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

Ce travail a été soutenu par le National Institutes of Health [Grant pas. R01NS069726] et l&#39;Association Grant American Heart in Aid Grant [Grant pas. 13GRNT17020004] pour SD.

Note: Des souris (C57BL / 6J, mâle) ans 10-12 semaines fussent utilisés dans cette étude. Toutes les procédures ont été effectuées en conformité avec le Guide NIH pour le soin et l&#39;utilisation des animaux de laboratoire et ont été approuvés par l&#39;Université du Missouri institutionnel de protection des animaux et l&#39;utilisation Comité (IACUC). 1. Pré-chirurgie <ol><li> La veille de l&#39;intervention autoclave et stériliser tous les instruments chirurgic...

<ol>
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Time course of hemodynamic consequences.</a> J Cereb Blood Flow Metab. 6 (2), 184-194 (1986).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+protective+role+of+PBEF+in+a+mouse+model+of+cerebral+ischemia.">Neuronal protective role of PBEF in a mouse model of cerebral ischemia.</a> J Cereb Blood Flow Metab. 30 (12), 1962-1971 (2010).</li><li>Li, H., Zhang, N., Sun, G., Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+the+group+I+mGluRs+reduces+acute+brain+damage+and+improves+long-term+histological+outcomes+after+photothrombosis-induced+ischaemia.">Inhibition of the group I mGluRs reduces acute brain damage and improves long-term histological outcomes after photothrombosis-induced ischaemia.</a> ASN Neuro. 5 (3), 195-207 (2013).</li><li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological,+cellular+and+behavioral+assessments+of+stroke+outcomes+after+photothrombosis-induced+ischemia+in+adult+mice.">Histological, cellular and behavioral assessments of stroke outcomes after photothrombosis-induced ischemia in adult mice.</a> BMC Neurosci. 15, 58 (2014).</li><li>Wang, T., Cui, W., Xie, Y., Zhang, W., Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+Volume+of+the+Focal+Cerebral+Ischemic+Lesion+through+Photothrombosis.">Controlling the Volume of the Focal Cerebral Ischemic Lesion through Photothrombosis.</a> American Journal of Biomedical Sciences. 2 (1), 33-42 (2010).</li><li>Schroeter, M., Jander, S., Stoll, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+induction+of+focal+cerebral+ischemia+in+mice+by+photothrombosis+of+cortical+microvessels:+characterization+of+inflammatory+responses.">Non-invasive induction of focal cerebral ischemia in mice by photothrombosis of cortical microvessels: characterization of inflammatory responses.</a> J Neurosci Methods. 117 (1), 43-49 (2002).</li><li>Boquillon, M., Boquillon, J. 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Development of a model in the mouse.</a> Stroke. 31 (1), 208-213 (2000).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+simplified+spinal+cord+ischemia+model+in+mice.">Development of a simplified spinal cord ischemia model in mice.</a> J Neurosci Methods. 189 (2), 246-251 (2010).</li><li>Labat-gest, V., Tomasi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photothrombotic+ischemia:+a+minimally+invasive+and+reproducible+photochemical+cortical+lesion+model+for+mouse+stroke+studies.">Photothrombotic ischemia: a minimally invasive and reproducible photochemical cortical lesion model for mouse stroke studies.</a> J Vis Exp. (76), (2013).</li><li>Lu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+imaging+of+photothrombotic+stroke+in+conscious+and+freely+moving+rats.">Induction and imaging of photothrombotic stroke in conscious and freely moving rats.</a> J Biomed Opt. 19 (9), 96013 (2014).</li><li>Seto, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+ischemic+stroke+in+awake+freely+moving+mice+reveals+that+isoflurane+anesthesia+can+mask+the+benefits+of+a+neuroprotection+therapy.">Induction of ischemic stroke in awake freely moving mice reveals that isoflurane anesthesia can mask the benefits of a neuroprotection therapy.</a> Front Neuroenergetics. 6 (1), (2014).</li><li>Bell, M. 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Dans cette étude, nous avons décrit un modèle de photothrombotique SC ischémie. Grâce aux progrès en génie génétique, il ya eu une forte augmentation des souris transgéniques disponible dans le commerce qui a permis d&#39;étudier l&#39;impact des gènes spécifiques impliqués dans la physiopathologie ischémique dans la SC. Le but de l&#39;étude était de développer un modèle de souris reproductible de ischémie de la moelle épinière. Ici nous avons adapté un modèle PT corticale pour ...

Traumatisme de la moelle épinière (SCI) est un état clinique dévastatrice qui affecte les fonctions sensori-motrices et autonomes de la SC. SCI patients survivants se retrouvent souvent avec une paraplégie débilitante qui affecte considérablement leurs activités et de la qualité de la vie quotidienne 1. SCI modèles expérimentaux ont été un outil indispensable dans la recherche scientifique pour comprendre la physiopathologie de la SCI et les processus de réparation de neurones associés. Ces modèles ont également été utilisés pour tester l&#39;efficacité préclinique de diverses interventions neuroprotectrices expérimentales qui visent à la récupération fonctionnelle. Actuellement, la majorité des modèles SCI dans la pratique emploient l&#39;utilisation de la force physique brutale de perturber mécaniquement et blesser le SC. Ces méthodes comprennent contusion, compression, la dislocation et transection du SC 2. Il a été suggéré que, après l&#39;insulte mécanique primaire une lésion secondaire sous la forme d&#39;ensembles d&#39;ischémie dans la SC blessé<html> 3,4. L&#39;étiologie de l&#39;ischémie secondaire comprend une vaste dégénérescence des tissus, l&#39;hémorragie parenchymateuse et parfois par l&#39;obstruction des vaisseaux sanguins par un œdème des tissus 5-7. À la suite de la lésion secondaire l&#39;intégrité de SC est en outre affectée, les neurones et les cellules gliales sont gravement altérées dans la fonction et la viabilité et l&#39;apoptose qui conduit à l&#39;infarctus croissance au cours de la phase chronique de la blessure, analogue à la croissance de pénombre ischémique suite d&#39;un AVC 8,9. Plusieurs mécanismes comme excitotoxicité, production de radicaux libres, et l&#39;inflammation ont été signalés à être responsable de la mort cellulaire ischémique suivant SCI 10,11. En outre, SC ischémie est une complication grave de l&#39;anévrisme aortique thoraco-abdominaux chirurgies de réparation qui conduisent souvent à la paraplégie chez les patients 12,13. En dépit de cet impact clinique haute très peu de modèles de ischémie de la moelle épinière avec une reproductibilité élevée sont actuellement disponibles. nt &quot;&gt; Photothrombosis (PT) est une méthode couramment utilisée pour l&#39;induction d&#39;une ischémie focale dans le cerveau 14-20. La technique est assez non-invasif, hautement reproductible et produit une lésion ischémique focal précis dans la zone exposée du cerveau 17 -21. Ceci est réalisé par administration systémique de colorants photoactifs tels que le rose Bengale (RB) 16-20,22 érythrosine B ou 23 suivie par une irradiation localisée de vaisseaux sanguins avec une source de lumière appropriée. photoactivation du colorant provoque la génération de radicaux libres qui perturber l&#39;intégrité de l&#39;endothélium vasculaire lisse, et provoquent les plaquettes d&#39;accumuler, qui forme ensuite un thrombus. L&#39;obstruction de la circulation sanguine par les résultats de thrombus dans un infarctus dans la région fournies par le navire 24. En raison de la facilité de contrôle de la l&#39;intensité et la durée de l&#39;irradiation de cette procédure donne un infarctus très uniforme et reproductible. En outre, ce procédé peut être utilisé pour induire un infarctust à divers emplacements anatomiques permettant spatiale (par exemple, la matière grise vs. matière blanche) compréhension de l&#39;effet de l&#39;ischémie. L&#39;objectif de la présente étude est de développer un modèle facile et hautement reproductible de SC ischémie chez les souris. Nous avons décrit la procédure d&#39;un modèle d&#39;ischémie PT de SC chez des souris. Les résultats de l&#39;histologie et immunomarquage ont démontré que PT peut induire efficacement SC du myocarde, une perte neuronale et une gliose réactive.

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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.

Le but de cette étude était de produire ischémie de la moelle épinière chez des souris en utilisant un modèle de PT. Après la région désirée de l&#39;os au-dessus de la moelle épinière (T10 - T12) a été amincie, Rose Bengale a été injecté à travers le sinus rétro-orbital itinéraire, et l&#39;ischémie a été induite par PT Figure 1A, B correspond à la souris placée dans un instrument chirurgical sur mesure. plate-forme au cours de la chirurgie. La souris a été maintenu en place ...

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 induite par ischémie focale comme un modèle de lésion médullaire chez la souris

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

Aiguë et chronique Test tactile sensorielle après un traumatisme médullaire chez le rat

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

Recherche

Médecine

Controlled Cervical Laceration Injury in Mice

Contrôlé blessures de lacération du col chez la souris

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

Un modèle de contusion de graves lésions de la moelle épinière chez les 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

Ischémie-reperfusion modèle de lésions rénales aiguës et post blessures fibrose chez la souris

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

Reproductible paraplégie par l&#39;aorte thoracique occlusion dans un modèle murin de la moelle épinière ischémie-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

Contrôlée corticale modèle d&#39;impact pour 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, ...

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

Un système de radio-télémétrique de surveiller la fonction cardiovasculaire chez le rat avec la moelle épinière Transection et embryonnaires souches neurales greffes cellulaires

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 Transplantation de cellules souches dans la partie expérimentale Modèle contusion de la moelle épinière des blessures

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

Calibré Forceps modèle de compression de la moelle épinière des blessures

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

Un nouveau-né de la moelle épinière de souris Compression Modèle de lésion

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