Following ischemia reperfusion injury, inflamed macrophages switch from oxidative phosphorylation to glycolysis; in contrast, macrophages associated with tissue repair rely on oxidative phosphorylation. The triggers inducing macrophage reparative behavior following inflammation are not well understood, but may rely on available cellular energy. Based on our previous work demonstrating laser enhanced mitochondrial activity and reduced apoptosis in injured kidneys, we hypothesized that low level light laser energy may speed macrophage inflammatory activity with more rapid resolution of inflammation. To observe macrophage behavioral dynamics, we used optically transparent zebrafish, which expressed the mpeg1 transgene, a macrophage marker. Zebrafish were exposed to 35 [mu]M dihydroethidium, a red fluorescent superoxide indicator and imaged live via confocal microscopy to quantify ROS pixel intensity by time-lapsed imaging, post- tail injury. Macrophages expressed the green fluorescent protein dendra2 and were individually tracked via a keyhole tracking algorithm performed on MATLAB. When compared to untreated zebrafish (n=6), laser treated (n=8, 635nm,3 J/cm²), demonstrated significantly decreased ROS intensity at 60 (T1), (8,688±1461 vs 10,392±1040,p=0.04 ) 90,(T2, 8490±1300 vs 10,311±976,p=0.02), 120(T3, 8,397±1264 vs 10,323±833,p=0.01) and 150 minutes (T4, 8,3998±1255 vs 10,301±1000,p=0.01). There was a trend for laser- induced increased macrophage velocity(T4, 5.6±0.7 vs 5.2±0.7 [mu]m/min)and more meandering behavior, 12.77±9 vs 6.3±1.8 segments, p=0.15. Macrophages of laser treated zebrafish were more rapidly recruited to the wound at every timepoint studied: T1 (6 ±1 vs 4±3, p=0.006), T2 (9±2 vs 6±3, p=0.03), T3 ( 14±3 vs 9±5, p=0.03), T4 (17±4 vs 12±6, p=0.05) and subsequently left the wound 5 times more frequently, p=0.04. These data show laser light energy speed macrophage entry and exit of injured tissues, reducing ROS, and may provide a non-invasive strategy to mitigate tissue injury following ischemia reperfusion.

CITATION INFORMATION: Paredes A, Benavidez D, Patil R, Khalil B, Mitchell M, Cheng J, Mangos S, Bartholomew A. Low Level Light Laser Treatment Reduces Macrophage Retention and ROS Levels Following Tissue Injury. Am J Transplant. 2017;17 (suppl 3).

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