Lung tissue bioenergetics and caspase activity in rodents

Abstract

Doc number: 12

Abstract

Background: This study aimed to establish a suitable in vitro system for investigating effects of respiratory pathogens and toxins on lung tissue bioenergetics (cellular respiration and ATP content) and caspase activity. Wistar rats and C57Bl/6 mice were anesthetized by sevoflurane inhalation. Lung fragments were then collected and incubated at 37°C in a continuously gassed (with 95% O ₂ :5% CO₂ ) Minimal Essential Medium (MEM) or Krebs-Henseleit buffer. Phosphorescence O₂ analyzer that measured dissolved O₂ concentration as a function of time was used to monitor the rate of cellular mitochondrial O₂ consumption. Cellular ATP content 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. Lung histology and immunostaining with anti-cleaved caspase-3 antibody were also performed.

Results: For Wistar rats, the values of k _c and ATP for 0 < t ≤ 7 h (mean ± SD) were 0.15 ± 0.02 μM O₂ min^-1 mg^-1 (n = 18, coefficient of variation, Cv = 13%) and 131 ± 69 pmol mg^-1 (n = 16, Cv = 53%), respectively. The AMC peak areas remained relatively small despite a ~5-fold rise over 6 h. Good tissue preservation was evident despite time-dependent increases in apoptotic cells. Lung tissue bioenergetics, caspase activity and structure were deleterious in unoxygenated or intermittently oxygenated solutions. Incubating lung tissue in O ₂ depleted MEM for 30 min or anesthesia by urethane had no effect on lung bioenergetics, but produced higher caspase activity.

Conclusions: Lung tissue bioenergetics and structure could be maintained in vitro in oxygenated buffer for several hours and, thus, used as biomarkers for investigating respiratory pathogens or toxins.