Ce travail a été soutenu par le National Institutes of Health [Grant pas. R01NS069726] et l&#39;Association Grant American Heart in Aid Grant [Grant pas. 13GRNT17020004] pour SD.

Note: Des souris (C57BL / 6J, mâle) ans 10-12 semaines fussent utilisés dans cette étude. Toutes les procédures ont été effectuées en conformité avec le Guide NIH pour le soin et l&#39;utilisation des animaux de laboratoire et ont été approuvés par l&#39;Université du Missouri institutionnel de protection des animaux et l&#39;utilisation Comité (IACUC). 1. Pré-chirurgie <ol><li> La veille de l&#39;intervention autoclave et stériliser tous les instruments chirurgicaux. Enveloppez les instruments et l&#39;autoclave à 121 o C à 15 psi pendant 30 min suivie par 30 minutes de séchage (121 o C, 15 psi, 30/30 cycle). Placez les instruments dans un environnement propre et stérile jusqu&#39;à l&#39;utilisation. </li><li> Préparer une solution fraîche Rose Bengale (RB) solution (20 mg / ml dans une solution saline stérile) à chaque fois avant la chirurgie. Pour dissoudre complètement RB, vortex le tube puis suivi par ultrasons pendant 5 min à 50/60 Hz avec une puissance de sortie de 19 W. Enrouler le tube dans une feuille d&#39;aluminium et de la protéger de la lumière jusqu&#39;à nouvel utilisé pendant surGery. </li><li> Préparer un mélange de kétamine / xylazine dans une solution saline stérile. Ajouter 125 pi de xylazine (concentration du stock: 20 mg / ml) et 325 pi de ketmaine (concentration du stock: 100 mg / ml) et 550 pi de solution saline stérile pour obtenir un volume final de 1 ml de mélange anesthésique. </li><li> Préchauffer le coussin chauffant homéothermes. </li><li> Préchauffer la lampe à halogénure métallique (de source de lumière pour FN1 épi-microscope à fluorescence) pendant 30 minutes pour stabiliser la puissance de la lampe. </li><li> Ajuster la taille de la région éclairée à un diamètre de 1 mm en utilisant objectif 10X et un réticule en ajustant le diaphragme de champ dans le FN1 debout epi-microscope à fluorescence. </li></ol> 2. Procédure chirurgicale <ol><li> Anesthésier les souris avec une dose de kétamine (130 mg / kg B. poids.) Et de xylazine (10 mg / kg B. poids.). Basé sur le cocktail dans 1.3, 4 pi par g de souris B. poids. sont nécessaires. Stériliser le site d&#39;injection avec un tampon imbibé d&#39;alcool et d&#39;administrer les anesthésiques par voie intra-peritoneal (IP) itinéraire. Prenez soin de ne pas injecter les anesthésiques dans les vaisseaux sanguins ou le muscle car cela retarderait l&#39;induction et la récupération de l&#39;animal. </li><li> Appliquer une pommade artificielle déchirure aux deux yeux de la souris pour éviter le dessèchement et placez l&#39;animal sur le coussin chauffant pour prévenir l&#39;hypothermie. </li><li> Préparation des animaux <ol><li> Vérifiez l&#39;animal pour le bon niveau de l&#39;anesthésie chirurgicale en utilisant la réponse orteil de pincement. </li><li> Une fois que l&#39;animal a atteint le niveau de l&#39;anesthésie chirurgicale, couper les poils sur la face dorsale autour de la ligne médiane de l&#39;animal à l&#39;aide d&#39;une tondeuse électrique. Frotter le site chirurgical avec de l&#39;éthanol à 70% puis avec une solution de bétadine trois fois. Couvrir le site avec un champ opératoire stérile jusqu&#39;à la prochaine étape. </li></ol></li><li> Intervention chirurgicale pour fluidifier l&#39;os pour exposer la moelle épinière <ol><li> Placez la souris dans une position couchée sur le coussin chauffant homéothermes sur la plate-forme chirurgicale (figure 1A). Fixer correctement la sourisposture aide d&#39;une pince de museau à maintenir une région du cou allongé (figure 1A, B). </li><li> Faire une incision (environ 1 cm de long) à l&#39;aide des ciseaux chirurgicaux le long de la ligne médiane dorsale étendant des vertèbres thoraciques T9 à T12. Écarter la peau pour exposer zone chirurgicale. </li><li> Avec un scalpel, effacer soigneusement le muscle pour exposer les épines dorsales au T9 - T12 vertèbres. Arrêter le saignement dans chaque étape en appliquant une légère pression avec un coton-tige stérile. T10 séparé - T12 vertèbres du muscle environnant et les fixer à l&#39;aide d&#39;une pince vertébrale à stabiliser et empêcher tout déplacement (figure 1A, B). </li><li> Utiliser un foret à grande vitesse avec l&#39;os de polissage foret, soigneusement et délicatement mince la surface dorsale de T10 ou T11 vertèbre de visualiser la veine postérieure de la moelle et d&#39;autres petits vaisseaux à la surface dorsale de la SC (figure 1C). </li><li> Pour éviter un endommagement thermique du fait de la chaleur générée durant la procédure d&#39;amincissement,appliquer un courant doux et constant de solution saline normale avec aspiration constante pour enlever les débris. </li><li> En utilisant un scalpel soigneusement lisser la surface de l&#39;os jusqu&#39;à ce que la cuve principale est clairement visible. Prenez soin de ne pas endommager la moelle épinière au cours de ce processus. </li><li> Une fois que le vaisseau sanguin est visualisée, RB administrer à une dose de 30 mg / kg (poids corporel) par le sinus rétro-orbital itinéraire à l&#39;aide d&#39;une seringue à insuline. </li><li> Mesurer le débit sanguin à l&#39;aide d&#39;un débitmètre Laser Doppler après 3 min après injection RB si nécessaire (figure 2A, B). Maintenir l&#39;asepsie pendant toute la procédure. </li></ol></li></ol> 3. Induction de PT <ol><li> Placez l&#39;animal sur une scène réglable en position XY sur un Lab-Jack qui peut ajuster la hauteur. Ajustez la position de la souris pour la région exposée de T11 moelle épinière est directement sous l&#39;objectif du microscope 10x FN1 épifluorescence (figure 3A). </li><li> Régler la puissance de la lisource de lutte à 12% et irradier la région T11 avec un diamètre de 0,75 mm au milieu de la moelle épinière amincie (Note: cette région contient la partie postérieure veine vertébrale et d&#39;autres capillaires) avec une lumière verte (longueur d&#39;onde 540-580 nm, ce qui est obtenu par le cube de filtre dans le microscope) à travers l&#39;objectif 10X pendant 2 min. Prendre des images au début et à la fin de l&#39;irradiation (figure 3B, C) ​​et enregistrer le temps de l&#39;expérience à ce stade. </li><li> Mesurer de nouveau le débit sanguin pendant 10 min si nécessaire en plaçant la sonde laser Doppler dans la même position au-dessus de la moelle épinière que dans 2.4.8 (figure 2A, B). </li><li> Après vérification de l&#39;irradiation de toute hémorragie et si aucun trouvé procéder à suturer l&#39;animal. Suturer l&#39;aponévrose superficielle avec les muscles sur le côté soit de la moelle épinière en utilisant une suture absorbable ou 4-0 taille soie suture. Prenez soin de ne pas endommager le SC exposée. Suturer la peau avec une suture de soie 4-0. Appliquer Bétadine ou de l&#39;iodesur les bords de la peau après la suture. </li></ol> 4. Post-chirurgie soins <ol><li> Après suture, placez l&#39;animal sur le coussin chauffant pour la récupération. Après la récupération examiner les animaux pour des signes de déficits neurologiques en observant le mouvement des deux membres postérieurs. Ne pas laisser l&#39;animal sans surveillance tant qu&#39;il a repris conscience suffisante pour maintenir décubitus sternal. </li><li> Transférer les animaux dans la cage de la maison. Ne retournez pas l&#39;animal qui a subi une intervention chirurgicale à la compagnie d&#39;autres animaux jusqu&#39;à guérison complète. </li><li> Vérifiez les animaux à intervalles réguliers. En cas de déficits neurologiques graves, fournir des soins appropriés, comme l&#39;évacuation de la vessie, de l&#39;administration d&#39;analgésiques (buprénorphine, de 0,05 à 0,1 mg / kg). Vérifier la déshydratation et à administrer par voie sous cutanée de solution saline normale en cas sévère. Habituellement, la buprénorphine (0,1 mg / kg) est administrée après la suture pour soulager la douleur dans le site chirurgical. </li><li> Si les animaux ne sont pas immédiatementeuthanasiés après la chirurgie, nous allons mettre l&#39;alimentation à haute teneur en eau sur le sol de la cage, de sorte que les animaux peuvent atteindre la nourriture facilement. </li></ol> 5. Transcardial perfusion, la coloration de Nissl et immunocoloration <ol><li> Transcardiaque perfuser l&#39;animal de 17 à 20 comme décrit précédemment. <ol><li> Anesthésier l&#39;animal, comme décrit plus haut dans le protocole et perfuser transcardiaque avec une solution saline tamponnée phosphate (PBS, pH 7,4), suivie par de la glace froide 4% de paraformaldehyde (PFA) dans du PBS. </li><li> Après la perfusion, retirer la moelle épinière (SC) et post-fixer dans 4% PFA dans du PBS à 4 ° CO / N. Transférer le SC fixe dans PBS avec 30% de saccharose et le garder pour 2 - 3 jours jusqu&#39;à ce qu&#39;il coule au fond du tube. </li><li> L&#39;utilisation d&#39;un cryostat coupé la moelle épinière en 30 sections um d&#39;épaisseur et les placer en série sur des lamelles de verre recouvertes de gélatine ou dans une plaque de 48 puits avec 0,01 M de PBS. </li></ol></li><li> Coloration Nissl: Pour inspecter le dommage causé par PT effectuer une Nissl staining sur des sections de la moelle épinière comme décrit précédemment 17-20. <ol><li> En bref, de recueillir chaque cinquième tranche de la moelle épinière sur les lames de verre et des taches avec 0,25% violet de crésyl. Prenez des photos de coupes colorées (figure 4). </li></ol></li><li> L&#39;immunocoloration: Comme il est décrit précédemment en utilisant un procédé de coupe flottant 17,18,20. <ol><li> En bref, colorer les sections de la moelle épinière par incubation O / N à 4 ° C avec de lapin anti-glie protéine acide fibrillaire (GFAP) d&#39;anticorps polyclonaux (1: 300), anticorps de lapin-NeuN contre (1: 300), et de lapin anti- Iba1 anticorps (1: 500) suivi d&#39;âne anti-lapin Alexa 568-conjugué IgG (1: 400) les anticorps secondaires pendant 4 h à température ambiante. Prenez des photos avec un microscope à fluorescence (figure 5). </li></ol></li></ol>

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The authors have nothing to disclose.

Dans cette étude, nous avons décrit un modèle de photothrombotique SC ischémie. Grâce aux progrès en génie génétique, il ya eu une forte augmentation des souris transgéniques disponible dans le commerce qui a permis d&#39;étudier l&#39;impact des gènes spécifiques impliqués dans la physiopathologie ischémique dans la SC. Le but de l&#39;étude était de développer un modèle de souris reproductible de ischémie de la moelle épinière. Ici nous avons adapté un modèle PT corticale pour ...

Traumatisme de la moelle épinière (SCI) est un état clinique dévastatrice qui affecte les fonctions sensori-motrices et autonomes de la SC. SCI patients survivants se retrouvent souvent avec une paraplégie débilitante qui affecte considérablement leurs activités et de la qualité de la vie quotidienne 1. SCI modèles expérimentaux ont été un outil indispensable dans la recherche scientifique pour comprendre la physiopathologie de la SCI et les processus de réparation de neurones associés. Ces modèles ont également été utilisés pour tester l&#39;efficacité préclinique de diverses interventions neuroprotectrices expérimentales qui visent à la récupération fonctionnelle. Actuellement, la majorité des modèles SCI dans la pratique emploient l&#39;utilisation de la force physique brutale de perturber mécaniquement et blesser le SC. Ces méthodes comprennent contusion, compression, la dislocation et transection du SC 2. Il a été suggéré que, après l&#39;insulte mécanique primaire une lésion secondaire sous la forme d&#39;ensembles d&#39;ischémie dans la SC blessé<html> 3,4. L&#39;étiologie de l&#39;ischémie secondaire comprend une vaste dégénérescence des tissus, l&#39;hémorragie parenchymateuse et parfois par l&#39;obstruction des vaisseaux sanguins par un œdème des tissus 5-7. À la suite de la lésion secondaire l&#39;intégrité de SC est en outre affectée, les neurones et les cellules gliales sont gravement altérées dans la fonction et la viabilité et l&#39;apoptose qui conduit à l&#39;infarctus croissance au cours de la phase chronique de la blessure, analogue à la croissance de pénombre ischémique suite d&#39;un AVC 8,9. Plusieurs mécanismes comme excitotoxicité, production de radicaux libres, et l&#39;inflammation ont été signalés à être responsable de la mort cellulaire ischémique suivant SCI 10,11. En outre, SC ischémie est une complication grave de l&#39;anévrisme aortique thoraco-abdominaux chirurgies de réparation qui conduisent souvent à la paraplégie chez les patients 12,13. En dépit de cet impact clinique haute très peu de modèles de ischémie de la moelle épinière avec une reproductibilité élevée sont actuellement disponibles. nt &quot;&gt; Photothrombosis (PT) est une méthode couramment utilisée pour l&#39;induction d&#39;une ischémie focale dans le cerveau 14-20. La technique est assez non-invasif, hautement reproductible et produit une lésion ischémique focal précis dans la zone exposée du cerveau 17 -21. Ceci est réalisé par administration systémique de colorants photoactifs tels que le rose Bengale (RB) 16-20,22 érythrosine B ou 23 suivie par une irradiation localisée de vaisseaux sanguins avec une source de lumière appropriée. photoactivation du colorant provoque la génération de radicaux libres qui perturber l&#39;intégrité de l&#39;endothélium vasculaire lisse, et provoquent les plaquettes d&#39;accumuler, qui forme ensuite un thrombus. L&#39;obstruction de la circulation sanguine par les résultats de thrombus dans un infarctus dans la région fournies par le navire 24. En raison de la facilité de contrôle de la l&#39;intensité et la durée de l&#39;irradiation de cette procédure donne un infarctus très uniforme et reproductible. En outre, ce procédé peut être utilisé pour induire un infarctust à divers emplacements anatomiques permettant spatiale (par exemple, la matière grise vs. matière blanche) compréhension de l&#39;effet de l&#39;ischémie. L&#39;objectif de la présente étude est de développer un modèle facile et hautement reproductible de SC ischémie chez les souris. Nous avons décrit la procédure d&#39;un modèle d&#39;ischémie PT de SC chez des souris. Les résultats de l&#39;histologie et immunomarquage ont démontré que PT peut induire efficacement SC du myocarde, une perte neuronale et une gliose réactive.

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			<td height="21" style="height:21px;width:216px;">&#160;Rose Bengal</td>
			<td style="width:233px;">Sigma-Aldrich</td>
			<td style="width:180px;">330000</td>
			<td style="width:421px;">20 mg/ml in sterile saline</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>NDC-0536-6550-91</td>
			<td>83% white petrolatum</td>
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			<td height="21" style="height:21px;">Ethanol</td>
			<td>Decon labs.Inc</td>
			<td>2716</td>
			<td>70% ethanol for disinfection</td>
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			<td height="21" style="height:21px;">Metal halide lamp</td>
			<td>EXFO, Canada</td>
			<td>X-Cite 120 PC&#160;</td>
			<td>Set power at 12%</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Spring scissors&#160;</td>
			<td>Fine Science Tool</td>
			<td>15000-10</td>
			<td>for minor dissection</td>
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			<td height="21" style="height:21px;">Scissors (angled to side)</td>
			<td>Fine Science Tool</td>
			<td>14063-011</td>
			<td>No. 3 handle</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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		<tr height="21">
			<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>
			<td>Fine Science Tool</td>
			<td>11151-10</td>
			<td>for holding tissue</td>
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			<td height="21" style="height:21px;">Needle holder&#160;</td>
			<td>Fine Science Tool</td>
			<td>12002-12</td>
			<td>for suturing</td>
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			<td height="21" style="height:21px;">Tissue adhesive glue</td>
			<td>3M Vetbond</td>
			<td>1469SB</td>
			<td>to adhere to edges of the cut skin</td>
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			<td height="21" style="height:21px;">Monofilament polypropylene&#160;</td>
			<td>USSC Sutures</td>
			<td>VP-521</td>
			<td>Size = 4-0 (for fascia)</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>
			<td>with bone polishing drill bit</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.

Le but de cette étude était de produire ischémie de la moelle épinière chez des souris en utilisant un modèle de PT. Après la région désirée de l&#39;os au-dessus de la moelle épinière (T10 - T12) a été amincie, Rose Bengale a été injecté à travers le sinus rétro-orbital itinéraire, et l&#39;ischémie a été induite par PT Figure 1A, B correspond à la souris placée dans un instrument chirurgical sur mesure. plate-forme au cours de la chirurgie. La souris a été maintenu en place ...

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 induite par ischémie focale comme un modèle de lésion médullaire chez la souris

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 vues.  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. 

Vidéo: 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.

Aiguë et chronique Test tactile sensorielle après un traumatisme médullaire chez le rat

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

Recherche

Médecine

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.

Contrôlé blessures de lacération du col chez la souris

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 modèle de contusion de graves lésions de la moelle épinière chez les rats

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.

Ischémie-reperfusion modèle de lésions rénales aiguës et post blessures fibrose chez la souris

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.

Reproductible paraplégie par l&#39;aorte thoracique occlusion dans un modèle murin de la moelle épinière ischémie-reperfusion

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.

Contrôlée corticale modèle d&#39;impact pour 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, ...

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 système de radio-télémétrique de surveiller la fonction cardiovasculaire chez le rat avec la moelle épinière Transection et embryonnaires souches neurales greffes cellulaires

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 Transplantation de cellules souches dans la partie expérimentale Modèle contusion de la moelle épinière des blessures

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.

Calibré Forceps modèle de compression de la moelle épinière des blessures

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 nouveau-né de la moelle épinière de souris Compression Modèle de lésion

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