Vorremmo riconoscere il sostegno finanziario per questo lavoro dal Canadian Institutes of Health Research (CIHR), Family Foundation Krembil, il Halbert cattedra di riparazione Neural and Regeneration, Phillip e Peggy DeZwirek, e Gordon Yao per il contributo alla Figura 2 . Klaus Zweckberger è stato finanziato da una sovvenzione della &quot;Deutsche Forschungsgesellschaft&quot; (DFG).

NOTA: La seguente protocollo sperimentale è stato approvato dal comitato cura degli animali della University Health Network (Toronto, Canada), ed è in conformità con le politiche stabilite nella guida per la cura e l&#39;uso di animali da esperimento preparata dal Consiglio canadese della cura degli animali . 1. cervicale Contusione Aneurysm Clip / compressione Modello <ol><li> Prima di strumenti autoclave chirurgia e mantenere condizioni di sterilità durante tutta la procedura surgcial mettendo gli strumenti in un bagno di alcool al 70%. </li><li> Anestetizzare ratti Wistar (250-270 g) con una combinazione di ossigeno (O 2), protossido di azoto (N 2 O) (1: 1), e 1,8-2,2% isoflurano e sostenere la respirazione spontanea mediante una maschera di anestesia gas. Per l&#39;induzione dell&#39;anestesia iniziare con 5% isoflurano per 1 minuto e ridurre in seguito. Prima di iniziare l&#39;intervento chirurgico, il controllo di profondità dell&#39;anestesia, dando uno stimolo doloroso (ad es. Le zampe). Applicare pomate grassenegli occhi per evitare secchezza e per prevenire le infezioni successive. </li><li> Mettere i ratti su un cuscino di riscaldamento (37 ° C) e fissare la testa in un telaio stereotassico. </li><li> Radere la regione chirurgica attorno alla colonna vertebrale cervicale e disinfettare con povidone iodio e il 70% di alcol. </li><li> Eseguire un&#39;incisione mediana sopra la spina dorsale della vertebra cervicale (C2), raggiungendo al processus spicco della toracico vertebrale corpo 2 (T2). </li><li> Tagliare lo strato esterno dei muscoli vertebrali direttamente sulla linea mediana (per evitare sanguinamenti) in direzione cranio-caudale e ulteriormente sezionare gli strati muscolari più profondi senza mezzi termini, fino a raggiungere il spinosus processus e le lamine. Inserisci divaricatori. </li><li> Orientare il processus spinosus spicco del corpo vertebra T2 ad osservare i livelli previsti per laminectomia. Dopo l&#39;identificazione e la micro-chirurgica la preparazione delle lamine selezionato, tagliare il flavae ligamenti per allentare le lamine e la spinosus processus. Infine, cut attraverso le lamine laterali con un Clipper ossea al midollo spinale e rimuovere quelli delicatamente, evitando la compressione del midollo spinale stesso. NOTA: I più comuni sono i livelli C5 / 6, C6 / 7, o C7 / T1. Il tasso di mortalità perioperatoria aumenta il più rostrale il livello di pregiudizio. Sanguinamento dal seno venoso paravertebrale è comune e può essere affrontato da compressione attento con la spugna. </li><li> Prima di inserire il clip per traumatizzare il cavo, identificare emergenti radici nervose per risparmiare loro di clipping (soprattutto a livello C5 / 6). </li><li> Per garantire la clip un&#39;induzione, allentare la dura ventrale dal lato dorsale dei corpi vertebrali con un gancio e preparare un corridoio per la clip. </li><li> Infine, inserire la clip aperta e farlo scattare chiuso (chiusura rapida) per ottenere un infortunio contusione. Clip forza di chiusura e la durata della chiusura a clip determinano l&#39;intensità del trauma e l&#39;entità della compressione. Comunemente utilizzati sono forze di clip di tra 15-35 g ed un clipping durata es 1 min. (Figura 1). </li><li> Dopo la rimozione della clip, adattare muscoli in 2 strati e chiudere la ferita. </li><li> Smettere di anestesia e lasciare che il risveglio degli animali sotto la vostra osservazione continua fino a quando non riprende conoscenza sufficiente decubito sternale. Infine, mettere il topo in una singola gabbia e seguire le linee guida di trattamento post-operatorio. </li><li> Poiché gli animali lottano con la gravità di questo tipo di lesioni, è necessario prestare particolare attenzione ai trattamenti post-operatori: <ol><li> Somministrare antidolorifici (buprenorfina e Meloxicam per 3 giorni e 5 giorni, rispettivamente e in base ai sintomi clinici). </li><li> Dare ulteriore sottocutanea soluzione salina per 3 giorni (2 volte al giorno, 5-10 millilitri (ml)). </li><li> Fornire antibiotici nell&#39;acqua potabile 2 giorni prima e entro 7 giorni dopo l&#39;intervento chirurgico (es moxifloxacina) </li><li> Premere la vescica urinaria 2-3 volte al giorno fino al recupero della funzione della vescica è costantely apparente. </li><li> Osservare deficit neurologici e condizioni fisiologiche degli animali operati, almeno una volta al giorno. </li></ol></li></ol> 2. Iniezione SAP e NPC (14 giorni dopo la lesione) <ol><li> Indurre l&#39;anestesia come descritto in 1.1. a 1,3, fissare la testa del topo in un telaio stereotassico, togliere i punti o clip ferita, e disinfettare la ferita e la zona chirurgica con povidone iodio e 70% di alcol. </li><li> Sezionare con cura i muscoli paravertebrali, inserire divaricatori, rimuovere il tessuto cicatriziale microscopicamente dalla dura, e ri-esporre il sito della lesione. </li><li> Preparare PAS in una concentrazione di 1% (w / v) effettuare un gel matrice extracellulare. SAP QL6 hanno un pH fisiologicamente compatibili e non devono essere tamponato prima dell&#39;iniezione. Per la visualizzazione dei SAP nel midollo spinale, usare un derivato fluorescente QL6 (QL6-FITC). </li><li> Iniettare SAP (5 microlitri (microlitri) al centro della lesione, distribuito in 2 porzioni, ciascuna2.5 bilaterali microlitri della linea mediana. Utilizzare una siringa Hamilton collegata al telaio stereotassico con un bicchiere micro capillare (100 micrometri (micron) di diametro esterno (OD)). Aprire la dura accuratamente con la punta di un ago appuntito, e inserire il capillare di vetro stereotactically 2 millimetri (mm) nel midollo spinale traumatizzato. </li><li> Dopo l&#39;iniezione 1/3 del volume, rimuovere l&#39;ago alla profondità 1,5 mm e dopo altri 1/3 a 1 mm. Dopo l&#39;iniezione dell&#39;intero volume, e prima della rimozione della siringa, attendere 5 minuti per stabilizzare la formazione di gel. </li><li> Per generare NPC, utilizzare topi adulti DsRed (o YFP topi positivi, verdi) e isolare e coltivare dalla zona paraventrical 4,18,21. </li><li> Valutare la vitalità di NPC di Trypan Blue colorazione che indicano la presenza di cellule vive ~ 90% nella sospensione cellulare. Diluire le cellule nel terreno di crescita (50 x 103 cellule vive / ml) e poi li usano per il trapianto di cellule. </li><li> Fare quattro 2 ml (8 microlitri vo totalelume, contenente 4 x 10 5 NPC) iniezioni intraspinali bilaterale a 2 mm rostrale e caudale del luogo di ferita. Dopo l&#39;apertura della dura, inserire il vetro micro capillare Hamilton 1,5 millimetri sotto la superficie dorsale del midollo spinale e iniettare 2 ml di sospensione cellulare. Scegliere una velocità di iniezione a circa 0,5 ml / min (min). </li><li> Alla fine di ogni iniezione e prima della rimozione del capillare fuori del cavo, attendere almeno 1 min permettendo tessuto estende per accogliere il nuovo volume cellulare. (Figura 2) </li></ol> 3. L&#39;impianto di subdurale pompe per la crescita Application Factor <ol><li> Al fine di arricchire il liquido cerebro-spinale (CSF) con fattori di crescita sostenendo la sopravvivenza cellulare, utilizzare pompe micro-osmotica diluitivi fattori di crescita sub-durally tutto 7-14 giorni, con un tasso di diluizione di 0,5 microlitri / hr, un diametro del catetere di 0,04 OD cm, e un volume serbatoio di 100 microlitri. </li><li> Scegli fattori di crescita preferiti(Ad esempio, il cervello derivato fattore di crescita (BDGF), fattore di crescita epidermico (EGF), fattore di crescita dei fibroblasti (FGF)), riempire le pompe 6-10 ore prima dell&#39;impianto, e equilibrare le pompe in un bagno d&#39;acqua a 37 ° C. </li><li> Subito dopo iniezioni NPC, preparare un incavo sottocutaneo per posizionare la pompa. Località preferiti sono i fianchi laterali della regione addominale thoraco- evitando grande fastidio locale causati dalla pompa stessa. </li><li> Per ottenere la più alta concentrazione di fattori di crescita, assicurarsi che la punta aperta del catetere finisce vicino al luogo della lesione. Pertanto, eseguire un salto-laminectomia del livello superiore o inferiore adiacente. Ad esempio, se il SCI è a C7 / T1, eseguire una piccola laminectomia C5. </li><li> Mettere la pompa nella cavità sottocutaneo, abbreviare il catetere alla lunghezza necessaria, e fissarlo con diversi punti di sutura (6.0) sui muscoli paravertebrali evitando qualsiasi movimento lussazione associata. </li><li> Dopo l&#39;apertura della dura (per esempio, in C5) con la punta affilatadi un ago, introdurre il catetere nello spazio subdurale e farlo scivolare in una direzione caudale senza alcuna resistenza e senza danneggiare il cavo. La lamina saltato di esempio C6 serve come punto supplementare di fissazione e la stabilizzazione del catetere. </li><li> Assicurarsi che il catetere senza intoppi e non si piega. </li><li> Chiudi muscoli di strato, e la pelle, con suture o clip. (Figura 3) </li><li> Smettere di anestesia e lasciare che il risveglio degli animali sotto la vostra osservazione continua fino a quando non riprende conoscenza sufficiente decubito sternale. Infine, riporli in una singola gabbia e seguire le linee guida di trattamento post-operatorio. </li><li> Fornire un trattamento speciale post-operatorio, anche se, gli animali potrebbero aver recuperato in parte in questo momento-point: <ol><li> Somministrare antidolorifici (Buprenorthine e Meloxicam per 3 giorni e 5 giorni, rispettivamente e in base ai sintomi clinici). </li><li> Fornire antibiotici in bottiglie d&#39;acqua fino a 2 giorni prima7 giorni dopo l&#39;intervento chirurgico (es moxifloxacina). </li><li> Continuare comprimendo la vescica urinaria, se ancora necessario. </li><li> Dare fluidi sottocutanei aggiuntive, se i topi appaiono disidratati. </li><li> Mantenere osservando la funzione neurologica e condizioni fisiologiche degli animali operati almeno una volta al giorno. </li><li> Somministrare trattamento immunosoppressione 2 giorni prima dell&#39;iniezione NPC e entro 7 giorni dopo transplanation (minociclina) e fino al sacrificio (Sandimmune), rispettivamente. </li></ol></li></ol> 4. Tissue Assessment <ol><li> Sacrifica animali alla fine del periodo di osservazione (ad es. 4 settimane dopo SCI) in anestesia profonda (5% isoflurano per 2-3 min) e li transcardially profumato con 50 ml freddo (4 ° C) seguito da soluzione fisiologica 150 ml freddo 4 % paraformaldeide in 0,1 M tampone fosfato salino (PBS). </li><li> Rimuovere il midollo spinale e metterlo in 4% paraformaldeide in 0,1 M tampone fosfato salino (PBS) per 24 ore. </li><li&gt; Mettere midollo spinale in soluzione di saccarosio al 25% per crioprotezione per 48 ore. </li><li> Fornire criosezioni longitudinali con spessore &lt;30 micron. </li><li> Per la colorazione immunoistochimica di tutti i cellulari-nuclei fornire un uso di sfondo DAPI (1: 1.000). DsRed NPC positivi appaiono di colore rosso, QL-6 FITC appare verde, e sia quindi non ha bisogno di essere tinto appositamente. (Figura 4). </li><li> Per la microscopia elettronica a scansione (SEM) lasciano i campioni in ammollo in glutaraldeide a 4 ° C per 2 ore, disidratano lentamente in 10 passi% di incremento di etanolo per 5 minuti e metterli in un liquido in pressione di CO 2 sifone per 1 ora. Ponteggi cappotto con oro utilizzando una verniciatura polverizzazione. Prendete le immagini con un microscopio elettronico a scansione Hitachi S-3400N. (Figura 5) </li></ol>

<ol>
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Covidien supplied the QL6 SAP for this study.

Questo protocollo è stato sviluppato per consentire al lettore di eseguire un modello di lesione cervicale nei ratti e di utilizzare un approccio di trattamento combinato con SAP e NPC favore di un migliore recupero dopo cervicale SCI. Soprattutto in confronto ad altri modelli traumi cervicali, come il (emi) -transection modello o modelli di peso goccia e concussione, il modello / compressione clip di contusione rappresenta sia principali meccanismi fisiopatologici traumi - contusione e per...

Più del 50% delle lesioni del midollo spinale sono legati alla colonna cervicale. Nella clinica impostazione due principali meccanismi patofisiologici sono descritti: il primo contusione del midollo spinale e successivamente, la compressione continua causata da fratture ossee, emorragie o gonfiore tessuto. Le clip da aneurisma contusione modello / compressione imita entrambi i meccanismi fisiopatologici: scattare la clip produce una contusione e la durata del ritaglio rappresenta la componente di compressione, ammettendo che la compressione in ambito clinico causate da fratture ossee, emorragie o gonfiore dei tessuti ultima più significativa più a lungo. La clip da aneurisma utilizzato viene modificato da una molla ad anello che garantisce esatte e riproducibili forza clipping. Soprattutto in confronto al emi-resezione o il modello contusione, questa clip aneurisma modello imita migliori ambienti clinici. Mentre i pazienti con lesioni toraciche soffrono di paraplegia, la maggior parte dei pazienti con inj cervicaleuries sono tetraplegico e completamente dipendente. La struttura anatomica del midollo cervicale, tuttavia, mostra differenze significative rispetto alla toracica o lombare della colonna vertebrale, e quindi, è rivolto in particolare in questo protocollo. Lo sviluppo di cavità intramidollari e cicatrici di tessuto sono ostacoli per il recupero e la rigenerazione. Per superare questi ostacoli l&#39;uso di materiale scaffold è un approccio promettente. Peptidi autoassemblanti possono essere iniettati direttamente nel epicentro della lesione. Ci si riuniscono in scaffolds nano-fibre ponte della cavità e migliorare l&#39;ambiente inibitorio, riducendo l&#39;infiammazione e spaventare i tessuti. Mentre i materiali rigidi provocano danni considerevoli del midollo spinale durante l&#39;impianto, i peptidi fluidi possono essere iniettati in modo sicuro e senza gravi danni aggiuntivi. Migliorare l&#39;ambiente inibitorio con peptidi autoassemblanti prima del trapianto di cellule staminali, quindi, sostenere cella integrazione, differenziazione e, infine, il recupero funzionale, dopo una lesione del midollo spinale cervicale.

<table border="0" cellpadding="0" cellspacing="0" width="541">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Name</td>
			<td style="width:268px;">Company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aneurysmal clip</td>
			<td>SharpTech</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical microscope</td>
			<td>Leica</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro injection system</td>
			<td>World Precision Instruments, Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small animal stereotaxic instrument</td>
			<td>David Kopf Instruments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hamilton syringe</td>
			<td>Hamilton company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Subdural pumps</td>
			<td>Alzet osmotic micro pump 1007D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical instrument</td>
			<td>Fine Science tools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane USP</td>
			<td>Pharmaceutical Partners of Canada Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.9% Sodium Chloride injection USP</td>
			<td>Baxter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7.5% Povidone iodine</td>
			<td>Purdue Pharma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Isopropyl alcohol USP</td>
			<td>GreenField Ethanol Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QL6 SAP</td>
			<td>Covidien</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.4% Trypan blue</td>
			<td>Gibco</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platelet-Derived Growth Factor (PDGF)</td>
			<td>Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epidermal Growth Factor (EGF)</td>
			<td>Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibroblast Growth Factor (FGF)</td>
			<td>Sigma</td>
		</tr>
	</tbody>
</table>

synergetic use of neural precursor cells and self-assembling peptides in experimental cervical spinal cord injury

Lesioni del midollo spinale (SCI) causano gravi danni neurologici e conseguenze psicologiche, economiche e sociali per i pazienti e le loro famiglie. Clinicamente, più del 50% del SCI influisce sulla colonna cervicale 1. Come conseguenza della lesione primaria, una cascata di meccanismi secondari tra cui l&#39;infiammazione, apoptosi, e demielinizzazione si verificano infine porta alla cicatrizzazione dei tessuti e sviluppo di cavità intramidollari 2,3. Entrambi rappresentano barriere fisiche e chimiche per il trapianto di cellule, l&#39;integrazione, e la rigenerazione. Pertanto, plasmare l&#39;ambiente inibitorio e bridging cavità per creare un ambiente favorevole per il trapianto di cellule e la rigenerazione è un promettente target terapeutico 4. Qui, un modello contusione / compressione cervicale SCI utilizzando una clip da aneurisma è descritto. Questo modello è più clinicamente rilevante rispetto ad altri modelli sperimentali, poiché transezione completa o rotture del cavo sono rare. Anche in CONFRONTOn al modello carico di caduta, quali in particolare i danni colonne dorso, la compressione circonferenziale del midollo spinale appare vantaggiosa. Clip forza di chiusura e la durata possono essere regolati per ottenere diverse gravità infortuni. Una molla anello facilita calibrazione precisa e costanza della clip di forza. In condizioni fisiologiche, peptidi autoassemblanti sintetici (SAP) di auto-assemblarsi in nanofibre e, quindi, sono attraenti per l&#39;applicazione in SCI 5. Possono essere iniettato direttamente nella lesione minimizzare danneggiamento del cavo. SAP sono strutture biocompatibili erigere ponteggi per colmare le cavità intramidollari e quindi, dotare il cavo danneggiato per i trattamenti rigenerativi. K2 (QL) 6K2 (QL6) è un romanzo SAP introdotto da Dong et a l. 6 In confronto ad altri peptidi, QL6 auto-assembla in β-sheets a pH neutro 6 0,14 giorni dopo SCI, dopo la fase acuta, SAP vengono iniettati nel centro della lesione e cellule precursori neurali (NPC) sono InjeCTED in colonne dorsali adiacenti. Al fine di sostenere la sopravvivenza delle cellule, il trapianto è combinato con la somministrazione continua subdurale di fattori di crescita da parte di micro pompe osmotiche per 7 giorni.

<html> Quando si esegue il procedimento sopra descritto, si otterrà un ponteggio SAP colmare la cavità e offrendo un miglioramento dell&#39;ambiente inibitorio, meno spaventare tessuti e l&#39;aumento di sopravvivenza NPC. Figura 4 mostra una sezione longitudinale di un ratto midollo spinale ottenuto al luogo di ferita sei settimane dopo SCI e 4 settimane dopo QL6 SAP iniezione e NPC trapianto. Peptidi QL6 sono state iniettate con successo nel cavo, aggregato nell&#39;epicentro e diffusi rostro-caudale...

Watch this Scientific Journal Video about Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury at JoVE.com

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

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. ...

synergetic-use-neural-precursor-cells-self-assembling-peptides

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Medicine

9.1K Views.  University Health Network, Krembil Neuroscience Centre. 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. 

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

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

Intraspinale trapianto di cellule cervicali targeting per corno ventrale nella Sclerosi Laterale Amiotrofica e le lesioni traumatiche del midollo spinale

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) ...

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Medicina

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 Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

Un modello contusivo unilaterale di lesioni del midollo spinale cervicale Uso della Impactor Infinite Horizon

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Controlled Cervical Laceration Injury in Mice

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

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

Controllata cervicale lesioni lacerazione nei topi

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

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination is disrupted by spinal cord injury (SCI), control of respiratory muscles innervated below the injury level is compromised1,2 leading to respiratory muscle dysfunction and pulmonary complications. These conditions are among the leading causes of death in patients with SCI3. Standard pulmonary function tests that assess respiratory motor function include spirometrical and maximum airway pressure outcomes: Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), Maximal Inspiratory Pressure (PImax) and Maximal Expiratory Pressure (PEmax)4,5. These values provide indirect measurements of respiratory muscle performance6. In clinical practice and research, a surface electromyography (sEMG) recorded from respiratory muscles can be used to assess respiratory motor function and help to diagnose neuromuscular pathology. However, variability in the sEMG amplitude inhibits efforts to develop objective and direct measures of respiratory motor function6. Based on a multi-muscle sEMG approach to characterize motor control of limb muscles7, known as the voluntary response index (VRI)8, we developed an analytical tool to characterize respiratory motor control directly from sEMG data recorded from multiple respiratory muscles during the voluntary respiratory tasks. We have termed this the Respiratory Motor Control Assessment (RMCA)9. This vector analysis method quantifies the amount and distribution of activity across muscles and presents it in the form of an index that relates the degree to which sEMG output within a test-subject resembles that from a group of healthy (non-injured) controls. The resulting index value has been shown to have high face validity, sensitivity and specificity9-11. We showed previously9 that the RMCA outcomes significantly correlate with levels of SCI and pulmonary function measures. We are presenting here the method to quantitatively compare post-spinal cord injury respiratory multi-muscle activation patterns to those of healthy individuals.

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment RMCA in Individuals with Chronic Spinal Cord Injury

The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis.

Valutazione delle vie respiratorie di attivazione muscolare Utilizzando respiratoria Motor Control Assessment (RMCA) in soggetti con lesioni spinali croniche

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination ...

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Respiratory failure is the leading cause of death following a cervical spinal cord injury. Having a reproducible, quantifiable, and reliable pre-clinical animal model of respiratory failure induced by a partial cervical injury will help to understand the subsequent respiratory and non-respiratory neuroplasticity and allow testing putative repair strategies.

Un modello murino di cervicale lesioni del midollo spinale per studiare post-lesionale respiratoria neuroplasticità

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Controllata corticale modello Impact per trauma cranico

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

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

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

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 ...

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 ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

Calibrato forcipe Modello di Spinal Cord Injury Compression

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...