Once the abdominal cavity is opened, using a cut along the median axis of the muscular wall (Fig.9), the investigator should note phenomena which will be disturbed later in the progress of the autopsy. Therefore the position of the organs, the presence of any adhesions or liquids in the cavity, will have to be observed.
Fig. 9 Opening of the abdominal wall (Click on the image for a larger version)
Variations of organ position (ptosis or situs inversus), or absence of an organ, are congenital anomalies and very rare occurrence. Adhesions between several organs, due to inflammatory or neoplastic processes, as well as amounts of liquids, more or less abundant, are more frequently seen.
The abundant presence of liquids in the abdominal cavity is an indication of a pathological condition, and, in this case, it is important to carefully record this phenomenon, in order to achieve an accurate diagnosis.
An abundant, clear, lemon-coloured liquid (ascites) is as a result of portal hypertension, or of some severe lesions: renal nephritis or nephrosclerosis, hepatic cirrhosis or cardiac lesion. These findings are also frequent in mice bearing generalized lymphoma or leukaemia, associated with the presence of abundant infiltration of neoplastic cells.
The presence of less or more abundant blood (hemoperitoneum) could be a consequence of abdominal blood vessel ruptures, or of inflammatory processes of the arteries (angitis, polyarteritis), trauma, or of spontaneous ruptures of aneurisms. In some cases, the hemoperitoneum can also be a consequence of a rupture of a vessel in a determined organ, affected by hepatic tumours or lymphoma/leukaemia.
The presence of blood in the abdominal cavity frequently produces an inflammatory reaction of the peritoneum, called peritonitis. Various types of peritonitis can be observed: the serum-fibrinous peritonitis or the purulent one, easily distinguishable on the basis of exudate peculiarities. The exudate will be serous with thin fibrinous filaments in the first case, and dense, creamy and yellowish in the second case.
In the abdominal cavity, mainly in the pelvic region, small masses of adipose necrotic tissue can also be found, attached by a connective peduncle to the abdominal wall. This adipose dark-coloured tissue can easily be confused with small glands or other structures.
Once the observation of the abdominal cavity is completed, the investigator can proceed to the extraction of the individual abdominal viscera and to their detailed examination.
Anatomical outline. The spleen is an organ of predominantly lympho-erythropoietic function. The parenchyma of this organ contains: a) a tissue with erythropoietic function called red pulp, constituted by vessels and cords of various types of red cells (haemocytoblasts, erythrocytes, leukocytes, megakaryocytes); b) a lymphoid tissue called white pulp.
The spleen is situated in the left superior abdominal quadrant; it has a lengthened, oval, slightly curved shape.
The ventral face is smooth and convex; the dorsal one is slightly concave, oriented towards the stomach, to which it is connected by means of the gastrosplenic ligament. Moreover, the hilum, through which the splenic vessels enter the organ, lies on the dorsal face of the spleen.
In a young-adult mouse the spleen measures approximately 15 millimetres in length, 3 millimetres in width, 2 millimetres in thickness. Its average weight is approximately of 100 mg. Under normal conditions, the spleen has a friable and soft consistency, a smooth surface covered by a thin and transparent capsule, and a dark-red colour. Small variations in shape and volume, or the presence of rare aberrant accessories nodules of splenic tissue, can be rarely observed in animals of different strains and ages. In the spleen of the C57Bl mice it has been noticed, with a certain frequency, the presence of black spots, mainly localized on the postero-lateral and anterior part of the organ; these areas are found in the red pulp and are due to the presence of melanin in the cells.
Removal and examination. The spleen will be the first visceral organ of the abdominal cavity to be extracted using tweezers to hold and then cut the hilum together with the gastrosplenic ligament (Fig. 10).
The splenectomy is a simple operation since the organ is easily detached without abundant haemorrhage.
During this extraction the pancreas can also be easily removed due to its intimate connection with the spleen.
The anatomo-pathological examination of the spleen must take into consideration volume, consistency, colour, margins and any evident lesion.
Fig. 10 Spleen extraction
There are lesions, like the lymphoma/leukaemia, in which the organ is enormously enlarged (splenomegaly) with a tense capsule, of a hard consistency, and of rose or greenish colour (chloroleukemia). In these cases, if a longitudinal cut of the organ is executed, the investigator will be able to find on the cut surface proliferation of red pulp (myeloid leukaemia), or presence of trabecular stripes, or many large lymphatic follicles easily visible on the red pulp (lymphoma). The presence of large haemorrhagic zones can be associated with the rupture of vessels.
An enlarged spleen of soft consistency and with an elastic capsule, through which a dark-red colour is visible, is an indication of infectious process at the moment of death. In this case, at the cut, the red pulp will appear hyperplastic, flowing, and easily removable with the scissors.
An enlargement is also seen in the so-called "spleen stasis" for the abundant presence of blood which pools in the organ. Among the causes that can produce this condition, it is worth remembering serious disturbances of the circulation caused by cardiopathies or pulmonary lesions.
Moreover the spleen can be affected by infarcts, which show some peculiar characteristics in relation to the period over which the lesion has been manifested. A strikingly triangular hemorrhagic area, with the base usually oriented toward the surface, suggests a recent infarct. On the contrary, a sclerotic cicatrix suggests that the lesion has taken place well before the death of the animal and, therefore, is not correlated with the cause of death.
Finally the presence of a circumscribed visible mass on the surface of the spleen, usually smooth, haemorrhagic and of dark-red colour is associated with vessel hyperplasia or neoplasia (angiomas).
The glandular pancreatic tissue in the mouse is completely enclosed in the mesenteric adipose tissue and does not appear as true compact organ as in the human being. Frequently, in the old animals, the pancreas is atrophic, because it is completely lacking in acini. The atrophy of the pancreas can also be the consequence of a chronic pancreatitis or nodular polyarteritis. In some cases, the pancreatic ducts can be found enlarged as cysts due to stenosis of the pancreatic duct, and its rupture may cause a disease such as fat necrosis (steatonecrosis).
Primary tumours are extremely rare in the mouse. Within these neoplasms, the benign types are represented by nodular hyperplasia, and by the so-called adenomatosis, very difficult to distinguish macroscopically. The malignant tumours, very often of epithelial origin (adenocarcinoma), are highly invasive, mainly in the mesentery and in the adjacent lymph nodes of the liver. Of course, all these tumours are detected more frequently after oncogenic drug treatment.
Anatomical outline. The oesophagus is a thin rectilinear tube passing through the pharynx to the stomach. For the two-thirds of its length, the oesophagus lies in the thoracic cavity, behind the trachea. Through the diaphragm, it enters into the abdominal cavity where it is linked to the stomach through the cardiac sphincter.
The stomach is a hollow organ with a bag shape that lies in the ventral part of the abdomen, located mainly in the superior left quadrant, and is partly covered by liver lobes.
The stomach is composed by two parts: the non-glandular and the glandular one delimited by a macroscopically visible edge. The non-glandular stomach includes the superior part of the organ (fundus), called forestomach, and is covered by squamous epithelium. The glandular stomach comprises the body of the organ and continues with the duodenum through the pyloric sphincter. The mucosa of the stomach is thick and with convoluted plica. Looking from the outside, the stomach shows a lesser and a greater curvature.
The intestine is approximately 40 centimetres long and comprises the small and the large intestine. The small intestine is divided in duodenum, jejunum, and jejunoileum. The duodenum starts from the stomach and has a horse shoe shape; it continues into the jejunum that represents the longest tract of the small intestine. The jejunoileum follows, and ends in the caecum. The large intestine comprises the caecum, which consists of a small bag located in the right inferior quadrant of the abdomen in the iliac fossa, and by the colon with its ascending portion (starting from the caecum), by a traverse short portion, and finally by the descending part. The rectum is the final portion of the intestine and it goes from the descending part of the colon to the anus.
Removal and examination. Once the external observation of the spleen and pancreas is completed, the investigator can proceed to the examination of the gut, including the stomach, and then to its extraction. To this aim, it is necessary to take into consideration the external aspect of the intestine and how it is arranged, in order to detect possible distension of the wall, usually caused by the presence of putrefactive gas, or colour variations, caused by enteritis, colitis, or for possible presence of blood.
At this is point, it is suggested that the investigator will first proceed to examination of the mesenteric lymph node.
With just one movement, the extraction of whole intestine and stomach can be achieved, starting with a single cut at the level of the rectum (Fig. 11).
Fig. 11 Stomach and gut extraction
If the investigator holds this segment of intestine (the cut rectum) with forceps and lifts upwards and superiorly , the various intestinal segments can be gradually extracted proceeding in a caudal-cranial direction, cutting the insertion of the mesentery from the vertebral column up to the stomach. If you make another cut at the level of the oesophagus, closed to the cardiac sphincter, the procedure is completed so that the stomach and the intestine can be removed in one piece and are then ready for a detailed examination. Before proceeding to the luminal examination, however, it is advisable to carry out one technical procedure consisting in pulling out the full intestine, to achieve this you must proceed by cutting the intestine insertion of the mesentery (Fig. 12).
Opening of the stomach and the intestine. The opening of intestine and stomach can be made either during the autopsy, with fresh organs, or after a few hours of formalin fixation. To pass to the internal examination of the gut lumen, the investigator must proceed with a single longitudinal cut up the intestine at the level of rectum, and from here continuing to the stomach, taking care that this cut be performed close to the mesentery insertion.
Fig. 12 Gut extracted and ready to be examined
The investigator is now able to investigate the contents of the intestine, wall thickness, colour, aspect of mucosa and its obvious lesions. In the glandular part of the stomach, particularly in DBA strain, it is possible to observe proliferative lesions of the mucosa, which can also be macroscopically diagnosed as a tumour. The mucosa of the large and small intestine appears reddish, hyperplastic and with fine haemorrhagic petechia or small ulcers in case of an inflammatory lesion (enteritis and colitis). Furthermore, in haemorrhagic gastritis there is an abundant presence of blood, which appears as a dark fluid.
The small intestine walls are normally scattered by small, white, protuberant nodules, called Peyer's patches, which, at the autopsy, can be erroneously diagnosed as small tumours or metastases. In generalized lymphoma, these patches, together with superficial and deep lymph nodes, can appear hardened in texture, and remarkably enlarged to become very big nodules of grey colour. Finally, in the advanced stage of this lesion, the Peyer 's patches can cover the entire intestine wall which will then appear uniformly thickened.
Digestive system tumours are frequent in the mouse and easy to diagnose. The epithelial malignant tumours (adenocarcinoma) are usually localized in the glandular part of the stomach and in the small and large intestine mucosa. They appear as large ulcerated masses with a distinct invasive character. More rarely the investigator may observe large tumours in the forestomach and small tumours, or polyps, usually localized in the duodenum of the DBA strain.
Anatomical outline. The liver is a large glandular organ which occupies a a large portion of the abdominal cavity of the mouse. With its superior convex surface, the liver adheres to the diaphragm, while its inferior concave surface is in contact with the stomach and the duodenum.
The liver has four large lobes which join themselves in the dorsal region around the hilum. It can be distinguished a median lobe, two lateral (one right and one left), and one caudal lobe, subdivided into dorsal and ventral half. A thin transparent capsule, called the Glisson capsule, covers the organ. At the macroscopic examination, the cut surface shows a granular aspect due to a typical lobular structure. In fact, the lobules constitute the most important entity of the liver and they are composed by several cords of epithelial cells, arranged in a radial pattern around a vessel.
Fig. 13 Liver extraction
The normal colour of the liver is dark-reddish. The liver is hard, but friable when one exerts pressure on the parenchyma. The weight of the liver is of approximately 1.34 g in a mouse of three months of age.
The cholecyst, also called gall bladder, is visible on the inferior surface of the organ where it appears like a small a bag of a few millimetres in diameter. The bile, produced by the liver, reaches the gall bladder via the hepatic duct, where it is concentrated; the bile reaches the intestine through the cystic and the common bile ducts.
Removal and examination. In order to remove the liver, the investigator cuts, with scissors, the falciform and coronary ligaments that keep the organ intimately connected with the diaphragm, taking care not to cause lesions (Fig. 13). Once the liver is isolated, it will be removed, together with the inferior vena cava and the hepatic vessels. The investigator will then pass to the external examination of the organ, taking care to describe the key characteristics such as volume, consistency, colour, and general aspects of the cut surface.
In some lesions, variation of the volume of the organ is often associated with modification of the liver margins: in the case of organ atrophy, such as acute atrophy of the liver and cirrhosis, the margins are thin and sharp; while they become large and round in all the lesions associated with hepatomegaly, such as hepatostasis, fatty degeneration, and inflammation.
Of course, an increase in liver volume is also observed in neoplasia, and it must be remembered that hepatomas are among the most frequent tumours observed in the mouse, particularly in the C3H strain. These tumours consist of globular masses of variable size, from a few millimetres to some centimetres, and are usually localized in one of the greater lobes of the liver; they are usually well circumscribed and of soft consistency; their colour is similar to that of normal liver tissue.
A liver enlargement can also be observed in the case of generalized leukaemia/lymphoma accompanied with an increase in firmness, and variation of the colour from rose to dark-greenish. In some cases, the presence of irregular nodules on the organ surface is observed.
If the investigator proceeds to make a longitudinal cut along the greater lobes, he/she will then be able to observe more carefully the structure and the consistency of the organ. Possible variations of the colour and aspect of the lobe can be seen.
In many types of lymphoma, the pathologist might observe a massive infiltration of neoplastic cells associated with a pronounced proliferation of the connective tissue, with the appearance of small islands or large stripes, which usually alter the normal architecture of the lobes.
In fatty degeneration, some yellowish areas, surrounded by dark-red parenchyma, are observed at the cut surface. On the contrary, in the condition of "stasis", the normal lobular structure seems preserved, even if a marked congestion is present, due to an abundant blood content in the lobes.
Once the liver observation is completed, the organ is turned with its inferior face towards the investigator to examine the gall bladder. The external characteristics of this organ should be recorded, such as shape, size, and, soon after having produced one small cut, the content of the organ and the state of the covering mucosa.
The volume of the gall bladder is increased by bile retention, mainly caused by the presence of stones or as a consequence of the pressure of abdominal tumour masses. Other conditions may cause a volume decrease; such as chronic inflammatory diseases, ductal obstruction, and tumours.
The gall bladder liquid content, when present in sufficient amount, should be drawn up into a pipette and examined. Usually, the liquid is more or less clear and of a brown colour. Very rarely, the investigator will note the presence of stones; these are small, with smooth irregular surfaces, of different colours from dark-brown to grey, due to their different contents. Finally, the mucosa should be examined. In normal conditions, it has a smooth aspect and shows an irregular, reddish surface due to inflammatory diseases only in few cases.
Once the abdominal cavity is freed of the organs previously removed, the investigator will be able to more easily examine the urinary apparatus.
Fig. 14 Kidney and ureter extraction
Anatomical outline.The urinary apparatus consists of kidneys, ureters, bladder and urethra. The kidneys are paired organs, located on the dorsal (posterior) wall of the abdomen, beside the vertebral column. They have a bean shape. In the concave medial margin of the organ, you can appreciate the presence of the hilum of the kidney, from which the main vessels and nerves exit. The ureter also originates from this same area, called the renal pelvis.
The variation in dimensions or position of these organs might be easily appreciated by comparing the two kidneys. In fact, the right is usually larger and heavier that the left one, and it is also positioned more cranially. The weight of the right kidney is approximately 210 milligrams, and that of the left is 200 milligrams. The kidneys show a hard compact consistency, and a reddish colour that sometimes turns to yellow.
The ureters are two thin small ducts that connect the kidneys to the bladder. Their main function is to allow the urine, produced by kidneys, to be collected in the bladder. The ureters end up separately in the posterior part of the bladder, close to the neck.
The bladder appears as a small oval bag, covered by a thin greyish wall, and lies in the anteromedial area of the abdominal cavity. In its inferior part, this bag shrinks and continues in the neck and then in the urethra.
The urethra consists in one medial small duct that goes from the neck of the bladder to the external opening of the penis in the male mouse, and in the fossa clitoridis in females. This tract represents the last part of the urinary system, through which the urine is eliminated.
Just for topographical reasons, the adrenal glands are described in this chapter, dealing with the urinary system, because these glands lie on the superior pole of the kidneys and are intimately joined to them by fibrous and fat tissue. In normal conditions, it would be very difficult to isolate and extract these organs for their small size, that do not usually exceeds the head of a pin.
Removal and examination of kidneys, adrenal glands and ureters.
An useful technique for extracting the kidneys and ureters altogether consists in grasping the inferior part of the ureter with forceps near to its opening (Fig. 14) and lifting upwards.
The ureter is then detached from its attachments to the dorsal body wall up to the renal pelvis; here the main vessels are cut so that the investigator might proceed to the removal; in this way the adrenal glands will be also removed.
Nephrosclerosis and glomerulosclerosis are the most common lesions of the mouse kidney, macroscopically detectable mainly in aged animals. In these cases, the organ will be reduced in volume, pale, and with a shrunken surface. In the terminal stage, the lesion may also cause an increased hardness of the whole organ, which is easily detected when the longitudinal cut is subsequently made; sometimes small cysts, filled with urine, can also be seen.
Hydronephrosis and pyonephrosis, on the contrary, may cause an enlargement of the kidney. In such a case, the investigator should make a careful examination of the ureters because some disease, such as inflammation, presence of stones or of tumours, can be closely correlated with this enlargement.
Of course, the enlargement of the kidney is also detectable in presence of tumours; rare in the mouse. The tumours of the kidney generally consist in a whitish nodule present just in one of the two organs. Finally, in leukaemia/lymphoma, a diffuse or nodular whitish tissue is detected, with the same characteristics observed in other organs when similarly affected (spleen, liver).
At macroscopic examination, the adrenal glands show some morphological differences correlated with the sex of the animal. In the females, the adrenals appear of an opaque pale colour, because of the high lipid content, while, in the male, they are often rose-coloured and translucent. The volume of the adrenals can also be greatly increased by the presence of a large nodular formation or tumour of variable dimensions and of dark colour.
Removal and examination of the bladder. The technique for the extraction of the bladder is identical in both sexes and is executed by grasping the highest point of the bladder with forceps and making a small cut with the scissors, as a keyhole, on the front wall. The liquid present in the cavity should be sucked up through the opening with a small pipette.
This operation will become necessary in case of vesical urinary retention, due to paresis or blocked outflow, due to the abundant amount of stagnant urine. In this case, the bladder walls are so distended that removal of the contents is very difficult. Therefore, after having emptied the organ of its contents, the investigator makes a cut at level of the neck, possibly in the lowest part.
With this technique, the prostate gland, which is very difficult to detect macroscopically, will then be removed.
Diseases that alter the shape and the volume of this organ (prostatic hypertrophy, tumours) are observed very rarely in the mouse; in these cases only, the investigator will be able to describe its macroscopic characteristics.
The female and male genital apparatuses will be examined separately, having specific configurations in each sex and different relationships with the surrounding organs.
Anatomical outline. The genital apparatus of the female mouse includes the uterus, the ovaries, the oviducts and the vagina.
The uterus is formed by a tubular median part (body of the uterus) and two lateral formations (lateral horns). The caudal part of the uterus body is called the neck.
The uterus is found in the inferior part of the abdominal cavity and adheres to the posterior wall of the abdomen. In particular, the body of the uterus is placed anteriorly to the urinary bladder and is covered by a serosa adherent to the posterior wall of the abdomen. In normal conditions the colour of the organ is greyish.
The ovaries lie close to the inferior pole of the kidneys and are tied to the posterior wall of the abdomen by means of the mesovarium. They are small bilateral spherical organs with smooth surfaces. During the period of the sexual maturity, the ovaries assume a nodular aspect due to the presence of follicles and corpora lutea.
Beyond their role as endocrine glands, the ovaries have the fundamental function of production of the oocytes that, once expelled, pass into the uterus through the oviducts, and there are fertilized.
The oviducts are two tight and small tubes, wrapped around themselves as a ball; they connect the ovaries to the horns of the uterus.
Finally, the vagina is a short channel that begins from the neck of the uterus and ends externally in front of the anal opening. In the abdominal cavity, the vagina lies in front of the rectum and behind the urethra.
Removal and examination.The Female genital apparatus is excised by making a cut on the median body of the uterus close to the neck (Fig. 15). From here, proceeding upwards, the mesometrium ligaments, that fix the organ to the posterior wall of the abdomen, are cut up to the level of the ovaries at the back.The investigator will then delicately cut those ligaments by which the ovaries are attached to the inferior poles of the kidneys, as well as from the mesovarium ligaments, with which the ovaries are joined to the posterior wall of the abdomen. Isolated organs are extracted and examined for shape, volume, consistency and obvious lesions.
Variations of uterine shape are very rare and are associated usually with congenital malformations. More frequently, the enlargement of this organ is associated with the presence of neoplasia (fibrosarcoma), or to the collection of pathological liquids (hydrometra, pyometra). In these latter cases the uterus will be markedly enlarged.
Fig. 15 Uterus Removal (Click on the image for a larger version)
The investigator will make some cuts on the surface of the organ to investigate the consistency, the thickness of the walls and the character of the contents. Moreover, enlargements are found in case of leukaemia/lymphoma or cystic hyperplasia of the endometrium.
The ovaries are then examined for their volume and surfaces. Tumours (luteoma, tubular-adenoma), cysts, leukaemia/lymphoma are some of the lesions that more frequently affect this organ and induce variations of shape or volume.
Anatomical outline. In the male mouse, the genital apparatus includes the testes, receptacula seminis, the epididymis, the penis and the preputial glands.
The testes are two organs of oval shape with a diameter of a few millimetres, situated at the side of the bladder and inside the scrotum. They are covered by a smooth and transparent membrane (tunica albuginea). Under normal conditions, they are greyish-white, of soft elastic consistency. The average weight is 103 mg for the right and 101 mg for the left testis.
The testes consist of numerous tubules covered with various cell layers and their main function is the production of mature sexual cells or spermatozoa. Moreover the testes produce male sexual hormones or androgens.
The spermatozoa produced from the testes pass into the urethra through the excretory ducts (epididymis and the deferent ducts).
The receptacula seminis are two bilateral structures that are found at the bladder sides and are in communication with the urethra. They are formed from numerous small secretion filled cavities. They have a curved flattened shape and a clear-grey colour. Owing to their vesicular conformation these formations have a soft consistency.
In the mouse, the preputial glands are included in the connective tissue, they are leaf shaped, and lie above the penis. They are of a dark-grey colour and a soft consistency.
Removal and examination. The investigator first of all must check the position of the organ, because, when the abdominal cavity is opened, the testes are often found outside the scrotum, leaning to the posterior wall of the abdomen and at the sides of the bladder; therefore, by grasping them delicately, they may be removed, cutting the excretory ducts close to their outlet in the membranous urethra (Fig. 16).
Fig. 16 Testis extraction
The investigator will then record the shape, the volume, the consistency, the presence of tumour masses and the characters of the epididymis. If a median longitudinal cut of the testes is made, he/she will also appreciate the colour, cut surface consistency, presence of inflammatory zones or of neoplasms.
A rupture of a blood vessel that accompanies the excretory ducts or the same testis usually causes the enlargement of the organ. In this cases the testis appears markedly enlarged with wide areas of blood infiltration, the remaining parenchyma is reduced to a small necrotic portion.
The receptacula seminis are often expanded and filled of serous liquid. This happens when an obstacle is present in the ejaculatory duct. In other cases, they are enlarged and filled of a yellowish liquid (pus), as it happens in the purulent inflammation. These lesions are of great diagnostic importance in the mouse, because they can be often the cause of peritonitis.
In order to complete the examination of the male genital apparatus, the investigator will then examine the penis and the preputial glands. The abscesses of these glands represent a rather common lesion in old animals and they can sometimes be detectable on the correspondent ulcerated skin.