Pathological examination method of mammalian laboratory animals

1. Basic requirements

(1) Background data records of laboratory animals

(1) Source, type, age, sex, original number, weight, clinical symptoms, etc. of laboratory animals.

(2) Time and place of necropsy, method of anesthesia, time, anesthetized person, method of sacrifice, dissected person, record person, temperature, humidity.

(3) Other indicators: the time of fasting (not water) before the animal is killed is 12h.

(2) Physical examination

Generally, experimental animals used for histological sampling should be isolated and quarantined for 7-10 days before being killed. Pathological autopsies for experimental and control animals depend on the requirements of different animal experiments. The physical examination items before the necropsy are as follows:

1. Developmental status

Whether the physical development is commensurate with the age and breed, and whether the development ratio of each part is normal, and there is no deformity.

2. Nutritional status

Fullness or weight loss, you can use your hands to touch the back and waist of the experimental animal during the test. When the nutrition is good, the back and waist are thick and the skin elasticity is good. When malnutrition occurs, the vertebrae of the back and waist are prominent, and the ribs are obvious.

3. Mental state

Experimental animals' autonomous activities, movements, responses to the outside world (slowness or hyperactivity), and gait.

4. Sensory organ

Whether the pupil of the eye is clear, etc., whether there is secretion, whether the eyelid is inflamed and red, the color of the bulbar conjunctiva changes, and whether there is flushing, paleness, yellow staining or cyanosis.

5. Respiratory system

Respiratory animals such as the number of breaths, rhythm, breathing difficulties; upper respiratory tract examination such as nasal secretions, sneezing and coughing; if necessary, the lungs can be checked by auscultation.

6. Digestive system

Observation of feeding and drinking, including loss of appetite, loss, hyperactivity and heterophilia, color and odor of oral mucosa. Whether there is vomiting, diarrhea, constipation, whether there is dirt around the anus, fecal quantity, hardness, color, smell, etc.

7. Coat and skin

Check skin color, temperature, elasticity, presence of trauma, abscesses, scabies, eczema, hair color, looseness, and loss of skin.

(3) Basic requirements for pathological materials

(1) Pathological examination should be carried out at different levels, with general appearance observation first, then necropsy observation, and then light microscope detailed examination.

(2) Normally choose the tissue at the border between the normal and the lesion, that is, include the lesion itself and the surrounding tissue.

(3) When the same organs of the animals in the control group are selected, the material selection parts should be as consistent as possible.

(4) When the obvious lesion is invisible to the naked eye, the specimen positions of each test group should be consistent.

(5) The selected tissue should include all hierarchical structures or important structures of the organs, such as the kidney should include cortex, medulla and renal pelvis.

(6) For large and lobed organs, multiple parts should be selected according to different tissues. Small organs can be obtained and fixed as a whole, such as lymph nodes, tonsils, and thyroid.

(7) The contents of gastrointestinal specimens should be washed away to prevent the contents from affecting tissue fixation and causing autolysis.

(8) The material taken should keep the integrity of the naked eye specimen as far as possible, and should not be too thick or too thin, generally about 3 ~ 5mm thick and 1.5 ~ 2cm2 in size.

(9) Do not squeeze when cutting tissue, use sharp knives, use less scissors, and do not use the parts that have been pressed by instrument clamps.

(10) The specimens should be familiar with the material and complete the whole process as quickly as possible, especially the self-lysable tissues such as intestine, brain, glands, etc.

(11) The necropsy record should be objective and detailed, and be described with images instead of the diagnosed disease name.

(12) Animals in the control group and the experimental group in the same experiment should be cross-necked, strictly conform to various conditions and operations, and try to avoid various possible interference factors.

Second, the basic operation of pathological necropsy

(1) Commonly used equipment and methods of use

1. scalpel

It is mainly used to cut and divide soft tissue. The blade should be clamped and installed with vascular forceps (needle-holding forceps) to avoid cutting fingers. There are four commonly used knife holding methods: bow holding type, gripping type, pen holding type, and reverse picking type.

2. Surgical scissors

For separating and cutting

3. Vascular forceps

Used to separate and clamp tissue, clamp suture needles and cloth towels.

4. Surgical forceps

Used to clamp tissue to facilitate dissection and suture.

5. Pull hook

For traction and exposure

(2) Basic operation

1. Cut

When it is on the same plane, first tighten the tissue, and the blade is perpendicular to the plane, with proper force, cut vertically once, the incision is neat and not skewed, oblique cutting and sawing are prohibited to reduce damage. When cutting multiple layers of tissue, it should generally be cut layer by layer to avoid damage to deep tissues and organs.

2. Tissue separation

Can use sharp separation method, using knife, scissors and other sharp instruments to cut directly, such as the separation of skin, mucous membrane, fine structure and tightly connected tissue; also can use blunt separation method, using split handle, hemostatic forceps, peeler, fingers, etc. Separation, such as separation of muscle, loose connective tissue, etc.

3. ligation

Tie tissues and blood vessels with silk knots and participate in suturing organs and skin.

4. Hemostasis

Massive hemorrhage leads to hemorrhage in the chest and abdomen, which obscures the visual field of the necropsy, which may affect and interfere with the identification and sampling of the lesion. Therefore, the anatomy of the animal sometimes requires hemostasis.

(1) Gauze block to stop bleeding. During the necropsy, in order to observe the nature and location of the lesion, distinguish tissues, nerves, and vascular access, and ooze out to a wider range of capillaries, gauze can be used for temporary hemostasis, but gauze should not be used to wipe the blood back and forth to avoid damage organization.

(2) Hemostatic method with forceps. First, hemostasis with a gauze block, see the bleeding point or blood vessel, then use the tip of the hemostatic forceps to vertically align the bleeding point, quickly and accurately clamp and twist, make the blood vessel occlude and stop bleeding.

(3) Ligation and hemostasis. Used for arterial bleeding or large blood vessel bleeding.

3. Procedures for autopsy

(1) Fixation of experimental animal carcasses

Usually fixed in the supine position.

(B) The sequence of necropsy

Mostly after the abdominal cavity, then the chest cavity, then the brain, spinal cord, bone marrow, skin muscles and so on. The material order is basically the same.

(3) Check content

1. position

Whether there is shift, ectopic

2. size

Volume increases, decreases, or enlarges

3. Color

The increase or decrease of the overall or partial color.

4. addenda

Check for bleeding, fluid accumulation, and adhesions.

5. texture

Hard, tough, soft, etc.

6. section

Juicy, foamy, bloody, pus, dry, etc.

7. Mucosal surface of hollow organ

Are there any bleeding, ulcers, thickening, bumps, etc.

(4) Examination and material extraction

1. Abdominal cavity and abdominal organs

Cut the xiphoid to the anterior abdominal wall between the anus along the midline of the abdomen, and then cut the lateral abdominal wall to the left and right sides along the lowest ribs to the sides of the spine, completely exposing the abdominal organs. Observe for effusion, blood, and inflammatory exudates. If any, use a pipette to measure the volume and check by centrifugal sediment smear. If necessary, culture the bacteria. Check whether the mucosa is smooth, whether there is congestion, blood stasis, bleeding, rupture, abscess, adhesion, tumor, parasite, etc., the tension of the diaphragm and whether there is rupture.

(1) The spleen. Check the size, thickness, hardness, traits, color, thickness, cracks, etc. Then cut the spleen in half along the long axis, the cut surface should be smooth, check the color of the splenic trabecula, red pulp, follicles, and the amount of bleeding on the cut surface.

(2) Pancreas. Check the color and hardness of the pancreas, and check the section for bleeding.

(3) Gastrointestinal. Check the size of the stomach and the color of the serosal surface of the gastrointestinal tract for adhesions, tumors, and parasitic nodules. Then cut the stomach, duodenum, jejunum, ileum, cecum, colon, and rectum along the great curvature of the stomach and the mesentery attachment. Observe the stomach for foreign bodies, the smell and traits of the contents, remove the contents, and check the color of the mucosa for congestion, bleeding, and suppuration. Inspect the intestinal canal by cutting and observing, observe the number and traits of intestinal contents, whether there is gas, blood, foreign bodies, parasites, whether the intestinal mucosa wall is thickened, edema, hyperemia, mucus volume, ulcer, necrosis Lymphatic tissue traits and inflammation.

(4) Kidney. First check whether the kidney size, hardness, coating is easy to peel off, the color and smoothness of the kidney surface, whether there are scars, bleeding changes. Then check the color of the cortex and medulla of the cut surface for stasis, bleeding, purulence, and infarction. Pay attention to observe whether the cut surface at the junction of cortex and medulla is bulge, as well as the characteristics of renal pelvis, ureter and renal lymph nodes, whether there are tumors and parasites.

(5) The liver. First check the size of the liver, the properties of the capsule, the thickness of the edges, the substantial hardness and color, and the properties of the liver lymph nodes, blood vessels, liver ducts, and so on. Then make a cut surface, check the bleeding volume, color, liver lobular traits, abscess, liver necrosis and other changes.

(6) Adrenal glands. Observe the shape, size, color and hardness, make longitudinal and transverse cuts, and check the color and cortex of the cortex and medulla for bleeding.

2. Pelvic organs

(1) The bladder. Check the size, urine volume and color of the bladder, mucosa for bleeding, inflammation and stones.

(2) Male reproductive organs. Check the testicles, epididymis, coagulation glands, prostate for adhesions, bleeding, edema, fluid accumulation, etc.

(3) Female reproductive organs. Cut the uterine horn along the dorsal side of the uterus, check the color, endometriosis, bleeding, inflammation, etc. of the endometrium, and observe the ovaries and fallopian tubes for adhesions, bleeding, edema, and fluid accumulation.

3. Thoracic cavity, oral cavity and its organs

Hold the xiphoid process of the sternum with tweezers, cut the connection between the diaphragm and the sternum, lift the sternum, cut the ribs of the left and right chest walls on both sides of the thoracic spine, remove the entire chest wall, open the chest cavity, and then take out the thymus and heart. Cut off the muscle connecting the inside of the two mandibular branches of the mandible with the tongue, separate the pharynx, larynx, trachea, and esophagus from the surrounding tissues, lift the trachea with tweezers, lift up, cut the connecting ligaments of the lung and pleura, and then The trachea, esophagus, and the entire lung are removed. If there are effusions, the quantity and traits should be observed, as much as possible, aspiration, volume measurement and smear, check pleural color, bleeding, congestion or adhesions.

(1) The heart. Cut the pericardium to expose the heart, pay attention to the gloss of the pericardium and the condition of the fluid in the pericardium, the size, shape, and epicardium of the heart. A straight line was cut from the entrance of the inferior vena cava to the right atrium, and the midpoint of this line was cut along the right edge of the heart to the apex of the heart, and parallel to the pulmonary artery at a distance of 1 cm from the apex to the right side of the ventricle; , Whether the tricuspid valve, pulmonary valve, and chordae are diseased. Cut the left atrium straight from the entrance of the left and right veins, cut along the left edge of the heart to the apex, and then cut the anterior wall of the left ventricle and the aorta parallel to the left side of the ventricle 1 cm away from the apex and check the mitral valve , Whether the aortic valve and tendon cord are diseased, and whether there is bleeding or infection in the left atrium and left ventricular wall. Cut the anterior descending branch and the circumflex branch from the mouth of the coronary artery. Find the right coronary artery trunk on the right side of the aortic root in the epicardium of the right ventricle. Like sclerosis and thrombosis.

(2) Mouth. Check the changes of the teeth, the color of the oral mucosa, the presence of trauma, ulcers and leukoplakia, and the tongue mucosa for bleeding, trauma and tongue coating.

(3) Throat. Observe the color of the larynx, epiglottis cartilage mucosa, the characteristics of the lymph nodes, and whether there is empyema in the larynx.

(4) Nasal cavity. Check the color of the nasal cavity and septal mucosa, for bleeding, inflammatory edema, nodules, erosion, ulcer perforation, scars, etc.

(5) Mandibular and cervical lymph nodes. Check the size, hardness, bleeding, and suppuration of lymph nodes in the jaw and neck.

(6) Trachea. Check the trachea for bleeding, mucus volume, etc.

(7) Lungs. Check the color of the lungs, for bleeding, inflammation, emphysema, lung atrophy, tumors, etc.

(8) Other organs. Thymus, thyroid, tonsils, etc., color, adhesion, bleeding, edema, etc.

4. Cranial cavity and brain

Take the monkey as an example, peel off the soft tissue at the cranial crest, follow the line from the arch of the eyebrow to the occipital protuberance at 0.5 cm, use an arch saw to cut the outer plate of the skull and the plate barrier around the wire, and then use the T-shaped chisel to gently open the inner Open the cranial cap, the dura mater covering the surface of the brain can be seen at this time, cut the dura mater to expose the brain tissue, and cut the dura mater from the front to the back at 0.5 cm above the cut end of the cranium The dura should be 1.5 cm long to prevent the occipital bone from damaging the occipital lobe brain tissue when the brain tissue is pushed backwards during brain extraction. Gently lift the dura mater and brain sickle to the rear, expose the brain tissue, use your fingers to reach the anterior cranial fossa from the front of the frontal bone, gently push the frontal lobe of the brain until you see the olfactory bulb on the sieve plate, cut the olfactory filament Contact with the olfactory bulb, pull the olfactory bulb and the brain together, and stop immediately when seeing the optic nerve and the optic chiasm, cut off the internal carotid artery and optic nerve in order near the base of the brain, and then pull the brain back, you can see the pituitary and funnel, continue to The brain is pulled back to cut off the nerves connected to the brain. From the ventral side of the brainstem, extend the scalpel into the occipital foramen, cut off the spinal cord, and then remove the brain. Rinse with running water and set aside. Fresh brain is very soft, easily deformed and contused. It must be supported by hand during the operation. After removing the brain, it should be wrapped with gauze immediately and stored in a fixing solution to avoid deformation.

Check the filling of the pia mater and dura mater, the amount and color of the fluid in the sulcus between the gyrus, and whether the convex or concave part of the brain surface is obvious. You can use the hand to touch the membrane to determine its hardness. Use a knife to make a horizontal section of the brain, retain the corpus callosum, expose the lateral ventricle, and pay attention to check the caudate nucleus for bleeding and softening foci. The number and nature of the contents of the lateral ventricle, then cut the brain in multiple places to check for changes.

4. Organ measurement and weighing

(1) After dissection, the organs should be weighed quickly to avoid differences in water evaporation, especially the small organs such as the adrenal glands.

(2) Before weighing the organs, try to remove the surrounding fat tissue and connective tissue, and use the filter paper to absorb the blood and body fluids on the surface of the organs, especially the smaller organs such as the adrenal gland, thyroid gland, and prostate. The organs are dry and lose water and lose weight.

(3) Before weighing the cavity organs, the fluid in the cavity should be removed, such as blood clots in the heart.