Necropsy Procedure

Initial characterization should include necropsies performed on immature and mature mice of both genders as well as mice sacrificed in a moribund condition or found dead (autolytic state permitting). Embryos and fetuses should be examined in those mouse lines with high fetal loss or developmental anomalies. Blood for serum chemistry evaluation should be collected immediately before euthanasia. The necropsy is performed in a systematic and orderly fashion. Tissues in each system are evaluated grossly and microscopically (Table 10.2). Mice are weighed then humanely euthanized. The animal's weight, sex, age, and identification number are recorded. The gross necropsy begins with an external examination of the external body surface, all orifices, cranial vault, external surface of the brain, the nasal cavity and sinuses, the thoracic, abdominal, and pelvic cavities and viscera. It is important to keep in mind that all tissues should be examined in situ before being dissected from the body. Samples for microbiological culture are taken before tissue samples are collected. The entire body is palpated for superficial swellings, enlarged organs, or masses. If an abnormality of the hair coat is noted, a hair sample is manually plucked. Forceps are not recommended, as this may damage the hair shaft. Hair samples are collected from the same area in every mouse in a study in order to keep collection techniques standard. The skin should be sampled from an area on the mouse that was not plucked to prevent artifactual changes in the hair follicles being studied.

Table 10.2 Tissues Collected from Genetically Engineered Mice for Potential Pathological Evaluation

Adrenals Aorta

Bone marrow

Eyes Femur Heart Ileum

Esophagus

Liver Lungs

Lymph nodes Mammary gland Muscle (thigh) Optic nerve *Gonads Pancreas

Pituitary Prostate Rectum Salivary gland Sciatic nerve Seminal vesicle Skin

Spinal cord

Spleen

Stomach

Thymus

Thyroid

Tongue

Trachea

Brain

Cecum

Colon

Jejunum Kidneys

Lacrimal glands

Duodenum Epididymis

Urinary bladder Uterus/vagina

The following organs will be weighed prior to partitioning and fixation: brain, heart, lungs, liver, kidneys, spleen, uterus, gonads, and adrenal gland. The pituitary will be weighed postfixation, and paired organs will be weighed separately. Organ to body weight percentages and organ to brain weight ratios will be calculated. The intestinal tract will be collected and the cecum separated from the colon. Intestinal specimens will be gently inflated with fixative by intraluminal injection and prepared as an intestinal roll (by placing it on an index card and rolling it in a flat spiral around a central toothpick). Care must be taken when collecting the pancreas, as it is firmly adhered to both the small and large intestine. The stomach is removed and also inflated with fixative. The liver should be the last abdominal organ removed. One should enter the thorax so as to use the diaphragm as a handle while the liver is removed. Upon removal of the liver, the salivary glands can be removed, and the heart, lungs, and trachea can be removed as one. The lungs should also be inflated with fixative to ensure proper fixation for histology. Lastly, the brain is collected as well as the spinal column if needed. It is important to carefully remove the meninges and then the brain from the skull. Tissues should be preserved in 4F-1G (modified Karnovskys formalin-gluteraldehyde fixative) for one week then transferred to 10% neutral buffered formalin for storage. Primary fixation in 4F-1G will permit ultrastructural analysis if indicated.

Proper trimming is important for proper orientation of tissue samples on the slides. This will ensure that the pathologist can identify normal tissue from any pathologic changes or lesions. Primary fixation in 4F-1G will permit ultrastructural analysis if indicated. Hematoxylin and Eosin (H&E) stain should be used on all histopathology samples. Special stains may be employed if needed to disclose certain pathological changes. Special stains include Congo red for amyloid, Trichrome for collagen, Von Kossa for calcium, and PAS for glycogen, especially in skeletal muscle and heart. Intracytochemistry for the different types of amyloid should be performed on the heart, if the data warrants it. Before trimming any bone samples, they must be decalcified in a hydrochloric acid decalcifying solution. After decalcifying, the samples must be rinsed with running water before being trimmed. The rinsing can take up to 3 to 4 h. Failure to properly rinse samples may result in inadequate staining.

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