Stofkova and Murakami Bioelectronic Medicine
https://doi.org/10.1186/s42234-019-0030-2(2019) 5:14

Bioelectronic Medicine

REVIEW

Open Access

Neural activity regulates autoimmune
diseases through the gateway reflex
Andrea Stofkova1,2 and Masaaki Murakami2*

Abstract
The brain, spinal cord and retina are protected from blood-borne compounds by the blood-brain barrier (BBB),
blood-spinal cord barrier (BSCB) and blood-retina barrier (BRB) respectively, which create a physical interface thattightly controls molecular and cellular transport. The mechanical and functional integrity of these unique structures
between blood vessels and nervous tissues is critical for maintaining organ homeostasis. To preserve the stability ofthese barriers, interplay between constituent barrier cells, such as vascular endothelial cells, pericytes, glial cells andneurons, is required. When any of these cells are defective, the barrier can fail, allowing blood-borne compounds toencroach neural tissues and cause neuropathologies. Autoimmune diseases of the central nervous system (CNS)
and retina are characterized by barrier disruption and the infiltration of activated immune cells. Here we review ourrecent findings on the role of neural activity in the regulation of these barriers at the vascular endothelial cell levelin the promotion of or protection against the development of autoimmune diseases. We suggest nervous system
reflexes, which we named gateway reflexes, are fundamentally involved in these diseases. Although their reflex arcs
are not completely understood, we identified the activation of specific sensory neurons or receptor cells to whichbarrier endothelial cells respond as effectors that regulate gateways for immune cells to enter the nervous tissue.
We explain this novel mechanism and describe its role in neuroinflammatory conditions, including models ofmultiple sclerosis and posterior autoimmune uveitis.

Introduction
The nervous system, with its central and peripheral divisions, is the major integration and command center forcontrolling all other body systems and organs. It processes
and transmits information from the internal and external
environments to produce an instant response in various
effector organs. The neural activity is organized into reflex
arcs, which are the basic functional units of the nervous
system and composed of (1) sensory receptors, whichmonitor the environment, (2) afferent nerve fibers, whichtransmit the monitoring signals to (3) integrating centers,such as central nervous system (CNS) nuclei, and (4) activated efferent nerve fibers, which regulate organ homeostasis (Levine 2007).
All homeostatic reactions in the body depend on nervous
reflexes and/or humoral factors including hormones, nutrients, metabolites, and ions, which are at the same time regulated by the neural system. However, unlike humoral* Correspondence: murakami@igm.hokudai.ac.jp
2
Division of Molecular Psychoimmunology, Institute for Genetic Medicine,
Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan
Full list of author information is available at the end of the article

factors, electrical impulses running through neural reflex
arcs enable instantaneous responses to inner and outerchanges. Thus, nervous reflexes are key to survival andhomeostasis (Kotas and Medzhitov 2015).
The presence of various neuronal receptors (e.g. for neurotransmitters, growth factors, etc.) on immunocompetentcells implies that neural reflexes also directly control
homeostasis of the immune system. In this context, theinflammatory reflex by which the vagus nerve triggers an
immunosuppressive signal to reduce an inflammatory response has been described (Andersson and Tracey 2012;
Tracey 2016; Pavlov and Tracey 2017). In the case of autoimmune diseases that result from an abnormal immune
response against self-tissue, immune homeostasis fails, andnervous reflexes including the inflammatory reflex regulate
the disease pathogenesis. A newly discovered mechanism
for this regulation is the gateway reflex, which occurs at
blood-nervous tissue barriers such as the blood-brain
barrier (BBB), blood-spinal cord barrier (BSCB) andblood-retina barrier (BRB). In gateway reflexes, a common
effector non-immune cell type is the barrier endothelial cellin the brain, spinal cord or retina and is activated by© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Stofkova and Murakami Bioelectronic Medicine(2019) 5:14

noradrenergic efferent pathways (Arima et al. 2012, 2015,2017; Stofkova et al. 2019).
In this review, we first present an overview of fundamental communication networks between neural pathways
and barrier endothelial cells with regard to the development and maintenance of the blood-nervous tissue barrier.
Second, we discuss a group of gateway reflexes that takeadvantage of the neuron-endothelial cell communications
to open or close immune cell gateways across the BBB,BSCB and BRB at specific vessels of the CNS and retina.

Neural-endothelial crosstalk
The first recognition that the phenotype of BBB endothelial cells is not intrinsic to these cells but acquired
from the neural microenvironment comes from a study
performed by Stewart and Wiley (1981). The transplantation of nonvascular tissue from embryonic quail brain
into the gut of chick embryos caused the newly formed
vascular endothelial cells originating from the host’s gut
to develop BBB characteristics. In contrast, the transplantation of embryonic quail mesodermal tissue to embryonic chick brain caused brain vessels vascularizing
the grafted mesoderm to not acquire structural or functional barrier features. Follow-up studies focused on
identifying the cells in the neural microenvironment that
contribute to the development and maintenance of thebarrier phenotype. Due to the anatomical proximity ofastrocytes to CNS endothelial cells, the earliest of thesestudies were performed on astrocytes during the induction of BBB properties by endothelial cells. Grafted
astrocytes as well as Müller cells to the anterior eyechamber were able to induce barrier characteristics to
non-neural vascular endothelial cells (Janzer and Raff
1987; Tout et al. 1993). Consistent with these observations, in vitro investigations showed the importance ofastrocytic endfeet for the formation of the BBB (Hayashi
et al. 1997). That study used a heterologous co-culture
system, in which rat fetal brain astrocytes were cultured
on one surface of a porous membrane and humanumbilical vein endothelial cells on the opposite surface,enabling the contact of endothelial cells with only astrocytic endfeet projections. This unique culture system revealed the development of barrier activity against inulin
and an upregulation of BBB-specific markers, includingtransferrin receptor, P-glycoprotein, brain-type glucose
transporter GLUT-1 and gamma-glutamyltranspeptidase(GGTP) on umbilical vein endothelial cells. Other studies have also proven the contribution of astrocytes to thebarrier phenotype and the importance of astrocytederived factors on the functional characteristics of BBB
endothelial cells (Haseloff et al. 2005; Abbott et al. 2006;
Broux et al. 2015).
Other cell types also contribute to the characteristics
of barrier endothelial cells, including neurons, pericytes,

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vascular smooth muscle cells (VSMCs), microglia and oligodendrocytes (Broux et al. 2015; Keaney and Campbell
2015; O’Brown et al. 2018; Miyamoto et al. 2014). In
addition, one study suggested that neurons have predominant effects on the acquisition of barrier phenotypes by
vascular endothelial cells (Tontsch and Bauer 1991). Here,the co-culture of cerebral capillary endothelial cells withastrocytes, glioma cells and cortical neurons showed that,
although both neurons and glial cells induced the activityof GGTP, an enzymatic marker of the BBB, in cerebral
capillary endothelial cells, the GGTP activity was higher inendothelial cell co-cultures with neurons or neuronalplasma membranes. Moreover, in early development of
the CNS, neurons play a critical role in the BBB formation
by cerebral endothelial cells.
Finally, it was reported that the invasion of blood vessels
in the mouse developing neural tube occurs at embryonicday (E) 10, which is consistent with neurogenesis starting
between E11-E17. At this stage, the newly formed cerebral
microvasculature is surrounded by neuroblasts and undifferentiated neurons, and gliogenesis occurs after functional
BBB formation, excluding the role of glia cells on BBB
development in vivo (Saunders et al. 1999; De Bock et al.
2014; O’Brown et al. 2018). Together, the above studies
suggest that neural signals are essential to establishing thebarrier function of vascular endothelial cells in the CNS.
Nevertheless, little is known about the cellular and molecular mechanisms for the communication between neurons and endothelial cells that establishes the BBB
properties of the CNS endothelium. Given the arrangement of neurons, glial cells and the CNS vasculature,
which are collectively referred to as the neurovascular unit
(NVU), neurons are not in direct contact with endothelial
cells (Iadecola 2017; Kisler et al. 2017). At the level of
CNS-penetrating arteries or arterioles, endothelial cells arecovered by the extracellular basement membrane andringed by VSMCs, which are surrounded by astrocytes. At
the capillary level, endothelial cells share a common
basement membrane with pericytes that are covered byastrocyte endfeet. In this assembly, neurons usually form
physical contacts with VSMCs, pericytes and astrocytes,
but not directly with endothelial cells (Iadecola 2017; Kisler
et al. 2017). However, neurons can communicate withendothelial cells by contact-independent strategies througha wide range of regulatory molecules including growth
factors, neurotransmitters, neuropeptides, semaphorins
and immunomodulating factors (Chavarría and Cárdenas2013). Much work has been done on the role of specificneurotransmitters and neuropeptides in neurovascular
coupling, a process by which local neuronal activity
controls blood flow in the CNS. There are several excellent
reviews on this subject (e.g. Drake and Iadecola 2007;
McConnell et al. 2017; Iadecola 2017; Kisler et al. 2017).
Here we focus on neuronal mechanisms that regulate theStofkova and Murakami Bioelectronic Medicine(2019) 5:14

barrier phenotype in vascular endothelial cells in the CNS
and retina.

Role of noradrenergic signaling in modulating
barrier endothelium
The catecholamines norepinephrine (NE) and epinephrine (EPI) are neurotransmitters widely distributed
throughout the CNS and play a role in CNS development, motor control, emotion, motivation, learning,memory formation and processing, as well as neuroendocrine and autonomic regulations (Kobayashi 2001).
Their receptors, alpha(α)- and beta(β)-adrenoceptors(ARs), are present on vascular endothelial cells in thebrain, spinal cord and retina (Hertz et al. 2010; Böhmer
et al. 2014; Haselton et al. 1998).
The central noradrenergic system significantly contributes to the development of the cerebral vasculature,including angiogenesis and the contractile function ofblood vessels (Buchholz et al. 2007). NE in the CNS
regulates BBB permeability of the vascular endothelium.
NE injection to the right lateral cerebral ventricle has
been shown to increase BBB permeability for sodium
fluorescein dose-dependently, most likely due to theNE-induced pinocytotic activity of endothelial cells via
α-ARs (Sarmento et al. 1991). Similar observations havebeen reported when the locus coeruleus, a major sourceof NE in the brain, was electrically stimulated. Specifically, BBB permeability was increased in a stimulation
frequency-dependent manner, and this effect wascompletely blocked by the α-AR antagonist phenoxybenzamine, but not by the β-AR antagonist pindolol(Sarmento et al. 1994). In addition, both NE- and EPIinduced leakage of the BBB has been confirmed using an
in vitro BBB system composed of brain microvessel
endothelial cells (Borges et al. 1994). In that study, theincreased monolayer permeability was shown to be mediated by α-ARs, while the activation of β-ARs wasobserved to decrease monolayer permeability. Thus,
these findings demonstrated opposite effects of ARs on
BBB functions: β-adrenergic innervation appears to
support BBB integrity, whereas α-adrenergic innervation
is associated with increased BBB permeability.
In the context of multiple sclerosis (MS), disturbances in
the noradrenergic system in the CNS have been describedby several studies. However, controversies concerning CNS
NE levels in patients with MS still exist. For example,
Barkhatova et al. (1998) reported increased NE levels in the
cerebrospinal fluid (CSF) of both relapsing-remitting andprogressive MS patients compared to controls. On theother hand, Markianos et al. (2009) observed a negative
correlation of the CSF level of methoxyhydroxyphenylglycol (MHPG), the major metabolite of NE, with the durationof the illness as well as with the number of relapses, possibly indicating diminishing central noradrenergic system

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activity with relapses. Moreover, autopsied brains from MS
patients showed a reduction in NE levels and astrocyte activation, along with a hypertrophy of tyrosine hydroxylase
positive neuronal cell bodies in the locus coeruleus compared to controls (Polak et al. 2011).
Experimental autoimmune encephalomyelitis (EAE), an
animal model of MS, is an invaluable tool for studying thepathogenesis of the BBB breakdown and the infiltration of
immune cells into the CNS. Many different models of EAE
have been developed based on the animal species, encephalitogenic antigen and technique used to induce the disease.
One of the most popular murine models is EAE induced
by a myelin-derived antigen, myelin oligodendrocyte glycoprotein 35–55 (MOG35–55), which is given emulsified incomplete Freund’s adjuvant (CFA) as a subcutaneous injection. The adoptive transfer of MOG-specific CD4+ T cells(pathogenic CD4+ T cells) from donor mice that were actively immunized by MOG to naïve recipient mice can also
induce EAE. This adoptive transfer EAE model allows
study of the autoimmune CNS inflammation induced specifically by pathogenic CD4+ T cell responses (Glatigny
and Bettelli 2018; Rangachari and Kuchroo 2013; Tanaka
et al. 2017a). Indeed, we showed that the MOG-tetramer+
cells in pathogenic CD4+ T cells, which are in vitro restimulated splenic CD4+ T cells isolated from active EAE mice(direct immunization of a MOG peptide in wild type mice),
are about 20% just before the transfer to wild type mice
(Arima et al. 2017). On the other hand, pathogenic CD4+
T cells prepared from MOG-specific TCR transgenic mice
(2D2 mice) are not MOG-tetramer+ cells (Sabatino et al.
2011). Therefore, we hypothesized that the majority of ourpathogenic CD4+ T cells that survived even after in vitro
restimulation are MOG-specific regardless of the MOGtetramer staining.
EAE models support clinical findings of altered NE
and EPI levels in patients with MS. Reduced cortical andspinal cord NE levels as well as neuronal damage andastrocyte activation in the locus coeruleus have been reported in EAE mice (Polak et al. 2011). In the lumbar or
lumbosacral spinal cord, the decreased NE levels in EAE
have been confirmed by other groups as well (Krenger
et al. 1986, 1989; Musgrave et al. 2011). On the other
hand, in the craniothoracal region of the spinal cord, NE
levels decreased only during attacks of chronicallyrelapsing allergic encephalomyelitis and returned tonormal in remission (Krenger et al. 1986). Regarding thebrainstem of mouse EAE, NE levels increased (Musgrave
et al. 2011), but in rat EAE they decreased (Krenger
et al. 1989). Despite this discrepancy among studies,
which might be due to differences in species or experimental settings, these results point to an altered central
noradrenergic system in MS and related animal models.
The importance of these pathologic alterations of thenoradrenergic system for EAE development has been

Stofkova and Murakami Bioelectronic Medicine(2019) 5:14

demonstrated either by increasing the synthesis of NE in
the CNS (Simonini et al. 2010; Li et al. 2018) or by treatment with AR antagonists (Arima et al. 2012; Brosnan
et al. 1985; Goldmuntz et al. 1986; Dimitrijević et al.
2007). Interestingly, modulating the noradrenergic system can attenuate EAE development. These findings are
particularly noteworthy, since a prolonged exposure ofcells to NE downregulates and desensitizes ARs, particularly in vitro (Stofkova et al. 2019; Akinaga et al. 2013;
Heck and Bylund 1997; Leeb-Lundberg et al. 1987).

Gateway reflexes: unique neural circuits that
regulate initiation and manifestation of
autoimmune diseases via alterations in CNS
endothelial cells
Gravity gateway reflex

The posture of the body in the gravitational environment of Earth requires steady muscular tonus to provide
accurate body configuration and verticality. Mechanically, this process involves the constant neural activationof anti-gravity muscles, which is controlled by a typicalself-regulatory feedback mechanism. For example, coping with gravity to maintain sitting or standing uprightmostly does not need any voluntary attention. The antigravity muscles basically act through a monosynaptic
stretch reflex consisting of an afferent proprioceptive
sensory nerve fiber from the muscle spindle, which has
its body in the dorsal root ganglia (DRG) of the spinal
cord and makes synapses in the ventral spinal cord withalpha motor neurons that innervate the same muscle(Rossi-Durand 2006).
Since the stretch reflexes of the anti-gravity muscles
are constantly active in everyday activities depending
upon posture, it is not surprising that this neural activity
has an impact on regional spinal cord blood flow and
NVU dynamics (Marcus et al. 1977). In healthy subjects,
such neural activity does not breach the BSCB. However,in the course of CNS autoimmune inflammation in thepresence of pathogenic CD4+ T cells, such as EAE,
neural impulses from the muscle spindle sensory
neurons to the spinal cord can dramatically alter theproperties of BSCB endothelial cells (Arima et al. 2012).
Specifically, we observed that this neural activity, particularly noradrenergic activity, is able to induce a proinflammatory phenotype of endothelial cells around thespinal cord via NF-κB activity. Thus, endothelial cells
upregulate NF-κB targets such as chemokines and IL-6
to accumulate pathogenic CD4+ T cells in a mannerdependent on the antigen specificity. Pathogenic CD4+
T cells express excessive amounts of cytokines including
IL-17, IFNγ, IL-6, and TNFα to simultaneously activate
two transcription factors, NF-κB and STAT3, in theregional endothelial cells, resulting in more chemokine
and IL-6 expression, a phenomenon known as the

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inflammation amplifier (originally the IL-6 amplifier)(Ogura et al. 2008; Atsumi et al. 2014; Nakagawa et al.
2015; Murakami et al. 2019). Activation of the inflammation amplifier at regional blood vessel sites increases
the BSCB permeability, allowing for the accumulation ofvarious immune cells in the 5th lumbar region (L5) of
the CNS. Interestingly, in this model system, systemic
inflammation is minimal and thereby has no impact on
the systemic endothelial permeability during the initial
phase of the disease development (Arima et al. 2012).
Because the signals transmitted to endothelial cells by
this neural pathway depend on the stretch reflex arc ofanti-gravity muscles and create gateways for immune
cells to cross the BSCB, we named this mechanism thegravity gateway reflex (Pavlov et al. 2018; Tanaka et al.
2017b; Kamimura et al. 2018a, b)(Fig. 1).
The stretch reflex plays a physiological role to counterbalance gravity under a homeostatic principle. Because the gravity gateway reflex is one of the responsesof the stretch reflex against gravity, the gravity gatewayreflex should exist under homeostatic conditions on
Earth. In other words, the gravity-triggered reflex arc
should have physiological roles for CNS homeostasis.
We hypothesized that the gravity-mediated L5 gateway
establishes an immune surveillance mechanism forpathogen infection and tumor development in the CNS
to keep a certain level of immune cells in the CNS.
To extend our understanding of the nature of the neuralstimuli underlying changes in barrier endothelial cells in
response to sensory nerve activations, we performed aseries of experiments in mice with pathogenic CD4+ T
cell transfer: 1) tail suspension experiments, in which thehind legs (soleus muscle) are freed from gravity stimulation (Morey-Holton and Globus 2002) and electrostimulated to test the relative contribution of peripheral
neurons to the gravity gateway reflex, and 2) the inhibitionof EPI signaling using α1-AR or β1-AR antagonists to investigate the role of sympathetic neurons in the gravity
gateway reflex.
The tail suspension model employed in the first series,
also known as the hindlimb-unloading model, is a welldocumented model of microgravity that mimics neuromuscular alterations during spaceflights. Since the soles of
the animal hind limbs are not in the contact with theground, there is no stimulus of the sensory fibers in antigravity hind limb muscles, such as the soleus, to activate
the postural reflexes required for the upright stance (DeDoncker et al. 2005; Kawano et al. 2007; Ohira et al.
2002). We found that this model with pathogenic CD4+ T
cell transfer caused reduced NF-κB and STAT3 activation
along with suppressed CCL20 expression and immune cell
infiltration in the dorsal blood vessels of the fifth lumbar
cord (L5). Interestingly, since these suspended EAE mice
were in a handstand position, CCL20 expression andStofkova and Murakami Bioelectronic Medicine(2019) 5:14

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Fig. 1 Gravity gateway reflex. In free-moving mouse (upper panel) with adoptive-transfer EAE, pathogenic CD4+ T cells infiltrate the spinal cordparenchyma at the fifth lumbar (L5) level. This process is triggered by soleus muscle contraction (1) that counteracts gravity to maintain stability
and body posture. Stimulated sensory nerves arising from the soleus muscle (2) and entering the L5 spinal cord through dorsal root stimulate
sympathetic nerves (3) at the L5 spinal cord level via an unidentified neural circuit (?). This leads to chemokine expression including CCL20 inregional dorsal vessels of the spinal cord (4), establishing an immune cell gateway for migration of pathogenic CD4+ T cells across the BSCB. In
tail-suspended mouse (lower panel) with adoptive-transfer EAE, soleus muscle relief from gravitational force (1’) prevents regional activation of
sensory- (2’) and sympathetic-nerves (3’), and infiltration of pathogenic CD4+ T cells in the L5 spinal cord (4’). Abbreviations: EAE, experimental
autoimmune encephalomyelitis; DRG, dorsal root ganglion; SG, sympathetic ganglion; NE, norepinephrine; BSCB, blood-spinal cord barrier

pathogenic CD4+ T cell accumulation increased in thecervical spinal cord blood vessels. Consistent with thesefindings, c-fos expression was downregulated in L5 DRG,
but upregulated in cervical spinal cord DRG.
It is possible that the hindlimb-unloading model itself is a stressor. However, one week of tail suspension did not cause any difference in the absoluteweight of the adrenals of suspended and control animals (Ohira et al. 2002). Thus, the stress induced bythis model is minimal. It is also possible that the animals can perceive some weight of the limbs, but thepostural reflex activity is at least partially prevented
by unloading the hind limbs. Importantly, electrostimulation, which mimics gravity responses via neuralpathways of the muscles, increased chemokine expression in the dorsal vessels of the L5, which strongly
suggests that a specific neural pathway triggered bygravity responses induces chemokine expression in
specific vessel sites of the L5 cord.
In the second series, we reasoned that somatic sensory
neurons innervating skeletal muscles interact with
peripheral sympathetic neurons that innervate specificdorsal vessels of the spinal cord, where immune cells
including pathogenic CD4+ T cells accumulate. c-fos

expression in the sympathetic ganglia at the L5 spinal
cord was significantly increased and correlated with
CCL20 expression at the L5 compared to the L1 spinal
cord level, where pathogenic CD4+ T cells do not preferentially enter the spinal cord parenchyma. We foundthat both α1-AR or β1-AR antagonists could suppress
EAE development as well as the CCL20 expression, NFκB activation, and immune cell infiltration around theL5 dorsal vessels (Arima et al. 2012).
Based on our observations, we concluded that thegravity gateway reflex is a somato-sympathetic reflex induced by proprioceptive stimuli applied to anti-gravity
muscles. Though our data indicate that proprioceptive
sensory nerve fibers from muscle spindles represent theafferent pathway and sympathetic nerve fibers represent
the efferent pathway of the gravity gateway reflex, thelocation of the reflex center or anatomical connections
between the DRG and sympathetic ganglia remain to beelucidated.
Electric gateway reflex

In view of the apparent gateway reflex effect with pathogenic CD4+ T cell infiltration at the L5 dorsal vessels after
electrostimulation described above (Arima et al. 2012), weStofkova and Murakami Bioelectronic Medicine(2019) 5:14

investigated the mechanism mediating this process. An obvious hypothesis was that the stimulation of sensory neurons, which carry information from different muscles via
the dorsal root of the spinal cord, would cause the formation of gateways for pathogenic CD4+ T cells through theupregulation of chemokines in the dorsal vessels of different spinal cord levels via a similar NE-NF-κB axis. To test
this hypothesis, we applied electrostimulation to the quadriceps and epitrochlearis/triceps brachii in tail suspended
mice. Interestingly, we observed an increased expression ofchemokines including CCL20 in the dorsal spinal vessels at
the level of entry of the respective sensory neurons. Indeed,
the stimulation of the quadriceps resulted in CCL20 expression at the 3rd lumbar (L3) dorsal spinal vessels and thestimulation of the epitrochlearis/triceps brachii triggered
CCL20 expression between the 5th cervical (C5) and 5ththoracic (T5) dorsal spinal vessels. Moreover, we also foundincreased c-fos expression in the L3 DRG and the DRG between C5 and T5, suggesting activation of sensory afferent
neurons in response to the electrostimulation, since thebodies of these neurons are located in the DRGs (Arimaet al. 2012). Together, these findings indicate that muscle
electrostimulation can activate a pro-inflammatory response (“electric gateway reflex”, Fig. 2) in the dorsal vesselsof the spinal cord, suggesting a possible therapeutic application of electrical muscle stimulation on pathological conditions such as MS. We might have a new delivery method
for immune cells to the CNS by the electric gateway reflex,
suggesting that the electric gateway reflex could be a formof bioelectronic medicine akin to the inflammatory reflex.

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Pain gateway reflex

Chronic neuropathic pain is one of the most common debilitating symptoms of MS. Many patients with MS usually
suffer from pain sensation in multiple areas of the body.
The most frequent is central neuropathic (“dysesthetic”)
extremity pain, back pain and trigeminal neuralgia (O’Connor et al. 2008). Moreover, MS patients are at a higher riskof developing complex regional pain syndrome (CRPS)
than the general population (Schwartzman et al. 2008). In
CRPS, even without a definable nerve injury, formerly
known as reflex sympathetic dystrophy syndrome (StantonHicks et al. 1995), sympathetically mediated pain often occurs and can be relieved by α-AR antagonists or a sympathetic ganglion block (Campbell and Meyer 2006). Studies
using primate models of neuropathic pain induced byspinal nerve ligation indicate that the mechanisms of thesympathetically mediated pain involve an adrenergic
sensitization of nociceptors. Specifically, it has been shownthat sympathetic nerve fibers sprout into superficial areasof the skin, where they come into close contact withsensory nociceptive terminals that express α1-ARs. In thisway, NE released from sympathetic terminals activatesnociceptive terminals to produce a nociceptor response(Campbell and Meyer 2006).
Given our findings that the function of the gravity
gateway reflex is related to the activation of peripheral
sympathetic nerves, we hypothesized that sympathetically mediated pain sensation in mice plays a role in thedevelopment of transfer EAE. Indeed, our study suggests
a bidirectional relationship between pain and activity ofFig. 2 Electronic gateway reflex. Artificial neural activations by weak electric stimulations can induce the gateway reflex at individual blood vessels in
the CNS. Electric stimulations to the triceps (1) induce chemokine upregulation at the dorsal vessels of the fifth cervical (C5) to fifth thoracic (T5) spinal
cord (2) via the inflammation amplifier, which is mediated by NE secretion. Likewise, electric stimulations to the quadriceps (1) trigger chemokine
upregulation at the L3 dorsal vessels (2), whereas the L5 gateway is formed by electric stimulation to the soleus muscles. Abbreviations: DRG, dorsal
root ganglion; SG, sympathetic ganglion; NE, norepinephrineStofkova and Murakami Bioelectronic Medicine(2019) 5:14

the sympathetic nervous system (Arima et al. 2015). Not
only can sympathetic nerve impulses trigger pain sensation, but sympathetic responses can be driven by pain
(Campbell and Meyer 2006; Burton et al. 2016). Thisconnection is particularly important in the situation of
disease remission, where pain-mediated sympathetic responses may reactivate quiescent neuroinflammation.
We found that neuropathic pain in EAE-recovered micetriggered relapse via sensory nociceptive-sympathetic
coupling in a similar manner as the gravity gateway reflex(Arima et al. 2015). In that study, we employed a chronic
constriction injury (CCI) model of neuropathic pain,
which was induced by a loose ligation of the infraorbital
sensory nerve from the maxillary division of the trigeminal
nerve. The trigeminal neuropathic pain was proposed torequire activation of the anterior cingulate cortex (ACC)
for the development of pain-associated behavior and thalamic sensory neuron activity (Moon et al. 2017). The
ACC is a key cortical structure important not only foracute pain but also for neuropathic pain processing andperception (Vogt 2005; Moon et al. 2017). A positive correlation between the activity of the ACC and pain-induced
sympathetic vasoconstrictor reflexes as well as sympathetic responses that occur during pain anticipation havebeen reported in humans, suggesting functional links between the ACC, central sympathetic pathways and pain
experience (Seifert et al. 2013). This involvement of the
ACC in the regulation of autonomic processes can be attributed to its anatomical connections with brainstem
structures (Critchley et al. 2003; Luu and Posner 2003).
CCI and capsicin stimulation both activate the ACC in
EAE mice with remission (Arima et al. 2015). This
activation is followed by increased neuronal activity insympathetic ganglia at the whole spinal cord level and development of EAE relapse due to the presence of major
histocompatibility complex class II+ (MHC II+) CD11b +cells in the L5 cord. In EAE-recovered animals, we haveobserved an increased number of MHC class II + CD11b +cells with MOG-antigen presentation ability and of peripheral origin around the L5 spinal cord (Arima et al. 2015).
Subsequently, after pain induction, many immune cells including MOG-specific CD4 + T cells have been found to
infiltrate parenchyma of the L5 spinal cord following theaccumulation of MHC class II + CD11b + cells at two ventral vessel sites of the spinal cord. These findings led us to
examine the role of MHC class II + CD11b + cells in paininduced EAE relapse. We found that in response to NE
from sympathetic activation around the vessels, these cells
expressed a chemokine, CX3CL1, which accumulates thesame cells through β1- and β2-mediated signaling at thevessel sites. Additionally, by directly injecting an antagonist
of NMDA receptors, MK801, or agonist of NMDA
receptors, L-Homocysteic acid, to the ACC, we found theaccumulation of MHC class II+ CD11b + cells at the

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ventral vessels in the L5 cord is at least in part dependent
on ACC activity. We then analyzed the involvement ofdownstream sympathetic pathways on the infiltration of
MHC class II + CD11b + cells around the ventral vessels of
the spinal cord using the β1-AR antagonist atenolol or 6OHDA-mediated sympathectomy. Both strategies led tosystemic suppression of the sympathetic nervous systemand protected the L5 spinal cord area from MHC class II +CD11b + cell infiltration and EAE relapse. Our analyses of
the afferent sensory pathway using various pharmacological or genetic manipulations showed that activatedneurons processing nociceptive information from the maxillary region to the brain express TRPV1 and Nav1.8(Arima et al. 2015). Consistent with the importance of the
TRPV1+ sensory pathway for EAE relapse is the dependency of chronic pain sensation on the pathway (Huang
et al. 2019). Thus, neuropathic pain-mediated activation of
the ACC with subsequent stimulation of the sympathetic
nerves distributed around the ventral vessels of spinal
cords exacerbates quiescent neuroinflammation in the
spinal cord by opening gateways for pathogenic CD4 + T
cells to enter the CNS in the presence of MHC class II +CD11b + peripheral-derived activated monocytes. Considering the similar nature of this mechanism to the gravity
gateway reflex, we named it the “pain gateway reflex”(Fig. 3) (Arima et al. 2015; Tanaka et al. 2017b; Kamimura
et al. 2018a, b). The proposed arc of this reflex is composed of sensory nociceptive neurons expressing TRPV1and Nav1.8 as the afferent pathway, the ACC with its connections to autonomic nuclei in the brainstem as the center, and sympathetic neurons as the efferent pathway.
In the pain gateway reflex, both MHC class II + CD11b +activated monocytes and endothelial cells of spinal cord
ventral vessels appear to be the initial effector cells.
Stress gateway reflex

The hypothalamus plays a key role in orchestrating endocrine, autonomic, and behavioral responses to stress. With
regard to autonomic responses, communications between
the hypothalamus and autonomic centers in the brainstemand spinal cord are bidirectional. Moreover, ascending
projections from the autonomic nuclei to the hypothalamus and descending projections in the opposite direction
may use the same pathways. Most viscerosensory signals,
which are related to autonomic functions such as cardiovascular, respiratory, osmolarity, gastric motility, and foodintake, reach the hypothalamus mainly through the nucleus of the solitary tract (NTS), which receives signals
from the vagus nerve and glossopharyngeal nerve. Somatosensory signals, including nociception, project to the hypothalamus as well. Especially neurons in the lateralhypothalamus at the level of the paraventricular nucleus(PVN) appear to be crucial, as they significantly increase cfos expression after painful stimuli (Palkovits 1999;

Stofkova and Murakami Bioelectronic Medicine(2019) 5:14

Page 8 of 15

Fig. 3 Pain gateway reflex. In a transfer EAE-recovered mouse, induction of trigeminal pain by ligation of the infraorbital sensory nerve causes
EAE relapse. In this condition, pathway carrying nociceptive information activates the ACC (1), a pain-processing area of the brain, which in turnstimulates sympathetic centers (2) and their neural output to the ventral spinal vessels (3). NE release around these vessels induces chemokine
CX3CL1 expression from activated monocytes and endothelial cells (4). CX3CL1 recruits these activated monocytes around the ventral vessels in
an autocrine/paracrine manner (5). Because the L5 spinal cord contains a high number of activated monocytes in EAE-recovered mice, the L5
region is most affected in terms of the accumulation of activated monocytes. Activated monocytes present MOG antigens to reactivate circulating
pathogenic CD4+ T cells (6), thus causing their accumulation in the spinal cord and EAE relapse (7). Abbreviations: EAE, experimental autoimmune
encephalomyelitis; ACC, anterior cingulate cortex; NE, norepinephrine; MOG, myelin oligodendrocyte glycoprotein; TG, trigeminal ganglion

Benarroch 2005). The lateral hypothalamus is also suggested to relay viscero- and somatosensory signals to otherhypothalamic nuclei and limbic nuclei, and vice versa. In
this way, the lateral hypothalamus serves as an integration
center in long-loop autonomic reflexes for mediating cardiovascular, behavioral, and endocrine responses elicitedfrom the insular cortex, limbic areas, and medial hypothalamic nuclei, i.e. the arcuate nucleus (ARC), ventral medial
nucleus (VMN), dorsal medial nucleus (DMN), anterior
hypothalamic area (AHP), and PVN (Palkovits 1999).
The gateway reflexes use the same neuronal pathways as
reflexes activated by natural physiological stimuli (e.g.
gravitational and nociceptive) to breach the BSCB and exacerbate EAE outcomes. However, if gateway reflexes
utilize pathways of vital autonomic reflexes like cardiovascular or gastrointenstinal reflexes, they may not exacerbate EAE, but they could compromise vital functions to
the extent of causing sudden death (Arima et al. 2017).
We employed two stress conditions including a light sleep
and a hostile environment, both of which can be experienced in normal social life. Importantly, these conditions
cause discomfort, but they are not life-threatening (Arimaet al. 2017). Nevertheless, in mice with transferred pathogenic CD4+ T cells under these chronic stress conditions,
we observed severe gastrointestinal inflammation andheart failure including catabolism of vital tissues without
the presence of typical EAE symptoms such as tail or leg
paralysis. Importantly, in these mice, pathogenic CD4+ T

cells and MHC class II+ cells accumulated at two symmetrical brain vessel sites of the boundary region of the thirdventricle (3 V), thalamus, and dentate gyrus (DG), but notin the L5 spinal cord, which is where they accumulated in
unstressed EAE animals undergoing the gravity gatewayreflex. We hypothesized that the infiltration of immune
cells at the specific sites of the brain might be due to upregulated chemokine expressions in vascular endothelial
cells of regional vessels followed by the formation of gateways for immune cells to pass through the BBB. To assess
this possibility, we tested several neutralizing antibodies
against chemokines known to recruit pathogenic CD4+ T
cells and MHC class II+ antigen presenting cells. An antiCCL5 antibody was shown to prevent the mortality of
EAE mice under chronic stress. Consistent with thisobservation, we found that chronic stress could induce
CCL5 expression at the specific vessels as well as activate
PVN noradrenergic neurons. Moreover, many neurons in
the DMH (dorsomedial hypothalamic nucleus) and AHP
were activated in mice with stress plus pathogenic CD4+
T cells compared to mice with stress or pathogenic CD4+
T cells alone. Neural tracing experiments showed direct
neural connections between the specific vessels and neurons in the PVN or DMH/AHP. To study the functional
importance of noradrenergic neurons at the vessels, we
performed chemical sympathectomy. The microinjectionof 6-hydroxydopamine at the vessels, which depleted tyrosine hydroxylase sympathetic neurons and phospho-CREB

Stofkova and Murakami Bioelectronic Medicine(2019) 5:14

signals, suppressed the microinflammation development
and the fatal outcome in mice with stress plus pathogenic
CD4+ T cells. To study the functional importance of neurons in the PVN and DMH/AHP, we performed electric
cauterization of the brain regions, finding the fatal phenotype was a suppressed in mice with stress plus pathogenic
CD4+ T cells (Arima et al. 2017). We are also planning toperform optogenetic and chemogenetic experiments toclarify the functions of the neural connections and to
identify neural markers.
Importantly, activation of the DMH/AHP was associatedwith descending ATPergic projections arising from thespecific inflamed brain vessel sites in the presence of
pathogenic CD4+ T cells and MHC class II+ antigen presenting cells. Stimulation of this pathway was associatedwith the sudden death of EAE mice exposed to stressconditions, because the intracranial injection of A438079,
an ATP P2RX7 receptor antagonist, at the specific vessels
could suppress the DMH/AHP activation and the mortality
caused by severe gastrointestinal bleeding and heart failure(Fig. 4) (Arima et al. 2017). ATP is a fast excitatory neurotransmitter, and its function as a cotransmitter in central
and peripheral noradrenergic neurons is well established(Zimmermann 1994; Burnstock 2006). Furthermore, ATP

Page 9 of 15

as a neurotransmitter was reported to act on postsynaptic
P2X receptors to modulate the excitability of DMH/AHP
neurons (Matsumoto et al. 2004; Burnstock 2007). Activation of the DMH/AHP has been shown to stimulate intestinal motility, heart rate and blood pressure through vagal
cholinergic, nonvagal cholinergic and noncholinergic
pathways (Greenwood and DiMicco 1995). We found that
activation of the vagal motor nerves most likely mediatedby a DMH–DMX (dorsal motor vagal nucleus) pathway
was critical for the development of the fatal gastrointestinal
failure under stress conditions in EAE mice, because
vagotomy suppressed the stress-EAE phenotype withsudden death. We also found cholinergic activation in epithelial cells in the upper gastrointestinal tract was affected.
Additionally, enhanced activation of the NTS, which receives afferent inputs from the vagus nerve, indicated increased vagal sensory signaling to the brain in mice understress conditions and pathogenic CD4+ T cell transfer(Arima et al. 2017). These vagal afferent inputs to the NTS
likely arise from baroreceptors, since the baroreflex ischronically activated in conditions of high blood pressureand heart rate as a compensatory response (Lohmeier andIliescu 2015). The increased neural activity associated withthese autonomic long-loop reflexes controlling blood

Fig. 4 Stress gateway reflex. Hostile environment induced by a specific cage with water and a free rotation wheel causes chronic stress associatedwith sleep disorder. In a mouse with adoptive-transfer EAE, this chronic stress stimulates noradrenergic neurons in the PVN (1), which then project tobilateral specific vessels surrounded by the third ventricle (3V), dentate gyrus (DG) and thalamus (TH) (2) to upregulate chemokines including CCL5 andrecruit pathogenic CD4+ T cells plus MHC class II+ monocytes. The resulted microinflammation activates a neural pathway involving the DMH/AHP
and DMX via ATP production (3). Hyperactivation of the vagus pathway particularly distributed the upper gastrointestinal (GI) tract causes a severe
damage in the upper GI tract followed by a heart failure with sudden death (5). Abbreviations: AHP, anterior hypothalamic area; EAE, experimental
autoimmune encephalomyelitis; PVN, paraventricular nucleus of the hypothalamus; DMH, dorsomedial nucleus of the hypothalamus; DMX, dorsal
motor nucleus of the vagus nerve; NTS, nucleus tractus solitarii; 4V, fourth ventricle

Stofkova and Murakami Bioelectronic Medicine(2019) 5:14

pressure during chronic stress might explain the activationof noradrenergic PVN neurons projecting to specific vessels in the area of the 3 V, thalamus, and DG, resulting in
BBB breakdown and the formation of immune cell gateways. Microinflammation in the boundary region of the 3V, thalamus, and DG appears to be critical, given the lethal effect of the intracranial injection of proinflammatory cytokines such as IL-6 plus IL-17A, IFNγ
plus IL-17A, or pathogenic CD4+ T cells plus antigenpresenting cells to this brain area. Importantly, none ofstress, the transfer of pathogenic CD4+ T cells alone, orthe combination of stress with the transfer of activatedCD4+ T cells directed against non-CNS antigens such as
ovalbumin or interphotoreceptor retinoid-binding protein(IRBP) were able to cause the fatal phenotype (Arimaet al. 2017).
Circulating EPI could affect the phenotype of endothelial cells in the brain, but because it is also a strong vasoconstrictive agent it can also prevent gastrointestinal
bleeding. In fact, endoscopic injection of EPI is widelyused in clinical practice in patients with actively bleeding
ulcers (Lin et al. 1999; Liou et al. 2006; Vergara et al.
2014). Therefore, it is important to investigate whether
peripheral levels of catecholamines, which are released
from the adrenals in response to chronic stress, contributeto the gastrointestinal failure. We performed experiments
using two antagonists of the corticosteroid receptor, mifepristone and guggulsterone, on mice in a stress condition
after the microinjection of cytokines at specific vessels(Arima et al. 2017). The antagonists had no effect on thecytokine-mediated fatal phenotype, which suggests the
hypothalamic–pituitary–adrenal axis has a minimum role.
We named this autonomic long-loop reflex circuit
exploited by stress-mediated brain microinflammation
the “stress gateway reflex”. To summarize our current
understanding of this fatal gateway reflex, our findings
support the proposal that overactivation of central noradrenergic signaling induced by stress accounts for a
pro-inflammatory phenotype of the BBB endothelium at
specific brain vessel sites characterized by increased
CCL5 expression and triggers the infiltration of pathogenic CD4+ T cells and activated MHC class II+ monocytes. The microinflammation induced by pathogenic
CD4+ T cell activation stimulates fast excitatory
ATPergic neurons descending the DMH/AHP, which inturn activates efferent (motor) vagal outflow to theupper gastrointestinal tract. Constant stimulation of thegateway reflex under chronic stress leads to gastricinflammation, gastrointestinal bleeding, hyperkalemia,
and eventually heart failure (Arima et al. 2017; Kamimura et al. 2018a, b).
Our findings are in contrast with previous studies thatsuggested an anti-inflammatory role of the vagus nerve inmurine and rat colitis models (Ghia et al. 2006, 2007;

Page 10 of 15

Meregnani et al. 2011) or murine postoperative ileus (deJonge et al. 2005). These studies are in parallel with promising results of bioelectronic medicine targeting the vagus
nerve of patients with rheumatoid arthritis, inflammatory
bowel disease, and Crohn’s disease (Merrill et al. 2006;
Schwartz et al. 2008; González et al. 2019; Engineer et al.
2019; Koopman et al. 2016; Bonaz et al. 2016). The discrepancy is most likely due to the fact that the activatedvagus fibers are different. It is possible that stress gatewayreflex activates only vagus fibers distributed on the uppergastrointestinal tract, while bioelectronic medicine stimulates almost all vagus nerves. Consistent with this hypothesis, vagus nerves with mechanosensor PIEZO+ alone are
critical for the baroreceptor reflex (Zeng et al. 2018), whileP2ry1 and Npy2r vagal sensory neuron types evoke distinct
effects on breathing (Chang et al. 2015). Moreover, vagal
GPR65 neurons control gut motility, and GLP1R neurons
detect stomach and intestine stretch (Williams et al. 2016).
Therefore, it is possible that there exist specific functional
markers of immunosuppressive vagus fibers and/or vagus
fibers that distribute or function only on the upper gastrointestinal tract. Further studies are required to gain broader
insight of the effect of vagus nerve stimulation on autoimmune diseases, particularly those associated withchronic stress and CNS inflammation.
The suggested arcs of the stress gateway reflex consistof two branches. The first branch is composed of (1) interoceptors distributed in blood vessels and the heart,(2) an afferent pathway represented by sensory vagal
nerves transmitting information from the interoceptors
to (3) the NTS and PVN centers in the brain, (4) an efferent pathway represented by noradrenergic neurons
ascending from the PVN, and (5) endothelial cells of thespecific vessel sites at the boundary area of the 3 V, thalamus, and DG that function as effectors. The second
branch of the stress gateway reflex starts with (1) afferent ATP-producing neurons at the boundary area of the
3 V, thalamus, and DG, which are activated by microinflammatory stimuli projecting to (2) the reflex center inthe DMH/AHP, which then transmits signals to the
DMX to activate (3) efferent vagal nerves, resulting inincreased gastrointestinal secretory and motor functions
mediated by (4) local gastrointestinal cells.
Light gateway reflex

When bright light enters the eyes, the pupils decrease insize to reduce the amount of light reaching the retina.
On the contrary, under dim light, the pupils enlarge toallow as much light as possible to reach the retina. Thismechanism, known as the pupillary light reflex, is a
result of interactions between neurons and iris smoothmuscle cells. Under photopic light condition, photoreceptive pigments in the photoreceptors and intrinsicallyphotosensitive retinal ganglion cells (ipRGCs) absorb

Stofkova and Murakami Bioelectronic Medicine(2019) 5:14

visible light photons and convert this light energy into
nerve impulses. These impulses are then transmitted
via the optic nerve pathway to the pupillary light reflex
center in the midbrain: EdingerWestphal nuclei.
Efferent projections from these nuclei travel to the ciliary ganglion, where parasympathetic postganglionic
neurons of the oculomotor nerve extend to innervate
the pupillary sphincter muscles leading to pupillary
constriction. In mesopic and scotopic light conditions,the pupil enlargement is under sympathetic influenceon the iris dilator muscle and consists of a threeneuron arc extending from the retinohypothalamic
tract through the ciliospinal center of Budge in theintermediolateral gray column of the spinal cord at
C8–T1 to the superior cervical ganglion (SCG), fromwhich sympathetic postganglionic neurons travel to andinnervate the iris dilator muscle to cause pupil dilation(McDougal and Gamlin 2015; Hall and Chilcott 2018;
Belliveau and Dossani 2019).
Using an animal model of posterior autoimmune uveitis, experimental autoimmune uveoretinitis (EAU), we
investigated whether the pupillary light reflex pathway
influences the infiltration of immune cells into the retina. To avoid the disruption of circadian rhythms, we
stimulated mice with mesopic light of 2 lx or photopic
light of 230 lx only in the light phase of the day. In thedark phase, all mice were exposed to 0 lx condition. Furthermore, these light interventions were applied only
four days, starting from the first appearance of CD4+ T
cells in the eyes after the onset of the clinical phase of
EAU (Stofkova et al. 2019).
We observed that photopic and mesopic light cancontrol retinal vascular endothelial cells via neural
regulation. Indeed, mice housed under photopic lightduring EAU development exhibited a downregulationof NF-κB and STAT3 activity in the retinal endothelium, decreased gene expressions of various chemokinesand cytokines that attract and/or activate immune cells
in the retina, and reduced EAU development compared
to EAU mice housed under mesopic light. Consistently,
EAU mice exposed to photopic light showed lower
numbers of CD4+, CD8+ and CD11b + cells in the retina and around the central retinal vessels within the
optic nerve head in comparison to EAU mice exposed
to mesopic light.
Given our previous data demonstrating the involvement of sympathetic ganglia in gravity-, electric-, andpain-gateway reflexes and the fact that the SCG is
physiologically activated under mesopic light and inhibited under photopic light by the pupillary light reflex, we
hypothesized that changes in the retinal endothelium
observed in EAU mice housed under different light intensities are related to the activity of sympathetic neurons from the SCG. To test this hypothesis, wePage 11 of 15

performed unilateral surgical ablation of the SCG (SCGX) to eliminate sympathetic inputs to the eye, and we
immunized mice with interphotoreceptor retinoidbinding protein peptide 1–20 (IRBP1–20) to induce EAU.
However, mesopic light- and photopic light-exposed
mice with SCG-X developed EAU in both eyes without
significant lateralization of immune cell infiltrates to either eye, suggesting that peripheral sympathetic nerves
do not mediate the phenotype of the BRB endothelium
during EAU (Fig. 5) (Stofkova et al. 2019). This finding
is consistent with the fact that retinal vessels receive
minimal sympathetic innervation (McDougal and Gamlin 2015). Since retinal neurons, including amacrine cells
and horizontal cells have been shown to produce NE
and EPI (Park et al. 1988; Hadjiconstantinou et al. 1983;
Ishimoto et al. 1989; Chen et al. 1999), we assessed NE
and EPI levels in the eyes in response to photopic andmesopic light in EAU mice. We found a significant increase in NE and EPI levels in the eyes, but not in theserum of mice exposed to photopic light when compared
to mesopic light. In addition, we also observed an upregulation of dopamine β-hydroxylase (DBH), an enzyme
necessary for NE and EPI synthesis, in the inner nuclear
layer (INL) of the retina, which is composed of amacrine, horizontal and bipolar cells. Simultaneously, we
also detected the downregulation of α1-AR expression in
retinal endothelial cells in EAU mice exposed to photopic light. We assume that this downregulation is due
to high NE and EPI levels in the eyes, since AR ligands
downregulate AR expression (Stofkova et al. 2019;
Akinaga et al. 2013; Heck and Bylund 1997; LeebLundberg et al. 1987). Consistent with this effect, pharmacological or genetic inhibition of α1-AR or α1A-AR
reduced the activity of noradrenergic signaling to suppress
pro-inflammatory cytokine/chemokine expressions in theretina and EAU development under mesopic light condition (Stofkova et al. 2019).
We therefore concluded that NE/EPI-producing neurons after photopic light treatment in the retina interact
with retinal endothelial cells to inhibit the formation of
immune cell gateways during EAU. Since this mechanism, like the gateway reflexes observed in EAE, requires
the neural stimulation of effector barrier endothelial
cells, we named it the “light gateway reflex” (Fig. 5).
However, unlike the pro-inflammatory gateway reflexes associated with EAE, the light gateway reflex isan anti-inflammatory reflex and seems to be integrated entirely within the retina independently of thecentral or peripheral nervous system. Thus, we foundan unprecedented mechanism by which retinal neuralactivity mediated by light can regulate the local
homeostasis and defend the local tissue against invading
immune cells without utilizing other ocular reflexes related to SCG activity.

Stofkova and Murakami Bioelectronic Medicine(2019) 5:14

Page 12 of 15

Fig. 5 Light gateway reflex. Exposure to photopic light in actively induced EAU attenuates retinal inflammation. This process is mediated throughlight-induced expression of DBH, an enzyme essential for the synthesis of NE and EPI, by retinal neurons located in the INL (1). The increased NE
and EPI levels down-regulate retinal α1AAR expression, which is followed by reduced NF-κB and STAT3 activation and decreased chemokine andIL-6 expressions in the retina (2)(3). This protects the BRB integrity and suppresses recruitment of immune cells including IRBP-specific pathogenic
CD4+ T cells in the retina. Abbreviations: EAU, experimental autoimmune uveoretinitis; DBH, dopamine beta-hydroxylase; INL, inner nuclear layer;
NE, norepinephrine, EPI: epinephrine; α1AAR, alpha-1A adrenoceptor; BRB, blood-retina barrier; IRBP, interphotoreceptor retinoid-binding protein

Conclusion
We here shed light on new aspects of neuroinflammation and how neural activity can modulate the formationof immune cell gateways to initiate and progress the development of autoimmune diseases of the CNS and retina. The principle of this process is called the gatewayreflex, which can be defined as neural circuits regulating
the entry of immune cells to the CNS and retina by
modulating the barrier phenotype of vascular endothelial
cells. We have characterized four pro-inflammatory gateway reflexes that promote the development and outcomeof experimental models of MS: gravity-, electric-, pain-,and stress-gateway reflexes. All these reflexes should be
utilized as neural pathways of physiological reflexes thatserve to keep body homeostasis, particularly in the absence of autoreactive CD4+ T cells, which are known toincrease with age and/or infection. Despite the reflexes
using distinct viscero- and somatosensory stimuli and afferent pathways for their activation, the efferent pathway
resulting in the pro-inflammatory phenotype of endothelial cells is mainly noradrenergic. We also characterized
one anti-inflammatory reflex, the light gateway reflex,
which protects against the development of an experimental model of posterior autoimmune uveitis andmaintains the anti-inflammatory phenotype of the BRB

endothelium. The light gateway reflex is unique in thatit involves retinal neural circuits to prevent BRB breakdown, and the anti-inflammatory response in this reflex
is mediated by the inhibition of noradrenergic signaling
in retinal vascular endothelial cells. The molecularmechanism of the light gateway reflex could be a generalmechanism that suppresses the formation of immune
cell gateways seen in other gateway reflexes, all of which
are associated with the transient development of diseases. We could just easily find a negative feedback
mechanism in the EAU system, because many neuralcells in retina tissues make the responses drastically.
It is reasonable to speculate that regional neural activity
at specific vessels can modulate the development of otherautoimmune diseases beyond those affecting nervous
tissues. An example from clinical research suggesting theimportance of neural activity in autoimmune diseases is
the sparing effect of hemiplegia in several rheumatoid
diseases, particularly in patients with rheumatoid arthritis,
psoriatic arthritis, polymyalgia rheumatica or systemicsclerosis (Ughi et al. 2015). In these rheumatic patients,
paretic limbs have been shown to exhibit a marked remission of arthritic signs, including diminished erosivechanges and decreased skin temperature (Keyszer et al.
2004), suggesting that a loss of neural activity can protect

Stofkova and Murakami Bioelectronic Medicine(2019) 5:14

against rheumatoid inflammation. Thus, the next crucial
step is to elucidate whether the concept of the gatewayreflex is a fundamental phenomenon that also plays a rolein the recruitment of immune cells to other tissues in thebody.
Abbreviations
3 V: Third ventricle; ACC: Anterior cingulate cortex; AHP: Anterior
hypothalamic area; AR: Adrenoceptor; BBB: Blood-brain barrier; BRB: Bloodretina barrier; BSCB: Blood-spinal cord barrier; CCI: Chronic constriction injury;
CNS: Central nervous system; CRPS: Complex regional pain syndrome;
CSF: Cerebrospinal fluid; DG: Dentate gyrus; DMH: Dorsomedial hypothalamic
nucleus; DMX: Dorsal motor vagal nucleus; DRG: Dorsal root ganglia;
E: Embryonic day; EAE: Experimental autoimmune encephalomyelitis;
EAU: Experimental autoimmune uveoretinitis; EPI: Epinephrine;
GGTP: Gamma-glutamyltranspeptidase; INL: Inner nuclear layer;
ipRGCs: Intrinsically photosensitive retinal ganglion cells;
IRBP: Interphotoreceptor retinoid-binding protein;
MHPG: Methoxyhydroxyphenylglycol; MOG: Myelin oligodendrocyte
glycoprotein; MS: Multiple sclerosis; NE: Norepinephrine; NTS: Nucleus of thesolitary tract; NVU: Neurovascular unit; PVN: Paraventricular nucleus;
SCG: Superior cervical ganglion; VSMCs: Vascular smooth muscle cells
Acknowledgements
We appreciate the outstanding experimental contributions by Dr. Y. Arima to
the studies described here, the excellent technical assistance by Ms. Ezawa
and Ms. Nakayama, and thank Ms. Fukumoto for her excellent technical andsecretarial assistance. We thank Dr. P. Karagiannis (CiRA, Kyoto University,
Kyoto, Japan) for carefully reading the manuscript.
Authors’ contributions
AS and MM wrote the manuscript. Both authors read and approved the final
manuscript.
Funding
This work was supported by grant funds provided to Dr. Andrea Stofkova(JSPS Postdoctal Fellowship for Foreign Researchers, GACR 18-11795Y, PRIMUS/17/MED/7, and Progress Q35) and to Dr. Masaaki Murakami (KAKENHI,
the Joint Usage/Research Center Institute for Genetic Medicine, Hokkaido
University, Takeda Science Foundation).
Availability of data and materials
Data sharing not applicable to this article.
Ethics approval and consent to participate
All animal experiments were performed following the guidelines of theInstitutional Animal Care and Use Committees of the Graduate School of
Frontier Biosciences and Graduate School of Medicine, Osaka University, andthe Institute for Genetic Medicine, Hokkaido University. The protocols foranimal experiments were approved by the Institutional Animal Care and Use
Committees of the Graduate School of Frontier Biosciences and Graduate
School of Medicine, Osaka University, and Hokkaido University.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Physiology, Third Faculty of Medicine, Charles University,
Prague, Czech Republic. 2Division of Molecular Psychoimmunology, Institutefor Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo
060-0815, Japan.

Page 13 of 15

Received: 21 May 2019 Accepted: 18 July 2019

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