A novel ex-vivo isolated rabbit heart preparation to explore the cardiac effects of cervical and cardiac vagus nerve stimulation

Abstract

The cardiac responses to vagus nerve stimulation (VNS) are still not fully understood, partly due to uncontrollable confounders in the in-vivo experimental condition. Therefore, an ex-vivo Langendorff-perfused rabbit heart with intact vagal innervation is proposed to study VNS in absence of cofounding anesthetic or autonomic influences. The feasibility to evoke chronotropic responses through electrical stimulation ex-vivo was studied in innervated isolated rabbit hearts (n = 6). The general nerve excitability was assessed through the ability to evoke a heart rate (HR) reduction of at least 5 bpm (physiological threshold). The excitability was quantified as the charge needed for a 10-bpm HR reduction. The results were compared to a series of in-vivo experiments rabbits (n = 5). In the ex-vivo isolated heart, the baseline HR was about 20 bpm lower than in-vivo (158 ± 11 bpm vs 181 ± 19 bpm). Overall, the nerve remained excitable for about 5 h ex-vivo. The charges required to reduce HR by 5 bpm were 9 ± 6 µC and 549 ± 370 µC, ex-vivo and in-vivo, respectively. The charges needed for a 10-bpm HR reduction, normalized to the physiological threshold were 1.78 ± 0.8 and 1.22 ± 0.1, in-vivo and ex-vivo, respectively. Overall, the viability of this ex-vivo model to study the acute cardiac effects of VNS was demonstrated.

Figure 1

Surgical window showing the dissection of the right vagus nerve (VN) at the cervical level for instrumentation with cuff electrodes in a rabbit. The carotid artery runs parallelly to the cervical VN. Cr cranial, cd caudal.

Figure 2

(a) Schematic of a cuff electrode wrapped around the cervical VN at mid-cervical level, approximately 4–5 cm cranial to the cardiac branching point of the superior cardiac branch. (b) Surgical window of a rabbit instrumented with the cuff electrode placed at the mid-cervical level. Cr cranial, cd caudal, CB cardiac branch, VN vagus nerve.

Figure 3

Surgical dissection of the vagus nerve (VN) for ex-vivo experiments including superior cardiac branch (CB). Here, the VN is shown from its caudal half of the cervical level to caudal to the superior CB. The heart is exposed after opening of the pericardium. Cr cranial, cd caudal, PA pulmonary artery, RA right atrium, RV right ventricle.

Figure 4

(a) Schematic showing the placement of needle electrodes. (b) Isolated innervated rabbit heart mounted to the working heart system. A pair of needle electrodes is placed close to the superior cardiac branch (cathode) and cranial to the cervical VN (anode). ECG leads were placed into the right atrium for heart rate detection. cr cranial, cd caudal, CB cardiac branch, ECG electrocardiogram, VN vagus nerve.

Figure 5

Schematic of the cardiac-synchronized stimulation. (a) Electrocardiogram (ECG) trace along with a stimulation burst with respect to the R-peak highlighted by the dashed lines in the ECG. (b) Detailed view of biphasic pulses applied per stimulation burst. C current amplitude, PW pulse width, NP number of pulses, F frequency, D delay between R-peak and stimulation onset.

Figure 6

Comparison of the baseline heart rate in-vivo versus ex-vivo before and after heart explantation. Data are represented as mean and standard deviation (SD). The “*” symbol indicates statistical significance. Bpm: beats per minute. In-vivo: heart rate measurements after instrumentation in-vivo, in-situ: heart rate measurements prior to heart explantation in the ex-vivo animals, ex-vivo: after heart explantation in Langendorff mode, HR heart rate.

Figure 7

Comparison of the normalized charges required to reduce heart rate by 10 bpm ex-vivo versus in-vivo. Data represent the mean values from five experiments in-vivo ex-vivo, and six experiments ex-vivo. Data are presented as mean and standard deviation (SD).

Figure 8

Representative ECG recordings for in-vivo (a,b) and ex-vivo VNS (c,d), each represented without and with overshoot. Curves a and c represent the ECG curves without overshoot in-vivo (a) and ex-vivo (c), respectively (“post stimulation”). Curves (b,d) represent the ECG curves with overshoot in-vivo (b) and ex-vivo (d), respectively after stimulation was turned off. The onset of stimulation is demonstrated by the decrease of heart rate in this ECG, whereas the stop of the stimulation is highlighted increasing heart rate. Overshoots are defined as an immediate increase of heart rate by at least 5 bpm above baseline after stimulation was turned off, followed by a return of heart rate back to the actual baseline.