Transcutaneous Auricular Vagus Nerve Stimulation Protects Endotoxemic Rat from Lipopolysaccharide-Induced Inflammation

Background. Transcutaneous auricular vagus nerve stimulation (ta-VNS) could evoke parasympathetic activities via activating the brainstem autonomic nuclei, similar to the effects that are produced after vagus nerve stimulation (VNS). VNS modulates immune function through activating the cholinergic anti-inflammatory pathway. Methods. VNS, ta-VNS, or transcutaneous electrical acupoint stimulation (TEAS) on ST36 was performed to modulate the inflammatory response. The concentration of serum proinflammatory cytokines and tissue NF-kappa B p65 (NF-κB p65) were detected in endotoxaemia affected anesthetized rats. Results. Similar to the effect of VNS, ta-VNS suppressed the serum proinflammatory cytokines levels, such as tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) as well as NF-kappa B p65 expressions of lung tissues. ST36 stimulation also decreases LPS-induced high TNF-α level and NF-κB signal, but it did not restrain proinflammatory cytokine IL-1β and IL-6. Neither ta-VNS nor ST36 stimulation could suppress LPS-induced TNF-α and NF-κB after vagotomy or with α7nAChR antagonist injection. Conclusions. The present paper demonstrated that ta-VNS could be utilized to suppress LPS-induced inflammatory responses via α7nAChR-mediated cholinergic anti-inflammatory pathway.


Introduction
Inflammation is a local, protective response to microbial invasion or injury, which must be fine-tuned and regulated precisely [1]. Several potent cytokines including tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and transforming growth factor-beta (TGF-β) are produced by activated macrophages and other immune cells, as necessary and sufficient mediators involved in local and systemic inflammation [2,3]. Overproduction of cytokines leads to systematic inflammation and tissue injury. If the overproduced cytokines spread into the bloodstream, dangerous inflammatory responses will be induced. In the past ten years, abundant studies have been focused on "the cholinergic anti-inflammatory pathway," namely, the efferent vagus nerve which inhibits proinflammatory cytokine production and protects against systemic inflammation via a α7nAChR-dependent pathway [4]. Vagus nerve stimulation (VNS) prevents the occurrence and development of inflammation effectively via activating the cholinergic anti-inflammatory pathway. VNS and acetylcholine (Ach) attenuated the release of cytokines significantly and improved survival in lethal endotoxemia or sepsis models [5,6]. For instance, in a rat model of lethal endotoxamia, electrical stimulation of the efferent vagus nerve decreases serum and hepatic TNF levels [6]. Moreover, VNS inhibited all lipopolysaccharide-(LPS-) induced procoagulant responses strongly, attenuated the fibrinolytic response more modestly, and improved hepatic Ach levels significantly in endotoxemia rats [7]. VNS attenuated the LPS-induced increases of the plasma and splenic proinflammatory cytokines, rather than influencing the antiinflammatory cytokine IL-10 [8]. In addition, activation of this neural immune-modulatory pathway by electrical 2 Evidence-Based Complementary and Alternative Medicine stimulation of vagus nerve also protects animals from various circumstances, such as ischemia-reperfusion injury, hypovolemic hemorrhagic shock, heart failure, and myocardial ischemia/reperfusion [9][10][11][12].
It was demonstrated that the transcutaneous auricular vagus nerve stimulation (ta-VNS) induced a series of parasympathetic activities [13][14][15][16][17]. Auricular branch of vagus nerve is a special vagal branch that innervates the body surface which could not be found on the other parts of the body [18], mainly innervating the cymba conchae and cavum conchae within the auricle. Our previous studies indicated that there is an intimate connection between auricular concha, the nucleus tractus solitarius (NTS), dorsal motor nucleus of the vagus nerve (DMN), and vagus nerve, which constructs the pathway of the auricular-vagal reflex. Accordingly, there might be some kinds of connection between auricular concha and efferent vagus nerve. In the present study, we reasoned that ta-VNS may have a role in activating the vagus nerve-based cholinergic anti-inflammatory pathway.
Here, the effect of ta-VNS on proinflammatory cytokines and NF-kappa B p65 was explored to clarify the mechanism of ta-VNS underlying regulating inflammatory diseases.

Animals.
Male Sprague Dawley rats (12 weeks old) were used in the present study, weighing 275-350 g, supplied by China Academy of Military Science. Rats were housed in groups of 5-6 in standard polycarbonate cages at ambient temperature (22 • C) and allowed access to food and water ad libitum. Lights were set to an automated 07:00 on and 19:00 off light-dark cycle, and all animal experiments were done between 08:00 and 11:00 a.m. Rats received care consistent with the National Institutes of Health Guide for the Care and use of Laboratory Animals, and the experiments were conducted in accordance with a protocol approved by the Institutional Animal Care and Use Committee of China Academy of Chinese Medical Sciences.

Experimental Protocols.
The present study consisted of 4 main parts. (1) The first detects the effect of ta-VNS, VNS, or transcutaneous electrical acupoint stimulation (TEAS) on ST36 on LPS-induced serum cytokine response. In this part, rats were randomly divided into 5 groups of twelve each: (a) saline-treated animals (NS), (b) endotoxemia model rats (LPS), (c) endotoxemia model rats receiving treatment of ta-VNS (ta-VNS), (d) endotoxemia model rats receiving treatment of VNS (VNS) and (e) endotoxemia model rats received treatment of TEAS on ST36 (ST36). (2) The second detects the effect of ta-VNS, VNS, or TEAS on ST36 on LPS-induced pulmonary NF-κB p65 expression. In this part, rats were randomly divided into 6 groups, which consisted of the 5 groups aforementioned, and a group of salinetreated animals received treatment of ta-VNS (NS+ta-VNS).

Endotoxemia
Model. LPS is an endotoxin derived from cell wall of gram-negative bacteria, and systemic injection of LPS results in various symptoms of bacterial infection including fever and inflammation [19]. Rats were injected intravenously with lipopolysaccharide (LPS, Escherichia coli 0111:B4; Sigma, 5 mg/kg), dissolved in sterile, pyrogenfree saline that was sonicated for 30 minutes immediately before use. Rats (n = 12 per group) were killed 2 hours after LPS injection (Figure 1), and the blood was collected from abdominal aorta, allowed to clot for 2 hours at room temperature, and then centrifuged at room temperature for 15 minutes at 2000 rpm. Serum samples were stored at −20 • C before cytokine analysis. Lung samples were rapidly excised, rinsed of blood with normal saline, placed into liquid nitrogen immediately and then frozen and stored at −80 • C till measurement of NF-κB p65 expression.

Electrical Vagus Nerve Stimulation.
Rats were anaesthetized with urethane (1 g/kg, intraperitoneally). A midline cervical incision was made to expose the left cervical branch of the vagus nerve. The left carotid sheath was isolated. After blunt preparation, the left vagus nerve trunk was carefully freed from surrounding tissue, separated from the carotid artery trunks, and placed on a custom-made bipolar platinum electrode connected via an isolation unit to a stimulator (SEN-7203, Nihon Kohden). All the exposed nerves were protected from dehydration by covering warm paraffin mineral oil tampons [6]. One and a half hour after LPS administration, constant electrical current stimuli with parameter of 1 mA, 10 Hz, 1 ms were turned on for 20 min (Figure 1(a)).

Transcutaneous Electrical Acupoint Stimulation (TEAS) on ST36.
Transcutaneous surface electrodes were placed bilaterally on the depilatory rat skin at Zusanli (ST36). The ST36 points are located at 5 mm lateral to the anterior tubercle of the tibia and 10 mm below the knee joints. Bilateral surface electrodes at hind limbs were connected via an isolation unit to a stimulator (SEN-7203, Nihon Kohden), and the points were stimulated with the same parameter aforementioned. One and a half hour after LPS administration, the stimulation started and lasted for 20 min (Figure 1). Stimulation intensity was adjusted to a level that elicited a slight muscle twitch at the stimulated site and was limited to a maximum of 1 mA to minimize animal discomfort.

Transcutaneous Auricular Vagus Nerve Stimulation (ta-VNS).
Transcutaneous surface electrodes were placed bilaterally on the auricular concha, which mainly includes cymba conchae and cavum conchae of the auricular. Bilateral surface electrodes at auricular concha were connected via an isolation unit to a stimulator (SEN-7203, Nihon Kohden), and the auricular conchae on both sides were stimulated with the same parameter. One and a half hour after LPS administration, the stimulation started and lasted for 20 min (Figure 1). Stimulation intensity was adjusted to a level that elicited a slight twitch of the auricle and was limited to maximum of 1 mA to minimize animal discomfort.

Vagotomy.
Vagotomy was performed before LPS administration ( Figure 1(b)). In vagotomized animals, following a ventral cervical midline incision, bilateral vagus trunks were exposed and separated from the common carotid artery, ligated with a 4-0 silk suture.

Cytokine Analysis.
Abdominal aortic blood was collected two hours after LPS administration, allowed to clot for 2 h at room temperature, and centrifuged for 20 min at 2500 rpm. Serum TNF-α, IL-1β, and IL-6 concentrations were analyzed, respectively, by TNF-α, IL-1β, and IL-6 ELISA kits (R&D Systems) following the manufacturer's instructions.
2.11. NF-κB Immune-Histochemistry. NF-κB p65 immunohistochemistry staining was performed as described previously [22] to evaluate the lung tissues inflammatory response. Briefly, tissue sections were deparaffinized with xylene and rehydrated through graded series of alcohols. Tissue sections were rinsed in PBS, pretreated with citrate buffer at 93 • C, blocked with PBS containing 2% BSA, and then incubated with a primary antibody reactive against rabbitactivated p65 subunit of NF-κB (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA). Washed sections were incubated for 10 min with secondary goat anti-rabbit IgG biotin. The reaction product was visualized with DAB chromogenic agent. The sections were counterstained with hematoxylin stain. Slides were analysed on a light microscope (Olympus BX60) using an ImagePro Plus Imaging System (Universal Imaging).

Statistical Analysis.
All the data in the present study were expressed as means ± SEM and analyzed by one-way  ANOVA with SPSS software. The two-tailed Student's ttest was used to compare mean values between two groups. Pvalues < 0.05 were considered significant.

The Effect of ta-VNS or TEAS on ST36 on Serum TNFα Level Was
Blocked by α-BGT Administration. The above results showed that ta-VNS has similar effects to VNS on cytokine levels. Previous studies show that VNS-activated "cholinergic anti-inflammatory pathway" regulates systemic inflammatory responses via α7nAChR, hereby ta-VNS may have the same effect. To test this hypothesis, we pretreated animals with the α7nAChR antagonist α-BGT. LPS injection induced profound rise in the concentration of serum TNF-α. Either ta-VNS or TEAS on ST36 failed to inhibit TNF-α level after α-BGT administration (Figure 3).

Effect of ta-VNS or TEAS on ST36 on Serum TNF-α Was
Blunted by Vagotomy. To examine the mechanism of ta-VNS in "cholinergic anti-inflammatory pathway," we pretreated animals with vagotomy. The result indicated that intravenous injection of LPS elicited a rapid raise of TNF-α level. Neither ta-VNS nor TEAS on ST36 was effective on inhibiting TNF-α level after vagotomy (Figure 4). increased expression of NF-κB p65 in lung tissues (Figures 4  and 5). Both electrical VNS and ta-VNS strongly inhibited LPS-induced NF-κB p65 (n = 10, P < 0.01, Figures 4 and 5). TEAS on ST36 did not have the same effect (Figures 5 and 6).

Effect of ta-VNS or TEAS on ST36 on NF-κB p65 Was
Blunted by Vagotomy. We pretreated animals with vagotomy. The result indicated that ta-VNS or ST36 failed to inhibit the expressions of NF-κB p65 after vagotomy (Figure 7).

Discussion
Here, we reported our original study that auricular concha stimulation is also a potent anti-inflammatory stimulus that can modulate immune factors in endotoxemia rat model. The present study demonstrates that ta-VNS may have an  Previous study demonstrated that the cholinergic antiinflammatory pathway is a α7nAChR-dependent, vagus nerve-mediated pathway [1]. It can inhibit macrophage activation through parasympathetic outflow, which functions as an anti-inflammatory pathway in systemic and local inflammation. Inflammatory signals stimulate sensory fibers that ascend in the vagus nerve to synapse in the NTS and then activate efferent fibers in the vagus nerve to suppress peripheral cytokine release through alpha7nAChR.
The most important cytokine involved is TNF-α, which activates other proinflammatory cytokines such as IL-1β, IL-6, and high mobility group B1 (HMGB1) and amplifies other inflammatory mediators. VNS has been demonstrated to inhibit proinflammatory cytokine production [23][24][25], especially the release and synthesis of TNF-α. The present study indicates that VNS decreases LPS-induced TNF-α, IL-1, and IL-6 in circulation. And ta-VNS reduced the levels of proinflammatory cytokines TNF-α, IL-1β, and IL-6, which is similar to the effect of VNS. After administration of α7nAChR antagonist α-BGT, ta-VNS failed to attenuate serum TNF-α level, which is consistent with previous reports [6-10, 20, 23]. This result indicated that α7nAChR played a critical role in anti-inflammatory effect of ta-VNS. The present study also demonstrated that vagotomy exacerbated serum TNF responses to inflammatory stimulation, sensitized animals to the lethal effects of endotoxin, and abolished the anti-inflammatory effect of ta-VNS. The results indicated that ta-VNS fails to suppress excessive cytokine response characterized by exaggerated TNF-α level if there is deficiency in either the α7nAChR subunit or vagus nerve. NF-κB is a master transcription factor controlling the expression of a wide range of proinflammatory genes [26][27][28]. Previous studies reported that NF-κB is involved in TNF-α genetic activation and TNF-α production [29,30]. In the present study, both western blot data and immunehistochemical results indicated that ta-VNS suppresses the  LPS-induced NF-κB expression in rat lung tissue (Figures 4  and 5), which mimicking the effects of VNS [25,31]. In vagotomy animals, ta-VNS failed to inhibit increased NF-κB expression, suggesting ta-VNS functions in situations that require intact vagus nerve.
Auricular acupuncture, as a special form of acupuncture, has been used for the treatment of different disorders for centuries in China. Our research group previously demonstrated  that auricular acupuncture stimulation could activate neurons of NTS and upregulate vagal tune, to decrease MAP and HR [35], to trigger gastric motility [36]. Our previous studies also demonstrated that TEAS of auricular concha could activate the parasympathetic nervous system and mimic the effect of VNS to suppress epileptic seizures [18]. In the present study, the results showed that ta-VNS inhibited proinflammatory cytokine levels and suppressed NF-κB expressions in endotoxaemia rats (Figures 1, 4, and 5), which is similar to the effect of VNS. However, vagotomy or α7nAChR antagonist α-BGT could diminish the effect of ta-VNS on the anti-inflammatory responses, suggesting that auricular acupuncture may perform an anti-inflammatory effect via cholinergic anti-inflammatory pathway.
In general (Figure 8), VNS directly activates the cholinergic anti-inflammatory pathway via stimulating efferent vagus nerve. As the peripheral branch [18], auricular branch of vagus nerve (ABVN) innervates the auricular concha and the external auditory meatus. Stimulation of the ABVN region could evoke parasympathetic excitation [37][38][39]. Acupuncture in the area of auricular concha may increase discharge of NTS [18], as the central terminal nuclear for afferent vagal fibers, which primarily transmit signals from local inflammation lesion [4,40]. Thus, we hypothesize that ABVN could be evoked by ta-VNS, and the activated signals ascend with vagal input to the NTS. The signals are processed within the NTS, and the integrated output signal is carried by efferent vagus nerve to inhibit inflammatory responses. TEAS on the acupoint of ST36 activates the somatic fiber endings around ST36 point, which send the acupuncture signals to the spinal cord via somatic sensory nerve fibers. In the spinal cord, the nerve impulses are delivered to the NTS by the secondary order neurons, where the signals were processed. Ultimately, the increased efferent vagal output activates the cholinergic anti-inflammatory pathway.

Conclusions
The results presented here demonstrate that ta-VNS plays an important role in immuneregulation, through the activation of the cholinergic anti-inflammatory pathway and the downregulation of proinflammatory cytokine expressions and NF-κB activities. VNS and TEAS on ST36 might suppress the inflammatory responses via different mechanisms.