The authors declare that they have no competing financial interests.

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 &#8212; experimental study). All experiments are done in compliance with Czech law 246/1992 Sb. &#34;On the protection of animals against maltreatment, as amended&#34;.&#160;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
<ol>
	<li>Prepare the PCB using a third-party PCB manufacturing service. The complete printed circuit board design is provided in the supplementary file &#34;gerber_implant.7z&#34;. The schematic diagram is provided in Figure 1.</li>
	<li>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 &#956;L.</li>
	<li>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 &#34;bom_implantabledevice.csv&#34; for the assignment of components to their numbers.</li>
	<li>Use a PCB hot air gun station at 260 &#176;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.</li>
	<li>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).</li>
	<li>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 &#34;bom_implantabledevice.csv&#34; for the assignment of components to their numbers.</li>
	<li>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.</li>
	<li>Check the PCB for any short circuits visually. If any short circuit is found, remove it with a soldering iron.</li>
	<li>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.</li>
	<li>Design and manufacture the electrode. The electrode design is provided in the supplementary file &#34;gerber_electrodes.7z&#34;. 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)</li>
	<li>Prepare the antenna by using 7 cm of enameled wire and scraping off 3 mm of the enamel from one end (Figure 4e)</li>
	<li>Connect the PICkit 3 programmer to the PCB (Figure 4b-c)
	<ol>
		<li>Connect pads 6 and 7, according to Figure 5, to pins 2 and 3 of the PICkit programmer, respectively.</li>
		<li>Connect pads TP1, TP2 and TP3 (refer to Figure 3) to pins 1, 5 and 4 of the PICkit programmer, respectively</li>
	</ol>
	</li>
	<li>Plug in the PICkit 3 programmer into the USB port of a computer with MPLAB IPE software installed.</li>
	<li>Run the MPLAB IPE software and program the firmware into the microcontroller.
	<ol>
		<li>Run the MPLAB IPE v3.61. Select &#34;Settings | Advanced Mode&#34;</li>
		<li>Into the Password field, enter the default password which is &#39;microchip&#39;. Click &#34;Log on&#34;. A tab with different panels on the left will appear.</li>
		<li>On the top left, click &#34;Operate&#34;, then in top middle part of the screen, click &#34;Device field&#34; and type in &#34;PIC16LF1783&#34;. Click &#34;Apply&#34;.</li>
		<li>Select panel &#34;Power&#34; on the left (Figure 6).</li>
		<li>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).</li>
		<li>Click the checkbox &#34;Power Target Circuit&#34; from &#34;Tool&#34; (Figure 7).</li>
		<li>Click the &#34;Operate&#34; tab on the left (Figure 6).</li>
		<li>Click &#34;Connect&#34;.</li>
		<li>Download the supplementary file &#34;IMPLANTABLE_V2.X.production.hex&#34; and note its location on the hard drive. In the IPE software, find the Source line and click the &#34;Browse&#34; button near it (Figure 8).</li>
		<li>Click Program. Wait till the software says that the software has been successfully downloaded to the microcontroller (Figure 9).</li>
	</ol>
	</li>
	<li>Desolder the wires soldered to pads TP1, TP2 and TP3 (Figure 3) as well as wires soldered to pads 6 and 7 (Figure 5).</li>
	<li>Connect the PCB to all other electrical components except for the battery (Figure 10a).
	<ol>
		<li>Solder the wireless charging/communication coil to pads 2 and 3 according to Figure 8. The polarity is not important.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>Wind the antenna around the device in a spiral (Figure 10b)</li>
	<li>Cut a 32 mm long piece of a heat-shrinkable tubing with an internal diameter of 9.5 mm.</li>
	<li>Place the coil on the PCB. Refer to Figure 7b for the correct placement.</li>
	<li>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.</li>
	<li>Heat the tubing with a hot air gun to 150 &#176;C to shrink and then allow it to cool (Figure 10d).</li>
	<li>Apply epoxy glue to the left end to seal one side of the tubing (Figure 10e).</li>
	<li>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.</li>
	<li>Wait for at least 24 h for the glue to harden and fully cure.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Wireless Charger/Transmitter Design
<ol>
	<li>The PCB design is provided in the supplementary file &#34;gerber_transmitter.7z&#34;. Use the same manufacturing process as for the implantable device. The schematic diagram is provided in Figure 12.</li>
	<li>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 &#956;L.</li>
	<li>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 &#34;bom_transmitterdevice.csv&#34; for the assignment of components to their numbers.</li>
	<li>Use a PCB hot air gun station preset to 260 &#176;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.</li>
	<li>Repeat the steps 2.3&#8211;2.4&#160;for the bottom side of the device. Follow a similar procedure as during the manufacturing of the implantable device.</li>
	<li>Create a coil with 3 turns of AWG18 enameled wire (Figure 14c) and connect it to pads COIL1 and COIL2 (Figure 13).</li>
	<li>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.</li>
	<li>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.</li>
	<li>Plug in the PICkit 3 programmer into the USB port of a computer with MPLAB IPE software installed.</li>
	<li>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.
	<ol>
		<li>Run the MPLAB IPE v3.61. Select &#34;Settings | Advanced Mode&#34;.</li>
		<li>Into password box, enter the default password which is &#39;microchip&#39;. Click&#160;&#34;Log on&#34;. A tab with different panels on the left will appear.</li>
		<li>On the top left, click &#34;Operate&#34;, then in the top middle part of the screen, click the &#34;Device&#34; and type in &#34;PIC16LF1783&#34;. Click &#34;Apply&#34;.</li>
		<li>Select panel &#34;Power&#34; on the left</li>
		<li>Change the VDD voltage value to 3.3.</li>
		<li>Click the checkbox &#34;Power Target Circuit&#34; from &#34;Tool&#34;.</li>
		<li>Click the &#34;Operate&#34; tab on the left.</li>
		<li>Click &#34;Connect&#34;.</li>
		<li>Download the supplementary file &#34;IMPLANTABLE_V2_TRANSMITTER.X.production.hex&#34; and note its location on the hard drive. In the IPE software, find the Source line and click the &#34;Browse&#34; button near to it.</li>
		<li>Click &#34;Program&#34;. Wait till the software says that the software was downloaded to the microcontroller successfully.</li>
	</ol>
	</li>
	<li>Desolder the wires soldered to pads TP1 to TP5</li>
	<li>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.</li>
	<li>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.</li>
	<li>Open the PuTTY software and set it up (Figure 15).
	<ol>
		<li>Open the PuTTY software. Select &#34;Serial&#34; as connection type.</li>
		<li>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.</li>
		<li>Enter &#34;38400&#34; as Speed. Click &#34;Open&#34;. The charger/transmitter device is now ready to be used. Press H key for help.</li>
	</ol>
	</li>
</ol>
3. Endoscopic Implantation
<ol>
	<li>Use a live mini pig as an in vivo model, adult (8-36 months), 20-30 kg weight.
	<ol>
		<li>Let the pig fast for 24 h prior to the procedure.</li>
		<li>Allow clear liquids ad libitum.</li>
		<li>Administer intramuscular tiletamine (2 mg/kg), zolazepam (2 mg/kg) and ketamine (11 mg/kg) as a premedication.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Grasp the device externally with a snare. After that, insert it into the stomach, then release it.</li>
	<li>Extract the endoscope, equip it with a dissection cap (15.5 mm), and then re-insert it to the stomach.</li>
	<li>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).</li>
	<li>Make a horizontal incision to create an opening in the submucosa using an electrosurgical knife with a knob-shaped tip.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Use an over the scope clip to secure the device in place inside the submucosal pocket and prevent any migration or dislodging.</li>
</ol>
4. Experiment &#8212; After Implantation
<ol>
	<li>After successful implantation, place the charger/transmitter coil in proximity of the implanted device.</li>
	<li>Plug in the RTL2832 dongle in the PC.</li>
	<li>Run the HDSDR software and set the center frequency to 432 MHz.
	<ol>
		<li>Open the HDSDR software (Figure 15) for correct settings and PuTTY software (Figure 16). In the HDSDR software, click &#34;Options | Select Input | ExtIO&#34;.</li>
		<li>Select Bandwidth &#8212; &#34;960000&#34;. Select the LO frequency to 431.95 MHz. Select the Tune frequency to 432.00 MHz.</li>
	</ol>
	</li>
	<li>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).</li>
</ol>
5. Euthanasia after the Experiment
<ol>
	<li>Use an anesthetic overdose for euthanasia (lethal dose of thiopental and KCl).</li>
</ol>

<ol>
	<li>Abell, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+electrical+stimulation+for+medically+refractory+gastroparesis.">Gastric electrical stimulation for medically refractory gastroparesis.</a> Gastroenterology. 125 (2), 421-428 (2003).</li><li>O'Grady, G., Egbuji, J., Du, P., Cheng, L. K., Pullan, A. J., Windsor, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+gastric+electrical+stimulation+for+the+treatment+of+gastroparesis:+a+meta-analysis.">High-frequency gastric electrical stimulation for the treatment of gastroparesis: a meta-analysis.</a> World J Surg. 33 (8), 1693-1701 (2009).</li><li>Chu, H., Lin, Y., Zhong, L., McCallum, R. W., Hou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+high-frequency+gastric+electrical+stimulation+for+gastroparesis.">Treatment of high-frequency gastric electrical stimulation for gastroparesis.</a> J Gastroenterol Hepatol. 27 (6), 1017-1026 (2012).</li><li>Rodr&#237;guez, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+stimulation+therapy+of+the+lower+esophageal+sphincter+is+successful+in+treating+GERD:+final+results+of+open-label+prospective+trial.">Electrical stimulation therapy of the lower esophageal sphincter is successful in treating GERD: final results of open-label prospective trial.</a> Surg Endosc. 27 (4), 1083-1092 (2013).</li><li>Ellis, F., Berne, T. V., Settevig, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevention+of+experimentally+induced+reflux+by+electrical+stimulation+of+the+distal+esophagus.">The prevention of experimentally induced reflux by electrical stimulation of the distal esophagus.</a> Am J Surg. 115, 482-487 (1968).</li><li>Rinsma, N. F., Bouvy, N. D., Masclee, A. A. M., Conchillo, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+Stimulation+Therapy+for+Gastroesophageal+Reflux+Disease.">Electrical Stimulation Therapy for Gastroesophageal Reflux Disease.</a> J Neurogastroenterol. 20 (3), 287-293 (2014).</li><li>Medtronic Inc, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Enterra Therapy 3116 - Gastric Electrical Stimulation System. , (2016).</li><li>Rodriguez, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-year+results+of+intermittent+electrical+stimulation+of+the+lower+esophageal+sphincter+treatment+of+gastroesophageal+reflux+disease.">Two-year results of intermittent electrical stimulation of the lower esophageal sphincter treatment of gastroesophageal reflux disease.</a> Surgery. 157 (3), 556-567 (2015).</li><li>Hajer, J., Nov&#225;k, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+Autonomous+Endoscopically+Implantable+Submucosal+Microdevice+Capable+of+Neurostimulation+in+the+Gastrointestinal+Tract.">Development of an Autonomous Endoscopically Implantable Submucosal Microdevice Capable of Neurostimulation in the Gastrointestinal Tract.</a> Gastroent Res Pract. , 8098067 (2017).</li><li>Deb, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+innovative+techniques+for+the+endoscopic+implantation+and+securing+of+a+novel,+wireless,+miniature+gastrostimulator+(with+videos).">Development of innovative techniques for the endoscopic implantation and securing of a novel, wireless, miniature gastrostimulator (with videos).</a> Gastrointest. Endosc. 76 (1), 179-184 (2012).</li><li>Jiang, G., Zhou, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Technology advances and challenges in hermetic packaging for implantable medical devices. , (2017).</li><li>Vonthein, R., Heimerl, T., Schwandner, T., Ziegler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+stimulation+and+biofeedback+for+the+treatment+of+fecal+incontinence:+a+systematic+review.">Electrical stimulation and biofeedback for the treatment of fecal incontinence: a systematic review.</a> Int J Colorectal Dis. 28 (11), 1567-1577 (2013).</li></ol>

This work was supported by the Research Project PROGRES-Q28, and awarded by Charles University in Prague. The authors thank to Ass. Prof. Jan Mart&#237;nek, Ph.D. and PIGMOD centre.

The design of the implantable device should primarily focus on the overall size of the device, achievable stimulation profiles (maximum voltage, maximum deliverable current, length of pulses and pulse frequency). Main limitation from the hardware perspective is the size and availability of suitable components. To minimize the overall size, surface mount components are preferred because of their compact packaging. The best solution would be to integrate bare chip dies on the substrate. However, this is limited by both the availability of bare die packaging option for components and the accessibility of the wire bonding technology. Second important parameter is the battery. Lithium batteries are preferred because of its high energy density. Also, the nominal voltage of 3.7 V is beneficial. The major benefit of the presented hardware topology is its small size and minimum invasiveness. Compared to the current solutions7,8, the presented solution is a magnitude smaller and can be implanted directly to the submucosa, without need for external leads and subcutaneous implantation of the neurostimulator.
Except for the hardware itself, in future, additional attention needs to be given to the device enclosure. The first point is the biocompatibility and hermeticity11 to avoid possible rejection of the implant. The other is the fixation of the device in the submucosa to avoid unwanted migration of the implant.
The most critical steps during endoscopic implantation is the capturing of the device and its placement into the submucosal pocket. The limitation is the size of the pocket, which must be, from the observations, approximately at least twice as large as the device to be implanted. Next issue is the correct orientation of the implant inside the pocket. With the respect to the technical difficulty of the endoscopic procedure, this method is dedicated to experts with experience with tunnel dissection or peroral endoscopic myotomy (POEM).
The next problematic part is the closure of the pocket which is relatively difficult using the over the scope clip. However, the use of this type of clip prevents the migration and rejection of the device. Limitations of this technique from hardware point of view include the hardware development equipment to solder with required accuracy. The device is designed to withstand during the surgery and a short time afterwards. Thus, with current enclosure, it is not designed to stay for prolonged periods of time inside the body. Also, the material of the enclosure is not biocompatible which represents a high risk of rejection of the implant in case of a survival experiment. This technique can be further developed, especially in terms of the development of biocompatible and hermetic enclosure which is essential for survival model experiments. Next, the functionality of multiple integrated circuits can be concentrated into a single application specific integrated circuit. Similarly, smaller surface mount components can be used to make the device more compact. The next possible direction of this research may lead to development of novel endoscopical methods for the treatment of other gastrointestinal diseases such as GERD, incontinence or sphincter dysfunctions12.

Gastric dysmotility can be a sign of several relatively common diseases such as gastroparesis, which is usually characterized by a chronic progression and imposes rather severe consequences on the social, work-related, and physical status of the patient. Most cases of gastroparesis are usually diabetic or idiopathic in origin and are often resistant to available medication1. Patients afflicted with this condition most commonly present with nausea and repeated vomiting. Based on previous research, it is known that the application of high-frequency low-energetic electrical stimulation can help to effectively moderate and alleviate the symptoms of gastric dysmotility1,2.
Based on previous studies, it is proven that high-frequency gastric electrical stimulation can significantly improve the symptoms and gastric emptying3. It has also been shown that lower esophageal sphincter neurostimulator therapy is safe and effective for the treatment of gastroesophageal reflux disease (GERD), reducing the acid exposure and eliminating daily proton-pump inhibitor (PPI) usage without stimulation related adverse effects4. Before human trials, first studies were performed in animal models (canine models5). Based on these studies, electrical stimulation of the lower esophageal sphincter (LES, 20 Hz, pulse width of 3 ms) caused a prolonged contraction of the LES5. Similar effects of high (20 Hz, pulse width of 200 &#956;s) and low (6 cycles/min, pulse width of 375 ms) frequency electrical stimulation on LES in GERD patients were investigated. Both high and low frequency stimulation were effective6. However, currently, there are only two neurostimulation devices for gastric or esophageal stimulation available on the market7,8. In those devices, the electrodes can be implanted surgically, laparoscopically or robotically. The device itself is implanted subcutaneously. This requires general anaesthesia and have a bulky device fitted, using intramuscular catheters which allow for the stimulation of the gastric or esophageal muscle tissue. So, the option of using a wirelessly communicating device implanted into the gastric submucosal layer would represent a definite advantage and improvement in patient comfort. As stated in the previous research9,10, it was proven that an implantation of a miniature neurostimulator into submucosa is possible. For the endoscopic submucosal implantation, we use a technique called endoscopic submucosal pocketing (ESP), based on endoscopic submucosal tunnel dissection10. The goal of this research is to further improve this concept of an implantable neurostimulator, primarily in the scope of power management (specifically the wireless recharging capability), conformity with respective laws and regulations for wireless communication links in medical implantable devices and possibility of bipolar neurostimulation. Next, the presented microneurostimulator is capable of bidirectional communication and the stimulation parameters can be changed in real-time, even while the device is implanted.
This technique is suitable for teams with a therapeutic endoscopist experienced in endoscopic pocketing or tunnel dissections. Next, a hardware and embedded software designer with experience in building hardware prototypes with microcontrollers and radio frequency circuits using surface mount technology is needed. For building the hardware prototypes, a lab equipped with a reflow soldering station and basic equipment for electrical measurements (at least a digital multimeter, an oscilloscope, a spectrum analyzer and PICkit3 programmer) is required.

<table><tbody><tr><td>EIA 0402 ceramic capacitor 1.8 pF</td><td>AVX</td><td>04025U1R8BAT2A</td><td>1 pc</td></tr><tr><td>EIA 0402 ceramic capacitor 100 nF</td><td>TDK</td><td>CGA2B3X7R1H104K050BB</td><td>7 pcs</td></tr><tr><td>EIA 0402 ceramic capacitor 100 pF</td><td>Murata Electronics</td><td>GRM1555C1H101JA01D</td><td>1 pc</td></tr><tr><td>EIA 0402 thick film resistor 10 k&amp;Omega;</td><td>Vishay</td><td>CRCW040210K7FKED</td><td>1 pc</td></tr><tr><td>EIA 0402 ceramic capacitor 10 nF</td><td>Murata Electronics</td><td>GRM155R71C103KA01D</td><td>3 pcs</td></tr><tr><td>EIA 0402 ceramic capacitor 10 pF</td><td>Murata Electronics</td><td>GJM1555C1H100JB01D</td><td>3 pc</td></tr><tr><td>EIA 0402 ceramic capacitor 12 pF</td><td>Murata Electronics</td><td>GJM1555C1H120JB01D</td><td>2 pcs</td></tr><tr><td>EIA 0402 ceramic capacitor 18 pF</td><td>KEMET</td><td>C0402C180J3GACAUTO</td><td>2 pcs</td></tr><tr><td>EIA 0402 resistor 1 m&amp;Omega;</td><td>Vishay</td><td>MCS04020C1004FE000</td><td>2 pcs</td></tr><tr><td>EIA 0402 resistor 1 k&amp;Omega;</td><td>Yageo</td><td>RC0402FR-071KL</td><td>1 pc</td></tr><tr><td>EIA 0402 ceramic capacitor 1 nF</td><td>Murata Electronics</td><td>GRM1555C1H102JA01D</td><td>3 pcs</td></tr><tr><td>EIA 0603 ceramic capacitor 2.2 uF</td><td>Murata Electronics</td><td>GCM188R70J225KE22D</td><td>2 pcs</td></tr><tr><td>EIA 0402 resistor 220 k&amp;Omega;</td><td>Vishay</td><td>CRCW0402220KJNED</td><td>5 pcs</td></tr><tr><td>0805 22 uH inductor</td><td>TDK</td><td>MLZ2012N220LT000</td><td>1 pc</td></tr><tr><td>EIA 0402 resistor 330 k&amp;Omega;</td><td>Vishay</td><td>CRCW0402330KFKED</td><td>1 pc</td></tr><tr><td>EIA 0603 ceramic capacitor 4.7 uF</td><td>TDK</td><td>C1608X6S1C475K080AC</td><td>1 pc</td></tr><tr><td>EIA 0402 resistor 470 &amp;Omega;</td><td>Vishay</td><td>RCG0402470RJNED</td><td>1 pc</td></tr><tr><td>EIA 0402 resistor 470 k&amp;Omega;</td><td>Vishay</td><td>CRCW0402470KJNED</td><td>1 pc</td></tr><tr><td>EIA 0603 inductor 470 nH</td><td>Murata Electronics</td><td>LQW18ANR47G00D</td><td>1 pc</td></tr><tr><td>EIA 0402 resistor 47 k&amp;Omega;</td><td>Murata Electronics</td><td>CRCW040247K0JNED</td><td>2 pcs</td></tr><tr><td>27.0000 MHz crystal 5032</td><td>AVX / Kyocera</td><td>KC5032A27.0000CMGE00</td><td>1 pc</td></tr><tr><td>EIA 0402 capacitor 6.8 pF</td><td>Murata Electronics</td><td>GJM1555C1H6R8CB01D</td><td>1 pc</td></tr><tr><td>EIA 0402 inductor 82 nH</td><td>EPCOS / TDK</td><td>B82498F3471J</td><td>1 pc</td></tr><tr><td>ABS05 32.768 kHz crystal</td><td>ABRACON</td><td>ABS05-32.768KHZ-T</td><td>1 pc</td></tr><tr><td>CDBU00340-HF schottky diode</td><td>COMCHIP technology</td><td>CDBU00340-HF</td><td>2 pcs</td></tr><tr><td>CG-320S Li-Ion pinpoint battery</td><td>Panasonic</td><td>CG-320S</td><td>1 pc</td></tr><tr><td>HSMS282P schottky diode rectifier</td><td>Broadcom / Avago</td><td>HSMS-282P-TR1G</td><td>1 pc</td></tr><tr><td>MAX8570 step-up converter</td><td>Maxim Integrated</td><td>MAX8570EUT+T</td><td>1 pc</td></tr><tr><td>MICRF113 RF transmitter</td><td>Microchip Technology</td><td>MICRF113YM6-TR</td><td>1 pc</td></tr><tr><td>4.3 V Zener diode</td><td>ON Semiconductor</td><td>MM3Z4V3ST1G</td><td>1 pc</td></tr><tr><td>OPA237 operational amplifier</td><td>Texas Instruments</td><td>OPA237N</td><td>1 pc</td></tr><tr><td>PIC16LF1783 8-bit microcontroller</td><td>Microchip Technology</td><td>PIC16LF1783-I/ML</td><td>1 pc</td></tr><tr><td>TPS70628 low-drop regulator</td><td>Texas Instruments</td><td>TPS70628DBVT</td><td>1 pc</td></tr><tr><td>EIA 1206 thick film resistor 0 &amp;Omega;</td><td>Yageo</td><td>RC1206JR-070RL</td><td>2 pcs</td></tr><tr><td>EIA 0603 thick film resistor 0 &amp;Omega;</td><td>Yageo</td><td>RC0603JR-070RL</td><td>1 pc</td></tr><tr><td>EIA 0402 thick film resistor 100 k&amp;Omega;</td><td>Yageo</td><td>RC0402FR-07100KL</td><td>1 pc</td></tr><tr><td>EIA 0603 thick film resistor 100 k&amp;Omega;</td><td>Yageo</td><td>RC0603FR-07100KL</td><td>1 pc</td></tr><tr><td>EIA 0805 ceramic capacitor 100 nF</td><td>KEMET</td><td>C0805C104K5RAC7210</td><td>2 pcs</td></tr><tr><td>EIA 0402 thick film resistor 10 k&amp;Omega;</td><td>Yageo</td><td>RC0402JR-0710KL</td><td>1 pc</td></tr><tr><td>EIA 1206 ceramic capacitor 10 nF</td><td>Samsung</td><td>CL31B103KHFSW6E</td><td>2 pcs</td></tr><tr><td>EIA 0402 thick film resistor 1 k&amp;Omega;</td><td>Yageo</td><td>RC0402JR-071KL</td><td>2 pcs</td></tr><tr><td>EIA 0402 thick film resistor 220 &amp;Omega;</td><td>Yageo</td><td>RC0402JR-07220RL</td><td>2 pcs</td></tr><tr><td>EIA 0402 ceramic capacitor 220 nF</td><td>TDK</td><td>C1005X5R1C224K050BB</td><td>1 pc</td></tr><tr><td>EIA 1206 ceramic capacitor 22 nF</td><td>TDK</td><td>C3216X7R2J223K130AA</td><td>2 pcs</td></tr><tr><td>SMC B tantalum capacitor 22 uF</td><td>AVX</td><td>TPSB226K010T0700&amp;nbsp;</td><td>1 pc</td></tr><tr><td>EIA 0402 thick film resistor 27 &amp;Omega;</td><td>Yageo</td><td>RC0402FR-0727RL</td><td>2 pcs</td></tr><tr><td>EIA 1206 thick film resistor 3.3 &amp;Omega;</td><td>Yageo</td><td>RC1206JR-073K3L</td><td>3 pcs</td></tr><tr><td>SOT23 3.3V zener diode</td><td>ON Semiconductor</td><td>BZX84C3V3LT1G</td><td>1 pc</td></tr><tr><td>SMC A tantalum capacitor 4.7uF</td><td>KEMET</td><td>T491A475M016AT</td><td>2 pcs</td></tr><tr><td>EIA 0603 thick film resistor 470 &amp;Omega;</td><td>Yageo</td><td>RC0603JR-07470RL</td><td>2 pcs</td></tr><tr><td>EIA 1206 ceramic capacitor 470 nF</td><td>KEMET</td><td>C1206C471J5GACTU</td><td>3 pcs</td></tr><tr><td>Electrolytic capacitor 470 uF</td><td>Panasonic</td><td>EEE-1CA471UP</td><td>3 pcs</td></tr><tr><td>EIA 0402 ceramic capacitor 47 pF</td><td>AVX</td><td>04025A470JAT2A</td><td>2 pcs</td></tr><tr><td>0603 GREEN LED</td><td>Lite-On Inc.</td><td>LTST-C191KGKT</td><td>1 pc</td></tr><tr><td>0603 RED LED</td><td>Lite-On Inc.</td><td>LTST-C191KRKT</td><td>1 pc</td></tr><tr><td>16 MHz CX3225 crystal</td><td>EPSON</td><td>FA-238 16.0000MB-C3</td><td>1 pc</td></tr><tr><td>0805 ferrite bead</td><td>Wurth Electronics Inc.</td><td>742792040</td><td>1 pc</td></tr><tr><td>IR2110SO FET driver</td><td>Infineon Technologies</td><td>IR2110SPBF</td><td>1 pc</td></tr><tr><td>FT230XS USB to seri&amp;aacute;l converter</td><td>FTDI Ltd.</td><td>FT230XS-R</td><td>1 pc</td></tr><tr><td>Mini USB connector</td><td>EDAC Inc.</td><td>690-005-299-043</td><td>1 pc</td></tr><tr><td>PIC16F1783 8-bit microcontroller</td><td>Microchip Technology</td><td>PIC16F1783-I/ML</td><td>1 pc</td></tr><tr><td>REG1117 3.3 V regulator SOT223</td><td>Texas Instruments</td><td>REG1117-3.3/2K5</td><td>1 pc</td></tr><tr><td>Schottky SMB diode rectifier</td><td>STMicroelectronics</td><td>STPS3H100UF</td><td>1 pc</td></tr><tr><td>SMB package TVS diode</td><td>Littelfuse Inc.</td><td>1KSMBJ6V8</td><td>1 pc</td></tr><tr><td>IRLZ44NPBF N-channel MOSFET</td><td>Infineon Technologies</td><td>IRLZ44NPBF</td><td>2 pcs</td></tr><tr><td>RTL2832U receiver dongle</td><td>EVOLVEO</td><td>Mars</td><td>1 pc</td></tr><tr><td>PICkit 3</td><td>Microchip Technology</td><td>PICkit 3</td><td>1 pc</td></tr><tr><td>Mini USB to USB A cable</td><td>OEM</td><td>Mini USB to USB-A</td><td>1 pc</td></tr><tr><td>Printed circuit board, implantable device</td><td>---</td><td>Manufacture with the provided supplementary file</td><td>1 pc</td></tr><tr><td>Printed circuit board, transmitter/receiver device</td><td>---</td><td>Manufacture with the provided supplementary file</td><td>1 pc</td></tr><tr><td>Printed circuit board, implantable device</td><td>---</td><td>Manufacture with the provided supplementary file</td><td>1 pc</td></tr><tr><td>AWG18 wire</td><td>Alpha Wire</td><td>3055 BK001</td><td>2 m</td></tr><tr><td>AWG42 wire</td><td>Daburn Electronics</td><td>2420/42 BK-100</td><td>1 m</td></tr><tr><td>Olympus GIFQ-160</td><td>Olympus</td><td>N/A (part is obsoleted)</td><td>1 pc</td></tr><tr><td>Single-use electrosurgical knife with knob-shaped tip and integrated jet function</td><td>Olympus</td><td>KD-655L</td><td>1 pc</td></tr><tr><td>Single-use oval electrosurgical snare</td><td>Olympus</td><td>SD-210U-15</td><td>1 pc</td></tr><tr><td>15.5 mm lens hood</td><td>FujiFilm</td><td>DH-28GR</td><td>1 pc</td></tr><tr><td>Injection therapy needle catheter</td><td>Boston Scientific</td><td>25G</td><td>1 pc</td></tr><tr><td>Alligator law grasping forceps</td><td>Olympus</td><td>FG-6L-1</td><td>1 pc</td></tr><tr><td>Instant Mix 5 min epoxy</td><td>Loctite</td><td>N/A</td><td>1 pc</td></tr><tr><td>Heat shrinkable tubing, inside diameter 9.5 mm</td><td>TE Connectivity</td><td>RNF-100-3/8-X-STK</td><td>1 pc</td></tr><tr><td>ChipQuik solder paste</td><td>Chip Quik</td><td>SMD4300AX10</td><td>1 pc</td></tr></tbody></table>

autonomous and rechargeable microneurostimulator endoscopically implantable into the submucosa

Gastric dysmotility can be a sign of common diseases such as longstanding diabetes mellitus. It is known that the application of high-frequency low-energetic stimulation can help to effectively moderate and alleviate the symptoms of gastric dysmotility. The goal of the research was the development of a miniature, endoscopically implantable device to a submucosal pocket. The implantable device is a fully customized electronic package which was specifically designed for the purpose of experiments in the submucosa. The device is equipped with a lithium-ion battery which can be recharged wirelessly by receiving an incident magnetic field from the charging/transmitting coil. The uplink communication is achieved in a MedRadio band at 432 MHz. The device was endoscopically inserted into the submucosal pocket of a live domestic pig used as an in vivo model, specifically in the stomach antrum. The experiment confirmed that the designed device can be implanted into the submucosa and is capable of bidirectional communication. The device can perform bipolar stimulation of muscle tissue.

Figure 17 shows that an endoscopic placement of the gastric neurostimulator into a pocket in submucosa as well as proper placement of the electrodes to the muscular layer was successful. The dimensions of the device (Figure 10) are 35 x 15 x 5 mm3 while the weight is 2.15 g. Figure 17 shows the circuit diagram of the device showing that the device comprises of 6 different modules which are connected together. Figure 3 shows the PCB layout and component placement in the device. Figure 18 shows that in order to implant the device into the submucosal layer, a technique called an endoscopic submucosal pocket9,10 (ESP) was used. The stimulator was attached near the muscular layer (muscularis propria) where it is theoretically the optimal stimulation depth. Creating the submucosal pocket and implanting of the gastric neurostimulator endoscopically took 20&#8211;30 min. During this procedure, there is no intraprocedural complication such as perforation or severe bleeding. Migration of the device in the stomach could not be determined because the experiment was non-survival. After the implantation, bidirectional communication link with the implantable device was established with an external device shown in Figure 14. The approximate distance between the charger/programmer coil and the implant was 10 cm. The achieved signal-to-noise (SNR) ratio with RTL2832 based software-defined-radio (SDR) receiver was over 40 dB.&#160;
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57268/57268fig1v2.jpg" /> 
Figure 1: Schematic diagram of the implantable device. The Figure shows how different components and circuit parts are connected in the implantable device. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/57268fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57268/57268fig2v2.jpg" /> 
Figure 2: Fabrication of the implantable device - PCB assembly. (a) PCB, top view. (b) Solder paste applied to top layer. (c) An example of hand placement of 0402 capacitor. (d) Solder paste applied to bottom layer. (e) Fully populated top side of the PCB. (f) Fully populated bottom side of the PCB <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/57268fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57268/57268fig3v2.jpg" /> 
Figure 3: Design of the implantable device. (a) Top copper layer of the PCB. (b) Component names on the top layer. (c) Bottom copper layer of the PCB. (d) Component names on the bottom layer. (e) Composite picture of all PCB layers <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/57268fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57268/57268fig4v2.jpg" /> 
Figure 4: Fabrication of the implantable device &#8212; preparation of other parts. (a) Hot air flow of the bottom side of the PCB. (b) Programming wires soldered to the PCB. (c) PCB connected to the programmer. (d) Wireless charging coil. (e) 432 MHz antenna. (f) Stimulation electrodes with two wires attached <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/57268fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57268/57268fig5v2.jpg" /> 
Figure 5: Recommended solder joint placement for the external components of the implantable device. The picture shows where the coil, antenna, battery and electrodes should be soldered. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/57268fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57268/57268fig6v2.jpg" /> 
Figure 6: Establishing a connection with the implantable device &#8212; important settings mentioned in the text are marked with red arrows. This picture is from the MPLAB IPE software, a screen which shows how to determine that the microcontroller inside the implantable device is correctly communicating with the PICkit programmer is provided. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/57268fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57268/57268fig7v2.jpg" /> 
Figure 7: Power settings of the software used for programming &#8212; important settings mentioned in the text are marked with red arrows. This is picture from the MPLAB IPE software. It shows how to properly power the implantable device for programming <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/57268fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57268/57268fig8v2.jpg" /> 
Figure 8: Choosing a correct programming file for the implantable device. The picture shows which button to click in order to load the supplementary .hex file correctly. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/57268fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/57268/57268fig9v2.jpg" /> 
Figure 9: Process of programming the firmware into the implantable device. The picture shows which button to press to program the software into the implantable device. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/57268fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/57268/57268fig10v2.jpg" /> 
Figure 10: Fabrication of the implantable device &#8212; final assembly. (a) Wireless charging coil, stimulation electrodes and antenna soldered to the PCB, together with battery. (b) Stacked implant. (c) Transparent heat shrinkable tubing put over the PCB. (d) Shrinking of the tubing with hot air. (e) Tubing fully shrinked and ends glued. (f) Finalized implantable device <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/57268fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/57268/57268fig11v2.jpg" /> 
Figure 11: Typical output stimulation pattern of the device as displayed on the DSOX1102G oscilloscope. After programming of the implantable device, soldering of the electrodes and the battery, output stimulation pattern similar to the one displayed in the figure should appear at the electrodes. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/57268fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/57268/57268fig12v2.jpg" /> 
Figure 12: Schematic diagram of the wireless charger/transmitter device. The figure is analogical to&#160;Figure 1.&#160;Shown here is the internal workings of the wireless charger/transmitter device <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/57268fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/57268/57268fig13v2.jpg" /> 
Figure 13: Design of the charger/transmitter device. (a) Top copper layer of the PCB. (b) Component names on the top layer. (c) Bottom copper layer of the PCB. (d) Component names on the bottom layer. (e) Composite picture of all PCB layers <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/57268fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/57268/57268fig14v2.jpg" /> 
Figure 14: Fabrication of the wireless charger/transmitter device. (a) Completed PCB, top side (b) Completed bottom side of the PCB (c) Mechanical design of the wireless transmitter/charger coil (d) One possible embodiment of the finalized charger/transmitter device <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/57268fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 15" class="xfigimg" src="/files/ftp_upload/57268/57268fig15v2.jpg" /> 
Figure 15: Correct settings of the HDSDR software. The HDSDR software is used together with the RTL2832U based USB receiving dongle as a spectrum analyzer to display the radio spectrum. In this case, it is used to receive the answer from the implant transmitted at approximately 432 MHz. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/57268fig15large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 16" class="xfigimg" src="/files/ftp_upload/57268/57268fig16v2.jpg" /> 
Figure 16: Correct settings of the PuTTY software. The PuTTY software is used for communication with the charger/transmitter device. It must be correctly configured in order to display correct data to the user. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/57268fig16large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 17" class="xfigimg" src="/files/ftp_upload/57268/57268fig17v2.jpg" /> 
Figure 17: Endoscopical implantation of the implantable device and checking if it is working. (a) In vivo model in animal endoscopic unit. (b) Insertion of the endoscope by the standard way into the in vivo model. (c) Implantable device prototype grasped with a snare. (d) Process of establishing bidirectional wireless link with the implantable device. (e) HDSDR software. (f) Detail of OOK modulated data transmitted by the implant. (g) X-ray &#8212; device position check. (h) X-ray scan of the implant area, the device as well as over the scope clip is clearly visible. (i) Detailed device view. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/57268fig17large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 18" class="xfigimg" src="/files/ftp_upload/57268/57268fig18v2.jpg" /> 
Figure 18: View of device implantation and endoscopic technique. (a) Submucosal injection with methylene blue. (b) Submucosal incision (an entrance for the submucosal pocket formation). (c) Tunnelisation of the submucosal pocket. (d-f) Disrupting, dilating, and dissecting the submucosal layer. (g,&#160;h) Device implantation. (i) Closing the entry with over the scope clip. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/57268fig18large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: gerber_implant.7z. Zip archive with files required to manufacture the printed circuit board of the implantable device. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/gerber_implant.7z" target="_blank">Please click here to download this file</a>
Supplementary File 2: gerber_transmitter.7z. Zip archive with files required to manufacture the printed circuit board of the charger/transmitter device. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/gerber_transmitter.7z" target="_blank">Please click here to download this file</a>
Supplementary File 3: gerber_electrodes.7z. Zip archive with files required to manufacture the electrodes. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/gerber_electrodes.7z" target="_blank">Please click here to download this file</a>
Supplementary File 4: IMPLANTABLE_V2.X.production.hex. Firmware for the implantable device. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/IMPLANTABLE_V2.X.production.hex" target="_blank">Please click here to download this file</a>
Supplementary File 5: IMPLANTABLE_V2_TRANSMITTER.X.production.hex. Firmware for the charger/transmitter device. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/IMPLANTABLE_V2_TRANSMITTER.X.production.hex" target="_blank">Please click here to download this file</a>
Supplementary File 6: bom_implantabledevice.csv. Bill of material (BOM) file describing the assignment of component values to specific components on the PCB of the implantable device. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/bom_implantabledevice.csv" target="_blank">Please click here to download this file</a>
Supplementary File 7: bom_transmitterdevice.csv. BOM file describing the assignment of component values to specific components on the PCB of the charger/transmitter device. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57268/bom_transmitterdevice.csv" target="_blank">Please click here to download this file</a>

Watch this Scientific Journal Video about Autonomous and Rechargeable Microneurostimulator Endoscopically Implantable into the Submucosa at JoVE.com

Autonomous and Rechargeable Microneurostimulator Endoscopically Implantable into the Submucosa

The application of high-frequency low-energetic stimulation can alleviate the symptoms of gastric dysmotility. In this research, a miniature, endoscopically implantable and wirelessly rechargeable device which is implanted into a submucosal pocket is presented. Successful both-way communication and stimulation control were achieved during an experiment on live pig.

Gastric dysmotility can be a sign of common diseases such as longstanding diabetes mellitus. It is known that the application of high-frequency ...

autonomous-rechargeable-microneurostimulator-endoscopically

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.4K Views.  Charles University, Prague, Czech Republic. Het belangrijkste voordeel van deze techniek is dat het een aanzienlijk minder invasieve aanpak biedt. Over het algemeen, individuen nieuw op deze methode zal worstelen, omdat de implanteerbare apparaat ontwikkeling is een discipline die ervaring vereist, zowel in hardware en software ontwerp. Visuele demonstratie van deze methode is van cruciaal belang omdat de endoscopische submucosale implantatie een experimentele procedure is die momenteel niet in de dagelijkse praktijk wordt gebruikt.

Video: Autonomous and Rechargeable Microneurostimulator Endoscopically Implantable into the Submucosa

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

Construction of a Wireless-Enabled Endoscopically Implantable Sensor for pH Monitoring with Zero-Bias Schottky Diode-based Receiver

Ambulatory pH monitoring of pathological reflux is an opportunity to observe the relationship between symptoms and exposure of the esophagus to acidic or non-acidic refluxate. This paper describes a method for the development, manufacturing, and implantation of a miniature wireless-enabled pH sensor. The sensor is designed to be implanted endoscopically with a single hemostatic clip. A fully passive rectenna-based receiver based on a zero-bias Schottky diode is also constructed and tested. To construct the device, a two-layer printed circuit board and off-the-shelf components were used. A miniature microcontroller with integrated analog peripherals is used as an analog front end for the ion-sensitive field-effect transistor (ISFET) sensor and to generate a digital signal which is transmitted with an amplitude shift keying transmitter chip. The device is powered by two primary alkaline cells. The implantable device has a total volume of 0.6 cm3 and a weight of 1.2 grams, and its performance was verified in an ex vivo model (porcine esophagus and stomach). Next, a small footprint passive rectenna-based receiver which can be easily integrated either into an external receiver or the implantable neurostimulator, was constructed and proven to receive the RF signal from the implant when in proximity (20 cm) to it. The small size of the sensor provides continuous pH monitoring with minimal obstruction of the esophagus. The sensor could be used in routine clinical practice for 24/96 h esophageal pH monitoring without the need to insert a nasal catheter. The &#34;zero-power&#34; nature of the receiver also enables the use of the sensor for automatic in-vivo calibration of miniature lower esophageal sphincter neurostimulation devices. An active sensor-based control enables the development of advanced algorithms to minimize the used energy to achieve a desirable clinical outcome. One of the examples of such an algorithm would be a closed-loop system for on-demand neurostimulation therapy of gastroesophageal reflux disease (GERD).

The manuscript presents a miniature implantable pH sensor with ASK modulated wireless output together with a fully passive receiver circuit based on zero-bias Schottky diodes. This solution can be used as a basis in the development of in vivo calibrated electrostimulation therapy devices and for ambulatory pH monitoring.

Ambulatory pH monitoring of pathological reflux is an opportunity to observe the relationship between symptoms and exposure of the esophagus to acidic or ...

Bioengineering

Intra-Operative Neural Monitoring of Thyroid Surgery in a Porcine Model

Intraoperative injury to the recurrent laryngeal nerve (RLN) can cause vocal cord paralysis, which interferes with speech and can potentially interfere with breathing. In recent years, intraoperative neural monitoring (IONM) has been widely adapted as an adjunct technique to localize the RLN, detect RLN injury, and predict vocal cord function during the operations. Many studies have also used animal models to investigate new applications of IONM technology and to develop reliable strategies for preventing intraoperative RLN injury. The aim of this article is to introduce a standard protocol for using a porcine model in IONM research. The article demonstrates the procedures for inducing general anesthesia, performing tracheal intubation, and experimental design to investigate the electrophysiological characteristics of RLN injuries. Applications of this protocol can improve overall efficacy in implementing the 3R principle (replacement, reduction and refinement) in porcine IONM studies.

This study aims to develop a standard protocol of intra-operative neural monitoring of thyroid surgery in a porcine model. Here, we present a protocol to demonstrate general anesthesia, to compare different types of electrodes, and to investigate the electrophysiological characteristics of the normal and injured recurrent laryngeal nerves.

Intraoperative injury to the recurrent laryngeal nerve (RLN) can cause vocal cord paralysis, which interferes with speech and can potentially interfere ...

Medicine

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas.
The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable&#160;electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days.
This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of ...

An Implantable System For Chronic In Vivo Electromyography

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in assessing neuromuscular function. Chronic recording of EMG activity reflecting a muscle&#8217;s reinnervation status after nerve injury has been limited, due to the invasive nature of traditional EMG recording techniques. In this regard, an implantable system is designed for long-term, in vivo EMG recording and nerve stimulation. It has been applied and tested in a study on reinnervation of laryngeal muscles. This system consists of 1) two bipolar electrode nerve cuffs and leads for stimulating each of two nerves: the recurrent laryngeal nerve (RLN) and internal branch of the superior laryngeal nerve (SLN); 2) two EMG recording electrodes and leads for each of the two laryngeal muscles: posterior cricoarytenoid (PCA) muscle and thyroarytenoid-lateral cricoarytenoid (TA-LCA) muscle complex; and 3) a skin receptacle interfacing all implanted lead terminals to an external recording preamplifier and stimulator using a connection cable. The wire leads are Teflon-coated, multi-filament, type 316 stainless steel. They are coiled and can stretch during body movement of the awake animal to prevent lead breakage and electrode migration. This system is implanted during an aseptic surgery. Afterwards, baseline EMG recordings are performed before the RLN is transected in the second surgery to study muscle reinnervation. Throughout the study, multiple physiological sessions are conducted in the anesthetized animal to obtain evoked and spontaneous EMG activity that reflects the reinnervation status of laryngeal muscles. The system is compact, free of infection over the course of the study, and highly durable. This implantable system can provide a reliable platform for research in which long-term recording or nerve stimulation is required in an anesthetized or freely moving animal.&#160;

Presented here is a protocol for the manufacturing of an implantable system for in vivo chronological recording of evoked and spontaneous electromyographic potentials. The system is applied to the investigation of reinnervation of laryngeal muscles following nerve injury. &#160;&#160;

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in ...

Microinjectrode System for Combined Drug Infusion and Electrophysiology

This microinjectrode system is designed for drug infusion, electrophysiology, and delivery and retrieval of experimental probes, such as microelectrodes and nanosensors, optimized for repeated use in awake, behaving animals. The microinjectrode system can be configured for multiple purposes: (1) simple arrangement of the cannula for placement of an experimental probe that would otherwise be too fragile to penetrate the dura mater, (2) microfluidic infusion of a drug, either independently or coupled to a cannula containing an experimental probe (i.e., microelectrode, nanosensor). In this protocol we explain the step by step construction of the microinjectrode, its coupling to microfluidic components, and the protocol for use of the system in vivo. The microfluidic components of this system allow for delivery of volumes on the nanoliter scale, with minimal penetration damage. Drug infusion can be performed independently or simultaneously with experimental probes such as microelectrodes or nanosensors in an awake, behaving animal. Applications of this system range from measuring the effects of a drug on cortical electrical activity and behavior, to understanding the function of a specific region of cortex in the context of behavioral performance based on probe or nanosensor measurements. To demonstrate some of the capabilities of this system, we present an example of muscimol infusion for reversible inactivation of the frontal eye field (FEF) in rhesus macaque during a working memory task.

We present a microinjectrode system designed for electrophysiology and assisted delivery of experimental probes (i.e., nanosensors, microelectrodes), with optional drug infusion. Widely available microfluidic components are coupled to a cannula containing the probe. A step-by-step protocol for microinjectrode construction is included, with results during muscimol infusion in macaque cortex.

This microinjectrode system is designed for drug infusion, electrophysiology, and delivery and retrieval of experimental probes, such as microelectrodes ...

Implantation and Control of Wireless, Battery-free Systems for Peripheral Nerve Interfacing

Peripheral nerve interfaces are frequently used in experimental neuroscience and regenerative medicine for a wide variety of applications. Such interfaces can be sensors, actuators, or both. Traditional methods of peripheral nerve interfacing must either tether to an external system or rely on battery power that limits the time frame for operation. With recent developments of wireless, battery-free, and fully implantable peripheral nerve interfaces, a new class of devices can offer capabilities that match or exceed those of their wired or battery-powered precursors. This paper describes methods to (i) surgically implant and (ii) wirelessly power and control this system in adult rats. The sciatic and phrenic nerve models were selected as examples to highlight the versatility of this approach. The paper shows how the peripheral nerve interface can evoke compound muscle action potentials (CMAPs), deliver a therapeutic electrical stimulation protocol, and incorporate a conduit for the repair of peripheral nerve injury. Such devices offer expanded treatment options for single-dose or repeated dose therapeutic stimulation and can be adapted to a variety of nerve locations.

This is a protocol for the surgical implantation and operation of a wirelessly powered interface for peripheral nerves. We demonstrate the utility of this approach with examples from nerve stimulators placed on either the rat sciatic or phrenic nerve.

Peripheral nerve interfaces are frequently used in experimental neuroscience and regenerative medicine for a wide variety of applications. Such interfaces ...

A Cost-Effective, Versatile Probe Implant System for Electrophysiology in Mice

The DREAM Implant: A Lightweight, Modular, and Cost-Effective Implant System for Chronic Electrophysiology in Head-Fixed and Freely Behaving Mice

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. However, current methods for chronically implanting probes present steep trade-offs between cost, ease of use, size, adaptability, and long-term stability.
This protocol introduces a novel chronic probe implant system for mice called the DREAM (Dynamic, Recoverable, Economical, Adaptable, and Modular), designed to overcome the trade-offs associated with currently available options. The system provides a lightweight, modular and cost-effective solution with standardized hardware elements that can be combined and implanted in straightforward steps and explanted safely for recovery and multiple reuse of probes, significantly reducing experimental costs.
The DREAM implant system integrates three hardware modules: (1) a microdrive that can carry all standard silicon probes, allowing experimenters to adjust recording depth across a travel distance of up to 7 mm; (2) a three-dimensional (3D)-printable, open-source design for a wearable Faraday cage covered in copper mesh for electrical shielding, impact protection, and connector placement, and (3) a miniaturized head-fixation system for improved animal welfare and ease of use. The corresponding surgery protocol was optimized for speed (total duration: 2 h), probe safety, and animal welfare.
The implants had minimal impact on animals&#39; behavioral repertoire, were easily applicable in freely moving and head-fixed contexts, and delivered clearly identifiable spike waveforms and healthy neuronal responses for weeks of post-implant data collection. Infections and other surgery complications were extremely rare.
As such, the DREAM implant system is a versatile, cost-effective solution for chronic electrophysiology in mice, enhancing animal well-being, and enabling more ethologically sound experiments. Its design simplifies experimental procedures across various research needs, increasing accessibility of chronic electrophysiology in rodents to a wide range of research labs.

Here, we introduce a lightweight, cost-effective probe implant system for chronic electrophysiology in rodents optimized for ease of use, probe recovery, experimental versatility, and compatibility with behavior.

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. ...

Automated Periodic Stimulation Electrode Installation: A Technique to Surgically Implant An Automated Stimulation Device on the Vagus Nerve in a Porcine Model

Source: Wu, C. W., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/57919">Intra-Operative Neural Monitoring of Thyroid Surgery in a Porcine Model.</a> J. Vis. Exp. (2019).
In this video, we demonstrate the procedure for the installation of an automated periodic stimulation electrode on the vagus nerve for monitoring nerves during thyroid surgeries in a porcine model. This helps in the correct identification of the target nerve during surgical procedures.

Source: Wu, C. W., et al. Intra-Operative Neural Monitoring of Thyroid Surgery in a Porcine Model. J. Vis. Exp. (2019).
In this video, we demonstrate the ...

Autonomous and Rechargeable Microneurostimulator Endoscopically Implantable into the Submucosa

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video