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Jan 2021
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Optogenetic Targeting of Mouse Vagal Afferents Using an Organ-specific, Scalable, Wireless Optoelectronic Device
使用器官特异性、可扩展的无线光电设备对小鼠迷走神经传入进行光遗传学靶向研究   

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Abstract

Optogenetics has the potential to transform the study of the peripheral nervous system (PNS), but the complex anatomy of the PNS poses unique challenges for the focused delivery of light to specific tissues. This protocol describes the fabrication of a wireless telemetry system for studying peripheral sensory pathways. Unlike existing wireless approaches, the low-power wireless telemetry offers organ specificity via a sandwiched pre-curved tether, and enables high-throughput analysis of behavioral experiments with a channel isolation strategy. We describe the technical procedures for the construction of these devices, the wireless power transmission (TX) system with antenna coils, and their implementation for in vivo experimental applications. In total, the timeline of the procedure, including device fabrication, implantation, and preparation to begin in vivo experimentation can be completed in ~2-4 weeks. Implementation of these devices allows for chronic (>1 month) wireless optogenetic manipulation of peripheral neural pathways in freely behaving animals navigating homecage environments (up to 8).

Keywords: Wireless optogenetics (无线光遗传学), Vagal sensory pathway (迷走神经感觉通路), Device implantation (装置植入), Implantable optoelectronics (植入式光电器件), Wireless power transmission (无线电力传输)

Background

Optogenetics has transformed neuroscience by enabling the targeted manipulation of genetically defined neural cell types (Deisseroth, 2011 and 2015). This typically involves expressing a light-activated ion channel using a Cre driver mouse and implanting an optical fiber in the brain (Park et al., 2015b, 2015a and 2016). In this way, it is possible to activate or inactivate specific cell types with light and thereby test their causal role in behavior (Kim et al., 2013; Montgomery et al., 2015; Shin et al., 2017).


Optogenetics also has the potential to transform the study of the PNS, but the complex anatomy of the PNS poses unique challenges for the focused delivery of light to specific tissues (Towne et al., 2013; Montgomery et al., 2016; Maimon et al., 2018). The PNS consists of sensory and motor neurons, with cell bodies located in widely distributed peripheral ganglia. The major sensory ganglia include dorsal root ganglia, which consist of clusters of cell bodies of spinal sensory neurons that are organized bilaterally along the length of the spine, and nodose ganglia, which contain the cell bodies of vagal sensory neurons (Berthoud and Neuhuber, 2000).


These two sensory systems broadly innervate a range of peripheral organs that differ in their size, shape, location, and accessibility, necessitating individualized strategies for light delivery to different tissues (Berthoud, 2008; de Lartigue, 2016; Williams et al., 2016). Moreover, some of these organs are highly mobile during normal physiology or behavior (e.g., movement of the intestine during digestion), which further complicates the positioning and anchoring of optical fibers. Thus, unlike in the brain, it is a considerable challenge to develop robust strategies for the delivery of light selectively to peripheral tissues in freely behaving animals.


In principle, it is possible to target peripheral tissues with light using a wired fiber optic that stimulates the animal via the back or rectum (Caggiano et al., 2016; Hibberd et al., 2018), analogous to the intracranial fiber optics commonly used in the brain. While this approach has the advantage that it can be readily coupled with off-the-shelf components (e.g., lasers and LEDs), the abdominal viscera differs from the brain, in that it lacks a stable interface for securing large fiber optics and preventing motion. Moreover, the inflexible nature of most optical fibers can cause the shearing of tissues and nerves, or of the fiber itself, during normal animal movement. For this reason, wired fiber optics have been used less frequently for peripheral optogenetics in behaving animals (Maimon et al., 2017 and 2018; Towne et al., 2013).


An alternative approach is to implant a wireless probe that internalizes the light source, thereby bypassing physical constraints associated with fiber optic cables. Strategies for such wireless, radio-frequency(RF)-powered devices have recently been described (Montgomery et al., 2015 and 2016; Park et al., 2015a and 2015b), but it remains a major challenge to achieve long-term, organ-restricted illumination, without impeding normal behavior or physiology. For example, a wirelessly powered micro-light emitting diode (µLED) has been secured to the rat bladder using a circumferential elastomer sleeve (Mickle et al., 2019), but this device impedes normal organ expansion. A wireless flexible device that sutures a µLED onto the heart surface has been reported (Gutruf et al., 2019), but it has not been demonstrated to function for more than eight days, precluding most behavioral studies. More generally, affixing any µLED to a target organ surface results in light back-scatter and non-specific optogenetic illumination of nearby tissues.


Recently, we developed and published a new class of implantable wireless devices, demonstrating their applicability to study the vagus nerve (Kim et al., 2021). This new wireless optogenetic implant delivers light to nerve endings from inside the stomach, via a pre-curved tether. This intragastric light delivery enables illumination of stomach innervating nerves, with much greater specificity than µLEDs affixed to the organ surface, and could in principle be extended to study other organs, such as the intestine. Here, we describe the technical procedures for the fabrication of these devices and their in vivo applications.

Materials and Reagents

  1. Pipet tips (VWR, catalog number: 613-0239)

  2. Glass slide (Brain Research Laboratories, catalog numbers: 5075 and 3040)

  3. Copper/Polyimide (Cu/PI) film (DupontTM Pyralux®, catalog number: AC181200RY)

  4. Wescorp anti-static high-temp Polyimide tape (Desco, catalog number: 81270)

  5. Photoresistor (AZ®, catalog number: AZ1518)

  6. Cored wire for soldering (Multicore, catalog number: 397952)

  7. Electrical components [Supplementary Figure 1 from previous work (Kim et al., 2021)]

  8. Gauze (Medique, catalog number: 60673)

  9. 6-0 Silk suture (Braintree Scientific, catalog number: NC9201232)

  10. 3-0 PGA sutures (Pro Advantage, catalog number: P420493)

  11. 5-0 PGA suture (Stoelting, catalog number: 50495)

  12. 4-0 Nylon sutures (Pro Advantage, catalog number: P420661)

  13. Betadine (Fisher Scientific, catalog number: 19-027132)

  14. 70% Isopropyl Alcohol (Fisher, catalog number: 19-027026)

  15. 2-propanol (Fisher Chemical, catalog number: 190971)

  16. Methyl Alcohol, Anhydrous (Macron, catalog number: 3016-16)

  17. Acetone (Macron, catalog number: 2440-16)

  18. Developer solution (AZ®, AZ Developer 1:1)

  19. Copper etchant (Alfa AesarTM, catalog number: Z03E099)

  20. Flux no-clean lead-free (Chip Quik, Inc., catalog number: SMD291NL)

  21. Polydimethylsiloxane (PDMS) (Dow®, SylgardTM 184 Silicone Elastomer Kit)

  22. DAPI Fluoromount-G (SouthernBiotech, catalog number: 0100-20)

  23. Ketoprofen (Sigma, catalog number: K2012-5G)

  24. Isoflurane (Piramal NDC 66794-017-10)

  25. Material cleaning (see Recipes)

  26. Electrical sample cleaning (see Recipes)

  27. Photoresistor spin coating (see Recipes)

  28. UV-lithography and wet-etching (see Recipes)

  29. PDMS preparation (see Recipes)

Equipment

  1. Scissors (Fine Science Tools, catalog number: 14090-09)

  2. Magnetic retractors (Fine Science Tools, catalog number: 18200-20)

  3. Spatula (Fine Science Tools, catalog number: 10091-12)

  4. Graefe knife (Fine Science Tools, catalog number: 10059)

  5. Graefe forceps (Fine Science Tools, catalog number: 11153-10)

  6. Bonn artery scissors, ball tip (Fine Science Tools, catalog number: 14086-09)

  7. Ring forceps (Fine Science Tools, catalog number: 11103-09)

  8. 32G needle (Hamilton, 75SN syringe, catalog number: 87908)

  9. Fine-tipped Dumont forceps (Fine Science Tools, catalog number: 11251-20)

  10. Flat-tipped forceps (Fine Science Tools, catalog number: 11220-21)

  11. Dumont forcep (Fine Science Tools, Dumont #5SF Forceps)

  12. Sterile disposable scalpels (TruMed©, 190618)

  13. Spin-coat (SCS, G3P-8)

  14. Isotemp stirring hotplate (Fisher Scientific, SP88850200)

  15. UV lithography (EV Group, EVG610)

  16. Stereo microscope (AVEN, SPZT 50)

  17. Soldering machine (Weller, WD1002/WP80)

  18. Milligram balance (Intelligent Weighing Technology, PM-300)

  19. Vacuum pump (Across International, EasyVac-9)

  20. Vacuum oven (Across International, Accu Temp 1.9)

  21. Oven (Quincy La, Inc., Model 30 Lab Oven)

  22. RF generator (FEIG Electronic, ID ISC.LRM2500-A)

  23. Dynamic antenna tuner, matching board (FEIG Electronic, ID ISC.DAT)

  24. Nanoject II Auto-Nanoliter Injector (Drummond Scientific Company, 3-000-204)

  25. Laser-scanning confocal microscope (Olympus, FV1200)

  26. Epifluorescent microscope (Nikon, Eclipse E600)

Software

  1. ISO Start2017 v9.9.10 (FEIG Electronic GmbH)

  2. BioDAQ Viewer v. 2.2.01 (Research Diets)

Procedure

  1. Pattern preparation at cleanroom facility (Video 1)

    1. Prepare a flexible Cu/PI bilayer film, mounted onto a glass slide, and secured by polyimide tape (Figure 1A).

    2. Follow the material cleaning recipe (see Recipe 1).

    3. Deposit a 2.5 μm thickness photoresist layer on the Cu/PI film, using a spin coater.

    4. Bake the sample for 1 min at 105°C.

    5. Wait for the surface of the sample to cool to room temperature for 3 min.

    6. Follow UV lithography and wet-etching recipe (Figure 1B–1C and see Recipe 4).

    7. Follow the material cleaning recipe (see Recipe 1).


    Video 1. Fabrication of the device substrate


  2. Electrical components soldering at a standard laboratory facility (Video 2)

  1. Mount components on the pattern, using a soldering machine [Supplementary Figure 1 from previous work (Kim et al., 2021)].

  2. Follow the electrical sample cleaning recipe (see Recipe 2).

  3. Use a scalpel to cut the boundary of the sample from the Cu/PI bilayer film (Figure 1F–1H).

  4. Confirm device operation within the wireless power transfer system (Figure 1G).


    Video 2. Device fabrication


  1. Encapsulation at a standard laboratory facility (Video 2)

    1. Follow PDMS preparation recipe (see Recipe 5).

    2. Apply PDMS to the device and remove bubbles using the vacuum oven at room temperature.

      Refer to Notes in B section when you make the pre-curved structure (Figure 1H).

    3. Cure the sample in the oven at 80°C for 1 h.

    4. If needed, use a scalpel to trim the boundaries of the sample encapsulated in PDMS.

    5. Perform device quality control before implanting the device in the animal (Figure 1I).



    Figure 1. Device fabrication procedures.

    A–D. Through the UV lithography and wet-etching procedure, a pattern including pads and interconnections on the Cu/PI bilayer film is defined. E–F. All electrical components are soldered using solder flux and solder wires. In particular, the μLED tether is covered with another Cu/PI bilayer film to increase durability. G. Before the encapsulation step, the device operation with wireless power is confirmed. H. The tether is coated with PDMS in a curved structure (pre-curved), helping to decrease the stress of natural movements inside the stomach. I. This results in a soft, flexible, and lightweight (~380 mg), wireless gastric optogenetic implant. Scale bars: 1 cm.


  2. The Wireless power transfer system setup

    1. Setting up the hardware (Figure 2A):

      1. Connect the power supply to the RF generator (Figure 2B).

      2. Connect the RF generator to the matching board, via the RF cable (Figure 2C).

      3. Attach the antenna to the matching board (Figure 2D). The coil antenna is made of copper sheet (Onlinemetats.com) with dimensions of 1 inch by 30 inch. The antenna is designed so that it resonates at 13.56 MHz. Measuring the antenna using a vector network analyzer allows matching the antenna's resonant frequency at 13.56MHz. Connect capacitors in parallel to the coil, to adjust the resonancy of the antenna.



      Figure 2. Hardware setup for wireless power transfer system.

      A. Components of the wireless power TX system. B. Power supply connection for the RF generator. C. Connection between the RF generator and matching board via RF coax cable. D. Attach the TX antenna to the matching board.


    2. Software manipulation (ISO Start2017 v9.9.10):

      1. Connect the RF generator to the PC, via a USB cable (Figure 3A).

      2. Detect the antenna with “Host mode” (Figure 3B).

      3. Set the TX power level (Figure 3C).

      4. Tune the antenna using the dynamic antenna tuner function, so that the impedance of the antenna is adjusted to 50 Ω at 13.56 MHz (Figure 3D–3E).

      5. Check the tuning status (Figure 3F).

    3. Confirm the wireless power transfer system using a sample device (Figure 3G).



    Figure 3. Software manual.

    A. First, connect the RF generator to the PC for control via the software. B. Detect the antenna for powering via the RF generator. C. Set the TX power level. D. Perform antenna tunning to adjust impedance to 50 Ω, for maximum power transmission. E. Confirm impedance of 50 Ω with the vector network analyzer. F. Check the tuning status. G. Place the device in the cage (wireless power transfer system) and confirm light illumination from the device.


  3. Nodose ganglion injection (Calik et al., 2014)

    1. Under surgical anesthesia (isoflurane, Piramal NDC 66794-017-10), shave the animal’s neck area with a small hair trimmer (Wahl BravMini+).

    2. Administer ketoprofen analgesic subcutaneously.

    3. Sterilize with three alternating scrubs of betadine and alcohol.

    4. Place the animal on its back and lay sterile drape over the neck. The drape should have an opening in the center to allow for incision.

    5. Using saw tooth forceps, lift the skin and make a 1.5 cm midline incision from the clavicle to the jaw area, to expose the neck muscles.

    6. Separate the platysma, sternohyoideus, and omohyoideus muscles using blunt dissection methods, such as inserting a closed scissor tip into the connective tissue and opening the scissors, to break apart the connective tissue. Take care not to damage the carotid artery, as the procedure will be terminal if the artery is nicked.

    7. Utilize magnetic retractors to pull aside the muscle tissue and expose the carotid artery.

    8. The vagus nerve lies inside the carotid sheath. Use a small spatula, to separate the nerve and blood vessel, and a Graefe knife with the other hand, to cut open the sheath.

    9. Trace the nerve up to the jugular foramen. Depending on the animal, the ganglion might lie inside the foramen. Loop a silk suture underneath the nerve, and secure the suture onto a magnetic post. This will help to visualize the ganglion, and provide tension for injecting the virus.

    10. After visualizing the ganglion, inject the virus (200 nL) with a glass micropipette attached to a Nanoject II.

    11. Remove retractors and place muscle tissue back into its original position.

    12. Close the skin with interrupted stitches (use a 6-0 silk suture).


  4. Stomach-targeting device implantation (Video 3)

    1. Under surgical anesthesia (isoflurane, Piramal NDC 66794-017-10), shave the animal’s ventral side with a small hair trimmer (Wahl BravMini+).

    2. Administer ketoprofen analgesic subcutaneously.

    3. Sterilize with three alternating scrubs of betadine and 70% isopropyl alcohol.

    4. Place the animal on its back and lay sterile rodent surgical drapes over its abdomen. The drape should have an opening in the center to allow for incision.

    5. Using Graefe forceps, lift the skin and make a 2 cm incision along the abdominal midline to expose the abdominal muscle tissue.

    6. Lift the muscle layer using Graefe forceps and snip open a small hole. While holding up the abdominal muscle with forceps, insert scissors with ball tip (to avoid damage to the underlying tissue) and make an incision from the mid-abdomen extending to the xiphoid cartilage (Figure 4A).

    7. Pull the stomach outside of the abdomen using ring forceps, and place the stomach onto gauze soaked with sterile saline. This keeps the stomach hydrated.

    8. Utilize retractors to keep the incision site open.

    9. Puncture two adjacent holes in the stomach fundus using a 32-G needle. The holes need to be within several millimeters of each other. Enough tissue is needed for suturing around the LED tether entry and exit, but not too far apart which could restrict stomach expansion.

    10. Thread the LED tether in one hole and out the other, using fine-tipped Dumont forceps (Figure 4B). Blunt, flat-tipped forceps are also useful for handling the tether during this step.

    11. With the µLED inside the stomach, secure the tether in place with purse-string sutures (5-0 PGA) (Figure 4C).

    12. Ensure that the implantation area will not come into contact with the liver, otherwise the liver will form scar tissue and fuse with the surgical area.

    13. Place the device harvester in the lower abdominal cavity and place the stomach back into its normal orientation.

    14. Close the abdominal wall with interrupted stitches using absorbable suture (PGA 3-0 sutures) and the skin with non-absorbable suture (Nylon 4-0 sutures) (Figure 4D).

    15. Mice receive analgesics during the surgery and daily post-operative care.



    Figure 4. Step-by-step procedures for device implantation.

    A. Stomach exposal for the tether insertion. B. Conceptual illustration of insertion of the gastric optoelectronic device into the stomach. C. Insertion of the tether, including μLED, into the stomach. D. Checking the device operation after implantation surgery.


    Video 3. Surgery procedure

    (This video was made at Baylor College of Medicine. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committees at Baylor College of Medicine under protocol AN-6598.)


    Video 4. Post-surgery validation


  5. Troubleshooting

    1. When bleeding occurs:

      We implant the device away from blood vessels and do not encounter excessive bleeding. If there is blood, it is minimal and it can be cleaned with a sterile cotton tip. Defining a typical successful surgery encompasses the animal being returned to a cage and no postoperative complications or animal deaths occur.

      We have not previously experienced severe complications with stomach implants. As with any surgical procedure, it is advisable to monitor body weights for 7–10 days postoperatively. Analgesics are provided during the surgery and daily if the animals lose weight or exhibit discomfort. We euthanize mice if they fall below 80% of their starting body weight.

    2. Rate of animal deaths during surgery and after post-surgery:

      Success rate is virtually 100% for stomach implants (video 4). The most severe procedure is the injection of a virus into the nodose ganglion. We recommend injecting the virus during a separate surgical procedure. The most common cause of death is bleeding during the surgery, if a blood vessel is punctured. There is a high survival rate (95%) if the mouse wakes up from a unilateral virus injection surgery and care is taken to not damage neck muscles. There is about a 15-20% mortality rate with bilateral virus injections. We believe this is due to bilateral nerve damage and breathing difficulty.

Data analysis

  1. Device test at a standard laboratory facility

    1. Turn on the wireless power transfer system.

    2. Check the device operation at all positions and heights in the cage [Supplementary Figures 11 and 12 from previous work (Kim et al., 2021)].


  2. Device quality control at a standard laboratory facility

    1. Follow device test step (1st test).

    2. Immerse the device in 10% PBS solution for 24 h [Supplementary Figure 6 from previous work (Kim et al., 2021)].

    3. Follow device test step under this immersion condition (2nd test).

    4. After the electrical sample cleaning recipe, follow the device test step (3rd test).


  3. Device operation in an animal model

    1. Turn on the wireless power transfer system.

    2. Place a device-implanted animal into a cage.

    3. Check the indicator LED in the abdominal cavity.

    4. Experimental results are in Figures 3 and 4 from previous work (Kim et al., 2021).


  4. Proof of virus expression

    1. After experiments, anesthetize (Beuthanasia, 320 mg/kg, delivered intraperitonially) mice and perform intracardiac perfusion with 1× PBN or saline, not with heparin or EDTA (Gage et al., 2012), followed by 4% paraformaldehyde.

    2. Extract the brain, nodose ganglion, and stomach, post-fix in 4% paraformaldehyde overnight, and cryoprotect in PBS containing 30% sucrose, until the tissues sink to the bottom in the sucrose solution.

    3. Collect coronal cryostat sections for the brain, nodose ganglion, and stomach tissues (30, 20, and 10 μm thick), and mount them directly onto microscope slides.

    4. Coverslip samples using DAPI Fluoromount-G mounting medium (SouthernBiotech).

    5. Experimental results are in Figures 3c-e from previous work (Kim et al., 2021).


Notes

  1. Soldering of electrical components

    1. Apply solder wires onto the pads of the pattern.

    2. Attach the components to the pads using a small amount of solder flux and solder wires.

    3. Before covering the tether with a sandwich Cu/PI film layer, make a tiny hole with a scalpel near the μLED position, for defined μLED illumination.


  2. Encapsulation of pre-curved structure

    1. Hold the device using clips, to shape the device as a pre-curved design; then, apply a small amount of PDMS using a pipet tip (Figure 1H).

    2. Cure the tether part, including the pre-curved structure near the μLED.

    3. Dip the rest of the device into the PDMS, and then cure it in the oven.

Recipes

  1. Material cleaning

    1. Rinse with acetone for 10 s.

    2. Rinse with methanol for 10 s.

    3. Rinse with isopropanol for 20 s.

    4. Rinse with distilled water (Singly Distilled, Laboratory Grade) for 1 min.

    5. Dry samples on the hot plate at 105°C until fully dried.

  2. Electrical sample cleaning

    1. Immerse the sample in isopropanol for 5 min.

    2. Immerse the sample in distilled water for 5 min.

    3. Rinse with distilled water for 1 min.

    4. Dry samples in the oven at 80°C until fully dried.

  3. Photoresistor spin coating

    1. Place 1 mL of photoresist onto the center of the substrate without bubbles.

    2. Spin-coat at 4,000 r.p.m. for 20 s.

  4. UV-lithography and wet-etching

    1. Align the sample and the pattern mask carefully.

    2. Illuminate with 100 mJ/cm2 intensity-UV lights to lithograph patterns for pads and interconnections.

    3. Immerse in the developer for 20 s, and wash with distilled water.

    4. Immerse in the copper etchant for 7 min.

    5. Follow the material cleaning recipe.

  5. PDMS preparation

    1. Mix the PDMS kit gently at a 10:1 ratio.

    2. Place the mixed PDMS in a vacuum oven at room temperature, until all the bubbles have been removed.

    3. Vent the air to normal pressure values.

Acknowledgments

This work was supported by grants from the interdisciplinary X-Grants Program, part of the President’s Excellence Fund at Texas A&M University, 2018 NARSARD Young Investigator Awards from Brain & Behavior Foundation, and National Science Foundation Engineering Research Center for Precise Advanced Technologies and Health Systems for Underserved Populations (PATHS-UP; EEC-164851). We thank Dr. Carlos Campos from the University of Washington for in vivo experiments. This protocol was adapted from previous work (Kim et al., 2021).

Competing interests

The subject matter of the manuscript is protected by Texas A&M Technology Commercialization (TTC) Ref. & Title (5687TEES21, Implantable Devices and Techniques for the Treatment of Obesity).

Ethics

All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committees at Baylor College of Medicine under protocol AN-6598.

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简介

[摘要]光遗传学有可能改变周围神经系统 (PNS) 的研究,但 PNS 的复杂解剖结构对将光集中传递到特定组织提出了独特的挑战。该协议描述了用于研究外围感觉通路的无线遥测系统的制造。与现有的无线方法不同,低功率无线遥测通过夹在中间的预弯曲系绳提供器官特异性,并通过通道隔离策略对行为实验进行高通量分析。我们描述了构建这些设备的技术程序、带有天线线圈的无线电力传输 (TX) 系统,以及它们在体内实验应用中的实现。总的来说,该程序的时间表,包括设备制造、植入和准备开始体内实验,可以在 ~ 2-4 周内完成。这些设备的实施允许在自由行为的动物(最多 8 个)中对周围神经通路进行慢性(>1 个月)无线光遗传学操作。


[背景]光遗传学通过对基因定义的神经细胞类型进行有针对性的操作,改变了神经科学( Deisseroth ,2011 和 2015)。这通常涉及使用 Cre 驱动鼠标表达光激活离子通道并将光纤植入大脑(Park等人,2015b、2015a 和 2016)。通过这种方式,可以用光激活或灭活特定细胞类型,从而测试它们在行为中的因果作用(Kim等人,2013;Montgomery等人,2015;Shin等人,2017)。
光遗传学也有可能改变 PNS 的研究,但 PNS 的复杂解剖结构对将光集中传递到特定组织提出了独特的挑战(Towne等人,2013;Montgomery等人,2016; Maimon 等人,2018)。 PNS 由感觉和运动神经元组成,细胞体分布在广泛分布的外周神经节中。主要的感觉神经节包括背根神经节,它由沿脊柱长度两侧排列的脊髓感觉神经元的细胞体簇组成,以及包含迷走神经感觉神经元细胞体的结节神经节(Berthoud 和Neuhuber ,2000 )。
这两种感觉系统广泛地支配着一系列在大小、形状、位置和可及性方面不同的外周器官,因此需要个性化的策略来将光传递到不同的组织(Berthoud,2008;de Lartigue ,2016;Williams等人,2016) .此外,这些器官中的一些在正常生理或行为期间是高度可移动的(例如,在消化期间肠的运动),这进一步使光纤的定位和锚定复杂化。因此,与大脑不同,开发稳健的策略以选择性地将光传递到自由行为动物的外周组织是一项相当大的挑战。
Caggiano )用光靶向外周组织 等人,2016; Hibberd等人,2018 年),类似于大脑中常用的颅内光纤。虽然这种方法的优点是它可以很容易地与现成的组件(例如,激光和 LED)耦合,但腹部内脏与大脑不同,因为它缺乏用于固定大型光纤和防止运动。此外,大多数光纤的不灵活特性会在正常动物运动期间导致组织和神经或光纤本身的剪切。出于这个原因,有线光纤很少用于行为动物的外围光遗传学( Maimon 等人,2017 年和 2018 年; Towne等人,2013 年)。
另一种方法是植入一个无线探头,将光源内部化,从而绕过与光纤电缆相关的物理限制。最近已经描述了这种无线、射频( RF) 供电设备的策略(Montgomery等人,2015 和 2016 年;Park等人,2015a 和 2015b),但实现长期、器官限制照明,不妨碍正常行为或生理。例如,无线供电的微型发光二极管 (µLED) 已使用环形弹性体套管固定在大鼠膀胱上(Mickle等人,2019 年),但该装置阻碍了正常的器官扩张。已经报道了一种将 µLED 缝合到心脏表面的无线柔性设备( Gutruf et al ., 2019),但它的功能没有被证明超过八天,排除了大多数行为研究。更一般地说,将任何 µLED 固定到目标器官表面会导致光反向散射和附近组织的非特异性光遗传学照明。
最近,我们开发并发布了一类新的可植入无线设备,展示了它们在研究迷走神经方面的适用性(Kim等人,2021 年)。这种新的无线光遗传学植入物通过预先弯曲的系绳从胃内部向神经末梢传递光。这种胃内光传输能够照亮胃支配神经,比贴在器官表面的 µLED 具有更高的特异性,并且原则上可以扩展到研究其他器官,例如肠道。在这里,我们描述了制造这些设备及其体内应用的技术程序。

关键字:无线光遗传学, 迷走神经感觉通路, 装置植入, 植入式光电器件, 无线电力传输



材料和试剂


1. 移液器吸头(VWR,目录号:613-0239)
2. 载玻片(Brain Research Laboratories,目录号:5075 和 3040)
3. 铜/聚酰亚胺 (Cu/PI) 薄膜 ( Dupont TM Pyralux ® ,目录号:AC181200RY)
4. Wescorp抗静电高温聚酰亚胺胶带( Desco ,目录号:81270)
5. 光敏电阻(AZ ® ,目录号:AZ1518)
6. 焊接用芯线(多芯,目录号:397952)
7. 电气元件 [以前工作的补充图 1 ( Kim et al ., 2021 )]
8. 纱布( Medique ,目录号:60673)
9. 6-0丝线(Braintree Scientific,目录号:NC9201232)
10. 3-0 PGA 缝合线(Pro Advantage,目录号:P420493)
11. 5-0 PGA缝合线( Stoelting ,目录号:50495)
12. 4-0 尼龙缝线(Pro Advantage,目录号:P420661)
13. Betadine(Fisher Scientific,目录号:19-027132)
14. 70%异丙醇(Fisher,目录号:19-027026)
15. 2-丙醇(Fisher Chemical,目录号:190971)
16. 无水甲醇(Macron,目录号:3016-16)
17. 丙酮(Macron,目录号:2440-16)
18. 开发者解决方案(AZ ® , AZ Developer 1:1)
19. 铜蚀刻剂(Alfa Aesar TM ,目录号:Z03E099)
20. 助焊剂免清洗无铅(Chip Quik ,Inc.,目录号:SMD291NL)
21. 聚二甲基硅氧烷 (PDMS) (Dow ® , Sylgard TM 184 Silicone Elastomer Kit)
22. DAPI Fluoromount -G( SouthernBiotech ,目录号:0100-20)
23. 酮洛芬(Sigma,目录号:K2012-5G)
24. 异氟醚(Piramal NDC 66794-017-10)
25. 材料清洁(见食谱)
26. 电样品清洗(见配方)
27. 光敏电阻旋涂(见配方)
28. 紫外光刻和湿法蚀刻(见配方)
29. PDMS 制备(见食谱)


设备


1. 剪刀(精细科学工具,目录号:14090-09)
2. 磁性牵开器(Fine Science Tools,目录号:18200-20)
3. 抹刀(精细科学工具,目录号:10091-12)
4. Graefe刀(Fine Science Tools,目录号:10059)
5. Graefe镊子(Fine Science Tools,目录号:11153-10)
6. 波恩动脉剪刀,球尖(Fine Science Tools,目录号:14086-09)
7. 环形镊子(Fine Science Tools,目录号:11103-09 )
8. 32G针头(Hamilton,75SN注射器,目录号:87908)
9. 细尖杜蒙钳(Fine Science Tools,目录号:11251-20)
10. 平头镊子(Fine Science Tools,目录号:11220-21)
11. Dumont镊子(精细科学工具,Dumont #5SF 镊子)
12. 无菌一次性手术刀 ( TruMed ©, 190618)
13. 旋涂 (SCS, G3P-8)
14. Isotemp搅拌加热板 (Fisher Scientific, SP88850200)
15. 紫外光刻(EV Group,EVG610)
16. 立体显微镜(AVEN,SPZT 50)
17. 焊锡机 (Weller, WD1002/WP80)
18. 毫克天平(智能称重技术,PM-300)
19. 真空泵(Across International,EasyVac-9)
20. 真空烤箱(Across International, Accu Temp 1.9)
21. 烤箱(Quincy La, Inc.,30 型实验室烤箱)
22. 射频发生器(FEIG Electronic,ID ISC.LRM2500-A)
23. 动态天线调谐器,匹配板(FEIG Electronic,ID ISC.DAT)
24. Nanoject II 自动纳升注射器(Drummond Scientific Company,3-000-204)
25. 激光扫描共聚焦显微镜(Olympus,FV1200)
26. 荧光显微镜(尼康,Eclipse E600)


软件 


1. ISO Start2017 v9.9.10 (FEIG Electronic GmbH)
2. BioDAQ Viewer v. 2.2.01(研究饮食)


程序


A. 洁净室设施的图案准备(视频 1)
1. 准备一个灵活的 Cu/PI 双层薄膜,安装在载玻片上,并用聚酰亚胺胶带固定(图 1A)。
2. 遵循材料清洁配方(参见配方 1)。
3. 使用旋涂机在 Cu/PI 薄膜上沉积 2.5 μ m厚的光刻胶层。
4. 105°C 下烘烤 1 分钟。
5. 等待样品表面冷却至室温 3 分钟。
6. 遵循 UV 光刻和湿法蚀刻配方(图 1B - 1C,参见配方 4)。
7. 遵循材料清洁配方(参见配方 1)。




视频 1. 器件基板的制造


B. 在标准实验室设施中焊接电气元件(视频 2)
1. 使用焊接机将组件安装在图案上 [以前工作的补充图 1( Kim等人,2021 年) ]。
2. 遵循电气样品清洁配方(参见配方 2)。
3. 使用手术刀从 Cu/PI 双层膜上切割样品的边界(图 1F - 1H)。
4. 确认无线电力传输系统内的设备操作(图 1G) 。




视频 2. 设备制造


C. 在标准实验室设施中封装(视频 2)
1. 遵循 PDMS 制备配方(参见配方 5)。
2. 将 PDMS 应用于设备并在室温下使用真空烘箱去除气泡。
制作预弯曲结构时,请参阅 B 部分中的注释(图 1H)。
3. 在 80°C 的烘箱中固化样品 1 小时。
4. 如果需要,使用手术刀修剪封装在 PDMS 中的样品的边界。
5. 在将设备植入动物之前执行设备质量控制(图 1I)。




图 1. 设备制造程序。 
A – D. 通过紫外光刻和湿法蚀刻程序,在 Cu/PI 双层膜上定义了包括焊盘和互连的图案。 E – F. 所有电气元件均使用助焊剂和焊锡丝焊接。特别是, μLED系绳被另一个 Cu/PI 双层膜覆盖以增加耐用性。 G. 在封装步骤之前,确认设备使用无线电力操作。 H. 系绳以弯曲结构(预弯曲)涂有 PDMS,有助于减少胃内自然运动的压力。 I. 这产生了一种柔软、灵活且重量轻(约 380 毫克)的无线胃光遗传学植入物。比例尺:1 厘米。


D. 无线电力传输系统设置
1. 设置硬件(图 2A):
a. 将电源连接到射频发生器(图 2B)。
b. 通过射频电缆将射频发生器连接到匹配板(图 2C)。
c. 将天线连接到匹配板(图 2D)。线圈天线由尺寸为 1 英寸 x 30 英寸的铜板 (Onlinemetats.com) 制成。天线的设计使其以 13.56 MHz 共振。使用矢量网络分析仪测量天线可以匹配 13.56MHz 的天线谐振频率。将电容器并联到线圈,以调整天线的谐振。




图 2. 无线电力传输系统的硬件设置。 
A. 无线电力 TX 系统的组件。 B. 射频发生器的电源连接。 C. 通过射频同轴电缆连接射频发生器和匹配板。 D. 将 TX 天线连接到匹配板上。


2. 软件操作(ISO Start2017 v9.9.10):
a. 通过 USB 电缆将射频发生器连接到 PC(图 3A)。
b. 使用“主机模式”检测天线(图 3B)。
c. 设置 TX 功率电平(图 3C)。
d. 使用动态天线调谐器功能调谐天线,使天线的阻抗在 13.56 MHz 时调整为 50 Ω(图 3D - 3E)。
e. 检查调整状态(图 3F)。
3. 使用示例设备确认无线电力传输系统(图 3G)。




图 3. 软件手册。 
A. 首先,将射频发生器连接到PC,通过软件进行控制。 B. 检测通过射频发生器供电的天线。 C. 设置 TX 功率电平。 D. 执行天线调谐以将阻抗调整为 50 Ω,以实现最大功率传输。 E. 用矢量网络分析仪确认 50 Ω 的阻抗。 F. 检查调谐状态。 G. 将设备放入笼子(无线电源传输系统)并确认设备的照明。


E. 结节神经节注射( Calik 等人,2014)
1. 在手术麻醉下(异氟醚,Piramal NDC 66794-017-10),用小毛发修剪器(Wahl BravMini +)剃毛动物的颈部区域。
2. 皮下给予酮洛芬镇痛剂。
3. 用 3 次交替使用 betadine 和酒精的擦洗剂进行消毒。
4. 将动物放在它的背上,并在脖子上放置无菌悬垂。悬垂应该在中心有一个开口,以允许切口。
5. 使用锯齿钳,提起皮肤,从锁骨到下颌区域做一个 1.5 厘米的中线切口,露出颈部肌肉。
6. 分离颈阔肌、胸骨舌骨肌和omohyoideus肌肉,例如将闭合的剪刀尖端插入结缔组织并打开剪刀,以分解结缔组织。注意不要损坏颈动脉,因为如果动脉被划伤,手术将是终点。
7. 利用磁性牵开器拉开肌肉组织并暴露颈动脉。
8. 迷走神经位于颈动脉鞘内。用小抹刀将神经和血管分开,另一只手用Graefe刀切开鞘。
9. 将神经追踪到颈静脉孔。根据动物的不同,神经节可能位于孔内。将丝线缝合在神经下方,并将缝合线固定在磁性柱上。这将有助于可视化神经节,并为注射病毒提供张力。
10. 可视化神经节后,用连接到Nanoject II的玻璃微量移液器注射病毒(200 nL )。
11. 取下牵开器并将肌肉组织放回其原始位置。
12. 用间断的针迹关闭皮肤(使用 6-0 丝线缝合)。


F. 胃靶向装置植入(视频 3)
1. BravMini +)剃毛动物的腹侧。
2. 皮下给予酮洛芬镇痛剂。
3. 用 3 次交替的 betadine 和 70% 异丙醇擦洗消毒。
4. 将动物放在它的背上,并在其腹部放置无菌啮齿动物手术窗帘。悬垂应该在中心有一个开口,以允许切口。
5. 使用Graefe钳,提起皮肤并沿腹部中线做一个 2 厘米的切口,以暴露腹部肌肉组织。
6. Graefe镊子提起肌肉层并剪开一个小孔。在用镊子夹住腹部肌肉的同时,插入带球尖的剪刀(以避免损坏底层组织),并从中腹部切开一个切口,延伸到剑突软骨(图 4A)。
7. 使用环钳将胃拉出腹部,并将胃放在用无菌盐水浸泡的纱布上。这样可以保持胃的水分。
8. 利用牵开器保持切口部位开放。
9. 使用 32-G 针在胃底穿刺两个相邻的孔。这些孔需要彼此相距几毫米。需要足够的组织来缝合 LED 系绳入口和出口周围,但不要相距太远,以免限制胃扩张。
10. 使用细尖的 Dumont 镊子将 LED 系绳穿入一个孔并穿出另一个孔(图 4B)。在此步骤中,钝的平头镊子也可用于处理系绳。
11. 将 µLED 置于胃内,用荷包缝合线 (5-0 PGA) 将系绳固定到位(图 4C)。
12. 确保植入区域不会与肝脏接触,否则肝脏会形成疤痕组织并与手术区域融合。
13. 将设备收割机放在下腹腔中,并将胃放回其正常方向。
14. 使用可吸收缝合线(PGA 3-0 缝合线)和不可吸收缝合线(尼龙 4-0 缝合线)的皮肤(图 4D)关闭腹壁。
15. 小鼠在手术和日常术后护理期间接受镇痛剂。




图 4. 设备植入的分步程序。 
A. 用于系绳插入的胃外露。 B. 将胃光电装置插入胃的概念图。 C. 将包括μLED在内的系绳插入胃中。 D. 植入手术后检查设备运行情况。




视频 3. 手术过程
(该视频是在贝勒医学院制作的。所有动物护理和实验程序均由贝勒医学院的机构动物护理和使用委员会根据协议 AN-6598 批准。)




视频 4. 术后验证


G. 故障排除
1. 当出血发生时:
我们将设备植入远离血管的地方,不会出现过多出血。如果有血,那是极少的,可以用无菌棉签清洗。定义典型的成功手术包括将动物送回笼子,并且不会发生术后并发症或动物死亡。
我们以前没有经历过胃植入物的严重并发症。与任何外科手术一样,建议术后7-10 天监测体重。如果动物体重减轻或出现不适,则在手术期间和每天提供镇痛剂。如果小鼠体重低于起始体重的 80%,我们会对它们实施安乐死。
2. 手术期间和手术后的动物死亡率:
胃植入物的成功率几乎是 100%(视频 4)。最严重的程序是将病毒注射到结节神经节中。我们建议在单独的手术过程中注射病毒。如果血管被刺破,最常见的死亡原因是手术期间出血。如果小鼠从单侧病毒注射手术中醒来,并且注意不损伤颈部肌肉,则存活率很高 (95%)。双侧病毒注射的死亡率约为 15-20%。我们认为这是由于双侧神经损伤和呼吸困难。


数据分析


A. 在标准实验室设施进行设备测试
1. 打开无线电力传输系统。
2. 检查笼中所有位置和高度的设备操作[以前工作的补充图 11 和 12 ( Kim等人,2021 年) ]。


B. 标准实验室设施的设备质量控制
1. 遵循设备测试步骤(第一次测试)。
2. 将设备浸入 10% PBS 溶液中 24 小时 [以前工作的补充图 6 ( Kim等人,2021 年) ]。
3. 在此浸没条件下执行设备测试步骤(第 2 次测试)。
4. 在电气样品清洁配方之后,按照设备测试步骤(第 3次测试)进行操作。


C. 动物模型中的设备操作
1. 打开无线电力传输系统。
2. 将植入设备的动物放入笼子中。
3. 检查腹腔中的 LED 指示灯。
4. 实验结果在图 3 和图 4 中来自之前的工作 ( Kim et al ., 2021) 。


D. 病毒表达的证明
1. 实验后,麻醉( Beuthanasia ,320 mg/kg,腹膜内给药)小鼠并用 1 × PBN 或生理盐水进行心内灌注,而不是肝素或 EDTA (Gage等人,2012) ,然后是 4% 多聚甲醛。
2. 提取大脑、结节神经节和胃,在 4% 多聚甲醛中过夜后固定,并在含有 30% 蔗糖的 PBS 中冷冻保护,直到组织在蔗糖溶液中沉到底部。
3. 收集大脑、结节神经节和胃组织(30、20 和 10 μm厚)的冠状低温恒温器切片,并将它们直接安装在显微镜载玻片上。
4. 使用 DAPI Fluoromount -G 封固剂 ( SouthernBiotech ) 的盖玻片样品。
5. 实验结果在之前工作的图 3c-e 中 ( Kim et al ., 2021) 。


笔记 


A. 电气元件的焊接
1. 将焊锡丝涂在图案的焊盘上。
2. 使用少量助焊剂和焊线将组件连接到焊盘上。
3. 在μ LED位置附近制作一个小孔,用于定义的μ LED照明。


B. 预弯曲结构的封装
1. 使用夹子夹住设备,将设备塑造成预先弯曲的设计;然后,使用移液器尖端应用少量 PDMS(图 1H)。
2. μ LED附近的预弯曲结构。
3. 将设备的其余部分浸入 PDMS,然后在烤箱中固化。


食谱


1. 材料清洗
a. 用丙酮冲洗 10 s。
b. 用甲醇冲洗 10 秒。
c. 用异丙醇冲洗 20 秒。
d. 用蒸馏水(单蒸馏,实验室级)冲洗 1 分钟。
e. 在 105°C 的热板上干燥样品直至完全干燥。
2. 电样品清洗
a. 将样品浸入异丙醇中 5 分钟。
b. 将样品浸入蒸馏水中 5 分钟。
c. 用蒸馏水冲洗 1 分钟。
d. 在 80°C 的烘箱中干燥样品直至完全干燥。
3. 光敏电阻旋涂
a. 将 1 mL 的光刻胶放在基板的中心,没有气泡。
b. rpm的速度旋涂。 20 秒。
4. 紫外光刻和湿法蚀刻
a. 仔细对齐样品和图案掩模。
b. 用 100 mJ /cm 2强度的紫外灯照射,以平版印刷图案,用于焊盘和互连。
c. 浸入显影液中 20 s,用蒸馏水洗涤。
d. 浸入铜蚀刻剂中 7 分钟。
e. 遵循材料清洁配方。
5. 聚二甲基硅氧烷制备
a. 以 10:1 的比例轻轻混合 PDMS 套件。
b. 将混合的 PDMS 置于室温下的真空烘箱中,直到所有气泡都被去除。
c. 将空气排放到正常压力值。


致谢


这项工作得到了跨学科 X-Grants 计划的资助,该计划是德克萨斯 A&M 大学总统卓越基金的一部分、大脑与行为基金会 2018 年 NARSARD 青年研究员奖以及国家科学基金会精密先进技术和卫生系统工程研究中心对于服务不足的人群(PATHS-UP;EEC-164851)。我们感谢华盛顿大学的 Carlos Campos 博士进行体内实验。该协议改编自之前的工作(Kim等人,2021)。


利益争夺


手稿的主题受 Texas A&M Technology Commercialization (TTC) Ref. 的保护。和标题(5687TEES21,用于治疗肥胖症的植入设备和技术)。


伦理


所有动物护理和实验程序均由贝勒医学院的机构动物护理和使用委员会根据协议 AN-6598 批准。


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引用:Hong, S., Kim, W. S., Han, Y., Cherukuri, R., Jung, H., Campos, C., Wu, Q. and Park, S. I. (2022). Optogenetic Targeting of Mouse Vagal Afferents Using an Organ-specific, Scalable, Wireless Optoelectronic Device. Bio-protocol 12(5): e4341. DOI: 10.21769/BioProtoc.4341.
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