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Detection of Mitochondrial Reactive Oxygen Species in Living Rat Trigeminal Caudal Neurons

  • Lee, Hae In (Dept. of Oral Physiology, College of Dentistry, Institute of Wonkwang Biomaterial and Implant, Wonkwang University) ;
  • Chun, Sang Woo (Dept. of Oral Physiology, College of Dentistry, Institute of Wonkwang Biomaterial and Implant, Wonkwang University)
  • Received : 2015.05.29
  • Accepted : 2015.06.15
  • Published : 2015.06.30

Abstract

Growing evidence suggests that mitochondrial reactive oxygen species (ROS) are involved in various pain states. This study was performed to investigate whether ROS-induced changes in neuronal excitability in trigeminal subnucleus caudalis are related to ROS generation in mitochondria. Confocal scanning laser microscopy was used to measure ROS-induced fluorescence intensity in live rat trigeminal caudalis slices. The ROS level increased during the perfusion of malate, a mitochondrial substrate, after loading of 2',7'-dichlorofluorescin diacetate ($H_2DCF-DA$), an indicator of the intracellular ROS; the ROS level recovered to the control condition after washout. When pre-treated with phenyl N-tert-butylnitrone (PBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidene-1-oxyl (TEMPOL), malate-induced increase of ROS level was suppressed. To identify the direct relation between elevated ROS levels and mitochondria, we applied the malate after double-loading of $H_2DCF-DA$ and chloromethyl-X-rosamine (CMXRos; MitoTracker Red), which is a mitochondria-specific fluorescent probe. As a result, increase of both intracellular ROS and mitochondrial ROS were observed simultaneously. This study demonstrated that elevated ROS in trigeminal subnucleus caudalis neuron can be induced through mitochondrial-ROS pathway, primarily by the leakage of ROS from the mitochondrial electron transport chain.

Acknowledgement

Supported by : 원광대학교

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