**5. Pain‐mediated gateway reflex**

**4. Electric stimulation‐mediated gateway reflex**

36 Trending Topics in Multiple Sclerosis

Neural activations can be artificially induced by various methods including electric stimula‐ tions and treatment with reagents. We wondered if electric stimulation of different muscles could create gateways in blood vessels at distinct positions via regional neural activations. As discussed earlier, weak electric stimulation to the soleus muscles restored chemokine expres‐ sion at the L5 dorsal vessels of tail‐suspended mice, because the cell body of sensory neurons in the soleus muscles is mainly located in the L5 DRG. We applied this methodology to other muscles. As expected, electric stimulations to thigh muscles including the quadriceps, which are known to be regulated by L3 DRG neurons, led to an increased expression of chemokines in L3 dorsal vessels in mice, while electric stimulations of upper regions, such as forefoot muscles including the epitrochlearis and triceps brachii, resulted in an up‐regulation of chemokines in the fifth cervical to fifth thoracic cord dorsal vessels, which is where the DRG

**Figure 2.** Electric stimulation‐mediated gateway reflex. Artificially induced neural activation by weak electric stimula‐ tion followed by sensory‐sympathetic cross talk can trigger the gateway reflex. Electric pulses to the triceps induce chemokine up‐regulation at the dorsal vessels of the fifth cervical (C5) to fifth thoracic (T5) spinal cord through activa‐ tion of the inflammation amplifier (Amp). Similarly, stimulation of the quadriceps induces a gateway at the dorsal ves‐ sels of the third lumbar (L3) cord, whereas the gateway dorsal vessels of the L5 cord are created by electric pulses to

the soleus muscles.

It is reported that many MS patients experience pain, and some patients take pain relief medication to improve their quality of life [43]. Pain is a common symptom in many diseases and disorders, but it is often considered a symptomatic effect that arises from tissue damage caused by the disease. However, it is known that pain triggers neural inputs via sensory neurons expressing nociceptors such as TRPV1 [44, 45]. Therefore, we assumed that a pain‐ mediated gateway reflex might exist, which would affect the disease status of MS and EAE. In EAE, modulations of nociception (pain sensation) are reported during the disease devel‐ opment [46]. Adoptive transfer of MOG reactive, pathogenic CD4+ T cells induces a transient EAE within a week after transfer, followed by a remission phase. Mice that experienced the first episode of EAE and are in the remission phase, which we termed EAE‐recovered mice, never develop EAE symptoms again under normal conditions. We experimentally induced pain sensation in these EAE‐recovered mice by ligating the middle branch of the trigeminal nerves, which has been reported as being composed of only sensory neurons [47]. Pain induction at the time of pathogenic T‐cell transfer resulted in a persistence of EAE symptoms and caused considerable delay of the remission phase. On the contrary, suppressing pain by medication inhibited EAE development in several mouse models including an active immu‐ nization model in SJL mice. Because a majority of MS progresses with relapse and it is reported that pain is more intense in MS patients with higher disease scores [48], we examined whether pain induction triggers EAE relapse in mice. EAE‐recovered mice were subjected to trigeminal nerve ligation to induce pain sensation. In separate experiments, pain‐processing nerves were also chemically activated by the injection of capsaicin and substance P at the cheek or forefeet of EAE‐recovered mice. All these treatments induced relapse of EAE, indicating that pain is not a simple by‐product of the disease, but significantly contributes to the development and EAE relapse [13]. Similar to the gravity‐mediated gateway reflex, a sensory‐sympathetic pathway is involved in the pain‐mediated EAE relapse, because pharmacological or genetic inhibition of pain‐processing molecules such as TRPV1 and Nav1.8, and chemical ablation of sympathetic neurons by 6‐hydroxydopamine (6‐OHDA) suppressed EAE relapse induced by trigeminal nerve ligation. Interestingly, stress‐mediated events, including immobilization stress and forced swimming stress (about 20 minutes/day), did not trigger EAE relapse although serum corticosterone, norepinephrine, and epinephrine were induced at a similar level to pain induction, suggesting that specific sensory‐sympathetic nerve pathways, rather than systemic hormonal stress responses, mediate the relapse of EAE (**Figure 3**).

**Figure 3.** Pain‐mediated gateway reflex. Pain‐induced sensory neuron activation results in the activation of specific sympathetic neurons that control norepinephrine (NE) expression around the ventral vessels of every level of the spi‐ nal cords. This system is regulated by the anterior cinculate cortex (ACC) in the brain. Because the fifth lumbar (L5) spinal cord abundantly contains MHC class II high activated monocytes, this region is affected significantly during pain sensation. NE from pain‐specific sympathetic neurons at the ventral vessels induces the production of CX3CL1 from the activated monocytes, further recruiting these cells in an auto/paracrine manner. The MHC class II high acti‐ vated monocytes are able to present MOG antigens to pathogenic CD4+ T cells, which in turn activate the inflamma‐ tion amplifier (Amp) in regional endothelial cells and subsequently cause a relapse in the disease.

During the first episode of EAE, MOG‐reactive CD4+ T cells enter the CNS from the dorsal vessels of the L5 spinal cord [12]. The transferred CD4+ T cells are then found at the upper levels of the spinal cord and brain, which matches typical clinical manifestations of EAE including the initial tail tip weakness and subsequent ascending paralysis. Intriguingly, after pain induction in apparently normal EAE‐recovered mice, the relapse also starts from the loss of tonicity of the tail tip, suggesting that the L5 cord could again be a gateway for relapse. However, an immunohistochemical examination of the L5 spinal cord from EAE‐recovered mice showed differences with naïve mice, with many MHC class II high monocytes around the meningeal region. After pain induction, these cells accumulated at the L5 ventral vessels, but not dorsal vessels within a day. This accumulation is dependent on a chemokine CX3CL1, which is secreted from the MHC class II high monocytes themselves and astrocytes after norepinephrine stimulation. Therefore, the pain‐mediated gateway reflex induces norepi‐ nephrine secretion from sympathetic neurons around the L5 ventral vessels and subsequent auto/paracrine induction of CX3CL1 followed by MHC class II high monocyte accumulations. Because these monocytes are able to present MOG peptides, circulating MOG‐reactive pathogenic CD4+ T cells can recognize the L5 ventral vessels as an entry site to the CNS. Indeed, a depletion of CD4+ T cells including pathogenic ones from EAE‐recovered mice abrogated the clinical symptoms of EAE relapse (i.e. paralysis), but the accumulation of MHC class II high monocytes around the L5 ventral vessels remained intact. These results suggested that the activated monocyte accumulation is an upstream event relative to pathogenic CD4+ T‐cell invasion and required for EAE relapse induced by pain sensation [13].

The sympathetic nerve activation caused by pain appeared to be relatively systemic compared with that via the sensory pathway, since the expression levels of neural activation marker c‐ Fos increased in certain nerve cells in sympathetic ganglions at all spinal levels investigated, which is in contrast to the gravity‐mediated gateway reflex in which the L5 sympathetic ganglion alone showed the highest expression of c‐Fos. However, the pain‐mediated gateway reflex affects blood vessels at the ventral sides of the L5 spinal cord. Why does the pain‐ mediated gateway reflex influence the L5 level alone, similar to the gravity‐mediated gateway reflex? We assume that this property is related to the fact that the L5 cord is the first lesion in the transfer EAE model. Even at the remission phase of transfer EAE, MHC class II high monocytes that have infiltrated in the CNS during the initial episode remained mostly in the L5 spinal cord despite the disappearance of clinical symptoms [13]. Otherwise, gravity‐ mediated neural inputs might make the L5 environment different from other CNS regions. Whichever the explanation, we found that pain induction similarly triggers sympathetic neural activation to some neuron cells of every spinal level, but the L5 spinal cord responded strongest, most likely due to an abundance of activated monocytes.

It is known that pain‐sensing neurons activate sympathetic neurons at least partially via the anterior cingulate cortex (ACC) in the somatosensory area of the brain [49, 50]. Blockade of this sensory‐sympathetic connection by the injection of an N‐methyl‐D‐aspartate (NMDA) receptor antagonist at the ACC suppressed the accumulation of MHC class II high monocytes at the L5 ventral vessels even after pain induction, and conversely, injection of an NMDA agonist at the ACC induced the accumulation of these cells without pain stimulation in EAE‐ recovered mice. These results clearly suggest that the sensory‐sympathetic connection at the ACC is involved in pain‐mediated EAE relapse. Thus, we showed a third pain‐mediated gateway reflex where neural signals travel from a TRPV1 and Nav1.8‐dependent sensory circuit to sympathetic neurons via the ACC, reaching the dorsal vessels of the spinal cords [13] (**Figure 3**).

The study of the pain‐mediated gateway reflex also highlights the importance of MHC class II high monocytes for EAE relapse. Parabiosis experiments, in which two different congenically marked mice are joined together surgically to share blood circulation, revealed that MHC class II high monocytes that accumulated at the L5 ventral vessels were derived from the peripheral blood stream during the first EAE development and stayed in the CNS, but not from resident microglia in the CNS [13]. These infiltrated MHC class II high monocytes survive in the CNS during the remission phase and play a pathogenic role upon relapse when the mice have a pain sensation. Therefore, in addition to controlling neural pathways and/or molecular machinery of the gateway reflexes, MHC class II high monocytes could also be a potential cellular target for treatment of relapse‐remitting MS and progressive‐relapsing MS.
