Posttrauma cotreatment with lithium and valproate: reduction of lesion volume, attenuation of blood-brain barrier disruption, and improvement in motor coordination in mice with traumatic brain injury

Laboratory investigation

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Object

Although traumatic brain injury (TBI) is the leading cause of death and morbidity in young adults, no effective pharmaceutical treatment is available. By inhibiting glycogen synthase kinase–3 (GSK-3) and histone deacetylases (HDACs), respectively, lithium and valproate (VPA) have beneficial effects in diverse neurodegenerative diseases. Furthermore, in an excitotoxic neuronal model and in animal models of amyotrophic lateral sclerosis, Huntington disease, and stroke, combined treatment with lithium and VPA produces more robust neuroprotective effects than treatment with either agent alone. Building on previous work that establishes that therapeutic doses of either lithium or VPA have beneficial effects in mouse models of TBI, this study evaluated the effects of combined treatment with subeffective doses of lithium and VPA in a mouse model of TBI.

Methods

Male C57BL/6 mice underwent TBI and were subsequently treated with lithium, VPA, or a combination of lithium and VPA 15 minutes post-TBI and once daily thereafter for up to 3 weeks; all doses were subeffective (1 mEq/kg of lithium and 200 mg/kg of VPA). Assessed parameters included lesion volume via H & E staining; blood-brain barrier (BBB) integrity via immunoglobulin G extravasation; neurodegeneration via Fluoro-Jade B staining; motor coordination via a beam-walk test; and protein levels of acetylhistone H3, phospho-GSK-3β, and β-catenin via Western blotting.

Results

Posttrauma treatment with combined subeffective doses of lithium and VPA significantly reduced lesion volume, attenuated BBB disruption, and mitigated hippocampal neurodegeneration 3 days after TBI. As expected, subeffective doses of lithium or VPA alone did not have these beneficial effects. Combined treatment also improved motor coordination starting from Day 7 and persisting at least 21 days after TBI. Acetylation of histone H3, an index of HDAC inhibition, was robustly increased by the combined treatment 3 days after TBI.

Conclusions

Cotreatment with subeffective doses of lithium and VPA significantly attenuated TBI-induced brain lesion, BBB disruption, and neurodegeneration, and robustly improved long-term functional recovery. These findings suggest that potentiating histone acetylation by HDAC inhibition is probably part of the mechanism underlying the beneficial effects associated with this combined treatment for TBI. Because both lithium and VPA have a long history of safe clinical use, the results suggest that using a combination of these 2 agents at subtherapeutic doses to treat patients with TBI may also reduce side effects and enhance tolerability.

Abbreviations used in this paper:Ac-H3 = acetylhistone H3; BBB = blood-brain barrier; CCI = controlled cortical impact; FJB = Fluoro-Jade B; GSK-3 = glycogen synthase kinase–3; HDAC = histone deacetylase; IgG = immunoglobulin G; MMP-9 = matrix metalloproteinase–9; TBI = traumatic brain injury; VPA = valproate.

Article Information

Address correspondence to: De-Maw Chuang, Ph.D., Molecular Neurobiology Section, National Institute of Mental Heath, National Institutes of Health, 10 Center Dr., MSC-1363, Bethesda, MD 20892. email: chuang@mail.nih.gov.

Please include this information when citing this paper: published online July 12, 2013; DOI: 10.3171/2013.6.JNS13135.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Combined treatment with subeffective doses of lithium and VPA reduced lesion volume 3 days post-TBI. H & E staining was performed to evaluate TBI-induced lesion volume. Representative H & E staining showed that TBI resulted in lesions in the ipsilateral cortex and hippocampus. A: Treatment with 300 mg/kg VPA significantly reduced lesion volume 3 days after TBI (n = 8/group; *p < 0.05). B: Treatment with lithium at 1 mEq/kg or VPA at 200 mg/kg had no effect on lesion volume. Combined treatment with these subeffective doses of lithium and VPA significantly reduced lesion volume (B; n = 9/group; *p < 0.05). Data throughout are expressed as the mean ± SEM.

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    Combined treatment with subeffective doses of lithium and VPA attenuated BBB disruption 3 days after TBI. IgG staining was used to determine BBB disruption. Representative photographs (upper row) showing TBI increased the immunepositive signal in the area close to injury. The immune reactivity in monotreatment groups with either lithium or VPA was similar to that in the saline-treated group, whereas combined treatment significantly reduced the area of IgG-positive staining. Quantitative analysis showed that the relative optical density of IgG staining was robustly reduced after combined treatment, compared with the saline-treated group (n = 8/group; *p < 0.05), whereas treatment with lithium or VPA alone did not affect relative optical density.

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    Combined treatment with subeffective doses of lithium and VPA reduced the number of degenerating neurons 3 days post-TBI. Neuronal degeneration was evaluated using FJB staining. Representative microphotographs (upper row) showing FJB staining in the hippocampal dentate gyrus 3 days after injury. In the saline-treated group, many FJB-positive cells were visualized. Although fewer FJB-positive cells were observed in response to lithium or VPA treatment alone, the combined treatment group showed the fewest FJB-positive cells. Compared with the saline-treated group, quantitative analysis showed that the number of FJB-positive cells was significantly reduced in the combined treatment group (n = 9/group; *p < 0.05). Nuclei were stained with 4,6-diamino-2-phenylindole, as shown with blue fluorescence. Bar = 50 μm.

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    Combined treatment with subeffective doses of lithium and VPA improved beam-walk performance after TBI. The beam-walk test was performed 1, 3, 7, 14, and 21 days after injury to assess motor coordination. TBI markedly increased the number of foot faults in all groups examined. A slow recovery from 7 to 21 days postinjury was seen in all groups. Compared with the saline-treated group, a significant reduction in the number of foot faults was observed in the combined treatment group 7, 14, and 21 days after injury (n = 8/group for all drug treatment groups; n = 6/group for the sham and saline groups; *p < 0.05; ***p < 0.001). Treatment with VPA alone did not affect the number of foot faults at any time point, whereas the lithium-treated group showed a reduced number of foot faults compared with the saline-treated group on Days 14 and 21 after injury.

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    Combined treatment with subeffective doses of lithium and VPA increased levels of Ac-H3 3 days after TBI. The Ac-H3 levels remained unchanged in the ipsilateral cortex in the saline-treated group compared with the sham-operated group. After treatment with lithium or VPA alone, levels of Ac-H3 increased slightly. Combined treatment significantly increased Ac-H3 levels compared with the saline-treated group (upper). Quantified data showed a significant increase in Ac-H3 levels after combined treatment compared with the saline-treated group (lower; n = 6/group; *p < 0.05). Levels of phospho-GSK-3β (p-GSK-3β) and β-catenin were unchanged after injury or any treatment. The β actin level was measured as a loading control.

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