Abstract
Purpose
The effects of mild and moderate hypothermic therapy on cerebral injury are still controversial. Our hypothesis is that mild and moderate hypothermia should have some effects on neurons themselves if they really have protective effects. By using a nearly pure neuronal culture, we evaluated the effects and mechanism of hypothermia against hypoxic insult.
Methods
A nearly pure neuronal culture from cortices of 18-day-old Wister rats was used. The neurons were exposed to below 1% oxygen at 3 different temperatures (30, 33 and 37°C). First, cell viability was measured by assessing viable neurons with trypan blue. Second, to evaluate the mechanism, the extracellular glutamate concentration was measured by high-performance liquid chromatography after hypoxia; cell viability after exposure to extrinsic glutamate was also evaluated. Next, mitochondrial membrane potential was estimated, by monitoring aggregation of MitoCapture™, and the percentage of apoptotic cells was evaluated by staining with Hoechst 33342 and propidium iodide.
Results
After 24-h hypoxic insult, cell viability at 30 and 33°C was significantly higher than at 37°C. There was no significant difference between extracellular concentrations of glutamate after hypoxia or cell viability after glutamate exposure among the 3 temperature groups. In moderate hypothermia, the number of neurons with mitochondrial injury and the percentage of apoptotic cells were significantly reduced.
Conclusion
Mild and moderate hypothermia inhibited hypoxic neuronal cell death. The mechanism of this effect may be related to protection of mitochondrial function, presumably followed by inhibition of apoptosis, at least in moderate hypothermia.
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References
Kawaguchi M, Furuya H, Patel PM. Neuroprotective effects of anesthetic agents. J Anesth. 2005;19:150–6.
Grocott HP, Yoshitani K. Neuroprotection during cardiac surgery. J Anesth. 2007;21:367–77.
Zhao H, Steinberg GK, Sapolsky RM. General versus specific actions of mild-moderate hypothermia in attenuating cerebral ischemic damage. J Cereb Blood Flow Metab. 2007;27:1879–94.
Miyazawa T, Tamura A, Fukui S, Hossmann KA. Effect of mild hypothermia on focal cerebral ischemia. Review of experimental studies. Neurol Res. 2003;25:457–64.
Bernard SA, Gray TW, Buist MD, Jones BM, Silvester W, Gutteridge G, Smith K. Treatment of comatose survivors of out-of-hospital cardiac arrest with induced hypothermia. N Engl J Med. 2002;346:557–63.
Hypothermia after Cardiac Arrest Study G. Mild therapeutic hypothermia to improve the neurologic outcome after cardiac arrest. N Engl J Med. 2002;346:549–56.
Gluckman PD, Wyatt JS, Azzopardi D, Ballard R, Edwards AD, Ferriero DM, Polin RA, Robertson CM, Thoresen M, Whitelaw A, Gunn AJ. Selective head cooling with mild systemic hypothermia after neonatal encephalopathy: multicentre randomised trial. Lancet. 2005;365:663–70.
Shankaran S, Laptook AR, Ehrenkranz RA, Tyson JE, McDonald SA, Donovan EF, Fanaroff AA, Poole WK, Wright LL, Higgins RD, Finer NN, Carlo WA, Duara S, Oh W, Cotten CM, Stevenson DK, Stoll BJ, Lemons JA, Guillet R, Jobe AH, National Institute of Child Health, Human Development Neonatal Research. Whole-body hypothermia for neonates with hypoxic–ischemic encephalopathy. N Engl J Med. 2005;353:1574–84.
Clifton GL, Miller ER, Choi SC, Levin HS, McCauley S, Smith KR Jr, Muizelaar JP, Wagner FC Jr, Marion DW, Luerssen TG, Chesnut RM, Schwartz M. Lack of effect of induction of hypothermia after acute brain injury. N Engl J Med. 2001;344:556–63.
Todd MM, Hindman BJ, Clarke WR, Torner JC. Intraoperative Hypothermia for Aneurysm Surgery Trial. Mild intraoperative hypothermia during surgery for intracranial aneurysm. N Engl J Med. 2005;352:135–45.
Uchino H, Kuroda Y, Morota S, Hirabayashi G, Ishii N, Shibasaki F, Ikeda Y, Hansson MJ, Elmer E. Probing the molecular mechanisms of neuronal degeneration: importance of mitochondrial dysfunction and calcineurin activation. J Anesth. 2008;22:253–62.
Chan PH. Mitochondria and neuronal death/survival signaling pathways in cerebral ischemia. Neurochem Res. 2004;29:1943–9.
Zhao H, Yenari MA, Cheng D, Sapolsky RM, Steinberg GK. Biphasic cytochrome c release after transient global ischemia and its inhibition by hypothermia. J Cereb Blood Flow Metab. 2005;25:1119–29.
Xie C, Markesbery WR, Lovell MA. Survival of hippocampal and cortical neurons in a mixture of MEM+ and B27-supplemented neurobasal medium. Free Radic Biol Med. 2000;28:665–72.
Hisano K, Watanabe M, Morimoto Y. Protective effects of free radical scavenger edaravone against glutamate neurotoxicity in nearly pure neuronal culture. J Anesth. 2009;23:363–9.
Matsumoto M, Togashi H, Kaku A, Kanno M, Tahara K, Yoshioka M. Cortical GABAergic regulation of dopaminergic responses to psychological stress in the rat in the rat dorsolateral striatum. Synapse. 2005;56:117–21.
Bossenmeyer-pourie C, Koziel V, Daval J-L. Effects of hypothermia on hypoxia-induced apoptosis in cultured neurons from developing rat forebrain: comparison with preconditioning. Pediatr Res. 2000;47:385–91.
Busto R, Globus MY-T, Dietrich WD, Martinez D, Valdes I, Ginsberg MD. Effect of mild hypothermia on ischemia-induced release of neurotransmitters and free fatty acids in rat brain. Stroke. 1989;20:904–10.
Winfee CJ, Connolly ES, Fiore AJ, Solomon RA. Mild hypothermia reduces penumbral glutamate levels in the rat focal cerebral ischemia model. Neurosurgery. 1996;38:1216–22.
Berger R, Jensen A, Hossmann KA, Paschen W. Effect of mild hypothermia during and after transient in vitro ischemia on metabolic disturbances in hippocampal slices at different stages of development. Dev Brain Res. 1998;105:67–77.
Bruno VM, Goldberg MP, Dugan LL, Giffard RG, Choi DW. Neuroprotective effect of hypothermia in cortical cultures exposed to oxygen–glucose deprivation or excitatory amino acids. J Neurochem. 1994;63:1398–406.
Asai S, Zhao H, Kohno T, Takahashi Y, Nagata T, Ishikawa K. Quantitative evaluation of extracellular glutamate concentration in postischemic glutamate re-uptake, dependent on brain temperature, in the rat following severe global brain ischemia. Brain Res. 2000;864:60–8.
Arai H, Uto A, Ogawa Y, Sato K. Effect of low temperature on glutamate-induced intracellular calcium accumulation and cell death in cultured hippocampal neurons. Neurosci Lett. 1993;163:132–4.
Bickler PE, Buck LT, Hansen BM. Effects of isoflurane and hypothermia on glutamate receptor-mediated calcium influx in brain slices. Anesthesiology. 1994;81:1461–9.
Yenari MA, Iwayama S, Cheng D, Sun GH, Fujimura M, Morita-Fujimura Y, Chan PH, Steinberg GK. Mild hypothermia attenuates cytochrome c release but does not alter Bcl-2 expression or caspase activation after experimental stroke. J Cereb Blood Flow Metab. 2002;22:29–38.
Belkhiri A, Dar AA, Zaika A, Kelley M, El-Rifai W. t-Darpp promotes cancer cell survival by up-regulation of Bcl2 through Akt-dependent mechanism. Cancer Res. 2008;68:395–403.
Zhu C, Wang X, Cheng X, Qiu L, Xu F, Simbruner G, Blomgren K. Post-ischemic hypothermia-induced tissue protection and diminished apoptosis after neonatal cerebral hypoxia–ischemia. Brain Res. 2004;996:67–75.
Xu L, Yenari MA, Steinberg GK, Giffard RG. Mild hypothermia reduces apoptosis of mouse neurons in vitro early in the cascade. J Cereb Blood Flow Metab. 2002;22:21–8.
Shibano T, Morimoto Y, Kemmotsu O, Shikama H, Hisano K, Hua Y. Effects of mild and moderate hypothermia on apoptosis in neuronal PC12 cells. Br J Anaesth. 2002;89:301–5.
Acknowledgments
The authors thank Professor M. Yoshioka and former Assistant Professor M. Matshumoto, Department of Neuropharmacology in our university, for their teaching of HPLC measurement. The authors also thank Ms Naoko Kimura for her technical assistance.
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Hua, Y., Hisano, K. & Morimoto, Y. Effect of mild and moderate hypothermia on hypoxic injury in nearly pure neuronal culture. J Anesth 24, 726–732 (2010). https://doi.org/10.1007/s00540-010-0999-x
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DOI: https://doi.org/10.1007/s00540-010-0999-x