All experiments were performed using adult mongrel dogs (n = 10; all males; weights, 15–25 kg). Adult mongrel dogs were purchased from International Center Laboratory Animal (Seoul, Korea) and were housed in the Seoul National University Hospital Biomedical Research Institute. We only included male mongrel dogs in this study, and use of male mongrel dogs is usually accepted study design in cardiac model. Further study will be performed to find sex differences in GP distribution and innervation. General anesthesia was induced with Zoletil^® (intravenously injected, 5 mg/kg; combination of tiletamine/zolazepam, Virbac S/A, Carros, France) and maintained with isoflurane gas (1%–2% in oxygen). The animals were intubated and mechanically ventilated. Core body temperature was maintained at 36.5°C–37°C. A limb lead electrocardiogram was continuously monitored during surgical procedure. Under sterile surgical conditions, a left or right lateral thoracotomy was performed at the fourth intercostal space, followed by a pericardiotomy. After surgical procedure, meloxicam was intravenously injected (0.2 mg/kg) for 3 days to relief pain and discomfort in animals. Dogs were monitored daily. At the end of the study, animals were fully anesthetized with Zoletil^® and isoflurane gas and euthanized by induced ventricular fibrillation using direct contact of 9 V battery to ventricle.

CTB was used as a retrograde neuronal tracer that is taken up by nerve terminals and retrogradely transported along the axon at a rate 6.3 mm/hr.18 It specifically binds to GM 1 gangliosides which are concentrated on the synaptic membranes of nerve cells.17 Fig. 1A shows how CTB identifies the origins of neurons innervating a particular muscle, schematically. CTB is not transferred trans-synaptically; and thus remains in vesicles and appears as granules in the neuron cell body.17 CTB conjugated with Alexa Fluor (CTB-AF) is a highly sensitive and reliable retrograde tracer used for identifying the origins of neurons innervating a particular muscle, or examining multiple pathways within nerve tissues. In this study, CTB-AF 488 (green), and CTB-AF 555 or 594 (red) (Invitrogen, Carlsbad, CA, USA) were directly injected into the atrium or GPs. We then examined the retrogradely-transported CTB-AF in GP for the purpose of evaluating which GPs innervated to a particular atrial area or connected with other GPs. The tracer was dissolved in phosphate buffered saline (0.1% concentration [500 µg dissolved in 500 µL buffer]).17

The tracers were injected after surgically exposing the cardiac atria. The following two sets of experiments were performed. In Experiment 1, CTB-AF was injected into the targeted atrial area to determine the patterns of atrial innervation by GPs (Fig. 1B). In Experiment 2, CTB-AF was injected into epicardial fat pads containing the ganglia for the purpose of characterizing the neuroanatomical interconnections among different GPs (Fig. 1C). In both experiments, CTB-AF was carefully injected to avoid leakage of the solution into the surrounding tissues. Table 1 describes the injection sites and the types of fluorescence used in each experimental animal. After injection, the chest wall was closed.

Experiment 1 was performed using six mongrel dogs. CTB-AF was injected into four atrial sites as illustrated in Fig. 1B. These included right atrial (RA) sites (RA free wall [RAFW] and the RA appendage [RAA]) and left atrial (LA) sites (LA free wall [LAFW] and the LA appendage [LAA]). A total of 500 µL of the tracers was injected into each area (20 injections of 25 µL per injection) using a Hamilton Neuros syringe (Hamilton, Reno, NV, USA). One dog was labeled with a single fluorescent tracer injected into the RAFW. Five of the 6 dogs were double-labeled, of which CTB-AF was injected into the RAFW and RAA of three dogs, and into the LAFW and LAA of two dogs. Each site was labeled with a different color of fluorescence.

Experiment 2 was conducted using four dogs. Two dogs underwent a right lateral thoracotomy, and CTB-AF was injected into right-sided fat pads, which included the anterior right GP (ARGP) and inferior right GP (IRGP) (green and red-fluorescence, respectively). The ARGP located between the caudal end of the sinoatrial node and the right superior pulmonary vein-atrial junction. The IRGP located at the junction of the inferior vena cava and both atria. The other two dogs underwent a left lateral thoracotomy, and tracers were injected into left-sided fat pads, which included the superior left GP (SLGP) and ligament of Marshall (LOM) (green and red-fluorescence, respectively). The SLGP located adjacent to the left superior pulmonary vein-atrial junction between the LAA and left pulmonary vein. The LOM located between both left pulmonary veins and LAA. Fig. 1C shows the anatomic location of each GP. A total of 250 µL of CTB-AF was injected in each GP using a 1 mL insulin syringe (BD Ultra-FineTM II; Becton Dickinson, Franklin Lakes, NJ, USA).

Previous studies had reported that maximum numbers of CTB-labeled ganglion cells were observed 72 hours after injection, and there was no striking change in the distribution of the labeled neurons until 1 week.18 In this study, the tissues used for analyses were harvested either 4 or 7 days after injection. The following 5 GP were harvested from the animals and analyzed: superior vena cava-aorta fat pad (SVC-Ao FP), ARGP, IRGP, SLGP and LOM. Tissues were fixed in 4% formalin for one hour, then stored in 70% alcohol and embedded into paraffin blocks. The paraffinized tissue blocks were horizontally divided into 6 segments, and 4 μm thick sections were obtained from each segment. The first sections as representative sections of each segment were examined with a laser-scanning confocal microscope (LSM 710; Zeiss, Oberkochen, Germany) to detect CTB-labeled neuronal cells.

Then, immunohistochemical staining was done using anti-tyrosine hydroxylase (TH) (Accurate Chemical, Westbury, NY, USA) and anti-choline acetyltransferase (ChAT) (Chemicon, Billerica, MA, USA) for characterizing of CTB-labeled neuronal cells. TH is a marker for adrenergic neurons, and ChAT is a marker for cholinergic neurons.

Data were analyzed using both qualitative and semi-quantitative methods. First, we observed the presence of CTB-AF in a particular GP defined as a fat pad which included the ganglia. For the semi-quantitative analysis, ganglia that included CTB-labeled neurons were manually counted in the representative section of each segment by two independent investigators. If there was a disagreement between the two investigators, the case was assessed by a third independent investigator to reach a consensus. We used ZEN 2009 Light Edition software (Zeiss) for analyzing confocal microscopic images and Image J (version 1.47; National Institutes of Health, Bethesda, MA, USA) for cell count. The percentage of labeled ganglia was estimated as the number of CTB-labeled ganglia, compared to the total number of ganglia in a particular GP.

All numerical data are expressed as the mean ± standard deviation (SD). SPSS version 19.0 (IBM Corp., Armonk, NY, USA) was used for all statistical analyses. For semi-quantitative analyses, the number of CTB-labeled ganglia and the total number of ganglia are presented as their pooled numbers in each atrial or GP injection site across all experimental animals.

The protocol for this study was approved by the Institutional Animal Care and Use Committee of the Seoul National University Hospital; and animal were maintained in the facility accredited AALAC International (#001169) in accordance with Guide for the Care and Use of Laboratory Animals 8th edition, NRC (2010).

A total of 5 GPs were inspected in each animal to identify the presence of fluorescence. CTB-labeling was found in the neuronal cell bodies located in ganglia. Most neuronal cells in ganglia showed a cholinergic nature, as indicated by the presence of ChAT-positive cells (Fig. 4). The distribution of CTB-labeled ganglia varied in each animal (Table 1). Fig. 4 shows the representative microscopic images of immunohistochemical staining and CTB-labeled neuronal cells in the ganglia of LOM. ChAT staining shows the cholinergic nature of neuronal cells in ganglia and TH staining shows the adrenergic axon bundles within the same ganglia (Fig. 4A and B). CTB-labeled cells with red-fluorescence from the LAA and with green-fluorescence from the LAFW identified in a ganglia of LOM (Fig. 4C and D). The fluorescence is mainly observed in the cell bodies of cholinergic neurons in ganglia which were also ChAT-positive.

An analysis for the presence of neuronal tracer in a particular GP showed that GPs in either right or left-side were innervating to both the same and opposite side of atrium. In other words, GPs projected their axons widely to both the nearby and distant atrial areas. The RA tended to be dominantly innervated by right-sided GPs such as the ARGP and IRGP. CTB-AF injected into the RAFW was mainly observed in the ARGP and IRGP (4 of 4 dogs and 2 of 4 dogs, respectively). In one animal, CTB-AF from the RAFW was also observed in the SLGP, LOM, and SVC-Ao FP. CTB-AF from the RAA was detected in the ARGP and IRGP (3 of 3 dogs and 2 of 3 dogs, respectively). Additionally, the SVC-Ao FP (2 of 3 dogs) and SLGP (1 of 3 dogs) projected innervating neurons to the RAA. The LA tended to be innervated by right-sided GPs (2 of 2 dogs). Left-sided GPs innervated both the LAFW (LOM in 1 of 2 dogs, and the SLGP in 2 of 2 dogs) and LAA (LOM, in 1 of 2 dogs and the SLGP in 1 of 2 dogs), although these innervations occurred less frequently compared to right-sided GP innervations.

Our semi-quantitative analysis provided more detailed information concerning the predominance of right-sided GPs in atrial innervation. Based on the absolute numbers of CTB-positive ganglia, right-sided GPs were numerically predominant in atrial innervation (Fig. 5). The axons of right-sided GPs were widely projected to both RA and LA. However, a small number of neurons directly originating from left-sided GPs also innervated RA areas. While right-sided GPs showed dominance in atrial innervation, some atrial areas were also innervated by GPs located in the opposite side of an injected atrial area. Based on the proportion of CTB-labeled ganglia in each GP, all 4 atrial areas appeared to be primarily innervated by GPs located the same side of the injection sites; showing that atrial innervation by GPs had a tendency for laterality.

In 5 of 6 animals, two distinct atrial areas on the same side were injected with different types of fluorescence conjugated tracers. In those double-labeled animals, both dual-fluorescence labeled ganglia and dual-fluorescence labeled neuron cell bodies were observed (Fig. 4E).

We examined 5 GPs in 4 experimental animals, and the numbers of CTB-labeled ganglia and total ganglia found in those animals are presented in Table 1. As shown in Fig. 6, CTB-labeled neuronal cells were widely distributed throughout 4 GPs, regardless of where the CTB-AF injected GP was located. ARGP projected the largest numbers of innervating neurons to the IRGP, SLGP, and LOM (Fig. 7). Fig. 7 shows neuronal cells that were labeled with CTB-AF transported from different GPs. In Experiment 2, all animals were double-labeled with two different types of CTB-AF (488 and 555) that were injected into the ARGP and IRGP or SLGP and LOM, respectively. Similar to Experiment 1, dual-fluorescence labeled ganglia and neuronal cells were also found (Fig. 7B).