The use of diagnostic frequency continuous ultrasound to improve microcirculatory function after ischemia-reperfusion injury

Abstract

Objective: Damage to the circulatory system resulting from ischemia-reperfusion injury (I/R injury) occurs during heart attacks and hemorrhagic shock. The authors report a method for mitigating microcirculatory injury, using diagnostic frequency continuous-mode ultrasound and how effects are influenced by nitric oxide production impairment.

Methods: Five groups of hamsters were studied using the dorsal skin fold window chamber: (1) I/R; (2) I/R + ultrasound during ischemia; (3) I/R + ultrasound after ischemia; (4) I/R + N(omega)-nitro-L-arginine methyl ester (L-NAME); and (5) I/R + L-NAME + ultrasound. Functional capillary density (FCD) and microvascular diameter, flow velocity, and flow were monitored. During the exposures 2.49 MHz continuous ultrasound was used.

Results: Significant improvements in animals exposed to ultrasound after ischemia were found at 24 h of reperfusion in FCD, arteriolar diameter, and arteriolar and venular flow velocity and flow. Animals exposed to ultrasound during ischemia showed significantly improved FCD. L-NAME treatment reduced the improvement of microvascular function, compared to animals exposed after ischemia.

Conclusions: The use of continuous-mode diagnostic frequency ultrasound is beneficial in preventing long-term ischemia-reperfusion effects in the microcirculation as shown by the return of microvascular parameters to baseline values, an effect not attained in the absence of ultrasound treatment. The effects may be in part due to the production of nitric oxide consequent to locally induced shear stress effects by ultrasound exposure.

Figure 1

Schematic outline of the experimental protocol for each group. US, ultrasound.

Figure 2

Changes in functional capillary density (FCD) due to ischemia–reperfusion injury and exposure to ultrasound. FCD of groups receiving ultrasound exposure, for 10 min during ischemia and for 20 min after ischemia showed a significant increase from I/R at 24 h. Data are presented as means ± standard deviations. I/R, I/R group; D, I/R + ultrasound during ischemia group; A, I/R + ultrasound after ischemia group. * – p < .05 vs. I/R.

Figure 3

Changes in arteriolar and venular diameters due to ischemia–reperfusion injury and exposure to ultrasound. Top: I/R + ultrasound after ischemia animals showed significant increase of arteriolar diameters at 24 h, compared to both I/R and I/R + ultrasound during ischemia animals. Bottom: No significant venular diameter difference from I/R was found for either of the groups exposed to ultrasound. Values are presented as means ± standard deviations. I/R, I/R group; D, I/R + ultrasound during ischemia; A, I/R + ultrasound after ischemia group. * * * – p < .001 vs. I/R. ∧∧∧ – p < .001 vs. IR + ultrasound during ischemia.

Figure 4

Changes in arteriolar and venular flow velocity due to ischemia–reperfusion injury and ultrasound exposure. Top: Arteriolar flow velocity of I/R + ultrasound after ischemia animals significantly increased at 24 h, compared to both I/R and I/R + ultrasound during ischemia animals. Bottom: I/R + ultrasound after ischemia animals showed significant increases in venular flow velocity at 24 h, compared to the I/R group. Values are presented as means ± standard deviations. I/R, I/R group; D, I/R + ultrasound during ischemia group; A, I/R + ultrasound after ischemia group. +++ – p < .001 vs. I/R 0.5 h assessment. ββ – p < .01 vs. I/R + ultrasound during ischemia 0.5 h assessment. Φ – p < .05 vs. I/R + ultrasound after ischemia 0.5 h assessment. * * * – p < .001 vs. I/R. ∧∧∧ – p < .001 vs. I/R + ultrasound during ischemia. * – p < .05 vs. I/R.

Figure 5

Calculated changes in arteriolar and venular flow due to ischemia–reperfusion injury and ultrasound exposure. Top: In the I/R + ultrasound after ischemia group arteriolar flow significantly increased at 24 h, compared to both the I/R and the I/R + ultrasound during ischemia groups. Bottom: Venular flow significantly increased at 24 h, for the I/R + ultrasound after ischemia group compared to the I/R group. Values are presented as means ± standard deviations. I/R, I/R group; D, I/R + ultrasound during ischemia group; A, I/R + ultrasound after ischemia group. +++ – p < .001 vs. I/R 0.5 h assessment. β – p < .05 vs. I/R + ultrasound during ischemia 0.5 h assessment. Φ – p < .05 vs. I/R + ultrasound after ischemia 0.5 h assessment. * * * – p < .001 vs. I/R. ∧∧∧ – p < .001 vs. I/R + ultrasound during ischemia. ∇∇ – p < .01 vs. I/R + ultrasound after ischemia 0.5 h assessment. ΨΨΨ – p < .001 vs. I/R + ultrasound after ischemia 2 h assessment. * – p < .05 vs. I/R.

Figure 6

Changes in functional capillary density (FCD) due to ischemia–reperfusion injury, exposure to ultrasound, and NO production inhibition using l-NAME. FCD of the I/R + l-NAME group is greater than that of the I/R + l-NAME + ultrasound group, at 0.5 and 24 h, without statistical significance. Data are presented as means ± standard deviation. I/R, I/R group; I/RL, I/R + l-NAME group; UL, I/R + l-NAME + ultrasound group.

Figure 7

Changes in arteriolar and venular diameters due to ischemia–reperfusion injury, exposure to ultrasound, and NO production inhibition using l-NAME. Top: Most arteriolar diameters dilated compared to baseline values. Bottom: Venular diameters also show mostly dilation at all observation time points compared to baseline values, without statistical significance. Values are presented as means ± standard deviation. I/R, I/R group; I/RL, I/R + l-NAME group; UL, I/R + l-NAME + ultrasound group.

Figure 8

Changes in arteriolar and venular flow velocity due to ischemia–reperfusion injury, exposure to ultrasound, and NO production inhibition using l-NAME. Top: The influence of l-NAME is seen in the 0.5-h measurement, with a significant decrease compared to I/R. Arteriolar velocity exhibited an increase from 0.5 h through the 24-h time point. A significant increase was seen at 24 h in I/R + l-NAME + ultrasound animals, compared to I/R + l-NAME. Bottom: Venular velocity of both groups was highest at the 24-h time point but not significantly different. Values are presented as means ± standard deviation. I/R, I/R group; I/RL, I/R + l-NAME group; UL, I/R + l-NAME + ultrasound group. #p < .05 vs. I/R. * – p < .05 vs. I/R + l-NAME. ∇∇ – p < .01 vs. I/R + l-NAME + ultrasound 0.5-h assessment.

Figure 9

Changes in arteriolar and venular flow due to ischemia–reperfusion injury, exposure to ultrasound, and NO production inhibition using l-NAME. Top: Arteriolar flow increased in I/R + l-NAME + ultrasound animals, while the increase at 2 h was not maintained through 24 h in I/R + l-NAME animals. Statistical significance was seen at 24 h for I/R + l-NAME + ultrasound compared to I/R + l-NAME animals. Bottom: Venular flow exhibited a stepwise increase for I/R + l-NAME animals, while the decrease at 2 h increased at 24 h for ultrasound exposed animals. No significant differences were seen between the groups. Values are presented as means ± standard deviation. I/R, I/R group; I/RL, I/R + l-NAME group; UL, I/R + l-NAME + ultrasound group. ** – p < .01 vs. I/R + l-NAME ∇∇ – p < .01 vs. I/R + L-NAME + ultrasound 2-h assessment. +++ – p < .001 vs. I/R + l-NAME + ultrasound 0.5-h assessment.