This work was supported by the American Heart Association Scientist Development Grant and the Foundation for Anesthesia Education and Research (MLJ). We would like to thank Talaignair N. Venkatraman PhD for his assistance with magnetic resonance imaging.

Etica Dichiarazione: Questo protocollo è stato approvato dal Comitato Duke University Istituzionale cura degli animali ed uso e segue tutte le linee guida per l&#39;uso etico degli animali. 1. Preparazione delle attrezzature <ol><li> Sterilizzare gli strumenti chirurgici prima della chirurgia. </li><li> Disinfettare l&#39;apparato stereotassico con il 70% di etanolo. </li><li> Accendere bagnomaria e mantenere la temperatura dell&#39;acqua a 42 ° C. </li><li> Sciogliere Type IV-S clostridiale collagenasi in soluzione fisiologica ad una concentrazione di 0,075 U per 0.4 ml. </li></ol> 2. Collagenase iniezione Modello <ol><li> Pesare il mouse. </li><li> Anestetizzare il mouse in una camera di induzione con 5% isoflurano nel 30% O 2/70% N 2. Anestesia adeguata viene segnalato dopo circa 2 minuti quando la respirazione del mouse hanno rallentato a 1 al secondo. </li><li> Intubare la trachea con una G catetere endovenosa da 30 mm 20. </li><li> Collegare il catetere ad unroditore ventilatore e ventilare meccanicamente i polmoni 1,6% isoflurano nel 30% O 2/70% N 2 a una velocità di 105 respiri al minuto, con un volume corrente erogato di 0,75 ml .. </li><li> Shave il cuoio capelluto con un rasoio elettronico. Una volta che il mouse è anestetizzato e intubato, spostarlo in una stazione di lavoro diversa per la rasatura e poi tornò al banco chirurgico. </li><li> Fissare la testa in una cornice stereotassica, e livellare la testa con sutura sia coronale e sagittale come punti di riferimento. </li><li> Applicare una pomata oftalmica per gli occhi. </li><li> Inserire una sonda di temperatura rettale. Mantenere la temperatura rettale a 37,0 ± 0,2 ° C con un sottoscocca letto ad acqua circolante. </li><li> Pulire l&#39;area chirurgica con betadine seguita con il 70% di etanolo e ripetere 3 volte. </li><li> Fare un 1 centimetro linea mediana del cuoio capelluto incisione e pulire periostio lateralmente con un applicatore con punta di cotone sterile per esporre bregma. </li><li> Drill 1 millimetro di diametro bava foro 2,2 millimetri lasciato latrale di bregma con un trapano raffreddato ad acqua. </li><li> Ruotare collagenasi flaconcino 5 volte, poi lavare una siringa da 0,5 ml con il 25 ago G (collegato al telaio stereotassico) con 0,5 ml soluzione di collagenasi 5 volte (Lascia 0,5 ml di soluzione di collagenasi in siringa dopo l&#39;ultimo lavaggio). </li><li> Allineare la punta dell&#39;ago con foro cranico poi espellere 0,1 microlitri dalla siringa e pulire l&#39;ago smusso con il rasoio di disfarsi. </li><li> Utilizzando un micromanipolatore, avanzare l&#39;ago 3 millimetri in profondità per corteccia e lasciare immobile per 30 sec. </li><li> Iniettare 0,4 ml oltre 90 sec. </li><li> Diminuire isoflurano al 1% e lasciare l&#39;ago immobile per 5 min. </li><li> Ritirare l&#39;ago lentamente. </li><li> Applicare 1-2 gocce di 0,25% bupivacaina per via sottocutanea e suturare la pelle. </li><li> Spegnere isoflurano vaporizzatore e togliere il mouse dal telaio stereotassico. </li><li> Lasciare il mouse per recuperare ventilazione spontanea con successiva estubazione tracheale. </li><li> Ritorno mouse in una gabbia pulita e consentire l&#39;accesso gratuito alcibo e acqua. </li></ol> 3. Autologo Modello Iniezione di sangue <ol><li> Seguire i passaggi 2,1-2,11 per il modello iniezione di collagenasi. </li><li> Disegna 50 ml di soluzione fisiologica sterile in una siringa da 30 G 50 microlitri. </li><li> Collegare la siringa microlitri con un tubo PE10 70 centimetri. </li><li> Espellere tutta la soluzione fisiologica dalla siringa microlitro in PE10 tubi di tubi completamente de-aria. </li><li> Estrarre il microlitro siringa pistone 1 mm fare una bolla d&#39;aria all&#39;apertura distale del tubo-microlitro apparecchi siringa PE10 evitare miscela di soluzione salina e sangue durante le procedure successive. </li><li> Pulire la coda regione centrale arteria distale del mouse con etanolo al 70%, e tagliare l&#39;arteria con un rasoio a 0.5 a 1 cm dalla punta della coda. </li><li> Raccogliere 40 ml di sangue dalla coda tagliata nel tubo-microlitro apparecchi siringa PE10. Nota: che l&#39;eparina non viene utilizzato nell&#39;ago, tubi, o mouse. </li><li> Attaccare la siringa microlitri sul injepompa ction. </li><li> Collegare la porzione cannula metallica di un ago 27 G all&#39;estremità del tubo PE10, e fissare l&#39;ago a un micromanipolatore sul telaio stereotassico. </li><li> Espellere 2 ml di sangue su 27 G ago e pulire l&#39;ago smusso con il rasoio di disfarsi. </li><li> Allineare la punta dell&#39;ago con foro cranico e inserire aghi 3 mm di profondità di corteccia. </li><li> Iniettare 35 ml di sangue autologo ad una velocità di 2 ml al minuto. </li><li> Diminuire isoflurano al 1% e lasciare l&#39;ago immobile per 10 min. </li><li> Ritirare l&#39;ago oltre 30 sec. </li><li> Applicare 1-2 gocce di 0,25% bupivacaina per via sottocutanea e suturare la pelle. </li><li> Spegnere isoflurano vaporizzatore e togliere il mouse dal telaio stereotassico. </li><li> Lasciare il mouse per recuperare ventilazione spontanea con successiva estubazione. </li><li> Ritorno mouse in una gabbia pulita e permettere il libero accesso a cibo e acqua. </li></ol> 4. Sham Funzionamento <ol><li> Seguire le stesse procedure di collagenasi injectioModello n, tranne senza iniezione dopo l&#39;inserimento dell&#39;ago. </li></ol> 5. Assistenza post-operatorio <ol><li> Iniettare 0,5 ml di soluzione salina normale sottocutanea la sera della procedura chirurgica sul retro del collo dell&#39;animale. </li><li> Fornire cibo ammorbidito con acqua e cibo gel in piccole tazze di plastica posti sul pavimento della gabbia. Sostituire il cibo al giorno per 7 giorni. </li><li> Verificare la perdita di peso, la guarigione delle ferite, e segni di disagio al giorno per 7 giorni. </li><li> Se sono necessari intervalli di recupero superiori a 7 giorni, rimozione di sutura può essere effettuata in luce anestesia inalatoria (circa 1% isoflurano nel 30% O 2/70% N 2), se necessario. </li></ol>

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Lei, Sheng, Wang, Lascola, Warner, and Laskowitz have no conflicts of interest to declare. James received grant funding by American Heart Association, National Institutes of Health, and Cephalogics.

Nonostante emergenti ricerca preclinica e grandi studi clinici che ne derivano per i promettenti terapie 15-18, non ci sono interventi farmacologici dimostrato di migliorare il risultato in ICH, e la cura rimane in gran parte di supporto. Elenchi di possibili terapie possono essere generati da tecnologie ad alto rendimento, come il lavoro trascrittomica e proteomica. Mentre queste tecnologie continuano a progredire la nostra conoscenza di potenziali bersagli terapeutici, in avanti e all&#39;indietro la traduz...

Emorragia intracerebrale (ICH) è una forma relativamente comune di malattia cerebrovascolare con circa il 40-50% dei pazienti colpiti muore entro 30 giorni 1. Purtroppo, piccolo miglioramento è stato fatto nel tasso di mortalità negli ultimi 20 anni 2. I rapporti del National Institutes of Health 3 e linee guida dell&#39;American Heart Association 4 hanno sottolineato l&#39;importanza di sviluppare modelli clinicamente rilevanti di ICH per estendere la comprensione della fisiopatologia e sviluppare obiettivi per nuovi approcci terapeutici. Esistono diversi modelli di imitare ICH umano 5. Come comprensione della fisiopatologia ICH matura, è diventato evidente che una varietà di modelli può essere utilizzato per esaminare i diversi aspetti della malattia. Modelli usati in precedenza includono murino angiopatia amiloide 6, inserimento microballoon intraparenchimale e l&#39;inflazione 7, e il sangue arterioso direttoinfiltrazione 8,9. Lobare emorragia da angiopatia amiloide è stato modellato con l&#39;uso di topi transgenici e rappresenta un distinto ICH sottotipo. Modelli Microballoon imitano effetto massa acuta da ematoma ma non riescono a catturare risposta cellulare del cervello per la presenza di sangue. Infine, diretta infiltrazione di sangue arterioso sottopone il cervello a pressioni arteriose dalla arteria femorale. Così, questo modello imita la pressione arteriosa e la presenza di sangue, ma non sottoporre il cervello a danno microvascolare da piccola rottura del vaso sanguigno. Inoltre, questo modello ha intrinsecamente elevata variabilità. È interessante notare che i ratti spontaneamente ipertesi 10 si sviluppano spontaneamente ICH man mano che invecchiano. Studio di questi animali dopo sviluppo ICH può mimare la malattia in presenza di uno dei principali comorbidità predisponenti esseri umani di ICH. Mentre esistono questi altri modelli, iniezione intrastriatali di Clostridial collagenasi 11 o iniezione instrastiatal di unsangue intero utologous 12 sono, attualmente, i due modelli più comuni utilizzati nella ricerca preclinica ICH. Selezione del modello ICH deve essere effettuata sulla base dell&#39;obiettivo della questione sperimentale, compresa la selezione delle specie e metodo per indurre la formazione di ematomi. Ad esempio, i maiali sono animali di grandi dimensioni con volumi relativamente grandi bianca del cervello questione rispetto ai topi. Così, i modelli suina sono adatti per studiare la materia bianca fisiopatologia seguente ICH. Al contrario, il cervello di roditori sono materia grigia in gran parte, ma i sistemi transgenici fanno roditori utile per valutare i meccanismi molecolari di danno e di recupero dopo ICH. Ogni modello ha i suoi punti di forza intrinseci e di debolezza (Tabella 1), che dovrebbe essere attentamente valutata prima della sperimentazione. I seguenti protocolli dimostrano il sangue e di iniezione di collagenasi modelli autologhe nei topi. Questi modelli sono stati entrambi tradotti da modelli originariamente sviluppati nei ratti13,14 e consentire l&#39;utilizzo di ampiamente disponibili tecnologia transgenica per esplorare meccanismi molecolari associati alla morte cellulare dopo ICH. Entrambi rappresentano nettamente diversi meccanismi di lesione da ICH umana, ed entrambi hanno risultato atteso nettamente differenti in termini di misure comportamentali e istologici. Così, certe ipotesi possono prestarsi ad uno o l&#39;altro, ma molte idee possono richiedere convalida in entrambi i modelli. Tabella 1. Confronto delle caratteristiche dei modelli di emorragia intracerebrale iniezione di sangue autologo-collagenasi e. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td style="width:197px;"></td><td style="width:80px;"> Collagenase iniezione </td><td style="width:66px;"> Iniezione di sangue </td></tr><tr><td style="width:197px;"> Facilità d&#39;uso </td><td style="width:80px;"> + + + </td><td style="width:66px;"> + + </td></tr><tr&gt; <td style="width:197px;"> Riproducibilità </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;"> Controllo di emorragia Size </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + + </td></tr><tr><td style="width:197px;"> Reflusso di sangue </td><td style="width:80px;"> + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;"> Simula malattie umane </td><td style="width:80px;"> + </td><td style="width:66px;"> - </td></tr><tr><td style="width:197px;"> Semplicità </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;"> Usare in più specie </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr></tbody></table>

<table border="0" cellpadding="0" cellspacing="0" width="416">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Name</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalog Number</td>
			<td style="width:88px;">Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Stereotactic frame</td>
			<td style="width:88px;">Stoelting Co.</td>
			<td style="width:88px;">51603</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Probe holder with corner clamp</td>
			<td style="width:88px;">Stoelting Co.</td>
			<td style="width:88px;">51631</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Mini grinder</td>
			<td style="width:88px;">Power Glide</td>
			<td style="width:88px;">Model 60100002</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">0.5 &#181;l Hamilton syringe</td>
			<td style="width:88px;">Hamilton Co.</td>
			<td style="width:88px;">86259</td>
			<td style="width:88px;">25 gauge needle</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">50 &#181;l Hamilton syringe</td>
			<td style="width:88px;">Hamilton Co</td>
			<td style="width:88px;">7637-01</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">26G Hamilton needle</td>
			<td style="width:88px;">Hamilton Co</td>
			<td style="width:88px;">7804-03</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Syringe pump</td>
			<td style="width:88px;">KD Scientific</td>
			<td style="width:88px;">Model 100</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Heat therapy water pump</td>
			<td style="width:88px;">Gaymar Industries, Inc.</td>
			<td style="width:88px;">Model# TP650</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Circulating waterbed</td>
			<td style="width:88px;">CMS Tool &#38; Die, Inc.</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Rodent ventilator</td>
			<td style="width:88px;">Harvard Apparatus</td>
			<td style="width:88px;">Model 683</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Isoflurane vaporizer</td>
			<td style="width:88px;">Drager</td>
			<td style="width:88px;">Vapor 19.1</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Air flowmeter</td>
			<td style="width:88px;">Cole Parmer</td>
			<td style="width:88px;">Model PMR1-010295</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Induction chamber</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
			<td style="width:88px;">Self made</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Otoscope</td>
			<td style="width:88px;">Welch Allyn</td>
			<td style="width:88px;">22820</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">intravenous catheter</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">381534</td>
			<td style="width:88px;">20-gauge, 1.16 inch Insyte-W</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Isoflurane</td>
			<td style="width:88px;">Baxter Healthcare Corporation</td>
			<td style="width:88px;">NDC10019-360-69</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Collagenase Type IV-S</td>
			<td style="width:88px;">Sigma</td>
			<td style="width:88px;">C1889</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Polyethylene tubing PE20</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">427406</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Polyethylene tubing PE10</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">427401</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">30G 1 inch needle</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">305128</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">27G 1 1/4 inch needle</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">305136</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Surgical scissors</td>
			<td style="width:88px;">Miltex</td>
			<td style="width:88px;">21-539</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Forceps</td>
			<td style="width:88px;">Miltex</td>
			<td style="width:88px;">17-307</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Needle holder</td>
			<td style="width:88px;">Boboz</td>
			<td style="width:88px;">RS-7840</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Monofilament suture</td>
			<td style="width:88px;">Ethicon</td>
			<td style="width:88px;">8698</td>
			<td style="width:88px;">Size 5-0</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Indicating controller</td>
			<td style="width:88px;">YSI</td>
			<td style="width:88px;">73ATD</td>
			<td style="width:88px;"></td>
		</tr>
	</tbody>
</table>

intrastriatal injection of autologous blood or clostridial collagenase as murine models of intracerebral hemorrhage

Emorragia intracerebrale (ICH) è una comune forma di malattia cerebrovascolare ed è associata a significativa morbidità e mortalità. La mancanza di un trattamento efficace e il fallimento di grandi studi clinici volti a emostasi e la rimozione di coaguli dimostrare la necessità di ulteriori indagini meccanismo-driven di ICH. Questa ricerca può essere effettuata attraverso il quadro fornito dai modelli preclinici. Due modelli murini in uso popolare includono intrastriatali (gangli basali) iniezione di entrambi sangue intero autologo o collagenasi clostridiale. Poiché, ogni modello rappresenta nettamente diverse caratteristiche fisiopatologiche relative a ICH, l&#39;uso di un particolare modello può essere selezionato in base a ciò aspetto della malattia è da studiare. Ad esempio, l&#39;iniezione di sangue autologo rappresenta più accuratamente la risposta del cervello alla presenza di sangue intraparenchimale, e può più strettamente replicare lobare emorragia. Clostridial iniezione di collagenasi rappresenta la maggior precisione le srottura del vaso centro commerciale ed ematoma evoluzione caratteristica emorragie profonde. Così, ogni modello si traduce in diversa formazione di ematomi, la risposta neuroinfiammatorie, sviluppo edema cerebrale, e gli esiti neurocomportamentali. La robustezza di un intervento terapeutico presunto può essere meglio valutata utilizzando entrambi i modelli. In questo protocollo, l&#39;induzione di ICH utilizzare entrambi i modelli, immediata dimostrazione post-operatorio del pregiudizio e tecniche di cura post-operatorie precoci sono dimostrati. Entrambi i modelli provocare lesioni riproducibili, volumi ematoma, e deficit neurocomportamentali. A causa della eterogeneità dei ICH umana, sono necessari diversi modelli preclinici di esplorare a fondo i meccanismi fisiopatologici e testare potenziali strategie terapeutiche.

A causa delle differenze nella formazione di ematomi (Figura 1), svolta ipsilaterale è mostrata subito dopo la sveglia per sangue autologo topi iniettati ed entro 2-4 ore dopo l&#39;iniezione di collagenasi, l&#39;espansione ematoma si verifica (Figura 2). Assenza di rotazione omolaterale dovrebbe sollevare preoccupazione per l&#39;assenza di lesioni gravi. Il primo giorno pregiudizio postale, topi in entrambi i modelli dovrebbero dimostrare significativi deficit neurologici (F...

Watch this Scientific Journal Video about Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage at JoVE.com

Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

Iniezione intrastriatali di sangue autologo o Clostridial Collagenase come modelli murini di emorragia intracerebrale

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective ...

intrastriatal-injection-autologous-blood-or-clostridial-collagenase

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JoVE Journal

Medicine

14.6K Views.  Duke University. Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase. 

Video: Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Fixed Volume or Fixed Pressure: A Murine Model of Hemorrhagic Shock

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, 2, 3, 4, 5 These signaling events can also lead to sepsis and/or multiple organ dysfunction (MOD). 6, 7, 8, 9 It is critical then to investigate the causes of suppressed immune function and detrimental signaling cascades in order to develop more effective ways to help patients who suffer from traumatic injuries. 10 This fixed pressure Hemorrhagic Shock (HS) procedure, although technically challenging, is an excellent resource for investigation of these pathophysiologic conditions. 11, 12, 13 Advances in the assessment of biological systems, i.e. Systems Biology have enabled the scientific community to further understand complex physiologic networks and cellular communication patterns. 14 Hemorrhagic Shock has proven to be a vital tool for unveiling these cellular communication patterns as they relate to immune function. 15, 16, 17, 18 This procedure can be mastered! This procedure can also be used as either a fixed volume or fixed pressure approach. We adapted this technique in the murine model to enhance research in innate and adaptive immune function. 19, 20, 21 Due to their small size HS in mice presents unique challenges. However due to the many available mouse strains, this species represents an unparalleled resource for the study of the biologic responses. The HS model is an important model for studying cellular communication patterns and the responses of systems such as hormonal and inflammatory mediator systems, and danger signals, i.e. DAMP and PAMP upregulation as it elicits distinct responses that differ from other forms of shock. 22, 23, 24, 25 The development of transgenic murine strains and the induction of biologic agents to inhibit specific signaling have presented valuable opportunities to further elucidate our understanding of the up and down regulation of signal transduction after severe blood loss, i.e. HS and trauma 26, 27, 28, 29, 30. 
There are numerous resuscitation methods (R) in association with HS and trauma. 31, 32, 33, 34 A fixed volume resuscitation method of solely lactated ringer solution (LR), equal to three times the shed blood volume, is used in this model to study endogenous mechanisms such as remote organ injury and systemic inflammation. 35, 36, 38 This method of resuscitation is proven to be effective in evaluating the effects of HS and trauma 38, 39.

The Hemorrhagic Shock model has been a reliable and reproducible resource facilitating the identification and understanding of signaling cascades associated with inflammation and end-organ damage after trauma. This article provides a step-by-step description of surgical and mechanical aspects associated with the Hemorrhagic Shock experimental procedure in mice.

Volume fisso o fisso di pressione: un modello murino di shock emorragico

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, ...

Ricerca

Medicina

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Legatura dell&#39;arteria coronarica e di iniezione intramiocardico in un modello murino di infarto

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

Modellazione emorragia intracerebrale in Mouse: iniezione di sangue autologo o collagenasi batterica

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Un modello murino di emorragia subaracnoidea

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

Stereologica e Citometria a Flusso Caratterizzazione dei leucociti sottopopolazioni in modelli di transitori o permanenti Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

Il Coniglio Blood-shunt modello per lo studio di acuta e sequele tardive di emorragia subaracnoidea: Aspetti tecnici

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

Ferrico I modelli murini Trombosi indotta da cloruri

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Ultrasuono-guida di iniezione intracardiaca di cellule staminali mesenchimali umane per aumentare Homing all'intestino per l'utilizzo in modelli murini di malattie intestinali infiammatorie sperimentali

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator.
Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage.
This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

Nervo stimolatore-guida iniezione di cellule staminali autologhe vicino il nervo laringeo ricorrente di sinistra equina

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

Puntura della vena succlavia come metodo alternativo di raccolta del campione di sangue in ratti

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...