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

All animals received humane care in compliance with the European Convention on Animal Care. Surgical procedures were performed in accordance with the Swiss Animal Protection Law after obtaining permission of the State Veterinary Office, Fribourg, approved by the Swiss Federal Veterinary Office, Switzerland.
1. Heart Harvesting
NOTE: All surgical interventions were performed under isoflurane anesthesia. Efforts were made to diminish animal suffering. In particular, all animals received a subcutaneous injection of 0.1 mg/kg buprenorphine pre-anesthesia. The surgical protocol for inducting&#160;myocardial&#160;infarction has been previously described elsewhere13.
<ol>
	<li>Perform a sternotomy on a myocardially infarcted animal under anesthesia (intubated animal, 2.5% isoflurane, proper anesthesia confirmed by paw pinch reflex).
	<ol>
		<li>Open the animal by cutting the skin and then the muscles with surgical scissors. Cut the ribs on the left and right, and then remove the chest.</li>
		<li>Remove the adhesions remaining after the previous surgery (LAD ligation). Cut the aorta and remove the heart. Put the tissue in 1 M KCl (in PBS), and then wash it with PBS.</li>
	</ol>
	</li>
</ol>
2. Tissue Preparation
<ol>
	<li>Put the infarcted heart longitudinally in an acrylic rat heart matrix. Keep it at -20 &#176;C for 1 hr.</li>
	<li>Cut the heart directly in the matrix using a razor blade with a transversal. Ensure each slice is 2 mm thick. Cut approximately 5 - 7 slides for each heart (based on the remodeling level); this is called systematic sampling.</li>
	<li>At this step, performing the 2,3,5-triphenyltetrazolium chloride (TTC) staining is optional (keep the orientation of the heart slices base-to-apex).
	<ol>
		<li>Incubate in 1% TTC in PBS for 50 min at 37 &#176;C. Incubate it in 4% paraformaldehyde (PFA) for 20 min to 1 hr. Put the sections between two glass plates with 2-mm spacers and take pictures with a stereological microscope coupled with a camera at 15X magnification. 
		CAUTION! Paraformaldehyde is toxic.</li>
	</ol>
	</li>
	<li>Slide freezing (1st option)
	<ol>
		<li>Put each slide in a plastic mold (10 x 10 x 5 mm) with mounting medium for cryotomy (optimal cutting temperature, OCT), maintain the orientation (place the apex-oriented side of the section down on the bottom of the mold). Freeze the blocks with 2-methylbutane vapors under liquid nitrogen cooling for 10 - 15 min. Finally, store the tissue at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Paraffinization (2nd option)
	<ol>
		<li>Place each slide in embedding cassettes (maintain the orientation by placing the apex-oriented side of the section down on the bottom of the cassette) and then in 4% PFA for 24 hr. Put it in a tissue processor machine overnight.
		<ol>
			<li>Incubate it in ethanol 70% for 2 hr. Incubate it in ethanol 95% for 2 hr. Incubate it in ethanol 100% for 3 hr. Incubate it in xylol for 4 hr. Incubate it in paraffin (molten at 60 &#176;C in an oven) for 5 hr. Finally, make blocks by embedding each heart slide in paraffin.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Masson-Goldner Trichrome Staining
<ol>
	<li>Make tissue sections from paraffin blocks with a manual microtome (thickness: 5 &#181;m) or from OCT blocks with a cryostat set to -18 &#176;C (thickness: 7 &#181;m).</li>
	<li>Stain one slide from each heart part for each rat (3 - 4 transversal sections per slide) with Masson-Goldner trichrome staining (see annex). 
	NOTE: Start from this step for the paraffin sections.
	<ol>
		<li>Melt the slides at 60 &#176;C in an oven. Under a fume hood, deparaffinize them in xylol twice for 10 min each. Rehydrate them in 100% ethanol, 95% ethanol, 70% ethanol, and distilled water for 3 min each. 
		NOTE: Start from this step for the cryosections.</li>
		<li>Fix them in Bouin solution overnight. Then, rinse them in running tap water for 10 - 15 min. Rinse them in distilled water. Incubate them in Mayer&#39;s hematoxylin for 3 min.</li>
		<li>Remove the slides and leave them in distilled water for 5 min. Then, incubate them in acid Fuchsin-Ponceau for 5 min. Rinse them in 1% acetic acid for 1 min.</li>
		<li>Next, incubate them in phosphomolybdic acid Orange G for 1 min. Rinse them in 1% acetic acid for 1 min, incubate them in light green dye for 10 min, and rinse them in 1% acetic acid for 1 min.</li>
		<li>Dehydrate them in 70% ethanol (30 sec), 95% ethanol (30 sec), and 100% ethanol (5 min). Put one drop of a resinous mounting medium on the tissue sections, cover them with coverslips, and let them dry.</li>
	</ol>
	</li>
</ol>
4. Infarct Size Analysis
<ol>
	<li>Acquire one image from each slide on a stereomicroscope (15X magnification) coupled with a camera. Photograph a ruler with the same settings.</li>
	<li>Use image analysis software to measure the scar thickness in the middle of the infarct, the septum thickness, the left ventricle (LV) cavity area, the infarct area, and the LV tissue area using.
	<ol>
		<li>Set the scale with the ruler picture (Figure 1A).
		<ol>
			<li>Click on Measurements then select Set Conversion Factor. Draw a line on the calibration bar. Right click on the picture and click on End calibration. Note the bar value, and then click OK.</li>
		</ol>
		</li>
		<li>Select the LV from the stained heart picture (Figure 1B). Use the Multiple segment tool. Select the LV point-by-point, and then right click Copy/Paste anywhere.</li>
		<li>Measure the scar and septum thickness using the single segment measurement tool (Figure 1C).</li>
		<li>Automatically detect the LV cavity, infarct, and LV areas using the automatic area measurement tool in RGB mode (Figure 1D).
		<ol>
			<li>Click on the tool. Activate Only Borders and Contiguous in RGB mode. Finally, click inside the area of interest to detect it. If necessary, use precision tools.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Calculate the infarct size as the ratio of the infarct area to the LV area.</li>
	<li>Calculate the expansion index as follows: [LV cavity area / whole LV area] / [infarct thickness / septum thickness], with the whole LV area including both the LV cavity and LV tissue areas.</li>
	<li>Finally, calculate an &#34;average&#34; for each heart as follows: [average of expansion index from the infarcted slides] * [Number of infarcted slides / Total number of slides].</li>
</ol>

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G., 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=Plasma-functionalized+electrospun+matrix+for+biograft+development+and+cardiac+function+stabilization.">Plasma-functionalized electrospun matrix for biograft development and cardiac function stabilization.</a> Acta Biomater. 10, 2996-3006 (2014).</li><li>Landa, N., 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=Effect+of+injectable+alginate+implant+on+cardiac+remodeling+and+function+after+recent+and+old+infarcts+in+rat.">Effect of injectable alginate implant on cardiac remodeling and function after recent and old infarcts in rat.</a> Circulation. 117, 1388-1396 (2008).</li><li>Valente, 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=Optimized+heart+sampling+and+systematic+evaluation+of+cardiac+therapies+inmouse+models+of+ischemic+injury:+Assessment+of+cardiacremodeling+and+semi-automated+quantification+of+myocardial+infarctsize.">Optimized heart sampling and systematic evaluation of cardiac therapies inmouse models of ischemic injury: Assessment of cardiacremodeling and semi-automated quantification of myocardial infarctsize.</a> Curr. 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The authors have nothing to disclose.

Critical Steps within the Protocol
Fibrotic tissue can be accurately assessed in a chronic MI rat model using systematic sampling of the harvested heart and image analyses of trichromatic-stained histological sections obtained from base to apex. Two steps are particularly important for successful protocol implementation. First, the use of KCl for heart harvesting allows the cardiac muscle to be maintained in a relaxed state. This step is important for comparisons of infarct di...

Myocardial infarction (MI) is a leading cause of death and disability worldwide. Coronary heart disease is the main cause; MI results from ischemia consecutive to coronary events such as occlusion. When reperfusion is not performed within the first 6 hr, ischemia induces irreversible myocardial necrosis. In patients, the characterization of MI relies on different diagnostic tools, including clinical signs, electrocardiography, assessment of plasma levels of biomarkers, echocardiography, MRI imaging, and histological analyses1. Acute and chronic MI are classified as two different phases of injury according to the timing of the myocardial necrosis relative to the time of the coronary occlusion. The acute phase, occurring during the first 7 days, is associated with the loss of cardiomyocytes, extensive inflammation, and the recruitment of fibroblasts. The sub-acute phase, characterized by healing of the cardiac tissue and the formation of a scar, occurs between 1 and 4 - 6 weeks. Expansion of the infarct, ventricle wall thinning, and ventricle dilatation characterize the chronic phase. Extensive remodeling of the left ventricle progressively results in severe heart failure2.
MI induced by permanent left anterior descending artery (LAD) ligation represents the standard rodent model of chronic myocardial infarction. The coronary ligature mimics the coronary occlusion. The size of the infarct depends on the site of the ligature. Characterization of myocardial ischemic injury in a rodent model is classically performed using biomarker plasma levels, such as troponin I and T3, echocardiography, MRI, and histology4,5. Biomarker levels are correlated with the extent of cardiomyocyte death. Echocardiography evaluates the left ventricular function impairment resulting from regional wall motion abnormalities. In addition, non-invasive imaging techniques, such as MRI or high-resolution echocardiography, allow the assessment of the reduction in wall motion, the volume of the scar area with reduced perfusion and viable myocardium, and the wall thinning. LV dimensions permit the accurate evaluation of infarct size. Finally, the quantification of viable and dead myocardium can be performed postmortem using specific stains of histological sections of harvested hearts and allows verification of the infarct size (IS). Another important feature is the evaluation of the infarct expansion index (EI)6. The EI is associated with the transmural infarct and starts within the first 3 days. The EI is characterized by a progressive reduction in wall thickness, an increase in the LV cavity size, and consequent changes in LV shape.
In order to evaluate the therapeutic efficacy of novel treatments--in particular, the regenerative strategies based on cells, matrices, and gene delivery-accurate assessment of MI in rodents is of paramount importance. When measured on a single cross section obtained at the papillary muscle level, the IS size may be biased due to the large variability that exists in infarct development following LAD ligation; the apex infarct might be then occulted. Importantly, more accurate methodologies to determined MI size have been described for mice7-9 or rats10. Nevertheless, IS is insufficient to accurately quantify LV remodeling or therapeutically induced reductions (or preventions) of the remodeling. Indeed, IS is commonly expressed as a percentage of total LV volume assessed on cross sections of the heart. Although this method is valid for acute MI, the thinning of the LV wall occurring during remodeling remains under-evaluated11,12. A complete morphometric quantification of infarct size and structural changes should quantify several parameters, such as endocardial and epicardial lengths and diameters, as well as infarct and healthy areas. We describe a methodological approach to accurately assess MI and remodeling in a chronic rat model.

<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>
			<td style="width:136px;">Carl Roth</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">Bersoft Image measurement Software</td>
			<td style="width:119px;"></td>
			<td style="width:136px;">Bersoft.com</td>
			<td style="width:273px;">Licensed software</td>
		</tr>
	</tbody>
</table>

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.

Six weeks post-LAD ligation, hearts were harvested from Lewis rats. 2-mm tissue sections were obtained from apex to base. A TTC staining procedure was performed to visualize the infarct area, which appears in white, and the healthy myocardium, which appears in red (Figure 2). Depending on the site of ligation of the LAD, the infarct size varies. For large MI, transmural infarcts were observed from apex to base (Figure 2A). Smaller infarcts presented white...

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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.

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

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14.4K Views.  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. 

Video: 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.

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

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.

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).

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.

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.

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.

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.

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.

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;

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