| BackgroundsBenign prostatic hyperplasia (BPH) is a common disease of aged men, featured with progressive dysuria. BPH is one of the main reasons of bladder outlet obstruction because the urethra may be stressed and may lead to retention, hydronephrosis and even bladder and renal dysfunction which may severely reduce life quality.There are several methods for BPH treatment, including medication, surgery and minimally invasive therapy. With the emerging and fast developing of novel ultrasound therapy techniques in recent years, more and more studies focused on ablation of BPH using therapeutic ultrasound (TUS), including high intensity focused ultrasound (HIFU) and histotripsy.Acoustic cavitation is one of the major physical effects of ultrasound. Microbubble (MB) enhanced ultrasound (MEUS) cavitation can induce shockwave, micro-jets and other mechanical effects, which may lead to microvascular disruption, hemorrhage and thrombosis. Since MB can only travel in blood vessels, like red blood cell, the damage effects of MEUS mainly happen to the vascular wall. BPH is a hyperplastic disease with abundant neovasculature which happens in inner gland. The neovasculature usually grows with many developmental defects and it is physically vulnerable. This makes the mechanical disruption possible by using MEUS. In the previous study, we found MEUS can make significant damage to prostate microvessel. However, the impact of MEUS vascular effect on prostate blood perfusion and the probability of prostate ablation using MEUS had never been investigated.Objectives1. To study the effect of pulsed TUS combined with circulating MBs on prostatic microvasculature and the whole prostatic blood perfusion in a canine model, and to explore its related pathological mechanisms.2. To explore the pathological process of the prostate after treatment of pulsed TUS combined with circulating MBs.Materials and Methods1. MaterialsCZ-960digital ultrasonic cavitation therapy device was an instrument for emission of pulsed TUS, equipped with a transducer with frequency of831kHz. Pulse repetition frequency, pulse length, and peak acoustic pressure were adjustable.Two diagnostic ultrasound systems were applied for contrast enhanced ultrasound (CEUS) with a low mechanical index (MI). They were Philips iU22ultrasound system, equipped with an L12-5linear probe, and Siemens Acuson S2000ultrasound system, equipped with a9L4linear probe. Both of them were able to perform acoustic quantitative analysis.Microbubbles, named Zhifuxian, consisted of perfluoropropane gas encapsulated by a lipid-shell, and played roles of both contrast agents in CEUS and cavitation nuclei in the treatment. The MBs were measured to be2-10μm in diameter,98%of which were less than8μm. And the concentration was (4-9)×109/ml.2. MethodsFirst part:3adult male dogs were generally anesthetized and their prostates were surgically exposed. The transducer of the ultrasonic cavitation therapy device was held directly and perpendicularly above the prostate, with only half part of one prostate sonicated by pulsed therapeutic ultrasound, leaving the other part as the control. The prostates was treated for10min while MBs at0.1ml/kg were intravenously injected. After that,2%Evans blue (EB) dye solution was injected. Then the prostates were harvested30min later and cut into2parts for comparison of the EB perfusion between the sonicated area and the control area.20dogs were randomly assigned into3groups:ultrasound combined with MB group (US+MB, n=10), ultrasound group (US, n=5) and MB group (MB, n=5). In US+MB group, the whole prostates were sonicated perpendicularly for10minutes by the pulsed ultrasound transducer, as diluted MB suspension (0.1ml/kg MBs diluted into10ml saline) was infused for approximately8minutes. In US group, the same ultrasound treatment was applied without injection of MBs, replaced by the same dose of saline. In MB group, sham ultrasound was applied when the same dose of MB was injected. CEUS was performed both before and after the treatment in each group and acoustic quantitative analysis was performed for assessment of prostate blood perfusion.After all the treatment procedure, the prostates were harvested for pathological examination including gross observation, light microscope (LM) and transmission electron microscope (TEM), in order to reveal the related mechanism.Second part:18dogs were divided into3groups following the same treatment protocols above. Each group included3different time points (immediately,2and4days after the treatment). All the prostates were harvested following the above time points for pathological examination including gross observation, LM and TEM, in order to find out the pathological process. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay of the prostate samples from the dogs which sacrificed immediately after treatment was performed for detection of cellular apoptosis.Results1. After treatment by pulsed TUS with circulating MBs, EB tracing resulted that the sonicated area of the prostate was dark red without EB infusion, while the control area was dyed blue homogeneously, indicating that EB perfusion of the sonicated area reduced significantly. After treatment in US+MB group, the time intensity curve (TIC) obtained from CEUS showed that the ascending and descending parts became cliffy, with the plateau around the peak disappeared or unapparent. Time to peak was14.81±5.15s after treatment, shorter than that before treatment (28.55±10.88s)(P<0.05), and area under curve reduced from2083.87±596.22dBs before treatment to1635.63±524.65dBs (P<0.05), while there was no significant difference of peak intensity. However, no significant difference was found in the TIC morphology and acoustic quantitative analysis both within US group and MB group. Pathological examinations revealed in US+MB group that small blood vessels were destroyed, and plenty of red blood cells leaked out into the interstitials and glandular lumen. In US group and MB group, no visual changes of microvasculature were found.2. Immediately after the treatment of MEUS, disruption of microvasculature was found, showing the breakdown of blood vessel walls and the thrombosis. Cellular apoptosis of the prostate was found by TEM. Two days later, cellular necrosis and infiltration of inflammatory cells could be seen. Four days later, there was complete necrosis in some parts of the prostate. In US group, no significant changes of microvasculature but cellular organelle swelling were found immediately after US insonation, and there was a recovery two and four days later. In MB group, there were no pathological changes through the whole procedure. The apoptosis index (AI) in US group was (22.54±5.86)%, higher than that of (5.18±1.19)%in MB group (P<0.05). The AI in US+MB group was up to (52.72±6.29)%, significantly higher than that in US group and MB group (P<0.05), respectively.Conclusions1. TUS combined with circulating MBs can destroy prostatic microvasculature and greatly reduce blood perfusion.2. TUS can induce apoptosis of prostatic cells. MBs enhanced therapeutic ultrasound cavitation can significantly promote the cellular apoptosis.3. TUS combined with circulating MBs can lead to local necrosis of prostate tissue, and might be a potential ablation application on BPH. |
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