
ADENOSINE AND I-R INJURY IN RABBITS
455
Fig 1. Typical records of nervemediated contractions induced by EFS (5 and 30 Hz) or KCl (100 mmol/L) in jejunal segments of rabbits treated with ADO or SS and submitted to intestinal I or I/R. EFS, electrical field stimulation; ADO, adenosine; SS, saline solution; I, ischemia; R, reperfusion. 5 minutes before I. In the I/R group, six rabbits were treated with SS and six with ADO (15 mg · kg⫺1) injected into the femoral vein 5 minutes before to I and 5 minutes before R and then, 55 minutes after R. After I or I/R, jejunal segments (2 cm) isolated from sacrificed rabbits were 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 of 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) were performed by means of platinum electrodes connected to an S88 electrical stimulator (Grass, USA).6 – 8 Responses to EFS and KCl were recorded by means of forcedisplacement transducers connected via a bridge amplifier to a analog/digital recording system (AD Instruments, USA).6 – 8 Data on contractile responses were submitted to statistical analyses using one-way analysis of variance and Student t test.6 – 8 We also perform histological analyses on jejunal pieces embedded in paraffin, cut into thin slices, and stained by hematoxylin and eosin using optical microscopy.7,8
RESULTS
Figure 1 shows that EFS (5 and 30 Hz) and KCl produced contractile responses in jejunal segments of all groups: sham, I ⫹ SS, I ⫹ ADO, I/R ⫹ SS, I/R ⫹ ADO. However, the amplitude of these contractions was similar to sham in Table 1. Values of Amplitude of Nerve-Mediated Contractions (Expressed in Grams of Tension) Induced by Electrical Field Stimulation (5 and 30 Hz) or KCl (100 mmol/L) in Jejunal Segments of Rabbits Treated With ADO or SS and Submitted to Intestinal I or I/R Groups
5 Hz
30 Hz
KCl
I ⫹ SS I ⫹ ADO I/R ⫹ SS I/R ⫹ ADO
1.03 ⫾ 0.08 1.93 ⫾ 0.14* 1.19 ⫾ 0.10 1.21 ⫾ 0.12
1.83 ⫾ 0.09 2.98 ⫾ 0.25* 2.19 ⫾ 0.15 2.39 ⫾ 0.22
2.21 ⫾ 0.18 3.38 ⫾ 0.24* 2.12 ⫾ 0.13 2.32 ⫾ 0.38
Data corresponding to mean ⫾ standard error of the mean (n ⫽ 6). SS, saline solution; ADO, adenosine; I, ischemia; R, reperfusion. *Statistically different of SS (P ⬍ 0.05).
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TAHA, MIRANDA-FERREIRA, SIMÕES ET AL
Fig 2. Histological aspects of jejunal segments of rabbit treated with saline solution or adenosine and submitted to intestinal ischemia. The images show the longitudinal muscle (LM), circular muscle (CM), epithelial layer (E), and enteric nerves (black arrows). (Hematoxylin and eosin, 400⫻).
the I ⫹ ADO group, but reduced in I ⫹ SS, IR ⫹ SS, and I/R ⫹ ADO cohorts (Fig 1 and Table 1). Histological analysis showed a loss of structural integrity of enteric nerves in the jejunal of I ⫹ SS, I/R ⫹ SS, and I/R ⫹ ADO groups, but not I ⫹ ADO (Fig 2). DISCUSSION
Our results showed that the motility stimulated by transmitters released by enteric nerves and amount of enteric nerves were significantly reduced in jejunal segments of rabbits undergoing intestinal I and I/R. However, these dysfunctions in I, but not in R, were reduced or absent in rabbits treated with ADO. These results suggested that the activation of AR by ADO attenuates or prevents the motor and neural dysfunctions caused by intestinal I, but not R. The deleterious effects of R on intestines exacerbate the I injury.14 Molecular mechanisms involved in the I/R injury are little known, but they involve formation of free oxygen radicals and consequent oxidative stress alterations of calcium flux, and activation of phospholipase A2.9,10,14 The stressed or injured tissues release endogenous ADO, which blocks potentially destructive inflammatory cascades, decreasing activation of platelets, leukocytes, and endothelial cells,9,10 responses that are mediated mainly by A2A and A2B receptors.9,10 Recently, Hart et al showed that compared to wild-type mice, the intestinal I-R injury was increased in transgenic mice with selective knockout of A2B receptors, suggesting their protective role in I/R injury.11 They also showed that selective inhibition of A2B receptors in wild-type mice produced an increment in intestinal inflammation and injury during I-R. In addition, treatment with agonist of A2B receptors (BAY 60-6583) protected wild-type mice from intestinal injury, inflammation, and permeability dysfunction, whereas their therapeutic effects were abolished following targeted gene deletion of A2B receptors. Similarly, Haskó et al12 showed that preand posttreatment with a selective agonist of A2A receptors CGS-21680 protected lungs against lesions caused by I. ADO reduced the expression of adhesion molecules and the release of pro-inflammatory mediators: for example, reactive oxygen species, elastase, and tumor necrosis factor-alpha.13 Synthetic A2A and A2B selective agonists are currently under-
going preclinical testing for the treatment of I-R injury and other pathological conditions, including allergen-induced inflammation, autoimmune diseases, and sepsis.13 It is possible that these molecular mechanisms may be involved in the protective effects of ADO against cellular lesions caused by I, but not by R, as observed in present study. In conclusion, treatment with ADO was able to attenuate the motor and neural dysfunctions of small bowel caused by I, but not by R, in rabbits. REFERENCES 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. Abbracchio MPA, Burnstock G: Purinergic signaling: pathophysiological roles. Jpn J Pharmacol 78:113, 1998 10. Burnstock G: Purinergic signaling. B J Pharmacol 147:S172, 2006 11. Hart ML, Jacobi B, Schittenhelm J, et al: Cutting edge: A2B adenosine receptor signaling provides potent protection during intestinal ischemia/reperfusion injury. J Immunol 182:3965, 2009 12. Haskó G, Xu DZ, Lu Q, et al: Adenosine A2A receptor activation reduces lung injury in trauma/hemorrhagic shock. Crit Care Med 34:1119, 2006 13. Sullivan GW: Adenosine A2A receptor agonists as antiinflammatory agents. Curr Opin Investig Drugs 4:1313, 2003 14. Sies H, de Groot H: Role of reactive oxygen species in cell toxicity. Toxicol Lett 64:547, 1992
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0041-1345/10/$–see front matter doi:10.1016/j.transproceed.2010.01.019 454
injured tissues release endogenous ADO, which blocks potentially destructive inflammatory cascades, decreasing activation of platelets, leukocytes, and endothelial cells.9,10 We sought to investigate whether ADO attenuated or prevented intestinal I/R injury, by analyzing the effects of ADO treatment on the motility and histology of jejunal segments of rabbits undergoing I/R. MATERIALS AND METHODS Male New Zealand rabbits (2.5–3.0 kg) were anesthetized (ketamine 60 mg · kg⫺1 and xylazine 40 mg · kg⫺1, intravenously) and submitted to occlusion of superior mesenteric artery using a metallic clip for 60 minutes (I) followed by blood recirculation (R) for 120 minutes following removal of the clip. Twelve rabbits underwent only I (I group) and 12, I and R (I/R group). We also included a sham group (n ⫽ 6). In the I group, six rabbits were treated with saline solution 0.9% (SS) and six with ADO (15 mg · kg⫺1) injected into the femoral vein From the Departments of Pharmacoloy, Biochemistry, Morphology and Surgery of the Federal University of São Paulo (UNIFESP) and Federal University of Great Dourados (UFGD), São Paulo, Brazil. Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil. Address reprint requests to Dr Murched Omar Taha, Department of Surgery, UNIFESP/EPM, Rua Botucatu, 740, CEP 04023-900, São Paulo-SP, Brazil. E-mail: [email protected] © 2010 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 42, 454 – 456 (2010)
ADENOSINE AND I-R INJURY IN RABBITS
455
Fig 1. Typical records of nervemediated contractions induced by EFS (5 and 30 Hz) or KCl (100 mmol/L) in jejunal segments of rabbits treated with ADO or SS and submitted to intestinal I or I/R. EFS, electrical field stimulation; ADO, adenosine; SS, saline solution; I, ischemia; R, reperfusion. 5 minutes before I. In the I/R group, six rabbits were treated with SS and six with ADO (15 mg · kg⫺1) injected into the femoral vein 5 minutes before to I and 5 minutes before R and then, 55 minutes after R. After I or I/R, jejunal segments (2 cm) isolated from sacrificed rabbits were 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 of 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) were performed by means of platinum electrodes connected to an S88 electrical stimulator (Grass, USA).6 – 8 Responses to EFS and KCl were recorded by means of forcedisplacement transducers connected via a bridge amplifier to a analog/digital recording system (AD Instruments, USA).6 – 8 Data on contractile responses were submitted to statistical analyses using one-way analysis of variance and Student t test.6 – 8 We also perform histological analyses on jejunal pieces embedded in paraffin, cut into thin slices, and stained by hematoxylin and eosin using optical microscopy.7,8
RESULTS
Figure 1 shows that EFS (5 and 30 Hz) and KCl produced contractile responses in jejunal segments of all groups: sham, I ⫹ SS, I ⫹ ADO, I/R ⫹ SS, I/R ⫹ ADO. However, the amplitude of these contractions was similar to sham in Table 1. Values of Amplitude of Nerve-Mediated Contractions (Expressed in Grams of Tension) Induced by Electrical Field Stimulation (5 and 30 Hz) or KCl (100 mmol/L) in Jejunal Segments of Rabbits Treated With ADO or SS and Submitted to Intestinal I or I/R Groups
5 Hz
30 Hz
KCl
I ⫹ SS I ⫹ ADO I/R ⫹ SS I/R ⫹ ADO
1.03 ⫾ 0.08 1.93 ⫾ 0.14* 1.19 ⫾ 0.10 1.21 ⫾ 0.12
1.83 ⫾ 0.09 2.98 ⫾ 0.25* 2.19 ⫾ 0.15 2.39 ⫾ 0.22
2.21 ⫾ 0.18 3.38 ⫾ 0.24* 2.12 ⫾ 0.13 2.32 ⫾ 0.38
Data corresponding to mean ⫾ standard error of the mean (n ⫽ 6). SS, saline solution; ADO, adenosine; I, ischemia; R, reperfusion. *Statistically different of SS (P ⬍ 0.05).
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TAHA, MIRANDA-FERREIRA, SIMÕES ET AL
Fig 2. Histological aspects of jejunal segments of rabbit treated with saline solution or adenosine and submitted to intestinal ischemia. The images show the longitudinal muscle (LM), circular muscle (CM), epithelial layer (E), and enteric nerves (black arrows). (Hematoxylin and eosin, 400⫻).
the I ⫹ ADO group, but reduced in I ⫹ SS, IR ⫹ SS, and I/R ⫹ ADO cohorts (Fig 1 and Table 1). Histological analysis showed a loss of structural integrity of enteric nerves in the jejunal of I ⫹ SS, I/R ⫹ SS, and I/R ⫹ ADO groups, but not I ⫹ ADO (Fig 2). DISCUSSION
Our results showed that the motility stimulated by transmitters released by enteric nerves and amount of enteric nerves were significantly reduced in jejunal segments of rabbits undergoing intestinal I and I/R. However, these dysfunctions in I, but not in R, were reduced or absent in rabbits treated with ADO. These results suggested that the activation of AR by ADO attenuates or prevents the motor and neural dysfunctions caused by intestinal I, but not R. The deleterious effects of R on intestines exacerbate the I injury.14 Molecular mechanisms involved in the I/R injury are little known, but they involve formation of free oxygen radicals and consequent oxidative stress alterations of calcium flux, and activation of phospholipase A2.9,10,14 The stressed or injured tissues release endogenous ADO, which blocks potentially destructive inflammatory cascades, decreasing activation of platelets, leukocytes, and endothelial cells,9,10 responses that are mediated mainly by A2A and A2B receptors.9,10 Recently, Hart et al showed that compared to wild-type mice, the intestinal I-R injury was increased in transgenic mice with selective knockout of A2B receptors, suggesting their protective role in I/R injury.11 They also showed that selective inhibition of A2B receptors in wild-type mice produced an increment in intestinal inflammation and injury during I-R. In addition, treatment with agonist of A2B receptors (BAY 60-6583) protected wild-type mice from intestinal injury, inflammation, and permeability dysfunction, whereas their therapeutic effects were abolished following targeted gene deletion of A2B receptors. Similarly, Haskó et al12 showed that preand posttreatment with a selective agonist of A2A receptors CGS-21680 protected lungs against lesions caused by I. ADO reduced the expression of adhesion molecules and the release of pro-inflammatory mediators: for example, reactive oxygen species, elastase, and tumor necrosis factor-alpha.13 Synthetic A2A and A2B selective agonists are currently under-
going preclinical testing for the treatment of I-R injury and other pathological conditions, including allergen-induced inflammation, autoimmune diseases, and sepsis.13 It is possible that these molecular mechanisms may be involved in the protective effects of ADO against cellular lesions caused by I, but not by R, as observed in present study. In conclusion, treatment with ADO was able to attenuate the motor and neural dysfunctions of small bowel caused by I, but not by R, in rabbits. REFERENCES 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. Abbracchio MPA, Burnstock G: Purinergic signaling: pathophysiological roles. Jpn J Pharmacol 78:113, 1998 10. Burnstock G: Purinergic signaling. B J Pharmacol 147:S172, 2006 11. Hart ML, Jacobi B, Schittenhelm J, et al: Cutting edge: A2B adenosine receptor signaling provides potent protection during intestinal ischemia/reperfusion injury. J Immunol 182:3965, 2009 12. Haskó G, Xu DZ, Lu Q, et al: Adenosine A2A receptor activation reduces lung injury in trauma/hemorrhagic shock. Crit Care Med 34:1119, 2006 13. Sullivan GW: Adenosine A2A receptor agonists as antiinflammatory agents. Curr Opin Investig Drugs 4:1313, 2003 14. Sies H, de Groot H: Role of reactive oxygen species in cell toxicity. Toxicol Lett 64:547, 1992