Acoustic Nanodroplet Vaporization Attenuates Ischemia-reperfusion Injury In Hindlimb Of Rats

Objection:Ischemia-reperfusion injury(IRI)refers to the finding that postischemic reestablishment of vascular perfusion may result in tissue damage above and beyond that directly resulting from the ischemia itself.Increasing evidence has shown that reactive oxygen species(ROS)as well as the downstream activated cytokines may play major roles in IR injury.If it can effectively reduce the generation of ROS after reperfusion,that is to say,it can reduce IRI while restoring blood supply reconstruction,which will greatly improve the prognosis of patients.Commercially available microbubbles(MBs)can be compressed into liquid nanodroplets(NDs)at room temperatures and reactivated with ultrasound(US).It has been reported that it can scavenging oxygen around during the phase transition of NDs into MBs.We hypothesized that the acoustic phase transition of NDs could alleviate the IR injury by oxygen scavenging,thus reducing the reaction between ROS and oxygen,and protecting the IR injury.The study was aim to verify the scavenging effect of dissolved oxygen in vitro experiment,and try to observe its effect of reducing IRI in rat lower limb ischemia-reperfusion model.Method:1.Nanodroplet formulation and sizing:NDs were formulated according to a protocol adapted from Professor Porter's laboratory.MB and ND were sized by using Nano-ZS instrument.2.In Vitro Experimental:Nanodroplets were exposed to ultrasound using a Vevo 2100 high frequency small animal ultrasound machine in either a setup with static fluid or a flow phantom.Dissolved oxygen was measured before and after ultrasound exposure using DO sensors.3.In Vivo Experimental:Male Sprague Dawley rats were selected to modeling ischemia-reperfusion injury in the hind limb,arterial clamp were used to occlude and regain blood perfusion of right hind limbs of the rats,time for occlusion was 4 hours and 12 hours reperfusion.Thirty-three SD rats were divided into 4 groups(n=8-9):Sham group-sham operation;IRI group-ischemia reperfusion injury;NDs group-ischemia and reperfusion accompanied with US+NDs intervention;Saline group-ischemia and reper-fusion accompanied with US+saline intervention.At the end of the trial,samples of skeletal muscle in each group were taken for the following tests:(1)Determination of skeletal muscle tissue oxidative stress indicators such as MDA(Malondialdehyde),SOD(Superoxide dismutase),CAT(Catalase),GSH(Glutathione);(2)Measurement of skeletal muscle MPO(Myeloperoxidase)activity;(3)The quantitative analysis for the caspase3 in skeletal were detected at the protein expression level;(4)Immunohistochemical staining was used to observe the HMGB1 expression(qualitative and localization),TUNEL was used for cell apoptosis assay;(5)Compare the changes of hemodynamic indexes in each group.Result:1.Definity MBs(DMBs)were able to condense into Definity NDs(DNDs).The formed NDs were smaller than microbubbles and ranged in size from approximately 68.0 to 295.4nm,and the largest concentration in the 100 nm range.2.In vitro studies:In a flow phantom,a 3.8%decrease in DO was measured by the downstream sensor when after the Ultrasonic irradiation(transmit power 100%).In a static fluid system,the DO content was reduced by 3.9%when droplets were exposed to ultrasound(transmit power 100%).For all trials without ultrasound,the DO sensor did not detect a substantial change in DO for the duration of the experiments.3.The level of MDA in NDs group was lower than IRI and Saline groups(t=6.974,P<0.001;t=5.768,P<0.001),while the levels of GSH?CAT?and SOD in NDs group were respectively significantly higher than those in Saline and IRI groups.The MPO activity in NDs group was significantly lower than Saline group(t=9.486,P<0.0001)and IRI group(t=8.689,P<0.0001).Immunohistochemical results showed the expression of inflammation associated proteins-HMGB1 in the NDs group and sham group were significantly lower than the other groups.Tunel staining and caspase3 western blot results indicated apoptosis of skeletal muscles were observed after ischemia/reperfusion,but the NDs intervention could significantly reduce the proportion of apoptosis by showing a significant difference compared with Saline group(t=6.411,P=0.003)and IRI group(t=7.107,p=0.002).Conclusion:Ultrasound-mediated conversion of perfluorocarbon droplets into microbubbles scavenged dissolved oxygen.The acoustic phase transition of nanodroplets in the ultrasound field can reduce the generation of ROS and oxidative stress.It also decreased tissue inflammation and cell apoptosis followed ROS burst.and improved hemodynamics abnormality after IRI.This novel approach may be of clinical value in myocardial reperfusion therapy.