Int J Biol Sci 2014; 10(8):873-881. doi:10.7150/ijbs.8868
Intermittent Hypothermia Is Neuroprotective in an in vitro Model of Ischemic Stroke
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.
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 http://www.ijbs.com/v10p0873.htm