Premium
Halogenated Anesthetics Loaded‐Lipid Emulsions Preconditioning Effect in Canine Primary Hepatocytes: In Vitro Study
Author(s) -
Correa Natalini Claudio,
Ellrich Patricia
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.00345
Subject(s) - sevoflurane , isoflurane , inhalation , chemistry , volatile anesthetic , hypoxia (environmental) , viability assay , in vitro , anesthesia , pharmacology , medicine , biochemistry , oxygen , organic chemistry
/OBJECTIVE/HYPOTHESIS Emulsification of halogenated anesthetics with lipids or perfluorocarbon allow for systemic delivery, as an alternative to inhalation. The emulsification may change the pharmacologic effects of these agents presenting some advantages. Both sevoflurane and isoflurane may produce preconditioning effects that have been demonstrated with the inhaled and emulsified forms. We hypothesize that these emulsified anesthetics, when added to the cultured primary dog hepatocytes before a hypoxic event, would produce preconditioning effects by reducing apoptosis and increasing viability. METHODS Primary canine hepatocytes were cultured in four 96‐well plates and incubated at 37 o C 5% CO 2 /95% air for 12 hours. After cell attachment, Lipoid 10%‐based emulsified isoflurane (EI) 15% v/v or sevoflurane (ES) 15% v/v were added at 0.3µL, 0.6µL and 1.2µL per well. On each plate, Lipoid 10% at 0.3µL, 0.6µL and 1.2µL and a no‐treatment control were included. All four plates were submitted initially to the treatments for 30 minutes at 37°C 5% CO 2 and a baseline data was recorded by an endpoint study. After that, they were divided into groups of hypoxia (1% O 2 ) of 30 minutes or 120 minutes in a Billups‐Rothenberg chamber at 37°C. A kinetic study was conducted for 3 hours at 37C 5% CO 2 ,following the hypoxic period. After the data was collected, the plates were returned to the incubator and at the end of 24 hours an endpoint study was performed. Each group of hypoxia had one plate for viability response by a resazurin‐based reagent, and another for apoptotic response by a caspase 3/7 fluorogenic substrate. Linear Mixed Model was performed for repeated measures and multiple comparison adjustment with Tukey, p <0.05. RESULTS When placed in hypoxia for 120 minutes, the hepatocytes on all EI and ES treatments had a significantly decreased apoptotic response at 24h in comparison to Lipoid 10% and control. At 24h after 30 minutes of hypoxia (1% O 2 ), EI at 1.2 µL, ES 0.3 µL and 0.6 µL were significantly lower than control and Lipoid 10%. There was no significant difference between EI and ES after 30 or 120 minutes of hypoxia in comparison to control for viability response. CONCLUSION Lipoid 10%‐based emulsified isoflurane and sevoflurane 15% v/v significantly diminish the apoptotic response in hepatocytes submitted to 120 minutes of hypoxia and 24h of reoxygenation, also maintaining but not increasing the viability after 30 or 120 minutes of hypoxia (1% O 2 ). Cellular damage is pronounced with periods >30 minutes at oxygen supply ≤4%, justifying the preconditioning effect to be superior on a 120‐minute period of hypoxia when compared to 30 minutes. Both isoflurane and sevoflurane when emulsified in Lipoid 10% are effective to preconditioning cultured canine hepatocytes submitted to 1% oxygen for 120 minutes reducing the apoptotic process.