Early iNOS impairment and late eNOS enhancement during reperfusion following 2.49 MHz continuous ultrasound exposure after ischemia

Abstract

Objective: Ischemia reperfusion (IR) injury, occurring during heart attacks, hemorrhagic shock, and bypass and transplant surgeries, impairs microcirculatory function and nitric oxide (NO) synthesis. We report the regulation of endothelial and inducible NO synthase (eNOS and iNOS) proteins as a consequence of the application of continuous mode diagnostic frequency ultrasound application following IR injury.

Methods: Animals were assigned to one of five groups for microcirculatory assessment or Western blot analysis (WB) as follows: (1) IR+iNOS inhibition (1400W); and (2) IR+1400W+ultrasound for microcirculatory assessment, (3) Control; (4) IR; and (5) IR+ultrasound for WB. Functional capillary density and microvascular diameter, flow velocity, and flow were monitored for microcirculatory assessment. Skin tissue samples were harvested for WB. 2.49MHz continuous ultrasound was used for application.

Results: Both the inhibition of iNOS alone and iNOS inhibition with ultrasound irradiation positively influenced the microcirculation of observed animals relative to baseline values. Ultrasound exposure resulted in a significant production of eNOS protein in skin tissue harvested 24h into reperfusion (p<0.01). iNOS levels from the same tissue of ultrasound exposed animals were found to be significantly decreased 0.5h into reperfusion (p<0.05).

Conclusion: Protection from lasting IR injury effects in the microcirculation, with continuous mode diagnostic frequency ultrasound, results from augmented eNOS protein levels during late reperfusion. Ultrasound inhibited iNOS protein production during early reperfusion may also confer protection from IR injury.

Figure 1

Schematic outline of the microvascular and chamber tissue harvest experimental protocol for each group, not including control group. US, ultrasound.

Figure 2

Western blot analysis of NOS protein content in 3 individual hamster chamber tissue samples of animals subjected to ischemia reperfusion injury with or without ultrasound exposure. Tissue was harvested at 0.5, 2, or 24 h of reperfusion. β-Tubulin band intensities of samples are not shown. Left Panel: eNOS protein content. Right Panel: iNOS protein content. IR, IR group; IR&US, IR+ultrasound group.