Involvement of Degenerating 21.5 kDa Isoform of Myelin Basic Protein in the Pathogenesis of the Relapse in Murine Relapsing-Remitting Experimental Autoimmune Encephalomyelitis and MS Autopsied Brain.

Multiple sclerosis (MS) is the chronic inflammatory demyelinating disease of the CNS. Relapsing-remitting MS (RRMS) is the most common type of MS. However, the mechanisms of relapse and remission in MS have not been fully understood. While SJL mice immunized with proteolipid protein (PLP) develop relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE), we have recently observed that some of these mice were resistant to the active induction of relapsing EAE after initial clinical and histological symptoms of EAE with a severity similar to the relapsing EAE mice. To clarify the mechanism of relapsing, we examined myelin morphology during PLP139-151-induced RR-EAE in the SJL mice. While RR-EAE mice showed an increased EAE severity (relapse) with CNS inflammation, demyelination with abnormal myelin morphology in the spinal cord, the resistant mice exhibited a milder EAE phenotype with diminished relapse. Compared with the RR-EAE mice, the resistant mice showed less CNS inflammation, demyelination, and abnormalities of the myelin structure. In addition, scanning electron microscopic (SEM) analysis with the osmium-maceration method displayed ultrastructural abnormalities of the myelin structure in the white matter of the RR-EAE spinal cord, but not in that of the resistant mice. While the intensity of myelin staining was reduced in the relapsing EAE spinal cord, immunohistochemistry and immunoblot analysis revealed that the 21.5 kDa isoform of degenerating myelin basic protein (MBP) was specifically induced in the relapsing EAE spinal cord. Taken together, the neuroinflammation-induced degenerating 21 kDa isoform of MBP sheds light on the development of abnormal myelin on the relapse of MS pathogenesis.

Systemic Inflammation Leads to Changes in the Intracellular Localization of KLK6 in Oligodendrocytes in Spinal Cord White Matter.

Axonal injury and demyelination occur in demyelinating diseases, such as multiple sclerosis, and the detachment of myelin from axons precedes its degradation. Paranodes are the areas at which each layer of the myelin sheath adheres tightly to axons. The destruction of nodal and paranodal structures during inflammation is an important pathophysiology of various neurological disorders. However, the underlying pathological changes in these structures remain unclear. Kallikrein 6 (KLK6), a serine protease produced by oligodendrocytes, is involved in demyelinating diseases. In the present study, we intraperitoneally injected mice with LPS for several days and examined changes in the localization of KLK6. Transient changes in the intracellular localization of KLK6 to paranodes in the spinal cord were observed during LPS-induced systemic inflammation. However, these changes were not detected in the upper part of brain white matter. LPS-induced changes were suppressed by minocycline, suggesting the involvement of microglia. Moreover, nodal lengths were elongated in LPS-treated wild-type mice, but not in LPS-treated KLK6-KO mice. These results demonstrate the potential involvement of KLK6 in the process of demyelination.

Dataset on the effect of knockout of KLK8 in social memory.

The data presented in this article have been produced as supporting data of the original research article titled "Impaired social discrimination behavior despite normal social approach by kallikrein-related peptidase 8 knockout mouse" (Nakazawa et al., 2019). Sociability and recognition of conspecifics and discrimination among conspecifics (social memory) is fundamental for pair bonding, to create social hierarchy, and eventually establish affiliated societies in social animals, including humans. It has been speculated that the processes of cognition, attention and memory, which are largely mediated by the hippocampus, contribute to social behavior. However, the molecular basis of social behavior remains elusive. This article presents a dataset of behavior-related KLK8-NRG1-ErbB signaling changes in the hippocampus and the effect of activation of ErbB signaling on social behavior.

Impaired social discrimination behavior despite normal social approach by kallikrein-related peptidase 8 knockout mouse.

For social mammals, recognition of conspecifics and discrimination of each other (social memory) is crucial to living in a stable colony. Here, we investigated whether kallikrein-related peptidase 8 (KLK8)-neuregulin 1 (NRG1)-ErbB signaling is crucial for social discrimination behavior using the social discrimination three chamber behavioral test. Klk8 knockout mice (NRG1-deactivated mice) exhibited normal social approach but impaired social discrimination. Intraventricular injection of recombinant NRG1177-246 into Klk8 knockout mice reversed this impaired social discrimination. This study reveals that KLK8 is a key regulator of NRG1-ErbB signaling, which contributes to social discrimination behavior.

Pathological changes in mice with long term cuprizone administration.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). In MS, a long disease duration is known to be a strong risk factor for converting the clinical course of the disease from relapse remitting MS to secondary progressing MS. There is a hypothesis that long sustained demyelination may exhaust neurons, however, pathological changes induced in neurons following demyelination remain unknown. Cuprizone administration can induce and sustain demyelination in the mouse CNS. We examined pathological changes in mice following long sustained demyelination caused by up to 34-week cuprizone administration. Twelve-week cuprizone administration induced severe demyelination in the cerebral cortex, corpus callosum and deep cerebellar nuclei. Demyelination persisted up to 34 weeks, as shown by myelin basic protein immunohistochemistry. In contrast, cuprizone administration developed demyelination in the striatum by week 34. In these demyelinated regions, no neuronal loss was observed. However, in the striatum and deep cerebellar nuclei, cuprizone-induced demyelination changed the intracellular distribution of parvalbumin (PV). Furthermore, in the striatum, there was an increase in PV in the demyelinated axons and most PV immunoreactivity did not co-localize with SMI32 immunoreactivity in mice with 34-week cuprizone administration. Further, mice with 34-week cuprizone administration showed motor coordination dysfunction in the balance beam test. However, 12-week withdrawal from the cuprizone diet induced remyelination in the regions and motor coordination dysfunction recovered. These results indicate that 34-week cuprizone administration induces and sustains demyelination and results in reversible motor coordination dysfunction. The change of intracellular PV distribution suggests that PV may protect demyelinated axons by Ca2+ buffering. This model may be useful to investigate pathological and behavioral changes following demyelination in the CNS.

Kallikrein 6 secreted by oligodendrocytes regulates the progression of experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), and experimental autoimmune encephalomyelitis (EAE) is a well-established animal model of the disease. Here, we examined the pathophysiological role of Kallikrein 6 (Klk6), a serine protease produced by oligodendrocytes (OLs), in EAE using Klk6 knockout (Klk6-/-) mice. Compared with Klk6+/+ (wild-type) mice, Klk6-/- mice showed milder EAE symptoms, including delayed onset and milder paralysis. Loss of Klk6 suppressed matrix metalloprotease-9 expression and diminished the infiltration of peripheral inflammatory cells into the CNS by decreasing blood-brain barrier (BBB) permeability and reducing expression levels of inflammatory cytokines, chemokines and their receptors. Scanning electron microscopic analysis revealed demyelination characterized by myelin detachment from the axons in the early phase of EAE progression (days 3-7) in Klk6+/+ mice but not in Klk6-/- mice. Interestingly, anti-MOG (myelin oligodendrocyte glycoprotein) autoantibody was also detected in the cerebrospinal fluid (CSF) and spinal cord on day 3 after MOG immunization. Furthermore, treatment of primary cultured OLs with anti-MOG autoantibody induced oligodendroglial morphological changes and increases in myelin basic protein and Klk6 expression. We also developed a novel enzyme-linked immunoabsorbent assay method for detecting activated KLK6 in human CSF. In human autopsy brain samples, expression of active KLK6 was detected in OLs using an antibody that specifically recognizes the protein's activated form. Taken together, our findings demonstrate that Klk6 secreted by OLs plays a critical role in the pathogenesis of EAE/MS and that it might serve as a potential therapeutic target for MS.

Yokukansan Reduces Cuprizone-Induced Demyelination in the Corpus Callosum Through Anti-inflammatory Effects on Microglia.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). The release of inflammatory cytokines and pro-oxidant molecules from microglia has been shown to play a key role in the pathology of MS. Thus, suppression of microglial cell activation is an attractive therapeutic option. Yokukansan, a traditional Japanese herbal medicine, has been shown to suppress microglial activity in the CNS. However, whether or not yokukansan reduces demyelination observed in the CNS during MS remains unknown. In this study, female C57BL/6 mice were fed a diet containing 0.2% cuprizone (bis-cyclohexanone oxaldihydrazone) to induce demyelination in the corpus callosum. We investigated whether or not yokukansan reduces cuprizone-induced demyelination using immunohistochemical analyses. Furthermore, we examined the in vitro anti-inflammatory effects of yokukansan on LPS-stimulated BV2 cells, a murine microglial cell line. Luxol fast blue staining and immunostaining for myelin basic protein demonstrated that yokukansan reduces demyelination of the corpora callosa of cuprizone-fed mice. In addition, yokukansan significantly decreased the number of activated microglial cells in the corpora callosa of cuprizone-fed mice. Furthermore, treatment with 500 µg/ml yokukansan suppressed the expression of interleukin-1ß and inducible nitric-oxide synthase mRNA and protein in LPS-stimulated BV2 cells. These findings suggest that yokukansan reduces demyelination owing to anti-inflammatory effects on microglia. As yokukansan has few adverse effects, yokukansan has the potential to be a novel option to treat MS.

Microglia support ATF3-positive neurons following hypoglossal nerve axotomy.

Microglia are essential in developmental processes and maintenance of neuronal homeostasis. Experimental axotomy of motor neurons results in neurodegeneration, and microglia in motor nuclei become activated and migrate towards injured neurons. However, whether these activated microglia are protective or destructive to neurons remains controversial. In the present study, we transected the hypoglossal nerve in BALB/c mice, causing activating transcription factor 3 (ATF3) and growth associated protein 43 (GAP43) induction, and partial neuronal death. Inhibition of microglial accumulation by minocycline administration impaired microglial accumulation, decreased GAP43 mRNA expression, and reduced motor neuron survival. Expression of ATF3 contributed to nerve regeneration, and increased within 6 h after axotomy, prior to microglial migration. Further, microglial contact with neuronal cell bodies was associated with neuronal ATF3 expression. Colchicine administration blocked lesion-induced ATF3 transcription in axotomized neurons and microglial accumulation. In addition, perineuronal microglia-derived ciliary neurotrophic factor (CNTF) increased, indicating that perineuronal microglia in the hypoglossal nucleus protect axotomized motor neurons by releasing trophic factors. We also observed that microglia secrete CNTF and that neurons have CNTFRα and can respond to it in vitro. CNTF promote neurite elongation and neuronal survival of primary cultured neurons. Microglia make contact through unknown neuronal signals that are possibly regulated by ATF3 in hypoglossal nucleus. Moreover, they play important roles in regenerating motor neurons and are potential new therapeutic targets for motor neuron diseases.

Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.

Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited in vitro myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a dissociated monolayer and feeder-free culture system have the potential to generate oligodendrocyte progenitor cells and mature oligodendrocytes in vitro and in vivo. This culture method could be applied to prepare large amounts of oligodendrocyte progenitor cells and mature oligodendrocytes in a relatively short amount of time.

Important role of kallikrein 6 for the development of keratinocyte proliferative resistance to topical glucocorticoids.

One of the major adverse effects of topical glucocorticoids is cutaneous atrophy often followed by development of resistance to steroids (tachyphylaxis). Previously we showed that after two weeks, interfollicular mouse keratinocytes acquired resistance to anti-proliferative effects of glucocorticoid fluocinolone acetonide (FA). One of the top genes activated by FA during tachyphylaxis was Klk6 encoding kallikrein-related peptidase 6, known to enhance keratinocyte proliferation. KLK6 was also strongly induced by chronic glucocorticoids in human skin. Double immunostaining showed that KLK6+ keratinocytes, localized in suprabasal layer of mouse skin, were frequently adjacent to proliferating 5-bromo-2'-deoxyuridine-positive basal keratinocytes. We used KLK6 knockout (KO) mice to evaluate KLK6 role in skin regeneration after steroid-induced atrophy. KLK6 KOs had thinner epidermis and decreased keratinocyte proliferation. The keratinocytes in wild type and KLK6 KO epidermis were equally sensitive to acute anti-proliferative effect of FA. However, the development of proliferative resistance during chronic treatment was reduced in KO epidermis. This was not due to the changes in glucocorticoid receptor (GR) expression or function as GR protein level and induction of GR-target genes were similar in wild type and KLK6 KO skin. Overall, these results suggest a novel mechanism of epidermal regeneration after glucocorticoid-induced atrophy via KLK6 activation.

Abnormal morphology of myelin and axon pathology in murine models of multiple sclerosis.

Demyelination and axonal damage are responsible for neurological deficits in multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system. However, the pathology of axonal damage in MS is not fully understood. In this study, histological analysis of morphological changes of axonal organelles during demyelination in murine models was investigated by scanning electron microscopy (SEM) using an osmium-maceration method. In cuprizone-induced demyelination, SEM showed typical morphology of demyelination in the corpus callosum of mouse brain. In contrast, SEM displayed variations in ultrastructural abnormalities of myelin structures and axonal organelles in spinal cord white matter of experimental autoimmune encephalomyelitis (EAE) mice, an animal model of MS. Myelin detachment and excessive myelin formation were observed as typical morphological myelin abnormalities in EAE. In addition, well-developed axoplasmic reticulum-like structures and accumulated mitochondria were observed in tortuous degenerating/degenerated axons and the length of mitochondria in axons of EAE spinal cord was shorter compared with naïve spinal cord. Immunohistochemistry also revealed dysfunction of mitochondrial fusion/fission machinery in EAE spinal cord axons. Moreover, the number of Y-shaped mitochondria was significantly increased in axons of the EAE spinal cord. Axonal morphologies in myelin basic protein-deficient shiverer mice were similar to those in EAE. However, shiverer mice had "tortuous" (S-curve shaped mitochondria) and larger mitochondria compared with wild-type and EAE mice. Lastly, analysis of human MS patient autopsied brains also demonstrated abnormal myelin structures in demyelinating lesions. These results indicate that morphological abnormalities of myelin and axonal organelles play important role on the pathogenesis of axonal injury in demyelinating diseases.

Interferon regulatory factor 7 participates in the M1-like microglial polarization switch.

Microglia are generally considered the immune cells of the central nervous system. Recent studies have demonstrated that under specific polarization conditions, microglia develop into two different phenotypes, termed M1-like and M2-like microglia. However, the phenotypic characteristics of M1-like- and M2-like-polarized microglia and the mechanisms that regulate polarization are largely unknown. In this study, we characterized lipopolysaccharide-treated M1-like and IL-4-treated M2-like microglia and investigated the mechanisms that regulate phenotypic switching. The addition of M2-like microglial conditioned medium (CM) to primary neurons resulted in an increase in neurite length when compared with neurons treated with M1-like microglial CM, possibly because of the enhanced secretion of neurotrophic factors by M2-like microglia. M1-like microglia were morphologically characterized by larger soma, whereas M2-like microglia were characterized by long processes. M2-like microglia exhibited greater phagocytic capacity than M1-like microglia. These features switched in response to polarization cues. We found that expression of interferon regulatory factor 7 (IRF7) increased during the M2-like to M1-like switch in microglia in vitro and in vivo. Knockdown of IRF7 using siRNA suppressed the expression of M1 marker mRNA and reduced phosphorylation of STAT1. Our findings suggest that IRF7 signaling may play an important role in microglial polarization switching.

Mechanisms of remyelination: recent insight from experimental models.

Oligodendrocytes and myelin play essential roles in the vertebrate central nervous system. Demyelination disrupts saltatory nerve conduction, leading to axonal degeneration and neurological disabilities. Remyelination is a regenerative process that replaces lost myelin. However, remyelination is disrupted in demyelinating diseases such as multiple sclerosis, at least partially, due to the failure of oligodendrocyte precursor cells to differentiate into myelinating oligodendrocytes. Understanding the molecular and cellular mechanisms that impact the differentiation of oligodendrocytes and myelination may help in the development of novel therapeutic strategies for demyelinating diseases. In this review, we focus on the molecular mechanisms controlling the differentiation of oligodendrocytes during remyelination, and we discuss the function of astrocytes and microglia in animal models of demyelinating diseases.

Local mineralocorticoid receptor activation and the role of Rac1 in obesity-related diabetic kidney disease.

BACKGROUND/AIMS: Obesity and diabetes are intimately interrelated, and are independent risk factors for kidney disease. Overactivation of mineralocorticoid receptor (MR) is implicated in end organ damage of both pathologies. But the underlying mechanism of MR activation in kidney remains uncertain. We explored the involvement of Rac1, which we previously identified as a ligand-independent MR activator, in renal MR activation in vitro and in vivo. METHODS: We evaluated the MR activity and Rac1 activity under high-glucose stimulation using luciferase reporter system and glutathione S-transferase pull-down assay in cultured mesangial cells. To elucidate the role of Rac1 in vivo, we employed KKA(y), a mouse model of obesity-related type 2 diabetes, which spontaneously developed massive albuminuria and distinct glomerular lesions accompanied by increased plasma aldosterone concentration. RESULTS: High-glucose stimulation increased Rac1 activity and MR transcriptional activity in cultured mesangial cells. Overexpression of constitutively active Rac1 activated MR, and glucose-induced MR activation was suppressed by overexpression of dominant negative Rac1 or Rac inhibitor EHT1864. In KKA(y), renal Rac1 was activated, and nuclear MR was increased. EHT1864 treatment suppressed renal Rac1 and MR activity and mitigated renal pathology of KKA(y) without changing plasma aldosterone concentration. CONCLUSION: Our results suggest that MR activation plays an important role in the nephropathy of KKA(y) mice, and that glucose-induced Rac1 activation, in addition to hyperaldosteronemia, contributes to their renal MR activation. Along with MR blockade, Rac inhibition may potentially be a preferred option in the treatment of nephropathy in obesity-related diabetic patients.

Renin inhibition ameliorates renal damage through prominent suppression of both angiotensin I and II in human renin angiotensinogen transgenic mice with high salt loading.

BACKGROUND: The renin-angiotensin-aldosterone system (RAAS) plays pivotal roles in the pathogenesis of chronic kidney disease (CKD) progression. Aliskiren, a direct renin inhibitor, inhibits the rate-limiting step of the RAAS without any alternative pathway. It is proven to reduce albuminuria in CKD patients treated with angiotensin blockade. However, there are few reports which evaluate the advantage of aliskiren as the first-line drug against CKD progression in RAAS-activated hypertensive patients. METHODS: Tsukuba hypertensive mice (THM), double transgenic mice carrying both the human renin and human angiotensinogen genes, were fed a high-salt diet and treated with hydraladine, ramipril and aliskiren for 10 weeks. Blood pressure and urinary albumin excretion were measured every 2 weeks during the experimental period. We evaluated renal histological changes and gene expression. Plasma angiotensin concentration was measured to evaluate the RAAS inhibitory effect. RESULTS: High-salt-loaded THM showed severe hypertension and renal injury. All antihypertensive drugs suppressed blood pressure and prevented renal disease progression. RAAS blockade showed a higher renoprotective effect than hydraladine despite an equivalent blood pressure lowering effect. Aliskiren exhibited even stronger renoprotection than ramipril. Plasma angiotensin concentration was increased in THM fed both normal salt and high salt. Hydraladine did not alter the plasma angiotensin concentration. Ramipril significantly decreased angiotensin II concentration. Aliskiren treatment almost completely suppressed angiotensin I and resulted in lower angiotensin II concentration than ramipril treatment. CONCLUSION: Aliskiren prevents renal disease progression by suppressing both angiotensin I and II in RAAS-activated pathology. Our data suggest the application of a renin inhibitor for preventing kidney disease progression in CKD patients.

Differences in Kallikrein-related peptidase mRNA sequences and KLK6 enzyme activity between mouse strains.

Several studies have reported differences in physiological and pathological phenotypes between different strains of experimental mice, such as environment-based behavior, skin damage, damage in response to toxins and nervous system injury. However, the mechanisms underlying these differences have not yet been fully elucidated. We have been studying the function of kallikrein-related peptidases (KLKs), serine proteases known to serve a variety of functions. In this study, we focused on differences in KLKs between C57BL/6 mice and 129 mice. Among 13 KLKs genes examined, 12 KLKs showed differences in the mRNA coding region sequence and 7 KLKs showed different deduced amino acid sequences of their proteins when comparing C57BL/6 and 129 mice. KLK6 protein from 129 mice had six amino acid differences compared with that from C57BL/6 mice. KLK6 protein from 129 mice showed reduced SDS-PAGE mobility compared with that from C57BL/6 mice. Moreover, recombinant KLK6 protein from 129 mice had a higher optimum pH and >15 times higher hydrolytic enzymatic activity for several substrates than that from C57BL/6 mice. These results suggest that KLKs may contribute to the genetic basis of the differences between mouse strains.

Minocycline reduces remyelination by suppressing ciliary neurotrophic factor expression after cuprizone-induced demyelination.

Remyelination is disrupted in demyelinating diseases such as multiple sclerosis, but the underlying pathogenetic mechanisms are unclear. In this study, we employed the murine cuprizone model of demyelination, in which remyelination occurs after removal of the toxin from the diet, to examine the cellular and molecular changes during demyelination and remyelination. Microglia accumulated in the corpus callosum during weeks 2-4 of the cuprizone diet, and these cells remained activated 2 weeks after the change to the normal diet. To examine the role of microglia in remyelination, mice were treated with minocycline to inactivate these cells after cuprizone-induced demyelination. Minocycline treatment reduced the number of CC1-positive oligodendrocytes, as well as levels of myelin basic protein (MBP) and CNPase in the remyelination phase. The expression of CNTF mRNA in the corpus callosum increased after 4 weeks on the cuprizone diet and remained high 2 weeks after the change to the normal diet. Minocycline suppressed CNTF expression during the remyelination phase on the normal diet. Primary culture experiments showed that CNTF was produced by microglia in addition to astrocytes. In vitro, CNTF directly affected the differentiation of oligodendrocytic cells. These findings suggest that minocycline reduces remyelination by suppressing CNTF expression by microglia after cuprizone-induced demyelination.

Three-dimensional ultra-structures of myelin and the axons in the spinal cord: application of SEM with the osmium maceration method to the central nervous system in two mouse models.

Axonal injury and demyelination are observed in demyelinating diseases such as multiple sclerosis. However, pathological changes that underlie these morphologies are not fully understood. We examined in vivo morphological changes using a new histological technique, scanning electron microscopy (SEM) with osmium maceration method to observe three-dimensional structures such as myelin and axons in the spinal cord. Myelin basic protein-deficient shiverer mice and mice with experimental autoimmune encephalomyelitis (EAE) were used to visualize how morphological changes in myelin and axons are induced by dysmyelination and demyelination. SEM revealed following morphological changes during dysmyelination of shiverer mice. First, enriched mitochondria and well-developed sER in axons were observed in shiverer, but not in wild-type mice. Second, the processes from some perinodal glial cells ran parallel to internodes of axons in addition to the process that covered the nodal region of the axon in shiverer mice. Last, this technique left myelin and axonal structures undisturbed. Moreover, SEM images showed clear variations in the ultrastructural abnormalities of myelin and axons in the white matter of the EAE spinal cord. This technique will be a powerful tool for identifying the mechanisms underlying the pathogenesis in demyelination.