Effectiveness of pre-anesthetic video information on patient anxiety and economical aspects.

BACKGROUND: Adequate preoperative information can lessen patient anxiety. Delivering sufficient information during a personal interview, however, is time consuming, and therefore a relevant economical aspect. We investigated whether video information given to the patient before the pre-anesthetic interview has an influence on the patient's anxiety and the duration of the interview. METHOD: We randomized 302 patients undergoing different types of anesthesia. In all, 151 patients watched a short video with general information about the anticipated anesthesia procedure. Afterward, all patients had a standard pre-anesthetic interview. Patients' anxiety and satisfaction with pre-anesthesia care were assessed after the interview using a visual analogue scale. The duration of the interview was documented. Student t-test and P < 0.05 for differences between the groups. RESULTS: There was no difference in gender, age, ASA physical status, previous anesthesia experience, and the planned anesthesia procedure between the two groups. No difference in anxiety and satisfaction with pre-anesthesia care was observed. The duration of the pre-anesthetic interview was also not different between the groups. DISCUSSION: Preoperative multimedia information did not reduce anxiety or increase the patient satisfaction undergoing anesthesia. The video containing general information did not save time in the pre-anesthetic interview.

Blocking of plasma kallikrein ameliorates stroke by reducing thromboinflammation.

OBJECTIVE: Recent evidence suggests that ischemic stroke is a thromboinflammatory disease. Plasma kallikrein (PK) cleaves high-molecular-weight kininogen to release bradykinin (BK) and is a key constituent of the proinflammatory contact-kinin system. In addition, PK can activate coagulation factor XII, the origin of the intrinsic coagulation cascade. Thus, PK triggers 2 important pathological pathways of stroke formation, thrombosis and inflammation. METHODS: We investigated the consequences of PK inhibition in transient and permanent models of ischemic stroke. RESULTS: PK-deficient mice of either sex challenged with transient middle cerebral artery occlusion developed significantly smaller brain infarctions and less severe neurological deficits compared with controls without an increase in infarct-associated hemorrhage. This protective effect was preserved at later stages of infarctions as well as after permanent stroke. Reduced intracerebral thrombosis and improved cerebral blood flow could be identified as underlying mechanisms. Moreover, blood-brain barrier function was maintained in mice lacking PK, and the local inflammatory response was reduced. PK-deficient mice reconstituted with PK or BK again developed brain infarctions similar to wild-type mice. Important from a translational perspective, inhibition of PK in wild-type mice using a PK-specific antibody was likewise effective even when performed in a therapeutic setting up to 3 hours poststroke. INTERPRETATION: PK drives thrombus formation and inflammation via activation of the intrinsic coagulation cascade and the release of BK but appears to be dispensable for hemostasis. Hence, PK inhibition may offer a safe strategy to combat thromboembolic disorders including ischemic stroke.

The two-pore domain potassium channel KCNK5 deteriorates outcome in ischemic neurodegeneration.

Potassium channels can fulfill both beneficial and detrimental roles in neuronal damage during ischemic stroke. Earlier studies have characterized a neuroprotective role of the two-pore domain potassium channels KCNK2 (TREK1) and KCNK3 (TASK1). Protective neuronal hyperpolarization and prevention of intracellular Ca(2+) overload and glutamate excitotoxicity were suggested to be the underlying mechanisms. We here identify an unexpected role for the related KCNK5 channel in a mouse model of transient middle cerebral artery occlusion (tMCAO). KCNK5 is strongly upregulated on neurons upon cerebral ischemia, where it is most likely involved in the induction of neuronal apoptosis. Hypoxic conditions elevated neuronal expression levels of KCNK5 in acute brain slices and primary isolated neuronal cell cultures. In agreement, KCNK5 knockout mice had significantly reduced infarct volumes and improved neurologic function 24 h after 60 min of tMCAO and this protective effect was preserved at later stages of infarct development. KCNK5 deficiency resulted in a significantly reduced number of apoptotic neurons, a downregulation of pro-apoptotic and upregulation of anti-apoptotic factors. Results of adoptive transfer experiments of wild-type and Kcnk5 (-/-) immune cells into Rag1 (-/-) mice prior to tMCAO exclude a major role of KCNK5 in poststroke inflammatory reactions. In summary, KCNK5 expression is induced on neurons under ischemic conditions where it most likely exerts pro-apoptotic effects. Hence, pharmacological blockade of KCNK5 might have therapeutic potential in preventing ischemic neurodegeneration.

FTY720 ameliorates acute ischemic stroke in mice by reducing thrombo-inflammation but not by direct neuroprotection.

BACKGROUND AND PURPOSE: Lymphocytes are important players in the pathophysiology of acute ischemic stroke. The interaction of lymphocytes with endothelial cells and platelets, termed thrombo-inflammation, fosters microvascular dysfunction and secondary infarct growth. FTY720, a sphingosine-1-phosphate receptor modulator, blocks the egress of lymphocytes from lymphoid organs and has been shown to reduce ischemic neurodegeneration; however, the underlying mechanisms are unclear. We investigated the mode of FTY720 action in models of cerebral ischemia. METHODS: Transient middle cerebral artery occlusion (tMCAO) was induced in wild-type and lymphocyte-deficient Rag1(-/-) mice treated with FTY720 (1 mg/kg) or vehicle immediately before reperfusion. Stroke outcome was assessed 24 hours later. Immune cells in the blood and brain were counted by flow cytometry. The integrity of the blood-brain barrier was analyzed using Evans Blue dye. Thrombus formation was determined by immunohistochemistry and Western blot, and was correlated with cerebral perfusion. RESULTS: FTY720 significantly reduced stroke size and improved functional outcome in wild-type mice on day 1 and day 3 after transient middle cerebral artery occlusion. This protective effect was lost in lymphocyte-deficient Rag1(-/-) mice and in cultured neurons subjected to hypoxia. Less lymphocytes were present in the cerebral vasculature of FTY720-treated wild-type mice, which in turn reduced thrombosis and increased cerebral perfusion. In contrast, FTY720 was unable to prevent blood-brain barrier breakdown and transendothelial immune cell trafficking after transient middle cerebral artery occlusion. CONCLUSIONS: Induction of lymphocytopenia and concomitant reduction of microvascular thrombosis are key modes of FTY720 action in stroke. In contrast, our findings in Rag1(-/-) mice and cultured neurons argue against direct neuroprotective effects of FTY720.

The phosphodiesterase-4 inhibitor rolipram protects from ischemic stroke in mice by reducing blood-brain-barrier damage, inflammation and thrombosis.

Blood-brain-barrier (BBB) disruption, inflammation and thrombosis are important steps in the pathophysiology of acute ischemic stroke but are still inaccessible to therapeutic interventions. Rolipram specifically inhibits the enzyme phosphodiesterase (PDE) 4 thereby preventing the inactivation of the intracellular second messenger cyclic adenosine monophosphate (cAMP). Rolipram has been shown to relief inflammation and BBB damage in a variety of neurological disorders. We investigated the therapeutic potential of rolipram in a model of brain ischemia/reperfusion injury in mice. Treatment with 10mg/kg rolipram, but not 2 mg/kg rolipram, 2 h after 60 min of transient middle cerebral artery occlusion (tMCAO) reduced infarct volumes by 50% and significantly improved clinical scores on day 1 compared with vehicle-treated controls. Rolipram maintained BBB function upon stroke as indicated by preserved expression of the tight junction proteins occludin and claudin-5. Accordingly, the formation of vascular brain edema was strongly attenuated in mice receiving rolipram. Moreover, rolipram reduced the invasion of neutrophils as well as the expression of the proinflammatory cytokines IL-1ß and TNFα but increased the levels of TGFß-1. Finally, rolipram exerted antithrombotic effects upon stroke and fewer neurons in the rolipram group underwent apoptosis. Rolipram is a multifaceted antiinflammatory and antithrombotic compound that protects from ischemic neurodegeneration in clinically meaningful settings.

Regulatory T cells are strong promoters of acute ischemic stroke in mice by inducing dysfunction of the cerebral microvasculature.

We have recently identified T cells as important mediators of ischemic brain damage, but the contribution of the different T-cell subsets is unclear. Forkhead box P3 (FoxP3)-positive regulatory T cells (Tregs) are generally regarded as prototypic anti-inflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. In the present study, we examined the role of Tregs after experimental brain ischemia/reperfusion injury. Selective depletion of Tregs in the DEREG mouse model dramatically reduced infarct size and improved neurologic function 24 hours after stroke and this protective effect was preserved at later stages of infarct development. The specificity of this detrimental Treg effect was confirmed by adoptive transfer experiments in wild-type mice and in Rag1(-/-) mice lacking lymphocytes. Mechanistically, Tregs induced microvascular dysfunction in vivo by increased interaction with the ischemic brain endothelium via the LFA-1/ICAM-1 pathway and platelets and these findings were confirmed in vitro. Ablation of Tregs reduced microvascular thrombus formation and improved cerebral reperfusion on stroke, as revealed by ultra-high-field magnetic resonance imaging at 17.6 Tesla. In contrast, established immunoregulatory characteristics of Tregs had no functional relevance. We define herein a novel and unexpected role of Tregs in a primary nonimmunologic disease state.

Kininogen deficiency protects from ischemic neurodegeneration in mice by reducing thrombosis, blood-brain barrier damage, and inflammation.

Thrombosis and inflammation are hallmarks of ischemic stroke still unamenable to therapeutic interventions. High-molecular-weight kininogen (KNG) is a central constituent of the contact-kinin system which represents an interface between thrombotic and inflammatory circuits and is critically involved in stroke development. Kng(-/-) mice are protected from thrombosis after artificial vessel wall injury and lack the proinflammatory mediator bradykinin. We investigated the consequences of KNG deficiency in models of ischemic stroke. Kng(-/-) mice of either sex subjected to transient middle cerebral artery occlusion developed dramatically smaller brain infarctions and less severe neurologic deficits without an increase in infarct-associated hemorrhage. This protective effect was preserved at later stages of infarction as well as in elderly mice. Targeting KNG reduced thrombus formation in ischemic vessels and improved cerebral blood flow, and reconstitution of KNG-deficient mice with human KNG or bradykinin restored clot deposition and infarct susceptibility. Moreover, mice deficient in KNG showed less severe blood-brain barrier damage and edema formation, and the local inflammatory response was reduced compared with controls. Because KNG appears to be instrumental in pathologic thrombus formation and inflammation but dispensable for hemostasis, KNG inhibition may offer a selective and safe strategy for combating stroke and other thromboembolic diseases.

C1-inhibitor protects from brain ischemia-reperfusion injury by combined antiinflammatory and antithrombotic mechanisms.

BACKGROUND AND PURPOSE: Inflammation and thrombosis are pathophysiological hallmarks of ischemic stroke still unamenable to therapeutic interventions. The contact-kinin system represents an interface between inflammatory and thrombotic circuits and is involved in stroke development. C1-inhibitor counteracts activation of the contact-kinin system at multiple levels. We investigated the therapeutic potential of C1-inhibitor in models of ischemic stroke. METHODS: Male and female C57Bl/6 mice and rats of different ages were subjected to middle cerebral artery occlusion and treated with C1-inhibitor after 1 hour or 6 hours. Infarct volumes and functional outcomes were assessed between day 1 and day 7, and findings were validated by magnetic resonance imaging. Blood-brain barrier damage, thrombus formation, and the local inflammatory response were determined poststroke. RESULTS: Treatment with 15.0 U C1-inhibitor, but not 7.5 U, 1 hour after stroke reduced infarct volumes by ≈60% and improved clinical scores in mice of either sex on day 1. This protective effect was preserved at later stages of infarction as well as in elderly mice and in another species, ie, rats. Delayed C1-inhibitor treatment still improved clinical outcome. Blood-brain barrier damage, edema formation, and inflammation were significantly lower compared with controls. Moreover, C1-inhibitor showed strong antithrombotic effects. CONCLUSIONS: C1-inhibitor is a multifaceted antiinflammatory and antithrombotic compound that protects from ischemic neurodegeneration in clinically meaningful settings.

α(2)-adrenoceptors do not mediate neuroprotection in acute ischemic stroke in mice.

We assessed the neuroprotective potential of α(2)-adrenoceptors in ischemic stroke using mice with targeted deletions of individual α(2)-adrenoceptor subtypes (α(2A)(-/-), α(2B)(-/-), α(2C)(-/-), α(2A/C)(-/-)). The effects of the α(2)-adrenoceptor agonist clonidine were studied in parallel. Focal cerebral ischemia was induced with or without clonidine pretreatment by transient middle cerebral artery occlusion. Neurologic outcome and infarct volumes were evaluated on day 1. Cerebral blood flow (CBF) and mean arterial pressure were determined. α(2)-Adrenoceptor null mice did not display larger infarct volumes compared with wild-type (WT) mice under basal conditions (P>0.05). In line with this finding, pretreatment with clonidine did not protect from ischemic brain damage in WT mice or α(2A)(-/-), α(2B)(-/-), and α(2C)(-/-) mice. Clonidine induced smaller infarct volumes only in α(2A/C)(-/-) mice (P<0.05), but this did not translate into improved neurologic function (P>0.05). Importantly, while clonidine caused a significant decrease in arterial blood pressure in all groups, it had no blood pressure lowering effect in α(2A/C)(-/-) mice, and this correlated with higher CBF and smaller infarct volumes in this group. In summary, we could not demonstrate a neuroprotective function of α(2)-adrenoceptors in focal cerebral ischemia. Careful controlling of physiological parameters relevant for stroke outcome is recommended in experimental stroke studies.

Deficiency of vasodilator-stimulated phosphoprotein (VASP) increases blood-brain-barrier damage and edema formation after ischemic stroke in mice.

BACKGROUND: Stroke-induced brain edema formation is a frequent cause of secondary infarct growth and deterioration of neurological function. The molecular mechanisms underlying edema formation after stroke are largely unknown. Vasodilator-stimulated phosphoprotein (VASP) is an important regulator of actin dynamics and stabilizes endothelial barriers through interaction with cell-cell contacts and focal adhesion sites. Hypoxia has been shown to foster vascular leakage by downregulation of VASP in vitro but the significance of VASP for regulating vascular permeability in the hypoxic brain in vivo awaits clarification. METHODOLOGY/PRINCIPAL FINDINGS: Focal cerebral ischemia was induced in Vasp(-/-) mice and wild-type (WT) littermates by transient middle cerebral artery occlusion (tMCAO). Evan's Blue tracer was applied to visualize the extent of blood-brain-barrier (BBB) damage. Brain edema formation and infarct volumes were calculated from 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain slices. Both mouse groups were carefully controlled for anatomical and physiological parameters relevant for edema formation and stroke outcome. BBB damage (p<0.05) and edema volumes (1.7 mm(3)±0.5 mm(3) versus 0.8 mm(3)±0.4 mm(3); p<0.0001) were significantly enhanced in Vasp(-/-) mice compared to controls on day 1 after tMCAO. This was accompanied by a significant increase in infarct size (56.1 mm(3)±17.3 mm(3) versus 39.3 mm(3)±10.7 mm(3), respectively; p<0.01) and a non significant trend (p>0.05) towards worse neurological outcomes. CONCLUSION: Our study identifies VASP as critical regulator of BBB maintenance during acute ischemic stroke. Therapeutic modulation of VASP or VASP-dependent signalling pathways could become a novel strategy to combat excessive edema formation in ischemic brain damage.

Phosducin influences sympathetic activity and prevents stress-induced hypertension in humans and mice.

Hypertension and its complications represent leading causes of morbidity and mortality. Although the cause of hypertension is unknown in most patients, genetic factors are recognized as contributing significantly to an individual's lifetime risk of developing the condition. Here, we investigated the role of the G protein regulator phosducin (Pdc) in hypertension. Mice with a targeted deletion of the gene encoding Pdc (Pdc-/- mice) had increased blood pressure despite normal cardiac function and vascular reactivity, and displayed elevated catecholamine turnover in the peripheral sympathetic system. Isolated postganglionic sympathetic neurons from Pdc-/- mice showed prolonged action potential firing after stimulation with acetylcholine and increased firing frequencies during membrane depolarization. Furthermore, Pdc-/- mice displayed exaggerated increases in blood pressure in response to post-operative stress. Candidate gene-based association studies in 2 different human populations revealed several SNPs in the PDC gene to be associated with stress-dependent blood pressure phenotypes. Individuals homozygous for the G allele of an intronic PDC SNP (rs12402521) had 12-15 mmHg higher blood pressure than those carrying the A allele. These findings demonstrate that PDC is an important modulator of sympathetic activity and blood pressure and may thus represent a promising target for treatment of stress-dependent hypertension.

Genetic dissection of alpha2-adrenoceptor functions in adrenergic versus nonadrenergic cells.

Alpha(2)-adrenoceptors mediate diverse functions of the sympathetic system and are targets for the treatment of cardiovascular disease, depression, pain, glaucoma, and sympathetic activation during opioid withdrawal. To determine whether alpha(2)-adrenoceptors on adrenergic neurons or alpha(2)-adrenoceptors on nonadrenergic neurons mediate the physiological and pharmacological responses of alpha(2)-agonists, we used the dopamine beta-hydroxylase (Dbh) promoter to drive expression of alpha(2A)-adrenoceptors exclusively in noradrenergic and adrenergic cells of transgenic mice. Dbh-alpha(2A) transgenic mice were crossed with double knockout mice lacking both alpha(2A)- and alpha(2C)-receptors to generate lines with selective expression of alpha(2A)-autoreceptors in adrenergic cells. These mice were subjected to a comprehensive phenotype analysis and compared with wild-type mice, which express alpha(2A)- and alpha(2C)-receptors in both adrenergic and nonadrenergic cells, and alpha(2A)/alpha(2C) double-knockout mice, which do not express these receptors in any cell type. We were surprised to find that only a few functions previously ascribed to alpha(2)-adrenoceptors were mediated by receptors on adrenergic neurons, including feedback inhibition of norepinephrine release from sympathetic nerves and spontaneous locomotor activity. Other agonist effects, including analgesia, hypothermia, sedation, and anesthetic-sparing, were mediated by alpha(2)-receptors in nonadrenergic cells. In dopamine beta-hydroxylase knockout mice lacking norepinephrine, the alpha(2)-agonist medetomidine still induced a loss of the righting reflex, confirming that the sedative effect of alpha(2)-adrenoceptor stimulation is not mediated via autoreceptor-mediated inhibition of norepinephrine release. The present study paves the way for a revision of the current view of the alpha(2)-adrenergic receptors, and it provides important new considerations for future drug development.

Blockade of bradykinin receptor B1 but not bradykinin receptor B2 provides protection from cerebral infarction and brain edema.

BACKGROUND AND PURPOSE: Brain edema is detrimental in ischemic stroke and its treatment options are limited. Kinins are proinflammatory peptides that are released during tissue injury. The effects of kinins are mediated by 2 different receptors (B1 and B2 receptor [B1R and B2R]) and comprise induction of edema formation and release of proinflammatory mediators. METHODS: Focal cerebral ischemia was induced in B1R knockout, B2R knockout, and wild-type mice by transient middle cerebral artery occlusion. Infarct volumes were measured by planimetry. Evan's blue tracer was applied to determine the extent of brain edema. Postischemic inflammation was assessed by real-time reverse-transcriptase polymerase chain reaction and immunohistochemistry. To analyze the effect of a pharmacological kinin receptor blockade, B1R and B2R inhibitors were injected. RESULTS: B1R knockout mice developed significantly smaller brain infarctions and less neurological deficits compared to wild-type controls (16.8+/-4.7 mm(3) vs 50.1+/-9.1 mm(3), respectively; P<0.0001). This was accompanied by a dramatic reduction of brain edema and endothelin-1 expression, as well as less postischemic inflammation. Pharmacological blockade of B1R likewise salvaged ischemic tissue (15.0+/-9.5 mm(3) vs 50.1+/-9.1 mm(3), respectively; P<0.01) in a dose-dependent manner, even when B1R inhibitor was applied 1 hour after transient middle cerebral artery occlusion. In contrast, B2R deficiency did not confer neuroprotection and had no effect on the development of tissue edema. CONCLUSIONS: These data demonstrate that blocking of B1R can diminish brain infarction and edema formation in mice and may open new avenues for acute stroke treatment in humans.

The anesthetic effects of etomidate: species-specific interaction with alpha 2-adrenoceptors.

BACKGROUND: The IV anesthetic, etomidate, has structural and clinical similarities to specific alpha2-adrenoceptor agonists such as dexmedetomidine. We investigated whether the sedative effects of etomidate may be mediated by alpha2-adrenoceptors. METHODS: The anesthetic potency of etomidate (1-20 microM) was determined in Xenopus laevis tadpoles in the absence and presence of the specific alpha2-adrenoceptor antagonist atipamezole (10 microM). Anesthesia was defined as loss of righting reflex. Nonlinear logistic regression curves were fitted to the data and half-maximal effective concentrations and the slopes of the curves were calculated. Additionally, sedative/ hypnotic effects of etomidate (8 mg/kg IP) were studied by rotarod test in wild-type (WT) mice and mice carrying targeted deletions of the alpha2A-adrenoceptor gene (alpha2A-KO). Data are presented as mean +/- sem. RESULTS: The fraction of anesthetized tadpoles increased with increasing concentrations of etomidate. Atipamezole significantly increased the half-maximal effective concentration of etomidate (4.5 +/- 0.2 microM; slope: 2.6 +/- 0.3) to 8.4 +/- 0.4 microM (slope: 2.3 +/- 0.3). Etomidate resulted in time-dependent sedative effects in all mice, as assessed by rotarod performance. In WT mice, the sedative effects of etomidate were not decreased by atipamezole (2 mg/kg). Consistently, etomidate-induced sedation was not reduced in alpha2A-KO animals compared with WT mice. CONCLUSIONS: The sedative effects of etomidate exhibit a species-specific interaction with alpha2-adrenoceptors. Although the decrease in potency of etomidate by atipamezole may be caused by an interaction with alpha2-adrenoceptors in X. laevis tadpoles, results in mice indicate that the hypnotic effect of etomidate does not require alpha2-adrenoceptors.

Heterozygous alpha 2C-adrenoceptor-deficient mice develop heart failure after transverse aortic constriction.

OBJECTIVE: Feedback regulation of norepinephrine release from sympathetic nerves is essential to control blood pressure, heart rate and contractility. Recent experiments in gene-targeted mice have suggested that alpha(2C)-adrenoceptors may operate in a similar feedback mechanism to control the release of epinephrine from the adrenal medulla. As heterozygous polymorphisms in the human alpha(2C)-adrenoceptor gene have been associated with cardiovascular disease including hypertension and chronic heart failure, we have sought to characterize the relevance of alpha(2C)-gene copy number for feedback control of epinephrine release in gene-targeted mice. METHODS: Adrenal catecholamine release, basal hemodynamics and susceptibility to develop heart failure after transverse aortic constriction were tested in mice with two copies (+/+), one copy (+/-) or no functional alpha(2C)-adrenoceptor gene (alpha(2C)-/-). RESULTS: Heterozygous alpha(2C)-receptor deletion (alpha(2C)+/-) resulted in a 43% reduction of adrenal alpha(2C) mRNA copy number and in a similar decrease in alpha(2)-receptor-mediated inhibition of catecholamine release from isolated adrenal glands in vitro. Urinary excretion of epinephrine was increased by 74+/-15% in alpha(2C)+/- and by 142+/-23% in alpha(2C)-/- mice as compared with wild-type control mice. Telemetric determination of cardiovascular function revealed significant tachycardia but no hypertension in alpha(2C)-adrenoceptor-deficient mice. alpha(2C)+/- mice were more susceptible to develop cardiac hypertrophy, failure and mortality after left-ventricular pressure overload than alpha(2C)+/+ mice. CONCLUSION: Adrenal alpha(2)-mediated feedback regulation of epinephrine secretion differs fundamentally from sympathetic feedback control. A single adrenoceptor subtype, alpha(2C), operates without a significant receptor reserve to prevent elevation of circulating epinephrine levels. This genetic model may provide an experimental basis to study the pathophysiology of alpha(2C)-adrenoceptor dysfunction in humans.

The effects of dexmedetomidine on perinatal excitotoxic brain injury are mediated by the alpha2A-adrenoceptor subtype.

We performed the current study in mice lacking individual alpha2-adrenoceptor subtypes to elucidate the contribution of alpha(2)-adrenoceptor subtypes to the neuroprotective properties of dexmedetomidine in a model of perinatal excitotoxic brain injury. On postnatal Day 5, wild-type mice and mice lacking alpha2A-adrenoceptor (alpha2A-KO) or alpha2C-adrenoceptor subtypes (alpha2C-KO) were randomly assigned to receive dexmedetomidine (3 microg/kg) or phosphate-buffered saline intraperitoneally. Thirty minutes after the intraperitoneal injection, the glutamatergic agonist ibotenate (10 microg) was intracerebrally injected, producing transcortical necrosis and white matter lesions that mimic perinatal human hypoxic-like lesions. Quantification of the lesions was performed on postnatal Day 10 by histopathologic examination. Dexmedetomidine reduced mean lesion size in the cortex of wild-type mice and alpha2C-KO mice by 44% and 49%, respectively. Ibotenate-induced white matter lesions were reduced by 71% (wild-type mice) and 75% (alpha2C-KO mice) after pretreatment with dexmedetomidine. In contrast, in alpha2A-KO mice, dexmedetomidine did not protect against the cortical excitotoxic insult, and white matter lesions were even more pronounced (82% increase of mean lesion size). Dexmedetomidine provides potent neuroprotection in a model of perinatal excitotoxic brain damage. This effect was completely abolished in alpha2A-KO mice, suggesting that the neuroprotective effect is mediated via the alpha2A-adrenoceptor subtype.

Alpha2C-adrenoceptor polymorphism is associated with improved event-free survival in patients with dilated cardiomyopathy.

AIMS: The sympathetic nervous system plays a central role in cardiac growth but its overstimulation is associated with increased mortality in patients with chronic heart failure. Pre-synaptic alpha2-adrenoceptors are essential feedback regulators to control the release of norepinephrine from sympathetic nerves. In this study we tested whether a deletion polymorphism in the human alpha2C-adrenoceptor gene (alpha2CDel322-325) affects progression of heart failure in patients with dilated cardiomyopathy (DCM). METHODS AND RESULTS: We genotyped and phenotyped 345 patients presenting with DCM in the heart transplant unit of the German Heart Institute, starting in 1994. Patients were treated according to guidelines (99% ACEI, 76% beta-blockers) and were followed until December 2002 or until a first event [death, heart transplantation, or implantation of a left ventricular assist device (LVAD) for a life-threatening condition] occurred. Mean follow-up time was 249 weeks (4.9 years) in event-free patients and 104 weeks (2 years) in patients with events. During follow-up, 51% of the patients exhibited an event: death (18%), implantation of LVAD as bridging for transplantation (7%), or heart transplantation (25%). By Kaplan-Meier analysis, DCM patients with the deletion variant Del322-325 in the alpha2C-adrenoceptor showed significantly decreased event rates (P=0.0043). Cox regression analysis revealed that the presence of the deletion was associated with reduced death rate (relative risk: 0.129, 95% CI: 0.18-0.9441, P=0.044) and event rates (relative risk: 0.167, 95% CI: 0.041-0.685, P=0.012). CONCLUSION: Alpha2C-adrenoceptor deletion may be a novel, strong, and independent predictor of reduced event rates in DCM patients treated according to guidelines.

Pulmonary hypertension and right heart failure in pituitary adenylate cyclase-activating polypeptide type I receptor-deficient mice.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP), acting via 3 different G protein-coupled receptors, has been implicated in the regulation of several homeostatic systems in the body, including cardiopulmonary control. To define the physiologic role of the PACAP-preferring type I receptor, PAC1, in cardiopulmonary function, we developed a mutant mouse strain lacking functional PAC1 receptors. METHODS AND RESULTS: When PAC1-deficient mice were crossed onto a C57BL/6 background, almost all mutants died during the second postnatal week. Whereas mutant mice were indistinguishable from their wild-type littermates at birth, they showed progressive weakness and died from rapidly developing heart failure. Right ventricles of PAC1 mutants were massively dilated and showed cardiac myocyte hypertrophy, whereas left ventricular structure was unaltered. On direct cardiac catheterization, right ventricular pressure was elevated by 45% in PAC1-deficient mice, indicating increased pulmonary artery pressure, as no malformations were detected in the valves or outflow tract of the right ventricle. Consistent with elevated pulmonary pressure, lung capillary density was decreased by 30% and small pulmonary arteries of mutant mice had significant vascular smooth muscle cell hypertrophy compared with wild-type mice. CONCLUSIONS: Whereas PACAP induces vasodilation in isolated pulmonary vessels in wild-type mice, the absence of its specific receptor PAC1 causes pulmonary hypertension and right heart failure after birth. These in vivo findings demonstrate the crucial importance of PAC1-mediated signaling for the maintenance of normal pulmonary vascular tone during early postnatal life.

alpha2-adrenergic receptor subtypes - novel functions uncovered in gene-targeted mouse models.

The family of adrenergic receptors contains nine different subtypes of G protein-coupled receptors which mediate the biological effects of adrenaline and noradrenaline. With few exceptions, the full therapeutic potential of subtype-selective therapy has not yet been explored for the group of adrenergic receptors. In the absence of sufficiently subtype-selective ligands which can distinguish between individual receptor subtypes of the adrenergic family, gene-targeted mouse models with deletions in these receptor genes have recently been generated and characterized. These genetic mouse models have helped to assign specific pharmacological effects of alpha(2)-receptor agonists or antagonists to individual receptor subtypes. However, some unexpected and novel functions of alpha(2)-adrenergic receptors were also uncovered in these mouse models: Presynaptic control of catecholamine release from adrenergic nerves in the central and sympathetic nervous system may be regulated by three different alpha(2)-receptor subtypes, alpha(2A), alpha(2B), and alpha(2C). A similar feedback loop also controls the release of catecholamines from the adrenal gland. alpha(2B)-receptors are not only involved in regulating vascular tone in the adult organism, but they are essential for the development of the vascular system of the placenta during prenatal development. The challenge will now be to generate strategies to identify whether the findings obtained in gene-targeted mice may predict the action of receptor subtype-selective drugs in humans.

Cardiac hypertrophy is associated with decreased eNOS expression in angiotensin AT2 receptor-deficient mice.

Angiotensin II receptors play an essential role in cardiovascular physiology and disease. The significance of angiotensin type II (AT2) receptors in cardiac disease still remains elusive. Thus, we tested in gene-targeted mice whether AT2 receptors modulate cardiac function and remodeling after experimental myocardial injury. To generate myocardial infarcts of reproducible size, a cryolesion was generated at the free wall of the left ventricle of wild-type mice (Agtr2+/Y) and mice carrying a deletion of the AT2 receptor gene (Agtr2-/Y). Postinjury remodeling was followed up for 4 weeks after cryoinjury. The cryoprocedure led to an increased heart weight/body weight ratio and heart weight/tibia length ratio in AT2-deficient mice compared with control mice. Morphometric analysis revealed a significant increase in myocyte cross-sectional area after cardiac injury (infarct vs sham Agtr2+/Y, +53%; vs Agtr2-/Y, +95%). Expression of endothelial nitric oxide synthase (eNOS) was significantly lower in hearts from Agtr2-/Y than from Agtr2+/Y mice. eNOS downregulation was accompanied by a decrease in cardiac cGMP levels in Agtr2-/Y mice. In isolated murine cardiomyocytes, angiotensin II induced eNOS expression through AT2 receptors, and inhibition of NO production by NG-nitro-l-arginine methyl ester abolished the antihypertrophic effect of AT2 on cardiac myocytes. Our results demonstrate in a genetic mouse model that angiotensin II AT2 receptors exert an antihypertrophic effect in cardiac remodeling after myocardial cryoinjury and link the expression of cardiac eNOS to AT2 receptor activation.