Meszaros 2014 Abstract MiP2014
|Methodological aspects of assessing mitochondrial function in small intestinal mucosal samples of rats and guinea pigs.|
Although the renaissance of functional mitochondrial studies targets many tissues and organs, small intestinal mitochondria are less frequently examined. Nevertheless, the integrity of small intestinal mucosa is potentially threatened in various local and systemic pathologies, such as ischemia-reperfusion, septic shock or inflammatory bowel diseases. Mitochondrial dysfunction is critically involved in the pathomechanism of these diseases. Furthermore, timely restitution of adequate mitochondrial function can be a key step towards potentially effective therapeutic strategies.
Our goal was to provide firm experimental data for functional mitochondrial investigations, and as a first step, we aimed to test and validate the available methods used for isolation of small intestinal mitochondria in rodents [1,2]. Whole thickness small intestinal mucosal samples of rats and guinea pigs were used, and mitochondria were isolated according to published protocols using chelating agents and differential centrifugation. The assessment of the functional state of isolated mitochondria was performed by means of high-resolution respirometry (Oroboros Oxygraph-2k). The integrity of the outer mt-membrane was tested with cytochrome c addition and photometric assay for mitochondrial swelling, while membrane potential changes were monitored by safranin fluorescence measurements.
In case of rat samples, low respiratory control rates and extremely high cytochrome c responses were found, and swelling of mitochondria indicated serious damage of the outer mt-membrane. In contrast, guinea pig mitochondria were presented with good respiratory control, low cytochrome c response and baseline swelling parameters.
Based on high-resolution respirometry we conclude that using the accessible, published methodologies, only functionally impaired mitochondria can be isolated from the rat. However, high quantities of intact and well coupled mitochondria can be obtained from the guinea pig. These mitochondria are suitable for further focused studies. Further methodological investigations, possible modifications or even new protocols are needed in order to clarify the cause of this significant interspecies difference.
Labels: MiParea: Respiration, Instruments;methods
Organism: Rat, Guinea pig
Preparation: Isolated mitochondria
Regulation: Cyt c, mt-Membrane potential
HRR: Oxygraph-2k, O2k-Fluorometer Event: C2, Oral MiP2014
1-Inst Surgical Research, Univ Szeged, Hungary; 2-Dep Medical Biochem, Semmelweis Univ, Budapest, Hungary. - [email protected]
References and acknowledgements
Supported by grants OTKA K104656, TAMOP-4.2.2A-11/1-KONV-2012-0035, TAMOP-4.2.2A-11/1/KONV-2012-0073, TAMOP-4.2.4.A/2-11/1-2012-0001 ‘National Excellence Program’.
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