Background: Evidence is accumulating that formyl-peptide receptor (FPR) signaling constitutes an important danger signal that guide neutrophils to sites of inflammation. This study aimed to investigate in vivo real-time neutrophil recruitment using two-photon laser-scanning microscopy (TPLSM) in response to FPR1 blockade during hepatic ischemia-reperfusion (I/R) injury.
Methods: Male LysM-eGFP mice (neutrophil: eGFPhi) were subjected to 60 min of 70% partial warm hepatic I/R. They were pretreated with 50 Μg FPR1 antagonist cyclosporine H (CsH) or vehicle 30 min before ischemia. Mice received intravenous injection of TRITC-albumin just prior to imaging for visualizing the microvasculature. Left lateral lobe of the liver was imaged 6 h after I/R using an upright microscope (BX61; Olympus) and an FV1000MPE laser-scanning microscope system. In each group (n=5/group), 10-minute videos were recorded at low (×10) or high (×25) magnification. For in vitro study, blood and liver samples were collected 6 h after reperfusion.
Results: At low magnification, 4-6 hepatic lobules were visualized. The number of adherent neutrophils per field of view in CsH-treated I/R group was lower than that in non-treated I/R group (321.4 ± 40.9 vs. 630.8 ± 106.3, p<0.05). Time-lapse imaging revealed the reduction of their crawling velocity in CsH-treated group (3.84 ± 0.17 vs. 4.74 ± 0.20 Μm/min, p<0.001). High magnification images could reveal patchy distribution of non-perfused (necrotic) areas. In CsH-treated group, the number and crawling velocity of neutrophil in non-perfused area were significantly lower compared to non-treated group (Figure 1). CsH treatment decreased sALT levels, as compared with no treatment (9,434 ± 2,856 vs. 16,902 ± 2,595 U/L, p<0.05). These findings were correlated with histological damage of the liver.
Conclusions: TPLSM was a powerful tool for investigating the dynamic mechanism of hepatic I/R injury from the biological aspect. Using TPLSM, it was revealed that blockade of FPR1 signaling attenuated hepatic I/R injury by inhibiting neutrophil recruitment, in particular, in non-perfused area.
To cite this abstract in AMA style:Honda M, Takeichi T, Asonuma K, Inomata Y. Intravital Two-Photon Imaging of Neutrophil Recruitment Reveals an Efficacy of FPR1 Blockade To Attenuate the Hepatic Ischemia-Reperfusion Injury [abstract]. Am J Transplant. 2013; 13 (suppl 5). https://atcmeetingabstracts.com/abstract/intravital-two-photon-imaging-of-neutrophil-recruitment-reveals-an-efficacy-of-fpr1-blockade-to-attenuate-the-hepatic-ischemia-reperfusion-injury/. Accessed May 7, 2021.
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