Diese Arbeit wurde von den National Institutes of Health [Gabe nicht unterstützt. R01NS069726] und der American Heart Association in Grants Beihilfen [Zuschuss Nr. 13GRNT17020004] auf SD.

Hinweis: Die Mäuse (C57BL / 6J, männlich) im Alter 10 bis 12 Wochen um waren in dieser Studie verwendet. Alle Verfahren wurden in Übereinstimmung mit der NIH Leitfaden für die Pflege und Verwendung von Labortieren durchgeführt und wurden von der University of Missouri Institutional Animal Care und Verwenden Committee (IACUC) zugelassen. 1. Pre-Chirurgie <ol><li> Der Tag vor der Operation Autoklaven sterilisieren und alle chirurgischen Instrumenten. Wickeln Sie die Instrumente und Autoklav bei 121 ° C bei 15 psi für 30 Minuten, gefolgt von 30 min zum Trocknen (121 o C, 15 psi, 30/30 Zyklus). Legen Sie die Instrumente in einem sauberen und sterilen Umgebung bis zur weiteren Verwendung. </li><li> Bereiten Sie frische Rose Bengal (RB) Lösung (20 mg / ml in steriler Kochsalzlösung), jedes Mal vor der Operation. Um sich vollständig aufzulösen RB, Wirbel die dann durch Ultraschallbehandlung für 5 min bei 50/60 Hz mit einer Ausgangsleistung von 19 W. Wickeln Sie das Rohr in Aluminiumfolie und vor Lichtröhre bis auf weiteres während sur verwendetgery. </li><li> Bereiten Sie eine Mischung von Ketamin / Xylazin in steriler Kochsalzlösung. Fügen Sie 125 ul von Xylazin (Stammkonzentration: 20 mg / ml) und 325 &amp; mgr; l ketmaine (Stammkonzentration: 100 mg / ml) und 550 &amp; mgr; l steriler Kochsalzlösung, um ein Gesamtvolumen von 1 ml des Anästhetikums Mischung zu machen. </li><li> Pre-warm die homeothermic Heizkissen. </li><li> Pre-warm die Metallhalogenlampe (Lichtquelle für FN1 Epi-Fluoreszenzmikroskop) für 30 Minuten, um die Lampenleistung zu stabilisieren. </li><li> Anpassen der Größe des beleuchteten Bereichs auf einen Durchmesser von 1 mm unter Verwendung von 10X-Objektiv und einer Strichplatte durch Einstellung der Feldblende in der aufrechten FN1 Epi-Fluoreszenzmikroskop. </li></ol> 2. Chirurgisches Verfahren <ol><li> Betäuben die Maus mit einer Dosis von Ketamin (130 mg / kg B. Gew.) Und Xylazin (10 mg / kg B. Gew.). Auf der Grundlage der Cocktail in 1.3, 4 &amp; mgr; l pro g Maus B. Gew. werden gebraucht. Sterilisieren Sie die Injektionsstelle mit einem Alkoholtupfer und Verwaltung der Anästhesie durch den inner-peritoneal (IP) Route. Achten Sie darauf, die Betäubungsmittel in die Blutgefäße oder Muskel injizieren, da dies würde die Ein- und Ausleitung des Tieres zu verzögern. </li><li> Künstliche Tränen Salbe auf beiden Augen von der Maus, um ein Austrocknen zu verhindern und legen Sie das Tier auf dem Heizkissen, um Unterkühlung zu verhindern. </li><li> Aufbereiten von tierischen <ol><li> Überprüfen Sie das Tier für die ordnungsgemäße chirurgische Ebene der Anästhesie durch Verwendung des toe Prise Antwort. </li><li> Sobald das Tier chirurgischen Narkosetiefe erreicht hat, und klemmen die Haare auf der Rückenoberfläche um Mittellinie des Tieres mit einem elektrischen Haarschneider. Scheuern die Operationsstelle mit 70% Ethanol, gefolgt von Betadin-Lösung dreimal. Decken Sie die Website mit einem sterilen Operationstuch bis zum nächsten Schritt. </li></ol></li><li> Chirurgisches Verfahren, um dünne Knochen um das Rückenmark freizulegen <ol><li> Platzieren Sie die Maus in Bauchlage über die homeothermic Heizkissen auf der chirurgischen Plattform (Abbildung 1A). Die Maus richtig zu sichernHaltung mit einem Schnauzen Klemme einen länglichen Halsbereich (1A, B) zu halten. </li><li> Einen Einschnitt (etwa 1 cm Länge) unter Verwendung von chirurgischen Scheren entlang der dorsalen Mittellinie, die sich von der Brustwirbel T9 bis T12. Abnehmen der Haut, OP-Bereich freizulegen. </li><li> Mit einem Skalpell vorsichtig deaktivieren Sie das Muskel, um die Rückenstacheln bei T9 aussetzen - T12 Wirbel. Blutungen stoppen in jedem Schritt durch leichten Druck mit sterilen Wattestäbchen. Separate T10 - T12 Wirbel von der umgebenden Muskeln und sichern Sie diese mit Hilfe eines Wirbelklemme zu stabilisieren und jede Bewegung verhindert wird (Abbildung 1A, B). </li><li> Verwendung eines Hochgeschwindigkeitsbohrer mit Knochen Polieren Bohrer, vorsichtig und sanft dünne der dorsalen Oberfläche des T10 oder T11 Wirbels der hinteren Rückenader und andere kleine Schiffe auf der dorsalen Oberfläche des SC (1C) zu visualisieren. </li><li> Zu thermischen Schäden aufgrund von Wärme während der Ausdünnung Prozedur erzeugt verhindern,eine milde und konstanten Strom von normaler Kochsalzlösung mit konstanter Saugleistung, um die Trümmer zu entfernen. </li><li> Mit einem Skalpell vorsichtig glatt die Knochenoberfläche, bis der Hauptbehälter ist deutlich sichtbar. Darauf achten, dass das Rückenmark bei diesem Vorgang beschädigt werden. </li><li> Sobald das Blutgefäß visualisiert verabreichen RB bei einer Dosis von 30 mg / kg (Körpergewicht) durch retro-orbitalen Sinus Route unter Verwendung einer Insulinspritze. </li><li> Messen Sie den Blutfluss mit Hilfe eines Laser-Doppler-Durchflussmesser nach 3 min folgenden RB Injektion bei Bedarf (2A, B). Pflegen Asepsis während gesamte Verfahren. </li></ol></li></ol> 3. Induktion von PT <ol><li> Legen Sie das Tier auf einem XY-Position einstellbar Stufe über einen Lab-Jack, der die Höhe justieren kann. Einstellen der Position der Maus, so dass die freigelegten Bereich T11 Rückenmark ist direkt unter dem 10X-Objektiv des FN1 Epi-Fluoreszenzmikroskop (3A). </li><li> Stellen Sie die Macht des liGHT Quelle bei 12% und bestrahlen die Region T11 mit einem Durchmesser von 0,75 mm in der Mitte des ausgedünnten Rückenmark (Anmerkung: Diese Region umfasst die hintere Rücken Vene und anderen Kapillaren) mit grünem Licht (Wellenlänge 540-580 nm, das ist durch den Filterwürfel in dem Mikroskop) durch die 10X-Objektiv für 2 min erreicht wird. Bilder aufnehmen, am Anfang und Ende des Bestrahlungs (3B, C) ​​und Aufzeichnen der Zeit des Experiments an dieser Stelle. </li><li> Messung der Blutfluss wieder für 10 min bei Bedarf, indem der Laser-Doppler-Sonde an der gleichen Position über dem Rückenmark, wie in 2.4.8 (2A, B). </li><li> Nach der Bestrahlung Prüfung für jede Blutung und wenn keiner gefunden, Annähen des Tieres gehen. Naht der oberflächliche Faszie zusammen mit den Muskeln auf der jeder Seite des Rückenmarks mit einem resorbierbaren Naht oder 4-0 Größe Seidenfaden. Achten Sie darauf, um die freiliegende SC beschädigen. Naht der Haut mit 4-0 Seidennaht. Bewerben Betadine oder Jodzu den Rändern der Haut nach dem Vernähen. </li></ol> 4. Post-Chirurgie Pflege <ol><li> Nach dem Nähen, legen Sie das Tier auf dem Heizkissen für die Wiederherstellung. Nach der Wiederherstellung überprüfen Sie die Tiere auf Anzeichen von neurologischen Defiziten durch die Beobachtung der Bewegung der beiden hinteren Extremitäten. Stellen Sie das Tier nicht unbeaufsichtigt lassen, bis er wieder zu sich kam, um ausreichende Brustlage zu halten. </li><li> Übertragen Sie die Tiere auf den Käfig. Stellen Sie das Tier, das der Operation an die Firma von anderen Tieren unterzogen wurde, bis vollständig erholt nicht zurück. </li><li> Überprüfen Sie die Tiere in regelmäßigen Abständen. Im Falle von schweren neurologischen Defiziten, bieten die richtige Pflege wie Evakuierung von Blase, Verabreichung von Analgetika (Buprenorphin, 0,05 bis 0,1 mg / kg). Überprüfen Sie für die Dehydratisierung und verwalten normaler Kochsalzlösung subkutan in schweren Fall. Üblicherweise wird Buprenorphin (0,1 mg / kg) nach dem Vernähen zu Schmerzen im Operationsstelle zu lindern, verabreicht werden. </li><li> Wenn Tiere nicht sofortnach der Operation getötet, werden wir mit hohem Wassergehalt Ernährung auf dem Käfigboden setzen, damit die Tiere die Nahrung leicht zu erreichen. </li></ol> 5. transkardialer Perfusion, Nissl-Färbung und Immunfärbung <ol><li> Transkardial perfundieren das Tier wie zuvor 17-20 beschrieben. <ol><li> Das Tier zu betäuben, wie zuvor in dem Protokoll beschrieben und transkardial mit Phosphatpuffer-Salzlösung (PBS, pH 7,4), gefolgt von eiskaltem 4% Paraformaldehyd (PFA) in PBS perfundiert. </li><li> Nach Durchblutung, entfernen Sie das Rückenmark (SC) und post-fix it in 4% PFA in PBS bei 4 ° CO / N. Übertragen Sie die Fest SC in PBS mit 30% Saccharose und halten Sie sie für 2 - 3 Tage, bis es auf den Boden der Röhre sinkt. </li><li> Verwendung eines Kryostaten geschnitten das Rückenmark in 30 &amp; mgr; m dicke Abschnitte und sie seriell auf eine Gelatine-beschichtete Glasobjektträger oder in einem 48-Well-Platte mit 0,01 M PBS. </li></ol></li><li> Nissl-Färbung: Um die Verletzungsgefahr durch PT verursacht inspizieren führen eine Nissl staining auf Rückenmarksabschnitte wie zuvor beschrieben, 17-20. <ol><li> Kurz gesagt, sammeln jeden fünften Rückenmark Scheibe auf den Glasobjektträger und Färbung mit 0,25% Kresylviolett. Nehmen Sie Bilder von den gefärbten Schnitten (Abbildung 4). </li></ol></li><li> Immunfärbung: Wie vorher mit einem schwimmenden Abschnitt Verfahren 17,18,20 beschrieben. <ol><li> Kurz gesagt, färben die Rückenmarkschnitte durch Inkubieren O / N bei 4 ° C mit Kaninchen-anti-Glia fibrilläres saures Protein (GFAP) polyklonalem Antikörper (1: 300), Kaninchen-anti-NeuN Antikörper (1: 300), und Kaninchen-anti- Iba1 Antikörper (1: 500), gefolgt von Esel-Anti-Kaninchen Alexa 568-konjugierten IgG (1: 400) Sekundärantikörper für 4 h bei RT. Bilder aufnehmen, mit einem Fluoreszenzmikroskop (Abbildung 5). </li></ol></li></ol>

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The authors have nothing to disclose.

In dieser Studie haben wir beschrieben, eine photothrombotic Modell SC Ischämie. Aufgrund der Fortschritte in der Gentechnik hat es eine Schwankung in der im Handel erhältlichen transgenen Mäuse, die es möglich gemacht hat, um die Auswirkungen von spezifischen Genen in das ischämische Pathophysiologie im SC beteiligt studieren. Das Ziel der Studie war es, eine reproduzierbare Mausmodell der Ischämie des Rückenmarks zu entwickeln. Hier angepasst wir eine kortikale PT Modell SCI in Mäusen zu induz...

Traumatische Rückenmarksverletzungen (SCI) ist eine verheerende klinischen Zustand, die die sensomotorische und autonome Funktionen des SC. Patienten überleben SCI sind oft mit schwächenden Querschnittslähmung, die ihre täglichen Aktivitäten und die Lebensqualität erheblich beeinträchtigt 1 verlassen. Experimental SCI Modelle wurden zu einem unverzichtbaren Werkzeug in der wissenschaftlichen Forschung, um die Pathophysiologie der SCI und die damit verbundenen neuronalen Reparaturprozesse zu verstehen. Diese Modelle wurden auch verwendet, um die Wirksamkeit von verschiedenen präklinischen experimentellen neuroprotektive Eingriffe, bei funktionellen Erholung abzielen testen. Derzeit Mehrheit der SCI-Modelle in der Praxis beschäftigen die Verwendung von physikalischen stumpfe Gewalt mechanisch stören und schädigen die SC. Diese Methoden umfassen Quetschung, Kompression, Dislokation und Durchtrennung des SC 2. Es wurde vorgeschlagen, dass nach der primären mechanischen Insult eine sekundäre Verletzungen in Form von Sätzen in Ischämie im verletzten SC<html> 3,4. Die Ätiologie der sekundären Ischämie beinhaltet umfangreiche Gewebedegeneration, parenchymale Blutungen und manchmal durch Verstopfung von Gefäßen durch Gewebsödeme 5-7. Als Folge der Sekundärverletzung die Integrität SC ist weiter beeinflusst werden Neuronen und Gliazellen stark in Funktion und Lebensfähigkeit beeinträchtigen und Apoptose die an das Wachstum in der chronischen Phase der Schädigung Infarkt führt analog zu unterziehen, um das Wachstum von ischämischen Penumbra nach Schlaganfall 8,9. Mehrere Mechanismen wie Exzitotoxizität, Produktion von freien Radikalen und Entzündungen berichtet worden, für die ischämischen Zelltod nach SCI 10,11 verantwortlich. Darüber hinaus ist SC Ischämie eine ernste Komplikation der thorakoabdominale Aortenaneurysma Reparatur Operationen, die oft zu Querschnittslähmung bei den Patienten, 12,13. Trotz dieser hohen klinischen Auswirkungen gibt sehr wenige Modelle von Rückenmark-Ischämie mit hoher Reproduzierbarkeit verfügbar. nt &quot;&gt; Photothrombosis (PT) ist ein allgemein verwendetes Verfahren für die Induktion einer fokalen Ischämie im Gehirn 14-20. Die Technik ist ziemlich nicht-invasiven, hoch reproduzierbar und ergibt eine genaue fokalen ischämischen Läsion in dem belichteten Bereich des Gehirns 17 -21. Dies wird durch die systemische Verabreichung von photoaktiven Farbstoffen wie Rose Bengal (RB) 16-20,22 oder Erythrosin B erzielt 23, gefolgt von lokalisierten Bestrahlung der Blutgefäße mit der richtigen Lichtquelle. Die Photoaktivierung des Farbstoffs bewirkt die Erzeugung von freien Radikalen, stören die Integrität des glatten vaskulären Endothel, und bewirken, daß die Plättchen zu sammeln, die anschließend bildet einen Thrombus. die Behinderung der Blutströmung durch den Thrombus führt zu einem Infarkt in der von dem Behälter 24 zugeführt Bereich. Aufgrund der Kontrolle über die Leichtigkeit Intensität und Dauer der Bestrahlung dieses Verfahren ergibt sich eine sehr gleichmäßige und reproduzierbare Infarkt. Darüber hinaus kann dieses Verfahren verwendet, um eine infarc induzierent an verschiedenen anatomischen Stellen ermöglicht räumliche (zB graue Substanz vs. weißen Substanz) Verständnis der Wirkung der Ischämie. Das Ziel der vorliegenden Studie ist es, eine einfache und hoch reproduzierbare Modell SC-Ischämie bei Mäusen zu entwickeln. Beschrieben wir die Prozedur eines PT-Modell SC Ischämie in Mäusen. Ergebnisse aus Histologie und Immunfärbung nachgewiesen, dass PT effektiv induzieren SC Infarkt, Neuronenverlust und reaktive Gliose.

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			<td height="21" style="height:21px;width:216px;">&#160;Rose Bengal</td>
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			<td>Jackson lab</td>
			<td>664</td>
			<td>22-25g</td>
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			<td>NDC-50989-234-11</td>
			<td>100 mg/ml</td>
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			<td>Abott Laboratories</td>
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			<td>X-Cite 120 PC&#160;</td>
			<td>Set power at 12%</td>
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			<td height="21" style="height:21px;">Spring scissors&#160;</td>
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			<td>for minor dissection</td>
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			<td>for removing muscle</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Scalpel blade</td>
			<td>Feather</td>
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			<td height="21" style="height:21px;">Forceps (curved)</td>
			<td>Fine Science Tool</td>
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			<td>3M Vetbond</td>
			<td>1469SB</td>
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			<td height="21" style="height:21px;">Monofilament polypropylene&#160;</td>
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			<td height="21" style="height:21px;">Perma-hand silk</td>
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			<td>Fine Science Tool</td>
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			<td height="21" style="height:21px;">Lab-Jack</td>
			<td>Fisher scientific&#160;</td>
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photothrombosis-induced focal ischemia as a model of spinal cord injury in mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Das Ziel dieser Studie war es, das Rückenmark Ischämie bei Mäusen mit einem PT-Modell zu erzeugen. Nach der gewünschten Knochenbereich über dem Rückenmark (T10 - T12) wurde verdünnt wurde Rose Bengal durch retro-orbitalen Sinus Weg injiziert, und Ischämie wurde von PT induziert 1A, B zeigen die Maus positioniert eine maßgeschneiderte chirurgisch. Plattform während der Operation. Die Maus wurde anstelle von einer Schnauze Klammer gehalten und zwei verstellbare wirbelKlemmen zur Stabilisierung d...

Watch this Scientific Journal Video about Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice at JoVE.com

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Photothrombosis induzierte fokale Ischämie als ein Modell der Rückenmarksverletzungen in Mäusen

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

photothrombosis-induced-focal-ischemia-as-model-spinal-cord-injury

Research

JoVE Journal

Medicine

13.1K Aufrufe.  University of Missouri. Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice. 

Serie: Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Akute und chronische taktile Sensorische Testung nach Verletzungen des Rückenmarks bei der Ratte

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

Forschung

Medizin

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Controlled Cervical Laceration Verletzungen in Mäuse

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

Eine Prellung Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischämie-Reperfusion-Modell des akuten Nierenschädigung und nach der Verletzung Fibrose bei Mäusen

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

Reproduzierbare Paraplegie von thorakalen Aorta Occlusion in einem Mausmodell der Spinal Cord Ischämie-Reperfusion

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Controlled Cortical Impact-Modell für traumatische Hirnverletzung

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

Ein Radio-Telemetrie-System zur Herz-Kreislauf-Funktion bei Ratten mit Rückenmarksdurchtrennung und embryonalen Neural Stem Cell Grafts überwachen

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Neural Stem Cell Transplantation in Experimental Gequetscht Modell von Rückenmarksverletzungen

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

Calibrated Zange Modell Spinal Cord Injury Compression

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Ein Neonatal Maus Spinal Cord Injury Kompression Modell

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