Creatine Protects the Immature Brain From Hypoxic-Ischemic Injury

Abstract

Objective: We tested the neuroprotective effects of creatine against hypoxic-sichemic injury in hte immature brain.

Methods: Hippocampal slices were prepared from fetal guinea pigs at 0.9 gestation and incubated in artificial cerebrospinal fluid (aCSF) equilibrated with carbogen. Slices were subjected to oxygen-glucose deprivation (OGD) for 30 or 40 minutes. Two hours after OGD, adenosine triphosphate (ATP) and protein synthesis were analyzed. Creatine (3 mM) was applied to tissue slices of the study groups 2 hours before the insult. In a second set of experiments 7-day-old Wistar rats were anesthetized, and the left carotid artery was ligated. After 1 hour of recovery the pups were subjected to a hypoxic gas mixture (8% oxygen and 92% nitrogen) for 80 minutes. Seven days later the brains of the neonates were removed and analyzed for hypoxic-ischemic injury. The rat pups of the test group were treated with creatine (3 g/kg subcutaneously) before (-64 hours, -40 hours, and -16 hours) and after (+3 hours) the hypoxic-ischemic insult, with zero time corresponding to thes tart of hypoxia, whereas the animals of the control group received the solvent.

Results: Creatine significantly improved the recovery of protein synthesis 2 hours after OGD in hippocampal slices but had no effect on ATP levels. Whereas seven animals of the control group developed severe cystic cerebral infraction, only mild to moderate damage was observed in the rat pups of the study group. In contrast to creatine-treated pups, the volume of the ipsilateral hemisphere was considerably smaller than that of the contralateral one in control animals (104 ± 22 versus 138 ± 14 mL, P < .001). Except at had received cratine. This was also true for the evaluated subfileds in the hippocampus.

Conclusion: We conclude that creatine potects the immature brain from hypoxic-ischemic injury.

Keywords

Cretine neuroprotection brain hippocampus ischemia htpoxia

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References

1. Volpe JJ . Neurology of the newborn. Philadelphia: Saunders, 1995.

2. Berger R , Garnier Y . Pathophysiology of perinatal brain damage. Brain Res Brain res Rev 1999;30:107-134.

3. McDonald JW , Johnston MV . Physiological and pathophysiological roles of excitatory amino acids during central nervous system development. Brain Res Brain Res Rev 1990;15:41-70.

4. Woznicki DT , Walker JB . Utilization of cyclocreatine phosphate, an analogue of creatine phosphate, by mouse brain during ischemia and its sparing action on brain energy reserves. J Neurochem 1980;34:1247-1253.

5. Holtzman D , Tolgitti A , Khait I , Jense F . Creatine increases survival and suppresses seizures in the hypoxic immature rat. Pediatr Res 1998;44:410-444.

6. Clark JF . Creatine and phosphocreatine: A review of their use in exercise and sports. J Athletic Train 1997;32:45-51.

7. Earnest CP , Almada AL , Mitchell TL . High-performance capillary electrophoresis-pure creatine monohydrate reduces blood lipids in men and women. Clin Sci (Colch) 1996;91:113-118.

8. Davis BM , Miller RK , Brent RL , Koszalka TR . Materno-fetal transport of creatine in the rat. J Neurochem 1995;64:742-748.

9. Dechent P , Pouwels PJ , Wilken B , Hanefeld F , Frahm J . Increase of total creatine in human brain after oral supplementation of creatine-monohydrate. Am J Physiol 1999;277:R698-R704.

10.

10. Hemmer W , Wallimann T . Functional aspects of creatine kinase in brain. Dev Neurosci 1993;15:249-260.

11.

11. Chen L , Roberts R , Friedman DL . Expression of brain-type creatine kinase and ubiquitous mitochondrial creatine kinase in the fetal rat brain: Evidence for a nuclear energy shuttle. J Comp Neurol 1995;363:389-401.

12.

12. Bessman SP , Geiger PJ . Transport of energy in muscle: The phosphorylcreatine shuttle. Science 1981;211:448-452.

13.

13. Wallimann T , Wyss M , Brdiczka D , Nicolay K , Eppenberger HM . Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating enegy demands: The `phosphocreatine circuit' for cellular energy homeostasis. Biochem J 1992;281:21-40.

14.

14. Lipton P , Whittingham TS . Reduced ATP concentration as a basis for synaptic transmission failure during hypoxia in the in vitro guinea-pig hippocampus. J Physiol 1982;325:51-65.

15.

15. Luhmann HJ , Heinemann U . Hypoxia-induced functional alterations in adult rat neocortex. J Neurophysiol 1992;67:798-811.

16.

16. Okada Y , Yoneda K . Effects of accumulation of phosphocreatine on the survival time of thin hippocampal slices from the guinea pig during deprivation of both oxygen and glucose. Neurosci Lett 1983;41:119-124.

17.

17. Wilken B , Ramirez JM , Probst I , Richter DW , Hanefeld F . Creatineprotects the central respiratory network of mammals under anoxic conditions. Pediatr Res 1998;43:8-14.

18.

18. Artru AA , Michenfelder JD . Cyclocreatine phosphate, an analogue of creatine phosphate, does not improve hypoxic tolerance in mice. J Neurochem 1982;39:1198-1200.

19.

19. Hossmann KA . Disturbances of cerebral protein synthesis and ischemic cell death. Prog Brain Res 1993;96:167-177.

20.

20. Berger R , Djuricic B , Jensen A , Hossmann KA , Paschen W . Ontogenetic differences in energy metabolism and inhibition of protein synthesis in hippocampal slices during in vitro ischemia and 24 hours of recovery. Brain Res Dev Brain Res 1996;91:281-291.

21.

21. Djuricic B , Berger R , Paschen W . Protein synthesis and energy metabolism in hippocampal slices during extended (24 hours) recovery following different periods of ischemia. Metab Brain Dis 1994;9:377-389.

22.

22. Berger R , Jensen A , Paschen W . Metabolic disturbances in hippocampal slices of fetal guinea pigs during and after oxygen-glucose deprivation: Is nitricoxide involved? Neurosci Lett 1998;245:163-166.

23.

23. Lowry OH , Rosenbrough NH , Farr AL , Randall R . Protein measurement with the folin phelon reagent. J Biol Chem 1951;193:265-275.

24.

24. Levine S . Anoxic-ischemic encephalopathy in rats. Am J Pathol 1960;36:1-17.

25.

25. Rice JE , Vannucci RC , Brierley JB . The influence of immaturity on hypoxic-ischemic brain damagein the rat. Ann Neurol 1981;9:131-141.

26.

26. Holtzman D , Khait I , Mulkern R , et al. In vivo development of brain phosphocreatine in normal and creatine-treated rabbit pups. J Neurochem 1999;73:2477-2484.

27.

27. Stöckler S , Hanefeld F , Frahm J . Creatine replacement therapy in guanidinoacetate methyltransferase deficiency, a novel inborn error of metabolism. Lancet 1996;348:789-790.

28.

28. Stöckler S , Holzbach U , Hanefeld F , et al. Creatine deficiency in the brain: A new, treatable inborn error of metabolism. Pediatr Res 1994;36:409-413.

29.

29. Bona E , Aden U , Gilland E , Fredholm BB , Hagberg H . Neonatal cerebral hypoxia-ischemia: The effect of adenosine receptor antagonists. Neuropharmacology 1997;36:1327-1338.

30.

30. Mies G , Ishimaru S , Xie Y , Seo K , Hossmann KA . Ischemic thresholds of cerebral protein synthesis and energy state following middle cerebral artery occlusion in rat. J Cereb Blood Flow Metab 1991;11:753-761.

31.

31. Brown AW , Brieley JB . Anoxic-ischaemic cell change in rat brain light microscopic and fine-structural observations. J Neurol Sci 1972;16:59-84.

32.

32. Berger R , Lehmann T , Karcher J , Garnier Y , Jensen A . Low dose flunarizine protects the fetal brain from ischemic injury in sheep. Pediatr Res 1998;44:277-282.

33.

33. Flogel U , Decking UK , Godecke A , Schrader J . Contribution of NO to ischemia-reperfusion injury in the saline-perfused heart: A study in endothelial NO synthase knockout mice. J Mol Cell Cardiol 1999;31:827-836.

34.

34. Holtzman D , Tsuji M , Wallimann T , Hemmer W . Functional maturation of creatine kinase in rat brain. Dev Neurosci 1993;15:261-270.

35.

35. Carter AJ , Muller RE , Pschorn U , Stransky W . Preincubation with creatine enhances levels of creatine phosphate and prevents anoxic damage in rat hippocampal slices. J Neurochem 1995;64:2691-2699.

36.

36. Kleihues P , Hossmann KA , Pegg AE , Kobayashi K , Zimmermann V . Resuscitation of the monkey brain after one hour complete ischemia. III. Indications of metabolic recovery. Brain Res 1975;95:61-73.

37.

37. Bodsch W , Takahashi K , Barbier A , Ophoff BG , Hossmann KA . Cerebral protein synthesis and ischemia. Prog Brain Res 1985;63:197-210.

38.

38. Thilmann R , Xie Y , Kleihues P , Kiessling M . Persistent inhibition of protein synthesis precedes delayed neuronal death in postischemic gerbil hippocampus. Acta Neuropathol (Berl) 1986;71:88-93.

39.

39. Widmann R , Kuroiwa T , Bonnekoh P , Hossmann KA . [14C]leucine incorporation into brain proteins in gerbils after transient ischemia: Relationship to selective vulnerability of hippocampus. J Neurochem 1991;56:789-796.

40.

40. Hartmann JF , Becker RA , Cohen MM . Cerebral ultrastructure in experimental hypoxia and ischemia. Monogr Neural Sci 1973;1:50-64.

41.

41. Kirino T , Sano K . Fine structural nature of delayed neuronal death following ischemia in the gerbil hippocampus. Acta Neuropathol (Berl) 1984;62:209-218.

42.

42. Hossmann KA , Widmann R , Wiessner C , Dux E , Djuricic B , Röhn G . Protein synthesis after global cerebral ischemia and selective vulnerability. In: Krieglstein J , Oberpichler-Schwenk H , eds. Pharmacology of cerebral ischemia. Stuttgart: Wissenschaftliche Verlagsgesellschaft mbH, 1992:289-299.

43.

43. Degarcia DJ , Neumar RW , White BC , Krause GS . Global ischemia and reperfusion—modifications in eukaryotic initiation factors associated with the inhibition of translation initiation. J Neurochem 1996;67:2005-2012.

44.

44. Dobbing J , Sands J . Comparative aspects of the brain growth spurt. Early Hum Dev 1979;3:79-83.

45.

45. Tian R , Nascimben L , Kaddurah-Daouk R , Ingwall JS . Depletion of energy reserve via the creatine kinase reaction during the evolution of heart failure in cardiomyopathic hamsters. J Mol Cell Cardiol 1996;28:755-765.

46.

46. Hattori H , Morin AM , Schwart PH , Fujikawa DG , Wasterlain CG . Posthypoxic treatment with MK-801 reduces hypoxic-ischemic damage in the neonatal rat. Neurology 1989;39:713-718.

47.

47. Xu CJ , Klunk WE , Kanfer JN , Xiong Q , Miller G , Pettegrew JW . Phosphocreatine-dependent glutamate uptake by synaptic vesicles. A comparison with ATP-dependent glutamate uptake. J Biol Chem 1996;271:13435-13440.

48.

48. Palmer C , Towfighi J , Roberts RL , Heitjan DF . Allopurinol administered after inducing hypoxia-ischemia reduces brain injury in 7-day-old rats. Pediatr Res 1993;33:405-411.

49.

49. Hamada Y , Hayakawa T , Hattori H , Mikawa H . Inhibitor of nitric oxide synthesis reduces hypoxic-ischemic brain damage in the neonatal rat. Pediatr Res 1994;35:10-104.

50.

50. Matthews RT , Yang L , Jenkins BG , Ferrante RJ , Rosen BR , Kaddurah-Daouk R , Beal MF . Neuroprotective effects of creatine and cyclocreatine in animal models of Huntington's disease. J Neurosci 1998;18:156-163.

51.

51. O'Gorman E , Beutner G , Dodler M , Koretsky AP , Brdiczka D , Wallimann T . The role of creatine kinase in inhibition of mitochondrial permeability transition. FEBS Lett 1997;414:253-257.

52.

52. Sullivan PG , Geiger JD , Mattson MP , Scheff SW . Dietary supplement creatine protects against traumatic brain injury. Ann Neurol 2000;48:723-729.