Name: Autor em destaque: Estratégias de imagem multimodal para otimizar a administração de medicamentos e a detecção precoce no tratamento do glioblastoma
Uploaded: 2024-03-01T14:15:00
Description: Watch this Scientific Journal Video about Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model at JoVE.com

Gostaríamos de agradecer ao Stanford Wu Tsai Neuroscience Microscopy Service, ao Stanford Center for Innovations in In Vivo Imaging (SCi3) - centro de imagens de pequenos animais, ao NIH S10 Shared Instrumentation Grant (S10RR026917-01, PI Michael Moseley, Ph.D.) e ao Stanford Preclinical Imaging Facility at Porter Drive por fornecer o equipamento e a infraestrutura para este projeto. Este trabalho foi apoiado por uma bolsa do Instituto Nacional de Saúde da Criança e Desenvolvimento Humano, bolsa número R01HD103638. Agradecemos ao Grupo Schnitzer, Universidade de Stanford; o laboratório Zuo, da Universidade de Santa Cruz; e o Neurovascular Imaging Laboratory, Boston Photonic Center, University of Boston, para discussões educacionais sobre imagens de dois fótons e modelos de janela craniana.

O procedimento animal descrito neste protocolo está de acordo com as exigências do Painel Administrativo de Cuidados com Animais de Laboratório (APLAC).
1. Cultura celular
<ol><li>Preparação do exaustor<ol><li>Lave as mãos, use luvas e jaleco. Ligue o armário de segurança biológica e ajuste o nível da faixa para uma altura de abertura apropriada. Deixe o capuz purgar por 3-5 min. Borrife a área da coifa com etanol 70% e limpe-a com papel higiênico.</li>
		<li>Bor...

Rastreamento em tempo real de nanopartículas em tumores cerebrais usando 2P-IVM em camundongos

<ol>
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Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intubation-free+in+vivo+imaging+of+the+tracheal+mucosa+using+two-photon+microscopy.">Intubation-free in vivo imaging of the tracheal mucosa using two-photon microscopy.</a> Sci Rep. 7 (1), 694 (2017).</li><li>Zong, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+two-photon+calcium+imaging+in+freely+moving+mice.">Large-scale two-photon calcium imaging in freely moving mice.</a> Cell. 185 (7), 1240-1256.e30 (2022).</li><li>Takasaki, K., Abbasi-Asl, R., Waters, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superficial+bound+of+the+depth+limit+of+two-photon+imaging+in+mouse+brain.">Superficial bound of the depth limit of two-photon imaging in mouse brain.</a> eNeuro. 17 (1), (2019).</li><li>Birkner, A., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+two-photon+imaging+in+vivo+with+a+red-shifted+calcium+indicator.">Deep two-photon imaging in vivo with a red-shifted calcium indicator.</a> Methods Mol Biol. 1929, 15-26 (1929).</li><li>Busche, M. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+with+cyclic+AMP+activators+reduces+glioblastoma+growth+and+invasion+as+assessed+by+two-photon+microscopy.">Treatment with cyclic AMP activators reduces glioblastoma growth and invasion as assessed by two-photon microscopy.</a> Cells. 10 (3), 556 (2021).</li><li>Jiang, L. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+detection+of+fibrillar+collagen+deposition+associated+with+vascular+elements+in+glioblastoma+multiforme+by+using+multiphoton+microscopy.">Label-free detection of fibrillar collagen deposition associated with vascular elements in glioblastoma multiforme by using multiphoton microscopy.</a> J Microsc. 265 (2), 207-213 (2017).</li><li>Chen, Z., Ross, J. L., Hambardzumyan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+2-photon+imaging+reveals+distinct+morphology+and+infiltrative+properties+of+glioblastoma-associated+macrophages.">Intravital 2-photon imaging reveals distinct morphology and infiltrative properties of glioblastoma-associated macrophages.</a> Proc Natl Acad Sci U S A. 116 (28), 14254-14259 (2019).</li><li>Zhang, Y. 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Apresentamos um método para rastreamento in vivo de NP em tempo real usando 2P-IVM através de uma janela craniana para avaliar a liberação tumoral de NPs de óxido de ferro marcadas com fluorescência. A técnica cirúrgica para este procedimento requer mão firme e habilidades cirúrgicas experimentais avançadas. É aconselhável praticar o uso de carcaças ou objetos simuladores antes de avançar para a experiência com animais vivos. Como alternativa, Hoeferlin e col. implementaram uma broca robótica pa...

A microscopia intravital de dois fótons (2P-IVM) é uma técnica de imagem por fluorescência que permite a visualização de tecidos vivos1.
Desenvolvida pela primeira vez na década de 1990, a 2P-IVM tem sido utilizada para análise in vivo da retina2, rim3, intestino delgado4, cóclea5, coração6, traqueia7 e cérebro em vários modelos pré-clínicos 8,9. No campo da neurociência, a 2P-IVM tem ganhado importância como técnica de obtenção de imagens em tempo real do cérebro saudável em animaisacordados10, além de estudar doenças do sistema nervoso comoAlzheimer11,Parkinson12 e glioblastoma (GBM)13,14,15,16.
2P-IVM oferece uma solução elegante para estudar o microambiente tumoral durante o desenvolvimento do GBM. Enquanto alguns estudos anteriores se concentraram em modelos in vitro17 e ex vivo 18, outros implementaram modelos ortotópicos19 e xenotrópicos20 in vivo para examinar GBM. Madden e col. realizaram imagens nativas da linhagem celular de glioma de rato CNS-1 em um modelo de camundongo13. Ricard e col. realizaram a administração intravenosa de um fluoróforo para realçar os vasos sanguíneos na região tumoral em 2P-IVM, utilizando um modelo murino ortotópico GL261-DsRed14.
Aqui, aplicamos 2P-IVM para rastrear a liberação tumoral de nanopartículas de óxido de ferro (NP) marcadas fluorescentes em um modelo ortotópico de GBM em camundongos. Usando uma janela craniana, este método nos permite estudar a distribuição espaço-temporal em tempo real de NPs no cérebro em detalhes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% sodium chloride infusion solution</td>
			<td>Baxter Corp</td>
			<td>533-JB1301P</td>
			<td></td>
		</tr>
		<tr>
			<td> 
			Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringes</td>
			<td>BD</td>
			<td>Luer-Lok syringe, REF309628</td>
			<td></td>
		</tr>
		<tr>
			<td>10%&#160;FBS</td>
			<td>Thermo fisher</td>
			<td>Cytiva SH30910.03HI</td>
			<td></td>
		</tr>
		<tr>
			<td>10% DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>2-photon microscope</td>
			<td>Prairie Technologies, Bruker</td>
			<td>Prairie Ultima IV</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol applicators, 70%</td>
			<td>Medline Industries, LP</td>
			<td>MDS093810</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol, spray bottle</td>
			<td>Decon Labs Inc</td>
			<td>Decon SaniHol, 04-355-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Reynold Brands</td>
			<td>Reynold Wrap non-stick aluminum foil</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia machine</td>
			<td>Patterson Scientific</td>
			<td>SAS3</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia monitoring&#160;</td>
			<td>Kent Scientific&#160;</td>
			<td>MouseSTAT Jr. Rodent Pulsoxymeter</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100x), liquid</td>
			<td>Invitrogen</td>
			<td>15240-096</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine applicators</td>
			<td>Professional Disposables International, Inc</td>
			<td>S41125</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy punch, 5 mm&#160;</td>
			<td>Miltex</td>
			<td>Size 5</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine sustained release</td>
			<td>Zoo Pharm</td>
			<td>Bup SR Lab, 1.0 mg/mL</td>
			<td>A generic drug can be used instead.&#160;</td>
		</tr>
		<tr>
			<td>C6 rat glioma cell line</td>
			<td>ATCC (American Tissue Culture Collection)</td>
			<td>CCL-107</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannulas</td>
			<td>BD</td>
			<td>16 G, 1.1/2&#8221;, 30 G, 1&#8221;</td>
			<td></td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Pfizer&#160;</td>
			<td>Rimadyl, 50 mg/ml</td>
			<td>A generic drug can be used instead.&#160;</td>
		</tr>
		<tr>
			<td>Cefazoline</td>
			<td>Sagent Pharmaceuticals</td>
			<td>25021-101-10, 1 g/vial</td>
			<td>A generic drug can be used instead.&#160;</td>
		</tr>
		<tr>
			<td>Cell strainer, 40 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>87711</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton tip applicators, 6&#8221;&#160;</td>
			<td>Dyad Medical Sourcing, LLC</td>
			<td>HCS1005</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>Stoelting Co</td>
			<td>51459</td>
			<td>Dental cement kit, clear, 2 components</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Bimeda</td>
			<td>138RX, 2 mg/mL</td>
			<td>A generic drug can be used instead.&#160;</td>
		</tr>
		<tr>
			<td>DietGel</td>
			<td>ClearH2O</td>
			<td>Recovery, 72-06-502</td>
			<td></td>
		</tr>
		<tr>
			<td>Drape&#160;</td>
			<td>Cardinal Health</td>
			<td>Bio Shield Wrap</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Saeyang Microtech</td>
			<td>Escort Pro, B08350</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill tips&#160;</td>
			<td>Hager &#38; Meissinger GmbH</td>
			<td>REF310104001001005</td>
			<td>Size 005, US 1/4</td>
		</tr>
		<tr>
			<td>FIJI imaging analysis software</td>
			<td>National Institute of Health</td>
			<td>https://imagej.net/software/fiji/</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>13-812-41</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Fisher HealthCare</td>
			<td>Sterile Cotton Gauze Pad, 4 x 4&#8221;, 22-415-469</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelfoam&#160;</td>
			<td>Ethicon Inc.&#160;</td>
			<td>Surgifoam absorbable gelatin sponge, Ref. 1972</td>
			<td></td>
		</tr>
		<tr>
			<td>Germinator&#160;</td>
			<td>Cellpoint Scientific&#160;</td>
			<td>Germinator 500, No. 11688</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips, 5 mm diameter</td>
			<td>Fisher Scientific</td>
			<td>Menzel Cover glass&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves, non-sterile</td>
			<td>Fisher Scientific</td>
			<td>Nitrile powder-free medical examination gloves</td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves, sterile</td>
			<td>Medline Industries, LP</td>
			<td>MDS104070</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair removal cream</td>
			<td>Church &#38; Dwight</td>
			<td>Nair Hair remover lotion&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton Company Inc</td>
			<td>Gastight #1701, 10 &#181;L</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS without Ca, Mg</td>
			<td>Fisher Scientific</td>
			<td>PI88284</td>
			<td></td>
		</tr>
		<tr>
			<td>Head bar</td>
			<td>Hongway</td>
			<td>5 mm inner diameter O-rings</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Stoelting Co.&#160;</td>
			<td>Rodent warmer X2</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td>Exel International Medical Products&#160;</td>
			<td>29G x 1/2&#8243;</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron oxide nanoparticles</td>
			<td>Covis Pharma GmbH</td>
			<td>Feraheme ferumoxytol injection, 510 mg/17 mL, 59338077501</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Dechra</td>
			<td>26675-46-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Jackson Laboratories</td>
			<td>NSG, Strain 005557</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope (surgery)</td>
			<td>Seiler Medical</td>
			<td>Seiler IQ Q-100-220</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoparticles</td>
			<td>Custom</td>
			<td></td>
			<td>Iron oxide nanoparticles (Ferumoxytol) labeled with fluorescein isothiocyanate</td>
		</tr>
		<tr>
			<td>Ophtalmic ointment</td>
			<td>Major pharmaceuticals</td>
			<td>Lubrifresh P.M. nighttime ointment, 203964</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>Linde Gas &#38; Equipment Inc.&#160;</td>
			<td>High Pressure Steel K Style Cylinder, 249CF, 2000PSIG, CGA 540</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cups</td>
			<td>Georgia-Pacirif Consumer Products</td>
			<td>Dixie Portion Cup, 2 oz., Plastic, Clear, PK2400</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Braintree Scientific</td>
			<td>50-195-5494</td>
			<td></td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Ohaus Corp</td>
			<td>CR2200</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Integra Life Sciences Production Corp</td>
			<td>Integra Miltex Stainless steel disposable scalpel</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fisher Scientific</td>
			<td>13-804-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Sealant</td>
			<td>Henkel Corp</td>
			<td>Loctite 4014</td>
			<td></td>
		</tr>
		<tr>
			<td>Single use lab gown</td>
			<td>High Tech Conversions</td>
			<td>17-444-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic frame</td>
			<td>Stoelting Co.&#160;</td>
			<td>Stoelting New Standard TM</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Vacuum Bottle Top Filtration Systems</td>
			<td>Fisher Scientific</td>
			<td>S2GPU05RE, MilliporeSigma NO.:S2GPU05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Styrofoam box</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical gloves</td>
			<td>Cardinal Health</td>
			<td>19-163-108</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical glue, 3M Vetbond tissues adhesive</td>
			<td>3M Animal Care Prodcuts</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Tail vein cathether</td>
			<td>Custom</td>
			<td></td>
			<td>Consists of two 30 G cannulas connected with sillicone tubing&#160;</td>
		</tr>
		<tr>
			<td>TrypLE Express (1x), no phenol red</td>
			<td>Invitrogen</td>
			<td>12604-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraviolett torch</td>
			<td>Spring sunshine technology</td>
			<td>Consciot</td>
			<td></td>
		</tr>
	</tbody>
</table>

two-photon intravital microscopy of glioblastoma in a murine model

A administração intravenosa de terapêutica do câncer para tumores cerebrais é limitada pela barreira hematoencefálica. Um método para obter imagens diretas do acúmulo e distribuição de macromoléculas em tumores cerebrais in vivo aumentaria muito nossa capacidade de entender e otimizar a liberação de fármacos em modelos pré-clínicos. Este protocolo descreve um método para rastreamento in vivo em tempo real de nanopartículas fluorescentes marcadas por via intravenosa com microscopia intravital de dois fótons (2P-IVM) em um modelo de glioblastoma (GBM) em camundongos.
O protocolo contém uma descrição do procedimento em várias etapas, incluindo anestesia e analgesia de animais experimentais, criação de uma janela craniana, implante de células GBM, colocação de uma barra de cabeça, realização de estudos de 2P-IVM e cuidados pós-cirúrgicos para estudos de seguimento de longo prazo. Mostramos sessões representativas de imagens 2P-IVM e análise de imagens, examinamos as vantagens e desvantagens dessa tecnologia e discutimos possíveis aplicações.
Este método pode ser facilmente modificado e adaptado para diferentes questões de pesquisa no campo da imagem cerebral pré-clínica in vivo .

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-time imaging of the healthy brain in awake animals10, as well as studying diseases of the nervous system such as Alzheimer&#39;s11, Parkinson&#39;s12 and glioblastoma (GBM)13,14,15,16.
2P-IVM offers an elegant solution for studying the tumor microenvironment during the development of GBM. While some previous studies focused on in vitro17 and ex vivo models18, others implemented orthotopic19 and xenotropic20&#160;in vivo models for examining GBM. Madden et al. performed native imaging of CNS-1 rat glioma cell line in a mouse model13. Using an orthotopic GL261-DsRed murine model, Ricard et al. performed an intravenous administration of a fluorophore to enhance the blood vessels in the tumor region in 2P-IVM14.
Here, we apply 2P-IVM for tracking the tumor delivery of fluorescent-labeled iron oxide nanoparticles (NP) in an orthotopic mouse model of GBM. Using a cranial window, this method allows us to study the real-time spatiotemporal distribution of NPs in the brain in detail.

Autor em destaque: Estratégias de imagem multimodal para otimizar a administração de medicamentos e a detecção precoce no tratamento do glioblastoma

Microscopia Intravital de Dois Fótons de Glioblastoma em Modelo Murino

Glioblastoma is one of the most common primary brain cancer and is associated with fast growth and poor life expectancy. Developing effective therapies for glioblastoma remains a major challenge since the delivery of therapeutics is limited by the blood brain barrier. A method to directly image the accumulation and distribution of macromolecules in the brain would greatly enhance our ability to understand and optimize drug delivery.Two-photon microscopy offers an elegant solution for studying the real-time distribution of nanoparticles in the preclinical model of glioblastoma. By modifying the nanoparticle used in this study, it's possible to investigate the delivery of different drug candidates and combination therapies on a cellular and molecular level. In addition to two-photon intravital microscopy, the iron oxide component of these nanoparticles allow for multimodal imaging with MRI or MPI.For the future, imaging facilities could serve in the realization of early detection to cancer, thereby leading to more effective therapies against malignant neoplasia. One path to achieve this is to merge two-photon intravital microscopy with other imaging modalities such as positron emission tomography and photoacoustic ultrasound.

Aqui, realizamos cirurgia de janela craniana e enxertamos células C6 em um modelo de GBM em camundongos NSG (n = 5). Uma vedação adequada entre todos os componentes envolvidos na criação da janela (Figura 1A) garantirá a durabilidade das janelas para obtenção de imagens a longo prazo e, adicionalmente, reduzirá a morbidade. Usando o estágio adaptado para 2P-IVM in vivo (Figura 2), pudemos obter imagens de animais sob anestesia por até 2 h sem...

Watch this Scientific Journal Video about Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model at JoVE.com

Apresentamos uma nova abordagem para microscopia de dois fótons da liberação tumoral de nanopartículas de óxido de ferro marcadas fluorescente para glioblastoma em um modelo de camundongo.

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the ...

O glioblastoma é um dos cânceres cerebrais primários mais comuns e está associado ao crescimento rápido e à baixa expectativa de vida. O desenvolvimento de terapias eficazes para glioblastoma continua sendo um grande desafio, uma vez que a administração de terapêuticas é limitada pela barreira hematoencefálica. Um método para obter imagens diretas do acúmulo e distribuição de macromoléculas no cérebro aumentaria muito nossa capacidade de entender e otimizar a entrega de drogas.A microscopia de dois fótons oferece uma solução elegante para estudar a distribuição em tempo real de nanopartículas no modelo pré-clínico de glioblastoma. Ao modificar a nanopartícula usada neste estudo, é possível investigar a entrega de diferentes candidatos a fármacos e terapias combinadas em nível celular e molecular. Além da microscopia intravital de dois fótons, o componente óxido de ferro dessas nanopartículas permite imagens multimodais com RM ou IPM.Para o futuro, os serviços de imagem poderão servir na realização da detecção precoce do câncer, levando a terapias mais eficazes contra neoplasias malignas. Um caminho para conseguir isso é mesclar a microscopia intravital de dois fótons com outras modalidades de imagem, como tomografia por emissão de pósitrons e ultrassom fotoacústico.

two-photon-intravital-microscopy-of-glioblastoma-in-a-murine-model

Research

JoVE Journal

Cancer Research

Two-Photon Intravital Microscopy of Glioblastoma in a Murine 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.

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.