Name: Yazar Gündemi: Glioblastoma Tedavisinde İlaç Dağıtımını ve Erken Teşhisi Optimize Etmek için Multimodal Görüntüleme Stratejileri
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

Stanford Wu Tsai Nörobilim Mikroskopi Servisi'ne, Stanford In Vivo Görüntülemede Yenilikler Merkezi'ne (SCi3) - küçük hayvan görüntüleme merkezine, NIH S10 Paylaşılan Enstrümantasyon Hibesi'ne (S10RR026917-01, PI Michael Moseley, Ph.D.) ve Porter Drive'daki Stanford Klinik Öncesi Görüntüleme Tesisi'ne bu proje için ekipman ve altyapı sağladıkları için teşekkür ederiz. Bu çalışma, Ulusal Çocuk Sağlığı ve İnsani Gelişme Enstitüsü'nün R01HD103638 numaralı hibesi ile desteklenmiştir. Schnitzer Group, Stanford Üniversitesi'ne teşekkür ederiz; Zuo laboratuvarı, Santa Cruz Üniversitesi; ve Nörovasküler Görüntüleme Laboratuvarı, Boston Fotonik Merkezi, Boston Üniversitesi, iki foton görüntüleme ve kraniyal pencere modelleri üzerine eğitim tartışmaları için.

Bu protokolde açıklanan hayvan prosedürü, Laboratuvar Hayvanları Bakımı İdari Paneli'nin (APLAC) gerekliliklerine uygundur.
1. Hücre kültürü
<ol><li>Davlumbazın hazırlanması<ol><li>Ellerinizi yıkayın, eldiven ve laboratuvar önlüğü giyin. Biyolojik güvenlik kabinini açın ve kanat seviyesini uygun bir açılma yüksekliğine ayarlayın. Davlumbazın 3-5 dakika kurumasına izin verin. Davlumbaz bölgesine %70 etanol püskürtün ve kağıt mendille silin...

Farelerde 2P-IVM Kullanarak Beyin Tümörlerinde Nanopartiküllerin Gerçek Zamanlı Takibi

<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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+two-photon+calcium+imaging+of+hippocampal+neurons+in+Alzheimer+mouse+models.">In vivo two-photon calcium imaging of hippocampal neurons in Alzheimer mouse models.</a> Methods Mol Biol. 1750, 341-351 (2018).</li><li>Li, L., 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=A+sensitive+two-photon+probe+to+selectively+detect+monoamine+oxidase+B+activity+in+Parkinson's+disease+models.">A sensitive two-photon probe to selectively detect monoamine oxidase B activity in Parkinson's disease models.</a> Nat Commun. 5, 3276 (2014).</li><li>Madden, K. S., Zettel, M. L., Majewska, A. K., Brown, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+tumor+imaging:+live+imaging+of+glioma+by+two-photon+microscopy.">Brain tumor imaging: live imaging of glioma by two-photon microscopy.</a> Cold Spring Harb Protoc. 2013 (3), (2013).</li><li>Ricard, C., 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=Phenotypic+dynamics+of+microglial+and+monocyte-derived+cells+in+glioblastoma-bearing+mice.">Phenotypic dynamics of microglial and monocyte-derived cells in glioblastoma-bearing mice.</a> Sci Rep. 6, 26381 (2016).</li><li>Soub&#233;ran, A., 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=Effects+of+VEGF+blockade+on+the+dynamics+of+the+inflammatory+landscape+in+glioblastoma-bearing+mice.">Effects of VEGF blockade on the dynamics of the inflammatory landscape in glioblastoma-bearing mice.</a> J Neuroinflammation. 16 (1), 191 (2019).</li><li>Zhang, 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=Real-time+vivo+reveals+specific+nanoparticle+target+binding+in+a+syngeneic+glioma+mouse+model.">Real-time vivo reveals specific nanoparticle target binding in a syngeneic glioma mouse model.</a> Nanoscale. 14 (15), 5678-5688 (2022).</li><li>Wartchow, K. 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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H., 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=Long-term+optical+access+to+an+estimated+one+million+neurons+in+the+live+mouse+cortex.">Long-term optical access to an estimated one million neurons in the live mouse cortex.</a> Cell Rep. 17 (12), 3385-3394 (2016).</li><li>K&#305;l&#305;&#231;, K., 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=Chronic+cranial+windows+for+long+term+multimodal+neurovascular+imaging+in+mice.">Chronic cranial windows for long term multimodal neurovascular imaging in mice.</a> Front Physiol. 11, 612678 (2021).</li><li>Helm, P. J., Ottersen, O. P., Nase, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+optical+properties+of+the+mouse+cranium-implications+for+in+vivo+multi+photon+laser+scanning+microscopy.">Analysis of optical properties of the mouse cranium-implications for in vivo multi photon laser scanning microscopy.</a> J Neurosci Methods. 178 (2), 316-322 (2009).</li><li>Lu, W., Sun, Q., Wan, J., She, Z., Jiang, X. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+albumin-conjugated+pegylated+nanoparticles+allow+gene+delivery+into+brain+tumors+via+intravenous+administration.">Cationic albumin-conjugated pegylated nanoparticles allow gene delivery into brain tumors via intravenous administration.</a> Cancer Res. 66 (24), 11878-11887 (2006).</li><li>Zhang, B., 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=LDLR-mediated+peptide-22-conjugated+nanoparticles+for+dual-targeting+therapy+of+brain+glioma.">LDLR-mediated peptide-22-conjugated nanoparticles for dual-targeting therapy of brain glioma.</a> Biomaterials. 34 (36), 9171-9182 (2013).</li><li>Xin, H., 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=Enhanced+anti-glioblastoma+efficacy+by+PTX-loaded+PEGylated+poly(&#603;-caprolactone)+nanoparticles:+In+vitro+and+in+vivo+evaluation.">Enhanced anti-glioblastoma efficacy by PTX-loaded PEGylated poly(&#603;-caprolactone) nanoparticles: In vitro and in vivo evaluation.</a> Int J Pharm. 402 (1-2), 238-247 (2010).</li><li>Valable, S., 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=In+vivo+MRI+tracking+of+exogenous+monocytes/macrophages+targeting+brain+tumors+in+a+rat+model+of+glioma.">In vivo MRI tracking of exogenous monocytes/macrophages targeting brain tumors in a rat model of glioma.</a> Neuroimage. 40 (2), 972 (2008).</li><li>Jia, Y., 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=Phototheranostics:+Active+targeting+of+orthotopic+glioma+using+biomimetic+proteolipid+nanoparticles.">Phototheranostics: Active targeting of orthotopic glioma using biomimetic proteolipid nanoparticles.</a> ACS Nano. 13 (1), 386-398 (2021).</li><li>Asan, L., 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=Cellular+correlates+of+gray+matter+volume+changes+in+magnetic+resonance+morphometry+identified+by+two-photon+microscopy.">Cellular correlates of gray matter volume changes in magnetic resonance morphometry identified by two-photon microscopy.</a> Sci Rep. 11, 4234 (2021).</li></ol>

Floresan etiketli demir oksit NP'lerin tümör iletimini değerlendirmek için bir kraniyal pencereden 2P-IVM kullanarak gerçek zamanlı in vivo NP takibi için bir yöntem sunuyoruz. Bu prosedür için cerrahi teknik, sabit bir el ve ileri deneysel cerrahi beceriler gerektirir. Canlı hayvan deneyimine geçmeden önce karkaslar veya hayaletler kullanarak pratik yapmanız önerilir. Alternatif olarak, Hoeferlin ve ark. termal hasarı azaltmak, cerrahi teknik değişkenliğini en aza indirmek ve cerrahiyi standa...

İki fotonlu intravital mikroskopi (2P-IVM), canlı doku1'in görüntülenmesini sağlayan bir floresan görüntüleme tekniğidir.
İlk olarak 1990'larda geliştirilen 2P-IVM, çeşitli klinik öncesi modellerde retina2, böbrek3, ince bağırsak4, koklea5, kalp6, trakea7 ve beynin in vivo analizi için kullanılmıştır 8,9. Sinirbilim alanında, 2P-IVM, uyanık hayvanlarda sağlıklı beynin gerçek zamanlı görüntülenmesi için bir teknik olarakönem kazanmıştır 10 ve Alzheimer11, Parkinson12 ve glioblastoma (GBM) gibi sinir sistemi hastalıklarını incelemenin yanı sıra13,14,15,16.
2P-IVM, GBM'nin gelişimi sırasında tümör mikroçevresini incelemek için zarif bir çözüm sunar. Daha önceki bazı çalışmalar in vitro17 ve ex vivo modeller18'e odaklanırken, diğerleri GBM'yi incelemek için ortotopik19 ve ksenotropik20 in vivo modelleri uyguladı. Madden ve ark. bir fare modelinde CNS-1 sıçan glioma hücre hattının doğal görüntülemesini gerçekleştirdi13. Ortotopik bir GL261-DsRed murin modeli kullanarak, Ricard ve ark. 2P-IVM14'te tümör bölgesindeki kan damarlarını geliştirmek için intravenöz bir florofor uygulaması gerçekleştirdi.
Burada, GBM'nin ortotopik bir fare modelinde floresan etiketli demir oksit nanopartiküllerinin (NP) tümör iletimini izlemek için 2P-IVM uyguluyoruz. Bir kraniyal pencere kullanarak, bu yöntem beyindeki NP'lerin gerçek zamanlı uzay-zamansal dağılımını ayrıntılı olarak incelememizi sağlar.

<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

İntravenöz olarak uygulanan kanser terapötiklerinin beyin tümörlerine verilmesi, kan-beyin bariyeri ile sınırlıdır. Beyin tümörlerinde makromoleküllerin birikimini ve dağılımını in vivo olarak doğrudan görüntülemek için bir yöntem, klinik öncesi modellerde ilaç dağıtımını anlama ve optimize etme yeteneğimizi büyük ölçüde artıracaktır. Bu protokol, bir fare glioblastoma (GBM) modelinde iki fotonlu intravital mikroskopi (2P-IVM) ile intravenöz olarak uygulanan floresan etiketli nanopartiküllerin gerçek zamanlı in vivo takibi için bir yöntemi açıklar.
Protokol, deney hayvanlarının anestezi ve analjezisi, bir kraniyal pencere oluşturma, GBM hücre implantasyonu, bir kafa çubuğu yerleştirme, 2P-IVM çalışmaları yürütme ve uzun süreli takip çalışmaları için cerrahi sonrası bakım dahil olmak üzere prosedürün çok aşamalı bir tanımını içerir. Temsili 2P-IVM görüntüleme seansları ve görüntü analizi gösteriyoruz, bu teknolojinin avantaj ve dezavantajlarını inceliyoruz ve potansiyel uygulamaları tartışıyoruz.
Bu yöntem, in vivo preklinik beyin görüntüleme alanındaki farklı araştırma soruları için kolayca değiştirilebilir ve uyarlanabilir.

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.

Yazar Gündemi: Glioblastoma Tedavisinde İlaç Dağıtımını ve Erken Teşhisi Optimize Etmek için Multimodal Görüntüleme Stratejileri

Bir Murin Modelinde Glioblastomun İki Fotonlu İntravital Mikroskobu

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.

Burada kraniyal pencere cerrahisi uyguladık ve GBM'nin NSG fare modelinde C6 hücrelerini aşıladık (n=5). Pencerenin oluşturulmasında yer alan tüm bileşenler arasında uygun bir sızdırmazlık (Şekil 1A), pencerelerin uzun süreli görüntüleme için dayanıklılığını sağlayacak ve ayrıca morbiditeyi azaltacaktır. İn vivo 2P-IVM için uyarlanmış aşamayı kullanarak (Şekil 2), anestezi altındaki hayvanları herhangi bir büyük ha...

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

Bir fare modelinde floresan etiketli demir oksit nanopartiküllerinin glioblastoma tümör iletiminin iki foton mikroskobu için yeni bir yaklaşım sunuyoruz.

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

Glioblastoma en yaygın primer beyin kanserlerinden biridir ve hızlı büyüme ve kötü yaşam beklentisi ile ilişkilidir. Glioblastoma için etkili tedaviler geliştirmek, terapötiklerin verilmesi kan beyin bariyeri ile sınırlı olduğu için büyük bir zorluk olmaya devam etmektedir. Beyindeki makromoleküllerin birikimini ve dağılımını doğrudan görüntülemek için bir yöntem, ilaç dağıtımını anlama ve optimize etme yeteneğimizi büyük ölçüde artıracaktır.İki foton mikroskobu, glioblastomun klinik öncesi modelinde nanopartiküllerin gerçek zamanlı dağılımını incelemek için zarif bir çözüm sunar. Bu çalışmada kullanılan nanopartikülü değiştirerek, farklı ilaç adaylarının ve kombinasyon tedavilerinin hücresel ve moleküler düzeyde uygulanmasını araştırmak mümkündür. İki fotonlu intravital mikroskopiye ek olarak, bu nanopartiküllerin demir oksit bileşeni, MRI veya MPI ile multimodal görüntülemeye izin verir.Gelecekte, görüntüleme tesisleri kansere erken teşhisin gerçekleştirilmesinde hizmet edebilir ve böylece malign neoplaziye karşı daha etkili tedavilere yol açabilir. Bunu başarmanın bir yolu, iki fotonlu intravital mikroskopiyi pozitron emisyon tomografisi ve fotoakustik ultrason gibi diğer görüntüleme yöntemleriyle birleştirmektir.

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.