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Exposure is key to message effects. No effects can ensue if a health, political, or commercial message is not noticed. Yet, existing research in communication, advertising, and related disciplines often measures 'opportunities for exposure' at an aggregate level, whereas knowing whether recipients were 'actually exposed' to a message requires a micro-level approach. Micro-level research, on the other hand, focuses on message processing and retention, takes place under highly controlled laboratory conditions with forced message exposure, and largely ignores how recipients attend selectively to messages under more natural conditions. Eye-tracking enables us to assess actual exposure, but its previous applications were restricted to screen-based reading paradigms lacking ecological validity or field studies that suffer from limited experimental control. Our solution is to measure eye-tracking within an immersive VR environment that creates the message delivery and reception context. Specifically, we simulate a car ride down a highway alongside which billboards are placed. The VR headset (HP Omnicept Pro) provides an interactive 3D view of the environment and holds a seamlessly integrated binocular eye tracker that records the drivers' gaze and detects all fixations on the billboards. This allows us to quantify the nexus between exposure and reception rigorously, and to link our measures to subsequent memory, i.e., whether messages were remembered, forgotten, or not even encoded. An empirical study shows that incidental memory for messages differs based on participants' gaze behavior while passing the billboards. The study further shows how an experimental manipulation of attentional demands directly impacts drivers' gaze behavior and memory. We discuss the large potential of this paradigm to quantify exposure and message reception in realistic communication environments and the equally promising applications in new media contexts (e.g., the Metaverse).
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Publicidad , Realidad Virtual , Humanos , Tecnología de Seguimiento Ocular , Comunicación , Medios de Comunicación de MasasRESUMEN
A sensitive and accurate analytical method was developed and validated to detect bambermycin, a commonly used antibiotic in animal feed and livestock. The presence of bambermycin residues in food products can pose health risks to consumers, emphasizing the need for a sensitive and accurate analytical method. A reversed-phase analytical column was utilized with a mobile phase comprising 0.005 mol/L ammonium acetate in 5% acetonitrile (A) and 0.005 mol/L ammonium acetate in 95% acetonitrile (B) to achieve effective chromatographic separation. Quantitative determination of bambermycin in various samples, including beef, pork, chicken, milk, eggs, flatfish, eel, and shrimp, was performed using ultra-high-performance liquid chromatography-tandem mass spectrometry. Sample extraction involved a mixture of methanol and a 25% ammonium hydroxide solution, followed by low-temperature purification and phospholipid removal utilizing a Phree cartridge. The method exhibited a satisfactory recovery rate ranging from 69% to 100%. Validation results demonstrated the reliability, robustness, and accuracy of the method, exhibiting good linearity, precision, and recovery. This validated method can be applied for routine analysis of bambermycin residues, assisting in the development of effective monitoring and control measures to ensure the safety of livestock and aquatic products.
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Bambermicinas , Animales , Bovinos , Cromatografía Líquida de Alta Presión/métodos , Ganado , Espectrometría de Masas en Tándem/métodos , Reproducibilidad de los Resultados , Contaminación de Alimentos/análisis , Inocuidad de los Alimentos , Acetonitrilos/química , Extracción en Fase SólidaRESUMEN
PURPOSE: The purpose of this study was to comprehensively understand and describe the meaning of physical activity for managing menopausal symptoms in middle-aged women. METHODS: This study targeted middle-aged women with menopausal symptoms who participated in regular exercise at least three times a week for more than 12 weeks. Nine participants were individually interviewed via in-depth face-to-face interviews, and participatory observation was also employed. Colaizzi's phenomenological qualitative research method was applied for analysis. RESULTS: Participants were asked, "What does it means to participate in physical activity at this time of your life?" Fourteen codes, six themes, and three theme clusters were derived for the meaning of physical activity for managing menopausal symptoms these middle-aged women. The six themes were "reviving the exhausted body and mind," "being free from the yoke of pain," "being settled in life," "finding oneself and becoming altruistic," "striving while anticipating change," and "equipping the body and mind." The three theme clusters were "overcoming my past pain," "taking the initiative for today's life," and "moving towards new change." CONCLUSION: The narratives revealed that physical activity allowed women to overcome menopausal symptoms, the burden of relationships, and stress, thereby enabling them to make positive changes in their lives and have expectations for the future. Thus, physical activity was a positive force in a healthy menopausal transition for women with menopausal symptoms. The findings of this study can be used to encourage physical activity in peri-menopausal women and to develop physical activity programs for managing menopausal symptoms.
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Background: Ginseng contains three active components: ginsenosides, gintonin, and polysaccharides. After the separation of 1 of the 3 ingredient fractions, other fractions are usually discarded as waste. In this study, we developed a simple and effective method, called the ginpolin protocol, to separate gintonin-enriched fraction (GEF), ginseng polysaccharide fraction (GPF), and crude ginseng saponin fraction (cGSF). Methods: Dried ginseng (1 kg) was extracted using 70% ethanol (EtOH). The extract was water fractionated to obtain a water-insoluble precipitate (GEF). The upper layer after GEF separation was precipitated with 80% EtOH for GPF preparation, and the remaining upper layer was vacuum dried to obtain cGSF. Results: The yields of GEF, GPF, and cGSF were 14.8, 54.2, and 185.3 g, respectively, from 333 g EtOH extract. We quantified the active ingredients of 3 fractions: L-arginine, galacturonic acid, ginsenosides, glucuronic acid, lysophosphatidic acid (LPA), phosphatidic acid (PA), and polyphenols. The order of the LPA, PA, and polyphenol content was GEF > cGSF > GPF. The order of L-arginine and galacturonic acid was GPF >> GEF = cGSF. Interestingly, GEF contained a high amount of ginsenoside Rb1, whereas cGSF contained more ginsenoside Rg1. GEF and cGSF, but not GPF, induced intracellular [Ca2+]i transient with antiplatelet activity. The order of antioxidant activity was GPF > GEF = cGSF. Immunological activities (related to nitric oxide production, phagocytosis, and IL-6 and TNF-α release) were, in order, GPF > GEF = cGSF. The neuroprotective ability (against reactive oxygen species) order was GEF > cGSP > GPF. Conclusion: We developed a novel ginpolin protocol to isolate 3 fractions in batches and determined that each fraction has distinct biological effects.
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BACKGROUND: We recently showed that gintonin, an active ginseng ingredient, exhibits antibrain neurodegenerative disease effects including multiple target mechanisms such as antioxidative stress and antiinflammation via the lysophosphatidic acid (LPA) receptors. Amyotrophic lateral sclerosis (ALS) is a spinal disease characterized by neurodegenerative changes in motor neurons with subsequent skeletal muscle paralysis and death. However, pathophysiological mechanisms of ALS are still elusive, and therapeutic drugs have not yet been developed. We investigate the putative alleviating effects of gintonin in ALS. METHODS: The G93A-SOD1 transgenic mouse ALS model was used. Gintonin (50 or 100 mg/kg/day, p.o.) administration started from week seven. We performed histological analyses, immunoblot assays, and behavioral tests. RESULTS: Gintonin extended mouse survival and relieved motor dysfunctions. Histological analyses of spinal cords revealed that gintonin increased the survival of motor neurons, expression of brain-derived neurotrophic factors, choline acetyltransferase, NeuN, and Nissl bodies compared with the vehicle control. Gintonin attenuated elevated spinal NAD(P) quinone oxidoreductase 1 expression and decreased oxidative stress-related ferritin, ionized calcium-binding adapter molecule 1-immunoreactive microglia, S100ß-immunoreactive astrocyte, and Olig2-immunoreactive oligodendrocytes compared with the control vehicle. Interestingly, we found that the spinal LPA1 receptor level was decreased, whereas gintonin treatment restored decreased LPA1 receptor expression levels in the G93A-SOD1 transgenic mouse, thereby attenuating neurological symptoms and histological deficits. CONCLUSION: Gintonin-mediated symptomatic improvements of ALS might be associated with the attenuations of neuronal loss and oxidative stress via the spinal LPA1 receptor regulations. The present results suggest that the spinal LPA1 receptor is engaged in ALS, and gintonin may be useful for relieving ALS symptoms.
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BACKGROUND: Gintonin is a ginseng-derived exogenous G-protein-coupled lysophosphatidic acid (LPA) receptor ligand, which exhibits in vitro and in vivo functions against Alzheimer disease (AD) through lysophosphatidic acid 1/3 receptors. A recent study demonstrated that systemic treatment with gintonin enhances paracellular permeability of the blood-brain barrier (BBB) through the LPA1/3 receptor. However, little is known about whether gintonin can enhance brain delivery of donepezil (DPZ) (Aricept), which is a representative cognition-improving drug used in AD clinics. In the present study, we examined whether systemic administration of gintonin can stimulate brain delivery of DPZ. METHODS: We administered gintonin and DPZ alone or coadministered gintonin with DPZ intravenously or orally to rats. Then we collected the cerebral spinal fluid (CSF) and serum and determined the DPZ concentration through liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. RESULTS: Intravenous, but not oral, coadministration of gintonin with DPZ increased the CSF concentration of DPZ in a concentration- and time-dependent manner. Gintonin-mediated enhancement of brain delivery of DPZ was blocked by Ki16425, a LPA1/3 receptor antagonist. Coadministration of vascular endothelial growth factor (VEGF) + gintonin with DPZ similarly increased CSF DPZ concentration. However, gintonin-mediated enhancement of brain delivery of DPZ was blocked by axitinip, a VEGF receptor antagonist. Mannitol, a BBB disrupting agent that increases the BBB permeability, enhanced gintonin-mediated enhancement of brain delivery of DPZ. CONCLUSIONS: We found that intravenous, but not oral, coadministration of gintonin facilitates brain delivery of DPZ from plasma via LPA1/3 and VEGF receptors. Gintonin is a potential candidate as a ginseng-derived novel agent for the brain delivery of DPZ for treatment of patients with AD.
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In this study, we developed a simple screening procedure for the determination of 18 anthelmintics (including benzimidazoles, macrocyclic lactones, salicylanilides, substituted phenols, tetrahydropyrimidines, and imidazothiazoles) in five animal-derived food matrices (chicken muscle, pork, beef, milk, and egg) using liquid chromatography-tandem mass spectrometry. Analytes were extracted using acetonitrile/1% acetic acid (milk and egg) and acetonitrile/1% acetic acid with 0.5 mL of distilled water (chicken muscle, pork, and beef), and purified using saturated n-hexane/acetonitrile. A reversed-phase analytical column and a mobile phase consisting of (A) 10 mM ammonium formate in distilled water and (B) methanol were used to achieve optimal chromatographic separation. Matrix-matched standard calibration curves (R 2 ≥0.9752) were obtained for concentration equivalent to ×1/2, ×1, ×2, ×3, ×4, and ×5 fold the maximum residue limit (MRL) stipulated by the Korean Ministry of Food and Drug Safety. Recoveries of 61.2-118.4%, with relative standard deviations (RSDs) of ≤19.9% (intraday and interday), were obtained for each sample at three spiking concentrations (×1/2, ×1, and ×2 the MRL values). Limits of detection, limits of quantification, and matrix effects were 0.02-5.5 µg/kg, 0.06-10 µg/kg, and -98.8 to 13.9% (at 20 µg/kg), respectively. In five samples of each food matrix (chicken muscle, pork, beef, milk, and egg) purchased from large retailers in Seoul that were tested, none of the target analytes were detected. It has therefore been shown that this protocol is adaptable, accurate, and precise for the quantification of anthelmintic residues in foods of animal origin.
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Herein, an analytical method was developed for simultaneous determination of 12 anthelmintics (closantel, niclosamide, nitroxynil, rafoxanide, cymiazole, fluazuron, levamisole, morantel, praziquantel, pyrantel, thiophanate, and trichlorfon) in fishery products (eel, flatfish, and shrimp) using liquid-liquid extraction coupled with liquid chromatography-tandem mass spectrometry. A reversed-phase analytical column was then used to separate the analytes from various matrices. Linear matrix-matched calibration curves were generated with coefficients of determination ≥ 0.9935. Recovery rates at three spiking levels (5, 10, and 20 µg/kg) ranged between 61.58% and 119.37% with relative standard deviations ≤ 19.05%. Limits of detection were in the range of 0.3-1.6 µg/kg, whereas limits of quantification ranged between 1.0 and 5.0 µg/kg. The matrix effect was moderate with values ranging from -99.47% to 51.98%. Matrices procured from large markets tested negative for the 12 anthelmintics. The developed method proved amenable to real sample testing and can be used for simultaneous determination of target analytes in aquatic products.
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Cromatografía Líquida de Alta Presión , Explotaciones Pesqueras , Análisis de los Alimentos/métodos , Contaminación de Alimentos/análisis , Espectrometría de Masas en Tándem , Drogas Veterinarias/análisis , Residuos de Medicamentos/análisis , Límite de Detección , Extracción Líquido-Líquido , Alimentos Marinos/análisis , Factores de TiempoRESUMEN
The accumulation of antimicrobial residues in edible animal products and aquaculture products could pose health concerns to unsuspecting consumers. Hence, this study aimed to develop a validated method for simultaneous quantification of chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in beef, pork, chicken, shrimp, eel, and flatfish using a quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Primary-secondary amine (PSA) and MgSO4 were used for sample purification. The analytes were separated on a reversed-phase analytical column. The coefficients of determination for the linear matrix-matched calibration curves were ≥0.9941. Recovery rates ranged between 64.26 and 116.51% for the four analytes with relative standard deviations (RSDs) ≤ 18.05%. The calculated limits of detection (LODs) and limits of quantification (LOQs) were 0.005-3.1 and 0.02-10.4 µg/kg, respectively. The developed method was successfully applied for monitoring samples obtained from local markets in Seoul, Republic of Korea. The target residues were not detected in any tested matrix. The designed method was versatile, sensitive, and proved suitable for quantifying residues in animal-derived products.
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Gintonin, a novel ginseng-derived lysophosphatidic acid receptor ligand, improves brain functions and protects neurons from oxidative stress. However, little is known about the effects of gintonin against Pb-induced brain maldevelopment. We investigated the protective effects of gintonin on the developing cerebellum after prenatal and postnatal Pb exposure. Pregnant female rats were randomly divided into three groups: control, Pb (0.3% Pb acetate in drinking water), and Pb plus gintonin (100 mg/kg, p.o.). Blood Pb was increased in dams and pups; gintonin treatment significantly decreased blood Pb. On postnatal day 21, the number of degenerating Purkinje cells was remarkably increased while the number of calbindin-, GAD67-, NMDAR1-, LPAR1-immunoreactive intact Purkinje cells, and GABA transporter 1-immunoreactive pinceau structures were significantly reduced in Pb-exposed offspring. Following Pb exposure, gintonin ameliorated cerebellar degenerative effects, restored increased pro-apoptotic Bax, and decreased anti-apoptotic Bcl2. Gintonin treatment attenuated Pb-induced accumulation of oxidative stress (Nrf2 and Mn-SOD) and inflammation (IL-1ß and TNFα,), restoring the decreased cerebellar BDNF and Sirt1. Gintonin ameliorated Pb-induced impairment of myelin basic protein-immunoreactive myelinated fibers of Purkinje cells. Gintonin attenuated Pb-induced locomotor dysfunctions. The present study revealed the ameliorating effects of gintonin against Pb, suggesting the potential use of gintonin as a preventive agent in Pb poisoning during pregnancy and lactation.
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Lactancia/metabolismo , Intoxicación por Plomo , Exposición Materna/efectos adversos , Panax/química , Extractos Vegetales/farmacología , Células de Purkinje/metabolismo , Animales , Femenino , Intoxicación por Plomo/tratamiento farmacológico , Intoxicación por Plomo/embriología , Intoxicación por Plomo/patología , Extractos Vegetales/química , Embarazo , Células de Purkinje/patología , RatasRESUMEN
Gintonin is a newly discovered ingredient of ginseng and plays an exogenous ligand for G protein-coupled lysophosphatidic acid receptors. We previously showed that gintonin exhibits diverse effects from neurotransmitter release to improvement of Alzheimer's disease-related cognitive dysfunctions. However, previous studies did not show whether gintonin has protective effects against environmental heavy metal. We investigated the effects of gintonin-enriched fraction (GEF) on methylmercury (MeHg)-induced neurotoxicity and learning and memory dysfunction and on organ MeHg elimination. Using hippocampal neural progenitor cells (hNPCs) and mice we examined the effects of GEF on MeHg-induced hippocampal NPC neurotoxicity, on formation of reactive oxygen species (ROS), and on in vivo learning and memory functions after acute MeHg exposure. Treatment of GEF to hNPCs attenuated MeHg-induced neurotoxicity with concentration- and time-dependent manner. GEF treatment inhibited MeHg- and ROS inducer-induced ROS formations. Long-term treatment of GEF also improved MeHg-induced learning and memory dysfunctions. Oral administration of GEF decreased the concentrations of MeHg in blood, brain, liver, and kidney. This is the first report that GEF attenuated MeHg-induced in vitro and in vivo neurotoxicities through LPA (lysophosphatidic acids) receptor-independent manner and increased organ MeHg elimination. GEF-mediated neuroprotection might achieve via inhibition of ROS formation and facilitation of MeHg elimination from body.
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Disfunción Cognitiva/tratamiento farmacológico , Compuestos de Metilmercurio/toxicidad , Panax/química , Extractos Vegetales/uso terapéutico , Animales , Disfunción Cognitiva/inducido químicamente , Femenino , Ratones , Ratones Endogámicos C57BL , Receptores del Ácido LisofosfatídicoRESUMEN
Spinal cord injury (SCI) causes maladaptive changes to nociceptive synaptic circuits within the injured spinal cord. Changes also occur at remote regions including the brain stem, limbic system, cortex, and dorsal root ganglia. These maladaptive nociceptive synaptic circuits frequently cause neuronal hyperexcitability in the entire nervous system and enhance nociceptive transmission, resulting in chronic central neuropathic pain following SCI. The underlying mechanism of chronic neuropathic pain depends on the neuroanatomical structures and electrochemical communication between pre- and postsynaptic neuronal membranes, and propagation of synaptic transmission in the ascending pain pathways. In the nervous system, neurons are the only cell type that transmits nociceptive signals from peripheral receptors to supraspinal systems due to their neuroanatomical and electrophysiological properties. However, the entire range of nociceptive signaling is not mediated by any single neuron. Current literature describes regional studies of electrophysiological or neurochemical mechanisms for enhanced nociceptive transmission post-SCI, but few studies report the electrophysiological, neurochemical, and neuroanatomical changes across the entire nervous system following a regional SCI. We, along with others, have continuously described the enhanced nociceptive transmission in the spinal dorsal horn, brain stem, thalamus, and cortex in SCI-induced chronic central neuropathic pain condition, respectively. Thus, this review summarizes the current understanding of SCI-induced neuronal hyperexcitability and maladaptive nociceptive transmission in the entire nervous system that contributes to chronic central neuropathic pain.
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Dolor Crónico/etiología , Dolor Crónico/fisiopatología , Neuralgia/etiología , Neuralgia/fisiopatología , Neuronas/metabolismo , Traumatismos de la Médula Espinal/complicaciones , Traumatismos de la Médula Espinal/fisiopatología , Animales , Humanos , Inhibición Neural/fisiología , Especificidad de ÓrganosRESUMEN
BACKGROUND: Gintonin is a ginseng-derived exogenous ligand of the G protein-coupled lysophosphatidic acid (LPA) receptor. We previously reported that gintonin stimulates gliotransmitter release in primary cortical astrocytes. Astrocytes play key roles in the functions of neurovascular systems. Although vascular endothelial growth factor (VEGF) is known to influence the normal growth and maintenance of cranial blood vessels and the nervous system, there is little information about the effect of gintonin on VEGF regulation in primary astrocytes, under normal and hypoxic conditions. METHODS: Using primary cortical astrocytes of mice, the effects of gintonin on the release, expression, and distribution of VEGF were examined. We further investigated whether the gintonin-mediated VEGF release protects astrocytes from hypoxia. RESULTS: Gintonin administration stimulated the release and expression of VEGF from astrocytes in a concentration- and time-dependent manner. The gintonin-mediated increase in the release of VEGF was inhibited by the LPA1/3 receptor antagonist, Ki16425; phospholipase C inhibitor, U73122; inositol 1,4,5-triphosphate receptor antagonist, 2-APB; and intracellular Ca2+ chelator, BAPTA. Hypoxia further stimulated astrocytic VEGF release. Gintonin treatment stimulated additional VEGF release and restored cell viability that had decreased due to hypoxia, via the VEGF receptor pathway. Altogether, the regulation of VEGF release and expression and astrocytic protection mediated by gintonin under hypoxia are achieved via the LPA receptor-VEGF signaling pathways. CONCLUSION: The present study shows that the gintonin-mediated regulation of VEGF in cortical astrocytes might be neuroprotective against hypoxic insults and could explain the molecular basis of the beneficial effects of ginseng on the central nervous system.
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BACKGROUND: Ginseng is a traditional herbal medicine for human health. Ginseng contains a bioactive ligand named gintonin. The active ingredient of gintonin is lysophosphatidic acid C18:2 (LPA C18:2). We previously developed a method for gintonin-enriched fraction (GEF) preparation to mass-produce gintonin from ginseng. However, previous studies did not show the presence of other bioactive lipids besides LPAs. The aim of this study was to quantify the fatty acids, lysophospholipids (LPLs), and phospholipids (PLs) besides LPAs in GEF. METHODS: We prepared GEF from white ginseng. We used gas chromatography-mass spectrometry for fatty acid analysis and liquid chromatography-tandem mass spectrometry for PL analysis, and quantified the fatty acids, LPLs, and PLs in GEF using respective standards. We examined the effect of GEF on insulin secretion in INS-1 cells. RESULTS: GEF contains about 7.5% linoleic (C18:2), 2.8% palmitic (C16:0), and 1.5% oleic acids (C18:1). GEF contains about 0.2% LPA C18:2, 0.06% LPA C16:0, and 0.02% LPA C18:1. GEF contains 0.08% lysophosphatidylcholine, 0.03% lysophosphatidylethanolamine, and 0.13% lysophosphatidylinositols. GEF also contains about 1% phosphatidic acid (PA) 16:0-18:2, 0.5% PA 18:2-18:2, and 0.2% PA 16:0-18:1. GEF-mediated insulin secretion was not blocked by LPA receptor antagonist. CONCLUSION: We determined four characteristics of GEF through lipid analysis and insulin secretion. First, GEF contains a large amount of linoleic acid (C18:2), PA 16:0-18:2, and LPA C18:2 compared with other lipids. Second, the main fatty acid component of LPLs and PLs is linoleic acid (C18:2). Third, GEF stimulates insulin secretion not through LPA receptors. Finally, GEF contains bioactive lipids besides LPAs.
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Astrocytes are a unique brain cell-storing glycogen and express lysophosphatidic acid (LPA) receptors. Gintonin is a ginseng-derived exogenous G protein-coupled LPA receptor ligand. Accumulating evidence shows that astrocytes serve as an energy supplier to neurons through astrocytic glycogenolysis under physiological and pathophysiological conditions. However, little is known about the relationships between LPA receptors and astrocytic glycogenolysis or about the roles of LPA receptors in hypoxia and re-oxygenation stresses. In the present study, we examined the functions of gintonin-mediated astrocytic glycogenolysis in adenosine triphosphate (ATP) production, glutamate uptake, and cell viability under normoxic, hypoxic, and re-oxygenation conditions. The application of gintonin or LPA to astrocytes induced glycogenolysis in concentration- and time-dependent manners. The stimulation of gintonin-mediated astrocytic glycogenolysis was achieved through the LPA receptor-Gαq/11 protein-phospholipase C-inositol 1,4,5-trisphosphate receptor-intracellular calcium ([Ca2+]i) transient pathway. Gintonin treatment to astrocytes increased the phosphorylation of brain phosphorylase kinase, with sensitive manner to K252a, an inhibitor of phosphorylase kinase. Gintonin-mediated astrocytic glycogenolysis was blocked by isofagomine, a glycogen phosphorylase inhibitor. Gintonin additionally increased astrocytic glycogenolysis under hypoxic and re-oxygenation conditions. Moreover, gintonin increased ATP production, glutamate uptake, and cell viability under the hypoxic and re-oxygenation conditions. Collectively, we found that the gintonin-mediated [Ca2+]i transients regulated by LPA receptors were coupled to astrocytic glycogenolysis and that stimulation of gintonin-mediated astrocytic glycogenolysis was coupled to ATP production and glutamate uptake under hypoxic and re-oxygenation conditions, ultimately protecting astrocytes. Hence, the gintonin-mediated astrocytic energy that is modulated via LPA receptors helps to protect astrocytes under hypoxia and re-oxygenation stresses.
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Astrocitos/metabolismo , Astrocitos/patología , Glucogenólisis/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Oxígeno/farmacología , Panax/química , Receptores del Ácido Lisofosfatídico/metabolismo , Estrés Fisiológico , Adenosina Trifosfato/biosíntesis , Animales , Astrocitos/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Ácido Glutámico/metabolismo , Glucógeno Sintasa/metabolismo , Ligandos , Lisofosfolípidos/farmacología , Ratones , Modelos Biológicos , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacosRESUMEN
Celecoxib is a non-steroidal anti-inflammatory drug that selectively inhibits cyclooxygenase-2 and is prescribed for severe pain and inflammation. The excellent therapeutic effects of celecoxib mean that it is frequently used clinically, including for women of child-bearing age. However, the prenatal effects of this compound have not been studied extensively in vertebrates. The present study examined the developmental toxicity of celecoxib using a frog embryo teratogenic assay-Xenopus (FETAX). In addition, we examined its effects on cell migration using co-cultures of human umbilical vein endothelial cells and 10T1/2â¯cells. These studies revealed that celecoxib induced concentration-dependent mortality and various malformations of the Xenopus internal organs, including gut miscoiling, haemorrhage, and oedema. Celecoxib also downregulated the expression of vascular wall markers (Msr and alpha smooth muscle actin) and other organ-specific markers (Nkx2.5, Cyl104 and IFABP). In vitro co-culture studies revealed that celecoxib inhibited pericyte migration and differentiation into vascular smooth muscle cells. In conclusion, celecoxib was both toxic and teratogenic in Xenopus embryos, where it produced serious heart and vessel malformation by inhibiting vascular wall maturation and vascular network formation.
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Celecoxib/toxicidad , Teratógenos/toxicidad , Xenopus laevis/embriología , Animales , Antiinflamatorios no Esteroideos/toxicidad , Biomarcadores , Vasos Sanguíneos/anomalías , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/embriología , Celecoxib/administración & dosificación , Movimiento Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Embrión no Mamífero/efectos de los fármacos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Xenopus laevis/fisiologíaRESUMEN
The neurons in the trigeminal mesencephalic nucleus (Vmes) innervate jaw-closing muscle spindles and periodontal ligaments, and play a crucial role in the regulation of jaw movements. Recently, it was shown that many boutons that form synapses on them are immunopositive for glycine (Gly+), suggesting that these neurons receive glycinergic input. Information about the glycine receptors that mediate this input is needed to help understand the role of glycine in controlling Vmes neuron excitability. For this, we investigated the expression of glycine receptor subunit alpha 3 (GlyRα3) and gephyrin in neurons in Vmes and the trigeminal motor nucleus (Vmo), and the Gly+ boutons that contact them by light- and electron-microscopic immunocytochemistry and quantitative ultrastructural analysis. The somata of the Vmes neurons were immunostained for GlyRα3, but not gephyrin, indicating expression of homomeric GlyR. The immunostaining for GlyRα3 was localized away from the synapses in the Vmes neuron somata, in contrast to the Vmo neurons, where the staining for GlyRα3 and gephyrin were localized at the subsynaptic zones in somata and dendrites. Additionally, the ultrastructural determinants of synaptic strength, bouton volume, mitochondrial volume, and active zone area, were significantly smaller in Gly+ boutons on the Vmes neurons than in those on the Vmo neurons. These findings support the notion that the Vmes neurons receive glycinergic input via putative extrasynaptic homomeric glycine receptors, likely mediating a slow, tonic modulation of the Vmes neuron excitability.
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Neuronas/metabolismo , Terminales Presinápticos/metabolismo , Receptores de Glicina/metabolismo , Núcleo Motor del Nervio Trigémino/citología , Animales , Proteínas Portadoras/metabolismo , Proteínas Portadoras/ultraestructura , Dendritas/ultraestructura , Masculino , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Microscopía Confocal , Microscopía Inmunoelectrónica , Neuronas/ultraestructura , Terminales Presinápticos/ultraestructura , Ratas , Ratas Sprague-Dawley , Receptores de Glicina/ultraestructura , Núcleo Motor del Nervio Trigémino/diagnóstico por imagenRESUMEN
Spinal cord injury (SCI) frequently results in chronic neuropathic pain (CNP). However, the understanding of brain neural circuits in CNP modulation is unclear. The present study examined the changes of ventral tegmental area (VTA) putative GABAergic and dopaminergic neuronal activity with CNP attenuation in rats. SCI was established by T10 clip compression injury (35 g, 1 min) in rats, and neuropathic pain behaviors, in vivo extracellular single-cell recording of putative VTA gamma-aminobutyric acid (GABA)/dopamine neurons, extracellular GABA level, glutamic acid decarboxylase (GAD), and vesicular GABA transporters (VGATs) were measured in the VTA, respectively. The results revealed that extracellular GABA level was significantly increased in the CNP group (50.5 ± 18.9 nM) compared to the sham control group (10.2 ± 1.7 nM). In addition, expression of GAD65/67, c-Fos, and VGAT exhibited significant increases in the SCI groups compared to the sham control group. With regard to neuropathic pain behaviors, spontaneous pain measured by ultrasound vocalizations (USVs) and evoked pain measured by paw withdrawal thresholds showed significant alteration, which was reversed by intravenous (i.v.) administration of morphine (0.5-5.0 mg/kg). With regard to in vivo electrophysiology, VTA putative GABAergic neuronal activity (13.6 ± 1.7 spikes/sec) and putative dopaminergic neuronal activity (2.4 ± 0.8 spikes/sec) were increased and decreased, respectively, in the SCI group compared to the sham control group. These neuronal activities were reversed by i.v. administration of morphine. The present study suggests that chronic increase of GABAergic neuronal activity suppresses dopaminergic neuronal activity in the VTA and is responsible for negative emotion and motivation for attenuation of SCI-induced CNP.
Asunto(s)
Neuralgia/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Área Tegmental Ventral/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Dolor Crónico/etiología , Dolor Crónico/metabolismo , Masculino , Neuralgia/etiología , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/complicacionesRESUMEN
Peripheral nerve injury often causes neuropathic pain and is associated with changes in the expression of numerous proteins in the dorsal horn of the spinal cord. To date, proteomic analysis method has been used to simultaneously analyze hundreds or thousands of proteins differentially expressed in the dorsal horn of the spinal cord in rats or dorsal root ganglion of rats with certain type of peripheral nerve injury. However, a proteomic study using a mouse model of neuropathic pain could be attempted because of abundant protein database and the availability of transgenic mice. In this study, whole proteins were extracted from the ipsilateral dorsal half of the 4th-6th lumbar spinal cord in a mouse model of spared nerve injury (SNI)-induced neuropathic pain. In-gel digests of the proteins size-separated on a polyacrylamide gel were subjected to reverse-phase liquid-chromatography coupled with electrospray ionization ion trap tandem mass spectrometry (MS/MS). After identifying proteins, the data were analyzed with subtractive proteomics using ProtAn, an in-house analytic program. Consequently, 15 downregulated and 35 upregulated proteins were identified in SNI mice. The identified proteins may contribute to the maintenance of neuropathic pain, and may provide new or valuable information in the discovery of new therapeutic targets for neuropathic pain.
RESUMEN
Activated leukocyte cell adhesion molecule (ALCAM), a member of type I transmembrane immunoglobulin superfamily of cell adhesion molecule, is expressed in the surface membrane of various cell types including neurons. In the spinal cord dorsal horn (DH), the first gate for the sensory and pain transmission to the brain, the expression and function of ALCAM have not been known yet. Therefore, we here investigate the synaptic function of ALCAM in the substantia gelatinosa (lamina II) of the spinal DH, as well as its expression in the DH. Bath-application of ALCAM/Fc or CD6/Fc, the recombinant human IgG1-Fc chimeric proteins, specifically potentiated C-fiber-mediated excitatory synaptic transmission and predominantly increased spontaneous release of glutamate. In addition, the development of long-term potentiation, a form of synaptic plasticity, at excitatory synapses was significantly inhibited in the presence of the recombinant proteins. The functional roles of ALCAM in the spinal DH were further supported by immunohistochemical analysis; it showed that ALCAM intensely expressed through laminae I/II with the exception of lateral portion of the dorsal part of inner lamina II and distinctly co-localized with molecular markers of C-fibers, such as peptidergic calcitonin gene-related protein and transient receptor potential vanilloid type 1 and non-peptidergic isolectin B4. This study, for the first time, suggests the modulatory roles of ALCAM in the excitatory synaptic transmission and plasticity in the rat spinal DH.