Human cancer and response to therapy is better represented in orthotopic animal models. In this video, human ovarian tumor cells expressing luciferase are injected directly into the mouse bursa where each ovary is enclosed. Therapeutic drugs are then administered by oral gavage to deliver the drug directly to the stomach.Luciferian.
A substrate of firefly luciferase is injected causing the luciferase expressing cells to generate bioluminescence signals. The bioluminescent signals allow tumor growth, distribution, and regression to be tracked by in vivo imaging of individual animals. Imaging results indicate that tumor cells injected into the ovary, remained in the ovary of the left mouse and spread into the peritoneal cavity.
A major site of metastatic spread for human ovarian cancer in the right mouse. The main advantage of an orthotopic animal model over conventional xenograft models like subcutaneous or intraperitoneal injections of tumor cells is that it is better represented for human cancer and thereby provides a better understanding of human cancer in response to therapy. This procedure requires assistance from a second person.
All surgical procedures are conducted under aseptic conditions, including surgical attire and sterile surgical instruments. Syringe and needles confirm that the animal is under an acceptable plane of anesthesia by performing a toe pinch with forceps or fingers to the animal's hind paws. If there is pedal reflex, wait for a deeper plane of anesthesia until the animal is unresponsive to this procedure.
Position the animal dorsal side up on a sterile gauze pad with its head facing away. Locate the incision point to the left or right of the midline and above the ovaries. Then wet the area with 70%dec ethanol using forceps.
Lift the wetted skin and use scissors to make a small incision at the dorsal medial position directly above the ovarian fat pad. The ovarian fat pad should be visible beneath the surface of the peritoneal wall and is easily recognizable by its white color in contrast to the dark pink tissue surrounding it. Next, gently lift the peritoneal wall lining and make a small vertical incision.
As before. Place a sterile soaked saline gauze pad on the midline adjacent to the incision. Locate the ovarian fat pad.
Gently pull it out and rest it on the gauze. Stabilize the ovary by clamping the fat pad with a bulldog clip under a dissecting microscope, position the ovary to find the ov duct. Then insert the 30 gauge needle of a syringe loaded with five microliters of ocar.
Five human ovarian cancer cells expressing firefly luciferase into the oviduct bend leading to the bursa. When the needle is inserted into the proper position, it should be visible under the bursa with one person maintaining the position of the needle. A second person should gently push the plunger of the syringe to inject the cell suspension into the space between the bursa and the ovary.
Remove the needle quickly to seal the puncture site. Taking care not to tear the bursa and tubule. The bursa should appear slightly distended.
Release the fat pad from the bulldog clip and gently replace the reproductive tract and fat pad back into the peritoneal cavity. Pull the upper peritoneal lining over the lower lining to gently close the body wall. Close the skin with surgical staples.
Place the recovering animal back in its cage and provide a safe heat source to avoid hypothermia and speed up recovery. Monitor the breathing rate and ease the return of muscle tone and the ability to voluntarily move. Oral gavage is performed Using an 18 to 20 gauge gavage needle, the gavage needle should be no longer than the distance from the top of the animal's head to its last rib.
Begin this procedure by gently grasping the skin over the mouse's shoulders with thumb and fingers. The restraint should be just from enough for the forelimbs to extend to each side out of the way. The animal should not be able to grasp at the needle.
Gently pull back the animal's head as shown here, forming a straight line through the neck and esophagus. Support the animal's back with the inside of the thumb gently and without force. Insert the gavage needle at either side of the mouth and over the tongue.
In one smooth motion, the needle should pass against the roof of the mouth and down the esophagus. Gravity should guide the needle down and there should be no resistance. If there is any resistance, remove the needle and start again.
The animals gag and swallow reflex may be triggered during insertion. However, there should be no gasping from the animal. When the needle is in place, ensure that the mouse is breathing by checking the movement of the nostrils and chest.
Then slowly push the plunger of the syringe to dispense the dose volume. Gently remove the gavage needle at the same angle and pathway as it was inserted. Return the animal to its cage and monitor breathing and behavior for five to 10 minutes.
Here the IVUS Spectrum Imaging System is used to monitor the behavior of cells injected into the intra bursal cavity experiments. Using the system typically have a timeframe of four to 16 weeks from the time of tumor implantation. Load the LUCIFERIAN solution into a syringe with an attached 30 gauge needle.
Verify the depth of anesthesia of an isof fluorine, anesthetized mouse by toe pinch. Then deliver 200 microliters of prepared Luciferian solution via IP injection. Then place the Lucifer injected animal back into the induction chamber.
Record the time of injection in order to keep the time consistent between Lucifer injection and imaging performance. Throughout the study, place the anesthetized animal, dorsal or ventral side up on the imaging stage. Position the animal's nose and a nose cone to maintain proper anesthesia induction.
Make sure the animal is within the point of interest grid as indicated by green illumination. Set the imaging system to the appropriate settings. Be sure to initialize the system before acquiring the first animal image.
Close the door of the imaging chamber at the appropriate time. Acquire the image. Remove the animal from the imaging chamber and place it back into its cage.
Monitor the animal's recovery. As before O carv, five cells expressing luciferase were injected intra Sally into the right ovary of a mouse. And in vivo imaging was performed.
Images were taken 13, 17, and 22 days after the injection. Using the IVUS spectrum imaging system, the dorsal side is shown in the upper panel and the ventral side is in the lower panel. Note, the peritoneal spread of tumor cells.
22 days following injection injection of luciferase expressing cells. And in vivo imaging technology allow for a non-invasive means of tracking tumor growth and distribution in individual animals. Human cancer and response to therapy is better represented in an orthotopic animal model.
The techniques presented here are useful in understanding the development and spread of ovarian cancer and in assessing novel therapeutic regimens that may ultimately improve the outcome of patients with this deadly disease.
