A Real-Time Wearable Electromyography Measurement System for Small Animals

Approval of all ethical and experimental procedures and protocols was granted by the Institutional Animal Care and Use Committee under Application No. CGU1-2021-IA0041. A 7-week-old male Sprague-Dawley rat was used in this study. The details of the reagents and equipment are listed in the Table of Materials.

1. Animal preparation

1. Prepare rats at the age of 7 weeks. For safety, house rats of similar weights together, with a maximum of three rats per cage.
2. Ensure the rats adapt without stress before surgery. Provide water and food consistently, and replace the bedding periodically.
3. During the adaptation period (about 7 days), train the animals to run on a treadmill at a speed of 16-25 m/min for 5 min each day to help them acclimate to the treadmill.
   NOTE: If a rat stops or refuses to move as intended, gently tap its buttocks for encouragement.

2. Electrode preparation

1. Prepare five stainless steel wires coated with polytetrafluoroethylene, four of them at a length of 140 mm and one at a length of 60 mm.
2. Tie three overhand knots on a 140 mm long wire, placing them at 40 mm and 100 mm relative to the knot (Figure 1A).
   NOTE: During the insertion of the wire electrode into the muscle, it can be challenging to ensure precise contact and to keep the exposed part of the electrode properly positioned. To address this, a blocking knot is created in the wire to prevent further insertion when pulled, ensuring that the electrode remains stable within the muscle.
3. For the 140 mm wires, leave a 2 mm gap from the knot on the short side, then partially strip off the insulation using a soldering iron for 2 mm. Fully strip the insulation from the end of the long side by 2 mm. For the 60 mm electrodes, completely remove the insulation at one end by 2 mm and at the other end by 10 mm (Figure 1).
   NOTE: The tip of the soldering iron should be thoroughly cleaned to prevent contamination.
4. Fix the wire by soldering it to the connector accessory after fully removing the insulation (Figure 2B,C).
5. Assemble the metal part to which the wire is connected into the plastic housing (Figure 2D,E).
6. Attach the connector to a 3D-printed plastic guide (see Table of Materials) that includes holes for securing it to the skin. To prevent the soldered part from contacting the incision site, apply biocompatible cyanoacrylate (Figure 2F).
7. Cut a 0.2 mm thick nickel plate and bend it to wrap around the connector, shielding it to prevent potential damage by the animal.

3. Surgery

1. Place the rat in an induction chamber (custom fabricated using a transparent synthetic resin) and administer anesthesia using a mixture of 70% nitrous oxide and 30% oxygen, with an isoflurane concentration of 2%-3%. Prepare the necessary surgical instruments (Figure 3).
2. Remove the fur using an electric hair clipper to prevent it from entering the incision during surgery (Figure 4A).
3. Make an incision of about 10-15 mm using surgical scissors on the skin where the target muscle to be measured is located. Since the fascia lies beneath the skin, and muscles below that, first use scissors to cut the skin, then remove the fascia by gently wiping it away with a clean cloth.
4. Make an incision of 5-10 mm around the lumbar region where the connector will be attached.
5. Use surgical scissors to create a passage under the skin for the wire to pass from the connector to the target muscle position (Figure 4B).
6. Pass disinfected forceps from the muscle position, under the skin, toward the incision site on the lumbar side.
7. Prepare the electrode and the connector from the previous stage. Pinch the wire electrodes at the incision site on the lumbar side using the forceps and carefully pull them through to the incision at the target muscle. Once the wire has been pulled out to the location of the target muscle, refer to Figure 4C.
8. As shown in Figure 4D, use a suture needle to penetrate the electrode wire into the target muscle.
   NOTE: The target muscle for this experiment is the medial gastrocnemius (MG), which is one of the key muscles for analyzing motor tasks¹⁷. Since the length of the MG is approximately 18 mm in 7-week-old rats, electrodes should be placed around the muscle's center, with a spacing of about 5 mm, to minimize muscle damage and ensure accurate EMG measurements. It is recommended that the length of the wire penetrating the muscle exceeds 5 mm to ensure that the part with the insulation removed is in good contact with the muscle.
9. Guide the wire that has passed through and exited the muscle back to its insertion point and secure it with an overhand knot. Cut the remaining wire (Figure 4E).
10. After installing the wire electrode in the target muscle, suture the incision site with 4-0 suture thread.
11. Suture the incision under the connector. Ensure that the entire wire is inserted into the body and securely sutured (Figure 5A).
12. Secure the connector to the animal's dorsal skin using a suture thread, as shown in Figure 5B.
    NOTE: Ensure the shield is securely fastened to prevent the animal from chewing on the connector.
13. Once the surgical operation is complete, discontinue anesthesia and prepare a cage for the animal's recovery.
14. Turn on a heat lamp to prevent the animal's body temperature from dropping, and monitor the surgical site for any abnormalities, such as signs of infection, inflammation, or swelling. Disinfect the incision site thoroughly and apply ointment if necessary. Once the rat has recovered from anesthesia, remove the heat lamp.
15. Monitor the rat's behavior, including appetite, drinking, and urination.
    NOTE: After the surgery, allow the animal to rest for about 2 days before conducting any tests.

4. Printed circuit board (PCB) design and fabrication

1. Design the circuit for EMG measurement and digitization to include two analog amplifier integrated circuits (ICs) and a microcontroller unit (MCU). Ensure the amplifier has a bandwidth from 5-740 Hz and a gain of 1300 V/V.
   NOTE: Modify the amplifier gain and bandwidth according to the specific requirements of the animal if necessary¹⁸.
2. Include an integrated Bluetooth module in the MCU for wireless communication to transmit the measured data to the host device. A chip antenna operating at 2.4 GHz was used for this experiment.
3. Utilize a rechargeable 3.7 V Li-po battery along with a dedicated power management IC for this study.
4. Fabricate a PCB for the above ICs on an FR4 substrate. Create the PCB in various forms to meet specific requirements. Refer to Supplementary Figure 1, Supplementary Figure 2, and Supplementary Figure 3.
5. Populate the electronic components on the PCB either by soldering or using a heat gun.

5. Software configuration

1. Firmware
   1. Prepare the firmware (Supplementary Coding File 1) to configure a 2-channel ADC with 8-bit resolution and a sampling rate of 2 kSPs. Format the digitally converted data into an array and transmit it via Bluetooth Low Energy (BLE) using the designated software development tool.
   2. Upload the firmware to the MCU prior to deployment.
2. Real-time monitoring program
   1. Prepare a program to plot the data received via BLE in real-time. For this study, a Python-based plotting application is developed, which also stores the raw data in a specified location (Supplementary Coding File 2).
      NOTE: The system can experience a sampling buffer delay of at most 50 ms and a wireless communication delay of approximately 10 ms^19,20.

6. Treadmill test

1. Connect the battery to the prepared circuit and attach the PCB to the connector, as shown in Figure 6A.
2. Execute the monitoring program on the PC and establish wireless connectivity with the circuit PCB.
3. Place the rat on the treadmill and monitor the EMG waveform. For this study, the treadmill is set at a speed of 25 m/min (Figure 6B).

7. Data analysis

1. Load the EMG data stored in .CSV format.
2. Apply signal processing methods to remove unwanted noise and interference signals.
   NOTE: Envelope detection and spectral analysis can be further performed to analyze muscle fatigue and symmetry in motor activity.