Pulsed ultrasound attenuates the hyperglycemic exacerbation of myocardial ischemia-reperfusion injury

Eric J Charles, Yikui Tian, Aimee Zhang, Di Wu, J Hunter Mehaffey, Joseph C Gigliotti, Alexander L Klibanov, Irving L Kron, Zequan Yang, Eric J Charles, Yikui Tian, Aimee Zhang, Di Wu, J Hunter Mehaffey, Joseph C Gigliotti, Alexander L Klibanov, Irving L Kron, Zequan Yang

Abstract

Objective: Acute hyperglycemia during myocardial infarction worsens outcomes in part by inflammatory mechanisms. Pulsed ultrasound has anti-inflammatory potential in bone healing and neuromodulation. We hypothesized that pulsed ultrasound would attenuate the hyperglycemic exacerbation of myocardial ischemia-reperfusion injury via the cholinergic anti-inflammatory pathway.

Methods: Acute hyperglycemia was induced in wild-type C57BL6 or acetylcholine-receptor knockout (α7nAChR-/-) mice by intraperitoneal injection of glucose. Pulsed ultrasound (frequency 7 MHz, bursting mechanical index 1.2, duration 1 second, repeated every 6 seconds for 2 minutes, 20-second total exposure) was performed at the spleen or neck after glucose injection. Separate mice underwent vagotomy before treatment. The left coronary artery was occluded for 20 minutes, followed by 60 minutes of reperfusion. The primary end point was infarct size in explanted hearts.

Results: Splenic pulsed ultrasound significantly decreased infarct size in wild-type C57BL6 mice exposed to acute hyperglycemia and myocardial ischemia-reperfusion injury (5.2% ± 4.4% vs 16.9% ± 12.5% of risk region, P = .013). Knockout of α7nAChR abrogated the beneficial effect of splenic pulsed ultrasound (22.2% ± 12.1%, P = .79 vs control). Neck pulsed ultrasound attenuated the hyperglycemic exacerbation of myocardial infarct size (3.5% ± 4.8%, P = .004 vs control); however, the cardioprotective effect disappeared in mice that underwent vagotomy. Plasma acetylcholine, β2 adrenergic receptor, and phosphorylated Akt levels were increased after splenic pulsed ultrasound treatment.

Conclusions: Pulsed ultrasound treatment of the spleen or neck attenuated the hyperglycemic exacerbation of myocardial ischemia-reperfusion injury leading to a 3-fold decrease in infarct size. Pulsed ultrasound may provide cardioprotection via the cholinergic anti-inflammatory pathway and could be a promising new nonpharmacologic, noninvasive therapy to reduce infarct size during acute myocardial infarction and improve patient outcomes.

Keywords: cholinergic anti-inflammatory pathway; myocardial ischemia-reperfusion injury; pulsed ultrasound.

Copyright © 2019 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1
Figure 1
Upper panel: Experimental protocol and temporal manipulations of the mice. Lower panel: Ultrasound probe positions and locations of the targets (spleen or neck structures) before application of pulsed or B-mode ultrasound.i.p., intraperitoneal; pUS, pulsed ultrasound; B-US, B-mode ultrasound; LCA, left coronary artery; TTC, 1% 2,3,5-triphenyltetrazolium.
Figure 2
Figure 2
Splenic pUS attenuated hyperglycemic exacerbation of myocardial infarct size in a mouse model of ischemia-reperfusion injury, however no cardioprotective effect seen in α7nAChR−/− mice. Representative heart slices shown are from approximately the same level of the left ventricle in an area perfused primarily by the left coronary artery. *p=0.007 vs. Wild-type Control, ^p=0.013 vs. Wild-type HG, #p=0.004 vs. α7nAChR−/− Control, +p=0.79 vs. α7nAChR−/−+HG. Control, no hyperglycemia; HG, mice with acute hyperglycemia;HG+Splenic pUS, mice with acute hyperglycemia treated with pulsed ultrasound at the spleen; Wild-type, wild-type mice;α7nAChR−/−, α7 nicotinic acetylcholine receptor knockout mice. pUS, pulsed ultrasound.
Figure 3
Figure 3
Neck pUS attenuated hyperglycemic exacerbation of myocardial infarct size in a mouse model of ischemia-reperfusion injury, however no cardioprotective effect seen in mice who underwent cervical or GEJ vagotomy. The cardioprotective effect of neck pUS persisted for up to 24 hours, however treatment with neck pUS after initiation of reperfusion was not protective. *p=0.007 vs. WT Control, ^p=0.004 vs. HG, #p=0.99 vs. HG. +p=0.7 vs. HG+Neck pUS and p=0.02 vs. HG+Neck BUS 24 hrs prior. ∞p=0.5 vs. HG. WT Control, wild-type control mice; HG, wild-type mice with acute hyperglycemia; HG+Neck pUS, wild-type mice with acute hyperglycemia treated with pulsed ultrasound at the neck;HG+Cervical Vagotomy+Neck pUS, wild-type mice with acute hyperglycemia who underwent cervical vagotomy and were treated with pulsed ultrasound at the neck; HG+GEJ Vagotomy+Neck pUS, wild-type mice with acute hyperglycemia who underwent gastro-esophageal junction vagotomy and were treated with pulsed ultrasound at the neck; HG+Neck pUS 24 hrs prior, wild-type mice with acute hyperglycemia treated with pulsed ultrasound at the neck 24 hours prior to LCA occlusion; HG+Neck BUS 24 hrs prior, wild-type mice with acute hyperglycemia treated with control B-mode ultrasound at the neck 24 hours prior to LCA occlusion;HG+Neck pUS Post-reperfusion, wilt-type mice with acute hyperglycemia treated with pulsed ultrasound at the neck 10 minutes after the start of reperfusion pUS, pulsed ultrasound;GEJ, gastro-esophageal junction.
Figure 4
Figure 4
Splenic and neck pUS decreased splenic levels of acetylcholine and increased plasma levels in mice. Spleen tissue: *p=0.03 vs. Splenic BUS. Plasma: ^p=0.02 vs. Splenic BUS, #p=0.001 vs. Splenic BUS. Splenic BUS, wild-type mice treated with B-mode ultrasound at the spleen; Splenic pUS, wild-type mice treated with pulsed ultrasound at the spleen;Neck pUS, wild-type mice treated with pulsed ultrasound at the neck. pUS, pulsed ultrasound.
Figure 5
Figure 5
(A) Splenic and neck pUS increased β2AR protein expression in splenic tissue of mice. *p=0.008 vs. Splenic BUS, ^p=0.003 vs. Splenic BUS. (B) pAkt protein expression in splenic tissue increased after treatment with pUS at the spleen and decreased after treatment at the neck in mice. #p=0.002 vs. Splenic BUS, +p=0.015 vs. Splenic BUS. Splenic BUS, wild-type mice treated with B-mode ultrasound at the spleen; Splenic pUS, wild-type mice treated with pulsed ultrasound at the spleen; Neck pUS, wild-type mice treated with pulsed ultrasound at the neck.β2AR, β2 adrenergic receptor;pAkt, phosphorylated Akt; pUS, pulsed ultrasound.
Figure 6
Figure 6
Proposed cholinergic anti-inflammatory pathway involved in the cardioprotective effect of neck and splenic pUS via β2AR-α7nAChR-pAkt mechanism. ACh, acetylcholine;α7nAChR, α7 nicotinic acetylcholine receptor;β2AR, β2 adrenergic receptor;IL-10, interleukin 10; Norepi, norepinephrine; pAkt, phosphorylated Akt.

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