Miyake, T., Yokoyama, Y., Kokuryo, T., Mizutani, T., Imamura, A., & Nagino, M
Division of Surgical Oncology, Department of Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan, Inter Medical Co, Ltd, Nagoya, Japan.
General Fluorescence Microscopy
The aim of this study was to determine the intrahepatic kinetics of different types of nitric oxide (NO) synthase, such as endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS), during repeated ischemia/reperfusion (I/R). Three different protocols of hepatic I/R in rats were designed as follows: 60 min of ischemia and 30 min of reperfusion (I/R 60/30); 5 min of ischemia and 5 min of reperfusion (I/R 5/5); and repeating 15 min of ischemia and 5 min of reperfusion for four cycles (I/R 15/5 × 4). Intrahepatic NO levels were measured using a selective NO sensor. Changes in hepatic microcirculation during I/R 5/5 were investigated using intravital microscopy. Hepatic expression of eNOS, phospho-eNOS, and iNOS were evaluated during repeated I/R by Western blot, reverse transcription polymerase chain reaction, and immunohistochemistry. During I/R 60/30, intrahepatic NO levels gradually increased and then reached a plateau approximately 15 min after starting ischemia. During I/R 5/5, the sinusoids after 5 min reperfusion were dilated compared with the sinusoids before ischemia. The expression of phospho-eNOS during I/R 15/5 × 4 markedly increased during the first ischemia, and then the levels attenuated during the subsequent repeating I/R cycles; however, the expression of iNOS gradually increased, as observed by Western blot, reverse transcription polymerase chain reaction, and immunohistochemical analysis. An impact of NO production by phospho-eNOS activation during the superacute phase of I/R was also confirmed using pharmacologic NOS inhibitors. Our results firstly demonstrated an altered activation of the phospho-eNOS system and iNOS over the course of repeated hepatic I/R.
… “The liver surface was epi-illuminated with an LED excitation system (CoolLED pE; CoolLED Ltd, Andover, UK) with”…
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The Journal of Surgical Research
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