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Published in final edited form as: J Med Biol Eng. 2013 ; 33(1): 103–110.
In vivo Sonothrombolysis of Ear Marginal Vein of Rabbits Monitored with High-frequency Ultrasound Needle Transducer Ruimin Chen1,†, Dong-Guk Paeng2,†, Kwok Ho Lam1, Qifa Zhou1,*, K. Kirk Shung1, Naoki Matsuoka3, and Mark S. Humayun4 1NIH
Resource Center for Medical Ultrasonic Transducer Technology, Department of Biomedical Engineering, University of Southern California, Los Angeles, CA 90089, USA
2Department
of Ocean System Engineering, Interdisciplinary Postgraduate Program in Biomedical Engineering, Jeju National University, Jeju, Korea 690-756
3Division
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of Ophthalmology and Visual Science, Graduate School of Medical and Dental Sciences, Niigata University, Niigata 950-2181, Japan
4Doheny
Eye Institute, University of Southern California, Los Angeles, CA 90033, USA
Abstract
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Ultrasound (US) is known to enhance thrombolysis when thrombolytic agents and/or microbubbles are injected into the targeted vessels. In this research, high-intensity US (1 MHz, 7 W/cm2, 30 % duty cycle) was applied in vivo to the ear marginal vein of three rabbits which was occluded by either laser photothrombosis or thrombin, right after the injection of 0.3~0.6 cc of microbubbles (13 × 108 bubbles/ml of concentration) through the other ear vein without using any thrombolytic agent. To determine the effect of the sonothrombolysis, the blood flow velocity near the occlusion site in the vein was measured by a custom-made 40-MHz US needle transducer and its corresponding Doppler US system. The Doppler spectra show that the blood flow velocity recovered from total occlusion after three 10-minute high-intensity US treatments. Fluorescein angiography was employed to confirm the opening of the vessel occlusion. A control study of three rabbits with only the microbubble injection showed no recovery on the occlusion in 3 hours. The results show that the sonothrombolysis in the rabbit ear marginal vein can be achieved with microbubbles only. The results of cavitation measurements indicate that the mechanism of sonothrombolysis is probably due to the cavitation induced by the microbubbles. Without the need of applying any thrombolytic agent, high-intensity US has high potential for therapies targeting on small blood vessels.
Keywords Therapeutic ultrasound; Sonothrombolysis; Rabbit ear marginal vein; Microbubbles
*
Corresponding author: Qifa Zhou, Tel: +1-213-7409475, [email protected]. †These authors contributed equally to this work
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1. Introduction NIH-PA Author Manuscript
The use of thrombolytic therapy has attracted a lot of attention in clinical trials and has proven its value in treating common thrombotic diseases. The therapy is applied to restore the blood flow and oxygenation for body organs by removing blood clots inside blood vessels. Among various thrombolytic methods, ultrasound (US) thrombolysis has been found to be promising for facilitating recanalization of occluded arteries, especially for patients with acute strokes [1,2]. Combined with thrombolytic agents and microbubbles, sonothrombolysis helps the disintegration and dissolution of blood clots [3–5]. Many sonothrombolysis studies have been conducted in the laboratory using animal models [6–8]. For human applications, pilot studies of sonothrombolysis for stroke patients have been pursued [9].
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Although the process of sonothrombolysis can be enhanced when microbubbles are used in conjunction with thrombolytic agents [10], the use of chemical agents is constrained by numerous patient factors which might cause severe adverse effects, sometimes even fatal [11]. To eliminate the use of chemical agents and their associated adverse effects, US has been applied to thrombolysis with microbubbles alone [12]. There has also been evidence demonstrating that US alone can induce nonenzymatic thrombolysis in vitro and in clinical settings [13–15]. Although a number of in vitro sonothrombolysis experiments have been reported for US enhancement of thrombolysis [16–21], few in vivo studies have been conducted without using chemical agents [22]. Most studies have been performed on arteries. In the present study, in vivo sonothrombolysis without using thrombolytic agents was performed in a rabbit marginal ear vein. Only non-specific or targeted microbubbles were used for enhancing the efficiency of sonothrombolysis. During the process, the sonothrombolysis effect on blood flow in veins was graphically recorded in pulsed-wave (PW) Doppler mode using a custommade 40-MHz US needle transducer and its corresponding Doppler US system. The mechanism of sonothrombolysis was examined through in vitro measurements of cavitation.
2. Methods 2.1 Animals and treatment protocol
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Three pigmented rabbits obtained from Irish Farms (Norco, CA, USA), each weighing from 2 to 3 kg, were used in this study. The rabbit is a good animal model for thrombolytic therapy because it is relatively cheap and experimental procedures are well-established [23]. The rabbit marginal ear vein is a large convex vessel passing along the ear edge. This vein can be easily observed under a microscope through a thin layer of auricular cartilage and epithelium, which makes it accessible for manipulations either from outside or inside [24]. All animal procedures were carried out in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and with the Institutional Animal Care and Use Committee of University of Southern California. Initial sedation was induced with intramuscular injection of ketamine hydrochloride (25 mg/kg) and xylazine hydrochloride (6 mg/kg). Rabbits remained
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anesthetized throughout the entire experiment. After the experiment was concluded, the rabbits were euthanized with an intracardiac injection of sodium pentobarbital (120 mg/kg).
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2.2 Vascular occlusion One of the most reliable models of creating vein occlusion in rabbits is laser photothrombosis [25]. The photosensitizer Rose Bengal (tetrachlorotetraiodo fluorescein sodium) was injected through the vein of an ear to the targeted vessel by an argon laser to create an isolated vein occlusion without occluding the artery. The size of the laser spot was about 500 μm and the laser power was set to 100–300 mW with a duration of 200–1000 ms. The laser photothrombosis occlusion resulted in the deformation and damage of the vessel wall, which differs from the natural process of occlusion. The laser photothrombosis occlusion inside a marginal ear vein is shown in Fig. 1. In order to avoid the deformation and damage of the vessel wall, another occlusion model was applied by injecting 0.04 ml (200U) of Bovine Thrombin (BioPharm Laboratories, Alpine, UT, USA) either inside the clamped vessel by a 30-gauge needle or outside the vessel after stopping the blood flow of the targeted vessel using clamps, as shown in Fig. 2. Fluorescein angiography was used before and after occlusion to confirm the total occlusion, as shown in Figs. 1 and 2.
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2.3 High-frequency needle transducer and high-frequency ultrasound doppler system Since the rabbit ear marginal vein is too shallow for any conventional US probe to measure the blood flow speed and too small to locate and detect, a high-frequency US transducer with a high resolution and high sensitivity was used to measure blood flow velocity more easily. Lead magnesium niobate-lead titanate (PMN-PT) single crystal (33% PT content) (H. C. Materials Corporation, Bolingbrook, IL, USA) was selected as an active transducer element due to its high electromechanical coupling coefficient, low dielectric loss, and high dielectric constant [26]. These features make it an ideal candidate for fabricating very sensitive, small-aperture, and high-frequency transducers. A polarized PMN-PT plate was lapped to 45 μm for achieving the desired working frequency. A matching layer made by mixing 2–3 μm silver particles (Sigma-Aldrich Inc., St Louis, MO, USA) with Insulcast 501 and Insulcure 9 (American Safety Technologies, Roseland, NJ, USA) was cured over the PMN-PT sample.
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To optimize the transducer performance, the matching layer was lapped to 9 μm. A 3-mmthick conductive backing material, E-solder 3022 (VonRoll Isola, New Haven, CT, USA), was cured on the other side of the PMN-PT sample. Active element plugs were diced out at 0.4 × 0.4 mm aperture size using a mechanical dicing saw (Tcar 864-1 Thermocarbon, Inc., Casselberry, FL, USA). The transducer stack was then housed within a polyimide tube with an inner diameter of 0.57 mm (MedSource Technologies, Trenton, GA, USA) using Epotek 301 (Epoxy Technology Inc., Billerica, MA, USA). The polyimide tube was used to provide electrical isolation from the metal housing of the 20-gauge needle (20 GA 1-1/2″, McMaster-Carr Supply Company, Atlanta, GA, USA), whose inner diameter was 0.66 mm and outer diameter was 0.91 mm. An electrical connector was fixed to the conductive backing using conductive epoxy. An electrode was sputtered across the silver matching layer and the needle housing to form a common ground connection. Vapor-deposited parylene with a thickness of 12 μm was used to coat the aperture and the needle housing.
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The signal-to-noise ratio (SNR) of the Doppler system significantly affects blood flow measurement in the rabbit marginal ear vein because of the small backscatter coefficient of blood. Therefore, a customized high-frequency PW Doppler system was developed to achieve a high SNR [12]. The acquired digitized Doppler signals were converted into a realtime directional spectrogram using Labview software (National Instruments Corp., Austin, TX, USA). Further off- line analysis was conducted using Matlab software (The MathWorks Inc., Natick, MA, USA). The experimental arrangement is shown in Fig. 3. The details of the system and software can be found in [26]. 2.4 Blood flow estimation Estimation of blood flow velocity by the Doppler US method has been widely used [27,28]. The blood flow velocity can be determined as [28]:
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where Δf is the measured Doppler shift frequency, fo is the center frequency of the transducer, c is the sound velocity in a medium, and θ is the angle between the US beam and the flow [26]. In the Doppler US measurements, fo was 40 MHz, c was 1,500 m/s, and θ was about 45°. 2.5 Therapeutic ultrasound system A Sonitron 2000 system (Artison Corp., Inola, OK, USA) was used to generate highintensity US for clot dissolution in rabbit vessels in vivo. The system was set to a frequency of 1 MHz with 7-W/cm2 intensity (ISATA) and a 30% duty cycle. US was applied at an occlusion site for 10 minutes after the injection of 0.3–0.6 cc of microbubbles (Artison Corp., Inola, OK, USA), composed of a lipid shell with a polymer stabilizer and filled with a perfluorocarbon gas core, with a concentration of 13 × 108 bubbles/ml and a mean diameter of 2.4 μm through the other ear vein. The diameter of the probe tip was about 1 mm. 2.6 Therapeutic ultrasound procedures
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The skin above the rabbit marginal ear vein was removed surgically to allow closer contact of the Doppler transducer and high-intensity transducer with the vessel so as to increase the sensitivity, as shown in Fig. 3. Exposure of the vessel was also helpful to observe the microbubbles and blood flow using a microscope. US gel (Aloe-Sound Gel Plus, Rich-Mar Corporation, Canada) was applied over the exposed vessel in order to minimize the thermal effects induced by sonothrombolysis. Blood flow velocities at the occlusion site and upstream and downstream from the occlusion site were measured using the Doppler system before and after occlusion. High-intensity US was applied at the occlusion site for 10 minutes using the Sonitron system after the microbubbles had been injected through the ear vein. The temperature change was monitored by a thermocouple probe (DP460 Thermocouple, Omega Engineering Inc., Stamford, CT, USA) in the coupling medium (US gel) at the treatment site. After high-intensity US insonation, blood flow velocity was measured by the Doppler system at the treatment site to confirm the opening of the
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occlusion. All measurements were performed in a quiet room with stable temperature (16.8 °C).
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2.7 In vitro measurements of cavitation In vitro measurements of cavitation were performed to investigate the mechanism of sonothrombolysis. The experimental setup is shown in Fig. 4. 20 cc of microbubbles were injected into a transfer pipette (Samco Scientific, San Fernando, CA, USA) filled with distilled deionized (DI) water. The tip of the Sonitron probe was directly dipped into microbbles to generate cavitation. A 5-MHz ultrasonic transducer (Panametrics V308, Olympus NDT, MA, USA) with a −6 dB bandwidth of 75 % was used as a receiver to capture the cavitation signal and directly connected to a digital oscilloscope (LeCroy LC534, LeCroy Corporation, Chest- nut Ridge, NY) for data visualization and acquisition. A Hamming window was used for the fast Fourier transformation (FFT), and an 1:31 weighted average was used to obtain the FFT spectrum. The transducer was perpendicularly placed 6 cm away from the Sonitron probe, in which the distance is the focal length of the transducer. The system was set at a frequency of 1 MHz with a 1-W/cm2 intensity (ISATA).
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3. Results Figure 5 shows the pulse-echo waveform and frequency spectrum of the needle transducer from the quartz reflector in the DI water. When the transducer was excited by a highbandwidth pulser/receiver (PR5900, Olympus NDT Inc., Waltham, MA, USA) with a 1-J energy setting, the maximum output voltage (Vp-p) of the unamplified echo signal was close to 2.0 V. The center frequency of the transducer was about 40 MHz and the bandwidth at −6 dB was measured to be approximately 54%. The measured two-way insertion loss of the needle transducer is −16 dB at 40 MHz. The outstanding performance implies that the customized 40-MHz needle transducer should have sufficient sensitivity and resolution for detecting weak Doppler signals from small vessels, based on a previous study [29].
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As mentioned, the blood flow velocity was measured by the customized 40-MHz Doppler system. The measurement was performed before and after US insonation to determine the recovery situation of the occlusion site. Three sites (the occlusion site, 1 mm from the occlusion site towards upstream, and 1 mm from the occlusion site towards downstream) in the marginal vein (~ 8 cm away from the base of the ear) were monitored. Before occlusion, the blood flow velocity was found to be about 4 cm/s, as shown in the Doppler spectrogram (Fig. 6). After occlusion by laser photothrombosis, the flow velocity at the sites was measured. The results are shown in Fig. 7. The flow velocity at the upstream and downstream from the occlusion site decreased to 2–3 cm/s. The signal of the spectrogram at the occlusion site was not from the blood flow, but from the system noise, which could be easily differentiated by hearing the sound in the attached audio file. After three highintensity US insonations for 10 minutes at the occlusion site, the blood flow velocity was found to recover to 2–3 cm/s at the occlusion site and 4 cm/s at the upstream and downstream, as shown in Fig. 8. The results show that the customized high- frequency Doppler transducer can monitor the recanalization of occluded veins. For the thermal safety of the tissue and vessel, there was a pause of 1–2 minutes between each US insonation. Fluorescein angiography was used to confirm the total occlusion by laser photothrombosis J Med Biol Eng. Author manuscript; available in PMC 2014 October 27.
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and the opening of the occlusion by sonothrombolysis. During the treatment, the temperature at the site was found to increase from 21.1 °C to 30.3 °C, as shown in Fig. 9. The initial low temperature (21.1 °C) was considered to be due to the coupling medium. The temperature gradually increased with treatment time and remained at around 30 °C after 3 minutes. The temperature change should not induce significant US thermal bioeffects. For the other rabbit, whose ear marginal vein was occluded by thrombin, the occlusion could also be opened by sonothrombolysis, as shown in Table 1. As a control study, the other ear of each rabbit with occluded ear marginal veins was not subjected to US insonation treatment after microbubbles were injected through the other ear vein. It was found that the blood flow velocity did not change and the occlusion was not opened even after 3 hours. The results of the in vitro measurements of cavitation are shown in Fig. 10. Although there were harmonics generated by microbubbles in the DI water, the magnitude of the harmonic signal generated by the microbubbles when the Sonitron probe was turned on was much higher and additional sub-harmonics appeared at about every 500 kHz. After 1 minute, the magnitude of harmonics decreased and sub-harmonics disappeared (Fig. 10c).
4. Discussion NIH-PA Author Manuscript
This study demonstrates that high-intensity US insonation in combination with microbubbles at the occlusion site can reopen an occlusion in the ear marginal vein of rabbits in vivo without the application of any thrombotic agent. The veins in the present study were occluded by either laser photothrombosis or thrombin. The Doppler spectra confirmed that the blood flow recovered from the total occlusion after three 10-minute highintensity US insonations. Few in vivo experiments have demonstrated occlusion reopening by sonothrombolysis with microbubbles alone [21].
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The mechanisms for sonothrombolysis may include thermal effects and/or mechanical effects. In this study, thermal effects may be minimal since the temperature increment was small during a section of a 10-minute US insonation. Thrombolysis was reported to be enhanced at temperatures over 37 °C when a thrombolytic agent was added [30]. Thus, the possibility of thermal effects for thrombolysis is low at 30 °C. As shown in Fig. 10, strong harmonic signals were generated by US insonation when there were microbubbles in the tube. This indicates that the mechanical effects are likely to be the main cause of the sonothrombolysis effect. Since chemical agents were excluded in the in vitro measurements of cavitation, the results suggest that the mechanism of sonothrombolysis in this study is probably due to the inertial cavitation induced by the microbubbles. These experimental results show good agreement with previously reported results [3–5]. Even though in vivo harmonic signals could not be measured in this experimental setup, it can be assumed that the cavitation exists in the rabbit vein in vivo during the US insonation considering the results from in vitro experiments of higher harmonic signals, as shown in Fig. 10. Although the vessel wall occluded using laser photothrombosis was deformed, the occlusion site was smaller than 1 mm in length. When thrombin was used, the vessel wall was not deformed after blood flow stoppage by clamps. However, the length of the occlusion was a few mm. Regardless of the occlusion method, the results show that sonothrombolysis can
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recanalize veins with microbubbles only. Bioeffects or fatality may be reduced if no thrombolytic agent is used for sonothrombolysis, making the proposed system useful for clinical applications. The vascular network of the rabbit ear is composed of an auricular artery in the center and marginal veins, which are connected by numerous arteriovenous loops. Venous thrombus would not result in the total blockage of the blood flow in the marginal vein, but in a local blockage of the flow near the thrombosis site. Therefore, the blood flow of the proximal and distal vessels may enhance thrombolysis at the thrombosis site in the marginal vein. It is also known that pulsatile flow prevents blood coagulation inside a vessel and enhances thrombolysis [31]. Although the occlusion site was in the marginal vein, the surrounding arterioles may have enhanced the thrombolysis. According to a study on rabbit retinal vessels [29], both occlusion and revascularization depend on the anatomy and network of the rabbit retinal vasculature and blood flow speed. The vascular structure of the rabbit ear and the corresponding blood circulation system may also affect the results of sonothrombolysis. Further research is required for a better understanding of the effects of the blood circulation system on thrombolysis.
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Even though the results show that an occlusion can be reopened by US insonation without a thrombolytic agent in the rabbit ear marginal vein, care must be taken in interpreting the results. The method should be applied to other rabbit vessels, other animal models, or even human applications, as they have different blood circulation systems. The results reported in this study are preliminary and have to be confirmed by further studies on a wider range of animals for statistical analysis. A relatively invasive procedure was used in this study, where the vessels were exposed for easier experimental observation and more effective US insonation. This may be eliminated by designing better experimental protocols.
5. Conclusion
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Sonothrombolysis, combined with the injection of microbubbles and without a thrombolytic agent, of rabbit ear veins was studied in vivo. The occluded vessels at ear marginal veins induced by either laser photothrombosis or thrombin were opened after three 10-minute US insonations. Blood flow velocity measured by a 40-MHz Doppler system indicates recovery at the occlusion site after high-intensity US insonation with microbubbles injected through the other ear vein. Fluorescein angiography confirmed the opening of the occlusion site. The results presented in this paper suggest the potential use of high-intensity US for small vascular therapies such as the treatment of the retinal vein occlusion [32]. Sonothrombolysis without any thrombolytic agent minimizes unwanted bioeffects caused by the thrombolytic agent, making it a safer approach clinically.
Acknowledgments This work was supported by the National Institutes of Health under grant P41-EB2182 and the National Research Foundation of Korea (NRF) under a grant funded by the Korean government (MEST) (NRF-2011-0017984). The authors would like to thank Mr. Louis Fox for a private grant and Lina Flores and Fernando Gallardo for their technical support.
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Figure 1.
Exposed rabbit ear marginal vein occlusion created by laser photothrombosis and blood flow direction (left). Confirmation of the targeted vein vessel occlusion by fluorescein angiography (right).
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NIH-PA Author Manuscript Figure 2.
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Thrombin occlusion created by clamps after exposure of rabbit ear marginal vein (left). Confirmation of the vein vessel occlusion after thrombin injection by fluorescein angiography (right).
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Figure 3.
Block diagram of the experimental setup.
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Figure 4.
In vitro cavitation measurement experimental setup.
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NIH-PA Author Manuscript Figure 5.
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Measured pulse-echo waveform and frequency spectrum of the 40-MHz needle transducer.
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Figure 6.
Doppler spectrogram of the rabbit ear marginal vein before occlusion.
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Figure 7.
Doppler spectrogram of the rabbit ear marginal vein after laser photothrombolysis occlusion.
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Figure 8.
Doppler spectrogram of the rabbit ear marginal vein after opening of the occlusion by highintensity US insonation right after microbubble injection.
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Figure 9.
Temperature changes of the occluded rabbit ear marginal vein produced by US insonation.
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Figure 10.
FFT spectra of cavitation in (a) DI water, (b) with microbubbles, and (c) with microbubbles one minute after turning on the US.
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Table 1
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Blood flow velocities measured from Doppler spectrum at various sites before and after sonothrombolysis (mean ± standard deviation in cm/s). Rabbit # / Occlusion method
Time
Upstream
Occlusion site
Downstream
Before
2.3 ± 0.1
N/A
3.3 ± 0.8
1 / Laser After
4.0 ± 0.2
2.2 ± 0.5
4.0 ± 0.3
Before
2.7 ± 0.6
N/A
4.3 ± 0.3
2 / Laser After
3.5 ± 0.5
3.4 ± 0.7
4.7 ± 0.5
Before
3.5 ± 0.3
N/A
3.7 ± 0.4
After
4.3 ± 0.6
3.6 ± 0.9
4.0 ± 0.4
3 / Thrombin
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Published in final edited form as: J Med Biol Eng. 2013 ; 33(1): 103–110.
In vivo Sonothrombolysis of Ear Marginal Vein of Rabbits Monitored with High-frequency Ultrasound Needle Transducer Ruimin Chen1,†, Dong-Guk Paeng2,†, Kwok Ho Lam1, Qifa Zhou1,*, K. Kirk Shung1, Naoki Matsuoka3, and Mark S. Humayun4 1NIH
Resource Center for Medical Ultrasonic Transducer Technology, Department of Biomedical Engineering, University of Southern California, Los Angeles, CA 90089, USA
2Department
of Ocean System Engineering, Interdisciplinary Postgraduate Program in Biomedical Engineering, Jeju National University, Jeju, Korea 690-756
3Division
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of Ophthalmology and Visual Science, Graduate School of Medical and Dental Sciences, Niigata University, Niigata 950-2181, Japan
4Doheny
Eye Institute, University of Southern California, Los Angeles, CA 90033, USA
Abstract
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Ultrasound (US) is known to enhance thrombolysis when thrombolytic agents and/or microbubbles are injected into the targeted vessels. In this research, high-intensity US (1 MHz, 7 W/cm2, 30 % duty cycle) was applied in vivo to the ear marginal vein of three rabbits which was occluded by either laser photothrombosis or thrombin, right after the injection of 0.3~0.6 cc of microbubbles (13 × 108 bubbles/ml of concentration) through the other ear vein without using any thrombolytic agent. To determine the effect of the sonothrombolysis, the blood flow velocity near the occlusion site in the vein was measured by a custom-made 40-MHz US needle transducer and its corresponding Doppler US system. The Doppler spectra show that the blood flow velocity recovered from total occlusion after three 10-minute high-intensity US treatments. Fluorescein angiography was employed to confirm the opening of the vessel occlusion. A control study of three rabbits with only the microbubble injection showed no recovery on the occlusion in 3 hours. The results show that the sonothrombolysis in the rabbit ear marginal vein can be achieved with microbubbles only. The results of cavitation measurements indicate that the mechanism of sonothrombolysis is probably due to the cavitation induced by the microbubbles. Without the need of applying any thrombolytic agent, high-intensity US has high potential for therapies targeting on small blood vessels.
Keywords Therapeutic ultrasound; Sonothrombolysis; Rabbit ear marginal vein; Microbubbles
*
Corresponding author: Qifa Zhou, Tel: +1-213-7409475, [email protected]. †These authors contributed equally to this work
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1. Introduction NIH-PA Author Manuscript
The use of thrombolytic therapy has attracted a lot of attention in clinical trials and has proven its value in treating common thrombotic diseases. The therapy is applied to restore the blood flow and oxygenation for body organs by removing blood clots inside blood vessels. Among various thrombolytic methods, ultrasound (US) thrombolysis has been found to be promising for facilitating recanalization of occluded arteries, especially for patients with acute strokes [1,2]. Combined with thrombolytic agents and microbubbles, sonothrombolysis helps the disintegration and dissolution of blood clots [3–5]. Many sonothrombolysis studies have been conducted in the laboratory using animal models [6–8]. For human applications, pilot studies of sonothrombolysis for stroke patients have been pursued [9].
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Although the process of sonothrombolysis can be enhanced when microbubbles are used in conjunction with thrombolytic agents [10], the use of chemical agents is constrained by numerous patient factors which might cause severe adverse effects, sometimes even fatal [11]. To eliminate the use of chemical agents and their associated adverse effects, US has been applied to thrombolysis with microbubbles alone [12]. There has also been evidence demonstrating that US alone can induce nonenzymatic thrombolysis in vitro and in clinical settings [13–15]. Although a number of in vitro sonothrombolysis experiments have been reported for US enhancement of thrombolysis [16–21], few in vivo studies have been conducted without using chemical agents [22]. Most studies have been performed on arteries. In the present study, in vivo sonothrombolysis without using thrombolytic agents was performed in a rabbit marginal ear vein. Only non-specific or targeted microbubbles were used for enhancing the efficiency of sonothrombolysis. During the process, the sonothrombolysis effect on blood flow in veins was graphically recorded in pulsed-wave (PW) Doppler mode using a custommade 40-MHz US needle transducer and its corresponding Doppler US system. The mechanism of sonothrombolysis was examined through in vitro measurements of cavitation.
2. Methods 2.1 Animals and treatment protocol
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Three pigmented rabbits obtained from Irish Farms (Norco, CA, USA), each weighing from 2 to 3 kg, were used in this study. The rabbit is a good animal model for thrombolytic therapy because it is relatively cheap and experimental procedures are well-established [23]. The rabbit marginal ear vein is a large convex vessel passing along the ear edge. This vein can be easily observed under a microscope through a thin layer of auricular cartilage and epithelium, which makes it accessible for manipulations either from outside or inside [24]. All animal procedures were carried out in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and with the Institutional Animal Care and Use Committee of University of Southern California. Initial sedation was induced with intramuscular injection of ketamine hydrochloride (25 mg/kg) and xylazine hydrochloride (6 mg/kg). Rabbits remained
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anesthetized throughout the entire experiment. After the experiment was concluded, the rabbits were euthanized with an intracardiac injection of sodium pentobarbital (120 mg/kg).
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2.2 Vascular occlusion One of the most reliable models of creating vein occlusion in rabbits is laser photothrombosis [25]. The photosensitizer Rose Bengal (tetrachlorotetraiodo fluorescein sodium) was injected through the vein of an ear to the targeted vessel by an argon laser to create an isolated vein occlusion without occluding the artery. The size of the laser spot was about 500 μm and the laser power was set to 100–300 mW with a duration of 200–1000 ms. The laser photothrombosis occlusion resulted in the deformation and damage of the vessel wall, which differs from the natural process of occlusion. The laser photothrombosis occlusion inside a marginal ear vein is shown in Fig. 1. In order to avoid the deformation and damage of the vessel wall, another occlusion model was applied by injecting 0.04 ml (200U) of Bovine Thrombin (BioPharm Laboratories, Alpine, UT, USA) either inside the clamped vessel by a 30-gauge needle or outside the vessel after stopping the blood flow of the targeted vessel using clamps, as shown in Fig. 2. Fluorescein angiography was used before and after occlusion to confirm the total occlusion, as shown in Figs. 1 and 2.
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2.3 High-frequency needle transducer and high-frequency ultrasound doppler system Since the rabbit ear marginal vein is too shallow for any conventional US probe to measure the blood flow speed and too small to locate and detect, a high-frequency US transducer with a high resolution and high sensitivity was used to measure blood flow velocity more easily. Lead magnesium niobate-lead titanate (PMN-PT) single crystal (33% PT content) (H. C. Materials Corporation, Bolingbrook, IL, USA) was selected as an active transducer element due to its high electromechanical coupling coefficient, low dielectric loss, and high dielectric constant [26]. These features make it an ideal candidate for fabricating very sensitive, small-aperture, and high-frequency transducers. A polarized PMN-PT plate was lapped to 45 μm for achieving the desired working frequency. A matching layer made by mixing 2–3 μm silver particles (Sigma-Aldrich Inc., St Louis, MO, USA) with Insulcast 501 and Insulcure 9 (American Safety Technologies, Roseland, NJ, USA) was cured over the PMN-PT sample.
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To optimize the transducer performance, the matching layer was lapped to 9 μm. A 3-mmthick conductive backing material, E-solder 3022 (VonRoll Isola, New Haven, CT, USA), was cured on the other side of the PMN-PT sample. Active element plugs were diced out at 0.4 × 0.4 mm aperture size using a mechanical dicing saw (Tcar 864-1 Thermocarbon, Inc., Casselberry, FL, USA). The transducer stack was then housed within a polyimide tube with an inner diameter of 0.57 mm (MedSource Technologies, Trenton, GA, USA) using Epotek 301 (Epoxy Technology Inc., Billerica, MA, USA). The polyimide tube was used to provide electrical isolation from the metal housing of the 20-gauge needle (20 GA 1-1/2″, McMaster-Carr Supply Company, Atlanta, GA, USA), whose inner diameter was 0.66 mm and outer diameter was 0.91 mm. An electrical connector was fixed to the conductive backing using conductive epoxy. An electrode was sputtered across the silver matching layer and the needle housing to form a common ground connection. Vapor-deposited parylene with a thickness of 12 μm was used to coat the aperture and the needle housing.
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The signal-to-noise ratio (SNR) of the Doppler system significantly affects blood flow measurement in the rabbit marginal ear vein because of the small backscatter coefficient of blood. Therefore, a customized high-frequency PW Doppler system was developed to achieve a high SNR [12]. The acquired digitized Doppler signals were converted into a realtime directional spectrogram using Labview software (National Instruments Corp., Austin, TX, USA). Further off- line analysis was conducted using Matlab software (The MathWorks Inc., Natick, MA, USA). The experimental arrangement is shown in Fig. 3. The details of the system and software can be found in [26]. 2.4 Blood flow estimation Estimation of blood flow velocity by the Doppler US method has been widely used [27,28]. The blood flow velocity can be determined as [28]:
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where Δf is the measured Doppler shift frequency, fo is the center frequency of the transducer, c is the sound velocity in a medium, and θ is the angle between the US beam and the flow [26]. In the Doppler US measurements, fo was 40 MHz, c was 1,500 m/s, and θ was about 45°. 2.5 Therapeutic ultrasound system A Sonitron 2000 system (Artison Corp., Inola, OK, USA) was used to generate highintensity US for clot dissolution in rabbit vessels in vivo. The system was set to a frequency of 1 MHz with 7-W/cm2 intensity (ISATA) and a 30% duty cycle. US was applied at an occlusion site for 10 minutes after the injection of 0.3–0.6 cc of microbubbles (Artison Corp., Inola, OK, USA), composed of a lipid shell with a polymer stabilizer and filled with a perfluorocarbon gas core, with a concentration of 13 × 108 bubbles/ml and a mean diameter of 2.4 μm through the other ear vein. The diameter of the probe tip was about 1 mm. 2.6 Therapeutic ultrasound procedures
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The skin above the rabbit marginal ear vein was removed surgically to allow closer contact of the Doppler transducer and high-intensity transducer with the vessel so as to increase the sensitivity, as shown in Fig. 3. Exposure of the vessel was also helpful to observe the microbubbles and blood flow using a microscope. US gel (Aloe-Sound Gel Plus, Rich-Mar Corporation, Canada) was applied over the exposed vessel in order to minimize the thermal effects induced by sonothrombolysis. Blood flow velocities at the occlusion site and upstream and downstream from the occlusion site were measured using the Doppler system before and after occlusion. High-intensity US was applied at the occlusion site for 10 minutes using the Sonitron system after the microbubbles had been injected through the ear vein. The temperature change was monitored by a thermocouple probe (DP460 Thermocouple, Omega Engineering Inc., Stamford, CT, USA) in the coupling medium (US gel) at the treatment site. After high-intensity US insonation, blood flow velocity was measured by the Doppler system at the treatment site to confirm the opening of the
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occlusion. All measurements were performed in a quiet room with stable temperature (16.8 °C).
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2.7 In vitro measurements of cavitation In vitro measurements of cavitation were performed to investigate the mechanism of sonothrombolysis. The experimental setup is shown in Fig. 4. 20 cc of microbubbles were injected into a transfer pipette (Samco Scientific, San Fernando, CA, USA) filled with distilled deionized (DI) water. The tip of the Sonitron probe was directly dipped into microbbles to generate cavitation. A 5-MHz ultrasonic transducer (Panametrics V308, Olympus NDT, MA, USA) with a −6 dB bandwidth of 75 % was used as a receiver to capture the cavitation signal and directly connected to a digital oscilloscope (LeCroy LC534, LeCroy Corporation, Chest- nut Ridge, NY) for data visualization and acquisition. A Hamming window was used for the fast Fourier transformation (FFT), and an 1:31 weighted average was used to obtain the FFT spectrum. The transducer was perpendicularly placed 6 cm away from the Sonitron probe, in which the distance is the focal length of the transducer. The system was set at a frequency of 1 MHz with a 1-W/cm2 intensity (ISATA).
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3. Results Figure 5 shows the pulse-echo waveform and frequency spectrum of the needle transducer from the quartz reflector in the DI water. When the transducer was excited by a highbandwidth pulser/receiver (PR5900, Olympus NDT Inc., Waltham, MA, USA) with a 1-J energy setting, the maximum output voltage (Vp-p) of the unamplified echo signal was close to 2.0 V. The center frequency of the transducer was about 40 MHz and the bandwidth at −6 dB was measured to be approximately 54%. The measured two-way insertion loss of the needle transducer is −16 dB at 40 MHz. The outstanding performance implies that the customized 40-MHz needle transducer should have sufficient sensitivity and resolution for detecting weak Doppler signals from small vessels, based on a previous study [29].
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As mentioned, the blood flow velocity was measured by the customized 40-MHz Doppler system. The measurement was performed before and after US insonation to determine the recovery situation of the occlusion site. Three sites (the occlusion site, 1 mm from the occlusion site towards upstream, and 1 mm from the occlusion site towards downstream) in the marginal vein (~ 8 cm away from the base of the ear) were monitored. Before occlusion, the blood flow velocity was found to be about 4 cm/s, as shown in the Doppler spectrogram (Fig. 6). After occlusion by laser photothrombosis, the flow velocity at the sites was measured. The results are shown in Fig. 7. The flow velocity at the upstream and downstream from the occlusion site decreased to 2–3 cm/s. The signal of the spectrogram at the occlusion site was not from the blood flow, but from the system noise, which could be easily differentiated by hearing the sound in the attached audio file. After three highintensity US insonations for 10 minutes at the occlusion site, the blood flow velocity was found to recover to 2–3 cm/s at the occlusion site and 4 cm/s at the upstream and downstream, as shown in Fig. 8. The results show that the customized high- frequency Doppler transducer can monitor the recanalization of occluded veins. For the thermal safety of the tissue and vessel, there was a pause of 1–2 minutes between each US insonation. Fluorescein angiography was used to confirm the total occlusion by laser photothrombosis J Med Biol Eng. Author manuscript; available in PMC 2014 October 27.
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and the opening of the occlusion by sonothrombolysis. During the treatment, the temperature at the site was found to increase from 21.1 °C to 30.3 °C, as shown in Fig. 9. The initial low temperature (21.1 °C) was considered to be due to the coupling medium. The temperature gradually increased with treatment time and remained at around 30 °C after 3 minutes. The temperature change should not induce significant US thermal bioeffects. For the other rabbit, whose ear marginal vein was occluded by thrombin, the occlusion could also be opened by sonothrombolysis, as shown in Table 1. As a control study, the other ear of each rabbit with occluded ear marginal veins was not subjected to US insonation treatment after microbubbles were injected through the other ear vein. It was found that the blood flow velocity did not change and the occlusion was not opened even after 3 hours. The results of the in vitro measurements of cavitation are shown in Fig. 10. Although there were harmonics generated by microbubbles in the DI water, the magnitude of the harmonic signal generated by the microbubbles when the Sonitron probe was turned on was much higher and additional sub-harmonics appeared at about every 500 kHz. After 1 minute, the magnitude of harmonics decreased and sub-harmonics disappeared (Fig. 10c).
4. Discussion NIH-PA Author Manuscript
This study demonstrates that high-intensity US insonation in combination with microbubbles at the occlusion site can reopen an occlusion in the ear marginal vein of rabbits in vivo without the application of any thrombotic agent. The veins in the present study were occluded by either laser photothrombosis or thrombin. The Doppler spectra confirmed that the blood flow recovered from the total occlusion after three 10-minute highintensity US insonations. Few in vivo experiments have demonstrated occlusion reopening by sonothrombolysis with microbubbles alone [21].
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The mechanisms for sonothrombolysis may include thermal effects and/or mechanical effects. In this study, thermal effects may be minimal since the temperature increment was small during a section of a 10-minute US insonation. Thrombolysis was reported to be enhanced at temperatures over 37 °C when a thrombolytic agent was added [30]. Thus, the possibility of thermal effects for thrombolysis is low at 30 °C. As shown in Fig. 10, strong harmonic signals were generated by US insonation when there were microbubbles in the tube. This indicates that the mechanical effects are likely to be the main cause of the sonothrombolysis effect. Since chemical agents were excluded in the in vitro measurements of cavitation, the results suggest that the mechanism of sonothrombolysis in this study is probably due to the inertial cavitation induced by the microbubbles. These experimental results show good agreement with previously reported results [3–5]. Even though in vivo harmonic signals could not be measured in this experimental setup, it can be assumed that the cavitation exists in the rabbit vein in vivo during the US insonation considering the results from in vitro experiments of higher harmonic signals, as shown in Fig. 10. Although the vessel wall occluded using laser photothrombosis was deformed, the occlusion site was smaller than 1 mm in length. When thrombin was used, the vessel wall was not deformed after blood flow stoppage by clamps. However, the length of the occlusion was a few mm. Regardless of the occlusion method, the results show that sonothrombolysis can
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recanalize veins with microbubbles only. Bioeffects or fatality may be reduced if no thrombolytic agent is used for sonothrombolysis, making the proposed system useful for clinical applications. The vascular network of the rabbit ear is composed of an auricular artery in the center and marginal veins, which are connected by numerous arteriovenous loops. Venous thrombus would not result in the total blockage of the blood flow in the marginal vein, but in a local blockage of the flow near the thrombosis site. Therefore, the blood flow of the proximal and distal vessels may enhance thrombolysis at the thrombosis site in the marginal vein. It is also known that pulsatile flow prevents blood coagulation inside a vessel and enhances thrombolysis [31]. Although the occlusion site was in the marginal vein, the surrounding arterioles may have enhanced the thrombolysis. According to a study on rabbit retinal vessels [29], both occlusion and revascularization depend on the anatomy and network of the rabbit retinal vasculature and blood flow speed. The vascular structure of the rabbit ear and the corresponding blood circulation system may also affect the results of sonothrombolysis. Further research is required for a better understanding of the effects of the blood circulation system on thrombolysis.
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Even though the results show that an occlusion can be reopened by US insonation without a thrombolytic agent in the rabbit ear marginal vein, care must be taken in interpreting the results. The method should be applied to other rabbit vessels, other animal models, or even human applications, as they have different blood circulation systems. The results reported in this study are preliminary and have to be confirmed by further studies on a wider range of animals for statistical analysis. A relatively invasive procedure was used in this study, where the vessels were exposed for easier experimental observation and more effective US insonation. This may be eliminated by designing better experimental protocols.
5. Conclusion
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Sonothrombolysis, combined with the injection of microbubbles and without a thrombolytic agent, of rabbit ear veins was studied in vivo. The occluded vessels at ear marginal veins induced by either laser photothrombosis or thrombin were opened after three 10-minute US insonations. Blood flow velocity measured by a 40-MHz Doppler system indicates recovery at the occlusion site after high-intensity US insonation with microbubbles injected through the other ear vein. Fluorescein angiography confirmed the opening of the occlusion site. The results presented in this paper suggest the potential use of high-intensity US for small vascular therapies such as the treatment of the retinal vein occlusion [32]. Sonothrombolysis without any thrombolytic agent minimizes unwanted bioeffects caused by the thrombolytic agent, making it a safer approach clinically.
Acknowledgments This work was supported by the National Institutes of Health under grant P41-EB2182 and the National Research Foundation of Korea (NRF) under a grant funded by the Korean government (MEST) (NRF-2011-0017984). The authors would like to thank Mr. Louis Fox for a private grant and Lina Flores and Fernando Gallardo for their technical support.
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Figure 1.
Exposed rabbit ear marginal vein occlusion created by laser photothrombosis and blood flow direction (left). Confirmation of the targeted vein vessel occlusion by fluorescein angiography (right).
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NIH-PA Author Manuscript Figure 2.
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Thrombin occlusion created by clamps after exposure of rabbit ear marginal vein (left). Confirmation of the vein vessel occlusion after thrombin injection by fluorescein angiography (right).
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Figure 3.
Block diagram of the experimental setup.
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Figure 4.
In vitro cavitation measurement experimental setup.
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NIH-PA Author Manuscript Figure 5.
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Measured pulse-echo waveform and frequency spectrum of the 40-MHz needle transducer.
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Figure 6.
Doppler spectrogram of the rabbit ear marginal vein before occlusion.
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Figure 7.
Doppler spectrogram of the rabbit ear marginal vein after laser photothrombolysis occlusion.
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Figure 8.
Doppler spectrogram of the rabbit ear marginal vein after opening of the occlusion by highintensity US insonation right after microbubble injection.
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Figure 9.
Temperature changes of the occluded rabbit ear marginal vein produced by US insonation.
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Figure 10.
FFT spectra of cavitation in (a) DI water, (b) with microbubbles, and (c) with microbubbles one minute after turning on the US.
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Table 1
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Blood flow velocities measured from Doppler spectrum at various sites before and after sonothrombolysis (mean ± standard deviation in cm/s). Rabbit # / Occlusion method
Time
Upstream
Occlusion site
Downstream
Before
2.3 ± 0.1
N/A
3.3 ± 0.8
1 / Laser After
4.0 ± 0.2
2.2 ± 0.5
4.0 ± 0.3
Before
2.7 ± 0.6
N/A
4.3 ± 0.3
2 / Laser After
3.5 ± 0.5
3.4 ± 0.7
4.7 ± 0.5
Before
3.5 ± 0.3
N/A
3.7 ± 0.4
After
4.3 ± 0.6
3.6 ± 0.9
4.0 ± 0.4
3 / Thrombin
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