Model of Ischemia/Reperfusion Injury in Rat Brain Isolated Mitochondria
Authors: Pille Johannes A. , Salzman Michele M. , Sonju Anna A. , Lotze Felicia P. , Hees Josephine E. , Cleveland William J. , Balzer Claudius , Riess Matthias L. 
Published: June 2021

INTRODUCTION

When ischemia occurs, reperfusion is crucial for the survival of the affected tissue. However, the combination of ischemia and reperfusion can cause decreased pH, increased Ca2+, as well as formation of reactive oxygen species (ROS) [e.g., hydrogen peroxide (H2O2)] in the cell, affecting in turn mitochondrial function (e.g., impaired electron transport, ATP synthesis, oxygen consumption). Several of our postconditioning strategies have been found to attenuate ischemia/reperfusion (IR) injury, and to specifically improve mitochondrial function. Yet, it is unclear if the improved mitochondrial function is due to a direct postconditioning effect on mitochondria, or if the improved mitochondrial function is a result of a generally improved cell metabolism. Therefore, to simulate a component of the metabolic stress, namely ROS, occurring to mitochondria during cerebral IR injury, this study focused on injuring rat brain isolated mitochondria by exposing them to H2O2.

METHODS

Male Sprague‐Dawley rats were euthanized, and their brains removed. Mitochondria were isolated by differential centrifugation and Percoll gradients, then kept on ice to slow their bioenergetics prior to any experimental treatments. To simulate exposure to ROS during ischemia, mitochondria were treated with 200μM H2O2 for 10 min at room temperature (~23°C); controls were kept at room temperature for the same duration. The samples were then diluted ½ with experimental buffer to simulate reperfusion and placed back on ice. ATP synthesis was then measured in a luminometer using a firefly luciferase enzymatic assay. Oxygen consumption was measured by closed cell respirometry with an oxygen meter. In both assays, Complex I and Complex II were examined; Complex I with the substrates glutamate and malate, Complex II with the substrate succinate and the Complex I inhibitor rotenone. Statistics: Data are expressed as mean ± standard error of the mean (SEM). Unpaired 2‐tailed Student's t‐test, α=0.05, * vs control.

RESULTS

H2O2 treatment significantly impaired mitochondrial ATP synthesis and oxygen consumption for Complex I and oxygen consumption for Complex II (table). However, for Complex II, ATP synthesis did not show a significant difference.

CONCLUSIONS

This work shows that the effect of increased ROS (i.e., H2O2) on mitochondrial function, which occurs during IR injury, can be effectively simulated by this model. Future work will utilize this model to test whether our post conditioning strategies work directly on mitochondria during IR injury.

Support or Funding Information

This work was supported by institutional funds, NIH grant (5R01 HL123227), and a Merit Review Award (I01 BX003482) from the U.S. Department of Veterans Affairs Biomedical Laboratory R&D Service.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.