Lung tissue bioenergetics and caspase activity in rodents
1 Department of Pediatrics, United Arab Emirates University, P.O. Box 17666, Al Ain, UAE
2 Department of Medicine, United Arab Emirates University, P.O. Box 17666, Al Ain, UAE
3 Department of Pathology, United Arab Emirates University, P.O. Box 17666, Al Ain, UAE
4 Department of Microbiology, University of Iowa, Iowa City, IA, 52242, USA
5 Department of Pathology and Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, IA, 52242, USA
BMC Research Notes 2013, 6:12 doi:10.1186/1756-0500-6-12Published: 12 January 2013
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% O2:5% 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.
For Wistar rats, the values of kc 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 O2 depleted MEM for 30 min or anesthesia by urethane had no effect on lung bioenergetics, but produced higher caspase activity.
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.