The mouse hind limb ischemia (HLI) model is well established for studying collateral vessel formation and testing therapies for peripheral arterial disease, but there is a lack of quantitative techniques for intravitally analyzing blood vessel structure and function. To address this need, non-invasive, quantitative optical imaging techniques were developed to assess the time-course of recovery in the mouse HLI model. Hyperspectral imaging and optical coherence tomography (OCT) were used to non-invasively image hemoglobin oxygen saturation and microvessel morphology plus blood flow, respectively, in the anesthetized mouse after induction of HLI. Hyperspectral imaging detected significant increases in hemoglobin saturation in the ischemic paw as early as 3 days after femoral artery ligation (P < 0.01), and significant increases in distal blood flow were first detected with OCT 14 days postsurgery (P < 0.01). Intravital OCT images of the adductor muscle vasculature revealed corkscrew collateral vessels characteristic of the arteriogenic response to HLI. The hyperspectral imaging and OCT data significantly correlated with each other and with laser Doppler perfusion imaging (LDPI) and tissue oxygenation sensor data (P < 0.01). However, OCT measurements acquired depth-resolved information and revealed more sustained flow deficits following surgery that may be masked by more superficial measurements (LDPI, hyperspectral imaging). Therefore, intravital OCT may provide a robust biomarker for the late stages of ischemic limb recovery. This work validates non-invasive acquisition of both functional and morphological data with hyperspectral imaging and OCT. Together, these techniques provide cardiovascular researchers an unprecedented and comprehensive view of the temporal dynamics of HLI recovery in living mice.
Keywords: hyperspectral imaging; intravital imaging; ischemia; optical coherence tomography; peripheral arterial disease.