title: Intravital imaging of neutrophil recruitment in hepatic ischemia-reperfusion injury in mice.
abstract: BACKGROUND
  Neutrophils are considered responsible for the pathophysiologic changes during hepatic ischemia-reperfusion (I/R) injury; however, few studies have examined real-time intravital neutrophil recruitment. Here, we show a method for imaging the neutrophil recruitment in hepatic I/R injury using two-photon laser scanning microscopy (TPLSM).
  
  
  METHODS
  LysM-eGFP mice were subjected to 45 min of partial warm hepatic ischemia followed by reperfusion. Mice received an intravenous injection of tetramethylrhodamine isothiocyanate-labeled albumin to visualize the microvasculature. Using time-lapse TPLSM technique, we directly observed the behavior of neutrophils in I/R injury.
  
  
  RESULTS
  At low magnification, four to six hepatic lobules could be visualized. The number of adherent neutrophils continued to increase for 4 hr after reperfusion, whereas their crawling velocity reached a maximum of 2 hr after reperfusion and then decreased gradually. High-magnification images revealed the presence or absence of blood circulation in sinusoids. Six hours after control operation or reperfusion, circulation was maintained in all sinusoids in the control group, whereas spotty nonperfused areas accompanied by neutrophil infiltration could be observed in the I/R group. Adherent neutrophils in perfused areas in the I/R group had more elongated shapes and moved more quickly than those in nonperfused areas and in the control group. Some hepatocytes affected by I/R injury showed the changes in their size and fluorescent intensity, which could attract neutrophils.
  
  
  CONCLUSIONS
  TPLSM was successfully used for intravital imaging of hepatic I/R injury in mice and has potential for a wide range of applications to investigate the mechanism of I/R injury.
authors: Department of Transplantation and Pediatric Surgery, Postgraduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan.
authors: Masaki
authors: M
authors: Honda
authors: Takayuki
authors: T
authors: Takeichi
authors: Katsuhiro
authors: K
authors: Asonuma
authors: Koji
authors: K
authors: Tanaka
authors: Masato
authors: M
authors: Kusunoki
authors: Yukihiro
authors: Y
authors: Inomata
mesh_terms: D000818:Animals; D016923:Cell Death; D054360:Chemokine CXCL1 / Q000378:metabolism; D054426:Chemokine CXCL2 / Q000378:metabolism; D004195:Disease Models, Animal; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D007113:Immunity, Innate; D008099:Liver / Q000098:blood supply* / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D036641:Microscopy, Fluorescence, Multiphoton; D009113:Muramidase / Q000235:genetics; D020556:Neutrophil Infiltration; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic; D015427:Reperfusion Injury / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D012235:Rhodamines; D013997:Time Factors; D052096:Warm Ischemia
chemical_list: D054360:Chemokine CXCL1; D054426:Chemokine CXCL2; C475895:Cxcl1 protein, mouse; C404915:Cxcl2 protein, mouse; D005456:Fluorescent Dyes; D012235:Rhodamines; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C009434:tetramethylrhodamine isothiocyanate; D009113:Muramidase