title: Development of a novel neuroprotective strategy: combined treatment with hypothermia and valproic acid improves survival in hypoxic hippocampal cells.
abstract: 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 μM cobalt chloride (CoCl(2)), 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 μM CoCl(2); (3) 200 μM CoCl(2) plus 1 mmol/L VPA; (4) 200 μM CoCl(2) plus 32°C hypothermia; and (5) 200 μM CoCl(2) 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-1α, phospho-GSK-3β, β-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-1α and phospho-GSK-3β expression were synergistically affected by the combination treatment, whereas high-mobility group box-1 was increased by VPA treatment, and inhibited by the hypothermia.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Surgery, University of Michigan, Ann Arbor, MI.
authors.forename: Guang
authors.initials: G
authors.lastname: Jin
authors.affiliation: Department of Surgery, University of Michigan, Ann Arbor, MI.
authors.forename: Baoling
authors.initials: B
authors.lastname: Liu
authors.affiliation: Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital/Harvard Medical School, Charlestown, MA.
authors.forename: Zerong
authors.initials: Z
authors.lastname: You
authors.affiliation: Department of Surgery, University of Michigan, Ann Arbor, MI.
authors.forename: Ted
authors.initials: T
authors.lastname: Bambakidis
authors.affiliation: Department of Surgery, University of Michigan, Ann Arbor, MI.
authors.forename: Simone E
authors.initials: SE
authors.lastname: Dekker
authors.affiliation: Department of Surgery, University of Michigan, Ann Arbor, MI.
authors.forename: Jake
authors.initials: J
authors.lastname: Maxwell
authors.affiliation: Department of Surgery, University of Michigan, Ann Arbor, MI.
authors.forename: Ihab
authors.initials: I
authors.lastname: Halaweish
authors.affiliation: Department of Surgery, University of Michigan, Ann Arbor, MI.
authors.forename: Durk
authors.initials: D
authors.lastname: Linzel
authors.affiliation: Department of Surgery, University of Michigan, Ann Arbor, MI. Electronic address: alamh@med.umich.edu.
authors.forename: Hasan B
authors.initials: HB
authors.lastname: Alam
mesh_terms: D000107:Acetylation; D000818:Animals; D015687:Cell Hypoxia / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003035:Cobalt / Q000633:toxicity; D003131:Combined Modality Therapy; D004195:Disease Models, Animal; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D024243:HMGB1 Protein / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D056572:Histone Deacetylase Inhibitors / Q000008:administration & dosage*; D006657:Histones / Q000378:metabolism; D007036:Hypothermia, Induced; D002534:Hypoxia, Brain / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors; D051379:Mice; D018696:Neuroprotective Agents / Q000008:administration & dosage*; D010766:Phosphorylation; D014635:Valproic Acid / Q000008:administration & dosage*; D051176:beta Catenin / Q000378:metabolism
chemical_list: C495267:CTNNB1 protein, mouse; D024243:HMGB1 Protein; C582808:HMGB1 protein, mouse; C497443:Hif1a protein, mouse; D056572:Histone Deacetylase Inhibitors; D006657:Histones; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D018696:Neuroprotective Agents; D051176:beta Catenin; D003035:Cobalt; D014635:Valproic Acid; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3; C018021:cobaltous chloride