Non-Invasive Contrast Ultrasound Imaging Of Abnormal Microvascular Perfusion and Reduced Functional Blood Volume In Sickle Cell Disease

Vaso-occlusive events play a role in the pathophysiology of sickle cell disease (SCD) and are a potential target for new therapies. There is strong evidence that platelet and endothelial activation as well as microvascular dysfunction contribute to this process. Techniques capable of spatially and temporally assessing basal or provoked tissue hypoperfusion in animal models or in patients with SCD would be valuable for evaluating new therapies. Contrast enhanced ultrasound (CEU) is a non-invasive imaging technique that relies on the ultrasound detection of microbubble contrast agents during their microvascular transit. In this study we hypothesized that CEU perfusion imaging would provide a biologic readout for abnormalities in skeletal muscle perfusion in a murine model of SCD.

NY1DD mice with either homozygous (n=16) or heterozygous (n=18) gene deletion of murine β globin that also transgenically express human α and β^sglobin were studied at 8-10 weeks of age, while C57/BL6 mice (n=7) were used as controls. Animals were studied under basal conditions only if they appeared healthy and not in distress or pain crisis equivalent. Mice were anesthetized with inhaled isoflurane and a jugular vein was cannulated for contrast administration. CEU perfusion imaging of the proximal hindlimb adductor muscles and the myocardium was performed using continuous intravenous infusions of lipid-shelled decafluorobutane microbubbles. Time-activity curves from real-time imaging following a disruptive high power ultrasound pulse sequence was used to quantify both microvascular blood flow (MBF) and its parametric components: functional microvascular blood volume (MBV) and flux rate (β). Skeletal muscle CEU was performed at rest and during electrostimulated contractile exercise at 1 Hz. Spatial analysis of perfusion was done to assess average percent area of perfusion per areas of tissue. High-frequency echocardiography was performed to evaluate LV dimensions and stroke volume.

Skeletal muscle MBF was significantly lower in NY1DD mice compared to controls, the extent of which was similar for the two NY1DD cohorts (MBF= 1.11±0.84, 0.58±0.77, 0.48±0.35 for control, homozygous, and heterozygous NY1DD mice, respectively; Kruskall-Wallis p<0.05). Microbubble microvascular flux rate was similarly reduced by approximately half in both NY1DD groups compared to controls (p<0.01). Skeletal muscle MBV was reduced only in the homozygous NY1DD mice (approximately 70% the volume of controls). Spatial analysis of MBV revealed coarse rather than diffuse spatial hypoperfusion in the homozygous NY1DD mice, indicating lack of perfusion downstream from large intramuscular vascular units. Electrostimulated exercise produced a significant increase in skeletal muscle MBF in all groups, although hyperemic blood flow remained 60-70% lower than controls for both NY1DD groups. In contrast to skeletal muscle perfusion, myocardial MBF was increased in NY1DD mice, largely due to increased flux rate. Echocardiography demonstrated a similar stroke volume and cardiac output between animals.

CEU perfusion imaging can be used to non-invasively detect microvascular abnormalities in murine models of SCD. Even under basal resting conditions, there is a reduction in skeletal muscle MBF. The reduction in microvascular flux rate under basal conditions and presence of MBF reserve in mice with SCD strongly suggest that abnormalities in MBF at rest were to a large extent functional in nature rather than obstructive. From spatial analysis, this appears to involve medium to large intravascular units. CEU will likely provide important insight into the pathophysiology of worsening vaso-occlusive events during crisis and may also potentially be used to assess the efficacy of new treatments for SCD.

No relevant conflicts of interest to declare.