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Lab Animal Research
 

Diagnostic Necropsy and Tissue Harvest

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Necropsy-meaning postmortem examination of organs-and tissue harvest are integral components of many lab experiments, which rely on the final data collection time points gathered from the analyses of the harvested organs. Therefore, it is important to learn the correct diagnostic necropsy and tissue harvesting technique, as the procedure can significantly impact the quality of the samples collected. This presentation will review the method of dissection and extraction of abdominal, reproductive, and thoracic organs from lab animals.

Before starting with the procedure, a form should be initiated that includes a complete history of the animal: animal identification number, gender, housing conditions, date of birth, date of death, study/protocol number, and the name of the Principle Investigator.

Next, prepare the dissection area. Lay down a bench covering, dissection tray, instruments designated for necropsy, which includes scissors, forceps, sterile scalpel blade, bone cutters, blunt probe, small spatula and non-absorbable suture material. Organ and tissue harvest for histological examination require that the tissues be preserved in a fixative solution. Unless otherwise specified, the fixative most commonly used is 10% neutral buffered formalin. Note that the tissue samples should be 0.5-1 cm in thickness to allow sufficient penetration of the fixative solution. Fixation prevents decay, autolysis, and putrefaction; stops any ongoing biochemical reactions; and may increase the mechanical strength and stability of the treated tissues.

The method of euthanasia of the animal can also impact the quality of the samples. The most commonly used euthanasia method for mice and rats is an overdose of carbon dioxide gas. The animals are left in their home cage, which is placed, into a chamber, and CO2 is gradually introduced into the chamber at a displacement rate of 10-30% of the chamber volume/min. This causes the animals to lose consciousness prior to the perception of pain that is associated with nociceptor activation by carbonic acid. The flow is then maintained in the chamber till respiratory arrest has occurred and the animal is dead.

Following euthanasia, first perform an initial external examination by visually inspecting the carcass for lesions and masses, excessive tooth growth, or any staining of the fur at the mouth, nares, ears, eyes and the anal and genital openings. If warranted, perform a tape test, skin scraping, and pelt exams to detect external parasites. Once the external exam has been completed, it is time to start the necropsy, beginning with the abdominal cavity. Note that before removing any tissue samples it is important to observe the organ in situ.

To begin excising the skin, place a small cut just anterior to the pelvis in females and above the prepuce in males. Then, using the blunt dissection technique, loosen the skin from the fascia and muscle. Blunt dissection is a technique in anatomical dissection in which tissues are separated and underlying structures exposed without cutting. In this technique, the scissors are used to spread tissues apart rather than to cut them apart. The closed tips are pushed into tissue and then opened to split tissue along natural planes. This process requires patience and a delicate touch, as the stretching of the tissue can result in damage to adjacent organs and blood vessels. Then extend the cut to the chin. Next make, transverse cuts anterior to the hind limbs and posterior to the forelimbs, and use blunt dissection to expose the cervical area and chest. Extra care should be exercised to avoid rupturing the jugular and carotid vessels in the neck.

In females, with the skin excised, observe the mammary tissue from the top of the sternum at the manubrium to the genital opening on the ventral surface; extending laterally on both sides. Lactating or pregnant females will have increased mammary tissue volume and milk may be present. To excise the mammary glands, grasp the tissue edge with forceps and use blunt dissection to loosen attachments to the skin. Once the gland has been separated, it can be placed in a fixative solution for subsequent histological analysis.

At this stage, you can observe submandibular salivary glands, which are paired and located at the mandible -- extending along the neck to the manubrium sternum. Removal of these glands requires blunt dissection and extra care when working in the cervical region to prevent rupture of blood vessels. Once freed from the underlying muscles, lift up the glands and sever any residual attachments. The glands require at least one cut to allow penetration of the fixative.

In addition, you can see the trachea, which extends from the epiglottis to the bifurcation of the bronchi. It is a ridged, cartilaginous tube that is flexible. While normally clear, euthanasia with CO2 can cause fluid accumulation in the lungs and trachea that looks like a frothy clear fluid. Subcutaneous fat will also be present in this area. Evaluate it for quantity and deposition. An obese animal may have a large amount of fat with the skin feeling thickened.

Prior to opening the body cavity, observe the abdominal, intercostal, and exposed neck muscles, and limbs for any thickening, masses or discoloration. To open the body cavity, first make a small transverse cut at the most caudal point of the exposed abdominal muscle. Then lift the muscle away from the organs, and cut along the linea alba to the xiphoid. Next, dissect the muscles laterally from the midline to just above the hind limbs on both sides. Lastly, cut along the curve of the ribs on both sides. Once the body cavity has been opened, evaluate the quantity and accumulation of abdominal fat. A healthy animal will have abdominal fat pads and some fat along the dorsal surface in the cavity. Observe the color of this tissue and note any abnormalities.

Now we are ready to begin the harvest of abdominal organs. Begin by locating the spleen, which is dark red and located along the lower curvature of the stomach. It should be regular in shape and with a slightly matte surface. To remove the spleen, lift the organ and snip the attachments to the stomach.

The stomach is located at the distal end of the esophagus. It appears to be two-toned, differentiating the muscular and glandular portions. Feel it for presence of food. An empty stomach should be noted, as it can be indicative of illness. Do not cut the stomach, as the contents will contaminate the organs in the abdominal cavity.

The small intestines are connected distally and inferior to the stomach. There are three distinct sections of the small intestine. First is the duodenum - a shorter section from the posterior stomach sphincter to the start of the jejunum. The bile duct enters here and the pancreatic tissue is more firmly attached to this portion of the small intestine. The jejunum is the center portion. Peyer's Patches, which are small oval areas composed of lymphoid tissue, can be observed on the surface of the jejunum and ileum. The ileum is the longest portion of the small intestine that terminates at the cecum - located at the junction of the small and large intestines. The cecum appears greenish in color and is very soft.

The large intestine continues from the cecum to the anus. It is readily identifiable as fecal pellets can be visualized within the lumen of this structure. The small and large intestines are anchored to the body by the mesentery, a membrane containing blood vessels, fat and lymph nodes. This should be examined for enlarged lymph nodes and any masses prior to removal of the intestinal tract. The pancreas is a diffuse organ located posterior to the stomach. It is a light tan to gray color and composed of multiple small lobes with irregular edges. To harvest pancreas, grasp the organ and gently tease it from the surrounding mesenteric tissue. This must be done prior to the removal of the intestinal tract.

To remove the entire tract as one piece beginning with the stomach and extending to the anus, first place a ligature at the rectum and then make a cut through the large intestine just anterior to the anus. Next, place a ligature at the junction of the esophagus and stomach, following which the entire intestinal tract can be lifted and any membranous attachments severed. The whole thing can then be separated from the mesentery, cut into sections and fixed.

The kidneys are paired organs located against the muscles of the back. They should be the size and color of a dark kidney bean and have a smooth surface. Immediately anterior to the kidney is the adrenal gland appearing as a small light pink nodule.

To remove a kidney, isolate it using a forceps and cut between the organ and the ureter. The tough outer layer can then be peeled off to examine the surface. Cut one kidney in half along the long axis and the other transversely. Any grit within may indicate the presence of crystals or mineral deposits. Lastly, remove the liver, which is a dark red in color and its margins should be smooth with a crisp edge. Care should be taken when handling the liver, as it is a friable tissue. Any disruption to the integrity of the organ will result in blood leaking into the body cavity and obscure other organs.

To remove the liver, first gently reflect the lobes away from the diaphragm and make a cut through the blood vessels anterior to the structure. Next, reflect the liver back toward the diaphragm and grasp the fibrous node that connects all the lobes centrally. Lastly, lift the organ while severing all attachments to the intestinal tract and stomach.

The female reproductive system consists on the uterus and the ovaries. The uterus is a short Y-shaped structure with horns extending in both directions. The horns terminate at the fallopian tubes and the ovaries -- located just below the kidneys. The ovaries will have a rough surface due to different maturation stages of the follicles.

To remove the ovaries, cut the arterial attachments anteriorly and the fallopian tube posteriorly. To remove the uterus, gently grasp and cut below the cervix. After the cut, lift the body and horns of the uterus breaking any attachments in the body cavity.

In males, you can also observe the preputial glands located just anterior to the prepuce. They appear large and are a gray to yellowish color with a foamy appearance. Removal and fixing of these glands is similar to submandibular glands. In addition, the male reproductive system consists of seminal vesicles, the prostate gland and testes. The seminal vesicles are white, "ram's horn shaped" structures located anterior to the urinary bladder and attached on the midline at the prostate gland.

The prostate gland -- generally light tan in color -- is located surrounding the urinary bladder at the base. To visualize the testes, grasp the abdominal fat pads located in the lower abdomen and pull them anteriorly. This will pull the testes from the scrotum to allow examination. The surface should be smooth with fine vascularization evident on the surface.

The epididymis is along the lower margin of the testis and tapers toward the top. The vas deferens is attached to the end of the epididymis and leads back to the prostate. To remove the testes, cut the attachment at the scrotum and cut the vas deferens. To remove the prostate and seminal vesicles, grasp the base of the urinary bladder and lift while severing the attachments beneath the prostate.

Moving on to the organs within the thoracic cavity. To expose the vital structures, remove the diaphragm from the attachment to the ribs. Next, cut through the rib cage laterally on both sides up to the top of the manubrium sternum, and then reflect the bones cranially to visualize the thoracic organs.

The lungs are normally a bright pink color, spongy in texture with a smooth surface. However euthanasia with CO2 can cause pulmonary hemorrhages resulting in dark red splotches that can cover the entire lung surfaces.

The heart is dark red and the ventricles are muscular, which feel firm to the touch. The atria are darker red in color and sit at the top of the ventricles. They are much less muscular and appear flaccid. A thin translucent membrane called the pericardial sac surrounds the heart.

The thymus is located anterior to the heart and sits over the trachea. It should be smooth in texture. You can see the trachea as described before, and the esophagus is a very thin tube that lies directly behind the trachea and behind the heart and passes through the diaphragm to the stomach.

To remove the organs in the thoracic cavity, begin by grasping the trachea just above the thymus and make a perpendicular cut just anterior to the forceps. While maintaining the grasp, lift the trachea up caudally and snip any attachments of the lungs to the spinal surface in the rib cage. The esophagus may need to be cut to be able to lift the heart and lungs free of the chest cavity

After the heart is removed for the body, flush it with saline to remove residual blood and clots, or fill it with the fixative through the aorta. Once the lungs are excised, placed a loose ligature around the trachea. Next, thread a needle attached to a syringe containing the fixative into the lumen of the trachea, and tighten the ligature. Then inject the fixative until the lungs are inflated. Lastly, remove the needle and tighten the ligature further to prevent leakage.

With this breath of knowledge regarding the rodent necropsy and tissue harvest, let's look at some of the current lab experiments involving these procedures.

Diagnostic necropsy is a common endpoint in cancer metastasis experiments. In this example, the investigators injected cancer cells into the rodent's spleen Then, thirty to sixty days later, they conducted necropsy, which revealed significant liver metastases.

Tissue extraction is often followed by histological analysis, which helps in the study of microscopic anatomy of the sample. By following the protocol of fixation, embedding, sectioning and staining of tissues, researchers are able to study the microscopic structures in these organs, and uncover the effect of genetic or pharmacological interventions at atomic level.

At times researchers remove tissues to study physiological processes, like angiogenesis. Here, the experimenters exteriorized a rat's mesenteric tissue to stimulate vascular growth. Then, they harvested a few sections, and treated them with the markers that stain the cells of any blood vessel, to study the cellular dynamics involved in vascular network growth.

You've just watched JoVE's video detailing the steps of diagnostic necropsy and tissue harvest in lab animals. It is important to use proper techniques for organ removal and preservation, so that the extraction procedure has no effect on the interpretation of the data collected. As always, thanks for watching!

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