Cerebral Endothelium Research Articles

Interferon-γ Safeguards Blood-Brain Barrier during Experimental Autoimmune Encephalomyelitis

The function of blood-brain barrier is often disrupted during the progression of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the molecular mechanism of blood-brain barrier modulation during neuroinflammation remains unclear. Herein, we show that the expression of interferon-γ (IFNγ) receptor on endothelial cells (ECs) protected mice from the brain inflammation during EAE. IFNγ stabilized the integrity of the cerebral endothelium and prevented the infiltration of leukocytes into the brain. Further analysis revealed that IFNγ increased the expression of tight junction proteins zonula occludens protein 1 and occludin, as well as membranous distribution of claudin-5, in brain ECs. Silencing claudin-5 abolished the IFNγ-mediated improvement of EC integrity. Taken together, our results show that IFNγ, a pleiotropic proinflammatory cytokine, stabilizes blood-brain barrier integrity and, therefore, prevents brain inflammation during EAE.

Cerebral vasculopathy in children with sickle cell anemia.

Sickle cell anemia (SCA)-associated cerebral vasculopathy and moyamoya is a unique entity reflecting the abnormal interactions between sickled red blood cells (RBCs) and the cerebral arterial endothelium. Endothelial injury, coagulation activation, and the inflammatory response generated by sickled RBCs are implicated in the development of cerebral vasculopathy, but the pathophysiology remains incompletely understood. SCA-specific screening and treatment guidelines have successfully reduced the incidence of overt strokes in this high-risk population. However, despite aggressive hematological management, many children with cerebral vasculopathy due to SCA have progressive vasculopathy and recurrent strokes; therefore, more effective therapies, such as revascularization surgery and curative hematopoietic stem cell transplant, are urgently needed.

Role of soluble epoxide hydrolase in age-related vascular cognitive decline

P450 eicosanoids are important regulators of the cerebral microcirculation, but their role in cerebral small vessel disease is unclear. We tested the hypothesis that vascular cognitive impairment (VCI) is linked to reduced cerebral microvascular eicosanoid signaling. We analyzed human brain tissue from individuals formerly enrolled in the Oregon Brain Aging Study, who had a history of cognitive impairment histopathological evidence of microvascular disease. VCI subjects had significantly higher lesion burden both on premortem MRI and postmortem histopathology compared to age- and sex-matched controls. Mass spectrometry-based eicosanoid analysis revealed that 14,15-dihydroxyeicosatrienoic acid (DHET) was elevated in cortical brain tissue from VCI subjects. Immunoreactivity of soluble epoxide hydrolase (sEH), the enzyme responsible for 14,15-DHET formation, was localized to cerebral microvascular endothelium, and was enhanced in microvessels of affected tissue. Finally, we evaluated the genotype frequency of two functional single nucleotide polymorphisms of sEH gene EPHX2 in VCI and control groups. Our findings support a role for sEH and a potential benefit from sEH inhibitors in age-related VCI.

Chronic aldosterone administration causes Nox2-mediated increases in reactive oxygen species production and endothelial dysfunction in the cerebral circulation.

An elevated plasma aldosterone level is an independent cardiovascular risk factor. Although excess aldosterone promotes cardiovascular disease, no studies have examined the effect of increased plasma aldosterone on the cerebral circulation. A major source of vascular reactive oxygen species (ROS) during cardiovascular disease is the NADPH oxidases. Because Nox2-containing NADPH oxidase (Nox2 oxidase) is highly expressed in the cerebral endothelium, we postulated that it might contribute to ROS generation and vascular dysfunction in response to aldosterone. Here, we examined the effect of aldosterone and Nox2 oxidase on ROS production and endothelial dysfunction in the cerebral circulation, and whether the effects of aldosterone are exacerbated in aged mice. In adult (average age ∼24-25 weeks) wild-type and Nox2-deficient (Nox2(/y)) mice, neither vehicle nor aldosterone (0.28 mg/kg per day for 14 days) affected blood pressure (measured using tail-cuff). By contrast, aldosterone treatment reduced dilation of the basilar artery (measured using myography) to the endothelium-dependent agonist acetylcholine in wild-type mice (P < 0.05), but had no such effect in Nox2(/y) mice (P > 0.05). Aldosterone increased basal and phorbol dibutyrate-stimulated superoxide production (measured using L-012-enhanced chemiluminesence) in cerebral arteries from wild-type but not from Nox2(/y) mice. In aged wild-type mice (average age ∼70 weeks), aldosterone treatment increased blood pressure, but had a similar effect on cerebral artery superoxide levels as in adult wild-type mice. These data indicate that Nox2 oxidase mediates aldosterone-induced increases in ROS production and endothelial dysfunction in cerebral arteries from adult mice independently of blood pressure changes. Aldosterone-induced hypertension is augmented during aging.

Microvascular pathology and morphometrics of sporadic and hereditary small vessel diseases of the brain.

Small vessel diseases (SVDs) of the brain are likely to become increasingly common in tandem with the rise in the aging population. In recent years, neuroimaging and pathological studies have informed on the pathogenesis of sporadic SVD and several single gene (monogenic) disorders predisposing to subcortical strokes and diffuse white matter disease. However, one of the limitations toward studying SVD lies in the lack of consistent assessment criteria and lesion burden for both clinical and pathological measures. Arteriolosclerosis and diffuse white matter changes are the hallmark features of both sporadic and hereditary SVDs. The pathogenesis of the arteriopathy is the key to understanding the differential progression of disease in various SVDs. Remarkably, quantification of microvascular abnormalities in sporadic and hereditary SVDs has shown that qualitatively the processes involved in arteriolar degeneration are largely similar in sporadic SVD compared with hereditary disorders such as cerebral autosomal arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Important significant regional differences in lesion location within the brain may enable one to distinguish SVDs, where frontal lobe involvement appears consistently with almost every SVD, but others bear specific pathologies in other lobes, such as the temporal pole in CADASIL and the pons in pontine autosomal dominant microangiopathy and leukoencephalopathy or PADMAL. Additionally, degenerative changes in the vascular smooth muscle cells, the cerebral endothelium and the basal lamina are often rapid and more aggressive in genetic disorders. Further quantification of other microvascular elements and even neuronal cells is needed to fully characterize SVD pathogenesis and to differentiate the usefulness of vascular interventions and treatments on the resulting pathology.

Cerebral malaria: gamma-interferon redux.

There are two theories that seek to explain the pathogenesis of cerebral malaria, the mechanical obstruction hypothesis and the immunopathology hypothesis. Evidence consistent with both ideas has accumulated from studies of the human disease and experimental models. Thus, some combination of these concepts seems necessary to explain the very complex pattern of changes seen in cerebral malaria. The interactions between malaria parasites, erythrocytes, the cerebral microvascular endothelium, brain parenchymal cells, platelets and microparticles need to be considered. One factor that seems able to knit together much of this complexity is the cytokine interferon-gamma (IFN-γ). In this review we consider findings from the clinical disease, in vitro models and the murine counterpart of human cerebral malaria in order to evaluate the roles played by IFN-γ in the pathogenesis of this often fatal and debilitating condition.

Erythropoietin increases bioavailability of tetrahydrobiopterin and protects cerebral microvasculature against oxidative stress induced by eNOS uncoupling.

This study was designed to determine whether treatment with erythropoietin (EPO) could protect cerebral microvasculature against the pathological consequences of endothelial nitric oxide (NO) synthase uncoupling. Wild-type and GTP cyclohydrolase I (GTPCH-I)-deficient hph1 mice were administered EPO (1000 U/kg/day, s.c., 3days). Cerebral microvessels of hph1 mice demonstrated reduced tetrahydrobiopterin (BH4) bioavailability, increased production of superoxide anions and impaired endothelial NO signaling. Treatment of hph1 mice with EPO attenuated the levels of 7,8-dihydrobiopterin, the oxidized product of BH4, and significantly increased the ratio of BH4 to 7,8-dihydrobiopterin. Moreover, EPO decreased the levels of superoxide anions and increased NO bioavailability in cerebral microvessels of hph1 mice. Attenuated oxidation of BH4 and inhibition of endothelial NO synthase uncoupling were explained by the increased expression of antioxidant proteins, manganese superoxide dismutase, and catalase. The protective effects of EPO observed in cerebral microvessels of hph1 mice were also observed in GTPCH-I siRNA-treated human brain microvascular endothelial cells exposed to EPO (1U/mL or 10U/mL; 3days). Our results suggest that EPO might protect the neurovascular unit against oxidative stress by restoring bioavailability of BH4 and endothelial NO in the cerebral microvascular endothelium. We demonstrate that treatment with erythropoietin (EPO) could protect cerebral microvasculature against the pathological consequences of endothelial nitric oxide (NO) synthase uncoupling. Our results suggest that EPO might protect the neurovascular unit against oxidative stress by restoring bioavailability of tetrahydrobiopterin (BH4) and endothelial nitric oxide.

Oligodendrocyte precursor cells support blood-brain barrier integrity via TGF-β signaling.

Trophic coupling between cerebral endothelium and their neighboring cells is required for the development and maintenance of blood-brain barrier (BBB) function. Here we report that oligodendrocyte precursor cells (OPCs) secrete soluble factor TGF-β1 to support BBB integrity. Firstly, we prepared conditioned media from OPC cultures and added them to cerebral endothelial cultures. Our pharmacological experiments showed that OPC-conditioned media increased expressions of tight-junction proteins and decreased in vitro BBB permeability by activating TGB-β-receptor-MEK/ERK signaling pathway. Secondly, our immuno-electron microscopic observation revealed that in neonatal mouse brains, OPCs attach to cerebral endothelial cells via basal lamina. And finally, we developed a novel transgenic mouse line that TGF-β1 is knocked down specifically in OPCs. Neonates of these OPC-specific TGF-β1 deficient mice (OPC-specific TGF-β1 partial KO mice: PdgfraCre/Tgfb1flox/wt mice or OPC-specific TGF-β1 total KO mice: PdgfraCre/Tgfb1flox/flox mice) exhibited cerebral hemorrhage and loss of BBB function. Taken together, our current study demonstrates that OPCs increase BBB tightness by upregulating tight junction proteins via TGF-β signaling. Although astrocytes and pericytes are well-known regulators of BBB maturation and maintenance, these findings indicate that OPCs also play a pivotal role in promoting BBB integrity.

Differential effects of indoxyl sulfate and inorganic phosphate in a murine cerebral endothelial cell line (bEnd.3).

Endothelial dysfunction plays a key role in stroke in chronic kidney disease patients. To explore the underlying mechanisms, we evaluated the effects of two uremic toxins on cerebral endothelium function. bEnd.3 cells were exposed to indoxyl sulfate (IS) and inorganic phosphate (Pi). Nitric oxide (NO), reactive oxygen species (ROS) and O2•– were measured using specific fluorophores. Peroxynitrite and eNOS uncoupling were evaluated using ebselen, a peroxide scavenger, and tetrahydrobiopterin (BH4), respectively. Cell viability decreased after IS or Pi treatment (p < 0.01). Both toxins reduced NO production (IS, p < 0.05; Pi, p < 0.001) and induced ROS production (p < 0.001). IS and 2 mM Pi reduced O2•– production (p < 0.001). Antioxidant pretreatment reduced ROS levels in both IS- and Pi-treated cells, but a more marked reduction of O2•– production was observed in Pi-treated cells (p < 0.001). Ebselen reduced the ROS production induced by the two toxins (p < 0.001); suggesting a role of peroxynitrite in this process. BH4 addition significantly reduced O2•– and increased NO production in Pi-treated cells (p < 0.001), suggesting eNOS uncoupling, but had no effect in IS-treated cells. This study shows, for the first time, that IS and Pi induce cerebral endothelial dysfunction by decreasing NO levels due to enhanced oxidative stress. However, Pi appears to be more deleterious, as it also induces eNOS uncoupling.

Endothelial progenitor cells augment collateralization and hemodynamic rescue in a model of chronic cerebral ischemia.

Surgical flow augmentation for treatment of cerebral hemodynamic impairment remains controversial. Here, we investigated the benefit of endothelial progenitor cell (EPC) treatment in a rat model of chronic cerebral hypoperfusion. At repeated time points after 3-vessel occlusion (3-VO), animals were treated with 1 × 10(6) DiI-labeled (a) ex vivo-expanded embryonic-EPC (e-EPC), (b) cyclic AMP-differentiated embryonic-endothelial progenitor-derived cells (e-EPDC as biologic control) or, (c) saline. The cerebrovascular reserve capacity (CVRC) was assessed immediately before and on days 7 and 21 after 3-VO. Structural effects were assessed by latex perfusion, immunohistochemistry, and intravital fluorescence video microscopy on day 21. Three-vessel occlusion resulted in a significant impairment of the CVRC with better functional recovery after treatment with e-EPC (16.4±8%) compared with e-EPDC (3.7±8%) or saline (6.4±9%) by day 21 (P<0.05), which was paralleled by a significant increase in the vessel diameters of the anterior Circle of Willis, a significantly higher number of leptomeningeal anastomoses and higher parenchymal capillary density in e-EPC-treated animals. Interestingly, despite in vivo interaction of e-EPC with the cerebral endothelium, e-EPC incorporation into the cerebral vasculature was not observed. Our results suggest that EPC may serve as a novel therapeutic agent in clinical trials for nonsurgical treatment of chronic cerebral hemodynamic impairment.

TRPA1 mediates NADPH oxidase‐dependent cerebral artery dilation (1079.1)

Activation of the Ca2+‐permeable transient receptor potential (TRP) ankyrin (A) channel TRPA1 in the cerebral artery endothelium with electrophilic compounds induces dilation. Endogenous agonists for endothelial TRPA1 have not been reported. We hypothesized that H2O2 is an endogenous agonist for TRPA1 in this tissue. H2O2 can be generated by NADPH oxidase (NOX) activity via production of a O2‐ intermediate or directly by NOX isoform NOX4. We find that activation of NOX with its substrate, NADPH, increases TRPA1 activity and induces dilation of pressurized cerebral arteries (80 mmHg) that is blocked by TRPA1 inhibitor HC‐030031 (HC) and H2O2 degradation by catalase. In the presence of iron, H2O2 is converted to highly reactive oxygen radicals, such as OH•, by the Fenton reaction. To distinguish between direct effects of H2O2 vs. those of OH• or OH• metabolites we inhibited the Fenton reaction by chelating iron with deferoxamine. Deferoxamine attenuated NADPH‐induced TRPA1 activation and vasodilation, suggesting that OH• formation is required for NOX‐dependent TRPA1 activation. OH• peroxidizes membrane lipids yielding lipid peroxidation products (LPP). The LPP 4‐hydroxynonenal increased TRPA1 activity and induced cerebral artery dilation that was blocked by HC. Together, these data suggest that NOX‐dependent LPP generation activates TRPA1 in cerebral artery endothelial cells to induce vasodilation.Grant Funding Source: Supported by HL091905

PACAP Enhances Barrier Properties of Cerebral Microvessels

Cerebral microvascular endothelial cells-coming in contact with pericytes and astrocytes-constitute the structural basis of the blood-brain barrier (BBB). The continuous belt of interendothelial tight junctions (TJs) and the presence of specific transport systems, enzymes, and receptors in the brain endothelium regulate the molecular and cellular traffic into the central nervous system. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide having several cellular protective effects. However, little is known about the effects of PACAP on the cerebral endothelium and BBB functions. Here, we show that PACAP has no significant pro-survival role in cerebral microvascular endothelial cells; however, it improves the barrier properties of the brain endothelium. PACAP induces an increase in the transendothelial electrical resistance, which is the most important marker of the tightness of the TJs. Moreover, PACAP has a protective role against glucose deprivation- and oxidative stress-induced junctional damage in microvascular brain endothelial cells.

Abstract 133: Inhibition of TRPV4 is Protective of the Brain and Cerebral Circulation During Ischemic Stroke

Background: Transient receptor potential vanilloid 4 (TRPV4) channels are highly calcium permeable channels expressed in brain and cerebral endothelium and are regulated by a diverse array of stimuli including shear stress, cell swelling and second messengers. We hypothesized that inhibition of TRPV4 during postischemic reperfusion would be protective of the brain and cerebral endothelium. Methods: Transient MCAO was used for all experiments. Infarction (by TTC) was measured in TRPV4 knockout mice (n=3) or C57B mice treated upon reperfusion with the TRPV4 inhibitor HC067047 (1mg/kg; n=7) or vehicle (n=8) after 90 minutes of ischemia with 24 hours of reperfusion. To determine the effect of TRPV4 inhibition on BBB permeability, hyperglycemic male Wistar rats (3 days by STZ) were treated with HC067047 (1mg/kg; n=6) or vehicle (n=6) upon reperfusion after 2 hours of ischemia and edema measured at 2 hours by wet:dry weights. Lastly, basal tone of MCAs isolated from male Wistar rats after 2 hours of ischemia with 2 hours of reperfusion (MCAO, n=7) or sham controls (Sham, n=6) was measured before and after addition of HC067047 (1μM) at 75 mmHg. Results: Infarct volume was significantly reduced in TRPV4 knockout mice and with HC067047 treatment vs. vehicle (17.8 ± 2.8% for KO, 22.9 ± 2.7% for HC067047 vs. 39.8 ± 3.3% for vehicle; p&lt;0.05). Hyperglycemic stroke caused considerable edema formation the ipsilateral vs. contralateral brain in vehicle-treated rats (81.38 ± 0.21% vs. 78.64 ± 0.12%; p&lt;0.01) that was prevented by treatment with HC067047 (78.58 ± 0.22% vs. 78.24 ± 0.11%;p&gt;0.05). MCAO diminished basal tone of MCAs vs. sham (16.3 ± 3.3 vs. 26.4 ± 2.8%; p&lt;0.05) that was restored by HC067047 (25.9 ± 3.0%; p&lt;0.05). The constriction to HC067047 did not occur in MCA that were denuded of endothelium or from sham animals. Conclusion: These results suggest that postischemic reperfusion activates TRPV4 in cerebral endothelium to cause dilation and BBB permeability. The decreased infarct size after TRPV4 inhibition suggests that activation of TRPV4 during ischemia contributes to neuronal cell death. Thus, inhibition of TRPV4 may be an effective treatment for acute ischemic stroke.

Cerebral endothelial function determined by cerebrovascular reactivity to L-arginine.

Endothelium forms the inner cellular lining of blood vessels and plays an important role in many physiological functions including the control of vasomotor tone. Cerebral endothelium is probably one of the most specific types but until recently it was impossible to determine its function. In this review, the role of cerebrovascular reactivity to L-arginine (CVR-L-Arg) for assessment of cerebral endothelial function is discussed. L-Arginine induces vasodilatation through enhanced production of nitric oxide (NO) in the cerebral endothelium. Transcranial Doppler sonography is used for evaluation of cerebral blood flow changes. The method is noninvasive, inexpensive, and enables reproducible measurements. CVR-L-Arg has been compared to flow-mediated dilatation as a gold standard for systemic endothelial function and intima-media thickness as a marker for morphological changes. However, it seems to show specific cerebral endothelial function. So far CVR-L-Arg has been used to study cerebral endothelial function in many pathological conditions such as stroke, migraine, etc. In addition CVR-L-Arg has also proven its usefulness in order to show potential improvement after pharmacological interventions. In conclusion CVR-L-Arg is a promising noninvasive research method that could provide means for evaluation of cerebral endothelial function in physiological and pathological conditions.

Reactive astrocytes promote adhesive interactions between brain endothelium and endothelial progenitor cells via HMGB1 and beta-2 integrin signaling

Endothelial progenitor cells (EPCs) may contribute to neurovascular repair after stroke and neurodegeneration. A key step in this process should involve adhesive interactions between EPCs and the targeted cerebral endothelium. Here, we tested the hypothesis that reactive astrocytes may play a critical role in enhancing adhesive interactions and transmigration of EPCs across cerebral endothelial cells. Transiently seeding EPCs onto a monolayer of RBE.4 rat brain endothelial cells resulted in a time-dependent adherence between the two cell types. Blocking β2 integrins on EPCs or blocking the receptor for advanced glycation endproducts (RAGE) on endothelial cells significantly decreased EPC-endothelial adherence. Next, we tested whether reactive astrocytes can enhance this process by growing EPCs, brain endothelial cells and astrocytes together in a transwell co-culture system. The presence of reactive astrocytes in the lower chamber significantly promoted adherence between EPCs and endothelial cells in the upper chamber. This process involved the release of soluble HMGB1 from reactive astrocytes that then upregulated endothelial expression of RAGE via Egr1 signaling. Directly adding HMGB1 to the transwell system also promoted EPC-endothelial adhesion and accelerated EPC transmigration into the lower chamber. These initial findings provide proof-of-concept that reactive astrocytes promote crosstalk between cerebral endothelium and EPCs. Further investigation of this phenomenon may lead to a better understanding of cell–cell interactions required for neurovascular recovery after stroke.

Crosstalk between cerebral endothelium and oligodendrocyte

It is now relatively well accepted that the cerebrovascular system does not merely provide inert pipes for blood delivery to the brain. Cerebral endothelial cells may compose an embedded bunker of trophic factors that contribute to brain homeostasis and function. Recent findings suggest that soluble factors from cerebral endothelial cells nourish neighboring cells, such as neurons and astrocytes. Although data are strongest in supporting mechanisms of endothelial-neuron and/or endothelial-astrocyte trophic coupling, it is likely that similar interactions also exist between cerebral endothelial cells and oligodendrocyte lineage cells. In this mini-review, we summarize current advances in the field of endothelial-oligodendrocyte trophic coupling. These endothelial-oligodendrocyte interactions may comprise the oligovascular niche to maintain their cellular functions and sustain ongoing angiogenesis/oligodendrogenesis. Importantly, it should be noted that the cell-cell interactions are not static-the trophic coupling is disturbed under acute phase after brain injury, but would be recovered in the chronic phase to promote brain remodeling and repair. Oligodendrocyte lineage cells play critical roles in white matter function, and under pathological conditions, oligodendrocyte dysfunction lead to white matter damage. Therefore, a deeper understanding of the mechanisms of endothelial-oligodendrocyte trophic coupling may lead to new therapeutic approaches for white matter-related diseases, such as stroke or vascular dementia.

Aetiopathogenesis of cerebral ischemia in young Turkish patients

Aetiopathogenesis of cerebral ischemia in young Turkish patients

P-Selectin-Mediated Monocyte–Cerebral Endothelium Adhesive Interactions Link Peripheral Organ Inflammation To Sickness Behaviors

Sickness behaviors, such as fatigue, mood alterations, and cognitive dysfunction, which result from changes in central neurotransmission, are prevalent in systemic inflammatory diseases and greatly impact patient quality of life. Although, microglia (resident cerebral immune cells) and cytokines (e.g., TNFα) are associated with changes in central neurotransmission, the link between peripheral organ inflammation, circulating cytokine signaling, and microglial activation remains poorly understood. Here we demonstrate, using cerebral intravital microscopy, that in response to liver inflammation, there is increased monocyte specific rolling and adhesion along cerebral endothelial cells (CECs). Peripheral TNFα-TNFR1 signaling and the adhesion molecule P-selectin are central mediators of these monocyte-CEC adhesive interactions which were found to be closely associated with microglial activation, decreased central neural excitability and sickness behavior development. Similar monocyte-CEC adhesive interactions were also observed in another mouse model of peripheral organ inflammation (i.e., 2,4-dinitrobenzene sulfonic acid-induced colitis). Our observations provide a clear link between peripheral organ inflammation and cerebral changes that impact behavior, which can potentially allow for novel therapeutic interventions in patients with systemic inflammatory diseases.

Sulforaphane preconditioning of the Nrf2/HO-1 defense pathway protects the cerebral vasculature against blood–brain barrier disruption and neurological deficits in stroke

Disruption of the blood–brain barrier (BBB) and cerebral edema are the major pathogenic mechanisms leading to neurological dysfunction and death after ischemic stroke. The brain protects itself against infarction via activation of endogenous antioxidant defense mechanisms, and we here report the first evidence that sulforaphane-mediated preactivation of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream target heme oxygenase-1 (HO-1) in the cerebral vasculature protects the brain against stroke. To induce ischemic stroke, Sprague-Dawley rats were subjected to 70min middle cerebral artery occlusion (MCAo) followed by 4, 24, or 72h reperfusion. Nrf2 and HO-1 protein expression was upregulated in cerebral microvessels of peri-infarct regions after 4–72h, with HO-1 preferentially associated with perivascular astrocytes rather than the cerebrovascular endothelium. In naïve rats, treatment with sulforaphane increased Nrf2 expression in cerebral microvessels after 24h. Upregulation of Nrf2 by sulforaphane treatment prior to transient MCAo (1h) was associated with increased HO-1 expression in perivascular astrocytes in peri-infarct regions and cerebral endothelium in the infarct core. BBB disruption, lesion progression, as analyzed by MRI, and neurological deficits were reduced by sulforaphane pretreatment. As sulforaphane pretreatment led to a moderate increase in peroxynitrite generation, we suggest that hormetic preconditioning underlies sulforaphane-mediated protection against stroke. In conclusion, we propose that pharmacological or dietary interventions aimed to precondition the brain via activation of the Nrf2 defense pathway in the cerebral microvasculature provide a novel therapeutic approach for preventing BBB breakdown and neurological dysfunction in stroke.

Abstract 351: Mineralocorticoid Receptor Antagonism During the Development of Hypertension Improves Cerebral Artery Endothelium Dependent Dilation Even After Treatment Withdrawal

Hypertension causes cerebral artery remodeling and increases the risk of stroke. Renin angiotensin system blockade during the development of hypertension has therapeutic effects even after treatment withdrawal. Mineralocorticoid receptor (MR) activation has been implicated in artery remodeling and impaired endothelial function. The possibility that there is a critical therapeutic window for MR antagonism has not been investigated. We hypothesized that temporary MR antagonism while hypertension develops would improve endothelium dependent dilation even after treatment withdrawal. Six-week-old male stroke prone spontaneously hypertensive rats (SHRSP) were treated with spironolactone (Spir; 25mg/kg/day, n=8) from 6-12 weeks then aged to 18 weeks. Age-matched untreated SHRSP were controls (n=8). The middle cerebral artery (MCA) was removed and mounted on a pressure myograph, endothelium dependent dilation was assessed by intralumenal perfusion of 2-methylthioadenosine 5′-triphosphate (2-MeS-ATP), or uridine 5′-triphosphate (UTP). Spir had no effect on blood pressure. The MCA diameter after the development of tone was not different between the groups (197.7 ± 8.7 vs. 199.1 ± 4.1 μm, Spir vs. control). Spir treatment increased nitric oxide (NO) mediated MCA dilation in response to 2-MeS-ATP (% change from baseline; 50.9 ± 5.0 vs. 28.0 ± 4.3, Spir vs. control, p&lt;0.05 ANOVA). Spir treatment also increased UTP mediated dilation (% change from baseline; 48.4 ± 6.0 vs. 37.4 ± 6.7, Spir vs. control, p&lt;0.05 ANOVA), and increased the MCAs sensitivity to UTP as evidenced by a reduction in the Log EC50 for UTP (-9.2 ±0.1 vs. -8.7±0.2 Spir vs. control, p&lt;0.05 t-test). There was no difference in the response to sodium nitroprusside between the two groups (% change from baseline; 42.5 ± 7.2 vs. 47.0 ± 7.6, Spir vs. control). These results suggest that MR antagonism improves both NO and endothelium derived relaxing factor mediated dilation in MCAs when the antagonist is administered while hypertension develops. Intriguingly, the improved endothelial function persists even after several weeks of treatment withdrawal suggesting there may be a critical time window during which endothelial function can be permanently improved in cerebral arteries from SHRSP.