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Stretch and/or oxygen glucose deprivation (OGD) in an in vitro traumatic brain injury (TBI) model induces calcium alteration and inflammatory cascade.

Salvador E, Burek M, Förster CY - Front Cell Neurosci (2015)

Bottom Line: Furthermore, reduction of cell membrane integrity decreased tight junction proteins claudin-5 and occludin expression.Also, since cell damage requires an increased uptake of glucose, expression of glucose transporter glut1 was found to increase at the mRNA level after OGD.Astrocytes potentiate these effects on calcium level in cEND cells.

View Article: PubMed Central - PubMed

Affiliation: Klinik und Poliklinik für Anästhesiologie, Zentrum für Operative Medizin der Universität Würzburg Würzburg, Germany.

ABSTRACT
The blood-brain barrier (BBB), made up of endothelial cells of capillaries in the brain, maintains the microenvironment of the central nervous system. During ischemia and traumatic brain injury (TBI), cellular disruption leading to mechanical insult results to the BBB being compromised. Oxygen glucose deprivation (OGD) is the most commonly used in vitro model for ischemia. On the other hand, stretch injury is currently being used to model TBI in vitro. In this paper, the two methods are used alone or in combination, to assess their effects on cerebrovascular endothelial cells cEND in the presence or absence of astrocytic factors. Applying severe stretch and/or OGD to cEND cells in our experiments resulted to cell swelling and distortion. Damage to the cells induced release of lactate dehydrogenase enzyme (LDH) and nitric oxide (NO) into the cell culture medium. In addition, mRNA expression of inflammatory markers interleukin (I L)-6, IL-1α, chemokine (C-C motif) ligand 2 (CCL2) and tumor necrosis factor (TNF)-α also increased. These events could lead to the opening of calcium ion channels resulting to excitotoxicity. This could be demonstrated by increased calcium level in OGD-subjected cEND cells incubated with astrocyte-conditioned medium. Furthermore, reduction of cell membrane integrity decreased tight junction proteins claudin-5 and occludin expression. In addition, permeability of the endothelial cell monolayer increased. Also, since cell damage requires an increased uptake of glucose, expression of glucose transporter glut1 was found to increase at the mRNA level after OGD. Overall, the effects of OGD on cEND cells appear to be more prominent than that of stretch with regards to TJ proteins, NO, glut1 expression, and calcium level. Astrocytes potentiate these effects on calcium level in cEND cells. Combining both methods to model TBI in vitro shows a promising improvement to currently available models.

No MeSH data available.


Related in: MedlinePlus

Microscopic examination of unstretched and stretched cEND cells. Under the light microscope, unstretched cEND cells are compact and spindle-shaped while severely stretched cells appear swollen, deformed, and retracted (indicated by arrows). Meanwhile, fluorescence microscopy shows that more cells take up the viability stain with increased stretch. Magnification 100X.
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Figure 1: Microscopic examination of unstretched and stretched cEND cells. Under the light microscope, unstretched cEND cells are compact and spindle-shaped while severely stretched cells appear swollen, deformed, and retracted (indicated by arrows). Meanwhile, fluorescence microscopy shows that more cells take up the viability stain with increased stretch. Magnification 100X.

Mentions: Control cells which were not subjected to injury appear as regularly shaped cEND cells without any indication of cell swelling or distortion (Figure 1). On the other hand, deformation could be observed for cells subjected to low, moderate, and severe stretch (Figure 1). After the cells were stretched with a peak pressure between 3.5 and 4.5 psi corresponding to severe stretch, they appeared markedly retracted, swollen, and deformed with notable intercellular spaces (Figure 1). Moreover, when viability stain (100 nm final concentration) was applied to the cells, the dye was excluded from most of the control cells, thus, only a few of the cells were stained (Figure 1). However, most of the cells that were subjected to stretch took up the dye (Figure 1). The viability stain used is a dye impermeant to healthy cells that becomes permeant when the plasma membrane integrity of the cells is compromised.


Stretch and/or oxygen glucose deprivation (OGD) in an in vitro traumatic brain injury (TBI) model induces calcium alteration and inflammatory cascade.

Salvador E, Burek M, Förster CY - Front Cell Neurosci (2015)

Microscopic examination of unstretched and stretched cEND cells. Under the light microscope, unstretched cEND cells are compact and spindle-shaped while severely stretched cells appear swollen, deformed, and retracted (indicated by arrows). Meanwhile, fluorescence microscopy shows that more cells take up the viability stain with increased stretch. Magnification 100X.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4543908&req=5

Figure 1: Microscopic examination of unstretched and stretched cEND cells. Under the light microscope, unstretched cEND cells are compact and spindle-shaped while severely stretched cells appear swollen, deformed, and retracted (indicated by arrows). Meanwhile, fluorescence microscopy shows that more cells take up the viability stain with increased stretch. Magnification 100X.
Mentions: Control cells which were not subjected to injury appear as regularly shaped cEND cells without any indication of cell swelling or distortion (Figure 1). On the other hand, deformation could be observed for cells subjected to low, moderate, and severe stretch (Figure 1). After the cells were stretched with a peak pressure between 3.5 and 4.5 psi corresponding to severe stretch, they appeared markedly retracted, swollen, and deformed with notable intercellular spaces (Figure 1). Moreover, when viability stain (100 nm final concentration) was applied to the cells, the dye was excluded from most of the control cells, thus, only a few of the cells were stained (Figure 1). However, most of the cells that were subjected to stretch took up the dye (Figure 1). The viability stain used is a dye impermeant to healthy cells that becomes permeant when the plasma membrane integrity of the cells is compromised.

Bottom Line: Furthermore, reduction of cell membrane integrity decreased tight junction proteins claudin-5 and occludin expression.Also, since cell damage requires an increased uptake of glucose, expression of glucose transporter glut1 was found to increase at the mRNA level after OGD.Astrocytes potentiate these effects on calcium level in cEND cells.

View Article: PubMed Central - PubMed

Affiliation: Klinik und Poliklinik für Anästhesiologie, Zentrum für Operative Medizin der Universität Würzburg Würzburg, Germany.

ABSTRACT
The blood-brain barrier (BBB), made up of endothelial cells of capillaries in the brain, maintains the microenvironment of the central nervous system. During ischemia and traumatic brain injury (TBI), cellular disruption leading to mechanical insult results to the BBB being compromised. Oxygen glucose deprivation (OGD) is the most commonly used in vitro model for ischemia. On the other hand, stretch injury is currently being used to model TBI in vitro. In this paper, the two methods are used alone or in combination, to assess their effects on cerebrovascular endothelial cells cEND in the presence or absence of astrocytic factors. Applying severe stretch and/or OGD to cEND cells in our experiments resulted to cell swelling and distortion. Damage to the cells induced release of lactate dehydrogenase enzyme (LDH) and nitric oxide (NO) into the cell culture medium. In addition, mRNA expression of inflammatory markers interleukin (I L)-6, IL-1α, chemokine (C-C motif) ligand 2 (CCL2) and tumor necrosis factor (TNF)-α also increased. These events could lead to the opening of calcium ion channels resulting to excitotoxicity. This could be demonstrated by increased calcium level in OGD-subjected cEND cells incubated with astrocyte-conditioned medium. Furthermore, reduction of cell membrane integrity decreased tight junction proteins claudin-5 and occludin expression. In addition, permeability of the endothelial cell monolayer increased. Also, since cell damage requires an increased uptake of glucose, expression of glucose transporter glut1 was found to increase at the mRNA level after OGD. Overall, the effects of OGD on cEND cells appear to be more prominent than that of stretch with regards to TJ proteins, NO, glut1 expression, and calcium level. Astrocytes potentiate these effects on calcium level in cEND cells. Combining both methods to model TBI in vitro shows a promising improvement to currently available models.

No MeSH data available.


Related in: MedlinePlus