Time course of neuroprotection induced by in vivo normobaric hyperoxia preconditioning and angiogenesis factors

Document Type : Original Article


1 Department of Physiology, Faculty of Biological Sciences, Shahid Beheshti University, G.C., Tehran, Iran

2 Institute for Cognitive and Brain Sciences, Shahid Beheshti University, G.C., Tehran, Iran


Objective(s):Every year, a large number of people lose their lives due to stroke. Stroke is the second leading cause of death worldwide. Surprisingly, recent studies have shown that preconditioning with hyperoxia (HO) increases tissue tolerance to ischemia, ultimately reducing damages caused by stroke. Addressed in this study are beneficial contributions from HO preconditioning into reduced harm to be incurred by the attack, as well as its effect on the expression levels of vascular endothelial growth factor (VEGF) and endostatin.
Materials and Methods: A set of experiments was conducted where a number of rats were divided into three groups. The animals in the first group received 90% oxygen for 4 hr a day, for 6 days. The second group was housed in room air and the third group was a sham (surgical stress). After 60 min of ischemia, 24 hr blood flow, neurological deficit score (NDS) and infarct volume (IV) in the group MCAO (Middle Cerebral Artery Occlusion) were investigated. Immediately following a 48 hr HO pre-treatment, sampling was performed to measure the expression levels of VEGF and endostatin.
Results: Preconditioning with alternating HO led to reduced infarct volume and NDS. Moreover, pre-treatment with HO resulted in increased VEGF expression while decreasing endostatin.
Conclusion: Although further studies are deemed necessary to clarify the mechanisms of ischemic tolerance, apparently, somewhat intermittent hyperoxia can be associated with positive impacts by increasing VEGF and decreasing expression of endostatin.


1. Kochanek KD, Xu J, Murphy SL, Minino AM, Kung HC. Deaths: final data for 2009. Natl Vit Stat Rep 2011; 60:1-116.
2. Eltzschig HK, Eckle T. Ischemia and reperfusion--from mechanism to translation. Nat Med 2011; 17:1391-1401.
3. Margaritescu O, Pirici D, Margaritescu C. VEGF expression in human brain tissue after acute ischemic stroke. Rom J Morphol Embryol 2011; 52:1283-1292.
4. Shin HK, Dunn AK, Jones PB, Boas DA, Lo EH, Moskowitz MA, et al. Normobaric hyperoxia improves cerebral blood flow and oxygenation, and inhibits peri-infarct depolarizations in experimental focal ischaemia. Brain 2007; 130:1631-1642.
5. Bigdeli MR, Hajizadeh S, Froozandeh M, Rasulian B, Heidarianpour A, Khoshbaten A. Prolonged and intermittent normobaric hyperoxia induce different degrees of ischemic tolerance in rat brain tissue. Brain Res 2007; 1152:228-233.
6. Nasrniya S, Bigdeli MR. Ischemic tolerance induced by normobaric hyperoxia and evaluation of group I and II metabotropic glutamate receptors. Curr Neurovasc Res 2013; 10:21-28.
7. Thom SR. Functional inhibition of leukocyte B2 integrins by hyperbaric oxygen in carbon monoxide-mediated brain injury in rats. Toxicol Appl Pharmacol 1993; 123:248-256.
8. Mink RB, Dutka AJ. Hyperbaric oxygen after global cerebral ischemia in rabbits reduces brain vascular permeability and blood flow. Stroke 1995; 26:2307-2312.
9. Badr AE, Yin W, Mychaskiw G, Zhang JH. Effect of hyperbaric oxygen on striatal metabolites: a microdialysis study in awake freely moving rats after MCA occlusion. Brain Res 2001; 916:85-90.
10. Bigdeli MR. Neuroprotection caused by hyperoxia preconditioning in animal stroke models. ScientificWorldJournal 2011; 11:403-421.
11. Bigdeli MR, Rasoulian B, Meratan AA. In vivo normobaric hyperoxia preconditioning induces different degrees of antioxidant enzymes activities in rat brain tissue. Eur J Pharmacol 2009; 611:22-29.
12. Mohammadi E, Bigdeli MR. Effects of preconditioning with normobaric hyperoxia on Na(+)/Ca(2)(+) exchanger in the rat brain. Neuroscience 2013; 237:277-284.
13. Arenillas JF, Sobrino T, Castillo J, Davalos A. The role of angiogenesis in damage and recovery from ischemic stroke. Curr Treat Options Cardiovasc Med 2007; 9:205-212.
14. Navaratna D, Guo S, Arai K, Lo EH. Mechanisms and targets for angiogenic therapy after stroke. Cell Adh Mig 2009; 3:216-223.
15. Marti HJ, Bernaudin M, Bellail A, Schoch H, Euler M, Petit E, et al. Hypoxia-induced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia. Am J Pathol 2000; 156:965-976.
16. Taguchi A, Soma T, Tanaka H, Kanda T, Nishimura H, Yoshikawa H, et al. Administration of CD34+ cells after stroke enhances neurogenesis via angiogenesis in a mouse model. J Clin Invest 2004; 114:330-338.
17. Hoeben A, Landuyt B, Highley MS, Wildiers H, Van Oosterom AT, De Bruijn EA. Vascular endothelial growth factor and angiogenesis. Pharmacol Rev 2004; 56:549-580.
18. Jin K, Mao XO, Batteur SP, McEachron E, Leahy A, Greenberg DA. Caspase-3 and the regulation of hypoxic neuronal death by vascular endothelial growth factor. Neuroscience 2001; 108:351-358.
19. Dhanabal M, Ramchandran R, Waterman MJ, Lu H, Knebelmann B, Segal M, et al. Endostatin induces endothelial cell apoptosis. J Biol Chem 1999; 274:11721-11726.
20. Ohab JJ, Fleming S, Blesch A, Carmichael ST. A neurovascular niche for neurogenesis after stroke.  J Neurosci 2006; 26:13007-13016.
21. Kim YM, Hwang S, Kim YM, Pyun BJ, Kim TY, Lee ST, et al. Endostatin blocks vascular endothelial growth factor-mediated signaling via direct interaction with KDR/Flk-1. J Biol Chem 2002; 277:27872-27879.
22. Ema M, Taya S, Yokotani N, Sogawa K, Matsuda Y, Fujii-Kuriyama Y. A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1alpha regulates the VEGF expression and is potentially involved in lung and vascular development. Proc Natl Acad Sci U S A 1997; 94:4273-4278.
23. Josko J, Mazurek M. Transcription factors having impact on vascular endothelial growth factor (VEGF) gene expression in angiogenesis. Med Sci Monit 2004; 10:RA89-98.
24. Kwak DJ, Kwak SD, Gauda EB. The effect of hyperoxia on reactive oxygen species (ROS) in rat petrosal ganglion neurons during development using organotypic slices. Pediatr Res 2006; 60:371-376.
25. Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 1989; 20:84-91.
26. Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C, Sharp FR. A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab 1990; 10:290-293.
27. Zemke D, Smith JL, Reeves MJ, Majid A. Ischemia and ischemic tolerance in the brain: an overview. Neurotoxicology 2004; 25:895-904.
28. Downey JM, Cohen MV, Ytrehus K, Liu Y. Cellular mechanisms in ischemic preconditioning: the role of adenosine and protein kinase C. Ann N Y Acad Sci 1994; 723:82-98.
29. Bostek CC. Oxygen toxicity: an introduction. AANA J 1989; 57:231-237.
30. Bitterman N. CNS oxygen toxicity. Undersea Hyperb Med 2004; 31:63-72.
31. Demchenko IT, Welty-Wolf KE, Allen BW, Piantadosi CA. Similar but not the same: normobaric and hyperbaric pulmonary oxygen toxicity, the role of nitric oxide. Am J Physiol Lung Cell Mol Physiol 2007; 293:L229-238.
32. Liu S, Zhen G, Meloni BP, Campbell K, Winn HR. Rodent stroke model guidelines for preclinical stroke trials (1st Edition). J Exp Stroke Transl Med 2009; 2:2-27.
33. Rousselet E, Kriz J, Seidah NG. Mouse model of intraluminal MCAO: cerebral infarct evaluation by cresyl violet staining. J Vis Exp 2012; pii:4038.
34. Lee SH, Kim YJ, Lee KM, Ryu S, Yoon BW. Ischemic preconditioning enhances neurogenesis in the subventricular zone. Neuroscience 2007; 146:1020-1031.
35. Dawson DA, Furuya K, Gotoh J, Nakao Y, Hallenbeck JM. Cerebrovascular hemodynamics and ischemic tolerance: lipopolysaccharide-induced resistance to focal cerebral ischemia is not due to changes in severity of the initial ischemic insult, but is associated with preservation of microvascular perfusion. J Cereb Blood Flow  Metab 1999; 19:616-623.
36. Gustavsson M, Mallard C, Vannucci SJ, Wilson MA, Johnston MV, Hagberg H. Vascular response to hypoxic preconditioning in the immature brain. J Cereb Blood Flow Metab 2007; 27:928-938.
37. Li S, Zhang Y, Shao G, Yang M, Niu J, Lv G, Ji X. Hypoxic preconditioning stimulates angiogenesis in ischemic penumbra after acute cerebral infarction. Neural Regen Res 2013; 8:2895-2903.
38. Veltkamp R, Siebing DA, Sun L, Heiland S, Bieber K, Marti HH, et al. Hyperbaric oxygen reduces blood-brain barrier damage and edema after transient focal cerebral ischemia. Stroke 2005; 36:1679-1683.
39. Li JS, Zhang W, Kang ZM, Ding SJ, Liu WW, Zhang JH, et al. Hyperbaric oxygen preconditioning reduces ischemia-reperfusion injury by inhibition of apoptosis via mitochondrial pathway in rat brain. Neuroscience 2009; 159:1309-1315.
40. Liu S, Liu W, Ding W, Miyake M, Rosenberg GA, Liu KJ. Electron paramagnetic resonance-guided normobaric hyperoxia treatment protects the brain by maintaining penumbral oxygenation in a rat model of transient focal cerebral ischemia. J Cereb Blood Flow Metab 2006; 26:1274-1284.
41. van Bruggen N, Thibodeaux H, Palmer JT, Lee WP, Fu L, Cairns B, et al. VEGF antagonism reduces edema formation and tissue damage after ischemia/reperfusion injury in the mouse brain.  J Clin Invest 1999; 104:1613-1620.
42. Malek M, Duszczyk M, Zyszkowski M, Ziembowicz A, Salinska E. Hyperbaric oxygen and hyperbaric air treatment result in comparable neuronal death reduction and improved behavioral outcome after transient forebrain ischemia in the gerbil. Exp Brain Res 2013 ; 224:1-14.
43. Ostrowski RP, Graupner G, Titova E, Zhang J, Chiu J, Dach N, et al. The hyperbaric oxygen preconditioning-induced brain protection is mediated by a reduction of early apoptosis after transient global cerebral ischemia. Neurobiol Dis 2008; 29:1-13.
44. Dhanabal M, Volk R, Ramchandran R, Simons M, Sukhatme VP. Cloning, expression, and in vitro activity of human endostatin. Biochem Biophys Res Commun 1999; 258:345-352.
45. Chao CC, Ma YL, Lee EH. Brain-derived neurotrophic factor enhances Bcl-xL expression through protein kinase casein kinase 2-activated and nuclear factor kappa B-mediated pathway in rat hippocampus. Brain Pathol 2011; 21:150-162.
46. Zhang X, Xiong L, Hu W, Zheng Y, Zhu Z, Liu Y, et al. Preconditioning with prolonged oxygen exposure induces ischemic tolerance in the brain via oxygen free radical formation. Canad J Anaesth 2004; 51:258-263.
47. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007; 39:44-84.
48. Gu GJ, Li YP, Peng ZY, Xu JJ, Kang ZM, Xu WG, et al. Mechanism of ischemic tolerance induced by hyperbaric oxygen preconditioning involves upregulation of hypoxia-inducible factor-1alpha and erythropoietin in rats. J Appl Physiol 2008 ; 104:1185-1191.
49. Peng Z, Ren P, Kang Z, Du J, Lian Q, Liu Y, et al. Up-regulated HIF-1alpha is involved in the hypoxic tolerance induced by hyperbaric oxygen preconditioning. Brain Res 2008; 1212:71-78.
50. Ren P, Kang Z, Gu G, Liu Y, Xu W, Tao H, et al. Hyperbaric oxygen preconditioning promotes angiogenesis in rat liver after partial hepatectomy. Life Sci 2008; 83:236-241.
51. Liu W, Liu K, Tao H, Chen C, Zhang JH, Sun X. Hyperoxia preconditioning: the next frontier in neurology? Neurol Res 2012; 34:415-421.