MiP2005: Session 7

Mitochondrial Physiology Network 10.9: 88 (2005) - download pdf

 

The preparation procedure does not influence significantly the function and morphology of liver mitochondria from control and endotoxic rats.

Susanne Haindl1, L Gille2, H Nohl2, S Bahrami1, S Nürnberger3, E Gnaiger4, H Redl1, AV Kozlov1

1Ludwig Boltzmann Inst. Experimental Clinical Traumatology and Research Ctr. AUVA, Vienna; 2Research Inst. Pharmacology Toxicology, University of Veterinary Medicine, Vienna; 3Dept. Traumatology, Medical University of Vienna; 4D. Swarovski Research Lab., Dept. Transplant Surgery, Innsbruck Medical University, Innsbruck, Austria. – susanne.haindl@lbitrauma.org

    Endotoxic shock is a condition in which the cardiovascular system fails to perfuse tissues adequately and cells fail to extract oxygen from blood. In each cell the mitochondria are the main oxygen sink and are thus responsible for building up an gradient which contributes to oxygen extraction from the blood. It is important, therefore, to determine adequately mitochondrial function during endotoxic shock and in other pathological states. A major problem is the isolation of mitochondria from tissues, which can be accompanied by a selective loss of a mitochondrial population with potential different size in control and experimental animals. The aim of this study was to check  whether the preparation procedure exerts an influence to mitochondrial functions and morphology. Sprague Dawley rats were treated either with LPS 8 mg/kg (i.v. or i.p.). Untreated rats were used as a control. After 16 hours the livers were collected, homogenized, mitochondria were prepared and used in three experiments. The first  experiment aimed at the comparison of mitochondrial respiratory parameters measured directly in liver homogenates and in isolated mitochondria using high-resolution respirometery (OROBOROS Oxygraph-2k). In these experiments we did not observe any significant difference between those parameters in homogenate and in mitochondria. This indicates that the isolated mitochondria represent the entire mitochondrial pool in liver homogenate. The second experiment aimed at the comparison of mitochondrial morphology in liver tissue and isolated mitochondria by means of electron microscopy. Staining was performed with uranyl acetate and lead citrate. The results showed that the swelling of mitochondria, typically observed in liver slices during endotoxic shock can clearly be seen in isolated mitochondria. In contrast, no swelling was observed in mitochondria isolated from control animals. This demonstrates that the morphological structure of mitochondria was not changed significantly during isolation procedure. The third experiment aimed at the comparison of the quality of mitochondrial preparations from control and LPS treated animals. We performed the analysis of typical respiratory chain markers (cyt b, cyt c1, cyt c and cyt a) in control mitochondria and mitochondria isolated from LPS treated rats by means of optical redox-difference spectroscopy using a dual-wavelength photometer. There was no significant difference between isolated mitochondria from control and LPS treated animals, indicating that there was no group-specific contamination with non-mitochondrial organelles. Together all these data suggest that the isolation of rat liver mitochondria from control and LPS treated rats does not significantly influence mitochondrial function and morphology and represent the whole mitochondrial pool in liver tissue.


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