Comparison of Carbonyl Levels in Young and Old Rats

Abstract

Reactive oxygen species (ROS) are a natural by-product of aerobic cellular metabolism. They are highly reactive molecules which cause oxidative damage, such as carbonylation, to proteins, lipids and DNA. Because of the high metabolism and energy demand of skeletal muscle, it is especially susceptible to carbonylation. Thus the level of carbonylation can be used as a surrogate of the amount of oxidative damage to the tissue. This research uses fluorescent immuno-labeling to visualize the carbonyls within skeletal muscle cross sections in order to compare the carbonyl levels in young and old skeletal muscles. The comparative carbonyl levels are determined by the relative fluorescent intensity of the tissue. In addition, the carbonyl levels in mitochondria and the rest of the subcellular regions can be determined by observing the carbon-associated fluorescence in regions identified by labeling mitochondria with a separate probe. At the beginning of the project, we found one complication was that rat skeletal muscle tissue have high levels of native fluorescence that interfere with carbonyl detection. In order to eliminate this complication, a method was developed to photo-bleach the native fluorescence prior to labeling the carbonyls. Our preliminary results suggest that there was a difference in the levels of oxidative damage in specific areas of the cell. Preliminary results also indicate that older muscle tissue has accumulated a higher level of carbonylation and therefore more oxidative damage. Since with muscle aging the ability to produce force and muscle mass both decrease, these results suggest a correlation of oxidative damage with muscle changes attributed to the aging process. Greater understanding of aging mechanisms will hopefully give rise to the future development of interventional techniques.