gms | German Medical Science

Objective: Until date, mechanisms underlying the intravascular inflammatory response post subarachnoid hemorrhage (SAH) remain unclear. Interleukin-6 (IL-6) has been implicated to play a crucial role in the inflammatory response post SAH. Here, we aimed to show the time-lapse of the expression and localization of this molecule following SAH in mice. The impact of IL-6 on blood-brain barrier (BBB) properties has been analysed in vitro.

Method: SAH was induced in wildtype mice by the insertion of a filament perforating the Willis Circle. Our analyses were performed on day 1 to 7 post SAH. In order to determine the endothelial cell (EC)-specific expression and distribution of IL-6, brain capillaries from SAH and sham-operated mice have been isolated. IL-6 vesicles were stained by immune-fluorescence techniques and gene expression of this cytokine was analysed by real-time qPCR. In addition, the impact on EC integrity mediated by IL-6 recombinant protein with and without the IL-6 antibody tocilizumab was determined using microvascular brain ECs, cEND. For this, electrophysiological and molecular methodologies were applied.

Results: We observed a time-dependent increase of IL-6 transcripts of approximately 3.5-fold in brain capillaries isolated from mice brains on day 4 and 7 post SAH versus controls. IL-6 overexpression was accompanied by a time-dependent down-regulation of important cellular contacts, namely claudin-1 and -5 and occludin. We observed a destabilization of the cEND cell monolayer by a decrease of EC resistance to 60% of the untreated ECs and by a down-regulation of TJ expression in response to IL-6 treatment (25 ng/ml). Claudin-5 and occludin were down-regulated by approximately 40% of the untreated control cells. Deregulated barrier properties of cEND cells could be partially restored by a subsequent incubation of cEND cells with IL-6 and tocilizumab (up to approximately 90% of the control). Moreover, cEND cell proliferation and their ability to form tubes were diminished.

Conclusions: Investigation of mechanisms leading to BBB dysfunction remains fundamental to improve the outcome of SAH patients. The inflammatory response in brain vasculature is mediated by the overexpression of pro-inflammatory cytokines, as we have demonstrated for IL-6. This leads to a progressive reduction of important barrier tightening molecules in vivo and in our BBB in vitro model. Our here presented results suggest that targeting IL-6 by the usage of tocilizumab might potentially prevent BBB dysregulations.