Abstract Body (Do not enter title and authors here): Background: There is growing interest in understanding microbubble (MB) behavior under ultrasound (US) in complex in vivo environments, to optimize safety and efficacy for various clinical applications, e.g., large molecule drug delivery, temporary blood-brain-barrier opening, and thrombus disintegration. However, there have been few mechanistic in vivo studies due to the challenges of visualizing vibrating MBs in the circulation at high temporal resolutions. Our study elucidates MB interactions with surrounding microvessels using ultra-fast intravital imaging of the rat cremaster muscle.
Hypothesis: MB vibration-induced stress deforms surrounding microvessels in vivo. Deformations are influenced by not only the US parameters, but also the biomechanical properties of the vessel wall.
Methods: Definity® was injected via the femoral artery of anesthetized rats and imaged in externalized cremaster muscle using an ultrafast microscope (up to 12 Mfps). Concurrently, a single US pulse of 6-10 cycles [1 MHz, peak negative pressures (PNP) 0.5-2.0 MPa] was delivered. Images were analyzed to calculate MB and vessel dimensions and to perform Fourier analyses. Stress-strain curves were generated using normal stress exerted by the MB (derived from the linearized Euler’s equation) and measured circumferential vessel strain (Diameter/initial Diameter-1).
Results: MBs expanded in both circular and elliptical shapes (Fig 1A), and vibrated nonlinearly under US. Vessels vibrated in-phase with the MBs at higher PNP (Fig 1B). Fourier analyses revealed nonlinear vessel vibration (Fig 1C) at fundamental and harmonic US frequencies. Calculated normal stress exerted by the MB of three MB-vessel pairs displayed linear relationships with measured circumferential vessel strain (Fig 1D), with slopes (representing Young’s modulus, or stiffness) between 0.6-1.7 MPa.
Conclusions: We demonstrate, for the first time, microvascular harmonic behavior in response to MB vibration under US; the associated bioeffects warrant further investigation. Finally, we introduce a novel method for measuring in vivo microvessel stiffness using MBs as mechanical probes, where vessel stiffness is represented by the slope of the presented stress-strain curve.