Neuropeptide Y may participate in the pathogenesis of cervical vertigo

2.1 Animals and experimental design

White rabbits were purchased from Jinan xilingjiao biotechnology co. LTD. Sixty adult healthy male New Zealand White rabbits weighing 2 kg to 2.5 kg were equally and randomly divided into five groups: the blank control (n = 12), superior cervical sympathetic ganglia (SCSG) stimulation (n = 12), SCSG sham-operated control (n = 12), inferior cervical sympathetic ganglia (ICSG) stimulation (n = 12), and ICSG sham-operated control (n = 12) groups. All animal procedures were conducted in accordance with the National Institutes Health guideline and approved by the Animal Experimentation Ethic Committee of Shandong University.

2.2 Electrical stimulation of cervical sympathetic ganglia

Animals were anesthetized with an intraperitoneal injection of 10% chloral hydrate (150 mg/kg body weight) and placed in the supine position on the experimental table. This experiment was conducted under sterile conditions to eliminate disturbance from bacteria or other microorganisms. After the administration of chloral hydrate, the cervical hair was shaved, and the corresponding SCSG or ICSG was exposed unilaterally through a flank incision on the neck. Subsequently, the corresponding ganglion was isolated from the surrounding tissue by using rubber gloves with a stimulating electrode being needled inside (Fig. 1A).

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Fig. 1

Electrical stimulation of cervical sympathetic ganglion (CSG) in rabbits. A. A stimulating electrode was stabbed into the exposed cervical sympathetic ganglion; B. Parameters regarding the electrical stimulation were presented.

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The electrical stimuli (i.e., square-wave pulses of 0.5 ms pulse width at 30.0 Hz and 10.0 V for 5 min) applied in the SCSG and the ICSG groups were the same (Fig. 1B). These stimulus pulses were delivered from a pulse generator (SEN-7103, Nihon Kohden). The rabbits in the corresponding control group experienced the same surgery for cervical sympathetic ganglion exposure and electrode penetration without electrical stimulation.

2.3 Specimen collection and cryosection

Specimens were collected and disposed using the aforementioned method with modifications (Zuo et al., 2014). Immediately after electrical stimulation, the animals were sacrificed using a rapid left ventricular perfusion with physiological saline (37 °C, 300–400 mL) followed by slow perfusion with 4% paraformaldehyde at room temperature. After 1 h perfusion, the ipsilateral cervical spinal ganglia (C1–C8) were dissected and postfixed in 4% paraformaldehyde for 4 h at room temperature. Subsequently, the specimens were successively immersed into 10%, 20%, and 30% sucrose solutions for gradient dehydration until the specimens completely sank. Cryosections at thickness of 8 μm were sliced along the long axis of the ganglia. Three slides of each specimen were chosen from the front, middle, and back of the ganglia for further immunohistochemistry experiments.

2.4 Immunohistochemistry

The immunohistochemistry detection for NPY expression was conducted using the PV-9003 Polink-2 plus® polymer HRP detection system (ZSGB-Bio, Beijing, China) following the manufacturer’s instructions. Cryosections stored at −20 °C were placed aside for 5 min at room temperature, postfixed with 4% paraformaldehyde for 15 min, incubated with 3% H₂O₂ for 10 min at 37 °C, and washed thrice with PBS buffer. Slides were incubated with goat anti-NPY polyclonal primary antibody (Abcam, USA) at 4 °C overnight. The next day, the section were washed thrice with PBS buffer and successively incubated with polymer adjuvant for 20 min at 37 °C and secondary HRP-conjugated antigoat IgG antibody for 1 h (Sigma, USA). Afterward, the sections were immersed for 3 min in the 3,3ʹ-diaminobenzidine coloring substrate (0.4 mg/mL, DAKO, USA), rinsed with water, counterstained with hematoxylin, dehydrated, and sealed with neutral balsam. All sections were observed under a microscope. Positive areas were stained brown, and the Image-Pro Plus software was used to quantify the average optical density. Ten randomly selected discontinuous fields (200×) per slide were evaluated.

2.5 Statistical analysis

Data were expressed as mean ± SD. Statistical analysis was performed using the SPSS 13.0 (SPSS, Chicago, IL). The values of optical density were compared using the Student’s T-test. P < 0.05 was considered statistically significant.

3.1 Low NPY level in normal cervical spinal ganglia

NPY immunohistochemical detection was performed to detect the level of NPY in cervical spinal cord ganglia under physiological conditions. As shown in Figs. 2A and 3A, only a few cells showed positive nucleus staining, indicating that NPY was weakly expressed in all cervical ganglia (C1–C8).

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Fig. 2

Immunohistochemistry of neuropeptide Y in the C2–C5 cervical spinal ganglia at hours after the electrical stimulation of the superior cervical sympathetic ganglion (SCSG). Representative immunohistochemistry images (200×) of neuropeptide Y in the blank control (A, C2), sham-operated (B, C2), and SCSG-stimulated (C, C2; D, C3; E, C4; F, C5) groups. Brown-colored cells in nuclei are neuropeptide Y-positive cells.

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3.2 The mechanical stimuli cannot affect the expression of NPY in cervical spinal ganglia

Ganglion exposure and electrode placement were external mechanical stimuli to the body and may disturb the results in this study. A sham-operated group was established to eliminate the influence of surgery and electrode placement on NPY. Subsequently, the expression levels of NPY in the corresponding ipsilateral cervical spinal ganglia were detected by immunohistochemistry. As shown in Figs. 2B and 3B, few NPY-positive cells were observed in the corresponding sham-operated groups. Quantification analysis revealed that no significant difference was found in the NPY expressions between the sham-operated and blank control groups, indicating that the mechanical stimuli in the cervical sympathetic ganglion did not affect the expression of NPY in the corresponding cervical ganglion.

3.3 The electrical stimuli increases the expression of NPY in some cervical spinal ganglia

The SSCG or ISCG suffered from electrical stimulation because the mechanical stimuli did not affect the NPY expression level, and the expression of NPY in cervical spinal ganglion was detected using immunohistochemistry. As shown in Figs. 2C–E and 4A, the SCSG stimulation group had more NPY-positive cells in C2, C3, C4, and C5 compared with the sham-operated group, and the increases in C2 and C3 were the most evident. As shown in Figs. 3C–D and 4B, the ICSG stimulation group had more NPY-positive cells in C6, C7, and C8 compared with the sham-operated group, and those in C6 and C7 presented significant increases. These results suggested that electrical stimulation can increase the expression levels of NPY in certain cervical spinal ganglia, which were primarily controlled by the corresponding cervical sympathetic ganglion.

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Fig. 3

Immunohistochemistry of neuropeptide Y in C6–C8 cervical spinal ganglia after the electrical stimulation of the inferior cervical sympathetic ganglion (ICSG). Representative immunohistochemistry images (200×) of neuropeptide Y in the blank control (A, C6), sham-operated (B, C6), and ICSG-stimulated (C, C6; D, C7; E, C8) groups. Brown-colored cells in nuclei are neuropeptide Y-positive cells.

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Fig. 4

Quantification analysis of neuropeptide Y in the ipsilateral cervical spinal ganglia (C1–C8) after electrical stimulation of unilateral superior (A) and inferior (B) cervical sympathetic ganglion.

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