Abstract 19703: Real-Time Microscopic Assessment of Ischemia-Reperfusion with Intravital Autofluorescence Imaging
Background: Real-time microscopic characterization of ischemia and reperfusion (IR) in vivo is essential to improve our understanding of the underlying pathogenesis of IR injury, but histopathological examination post reperfusion remains a common means to assess IR. The mitochondrial flavoprotein switches between non-fluorescent and fluorescent state as it undergoes redox change. We herein sought to visualize IR microscopically in a real-time and label-free manner in vivo based on the autofluorescence of this intrinsic indicator.
Methods: Intravital autofluorescence microscopy was employed to image hepatic IR in vivo on a rat model. Time-lapse autofluorescence images of the liver tissue and time-varying intensity of the autofluorescence were recorded continuously on liver subject to short and long durations of ischemia. FITC-dextran was injected intravenously to highlight sinusoids with circulating blood.
Results: The autofluorescence decreased by ~30% immediately after ischemia, but restored gradually after reperfusion. The recovery rate depended strongly on the duration of ischemia: the intensity restored to the baseline within 10 min for 20-min ischemia whereas the recovery was significantly slower (> 30 min) for 60-min ischemic time. Results of complementary experiments on cultured hepatocytes in vitro including (1) emission spectral measurements, (2) autofluorescence imaging on cells undergoing simulated IR, and (3) inhibitory assay all support strongly that the autofluorescence change observed during IR is associated with hypoxia-reoxygneartion of tissue. The intravital imaging further revealed that the retarded autofluorescence recovery is associated with delayed reoxygenation that was caused by impaired microcirculation.
Conclusions: Our work demonstrates that the autofluorescence of mitochondrial flavoprotein is a sensitive endogenous indicator that enables visualization of the hypoxia-reoxygenation of tissues associated with IR at the microscopic level in real time. This approach should be extendible to investigate IR of other vital organs.
- © 2012 by American Heart Association, Inc.