OBJECT Delayed ischemic neurological deficits (DINDs) and cerebral vasospasm (CVS) are responsible fora poor outcome in patients with aneurysmal subarachnoid hemorrhage (SAH), most likely because of a decreased availability of nitric oxide (NO) in the cerebral microcirculation. In this study, the authors examined the effects of treatment with the NO donor molsidomine with regard to decreasing the incidence of spasm-related delayed brain infarctions and improving clinical outcome in patients with SAH. METHODS Seventy-four patients with spontaneous aneurysmal SAH were included in this post hoc analysis. Twenty-nine patients with SAH and proven CVS received molsidomine in addition to oral or intravenous nimodipine. Control groups consisted of 25 SAH patients with proven vasospasm and 20 SAH patients without. These patients received nimodipine therapy alone. Cranial computed tomography (CCT) before and after treatment was analyzed for CVS-related infarcts. A modified National Institutes of Health Stroke Scale (mNIHSS) and the modified Rankin Scale (mRS) were used to assess outcomes at a 3-month clinical follow-up. RESULTS Four of the 29 (13.8%) patients receiving molsidomine plus nimodipine and 22 of the 45 (48%) patients receiving nimodipine therapy alone developed vasospasm-associated brain infarcts (p < 0.01). Follow-up revealed a median mNIHSS score of 3.0 and a median mRS score of 2.5 in the molsidomine group compared with scores of 11.5 and 5.0, respectively, in the nimodipine group with CVS (p < 0.001). One patient in the molsidomine treatment group died, and 12 patients in the standard care group died (p < 0.01). CONCLUSIONS In this post hoc analysis, patients with CVS who were treated with intravenous molsidomine had a significant improvement in clinical outcome and less cerebral infarction. Molsidomine offers a promising therapeutic option in patients with severe SAH and CVS and should be assessed in a prospective study.

Background: This manuscript addresses the multilevel role of dietary nitrate and nitrite in health and diseases because of increased interest in dietary supplementation.Method: Review of research devoted to both beneficial but also possible deleterious effects of uncontrolled indigestion of red beet-root juice and other sources of high concentrated nitrate and nitrite.Results: The body of evidence strongly suggested that persons using nitrate/nitrite supplementation and particularly patients treated with sodium nitrite, having kidney disease or impaired methemoglobin reeducates activity, should closely monitor nitrate/nitrite indigestion.Conclusion: Oral intake of red beet-root juice, green leaved veggies, cured meat, as well as urine excretion of nitrate/nitrite, methemoglobin reeducates activity should be carefully assessed especially in patients treated with sodium nitrite.

BACKGROUND: A case of hyperacute vasospasm, indicating a poor prognosis after aneurysmal subarachnoid hemorrhage (SAH), is reported, and a review is presented of the literature addressing use of nitric oxide (NO) donors in cases of refractory vasospasm and recurrent delayed cortical ischemias (DCI). CASE DESCRIPTION: A 65-year-old woman was admitted within 1 hour after aneurysmal SAH (Hunt and Hess grade III, Fisher modified by Frontera grade IV). A hyperacute vasospasm had been confirmed arteriographically, the right middle cerebral artery (MCA) aneurysm was immediately coiled and a standard antivasospastic therapy was started. Within 48 hours, the patient developed cerebral vasospasm with DCI. Because the standard therapy failed to control clinical symptoms and to address severe vasospasm, an individualized rescue treatment with NO donors was initiated. A continuous intravenous molsidomine infusion was started and clinical stabilization was achieved for a week (Hunt and Hess grade I; World Federation of Neurological Surgeons grade I; Glasgow Coma Scale score, 15) after which vasospasm and DCI recurred. During a subsequent DCI, we escalated NO donor therapy by adding intraventricular boluses of sodium nitroprusside (SNP). Over the course of the following 22 days, 7 transient DCIs (Glasgow Coma Scale score, 8) were treated with boluses of SNP during continued molsidomine therapy and each time vasospasm and DCI were completely reversed. Despite initial poor prognosis, the clinical outcome was excellent; at 3, 6, and 12 months follow-up the patient's modified National Institutes of Health-Stroke Scale and modified Rankin Scale scores were 0, with no cognitive deficits.CONCLUSIONS: The review of the literature suggested that combined intravenous molsidomine with intraventricular SNP treatment reversed refractory, recurrent vasospasm and DCIs probably by addressing the hemoglobin NO sink effect, NO depletion, and decreased NO availability after aneurysmal SAH.

HomeJournal of the American Heart AssociationVol. 5, No. 7Dietary Nitrate and the Epidemiology of Cardiovascular Disease: Report From a National Heart, Lung, and Blood Institute Workshop Open AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citations ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toOpen AccessResearch ArticlePDF/EPUBDietary Nitrate and the Epidemiology of Cardiovascular Disease: Report From a National Heart, Lung, and Blood Institute Workshop Amrita Ahluwalia, PhD, Mark Gladwin, MD, Gary D. Coleman, PhD, Norman Hord, PhD, MPH, RD, George Howard, DrPH, Daniel B. Kim‐Shapiro, PhD, Martin Lajous, MD, PhD, Filip J. Larsen, PhD, David J. Lefer, PhD, Leslie A. McClure, PhD, Bernard T. Nolan, PhD, Ryszard Pluta, MD, PhD, Alan Schechter, MD, Chia‐Yih Wang, PhD, Mary H. Ward, PhD and Jane L. Harman, DVM, MS, PhD Amrita AhluwaliaAmrita Ahluwalia The William Harvey Research Institute, Barts & The London Medical School, Queen Mary University of London, UK Search for more papers by this author , Mark GladwinMark Gladwin Vascular Medicine Institute, Pittsburgh University, Pittsburgh, PA Search for more papers by this author , Gary D. ColemanGary D. Coleman University of Maryland, College Park, MD Search for more papers by this author , Norman HordNorman Hord Oregon State University, Corvallis, OR Search for more papers by this author , George HowardGeorge Howard University of Alabama at Birmingham, Birmingham, AL Search for more papers by this author , Daniel B. Kim‐ShapiroDaniel B. Kim‐Shapiro Wake Forest University, Winston‐Salem, NC Search for more papers by this author , Martin LajousMartin Lajous Nacional de Salud Pública de Mexico, Mexico, Albania Search for more papers by this author , Filip J. LarsenFilip J. Larsen Karolinska Institute, Stockholm, Sweden Search for more papers by this author , David J. LeferDavid J. Lefer Louisiana State University Health Sciences Center, New Orleans, LA Search for more papers by this author , Leslie A. McClureLeslie A. McClure Dornsife School of Public Health at Drexel University, Philadelphia, PA Search for more papers by this author , Bernard T. NolanBernard T. Nolan US Geological Survey, Reston, VA Search for more papers by this author , Ryszard PlutaRyszard Pluta National Institute of Neurological Disorders and Stroke, Bethesda, MD Search for more papers by this author , Alan SchechterAlan Schechter National Institute for Diabetes and Digestive and Kidney Diseases, Bethesda, MD Search for more papers by this author , Chia‐Yih WangChia‐Yih Wang National Center for Health Statistics, CDC, Hyattsville, MD Search for more papers by this author , Mary H. WardMary H. Ward National Cancer Institute, Rockville, MD Search for more papers by this author and Jane L. HarmanJane L. Harman Division of Cardiovascular Sciences, National Heart, Lung, and Blood Institute, Bethesda, MD Search for more papers by this author Originally published6 Jul 2016https://doi.org/10.1161/JAHA.116.003402Journal of the American Heart Association. 2016;5:e003402IntroductionIn view of continuing unanswered questions regarding the geographical and demographic distribution of cardiovascular disease, and recent discoveries about the effects of dietary nitrate on cardiovascular physiology, the National Heart, Lung, and Blood Institute (NHLBI) convened a workshop to identify approaches to address how best to incorporate the study of nitrate exposures into ongoing studies of cardiovascular epidemiology. The NHLBI invited speakers who had made recent contributions to the study of the functions of nitrate on the cardiovascular system, on the occurrence of nitrate in foods and drinking water, or who had expert knowledge of cardiovascular surveys with wide geographical variability and therefore the greatest potential variability in dietary and drinking water nitrate. Because of the history of research on the possible carcinogenicity of nitrite, an expert in this field was also invited. The following document is a synthesis of the material presented and discussed and of literature cited at the workshop. The workshop from which this article is derived was funded and convened by the NHLBI.From Dietary Nitrate to Nitric OxideNitrate (NO3−) is an essential plant nutrient found in soil after the fixation of atmospheric nitrogen by the action of lightning or soil microbes. As a component of plants, nitrate enters the human diet mainly through the consumption of vegetables whereas nitrite (NO2−) enters the diet through consumption of processed foodstuffs, particularly resulting from use in meat preservation.1 Dietary nitrate and nitrite confer physiological effects that have been observed by physicians for over 2000 years, and which have now been attributed to the action of nitric oxide (NO) through the recently discovered endogenous nitrate‐nitrite‐nitric oxide pathway.2 Nitric oxide was identified as an important biologically active molecule in the late 1980s as the elusive “endothelium‐derived relaxation factor.” Soon thereafter, nitric oxide was recognized as a signaling molecule involved in a vast number of physiologic processes, including regulation of blood flow and blood pressure. Nitric oxide–mediated signaling is also used to protect the heart against cellular injury or death and helps regulate mitochondrial respiration by its reversible inhibition of cytochrome c oxidase.For many years after the discovery of nitric oxide's biological role, it was assumed that the body's supply of nitric oxide was produced solely by the action of nitric oxide synthase on the amino acid, l‐arginine, so that dietary protein was the ultimate source of nitric oxide. The production of nitric oxide from l‐arginine requires molecular oxygen and a number of other critical factors, including tetrahydrobiopterin. Nitric oxide is rapidly oxidized to nitrite and nitrate, 2 reaction products that were thought to be metabolically inactive and destined only for excretion in the urine. These assumptions were wrong on 2 counts: Although oxidation of nitric oxide to nitrate and nitrite acutely terminates nitric oxide bioactivity, it is now recognized that there is a pathway that recycles these anions back into nitric oxide within the body,3 providing another important source. Because this pathway uses nitrate to produce nitrite and then nitric oxide, we now know that dietary nitrate, obtained mostly through dietary vegetable content, is an important source of the body's supply of nitric oxide. And very importantly, this pathway for producing nitric oxide from the reduction of nitrite does not require oxygen and is critical under conditions of relative hypoxia, when the vasodilatory effect of nitric oxide is most needed.Nitrate Absorption and Enterosalivary Reduction CycleNitrate occurs in the diet mostly as a component of green leafy and root vegetables. After the ingestion of a nitrate‐rich meal, this anion is almost 100% absorbed through the gastrointestinal tract. Nitrate is then extracted from the circulation by the salivary glands and actively secreted into the saliva, such that salivary nitrate concentration may be greater than 10‐fold that found in plasma.4 Analogous to the action of bacteria in the soil, bacteria in the oral cavity employ this salivary nitrate in their respiration pathway, using intrinsic nitrate reductase enzymes to reduce it to nitrite.5 Higher‐order animals are not capable of reducing nitrate to nitrite, so this crucial chemical reduction step by the oral microbiome is a marvelous example of vertebrate‐bacteria commensalism. The resulting microbial‐produced nitrite is swallowed, whereupon much of it is absorbed through the gastrointestinal tract as nitrite and some of it is reduced to nitric oxide in the stomach; the nitric oxide so produced contributes to gastric mucosa integrity and provides protection against colonization of the stomach by infectious agents.6, 7, 8Plasma nitrate levels rise immediately after a nitrate‐rich meal, with a half‐life of ≈5 hours, and plasma nitrite levels subsequently rise in parallel, slightly delayed by the enterosalivary cycling for the reduction of nitrate to nitrite.9, 10 Practitioners of ancient Chinese medical arts demonstrated empirical knowledge of this enterosalivary cycle: These healers wrote that while administering saltpeter (potassium nitrate) for heart pain, it is important for the patient to swallow any saliva that is produced.2 It has also been demonstrated that the salivary reduction of nitrate to nitrite can be inhibited by antiseptic mouthwash, which kills the necessary tongue microorganisms; the formation of nitric oxide in the stomach can also be lowered by the use of proton pump inhibitors, which diminish stomach acidity.11, 12In the stomach, some of the swallowed nitrite can alternatively be protonated to HNO2, which, in turn, spontaneously yields dinitrogen trioxide (N2O3), nitrogen dioxide (NO2), and nitric oxide (NO). The N2O3 so formed is a powerful nitrosating agent capable of donating a nitrosonium cation (NO+) to secondary and tertiary amines to form potentially carcinogenic N‐nitrosamines.13 Under ingestion conditions lacking food components that protect against nitrosation reactions, HNO2 can be protonated to H2NO2, which reacts with amides to form N‐nitrosamides. These mechanisms of endogenous nitrosation may contribute substantially to human exposure to N‐nitroso compounds.14The Production of Nitric Oxide From Circulating NitriteMost of the swallowed salivary nitrite is absorbed in the gastrointestinal tract and enters the circulation, becoming available to all tissues of the body. Nitrite is now available for reduction to the active messenger, nitric oxide, especially under conditions of physiological or pathological hypoxia, when nitric oxide cannot be produced from l‐arginine by nitric oxide synthases. The nitrite anion can be considered a circulating storage pool for nitric oxide bioactivity15 that regulates hypoxic vasodilation16 and the cellular resilience to low oxygen and ischemia.17In addition to their roles as oxygen transporters, hemoglobin and myoglobin function as allosterically regulated nitrite reductases under hypoxic conditions.16, 18, 19 Under conditions of hypoxia, blood nitrite reacts with deoxyhemoglobin to generate nitric oxide to cause vasodilation. Deoxymyoglobin serves a similar function in muscle. This effective nitrite reductase activity in the vascular space by red cells comprises an important component of the body's overall nitric oxide production. In the heart, nitric oxide availability by the nitric oxide synthase pathway is induced by exercise training; however, the increase in nitric oxide synthase is also associated with increased levels of myocardial nitrite storage. During subsequent periods of hypoxia, a condition under which nitric oxide synthase activity is inhibited, this stored nitrite is available for rapid reduction to nitric oxide to protect the heart from myocardial ischemia,20 and deoxymyoglobin has been implicated as a key nitrite reductase involved in this process.21A number of cellular enzymes/proteins regulate nitrite reduction to nitric oxide at different oxygen tensions and with organ system specificity. It is proposed that the nitrate‐nitrite‐nitric oxide pathway represents a fundamentally conserved pathway for energetics and signaling in biology. The role of molybdenum‐containing enzymes and heme‐globin superfamily proteins seem to be especially important as nitrite reductases: xanthine oxidoreductase, aldehyde oxidase, mitochondrial amidoxime reductase, and sulphite oxidase; the four mammalian molybedenum containing enzymes have all individually been shown to possess significant nitrite reductase activity.22, 23, 24 Similarly, all of the mammalian globins, hemoglobin, myoglobin, neuroglobin, cytoglobin, and the plant and Drosophila hemoglobin, are the subject of active current study.25 Studies of cytochrome C, neuroglobin, and plant hemoglobins have identified a role for heme coordination in the control of nitrite reduction to nitric oxide (ie, 6‐to‐5 coordinate regulation of nitrite binding and reduction).25 Interestingly, the predominance of these two key, but distinct, types of nitrite reductase seems to depend upon whether the activity relates to nitrite reduction in health (globins) or nitrite reduction in disease (xanthine oxidoreductase).26Physiological Effects of Exogenous Nitrate and NitriteBecause nitrate and its reduction product, nitrite, are reservoirs for nitric oxide production, many of the very important roles played by nitric oxide—including its antithrombotic and immune modulatory effect and its role in cytoprotection and vasodilation—have now been shown to be effects likewise occurring with provision of exogenous sources of nitrate and nitrite. In animal models of myocardial infarction,17, 27 heart failure,28 pulmonary hypertension,29, 30, 31 and vascular hypertrophy,32 dietary provision of nitrate or nitrite improves outcome in terms of infarct size, cardiac function and hypertrophy, pulmonary arterial pressures, and cardiac and vascular hypertrophy, respectively.Perhaps the most well‐reported and consistent observation with oral nitrate ingestion is that it lowers blood pressure. The first demonstration of this phenomenon came from a study using sodium nitrate salt solution to deliver nitrate in 17 healthy volunteers, in whom significant reductions in diastolic blood pressure (3.5 mm Hg) were evident with a dose that approximated a nitrate‐rich vegetable meal, such as a lettuce salad.33 In 2008, it was demonstrated that ingestion of nitrate‐rich beetroot juice in 14 healthy volunteers caused decreases in both systolic (≈10 mm Hg) and diastolic (≈8 mm Hg) pressures.34 Further studies demonstrated a dose‐response relationship in 19 healthy volunteers with a 4‐mmol dose representing a threshold dose with little blood pressure (BP)‐lowering effect. In contrast, in 14 patients with hypertension, delivery of just below this threshold dose (3.5 mmol) caused BP lowering similar in magnitude to that which might be achieved with antihypertensive pharmacotherapy (≈12/8 mm Hg), intimating possibly an increased potency in disease scenarios. Recent studies in 64 hypertensive patients both drug naïve and those on multiple medications demonstrate that the effects of a once‐daily dose are sustained over the long term. In this study, a single dose of dietary nitrate (beetroot juice) was consumed once‐daily for 4 weeks and the BP‐lowering effects sustained for the duration of ingestion.35 Whereas these studies together suggest clinically relevant levels of BP lowering in both healthy volunteers and hypertensive patients, further studies both of longer duration and in larger cohorts to determine the general clinical translatability across the diverse hypertensive patient profile would be of value.Ingestion of inorganic nitrate as a supplement has also captured the attention of the sports and exercise fraternity. Administration of inorganic nitrate increases the efficiency of oxidative metabolism, evident as reduced oxygen consumption, whether the body is at rest or at maximal exertion of large muscle groups.36, 37, 38 This effect is coupled to enhanced mitochondrial respiratory efficiency in human skeletal muscle and a reduced proton leak across the inner mitochondrial membrane.39 The reduction in metabolic rate seems to be strongly influenced by the active uptake of nitrate in saliva.37 Recently, based on studies showing improvement in the efficiency of oxidative metabolism, there has been a proliferation of sports supplements containing high amounts of nitrate that claim to boost athletic performance.40Dietary supplementation with nitrite also inhibits platelet reactivity and increases bleeding time, and as such, the nitrate‐nitrite‐nitric oxide pathway may provide a way to modulate blood clotting processes pharmacologically. Nitric oxide inhibits aggregation of platelets and, as shown recently,41 reduction of nitrite at 0.1 μmol/L by red cells consequently inhibits platelet aggregation and ATP release, decreases P‐selectin, and increases cGMP levels in human platelets ex vivo with various agonists, such as ADP and collagen. This inhibitory effect of nitrite on platelet aggregation is enhanced by deoxygenation of the red cells; this inhibition of platelet aggregation is, in turn, blocked by a nitric oxide inhibitor. Because erythrocytic hemoglobin must be partially deoxygenated in order to reduce nitrite to NO, this suggests a basis for the known differences in arterial and venous blood clotting. In murine models, platelet reactivity is inversely correlated with plasma levels of these 2 anions, especially nitrite levels. Dietary nitrate administration to healthy human subjects results in a reduction in platelet reactivity assessed ex vivo in response to ADP and collagen,42 an effect that is critically dependent upon the enterosalivary circuit and elevation of circulating nitrite levels.34Exogenous nitrite as a source of nitric oxide has recently emerged as a promising therapy to attenuate myocardial injury and improve cardiac performance in the setting of heart failure. In most cardiovascular diseases, including heart failure, endothelial nitric oxide synthase activity is significantly attenuated, resulting in depletion of both nitric oxide and nitrite within the myocardium; and this loss of nitric oxide–mediated signaling contributes to the pathogenesis of acute myocardial infarction and heart failure. However, it has been demonstrated that oral administration of sodium nitrite protects mice against myocardial ischemia‐reperfusion injury, as well as against chronic cardiac hypertrophy and heart failure in mice subjected to pressure overload. Infused nitrite similarly protected mice against both hepatic and cardiac ischemia‐reperfusion injury.17 More recently, efforts to translate these latter observations to the clinical setting have produced mixed results in patients presenting with ST‐elevated myocardial infarction. Intravenous infusion of sodium nitrite before primary percutaneous coronary intervention (PPCI) proffered no benefit in terms of infarct size,43 but intracoronary administration44 resulted in reductions in infarct size in a subgroup of patients classified with an occluded culprit artery at the time of PPCI.With respect to heart failure, very recent research indicates positive effects of dietary nitrate, through beetroot juice delivery, on exercise capacity45 and endurance46 effects that may be related to improvements specifically in skeletal muscle activity.47 In a related manner, evidence suggests potential for the nitrate‐nitrite‐nitric oxide pathway in the therapeutics of pulmonary hypertension. Indeed, inhaled nitrite reverses hypoxic neonatal pulmonary hypertension in sheep48 and dietary nitrite and nitrate exert similar effects in mice with pulmonary hypertension induced either by hypoxia or bleomycin.30 In humans, inhaled and oral nitrite may be able to prevent and reverse established pulmonary arterial hypertension29; phase 2 proof‐of‐concept trials for this indication are currently in progress in the United States and Europe.The effects of nitrate‐rich beetroot juice on aging of the human cardiovascular system has been investigated. A high nitrate diet in older persons did not alter global cerebral perfusion, but did lead to increased regional cerebral perfusion in frontal lobe white matter.49 In older chronic obstructive pulmonary disease patients given beetroot juice, exercise time at constant work load was significantly longer compared to those given placebo.50 However, a significant lowering of BP was not observed in older adults with controlled hypertension undergoing supervised exercise therapy while consuming a high‐nitrate beverage compared to those on supervised exercise consuming a placebo beverage. All subjects underwent supervised exercise training 3 times per week for 6 weeks. Exercise lowered BP and improved other vascular measures, but dietary nitrate had no additional beneficial effects.51Exogenous administration of nitrite may have a role in attenuation of the ischemic injury to the brain poststroke. Since its discovery, nitric oxide has captured the attention of neuroscientists because of its many roles in the brain: the regulation of brain blood flow,52, 53 the inhibition of platelet activation as a crucial event in cerebral embolism, cerebral ischemia,54 mediation of inflammatory response,55 limitation of reperfusion injury,56 activity as a reactive oxygen species scavenger,57 role in synapse‐less neurotransmission,58 and as a regulator of blood–brain barrier permeability in brain tumors.59 Early investigations focused on the presence and activity of various nitric oxide synthase enzymes in the brain.60 Subsequent to recent elucidation of endogenous nitric oxide production pathways, interest has shifted toward brain‐specific effects of reduction of nitrite to nitric oxide that depends on local hypoxia61 and the presence of deoxygenated hemoglobin, neuroglobin,62, 63 and other enzymes.64 Several seminal studies have confirmed the feasibility of exogenous nitrite or nitrate as a physiological source of nitric oxide to limit ischemic damage to brain after stroke and reperfusion.16, 65, 66, 67, 68Dietary Nitrate and Potential Cardiovascular BenefitsThe aforementioned trials demonstrating beneficial effects of exogenous nitrate and nitrite suggest that a habitual ingestion of high‐nitrate‐containing foods may have beneficial effects on cardiovascular disease risk. Humans are exposed to nitrate on a daily basis through their diet because vegetables are a rich source of nitrate, with especially high amounts of nitrate found in green leafy vegetables and root vegetables. Accordingly, studies have demonstrated efficacy of oral nitrate delivered through dietary components, such as beetroots, as demonstrated in the experiments described previously.Epidemiological studies have consistently found that fruit‐and‐vegetable–rich diets are associated with lower BP and lower risk of ischemic stroke and ischemic heart disease.69, 70, 71 Although these effects have been demonstrated in many populations, the exact mechanism of this protection remains unknown. Epidemiological studies that have sufficient numbers of persons and adequate dietary data to examine the protective effects of various categories of fruits and vegetables find that green leafy vegetables appear to confer the highest degree of protection against cardiovascular disease.69 Green leafy vegetables are also the major source of nitrate in the diet of most Americans.72There is ecological evidence for a hypothesis that the cardioprotective benefit of vegetables may be conferred by nitrate. For example, a traditional Japanese diet is very high in nitrate from vegetables, and Japan has historically lower rates of coronary heart disease than the United States.73 In contrast, India, a country with a high incidence of coronary heart disease, has low levels of nitrate in the traditional diet, similar to a typical US diet.74 Usual dietary consumption of nitrate in the United States is estimated to be between 40 and 100 mg/day, with nitrite ingestion at much lower levels (0 to 20 mg/day).72 Dietary patterns associated with BP lowering, such as the Dietary Approaches to Stop Hypertension diet, used vegetable combinations with ≈160 mg of nitrate/day75 and could contain dietary nitrate concentrations ranging from 174 to 1222 mg/day depending upon whether low‐ or high‐nitrate food choices were used.72 As such, it is biologically plausible that habitual dietary nitrate concentration may, in part, explain population risk for cardiovascular disease.Although 80% to 85% of nitrate is derived from vegetables, nitrite is obtained mostly through cured meats in the diet. For centuries, meats have been preserved using nitrite to protect against botulism and to confer a red color to meat. Responding to concerns about the potential for formation of N‐nitrosamines, US food regulations in 1978 and 1986 lowered the level of residual nitrite allowable in processed meat.76 These revisions succeeded in substantially lowering nitrite level in cured meat: Surveys during 1971–1972 yielded mean nitrite levels between 26 and 64 parts per million (ppm)77; by 2012, mean nitrite in US cured meat products had declined to a range of 0.1 to 11 ppm.78 In a 1975 study, notwithstanding the higher levels of nitrite in processed meat of that era, White calculated that 80% of nitrite that entered the stomach was derived from saliva, rather than directly from the diet.77To better assess the relation between dietary nitrate and nitrite and cardiovascular disease, cohort studies with both accurate dietary nitrate assessment and cardiovascular event characterization are needed. Two studies that collect dietary and health data from a large, geographically diverse sample of the US population were reviewed at the current workshop. The Reasons for Geographic and Racial Differences in Stroke (REGARDS) Study and The National Health and Nutrition Examination Surveys (NHANES) may offer opportunities to study the epidemiological relation between dietary nitrate intake and coronary risk and outcomes. REGARDS is following 30 239 black and white participants age 45+, sampled from residents in 60.3% of US counties.79 Follow‐up for cardiovascular case ascertainment is conducted by means of hospitalization abstractions and death certificate examination. NHANES assesses the health and nutritional status from a cross‐sectional sample of adults and children in the United States, combining personal interviews with standardized physical examinations and laboratory tests.80 Every year, a nationally representative sample of ≈5000 individuals of all ages completes the survey. There is no follow‐up for subsequent clinical events, but, annually, all current and past survey participants are matched with the National Death Index to ascertain eventual cause of death. In addition, NHANES data are linked to Medicare administrative records so that information on cardiovascular events among older adults may also be examined.However, exposure estimates for dietary nitrate and nitrite in cohorts such as these remain difficult because of the lack of widely applicable dietary databases that include the nitrate and nitrite content of common foods.81 Compared to other dietary nutrients, there can be substantial variability in nitrate composition between samples of the same vegetable species. During growth, the plant takes up nitrate from the roots and accumulates varying amounts of nitrate in different parts of the plant. Overall, nitrate flux in plants is controlled both by internal nutritional status and by external abiotic factors, including water, light, and external nitrate concentration. Variability in nitrate content of a consumed vegetable can result from varietal differences, differences in growing temperature and amounts of rainfall or irrigation, the amount of nitrogen‐containing fertilizers or amount of nitrate in irrigation water, and differences in postharvest storage and handling.82 Such factors of variability will, for example, inevitably result in vastly different nitrate content of any 1 vegetable from 1 farm to another let alone from 1 state to another. Analyses that evaluate health outcomes within any cohort as a function of habitual dietary nitrate ingestion may need to incorporate contemporaneous measures of this variability when assessing the nitrate composition of dietary components.Nitrite and Infant MethemoglobinemiaNotwithstanding the likely cardiovascular benefits of nitrate from dietary vegetables, high levels of dietary nitrate or nitrite can be problematic for very young infants. In the newborn, both serum nitrate and nitrite are at very low concentrations for the first few weeks of their lives.83 It is well established that excessive gastrointestinal nitrite absorption puts young infants at risk of methemoglobinemia (met‐Hb) because of a neonate's high proportion of fetal hemoglobin and relative lack of metHb‐reducing capacity.84 Very young infants, with their undeveloped oral microbiome, do not produce nitrite from salivary nitrate.83 But preformed nitrite from improperly stored vegetable purees fed to young infants, or nitrite formed in their stomach from overgrowth of nitrate‐reducing bacteria, can cause MetHb. The higher pH of a young infant's stomach allows its colonization by nitrate‐reducing bacteria usually found in the lower gastrointestinal tract,84 thereby increasing the likelihood of excessive nitrite absorption when high‐nitrate vegetable purees are fed or when high‐nitrate water is used to prepare infant feeding formula.85, 86, 87, 88 This “well‐water cyanosis” has been found to occur only in the first few weeks of life, in infants with gastric pH >4, and in whom the upper gastrointestinal tract has been populated by nitrate‐reducing organisms.89Contrary to the situation for absorbed nitrite healthy infants appear to be quite tolerant of orally ingested nitrate in the absence of bacterial contamination or coinfection. In fact, some commercially prepared infant formulas have been found to exceed the World Health Organization recommended standard for adult nitrate exposure.90 A review conducted by the Canadian Food and Drug Directorate cited studies in which healthy infants as young as 3.5 to 8 months, when fed high‐nitrate vegetables, absorbed the nitrate rapidly in the upper gastrointestinal tract and excreted them in the urine.91 This review concluded, however, that because of their unique physiology and predilection to upper gastrointestinal bacterial colonization, infants young

Animal modeling of human disease has a long history but remains controversial especially when using a sensitive species. Despite these controversies, in a stroke research, a nonprimate model has been recognized as a most successful and useful for developing new treatments. Among stroke models, a nonhuman primate model of delayed cerebral vasospasm after subarachnoid blood clot placement has a very unique position as it revealed important pathomechanisms and led to development of several crucial clinical trials. In 1989, we adopted this model to study pathophysiology and develop a treatment against delayed cerebral vasospasm after intracranial aneurysm rupture (aSAH). In this chapter, we presented detailed descriptions of the animal treatment according to the National Institutes of Health guidance, techniques of anesthesia, cerebral arteriography, suboccipital puncture for cerebrospinal fluid collection, a surgical clot placement along the middle cerebral artery as well as postoperative care, euthanasia, and autopsy. Moreover, to accommodate the recent clinical findings, strongly suggesting a limited role of delayed cerebral vasospasm on the outcome of aSAH, we proposed a modification of the model, which addressed some mechanisms of ultra and early damage to the brain evoked by an intracranial aneurysm rupture.

Despite ongoing extensive and promising research to prevent and treat cerebrovascular vasospasm and delayed ischemic neurological deficits (DIND) after aneurysmal subarachnoid hemorrhage (aSAH), clinical outcomes remain unsatisfying. Neuroprotective strategies developed in basic science research laboratories need to be translated from bench-to-bedside using appropriate animal models. While a primate model is widely accepted as the best animal model mimicking development of delayed cerebral vasospasm after aSAH, its worldwide usage has dramatically decreased because of ethical and financial limitations. However, the use of primate models of subarachnoid hemorrhage (SAH) remains a recommended bridge for translation of early preclinical studies in rodents to human clinical trials. This paper discusses the technical aspects as well as advantages and disadvantages of a blood clot placement model of subarachnoid hemorrhage in non-human primates.

Aneurismal subarachnoid hemorrhage (SAH) is relatively rare form of hemorrhagic stroke, which produces significant social and medical challenges. As it affects people in their high productivity age and leaves 50 % of them dead and almost 70 % of survivors disabled, many of them severely, the reasons of such a dismal outcome have been intensively researched all over the world. Nevertheless, despite more than a half a century of clinical and scientific effort and dramatic improvement of surgical repair of aneurysms, the causes of poor outcome remain enigmatic. Introduction of numerous in vitro and in vivo models to study the unleashed by SAH mechanisms that injured the brain significantly advanced our understanding of biology of cerebral vessels, brain responses to intracranial pressure changes, and the presence of blood clot in subarachnoid space. One of the most important animal models that significantly contributed to those advances has been a non-human primate model introduced at the Bryce Weir laboratory in the University of Alberta, Canada, in 1984. Since then, this model, with some modifications, has been successfully used in several animal laboratories in the USA, Canada, and Japan. We present the model characteristics and describe in details medical, surgical, imagining techniques that we have used at the Surgical Neurology Branch of the National Institute of Neurological Disorders and Stroke from 1989.

Brain injury of diverse etiology is frequently encountered clinically. Management, thus, is diverse. Transient global ischemic (TGI) brain injury may result from cardiac arrest where cerebral perfusion diminishes to the point that blood supply can no longer meet the metabolic demand of the brain or from aneurysmal subarachnoid hemorrhage where hemorrhage from an intracranial aneurysm elevates the intracranial pressure above the blood pressure leading to momentary perfusion arrest. Though there are commonalities and differences in the etiology and pathogenesis of these two brain injuries, whether they required differential management or they can be grouped together is not clear. Some risk factors of TGI and SAH are shared and common and others are unique to the injury. Shared risk factors include high blood pressure, smoking, alcohol abuse, and stress. The risk factors unique to TGI are clinical conditions such as cardiac arrest (major cause), shock that creates prolonged hypoxia or hypoglycemia, pathologically elevated cerebral metabolic rate, or decreased cerebral perfusion pressure. The risk factor unique to SAH is the presence of an intracranial aneurysm. Other contrasting risk factors are age and gender; whereas old age increases the risk of TGI, SAH occurs in a younger population with an average age of 52–55 years. Women harbor significantly more intracranial aneurysms than men and consequently are more frequently the victims of SAH. Although the average age of women with SAH is greater than men, the outcome is similar [1]. In contrast, more men than women are at risk of ischemic stroke and women with ischemic stroke are usually older and more likely to die of stroke than men [2]. Brain injury after TGI can be separated into an initial ischemic phase that lasts for the duration that the brain blood supply remains reduced (usually ≤10 minutes, otherwise death is inevitable) and the reperfusion phase that begins immediately after reconstitution of cerebral blood supply (>10 minutes). The initial phase of brain injury after SAH is more complex and lasts for 48 to 72 hours. A complex series of events occurs during this initial (early) phase, including blood-induced mechanical trauma, oxyhemoglobin (released upon degradation of blood) induced oxidative stress, inflammation, and ischemia [3]. A delayed phase of brain injury, unique to SAH, develops 3–7 days after SAH. This injury is characterized by angiographic vasospasm and delayed cerebral ischemia [4]. In order to help understand the management of brain injury after TGI and SAH, this special issue compares and contrasts the various mechanisms of brain injury after TGI and SAH. It presents two original research articles and 7 reviews. The research article by C. S. Jung et al. studies the correlation of serum and CSF injury markers with ischemic events in SAH patients and that by S. O. Eicker et al. compares neuroprotective qualities of vascular endothelial drive growth factor (VEGF) against stroke and cerebral vasospasm after SAH. F. A. Sehba and R. M. Pluta review the existing TGI and aSAH animal models and present a modified aSAH model which effectively mimics the disease and has the potential of becoming a better resource for studying the brain injury mechanisms and developing a treatment. M. A. Kamp et al. review the mechanisms and clinical significance of the alteration in calcium and potassium channel after SAH and TGI. M. K. Tso and R. L. Macdonald review preclinical studies on microvascular changes and their therapeutic modification following SAH and TGI. N. Plesnila compares and contrasts pathophysiological events occurring in experimental models of SAH and TGI and evaluates the contribution and importance of global cerebral ischemia in the pathophysiology of SAH. M. Koide et al. summarize the current knowledge regarding the impact of SAH and global ischemia on neurovascular communication. J. A. Frontera reviews clinical trials in cardiac arrest and SAH and concludes that clinical trials in SAH assessing acute brain injury are conducted and that these trials may receive benefit from interventions identified successfully against brain injury following cardiac arrest. We hope that the present special issue stimulates further research in this topic and brings to attention the information that would provide a better understanding of the management of TGI and SAH patients. Fatima A. Sehba Ryszard M. Pluta R. Loch Macdonald

The discovery of tissue plasminogen activator to treat acute stroke is a success story of research on preventing brain injury following transient cerebral ischemia (TGI). That this discovery depended upon development of embolic animal model reiterates that proper stroke modeling is the key to develop new treatments. In contrast to TGI, despite extensive research, prevention or treatment of brain injury following aneurysmal subarachnoid hemorrhage (aSAH) has not been achieved. A lack of adequate aSAH disease model may have contributed to this failure. TGI is an important component of aSAH and shares mechanism of injury with it. We hypothesized that modifying aSAH model using experience acquired from TGI modeling may facilitate development of treatment for aSAH and its complications. This review focuses on similarities and dissimilarities between TGI and aSAH, discusses the existing TGI and aSAH animal models, and presents a modified aSAH model which effectively mimics the disease and has a potential of becoming a better resource for studying the brain injury mechanisms and developing a treatment.

In preclinical studies, infusion of sodium nitrite delivers nitric oxide (NO) as treatment of vasospasm after subarachnoid hemorrhage. We evaluated safety and toxicity of intravenous nitrite administration in healthy volunteers infused with increasing doses of sodium nitrite for 48 h. Twelve volunteers (5 men, 7 women; mean age was 38.8 years, range 27-56 years) participated in the study. The starting sodium nitrite dose was 4.2 mg/kg/h, and it was doubled for each subsequent volunteer up to a maximal dose of 533.8 mg/kg/h at which a clinically silent dose-limiting toxicity (DLT) was observed. Toxicity included a transient decrease of mean arterial blood pressure or asymptomatic increase of methemoglobin level above 5%. The maximal tolerated dose (MTD) was 267 mg/kg/h. S-Nitrosothiols increased significantly in plasma, confirming in vivo sodium nitrite reduction to NO and encouraging its use against vasospasm and ischemia-reperfusion injury to the brain, kidneys, liver, and heart.

Aneurysmal subarachnoid hemorrhage (aSAH) is a medical emergency that accounts for 5% of all stroke cases. Individuals affected are typically in the prime of their lives (mean age 50 years). Approximately 12% of patients die before receiving medical attention, 33% within 48 h and 50% within 30 days of aSAH. Of the survivors 50% suffer from permanent disability with an estimated lifetime cost more than double that of an ischemic stroke. Traditionally, spasm that develops in large cerebral arteries 3-7 days after aneurysm rupture is considered the most important determinant of brain injury and outcome after aSAH. However, recent studies show that prevention of delayed vasospasm does not improve outcome in aSAH patients. This finding has finally brought in focus the influence of early brain injury on outcome of aSAH. A substantial amount of evidence indicates that brain injury begins at the aneurysm rupture, evolves with time and plays an important role in patients' outcome. In this manuscript we review early brain injury after aSAH. Due to the early nature, most of the information on this injury comes from animals and few only from autopsy of patients who died within days after aSAH. Consequently, we began with a review of animal models of early brain injury, next we review the mechanisms of brain injury according to the sequence of their temporal appearance and finally we discuss the failure of clinical translation of therapies successful in animal models of aSAH. (C) 2012 Elsevier Ltd. All rights reserved.

BACKGROUND AND PURPOSE:For many years survival and neurological functionality of patients were the main outcome measures after treatment of intracranial aneurysms. But, the variable outcomes of patients operated on in a delayed fashion or before the aneurysm rupture indicate that more precise measures are needed for assessment of not only the neurological but also the neuropsychological outcome. However, development and testing of such new tools requires better understanding of pathomechanisms of neurobehavioral changes evoked by aneurysmal subarachnoid hemorrhage (aSAH), which can be achieved using animal models.METHODS:We reviewed and selected (1) animal models developed to investigate delayed cerebral vasospasm that could be useful for examining effects of brain injury evoked by aSAH and (2) a battery of neurobehavioral animal testing that can be used for assessment of patients after aSAH.RESULTS:For every species used as an aSAH model, a battery of neurobehavioral test exists.CONCLUSION:Albeit some limitations must be recognized, research using animal models of SAH should continue to play a critical role in assessment of cognitive and behavioral functions after aSAH.

Infusion of sodium nitrite could provide sustained therapeutic concentrations of nitric oxide (NO) for the treatment of a variety of vascular disorders. The study was developed to determine the safety and feasibility of prolonged sodium nitrite infusion.Healthy volunteers, aged 21 to 60 years old, were candidates for the study performed at the National Institutes of Health (NIH; protocol 05-N-0075) between July 2007 and August 2008. All subjects provided written consent to participate. Twelve subjects (5 males, 7 females; mean age, 38.8±9.2 years (range, 21-56 years)) were intravenously infused with increasing doses of sodium nitrite for 48 hours (starting dose at 4.2 µg/kg/hr; maximal dose of 533.8 µg/kg/hr). Clinical, physiologic and laboratory data before, during and after infusion were analyzed.The maximal tolerated dose for intravenous infusion of sodium nitrite was 267 µg/kg/hr. Dose limiting toxicity occurred at 446 µg/kg/hr. Toxicity included a transient asymptomatic decrease of mean arterial blood pressure (more than 15 mmHg) and/or an asymptomatic increase of methemoglobin level above 5%. Nitrite, nitrate, S-nitrosothiols concentrations in plasma and whole blood increased in all subjects and returned to preinfusion baseline values within 12 hours after cessation of the infusion. The mean half-life of nitrite estimated at maximal tolerated dose was 45.3 minutes for plasma and 51.4 minutes for whole blood.Sodium nitrite can be safely infused intravenously at defined concentrations for prolonged intervals. These results should be valuable for developing studies to investigate new NO treatment paradigms for a variety of clinical disorders, including cerebral vasospasm after subarachnoid hemorrhage, and ischemia of the heart, liver, kidney and brain, as well as organ transplants, blood-brain barrier modulation and pulmonary hypertension.http://www.clinicaltrials.gov; NCT00103025.

Cerebral vessels may regulate cerebral blood flow by responding to changes in carbon dioxide (CO2) through nitric oxide (NO) production. To better determine the role of NO production by human adult cerebral microvascular endothelial cells and human fetal astrocytes under different CO2 conditions, we studied endothelial cell and astrocyte production of NO under hypo-, normo- and hypercapnic conditions. Human cerebral endothelial cell and fetal astrocyte cultures were exposed to hypocapnic (pCO2 21.7 ± 6.7 mmHg), normocapnic (pCO2 40.1 ± 0.9 mmHg) and hypercapnic (pCO2 56.3 ± 8.7 mmHg) conditions. NO production was recorded continuously over 24 hours with stable pH. N-nitro-l-arginine [NLA; a nitric oxide synthase (NOS) inhibitor] and l-arginine (substrate for NO production via NOS) were used to further define the role of NOS in chemoregulation. NO levels in endothelial cells increased during hypercapnia by 36% in 8 hours and remained 25% above baseline. NO increase in astrocytes was 30% after 1 hour but returned to baseline at 8 hours. NLA blocked NO increase in endothelial cells under hypercapnia. During hypocapnia, NO levels in the endothelial cells decreased by 30% at 8 hours but were unchanged in astrocytes. l-arginine prevented NO decrease in endothelial cells under hypocapnia. NO changes in the endothelial cells correlated with changes in pCO2 (R = 0.99) and were independent of pH. This study suggests that cerebral endothelial cells and astrocytes release NO under normocapnic conditions and NO production is increased during hypercapnia and decreased during hypocapnia independent of pH. Further, this demonstrates that endothelial cells may play a pivotal role in chemoregulation by modulating NOS activity.

Reduced intra- and perivascular availability of nitric oxide (NO) significantly contributes to the multifactorial pathophysiology of cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH). The short half-life of NO demands its therapeutic substitution via NO donors. Classic NO donors such as sodium nitroprusside and nitroglycerin cannot be used as routine therapeutics because of serious side effects. Thus, a new generation of NO donors has been the subject of experimental investigations to avoid the drawbacks of the classic drugs. The purpose of this paper is to review the characteristics of different NO donors with regard to their promise and potential consequences in treating cerebral vasospasm. Additional novel concepts to increase NO concentrations, such as the activation of endothelial nitric oxide synthase (eNOS), are discussed.

OBJECT:Subarachnoid hemorrhage (SAH)-induced vasospasm is a significant underlying cause of aneurysm rupture-related morbidity and death. While long-term intravenous infusion of sodium nitrite (NaNO(2)) can prevent cerebral vasospasm after SAH, it is not known if the intravenous administration of this compound can reverse established SAH-induced vasospasm. To determine if the intravenous infusion of NaNO(2) can reverse established vasospasm, the authors infused primates with the compound after SAH-induced vasospasm was established. METHODS:Subarachnoid hemorrhage-induced vasospasm was created in 14 cynomolgus macaques via subarachnoid implantation of a 5-ml blood clot. On Day 7 after clot implantation, animals were randomized to either control (saline infusion, 5 monkeys) or treatment groups (intravenous NaNO(2) infusion at 300 μg/kg/hr for 3 hours [7 monkeys] or 8 hours [2 monkeys]). Arteriographic vessel diameter was blindly analyzed to determine the degree of vasospasm before, during, and after treatment. Nitric oxide metabolites (nitrite, nitrate, and S-nitrosothiols) were measured in whole blood and CSF. RESULTS:Moderate-to-severe vasospasm was present in all animals before treatment (control, 36.2% ± 8.8% [mean ± SD]; treatment, 45.5% ± 12.5%; p = 0.9). While saline infusion did not reduce vasospasm, NaNO(2) infusion significantly reduced the degree of vasospasm (26.9% ± 7.6%; p = 0.008). Reversal of the vasospasm lasted more than 2 hours after cessation of the infusion and could be maintained with a prolonged infusion. Nitrite (peak value, 3.7 ± 2.1 μmol/L), nitrate (18.2 ± 5.3 μmol/L), and S-nitrosothiols (33.4 ± 11.4 nmol/L) increased significantly in whole blood, and nitrite increased significantly in CSF. CONCLUSIONS:These findings indicate that the intravenous infusion of NaNO(2) can reverse SAH-induced vasospasm in primates. Further, these findings indicate that a similar treatment paradigm could be useful in reversing cerebral vasospasm after aneurysmal SAH.

BACKGROUND:In human autopsy studies, 70% to 80% of patients with aneurysmal subarachnoid hemorrhage (SAH) showed infarcts in cerebral cortex covered by subarachnoid blood. Thus far, no animal model of SAH is known to produce this peculiar infarct pattern, and its pathogenesis remains enigmatic. OBJECTIVE:To investigate whether such infarcts occur in the clot model of SAH in primates. METHODS:We performed a retrospective pathological review of 16 primate brains. In 13 cynomolgus monkeys, a blood clot was placed around the middle cerebral artery after additional removal of the arachnoid membrane from the basal surface of the frontal and temporal cortexes. Three animals underwent sham surgery without placement of a blood clot (controls). The brains were harvested between days 1 and 28 after SAH and examined by a neuropathologist blinded to study group. RESULTS:We identified 2 types of cortical infarcts. A band of selective cortical laminar necrosis parallel to the cortical surface ("horizontal") was found in 5 animals. The second category of cortical lesions had a "vertical" extension. It included wedge-shaped (n = 2) or pillarlike (n = 2) necrosis. Both horizontal and vertical infarcts were located exclusively in areas adjacent to subarachnoid blood. The presence of a cortical infarct did not correlate with the degree of middle cerebral artery vasospasm (r2 = .24, P = .13). CONCLUSION:The presence of cortical infarcts suggests that a modified nonhuman primate model of SAH is suitable to examine the pathogenesis of proximal vasospasm and permits investigation of cortical lesions similar to those reported in patients after SAH. Furthermore, it indicates that direct effects of the blood clot on the brain and microcirculation contribute to the development of cortical infarcts after SAH.

Delayed vasospasm that develops 3–7 days after aneurysmal subarachnoid hemorrhage (SAH) has traditionally been considered the most important determinant of delayed ischemic injury and poor outcome. Consequently, most therapies against delayed ischemic injury are directed towards reducing the incidence of vasospasm. The clinical trials based on this strategy, however, have so far claimed limited success; the incidence of vasospasm is reduced without reduction in delayed ischemic injury or improvement in the long-term outcome. This fact has shifted research interest to the early brain injury (first 72 h) evoked by SAH. In recent years, several pathological mechanisms that activate within minutes after the initial bleed and lead to early brain injury are identified. In addition, it is found that many of these mechanisms evolve with time and participate in the pathogenesis of delayed ischemic injury and poor outcome. Therefore, a therapy or therapies focused on these early mechanisms may not only prevent the early brain injury but may also help reduce the intensity of later developing neurological complications. This manuscript reviews the pathological mechanisms of early brain injury after SAH and summarizes the status of current therapies.