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R E S E A R C H Open Access
Electrostimulation of the carotid sinus
nerve in mice attenuates inflammation via
glucocorticoid receptor on myeloid
immune cells
Aidan Falvey
1
, Fabrice Duprat
1
, Thomas Simon
1
, Sandrine Hugues-Ascery
2
, Silvia V. Conde
3
,
Nicolas Glaichenhaus
1
and Philippe Blancou
1*
Abstract
Background: The carotid bodies and baroreceptors are sensors capable of detecting various physiological parameters
that signal to the brain via the afferent carotid sinus nerve for physiological adjustment by efferent pathways. Because
receptors for inflammatory mediators are expressed by these sensors, we and others have hypothesised they could
detect changes in pro-inflammatory cytokine blood levels and eventually trigger an anti-inflammatory reflex.
Methods: To test this hypothesis, we surgically isolated the carotid sinus nerve and implanted an electrode, which
could deliver an electrical stimulation package prior and following a lipopolysaccharide injection. Subsequently, 90 min
later, blood was extracted, and cytokine levels were analysed.
Results: Here, we found that carotid sinus nerve electrical stimulation inhibited lipopolysaccharide-induced tumour
necrosis factor production in both anaesthetised and non-anaesthetised conscious mice. The anti-inflammatory effect of
carotid sinus nerve electrical stimulation was so potent that it protected conscious mice from endotoxaemic shock-induced
death. In contrast to the mechanisms underlying the well-described vagal anti-inflammatory reflex, this phenomenon does
not depend on signalling through the autonomic nervous system. Rather, the inhibition of lipopolysaccharide-induced
tumour necrosis factor production by carotid sinus nerve electrical stimulation is abolished by surgical removal of the
adrenal glands, by treatment with the glucocorticoid receptor antagonist mifepristone or by genetic inactivation of the
glucocorticoid gene in myeloid cells. Further, carotid sinus nerve electrical stimulation increases the spontaneous discharge
activity of the hypothalamic paraventricular nucleus leading to enhanced production of corticosterone.
Conclusion: Carotid sinus nerve electrostimulation attenuates inflammation and protects against lipopolysaccharide-
induced endotoxaemic shock via increased corticosterone acting on the glucocorticoid receptor of myeloid immune cells.
Theseresultsprovidearationalefortheuseofcarotidsinus nerve electrostimulation as a therapeutic approach for
immune-mediated inflammatory diseases.
Keywords: Bioelectronic medicine, Carotid body, Carotid sinus nerve, Corticosterone, Electrostimulation, Immunology
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* Correspondence: Blancou@ipmc.cnrs.fr
1
Université Côte d’Azur, CNRS, Institut de Pharmacologie Moléculaire et
Cellulaire, Valbonne, France
Full list of author information is available at the end of the article
Falvey et al. Journal of Neuroinflammation (2020) 17:368
https://doi.org/10.1186/s12974-020-02016-8
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Introduction
Inflammation is part of the complex biological response of
body tissues to harmful stimuli—pathogens, damaged cells
or irritants. It involves the recruitment of immune cells
and the production of soluble molecules including pro-
inflammatory cytokines—tumour necrosis factor (TNF),
interleukin (IL)-1α,IL-1β, IL-6 and IL-12. These cytokines
eventually act on both immune and non-immune cell types
by signalling through specific surface receptors. While in-
flammation could be viewed as a protective mechanism,
pro-inflammatory cytokines may also cause tissue injury
and have a deleterious effect. This occurs during endotoxic
shock which results from a severe, generalised inflamma-
tory response induced by bloodstream infection with
gram-negative bacteria. It also occurs in immune-mediated
inflammatory diseases (IMIDs) such as rheumatoid arth-
ritis (RA), inflammatory bowel disease (IBD) and systemic
lupus erythematosus (SLE) [1]. It is therefore not surprising
that several neuro-hormonal anti-inflammatory pathways
have been identified. At least two neuro-hormonal anti-
inflammatory pathways have been described: the activation
of the hypothalamic-pituitary-adrenal (HPA) axis and the
vagal anti-inflammatory reflex [2].
The HPA is activated by several stimuli including
psychological stress, which activate the paraventricular nu-
cleus (PVN) of the hypothalamus and eventually the re-
lease of cortisol-releasing hormone (CRH) into the anterior
pituitary. In turn, CRH induces the release of adrenocorti-
cotrophic hormone (ACTH) into the blood which stimu-
lates the production of glucocorticoids by the cortex of the
adrenal glands. Glucocorticoids are potent anti-
inflammatory molecules, and their effect is mediated via
signalling by the glucocorticoid receptor (GR) which is
expressed by almost all cells in the body and in particular
innate immune cells [3].
While the inhibition of pro-inflammatory cytokine pro-
duction by immune cells is mediated by glucocorticoids
when the HPA axis is activated, the vagal anti-
inflammatory reflex relies on the binding of acetylcholine
(ACh) on nicotinic ACh receptors (nAChR). The vagal
anti-inflammatory reflex primarily involves the vagus nerve,
synapsing at the coeliac ganglion, and the release of nor-
epinephrine by sympathetic nerve fibres that project to the
spleen. Norepinephrine binds to β2 adrenergic receptor
(AR) at the surface of CD4
+
T cells, eventually inducing the
release of ACh and the inhibition of pro-inflammatory
cytokine production by spleen macrophages through a
nAChR-dependent mechanism [4]. In recent years, efforts
to convert this inflammatory reflex into a therapeutic have
been conducted via electrical activation of the vagus nerve.
This form of therapeutic can be described as bioelectronic
medicine, and clinical trials have been performed to investi-
gate its anti-inflammatory properties in IMIDs [5,6]. Par-
tially, due to the studies on the inflammatory reflex,
bioelectronic medicine has seen a resurgence in recent
years [7]. There is a lot of interest in discovering the anti-
inflammatory potential of additional nerves. One interesting
target is potentially the carotid sinus nerve (CSN).
The CSN is connected to the carotid body (CB) and
baroreceptors that project to the brain [8]. The CB is a
paraganglion located bilaterally in the neck at the bifur-
cation of the carotid artery into the internal and external
arteries. It is a polymodal sensor and is capable of detecting
oxygen and carbon dioxide concentration in the blood and
insulin [8,9]. Baroreceptors are sensors located in the ca-
rotid sinus and in the aortic arch, which sense the blood
pressure and relay the information to the brain via the
CSN or the aortic depressor nerve, respectively. Once stim-
uli are detected by the CB or baroreceptors, they signal via
the CSN to the brain to modulate these physiological vari-
ables as required. In recent years, it is becoming increas-
ingly evident that both the CB and the baroreceptors can
detect inflammation. For example, the CB can detect cyto-
kines—TNF, IL-1B and IL-6 [10–12]—and pathogenic
components—lipopolysaccharide (LPS) and zymosan [11,
13,14]. This detection causes activation of the CB [10,12],
as well as inducing signalling in the CSN [11,15]. Addition-
ally, baroreceptors also express receptors for inflammatory
mediators, and stimulation of immune receptors with their
cognate ligands would lead to activation of C-fibre neurons
[16]. Most importantly, there is preliminary evidence sug-
gesting that both the CB [17] and the baroreceptors [18]
signalling to the brain could be anti-inflammatory in rats.
In line with these results, it was found that bilateral re-
moval of the CSN in rats reduced survival when these ani-
mals are exposed to high bacterial load [19]. Overall, the
evidence suggests that selective activation of the CSN may
attenuate inflammation. Therefore, we hypothesised that
electrical activation of the CSN in mice will attenuate in-
flammation and protect against IMIDs.
Methods
Animals
Female 6–7-week-old C57BL/6 mice were purchased from
Charles River, France. All experiments were conducted on
mice age 8–10 weeks old. LysM-Cre:GR
fl/fl
mice were
backcrossed more than 12 times onto the C57BL/6 back-
ground and confirmed to be double-positive by genomic
PCR (GoTaq Green Master Mix, Promega). All mice were
given access to food and water ad libitum and maintained
on a 12-h light and dark schedule.
Reagents
Lipopolysaccharide (LPS) from Escherichia coli (O127:B8)
(Sigma-Aldrich) was aliquoted to 5 mg/ml. The aliquots
were frozen at −20 °C and defrosted to be prepared fresh
with PBS to a concentration of 100 μgin200μlintraperito-
neally (IP). Prior to surgery, buprecare (Axience, Centravet,
Falvey et al. Journal of Neuroinflammation (2020) 17:368 Page 2 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
France) was injected at 0.2 mg/kg IP, and the following day,
mice were injected with an additional 50 μlsubcutaneously.
Dolethal (Vétoquinol, Centravet, France) was used to
induce lethal unconsciousness prior to intracardial blood
extraction. Atropine, hexamethonium, propranolol and
mifepristone (all from Sigma-Aldrich) were used at a con-
centration of 1 mg/kg, 10 mg/kg, 2.5 mg/kg and 80 mg/kg,
respectively. All were diluted in PBS except for mifepristone
which was diluted in a PBS/DMSO mix (10%) (Sigma-Al-
drich). All were injected IP in 200 μl.
Acute CSN stimulation
Mice were injected IP with buprecare (100 μl—Axience,
Centravet, France), as an analgesic, at least 10 min prior to
surgery. Isoflurane (Piramal, CSP, France) mixed with air
at 2.5% was used to induce mice into unconsciousness.
The left carotid artery bifurcation was exposed, and the
hypoglossal nerve was located. A small muscle covering
the hypoglossal nerve was located and cut solely. The
CSN was located via its connection to the CB.
In sham and experimental mice, home-made nichrome
electrodes made of two individual wires (A&M Systems)
were placed under and around the left CSN. Experimental
mice only received electrostimulation, and sham mice did
not. For all acute experiments, electrical stimulation was
administered by a Plexon stimulator (PlexStim Electrical
Stimulator System) as rectangular charged-balanced bi-
phasic pulses with 200 to 600 μA pulse amplitude, 100 μs
pulse width (positive and negative) at 5 or 10 Hz fre-
quency for 2 min, 5 min before and after a 100-μgLPS
(Sigma-Aldrich) IP injection in 200 μl of PBS (Fig. 1a–d).
Organ excision and drug injection were performed 20 and
30 min prior to LPS injection, respectively (Fig. 1b–d). As
previously observed by us and others, LPS-induced serum
TNF levels vary from one experiment to another due to
the time of the day it was injected or the LPS lot-to-lot
variations. A variety of stimulation patterns were shown
to be effective; however, depending on the manufactured
electrodes, the optimal stimuli varied. Typically, the lowest
impact stimulation pattern that was effective was chosen
to decrease the likelihood of electrical spread to other
nerves. Exact stimulation patterns are outlined per experi-
ment (Fig. 1). Once stimulation was complete, mice were
sutured and allowed to recover in a heated cage. Blood
was collected 90 min after LPS injection retro-orbitally
just before being sacrificed.
Chronic CSN stimulation
Surgery was performed as described for acute CSN stimu-
lation. An additional step was added to enable further an-
choring of the physiological glue (Kwik-cast & Kwik-sil,
World Precision Instruments) to ensure the wires were
kept safely in place. The animal was cervically sutured and
gently flipped to place dental cement (Super Bond C&B)
around the electrode end to build the head cap, prior to
placing the mouse in a heated cage for recovery. The
following day, mice were given 4 μgofmorphine(Bur-
precare) subcutaneously, and the subsequent 2 days
were undisturbed. Mice were placed into individual
cages, and a stimulating wire was connected to their
head caps when needed.
Inhibition of LPS-induced TNF release was assessed
following timeline F (Fig. 1f) in two independent co-
horts. Mice were electrically stimulated and injected
with LPS as described above. Ninety minutes after LPS
injection, mice were given Dolethal (Vétoquinol, Centra-
vet, France) before blood was extracted intracardially.
Survival to lethal LPS injection was assessed following IP
injection of 1 mg of LPS. Conscious stimulation was per-
formed as described, following timeline G (Fig. 1g). For this
experiment, however, on the first day, mice were stimulated
(200 μA, 5Hz, 0.1ms) for 5 min at 11:00 and again at 18:
00. LPS was injected at 13:00, the mice were watched for
the next 72 h and when a mouse died the hour was noted.
Organs excision and vagus nerve resection
The spleen excision was performed before electrode im-
plantation by tying a suture knot onto the three major
blood vessels entering the spleen and then gently separat-
ing out the spleen. In a separate experiment, the entire ad-
renal glands, bilaterally, were solely cauterised by a
biological cauterising tool. In another separate experi-
ment, prior to CSN stimulation, the left cervical vagus
nerve in the vicinity of the carotid bifurcation was excised
unilaterally. All mice were adequately sutured as required.
Arterial blood pressure measurement
Mice were anaesthetised by isoflurane inhalation, and
their body temperature was kept constant with a
temperature controller (ATC2000, World Precision In-
strument). Blood pressure was measured through a pres-
sure catheter (outside diameter of 0.61 mm) inserted into
the carotid artery. Pressure was measured with a BP-100
intravascular blood pressure transducer and acquired at
100 Hz (iWorx 214); data were acquired and analysed
using LabScribe2 (iWorx Systems Inc, USA). Final traces
are the mean values of data after decimation to 10 Hz.
Vagus stimulation
Similar methods as described for CSN stimulation were
used, except the cervical vagus nerve was isolated and dual
nichrome wires were placed underneath it. Electrical
stimulation (2 min, 600 μA, 10 Hz, 0.1 ms) was applied, 5
min before and after an IP LPS (100 μg) injection. Animals
were allowed to recover as described in the “Acute CSN
stimulation”section. Additionally, 30 min prior to LPS in-
jection, atropine (1 mg/kg), hexamethonium (10 mg/kg)
and propranolol (2.5 mg/kg) or sham PBS was injected IP.
Falvey et al. Journal of Neuroinflammation (2020) 17:368 Page 3 of 12
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Fig. 1 Timelines for experimental protocols. Eight to 10-week-old C57BL/6 mice were used in various experimental protocols. Prior to CSN surgery, some
experiments had organ/nerve excisions (b), and in others, blocking drugs were administered (c,d). In all instances with recovery, mice recovered in a
heated cage (a–d,f,g). LPS was administered IP (200 μl) in all instances at 100 μg(a–d,f,exceptg) when a lethal dose of LPS was used (1 mg–200 μl).
When stimulation was required (a–g), it was administered at 600 μA + 10 Hz, 400 μA + 10 Hz or 200 μA + 5 Hz and 0.1 ms in all instances. Stimulation
occurred during unconsciousness (a–e) and consciousness (f,g). Experiments were typically conducted at least twice with an nnumber of 6–8
Falvey et al. Journal of Neuroinflammation (2020) 17:368 Page 4 of 12
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Paraventricular nucleus recording
Mice were anaesthetised with ketamine (130 mg/kg, Imal-
gene 1000) and xylazine (10 mg/kg) injection IP. The CSN
was isolated as described above, and physiological glue was
used to ensure electrodes remained in place during the
stereotaxic surgery. The mice were placed in a stereotaxic
frame (SR-6, Narishige, Japan), and a craniotomy to target
the hypothalamic PVN was performed. Stereotaxic coordi-
nates for the PVN were determined from the Paxinos and
Watson rat brain atlas (2013), and stainless-steel recording
electrodes (platinum/iridium wires) were implanted into
the area of the PVN—which was confirmed later by dye
placement. A baseline recording of PVN activity for 5 min
was recorded, and subsequently, continuous stimulation
(600 μA, 10 Hz, 0.1 ms) of the CSN was started, and PVN
activity was recorded for 5 min using a multichannel system
(Multi Channel Systems MCS GmbH, Germany). Mice
were sacrificed at the end of the experiment, the brain was
removed and immediately sectioned using a vibratome to
view dye placement in the sections.
Cytokines and corticosterone assays
Routinely, a TNF and corticosterone were measured by
ELISA (DY410 R&D Systems and ADI-900-097, Enzo
LifeSciences) as described by the manufacturers. The
Meso Scale Discovery kit (V-PLEX Plus Proinflamma-
tory Panel1 Mouse Kit) was used to assess a wide variety
of cytokines (Fig. 1).
Statistics
Data is pooled, and individual points represent one ani-
mal; results are expressed as means ± standard deviation
(SD). The unpaired ttest was used if results were shown
to have a normal distribution (Shapiro-Wilks test); if
not, a Mann-Whitney test was used. When the compari-
son between more than two experimental groups was
necessary, the one-way ANOVA with Tukey post hoc
test was used if the results were shown to have a normal
distribution (Shapiro-Wilks test); if not, a Kruskal-Wallis
with Dunn’s post hoc test was used. In all instances, *p<
0.05, **p< 0.01, ***p< 0.001 and ****p< 0.0001.
Results
CSN electrostimulation inhibits LPS-induced production of
pro-inflammatory cytokines
To confirm that we could activate the CSN by electrical
stimulation, we placed an electrode underneath the CSN
in anaesthetised mice. Applying electrical stimulation in-
duced a transient increase in breath rate (Fig. 2a: p= **,
one-way ANOVA, n= 7), therefore confirming that the
electrode was correctly positioned and that it can mimic
chemoreceptor activation. To test whether our chosen
stimulation packages (600 μA, 10 Hz, 0.1 ms and 200 μA,
5 Hz, 0.1 ms) induced baroreceptor CSN activity, in
addition to the confirmed chemoreceptor activity, we
measured the arterial blood pressure from the carotid ar-
tery before, during and after stimulation (Fig. 2b, n=3).A
transient decrease in arterial blood pressure was recorded
following 1 mA 30 Hz electrical stimulation of the CSN (p
= ***, unpaired ttest, n= 3) further confirming the loca-
tion of the CSN. Interestingly, when a lower electrical
stimulation was applied, 600 μA, 10 Hz or 200 μA, 5 Hz
carotid blood pressure was not affected by CSN stimula-
tion. This result suggests that we are solely inducing a
chemoreceptor response from the CB/CSN.
To further investigate the impact of CSN electrical
stimulation on LPS-induced TNF production, we injected
LPS into anaesthetised mice, applied or not electrical
stimulation and measured TNF serum levels 90 min later.
Compared to sham-stimulated mice, CSN electrical
stimulation significantly reduced the serum levels of TNF
(Fig. 1a for timeline; Fig. 3a: p= ****; unpaired ttest; n=
20–21), IL-1β(Fig. 3b: p= **, Mann-Whitney, n=16–21),
IL-6 (Fig. 3c; p= ****, Mann-Whitney, n=22–24) and IL-
12p70 (Fig. 3d; p= ****; Mann-Whitney, n=21–24). We
confirmed that these results were not due to current leak-
age by repeating these experiments when the CSN/elec-
trodes were surrounded by oil (Fig. 3e; p= **; unpaired t
test; n= 19) and upon unilateral vagus excision (Fig. 1b
for timeline; Fig. 3f; p= *; unpaired ttest, n=10–12). We
also found that the effect of CSN stimulation was medi-
ated by an afferent signal to the brain as the impact on
TNF is prevented by an efferent cut of the CSN (Fig. 3g: p
= **, unpaired ttest, n=7–9).
CSN electrostimulation attenuates inflammation
independently of the vagus nerve
Having shown that CSN electrical stimulation inhibited
LPS-induced TNF production, we investigated the under-
lying mechanisms by using pharmacological antagonists.
Since previous studies have shown that the inhibition of
LPS-induced TNF secretion can be attenuated by signalling
through acetylcholine receptor (AChR) and by β2adrener-
gic receptor (AR) [4], we tested the impact of antagonism
against these pathways examining CSN inhibition of LPS-
induced cytokine release. To this aim, atropine, hexame-
thonium and propranolol which are respectively muscar-
inic, nicotinic AChR and β1/β2 AR antagonists were used
(Fig. 1c for timeline). We first confirmed that these antago-
nists were effective at the doses used by showing that they
prevented the decrease of LPS-induced TNF release follow-
ing vagus nerve stimulation (Fig. 1d for timeline; Fig. 4a: p
> 0.05, unpaired ttest, n=10–17). We then stimulated the
CSN in the presence of atropine, hexamethonium and pro-
pranolol and found that the inhibition of LPS-induced
TNF production was abolished neither by atropine (Fig. 4b:
p= ***, unpaired ttest, n=12–16) nor by hexamethonium
(Fig. 4c: p= **, unpaired ttest, n=9–12) nor by
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Fig. 2 Electrostimulation of the CSN increases breath rate and decreases arterial blood pressure in mice. Eight to 10-week-old C57BL/6 mice were
obtained, and CSN isolation surgery was performed. aBreath rate was recorded for 5 min, and the average breath per minute was calculated at
baseline, during stimulation (200 μA, 5 Hz) and immediately after stimulation. Breath rate in mice before, during and after stimulation. Each
individual point represents an animal, and data is expressed as means ± SD. bThe mean carotid pressure recorded in anaesthetised mice (n=3)
before and during CSN electrostimulation with either 1 mA (left), 600 μA (middle) or 200 μA (right) current amplitude. Traces are averaged values
l
l
l
l
l
l
l
β
Fig. 3 CSN electrostimulation attenuates inflammation independently of the vagus nerve. a–gEight to 10-week-old C57BL/6 mice were
anaesthetised; CSN was isolated and either cut (g) of left intact (a–f). Electrical stimulation was applied at 600 μA, 10 Hz (a–e) or 200 μA, 5 Hz (f,g)
5 min before and after IP LPS injection (100 μg). Blood was collected 90 min after LPS injection for serum analysis by Meso Scale Discovery (a–d)
or ELISA (e–g). Impact of electrical activation of the CSN on aLPS-induced serum TNF levels, bIL-1β,cIL-6 and dIL-12p70. eImpact of electrical
activation of the CSN on LPS-induced serum TNF levels in the presence of oil. fImpact of unilateral left vagal removal on LPS-induced serum TNF
levels following left CSN electrostimulation. All individual points represent one animal, and data is expressed as means ± SD. gImpact of afferent
CSN stimulation on LPS-induced serum TNF levels
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propranolol (Fig. 4d: p= **, unpaired ttest, n=13–14).
LPS-induced TNF production was decreased by CSN
stimulation after the surgical removal of the spleen (Fig. 1b
for timeline; Fig. 4e: p=*,unpairedttest, n= 14). Overall,
these results indicate that the effect of CSN stimulation is
independent of the cholinergic anti-inflammatory pathway.
The inhibitory effect of CSN stimulation on LPS-induced
TNF production is dependent on the expression of GR by
myeloid immune cells
The HPA axis is a major neuroendocrine system that regu-
lates immune response via the production of glucocorti-
coids by the cortex of the adrenal glands. To explore
whether the inhibition of LPS-induced TNF production by
CSN electrical stimulation could be dependent on the HPA
axis, we applied electrical stimulation to the CSN and mea-
sured the serum levels of corticosterone. We found that
CSN stimulation significantly increased the production of
corticosterone (Fig. 1a for timeline; Fig. 5a: p= **, unpaired
ttest, n=21–24). To investigate whether the ability of
CSN electrical stimulation to inhibit LPS-induced TNF
secretion was mediated by corticosterone, we bilaterally
removed the adrenal gland in mice. While CSN electric
stimulation did inhibit LPS-induced TNF secretion in
sham-operated mice, adrenalectomy completely abolished
this phenomenon (Fig. 1b for timeline; Fig. 5b, left: p=
0.4438, unpaired ttest, n=19–20). We confirmed that
serum corticosterone levels were reduced by adrenalectomy
(Fig. 1b for timeline; Fig. 5b, right: p= 0.9097, unpaired t
test, n=7–8). Since adrenal gland resection can affect other
hormones than glucocorticoids, such as adrenaline, we ad-
ministered mifepristone as a GR antagonist to mice. Treat-
ment with the GR antagonist mifepristone also abolished
the ability of CSN electric stimulation to inhibit LPS-
Fig. 4 Attenuation of inflammation mediated via CSN stimulation does not utilise the vagal anti-inflammatory reflex. Eight to 10-week-old C57BL/
6 mice were obtained, and vagus nerve (a) or CSN (b–e) isolation surgery was performed, followed by electrical stimulation 200–600 μA, 5–10 Hz.
Thirty minutes prior to surgery, sham vehicle (PBS), hexamethonium (10 mg/kg) (a,c), atropine (1 mg/kg) (b) or propranolol (2.5 mg/kg) (a,d)
were administered IP. Blood was collected 90 min after an IP LPS injection (100 μg) for serum analysis by ELISA. eThe spleen was surgically
removed, and the CSN electrostimulation was applied or not. LPS-induced serum TNF release was evaluated by ELISA. a–eEach individual point
represents an animal, and data is expressed as means ± SD
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induced TNF production (Fig. 1c for timeline; Fig. 5c: p=
0.4366, Mann-Whitney, n=30–31). As myeloid cells, and
more specifically macrophages, are the main source of TNF
in LPS-injected mice, we investigated whether the inhib-
ition of LPS-induced TNF production by CSN stimulation
required the expression of GR by myeloid cells. To test this,
we used LysM-Cre:GR
fl/fl
mice in which myeloid cells are
selectively deficient in GR. While CSN electrical stimulation
did inhibit LPS-induced TNF production in GR
loxp/loxp
lit-
termates (Fig. 1afortimeline;Fig.5d: p= **, unpaired ttest,
n=13–14), it had no impact on LysM-Cre:GR
fl/fl
mice
(Fig. 5d: p= 0.0963, unpaired ttest, n=17–18) further
demonstrating that GR signalling in myeloid cells was re-
quired for the inhibitory effect of CSN electrical stimula-
tion. We further investigated the connection between the
CB and the HPA axis by assessing the neural connection
between the CSN and the PVN of the hypothalamus. We
found that CSN stimulation did increase the activity in the
PVN compared to baseline levels of activity (Fig. 1efor
timeline; Fig. 6b: p= **, Mann-Whitney, n= 7). Altogether
it is evident that CSN stimulation is activating the PVN
which in-turn triggers the HPA axis and ultimately causes
increased production of corticosterone, which inhibits LPS-
induced inflammation by a GR-dependent and myeloid im-
mune cell mechanism.
CSN stimulation in conscious animals attenuates LPS-
induced TNF production and increases survival to
endotoxaemic shock
It is well documented that anaesthesia causes an anti-
inflammatory effect [20]; therefore, it was necessary to
confirm the previous results in fully conscious, non-
TNF pg/ml
TNF pg/ml
Fig. 5 CSN electrostimulation attenuates inflammation via glucocorticoids signalling in myeloid cells. C57BL/6 mice (a–c) or LysM-Cre:GR
fl/fl
mice
(d) were obtained, aged 8–10 weeks, and CSN isolation and stimulation (600 μA, 10 Hz) were conducted. An LPS IP injection (100 μg) was
administered in all animals; 90 min later, blood was collected for serum analysis by assay. aCorticosterone was measured in sham and CSN
electrostimulated animals. bImpact of bilateral adrenal gland removal prior to CSN isolation and stimulation on LPS-induced serum TNF levels
and serum corticosterone levels. cImpact of mifepristone (80 mg/kg) or vehicle (10% DMSO) IP administration prior to CSN isolation and
stimulation on LPS-induced serum TNF levels. dLysM-Cre:GR
fl/fl
mice and their littermate controls, GR
loxp/loxp
, were obtained, and both received
electrostimulation of the CSN. LPS-induced serum TNF levels were measured by ELISA. All individual traces, or points, represent one animal. The
means are represented as ± SD
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anaesthetised animals. To this aim, we implanted elec-
trodes onto the CSN and allowed the mice to recover.
Mice were then injected with LPS, and electrical stimula-
tion was applied. As observed in anaesthetised animals,
CSN stimulation in conscious animals significantly de-
creased TNF concentration in the blood (Fig. 1f for time-
line; Fig. 7a: p= ***, Mann-Whitney, n=23–29).
Furthermore, to replicate our results obtained in anaesthe-
tised LysM-Cre:GR
fl/fl
mice, LysM-Cre:GR
fl/fl
mice and
their littermate controls, GR
loxp/loxp
,wereelectrostimu-
lated. Littermate controls had decreased TNF concentra-
tion following CSN electrostimulation (Fig. 1f for timeline;
Fig. 7b: p= ***, unpaired ttest, n=6–7), whereas CSN-
stimulated LysM-Cre:GR
fl/fl
did not show a decrease in
TNF concentration compared to sham-stimulated mice
(Fig. 7b: p=0.888,Mann-Whitney,n=13–14).
We next investigated whether the inhibition of LPS-
induced TNF production in conscious mice translates
into increased survival following an endotoxaemic shock.
We implanted electrodes underneath the CSN and
allowed the mice to recover for several days. We then
injected these mice with a lethal dose of LPS and applied
electrical stimulation twice a day for 3 days. Conscious
CSN stimulation of mice was protective against endotox-
aemic shock-induced death (Fig. 1g for timeline; Fig. 7c:
p= **, Gehan-Breslow-Wilcoxon test, n=13–16).
Overall, our results suggest that CSN stimulation may
prove to be a successful therapeutic for inflammatory
disorders.
Discussion
Here, we found that CSN electrical stimulation attenuates
the production of pro-inflammatory cytokines via the in-
creased production of corticosterone and a mechanism that
is dependent on GR signalling in myeloid cells. This
phenomenon was observed in both anaesthetised and non-
anaesthetised conscious mice. Furthermore, CSN electrical
stimulation translated into physiological benefit and pro-
tected mice from endotoxaemic shock-induced death, an
observation that may be of clinical interest. Most import-
antly, the inhibition of LPS-induced TNF production by
CSN electrical stimulation was not an artefact due to elec-
trical spread to the vagus nerve. Interestingly, we also dem-
onstrated that the stimulation is a purely afferent response.
It should be noted, however, that this afferent signal to
decrease inflammation could potentially be mediated via
the chemoreceptor or the baroreceptor sensors [8]. How-
ever, since the stimulation electrical parameters we used
to decrease inflammation are sufficient to initiate the
chemoreceptor response (Fig. 2a) but not the baroreflex
response (Fig. 2b), it suggests that CSN electrostimulation
is decreasing inflammation via a chemoreceptor reflex
Fig. 6 Increased PVN activity during CSN stimulation. Eight to 10-week-old C57BL/6 mice were obtained, and the CSN was isolated and
stimulated (600 μA, 10 Hz). Electrical activity recordings of the PVN of the hypothalamus were taken using a stereotaxic frame. Impact of CSN
electrostimulation on PVN activity was evaluated. aA representative trace of activity in the PVN during CSN stimulation. bThe average discharge
was acquired per minute for baseline recordings and during stimulation. All individual traces or points represent one animal. The means are
represented as ± SD. cAfter recording, the frontal plane of a representative brain was injected with dye to show the location of the recording
electrode and a zoomed in image of the PVN. Both images shown are representative of the whole
Falvey et al. Journal of Neuroinflammation (2020) 17:368 Page 9 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
rather than the baroreceptor reflex. In line with these re-
sults, Brognara et al. showed that baroreflex stimulation
did not impact serum, spleen and heart TNF levels follow-
ing IP LPS injection [18].
In a recent paper, Santos-Almeida et al. demonstrated
that CSN electrical stimulation in conscious rats attenu-
ates inflammation and inhibits LPS-induced TNF pro-
duction [17]. According to these authors, the inhibition
of LPS-induced TNF production is dependent on signal-
ling through both the AChR and the β1/β2 AR. They
concluded that the CB is the afferent source of the vagal
anti-inflammatory reflex as previously proposed [21]. In
striking contrast, we found here that the inhibition of
LPS-induced TNF production by CSN electrical stimula-
tion in mice is neither abolished by the nAChR antagon-
ist hexamethonium nor by the β1/β2 AR antagonist
propranolol. These latter results led us to conclude that
the mechanisms underlying the anti-inflammatory effect
of CSN electrical stimulation are different from those in-
volved in the vagal anti-inflammatory reflex. In line with
this conclusion, the inhibition of LPS-induced TNF pro-
duction by CSN electrical stimulation is not abolished
by surgical removal of the spleen. Moreover, we also ob-
served that the inhibition of LPS-induced cytokine secre-
tion affects not only TNF and IL-6 but also IL-1 and IL-
12p70 (Fig. 2a–d), which is not the case for vagus nerve
stimulation (VNS) (personal communication). In conclu-
sion, by contrast to VNS, the release of corticosteroids
by CSN electrostimulation mediates its effects in a rather
non-specific manner.
One possible explanation of the discrepancies between
our results and those reported by Santos-Almeida et al.
is that the physiological pathways that are mobilised by
CSN electrical stimulation are different in mice and rats
Fig. 7 Conscious CSN stimulation is protective against LPS-induced shock. Eight to 12-week-old wild-type and transgenic C57BL/6 mice
underwent surgery 4 days prior to stimulation (a–c). a,bStimulation (200 μA, 5 Hz, 2 × 2 min) was applied on freely moving mice, and LPS (5
mg/kg) was injected IP. All individual points represent one animal, and the means are expressed as ± SD. aImpact of CSN electrostimulation in
conscious animals on LPS-induced serum TNF levels. bLysM-Cre:GR
fl/fl
mice and littermate controls underwent the same procedure as in a.c
Wild-type C57BL/6 mice were implanted with an electrode on their CSN and stimulated (n= 16, 200 μA, 5 Hz, 5 min) or not (n=13–16) twice a
day for the next 3 days. A lethal dose of LPS (20 mg/kg) was administered to the mice (1 mg) IP. Animal survival was monitored
Falvey et al. Journal of Neuroinflammation (2020) 17:368 Page 10 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
[17]. While this seems unlikely because of the phylogen-
etic proximity of these two species, it is noteworthy that
fundamental differences in cytokine receptor profiles in
CB have been reported between mice and rats [22–24].
Another explanation may be related to the differences in
experimental procedures and more specifically to elec-
trical stimulation parameters and regiments. In this re-
spect, it is noteworthy that Santos-Almeida et al. have
used a stronger than necessary signal and a protracted
duration for activating the CSN [17]. We used amplitude
of stimulation between 200 and 600 μA whereas Santos-
Almeida et al. used 1-mA stimulation amplitude, which
could lead to effects through the vagus nerve in addition
to the CB-PVN. For example, this may have resulted in
the artefactual recruitment of neural fibres in the vagus
nerve and the subsequent triggering of the vagal anti-
inflammatory reflex. We believe that this is a reasonable
possibility as it was previously reported that a single
electrical pulse is sufficient to activate the vagus nerve
[25]. Furthermore, it is necessary to consider that their
electrodes used were made of naked wires wrapped
around the internal carotid artery, and this adds further
likelihood of activating the close in proximity cervical
vagus nerve. Regarding these points, we believe it is a
reasonable possibility to suggest that there has been
electrical spread to the vagus nerve instigating the vagal
anti-inflammatory reflex. Unilateral removal of the vagus
nerve might have confirmed that the attenuation of in-
flammation by rat CSN stimulation was independent of
the vagus nerve. This control experiment would ensure
that their effect of CSN stimulation did not occur due to
electrical spread to the close in proximity vagus nerve,
confirming or not the possibility of species differences.
Considering that CSN stimulation in mice is independ-
ent of the vagal anti-inflammatory reflex, we investigated
other potential mechanisms for the effect of CSN stimula-
tion. One possible mechanism for the attenuation of pro-
inflammatory cytokines in mice by CSN electrostimula-
tion is the HPA axis. There is evidence of a connection be-
tween the CB and the PVN of the hypothalamus—perhaps
electrical activation of the CSN activates the PVN and in-
stigates the HPA axis [26–28]. We first demonstrated that
bilateral removal of the adrenal glands prevented the effect
of CSN stimulation. Furthermore, we have shown that
CSN electrostimulation is increasing serum corticosterone
concentration. This result suggests that corticosterone is
the ultimate mediator of the effect of CSN stimulation.
This was confirmed by showing that the inhibition of
LPS-induced TNF production by CSN electrical stimula-
tion is abolished by treatment with the GR antagonist
mifepristone or by inactivation of the GR signalling in
myeloid cells. Overall, these results confirm that CSN
stimulation acts via corticosterone activating GR on mye-
loid cells. It is known that there is a connection between
the CB and the PVN [26–28]. The evidence, however, is
effect-based or relying on c-fos studies. We confirmed the
link between the CB and the PVN using an electrophysio-
logical approach.
A method for conscious stimulation of the mouse CSN
was developed, and it was demonstrated that conscious
CSN stimulation can attenuate LPS-induced TNF produc-
tion (Fig. 7a). Additionally, it was investigated if CSN stimu-
lation in LysM-Cre:GR
fl/fl
produced an effect. It was found
that there was no difference between sham-stimulated and
CSN-stimulated LysM-Cre:GR
fl/fl
mice; this result indicates
that the pathway for the effect of conscious CSN stimula-
tion is concordant with that for anaesthetised stimulation.
We additionally investigated if this mitigation of in-
flammation by CSN electrostimulation could translate
into physiological benefit—using a model of endotoxae-
mic shock by lethal LPS injection. To this aim, a method
for conscious stimulation of the mouse CSN was devel-
oped, and it was demonstrated that conscious CSN
stimulation can attenuate LPS-induced TNF production
through the GR signalling in myeloid cells (Fig. 7b). We
also found that CSN stimulation significantly increased
the chance of survival in mice (Fig. 7c). This enhanced
survival is likely to result from decreased production of
pro-inflammatory cytokines such as TNF, IL-6, IL-12
and IL-1β, which play a critical role at recruiting and ac-
tivating immune cells. For example, IL-12 activates nat-
ural killer (NK) cells while both IL-1βand IL-6 induce
pyrogenic activity. TNF promotes the loosening of tight
junctions between endothelial cells resulting in fluid loss
and multiple organ failure [29]. Endotoxaemic shock is
representative of immune system dysregulation, and as
CSN stimulation can endow protection onto mice
against it, it suggests that CSN stimulation may prove ef-
fective against additional IMIDs.
Conclusion
CSN stimulation in mice activates the HPA axis enhan-
cing the production of corticosterone, which in turn acti-
vates GR on myeloid immune cells ultimately mediating a
decrease in inflammation, which aids survival in mice
against endotoxaemic shock by lethal LPS injection. It
may represent an interesting option in the anti-
inflammatory bioelectronic medicine field as it is the first
instance of an anti-inflammatory pathway using the HPA
axis, which would be particularly interesting for patients
requiring long-term administration of glucocorticoids.
Abbreviations
ACh: Acetylcholine; AChR: Acetylcholine receptor;
ACTH: Adrenocorticotrophic hormone; AR: Adrenergic receptor; CB: Carotid
body; CRH: Cortisol-releasing hormone; CSN: Carotid sinus nerve;
GR: Glucocorticoid receptor; HPA: Hypothalamic-pituitary adrenal;
IBD: Inflammatory bowel disease; IL: Interleukin; IMID: Immune-mediated
inflammatory disorders; IP: Intraperitoneal; LPS: Lipopolysaccharide;
nAChR: Nicotinic acetylcholine receptor; NK: Natural killer;
Falvey et al. Journal of Neuroinflammation (2020) 17:368 Page 11 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
PVN: Paraventricular nucleus; RA: Rheumatoid arthritis; SD: Standard
deviation; SLE: Systemic lupus erythematosus; TNF: Tumour necrosis factor
Acknowledgements
Aidan Falvey was a recipient of the LABEX SignaLife PhD fellowship 2015.
Authors’contributions
AF—daily project management, all CSN surgeries + assays, experiment
design. FD—aided blood pressure recording experiment (Fig. 2b). TS—aided
experiment design, aided vagus experiments. SHA—aided paraventricular
recording (Fig. 6). SVC—aided experiment design, taught AF surgery on rat
CSN. NG—aided project management. PB—overall project manager,
corresponding author, experiment design. The author(s) read and approved
the final manuscript.
Funding
This work was supported by the LABEX SIGNALIFE (#ANR-11-LABX-0028-01)
and the FHU OncoAge.
Availability of data and materials
Not applicable.
Ethics approval and consent to participate
All studies conducted within this manuscript have obtained ethical approval
and comply with the regulations set forth by both local (Alpes-Maritimes)
and national (France) commissions for the treatment of experimental
animals.
Consent for publication
All authors of this manuscript consent to the publication of these results.
Competing interests
The authors and this manuscript have no conflict of interest nor competing
interests.
Author details
1
Université Côte d’Azur, CNRS, Institut de Pharmacologie Moléculaire et
Cellulaire, Valbonne, France.
2
E-Phy-Science, Valbonne, France.
3
CEDOC,
NOVA Medical School, Faculdade de Ciências Médicas, Universidade NOVA
de Lisboa, Lisboa, Portugal.
Received: 26 June 2020 Accepted: 29 October 2020
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