Il lavoro di questo manoscritto è stato sostenuto da finanziamenti al dottor Neal del Programma Pilot Project dell&#39;Istituto di Medicina Vascolare in emostasi e biologia vascolare (P3HVB) e della borsa di ricerca AAST. Questo lavoro è supportato da US National Institutes of Health grante 1 R35 GM119526-01 e UM1HL120877-01.

I topi sono stati ospitati in conformità con le norme di cura degli animali in condizioni specifiche per la cura degli animali, con 12 ore di ciclo chiaro e scuro, e accesso libero a tutti i pazienti, in conformità con l&#39;Università di Pittsburgh (Pittsburgh, PA) e National Institutes of Health (NIH; Bethesda, MD). L&#39;alimentazione standard e l&#39;acqua. Tutti gli esperimenti sugli animali sono stati approvati e condotti secondo le linee guida stabilite dal comitato di ricerca e cura degli animali presso l&#39;Università di Pittsburgh. 1. Installazione del campo chirurgico e dello strumento <ol><li> Sterilizzare tutti gli strumenti chirurgici, sutura, garza, applicatori a punta di cotone, tubi e raccordi di tubi prima della procedura. <ol><li> Sterilizzare strumenti chirurgici, sutura, garza e applicatori a punta di cotone in autoclave. Sterilizzare connettori tubi e tubi con ossido di etilene. </li></ol></li><li> Campo chirurgico <ol><li> Accendere un tappetino di riscaldamento circolante e impostare a 37 ° C. Mettere un surgiCalotta blu su di esso e poi un drappo sterile in cima al tampone blu chirurgico. </li><li> Aprire tutti gli strumenti sterilizzati sul tetto sterile. Utilizzare guanti sterili per evitare la rottura della sterilità durante questo passaggio. </li><li> Riempire una ciotola in acciaio inox con il 70% di etanolo e mettere da parte. Questo sarà usato per pulire gli strumenti tra gli animali. </li><li> Attivare lo sterilizzatore a micropiuma e lasciare raffreddare a 150 ° C. Questo sarà utilizzato anche per pulire gli strumenti tra gli animali. Se effettui interventi su più di 5 topi, assicuratevi di cambiare gli strumenti in un nuovo kit sterile. </li></ol></li><li> Configurazione del trasduttore <ol><li> Collegare un trasduttore sterile, tubo PE-50, due aghi 23G e un luer maschio-maschio e un rubinetto a tre vie. 6 </li><li> Calibrare e rimuovere il trasduttore per istruzioni del produttore. </li></ol></li></ol> Procedura chirurgica di Lacerazione del fegato <ol><li> Induzione e posizionamento di anestesia </strong&gt; <ol><li> Iniettare il pentobarbital sodico intraperitonealmente alla dose di 70 mg / kg. L&#39;anestesia dovrebbe avere effetto tra 5-10 minuti; Valutare la profondità dell&#39;anestesia con un pizzico di punta. Se il mouse ha risposta al pizzico del piede, è necessario un tempo aggiuntivo o anestesia. Se durante la procedura è necessaria un&#39;anestesia aggiuntiva, completare pentobarbital sodico. Non somministrare integratori in quantità superiori a 0,05 ml per prevenire il sovradosaggio. </li><li> Dopo che il mouse è completamente sotto l&#39;anestesia, posizionare il mouse supino su una scheda chirurgica. Fissare tutti i quattro arti del mouse sulla scheda con nastro. </li><li> Rasare l&#39;addome e gli ingombri bilaterali con un rasoio. </li><li> Immergere la garza sterile con betadina e applicare all&#39;addome e alle intestine bilaterali per la chirurgia. Per esperimenti di sopravvivenza, preparare l&#39;addome e gli inguine con betadina seguita da etanolo per un totale di tre cicli di preparazione. </li><li> Inserire una sonda di temperatura rettale per monitorare la temperatura del cuore durante tutta la procedura. Tenere il cTemperatura del minerale tra 35-37 ° C. </li></ol></li><li> Arteria femorale e cannulazione venosa <ol><li> Per il set-up del catetere venoso: riempire il tubo PE-10, un ago da 30 G e un rubinetto a tre vie con la soluzione di Lactated Ringer da una borsa IV. </li><li> Per il set-up del catetere arterioso: riempire il tubo PE-10 e l&#39;ago 30G con salina eparinizzata (diluizione 1:10 di 1.000 U di eparina). È necessaria la salina eparina per prevenire il coagulazione. </li><li> Posizionare il mouse sotto un microscopio di dissezione. </li><li> Fai un&#39;incisione longitudinale di 4-5 millimetri sopra il muscolo dell&#39;inguine utilizzando le forbici chirurgiche Iris. Usando le pinze Dumont afferrare il tessuto adiposo collegato al muscolo adductor e tirare lateralmente per un&#39;esposizione pulita del fascio femorale. Non dissectare attraverso il tessuto adiposo in quanto ciò porterà a lesioni vascolari. </li><li> Separare con cura il nervo dall&#39;arteria e dalla vena con la pinza Dumont. C&#39;è un rilievo di grasso adiacente al nervo. Prendi questo con un Dumont forcep anD tirare lateralmente; Questo tira fuori il nervo dall&#39;arteria creando un piano per la dissezione. Con l&#39;altro Dumont forcep si disseccano con estrema facilità il tessuto connettivo tra il nervo e l&#39;arteria. <ol><li> Non afferrare il nervo durante questa parte della dissezione. </li></ol></li><li> Loop tre suture di seta 6-0 intorno all&#39;arteria e vena prossimale al profunda femoris decollare. <ol><li> Posizionare la sutura 1 in modo più prossimale e lasciare sciolta. </li><li> Posizionare la sutura 2 più distalmente e legare immediatamente. </li><li> Posizionare la sutura 3 tra sutura 1 e 2 e lasciarla allentata. </li></ol></li><li> Fare un&#39;arteriotomia sulla superficie ventrale del vaso. L&#39;uso di forbici microvascolari è raccomandato per evitare l&#39;attraversamento della vasca per evitare l&#39;arteriotomia. </li><li> Inserire il catetere a tre vie nell&#39;arteria. Fissare immediatamente la Sutura 1 e 2 per fissare il catetere in posizione. </li><li> Collegare il catetere a tre vie al trasduttore e raccogliere i dati della pressione sanguigna di base. </li><li> Ripetere i passaggi 2.2.4 - 2.2.6 sull&#39;inguine opposto. Canottare la vena femorale in modo simile all&#39;arteria. Eseguire una venotomia sulla superficie ventrale del vaso seguita dall&#39;inserzione del catetere. Questo catetere può essere utilizzato per somministrazione di fluidi o farmaci. </li></ol></li><li> Lacerazione del fegato <ol><li> Pre-pesare un tubo contenente 0,5 mL di PBS, tre triangoli di assorbimento e una barca di pesatura per mouse. </li><li> Effettuare un&#39;incisione laparotomia midline ventrale partendo dal processo xiphoid e estendentesi caudalmente per consentire l&#39;esposizione del fegato completamente. </li><li> Inserire un triangolo di assorbimento nell&#39;addome contro la parete addominale destra. Ripetere sul lato sinistro. <ol><li> Assicurarsi che il triangolo di assorbimento sia lontano dal fegato per evitare un effetto emostatico di imballaggio dopo che il fegato è lacerato. </li></ol></li><li> Prendete con attenzione il lobo sinistro del fegato e lacerate il 75% del lobo con le forbici chirurgiche Iris. Mettere il laceratoSegmento d in un tubo contenente PBS. </li><li> Chiudere la parete addominale con le graffette tramite un applicatore di graffette. Afferrare la pelle e il muscolo insieme e sparare il fiocco. Fai questo il più rapidamente possibile dopo lacerazione del fegato per evitare la perdita di sangue al di fuori dell&#39;addome. Negli esperimenti di sopravvivenza, l&#39;addome è chiuso in due strati. Una sutura assorbibile in esecuzione per il muscolo seguita da uno strato di sutura non assorbibile per la pelle fornisce una chiusura adeguata. </li><li> Per i topi che sono per il tempo di sopravvivenza più di 30 minuti i cateteri femorali devono essere rimossi, l&#39;arteria e la vena legati con la sutura 3 dalla fase 2.2.6. I ginocchi laterali sono quindi chiusi in due strati come descritto nel passaggio precedente. </li><li> Dopo un periodo di tempo specifico per l&#39;emorragia (da 30 minuti a 72 h), togliere le graffette. Rimuovere i triangoli di assorbimento e mettere in corrispondenti barche pesate pre-pesate. Utilizzare ulteriori triangoli di assorbimento per assorbire eventuali sangue non assorbito. </li><li&gt; Filtri di assorbimento del peso per calcolare la perdita totale del sangue. </li></ol></li><li> Cura postoperatoria <ol><li> Lasciare i topi che devono essere sacrificati a 30 minuti sulla scheda chirurgica e sotto costante monitoraggio e sotto anestesia completa fino al momento del sacrificio. I topi vengono eutanizzati con una combinazione di puntura cardiaca e un sovradosaggio di isoflurano inalato. </li><li> Metti i topi che sono per più tempo di sopravvivenza punti in una gabbia di recupero in cima a un rilievo di riscaldamento circolante con acqua. Monitorare costantemente i topi durante il recupero e non lasciare incustoditi fino a quando non riesumano la coscienza per mantenere la ricostruzione sternale. Rimettere il mouse allo spazio di gabbia con altri topi solo dopo aver recuperato dall&#39;anestesia. </li><li> Somministrare analgesia postoperatoria con 0.1 mg / kg di buprenex mediante iniezione sottocutanea una volta svegliata da anestesia e ogni 12 h dopo fino all&#39;ora del sacrificio. </li><li> Consentire ai topi di accedere gratuitamente al cibo e all&#39;acqua dopo che saranno restituiti al loroNormali gabbie post-operative. </li><li> Al momento del sacrificio per topi di sopravvivenza, l&#39;anestesia viene eseguita con isoflurano inalato. Una volta sottoposta a anestesia, il sangue viene raccolto tramite una punzonatura cardiaca destra, la perdita di sangue viene registrata come sopra descritto e infine l&#39;eutanasia è assicurata con un sovradosaggio di isoflurano. </li></ol></li></ol>

<ol>
	<li>Chang, R., Cardenas, J. C., Wade, C. E., Holcomb, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+understanding+of+trauma-induced+coagulopathy.">Advances in the understanding of trauma-induced coagulopathy.</a> Blood. 128 (8), 1043-1049 (2016).</li><li>Kutcher, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+paradigm+shift+in+trauma+resuscitation:+evaluation+of+evolving+massive+transfusion+practices.">A paradigm shift in trauma resuscitation: evaluation of evolving massive transfusion practices.</a> JAMA surgery. 148 (9), 834-840 (2013).</li><li>Tsukamoto, T., Pape, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+for+Trauma+Research.">Animal Models for Trauma Research.</a> Shock. 31 (1), 3-10 (2009).</li><li>Darwiche, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudofracture:+an+acute+peripheral+tissue+trauma+model.">Pseudofracture: an acute peripheral tissue trauma model.</a> J Vis Exp. (50), (2011).</li><li>Lomas-Niera, J. L., Perl, M., Chung, C. -. S., Ayala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shock+and+Hemorrhage:+an+Overview+of+Animal+Models.">Shock and Hemorrhage: an Overview of Animal Models.</a> Shock. 24, 33-39 (2005).</li><li>Kohut, L. K., Darwiche, S. S., Brumfield, J. M., Frank, A. M., Billiar, T. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+a+topical+bovine-derived+thrombin+solution+as+a+hemostatic+agent+in+a+rodent+model+of+hepatic+injury.">Efficacy of a topical bovine-derived thrombin solution as a hemostatic agent in a rodent model of hepatic injury.</a> Can J Vet Res. 14 (14), 303-308 (2015).</li><li>Sheppard, F. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Nonhuman+Primate+(Rhesus+Macaque)+Model+of+Uncontrolled+Traumatic+Liver+Hemorrhage.">Development of a Nonhuman Primate (Rhesus Macaque) Model of Uncontrolled Traumatic Liver Hemorrhage.</a> Shock. 44, 114-122 (2015).</li><li>Nemzek-Hamlin, J. A., Hwang, H., Hampel, J. A., Yu, B., Raghavendran, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+murine+model+of+blunt+hepatic+trauma.">Development of a murine model of blunt hepatic trauma.</a> Comp Med. 63 (5), 398-408 (2013).</li><li>Vogel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-derived+HMGB1+is+a+critical+mediator+of+thrombosis.">Platelet-derived HMGB1 is a critical mediator of thrombosis.</a> J Clin Invest. 125 (12), (2015).</li><li>Modery-Pawlowski, C. L., Tian, L. L., Ravikumar, M., Wong, T. L., Sen Gupta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+hemostatic+capabilities+of+a+functionally+integrated+platelet-mimetic+liposomal+nanoconstruct.">In vitro and in vivo hemostatic capabilities of a functionally integrated platelet-mimetic liposomal nanoconstruct.</a> Biomaterials. 34 (12), 3031-3041 (2013).</li></ol>

Gli autori non hanno interessi concorrenti finanziari da dichiarare.

Il modello di lacerazione del fegato murino qui descritto fornisce un modello affidabile e coerente di emorragia incontrollata. Questo modello è semplice da eseguire ma ci sono importanti passaggi che richiedono una considerazione meticolosa. La parte più tecnicamente impegnativa del modello è la cannulazione dei vasi femorali per il monitoraggio emodinamico e la somministrazione di fluido / farmaco. La cura deve essere presa durante la dissezione del nervo e dell&#39;arteriotomia / venotomia. È importante non tocca...

Il trauma è la principale causa di morte e disabilità tra i giovani in tutto il mondo. 1 L&#39; emorragia non controllata rimane una causa principale di mortalità tra i pazienti con traumi gravemente feriti. 2 La gestione del paziente traumatico emorragico è duplice: il controllo del sanguinamento chirurgico, la rianimazione e la sostituzione del sangue perduto. I modelli animali di shock emorragico sono stati la pietra angolare nella ricerca del trauma e possono essere utilizzati nella valutazione della fisiopatologia e del trattamento dello shock traumatico / emorragico. 3 , 4 Gli shock nei modelli animali possono essere ottenuti ampiamente da due metodi: emorragia controllata e emorragia incontrollata. 5 , 6 L&#39; emorragia controllata viene eseguita rimuovendo un volume fisso di sangue o rimuovendo il sangue per ottenere una certa pressione sanguigna (pressione fissa). Mentre ilSe sono utili nella valutazione nei meccanismi e nelle alterazioni immunitarie dello shock emorragico, non sono applicabili alla prova degli agenti emostatici e non mimano lo scenario clinico di emorragia dopo traumi. A questo punto abbiamo cercato di sviluppare un modello di emorragia incontrollata che ci permetterebbe di verificare i cambiamenti emostatici e gli agenti pro-coagulanti in un modello murino. Il fegato è un&#39;opzione attraente per l&#39;emorragia incontrollata in parte a causa della doppia alimentazione del sangue al fegato ed è uno degli organi intrabdominali più comuni in entrambi i traumi sbarrati e penetranti. Data l&#39;elevata rilevanza clinica, il fegato è stato utilizzato come modello di emorragia incontrollata, più comunemente nei modelli ratto e suina ma recentemente anche nei primati. 7 , 8 , 9 , 10 , 11 , 12 </suP&gt; I modelli murini hanno anche incluso lesioni epatiche, ad esempio un modello di schiacciamento o un trauma sordo; Tuttavia, questi modelli non provocano uno shock emorragico secondario alla lesione epatica. 13 , 14 I modelli ratto e sanguigni di emorragia epatica incontrollata, pur valorizzando le pratiche di rianimazione e il monitoraggio emodinamico, sono meno vantaggiose rispetto a un modello murino per varie ragioni come il costo, il numero di animali utilizzati e soprattutto la mancanza relativa di linee transgeniche disponibili per l&#39;analisi Di specifiche segnalazioni cellulari e molecolari. L&#39;attuale modello murino condivide importanti somiglianze con i modelli di emorragia epatica esistenti, tra cui lacerazione epatica standardizzata, la quantificazione della perdita di anima, il monitoraggio emodinamico e la capacità di eseguire l&#39;analisi di sopravvivenza. Molti modelli esistenti incorporano solo alcuni di questi aspetti, mentre il nostro modello è stato sviluppato per misurare molte delle variazioni fisiologicheSimultaneamente e in topi multipli. Inoltre, lo sviluppo di un modello murino apre la porta a indagini oltre la rianimazione e nei più grandi meccanismi di fisiopatologia in emorragia incontrollata con il potenziale di un modello a costi elevati e ad alto rendimento utilizzando tecniche avanzate molecolari.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>SS/45 dumonts</td>
			<td>Fine Science Tools</td>
			<td>11203-25</td>
		</tr>
		<tr>
			<td>surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14068-12</td>
		</tr>
		<tr>
			<td>hemostats</td>
			<td>Fine Science Tools</td>
			<td>13009-12</td>
		</tr>
		<tr>
			<td>microscissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
		</tr>
		<tr>
			<td>0.8mm curved forceps</td>
			<td>Fine Science Tools</td>
			<td>11009-13</td>
		</tr>
		<tr>
			<td>suture reel 6-0</td>
			<td>Fine Science Tools</td>
			<td>18020-60</td>
		</tr>
		<tr>
			<td>suture 4-0 silk w/ needle</td>
			<td>Owens Minor</td>
			<td>K188H</td>
		</tr>
		<tr>
			<td>gauze 4x4</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>cotton-tip applicator</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>30G needle</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>23G needle</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>10cc syringe</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>50cc conical tube</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>1cc syringe w/ 25G needle</td>
			<td>Fisher Scientific</td>
			<td>14-826-88</td>
		</tr>
		<tr>
			<td>Polyethylene 10 tubing 100`(PE-10)</td>
			<td>Fisher Scientific</td>
			<td>14-170-12P</td>
		</tr>
		<tr>
			<td>Polyethylene 50 tubing 100`(PE-50)</td>
			<td>Fisher Scientific</td>
			<td>14-170-12B</td>
		</tr>
		<tr>
			<td>3-way stopcock</td>
			<td>Fisher Scientific</td>
			<td>NC9779127</td>
		</tr>
		<tr>
			<td>surgical blue pad</td>
			<td>Fisher Scientific</td>
			<td>50-7105</td>
		</tr>
		<tr>
			<td>Sterile Field dressings</td>
			<td>Fisher Scientific</td>
			<td>NC9517505</td>
		</tr>
		<tr>
			<td>tape rolls 1&#34;</td>
			<td>Corporate Express</td>
			<td>MMM26001</td>
		</tr>
		<tr>
			<td>straight side wide mouth jars</td>
			<td>VWR</td>
			<td>159000-058</td>
		</tr>
		<tr>
			<td>stainless steel tray 8&#34; x 11&#34;</td>
			<td>VWR</td>
			<td>62687-049</td>
		</tr>
		<tr>
			<td>male-male leur lock 3-way</td>
			<td>VWR</td>
			<td>20068-909</td>
		</tr>
		<tr>
			<td>sterilization pouch 3&#34;x8&#34;</td>
			<td>VWR</td>
			<td>24008</td>
		</tr>
		<tr>
			<td>sterilization pouch 5&#34;x10&#34;</td>
			<td>VWR</td>
			<td>24010</td>
		</tr>
		<tr>
			<td>absorption triangles</td>
			<td>Fine Science Tools</td>
			<td>18105-03</td>
		</tr>
		<tr>
			<td>7mm wound clip applier</td>
			<td>Fisher Scientific</td>
			<td>E0522687</td>
		</tr>
		<tr>
			<td>1000 7mm wound clips</td>
			<td>Fisher Scientific</td>
			<td>E0522687</td>
		</tr>
		<tr>
			<td>betadine (4oz)</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>sterile gloves</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>eppendorfs&#160;</td>
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			<td>1/2cc Lo-Dose insulin syringe</td>
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			<td>lactated ringers</td>
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			<td>Digi-Med BPA-400 analyzer &#38; systems integrator</td>
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			<td>TXD-310 (Digi-Med Transducer)&#160;</td>
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			<td>Kent Scientific</td>
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	</tbody>
</table>

uncontrolled hemorrhagic shock modeled via liver laceration in mice with real time hemodynamic monitoring

L&#39;emorragia non controllata è una causa importante delle morti evitabili tra i pazienti traumatici. Abbiamo sviluppato un modello murino di emorragia incontrollata attraverso una lacerazione epatica che comporta una consistente perdita di sangue, alterazioni emodinamiche e sopravvivenza. I topi subiscono una resezione standardizzata del lobo sinistro e medio del fegato. Sono autorizzati a sanguinare senza intervento meccanico. Gli agenti emostatici possono essere somministrati come terapia di pre-trattamento o di soccorso a seconda dell&#39;interesse dell&#39;investigatore. Durante l&#39;emorragia, viene eseguito il monitoraggio emodinamico in tempo reale attraverso una linea arteriosa sinistra femorale. I topi vengono poi sacrificati, la perdita di sangue viene quantificata, il sangue viene raccolto per ulteriori analisi e gli organi vengono raccolti per l&#39;analisi del danno. È descritto il disegno sperimentale per consentire la sperimentazione simultanea di più animali. L&#39;emorragia epatica come modello di emorragia incontrollata esisteN la letteratura, principalmente nei modelli ratto e porcino. Alcuni di questi modelli utilizzano il monitoraggio emodinamico o quantificano la perdita di sangue ma mancano la coerenza. Il presente modello comprende la quantificazione della perdita di sangue, il monitoraggio emodinamico in tempo reale in un modello murino che offre il vantaggio di utilizzare linee transgeniche e un meccanismo ad alto rendimento per studiare ulteriormente i meccanismi patofisiologici in emorragia incontrollata.

Il modello di lacerazione del fegato produce una perdita di sangue riproducibile e coerente nei topi. La figura 1A mostra il peso coerente del fegato lacerato che può essere ottenuto con una deviazione standard di soli 0,02 g. Questa coerenza nel peso fegato lacerato consente la capacità di riprodurre il modello tra i topi e in differenti configurazioni sperimentali come diversi protocolli resuscitativi. Inoltre, il peso riproducibile del fegato lacerato, con un errore...

Watch this Scientific Journal Video about Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring at JoVE.com

Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring

L&#39;emorragia non controllata, una causa importante della mortalità tra i pazienti traumatici, può essere modellata utilizzando una lacerazione epatica standard in un modello murino. Questo modello provoca una consistente perdita di sangue, la sopravvivenza e consente di testare agenti emostatici. Questo articolo fornisce il processo passo per passo per eseguire questo prezioso modello.

Scossa emorragica non controllata modellata mediante Lacerazione del fegato nei topi con monitoraggio emodinamico in tempo reale

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via ...

uncontrolled-hemorrhagic-shock-modeled-via-liver-laceration-mice-with

Research

JoVE Journal

Medicine

8.5K Views.  University of Pittsburgh.  L'emorragia non controllata, una causa importante della mortalità tra i pazienti traumatici, può essere modellata utilizzando una lacerazione epatica standard in un modello murino. Questo modello provoca una consistente perdita di sangue, la sopravvivenza e consente di testare agenti emostatici. Questo articolo fornisce il processo passo per passo per eseguire questo prezioso modello. 

Video: Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring

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, ...

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Medicina

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Controllata cervicale lesioni lacerazione nei topi

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

In tempo reale citotossicità saggi in Human Sangue intero

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

Monitoraggio della sistemici e epatica parametri emodinamici nei topi

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

Analisi in tempo reale pressione-volume di infarto miocardico acuto nei topi

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

Complete and Partial Aortic Occlusion for the Treatment of Hemorrhagic Shock in Swine

Hemorrhage remains the leading cause of preventable deaths in trauma. Endovascular management of non-compressible torso hemorrhage has been at the forefront of trauma care in recent years. Since complete aortic occlusion presents serious concerns, the concept of partial aortic occlusion has gained a growing attention. Here, we present a large animal model of hemorrhagic shock to investigate the effects of a novel partial aortic balloon occlusion catheter and compare it with a catheter that works on the principles of complete aortic occlusion. Swine are anesthetized and instrumented in order to conduct controlled fixed-volume hemorrhage, and hemodynamic and physiological parameters are monitored. Following hemorrhage, aortic balloon occlusion catheters are inserted and inflated in the supraceliac aorta for 60 min, during which the animals receive whole-blood resuscitation as 20% of the total blood volume (TBV). Following balloon deflation, the animals are monitored in a critical care setting for 4 h, during which they receive fluid resuscitation and vasopressors as needed. The partial aortic balloon occlusion demonstrated improved distal mean arterial pressures (MAPs) during the balloon inflation, decreased markers of ischemia, and decreased fluid resuscitation and vasopressor use. As swine physiology and homeostatic responses following hemorrhage have been well-documented and are like those in humans, a swine hemorrhagic shock model can be used to test various treatment strategies. In addition to treating hemorrhage, aortic balloon occlusion catheters have become popular for their role in cardiac arrest, cardiac and vascular surgery, and other high-risk elective surgical procedures.

Here, we present a protocol demonstrating a hemorrhagic shock model in swine that uses aortic occlusion as a bridge to definitive care in trauma. This model has application in testing a wide range of surgical and pharmacological therapeutic strategies.

Completa e parziale occlusione aortica per il trattamento dello Shock emorragico nei suini

Hemorrhage remains the leading cause of preventable deaths in trauma. Endovascular management of non-compressible torso hemorrhage has been at the ...

Measurement of the Hepatic Venous Pressure Gradient and Transjugular Liver Biopsy

Here we provide a detailed protocol describing the clinical procedure of hepatic venous pressure gradient (HVPG) measurement in patients with advanced chronic liver disease followed by an instruction for transjugular biopsy. Under local anesthesia and ultrasound guidance, a catheter introducer sheath is placed in the right internal jugular vein. Using&#160;fluoroscopic guidance, a balloon catheter is advanced into the inferior vena cava (IVC) and inserted into a large hepatic vein. Correct and sufficient wedge position of the catheter is ensured by injecting contrast media while the balloon is blocking the outflow of the cannulated hepatic vein. After calibrating the external pressure transducer, continuous pressure recordings are obtained with triplicate recordings of the wedged hepatic venous pressure (WHVP) and free hepatic venous pressure (FHVP). The difference between FHVP and WHVP is referred to as HVPG, with values &#8805;10 mm Hg indicating clinically significant portal hypertension (CSPH). Before removing the catheter, pressure readings obtained in the IVC at the same level, as well as the right atrial pressure are recorded.
Finally, a transjugular liver biopsy can be obtained via the same vascular route. Different systems are available; however, core biopsy needles are preferred over aspiration needles, especially for cirrhotic livers. Again, under fluoroscopic guidance a biopsy needle introducer sheath is advanced into an hepatic vein. Next, the transjugular biopsy needle is gently advanced through the introducer sheath: (i) in case of aspiration biopsy, the needle is advanced into the liver parenchyma under aspiration and then removed quickly, or (ii) in case of a core biopsy, the cutting-mechanism is triggered inside the parenchyma. Several separate passages can be safely performed to obtain sufficient liver specimens via transjugular biopsy. In experienced hands, the combination of these procedures takes about 30-45 min.

Here, we present a protocol for measurement of hepatic venous pressure gradient (HVPG),the gold standard to diagnose clinically significant portal hypertension. Moreover, we describe how to perform a transjugular liver biopsy within the same session.

Misurazione del gradiente di pressione venosa epatica e della biopsia epatica transgiugulare

Here we provide a detailed protocol describing the clinical procedure of hepatic venous pressure gradient (HVPG) measurement in patients with advanced ...

Standardized Hemorrhagic Shock Induction Guided by Cerebral Oximetry and Extended Hemodynamic Monitoring in Pigs

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an insufficient oxygen supply and irreversible organ failure. The brain exerts only limited compensation capacities and is particularly at high risk of severe hypoxic damage.This article demonstrates the reproducible induction of life-threatening hemorrhagic shock in a porcine model by means of calculated blood withdrawal. We titrate shock induction guided by near-infrared spectroscopy&#160;and extended hemodynamic monitoring to display systemic circulatory failure, as well as cerebral microcirculatory oxygen depletion. In comparison to similar models that primarily focus on predefined removal volumes for shock induction, this approach highlights a titration by means of the resulting failure of macro- and microcirculation.

Hemorrhagic shock is a severe complication in seriously injured patients, which leads to life-threatening oxygen undersupply. We present a standardized method to induce hemorrhagic shock via blood withdrawal in pigs that is guided by hemodynamics and microcirculatory cerebral oxygenation.

Induzione da shock emorragico standardizzato guidato da ossimetria cerebrale e monitoraggio emodinamico esteso esteso nei suini

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an ...

Real-Time Monitoring of Neurocritical Patients with Diffuse Optical Spectroscopies

Neurophysiological monitoring is an important goal in the treatment of neurocritical patients, as it may prevent secondary damage and directly impact morbidity and mortality rates. However, there is currently a lack of suitable non-invasive, real-time technologies for continuous monitoring of cerebral physiology at the bedside. Diffuse optical techniques have been proposed as a potential tool for bedside measurements of cerebral blood flow and cerebral oxygenation in case of neurocritical patients. Diffuse optical spectroscopies have been previously explored to monitor patients in several clinical scenarios ranging from neonatal monitoring to cerebrovascular interventions in adults. However, the feasibility of the technique to aid clinicians by providing real-time information at the bedside remains largely unaddressed. Here, we report the translation of a diffuse optical system for continuous real-time monitoring of cerebral blood flow, cerebral oxygenation, and cerebral oxygen metabolism during intensive care. The real-time feature of the instrument could enable treatment strategies based on patient-specific cerebral physiology rather than relying on surrogate metrics, such as arterial blood pressure. By providing real-time information on the cerebral circulation at different time scales with relatively cheap and portable instrumentation, this approach may be especially useful in low-budget hospitals, in remote areas and for&#160;monitoring in open fields (e.g., defense and sports).

Presented here is a protocol for non-invasively monitoring cerebral hemodynamics of neurocritical patients in real-time and at the bedside using diffuse optics. Specifically, the proposed protocol uses a hybrid diffuse optical systems to detect and display real-time information on cerebral oxygenation, cerebral blood flow and cerebral metabolism.

Monitoraggio in tempo reale di pazienti neurocritici con spettroscopie ottiche diffuse

Neurophysiological monitoring is an important goal in the treatment of neurocritical patients, as it may prevent secondary damage and directly impact ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

Lesione da ischemia-riperfusione dell'organo simulando cambiamenti emodinamici nel modello di trapianto di fegato di ratto

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...