The experimental protocol received bioethical agreement (No. 311 from 2022) and was approved by the Bioethics Committee of the University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, in compliance with both national (Law No. 43 of 2014) and European (EU Directive No 63 from 2010) legislation. See the Table of Materials for details about all materials and instruments used in this protocol.
1.&#160;Necropsy protocol
NOTE: It is recommended that the same necropsy protocol be used for all the species presented due to easier access it is when performed2,12. The following steps represent the necropsy performed in a medium-sized dog. Adapt the steps when performing necropsies on different subjects.
<ol>
	<li>Weigh the subject and place it on the necropsy table on its left side (Figure 1A). Proceed to the external examination of the subject (by inspecting and palpation of the cutis and mucosas, noting all the changes in color, consistency, and volume).</li>
	<li>Use a knife (or a scalpel) and make an incision of 7 cm in the right axilla (Figure 1A).</li>
	<li>Locate the coxofemoral junction (Figure 1A,B). Open the capsule with a knife. Cut the ligament of the head of the femur (Figure 1B).</li>
	<li>Use the knife to extend the skin incision in the axilla cranial to the mandibular symphysis and caudally to the perineum (Figure 1B).</li>
	<li>Remove the skin with the scalpel and expose the thoracic and abdominal wall from the ventral midline to the level of the vertebral transverse processes (Figure 1B).</li>
	<li>With the scalpel and forceps, create a flap through the abdominal wall, ventrocaudal to the right costal arch. Open the abdominal cavity with scissors-follow the caudal aspect of the right hypochondrium (Figure 1B).</li>
	<li>Extend the incision caudally along the right paralumbar area until the external iliac crest. Afterward, extend the incision until the pubic insertion of the linea alba.</li>
	<li>Briefly, examine the abdominal organs in situ (by noting any changes in changes in color, consistency, volume, and position of the organs).</li>
	<li>With the scalpel and forceps, puncture the diaphragm at the highest point (Figure 1B).</li>
	<li>Enter the thoracic cavity by removing the right rib cage with two longitudinal sections using bone-cutting forceps. Section the parasternal cartilaginous segment of the ribs. Next, section the dorsal segment of the ribs, proximal to the costovertebral joints (Figure 1C).</li>
	<li>Examine the heart in situ (Figure 1C). Notice the size, position, color, connections with the adjacent tissues, and by palpation, any changes in the consistency9. 
	NOTE: If a thrombosis or congenital heart disease is present, the heart must be dissected in situ (this approach allows complete examination of the large vessels).</li>
	<li>Use scissors to examine and open the pericardial cavity with a longitudinal section from the base to the apex of the heart. Examine the pericardial cavity and the epicardium (noting any changes in color, consistency, and volume)12.</li>
	<li>Release the heart with scissors, yielding transverse sections of the aorta, pulmonary trunk, and both vena cavae. Section the superior vena cava at least 1 cm above the entrance into the right atrium to preserve the crista terminalis11,13.</li>
	<li>Examine the external contour of the organ, the appearance of the epicardium, the appearance of the myocardium by the transparency of the epicardium, and the external appearance of the large vessels (Figure 2A). 
	NOTE: In general, the opening and examination of the pericardium present some differences among the species. The examination of the pericardial cavity starts with an external inspection and assessment through the transparency of the pericardium of the overall pericardial content1,2. In a cat or dog, the pericardium typically contains about 0.25 mL/kg of thin, clear, translucent to light yellow fluid9. If there are any pathologic effusions, they should be collected with a sterile syringe or vacutainer tube.</li>
</ol>
2. Dissection protocol
NOTE: Several necropsy techniques are used for the heart, each with several advantages. For this protocol, two of the most used techniques were chosen: 1) the &#34;inflow-outflow method,&#34; which allows a better examination of the valves and the endocardium and is a protocol used for most of the species2,11,12,16, and 2) the &#34;four chambers dissection&#34;/&#34;echocardiographic plane&#34; technique, typically used for cats or small-sized dogs1,17.
<ol>
	<li>The &#34;inflow-outflow method&#34; for cardiac dissection2,11,12,16
	<ol>
		<li>Place the heart with the atrial face upward (Figure 2A).</li>
		<li>Use scissors and toothless forceps to broadly cut from the caudal vena cava along into the right atrium (Figure 2B). Continue the cut to the right auricular appendage to expose the crista terminalis/sinoatrial node (SA node). Inspect the tricuspid valve from the dorsal view.</li>
		<li>Cut a section from the right atrium to the junction between the right ventricle and the interventricular septum (Figure 2C). Display the right atrioventricular valve.</li>
		<li>Section the right ventricle free wall along the interventricular septum. Continue the section until the origin of the pulmonary trunk (Figure 2C,D).</li>
		<li>Thoroughly examine the right atrioventricular valves, including the chordae tendineae, and the papillary muscles (Figure 2D). Cut the chordae tendineae.</li>
		<li>Place forceps through the pulmonary trunk and cut the trunk longitudinally (Figure 2E). Examine the pulmonary outflow tract, pulmonary trunk origin, and semilunar valves.</li>
		<li>Cut the left atrium from the pulmonary veins to the tip of the left auricle (Figure 2F). Inspect the bicuspid (mitral) valve from the dorsal view.</li>
		<li>Place the heart with the interventricular septum facing downward (Figure 2F). Make a large incision in the ventricular free wall from the base of the ventricle to the cardiac apex. Display the left atrioventricular valve completely.</li>
		<li>Cut the chordae tendineae of the cusp of the left atrioventricular valve.</li>
		<li>Place forceps through the left ventricular outflow tract and use it as guidance to open the aorta (Figure 2G).</li>
		<li>Use saline solution to remove all the blood clots. Once all the blood clots are removed, assess and compare cardiac weight against the body mass or standard cardiac weight tables (Table 1)11,13.</li>
		<li>Weigh the heart.</li>
		<li>If only the SA node is the point of interest, after the left ventricle examination, place the entire heart entirely in 10% neutral-buffered formalin (NBF) for 24 h, and trim the right atrium to sample the crista terminalis. 
		NOTE: By this technique, the left and right ventricles can be easily appreciated. The ratio between the left and right ventricular wall thickness is 3:1, with small differences by species and breed; for the animal fetus heart, the ratio is 1:112.</li>
	</ol>
	</li>
</ol>
3. Sampling protocol for histology16
<ol>
	<li>With a scalpel, make two longitudinal, parallel cuts (approximately 5 mm apart) involving the right atrium, the tricuspid valve, and the right ventricular free wall (Figure 2I)</li>
	<li>Make two longitudinal parallel cuts (approximately 5 mm apart) involving the left atrium, mitral valve, dorsal papillary muscle, and left ventricle (Figure 2H).</li>
	<li>Within the dorsal upper-third of the heart, transversally section the base of the heart following a line, including the interventricular septum, right atrium, aorta, aortic valve, and tricuspid valve (Figure 2J).</li>
	<li>Make two longitudinal parallel cuts involving the base of the aorta and the interventricular septum (Figure 2J).</li>
	<li>Trim the right atrium around the crista terminalis, and provide several parallel sections oriented transversally on the longitudinal axis of the crista (approximately 3-4 mm apart) to obtain six pieces of the SA node (Figure 3, Figure 4I, and Figure 5H).</li>
	<li>Trim the obtained tissues in histological cassettes. 
	NOTE: Recommended blocking protocol: step 3.1.-histological block 1 (Figure 6 shows the resulting slide from this block.), step 3.2.-histological block 2 (Figure 7 shows the resulting slide from this block), step 3.4.-histological block 3 (Figure 8 shows the resulting slide from this block), step 3.5.-histological block 4 (Figure 9 shows the resulting slide from this block).</li>
	<li>Immerse the tissues in 10% NBF for at least 48 h.</li>
</ol>
4. Sampling of coronary arteries3,10,14
<ol>
	<li>Use forceps and a scalpel to dissect the coronary arteries from the cardiac paraconal groove (Figure 4K). Fix the coronary artery overnight in 10% NBF.</li>
	<li>If needed, decalcify adequately in a 1:1 mixture of 8% formic acid and hydrochloric acid.</li>
	<li>Make multiple transverse cuts at 3 mm intervals. Place the sections in a cassette and then put the cassette in 10% NBF until embedding. 
	NOTE: Pigs can be used as a cardiac model for humans, especially when it comes to coronary heart disease. Hence, supplementary samples and histological sections of coronary arteries are recommended3,10,14.</li>
</ol>
5. The &#34;four chambers&#34;/&#34;echocardiographic plane&#34; dissection technique of the heart1,17
NOTE: The four-chamber dissection technique consists of a cut from the base to the apex of the heart to obtain the standard view1. The &#34;four-chamber&#34; technique is best applied to fixed tissues.
<ol>
	<li>Place the whole heart into 10% NBF for at least 48 h.</li>
	<li>After the heart is fixed in 10% NBF for at least 48 h (Figure 10A), place two toothless straight dissection forceps, one through the cranial cava-right atrium-tricuspid valve-right ventricle, and the second through the pulmonary vein-left atrium-mitral valve-left ventricle (Figure 10B).</li>
	<li>Use the space between the forceps arms as guidance for the cutting blade from the base to the apex of the heart (Figure 10C). Provide an additional longitudinal section approximately 5 mm apart from the section described in step 5.2. on the cardiac segment, which includes the aorta.</li>
	<li>With a transversal incision, completely separate the cardiac base from the apex. Place each tissue segment in a histological cassette. 
	NOTE: Recommended blocking protocol: the cardiac apex-histological block 5 (Figure 11A shows the resulting slide from this block), the cardiac base-histological block 6 (Figure 11B shows the resulting slide from this block).</li>
	<li>Place the cassettes in 10% NBF until embedding. Fix all the cardiac samples in 10% NBF (more than 10 times the volume of tissue), routinely process them in paraffin wax, section at 4 &#181;m thickness, and stain with hematoxylin and eosin stain (H&#38;E stain) following a previously described protocol10. 
	NOTE: If myocarditis is suspected, &#34;a piece of fresh heart tissue should be retained under sterile conditions with sterile surgical material and sterile sample container for microbiological and viral studies&#34;13.</li>
</ol>
6. Photographic documentation
NOTE: Photographic documentation is an optional step in the necropsy examination. However, photography and video recordings are essential to have an &#34;accurate documentation of normal and abnormal anatomy&#34;3.
<ol>
	<li>For proper photographic documentation, take digital images while performing &#34;progressive dissection of cardiac tissues&#34;13. Refer to the guidelines for photographic documentation and photograph15&#160;the general view-anterior and posterior; a slice of the heart; and any suspected lesions.</li>
</ol>

<ol>
	<li>Ashworth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathology of Heart Disease in the Fetus, Infant and Child: Autopsy, Surgical and Molecular Pathology. , (2019).</li><li>Barone, R., Vigot, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anatomie Compar&#233;e des Mammif&#232;res Domestiques: Angiologie. , (2011).</li><li>Basso, 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=Guidelines+for+autopsy+investigation+of+sudden+cardiac+death:+2017+update+from+the+Association+for+European+Cardiovascular+Pathology.">Guidelines for autopsy investigation of sudden cardiac death: 2017 update from the Association for European Cardiovascular Pathology.</a> Virchows Archiv. 471 (6), 691-705 (2017).</li><li>Bishop, S. P., Fox, P. R., Sisson, D., Moi&#776;se, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+37+-+Necropsy+Techniques+for+the+Heart+and+Great+Vessels.">Chapter 37 - Necropsy Techniques for the Heart and Great Vessels.</a> Textbook of Canine and Feline Cardiology: Principles and Clinical Practice,.2nd ed. , 846-848 (1999).</li><li>Joseph, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ratio+between+the+heart-weight+and+body+weight+in+various+animals.">The ratio between the heart-weight and body weight in various animals.</a> Journal of Experimental Medicine. 10 (4), 521-522 (1908).</li><li>Keenan, C. M., Vidal, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+morphologic+evaluation+of+the+heart+in+the+laboratory+dog+and+monkey.">Standard morphologic evaluation of the heart in the laboratory dog and monkey.</a> Toxicologic Pathology. 34 (1), 67-74 (2006).</li><li>Latimer, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability+in+body+and+organ+weights+in+the+newborn+dog+and+cat+compared+with+that+in+the+adult.">Variability in body and organ weights in the newborn dog and cat compared with that in the adult.</a> The Anatomical Record. 157 (3), 449-456 (1967).</li><li>Lee, J. C., Taylor, F. N., Downing, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+ventricular+weights+and+geometry+in+newborn,+young,+and+adult+mammals.">A comparison of ventricular weights and geometry in newborn, young, and adult mammals.</a> Journal of Applied Physiology. 38 (1), 147-150 (1975).</li><li>McDonough, S. P., Southard, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Necropsy Guide for Dogs, Cats, and Small Mammals. , 9 (2016).</li><li>Prophet, E. B., Mills, B., Arrington, J. B., Sobin, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Methods in Histotechnology. , 29-58 (1992).</li><li>Robinson, W. F., Robinson, N. A., Grant Maxie, M., Saunders, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+1+-+Cardiovascular+System.">Chapter 1 - Cardiovascular System.</a> Jubb, Kennedy &amp; Palmer's Pathology of Domestic Animals, 6th ed. 3, 12-49 (2016).</li><li>Tabaran, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Autopsie et Medecine Legale: Guide de Travaux Pratiques. , 21-86 (2020).</li><li>. Toxpath.org Available from: <a target="_blank" href="https://www.toxpath.org/Trimming-N-Collection-Training.asp">https://www.toxpath.org/Trimming-N-Collection-Training.asp</a> (2021)</li></ol>

The authors have no conflicts of interest to disclose.

When performing the current protocol, several critical steps should be carefully considered for consistent results. In young animals, cardiac measurements are different from those in adults (including ventricular wall thickness), and generally, the heart represents a greater proportion of body weight11,12. The degree of ventricular hypertrophy can be quantified by applying a general weight formula, the ratio between the left ventricle plus septum divided by the f...

Due to the complex anatomy and early rigor, which can interfere with assessing the cardiac wall thickness, the gross examination of the heart is challenging and prone to several technique-related or interpretation errors. This is amplified by the interspecific morphological variations and by the fact that many clinical, major cardiac pathologies (including early cases of coronary heart diseases, fibrosis, arteritis, and amyloidosis) are not associated with any gross changes, being, in essence, histological pathologies. A standardized dissection and histological sample harvesting protocol can bring consistency between observers and, at the same time, comparability and reproducibility of experimental studies of cardiovascular diseases.
The samples were collected from two dogs (Canis lupus familiaris) (a 3-year-old, male French Bulldog, and an 8-year-old, female mixed-breed), a cat (Felis catus) (a 6-year-old, male European shorthair), a domestic pig (Sus scrofa domesticus) (a 1-year-old, male Large White), and a cow (Bos taurus) (a 2-month-old, female Holstein).&#160;Each of the chosen species has particular use as a cardiovascular model for a different disease; for example, dogs are a preferred model for arrhythmia modeling; cats are preferred for hypertrophic cardiomyopathy (HCM), as it is the species with the highest prevalence of HCM; pigs are used as a model for acute myocardial infarction; and ruminants are used as a model for intoxication due to their exposure to toxins that can easily be found on meadows11.
In this manuscript, one necropsy protocol and two dissection protocols of the heart are presented, designed to improve the gross and histological examination of the heart in experimental cardiovascular diseases. The described protocols were developed based on information from veterinary textbooks1,2,4,5,6,7,8,9,10,11,12, journal literature3,13,14, official documents15, and webinars16,17. The samples used in this study were harvested from cadavers submitted to the Pathology Department of the USAMV Cluj-Napoca for routine autopsy diagnostic.

<table><tbody><tr><td>0.9% saline solution</td><td>B. BRAUN MELSUNGEN AG</td><td>W04479004</td><td>For washing all the blood, and blood clots from the heart.</td></tr><tr><td>10% neutral buffered formalin (NBF)</td><td>&amp;nbsp;Q Path</td><td>11699404</td><td>Materials for collecting histopathology samples.</td></tr><tr><td>Bone cutting forceps</td><td>HELEN SRL</td><td>LS109HV</td><td>Sturdy instrument for cutting bone.</td></tr><tr><td>Cutting board</td><td>Ambition</td><td>86304</td><td>For an easier manipulation, and cutting the organs.</td></tr><tr><td>Decalcifying solution</td><td>Thermo Scientific TBD-2</td><td>6764004</td><td>1:1 mixture of 8% formic acid and hydrochloric acid.</td></tr><tr><td>Digital camera</td><td>Canon Inc.</td><td>PowerShot SX540 HS</td><td>For photographic, and videographic documentation.</td></tr><tr><td>Forceps</td><td>MKD-Medicale</td><td>15-430</td><td>Dissection instruments.</td></tr><tr><td>Histological cassettes</td><td>&amp;nbsp;Q Path</td><td>720-2215</td><td>Materials for collecting histopathology samples.&lt;br/&gt; Dimensions: 3 &amp;times; 2.5 &amp;times; 0.4 cm</td></tr><tr><td>Knife</td><td>TEHNO FOOD COM SERV SRL</td><td>D2006/15</td><td>Sharp blade for cutting soft tissue.</td></tr><tr><td>Latex gloves</td><td>MKD-Medicale</td><td>SANTEX-S</td><td>Protection equipment.</td></tr><tr><td>Mask</td><td>MKD-Medicale</td><td>21221</td><td>Protection equipment.</td></tr><tr><td>Petri dishes</td><td>MKD-Medicale</td><td>0598-1V</td><td>Materials for collecting ancillary testing samples.</td></tr><tr><td>Plastic recipients</td><td>Corning Gosselin</td><td>TP200-02</td><td>Materials for collecting histopathology samples.</td></tr><tr><td>Scale</td><td>ESPERANZA</td><td>MEEKS008</td><td>For weighing the organs.</td></tr><tr><td>Scale</td><td>White Deals</td><td>72</td><td>For weighing the subjects.</td></tr><tr><td>Scalpel</td><td>MKD-Medicale</td><td>10322E</td><td>Sharp blade for cutting soft tissue.</td></tr><tr><td>Scissors</td><td>MKD-Medicale</td><td>13-260</td><td>Dissection instruments.</td></tr><tr><td>Scrub</td><td>MKD-Medicale</td><td>410100-52</td><td>Protection equipment.</td></tr><tr><td>Syringes</td><td>MKD-Medicale</td><td>10573EU</td><td>Materials for collecting ancillary testing samples.</td></tr></tbody></table>

dissection techniques and histological sampling of the heart in large animal models for cardiovascular diseases

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried out in large animals. Considering the complex anatomy of the heart, interspecies differences, and the types of compensatory and pathological reactions, consistent protocols are challenging to implement. The utilization of multiple dissection protocols is usually adapted to suit the prosector's experience, and personal preference continues to be a source of experimental and interobserver variability. Here, the aim is to present the main anatomical features and landmarks, dissection protocols, and histological sampling standards of the heart in some commonly used species (including dogs, pigs, ruminants, and cats) as models of cardiovascular diseases.
Two standard gross examination protocols are presented here. First, the inflow-outflow method, which follows the physiological blood flow direction through the heart and large vessels (frequently used in dogs, ruminants, and pigs), and second, the four-chamber dissection technique (exemplified in cats). Both techniques can be adapted to any species in certain experimental circumstances. The sampling protocols include all the areas of interest (sinoatrial node, ventricles, interventricular septum, atria, valves, and aorta), and if properly carried out, improve both the reproducibility and reliability of experimental studies.

This protocol was used to visualize anatomical features and collect samples for histological examination of the heart in four different species (dog, cat, pig, and cow). The necropsy protocol was repeated in each of the above-mentioned species but illustrated only in dogs. The necropsy protocol begins with an extensive external examination of the body (Figure 1A) (including the skin, explorable lymph nodes, and exterior mucosa), measuring the overall weight, and scoring the general state of ...

Watch this Scientific Journal Video about Dissection Techniques and Histological Sampling of the Heart in Large Animal Models for Cardiovascular Diseases at JoVE.com

Dissection Techniques and Histological Sampling of the Heart in Large Animal Models for Cardiovascular Diseases

Due to the complex anatomy, a consistently reproducible dissection and harvesting protocol for cardiac samples can be challenging to implement. This manuscript presents the key elements of some standard cardiac dissection protocols, highlighting both the gross examination approaches and the sampling sites commonly used for histopathologic examination.

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried ...

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7.0K Views.  University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Due to the complex anatomy, a consistently reproducible dissection and harvesting protocol for cardiac samples can be challenging to implement. This manuscript presents the key elements of some standard cardiac dissection protocols, highlighting both the gross examination approaches and the sampling sites commonly used for histopathologic examination. 

Video: Dissection Techniques and Histological Sampling of the Heart in Large Animal Models for Cardiovascular Diseases

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Bioengineering

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes &#8211; a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user&#8217;s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.

Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data acquisition.

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

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 ...

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a considerable proportion of coronary artery disease patients remain symptomatic. Gene therapy-mediated therapeutic angiogenesis offers a novel therapeutic method for improving myocardial perfusion and relieving symptoms. Gene therapy with different angiogenic factors has been studied in few clinical trials. Due to the novelty of the method, the progress of myocardial gene therapy is a continuous path from bench to bedside. Therefore, large animal models are needed for evaluating the safety and efficacy. The more the large animal model identifies the original disease and the endpoints used in clinics, the more predictable outcomes are from clinical trials. Here, we introduce a large animal model for evaluating the efficacy of the gene therapy in the ischemic porcine heart. We use clinically relevant imaging methods such as ultrasound imaging and 15H2O-PET. For targeting the gene transfers into the desired area, electroanatomical mapping is used. The aim of this method is: (1) to mimic chronic coronary artery disease, (2) to induce therapeutic angiogenesis at hypoxic areas of the heart, and (3) to evaluate the safety and efficacy of the gene therapy by using relevant endpoints.

Myocardial gene therapy for ischemic heart disease holds great promise for future therapeutics. Here, we introduce a large animal model for evaluating the efficacy of gene therapy in the ischemic heart.

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Biology

Revealing Murine Pulmonary Veins Myocardial Architecture

Microdissection and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins

Pulmonary veins (PVs) are the major source of ectopic beats in atrial arrhythmias and play a crucial role in the development and progression of atrial fibrillation (AF). PVs contain myocardial sleeves (MS) composed of cardiomyocytes. MS are implicated in the initiation and maintenance of AF, as they preserve similarities to the cardiac working myocardium, including the ability to generate ectopic electrical impulses. Rodents are widely used and may represent excellent animal models to study the pulmonary vein myocardium since cardiomyocytes are widely present all over the vessel wall. However, precise microdissection and preparation of murine PVs is challenging due to the small organ size and intricate anatomy.
We demonstrate a microscopy-guided microdissection protocol for isolating the murine left atrium (LA) together with the PVs. Immunofluorescence staining using cardiac Troponin-T (cTNT) and connexin 43 (Cx43) antibodies is performed to visualize the LA and PVs in full length. Imaging at 10x and 40x magnification provides a comprehensive view of the PV structure as well as detailed insights into the myocardial architecture, particularly highlighting the presence of connexin 43 within the MS.

Author Spotlight: Developing a Translational Model for Atrial Fibrillation Research Across Species 

This protocol demonstrates microscopy-guided isolation and immunofluorescence staining of murine pulmonary veins. We prepare tissue samples containing the left atrium, pulmonary veins, and the corresponding lungs and stain them for cardiac Troponin T and Connexin 43.

Pulmonary veins (PVs) are the major source of ectopic beats in atrial arrhythmias and play a crucial role in the development and progression of atrial ...

Dissection Techniques and Histological Sampling of the Heart in Large Animal Models for Cardiovascular Diseases

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Chapters in this video

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

An Isolated Working Heart System for Large Animal Models

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Microdissection and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins