“…It is based on measurement of the electrical resistance across the monolayers that form the experimental cerebral barrier and indicates the extent of tight junctional unity between neighbouring endothelial cells. Hence, it is considered as a strong and reliable indicator of the tightness of the monolayer (Srinivasan and Kolli 2019 ; Stone et al 2019 ). The measurement of the amount of solute crossing the cellular barrier, on the other hand, provides invaluable information on the extent of openings between tight junctions and therefore serves as an important marker for the functional status of the BBB (Helms et al 2016 ; Zucco et al 2005 ; Sun et al 2021 ).…”
To investigate whether therapeutic hypothermia augments the restorative impact of protein kinase C-β (PKC-β) and Nox2 inhibition on an in vitro model of human blood–brain barrier (BBB). Cells cultured in normoglycaemic (5.5 mM) or hyperglycaemic (25 mM, 6 to 120 h) conditions were treated with therapeutic hypothermia (35 °C) in the absence or presence of a PKC-β inhibitor (LY333531, 0.05 μM) or a Nox2 inhibitor (gp91ds-tat, 50 μM). BBB was established by co-culture of human brain microvascular endothelial cells (HBMECs) with astrocytes (HAs) and pericytes. BBB integrity and function were assessed via transendothelial electrical resistance (TEER) and paracellular flux of sodium fluorescein (NaF, 376 Da). Nox activity (lucigenin assay), superoxide anion production (cytochrome-C reduction assay), cellular proliferative capacity (wound scratch assay) and actin cytoskeletal formation (rhodamine-phalloidin staining) were assessed both in HBMECs and HAs using the specific methodologies indicated in brackets. Therapeutic hypothermia augmented the protective effects of PKC-β or Nox2 inhibition on BBB integrity and function in experimental setting of hyperglycaemia, as evidenced by increases in TEER and concomitant decreases in paracellular flux of NaF. The combinatory approaches were more effective in repairing physical damage exerted on HBMEC and HA monolayers by wound scratch and in decreasing Nox activity and superoxide anion production compared to sole treatment regimen with either agent. Similarly, the combinatory approaches were more effective in suppressing actin stress fibre formation and maintaining normal cytoskeletal structure. Therapeutic hypothermia augments the cerebral barrier-restorative capacity of agents specifically targeting PKC-β or Nox2 pathways.
“…It is based on measurement of the electrical resistance across the monolayers that form the experimental cerebral barrier and indicates the extent of tight junctional unity between neighbouring endothelial cells. Hence, it is considered as a strong and reliable indicator of the tightness of the monolayer (Srinivasan and Kolli 2019 ; Stone et al 2019 ). The measurement of the amount of solute crossing the cellular barrier, on the other hand, provides invaluable information on the extent of openings between tight junctions and therefore serves as an important marker for the functional status of the BBB (Helms et al 2016 ; Zucco et al 2005 ; Sun et al 2021 ).…”
To investigate whether therapeutic hypothermia augments the restorative impact of protein kinase C-β (PKC-β) and Nox2 inhibition on an in vitro model of human blood–brain barrier (BBB). Cells cultured in normoglycaemic (5.5 mM) or hyperglycaemic (25 mM, 6 to 120 h) conditions were treated with therapeutic hypothermia (35 °C) in the absence or presence of a PKC-β inhibitor (LY333531, 0.05 μM) or a Nox2 inhibitor (gp91ds-tat, 50 μM). BBB was established by co-culture of human brain microvascular endothelial cells (HBMECs) with astrocytes (HAs) and pericytes. BBB integrity and function were assessed via transendothelial electrical resistance (TEER) and paracellular flux of sodium fluorescein (NaF, 376 Da). Nox activity (lucigenin assay), superoxide anion production (cytochrome-C reduction assay), cellular proliferative capacity (wound scratch assay) and actin cytoskeletal formation (rhodamine-phalloidin staining) were assessed both in HBMECs and HAs using the specific methodologies indicated in brackets. Therapeutic hypothermia augmented the protective effects of PKC-β or Nox2 inhibition on BBB integrity and function in experimental setting of hyperglycaemia, as evidenced by increases in TEER and concomitant decreases in paracellular flux of NaF. The combinatory approaches were more effective in repairing physical damage exerted on HBMEC and HA monolayers by wound scratch and in decreasing Nox activity and superoxide anion production compared to sole treatment regimen with either agent. Similarly, the combinatory approaches were more effective in suppressing actin stress fibre formation and maintaining normal cytoskeletal structure. Therapeutic hypothermia augments the cerebral barrier-restorative capacity of agents specifically targeting PKC-β or Nox2 pathways.
“…TEER measurement is a rapid and non-invasive quantitative method to assess the permeability of the BBB in vitro ( Liang and Yoon, 2021 ; Srinivasan and Kolli, 2019 ). TEER translates the ionic resistance of the barrier in Ω·cm 2 units.…”
Section: In Vitro
Models Of the Bbbmentioning
confidence: 99%
“… Figure 6 TEER measurement approaches (A) TEER measurement using Ohm’s law method with simplified equivalent circuit. (B) TEER measurement method based on impedance spectroscopy with the component of the impedance ( Srinivasan and Kolli, 2019 ). …”
Section: In Vitro
Models Of the Bbbmentioning
confidence: 99%
“… (B) TEER measurement method based on impedance spectroscopy with the component of the impedance ( Srinivasan and Kolli, 2019 ). …”
“…BMEC identity is confirmed by cytochemical analysis showing the coexpression of PECAM1, TJP1, OCLN, CLDN5 and SLC2A1, and the BBB phenotype is confirmed by the presence of physiologic range TEER values (2,000 Ω × cm 2 ), angiogenic potential, and measurable transporter activity [72]. Coculturing BMEC with neural progenitor cells, pericytes, or astrocytes further enhances BBB function, with the combination of BMEC, pericytes, and astrocytes providing the greatest TEER values (∼5,000 Ω × cm 2 ) [116]. These protocols have been used to generate BMEC from patients with Huntington disease and schizophrenia with 22q11 deletion syndrome [67, 71] and to test the effects of various infectious agents on BBB function [112, 117].…”
Section: Ex Vivo Models Of Bmec Function In Schizophreniamentioning
<b><i>Background:</i></b> Despite decades of research, little clarity exists regarding pathogenic mechanisms related to schizophrenia. Investigations on the disease biology of schizophrenia have primarily focused on neuronal alterations. However, there is substantial evidence pointing to a significant role for the brain’s microvasculature in mediating neuroinflammation in schizophrenia. <b><i>Summary:</i></b> Brain microvascular endothelial cells (BMEC) are a central element of the microvasculature that forms the blood-brain barrier (BBB) and shields the brain against toxins and immune cells via paracellular, transcellular, transporter, and extracellular matrix proteins. While evidence for BBB dysfunction exists in brain disorders, including schizophrenia, it is not known if BMEC themselves are functionally compromised and lead to BBB dysfunction. <b><i>Key Messages:</i></b> Genome-wide association studies, postmortem investigations, and gene expression analyses have provided some insights into the role of the BBB in schizophrenia pathophysiology. However, there is a significant gap in our understanding of the role that BMEC play in BBB dysfunction. Recent advances differentiating human BMEC from induced pluripotent stem cells (iPSC) provide new avenues to examine the role of BMEC in BBB dysfunction in schizophrenia.
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