Ultrasonic Shockwave

This was an observation retrospective secondary analysis of a previous study, registered with the number NCT03082326 and approved by the ethics committee of West China Hospital of Sichuan University (2017 (200)). The previous study enrolled 181 consecutive admitted patients who met the criteria for shock and aimed to describe and analyse the physiopathologic characteristics of shock patients assessed by critical care ultrasound on ICU admission. The following are some details of the previous study: right after patients enrolled, the completed lung ultrasound examination was required as part of a complete ultrasound assessment examination. The examinations were performed by a board certificated physician who had completed a full critical care ultrasound (CCUS) training course and had more than a half-year of critical care ultrasonic performance experience. The investigators recorded the ultrasonic data, which were blinded to the treatment team, and assessed the outcome. The data consisted of clinical and ultrasonic variables that were entered into the database after the patient’s discharge or death.
In the current study, we extracted and listed the patients’ demographics, clinical characteristics, prognosis, and the LUSS as part of the indicators in this study. We then established a bivariate logistic regression model to identify the correlation between the LUSS on admission and the 28-day mortality and divided the patients into LUSS quartiles. The COX model was employed to investigate the multiplicative relationship between the predictors and the hazards.

Evans blue (EB) was used as an indicator to show the location and extent of BBB opening in rat brains since EB cannot penetrate an intact BBB. Immediately after the injection of 0.5 ml of 3% EB via the rats’ tail vein, 100 shockwave iterations were applied (intensity level 5, pulse repetition frequency (PRF) 5 Hz, N = 8). The following process was modified from a previous study^{27 (link)}. The original two hour interval time for euthanization was extended to three hours. Three hours after the shockwave application, the rats were sacrificed and the brains were harvested for histological analysis^{27 (link)}. The intensity levels for each parameter are shown in Table 1.Table 1

Major shockwave parameters of the intensity levels.

Intensity level	0.1	1	2	3	4	5	10	20
Negative peak pressure (MPa)	−4.2	−7.3	−7.92	−8.54	−9.17	−9.79	−12.9	−18.7
Positive peak pressure (MPa)	5.4	11.8	13.66	15.51	17.37	19.22	28.5	77.7
Energy flux density (Total) (mJ/mm2)	0.03	0.1	0.13	0.16	0.18	0.21	0.35	0.82

To evaluate the performance of shockwave applications, the successful (visible) BBB opening rate is defined as number of rats with BBB opening after shockwave treatment over the total number of rats receiving shockwave treatment. To define BBB opening, the Evans blue stained area of the histology sections were further analyzed using a color histogram of Image J. The delta blue and delta red in the proposed study were the differences in pixels of blue and red color in RGB-image system that were used to evaluate the staining level of Evans blue and the red blood cell extravasation level between the shockwave applied side and untreated side. Both of them were assessed using student’s t-test and one-way ANOVA and post-hoc analysis, wherein p < 0.05 for the delta blue between the shockwave applied side and untreated side.
To confirm the reversibility of the shockwave-induced BBB opening, 0.5 ml of 3% EB was injected via the rats’ tail vein 24 hrs after applying 100 shockwave iterations with all other conditions remaining constant (N = 5). The rats were then sacrificed for brain sectioning 3 hours after the EB injection.
Traditionally, ultrasound contrast agent (UCA) is used together with HIFU to induce BBB opening in the brain by enhancing cavitation^{28 (link),29 (link)}. The effect of UCA on shockwaves was investigated by adding various concentrations of SonoVue UCA from 2.5 × 10² to 2.5 × 10⁸ microbubbles/kg body weight (the clinical concentration is 3–15 × 10⁶ microbubbles/kg) through the tail vein immediately before shockwave treatment (N = 5).

Accurately weigh 5 g powder to be tested and placed in a 50 mL conical flask, 10 mL of 70% methanol was added, the plug was covered, its quality was recorded, and the water temperature was controlled at 40°C. The ultrasonic was 45 min and stirred every 15 min with ultrasonic power of 50 kHz. Let it sit overnight, ultrasound again for 45 min, cool it at room temperature, weigh the weight again, use 70% methanol to make up the weight lost, mix well, transfer it to 15 mL centrifuge tube, centrifuged at 4,000 rpm for 10 min, the supernatant was filtered by 0.22 μm microporous membrane to obtain the test solution.