Lung tissue bioenergetics and caspase activity in rodents

BMC Research Notes

Abdul-Kader Souid³  Steven M Varga⁵  Stacey M Hartwig²  Thachillath Pramathan³  Aws R Othman³  Alia Albawardi⁴  Saeeda Almarzooqi⁴  Ali S Alfazari¹  Mohammed T Alsamri³  Ahmed R Alsuwaidi³ 

[1] Department of Medicine, United Arab Emirates University, P.O. Box 17666, Al Ain, UAE;Department of Microbiology, University of Iowa, Iowa City, IA, 52242, USA;Department of Pediatrics, United Arab Emirates University, P.O. Box 17666, Al Ain, UAE;Department of Pathology, United Arab Emirates University, P.O. Box 17666, Al Ain, UAE;Department of Pathology and Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, IA, 52242, USA

关键词: Apoptosis;    Cytotoxicity;    In vitro;   

Others  :  1145043
DOI  :  10.1186/1756-0500-6-12

 received in 2012-07-26, accepted in 2013-01-10,  发布年份 2013

【 摘 要 】

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% 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.

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 O₂ 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.

【 授权许可】

   
2013 Alsuwaidi et al.; licensee BioMed Central Ltd.

【 图 表 】

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