Co-expression of estrogen receptor and nerve growth factor in rat intrinsic cardiac ganglia
Shaochun Zhang, Xiaoliu Liu, Guirong Cheng, Yane Xiong

Department of Anatomy, Medical College, Wuhan University of Science and Technology, Wuhan  430081, Hubei Province, China

Shaochun Zhang, Experimentalist, Department of Anatomy, Medical College, Wuhan University of Science and Technology, Wuhan  430081, Hubei Province, China

Corresponding author: Xiaoliu Liu, Master, Associate professor, Department of Anatomy, Medical College, Wuhan University of Science and Technology, Wuhan  430081, Hubei Province, China   
xiaoliu99611@163.com

Abstract
BACKGROUND: Previous studies have shown that neurons expressing estrogen receptor and nerve growth factor exist in the intrinsic cardiac ganglia in rats. However, it remains to be shown whether estrogen receptor and nerve growth factor are co-expressed within these cells.
OBJECTIVE: To determine whether estrogen receptor and nerve growth factor are co-expressed in intrinsic cardiac ganglia.
DESIGN, TIME AND SETTING: This cellular morphology observational study was performed at the Immunohistochemistry Department, Medicine School, Wuhan University of Science and Technology, between March and July in 2007.
MATERIALS: Mouse anti-estrogen receptor and rabbit anti-nerve growth factor polyclonal antibody, biotinylated goat anti-mouse IgG, and biotinylated goat anti-rabbit IgG were provided by Wuhan Boster, China.
METHODS: Ten healthy, Wistar rats were included in the present study. Ten sections of in-trinsic cardiac ganglia from the atrial posterior wall were randomly selected from each rat to perform estrogen receptor and nerve growth factor double-labeling immunohistochemical staining.
MAIN OUTCOME MEASURES: Expression of estrogen receptor and nerve growth factor in intrinsic cardiac ganglia of rats.
RESULTS: Immunohistochemistry results demonstrated expression of estrogen receptor and nerve growth factor in rat intrinsic cardiac ganglia, and double-labeling revealed co-expression of estrogen receptor and nerve growth factor in intrinsic cardiac ganglial cells.
CONCLUSION: Estrogen receptor and nerve growth factor were shown to be co-expressed in rat intrinsic cardiac ganglial cells.
Key Words: estrogen receptor; nerve growth factor; intrinsic cardiac ganglia; immunohistochemistry; co-experession

INTRODUCTION
  
Previous studies have primarily focused on estrogen receptor (ER) and nerve growth factor (NGF) expression with regard to myocardial action[1]. However, intrinsic car-diac ganglia (ICG) are considered to be the main components of the intrinsic cardiac nervous system and play a pivotal role in modulation of cardiac function. Previous studies have shown the distribution and interaction of substances, such as ER and its receptor[2], androgen and its receptor[3], NGF[4], and interleukin-6[5] in the ICG. These studies provided a morphological basis for ICG functional studies. The present study aimed to determine whether ER and NGF are co-expressed in the rat ICG.

MATERIALS AND METHODS

Design
Utilizing double-labeling immunocyto-chemical staining methods, a cellular mor-phology observational study was designed.
Time and setting
Experiments were performed at the Immu-nohistochemistry Department, Medicine School, Wuhan University of Science and Technology, China, between March and July in 2007.
Materials
Ten healthy, Wistar rats, of clean grade, irrespective of gender, aged 2–3 months, and weighing 180–250 g, were provided by the Experimental Animal Center, Tongji Medical College of Huazhong University of Science and Technology, China (License No. SCXK(E)2004-0007). All experimental pro-tocols were in accordance with the Guid-ance Suggestions for the Care and Use of Laboratory Animals formulated by the Min-istry of Science and Technology of the People’s Republic of China[6].
The main reagents and instruments used are as follows: 

Method
Harvesting specimens
The rats were anesthetized via an intraperitoneal injec-tion of 0.4% pentobarbital (1 mL/100 g), thoracic surgery was performed, and then the rats were rapidly perfused with 200 mL warm, physiological saline for 5–10 minutes, followed by 200 mL 4% paraformalde-hyde/phosphate-buffered saline (pH 7.4). Because the ICG are scattered around the cardiac muscles below the adventitia of atrial posterior wall in normal rats, this study used the atrial posterior wall above sulcus for vena cava. This was fixed for 2 hours with 4% paraformalde-hyde/phosphate-buffered saline and placed overnight at 4 °C in 20% sucrose/ phosphate-buffered saline (pH 7.4). The tissue was then cut into 15-μm thick serial sections using a cryostat, and the tissue was prepared for ER and NGF double-labeling immunohistochemical staining fol-lowing microwave antigen retrieval.
ER and NGF double-labeled immunohistochemistry
Five sections were selected from 6 rats (30 sections in total), and these sections were rinsed with phos-phate-buffered saline, followed by mouse anti-ER and rabbit anti-NGF polyclonal antibody    (1: 100) overnight at 4 °C. Subsequently, the sections were incubated in biotinylated goat anti-mouse IgG and biotinylated goat anti-rabbit IgG (1: 100) for 30 min-utes at 37 °C, followed by streptavidin-horseradish peroxidase complex (1: 200) for 30 minutes at 37 °C, diaminobenzidine coloration, conventional dehydra-tion, transparency, and mounting with a neutral gum.  
Double-labeled immunohistochemistry
Five sections were selected from each of the remaining four rats (20 sections in total). The sections were then rinsed with phosphate-buffered saline and incubated in mouse anti-ER polyclonal antibody (1: 100) overnight at  4 °C, followed by biotinylated goat anti-mouse IgG     (1: 100) for 30 minutes at 37 °C, streptavidin-horseradish peroxidase complex (1: 200) for 30 minutes at 37 °C, DAB coloration, phosphate-buffered saline washing, rabbit anti-NGF overnight at 4 °C, biotinylated goat anti-rabbit IgG (1: 100) for 30 minutes at 37 °C, strepta-vidin-alkaline phosphatase complex (1: 200) for 30 min-utes at 37 °C, fast-red counterstain, conventional dehy-dration, transparency, and mounting with a neutral gum.  
Negative control trials
Primary antibody was removed or normal goat serum was used for incubation, and no immunological reaction occurred. Coloration mixture control trial: following the first series of staining protocols, the sections were rinsed with phosphate-buffered saline and then used for a second series of staining protocols. The results were negative due to lack of primary antibody, which indicated no interference between the two series of immunological reactions and immune specificity.
Main outcome measures
Expression and co-existence of ER and NGF in the rat ICG.

RESULTS

ICG distributions
The ICG was distributed in a sparse manner surrounding the cardiac muscles and below the adventitia of the atrial posterior wall in normal rats. The ICG comprised various sized ganglia and various numbers of neurons. The ER- and NGF-positive cells were scattered throughout, and those containing a nucleus were quantified.
ER-positive cells in the ICG
ER-positive cells were observed in the ICG. The ER im-muno-positive substances were primarily located in the nuclei, and the cytoplasm and processes were slightly stained. The positive cells were round or oval and of varying sizes, and a total of 450 positive neurons were quantified, which accounted for 60% of the total number of ganglial cells (450/750, subsequent to hematoxy-lin-eosin staining, 750 ganglial cells with blue nuclei were quantified; Figure 1).

NGF-positive cells in the ICG
NGF-positive cells were observed in the ICG. The NGF immuno-positive substances were primarily located in the nuclei. Although the cytoplasm lacked staining, some positive cells exhibited a slightly stained cytoplasm. The positive cells were round or oval and of varying sizes. A total of 420 positive neurons was quantified, which ac-counted for 65% of the total number of ganglial cells (420/645, subsequent to hematoxylin-eosin staining, 645 ganglial cells with blue nuclei were quantified; Figure 2).

ER/NGF double-labeled cells in the ICG
In the ICG, three types of labeling were observed. ER expression was located in the nucleus; NGF expression was located in the cytoplasm; ER/NGF double-labeled cells exhibited ER expression in the nucleus and NGF expression in the cytoplasm. There were approximately 225–300 double-labeled cells, which accounted for 30%-40% of total labeled neurons (Figure 3).

DISCUSSION

The intrinsic cardiac nervous system contains a wide dis-tribution of intrinsic ganglia, scattered neurons, and nerve fiber-containing nerve plexus. The ICG is the predominant component of the intrinsic cardiac nervous system and plays a pivotal role in the modulation of cardiac function. ICG are spread throughout the entire heart and proximal myocardium below the epicardium, with a greater number in the atrium than in the ventricle, and a large number in the atrial posterior wall, interatrial septum, and coronary sulcus. Therefore, positively stained ICG cells were quan-tified in the atrial posterior wall, thereby providing novel experimental evidence for complex functions of the intrin-sic cardiac nervous system.
ER mediated action modulates target gene transcription of neurons. Estrogen, through action with the ER, acts on the nervous system by influencing neuronal growth, de-velopment, plasticity, and survival, as well as neuro-transmitter synthesis[7]. In ovariectomized, adult, female rats, the number and density of dendritic spines are dramatically reduced in pyramidal cells of hippocampal CA1 region, but estrogen intervention can ameliorate this decreasing trend[8]. In cultured hippocampal cells, es-trogen intervention promotes the release of brain-derived neurotrophic factor and accordingly increases the num-ber and density of dendritic spines[9]. By binding to the α-receptor, which is a nuclei receptor-mediated mecha-nism, estrogen increases synaptic number and density, thereby promoting synapse appearance[10]. This sug-gests that estrogen induces dendritic spine formation. Moreover, estrogen decreases apoptosis and promotes neural regeneration in the developing cerebral cortex[11]. Estrogen has been shown to maintain growth, develop-ment, and survival of in vitro-cultured neurons that do not express NGF[12], which suggests an association between estrogen and NGF. In the present study, immunohisto-chemistry results demonstrated ER-positive cells in the LCG, which indicates the existence of estrogen binding sites in LCG. These results suggest that estrogen could provide protective effects to the intrinsic cardiac nervous system through the ER.
NGF is important for neuronal differentiation and sur-vival[13], in particular for survival of sympathetic neurons and sensory neurons in the peripheral nervous system[14]. A stable intrinsic cardiac environment depends on bal-anced input between the sympathetic and parasympa-thetic nervous systems. NGF controls synapse trans-mission between the autonomic nervous system and the myocardium, and it also affects the strength and number of synapse binding sites[15]. NGF rapidly modulates synaptic transmission between sympathetic neurons and myocardium, and this action is mediated by the protein tyrosine kinase receptor[16]. Previous results have shown that NGF is expressed within a short period following acute myocardial ischemia and reperfusion. In addition, endogenous NGF protect neurons from damage, which is been shown to be indirectly mediated by serine stretch protein kinase through adenosine[17]. In the present study, immunohistochemistry results demonstrated NGF ex-pression in the ICG, which suggested a neurotrophic effect for NGF in the ICG.
The central nervous system widely expresses estrogen and neurotrophic factor receptors, and co-expression of these two receptors could lead to the interaction of two signal transmission pathways that are mediated by estrogen. This suggests a complex interaction between estrogen and NGF[18]. Immunohistochemistry results demonstrated that ER and NGF are co-expressed in ICG cells, which suggested an interaction between the two receptors for the maintenance of ICG structure and function. Further studies are required to determine the interaction mechanisms between ER and NGF.

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 (Edited by Liu XL/Yang Y/Song LP)