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><tbody><tr><td>Stereotactic frame</td><td>Stoelting Co.</td><td>51603</td><td></td></tr><tr><td>Probe holder with corner clamp</td><td>Stoelting Co.</td><td>51631</td><td></td></tr><tr><td>Mini grinder</td><td>Power Glide</td><td>Model 60100002</td><td></td></tr><tr><td>0.5 &amp;mu;l syringe</td><td>Microliter</td><td>86259</td><td>25 G needle</td></tr><tr><td>5 &amp;mu;l syringe</td><td>Microliter</td><td>7637-01</td><td></td></tr><tr><td>30 G microliter syringe</td><td>Microliter</td><td>7762-03</td><td></td></tr><tr><td>Syringe pump</td><td>KD Scientific</td><td>Model 100</td><td></td></tr><tr><td>Heat therapy water pump</td><td>Gaymar Industries, Inc.</td><td>Model# TP650</td><td></td></tr><tr><td>Circulating waterbed</td><td>CMS Tool &amp;amp; Die, Inc.</td><td></td><td></td></tr><tr><td>Rodent ventilator</td><td>Harvard Apparatus</td><td>Model 683</td><td></td></tr><tr><td>Isoflurane vaporizer</td><td>Drager</td><td>Vapor 19.1</td><td></td></tr><tr><td>Air flowmeter</td><td>Cole Parmer</td><td>Model PMR1-010295</td><td></td></tr><tr><td>Induction chamber</td><td></td><td></td><td>Self made</td></tr><tr><td>Otoscope</td><td>Welch Allyn</td><td>22820</td><td></td></tr><tr><td>Intravenous catheter</td><td>Becton-Dickinson</td><td>381534</td><td>20 G, 1.16 inch Insyte-W</td></tr><tr><td>Isoflurane</td><td>Baxter Healthcare Corporation</td><td>NDC10019-360-69</td><td></td></tr><tr><td>Collagenase Type IV-S</td><td>Sigma</td><td>C1889</td><td></td></tr><tr><td>Polyethylene tubing PE10</td><td>Becton-Dickinson</td><td>427401</td><td></td></tr><tr><td>27 G 1 1/4 inch needle</td><td>Becton-Dickinson</td><td>305136</td><td></td></tr><tr><td>Surgical scissors</td><td>Miltex</td><td>21-539</td><td></td></tr><tr><td>Forceps</td><td>Miltex</td><td>17-307</td><td></td></tr><tr><td>Needle holder</td><td>Boboz</td><td>RS-7840</td><td></td></tr><tr><td>Monofilament suture</td><td>Ethicon</td><td>8698</td><td>Size 5-0</td></tr><tr><td>Indicating controller</td><td>YSI</td><td>73ATD</td><td></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

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Medicine

14.7K 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

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model. While ICH
accounts for 10-15% of all strokes, there remains no specific
effective therapy. The autologous blood injection model in mice
involves the stereotaxic injection of arterial blood into the basal
ganglia mimicking a spontaneous hypertensive hemorrhage in man. The
response to hemorrhage can then be studied in vivo and the
neurobehavioral deficits quantified, allowing for description of the
ensuing pathology and the testing of potential therapeutic agents. The
procedure described in this protocol uses the double injection
technique to minimize risk of blood reflux up the needle track, no
anticoagulants in the pumping system, and eliminates all dead space
and expandable tubing in the system.

The autologous blood injection model of intracerebral hemorrhage in mice described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.

Eigenblut Injection zu Spontane intrazerebrale Blutungen in Mäuse-Modell

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

Forschung

Neurowissenschaften

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.

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

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 ...

Medizin

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 ...

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and contributes significantly to the development of vascular dementia. Few models of focal white matter stroke exist and this lack of appropriate models has hampered understanding of the neurobiologic mechanisms involved in injury response and repair after this type of stroke. The main limitation of other subcortical stroke models is that they do not focally restrict the infarct to the white matter or have primarily been validated in non-murine species. This limits the ability to apply the wide variety of murine research tools to study the neurobiology of white matter stroke. Here we present a methodology for the reliable production of a focal stroke in murine white matter using a local injection of an irreversible eNOS inhibitor. We also present several variations on the general protocol including two unique stereotactic variations, retrograde neuronal tracing, as well as fresh tissue labeling and dissection that greatly expand the potential applications of this technique. These variations allow for multiple approaches to analyze the neurobiologic effects of this common and understudied form of stroke.

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

Ein vielseitiges Mausmodell von Subcortical Weiß Matter Stroke für das Studium der axonalen Degeneration and White Matter Neurobiologie

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Intra-arterielle Lieferung von neuronalen Stammzellen an das Ratten- und Maushirn: Anwendung auf zerebrale Ischämie

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

Doppelte Direkteinspritzung von Blut in die Cisterna Magna als Modell der subarachnoiden Blutung

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

Massive Pontine Hemorrhage by Dual Injection of Autologous Blood

We provide a protocol to establish a massive pontine hemorrhage model in a rat. Rats weighing about 250 grams were used in this study. One hundred microliters of autologous blood was taken from the tail vein and stereotaxically injected into the pons. The injection process was divided into 2 steps: First, 10 &#181;L of blood was injected into a specific location, anteroposterior position (AP) -9.0 mm; lateral (Lat) 0 mm; vertical (Vert) -9.2 mm, followed by a second injection of the residual blood located at AP -9.0 mm; Lat 0 mm; Vert -9.0 mm with a 20-minute interval. The balance beam test, limb placement test, and the modified Voestch neuroscore were used to evaluate neurological function. Magnetic Resonance Imaging (MRI) was used to assess the volume of hemorrhage in vivo. The symptoms of this model were in line with patients with massive pontine hemorrhage.

We present a protocol to establish a massive pontine hemorrhage model in a rat via dual injection of autologous blood.

Massive Pontinenblutung durch Doppelinjektion von Eigenblut

We provide a protocol to establish a massive pontine hemorrhage model in a rat. Rats weighing about 250 grams were used in this study. One hundred ...

Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely afflicting a fairly young population. Several animal models of SAH have been developed to investigate the pathophysiological mechanisms behind SAH and to test pharmacological interventions. The pre-chiasmatic, single injection model in the rat presented in this article is an experimental model of SAH with a predetermined blood volume. Briefly, the animal is anesthetized, intubated, and kept under mechanical ventilation. Temperature is regulated with a heating pad. A catheter is placed in the tail artery, enabling continuous blood pressure measurement as well as blood sampling. The atlantooccipital membrane is incised and a catheter for pressure recording is placed in the cisterna magna to enable intracerebral pressure measurement. This catheter can also be used for intrathecal therapeutic interventions. The rat is placed in a stereotaxic frame, a burr hole is drilled anteriorly to the bregma, and a catheter is inserted through the burr hole and placed just anterior to the optic chiasm. Autologous blood (0.3 mL) is withdrawn from the tail catheter and manually injected. This results in a rise of intracerebral pressure and a decrease of cerebral blood flow. The animal is kept sedated for 30 min and given subcutaneous saline and analgesics. The animal is extubated and returned to its cage. The pre-chiasmatic model has a high reproducibility rate and limited variation between animals due to the pre-determined blood volume. It mimics SAH in humans making it a relevant model for SAH research.

Subarachnoid hemorrhage continues to carry a high burden of mortality and morbidity in man. To facilitate further research into the condition and its pathophysiology, a pre-chiasmatic, single injection model is presented.

Prächiasmatische, einmalige Injektion von Eigenblut zur Induktion einer experimentellen Subarachnoidalblutung in einem Rattenmodell

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely ...

Inducing Massive Pontine Hemorrhage in a Rat Model by Autologous Blood Injection

<a href='https://app.jove.com/v/62089/massive-pontine-hemorrhage-by-dual-injection-of-autologous-blood'>Source: Tang, X. et. al., Massive Pontine Hemorrhage by Dual Injection of Autologous Blood. J. Vis. Exp. (2021)</a>This protocol describes the controlled infusion of autologous blood into the pontine region of a rat's brain to model massive pontine hemorrhage. The purpose is to simulate a localized brain bleed by increasing localized pressure.

Source: Tang, X. et. al., Massive Pontine Hemorrhage by Dual Injection of Autologous Blood. J. Vis. Exp. (2021)This protocol describes the controlled ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Cerebrovascular Diseases

Establishing a Subarachnoid Hemorrhage Rat Model Induced by Autologous Blood Injection

To place the laser Doppler probe, make a 15-millimeter incision caudally in the midline, starting just anterior to the eyes. After removing the connective tissue and the muscles with forceps, use the end of a sterile cotton swab as a regime to identify the bregma and the coronal sutures.
After placing the armed retractor, place a 25-gauge spinal needle in the stereotaxic frame exactly on the bregma and note the position. Next, remove the needle from the bregma. Move the frame anteriorly by 6.5 millimeters, then replace the needle in the midline to mark the side of drilling.
Drill until the dura mater is identified below the bone. Using straight forceps, gently remove the bone fragments, then fill the cavity with bone wax. Next, 3 to 4 millimeters lateral to the right of the bregma and just anterior to the coronal suture for the laser Doppler, drill another hole taking care not to penetrate the dura mater.
Look for the vessels where the laser Doppler can measure the blood flow. Place the laser Doppler and check the values. A minimum value of 100 flux units is required. If the values remain acceptable after removing the microscope, add 1 drop of glue to fix the probe. Recheck to confirm whether the value is above 80 flux units.
To induce subarachnoid hemorrhage, insert the needle gently through the skull in the midline between the hemispheres until resistance is felt due to the base of the skull. Retract the needle by 1 millimeter to ensure correct placement just anteriorly to the optic chiasm. To ensure the most homogeneous result when injecting the blood, turn the needle clockwise by 90 degrees such that the needle tip points to the right. Then, remove the stiletto.
After a 15-minute equilibration, adjust the level of anesthesia to obtain a mean arterial blood pressure in the range 80 to 100 millimeters of mercury. Then, perform a blood gas analysis to confirm that the pH, partial pressure of carbon dioxide and partial pressure of oxygen are within physiological parameters.
Next, using a 1-milliliter syringe with a blunt 23-gauge needle, withdraw 500 microliters of blood from the tail catheter. To avoid injection of air, fill the dead space of the spinal needle chamber with blood. After adjusting the volume of the blood in the syringe to 300 microliters, connect the syringe to the spinal needle. Then, grasping firmly around the syringe, inject the blood manually to surpass mean arterial pressure. Observe a steep rise in the intracerebral pressure and a concurrent steep fall in the cerebral blood flow.