The Preliminary Research On The Feasibility Of Blocking Splenic Blood Perfusion By Using Microbubbles Enhanced Focused Low－frequency Pulsed Ultrasound In Rabbits

Backgrounds:Spleen is the biggest immune organ of the body which has important functions of antiinfection and anti-tumor.Spleen is also related to endocrine system and digestion system. Hypersplenism is the most common disease of spleen.Splenectomy, partial splenic embolization(PSE) and splenic radiofrequency ablation(RFA) are traditional treatments.However, these methods have certain side effects and their efficacy is not satisfactory. No physical methods targeting at hypersplenism shave been reported yet.Ultrasonic cavitation effect refers to a series of dynamic processes of microbubbles in liquid during ultrasonication, e.g., shock, distension, contraction and implosion, with accompanying energy release behaviors such as transient high temperature, high pressure, shock wave, electrical discharge and microefflux, which is a major physical effect of ultrasound besides thermal effect. Microbubbles act as an effective cavitation core, incuring significant cavitation effect under appropriate ultrasonic impulse excitation. Cavitation released mechanical energy (non-thermal effect) has the potential of targeted tissue ablation, thus making possible damage of new vessels and lead to bleeding and prothrombin.ObjectiveIt is known that ultrasound convitation enhanced by microbubble may lead to significant damages on vasculars. By using a therapeutic US device in the presence of a lipid MB,we will investigate the feasibility of blood perfusion obstruction of normal rabbit spleens by microbubble (MB) mediated ultrasound(US) cavitation. Contrast ultrasonography and AD analyses were performed on the spleens at before and 0 min to 1h after treatment to evaluate the blood perfusion. Histologic analysis of the regional spleen was performed.Materials and Methods36 healthy New Zealand white rabbits were divided into four groups randomly, i.e, the prothrombin plus ultrasound plus microbubble group (PUSMB), the ultrasound plus microbubble group (USMB), the simple ultrasound group (US), the simple microbubble group (MB).Each group contained 9 rabbits. In the PUSMB group, ultrasound insonation and intravenous microbubble injection were both applied after injection of prothrombin. In the USMB group, ultrasound insonation and intravenous microbubble injection were both applied without prothrombin. In the US group, only US exposure was applied.sham US exposure and MB IV administration were applied in the MB group.Sliced off abdomen and exposed the spleen. The spleens in vivo of 36 healthy New Zealand white rabbits were insonated by using a therapeutic US device in the presence of a lipid MB. The device delivered pulsed US at 1.2 MHz of frequency, 4.6 MPa of peak acoustic pressure. Both US and MB were applied in the experimental group in PUSMB group and USMB group. Rabbits were treated with US or MB only served as the controls. Contrast ultrasonography and Mean Echo analyses were performed on the spleens at before and 0 min to 1h after treatment to evaluate the spleen blood perfusion. Histologic analysis of the regional spleen was performed.To evaluate the spleen blood perfusion,the spleens of rabbits from each group were firstly imaged by contrast enhanced ultrasonography (CEU) using a commercial diagnostic US system, Phillps IU22 with 9L high-frequency linear array probe (5-9 MHz). The CEU was performed using a low mechanic index (MI=0.13) CHI contrast modality followed by intravenous bolus injection of a lipid MB suspension (Zhifuxian) at 0.02 ml/kg. Depth, gain and other settings were kept the same during all the contrast studies. Once the spleen was targeted, the focused therapeutic US transducer was manually held against it. Then the therapeutic US was used to insonate the spleen for 5 min followed by intravenous injection of the original MB suspension (Zhifuxian) at 0.1 ml/kg. The original MB suspension was diluted into a total 5 ml volume of saline solution. The diluted microbubble solution was injected constantly at approximately 1 ml/min during the US treatment. After treatment, the spleens were imaged again on the same cut plane by using CEU at post 0 min, 30 min and 60 min. Then, three animals of every group were sacrificed immediately after treatment and their targeted spleen specimens were harvested for histological examination.ResultsThe contrast enhancement of all the spleens parenchyma was homogenous without perfusion defect in every group before treatment. From 0 min after treatment, A significant perfusion defect (no enhancement) was formed in the PUSMB and USMB group. From post 30 min on to 60 min, the perfusion of the defective spleen region began a slow process of recanalization in which the perfusion gradually restored in the former defective region in USMB group,which didn't happen in PUSMB group. In comparison, the spleen parenchyma and vessels were well perfused and no visible contrast perfusion defect was found in the two control groups from baseline to 1h after treatment.Before treatment, the contrast Mean Echo of ROIs, ranged from21.2-23.1(dB), in all groups had no significant statistical difference. After treatment, the Mean Echo of ROI dropped significantly to 13.8±1.92 and 12.4±1.89 at 0 min post-treatment in the PUSMB group and USMB group, while gradually restored to 14.08±1.58 in 1 hour in the USMB group and gradually decreased to 6.75±1.73 in the PUSMB group. In contrast, the Mean Echo of all the time points, ranged from 21.7 to22.8, in the US group and the MB group had no statistical difference.In histologic examination,the spleens of the sham group appear to be normal.The H.E. sections from the US group demonstrate only some occasionally minor vascular hyperemia and focal hemorrhage without other abnormalities.Specimens from the US+MB group demonstrate massive, diffused and severe hemorrhage, sinusoidal hyperemia, microvascular rupture, endothelial damage and hematoma formation. There are also some focal necrotic hepatocytes.Conclusions:Regional blood perfusion of normal spleen can be temporarily blocked by microbubble enhanced focused ultrasound with a high acoustic pressure,which can be enhanced and pronged by prothrombin.