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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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-....

Protocol

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

  1. Preparation of hood
    1. Wash hands, wear gloves and a lab coat. Turn on the biological safety cabinet and set the sash level to an appropriate opening height. Let the hood purge for 3-5 min. Spray the hood area with 70% ethanol and wipe it down with tissue paper.
    2. Spray all reagents w.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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....

Materials

NameCompanyCatalog NumberComments
0.9% sodium chloride infusion solutionBaxter Corp533-JB1301P

Dulbecco's Modified Eagle Medium
Invitrogen11965-092
1 mL syringesBDLuer-Lok syringe, REF309628
10% FBSThermo fisherCytiva SH30910.03HI
10% DMSOSigma-AldrichD8418-50ML
2-photon microscopePrairie Technologies, BrukerPrairie Ultima IV
Alcohol applicators, 70%Medline Industries, LPMDS093810
Alcohol, spray bottleDecon Labs IncDecon SaniHol, 04-355-122
Aluminum foilReynold BrandsReynold Wrap non-stick aluminum foil
Anesthesia machinePatterson ScientificSAS3
Anesthesia monitoring Kent Scientific MouseSTAT Jr. Rodent Pulsoxymeter
Antibiotic-Antimycotic (100x), liquidInvitrogen15240-096
Betadine applicatorsProfessional Disposables International, IncS41125
Biopsy punch, 5 mm MiltexSize 5
Buprenorphine sustained releaseZoo PharmBup SR Lab, 1.0 mg/mLA generic drug can be used instead. 
C6 rat glioma cell lineATCC (American Tissue Culture Collection)CCL-107
CannulasBD16 G, 1.1/2”, 30 G, 1”
CarprofenPfizer Rimadyl, 50 mg/mlA generic drug can be used instead. 
CefazolineSagent Pharmaceuticals25021-101-10, 1 g/vialA generic drug can be used instead. 
Cell strainer, 40 µmFisher Scientific87711
Cotton tip applicators, 6” Dyad Medical Sourcing, LLCHCS1005
Dental cementStoelting Co51459Dental cement kit, clear, 2 components
DexamethasoneBimeda138RX, 2 mg/mLA generic drug can be used instead. 
DietGelClearH2ORecovery, 72-06-502
Drape Cardinal HealthBio Shield Wrap
DrillSaeyang MicrotechEscort Pro, B08350
Drill tips Hager & Meissinger GmbHREF310104001001005Size 005, US 1/4
FIJI imaging analysis softwareNational Institute of Healthhttps://imagej.net/software/fiji/
ForcepsFisher Scientific13-812-41
GauzeFisher HealthCareSterile 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 diameterFisher ScientificMenzel Cover glass 
Gloves, non-sterileFisher ScientificNitrile powder-free medical examination gloves
Gloves, sterileMedline Industries, LPMDS104070
Hair removal creamChurch & DwightNair Hair remover lotion 
Hamilton syringeHamilton Company IncGastight #1701, 10 µL
HBSS without Ca, MgFisher ScientificPI88284
Head barHongway5 mm inner diameter O-rings
Heating padStoelting Co. Rodent warmer X2
Insulin syringesExel International Medical Products 29G x 1/2″
Iron oxide nanoparticlesCovis Pharma GmbHFeraheme ferumoxytol injection, 510 mg/17 mL, 59338077501
IsofluraneDechra26675-46-7
MiceJackson LaboratoriesNSG, Strain 005557
Microscope (surgery)Seiler MedicalSeiler IQ Q-100-220
NanoparticlesCustomIron oxide nanoparticles (Ferumoxytol) labeled with fluorescein isothiocyanate
Ophtalmic ointmentMajor pharmaceuticalsLubrifresh P.M. nighttime ointment, 203964
OxygenLinde Gas & Equipment Inc. High Pressure Steel K Style Cylinder, 249CF, 2000PSIG, CGA 540
Plastic cupsGeorgia-Pacirif Consumer ProductsDixie Portion Cup, 2 oz., Plastic, Clear, PK2400
Polyethylene tubingBraintree Scientific50-195-5494
ScaleOhaus CorpCR2200
ScalpelIntegra Life Sciences Production CorpIntegra Miltex Stainless steel disposable scalpel
ScissorsFisher Scientific13-804-18
SealantHenkel CorpLoctite 4014
Single use lab gownHigh Tech Conversions17-444-081
Stereotactic frameStoelting Co. Stoelting New Standard TM
Sterile Vacuum Bottle Top Filtration SystemsFisher ScientificS2GPU05RE, MilliporeSigma NO.:S2GPU05RE
Styrofoam boxN/AN/A
Surgical glovesCardinal Health19-163-108
Surgical glue, 3M Vetbond tissues adhesive3M Animal Care Prodcuts1469SB
Tail vein cathetherCustomConsists of two 30 G cannulas connected with sillicone tubing 
TrypLE Express (1x), no phenol redInvitrogen12604-039
Ultraviolett torchSpring sunshine technologyConsciot

References

  1. Denk, W., Strickler, J. H., Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science. 248 (4951), 73-76 (1990).
  2. Wang, Z., McCracken, S., Williams, P. R. Transpupillary two-photon in vivo imaging of the mouse retina.

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