Temporal Assessment of Regional and Remote Non Tumor Microvascular Response to High Dose Radiation Therapy Using Contrast Enhanced Ultrasound

Abstract

High-dose radiation therapy (RT) produces multifactorial microvascular injury in both tumor targets and normal tissue within the RT field. Non-invasive quantification of microvascular perfusion would be useful for understanding protective strategies for reducing normal tissue injury as well assessing for treatment response or resistance. We hypothesized that contrast-enhanced ultrasound (CEU) could characterize alterations in microvascular blood flow (MBF) and functional microvascular blood volume (MBV) that occur in normal tissue after RT. Proximal hind limb skeletal muscle of C57BL/6J mice were irradiated in a single fraction using 6 MV photons, 1 cm bolus, and a dynamic wedge, creating an increasing dose gradient in a rostral-caudal direction. CEU during intravenous infusion of microbubble contrast was performed at day 1 and 8 after RT in muscle exposed to high-dose (HD) RT (15 Gy), an immediately adjacent area of lower-dose (LD) RT (12 Gy), and a region on the unirradiated contralateral limb. Control mice not undergoing any RT were also studied. CEU time-intensity data were analyzed to quantify MBF, microvascular blood transit rate (β), and MBV. Two-sided paired t-tests were used for comparisons with significance placed at p ≤ 0.017 per Bonferroni correction when comparing three groups and p ≤ 0.05 when comparing two groups. On day 1, there was a significant reduction in MBF in the RT versus unirradiated contralateral limbs, the degree of which was dose-dependent (0.51 ± 0.24, 0.66 ± 0.30, and 0.78 ± 0.39 ml/min/g in the HDRT, LDRT, and unirradiated limbs, respectively; HDRT vs. LDRT, p = 0.017; HDRT vs. unirradiated limbs, p = 0.007). The reduction in MBF in RT limbs was attributable primarily to reduction in β. On day 8, MBF in the RT limbs remained reduced to a similar degree (0.42 ± 0.18, and 0.47 ± 0.26 ml/min/g for HDRT and LDRT, respectively), which again was attributable primarily to reduced β. In the unirradiated contralateral limbs, there was a decrease in MBF between day 1 and 8 (0.78 ± 0.39 to 0.48 ± 0.40 ml/min/g) which at day 8 was significantly lower than that seen in untreated mice (0.99 ± 0.69 ml/min/g, p = 0.04). Scatter radiation dose in the unirradiated contralateral limbs was 15 cGy per in vivo dosimetry. CEU perfusion imaging can spatially and temporally quantify dose-dependent radiation-induced microvascular alterations. The perfusion deficit is due largely to reduced microvascular flux rate, suggesting that vasomotor dysregulation plays a greater role than vaso-occlusion or microvascular rarefaction. CEU also has identified a delayed reduction in perfusion at sites distant from local RT suggesting a systemic, or abscopal, microvascular response to high-dose RT that warrants further investigation.