Original ContributionSulforaphane preconditioning of the Nrf2/HO-1 defense pathway protects the cerebral vasculature against blood–brain barrier disruption and neurological deficits in stroke
Graphical abstract
Introduction
Stroke is one of the most common causes of adult disability and death, with about 80% of all incidents triggered by ischemic events [1]. After focal ischemia, progressive cell death occurs in the brain during the first few hours with gradual recruitment of surrounding peri-infarct regions [2]. The central core of stroke-affected areas is subjected to a marked reduction in oxygen levels and blood flow as well as rapid necrosis, whereas peripheral areas exhibiting modest or moderate oxygen and perfusion deficits are potentially salvageable. These areas, at risk of infarction but not yet irreversibly damaged, form the ischemic penumbra and are the current focus of recanalization and neuroprotective therapies in stroke [3].
Microvascular endothelial cells constitute the blood–brain barrier (BBB) and surrounding astrocytic end-feet regulate barrier permeability, transport, and metabolic functions [4]. The structural and functional relationship between endothelial cells and astrocytes in the brain has been defined as the gliovascular complex [5], and reactive oxygen and nitrogen species function as triggers of BBB breakdown and cerebral edema, the major pathogenic mechanism leading to death after stroke [6], [7].
Heme oxygenases (HO) provide important endogenous defenses against oxidative injury in the brain during ischemia and inflammation [8], [9]. Delivery of the HO-1 gene via adenoviral transfection or dexamethasone-loaded peptide vesicles [10], [11] protects the brain against ischemic injury. Moreover, upregulation of HO-1 in the brain following ischemic preconditioning only protects against ischemic injury in wild-type but not HO-1–/– mice [12] while treatment with the HO-1 inhibitor ZnPP attenuates protection against ischemic brain injury [13]. Constitutive HO-2 and inducible HO-1 are rate-limiting enzymes in the oxidative degradation of pro-oxidant heme to the vasodilator carbon monoxide (CO), free iron, biliverdin, and the chain-breaking antioxidant bilirubin [8], [14], [15]. Intracellular generation of CO and/or bilirubin via heme oxygenases protects peripheral tissues and the brain against oxidative stress [16], [17]. Notably, low doses of CO protect against focal transient cerebral ischemia [18] and pretreatment of rats with CORM-3, a water-soluble CO-releasing molecule, reduces brain injury following hemorrhagic stroke [19].
A number of different cellular stressors induce HO-1 expression via redox-sensitive transcription factors, including nuclear factor erythroid 2-related factor 2 (Nrf2) [20], [21]. Nrf2 is a member of the cap ‘n' collar family and is normally targeted for proteasomal degradation via its cytosolic binding protein Kelch-like ECH associated protein 1 (Keap1) [22], [23], [24]. Under conditions of oxidative, nitrosative or electrophilic stress Nrf2 accumulates in the nucleus and together with small Maf proteins binds to antioxidant response element (ARE) regions in the promoter of antioxidant proteins such as HO-1 and Nrf2 itself [25], [26].
HO-1 expression is induced in the brain of rats following hypoxia and ischemia–reperfusion injury [27], [28], yet little is known about expression of Nrf2 and heme oxygenases in the gliovascular complex of cerebral microvessels following stroke. Although there are limited reports of HO-1 detection in blood vessels of the infarct region after global cerebral ischemia in rats [29] and constitutive HO-2 expression in vessels of naïve brain [27], to our knowledge there are no reported studies of the temporal or spatial distribution of Nrf2, HO-1, and HO-2 between endothelial cells and perivascular astrocytes in stroke.
In the present study, we used an experimental model of ischemic stroke to monitor the time course of Nrf2 and HO-1 induction in core and peri-infarct regions of stroke-affected and contralateral hemispheres of the rat brain after ischemia–reperfusion injury. HO-1 and Nrf2 expression in the gliovascular complex of cerebral microvessels was correlated with levels of nitrosative stress, BBB disruption, lesion progression (MRI), and functional behavioral outcomes. Pretreatment with sulforaphane, a dietary isothiocyanate and well-characterized inducer of Nrf2 and HO-1 expression [30], upregulated antioxidant defenses in the brain and significantly attenuated functional and behavioral deficits after stroke.
Section snippets
Material and methods
All general consumables were purchased from Sigma, unless otherwise stated.
Temporal and spatial induction of HO-1 after cerebral ischemia–reperfusion
Ischemia–reperfusion induced substantial brain injury (Supplementary Fig. 1) and increased HO-1 protein expression in the ipsilateral stroke-affected hemisphere (Fig. 1), whereas HO-1 immunoreactivity was low in the contralateral unaffected hemisphere, in sham and naïve brains (data not shown). A zone of HO-1 induction surrounding the infarct core was observed after 24 h reperfusion, as highlighted by GFAP costaining, while after 72 h HO-1 was detected in both peri-infarct and infarct core
Discussion
In this study, we report the first temporal and spatial distribution of Nrf2 and HO-1 in the gliovascular complex of cerebral microvessels following ischemic stroke, and demonstrate that preactivation of the Nrf2 pathway in perivascular astrocytes and brain endothelium before stroke with sulforaphane significantly ameliorates BBB disruption, lesion progression, and neurological dysfunction. We further established that induction of Nrf2 and HO-1 in the brain is associated with moderate
Conclusions
Due to the pivotal role played by oxidative stress in the early phases following cerebral ischemia, antioxidants have been widely regarded as protective agents against stroke [72]. Nevertheless, experimental and clinical studies have provided conflicting results on the efficacy of antioxidants, e.g., vitamins and ROS scavengers, to prevent stroke incidence or mortality [73]. Importantly, exogenous antioxidants and ROS scavengers may inhibit physiological redox signaling pathways and the
Acknowledgments
This work was supported by the Henry Smith Charity, UK (RG20092511), and British Heart Foundation (FS/09/056). We also thank the British Heart Foundation for funding the purchase of the 7 T MRI scanner used in this study.
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