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

Summary

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.

Abstract

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.

Introduction

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 surgery^2,3. 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 results^5,6,7,8. 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 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.

Protocol

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

1. 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: Use cells that are stably transduced to express firefly luciferase for bioluminescence imaging to enable cancer recurrence monitoring after resection surgery¹¹ (see step 4).
   NOTE: The cell line and the medium mentioned above were used in this study.
2. Grow cells at 37 °C with 5% CO₂. Passage cells aseptically in a hood at <80% confluency. Use low-passage cells in the logarithmic growth phase for optimal in vivo results.
3. Lift the adherent cells with 0.5 mg/mL of trypsin in PBS with 10 mM EDTA; 2 mL for a T75 flask. When lifted, add medium to inactivate trypsin, and wash the cells in PBS.
4. Count the cells using a hemocytometer. Dilute the cells in PBS for injection. For 66cl4 mammary cancer cells, inject 1 x 10⁵ cells in 20 μL of PBS per mouse.
5. Place the cells on ice prior to the injection.

2. Generate a mouse model of breast cancer

1. Use 4% isoflurane to anesthetize the mouse in an induction chamber. Then, maintain anesthesia with 2%-3% isoflurane using a nose cone. Confirm proper anesthetization by lack of response to toe pinch.
2. Prepare the injection site by wiping the fourth left mammary fatpad area using a single-use alcohol swab.
3. Draw up tumor cells (see step 1.4) into a 25 μL Hamilton syringe attached to a sterile 27 G hypodermic needle.
4. Inject the cells into the fourth left mammary fatpad. Use forceps to secure and lift the skin. Inject approximately 1 mm from the nipple.
5. OPTIONAL: If cells are tagged with luciferase, confirm successful injection of tumor cells by bioluminescence imaging. Inject 100 μL of 150 mg/kg D-luciferin into the lateral tail vein of the anesthetized mice using a 0.5 mL insulin syringe with a 30 G hypodermic needle.
6. OPTIONAL: Place the mouse in a bioluminescence imaging system with the mammary fatpad facing up. Wait for 2 min from the luciferin injection for optimal tissue uptake of luciferin, then image for 10 s.
7. Place the mouse in a clean cage and allow it to recover from anesthesia.
8. Continue to monitor animal welfare as per the institutional animal ethics guidelines.

3. Induce stable anesthesia with intravenous delivery of propofol

1. Monitor growth of the primary tumor using caliper measurement and calculate the tumor volume using the equation: Volume (mm³) = (length x (width)² ÷ 2).
2. Perform tumor resection surgery on mice when the primary tumor reaches the required volume (here, 80-90 mm³).
3. Set up an automated syringe pump with a 30 G 1 mL insulin syringe containing propofol formulation (2% Lipuro propofol) (Figure 1A).
4. Induce anesthesia of the mouse in an induction chamber with 3% sevoflurane or isoflurane.
   NOTE: Here, sevoflurane was used as this is the predominant volatile used clinically.
5. Transfer the mouse to a 37 °C heating pad for the duration of the surgery. Briefly maintain anesthesia with 2%-3% sevoflurane using a nose cone.
6. Apply aqueous lubricant to the eyes to prevent drying. 
7. Inject 0.05 mg/kg of buprenorphine subcutaneously for analgesia.
8. To prepare for surgery, shave the abdomen and prepare the skin for surgery with an iodine-povidone solution. Wipe the skin using an alcohol or sterile wipe.
9. To deliver propofol-based TIVA, cannulate the lateral tail vein using a sterile 30 G hypodermic needle attached to a sterile polyurethane catheter. Confirm correct placement by blood flashback into the catheter (Figure 1B). Adjust the delivery of sevoflurane as required during intravenous cannulation to maintain a stable depth of anesthesia demonstrated by loss of corneal and pedal reflex, and respiratory rate < 100 breaths per min.
10. Commence propofol-TIVA by administering 2% propofol as an initial bolus of 27 mg/kg for over 1 min. Cease sevoflurane administration.
11. Continue the infusion of propofol at a maintenance rate of 2.2-4.0 mg/kg/min to maintain a stable depth of anesthesia for the duration of the surgery (Figure 1C).

4. Resect the primary tumor

1. Make a straight 1 cm incision inferior to the tumor in the region of the left fourth mammary fatpad. Carefully resect the tumor and the left inguinal lymph node using dissection with blunt forceps.
2. OPTIONAL: If using luciferase-tagged tumor cells, use bioluminescence imaging to confirm clear surgical margins. Inject 150 mg/kg D-luciferin into the lateral tail vein, wait for 2 min, and then image for 60 s using a bioluminescence imaging system. If a residual tumor is identified, resect additional tissue from the mammary fatpad and re-image to achieve clear margins.
3. Ensure hemostasis at the surgical site and close the skin using 5-0 nylon sutures. Ensure that the fur does not enter the surgical site.
4. At the conclusion of resection surgery, cease anesthesia. Place the mouse in a clean cage on a 37 °C heating pad and allow it to recover from anesthesia.
5. Monitor every 15 min after anesthesia until the mouse has returned to normal alertness. Then, monitor the mouse every 12 h for 48 h after surgery.
6. Administer 0.05 mg/kg of buprenorphine subcutaneously every 12 h for 48 h after surgery.
7. After 7-10 days, remove sutures using sterile curved stitch cutters under brief sevoflurane or isoflurane anesthesia.

5. Track cancer recurrence with in vivo imaging

1. Use bioluminescence imaging to track cancer recurrence after resection surgery non-invasively. Use a bioluminescence imaging system to monitor the mice once per week for evidence of primary tumor recurrence or distant recurrence, commencing the week following surgery.
2. Induce anesthesia of the mouse in an induction chamber with 4% isoflurane. Then, transfer the mouse to a 37 °C heating pad for the duration of anesthesia and maintain anesthesia with 2%-4% isoflurane using a nose cone.
3. Apply aqueous lubricant to the eyes to prevent drying.
4. Inject D-luciferin 150 mg/kg into the lateral tail vein. Wait for 2 min, then measure bioluminescence over a 60 s exposure to detect recurrence of the primary tumor or distant metastasis.
5. If the primary tumor recurs and becomes palpable, commence monitoring of tumor growth using caliper measurements.
6. At the end of the experiment, humanely kill the mice according to the approved protocol. Here, CO₂ was used, followed by cervical dislocation.

Results

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...

Discussion

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<...

Materials

Name	Company	Catalog Number	Comments
0.9% saline	Fresnius Kabi	AUST R 197198
Artery forceps	Proscitech	TS1322-140
Buprenorphine	Temgesic	TEMG I
Heated surgical mat	Custom	-
Hypodermic needle (30 G, 1 mL insulin syringe)	Terumo	NN3013R
IVIS Lumina	PerkinElmer	126274
Luciferin	Promega	P1041/2/3
Polyurethane catheter	Intramedic	427401
Povidone Iodine	Betadine	AUST R 29562
Propofol Lipuro, 2%	Braun	3521490
Sevoflurane	Baxter	ANZ2L9117
Sevoflurane vaporiser	Vetquip	VQ1334
Sterile gauze	Multigate Medical Products	11-600A
Surgical scissors	Proscitech	TS1044
Sutures, 5-0 nylon	Dynek	V504
Syringe pump	Harvard Apparatus	70-4500
Syringes (1 mL)	Terumo	SS+01T