barrier destruction and statistical analysis after teer measurement in an in vitro blood-brain barrier model

Mesure de la résistance transendothéliale pour évaluer l’intégrité de la barrière cellulaire

BBB is a unique biological interface between blood circulation and nerve tissue, which is composed of vascular endothelial cells, pericytes, astrocytes, neurons, and other cellular structures1. The flow of ions, chemicals, and cells between the blood and the brain is strictly regulated by this barrier. This homeostasis safeguards the nervous tissues against toxins and pathogens while also enabling the appropriate operation of the brain&#39;s nerves2,3. Maintaining the integrity of the BBB can effectively prevent the development and progression of disorders affecting the central nervous system, such as neuronal dysfunction, edema, and neuroinflammation4. However, the unique physiological properties of the BBB prevent more than 98% of small molecule medications and 100% of macromolecular pharmaceuticals from entering the central nervous system5. Therefore, increasing the penetration of medications through the BBB during the development of drugs for the central nervous system is essential for achieving therapeutic efficacy6,7. Even though computer simulation screening of substrates has significantly raised the probability of drug candidates crossing the BBB, reliable and affordable in vitro/in vivo BBB models are still needed to meet the needs of scientific research8.
A quick and affordable technique for high-throughput drug screening is the in vitro model9. To shed light on the fundamental processes of medicines&#39; effects on BBB function and their part in the development and progression of disease, a series of simplified in vitro BBB models has been created. At present, the common in vitro BBB models are the monolayer, co-culture, dynamic, and microfluidic models10,11,12, constructed by vascular endothelial cells and astrocytes, pericytes, or microglia13,14. Although 3D cell cultures are morein line with the physiological structure of BBB15, their application as a means of drug screening for BBB is still constrained by their intricate design and subpar reproducibility. In contrast, the monolayer in vitro model is the one most frequently used to research the BBB and is applicable for determining the expression of membrane transporters and tight junction proteins in particular cells.
Transmembrane electrical resistance (TEER) measurement is a technique to evaluate and monitor the layer of cells across the resistance and evaluate the cell integrity and permeability of the barrier. By simultaneously inserting two electrodes into the growth medium or buffer solution on either side of the monolayer, it is possible to measure the alternating current or electrical impedance through the cell&#39;s compact layer16,17. In order to determine whether the in vitro BBB model has been properly created, the measurement of TEER will usually be employed as the gold standard18. On the other hand, the trend of medication action on BBB permeability can be accurately predicted by measuring the change in electrical resistance of the cell layer after drug involvement19. For example, Feng et al. discovered that catalpol (the primary active monomer of rehmanniae) could effectively reverse the lipopolysaccharide-induced down-regulation of tight junction proteins in the BBB and raise the TEER value of the mouse brain endothelial cell layer20.
The neuroinflammatory response is usually the main cause of BBB homeostasis imbalance21. Hypoxic treatment to induce neuroinflammatory injury is the main method to destroy the blood-brain barrier, mainly including physical methods and chemical reagent methods. The former primarily utilizes a three-gas incubator to vary the oxygen content in the cell growth environment to simulate hypoxic conditions22,while the latter is achieved by artificially introducing deoxy reagents such as CoCl2 to the cell culture medium23. The cells will remain in a deoxygenated condition if Fe2+ is substituted for Co2+ in the heme. If Fe2+ is substituted for Co2+ in the catalytic group, proline hydroxylase and aspartate hydroxylase activity will be inhibited, resulting in an accumulation of hypoxia-inducible factor-1&#945; (HIF-1&#945;)24. Under persistent hypoxia, the dephosphorylation of HIF-1&#945; in the cytoplasm triggers cell death and activates vascular endothelial growth factor, which ultimately raises vascular permeability. In previous studies25,26, it has been well demonstrated that hypoxia can significantly reduce the expression of endothelial tight junction proteins to increase the permeability of BBB. In this study, the time-resistance curve of bEnd.3 cells seeded in 24-well plates were measured in order to create a straightforward BBB model. Using this model, we characterized the changes in cell TEER after CoCl2 intervention in order to construct a cell model that can be used to screen drugs for BBB protection.

Pleins feux sur l’auteur : Applications de la détection TEER pour évaluer l’intégrité de la barrière cellulaire 

Enregistrement de l’intégrité fonctionnelle de la barrière sur les cellules endothéliales vasculaires bEnd.3 via la détection de résistance électrique transendothéliale

Watch this Scientific Journal Video about Barrier Destruction and Statistical Analysis After TEER Measurement in an In Vitro Blood-Brain Barrier Model at JoVE.com

Barrier Destruction and Statistical Analysis After TEER Measurement in an In Vitro Blood-Brain Barrier Model  

Destruction de la barrière et analyse statistique après mesure TEER dans un modèle in vitro de barrière hémato-encéphalique

Commencez par cultiver les cellules BEND3, puis divisez les puits en groupe témoin et en groupe chlorure de cobalt. Ajouter 200 microlitres de milieu de culture dans la chambre supérieure du groupe témoin et un milieu de culture contenant 300 micromolaires de chlorure de cobalt dans le groupe chlorure de cobalt. Incuber les plaques à 37 degrés Celsius et détecter les valeurs de résistance à 12 et 24 heures d’incubation à l’aide de la méthode TEER.Utilisez un logiciel commercial pour cartographier la tendance des valeurs TEER de la barrière cellulaire. Utilisez un logiciel d’analyse statistique pour analyser la différence de valeurs de résistance entre les cellules traitées au chlorure de cobalt et les cellules témoins. L’ajout de 300 micromolaires de chlorure de cobalt a entraîné une diminution significative des valeurs TEER par rapport au témoin aux points de temps de 12 heures et de 24 heures.Après 24 heures, la valeur TEER du groupe 300 micromolaires de chlorure de cobalt a diminué par rapport au groupe témoin, indiquant une rupture de la barrière cellulaire induite par des conditions hypoxiques dues au chlorure de cobalt.

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Research

JoVE Journal

Neuroscience

Barrier Destruction and Statistical Analysis After TEER Measurement in an In Vitro Blood-Brain Barrier Model

141 vues. Commencez par cultiver les cellules BEND3, puis divisez les puits en groupe témoin et en groupe chlorure de cobalt. Ajouter 200 microlitres de milieu de culture dans la chambre supérieure du groupe témoin et un milieu de culture contenant 300 micromolaires de chlorure de cobalt dans le groupe chlorure de cobalt. Incuber les plaques à 37 degrés Celsius et détecter les valeurs de résistance à 12 et 24 heures d’incubation à l’aide de la méthode TEER.Utilisez un logiciel comm...

Vidéo: Barrier Destruction and Statistical Analysis After TEER Measurement in an In Vitro Blood-Brain Barrier Model  

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating the interaction between restricted transit of harmful substances, nutrient absorption, and metabolite clearance in the brain, the BBB is essential in preserving central nervous system homeostasis. Building in vitro models of the BBB is a valuable tool for exploring the pathophysiology of neurological disorders and creating pharmacological treatments. This study describes a procedure for creating an in vitro monolayer BBB cell model by seeding bEnd.3 cells into the upper chamber of a 24-well plate. To assess the integrity of cell barrier function, the conventional epithelial cell voltmeter was used to record the transmembrane electrical resistance of normal cells and CoCl2-induced hypoxic cells in real-time. We anticipate that the above experiments will provide effective ideas for the creation of in vitro models of BBB and drugs to treat disorders of central nervous system diseases.

This protocol describes a dependable and efficient in vitro model of the brain blood barrier. The method uses mouse cerebral vascular endothelial cells bEnd.3 and measures transmembrane electrical resistance.

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating ...