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* These authors contributed equally
We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.
The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM).
The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications.
This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.
Two-photon intravital microscopy (2P-IVM) is a fluorescence imaging technique that allows the visualization of living tissue1.
First developed in the 1990s, 2P-IVM has been used for in vivo analysis of the retina2, kidney3, small intestine4, cochlea5, heart6, trachea7, and the brain in various preclinical models8,9. In the field of neuroscience, 2P-IVM has gained importance as a technique for real-....
The animal procedure described in this protocol is in accordance with the requirements of the Administrative Panel on Laboratory Animal Care (APLAC).
1. Cell culture
Here, we performed cranial window surgery and engrafted C6 cells in an NSG mouse model of GBM (n = 5). A proper seal between all components involved in the creation of the window (Figure 1A) will ensure the windows' durability for long-term imaging and, additionally, reduce morbidity. Using the stage adapted for in vivo 2P-IVM (Figure 2), we could image animals under anesthesia for up to 2 h without any major motion artifacts. Approximately 10 min a.......
We present a method for real-time in vivo NP tracking using 2P-IVM through a cranial window to evaluate the tumor delivery of fluorescent-labeled iron oxide NPs. The surgical technique for this procedure requires a steady hand and advanced experimental surgical skills. It is advisable to practice using carcasses or phantoms before moving forward to live animal experience. As an alternative, Hoeferlin et al. implemented a robotic drill to reduce thermal damage, minimize surgical technique variability, and standar.......
We would like to thank the Stanford Wu Tsai Neuroscience Microscopy Service, the Stanford Center for Innovations in In Vivo Imaging (SCi3) - small animal imaging center, NIH S10 Shared Instrumentation Grant (S10RR026917-01, PI Michael Moseley, Ph.D.), and Stanford Preclinical Imaging Facility at Porter Drive for providing the equipment and infrastructure for this project. This work was supported by a grant from the National Institute for Child Health and Human Development, grant number R01HD103638. We would like to thank the Schnitzer Group, Stanford University; the Zuo lab, University of Santa Cruz; and the Neurovascular Imaging Laboratory, Boston Photonic Center, Un....
Name | Company | Catalog Number | Comments |
0.9% sodium chloride infusion solution | Baxter Corp | 533-JB1301P | |
Dulbecco's Modified Eagle Medium | Invitrogen | 11965-092 | |
1 mL syringes | BD | Luer-Lok syringe, REF309628 | |
10% FBS | Thermo fisher | Cytiva SH30910.03HI | |
10% DMSO | Sigma-Aldrich | D8418-50ML | |
2-photon microscope | Prairie Technologies, Bruker | Prairie Ultima IV | |
Alcohol applicators, 70% | Medline Industries, LP | MDS093810 | |
Alcohol, spray bottle | Decon Labs Inc | Decon SaniHol, 04-355-122 | |
Aluminum foil | Reynold Brands | Reynold Wrap non-stick aluminum foil | |
Anesthesia machine | Patterson Scientific | SAS3 | |
Anesthesia monitoring | Kent Scientific | MouseSTAT Jr. Rodent Pulsoxymeter | |
Antibiotic-Antimycotic (100x), liquid | Invitrogen | 15240-096 | |
Betadine applicators | Professional Disposables International, Inc | S41125 | |
Biopsy punch, 5 mm | Miltex | Size 5 | |
Buprenorphine sustained release | Zoo Pharm | Bup SR Lab, 1.0 mg/mL | A generic drug can be used instead. |
C6 rat glioma cell line | ATCC (American Tissue Culture Collection) | CCL-107 | |
Cannulas | BD | 16 G, 1.1/2”, 30 G, 1” | |
Carprofen | Pfizer | Rimadyl, 50 mg/ml | A generic drug can be used instead. |
Cefazoline | Sagent Pharmaceuticals | 25021-101-10, 1 g/vial | A generic drug can be used instead. |
Cell strainer, 40 µm | Fisher Scientific | 87711 | |
Cotton tip applicators, 6” | Dyad Medical Sourcing, LLC | HCS1005 | |
Dental cement | Stoelting Co | 51459 | Dental cement kit, clear, 2 components |
Dexamethasone | Bimeda | 138RX, 2 mg/mL | A generic drug can be used instead. |
DietGel | ClearH2O | Recovery, 72-06-502 | |
Drape | Cardinal Health | Bio Shield Wrap | |
Drill | Saeyang Microtech | Escort Pro, B08350 | |
Drill tips | Hager & Meissinger GmbH | REF310104001001005 | Size 005, US 1/4 |
FIJI imaging analysis software | National Institute of Health | https://imagej.net/software/fiji/ | |
Forceps | Fisher Scientific | 13-812-41 | |
Gauze | Fisher HealthCare | Sterile Cotton Gauze Pad, 4 x 4”, 22-415-469 | |
Gelfoam | Ethicon Inc. | Surgifoam absorbable gelatin sponge, Ref. 1972 | |
Germinator | Cellpoint Scientific | Germinator 500, No. 11688 | |
Glass coverslips, 5 mm diameter | Fisher Scientific | Menzel Cover glass | |
Gloves, non-sterile | Fisher Scientific | Nitrile powder-free medical examination gloves | |
Gloves, sterile | Medline Industries, LP | MDS104070 | |
Hair removal cream | Church & Dwight | Nair Hair remover lotion | |
Hamilton syringe | Hamilton Company Inc | Gastight #1701, 10 µL | |
HBSS without Ca, Mg | Fisher Scientific | PI88284 | |
Head bar | Hongway | 5 mm inner diameter O-rings | |
Heating pad | Stoelting Co. | Rodent warmer X2 | |
Insulin syringes | Exel International Medical Products | 29G x 1/2″ | |
Iron oxide nanoparticles | Covis Pharma GmbH | Feraheme ferumoxytol injection, 510 mg/17 mL, 59338077501 | |
Isoflurane | Dechra | 26675-46-7 | |
Mice | Jackson Laboratories | NSG, Strain 005557 | |
Microscope (surgery) | Seiler Medical | Seiler IQ Q-100-220 | |
Nanoparticles | Custom | Iron oxide nanoparticles (Ferumoxytol) labeled with fluorescein isothiocyanate | |
Ophtalmic ointment | Major pharmaceuticals | Lubrifresh P.M. nighttime ointment, 203964 | |
Oxygen | Linde Gas & Equipment Inc. | High Pressure Steel K Style Cylinder, 249CF, 2000PSIG, CGA 540 | |
Plastic cups | Georgia-Pacirif Consumer Products | Dixie Portion Cup, 2 oz., Plastic, Clear, PK2400 | |
Polyethylene tubing | Braintree Scientific | 50-195-5494 | |
Scale | Ohaus Corp | CR2200 | |
Scalpel | Integra Life Sciences Production Corp | Integra Miltex Stainless steel disposable scalpel | |
Scissors | Fisher Scientific | 13-804-18 | |
Sealant | Henkel Corp | Loctite 4014 | |
Single use lab gown | High Tech Conversions | 17-444-081 | |
Stereotactic frame | Stoelting Co. | Stoelting New Standard TM | |
Sterile Vacuum Bottle Top Filtration Systems | Fisher Scientific | S2GPU05RE, MilliporeSigma NO.:S2GPU05RE | |
Styrofoam box | N/A | N/A | |
Surgical gloves | Cardinal Health | 19-163-108 | |
Surgical glue, 3M Vetbond tissues adhesive | 3M Animal Care Prodcuts | 1469SB | |
Tail vein cathether | Custom | Consists of two 30 G cannulas connected with sillicone tubing | |
TrypLE Express (1x), no phenol red | Invitrogen | 12604-039 | |
Ultraviolett torch | Spring sunshine technology | Consciot |
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