Questo lavoro è stato sostenuto dal National Institutes of Health [di Grant no. R01NS069726] e l&#39;American Heart Association Grant nel concedere aiuti [Concessione n. 13GRNT17020004] per SD.

Nota: I topi (C57BL / 6J, maschio) di età compresa 10 - 12 settimane sono state utilizzate in questo studio. Tutte le procedure sono state eseguite in conformità con la guida NIH per la cura e l&#39;uso di animali da laboratorio e sono stati approvati dalla University of Missouri Istituzionale Animal Care and Use Committee (IACUC). 1. Pre-Surgery <ol><li> Il giorno prima dell&#39;intervento autoclave e sterilizzare tutti gli strumenti chirurgici. Avvolgere gli strumenti e autoclave a 121 ° C a 15 psi per 30 minuti seguiti da 30 minuti di essiccazione (121 o C, 15 psi, 30/30 ciclo). Mettere gli strumenti in un ambiente pulito e sterile fino a nuovo uso. </li><li> Preparare soluzione fresca Rosa Bengala (RB) (20 mg / ml in soluzione fisiologica sterile) ogni volta prima della chirurgia. Per sciogliere completamente RB, vortex il tubo poi seguita da ultrasuoni per 5 min a 50/60 Hz con potenza di uscita di 19 W. Avvolgere il tubo in un foglio di alluminio e proteggerlo dalla luce fino a nuovo utilizzata durante surgery. </li><li> Preparare una miscela di ketamina / xylazina in soluzione fisiologica sterile. Aggiungere 125 ml di xylazina (concentrazione magazzino: 20 mg / ml) e 325 ml di ketmaine (concentrazione magazzino: 100 mg / ml) e 550 ml di soluzione fisiologica sterile per fare un volume finale di 1 ml di miscela anestetica. </li><li> Pre-riscaldare il rilievo di riscaldamento omeotermi. </li><li> Pre-riscaldare la lampada ad alogenuri metallici (sorgente luminosa per microscopio FN1 epi-fluorescenza) per 30 minuti per stabilizzare la potenza della lampada. </li><li> Regolare la dimensione della regione illuminata ad un diametro di 1 mm con obiettivo 10X e un reticolo regolando il diaframma di campo nel FN1 verticale microscopio a epifluorescenza. </li></ol> 2. Procedura chirurgica <ol><li> Anestetizzare il mouse con una dose di ketamina (130 mg / kg B. wt.) E xilazina (10 mg / kg B. wt.). Sulla base del cocktail in 1.3, 4 ml per g di topo B. wt. sono necessari. Sterilizzare il sito di iniezione con un batuffolo imbevuto di alcool e somministrare anestetici attraverso intra-peritoneal (IP) del percorso. Fare attenzione a non iniettare anestetici nei vasi sanguigni o muscolare come questo potrebbe ritardare l&#39;induzione e il recupero dell&#39;animale. </li><li> Applicare lacrima artificiale unguento per entrambi gli occhi del mouse per prevenire l&#39;essiccazione e posizionare l&#39;animale sulla piastra elettrica per prevenire l&#39;ipotermia. </li><li> Preparazione di animali <ol><li> Controllare l&#39;animale per il corretto livello anestesia chirurgica utilizzando la risposta pinch punta. </li><li> Una volta che l&#39;animale ha raggiunto il livello chirurgico di anestesia, agganciare i capelli sulla superficie dorsale attorno linea mediana dell&#39;animale utilizzando un trimmer capelli elettrici. Scrub il sito chirurgico con il 70% di etanolo seguita da soluzione betadine tre volte. Coprire il sito con un telo chirurgico sterile fino al passaggio successivo. </li></ol></li><li> Procedura chirurgica per assottigliare l&#39;osso per esporre il midollo spinale <ol><li> Posizionare il mouse in posizione prona sul pad riscaldamento omeotermi sulla piattaforma chirurgica (Figura 1A). Correttamente garantire il mousepostura utilizzando un morsetto muso di mantenere una regione del collo allungato (Figura 1A, B). </li><li> Fare un&#39;incisione (lunghezza di circa 1 cm) con le forbici chirurgiche lungo la linea mediana dorsale, che si estende dalle vertebre toraciche T9 a T12. Spostare la pelle per esporre area chirurgica. </li><li> Usando un bisturi, cancellare con attenzione il muscolo per esporre le spine dorsali a T9 - T12 vertebre. Fermare le emorragie in ogni passo applicando una leggera pressione con un batuffolo di cotone sterile. T10 separato - T12 vertebre dal muscolo circostante e fissarli utilizzando un morsetto vertebrale stabilizzare e impedire qualsiasi movimento (Figura 1A, B). </li><li> Utilizzando un trapano ad alta velocità con l&#39;osso di lucidatura punta, attenzione e delicatezza sottile la superficie dorsale del T10 o T11 vertebra di visualizzare la vena dorsale posteriore e altri piccoli vasi sulla superficie dorsale del SC (Figura 1C). </li><li> Per evitare danni termici a causa del calore generato durante il procedimento diradamento,applicare una leggera corrente e costante di soluzione fisiologica con costante aspirazione per rimuovere i detriti. </li><li> Usando un bisturi liscia accuratamente la superficie ossea fino vaso principale è chiaramente visibile. Fare attenzione a non danneggiare il midollo spinale durante questo processo. </li><li> Una volta che il vaso sanguigno viene visualizzato, somministrare RB alla dose di 30 mg / kg (di peso corporeo) attraverso route seno retro-orbitale utilizzando una siringa da insulina. </li><li> Misurare il flusso sanguigno usando un flussometro Laser Doppler dopo 3 min dopo l&#39;iniezione RB se necessario (Figura 2A, B). Mantenere asepsi durante tutta la procedura. </li></ol></li></ol> 3. Induzione di PT <ol><li> Posto l&#39;animale in una fase regolabile posizione XY per un laboratorio di Jack, che può regolare l&#39;altezza. Regolare la posizione del mouse così regione esposta T11 midollo spinale è direttamente sotto l&#39;obiettivo 10X del microscopio FN1 epi-fluorescenza (Figura 3A). </li><li> Impostare la potenza del lifonte ght a 12% e irradiare la regione T11 con un diametro di 0,75 mm mezzo assottigliata midollo spinale (Nota: questa regione comprende la vena vertebrale posteriore e altri capillari) con una luce verde (lunghezza d&#39;onda di 540-580 nm, che è raggiunto dal cubo filtro microscopio) attraverso l&#39;obiettivo 10X per 2 min. Prendere immagini all&#39;inizio e alla fine di irradiazione (Figura 3B, C) ​​e registrare il tempo dell&#39;esperimento a questo punto. </li><li> Misurare nuovamente il flusso di sangue per 10 minuti, se necessario, posizionando la sonda laser Doppler nella stessa posizione sopra il midollo spinale come in 2.4.8 (Figura 2A, B). </li><li> Dopo il check irradiazione per qualsiasi emorragia e se nessuno trovato procedere alla sutura all&#39;animale. Suturare la fascia superficiale insieme con i muscoli sul entrambi i lati del midollo spinale con una sutura assorbibile o 4-0 dimensioni sutura di seta. Fate attenzione a non danneggiare la SC esposta. Suturare la pelle con 4-0 sutura di seta. Applicare Betadine o iodioai bordi della pelle dopo la sutura. </li></ol> 4. post-chirurgia Cura <ol><li> Dopo sutura, posizionare l&#39;animale sulla rampa di riscaldamento per il recupero. Dopo il recupero controllare gli animali per i segni di deficit neurologici osservando il movimento di entrambi gli arti posteriori. Non lasciare l&#39;animale incustodito fino a quando non ha ripreso conoscenza sufficiente a mantenere decubito sternale. </li><li> Trasferire gli animali alla gabbia di casa. Non rispedire l&#39;animale che ha subito un intervento chirurgico per la compagnia di altri animali fino alla completa guarigione. </li><li> Controllare gli animali ad intervalli regolari. In caso di gravi deficit neurologici, fornire cure adeguate, come l&#39;evacuazione di vescica, la somministrazione di analgesici (buprenorfina, 0,05-0,1 mg / kg). Verificare la presenza di disidratazione e amministrare normale per via sottocutanea di soluzione salina in caso grave. Di solito, la buprenorfina (0,1 mg / kg) sarà somministrato dopo la sutura per alleviare il dolore nel sito chirurgico. </li><li> Se gli animali non sono immediatamenteeutanasia dopo l&#39;intervento chirurgico, metteremo alto contenuto di acqua dieta sul pavimento della gabbia, così gli animali possono raggiungere il cibo facilmente. </li></ol> 5. transcardial perfusione, Nissl colorazione e Immunostaining <ol><li> Transcardiaca profumato l&#39;animale come descritto in precedenza 17-20. <ol><li> Anestetizzare l&#39;animale come descritto in precedenza nel protocollo e transcardially profumato con tampone fosfato salino (PBS, pH 7,4), seguito da ghiacciata 4% paraformaldeide (PFA) in PBS. </li><li> Dopo perfusione, rimuovere il midollo spinale (SC) e post-fissarlo in 4% PFA in PBS a 4 ° CO / N. Trasferire il SC fisso in PBS con il 30% di saccarosio e tenerlo per 2 - 3 giorni fino affonda al fondo della provetta. </li><li> Utilizzando un criostato tagliare il midollo spinale in 30 micron sezioni spesse e metterli in serie su una gelatina rivestita vetrini o in una piastra a 48 pozzetti con 0,01 M PBS. </li></ol></li><li> Colorazione Nissl: per ispezionare il danno causato dalle PT eseguire un Nissl staining su sezioni del midollo spinale come precedentemente descritto 17-20. <ol><li> In breve, raccogliere ogni quinta fetta midollo spinale sui vetrini e macchia con 0,25% violetto cresolo. Prendere immagini delle sezioni colorate (Figura 4). </li></ol></li><li> Immunocolorazione: Come descritto in precedenza utilizzando un galleggiante metodo della sezione 17,18,20. <ol><li> Brevemente, macchiare sezioni midollo spinale incubando O / N a 4 ° C con coniglio anti-glia proteina acidica fibrillare (GFAP) anticorpo policlonale (1: 300), coniglio anti-NeuN anticorpi (1: 300), e anti-rabbit anticorpi Iba1 (1: 500) seguita da asino coniglio anti Alexa 568-coniugato IgG (1: 400) anticorpi secondari per 4 ore a temperatura ambiente. Prendere immagini con un microscopio a fluorescenza (Figura 5). </li></ol></li></ol>

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The authors have nothing to disclose.

In questo studio, abbiamo descritto un modello photothrombotic di SC ischemia. Grazie ai progressi nel campo dell&#39;ingegneria genetica si è registrato un aumento in commercio topi transgenici che ha reso possibile lo studio l&#39;impatto di specifici geni coinvolti nella patofisiologia ischemico nel SC. Lo scopo dello studio è stato quello di sviluppare un modello riproducibile murino di ischemia del midollo spinale. Qui abbiamo adattato un modello corticale PT per indurre SCI nei topi. Dopo l&#39;...

Traumatica lesioni del midollo spinale (SCI) è una condizione clinica devastante che colpisce le funzioni sensomotorie e autonome della SC. I pazienti che sopravvivono SCI sono spesso lasciati con debilitante paraplegia che colpisce in modo significativo le loro attività quotidiane e la qualità della vita 1. SCI modelli sperimentali sono stati uno strumento indispensabile per la ricerca scientifica per capire la fisiopatologia della SIC e processi di riparazione neurale associati. Questi modelli sono stati utilizzati anche per testare l&#39;efficacia preclinica di vari interventi neuroprotettivi sperimentali che mirano a recupero funzionale. Attualmente, la maggior parte dei modelli di SCI, in pratica, impiegare l&#39;uso della forza fisica contundente di interrompere meccanicamente e ferire la SC. Questi metodi includono contusione, la compressione, la dislocazione e recisione della SC 2. È stato suggerito che dopo l&#39;insulto meccanico primario un danno secondario sotto forma di insiemi in ischemia nel SC lesa<html> 3,4. L&#39;eziologia di ischemia secondaria comprende una vasta degenerazione dei tessuti, emorragia parenchimale e talvolta da ostruzione dei vasi sanguigni da parte del tessuto edema 5-7. Come risultato della lesione secondaria l&#39;integrità del SC è ulteriormente influenzata, neuroni e cellule gliali sono gravemente compromessa in funzione e vitalità e apoptosi che porta a infarto crescita durante la fase cronica di lesioni, analoga alla crescita della penombra ischemica ictus seguente 8,9. Diversi meccanismi come eccitotossicità, produzione di radicali liberi, e infiammazione sono stati segnalati per essere responsabile della morte cellulare ischemica seguente SCI 10,11. Inoltre, SC ischemia è una grave complicanza di toraco-aneurisma dell&#39;aorta addominale ambulatori di riparazione che spesso portano a paraplegia nei pazienti 12,13. Nonostante così alto impatto clinico sono attualmente disponibili pochissimi modelli di ischemia del midollo spinale con elevata riproducibilità. nt &quot;&gt; Photothrombosis (PT) è un metodo comunemente utilizzato per l&#39;induzione di ischemia focale nel cervello 14-20. La tecnica è abbastanza non invasiva, altamente riproducibili e produce una precisa focale lesione ischemica sulla superficie esposta del cervello 17 -21. Ciò si ottiene con la somministrazione sistemica di coloranti fotoattivi come Rosa Bengala (RB) 16-20,22 o Eritrosina B 23 seguita da irradiazione localizzata di vasi sanguigni con sorgente luminosa corretta. Fotoattivazione del colorante provoca la generazione di radicali liberi che compromettere l&#39;integrità dell&#39;endotelio vascolare liscia, e causano le piastrine di accumulare, che forma successivamente un trombo. L&#39;ostruzione del flusso di sangue dai risultati trombo in un infarto nella regione fornito dal recipiente 24. Grazie alla facilità di controllo della intensità e durata dell&#39;irradiazione questa procedura produce un infarto molto uniforme e riproducibile. Inoltre, questo metodo può essere impiegato per indurre un infartot in varie sedi anatomiche abilitazione spaziale (ad esempio, materia grigia vs. materia bianca) comprensione degli effetti di ischemia. Lo scopo del presente studio è di sviluppare un modello semplice e altamente riproducibile di SC ischemia nei topi. Abbiamo descritto la procedura di un modello PT di SC ischemia nei topi. I risultati di istologia e immunostaining dimostrato che TP può effettivamente indurre SC miocardico, perdita neuronale e gliosi reattiva.

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			<td height="21" style="height:21px;width:216px;">&#160;Rose Bengal</td>
			<td style="width:233px;">Sigma-Aldrich</td>
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			<td height="21" style="height:21px;">C57Bl6/J</td>
			<td>Jackson lab</td>
			<td>664</td>
			<td>22-25g</td>
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			<td>VEDCO</td>
			<td>NDC-50989-996-06</td>
			<td>100 mg/ml</td>
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			<td height="21" style="height:21px;">Xylazine&#160;</td>
			<td>VEDCO</td>
			<td>NDC-50989-234-11</td>
			<td>100 mg/ml</td>
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			<td height="21" style="height:21px;">Betadine solution</td>
			<td>Purdue</td>
			<td>NDC-67618-150-01</td>
			<td>10% povidone iodine topical solution</td>
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			<td height="21" style="height:21px;">Normal saline</td>
			<td>Abott Laboratories</td>
			<td>04930-04-10</td>
			<td>For diluting RB, anaesthesia and for preventing tissue from drying</td>
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			<td>Decon labs.Inc</td>
			<td>2716</td>
			<td>70% ethanol for disinfection</td>
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			<td>EXFO, Canada</td>
			<td>X-Cite 120 PC&#160;</td>
			<td>Set power at 12%</td>
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			<td height="21" style="height:21px;">Spring scissors&#160;</td>
			<td>Fine Science Tool</td>
			<td>15000-10</td>
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			<td>14063-011</td>
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			<td height="21" style="height:21px;">Standard scalpel</td>
			<td>Fine Science Tool</td>
			<td>10003-12</td>
			<td>for removing muscle</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Scalpel blade</td>
			<td>Feather</td>
			<td>2976</td>
			<td>No. 10</td>
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			<td height="21" style="height:21px;">Forceps (curved)</td>
			<td>Fine Science Tool</td>
			<td>11150-10</td>
			<td>for holding tissue</td>
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			<td height="21" style="height:21px;">Forceps (straight)</td>
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			<td>11151-10</td>
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			<td height="21" style="height:21px;">Tissue adhesive glue</td>
			<td>3M Vetbond</td>
			<td>1469SB</td>
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			<td height="21" style="height:21px;">Monofilament polypropylene&#160;</td>
			<td>USSC Sutures</td>
			<td>VP-521</td>
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			<td height="21" style="height:21px;">Perma-hand silk</td>
			<td>Ethicon</td>
			<td>683G</td>
			<td>Size = 4-0 (for skin)</td>
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			<td height="21" style="height:21px;">Micro drill</td>
			<td>Roboz Surgical Instrument Co. Inc.</td>
			<td>RS-6300</td>
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			<td height="21" style="height:21px;">Laser doppler flowmeter</td>
			<td>Moor Instruments</td>
			<td>moorVMS-LDF1</td>
			<td>for monitoring change in blood flow</td>
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			<td height="21" style="height:21px;">Heating pad</td>
			<td>Fine Science Tool</td>
			<td>21052-00</td>
			<td>to prevent hypothermia</td>
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			<td height="21" style="height:21px;">Lab-Jack</td>
			<td>Fisher scientific&#160;</td>
			<td>14-673-50</td>
			<td>4x4 in plate to adjust the height of the animal</td>
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			<td height="21" style="height:21px;">X-Y gliding stage&#160;</td>
			<td>Amscope</td>
			<td>GT100</td>
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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.

Lo scopo di questo studio è stato quello di produrre ischemia del midollo spinale nei topi utilizzando un modello PT. Dopo la regione desiderata di osso sopra del midollo spinale (T10 - T12) è stato diluito, Rosa Bengala stato iniettato attraverso route seno retro-orbitale, e ischemia è stata indotta dal PT Figura 1A, B mostra il mouse posizionato in una chirurgica misura. piattaforma durante l&#39;intervento chirurgico. Il topo è tenuta in posizione da un morsetto muso e due vertebrato regolabile m...

Watch this Scientific Journal Video about Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice at JoVE.com

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

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) ...

photothrombosis-induced-focal-ischemia-as-model-spinal-cord-injury

Research

JoVE Journal

Medicine

13.1K Views.  University of Missouri. 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. 

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

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 ...

Ricerca

Medicina

Controlled Cervical Laceration Injury in Mice

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

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

Controllata cervicale lesioni lacerazione nei topi

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

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 ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-riperfusione Modello di danno renale acuta e post Infortunio fibrosi nei topi

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

Riproducibile Paraplegia da Toracica aortica occlusione in un modello murino di Spinal Cord ischemia-riperfusione

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

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 ...

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 ...