We demonstrate for first time that ultrasound exposure at therapeutic frequency in continuous or pulsed mode, is able to reduce hepatic injury in the setting of partial hepatectomy with vascular exclusion. Importantly, this study enlightened that therapy based on pulsed ultrasonography at 0. 8 MHz results in two effects: a) protection against I/R injury associated with lessening of hepatic IL-1β, and b) an enhancement of liver regeneration after 6 hours of reperfusion. These postsurgical results were obtained by applying non-invasive pulsed-wave ultrasound during short periods on the upper right portion of the rat abdomen, before and after surgery. Since more than 40 years, ultrasound has been applied as a therapy that favors healing. Also, ultrasound enhances diverse growth factors associated with angiogenesis, and it has been demonstrated to diminish inflammatory response following a surgery. By applying ultrasound in tissues, the pressure wave generates heat, cavitation or mechanical forces that are ultimately responsible for the well-known biological effects of ultrasound.

As a result of ultrasound waves propagation across tissues, endothelial cells and blood absorb part of incident field, thus producing localized effects. Our results point to a preservation of tissue structure and reduction in tissue inflammation subsequent to I/R injury in liver, which is in accordance with other studies that demonstrated ultrasound regimen also protect different tissues against I/R injury. It has been previously demonstrated that ultrasound waves induce generation of growth factors (FGF and VEGF) and modulate several cytokines that play a lead role in inflammation (TNFα, IL-6, IL-8, IL-1β, IL-2,). The present study showed that pulsed ultrasound increased HGF, which could be related to liver regeneration. As far as our knowledge is concerned, previously no effects of ultrasound on HGF have been described. Also, it has been evidenced that reduction of hepatic injury afforded by ultrasound therapy (in the continuous or pulsed mode) was associated with a reduction in the hepatic levels of IL-1β. In addition to the anti-inflammatory effects of ultrasound, it has also been described that injury caused by oxidative stress throughout post-ischemic reperfusion can be attenuated by intermittent pulses of ultrasound. In line with this, occurring upon application of ultrasound waves after I/R in hamsters, reduced lipid peroxide formation was registered in pheripheral blood.

In the conditions evaluated herein, neither continuous nor pulsed ultrasound was able to reduce oxidative stress. It should be noticed that in our conditions we assessed oxidative stress markers in hepatic tissue from rats, indicating differences in ultrasound waves effects depending on tissues and animal species. Findings from the present study point to different effects of ultrasound depending of the application mode and frequencies used. Previous studies suggest that there are differences between animal species in the responses of tissues to continuous and pulsed ultrasound. Continuous ultrasound causes tissue temperature increases that can results in decreased pain, increased blood flow, and reduction of subacute and chronic inflammation, among other effects. Pulsed ultrasound has minimal thermal effects and has be found to have a range of biological effects on tissues, including accelerating soft-tissue regeneration,35 and also inhibiting inflammatory responses. 36 However, the cellular and molecular mechanisms underlying ultrasound biological effects in vivo either in continuous or pulsed mode remains obscure. Results presented herein point to treatment with continuous and pulsed ultrasound lead to an anti-inflammatory effect in liver tissue undergoing I/R injury, in both cases associated with reduction in IL-1β. In addition to this effect, pulsed ultrasound elicited a hepatic regenerative response. The target of pulsed ultrasound on soft-tissue regeneration has covered a wide range of cells and organs, including fibroblasts, myoblasts, epithelial cells, chondrocytes and cartilage, inter-vertebral discs, ligaments, and tendons. 37 Until date there are not been reported upregulation of liver regeneration due to ultrasound waves. Regarding differential effects of ultrasound frequencies, it is important consider that depth at which ultrasound can penetrate in tissues is frequency dependent. The rate of absorption and therefore the attenuation increases as the frequency of ultrasound waves increases. The lower frequency, the less the energy is absorbed in the superficial tissues, and thus the deeper it penetrates.

In our hands, results seem to indicate that the protective effect of ultrasound against inflammatory response in hepatic I/R injury does not depend on the degree of penetration of the ultrasound waves in the tissue. On the opposite, the degree of penetration could be important to achieve a regenerative response with pulsed ultrasound, since this effect was achieved only with the frequency that allows the effect of ultrasound at greater depth. Undoubtedly, mechanisms underlying protective effects of ultrasound treatment to reduce inflammation and improve regeneration in the surgical situation of partial hepatectomy with vascular exclusion should be investigated in depth. In conclusion, we report for first time that ultrasound waves reduce injury and improve regeneration in livers undergoing partial hepatectomy with vascular exclusion. To date, there are no reports in the literature about the role of ultrasound-based therapy in hepatic I/R. Evidence in the present study suggests that application of ultrasound in pulsed mode could be considered as a therapy to improve post-surgical results in liver surgery. Taking into account that ultrasound is a therapy that does not involve the use of drugs, non-invasive, portable, simple and inexpensive, it could be translated in the short term into a clinical application.