Nina J. Solenski

Mechanisms of ischemic neuroprotection by acetyl-L-carnitine.

Abstract: Acetyl‐l‐carnitine is a naturally occurring substance that, when administered at supraphysiologic concentrations, is neuroprotective in several animal models of global and focal cerebral ischemia. Three primary mechanisms of action are supported by neurochemical outcome measures performed with these models and with in vitro models of acute neuronal cell death. The metabolic hypothesis is based on the oxidative metabolism of the acetyl component of acetyl‐l‐carnitine and is a simple explanation for the reduction in postischemic brain lactate levels and elevation of ATP seen with drug administration. The antioxidant mechanism is supported by reduction of oxidative stress markers, for example, protein oxidation, in both brain tissue and cerebrospinal fluid. The relatively uncharacterized mechanism of inhibiting excitotoxicity could be extremely important in both acute brain injury and chronic neurodegenerative disorders. New experiments performed with primary cultures of rat cortical neurons indicate that the presence of acetyl‐l‐carnitine significantly inhibits both acute and delayed cell death following exposure to NMDA, an excitotoxic glutamate antagonist. Finally, several other mechanisms of action are possible, including a neurotrophic effect of acetyl‐l‐carnitine and inhibition of mitochondrial permeability transition. While the multiple potential mechanisms of neuroprotection by acetyl‐l‐carnitine limit an accurate designation of the most important mode of action, they are compatible with the concept that several brain injury pathways must be inhibited to optimize therapeutic efficacy.

Differential Hydroxylation of Salicylate in Core and Penumbra Regions During Focal Reversible Cerebral Ischemia

BACKGROUND AND PURPOSEnFree radical-mediated damage during and/or after cerebral ischemia is thought to participate in the elaboration of stroke-related injury. To elucidate the role of this mechanism in cerebral damage, the study presented herein sought to clarify the spatial and temporal features of the free radical response to transient ischemia. With use of a reproducible model of in vivo focal ischemia/reperfusion, the time course of salicylate hydroxylation was measured in ischemic core and penumbra regions.nnnMETHODSnTransient focal cerebral ischemia was produced in Sprague-Dawley rats by occluding both carotid arteries and one middle cerebral artery for 3 hours, followed by reperfusion. Cerebral reperfusion was confirmed by visual inspection and iodo[14C]antipyrine autoradiography. A microdialysis probe was placed stereotactically in either the ischemic core or ischemic penumbra of the frontoparietal cortex; the probe was perfused with salicylate, and dialysate samples were analyzed by high-performance liquid chromatography for salicylate hydroxylation products.nnnRESULTSnSalicylate hydroxylation was significantly increased during ischemia and was further increased during 6 hours of reperfusion in the penumbra compared with sham controls. In comparison, a delayed increase in hydroxylation was observed within the ischemic core region only after 3 hours of reperfusion.nnnCONCLUSIONnA differential generation of salicylate hydroxylation occurs in core and penumbra regions in association with focal ischemia/reperfusion of the rat neocortex. The early and progressive response in the penumbra suggests that free radical mechanisms may be continuously active in the aggravation of injury in the ischemic penumbra during ischemia and reperfusion. In contrast, the relatively delayed onset of hydroxylation in the core region indicates that this mechanism participates primarily in the late stages of ischemic injury in densely ischemic tissue. These findings are consistent with the concept that the role of free radicals in cerebral injury may differ qualitatively and/or quantitatively in areas of total and partial cerebral perfusion.

Nitric oxide impairs mitochondrial movement in cortical neurons during hypoxia

Cortical nitric oxide (NO) production increases during hypoxia/ischemia in the immature brain and is associated with both neurotoxicity and mitochondrial dysfunction. Mitochondrial redistribution within the cell is critical to normal neuronal function, however, the effects of hypoxia on mitochondrial dynamics are not known. This study tested the hypothesis that hypoxia impairs mitochondrial movement via NO‐mediated pathways. Fluorescently labeled mitochondria were studied using time‐lapse digital video microscopy in cultured cortical neurons exposed either to hypoxia/re‐oxygenation or to diethyleneamine/nitric oxide adduct, DETA‐NO (100–500u2003µm). Two NO synthase inhibitors, were used to determine NO specificity. Mitochondrial mean velocity, the percentage of movement (i.e. the time spent moving) and mitochondrial morphology were analyzed. Exposure to hypoxia reduced mitochondrial movement to 10.4u2003±u20031.3% at 0u2003h and 7.4u2003±u20031.7% at 1u2003h of re‐oxygenation, versus 25.6u2003±u20031.4% in controls (pu2003<u20030.05). Mean mitochondrial velocity (µmu2003s−1) decreased from 0.374u2003±u20030.01 in controls to 0.146u2003±u20030.01 at 0u2003h and 0.177u2003±u20030.02 at 1u2003h of re‐oxygenation (pu2003<u20030.001). Exposure to DETA‐NO resulted in a significant decrease in mean mitochondrial velocity at all tested time points. Treatment with NG‐nitro‐l‐arginine methyl ester (l‐NAME) prevented the hypoxia‐induced decrease in mitochondrial movement at 0u2003h (30.1u2003±u20031.6%) and at 1u2003h (26.1u2003±u20039%) of re‐oxygenation. Exposure to either hypoxia/re‐oxygenation or NO also resulted in the rapid decrease in mitochondrial size. Both hypoxia and NO exposure result in impaired mitochondrial movement and morphology in cultured cortical neurons. As the effect of hypoxia on mitochondrial movement and morphology can be partially prevented by a nitric oxide synthase (NOS) inhibitor, these data suggest that an NO‐mediated pathway is at least partially involved.

R(+) pramipexole as a mitochondrially focused neuroprotectant: Initial early phase studies in ALS

R(+) pramipexole (PPX) is a lipophilic cation that concentrates into brain and mitochondria and efficiently scavenges reactive oxygen and nitrogen species (RONS). Under the auspices of a Physician‐Sponsor IND, R(+)PPX was dosed to small numbers of ALS patients for tolerability and safety while efficacy measures were also collected. The purpose of this paper is to describe the outcomes of these initial clinical studies. In a futility design study, 30 patients with early SALS were evaluated monthly for ALSFRS‐R scores and FVC measurements for three months during lead‐in, followed by open‐label dosing at 30u2005mg/day of R(+)PPX for the next six months. In the dose escalation study, 10 subjects with early ALS received daily doses of R(+)PPX from 10u2005mg t.i.d. to 100u2005mg t.i.d. over seven weeks. In the open‐label extension analysis, subjects from the initial studies were treated with 30u2005mg/day for at least six months, then switched to 60u2005mg/day. R(+)PPX was tolerated well in all studies. In the futility study, slopes of decline in ALSFRS‐R scores and neurophysiological index (NI) values yielded non‐significant reductions during treatment. In the dose‐escalation study, all subjects increased daily R(+)PPX intake safely to 100u2005mg t.i.d. Markers of ALS did not change (ALSFRS‐R) or improved (FVC). Trough and peak plasma (PPX) increased linearly with dosing, and several subjects achieved plasma (PPX) >1u2005µM. In the open‐label extension protocol, changing from 30 to 60u2005mg/day caused a non‐significant 17% reduction in slope of decline of ALSFRS‐R. It was concluded that R(+)PPX was tolerated well in long‐term dosing at 30 and 60u2005mg/day. Encouraging but non‐significant effects of R(+)PPX on ALS decline were observed. High doses of R(+)PPX were tolerated well and yielded neuroprotective plasma levels. These findings support longer‐term testing of higher R(+)PPX doses as a potential disease‐altering therapy for SALS.

Neurotoxic nitric oxide rapidly depolarizes and permeabilizes mitochondria by dynamically opening the mitochondrial transition pore.

Exposure of SH-SY5Y neuroblastoma or rat cortical neurons to diethylenetriamine-NO (DETA-NO) rapidly depolarized mitochondria. In SH-SY5Y DETA-NO activated caspase 3 and produced cell death. Mitochondrial depolarization in SH-SY5Y was visualized both with JC-1 accumulation and as dequenching of calcein fluorescence in mitochondria initially loaded with calcein-AM and tetramethylrhodamine methyl ester (TMRM). Calcein/TMRM-visualized mitochondrial depolarization was prevented by cyclosporin A (CsA) or approximately two-fold increased levels of BclXL protein. Dynamic imaging of mitochondrial potential (Deltapsi M) with TMRM showed that DETA-NO induced cycles of mitochondrial depolarization/repolarization (flickering). Fifteen-30 min of DETA-NO exposure caused high-frequency flickering with small peak size; 2 h of DETA-NO produced large peaks with prolonged depolarization. NO-induced flickering but not that from Bax was blocked by the calcium uniporter antagonist Ru360. Our findings show rapid-onset, dynamic regulation of Deltapsi M by NO, implying that neuroprotective therapies for brain ischemia target cell death processes downstream of effects of NO on mitochondria.

Overlapping Features of Eclampsia and Postpartum Angiopathy

IntroductionPostpartum cerebral angiopathy (PPA) is considered one of a diverse group of rare conditions termed “reversible cerebral vasoconstriction syndromes”. Existing literature suggest considerable overlap in the manifestations of eclampsia and PPA.MethodsRetrospective case series review of PPA and eclampsia from a single neurosciences intensive care unit patient log identified over a consecutive 18-month period. A MEDLINE search (using OVID) of the English literature from 1950 through October 2008 was also performed.ResultsFour patients who meet the obstetrical criteria for eclampsia and four patients whose clinical and radiographic features were consistent with PPA were identified. Twenty-eight patients with PPA were identified from the literature and showed significant clinical and radiographic overlap without cohort.ConclusionGiven the overlapping clinical, laboratory, and radiographical features of eclampsia and PPA, it is probable they share a similar underlying pathophysiological mechanism and represent different clinical expressions of the same pregnancy-related disorder. The obstetrical definition of eclampsia may be to strict when applied in the neurosciences intensive care unit.

Nitric-oxide-induced depolarization of neuronal mitochondria: implications for neuronal cell death

Nitric oxide (NO(*)) has known toxic effects on central nervous system neurons. This study characterized the effect of NO(*) on mitochondrial membrane changes by exploring the relationship among NO(*), excitatory receptor activation, and the induction of peroxynitrite, a highly toxic NO(*) reactant, to neuronal injury. Cultured rat cortical neurons were exposed to the NO(*) generator, diethylenetriamine/nitric oxide adduct, and were examined for signs of cell death, mitochondrial membrane potential changes (Deltapsi(m)), and the induction of a mitochondrial permeability transition (MPT). Neurons were also examined for nitrotyrosine (NT) immunoreactivity, a marker of reactive nitrogen species (RNS) formation. Neurons exposed to NO(*) or to N-methyl-D-aspartate (NMDA) exhibited similar rapid depolarization of mitochondria, which was prevented by an NMDA receptor antagonist. Electrophysiological studies demonstrated NO(*) potentiation of NMDA-induced NMDA receptor currents. NO(*) and NMDA-treated neurons had evidence of mitochondrial-specific NT immunoreactivity that was prevented by a SOD/catalase mimetic (EUK-134). EUK-134 treatment reduced both NO(*) and NMDA-induced NT formation and neuronal cell death. EUK-134 did not prevent NO-induced Deltapsi(m) but partially prevented NMDA-induced Deltapsi(m) loss. Although NO(*) and NMDA both induced MPT and MPT inhibitors prevented NO-induced Deltapsi(m), they did not result in significant neuroprotection, in contrast to treatment designed to decrease peroxynitrite formation. These data suggest that NO-induced NMDA receptor activation is closely linked to intramitochondrial NO-peroxynitrite/RNS formation and thereby acts as a major mediator of neuronal cell death.

The TeleStroke Mimic (TM)-Score: A Prediction Rule for Identifying Stroke Mimics Evaluated in a Telestroke Network

Background Up to 30% of acute stroke evaluations are deemed stroke mimics (SM). As telestroke consultation expands across the world, increasing numbers of SM patients are likely being evaluated via Telestroke. We developed a model to prospectively identify ischemic SMs during Telestroke evaluation. Methods and Results We analyzed 829 consecutive patients from January 2004 to April 2013 in our internal New England–based Partners TeleStroke Network for a derivation cohort, and 332 cases for internal validation. External validation was performed on 226 cases from January 2008 to August 2012 in the Partners National TeleStroke Network. A predictive score was developed using stepwise logistic regression, and its performance was assessed using receiver‐operating characteristic (ROC) curve analysis. There were 23% SM in the derivation, 24% in the internal, and 22% in external validation cohorts based on final clinical diagnosis. Compared to those with ischemic cerebrovascular disease (iCVD), SM had lower mean age, fewer vascular risk factors, more frequent prior seizure, and a different profile of presenting symptoms. The TeleStroke Mimic Score (TM‐Score) was based on factors independently associated with SM status including age, medical history (atrial fibrillation, hypertension, seizures), facial weakness, and National Institutes of Health Stroke Scale >14. The TM‐Score performed well on ROC curve analysis (derivation cohort AUC=0.75, internal validation AUC=0.71, external validation AUC=0.77). Conclusions SMs differ substantially from their iCVD counterparts in their vascular risk profiles and other characteristics. Decision‐support tools based on predictive models, such as our TM Score, may help clinicians consider alternate diagnosis and potentially detect SMs during complex, time‐critical telestroke evaluations.

Inhibition of nitric oxide generation and lipid peroxidation attenuates hemolysate-induced injury to cerebrovascular endothelium.

SummaryThe mechanisms of hemolysate-induced cerebral injury following subarachnoid hemorrhage are just beginning to be clarified. This study examined the injurious effects of hemolysate on endothelial cells derived from bovine middle cerebral arteries, and evaluated the roles of lipid peroxidation and nitric oxide production in this type of damage. Cultured endothelial cells were grown to confluency on gelatin-coated plates. The cells were characterized as endothelial cells on the basis of morphology, Factor VIII-related antigen staining, and low density lipoprotein (LDL) uptake. Additional cells were grown to confluency on collagen-coated well inserts, and were treated with hemolysate for 24 hours. Prior to hemolysate exposure, cells were treated with: a) an inhibitor of iron-dependent lipid peroxidation (tirilazad mesylate 100 μM), or b) an inhibitor of nitric oxide synthase (either N-nitro-L-arginine: NLA 300 μM, or aminoguanidine: AG at 1.5, 7.5, 15 or 150 μM). Permeability of the tracer, U-14C-sucrose, across the layer of endothelial cells was examined over a 24 hour period. Hemolysate induced a significant increase in the permeability across the endothelial cell layer. Pretreatment with tirilazad mesylate, NLA, or AG attenuated significantly hemolysate-induced changes in the endothelial cell barrier.These findings indicate that free radical generation and lipid peroxidation are critical participants in hemolysate-induced injury to the barrier function of the cerebrovascular endothelium. In addition, the results indicate that endothelial cells provide an adequate source of nitric oxide to damage their own cellular function. Finally, these findings strongly implicate free radical mechanisms in endothelial damage associated with subarachnoid hemorrhage.

A low-cost, tablet-based option for prehospital neurologic assessment: The iTREAT Study

Objectives: In this 2-center study, we assessed the technical feasibility and reliability of a low cost, tablet-based mobile telestroke option for ambulance transport and hypothesized that the NIH Stroke Scale (NIHSS) could be performed with similar reliability between remote and bedside examinations. Methods: We piloted our mobile telemedicine system in 2 geographic regions, central Virginia and the San Francisco Bay Area, utilizing commercial cellular networks for videoconferencing transmission. Standardized patients portrayed scripted stroke scenarios during ambulance transport and were evaluated by independent raters comparing bedside to remote mobile telestroke assessments. We used a mixed-effects regression model to determine intraclass correlation of the NIHSS between bedside and remote examinations (95% confidence interval). Results: We conducted 27 ambulance runs at both sites and successfully completed the NIHSS for all prehospital assessments without prohibitive technical interruption. The mean difference between bedside (face-to-face) and remote (video) NIHSS scores was 0.25 (1.00 to −0.50). Overall, correlation of the NIHSS between bedside and mobile telestroke assessments was 0.96 (0.92–0.98). In the mixed-effects regression model, there were no statistically significant differences accounting for method of evaluation or differences between sites. Conclusions: Utilizing a low-cost, tablet-based platform and commercial cellular networks, we can reliably perform prehospital neurologic assessments in both rural and urban settings. Further research is needed to establish the reliability and validity of prehospital mobile telestroke assessment in live patients presenting with acute neurologic symptoms.