Protection of Microvascular Liver Perfusion with 5-Hydroxymethylfurfural in Sickle Cell Disease Mice Following Hypoxia-Induced Vasoocclusion

Introduction: Sickle cell disease (SCD) is a debilitating genetic disorder, and the resultant "sickling" deformity of red blood cells leads to acute vasoocclusive (VOC) events and chronic disease in multiple organs. Sickle cell hepatopathy, arising from VOC events in the hepatic sinusoids, which can lead to fatal sickle cell intrahepatic cholestasis, is estimated to occur in approximately 10% of the SCD population. Using contrast-enhanced ultrasound (CEUS), we measured microvascular liver perfusion (MVLP) in SCD and control mice at basal levels to determine if noninvasive CEUS can be utilized to assess the underlying extent of disease and also investigated the effect of hypoxia-induced VOC with and without treatment with an antisickling agent, 5-hyroxymethylfurfural (5-HMF).

Methods: Townes sickle cell mice (SCD), homozygous for hα:β^s -globulin, approximately 7−9 weeks of age (n=28), and control mice, homozygous for hα:β-globulin (n=11), were used in these studies. CEUS perfusion imaging (Vevo^® 2100) was performed on a central cross-section of the liver at the renal artery. Contrast agent was administered as an intravenous bolus via tail vein to anesthetized mice (isoflurane with ~21% O₂); peak enhancement (PE) was analyzed with VisualSonics software. CEUS measurements were obtained at baseline and following either (1) hypoxia, 60 minutes with 5.5.% O₂ followed by ~60 minutes of reoxygenation at room oxygen (~21% O₂) or (2) normoxia, ~120 minutes at room oxygen. 5-HMF at 20 and 200 mg/kg PO or vehicle was administered following baseline PE measurement and approximately 30 minutes before start of hypoxia.

Results: MVLP in SCD (n=28) was significantly reduced by approximately 40% compared with controls (n=11) at baseline (PE of 14.0±0.7 linear arbitrary units [l.a.u.] vs. 23.6±2.1 l.a.u.), respectively, P<0.001 [Student t test]). Normoxic SCD maintained similar PE to baseline levels (Table 1); however, hypoxia significantly reduced MVLP by 49% in SCD mice. In contrast, hypoxia had no significant effect in control mice. 5-HMF at 20 and 200 mg/kg resulted in a dose-dependent increase in posthypoxia MVLP. 5-HMF at 200 mg/kg was not significantly different from baseline PE, and 5-HMF at 20 mg/kg increased MVLP by approximately 50% compared with the vehicle group posthypoxia (26% vs 49% reduction in MVLP, respectively). Pathologic evaluation of naive SCD formalin-fixed liver tissues (n=10) showed congestion, necrosis, hepatocellular hypertrophy, and extramedullary hematopoiesis.

Summary: CEUS measured lower basal levels of MVLP in SCD compared with control mice, which correlated with pathologic findings of congestion and necrosis in the livers of SCD mice. The hypoxia-induced VOC decrease in MVLP was present only in the SCD mice; no effect was observed in control mice. Treatment with the antisickling agent, 5-HMF, dose-dependently ameliorated the hypoxia-induced VOC decrease in MVLP in SCD mice. Based on these results, CEUS may be considered as a noninvasive method to measure acute and chronic organ perfusion changes for evaluating new therapeutics for sickle cell-mediated VOC events and end-organ damage.