RESUMEN
The blood-brain barrier (BBB) is a large regulatory and exchange interface between the brain and peripheral circulation. We propose that changes of the BBB contribute to many pathophysiological processes in the brain of subjects with chronic sleep restriction (CSR). To achieve CSR that mimics a common pattern of human sleep loss, we quantified a new procedure of sleep disruption in mice by a week of consecutive sleep recording. We then tested the hypothesis that CSR compromises microvascular function. CSR not only diminished endothelial and inducible nitric oxide synthase, endothelin1, and glucose transporter expression in cerebral microvessels of the BBB, but it also decreased 2-deoxy-glucose uptake by the brain. The expression of several tight junction proteins also was decreased, whereas the level of cyclooxygenase-2 increased. This coincided with an increase of paracellular permeability of the BBB to the small tracers sodium fluorescein and biotin. CSR for 6 d was sufficient to impair BBB structure and function, although the increase of paracellular permeability returned to baseline after 24 h of recovery sleep. This merits attention not only in neuroscience research but also in public health policy and clinical practice.
Asunto(s)
Barrera Hematoencefálica/fisiopatología , Encéfalo/fisiopatología , Privación de Sueño/fisiopatología , Sueño/fisiología , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Regulación hacia Abajo , Células Endoteliales/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Permeabilidad , Privación de Sueño/metabolismoRESUMEN
Sleep disturbance in patients with multiple sclerosis is prevalent and has multifactorial causes. In mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, we determined the dynamic changes of sleep architecture and the interactions between sleep changes and EAE symptoms. The changes of sleep patterns were mainly reflected by altered sleep stage distribution and increased sleep fragmentation. Increased waking and decreased non-rapid eye movement sleep occurred after EAE onset and persisted through the symptomatic phase. There also was increased sleep state transition, indicating a reduction of sleep cohesiveness. Furthermore, the extent of sleep fragmentation correlated with the severity of disease. This is the first study of sleep characteristics in EAE mice demarcating specific changes related to the autoimmune disorder without confounding factors such as psychosocial impact and treatment effects. The reduction of sleep efficiency and cohesiveness supports the notion that enhancing sleep might facilitate the recovery of mice from EAE, pertinent to the multimodality treatment of multiple sclerosis.
Asunto(s)
Encefalomielitis Autoinmune Experimental/fisiopatología , Privación de Sueño/fisiopatología , Animales , Encefalomielitis Autoinmune Experimental/complicaciones , Femenino , Ratones , Privación de Sueño/complicacionesRESUMEN
Leptin, a pleiotropic adipokine, crosses the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) from the periphery and facilitates experimental autoimmune encephalomyelitis (EAE). EAE induces dynamic changes of leptin receptors in enriched brain and spinal cord microvessels, leading to further questions about the potential roles of endothelial leptin signaling in EAE progression. In endothelial leptin receptor specific knockout (ELKO) mice, there were lower EAE behavioral scores in the early phase of the disorder, better preserved BSCB function shown by reduced uptake of sodium fluorescein and leukocyte infiltration into the spinal cord. Flow cytometry showed that the ELKO mutation decreased the number of CD3 and CD45 cells in the spinal cord, although immune cell profiles in peripheral organs were unchanged. Not only were CD4(+) and CD8(+) T lymphocytes reduced, there were also lower numbers of CD11b(+)Gr1(+) granulocytes in the spinal cord of ELKO mice. In enriched microvessels from the spinal cord of the ELKO mice, the decreased expression of mRNAs for a few tight junction proteins was less pronounced in ELKO than WT mice, as was the elevation of mRNA for CCL5, CXCL9, IFN-γ, and TNF-α. Altogether, ELKO mice show reduced inflammation at the level of the BSCB, less leukocyte infiltration, and better preserved tight junction protein expression and BBB function than WT mice after EAE. Although leptin concentrations were high in ELKO mice and microvascular leptin receptors show an initial elevation before inhibition during the course of EAE, removal of leptin signaling helped to reduce disease burden. We conclude that endothelial leptin signaling exacerbates BBB dysfunction to worsen EAE.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Leptina/sangre , Leucocitos/inmunología , Receptores de Leptina/metabolismo , Médula Espinal/inmunología , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/irrigación sanguínea , Encéfalo/inmunología , Citocinas/inmunología , Células Endoteliales/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/metabolismo , Receptores de Leptina/genética , Índice de Severidad de la Enfermedad , Transducción de Señal , Médula Espinal/irrigación sanguínea , Uniones Estrechas/inmunologíaRESUMEN
This study was aimed to evaluate the effectiveness of solution form of 17% ethylenediaminetetraacetic acid (EDTA) on removing smear layer of root canals at different exposure time periods and to provide scientific basis for EDTA as a choice of root canal irrigation in clinical practice. Twenty-five single-rooted teeth were randomly divided into 5 groups: control group (group A) was given 2.5% NaOCl, and 4 experimental groups were given 2.5% NaOCl and 17% EDTA, including groups B, C, D and E with exposure time of 1, 3, 5 and 7 min, respectively. After preparation of the root canals, the teeth were split along their longitudinal axis, and the root sections were examined under scanning electron microscope for evaluation of smear layer removal and erosion on the surface of the root canal walls. The specimens in group B showed presence of smear layer on the walls of the root canal with no statistical difference from that in group A (P>0.05). In groups C and D, partial removal of smear layer was obtained, and there was no significant difference between the two groups (P>0.05), but there was significant difference in removal of smear layer between group C and group B (P<0.05). Root canal walls in group E specimens showed almost complete removal of smear layer, and the removal of smear layer was significantly different from that in group D (P<0.01). There was no significant change in the structure of the surface of root canal for each sample. It was concluded that combined irrigation with 17% EDTA and 2.5% NaOCl could remove the smear layer with no significant alteration in dentinal structure when the chelating agent was applied for 7 min. At 3 and 5 min of application, partial removal of smear layer was observed and at 1 min negligible removal of smear layer was achieved.
Asunto(s)
Ácido Edético/uso terapéutico , Irrigantes del Conducto Radicular/uso terapéutico , Capa de Barro Dentinario , Hipoclorito de Sodio/uso terapéutico , Adolescente , Diente Premolar/cirugía , Diente Premolar/ultraestructura , Quelantes/uso terapéutico , Humanos , Microscopía Electrónica de Rastreo , Preparación del Conducto Radicular/métodos , Soluciones , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
Hyperleptinemia is usually associated with obesity and leptin resistance. Endothelial cell leptin receptor knockout (ELKO) mice without a signaling membrane-bound leptin receptor in endothelia, however, have profound hyperleptinemia without signs of leptin resistance. Leptin mRNA in adipose tissue was unchanged. To test the hypothesis that the ELKO mutation results in delayed degradation and slowed excretion, we determined the kinetics of leptin transfer in groups of ELKO and wildtype mice after intravenous bolus injection of (125) I-leptin and the reference substance (131) I-albumin. The degradation pattern of (125) I-leptin in serum and brain homogenates at different time points between 10 and 60 min was measured by HPLC and acid precipitation. Although ELKO mice had reduced uptake of (125) I-leptin uptake by the brain and several peripheral organs, leptin was more stable in blood and tissue. There was no change in the rate of renal excretion. ELISA showed that serum soluble leptin receptor, known to antagonize leptin transport, had a 400-fold increase, probably contributing to the hyperleptinemia and reduced tissue uptake. Thus, the ELKO mutation unexpectedly increased the stability of leptin but suppressed its tissue uptake. These changes probably contribute to the known partial resistance of the ELKO mice to diet-induced obesity.
Asunto(s)
Leptina/sangre , Receptores de Leptina/deficiencia , Tejido Adiposo/metabolismo , Animales , Transporte Biológico Activo , Encéfalo/metabolismo , Células Endoteliales/metabolismo , Riñón/metabolismo , Leptina/metabolismo , Ratones , Ratones Noqueados , Obesidad/etiología , Obesidad/metabolismo , Estabilidad Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Leptina/genética , Receptores de Leptina/metabolismo , Albúmina Sérica/metabolismo , Distribución TisularRESUMEN
Leptin is commonly thought to play a detrimental role in exacerbating experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis. Paradoxically, we show here that astrocytic leptin signaling has beneficial effects in reducing disease severity. In the astrocyte specific leptin receptor knockout (ALKO) mouse in which leptin signaling is absent in astrocytes, there were higher EAE scores (more locomotor deficits) than in the wildtype counterparts. The difference mainly occurred at a late stage of EAE when wildtype mice showed signs of recovery whereas ALKO mice continued to deteriorate. The more severe symptoms in ALKO mice coincided with more infiltrating cells in the spinal cord and perivascular brain parenchyma, more demyelination, more infiltrating CD4 cells, and a lower percent of neutrophils in the spinal cord 28 days after EAE induction. Cultured astrocytes from wildtype mice showed increased adenosine release in response to interleukin-6 and the hippocampus of wildtype mice had increased adenosine production 28 days after EAE induction, but the ALKO mutation abolished the increase in both conditions. This indicates a role of astrocytic leptin in normal gliotransmitter release and astrocyte functions. The worsening of EAE in the ALKO mice in the late stage suggests that astrocytic leptin signaling helps to clear infiltrating leukocytes and reduce autoimmune destruction of the CNS.
Asunto(s)
Astrocitos/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Receptores de Leptina/genética , Adenosina/análisis , Adenosina/metabolismo , Animales , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Hipocampo/patología , Leucocitos/metabolismo , Ratones , Ratones Noqueados , Receptores de Leptina/metabolismo , Médula Espinal/patologíaRESUMEN
BACKGROUND/AIMS: Acute phase C-reactive protein (CRP), elevated in obesity and inflammation, is a major binding protein for leptin. It is thought that CRP contributes to leptin resistance by preventing leptin from crossing the blood-brain barrier (BBB). Here we determined how CRP interacts with the BBB and whether it deters leptin from reaching CNS targets. METHODS: BBB permeability, compartmental distribution, tracer stability, and expression of tight junction protein and inflammatory marker were determined. RESULTS: CRP was stable in blood, but did not permeate the BBB in trace amounts. However, it increased paracellular permeability at a higher dose. Agouti viable (A(vy)) mice with adult-onset obesity show higher CRP entry into the brain. CRP did not permeate hCMEC/D3 cells nor change zona occludin-1 or cyclooxygenase-2 expression. An intermediate dose of CRP had no effect on leptin transport across the BBB after co-treatment. Thus, acute interactions between CRP and leptin at the BBB level were negligible and did not explain the leptin resistance seen in obesity. CONCLUSIONS: The interactions of CRP and the BBB are a two-phase process, with increased paracellular permeability at a high dose that enables its entry into the CNS and serves to induce reactive gliosis and impair CNS function.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Proteína C-Reactiva/metabolismo , Permeabilidad Capilar , Sistema Nervioso Central/metabolismo , Inflamación/metabolismo , Obesidad/metabolismo , Animales , Leptina/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
An effective long-term cell therapy for skeletal muscle regeneration requires donor contribution to both muscle fibers and the muscle stem cell pool. Although satellite cells have these abilities, their therapeutic potential so far has been limited due to their scarcity in adult muscle. Myogenic progenitors obtained from Pax3-engineered mouse embryonic stem (ES) cells have the ability to generate myofibers and to improve the contractility of transplanted muscles in vivo, however, whether these cells contribute to the muscle stem cell pool and are able to self-renew in vivo are still unknown. Here, we addressed this question by investigating the ability of Pax3, which plays a critical role in embryonic muscle formation, and Pax7, which is important for maintenance of the muscle satellite cell pool, to promote the derivation of self-renewing functional myogenic progenitors from ES cells. We show that Pax7, like Pax3, can drive the expansion of an ES-derived myogenic progenitor with significant muscle regenerative potential. We further demonstrate that a fraction of transplanted cells remains mononuclear, and displays key features of skeletal muscle stem cells, including satellite cell localization, response to reinjury, and contribution to muscle regeneration in secondary transplantation assays. The ability to engraft, self-renew, and respond to injury provide foundation for the future therapeutic application of ES-derived myogenic progenitors in muscle disorders.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Factor de Transcripción PAX7/metabolismo , Factores de Transcripción Paired Box/metabolismo , Animales , Células Cultivadas , Células Madre Embrionarias/trasplante , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Factor de Transcripción PAX3 , Factor de Transcripción PAX7/genética , Factores de Transcripción Paired Box/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Our previous study verified a sex difference of anti-hyperalgesia in rats and anti-allodynia in mice induced by intrathecal oxytocin (OT). In the model of intraplantar carrageenan-induced inflammatory hyperalgesia, intrathecal OT injection induced a substantial anti-hyperalgesia in male rats even at a low dose (0.125 nmol). In contrast, female rats only responded to an extremely high dose (1.25 nmol). This sex difference concurs with a lower expression of OT receptors and higher expression of insulin-regulated aminopeptidase (IRAP; OT degrading enzyme) in the spinal cords of female rats. In this study, we further determined the role of female hormones in this sex difference by using ovariectomized rats. Our results show that a low dose of intrathecal OT caused a significant anti-hyperalgesia effect in ovariectomized female rats, similar to that seen in male rats. Ovariectomy did not cause any change of paw edema except at the late stage of convalescence when compared with the sham-operated group. Ovariectomy-induced faster recovery from edema but did not affect the severity of hyperalgesia. Moreover, there was a similar amount of IRAP expression in ovariectomized and sham rats. When estradiol (E2) was given together with OT, OT-induced anti-hyperalgesia was abolished at the developmental stage of hyperalgesia in ovariectomized rats. These results show an inhibitory role of female hormones generated from ovaries (mainly estrogen) in the sex difference of anti-hyperalgesia induced by OT. This study suggests the feasibility of a novel OT-based remedy to treat hyperalgesia in men and in menopausal women no receiving hormonal supplements.
Asunto(s)
Hiperalgesia , Oxitocina , Animales , Edema/metabolismo , Estrógenos/metabolismo , Estrógenos/farmacología , Femenino , Humanos , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Masculino , Ratones , Ovariectomía , Oxitocina/metabolismo , Ratas , Ratas Sprague-Dawley , Médula Espinal/metabolismoRESUMEN
Our previous studies documented that interleukin-15 receptor α (IL-15Rα) knockout (KO) mice exhibited hyperactivity, memory impairment, and desperate behavior, which are core features of schizophrenia and depression. Due to the overlapping symptomology and pathogenesis observed for schizophrenia and depression, the present study attempted to determine whether IL-15Rα was associated with the risk of schizophrenia or depression. One hundred fifty-six participants, including 63 schizophrenia patients, 29 depressive patients, and 64 age-matched healthy controls, were enrolled in the study. We investigated the circulating levels of soluble IL-15Rα and analyzed potential links between the IL-15Rα levels and clinical symptoms present in schizophrenia or depressive patients. We observed reduced serum IL-15Rα levels in schizophrenia patients, but not depressive patients compared with controls. Moreover, a significant negative association was observed between the circulating IL-15Rα levels and excited phenotypes in the schizophrenia patients. The IL-15Rα KO mice displayed pronounced pre-pulse inhibition impairment, which was a typical symptom of schizophrenia. Interestingly, the IL-15Rα KO mice exhibited a remarkable elevation in the startle amplitude in the startle reflex test compared to wild type mice. These results demonstrated that serum levels of soluble IL-15Rα were reduced in schizophrenia and highlighted the relationship of IL-15Rα and the excited phenotype in schizophrenia patients and mice.
RESUMEN
Interleukin-15 (IL15) is a cytokine produced by normal brain, but the functions of the IL15 system in normal adults are not yet clear. The hypothesis that the hippocampal IL15 system is essential for memory consolidation was tested by use of IL15Ralpha knock-out mice in behavioral, biochemical, immunohistological, and electron microscopic analyses. The knock-out mice showed deficits in memory, determined by the Stone T-maze and fear conditioning. In their hippocampi, the concentration of GABA was significantly lower. There were region-specific changes of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), with increased GAD-67-immunopositive interneurons in the stratum oriens of the CA1 region of the hippocampus, accompanied by nonsignificant reduction of GAD-67 synapses in the CA3 region. Western blotting showed an increase of GAD-65, but not GAD-67, in the hippocampal homogenate. The ultrastructure of the hippocampus remained intact in the knock-out mice. To further test the hypothesis that IL15 directly modulates GABA turnover by reuptake mechanisms, the dose-response relationship of IL15 on (3)H-GABA uptake was determined in two neuronal cell lines. The effective and nontoxic dose was further used in the synaptosomal uptake studies. IL15 decreased the uptake of (3)H-GABA in synaptosomes from the forebrain of wild-type mice. Consistent with this, IL15Ralpha knock-out mice had increased synaptosomal uptake of (3)H-GABA. Overall, the results show novel functions of a unique cytokine in normal hippocampal activity by regulating GABA transmission.
Asunto(s)
Hipocampo/fisiología , Subunidad alfa del Receptor de Interleucina-15/fisiología , Memoria/fisiología , Neurotransmisores/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Células Cultivadas , Condicionamiento Psicológico , Miedo , Glutamato Descarboxilasa/metabolismo , Hipocampo/ultraestructura , Interleucina-15/farmacología , Interleucina-15/fisiología , Subunidad alfa del Receptor de Interleucina-15/genética , Aprendizaje por Laberinto , Ratones , Ratones Noqueados , Neuronas/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructura , Sinaptosomas/metabolismoRESUMEN
Interleukin (IL)-15 receptors are present in the cerebral endothelia composing the blood-brain barrier where they show robust up-regulation by neuroinflammation. To determine how IL15 receptor subunits participate in the endocytosis and intracellular trafficking of IL15, we performed confocal microscopic imaging and radioactive tracer uptake assays in primary brain microvessel endothelial cells and related cell lines transfected with modulatory molecules. By immunostaining and co-localization studies with organelle markers, we showed that IL15 was rapidly endocytosed via lipid rafts and was directed to diverse intracellular pathways. During the course of intracellular trafficking, Alexa dye-conjugated IL15 was partially co-localized with both the specific receptor IL15Rα and the co-receptor IL2Rγ. However, deletion of one of the receptor subunits had only a minor effect in slowing IL15 uptake when primary brain microvessel endothelial cells from the receptor knockout mice were compared with those from wildtype mice. IL15 was trafficked to early, recycling, and late endosomes, to the Golgi, and to lysosomes. The diffuse distribution suggests that IL15 activates multiple endothelial signaling events.
Asunto(s)
Corteza Cerebral/metabolismo , Endocitosis/fisiología , Endotelio Vascular/metabolismo , Mediadores de Inflamación/metabolismo , Interleucina-15/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Ciclo Celular/genética , Línea Celular , Células Cultivadas , Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/patología , Endotelio Vascular/patología , Humanos , Mediadores de Inflamación/fisiología , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transporte de Proteínas/genética , Ratas , Receptores de Interleucina-15/deficiencia , Receptores de Interleucina-15/genética , Receptores de Interleucina-15/metabolismo , Factores de TiempoRESUMEN
Interleukin (IL)-15 and its receptors are induced by tumor necrosis factor α (TNF) in the cerebral endothelial cells composing the blood-brain barrier, but it is not yet clear how IL-15 modulates endothelial function. Contrary to the known induction of JAK/STAT3 signaling, here we found that nuclear factor (NF)- κB is mainly responsible for IL-15 actions on primary brain microvessel endothelial cells and cerebral endothelial cell lines. IL-15-induced transactivation of an NFκB luciferase reporter resulted in phosphorylation and degradation of the inhibitory subunit IκB that was followed by phosphorylation and nuclear translocation of the p65 subunit of NFκB. An IκB kinase inhibitor Bay 11-7082 only partially inhibited IL-15-induced NFκB luciferase activity. The effect of IL-15 was mediated by its specific receptor IL-15Rα, since endothelia from IL-15Rα knockout mice showed delayed nuclear translocation of p65, whereas those from knockout mice lacking a co-receptor IL-2Rγ did not show such changes. At the mRNA level, IL-15 and TNF showed similar effects in decreasing the tight junction protein claudin-2 and increasing the p65 subunit of NFκB but exerted different regulation on caveolin-1 and vimentin. Taken together, NFκB is a major signal transducer by which IL-15 affects cellular permeability, endocytosis, and intracellular trafficking at the level of the blood-brain barrier.
Asunto(s)
Células Endoteliales/metabolismo , Interleucina-15/metabolismo , FN-kappa B/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Caveolina 1/metabolismo , Células Cultivadas , Cerebelo/citología , Claudinas/metabolismo , Activación Enzimática , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/metabolismo , Interleucina-15/genética , Subunidad alfa del Receptor de Interleucina-15/deficiencia , Subunidad alfa del Receptor de Interleucina-15/genética , Subunidad alfa del Receptor de Interleucina-15/metabolismo , Ratones , Ratones Noqueados , Fosforilación , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Factor de Transcripción ReIA/metabolismo , Activación Transcripcional , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Vimentina/metabolismoRESUMEN
Impairment in blood-to-brain transport of leptin is a major cause as well as consequence of obesity. Leptin crosses the blood-brain barrier by transcytosis rather than undergoing intracellular degradation. Results from previous studies have indicated that the membrane juxtapositional cytoplasmic sequence of the leptin receptor ObR is responsible for leptin transport. To identify the specific structural domains, we generated a series of ObR truncates with different lengths of the intracellular sequence, overexpressed them in 3 types of mammalian cells including cerebral endothelia, and quantified leptin binding and endocytosis. All mutant ObRs were able to bind and mediate the internalization of leptin. Surprisingly, ObR860, a construct with no cytoplasmic sequence, could act like the classical ObRa transporter in internalizing leptin. There were some cell type-dependent variations in the intracellular trafficking of Alexa-labeled leptin when mediated by ObR860 or ObRa because of differential involvement of membrane microdomains, as shown by use of the clathrin inhibitor chlorpromazine and the dynamin inhibitor Dynasore. The clathrin- and dynamin-mediated endocytosis of leptin contrasts with the lack of effect of the caveolae inhibitors nystatin and filipin. Thus, leptin-induced internalization of the ligand-receptor complex can occur without specific sorting signals in the cytoplasmic region of ObR. This novel finding may have significant implications for leptin transport.
Asunto(s)
Leptina/metabolismo , Mutación , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Leptina/metabolismo , Animales , Línea Celular , Citoplasma/metabolismo , Endocitosis , Ratones , Transporte de Proteínas , Receptores de Leptina/genéticaRESUMEN
Astrocytic leptin receptors (ObR) can be up-regulated in conditions such as adult-onset obesity. To determine whether the levels and subtypes of astrocytic ObR modulate leptin transport, we co-cultured hCMEC/D3 human brain endothelial cells and C6 astrocytoma cells in the Transwell system, and tested leptin permeation from apical to basolateral chambers. In comparison with hCMEC alone, co-culture of C6 cells reduced the permeability of paracellular markers and leptin. Unexpectedly, ObRb over-expression in C6 cells increased leptin permeation whereas ObRa over-expression showed no effect when compared with the control group of pcDNA-transfected C6 cells. By contrast, the paracellular permeability to the sodium fluorescein control was unchanged by over-expression of ObR subtypes. Leptin remained intact after crossing the monolayer as shown by HPLC and acid precipitation, and this was not affected by C6 cell co-culture or the over-expression of different ObR subtypes. Thus, increased expression of ObRb (and to a lesser extent ObRe) in C6 cells specifically increased the permeation of leptin across the hCMEC monolayer. Consistent with the evidence that the most apparent regulatory changes of ObR during obesity and inflammation occur in astrocytes, the results indicate that astrocytes actively regulate leptin transport across the blood-brain barrier, a mechanism independent of reduction of paracellular permeability.
Asunto(s)
Astrocitos/metabolismo , Células Endoteliales/metabolismo , Leptina/metabolismo , Receptores de Leptina/fisiología , Albúminas/metabolismo , Animales , Astrocitos/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Encéfalo/citología , Línea Celular Transformada , Cromatografía Líquida de Alta Presión/métodos , Técnicas de Cocultivo , Células Endoteliales/efectos de los fármacos , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Isótopos de Yodo/metabolismo , Leptina/farmacología , Ratones , Receptores de Leptina/genética , Rodaminas/metabolismo , Factores de Tiempo , Transfección/métodos , Regulación hacia Arriba/fisiologíaRESUMEN
Interleukin (IL)-15 and its receptors in cerebral microvascular endothelial cells play an important role in mediating neuroinflammatory signaling across the blood-brain barrier. Although alternative splice variants of IL15Ralpha (the specific receptor) are seen in immune cells, the presence and functions of splice variants have not been studied in the cerebral endothelia that compose the blood-brain barrier. In this study, we identified five splice variants from mouse cerebral capillaries by RT-PCR, cloning, and DNA sequencing, and performed domain analysis. Four of these isoforms have never been described in any tissue. All isoforms were detected by qPCR in enriched mouse cerebral microvessels and their expression was increased by tumor necrosis factor treatment in vivo. To determine their functions, plasmids encoding individual isoforms were transfected into RBE4 cerebral endothelial cells. All of these predicted alkalinic proteins were expressed and most showed post-translational modifications. There were variations in their subcellular distribution. Only the full length IL15Ralpha and to a lesser degree isoform alpha1 were trafficked to the cell surface 24 h after over-expression. As shown by a luciferase reporter for signal transducer and activator of transcription (STAT)-3, over-expression of isoforms alpha2 and alpha4 reduced basal STAT3 activation. In comparison with the control, over-expression of the full length IL15Ralpha had a greater effect in increasing IL15-induced STAT3 transactivation than other isoforms. The results show that IL15 signaling in cerebral endothelia is probably an orchestrated effect of all IL15Ralpha splice variants that determine the eventual outcome by differential regulation.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Corteza Cerebral/irrigación sanguínea , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Microvasos/metabolismo , Animales , Barrera Hematoencefálica/citología , Endotelio Vascular/citología , Femenino , Interleucina-15/fisiología , Ratones , Ratones Endogámicos C57BL , Microvasos/citología , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , ARN Mensajero/biosíntesis , Receptores de Interleucina-15/biosíntesis , Receptores de Interleucina-15/genética , Transducción de SeñalRESUMEN
BACKGROUND/AIMS: The transcription factor NFkappaB is a major mediator of lipopolysaccharide (LPS) signaling. We determined the role of NFkappaB activation in regulatory changes of the P-glycoprotein (Pgp) drug efflux transporter at the blood-brain barrier (BBB) and proinflammatory cytokine receptors. METHODS: We treated NFkappaB knockout and wildtype mice with LPS or vehicle, obtained enriched cerebral microvessels, and determined target mRNA by qPCR for MDR1a/b, IL15Ralpha, IL2 Ralpha, IL2Rgamma, LIFR, gp130, and TNFR1/2, and protein expression by western blotting for P-gp, IL15Ralpha, IL2Rgamma, LIFR, and gp130. RESULTS: The effects of LPS on the transporters and cytokine receptors showed differences between wildtype and NFkappaB knockout mice, and between mRNA and protein changes. NFkappaB not only mediated the LPS-induced increase of MDR1b, IL2Rgamma, and TNFR2 mRNA in the wildtype mice, but it showed opposite effects by elevating IL15Ralpha and TNFR1 mRNA and decreasing IL2Ralpha in the knockout mice. Although basal vinblastine uptake was unchanged in the NFkappaB knockout mice, LPS induced an increase of the uptake (depressed efflux transport) greater than that seen in the wildtype mice, indicating that NFkappaB helps to maintain Pgp efflux transporter function. CONCLUSION: The results show differential involvement of NFkappaB signaling in response to LPS at the BBB.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/inmunología , Barrera Hematoencefálica/inmunología , Inflamación/inmunología , FN-kappa B/genética , FN-kappa B/inmunología , Receptores de Citocinas/inmunología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Antineoplásicos Fitogénicos/farmacocinética , Barrera Hematoencefálica/metabolismo , Lipopolisacáridos/inmunología , Ratones , Ratones Noqueados , Mutación , ARN Mensajero/genética , Receptores de Citocinas/genética , Receptores de Interleucina-15/genética , Receptores de Interleucina-15/inmunología , Vinblastina/farmacocinéticaRESUMEN
The interactions between the cytokine interleukin (IL)-15 and the classical neurotransmitter GABA have been shown in IL15Rα receptor knockout mice by observations of memory deficits and reduction of GABA. To test the hypothesis that IL15 affects anxiety-like behavior, knockout mice without IL15, IL15Rα, or the co-receptor IL2Rγ were subjected to open-field and elevated plus maze tests. All three strains showed reduction of anxiety, with greater changes in the IL15Rα knockout mice than in the IL15 or IL2Rγ knockout mice. This unexpected observation is opposite to the reported increase of anxiety in mice lacking other proinflammatory cytokines or their receptors. The reduced anxiety was not associated with changes in associated serum cytokines. However, Western blotting, qPCR, and immunohistochemistry all showed that IL15Rα knockout mice had mild microgliosis and astrogliosis in the hippocampus. To determine whether this gliosis plays a role in decreasing anxiety, IL15Rα knockout mice were treated with minocycline, but this did not cause a change in open field performance. To determine whether IL15 plays a direct role in anxiety, wildtype mice were treated with IL15 by intraperitoneal injection. This also failed to cause a change in open field behavior under the experimental conditions tested. Thus, IL15Rα is essential for normal anxiety-like behavior, but inhibition of gliosis in the fearless IL15Rα knockout mice or IL15 treatment of normal mice did not acutely modulate behavioral performance as tested.
Asunto(s)
Ansiedad/fisiopatología , Ansiedad/psicología , Receptores de Interleucina-15/fisiología , Animales , Antibacterianos/farmacología , Conducta Animal/fisiología , Western Blotting , Citocinas/sangre , Gliosis/genética , Gliosis/patología , Hipocampo/patología , Inmunohistoquímica , Interleucina-15/sangre , Subunidad alfa del Receptor de Interleucina-15/biosíntesis , Subunidad alfa del Receptor de Interleucina-15/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Minociclina/farmacología , Actividad Motora/efectos de los fármacos , Receptores de Interleucina-15/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The possible role of astrocytes in the regulation of feeding has been overlooked. It is well-established that the endothelial cells constituting the blood-brain barrier transport leptin from blood to brain and that hypothalamic neurons respond to leptin to induce anorexic signaling. However, few studies have addressed the role of astrocytes in either leptin transport or cellular activation. We recently showed that the obese agouti viable yellow mouse has prominent astrocytic expression of the leptin receptor. In this study, we test the hypothesis that diet-induced obesity increases astrocytic leptin receptor expression and function in the hypothalamus. Double-labelling immunohistochemistry and confocal microscopic analysis showed that all astrocytes in the hypothalamus express leptin receptors. In adult obese mice, 2 months after being placed on a high-fat diet, there was a striking increase of leptin receptor (+) astrocytes, most prominent in the dorsomedial hypothalamus and arcuate nucleus. Agouti viable yellow mice with their adult-onset obesity showed similar changes, but the increase of leptin receptor (+) astrocytes was barely seen in ob/ob or db/db mice with their early-onset obesity and defective leptin systems. The marked leptin receptor protein expression in the astrocytes, shown with several antibodies against different receptor epitopes, was supported by RT-PCR detection of leptin receptor-a and -b mRNAs in primary hypothalamic astrocytes. Unexpectedly, the protein expression of GFAP, a marker of astrocytes, was also increased in adult-onset obesity. Real-time confocal imaging showed that leptin caused a robust increase of calcium signalling in primary astrocytes from the hypothalamus, confirming their functionality. The results indicate that metabolic changes in obese mice can rapidly alter leptin receptor expression and astrocytic activity, and that leptin receptor is responsible for leptin-induced calcium signalling in astrocytes. This novel and clinically relevant finding opens new avenues in astrocyte biology.
Asunto(s)
Astrocitos/metabolismo , Hipotálamo/metabolismo , Obesidad/metabolismo , Receptores de Leptina/metabolismo , Animales , Astrocitos/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Dieta/efectos adversos , Hipotálamo/efectos de los fármacos , Hipotálamo/patología , Leptina/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/etiología , Obesidad/patología , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Urocortin is a member of the corticotropin-releasing hormone (CRH) family of peptides. In the brain, its potent suppression of food intake is mediated by CRH receptors (CRHR). Urocortin also participates in the regulation of anxiety, learning, memory, and body temperature, and it shows neuroprotection. This review will summarize the location of urocortin-producing neurons and their projections, the pharmacological evidence of its actions in the CNS, and information acquired from knockout mice. Urocortin interacts with leptin, neuropeptide Y, orexin, and corticotropin in the brain. Also produced by the GI tract, heart, and immune cells, urocortin has blood concentrations ranging from 13 to 152 pg/ml. Blood-borne urocortin stimulates the cerebral endothelial cells composing the blood-brain barrier and crosses the blood-brain barrier by a unique transport system. Overall, urocortin acts on a broad neuronal substrate as a neuromodulator important for basic survival.