Effects of Low‐Intensity Ultrasound on Gramicidin D‐Induced Erythrocyte Edema

Objectives To determine whether low‐intensity ultrasound (US) can reduce red blood cell (RBC) edema and, if so, whether the US activity is associated with aquaporin 1 (AQP‐1), a water channel in the cell membrane. Methods Red blood cell edema was induced by gramicidin D treatment at 40 ng/mL for 20 minutes and evaluated by a hematocrit assay. Low‐intensity continuous wave US at 1 MHz was applied to RBCs for the last 10 minutes of gramicidin D treatment. To determine whether US activity was associated with AQP‐1, RBCs were treated with 40 μM mercuric chloride (HgCl2), an AQP‐1 inhibitor, for 20 minutes at the time of gramicidin D treatment. Posttreatment morphologic changes in RBCs were observed by actin staining with phalloidin. Results Red blood cell edema increased significantly with gramicidin D at 20 (1.8%), 40 (6.7%), 60 (16.7%), and 80 (11.3%) ng/mL, reaching a peak at 60 ng/mL, compared to the control group (20 ng/mL, P = .019; 40, 60, and 80 ng/mL, P < .001). No significant RBC hemolysis was observed in any group. Edema induced by gramicidin D at 40 ng/mL was significantly reduced by US at 30 (3.4%; P = .003), 70 (4.4%; P = .001), and 100 (2.9%; P = .001) mW/cm2. Subsequent experiments showed that edema reduction by US ranged from 7% to 10%. Cotreatment with HgCl2 partially reversed the US effect and showed a significantly different level of edema compared to gramicidin D‐alone and US‐cotreated groups (P = .001). These results were confirmed by microscopic observation of RBC morphologic changes. Conclusions Low‐intensity US could reduce gramicidin D–induced RBC edema, and its effect appeared to at least partly involve regulation of AQP‐1 activity. These results suggest that low‐intensity US can be used as an alternative treatment to control edema and related disorders.