Intravital Imaging of Neutrophil Recruitment in intestinal Ischemia-Reperfusion Injury in Mice using two-photon laser scanning microscopy

Research Project

Project/Area Number	26461918
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Multi-year Fund
Section	一般
Research Field	General surgery
Research Institution	Kumamoto University
Principal Investigator	Honda Masaki 熊本大学, 医学部附属病院, 非常勤診療医師 (80573609)
Co-Investigator(Kenkyū-buntansha)	武市卒之熊本大学, 医学部附属病院, 非常勤診療医師 (00380999) 猪股裕紀洋熊本大学, 大学院生命科学研究部(医), 教授 (50193628)
Project Period (FY)	2014-04-01 – 2017-03-31
Project Status	Completed (Fiscal Year 2016)
Budget Amount *help	¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000) Fiscal Year 2016: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000) Fiscal Year 2015: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000) Fiscal Year 2014: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Keywords	小腸虚血再灌流障害 / 2光子励起顕微鏡 / 好中球動態 / 生体内イメージング / 微小循環 / 2光子励起レーザー顕微鏡 / 小腸移植 / 小腸虚血再灌流傷害
Outline of Final Research Achievements	Activated neutrophils induce local inflammation and result in pathophysiologic changes during intestinal ischemia-reperfusion (I/R) injury. However, few studies have examined real-time intravital neutrophil recruitment. Here, we show a method for imaging and dynamic change of the neutrophil recruitment in intestinal I/R injury using two-photon laser scanning microscopy (TPLSM). LysM-eGFP mice were subjected to 45 min of warm intestinal ischemia followed by reperfusion. Using time-lapse TPLSM technique, we directly observed the behavior of neutrophils in intestinal I/R injury. At low magnification, the number of neutrophils per field of view continued to increase for 4 hour after reperfusion. High magnification images revealed the presence or absence of blood circulation. TPLSM imaging could reveal the real-time neutrophil recruitment in intestinal I/R injury. Z-stack imaging was useful to evaluate pathophysiological changes of intestinal wall.