A modelling approach to study the effects of connexin43 modulation on the blood-brain barrier protection following central nervous system injuries

Abstract

The gap junction protein, Connexin 43 (Cx43), has been identified as a potential target in preventing vascular leakage and the spread of damage following CNS injury in a number of animal models. The animal models, however, are limited by the uncertainties in the relevance to human wound healing. This study aimed to establish a human in vitro injury model for investigation of the neuroprotective effects of Cx43 on the blood-brain barrier (BBB) following injury. The Electric Cell-substrate Impedance Sensing (ECIS) is an approach producing highly reproducible wounding models. It is an impedance-based system which monitors cellular behaviours including cell proliferation, migration and injury response in a real-time fashion. This study characterized wound healing assay using cultured human cerebral microvascular endothelial cells (hCMVEC) with two ECIS array types, the single electrode (8W1E) and the multiple electrode (8W10E+) arrays, in terms of cell remodelling, barrier functions and inflammatory response. Wounding produced by these two models are fundamentally different. The 8W1E model more closely mimics the microvascular disruption following CNS injuries whereas the 8W10E+ model induced an open wound without any necrotic tissue. Following wounding, the endothelial junctional proteins were dynamically regulated to mediate cell remodelling following wounding on both arrays. Nevertheless, the endothelial barrier strength was partially restored following wounding. Analysis of the pro-inflammatory cytokine profile revealed that an inflammatory response was not detectable in the 8W1E model due to the small injury size. Moreover, it is believed that the 8W10E+ model is not capable of inducing inflammatory reaction because cellular components were completely detached from the wounding site. The Cx43 protein has a pivotal role in regulating barrier integrity and cell remodelling during wound healing. Cx43 is promptly upregulated following wounding, and shows adhesive property that could have contributed to inadequate recovery of barrier functions. These findings demonstrate factors that contribute to the impaired barrier function following CNS injuries. In addition, the established ECIS models can be applied to other cell cultures which will advance our understanding in the CNS injury response.