The role of Nrf2 in protection of the neurovascular unit following stroke

Student thesis: Doctoral ThesisDoctor of Philosophy


Cerebral stroke contributes to approximately 10% of deaths worldwide and is the most common cause of adult morbidity. The cost of stroke to the UK is approximately £8.9 billion per annum, of which 5% accounts for costs incurred by the National Health Service. About 85% of cerebral stroke patients experience cerebral ischaemia-reperfusion injury, with recombinant tissue-plasminogen activator (rt-PA) being the only approved form of treatment. rtPA treatment is only efficacious within in a limited time window (3 – 4.5h) after the onset of ischaemia, however, it is coupled with severe side-effects. This thus warrants the requirement of further research into improved therapeutic strategies to protect the brain against ischaemia-induced damage.

Accumulating evidence implicates a major role for oxidative stress in the pathophysiology of stroke, resulting in the loss of cells that form the neurovascular unit. Research currently focuses on protecting astrocytes and neurons, but has failed to identify the need for protecting of the cerebrovascular endothelium, important for the maintenance of brain function and environment. The redox sensitive transcription factor NF-E2 related factor 2 (Nrf2) orchestrates an adaptive response in upregulating phase II detoxifying enzymes and antioxidant stress proteins. However, little is known about the temporal and spatial distribution of Nrf2 in different brain cell types after cerebral ischaemia and reperfusion injury. Furthermore, post-stroke administration of the dietary isothiocyanate sulforaphane (SFN), an Nrf2 inducer, has been shown to protect the ischaemic brain. However, the effects of sulforaphane pre-conditioning in vivo remain to be elucidated.

Male Sprague-Dawley rats (260 – 300g) were subjected to 70 min middle cerebral artery occlusion (MCAo) and reperfused for 4, 24 or 72h. Paraformaldehyde perfused-fixed brains were then excised and cryosectioned to obtain 10µm coronal brain sections. Total cellular and nuclear/cytoplasmic Nrf2 content was calculated using a novel quantitative immunohistochemical technique, based on the initial rate of 3,3’-diaminobenzidine (DAB) polymer formation, following the reaction of DAB with H2O2. Cytoplasmic Nrf2 content increased in cerebral cells after 4h reperfusion injury, whereas a significant increase in nuclear content was observed after 24h reperfusion injury. Total cellular Nrf2 content was also greater in stroke-affected regions 24h after 70 min MCAo and decreased in all regions of the ischaemic brain after 72h. To determine the effects of sulforaphane (SFN) treatment on Nrf2 in vivo, rats were administered SFN (5 mg/kg. i.p.) for 1, 2, 4 and 24h, or pre-treated with SFN 1h prior to 70 min MCAo and 24h reperfusion injury. SFN pre-treatment significantly reduced Nrf2 protein content in rats after 24h reperfusion injury. Furthermore, SFN treatment of naïve rats revealed a significant increase in Nrf2 protein content within 1h of administration.

To further elucidate the role of Nrf2 mediated protection in the cerebral endothelium, the mouse brain endothelial cell line, bEnd.3, was used as an in vitro model of the blood-brain barrier (BBB). SFN time- (4 – 24 h) and dose-dependently (0.5 – 2.5 µM) increased protein expression of HO-1 and NQO1, two Nrf2-regulated proteins. Furthermore, SFN (2.5µM) also induced Nrf2 nuclear translocation, 1 – 4h after treatment. bEnd.3 cells deprived of oxygen and glucose, as an in vitro model of cerebral ischaemia, revealed a time dependent increase in cell death, which was attenuated in cells pre-treated with SFN (2.5µM) for 12h.

This study thus reports the first quantitative analysis of the temporal and spatial distribution of Nrf2 following ischaemia and reperfusion injury. We provide evidence that SFN administration in vivo rapidly increases expression of Nrf2 and its target enzymes, and that pre-treatment protects the brain against oxygen-glucose deprivation. Our in vitro studies revealed SFN stimulates Nrf2-mediated HO-1 and NQO1 expression, conferring protection to the brain endothelium against ischaemia-induced damage. Thus, SFN pre-conditioning may provide a potential therapeutic strategy to limit BBB permeability and associated neurological deficits after cerebral stroke.
Date of Award1 Sept 2014
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorGiovanni Mann (Supervisor), Paul Fraser (Supervisor) & Richard Siow (Supervisor)

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