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Heterogeneity of spinal cord pathology in multiple sclerosis and variants: A study of postmortem specimen from 13 Asian patients

Zhenxin LI, Chuanzhen LU, Yin WANG, *Yoshio HASHIZUME, *Mari YOSHIDA

Department of Neurology, Huashan Hospital, Shanghai, China; *Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Japan

Abstract

The spinal cord is a predominant site of lesions in multiple sclerosis (MS) and its variants, particularly in Asian populations. We investigated postmortem spinal cords obtained from 4 patients with acute MS, 9 patients with chronic MS and 4 controls. A total of 387 spinal cord segments were examined to study the histopathology of myelin, axons, glia, and immune reactions with routine histopathological stains and immunohistochemistry. The cross-sectional areas of the spinal cord and demyelinating plaques were measured and correlations with disease duration were sought. We found a high incidence of spinal cord lesions with highly heterogeneous pathology, including diverse demyelinating plaques and necrosis in acute patients, and Wallerian degeneration in chronic patients. The cervical and upper thoracic cords most frequently had demyelinating plaques in the lateral and dorsal columns and in the dorsal horn with various topographic patterns. In acute patients, coagulation necrosis and edema with various topographic patterns. In acute patients, coagulation necrosis and edema. In acute patients, coagulation necrosis and edema In acute patients, coagulation necrosis and edemaoagulation necrosis and edemaand edema were predominant. �e�ere a�ona�� in��r�� wit�� in�on�i�tent dem��e��ination and imm�ne �e�� in��tration�e�ere a�ona�� in��r�� wit�� in�on�i�tent dem��e��ination and imm�ne �e�� in��tration was detected in the swollen spinal cords. In chronic patients, the average cross-sectional area wasIn chronic patients, the average cross-sectional area washe average cross-sectional area was negatively correlated to disease duration. The proportion of demyelinating lesions correlated with disease duration in the cervical cord. The load of cervical cord lesions is a reliable indicator of the progression of MS. Axonal loss and disorganization were the crucial elements responsible for spinal cord atrophy. We concluded that there are multiple causes of axonal injury that should be considered in future therapeutic strategies. Our observations provide further knowledge of the morphology of spinal cord lesions in MS and variants among Asians.

Neurology Asia 2006; 11 : 111 – 121

Address correspondence to: Dr Li Zhenxin, Department of Neurology, Huashan Hospital, 32 Wulumuqi Road, 200040 Shanghai, P. R. China. Tel.: +86-21-62489999, Fax: +86-21-62483421 E-mail: [email protected]

INTRODUCTION

Multiple sclerosis (MS) is the most common idiopat��i� inflammator�� dem��e��inating di�ea�e of the central nervous system (CNS). The classic form of MS usually follows a relapsing and remitting course at the initial stage and then progresses to permanent disability. In addition, rare forms (neuromyelitis optica, acute MS, and �on�entri� ��ero�i�) are identi��ed a� M� �ariant� because they share a wide range of common histopathological features such as the presence of multiple perivenous loss of normal myelin, relative preservation of axons, and perivascular ��ff� of inflammator�� e��.1-3 Demyelinating plaques occur mainly in periventricular white matter, brainstem, optic nerves, and spinal cord.2,4,8 Spinal cord is the predominant site of lesions in MS. Dysfunctions such as severe paralysis, spasticity, chronic pain, and sphincter disturbance are attributed to spinal cord lesions.5-8 Selective

attack of the spinal cord and optic nerve, with a relative sparing of the remainder of the CNS, causes a distinct form of demyelinating disease known as Devic disease or neuromyelitis optica (NMO). NMO is commonly believed to be a variant of MS.9,10 The spinal cord lesions in NMO are likely to be necrotic and cystic, frequently extending over several segments in a continuous fashion, which indicates distinct underlyinges distinct underlying distinct underlying immunopathogenesis from prototypic MS.11-13 Studies from Japan and other Asian countries have consistently found an incidence of either NMO or even spinal cord involvement in MS that is much higher than that reported in Western countries.14-18

MRI studies have found that spinal cord studies have found that spinal cordtudies have found that spinal cord abnormalities are present in 80% to 90% of MS patients19-21, even in patients without spinal cord symptoms.22 The importance of spinal cordThe importance of spinal corde importance of spinal cord atrophy and axonal damage in the development ofand axonal damage in the development of in the development of

Neurology Asia December 2006

112

permanent f�n�tiona�� de��it ��a� �een ��ig��ig��teds been highlighted highlighted repeatedly..23-27 The association between clinical status and the extent of abnormalities found in spinal cord by MRI, however, is generally poor �e�a��e of ��imited ��i�topat��o��ogi�a�� pe�i��it�� and the complexity of pathogenesis.28,29 Thus, there is a pre��ing need to identif�� pe�i�� i�topat��o��ogi�a�� features corresponding to abnormalities of spinal cord found by MRI for the different pathogenic mechanisms that may be involved in MS and variants. We made an extensive histopathological examination of 13 postmortem spinal cords and provide a detailed account of demyelination, inflammation, and parti��ar��, a�ona�� pat��o��og�� in MS and variants.

METHODS

Subjects

We studied the early postmortem tissues of 13 patients (age at death: 10 to 74 years, mean: 54.3 years; 8 women and 5 men) with MS or MS variants. The tissues were obtained from the Institute for Medical Science of Aging, Aichi Medical University, Japan, and the Institute of Neurology, Fudan University, China. Clinical and pathological background data are summarized in Table 1. In this series, spinal symptoms or signs were almost invariably present in the natural courses of disease of our patients and were identi��ed in 9 patient� a� t��e predominant feat�re of the disease. Single or bilateral involvement of the optic nerve was seen in 10 patients and �rain �tem in�o��ement in 9 patient�. T��e ��na�� clinicopathological diagnoses of the 13 patientsdiagnoses of the 13 patients were: secondary progressive MS (n=9), Devic-type MS (n=2), and acute MS (n=2). The patients were placed in the acute group (n=4) or chronic group (n=9) according to disease duration (17 days to 26 years). Four control samples from patients without evidence of neurological disease or neuropathological abnormalities were included in this study (Table 1).

Neuropathology

All tissues were acquired at autopsy within 8 hours after deat�� and were ��ed in �� forma��de��de20% formaldehyde% formaldehyde for over 2 weeks. Specimens from the cervical, thoracic, lumbar and sacral cords of the 13 patients and 4 controls were dissected transaxially into 5-mm segments. Representative cord segmentsRepresentative cord segments cord segments were em�edded in paraf��n. �eria�� 8-µm thick slices were stained for general histopathology with haematoxylin and eosin (H-E), for myelin with Luxol fast blue and cresyl violet (Klüver-

Barrera), and for axons with silver impregnation (Bodian).

Immunohistochemistry

Immunohistochemical staining was done on adjacent serial sections with a standard labeled strept-avidin-biotin (LSAB) procedure and a DAB developing system. Sections were deparaffinized and hydrated gradually. For amyloid beta-precursor protein (β-APP) staining, sections were immersed in 70% formic acid for 20 min at room temperature, followed by a 5-min washing with distilled water. All sections were pre-incubated in 3% H2O2 menthanol for 30 min to block endogenous hydrogen peroxide and then rinsed in 0.01% phosphate buffered saline (PBS). Primary antibodies were applied against the following targets: β-APP (Mouse anti-Amyloid beta-Precursor Protein, Zymed La�oratorie�, In�., U�A), ne�ro��ament (Mo��e anti-��kDa ne�ro��ament protein, DAKO), GFAP (Rabbit anti-GFAP, DAKO), HLA-DP, DQ, DR (Mouse anti-HLA-DP, DQ, DR, DAKO), CD68 (Mouse anti-CD68/KP1, DAKO), CD20 (Mouse anti-CD20/L26, DAKO), CD45RO (Mouse anti-CD45RO/UCHL1, DAKO), and CD8 (Mouse anti-CD8, DAKO). Non-immune serum from isotype-matched species, and only PBS without primary antibody, were applied as negative control reagents.

Morphometry and statistical analysis

Sections of cord segments stained by Luxol fast blue and cresyl violet were used for quantitative analysis. Digital images were captured by directly scanning sections at identical resolution (300 dpi) with a laser scanner attached to an Apple Power Mac PC. The cross-sectional area of each section from patients and controls was measured automatically with NIH Image software. The area occupied by demyelinating plaques was also measured in sections containing plaques, and the percentage of the total area was calculated. Student t-test was applied for comparisons. Correlations were assessed by regression analysis. Te�t� were ��a��i��ed a� �igni��ant if t��e P value was < 0.05.

RESULTS

Morphology of the spinal cord in MS and variants

Severe swelling throughout the length of the spinal cord was found in the cords from 3 patients

113

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114

(patients 1 to 3) with an acute monophasic course, whereas shrinkage to various degrees appeared in the spinal cords from 9 patients with secondary progressive courses, with the most severe instance in patient 6. Microscopically, three main pathological patterns (demyelination, necrosis or cavities and Wallerian degeneration) were observed in the cord segments from the 13 patients. The number of cord segments containing different types of lesions is listed in Table 2. Demyelinating plaques disseminated in 130 of 298 (43.6%) cord segments from 9 patients (3 acute, 6 chronic). Fifty-six of 82 (68%) cervical segments and 62 of 127 (49%) thoracic segments were affected by demyelination, whereas only 17.5% of lumbar and 0.6% of sacral segments were affected. Demyelinating plaques were distributed randomly in the lateral, dorsal, and anterior columns, the dorsal, anterior, and lateral horns, and the central canal, with frequencies shown in Figure 1�� The lateral columns (109 of 130 segments) were most frequently occupied by demyelinating plaques followed by dorsal columns (105 of 130 segments) and dorsal horns (100 of 130 segments). Demyelinating plaques in the thoracic segments were often located around the central canal (38 of 130 segments) and protruded into the lateral horn (29 of 130 segments). Subpial demyelinating plaques, having a wedge-��ike or ring-��ike pro��e, were fo�nd in 42 of 130 segments from 3 patients (patients 1, 4, 13). Typical hemi-transverse and nearly entire transverse demyelinating plaques were seen only in 6 segments from the upper thoracic cord of patient 1. Adjacent plaques often merged into a coalesced plaque with irregular boundary, around which some isolated plaques were scattered. Demyelinating plaques in different segments from the same patient were of similar stage. Active demyelinating plaques (Figure 2)2)) typically contained Luxol fast blue-positive myelin debris, and abundant macrophages, with HLA-DP, DQ, DR and CD68 immunoreactivity (Figure 2B, 2H),2B, 2H),B, 2H), were found in 125 of 130 segments from 3 acute3 acute and 6 chronic patients. Early active demyelinatingpatients. Early active demyelinating p��aq�e� were ��ig�� edemato�� and i��-de��ned (Figure 2A); perivascular and parenchyma2A); perivascular and parenchymaA); perivascular and parenchyma in��tration �� mp��o��te� wa� in�on�pi��o�� in the 4 acute patients. Macrophages were present. In the chronic group, active demyelinating plaques were also common, but the edema was moderate and �ommon�� on��ned to t��e margin (Figure 2E). The number and composition of2E). The number and composition ofE). The number and composition of perivascular lymphocytes were highly variable among patients. Furthermore, the distribution of

peri�a��ar in��tration wa� far �roader t��an t��e dem��e��inated area. Ina�ti�e p��aq�e�, de��ned ��, de��ned ��de��ned �� inten�e�� ro�� a�trog��io�i� and in�on�pi��o��and inconspicuous in��tration of inflammator�� e��, were �een in ��inflammator�� e��, were �een in �� cells, were seen in 20were seen in 20 of 130 segments in 3 patients. Coagulation necrosis and cavities (Figure 3)3)) developed in 64 of 298 segments (21.5%) from 3 patients with acute progressive courses and 1 secondary progressive MS patient with a malignant progress subsequent to an initial relapsing and remitting course. In acute patients, the necrotic lesion extended over a long length, even over theover a long length, even over theover thethe entire spinal cord in patient 2 and cavities formed spinal cord in patient 2 and cavities formed in patient 2 and cavities formed and cavities formedand cavities formed often in the lower part of the cord (Figure 3A, 3B). The normal architecture of the spinal cord in cross section was damaged, and the neuropil disintegrated into small fragments, leading to an intensely eosinophilic appearance. Wallerian degeneration in the lateral and dorsal columns, where the long ascending and descending tracts pass, was observed in 112 of 298 segments (37.6%). Lesions in lateral columns (Figure 3C) were more frequent than in dorsal columns (Figure 3D, 3E, 3F), which were detected only in patient 8. Usually, the lesions were symmetrical, but in patient 9, a unilateral Wallerian degeneration inWallerian degeneration inallerian degeneration in the lateral column was observed..

Axonal pathology

Axonal abnormalities varied in the demyelinating plaques at different stages and included axonal ovoids, axonal disorganization, and axonal loss. In early active plaques, demyelinated axons had an increased diameter and reduced argyrophilia (Figure 2C). Abundant axonal ovoids with intense2C). Abundant axonal ovoids with intenseC). Abundant axonal ovoids with intense β-APP immunoreactivity were seen (Figure 2D).2D).D). In chronic active plaques, the normal arrangement of axons was destroyed and remodeled to form a me��work pro��e into w��i�� n�mero�� macrophages invaded (Figure 2I). Argyrophilic2I). ArgyrophilicI). Argyrophilic o�oid�, ��a�ing inten�e ne�ro��ament ��t mi��d β-APP immunoreactivity (Figure 2J), were as2J), were asJ), were as frequent as in acute plaques. Conspicuous loss of a�on�, in parti��ar �ma�� er�, wa� dete�ted in chronic inactive plaques. Axons were broken down into debris with a weak argyrophilic nature and de�rea�ed ne�ro��ament imm�norea�ti�it�� in the necrotic areas. In the area of Wallerian degeneration, the reduction of axon density was noticeable (Figure 3E��. The changes of axonal arrangement and argyrophilic nature were unremarkable.

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Correlation of cross-sectional area and demyelinating area

T��ere were no �igni��ant �orre��ation� �etween age at death and average cross-sectional area. Compared to controls, the average cross-sectional area of cervical and thoracic segments increased �igni��ant�� in t��e a��te gro�p (P < 0.01). In the chronic group, however, the average cross-�e�tiona�� area wa� red��ed �igni��ant�� in t��e cervical, thoracic, and lumbar cords (P < 0.01) but not in the sacral cord (Figure 4A). The extent of atrophy in either cervical or thoracic cord as refle�ted �� t��e a�erage �ro��-�e�tiona�� area correlated negatively with the disease duration (Figure 4B). The total area of demyelination as a percentage of the cross-sectional area in each segment varied from 2.4% to 98.6%, with an average of 33.2%. In 6 patients from the chronic group, the disease duration correlated well with the proportion of demyelinating lesions in the cervical cord but not in the thoracic cord (Figure 4C). The total area of demyelinating lesions in t��e �pina�� ord ��ad no �igni��ant �orre��ation wit�� the disease duration and no correlation with the average cross-sectional area of the spinal cord.

DISCUSSION

Our study found a high prevalence of spinal cord involvement in both MS and variants that corresponds to findings from early clinicopathological and neuroimaging studies.19-

21,30 Coexistence of single or bilateral optic nerve and optic chiasm lesions was frequent in our study, which is consistent with clinicopathological features in Asian patients.14-16,31,32 Furthermore, it was common that parts of the CNS rather than spinal cord and optic nerve were compromised, among which the brain stem was the most frequent site. A recent study in Japan33 found that 31% of Japanese patients with MS had optic-spinal in�o��ement, ��t on�� 8.5�� ati��ed t��e �riteria for the pure optic-spinal variant of MS (normal magnetic resonance scan of the head, apart from optic nerve abnormalities, on repeated examination, and 5 or more years of follow-up). Pure spinal cord and optic nerve involvement is also rare in Asian patients, particularly in patients with long disease duration. However, in our study, demyelinating lesions were limited to the spinal cord in patients 4 and 13 in our series. Highly he te rogeneous pa thologica l changes, derived from different combinations of demyelination, degeneration, necrosis,


116

Figure 1: (A) Diagram of the topographic pattern of demyelinating plaques on a cross-section of the spinal cord. (B) Frequency distribution histogram of various anatomic structures occupied by demyelinating plaques. LC: lateral column; DC: dorsal column; DH: dorsal horn; AC: anterior column: AH: anterior horn; CC: central canal; LH: lateral horn; T, Transverse; Hemi-T: hemi-transverse.

inflammation, and glial reactions, were disseminated over a wide area of spinal cord, and were markedly different among patients. A variety of patterns of topographic distribution have been described in the present study and in earlier morphology reports8, and the consistent point is that the cervical and thoracic cord is usually involved, whereas the lumbar and sacral cord is only affected occasionally. Structures such as the lateral or dorsal columns, where the long ascending and descending tracts cluster, are the most vulnerable to demyelination.20,34 A high incidence of demyelination in grey matter was found in our study, particularly in the dorsal horns, which are recognized as crucial areas for afferent nociceptive stimuli. The lesions in the dorsal horns may be responsible for numerous sensory symptoms in MS patients. Despite extensive involvement of white and grey matter, most motor and sensory disturbances, as initial symptoms, occurred asymmetrically even in acute patients. With progression of the disease, paraparesis usually develops in an ascending dire�tion pro�a�� refle�ting t��e a��m��ation of spinal cord lesions. Bladder and bowel disturbances usually develop at the stage of permanent disability. Therefore, we assume that the occurrence of spinal cord lesions in MS is usually isolated and asynchronous at the very beginning and accumulates as the disease progresses. Coagulation necrosis of the spinal cord was common not only in patients with Devic-type

MS but also in other acute MS patients and even occurred in a patient with a chronic course. The remainder of the CNS, however, had inconspicuous necrosis but extensive demyelination in some patients. Coagulation necrosis may be just a consequence of the “bystander” effect of inflammation. In addition, because inherent limitation of the pia matter and vertebral canal makes the spinal cord vulnerable to the edema that always accompanied acute demyelination, the increased intramedullary pressure could have caused a series of injuries, such as circulatory disturbances and mechanical compression that leads to necrosis. Comparative studies of MRI records and histopathology35,36 have found a very good correlation of the abnormal focal signal intensity with demyelination. Recent MRI studies of MS patients have highlighted the potential importance of spinal cord atrophy and axonal loss in the development of disability.34-38 The morp��o��ogi�a�� nding� in o�r �t�d�� o��d �e helpful for interpreting in vivo MRI results. Our �tati�ti�a�� ana��i� ��a� ��own t��at �igni��ant spinal atrophy occurred not only in the cervical but also in the thoracic region in the chronic group and correlated well with the disease duration. At least 3 factors may be responsible for spinal cord atrophy: aging, demyelinating load, and axonal pathology. One study of spinal cord aging found a negative correlation between age and atrophy.39 In o�r �t�d��, no �igni��ant �orre��ation wa� fo�nd between age at death and the average cross-

117

Figure 2: Active demyelinating plaques in the spinal cord. (A) Early active plaque from anterior column of cervical cord of patient 3 had myelin vacuolation, an inconspicuous perivascular cuff of lymphocytes, �e�ere edema, and i��-de��ned margin�. (K��ü�er-Barrera �tain, magni��ation ×100) (B) Over-abundant macrophages immunoreactive for HLA-DR, DP, DQ in the edematous lesion parenchyma. (LSAB stain, magni��ation ×100) (C) Demyelinated axons with swellings and ovoids and reduced argyrophilia are apparent in t��e ear�� a�ti�e p��aq�e. (Bodian �tain, magni��ation ×100) (D) Numerous swollen axons and ovoids immunoreactive for β-APP. (L�AB �tain, magni��ation ×100) (E) Active plaque in chronic M� from t��e dor�a�� o��mn of t��e t��ora�i� �ord of patient 5. Peri�a��ar ��ff of inflammator�� e�� and myelin vacuolation are predominantly at the edge of the demyelinating plaque. (Klüver-Barrera �tain, magni��ation ×1��) (F) In��trating inflammator�� e�� aro�nd �e��e�� were main�� CD8+ T-�e��. (L�AB �tain, magni��ation ×��) (G) A��mo�t no CD4+ T-�e�� in��trate into t��e ��e�ion in patient 5. 5.. (L�AB �tain, magni��ation ×100) (H) Macrophages with CD68 immunoreactivity also apparently cluster in t��e ��roni� a�ti�e p��aq�e. (L�AB �tain, magni��ation ×100) (I) Disarrangement of axons forming a me��work. (Bodian �tain, magni��ation ×100) (J) A minority of axonal ovoids have immunoreactivity for β-APP in ��roni� a�ti�e p��aq�e. (L�AB �tain, magni��ation in ��roni� a�ti�e p��aq�e. (L�AB �tain, magni��ation ��roni� a�ti�e p��aq�e. (L�AB �tain, magni��ation a�ti�e p��aq�e. (L�AB �tain, magni��ation�ti�e p��aq�e. (L�AB �tain, magni��ation ×100)..


118

Figure 3: Cavities, coagulation necrosis, and Wallerian degeneration. (A) Lumbar cord from patient 1 with transverse coagulation necrosis and a round cavity on the right side. (Klüver-Barrera stain) (B) Intensely eo�inop��i��i� ne�ropi�� aro�nd t��e �ame �a�it��. (H-E �tain, magni��ation ×40) (C) Wallerian degeneration in the bilateral lateral columns is common in chronic patients as seen in the lumbar cord of patient 7 (indicated byindicated by *). (Klüver-Barrera stain) (D) In patient 6, Wallerian degeneration is seen in the dorsal. (Klüver-Barrera stain) (D) In patient 6, Wallerian degeneration is seen in the dorsal column of the cervical cord (indicated by(indicated byindicated by *), wit�� i�o��ated and �onfl�ent dem��e��inating p��aq�e� in t��e, wit�� i�o��ated and �onfl�ent dem��e��inating p��aq�e� in t��e cross section. (Klüver-Barrera stain) (E) The density of axons in the region of Wallerian degeneration is remarkably reduced compared to the adjacent normal white matter (indicated by(indicated byindicated by *). (Bodian stain,. (Bodian stain, magni��ation ×40) (F) Myelin loss also occurs in the region of Wallerian degeneration with a lack of in��tration �� inflammator�� e��. (K��ü�er-Barrera �tain; magni��ation ×40)

sectional area. A possible explanation for this discrepancy is that the initial symptoms in most of the patients in our study began at 40 to 60 years of age. Thus age-related atrophy may have made a mild contribution to spinal cord atrophy. A second assumption was that because demyelinating plaques lead to the remodeling of spinal cord architecture and accumulate with progress of the disease, spinal cord atrophy occurred because of

extensive loss of myelin and astrogliosis. Our statistical analysis, however, found no relation between the total demyelinated area and spinal �ord atrop�� a� refle�ted �� t��e a�erage �ro��-sectional area in chronic MS, although the area of demyelinating plaques correlated with disease duration in the cervical cord. The demyelinating load in the spinal cord made no contribution to the shrinkage of the spinal cord, but in the cervical

119

Figure 4: (A) Comparison of the average cross-sectional areas between acute and chronic groups and controls. �igni��ant differen�e� (P < 0.01) exist only in the cervical and thoracic cord, with an increased cross-�e�tiona�� area in t��e a��te gro�p. In t��e ��roni� gro�p, a �igni��ant red��tion of a�erage �ro��-�e�tiona�� area occurs in the cervical, thoracic and lumbar cord (P < 0.01). (B) Negative correlation between the average cross-sectional area and disease duration exists in cervical (Rsqr. = 0.505, P = 0.032) and thoracic cord (Rsqr. = 0.512, P = 0.030) in the chronic group. (C) Correlation of disease duration with total demyelinated area as a fraction of the total cross-sectional area in 6 patients of the chronic group (Rsqr. = 0.939, P = 0.0014).


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�ord t��e ��oad refle�ted t��e progre��ion of di�ea�e, as expected from the results of MRI studies.19,26,36 Hence, axonal pathology was the most likely cause of spinal cord atrophy. This conclusion ��a� �een �on��rmed repeated�� po�tmortem reviews, animal models, and MRI studies.23,24,37,38 Permanent impairment of neurological functions correlates best with axonal rather than myelin loss. Our study of acute patients supports and extends the established consensus that acute axonal damage identi��ed �� β-APP immunoreactivity occurs in the very early stage of the disease23,40,41 even before demyelinating plaques are formed, with a paucity of immune cells. A study of patients with acute demyelination mimicking a tumor has fo�nd a �imi��ar pro��e in t��e �rain.42 Dissociation of axonal injury, demyelination, and immune �e�� in��tration �ontradi�t� t��e genera�� e��ief t��at axonal injury is a sequel to demyelination and immune attack orchestrated by T cells (mainly CD4). Given that acute patients generally have marked swelling, necrosis, and even cavities in the spinal cord, it is reasonable that axonal damage in t��e �pina�� ord i� independent of inflammation and demyelination. The inherent anatomic features of the spinal cord and vertebral canal make the spinal cord more vulnerable to compressive and ischemic insults than other parts of the CNS. Therefore, heterogeneous pathogenesis of axonal damage in the spinal cord has to be considered, particularly at the acute stage. Furthermore, since axonal damage precedes demyelination, it can be postulated that axonal injury triggers demyelination, so-called Inside-Out model.42 The degree of secondary demyelination may depend on the extent of acute axonal damage and the period over which it occurs. The morphological similarity between spinal cord injury and spinal cord lesions of MS as well as studies of the experimental allergic encephalomyelitis model, have provided evidence for the Inside-Out process.43 In the chronic patients we studied, although the demyelinating plaques contained only an occasional β-APP immunoreactive a�on, t��e e�i�ten�e of ne�ro��ament-po�iti�e abnormal axons, remodeled axonal architecture, and �igni��ant a�on ��o�� indi�ated t��at a�ona�� damage persisted throughout the course of the disease. Axonal regeneration would always be truncated due to the spatial limitations and alterations of the microenvironment in the spinal cord. Thus, any attempt at remyelination would be aborted. How to prevent repeated insults to axons and promote axonal regeneration are the

keys for effective remyelination. Establishment of favorable microenvironment suitable for axonal growth and preservation of the matrix architecture are also essential.

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