Role of morphine preconditioning and nitric oxide following brain ischemia reperfusion injury in mice

Document Type : Original Article


1 Physiology Research Center, Physiology Department, Faculty of Medicine Iran University of Medical Sciences, Tehran, Iran Astronautics Research Institute, Iranian Space Research Center, Tehran, Iran

2 Physiology Research Center, Physiology Department, Faculty of Medicine Iran University of Medical Sciences, Tehran, Iran

3 Department of Anatomy, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran

4 Department of Physiology, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

5 Nutrition and Food Technology Research Institute, Faculty of Nutrition Science and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran

6 Department of Physiology and Physiology Research Center, Iran University of Medical Sciences, Tehran, Iran


Objective(s): Morphine dependence (MD) potently protects heart against ischemia reperfusion (IR) injury through specific signaling mechanisms, which are different from the pathways involved in acute morphine treatment or classical preconditioning. Since opioid receptor density changes post cerebral ischemia strongly correlated with brain histological damage, in the present study, we tried to elucidate the possible role of opioid receptors in IR injury among morphine-dependent mice.
Materials and Methods: Accordingly, incremental doses (10 mg/kg/day to 30 mg/kg/day) of morphine sulphate were subcutaneously administered for 5 days before global brain ischemia induction through bilateral common carotid artery occlusion. Animals were received naloxone (5 mg/kg) or L-NAME (20 mg/kg) 30 min after the last morphine dose. Twenty four hr after the ischemia induction, Retention trial of passive avoidance test and western blot analysis were done. histological analysis (TUNEL and NISSL staining) performed 72 hr after ischemia.
Results: MD improved post ischemia memory performance (P<0.01) and neuronal survival (P<0.001) and decreased apoptosis (P<0.05) in region I of hippocampus (CA1[F1] [M2]  region) in mouse. Treatment with naloxone or L-NAME abolished all MD aforementioned effects.
Conclusion: Results of the present study suggested that opioid receptors activation in the early hr post ischemia is crucial for MD-induced hippocampus tolerance against IR injury. Opioid receptor-dependent balance of NO production was another key factor in MD-induced protection. Further studies are required to determine the effect of MD on opioid receptor changes after ischemia and its correlation with MD-induced protection. 


1.   Kinlay S. Changes in stroke epidemiology, prevention, and treatment. Circulation 2011; 124:494-496.
2.   Blanco M,  Castillo J. Stroke in 2012: Major advances in the treatment of stroke. Nat Rev Neurol 2013; 2: 68-70.
3.   Bendel O,Alkass K, Bueters T, von Euler M, von Euler G. Reproducible loss of CA1 neurons following carotid artery occlusion combined with halothane-induced hypotension. Brain Res 2005; 1033:135-142.
4.   Ferrara A,El Bejaoui S, Seyen S, Tirelli E, Plumier JC. The usefulness of operant conditioning procedures to assess long-lasting deficits following transient focal ischemia in mice. Behav Brain Res 2009; 205:525-534.
5.   Stenzel-Poore MP, Stevens SL, King JS, Simon RP. Preconditioning reprograms the response to ischemic injury and primes the emergence of unique endogenous neuroprotective phenotypes: a speculative synthesis. Stroke 2007; 38: 680-685.
6.   Dirnagl U, BeckerK, Meisel A. Preconditioning and tolerance against cerebral ischaemia: from experimental strategies to clinical use. Lancet Neurol 2009; 8:398-412.
7.   Dirnagl U, Becker K, Meisel A. Preconditioning and tolerance against cerebral ischaemia: from experimental strategies to clinical use. Lancet Neurol 2009; 8: 398-412.
8.   Obrenovitch TP. Molecular physiology of preconditioning-induced brain tolerance to ischemia. Physiol Rev 2008; 88: 211-247.
9.   Andoh T, Chock PB, Chiueh CC. Preconditioning-mediated neuroprotection: role of nitric oxide, cGMP, and new protein expression. Ann N Y Acad Sci 2002; 962: 1-7.
10. Correia SC, Carvalho C, Cardoso S, Santos RX, Santos MS, Oliveira CR, et al. Mitochondrial preconditioning: a potential neuroprotective strategy. Front Aging Neurosci 2010; 2:211-218
11. Yellon DM, Dana A. The preconditioning phenomenon: A tool for the scientist or a clinical reality? Circ Res 2000; 87:543-550.
12. Thompson JW, Dave KR, Young JI, Perez-Pinzon MA. Ischemic preconditioning alters the epigenetic profile of the brain from ischemic intolerance to ischemic tolerance. Neurotherapeutics 2013; 10:789-797.
13. Peart JN, Gross ER, Gross GJ. Opioid-induced preconditioning: recent advances and future perspectives. Vascul Pharmacol 2005; 42: 211-218.
14. He X, Sandhu HK, Yang Y, Hua F, Belser N, Kim DH and Xia Y. Neuroprotection against hypoxia/ischemia: delta-opioid receptor-mediated cellular/molecular events. Cell Mol Life Sci 2013; 70: 2291-2303.
15. Narita M, Kuzumaki N, Miyatake M, Sato F, Wachi H, Seyama Y,et al. Role of delta-opioid receptor function in neurogenesis and neuroprotection. J Neurochem 2006; 97:1494-1505.
16. Charron C, MessierC, Plamondon H. Neuroprotection and functional recovery conferred by administration of kappa- and delta 1-opioid agonists in a rat model of global ischemia. Physiol Behav 2008; 93:502-511.
17. Peart JN, Gross GL. Morphine-tolerant mice exhibit a profound and persistent cardioprotective phenotype. Circulation 2004; 109:1219-1222.
18. Peart JN, Gross GL. Chronic exposure to morphine produces a marked cardioprotective phenotype in aged mouse hearts. Exp Gerontol 2004; 39:1021-1026.
19. Peart JN, Gross GJ. Cardioprotective effects of acute and chronic opioid treatment are mediated via different signaling pathways. Am J Physiol Heart Circ Physiol 2006; 291:H1746-1753.
20. Habibey R, Pazoki-Toroudi H. Morphine dependence protects rat kidney against ischaemia-reperfusion injury. Clin Exp Pharmacol Physiol 2008; 35:1209-1214.
21. Habibey R, Ajami M, Ebrahimi SA, Hesami A, Babakoohi S, Pazoki-Toroudi H. Nitric oxide and renal protection in morphine-dependent rats. Free Radic Biol Med 2010; 49:1109-1118.
22. He Dong, Xiangyu Ji, Dong Wang, Yueyi Ren, Shiduan Wang, Jianfang Song. Effect of morphine preconditioning on neuronal apoptosis following cerebral ischemia/reperfusion injury. Neural Regen Res 2010; 5:1144-1149.
23. Boutin H, Catherine A, Mackenzie ET, Jauzac P, Dauphin F. Long-term alterations in mu, delta and kappa opioidergic receptors following middle cerebral artery occlusion in mice. Acta Neuropathol 2007; 114:491-500.
24. Boutin H,Dauphin F, Jauzac P, MacKenzie ET. Exofocal alterations in opioidergic receptor densities following focal cerebral ischemia in the mouse. Exp Neurol 2000; 164:314-321.
25. Boutin H,Jauzac P, MacKenzie ET, Dauphin F. Potential use of early alterations in mu and delta opioid receptors as a predictive index for delayed brain ischemic damage. Neurobiol Dis 2003; 13:63-73.
26. Zare Mehrjerdi F,Aboutaleb N, Habibey R, Ajami M, Soleimani M, Arabian M, et al. Increased phosphorylation of mTOR is involved in remote ischemic preconditioning of hippocampus in mice. Brain Res 2013; 14; 1526:94-101.
27. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992; 119: 493-501.
28. Mihara M, Uchiyama M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 1978; 86: 271-278.
29. Honorio JE Jr, Vasconcelos GS, Rodrigues FT, Sena Filho JG, Barbosa-Filho JM, Aguiar CC, et al. Monocrotaline: histological damage and oxidant activity in brain areas of mice. Oxid Med Cell Longev 2012; 2012:541-547.
30. F Motamedi, M Ghasemi, FG Davoodi, N Naghdi. Comparison of learning and memory in morphine dependent rats using different behavioral models. Iran J Pharm Res 2010; 21:225-230.
31. Ajami M, Eghtesadi S, Razaz JM, Kalantari N, Habibey R, Nilforoushzadeh MA, et al. Expression of Bcl-2 and Bax after hippocampal ischemia in DHA + EPA treated rats. Neurol Sci 2011; 32:811-88.
32. Rehni AK, Singh TG, Jaggi AS, Singh N.Pharmacological preconditioning of the brain: a possible interplay between opioid and calcitonin gene related peptide transduction systems. Pharmacol Rep 2008; 60:904-913.
33. Loh KP, Huang SH, De Silva R, Tan BK, Zhu YZ. Oxidative stress: apoptosis in neuronalinjury. Curr Alzheimer Res 2006; 3:327-37.
34. Frassdorf J, Weber NC, Obal D, Toma O, Müllenheim J, Kojda G, et al. Morphine induces late cardioprotection in rat hearts in vivo: the involvement of opioid receptors and nucleartranscription factor kappaB. Anesth Analg 2005; 101: 934-941.
35. Huang PL. Nitric oxide and cerebral ischemic preconditioning. Cell Calcium 2004; 36:323-329.
36. Strijbos PJ. Nitric oxide in cerebral ischemic neurodegeneration and excitotoxicity. Crit Rev Neurobiol 1998; 12:223-243.
37. Goligorsky MS, Brodsky SV, Noiri E. Nitric oxide in acute renal failure: NOS versus NOS. Kidney Int 2002; 61:855-861.