Epidermal Growth Factor Receptors in Vascular Endothelial Cells Contribute to Functional Hyperemia in the Brain.

Functional hyperemia-activity-dependent increases in local blood perfusion-underlies the on-demand delivery of blood to regions of enhanced neuronal activity, a process that is crucial for brain health. Importantly, functional hyperemia deficits have been linked to multiple dementia risk factors, including aging, chronic hypertension, and cerebral small vessel disease (cSVD). We previously reported crippled functional hyperemia in a mouse model of genetic cSVD that was likely caused by depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) in capillary endothelial cells (EC) downstream of impaired epidermal growth factor receptor (EGFR) signaling. Here, using EC-specific EGFR-knockout (KO) mice, we directly examined the role of endothelial EGFR signaling in functional hyperemia, assessed by measuring increases in cerebral blood flow in response to contralateral whisker stimulation using laser Doppler flowmetry. Molecular characterizations showed that EGFR expression was dramatically decreased in freshly isolated capillaries from EC-EGFR-KO mice, as expected. Notably, whisker stimulation-induced functional hyperemia was significantly impaired in these mice, an effect that was rescued by administration of PIP2, but not by the EGFR ligand, HB-EGF. These data suggest that the deletion of the EGFR specifically in ECs attenuates functional hyperemia, likely via depleting PIP2 and subsequently incapacitating Kir2.1 channel functionality in capillary ECs. Thus, our study underscores the role of endothelial EGFR signaling in functional hyperemia of the brain.

The yin and yang of KV channels in cerebral small vessel pathologies.

Cerebral SVDs encompass a group of genetic and sporadic pathological processes leading to brain lesions, cognitive decline, and stroke. There is no specific treatment for SVDs, which progress silently for years before becoming clinically symptomatic. Here, we examine parallels in the functional defects of PAs in CADASIL, a monogenic form of SVD, and in response to SAH, a common type of hemorrhagic stroke that also targets the brain microvasculature. Both animal models exhibit dysregulation of the voltage-gated potassium channel, KV 1, in arteriolar myocytes, an impairment that compromises responses to vasoactive stimuli and impacts CBF autoregulation and local dilatory responses to neuronal activity (NVC). However, the extent to which this channelopathy-like defect ultimately contributes to these pathologies is unknown. Combining experimental data with computational modeling, we describe the role of KV 1 channels in the regulation of myocyte membrane potential at rest and during the modest increase in extracellular potassium associated with NVC. We conclude that PA resting membrane potential and myogenic tone depend strongly on KV 1.2/1.5 channel density, and that reciprocal changes in KV channel density in CADASIL and SAH produce opposite effects on extracellular potassium-mediated dilation during NVC.

Astrocyte Ca2+ Signaling Drives Inversion of Neurovascular Coupling after Subarachnoid Hemorrhage.

Physiologically, neurovascular coupling (NVC) matches focal increases in neuronal activity with local arteriolar dilation. Astrocytes participate in NVC by sensing increased neurotransmission and releasing vasoactive agents (e.g., K(+)) from perivascular endfeet surrounding parenchymal arterioles. Previously, we demonstrated an increase in the amplitude of spontaneous Ca(2+) events in astrocyte endfeet and inversion of NVC from vasodilation to vasoconstriction in brain slices obtained from subarachnoid hemorrhage (SAH) model rats. However, the role of spontaneous astrocyte Ca(2+) signaling in determining the polarity of the NVC response remains unclear. Here, we used two-photon imaging of Fluo-4-loaded rat brain slices to determine whether altered endfoot Ca(2+) signaling underlies SAH-induced inversion of NVC. We report a time-dependent emergence of endfoot high-amplitude Ca(2+) signals (eHACSs) after SAH that were not observed in endfeet from unoperated animals. Furthermore, the percentage of endfeet with eHACSs varied with time and paralleled the development of inversion of NVC. Endfeet with eHACSs were present only around arterioles exhibiting inversion of NVC. Importantly, depletion of intracellular Ca(2+) stores using cyclopiazonic acid abolished SAH-induced eHACSs and restored arteriolar dilation in SAH brain slices to two mediators of NVC (a rise in endfoot Ca(2+) and elevation of extracellular K(+)). These data indicate a causal link between SAH-induced eHACSs and inversion of NVC. Ultrastructural examination using transmission electron microscopy indicated that a similar proportion of endfeet exhibiting eHACSs also exhibited asymmetrical enlargement. Our results demonstrate that subarachnoid blood causes a delayed increase in the amplitude of spontaneous intracellular Ca(2+) release events leading to inversion of NVC. Significance statement: Aneurysmal subarachnoid hemorrhage (SAH)--strokes involving cerebral aneurysm rupture and release of blood onto the brain surface--are associated with high rates of morbidity and mortality. A common complication observed after SAH is the development of delayed cerebral ischemia at sites often remote from the site of rupture. Here, we provide evidence that SAH-induced changes in astrocyte Ca(2+) signaling lead to a switch in the polarity of the neurovascular coupling response from vasodilation to vasoconstriction. Thus, after SAH, signaling events that normally lead to vasodilation and enhanced delivery of blood to active brain regions cause vasoconstriction that would limit cerebral blood flow. These findings identify astrocytes as a key player in SAH-induced decreased cortical blood flow.

SAH-induced MMP activation and K V current suppression is mediated via both ROS-dependent and ROS-independent mechanisms.

Voltage-gated potassium (K V) channels regulate cerebral artery tone and have been implicated in subarachnoid hemorrhage (SAH)-induced pathologies. Here, we examined whether matrix metalloprotease (MMP) activation contributes to SAH-induced K V current suppression and cerebral artery constriction via activation of epidermal growth factor receptors (EGFRs). Using patch clamp electrophysiology, we observed that K V currents were selectively decreased in cerebral artery myocytes isolated from SAH model rabbits. Consistent with involvement of enhanced MMP and EGFR activity in SAH-induced K V current suppression, we found that: (1) oxyhemoglobin (OxyHb) and/or the exogenous EGFR ligand, heparin-binding EGF-like growth factor (HB-EGF), failed to induce further K V current suppression after SAH and (2) gelatin zymography detected significantly higher MMP-2 activity after SAH. The removal of reactive oxygen species (ROS) by combined treatment with superoxide dismutase (SOD) and catalase partially inhibited OxyHb-induced K V current suppression. However, these agents had little effect on OxyHb-induced MMP-2 activation. Interestingly, in the presence of a broad-spectrum MMP inhibitor (GM6001), OxyHb failed to cause K V current suppression. These data suggest that OxyHb suppresses K V currents through both ROS-dependent and ROS-independent pathways involving MMP activation. The ROS-independent pathway involves activation of MMP-2, whereas the ROS-dependent pathway involves activation of a second unidentified MMP or ADAM (a disintegrin and metalloprotease domain).

Activation of TRPV4 channels does not mediate inversion of neurovascular coupling after SAH.

Neurovascular coupling (NVC) allows increased blood flow to metabolically active neurons and involves the Ca²âº -dependent release of vasodilator influences by astrocyte endfeet that encase parenchymal arterioles. We previously reported inversion of NVC from dilation to constriction in brain slices from subarachnoid hemorrhage (SAH) model rats. Corresponding to NVC inversion, there was a marked increase in the amplitude of spontaneous Ca²âº oscillations in astrocyte endfeet. Calcium-permeable transient receptor potential vanilloid (TRPV)-4 channels have been reported in astrocyte endfeet, and activators of these channels enhance Ca²âº oscillations in healthy animals. Here, we examined the role of TRPV4 channels in the development of high-amplitude spontaneous Ca²âº oscillations in astrocyte endfeet and the inversion of neurovascular coupling after SAH. Treatment of brain slices with the TRPV4 channel antagonist, HC-067047 (10 µM), did not alter the amplitude of spontaneous Ca²âº oscillations after SAH. In addition, HC-067047 did not inhibit or change SAH-induced inversion of neurovascular coupling. In summary, TRPV4 channels do not appear to be involved in the inversion of neurovascular coupling after SAH. Further studies examining the impact of SAH on additional Ca²âº signaling pathways in astrocytes are likely to reveal valuable insights into new therapeutic strategies to advance SAH treatments.

Inversion of neurovascular coupling by subarachnoid blood depends on large-conductance Ca2+-activated K+ (BK) channels.

The cellular events that cause ischemic neurological damage following aneurysmal subarachnoid hemorrhage (SAH) have remained elusive. We report that subarachnoid blood profoundly impacts communication within the neurovascular unit-neurons, astrocytes, and arterioles-causing inversion of neurovascular coupling. Elevation of astrocytic endfoot Ca(2+) to ∼400 nM by neuronal stimulation or to ∼300 nM by Ca(2+) uncaging dilated parenchymal arterioles in control brain slices but caused vasoconstriction in post-SAH brain slices. Inhibition of K(+) efflux via astrocytic endfoot large-conductance Ca(2+)-activated K(+) (BK) channels prevented both neurally evoked vasodilation (control) and vasoconstriction (SAH). Consistent with the dual vasodilator/vasoconstrictor action of extracellular K(+) ([K(+)](o)), [K(+)](o) <10 mM dilated and [K(+)](o) >20 mM constricted isolated brain cortex parenchymal arterioles with or without SAH. Notably, elevation of external K(+) to 10 mM caused vasodilation in brain slices from control animals but caused a modest constriction in brain slices from SAH model rats; this latter effect was reversed by BK channel inhibition, which restored K(+)-induced dilations. Importantly, the amplitude of spontaneous astrocytic Ca(2+) oscillations was increased after SAH, with peak Ca(2+) reaching ∼490 nM. Our data support a model in which SAH increases the amplitude of spontaneous astrocytic Ca(2+) oscillations sufficiently to activate endfoot BK channels and elevate [K(+)](o) in the restricted perivascular space. Abnormally elevated basal [K(+)](o) combined with further K(+) efflux stimulated by neuronal activity elevates [K(+)](o) above the dilation/constriction threshold, switching the polarity of arteriolar responses to vasoconstriction. Inversion of neurovascular coupling may contribute to the decreased cerebral blood flow and development of neurological deficits that commonly follow SAH.

Evidence against a systemic arterial defect in patients with inflammatory bowel disease.

BACKGROUND: Despite increasing interest in local microvascular alterations associated with inflammatory bowel disease (IBD), the potential contribution of a primary systemic vascular defect in the etiology of IBD is unknown. We compared reactivity of large diameter mesenteric arteries from segments affected by Crohn disease (CD) or ulcerative colitis (UC) to an uninvolved vascular bed in both IBD and control patients. METHODS: Mesenteric and omental arteries were obtained from UC, CD, and non-IBD patients. Isometric arterial contractions were recorded in response to extracellular potassium (K(+)) and cumulative additions of norepinephrine (NE). In addition, relaxation in response to pinacidil, an activator of adenosine triphosphate-sensitive K(+) channels was examined. RESULTS: Contraction to K(+) and sensitivity to NE were not significantly different in arteries from CD, UC, and controls. Relaxation to pinacidil was also similar between groups. CONCLUSIONS: Potassium-induced contractions and sensitivity to NE and pinacidil were not significantly different in large diameter mesenteric and omental arteries obtained from IBD patients. Furthermore, there was no significant difference in the sensitivity to K(+), NE, and pinacidil between mesenteric and omental arteries of CD and UC patients and those from non-IBD patients. Our results suggest an underlying vascular defect systemic to CD or UC patients is unlikely to contribute to the etiology of IBD.

Impact of subarachnoid hemorrhage on parenchymal arteriolar function.

Intracerebral or parenchymal arterioles play an important role in the regulation of both global and regional blood flow within the brain. Brain cortex lacks significant collateral sources of blood and thus is at risk if blood flow through parenchymal arterioles is restricted. Increasingly, evidence is accumulating that abnormal parenchymal arteriolar constriction contributes to the development of neurological deficits caused by subarachnoid hemorrhage (SAH). For example, parenchymal arterioles isolated from SAH model rats exhibit enhanced constriction in response to increased intravascular pressure. This increased pressure-dependent constriction or myogenic tone would result in a shift in the cerebral autoregulatory response and decreased cerebral perfusion. Here, we summarize our current knowledge regarding cellular mechanisms contributing to enhanced contractility of parenchymal arteriolar myocytes following SAH. Our studies demonstrated that SAH-induced membrane potential depolarization involving altered K(+) homeostasis leads to enhanced voltage-dependent Ca(2+) channel activity, increased smooth muscle cytosolic Ca(2+), and parenchymal arteriolar constriction. In summary, emerging evidence demonstrates that SAH can profoundly affect parenchymal arteriolar tone, promoting decreased cortical blood flow and compromised neuronal viability.

SAH-induced suppression of voltage-gated K(+) (K (V)) channel currents in parenchymal arteriolar myocytes involves activation of the HB-EGF/EGFR pathway.

Potassium channels play an important role in the regulation of arterial tone, and decreased activity of these ion channels has been linked to pial artery vasospasm after subarachnoid hemorrhage (SAH). Our previous work has shown that acute application of a blood component, oxyhemoglobin, caused suppression of voltage-gated K(+) (K(V)) channels through heparin-binding epidermal growth factor-like growth factor (HB-EGF)-mediated activation of epidermal growth factor receptor (EGFR). Using patch clamp electrophysiology, we have now examined whether this pathway of K(V) channel suppression is activated in parenchymal arteriolar myocytes following long-term in vivo exposure to subarachnoid blood. We have found that K(V) currents, but not large conductance Ca(2+) activated or inwardly rectifying K(+) channel currents, were decreased in parenchymal arteriolar myocytes freshly isolated from day 5 SAH model rabbits. Interestingly, parenchymal arteriolar myocytes from control animals were more sensitive to exogenous HB-EGF (half-maximal inhibitory concentration [IC(50)] 0.2 ± 0.4 ng/ml) compared to pial arterial myocytes (IC(50) 2.4 ± 1.3 ng/ml). However, HB-EGF and oxyhemoglobin failed to decrease K(V) currents in parenchymal arteriolar myocytes from SAH animals, consistent with EGFR activation and K(V) current suppression by SAH. These data suggest that HB-EGF/EGFR pathway activation contributes to K(V) current suppression and enhanced parenchymal arteriolar constriction after SAH.

Subarachnoid blood converts neurally evoked vasodilation to vasoconstriction in rat brain cortex.

The matching of blood flow to regional brain function, called functional hyperemia or neurovascular coupling, involves the coordinated activity of neurons, astrocytes, and parenchymal arterioles. Under physiological conditions, localized neuronal activation leads to elevated astrocyte endfoot Ca(2+) and vasodilation, resulting in an increase in cerebral blood flow. In this study, we examined the impact of subarachnoid hemorrhage (SAH) on neurovascular coupling. SAH model rats received two injections of autologous blood into the cisterna magna 24 h apart. Cortical brain slices from SAH model animals were prepared 4 days after the initial blood injection. Arteriolar diameter and astrocyte endfoot Ca(2+) were simultaneously measured using two-photon microscopy. As expected, neuronal activity evoked by electrical field stimulation (EFS) caused an elevation in endfoot Ca(2+) and vasodilation in brain slices from control animals. However, in brain slices from SAH animals, EFS induced a similar increase in astrocyte endfoot Ca(2+) that caused arteriolar constriction rather than vasodilation. Vasoconstriction was observed in approximately 90% of brain slices from SAH animals in response to EFS, with 40% exhibiting a sustained vasoconstriction, 30% exhibiting a transient vasoconstriction -(diameter restored within 1 min after EFS), and 20% responded with a biphasic response (brief vasodilation followed by -vasoconstriction). This inversion of neurovascular coupling may play a role in the development of neurological deficits following SAH.

Epidermal growth factor receptors in vascular endothelial cells contribute to functional hyperemia in the brain.

Functional hyperemia - activity-dependent increases in local blood perfusion - underlies the on-demand delivery of blood to regions of enhanced neuronal activity, a process that is crucial for brain health. Importantly, functional hyperemia deficits have been linked to multiple dementia risk factors, including aging, chronic hypertension, and cerebral small vessel disease (cSVD). We previously reported crippled functional hyperemia in a mouse model of genetic cSVD that was likely caused by depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) in capillary endothelial cells (EC) downstream of impaired epidermal growth factor receptor (EGFR) signaling. Here, using EC-specific EGFR-knockout (KO) mice, we directly examined the role of endothelial EGFR signaling in functional hyperemia, assessed by measuring increases in cerebral blood flow in response to contralateral whisker stimulation using laser Doppler flowmetry. Molecular characterizations showed that EGFR expression was dramatically decreased in freshly isolated capillaries from EC-EGFR-KO mice, as expected. Notably, whisker stimulation-induced functional hyperemia was significantly impaired in these mice, an effect that was rescued by exogenous administration of PIP2, but not by the EGFR ligand, HB-EGF. These data suggest that the deletion of the EGFR specifically in ECs depletes PIP2 and attenuates functional hyperemia, underscoring the central role of the endothelial EGFR signaling in cerebral blood flow regulation.

Cardiac reactive oxygen species after traumatic brain injury.

BACKGROUND: Cardiovascular complications after traumatic brain injury (TBI) contribute to morbidity and mortality and may provide a target for therapy. We examined blood pressure and left ventricle contractility after TBI, and tested the hypothesis that ß-adrenergic blockade would decrease oxidative stress after TBI. MATERIAL AND METHODS: Rodents received fluid-percussion injury or sham surgery, confirmed with magnetic resonance imaging (MRI) and histopathology. We followed recovery with sensorimotor coordination testing and blood pressure measurements. We assessed left ventricular ejection fraction using ECG-gated cardiac MRI and measured myocardial reactive oxygen species (ROS) with dihydroethidium. We randomized additional TBI and sham animals to postoperative treatment with propranolol or control, for measurement of ROS. RESULTS: Blood pressure and cardiac contractility were elevated 48 h after TBI. Myocardial tissue sections showed increased ROS. Treatment with propranolol diminished ROS levels following TBI. CONCLUSIONS: TBI is associated with increased cardiac contractility and myocardial ROS; decreased myocardial ROS after ß-blockade suggests that sympathetic stimulation is a mechanism of oxidative stress.

Fundamental increase in pressure-dependent constriction of brain parenchymal arterioles from subarachnoid hemorrhage model rats due to membrane depolarization.

Intracerebral (parenchymal) arterioles are morphologically and physiologically unique compared with pial arteries and arterioles. The ability of subarachnoid hemorrhage (SAH) to induce vasospasm in large-diameter pial arteries has been extensively studied, although the contribution of this phenomenon to patient outcome is controversial. Currently, little is known regarding the impact of SAH on parenchymal arterioles, which are critical for regulation of local and global cerebral blood flow. Here diameter, smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)), and membrane potential measurements were used to assess the function of intact brain parenchymal arterioles isolated from unoperated (control), sham-operated, and SAH model rats. At low intravascular pressure (5 mmHg), membrane potential and [Ca(2+)](i) were not different in arterioles from control, sham-operated, and SAH animals. However, raising intravascular pressure caused significantly greater membrane potential depolarization, elevation in [Ca(2+)](i), and constriction in SAH arterioles. This SAH-induced increase in [Ca(2+)](i) and tone occurred in the absence of the vascular endothelium and was abolished by the L-type voltage-dependent calcium channel (VDCC) inhibitor nimodipine. Arteriolar [Ca(2+)](i) and tone were not different between groups when smooth muscle membrane potential was adjusted to the same value. Protein and mRNA levels of the L-type VDCC Ca(V)1.2 were similar in parenchymal arterioles isolated from control and SAH animals, suggesting that SAH did not cause VDCC upregulation. We conclude that enhanced parenchymal arteriolar tone after SAH is driven by smooth muscle membrane potential depolarization, leading to increased L-type VDCC-mediated Ca(2+) influx.

Incomplete reprogramming after fusion of human multipotent stromal cells and bronchial epithelial cells.

Bone marrow-derived progenitor cells can fuse with cells of several different tissues, including lung, especially following injury. Despite many reports of cell fusion, few studies have examined the function of the resulting hybrid cells. We cocultured human multipotent stromal cells (hMSCs) and normal human bronchial epithelial cells (NHBEs) and observed the formation of hMSC/NHBE heterokaryons. The heterokaryons expressed several proteins characteristic of epithelial cells, such as keratin and occludin. Hybrid cells also expressed the mRNAs and proteins for 2 important ion channels that maintain bronchial and alveolar fluid balance: the cystic fibrosis transmembrane conductance regulator (CFTR) and the amiloride-sensitive epithelial Na(+) channel (ENaC). By immunocytochemistry, CFTR was expressed in many hybrid cells but was absent or low in others. Whole-cell patch-clamp recordings demonstrated a glibenclamide-sensitive current in the presence of barium chloride, consistent with functional CFTR channels, in control NHBEs and hMSC/NHBE heterokaryons. Total cell capacitance measurements showed that the membrane surface area of heterokaryons was similar to that of NHBEs. Heterokaryons expressed the α- and γ-ENaC subunits but did not express the ß-ENaC subunit, indicating the inability to form a complete ENaC channel. In addition, hybrid cells formed by the fusion of hMSCs with immortalized bronchial cells that expressed CFTR ΔF508 did not lead to reprogramming of the hMSC nucleus and expression of wild-type CFTR mRNA. Our data show that reprogramming can be incomplete following fusion of adult progenitor cells and somatic cells and may lead to altered cell function.

Impact of subarachnoid hemorrhage on local and global calcium signaling in cerebral artery myocytes.

BACKGROUND: Ca2+ signaling mechanisms are crucial for proper regulation of vascular smooth muscle contractility and vessel diameter. In cerebral artery myocytes, a rise in global cytosolic Ca2+ concentration ([Ca2+]i) causes contraction while an increase in local Ca2+ release events from the sarcoplasmic reticulum (Ca2+ sparks) leads to increased activity of large-conductance Ca2+-activated (BK) K+ channels, hyperpolarization and relaxation. Here, we examined the impact of SAH on Ca2+ spark activity and [Ca2+]i in cerebral artery myocytes following SAH. METHODS: A rabbit double injection SAH model was used in this study. Five days after the initial intracisternal injection of whole blood, small diameter cerebral arteries were dissected from the brain for study. For simultaneous measurement of arterial wall [Ca2+]i and diameter, vessels were cannulated and loaded with the ratiometric Ca2+ indicator fura-2. For measurement of Ca2+ sparks, individual myocytes were enzymatically isolated from cerebral arteries and loaded with the Ca2+ indicator fluo-4. Sparks were visualized using laser scanning confocal microscopy. RESULTS: Arterial wall [Ca2+]i was significantly elevated and greater levels of myogenic tone developed in arteries isolated from SAH animals compared with arteries isolated from healthy animals. The L-type voltage-dependent Ca2+ channel (VDCC) blocker nifedipine attenuated increases in [Ca2+]i and tone in both groups suggesting increased VDCC activity following SAH. Membrane potential measurement using intracellular microelectrodes revealed significant depolarization of vascular smooth muscle following SAH. Further, myocytes from SAH animals exhibited significantly reduced Ca2+ spark frequency (~50%). CONCLUSIONS: Our findings suggest decreased Ca2+ spark frequency leads to reduced BK channel activity in cerebral artery myocytes following SAH. This results in membrane potential depolarization, increased VDCC activity, elevated [Ca2+]i and decreased vessel diameter. We propose this mechanism of enhanced cerebral artery myocyte contractility may contribute to decreased cerebral blood flow and development of neurological deficits in SAH patients.

Impaired Cerebral Autoregulation After Subarachnoid Hemorrhage: A Quantitative Assessment Using a Mouse Model.

Subarachnoid hemorrhage (SAH) is a common form of hemorrhagic stroke associated with high rates of mortality and severe disability. SAH patients often develop severe neurological deficits days after ictus, events attributed to a phenomenon referred to as delayed cerebral ischemia (DCI). Recent studies indicate that SAH-induced DCI results from a multitude of cerebral circulatory disturbances including cerebral autoregulation malfunction. Cerebral autoregulation incorporates the influence of blood pressure (BP) on arterial diameter in the homeostatic regulation of cerebral blood flow (CBF), which is necessary for maintaining constant brain perfusion during physiological swings in systemic BP. In this study, we quantitatively examined the impact of SAH on cerebral autoregulation using a mouse endovascular perforation model and a newly developed approach combining absolute and relative CBF measurements. This method enables a direct quantitative comparison of cerebral autoregulation between individual animals (e.g., SAH vs. control or sham-operated mice), which cannot be done solely using relative CBF changes by laser Doppler flowmetry. Here, absolute CBF was measured via injection of fluorescent microspheres at a baseline BP. In separate groups of animals, in vivo laser Doppler flowmetry was used to measure relative CBF changes over a range of BP using phlebotomy and the pressor phenylephrine to lower and raise BP, respectively. Absolute CBF measurements from microspheres were then used to calibrate laser Doppler measurements to calculate the relationship between CBF and BP, i.e., "cerebral autoregulation curves." Un-operated and sham-operated groups exhibited similar cerebral autoregulatory curves, showing comparable levels of relatively constant CBF over a range of BP from ~80 mmHg to ~130 mmHg. In contrast, SAH animals exhibited a narrower autoregulatory range of BP, which was primarily due to a decrease in the upper limit of BP whereby cerebral autoregulation was maintained. Importantly, SAH animals also exhibited a marked decrease in CBF throughout the entire range of BP. In sum, this study provides evidence of the dramatic reduction in cortical CBF and the diminished range of autoregulation after SAH. Furthermore, this novel methodology should pave the way for future studies examining pathological mechanisms and/or therapeutic strategies targeting impaired cerebral autoregulation, a pathology common to many cardiovascular and cerebrovascular disorders.

Endocrine Dysfunction Following Stroke.

Endocrine dysfunction is known to occur after traumatic brain injury. The purpose of this study was to examine the incidence of various endocrine dysfunctions after a stroke. The Taiwan National Health Insurance Research Database (NHIRD) was searched from 2001 to 2011 for patients with a diagnosis of stroke. Stroke patients were matched by diagnosis date, age, and sex to patients without a stroke. Cox proportional hazards regression analyses were performed to compare the incidence of goiter, acquired hypothyroidism, thyroiditis, pituitary dysfunction, and disorders of the adrenal glands between stroke and non-stroke patients. There were 131,951 patients in the stroke group, and 131,951 in the matched non- stroke group (mean age 66.1 ± 14.9 years). Stroke patients had significantly higher risk of acquired hypothyroidism (crude hazard ratio [cHR] = 1.65, 95% confidence interval [CI]: 1.44, 1.90; adjusted hazard ratio [aHR] = 1.65, 95% CI: 1.42, 1.91), pituitary dysfunction (cHR = 2.32, 95% CI: 1.79, 2.99; aHR = 1.92, 95% CI: 1.46, 2.52), and disorders of the adrenal glands (cHR = 1.79, 95% CI: 1.52, 2.12; aHR =1.62, 95% CI: 1.36, 1.92) than non-stroke patients. Pituitary dysfunction and disorders of the adrenal glands were found in both hemorrhagic stroke and ischemic stroke patients, while hypothyroidism was seen in ischemic stroke patients only. No significant association was found for goiter and thyroiditis. In conclusions, stroke survivors have an approximately 2-fold increased risk of developing acquired hypothyroidism, pituitary dysfunction, or disorders of the adrenal glands. These risks should be taken into account in the management of patients who have ischemic or hemorrhagic strokes. Graphical Abstract.

Differential restoration of functional hyperemia by antihypertensive drug classes in hypertension-related cerebral small vessel disease.

Dementia resulting from small vessel diseases (SVDs) of the brain is an emerging epidemic for which there is no treatment. Hypertension is the major risk factor for SVDs, but how hypertension damages the brain microcirculation is unclear. Here, we show that chronic hypertension in a mouse model progressively disrupts on-demand delivery of blood to metabolically active areas of the brain (functional hyperemia) through diminished activity of the capillary endothelial cell inward-rectifier potassium channel, Kir2.1. Despite similar efficacy in reducing blood pressure, amlodipine, a voltage-dependent calcium-channel blocker, prevented hypertension-related damage to functional hyperemia whereas losartan, an angiotensin II type 1 receptor blocker, did not. We attribute this drug class effect to losartan-induced aldosterone breakthrough, a phenomenon triggered by pharmacological interruption of the renin-angiotensin pathway leading to elevated plasma aldosterone levels. This hypothesis is supported by the finding that combining losartan with the aldosterone receptor antagonist eplerenone prevented the hypertension-related decline in functional hyperemia. Collectively, these data suggest Kir2.1 as a possible therapeutic target in vascular dementia and indicate that concurrent mineralocorticoid aldosterone receptor blockade may aid in protecting against late-life cognitive decline in hypertensive patients treated with angiotensin II type 1 receptor blockers.

Ca(v)1.2 splice variant with exon 9* is critical for regulation of cerebral artery diameter.

L-type voltage-dependent Ca(2+) channels (VDCCs) are essential for numerous processes in the cardiovascular and nervous systems. Alternative splicing modulates proteomic composition of Ca(v)1.2 to generate functional variation between channel isoforms. Here, we describe expression and function of Ca(v)1.2 channels containing alternatively spliced exon 9* in cerebral artery myocytes. RT-PCR showed expression of Ca(v)1.2 splice variants both containing (alpha(1)C(9/9*/10)) and lacking (alpha(1)C(9/10)) exon 9* in intact rabbit and human cerebral arteries. With the use of laser capture microdissection and RT-PCR, expression of mRNA for both alpha(1)C(9/9*/10) and alpha(1)C(9/10) was demonstrated in isolated cerebral artery myocytes. Quantitative real-time PCR revealed significantly greater alpha(1)C(9/9*/10) expression relative to alpha(1)C(9/10) in intact rabbit cerebral arteries compared with cardiac tissue and cerebral cortex. To demonstrate a functional role for alpha(1)C(9/9*/10), smooth muscle of intact cerebral arteries was treated with antisense oligonucleotides targeting alpha(1)C(9/9*/10) (alpha(1)C(9/9*/10)-AS) or exon 9 (alpha(1)C-AS), expressed in all Ca(v)1.2 splice variants, by reversible permeabilization and organ cultured for 1-4 days. Treatment with alpha(1)C(9/9*/10)-AS reduced maximal constriction induced by elevated extracellular K(+) ([K(+)](o)) by approximately 75% compared with alpha(1)C(9/9*/10-)sense-treated arteries. Maximal constriction in response to the Ca(2+) ionophore ionomycin and [K(+)](o) EC(50) values were not altered by antisense treatment. Decreases in maximal [K(+)](o)-induced constriction were similar between alpha(1)C(9/9*/10)-AS and alpha(1)C-AS groups (22.7 + or - 9% and 25.6 + or - 4% constriction, respectively). We conclude that although cerebral artery myocytes express both alpha(1)C(9/9*/10) and alpha(1)C(9/10) VDCC splice variants, alpha(1)C(9/9*/10) is functionally dominant in the control of cerebral artery diameter.