Cannabinoid agonists, including marijuana containing delta-9-tetrahydrocannabinol (THC), are found rewarding by humans. In addition to human self-reports and experimental studies that show marijuana is rewarding, contributions from preclinical studies also have implicated cannabinoid receptors in reward-motivated behavior. One way to assess these preclinical effects of cannabinoids is intracranial self-stimulation (ICSS), where an animal performs a response to receive electrical stimulation of a specific brain area or circuit known to be involved in reward. Drugs of abuse, such as psychomotor stimulants, facilitate responding for ICSS. While a few studies have shown facilitating effects of cannabinoids in rats, several have shown the opposite effect, and no studies so far have evaluated cannabinoids in mouse ICSS. Furthermore there are no studies evaluating specific inhibitors of endocannabinoid catabolic enzymes in ICSS in any species. In these studies we assessed the cannabinoid agonist THC, as well as the fatty acid amide hydrolase (FAAH) inhibitor, PF-3845, the monoacylglycerol lipase (MAGL) inhibitor JZL184, and the combined FAAH/MAGL inhibitor SA-57 in ICSS of the medial forebrain bundle in C57BL/6 mice. Additionally, we assessed the psychomotor stimulant cocaine as a positive control to facilitate ICSS. These studies were complimented with spontaneous locomotor activity and food-maintained operant experiments to assess the sensitivity of ICSS to cannabinoids. Additionally, brain endocannabinoid levels were measured in brain regions associated with the mesolimbic system after enzyme inhibitor treatments. THC, JZL184, and SA-57 all produced time-dependent reductions in ICSS that were mediated through CB1 receptors, as they were blocked by pre-treatment with the CB1 antagonist rimonabant, but not with the CB2 antagonist SR144528. PF-3845 also reduced ICSS, but did so independent of CB1 and CB2 receptors, and only with one dose (30.0 mg/kg) that has not been assessed previously in vivo. We showed that ICSS was more sensitive to the rate-reducing effects of cannabinoids than other measures of behavior with motor components including spontaneous locomotor activity and operant nose-poking for food, and that the reduction of ICSS produced by both JZL184 and SA-57 is accompanied by increases in 2-AG in mesolimbic brain areas. Thus, cannabinoids do not facilitate ICSS in C57BL/6 mice over a range of doses and pre-treatment times, similar to most studies with rats. These data suggest that cannabinoids may produce rewarding effects through non-mesolimbic areas of the brain.

Atherosclerosis is a disease characterized by plaque formation due to an accumulation of fat, cholesterol, and immune cells in the walls of arteries. If a plaque ruptures, an occlusive thrombosis may form that causes either a heart attack or stroke. Macrophages express CB-2 receptors, and are one type of immune cell that plays a role in plaque destabilization and rupture. Endocannabinoids anandamide (AEA) and 2-arachidonyl glycerol (2-AG) have been found to have activity on CB-1 and CB-2 receptors throughout the body and immune system. In this study, we investigated several sample preparation options for the LC-MS quantification of AEA and 2-AG from plasma and aortic tissue. The extractions considered included liquid–liquid (LLE), solid-phase (SPE), and supported liquid (SLE). Some extraction protocols yielded high analyte recovery and prevention of 1-AG/2-AG isomerization. Our results indicate that a liquid-liquid extraction using toluene yields the highest recovery for both analytes, coupled with low ionization suppression in the mass spectrometer. This extraction and corresponding LC-MS/MS assay provides a simple, high throughput mechanism for the quantification of 2-AG and AEA in matrices relevant to the study of endocannabinoids’ role in atherosclerosis.

Decades of research has provided evidence for the role of the endocannabinoid system in human health and disease. This versatile system, consisting of two receptors (CB1 and CB2), their endogenous ligands (endocannabinoids), and metabolic enzymes has been implicated in a wide variety of disease states, ranging from neurological disorders to cancer. CB2 has gained much interest for its beneficial immunomodulatory role that can be obtained without eliciting psychotropic effects through CB1. Recent studies have shed light on a protective role of CB2 in cardiovascular disease, an ailment which currently takes more lives each year in Western countries than any other disease or injury. By use of CB2 knockout mice and CB2-selective ligands, knowledge of how CB2 signaling affects atherosclerosis and ischemia has been acquired, providing a major stepping stone between basic science and translational clinical research. Here, we summarize the current understanding of the endocannabinoid system in human pathologies and provide a review of the results from preclinical studies examining its function in cardiovascular disease, with a particular emphasis on possible CB2-targeted therapeutic interventions to alleviate atherosclerosis.

O fígado desempenha um papel central na disposição metabólica de vários agentes químicos endógenos e exógenos, incluindo quase todos os fármacos. Neste processo de biotransformação pode ocorrer a formação de metabólitos intermediários altamente reativos que se não forem convenientemente eliminados podem interagir com macromoléculas celulares lesando o órgão. A hepatotoxicidade idiossincrática associada ao uso de antiepilépticos aromáticos (AEA) é bem conhecida e tem sido atribuída ao acúmulo de intermediários tóxicos (areno-óxidos) formados durante a bioativação hepática. Embora a participação de processos imunológicos no mecanismo de ação tóxica dos AEA tenha sido demonstrada, existe a possibilidade de mecanismos adjuvantes envolvendo a toxicidade mitocondrial, evento ainda não explorado na literatura científica. Neste estudo avaliou-se, in vitro, o efeito dos AEA: carbamazepina, fenitoína e fenobarbital, bem como dos seus respectivos metabólitos na função mitocondrial e na indução do estresse oxidativo em mitocôndrias de fígado de rato, como possível mecanismo de ação hepatotóxica desses fármacos. Sistema microssomal hepático de rato foi utilizado para bioativação dos fármacos e produção dos respectivos metabólitos in vitro. Sem a bioativação, somente o fenobarbital (em concentrações elevadas) apresentou efeitos inibidores sobre o estado 3 da respiração, síntese de ATP e potencial de membrana, sem, contudo induzir o estresse oxidativo. Quando bioativados, todos os fármacos apresentaram efeitos sobre a função mitocondrial através de processo mediado por estresse oxidativo. Todos os fármacos bioativados afetaram a respiração mitocondrial, causando diminuição do consumo de oxigênio no estado 3, diminuição do RCR e aumento do consumo de oxigênio no estado 4. Foram também evidenciadas alterações na captação/liberação de cálcio, inibição da síntese de ATP, diminuição do potencial de membrana e inibição do intumescimento mitocondrial induzido pelo cálcio. A oxidação de proteínas e lipídeos mitocondriais foi demonstrada pela formação de proteínas carboniladas, diminuição de proteínas com grupamentos sulfidrila, aumento de malondialdeído (MDA) e pela oxidação da cardiolipina. O sistema de defesa antioxidante mitocondrial também foi afetado, como evidenciado pela diminuição da relação GSH/GSSG (glutationa reduzida/glutationa oxidada). Os resultados sugerem fortemente a participação do dano mitocondrial, mediado pelo estresse oxidativo causado pelos metabólitos dos AEA, no desenvolvimento da hepatotoxicidade idiossincrática induzida por esses fármacos. / The liver plays a central role in the metabolic disposition of various endogenous and exogenous chemicals, including almost all drugs. During the biotransformation process, highly reactive metabolites can be produced, and if they are not detoxified, they can interact with cellular macromolecules and cause organ injury. Idiosyncratic hepatotoxicity is a well-known complication associated with aromatic antiepileptic drugs (AAED), and it has been suggested to occur due to the accumulation of toxic arene oxide metabolites. Although the participation of an immune process in the toxic action mechanism of AAED has been demonstrated, adjuvant mechanisms involving mitochondrial toxicity is also possible and such event has not been studied yet. Therefore, we investigated, in vitro, the effects of carbamazepine (CB), phenytoin (PT), phenobarbital (PB) and their respective metabolites on the hepatic mitochondrial function as well as their ability to induce oxidative stress in rat liver mitochondria, as a possible hepatotoxic action mechanism. The murine hepatic microsomal system was used to bioactivate the drugs and to produce the anticonvulsant metabolites in vitro. As an unaltered drug, only phenobarbital (in high concentrations) presented inhibitory effects on state 3 respiration, ATP synthesis, and membrane potential; however, it did not induce oxidative stress. All the bioactivated drugs affected mitochondrial function through an oxidative stress-mediated process. All the bioactivated drugs affected mitochondrial function causing decrease in state 3 respiration, decrease in RCR and increase in state 4 respiration. They also caused impairment of Ca+2 uptake /release, decrease in ATP synthesis, decrease in membrane potential and inhibition of calcium-induced swelling. Oxidation of proteins and lipids was evidenced by carbonil proteins formation, decrease in thiol proteins, increase in malonaldehyde (MDA) and cardiolipin oxidation. The mitochondrial antioxidant defense system was also affected, as evidenced by the decreased GSH/GSSG ratio (reduced glutathione/oxidized glutathione). Results strongly suggest the involvement of mitochondrial damage, which is mediated by the oxidative stress caused by the AAED metabolites, in the development of AAED-induced idiosyncratic hepatotoxicity.

O fígado desempenha um papel central na disposição metabólica de vários agentes químicos endógenos e exógenos, incluindo quase todos os fármacos. Neste processo de biotransformação pode ocorrer a formação de metabólitos intermediários altamente reativos que se não forem convenientemente eliminados podem interagir com macromoléculas celulares lesando o órgão. A hepatotoxicidade idiossincrática associada ao uso de antiepilépticos aromáticos (AEA) é bem conhecida e tem sido atribuída ao acúmulo de intermediários tóxicos (areno-óxidos) formados durante a bioativação hepática. Embora a participação de processos imunológicos no mecanismo de ação tóxica dos AEA tenha sido demonstrada, existe a possibilidade de mecanismos adjuvantes envolvendo a toxicidade mitocondrial, evento ainda não explorado na literatura científica. Neste estudo avaliou-se, in vitro, o efeito dos AEA: carbamazepina, fenitoína e fenobarbital, bem como dos seus respectivos metabólitos na função mitocondrial e na indução do estresse oxidativo em mitocôndrias de fígado de rato, como possível mecanismo de ação hepatotóxica desses fármacos. Sistema microssomal hepático de rato foi utilizado para bioativação dos fármacos e produção dos respectivos metabólitos in vitro. Sem a bioativação, somente o fenobarbital (em concentrações elevadas) apresentou efeitos inibidores sobre o estado 3 da respiração, síntese de ATP e potencial de membrana, sem, contudo induzir o estresse oxidativo. Quando bioativados, todos os fármacos apresentaram efeitos sobre a função mitocondrial através de processo mediado por estresse oxidativo. Todos os fármacos bioativados afetaram a respiração mitocondrial, causando diminuição do consumo de oxigênio no estado 3, diminuição do RCR e aumento do consumo de oxigênio no estado 4. Foram também evidenciadas alterações na captação/liberação de cálcio, inibição da síntese de ATP, diminuição do potencial de membrana e inibição do intumescimento mitocondrial induzido pelo cálcio. A oxidação de proteínas e lipídeos mitocondriais foi demonstrada pela formação de proteínas carboniladas, diminuição de proteínas com grupamentos sulfidrila, aumento de malondialdeído (MDA) e pela oxidação da cardiolipina. O sistema de defesa antioxidante mitocondrial também foi afetado, como evidenciado pela diminuição da relação GSH/GSSG (glutationa reduzida/glutationa oxidada). Os resultados sugerem fortemente a participação do dano mitocondrial, mediado pelo estresse oxidativo causado pelos metabólitos dos AEA, no desenvolvimento da hepatotoxicidade idiossincrática induzida por esses fármacos. / The liver plays a central role in the metabolic disposition of various endogenous and exogenous chemicals, including almost all drugs. During the biotransformation process, highly reactive metabolites can be produced, and if they are not detoxified, they can interact with cellular macromolecules and cause organ injury. Idiosyncratic hepatotoxicity is a well-known complication associated with aromatic antiepileptic drugs (AAED), and it has been suggested to occur due to the accumulation of toxic arene oxide metabolites. Although the participation of an immune process in the toxic action mechanism of AAED has been demonstrated, adjuvant mechanisms involving mitochondrial toxicity is also possible and such event has not been studied yet. Therefore, we investigated, in vitro, the effects of carbamazepine (CB), phenytoin (PT), phenobarbital (PB) and their respective metabolites on the hepatic mitochondrial function as well as their ability to induce oxidative stress in rat liver mitochondria, as a possible hepatotoxic action mechanism. The murine hepatic microsomal system was used to bioactivate the drugs and to produce the anticonvulsant metabolites in vitro. As an unaltered drug, only phenobarbital (in high concentrations) presented inhibitory effects on state 3 respiration, ATP synthesis, and membrane potential; however, it did not induce oxidative stress. All the bioactivated drugs affected mitochondrial function through an oxidative stress-mediated process. All the bioactivated drugs affected mitochondrial function causing decrease in state 3 respiration, decrease in RCR and increase in state 4 respiration. They also caused impairment of Ca+2 uptake /release, decrease in ATP synthesis, decrease in membrane potential and inhibition of calcium-induced swelling. Oxidation of proteins and lipids was evidenced by carbonil proteins formation, decrease in thiol proteins, increase in malonaldehyde (MDA) and cardiolipin oxidation. The mitochondrial antioxidant defense system was also affected, as evidenced by the decreased GSH/GSSG ratio (reduced glutathione/oxidized glutathione). Results strongly suggest the involvement of mitochondrial damage, which is mediated by the oxidative stress caused by the AAED metabolites, in the development of AAED-induced idiosyncratic hepatotoxicity.

Increasing awareness of sustainability in the concrete industry forces structural design and executionto focus on avoiding costly and unpredictable maintenance action, instead paying higher attention todamage prevention by direct actions on early stages of production. One of such approaches, whichdeals with the problem of freezing and thawing deterioration, is intentional air bubble introductionto the concrete mix. However, the mechanism of air entrainment in concrete can be negativelyaffected on different stages of production by many factors including cement type, admixture dosage,casting conditions or mixing procedure. Therefore, reliable tools for the end-product evaluationought to be considered. The experimental study, presented in this work, focuses on understandingthe blast furnace slag (BFS) influence as well as admixtures’ dosage effect on pore structure ofhardened concrete. Three types of cement were evaluated, including ordinary portland cement(OPC) and two types of CEM III cements with different BFS percentage. The optimal amountsof air entraining agent (AEA) and super plasticizer (SP) were chosen and later reduced in orderto evaluate their impact on total air content, spacing factor and specific surface of the air voids.The main method chosen for this evaluation was the use of an office flat-bed scanner to acquireimages and application of BubbleCounter software for the analysis of the air void structure. Thisapproach is based on linear traverse method and requires special surface treatment for contrastenhancement. Specimens for the analysis were cut from hardened concrete cubes and polishedto achieve a flat surface. The samples were later treated with black ink and zinc oxide paste toachieve a clear contrast between white voids and black paste/aggregate area. In order to estimatethe accuracy of this method, more conventional tools such as pressurised gauge method and air voidanalyser were applied for comparison. Resulted mixtures showed significant differences in air voidproperties between OPC and BFS containing concrete, with the latter being less affected by AEAdosage reduction. Changes in spacing factor and specific surface were also registered; however,their deterioration did not follow the same pattern as that of total air content. No significantdeviation between the two cements containing BFS was observed. An interesting effect of the usedpolycarboxylate ether SP on the AEA reactivity was registered, showing deterioration of air voidproperties with the decrease of plasticizer amount. Comparison of the results from different air voidanalysis methods, suggested an overall agreement on the measured air void system changes due tochanging the AEA content. However, the BubbleCounter software tended to slightly overestimatethe material’s resistance to freeze and thaw phenomenon, giving the most optimistic values inspacing factor and specific surface of air voids. / Betongindustrins ökande medvetenhet om hållbarhet leder till att man inom dimensionering ochutförande fokuserar mot att undvika kostnadskrävande och oförutsedda underhållsåtgärder ochistället lägga större vikt på att förebygga skador i produktionens tidiga skeden. En av dessaåtgärder, som hanterar problemet frostnedbrytning, är en medveten inblandning av luftbubblor ibetongen. Mekanismen för att skapa luftporssystemet kan emellertid bli negativt påverkad underolika skeden av produktionen av många faktorer såsom cementtyp, tillsatsmedelsdos, gjutvillkoroch blandningsordning. Därför behöver man reflektera över pålitliga verktyg för utvärderingenav slutprodukten. Den experimentella studien, som presenteras i detta arbete, fokuserar motförståelse hur slagg och tillsatsmedelsdos påverkar den hårdnade betongens luftporssystem. Tretyper av cement utvärderades, dels ett normalt portlandcement, dels två typer av CEM III-cementmed olika andelar av slagg. Optimala mängder av luftporbildare och flyttillsatsmedel valdesmen reducerades senare för att undersöka deras inverkan på totalt luftinnehåll samt luftporernasavståndsfaktor och specifika yta. Den huvudsakliga metoden som valdes för denna utvärderingvar en flatbäddsscanner (kontorsmodell) för att ta bilder och användningen av en programvaravid namn BubbleCounter för att analysera luftporssystemet. Detta tillvägagångssätt baseras påanalys av tvärgående linjer och kräver en speciell behandling av ytan för att åstadkomma kontraster.Provkroppar för analysen sågades ut ur hårdnade betongkuber och polerades för att erhålla en jämnyta. Provkropparna var senare behandlade med svart bläck och zinkoxidpasta för att åstadkomma entydlig kontrast mellan de vita porerna och den svarta ytan av cementpasta och ballast. För att studeranoggrannheten hos denna metod användes som jämförelse även mer konventionella metoder sommätningar med trycksatta givare och luftporsanalys. De framtagna blandningarna visade signifikantaskillnader i luftporernas egenskaper mellan betong med normalt portlandcement och betong medslaggcement, där den senare påverkades i mindre grad av reduktioner i dosen luftporbildare.Förändringar I avståndsfaktor och specifik yta noterades också men försämringen följde inte sammamönster som den för totala luftinnehållet. Ingen signifikant skillnad mellan de två cementeninnehållande slagg kunde observeras. En intressant inverkan av det använda polykarboxylateterbaseradeflyttillsatsmedlet på luftporbildarens reaktivitet noterades. Den visade en försämringav luftporernas egenskaper vid en reduktion av mängden flyttillsatsmedel. En jämförelse avresultaten från de olika metoderna för luftporsanalys indikerade en övergripande överensstämmelsegällande de uppmätta luftporssystemens förändring p.g.a. förändringar i mängden luftporbildare.Programvaran BubbleCounter tenderade emellertid att något överskatta materialets motstånd motfrostnedbrytning med de mest optimistiska värdena för luftporernas avståndsfaktor och specifikayta.

Fire resistance is becoming increasingly important along with the development of new concrete types with high strength and dense structure with reduced porosity. Such concrete types are susceptible to fire spalling and extensive crack formation. At the moment, there are a limited number of methods for enhancement of fire resistance of existing structures, which could be applied in underground structures with restricted space and limited air exchange, such as tunnels, underground garages or nuclear powerplants. This work is focused on the development of two methods, and both are dealing with porous structure modification. The first method is intentional heat treatment (IHT) method, suitable for the enhancement of fire resistance of existing structures. The second method emphasized the design of air-entrained concrete (AeA-FiResCrete) with the use of “new generation” air-entraining agents suitable for enhancement of fire resistance of newly designed concrete. Testing of compressive strength, porous structure modification was completed by the analysis of “moisture clog,” which contributes to explosive spalling and extensive cracking. The efficiency of developing methods was verified during large-scale testing according to modified ISO834 (m-ISO) curve. No extensive crack formation or explosive spalling was observed during the exposure period during the large-scale testing of slabs with the applied IHT method. The total thickness of the IHT method with configuration IHT200/2, composed of IHT zone and IHT transition zone, penetrated to the depth of 25,5 to 43,0 mm depending upon various concrete types. Moisture clog in AeA-FiResCrete was more significant than in the case of slabs with applied IHT method, and it could be concluded that the IHT method enhances fire resistance of concrete exposed to elevated temperatures without influencing its compressive strength and durability. Results from AeA-FiResCrete testing showed only a slight improvement of its fire resistance.