Instructions

 

This application will guide you through the anatomic dissection of the mouse and show a systematic approach to properly examine and collect tissues for histologic examination. The goal is to provide you with a better understanding of the mouse necropsy than could be obtained by images, video, diagrams etc.  The only thing more realistic is actually doing a necropsy. Note that this is a general approach and depending on the reason for performing the necropsy, other approaches or modifications may be necessary.

 

The virtual necropsy is organized into steps. Follow the directions below  (in blue text) to proceed through the dissection.

 

Check the mouse's ID, strain, color, age and sex information to make sure that they match the mouse being necropsied.

 

Record the mouse's weight.

 

Before dissection, saturate the hair with alcohol. Note: Be careful not to wet any areas that may need to be cultured later, such as around the mouth or nose. Also, all tissues and organs should be examined in situ before being dissected from the body.

 

Examine and palpate the entire body for superficial swellings or abnormalities. If found collect representative samples including normal tissue margins.

Click on the Gold "Enter" button to begin.

 

The necropsy begins with the  mouse in lateral recumbency. Click on the "1" button.

 

A skin incision is made as outlined in blue from above the medial canthus of the eye to the ramus of the mandible. The skin is reflected ventrally in the direction of the red arrow. Hold down the mouse button and drag the cursor along the arrow to reflect the skin. This is completed when the "2" button appears.

 

The globe is elevated with forceps by grabbing the conjunctival tissue around the eye. To elevate the eye, click on the "2" button and the underlying Harderian gland can be seen.

 

Trim the tissue beneath the forceps and around the eye with scissors to remove the Harderian gland attached to the eye. The intraorbital lacrimal gland lies superficial to the Harderian gland in the lateral corner of the orbit and may also be removed. Hold down the mouse button and drag the cursor from the eye to the blue circle to remove. This is completed when the "3" button appears.

 

Click on the "3" button. The flat, light tan exorbital lacrimal gland is located caudal and ventral to the eye. To remove, hold down the mouse button and drag the cursor from the outlined gland in the direction of the red arrow. This is completed when the "4" button appears.

 

Click on the "4' button. The diffuse, pink parotid salivary gland is located ventral to the base of the ear.  To remove the parotid gland, follow directions as shown until the "5" button appears. This completes the lateral recumbency dissection.

 

Click on the "5" button to place the mouse in dorsal recumbency. Wet the ventral surface of the body with alcohol.

 

A midline skin incision extending from the caudal abdomen to the lower jaw is made. Be careful not to cut the superficial clitoral glands (female) or the preputial glands (male) located cranial to the external genitalia. To make the incision, hold down the mouse button and drag the cursor along the red arrow. This is completed when the "6" button appears.

 

Click on the "6" button. The skin is reflected by applying lateral traction and then bluntly dissecting away the subcutaneous tissue along with the skin from the underlying musculature. To do this, hold down the mouse button and drag along the red arrow. This is complete when the "7" button appears.

 

Click on the "7" button. The paired submaxillary salivary glands, sublingual salivary glands and the cervical lymph nodes are removed from the ventral cervical region.  The submaxillary glands are the largest of these tissues and lies caudal to the sublingual glands. The lymph nodes are cranial to the sublingual glands. To remove these tissues, follow directions as shown until the "8" button appears.

 

In female animals locate a nipple on the reflected abdominal skin. Cut a 1 cm diameter area out of the skin including the nipple and underlying mammary tissue.

 

Click on the "8" button. Make an incision into the abdominal cavity just cranial to the external genitalia. Extend the incision to the rib cage by cutting abdominal musculature on both sides close to reflected skin (blue lines). The abdominal musculature is reflected over the thorax to expose the abdominal contents. To do this, hold down the mouse button and drag along the red arrow. This is complete when the "9'" button appears.

 

Click on the "9" button. To further examine the abdominal viscera, reflect the fat overlying the reproductive system (uterus). To reflect the fat, follow directions as shown until the "10" button appears.

 

Click on the "10" button. Cut away the reflected flap of abdominal wall away from the caudal margins of the thoracic cavity. Grasp the xiphoid process at the caudal end of the sternum and separate the diaphragm from the overlying ribs. Cut along both sides of the rib cage close to the reflected skin (blue lines). Extend each incision cranially through the first rib. Maintain upward tension and cut the muscles on each side of the trachea.  The sternum should be saved along with ribs to the costochondral junction. If warranted, following decalcification, the bone marrow can be examined with the sternum. To remove the sternum and ribcage, hold down the mouse button and drag along the red arrow. This is complete when the "11" button appears.  Note the trachea and thoracic viscera are now exposed for examination.

 

Click on the "11" button. A blunt needle attached to a 3 cc syringe containing fixative is inserted into the trachea through a small incision. Be careful not to completely transect the trachea. Clamp forceps around the needle and inject enough fixative to fill all lung lobes to full inflation. Do not over distend the lungs. To perfuse the lungs click on the needle.

 

A ligature is placed around the trachea distal to the needle. To remove the needle click on it again.

 

Click on the "12" button. Insert scissors into the corner of the mouth and cut through skin, masseter muscle, and mandible (blue lines). Pull the jaw downward and examine the tongue, teeth, and rest of oral cavity. To do this,  hold down on the mouse button and drag along the red arrow. This is complete when the "13" button appears.

 

Click on the "13" button. Continue dissecting  the trachea and esophagus from the cervical region and from the attachments to the dorsal chest wall. Cut the remaining attachments as far as the diaphragm until the trachea, esophagus, heart, lungs and accompanying structures are freed. To remove the mandible and the thoracic viscera, hold down on the mouse button and drag along the red arrow. This is complete when the "14" button appears.

The reddish-brown thyroid glands are located just below the larynx adjacent to the trachea. The parathyroid glands are white and embedded in the anterolateral poles of each thyroid. They are small in the mouse and may not be readily apparent grossly. The mediastinal lymph nodes are located ventral to the trachea at the level of the thymus. The bronchial lymph nodes are situated at the bifurcation of the trachea. Further dissection may be necessary. However, for routine processing the mandible and thoracic viscera can be placed in fixative together.

 

Click on the "14" button. The liver is reflected cranially to expose the stomach. The spleen lies adjacent to the greater curvature of the stomach (Red arrow). Grasp the greater curvature of the stomach and apply traction caudally. Sever the membrane between the stomach and the caudate lobe of the liver. If the stomach has not already been freed from the distal esophagus, sever the attachment. Click on the "15" button to do this.

 

The pancreas is located at the hilus of the spleen and should be removed with it. To remove the spleen and the associated pancreas, hold down the mouse button and drag along the red arrow. This is complete when the "16" button appears.

 

Click on the "16" button. Maintain tension caudally and severe the mesentery attaching the intestines to the dorsal wall. To do this and reflect the GI tract caudally, hold down the mouse button and drag along the red arrow. This is complete when the "17" button appears. Note the kidneys and the adrenal glands at their cranial poles are now exposed.  The ovaries and distal uterine horns are also evident caudo-laterally to the kidneys.

 

Click on the "17" button. Cut any remaining vasculature or connective tissue attachments to the liver and remove it. Hold down the mouse button and drag along the red arrow. This is complete when the "18" button appears.

 

Click on the "18" button. The adrenal glands and kidneys can now be examined. Grasp the connective tissue holding the two kidneys together. Apply upward tension and gently separate the kidneys with attached adrenal glands from the dorsal body wall. To do this, hold down the mouse button and drag along the red arrow. This is complete when the "19" button appears. If necessary, to identify right from left kidney, section the right kidney transversely to the cranio-caudal axis and the left kidney parallel to the cranio-caudal axis.

At this point, you can click on the "Male" button to perform dissection of the male reproductive tract. You will then come back to the female mouse. Alternatively, click on the "19' button to skip the male dissection and continue with the female (See Below).

 

Male:

Click on the "Male" button.

 

Hold down the mouse button and drag along the red arrow to make the initial skin incision. This is complete when the "A" button appears.

 

Click on the "A" button. To reflect the skin and attached subcutaneous tissue laterally, hold down the mouse button and drag along the red arrow. This is complete when the "B" button appears.

 

Click on the "B" button. Hold down the mouse button and drag along the red arrow to reflect the incised abdominal wall (blue lines) over the thorax. This is complete when the "C" button appears.

 

Click on the "C" button. To visualize the male reproductive tract, reflect the intestines cranially. Hold down the mouse button and drag along the red arrow. This is complete when the "D" button appears.

 

Displace the testicles into the abdominal cavity and sever the connection between the tail of the epididymis and the scrotum. Examine the urinary bladder, prostate gland, seminal vesicles, coagulating glands, and testicles.

 

Insert one blade of the scissors into the pelvic canal just craniolateral to the penis and cut the pubis. Repeat on the other side.

 

Gently express any remaining urine from the bladder , then inject fixative into the lumen.

 

Make circumferential incision around the penis including the preputial glands. While applying traction dissect the connective tissue around the urethra and the prostate.

 

Click on the "D" button. Hold down the mouse button and drag along the red arrow to remove the intact male reproductive tract. This is complete when the "Female" and "20" button appear.

At this point, to go back to the female necropsy, you can click on the "Female" button. Alternatively to skip the dissection of the female reproductive tract click on the "20" button (See below).

 

Female:

Click on the "Female" button.

 

Insert one blade of  scissors into the pelvic canal just craniolateral to the vulva and cut the pubis. Repeat on the other side.

 

Gently express any remaining urine from the bladder , then inject fixative into the lumen.

 

Make a circumferential incision around the skin of the vulva (including the clitoral glands) separating the vulva from the anus. Grasp the end of the vagina and apply traction while dissecting the connective tissue supporting the uterine horns and ovaries. Cut the ligament attaching the ovaries to the kidneys.

 

To remove the excised female reproductive tract, hold down the mouse button and drag along the red arrows. This is complete when the “20”  button appears.

 

The GI tract is perfused by injecting fixative at multiple sites along the tract (sufficient to fix that region). (This can be done in situ or after the GI tract is removed.) To insert the needle, Click on the “20” button. To see the perfused GI tract, click on the needle.

 

To remove the needle, click on it again.

 

Cut any remaining attachments to the dorsal wall, and dissect around the anus to remove the entire GI tract. To do this, hold down the mouse button and drag along the red arrow.  This is complete when the “21” button appears. The GI tract is uncoiled by severing the mesentery. The mesenteric lymph nodes are embedded in the mesentery close to the ileo-cecal junction. The tract is  laid out “serpentine-fashion” with the stomach in the upper right corner (as shown).

 

To remove the head from the body, sever the joint capsule (between red arrows) to expose the atlanto-occipital joint at the base of the skull. To do this, click on the “21” button.

 

Continue dissection separating the atlas and skull and sever the spinal cord. To remove the head click on the “22” button.

 

Click on the “23” button. The head is shown with the skin reflected from lateral recumbency dissection. The skin and musculature is removed from the skull to the tip of the nose.  Insert one blade of  scissors into the foramen magnum (large opening at the base of the skull) and cut the lateral surface of the cranium above the opening of the ear canal.  Repeat on the other side. Extend these incisions in an anterior direction to the level of the nose. Reflect the cranium in an anterior direction cutting dura mater to release the bone.

To do this, hold down the mouse button and drag along the Red arrow. This is complete when the “24’” button appears.

The brain is now exposed and can be examined.

 

Click on the “24” button. To remove the brain point the nose downward. To separate the brain from the cranial cavity, cut the cranial nerves on the ventral aspect of the brain (between the brain and cranial cavity). To remove the brain, hold down the mouse button and drag along the red arrow. This is complete when the “25” button appears.

 

Click on the “25” button. The pituitary gland is located in the center of the base of the cranial cavity. It can be left in the skull for fixation or removed. To remove, hold down the mouse button and drag along the red arrow. This is complete when the “26” button appears.

 

The nasal passages and ear canals are normally sectioned after the  skull is fixed and decalcified.

 

Click on the “26” button.

 

The sciatic nerve is located parallel and caudal to the femur (red arrow). Bluntly dissect through the muscle caudal to the femur. To expose the sciatic nerve, click on the “27” button. 

 

Cut out approximately .5 cm of nerve and adjacent muscle. To do this, Hold down the mouse button and drag along the red arrow. This is complete when the “28” button appears.

 

In addition to the sternum, the proximal femur can also be collected for examination of bone marrow.

To place the body in ventral recumbency, click on the “28” button.

 

Hold down the mouse button and drag along the red arrow to reflect the skin and subcutaneous tissue from the spinal column. This is complete when the “29” button appears.

 

Click on the “29” button.

Remove the musculature surrounding the spine with any excess ribs. Cut parallel on each side, down the spinal column. To do this, hold down the mouse button and drag along the red arrows. To examine the spinal cord, the vertebral column is decalcified following fixation.

 

THE END

 

Acknowledgement: This application was prepared with the assistance of Dr. Fon Chang, University Of California, Davis.

 

Reference: Necropsy Guide: Rodents and Rabbits. D.B. Feldman and J.C. Seely, CRC Press, 1988.