Resveratrol Preconditioning Induces a Novel Extended Window of Ischemic Tolerance in the Mouse Brain
Background and Purpose—Prophylactic treatments that afford neuroprotection against stroke may emerge from the field of preconditioning. Resveratrol mimics ischemic preconditioning, reducing ischemic brain injury when administered 2 days before global ischemia in rats. This protection is linked to silent information regulator 2 homologue 1 (Sirt1) and enhanced mitochondrial function possibly through its repression of uncoupling protein 2. Brain-derived neurotrophic factor (BDNF) is another neuroprotective protein associated with Sirt1. In this study, we sought to identify the conditions of resveratrol preconditioning (RPC) that most robustly induce neuroprotection against focal ischemia in mice.
Methods—We tested 4 different RPC paradigms against a middle cerebral artery occlusion model of stroke. Infarct volume and neurological score were calculated 24 hours after middle cerebral artery occlusion. Sirt1-chromatin binding was evaluated by ChIP-qPCR. Percoll gradients were used to isolate synaptic fractions, and changes in protein expression were determined via Western blot analysis. BDNF concentration was measured using a BDNF-specific ELISA assay.
Results—Although repetitive RPC induced neuroprotection from middle cerebral artery occlusion, strikingly one application of RPC 14 days before middle cerebral artery occlusion showed the most robust protection, reducing infarct volume by 33% and improving neurological score by 28%. Fourteen days after RPC, Sirt1 protein was increased 1.5-fold and differentially bound to the uncoupling protein 2 and BDNF promoter regions. Accordingly, synaptic uncoupling protein 2 level decreased by 23% and cortical BDNF concentration increased 26%.
Conclusions—RPC induces a novel extended window of ischemic tolerance in the brain that lasts for at least 14 days. Our data suggest that this tolerance may be mediated by Sirt1 through upregulation of BDNF and downregulation of uncoupling protein 2.
With few and limited clinically approved treatments, ischemic stroke remains a devastating pathology that plagues the world population.1 Our ability to predict those who are predisposed to or at risk for stroke has significantly improved in recent years, making prophylactic treatment a viable option for neuroprotection. There is an identifiable population in the millions that are at an increased risk for stroke and are ideal candidates for prophylactic therapeutic intervention. Indeed, studies have demonstrated the immense potential of a prophylactic approach, using forthcoming pharmacological agents derived from the field of ischemic preconditioning (IPC).
IPC refers to the ability of a brief, nondamaging ischemic episode to induce tolerance against a subsequent, damaging ischemic insult.2 Ischemic tolerance is characterized to have an early window of protection, which is activated immediately and lasts for several hours, as well as a late window of protection, which is active 2 days later.2 Several pharmacological agents can mimic the effects of IPC, including resveratrol.3 Resveratrol is a polyphenol synthesized naturally in several species of plants.4 The therapeutic potential of resveratrol has been widely investigated in many pathological conditions,5 although the investigation of resveratrol as an agent in stroke treatment is still in its infancy.
Similar to IPC, resveratrol preconditioning (RPC) induces ischemic tolerance, at least in part, by activation of silent information regulator 2 homologue 1 (Sirt1).3,6 Sirt1 is a versatile NAD+-dependent deacetylase that acts on both histone and nonhistone proteins to regulate many biological processes. Additionally, Sirt1 has a host of neuroprotective properties in pathological states, such as neurological disorders.7 More recently, Sirt1 has been implicated in cerebral ischemia, fostering ischemic tolerance through common targets, such as HIF, PGC1α, and p53.8 Additional targets of Sirt1 include mitochondrial uncoupling protein 2 (UCP2), a proton channel found in the inner mitochondrial membrane that uncouples oxidative phosphorylation,6 and brain-derived neurotrophic factor (BDNF),9 a growth factor involved in many neuronal processes, including synaptic plasticity.
Our previous work demonstrates that RPC reduces ischemic brain injury when administered 2 days before cardiac arrest in rats6 as well as middle cerebral artery occlusion (MCAo) in mice.9a In terms of neuronal function, ischemic tolerance was associated with an increase in Sirt1 activity, a decrease in UCP2, and enhanced mitochondrial function in the hippocampus. The goal of this study was to identify the conditions of RPC that most robustly induce neuroprotection against focal cerebral ischemia in mice and investigate further the contribution of the Sirt1 signaling axis.
Materials and Methods
Full descriptions of the methods sections can be found in the online-only Data Supplement.
Animals and Treatments
All experiments were approved by the Institutional Animal Care and Use Committee of the University of Miami and were in accordance with institutional regulations. Eight- to 12-week-old C57Bl/6J male mice obtained from The Jackson Laboratory (n=108) were randomly assigned to different treatment groups. Mice were injected IP with 10 mg/kg transresveratrol (3,4′,5-trihydroxy-trans-stilbene; Sigma) in 1.5% dimethyl sulfoxide (DMSO; Sigma) and 0.9% saline (Hospira Inc; RPC) or the control 1.5% DMSO and 0.9% saline (Veh). All injections, experimental procedures, and analysis throughout this study were conducted in a blinded fashion.
Focal Cerebral Ischemia by Reversible MCAo
Right-side MCAo was produced as previously described.10 Cerebral blood flow was monitored using a Laser-Doppler (Perimed System, Stockholm, Sweden) probe. MCAo was induced by inserting a silicone-coated 8-0 monofilament nylon surgical suture into the internal carotid artery and circle of Willis via the proximal external carotid. After 60 minutes of MCAo, the suture was removed.
Neurological Scoring and Infarct Volume
Chromatin Immunoprecipitation and Subsequent Quantitative PCR
Mouse brain cortices were dissected, frozen, pulverized in liquid nitrogen, and resuspended in cross-linking solution (1% formaldehyde). Nuclei were lysed, chromatin was sonicated to 200 to 500 bp fragments, centrifuged, and diluted in ChIP dilution buffer. The sonicated, cleared chromatin supernatant was used for immunoprecipitation with 5 μg of anti-Sirt1 antibody or control IgG overnight at 4°C. Beads were washed sequentially with 1 mL each of low salt wash, high salt wash, LiCl wash, and TE wash. Beads were resuspended in elution buffer and cross-linking was reversed. DNA was purified by phenol–chloroform extraction and EtOH precipitation. Quantitative real-time PCR was performed using SYBR Green reagent (Roche) in the LightCycler 480 II (Roche Applied Science) and analyzed with the ΔΔCT method. DNA-relative enrichment was determined by normalizing to input genomic DNA and preserum IgG as background.
Isolation of Synaptosomes via Percoll Gradient Fractionation
Percoll gradients were used to isolate synaptosomes according to Dunkley and colleagues12 with slight modifications. Mice were decapitated under isoflurane anesthesia and their cortices immersed in isolation medium at 4°C. Tissue was chopped, homogenized, diluted to give 10% wt/vol and centrifuged at 500g for 5 minutes (Sorvall RC5 centrifuge, Newton CT). The supernatant was layered on a Percoll (Sigma) gradient and centrifuged at 32 500g for 5 minutes. Synaptosomes were collected between 23% to 15% and 15% to 10% Percoll junctions, washed once with isolation medium by centrifugation at 15 000g for 10 minutes and once with 0.25 mol/L sucrose. The final pellet was resuspended in radioimmunoprecipitation assay buffer and processed for protein analysis according to the Western blot protocol.
Mouse cortices were homogenized in radioimmunoprecipitation assay buffer. Protein concentration was measured by Bradford assay (BioRad). Equal amounts of protein were run on 12% SDS-polyacrylamide gels and transferred to a nitro-cellulose membrane (BioRad). The next steps in order were block 2 hours at room temperature; probe with primary antibody overnight at 4°C; incubate secondary antibody for 1 hour at RT; addition of substrate for 3 minutes; protein bands detected with x-ray film (Denville Scientific). Films were analyzed densitometrically using Image J. Two normalized values (for the same sample) from different blots were averaged to give the values presented.
BDNF ELISA Assay
BDNF concentration was quantified using a ChemiKineTM BDNF Sandwich ELISA Kit (Millipore) according to the manufacturer’s instructions.
All data are represented as mean±SEM. For statistical analysis, Student’s t-tests were used to compare differences between 2 groups. Differences were considered significant at the 95% confidence interval (*P<0.05, **P<0.01).
Repetitive RPC Treatment Confers Ischemic Tolerance Against Focal Ischemia
Based on our previous findings that RPC induces ischemic tolerance when administered 2 days before injury,3,6 we sought to evaluate RPC in the context of focal cerebral ischemia using the MCAo mouse model. Our first hypothesis was that repetitive RPC over an extended period of time induces ischemic neuroprotection. To this end, mice were given RPC or Veh every other day for 14 days before MCAo, mimicking the standard preconditioning paradigm repeated 7 times (Figure 1A). Animals that did not display ≥80% reduction in cerebral blood flow were excluded—full documentation of cerebral blood flow, exclusion, and mortality can be found in Table. Compared with Veh, RPC significantly reduced infarct volume by 20% (P<0.01, Veh n=12, RPC n=12; Figure 1B). Next, we hypothesized that increasing the frequency of RPC treatment could enhance this protection. Thus, mice were given RPC or Veh every day for 14 days before MCAo (Figure 1C). Similar to every other day treatment, everyday treatment significantly reduced infarct volume by 27% compared with Veh (P<0.01, Veh n=12, RPC n=14; Figure 1D). From this, we conclude that repetitive RPC induces ischemic tolerance against focal ischemia.
RPC Induces a Novel Extended Window of Ischemic Tolerance Against Focal Ischemia
Because there was no significant difference between the protection afforded by every other day and every day treatment, we rationalized that perhaps less frequent RPC could also provide ischemic tolerance. Our previous studies show that IPC induces ischemic tolerance that lasts for at least 4 days in organotypic slice cultures.3 Therefore, our next hypothesis was that intermittent RPC could induce an extended window of ischemic tolerance. To test this hypothesis, we used a geometric growth pattern of intermittent RPC consisting of 4 applications at intervals of 1, 2, 4, and 8 days, with 14 days between the last treatment and MCAo (Figure 2A). Strikingly, compared with Veh, intermittent RPC significantly reduced infarct volume by 24% (P<0.01, Veh n=15, RPC n=16; Figure 2B). Before any conclusions can be drawn from this intermittent RPC paradigm, an important distinction to make is whether it is the culmination of 4 applications of RPC that give rise to this long-lasting ischemic tolerance or if simply one application of RPC can induce ischemic tolerance for this extended period of time. To test this, mice were given RPC or Veh once, 14 days before MCAo (Figure 2C). Remarkably, one application of RPC 14 days before MCAo significantly reduced infarct volume by 33% compared with Veh (P<0.01, Veh n=9, RPC n=10; Figure 2D). To confirm that this reduction in infarct volume was accompanied by improved neurological performances, we calculated neurological score 24 hours after MCAo and found that compared with Veh, RPC significantly improved this functional outcome measure by 28% (P<0.05; Figure 2D). From these results, we conclude that a single application of RPC induces a novel extended window of ischemic tolerance that lasts for at least 14 days.
Sirt1 and Its Targets Are Augmented 14 Days Following RPC
Given that Sirt1 is a well-established target of resveratrol and that in our previous studies blockade of Sirt1 ablates ischemic tolerance,3,6 we investigated whether changes in Sirt1 expression occurred in this extended window of ischemic tolerance. Fourteen days after a single application of RPC, cortical Sirt1 levels were significantly increased 1.5-fold of Veh (P<0.01, n=10; Figure 3A). To assess whether this increase had functional implications, we turned our attention to putative chromatin binding sites for Sirt1. Based on known interactions with Sirt113,14 and our previous studies implicating BDNF9 and UCP26 in ischemic tolerance, we evaluated Sirt1 binding to promoter regions within the BDNF and UCP2 genes. Chromatin immunoprecipitation and subsequent qPCR 14 days after a single application of RPC revealed that Sirt1 binding to both the BDNF and UCP2 promoters was significantly enhanced, 2.66-fold and 1.14-fold, respectively (BDNF P<0.01, UCP2 P<0.05, n=3; Figure 3B). This enhancement was not seen in Veh-treated mice. Accordingly, we found that cortical BDNF levels were significantly increased by 27% (P<0.05, n=7) and synaptic UCP2 levels were significantly decreased by 23% (P<0.05, n=6) compared with Veh 14 days after a single application of RPC (Figure 3C and 3D). These data suggest that Sirt1 may be modulating BDNF and UCP2 expression at 14 days where ischemic tolerance is observed.
The major finding of this study—that a single application of RPC can protect against focal ischemia 14 days later—is both novel and intriguing. To our knowledge, this is the first evidence that a single pharmacological preconditioning treatment can induce a state of long-lasting ischemic tolerance. This is a new extended window of preconditioning beyond the 2 to 3 day time point or classical late window commonly studied.
In work before this study, evidence hinted that an extended window may exist. In organotypic slices, we showed that one application of IPC affords protection that lasts at least 4 days.3 Additionally, a paradigm of repetitive hypoxia preconditioning (varying O2 exposure over 2 weeks) conferred protection from focal ischemia for ≤2 months after treatment.15 Elucidating the long-term effects of preconditioning will provide a framework for the development of novel clinical applications in the future. The major argument for resveratrol’s immense potential is that it has already been deemed safe for clinical use in humans.16
Resveratrol either directly or indirectly activates or upregulates Sirt1.17,18 Previously we have shown increased Sirt1 to be a common mechanism of preconditioning-mediated protection, where protection was lost in the presence of the Sirtuin inhibitor Sirtinol.3 The proposed acute mechanisms for Sirt1-mediated ischemic tolerance include modulating the neuroinflammatory response via NF-κB,19 reducing/inhibiting proapoptotic factors, such as p53,20 as well as enhancing mitochondrial function via PGC1α21 and UCP2.6 Here, we found Sirt1 to be upregulated at 14 days after a single RPC treatment. A chronic upregulation of Sirt1 could promote changes in gene expression that are maintained overtime that give rise to this extended window of ischemia tolerance. In line with this notion, 14 days after RPC, we find enhanced Sirt1 binding to UCP2 and BDNF promoter regions.
As mentioned earlier, Sirt1 is known to negatively regulate UCP2 by binding directly to its promoter.14 This is supported in our current study as we observed a decrease in synaptic UCP2 protein levels. In our previous studies, we observed a reduction in UCP2 2 days after RPC in rats where protection from cardiac arrest was seen. Neuroprotection and decreased UCP2 were also linked to enhanced mitochondrial efficiency.6 In another line of evidence, UCP2 knockout mice were found to be resistant to permanent focal ischemia.22 In these mice, protection was attributed to increased antioxidant defenses and a reduction in oxidative damage. It should be noted that UCP2’s effects in regards to cerebral ischemia are still somewhat controversial. Other groups have shown that overexpression of UCP2 is also protective against cerebral ischemia23 and that the same UCP2 knockout mice mentioned above were actually more susceptible to a model of transient focal ischemia.24 Discrepancies could be as a result of variation across species, genetic compensation in knockout mouse models, the type of ischemia model used, or choice of preconditioning agent.
Sirt1 is also known to upregulate BDNF expression; the proposed mechanisms are indirect and include Sirt1 deacety lation of MeCP2 (which regulates expression of BDNF by binding at its promoter)25 and Sirt1 inhibition of miR-134 expression (a micro-RNA that downregulates expression of BDNF).13 In addition, binding of Sirt1 to the BDNF promoter has been identified in the mouse cerebellum, suggesting that a direct interaction might also occur.26 Moreover, BDNF was decreased in the brains of Sirt1 null mice.27 BDNF is an important growth factor that promotes survival and growth of existing neurons, as well as the differentiation of new ones. It also impacts synaptic plasticity and learning and memory.28 Our previous work shows enhanced BDNF as a mechanism for ischemia tolerance after IPC or protein kinase C epsilon–induced preconditioning,9 in which increased BDNF was associated with a delay in anoxic depolarization. Another group shows that intravenous BDNF administration after ischemia improves recovery possibly through a mechanism centered on neurogenesis.29 Given that all of these changes seem to occur homogeneously throughout the cortex, we expect them to impact both the core and penumbral regions of the infarct.
The fact that one dose of resveratrol has protective effects that are apparent 2 weeks later suggests a promising prophylactic avenue. The validity of a preconditioning approach in the clinic was nicely described by Dirnagl and colleagues30 who defined clinical scenarios where a preconditioning approach could have a direct impact. Resveratrol also protects against stroke when administered post-injury. Although not tested as a postconditioning paradigm, future studies are needed to test whether resveratrol could be used in a postconditioning setting. Also future studies can test whether pre- and post-resveratrol treatment in tandem can enhance neuroprotection.
Resveratrol has been used in several clinical trials and deemed safe for use in humans at doses up to a few grams per day. Our finding that tachyphylaxis to RPC did not occur when administered on an everyday basis suggests that multiple administrations are feasible, if required. Additionally, oral administration of resveratrol as a neuroprotective intervention in rodents has been validated by several groups and seems similar to that of intraperitoneal or intravenous administration.31 Taken together, our current findings build on the body of preclinical evidence for resveratrol as a potential treatment in the stroke clinic.
Sources of Funding
National Institute of Health Grants NS45676, NS054147, NS34773 (M.A. Perez-Pinzon), 1F31NS089356-01 (K.B. Koronowski), and 5R01NS073779-04 (K.R. Dave) and American Heart Association Grant 13POST16720001 (J.T. Neumann).
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.115.009876/-/DC1.
- Received April 24, 2015.
- Revision received May 21, 2015.
- Accepted June 11, 2015.
- © 2015 American Heart Association, Inc.
- Go AS,
- Mozaffarian D,
- Roger VL,
- Benjamin EJ,
- Berry JD,
- Borden WB,
- et al
- Neumann JT,
- Thompson JW,
- Raval AP,
- Cohan CH,
- Koronowski KB,
- Perez-Pinzon MA.
- Narayanan SV,
- Dave KR,
- Saul I,
- Perez-Pinzon MA.
- Dai H,
- Kustigian L,
- Carney D,
- Case A,
- Considine T,
- Hubbard BP,
- et al
- Yeung F,
- Hoberg JE,
- Ramsey CS,
- Keller MD,
- Jones DR,
- Frye RA,
- et al
- de Bilbao F,
- Arsenijevic D,
- Vallet P,
- Hjelle OP,
- Ottersen OP,
- Bouras C,
- et al
- Haines BA,
- Mehta SL,
- Pratt SM,
- Warden CH,
- Li PA.
- Schäbitz WR,
- Steigleder T,
- Cooper-Kuhn CM,
- Schwab S,
- Sommer C,
- Schneider A,
- et al