AUTHOR=Aoki Tomoaki , Okuma Yu , Becker Lance B. , Hayashida Kei , Shinozaki Koichiro 

TITLE=Methodological Issue of Mitochondrial Isolation in Acute-Injury Rat Model: Asphyxia Cardiac Arrest and Resuscitation

JOURNAL=Frontiers in Medicine

VOLUME=8

YEAR=2021

URL=https://www.frontiersin.org/journals/medicine/articles/10.3389/fmed.2021.666735

DOI=10.3389/fmed.2021.666735

ISSN=2296-858X

ABSTRACT=<p><bold>Background:</bold> Identification of the mechanisms underlying mitochondrial dysfunction is key to understanding the pathophysiology of acute injuries such as cardiac arrest (CA); however, effective methods for measurement of mitochondrial function associated with mitochondrial isolation have been debated for a long time. This study aimed to evaluate the dysregulation of mitochondrial respiratory function after CA while testing the sampling bias that might be induced by the mitochondrial isolation method.</p><p><bold>Materials and Methods:</bold> Adult rats were subjected to 10-min asphyxia-induced CA. 30 min after resuscitation, the brain and kidney mitochondria from animals in sham and CA groups were isolated (<italic>n</italic> = 8, each). The mitochondrial quantity, expressed as protein concentration (isolation yields), was determined, and the oxygen consumption rates were measured. ADP-dependent (state-3) and ADP-limited (state-4) respiration activities were compared between the groups. Mitochondrial quantity was evaluated based on citrate synthase (CS) activity and cytochrome c concentration, measured independent of the isolation yields.</p><p><bold>Results:</bold> The state-3 respiration activity and isolation yield in the CA group were significantly lower than those in the sham group (brain, <italic>p</italic> &lt; 0.01; kidney, <italic>p</italic> &lt; 0.001). The CS activity was significantly lower in the CA group as compared to that in the sham group (brain, <italic>p</italic> &lt; 0.01; kidney, <italic>p</italic> &lt; 0.01). Cytochrome c levels in the CA group showed a similar trend (brain, <italic>p</italic> = 0.08; kidney, <italic>p</italic> = 0.25).</p><p><bold>Conclusions:</bold> CA decreased mitochondrial respiration activity and the quantity of mitochondria isolated from the tissues. Owing to the nature of fragmented or damaged mitochondrial membranes caused by acute injury, there is a potential loss of disrupted mitochondria. Thus, it is plausible that the mitochondrial function in the acute-injury model may be underestimated as this loss is not considered.</p>