Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release.

Microglial cells react early to a neurotoxic insult. However, the bioactive factors and the cell-cell interactions leading to microglial activation and finally to a neuroprotective or neurodegenerative outcome remain to be elucidated. Therefore, we analyzed the microglial reaction induced by methylmercury (MeHgCl) using cell cultures of different complexity. Isolated microglia were found to be directly activated by MeHgCl (10(-10) to 10(-6) M), as indicated by process retraction, enhanced lectin staining, and cluster formation. An association of MeHgCl-induced microglial clusters with astrocytes and neurons was observed in three-dimensional cultures. Close proximity was found between the clusters of lectin-stained microglia and astrocytes immunostained for glial fibrillary acidic protein (GFAP), which may facilitate interactions between astrocytes and reactive microglia. In contrast, immunoreactivity for microtubule-associated protein (MAP-2), a neuronal marker, was absent in the vicinity of the microglial clusters. Interactions between astrocytes and microglia were studied in cocultures treated for 10 days with MeHgCl. Interleukin-6 release was increased at 10(-7) M of MeHgCl, whereas it was decreased when each of these two cell types was cultured separately. Moreover, addition of IL-6 to three-dimensional brain cell cultures treated with 3 x 10(-7) M of MeHgCl prevented the decrease in immunostaining of the neuronal markers MAP-2 and neurofilament-M. IL-6 administered to three-dimensional cultures in the absence of MeHgCl caused astrogliosis, as indicated by increased GFAP immunoreactivity. Altogether, these results show that microglial cells are directly activated by MeHgCl and that the interaction between activated microglia and astrocytes can increase local IL-6 release, which may cause astrocyte reactivity and neuroprotection.

Nutrición enteral en el paciente neurológico: ¿es suficiente el contenido en vitamina D en las fórmulas de uso habitual?

INTRODUCTION Vitamin D deficiency produces inadequate bone mineralization, proximal muscle weakness, abnormal gait and increased risk of falls and fractures. Moreover, in epidemiological studies, has been associated with increased risk of cancer, autoimmune diseases, type 1 and 2 diabetes, rheumatoid arthritis, multiple sclerosis, infectious diseases, cardiovascular diseases and depression. When synthesis through the skin by sun exposure is not possible and the patient can not eat by mouth, as in the advanced stages of various neurological diseases, the supply of vitamin D has to be done by enteral nutrition. OBJECTIVES The aim of this study is to review the role of vitamin D in a common group of neurological conditions that often require artificial nutrition and analyze whether the vitamin D of different enteral nutrition formulas is adequate to meet the needs of this group of patients. RESULTS Numerous studies have shown the association between vitamin D deficiency and increased incidence of dementia, stroke and other neurodegenerative diseases. Interventions aimed to increase levels of vit. D and its effects on functional (falls, pain, quality of life) and cardiovascular goals (cardiovascular death, stroke, myocardial infarction, cardiovascular risk factors) have obtained as highlight data a clear reduction of falls and fractures, while the evidence for the other parameters studied is still limited and inconsistent. The content of calcium and vitamin D of enteral formulas is legislated in our country. The total amount of vitamin D for a daily intake of 1,500-2,000 kcal ranges between 300 and 1,600 IU/d (mean ± SD: 32.9 ± 8.5 mg/100 kcal) in the complete formulas for enteral nutrition most commonly used. 50% of the diets studied, for an intake of 2,000 kcal/d, and 90% for an intake of 1,500 kcal/d, provide less than 600 IU/d of vitamin D. DISCUSSION Some revised recently guidelines published recommendations of daily intake of vitamin D. The document published by the U.S. Institute of Medicine recommended for adults between 19 and 70 years, 600 IU/d and up from 70, proposes 800 IU/d of vitamin D. These amounts are deemed insufficient by other scientific societies to state that to achieve blood levels of 25 (OH) D equal or greater than 30 ng/ml may be required a daily intake of 1,500-2,000 IU and a number two or three times higher if previous deficiency exists. CONCLUSIONS Further controlled studies are needed to ascertain which is the appropriate dose of vitamin D in advanced stages of neurological disease, where sun exposure is difficult and unlikely. We suggest that the vitamin D content should probably be reconsidered in enteral nutrition formulas, which, in light of recent publications appear as clearly insufficient for standard energy intakes (1,500-2,000 kcal).

Neurological complications of infective endocarditis: risk factors, outcome, and impact of cardiac surgery: a multicenter observational study.

BACKGROUND The purpose of this study was to assess the incidence of neurological complications in patients with infective endocarditis, the risk factors for their development, their influence on the clinical outcome, and the impact of cardiac surgery. METHODS AND RESULTS This was a retrospective analysis of prospectively collected data on a multicenter cohort of 1345 consecutive episodes of left-sided infective endocarditis from 8 centers in Spain. Cox regression models were developed to analyze variables predictive of neurological complications and associated mortality. Three hundred forty patients (25%) experienced such complications: 192 patients (14%) had ischemic events, 86 (6%) had encephalopathy/meningitis, 60 (4%) had hemorrhages, and 2 (1%) had brain abscesses. Independent risk factors associated with all neurological complications were vegetation size ≥3 cm (hazard ratio [HR] 1.91), Staphylococcus aureus as a cause (HR 2.47), mitral valve involvement (HR 1.29), and anticoagulant therapy (HR 1.31). This last variable was particularly related to a greater incidence of hemorrhagic events (HR 2.71). Overall mortality was 30%, and neurological complications had a negative impact on outcome (45% of deaths versus 24% in patients without these complications; P<0.01), although only moderate to severe ischemic stroke (HR 1.63) and brain hemorrhage (HR 1.73) were significantly associated with a poorer prognosis. Antimicrobial treatment reduced (by 33% to 75%) the risk of neurological complications. In patients with hemorrhage, mortality was higher when surgery was performed within 4 weeks of the hemorrhagic event (75% versus 40% in later surgery). CONCLUSIONS Moderate to severe ischemic stroke and brain hemorrhage were found to have a significant negative impact on the outcome of infective endocarditis. Early appropriate antimicrobial treatment is critical, and transitory discontinuation of anticoagulant therapy should be considered.

Genetic variation in the Cytbgene of human cerebral Taenia soliumcysticerci recovered from clinically and radiologically heterogeneous patients with neurocysticercosis

Neurocysticercosis (NC) is a clinically and radiologically heterogeneous parasitic disease caused by the establishment of larval Taenia solium in the human central nervous system. Host and/or parasite variations may be related to this observed heterogeneity. Genetic differences between pig and human-derived T. solium cysticerci have been reported previously. In this study, 28 cysticerci were surgically removed from 12 human NC patients, the mitochondrial gene that encodes cytochrome b was amplified from the cysticerci and genetic variations that may be related to NC heterogeneity were characterised. Nine different haplotypes (Ht), which were clustered in four haplogroups (Hg), were identified. Hg 3 and 4 exhibited a tendency to associate with age and gender, respectively. However, no significant associations were found between NC heterogeneity and the different T. solium cysticerci Ht or Hg. Parasite variants obtained from patients with similar NC clinical or radiological features were genetically closer than those found in groups of patients with a different NC profile when using the Mantel test. Overall, this study establishes the presence of genetic differences in the Cytb gene of T. solium isolated from human cysticerci and suggests that parasite variation could contribute to NC heterogeneity.

Impact of obesity-related genes in Spanish population

Background: The objective was to investigate the association between BMI and single nucleotide polymorphisms previously identified of obesity-related genes in two Spanish populations. Forty SNPs in 23 obesity-related genes were evaluated in a rural population characterized by a high prevalence of obesity (869 subjects, mean age 46 yr, 62% women, 36% obese) and in an urban population (1425 subjects, mean age 54 yr, 50% women, 19% obese). Genotyping was assessed by using SNPlex and PLINK for the association analysis. Results: Polymorphisms of the FTO were significantly associated with BMI, in the rural population (beta 0.87, p-value <0.001). None of the other SNPs showed significant association after Bonferroni correction in the two populations or in the pooled analysis. A weighted genetic risk score (wGRS) was constructed using the risk alleles of the Tag-SNPs with a positive Beta parameter in both populations. From the first to the fifth quintile of the score, the BMI increased 0.45 kg/m2 in Hortega and 2.0 kg/m2 in Pizarra. Overall, the obesity predictive value was low (less than 1%). Conclusion: The risk associated with polymorphisms is low and the overall effect on BMI or obesity prediction is minimal. A weighted genetic risk score based on genes mainly acting through central nervous system mechanisms was associated with BMI but it yields minimal clinical prediction for the obesity risk in the general population.

Repeating with the right hemisphere: reduced interactions between phonological and lexical-semantic systems in crossed aphasia?

Knowledge on the patterns of repetition amongst individuals who develop language deficits in association with right hemisphere lesions (crossed aphasia) is very limited. Available data indicate that repetition in some crossed aphasics experiencing phonological processing deficits is not heavily influenced by lexical-semantic variables (lexicality, imageability, and frequency) as is regularly reported in phonologically-impaired cases with left hemisphere damage. Moreover, in view of the fact that crossed aphasia is rare, information on the role of right cortical areas and white matter tracts underpinning language repetition deficits is scarce. In this study, repetition performance was assessed in two patients with crossed conduction aphasia and striatal/capsular vascular lesions encompassing the right arcuate fasciculus (AF) and inferior frontal-occipital fasciculus (IFOF), the temporal stem and the white matter underneath the supramarginal gyrus. Both patients showed lexicality effects repeating better words than non-words, but manipulation of other lexical-semantic variables exerted less influence on repetition performance. Imageability and frequency effects, production of meaning-based paraphrases during sentence repetition, or better performance on repeating novel sentences than overlearned clichés were hardly ever observed in these two patients. In one patient, diffusion tensor imaging disclosed damage to the right long direct segment of the AF and IFOF with relative sparing of the anterior indirect and posterior segments of the AF, together with fully developed left perisylvian white matter pathways. These findings suggest that striatal/capsular lesions extending into the right AF and IFOF in some individuals with right hemisphere language dominance are associated with atypical repetition patterns which might reflect reduced interactions between phonological and lexical-semantic processes.

Efficacy of lisdexamfetamine dimesylate throughout the day in children and adolescents with attention-deficit/hyperactivity disorder: results from a randomized, controlled trial.

Lisdexamfetamine dimesylate (LDX) is a long-acting, prodrug stimulant therapy for patients with attention-deficit/hyperactivity disorder (ADHD). This randomized placebo-controlled trial of an optimized daily dose of LDX (30, 50 or 70 mg) was conducted in children and adolescents (aged 6-17 years) with ADHD. To evaluate the efficacy of LDX throughout the day, symptoms and behaviors of ADHD were evaluated using an abbreviated version of the Conners' Parent Rating Scale-Revised (CPRS-R) at 1000, 1400 and 1800 hours following early morning dosing (0700 hours). Osmotic-release oral system methylphenidate (OROS-MPH) was included as a reference treatment, but the study was not designed to support a statistical comparison between LDX and OROS-MPH. The full analysis set comprised 317 patients (LDX, n = 104; placebo, n = 106; OROS-MPH, n = 107). At baseline, CPRS-R total scores were similar across treatment groups. At endpoint, differences (active treatment - placebo) in least squares (LS) mean change from baseline CPRS-R total scores were statistically significant (P < 0.001) throughout the day for LDX (effect sizes: 1000 hours, 1.42; 1400 hours, 1.41; 1800 hours, 1.30) and OROS-MPH (effect sizes: 1000 hours, 1.04; 1400 hours, 0.98; 1800 hours, 0.92). Differences in LS mean change from baseline to endpoint were statistically significant (P < 0.001) for both active treatments in all four subscales of the CPRS-R (ADHD index, oppositional, hyperactivity and cognitive). In conclusion, improvements relative to placebo in ADHD-related symptoms and behaviors in children and adolescents receiving a single morning dose of LDX or OROS-MPH were maintained throughout the day and were ongoing at the last measurement in the evening (1800 hours).

Pharmacological blockade of either cannabinoid CB1 or CB2 receptors prevents both cocaine-induced conditioned locomotion and cocaine-induced reduction of cell proliferation in the hippocampus of adult male rat.

Addiction to major drugs of abuse, such as cocaine, has recently been linked to alterations in adult neurogenesis in the hippocampus. The endogenous cannabinoid system modulates this proliferative response as demonstrated by the finding that pharmacological activation/blockade of cannabinoid CB1 and CB2 receptors not only modulates neurogenesis but also modulates cell death in the brain. In the present study, we evaluated whether the endogenous cannabinoid system affects cocaine-induced alterations in cell proliferation. To this end, we examined whether pharmacological blockade of either CB1 (Rimonabant, 3 mg/kg) or CB2 receptors (AM630, 3 mg/kg) would affect cell proliferation [the cells were labeled with 5-bromo-2'-deoxyuridine (BrdU)] in the subventricular zone (SVZ) of the lateral ventricle and the dentate subgranular zone (SGZ). Additionally, we measured cell apoptosis (as monitored by the expression of cleaved caspase-3) and glial activation [by analyzing the expression of glial fibrillary acidic protein (GFAP) and Iba-1] in the striatum and hippocampus during acute and repeated (4 days) cocaine administration (20 mg/kg). The results showed that acute cocaine exposure decreased the number of BrdU-immunoreactive (ir) cells in the SVZ and SGZ. In contrast, repeated cocaine exposure reduced the number of BrdU-ir cells only in the SVZ. Both acute and repeated cocaine exposure increased the number of cleaved caspase-3-, GFAP- and Iba1-ir cells in the hippocampus, and this effect was counteracted by AM630 or Rimonabant, which increased the number of BrdU-, GFAP-, and Iba1-ir cells in the hippocampus. These results indicate that the changes in neurogenic, apoptotic and gliotic processes that were produced by repeated cocaine administration were normalized by pharmacological blockade of CB1 and CB2. The restorative effects of cannabinoid receptor blockade on hippocampal cell proliferation were associated with the prevention of the induction of conditioned locomotion but not with the prevention of cocaine-induced sensitization.

Combining rimonabant and fentanyl in a single entity: preparation and pharmacological results.

Based on numerous pharmacological studies that have revealed an interaction between cannabinoid and opioid systems at the molecular, neurochemical, and behavioral levels, a new series of hybrid molecules has been prepared by coupling the molecular features of two wellknown drugs, ie, rimonabant and fentanyl. The new compounds have been tested for their affinity and functionality regarding CB1 and CB2 cannabinoid and μ opioid receptors. In [(35)S]-GTPγS (guanosine 5'-O-[gamma-thio]triphosphate) binding assays from the post-mortem human frontal cortex, they proved to be CB1 cannabinoid antagonists and μ opioid antagonists. Interestingly, in vivo, the new compounds exhibited a significant dual antagonist action on the endocannabinoid and opioid systems.

Localization of peroxisome proliferator-activated receptor alpha (PPARα) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) in cells expressing the Ca(2+)-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus.

The N-acylethanolamines (NAEs), oleoylethanolamide (OEA) and palmithylethanolamide (PEA) are known to be endogenous ligands of PPARα receptors, and their presence requires the activation of a specific phospholipase D (NAPE-PLD) associated with intracellular Ca(2+) fluxes. Thus, the identification of a specific population of NAPE-PLD/PPARα-containing neurons that express selective Ca(2+)-binding proteins (CaBPs) may provide a neuroanatomical basis to better understand the PPARα system in the brain. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the co-existence of NAPE-PLD/PPARα and the CaBPs calbindin D28k, calretinin and parvalbumin in the rat hippocampus. PPARα expression was specifically localized in the cell nucleus and, occasionally, in the cytoplasm of the principal cells (dentate granular and CA pyramidal cells) and some non-principal cells of the hippocampus. PPARα was expressed in the calbindin-containing cells of the granular cell layer of the dentate gyrus (DG) and the SP of CA1. These principal PPARα(+)/calbindin(+) cells were closely surrounded by NAPE-PLD(+) fiber varicosities. No pyramidal PPARα(+)/calbindin(+) cells were detected in CA3. Most cells containing parvalbumin expressed both NAPE-PLD and PPARα in the principal layers of the DG and CA1/3. A small number of cells containing PPARα and calretinin was found along the hippocampus. Scattered NAPE-PLD(+)/calretinin(+) cells were specifically detected in CA3. NAPE-PLD(+) puncta surrounded the calretinin(+) cells localized in the principal cells of the DG and CA1. The identification of the hippocampal subpopulations of NAPE-PLD/PPARα-containing neurons that express selective CaBPs should be considered when analyzing the role of NAEs/PPARα-signaling system in the regulation of hippocampal functions.

Insulin and NPY pathways and the control of GnRH function and puberty onset.

Energy balance exerts a critical influence on reproductive function. Leptin and insulin are among the metabolic factors signaling the nutritional status of an individual to the hypothalamus, and their role in the overall modulation of the activity of GnRH neurons is increasingly recognized. As such, they participate to a more generalized phenomenon: the signaling of peripheral metabolic changes to the central nervous system. The physiological importance that the interactions occurring between peripheral metabolic factors and the central nervous system bear for the control of food intake is increasingly recognized. The central mechanisms implicated are the focus of attention of very many research groups worldwide. We review here the experimental data that suggest that similar mechanisms are at play for the metabolic control of the neuroendocrine reproductive function. It is appearing that metabolic signals are integrated at the levels of first-order neurons equipped with the proper receptors, ant that these neurons send their signals towards hypothalamic GnRH neurons which constitute the integrative element of this network.

Lipid changes in HIV-patients switching to the coformulated single tablet FTC/RPV/TDF (Eviplera®). Efficacy and safety analysis. GeSida Study 8114.

INTRODUCTION Rilpivirine (RPV) has a better lipid profile than efavirenz (EFV) in naïve patients (1). Switching to RPV may be convenient for many patients, while maintaining a good immunovirological control (2). The aim of this study was to analyze lipid changes in HIV-patients at 24 weeks after switching to Eviplera® (emtricitabine/RPV/tenofovir disoproxil fumarate [FTC/RPV/TDF]). MATERIALS AND METHODS Retrospective, multicentre study of a cohort of asymptomatic HIV-patients who switched from a regimen based on 2 nucleoside reverse transcriptase inhibitors (NRTI)+protease inhibitor (PI)/non nucleoside reverse transcriptase inhibitor (NNRTI) or ritonavir boosted PI monotherapy to Eviplera® during February-December, 2013; all had undetectable HIV viral load for ≥3 months prior to switching. Patients with previous failures on antiretroviral therapy (ART) including TDF and/or FTC/3TC, with genotype tests showing resistance to components of Eviplera®, or who had changed the third drug of the ART during the study period were excluded. Changes in lipid profile and cardiovascular risk (CVR), and efficacy and safety at 24 weeks were analyzed. RESULTS Among 305 patients included in the study, 298 were analyzed (7 cases were excluded due to lack of data). Men 81.2%, mean age 44.5 years, 75.8% of HIV sexually transmitted. 233 (78.2%) patients switched from a regimen based on 2 NRTI+NNRTI (90.5% EFV/FTC/TDF). The most frequent reasons for switching were central nervous system (CNS) adverse events (31.0%), convenience (27.6%) and metabolic disorders (23.2%). At this time, 293 patients have reached 24 weeks: 281 (95.9%) have continued Eviplera®, 6 stopped it (3 adverse events, 2 virologic failures, 1 discontinuation) and 6 have been lost to follow up. Lipid profiles of 283 cases were available at 24 weeks and mean (mg/dL) baseline vs 24 weeks are: total cholesterol (193 vs 169; p=0.0001), HDL-c (49 vs 45; p=0.0001), LDL-c (114 vs 103; p=0.001), tryglycerides (158 vs 115; p=0.0001), total cholesterol to HDL-c ratio (4.2 vs 4.1; p=0.3). CVR decreased (8.7 vs 7.5%; p= 0.0001). CD4 counts were similar to baseline (653 vs 674 cells/µL; p=0.08), and 274 (96.8%) patients maintained viral suppression. CONCLUSIONS At 24 weeks after switching to Eviplera®, lipid profile and CVR improved while maintaining a good immunovirological control. Most subjects switched to Eviplera® from a regimen based on NNRTI, mainly EFV/FTC/TDF. CNS adverse events, convenience and metabolic disorders were the most frequent reasons for switching.

Efficacy of prescribed injectable diacetylmorphine in the Andalusian trial: Bayesian analysis of responders and non-responders according to a multi domain outcome index.

BACKGROUND The objective of this research was to evaluate data from a randomized clinical trial that tested injectable diacetylmorphine (DAM) and oral methadone (MMT) for substitution treatment, using a multi-domain dichotomous index, with a Bayesian approach. METHODS Sixty two long-term, socially-excluded heroin injectors, not benefiting from available treatments were randomized to receive either DAM or MMT for 9 months in Granada, Spain. Completers were 44 and data at the end of the study period was obtained for 50. Participants were determined to be responders or non responders using a multi-domain outcome index accounting for their physical and mental health and psychosocial integration, used in a previous trial. Data was analyzed with Bayesian methods, using information from a similar study conducted in The Netherlands to select a priori distributions. On adding the data from the present study to update the a priori information, the distribution of the difference in response rates were obtained and used to build credibility intervals and relevant probability computations. RESULTS In the experimental group (n = 27), the rate of responders to treatment was 70.4% (95% CI 53.287.6), and in the control group (n = 23), it was 34.8% (95% CI 15.354.3). The probability of success in the experimental group using the a posteriori distributions was higher after a proper sensitivity analysis. Almost the whole distribution of the rates difference (the one for diacetylmorphine minus methadone) was located to the right of the zero, indicating the superiority of the experimental treatment. CONCLUSION The present analysis suggests a clinical superiority of injectable diacetylmorphine compared to oral methadone in the treatment of severely affected heroin injectors not benefiting sufficiently from the available treatments. TRIAL REGISTRATION Current Controlled Trials ISRCTN52023186.

The systemic administration of oleoylethanolamide exerts neuroprotection of the nigrostriatal system in experimental Parkinsonism.

Oleoylethanolamide (OEA) is an agonist of the peroxisome proliferator-activated receptor α (PPARα) and has been described to exhibit neuroprotective properties when administered locally in animal models of several neurological disorder models, including stroke and Parkinson's disease. However, there is little information regarding the effectiveness of systemic administration of OEA on Parkinson's disease. In the present study, OEA-mediated neuroprotection has been tested on in vivo and in vitro models of 6-hydroxydopamine (6-OH-DA)-induced degeneration. The in vivo model was based on the intrastriatal infusion of the neurotoxin 6-OH-DA, which generates Parkinsonian symptoms. Rats were treated 2 h before and after the 6-OH-DA treatment with systemic OEA (0.5, 1, and 5 mg/kg). The Parkinsonian symptoms were evaluated at 1 and 4 wk after the development of lesions. The functional status of the nigrostriatal system was studied through tyrosine-hydroxylase (TH) and hemeoxygenase-1 (HO-1, oxidation marker) immunostaining as well as by monitoring the synaptophysin content. In vitro cell cultures were also treated with OEA and 6-OH-DA. As expected, our results revealed 6-OH-DA induced neurotoxicity and behavioural deficits; however, these alterations were less severe in the animals treated with the highest dose of OEA (5 mg/kg). 6-OH-DA administration significantly reduced the striatal TH-immunoreactivity (ir) density, synaptophysin expression, and the number of nigral TH-ir neurons. Moreover, 6-OH-DA enhanced striatal HO-1 content, which was blocked by OEA (5 mg/kg). In vitro, 0.5 and 1 μM of OEA exerted significant neuroprotection on cultured nigral neurons. These effects were abolished after blocking PPARα with the selective antagonist GW6471. In conclusion, systemic OEA protects the nigrostriatal circuit from 6-OH-DA-induced neurotoxicity through a PPARα-dependent mechanism.

Spatial analysis of the relationship between mortality from cardiovascular and cerebrovascular disease and drinking water hardness.

Previously published scientific papers have reported a negative correlation between drinking water hardness and cardiovascular mortality. Some ecologic and case-control studies suggest the protective effect of calcium and magnesium concentration in drinking water. In this article we present an analysis of this protective relationship in 538 municipalities of Comunidad Valenciana (Spain) from 1991-1998. We used the Spanish version of the Rapid Inquiry Facility (RIF) developed under the European Environment and Health Information System (EUROHEIS) research project. The strategy of analysis used in our study conforms to the exploratory nature of the RIF that is used as a tool to obtain quick and flexible insight into epidemiologic surveillance problems. This article describes the use of the RIF to explore possible associations between disease indicators and environmental factors. We used exposure analysis to assess the effect of both protective factors--calcium and magnesium--on mortality from cerebrovascular (ICD-9 430-438) and ischemic heart (ICD-9 410-414) diseases. This study provides statistical evidence of the relationship between mortality from cardiovascular diseases and hardness of drinking water. This relationship is stronger in cerebrovascular disease than in ischemic heart disease, is more pronounced for women than for men, and is more apparent with magnesium than with calcium concentration levels. Nevertheless, the protective nature of these two factors is not clearly established. Our results suggest the possibility of protectiveness but cannot be claimed as conclusive. The weak effects of these covariates make it difficult to separate them from the influence of socioeconomic and environmental factors. We have also performed disease mapping of standardized mortality ratios to detect clusters of municipalities with high risk. Further standardization by levels of calcium and magnesium in drinking water shows changes in the maps when we remove the effect of these covariates.