En benchtop tilgang til placeringsspecifik blodhjernebarriereåbning ved hjælp af fokuseret ultralyd i en rottemodel
Summary
Fokuseret ultralyd med mikroboble midler kan åbne blodhjerne barrieren fokalt og forbigående. Denne teknik er blevet brugt til at levere en bred vifte af agenter på tværs af blodhjerne barrieren. Denne artikel indeholder en detaljeret protokol for den lokaliserede levering til gnaverhjernen med eller uden MR-vejledning.
Abstract
Stereotaxic kirurgi er guldstandarden for lokaliseret medicin og gen levering til gnaver hjernen. Denne teknik har mange fordele i forhold til systemisk levering, herunder præcis lokalisering til et mål hjerne region og reduktion af off target bivirkninger. Stereotaxisk kirurgi er imidlertid meget invasiv, hvilket begrænser dens translationelle effekt, kræver lange restitutionstider og giver udfordringer, når den målrettes mod flere hjerneområder. Fokuseret ultralyd (FUS) kan bruges i kombination med cirkulerende mikrobobler til forbigående at åbne blodhjernebarrieren (BBB) i millimeterstore regioner. Dette muliggør intrakraniel lokalisering af systemisk leverede agenter, der normalt ikke kan krydse BBB. Denne teknik giver et noninvasive alternativ til stereotaxic kirurgi. Til dato er denne teknik dog endnu ikke blevet udbredt i neurovidenskabslaboratorier på grund af den begrænsede adgang til udstyr og standardiserede metoder. Det overordnede mål med denne protokol er at give en bænktop tilgang til FUS BBB åbning (BBBO), der er overkommelig og reproducerbar og kan derfor let vedtages af ethvert laboratorium.
Introduction
På trods af de mange opdagelser i grundlæggende neurovidenskab forbliver antallet af nye behandlinger for neuroudviklingsmæssige og neurodegenerative lidelser relativt begrænset1,2. En dybere forståelse af de gener, molekyler og cellulære kredsløb, der er involveret i neurologiske lidelser, har foreslået lovende behandlinger, der ikke kan realiseres hos mennesker med nuværende teknikker3. Effektive behandlinger er ofte begrænset af behovet for at være hjernepenetrable og stedsspecifikke4,5,6,7,8. Eksisterende metoder til lokaliseret lægemiddellevering til specifikke hjerneregioner (f.eks. levering via injektion eller kanyle) er imidlertid invasive og kræver en åbning, der skal foretages i kraniet9. Invasiviteten af denne operation forhindrer rutinemæssig brug af lokaliseret levering til den menneskelige hjerne. Derudover er vævsskader og de resulterende inflammatoriske reaktioner allestedsnærværende confounds for grundlæggende og prækliniske undersøgelser, der er afhængige af intracerebral injektion10. Evnen til ikke-invasivt at levere agenter på tværs af blodhjernebarrieren (BBB) og målrette dem mod specifikke hjerneregioner kan have en enorm indflydelse på behandlinger for neurologiske lidelser, samtidig med at det giver et kraftfuldt undersøgelsesværktøj til præklinisk forskning.
En metode til målrettet transport på tværs af BBB med minimal vævsskade er transkranial fokuseret ultralyd (FUS) sammen med mikrobobler til fokalt og forbigående åbne BBB11,12,13,14,15,16. FUS BBB åbning har fået den seneste opmærksomhed til behandling af neurodegenerative lidelser, slagtilfælde og gliom ved at lokalisere terapeutiske til at målrette hjernen regioner såsom neurotrofiske faktorer17,18,19, genterapier20,21,22, antistoffer23, neurotransmittere24, og nanopartikler25,26,27,28,29. Med sin brede vifte af applikationer og sin noninvasive natur30,31, FUS BBB åbning er et ideelt alternativ til rutinemæssige stereotaxic intrakranielle injektioner. På grund af sin nuværende anvendelse hos mennesker30,32, kan prækliniske undersøgelser ved hjælp af denne teknik betragtes som meget translationelle. Fus BBB-åbningen er dog endnu ikke en veletableret teknik inden for grundforskning og præklinisk forskning på grund af manglende tilgængelighed. Derfor leverer vi en detaljeret protokol for en bænktoptilgang til FUS BBB-åbning som udgangspunkt for laboratorier, der er interesseret i at etablere denne teknik.
Disse undersøgelser blev udført med enten en høj effekt luftstøttet FUS specifik ultralyd transducer eller en lav effekt dæmpet fokuseret ultralyd nedsænkning transducer. Transducerne blev drevet af en RF-effektforstærker designet til reaktive belastninger og en standard bænktopfunktionsgenerator. Nærmere oplysninger om disse varer findes i tabellen over materialer.
Protocol
Representative Results
Discussion
Her beskrev vi en benchtop tilgang til mikroboble assisteret FUS BBB åbning med alternative tilgange, herunder to forskellige transducere og metoder til intrakraniel målretning med og uden MR-vejledning. For at etablere MRI-guidet FUS BBB-åbning i laboratoriet er der i øjeblikket mulighed for at købe fremragende brugsklare enheder, der giver meget standardiserede og reproducerbare resultater med brugervenlige grænseflader. Men mange laboratorier er ikke forberedt på omkostningerne ved sådanne instrumenter. Derfor…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Denne forskning blev delvist støttet af et NSF EPSCoR Research Infrastructure-tilskud til Clemson University (1632881). Derudover blev denne forskning delvist støttet af Civitan International Research Center, Birmingham, AL. Forfatterne taknemmeligt anerkende brugen af de tjenester og faciliteter ved University of Alabama i Birmingham Small Animal Imaging Shared Facility Grant [NIH P30 CA013148]. Forfatterne anerkender Rajiv Chopra for hans støtte og vejledning.
Materials
| Bubble shaker | Lantheus Medical Imaging | VMIX | VIALMIX, actiation device used to activate Definity microbubbles |
| Catheter plug/ Injection cap | SAI infusion technologies | Part Number: IC | Catheter plug/ Injection cap |
| Evans blue dye | Sigma | E2129-10G | Evans blue dye |
| Function generator | Tektronix | AFG3022B | Dual channel, 250MS/s, 25MHz |
| FUS transducer, 1.1MHz | FUS Instruments | TX-110 | 1 MHz MRI-compatible spherically focused ultrasound transducer with a hydrophone |
| Heating pad for Mice and Rats | Kent Scientific | PS-03 | Heating pad- PhysioSuite for Mice and Rats |
| Infusion pump | KD Scientific | 780100 | KDS 100 Legacy Single Syringe Infusion Pump |
| Kapton tape | Gizmo Dorks | https://www.amazon.com/dp/B01N1GGKRC/ ref=cm_sw_em_r_mt_dp_U_GbR7Db56HKD91 |
Gizmo Dorks Kapton Tape (Polyimide) for 3D Printers and Printing, 8 x 8 inches, 10 Sheets per Pack |
| Low power immersion transducer, 1MHz | Olympus | V303-SU | Immersion Transducer, 1 MHz, 0.50 in. Element Diameter, Standard Case Style, Straight UHF Connector, F=0.80IN PTF |
| Magnet sets | WINOMO | https://www.amazon.com/dp/B01DJZQJBG/ ref=cm_sw_em_r_mt_dp_U_JYQ7DbM32E5QC |
WINOMO 15mm Sew In Magnetic Bag Clasps for Sewing Scrapbooking – 10 Sets |
| RF amplifier | E&I | A075 | 75W |
| Tail vein catheter | BD | 382512/ Fisher Item: NC1228513 | 24g BD Insyte Autoguard shielded IV catheters (non-winged) |
| Ultrasound contrast microbubbles | Lantheus Medical Imaging | DE4, DE16 | DEFINITY (Perflutren Lipid Microsphere) |
| Ultrasound gel | Aquasonic | https://www.amazon.com/dp/B07FPQDM4F/ ref=cm_sw_em_r_mt_dp_U_D6Q7Db3J9QP7P |
Ultrasound Gel Aquasonic 100 Transmission 1 Liter Squeeze Bottle |
| Winged infusion sets, 22ga. | Fisher Healthcare | 22-258087 | Terumo Surflo Winged Infusion Sets |
| motor controller software | N/A | N/A | custom software written in LabView for controlling the Velmex motor controller |
| runtime environment for the motor controller software | National Instruments | LabView runtime engine version 2017 or better | https://www.ni.com/en-us/support/downloads/software-products/download.labview.html |
| 3 axis Linear stage actuator (XYZ positioner) | Velmex | ||
| bolts | Velmex | MB-1 | BiSlide Bolt 1/4-20×3/4" Socket cap screw (10 pack), Qty:3 |
| motor controller | Velmex | VXM-3 | Control,3 axis programmable stepping motor control, Qty:1 |
| mounting cleats | Velmex | MC-2 | Cleat, 2 hole BiSlide, Qty:6 |
| mounting cleats | Velmex | MC-2 | Cleat, 2 hole BiSlide, Qty:2 |
| usb to serial converter | Velmex | VXM-USB-RS232 | USB to RS232 Serial Communication Cable 10ft, Qty:1 |
| x-axis linear stage | Velmex | MN10-0100-M02-21 | BiSlide, travel=10 inch, 2 mm/rev, limits, NEMA 23, Qty:1 |
| x-axis stepper motor | Velmex | PK266-03A-P1 | Vexta Type 23T2, Single Shaft Stepper Motor, Qty:1 |
| y-axis linear stage | Velmex | MN10-0100-M02-21 | BiSlide, travel=10 inch, 2 mm/rev, limits, NEMA 23, Qty:1 |
| y-axis stepper motor | Velmex | PK266-03A-P1 | Vexta Type 23T2, Single Shaft Stepper Motor, Qty:1 |
| z-axis damper | Velmex | D6CL-6.3F | D6CL Damper for Type 23 Double Shaft Stepper Motor, Qty:1 |
| z-axis linear stage | Velmex | MN10-0100-M02-21 | BiSlide, travel=10 inch, 2 mm/rev, limits, NEMA 23, Qty:1 |
| z-axis stepper motor | Velmex | PK266-03B-P2 | Vexta Type 23T2, Double Shaft Stepper Motor, Qty:1 |
| 3D printable files | |||
| Immersion transducer mount and pointer | https://www.tinkercad.com/things/cRgTthGXSRq | ||
| Stereotaxic frame | https://www.tinkercad.com/things/ilynoQcdqlH | ||
| Stereotaxic frame holder | https://www.tinkercad.com/things/aZNgqhBOHAX | ||
| 9.4T small bore animal MRI | Bruker | Bruker BioSpec 94/20 | ParaVision version 5.1 |
| AAV9-hsyn-GFP | Addgene | ||
| Cream hair remover | Church & Dwight | Nair cream | |
| gadobutrol MRI contrast agent | Bayer | Gadavist (Gadobutrol, 1mM/mL) | |
| Stereotactic frame | Stoelting | #51500 | not MRI compatible |
| turnkey FUS delivery device | FUS Instruments | RK-300 | ready to use MRI compatible FUS for rodents |
References
- Markou, A., Chiamulera, C., Geyer, M. A., Tricklebank, M., Steckler, T. Removing obstacles in neuroscience drug discovery: the future path for animal models. Neuropsychopharmacology. 34 (1), 74-89 (2009).
- Schoepp, D. D. Where will new neuroscience therapies come from. Nature Reviews. Drug Discovery. 10 (10), 715-716 (2011).
- Insel, T. R., Landis, S. C. Twenty-five years of progress: the view from NIMH and NINDS. Neuron. 80 (3), 561-567 (2013).
- Bicker, J., Alves, G., Fortuna, A., Falcão, A. Blood-brain barrier models and their relevance for a successful development of CNS drug delivery systems: a review. European Journal of Pharmaceutics and Biopharmaceutics. 87 (3), 409-432 (2014).
- Pardridge, W. M. The blood-brain barrier: bottleneck in brain drug development. NeuroRx: the journal of the American Society for Experimental NeuroTherapeutics. 2 (1), 3-14 (2005).
- Millan, M. J., Goodwin, G. M., Meyer-Lindenberg, A., Ove Ögren, S. Learning from the past and looking to the future: Emerging perspectives for improving the treatment of psychiatric disorders. European Neuropsychopharmacology. 25 (5), 599-656 (2015).
- Correll, C. U., Carlson, H. E. Endocrine and metabolic adverse effects of psychotropic medications in children and adolescents. Journal of the American Academy of Child and Adolescent Psychiatry. 45 (7), 771-791 (2006).
- Girgis, R. R., Javitch, J. A., Lieberman, J. A. Antipsychotic drug mechanisms: links between therapeutic effects, metabolic side effects and the insulin signaling pathway. Molecular Psychiatry. 13 (10), 918-929 (2008).
- Patel, M. M., Goyal, B. R., Bhadada, S. V., Bhatt, J. S., Amin, A. F. Getting into the brain: approaches to enhance brain drug delivery. CNS Drugs. 23 (1), 35-58 (2009).
- McCluskey, L., Campbell, S., Anthony, D., Allan, S. M. Inflammatory responses in the rat brain in response to different methods of intra-cerebral administration. Journal of Neuroimmunology. 194 (1-2), 27-33 (2008).
- Thanou, M., Gedroyc, W. MRI-Guided Focused Ultrasound as a New Method of Drug Delivery. Journal of drug delivery. 2013, 616197 (2013).
- Burgess, A., Hynynen, K. Noninvasive and targeted drug delivery to the brain using focused ultrasound. ACS Chemical Neuroscience. 4 (4), 519-526 (2013).
- Burgess, A., Shah, K., Hough, O., Hynynen, K. Focused ultrasound-mediated drug delivery through the blood-brain barrier. Expert Review of Neurotherapeutics. 15 (5), 477-491 (2015).
- Shin, J., et al. Focused ultrasound-mediated noninvasive blood-brain barrier modulation: preclinical examination of efficacy and safety in various sonication parameters. Neurosurgical Focus. 44 (2), 15 (2018).
- Bing, C., et al. Characterization of different bubble formulations for blood-brain barrier opening using a focused ultrasound system with acoustic feedback control. Scientific Reports. 8 (1), 7986 (2018).
- Hynynen, K., McDannold, N., Vykhodtseva, N., Jolesz, F. A. Noninvasive MR imaging-guided focal opening of the blood-brain barrier in rabbits. Radiology. 220 (3), 640-646 (2001).
- Baseri, B., et al. Activation of signaling pathways following localized delivery of systemically administered neurotrophic factors across the blood-brain barrier using focused ultrasound and microbubbles. Physics in Medicine and Biology. 57 (7), 65-81 (2012).
- Rodríguez-Frutos, B., et al. Enhanced brain-derived neurotrophic factor delivery by ultrasound and microbubbles promotes white matter repair after stroke. Biomaterials. 100, 41-52 (2016).
- Karakatsani, M. E., et al. Amelioration of the nigrostriatal pathway facilitated by ultrasound-mediated neurotrophic delivery in early Parkinson’s disease. Journal of Controlled Release. 303, 289-301 (2019).
- Lin, C. -. Y., et al. Non-invasive, neuron-specific gene therapy by focused ultrasound-induced blood-brain barrier opening in Parkinson’s disease mouse model. Journal of Controlled Release. 235, 72-81 (2016).
- Long, L., et al. Treatment of Parkinson’s disease in rats by Nrf2 transfection using MRI-guided focused ultrasound delivery of nanomicrobubbles. Biochemical and Biophysical Research Communications. , (2016).
- Fan, C. -. H., Lin, C. -. Y., Liu, H. -. L., Yeh, C. -. K. Ultrasound targeted CNS gene delivery for Parkinson’s disease treatment. Journal of Controlled Release. 261, 246-262 (2017).
- Kinoshita, M., McDannold, N., Jolesz, F. A., Hynynen, K. Targeted delivery of antibodies through the blood-brain barrier by MRI-guided focused ultrasound. Biochemical and Biophysical Research Communications. 340 (4), 1085-1090 (2006).
- Todd, N., et al. Modulation of brain function by targeted delivery of GABA through the disrupted blood-brain barrier. Neuroimage. 189, 267-275 (2019).
- Nance, E., et al. Non-invasive delivery of stealth, brain-penetrating nanoparticles across the blood-brain barrier using MRI-guided focused ultrasound. Journal of Controlled Release. 189, 123-132 (2014).
- Mulik, R. S., et al. Localized delivery of low-density lipoprotein docosahexaenoic acid nanoparticles to the rat brain using focused ultrasound. Biomaterials. 83, 257-268 (2016).
- Lin, T., et al. Blood-Brain-Barrier-Penetrating Albumin Nanoparticles for Biomimetic Drug Delivery via Albumin-Binding Protein Pathways for Antiglioma Therapy. ACS Nano. 10 (11), 9999-10012 (2016).
- Timbie, K. F., et al. MR image-guided delivery of cisplatin-loaded brain-penetrating nanoparticles to invasive glioma with focused ultrasound. Journal of Controlled Release. 263, 120-131 (2017).
- Fan, C. -. H., et al. SPIO-conjugated, doxorubicin-loaded microbubbles for concurrent MRI and focused-ultrasound enhanced brain-tumor drug delivery. Biomaterials. 34 (14), 3706-3715 (2013).
- Mainprize, T., et al. Blood-Brain Barrier Opening in Primary Brain Tumors with Non-invasive MR-Guided Focused Ultrasound: A Clinical Safety and Feasibility Study. Scientific Reports. 9 (1), 321 (2019).
- Chen, K. -. T., Wei, K. -. C., Liu, H. -. L. Theranostic Strategy of Focused Ultrasound Induced Blood-Brain Barrier Opening for CNS Disease Treatment. Frontiers in Pharmacology. 10, 86 (2019).
- Lipsman, N., et al. Blood-brain barrier opening in Alzheimer’s disease using MR-guided focused ultrasound. Nature Communications. 9 (1), 2336 (2018).
- . Compound Administration I Available from: https://www.jove.com/science-education/10198/compound-administration-i (2020)
- Liu, H. -. L., et al. Magnetic resonance imaging enhanced by superparamagnetic iron oxide particles: usefulness for distinguishing between focused ultrasound-induced blood-brain barrier disruption and brain hemorrhage. Journal of Magnetic Resonance Imaging. 29 (1), 31-38 (2009).
- Geiger, B. M., Frank, L. E., Caldera-Siu, A. D., Pothos, E. N. Survivable stereotaxic surgery in rodents. Journal of Visualized Experiments. (20), e880 (2008).
- Marty, B., et al. Dynamic study of blood-brain barrier closure after its disruption using ultrasound: a quantitative analysis. Journal of Cerebral Blood Flow and Metabolism. 32 (10), 1948-1958 (2012).
- Alonso, A., Reinz, E., Fatar, M., Hennerici, M. G., Meairs, S. Clearance of albumin following ultrasound-induced blood-brain barrier opening is mediated by glial but not neuronal cells. Brain Research. 1411, 9-16 (2011).
- McDannold, N., Zhang, Y., Vykhodtseva, N. Blood-brain barrier disruption and vascular damage induced by ultrasound bursts combined with microbubbles can be influenced by choice of anesthesia protocol. Ultrasound in Medicine & Biology. 37 (8), 1259-1270 (2011).
- McDannold, N., Zhang, Y., Vykhodtseva, N. The Effects of Oxygen on Ultrasound-Induced Blood-Brain Barrier Disruption in Mice. Ultrasound in Medicine & Biology. 43 (2), 469-475 (2017).
- O’Reilly, M. A., Muller, A., Hynynen, K. Ultrasound insertion loss of rat parietal bone appears to be proportional to animal mass at submegahertz frequencies. Ultrasound in Medicine & Biology. 37 (11), 1930-1937 (2011).
- Abrahao, A., et al. First-in-human trial of blood-brain barrier opening in amyotrophic lateral sclerosis using MR-guided focused ultrasound. Nature Communications. 10 (1), 4373 (2019).
