Cerebrovascular Constriction Research Articles

Abstract 345: Occult Antiphospholipid Syndrome Presenting as Subdural Hematoma: A Rare Manifestation of Arterial Hypercoagulability

Objective To describe a case of antiphospholipid syndrome (APS) which presented with subdural hemorrhage, a rarely described manifestation of a commonly hypercoagulable disorder. Background APS is a common arterial and venous hypercoagulable state—though rare in the general population with an incidence of 5 per 100,000 individuals. It most commonly presents with systemic ischemic insults, commonly to the brain in the form of ischemic stroke. This case highlights a known rare manifestation of APS: intracranial hemorrhage. Design/Methods: N/A Results Patient is a 49 year old female with history of end‐stage renal disease who initially presented with altered mentation and hypertension. Her GCS was 7 upon arrival prompting intubation. CT head at that time revealed acute left parietal subdural hematoma. There was no clear history of trauma per the patient's family, and the patient was unable to provide any additional history of their own at that time. The etiology of her hemorrhage, in the acute setting, was not clearly discerned. She was managed in the ICU for several days, however she remained somnolent and unable to participate in examination despite removal of sedating agents. A repeat CT scan of the head was done at that time which revealed multifocal hypodensities consistent with late acute‐early subacute ischemic stroke. Subsequent CT angiography of the head and neck was negative for large vessel occlusive disease, extracranial atherosclerotic disease, vasospasm or other clear cerebrovascular constriction. Other embolic stroke workup including transthoracic and transesophageal echocardiogram was negative. At this point, a broader workup for embolic stroke of unclear source was launched. These studies included hypercoagulablity labs were sent and ultimately returned positive with elevated anticardiolipin and beta‐2 microglobulin consistent with an antiphospholipid antibody syndrome, prompting the initiation of anticoagulation. The patient has since improved clinically and is no longer mechanically ventilated, though does still require significant assistance with activities of daily living. Conclusions While rare, patients with otherwise unexplained hemorrhage should be investigated for possible immune‐mediated mechanisms for stroke—both ischemic and hemorrhagic.

Progesterone activation of β1-containing BK channels involves two binding sites

Progesterone (≥1 µM) is used in recovery of cerebral ischemia, an effect likely contributed to by cerebrovascular dilation. The targets of this progesterone action are unknown. We report that micromolar (µM) progesterone activates mouse cerebrovascular myocyte BK channels; this action is lost in β1-/- mice myocytes and in lipid bilayers containing BK α subunit homomeric channels but sustained on β1/β4-containing heteromers. Progesterone binds to both regulatory subunits, involving two steroid binding sites conserved in β1-β4: high-affinity (sub-µM), which involves Trp87 in β1 loop, and low-affinity (µM) defined by TM1 Tyr32 and TM2 Trp163. Thus progesterone, but not its oxime, bridges TM1-TM2. Mutation of the high-affinity site blunts channel activation by progesterone underscoring a permissive role of the high-affinity site: progesterone binding to this site enables steroid binding at the low-affinity site, which activates the channel. In support of our model, cerebrovascular dilation evoked by μM progesterone is lost by mutating Tyr32 or Trp163 in β1 whereas these mutations do not affect alcohol-induced cerebrovascular constriction. Furthermore, this alcohol action is effectively counteracted both in vitro and in vivo by progesterone but not by its oxime.

FRI086 Statin Use After Lobar Intracerebral Hemorrhage- Is It Safe?

Abstract Disclosure: D. Vedamurthy: None. M. Ambrosino: None. K. Suri: None. D. Soffer: None. D. Jacoby: None. Background: Observational studies and randomized clinical trials have yielded conflicting data on the role of statins in causing intracerebral hemorrhage. It is unclear whether it is safe to continue statins in patients who have atherosclerotic cardiovascular disease (ASCVD) when they sustain an intracerebral hemorrhage (ICH). It is also not clear whether this risk of ICH (if present) relates to statin use or “too low” levels of LDL-C. Case: 75-year-old female with history of hypertension, hyperlipidemia, and a coronary artery calcium score of 450, presented to the hospital with severe headache and was diagnosed to acute right ICH. She did not smoke, use street drugs or drink excess alcohol. She did not have a family history of premature ASCVD. Her other medication included amlodipine, rosuvastatin and aspirin. Her lipid panel at hospitalization was TC 130 mg/dl, HDL 38 mg/dl, TG 137 mg/dl and LDL-C 70 mg/dl. Imaging studies were negative for cortical venous thrombosis, reversible cerebrovascular constriction, vasculitis, aneurysm, or AV fistula as etiologies. TEE was negative for cardioembolic source. Her hypertension medications were optimized, and she was discharged without any statin therapy. She fortunately recovered with mild deficits. She enrolled in an open-label randomized study- SATURN- which aims to assess safety of statin use in recurrent ICH. Her lipid panel while off statins showed a TC 274 mg/dl, TG 159 mg/dl, markedly elevated LDL-C of 197 mg/dl, and HDL-C 47 mg/dl. She presents to the lipid clinic for risk reduction. Early studies suggested increased ICH related to very low LDL-C levels, especially in the East Asian population. Concerns about statin use and ICH sparked after post-hoc analyses of SPARCL. Data from mendelian randomization studies and meta-analysis have failed to show convincing associations between statin use and ICH. Certain genetic variants, especially related to Apo E, have been shown to enhance this risk for ICH. There is a diversity in management of patients who have sustained an ICH (especially the lobar type as opposed to deep). An open label randomized trial (SATURN) is currently recruiting patients to answer this unresolved question. The dilemma is compounded in patients who have comorbid ASCVD. There are signals that PCSK9 inhibitors may be safer in terms of less propensity to cause intracerebral hemorrhage, but more data is needed. Moreover, the very low LDL-C levels achieved with use of PCSK-9 inhibitors may be concerning. Conclusion: There are no clear major society guidelines to advice clinicians on the best way to manage patients who have sustained a lobar intracerebral hemorrhage and have a compelling reason to be on statins for lipid lowering therapy to treat ASCVD. There are no clear guidelines on how low is “too low” as far as LDL-C targets are concerned. We present this case study as a call for more robust and expedient research in this field. Presentation: Friday, June 16, 2023

Osteopontin in post-subarachnoid hemorrhage pathologies.

Rupture of intracranial aneurysms causes subarachnoid hemorrhage (SAH), of which the treatment remains the most difficult among cerebrovascular disorders even in this modern medical era. Following successful surgical ablation of ruptured intracranial aneurysms, other conditions may be encountered including delayed cerebral ischemia and chronic hydrocephalus, in addition to early brain injury. Osteopontin (OPN) is one of matricellular proteins that have cytokine-like effect on various cells and act as secretory extracellular matrix proteins between cells. The complexity of OPN functions is attributed to its several isoforms, cleavage sites and functional changes determined by its differing isoforms following various cleavages or other post-translational modifications. Notably, OPN functions beneficially or harmfully in accordance with the context of OPN upregulation. In the field of aneurysmal SAH, OPN has exerted neuroprotective effects against early brain injury and delayed cerebral ischemia by suppressing apoptosis of neurons, disruption of blood-brain barrier, and/or cerebrovascular constriction, while excessive and prolonged secretion of OPN can be harmful through the occurrence of chronic hydrocephalus requiring shunt surgery. This is a review article that is focused on OPN's potential roles in post-SAH pathologies.

Celastrol Dilates and Counteracts Ethanol-Induced Constriction of Cerebral Arteries.

The increasing recognition of the role played by cerebral artery dysfunction in brain disorders has fueled the search for new cerebrovascular dilators. Celastrol, a natural triterpene undergoing clinical trials for treating obesity, exerts neuroprotection, which was linked to its antioxidant/anti-inflammatory activities. We previously showed that celastrol fit pharmacophore criteria for activating calcium- and voltage-gated potassium channels of large conductance (BK channels) made of subunits cloned from cerebrovascular smooth muscle (SM). These recombinant BK channels expressed in a heterologous system were activated by celastrol. Activation of native SM BK channels is well known to evoke cerebral artery dilation. Current data demonstrate that celastrol (1-100 µM) dilates de-endothelialized, ex vivo pressurized middle cerebral arteries (MCAs) from rats, with EC50 = 45 µM and maximal effective concentration (Emax)= 100 µM and with MCA diameter reaching a 10% increase over vehicle-containing, time-matched values (P < 0.05). A similar vasodilatory efficacy is achieved when celastrol is probed on MCA segments with intact endothelium. Selective BK blocking with 1 μM paxilline blunts celastrol vasodilation. Similar blunting is achieved with 0.8 mM 4-aminopirydine, which blocks voltage-gated K+ channels other than BK. Using an in vivo rat cranial window, we further demonstrate that intracarotid injections of 45 μM celastrol into pial arteries branching from MCA mimics celastrol ex vivo action. MCA constriction by ethanol concentrations reached in blood during moderate-heavy alcohol drinking (50 mM), which involves SM BK inhibition, is both prevented and reverted by celastrol. We conclude that celastrol could be an effective cerebrovascular dilator and antagonist of alcohol-induced cerebrovascular constriction, with its efficacy being uncompromised by conditions that disrupt endothelial and/or BK function. SIGNIFICANCE STATEMENT: Our study demonstrates for the first time that celastrol significantly dilates rat cerebral arteries both ex vivo and in vivo and both prevents and reverses ethanol-induced cerebral artery constriction. Celastrol actions are endothelium-independent but mediated through voltage-gated (KV) and calcium- and voltage-gated potassium channel of large conductance (BK) K+ channels. This makes celastrol an appealing new agent to evoke cerebrovascular dilation under conditions in which endothelial and/or BK channel function are impaired.

Reversal of cerebrovascular constriction in experimental cerebral malaria by L-arginine

Vascular dysfunction associated with low nitric oxide (NO) biavailability and low plasma L-arginine levels is observed in both human and experimental cerebral malaria (ECM). In ECM, cerebrovascular constriction results in decreased pial blood flow and hypoxia, and administration of NO donors reverses constriction and increases survival. Supplementation of L-arginine, the substrate for NO synthesis by NO synthases, has been considered as a strategy to improve vascular health and act as adjunctive therapy in human severe malaria. We investigated the effect of L-arginine supplementation on pial vascular tonus of mice with ECM after direct superfusion on the brain surface or systemic delivery. Pial arteriolar diameters of Plasmodium berghei-infected mice with implanted cranial windows were measured using intravital microscopy methods, before and after L-arginine administration. Systemic delivery of L-arginine was performed intravenously, at 10, 50, 100 and 200 mg/kg, as bolus injection or slowly through osmotic pumps, combined or not with artesunate. Direct superfusion of L-arginine (10−7M, 10−5M and 10−3M) on the brain surface of mice with ECM resulted in immediate, consistent and dose-dependent dilation of pial arterioles. ECM mice showed marked cerebrovascular constriction that progressively worsened over a 24 h-period after subcutaneous saline bolus administration. L-arginine administration prevented the worsening in pial constriction at all the doses tested, and at 50 mg/kg and 100 mg/kg it induced temporary reversal of vasoconstriction. Slow, continuous delivery of L-arginine by osmotic pumps, or combined bolus administration of artesunate with L-arginine, also prevented worsening of pial constriction and resulted in improved survival of mice with ECM. L-arginine ameliorates pial vasoconstriction in mice with ECM.

Intracranial and Extracranial Neurovascular Manifestations of Takayasu Arteritis.

Takayasu arteritis is a rare, large-vessel vasculitis that presents with symptoms related to end-organ ischemia. While the extracranial neurovascular manifestations of Takayasu arteritis are well-established, little is known regarding the intracranial manifestations. In this study, we characterize the intracranial and cervical neurovascular radiologic findings in patients with Takayasu arteritis. Patients with Takayasu arteritis who presented to our institution between 2001 and 2016 with intracranial and/or cervical vascular imaging were included in this study. Images were evaluated for the presence of vascular abnormalities, including intracranial or extracranial stenosis, vessel-wall thickening, dissection, subclavian steal, aneurysms, infarcts, and hemorrhages. Descriptive analyses are reported. Seventy-nine patients with Takayasu arteritis met the criteria for inclusion in this study. The most common presenting neurologic symptoms were headache (32.9%) and dizziness (15.2%). Intracranial and extracranial vascular imaging was performed in 84.8% and 89.9% of patients, respectively. Among patients with intracranial vascular imaging, 3 (3.9%) had intracranial aneurysms, 3 (3.9%) had acute large-vessel occlusion, 6 (7.6%) had intracranial vasculitis, and 1 (1.3%) had reversible cerebrovascular constriction syndrome. Among patients with cervical vascular imaging, 42 (53.1%) had some degree of narrowing of the common carotid artery and 18 (22.8%) had narrowing of the ICAs. Seventeen patients (23.6%) had subclavian steal. Intracranial vascular abnormalities in patients with Takayasu arteritis presenting with neurologic symptoms are not rare, with cerebral vasculitis seen in 7.8% of patients, and stroke secondary to large-vessel occlusion, in 3.9% of patients. Cervical vascular manifestations of Takayasu arteritis were present in most patients in our study.

Endothelial Nitric Oxide Mediates Caffeine Antagonism of Alcohol-Induced Cerebral Artery Constriction.

Despite preventive education, the combined consumption of alcohol and caffeine (particularly from "energy drinks") continues to rise. Physiologic perturbations by separate intake of ethanol and caffeine have been widely documented. However, the biologic actions of the alcohol-caffeine combination and their underlying subcellular mechanisms have been scarcely studied. Using intravital microscopy on a closed-cranial window and isolated, pressurized vessels, we investigated the in vivo and in vitro action of ethanol-caffeine mixtures on cerebral arteries from rats and mice, widely recognized models to address cerebrovascular pathophysiology and pharmacology. Caffeine at concentrations found in human circulation after ingestion of one to two cups of coffee (10 µM) antagonized the endothelium-independent constriction of cerebral arteries evoked by ethanol concentrations found in blood during moderate-heavy alcohol intoxication (40-70 mM). Caffeine antagonism against alcohol was similar whether evaluated in vivo or in vitro, suggesting independence of systemic factors and drug metabolism, but required a functional endothelium. Moreover, caffeine protection against alcohol increased nitric oxide (NO•) levels over those found in the presence of ethanol alone, disappeared upon blocking NO• synthase, and could not be detected in pressurized cerebral arteries from endothelial nitric-oxide synthase knockout (eNOS(-/-)) mice. Finally, incubation of de-endothelialized cerebral arteries with the NO• donor sodium nitroprusside (10 µM) fully restored the protective effect of caffeine. This study demonstrates for the first time that caffeine antagonizes ethanol-induced cerebral artery constriction and identifies endothelial NO• as the critical caffeine effector on smooth muscle targets. Conceivably, situations that perturb endothelial function and/or NO• availability will critically alter caffeine antagonism of alcohol-induced cerebrovascular constriction without significantly disrupting endothelium-independent, alcohol-induced cerebral artery constriction itself.

Therapeutically Targeting Tumor Necrosis Factor-α/Sphingosine-1-Phosphate Signaling Corrects Myogenic Reactivity in Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is a complex stroke subtype characterized by an initial brain injury, followed by delayed cerebrovascular constriction and ischemia. Current therapeutic strategies nonselectively curtail exacerbated cerebrovascular constriction, which necessarily disrupts the essential and protective process of cerebral blood flow autoregulation. This study identifies a smooth muscle cell autocrine/paracrine signaling network that augments myogenic tone in a murine model of experimental SAH: it links tumor necrosis factor-α (TNFα), the cystic fibrosis transmembrane conductance regulator, and sphingosine-1-phosphate signaling. Mouse olfactory cerebral resistance arteries were isolated, cannulated, and pressurized for in vitro vascular reactivity assessments. Cerebral blood flow was measured by speckle flowmetry and magnetic resonance imaging. Standard Western blot, immunohistochemical techniques, and neurobehavioral assessments were also used. We demonstrate that targeting TNFα and sphingosine-1-phosphate signaling in vivo has potential therapeutic application in SAH. Both interventions (1) eliminate the SAH-induced myogenic tone enhancement, but otherwise leave vascular reactivity intact; (2) ameliorate SAH-induced neuronal degeneration and apoptosis; and (3) improve neurobehavioral performance in mice with SAH. Furthermore, TNFα sequestration with etanercept normalizes cerebral perfusion in SAH. Vascular smooth muscle cell TNFα and sphingosine-1-phosphate signaling significantly enhance cerebral artery tone in SAH; anti-TNFα and anti-sphingosine-1-phosphate treatment may significantly improve clinical outcome.

Association between reversible cerebral vasoconstriction syndrome and thrombotic thrombocytopenic purpura

BackgroundReversible cerebrovascular constriction syndrome (RCVS) presents with thunderclap headache and ischemic neurological deficits due to focal vasoconstrictions in major intracranial arteries. Reversibility of focal vasoconstrictions is the hallmark of RCVS. The commonest conditions associated with RCVS are a post-partum state and administration of vasoactive agents. MethodsWe describe a series of 3 cases of thrombotic thrombocytopenic purpura (TTP) who presented with thunderclap headache and focal neurological deficits. ResultsVascular imaging revealed focal areas of constrictions in multiple intracranial arteries. All cases were treated with plasmapheresis and serial transcranial Doppler (TCD) and magnetic resonance angiography revealed resolution of cerebral vasoconstriction, accompanied by clinical recovery. ConclusionRCVS should be considered in patients with TTP who presented with thunderclap headache. TCD is an important tool for monitoring the course of cerebral vasoconstriction in TTP.

Transdermal Glyceryl Trinitrate as an Effective Adjunctive Treatment with Artemether for Late-Stage Experimental Cerebral Malaria

Cerebral malaria (CM) is associated with low nitric oxide (NO) bioavailability, cerebrovascular constriction, occlusion, and hypoperfusion. Administration of exogenous NO partially prevents the neurological syndrome and associated vascular pathology in an experimental CM (ECM) mouse model. In this study, we evaluated the effects of transdermal glyceryl trinitrate in preventing ECM and, in combination with artemether, rescuing late-stage ECM mice from mortality. The glyceryl trinitrate and/or artemether effect on survival and clinical recovery was evaluated in C57BL/6 mice infected with P. berghei ANKA. NO synthase (NOS) expression in mouse brain was determined by Western blots. Mean arterial pressure (MAP) and pial arteriolar diameter were monitored using a tail-cuff blood pressure system and a cranial window preparation, respectively. Preventative administration of glyceryl trinitrate at 0.025 mg/h decreased ECM mortality from 67 to 11% and downregulated inducible NOS expression in the brain. When administered as adjunctive rescue therapy with artemether, glyceryl trinitrate increased survival from 47 to 79%. The adjunctive therapy caused a sustained reversal of pial arteriolar vasoconstriction in ECM mice, an effect not observed with artemether alone. Glyceryl trinitrate induced a 13% decrease in MAP in uninfected mice but did not further affect MAP in hypotensive ECM mice. Glyceryl trinitrate, when combined with artemether, was an effective adjunctive rescue treatment for ECM. This treatment ameliorated pial arteriolar vasospasm and did not significantly affect MAP. These results indicate that transdermal glyceryl trinitrate has potential to be considered as a candidate for adjunctive therapy for CM.

Abstract WP262: Therapeutically Targeting TNFa-S1P Signaling Restores Microvascular Reactivity after Experimental Subarachnoid Hemorrhage

Background: Subarachnoid hemorrhage (SAH) is characterized by an initial hemorrhagic and ischemic brain injury followed by delayed macro- and microvascular constriction. Large-artery vasospasm and enhanced microcirculatory myogenic tone may contribute to delayed cerebral ischemia. Although this implies that therapeutic interventions must specifically correct the SAH-induced myogenic tone enhancement, current therapeutic approaches non-selectively interfere with vasoconstriction and risk disrupting cerebral autoregulation. This may explain why most interventions do not improve clinical outcome. This study identifies the molecular basis for exacerbated cerebrovascular constriction and validates new targets for SAH treatment. Methods: Wild-type, tumor necrosis factor α (TNFα) knockout, sphingosine-1-kinase (Sphk1) knockout and inducible, smooth muscle cell-targeted TNFα knockout mice were used. SAH was created by injection of 80 μl of arterial blood into the prechiasmatic cistern. Myogenic tone in the olfactory artery was assessed with a myograph system. Standard procedures for fluorescent immunolocalization, Western blotting and assessment of apoptosis were used. Results: SAH increased myogenic tone and vascular wall TNFα expression, without enhancing overall vascular contractility in response to phenylephrine. Knockout of TNFα globally or smooth muscle cell-specifically prevented SAH-induced increased myogenic tone. Inhibition of TNFα-shedding (TAPI, 50 μmol/L) or receptor-binding (etanercept, 10 mg/ml) eliminated SAH-mediated myogenic tone augmentation. Cystic fibrosis transmembrane regulator (CFTR) protein expression was down-regulated in cerebral arteries after SAH, which was abolished by antagonism of TNFα. Genetic mouse models confirmed that S1P signaling mediates the myogenic tone augmentation in SAH. Finally, disrupting TNFα signaling attenuated neuronal apoptosis in SAH animals. Conclusion: We identify a novel smooth muscle cell autocrine/paracrine signaling network that augments myogenic tone in SAH. It links TNFα, CFTR and sphingosine-1-phosphate (S1P) signaling. Targeting TNFα and the S1P 2 receptor subtype are potential therapeutic options to improve clinical outcome in SAH.

Electromagnetic Treatment to Old Alzheimer's Mice Reverses β-Amyloid Deposition, Modifies Cerebral Blood Flow, and Provides Selected Cognitive Benefit

Few studies have investigated physiologic and cognitive effects of “long-term" electromagnetic field (EMF) exposure in humans or animals. Our recent studies have provided initial insight into the long-term impact of adulthood EMF exposure (GSM, pulsed/modulated, 918 MHz, 0.25–1.05 W/kg) by showing 6+ months of daily EMF treatment protects against or reverses cognitive impairment in Alzheimer's transgenic (Tg) mice, while even having cognitive benefit to normal mice. Mechanistically, EMF-induced cognitive benefits involve suppression of brain β-amyloid (Aβ) aggregation/deposition in Tg mice and brain mitochondrial enhancement in both Tg and normal mice. The present study extends this work by showing that daily EMF treatment given to very old (21–27 month) Tg mice over a 2-month period reverses their very advanced brain Aβ aggregation/deposition. These very old Tg mice and their normal littermates together showed an increase in general memory function in the Y-maze task, although not in more complex tasks. Measurement of both body and brain temperature at intervals during the 2-month EMF treatment, as well as in a separate group of Tg mice during a 12-day treatment period, revealed no appreciable increases in brain temperature (and no/slight increases in body temperature) during EMF “ON" periods. Thus, the neuropathologic/cognitive benefits of EMF treatment occur without brain hyperthermia. Finally, regional cerebral blood flow in cerebral cortex was determined to be reduced in both Tg and normal mice after 2 months of EMF treatment, most probably through cerebrovascular constriction induced by freed/disaggregated Aβ (Tg mice) and slight body hyperthermia during “ON" periods. These results demonstrate that long-term EMF treatment can provide general cognitive benefit to very old Alzheimer's Tg mice and normal mice, as well as reversal of advanced Aβ neuropathology in Tg mice without brain heating. Results further underscore the potential for EMF treatment against AD.

Impact of Aging and Life‐long Exercise on Cerebral Vasomotor Reactivity

This study investigated the impact of aging and life-long aerobic exercise training on cerebral vasomotor reactivity to changes in end-tidal CO2. We used transcranial Doppler to measure cerebral blood flow velocity (CBFV) in response to hypocapnia and hypercapnia induced by hyperventilation and CO2 rebreathing method. Eleven sedentary healthy young (SY, 27 ± 5 yr, 5 women), 10 sedentary seniors (SS, 72 ± 4 yr, 3 women) and 10 Masters Athletes (MA, 72 ± 6 yr, 1 woman) participated in this study. Compared with SY, resting CBFV was reduced and cerebrovascular resistance was increased in both SS and MA (P < 0.05). The relative changes in CBFV (%) in response to hypercapnia were greater in SS and MA than in SY (79 ± 21, 96 ± 23 vs. 56 ± 11 %, P < 0.01), but the absolute increases in CBFV were similar among 3 groups ( 39 ± 19, 41 ± 17 vs. 35 ± 8 cm/s, P > 0.2). During hypocapnia, both relative and absolute decreases in CBFV were less in SS and MA than in SY (P < 0.05). These decreases also were less in MA than in SS (P < 0.05). The cerebral vasomotor reactivity, in the sedentary and physically active elderly, was preserved or enhanced to hypercapnia, but reduced to hypocapnia. It suggests that there are probably defense mechanisms against chronic cerebrovascular constriction in the elderly which improve cerebral perfusion efficiently in hypercapnia and keep the brain of hypoperfusion in hypocapnia. This study was supported by R01HL102457-01.

New concepts regarding cerebral vasospasm: glial-centric mechanisms

Poor outcome in patients with cerebral vasospasm following subarachnoid hemorrhage remains a serious clinical problem. The current management with focus on the cerebrovascular constriction accounts for the use of "triple-H" therapy (hypertension, hypervolemia, and hemodilution) to enhance cerebral blood flow through constricted vessels. Recent work suggests that spreading depression (a stereotypical response of cerebral cortical tissue to noxious stimuli with subsequent oligemic blood flow) occurs in patients with cerebral vasospasm. A narrative review was conducted to examine the relationship between spreading depression and subarachnoid hemorrhage and to identify the anesthetic effects on the propagation of spreading depression. Following review of the literature, an underlying mechanism is advanced that cerebral vasospasm is not primarily a problem of the cerebral vasculature but a consequence of glial cell dysfunction following spreading depression - a glial-centric cause for vasospasm. Such a mechanism for vasospasm becomes manifest when spreading depression waves transition to peri-infarct depolarization waves - with protracted ischemic blood flow in compromised tissue. The extracellular microenvironment with high potassium and low nitric oxide tension can account for conducting vessel narrowing. The implication for clinical management is discussed supposing glial cell dysfunction is an underlying mechanism responsible for the vascular spasm.

Dihydropyridine-Insensitive Calcium Currents Contribute to Function of Small Cerebral Arteries

Although dihydropyridines are widely used for the treatment of vasospasm, their effectiveness is questionable, suggesting that other voltage-dependent calcium channels (VDCCs) contribute to control of cerebrovascular tone. This study therefore investigated the role of dihydropyridine-insensitive VDCCs in cerebrovascular function. Using quantitative PCR and immunohistochemistry, we found mRNA and protein for L-type (Ca(V)1.2) and T-type (Ca(V)3.1 and Ca(V)3.2) channels in adult rat basilar and middle cerebral arteries and their branches. Immunoelectron microscopy revealed both L- and T-type channels in smooth muscle cell (SMC) membranes. Using patch clamp electrophysiology, we found that a high-voltage-activated calcium current, showing T-type channel kinetics and insensitivity to nifedipine and nimodipine, comprised approximately 20% of current in SMCs of the main arteries and approximately 45% of current in SMCs from branches. Both components were abolished by the T-type antagonists mibefradil, NNC 55-0396, and efonidipine. Although nifedipine completely blocked vasoconstriction in pressurized basilar arteries, a nifedipine-insensitive constriction was found in branches and this increased in magnitude as vessel size decreased. We conclude that a heterogeneous population of VDCCs contributes to cerebrovascular function, with dihydropyridine-insensitive channels having a larger role in smaller vessels. Sensitivity of these currents to nonselective T-type channel antagonists suggests that these drugs may provide a more effective treatment for therapy-refractory cerebrovascular constriction.

The BK channel accessory β1 subunit determines alcohol-induced cerebrovascular constriction

Ethanol-induced inhibition of myocyte large conductance, calcium- and voltage-gated potassium (BK) current causes cerebrovascular constriction, yet the molecular targets mediating EtOH action remain unknown. Using BK channel-forming (cbv1) subunits from cerebral artery myocytes, we demonstrate that EtOH potentiates and inhibits current at Cai2+ lower and higher than ∼15μM, respectively. By increasing cbv1’s apparent Cai2+-sensitivity, accessory BK β1 subunits shift the activation-to-inhibition crossover of EtOH action to <3μM Cai2+, with consequent inhibition of current under conditions found during myocyte contraction. Knocking-down KCNMB1 suppresses EtOH-reduction of arterial myocyte BK current and vessel diameter. Therefore, BK β1 is the molecular effector of alcohol-induced BK current inhibition and cerebrovascular constriction.

Molecular evidence for the involvement of calcium sensitization in serotonin‐induced cerebrovascular constriction

The mechanisms contributing to serotonin (5‐HT)‐induced vasoconstriction of cerebral arteries are poorly characterized. This is important as abnormalities in control of cerebral blood flow by 5‐HT are implicated in subarachnoid hemorrhage and may be involved in migraine. Force generation by vascular smooth muscle cells depends on the balance of myosin light chain kinase (MLCK) and phosphatase (MLCP) activities that determine the level of myosin light chain (LC20) phosphorylation. Agonists that activate Rho kinase (ROK) and protein kinase C (PKC) can inhibit MLCP by phosphorylation of myosin light chain targeting subunit 1 (MYPT1) and CPI‐17, respectively. This inhibition of MLCP leads to increased phospho‐LC20 and force generation at constant [Ca2+]i, i.e. Ca2+ sensitization. Here, we have quantified LC20, MYPT1 and CPI‐17 phosphorylation in protein samples derived from pressurized segments of rat middle cerebral arteries using a novel, highly sensitive western blotting method. 5‐HT induced constriction and increased phospho‐LC20; both were reduced by ROK (H1152, 0.3 µM) or PKC (GF109203, 3 µM) inhibition. Phospho‐MYPT1 level was increased by 5‐HT, but not phospho‐CPI‐17. Our data indicate for the first time that Ca2+ sensitization via ROK‐mediated phosphorylation of MYPT1 resulting in enhanced LC20phosphorylation and force generation may contribute to 5‐HT‐induced cerebral vasoconstriction.

Arterial Smooth Muscle BK Channel Beta1 Subunits Determine Ethanol-Induced Cerebrovascular Constriction

Ethanol at concentrations obtained in circulation after binge drinking (≤50 mM) causes cerebrovascular constriction, which primarily results from ethanol-induced inhibition of arterial smooth muscle voltage- and calcium-gated potassium (BK) channels (Liu et al., 2004). Cerebrovascular myocyte BK channels are made of channel-forming α (encoded by KCNMA1) and smooth muscle-abundant β1 subunits (encoded by KCNMB1). After cloning α (“cbv1”, AY330293) and β1 (FJ154955) subunits from rat cerebral artery myocytes, we set to identify the molecular effector of ethanol-induced inhibition of channel activity and cerebrovascular constriction. Cbv1 and cbv1+β1 channels were expressed in Xenopus oocytes and channel steady-state activity (NPo) was recorded in inside-out (I/O) macropatches at a wide Ca2+i range (0.3-100 μM). Ethanol potentiated current at Ca2+i 30 μM. Beta1 subunits shifted the crossover for ethanol-induced macroscopic current potentiation to inhibition towards lower Ca2+i (≤3 μM). This shift was paralleled by a similar shift in NPo. To evaluate β1-modulation of ethanol action in native channels, we probed ethanol on BK channels in cerebral artery myocytes isolated from wt (C57BL/6) and KCNMB1 K/O mice. Recordings were performed in I/O patches, at Vm=-20, -40mV and Ca2+i=10 μM. In myocytes from wt mice (having BK made of α and β1 subunits) 50 mM ethanol significantly decreased NPo (-23.4±9%). In contrast, ethanol reversibly increased NPo in KCNMB1 K/O myocytes. Finally, we pressurized isolated mouse cerebral arteries and evaluated the impact of β1 subunit modulation of ethanol action on organ function. In vessels from wt animals ethanol caused a robust decrease in diameter (-14%). In contrast, KCNMB1 K/O vessels were resistant to this ethanol action. Our data indicate that BK β1 subunits are the functional targets mediating ethanol-induced cerebrovascular constriction. NIH Grant AA11560 (AMD).

Cholesterol-ethanol Interactions On Vascular Myocyte BK Channels: Contribution To Alcohol-induced Cerebrovascular Constriction

The activity of large conductance, calcium- and voltage-gated potassium (BK) channels critically limits the degree of vascular smooth muscle contraction, favoring cerebrovascular dilation (Brayden and Nelson, 1992). Ethanol at levels reached in circulation after moderate binge drinking (50 mM) inhibits the activity of cerebrovascular myocyte BK channels, leading to endothelium-independent cerebrovascular constriction (Liu et al., 2004). On the other hand, high cholesterol levels, which inhibit vascular smooth muscle BK channels (Bolotina et al., 1989), decrease vascular compliance, favoring vasoconstriction (Bukiya et al., 2008). Synergistic inhibition of cerebrovascular BK channels by cholesterol and ethanol would certainly have a profound negative impact on vascular compliance and dilation. Remarkably, such synergism on channel function has not been studied. Thus, we cloned BK subunits (channel-forming cbv1 and accessory, smooth muscle-abundant β1) from rat resistance-size cerebral arteries, reconstituted the channel complex into 1-palmitoyl-2-oleoyl phosphatidylethanolamine/1-palmitoyl-2-oleoyl phosphatidylserine (POPE/POPS) bilayers, and studied cholesterol modulation of ethanol action on channel steady-state activity (NPo). Acute exposure to 50 mM ethanol mildly yet significantly decreased BK NPo (−4 ± 0.8 % from control) without modifying channel unitary conductance. In the same bilayer type, incorporation of cholesterol at levels found in cell membranes (15 % w/w) also reduced BK NPo (-8.78 ± 5.2 % from control). Remarkably, 50 mM EtOH added to the cholesterol-containing bilayer resulted in a robust decrease in BK NPo (−36 ± 8.4 % from control). These data unveil a multiplicative inhibition of BK channel activity by alcohol and cholesterol. The kinetic and biophysical mechanisms of such synergism are currently being investigated.