High Intensity Focus Ultrasound Stimulation of Spleen Reduces Traumatic Bleeding in Mice

Abstract

Introduction: Uncontrolled hemorrhage is the most common preventable cause of traumatic death. Direct pressure or tourniquets limit extremity bleeding, but non-compressible hemorrhage requires surgical control. Systemic hemostatic therapies are limited. In preclinical models of hemorrhage, electrical vagus nerve stimulation (VNS) accelerates thrombosis and decreases traumatic hemorrhage in a spleen-dependent manner. Here, we demonstrate that non-invasive high intensity focused ultrasound (HIFU) stimulation targeting the spleen significantly reduces bleeding in a murine tail transection model. Methods: Adult male C57BL/6J mice were anesthetized (ketamine/xylazine), placed in the right lateral decubitus position, and shaved after identifying the spleen by anatomical markers and palpation. The ultrasound probe was directed at the splenic hilum, followed by 1 min stimulation (1.1 MHz, 200 mV/pulse, 150 burst cycles, 500 μs burst period), a 30 s rest, and 1 min stimulation for a 5 min total stimulation. Animal tails were warmed in water (37°C, 5 min), transected 2 mm from the tip, and bled into water until hemorrhage stops for a minimum of 10 s to record the duration of bleeding (bleeding time). Results: Compared with sham stimulation, HIFU significantly reduces bleeding time after tail transection (Sham = 110.5 ± 7.7 s vs HIFU = 72.9 ± 6.6 s, Mean ± SEM, n = 10/group, p < 0.01 (two-tailed t-test). Conclusion: These data demonstrate that HIFU stimulation of the spleen provides a novel method to reduce traumatic hemorrhage. HIFU warrants additional study in traumatic and surgical bleeding, and as a treatment for bleeding disorders.