Molecular Imaging of Ischemia and Reperfusion in Vivo with Mitochondrial Autofluorescence

Abstract

Ischemia and reperfusion (IR) injury constitutes a pivotal mechanism of tissue damage in pathological conditions such as stroke, myocardial infarction, vascular surgery, and organ transplant. Imaging or monitoring of the change of an organ at a molecular level in real time during IR is essential to improve our understanding of the underlying pathophysiology and to guide therapeutic strategies. Herein, we report molecular imaging of a rat model of hepatic IR with the autofluorescence of mitochondrial flavins. We demonstrate a revelation of the histological characteristics of a liver in vivo with no exogenous stain and show that intravital autofluorescent images exhibited a distinctive spatiotemporal variation during IR. The autofluorescence decayed rapidly from the baseline immediately after 20-min ischemia (approximately 30% decrease in 5 min) but recovered gradually during reperfusion (to approximately 99% of the baseline 9 min after the onset of reperfusion). The autofluorescent images acquired during reperfusion correlated strongly with the reperfused blood flow. We show further that the autofluorescence was produced predominantly from mitochondria, and the distinctive autofluorescent variation during IR was mechanically linked to the altered balance between the flavins in the oxidized and reduced forms residing in the mitochondrial electron-transport chain. Our approach opens an unprecedented route to interrogate the deoxygenation and reoxygenation of mitochondria, the machinery central to the pathophysiology of IR injury, with great molecular specificity and spatiotemporal resolution and can be prospectively translated into a medical device capable of molecular imaging. We envisage that the realization thereof should shed new light on clinical diagnostics and therapeutic interventions targeting IR injuries of not only the liver but also other vital organs including the brain and heart.

Autofluorescence‐based optical biopsy: An effective diagnostic tool in hepatology

“…NAD(P)H and flavins have been confirmed as AF biomarkers of organ functionality, suitable for application during surgery or transplantation . Notably, both AF‐based spectroscopy and imaging have allowed real‐time, in situ monitoring of transient events (Figure ), otherwise undetectable or requiring the collection of sequential samples from far more numerous organs to perform ex vivo biochemical and histological analyses. Additionally, cell energy metabolism and redox balance could be upset during specimen collection, at least as an immediate consequence of the oxygen supply interruption .…”

“…Intravital AF images of rat liver tissue, showing mitochondrial flavin AF , along with the temporal variation in emission intensity during ischaemia and reperfusion. Reproduced with permission, from Lu et al (2014)…”

Understanding liver immunology using intravital microscopy

“…Interestingly, even without employing exogenous fluorescent agents, it is possible to assess hepatocyte metabolic status in vivo by visualizing mitochondria autofluorescence (λ excitation = 445 nm and λ emission = 458−630 nm), which strongly correlates with tissue perfusion status [43]. In fact, autofluorescence recovery strongly correlated with the restoration of microcirculatory blood flow during reperfusion.…”

Label-free microscopy and stress responses reveal the functional organization of Pseudodiaptomus marinus copepod myofibrils

“…The cellular autofluorescence of flavins is a sensitive indicator of their oxygenation, decreasing when cells are placed in an hypoxic medium and restoring gradually to the base line after the cells were perfused with a normoxic medium (Lu et al, 2014). In copepods, the absolute levels of the flavin TPEF signals cannot provide information about muscle oxygenation after the different stresses applied because the absorbance of the cuticle which affects these levels can be temperature or ionic strength dependent.…”

“…The mean cellular NAD(P)H fluorescence level in Xenopus skeletal muscle fibers depends upon oxygen availability (Hogan et al, 2005). Flavins that switch between the fluorescent oxidized form and the nonfluorescent reduced form allow imaging the deoxygenation and reoxygenation of mitochondria during ischemia reperfusion in mouse hepatocytes (Lu et al, 2014). The distribution of reduced NAD(P)H fluorescence in mouse skeletal muscle reveals the existence of intersarcomeric populations of mitochondria (Rothstein et al, 2005).…”