The work was supported by grants from NIH/NINDS (NS054883), Craig H. Neilsen Foundation (280072), and the Veterans Administration and Canadian Spinal Research Organization. We thank the Rat Resource and Research Center, University of Missouri, Columbia, Missouri, for providing GFP rats.

Tutti i protocolli di animali sono stati approvati dalla cura e l&#39;uso Comitato Istituzionale Animal (IACUC). Linee guida NIH per il laboratorio la cura e la sicurezza degli animali sono rigorosamente seguite. Gli animali con procedure chirurgiche sono stati adeguatamente trattati per ridurre al minimo il dolore e il disagio. 1. midollo spinale Curgery e innesto cellulare <ol><li> Strumenti chirurgici autoclave prima di tutti gli interventi chirurgici. Utilizzare Hot Bead Sterilizzatore (Fine Science Tools) per rimuovere gli agenti patogeni e contaminanti microbici dagli strumenti tra procedure su animali diversi. Utilizzare guanti sterili chirurgici, abito e tende durante l&#39;intervento chirurgico. Impiegare tecnica chirurgica asettica per ogni procedura chirurgica. </li><li> Anestetizzare femminile Fischer 344 ratti con una combinazione (2 ml / kg) di ketamina (25 mg / ml), xilazina (1,3 mg / ml), e acepromazina (0,25 mg / ml) somministrato per via intraperitoneale (ip). </li><li> Radere la zona posteriore e pulire la pelle ripetutamente con Betadine ed etanolo.</li><li> Tagliare la pelle con una lama # 10 e senza mezzi termini sezionare muscoli strati. Utilizzare un bisturi per isolare le vertebre, esporre la vertebra T3, ed eseguire una laminectomia dorsale usando una punta fine rongeurs. </li><li> Incidere la dura longitudinalmente e transetto il midollo spinale a livello T4 utilizzando una combinazione di iridectomia forbici e microaspirazione per creare un gap rostrocaudale circa 1 mm tra i monconi del midollo spinale. </li><li> Attendere circa 1 - 2 minuti fino a quando il sanguinamento completamente ferma, poi i muscoli di sutura con 3-0 Vycril e chiudere la pelle con clip ferita. </li><li> Iniettare Ringer lattato (5 ml), buprenorfina (0,035 mg / kg), e ampicillina (33 mg / kg) per via sottocutanea immediatamente dopo l&#39;intervento chirurgico e mantenere i ratti in un incubatore caldo fino svegli. </li><li> Iniettare la soluzione Ampicillin una volta al giorno fino a 10 giorni, e buprenorfina due volte al giorno per 3 giorni o fino a quando i segni di dolore e di sofferenza scompaiono di Ringer e. Non restituire un animale per la compagnia di altri animali fino a fUlly recuperato. </li><li> Svuotare manualmente la vescica due volte al giorno per circa due settimane fino all&#39;istituzione della vescica svuotamento riflesso, e quindi svuotare la vescica una volta al giorno per tutta la sopravvivenza, se necessario. </li><li> Due settimane dopo, ri-anestetizzare ratti SCI come descritto sopra e ri-esporre il sito cordone lesione spinale. Mantenere la dura chiusa per mantenere le cellule impiantate nel sito della lesione. </li><li> Iniettare 10 ml di soluzione di cella (3,5 × 10 5 ml), raccolti da E14 GFP onnipresente embrioni transgenici ratto e incorporato in fibrinogeno e trombina 5,6, nel epicentro della cavità lesione e l&#39;interfaccia rostrale e caudale della lesione con iniezione multipla siti, usando una micropipetta di vetro tirato con un diametro interno di 40 micron, collegato ad una siringa Hamilton. </li><li> Suturare gli strati muscolari e chiudere la pelle con clip ferita. </li><li> Eseguire l&#39;intervento chirurgico dopo l&#39;iniezione e la cura della vescica, come descritto nei passaggi 1,6-1,8. </li><li> Una settimana essereribalta perfusione, iniettare 0,5% Fluorogold (FG, 0,4 ml di acqua distillata) per via intraperitoneale per etichetta retrograda neuroni simpatici pregangliari nel midollo spinale 7. </li></ol> 2. Trasmettitore impianto <ol><li> Bagnare transmittersin soluzione di glutaraldeide al 2% (4 ml di 50% glutaraldhyde in 96 ml di acqua distillata) per almeno 1 - 2 ore (fino a 10 ore) a temperatura ambiente per la sterilizzazione. </li><li> Lavare accuratamente con trasmettitori sterile allo 0,9% soluzione salina 3 volte e memorizzarli in soluzione salina fino al momento dell&#39;uso (non più di 1 ora). </li><li> Otto settimane dopo il trapianto di cellule E14 (10 settimane lesioni post), reanesthetize i ratti che hanno subito un SIC, con o senza innesto di cellule e ratti di controllo ingenui. </li><li> Radere la zona addominale e arti posteriori. Pulire la pelle con Betadine. Posizionare il ratto sul tavolo operatorio in posizione supina. </li><li> Incidere la pelle sulla coscia addome e interno ventrale sul lato destro con una lama # 15. </li><li> Tagliare le stessuti connettivi ubcutaneous per esporre il fascio di vasi femorali ed il nervo utilizzando una piccola forbice. </li><li> Separare l&#39;arteria femorale dalla vena e il nervo utilizzando una pinza sottile con punte curve. </li><li> Mettere tre punti di sutura in seta sotto l&#39;arteria e fare un nodo allentato in ogni sutura. </li><li> Applicare 0,1 ml di lidocaina (2%) alla superficie dell&#39;arteria per suscitare vasodilatazione per la successiva cateterizzazione. </li><li> Fissare il vaso distale con un nodo di seta permanente e blocco temporaneo prossimalmente allungando una sutura di seta allentato. </li><li> Puntura dell&#39;arteria utilizzando un ago curvo calibro 20 e inserire la punta del catetere telemetro (lunga 8 cm) utilizzando uno strumento di inserimento del catetere. </li><li> Inserire il catetere rostralmente fino a 4 cm determinando così la punta in aorta toracica. </li><li> Ancorare il catetere all&#39;interno del vaso legando tre suture in seta intorno all&#39;arteria femorale. </li><li> Fare una tasca sottocutanea lungo il fianco tra il bordo caudale del torace e tche proroga la maggior parte craniale della gamma di ginocchio con un paio di forbici spuntate. </li><li> Inserire il corpo del trasmettitore nella tasca e sutura al tessuto connettivo che circonda il trasmettitore per evitare movimenti eccessivi. </li><li> Suturare la pelle con n ° 6 filo di seta. </li><li> Iniettare Ringer lattato (5 ml), buprenorfina (0,035 mg / kg), e ampicillina (33 mg / kg) per via sottocutanea immediatamente dopo l&#39;intervento chirurgico e mantenere i ratti in un incubatore caldo fino svegli. </li></ol> 3. basali di pressione arteriosa media (MAP) e frequenza cardiaca (HR) di registrazione <ol><li> Già il giorno successivo trasmettitore impianto, mettere un singolo animale sul pad ricevitore e accendere il trasmettitore. Attendere per circa 10 - 15 minuti per abituare l&#39;animale e stabilizzare i parametri cardiovascolari. </li><li> Record MAP riposo e HR, che derivano dalla pressione del polso arterioso con un sistema di acquisizione dati informatizzata per almeno 1 ora. Raccogliere i dati ogni 5 secondi. </li><li> Monitorare l&#39;unaimals rimuovono continuamente e punti dati durante il verificarsi di spasmi visibili. Per ogni animale, punti dati medi per ottenere valori medi. </li></ol> 4.-retto distensione indotta autonomica disreflessia <ol><li> Trattenere ratti NSC-innestate o di controllo SCI in un asciugamano fornito con pellet di cibo all&#39;interno del ricevitore trasmettitore. Di solito i ratti possono rimanere cooperativo durante la procedura. </li><li> Inserire un catetere a palloncino con punta in lattice nel retto per circa 2 cm e fissarlo alla coda con nastro 8. </li><li> Accendere il trasmettitore e attendere 10 - 15 minuti permette la pressione del sangue per tornare alla preinsertion basale. </li><li> Indurre distensione del colon-retto dal gonfiaggio del palloncino lentamente oltre 10 secondi con 1,4 ml di aria per 1 min, a generare una pressione di circa 30 mmHg. </li><li> Record MAP e HR 1 minuti prima, 1 min durante e 1 min dopo distensione del colon-retto; dati di esempio ogni 3 sec durante la procedura di 3 min. </li><li>Eseguire 2 - 3 studi clinici per animali con almeno 15 min intervallo di recupero tra le due prove. </li><li> Animali Overdose (ip) con doppia combinazione anestesia dosaggio sopra descritto, se non ulteriore valutazione. Profumato animali con soluzione fisiologica seguita da paraformaldeide al 4%. </li><li> Per ogni animale, la media dei valori prima e durante rispettivamente distensione del colon-retto; calcolare la differenza tra il basale e distensione indotta MAP e HR modifiche per ciascuna prova; media dei 2 - 3 prove per ottenere valori medi. </li></ol> 5. Trasmettitore Pulizia <ol><li> Rimuovere il trasmettitore dal corpo animale dopo l&#39;anestesia, ma prima di perfusione. Bagnare immediatamente in un becher riempito con acqua distillata fino pulizia; evitare l&#39;essiccamento del dispositivo telemetro. </li><li> Trasferire il telemetro al 1% soluzione detergente enzimatica Terg-A-Zyme (10 g acqua / L) per 24 ore a temperatura ambiente. </li><li> Regel la punta del catetere trasmettitore con un ottuso ago 30 gaugecollegato alla siringa regel. </li><li> Asciugare con cura il trasmettitore con un panno morbido piegato e riporlo nel cassetto di plastica originale. </li></ol>

<ol>
	<li>Krassioukov, A. V., Furlan, J. C., Fehlings, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomic+dysreflexia+in+acute+spinal+cord+injury:+an+under-recognized+clinical+entity.">Autonomic dysreflexia in acute spinal cord injury: an under-recognized clinical entity.</a> J Neurotrauma. 20, 707-716 (2003).</li><li>Lindan, R., Joiner, E., Freehafer, A. A., Hazel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+and+clinical+features+of+autonomic+dysreflexia+in+patients+with+spinal+cord+injury.">Incidence and clinical features of autonomic dysreflexia in patients with spinal cord injury.</a> Paraplegia. 18, 285-292 (1980).</li><li>Inskip, J. A., Ramer, L. M., Ramer, M. S., Krassioukov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomic+assessment+of+animals+with+spinal+cord+injury:+tools,+techniques+and+translation.">Autonomic assessment of animals with spinal cord injury: tools, techniques and translation.</a> Spinal Cord. 47, 2-35 (2009).</li><li>Mayorov, D. N., Adams, M. A., Krassioukov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telemetric+blood+pressure+monitoring+in+conscious+rats+before+and+after+compression+injury+of+spinal+cord.">Telemetric blood pressure monitoring in conscious rats before and after compression injury of spinal cord.</a> J Neurotrauma. 18, 727-736 (2001).</li><li>Hou, S., Tom, V. J., Graham, L., Lu, P., Blesch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+restoration+of+cardiovascular+function+by+embryonic+neural+stem+cell+grafts+after+complete+spinal+cord+transection.">Partial restoration of cardiovascular function by embryonic neural stem cell grafts after complete spinal cord transection.</a> J Neurosci. 33, 17138-17149 (2013).</li><li>Lu, P., 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=Long-distance+growth+and+connectivity+of+neural+stem+cells+after+severe+spinal+cord+injury.">Long-distance growth and connectivity of neural stem cells after severe spinal cord injury.</a> Cell. 150, 1264-1273 (2012).</li><li>Akhavan, M., Hoang, T. X., Havton, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+detection+of+fluorogold-labeled+neurons+in+long-term+studies.">Improved detection of fluorogold-labeled neurons in long-term studies.</a> J Neurosci Methods. 152, 156-162 (2006).</li><li>Maiorov, D. N., Fehlings, M. G., Krassioukov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+severity+of+spinal+cord+injury+and+abnormalities+in+neurogenic+cardiovascular+control+in+conscious+rats.">Relationship between severity of spinal cord injury and abnormalities in neurogenic cardiovascular control in conscious rats.</a> J Neurotrauma. 15, 365-374 (1998).</li><li>Laird, A. S., Carrive, P., Waite, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+and+temperature+changes+in+spinal+cord+injured+rats+at+rest+and+during+autonomic+dysreflexia.">Cardiovascular and temperature changes in spinal cord injured rats at rest and during autonomic dysreflexia.</a> J Physiol. 577, 539-548 (2006).</li><li>Phillips, A. A., Krassioukov, A. V., Ainslie, P. N., Warburton, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baroreflex+function+after+spinal+cord+injury.">Baroreflex function after spinal cord injury.</a> J Neurotrauma. 29, 2431-2445 (2012).</li><li>Osborn, J. W., Taylor, R. F., Schramm, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+arterial+pressure+after+chronic+spinal+transection+in+rats.">Determinants of arterial pressure after chronic spinal transection in rats.</a> Am J Physiol. 256, 666-673 (1989).</li><li>Rabchevsky, A. G., 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=Effects+of+gabapentin+on+muscle+spasticity+and+both+induced+as+well+as+spontaneous+autonomic+dysreflexia+after+complete+spinal+cord+injury.">Effects of gabapentin on muscle spasticity and both induced as well as spontaneous autonomic dysreflexia after complete spinal cord injury.</a> Front Physiol. 3, 329 (2012).</li><li>Hou, S., Lu, P., Blesch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+supraspinal+vasomotor+pathways+and+autonomic+dysreflexia+after+spinal+cord+injury+in+F344+rats.">Characterization of supraspinal vasomotor pathways and autonomic dysreflexia after spinal cord injury in F344 rats.</a> Auton Neurosci. 176, 54-63 (2013).</li></ol>

Gli autori non hanno nulla da rivelare.

Tradizionalmente, un fluido riempito cannula viene inserito nell&#39;arteria e collegato ad un trasduttore di pressione per registrare parametri cardiovascolari come un&#39;istantanea terminale di ciascun animale 11. Per monitorare continuamente le prestazioni cardiovascolare per lungo tempo, sistemi radio-telemetrica sono impiegati in molti laboratori. Questo strumento più raffinato in grado di registrare la pressione arteriosa in coscienza, muoversi liberamente gli animali. Rispetto al fluido riempito cate...

La disfunzione cardiovascolare si verifica dopo la lesione del midollo spinale (SCI) ad alti livelli. Essa si manifesta in disordinata pressione sanguigna e la frequenza cardiaca a riposo, ipotensione ortostatica, ipotensione indotta da esercizio fisico, e disreflessia autonomo caratterizzato da episodi di ipertensione e bradicardia baroriflesso-mediata in risposta a stimoli sensoriali al di sotto del livello di pregiudizio 1,2. Questi sintomi interferiscono con la vita quotidiana dei pazienti del midollo spinale danneggiato. Pertanto, è importante stabilire strumenti efficaci per lo studio dei cambiamenti cardiovascolari negli animali con SCI e trattamenti sperimentali. Per studiare la funzione cardiovascolare negli animali, sono state utilizzate diverse tecniche per monitorare la pressione sanguigna e la frequenza cardiaca. Parametri cardiovascolari centrali possono essere registrati mediante inserimento cannula e telemetria, mentre non invasive coda polsini possono essere utilizzati per misurare la pressione del sangue periferico 3. Rispetto ad altri metodi, telemetria ha il vantaggio principale che permette la registrazione continua in animali liberi di muoversi e di monitoraggio a lungo termine della funzione cardiovascolare 4. In modelli animali di SCI, i cambiamenti della pressione del sangue periferico dopo stimolazione sperimentale non possono essere abbastanza grandi per essere rilevato. Di conseguenza, adatto tecnica di monitoraggio cardiaco dovrebbe essere selezionato per animali con SCI. Nel presente studio, un sistema radio-telemetrico è stato introdotto per monitorare la funzione cardiovascolare in ratti adulti dopo resezione completa del midollo spinale. I ratti hanno ricevuto innesti di ratto singenico giorno embrionale 14 (E14), le cellule staminali neurali del tronco encefalico-derivato (BS-NSC) nel sito della lesione. I ratti con lesioni e senza trapianto e ingenui, ratti infortunati serviti come controlli. La procedura di telemetria comprende trasmettitore sterilizzazione e impianto (Figura 1), la registrazione dei parametri cardiovascolari basali, risposte distensione indotta colorettali e pulizia trasmettitore estoccaggio.

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	<tbody>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:315px;">Table of Specific Materials/Equipment:</td>
			<td style="width:103px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:120px;">Reagents</td>
			<td rowspan="2" style="width:195px;">Company</td>
			<td rowspan="2" style="width:103px;">Catalogue</td>
			<td rowspan="2" style="width:164px;">Comment</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="62">
			<td height="83" rowspan="2" style="height:83px;width:120px;">Fibrinogen (rat)</td>
			<td rowspan="2" style="width:195px;">Sigma</td>
			<td rowspan="2" style="width:103px;">F6755-25MG</td>
			<td rowspan="2" style="width:164px;">2 hr at 37oC to dissovle</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="59">
			<td height="80" rowspan="2" style="height:80px;width:120px;">Thrombin (rat)</td>
			<td rowspan="2" style="width:195px;">Sigma</td>
			<td rowspan="2" style="width:103px;">T5772-100UN</td>
			<td rowspan="2" style="width:164px;">Dissovle in 10 mM CaCl2</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">1% Terg-A-Zyme</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:103px;">Z273287</td>
			<td style="width:164px;">Enzymatic solution for telemeter cleaning</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">Fluorogold</td>
			<td style="width:195px;">Fluorochrome</td>
			<td style="width:103px;"></td>
			<td style="width:164px;">Dissovle in distilled water and avoid light</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">Telemeter&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (PA-C40)</td>
			<td style="width:195px;">Data Sciences International</td>
			<td style="width:103px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:120px;">Telementric recording and analysis system</td>
			<td style="width:195px;">Data Sciences International</td>
			<td style="width:103px;"></td>
			<td style="width:164px;">Signal stimulator, Data Exchange Matrix, receivers, Ambient pressure reference monitor</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:120px;">Balloon-tipped catheter</td>
			<td style="width:195px;">Edward Lifesciences</td>
			<td style="width:103px;">111F7-P</td>
			<td style="width:164px;">For colorectal distension</td>
		</tr>
	</tbody>
</table>

a radio-telemetric system to monitor cardiovascular function in rats with spinal cord transection and embryonic neural stem cell grafts

Lesione alta del midollo spinale toracico o cervicale (SCI) può portare a disfunzioni cardiovascolari. Per monitorare i parametri cardiovascolari, abbiamo impiantato un catetere collegato ad un trasmettitore radio in arteria femorale di ratti che hanno subito una resezione del midollo spinale T4 con o senza innesto di cellule staminali neurali del tronco encefalico-embrionali derivate che esprimono la proteina fluorescente verde. Rispetto ad altri metodi come l&#39;inserimento cannula o la coda-bracciale, telemetria è vantaggioso per monitorare costantemente la pressione arteriosa e la frequenza cardiaca in animali liberi di muoversi. E &#39;anche in grado di lungo termine acquisizioni multiple di dati. Nei ratti midollo spinale feriti, sono stati registrati con successo dati cardiovascolari basali sotto condizione sfrenato e disreflessia autonomica in risposta alla distensione del colon-retto. Inoltre, i parametri cardiovascolari prima e dopo SCI possono essere confrontati nello stesso ratto se un trasmettitore viene impiantato prima una transezione del midollo spinale. Una limitazione del Telemet descrittoprocedura ry è che l&#39;impianto nell&#39;arteria femorale può influenzare l&#39;apporto di sangue al arti posteriori omolaterale.

Utilizzando la tecnica di telemetria sopra descritto, abbiamo registrato con successo parametri cardiovascolari in midollo spinale degli animali feriti. Negli animali con il solo SCI, MAP è stata significativamente ridotta, mentre HR aumentato rispetto agli animali naïve, in linea con le precedenti relazioni 9. Negli animali con BS-NSC innesto, MAP e HR avvicinato livelli misurati negli animali naïve (Figura 2). Durante la distensione del colon-retto, un topo era considerato dysreflexic se...

Watch this Scientific Journal Video about A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts at JoVE.com

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

Vi presentiamo un protocollo per l&#39;utilizzo di un sistema radio-telemetrico per registrare i parametri cardiovascolari in T4 midollo spinale ratti transected otto settimane successive tronco cerebrale embrionale delle cellule staminali neurali innesto nel sito della lesione. Telemetria è una tecnica avanzata per valutare con precisione la funzione cardiovascolare in coscienti liberi di muoversi midollo spinale topi feriti.

Un sistema radio-telemetria per monitorare la funzionalità cardiovascolare in ratti con midollo spinale e Transection embrionali staminali neurali Innesti cellulari

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

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10.4K Views.  Drexel University College of Medicine.  Vi presentiamo un protocollo per l'utilizzo di un sistema radio-telemetrico per registrare i parametri cardiovascolari in T4 midollo spinale ratti transected otto settimane successive tronco cerebrale embrionale delle cellule staminali neurali innesto nel sito della lesione. Telemetria è una tecnica avanzata per valutare con precisione la funzione cardiovascolare in coscienti liberi di muoversi midollo spinale topi feriti. 

Video: A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

Assessing Endothelial Vasodilator Function with the Endo-PAT 2000

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of the panoply of paracrine factors that it secretes, the endothelium regulates the contractile and proliferative state of the underlying vascular smooth muscle, as well as the interaction of the vessel wall with circulating blood elements. Because of its central role in mediating vessel tone and growth, its position as gateway to circulating immune cells, and its local regulation of hemostasis and coagulation, the the properly functioning endothelium is the key to cardiovascular health. Conversely, the earliest disorder in most vascular diseases is endothelial dysfunction.
In the arterial circulation, the healthy endothelium generally exerts a vasodilator influence on the vascular smooth muscle. There are a number of methods to assess endothelial vasodilator function. The Endo-PAT 2000 is a new device that is used to assess endothelial vasodilator function in a rapid and non-invasive fashion. Unlike the commonly used technique of duplex ultra-sonography to assess flow-mediated vasodilation, it is totally non-operator-dependent, and the equipment is an order of magnitude less expensive. The device records endothelium-mediated changes in the digital pulse waveform known as the PAT ( peripheral Arterial Tone) signal, measured with a pair of novel modified plethysmographic probes situated on the finger index of each hand. Endothelium-mediated changes in the PAT signal are elicited by creating a downstream hyperemic response. Hyperemia is induced by occluding blood flow through the brachial artery for 5 minutes using an inflatable cuff on one hand. The response to reactive hyperemia is calculated automatically by the system. A PAT ratio is created using the post and pre occlusion values. These values are normalized to measurements from the contra-lateral arm, which serves as control for non-endothelial dependent systemic effects. Most notably, this normalization controls for fluctuations in sympathetic nerve outflow that may induce changes in peripheral arterial tone that are superimposed on the hyperemic response.
In this video we demonstrate how to use the Endo-PAT 2000 to perform a clinically relevant assessment of endothelial vasodilator function.

A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000.

Valutazione della funzione endoteliale vasodilatatore con l&#39;Endo-PAT 2000

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of ...

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Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Acuta e Cronica test sensoriale tattile dopo lesione del midollo spinale in ratti

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

Un modello Contusione di gravi lesioni del midollo spinale nei ratti

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

Utilizzo sinergico delle cellule neurali e precursori peptidi autoassemblanti in Sperimentale Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Neural Stem Cell Transplantation in Sperimentale Contusivi Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Photothrombosis-indotta ischemia focale come un modello di lesioni del midollo spinale nei topi

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Un neonatale mouse Spinal Cord Injury compressione Modello

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Vena interposizione Modello: un modello adatto per studiare Bypass Graft pervietà

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

Studiando il segnale ipotalamico insulina ad intolleranza al glucosio periferico con un sistema di infusione del farmaco continuo nel cervello del Mouse

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

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

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

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

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

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