Open thoracic surgical implantation of cardiac pacemakers in rats

Abstract

Genetic engineering and implantable bioelectronics have transformed investigations of cardiovascular physiology and disease. However, the two approaches have been difficult to combine in the same species: genetic engineering is applied primarily in rodents, and implantable devices generally require larger animal models. We recently developed several miniature cardiac bioelectronic devices suitable for mice and rats to enable the advantages of molecular tools and implantable devices to be combined. Successful implementation of these device-enabled studies requires microsurgery approaches that reliably interface bioelectronics to the beating heart with minimal disruption to native physiology. Here we describe how to perform an open thoracic surgical technique for epicardial implantation of wireless cardiac pacemakers in adult rats that has lower mortality than transvenous implantation approaches. In addition, we provide the methodology for a full biocompatibility assessment of the physiological response to the implanted device. The surgical implantation procedure takes ~40 min for operators experienced in microsurgery to complete, and six to eight surgeries can be completed in 1 d. Implanted pacemakers provide programmed electrical stimulation for over 1 month. This protocol has broad applications to harness implantable bioelectronics to enable fully conscious in vivo studies of cardiovascular physiology in transgenic rodent disease models.

Extended Data Fig. 1 |. Assessment of surgery time.

a, The surgery workflow timeline is as follows: induction, intubation, incision, closure and extubation. b, For an experienced surgeon performing this procedure, the total time for surgery from induction to extubation is ~43 min, including initiation of anesthesia, intubation, incision, affixation of pacemaker, closure and extubation. Average times for an experienced surgeon to complete each section of the workflow timeline are provided. For operators just beginning to learn this technique, intubation may take several additional attempts. Additional time is required pre- and postoperatively to fulfill responsibilities such as preparation of equipment, retrieval and return of animals to the housing facility, administration of follow-up analgesic doses and recovery observation.

Extended Data Fig. 2 |. Open thoracic implantation technique for biventricular pacemaker implantation.

a, Electrodes of biventricular pacemaker positioned onto ventricle of Langendorff-perfused mouse heart. Scale bar, 5 mm. b, Electrodes of the biventricular pacemaker sutured onto left and right ventricles during implantation surgery. Scale bar, 5 mm. c, Anterior and d, cross-sectional CT visualization of the biventricular pacemaker placed within a rat’s anatomy.

Extended Data Fig. 3 |. Implantation of miniature battery-free wireless pacemakers using hydrogel adhesive.

a, This technique allows for pacemakers to be attached with adhesives. Following opening of the chest, the pacemaker is affixed to the epicardial surface of the ventricles with a soft injectable bioadhesive. UV light is used to illuminate the bioadhesive to cure and secure the electrode pad in place. Scale bar, 5 mm. b, ECG traces of animals implanted with pacemakers were recorded daily postoperation. Pacemakers were able to capture and drive the heart rhythm for up to 8 d postsurgery.

Fig. 1 |. Overview of surgical implantation of wireless battery-free miniature pacemakers in rodents.

a, Open thoracic implantation approach allows for full implantation of miniature, battery-free devices (pacemakers) for cardiac rate and rhythm therapy. b, Various wireless miniature pacemakers can be implanted using this technique: optical and electrical pacemakers (left), bioresorbable pacemakers (center) and biventricular pacemakers (right). Each device is composed of three primary components: the receiver, the electrode pad, and the serpentine connector. Scale bars, 5 mm.

Fig. 2 |. Surgical space setup

. a, Surgical station setup for pacemaker implantation surgery. From left to right: anesthesia induction chamber; tape; intubation equipment; 4% chlorhexidine gluconate solution (chl. hex. sol’n); clippers; spotlight; nose cone; mini spotlights; ECG electrodes; ECG recording laptop; small-animal ventilator; heat lamp; sterile supplies and tools; hot bead sterilizer. All supplies are purchased sterilized. The sterile instrument area was set up on the dominant-hand side of the surgeon’s workspace. Globally illuminate the operating field with a large spotlight. Use at least two sources of small adjustable miniature spotlights to accurately illuminate the immediate incision area and to prevent shadowing that may obscure visualization during surgery. b, Intubation tools and stand, including: dental mirror; standard forceps; blunt curved stylet; 16-gauge catheter. For anterior neck cutdown, additional equipment is required: nose cone (not pictured); metal base plate; magnetic retractors. c, Tools and supplies required for pacemaker implantation surgery. Tools from left to right: standard forceps; microspatula; fine forceps; surgical scissors, sharp; Metzenbaum scissors, blunt/blunt; Goldstein retractor; Crile hemostat, curved; Castroviejo needle holder; Crile hemostat, straight; Halsey needle holder.

Fig. 3 |. Intubation technique for open thoracic approach and surgical table preparations before incision.

a, The open thoracic approach requires intubation and assisted ventilation of the unconscious animal before incision. From left to right: place the rat on the intubation stand so that the nose is positioned in the stand’s nose cone to maintain general anesthesia by inhalation. Secure the nose in place by threading the stand’s thread anterior to the teeth. Retract the rat’s tongue by pinching it with fingers or forceps. Blindly pass a 16-gauge catheter supported by a blunt stylet into the trachea. Confirm placement of the endotracheal tube by condensation on the dental mirror. b, The endotracheal tube of the intubated rat is connected by the catheter to the ventilator at 80 breaths per minute on pressure control with a peak inspiratory pressure limit of 14 cmH₂O to maintain anesthesia. c, Position the intubated rat on the surgical table covered with an absorbent pad. Shave and disinfect the chest area to the left of the sternum (shown in dotted gray outline). Place the ECG electrodes in the Lead II position to record ECGs and monitor heart rate throughout the procedure. Use a miniature spotlight to provide lighting into the thoracic cavity. Scale bar, 5 cm.

Fig. 4 |. Pacemaker implantation technique for attachment to ventricles.

a, Palpate for the fourth intercostal space, which is usually two to three intercostal spaces below the most maximum pulsation from the heart. b, Using scissors, make a 4 cm curvilinear skin incision along the curvature of the ribs over the fourth intercostal space (pictured) and then dissect through the chest wall muscle. c, Using Metzenbaum scissors, carefully enter the pleural space while taking care not to injure the lung (pictured) and then extend the incision through the intercostal space anterior and posteriorly. d, Place a rib-spreader retractor to hold the rib space open. e, Gently hold the lung away from the heart with a cotton swab Secure the cotton swab in place with hemostats. Clear the pericardium from the heart with a cotton swab. Using a 6–0 suture, place sutures through the epicardium to secure the electrode pad of the pacemaker. f, Create a subcutaneous pocket using scissors. Secure the receiver of the pacemaker in the subcutaneous pocket. Remove the retractor to make sure there is no tension or excess length of the electrode. Inset shows schematic illustration of suture technique for attachment of the electrode pad to the heart (see e). g, Close the intercostal space with simple intermittent absorbable 4–0 sutures so that the serpentine electrode intersects the intercostal space and the receiver coil rests in a subcutaneous pocket. h, Close the muscle and skin incisions with running nonabsorbable 4–0 suture. Scale bars, 2 mm.

Fig. 5 |. Pacemaker implantation technique for attachment to right atrium.

a, Palpate for the third intercostal space, which is usually two intercostal spaces below the point of maximum impulse from the heart. b, Using scissors, make a 3–4 cm curvilinear skin incision along the curvature of the ribs over the third intercostal space (pictured) and then dissect through the chest wall muscle. c, Using Metzenbaum scissors, carefully enter the pleural space while taking care not to injure the lung, and then extend the incision through the intercostal space anterior and posteriorly. d, Hold the lung away from the heart with a cotton swab. Secure the cotton swab in place with hemostats. Clear the pericardium from the heart using a cotton swab. e, Create a subcutaneous pocket with blunt dissection using Metzenbaum scissors while taking care to stay in the subcutaneous plane right under the dermis and avoid violating the peritoneum. Place the pacemaker receiver in the subcutaneous pocket. f, Using a 6–0 suture, place sutures through the right atrium to secure the pacemaker via the electrode pad. g, Close the intercostal space by placing a simple-interrupted 4–0 absorbable suture across the inferior and superior ribs while protecting the organs underneath with a metal spatula. The device electrode intersects the intercostal space. h, Close the intercostal space so that it is airtight. i, Close the muscle and skin incisions with running 4–0 nonabsorbable suture. Scale bars, 2 mm.

Fig. 6 |. Physiological effects of pacemaker implantation surgery.

a, Representative images of Masson’s trichrome-stained cross-sections of rat hearts implanted with pacemakers. Pink represents myocardium, blue represents fibrosis and white represents interstitial space. Scale bars, 1 mm. b, Volume fractions of interstitial space, collagen and myocardium in Sprague Dawley rats that were implanted with pacemakers or underwent Sham surgery. A significant increase in fibrosis—indicated by collagen %—was found 6 weeks following surgery in rats implanted with pacemakers. Kruskal–Wallis test. Post-hoc Dunn’s multiple comparison test. P values: *, 0.002; $, 0.105; #, 0.0101; &, 0.0442. c, Animal weight dropped immediately following surgery and was steadily regained—as expected—in the following weeks. d, Significant increase in levels of cardiac troponin 3–6 h following surgery compared with control (Cntl). Kruskal–Wallis test, **P, 0.0085. e, No significant differences in levels of BNP 45 before surgery (Cntl.), 3 weeks following surgery or compared with sham. Kruskal–Wallis test, P, 0.9114. f, No significant differences in ejection fraction before surgery (Cntl.), 1 week postoperation and 3 weeks following surgery, showing that the procedure does not impair cardiac mechanical function. Sham versus device implanted: Kruskal–Wallis test, P, 0.1058. Sham: Friedman test, P, 0.2731. Device implanted: Friedman test, P, 0.5216. Post-hoc Dunn’s multiple comparison test, α = 0.05. g, No significant differences in stroke volume before surgery (Cntl.), 1 week postoperation or 3 weeks following surgery, demonstrating that the procedure does not impair cardiac output. Sham versus device implanted: Kruskal–Wallis test, P, 0.1851. Sham: Friedman test, P, 0.9306. Device implanted: Friedman test, P, 0.3673. Post-hoc Dunn’s multiple comparison test, α = 0.05. In b–g, values are reported as mean ± standard deviation. In b, n = 3 biologically independent animals per group. In d,e, n = 4 or 6 biologically independent animals per group. In c, n = 6–12 biologically independent animals per day. In f,g, n = 4–5 biologically independent animals for each experimental group.

Fig. 7 |. Long-term in vivo pacing following pacemaker implantation.

As is standard in clinical pacemaking, we wirelessly powered the implanted pacemakers while recording an ECG to determine from the ECG signal pattern if the heart rhythm could be captured by the pacemaker. a, ECGs recorded from animals with pacemaker electrodes attached to the right atrium the day following surgery show pacing spikes at the P wave that drives increased heart rhythm. b, ECGs recorded from animals with pacemaker electrodes attached to the ventricle the day following surgery demonstrate paced QRS complexes that drive the increased heart rhythm. c, Live stimulation setup where stimulation was delivered via wireless inductive power transfer system and ECGs were recorded using LabChart software. ECGs recorded from nonanesthetized animals immediately following surgery show conversion from sinus rhythm to a paced rhythm when electrical stimuli were delivered by the pacemaker. d, ECG traces show conversion from the normal sinus rhythm to paced beats for up to 32 d following surgery when electrical stimuli were delivered by the implanted pacemaker. e, Explantation of a pacemaker 1 week after surgery shows that the device was well incorporated into the body at the subcutaneous and intercostal space. In addition, electrode pads were still well secured to the heart. Red arrows indicate when an electrical stimulus was applied to the heart via the electrodes. The strength of the electrical stimulus was adjusted for each animal as the minimum electrical energy required to see a capturing paced rhythm on the ECG. Scale bars, 2 mm.