The work was supported by the intramural research program of the NIH, National Institute on Aging, and by a Priority Research Centers Program grant from the National Research Foundation (NRF-2009-0093812) funded by the Ministry of Science, Information and Communication Technology, &amp; Future Planning, the Republic of Korea (H.T.). We thank Dr. Han-sol Park for putting "material" part together for the manuscript.

NOTA: l&#39;uso di topo palloncino modello di lesione e relative procedure, tra cui l&#39;iniezione di ricombinanti Srage e ultrasuoni studi ecografici sono stati approvati dal Comitato Animal Care e Usa (ACUC) del National Institute on Aging, NIH. 1. Preparativi pre-chirurgia <ol><li> Strumento, piattaforma chirurgica, e Dispositivi di Protezione Individuale. <ol><li> Vedi Materiali e Tabella di attrezzature per tutti gli strumenti chirurgici e dei reagenti utilizzati in questa procedura. Autoclavare tutti gli strumenti chirurgici prima di intervento chirurgico. NOTA: Nel caso di più interventi chirurgici eseguiti nello stesso giorno, usare un pre-riscaldato perle di vetro sterilizzatore per sterilizzare gli strumenti tra interventi chirurgici. </li><li> Sterilizzare la superficie della piattaforma operativa con il 70% di alcol. Indossare camice chirurgico, la copertura dei capelli, mascherina chirurgica, occhiali di sicurezza e guanti. Lasciare una scatola di guanti sterilizzati vicino alla piattaforma chirurgica per ulteriori noie. </li></ol></li><li> Preparazione del catetere a palloncino. <ol><li> Riempire siringa da 1 ml con acqua sterile e collegare la siringa di uno sterile a due vie rubinetto pieno d&#39;acqua. spingere delicatamente l&#39;acqua attraverso il rubinetto per riempire la porzione Luer-lock del catetere a palloncino e rimuovere le bolle d&#39;aria dall&#39;apertura del catetere. </li><li> gonfiare il palloncino Test per assicurare il palloncino può essere gonfiato da 0,02 ml di acqua. Conservare la punta del palloncino in un recipiente di acqua sterile prima della chirurgia per evitare la disidratazione e la contaminazione. </li></ol></li><li> Anestesia e preparazione degli animali. <ol><li> Pesare ratti maschi Wistar (400-450 g) e controllare il marchio auricolare per confermare l&#39;ID. Posizionare il topo in una camera di induzione, quindi accendere il vaporizzatore e regolare il livello isoflurano al 3,5-4,0% e il livello di ossigeno a 1,5-2,0 L. Controllare la profondità della sedazione controllando pedale ritiro riflesso, come pizzicare la punta o la coda dell&#39;animale. </li><li> Dopo aver raggiunto l&#39;anestesia ( <em&gt; cioè l&#39;animale privo di pedale ritiro reflex), trasferire l&#39;animale alla piattaforma chirurgica e agganciare il ratto con un cono che si connette a un vaporizzatore anestesia. Regolare la concentrazione isoflurano a 1.5-2.0 e ossigeno al 0,6-1,0 L. controllare periodicamente la profondità di sedazione pizzicando la punta o la coda dell&#39;animale per garantire che il ratto è completamente rilassato e immobile. </li><li> Dopo aver raggiunto il livello di anestesia chirurgica, radere la regione ventrale del collo dal mento verso il basso appena sopra lo sterno con tosatrici animali e tamponare la pelle rasata con alternanza di diluizione 1:10 di povidone-iodio e il 70% di etanolo per 3 volte. </li><li> Posizionare soluzione oftalmica sugli occhi aperti dell&#39;animale con un batuffolo di cotone per evitare che si secchi. Bagnare la lingua e la bocca dell&#39;animale con acqua potabile per evitare l&#39;essiccamento durante l&#39;operazione. </li><li> Coprire il ratto con un foglio chirurgica sterile e tagliare il foglio per esporre la regione del collo. Posto l&#39;animale in posizione supina ona riscaldata pad sulla piattaforma chirurgica con la testa verso il chirurgo. Fissare gli arti dell&#39;animale alla piattaforma chirurgica con del nastro. </li></ol></li></ol> 2. Procedure chirurgiche <ol><li> Dissezione e isolamento dell&#39;arteria carotidea sinistra <ol><li> Utilizzare un bisturi per fare una incisione diritta sotto il mento in una direzione verso la coda fino alla sommità dello sterno appena sopra la gabbia toracica. Utilizzare una pinza 7S di sezionare senza mezzi termini i tessuti ghiandolari salivari sotto la pelle per esporre lo strato muscolare. tessuti muscolari separati per accedere al sistema vascolare carotidea e il nervo vago nella regione del collo. </li><li> Senza mezzi termini sezionare tessuti circostanti la carotide, e con attenzione separano il nervo vago e la fascia vascolare per evitare danni. Continuamente sezionare fino alla biforcazione carotidea per esporre i rami interni ed esterni e l&#39;arteria carotide comune fino allo sterno. </li></ol></li><li> Preparazione di Arteriotomy <ol><li> Per retrazione e l&#39;emostasi, inserire una legatura nella sede più prossimale sulla arteria carotide comune sinistra, e un&#39;altra legatura nella posizione distale immediato alla biforcazione. </li><li> Collocare una sutura di seta 4.0 a livello dell&#39;arteria carotide esterna fino dalla biforcazione possibile per ritrarre l&#39;arteria verso la testa. Permanentemente legare piccole arterie utilizzando 4,0 suture di seta lungo l&#39;arteria carotide esterna compresa la faringea ascendente, occipitale e la tiroide superiore per evitare perdite arteriosa. </li><li> Collocare una sutura di seta intorno 4.0 carotide interna per evitare una significativa perdita di sangue retrograda. Di solito, la lunghezza disponibile del ramo interno è minima. Per visualizzare l&#39;arteria carotide interna, esporre l&#39;arteria carotide interna quanto possibile ritraendo delicatamente l&#39;arteria carotide sovrastante verso destra. </li></ol></li><li> L&#39;introduzione del catetere a palloncino <ol><li> Ritrarre la sutura prossimale sulla carotide comune unrtery e posizionare un morsetto arteriosa sulla nave per interrompere temporaneamente il flusso di sangue. Fai una piccola (1/3 a 1/4 della circonferenza dell&#39;arteria) incisione sulla carotide esterna in un luogo come distale alla sutura possibile. </li><li> Inserire delicatamente un catetere a palloncino gonfiato un-nell&#39;incisione e poi avanzare il catetere nel lume vicino al morsetto arteriosa sull&#39;arteria carotide comune. Rimuovere il morsetto, e far progredire ulteriormente il catetere a palloncino per l&#39;arco aortico, di circa 35-40 mm dal incisione. </li></ol></li><li> Balloon Injury <ol><li> gonfiare manualmente il catetere ad un volume di 0,02 ml attraverso la siringa attaccata e quindi bloccare il rubinetto tra la siringa e il catetere. Lentamente ritirare il catetere con rotazioni. Quando il catetere è vicino all&#39;incisione arteriotomic, sgonfiare il palloncino. Far avanzare il catetere nella posizione originale. </li><li> Ripetere 2.4.1 per un totale di 3 volte. Rimuovere con cautela il catetere dal lume delle arterie. </li></ol></li><li> Close Up ferita chirurgica <ol><li> Chiudere il arteriotomia con punti di sutura. Rilasciare l&#39;arteria carotide interna per ripristinare il flusso di sangue. Verificare la presenza di emorragia arteriosa o perdita. Se si verifica sanguinamento, applicare un pezzo di garza con una pressione della punta per fermare l&#39;emorragia. </li><li> Rimuovere il resto di suture e morsetti utilizzati per la procedura. Chiudere il tessuto ghiandolare con 6,0 punti di sutura. Chiudere la pelle con suture cutanee o clip ferita. Spegnere isoflurano e lasciare l&#39;animale con ossigeno puro per pochi minuti. </li></ol></li></ol> 3. La somministrazione di Therapeutics, Analgesici, cure post-intervento chirurgico, e l&#39;eutanasia <ol><li> La somministrazione di Therapeutics e analgesici. <ol><li> Dopo l&#39;intervento chirurgico, iniettare buprenorfina (0,03 mg / kg) per via intramuscolare. Iniettare 5 ml di pre-riscaldato per via sottocutanea soluzione salina sterile per compensare la perdita di sangue durante l&#39;intervento chirurgico. Iniettare Srage (0,5 ng / g) (terapie) nel peritoneo. </li><li> Inumidire di nuovo gli occhi con OPHpomata thalmic. Posizionare il topo sul pad di calore e monitorare l&#39;animale fino a quando non riprende conoscenza completa per mantenere decubito sternale. </li><li> Riportare il topo di una gabbia con lenzuola pulite e portare la gabbia torna al vivaio. Mettere una carta speciale di osservazione sulla gabbia per avvisare i veterinari presenti e bidelli. </li></ol></li><li> Post-chirurgia Cleanup <ol><li> Mettere materiale chirurgico utilizzati, inclusi i rilievi, tamponi, aghi ed altri sia in cestino o un apposito contenitore di smaltimento. Disinfettare la piattaforma di chirurgia e pulire gli strumenti chirurgici. </li></ol></li><li> Cure post-intervento chirurgico <ol><li> Somministrare buprenorfina (0,03 mg / kg) due volte al giorno (bid) durante il periodo post-operatorio 48 ore. Monitorare l&#39;animale da vicino durante il periodo post-operatorio a 48 ore per i segni comuni di disagio, come l&#39;immobilità, l&#39;incapacità di nutrire, postura hutched, pelliccia arruffata e smorfia. Monitorare le condizioni della ferita per arrossamento, gonfiore, deiscenza, e infezioni. NOTA:Ulteriori trattamenti possono essere prescritti in base a frequentare raccomandazione del veterinario. </li><li> Rimuovere la sutura o clip ferita in base a frequentare raccomandazione del veterinario. </li></ol></li><li> Eutanasia <ol><li> Due settimane dopo l&#39;intervento, l&#39;eutanasia i topi per isolare le arterie carotidi per la post-mortem isto-morfologico analisi 22. Posizionare il ratto in una camera contenente 5-20% isoflurano aria satura per almeno 2 minuti fino a completa cessazione della respirazione. Verificare la morte prima di dissezione degli organi e dei tessuti. </li></ol></li></ol>

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Gli autori dichiarano di non avere interessi finanziari concorrenti.

Ci sono stati due metodi utilizzati per gonfiare il palloncino per generare il pregiudizio che rimuove l&#39;endotelio murale nel lume carotide arteriose. Uno è quello di riempire la siringa attaccata con liquido 20, e l&#39;altro è di utilizzare la pressione dell&#39;aria 21. Noi preferiamo utilizzare la siringa a liquido in quanto il volume di liquido esatta (0,02 ml) sarà usato per ogni procedura. Questo rende un gonfiaggio preciso e riproducibile del pallone, portando ad un livello simile di...

L&#39;angioplastica è una procedura endovascolare utilizzato per allargare le arterie limitate o ostruite risultanti da condizioni patologiche come l&#39;aterosclerosi. Una complicazione comune di angioplastica è il post-operativa neointimale, o restenosi, che si verifica a causa di ferite chirurgiche e il conseguente rimodellamento vascolare infiammazione indotta. Queste condizioni portano alla proliferazione di cellule lisce vascolari, e molteplici conseguenze fisiopatologiche 1-3. Neointimale ri-addensa la nave, e si verifica in circa il 60% dei pazienti post-angioplastica entro il primo anno. Pertanto, restenosi è una grave battuta d&#39;arresto della procedura di angioplastica ampiamente utilizzato 4. Anche se l&#39;impianto dello stent farmaco-eluizione può aiutare a prevenire la restenosi, i candidati solo selezionati possono sottoporsi a questa procedura costosa 5. Sia animali e studi clinici hanno dimostrato che l&#39;infiammazione cronica generato da vasculesioni lar e / o ferita chirurgica serve come lo stimolo principale per la post-angioplastica 2,4 crescita neointimale. Il ratto carotidi balloon injury modello imita la situazione clinica e quindi serve come sistema modello prezioso per identificare i fattori cellulari che coinvolgono nel rimodellamento vascolare e proliferazione delle cellule vascolari 6-9. Questo modello di sistema è anche uno strumento molto utile per valutare e / o lo schermo per i farmaci e reagenti terapeutici che sopprimono la crescita neointimale in studi pre-clinici traslazionali 10-14. Rispetto al filo modello murino carotide pregiudizio 15 e il filo arteria femorale murino modello di lesione 16, il modello di balloon injury carotidea ratto ha i vantaggi di essere sufficientemente grandi dimensioni per la facilità di procedura chirurgica che facilita la riproducibilità della ferita inflitta. Può fornire un numero maggiore di cellule primarie (ad esempio cellule muscolari lisce vascolari, cellule endoteliali) per additionale in vitro studi per delineare il meccanismo molecolare che regola il rimodellamento vascolare. È importante sottolineare che, rispetto ai topi, ratti sono anche noti per essere un modello migliore per gli studi fisiologici e tossicologici 17. Anche se uno svantaggio o di limitazione del modello di ratto è la mancanza di modelli modificati e gene knockout genetiche, questo svantaggio può essere superato dalla disponibilità della sequenza genomica di ratto e il recente sviluppo di potenti strumenti di editing genomici come la tecnologia CRISPR-CAS che rende possibile manipolazione di vaste gamme di sequenze genomiche in diversi sistemi modello 18,19. Anche se il modello di ratto lesioni palloncino è stato utilizzato da più laboratori e vari protocolli completi sono stati pubblicati 20,21, questo protocollo si propone di fornire maggiori dettagli in preparazioni pre-chirurgia e può guidare i ricercatori nuovi a questa procedura per impostare questa pratica chirurgica. Sottolineiamo anche la cura post-operatorio di tegli animali che non permette solo post mortem analisi patologiche e istomorfologici degli effetti terapeutici su arteriosa rimodellamento, ma anche studi ultrasuoni ecografici di animali vivi 13,22.

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			<td height="21" style="height:21px;width:287px;">2 F Fogarty balloon embolectomy catheter&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:163px;">Edwards Lifesciences&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
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			<td height="21" style="height:21px;width:287px;">Standard scalpel</td>
			<td style="width:163px;">Fine Science Tools</td>
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			<td height="40" style="height:40px;width:287px;">Small&#160; curved forceps (Large radius Dumont#7shanks curved)&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:163px;">Fine Science Tools&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Large, medium and small micro-scissors</td>
			<td style="width:163px;">Roboz</td>
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			<td height="21" style="height:21px;width:287px;">Needles (20 G)&#160;&#160;</td>
			<td style="width:163px;">TycoHealthcare</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:287px;">Micro-surgery forceps with micro-blunted atraumatic tips</td>
			<td style="width:163px;">Fine Science Tools&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Atraumatic straight small arterial clamps&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:163px;">Fine Science Tools&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:287px;">Retractor&#160; with maximum spread 5.5 cm long blunt teeth</td>
			<td style="width:163px;">Fine Science Tools&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Silk suture (4.0 and&#160; 6.0 )</td>
			<td style="width:163px;">Fine Science Tools&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Syringe (1.0 ml)&#160;</td>
			<td style="width:163px;">BD&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Curity gauze sponges</td>
			<td style="width:163px;">AllegroMedical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Cotton tip applicators sterile and non-sterile</td>
			<td style="width:163px;">Puritan Medical Products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Compact hot bead sterilizer</td>
			<td style="width:163px;">Fine Science Tools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Self-regulating heating pad</td>
			<td style="width:163px;">Fine Science Tools&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">ADS200 anesthesia system/ventilator</td>
			<td style="width:163px;">Paragon Medical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Isoflurane (forane), liquid form</td>
			<td style="width:163px;">Baxter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Sodium chloride 0.9% (Saline)&#160;</td>
			<td style="width:163px;">Hospira</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:287px;">Buprenex (buprenorphine)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:163px;">Reckitt Benckiser Healthcare (UK) Ltd.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">70% alcohol</td>
			<td style="width:163px;">Fisher</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">1: 10 Betadine</td>
			<td style="width:163px;">Fisher</td>
		</tr>
	</tbody>
</table>

a rat carotid balloon injury model to test anti-vascular remodeling therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

Due settimane dopo l&#39;infortunio a palloncino, il ratto è eutanasia e le arterie carotidi sono isolate per l&#39;analisi isto-morfologica. Entrambi operato arteria carotide sinistra e l&#39;arteria destra non-operato sono sezionato, trasformato, e inclusi in paraffina. I campioni di paraffina sono poi ulteriormente sottili a sezione, e colorati con ematossilina-eosina (H &amp; E). analisi isto-morfologico vengono eseguite utilizzando un sistema di analisi di imaging digitale. I detta...

Watch this Scientific Journal Video about A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics at JoVE.com

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

Un ratto carotidea Pallone Lesioni modello per verificare Therapeutics ritocca Anti-vascolari

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

a-rat-carotid-balloon-injury-model-to-test-anti-vascular-remodeling

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8.6K Views.  National Institute on Aging. The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described. 

Video: A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Somministrazione intranasale di terapeutica del sistema nervoso centrale di topi Awake

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

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Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Modello Rat di emato-encefalica Turbativa Barriera per consentire mirato Therapeutics neurovascolare

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusi carotide di farmacocinetica e di farmacodinamica Analisi dei taxani nei topi

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

Vascolare Balloon Lesioni e intraluminale Amministrazione in Rat carotide

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

Retroductal ghiandola sottomandibolare instillazione e localizzati frazionato irradiazione in un modello murino di salivare Ipofunzione

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the industrialized world. New Zealand White (NZW) rabbits are widely used as an animal model for the study of atherosclerosis. They develop spontaneous lesions when fed with atherogenic diet; however, this requires long time of 4 - 8 months. To further enhance and accelerate atherogenesis, a combination of atherogenic diet and mechanical endothelial injury is often employed. The presented procedure for inducing atherosclerotic plaques in rabbits uses a balloon catheter to disrupt the endothelium in the left iliac artery of NZW rabbits fed with atherogenic diet. Such mechanical damage caused by the balloon catheter induces a chain of inflammatory reactions initiating neointimal lipid accumulation in a time dependent fashion. Atherosclerotic plaque following balloon injury show neointimal thickening with extensive lipid infiltration, high smooth muscle cell content and presence of macrophage derived foam cells. This technique is simple, reproducible and produces plaque of controlled length within the iliac artery. The whole procedure is completed within 20 - 30 min. The procedure is safe with low mortality and also offers high success in obtaining substantial intimal lesions. The procedure of balloon catheter induced arterial injury results in atherosclerosis within two weeks. This model can be used for investigating the disease pathology, diagnostic imaging and to evaluate new therapeutic strategies.

Animal models of atherosclerosis are essential to understand the mechanism and to investigate newer approaches to prevent plaque development or rupture, a leading cause of death in the industrialized world. This protocol uses a combination of balloon injury and cholesterol rich diet to induce atherosclerotic plaques in rabbit iliac artery.

Il modello del coniglio di aterosclerosi accelerata: una prospettiva metodologica della ferita dell'aerostato dell'arteria iliaca

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

Basato su palloncino ferita per indurre iperplasia Myointimal nell'Aorta addominale di Mouse

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

Un modello di ferita Drosophila In Vivo per lo studio della neurorigenerazione la periferica e del sistema nervoso centrale

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

Trombosi dell'arteria carotica canini cloruro ferrico-indotta: Un grande modello animale di danno vascolare

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Una lesione segmentale del palloncino segmentale controllato dalla pressione carotide del ratto con applicazione terapeutica periadventitiale

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...