(T0930-05-29) Effect of Propofol on the In Vitro Model of the Blood-Brain Barrier Based on Induced Pluripotent Stem Cell-Derived Human Brain Microvascular Endothelial Cells

Purpose: Recent reports show that lipophilic volatile anesthetic agents, including isoflurane and sevoflurane, increase the permeability of the blood-brain barrier (BBB) [1,2]. Propofol is an injectable anesthetic, which is also highly lipophilic. The objective of the present studies was to evaluate whether propofol at clinical concentrations also affects the integrity of the BBB. We measured the permeability of an in vitro BBB model, consisting of a monolayer of induced pluripotent stem cell (iPSC)-derived human brain microvascular endothelial cells (hBMECs), using stable isotope-labeled [¹³C]sucrose as a marker.

Methods: Due to the minimal aqueous solubility of propofol (124 mg/L), it was reconstituted in dimethyl sulfoxide (DMSO). To assess the cytotoxicity effect of DMSO at the final concentration of 0.2% (v/v), MTT assay was performed. Trans-endothelial electrical resistance (TEER) values were measured before starting the permeability experiment, after the completion of the experiment, and 24 hours after the completion of the experiment. Permeability studies were conducted in two sets of experiments in vitro using transwells: (1) Media-only treated hBMECs vs. DMSO-treated cells. (2) Propofol-treated hBMECs vs. DMSO- treated cells. After 2 hrs incubation with propofol in media, 1 mg/ml of the stable isotope-labeled [¹³C]sucrose was added to the top compartment of the transwells (corresponding to the luminal side of the BBB model). Subsequently, samples were collected from the lower chamber (corresponding to the abluminal or brain side) at different time points up to 120 min. Samples were analyzed on an AB Sciex 7500 QTRAP mass spectrometer attached to a Shimadzu UPLC system using an Acquity B.E.H. amide column (2.1 mm x 50 mm, 1.7 μm; Waters, Milford, MA, USA). Statistical analysis was performed using unpaired student t-tests, with a p-value < 0.05 considered significant. Immunocytochemistry (ICC) was performed on hBMECs from different treatment groups immediately after or 24 hours after the experiment to analyze tight junction protein expression. ZO-1 tight junction protein was stained with appropriate primary and secondary antibodies, and images were acquired on a Leica Stellaris 8 FALCON STED super-resolution confocal microscope.

Results: No significant change was observed in the viability of hBMECs between the 0.2% (v/v) DMSO treated group and the media-only treated group. TEER values were comparable for both DMSO-treated hBMECs and propofol-treated cells at different time points. No significant change was observed in the permeability coefficients of stable isotope-labeled [¹³C]sucrose as the permeability marker between DMSO-treated hBMECs and the media-only treated group. There was a statistically significant difference between the permeability coefficients of stable isotope-labeled [¹³C]sucrose in DMSO-treated hBMECs (4.78 x 10^-7 ± 1.56 x 10^-7 cm/s) and propofol-treated hBMECs (6.32 x 10^-7 ± 1.83 x 10^-7 cm/s) (p = 0.037, n = 5). Based on the staining pattern of tight junction proteins in the control hBMECs compared to the propofol-treated group, when stained immediately after the experiment, the tight junction proteins in the latter group were compromised. Additionally, the mean fluorescence intensity value was lower for the propofol treated group than for the DMSO treated group in the intensity analysis.

Conclusion: iPSC-derived hBMEC monolayer presented a suitable in vitro BBB model for permeability studies. 0.2% (v/v) DMSO has no significant cytotoxic effect on iPSC-derived hBMECs. Also, this concentration of DMSO does not compromise the integrity of BBB in iPSC-derived hBMECs. Propofol at the final concentration of 100 µM increases the leakiness of the BBB in iPSC-derived hBMECs. As the most commonly used IV general anesthetic agent, propofol decreases the expression of the tight junction protein ZO-1 in brain endothelium as detected by confocal imaging.

References: 1. Tetrault S, Chever O, Sik A and Amzica F (2008) Opening of the blood-brain barrier during isoflurane anesthesia. Eur J Neurosci 28:1330-1341.
2. Acharya NK, Goldwaser EL, Forsberg MM et al.(2015) Sevoflurane and Isoflurane induce structural changes in brain vascular endothelial cells and increase blood-brain barrier permeability: Possible link to postoperative delirium and cognitive decline. Brain Res 1620:29-41.


Permeability coefficients comparison of the stable isotope-labeled [13C] sucrose for propofol-treated hBMECs and DMSO-treated cells


A) ZO-1 tight junction protein expression in DMSO-treated hBMECs
B) ZO-1 tight junction protein expression in propofol-treated hBMECs