(T1330-04-27) Effect of Propofol on Lipid Membrane Fluidity and Blood-Brain Barrier Permeability

Purpose: We and others have shown that volatile anesthetic agents increase lipid membrane fluidity and blood-brain barrier (BBB) permeability (1). Propofol, an injectable anesthetic acting on GABA_A receptors, is highly lipophilic (logD7.4 of 4.2). Therefore, membrane and permeability effects similar to volatile anesthetics could be present. In fact, a recent publication showed increased permeability in an in vitro model of the BBB in the presence of propofol (2). The present study examined whether propofol will also increase the passive permeability of the BBB in vivo using [¹³C]sucrose as a highly sensitive and specific marker. In addition, we studied the effect of propofol compared to isoflurane on the fluidity of artificial lipid membranes by measuring fluorescence anisotropy and excimer/monomer ratios.

Methods: C57Bl/6J mice were anesthetized with ketamine:xylazine (100:10 mg/kg) intraperitoneally. Diprivan (propofol 10 mg/mL in lipid emulsion) was infused via tail vein catheter at a rate of 5 μL/min, corresponding to a dose rate of 120 mg/kg/h in a 25g mouse (n=5). Control groups received no infusion (ketamine/xylazine only, n=6) or an infusion of Intralipid (n=5). The jugular veins on both sides were exposed by skin incision. After 15 min of propofol or Intralipid infusion, we injected the hydrophilic marker [¹³C₁₂]sucrose (10 mg/kg) by IV bolus into one jugular vein. Blood samples were drawn after 1, 5, 10, 20, and 30 min from the contralateral jugular vein under ongoing infusion. At 29.5 min [¹³C₆]sucrose was injected as a vascular marker, followed by euthanization at 30 min and brain sampling. [¹³C₁₂]sucrose and [¹³C₆]sucrose were quantified in plasma and brain samples by our established LC-MS/MS technique. Brain uptake clearance, K_in, of [¹³C₁₂]sucrose was calculated from brain concentrations (corrected for vascular content) and plasma AUC. Expression of the tight junction proteins (TJs) (Claudin-5, Occludin, and ZO-1) was quantified and compared between the propofol-treated and control groups with a confocal microscope. An immunoblotting assay was conducted on the brain samples of each group, and the relative quantity of TJ proteins was compared between them. Finally, fluorescence anisotropy studies were performed on Dipalmitoylphosphatidylcholine (DPPC) lipid membranes labeled with 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-PC) in the presence of different concentrations of propofol or isoflurane to see the effect of these anesthetic agents on the fluidity of the lipid membranes. In vivo permeability, immunofluorescence, and immunoblotting studies were evaluated by One-Way ANOVA with Tukey post test.

Results: K_in values of [¹³C₁₂]sucrose increased under propofol infusion significantly to 0.119 ± 0.018 μL min^-1 g^-1 from 0.092 ± 0.007 μL min^-1 g^-1 under Intralipid (+29%, p < 0.05) and 0.0828 ± 0.013 μL min^-1 g^-1 under ketamine/xylazine only (+43%, p < 0.01). The expression of ZO-1 tight junction protein decreased under propofol infusion slightly but significantly (p < 0.05, n=6). However, expression of the other TJ proteins (Claudin-5 and Occludin) did not show any significant change under propofol infusion compared to the control groups. Propofol at 100 micromolar concentration decreased DPH-PC anisotropy in DPPC bilayers significantly to a similar extent as 1 millimolar isoflurane, which is in the clinical range of concentrations. However, only a slight decline was observed in the DPH-PC anisotropy of the lipid membranes in the presence of 10 micromolar propofol, which is in the high range of pharmacological concentrations.

Conclusion: While propofol at high micromolar concentrations shows effects on lipid membrane fluidity, the in vivo effect on BBB permeability is modest, with an increase of only 30-40 percent compared to the control conditions. In comparison, clinical concentrations of volatile anesthetic agents (isoflurane and sevoflurane) cause two-fold higher passive permeability at the BBB in vivo.

References: 1. Behnam Noorani, Ekram Ahmed Chowdhury, Faleh Alqahtani, Yeseul Ahn, Ehsan Nozohouri, Sumaih Zoubi, Dhavalkumar Patel, Laurence Wood, Juyang Huang, Muhammad Bilal Siddique, Abraham Al-Ahmad, Reza Mehvar and Ulrich Bickel
Journal of Pharmacology and Experimental Therapeutics February 24, 2023, JPET-AR-2022-001281; DOI: https://doi.org/10.1124/jpet.122.001281
2. Hughes JM, Neese OR, Bieber DD, Lewis KA, Ahmadi LM, Parsons DW, Canfield SG. The Effects of Propofol on a Human in vitro Blood-Brain Barrier Model. Front Cell Neurosci. 2022 May 11;16:835649. doi: 10.3389/fncel.2022.835649. PMID: 35634467; PMCID: PMC9132176.

Apparent Kin values of [13C12]Sucrose for propofol-treated and control groups

Mean Fluorescence Intensity (MFI) of tight junction proteins in cortex and hippocampus regions of the brain for experimental and control groups

The relative quantity of tight junction proteins for experimental and control groups