Preoperative Considerations (timing: 1 hr)
1. Prepare surgical table with dedicated areas for the induction chamber, intubation station, surgical workspace, ventilator, sterile surgical instrument and equipment area, adjustable lights, and a heating source (Fig. 2a). To aid in precise dissection, ensure that lighting is adequate. To prevent shadowing that may obscure visualization during surgery, use at least two sources of adjustable lighting to illuminate the operating field.
2. For convenience of transferring the animal once sedated, set the induction chamber next to the intubation station. For intubation, use a stand with an adjustable head positioning (Fig. 2b). In the case that an anterior neck cutdown is necessary for intubation, prepare a base plate with adjustable retractors that attach magnetically for visualization of the airway.
3. For ventilation, place the ventilator directly in front of the surgical workspace.
4. Autoclave all surgical instruments and prepare sterile packages of drapes, cotton tips, and gauze.
5. Set up the sterile instrument area on the dominant-hand side of the surgeon’s workspace (Fig. 2c).
6. Set the ventilator to 80 breaths per minute on pressure control with peak inspiratory pressure limit of 14 cmH2O.
7. Provide additional heat by an overhead heating lamp.
a. CRITICAL STEP: The Guide for the Care and Use of Laboratory Animals recommends using a recirculated heating pad under the animal during surgery to prevent hypothermia. However, because the metal pad interferes with the inductive power transfer for the devices, use an overhead heating source or exothermal pads instead.
Induction of anesthesia (timing: 5 min)
8. Induce general anesthesia using inhaled isoflurane vapors by placing the animal in an induction chapter with 3 to 4% of isoflurane (vol/vol) and an oxygen flow of 2 mL/min for several minutes until the rat becomes unconscious. Confirm loss of consciousness by gentle toe pinch. If movement is observed, wait until a deeper plane of anesthesia is achieved. If breathing is too slow, reduce the isoflurane level.
Blind orotracheal intubation (timing: 5 min)
9. After induction, place the rat on the intubation stand. Gently retract the rat’s tongue with forceps to put the airway on slight tension. Blindly pass the 16-gauge cannula, supported by a blunt curved stylet, through the vocal cords and into the trachea. The operator should feel the tactile feedback of the stylet running against the tracheal rings to confirm that the endotracheal tube is in the trachea. Remove the stylet while holding the endotracheal tube in place. Confirm the proper placement of the endotracheal tube by checking the presence of condensation on a dental mirror (Fig. 3a). If at least three intubation attempts are unsuccessful, use an anterior neck cutdown to directly visualize the trachea.
10. Transfer the rat to the surgical workspace and connect the cannula to the ventilator (Fig. 3b).
a. CRITICAL STEP: For rats, set the ventilator to 80 breaths/minute on pressure control with a peak inspiratory pressure limit of 14 cmH2O. For mice, set the ventilator to volume mode with 180 breaths/min, a tidal volume of 180 L, peak inspiratory pressure between 20 cmH2O, and a positive end-expiratory pressure of 4 cmH2O.
Preparation for surgery (timing: 2 min)
11. Place the animal in the right lateral decubitus position for left thoracotomy atrial implantation (Supplementary Video 1) or left lateral decubitus position for right thoracotomy for ventricular implantation (Supplementary Video 2).
12. Retract both forearms and secure them anteriorly and superiorly (Fig. 3C).
13. Administer a subcutaneous injection of buprenorphine (0.01–0.05 mg/kg) with saline for preoperative analgesia.
14. Using clippers, shave a 4 cm × 4 cm area over the left/right chest between the left/right axilla and the abdomen.
15. Disinfect the shaved region with 4% chlorhexidine gluconate solution. Use a cotton swab to scrub the area.
16. Place subdermal ECG needle electrodes in the Lead II configuration (left arm: ground; right arm: negative electrode; right leg: positive electrode).
a. CRITICAL STEP: Recording an ECG throughout the duration of the surgery enables monitoring of the animal’s health status and confirmation of capture during pacing threshold testing where the lowest voltage setting that can drive the heart rhythm is identified.
17. Lower the isoflurane vapors to 2% (vol/vol) during the surgery.
a. CAUTION: To prevent cardiotoxicity, use the minimum level of isoflurane that maintains a deep plane of anesthesia, which is especially important during longer procedures (>1 hour).
Thoracotomy and cardiac exposure (timing: 5 min)
18. Using sterile technique, don surgical gloves.
19. Cut an opening in a sterile drape approximately the size of the shaved area of the animal’s chest. Place the drape over the animal to expose the surgical field.
20. Palpate for the area of maximum pulsation of the heart to identify the ideal point of chest entry. This area is usually within the fourth intercostal space, which is about 5 to 10 mm lateral to the sternum (Fig. 4A, 5A). For atrial or ventricular implantations, two or three intercostal spaces below the maximum pulsation point was identified for incision, respectively.
21. Make a curvilinear incision with surgical scissors over the chest approximately 2 cm below the left axilla (Fig. 4B, 5B). Carry the dissection through the skin and the muscle layer until the ribs and intercostal muscle are directly visualized. During dissection, stop any bleeding encountered by applying direct pressure over the area with either sterile cotton swabs or forceps. Alternatively, use electrocautery for hemostasis during subcutaneous dissection.
22. To enter the chest, gently dissect the intercostal muscle with Metzenbaum scissors while grasping and retracting the rib above with forceps to lift the chest wall away from the lung (Fig. 4C, 5C). As the intercostal muscle is dissected away into a thin layer, visualize the sliding lung and the pleural space.
a. CAUTION: Take care to not injure the lung upon entry with the Metzenbaum scissors.
b. CRITICAL STEP: Dissect the intercostal muscle along the superior aspect of the ribs to minimize bleeding because the neurovascular bundles run along the inferior surfaces of the ribs.
23. Upon entry into the pleural space, carefully extend the incision through the intercostal muscle with Metzenbaum scissors to create a 2-3 cm opening along the desired intercostal space.
a. CAUTION: Take care to not encroach onto the sternum so that the left and right internal thoracic arteries and veins are not injured. To avoid harming any other structures during this entry, gently push the lungs away, and point the tips of the Metzenbaum scissors up and away from the chest cavity.
24. Place a small animal rib spreader to retract the intercostal space open. Push the lungs down using a microspatula to avoid injury to the lung (Fig. 4D, 5D).
25. Gently retract the left lung posteriorly and secure it in place with a cotton swab so that the target implantation area of the heart is positioned in view.
26. Gently open the pericardium and clear it away from the surface using forceps and cotton swabs. If more optimal positioning of the heart is required for device implantation, the heart can be gently maneuvered with cotton swabs or gauze.
Pacemaker placement (timing: 10 min)
27. Along the ventral aspect of the thoracotomy incision, use blunt dissection to create a subcutaneous pocket to house the pacemaker’s receiver (Fig. 5E).
28. Place the receiver of the pacemaker into the subcutaneous pocket so that the electrode pads are near the target implantation area and the thin stretchable connector that connects the electrode pads to the receiver intersects the intercostal space.
a. CRITICAL STEP: When selecting the location for suture attachment, consider anatomical landmarks of the heart to avoid iatrogenic injury of the coronary arteries that may result in myocardial infarction or excessive blood loss.
29. Secure the device to the atrium or ventricle using a small caliber non-absorbable monofilament 6-0 polypropylene suture. Thread the suture through the electrode pad and throw a shallow stitch through the epicardium (Fig. 4E-F, 5F) at the target site of implantation. Then, pass the suture through the same electrode again. Tie down the suture.
a. CRITICAL STEP: Before placing any sutures, ensure that the side of the electrode interfacing with the heart is the conductive region of the device.
30. Repeat the same steps for the adjacent electrode.
31. Briefly turn on the pacemaker with the power transfer system (Neurolux, Inc.) to visualize capture of the heart in the ECG to ensure successful contact at the tissue-electrode interface.
a. CAUTION: Pacing equipment are generally not sterile. Be sure to maintain a sterile field during this short test for capture.
b. CAUTIOIN: Perform more extensive threshold testing at the end of the operation after closure of the chest to minimize the amount of time during which the animal is anesthetized.
Thoracotomy closure (timing: 5 min)
32. Close the thoracotomy using an absorbable braided 4-0 PGA suture. Place sutures under the lower rib and over the upper rib at three or more points along the thoracotomy site in an interrupted fashion to make sure the lower and upper ribs are approximated together without air leaking through the incision (Fig. 4G, 5G-H). Then, re-approximate the chest muscle in the same way with interrupted sutures at points which alternate in position from the sutures closing the thoracic cavity.
a. CRITICAL STEP: Alternating the placement of the sutures re-approximating the thoracic cavity and the chest muscle will ensure a tight seal to restore the negative pressure in the thoracic cavity.
33. Re-approximate skin subcutaneous tissue with a running non-absorbable 4-0 nylon suture (Fig. 4H, 5I).
a. CRITICAL STEP: Ensure that the intra- and extrathoracic portions of the device are fully covered under the skin to minimize risk of infection and discomfort to the animal.
Extubation and regaining of consciousness (timing: 20 min)
34. Reduce isoflurane vapors to 0% (vol/vol) with maintained 100% oxygen flow at 2 L/min to allow the animal to recover from sedation. Keep the animal on the ventilator and under a heating lamp.
35. During this recovery time, perform more thorough pacemaker threshold testing.
36. Once the rat retracts its foot from a gentle toe pinch, extubate the animal and place it on a nose cone with 100% oxygen flow.
37. When the animal regains sternal recumbency, return the animal to a clean home cage with food and water. Monitor the animal for an additional 1-2 h in the home cage. Provide additional warmth with by a heat lamp or heating pad.
Post-operative blood draw (timing: 15 min per animal)
38. Collect blood and serum samples by performing a tail-vein blood draw to assess serology and biomarkers of myocardial infarction and heart failure. For myocardial infarction assessment, draw blood 3-6 hours after surgery. For heart failure, draw blood 3 weeks after surgery. For serology assessment, draw blood every 2 weeks after surgery.
39. Prepare a heating pad and a nose cone for maintenance of a light plane of anesthesia. Drape an absorbent pad over the heating pad.
40. Induce general anesthesia using inhaled isoflurane vapors by placing the animal in an induction chapter with 2 to 3% of isoflurane (vol/vol) and an oxygen flow of 2 mL/min for several minutes until the rat becomes unconscious.
41. Move the rat onto the surface of the heating pad with its nose placed in the nose cone. Confirm that the rat is unconscious using a toe pinch.
42. In order to dilate blood vessels, place the tail of the rat into a container of warm water for one minute. Dry the tail and wipe down with 70% ethanol.
43. Identify the lateral tail vein. Insert a 25-gauge sterile needle (or smaller) into the vein about two thirds of the way down the tail. If additional vein entry is needed, proceed up the tail proximally.
44. Gently pull the syringe to draw out a sufficient volume of blood.
a. CAUTION: A rat’s approximate total blood volume is 62 mL/kg. The maximum volume of blood drawn in one instance depends on the frequency of collection: 3% blood volume for every 3 days, 7.5% blood volume for every week, or 15% blood volume for every 2 weeks.
45. Once enough blood has been collected, remove the needle and apply gauze using slight pressure to ensure the bleeding has stopped.
46. Collect the blood into serum separator tubes.
47. Return the animal to its cage and monitor until recovery. Take care to ensure that any excessive bleeding is controlled.
48. For serum samples, spin the collected blood at 2400G for 10 minutes 4℃.
49. If necessary, store serum samples at 4℃ overnight or freeze at -20℃ for longer-term storage.
Recovery and postoperative monitoring (timing: 2+ days)
50. Monitor the animal two times per day until recovery is complete. Continue with subcutaneous analgesic doses of buprenorphine (0.05 mg/kg) with a 1:2 dilution of saline every 12 hours in the 48 hours postoperative period.
51. If the rats show any signs of distress, such as continued labored respiration more than 1 hour after surgery or perioral or perinasal porphyrin discharge, administer additional analgesics. If animals present extended signs of discomfort, consider euthanization with the consultation of animal research facility veterinarians.
Functional testing of pacemaker stimulation (timing: 30+ days)
52. Induce general anesthesia using inhaled isoflurane vapors by placing the animal in an induction chapter with 2 to 3% of isoflurane and 2 mL/min oxygen for several minutes until the rat becomes unconscious.
53. Attach subdermal electrocardiogram needle electrodes to the animal in the Lead II configuration (left arm: ground electrode; right arm: negative electrode; right leg: positive electrode).
54. Wirelessly power the pacemaker. Observe the ECG signal trace and identify whether the pacemaker is capturing the heart rhythm. If there is no capture, increase the power. If there is capture, lower the power to the minimum threshold for capture.
55. Once capture or device failure is confirmed, remove the ECG electrodes from the animal and gently return the animal to its cage.
Echocardiography (timing: 15 min per animal, every 2 weeks for >8 weeks)
56. Induce general anesthesia using inhaled isoflurane vapors by placing the animal in an induction chapter with 2 to 3% of isoflurane (vol/vol) and an oxygen flow of 2 mL/min for several minutes until the rat becomes unconscious.
57. Confirm loss of consciousness by gentle toe pinch.
58. Transfer the animal to the imaging stage. Affix the paws to ECG electrodes with tape to monitor the heart rate throughout recording.
a. CRITICAL STEP: Maintain the heart rate between 300-350 BPM by adjusting the level of isoflurane vapors. Too much variance in heart rates between animals can affect the comparison between parameters.
59. Using clippers, shave the chest hair to the left of the sternum. Apply a hair removal gel using a cotton swab to further remove chest hair from the shaved area.
a. CRITICAL STEP: Hairs on the chest may distort the ultrasound signal with noise. Ensure that as much as possible of the chest hair in the area of interest is removed.
60. Apply ultrasound gel to the shaved chest area.
61. Perform M-mode echocardiography of the left ventricle.
62. Analyze data using software to generate echocardiographic parameters.
Weight monitoring (timing: 2 min per animal, every 2 weeks for >8 weeks)
63. Weigh each animal at a consistent frequency every (i.e.: every 3 days) over several weeks.
Behavioral assessment (timing: 2 min per animal)
64. For the first 48 hours after surgery, monitor the animals’ behavior every 12 hours.
65. Grade the behavior of each animal on the following scale for behavior and reactivity to handling, respectively: Behavior: 1 - normal, 2 - minor changes, 3 - decreased activity/mobility, 4 - immobility. Reactivity to handling following surgery was scored as: 1 - normal, 2 - mild dysfunction, 3 - slow.
Assessment of biomarkers of myocardial infarction and heart failure (timing: 3-7 hrs per assay)
66. To assess myocardial infarction, collect serum samples 3-6 hours after surgery as described above.
67. To assess heart failure, collect serum samples 3-6 hours after surgery as described above.
68. Perform enzyme-linked immunosorbent assay for each sample.
Tissue collection (timing: 30 min per animal)
69. Deeply anesthetize the animal using inhaled isoflurane vapors by placing the animal in an induction chapter with 5% of isoflurane (vol/vol) at 2 mL/min oxygen flow for several minutes until the rat becomes unconscious.
70. Confirm adequate anesthesia by toe pinch.
71. Make an incision just below the sternum and enter the thoracic cavity through the diaphragm.
72. Harvest the heart by gripping the lungs and cutting at the base of the heart to achieve euthanasia by exsanguination.
73. Cannulate the excised heart via the aorta.
74. Retrograde perfuse University of Wisconsin cardioplegic solution followed by perfusion of neutral buffered formalin (10%).
75. Store the hearts in neutral buffered formalin (10%) for 24 hours. Then, switch the storage solution to 70% ethanol.
Histology (3+ days)
76. Embed heart samples in paraffin and section into cross sections.
77. Process samples with either Masson’s trichrome.
78. For Masson’s trichrome staining, quantify the volume fraction of myocardium, collage, and interstitial space near the site of device attachment using our custom MATLAB software.