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Lung tissue bioenergetics and caspase activity in rodents
Ahmed R Alsuwaidi1, Mohammed T Alsamri, Ali S Alfazari
1Department of Pediatrics, United Arab Emirates University, Al Ain, UAE.
BMC Research Notes
|January 15, 2013
Summary
This study developed an in vitro lung tissue model to assess cellular respiration and apoptosis. The model effectively maintains lung tissue bioenergetics and structure for several hours in oxygenated buffer.
Area of Science:
- Biomedical Engineering
- Cellular Biology
- Respiratory Physiology
Background:
- Investigating respiratory pathogens and toxins requires a reliable in vitro system.
- Lung tissue bioenergetics (cellular respiration, ATP content) and caspase activity are key indicators of cellular health.
- Previous methods lacked a suitable system for studying these parameters in lung tissue.
Purpose of the Study:
- To establish a robust in vitro system for evaluating the impact of respiratory pathogens and toxins on lung tissue.
- To measure cellular respiration, ATP content, and caspase activity in isolated lung tissue.
- To assess the viability and structural integrity of lung tissue in vitro over time.
Main Methods:
- Lung tissue fragments from Wistar rats and C57Bl/6 mice were incubated in oxygenated Minimal Essential Medium (MEM) or Krebs-Henseleit buffer.
- Cellular mitochondrial oxygen consumption was measured using a phosphorescence O2 analyzer.
- Adenosine triphosphate (ATP) content was quantified using the luciferin/luciferase system.
- Caspase-3 activity was monitored using a fluorogenic substrate (Ac-DEVD-AMC) and HPLC analysis.
- Lung histology and immunostaining for cleaved caspase-3 were performed.
Main Results:
- Oxygenated buffer maintained lung tissue bioenergetics and structure for up to 7 hours, despite some increase in apoptotic cells.
- Cellular mitochondrial oxygen consumption (kc) and ATP content were quantified.
- Caspase activity showed a modest increase over time in oxygenated conditions.
- Non-oxygenated or intermittently oxygenated conditions were detrimental to lung tissue bioenergetics and structure.
- Anesthesia by urethane did not affect bioenergetics but increased caspase activity.
Conclusions:
- The developed in vitro system effectively maintains lung tissue bioenergetics and structure.
- This system is suitable for investigating the effects of respiratory pathogens and toxins.
- Lung tissue bioenergetics and structure can serve as valuable biomarkers in such studies.
