Name: combined near-infrared fluorescent imaging and micro-computed tomography for directly visualizing cerebral thromboemboli
Uploaded: 2016-09-25T14:00:00
Description: Watch this Scientific Journal Video about Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli at JoVE.com

Dette arbejde blev støttet af Korea Healthcare Technology R &amp; D Project, Ministeriet for Sundhed og Velfærd (HI12C1847, HI12C0066), Bio &amp; Medical Technology Development Program (2010 til 0.019.862) og Global Research Lab (GRL) program (NRF-2015K1A1A2028228) af National Research Foundation, finansieret af den koreanske regering.

Alle dyreforsøg påvist i denne protokol er blevet revideret og godkendt af Dongguk University Ilsan Hospital Animal Care og brug Udvalg og gennemføres i overensstemmelse med de principper og procedurer, der er beskrevet i NIH Guide for pasning og anvendelse af dyr. 1. Fremstilling af Exogent Formed Clot mærket med Fluorescence Marker (figur 1) <ol><li> Bedøver en mus i en induktion kammer under anvendelse af 3% isofluran blandet med 30% oxygen (1,5 l / min 100% oxygen). Sikre tilstrækkelig dybde af anæs...

<ol>
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Vi demonstrerede anvendelsen af to komplementære molekylære billeddannelsesteknikker til direkte trombeimagografimiddel i forsøgsmodeller af embolisk slagtilfælde: en fibrin målrettet guld nanopartikel (FIB-GC-AUNP) til in vivo microCT-baserede billeddannelse, og en FXIIIa målrettet optisk afbildning probe for ex vivo fluorescerende billeddannelse. Efter intravenøs administration af FIB-GC-AuNPs, tromber blev synlige til CT som tætte strukturer, forårsaget af parti...

En i 6 personer vil have et slagtilfælde på et tidspunkt i deres levetid. Iskæmisk slagtilfælde er langt den mest almindelige slag type og tegner sig for omkring 80 procent af alle slagtilfælde sager. Fordi tromboemboli forårsager størstedelen af ​​disse iskæmiske slagtilfælde, er der en stigende interesse for direkte trombeimagografimiddel. Det blev anslået, at omkring 2 millioner hjerneceller dør under hvert minut af midterste cerebral arterie okklusion 1, der fører til sloganet &quot;Tid er Brain&quot;. Computertomografi (CT) undersøgelser kan gøres hurtigt, og er bredt tilgængelige; af denne grund, CT forbliver billeddannelse af valg for den første diagnose og behandling af hyperakut iskæmisk slagtilfælde. CT er særligt værdifuldt for at informere de kritiske tidlige beslutninger: administrere vævsplasminogenaktivator (TPA) til trombolyse og / eller triaging til endovaskulær blodprop-hentning 2. Nuværende CT-baserede trombeimagografimiddel kan imidlertid ikke serielt spore cerebral tromboemboli in vivo, fordi det bruger indirekte metoder til at demonstrere tromber: efter opacifikation af blodet pool ved jodholdige modsætning hertil er den tromber demonstreret som fyld fejl i karrene. Der er dosisgrænser og risici forbundet med den gentagen administration af jodholdige kontrastmidler, som udelukker gentagne billeddannelse af tromber på denne måde. Således er der et kritisk behov for en direkte billeddannelse metode til cerebral thromber hos patienter med slagtilfælde, for at tillade en hurtigere og bedre behandling beslutninger, der skal foretages. Vi foreslår at opnå dette ved at øge værdien af ​​CT, den aktuelt anvendte frontlinjen afbildningsmodalitet til slagtilfælde, ved anvendelse af en trombe-søger nanopartikelformige molekylær billeddannelse agent. Vi har vist anvendelsen af dette middel med micro-computertomografi (microCT), en høj opløsning ex vivo eller in vivo (små dyr) afbildning version af CT, der tillader erhvervelse hurtige data &lt;sup&gt; 3,4. Selv med den relativt dårlige bløde væv kontrast til rådighed for små dyr microCT (meget værre end tilgængelige fra human størrelse scannere), det billeddannende agent var i stand til at søge og markere tromber ved at gøre dem hyperdense på CT, en &#39;tæt fartøj tegnet »forstærket af molekylær billeddannelse. Som supplement til CT-teknik, har vores gruppe tidligere udviklet en optisk direkte trombe imaging teknik hjælp Cy5.5 nær-infrarødt fluorescerende (NIRF) sonde til at visualisere cerebral trombe byrde 5. Dette er en ex vivo teknik på obduktioner hjerner, men er meget følsom, og tjener til at bekræfte in vivo data i indstillingen forskning. Under både CT og NIRF baseret trombe-søger billeddannelsesteknikker giver os mulighed for at sammenligne disse teknikker til at opnå meget informative data om den rolle, thrombus og trombeimagografimiddel i processen med iskæmisk slagtilfælde udvikling. Here, beskriver vi en detaljeret protokol af et kombineret teknik med in vivo microCT og ex vivo NIRF billeddannelse til direkte visualisere tromboemboli i en musemodel af embolisk slagtilfælde. Disse enkle og robuste metoder er anvendelige til at fremme vores forståelse af trombotiske sygdomme ved at muliggøre nøjagtige in vivo vurdering af trombe byrde / distribution og karakterisering af dynamisk trombe evolution på en hurtig og kvantitativ måde in vivo under behandlingen efterfulgt af ex vivo-data, der tjener som en kontrol og reference standard til bekræftelse af in vivo resultater billeddannelse.

<table border="0" cellpadding="0" cellspacing="0" width="588">
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	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">Machines</td>
			<td style="width:219px;"></td>
			<td style="width:119px;"></td>
			<td style="width:96px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microCT</td>
			<td>NanoFocusRay, JeonJu, Korea</td>
			<td>NFR Polaris-G90</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NIRF imaging system</td>
			<td>Roper-scientific,Tucson, AZ</td>
			<td>coolsnap-Ez</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laser Doppler flowmeter</td>
			<td>Perimed, Stockholm, Sweden</td>
			<td>PeriFlux System 5000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical microscope</td>
			<td>Leica Microsystems, Seoul, Korea</td>
			<td>EZ4HD</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Inhalation anesthesia machine</td>
			<td>PerkinElmer, Massachusetts, USA</td>
			<td>XGI-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">NFR control</td>
			<td>NanoFocusRay, JeonJu, Korea</td>
			<td>NFR Polaris-G90</td>
			<td style="width:96px;">microCT control software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Lucion</td>
			<td>Infinitt, Seoul, Korea</td>
			<td>Lucion</td>
			<td style="width:96px;">3D render imaging software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lab chart 7</td>
			<td>ADInstruments, Colorado, USA</td>
			<td>Lab chart 7</td>
			<td>rCBF</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Image J software</td>
			<td>Wanye Rasband, NIH, USA</td>
			<td>1.49d</td>
			<td>imaging analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Devices/Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Infusion pump</td>
			<td>Harvard, Massachusetts, USA</td>
			<td>pump 22(55-2226)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Homeothermic blanket</td>
			<td>Panlab, Barcelona, Spain</td>
			<td>HB101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pocket cautery</td>
			<td>Daejong, Seoul, Korea</td>
			<td>DJE-39</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Brain matrice</td>
			<td>Ted pella, CA, USA</td>
			<td>15003</td>
			<td style="width:96px;">coronal section</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PE-50 tubing</td>
			<td>Natsume, Tokyo, Japan</td>
			<td>SP-45(PE-50)</td>
			<td style="width:96px;">I.D. 0.58 mm O.D. 0.96 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PE-10 tubing</td>
			<td>Natsume, Tokyo, Japan</td>
			<td>SP-10(PE-10)</td>
			<td style="width:96px;">I.D. 0.28mm O.D. 0.61 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">30 gauge needle</td>
			<td>sungshim-medical, Seoul, Korea</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe</td>
			<td>CPL-medical, Ansan, Korea</td>
			<td></td>
			<td>1 &#38; 3 cc</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gauze</td>
			<td>Panamedic, Cheonan, Korea</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tape</td>
			<td>Scotch, Seoul, Korea</td>
			<td>3M-810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro forceps</td>
			<td>Fine Science Tools, Vancouver, Canada</td>
			<td>&#160;11253-27</td>
			<td>Dumont #L5</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro scissor</td>
			<td>Fine Science Tools, Vancouver, Canada</td>
			<td style="width:119px;">15000-03</td>
			<td>Vannas spring</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissor</td>
			<td>Fine Science Tools, Vancouver, Canada</td>
			<td style="width:119px;">14084-08</td>
			<td>8.5 cm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Black silk suture</td>
			<td>Ailee, Busan, Korea</td>
			<td>SK6071, SK728</td>
			<td>6-0 and 7-0</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">meloxicam</td>
			<td>Yuhan, Seoul, Korea</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">vet ointment</td>
			<td>Novartis, Basel, Swiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10% Povidone-iodine (betadine)</td>
			<td>Firson, Cheon-an, Korea</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FeCl3</td>
			<td>Sigma, Missouri, United States</td>
			<td>157740-5G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TTC</td>
			<td>Amresco, Ohio, USA</td>
			<td>0765-100g</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isoflurane</td>
			<td>Hana-Pham, Gyeonggi, Korea</td>
			<td>Ifran</td>
			<td>100 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Welgene, Daegu, Korea</td>
			<td style="width:119px;">LB001-02</td>
			<td>500 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gold nanoparticles</td>
			<td></td>
			<td></td>
			<td>Synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">C15 optical agent</td>
			<td></td>
			<td></td>
			<td>Synthesis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue plasminogen activator</td>
			<td style="width:219px;">Boehringer Ingelheim, Biberach, Germany</td>
			<td style="width:119px;">rtPA(actilyse)</td>
			<td>20 mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal saline</td>
			<td>Daihan Pham, Seoul, Korea</td>
			<td>48N3AF3</td>
			<td>20 mL</td>
		</tr>
	</tbody>
</table>

combined near-infrared fluorescent imaging and micro-computed tomography for directly visualizing cerebral thromboemboli

Direkte trombe imaging visualiserer den egentlige årsag til tromboembolisk infarkt. At kunne billedet trombe direkte giver langt bedre undersøgelse af slagtilfælde end at lægge indirekte målinger, og vil være en kraftig og robust vaskulær forskning værktøj. Vi bruger en optisk billeddannelse tilgang, etiketter tromber med en molekylær billeddannelse blodprop markør - en Cy5.5 nær-infrarødt fluorescerende (NIRF) probe, der er kovalent bundet til fibrinstrengene af thromben af ​​fibrin-tværbinding enzymatiske virkning af aktiveret koagulationsfaktor Xllla under processen med koagel modning. En mikro-computertomografi (microCT) -baseret tilgang bruger trombe-søger guld nanopartikler (AuNPs) funktionaliserede at målrette hovedbestanddelen af ​​blodprop: fibrin. Dette papir beskriver en detaljeret protokol for den kombinerede in vivo microCT og ex vivo NIRF billeddannelse af tromboemboli i en musemodel af embolisk slagtilfælde. Vi viser, at in vivo &lt;/ em&gt; microCT og fibrin-målrettet glycol-chitosan AuNPs (FIB-GC-AuNPs) kan anvendes til at visualisere både in situ tromber og cerebral embolisk tromber. Vi beskriver også anvendelsen af in vivo microCT-baserede direkte trombeimagografimiddel til serielt overvåge de terapeutiske virkninger af vævsplasminogenaktivator-medieret trombolyse. Efter den sidste billeddannelse session, viser vi ved ex vivo NIRF billeddannelse omfanget og fordelingen af resterende tromboemboli i hjernen. Endelig beskriver vi kvantitativ billede analyser af microCT og NIRF billeddata. Den kombinerede teknik med direkte trombeimagografimiddel tillader to uafhængige fremgangsmåder til thrombe-visualisering, der skal sammenlignes: arealet af trombe-relaterede fluorescerende signal på ex vivo NIRF billeddannelse vs. mængden af hyperdense microCT tromber in vivo.

Baseline microCT billeder, opnået in vivo efter indgivelse FIB-GC-AUNP (10 mg / ml, 300 pi) efter 1 time efter embolisk slagtilfælde, klart visualiseret cerebral trombe i MCA - ACA bifurcation område af den distale arteria carotis interna (figur 6 ). Opfølgning microCT imaging viste ingen ændring i COW trombe med saltvand behandling. Men behandling med tPA viste en gradvis opløsning af COW trombe (blå pilespidser i figur 6). Dette fund vi...

Watch this Scientific Journal Video about Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli at JoVE.com

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Denne protokol beskriver anvendelsen af ​​kombineret nær-infrarødt fluorescerende (NIRF) billedbehandling og mikro-computertomografi (microCT) til visualisering cerebral tromboemboli. Denne teknik tillader kvantificering af trombe byrde og evolution. Den NIRF imaging teknik visualiserer fluorescensmærkede blodprop i udskårne hjerne, mens microCT teknik visualiserer blodprop inde levende dyr ved hjælp af guld-nanopartikler.

Kombineret Nær-infrarød Fluorescent Imaging og Micro-computertomografi for Direkte Visualisering Cerebral tromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Research

JoVE Journal

Medicine

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Retrograd perfusion og påfyldning af Mouse koronar kar som forberedelse til Micro Computed Tomography Imaging

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research

Near Infrared Optical Projection Tomography for Assessments of ÃƒÅ½Ã‚Â²-cell Mass Distribution in Diabetes Research

Near Infrared Optical Projection Tomography for Vurderinger af β-celle Mass Distribution i Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Osmotisk Drug Delivery til Iskæmisk bagbensknogler og perfusion af kar med Microfil for Micro-computertomografi Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

Tissue-simulering Phantoms til vurdering af potentielle nærinfrarøde fluorescensimagografi Applications i Breast Cancer Surgery

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Vurdering af virksomhedens rentabilitet for Human Fat Injektion i nøgne mus med Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Brug Micro-computertomografi for Vurdering af Tumor Udvikling og opfølgning af respons på behandling i en musemodel for lungekræft

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Skeletmuskulatur neurovaskulære kobling, Oxidative kapacitet og mikrovaskulære funktion med 'Et Stop Shop' nær-infrarød spektroskopi

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...

How to Administer Near-Infrared Spectroscopy in Critically ill Neonates, Infants, and Children

Sådan administreres nær-infrarød spektroskopi hos kritisk syge nyfødte, spædbørn og børn

Near infrared spectroscopy (NIRS) calculates regional tissue oxygenation (rSO2) using the different absorption spectra of oxygenated and deoxygenated ...

Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination

I nærheden af infrarød fotoimmunoterapi til musemodeller af pleural formidling

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination ...