Toll-Like Receptor 4 Plays a Significant Role in the Biochemical and Neurological Alterations Observed in Two Distinct Mice Models of Huntington's Disease.

Toll-like receptors (TLRs) are central players in innate immunity responses. They are expressed in glial cells and neurons, and their overactivation leads to the production of proinflammatory molecules, neuroinflammation, and neural damage associated with many neurodegenerative pathologies, such as Huntington's disease (HD). HD is an inherited disorder caused by a mutation in the gene coding for the protein Huntingtin (Htt). Expression of mutated Htt (mHtt) causes progressive neuronal degeneration characterized by striatal loss of GABAergic neurons, oxidative damage, neuroinflammatory processes, and impaired motor behavior. The main animal models to study HD are the intrastriatal injection of quinolinic acid (QA) and the transgenic B6CBA-Tg (HDexon1)61Gpb/1 J mice (R6/1). Those models mimic neuronal damage and systemic manifestations of HD. The objective of this work was to study the participation of TLR4 in the manifestations of neuronal damage and HD symptoms in the two mentioned models. For this purpose, C57BL6/J and TLR4-KO mice were administered with QA, and after that motor activity, and neuronal and oxidative damages were measured. R6/1 and TLR4-KO were mated to study the effect of low expression of TLR4 on the phenotype manifestation in R6/1 mice. We found that TLR4 is involved in motor activity, and neurological and oxidative damage induced by intrastriatal injection of QA, and the low expression of TLR4 causes a delay in the onset of phenotypic manifestations by the mHtt expression in R6/1 mice. Our results show that TLR4 is involved in both models of HD and focuses then as a therapeutic target for some deleterious reactions in HD.

Neuroprotective effect of dapsone in patients with aneurysmal subarachnoid hemorrhage: a prospective, randomized, double-blind, placebo-controlled clinical trial.

OBJECTIVE: In this study, the authors sought to define the differences in the incidence of delayed cerebral ischemia (DCI) between patients treated with dapsone and those treated with placebo. Secondary objectives were to define the clinical outcome at discharge and 3 months and the incidence of brain infarction. METHODS: A prospective, randomized, double-blind, placebo-controlled study was performed and included patients with aneurysmal subarachnoid hemorrhage (SAH) within 5 days from ictus who were candidates for aneurysm occlusion, and who had a Fisher grade of 3 or 4. Patients with sulfa or sulfone drug allergies, hemoglobin < 11 g/dl, known G6PD deficiency, and those refusing informed consent were excluded. A minimal relevant effect decrease of 35% in the incidence of DCI was established. Patients were randomly assigned to receive a regimen of dapsone 2.5 ml (100 mg) daily or a placebo (aluminum hydroxide suspension, 2.5 ml daily). Both groups received validated treatment for aneurysmal SAH. The appearance of DCI on CT was assessed in every patient at discharge and 3 months later. We used the chi-square test to compare the DCI incidence between both groups, and the Student t-test or nonparametric tests to compare quantitative variables. RESULTS: Overall, 48 patients (70.8% women and 29.2% men) were included. The mean age was 50 years (SD 14.28 years, range 18-72 years). Prerandomization and postrandomization characteristics were balanced, except for the necessity of intra-arterial nimodipine administration in patients treated with placebo (15.4% vs 45.5%, p = 0.029. The incidence of DCI, the primary endpoint, for the whole cohort was 43.8% and was significantly lower in the dapsone group (26.9% vs 63.6%, p = 0.011). In addition, the irreversible DCI incidence was lower in the dapsone group (11.5% vs 54.5%, p = 0.12). A favorable modified Rankin Scale score was more frequent in the dapsone group at discharge and at 3 months (76.9% vs 36.4%, p = 0.005 and 80% vs 38.9%, p = 0.019, respectively). Also, the brain infarction incidence was lower in the dapsone group (19.2% vs 63.6%, p = 0.001). There was no difference between groups regarding adverse events. CONCLUSIONS: Dapsone seems to play a role as a prophylactic agent in patients at high risk of developing DCI after aneurysmal SAH. A multicenter investigation is necessary to increase the study population and confirm the consistency of the results observed in this study.

Mast cells and histamine are involved in the neuronal damage observed in a quinolinic acid-induced model of Huntington's disease.

Huntington´s disease (HD) is a pathological condition that can be studied in mice by the administration of quinolinic acid (QUIN), an agonist of the N-methyl-d-aspartate receptor (NMDAR) that induces NMDAR-mediated cytotoxicity and neuroinflammation. Mast cells (MCs) participate in numerous inflammatory processes through the release of important amounts of histamine (HA). In this study, we aimed to characterize the participation of MCs and HA in the establishment of neural and oxidative damage in the QUIN-induced model of HD. C57BL6/J mice (WT), MC-deficient c-KitW-sh/W-sh (Wsh) mice and Wsh mice reconstituted by intracerebroventricular (i.c.v.) injection of 5 × 105 bone marrow-derived mast cells (BMMCs), or i.c.v. administered with HA (5 µg) were used. All groups of animals were intrastriatally injected with 1 µL QUIN (30 nmol/µL) and 3 days later, apomorphine-induced circling behavior, striatal GABA levels and the number of Fluoro-Jade positive cells, as indicators of neuronal damage, were determined. Also, lipid peroxidation (LP) and reactive oxygen species production (ROS), as markers of oxidative damage, were analyzed. Wsh mice showed less QUIN-induced neuronal and oxidative damage than WT and Wsh-MC reconstituted animals. Histamine administration restored the QUIN-induced neuronal and oxidative damage in the non-reconstituted Wsh mice to levels equivalent or superior to those observed in WT mice. Our results demonstrate that MCs and HA participate in the neuronal and oxidative damages observed in mice subjected to the QUIN -induced model of Huntington's disease.

Copper biodistribution after acute systemic administration of copper gluconate to rats.

Neurodegenerative disorders have been linked to the decrease of copper concentrations in different regions of the brain. Therefore, intake of micronutrient supplements could be a therapeutic alternative. Since the copper distribution profile has not been elucidated yet, the aim of this study was to characterize and to analyze the concentration profile of a single administration of copper gluconate to rats by two routes of administration. Male Wistar rats were divided into three groups. The control group received vehicle (n = 5), and the experimental groups received 79.5 mg/kg of copper orally (n = 4-6) or 0.64 mg/kg of copper intravenously. (n = 3-4). Blood, striatum, midbrain and liver samples were collected at different times. Copper concentrations were assessed using atomic absorption spectrophotometry. Copper concentration in samples from the control group were considered as baseline. The highest copper concentration in plasma was observed at 1.5 h after oral administration, while copper was quickly compartmentalized within the first hour after intravenous administration. The striatum evidenced a maximum metal concentration at 0.25 h for both routes of administration, however, the midbrain did not show any change. The highest concentration of the metal was held by the liver. The use of copper salts as replacement therapy should consider its rapid and discrete accumulation into the brain and the rapid and massive distribution of the metal into the liver for both oral and intravenous routes. Development of controlled-release pharmaceutical formulations may overcome the problems that the liver accumulation may imply, particularly, for hepatic copper toxicity.

Alcohol intake potentiates clozapine adverse effects associated to CYP1A2*1C in patients with refractory psychosis.

Clozapine (CLZ) is an atypical antipsychotic and the gold standard for refractory psychosis treatment. However, there is little information regarding pharmacogenetics of CLZ in patients with refractory psychosis and its clinical correlation with alcohol intake. Although neurological effects of CLZ in patients with concomitant alcohol intake are documented, its use is very common in patients with psychosis. We explored the impact of CYP1A2, CYP2D6, CYP2C19, and CYP3A4 genetic variants on CLZ pharmacokinetics and side effects, along with coffee/alcohol/tobacco consumption habits and clinical data of 48 adult patients with refractory psychosis on CLZ antipsychotic monotherapy. Relevant CYP variants in CLZ metabolism were evaluated by targeted genotyping and multiplex ligation-dependent probe amplification. CLZ and its main metabolite plasma concentrations were determined by high performance liquid chromatography. Biochemical and molecular data, along with other potential confounders, were included in the analysis by linear regression. Overall, CYP variants showed no effect on CLZ pharmacokinetics. The rs2069514 variant in homozygous genotype (also known as CYP1A2*1C/*1C) was associated with CLZ adverse reactions in Mexican patients with refractory psychosis (OR = 3.55 CI95  = 1.041-12.269, p = .043) and demonstrated that this effect is doubled by concomitant alcohol consumption (OR = 7.9 CI95  = 1.473-42.369, p = .016). Clinicians should be aware of this information before starting CLZ use, when treating patients with refractory psychosis, who are alcohol drinkers and carriers of this genetic variant in order to prevent CLZ-related adverse reactions. Nevertheless, our findings should be replicated in larger samples.

In vitro inhibition of brain phosphate-activated glutaminase by ammonia and manganese.

INTRODUCTION: As a consequence of the loss of liver function in chronic liver disease, increased levels of ammonia, manganese, and glutamine have been observed in the brain of hepatic encephalopathy patients. OBJECTIVE: In the present study, we explored phosphate activated glutaminase (PAG) activity in mitochondrial enriched fractions under treatment with ammonia and manganese. METHODS: We dissected out the brain cortex, striatum, and cerebellum of male Wistar rats 250-280 g weight; brain sections were pooled to obtain enriched mitochondrial fractions by differential centrifugation. Aliquots equivalent to 200 µg of protein were incubated with semi-log increasing concentrations of ammonia and/or manganese both as chloride salts (from 0 to 10 000 µM) and glutamine (4 mM) for 30 min. Then, the glutamate produced by the reaction was determined by HPLC coupled with fluorescence detection. RESULTS AND DISCUSSION: Both manganese and ammonia inhibited PAG in a concentration-dependent manner. Non-linear modeling was used to determine IC50 and IC20 for ammonia (120 µM) and manganese (2 mM). We found that PAG activity under the combination of IC20 of ammonia and manganese was equivalent to the sum of the effects of both substances, being PAG inhibition more pronounced in mitochondrial fractions from cerebellum. The PAG inhibition observed here could potentially explain a pathway for glutamine accumulation, by means of the inhibition of PAG activity as a consequence of increased concentrations of manganese and ammonia in the brain under liver damage conditions.

Clozapine and desmethylclozapine: correlation with neutrophils and leucocytes counting in Mexican patients with schizophrenia.

PURPOSE: The aim of present study is to measure plasma clozapine (CLZ) and N-desmethyl clozapine (DMC) as biomarkers to correlate drug concentrations with the appearance of preclinical adverse hematic effects. METHODS: A high-performance liquid chromatographic method, using a diode-array (ultraviolet) detector, was validated to obtain reliable concentrations of CLZ and DMC, its main metabolite, in plasma of 41 schizophrenic patients taking CLZ. Blood neutrophils and leucocytes counting were concurrently assessed as a proxy to subclinical adverse reactions. RESULTS: The analytical method employed was linear, reproducible, and stable to measure concentrations of CLZ between 30 and 1000 ng/mL, while 12.5-560 ng/mL of the metabolite. The method allowed us to correlate CLZ plasma concentrations, the time taking CLZ and CLZ dose as determinants of neutrophils' counting with a R2 = 0.447, using a multiple regression analysis model. Likewise, the correlation of leucocyte counting vs CLZ plasma levels and CLZ time, showed a R2 = 0.461. DMC correlated significantly with both neutrophils and leucocytes counting, but was excluded from the regression when CLZ concentration was included in the model. Finally, no other hematological adverse reactions were recorded. One patient presented a cardiovascular complication. The negative correlation between clozapine and neutrophil count observed in patients, suggest that CLZ itself, but not DMC, could be related to hematologic side-effects. CONCLUSION: The findings of this study, demonstrate for the first time, that plasma levels of CLZ and time taking the drug are independent determinants of blood neutrophils and leucocytes, so the monitoring of plasma CLZ may be useful in the clinic practice to determine safe dosing of the drug.

A Hypothesis of the Interaction of the Nitrergic and Serotonergic Systems in Aggressive Behavior Induced by Exposure to Lead.

The effects caused by exposure to lead (Pb) are still considered as a relevant health risk despite public policies aimed to restricting the use of this element. The toxicity limit in the blood (10 µg/dL, established by the Center for Disease Control and Prevention) has been insufficient to prevent adverse effects and even lower values have been related to neurobehavioral dysfunctions in children. Currently, there is not a safe limit of exposure to Pb. A large body of evidence points to environmental pollutant exposure as the cause of predisposition to violent behavior, among others. Considering the evidence by our group and others, we propose that Pb exposure induces alterations in the brain vasculature, specifically in nitric oxide synthases (NOS), affecting in turn the serotonergic system and leading to heightened aggressive behavior in the exposed individuals. This review article describes the consequences of Pb exposure on the nitrergic and serotonergic systems as well as its relationship with aggressive behavior. In addition, it summarizes the available therapy to prevent damage in gestation and among infants.

Essential fatty acid-rich diets protect against striatal oxidative damage induced by quinolinic acid in rats.

Essential fatty acids have an important effect on oxidative stress-related diseases. The Huntington's disease (HD) is a hereditary neurologic disorder in which oxidative stress caused by free radicals is an important damage mechanism. The HD experimental model induced by quinolinic acid (QUIN) has been widely used to evaluate therapeutic effects of antioxidant compounds. The aim of this study was to test whether the fatty acid content in olive- or fish-oil-rich diet prevents against QUIN-related oxidative damage in rats. Rats were fed during 20 days with an olive- or a fish-oil-rich diet (15% w/w). Posterior to diet period, rats were striatally microinjected with QUIN (240 nmol/µl) or saline solution. Then, we evaluated the neurological damage, oxidative status, and gamma isoform of the peroxisome proliferator-activated receptor (PPARγ) expression. Results showed that fatty acid-rich diet, mainly by fish oil, reduced circling behavior, prevented the fall in GABA levels, increased PPARγ expression, and prevented oxidative damage in striatal tissue. In addition none of the enriched diets exerted changes neither on triglycerides or cholesterol blood levels, nor or hepatic function. This study suggests that olive- and fish-oil-rich diets exert neuroprotective effects.

Glazed clay pottery and lead exposure in Mexico: Current experimental evidence.

OBJECTIVES: Lead exposure remains a significant environmental problem; lead is neurotoxic, especially in developing humans. In Mexico, lead in human blood is still a concern. Historically, much of the lead exposure is attributed to the use of handcrafted clay pottery for cooking, storing and serving food. However, experimental cause-and-effect demonstration is lacking. The present study explores this issue with a prospective experimental approach. METHODS: We used handcrafted clay containers to prepare and store lemonade, which was supplied as drinking water to pregnant rats throughout the gestational period. RESULTS AND DISCUSSION: We found that clay pots, jars, and mugs leached on average 200 µg/l lead, and exposure to the lemonade resulted in 2.5 µg/dl of lead in the pregnant rats' blood. Neonates also showed increased lead content in the hippocampus and cerebellum. Caspase-3 activity was found to be statistically increased in the hippocampus in prenatally exposed neonates, suggesting increased apoptosis in that brain region. Glazed ceramics are still an important source of lead exposure in Mexico, and our results confirm that pregnancy is a vulnerable period for brain development.

The neuroprotective effect of lovastatin on MPP(+)-induced neurotoxicity is not mediated by PON2.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of the pigmented dopaminergic neurons in the substantia nigra pars compacta with subsequent striatal dopamine (DA) deficiency and increased lipid peroxidation. The etiology of the disease is still unclear and it is thought that PD may be caused by a combination of genetic and environmental factors. In the search of new pharmacological options, statins have been recognized for their potential application to treat PD, due to their antioxidant effect. The aim of this work is to contribute in the characterization of the neuroprotective effect of lovastatin in a model of PD induced by 1-methyl-4-phenylpyridinium (MPP(+)). Male Wistar rats (200-250 g) were randomly allocated into 4 groups and administered for 7 days with different pharmacological treatments. Lovastatin administration (5 mg/kg) diminished 40% of the apomorphine-induced circling behavior, prevented the striatal DA depletion and lipid peroxides formation by MPP(+) intrastriatal injection, as compared to the group of animals treated only with MPP(+). Lovastatin produced no change in paraoxonase-2 (PON2) activity. It is evident that lovastatin conferred neuroprotection against MPP(+)-induced protection but this effect was not associated with the induction of PON2 in the rat striatum.

Copper and copper proteins in Parkinson's disease.

Copper is a transition metal that has been linked to pathological and beneficial effects in neurodegenerative diseases. In Parkinson's disease, free copper is related to increased oxidative stress, alpha-synuclein oligomerization, and Lewy body formation. Decreased copper along with increased iron has been found in substantia nigra and caudate nucleus of Parkinson's disease patients. Copper influences iron content in the brain through ferroxidase ceruloplasmin activity; therefore decreased protein-bound copper in brain may enhance iron accumulation and the associated oxidative stress. The function of other copper-binding proteins such as Cu/Zn-SOD and metallothioneins is also beneficial to prevent neurodegeneration. Copper may regulate neurotransmission since it is released after neuronal stimulus and the metal is able to modulate the function of NMDA and GABA A receptors. Some of the proteins involved in copper transport are the transporters CTR1, ATP7A, and ATP7B and the chaperone ATOX1. There is limited information about the role of those biomolecules in the pathophysiology of Parkinson's disease; for instance, it is known that CTR1 is decreased in substantia nigra pars compacta in Parkinson's disease and that a mutation in ATP7B could be associated with Parkinson's disease. Regarding copper-related therapies, copper supplementation can represent a plausible alternative, while copper chelation may even aggravate the pathology.

Protective effect of N,N'-dialkylated analogs of 4,4'-diaminodiphenylsulfone in a model of intrastriatal quinolinic acid induced-excitotoxicity.

The bacteriostatic agent 4,4'-diaminodiphenylsulfone or dapsone (DDS) and some of its N,N'-dialkylated analogs have shown anticonvulsant and neuroprotective properties in different experimental models. In this study, we tested the ability of five DDS analogs (N,N'-dimethyldapsone, N,N'-diethyldapsone, N,N'-dipropyldapsone, N,N'-dibutyldapsone and N,N'-ditosyldapsone) to attenuate quinolinic acid-induced toxicity in vivo. Male Wistar rats were treated with either DDS or analogs (12.5mg/kg and equimolar doses respectively) 30 min before quinolinic acid intrastriatal stereotaxic injection (240 nmol/µl). Six days after injury, circling behavior was evaluated by counting ipsilateral turns for 1h after apomorphine challenge (1mg/kg, sc). Twenty-four hours later, rats were sacrificed and their corpora striata were dissected out to determine GABA content. Hemotoxicity of the analogs was assessed as the ability to produce methemoglobin (MHb) in vivo. Blood was sampled from tail vein within 18 h after drugs administration. Methemoglobin levels were determined by visible spectrophotometry and mean profiles of MHb-percentage versus time were obtained. All of the analogs tested decreased the number of ipsilateral turns/hour, reducing up to 67% the turns counting (p<0.05) when compared to those induced in animals receiving quinolinic acid with no treatment. N,N'-dimethylated, N,N'-diethylated and N,N'-dibutylated analogs significantly prevented the decrease of intrastriatal GABA content (p<0.05). Methemoglobin produced by the administration of analogs was significantly lower than the levels of the group receiving dapsone (p<0.05). The neuroprotective effect of analogs and their diminished hemotoxicity make them potential candidates for therapeutic applications.

The transition metals copper and iron in neurodegenerative diseases.

Neurodegenerative diseases constitute a worldwide health problem. Metals like iron and copper are essential for life, but they are also involved in several neurodegenerative mechanisms such as protein aggregation, free radical generation and oxidative stress. The role of Fe and Cu, their pathogenic mechanisms and possible therapeutic relevance are discussed regarding four of the most common neurodegenerative diseases, Alzheimer's, Parkinson's and Huntington's diseases as well as amyotrophic lateral sclerosis. Metal-mediated oxidation by Fenton chemistry is a common feature for all those disorders and takes part of a self-amplifying damaging mechanism, leading to neurodegeneration. The interaction between metals and proteins in the nervous system seems to be a crucial factor for the development or absence of neurodegeneration. The present review also deals with the therapeutic strategies tested, mainly using metal chelating drugs. Metal accumulation within the nervous system observed in those diseases could be the result of compensatory mechanisms to improve metal availability for physiological processes.