(M0930-04-21) Evaluation of the Blood-Brain Barrier Protective Effects of Metformin after Mixed Exposures to Cigarette Smoke, e-Cigarette Vapor, and Stroke Conditions

Purpose: It has been demonstrated that usage of inhaled tobacco products with nicotine and stroke conditions each have distinct effects on the blood-brain barrier (BBB), leading to significant comorbidities. It was assumed that smoking and stroke would cause additive or synergistic BBB dysfunction because smoking is a risk factor for stroke and shares several similar pathological pathways. The harmful effects of cigarette smoke and e-cigarette vapor on BBB have already been established and have also been reported to be mitigated by Metformin (MF), a commonly prescribed antidiabetic drug (1. However, evaluation of the protective effect of MF on the impact of cigarette smoke, e-cigarette (JUUL) vapor, and dual exposure (cigarette smoke combined with e-cigarette (JUUL) vapor) along with stroke has not been investigated. Thus, the goal of this work was to assess this hypothesis in an in vitro blood-brain barrier (BBB) model. We also wanted to determine how MF was able to protect these detrimental effects utilizing a reliable and well-established bEnd3/astrocyte co-culture  BBB model.

Methods: bEnd3 cells passages 21–24 (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, MO, USA) supplemented by 10% FBS (Atlanta Biologicals, Minneapolis, MN, USA) and 1% each of non-essential amino acid and penicillin/streptomycin (PS) solution (Sigma Aldrich). Cells were then maintained in a humidified cell culture incubator at 37 °C and with 5% CO₂/95% air. Mouse primary astrocytes were obtained from the cerebral cortices of one-day-old CD-1 mouse pups (Charles Rivers Laboratory) according to the previously published methods (2). bEnd3/astrocyte co-cultured cells were exposed to Oxygen Glucose Deprivation (OGD) alone for 4 hours or combined with either 5% soluble cigarette smoke extract or 5% JUUL extracts or dual exposure for 5 days and pre-treated with metformin for 3 days. Eventually, we had nine different groups comprising of control, OGD alone, OGD with MF pretreatment, OGD combined with tobacco smoke extract (TSe) exposure, OGD combined with TSe exposure as well as MF pretreatment, OGD combined with JUUL exposure (ECe), OGD combined with ECe exposure and MF pretreatment, OGD combined with dual exposure (Mix), OGD combined with dual exposure as well as MF pretreatment. The integrity of the co-culture setup was carried out using two techniques: [1] through measurement of trans-endothelial electric resistance (TEER) of the transwell inserts and [2] by permeability assessment (apical to basolateral) of sodium fluorescein (NaF) (Sigma Aldrich), a low molecular weight BBB marker, according to a previously published method (3). Specially we used a quantitative platform to determine the harmful effects of stroke alone or combined with smoking on the BBB and study the protective effect of MF. Western blot (WB) analysis of relevant molecular targets was carried out using bEnd3 monolayer cells.

Results: According to our findings, OGD either by itself or in combination with TSe, ECe, or Mix exposure for five days had a detrimental effect on BBB permeability. In comparison to the control group, a decrease in TEER values and an increase in the permeability coefficient (PC) was observed in the TSe, ECe, or Mix exposure combined with OGD groups. MF pretreatment, however, protected the BBB from losing viability. Even after being exposed to OGD alone or in combination with nicotine exposure from various sources, the TEER and PC values obtained from MF pretreatment did not change significantly from the comparable control. ZO-1 protein expression was significantly down-regulated when OGD plus TSe were applied to bEnd3 cells. ZO-1 expression showed an increasing trend following MF pre-treatment, although this trend was not statistically significant. Interestingly, although we did not observe any reduction in the expression of occludin after ODG treatment, a significant decrease in its expression was observed after OGD combined with TSe and mixed exposure. Nevertheless, we only noticed a statistically significant increase in occludin expression following MF pretreatment when OGD exposure was given alone—increased expression was not observed when cells were exposed to OGD combined with TSe, ECe, or Mix exposure. Additionally, Claudin-5 expression was markedly downregulated following TSe exposure and mix exposure paired with OGD compared to the control and OGD alone. While MF was able to considerably upregulate Claudin-5 expression following TSe exposure in combination with OGD, its expression was not significantly upregulated following mix exposure in combination with OGD.

Conclusion: Overall, our findings have demonstrated that OGD in combination with TSe, ECe, or mix exposure adversely affects NaF permeability. A decrease in the TEER value was linked to the deterioration of the barrier. MF was able to offset the majority of these adverse effects by exhibiting different patterns of increased ZO-1, occludin, and Claudin-5 expression. Additional clarification will be provided by immunofluorescence analysis in future studies.

References: 1. Kadry H, Noorani B, Bickel U, Abbruscato TJ, Cucullo L. 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. Fluids Barriers CNS. 2021;18(1):28.
2. Du F, Qian ZM, Zhu L, Wu XM, Qian C, Chan R, et al. Purity, cell viability, expression of GFAP and bystin in astrocytes cultured by different procedures. Journal of cellular biochemistry. 2010;109(1):30-7.
3. Nozohouri S, Noorani B, Al-Ahmad A, Abbruscato TJ. Estimating Brain Permeability Using In Vitro Blood-Brain Barrier Models. Methods Mol Biol. 2021;2367:47-72.

Acknowledgements:

Funding: The work was supported by NIH grant R01NS117906 to TJA.
Declaration: Ethics approval and consent to participate all animal studies were approved by Texas Tech University Health Sciences Center Institute Animal Care and Use Committees (IACUC).

Conflict of interest: Authors declare there is no conflict of interest.


Fig 1. OGD alone (4h) or combined with TSe, ECe, or a mix of both negatively impacts BBB integrity, as demonstrated by TEER measurement. One-way ANOVA; n=3 biological replicates



Fig 2. OGD alone (4h) or combined with TSe, ECe, or a mix of both negatively impacts BBB integrity, as demonstrated by permeability coefficient (PC). One-way ANOVA; n=3 biological replicates