The Effect Of Microbubble Mediated Ultrasonic Cavitation On Prostate Permeability

BackgroundThe 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.ObjectiveThe 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. Methods1. 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).Results1. 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.Conclusion1. 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.