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Role of astrocytes in cerebrovascular protection provided by an opioid agonist, biphalin during ischemic stroke

Biphalin, a dimeric enkephalin analog (Tyr‐D‐Ala‐Gly‐Phe‐NH‐)2 with potent, nonselective opioid receptor agonist activity, has shown to be neuroprotective in both in vitro as well as in vivo ischemic stroke models. Here we aimed to understand the role of biphalin on blood‐brain barrier (BBB) integrity during ischemic stroke. In this study, we used mouse brain endothelial cells, bEnd.3 and primary astrocytes as monoculture and co‐culture in‐vitro BBB models. Restrictive BBB properties were evaluated by measuring trans‐endothelial electrical resistance (TEER) and [14C] sucrose paracellular permeability, and the redistribution of the tight junction protein occludin. The protective effect of biphalin on BBB integrity was assessed after exposing cells to oxygen‐glucose deprivation (OGD) (1% O2, 2 hours) and glutamate (2mM). Naltrexone, a non‐selective opioid antagonist was used to determine opioid receptors dependent activity of biphalin. Further, we measured the effect of biphalin on astrocytic glutamate uptake and on the expression of excitatory amino acid transporter (EAAT) to study the indirect role of astrocytes on biphalin mediated BBB protection. It was found that combined stress (2mM glutamate and 2 hours OGD) significantly increased the [14C] sucrose paracellular permeability in bEnd.3 cells as well as in a co‐culture cell model. Biphalin (100 nM) treatment attenuated the effect of the combined stress which was reversed by the opioid antagonist naltrexone (100 nM), suggesting the role of opioid receptors in biphalin activity. It was also observed that combined stress (2mM glutamate and 2 hours OGD) condition, significantly reduced the glutamate uptake by astrocytes however, biphalin treatment increased glutamate uptake in primary astrocytes, suggesting the role of astrocytes, increased buffering capacity, in biphalin meditated protection of the BBB during ischemic stroke.