A preparation of murine liver fragments for in vitro studies: liver preparation for toxicological studies
| Autor: | Saeeda Almarzooqi, Bayan Al-Dabbagh, Ali S Alfazari, Alia Albawardi, Abdul-Kader Souid |
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| Rok vydání: | 2012 |
| Předmět: |
Male
Cellular respiration Cytotoxicity Respiratory chain Caspase 3 Mitochondria Liver Apoptosis Biology In Vitro Techniques Bioenergetics General Biochemistry Genetics and Molecular Biology chemistry.chemical_compound Mice Adenosine Triphosphate Oxygen Consumption In vitro Respiration Toxicity Tests medicine Technical Note Animals Chromatography High Pressure Liquid Medicine(all) Biochemistry Genetics and Molecular Biology(all) General Medicine Molecular biology Luciferin Mice Inbred C57BL medicine.anatomical_structure Spectrometry Fluorescence Biochemistry chemistry Liver Hepatocyte Caspases Urea Energy Metabolism |
| Zdroj: | BMC Research Notes |
| ISSN: | 1756-0500 |
| Popis: | Background The aim of this study was to develop liver tissue preparation suitable for investigating toxins. Hepatocyte respiration, ATP content, urea synthesis, caspase activity and morphology were measured as a function of in vitro incubation time. Mice were anesthetized by sevoflurane inhalation. Small liver fragments were then rapidly excised and incubated at 37°C in Krebs-Henseleit buffer (continuously gassed with 95% O2: 5% CO2) for up to 6 h. Phosphorescence O2 analyzer was used to determine the rate of cellular mitochondrial O2 consumption (k c , μM O2 min-1 mg-1). Cellular ATP was measured using the luciferin/luciferase system. The caspase-3 substrate N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) was used to monitor intracellular caspase activity; cleaved AMC moieties (reflecting caspase activity) were separated on HPLC and detected by fluorescence. Findings Respiration was inhibited by cyanide, confirming the oxidation occurred in the respiratory chain. The values of k c (mean ± SD) for 0≤ t ≤6 h were 0.15 ± 0.02 μM O2 min-1 mg-1 (n = 18, coefficient of variation, CV = 13%), ATP content 131 ± 69 pmol mg-1 (1≤ t ≤6 h, n = 16, CV = 53%), synthesized urea 0.134 ± 0.017 mg/dL mg-1 in 50 min (0≤ t ≤6 h, n = 14, CV = 13%), and AMC peak area 62,540 ± 26,227 arbitrary units mg-1 (1≤ t ≤6 h, n = 3, CV = 42%). Hepatocyte morphology and organelles were reasonably persevered. Conclusions The described liver tissue preparation demonstrates stable hepatocyte structure, ultrastructure and biomarkers for up to 6 h, permitting in vitro studies. |
| Databáze: | OpenAIRE |
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