Ultrasound-Targeted Microbubble Cavitation with Sodium Nitrite Synergistically Enhances Nitric Oxide Production and Microvascular Perfusion

Abstract

Microvascular obstruction is a common repercussion of percutaneous coronary intervention for distal microembolization, ischemia-reperfusion injury and inflammation, which increases post-myocardial infarction heart failure and mortality. Ultrasound-targeted microbubble cavitation (UTMC) may resolve microvascular obstruction while activating endothelial nitric oxide synthase (eNOS) and increasing endothelium-derived nitric oxide (NO) bioavailability. Nitrite, a cardioprotective agent, offers an additional source of NO and potential synergy with UTMC. UTMC and nitrite co-therapy increased microvascular perfusion and NO concentration in a rat hindlimb model. Using N-nitro-L-arginine methyl ester for eNOS blockade, we found a three-way interaction effect between nitrite, UTMC and eNOS on microvascular perfusion and NO production. Modulating ultrasound peak negative acoustic pressure (0.33-1.5 MPa) significantly affected outcomes, while microbubble dosage (2 × 10⁸ bubbles/mL, 1.5 mL/h to 1 × 10⁹ bubbles/mL, 3 mL/h) did not. Nitrite co-therapy also protected against oxidative stress. Comparison of nitrite to sodium nitroprusside with UTMC revealed synergistic effects were specific to nitrite. Synergy between UTMC and nitrite holds therapeutic potential for cardiovascular disease.

Keywords: Microbubbles; Microvascular; Nitric oxide; Nitrite; Perfusion; Ultrasound.

Figure 1.. Effect of ultrasound-targeted microbubble cavitation (UTMC) nitrite combination therapy on microvascular blood volume:

Contrast-enhanced still-frame ultrasound images of the treated hindlimb gastrocnemius region are shown for all groups after UTMC (2×10⁸ MB/mL at 1.5 mL/hr for 2 min) at 1.5 MPa negative peak acoustic pressure. Images were taken 30 sec after burst in the reperfusion sequence at baseline, 3, 6, 10, and 30 min after the 2 min treatment period. Note the transient decrease in image intensity at 3 min for the UTMC group (red arrow). For the UTMC nitrite group, large increases in image intensity are seen at all time points after baseline.

Figure 2.. Changes in microvascular blood volume (MBV) and nitric oxide (NO) concentration after ultrasound-targeted microbubble cavitation (UTMC) combination therapy:

Changes in MBV (A and C) and NO concentration (B and D) are shown. Asterisk indicates p < 0.0001 compared to control. The transient decrease in MBV in the UTMC group at 3 min was reversed with the addition of nitrite, while addition of N-Nitro-L-Arginine methyl ester (LNAME) resulted in persistent and exacerbated decrease. UTMC with nitrite resulted in greatly increased NO over individual treatments. Addition of LNAME to UTMC resulted in decreased NO which was partially rescued by addition of nitrite. There was a significant three-way interaction effect between factors of UTMC, nitrite, and LNAME (p < 0.0001) for both change in MBV and NO concentration. All error bars indicate standard deviation.

Figure 3.. Effect of ultrasound (US) pressure and microbubble (MB) dosage on microvascular blood volume (MBV) and nitric oxide (NO) concentration during ultrasound-targeted microbubble cavitation (UTMC) nitrite therapy:

For animals receiving UTMC nitrite therapy, changes in MBV and NO concentration for varied US pressures with constant MB dosage of 1×10⁹ MB/mL, 3 mL/hr for 3 min (A and B) and varied MB doses at a constant 1.5 MPa peak negative US pressure (C and D) are shown. While there was a significant effect of varying US pressure on MBV and NO concentration, change in MB dosage for the levels shown was not significant. Asterisk (*) indicates p < 0.05 compared to other groups. Dagger (†) indicates all groups are significantly different from each other (p < 0.0005). Error bars indicate standard deviation.

Figure 4.. Protective effects of nitrite with ultrasound-targeted microbubble cavitation (UTMC) therapy on markers of oxidative stress:

Change in hydrogen peroxide concentration (A) show that addition of nitrite to UTMC prevented early increases in hydrogen peroxide seen in UTMC alone in the first seven minutes after treatment. Extent of protein carbonylation is presented as a concentration for each group (B), and as a carbonyl content ratio (C). The carbonyl content ratio was obtained by taking the ratio of carbonyl concentration of a muscle tissue sample within the US treatment region (gastrocnemius) to one outside the US treatment region in the same rat hindlimb (thigh). For each group in (B) and (C), n = 5 animals. Addition of nitrite with UTMC therapy resulted in no increase in carbonylation seen in UTMC without nitrite. Asterisk (*) signifies p < 0.005 against all other groups (left) or compared group (middle, right). Error bars indicate standard deviation. (Gastroc. = gastrocnemius)

Figure 5.. Contrast ultrasound imaging comparing nitric oxide (NO) donor efficacy at higher microbubble (MB) dose:

Contrast-enhanced still-frame ultrasound images are shown after 2 min of ultrasound-targeted microbubble cavitation (UTMC) at 1×10⁹ MB/mL at 3 mL/hr for 2 min and 1.5 MPa pressure. Images were taken 30 sec after burst in the reperfusion sequence at baseline (BL), 3, 6, 10, and 30 min after the 2 min treatment period. In the UTMC group, microvascular spasm is observed at 3 and 6 min after treatment (red arrow). This is also noted in the UTMC sodium nitroprusside (SNP) group, although spasm size is notably smaller. Microvascular spasm is absent in the UTMC nitrite group at all time points.

Figure 6.. Effect of sodium nitroprusside (SNP) compared against nitrite on microvascular blood volume (MBV) and nitric oxide (NO) concentration during ultrasound-targeted microbubble cavitation (UTMC):

Change in MBV (left) and NO concentration (right) are shown for UTMC (1×10⁹ MB/mL at 3 mL/hr for 2 min at 1.5 MPa pressure) and NO donor co-therapies. While a decrease in MBV with UTMC was observed at 3 min, this was mitigated with SNP and reversed with nitrite co-therapies. In addition, a significant increase in NO was only observed for UTMC with nitrite. Dagger (†) indicates all groups are significantly different (p < 0.005). Asterisk (*) indicates p < 0.0005 against other groups. Error bars indicate standard deviation.