Int J Biol Sci 2014; 10(8):873-881. doi:10.7150/ijbs.8868

Research Paper

Intermittent Hypothermia Is Neuroprotective in an in vitro Model of Ischemic Stroke

Sui-yi Xu1,2, Ya-fang Hu1, Wei-pin Li2, Yong-ming Wu1, Zhong Ji1, Sheng-nan Wang1, Ke Li3, Su-yue Pan1✉

1. Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China;
2. Department of Neurosurgery, Shenzhen Key Laboratory of Neurosurgery, Shenzhen Second People's Hospital, Shenzhen University 1st Affiliated Hospital, Shenzhen 518035, China;
3. Research Center of Clinical Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.


Objective: To investigate whether the intermittent hypothermia (IH) protects neurons against ischemic insult and the potential molecular targets using an in vitro ischemic model of oxygen glucose deprivation (OGD).

Methods: Fetal rat cortical neurons isolated from Day E18 rat embryos were subjected to 90-min OGD and hypothermia treatments during reoxygenation before examining the changes in microscopic morphology, cell viability, microtubule- associated protein 2 (MAP-2) release, intracellular pH value and calcium, reactive oxygen species (ROS) generation, mitochondrial membrane potential (△Ψm) and neuronal death using cell counting kit (CCK-8), enzyme-linked immunosorbent assay (ELISA), BCECF AM, Fluo-3 AM, DCFH-DA and dihydroethidium (DHE), JC-1 staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), respectively.

Results: 90-min OGD induced morphologic abnormalities, cell viability decline, MAP-2 release, intracellular acidosis, calcium overload, increased ROS generation, △Ψm decrease and cell death in primary neurons, which was partially inhibited by continuous hypothermia (CH) and intermittent hypothermia (IH). Interestingly, 6-h CH was insufficient to reduce intracellular calcium overload and stabilize mitochondrial membrane potential (△Ψm), while 12-h CH was effective in reversing the above changes. All IH treatments (6×1 h, 4×1.5 h or 3×2 h) effectively attenuated intracellular free calcium overload, inhibited ROS production, stabilized mitochondrial membrane potential (△Ψm) and reduced delayed cell death in OGD-treated cells. However, only IH intervals longer than 1.5 h appeared to be effective in preventing cell viability loss and intracellular pH decline.

Conclusion: Both CH and IH were neuroprotective in an in vitro model of ischemic stroke, and in spite of shorter hypothermia duration, IH could provide a comparable neuroprotection to CH.

Keywords: Ischemic stroke, Hypothermia, Neuroprotection, Primary neuronal culture.

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How to cite this article:
Xu Sy, Hu Yf, Li Wp, Wu Ym, Ji Z, Wang Sn, Li K, Pan Sy. Intermittent Hypothermia Is Neuroprotective in an in vitro Model of Ischemic Stroke. Int J Biol Sci 2014; 10(8):873-881. doi:10.7150/ijbs.8868. Available from