Background
Microvascular dysregulation, abnormal rheology, and vaso-occlusive events play a role in the pathophysiology of sickle cell disease (SCD). The aim of this study was to test the hypothesis that abnormalities in skeletal muscle perfusion in a murine model of SCD could be parametrically assessed by quantitative contrast-enhanced ultrasound perfusion imaging.
Methods
A murine model of moderate SCD without anemia produced by homozygous β-globin deletion replaced by human βs-globin transgene (NY1DD−/−; n = 18), heterozygous transgene replacement (NY1DD+/−; n = 19), and C57Bl/6 control mice ( n = 14) was studied. Quantitative contrast-enhanced ultrasound of the proximal hindlimb skeletal muscle was performed at rest and during contractile exercise (2 Hz). Time-intensity data were analyzed to measure microvascular blood volume (MBV), microvascular blood transit rate (β), and microvascular blood flow. Erythrocyte deformability was measured by elongation at various rotational shears.
Results
At rest, muscle MBV was similar between strains, whereas β was significantly ( P = .0015, analysis of variance) reduced to a similar degree in NY1DD−/− and NY1DD+/− compared with wild-type mice (0.24 ± 0.10, 0.16 ± 0.07, and 0.34 ± 0.14 sec −1 , respectively), resulting in a reduction in microvascular blood flow. During contractile exercise, there were no groupwise differences in β (1.43 ± 0.67, 1.09 ± 0.42, and 1.36 ± 0.49 sec −1 for NY1DD−/−, NY1DD+/−, and wild-type mice, respectively) or in microvascular blood flow or MBV. Erythrocyte deformability at high shear stress (≥5 Pa) was mildly reduced in both transgenic groups, although it was not correlated with blood flow or β.
Conclusions
Contrast-enhanced ultrasound in skeletal muscle revealed a lower microvascular blood transit rate in the NY1DD model of SCD and sickle trait but no alterations in MBV. The abnormality in microvascular blood transit rate was likely due to vasomotor dysfunction, because it was abrogated by contractile exercise and at rest was only weakly related to erythrocyte deformability.
Sickle cell disease (SCD) is a multisystem disorder caused by a single-nucleotide mutation at the sixth position of the red blood cell β-globin gene that substitutes valine for glutamic acid. The pathophysiology of SCD is complex and involves much more than simply the hemolytic anemia produced by hemoglobin polymerization. Dysregulation of microvascular perfusion is thought to play a key role in the myriad SCD complications, such as acute pain crisis, pulmonary hypertension, renal failure, priapism, and cognitive impairment. Abnormal rheology of sickled erythrocytes contributes to vascular-related complications. However, other mechanisms have been implicated, including vaso-occlusive microthrombosis. Abnormal vasomotor tone also occurs as a result of free hemoglobin, which scavenges nitric oxide (NO), release of vasoconstrictors from platelet activation, and oxidative stress, which can inactivate NO and promote smooth muscle constriction.
Noninvasive techniques that are capable of comprehensively assessing the status of the microcirculation are likely to yield additional insight into the vascular pathophysiology of SCD and could be used for preclinical and clinical testing of new therapies. Contrast-enhanced ultrasound (CEU) has been used extensively in humans and animal models to evaluate microvascular responses in various disease processes. A distinct advantage of CEU microvascular perfusion imaging in SCD is that it provides quantitative parametric information on both erythrocyte microvascular transit rate, which is influenced by blood rheology and resistance arteriolar tone, and microvascular blood volume (MBV), which may become abnormal by vaso-occlusion or increased precapillary terminal arteriolar tone. In this study, we examined the hypothesis that quantitative CEU perfusion imaging could detect skeletal muscle perfusion abnormalities and characterize abnormalities in red blood cell microvascular transit rate and MBV in a genetically modified murine model of moderate SCD under healthy noncrisis conditions.
Methods
Murine Model and Surgical Preparation
Studies were approved by the Animal Care and Use Committee at Oregon Health & Science University. As a model for SCD, NY1DD−/− mice (Jackson Laboratories, Bar Harbor, ME) were studied. These mice have a C57/BL6 background, are homozygous for a spontaneous deletion at the β-major-globin locus (βMDD), and carry human α- and βS-globin transgenes (αHβS[βMDD]). Mice that are heterozygous for the β-major-globin deletion (NY1DD+/−) were used as a model for sickle trait, and C57/BL6 mice were used as wild-type controls. Mice were studied at 8 to 14 weeks of age (NY1DD−/−, n = 18; NY1DD+/−, n = 19; wild-type, n = 10). Animals were anesthetized with inhaled isoflurane (1.0%–1.2%) mixed with room air and were kept euthermic with a heating pad. A jugular vein was cannulated for administration of microbubble contrast agents. All studies in SCD mice were performed on animals that were in apparent good health, without any behavioral evidence of pain or distress.
Microbubble Preparation
Lipid-shelled microbubbles were prepared by sonication of a decafluorobutane gas–saturated aqueous suspension of 2 mg/mL distearoylphosphatidylcholine and 1 mg/mL polyoxyethylene-40-stearate. Microbubble size distribution and concentration were measured by electrozone sensing (Multisizer III, Beckman Coulter).
CEU Perfusion Imaging
Contrast ultrasound perfusion imaging was performed using a linear-array transducer at a centerline frequency of 7 MHz (Sequoia 512; Siemens Medical Systems, Erlangen, Germany). The nonlinear fundamental signal component for microbubbles was detected using multipulse phase and amplitude modulation at a mechanical index of 0.18 and a dynamic range of 55 dB. Gain settings were optimized and held constant. Blood pool signal ( I B ) was measured from a region of interest placed in the left ventricular (LV) cavity at end-diastole during a microbubble intravenous infusion rate of 5 × 10 5 min −1 . The infusion rate was then increased to 1 × 10 7 min −1 , and the proximal hindlimb adductor muscles were imaged in a transverse plane halfway between the inguinal fold and the knee. Images were acquired at a frame rate of 20 Hz after a brief high-power (mechanical index, 1.0–1.1) destructive pulse sequence. Background-subtracted video intensity was calculated by digital subtraction of the first frame acquired immediately after the high-power sequence. Time-intensity data were fit to the function
y = A ( 1 – e – β t ) ,
( A ) / ( 1 . 06 × I B × F × 0 . 9 ) ,
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