The Role of Nitric Oxide during Sonoreperfusion of Microvascular Obstruction

Abstract

Rationale: Microembolization during PCI for acute myocardial infarction can cause microvascular obstruction (MVO). MVO severely limits the success of reperfusion therapies, is associated with additional myonecrosis, and is linked to worse prognosis, including death. We have shown, both in in vitro and in vivo models, that ultrasound (US) and microbubble (MB) therapy (termed "sonoreperfusion" or "SRP") is a theranostic approach that relieves MVO and restores perfusion, but the underlying mechanisms remain to be established. Objective: In this study, we investigated the role of nitric oxide (NO) during SRP. Methods and results: We first demonstrated in plated cells that US-stimulated MB oscillations induced a 6-fold increase in endothelial nitric oxide synthase (eNOS) phosphorylation in vitro. We then monitored the kinetics of intramuscular NO and perfusion flow rate responses following 2-min of SRP therapy in the rat hindlimb muscle, with and without blockade of eNOS with LNAME. Following SRP, we found that starting at 6 minutes, intramuscular NO increased significantly over 30 min and was higher than baseline after 13 min. Concomitant contrast enhanced burst reperfusion imaging confirmed that there was a marked increase in perfusion flow rate at 6 and 10 min post SRP compared to baseline (>2.5 fold). The increases in intramuscular NO and perfusion rate were blunted with LNAME. Finally, we tested the hypothesis that NO plays a role in SRP by assessing reperfusion efficacy in a previously described rat hindlimb model of MVO during blockade of eNOS. After US treatment 1, microvascular blood volume was restored to baseline in the MB+US group, but remained low in the LNAME group. Perfusion rates increased in the MB+US group after US treatment 2 but not in the MB+US+LNAME group. Conclusions: These data strongly support that MB oscillations can activate the eNOS pathway leading to increased blood perfusion and that NO plays a significant role in SRP efficacy.

Keywords: Microvascular obstruction; microbubbles; nitric oxide; sonothrombolysis; ultrasound.

Figure 1

Cell culture experimental setup for in vitro exposure of HUVECs to microbubbles and ultrasound.

Figure 2

(A) In vivo hindlimb model for SRP therapy. Intramuscular NO was measured using a needle probe, microvascular perfusion was assessed using an imaging transducer and therapeutic US was delivered with a therapeutic transducer positioned perpendicularly to the imaging probe (more details on the probes orientations can be found in supplemental Figure S2; (B) The kinetics of NO and perfusion rate following SRP were assessed for 30 min after 2 min of SRP therapy with and without LNAME; (C) MVO was created by injecting microclots (See supplemental Figure S1). Two successive 10 min SRP therapy sessions were performed with and without LNAME.

Figure 3

In vitro eNOS phosphorylation: (A) Western blot of phospho-eNOS (S1177) and total-eNOS for Ionomycin (+ control), No Treatment (- control) and MB+US therapy; p-eNOS/t-eNOS ratio increased following MB+US therapy (n=3); (B,C) p-eNOS/t-eNOS ratio without MB did not induce eNOS phosphorylation (n=3); (D) Trypan blue exclusion assay from supernatant and trypsinized cells and (E) metabolic activity measured by Alamar Blue fluorescence at 24 h, both showing that cells tolerated MB+US therapy; (F) immunostaining for p-eNOS (green) with Hoechst (blue) nuclear counter stain (* p<0.05)

Figure 4

(A) Intramuscular NO after LNAME injection (No US): Intramuscular NO level decreased steadily following i.v. LNAME injection at t=0 s in two rats, as expected; (B) Intramuscular NO following SRP in 4 rats with and without LNAME. After a transient initial decrease, NO rose steadily following 2 min of SRP therapy and was significantly higher than at baseline starting 13 min after treatment. This increase was completely blunted with LNAME (Sidak's multiple comparison).

Figure 5

(A) Hindlimb contrast ultrasound imaging, in the MB+US group and without microthrombi, taken 2 s into the burst reperfusion imaging video sequence at baseline, post SRP (t=2 min) and at t=3, 6, 10 and 30 min. Note the transient decrease in echo contrast in the treated area (circle) after SRP followed by recovery at t=3 min and improved perfusion above baseline after 6 min; (B) Blood volume and (C) microvascular flow rate during and following 2 min of SRP therapy in rats receiving MB+US and LNAME+MB+US. Typical video sequences can be found in supplemental video data.

Figure 6

Photographs of rear paws taken at baseline, after MVO, and after two sessions of SRP therapy. In the LNAME+MB+US animal, the left paw, originally pink, becomes cyanotic after injection of microthrombi. It remains cyanotic after two sessions of SRP therapy. In the second row, in the MB+US group (intact NO), the paw returns to pink in color after the first SRP treatment and remains pink after treatment 2. The right paw, which becomes ischemic after placement of the catheter in the femoral artery and remains ischemic throughout, is shown for comparison.

Figure 7

Contrast enhanced ultrasound images, taken 20 s after burst, for baseline, MVO and SRP treatments 1 and 2 for a rat in the LNAME + MB + US and MB + US groups. Reperfusion is near complete after SRP therapy in the MB + US rat, but not in the rat with NO blockade by LNAME.

Figure 8

Blood volume and flow rate calculated using contrast enhanced burst replenishment ultrasound imaging at different stages of sonoreperfusion therapy using MB+US with LNAME (n=9) and without LNAME (n=9).