Resveratrol Preconditioning Induces a Novel Extended Window of Ischemic Tolerance in the Mouse Brain

Abstract

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.

Keywords: BDNF; UCP2; cerebral ischemia; preconditioning; resveratrol.

Figure 1. Repetitive RPC induces ischemic tolerance against focal ischemia

A, every other day treatment schematic. B, every other day RPC reduces infarct volume. Veh n=12; RPC n=12; ** = p<0.01 compared to Veh. C, every day treatment schematic. D, every day RPC reduces infarct volume similar to every other day RPC. Veh n=12; RPC n=14; ** = p<0.01 compared to Veh. Individual arrows indicate a RPC or Veh treatment; MCAo is indicated by the black square; TTC is indicated by the checkered square. Representative TTC stained brain sections are shown on the left.

Figure 2. RPC induces a novel extended window of ischemic tolerance against focal ischemia

A, intermittent treatment schematic. B, intermittent RPC reduces infarct volume. Veh n=15; RPC n=16; ** = p<0.01 compared to Veh. C, single treatment schematic. D, a single application of RPC reduces infarct volume and improves neurological score. Veh n=9; RPC n=10; * = p<0.05; ** = p<0.01 compared to Veh. Individual arrows indicate a RPC or Veh treatment; MCAo is indicated by the black square; TTC and neurological scoring is indicated by the checkered square. Representative TTC stained brain sections are shown on the left.

Figure 3. Sirt1 and its targets are differentially regulated 14 days following a single application of RPC

A, cortical Sirt1 protein was increased 14 days following a single application of RPC. Representative bands for Sirt1 are shown below the quantification graph. ⸗ = non-adjacent bands from the same blot. n=10; ** = p<0.01 compared to Veh. B, ChIP-qPCR analysis revealed Sirt1 binding to BDNF and UCP2 promoter regions was enhanced 14 days following a single application of RPC. n=3; * = p<0.05; ** = p<0.01. C, cortical BDNF levels were increased 14 days following a single application of RPC. Values are expressed as pg/mg of protein. n=7; * = p<0.05. D, synaptic UCP2 protein levels were decreased 14 days following a single application of RPC. Values were normalized to the mitochondrial marker voltage VDAC. The functional UCP2 dimer (70 kDa) was used for quantification, no 35 kDa monomer band was detected. Representative band are shown below the quantification. n=6; * = p<0.05.