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.

Ethikerklärung: Dieses Protokoll wurde von der Duke University Institutional Animal Care und Verwenden Ausschuss gebilligt wurde und folgt alle Richtlinien für die ethische Nutzung von Tieren. 1. Vorbereitung der Ausrüstung <ol><li> Autoklaven werden die Operationswerkzeuge vor der Operation. </li><li> Desinfizieren Sie die stereotaktische Apparat mit 70% Ethanol. </li><li> Schalten Sie Wasserbad und halten die Wassertemperatur bei 42 ° C </li><li> Auflösen Typ IV-S Clostridien Kollagenase in physiologischer Kochsalzlösung in einer Konzentration von 0,075 U pro 0,4 &amp;mgr;. </li></ol> 2. Collagenase Injection Modell <ol><li> Wiegen Sie die Maus. </li><li> Anesthetize die Maus in eine Induktionskammer mit 5% Isofluran in 30% O 2/70% N 2. Adäquate Anästhesie ist nach ca. 2 min, wenn die Maus Atmung haben, 1 pro Sekunde verlangsamt signalisiert. </li><li> Intubate die Luftröhre mit einem 30 mm 20 G intravenösen Katheter. </li><li> Verbinden Sie den Katheter einNagetier Ventilator und mechanisch lüften die Lunge mit 1,6% Isofluran in 30% O 2/70% N 2 bei einer Rate von 105 Atemzügen pro Minute mit einem Tidalvolumen von 0,75 ml .. </li><li> Rasieren Sie die Kopfhaut mit einem elektronischen Rasierer. Sobald die Maus anästhesiert und intubiert, verschieben Sie es an einem anderen Arbeitsplatz für die Rasur und kehrte in die chirurgische Bank dann. </li><li> Sichern Sie den Kopf in einem stereotaktischen Rahmen, und ebnen den Kopf mit beiden koronalen und Pfeilnaht als Referenzpunkte. </li><li> Bewerben Augensalbe auf die Augen. </li><li> Legen Sie eine rektale Temperatursonde. Aufrechterhaltung Rektaltemperatur bei 37,0 ± 0,2 º C unter Verwendung eines zirkulierenden Wasserbettboden. </li><li> Wischen den Operationsbereich mit Betadin, gefolgt mit 70% Ethanol und 3-mal wiederholen. </li><li> Machen Sie eine 1 cm Mittellinie Kopfhauteinschnitt und wischen Knochenhaut seitlich mit einem sterilen Wattestäbchen zu Bregma aussetzen. </li><li> Drill 1 mm Durchmesser Bohrloch 2,2 mm links latBundes mit einem wassergekühlten Bohrer Bregma. </li><li> Drehen Kollagenase Fläschchen 5-mal, dann waschen eine 0,5 ul Spritze mit 25 G-Nadel (zum stereotaktischen Rahmen befestigt) mit 0,5 ul Collagenase-Lösung 5 mal Pinnwand (0,5 ul der Collagenase-Lösung in der Spritze nach dem letzten Waschgang). </li><li> Richten Nadelspitze mit Bohrloch dann vertreiben 0,1 ul aus Spritze und Nadel wischen Kegel mit gestochen zu verwerfen. </li><li> Mit einem Mikromanipulator, vorab die Nadel 3 mm tief, um Rinde und lassen für 30 Sekunden regungslos. </li><li> Inject 0,4 ul über 90 sek. </li><li> Verringern Isofluran auf 1% und lassen Nadel bewegungslos für 5 min. </li><li> Ziehen Nadel langsam. </li><li> Wenden 1-2 Tropfen 0,25% Bupivacain subkutan und Naht der Haut. </li><li> Schalten Sie Isofluran-Verdampfer und Maus zu entfernen aus der stereotaktischen Rahmen. </li><li> Lassen Sie Maus, um Spontanatmung mit anschließender Luftröhren Extubation erholen. </li><li> Zurück Maus an einen sauberen Käfig und ermöglichen den freien Zugang zuNahrung und Wasser. </li></ol> 3. Eigenblut Injection Modell <ol><li> Führen Sie die Schritte 2,1-2,11 für die Kollagenase Injektion Modell. </li><li> Draw 50 ul sterilem normaler Kochsalzlösung in eine 30 G 50 ul Spritze. </li><li> Verbinden Sie den Mikroliter-Spritze mit einer 70 cm PE10 Rohr. </li><li> Vertreibt alle normalen Kochsalzlösung aus Mikroliter-Spritze in PE10 Rohr komplett de-Luftschlauch. </li><li> Ziehen Sie den Mikroliterspritze Kolbens aus 1 mm, um eine Luftblase an der distalen Öffnung des röhren PE10 Mikroliterspritze Vorrichtung zu machen Gemisch aus Kochsalzlösung und Blut im späteren Verfahren zu vermeiden. </li><li> Wischen Sie den distalen Schwanzarterie zentralen Region des Maus mit 70% Ethanol, und schneiden Sie die Arterie mit einer Rasierklinge in 0,5 bis 1 cm auf die Schwanzspitze. </li><li> Sammeln Sie 40 ul Blut aus dem Schwanz in die PE10 Rohr-Mikroliterspritze Gerät geschnitten. Hinweis: das Heparin nicht in der Nadel, Schläuche oder Maus. </li><li> Befestigen Sie den Mikroliterspritze auf die injection Pumpe. </li><li> Verbinden der Metallkanüle Teil einer 27 G-Nadel auf das Ende der Röhre PE10 und sichern die Nadel in einem Mikromanipulator auf dem stereotaktischen Rahmen. </li><li> Vertreibt 2 ul Blut von 27 G-Nadel und wischen Nadel Kegel mit gestochen zu verwerfen. </li><li> Richten Nadelspitze mit Bohrloch und Nadel 3 mm tief, um Kortex. </li><li> Spritzen Sie 35 ul der autologen Blut mit einer Rate von 2 ul pro min. </li><li> Verringern Isofluran auf 1% und lassen Nadel bewegungslos für 10 min. </li><li> Ziehen Nadel über 30 sek. </li><li> Wenden 1-2 Tropfen 0,25% Bupivacain subkutan und Naht der Haut. </li><li> Schalten Sie Isofluran-Verdampfer und Maus zu entfernen aus der stereotaktischen Rahmen. </li><li> Lassen Sie Maus, um Spontanatmung mit anschließender Extubation erholen. </li><li> Zurück Maus an einen sauberen Käfig und lassen Sie freien Zugang zu Futter und Wasser. </li></ol> 4. Sham Betrieb <ol><li> Führen Sie die gleichen Verfahren für die Kollagenase injection-Modell, jedoch ohne Injektion nach Nadeleinführung. </li></ol> 5. Post-Chirurgie Pflege <ol><li> Injizieren von 0,5 ml physiologischer Kochsalzlösung subkutan in den Abend des chirurgischen Eingriffs an der Rückseite des Halses des Tieres. </li><li> Geben enthärtetem Wasser und Nahrung mit Gel-Essen in kleinen Plastikbecher auf dem Boden des Käfigs platziert. Ersetzen Sie das Essen täglich für 7 Tage. </li><li> Überprüfen Sie für die Gewichtsabnahme, Wundheilung, und Anzeichen von Unbehagen täglich für 7 Tage. </li><li> Wenn Erholungsintervalle von mehr als 7 Tage erforderlich sind, können Nahtentfernung unter Licht inhaliert Anästhesie (ca. 1% Isofluran in 30% O 2/70% N 2) durchgeführt werden, falls erforderlich. </li></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.

Trotz Schwellen präklinischen Forschung und der daraus resultierenden großen klinischen Studien für vielversprechende Therapeutika 15-18, es gibt keine pharmakologischen Interventionen gezeigt, dass Ergebnis in ICH verbessern und Pflege bleibt weitgehend unterstützend. Listen von möglichen Therapien können durch Hochdurchsatz-Technologien, wie Transkriptom-und Proteom-Arbeit erzeugt werden. Während diese Technologien weiter, unser Wissen über mögliche therapeutische Ziele voranzubringen, vorwärts un...

Intrazerebrale Blutung (ICH) ist eine relativ häufige Form von zerebrovaskulären Erkrankungen mit etwa 40-50% der betroffenen Patienten sterben innerhalb von 30 Tagen ein. Leider hat sich wenig Verbesserung der Sterblichkeit in den letzten 20 Jahre 2 gemacht. Berichte aus den National Institutes of Health 3 und Richtlinien der American Heart Association 4 betont die Bedeutung der Entwicklung klinisch relevanten Modelle von ICH, das Verständnis der Pathophysiologie zu erweitern und zu entwickeln, Ziele für neue therapeutische Ansätze. Einige Modelle gibt es für die menschliche ICH 5 imitieren. Wie Verständnis der Pathophysiologie ICH reift, hat es sich gezeigt, dass eine Vielzahl von Modellen verwendet werden, um verschiedene Aspekte der Krankheit zu untersuchen. Früher verwendete Modelle sind murine Amyloid-Angiopathie 6, intraparenchymal Mikroballon Einsetzen und Inflation 7 und direkten arteriellen BlutInfiltration 8,9. Lobäre Blutung aus Amyloid-Angiopathie ist mit der Verwendung von transgenen Mäusen, modelliert und stellt einen deutlichen ICH-Subtyp. Mikroballonmodelle imitieren akuten Massenwirkung von Hämatombildung, aber nicht zu zellulären Reaktion des Gehirns auf die Anwesenheit von Blut zu erfassen. Schließlich unterzieht direkten arteriellen Blut Infiltration des Gehirns, um den arteriellen Druck der Oberschenkelarterie. So ist dieses Modell imitiert arteriellen Druck und die Anwesenheit von Blut, aber nicht das Gehirn unterziehen, um mikrovaskulären Schädigung von kleinen Blutgefäß Bruch. Weiterhin hat dieses Modell inhärent hohe Variabilität. Interessant ist, spontan hypertensiven Ratten 10 entwickeln spontane ICH wie sie altern. Untersuchung dieser Tiere nach ICH-Entwicklung kann die Krankheit in Gegenwart eines der Hauptbegleiterkrankungen prädisponiert Menschen ICH imitieren. Während diese anderen Modelle gibt, intrastriatalen Injektion von Clostridien-Kollagenase 11 oder instrastiatal Injektion einutologous Vollblut 12 sind derzeit in der präklinischen Forschung ICH die beiden häufigsten Modelle. ICH Modellauswahl auf Basis der Ziel der experimentellen Frage, einschließlich Artenwahl und Art der Hämatombildung induziert werden. Zum Beispiel Schweine sind große Tiere mit relativ großen Volumen der weißen Substanz des Gehirns im Vergleich zu Mäusen. So sind Schweine Modelle geeignet, um der weißen Substanz Pathophysiologie folgende ICH studieren. Im Gegensatz dazu sind Nagetier Gehirn weitgehend grauen Substanz, aber transgene Systeme machen Nager nützlich, um die molekularen Mechanismen der Schädigung und Erholung nach ICH beurteilen. Jedes Modell hat seine eigenen Stärken und Schwächen (Tabelle 1), die vor dem Experiment sorgfältig betrachtet werden sollten. Die folgenden Protokolle zeigen die Eigenblutinjektion und Collagenase-Modellen bei Mäusen. Diese Modelle haben jeweils von Modelle, die ursprünglich bei Ratten entwickelt übersetzt13,14 und erlauben die Verwendung von transgenen Technologie weit verbreitet, um die molekularen Mechanismen mit Zelltod nach ICH verbunden zu erkunden. Beide stellen deutlich verschiedene Verletzungsmechanismen von menschlichen ICH, und beide haben deutlich unterschiedliche erwartete Ergebnis in Bezug auf Verhaltens-und histologische Maßnahmen. So sind bestimmte Hypothesen können sich auf ein Modell, über den anderen zu leihen, aber viele Ideen kann die Validierung in beiden Modellen erfordern. Tabelle 1. Vergleich der Eigenschaften von Kollagenase-und Eigenblutinjektion Hirnblutung Modelle. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td style="width:197px;"></td><td style="width:80px;"> Collagenase-Injection </td><td style="width:66px;"> Blutinjektion </td></tr><tr><td style="width:197px;"> Benutzerfreundlichkeit </td><td style="width:80px;"> + + + </td><td style="width:66px;"> + + </td></tr><tr&gt; <td style="width:197px;"> Reproduzierbarkeit </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;"> Kontrolle der Blutung Größe </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + + </td></tr><tr><td style="width:197px;"> Blut Reflux </td><td style="width:80px;"> + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;"> Simuliert Human Disease </td><td style="width:80px;"> + </td><td style="width:66px;"> - </td></tr><tr><td style="width:197px;"> Einfachheit </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;"> Verwendung in mehreren Arten </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr></tbody></table>

<table border="0" cellpadding="0" cellspacing="0" width="416">
	<tbody>
		<tr height="43">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Mini grinder</td>
			<td style="width:88px;">Power Glide</td>
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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

Intrazerebralen Blutungen (ICH) ist eine häufige Form von zerebrovaskulären Erkrankungen und ist mit einer signifikanten Morbidität und Mortalität verbunden. Mangel an wirksamen Behandlung und Miss großen klinischen Studien bei Blutstillung und Entfernung von Blutgerinnseln richtet zeigen die Notwendigkeit weiterer Mechanismus gesteuerte Untersuchung der ICH. Diese Forschung kann durch die Rahmen von präklinischen Modellen vorgesehen durchgeführt werden. Zwei Mausmodellen im populären Gebrauch sind intrastriatalen (Basalganglien) Injektion von autologem Vollblut oder Clostridien-Kollagenase. Seit Jedes Modell stellt deutlich unterschiedliche pathophysiologische Funktionen ICH Zusammenhang kann die Verwendung eines bestimmten Modells auf Basis welcher Aspekt der Krankheit untersucht werden soll gewählt werden. Zum Beispiel Eigenblutinjektion am genauesten entspricht der Reaktion des Gehirns auf die Anwesenheit von intraparenchymal Blut und kann lobäre Blutung am nächsten replizieren. Clostridien-Kollagenase-Injektion am genauesten stellt die sMall Gefäßruptur und Hämatome Evolution charakteristisch tiefen Blutungen. So, jeder Modellergebnisse in verschiedenen Hämatombildung, neuroinflammatorischen Antwort, Hirnödem Entwicklung und neurologisch-Ergebnisse. Robustheit eines angeblichen therapeutischen Intervention am besten mit beiden Modellen bewertet werden. In diesem Protokoll Induktion von ICH mit beiden Modellen unmittelbaren postoperativen Demonstration von Verletzungen und frühen postoperativen Pflege Techniken werden demonstriert. Beide Modelle führen zu reproduzierbaren Verletzungen, Hämatome Volumen und Neuropsychologische Defizite. Aufgrund der Heterogenität der menschlichen ICH werden mehrere präklinischen Modellen benötigt, um gründlich zu erforschen pathophysiologischen Mechanismen und Testen potentieller therapeutischer Strategien.

Aufgrund der Unterschiede in Hämatombildung (Abbildung 1), wird ipsilateral Wende sofort nach dem Aufwachen für Eigenblut injizierten Mäusen gezeigt und innerhalb von 2 - 4 Stunden nach der Kollagenase-Injektion, wie Hämatome Expansion auftritt (Abbildung 2). Fehlen ipsilateralen Wende sollte Sorge um Abwesenheit von erheblichen Verletzungen erhöhen. Am ersten Tag nach der Verletzung sollte Mäuse in beiden Modellen erhebliche neurologische Defizite (Abbildung 3) z...

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.

Intrastriataler Injektion von Eigenblut oder Clostridien-Kollagenase als Murine Modelle der Hirnblutung

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

Research

JoVE Journal

Medicine

14.6K Aufrufe.  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. 

Serie: 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.

Fixed Volume oder Feste Druck: einem Mausmodell des hämorrhagischen Schocks

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, ...

Forschung

Medizin

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.

Koronare Ligation und intramyokardialen Injection in einem Mausmodell der Infarkt

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.

Modeling Hirnblutung in Mice: Injektion von Eigenblut oder bakterielle Collagenase

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.

Einem Mausmodell der Subarachnoidalblutung

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.

Stereo und Durchflusszytometrie Charakterisierung von Leukozyten-Subpopulationen in Models der vorübergehend oder bleibend zerebrale Ischämie

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.

Das Kaninchen Blut-Shunt-Modell für die Untersuchung von akuten und Spätfolgen Subarachnoidalblutung: Technische Aspekte

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.

Ferric Chloride-induzierte Murine Thrombosis Models

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.

Ultraschall-geführte intrakardiale Injektion humaner mesenchymaler Stammzellen Homing in den Darm zur Verwendung in murinen Modellen von experimentellen entzündlichen Darmerkrankungen erhöhen

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.

Nerv Stimulator-gesteuerte Injektion von autologen Stammzellen in der Nähe der Pferde Links wiederkehrende Laryngeal Nerv

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.

Vene subclavia Punktion als eine Alternative Methode zur Probe Blutentnahme bei Ratten

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