Ali Razmara

Estrogen suppresses brain mitochondrial oxidative stress in female and male rats

Mitochondria are a major source of reactive oxygen species (ROS) and oxidative stress, key contributors to aging and neurodegenerative disorders. We report that gonadal hormones influence brain mitochondrial ROS production in both females and males. Initial experiments showed that estrogen decreases mitochondrial superoxide production in a receptor-mediated manner, as measured by MitoSOX fluorescence in differentiated PC-12 cells. We then assessed in vivo effects of gonadal hormones on brain mitochondrial oxidative stress in female and male rats. Brain mitochondria were isolated to measure a functional indicator of ROS, i.e., activity of the ROS-sensitive mitochondrial enzyme, aconitase. Gonadectomy of both males and females caused a decrease in aconitase activity, suggesting that endogenous gonadal hormones influence mitochondrial ROS production in the brain. In vivo treatment of gonadectomized animals with testosterone or dihydrotestosterone (DHT) had no effect, but estrogen replacement significantly increased aconitase activity in brain mitochondria from both female and male rats. This indicates that estrogen decreases brain mitochondrial ROS production in vivo. Sex hormone treatments did not affect protein levels of brain mitochondrial uncoupling proteins (UCP-2, 4, and 5). However, estrogen did increase the activity, but not the levels, of manganese superoxide dismutase (MnSOD), the mitochondrial enzyme that catalyzes superoxide radical breakdown, in brain mitochondria from both female and male rats. Thus, in contrast to the lack of effect of androgens on mitochondrial ROS, estrogen suppression of mitochondrial oxidative stress may influence neurological disease incidence and progression in both females and males.

Mitochondrial Effects of Estrogen are Mediated by ERα in Brain Endothelial Cells

Mitochondrial reactive oxygen species (ROS) and endothelial dysfunction are key contributors to cerebrovascular pathophysiology. We previously found that 17β-estradiol profoundly affects mitochondrial function in cerebral blood vessels, enhancing efficiency of energy production and suppressing mitochondrial oxidative stress. To determine whether estrogen specifically affects endothelial mitochondria through receptor mechanisms, we used cultured human brain microvascular endothelial cells (HBMECs). 17β-Estradiol treatment for 24 h increased mitochondrial cytochrome c protein and mRNA; use of silencing RNA for estrogen receptors (ERs) showed that this effect involved ERα, but not ERβ. Mitochondrial ROS were determined by measuring the activity of aconitase, an enzyme with an iron-sulfur center inactivated by mitochondrial superoxide. 17β-Estradiol increased mitochondrial aconitase activity in HBMECs, indicating a reduction in ROS. Direct measurement of mitochondrial superoxide with MitoSOX Red showed that 17β-estradiol, but not 17α-estradiol, significantly decreased mitochondrial superoxide production, an effect blocked by the ER antagonist, ICI-182,780 (fulvestrant). Selective ER agonists demonstrated that the decrease in mitochondrial superoxide was mediated by ERα, not ERβ. The selective estrogen receptor modulators, raloxifene and 4-hydroxy-tamoxifen, differentially affected mitochondrial superoxide production, with raloxifene acting as an agonist but 4-hydroxy-tamoxifen acting as an estrogen antagonist. Changes in superoxide by 17β-estradiol could not be explained by changes in manganese superoxide dismutase. Instead, ERα-mediated decreases in mitochondrial ROS may depend on the concomitant increase in mitochondrial cytochrome c, previously shown to act as an antioxidant. Mitochondrial protective effects of estrogen in cerebral endothelium may contribute to sex differences in the occurrence of stroke and other age-related neurodegenerative diseases.

Patterns and Predictors of Blood Pressure Treatment, Control, and Outcomes among Stroke Survivors in the United States.

BACKGROUND Expert consensus guidelines recommend antihypertensive treatment to lower secondary stroke risk, but patterns and predictors of blood pressure (BP) treatment and control among stroke survivors in the United States remain unknown. Understanding predictors of poor control can facilitate development of targeted strategies. METHODS We reviewed the prevalence and control of hypertension among adults 40 years or older with self-reported stroke who participated in the National Health and Nutrition Examination Surveys 1999-2004 with mortality follow-up through 2006. Predictors of poorly controlled BP (>140/90 mm Hg) and nontreatment were determined via logistic regression. Independent association between antihypertensive use and mortality was determined using Cox models. RESULTS Among 9145 participants, 490 reported previous stroke; 72% had known hypertension, 8% had undiagnosed hypertension, and 47% had poorly controlled BP. In multivariable analyses, age (odds ratio [OR] per year 1.06, 95% confidence interval [CI] 1.03-1.09), female sex (OR 1.70, 95% CI 1.12-2.57), non-Mexican Hispanic ethnicity (OR 4.54, 95% CI 1.76-11.70), black race (OR 3.15, 95% CI 1.59-6.25), hypercholesterolemia (OR 2.46, 95% CI 1.44-4.21), and diabetes (OR 1.96, 95% CI 1.16-3.33) were associated with poorly controlled BP. Obesity was associated with lower odds of poorly controlled BP (OR .51, 95% CI .26-.99). Non-Mexican Hispanic ethnicity (OR 7.37, 95% CI 2.25-24.10) and black race (OR 3.13, 95% CI 1.05-9.34) were predictors of nontreatment, whereas diabetes was linked to treatment (OR 3.57, 95% CI 1.21-10.43). There was no association between antihypertensive treatment and mortality after adjustment for demographics and comorbidities. CONCLUSIONS One in 2 stroke survivors in the United States has poorly controlled BP; the most vulnerable groups include women, non-Mexican Hispanics, blacks, diabetics, and older individuals. Understanding causes of this evidence-practice gap may assist in developing effective targeted interventions.

Teaching Video NeuroImages: periodic alternating nystagmus evident only in darkness.

An 81-year-old man with retinitis pigmentosa had severe, slowly progressive visual loss that began at age 20. Intermittent nystagmus was observed for 1 decade. Examination revealed severe retinal atrophy and periodic alternating nystagmus (PAN) that occurred only in darkness (video on the Neurology® Web site at www.neurology.org).

Depression Is Associated with a Higher Risk of Death among Stroke Survivors

BACKGROUND Poststroke depression is common, affecting approximately 1 in 3 stroke survivors. We aimed to evaluate the association between depression and mortality in adults with and without prior stroke. METHODS Using the National Health and Nutrition Examination Survey (NHANES) I Epidemiologic Follow-up Study (1982-1992), we investigated the association between depression and all-cause mortality among adults aged 25-74 years with and without prior stroke, and stroke mortality among stroke survivors, adjusting for covariates. RESULTS Among 9919 individuals, 121 (1.2%) reported prior stroke. The adjusted depression prevalence was 37.1% among stroke survivors and 17.3% among individuals without stroke. In persons aged 25-64 years, neither stroke nor depression was associated with all-cause mortality. The combination of depression and stroke was not associated with all-cause mortality (adjusted hazard ratio [HR] 2.83, 95% confidence interval [CI] .67-12.04). Among persons aged 65-74 years, depression alone (adjusted HR 1.24, 95% CI 1.04-1.47), stroke alone (adjusted HR 1.64, 95% CI 1.17-2.32), and the combination of depression and stroke (adjusted HR 2.28, 95% CI 1.79-2.90) were associated with all-cause mortality, consistent with an additive relationship. Among all ages, the combination of depression and stroke was associated with all-cause mortality (adjusted HR 1.93, 95% CI 1.28-2.92). Higher stroke mortality was only observed in those aged 65-74 years (adjusted HR 2.43, 95% CI 1.05-5.60). Compared with stroke survivors without depression, those with depression were ~35 times more likely to die from a stroke (adjusted HR 35.33, 95% CI 7.79-160.32). CONCLUSIONS The combination of prior stroke and depression is associated with higher all-cause mortality than either condition alone. The presence of depression after stroke increases stroke mortality 35-fold, highlighting the importance of identifying and treating depression among stroke survivors.

Vascular Neurologists as Directors of Stroke Centers in the United States

Background and Purpose— Hospital certification as primary and comprehensive stroke center is associated with improvement in care. We aimed to characterize the leadership at stroke centers nationwide to determine the proportion led by vascular neurologists, a board-recognized subspecialty focusing on stroke care. Methods— We identified hospitals in the United States holding primary and comprehensive stroke center designation as of September 2013. We contacted each hospital to identify the medical director and used data from relevant medical boards to determine specialization. Sex and date of medical school graduation were obtained from an online physician database. Results— Of the 1167 primary and 50 comprehensive stroke center hospitals certified by the Joint Commission (n=1114), Det Norske Veritas (n=68), and Healthcare Facilities Accreditation Program (n=35), we identified the director in 940 (77%). Leadership was most often by a neurologist (n=745; 79%) followed by physicians in emergency medicine (n=58; 6%) and internal medicine (n=17; 2%). Vascular neurologists (n=319) led about one-third of stroke centers. Directors were mostly men (n=764; 81%), with a median number of years after medical school graduation of 25 (interquartile range, 18–34). Comprehensive stroke centers were more likely than primary stroke centers to have leadership by vascular neurologist (77%, n=37 versus 32%, n=282; P<0.001). Conclusions— Vascular neurologist led about one-third of stroke centers. There is opportunity for vascular neurologists to increase their role in stroke center directorship.

Hospital and demographic characteristics associated with inpatient neurological services in the United States

OBJECTIVE To determine nationwide availability and factors associated with inpatient neurological services. PATIENTS AND METHODS Using the 2011 American Hospital Association survey, we determined the proportion of hospitals that provided inpatient neurological services. Demographic and household data from the 2010 national census and survey results were utilized to determine regional factors associated with the availability of inpatient neurologic services. Using rate ratios, the association was estimated using Poisson regression. Hospitals lacking emergency departments or with a bed size of less than 25 beds were excluded to focus on acute care facilities with the potential to have subspecialty services. RESULTS Of 3969 hospitals that completed the survey, 2017 (65%) provided inpatient neurological services. Hospitals with Joint Commission (JC) accreditation were 1.35 times more likely (95% CI: 1.16-1.57) to have inpatient neurological services. Compared to small hospitals (bed size 25-36), large hospitals (bed size 246-2264) were 4.53 times more likely (95% CI: 2.79-7.35) to provide inpatient neurological services. Hospitals that were the sole community provider or were non-federal governmental hospitals had a lower probability of providing inpatient neurological services with rate ratio of 0.65 (95% CI: 0.5-0.84) and 0.81 (95% CI: 0.7-0.94), respectively. CONCLUSIONS Approximately two-thirds of hospitals in this nationwide survey provided hospital-based neurological services. Larger hospitals and those with JC accreditation were more likely to provide neurological services, whereas small hospitals, sole community providers, and non-federal governmental hospitals were less likely to provide them.

History of Neuroprotection: Trials and Tribulations

Neuroprotection is a strategy of interference, antagonism, and slowing down the sequence of molecular pathophysiological processes eventually resulting in irreversible cerebral ischemia. Over the past two decades, neuroprotection in ischemic stroke has emerged as a central topic of intense experimental animal studies and clinical trials in humans. Although rigorous animal studies have provided the proof of principle that neuroprotection is achievable, the novel agents and mechanisms investigated in human clinical trials have consistently failed to demonstrate a significant beneficial effect. Here we survey key neuroprotective trials and consider the strengths and shortcomings of these studies. Agents and mechanisms considered include calcium channel blockers, glutamate antagonists, GABA agonists, antioxidants and free radical scavengers, nitric oxide signal-transduction, modulation of inflammation, hemodilution, hypothermia, albumin therapy, and magnesium therapy. These human trials of neuroprotection therapies have been disappointing, unlike successful acute stroke approaches using reperfusion therapies such as thrombolytics or clot-retrieving devices. We highlight how improved clinical trial design and translational strategies and lessons learned from these negative trials will guide future directions including better clinical trial design and patient selection, multiple agent-combination therapies, and pre-hospital intervention.

231 Gonadal Hormones Differentially Modulate Cerebrovascular Inflammation in Male Rats.

Activation of inflammatory pathways underlies cerebrovascular disease pathology. There is a well-described gender difference in stroke incidence between men and women. Also, testosterone and male gender are associated with increased stroke risk. Thus, we explored the impact of testosterone and estrogen on cerebrovascular inflammation using both in vivo and in vitro models of inflammation. We hypothesized that testosterone will augment and estrogen will suppress the expression of two vascular markers of cellular inflammation: cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Using four groups of male rats (intact, orchiectomized [ORX], estrogen-treated [ORXE], and testosterone-treated [ORXT]), we determined the effects of gonadal steroids on cerebrovascular inflammation after i.p. lipopolysaccharide (LPS) injections. Using Western analysis, the induction of inflammatory markers was decreased in blood vessels from estrogen-treated rats compared to intact or ORX males. In contrast, in cerebral vessels from testosterone-treated rats, there was significant augmentation in LPS-induced COX-2 and iNOS protein levels. Confocal microscopy was used for cellular localization of COX-2 and iNOS. ORXT rats showed increased COX-2 and iNOS immunoreactivity in endothelial and smooth muscle cells after LPS treatment. In vitro incubations with LPS of isolated pial vessels from the same animal groups demonstrated greater COX-2 induction in ORXT rats compared to ORX and ORXE. Increases in PGE2 production, a principal prostaglandin end-product of COX-2 enzymatic activity, were also greater in vessels from ORXT rats. In conclusion, testosterone and estrogen differentially contribute to cerebrovascular inflammation. Thus, this may contribute to differences in stroke morbidity and mortality between men and women. Presently, we are exploring mechanisms by which the gonadal hormones influence the proinflammatory signaling nuclear factor kappa-B (NFkB) pathway. Supported by NIH grant HL-50775.