Article Figures & Data
Figures
- Figure 1.
Diaphragm EMG electrode fabrication and assembly. A, Each EMG implant is composed of one plastic pedestal and five or six (if using an extra/backup electrode) gold pins. B, Deinsulate ∼2 mm of wire which will interface with a gold pin. Deinsulated portion of the wire is denoted with the bracket. C, Use the helping hands to hold the gold pin in an upright position. D, With a second helping hand, place the deinsulated end of the wire inside the gold pin. E, Solder the wire and gold pin with enough solder to fill the pin, but not spill out of the pocket. F, Use a multimeter to measure the resistance between gold pins on the same wire. Repeat for all recording and ground wires. Cut each wire in half. G, Tie an anchor knot on each recording wire opposite of the gold pin. Deinsulate the wire from the anchor knot to the distal end. H, Crimp a 31 G insulin needle onto the wire using a flat-blade screwdriver. I, Place each pin into the pedestal according to the configuration. We utilize Pinholes 1 and 2 (CH01) for the left hemidiaphragm, Pinholes 4 and 5 (CH02) for the right hemidiaphragm, Pinhole 3 for the ground, and Pinhole 6 for the extra, backup electrode (if used). It is vital to maintain a consistent configuration among produced implants to ensure correct identification of paired electrodes. J, Once assembled, tie suture around the pairs of electrodes to identify during surgery. Wires from electrodes in Pinholes 1 and 2 (CH01/left) will be identified with a size 0 suture; wires from electrodes in Pinholes 4 and 5 (CH02/right) will be identified with a size 3.0 suture. All recording electrodes will then be tied together with a larger size 0 suture to assist when tunneling the wire under the skin. K, Completed implant is shown with the cured epoxy both with and without using the 3-D–printed headcap holder. If using partial-thickness electrodes, steps found in subpanels G and H will not be performed.
- Figure 2.
Surgical placement of the diaphragm EMG implant. A, Create a midline incision over the skull, and clear the underlying connective tissue for a clear view of sutures on skull. B, Schematic depiction of location for screw placement. Use a 0.5-mm-diameter burr to drill into the skull at each location marked by “X”. C, Place self-tapping bone screws at each location, leaving the uppermost portion of the screw exposed. D, Once wires are tunneled from skull to abdomen, place implant epoxy-side down onto the skull between the screws. E, Use dental acrylic to secure the implant in place. Dashed lines indicate wires underneath the dental acrylic. F, Using the helping hands, tent the abdomen to view the diaphragm muscle. Insert electrodes at the black X's and advance behind the diaphragm and then back out. Gently pull until the anchor knot is touching the diaphragm and tie a second anchor knot. G, Repeat until two pairs of electrodes are placed in the diaphragm muscle. H, Schematic of the completed diaphragm EMG implant with subcutaneous wires extending from the secured implant to the abdomen and into the diaphragm muscle. Recording cables are plugged into the implant to record the EMG output. Dashed lines show subcutaneous wires from EMG implant to abdomen. Created in BioRender. Streeter (2024) BioRender.com/o298768.
- Figure 3.
Examples of processed diaphragm EMG signals. A, B, A representative, preprocessed unilateral diaphragm EMG trace during eupneic breathing and examples of varied data processing approaches for the signal across a compressed (A) and expanded (B) timeline. CV was calculated from the processed data prior to normalization and is noted for each data processing approach for the compressed timeline. RMS, root mean square.
- Figure 4.
Diaphragm EMG signals across respiratory behaviors. A, Representative raw bilateral diaphragm EMG traces for an awake, ad libitum behaving rodent across ventilatory and nonventilatory behaviors using the described implant. B, Integrated bilateral diaphragm EMG signals using the provided data pipeline for an awake, ad libitum behaving rodent across ventilatory and nonventilatory behaviors.
- Figure 5.
Bilateral diaphragm EMG across respiratory behaviors in an awake, ad libitum behaving rodent. A, Representative diaphragm EMG traces from the left and right hemidiaphragms during a 1 min recording in a ad libitum behaving animal showing various behaviors including (B) quiet breathing, (C) breathing during locomotion, (D) sniffing, and (E) return to quiet breathing.
- Figure 6.
Diaphragm EMG recordings over prolonged time periods. Representative diaphragm EMG recordings from (A) the baseline, (B) 2 weeks postimplant, (C) 4 weeks postimplant, and (D) 8 weeks postimplant. Representative diaphragm EMG recordings from a subset of animals have been collected during (E) the baseline and (F) 40 weeks postimplant. Note: panels A–D were collected with an amplification of 1,000×, and panels E and F were collected with an amplification of 100×.
- Figure 7.
Simultaneous collection of diaphragm EMG and whole-body plethysmography. Representative respiratory flow and right diaphragm EMG traces during (A) normoxia and (B) a maximal hypoxia (10.5%) and hypercapnia (7%) challenge.
Tables
Component Description Quantity Catalog # Company Sandpaper Sanding block 1 DSFM-F-ESF-10 3M Kimtech KimWipes Delicate task wipers 1 34155 Kimberly-Clark Professional Gold pins Six-channel electrode female socket 5 (6 if utilizing an extra, backup electrode) E363/0 Protech International Plastic pedestal Six-channel electrode pedestal 1 MS363 Protech International Recording wire Multistranded PFA-coated stainless steel wire (3) 36 cm sections of wire 793200 A-M Systems Grounding wire Stranded stainless steel wire (1) 16 cm section of wire AS631 Protech International Scalpel blade Size 10 scalpel blade 1 Amazon Lighter Pocket-style, classic lighter 1 Amazon 31 G needle Insulin syringe with ultrafine needle 5 NDC/HRI#: 08290-3249-10 Amazon Screwdriver Fine Science Tools Screwdriver Set, Nickel, six total 1 (size 1.8 or 2.3) NC9468697 Thermo Fisher Scientific Suture Braided silk suture (size 0 and 3-0) 1 ea Size 0: SUT-S 116
Size 3-0: SUT-S 110
Braintree Scientific Epoxy Devcon 2-ton epoxy 1 Devcon 31345 Amazon Multimeter Digital multimeter 1 AstroAI WH5000A Amazon Helping hands Helping hands platform and clips 1 OF-M4 OMNIFIXO Solder iron Digital soldering station 1 FX888D Amazon Solder 44 Flux Cored Wire (SN60PB40 3.3%/44) 1 2460400027 Kester Flux pen 951 Flux-Pen 1 8310000951 Kester Microscope Can be either stereo or surgical microscope 1 Mounting holder Six-channel mounting holder 1 MH-363 Protech International Component Description Quantity Catalog # Company Chlorhexidine scrub ChlorHex-Q Scrub 1 VINV-CLOR-SCRB Vedco Sterile saline Saline solution 0.9%—sterile 1 07-841-4550 Patterson Veterinary Scalpel handle Scalpel Handle #3 1 10003-12 Fine Science Tools Scalpel blade Size 10 1 Scissors Fine scissors 1 14558-11 Fine Science Tools Forceps Adson Forceps 1 11006-12 Fine Science Tools Curette Micro Curette 1 10082-15 Fine Science Tools Retractors Alm Retractors 1 17008-07 Fine Science Tools Drill Dremel Micro 1 8050 Dremel Collet for drill 3/32″ Rotary Tool Collet (Dremel 4485) 1 Amazon Drill bit Burrs for Micro Drill 1 19007-07 Fine Science Tools Bone screws Self-tapping bone screws 1 19010-10 Fine Science Tools Screw forceps Screw forceps 1 26100-00 Fine Science Tools Screwdriver Fine Science Tools Screwdriver Set, Nickel, six total 1 (size 1.5 or 1.8) NC9468697 Thermo Fisher Scientific Dental acrylic Dental acrylic powder 1 lb 8101 Patterson Dental Dental acrylic Dental acrylic liquid 16 oz 8502 Patterson Dental syringe 1 ml syringe 1 18 G needle 18 G needle 1 Hemostat Halsted-Mosquito Hemostat 1 13008-12 Fine Science Tools Rat-tooth forceps Extra Fine Graefe Forceps 1 11153-10 Fine Science Tools Spring scissors Student Vannas Spring Scissors 1 91500-09 Fine Science Tools Fine forceps Dumont #5SF Forceps 2 11252-00 Fine Science Tools Magnetic board QuadHands WorkBench with movable magnetic arms 1 Amazon Magnetic arms From QuadHands WorkBench 2 Amazon Alligator clips From QuadHands WorkBench 2 Amazon Suture Coated Vicryl Violet Braided Suture 1 J397H Ethicon - Table 3.
Surgical and postoperative complications and incidence rates associated with implanting chronic diaphragm EMGs and suggested steps to reduce each complication
Complication Incidence rate Mitigating solutions Pneumothorax/diaphragm tearing ∼2% - Use of minimal force to advance needle
- Use of diaphragm contraction to advance needle
- Ensure smooth needle/wire surface(s)
- Holding base of needle to thread needle and wire through diaphragm in a fluid movement
Idiopathic loss of unilateral diaphragm EMG signal ∼5% - Use of flux during implant fabrication
- Leaving slack outside the abdominal cavity to accommodate animal growth
Infection requiring additional treatment ∼3% - Proper sterile surgical technique
- Proper handling of sterile supplies
- Frequent (3 times/week) bedding/cage changes following implantation due to abdominal incision
- Use of Baytril and meloxicam postsurgery
Bleeding at site of headcap insertion (<24 h after implantation) ∼50% - Resolves spontaneously without intervention
Grooming-induced opening of abdominal incision ∼5% - Subcuticular suture technique
- Use of rodent Elizabethan collar
- Incidence decreases after buprenorphine treatment
Idiopathic dehiscence of incision caudal to headcap ∼2% - Interrupted absorbable suture caudal to implant
- Surgical glue to assist in wound closure
Incidence rates are based on observations from n = 105 surgeries, excluding the initial n = 10 surgeries, which were used for surgical training and optimization and refinement of the surgical approach.
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