TITLE: Protocol for vagus nerve stimulation in anesthetized pigs (subject-2)
AUTHORS: Oliver Armitage

[DESCRIPTION]
The Vagus nerve innervates a number of thoracic and visceral organs. Exogenous nervous signal, for example, Vagus nerve stimulation (VNS) provides a route to modulating their function for therapeutic purposes. However, the relationship between VNS parameters and organ responses is poorly understood as evidenced by a number of failed human trials, for example, in the heart failure space.
This study will contributes to our understanding of the relationship between VNS parameters and the organ responses. It does so by mapping VNS parameters (at a fine resolution) to their immediate neural and cardiac effects in 2 pigs under anaesthesia.

[STEPS]
SECTION: Surgery
3. We do the nerve preparation and device implantation. 





SECTION: Data Acquisition Validation
4. We validate the quality of the physiological and neural signals. 
SECTION: Equipment Setup
1. We do the setup of the BIOS A1 System (electrophysiology).
0.21. We install vital signs monitoring. [respiratory pressure, ECG, blood pressure]
* Install ECG surface electrodes on the right and left front legs.
 Install the pressure catheter in the femoral artery. 
SECTION: Surgery
3.3. Cuff installation:
Installation of cuff A most rostrally (record1)
Installation of cuff C most caudally
Installation of cuff B closer to cuff A (record2)

Distance from the center of cuff A to center of cuff C is 5.4mm. Distance from the center of cuff A to center of cuff B is 1.7mm. 
SECTION: Equipment Setup
1.1. Load BIOS Neurotool software for neural recording acquisition and stimulation.
SECTION: Equipment Setup
1.2. Place cuff electrodes in saline. Take impedance measurement to confirm system is functional. 
SECTION: Stimulation Protocol
5. Once we validated the quality of the data acquisition systems, we start the stimulation protocol. 
SECTION: Surgery
3.2. Make incision on the neck and expose 8cm of the left vagus nerve.
SECTION: Termination
6. Once testing is complete, the animal is terminated.
SECTION: Data Acquisition Validation
4.1. Visually validate the quality of the vitals. For the ECG, the r-peaks should be clearly visible to ensure a good extraction of the heart rate. 
SECTION: Data Acquisition Validation
4.2. Neural signals validated using BIOS neural analytics pipeline. Analyses looks for presence of underlying endogenous signals (e.g. respiration), afferent<>efferent velocity spectrum decomposition, among others.
SECTION: Animal Preparation
2. We prepare the animal before the surgery.
SECTION: Stimulation Protocol
5.1. Set the the following stimulation parameters in BIOS Neurotool software: 
 Pulse width: 500us
Train duration: 5s
Stimulation shape: monophasic
Polarity: anodic
SECTION: Stimulation Protocol
5.2. Conduct a grid search by doing the cartesian product of the frequency and current parameters. 
Frequency: {2Hz, 10Hz, 20Hz, 30Hz}
Current: {0.05mA, 0.5mA, 1mA, 1.5mA, 2.5mA}
After each stimulation, wait for the physiological signals to return to baseline. 
SECTION: Animal Preparation
2.3. Place the pig in supine position and mark incision.
SECTION: Animal Preparation
2.2. Intubate the animal and start mechanical ventilation.
SECTION: Animal Preparation
2.1. Animal under anesthesia breathing naturally.

SECTION: Surgery
3.1. Installation of the vitals monitoring (respiratory pressure, ECG, blood pressure).