An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery

This novel protocol describes surgical anesthesia in mice using propofol anesthesia. The method enables research into propofol anesthesia in mouse models of disease. The protocol allows the evaluation of total intravenous anesthesia and avoids the use of volatile anesthesia.

The protocol can be used to explore how propofol anesthesia impacts disease-related outcomes. For example, here we evaluate short and long-term cancer outcomes after surgery to remove the tumor. To begin, culture 66 CL4 Mirai mammary cancer cells that are stably transduced to express firefly luciferase in alpha MEM medium containing 10%FBS and 200 millimolar glutamine.

Incubate the cells at 37 degrees Celsius but 5%carbon dioxide. Subculture the cells when the cells reach approximately 80%confluency. When the cells are in a logarithmic growth phase with two milliliters of drips in EDTA solution, lift the adherent cells and count the cells using a hemocytometer.

Then dilute the cells in PBS and keep the diluted cell suspension on ice until the mouse is ready for injection. Once the mouse is completely anesthetized, wipe the fourth left mammary fat pad area with a single-use alcohol swab to prepare the injection site. Fill a 25 microliter Hamilton syringe attached to a sterile 27 gauge hypodermic needle with cancer cell suspension.

Use forceps to lift and secure the skin. Inject one times 10 to the fifth cells and 20 microliters of PBS into the fourth left mammary fat pad at approximately one millimeter from the nipple. If the cells are tagged with luciferase, inject 100 microliters of D-luciferin in the lateral tail vein of the anesthetized mouse using a 0.5 milliliter insulin syringe with a 30 gauge hypodermic needle.

Two minutes after the injection place the mouse in a bioluminescence imaging system with the mammary fat pad facing up and capture the image for 10 seconds. After imaging, place the mouse in the clean cage and allow it to recover from anesthesia. Monitor the growth of the primary tumor growth using a caliper.

Calculate the tumor volume and resect the tumor when the primary tumor reaches 80 to 90 cubic millimeters volume. Set up the automated syringe pump with a 30 gauge one milliliter insulin syringe containing two percent propofol formulation. Induce anesthesia in an induction chamber with three percent sevoflurane or isoflurane.

Then transfer the anesthetized mouse to a heating pad at 37 degrees Celsius and maintain the anesthesia with two to three percent sevoflurane or isoflurane using a nose cone. Adjust the delivery of sevoflurane as required during intravenous cannulation to maintain a stable depth of anesthesia demonstrated by loss of pedal reflex and respiratory rate less than 100 breaths per minute. Apply aqueous lubricant to the eyes to prevent drying of the cornea, and then inject 0.05 milligram per kilogram of buprenorphine subcutaneously.

To deliver the propofol based total intravenous anesthesia, cannulate the lateral tail vein using a sterile 30 gauge hypodermic needle attached to a sterile polyurethane catheter. Confirm correct placement by blood flashback into the catheter. Administer two percent propofol as an initial bolus of 27 milligrams per kilogram over one minute to begin total intravenous anesthesia.

Cease the sevoflurane administration. Maintain propofol infusion at a rate of 2.2 to 4.0 milligram per kilogram per minute for retaining stable anesthesia depth during the surgery. Shave the abdomen region and disinfect the surgical area with povidone-iodine solution.

Make a one centimeter incision inferior to the tumor in the left fourth mammary fat pad. Use forceps and scissors to dissect the tumor and the left inguinal lymph node. If luciferase-tagged tumor cells are used inject d-luciferin in the lateral tail vein as demonstrated and capture the images for 60 seconds to obtain clear margins.

If a residual tumor is identified, resect additional tissue from the mammary fat pad. Once hemostasis is achieved at the surgical site, close the skin using 5-0 nylon sutures. Wipe the wound with povidone-iodine solution.

Upon completion of the surgery, stop the anesthesia and place the mouse in a clean cage on a heating pad to allow it to recover from anesthesia. Monitor the mouse every 15 minutes until the mouse returns to normal alertness. Then monitor and provide analgesia every 12 hours for 48 hours after the surgery.

For primary tumor recurrence or distant recurrence assessment, monitor the mice once per week, commencing the week following surgery using a bioluminescence imaging system. Bioluminescence imaging was used to identify the distant tumor recurrence in the lungs after resection of the primary tumor. This model can be used to assess events that occurred during the perioperative period.

24 hours after cancer surgery under propofol, circulating plasma cytokines were evaluated. Practices required to rapidly cannulate the lateral tail vein and minimize sevoflurane exposure duration. The sevoflurane should gradually be down-titrated as the propofol bolus is administered to prevent over-sedation.

This method may be used to investigate the impact of propofol anesthesia in mouse models of cardiac surgery or critical illness, such as sepsis.