55 page Veterinary RODENT RAT NECROPSY Presentation CD

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RODENT NECROPSY TECHNIQUES, 2003, 55 pages

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RODENT NECROPSY TECHNIQUES Kenneth O. Jacobsen, DVM, MPH, DACVPM MAJ, VC, USA U.S. Army Medical Research Institute of Infectious Diseases Veterinary Medicine Division Fort Detrick, Maryland 21702-5011 RODENT NECROPSY TECHNIQUES Introduction Euthanasia Materials and instruments Topographical Anatomy and Necropsy Terminology Artifacts and Postmortem Changes Fixatives and Fixation Techniques Necropsy of the Rat Necropsy of the Mouse Necropsy of the Guinea Pig Necropsy of the Hamster RODENT NECROPSY TECHNIQUES Introduction Variety of approaches Experimental protocol Desire of pathologist Consistent technique Careful dissection RODENT NECROPSY TECHNIQUES Euthanasia Humane manner: refer to AVMA Panel on Euthanasia Not traumatic or cause tissue artifacts Method recorded on necropsy form RODENT NECROPSY TECHNIQUES Necropsy form RODENT NECROPSY TECHNIQUES Necropsy form RODENT NECROPSY TECHNIQUES Materials and Instruments Proper Personal Protective Equipment Performed in a vented enclosure Adequate lighting Basic items RODENT NECROPSY TECHNIQUES Materials and Instruments The following is a list of basic items that comprise a necropsy laboratory: Specimen containers rodent size ball-tipped feeding needle 10% neutral buffered formalin toothed tissue forceps serrated tissue forceps embedding bags protective gloves small (iris) forceps paper towels scalpel blades and handles 250 or 500 ml beaker dissecting scissors- 6 to 7 “ squeeze bottle of physiological saline small operating scissors squeeze bottle of 70% alcohol bone cutting forceps gauze sponges nail cutting forceps plastic disposal bags calipers hypodermic needles 16 to 25 gauge bone ronguers 1, 5, 10, and 20 ml syringes small spatula suture material 3/0, 1/0 tissue cassettes RODENT NECROPSY TECHNIQUES Topographic Anatomy and Necropsy Terminology Topographic Anatomy: method of indicating in descriptive terms the relative position of various body parts. Dorsal Distal Ventral Superficial Anterior Deep Posterior Volar Medial Plantar Lateral Cervical Proximal Thoracic RODENT NECROPSY TECHNIQUES Topographic Anatomy and Necropsy Terminology Planes of Dissection: Median Sagittal Transverse Frontal Coronal RODENT NECROPSY TECHNIQUES Topographic Anatomy and Necropsy Terminology Necropsy Terminology: precise observation, deliberatively vague, to allow scientific diagnosis during microscopic evaluation. Lesions Size and Number Color Shape Texture (consistency) Severity Weight / Volume RODENT NECROPSY TECHNIQUES Necropsy Terminology Lesions Abcess Laceration Adhesion Mass Alopecia Nodule Calculi Obstruction Crust Parasite Cyst Perforation Deformity Pigment Dilatation Prolapse Fluid Focus Foreign Fracture Thick Small Rupture RODENT NECROPSY TECHNIQUES Necropsy Terminology Size and Number Lesions should be measured in centimeters and in three dimensions Spherical lesions are best measured in diameter Terms such as focal, multifocal, or diffuse can be used RODENT NECROPSY TECHNIQUES Necropsy Terminology Color RODENT NECROPSY TECHNIQUES Necropsy Terminology Shape: Symmetry is very important in the consideration of tissue alteration. Paired organs can be compared to one another for detection of subtle changes. Compressed Nodular Confluent Papillary Cystic Pitted Depressed Punctate Flat Raised Irregular Round Linear Smooth Lobulated Spherical Oval Tubular RODENT NECROPSY TECHNIQUES Necropsy Terminology Texture (Consistency): the texture or consistency of a lesion is most revealing when it is cut open. The opened surface is referred to as the “cut surface”. The terms homogenous or heterogeneous are often used in conjunction with the appearance of the cut surface. Brittle Gritty Caseous Hard Firm Rubbery Friable Soft Gelatinous Watery RODENT NECROPSY TECHNIQUES Necropsy Terminology Severity Minimal Mild Moderate Marked RODENT NECROPSY TECHNIQUES Necropsy Terminology Weight / Volume: If organ weights or fluid volumes are to be measured, weights should be recorded in grams and volumes in milliliters. Organs should be weighed fresh and in a consistent manner. RODENT NECROPSY TECHNIQUES Artifacts and Postmortem Changes Artifacts: alterations induced in tissues that represent artificial change - poor tissue handling, improper moistening fluids, and poor fixation. RODENT NECROPSY TECHNIQUES Artifacts and Postmortem Changes Postmortem Changes: Autolysis is the result of two basic processes. release of of intrinsic tissue enzymes fermentation from bacterial flora Prevention (inhibit): fixation and cooling RODENT NECROPSY TECHNIQUES Fixatives and Fixation Techniques Fixatives: adequate fixation is crucial Neutral Buffered Formalin (NBF)- most common 10-20 X the amount of tissue fixed tissue no thicker than 5 mm 10% solution by volume buffered to a neutral pH contains Formaldehyde – highly toxic RODENT NECROPSY TECHNIQUES Fixation Techniques Fixation Techniques Immersion fixation Perfusion through vascular system Individual organ perfusion techniques RODENT NECROPSY TECHNIQUES Necropsy of the Mouse RODENT NECROPSY TECHNIQUES Step 1. Check animal’s identification number. Step 2. Record animal’s weight Step 3. Verify animal’s sex according to characteristics below Male: Long distance between anus and genital opening Nipples inconspicuous Pronounced scrotum Female: Short distance between anus and genital opening Conspicuous nipples Step 4. External examination. a. Observe entire body surface and orifices for abnormalities. b. Feel (palpate) the entire body for superficial swellings beginning from the head and proceeding posteriorly to the neck, chest, abdomen, and appendages. Palpate for enlarged organs or masses within the abdomen RODENT NECROPSY TECHNIQUES Step 5. Collection of the eye, Harderian gland, intraorbital lacrimal gland, exorbital lacrimal gland, parotid salivary gland. Autolytic changes occur early in the eye, therefore it should be collected first, along with neighboring glands on the side of the head. a. Lay the mouse on its side and make and incision form above and just in front of the eye to the convergence of the upper and lower jaws just posterior to the base of the ear. Reflect the skin ventrally. b. Press a small curved forceps deeply into the orbit and elevate the globe. c. Cut the tough fibrous tissue beneath the forceps with small scissors and remove the eye, accompanied by the pale, yellow-colored Harderian gland. RODENT NECROPSY TECHNIQUES In female mice, the Harderian gland secretes porphyrin, a pigment which accounts for the red color of nasal and/or ocular discharges. Secretion of pigment is greatly reduced in the Harderian glands of male mice. The intraorbital lacrimal gland is difficult to see with the naked eye in the mouse. It lies superficial to the Harderian gland in the lateral corner of the orbit and may adhere to the Harderian gland when the eye is excised. d. Identify the flat, light tan-colored exorbital lacrimal gland which is located just posterior and ventral to the eye. e. Remove and fix the more diffuse, pink parotid salivary gland which is located ventral to and just behind the base of the ear. RODENT NECROPSY TECHNIQUES Step 6. Collection of superficial masses. a. Remove any abnormal enlargement by cutting completely around it, being careful to include overlying skin, if possible, and at least some normal tissue surrounding the mass. Scrape the hair away from the skin because hairs dull the microtome knife which is used to section tissues in the preparation of slides. RODENT NECROPSY TECHNIQUES Step 7. Collection of cervical lymph nodes, submaxillary and sublingual salivary glands, mammary glands, preputial (or clitoral) glands. a. Lay the mouse on its back on a dissecting board and pin down the legs. Wetting the surface of the body with alcohol helps prevent loose hair from falling into the incisions. b. Stretch the skin in opposite directions with the thumb and forefinger and make a midline skin incision extending from the lower jaw to the vulva, or scrotum, respectively. Stretching the skin helps to prevent accidentally cutting into deeper structures. c. While exerting gentle outward traction on the skin with forceps, carefully incise the loose subcutaneous tissue and retract the skin on either side of the incision. RODENT NECROPSY TECHNIQUES d. Grasp the loose connective tissue just posterior to one of the paired submaxillary glands and bluntly dissect out the above gland together with the sublingual salivary gland and cervical lymph nodes. e. In female animals locate a nipple on the reflected abdominal skin and identify the whitish pink mammary tissue beneath the skin. Scrape the hair away from the nipple and cut a dime sized circular area out of the skin including the nipple. The clitoral glands are small, paired, flat, yellow masses of tissue located just anterior to the vulva. Both gland can be collected together by grasping the surrounding tissue with fine scissors. Corresponding structures in the male mouse are the preputial glands. These relatively large, flat, pale yellow glands are located on either side of the preputial opening. RODENT NECROPSY TECHNIQUES Step 8. Collection of the male or female reproductive tracts and urinary bladder Male a. Grasp the most posterior part of the abdominal wall and cut into the abdominal cavity just above the base of the penis. b. Cut the body wall on both sides close to the reflected skin being careful not to cut into any organs. Extend the incisions anteriorly to the rib cage and then reflect the cut portion of the abdominal wall over the chest. RODENT NECROPSY TECHNIQUES c. Pull both testicles out of the scrotum and sever the fibrous connection (gubernaculums testis) between the tail of the epididymis and the scrotum. Sever the vas deferens. Place the testicle with attached epididymis into fixative. d. Repeat the above procedure to collect the second testicle. e. Reflect the intestines anteriorly to reveal the urinary bladder, prostate gland, and seminal vesicles and coagulating glands. f. Insert one blade of a small scissors under the center of the pubis just above the base of the penis and cut through the cartilaginous connection (pubis symphysis). Separating the bones will reveal the underlying urethra and rectum. RODENT NECROPSY TECHNIQUES g. Dissect the skin away from the penis. Grasp the end of the penis with forceps and while pulling gently upward carefully dissect the connective tissue around the urethra and beneath the prostate gland. Place the excised male reproductive tract with attached urinary bladder onto a moistened paper towel. h. Place a loose ligature around the neck of the urinary bladder. Express most of the urine out of the bladder if it is distended and then tighten the ligature. Replace about half the volume by injecting formalin into the lumen using a small gauge needle. Fix the reproductive organs and urinary bladder. RODENT NECROPSY TECHNIQUES Female a. Grasp the most posterior part of the abdominal wall and cut into the abdominal cavity just above the vulva. b. Cut the body wall on both sides close to the reflected skin being careful not to cut into any organs. Extend the incision anteriorly to the rib cage and then reflect the cut portion of the abdominal wall over the chest. c. Insert one blade of a dissecting scissors under the center of the pubis and cut through the cartilaginous connection (pubic symphysis). Separating the bones will reveal the underlying vagina and rectum. RODENT NECROPSY TECHNIQUES d. Cut the skin between the vulva and anus and dissect free the posterior end of the vagina being careful not to cut into the rectum. Grasp the end of the vagina and while gently pulling upward sever the film-like connective tissue between the vagina and the rectum. Do not cut away the urinary bladder. Continue dissecting anteriorly to the area of the cervix and then cut the mesentery supporting the uterine horns up to the ovaries. Cut the supporting tissue between the ovaries and kidneys and transfer the entire reproductive tract onto a paper towel moistened with saline. RODENT NECROPSY TECHNIQUES Step 9. Collection of the spleen, pancreas, stomach, duodenum, jejunum, ileum, cecum, colon, rectum and mesenteric lymph nodes. a. Gently grasp the greater curvature of the stomach with serrated forceps, pull down, and carefully sever the filmy Membrane between the stomach and the caudate lobe of the liver. b. Sever the esophagus approximately 0.5 cm above the stomach. c. Grasp the esophagus with fine forceps close to the stomach and while maintaining tension, sever the bile duct and remaining connective tissue between the liver and the stomach. RODENT NECROPSY TECHNIQUES d. Maintain tension and sever the mesentery between the intestines and the dorsal body wall but do not cut the large blood vessels in this area. This should free the spleen and the entire gut except for its termination at the anus. Cut around the anus and lay the gut on a paper towel moistened with saline. e. The intestinal tract is most efficiently examined by stretching it to its full length. Sever the mesentery which binds the coils of the intestine. The mesenteric lymph nodes should remain imbedded in the mesentery close to the ileocecal junction. Once the mesenteric attachments are severed the entire gut may be laid out “serpentine-fashion”. RODENT NECROPSY TECHNIQUES Step 10. Collection of the liver, kidneys, and adrenal glands. a. The mouse liver can be excised intact by grasping the tough connective tissue under the median lobe with tissue forceps and while exerting tension posteriorly, sever the esophagus and blood vessels passing through the diaphragm. Cut any remaining connections to free the liver. Fix the liver intact or sectioned as specified. RODENT NECROPSY TECHNIQUES b. Once the liver is removed, the kidneys and adrenal glands are easily examined. The ureters may be difficult to identify unless they are distended. The adrenal glands lie close to the kidneys in mice and are larger in the female. If the adrenals are to be collected and/or weighed apart from the kidneys, carefully undercut each gland with a sharp scissors, lift out the gland, and trim the fat away from each gland on a moistened paper towel. RODENT NECROPSY TECHNIQUES c. If the adrenals are to remain with the kidneys, grasp the left renal artery and vein with the forceps close to the kidney and sever both vessels with a fine scissors medial to the forceps. Exert upward tension on the kidney with forceps and bluntly dissect the kidney away from its bed of perirenal fat. Continue dissecting anteriorly to include the adrenal gland with its surrounding fat. Grasp the kidney with a moistened gauze sponge and bisect it longitudinally with a sharp scalpel. The longitudinal cut distinguishes the left kidney from the right kidney. d. Collect the right adrenal gland and right kidney as above. Bisect the right kidney transversely to distinguish it from the left kidney in slide sections. RODENT NECROPSY TECHNIQUES Step 11. Collection of the sternum and rib (costochondral junction) a. Cut the reflected flap of abdominal wall away from the last ribs and xiphoid process of the sternum. b. Grasp the xiphoid process, slit the diaphragm under the sternum, and cut through the cartilaginous portion of the ribs on both sides close to the sternum. Extend each incision anteriorly through the first rib being careful to cut medial to the prominent jugular veins. Maintain upward tension and continue cutting the muscles on either side of the trachea up to the lower jaw. Sever the muscles attached to the lower jaw and remove the sternum with attached neck muscles. The full length of the trachea and opened thoracic cavity should be revealed at this point. RODENT NECROPSY TECHNIQUES Step 12. Examination of the oral cavity Insert a small scissors into the corner of the mouth and cut through the skin, masseter muscle, and lower jaw (mandible). Repeat on the opposite. Pull the jaw downward and examine the surface of the tongue, the teeth and the rest of the oral cavity. RODENT NECROPSY TECHNIQUES Step 13. Collection of the tongue, thyroid glands, parathyroid glands, larynx, esophagus, trachea, thymus, heart, lungs, and mediastinal and bronchial lymph nodes. a. Free all attachments of the mandible to the body but do not cut away the tongue at this point. Grasp the front of the mandible and pull downward while severing the soft palate and any remaining pharyngeal attachments. Continue dissecting the trachea and esophagus together away from the body and at the level of the first ribs carefully cut the attachments to the dorsal chest wall. Continue cutting all remaining attachments of the lungs down to the diaphragm until the trachea, heart, lungs, and accompanying structures can be freed. RODENT NECROPSY TECHNIQUES b. Just below the larynx, identify the reddish-brown thyroid glands. The isthmus of the thyroid is difficult to visualize in the mouse. The parathyroid glands are small, spherical white masses imbedded in the anterolateral poles of thyroid gland. The parathyroid glands are very small in the mouse and are better visualized with a magnifying lens or a dissecting microscope. c. Attempt to identify the mediastinal and bronchial lymph nodes. These nodes are particularly small in the mouse and difficult to locate unless they are enlarged. RODENT NECROPSY TECHNIQUES d. Place a loose ligature around the trachea 1 to 2 mm below the thyroid glands. Fill a 1.0cc syringe with fixative and attach a 25-gauge needle. Insert the needle into the opening of the trachea (glottis) and snug the ligature around the needle. Slowly inject sufficient fixative to enlarge all lobes of the lungs to a point corresponding to full inflation. (The margins of the lobes should remain sharp.) Do not overdistend the lungs. Note that the left lung consists of a single lobe as in the rat and hamster. Withdraw the needle and tighten the ligature. RODENT NECROPSY TECHNIQUES Step 14. Collection of the brain, pituitary gland, and turbinate bones. a. Turn the mouse over onto its ventral side and saturate the fur on the head with alcohol. Make an incision from between the eyes to behind the head and reflect the skin away from the cranium. b. Bend the mouse’s head down and cut through the dorsal muscles of the neck until the spinal cord can be seen entering the skull. c. Insert one blade of a small pointed scissors into the opening of the skull (foramen magnum) and cut the bone just above the opening of the ear canal being careful not to penetrate the brain. Repeat on the other side of the skull. RODENT NECROPSY TECHNIQUES d. Extend the bone incisions anteriorly to the level of the nose. Cut across the frontal bones covering the olfactory lobes. Lift the posterior cranium upward away from the head exposing the cerebellum and the cerebral hemispheres. If present, remove the tough membrane (dura mater) that surrounds the brain. Remnants of the dura usually need to be removed from the junction of the cerebral hemispheres and cerebellum. RODENT NECROPSY TECHNIQUES e. Sever the olfactory lobes with a fine scissors and then tilt the head upward and backward. Sever the optic nerves. At this point the brain should begin to fall backward exposing the remaining cranial nerve attachments. Carefully cut any remaining attachments and sever the brain stem (medulla oblongata) posterior to the cerebellum, freeing the brain. Handle the brain carefully and place it into fixative. f. Allow the pituitary gland to be fixed with the base of the skull so that the gland is not lost. Rodent Necropsy Techniques Step 15. Collection of the sciatic nerve and adjacent muscle. The sciatic nerve is located somewhat parallel and posterior to the femur in the thigh. Careful deep dissection will expose the white fibers of the nerve running down the leg. Cut out approximately 1 cm of nerve and include adjacent muscle. Step 16. Collection of the proximal femur with bone marrow. Bone marrow may be collected from the femur as well as from the sternum. Step 17. Collection of the vertebral column and spinal cord. It is tedious and too time consuming to dissect out the entire spinal cord from the bony vertebral column for routine necropsies. The simplest and most acceptable method of collection is to cut away as much muscle as possible from around the vertebral column and then to cut out segments of the spinal cord (cervical, thoracic, lumbar) by severing the vertebral column at intervals using bone forceps. Rodent Necropsy Techniques References 1. Necropsy Guide: Rodents and the Rabbit, Donald B. Feldman, DVM and John C. Seely, DVM, CRC Press, Boca Raton , Florida, 1988. 2. Guide to the Necropsy of the Mouse, Vincenzo Covelli, Division of Protection of Man and Ecosystems, ENEA, CR-Casaccia, via Anguillarese 301, 00060, Rome, Italy Rodent Necropsy Techniques References Web-based Resources for Rodent Necropsy Training Virtual Mouse Necropsy: http://www.geocities.com/virtualbiology/sq.html The Visible Mouse: http://ccm.ucdavis.edu/tvmouse/ Vincenzo Covelli - Guide to the Necropsy of the Mouse: http://www.eulep.org/Necropsy_of_the_Mouse/ University of Missouri: http://www.radil.missouri.edu/ Guides for organ sampling and trimming procedures in rats: http://www.item.fraunhofer.de/reni/trimming/TR_F.htm Questions Rodent Necropsy Techniques Quiz 1. What is the whitish-yellow glandular (auditory sebaceous glands) tissue at the entrance to the bony ear canal found in the Rat (Rattus norvegicus)? Ans. Zymbal gland Rodent Necropsy Techniques Quiz 1. How is the anatomic location of the thymus in the Guinea pig (Cavia porcellus) different from the mouse, rat, hamster and rabbit? Ans. The thymus is extrathoracic in the Guinea Pig Rodent Necropsy Techniques Quiz 1. Name the organ depicted in this picture of a mouse. Ans. Preputial gland Rodent Necropsy Techniques Quiz 1. Name the organ depicted in this picture of a rat. Ans. Extraorbital lacrimal gland