Targeted Blood-brain Barrier Disruption And Exogenous Gene Transfection By MRI-guided Focused Ultrasound-associated With Gene-loaded Microbubbles In Vivo

Part one: Targeted and reversible blood-brain barrier disruptionby MRI-guided focused ultrasound in miceOBJECTIVETo establish the mouse model of blood-brain barrier disruption byMRI-guided ultrasound-targeted microbubble destruction (UTMD) andfilter out the optimal ultrasound parameters for subsequent MRI-guidedUTMD-mediated gene transfection in vivoMETHODSFifteen Kunming mice were randomly divided into three groups andthe mice brains were irradiated respectively at different intensity of1.1W,2.2W,4.4W for40s by MRI-guided1.1MHz focused ultrasound andmicrobubbles. Contrast-enhanced MRI, EB staining and HE histologicalexamination were performed to learn about the extent of BBB opening atdifferent ultrasound intensity, the status of recovery at24h aftersonication and histological changes, then filter out the optimum ultrasound parameters with minimal damage.RESULTS1. Contrast-enhanced MRI: The contrast-enhanced MRI of mice brainswere scanned at0h and24h after sonication and the results showed that1.1W group wasn’t observed any obvious enhancement at two differenttime point;2.2W group appeared immediately a small range ofenhancement in sonicated region after sonication, which wasn’t seen24hlater;4.4W group displayed a wide range of enhancements in sonicatedregion after sonication, which even could be seen at24h after sonication,but with reduced range.2. EB staining: EB staining was performed immediately aftersonication. Blue staining wasn’t observed in the brain of1.1W group, butin2.2W,4.4W group. EB extravasation of4.4W group was more broadlydistributed and darker than that of2.2W group and these findingscorrelated well with the results of contrast-enhanced MRI.3. HE histological examination: No histological changes wereobserved in1.1W group. There were a small amount of erythrocytesextravasation observed at the targeted region of2.2W group, but noobvious signs of parenchymal damage were detected. There were extensiveerythrocytes extravasation, serious parenchymal necrosis and liquefactionand obvious interstitial edema observed at the targeted region of4.4Wgroup. CONCLUTIONMRI-guided UTMD could successfully induce the reversible andtargeted BBB disruption in mice and the parameters of1.1MHz,2.2W,40s was determined as the optimum ultrasound parameters with minimaldamage and lay the foundation for the subsequent gene transfectionexperiments.Part two: Targeted gene transfection across BBB by MRI-guidedfocused ultrasound-associated with pEGFP-N1-loaded microbubblesOBJECTIVETo investigate the feasibility of targeted gene transfer cross BBB byMRI-guided focused ultrasound-associated with pEGFP-N1-loadedmicrobubbles and analyse the site and amount of exogenous geneexpression.METHODSThe layer-by-layer assembly technique was applied to attach multiplelayers of pEGFP onto the surface of preformed lipid microbubbles. Sixtymice were randomly divided into five groups according to differenttreatment protocols, control group（Con group）, plasmid pEGFP group(Pgroup), pEGFP-loaded microbubbles group(P+M group), plasmid pEGFP+ultrasound group (P+U group), pEGFP-loaded microbubbles+ultrasoundgroup (P+U+M group). The corresponding treatment factor was added in each experimental group. Forty-eight hour later, the immunohistochemistryand immunofluorescence was used to detect the location of EGFPexpression, in addition, RT-PCR and western-blot analysis was performedto investigate the expression of EGFP mRNA and protein. Other90micereceiving ultrasound and pEGFP-loaded microbubbles were randomlydivided into0h/6h/12h/24h/48h/5d/7d/14d/28d group accordingto different observation durations. RT-PCR and western-blot analysis wasperformed to investigate the expression of EGFP mRNA and protein atdifferent time points.RESULTS1. The result of immunohistochemistry analysis of EGFP expressionrevealed several positive cells with brown-coloured cytoplasm were onlyobserved at the targeted region of P+U+M group, however, only blue-dyednuclei were shown at the contralateral (non-sonicated) region and the braintissues in other groups.2. The result of immunofluorescence analysis revealed EGFP (greenfluorescence) obviously colocalized with β-Tubulin III, a specific marker ofneurons (red fluorescence), in the cytoplasm of the neurons.3. RT-PCR and western-blot: EGFP could not be detected in the braintissue of con group, P group and P+M group. Although a small amount ofEGFP mRNA and protein was detected in the brain tissue of P+U group,there was no statistical significance. As expected, the EGFP mRNA and protein in the brain of P+U+M group were markedly enhanced, about15-fold and10-fold respectively than that of P+U group (P＜0.01). Thetranscription and translation of EGFP began within6h after sonication,increased significantly within24h, and peaked at48h, then reducedgradually, finally returned back to a much lower level at28d.CONCLUTIONThis part demonstrated the feasibility of targeted gene transfer crossBBB by MRI-guided UTMD, firstly pointed out the site of exogenous genetransfection was the cytoplasm of the neurons at the target region and thisprocess had a higher level of transcription and translation, in addition, theexogenous gene expression reached the climax at48h, then reducedgradually.Part three: Targeted gene transfection across BBB by MRI-guidedfocused ultrasound-associated with pEGFP-BDNF-loadedmicrobubbles and the underlying mechanismOBJECTIVETo investigate the feasibility of targeted gene pEGFP-BDNF transfercross BBB by MRI-guided UTMD and analyse whether the process oftransfection would affect the level of endogenous BDNF, then preliminarilyexplore the underlying process and mechanism. METHODSThirty-one mice were randomly divided into five groups according todifferent treatment protocols, control group （Con group）, Group1(pBDNF-EGFP-loaded microbubbles group), Group2(pEGFP-loadedmicrobubbles+ultrasound group), Group3(microbubbles+plasmidpBDNF-EGFP+ultrasound group), Group4(pBDNF-EGFP-loadedmicrobubbles+ultrasound group). Five mice of every group were used forwestern-blot to detect the level of BDNF at48h after treatment and theother6mice in Group4were used for frozen sections and transmissionelectron microscopy to explore the underlying process of gene transfectionat1h after sonication.RESULTS1. Western blot: The BDNF expressions at the target region of Group4were markedly enhanced, about20-fold than that at contralateral(non-sonicated) region (P＜0.01). As compared with the control group,there were no statistical significances of BDNF expression among the othergroups, including contralateral region of Group4, the target region ofGroup1and2, despite a slight increase in Group3.2. Frozen sections below the confocal microscope: After dyed by PI,the surface of DNA-loaded microbubbles presented red fluorescence. At1h after sonication, there was no obvious red fluorescence observed in thecontralateral brain except very few scattered red dots. However, throughoutthe target region, PI-dyed plasmid DNA had been uptake in the cytoplasm of the neurons, presenting numerous punctate regions of heterogeneousdistribution of red fluorescence.3. TEM examination: Large amount of small transparent vesicles wereobserved in the cytoplasm of the neurons at the targeted region, however nosignificant ultrastructural changes were viewed in neurons of thecontralateral non-sonicated brain.CONCLUTIONThis part demonstrated this method of MRI-guided UTMD couldpromote the exogenous gene BDNF highly expressed at the target regionand this process didn’t affect the level of endogenous BDNF at the targetregion or surrounding non-sonicated brain tissues. At1h after sonication,plasmid had been heterogeneously distributed in the cytoplasm of theneurons throughout the target region at higher concentrations, and largeamount of small transparent vesicles were observed in the cytoplasm of theneurons, suggesting the intracellular uptake of therapeutic macromoleculesvia vesicle-mediated endocytosis after sonication, but the underlyingprocess and mechanism need to be further studied. This experimentprovided a new evidence for the UTMD-induced endocytosis to improvethe uptake of macromolecular in vivo.