Contrast-Enhanced Ultrasound to Detect Early Microvascular Changes in Skeletal Muscle after High-Dose Radiation Treatment

The biological response of normal tissue to high-dose radiation treatment remains poorly understood. Alterations to the microenvironment, specifically the microvasculature, have been implicated as a significant contributor to tumoral cytotoxicity. We used contrast-enhanced ultrasound (CEU) perfusion imaging, which is uniquely suited to assess functional status of the microcirculation, to measure microvascular blood flow after high-dose irradiation to normal skeletal muscle tissue in a murine model. Proximal hindlimbs of wild-type C57Bl/6 mice were irradiated with a single fraction using 6 MV photons, 1 cm bolus and a dynamic wedge. Quantitative perfusion CEU imaging of the skeletal muscle was performed at days 1 and 8 postirradiation in three different regions of interest (ROIs): 1. 15 Gy external-beam irradiated leg; 2. 12 Gy irradiated 5 mm proximal area; 3. single ROI in the nonirradiated contralateral (CL) hindlimb. Perfusion imaging was also performed in the hindlimb of nonirradiated mice. CEU time-intensity data were analyzed to measure microvascular blood flow (MBF, also referred to as perfusion), and its parametric components of microvascular flux rate and functional microvascular blood volume (MBV). Plasma measurements of two potent vasoconstrictors, endothelin-1 and angiotensin II, were also performed to assess systemic response. CEU perfusion imaging values for the 12 and 15 Gy irradiated limb regions were pooled. At day 1, MBF in the irradiated limb was significantly lower than in the CL limb (P = 0.016) but quite similar to the nonirradiated mice. At day 8, both limbs of irradiated mice exhibited a trend towards lower MBF than the limbs of nonirradiated mice (28% decrease in mean MBF, P = 0.149 for CL; 39% decrease, P = 0.065 for irradiated limb). Compared to nonirradiated animals, the reduction in perfusion in irradiated limbs at day 8 may have been more influenced by the microvascular flux rate (25% decrease in the mean, P = 0.079) than the MBV (12% decrease in the mean, P = 0.328). Examination of vasoactive compounds revealed that the average plasma concentration for endothelin-1 at day 8 postirradiation was significantly higher in 14 irradiated animals than in 4 nonirradiated animals (3.07 pg/ ml vs. 2.51 pg/ml; P = 0.011). Up to day 8 after high-dose irradiation, flow deficits in irradiated muscle appear to be a consequence of increased vascular resistance more so than loss or functional de-recruitment of microvascular units.