Apocynin Improves Functional Microvascular Blood Flow in Sickle Cell Disease during Contractile Exercise

Background: Reactive oxygen species are thought to contribute to vascular dysfunction, including vaso-occlusion (VO), in sickle cell disease (SCD). It is unclear whether reduction of oxidative stress either prevents VO or improves microvascular blood flow (MBF) in key tissues. We hypothesized that apocynin, which is known to inhibit NADPH-oxidase, can reverse defects in skeletal muscle MBF at rest or during exercise. To test our hypothesis, quantitative perfusion imaging with contrast enhanced ultrasound (CEU) was performed in Townes SCD mice treated chronically with apocynin.

Methods: Townes SCD mice (S/S), a model of severe disease produced by transfer of human α, γ, and β^s globin genes, and control mice (A/A) were studied under normoxic conditions. On a separate day, mice were studied after exposure to hypoxia for 3 hrs at 8% O₂ with reoxygentation for 1 hr. For each strain, half of the mice were treated with apocynin (50mg/kg/day p.o.) for ≥6 weeks prior to study. Quantitative CEU of the proximal hindlimb adductor skeletal muscle was performed at rest and during contractile exercise produced by electrosimulation at 2 Hz. CEU was performed during a continuous infusion of lipid-shelled decafluorobutane microbubbles and time-intensity data was analyzed after a destructive pulse sequence to assess microvascular blood volume (A-value), microvascular blood flux rate (β), and MBF (Axβ). Hemoglobin levels were drawn prior to CEU studies.

Results: Blood hemoglobin was lower in untreated normoxic SCD than control mice (7.2±1.3 vs. 10.0±3.5 g/dL, p=0.04) and did not change significantly with either apocynin therapy or hypoxic conditions. Under normoxic conditions in untreated mice, SCD compared to control mice had higher resting skeletal muscle MBF (Ab: 5.2±3.4 vs 12.8±9.2 ml/min for A/A vs S/S, p=0.385) and microvascular flux rates (β: 0.10±0.04 vs 0.20±0.05 s^-1, p=0.02), although statistical significance was reached only for the latter. Exercise-mediated flow reserve was greater in A/A than S/S mice (9.3 vs 3.3, p=0.02), although peak hyperemic flow was similar. Apocynin did not alter resting perfusion under normoxic conditions, but did increase exercise MBF in S/S mice (72.16±35.3 vs. 35.72±24.98 ml/min for treated vs untreated; p=0.04). Hypoxic stress did not produce any obvious behavioral signs of systemic VO. Compared to normoxic conditions, hypoxic stress produced an increase in both resting and exercise MBF in all cohorts. Again, apocynin therapy increased exercise MBF in S/S mice after hypoxia, although in contrast to normoxic conditions statistical significance was not reached.

Conclusions: Under resting conditions, skeletal muscle perfusion in Townes SCD mice is increased compared to controls, which likely represents a compensatory response to anemia. This compensatory response is lost during exercise but is restored with chronic apocynin therapy. After hypoxic stress, there is an increase rather than decrease in muscle perfusion in Townes mice with only a minor effect of apocynin on exercise perfusion. These results suggest that anti-oxidant strategies in SCD could help normalize physiologic flow responses to exercise.