This work was supported by AI101263 from the National Institutes of Health (T.V.B), and by a grant from The Hartwell Foundation (M.L.M). M.L.M. is a member of the Duke Comprehensive Cancer Center.

NOTA: Tutte le procedure sperimentali che prevedono l&#39;uso di ratti sono stati fatti secondo protocolli approvati dalla cura e l&#39;uso degli animali Comitato della Duke University. 1. Preparazione del tracheale intubazione cannula <ol><li> Tagliare la fine dell&#39;ago di un 2 pollici 14 G angiocathether ago con taglienti. </li><li> Pinch il lume dell&#39;ago chiuso con pinze ad ago. </li><li> Tagliare sezione pizzicato fino al bordo della porzione aperta lumen con le pinze di taglio, e quindi tagliare i bordi su entrambi i lati della restante estremità a 30 - 45 ° angolo al bordo dell&#39;ago. </li><li> Posizionare la parte inferiore 3 - 4 mm della cannula nella zona aperta di pinze slip-joint diritte e tirare verso l&#39;alto fino a quando una leggera curva verso l&#39;alto è formata alla fine. NOTA: Ciò faciliterà dirigere il catetere nella trachea (Figura 1). </li><li> Utilizzare carta vetrata fine per lisciare i bordi all&#39;estremità della cannula. </li><li&gt; Torna lo strumento smussata nel tubo angiocatheter. Assicurarsi che il tubo è leggermente più corto del ago smussato. </li></ol> Procedure 2. Pre-chirurgiche <ol><li> Impostare il sito per la procedura inserendo un microscopio operatorio impostato a 10 volte di ingrandimento su tutta l&#39;area operativa. </li><li> Posizionare un pad di riscaldamento sul campo operatorio e coprire con un tampone assorbente pulita. </li><li> Impostare una frequenza cardiaca e un monitor ossigenazione del sangue nei pressi del campo operatorio. </li><li> Pesare il ratto per basato sul peso farmaci dosaggio (ad esempio, analgesia postoperatoria, antibiotici, o linfociti riducono anticorpi). NOTA: tipico ratti adulti pesano tra 350-450 g. </li><li> Sedate ratto con vaporizzato 3% isoflurano-O 2 (3 L / min) utilizzando una camera di induzione ventilato ad un sistema di evacuazione dei gas anestetico rifiuti contenenti carbone attivo. Lasciare 5 min per l&#39;induzione dell&#39;anestesia per fornire profonda anestesia necessaria per l&#39;intubazione endotracheale. </li><li> Somministrare Carprofen o Meloxicane (4,4 mg / kg) per via sottocutanea nel ratto prima di procedere con intubazione e chirurgia. </li><li> Utilizzare tagliatori elettrici a radersi il collo e il petto del topo anestetizzato prima di intubazione. </li></ol> 3. intubazione <ol><li> Preparare la configurazione intubazione strofinando una piccola quantità di lubrificante (ad esempio, gel KY) sull&#39;estremità della cannula. Accendere il ventilatore per iniziare il flusso di gas isoflurano attraverso il tubo connettore. </li><li> Trasferire il ratto all&#39;apparato intubazione e sospende il ratto sulla barra metallica dal suo incisivo superiore denti (Figura 2A). NOTA: alcuni apparecchi intubazione possono essere acquistati on-line. Il modello mostrato qui è fatto in casa utilizzando un foglio acrilico modellato attorno a sezioni di un pollice pezzo lombare 2 x 4 con una torcia butano, e rinforzi sono attaccati con cloroformio. Il bar (o filo metallico) è attaccato da fori di perforazione oltre nastro adesivo immessi sul side dell&#39;acrilico per evitare scheggiature o crepe. </li><li> Trans-illuminare il collo del ratto posizionando una fonte di luce ad alta intensità flessibile 1 - 2 cm dalla superficie ventrale del collo (Figura 2B). </li><li> Utilizzare un paio di pinze studente modello standard per tirare la linguetta leggermente verso l&#39;alto e verso il lato dei denti inferiori. Grip linguetta tra il pollice e l&#39;indice di una mano mentre si posiziona la superficie interna piatta di un lato della pinza contro l&#39;estremità inferiore della lingua. Premere ventralmente per esporre l&#39;epiglottide e l&#39;apertura della laringe (Figura 2C). </li><li> Visualizzare le corde vocali e guidare la rovesciata, estremità smussata della cannula intubazione anteriormente attraverso la glottide aperta nella trachea fino mozzo della angiocatheter tocca incisivi. </li><li> Togliere il mandrino metallico, e collegare il tubo dell&#39;anestesia all&#39;apertura angiocatheter per cominciare la ventilazione con isoflurano. NOTA: Il tipico flo ossigenotasso w al ventilatore è 3 L / min con il 3% isoflurano. <ol><li> Impostare il ventilatore ad una velocità di 60 respirazioni / min in una modalità ventilatore controllato volume raggiunge una pressione di ~ 12 - 14 mmHg. Utilizzare una pressione positiva di fine espirazione (PEEP) del 3 cm H 2 O. </li></ol></li><li> Osservare espansione parete toracica bilaterale in sincronia con il ventilatore per garantire il corretto posizionamento del tubo endotracheale. </li><li> Applicare veterinario consigliato occhio pomata agli occhi del ratto per prevenire la secchezza mentre sotto anestesia. </li><li> Applicare veterinario pomata sugli occhi del ratto per prevenire la secchezza mentre sotto anestesia. </li><li> Fissare il tubo endotracheale alla testa del ratto con una striscia di nastro di stoffa. Garantire un collegamento stabile tra il tubo endotracheale e la tubazione afflusso anestetico. </li><li> Attaccare l&#39;ossigenazione del sangue e il monitor della frequenza cardiaca al piede del ratto e iniziare il monitoraggio. Verificare il corretto anestesia confermando non vi è alcuna reazione ae-pizzico. </li></ol> 4. Toracotomia e Thymectomy <ol><li> Seguire tecnica asettica standard per l&#39;interezza della procedura. <ol><li> Pulire l&#39;area e il funzionamento tavolo di lavoro e disinfettare con una soluzione di etanolo al 70%. </li><li> Utilizzare guanti chirurgici sterili durante la procedura, e autoclave tutti gli strumenti ei materiali utilizzati durante la procedura. </li><li> Applicare Providone-iodio per l&#39;intera area del torace e lasciarlo asciugare. Poi pulire la superficie della pelle con il 70% di etanolo su garza. Coprire il ratto con involucro di plastica trasparente tagliare un buco per esporre il campo operatorio sterile. </li></ol></li><li> Identificare soprasternale nella regione toracica superiore. Effettuare un&#39;incisione di 2 cm mediana attraverso la pelle partenza 2 - 3 mm sopra il soprasternale e si estende distalmente linea mediana che attraversa lo sterno con le forbici Shea punta smussata. </li><li> Eseguire una sternotomia mediana 1,5 centimetri dal soprasternale di nuovo con le forbici Shea punta smussata. KEEP il bordo inferiore delle forbici appena sotto lo sterno e avanzare lentamente. </li><li> Inserire un piccolo divaricatore Alm appena sotto lo sterno separati e si apre per rivelare il muscoli cinghia pre-tracheale (sterno-ioideo e muscoli sterno-tiroideo). Separare i muscoli cinghia pre-tracheale con smussato pinze Graefe. NOTA: A questo punto, la trachea può essere visto, e il tubo di intubazione deve essere visualizzato all&#39;interno della trachea. </li><li> Posizionare le punte del piccolo divaricatore Alm sotto i muscoli cinghia separati e sterno. Aprire riavvolgitore per esporre la porzione superiore del timo. </li><li> Utilizzare pinza sottile Dumont per rilasciare i bordi laterali del tessuto del timo e esporre i lobi timici inferiori. </li><li> Estrarre il timo delicatamente superiormente al sito di incisione aperta, avendo cura di evitare il contatto con i vasi vena cava, succlavia e carotidi superiori, e per minimizzare i disagi del rivestimento pleurico delicato tra il timo ei polmoni. </li><li> Come tegli timica vasi sono sezionati e rivelati, utilizzare micro-forbici per dividerli. Usare un tampone di cotone per mantenere la pressione sui vasi di emostasi, se necessario. </li><li> Consegnare i lobi timici inferiori nell&#39;incisione e poi bruscamente lisare gli allegati posteriori. Rimuovere il timo intatta e controllare attentamente il timo rimosso in tutte le sezioni mancanti. NOTA: I piccoli linfonodi del torace possono essere visualizzati circonda il timo e sono spesso difficili da distinguere dal tessuto del timo. Questi saranno i nodi discreti rotondi con un aspetto simile al tessuto del timo, ma non sarà in continuità con il timo. </li><li> Rimuovere il divaricatore Alm e chiudere i muscoli sterno-ioideo e sternothyoid con due interrotto 5-0 suture Maxon. </li><li> Applicare 2 gocce di tessuto adesivo cianoacrilato grado chirurgico attraverso le suture per sigillare il mediastino sotto ventilazione a pressione positiva. NOTA: Questo diminuire l&#39;incidenza di pneumotorace ed ematoma. </li><li> Chiudere la sternum con due interrotto 4-0 punti di sutura di seta su un ago di taglio. Inserire l&#39;ago attraverso spazi fra le costolette, avendo cura di guidare l&#39;ago appena sotto lo sterno per evitare lo strato muscolare sottostante. </li><li> Chiudere lo strato di pelle con un running 4-0 Nylon sutura. Interrompere l&#39;isoflurano a questo punto di abbreviare il periodo di recupero post-operatorio anestesia. </li><li> Pulire il sito di incisione e la pelle circostante con garza salina-inumidito. </li><li> Applicare alcune gocce di bupivicaina (0,25%) per l&#39;anestesia locale, seguito da 1 - 2 gocce di colla cianoacrilato sopra l&#39;incisione per sigillare l&#39;incisione. </li><li> Continuare a ventilazione fino a quando il topo mostra segni di sforzo respiratorio indipendente e comincia a muoversi sue estremità. Estubare ratto e permettono di recuperare sotto stretta osservazione in una gabbia posta su un rilievo di riscaldamento. </li><li> Somministrare Buprenorfina (0,01-0,05 mg / kg) per via sottocutanea per il ratto subito dopo l&#39;intervento e ripetere ogni 8-12 ore per 48 ore post-operatorio (normalmente 5 dosi totali). A 24 e 48 ore dopo l&#39;intervento, l&#39;amministrazione Carprofen o Meloxicane (4,4 mg / kg) per via sottocutanea (3 dosi totali, comprese la dose pre-operatoria). </li><li> Continuare a monitorare fino a quando il topo si muove intorno alla gabbia. A tutti i segni di difficoltà respiratoria, effettuare re-intubazione e ri-esplorazione del torace. <ol><li> Non lasciare un animale incustodito fino a quando non ha ripreso conoscenza sufficiente per mantenere decubito sternale. </li><li> Non restituire un animale che ha subito un intervento chirurgico per la compagnia di altri animali fino alla completa guarigione. </li></ol></li><li> Rimuovere le suture strato di pelle 1 settimana dopo l&#39;intervento. </li></ol>

<ol>
	<li>Hard, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymectomy+in+the+neonatal+rat.">Thymectomy in the neonatal rat.</a> Lab. Anim. 9 (2), 105-110 (1975).</li><li>Kobayashi, 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+technique+for+complete+thymectomy+in+adult+rats.">A technique for complete thymectomy in adult rats.</a> J. Immunol. Methods. 1759 (94), 0 (1994).</li><li>Delrivi&#232;re, L., Gibbs, P., Kobayashi, E., Kamada, N., Gianello, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+technique+of+complete+thymectomy+in+adult+rats+without+tracheal+intubation.">New technique of complete thymectomy in adult rats without tracheal intubation.</a> Microsurgery. 18 (1), 6-8 (1998).</li><li>Walker, K. G., Jacques, B. C., Tweedle, J. R., Bradley, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simpler+technique+for+open+thymectomy+in+adult+rats.">A simpler technique for open thymectomy in adult rats.</a> J. Immunol. 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Educ. 28 (1-4), 29-34 (2004).</li><li>Weksler, B., Ng, B., Lenert, J., Burt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+method+for+endotracheal+intubation+in+the+rat.">A simplified method for endotracheal intubation in the rat.</a> J. Appl. Physiol. 76 (4), 1823-1825 (1994).</li><li>Jou, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+rat+intubation+using+a+new+oropharyngeal+intubation+wedge.">Simplified rat intubation using a new oropharyngeal intubation wedge.</a> J. Appl. Physiol. 89 (5), 1766-1770 (2000).</li><li>Rivard, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+intubation+and+ventilation+for+surgical+research.">Rat intubation and ventilation for surgical research.</a> J. Invest. Surg. 19 (4), 267-274 (2006).</li><li>Zhao, D., 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+model+of+isolated,+vascular+whole+thymus+transplantation+in+nude+rats.">A model of isolated, vascular whole thymus transplantation in nude rats.</a> Transplant Proc. 44 (5), 1394-1398 (2012).</li><li>AbuAttieh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+maturation+of+antibodies+requires+integrity+of+the+adult+thymus.">Affinity maturation of antibodies requires integrity of the adult thymus.</a> Eur. J. 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Immunol. 1 (Unit 1.10), (2001).</li><li>Siemionow, M., Izycki, D., Ozer, K., Ozmen, S., Klimczak, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+thymus+in+operational+tolerance+induction+in+limb+allograft+transplant+model.">Role of thymus in operational tolerance induction in limb allograft transplant model.</a> Transplantation. 81 (11), 1568-1576 (2006).</li><li>Satoh, 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=Immunosuppressive+effect+of+long-term+drainage+of+thoracic+duct+on+immunological+memory+in+adult+thymectomized+rats.+Transplant.">Immunosuppressive effect of long-term drainage of thoracic duct on immunological memory in adult thymectomized rats. 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The authors have nothing to disclose.

Il presente protocollo per completare timectomia fornisce un approccio mini-invasivo con una chiusura un&#39;incisione a tre strati con l&#39;applicazione colla chirurgica per ridurre al minimo le complicanze. La rimozione completa del timo è stata dimostrata dalla perdita di cellule T naive e IHC colorazione del tessuto linfoide mediastinica per citocheratina. La procedura di ratto adulto timectomia è stata complicata dalla mortalità da 1,5 - 20% a causa di complicazioni perioperatorie, ...

Dall&#39;inizio degli anni 1960, il timo è stato riconosciuto per il suo ruolo fondamentale nello sviluppo della tolleranza immunologica centrale. Roditore thymectomy ha dimostrato di essere una procedura essenziale nella definizione del ruolo del timo nella differenziazione dei linfociti, auto-tolleranza e immunotolleranza nella cornice del trapianto allogenico e metastasi tumorali. La rimozione del timo topo permette studi che coinvolgono la deplezione delle cellule T o il trasferimento adottivo di popolazioni di cellule T definiti senza il riemergere di cellule T naive nativi. Thymectomies nei roditori neonatali può essere realizzato utilizzando una tecnica di aspirazione con risultati affidabili 1. In ratti adulti, questa tecnica è associata ad un tasso di mortalità approssimativa 20% e spesso si traduce in un timectomia incompleta 2. Per ottenere sempre completa timectomia in ratti adulti, è necessaria l&#39;esposizione aperta del mediastino attraverso una sternotomia mediana. Tuttavia, questoprocedura è associata a complicazioni che includono lesioni tracheale, emorragia e pneumotorace che porta a un tasso di mortalità complessiva che vanno 1,5-6% 2- 4. Negli ultimi due decenni, il miglioramento delle tecniche di timectomia sono diminuiti complicanze perioperatorie e hanno migliorato i tassi di sopravvivenza. Intubazione endotracheale consentendo la ventilazione a pressione positiva ha diminuito i tassi di pneumotorace 5. Metodi di intubazione gamma precedentemente descritti dall&#39;esposizione aperta alla trachea a metodi meno invasivi che utilizzano la visualizzazione diretta delle corde vocali. Le complicanze associate alla procedura di intubazione tracheale includono infortunio, rottura delle corde vocali, non intenzionale intubazione esofagea, e emorragia derivante dalla puntura cardiaca o lacerazione della vena cava superiore. Inoltre, prossimità dei lobi timici inferiori al rivestimento pleurico può provocare pneumotorace. Qui si descrive una tecnica dithymectomy attraverso un mini-invasiva due centimetri incisione cutanea a seguito di un semplice metodo di intubazione endotracheale utilizzando un angiocatheter smussato-end e l&#39;illuminazione tracheale transcutanea. Il thymectomy comporta una sternotomia 1,5 centimetri ed una chiusura a tre strati con l&#39;applicazione della colla chirurgica per sigillare il mediastino e minimizzare l&#39;incidenza di emorragia e complicazioni respiratorie. Questo metodo assicura in maniera affidabile completa timectomia come evidenziato dalla scomparsa di cellule T naive CD4 + e CD8 + seguendo timectomia e l&#39;assenza di tessuto timico sul IHC colorazione. I tempi operativi e mortalità peri-procedurale sono ridotti al minimo.

<table border="0" cellpadding="0" cellspacing="0" width="718">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Name of Reagent/ Equipment</td>
			<td style="width:117px;">Company</td>
			<td style="width:152px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">2&#34; 14-gauge angiocatheter</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Operating microscope</td>
			<td style="width:117px;">Zeiss</td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Warming pad</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:283px;">Heart rate and blood oxygenation monitor for rodents with foot sensors</td>
			<td style="width:117px;">Harvard Apparatus</td>
			<td style="width:152px;">ST1 72-8010, ST1 72-8098 (Rat foot sensor)</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Intubation apparatus (plastic with metal bar at the top)</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;">See Figure 2&#160;</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:283px;">Small animal anesthesia system with induction box, isoflurane tank and O2 tank</td>
			<td style="width:117px;">Harvard Apparatus</td>
			<td style="width:152px;">ST1 72-6420</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:283px;">Small animal ventilator with tubing</td>
			<td style="width:117px;">CWE</td>
			<td style="width:152px;">12-02000 (ventilator)and 12-04000 (external valve assembly for mice/rats)</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">High-intensity Fiber-Optic Illuminator</td>
			<td style="width:117px;">Dolan Jenner</td>
			<td style="width:152px;">EEG 2823M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Student standard pattern forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">91100-16</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Fine straight scissors</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">14060-09</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Blunt-tipped Shea scissors</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">14105-12</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Small Alm retractor (for sternum)</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">17008-07</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Blunt Graefe forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">11050-10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Fine Dumont forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">11254-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">5-0 Maxon sutures</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">4-0 Silk sutures (with cutting needle)</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">6-0 Nylon suture</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cyanoacrylate glue (Endermil)</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Lubrication gel</td>
			<td style="width:117px;">Akorn Animal Health</td>
			<td style="width:152px;">NDC 17478-162-35</td>
			<td></td>
		</tr>
	</tbody>
</table>

complete thymectomy in adult rats with non-invasive endotracheal intubation

Thymectomy nei roditori neonatale è una procedura consolidata e affidabile per studi immunologici. Tuttavia, in ratti adulti, complicanze di emorragia e pneumotorace di interruzione pleurico può provocare un notevole tasso di mortalità. Questo protocollo è un metodo semplice di timectomia topo che utilizza un mini-sternotomia e intubazione endotracheale. Intubazione è compiuto con un metodo non-invasivo e facilmente riproducibile e consente di ventilazione a pressione positiva per evitare pneumotorace e una via aerea controllata che permette un tempo sufficiente per un&#39;attenta timo dissezione per minimizzare i disagi pleurico. A 1,5 centimetri un&#39;incisione sternale diminuisce il contatto con i vasi del mediastino e pleura, pur fornendo una visualizzazione completa di timo. Dopo l&#39;esposizione del mediastino, il timo viene rimosso per via smussa sotto ingrandimento. Lo spazio pleurico viene poi sigillata da sutura dei muscoli pre-tracheale seguita dall&#39;applicazione di colla chirurgica. Iltorace è poi chiusa da sutura dello sterno, seguita da sutura della pelle. Tutti thymectomies erano completo come evidenziato da immunoistochimica (IHC) colorazione del tessuto mediastinica, e l&#39;assenza di cellule T naive mediante citometria di flusso, e la procedura ha avuto un tasso di sopravvivenza del 96%. Questo metodo è adatto quando timectomia completo di complicazioni minime si desidera per ulteriori studi immunologici nei ratti adulti atimici.

Questa procedura è stata eseguita su ratti adulti Lewis (n = 26). Il tempo di funzionamento medio è stato di 15 ± 3 min. Non c&#39;era la mortalità intraoperatoria. Il tempo medio di intubazione - dal posizionamento del ratto su dell&#39;apparato intubazione alla costituzione di ventilazione - è stato di 45 ± 5 sec. 24 ratti avevano un pieno recupero dall&#39;operazione senza evidenza di difficoltà respiratorie o emorragia attraverso giornata post-operatoria (POD) 14. Un ratto sviluppato difficoltà respiratorie ...

Watch this Scientific Journal Video about Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation at JoVE.com

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

Thymectomy completa in ratti adulti con non-invasiva intubazione endotracheale

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

complete-thymectomy-adult-rats-with-non-invasive-endotracheal

Research

JoVE Journal

Immunology and Infection

16.4K Views.  Duke University Medical Center. Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described. 

Video: Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non invasiva di imaging di homing dei leucociti e la migrazione In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Ricerca

Immunologia e infezioni

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Non invasiva Imaging di candidosi disseminata nelle larve Zebrafish

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

Non invasiva di imaging ottico del sistema vascolare linfatico di un mouse

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Coltura Microglia dal Sistema Nervoso Centrale neonatale e adulti

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Non invasiva Modello di neuropatogeno<em&gt; Escherichia coli</em&gt; L&#39;infezione neonatale nel ratto

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Imaging non invasiva della risposta immunitaria innata in un modello di Zebrafish larvale<em&gt; Streptococcus iniae</em&gt; Infezione

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Un metodo non invasivo e tecnicamente non intensiva per l&#39;induzione e Fenotipizzazione Sperimentale polmonite batterica nei topi

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

Non invasivo<em&gt; In Vivo</em&gt; Fluorescenza ottica di imaging dell&#39;attività MMP infiammatoria utilizzando un agente fluorescente di imaging attivo

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

L&#39;analisi dei linfociti stravaso utilizzo di un<em&gt; In Vitro</em&gt; Modello del Sangue-Cervello umano Barriera

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

Un modo non invasivo per isolare e fenotipo le cellule della Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...