Purpose: The blood brain barrier (BBB) is a critical part of the central nervous system and helps to maintain its homeostasis. BBB disruption is a common phenomenon associated with all neurological diseases with lasting consequences. Recently effects of volatile anesthetic agents, in particular isoflurane, on BBB permeability have been postulated. Although the exact cause is still debatable, various studies have linked increases in permeability to disruption of tight junction proteins. In a study comparing the injectable anesthetic agent ketamine with isoflurane anesthesia and with unanesthetized mice, we found that isoflurane increases BBB permeability of the hydrophilic marker [13C12] sucrose by a factor of 2, and this effect was detected as early as 15-30 min after onset of anesthesia. Considering this rapid time course, we hypothesized that physicochemical effects on cell membranes may play a role. In fact, effects of anesthesia gases on the structure of artificial lipid membranes have been shown. Because to our knowledge a possible influence of volatile anesthetics on membrane permeability to hydrophilic small molecules has not been examined experimentally, the goal of the present work was to assess permeability by utilizing two blood-brain barrier permeability markers, [13C12] sucrose and sodium fluorescein, in a series of in vitro experiments. One experimental setup consisted of an in vitro BBB cell culture model in form of endothelial monolayers, the other setup used a biological cell membrane preparation, consisting of erythrocyte ghosts.
Methods: In vitro permeability studies on endothelial monolayers in transwell filter inserts were done using brain endothelial cells generated from human induced pluripotent stem cells (iPSCs). Permeability of [13C12]sucrose as well as fluorescein was measured under room air conditions at 37⁰C (control), or in presence of anesthesia gases, 3% isoflurane in N2O:O2 (70:30 v/v) at 37⁰C. Effects on tight junction proteins claudin-5 and occludin were assessed by immunocytochemistry, and trans endothelial electrical resistance (TEER) was measured at baseline and after exposure to the anesthesia agents for 6 hours. Cytotoxicity assay was also done to assess whether our experimental procedure was having any adverse effects on the cells. [13C12]sucrose was measured by LC-MS/MS, and fluorescein was quantified fluorometrically on a plate reader. A second set of in vitro experiments utilized erythrocyte ghosts generated from human red blood cells that were loaded with sodium fluorescein. In order to ensure that the loaded ghosts were stable over the time of experiment, stability of the loaded ghosts were assessed up to 12 days at 4⁰C and up to 8 hours at 37⁰C. Co-localization study were also done using fluorescein loaded erythrocyte ghosts whose membranes were stained with DiD dye. Based on acceptability of stability and co-localization study, loaded erythrocyte ghosts were subjected to anesthesia gas conditions as above for 6 hours, and the difference between control and anesthesia treated samples both before and after experiment were analyzed by flowcytometry.
Results: The in vitro permeability study utilizing human BBB endothelial stem cells showed a significant difference in permeability for both [13C12] sucrose and fluorescein p<0.01 . Permeability for control group in cm/min X 10-3 was found to be 0.0506 ± 0.001228 while that of the control group was found to be 0.04474 ± 0.0007564. The TEERs however were not found to differ significantly both before and after treatment between the two groups. The TEER for control group was found to be 1049 ± 50.09 Ω cm2 while anesthesia treated group was found to be 899.7 ± 70.95 Ω cm2 after the treatment. Similarly, immunocytochemistry of claudin and occludin did not indicate disruption in the barrier properties. The cytotoxicity assay also did not show any significant difference between control and treatment group. The fluorescein loaded erythrocyte ghosts were found to be stable over the period of time they were tested. Flowcytometry analysis of the erythrocyte ghosts showed similar fluorescein content of the ghosts in the control group and anesthesia group before the experiment, 16637 ± 1128 versus 16086 ± 1112, respectively. A significant decrease (p<0.05) in fluorescence intensity in the anesthesia treated group after 6 hours of treatment was observed when compared to control group 8779 ± 1744 and 15150 ± 1177, respectively.
Conclusion: The study provides evidence in support of our hypothesis that isoflurane at relatively low and clinically relevant concentrations may alter lipid membranes and this may result in enhanced barrier permeability. In the endothelial cell culture system, the lack of change in TEER and the stable appearance of tight junction proteins as judged by immunofluorescence argue against a major effect of isoflurane of tight junctions. The erythrocyte ghosts are a liposome-like system with a continuous lipid bilayer, with natural membrane proteins associated with the membrane. There are no intercellular junctions present membranes and any effect on membrane permeability of hydrophilic markers for which no transport systems is present should reflect underlying disturbance of the lipid bilayer and/or changes at the protein-lipid interfaces.
Behnam Noorani– Research Assistant, Texas Tech University Health Sciences Center
Faleh Alqahtani– Assistant Professor, King Saud University, Riyadh
Reza Mehvar– Professor, Chair of the Department of Biomedical and Pharmaceutical Sciences, Chapman University
Bickel Ulrich– Professor, Texas Tech University Health Sciences Center