Oral administration of the flavanol (−)‐epicatechin bolsters endogenous protection against focal ischemia through the Nrf2 cytoprotective pathway

Abstract

Consumption of flavan-3-ols, notably (−)-epicatechin (EC), has been highly recommended in complementary and alternative medicine (CAM) due to reports that flavan-3-ols boost antioxidant activity, support vascular function, and prevent cardiovascular disease. To date, in vivo efficacy and mechanisms of action for many CAM therapies, including EC, remain elusive in brain ischemia. In contrast to its purported direct antioxidant role, we hypothesized protection through activation of the endogenous transcriptional factor Nrf2. To screen cellular protection and investigate Nrf2 activation, we adopted a pretreatment paradigm using enriched primary neuronal cultures from mice and washed out EC prior to oxygen glucose deprivation to attenuate direct antioxidant effects. EC protected primary neurons from oxygen glucose deprivation by increasing neuronal viability (40.2 ± 14.1%) and reducing protein oxidation, effects that occurred concomitantly with increased Nrf2-responsive antioxidant protein expression. We also utilized wildtype and Nrf2 C57BL/6 knockout mice in a permanent model of focal brain ischemia to evaluate glial cell regulation and complex sensorimotor functioning. EC-treated wildtype mice displayed a reduction or absence of forelimb motor coordination impairments that were evident in vehicle-treated mice. This protection was associated with reduced anatomical injury (54.5 ± 8.3%) and microglia/macrophage activation/recruitment (56.4 ± 13.0%). The protective effects elicited by EC in both model systems were abolished in tissues and neuronal cultures from Nrf2 knockout mice. Together, these data demonstrate EC protection through Nrf2 and extend the benefits to improved performance on a complex sensorimotor task, highlighting the potential of flavan-3-ols in CAM approaches in minimizing subsequent stroke injury.

Efficacy of prophylactic flavan-3-ol in permanent focal ischemia in 12-mo-old mice

“…EC improves anatomical and functional outcomes in aging mice. We previously documented the efficacy of EC in young, healthy animals such that a single oral dose of 15 mg/kg 90 min before pdMCAO reduced infarct volume and functional recovery in WT but not in Nrf2 Ϫ/Ϫ mice (28). Therefore, the present study was conducted to determine whether the same prophylactic dose of EC also elicits protection in the aging brains of 12-mo-old mice.…”

“…Ϫ/Ϫ mice were randomized and administered either methylcellulose vehicle (WT ϭ 8; Nrf2 ϭ 9) or 15 mg/kg of freshly prepared EC (WT ϭ 7; Nrf2 ϭ 9) (Sigma Aldrich, St. Louis, MO) by gavage 90 min before permanent distal middle cerebral artery (MCA) occlusion (pdMCAO), as previously described (28,45). The pdMCAO procedure was performed under halothane anesthesia (4% induction and 2% maintenance, 1.5/3.0% oxygen/air balance).…”

“…These natural bioactive compounds are enriched in many food sources, thus representing a safe, noninvasive therapeutic option to bolster endogenous protection against vascular injury and stroke. Indeed, studies have documented improvements in vascular function (13,52,56,60), reduced the risk for cardiovascular disease and stroke (18,19,28,45), and heightened cognition following neurodegenerative conditions (35,36).…”

“…In response to injury and oxidative and xenobiotic stress, Nrf2 regulates the production of cytoprotective proteins that protect cells from the deleterious microenvironment (31,64) which persists in various pathological states (39, 40), including stroke (26,68). Interestingly, the beneficial effects of EC and resveratrol in protecting against vascular dysfunction and cerebral ischemia have been linked to Nrf2 induction (28,29,45,62,63).…”

“…Additionally, we have recently documented Nrf2-dependent improvements in anatomical outcomes and complex sensorimotor function in young mice after permanent cerebral ischemia (28). However, no studies have evaluated the prophylactic efficacy of EC in aging mice subjected to experimental stroke.…”