Therapeutic Hypothermia Protects In Vitro Brain Barrier from Ischaemic Damage through Attenuation of Inflammatory Cytokine Release and Apoptosis

Background: Ischaemic stroke disintegrates communications within a controlled ensemble of cells that constitutes the blood-brain barrier. Therapeutic hypothermia may act as a neuroprotectant during intra- and post-ischaemic injuries. Hence, this study focuses on the effects of oxygen-glucose deprivation mediated ischaemic injury on the barrier in the absence or presence of hypothermia (35oC).

Methods: An in vitro model of human blood-brain barrier, composed of human brain microvascular endothelial cells, astrocytes and pericytes, was subjected to oxygen-glucose deprivation (OGD) in the absence or presence of hypothermia in a time-dependent fashion. Following treatments, the integrity and function of blood-brain barrier was assessed by transendothelial electrical resistance (TEER) and paracellular flux of high and low molecular weight permeability markers i.e. Evan’s blue albumin (EBA) and sodium fluorescein (NaF), respectively. Levels of pro-inflammatory cytokines were assessed in all three cell lines using specific ELISA-based kits. To study the rate of apoptosis, caspase-3/7 enzyme activities and percentage of DNA fragmented cells (TUNEL) were measured.

Findings: Cells exposed to OGD alone displayed higher levels of both tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β). Concurrent use of hypothermia significantly decreased both cytokine levels in all three cell lines. Furthermore, hypothermia improved the blood-brain barrier integrity and function as ascertained by an increase in TEER and concomitant decreases in paracellular flux of EBA and NaF. Hypothermia also increased the expression of claudin-5 protein during intra-ischaemic phase which may contribute to improved barrier integrity and function. Lastly, intra- and post-ischaemic treatments of endothelial cells and astrocytes with hypothermia effectively reduced the rate of apoptosis as evidenced by decrease in TUNEL-positive cells and capase-3/7 enzyme activities.

Conclusion: Application of hypothermia during intra- and post-ischaemic periods of an ischaemic injury appears to protect cerebral barrier integrity and function.

Author(s): Manik Mathur and Ulvi Bayraktutan

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