Hong-Tao Wang1, Bo-Yuan Fan1#, Fei-Fei Su1, Di Zeng1, Tao Chen2* and Qiang-sun Zheng1*
1Department of Cardiology, Tangdu Hospital, Fourth Military Medical University, Baqiao District, Xi'an City, Shaanxi, China
2Department of Anatomy, Histology and Embryology, and K. K. Leung Brain Research Centre, Fourth Military Medical University, Xian, China
#Contribute equally to this paper
Received: 25 September, 2015; Accepted: 14 October, 2015; Published: 17 October, 2015
Qiang-Sun Zheng, Department of Cardiology, Tangdu Hospital, Fourth Military Medical University, Baqiao District, Xi'an City, Shaanxi,710038, China, Tel: +86-029-84777422; Fax: +86-029- 8477422; E-mail:
Tao Chen, Department of Anatomy, Histology and Embryology, and K. K. Leung Brain Research Centre, Fourth Military Medical University, Xian, China
Wang HT, Fan BY, Su FF, Zeng D, Chen T, et al. (2015) Autonomic Innervation from the Aortic Root Ventricular Ganglionated Plexi to the Pulmonary Vein: A Novel Pathway. J Cardiovasc Med Cardiol 2(2): 021-025. DOI: 10.17352/2455-2976.000018
© 2015 Wang HT, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Background: Autonomic nerve innervation pathway from the ventricular GP to the pulmonary veins (PV) remains unclear.
Aim:This study investigates the autonomic innervations from aortic root ventricular GP to the PVs. Nissl's staining and fluorescent dual label staining were performed to determine the neuron structure in the aortic root GP in five dogs. Avidin Biotin Complex (ABC) staining were performed to study the efferent autonomic pathway from the aortic root GP to the PVs.
Results:Adrenergic and cholinergic neurons were both present in the aortic root GP, with the majorities were cholinergic. ABC positive nerve fibers that contained both cholinergic and adrenergic neurotransmitters penetrated directly from the aortic root GP to the left PVs.
Conclusion: Autonomic innervation of the Left PVs is partly originated from the aortic root ventricular GP.
There are at least 7 ganglionated plexi (GP) located in the canine epicardium including 4 atrial GP and 3 ventricular GP .Several studies have demonstrated that most autonomic nerve fibers penetrated from the atrial GP to the pulmonary vein (PV) [2,3]. And the ventricular GP, has long been regarded only relative to the function of ventricle,eg. Blood pressure [4,5]. However, our previous study  demonstrated stimulation of the aortic root ventricular GP provoked AF in the absence of any extrinsic cardiac nerve activity. The aortic root ventricular GP was also an important element in the intrinsic neuronal loop that can increase the risk of AF in isolated heart models.
We hypothesized that some autonomic nerve fibers may directly project from the ventricular GP to the PVs except the atrial GP. And the goals of the present study were therefore two-fold. First, we determined the neuronal cell structure present in the aortic root GP by Nissl's staining and fluorescent staining to determine if existed neuronal cells that expressing dual neuronal markers. Second, we performed biotinylated dextran amine (BDA) tracing and the Avidin Biotin Complex (ABC) staining to study the specific efferent autonomic pathways from the aortic root GP to the PV.
Neuronal cell structure of the aortic root GP
Tissue preparation:Five adult mongrel dogs were anesthetized with sodium pentobarbital (30 mg/kg IV), then hearts were excised from each of the dog, and a cannula was infused into the aorta ascendens. This was followed by perfusion with 250ml of physiologic saline to clean the hearts. The tissues were perfused and fixed with 1L of 4% formalin, and then stored in 30% sucrose overnight after an additional post-fixation for 4h in 4% formalin. The tissues were then embedded in OCT (TissueTek) and cut into 20 mm sections at -25 °C with a cryostat (Kryostat 1720; Leitz, Mannheim, Germany).
Histochemistry and immunohistochemistry: The aortic root GP is embeded in adipose tissues surrounding the root of aorta and connected by the mesangial ligament as described in our previous study . Nissl's staining was used first to determine the location of the GPs in the aortic root GP. Fluorescent dual-labeling with antityrosine hydroxylase (TH) and anticholine acetyltransferase (ChAT) antibodies was performed to determine the neuron cell types in the GPs and to determine whether both adrenergic and cholinergic nerves co-existed within the GPs. Mouse anti-TH (1:500 dilution; Abcam) antibody was used to label the adrenergic nerves, and goat anti-ChAT (1:100 dilution; Millipore) antibody was used to label the cholinergic nerves. Fluorescein-labeled anti-mouse IgG was used as the secondary antibody. Fluorescein isothiocyanate (FITC) (1:50 dilution; KPL) was conjugated to TH, and Rhodamine-Labeled Anti-Goat IgG (1:50 dilution; KPL) was conjugated to ChAT. The dual-labeled sections were then visualized with a laser-scanning confocal microscope.
Autonomic innervation pathway from the aortic root GP to the PVs
Tissue preparation:5 adult mongrel dogs were anesthetized with the same method in protocol 1. The chest was opened using the median sternotomy method, and the cardiac pericardium was cut to expose the aortic root GP. Five microliters of 10% (w/v) biotinylated dextran amine (BDA) anterograde tracer (MW 10,000; lysine fixable; Molecular Probes, Invitrogen) dissolved in 0.05 M PBS was then injected into the aortic root GP with micropipettes. A total of 5 injection sites adjacent to the root of the right region of the aortic root GP were selected, and the micropipettes were used at each injection site for 5min. After BDA injection, the dogs were allowed to survive for 7 days, then were sacrificed, perfused, and fixed trans-aortically as described above.
Histochemistry and immunohistochemistry:The four PVs with injected BDA were cut into 10-μm-thick slices on a cryostat. Avidin-Horseradish peroxidase (HRP) complex (1:100) was first used to label BDA. The series of slices were incubated at room temperature for 2-3h, and BDA positive nerve fibers was showed by the reaction solution (Tris-HCl (0.05mol/L,pH7.6)50ml,DAB50mg,twice distilled water 50ml, Nickel ammonium sulfate solution 200μl). After DAB was dissolved, the incubated slices were put into the reaction solution, and 5μl of 0.03% H2O2 was add each time to allow better reaction. Finally, the slices were cleaned with PBS and were examined under a laser-scanning confocal microscope
Then the series of slices were processed for BDA, TH, and ChAT triple immunofluorescence histochemistry. Avidin-conjugated CY5 (1:50 dilution; Southern biotech) was used to label BDA, and both TH and ChAT were labeled as described above. Finally, the labeled slides were examined under a confocal laser-scanning microscope.
All data are expressed as means±SD. The LSD-t test was adopted to analyze the difference between two groups. Probability values ≤0.05 were considered significant.
Co-expression of adrenergic and cholinergic neurons in the aortic root GP
The aortic root GP were cut into three parts from right to left, and Nissl's staining was first performed to determine the location of the GPs. The results revealed that neurons are widely distributed in the GPs and mixed with fat cells, which present in small clusters of 3 to 15 cells, with most embedded in the right part of aortic root GP (Figure 1). Fluorescent double staining showed adrenergic and cholinergic neurons were both observed, but the majority of neurons in the GP were of the cholinergic subtype (Figures 2A, 2B). Moreover, a proportion of neurons expressed dual adreno-cholinergic phenotypes (Figure 2C). This suggested that the ventricular GP had the same neuron structure with the atrial GP.