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

Summary

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.

Abstract

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.

Introduction

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). 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 textbooks^{1,2,4,5,6,7,8,9,10,11,12}, journal literature^3,13,14, official documents¹⁵, and webinars^16,17. The samples used in this study were harvested from cadavers submitted to the Pathology Department of the USAMV Cluj-Napoca for routine autopsy diagnostic.

Protocol

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. 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 performed^2,12. The following steps represent the necropsy performed in a medium-sized dog. Adapt the steps when performing necropsies on different subjects.

1. 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).
2. Use a knife (or a scalpel) and make an incision of 7 cm in the right axilla (Figure 1A).
3. Locate the coxofemoral junction (Figure 1A,B). Open the capsule with a knife. Cut the ligament of the head of the femur (Figure 1B).
4. Use the knife to extend the skin incision in the axilla cranial to the mandibular symphysis and caudally to the perineum (Figure 1B).
5. 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).
6. 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).
7. 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.
8. Briefly, examine the abdominal organs in situ (by noting any changes in changes in color, consistency, volume, and position of the organs).
9. With the scalpel and forceps, puncture the diaphragm at the highest point (Figure 1B).
10. 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).
11. 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).
12. 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.
13. 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 terminalis^11,13.
14. 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 content^1,2. 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.

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 "inflow-outflow method," which allows a better examination of the valves and the endocardium and is a protocol used for most of the species^2,11,12,16, and 2) the "four chambers dissection"/"echocardiographic plane" technique, typically used for cats or small-sized dogs^1,17.

1. The "inflow-outflow method" for cardiac dissection^2,11,12,16
   1. Place the heart with the atrial face upward (Figure 2A).
   2. 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.
   3. 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.
   4. Section the right ventricle free wall along the interventricular septum. Continue the section until the origin of the pulmonary trunk (Figure 2C,D).
   5. Thoroughly examine the right atrioventricular valves, including the chordae tendineae, and the papillary muscles (Figure 2D). Cut the chordae tendineae.
   6. Place forceps through the pulmonary trunk and cut the trunk longitudinally (Figure 2E). Examine the pulmonary outflow tract, pulmonary trunk origin, and semilunar valves.
   7. 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.
   8. 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.
   9. Cut the chordae tendineae of the cusp of the left atrioventricular valve.
   10. Place forceps through the left ventricular outflow tract and use it as guidance to open the aorta (Figure 2G).
   11. 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.
   12. Weigh the heart.
   13. 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.

3. Sampling protocol for histology¹⁶

1. 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)
2. Make two longitudinal parallel cuts (approximately 5 mm apart) involving the left atrium, mitral valve, dorsal papillary muscle, and left ventricle (Figure 2H).
3. 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).
4. Make two longitudinal parallel cuts involving the base of the aorta and the interventricular septum (Figure 2J).
5. 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).
6. 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).
7. Immerse the tissues in 10% NBF for at least 48 h.

4. Sampling of coronary arteries^3,10,14

1. 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.
2. If needed, decalcify adequately in a 1:1 mixture of 8% formic acid and hydrochloric acid.
3. 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 recommended^3,10,14.

5. The "four chambers"/"echocardiographic plane" dissection technique of the heart^1,17

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 "four-chamber" technique is best applied to fixed tissues.

1. Place the whole heart into 10% NBF for at least 48 h.
2. 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).
3. 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.
4. 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).
5. 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 µm thickness, and stain with hematoxylin and eosin stain (H&E stain) following a previously described protocol¹⁰.
   NOTE: If myocarditis is suspected, "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"¹³.

6. Photographic documentation

NOTE: Photographic documentation is an optional step in the necropsy examination. However, photography and video recordings are essential to have an "accurate documentation of normal and abnormal anatomy"³.

1. For proper photographic documentation, take digital images while performing "progressive dissection of cardiac tissues"¹³. Refer to the guidelines for photographic documentation and photograph¹⁵ the general view-anterior and posterior; a slice of the heart; and any suspected lesions.

Results

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 ...

Discussion

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 weight^11,12. 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...

Materials

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