Catherine M. Davis

Mouse Schwann Cells Need Both NRG1 and Cyclic AMP to Myelinate

Genetically modified mice have been a major source of information about the molecular control of Schwann‐cell myelin formation, and the role of β‐neuregulin 1 (NRG1) in this process in vivo. In vitro, on the other hand, Schwann cells from rats have been used in most analyses of the signaling pathways involved in myelination. To correlate more effectively in vivo and in vitro data, we used purified cultures of mouse Schwann cells in addition to rat Schwann cells to examine two important myelin‐related signals, cyclic adenosine monophosphate (cAMP), and NRG1 and to determine whether they interact to control myelin differentiation. We find that in mouse Schwann cells, neither cAMP nor NRG1, when used separately, induced markers of myelin differentiation. When combined, however, they induced strong protein expression of the myelin markers, Krox‐20 and P0. Importantly, the level of cAMP signaling was crucial in switching NRG1 from a proliferative signal to a myelin differentiation signal. Also in cultured rat Schwann cells, NRG1 promoted cAMP‐induced Krox‐20 and P0 expression. Finally, we found that cAMP/NRG1‐induced Schwann‐cell differentiation required the activity of the cAMP response element binding family of transcription factors in both mouse and rat cells. These observations reconcile observations in vivo and on neuron‐Schwann‐cell cultures with studies on purified Schwann cells. They demonstrate unambiguously the promyelin effects of NRG1 in purified cells, and they show that the cAMP pathway determines whether NRG1 drives proliferation or induces myelin differentiation.

Mechanism of the sex difference in neuronal ischemic cell death

BACKGROUND Stroke risk and outcome are different in men and women. We hypothesized that this is partly due to an inherent difference in susceptibility to ischemia between neurons from male vs. female brains. We tested whether neurons from male rodents are more susceptible to in-vitro ischemia than cells from females, and if this is related to increased expression of soluble epoxide hydrolase (sEH). sEH contributes to neuronal cell death by inactivating neuroprotective epoxyeicosatrienoic acids (EETs). METHODS Rodent cortical neurons were cultured, and exposed to oxygen-glucose deprivation (OGD); then cell death was measured. EETs levels were determined by LC-MS/MS. Expression of sEH-encoding ephx2 was determined by qRT-PCR. Western blotting, immunocytochemistry, and hydrolase activity assay assessed protein expression and activity. RESULTS Cell death after OGD was higher in neurons from males vs. females, which correlated with higher ephx2 mRNA and stronger sEH immunoreactivity. However, EETs levels were similar in both sexes and pharmacological inhibition of the hydrolase domain of sEH did not abolish the sex difference in cell death. Genetic knockout of sEH in mice abolished the sex difference observed in neurons isolated from these mice after OGD. CONCLUSIONS Cultured cortical neurons from females are more resistant to ischemia than neurons from males. Neurons from females have less sEH activity compared to neurons from males at baseline, although sEH levels were not measured after OGD. While pharmacological inhibition of the hydrolase domain of sEH does not affect cell death, knockout of the gene encoding sEH eradicates the sex difference seen in wild-type neurons, suggesting a role for further study of the lesser-known phosphatase domain of sEH and its role in sexual dimorphism in neuronal sensitivity to ischemia.

Soluble Epoxide Hydrolase: Sex Differences and Role in Endothelial Cell Survival

Objective—Sex differences in cerebral ischemic injury are, in part, arrtibutable to the differences in cerebrovascular perfusion. We determined whether the brain microvascular endothelial cells (ECs) isolated from the female brain are more resistant to ischemic injury compared with male ECs, and whether the difference is attributable to lower expression of soluble epoxide hydrolase and higher levels of vasoprotective epoxyeicosatrienoic acids (EETs). We also determined whether protection by EETs is linked to the inhibition of rho-kinase (ROCK). Methods and Results—EC ischemic damage was measured after oxygen–glucose deprivation (OGD) using propidium iodide (PI) and cleaved caspase-3 labeling. Expression of soluble epoxide hydrolase was determined by quantitative polymerase chain reaction and immunocytochemistry, EETs levels by liquid chromatography-tandem mass spectrometry, and ROCK activity by ELISA. EC damage was higher in males compared with females, which correlated with higher soluble epoxide hydrolase mRNA, stronger immunoreactivity, and lower EETs compared with female ECs. Inhibition of soluble epoxide hydrolase abolished the sex difference in EC damage. ROCK activity was higher in male versus female ECs after OGD, and sex differences in EC damage and ROCK activity were abolished by 14,15-EET and ROCK inhibition. Conclusion—Sex differences in ischemic brain injury are, in part, attributable to differences in EET-mediated inhibition of EC ROCK activation after ischemia.

MicroRNA responses to focal cerebral ischemia in male and female mouse brain

Stroke occurs with greater frequency in men than in women across diverse ethnic backgrounds and nationalities. Work from our lab and others have revealed a sex-specific sensitivity to cerebral ischemia whereby males exhibit a larger extent of brain damage resulting from an ischemic event compared to females. Previous studies revealed that microRNA (miRNA) expression is regulated by cerebral ischemia in males; however, no studies to date have examined the effect of ischemia on miRNA responses in females. Thus, we examined miRNA responses in male and female brain in response to cerebral ischemia using miRNA arrays. These studies revealed that in male and female brains, ischemia leads to both a universal miRNA response as well as a sexually distinct response to challenge. Target prediction analysis of the miRNAs increased in male or female ischemic brain reveal sex-specific differences in gene targets and protein pathways. These data support that the mechanisms underlying sexually dimorphic responses to cerebral ischemia includes distinct changes in miRNAs in male and female brain, in addition to a miRNA signature response to ischemia that is common to both.

Role of endothelial soluble epoxide hydrolase in cerebrovascular function and ischemic injury.

Soluble Epoxide Hydrolase (sEH) is a key enzyme in the metabolism and termination of action of epoxyeicosatrienoic acids, derivatives of arachidonic acid, which are protective against ischemic stroke. Mice lacking sEH globally are protected from injury following stroke; however, little is known about the role of endothelial sEH in brain ischemia. We generated transgenic mice with endothelial-specific expression of human sEH (Tie2-hsEH), and assessed the effect of transgenic overexpression of endothelial sEH on endothelium-dependent vascular reactivity and ischemic injury following middle cerebral artery occlusion (MCAO). Compared to wild-type, male Tie2-hsEH mice exhibited impaired vasodilation in response to stimulation with 1 µM acetylcholine as assessed by laser-Doppler perfusion monitoring in an in-vivo cranial window preparation. No difference in infarct size was observed between wild-type and Tie2-hsEH male mice. In females, however, Tie2-hsEH mice sustained larger infarcts in striatum, but not cortex, compared to wild-type mice. Sex difference in ischemic injury was maintained in the cortex of Tie2-hsEH mice. In the striatum, expression of Tie2-hsEH resulted in a sex difference, with larger infarct in females than males. These findings demonstrate that transgenic expression of sEH in endothelium results in impaired endothelium-dependent vasodilation in the cerebral circulation, and that females are more susceptible to enhanced ischemic damage as a result of increased endothelial sEH than males, especially in end-arteriolar striatal region.

Mechanism of the Sex Difference in Endothelial Dysfunction after Stroke

Stroke, the number four cause of death in the USA, is a greatly debilitating event resulting from insufficient blood supply to the brain (cerebral ischemia). Endothelial dysfunction, primarily characterized by dampened endothelial-dependent vasodilation, is a major contributor to the development and outcome of stroke. This review discusses the role of soluble epoxide hydrolase, an enzyme responsible for the degradation of vasoprotective epoxyeicosatrienoic acids, in the context of the cerebral vasculature and its contribution to the sexual dimorphic nature of stroke.

Sex differences in brain proteomes of neuron‐specific STAT3‐null mice after cerebral ischemia/reperfusion

J. Neurochem. (2012) 121, 680–692.

Soluble Epoxide Hydrolase in Hydrocephalus, Cerebral Edema, and Vascular Inflammation After Subarachnoid Hemorrhage

Background and Purpose— Acute communicating hydrocephalus and cerebral edema are common and serious complications of subarachnoid hemorrhage (SAH), whose causes are poorly understood. Using a mouse model of SAH, we determined whether soluble epoxide hydrolase (sEH) gene deletion protects against SAH-induced hydrocephalus and edema by increasing levels of vasoprotective eicosanoids and suppressing vascular inflammation. Methods— SAH was induced via endovascular puncture in wild-type and sEH knockout mice. Hydrocephalus and tissue edema were assessed by T2-weighted magnetic resonance imaging. Endothelial activation was assessed in vivo using T2*-weighted magnetic resonance imaging after intravenous administration of iron oxide particles linked to anti–vascular cell adhesion molecule-1 antibody 24 hours after SAH. Behavioral outcome was assessed at 96 hours after SAH with the open field and accelerated rotarod tests. Results— SAH induced an acute sustained communicating hydrocephalus within 6 hours of endovascular puncture in both wild-type and sEH knockout mice. This was followed by tissue edema, which peaked at 24 hours after SAH and was limited to white matter fiber tracts. sEH knockout mice had reduced edema, less vascular cell adhesion molecule-1 uptake, and improved outcome compared with wild-type mice. Conclusions— Genetic deletion of sEH reduces vascular inflammation and edema and improves outcome after SAH. sEH inhibition may serve as a novel therapy for SAH.

Ultrasound stimulates formation and release of vasoactive compounds in brain endothelial cells

Stroke outcome is improved by therapeutic ultrasound. This benefit is presumed to be principally from ultrasound-mediated thrombolysis. We hypothesized that the therapeutic benefit of ultrasound in stroke may, in part, be mediated by the release of beneficial vasoactive substances. Accordingly, we investigated the effect of ultrasound on levels of cytochrome P-450, lipoxygenase, and cyclooxygenase metabolites of arachidonic acid as well as adenosine release and endothelial nitric oxide synthase (eNOS) phosphorylation in primary brain endothelial cells in vitro. Brain endothelial cells were exposed to 1.05-MHz ultrasound at peak rarefactional acoustic pressure amplitudes of 0.35, 0.55, 0.90, and 1.30 MPa. Epoxyeicosatrienoic acids (EETs), hydroxyeicosatetraenoic acids (HETEs), PGE2, adenosine, nitrate/nitrite, and eNOS phosphorylation were measured after ultrasound exposure. Levels of 8,9-EET, 11,12-EET, and 14,15-EET increased by 230 ± 28%, 240 ± 30%, and 246 ± 31% (P < 0.05), respectively, whereas 5-HETE and 15-HETE levels were reduced to 24 ± 14% and 10 ± 3% (P < 0.05), respectively, compared with cells not exposed to ultrasound. PGE2 levels were reduced to 56 ± 14% of control. Adenosine increased more than sixfold after ultrasound exposure compared with unstimulated cells (1.36 ± 0.22 vs. 0.37 ± 0.10 ng/ml, P < 0.05), nitrate/nitrite was below levels of quantification, and eNOS phosphorylation was not altered significantly. Our results suggest that ultrasound may enhance tissue perfusion during stroke by augmenting the generation of vasodilator compounds and inhibiting that of vasoconstrictors. Such regulation supports a beneficial role for therapeutic ultrasound in stroke independent of its effect on the occlusive thrombus.

Role of aromatase in sex-specific cerebrovascular endothelial function in mice

Stroke risk and outcome are strongly modified by estrogen. In addition to ovaries, estrogen is produced locally in peripheral tissue by the enzyme aromatase, and extragonadal synthesis becomes the major source of estrogen after menopause. Aromatase gene deletion in female mice exacerbates ischemic brain damage after stroke. However, it is not clear which cell type is responsible for this effect, since aromatase is expressed in multiple cell types, including cerebrovascular endothelium. We tested the hypothesis that cerebrovascular aromatase contributes to sex differences in cerebrovascular endothelial function. Cerebrocortical microvascular responses to the endothelium-dependent vasodilator ACh were compared between male and female wild-type (WT) and aromatase knockout (ArKO) mice by measuring laser-Doppler perfusion in vivo through a closed cranial window. Additional studies were performed in WT mice treated with the aromatase inhibitor fadrozole or vehicle. WT female mice had significantly greater responses to ACh compared with WT males (P < 0.001), which was associated with higher aromatase expression in female compared with male cerebral vessels (P < 0.05). ACh responses were significantly lower in ArKO compared with WT females (P < 0.05) and in WT females treated with fadrozole versus vehicle (P < 0.001). Conversely, ACh responses were significantly higher in ArKO versus WT males (P < 0.05). Levels of phosphorylated endothelial nitric oxide synthase (eNOS) were lower in ArKO versus WT female brains, but were not altered by aromatase deletion in males. We conclude that cerebrovascular endothelial aromatase plays an important and sexually dimorphic role in cerebrovascular function and that aromatase inhibitors in clinical use may have cardiovascular consequences in both males and females.