The study was supported by the Swiss National Foundation [SNF 310030-149986 to MNG], the University of Fribourg, and Fribourg Hospital.

Alle Tiere erhielten humane Pflege im Einklang mit der Europäischen Konvention zum Tierpflege. Chirurgische Eingriffe wurden in Übereinstimmung mit dem Schweizer Tierschutzgesetzes durchgeführt, nachdem die Erlaubnis des Staates Veterinäramt zu erhalten, Fribourg, genehmigt durch das Bundesamt für Veterinärwesen, Schweiz. 1. Herzernte HINWEIS: Alle chirurgischen Eingriffe wurden unter Isofluran-Narkose durchgeführt. Es wurden Anstrengungen unternommen Leiden der Tiere zu verringern. Insbesondere erhielten alle Tiere eine subkutane Injektion von 0,1 mg / kg Buprenorphin Präanästhesie. Das chirurgische Protokoll für Myokardinfarkt inducting wurde bereits an anderer Stelle 13 beschrieben. <ol><li> Führen Sie eine Sternotomie auf einem myocardial Infarkt Tier unter Narkose (intubiert Tier, 2,5% Isofluran, richtige von Pfote Prise Reflex bestätigt Anästhesie). <ol><li> Öffnen Sie das Tier durch die Haut schneiden und dann die Muskelnmit chirurgische Scheren. Schneiden Sie die Rippen auf der linken und rechten Seite, und dann auf die Brust zu entfernen. </li><li> Entfernen Sie die restlichen Adhäsionen nach der vorhergehenden Operation (LAD-Ligation). Schneiden Sie die Aorta und entfernen Sie das Herz. Legen Sie das Gewebe in 1 M KCl (in PBS), und dann waschen Sie es mit PBS. </li></ol></li></ol> 2. Gewebepräparation <ol><li> Setzen Sie das Infarkt Herz in Längsrichtung in einem Acrylrattenherz-Matrix. Halten Sie es bei -20 ° C für 1 Stunde. </li><li> Schneiden Sie das Herz direkt in der Matrix eine Rasierklinge mit einer Quer verwenden. Stellen Sie sicher, jede Scheibe ist 2 mm dick. Schneiden Sie ca. 5 bis 7 Rutschen für jedes Herz (basierend auf dem Umbau Ebene); dies ist eine systematische Probenahme genannt. </li><li> Bei diesem Schritt ist es, die 2,3,5-Triphenyltetrazoliumchlorid (TTC) Färbung durchführen optional (halten Sie die Ausrichtung des Herzens Scheiben Basis-Spitze). <ol><li> Inkubieren in 1% TTC in PBS für 50 min bei 37 ° C. Inkubieren es in 4% Paraformaldehyd (PFA) für 20 min bis 1 Std. Setzen Sie die Abschnitte between zwei Glasplatten mit 2-mm-Distanzscheiben und nehmen Sie Bilder mit einem stereo Mikroskop gekoppelt mit einer Kamera bei 15-facher Vergrößerung. VORSICHT! Paraformaldehyd ist giftig. </li></ol></li><li> Slide Einfrieren (1 st Option) <ol><li> Setzen Sie jede Folie in einer Kunststoff-Form (10 x 10 x 5 mm) mit Eindeckmittel für die Kryotomie (optimale Schnitttemperatur, OCT), halten Sie die Ausrichtung (der Ort, den Scheitel orientierte Seite des Abschnitts nach unten auf dem Boden der Form). Frieren Sie die Blöcke mit 2-Methyl Dämpfe unter Kühlung mit flüssigem Stickstoff 10 - 15 min. Schließlich lagern Sie das Gewebe bei -80 ° C. </li></ol></li><li> Paraffinization (2. Option) <ol><li> Platzieren Sie jede Folie in Kassetten Einbetten (halten Sie die Ausrichtung, indem die Spitze orientierte Seite des Abschnitts nach unten auf dem Boden der Kassette) und dann in 4% PFA für 24 Stunden. Legen Sie es in einem Gewebe-Prozessor-Maschine über Nacht. <ol><li> Inkubieren in 70% igem Ethanol für 2 Stunden. Inkubieren es in ethan95% für 2 Stunden ol. Inkubieren es in Ethanol 100% für 3 Stunden. Inkubieren es in Xylol 4 Stunden. Inkubieren es in Paraffin (geschmolzenes bei 60 ° C in einem Ofen) für 5 Std. Schließlich machen Blöcke durch jedes Herz Dia in Paraffin eingebettet wird. </li></ol></li></ol></li></ol> 3. Masson-Goldner Trichromfärbung <ol><li> Machen Sie Gewebeschnitte aus Paraffinblöcken mit einem manuellen Mikrotom (Dicke: 5 um) oder von Oktober Blöcke mit einem Kryostaten auf -18 ° C (Dicke: 7 &amp; mgr; m). </li><li> Stain eine Folie aus jedem Herzteil für jede Ratte (3 bis 4 Querschnitte pro Folie) mit Masson-Goldner Trichrom - Färbung (siehe Anhang). HINWEIS: Starten Sie von diesem Schritt für die Paraffinschnitten. <ol><li> Schmelzen die Objektträger bei 60 ° C in einem Ofen. Unter einem Abzug Deparaffinisieren sie in Xylol zweimal für jeweils 10 min. Rehydrieren sie in 100% Ethanol, 95% Ethanol, 70% Ethanol und destilliertem Wasser für 3 min pro. HINWEIS: Starten Sie von diesem Schritt für die Kryoschnittess. </li><li> Fix sie in Bouin-Lösung über Nacht. Dann spülen Sie Leitungswasser für 10 in Laufen - 15 min. Spülen Sie sie in destilliertem Wasser. Inkubieren sie in Mayers Hämatoxylin für 3 min. </li><li> Entfernen Sie die Folien und lassen Sie sie in destilliertem Wasser für 5 min. Dann brüten sie in Säure Fuchsin-Ponceau für 5 min. Spülen Sie sie in 1% Essigsäure für 1 min. </li><li> Als nächstes für 1 min sie in Phosphomolybdänsäure Orange G inkubiert. Spülen Sie sie in 1% Essigsäure für 1 min, brüten sie in hellgrünen Farbstoff für 10 min, und spülen sie in 1% Essigsäure für 1 min. </li><li> Dehydrieren sie in 70% Ethanol (30 sec), 95% Ethanol (30 sec) und 100% igem Ethanol (5 min). Geben Sie einen Tropfen eines harzigen Eindeckmedium auf den Gewebeschnitten, bedecken sie mit Deckgläsern, und lassen Sie sie trocknen. </li></ol></li></ol> 4. Infarktgrößenanalyse <ol><li> Erwerben Sie ein Bild von jeder Folie auf einem Stereomikroskop (15-facher Vergrößerung) gekoppelt mit einer Kamera. Fotografieren Sie ein Lineal mit der gleichen Einstellungs. </li><li> Verwenden der Bildanalyse-Software die Narbendicke in der Mitte des Infarkts, das Septum Dicke zu messen, den linken Ventrikel (LV) Hohlraumbereich, der Infarktbereich und dem Gewebe LV Bereich verwendet wird. <ol><li> Stellen Sie die Waage mit dem Lineal Bild (Abbildung 1A). <ol><li> Klicken Sie auf Messungen dann Set Umrechnungsfaktor wählen. Zeichnen Sie eine Linie auf der Kalibrierungs bar. Rechtsklick auf das Bild und klicken Sie auf End - Kalibrierung. Notieren Sie sich den Bar - Wert und dann auf OK klicken. </li></ol></li><li> Wählen Sie die LV aus dem gefärbten Herz Bild (Abbildung 1B). Verwenden Sie das Multiple - Segment - Tool. Wählen Sie den LV Punkt-für-Punkt, und dann einen Rechtsklick Kopieren / Einfügen überall. </li><li> Messen Sie die Narbe und Septum Dicke mit dem einzigen Segment Messwerkzeug (1C </strong&gt;). </li><li> Automatische Erkennung der LV Hohlraum, Infarkt und LV Bereiche der automatischen Bereich Messwerkzeug im RGB - Modus (Abbildung 1D) verwendet wird . <ol><li> Klicken Sie auf das Werkzeug. Aktivieren Sie nur Grenzen und Angrenzend im RGB - Modus. Schließlich, klicken Sie in den Bereich von Interesse, es zu erkennen. Falls erforderlich, Präzisionswerkzeuge. </li></ol></li></ol></li><li> Berechne die Infarktgröße als das Verhältnis der Infarktbereich zum Bereich LV. </li><li> Berechnen Sie den Erweiterungsindex wie folgt: [LV Hohlraumbereich / gesamte LV-Bereich] / [Infarktdicke / Septum Dicke], mit der gesamten LV-Bereich einschließlich der LV Hohlraum und LV Gewebebereiche. </li><li> Schließlich berechnen für jedes Herz ein &quot;durchschnittlich&quot; wie folgt: [durchschnittlich Erweiterungsindex aus den Infarkt Dias] * [Anzahl der Infarkt Dias / Gesamtzahl der Folien]. </li></ol>

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The authors have nothing to disclose.

Kritische Schritte im Rahmen des Protokolls Fibrotische Gewebe genau in einem chronischen MI Rattenmodell bewertet werden analysiert unter Verwendung von systematischen Probenahme des geernteten Herz und Bild von Trichromie-gefärbten histologischen Schnitten von der Basis bis zur Spitze erhalten. Zwei Schritte sind besonders wichtig für ein erfolgreiches Protokoll-Implementierung. Erstens ermöglicht die Verwendung von KCl für Herz Ernten des Herzmuskels in einem entspann...

Myokardinfarkt (MI) ist eine führende Ursache für Tod und Behinderung weltweit. Die koronare Herzkrankheit ist die Hauptursache; MI ergibt sich aus Ischämie in Folge einer koronaren Ereignissen wie Okklusion. Wenn Reperfusion nicht innerhalb der ersten 6 Stunden durchgeführt, induziert Ischämie irreversible myocardial necrosis. Bei Patienten, stützt sich die Charakterisierung von MI auf verschiedene Diagnose - Tools, einschließlich der klinischen Symptome, der Elektrokardiographie, die Beurteilung der Plasmaspiegel von Biomarkern, Echokardiographie, MRT - Bildgebung und histologische Analysen 1. nach dem Zeitpunkt der myokardialen Nekrose relativ zu der Zeit der Koronarverschluss akuten und chronischen MI als zwei verschiedene Phasen Verletzungs eingestuft. Die akute Phase, während der ersten 7 Tage stattfindet, wird mit dem Verlust von Kardiomyozyten, ausgedehnte Entzündungen, und die Rekrutierung von Fibroblasten. Die subakuten Phase durch Heilung des Herzgewebes und die Bildung einer Narbe gekennzeichnet, auftrittzwischen 1 und 4 - 6 Wochen. Erweiterung des Infarkts, Ventrikel Wandverdünnung und Ventrikel Dilatation charakterisieren die chronische Phase. Umfangreiche Neugestaltung des linken Ventrikels führt schrittweise in schwerer Herzinsuffizienz 2. MI durch permanente linken vorderen absteigenden Arterie (LAD) Ligatur stellt die Standard-Nagetiermodell von chronischem Myokardinfarkt induziert. Die koronare Ligatur ahmt die Koronarokklusion. Die Größe des Infarkts hängt von der Stelle der Ligatur. Charakterisierung von myokardialen ischämischen Verletzung in einem Nagetiermodell klassisch unter Verwendung von Biomarkern Plasmaspiegel, wie Troponin ausgeführt I und T 3, Echokardiographie, MRT und Histologie 4,5. Biomarkers Pegel werden mit dem Ausmaß der Kardiomyozyten Tod korreliert. Echokardiographie wertet die linke Beeinträchtigung der Ventrikelfunktion aus regionalen Wandbewegungsstörungen. Zusätzlich können nicht-invasive Bildgebungsverfahren, wie MRI oder hochauflösendeEchokardiographie, erlauben die Beurteilung der Verringerung der Wandbewegung, das Volumen des Narbenbereich mit reduzierten Perfusion und vitales Myokard und der Wandverdünnung. LV Abmessungen ermöglichen die genaue Auswertung der Infarktgröße. Schließlich kann die Quantifizierung der lebenden und toten Myokard durchgeführt werden post mortem spezifischen Flecken von histologischen Schnitten von geernteten Herzen verwendet und ermöglicht die Überprüfung der Infarktgröße (IS). Ein weiteres wichtiges Merkmal ist die Auswertung des Infarkts Erweiterungsindex (EI) 6. Die EI wird mit dem transmuralen Infarkt und beginnt innerhalb der ersten 3 Tagen verbunden. Die EI wird durch eine progressive Verringerung der Wandstärke, einer Erhöhung in der LV Hohlraumgröße und folglich Veränderungen in LV Form charakterisiert. Um die therapeutische Wirksamkeit von neuartigen Behandlungen zu bewerten - insbesondere die regenerative Strategien basierend auf Zellen, Matrizen und Genübertragung-genaue Beurteilung von MI bei Nagetieren ist von größter Bedeutung.Wenn auf einem einzigen Querschnitt an der Papillarmuskel Ebene erhalten gemessen, kann die IS Größe kann aufgrund der großen Variabilität vorgespannt werden, die nach LAD-Ligation in die Infarktentwicklung vorhanden ist; der Scheitel Infarkt abgedeckt werden, könnten dann. Wichtig sind für Mäuse 7-9 oder Ratten beschrieben, genauere Methoden zu bestimmt MI Größe 10. Dennoch ist nicht ausreichend, um genau LV Umbau oder therapeutisch induzierten Reduktionen (oder Verhinderungen) der Umbau zu quantifizieren. Tatsächlich wird üblicherweise als Prozentsatz der gesamten LV Volumen auf Querschnitte des Herzens beurteilt ausgedrückt. Obwohl dieses Verfahren zur Behandlung von akuter MI gültig ist, bleibt die Ausdünnung der LV Wand während des Umbaus auftretenden 11,12 untersucht. Eine vollständige morphometrische Quantifizierung der Infarktgröße und strukturelle Änderungen sollten einige Parameter, wie endokardialen und epikardialen Längen und Durchmesser sowie Infarkt und gesunde Bereiche zu quantifizieren. Wir beschreiben eine methodische ca.oach genau zu beurteilen, MI und Umbau in einem chronischen Rattenmodell.

<table border="0" cellpadding="0" cellspacing="0" width="775">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Acrylic rat heart matrix 2mm</td>
			<td style="width:119px;">72-5015</td>
			<td style="width:136px;">Harvard Appartus</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">INSPIRA ADVANCED VOLUME CONTROLLED VENTILATOR</td>
			<td style="width:119px;">HARVARD APPARATUS</td>
			<td style="width:136px;">557058</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">CATHETER INSYTE 14G</td>
			<td style="width:119px;">BD</td>
			<td style="width:136px;">381267</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">O.C.T</td>
			<td style="width:119px;">BDHA361603E</td>
			<td style="width:136px;">VWR</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">TTC</td>
			<td style="width:119px;">T8877-10G</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mayer hematoxylin</td>
			<td style="width:119px;">MHS32-1L</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Acid Fuchsin 
			CI 42685</td>
			<td style="width:119px;">F8129-50G</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ponceau Xylidin 
			CI 16150</td>
			<td style="width:119px;">P2395-25G</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Orange G 
			CI 16230</td>
			<td style="width:119px;">O3756-100G</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Light green 
			CI 42095</td>
			<td style="width:119px;">L5382-25G</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">KCl</td>
			<td style="width:119px;">P9333-500G</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Xylol</td>
			<td style="width:119px;">10315083</td>
			<td style="width:136px;">HoneyWell</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ethanol absolute</td>
			<td style="width:119px;">10303990</td>
			<td style="width:136px;">HoneyWell</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">2-methylbutane</td>
			<td style="width:119px;">M32631-1L</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Stereogical microscope</td>
			<td style="width:119px;">SM2800</td>
			<td style="width:136px;">Nikon</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Formaldehyde</td>
			<td style="width:119px;">99340</td>
			<td style="width:136px;">Reactolab</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Embedding cassette</td>
			<td style="width:119px;">K113.1</td>
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			<td height="41" style="height:41px;width:247px;">Bersoft Image measurement Software</td>
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			<td style="width:136px;">Bersoft.com</td>
			<td style="width:273px;">Licensed software</td>
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histological quantification of chronic myocardial infarct in rats

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ischemic injury. Following the initial acute phase, chronic remodeling of the left ventricle (LV) modifies the structure and function of the heart. Permanent coronary ligation in small animals has been widely used as a reference model for a chronic model of MI. Thinning of the infarcted wall progressively develops to transmural fibrosis. Histological assessment of infarct size is commonly performed; nevertheless, a standardization of the methods for quantification is missing. Indeed, important methodological aspects, such as the number of sections analyzed and the sampling and quantification methods, are usually not described and therefore preclude comparison across investigations. Too often, quantification is performed on a single section obtained at the level of the papillary muscles. Because novel strategies aimed at reducing infarct expansion and remodeling are under investigation, there is an important need for the standardization of accurate heart sampling protocols. We describe an accurate method to quantify the infarct size using a systematic sampling of harvested rat heart and image analyses of trichromatic stained histological sections obtained from base to apex. We also provide evidence that calculating the expansion index (EI) allowed for infarct size assessment, taking into account changes of the left ventricle throughout the remodeling.

Sechs Wochen nach der LAD-Ligation, Herzen wurden von Lewis-Ratten geerntet. 2-mm-Gewebeschnitte wurden von der Spitze zur Basis erhalten. Eine TTC - Färbung Verfahren wurde ausgeführt , um den Infarktbereich sichtbar zu machen, die in weiß erscheint, und das gesunde Myokard, die in rot (Abbildung 2) erscheint. Je nach der Stelle der Ligation der LAD variiert der Infarktgröße. Für große MI wurden transmurale Infarkte von der Spitze zur Basis (2A) ...

Watch this Scientific Journal Video about Histological Quantification of Chronic Myocardial Infarct in Rats at JoVE.com

Histological Quantification of Chronic Myocardial Infarct in Rats

The post-mortem assessment of myocardial infarction (MI) in rodents is based on quantification of the infarct on stained heart sections. We describe an accurate method to quantify the infarct size using systematic sampling of harvested rat hearts from base to apex and image analyses of trichrome-stained histological sections.

Die histologische Quantifizierung der chronischen Myokardinfarktes bei Ratten

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ...

histological-quantification-of-chronic-myocardial-infarct-in-rats

Research

JoVE Journal

Medicine

14.4K Aufrufe.  University of Fribourg. The post-mortem assessment of myocardial infarction (MI) in rodents is based on quantification of the infarct on stained heart sections. We describe an accurate method to quantify the infarct size using systematic sampling of harvested rat hearts from base to apex and image analyses of trichrome-stained histological sections. 

Serie: Histological Quantification of Chronic Myocardial Infarct in Rats

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

Akuter Myokardinfarkt bei Ratten

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

Forschung

Medizin

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 ...

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

Chronische Verengung des Ischiasnerv und Schmerzen Überempfindlichkeit Testing in Ratten

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

Beurteilung der Herzfunktion und Morphologie Myokardiale Mit Small Animal Look-Locker Inversion Recovery (SALLI) MRI in Ratten

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Ultrasonic Assessment of Myocardial Microstructure

Echocardiography is a widely accessible imaging modality that is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. Ultrasonic assessments of cardiac tissue can include analyses of backscatter signal intensity within a given region of interest. Previously established techniques have relied predominantly on the integrated or mean value of backscatter signal intensities, which may be susceptible to variability from aliased data from low frame rates and time delays for algorithms based on cyclic variation. Herein, we describe an ultrasound-based imaging algorithm that extends from previous methods, can be applied to a single image frame&#160;and accounts for the full distribution of signal intensity values derived from a given myocardial sample. When applied to representative mouse and human imaging data, the algorithm distinguishes between subjects with and without exposure to chronic afterload resistance. The algorithm offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

Echocardiography is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. We describe an ultrasound-based imaging algorithm that offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

Ultraschall-Bewertung der myokarddialen Mikrostruktur

Echocardiography is a widely accessible imaging modality that is commonly used to noninvasively characterize and quantify changes in cardiac structure and ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Harnblase Aufdehnung evozierte viszeromotorische Antworten als Modell für Bladder Pain in Mäuse

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

Echokardiographische und histologische Untersuchung der kardiale Morphologie in der Maus

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Bewertung des koronaren Flussreservats nach Myokard-Ischämie-Reperfusion bei Ratten

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Semi-Minimal Invasive Methode zur Induzieren von Myokardinfarkt bei Ratten und der Beurteilung der Herzfunktion durch ein isoliertes funktionierendes Herzsystem

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...