Development of a novel neuroprotective strategy: Combined treatment with hypothermia and valproic acid improves survival in hypoxic hippocampal cells

作者：Jin Guang; Liu Baoling; You Zerong; Bambakidis Ted; Dekker Simone E; Maxwell Jake; Halaweish Ihab; Linzel Durk; Alam Hasan B^*

来源：Surgery, 2014, 156(2): 221-228.

DOI：10.1016/j.surg.2014.03.038

Background. Therapeutic hypothermia and histone deacetylase inhibitors, such as valproic acid (VPA),.independently have been shown to have neuroprotective properties in models of cerebral ischemic and traumatic brain injury. However, the depth of hypothermia and the dose of VPA needed to achieve the desired result are logistically challenging. It remains unknown whether these two promising strategies can be combined to yield synergistic results. We designed an experiment to answer this question by subjecting hippocampal-derived HT22 cells to severe hypoxia in vitro.
Methods. Mouse hippocampal HT22 cells were exposed to 200 AM cobalt chloride (CoCl2), which created hypoxic conditions in vitro. Cells were incubated for 6 or 30 hours under the following conditions: (1) Dulbecco's Modified Eagle Medium; (2) 200 mu M CoCl2; (3) 200 mu M CoCl2 plus 1 mmol/L VPA; (4) 200 AM CoCl2 plus 32 degrees C hypothermia; and (5) 200 mu M CoCl2 plus both 1 mmol/L VPA and 32 C hypothermia. Cellular viability was evaluated by (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and lactate dehydrogenase release assays at 30 hours after treatment. Levels of acetylated histone H3, hypoxia-inducible factor-1 alpha, phospho-GSK-3 beta, beta-catenin, and high-mobility group box-1 were measured by Western blotting.
Results. High levels of acetylated histone H3 were detected in the VPA-treated cells. The release of lactate dehydrogenase was greatly suppressed after the combined hypothermia + VPA treatment (0.269 +/- 0.003) versus VPA (0.836 +/- 0.026) or hypothermia (0.451 +/- 0.005) treatments alone (n = 3, P = .0001). (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay showed that the number of viable cells was increased by 17.6 % when VPA and hypothermia were used in combination (n = 5, P = .0001). Hypoxia-inducible factor-la and phospho-GSK-3 beta expression were synergistically affected by the combination treatment, whereas high-mobility group box-1 was increased by VPA treatment, and inhibited by the hypothermia.
Conclusion. This is the first study to demonstrate that the neuroprotective effects of VPA and hypothermia are synergistic. This novel approach can be used to develop more effective therapies for the prevention of neuronal death.

• 出版日期2014-8