Name: an in vivo mouse model of total intravenous anesthesia during cancer resection surgery
Uploaded: 2021-06-08T14:30:00
Description: Watch this Scientific Journal Video about An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery at JoVE.com

The authors wish to thank members of the Cancer Neural-Immune Laboratory and Dr. Cameron Nowell at Monash Institute of Pharmaceutical Sciences, Monash University, Parkville. This work was supported by grants from National Health and Medical Research Council 1147498, the National Breast Cancer Foundation IIRS-20-025, the Australian and New Zealand College of Anaesthetists (ANZCA), Perpetual and CTC for Cancer Therapeutics.

All animal studies were undertaken under the approval of the Institutional Animal Care and Use Committee at Monash University. In this study, female Balb/c mice aged 6-8 weeks were used.
1. Prepare cancer cells
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	<li>Culture tumor cells in medium. Culture 66cl4 murine mammary cancer cells in alpha-MEM containing 10% FBS and 200 mM glutamine. Cells should text negative for mycoplasma. OPTIONAL:&#160;Use cells that are stably transduced to express firefly luciferase for bioluminescence ima...

<ol>
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This study reports on a protocol for administering total intravenous anesthesia (TIVA) with propofol in a mouse model of breast cancer that replicates key aspects of clinical practice for TIVA in patients requiring cancer surgery. The protocol allows investigation of both short-term and long-term clinically relevant outcomes after cancer surgery in a mouse model of cancer progression, including measurement of cytokine levels and cancer recurrence (Figure 2, and Figure 3<...

More than 60% of patients with cancer receive anesthesia for surgical resection1. Currently, there are no specific clinical guidelines that determine the choice of anesthesia used in cancer patients. Surveys of anesthesiologists indicate a preference for volatile-based anesthesia, including during cancer surgery2,3. However, there is a growing body of evidence that the use of propofol-based total intravenous anesthesia (TIVA) during cancer surgery may associate with improved postoperative outcomes (progression-free survival, overall survival) when compared to volatile anesthesia4. Subsequent clinical studies continue to report contradicting results5,6,7,8. These findings support the need for preclinical studies to better understand the mechanistic effects of different anesthetic agents on cancer-related outcomes.
However, in in vivo studies that model cancer surgery, anesthesia is frequently an incidental part of the procedure. The rationale for the choice of anesthesia is&#160;often not the focus of the experimental design, and its impact on cancer-related endpoints may not be evaluated. For example, in vivo studies that require maintenance of anesthesia for cancer surgery most commonly use inhaled volatile anesthesia9. Where propofol has been used in in vivo studies, it has been delivered by single bolus dosing with intraperitoneal delivery, which does not replicate clinical onco-anesthetic protocols10. That approach of propofol administration induces light anesthesia that is suitable for rapid procedures. However, it does not allow maintenance of anesthesia that is required for cancer resection surgery which may be protracted. Furthermore, the absorption kinetics of intraperitoneal delivery is distinct to clinical methods of administration.
A model of propofol-based TIVA for cancer resection surgery was developed to address this need. A protocol for sustained maintenance of anesthesia with titration of the anesthetic agent to allow response to the surgical stimulus was developed to replicate key aspects of anesthetic delivery to patients having cancer surgery. The resulting protocol is used with a mouse model of cancer to provide TIVA during cancer resection surgery. The effect on short-term and long-term cancer-related outcomes is evaluated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% saline</td>
			<td>Fresnius Kabi</td>
			<td>AUST R 197198</td>
			<td></td>
		</tr>
		<tr>
			<td>Artery forceps</td>
			<td>Proscitech</td>
			<td>TS1322-140</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Temgesic</td>
			<td>TEMG I</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated surgical mat</td>
			<td>Custom</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic needle (30 G, 1 mL insulin syringe)</td>
			<td>Terumo</td>
			<td>NN3013R</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina</td>
			<td>PerkinElmer</td>
			<td>126274</td>
			<td></td>
		</tr>
		<tr>
			<td>Luciferin</td>
			<td>Promega</td>
			<td>P1041/2/3</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyurethane catheter</td>
			<td>Intramedic</td>
			<td>427401</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone Iodine</td>
			<td>Betadine</td>
			<td>AUST R 29562</td>
			<td></td>
		</tr>
		<tr>
			<td>Propofol Lipuro, 2%</td>
			<td>Braun</td>
			<td>3521490</td>
			<td></td>
		</tr>
		<tr>
			<td>Sevoflurane</td>
			<td>Baxter</td>
			<td>ANZ2L9117</td>
			<td></td>
		</tr>
		<tr>
			<td>Sevoflurane vaporiser</td>
			<td>Vetquip</td>
			<td>VQ1334</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauze</td>
			<td>Multigate Medical Products</td>
			<td>11-600A</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Proscitech</td>
			<td>TS1044</td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures, 5-0 nylon</td>
			<td>Dynek</td>
			<td>V504</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>70-4500</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes (1 mL)</td>
			<td>Terumo</td>
			<td>SS+01T</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vivo mouse model of total intravenous anesthesia during cancer resection surgery

Anesthesia is a routine component of cancer care that is used for diagnostic and therapeutic procedures. The anesthetic technique has recently been implicated in impacting long-term cancer outcomes, possibly through modulation of adrenergic-inflammatory responses that impact cancer cell behavior and immune cell function. Emerging evidence suggests that propofol-based total intravenous anesthesia (TIVA) may be beneficial for long-term cancer outcomes when compared to inhaled volatile anesthesia. However, the available clinical findings are inconsistent. Preclinical studies that identify the underlying mechanisms involved are critically needed to guide the design of clinical studies that will expedite insight. Most preclinical models of anesthesia have been extrapolated from the use of anesthesia in in vivo research and are not optimally designed to study the impact of anesthesia itself as the primary endpoint. This paper describes a method for delivering propofol-TIVA anesthesia in a mouse model of breast cancer resection that replicates key aspects of clinical delivery in cancer patients. The model can be used to study mechanisms of action of anesthesia on cancer outcomes in diverse cancer types and can be extrapolated to other non-cancer areas of preclinical anesthesia research.

This method describes a model of total intravenous anesthesia (TIVA) with propofol during cancer resection surgery in mice. Propofol is delivered in this mouse model through an intravenous catheter using a syringe pump (Figure 1A,B) to replicate delivery of TIVA in the clinical setting of anesthesia for cancer surgery. Use of the syringe pump minimizes exposure to volatile anesthesia by allowing rapid conversion from initial induction by inhalational anesthesia to intravenou...

Watch this Scientific Journal Video about An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery at JoVE.com

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

This paper describes a method for modeling total intravenous anesthesia (TIVA) during cancer resection surgery in mice. The goal is to replicate key features of anesthesia delivery to patients with cancer. The method allows investigation of how anesthetic technique affects cancer recurrence after resection surgery.

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

Anesthesia is a routine component of cancer care that is used for diagnostic and therapeutic procedures. The anesthetic technique has recently been ...

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.

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