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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

Western blot and densitometric analyses of tight junction proteins in cEND cells. Values are the means (± SEM) of 4–5 independent experiments normalized to β-actin. Statistical significance was evaluated using One-Way ANOVA (Holm-Sidak method), *, ¥, $ p < 0.005 compared to no stretch control, OGD + Stretch + RO, and OGD + RO, respectively. OGD, oxygen glucose deprivation; RO, re-oxygenation.
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Figure 6: Western blot and densitometric analyses of tight junction proteins in cEND cells. Values are the means (± SEM) of 4–5 independent experiments normalized to β-actin. Statistical significance was evaluated using One-Way ANOVA (Holm-Sidak method), *, ¥, $ p < 0.005 compared to no stretch control, OGD + Stretch + RO, and OGD + RO, respectively. OGD, oxygen glucose deprivation; RO, re-oxygenation.

Mentions: Tight junction proteins are important in the maintenance of blood-brain barrier (BBB) permeability. Once the BBB is compromised or damaged, the expression of tight junction proteins changes. Therefore, we analyzed the expression of occludin and claudin-5 both at the protein and mRNA levels. Western blot and subsequent densitometric analyses show that occludin protein expression significantly decreased (p < 0.005) after subjecting cells to stretch as well as to OGD and a combination of both in comparison to no stretch control. On the other hand, claudin-5 protein expression decreased after subjecting cells to stretch as well as OGD, a combination of both, and with subsequent re-oxygenation (RO) compared to control but not significantly (Figure 6). Meanwhile, qPCR results show no change in the mRNA expression of occludin among the various treatments. In contrast, mRNA expression of claudin-5 in cells subjected to a combination of OGD and stretch, OGD with subsequent RO, and OGD combined with stretch and subsequent RO decreased significantly (p < 0.005) compared to cells subjected to stretch and made to recover for 15 min and OGD (Figure 7).


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)

Western blot and densitometric analyses of tight junction proteins in cEND cells. Values are the means (± SEM) of 4–5 independent experiments normalized to β-actin. Statistical significance was evaluated using One-Way ANOVA (Holm-Sidak method), *, ¥, $ p < 0.005 compared to no stretch control, OGD + Stretch + RO, and OGD + RO, respectively. 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 6: Western blot and densitometric analyses of tight junction proteins in cEND cells. Values are the means (± SEM) of 4–5 independent experiments normalized to β-actin. Statistical significance was evaluated using One-Way ANOVA (Holm-Sidak method), *, ¥, $ p < 0.005 compared to no stretch control, OGD + Stretch + RO, and OGD + RO, respectively. OGD, oxygen glucose deprivation; RO, re-oxygenation.
Mentions: Tight junction proteins are important in the maintenance of blood-brain barrier (BBB) permeability. Once the BBB is compromised or damaged, the expression of tight junction proteins changes. Therefore, we analyzed the expression of occludin and claudin-5 both at the protein and mRNA levels. Western blot and subsequent densitometric analyses show that occludin protein expression significantly decreased (p < 0.005) after subjecting cells to stretch as well as to OGD and a combination of both in comparison to no stretch control. On the other hand, claudin-5 protein expression decreased after subjecting cells to stretch as well as OGD, a combination of both, and with subsequent re-oxygenation (RO) compared to control but not significantly (Figure 6). Meanwhile, qPCR results show no change in the mRNA expression of occludin among the various treatments. In contrast, mRNA expression of claudin-5 in cells subjected to a combination of OGD and stretch, OGD with subsequent RO, and OGD combined with stretch and subsequent RO decreased significantly (p < 0.005) compared to cells subjected to stretch and made to recover for 15 min and OGD (Figure 7).

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