Cellular Effects of Halogenated Anesthetics Loaded‐Lipid Emulsions in Cultured Primary Dog Hepatocytes: In Vitro Study

Abstract

Objective/Hypothesis Emulsified halogenated anesthetics have been investigated as anesthetic agents as well as therapeutic agents as there is a potential cellular protective effect with these emulsions. The hypothesis of this study is that primary dog hepatocytes would present dissimilar responses in apoptosis and viability considering when exposed to isoflurane and sevoflurane emulsions. Methods : Emulsion preparation: In summary, after dissolving 1% Lipoid S75) in medium-chain triglycerides (15 mL) at 30 ºC under magnetic stirring, the solution will be cooled to 18 ºC and isoflurane or sevoflurane (15 mL) will be added just before emulsification. The aqueous phase containing polysorbate 80 (2%), sorbitol (2.5%), and water (64.5 mL) will be prepared under magnetic stirring at 30 ºC and cooled to 18 ºC before emulsification. The oily phase will be poured into the aqueous phase and emulsified using a high-shear mixer. The primary hepatocytes were cultured in six 96-well plates and incubated at 37°C in 5% CO2/95% air for 24 hours. After cell attachment the groups of plates were separated into Intralipid 20% and Lipoid 10%, and each group had one plate for viability assay with a resazurin-based reagent, other for apoptosis assay with a caspase 3/7 fluorogenic substrate and another for cytotoxicity assay with a lactate dehydrogenase (LDH) reagent. After all reagents were added, the emulsified isoflurane 15% (EI15) or 8% v/v (EI8), sevoflurane 15% (ES15) or 8% v/v (ES8) and lipid 10% or 20% v/v respectively were added in three different volumes (0.3µL, 0.6µL, and 1.2µL), where each volume had 6 replicates. A control group was added on each plate containing cellular culture and medium only. After the treatments were added, each plate was placed in a multimode plate reader for kinetic study during 240 minutes and 17 time-points for the viability and apoptosis reagents, and an endpoint for LDH assay. Linear Mixed Model analysis was performed with SAS 9.4 Software with Tukey test adjustment for repeated measures, with power 80% and alpha 0.05. Results No significant difference was observed for viability on EI8 and ES8 (Intralipid 20%-based).for apoptosis, EI8 and ES8 1.2µL had significantly higher RFU values compared to the other volumes within the same treatments. With EI15 and ES15 (Lipoid 10%-based), there were significant differences on viability response where the lipid 10% and ES15 presented higher RFU values compared to control and EI15. No significant difference was observed for apoptosis neither cytotoxicity between the loaded-lipid 10% groups. Conclusion : Lipoid 10%-based emulsions yield an increasing support of primary dog hepatocytes activity, in comparison to Intralipid 20%-based emulsions. Where emulsified sevoflurane 15% v/v present a superior response over emulsified isoflurane 15% v/v. The Intralipid 20% and Lipoid 10%-based emulsified anesthetics developed in this study, did not produce cellular toxicity on cultured primary dog hepatocytes for a period of 4 hours. Further studies are advocated on the cellular protective effects of isoflurane and sevoflurane loaded-lipid emulsions.