Visceral examination

A minimum of 50% of fetuses are to be examined for visceral alterations and a minimum of 50% for skeletal abnormalities. When a fresh tissue microdissection technique is being used for the visceral examination of rabbit fetuses, all fetuses should be examined for both visceral and skeletal abnormalities. 

The remaining fetuses are preserved for fixed visceral examination. This may be accomplished by serial sectioning (see Chapter 19) or by microdissection (see Chapter 20). 

The remaining fetuses are submitted to fresh soft tissue examination or preserved for fixed visceral examination (see Note 2). Various techniques are available typically based on Wilson s sections of the head and body or Wilson s sections of the head only with microdissection of the body. The techniques used for the preparation and examination of the fetuses are described in Chapters 16-21. 

Bouin s fixative has been used traditionally for the fixation of fetuses prior to fixed visceral examination. However, safety concerns over the use of picric acid have resulted in the use of alternatives such as Harrison s fixative (18). 

Fig. 4. Preparation of the fixed heart for visceral examination. Cut 1 Ventral view Open the right ventricle along the septum in baso-apical direction going trough the aorta and the truncus pulmonalis (cross pulmonary valve) dorsal view open right ventricle near the septum from the heart apex towards the right atrium (through tricuspidal valve). Cut2 Ventral view Open the left ventricle along the septum from the heart apex into the left atrium (through bicuspidal valve) dorsal view cut the wall of left ventricle from the heart apex towards the left atrium.

Infants—necropsy including visceral examination, and collection of organ weights and fixation of tissues, for details see Table 6. For a description of visceral examination, the reader is referred to Chapter 14 of this volume. 

Immediately after the external and visceral examinations and before the Alizarin Red S staining, the fetuses may be kept in thymol solution, for up to 7 days for rat, mouse, and minipig fetuses and 10 days for rabbit fetuses. Thymol has proven antibacterial activity (4-6). 

Stuckhardt JL, Poppe SM (1984) Fresh visceral examination of rat and rabbit fetuses used in... 

Both the fresh and fixed visceral examinations are destructive nonetheless, a trained fetal morphologist can at least reexamine the retained fixed organs and tissues. This is particularly of value for rodent specimens. Photographs of abnormalities can also be taken (see Note 2). 

The fetuses allocated to visceral examination (approximately half of each litter) are placed in the selected fixative (see Note 3) for at least 6 days. The remaining fetuses are processed for skeletal examination. 

The ICH M3 (1) guideline recommends external and visceral examinations for a minimum of six dams treated throughout the period of organogenesis in preliminary studies. Microdissection is well-suited to this approach, due to the speed with which results can be generated. 

In contrast to primates, which abort dead embryos, dead rodent embryos are resorbed and the implantation site is recorded as a resorption site. The numbers of living and dead fetuses, and the number of resorption sites, are counted, and fetal weight, sex, and external malformations are recorded. Fetuses can either be inspected fresh in evaluation of internal soft tissues (known as the Staples technique) or can be placed in a fixative (usually Bouin s solution) and sectioned at a later time (Wilson technique). Other fetuses are fixed in ethanol, cleared in potassium hydroxide, and the cartilage and bone are stained with Alcian Blue and Alizarin Red, respectively. The US FDA recommends that one-third of the rodent fetuses be subjected to visceral examination and that two-thirds be studied for abnormalities of cartilage and bone. The US Environmental Protection Agency (EPA) recommends that one-third to one-half of each litter be examined for skeletal anomalies. For rabbits, all fetuses are to be examined for both visceral and skeletal malformations. 

Faherty, J. F., B. A. Jackson and M. F. Greene Surface staining of 1 mm (Wilson) slices of fetuses for internal visceral examination. Stain Technology, 1972, 47(2) 53-58. 

Figure 7.1. Vomiting Mechanisms. The afferent nervous (peripheral and central) and humoral inputs converge to the medullary area. Here the signals are examined and integrated and may lead to emesis (expulsion of gastrointestinal contents). The efferent output involves respiratory muscles, visceral organs, cardiovascular system, visceral and cutaneous vasculature. The endogenous factors are released into...

Fitzhugh and Nelson (19) found that oxalic acid up to 1.2% of the diet did not affect growth or mortality rate of rats fed oxalic acid for 1 year. Microscopic pathological examination showed no major visceral damage, but some of the rats showed slight periportal hypertrophy of the hepatic cells along with slight centrolobular atrophy. 

Visceral Fetal Examinations. The examination of the abdominal and thoracic viscera of fetuses is performed either fresh without fixation ( Staples technique ) or after Bouin s fixation by making freehand razor blade sections ( Wilson s technique Wilson, 1965). Both techniques have advantages. The fresh examination technique, which may require less training for thorough proficiency, provides a more easily interpreted view of heart anomalies. The examination must be performed on the day the dam is terminated, however, so having a large number of litters to examine in one day requires that a large team of workers be committed to the task. 

Exposure of pregnant Alderley-Park rats to 1,4-dichlorobenzene via inhalation at levels up to 508 ppm for 6 hours per day on Gd 6-15 did not result in developmental effects in the offspring (Hodge et al. 1977). End points examined included the number of viable fetuses, fetal weight, litter weight, sex ratio, external abnormalities, and skeletal and visceral abnormalities. 

Two workers died while applying dry sodium metabisulfite in a ship hold. Postmortem examination showed diffuse pulmonary edema consistent with death secondary to asphyxia and visceral congestion. 

A 38-year-old man with a family history of cardiovascular and cerebrovascular disease makes an appointment for a routine physical examination with a physician he has not seen before. He explains that his father died young of a heart attack and that two paternal uncles have suffered strokes in their late 40s. Physical examination reveals yellowish lumps on his eyelids (xanthelasmas, which are often associated with a lipid disorder) and a resting blood pressure of 186/95 mm Hg. There is some excess visceral fat, and his body mass index calculates to 26.5. Total serum cholesterol (476 mg/dL) and triglycerides (288 mg/dL) are elevated and subsequent angiography reveals atherosclerotic restrictions of at least two coronary arteries. 

The fetuses are then examined. Full details of fetal examination are provided in other chapters of this book some techniques are performed on fresh fetuses, while other techniques require prior fixation of the fetuses. For rodents, one half of the fetuses in each litter should be examined for soft tissue (visceral) changes the remainder should be examined for skeletal changes. Rabbit fetuses should be examined for both soft tissue and skeletal abnormalities the heads from half of the fetuses should be examined by serial sectioning, with skeletal examination conducted on the remainder of the fetus. 

All live fetuses are examined viscerally and sexed at the time of caesarean section. 

The fetuses should be weighed, sexed, and examined for fresh visceral abnormalities before processing for skeletal examination. The litter sizes of minipigs are normally five or six fetuses. 

Key words Fetal examination, Visceral, Fresh soft tissue. Fixed tissue. Microdissection... 

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