RESUMEN
Disruption of the blood-brain barrier (BBB) leads to various neurovascular diseases. Development of therapeutics required to cross the BBB is difficult due to a lack of relevant in vitro models. We have developed a three-dimensional (3D) microfluidic BBB chip (BBBC) to study cell interactions in the brain microvasculature and to test drug candidates of neurovascular diseases. We isolated primary brain microvascular endothelial cells (ECs), pericytes, and astrocytes from neonatal rats and cocultured them in the BBBC. To mimic the 3D in vivo BBB structure, we used type I collagen hydrogel to pattern the microchannel via viscous finger patterning technique to create a matrix. ECs, astrocytes, and pericytes were cocultured in the collagen matrix. The fluid flow in the BBBC was controlled by a pump-free strategy utilizing gravity as driving force and resistance in a paper-based flow resistor. The primary cells cultured in the BBBC expressed high levels of junction proteins and formed a tight endothelial barrier layer. Addition of tumor necrosis factor alpha to recapitulate neuroinflammatory conditions compromised the BBB functionality. To mitigate the neuroinflammatory stimulus, we treated the BBB model with the glucocorticoid drug dexamethasone, and observed protection of the BBB. This BBBC represents a new simple, cost-effective, and scalable in vitro platform for validating therapeutic drugs targeting neuroinflammatory conditions.
Asunto(s)
Barrera Hematoencefálica , Técnicas de Cocultivo/instrumentación , Evaluación Preclínica de Medicamentos/instrumentación , Dispositivos Laboratorio en un Chip , Animales , Antiinflamatorios/farmacología , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Encéfalo/citología , Células Cultivadas , Técnicas de Cocultivo/métodos , Diseño de Equipo , Inflamación/metabolismo , Técnicas Analíticas Microfluídicas/instrumentación , Pericitos/citología , Pericitos/efectos de los fármacos , Pericitos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
We describe two Chinese families with a mild form of the myotonia congenita due to novel chloride channel (ClCN1) mutations. In one case, heterozygous I553F and H555N mutations were found. The patient shared the I553F mutation with his healthy father, and his mother had a history of mild myotonia when she was younger. In another family, autosomal dominant myotonia congenita was due to a L844F change. The physiological effects of the mutations were examined by using the two-electrode voltage-clamp technique after expression of the channels in Xenopus oocytes. All mutations drastically shifted the voltage required for half-maximal activation, more under conditions mimicking the homozygous situation, than under conditions mimicking the heterozygous situation. The larger effect was seen in the compound heterozygous situation combining the I553F and the H555N mutations. Our data suggest that myotonia congenita caused by CLCN1 mutations in Chinese have similar variable features to those found in the West.
Asunto(s)
Canales de Cloruro/genética , Mutación/genética , Mutación/fisiología , Miotonía/genética , Adolescente , Animales , China , Canales de Cloruro/fisiología , ADN Complementario/genética , Electromiografía , Electrofisiología , Ejercicio Físico/fisiología , Femenino , Humanos , Masculino , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Miotonía/patología , Examen Neurológico , Oocitos/metabolismo , Dolor/etiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , XenopusRESUMEN
Individual members of the RGK family of Ras-related GTPases, which comprise Rad, Gem/Kir, Rem and Rem2, have been implicated in important functions such as the regulation of voltage-gated calcium channel activity and remodeling of cell shape. The GTPase Kir/Gem inhibits the activity of calcium channels by interacting with the beta-subunit and also regulates cytoskeleton dynamics by inhibiting the Rho-Rho kinase pathway. In addition, Kir/Gem interacts with 14-3-3 and calmodulin, but the significance of this interaction on Kir/Gem function is poorly understood. Here, we present a comprehensive analysis of the binding of 14-3-3 and calmodulin to Kir/Gem. We show that 14-3-3, in conjunction with calmodulin, regulates the subcellular distribution of Kir/Gem between the cytoplasm and the nucleus. In addition, 14-3-3 and calmodulin binding modulate Kir/Gem-mediated cell shape remodeling and downregulation of calcium channel activity. Competition experiments show that binding of 14-3-3, calmodulin and calcium channel beta-subunits to Kir/Gem is mutually exclusive, providing a rationale for the observed regulatory effects of 14-3-3 and calmodulin on Kir/Gem localization and function.