Autonomous and Rechargeable Microneurostimulator Endoscopically Implantable into the Submucosa

All endoscopic procedures including animal subjects have been approved at the Institute of Animal Physiology and Genetics, Academy of Science Czech Republic (Biomedical Center PIGMOD), Libechov, Czech Republic (project Experiments in implantation of battery-less and battery devices into submucosa of the esophagus and stomach — experimental study). All experiments are done in compliance with Czech law 246/1992 Sb. "On the protection of animals against maltreatment, as amended". Transmitter device is not required to be sterilized, because it is an external device that is not in direct contact with the animal.

1. Implantable Device Design

1. Prepare the PCB using a third-party PCB manufacturing service. The complete printed circuit board design is provided in the supplementary file "gerber_implant.7z". The schematic diagram is provided in Figure 1.
2. Place the PCBs on a flat surface (Figure 2a). Use a solder paste dispenser with 0.6 mm needle and 60 psi pressure to manually dispense the soldering paste onto every metallic pad on the PCB. Begin with the top side of the PCB (Figure 2b). The total amount of soldering paste for both sides of the PCB should not exceed 15 μL.
3. With a pair of antistatic tweezers, place all components on the top layer of the PCB (Figure 2e). Use Figure 3 for component position and supplementary file "bom_implantabledevice.csv" for the assignment of components to their numbers.
4. Use a PCB hot air gun station at 260 °C to solder all components (Figure 4a). Wait until all the solder paste melts, then put away the hot air gun and allow the board cool to room temperature.
5. Turn the PCB over and dispense solder paste on the other side. Use the same needle and pressure as stated in 1.2 (Figure 2d).
6. As in step 1.3., place all components of the bottom layer of the PCB. Refer to Figure 3 for component position and the supplementary file "bom_implantabledevice.csv" for the assignment of components to their numbers.
7. Repeat the heating of the PCB with a hot air gun to solder all components on the bottom side. Use the same process as in step 1.4.
8. Check the PCB for any short circuits visually. If any short circuit is found, remove it with a soldering iron.
9. Manufacture the wireless charging/communication coil. Use 17 turns of AWG42 wire. The size of the coil is 26 x 13.5 mm² (Figure 4d). Twist the two output wires.
10. Design and manufacture the electrode. The electrode design is provided in the supplementary file "gerber_electrodes.7z". Use the same manufacturing process as in step 1.1. This PCB is fully completed after manufacturing, and no components are required to be soldered onto it. Solder two AWG42 wires to the small rectangular contacts (Figure 4f)
11. Prepare the antenna by using 7 cm of enameled wire and scraping off 3 mm of the enamel from one end (Figure 4e)
12. Connect the PICkit 3 programmer to the PCB (Figure 4b-c)
    1. Connect pads 6 and 7, according to Figure 5, to pins 2 and 3 of the PICkit programmer, respectively.
    2. Connect pads TP1, TP2 and TP3 (refer to Figure 3) to pins 1, 5 and 4 of the PICkit programmer, respectively
13. Plug in the PICkit 3 programmer into the USB port of a computer with MPLAB IPE software installed.
14. Run the MPLAB IPE software and program the firmware into the microcontroller.
    1. Run the MPLAB IPE v3.61. Select "Settings | Advanced Mode"
    2. Into the Password field, enter the default password which is 'microchip'. Click "Log on". A tab with different panels on the left will appear.
    3. On the top left, click "Operate", then in top middle part of the screen, click "Device field" and type in "PIC16LF1783". Click "Apply".
    4. Select panel "Power" on the left (Figure 6).
    5. Change the VDD voltage value to 2.55. This step is critical.
       Caution: Setting this value above 2.8 V will damage the board (Figure 7).
    6. Click the checkbox "Power Target Circuit" from "Tool" (Figure 7).
    7. Click the "Operate" tab on the left (Figure 6).
    8. Click "Connect".
    9. Download the supplementary file "IMPLANTABLE_V2.X.production.hex" and note its location on the hard drive. In the IPE software, find the Source line and click the "Browse" button near it (Figure 8).
    10. Click Program. Wait till the software says that the software has been successfully downloaded to the microcontroller (Figure 9).
15. Desolder the wires soldered to pads TP1, TP2 and TP3 (Figure 3) as well as wires soldered to pads 6 and 7 (Figure 5).
16. Connect the PCB to all other electrical components except for the battery (Figure 10a).
    1. Solder the wireless charging/communication coil to pads 2 and 3 according to Figure 8. The polarity is not important.
    2. Connect the antenna to pad 1 according to Figure 5. Connect the PCB electrodes to pads number 4 and 5 according to Figure 5. The polarity is not important.
17. Solder the CG-320 battery to pads 6 and 7 (Figure 5). The negative terminal of the battery must be soldered onto pad 7. Be careful while performing the next steps. The device is now powered and is sensitive to short circuits and contact with metallic objects.
18. To test the functionality of the wireless charging circuitry, all steps in part 2 need to be completed. After that, place the wireless charger/transmitter in close proximity of the device. Use a multimeter to measure the voltage of the battery. If the battery voltage is slowly rising (several mV per min), the charging function is working.
19. Wind the antenna around the device in a spiral (Figure 10b)
20. Cut a 32 mm long piece of a heat-shrinkable tubing with an internal diameter of 9.5 mm.
21. Place the coil on the PCB. Refer to Figure 7b for the correct placement.
22. Put the heat-shrinkable tubing over the device, coil and antenna. Only the electrodes should protrude from the tubing. Refer to Figure 7c for correct placement.
23. Heat the tubing with a hot air gun to 150 °C to shrink and then allow it to cool (Figure 10d).
24. Apply epoxy glue to the left end to seal one side of the tubing (Figure 10e).
25. Glue the electrode to the back side of the PCB with tubing. Also glue the other end of the tubing. Refer to Figure 10f for correct placement.
26. Wait for at least 24 h for the glue to harden and fully cure.
27. After the completion of the wireless charger/transmitter device, test the implantable device for water leaks by placing it into a 30 cm high column of saturated saline solution for 1 h. Any major leak can be spotted as a sudden drop of the battery voltage or the malfunction of the device caused by the saline solution shorting the electronics. After the test, the device is fully prepared to be implanted.
28. Test the stimulation function of the implant using an oscilloscope. Connect two measurement electrodes of the oscilloscope to the tin metal plated contact pads of the electrode on the implantable device. Observe the stimulation pattern on the oscilloscope screen. The correct stimulation pattern is given in Figure 11.

2. Wireless Charger/Transmitter Design

1. The PCB design is provided in the supplementary file "gerber_transmitter.7z". Use the same manufacturing process as for the implantable device. The schematic diagram is provided in Figure 12.
2. Place the PCBs on a flat surface. Use a solder paste dispenser with 0.6 mm needle and 60 psi pressure to manually dispense the soldering paste onto every metallic pad on the PCB. The total amount of solder paste dispensed on the PCB should not exceed 50 μL.
3. With a pair of antistatic tweezers, place all components on the top layer of the PCB. Consult Figure 13 for the component position and the supplementary file "bom_transmitterdevice.csv" for the assignment of components to their numbers.
4. Use a PCB hot air gun station preset to 260 °C to solder all components. Wait until all the solder paste melts, put away the hot air gun and allow the board to cool to room temperature.
5. Repeat the steps 2.3–2.4 for the bottom side of the device. Follow a similar procedure as during the manufacturing of the implantable device.
6. Create a coil with 3 turns of AWG18 enameled wire (Figure 14c) and connect it to pads COIL1 and COIL2 (Figure 13).
7. Make an aluminum heatsink for the power transistors (Figure 13, Q1 and Q2). The exact shape of the heatsink is not critical. One of the possible embodiments is shown in Figure 9d. In this case, the heatsink also forms an enclosure for the device.
8. Connect the PICkit 3 programmer to the assembled PCB. Connect pads TP1 to TP5 (Figure 13) with pins 1 to 5 of the PICkit programmer, respectively.
9. Plug in the PICkit 3 programmer into the USB port of a computer with MPLAB IPE software installed.
10. Run the MPLAB IPE software and program the firmware into the microcontroller. The process is the same as for the implantable device, except for the VDD voltage and file uploaded.
    1. Run the MPLAB IPE v3.61. Select "Settings | Advanced Mode".
    2. Into password box, enter the default password which is 'microchip'. Click "Log on". A tab with different panels on the left will appear.
    3. On the top left, click "Operate", then in the top middle part of the screen, click the "Device" and type in "PIC16LF1783". Click "Apply".
    4. Select panel "Power" on the left
    5. Change the VDD voltage value to 3.3.
    6. Click the checkbox "Power Target Circuit" from "Tool".
    7. Click the "Operate" tab on the left.
    8. Click "Connect".
    9. Download the supplementary file "IMPLANTABLE_V2_TRANSMITTER.X.production.hex" and note its location on the hard drive. In the IPE software, find the Source line and click the "Browse" button near to it.
    10. Click "Program". Wait till the software says that the software was downloaded to the microcontroller successfully.
11. Desolder the wires soldered to pads TP1 to TP5
12. Connect a 12 V power supply to the V- and V+ pads (Figure 5). The negative terminal has to be connected to the V- pad.
13. Plug in a mini-USB to USB-A cable to the X1 connector (Figure 5) and connect to a computer with PuTTy software pre-installed.
14. Open the PuTTY software and set it up (Figure 15).
    1. Open the PuTTY software. Select "Serial" as connection type.
    2. Enter COMx as a Serial line, where x is the number of the COM port of the device. If no other COM port device was installed, this number will be 1.
    3. Enter "38400" as Speed. Click "Open". The charger/transmitter device is now ready to be used. Press H key for help.

3. Endoscopic Implantation

1. Use a live mini pig as an in vivo model, adult (8-36 months), 20-30 kg weight.
   1. Let the pig fast for 24 h prior to the procedure.
   2. Allow clear liquids ad libitum.
   3. Administer intramuscular tiletamine (2 mg/kg), zolazepam (2 mg/kg) and ketamine (11 mg/kg) as a premedication.
   4. Apply intravenous thiopental ad effectum (5% solution) and inhalation anesthesia with isoflurane, N₂O and propofol injection. Proper anesthesia is confirmed by reflexes and muscle tone, eye position, palpebral reflex and pupillary reflex. Circulation, oxygenation, ventilation and body temperature are continuously monitored.
2. In order to perform the implantation and visualization, use an animal model dedicated endoscope. Insert it using the standard way into the in vivo model.
3. Grasp the device externally with a snare. After that, insert it into the stomach, then release it.
4. Extract the endoscope, equip it with a dissection cap (15.5 mm), and then re-insert it to the stomach.
5. In order to implant the device to submucosa, apply saline solution mixed with methylene blue into the submucosal layer using an injection therapy needle catheter (25 G).
6. Make a horizontal incision to create an opening in the submucosa using an electrosurgical knife with a knob-shaped tip.
7. Using the affixed cap, insert the cap into the newly created space, and with the use of an electrosurgical knife, continue disrupting, dilating, and dissecting the submucosal layer, creating a sufficiently large-enough pocket to insert the stimulation device.
8. Grasp the device which is lying freely inside the stomach with insertion and extraction loops and, using grasping forceps, navigate it into the submucosal pocket. Place the stimulation electrodes in contact with the muscularis propria using grasp forceps.
9. Use an over the scope clip to secure the device in place inside the submucosal pocket and prevent any migration or dislodging.

4. Experiment — After Implantation

1. After successful implantation, place the charger/transmitter coil in proximity of the implanted device.
2. Plug in the RTL2832 dongle in the PC.
3. Run the HDSDR software and set the center frequency to 432 MHz.
   1. Open the HDSDR software (Figure 15) for correct settings and PuTTY software (Figure 16). In the HDSDR software, click "Options | Select Input | ExtIO".
   2. Select Bandwidth — "960000". Select the LO frequency to 431.95 MHz. Select the Tune frequency to 432.00 MHz.
4. Transmit a Manchester coded sequence from the charger/transmitter by pressing the R key in the PuTTY terminal and receive the OOK modulated reply from the implant by observation of the main HDSDR window ( Figure 17e-f).

5. Euthanasia after the Experiment

1. Use an anesthetic overdose for euthanasia (lethal dose of thiopental and KCl).