Nicotine and electronic cigarette (E-Cig) exposure decreases brain glucose utilization in ischemic stroke.

Previous studies in our laboratory have shown that nicotine exposure decreases glucose transport across the blood-brain barrier in ischemia-reperfusion conditions. We hypothesize that nicotine can also dysregulate brain parenchymal glucose utilization by altering glucose transporters with effects on sensitivity to ischemic stroke. In this study, we investigated the effects of nicotine exposure on neuronal glucose utilization using an in vitro ischemic stroke model. We also tested the effects of e-Cig vaping on ischemic brain glucose utilization using an acute brain slice technique. Primary cortical neurons and brain slices were subjected to oxygen-glucose deprivation followed by reoxygenation to mimic ischemia-reperfusion injury. We estimated brain cell glucose utilization by measuring the uptake of [3 H] deoxy-d-glucose. Immunofluorescence and western blotting were done to characterize glucose transporters (GLUTs) and α7 nicotinic acetylcholine receptor (nAChR) expression. Furthermore, we used a glycolytic stress test to measure the effects of nicotine exposure on neuronal glucose metabolism. We observed that short- and long-term nicotine/cotinine exposure significantly decreased neuronal glucose utilization in ischemic conditions and the non-specific nAChR antagonist, mecamylamine reversed this effect. Nicotine/cotinine exposure also decreased neuronal GLUT1 and up-regulated α7 nAChR expression and decreased glycolysis. Exposure of mice to e-Cig vapor for 7 days likewise decreases brain glucose uptake under normoxic and ischemic conditions along with down-regulation of GLUT1 and GLUT3 expressions. These data support, from a cerebrovascular perspective, that nicotine and/or e-Cig vaping induce a state of glucose deprivation at the neurovascular unit which could lead to enhanced ischemic brain injury and/or stroke risk. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

In Vitro Modulation of Redox and Metabolism Interplay at the Brain Vascular Endothelium: Genomic and Proteomic Profiles of Sulforaphane Activity.

Sulforaphane (SFN) has been shown to protect the brain vascular system and effectively reduce ischemic injuries and cognitive deficits. Given the robust cerebrovascular protection afforded by SFN, the objective of this study was to profile these effects in vitro using primary mouse brain microvascular endothelial cells and focusing on cellular redox, metabolism and detoxification functions. We used a mouse MitoChip array developed and validated at the FDA National Center for Toxicological Research (NCTR) to profile a host of genes encoded by nuclear and mt-DNA following SFN treatment (0-5 µM). Corresponding protein expression levels were assessed (ad hoc) by qRT-PCR, immunoblots and immunocytochemistry (ICC). Gene ontology clustering revealed that SFN treatment (24 h) significantly up-regulated ~50 key genes (>1.5 fold, adjusted p < 0.0001) and repressed 20 genes (<0.7 fold, adjusted p < 0.0001) belonging to oxidative stress, phase 1 & 2 drug metabolism enzymes (glutathione system), iron transporters, glycolysis, oxidative phosphorylation (OXPHOS), amino acid metabolism, lipid metabolism and mitochondrial biogenesis. Our results show that SFN stimulated the production of ATP by promoting the expression and activity of glucose transporter-1, and glycolysis. In addition, SFN upregulated anti-oxidative stress responses, redox signaling and phase 2 drug metabolism/detoxification functions, thus elucidating further the previously observed neurovascular protective effects of this compound.

Conventional and electronic cigarettes dysregulate the expression of iron transporters and detoxifying enzymes at the brain vascular endothelium: In vivo evidence of a gender-specific cellular response to chronic cigarette smoke exposure.

It is well established that tobacco smoking is associated with vascular endothelial dysfunction in a causative and dose dependent manner primarily related to the tobacco smoke (TS) content of reactive oxygen species (ROS), nicotine, and oxidative stress (OS) -driven inflammation. Preclinical studies have also shown that nicotine (the principal e-liquid's ingredient used in e-cigarettes (e-Cigs) can also cause OS, exacerbation of cerebral ischemia and secondary brain injury. Likewise, chronic e-Cig vaping could be prodromal to vascular endothelial dysfunctions. Herein, we provide direct evidence that similarly to TS, e-Cig promotes mitochondrial depolarization in primary brain vascular endothelial cells as well as the vascular endothelial cell line bEnd3. In addition, both TS and e-Cig exposure upregulated the transmembrane iron exporter Slc40a1 (crucial to maintain cellular iron and redox homeostasis) and that of porphyrin importer Abcb6 (linked to accelerated atherosclerosis). We then investigated in vivo whether gender plays a role in how chronic TS affect vascular endothelial functions. Our results clearly show chronic TS exposure differentially impacts the expression levels of Phase-II enzymes as well as the iron transporters previously investigated in vitro. Although the physiological implications of the gender-specific differential responses to TS are not fully clear, they do demonstrate that gender is a risk factor that needs to be investigated when assessing the potential impact of chronic smoking and perhaps e-Cig vaping.

A convenient UHPLC-MS/MS method for routine monitoring of plasma and brain levels of nicotine and cotinine as a tool to validate newly developed preclinical smoking model in mouse.

BACKGROUND: A sensitive, rapid and selective UHPLC-MS/MS method has been developed and validated for the quantification of Nicotine (NT) and Cotinine (CN) using Continine-d 3 as internal standard (IS) as per FDA guidelines. Sample preparation involved simple protein precipitation of 20 µL mouse plasma or brain homogenate using acetonitrile at 1:8 ratio. Mass Spectrometer was operated in positive polarity under the multiple reaction-monitoring mode using electro spray ionization technique and the transitions of m/z 163.2 â†’ 132.1, 177.2 â†’ 98.0 and 180.2 â†’ 101.2 were used to measure the NT, CN and IS, respectively. The elution of NT, CN and IS are at 1.89, 1.77 and 1.76 min, respectively. This was achieved with a gradient mobile phase consisting of 5 mM ammonium bicarbonate, acetonitrile and methanol (3:1, v/v) at a flow rate of 0.3 mL/min on a Kinetex EVO C18 column. The method was validated with a lower limit of quantitation 3.0 ng/mL in mouse plasma and brain for both the analytes. RESULTS: A linear response function was established for the range of concentrations 3-200 (r > 0.995) for NT and 3-600 ng/mL (r > 0.995) for CN. The intra- and inter-day precision values met the acceptance criteria. NT and CN are stable in the battery of stability studies viz., stock solution, bench-top and auto-sampler. CONCLUSION: This method was successfully utilized to validate a newly developed preclinical smoking model in mice.

Blood-brain barrier disruption in diabetic mice is linked to Nrf2 signaling deficits: Role of ABCB10?

Blood-brain barrier (BBB) damage is a critical neurovascular complication of diabetes mellitus that adversely affects the CNS health and function. Previously, we showed the protective role of NF-E2 related factor-2 (Nrf2), a redox sensitive transcription factor, in regulation of BBB integrity. Given the pathogenic role of mitochondrial oxidative stress in diabetes-related microvascular complications, we focused on assessing: 1) the impact of diabetes on brain Nrf2 in correlation with BBB permeability and 2) Nrf2-dependent regulation of the mitochondrial transporter ABCB10, an essential player in mitochondrial function and redox balance at BBB endothelium. Using live animal fluorescence imaging, we demonstrated a strong increase in BBB permeability to 70kDa dextran in db/db diabetic mice that correlated with significant down-regulation of brain Nrf2 protein. Further, Nrf2 gene silencing in human BBB endothelial cells markedly suppressed ABCB10 protein, while Nrf2 activation by sulforaphane up-regulated ABCB10 expression. Interestingly, ABCB10 knockdown resulted in a strong-induction of Nrf2 driven anti-oxidant responses as evidenced by increased expression of Nrf2 and its downstream targets. Nrf2 or ABCB10 silencing elevated endothelial-monocyte adhesion suggesting an activated inflammatory cascade. Thus, our results demonstrate a novel mechanism of ABCB10 regulation driven by Nrf2. In summary, Nrf2 dysregulation and ABCB10 suppression could likely mediate endothelial oxidative/inflammatory stress and BBB disruption in diabetic subjects.

Offsetting the impact of smoking and e-cigarette vaping on the cerebrovascular system and stroke injury: Is Metformin a viable countermeasure?

Recently published in vitro and in vivo findings strongly suggest that BBB impairment and increased risk for stroke by tobacco smoke (TS) closely resemble that of type-2 diabetes (2DM) and develop largely in response to common key modulators such oxidative stress (OS), inflammation and alterations of the endogenous antioxidative response system (ARE) regulated by the nuclear factor erythroid 2-related factor (Nrf2). Preclinical studies have also shown that nicotine (the principal e-liquid's ingredient used in e-cigarettes) can also cause OS, exacerbation of cerebral ischemia and secondary brain injury. Herein we provide evidence that likewise to TS, chronic e-Cigarette (e-Cig) vaping can be prodromal to the loss of blood-brain barrier (BBB) integrity and vascular inflammation as well as act as a promoting factor for the onset of stroke and worsening of post-ischemic brain injury. In addition, recent reports have shown that Metformin (MF) treatment before and after ischemic injury reduces stress and inhibits inflammatory responses. Recent published data by our group revealead that MF promotes the activation of counteractive mechanisms mediated by the activation of Nrf2 which drastically reduce TS toxicity at the brain and cerebrovascular levels and protect BBB integrity. In this study we provide additional in vivo evidence showing that MF can effectively reduce the oxidative and inflammatory risk for stroke and attenuate post-ischemic brain injury promoted by TS and e-Cig vaping. Our data also suggest that MF administration could be extended as prophylactic care during the time window required for the renormalization of the risk levels of stroke following smoking cessation thus further studies in that direction are warrated.

New experimental models of the blood-brain barrier for CNS drug discovery.

INTRODUCTION: The blood-brain barrier (BBB) is a dynamic biological interface which actively controls the passage of substances between the blood and the central nervous system (CNS). From a biological and functional standpoint, the BBB plays a crucial role in maintaining brain homeostasis inasmuch that deterioration of BBB functions are prodromal to many CNS disorders. Conversely, the BBB hinders the delivery of drugs targeting the brain to treat a variety of neurological diseases. Area covered: This article reviews recent technological improvements and innovation in the field of BBB modeling including static and dynamic cell-based platforms, microfluidic systems and the use of stem cells and 3D printing technologies. Additionally, the authors laid out a roadmap for the integration of microfluidics and stem cell biology as a holistic approach for the development of novel in vitro BBB platforms. Expert opinion: Development of effective CNS drugs has been hindered by the lack of reliable strategies to mimic the BBB and cerebrovascular impairments in vitro. Technological advancements in BBB modeling have fostered the development of highly integrative and quasi- physiological in vitro platforms to support the process of drug discovery. These advanced in vitro tools are likely to further current understanding of the cerebrovascular modulatory mechanisms.

Hyperglycemia exacerbates antiretroviral drug combination induced blood-brain barrier endothelial toxicity.

In this study, we sought to investigate how concomitant hyperglycemia influences the impact of combination antiretroviral therapy on blood-brain barrier (BBB) endothelial function. Immortalized human brain microvascular endothelial cell line (hCMEC/D3) was exposed to azidothymidine (AZT; a nucleoside reverse transcriptase inhibitor) and/or indinavir (IND; protease inhibitor) in normal glycemic (5.5mM) or hyperglycemic (HG; 25mM) media containing D-glucose for 24-72h. Cellular reactive oxygen species (ROS) and mitochondria-specific superoxide levels were assayed in addition to membrane potential to determine the extent of mitochondrial dysfunction. Nrf2 expression was analyzed by immunofluorescence. Our results indicated a significant increase in BBB endothelial toxicity (decreased ATP) by HG and AZT+IND with progression of time (24-72h). Concurrent HG and antiviral drug combination synergistically elevated BBB endothelial ROS induced by either condition alone. Further, HG and AZT+IND mutually interact to elicit a pronounced increase in mitochondrial superoxide levels post 24h (vs. either condition alone or controls). In addition, HG and AZT+IND complemented each other to induce potential loss of mitochondrial membrane potential. While HG or AZT+IND alone for 24h increased Nrf2 nuclear distribution, co-exposure conditions induced a potential loss of Nrf2 expression/nuclear translocation in BBB endothelium. In summary, our data strongly suggest that antiretroviral drug combination potentially interacts with concomitant HG and triggers exacerbated mitochondrial dysfunction and BBB endothelial toxicity, possibly through dysregulation of Nrf2 signaling. Thus, this study warrants the critical need for safety evaluation and monitoring of neurovascular complications of HAART regimens in HIV-infected diabetic patient cohort.

Phytochemical and biological investigations of methanol extract of leaves of ziziphus mauritiana lam / Investigaciones fitoquímicas y biológicas de extractos metanólicos de hojas de ziziphus mauritiana lam

Seven compounds were isolated from the methanol extract of leaves of Ziziphus mauritiana. The structures of the isolated compounds were elucidated as y-fagarine (1), beta-sitosterol (2), stigmasterol (3), alfa-amyrin (4), lupeol (5), beta-amyrin (6) and betulin (7) by extensive spectroscopic studies and by co-TLC with authentic samples. This is the first report of isolation of compounds 1-7 from this plant species. The extractives from leaves were investigated for thrombolytic, membrane stabilizing, antimicrobial, antidiarrheal and analgesic potentials along with sleep inducing property on animal model. The thrombolytic and membrane stabilizing activities were assessed by using human erythrocyte comparing with standard streptokinase and acetylsalicylic acid, respectively. The methanolic extract reduced frequency of diarrheal faeces when compared to the standard loperamide. In castor oil induced diarrhea in mice a dose dependent decrease of gastrointestinal transits were observed. In radiant heat tail-flick method the methanol extract showed moderate antinociceptive activity, compared to standard morphine.

Siete compuestos fueron aislados del extracto metanólico de hojas de Ziziphus mauritiana. Las estructuras de los compuestos aislados fueron identificadas como y-fagarina (1), beta-sitosterol (2), estigmasterol (3), alfa-amirina (4), lupeol (5), beta-amirina (6) y betulina (7 ) por medio de extensivos estudios espectroscópicos y por co-TLC con muestras auténticas. Este es el primer informe de aislamiento de los compuestos 1-7 a partir de esta especie vegetal. Los extractos de las hojas fueron investigados por efectos trombolíticos, estabilizadores de membrana, antimicrobianos, antidiarreicos y potenciales analgésicos junto a la propiedad de inducir sueño en el modelo animal. Las actividades trombolíticos y la estabilización de la membrana se evaluaron mediante el uso de eritrocitos humanos comparando con estreptoquinasa estándar y el ácido acetilsalicílico, respectivamente. El extracto metanólico redujo la frecuencia de las heces diarreicas en comparación con el estándar de loperamida. En la inducción por aceite de ricino de diarrea en ratones, se observó una disminución dosis dependiente de los tránsitos gastrointestinales. En el método del coletazo producido por calor radiante, el extracto metabólico mostró una actividad antinociceptiva moderada, en comparación con la morfina estándar.

Clausenolide-1-methyl ether from Clausena heptaphylla W&A / Clausenolido-1-metil éter de Clausena heptaphylla W & A

From the stem bark of Clausena heptaphylla, the limonoid clausenolide-1-methyl ether (1) was isolated. The structure of the compound was elucidated by extensive spectroscopic studies, including 2D NMR and MS measurements and by comparison with spectroscopic and physical data from the literature. This is the first report of occurrence of clausenolide-1-methyl ether (1) in Clausena heptaphylla.

El limonoíde clausenolido-1-metil eter (1) fue aislado de corteza de Clausena heptaphylla. La estructura del compuesto fue determinada por metodos espectroscópicos, incluyendo 2D RMN, espectrometría de masas (EM) y comparación de los datos espectroscópicos y constantes físicas con los publicados en la literatura. Este es el primer informe del aislamiento de clausenolido-1-metil eter (1) de Clausena heptaphylla.

Preliminary antimicrobial activity and cytotoxicity of leaf extracts of mesua nagassarium (Burm.f. ) / Actividad antimicrobiana preliminar y citotoxicidad de extractos de hojas de Mesua nagassarium (Burm.f. )

In the present study the in vitro antimicrobial activity, along with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), of different extracts of leaves of Mesua nagassarium were evaluated against 13 pathogenic microorganisms. The methanol extract and its pet-ether and carbon tetrachloride soluble fractions showed the highest antimicrobial activity. The carbon tetrachloride soluble fraction showed the maximum inhibition zone of 24.33 mm against Bacillus megaterium with MIC and MBC values of 7.81 ug/ml and 250 ug/ml, respectively. Ciprofloxacin (30 ug/disc) was used as standard antimicrobial agent. In the Brine shrimp lethality bioassay, the crude methanol extract and its carbon tetrachloride soluble fraction showed significant cytotoxicity with LC50 of 2.99 and 1.74 ug/ml, respectively as compared vincristine sulphate (LC50 value 0.543 ug/ml).

En el presente estudio se evaluó la actividad antimicrobiana in vitro, incluyendo la concentración inhibitoria mínima (CIM) y la concentración bactericida mínima (CBM), de diferentes extractos obtenidos de hojas de Mesua nagassarium en 13 microorganismos patógenos. El extracto metanólico y sus fracciones solubles en éter de petróleo y tetracloruro de carbono, mostraron la mayor actividad antimicrobiana. La fracción de compuestos solubles en tetracloruro de carbono mostró la zona de inhibición máxima de 24.33 mm en Bacillus megaterium con valores de CIM y and CBM de 7.81 ug/ml y 250 ug/ml, respectivamente. Como agente antimicrobiano estándar se utilizó ciprofloxacina (30 ug/disco). En el bioensayo de mortalidad de Brine shrimp el extracto metanólico y su fracción soluble en tetracloruro de carbono mostraron importante citotoxicidad con CL50 de 2.99 y 1.74 ug/ml, respectivamente, comparadas con el sulfato de vincristina (CL50 0.543 ug/ml).

Chemical and biological investigations of Delonix regia (Bojer ex Hook.) Raf.

In this study, five compounds, lupeol (1), epilupeol (2), ß-sitosterol (3), stigmasterol (4) and p-methoxybenzaldehyde (5) were isolated from the petroleum ether and dichloromethane fractions of a methanolic extract of the stem bark of Delonix regia. Antimicrobial screening of the different extracts (15 µg mm-2) was conducted by the disc diffusion method. The zones of inhibition demonstrated by the petroleum ether, carbon tetrachloride and dichloromethane fractions ranged from 9-14 mm, 11-13 mm and 9-20 mm, respectively, compared to kanamycin standard with the zone of inhibition of 20-25 mm. In brine shrimp lethality bioassay, the carbon tetrachloride soluble materials demonstrated the highest toxicity with LC50 of 0.83 µg mL-1, while petroleum ether and dichloromethane soluble partitionates of the methanolic extract revealed LC50 of 14.94 and 3.29 µg mL-1, respectively, in comparison with standard vincristine sulphate with LC50 of 0.812 µg mL-1. This is the first report on compounds separation from D. regia, their antimicrobial activity and cytotoxicity.

Trans-isoferulic acid from Curcuma longa / Ácido trans-isoferulico en Curcuma Longa

Trans-isoferulic acid was isolated from the carbon tetrachloride soluble fraction of a methanol extract of the rhizomes of Curcuma longa (Zingiberaceae). The structure of the isolated compound was elucidated by comprehensive analysis of spectroscopic data. This is the first report of its occurrence from this plant.

El ácido trans-isoferulico fue aislado de la fracción soluble en tetracloruro de carbono del extracto metanólico de los rizomas de Curcuma Longa (Zingeraceae). La estructura del compuesto aislado fue elucidada por análisis de los datos espectroscópicos. Este es el primer reporte de su presencia en esta planta.