Name: photothrombosis-induced focal ischemia as a model of spinal cord injury in mice
Uploaded: 2015-07-16T15:00:00.000+00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice at JoVE.com

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.

<table><tbody><tr><td>Rose Bengal</td><td>Sigma-Aldrich</td><td>330000</td><td>20 mg/ml in sterile saline</td></tr><tr><td>C57BL/6J</td><td>Jackson lab</td><td>664</td><td>22 - 25 g</td></tr><tr><td>Ketamine&amp;nbsp;</td><td>VEDCO</td><td>NDC-50989-996-06</td><td>100 mg/ml</td></tr><tr><td>Xylazine&amp;nbsp;</td><td>VEDCO</td><td>NDC-50989-234-11</td><td>100 mg/ml</td></tr><tr><td>Betadine solution</td><td>Purdue</td><td>NDC-67618-150-01</td><td>10% povidone iodine topical solution</td></tr><tr><td>Normal saline</td><td>Abott Laboratories</td><td>04930-04-10</td><td>For diluting RB, anaesthesia and for preventing tissue from drying</td></tr><tr><td>Artificial tears ointment&amp;nbsp;</td><td>Rugby</td><td>NDC-0536-6550-91</td><td>83% white petrolatum</td></tr><tr><td>Ethanol</td><td>Decon labs.Inc</td><td>2716</td><td>70% ethanol for disinfection</td></tr><tr><td>Metal halide lamp</td><td>EXFO, Canada</td><td>X-Cite 120 PC&amp;nbsp;</td><td>Set power at 12%</td></tr><tr><td>Spring scissors&amp;nbsp;</td><td>Fine Science Tool</td><td>15000-10</td><td>for minor dissection</td></tr><tr><td>Scissors (angled to side)</td><td>Fine Science Tool</td><td>14063-011</td><td>No. 3 handle</td></tr><tr><td>Standard scalpel</td><td>Fine Science Tool</td><td>10003-12</td><td>for removing muscle</td></tr><tr><td>Scalpel blade</td><td>Feather</td><td>2976</td><td>No. 10</td></tr><tr><td>Forceps (curved)</td><td>Fine Science Tool</td><td>11150-10</td><td>for holding tissue</td></tr><tr><td>Forceps (straight)</td><td>Fine Science Tool</td><td>11151-10</td><td>for holding tissue</td></tr><tr><td>Needle holder&amp;nbsp;</td><td>Fine Science Tool</td><td>12002-12</td><td>for suturing</td></tr><tr><td>Tissue adhesive glue</td><td>3M Vetbond</td><td>1469SB</td><td>to adhere to edges of the cut skin</td></tr><tr><td>Monofilament polypropylene&amp;nbsp;</td><td>USSC Sutures</td><td>VP-521</td><td>Size = 4-0 (for fascia)</td></tr><tr><td>Perma-hand silk</td><td>Ethicon</td><td>683G</td><td>Size = 4-0 (for skin)</td></tr><tr><td>Micro drill</td><td>Roboz Surgical Instrument Co. Inc.</td><td>RS-6300</td><td>with bone polishing drill bit</td></tr><tr><td>Laser doppler flowmeter</td><td>Moor Instruments</td><td>moorVMS-LDF1</td><td>for monitoring change in blood flow</td></tr><tr><td>Heating pad</td><td>Fine Science Tool</td><td>21052-00</td><td>to prevent hypothermia</td></tr><tr><td>Lab-Jack</td><td>Fisher scientific&amp;nbsp;</td><td>14-673-50</td><td>4 x 4 in plate to adjust the height of the animal</td></tr><tr><td>X-Y gliding stage&amp;nbsp;</td><td>Amscope</td><td>GT100</td><td>for positioning the animal under microscope&amp;nbsp;&amp;nbsp;</td></tr></tbody></table>

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

13.3K 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

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Ischemia Photothrombotic: un modello fotochimico lesione corticale minimamente invasiva e riproducibile per mouse Studi Stroke

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

Ricerca

Medicina

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 ...

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 ...

A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between experimental animals. Several contusive SCI models have been developed to produce injuries using different mechanisms. The severity of the SCI is based on the height that a given weight is dropped, the injury force, or the spinal cord displacement. In the current study, we introduce a novel mouse contusive SCI device, the Louisville Injury System Apparatus (LISA) impactor, which can create a displacement-based SCI with high injury velocity and accuracy. This system utilizes laser distance sensors combined with advanced software to produce graded and highly-reproducible injuries. We performed a contusive SCI at the 10th thoracic vertebral (T10) level in mice to demonstrate the step-by-step procedure. The model can also be applied to the cervical and lumbar spinal levels.

We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

Un modello di lesioni del midollo spinale contusivo basato sullo spostamento tissutale nei topi

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between ...

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

This protocol describes how to create a precise laminectomy for induction of stable transection-type spinal cord injury in the mouse model, with minimal collateral damage for spinal cord injury research.

Induzione della lesione completa del midollo spinale di tipo transezione nei topi

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...

Neuroscienze

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with&#160;histological analysis are demonstrated.

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

Modellazione dell'ictus nei topi: lesioni corticali focali da fototrombosi

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, thereby improving the reproducibility of the experiments.
These methods need two requirements to be fulfilled: clarity of the surgical anatomical pathway and simplicity and convenience of the laboratory device. Crucially for the operator, a clear anatomical pathway provides minimally invasive exposure, which avoids additional damage to the experimental animal during the surgical procedures and allows the animal to maintain a consistent and stable anatomical morphology during the experiment.
In this study, the use of a novel integrated platform called the SCI coaxial platform for spinal cord injury in small animals to expose the T9 level spinal cord in a minimally invasive way and stabilize and immobilize the vertebra of mice using a vertebral stabilizer is researched, and, finally, a coaxial gravity impactor is used to contuse the spinal cord of mice to approach different degrees of T9 spinal cord injury. Finally, histological results are provided as a reference for the readers.

Minimally invasive techniques and a simple laboratory device improve the reproducibility of the spinal cord injury model by reducing operative damage to the experimental animals and allowing anatomical morphology maintenance. The method is worthwhile because the reliable results and reproducible procedure facilitate investigations of the mechanisms of disease reparation.

Stabilire un modello di lesione del midollo spinale per contusione murina basato su una tecnica minimamente invasiva

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, ...

Establishment of Central Cord Syndrome Model in C57BL/6J Mouse

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally invasive exposure approaches and reduce extra injury to experimental animals during operation, enabling the maintenance of consistent and stable anatomical morphology during experiments to minimize behavioral and histological differences between individuals to improve the reproducibility of experiments. In this study, the C6 level spinal cord was exposed using a spinal cord injury coaxial platform (SCICP) and combination with a minimally invasive technique. With the assistance of a vertebral stabilizator, we fixed the vertebrae and compressed the spinal cords of C57BL/6J mice with 5 g/mm2 and 10 g/mm2 weights with SCICP to induce different degrees of C6 spinal cord injury. In line with the previous description of CCS, the results reveal that the lesion in this model is concentrated in the gray matter around the central cord, enabling further research into CCS. Finally, histological results are provided as a reference for the readers.

The present protocol simulating central cord syndrome (CCS) in mice has improved repeatability and minimized operation damage to the experimental animals, avoiding disrupting the anatomical structure excessively. The strategy in this study is advantageous because it allows for research into injury mechanisms by producing consistent results.

Definizione del modello di sindrome del midollo centrale nel topo C57BL/6J

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally ...

Modeling Photothrombotic Stroke in Mouse Model: A Laser Illumination Technique Through Mouse Skull Following Photosensitive Dye Administration to Induce Photothrombosis

Source: Llovera, G. et al. <a href="https://www.jove.com/v/62536">Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis.</a> J. Vis. Exp. (2021)
This video demonstrates the procedure to generate photothrombotic stroke in a mouse model by laser illumination of an intact skull following administration of photosensitive dye.

Modellazione dell'ictus fototrombotico in un modello murino: una tecnica di illuminazione laser attraverso il cranio del topo dopo la somministrazione di colorante fotosensibile per indurre la fototrombosi

Source: Llovera, G. et al. Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis. J. Vis. Exp. (2021)
This video demonstrates the procedure ...

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Biologia

Rodent Models

Adult

Inducing a Spinal Cord Contusion in a Mouse Model Using a Spinal Cord Injury Coaxial Platform

<a href='https://app.jove.com/v/64538/establishing-mouse-contusion-spinal-cord-injury-model-based-on'>Source: Elzat, E. Y. Y., et al.,&#160;Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique. J. Vis. Exp.&#160;(2022).</a>This video demonstrates the method for inducing a contusion injury at the T9 level spinal cord in a mouse model, using a spinal cord injury coaxial platform. A weight is dropped on the T9 level spinal cord that creates a contusion that immediately causes tissue discoloration and swelling.