Wake-promoting effects of vagus nerve stimulation after traumatic brain injury: Upregulation of orexin-A and orexin receptor type 1 expression in the prefrontal cortex

Abstract

Orexins, produced in the lateral hypothalamus, are important neuropeptides that participate in the sleep/wake cycle, and their expression coincides with the projection area of the vagus nerve in the brain. Vagus nerve stimulation has been shown to decrease the amounts of daytime sleep and rapid eye movement in epilepsy patients with traumatic brain injury. In the present study, we investigated whether vagus nerve stimulation promotes wakefulness and affects orexin expression. A rat model of traumatic brain injury was established using the free fall drop method. In the stimulated group, rats with traumatic brain injury received vagus nerve stimulation (frequency, 30 Hz; current, 1.0 mA; pulse width, 0.5 ms; total stimulation time, 15 minutes). In the antagonist group, rats with traumatic brain injury were intracerebroventricularly injected with the orexin receptor type 1 (OX1R) antagonist SB334867 and received vagus nerve stimulation. Changes in consciousness were observed after stimulation in each group. Enzyme-linked immunosorbent assay, western blot assay and immunohistochemistry were used to assess the levels of orexin-A and OX1R expression in the prefrontal cortex. In the stimulated group, consciousness was substantially improved, orexin-A protein expression gradually increased within 24 hours after injury and OX1R expression reached a peak at 12 hours, compared with rats subjected to traumatic brain injury only. In the antagonist group, the wake-promoting effect of vagus nerve stimulation was diminished, and orexin-A and OX1R expression were decreased, compared with that of the stimulated group. Taken together, our findings suggest that vagus nerve stimulation promotes the recovery of consciousness in comatose rats after traumatic brain injury. The upregulation of orexin-A and OX1R expression in the prefrontal cortex might be involved in the wake-promoting effects of vagus nerve stimulation.

Full text

NEURAL REGENERATION RESEARCH www.nrronline.org
244
RESEARCH ARTICLE
Wake-promoting eects of vagus nerve stimulation
aer traumatic brain injury: upregulation of orexin-A
and orexin receptor type 1 expression in the prefrontal
cortex
*Correspondence to:
Zhen Feng,
fengzhenly@sina.com.
orcid:
0000-0003-0134-465X
(Zhen Feng)
doi: 10.4103/1673-5374.226395
Accepted: 2017-12-25
Xiao-yang Dong, Zhen Feng*
Department of Rehabilitation Medicine, the First Aliated Hospital of Nanchang University, Nanchang, Jiangxi Province, China
Funding: is study was supported by the Natural Science Foundation of China, No. 81260295 and the Graduate Student Innovation Fund of
Jiangxi Province of China, No. YC2015-S090.
Abstract
Orexins, produced in the lateral hypothalamus, are important neuropeptides that participate in the sleep/wake cycle, and their expres-
sion coincides with the projection area of the vagus nerve in the brain. Vagus nerve stimulation has been shown to decrease the amounts
of daytime sleep and rapid eye movement in epilepsy patients with traumatic brain injury. In the present study, we investigated whether
vagus nerve stimulation promotes wakefulness and aects orexin expression. A rat model of traumatic brain injury was established using
the free fall drop method. In the stimulated group, rats with traumatic brain injury received vagus nerve stimulation (frequency, 30 Hz;
current, 1.0 mA; pulse width, 0.5 ms; total stimulation time, 15 minutes). In the antagonist group, rats with traumatic brain injury were
intracerebroventricularly injected with the orexin receptor type 1 (OX1R) antagonist SB334867 and received vagus nerve stimulation.
Changes in consciousness were observed aer stimulation in each group. Enzyme-linked immunosorbent assay, western blot assay and
immunohistochemistry were used to assess the levels of orexin-A and OX1R expression in the prefrontal cortex. In the stimulated group,
consciousness was substantially improved, orexin-A protein expression gradually increased within 24 hours aer injury and OX1R expres-
sion reached a peak at 12 hours, compared with rats subjected to traumatic brain injury only. In the antagonist group, the wake-promoting
eect of vagus nerve stimulation was diminished, and orexin-A and OX1R expression were decreased, compared with that of the stim-
ulated group. Taken together, our findings suggest that vagus nerve stimulation promotes the recovery of consciousness in comatose
rats aer traumatic brain injury. e upregulation of orexin-A and OX1R expression in the prefrontal cortex might be involved in the
wake-promoting eects of vagus nerve stimulation.
Key Words: nerve regeneration; brain injury; orexin-A; orexin receptor type 1; vagus nerve stimulation; traumatic brain injury; wake-promoting;
coma; wakefulness; prefrontal cortex; neurotransmitter; neural regeneration
Graphical Abstract
Vagus nerve stimulation causes wake promotion by orexins pathways in traumatic brain injury-induced
coma rats
Introduction
Traumatic brain injury (TBI) has a high incidence world-
wide, and is associated with high rates of morbidity and
mortality. Recent progress in treating traumatic brain in-
juries has resulted in sharply reduced rates of mortality;
however, 14% of TBI patients remain in a long-term coma
or vegetative state following treatment. Furthermore, this
percentage is gradually increasing, resulting in a heavy bur-
den on society and the affected family members (Harvey,
2013; Durand et al., 2017). e current treatment regimen
for TBI-induced coma includes drug therapy, hyperbaric
oxygenation, music therapy, and medial nerve stimulation
(Kumaria and Tolias, 2012; Cossu, 2014; Kaelber et al.,
2016; Gray, 2017; Joseph et al., 2017). However, these treat-
Vagus nerve stimulation
Orexin receptor 1
antagonist SB334867
(intracerebroventricular
injection)
Wake promotion
Orexin-A expression
Orexin receptor
type 1 expression
Coma model
[Downloaded free from http://www.nrronline.org on Friday, May 4, 2018, IP: 84.209.170.222]

245
Dong XY, Feng Z (2018) Wake-promoting eects of vagus nerve stimulation aer traumatic brain injury: upregulation of orexin-A and orexin
receptor type 1 expression in the prefrontal cortex. Neural Regen Res 13(2):244-251. doi: 10.4103/1673-5374.226395
ments do not always result in the patient awakening, and
there remains a need for new methods of accelerating the
transition from a comatose or vegetative state to arousal.
Previous studies have shown that vagus nerve stimulation
(VNS) can reduce the amounts of daytime sleep and rapid
eye movement, thereby extending the amount of awake time
in patients with epilepsy caused by TBI (Malow et al., 2001;
Shi et al., 2013; Jain and Glauser, 2014; Neren et al., 2016).
VNS is a neurophysiologic method that has been extensively
used to treat refractory epilepsy, depression, and cognitive
disorders (Yuan and Silberstein, 2015). Recent studies have
shown that VNS affects the amounts of time spent awake
and asleep, and can decrease sleep duration. e vagus nerve
projects to several different brainstem regions, including
the locus coeruleus and the parabrachial nucleus. In the
parabrachial nucleus, the vagus nerve forms numerous con-
nections with bers that project to the basal forebrain, thala-
mus, hypothalamus, and cerebral cortex (Ansari et al., 2007;
Frangos and Komisaruk, 2017). Due to this widespread
connectivity, VNS has treatment potential for coma. In this
study, we evaluated whether VNS could promote grades I–
IV consciousness, as determined by observing sensory and
motor functions.
Orexin peptides (orexin-A and orexin-B) are produced by
the lateral hypothalamus and regulate feeding behavior, en-
ergy homeostasis, neuroendocrine activities and the sleep-
wake cycle by binding to orexin-1 and orexin-2 receptors
(Wu et al., 2007; Boss and Roch, 2015). e orexin receptor
type 1 (OX1R) is expressed in many regions of the brain, in-
cluding the cerebral cortex, prefrontal cortex, ventromedial
hypothalamic nucleus, and locus coeruleus. Orexin-A is one
of the most important neurotransmitters in the ascending
reticular activating system, participating in awareness and
the sleep-wake cycle. erefore, in the present study, we ex-
amined whether orexin-A and the OX1R are involved in the
wake-promoting eect of VNS.
Materials and Methods
Animals
A total of 120 specic-pathogen-free adult Sprague-Dawley
rats (half male and half female), weighing 250−300 g, were
obtained from the Institute of Laboratory Animals of Nan-
chang University of China, and housed in the Laboratory
Animal Center of the First Aliated Hospital of Nanchang
University of China. All rats were maintained under con-
trolled temperature and light conditions, and allowed free
access to food and water.
The study protocol was approved by the Animal Ethics
Committee of the First Aliated Hospital of Nanchang Uni-
versity (approval number: (2016)(003)). The experimental
procedures were in accordance with the National Institutes
of Health Guide for the Care and Use of Laboratory Animals
(NIH Publication No. 85-23, revised 1985).
Establishment of a TBI-induced coma model
e 120 rats were assigned to four dierent groups (n = 30
each), with 10 rats for each time point per group. In the con-
trol group, healthy rats received sham operation and anes-
thesia. In the TBI group, free-fall drop was used to establish
the model of TBI-induced coma (Feeney et al., 1981). In the
stimulated group, rats with TBI-induced coma were sub-
jected to VNS. In the antagonist group, comatose rats were
intracerebroventricularly injected with the OX1R antagonist
SB334867 and received VNS.
Rats in the TBI, stimulated and antagonist groups were
anesthetized by inhalation of diethyl ether, and then allowed
to breathe air spontaneously. Aer anesthesia, a 5-mm ver-
tical incision was made to expose the skull. The target for
impact was marked with a syringe needle at a spot 2 mm ad-
jacent to the le midline and 1 mm anterior to the coronal
suture. Next, a cylindrical impact hammer weighing 400 g
and of 2 cm diameter was dropped from a vertical height of
40–44 cm to produce a concave fracture of the skull (Feeney
et al., 1981). Following injury, the incision was closed, and
each animal was disinfected and placed in a cage.
Evaluation of sensory and motor functions
One hour aer the impact, the degree of consciousness was
evaluated on a scale of I–IV, based on sensory and motor
functions (I–VI consciousness scale) (Stephens and Levy,
1994). The levels of consciousness were as follows: level I:
normal activity as seen in the cage; level II: decreased activity;
level III: decreased activity accompanied by motor incoor-
dination; level IV: righting reflex could be elicited, and the
animal could stand up; level V: the righting reex was absent;
however, the animal could react to pain; level VI: the animal
showed no reaction to pain. Rats having consciousness lev-
els of V or VI for at least 30 minutes were deemed to be in a
coma state, and were used for the following procedures.
Intracerebroventricular injection of the OX1R antagonist
SB334867
Under sterile conditions, an injection catheter was inserted
into the le cerebral ventricle of each rat in the antagonist
group. Each rat was pretreated with gentamicin (0.1 mL/100
g body weight, intramuscular injection) and anesthetized
with 10% chloral hydrate (0.3 mL/100 g body weight, in-
Table 1 Eect of vagus nerve stimulation on the recovery of
consciousness in rats in a TBI-induced coma
Group Revived
Coma
Level IV Level V
Control 30(100.0)
TBI 8(26.7) 10(33.3) 12(40.0)
Stimulated 20(66.7) 8(26.7) 2(6.6)
Antagonist 12(40.0) 10(33.3) 8(26.7)
Data are expressed as number of rats recovered (%). Control group:
Healthy rats with sham operation and anesthesia. TBI group:
Free-fall drop used to establish the model of TBI-induced coma.
Stimulated group: Rats in a TBI-induced Coma subjected to vagus
nerve stimulation. Antagonist group: Comatose rats received
intracerebroventricular injection of the orexin receptor type 1
antagonist SB334867 and vagus nerve stimulation. Consciousness was
classied into six levels (consciousness scale of I–VI), with levels V and
VI dened as a coma state. TBI: traumatic brain injury.
[Downloaded free from http://www.nrronline.org on Friday, May 4, 2018, IP: 84.209.170.222]

246
Dong XY, Feng Z (2018) Wake-promoting eects of vagus nerve stimulation aer traumatic brain injury: upregulation of orexin-A and orexin
receptor type 1 expression in the prefrontal cortex. Neural Regen Res 13(2):244-251. doi: 10.4103/1673-5374.226395
traperitoneal injection) prior to surgery. e rats were po-
sitioned in a stereotaxic frame (ZS-B/S; Beijing Zhongshi
Dichuang Science and Technology Development Co., Ltd.,
Beijing, China). The following coordinates were used to
map the guide cannula: 1.0 mm posterior to the bregma, 1.5
mm lateral to the midline, and 4.5 mm ventral to the skull
surface, with the incisor bar 3.2 mm below the interauricu-
lar line. An injection catheter was inserted into the cerebral
ventricle of each rat in the antagonist group under sterile
conditions. The OXR1 inhibitor SB334867 (Tocris Biosci-
ence, Ellisville, MO, USA) was dissolved in a 60:40 dimethyl
sulfoxide solution and administrated at a dose of 10 mg/kg
body weight in a total volume of 5 μL. Aer awakening from
anesthesia, the rats were prepared for VNS.
VNS
Aer TBI and the rst evaluation of consciousness, VNS was
performed. Rats in the stimulated and antagonist groups were
treated with VNS using a low-frequency electrical stimulator
(ES-420; ITO Physiotherapy & Rehabilitation, Tokyo, Japan).
After establishment of the TBI-induced coma model and
prior to stimulation, the rats were intraperitoneally anesthe-
tized with 10% chloral hydrate (0.3 mL/100 g body weight).
Afterwards, the head and neck areas were disinfected with
Betadine and then shaved. A small incision was made on the
le ventral side of the neck adjoining the midline to approach
the left vagus nerve at the cervical level. We performed a
blunt dissection of the subcutaneous fat, salivary glands, ster-
nohyoid and sternocleidomastoid, and cut the carotid sheath,
including the vagus nerve and carotid artery. A 5-mm seg-
ment of the le vagus nerve was separated and attached to an
electrode. An ohmmeter was used to ensure that the electrode
had good contact with the vagus nerve. VNS was performed
with the following parameters: frequency, 30 Hz; current,
1.0 mA; pulse width, 0.5 ms; total stimulation time, 15 min-
utes. Following surgery, each animal received gentamicin
(0.1 mL/100 g body weight) by intramuscular injection. One
hour later, behavior and consciousness levels were observed
and evaluated based on previously described grading criteria
(Stephens and Levy, 1994). Rats in the TBI group underwent
a procedure identical to that used for the stimulated groups,
but without electrical stimulation.
Tissue extraction
Rats in the stimulated and antagonist groups, as well as rats
in the corresponding control and TBI-induced coma groups
were simultaneously euthanized with 10% chloral hydrate
at 6, 12 and 24 hours after TBI. Prefrontal cortical tissues
(within the frontal lobe) were removed and analyzed by
enzyme linked immunosorbent assay (ELISA), immunohis-
tochemistry and western blot assay to evaluate orexin-A and
OX1R expression.
ELISA
Five rats from each group were sacrificed at 6, 12 and 24
hours aer TBI. Tissue samples were tested using an ELISA
kit designed for detecting orexin-A protein (cE90607a 96
Tests; Uscn Life Science Inc., Wuhan, Hubei Province, Chi-
na). Optical density values were measured at 450 nm using
a microplate reader (Model 680, Bio-Rad, Hercules, CA,
USA). e concentration of orexin-A was calculated using a
standard curve.
Western blot assay
At 6, 12 and 24 hours after TBI, five rats from each group
were decapitated following intraperitoneal injection of 10%
chloral hydrate. Brains were carefully removed, and the pre-
frontal cortex was quickly dissected on ice. e tissue sam-
ples were homogenized using the Tissue Protein Extraction
Kit (CW0891; Beijing Kangwei Biotechnology Co., Ltd., Bei-
jing, China). Aerwards, the homogenates were centrifuged
at 12,000 × g for 10 minutes at 4°C. The total amount of
protein in each supernatant fraction was determined using
the Bio-Rad DC protein assay, and an aliquot of each super-
natant was removed and stored at −80°C. Equal amounts of
total supernatant protein in loading buffer were boiled for
5 minutes, and then separated on a 10% sodium dodecyl
sulfate/polyacrylamide gel. The separated proteins were
electrophoretically transferred onto polyvinylidene difluo-
ride membranes. The membranes were then blocked for 2
to 3 hours at room temperature with TBST buer (150 mM
NaCl, 20 mM Tris-HCl, pH 7.4, 0.1% Tween-20) containing
5% milk. The blots were then incubated overnight at 4°C
with rabbit anti-OX1R polyclonal antibody (1:200, ab68718;
Abcam, Hong Kong, China) and rabbit anti-rat β-actin
monoclonal antibody (1:400, CW0096; Beijing Kangwei).
Following incubation, the membranes were washed exten-
sively with TBST, and then incubated for 1 hour at room
temperature with horseradish peroxidase-conjugated goat
anti-rabbit IgG (H+L) (1:2,000; ZB-2301, Beijing Zhong-
shan Golden Bridge Biotechnology Co., Ltd., Beijing, China)
diluted in TBST containing 5% milk. After washing, the
blots were incubated with a chemiluminescence substrate
(32109, ECL Plus; Amersham Biosciences, Piscataway, NJ,
USA) and quantified using Image Lab software (Bio-Rad).
e blots were then stripped by incubation for 30 minutes
at 70°C in a solution containing 2% sodium dodecyl sulfate
and 100 mM β-mercaptoethanol in 62.5 mM Tris-HCl, pH
6.8. Subsequently, the blots were re-probed using rabbit an-
ti-β-actin monoclonal antibody (CW0096, Beijing Kangwei)
to monitor loading of the gel lanes. Western blot analyses of
OX1R in prefrontal cortical tissue were performed at 6, 12
and 24 hours aer TBI. e optical density measurements of
individual bands were normalized to the optical density of
β-actin.
Immunohistochemistry
e rats were anesthetized and decapitated at 6, 12 and 24
hours after TBI, and perfused through the heart with 4%
paraformaldehyde. Next, the brains were carefully removed,
and 40-µm-thick coronal sections were cut for examination.
e sections were rinsed with phosphate-buered saline and
treated with 0.3% hydrogen peroxide (H2O2) for 30 minutes.
Aerwards, the sections were rinsed three times for 5 minutes
[Downloaded free from http://www.nrronline.org on Friday, May 4, 2018, IP: 84.209.170.222]

247
Dong XY, Feng Z (2018) Wake-promoting eects of vagus nerve stimulation aer traumatic brain injury: upregulation of orexin-A and orexin
receptor type 1 expression in the prefrontal cortex. Neural Regen Res 13(2):244-251. doi: 10.4103/1673-5374.226395
each and then incubated with normal goat serum for 20 min-
utes. e sections were then incubated overnight at 4°C with
rabbit anti-OX1R antibody (1:200; ab68718, Abcam). Fol-
lowing incubation, the tissue sections were extensively rinsed
with phosphate-buered saline and then incubated with a bi-
otinylated goat anti-rabbit antibody. Finally, the sections were
reacted with diaminobenzidine and visualized under a light
microscope (BX511T-PHD-J11, Olympus, Tokyo, Japan).
Protein expression was quantified as a function of the
percentage and staining intensity of immunoreactive cells
(Soslow et al., 2000) as follows: A × B, where A represents
the percentage of positive cells (0–4, where 0 = 0–1%, 1 =
1–10%, 2 = 10–50%, 3 = 50–80%, 4 = 80–100%) and B rep-
resents the intensity of staining (0–3, where 0 = no signi-
cant staining, 1 = mild staining, 2 = moderate staining, 3 =
dark staining).
Statistical analysis
All data were analyzed with SPSS 17.0 software (SPSS,
Chicago, IL, USA). Western blot and ELISA data were ex-
pressed as the mean ± SD, and immunohistochemical data
as the mean rank. One-way analysis of variance followed by
Tukey’s test was used for comparison of western blot assay
and ELISA data. e Kruskal-Wallis test was used for com-
parison of immunohistochemical data. A value of P < 0.05
was considered statistically signicant.
Results
Evaluation of consciousness aer VNS
The degree of consciousness was evaluated using a dou-
ble-blind method 1 hour after the experiment ended. A
total of 20 rats died and were excluded from this study in
the TBI (3 rats), stimulated (8 rats) and antagonist (9 rats)
groups. Only 8 of the 30 rats in the TBI group re-awakened
from coma (level IV: 8; level V: 10; level VI: 12), while in the
stimulated group, 20 rats re-wakened (level II: 4; level III: 6;
level IV: 10; level V: 8; level VI: 2) and 10 rats remained in a
comatose state. Twelve rats in the antagonist group re-awak-
ened from coma (level III: 5; level IV: 7; level V: 10; level VI:
8). The number of rats that regained consciousness (levels
I–IV) in each group are shown in Table 1, revealing the
following order: TBI group < antagonist group < stimulated
group < control group.
VNS increased orexin-A expression in the prefrontal
cortex of rats with TBI-induced coma
ELISA showed that orexin-A expression diered in the var-
ious groups at 6, 12 and 24 hours, with the following trend:
antagonist group < control group < TBI group < stimulated
group (P < 0.05). Orexin-A expression in each of the groups
diered temporally as well, with the following trend: 6 hours
< 24 hours < 12 hours (P < 0.05; Figure 1).
VNS increased OX1R expression in the prefrontal cortex
of rats with TBI-induced coma
OX1R expression levels in the prefrontal cortex were mea-
sured by western blot assay. Signicant dierences in OX1R
expression were found among the four groups at 6, 12 and
24 hours. e relative levels of OX1R expression at 6 hours
displayed the following trends: control group < antagonist
group < TBI group < stimulated group (P < 0.05); at 12
hours: control group < TBI group < antagonist group <
stimulated group (P < 0.05); at 24 hours: antagonist group
< control group < TBI group < stimulated group (P < 0.05).
e mean level of OX1R expression was higher in the TBI
group than in the control group, and OX1R expression was
substantially higher in the stimulated group than in the TBI
group. Furthermore, the levels of OX1R expression after
injury were significantly different in the stimulated group
from those in the antagonist group. Within-group compar-
ison showed the following trends of OX1R expression at the
three time points among the four dierent groups: 6 hours <
12 hours (P < 0.05); 24 hours < 12 hours (P < 0.05); 6 hours
< 24 hours (P > 0.05) (Figure 2).
Positive immunostaining for OX1R was found in the cy-
toplasm and cell membranes of neurons in the prefrontal
cortex. OX1R-positive cells were present in all four groups,
and the data were analyzed using the Kruskal-Wallis H-test.
Samples of prefrontal cortex from the stimulated group
showed higher levels of OX1R expression (81.67) compared
with prefrontal cortex samples from the control group (40.56),
TBI group (59.67) or antagonist group (36.11) (P < 0.001).
No signicant dierences in OX1R expression were observed
within each group at the various time points (Figure 3).
Discussion
Previous studies have shown that TBI induces numerous
pathophysiological changes, including lipid peroxida-
tion, free radical formation, blood-brain barrier damage,
branched chain amino acid release, intracellular Ca2+ over-
load, oxidative stress, and arachidonic acid decomposition
(Singh et al., 2013; Elkind et al., 2015; Hiebert et al., 2015;
Lucke-Wold et al., 2015; Hue et al., 2016; Zhu et al., 2017).
It is currently believed that there are two main mechanistic
causes of TBI-induced coma: (i) impaired reticular activa-
tion system and (ii) changes in the levels of important neu-
rotransmitters that regulate the sleep/wake cycle (e.g., orex-
in-A, norepinephrine, 5-hydroxytryptamine and glutamate).
Previous studies showed that orexin levels are decreased in
the cerebrospinal uid of patients with narcolepsy resulting
from TBI during the rst 2 months aer the injury (Raman-
janeya et al., 2009; Jeter et al., 2013). In accordance with the
results of our previously published reports, we found in the
present study that orexin-A and OX1R expression levels in-
crease acutely aer injury and thereaer decrease over time
(Feng et al., 2015; Zhong et al., 2015; Feng and Du, 2016).
e increase in orexin expression in the rst several hours
aer TBI in our present study might be an acute stress re-
action. Alterations in OX1R expression during the first 24
hours after TBI can result from a reduced number of glial
cells, low blood pressure, an intracranial pressure change,
hypoxia and ischemia, changes in blood glucose levels, or
the release of neural specific nucleoprotein (Mihara et al.,
2011; Willie et al., 2012). Thus, the orexinergic system ap-
[Downloaded free from http://www.nrronline.org on Friday, May 4, 2018, IP: 84.209.170.222]

248
Dong XY, Feng Z (2018) Wake-promoting eects of vagus nerve stimulation aer traumatic brain injury: upregulation of orexin-A and orexin
receptor type 1 expression in the prefrontal cortex. Neural Regen Res 13(2):244-251. doi: 10.4103/1673-5374.226395
pears to play an important role in the pathophysiology of
TBI. In this study, we found increased levels of orexin-A and
OX1R expression in the prefrontal cortex of rats in the TBI
group compared with the control group. ese upregulated
levels of orexin-A and OX1R during the 24-hour period af-
Figure 1 Eect of vagus nerve stimulation on orexin-A expression in
the prefrontal cortex of rats in a TBI-induced coma (enzyme-linked
immunosorbent assay).
Data are expressed as the mean ± SD (n = 6 per group; one-way anal-
ysis of variance followed by Tukey’s test); *P < 0.05, vs. control group
and stimulated group; #P < 0.05, vs. antagonist group; †P < 0.05, vs. 6
hours aer TBI. Control group: Healthy rats with sham operation and
anesthesia. TBI group: Free-fall drop was used to establish the model
of TBI-induced coma. Stimulated group: TBI-induced comatose rats
subjected to vagus nerve stimulation. Antagonist group: Comatose rats
received intracerebroventricular injection of the orexin receptor type
1 antagonist SB334867 and vagus nerve stimulation. TBI: Traumatic
brain injury.
Figure 2 Eect of vagus nerve stimulation on OX1R protein
expression in the prefrontal cortex of rats in a TBI-induced coma
(western blot assay).
Dierent expression levels may be associated with the circadian rhythm
of hypothalamic orexin secretion. Data are expressed as the mean ± SD
(n = 6 per group; one-way analysis of variance). *P < 0.05, vs. control
and stimulated groups; #P < 0.05, vs. antagonist group; †P < 0.05, vs. 6
hours aer TBI. Control group: Healthy rats with sham operation and
anesthesia. TBI group: Free-fall drop was used to establish the model
of TBI-induced coma. Stimulated group: Rats in a TBI-induced coma
subjected to vagus nerve stimulation. Antagonist group: Comatose
rats received intracerebroventricular injection of the OX1R antagonist
SB334867 and vagus nerve stimulation. OX1R: Orexin receptor type 1;
TBI: traumatic brain injury.
Increased OX1R expression
was detected within the
cytoplasm of neurons in
the prefrontal cortex at 12
h aer TBI. Control group:
Healthy rats with sham op-
eration and anesthesia. TBI
group: TBI-induced coma.
Stimulated group: Rats
in a TBI-induced coma
subjected to vagus nerve
stimulation. Antagonist
group: Comatose rats re-
ceived intracerebroventric-
ular injection of the OX1R
antagonist SB334867 and
vagus nerve stimulation.
TBI: Traumatic brain inju-
ry; OX1R: orexin receptor
type 1; h: hours.
Figure 3 Eect of vagus nerve stimulation on OX1R expression in the prefrontal cortex of rats with TBI-induced coma (immunostaining,
light microscope, original magnication, 400×).
Control TBI Stimulated Antagonist
6 h
12 h
24 h
Control group TBI group
Stimulated group Antagonist group
6 12 24
Hours after coma induction
*#*
#
*† #†
†
†
Orexin-A expression (optical density)
Control group TBI group
Stimulated group Antagonist group
*
#
†
*†
†
†
*
#
OX1R (55 kDa)
β-Actin (45 kDa)
6 12 24
Hours after coma induction
Relative protein expression of OX1R
(optical density ratio to β-actin)
[Downloaded free from http://www.nrronline.org on Friday, May 4, 2018, IP: 84.209.170.222]

249
Dong XY, Feng Z (2018) Wake-promoting eects of vagus nerve stimulation aer traumatic brain injury: upregulation of orexin-A and orexin
receptor type 1 expression in the prefrontal cortex. Neural Regen Res 13(2):244-251. doi: 10.4103/1673-5374.226395
ter TBI might be a response to physiological stress, which is
important for protecting neurons during the early stages of
TBI.
Orexins (orexin-A and orexin-B) produced in the lateral
hypothalamus are hypothalamic peptides involved in food
intake, metabolic rate, growth hormone production, auto-
nomic function, and the sleep/wake cycle (Boss and Roch,
2015; Mieda, 2017; Walker and Lawrence, 2017). e orexin-
ergic system comprises many regions of the central nervous
system, including the cerebral cortex, thalamus, hypothala-
mus, brain stem, and limbic system. OX1R is expressed by
immunoreactive nerve ber projections that are extensively
distributed throughout the central nervous system, includ-
ing the hippocampus, dorsal raphe nucleus, arcuate nucleus,
anterior pretectal nucleus, tuberomammillary nucleus, and
raphe nucleus. Moreover, OX1R is most highly expressed
in the prefrontal cortex and locus coeruleus, which play key
roles in regulating wakefulness (Xu et al., 2013). A previous
report showed that an important function of the orexin-
ergic system is regulating the sleep/wake cycle by directly
activating various hypothalamic-cortical pathways (Kampe
et al., 2009). The prefrontal cortex has critically important
roles in advanced brain activities, including consciousness,
integration of information, and cognition. Orexinergic neu-
rons in the hypothalamus project into the prefrontal cortex
and excite central nervous system neurons (Xia et al., 2005).
Orexin-A-mediated activation of two G-protein coupled re-
ceptors, OX1R and OX2R, upregulates Ca2+ levels in the cy-
toplasm and induces the activation of phosphatases, second
messengers, and protein kinases (Kukkonen and Leonard,
2014; Shu et al., 2014; Kukkonen, 2016).
Since its approval for therapeutic use by the Food and
Drug Administration in 1997, VNS has been widely used
for treating refractory epilepsy, depression, and cognitive
disorders (Yuan and Silberstein, 2016, 2017; Ekmekci and
Kaptan, 2017; Fulton et al., 2017). While few studies have
investigated the relationship between VNS and wakefulness,
some evidence suggests that VNS can reduce daytime sleep
in patients with epilepsy caused by TBI.
For the following reasons, VNS might be a potentially
eective new method for improving the status of patients in
a TBI-induced coma: (1) Extensive brous projections. e
nucleus of the solitary tract receives the majority of vagal
aerent bers and projects into many brainstem regions, in-
cluding the locus coeruleus, parabrachial nucleus, thalamus,
basal forebrain, hypothalamus, and cerebral cortex (Ansari
et al., 2007). Electrical stimulation of the vagus nerve should
activate the ascending reticular activating system, which
plays a key role in promoting arousal, thereby alleviating
the comatose condition. (2) Inuence of related neurotrans-
mitters. Neurotransmitters such as orexin, noradrenaline,
glutamate and dopamine are known to have wake-promot-
ing eects. Recent studies suggest that some of the eects of
VNS may involve stimulation of the locus coeruleus to re-
lease noradrenaline throughout the central nervous system.
Noradrenaline significantly affects recovery from TBI by
promoting wakefulness and by inhibiting sleep (Smith et al.,
2005). Other reports show that VNS signicantly increases
extracellular noradrenaline levels in the hippocampus and
prefrontal cortex, as well as 5-HT levels in the dorsal raphe
nucleus and dopamine levels in the prefrontal cortex and
nucleus accumbens (Manta et al., 2013). (3) Anti-inflam-
matory eects. e cholinergic anti-inammatory pathway
that systemically inhibits pro-inammatory cytokine release
is well characterized. Vagal eerents are thought to regulate
systemic inammation by modulating the release of tumor
necrosis factor from macrophages. VNS has been shown
to decrease cerebral edema, thereby helping avoid further
damage to neurons aer TBI (Bonaz et al., 2013; Xiang et al.,
2015). (4) Increasing cerebral blood flow. Following VNS,
signicant increases in blood ow have been reported in the
le posterior limb of the internal capsule/medial putamen,
right dorsal anterior cingulate, right superior temporal gy-
rus, left cerebellum, and left dorsolateral prefrontal cortex
(Kosel et al., 2011; Conway et al., 2012). e enhanced blood
flow should improve neural survival and promote wake-
fulness. (5) Neurotrophic factors and synaptic plasticity.
Neurotrophic factors, such as brain-derived neurotrophic
factor and nerve growth factor, are crucial for neuronal
survival, development, function and synaptic plasticity, all
of which can be aected when previously inactive synapses
become functional. VNS rapidly activates the brain-derived
neurotrophic factor receptor TrκB and upregulates nerve
growth factor levels in the rat brain (Follesa et al., 2007;
Cossu, 2014). (6) Electrical activity of the brain. VNS has
been reported to increase slow-wave sleep prior to rapid eye
movement in rats and upregulate the frequencies of sleep
spindles, δ-waves and ponto-geniculo-occipital waves (Val-
des-Cruz et al., 2008). (7) Other mechanisms. VNS has been
reported to decrease damage to the blood-brain barrier,
modulate depolarization activity, upregulate endogenous
neurogenesis, and attenuate glutamate-mediated excitotox-
icity in models of TBI (Kumaria and Tolias, 2012).
Our previous studies showed that median nerve stimula-
tion promotes wakefulness by upregulating orexin-A and
OX1R in the prefrontal cortex and hypothalamus in rats
with TBI-induced coma (Feng et al., 2015; Zhong et al.,
2015; Feng and Du, 2016). erefore, our current study was
designed to explore the relationship between orexin-A levels
in the prefrontal cortex and VNS, and assess the wake-pro-
moting eect of VNS by evaluating the level of conscious-
ness using a I−VI grading scale. To our knowledge, there has
been no similar study reported to date.
In this study, only 8 of 30 rats re-awakened in the TBI
group, 20 of 30 rats re-awakened in the stimulated group,
and 12 of 30 rats recovered from a comatose state in the
antagonist group. ese results suggest that VNS promotes
wakefulness in rats with TBI-induced coma. We also found
that orexin-A levels were significantly upregulated in the
prefrontal cortex of rats in the stimulated group compared
with the other groups. Western blot assay and immunohis-
tochemistry studies revealed trends of increasing OX1R and
orexin-A expression in the stimulated group compared with
the other groups. We also found lower expression levels of
[Downloaded free from http://www.nrronline.org on Friday, May 4, 2018, IP: 84.209.170.222]

250
Dong XY, Feng Z (2018) Wake-promoting eects of vagus nerve stimulation aer traumatic brain injury: upregulation of orexin-A and orexin
receptor type 1 expression in the prefrontal cortex. Neural Regen Res 13(2):244-251. doi: 10.4103/1673-5374.226395
orexin-A and OX1R in the control group compared with
the stimulated group after VNS. Moreover, similar results
were observed at 6 and 12 hours in the group administered
an OX1R antagonist (SB334867). These findings suggest
that VNS directly impacts orexin-A and OX1R levels at 6,
12 and 24 hours. Within each group, levels of orexin-A and
OX1R were signicantly higher at 12 hours than at 6 or 24
hours; this may be associated with the rhythmic patterns
of orexin-A neurons. It has been reported that lateral hy-
pothalamic orexin-A neurons are rhythmic and innervated
by suprachiasmatic nucleus efferents, which are important
components of the arousal system. Orexin neuronal activity
is higher during the night than during the day (Belle et al.,
2014). Moreover, genes for orexin receptors are expressed
in mouse suprachiasmatic nucleus eerents, and OX1R be-
comes upregulated at dusk (Alo et al., 2017). is circadian
rhythmicity of orexin neurons might underlie the observed
changes in orexin-A and OX1R expression at the different
time points. In the present study, the time during the day
that the rats were killed was not constant, and was partially
dependent on individual dierences in recovery time follow-
ing TBI or VNS treatment.
Our study has some limitations that should be mentioned.
For example, we could have used electroencephalograms,
the Glasgow Coma Scale or the evoked potential test to ex-
amine the wake-promoting effects of VNS. Also, a larger
sample size and a more precise method of measuring TBI
could have been used. Additionally, we only examined
orexin-A and OX1R expression in the prefrontal cortex, al-
though other regions of the brain, such as the hypothalamus
and hippocampus, also participate in promoting wakeful-
ness. Further studies are needed to clarify how expression
of orexin-A and its receptor change following TBI-induced
coma, how VNS causes increased orexin-A expression, and
the mechanisms and pathways underlying these changes.
Despite the limitations, our study suggests that orexin-A
plays a key role in promoting consciousness, and that VNS
helps an animal recover consciousness from TBI-induced
coma by upregulating orexin-A and OX1R expression.
Additional wake-promoting mechanisms might be dis-
covered if other neurotransmitters and regions of the brain
related to orexin-A and/or wakefulness are studied to iden-
tify whether orexin-A acts as an “arousal switch”. Orexin-A
knockout animals could be used in future studies to provide
additional insight into the eects found in the current study.
In conclusion, upregulation of orexin-A and OX1R ex-
pression in the prefrontal cortex might contribute to the
wake-promoting effect of VNS in TBI-induced comatose
rats. Our ndings suggest that VNS is a promising method
for awakening patients in a TBI-induced coma. However,
further studies are required to test the clinical eects of VNS
and its possible complications.
Author contributions: XYD was responsible for study implementation,
data collection, data analysis and wrote the paper. ZF was responsible
for experiment design and supervision. Both authors approved the nal
version of the paper.
Conicts of interest: None declared.
Financial support: This study was supported by the Natural Science
Foundation of China, No. 81260295 and the Graduate Student Innova-
tion Fund of Jiangxi Province of China, No. YC2015-S090. Funders had
no involvement in the study design; data collection, management, analy-
sis, and interpretation; paper writing; or decision to submit the paper for
publication.
Research ethics: e study protocol was approved by the Animal Ethics
Committee of the First Affiliated Hospital of Nanchang University of
China (approval number (2016) (003)). e experimental procedure fol-
lowed the United States National Institutes of Health Guide for the Care
and Use of Laboratory Animal (NIH Publication No. 85-23, revised
1985).
Data sharing statement: Datasets analyzed during the current study
are available from the corresponding author on reasonable request.
Plagiarism check: Checked twice by ienticate.
Peer review: Externally peer reviewed.
Open access statement: is is an open access article distributed under
the terms of the Creative Commons Attribution-NonCommercial-Shar-
eAlike 3.0 License, which allows others to remix, tweak, and build upon
the work non-commercially, as long as the author is credited and the
new creations are licensed under identical terms.
References
Alo R, Avolio E, Mele M, Fazzari G, Carelli A, Facciolo RM, Canonaco M
(2017) Role of leptin and orexin-a within the suprachiasmatic nucleus
on anxiety-like behaviors in hamsters. Mol Neurobiol 54:2674-268
4.
Ansari S, Chaudhri K, Al Moutaery KA (2007) Vagus nerve stimula-
tion: indications and limitations. Acta Neurochir Suppl 97:281-286.
Belle MD, Hughes AT, Bechtold DA, Cunningham P, Pierucci M, Bur-
dakov D, Piggins HD (2014) Acute suppressive and long-term phase
modulation actions of orexin on the mammalian circadian clock. J
Neurosci 34:3607-3621.
Bonaz B, Picq C, Sinniger V, Mayol JF, Clarencon D (2013) Vagus
nerve stimulation: from epilepsy to the cholinergic anti-inammato-
ry pathway. Neurogastroenterol Motil 25:208-221.
Boss C, Roch C (2015) Recent trends in orexin research-2010 to 2015.
Bioorg Med Chem Lett 25:2875-2887.
Conway CR, Sheline YI, Chibnall JT, Bucholz RD, Price JL, Gangwani S,
Mintun MA (2012) Brain blood-ow change with acute vagus nerve
stimulation in treatment-refractory major depressive disorder. Brain
Stimul 5:163-171.
Cossu G (2014) Therapeutic options to enhance coma arousal after
traumatic brain injury: state of the art of current treatments to im-
prove coma recovery. Br J Neurosurg 28:187-198.
Durand E, Chevignard M, Ruet A, Dereix A, Jourdan C, Pradat-Diehl
P (2017) History of traumatic brain injury in prison populations: a
systematic review. Ann Phys Rehabil Med 60:95-101.
Ekmekci H, Kaptan H (2017) Vagus nerve stimulation. Open Access
Maced J Med Sci 5:391-394.
Elkind JA, Lim MM, Johnson BN, Palmer CP, Putnam BJ, Kirschen
MP, Cohen AS (2015) Efficacy, dosage, and duration of action of
branched chain amino acid therapy for traumatic brain injury. Front
Neurol 6:73.
Feeney DM, Boyeson MG, Linn RT, Murray HM, Dail WG (1981)
Responses to cortical injury: I. Methodology and local eects of con-
tusions in the rat. Brain Res 211:67-77.
Feng Z, Du Q (2016) Mechanisms responsible for the eect of median
nerve electrical stimulation on traumatic brain injury-induced coma:
orexin-A-mediated N-methyl-D-aspartate receptor subunit NR1 up-
regulation. Neural Regen Res 11:951-956.
Feng Z, Zhong YJ, Wang L, Wei TQ (2015) Resuscitation therapy for
traumatic brain injury-induced coma in rats: mechanisms of median
nerve electrical stimulation. Neural Regen Res 10:594-598.
Follesa P, Biggio F, Gorini G, Caria S, Talani G, Dazzi L, Puligheddu
M, Marrosu F, Biggio G (2007) Vagus nerve stimulation increases
norepinephrine concentration and the gene expression of BDNF and
bFGF in the rat brain. Brain Res 1179:28-34.
Frangos E, Komisaruk BR (2017) Access to vagal projections via cuta-
neous electrical stimulation of the neck: fMRI evidence in healthy
humans. Brain Stimul 10:19-27.
[Downloaded free from http://www.nrronline.org on Friday, May 4, 2018, IP: 84.209.170.222]

251
Dong XY, Feng Z (2018) Wake-promoting eects of vagus nerve stimulation aer traumatic brain injury: upregulation of orexin-A and orexin
receptor type 1 expression in the prefrontal cortex. Neural Regen Res 13(2):244-251. doi: 10.4103/1673-5374.226395
Fulton SP, Van Poppel K, McGregor AL, Mudigoudar B, Wheless JW
(2017) Vagus nerve stimulation in intractable epilepsy associated
with SCN1A gene abnormalities. J Child Neurol 32:494-498.
Gray SN (2017) An overview of the use of neurofeedback biofeedback
for the treatment of symptoms of traumatic brain injury in military
and civilian populations. Med Acupunct 29:215-219.
Harvey HH (2013) Reducing traumatic brain injuries in youth sports:
youth sports traumatic brain injury state laws, January 2009-Decem-
ber 2012. Am J Public Health 103:1249-1254.
Hiebert JB, Shen Q, immesch AR, Pierce JD (2015) Traumatic brain
injury and mitochondrial dysfunction. Am J Med Sci 350:132-138.
Hue CD, Cho FS, Cao S, Nicholls RE, Vogel Iii EW, Sibindi C, Aran-
cio O, Dale Bass CR, Meaney DF, Morrison Iii B (2016) Time
course and size of blood-brain barrier opening in a mouse model of
blast-induced traumatic brain injury. J Neurotrauma 33:1202-1211.
Jain SV, Glauser TA (2014) Effects of epilepsy treatments on sleep
architecture and daytime sleepiness: an evidence-based review of
objective sleep metrics. Epilepsia 55:26-37.
Jeter CB, Hergenroeder GW, Ward NH, 3rd, Moore AN, Dash PK (2013)
Human mild traumatic brain injury decreases circulating branched-
chain amino acids and their metabolite levels. J Neurotrauma
30:671-679.
Joseph B, Khan M, Rhee P (2017) Non-invasive diagnosis and treat-
ment strategies for traumatic brain injury: an update. J Neurosci Res
doi: 10.1002/jnr.24132.
Kaelber S, Pantcheva P, Borlongan CV (2016) Drug- and cell-based
therapies for targeting neuroinammation in traumatic brain injury.
Neural Regen Res 11:1575-1576.
Kampe J, Tschop MH, Hollis JH, Oldeld BJ (2009) An anatomic basis
for the communication of hypothalamic, cortical and mesolimbic
circuitry in the regulation of energy balance. Eur J Neurosci 30:415-
430.
Kosel M, Brockmann H, Frick C, Zobel A, Schlaepfer TE (2011)
Chronic vagus nerve stimulation for treatment-resistant depression
increases regional cerebral blood ow in the dorsolateral prefrontal
cortex. Psychiatry Res 191:153-159.
Kukkonen JP (2016) OX2 orexin/hypocretin receptor signal transduc-
tion in recombinant Chinese hamster ovary cells. Cell Signal 28:51-
60.
Kukkonen JP, Leonard CS (2014) Orexin/hypocretin receptor signal-
ling cascades. Br J Pharmacol 171:314-331.
Kumaria A, Tolias CM (2012) Is there a role for vagus nerve stimula-
tion therapy as a treatment of traumatic brain injury? Br J Neurosurg
26:316-320.
Lucke-Wold BP, Naser ZJ, Logsdon AF, Turner RC, Smith KE, Robson
MJ, Bailes JE, Lee JM, Rosen CL, Huber JD (2015) Amelioration of
nicotinamide adenine dinucleotide phosphate-oxidase mediated
stress reduces cell death aer blast-induced traumatic brain injury.
Transl Res 166:509-528.
Malow BA, Edwards J, Marzec M, Sagher O, Ross D, Fromes G (2001)
Vagus nerve stimulation reduces daytime sleepiness in epilepsy pa-
tients. Neurology 57:879-884.
Manta S, El Mansari M, Debonnel G, Blier P (2013) Electrophysiolog-
ical and neurochemical eects of long-term vagus nerve stimulation
on the rat monoaminergic systems. Int J Neuropsychopharmacol
16:459-470.
Mieda M (2017) e roles of orexins in sleep/wake regulation. Neuros-
ci Res 118:56-65.
Mihara Y, Dohi K, Yofu S, Nakamachi T, Ohtaki H, Shioda S, Aruga T
(2011) Expression and localization of the orexin-1 receptor (OX1R)
aer traumatic brain injury in mice. J Mol Neurosci 43:162-168.
Neren D, Johnson MD, Legon W, Bachour SP, Ling G, Divani AA
(2016) Vagus nerve stimulation and other neuromodulation meth-
ods for treatment of traumatic brain injury. Neurocrit Care 24:308-
319.
Ramanjaneya M, Conner AC, Chen J, Kumar P, Brown JE, Johren O,
Lehnert H, Stanfield PR, Randeva HS (2009) Orexin-stimulated
MAP kinase cascades are activated through multiple G-protein sig-
nalling pathways in human H295R adrenocortical cells: diverse roles
for orexins A and B. J Endocrinol 202:249-261.
Shi C, Flanagan SR, Samadani U (2013) Vagus nerve stimulation to
augment recovery from severe traumatic brain injury impeding con-
sciousness: a prospective pilot clinical trial. Neurol Res 35:263-276.
Shu Q, Hu ZL, Huang C, Yu XW, Fan H, Yang JW, Fang P, Ni L, Chen
JG, Wang F (2014) Orexin-A promotes cell migration in cultured rat
astrocytes via Ca2+-dependent PKCalpha and ERK1/2 signals. PLoS
One 9:e95259.
Singh IN, Gilmer LK, Miller DM, Cebak JE, Wang JA, Hall ED (2013)
Phenelzine mitochondrial functional preservation and neuropro-
tection aer traumatic brain injury related to scavenging of the lipid
peroxidation-derived aldehyde 4-hydroxy-2-nonenal. J Cereb Blood
Flow Metab 33:593-599.
Smith DC, Modglin AA, Roosevelt RW, Neese SL, Jensen RA, Brown-
ing RA, Clough RW (2005) Electrical stimulation of the vagus nerve
enhances cognitive and motor recovery following moderate fluid
percussion injury in the rat. J Neurotrauma 22:1485-1502.
Soslow RA, Dannenberg AJ, Rush D, Woerner BM, Khan KN, Mas-
ferrer J, Koki AT (2000) COX-2 is expressed in human pulmonary,
colonic, and mammary tumors. Cancer 89:2637-2645.
Stephens JR, Levy RH (1994) Eects of valproate and citrulline on am-
monium-induced encephalopathy. Epilepsia 35:164-171.
Valdes-Cruz A, Magdaleno-Madrigal VM, Martinez-Vargas D, Fer-
nandez-Mas R, Almazan-Alvarado S (2008) Long-term changes in
sleep and electroencephalographic activity by chronic vagus nerve
stimulation in cats. Prog Neuropsychopharmacol Biol Psychiatry
32:828-834.
Walker LC, Lawrence AJ (2017) e role of orexins/hypocretins in al-
cohol use and abuse. Curr Top Behav Neurosci 33:221-246.
Willie JT, Lim MM, Bennett RE, Azarion AA, Schwetye KE, Brody DL
(2012) Controlled cortical impact traumatic brain injury acutely dis-
rupts wakefulness and extracellular orexin dynamics as determined
by intracerebral microdialysis in mice. J Neurotrauma 29:1908-1921.
Wu HY, Tang CF, He H (2007) Orexin, orexin receptor and sports.
Zhongguo Zuzhi Gongcheng Yanjiu 11:7966-7969.
Xia JX, Chen XW, Cheng SY, Hu ZA (2005) Mechanisms of orexin
A-evoked changes of intracellular calcium in primary cultured corti-
cal neurons. Neuroreport 16:783-786.
Xiang YX, Wang WX, Xue Z, Zhu L, Wang SB, Sun ZH (2015) Electri-
cal stimulation of the vagus nerve protects against cerebral ischemic
injury through an anti-infammatory mechanism. Neural Regen Res
10:576-582.
Xu TR, Yang Y, Ward R, Gao L, Liu Y (2013) Orexin receptors:
multi-functional therapeutic targets for sleeping disorders, eating
disorders, drug addiction, cancers and other physiological disorders.
Cell Signal 25:2413-2423.
Yuan H, Silberstein SD (2015) Vagus nerve stimulation and headache.
Headache doi: 10.1111/head.12721
Yuan H, Silberstein SD (2016) Vagus nerve and vagus nerve stimula-
tion, a comprehensive review: part I. Headache 56:71-78.
Yuan H, Silberstein SD (2017) Vagus nerve stimulation and headache.
Headache 57 Suppl 1:29-33.
Zhong YJ, Feng Z, Wang L, Wei TQ (2015) Wake-promoting actions
of median nerve stimulation in TBI-induced coma: an investigation
of orexin-A and orexin receptor 1 in the hypothalamic region. Mol
Med Rep 12:4441-4447.
Zhu JQ, Hong J, Cui JZ, Wang KJ (2017) Adipose mesenchymal stem
cells for treatment of traumatic brain injury. Zhongguo Zuzhi Gong-
cheng Yanjiu 21:71-76.
（Copyedited by Patel B, Maxwell R, Wang J, Li CH, Qiu Y, Song LP,
Zhao M）
[Downloaded free from http://www.nrronline.org on Friday, May 4, 2018, IP: 84.209.170.222]