Interest in histone deacetylase (HDAC)-based therapeutics as a potential treatment for stroke has grown dramatically. The neuroprotection of HDAC inhibition may involve multiple mechanisms, including modulation of transcription factor acetylation independent of histones. The transcription factor Nrf2 has been shown to be protective in stroke as a key regulator of antioxidant-responsive genes. Here, we hypothesized that HDAC inhibition might provide neuroprotection against mouse cerebral ischemia by activating the Nrf2 pathway. We determined that the classic HDAC inhibitor trichostatin A increased neuronal cell viability after oxygen-glucose deprivation (from an OD value of 0.10±0.01 to 0.25±0.08) and reduced infarct volume in wild-type mice with stroke (from 49.1±3.8 to 21.3±4.6%). In vitro studies showed that HDAC inhibition reduced Nrf2 suppressor Keap1 expression, induced Keap1/Nrf2 dissociation, Nrf2 nuclear translocation, and Nrf2 binding to antioxidant response elements in heme oxygenase 1 (HO1), and caused HO1 transcription. Furthermore, we demonstrated that HDAC inhibition upregulated proteins downstream of Nrf2, including HO1, NAD(P)H:quinone oxidoreductase 1, and glutamate-cysteine ligase catalytic subunit in neuron cultures and brain tissue. Finally, unlike wild-type mice, Nrf2-deficient mice were not protected by pharmacologic inhibition of HDAC after cerebral ischemia. Our studies suggest that activation of Nrf2 might be an important mechanism by which HDAC inhibition provides neuroprotection.

Epidemiologic studies have shown that foods rich in polyphenols, such as flavanols, can lower the risk of ischemic heart disease; however, the mechanism of protection has not been clearly established. In this study, we investigated whether epicatechin (EC), a flavanol in cocoa and tea, is protective against brain ischemic damage in mice. Wild-type mice pretreated orally with 5, 15, or 30 mg/kg EC before middle cerebral artery occlusion (MCAO) had significantly smaller brain infarcts and decreased neurologic deficit scores (NDS) than did the vehicle-treated group. Mice that were posttreated with 30 mg/kg of EC at 3.5 hours after MCAO also had significantly smaller brain infarcts and decreased NDS. Similarly, WT mice pretreated with 30 mg/kg of EC and subjected to N-methyl-D-aspartate (NMDA)-induced excitotoxicity had significantly smaller lesion volumes. Cell viability assays with neuronal cultures further confirmed that EC could protect neurons against oxidative insults. Interestingly, the EC-associated neuroprotection was mostly abolished in mice lacking the enzyme heme oxygenase 1 (HO1) or the transcriptional factor Nrf2, and in neurons derived from these knockout mice. These results suggest that EC exerts part of its beneficial effect through activation of Nrf2 and an increase in the neuroprotective HO1 enzyme.

Hemoproteins undergo degradation during hypoxic/ischemic conditions, but the pro-oxidant free heme that is released cannot be recycled and must be degraded. The extracellular heme associates with its high-affinity binding protein, hemopexin (HPX). Hemopexin is shown here to be expressed by cortical neurons and it is present in mouse cerebellum, cortex, hippocampus, and striatum. Using the transient ischemia model (90-min middle cerebral artery occlusion followed by 96-h survival), we provide evidence that HPX is protective in the brain, as neurologic deficits and infarct volumes were significantly greater in HPX−/− than in wild-type mice. Addressing the potential protective HPX cellular pathway, we observed that exogenous free heme decreased cell survival in primary mouse cortical neuron cultures, whereas the heme bound to HPX was not toxic. Heme–HPX complexes induce HO1 and, consequently, protect primary neurons against the toxicity of both heme and prooxidant tert-butyl hydroperoxide; such protection was decreased in HO1−/− neuronal cultures. Taken together, these data show that HPX protects against heme-induced toxicity and oxidative stress and that HO1 is required. We propose that the heme–HPX system protects against stroke-related damage by maintaining a tight balance between free and bound heme. Thus, regulating extracellular free heme levels, such as with HPX, could be neuroprotective.

Background The enzyme cytosolic phospholipase A 2 alpha (cPLA 2 α) has been implicated in the progression of cerebral injury following ischemia and reperfusion. Previous studies in rodents suggest that cPLA 2 α enhances delayed injury extension and disruption of the blood brain barrier many hours after reperfusion. In this study we investigated the role of cPLA 2 α in early ischemic cerebral injury. Methods Middle cerebral artery occlusion (MCAO) was performed on cPLA 2 α +/+ and cPLA 2 α -/- mice for 2 hours followed by 0, 2, or 6 hours of reperfusion. The levels of cPLA 2 α, cyclooxygenase-2, neuronal morphology and reactive oxygen species in the ischemic and contralateral hemispheres were evaluated by light and fluorescent microscopy. PGE 2 content was compared between genotypes and hemispheres after MCAO and MCAO and 6 hours reperfusion. Regional cerebral blood flow was measured during MCAO and phosphorylation of relevant MAPKs in brain protein homogenates was measured by Western analysis after 6 hours of reperfusion. Results Neuronal cPLA 2 α protein increased by 2-fold immediately after MCAO and returned to pre-MCAO levels after 2 hours reperfusion. Neuronal cyclooxygenase-2 induction and PGE 2 concentration were greater in cPLA 2 α +/+ compared to cPLA 2 α -/- ischemic cortex. Neuronal swelling in ischemic regions was significantly greater in the cPLA 2 α +/+ than in cPLA 2 α -/- brains (+/+: 2.2 ± 0.3 fold vs. -/-: 1.7 ± 0.4 fold increase; P < 0.01). The increase in reactive oxygen species following 2 hours of ischemia was also significantly greater in the cPLA 2 α +/+ ischemic core than in cPLA 2 α -/- (+/+: 7.12 ± 1.2 fold vs. -/-: 3.1 ± 1.4 fold; P < 0.01). After 6 hours of reperfusion ischemic cortex of cPLA 2 α +/+ , but not cPLA 2 α -/- , had disruption of neuron morphology and decreased PGE 2 content. Phosphorylation of the MAPKs-p38, ERK 1/2, and MEK 1/2-was significantly greater in cPLA 2 a +/+ than in cPLA 2 α -/- ischemic cortex 6 hours after reperfusion. Conclusions ...

After the publication of this article, the following errors in the Materials and Methods section were noticed:On page 1953 in the Materials and Methods section it is erroneously stated that 'Cells were washed and incubated with rhodamine-conjugated, affinity-purified donkey anti-rat IgG (H þ L) and fluorescein isothiocyanate (FITC)-conjugated, affinitypurified goat anti-rabbit IfG (H þ L)'.Correction: Cells were washed and incubated with rhodamineconjugated, affinity-purified donkey anti-rabbit IgG (H þ L) and fluorescein isothiocyanate (FITC)-conjugated, affinity-purified goat anti-mouse IgG (H þ L).In Figure 5B the þ and -representation is incorrect as published.The corrected appears below. À / À and HO1 À / À mice were not significantly different from those of their vehicle-treated counterparts, but were significantly higher than those of the EC-treated WT mice. (C) Similarly, the neurologic deficit scores of EC-treated Nrf2 À / À and HO1 À / À mice at 24 hours after MCAO were significantly higher than those of the EC-treated WT mice and not significantly different from those of their vehicle-treated counterparts. *Po0.05 compared with EC-treated WT controls; NS, not significant. (D) Neurons were grown for 24 hours in serum-free medium with B27 supplement minus antioxidant in the presence and absence of tert-butyl hydroperoxide (t-BuOOH; 60 mmol/L) or H 2 O 2 (60 mmol/L), with or without EC (100 mmol/L), SnPPIX (10 mmol/L), or combinations thereof. Cell viability was assessed with the MTT assay. ***Po0.001 versus vehicle; À / À or Nrf2 À / À mice were incubated for 24 hours in Neurobasal medium alone (control) or in the presence or absence of t-BuOOH (60 mmol/L), EC (100 mmol/L), or combinations thereof. Cell viability was assessed with the MTT assay. ***Po0.001 versus control; NS, not significantly different versus t-BuOOH.

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Correction to: Journal of Cerebral Blood Flow & Metabolism (2009) 29, 953–964; doi: 10.1038/jcbfm.2009.19 After the publication of this article, errors in the Materials and Methods section were noticed. The section on immunofluorescence should read: IMMUNOCYTOFLUORESCENCE STAINING AND FLUORESCENCE MICROSCOPY Cultured embryonic neurons were grown on poly-D-lysine-coated glass coverslips for 10 days. The neurons then were permeabilized for 2 mins with 0.5% Triton X-100 followed by fixation with 3% paraformaldehyde for 20 mins at room temperature. The cells were incubated with primary antibodies to microtubule-associated protein (MAP2, a neuronal marker; Chemicon International) and to HPX for 30 mins, washed, and then incubated with rhodamine-conjugated, affinity-purified donkey anti-goat IgG (H+L) and FITC-conjugated, affinity-purified goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) secondary antibodies for 30 mins. For fluorescence microscopy, cells were observed by epifluorescence on a Nikon TE-200 microscope. Images were captured with a CoolSNAP HQ camera (Image Processing Solutions Inc., North Reading, MA, USA) with OpenLab software (Improvision Inc., Boston, MA, USA).