Background: The Blood Brain Barrier (BBB) is the major Neurovascular unit in the brain comprising of endothelial cells, astrocytes, and pericytes. Traumatic Brain Injury is characterized by loss of BBB integrity leading to increased barrier permeability, neuroinflammation, and poor clinical outcomes. Specific mediators released following injury lead to BBB gap junction degradation.

Aims: We hypothesize that astrocytes play a central role in restoration and maintenance of BBB integrity following trauma by upregulating expression of proteins that form gap junctions between endothelial cells and that this upregulation is delayed through a crosstalk feedback loop between astrocytes and endothelial cells.

Methods: Monocultures and co-cultures of HCMECs and astrocytes were incubated with trauma plasma and RNA was extracted at the end of an 8hr time course experiment. Expression of CLDN5, ZO-1 and GAPDH was assessed by RT-qPCR using Taqman chemistry and pre-validated primer probes sets.

Results: The co-cultures have a delayed expression of CLDN5 compared to the immediate upregulation in expression seen in astrocyte monocultures at 0.5hr. The CLDN5 co-cultures and astrocyte monoculture showed a robust spike in expression at 6hr, in contrast to ZO-1 co-cultures which showed no increased response above baseline. The astrocyte monoculture however, did present with a spike above baseline for ZO-1 expression at 6hr.

Conclusions: Comparison of expression data between the monoculture and co-cultures suggest that astrocytes are key players in restoration of BBB integrity after trauma via transcriptional upregulation of gap junction proteins such as CLDN5 and ZO-1. In co-culture, there appears to be mechanisms that prevent astrocytes from upregulating gap junction protein expression to restore BBB robustly for CLDN5. Also in co-culture, ZO-1 expression from astrocytes is muted compared to astrocyte monoculture. Future work will focus on mechanistic studies to delineate signaling pathways and transcriptional responses impacted by cross talk between endothelial cells and astrocytes affecting BBB integrity.

[Claudin-5 mRNA Expression]

[ZO-1 mRNA Expression]

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/astrocytes-are-central-contributors-in-restoration-of-the-blood-brain-barrier-via-transcriptional-upregulation-of-key-gap-junction-proteins-between-endothelial-cells/