TITLE: Measuring the Visceromotor Response in Rodents
AUTHORS: James Bayrer

[DESCRIPTION]
Protocol for measuring the visceromotor response (VMR) to colorectal distension in mice to investigate visceral nociception.

[BEFORE_START]
Ensure all equipment is functional and reagents available prior to start

[GUIDELINES]
Follow all institutional requirements for vertebrate animal work

[STEPS]
SECTION: EMG Wire Implantation
1. Prepare surgical area in accordance with institutional requirements.
- Clean and sterilize instruments
- Warm heating pad
- Check all equipment prior to starting
SECTION: Preparation for VMR
10. - Lightly anesthetize mouse with Isoflurane
- If using wired VMR, take down coil by carefully cutting sutures and unspooling wire. Take care not to cut the wire
- Lubricate disposable gavage needle with Surgilube or similar and administer 100 uL water enema to remove fecal pellets
- Insert balloon catheter to depth of 2 cm and secure in place by taping tubing to animal tail
- Place mouse into broomenator style restrainer and allow to wake up
SECTION: Colorectal Distension
11. - Follow manufactures instructions for recording EMG and pressure tracings
- We use a NeuroLog NL100AK headstage, Band-Pass Filter (NL125/6), Pressure Amp (NL108A), and Extracellular Recording Amplifier (NL104A) feeding into Spike2 for our wired recordings and the Data Sciences International Ponemah system for our wireless recordings
- Program barostat (Distended Series IIR) to deliver sequential balloon inflations of 20, 40, 50, 60, 70, and 80 mmHg for 20 s each with a 2 m rest period in between distensions
SECTION: EMG Wire Implantation
2. Prepare the mouse
- Anesthetize with 1.5% inhaled Isoflurane
- Shave neck and right abdomen
- Clean exposed skin with Betadine

SECTION: EMG Wire Implantation
3. Surgical incisions
- Make a small (< 1 cm) skin incision in right lower abdomen above the inguinal region to expose the abdominal muscle
- Turn over mouse and make small incision on nape of neck
SECTION: EMG Wire Implantation
4. Wire Placement
- Tunnel sterile cannula subcutaneously from neck incision along the side of the mouse and out the abdominal incision taking care to not injure/enter the abdominal compartment
- Thread two sterilized EMG wires (~20cm length) through cannula. Holding the wires from the abdominal side, withdraw the cannula through the neck incision leaving the wires in place
- Remove 2-3mm of insulation from the abdominal end of the wires and tunnel into the abdominal muscle utilizing a 27g needle one at a time, ensuring the wires are close to each but do not touch
- Secure wires using 6-0 non-dissolvable sutures
- Close skin incisions with interrupted sutures
- Coil the wires extending from the next into a flat disc and suture to nape of neck to prevent disruption
SECTION: Postoperative Care
9. - Provide analgesia per institutional surgical protocol
- Administer 1ml of warm, sterile saline subcutaneously to mitigate dehydration
- Animals should be singly housed to prevent surgical site and EMG wire disruption prior to VMR experiment
SECTION: EMG Wireless Transmitter Implantation
5. - Prepare surgical area in accordance with institutional requirements.
- Clean and sterilize instruments
- Warm heating pad
- Check all equipment prior to starting
SECTION: EMG Wireless Transmitter Implantation
6. Prepare the mouse
- Anesthetize with 1.5% inhaled Isoflurane
- Shave right abdomen
- Clean exposed skin with Betadine
SECTION: EMG Wireless Transmitter Implantation
7. Surgical incision
- Make a  ~ 1 cm skin incision in right lower abdomen above the inguinal region to expose the abdominal muscle sufficient to admit passage of the transmitter body
SECTION: EMG Wireless Transmitter Implantation
8. Transmitter Implantation
- Use blunt dissection to create a pocket in the right flank sufficient to house the transmitter. Take care to avoid making the pocket too large, which could result in seroma formation.
- Remove 2mm of insulation from each wire. Set aside insulation for later use.
- Using a 27 G needle, tunnel each wire individually through abdominal muscle. Wires should be placed closely together but should not touch
- Place insulation back on the exposed ends of each wire as they emerge from the muscle tissue and secure in place with 6-0 non-dissolvable sutures. Note: the insulation can be stretched with fine forceps to aid placement back into the wire.
- Suture the wires proximally to muscle entry to both secure the wires in place and to prevent moisture seepage into the transmitter.
- Close skin with either interrupted sutures or surgical staples