Name: photothrombosis-induced focal ischemia as a model of spinal cord injury in mice
Uploaded: 2015-07-16T15:00:00
Description: Watch this Scientific Journal Video about Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice at JoVE.com

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 Instrumen...

<ol>
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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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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;Rose Bengal</td>
			<td style="width:233px;">Sigma-Aldrich</td>
			<td style="width:180px;">330000</td>
			<td style="width:421px;">20 mg/ml in sterile saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57Bl6/J</td>
			<td>Jackson lab</td>
			<td>664</td>
			<td>22-25g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine&#160;</td>
			<td>VEDCO</td>
			<td>NDC-50989-996-06</td>
			<td>100 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine&#160;</td>
			<td>VEDCO</td>
			<td>NDC-50989-234-11</td>
			<td>100 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine solution</td>
			<td>Purdue</td>
			<td>NDC-67618-150-01</td>
			<td>10% povidone iodine topical solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal saline</td>
			<td>Abott Laboratories</td>
			<td>04930-04-10</td>
			<td>For diluting RB, anaesthesia and for preventing tissue from drying</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Artificial tears ointment&#160;</td>
			<td>Rugby</td>
			<td>NDC-0536-6550-91</td>
			<td>83% white petrolatum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Decon labs.Inc</td>
			<td>2716</td>
			<td>70% ethanol for disinfection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Metal halide lamp</td>
			<td>EXFO, Canada</td>
			<td>X-Cite 120 PC&#160;</td>
			<td>Set power at 12%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring scissors&#160;</td>
			<td>Fine Science Tool</td>
			<td>15000-10</td>
			<td>for minor dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissors (angled to side)</td>
			<td>Fine Science Tool</td>
			<td>14063-011</td>
			<td>No. 3 handle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard scalpel</td>
			<td>Fine Science Tool</td>
			<td>10003-12</td>
			<td>for removing muscle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel blade</td>
			<td>Feather</td>
			<td>2976</td>
			<td>No. 10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps (curved)</td>
			<td>Fine Science Tool</td>
			<td>11150-10</td>
			<td>for holding tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps (straight)</td>
			<td>Fine Science Tool</td>
			<td>11151-10</td>
			<td>for holding tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle holder&#160;</td>
			<td>Fine Science Tool</td>
			<td>12002-12</td>
			<td>for suturing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue adhesive glue</td>
			<td>3M Vetbond</td>
			<td>1469SB</td>
			<td>to adhere to edges of the cut skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monofilament polypropylene&#160;</td>
			<td>USSC Sutures</td>
			<td>VP-521</td>
			<td>Size = 4-0 (for fascia)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perma-hand silk</td>
			<td>Ethicon</td>
			<td>683G</td>
			<td>Size = 4-0 (for skin)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro drill</td>
			<td>Roboz Surgical Instrument Co. Inc.</td>
			<td>RS-6300</td>
			<td>with bone polishing drill bit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laser doppler flowmeter</td>
			<td>Moor Instruments</td>
			<td>moorVMS-LDF1</td>
			<td>for monitoring change in blood flow</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating pad</td>
			<td>Fine Science Tool</td>
			<td>21052-00</td>
			<td>to prevent hypothermia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lab-Jack</td>
			<td>Fisher scientific&#160;</td>
			<td>14-673-50</td>
			<td>4x4 in plate to adjust the height of the animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-Y gliding stage&#160;</td>
			<td>Amscope</td>
			<td>GT100</td>
			<td>for positioning the animal under microscope&#160;&#160;</td>
		</tr>
	</tbody>
</table>

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) ...

Research

JoVE Journal

Medicine

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

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

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

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

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

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

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

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

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

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

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 ...