This works was supported by funding from a Florida Health grant (Brain and spinal cord injury research fund) (KKW).

Alle Verfahren wurden von der Institutional Animal Care und Use Committee der University of Florida und in Übereinstimmung mit den National Institutes of Health Guide für die Pflege und Verwendung von Labortieren unter Protokolle # 201207692 genehmigt durchgeführt. 1. Animal Care <ol><li> Verwenden Sie 3-4 Monate alten männlichen C57BL / 6J-Mäuse. Geben Sie Bettwäsche, Nistmaterial, Nahrung und Wasser ad libitum. Halten Sie die Mäuse in einer Umgebungstemperatur gesteuert, um bei 20 bis 22 ° C mit konstanten 12-Stunden-Licht / 12-Stunden-Dunkel-Zyklen. </li></ol> 2. Pre-Impaktion Vorbereitung <ol><li> Bringen Sie eine maßgeschneiderte Silikon-Kautschuk beschichteten Metallspitze auf eine elektromagnetische stereotaxische Schlaggerät. Achten Sie darauf , die flache Unterseite der Spitze an die Oberfläche der Sondenspitze parallel ist (Abbildung 1A). </li><li> Anesthetize die Maus mit 4% Isofluran durch Aufrechterhaltung der Narkose von 2,5% Isofluran gefolgt. Überprüfen Sie die Anästhesie über den Durchflussmesser. Monitor die Anästhesie Niveau, bis das Tier erreicht eine chirurgische Ebene der Anästhesie durch den Verlust des Pedalrückzugsreflex zeigt. </li><li> Setzen Sie die Maus in Bauchlage auf einem Heizkissen. Verwenden Sie einen trichterförmigen Bugspitze der Maus unter Narkose zu halten. rasieren Sie vollständig den Kopf ein Trimmer. Verwenden Sie Petrolatum Augensalbe auf die Augen der Maus Trockenheit während der Narkose zu verhindern. </li></ol> 3. Auswirkungen Parameter Einstellung HINWEIS: Die Auswirkungen System enthält eine Steuerbox Schlagparameter einzustellen, eine Betätigungsvorrichtung der Impaktion und einen digitalen stereotaktischen Rahmen mit 3-Bewegungsachsen durchzuführen. <ol><li> die Geschwindigkeit der Schlagvorrichtung zu 4 m / s und die Verweilzeit auf 240 ms auf dem Steuerkasten voreingestellt. </li></ol> 4. Positionierung der Auswirkungen Zentrum <ol><li> Setzen Sie ein weiches Heizkissen unter dem Körper des Tieres in der Nähe von 39 ° C, um die Körpertemperatur zu halten. Montieren Sie die Maus in einem stereotaktischen Rahmen in einem prone-Position mit den stumpfen Enden Ohr Bars. </li><li> Senken Sie die Schlagspitze nahe an den Kopf der Maus durch die Z-Fahrer bewegt. Stellen Sie die flachen Schlagspitze (9 mm Durchmesser) durch die X- und Y-Bewegen-Treiber auf halbem Weg auf das Ziel über der Pfeilnaht Koordinaten. </li><li> Achten Sie darauf , eine Kante der Schlagspitze senkrecht zu einer gedachten horizontalen Linie zwischen den beiden Ohren (1C) gezogen wird parallel. Das Zentrum des Aufpralls entspricht dem zentralen Sagittalnaht auf halbem Weg zwischen interfrontal und lambdoid Nähten (interaurale 9 mm 0 mm Interaurale, seitlich 4,5 mm). </li></ol> 5. Auswirkungen Tiefeneinstellung <ol><li> Für eine korrekte Schlagtiefe eingestellt wurden, können zusätzliche Sondenspitze die isolierte Silikonkautschuk beschichtet Schlagspitze zu ersetzen. </li><li> Um sicherzustellen, dass es keine Verschiebung des Aufpralls Zentrum nach dem Einschalten Tipps, die X- und Y-Kanal auf der digitalen Stereotaxie-Bedienfeld auf Null vor dem Einschalten der Spitzen gesetzt. </li><li> Bewegen Sie den probe Spitze zur Mitte des Bereichs Auswirkungen durch manuell die X-und Y-Antriebe bewegen. </li><li> Clip Kontaktsensor an den Schwanz der Maus. </li><li> Bewegen Sie den Impaktor (Z-Antrieb) nach unten, bis die Sondenspitze die Oberfläche der Aufprallstelle berührt. </li><li> Stellen Sie den Z-Kanal auf dem stereotaktischen Bedienfeld auf Null. </li><li> Bewegen Sie die Schlagspitze zurück in den Einwirkungsbereich durch eine manuelle Einstellung der X- und Y-Treiber (nicht der Null-Tasten auf der digitalen Stereotaxie-Bedienfeld), bis X- und Y-Treiber sind Null (wobei die Schlagspitze zuvor positioniert wurde). </li><li> Ziehen Sie den Aktuator durch Bewegen des Rückzugs-Schalter auf dem Steuerkasten. Manuelles Verschieben der Impaktor nach unten (Z-Treiber) von 4 mm. </li></ol> 6. Auswirkungen <ol><li> Lösen Sie die Auswirkungen durch Klicken auf die Schlagschalter auf dem Steuerkasten und erreichen eine Verformungstiefe von 4 mm. </li></ol> 7. Post-Impaktion <ol><li> Messen Sie die Zeit von den Auswirkungen, bis der erste Atem Maus mit Hilfe einer Zeitschaltuhr. </li><li&gt; Das Tier aus dem Gerät entfernen und sie an einem warmen Pad platzieren, um ihre Körpertemperatur zu halten. Nicht das Tier unbeaufsichtigt lassen, bis es genügend Bewusstsein zu halten Brustlage wiedergewonnen hat. </li><li> Lassen Sie für die Wiederherstellung, bevor das Tier wieder in einen sauberen Käfig zurück. Verwenden Sie kein Tier an die Firma von anderen Tieren zurückkehren, bis sie vollständig erholt. </li><li> Bitte beachten und die Mäuse wiegen täglich. Wenn die Mäuse Anzeichen von Schmerzen zeigen, intraperitoneal sie mit Meloxicam injizieren mit einem 1 - 2 mg / kg alle 12 bis 24 Stunden. </li></ol> 8. Repetitive Impaction <ol><li> Geben Sie den Mäusen zusätzliche Verletzungen an den Tagen 4, 7 und 10 nach der ersten Verletzung (72 Stunden Intervall zwischen den Auswirkungen). </li></ol> 9. Immunhistochemie (IHC) <ol><li> transkardialer Perfusion <ol><li> Anesthetize die Mäuse über intraperitoneale Injektion mit 200 m / kg Pentobarbital. </li><li> Beurteilen und versichern chirurgische Ebene der Anästhesie durch eine Zehe Prise. Sekundeure die Maus in Rückenlage durch sanft die Vorderpfoten und Hinterpfoten auf einem Styropor-Arbeitsfläche innerhalb einer chemischen Abzugshaube Taping. </li><li> Machen Sie einen Schnitt durch die Haut entlang der Brustmittellinie von knapp unterhalb der Xiphoidbasis zum Schlüsselbein. Machen Sie zwei zusätzliche Hautschnitte am Xiphoidbasis und gehen seitlich entlang der Basis des ventralen Brustkorbs. </li><li> Öffnen Sie die Brusthöhle und setzen das Herz, indem sie durch die Brustmuskulatur und Brustkorb zu schneiden. </li><li> Sichern Sie das schlagende Herz mit stumpfen Pinzette und machen 1 - 2 mm Einschnitt im linken Ventrikel. </li><li> einfügen Sofort Butterfly-Nadel in den rechten Vorhof. Beginnen Sie mit der Infusion von 20 ml Kochsalzlösung durch die Spritze langsam schieben. </li><li> Wechseln Sie von Kochsalzlösung auf 4% Paraformaldehyd. Weiter Perfusion mit 20 ml Paraformaldehyd. </li><li> Enthaupten die Maus und entfernen Sie die Haut mit einer Schere. Isolieren Sie das Gehirn aus dem Schädel eines Knochenschneider. </li></ol></li><li> Cryostat sectioning <ol><li> Einbetten von Hirngewebe in optimaler Schnitttemperatur (OCT) Formulierung und Einfrieren bei -80 ° C. Legen Sie das Gehirn in den Kryostaten in sagittaler Ausrichtung. Schneiden Gehirnschnitte 5 &amp; mgr; m dick. </li></ol></li><li> Die Färbung <ol><li> Trocknen der gefrorenen Schnitte bei Raumtemperatur für 1 Std. </li><li> Inkubieren der Objektträger mit 100 ul 2% Ziegenserum und 0,1% Triton X-100 in phosphatgepufferter Kochsalzlösung (PBS) für 1 h bei RT. </li><li> Waschen Sie die Folien 3-mal mit 300 ul PBS. Dann Inkubieren der Objektträger mit Anti-GFAP (1: 200) oder anti-Ferritin-Antikörper (1: 200) getrennt über Nacht bei 4 ° C. </li><li> Waschen Sie die Folien 3-mal mit 300 ul PBS. Dann Inkubieren der Objektträger für 2 h bei Raumtemperatur mit Biotin-konjugiertem sekundärem Antikörper. </li><li> Waschen Sie die Folien 3-mal mit 300 ul PBS. Dann Inkubieren der Objektträger mit Avidin-Biotin-Komplex (ABC) Lösung (1:50) bei Raumtemperatur für 30 min. </li><li>Waschen Sie die Folien 3-mal mit 300 ul PBS. Dann Inkubieren in 3,3&#39;-Diaminobenzidin (DAB) Substratlösung (50 ml PBS, 10 &amp; mgr; l H 2 O 2, 10 mg DAB Tablette, Filter vor der Verwendung) für 5 bis 8 min. Den Objektträger unter dem Mikroskop, bis die positiven Zellen erscheinen. </li><li> Spülen Sie die Folien in langsam fließendem Leitungswasser für 5 min. Saubere Folien mit einem Labor-abzuwischen. Dann montieren Sie die Abschnitte mit Eindeckmedium und Deckglas. </li></ol></li></ol>

<ol>
	<li>Baugh, C. M., 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=Chronic+traumatic+encephalopathy:+neurodegeneration+following+repetitive+concussive+and+subconcussive+brain+trauma.">Chronic traumatic encephalopathy: neurodegeneration following repetitive concussive and subconcussive brain trauma.</a> Brain Imaging Behav. 6 (2), 244-254 (2012).</li><li>McKee, A. C., 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=Chronic+traumatic+encephalopathy+in+athletes:+progressive+tauopathy+after+repetitive+head+injury.">Chronic traumatic encephalopathy in athletes: progressive tauopathy after repetitive head injury.</a> J. Neuropathol Exp Neurol. 68 (7), 709-735 (2009).</li><li>Petraglia, A. L., Dashnaw, M. L., Turner, R. C., Bailes, J. 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M., 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=Functional+assessment+of+long-term+deficits+in+rodent+models+of+traumatic+brain+injury.">Functional assessment of long-term deficits in rodent models of traumatic brain injury.</a> RegenMed. 8 (4), 483-516 (2013).</li><li>Xiong, Y., Mahmood, A., Chopp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+traumatic+brain+injury.">Animal models of traumatic brain injury.</a> Nat Rev Neurosci. 14 (2), 128-142 (2013).</li><li>Luo, J., 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=Long-term+cognitive+impairments+and+pathological+alterations+in+a+mouse+model+of+repetitive+mild+traumatic+brain+injury.">Long-term cognitive impairments and pathological alterations in a mouse model of repetitive mild traumatic brain injury.</a> Front Neurol. , 5-12 (2014).</li><li>Yang, 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=Temporal+MRI+characterization,+neurobiochemical+and+neurobehavioral+changes+in+a+mouse+repetitive+concussive+head+injury+model.">Temporal MRI characterization, neurobiochemical and neurobehavioral changes in a mouse repetitive concussive head injury model.</a> Sci Rep. 10 (5), 11178 (2015).</li><li>Zhang, J., 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=Inhibition+of+monoacylglycerol+lipase+prevents+chronic+traumatic+encephalopathy-like+neuropathology+in+a+mouse+model+of+repetitive+mild+closed+head+injury.">Inhibition of monoacylglycerol lipase prevents chronic traumatic encephalopathy-like neuropathology in a mouse model of repetitive mild closed head injury.</a> J Cereb Blood Flow Metab. 35 (3), 443-453 (2015).</li><li>Petraglia, A. 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The authors have no financial interest to disclose.

Um Verletzungen des Gehirns morphologisch ähnlich dem klinischen Zustand, Post-Gehirnerschütterung Symptome erwartet imitieren. Post-Gehirnerschütterung Symptome in der Regel sind Kopfschmerzen, Schwindel, Schwindel, Müdigkeit, Gedächtnis und Schlafprobleme, Probleme sowie Angst und depressive Stimmung zu konzentrieren. Da somatische Symptome in Tiermodellen können noch nicht messbar sind, werden die Änderungen von motorischen und kognitiven Funktion und emotionale Verhalten als Kriterien für die Bewertung ratio...

Erschütternder, auch milde traumatische Hirnverletzungen (mTBI) genannt, ist die häufigste Auftreten von traumatischen Hirnverletzungen (TBI) und betrifft Millionen von Menschen in USA. Concussions kann schwierig zu diagnostizieren und es gibt keine spezifische Behandlung für Gehirnerschütterung. Es gibt eine wachsende Anerkennung und einige Hinweise darauf , dass eine geringe mechanische Trauma von Sportverletzungen, militärischen Kampf und andere körperlich eingreifenden Beschäftigungen haben kumulativ und chronische neurologische Konsequenzen 1,2. Allerdings gibt es immer noch ein Mangel an Wissen über Gehirnerschütterungen und deren Auswirkungen. Die derzeitigen Verfahren schränkt die Studien der Pathologie und Behandlung bei Menschen, da nur neurologische Beurteilung und Bildauswertung für die klinische Diagnose zur Verfügung stehen. Tiermodelle bieten ein Mittel Kommotio in einer effizienten, rigoros zu studieren, und kontrollierten Art und Weise mit der Hoffnung auf die weitere Diagnose und Behandlung von mTBI. Studien haben angepasst traditionellen TBIModelle wie kortikale Wirkung (CCI), Fluid-Schlagwirkung (FPI), Gewicht fallen Verletzungen gesteuert und Explosion Verletzungen mTBI durchzuführen und durch Änderung der Schädigungsparameter niedrige Verletzungsschweren stimulieren. Diese Modelle sind vorteilhaft zu verwenden, aufgrund ihrer Fähigkeit, Gehirntrauma morphologisch ähnlich dem klinischen Zustand zu replizieren; sie haben jedoch auch ihre eigenen Grenzen. Die Schwere der Verletzung durch einen Beschleunigungsverletzung (Gewichtsabfall) induziert ist oft sehr variabel. Die beiden Ergebnisse des milden CCI - Subarachnoidalblutung und fokale Quetschung - nicht vergleichbar mit typischen menschlichen Gehirnerschütterungen. CCI und FPI erfordern eine Kraniotomie, die klinisch nicht relevant ist, während Explosion Verletzungen ein umstrittener Modell in Bezug auf die unterschiedlichen Belichtungsposition und Spitzendruckmessungen sowie variable Sekundärverletzung während der Belichtung ist 3-6. Ein aktualisierter concussive Tiermodell das kann der präklinischen Forschung in die klinische setti übersetzenng ist in der Forschung notwendig. Die zentrale Frage mild TBI bei der Modellierung ist die experimentelle Verletzungsschwere zu definieren, die am ehesten mit der Verletzung in einer klinischen Umgebung repliziert. Vor kurzem entwickelte verschiedenen Forschungsgruppen des geschlossenen Kopfverletzung oder concussive Kopfverletzung (CHI) Modell 7-10. CHI ist eine Modifikation des CCI ohne Kraniotomie, aber es nutzt noch eine traditionelle elektronische magnetische Wirkung System einen Kopfaufprall zu erzeugen. Ein CHI kann durch Einstellen der Schlagparameter eine Gehirnerschütterung im Bereich von leichten bis mittelschweren induzieren. Verlust des Bewusstseins (LOC) kann durch Erfassen einer Abnahme der Atemfrequenz oder der transienten Beendigung des Atmens unmittelbar nach einem Aufprall zu beachten. Die Zeit des LOC wird verwendet, um die Schwere der Verletzung zu bestimmen. Dieses Papier enthält eine leicht verbesserte und aktualisierte Version eines wiederholten CHI (RCHI) Modell bei Mäusen, zusammen mit einer detaillierten Schritt-für-Schritt-Protokoll und repräsentative Ergebnisse. Die RCHI Modell Forschungsstrategien einre vorteilhaft bei der Bestimmung mTBI Effekte und mögliche Behandlungen, vor allem, da es keine einzelne Tiermodell der Nachahmung all der Erschütterung induzierten pathologischen Veränderungen fähig ist.

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			<td height="21" style="height:21px;width:216px;">anesthesia machine</td>
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			<td>perform impaction</td>
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			<td height="21" style="height:21px;width:216px;">Digital Stereotaxic instrument</td>
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			<td>mount mouse and positioning tips</td>
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			<td height="42" style="height:42px;width:216px;">Sicilone rubber-coated metal tip</td>
			<td style="width:188px;">Precision Tool &#38; Engineering, Gainesvill FL</td>
			<td style="width:137px;">custom-made</td>
			<td>impact tip</td>
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			<td height="21" style="height:21px;width:216px;">Lithium Ion All-in-One Trimmer</td>
			<td style="width:188px;">WAHL Home Products</td>
			<td style="width:137px;">9854-600</td>
			<td>shave mouse hair</td>
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			<td height="23" style="height:23px;width:216px;">paper clips</td>
			<td style="width:188px;">custom-made</td>
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			<td>probe tip</td>
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			<td height="21" style="height:21px;width:216px;">Cotton tipped applicators</td>
			<td style="width:188px;">MEDLINE</td>
			<td style="width:137px;">MDS202055</td>
			<td>scrub head with saline</td>
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			<td height="21" style="height:21px;">Tissue Tek O.C.T.</td>
			<td style="width:188px;">ASKURA FINETEK USA INC</td>
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			<td height="21" style="height:21px;width:216px;">anti-GFAP</td>
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			<td>antibody for IHC</td>
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			<td height="21" style="height:21px;width:216px;">anti Ferritin</td>
			<td style="width:188px;">Sigma</td>
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			<td>antibody for IHC</td>
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		<tr height="21">
			<td height="21" style="height:21px;">VECTASTAIN Elite ABC&#160; kit</td>
			<td style="width:188px;">Vector laboratories</td>
			<td style="width:137px;">PK-6100</td>
			<td>IHC detection system</td>
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			<td height="21" style="height:21px;width:216px;">Permount Mounting Medium</td>
			<td style="width:188px;">Fisher Scientific</td>
			<td style="width:137px;">SP15-100</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aperio XT ScanScope scanner</td>
			<td style="width:188px;">Leica Microsystems Inc,</td>
			<td style="width:137px;"></td>
			<td>slides scanning</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Leica AutoStainer XL</td>
			<td style="width:188px;">Leica the pathology Company</td>
			<td style="width:137px;">ST2010</td>
			<td>H&#38;E staining</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DAB&#160;</td>
			<td style="width:188px;">sigma</td>
			<td style="width:137px;">D3939</td>
			<td>IHC detection system</td>
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a repetitive concussive head injury model in mice

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of knowledge on the injury and its effects. To develop a better understanding of concussions, animals are often used because they provide a controlled, rigorous, and efficient model. Studies have adapted traditional animal models to perform mTBI to stimulate mild injury severity by changing the injury parameters. These models have been used because they can produce morphologically similar brain injuries to the clinical condition and provide a spectrum of injury severities. However, they are limited in their ability to present the identical features of injuries in patients. Using a traditional impact system, a repetitive concussive injury (rCHI) model can induce mild to moderate human-like concussion. The injury degree can be determined by measuring the period of loss of consciousness (LOC) with a sign of a transient termination of breathing. The rCHI model is beneficial to use for its accuracy and simplicity in determining mTBI effects and potential treatments.

In diesem Modell (Abbildung 1 AC), gab es kurze Perioden von Keuchen und flach respirations. Ein Verlust des Bewusstseins (unbewussten) als eine Abnahme der Atemfrequenz oder vorübergehende Beendigung der Atmung definiert, bevor eine normale Atmung wieder aufzunehmen. Ein Einfluss auf die Mitte des Kopfes verursacht kurzfristige Bewusstlosigkeit (7,5 ± 4,7, 7,8 ± 5,5, 10,2 ± 8,8, 9,5 ± 8,0 Sekunden bei jedem Aufprall getrennt, 1D). Gehirn der Maus z...

Watch this Scientific Journal Video about A Repetitive Concussive Head Injury Model in Mice at JoVE.com

A Repetitive Concussive Head Injury Model in Mice

Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner.

Ein Repetitive erschütternder Head Injury-Modell in Mäusen

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of ...

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JoVE Journal

Medicine

11.8K Aufrufe.  University of Florida. Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner.

Serie: A Repetitive Concussive Head Injury Model in Mice

The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared nerve injury (SNI) model induces symptoms of neuropathic pain such as mechanical allodynia i.e. pain due to tactile stimuli that do not normally provoke a painful response [1]. 
The SNI mouse model involves ligation of two of the three branches of the sciatic nerve (the tibial nerve and the common peroneal nerve), while the sural nerve is left intact [2]. The lesion results in marked hypersensitivity in the lateral area of the paw, which is innervated by the spared sural nerve. The non-operated side of the mouse can be used as a control. The advantages of the SNI model are the robustness of the response and that it doesn&#x2019;t require expert microsurgical skills.
The threshold for mechanical pain response is determined by testing with von Frey filaments of increasing bending force, which are repetitively pressed against the lateral area of the paw [3], [4]. A positive pain reaction is defined as sudden paw withdrawal, flinching and/or paw licking induced by the filament. A positive response in three out of five repetitive stimuli is defined as the pain threshold. 
As demonstrated in the video protocol, C57BL/6 mice experience profound allodynia as early as the day following surgery and maintain this for several weeks.

The Spared Nerve Injury SNI Model of Induced Mechanical Allodynia in Mice

The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.

Die Verschont Nerve Injury (SNI) Modell induzierte mechanische Allodynie in Mäusen

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared ...

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 ...

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

Renale Ischämie Reperfusionsschaden: Ein Maus-Modell der Schädigung und Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

Eine einfache Kritische große Femoral Defect Modell an der Maus

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

Facial Nerve Axotomie bei Mäusen: Ein Modell zur Motoneuron Reaktion auf eine Verletzung Studieren

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Die Quantifizierung der neurovaskulären Schutz Nach Repetitive hypoxische Präkonditionierung und Transient mittleren Hirnarterie Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

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.

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) ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Induktion von beschleunigter Atherosklerose bei Mäusen: Das "Wire-Injury"-Modell

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...