微泡介导超声空化对前列腺通透性影响的实验研究
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摘要
背景
前列腺常见疾病，包括前列腺炎、前列腺增生、前列腺癌在内，严重影响成年男性生存质量，其发病机制、病理生理学改变尚不十分清楚，治疗效果不理想，但不同的前列腺疾病之间存在着某些发病机制的联系。目前，研究显示前列腺炎症在前列腺增生的病理生理变化和前列腺癌进展中扮演关键性角色。基于前列腺炎症可能为前列腺增生、前列腺癌等其他前列腺疾病的风险因素或病理生理变化的始动机制，其治愈和预防处于最基本和最重要的地位。但是，前列腺炎患病率较高，复发率也较高，包括抗感染在内的药物治疗作为常见的基本治疗措施之一，疗效欠佳。一般认为，前列腺炎对抗生素等药物治疗具有相对耐受性。大部分的药物不能通过前列腺腺上皮从而在前列腺实质内达到有效治疗浓度。这可能与前列腺组织内具有某些特殊结构有关，可能存在实质性的解剖学屏障阻碍了抗生素从血液循环进入前列腺实质和前列腺液中。因此，我们认为血-前列腺屏障可能是由微血管内皮细胞、基底膜、肌纤维间质层、腺上皮细胞等构成，其组织结构和功能有待深入研究。正因其屏障功能，阻挡了某些药物进入前列腺腺体内部形成有效浓度来发挥治疗作用。同时组织炎症可能改变前列腺局部微环境。发生炎症时，前列腺腺体周围组织纤维增生，肌纤维间质层明显增厚，甚至出现脓腔周围组织纤维化，均对药物的屏障作用更为显著。因此，要攻克前列腺外途径给药疗效欠理想的难题，探寻新的治疗方法，如何增强前列腺组织通透性，如何有效开放血-前列腺屏障，将是解决前列腺疾病治疗的主要问题之一。
超声作为一种无创性的诊断手段虽被广泛地用于临床，但其空化效应在治疗领域的应用研究逐渐增多。近年来，微泡作为造影剂应用于超声造影检查中可以显著提高诊断的准确性，随着对微泡在超声场中产生振荡的特性的深入研究，伴随产生的生物学效应也越来越受到人们的关注。较多的关于超声造影剂的应用基础研究发现，当引入微泡后可以明显增强超声能量的吸收，显著降低超声空化阈值，增强其生物学效应，也正因此超声造影剂微泡由诊断转入治疗领域。在较低声压水平激发稳态空化，微泡通过线性和（或）非线性振荡产生的冲击波、微射流继而引发的剪切力导致声孔效应发生，组织细胞表面出现“可恢复性”小孔，细胞之间紧密连接短暂性开放，可引起组织通透性的增加并随即恢复，这也就为增加局部组织药物浓度和基因或药物靶向传输治疗某些疾病带来了新的希望，本课题也正是基于此展开的。
微泡作为一种高效的外源性空化核，经静脉注入血液循环后，可以显著提高体内空化核的数量和浓度，明显降低空化阈值，增强超声空化效应。而超声空化产生的显著的生物学效应，即声孔效应，导致细胞膜通透性增高，血管内皮细胞之间紧密连接开放已在较多的实验研究中被证实，但国内、外关于微泡介导超声空化能否增强前列腺通透性，能否有效开放血-前列腺屏障以及主要机制如何，相关报道很少，值得进一步研究。
目的
探讨微泡介导超声空化增强前列腺通透性的可行性、有效性、安全性及可恢复性。拟在实验动物水平，经静脉途径将超声造影剂微泡注入动物血液循环，设定能量参数的治疗超声靶向辐照前列腺，激励前列腺局部微循环内的微泡空化，利用超声空化所致的声孔效应开放血管内皮细胞之间紧密连接，继而再灌注的微泡在间歇式发射的超声激励下产生空化效应，进一步开放间质及腺上皮细胞间连接，进而开放整个血-前列腺屏障，增强前列腺组织通透性。旨在提出一种全新的微侵袭性改变前列腺组织通透性的物理方法，尝试解决经血流途径给药前列腺组织内药物浓度较低的难题，为有效增加前列腺间质及腺腔内药物浓度发挥更大的前列腺疾病治疗效应奠定前期的实验基础。
方法
1.选用健康、成年雄性新西兰白兔作为实验动物，随机分为5组：微泡联合超声组（MBUS组）、微泡联合超声处理后留观24hr组（MBUS24H组）、空白对照组（BC组）、单纯微泡组（MB组）、单纯超声组（US组），分别给予相应实验处理。
2.微泡介导超声空化实验仪器和材料：微泡超声空化治疗仪为新桥医院超声科实验室专用，该仪器可以发射脉冲式、非聚焦式超声能量，对微泡空化效率较高。本课题实验中采用频率为831KHz，峰值声压为2.4MPa，平均声强为0.3W/cm2。脂氟显脂质微泡由新桥医院超声科实验室研制，整体外观为乳白色凝乳状。作为空化核，其核心为全氟丙烷气体，平均粒径约为2μm，浓度约为9×1010/ml。本实验中采用的剂量为0.1ml/kg。
3.通过单位质量前列腺EB含量测定和大体标本EB渗出的观察，探讨微泡联合超声作用对前列腺通透性的影响。EB可作为微血管通透性增强的指示剂。通常情况下，EB在血液循环中与白蛋白稳定结合形成EB-白蛋白复合物，不能透过血管壁而进入血管外。因此可通过测定组织中EB含量，也即EB渗出量来评价组织通透性的变化。EB还可作为生物染色剂，如因通透性增强，EB渗出可致血管外渗出区域被蓝染。此外，HE染色光镜观察各组前列腺组织形态学改变。
4. EB作为示踪剂，可被激发出明亮的红色荧光，结合不同荧光标记显示前列腺组织微环境，使用激光共聚焦显微镜观测微泡联合超声作用前列腺后EB的渗出，探讨微泡介导超声空化增强前列腺通透性及开放血-前列腺屏障的主要机制。
5.硝酸镧示踪透射电子显微镜观测微泡联合超声作用于前列腺的超微组织结构改变，观测指示通透性改变的示踪剂-镧颗粒的沉积，进一步证实微泡介导超声空化增强前列腺通透性及开放血-前列腺屏障可行性、有效性和可恢复性，分析其主要机制。
6. TUNEL细胞原位检测分析BC组、MB组、US组、MBUS组、MBUS24H组细胞凋亡发生情况。通过对各组前列腺上皮细胞凋亡指数的比较，分析不同实验处理对前列腺组织是否造成损伤及损伤程度比较，初步评估微泡联合超声作用于前列腺的安全性。
7.数据的统计学分析：采用SPSS13.0统计分析软件，各组单位质量前列腺EB含量、凋亡指数均以均数±标准差（x±s）表示。梯度浓度系列EB溶液OD值和对应浓度分析调用线性回归分析程序。单因素方差分析用于各组的单位质量前列腺EB含量、凋亡指数的比较和分析，P<0.05具有统计学差异，P<0.001具有显著的统计学差异。
结果
1.在MBUS组中，单位质量前列腺EB含量为11.8876±0.4140μg/g，明显高于BC组、MB组、US组，表明微泡联合超声作用可以增强前列腺组织通透性。BC组、MB组、 US组的单位质量前列腺EB含量分别为4.8664±0.4166μg/g、5.1967±0.2251μg/g、5.0776±0.1761μg/g，组间比较无统计学差异，表明单纯微泡或单纯超声均对前列腺组织通透性无明显影响。MBUS24H组的单位质量前列腺EB含量为5.1773±0.3595μg/g，与BC组比较无统计学差异，而与MBUS组存在显著的统计学差异，表明微泡联合超声作用后24hr，增强的前列腺组织通透性可以复原，这也反映出微泡介导超声空化增强前列腺通透性具有可恢复性。BC组、MB组、US组和MBUS24H组测得的少量的EB含量可能与相同灌注条件下前列腺微循环内EB的少量残留有关，而MBUS组单位质量前列腺EB含量与这些值相比较，明显增多的部分应与EB渗出有关。
2. MBUS组大体标本整体观察可见前列腺组织明显蓝染，蓝染区分布均匀；横切面观察可见腺实质及包膜等结缔组织蓝染均匀。而BC、MB、US、MBUS24H组大体标本整体观察可见前列腺组织无明显蓝染；横切面显示腺实质均无明显蓝染，包膜等结缔组织部分区域内呈现不均匀的轻微蓝染，均与MBUS组的前列腺组织蓝染存在肉眼可见的差异。
3. HE染色光镜观察可见微泡联合超声作用后的即刻和24hr，前列腺组织结构未发生明显改变；单独给予微泡或超声作用对前列腺组织结构也无明显影响。
4.在MBUS组中，明亮的EB红色荧光广泛分布于血管外区域，包括间质组织及腺上皮，并有均匀分布于腺上皮基底侧及腺上皮细胞之间的趋势，甚至出现在腺上皮细胞内，这些与BC、MB、US组存在明显差异。在BC、MB、US组中，前列腺组织血管腔中未见EB红色荧光，这与EB随灌注液离开微循环有关；血管壁、间质组织、腺上皮区域内均未见明亮的EB红色荧光，显示EB未分布于这些部位。而在MBUS24H组中,血管腔、血管壁、间质组织、腺上皮区域内未显示明亮EB红色荧光，与BC、MB、US组相似，而与MBUS组存在明显差异。
5.在MBUS组中，镧颗粒不仅存在于血管腔内，可见黑色斑点状高电子密度沉积物附着在血管内皮细胞表面，还可以黑色线状沉积于内皮细胞连接之间，并可透过基底膜延伸入间质内；在腺上皮基底侧和腺上皮细胞之间也可见镧颗粒呈黑色线状高电子密度沉积。在BC、MB、US、MBUS24H组中，仅在血管腔内可见镧颗粒存在，血管内皮细胞形态完整，内皮细胞间连接紧密，基底膜完整，间质内、腺上皮基底侧和腺上皮细胞之间均未见镧颗粒沉积。
6. TUNEL细胞原位检测显示单独给予微泡或超声辐照，不会引起腺上皮细胞凋亡增多；微泡联合超声作用前列腺，腺上皮细胞凋亡增多，但尚未对腺上皮组织造成明显损伤，并在24hr后存在恢复趋势。
结论
1.微泡联合超声作用于兔前列腺，可以增强前列腺通透性，经24hr后这种改变可恢复至原有水平，而仅给予微泡静注或超声辐照对前列腺通透性未见明显影响。这可能与微泡介导超声空化所致的声孔效应有关，也即稳态空化致声孔效应可以增强兔前列腺通透性，并且这种通透性的改变具有可恢复性。
2. EB示踪激光共聚焦显微镜及硝酸镧示踪透射电子显微镜观测证实微泡联合超声作用可以增强兔前列腺组织通透性，有效开放血-前列腺屏障，其主要机制为微泡介导超声空化致声孔效应开放血管内皮细胞及腺上皮细胞之间的紧密连接，导致EB或镧颗粒这类示踪剂可以透过血管壁进入血管外间质组织，并且能够进入腺上皮近基底侧，甚至进入腺上皮细胞之间及细胞内，这种前列腺组织通透性增强的改变存在恢复趋势，并于24hr后逐渐恢复至原有状态，血-前列腺屏障的开放具有可恢复性。微泡联合超声作用于前列腺组织具有相对安全性。
Background
The prostate is the organ which is prone to male-specific diseases. Common prostatediseases, such as benign prostatic hyperplasia, prostate cancer and prostatitis, seriouslyaffect the survival and quality of life of adult males. At present, the etiology andpathophysiology of these diseases remain poorly understood. It has been reported thatprostatic inflammation plays a pivotal role in the pathophysiology of benign prostatichyperplasia, as well as in the development of prostate cancer. It is important to cureprostatitis and prevent its recurrence. In urologic practice, drugs, including antibiotics, areessential for the treatment of prostatitis syndrome. However, how to improve the efficacyis a difficult medical problem. Historically prostatitis has been relatively resistant toantimicrobial chemotherapy, for most drugs are unable to cross the prostate epithelium toreach effective therapeutic levels within the prostate gland. This may be due to the specialstructure of prostate tissue, where the circulation of antibiotics into tissues and prostatefluids is blocked by substantial anatomic barriers. Therefore, we believe that theblood-prostate barrier could be possibly made up of microvessel endothelial cells,basement membrane, fiber matrix layer, glandular epithelium, etc., its basic functioninvolves blocking of certain components in the blood circulation trafficking into theprostate glandular cavity, and preventing detrimental factors from retrograde spreadinginto stroma to invade male urogenital system via glandular epithelium. In addition to theblocking effect of the blood-prostate barrier, change of the prostate microenvironmentaroused by inflammation can reduce tissue permeability. Coupled with chronicinflammation, scars around the prostate acinar and tissues around the abscess develop intofibrosis, and fiber desmohemoblast is obviously thickened, imposing significant barrieractions on drug passage. As a result, it is crucial to increase the permeability of prostatetissues and maximally increase drug concentration in prostate tissues to achieve more powerful therapeutic effect.
As a biological effect of ultrasound, acoustic cavitation refers to an action thatmicrobubbles in a liquid, insonated by ultrasound, generate a series of dynamic processes,such as oscillation, expansion, shrinkage, implosion, etc., with multifarious patterns ofenergy release, such as transient high temperature, high pressure, shock wave, discharge,microstreaming. When cavitation takes place at the acoustics interface of tissue, physicalmechanical energy, accompanied by microstreaming, shock wave, etc., can destruct tightjunction between cells, and enhance membrane permeability, namely “sonoporation”,which could be self-healing. In general, the endogenous cavitation nuclei in vivo may beso insufficient, and the threshold of cavitation may be so high that acoustic cavitation cannot be aroused.
However, as highly effective exogenous cavitation nuclei, the ultrasound contrastagent microbubbles can significantly increase the quantity and concentration of cavitationnuclei in vivo by intravenous injection, consequently decreasing cavitation threshold andincreasing cavatition erosion. In recent years, an increasing number of researches haveutilized ultrasonic sonoporation to improve permeability of cell membranes or vascularendothelial cells in models such as cultured cell in vitro, myocardial tissues in vivo, andblood-brain barrier. However, few researches have investigated whether the ultrasonicsonoporation can open the blood-prostate barrier to enhance prostate permeability.
Objective
The aim of the study was to explore the impact of microbubble-enhanced ultrasoundon the prostate permeability and blood-prostate barrier, to investigate the feasibility,effectiveness, safety and recoverability of ultrasonic cavitation enhancing prostatepermeability. To apply the therapeutic ultrasound to irradiate the prostate of sexuallymature male rabbits as experimental animals, set ultrasound energy parameters to arousecavitation in perfusion peak of microbubbles via intravenous injection, It will pave theroad for a brand-new noninvasive physical method to alter prostate tissue permeability,thereby effectively increase drug concentration inside prostate stroma and glandular cavity,and improve therapeutic effect of prostate diseases.
Methods
1. Sexually mature male New Zealand white rabbits were randomly assigned into twoexperimental groups and three control groups. In the two experimental groups, theprostates were insonated using the therapeutic transducer with acoustic pressure of2.4MPa in the presence of circulating microbubbles (0.1ml/kg). The microbubbles wereinstantly injected into the ear veins during the insonation. The prostates were thenharvested instantly in one experimental group (MBUS group). In contrast, the prostates ofthe treated rabbits were harvested after24hr in the other experimental group (MBUS24Hgroup). Three other groups served as the controls. The2.4MPa ultrasound was applied tothe ultrasound-only group (US group) with the same dose of saline injection. Themicrobubble-only group (MB group) received sham ultrasound exposure with the sameamount of microbubbles injection. The blank control group (BC group) received shamultrasound exposure with the same dose of saline injection. The abdominal walls of rabbitswere cut to reveal their prostate under intravenous anesthesia. The ultrasound transducercould directly contact the back side of prostate, and irradiate the target region. To punctureaorta abdominalis and indwell the catheter to establish inflow path, and to cut openinferior vena cava as outflow path, we perfused the microcirculation of prostate beforeremoving the prostate specimen.
2. The therapeutic ultrasound transducer was operated at a frequency of831KHzwith the acoustic pressure output of2.4MPa. The transducer worked in an intermittentmode of6sec on and6sec off. The corresponding acoustic intensity came out to be0.3W/cm2. Lipid-coated microbubble, named Zhifuxian, was used for the nucleation ofcavitation. Zhifuxian was prepared by lyophilization of two lipid suspensions,1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and was agitated with perfluoropropane gas using ahigh-speed mechanical amalgamator. The microbubbles had a mean particle diameter of2μm and concentration of9×1010/ml. For the nucleation of cavitation,0.1ml/kgmicrobubbles were injected intravenously. The intravenous injection usually lasted9minfollowed by a3-ml saline flush.
3. As Evan blue (EB) is the indicator that reflects the alteration of microvascularpermeability, EB content per prostate unit mass was determined. The prostate tissue was submerged in formamide solution to extract EB. The value of optical density (OD) wasdetermined with a DU800UV/visible spectrophotometer. Based on the OD valuecorresponding to concentration gradient of EB solution, we analyzed the results by linearregression statistics to get the linear regression equation: y=0.125+28.181x (x: the value ofOD, y: EB concentration). Based on the OD value of EB extraction liquid and the linearregression equation, we calculated the concentration, and further got EB total contents inextraction liquid, and finally figured out EB content per prostate unit mass on behalf of EBexudation able to be compared in different groups. As an indicator, blue dyeing of EBexudation area may be visible to the naked eye. Experimental grouping and processingwere broadly in line with the determination of EB content, all by intravenous injection of2%EB solution at25mg/kg. After perfusing prostate microcirculation with0.01mol/LPBS, we excised the prostate to observe overall appearance and cross section of generalspecimens. By hematoxylin-eosin (HE) staining, we observed morphological changes ofthe prostate tissue in light microscope.
4. To explore the mechanism of prostate permeability to be enhanced by microbubblemediated ultrasonic cavitation, we apply laser scanning confocal microscopy (LSCM) toinvestigate EB exudation in the prostate tissue. As the tracer, EB was excited to showbright red fluorescence. Different fluorescence markers displayed the cytomembrane andnucleus of the prostate tissue. A laser scanning confocal microscope was used to surveyexistence and distribution of different fluorescence in frozen sections.
5. By virtue of transmission electron microscopy (TEM) with Lanthanum NitrateTracing, we investigated the ultrastructure alteration of prostate tissue, and the depositionof lanthanum particles to explore the feasibility, effectiveness, and recoverability ofultrasonic cavitation enhancing prostate permeability, combined with LSCM to explore themechanism of prostate tissue alteration.
6. To apply in situ cell detection kit for immunohistochemical detection andquantification of apoptosis at single cell level, based on TUNEL (terminal-deoxynucleotidyl transferase mediated nick end labeling) technology. We analysedTUNEL-positive cells, and compared apoptotic index (AI) in the BC, MB, US, MBUS,MBUS24H groups for the safety of microbubble mediated ultrasonic cavitation on prostatetissue.
7. All data are expressed as the mean±standard deviation values. Linear regressionwas used to analyze EB concentration. One-way analysis of variance was used to comparedifferences between groups. A p valve of0.05was considered statistically significant. Alldata were analyzed using SPSS software (SPSS, Inc., Chicago, IL, USA).
Results
1. EB content per unit mass of prostate was11.8876±0.414μg/g in the MBUS group,higher than in the BC, MB, US, and MBUS24H groups, and statistically significantdifferences were observed between the MBUS group and the other four groups. Whereas,no significant difference was found in BC, MB, US, and MBUS24H groups, thus thedatum of EB content in the four groups were possibly related to EB remained in prostatemicrocirculation under the same perfusion conditions, however the higher part of EBcontent in the MBUS group was due to EB exudation from blood vessel.
2. In MBUS group, the integral view of the prostates still remained obviously bluedyeing that was uniformly distributed in the glandular parenchyma of the cross section. Inthe BC, MB, and US groups, blue dyeing in the integral view greatly faded away, yetslight blue dyeing was located in the tunica or connective fascia, not distributed in theglandular parenchyma of the cross section, thus being consistent with less EB content inthe three groups, due to EB remains. In MBUS24H group, the results were significantlydifferent from those in the MBUS group, but similar to those in the BC group.
3. At the instant or24hr after insonation of ultrasound mediated by microbubble,prostate tissue, by HE staining, have no significantly morphological alteration under lightmicroscope. The action of microbubble or ultrasound on the prostate did not affectorganization structure.
4. The location of EB penetration was shown under LSCM. In MBUS group, EB redfluorescence was extensively distributed in the stroma and the base side of glandularepithelium, even in the intercellular space of glandular epithelium. However, in BC, MB,and US groups, EB red fluorescence was not observed in extravascular locations, such asstroma, glandular epithelium. In the MBUS24H group, no EB red fluorescence was alsoobserved in extravascular locations.
5. Since the deposition of La particles is shown as electron-dense in TEM. In the MBUS group, electron-dense of La particles appeared in both linear distribution betweenvascular endothelial cells and patchy remains in the vessel lumens, and even distributed inextravascular stroma and the intercellular space of glandular epithelium with the form ofblack lineament. Furthermore, in BC, MB and US groups, there was no linearelectron-dense to show distribution of La particles in stroma and the intercellular space ofglandular epithelium. No linear electron-dense was found in the extravascular locations ofthe MBUS24H group. In general, the results of TEM were consistent with those of LSCM.
6. TUNEL apoptosis detection suggested that apoptotic cells slightly increased inglandular epithelium after microbubble mediated ultrasound had irradiated the prostate,however, obvious injury of prostate tissue was not indicated. The action of microbubble orultrasound on the prostate did not promote apoptosis of prostate tissue.
Conclusion
1. Prostate permeability could be enhanced by microbubble-mediated ultrasoundirradiation, due to acoustic cavitation, rather than microbubble or ultrasound alone.Enhanced prostate permeability had been recovered to usually statue at24hr aftermicrobubble-mediated ultrasound irradiation.
2. Direct evidences of morphology by means of LSCM and TEM with lanthanumnitrate tracing, demonstrated the mechanism of the prostate permeability enhanced bymicrobubble-mediated ultrasonic insonation. the prostate permeability was affected byinsonation of ultrasound mediated by microbubble, and it was the possible mechanism thatultrasonic sonoporation induced by acoustic cavitation, which was embodied in theexudation of tracer, the opening of tight junctions between vascular endothelial orglandular epithelial cells at the instant of acoustic cavitation, and the reinstatement ofenhanced permeability at24hr after acoustic cavitation.
前列腺常见疾病，包括前列腺炎、前列腺增生、前列腺癌在内，严重影响成年男性生存质量，其发病机制、病理生理学改变尚不十分清楚，治疗效果不理想，但不同的前列腺疾病之间存在着某些发病机制的联系。目前，研究显示前列腺炎症在前列腺增生的病理生理变化和前列腺癌进展中扮演关键性角色。基于前列腺炎症可能为前列腺增生、前列腺癌等其他前列腺疾病的风险因素或病理生理变化的始动机制，其治愈和预防处于最基本和最重要的地位。但是，前列腺炎患病率较高，复发率也较高，包括抗感染在内的药物治疗作为常见的基本治疗措施之一，疗效欠佳。一般认为，前列腺炎对抗生素等药物治疗具有相对耐受性。大部分的药物不能通过前列腺腺上皮从而在前列腺实质内达到有效治疗浓度。这可能与前列腺组织内具有某些特殊结构有关，可能存在实质性的解剖学屏障阻碍了抗生素从血液循环进入前列腺实质和前列腺液中。因此，我们认为血-前列腺屏障可能是由微血管内皮细胞、基底膜、肌纤维间质层、腺上皮细胞等构成，其组织结构和功能有待深入研究。正因其屏障功能，阻挡了某些药物进入前列腺腺体内部形成有效浓度来发挥治疗作用。同时组织炎症可能改变前列腺局部微环境。发生炎症时，前列腺腺体周围组织纤维增生，肌纤维间质层明显增厚，甚至出现脓腔周围组织纤维化，均对药物的屏障作用更为显著。因此，要攻克前列腺外途径给药疗效欠理想的难题，探寻新的治疗方法，如何增强前列腺组织通透性，如何有效开放血-前列腺屏障，将是解决前列腺疾病治疗的主要问题之一。
超声作为一种无创性的诊断手段虽被广泛地用于临床，但其空化效应在治疗领域的应用研究逐渐增多。近年来，微泡作为造影剂应用于超声造影检查中可以显著提高诊断的准确性，随着对微泡在超声场中产生振荡的特性的深入研究，伴随产生的生物学效应也越来越受到人们的关注。较多的关于超声造影剂的应用基础研究发现，当引入微泡后可以明显增强超声能量的吸收，显著降低超声空化阈值，增强其生物学效应，也正因此超声造影剂微泡由诊断转入治疗领域。在较低声压水平激发稳态空化，微泡通过线性和（或）非线性振荡产生的冲击波、微射流继而引发的剪切力导致声孔效应发生，组织细胞表面出现“可恢复性”小孔，细胞之间紧密连接短暂性开放，可引起组织通透性的增加并随即恢复，这也就为增加局部组织药物浓度和基因或药物靶向传输治疗某些疾病带来了新的希望，本课题也正是基于此展开的。
微泡作为一种高效的外源性空化核，经静脉注入血液循环后，可以显著提高体内空化核的数量和浓度，明显降低空化阈值，增强超声空化效应。而超声空化产生的显著的生物学效应，即声孔效应，导致细胞膜通透性增高，血管内皮细胞之间紧密连接开放已在较多的实验研究中被证实，但国内、外关于微泡介导超声空化能否增强前列腺通透性，能否有效开放血-前列腺屏障以及主要机制如何，相关报道很少，值得进一步研究。
目的
探讨微泡介导超声空化增强前列腺通透性的可行性、有效性、安全性及可恢复性。拟在实验动物水平，经静脉途径将超声造影剂微泡注入动物血液循环，设定能量参数的治疗超声靶向辐照前列腺，激励前列腺局部微循环内的微泡空化，利用超声空化所致的声孔效应开放血管内皮细胞之间紧密连接，继而再灌注的微泡在间歇式发射的超声激励下产生空化效应，进一步开放间质及腺上皮细胞间连接，进而开放整个血-前列腺屏障，增强前列腺组织通透性。旨在提出一种全新的微侵袭性改变前列腺组织通透性的物理方法，尝试解决经血流途径给药前列腺组织内药物浓度较低的难题，为有效增加前列腺间质及腺腔内药物浓度发挥更大的前列腺疾病治疗效应奠定前期的实验基础。
方法
1.选用健康、成年雄性新西兰白兔作为实验动物，随机分为5组：微泡联合超声组（MBUS组）、微泡联合超声处理后留观24hr组（MBUS24H组）、空白对照组（BC组）、单纯微泡组（MB组）、单纯超声组（US组），分别给予相应实验处理。
2.微泡介导超声空化实验仪器和材料：微泡超声空化治疗仪为新桥医院超声科实验室专用，该仪器可以发射脉冲式、非聚焦式超声能量，对微泡空化效率较高。本课题实验中采用频率为831KHz，峰值声压为2.4MPa，平均声强为0.3W/cm2。脂氟显脂质微泡由新桥医院超声科实验室研制，整体外观为乳白色凝乳状。作为空化核，其核心为全氟丙烷气体，平均粒径约为2μm，浓度约为9×1010/ml。本实验中采用的剂量为0.1ml/kg。
3.通过单位质量前列腺EB含量测定和大体标本EB渗出的观察，探讨微泡联合超声作用对前列腺通透性的影响。EB可作为微血管通透性增强的指示剂。通常情况下，EB在血液循环中与白蛋白稳定结合形成EB-白蛋白复合物，不能透过血管壁而进入血管外。因此可通过测定组织中EB含量，也即EB渗出量来评价组织通透性的变化。EB还可作为生物染色剂，如因通透性增强，EB渗出可致血管外渗出区域被蓝染。此外，HE染色光镜观察各组前列腺组织形态学改变。
4. EB作为示踪剂，可被激发出明亮的红色荧光，结合不同荧光标记显示前列腺组织微环境，使用激光共聚焦显微镜观测微泡联合超声作用前列腺后EB的渗出，探讨微泡介导超声空化增强前列腺通透性及开放血-前列腺屏障的主要机制。
5.硝酸镧示踪透射电子显微镜观测微泡联合超声作用于前列腺的超微组织结构改变，观测指示通透性改变的示踪剂-镧颗粒的沉积，进一步证实微泡介导超声空化增强前列腺通透性及开放血-前列腺屏障可行性、有效性和可恢复性，分析其主要机制。
6. TUNEL细胞原位检测分析BC组、MB组、US组、MBUS组、MBUS24H组细胞凋亡发生情况。通过对各组前列腺上皮细胞凋亡指数的比较，分析不同实验处理对前列腺组织是否造成损伤及损伤程度比较，初步评估微泡联合超声作用于前列腺的安全性。
7.数据的统计学分析：采用SPSS13.0统计分析软件，各组单位质量前列腺EB含量、凋亡指数均以均数±标准差（x±s）表示。梯度浓度系列EB溶液OD值和对应浓度分析调用线性回归分析程序。单因素方差分析用于各组的单位质量前列腺EB含量、凋亡指数的比较和分析，P<0.05具有统计学差异，P<0.001具有显著的统计学差异。
结果
1.在MBUS组中，单位质量前列腺EB含量为11.8876±0.4140μg/g，明显高于BC组、MB组、US组，表明微泡联合超声作用可以增强前列腺组织通透性。BC组、MB组、 US组的单位质量前列腺EB含量分别为4.8664±0.4166μg/g、5.1967±0.2251μg/g、5.0776±0.1761μg/g，组间比较无统计学差异，表明单纯微泡或单纯超声均对前列腺组织通透性无明显影响。MBUS24H组的单位质量前列腺EB含量为5.1773±0.3595μg/g，与BC组比较无统计学差异，而与MBUS组存在显著的统计学差异，表明微泡联合超声作用后24hr，增强的前列腺组织通透性可以复原，这也反映出微泡介导超声空化增强前列腺通透性具有可恢复性。BC组、MB组、US组和MBUS24H组测得的少量的EB含量可能与相同灌注条件下前列腺微循环内EB的少量残留有关，而MBUS组单位质量前列腺EB含量与这些值相比较，明显增多的部分应与EB渗出有关。
2. MBUS组大体标本整体观察可见前列腺组织明显蓝染，蓝染区分布均匀；横切面观察可见腺实质及包膜等结缔组织蓝染均匀。而BC、MB、US、MBUS24H组大体标本整体观察可见前列腺组织无明显蓝染；横切面显示腺实质均无明显蓝染，包膜等结缔组织部分区域内呈现不均匀的轻微蓝染，均与MBUS组的前列腺组织蓝染存在肉眼可见的差异。
3. HE染色光镜观察可见微泡联合超声作用后的即刻和24hr，前列腺组织结构未发生明显改变；单独给予微泡或超声作用对前列腺组织结构也无明显影响。
4.在MBUS组中，明亮的EB红色荧光广泛分布于血管外区域，包括间质组织及腺上皮，并有均匀分布于腺上皮基底侧及腺上皮细胞之间的趋势，甚至出现在腺上皮细胞内，这些与BC、MB、US组存在明显差异。在BC、MB、US组中，前列腺组织血管腔中未见EB红色荧光，这与EB随灌注液离开微循环有关；血管壁、间质组织、腺上皮区域内均未见明亮的EB红色荧光，显示EB未分布于这些部位。而在MBUS24H组中,血管腔、血管壁、间质组织、腺上皮区域内未显示明亮EB红色荧光，与BC、MB、US组相似，而与MBUS组存在明显差异。
5.在MBUS组中，镧颗粒不仅存在于血管腔内，可见黑色斑点状高电子密度沉积物附着在血管内皮细胞表面，还可以黑色线状沉积于内皮细胞连接之间，并可透过基底膜延伸入间质内；在腺上皮基底侧和腺上皮细胞之间也可见镧颗粒呈黑色线状高电子密度沉积。在BC、MB、US、MBUS24H组中，仅在血管腔内可见镧颗粒存在，血管内皮细胞形态完整，内皮细胞间连接紧密，基底膜完整，间质内、腺上皮基底侧和腺上皮细胞之间均未见镧颗粒沉积。
6. TUNEL细胞原位检测显示单独给予微泡或超声辐照，不会引起腺上皮细胞凋亡增多；微泡联合超声作用前列腺，腺上皮细胞凋亡增多，但尚未对腺上皮组织造成明显损伤，并在24hr后存在恢复趋势。
结论
1.微泡联合超声作用于兔前列腺，可以增强前列腺通透性，经24hr后这种改变可恢复至原有水平，而仅给予微泡静注或超声辐照对前列腺通透性未见明显影响。这可能与微泡介导超声空化所致的声孔效应有关，也即稳态空化致声孔效应可以增强兔前列腺通透性，并且这种通透性的改变具有可恢复性。
2. EB示踪激光共聚焦显微镜及硝酸镧示踪透射电子显微镜观测证实微泡联合超声作用可以增强兔前列腺组织通透性，有效开放血-前列腺屏障，其主要机制为微泡介导超声空化致声孔效应开放血管内皮细胞及腺上皮细胞之间的紧密连接，导致EB或镧颗粒这类示踪剂可以透过血管壁进入血管外间质组织，并且能够进入腺上皮近基底侧，甚至进入腺上皮细胞之间及细胞内，这种前列腺组织通透性增强的改变存在恢复趋势，并于24hr后逐渐恢复至原有状态，血-前列腺屏障的开放具有可恢复性。微泡联合超声作用于前列腺组织具有相对安全性。
Background
The prostate is the organ which is prone to male-specific diseases. Common prostatediseases, such as benign prostatic hyperplasia, prostate cancer and prostatitis, seriouslyaffect the survival and quality of life of adult males. At present, the etiology andpathophysiology of these diseases remain poorly understood. It has been reported thatprostatic inflammation plays a pivotal role in the pathophysiology of benign prostatichyperplasia, as well as in the development of prostate cancer. It is important to cureprostatitis and prevent its recurrence. In urologic practice, drugs, including antibiotics, areessential for the treatment of prostatitis syndrome. However, how to improve the efficacyis a difficult medical problem. Historically prostatitis has been relatively resistant toantimicrobial chemotherapy, for most drugs are unable to cross the prostate epithelium toreach effective therapeutic levels within the prostate gland. This may be due to the specialstructure of prostate tissue, where the circulation of antibiotics into tissues and prostatefluids is blocked by substantial anatomic barriers. Therefore, we believe that theblood-prostate barrier could be possibly made up of microvessel endothelial cells,basement membrane, fiber matrix layer, glandular epithelium, etc., its basic functioninvolves blocking of certain components in the blood circulation trafficking into theprostate glandular cavity, and preventing detrimental factors from retrograde spreadinginto stroma to invade male urogenital system via glandular epithelium. In addition to theblocking effect of the blood-prostate barrier, change of the prostate microenvironmentaroused by inflammation can reduce tissue permeability. Coupled with chronicinflammation, scars around the prostate acinar and tissues around the abscess develop intofibrosis, and fiber desmohemoblast is obviously thickened, imposing significant barrieractions on drug passage. As a result, it is crucial to increase the permeability of prostatetissues and maximally increase drug concentration in prostate tissues to achieve more powerful therapeutic effect.
As a biological effect of ultrasound, acoustic cavitation refers to an action thatmicrobubbles in a liquid, insonated by ultrasound, generate a series of dynamic processes,such as oscillation, expansion, shrinkage, implosion, etc., with multifarious patterns ofenergy release, such as transient high temperature, high pressure, shock wave, discharge,microstreaming. When cavitation takes place at the acoustics interface of tissue, physicalmechanical energy, accompanied by microstreaming, shock wave, etc., can destruct tightjunction between cells, and enhance membrane permeability, namely “sonoporation”,which could be self-healing. In general, the endogenous cavitation nuclei in vivo may beso insufficient, and the threshold of cavitation may be so high that acoustic cavitation cannot be aroused.
However, as highly effective exogenous cavitation nuclei, the ultrasound contrastagent microbubbles can significantly increase the quantity and concentration of cavitationnuclei in vivo by intravenous injection, consequently decreasing cavitation threshold andincreasing cavatition erosion. In recent years, an increasing number of researches haveutilized ultrasonic sonoporation to improve permeability of cell membranes or vascularendothelial cells in models such as cultured cell in vitro, myocardial tissues in vivo, andblood-brain barrier. However, few researches have investigated whether the ultrasonicsonoporation can open the blood-prostate barrier to enhance prostate permeability.
Objective
The aim of the study was to explore the impact of microbubble-enhanced ultrasoundon the prostate permeability and blood-prostate barrier, to investigate the feasibility,effectiveness, safety and recoverability of ultrasonic cavitation enhancing prostatepermeability. To apply the therapeutic ultrasound to irradiate the prostate of sexuallymature male rabbits as experimental animals, set ultrasound energy parameters to arousecavitation in perfusion peak of microbubbles via intravenous injection, It will pave theroad for a brand-new noninvasive physical method to alter prostate tissue permeability,thereby effectively increase drug concentration inside prostate stroma and glandular cavity,and improve therapeutic effect of prostate diseases.
Methods
1. Sexually mature male New Zealand white rabbits were randomly assigned into twoexperimental groups and three control groups. In the two experimental groups, theprostates were insonated using the therapeutic transducer with acoustic pressure of2.4MPa in the presence of circulating microbubbles (0.1ml/kg). The microbubbles wereinstantly injected into the ear veins during the insonation. The prostates were thenharvested instantly in one experimental group (MBUS group). In contrast, the prostates ofthe treated rabbits were harvested after24hr in the other experimental group (MBUS24Hgroup). Three other groups served as the controls. The2.4MPa ultrasound was applied tothe ultrasound-only group (US group) with the same dose of saline injection. Themicrobubble-only group (MB group) received sham ultrasound exposure with the sameamount of microbubbles injection. The blank control group (BC group) received shamultrasound exposure with the same dose of saline injection. The abdominal walls of rabbitswere cut to reveal their prostate under intravenous anesthesia. The ultrasound transducercould directly contact the back side of prostate, and irradiate the target region. To punctureaorta abdominalis and indwell the catheter to establish inflow path, and to cut openinferior vena cava as outflow path, we perfused the microcirculation of prostate beforeremoving the prostate specimen.
2. The therapeutic ultrasound transducer was operated at a frequency of831KHzwith the acoustic pressure output of2.4MPa. The transducer worked in an intermittentmode of6sec on and6sec off. The corresponding acoustic intensity came out to be0.3W/cm2. Lipid-coated microbubble, named Zhifuxian, was used for the nucleation ofcavitation. Zhifuxian was prepared by lyophilization of two lipid suspensions,1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and was agitated with perfluoropropane gas using ahigh-speed mechanical amalgamator. The microbubbles had a mean particle diameter of2μm and concentration of9×1010/ml. For the nucleation of cavitation,0.1ml/kgmicrobubbles were injected intravenously. The intravenous injection usually lasted9minfollowed by a3-ml saline flush.
3. As Evan blue (EB) is the indicator that reflects the alteration of microvascularpermeability, EB content per prostate unit mass was determined. The prostate tissue was submerged in formamide solution to extract EB. The value of optical density (OD) wasdetermined with a DU800UV/visible spectrophotometer. Based on the OD valuecorresponding to concentration gradient of EB solution, we analyzed the results by linearregression statistics to get the linear regression equation: y=0.125+28.181x (x: the value ofOD, y: EB concentration). Based on the OD value of EB extraction liquid and the linearregression equation, we calculated the concentration, and further got EB total contents inextraction liquid, and finally figured out EB content per prostate unit mass on behalf of EBexudation able to be compared in different groups. As an indicator, blue dyeing of EBexudation area may be visible to the naked eye. Experimental grouping and processingwere broadly in line with the determination of EB content, all by intravenous injection of2%EB solution at25mg/kg. After perfusing prostate microcirculation with0.01mol/LPBS, we excised the prostate to observe overall appearance and cross section of generalspecimens. By hematoxylin-eosin (HE) staining, we observed morphological changes ofthe prostate tissue in light microscope.
4. To explore the mechanism of prostate permeability to be enhanced by microbubblemediated ultrasonic cavitation, we apply laser scanning confocal microscopy (LSCM) toinvestigate EB exudation in the prostate tissue. As the tracer, EB was excited to showbright red fluorescence. Different fluorescence markers displayed the cytomembrane andnucleus of the prostate tissue. A laser scanning confocal microscope was used to surveyexistence and distribution of different fluorescence in frozen sections.
5. By virtue of transmission electron microscopy (TEM) with Lanthanum NitrateTracing, we investigated the ultrastructure alteration of prostate tissue, and the depositionof lanthanum particles to explore the feasibility, effectiveness, and recoverability ofultrasonic cavitation enhancing prostate permeability, combined with LSCM to explore themechanism of prostate tissue alteration.
6. To apply in situ cell detection kit for immunohistochemical detection andquantification of apoptosis at single cell level, based on TUNEL (terminal-deoxynucleotidyl transferase mediated nick end labeling) technology. We analysedTUNEL-positive cells, and compared apoptotic index (AI) in the BC, MB, US, MBUS,MBUS24H groups for the safety of microbubble mediated ultrasonic cavitation on prostatetissue.
7. All data are expressed as the mean±standard deviation values. Linear regressionwas used to analyze EB concentration. One-way analysis of variance was used to comparedifferences between groups. A p valve of0.05was considered statistically significant. Alldata were analyzed using SPSS software (SPSS, Inc., Chicago, IL, USA).
Results
1. EB content per unit mass of prostate was11.8876±0.414μg/g in the MBUS group,higher than in the BC, MB, US, and MBUS24H groups, and statistically significantdifferences were observed between the MBUS group and the other four groups. Whereas,no significant difference was found in BC, MB, US, and MBUS24H groups, thus thedatum of EB content in the four groups were possibly related to EB remained in prostatemicrocirculation under the same perfusion conditions, however the higher part of EBcontent in the MBUS group was due to EB exudation from blood vessel.
2. In MBUS group, the integral view of the prostates still remained obviously bluedyeing that was uniformly distributed in the glandular parenchyma of the cross section. Inthe BC, MB, and US groups, blue dyeing in the integral view greatly faded away, yetslight blue dyeing was located in the tunica or connective fascia, not distributed in theglandular parenchyma of the cross section, thus being consistent with less EB content inthe three groups, due to EB remains. In MBUS24H group, the results were significantlydifferent from those in the MBUS group, but similar to those in the BC group.
3. At the instant or24hr after insonation of ultrasound mediated by microbubble,prostate tissue, by HE staining, have no significantly morphological alteration under lightmicroscope. The action of microbubble or ultrasound on the prostate did not affectorganization structure.
4. The location of EB penetration was shown under LSCM. In MBUS group, EB redfluorescence was extensively distributed in the stroma and the base side of glandularepithelium, even in the intercellular space of glandular epithelium. However, in BC, MB,and US groups, EB red fluorescence was not observed in extravascular locations, such asstroma, glandular epithelium. In the MBUS24H group, no EB red fluorescence was alsoobserved in extravascular locations.
5. Since the deposition of La particles is shown as electron-dense in TEM. In the MBUS group, electron-dense of La particles appeared in both linear distribution betweenvascular endothelial cells and patchy remains in the vessel lumens, and even distributed inextravascular stroma and the intercellular space of glandular epithelium with the form ofblack lineament. Furthermore, in BC, MB and US groups, there was no linearelectron-dense to show distribution of La particles in stroma and the intercellular space ofglandular epithelium. No linear electron-dense was found in the extravascular locations ofthe MBUS24H group. In general, the results of TEM were consistent with those of LSCM.
6. TUNEL apoptosis detection suggested that apoptotic cells slightly increased inglandular epithelium after microbubble mediated ultrasound had irradiated the prostate,however, obvious injury of prostate tissue was not indicated. The action of microbubble orultrasound on the prostate did not promote apoptosis of prostate tissue.
Conclusion
1. Prostate permeability could be enhanced by microbubble-mediated ultrasoundirradiation, due to acoustic cavitation, rather than microbubble or ultrasound alone.Enhanced prostate permeability had been recovered to usually statue at24hr aftermicrobubble-mediated ultrasound irradiation.
2. Direct evidences of morphology by means of LSCM and TEM with lanthanumnitrate tracing, demonstrated the mechanism of the prostate permeability enhanced bymicrobubble-mediated ultrasonic insonation. the prostate permeability was affected byinsonation of ultrasound mediated by microbubble, and it was the possible mechanism thatultrasonic sonoporation induced by acoustic cavitation, which was embodied in theexudation of tracer, the opening of tight junctions between vascular endothelial orglandular epithelial cells at the instant of acoustic cavitation, and the reinstatement ofenhanced permeability at24hr after acoustic cavitation.
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