This work was supported by the American Heart Association Scientist Development Grant and the Foundation for Anesthesia Education and Research (MLJ). We would like to thank Talaignair N. Venkatraman PhD for his assistance with magnetic resonance imaging.

Déclaration éthique: Ce protocole a été approuvé par le Comité de l&#39;Université Duke institutionnel de protection et d&#39;utilisation des animaux et suit toutes les directives pour l&#39;utilisation éthique des animaux. Une. Préparation de l&#39;équipement <ol><li> Autoclave les instruments chirurgicaux avant la chirurgie. </li><li> Désinfecter l&#39;appareil stéréotaxique avec 70% d&#39;éthanol. </li><li> Mettez bain d&#39;eau et maintenir la température de l&#39;eau à 42 ° C. </li><li> Dissoudre de type IV-S collagénase de Clostridium dans une solution saline normale à une concentration de 0,075 U par 0,4 ul. </li></ol> 2. Collagénase Injection Modèle <ol><li> Peser la souris. </li><li> Anesthésier la souris dans une chambre d&#39;induction avec 5% d&#39;isoflurane dans de 30% O 2 / N 2 à 70%. Anesthésie adéquate est signalée après environ 2 min quand la respiration de souris ont ralenti à 1 par seconde. </li><li> Intuber la trachée avec un G cathéter intraveineux 30 mm 20. </li><li> Branchez le cathéter à unrongeur ventilateur et ventiler mécaniquement les poumons avec 1,6% d&#39;isoflurane dans 30% O 2/70% N 2 à un taux de 105 respirations par minute avec un volume courant fourni de 0,75 ml .. </li><li> Raser le cuir chevelu avec un rasoir électronique. Une fois que la souris est anesthésiée et intubé, le déplacer vers un poste de travail différent pour le rasage et est ensuite retourné sur le banc chirurgicale. </li><li> Fixer la tête dans un cadre stéréotaxique, et le niveau de la tête avec suture coronale et sagittale deux points de référence. </li><li> Appliquer une pommade ophtalmique pour les yeux. </li><li> Insérez une sonde de température rectale. Maintenir la température rectale à 37,0 ± 0,2 ° C en utilisant un soubassement matelas d&#39;eau en circulation. </li><li> Essuyez la zone chirurgicale avec de la bétadine suivi avec 70% d&#39;éthanol et répéter 3 fois. </li><li> Faire une incision du cuir chevelu 1 cm de la ligne médiane et essuyez périoste latéralement avec un coton-tige stérile pour exposer bregma. </li><li> Drill 1 mm de diamètre trou de trépan de 2,2 mm à gauche latERALES à bregma avec une perceuse refroidi à l&#39;eau. </li><li> Tournez la collagénase flacon 5 fois, puis lavez une seringue de 0,5 pi avec 25 aiguille G (attaché au cadre stéréotaxique) avec 0,5 solution de collagénase pi 5 fois (Laissez 0,5 pi de solution de collagénase dans la seringue après le dernier lavage). </li><li> Alignez pointe de l&#39;aiguille avec trou de trépan, puis expulser 0,1 pi de la seringue et l&#39;aiguille de biseau essuyer avec un rasoir à jeter. </li><li> L&#39;utilisation d&#39;un micromanipulateur, avancer l&#39;aiguille 3 mm de profondeur de cortex et laisser immobile pendant 30 secondes. </li><li> Injecter 0,4 pi plus de 90 sec. </li><li> Diminuer l&#39;isoflurane à 1% et de laisser l&#39;aiguille immobile pendant 5 min. </li><li> Retirer l&#39;aiguille lentement. </li><li> Appliquer les 1 - 2 gouttes de bupivacaïne à 0,25% sous-cutanée et suturer la peau. </li><li> Désactiver l&#39;isoflurane vaporisateur et enlever la souris du cadre stéréotaxique. </li><li> Laissez la souris pour récupérer la ventilation spontanée avec trachéale ultérieure extubation. </li><li> Retour à la souris une cage propre et permettre le libre accès ànourriture et d&#39;eau. </li></ol> 3. Injection de sang autologue modèle <ol><li> Suivez les étapes 2.1 à 2.11 pour le modèle d&#39;injection de collagénase. </li><li> Dessinez 50 ul de solution saline normale stérile dans une seringue de 30 pi G 50. </li><li> Connectez la seringue microlitre avec un tube PE10 70 cm. </li><li> Expulser toute la solution saline normale de microlitre seringue dans PE10 tube de tube complètement hors d&#39;air. </li><li> Tirer la seringue piston microlitres à 1 mm pour faire une bulle d&#39;air à l&#39;ouverture distale de l&#39;appareil de la seringue tube microlitre PE10 pour éviter le mélange de solution saline et de sang au cours des procédures ultérieures. </li><li> Essuyez la région de l&#39;artère de la queue centrale distale de la souris avec 70% d&#39;éthanol, et couper l&#39;artère avec un rasoir de 0,5 à 1 cm à l&#39;extrémité de la queue. </li><li> Recueillir 40 pi de sang de la queue coupée dans le dispositif de seringue microlitre tube PE10. Remarque: l&#39;héparine qui n&#39;est pas utilisé dans l&#39;aiguille, le tube, ou une souris. </li><li> Fixer la seringue microlitre sur la Injepompe ction. </li><li> Connectez la partie de canule métallique d&#39;une aiguille 27 G à l&#39;extrémité du tube PE10, et fixer l&#39;aiguille à un micromanipulateur sur le cadre stéréotaxique. </li><li> Expulser 2 pl de sang sur 27 aiguille G et essuyez aiguille biseau avec un rasoir à jeter. </li><li> Alignez pointe de l&#39;aiguille avec trou de trépan et insérer l&#39;aiguille 3 mm de profondeur pour cortex. </li><li> Injecter 35 ul de sang autologue à un débit de 2 pi par minute. </li><li> Diminuer l&#39;isoflurane à 1% et de laisser l&#39;aiguille immobile pendant 10 min. </li><li> Retirer l&#39;aiguille en 30 sec. </li><li> Appliquer les 1 - 2 gouttes de bupivacaïne à 0,25% sous-cutanée et suturer la peau. </li><li> Désactiver l&#39;isoflurane vaporisateur et enlever la souris du cadre stéréotaxique. </li><li> Laissez la souris pour récupérer la ventilation spontanée avec extubation ultérieure. </li><li> Retour à la souris une cage propre et permettre l&#39;accès libre à la nourriture et de l&#39;eau. </li></ol> 4. Opération Sham <ol><li> Suivez les mêmes procédures pour la collagénase à injectern modèle, à l&#39;exception sans injection après l&#39;insertion de l&#39;aiguille. </li></ol> 5. Soins post-chirurgie <ol><li> Injecter 0,5 ml de solution saline normale voie sous-cutanée dans la soirée de l&#39;intervention chirurgicale à l&#39;arrière du cou de l&#39;animal. </li><li> Fournir de la nourriture ramolli avec de l&#39;eau et de la nourriture de gel dans de petites tasses en plastique placés sur le plancher de la cage. Remplacer la nourriture par jour pendant 7 jours. </li><li> Vérifiez la perte de poids, la cicatrisation, et des signes d&#39;inconfort par jour pendant 7 jours. </li><li> Si les intervalles de récupération de plus de 7 jours sont nécessaires, l&#39;enlèvement de fil de suture peut être réalisée sous anesthésie légère par inhalation (environ 1% d&#39;isoflurane dans de 30% O 2 / N 2 à 70%), si nécessaire. </li></ol>

<ol>
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Lei, Sheng, Wang, Lascola, Warner, and Laskowitz have no conflicts of interest to declare. James received grant funding by American Heart Association, National Institutes of Health, and Cephalogics.

Malgré la nouvelle recherche préclinique et grands essais cliniques qui en résultent pour la thérapeutique prometteuse 15-18, il n&#39;y a pas d&#39;interventions pharmacologiques démontrées pour améliorer les résultats dans le PCI, et les soins reste largement favorable. Listes des thérapies possibles peuvent être générées par les technologies à haut débit, tels que le travail transcriptomique et protéomique. Bien que ces technologies continuent à progresser notre connaissance des cibles th?...

L&#39;hémorragie intracérébrale (HIC) est une forme relativement fréquente de la maladie cérébro-vasculaire à environ 40-50% des patients atteints de mourir dans les 30 jours 1. Malheureusement, peu de progrès ont été réalisés dans le taux de mortalité au cours des 20 dernières années 2. Rapports des Instituts nationaux de la santé 3 et les lignes directrices de l&#39;American Heart Association 4 ont souligné l&#39;importance de développer des modèles cliniquement pertinentes du PCI pour étendre la compréhension de la physiopathologie et de développer des cibles pour de nouvelles approches thérapeutiques. Plusieurs modèles existent pour simuler ICH humaine 5. Que la compréhension de la physiopathologie de l&#39;ICH mûrit, il est devenu évident qu&#39;une variété de modèles peut être utilisé pour examiner les différents aspects de la maladie. Modèles utilisés précédemment comprennent murin angiopathie amyloïde 6, insertion de microballon intraparenchymateuse et l&#39;inflation 7, et le sang artériel directeinfiltration 8,9. Lobaire hémorragie de l&#39;angiopathie amyloïde a été modélisé à l&#39;utilisation de souris transgéniques et représente un sous-type distinct ICH. modèles de microballon imitent l&#39;effet de masse aigu de la formation d&#39;un hématome mais ne parviennent pas à capturer la réponse cellulaire du cerveau à la présence de sang. Enfin, l&#39;infiltration directe de sang artériel soumet le cerveau à partir de la pression artérielle de l&#39;artère fémorale. Ainsi, ce modèle imite les pressions artérielles et la présence de sang, mais ne pas soumettre le produit à une lésion cérébrale microvasculaire de petite rupture de vaisseau sanguin. En outre, ce modèle a intrinsèquement une forte variabilité. Fait intéressant, des rats spontanément hypertendus 10 développent ICH spontanée à mesure qu&#39;ils vieillissent. L&#39;étude de ces animaux après le développement ICH peut imiter la maladie, en présence de l&#39;un des principaux co-morbidités qui prédisposent l&#39;homme à l&#39;ICH. Bien que ces autres modèles existent, injection intrastriatale de la collagénase de Clostridium 11 ou par injection d&#39;un instrastiatalsang total utologous 12 sont, actuellement, les deux modèles les plus courants utilisés dans la recherche préclinique de l&#39;ICH. Sélection du modèle PCI doit être faite sur la base de l&#39;objectif de la question expérimentale, y compris la sélection des espèces et de la méthode d&#39;induire la formation d&#39;un hématome. Par exemple, les porcs sont de grands animaux avec des volumes relativement importants de la substance blanche du cerveau par rapport à des souris. Ainsi, les modèles porcins sont adaptées pour étudier la matière blanche physiopathologie suivante ICH. En revanche, les cerveaux de rongeurs sont largement matière grise, mais les systèmes transgéniques font rongeurs utile pour évaluer les mécanismes moléculaires des lésions et la récupération après l&#39;ICH. Chaque modèle a ses forces et ses faiblesses (tableau 1) inhérents, qui devrait être examinée avec soin avant l&#39;expérimentation. Les protocoles suivants mettent en évidence les modèles de sang et d&#39;injection de collagénase autologues chez la souris. Ces modèles ont chacun été traduits à partir de modèles développés à l&#39;origine chez les rats13,14 et permettre l&#39;utilisation de la technologie transgénique largement disponibles pour explorer les mécanismes moléculaires associés à la mort cellulaire après ICH. Représentent tous les deux très différents mécanismes de blessure de l&#39;ICH humaine, et les deux ont résultat attendu nettement différente en termes de mesures comportementales et histologiques. Ainsi, certaines hypothèses peuvent se prêter à un modèle sur l&#39;autre, mais de nombreuses idées peuvent être validés dans les deux modèles. Tableau 1. Comparaison des caractéristiques de la collagénase et modèles autologues hémorragie intracérébrale d&#39;injection de sang. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td style="width:197px;"></td><td style="width:80px;"> Collagénase injection </td><td style="width:66px;"> Injection de sang </td></tr><tr><td style="width:197px;"> Facilité d&#39;utilisation </td><td style="width:80px;"> + + + </td><td style="width:66px;"> + + </td></tr><tr&gt; <td style="width:197px;"> Reproductibilité </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;"> Contrôle de l&#39;hémorragie Taille </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + + </td></tr><tr><td style="width:197px;"> Le reflux du sang </td><td style="width:80px;"> + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;"> Simule maladies humaines </td><td style="width:80px;"> + </td><td style="width:66px;"> - </td></tr><tr><td style="width:197px;"> Simplicité </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;"> Utilisez des espèces multiples </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr></tbody></table>

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			<td height="43" style="height:43px;width:152px;">Name</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalog Number</td>
			<td style="width:88px;">Comments</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Stereotactic frame</td>
			<td style="width:88px;">Stoelting Co.</td>
			<td style="width:88px;">51603</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Probe holder with corner clamp</td>
			<td style="width:88px;">Stoelting Co.</td>
			<td style="width:88px;">51631</td>
			<td style="width:88px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Mini grinder</td>
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			<td style="width:88px;">Model 60100002</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">0.5 &#181;l Hamilton syringe</td>
			<td style="width:88px;">Hamilton Co.</td>
			<td style="width:88px;">86259</td>
			<td style="width:88px;">25 gauge needle</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:152px;">50 &#181;l Hamilton syringe</td>
			<td style="width:88px;">Hamilton Co</td>
			<td style="width:88px;">7637-01</td>
			<td style="width:88px;"></td>
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		<tr height="64">
			<td height="64" style="height:64px;width:152px;">26G Hamilton needle</td>
			<td style="width:88px;">Hamilton Co</td>
			<td style="width:88px;">7804-03</td>
			<td style="width:88px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Syringe pump</td>
			<td style="width:88px;">KD Scientific</td>
			<td style="width:88px;">Model 100</td>
			<td style="width:88px;"></td>
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		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Heat therapy water pump</td>
			<td style="width:88px;">Gaymar Industries, Inc.</td>
			<td style="width:88px;">Model# TP650</td>
			<td style="width:88px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Circulating waterbed</td>
			<td style="width:88px;">CMS Tool &#38; Die, Inc.</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Rodent ventilator</td>
			<td style="width:88px;">Harvard Apparatus</td>
			<td style="width:88px;">Model 683</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Isoflurane vaporizer</td>
			<td style="width:88px;">Drager</td>
			<td style="width:88px;">Vapor 19.1</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Air flowmeter</td>
			<td style="width:88px;">Cole Parmer</td>
			<td style="width:88px;">Model PMR1-010295</td>
			<td style="width:88px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Induction chamber</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
			<td style="width:88px;">Self made</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Otoscope</td>
			<td style="width:88px;">Welch Allyn</td>
			<td style="width:88px;">22820</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">intravenous catheter</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">381534</td>
			<td style="width:88px;">20-gauge, 1.16 inch Insyte-W</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Isoflurane</td>
			<td style="width:88px;">Baxter Healthcare Corporation</td>
			<td style="width:88px;">NDC10019-360-69</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Collagenase Type IV-S</td>
			<td style="width:88px;">Sigma</td>
			<td style="width:88px;">C1889</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Polyethylene tubing PE20</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">427406</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Polyethylene tubing PE10</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">427401</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">30G 1 inch needle</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">305128</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">27G 1 1/4 inch needle</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">305136</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Surgical scissors</td>
			<td style="width:88px;">Miltex</td>
			<td style="width:88px;">21-539</td>
			<td style="width:88px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Forceps</td>
			<td style="width:88px;">Miltex</td>
			<td style="width:88px;">17-307</td>
			<td style="width:88px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Needle holder</td>
			<td style="width:88px;">Boboz</td>
			<td style="width:88px;">RS-7840</td>
			<td style="width:88px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Monofilament suture</td>
			<td style="width:88px;">Ethicon</td>
			<td style="width:88px;">8698</td>
			<td style="width:88px;">Size 5-0</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Indicating controller</td>
			<td style="width:88px;">YSI</td>
			<td style="width:88px;">73ATD</td>
			<td style="width:88px;"></td>
		</tr>
	</tbody>
</table>

intrastriatal injection of autologous blood or clostridial collagenase as murine models of intracerebral hemorrhage

L&#39;hémorragie intracérébrale (HIC) est une forme commune de la maladie cérébro-vasculaire et est associée à une morbidité et une mortalité importantes. L&#39;absence de traitement efficace et l&#39;échec des grands essais cliniques visant à l&#39;hémostase et élimination des caillots de démontrer la nécessité d&#39;une enquête plus approfondie de mécanisme mécanique de l&#39;ICH. Cette recherche peut être effectuée par l&#39;intermédiaire du cadre prévu par les modèles précliniques. Deux modèles murins dans l&#39;usage populaire comprennent intrastriatale (ganglions de la base) injection de sang total autologue ou collagénase de Clostridium. Depuis, chaque modèle représente nettement différentes caractéristiques physiopathologiques liés au patrimoine culturel immatériel, l&#39;utilisation d&#39;un modèle particulier peut être choisi en fonction de ce que l&#39;aspect de la maladie est à l&#39;étude. Par exemple, l&#39;injection de sang autologue représente le plus fidèlement la réponse du cerveau à la présence de sang intraparenchymateuse, et peut reproduire le plus étroitement lobaire hémorragie. Injection de collagénase de Clostridium représente plus de précision les srupture de la cuve du centre commercial et hématome évolution caractéristique des hémorragies profondes. Ainsi, chaque modèle provoque la formation d&#39;un hématome différente, la réponse neuro-inflammatoire, le développement de l&#39;oedème cérébral, et les résultats neurocomportementaux. Robustesse d&#39;une intervention thérapeutique présumé peut être mieux évaluée en utilisant les deux modèles. Dans ce protocole, l&#39;induction de l&#39;ICH en utilisant les deux modèles, immédiate démonstration post-opératoire de blessures, et des techniques de soins post-opératoires précoces sont démontrées. Les deux modèles se traduisent par des blessures reproductibles, les volumes d&#39;hématome, et les déficits neurocomportementaux. En raison de l&#39;hétérogénéité de l&#39;ICH humaine, plusieurs modèles précliniques sont nécessaires pour explorer à fond les mécanismes physiopathologiques et tester des stratégies thérapeutiques potentielles.

En raison des différences dans la formation d&#39;un hématome (figure 1), tournant ipsilatéral est affiché immédiatement après le réveil pour les souris injectées de sang autologue et moins de 2 - 4 heures après l&#39;injection de collagénase, comme l&#39;expansion de l&#39;hématome se produit (figure 2). Absence de rotation ipsilatérale devrait soulever des inquiétudes pour absence de préjudice important. Le premier jour après des blessures, des souris dans les deux mod?...

Watch this Scientific Journal Video about Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage at JoVE.com

Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

Injection intrastriatale de sang autologue ou Clostridium collagénase comme modèles murins de Hémorragie intracérébrale

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective ...

intrastriatal-injection-autologous-blood-or-clostridial-collagenase

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14.6K vues.  Duke University. Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase. 

Vidéo: Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Fixed Volume or Fixed Pressure: A Murine Model of Hemorrhagic Shock

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, 2, 3, 4, 5 These signaling events can also lead to sepsis and/or multiple organ dysfunction (MOD). 6, 7, 8, 9 It is critical then to investigate the causes of suppressed immune function and detrimental signaling cascades in order to develop more effective ways to help patients who suffer from traumatic injuries. 10 This fixed pressure Hemorrhagic Shock (HS) procedure, although technically challenging, is an excellent resource for investigation of these pathophysiologic conditions. 11, 12, 13 Advances in the assessment of biological systems, i.e. Systems Biology have enabled the scientific community to further understand complex physiologic networks and cellular communication patterns. 14 Hemorrhagic Shock has proven to be a vital tool for unveiling these cellular communication patterns as they relate to immune function. 15, 16, 17, 18 This procedure can be mastered! This procedure can also be used as either a fixed volume or fixed pressure approach. We adapted this technique in the murine model to enhance research in innate and adaptive immune function. 19, 20, 21 Due to their small size HS in mice presents unique challenges. However due to the many available mouse strains, this species represents an unparalleled resource for the study of the biologic responses. The HS model is an important model for studying cellular communication patterns and the responses of systems such as hormonal and inflammatory mediator systems, and danger signals, i.e. DAMP and PAMP upregulation as it elicits distinct responses that differ from other forms of shock. 22, 23, 24, 25 The development of transgenic murine strains and the induction of biologic agents to inhibit specific signaling have presented valuable opportunities to further elucidate our understanding of the up and down regulation of signal transduction after severe blood loss, i.e. HS and trauma 26, 27, 28, 29, 30. 
There are numerous resuscitation methods (R) in association with HS and trauma. 31, 32, 33, 34 A fixed volume resuscitation method of solely lactated ringer solution (LR), equal to three times the shed blood volume, is used in this model to study endogenous mechanisms such as remote organ injury and systemic inflammation. 35, 36, 38 This method of resuscitation is proven to be effective in evaluating the effects of HS and trauma 38, 39.

The Hemorrhagic Shock model has been a reliable and reproducible resource facilitating the identification and understanding of signaling cascades associated with inflammation and end-organ damage after trauma. This article provides a step-by-step description of surgical and mechanical aspects associated with the Hemorrhagic Shock experimental procedure in mice.

Volume fixe ou pression fixe: un modèle murin de choc hémorragique

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, ...

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Médecine

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Ligature de l&#39;artère coronaire et injection intramyocardique dans un modèle murin d&#39;infarctus

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

Hémorragie intracérébrale chez la souris Modélisation: L&#39;injection de sang autologue ou collagénase bactérienne

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Un modèle murin d&#39;hémorragie sous-arachnoïdienne

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

Stéréologique et cytométrie de flux Caractérisation des sous-populations leucocytaires dans les modèles de transitoires ou permanentes ischémie cérébrale

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

Le lapin de sang-shunt modèle pour l&#39;étude de aiguë et les séquelles tardives de hémorragie méningée: Aspects techniques

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

Ferriques murin Thrombose modèles induits par Chlorure

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Guidée par échographie intracardiaque Injection des cellules souches mésenchymateuses d’augmenter le retour vers l’intestin pour une utilisation dans des modèles murins de maladies intestinales inflammatoires expérimentale

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator.
Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage.
This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

Injection guidée par stimulateur de nerf de cellules souches autologues près du nerf laryngé récurrent gauche équin

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

Ponction de la veine sous-clavière comme méthode Alternative de prélèvement d’échantillons de sang chez les Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...