TITLE: Protocol for chronic implantation of patch electrodes on the gastric muscle wall of the rat
AUTHORS: Robert  Phillips, Deborah  Jaffey, Terry  Powley

[DESCRIPTION]
Electrical stimulation is a potential therapy for gastric disorders.  Here we describe our surgical procedure for the implantation of patch electrodes on the muscle wall of the stomach of both male and female rats for use in chronic studies looking at the long-term effects of stimulation on physiology and behavior.  The surgery is well tolerated by the subjects as rats return to pre-surgical body weight and normal solid chow intake within 7 to 10 days post-surgery.  In our hands, rats implanted using the methods described below have been continuously run in feeding and motility experiments for 8 to 12 weeks with minimal to no loss of subjects.  When failure does occur, it is typically a result of back mount failure at the site of attachment of the overhead tether to the back mount.

[GUIDELINES]
All procedures described were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (8th ed., The National Academic Press, Washington, D.C.), and were approved by the Purdue University Animal Care and Use Committee.

[STEPS]
SECTION: Subjects
1. Male and/or female rats of the appropriate age and weight for the experimental design were used. Note: we have successfully implanted rats across a range of ages (3 to 6 months) and weights (250 to 550 grams).
SECTION: Subjects
2. Two days prior to surgery, start the rats on antibiotics (one 2 mg Baytril tablet per day).
SECTION: Setup
3. Don a fresh pair of sterile latex powder-free gloves.
SECTION: Setup
4. Sterilize surgical instruments using a hot bead sterilizer and place on a sterile surgical drape.
SECTION: Setup
5. Clean Surgisuite operating surface and heating pad with 70% ethanol and place a sterile drape over heating pad.
SECTION: Anesthetic Induction and Preparation
6. Fast rat overnight with only access to water.
SECTION: Anesthetic Induction and Preparation
7. Induce anesthesia using 5% isoflurane (Somnosuite anesthesia system).
SECTION: Anesthetic Induction and Preparation
8. Place rat on sterile drape covered heating pad and lower isoflurane to 2%.
SECTION: Anesthetic Induction and Preparation
9. Apply sterile lubricant to both eyes to prevent drying (Artificial Tears Ophthalmic Lubricant Ointment).
SECTION: Anesthetic Induction and Preparation
10. Shave hair from back between shoulder blades and entire abdomen.
SECTION: Anesthetic Induction and Preparation
11. Clean shaved area with 70% ethanol followed by povidone-iodine solution and allow to dry.
SECTION: Anesthetic Induction and Preparation
12. Inject glycopyrrolate (0.2 mg/ml, s.c.) to minimize secretions.
SECTION: Anesthetic Induction and Preparation
13. Inject buprenorphine (Buprenex, 0.01 mg/kg, s.c.) for analgesia.
SECTION: Anesthetic Induction and Preparation
14. Make a small incision using surgical scissors between the midline of the shoulder blades.
SECTION: Anesthetic Induction and Preparation
15. Change out sterile drape and don a fresh pair of sterile latex powder-free gloves.
SECTION: Anesthetic Induction and Preparation
16. Drape rat with opening in sterile drape to expose the abdomen.
SECTION: Median Laparotomy
17. Apply pressure to the hindfoot of all four limbs to ensure there is no pain response.
SECTION: Median Laparotomy
18. Ensure that body temp and respiration rate are normal.
SECTION: Median Laparotomy
19. Make a 3-4 cm incision in the upper midline of the abdomen using surgical scissors, just below the xyphoid process as a landmark.
SECTION: Median Laparotomy
20. Drape perimeter of incision with sterile gauze pads and wet gauze with body temp sterile saline solution.
SECTION: Mobilizing the Stomach
21. Using two wet sterile cotton swabs, gently lift the stomach out of the abdominal cavity and place on sterile wet gauze.
SECTION: Mobilizing the Stomach
22. Drape additional sterile gauze over stomach leaving only the electrode placement site exposed.
SECTION: Mobilizing the Stomach
23. Wet the gauze thoroughly with sterile, physiological saline warmed to body temperature.
SECTION: Mobilizing the Stomach
24. Note: continually wet the exposed region of the stomach throughout the surgical procedure using sterile saline warmed to body temperature ensuring that the muscle wall never dries out.
SECTION: Surgical Attachment of Patch Electrode
25. Swing surgical scope into place.
SECTION: Surgical Attachment of Patch Electrode
26. Place chronic EMG patch electrode (MicroProbes) on the muscle wall of the stomach at appropriate location and ensure that suture points will not pierce the underlying vasculature.  Note: prior to the start of the surgery check the impedance of the patch electrode with the electrode placed in physiological saline warmed to body temperature using an oscilloscope.
SECTION: Surgical Attachment of Patch Electrode
27. Using four stay sutures (8-0 nylon), one at each corner, attach the patch electrode to the serosal surface of the stomach muscle wall while being careful to avoid underlying blood vessels.
SECTION: Surgical Attachment of Patch Electrode
28. A fifth stay suture was routinely placed along the long edge of the patch electrode on the side opposite of the incoming lead wires.
SECTION: Surgical Attachment of Patch Electrode
29. Return the stomach to its natural position in the abdomen.
SECTION: Surgical Attachment of Patch Electrode
30. Using an oscilloscope, test patch the electrode again to ensure proper function.
SECTION: Exteriorizing Patch Electrode Leads
31. Roll rat onto its side.
SECTION: Exteriorizing Patch Electrode Leads
32. Starting at the incision site located between the shoulder blades, work a trocar (length 15 cm; I.D. 3 mm) between the skin and underlying muscle until roughly 2 cm past the rib cage and then puncture through muscle wall into abdominal cavity.
SECTION: Exteriorizing Patch Electrode Leads
33. Bring patch electrode leads through the trocar.
SECTION: Exteriorizing Patch Electrode Leads
34. Remove trocar leaving just the pins of the leads exposed between the shoulder blades.
SECTION: Exteriorizing Patch Electrode Leads
35. Return the rat to a supine position.
SECTION: Exteriorizing Patch Electrode Leads
36. Ensure that the stomach along with intestines are in their natural position.  If not, using wet sterile cotton swabs make the necessary adjustments to the organs.
SECTION: Exteriorizing Patch Electrode Leads
37. Ensure that the patch electrode leads are positioned so that they do not create any unnecessary torque on the stomach.
SECTION: Exteriorizing Patch Electrode Leads
38. Cover abdominal incision with wet sterile gauze.
SECTION: Exteriorizing Patch Electrode Leads
39. Roll rat into a prone position.
SECTION: Back Mount Construction and Implantation
40. Connect the exteriorized leads to a pedestal/pedestal back mount (P1 Technologies).  Note: prior to start of surgery, a piece of polyester mesh (3 cm x 3 cm) should be attached to the pedestal back mount using 3-0 silk suture.
SECTION: Back Mount Construction and Implantation
41. Place dummy pins in the remaining open slots in the pedestal/pedestal back mount.
SECTION: Back Mount Construction and Implantation
42. While wearing appropriate eye protection, secure pins in place with Fusio self-adhesive flowable composite and LED curing light.
SECTION: Back Mount Construction and Implantation
43. Using multiple layers of adhesive, slowly encapsulate the base of the pedestal back mount creating a watertight seal around the pins.
SECTION: Back Mount Construction and Implantation
44. Screw dust cap (P1 Technologies) onto pedestal sealing it from exposure to liquids (e.g, blood and saline).
SECTION: Back Mount Construction and Implantation
45. Place the back mount in position between the shoulder blades.
SECTION: Back Mount Construction and Implantation
46. Blunt dissect the skin away from underlying muscle as needed to make room for mesh.
SECTION: Back Mount Construction and Implantation
47. Suture the mesh attached to the back mount to the underlying muscle using multiple stay sutures (Surgipro II, monofilament polypropylene, 5-0).
SECTION: Back Mount Construction and Implantation
48. Return the skin to its natural lie and close around the pedestal of the back mount using interrupted sutures (Surgipro II, monofilament polypropylene, 3-0).
SECTION: Back Mount Construction and Implantation
49. Roll rat onto back exposing abdominal opening.
SECTION: Abdominal Closure
50. Ensure that organs and leads are in their appropriate positions.
SECTION: Abdominal Closure
51. Hydrate the rat by filling the abdominal cavity with 5-10 ml physiological sterile saline warmed to body temperature.
SECTION: Abdominal Closure
52. Close muscle with 5-0 polypropylene (Surgipro II) interrupted sutures.
SECTION: Abdominal Closure
53. Close the skin with 3-0 polypropylene (Surgipro II) interrupted sutures.
SECTION: Abdominal Closure
54. Reinforce skin sutures with staples.  NOTE: staples are removed 10-14 post surgery.
SECTION: Abdominal Closure
55. Using oscilloscope, check patency of patch electrode to ensure that no damage occurred during surgery.
SECTION: Abdominal Closure
56. Seal perimeter of dust cap using KWIK-SIL to ensure that the back mount is watertight during the healing process.
SECTION: Postoperative Care
57. Rats are brought out of anesthesia slowly by reducing the flow of isoflurane by 0.5% every 15 min.
SECTION: Postoperative Care
58. Once awake, rats are returned to their home cage and provided with a 5mg tablet of Baytril (2mg)/Rimadyl (2mg).
SECTION: Postoperative Care
59. A 5 mg Baytril/Rimadyl tablet is then provided once each day for the following three days post-surgery followed by a 5 mg Baytril tablet once a day for the next four days.
SECTION: Postoperative Care
60. Rats are housed individually to ensure proper healing of surgical sites.
SECTION: Postoperative Care
61. Ad lib water is provided throughout the duration of post-surgical recovery.
SECTION: Postoperative Care
62. Ad lib chow is provided starting one day post-surgery.
SECTION: Postoperative Care
63. Remove skin staples post-operative day 10-14.
SECTION: Postoperative Care
64. NOTE: Rats can be safely tethered immediately following surgery, however we have found that tethering the rats 14 days post-surgery results in optimal patency for long-term chronic use.