Growing evidence from in vitro studies supports that valproic acid (VPA), an anti-convulsant and mood-stabilizing drug, has neuroprotective effects. The present study investigated whether VPA reduces brain damage and improves functional outcome in a transient focal cerebral ischemia model of rats. Subcutaneous injection of VPA (300 mg/kg) immediately after ischemia followed by repeated injections every 12 h, was found to markedly decrease infarct size and reduce ischemiainduced neurological deficit scores measured at 24 and 48 h after ischemic onset. VPA treatment also suppressed ischemia-induced neuronal caspase-3 activation in the cerebral cortex. VPA treatments resulted in a time-dependent increase in acetylated histone H3 levels in the cortex and striatum of both ipsilateral and contralateral brain hemispheres of middle cerebral artery occlusion (MCAO) rats, as well as in these brain areas of normal, non-surgical rats, supporting the in vitro finding that VPA is a histone deacetylase (HDAC) inhibitor. Similarly, heat shock protein 70 (HSP70) levels were timedependently up-regulated by VPA in the cortex and striatum of both ipsilateral and contralateral sides of MCAO rats and in these brain areas of normal rats. Altogether, our results demonstrate that VPA is neuroprotective in the cerebral ischemia model and suggest that the protection mechanisms may involve HDAC inhibition and HSP induction. Keywords: acetylated histone 3, cerebral ischemia, heat shock protein 70, neuroprotection, rat, valproic acid. (De Sarno et al. 2002). In rat cortical neurons, long-term VPA treatment blocks glutamate-induced excitotoxicity (Hashimoto et al. 2002) and prolongs life span of these cortical cultures (Jeong et al. 2003). Similar to the effect of another mood stabilizing drug, lithium, VPA protects mature rat cerebellar granule cells in cultures from NMDA receptor-mediated excitotoxicity and this action is mimicked by other histone HDAC inhibitors such as butyrate and trichostatin A (Kanai et al. 2002). A growing body of reports also demonstrate that VPA is neuroprotective against a variety of other insults (Mark et al. 1995;Mora et al. 1999;Bown et al. 2000;Wang et al. 2003).Stroke is one of the leading causes of mortality and morbidity world-wide. Although our knowledge concerning the molecular and cellular pathophysiology of brain injury after focal ischemia has advanced greatly, the development of new treatment drugs for acute ischemic stroke has not progressed as rapidly. The use of intravenous recombinant Abbreviations used: HDAC, histone deacetylase; HSP70, heat shock protein 70; GSK-3b, glycogen synthase kinase-3b; MCAO, middle cerebral artery occlusion; rt-PA, recombinant tissue-type plasminogen activator; TTC, 2,3,5-triphenyltetrazolium chloride; VPA, valproic acid.

In the healthy adult brain, neurogenesis normally occurs in the subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Cerebral ischemia enhances neurogenesis in neurogenic and non‐neurogenic regions of the ischemic brain of adult rodents. This study demonstrated that post‐insult treatment with a histone deacetylase inhibitor, sodium butyrate (SB), stimulated the incorporation of bromo‐2′‐deoxyuridine (BrdU) in the SVZ, DG, striatum, and frontal cortex in the ischemic brain of rats subjected to permanent cerebral ischemia. SB treatment also increased the number of cells expressing polysialic acid–neural cell adhesion molecule, nestin, glial fibrillary acidic protein, phospho‐cAMP response element‐binding protein (CREB), and brain‐derived neurotrophic factor (BDNF) in various brain regions after cerebral ischemia. Furthermore, extensive co‐localization of BrdU and polysialic acid–neural cell adhesion molecule was observed in multiple regions after ischemia, and SB treatment up‐regulated protein levels of BDNF, phospho‐CREB, and glial fibrillary acidic protein. Intraventricular injection of K252a, a tyrosine kinase B receptor antagonist, markedly reduced SB‐induced cell proliferation detected by BrdU and Ki67 in the ipsilateral SVZ, DG, and other brain regions, blocked SB‐induced nestin expression and CREB activation, and attenuated the long‐lasting behavioral benefits of SB. Together, these results suggest that histone deacetylase inhibitor‐induced cell proliferation, migration and differentiation require BDNF–tyrosine kinase B signaling and may contribute to long‐term beneficial effects of SB after ischemic injury.

Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, is known to protect against cerebral ischemia. The effects of VPA on blood-brain barrier (BBB) disruption were investigated in rats subjected to transient middle cerebral artery occlusion (MCAO). Postischemic VPA treatment remarkably attenuated MCAO-induced BBB disruption and brain edema. Meanwhile, VPA significantly reduced MCAO-induced elevation of matrix metalloproteinase-9 (MMP-9), degradation of tight junction proteins, and nuclear translocation of nuclear factor-jB (NF-jB). Sodium butyrate, another HDAC inhibitor, mimicked these effects of VPA. Our findings suggest that BBB protection by VPA involves HDAC inhibition-mediated suppression of NF-jB activation, MMP-9 induction, and tight junction degradation.

Lithium and valproic acid (VPA) are two primary drugs used to treat bipolar mood disorder and have frequently been used in combination to treat bipolar patients resistant to monotherapy with either drug. Lithium, a glycogen synthase kinase-3 (GSK-3) inhibitor, and VPA, a histone deacetylase (HDAC) inhibitor, have neuroprotective effects. The present study was undertaken to demonstrate synergistic neuroprotective effects when both drugs were coadministered. Pretreatment of aging cerebellar granule cells with lithium or VPA alone provided little or no neuroprotection against glutamate-induced cell death. However, copresence of both drugs resulted in complete blockade of glutamate excitotoxicity. Combined treatment with lithium and VPA potentiated serine phosphorylation of GSK-3 ␣ and ␤ isoforms and inhibition of GSK-3 enzyme activity. Transfection with GSK-3␣ small interfering RNA (siRNA) and/or GSK-3␤ siRNA mimicked the ability of lithium to induce synergistic protection with VPA. HDAC1 siRNA or other HDAC inhibitors (phenylbutyrate, sodium butyrate or trichostatin A) also caused synergistic neuroprotection together with lithium. Moreover, combination of lithium and HDAC inhibitors potentiated ␤-catenin-dependent, Lef/Tcf-mediated transcriptional activity. An additive increase in GSK-3 serine phosphorylation was also observed in mice chronically treated with lithium and VPA. Together, for the first time, our results demonstrate synergistic neuroprotective effects of lithium and HDAC inhibitors and suggest that GSK-3 inhibition is a likely molecular target for the synergistic neuroprotection. Our results may have implications for the combined use of lithium and VPA in treating bipolar disorder. Additionally, combined use of both drugs may be warranted for clinical trials to treat glutamate-related neurodegenerative diseases.

Lithium, the major drug used to treat manic depressive illness, robustly protects cultured rat brain neurons from glutamate excitotoxicity mediated by N-methyl-D-aspartate (NMDA) receptors. The lithium neuroprotection against glutamate excitotoxiciy is long-lasting, requires long-term pretreatment and occurs at therapeutic concentrations of this drug. The neuroprotective mcchanisms involve inactivation of NMDA receptors, decreased expression of pro-apoptotic proteins, p53 and Bax, enhanced expression of the cytoprotective protein, Bcl-2, and activation of the cell survival kinase, Akt. In addition, lithium pretreatment suppresses glutamate-induced loss of the activities of Akt, cyclic AMP-response element binding protein (CREB), c-Jun - N-terminal kinase (JNK) and p38 kinase. Lithium also reduces brain damage in animal models of neurodegenerative diseases in which excitotoxicity has been implicated. In the rat model of stroke using middle cerebral artery occlusion, lithium markedly reduces neurologic deficits and decreases brain infarct volume even when administered after the onset of ischemia. In a rat Huntington's disease model, lithium significantly reduces brain lesions resulting from intrastriatal infusion of quinolinic acid, an excitotoxin. Our results suggest that lithium might have utility in the treatment of neurodegenerative disorders in addition to its common use for the treatment of bipolar depressive patients.

Neuroprotective properties of the mood stabilizer valproic acid (VPA) are implicated in its therapeutic efficacy. Heat shock protein 70 (HSP70) is a molecular chaperone, neuroprotective and anti-inflammatory agent. The present study aimed to investigate underlying mechanisms and functional significance of HSP70 induction by VPA in rat cortical neurons. VPA treatment markedly upregulated HSP70 protein levels, and this was accompanied by increased HSP70 mRNA levels and promoter hyperacetylation and activity. Other HDAC inhibitors -sodium butyrate, trichostatin A and Class I HDAC-specific inhibitors MS-275 and apicidin, -all mimicked the ability of VPA to induce HSP70. Pretreatment with PI3-kinase inhibitors or an Akt inhibitor attenuated HSP70 induction by VPA and other HDAC inhibitors. VPA treatment increased Sp1 acetylation, and a Sp1 inhibitor, mithramycin, abolished the induction of HSP70 by HDAC inhibitors. Moreover, VPA promoted the association of Sp1 with the histone acetyltransferases p300 and recruitment of p300 to the HSP70 promoter. Further, VPA-induced neuroprotection against glutamate excitotoxicity was prevented by blocking HSP70 induction. Taken together, the data suggest that the PI3-kinase/Akt pathway and Sp1 are likely involved in HSP70 induction by HDAC inhibitors, and induction of HSP70 by VPA in cortical neurons may contribute to its neuroprotective and therapeutic effects.

Background and Purpose The migratory efficiency of mesenchymal stem cells (MSC) toward cerebral infarct after transplantation is limited. Valproate (VPA) and lithium enhance in vitro migration of MSC by upregulating CXC chemokine receptor 4 and matrix metalloproteinase-9, respectively. Ability of VPA and lithium to promote MSC homing and to improve functional recovery was assessed in a rat model of cerebral ischemia. Methods MSC primed with VPA (2.5 mmol/L, 3 hours) and/or lithium chloride (2.5 mmol/L, 24 hours) were transplanted into rats 24 hours after transient middle cerebral artery occlusion (MCAO). Neurological function was assessed via rotarod test, Neurological Severity Score, and body asymmetry test for 2 weeks. Infarct volume was analyzed by MRI. The number of homing MSC and microvessel density in the infarcted regions were measured 15 days after MCAO using immunohistochemistry. Results Priming with VPA or lithium increased the number of MSC homing to the cerebral infarcted regions, and copriming with VPA and lithium further enhanced this effect. MCAO rats receiving VPA-primed and/or lithium-primed MSC showed improved functional recovery, reduced infarct volume, and enhanced angiogenesis in the infarcted penumbra regions. These beneficial effects of VPA or lithium priming were reversed by AMD3100, a CXC chemokine receptor 4 antagonist, and GM6001, a matrix metalloproteinase inhibitor, respectively. Conclusions Priming with VPA and/or lithium promoted the homing and migration ability of MSC, improved functional recovery, reduced brain infarct volume, and enhanced angiogenesis in a rat MCAO model. These effects were likely mediated by VPA-induced CXC chemokine receptor 4 overexpression and lithium-induced matrix metalloproteinase-9 upregulation.

Emerging evidence suggests that the mood stabilizers lithium and valproate (VPA) have broad neuroprotective and neurotrophic properties, and that these occur via inhibition of glycogen synthase kinase 3 (GSK-3) and histone deacetylases (HDACs), respectively. Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by impaired movement, cognitive and psychiatric disturbances, and premature death. We treated N171-82Q and YAC128 mice, two mouse models of HD varying in genetic backgrounds and pathological progressions, with a diet containing therapeutic doses of lithium, VPA, or both. Untreated, these transgenic mice displayed a decrease in levels of GSK-3β serine 9 phosphorylation and histone H3 acetylation in the striatum and cerebral cortex around the onset of behavioral deficits, indicating a hyperactivity of GSK-3β and HDACs. Using multiple well-validated behavioral tests, we found that co-treatment with lithium and VPA more effectively alleviated spontaneous locomotor deficits and depressive-like behaviors in both models of HD mice. Furthermore, compared with monotherapy with either drug alone, co-treatment more successfully improved motor skill learning and coordination in N171-82Q mice, and suppressed anxiety-like behaviors in YAC128 mice. This combined treatment consistently inhibited GSK-3β and HDACs, and caused a sustained elevation in striatal as well as cortical brain-derived neurotrophic factor and heat shock protein 70. Importantly, co-treatment markedly prolonged median survival of N171-82Q mice from 31.6 to 41.6 weeks. Given that there is presently no proven treatment for HD, our results suggest that combined treatment with lithium and VPA, two mood stabilizers with a long history of safe use in humans, may have important therapeutic potential for HD patients.