Comparative assessment of in vitro BBB tight junction integrity following exposure to cigarette smoke and e-cigarette vapor: a quantitative evaluation of the protective effects of metformin using small-molecular-weight paracellular markers.

BACKGROUND: The blood-brain barrier (BBB) plays a critical role in protecting the central nervous system (CNS) from blood-borne agents and potentially harmful xenobiotics. Our group's previous data has shown that tobacco smoke (TS) and electronic cigarettes (EC) affect the BBB integrity, increase stroke incidence, and are considered a risk factor for multiple CNS disorders. Metformin was also found to abrogate the adverse effects of TS and EC. METHODS: We used sucrose and mannitol as paracellular markers to quantitatively assess TS and EC's impact on the BBB in-vitro. Specifically, we used a quantitative platform to determine the harmful effects of smoking on the BBB and study the protective effect of metformin. Using a transwell system and iPSCs-derived BMECs, we assessed TS and EC's effect on sucrose and mannitol permeability with and without metformin pre-treatment at different time points. Concurrently, using immunofluorescence (IF) and Western blot (WB) techniques, we evaluated the expression and distribution of tight junction proteins, including ZO-1, occludin, and claudin-5. RESULTS: Our data showed that TS and EC negatively affect sucrose and mannitol permeability starting after 6 h and up to 24 h. The loss of barrier integrity was associated with a reduction of TEER values. While the overall expression level of ZO-1 and occludin was not significantly downregulated, the distribution of ZO-1 was altered, and discontinuation patterns were evident through IF imaging. In contrast to occludin, claudin-5 expression was significantly decreased by TS and EC, as demonstrated by WB and IF data. CONCLUSION: In agreement with previous studies, our data showed the metformin could counteract the negative impact of TS and EC on BBB integrity, thus suggesting the possibility of repurposing this drug to afford cerebrovascular protection.

In vivo and in vitro assessment of baseline blood-brain barrier parameters in the presence of novel nanoparticles.

PURPOSE: Nanoparticles have advantage as CNS drug delivery vehicles given they disguise drug permeation limiting characteristics. Conflicting toxicological data, however, is published with regard to blood-brain barrier integrity and gross mortality. METHODS: To address this issue two novel nanoparticle types: "emulsifying wax/Brij 78" and "Brij 72/Tween 80 nanoparticles were evaluated in vivo for effect on cerebral perfusion flow, barrier integrity, and permeability using the in situ brain perfusion technique. Additional evaluation was completed in vitro using bovine brain microvessel endothelial cells for effect on integrity, permeability, cationic transport interactions, and tight junction protein expression. RESULTS: In the presence of either nanoparticle formulation, no overall significant differences were observed for cerebral perfusion flow in vivo. Furthermore, observed in vitro and in vivo data showed no statistical changes in barrier integrity, membrane permeability, or facilitated choline transport. Western blot analyses of occludin and claudin-1 confirmed no protein expression changes with incubation of either nanoparticle. CONCLUSIONS: The nanoparticle formulations appear to have no effect on primary BBB parameters in established in vitro and in vivo blood-brain barrier models.