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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

Stretch and OGD alter calcium ion levels in cEND and cEND + C6 cells. Calcium concentration relative to that measured in unstretched cEND cells (100%). Values are the percentages of the means (± SEM) of 4 independent experiments. Statistical significance was evaluated using One-Way ANOVA (Holm-Sidak method), **p < 0.001 compared to unstretched cEND cells, s p < 0.001. OGD, oxygen glucose deprivation; RO, re-oxygenation.
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Figure 10: Stretch and OGD alter calcium ion levels in cEND and cEND + C6 cells. Calcium concentration relative to that measured in unstretched cEND cells (100%). Values are the percentages of the means (± SEM) of 4 independent experiments. Statistical significance was evaluated using One-Way ANOVA (Holm-Sidak method), **p < 0.001 compared to unstretched cEND cells, s p < 0.001. OGD, oxygen glucose deprivation; RO, re-oxygenation.

Mentions: Prior to subjecting cEND cells to stretch and/or OGD, glioma cells C6 were subjected to severe stretch and/or 4 h of OGD. Cell culture medium was collected from the C6 cells, added to the cEND cell culture and incubated for 24 h prior to further handling in order to generate interaction between endothelial cells and astrocyte-secreted factors. OGD and a combination of stretch and OGD significantly increased calcium concentration in both cEND cells and cEND cells incubated in medium of C6 cells subjected to stretch or OGD, compared to unstretched cells. On the other hand, stretch significantly decreased calcium concentration in both cEND cells and cEND cells with C6 medium compared to unstretched cEND cells. Incubation of cEND cells with medium from C6 cells subjected to OGD prior to OGD and stretch combined with OGD resulted to significantly higher calcium levels, suggesting influence of astrocytes on cEND cells (Figure 10). Meanwhile, both stretch injury and OGD significantly increased calcium concentration in C6 cells (Figure 11). Addition of the calcium blocker isradipine decreased calcium ion concentration in all samples (Data not shown).


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)

Stretch and OGD alter calcium ion levels in cEND and cEND + C6 cells. Calcium concentration relative to that measured in unstretched cEND cells (100%). Values are the percentages of the means (± SEM) of 4 independent experiments. Statistical significance was evaluated using One-Way ANOVA (Holm-Sidak method), **p < 0.001 compared to unstretched cEND cells, s p < 0.001. OGD, oxygen glucose deprivation; RO, re-oxygenation.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Stretch and OGD alter calcium ion levels in cEND and cEND + C6 cells. Calcium concentration relative to that measured in unstretched cEND cells (100%). Values are the percentages of the means (± SEM) of 4 independent experiments. Statistical significance was evaluated using One-Way ANOVA (Holm-Sidak method), **p < 0.001 compared to unstretched cEND cells, s p < 0.001. OGD, oxygen glucose deprivation; RO, re-oxygenation.
Mentions: Prior to subjecting cEND cells to stretch and/or OGD, glioma cells C6 were subjected to severe stretch and/or 4 h of OGD. Cell culture medium was collected from the C6 cells, added to the cEND cell culture and incubated for 24 h prior to further handling in order to generate interaction between endothelial cells and astrocyte-secreted factors. OGD and a combination of stretch and OGD significantly increased calcium concentration in both cEND cells and cEND cells incubated in medium of C6 cells subjected to stretch or OGD, compared to unstretched cells. On the other hand, stretch significantly decreased calcium concentration in both cEND cells and cEND cells with C6 medium compared to unstretched cEND cells. Incubation of cEND cells with medium from C6 cells subjected to OGD prior to OGD and stretch combined with OGD resulted to significantly higher calcium levels, suggesting influence of astrocytes on cEND cells (Figure 10). Meanwhile, both stretch injury and OGD significantly increased calcium concentration in C6 cells (Figure 11). Addition of the calcium blocker isradipine decreased calcium ion concentration in all samples (Data not shown).

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