A preparation of murine liver fragments for in vitro studies: liver preparation for toxicological studies

Abstract

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 (kc, 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 kc (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.

Figure 1

Liver tissue respiration, ATP content, caspase activity and urea synthesis in continuously oxygenated KH buffer. Liver specimens from a Taylor outbred mouse were incubated at 37°C in 50 mL KH buffer continuously gassed with 95% O₂: 5% CO₂ for up to ~6 h. At indicated time periods, samples were removed from the incubation medium and processed for measurements of O₂ consumption, ATP content, caspase activity and urea synthesis as described in Methods. Panel A: Representative runs of cellular mitochondrial O₂ consumption are shown; t = 0 corresponds to animal sacrifice. The rate of respiration (k, μM O₂ min^-1) was the negative of the slope of [O₂] vs. t. The additions of 10 mM NaCN and 50 μg/ml glucose oxidase are shown. PanelB: The values of k_c and ATP were plotted as a function of incubation time. Panel C: HPLC runs of caspase activity at 6 h. The retention time (R_t) for Ac-DEVD-AMC was ~2.5 min and AMC (representing caspase activity) ~4.8 min. Panel Cinsert: AMC peak areas are shown as a function of incubation time. PanelD: Liver specimens (~300 mg) were incubated at 37°C in 50 ml KH buffer supplemented with 10 mM NH₄Cl and 2.5 mM ornithine and continuously gassed with 95% O₂: 5% CO₂. At t = 0, 3 and 6 h, an aliquot of the solution was taken for urea determination. Insert (error bars are standard deviation of 2 independent experiments), liver specimens were incubated at 37°C in 50 ml KH buffer continuously gassed as above. At indicated time points, specimens (~80 mg) were placed in 1.0 mL KH buffer supplemented with 10 mM NH₄Cl and 2.5 mM ornithine. The reactions were allowed to continue at 37°C for 50 min with continuous gassing as above. At the end of the incubation period, the specimens were discarded and the solution was analyzed for urea.

Figure 2

Liver histology (oxygenated KH buffer). Normal hepatic architecture characterized by polygonal hepatocytes arranged in plates that radiate from central vein towards portal tracts. Hepatic plates are separated by thin sinusoids. Comparisons of hepatic architecture and cellular morphology in A to C demonstrate good preservation of cellular membranes and nuclear chromatin details over a period of 6 h. (H&E, 10x & 40x).

Figure 3

Electron microscopy images (oxygenated KH buffer). (A) TO mouse liver at 0 h demonstrating a hepatocyte with preserved architecture. Note hepatocyte nucleus (N) and numerous intact mitochondria (m). (B) TO mouse liver at 6 h demonstrating a preserved hepatocyte microarchitecture with only mild swelling of mitochondria (m). Note hepatocyte nucleus (N). (C) C57Bl/6 mouse liver at time 0 demonstrating a hepatocyte with preserved architecture. Note hepatocyte nucleus (N) and numerous intact mitochondria (m). (D) C57Bl/6 mouse liver at time 6 demonstrating a hepatocyte with swollen mitochondria (m). Note hepatocyte nucleus (N). Magnification 140,000.

Figure 4

Liver tissue respiration, ATP content and caspase activity in unoxygenated KH buffer. Liver specimens from a C57Bl/6 mouse were incubated at 37°C in 50 mL KH buffer for up to ~6 h. At indicated time periods, samples were removed from the incubation medium and processed for measurements of O₂ consumption, ATP content and caspase activity as described in Methods. Panel A: Representative runs of cellular mitochondrial O₂ consumption are shown; t = 0 corresponds to animal sacrifice. The rate of respiration (k, μM O₂ min^-1) was the negative of the slope of [O₂] vs. t. PanelB: The values of k_c and ATP are plotted as a function of incubation time. Panel C: HPLC runs of caspase activity at 6 h with and without the pancaspase inhibitor zVAD-fmk. Panel D: AMC peak areas (retention time, ~4.8 min) are shown as a function of incubation time.

Figure 5

Electron microscopy images (unoxygenated KH buffer). (A) Liver at time 0 demonstrating hepatocytes with preserved architecture. Note hepatocyte nucleus (N), numerous intact mitochondria (m), rough endoplasmic reticulum (rER), microvilli (mV) and cytoplasmic lipid vacuoles (L). (B) Liver at 6 h demonstrating hepatocytes with numerous cytoplasmic vesicles (v), rough endoplasmic reticulum (rER) and a nucleus (N). Some of the vesicles probably represent swollen disintegrating mitochondria under experimental conditions. Magnification 140,000.