Irreversible Electroporation: A Technique for Ablating Pancreatic Tumor in Mouse

Overview

This video describes the procedure for performing irreversible electroporation (IRE) for non-thermal ablation of pancreatic tumors in mice. Both subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various studies post-procedure.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the jove veterinary review board.

1. IRE of SQ Tumors

1. Prepare the materials required: square wave electroporator, safety foot switch, electrodes (needle array versus tweezer-trode), and adaptors. Keep sterile sutures (non-absorbable 4-0), alcohol swabs, hair clippers, depilatory cream, eye lubricant, cotton gauze, buprenorphine, and anesthetics also nearby.
2. Sterilize the tweezer or needle array electrodes using gas sterilization. Autoclaving is not recommended. Use a glass bead sterilizer to sterilize the electrodes between animals.
3. Follow steps to successfully induce anesthesia in mice once the SQ tumor implant reaches 5 mm diameter. Briefly, Administer the recipient mouse 0.05 - 1 mg/kg buprenorphine analgesic SQ, 30 min prior to surgery. Then, anesthetize the recipient mice with 2 - 3% isoflurane in oxygen (2 L/min) using a precision vaporizer (or other anesthetic agents). Keep mice on a 37 °C heating pad for the entirety of the procedure. Apply lubricant to the eyes to prevent desiccation. Test the depth of anesthesia by lack of startling reflex initially, and confirm surgical plane anesthesia by the lack of pedal reflex to a gentle toe pinch. Maintain the anesthesia during the entire surgical procedure.
4. Place the anesthetized mouse on its side to access the SQ tumor on the flank. Remove the hair using clippers and clean skin using an alcohol swab.
5. For SQ tumors, use 2-needle array electrodes. Elevate the skin directly under the tumor site using forceps and insert the electrodes through the skin parallel to the body making sure they do not penetrate the peritoneal cavity. Once through the skin, position the electrodes in such a way that they bracket the tumor.
6. Program the electroporator to deliver 100 µs pulses at a frequency of 1 Hz at 1,500 V/cm for a total of 150 pulses. Deliver the pulses using the foot pedal. Separate each set of 10 pulses by 10 s, in order to allow for heat dissipation and confirm the correct position of the electrodes.
   NOTE: Without the use of paralytic agents, mice will experience muscle contractions with IRE that can cause displacement of the electrodes unless manually secured.
7. Remove the electrodes after complete dosing, which should not exceed 200 s. Record the actual voltage delivered, which is displayed on the electroporator. Allow the mice to recover following IRE in its home cage with free access to food and water. Place the cage on a heating pad to facilitate recovery. Monitor vital signs such as breathing, perfusion etc., during the recovery process. Confirm the signs of righting reflex once the mouse recovers, and then return the cage can to regular housing. Administer buprenorphine 0.05 - 0.1 mg/kg 8 - 12 h after the surgery, and every 8 - 12 h after the procedure as needed for signs of pain.

2. IRE of Orthotopic Tumors

NOTE: IRE of orthotopic tumors involves a second survival surgery on the same mouse thus requiring special approval from local IACUC before beginning.

1. Prepare required materials: autoclave the surgical tools such as scissors, scalpels, needle drivers, pointed forceps, and flat tipped forceps. Keep the following nearby as well: sterile sutures (absorbable 6-0 and non-absorbable 4-0), alcohol swabs, hair clippers, depilatory cream, eye lubricant, cotton gauze, 10% povidone iodine solution, surgical drapes, heating pads, square wave electroporator, safety foot switch, electrodes and their adaptors, buprenorphine, and anesthetics.
2. Assess the mice for orthotopic tumor growth by in vivo bioluminescence imaging. Follow tumor growth by imaging starting on day 4 after orthotopic implantation, and perform imaging twice a week. On the day of imaging, administer (intraperitoneal injection) 30 mg/kg D-luciferin to an anesthetized (2 - 3% isoflurane in oxygen (2 L/min)) recipient mouse 10 min prior to imaging. Image for luciferase activity using the luminescence setting without any emission filters for a minimum of 5 s exposure in a luminescence imager with auto-fluorescence free heated stage and while still maintaining the anesthesia for the mice.
   NOTE: The orthotopic tumors are ideal for IRE treatment 8 to 10 days post implantation when the tumors are clearly visible and still confined to the pancreas as can be seen on the Luciferase luminescence images
3. Follow steps to locate the implanted tumor. Briefly, administer the recipient mouse 0.05 - 1 mg/kg buprenorphine analgesic SQ, 30 min prior to surgery. Anesthetize the recipient mice with 2 - 3% isoflurane in oxygen (2 L/min) using a precision vaporizer (or other anesthetic agents). Keep mice on a 37 °C heating pad for the entirety of the procedure. Apply lubricant to the eyes to prevent desiccation. Test the depth of anesthesia by lack of startling reflex initially, and confirm surgical plane anesthesia by the lack of pedal reflex to a gentle toe pinch. Maintain the anesthesia during the entire surgical procedure. Place the mouse on its back and gently turn it to its right side so that the left side of the abdomen is exposed. Remove the abdominal hair of the mouse using depilatory cream and clean with gauze to ensure no free hair enters the abdomen post incision. Prepare the left abdomen for surgery using 3 cycles of 10% povidone iodine followed by alcohol wipes to disinfect the skin. Using a sterile scalpel, make a 1.5-cm transverse or oblique incision in the skin, 1-cm to the left of the midline, below the ribcage, slightly medial to the spleen. Then, extend the incision through the abdominal musculature, mirroring the overlying superficial incision. Locate the spleen using flat tipped forceps and gently externalize it from the abdominal cavity. Retract the spleen using a sterile cotton applicator, and find the tail of the pancreas attached to the bottom of the spleen. Using flat-tipped forceps, retract the tail of the pancreas laterally. If the tumor is not easy to locate, use blunt nosed forceps to move the stomach and spleen gently to identify the tumor.
4. Externalize the tumor with blunt nosed forceps and capture the tumor tightly with the platinum electrodes of the tweezer-trode. Deliver the electroporation pulses as programmed in step 1.7 using the square wave electroporator in sets of 10 pulses controlled by the foot switch.
5. Keep the tumor externalized for at least 60 s post IRE to monitor for any signs of hemorrhage. Insert the tumor back into the abdominal cavity and close the incision as described. Briefly, Close the abdominal musculature using a 6-0 absorbable suture with either a continuous or interrupted stitch, and close the overlying skin using a 3-0 to 6-0 non-absorbable interrupted suture. Discontinue the anesthesia at this point. Allow the mouse to recover as described in 1.7.  Monitor the mouse until it recovers.
6. Monitor the effects of IRE on tumor growth using in vivo luciferase imaging as per step 2.2.

Materials

Name	Company	Catalog Number	Comments
ECM 830 square wave electroporator	Harvard Apparatus	BTX # 45-0002 ( 58018-004 )
2 needle array electrode	Harvard Apparatus	45-0167
Safety foot switch	Harvard Apparatus	45-0211
Platinum Tweezer-trode	Harvard Apparatus	45-0486
Isoflurane	Sigma-Aldrich, Inc.	792632
Lacrilube	Fisher Scientific	19090646
Buprenorphine	Fisher Scientific	NC1292810
D-luciferin	Perkin Elmer	122799
IVIS Spectrum In Vivo Imaging System	Perkin Elmer	124262
Mouse strain C57BL/6J	The Jackson Laboratory	000664/Black 6
BD Precisionglide syringe needles	Sigma-Aldrich, Inc.	Z192406
Alcohol Swab(70% isopropyl alcohol )	BD	326895
Disposable Scalpels, Sterile	VWR	21909
Cotton Tipped Applicators	VWR	89198
Suture Needle, 45 cm, Size 6-0	Harvard Apparatus	72-3308
Suture Needle, 45 cm, Size 4-0	Harvard Apparatus	72-3314
Povidone-iodine 10%	BD	29900-404
Disposable Warming Pad	KENT SCIENTIFIC CORP	TP-3E
Mouse Hair Clipper	KENT SCIENTIFIC CORP	CL8787
Surgical Drape	Harvard Apparatus	59-7421