Blood-brain barrier opening to large molecules does not imply blood-brain barrier opening to small ions.

Neuroimaging of exogenous tracer extravasation has become the technique of choice in preclinical and clinical studies of blood-brain barrier permeability. Such tracers have a larger molecular weight than small ions, neurotransmitters and many drugs. Therefore, it is assumed that tracer extravasation indicates both permeability to these and the cancelation of the electrical polarization across the barrier. Electrophysiological anomalies following intracarotideal administration of dehydrocholate, a bile salt causing extravasation of the albumin-binding tracer Evans blue, seemingly supported this. By contrast, electron microscopic studies suggested a different hierarchical pattern of blood-brain barrier dysfunction, a milder degree of impairment being characterized by increased function of the transcellular pathway and a severe degree by opening of the tight junctions. This would imply that the extravasation of macromolecules can occur before disruption of the electrical barrier. However, functional evidence for this has been lacking. Here, we further investigated the electrophysiological anomalies following intracarotideal application of dehydrocholate in rats and found that it caused focal cerebral ischemia by middle cerebral artery thrombosis, the electrophysiological recordings being characteristic of long-lasting spreading depolarization. These observations indicated that intracarotideal dehydrocholate is not a suitable model to study the isolated dysfunction of the blood-brain barrier. Second, we studied the topical application of dehydrocholate to the brain and the application of mannitol into the carotid artery. In both models, we found significant extravasation of Evans blue but no changes in either extracellular potassium or the CO(2)-dependent intracortical direct current deflection. The latter is assumed to depend on the proton gradient across the barrier in rats which we confirmed in additional experiments in vivo and in vitro. The stability of the extracellular potassium concentration and the CO(2)-dependent direct current deflection are two functional tests which indicate the integrity of the electrical barrier. Hence, our results provide functional evidence that the blood-brain barrier opening to large molecules does not necessarily imply the opening to small ions consistent with the hierarchy of damage in the previous electron microscopic studies.

Endothelin-1(1-31) induces spreading depolarization in rats.

BACKGROUND: The vasoconstrictor endothelin-1(1-21) (ET-1) seems to induce cerebral vasospasm after aneurismal subarachnoid hemorrhage (aSAH). Moreover, ET-1 causes spreading depolarization (SD) via vasoconstriction/ischemia. ET-1(1-31) is an alternate metabolic intermediate in the generation of ET-1. Our aim was to investigate whether endothelin-1(1-31) causes SD in a similar fashion to ET-1. METHOD: Increasing concentrations of either ET-1, ET-1(1-31) or vehicle were brain topically applied in 29 rats. Each concentration was superfused for one hour while regional cerebral blood flow (rCBF) and direct current electrocorticogram (DC-ECoG) were recorded. FINDINGS: In response to the highest concentration of 10(-6) M, all animals of both ET groups developed typical SD. At concentrations below 10(-6) M only ET-1 induced SD (n=14 of 19 rats). Thus, the efficacy of ET-1(1-31) to induce SD was significantly lower (P<0.001, two-tailed Fisher's Exact Test). CONCLUSIONS: Our findings suggest that ET-1(1-31) less potently induces SD compared to ET-1 which implicates that it is a less potent vasoconstrictor. Speculatively, it could be interesting to shift the metabolic pathway towards the alternate intermediate ET-1(1-31) after aSAH as an alternative strategy to ETA receptor inhibition. This could decrease ET-induced vasoconstriction and SD generation while a potentially beneficial basal ETA receptor activation is maintained.

A novel algorithm for the assessment of blood-brain barrier permeability suggests that brain topical application of endothelin-1 does not cause early opening of the barrier in rats.

There are a number of different experimental methods for ex vivo assessment of blood-brain barrier (BBB) opening based on Evans blue dye extravasation. However, these methods require many different steps to prepare the brain and need special equipment for quantification. We here report a novel, simple, and fast semiquantitative algorithm to assess BBB integrity ex vivo. The method is particularly suitable for cranial window experiments, since it keeps the spatial information about where the BBB opened. We validated the algorithm using sham controls and the established model of brain topical application of the bile salt dehydrocholate for early BBB disruption. We then studied spreading depolarizations in the presence and the absence of the vasoconstrictor endothelin-1 and found no evidence of early BBB opening (three-hour time window). The algorithm can be used, for example, to assess BBB permeability ex vivo in combination with dynamic in vivo studies of BBB opening.