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Immunohistological study of globoid cell leukodystrophy
Brain & Development 24 (2002) 284–290 www.elsevier.com/locate/braindev
Original article
Immunohistological study of globoid cell leukodystrophy Masahiro Itoh a, Masaharu Hayashi a,*, Yasunori Fujioka b, Kazuo Nagashima b, Yoshio Morimatsu a, Haruo Matsuyama a a
Department of Clinical Neuropathology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashi-dai, Fuchu-shi, Tokyo 183-8526, Japan b Department of Pathology, Hokkaido University School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo 060-8638, Japan Received 20 December 2001; received in revised form 15 March 2002; accepted 5 April 2002
Abstract We examined three autopsy cases of globoid cell leukodystrophy (GLD) with different survival, using immunohistochemistry and in situ nick end labeling (ISEL). The white matter lesion was pronounced in the corona radiata, corpus callosum and cerebellar peduncles in three cases, where a spongy state developed, in addition to the neuronal loss in the thalamus, cerebellum and inferior olivary nucleus. Ramified microglia, being immunoreactive for ferritin and HLA-DR alpha, were scattered in the white matter, and some of them also had immunoreactivity for TNF-alpha. Both the small-sized and large-sized globoid cells showed immunoreactivity for ferritin KP-1 and NCAM, while some of the small-sized globoid cells were also immunoreactive for HLA-DR alpha and TNF-alpha. As the survival became longer, the occurrence of the globoid cells decreased, however, they were commonly observed in the corpus callosum and cerebellar peduncle in three cases. T lymphocytes immunoreactive for LCA, UCHL-1 and CD3 were increased around the vessels in the white matter. ISEL stained nuclei of mononuclear cells in the white matter in two cases with short survival, although the cell origin was not verified. ISEL also visualized a few nuclei of the small-sized globoid cells in one case. On the other hand, immunostainings against cell death proteins such as bcl-2 family members and p53 failed to identify any significant changes. These data suggest that the immunological step and to a lesser extent the apoptotic process may partly be involved in the myelin breakdown and glial pathology in GLD, as reported in the twitcher mouse, a murine model of GLD. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Apoptosis; Globoid cell; Immune reaction; Krabbe disease; Leukodystrophy
1. Introduction Globoid cell leukodystrophy (GLD), also called Krabbe disease, is a progressive heredodegenerative disease caused by deficiency of the lysosomal enzyme galactocerebrosidase [1]. Psychosine (galactosylsphingosine), a toxic metabolite of galactocerebroside, accumulates in GLD causing degeneration of the myelin-producing cells, oligodendrocytes and Schwann cells [1]. Pathological features of the disease include severe axonal loss, myelin degeneration, astrocyte gliosis and formation of periodic acid schiff (PAS)-positive multinucleated giant cells, globoid cells, containing a granular cytoplasm filled with filamentous inclusion and fibrillated bodies [1,2]. The exact pathogenesis of myelin breakdown and the formation of globoid cells remains to be investigated, although immunological responses and apoptosis are speculated to be involved in the demyelination and/or oligodendrocyte depletion in the twitcher mouse, an * Corresponding author. Tel.: 181-42-325-3881, ext. 4703; fax: 181-42321-8678. E-mail address: [email protected] (M. Hayashi).
animal model of GLD [3,4]. We neuropathologically compared three cases of GLD with different survival lengths and tried to characterize the infiltrating glial cells, globoid cells and lymphocytes, using immunohistochemistry and in situ nick end labeling (ISEL).
2. Materials and methods The clinical subjects comprised three patients with clinicopathologically confirmed GLD (Table 1). Enzyme analysis was performed in all cases except case 1. Each brain was fixed in a 10% buffered formalin solution. The formalinfixed brain was cut coronally, embedded in paraffin, and then subjected to hematoxylin and eosin, Klu¨ver–Barrera, Bodian and Holzer staining. ISEL and immunohistochemical analysis was performed on paraffin blocks of the cerebral white matter, cerebellar white matter and pons. For ISEL, sections were stripped of proteins by incubation with 0.1% trypsin (Sigma, Missouri, USA) at 37 8C for 30 min. After rinsing with Tris-buffered saline, the sections were immersed in TdT buffer (30 mM Trizma base
0387-7604/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S03 87- 7604(02)0005 7-8
Case
Age (months)
Sex
Onset (months)
Psychomotor retardation (months)
Hypertonus (months)
Convulsions (months)
Galactocerebrosidase
Cause of death
Postmortem time (h)
Brain weight (g)
1 2 3
12 19 46
Female Male Female
3 3 0
3 11 10
4 16 24
3 16 24
NT a Decreased Decreased
Pneumonia Pneumonia Apnea
18 2 2
600 580 850
a
NT, not tested.
M. Itoh et al. / Brain & Development 24 (2002) 284–290
Table 1 Summary of cases
285
286
M. Itoh et al. / Brain & Development 24 (2002) 284–290
(pH 7.2), 140 mM sodium cacodylate, 1 mM cobalt chloride). TdT (0.3 units/ml) (Life Technologies, Inc., Maryland, USA) and digoxigenin-dUTP (Roche Diagnostics, Mannheim, Germany) in TdT buffer were then added to cover the sections, followed by incubation under a humid atmosphere at 37 8C for 60 min. The reaction was terminated by transferring the slides to TB buffer (300 mM sodium chloride, 30 mM sodium citrate) at room temperature for 15 min. After extensive rinsing in Tris-buffered saline, the sections were treated with a 1:500 dilution of alkaline phosphatase-labeled anti-digoxigenin antibodies (Roche Diagnostics, Mannheim, Germany) at room temperature for 1 h. 4-Nitroblue-tetrazolium-chloride/5bromine-4-chloride-3-indolyl-phosphate (Sigma, Missouri, USA) visualized the reaction. For immunohistochemistry, 6 mm thick sections were cut, deparaffinized, heated by a microwave oven in a citrate solution for 20 min, and then washed three times in Tris-buffered saline (pH 7.6). In order to abolish endogenous peroxidase activity, the sections were quenched with 1% hydrogen peroxide for 15 min, and then rinsed with Tris-buffered saline. Mouse monoclonal antibodies to bcl-2, p53, GFAP, vimentin, HLA-DR alpha, KP-1 (CD68), L26 (CD20), LCA, UCHL-1, VCAM-1 (DAKO, Glostrup, Denmark), ICAM-1 (Zymed Laboratories, Inc., California, USA), and NCAM (Santa Cruz Biotechnology, Inc., California, USA), and rabbit polyclonal antibodies to bcl-x, bax (Santa Cruz Biotechnology, Inc., California, USA), ferritin, CD3 (DAKO, Glostrup, Denmark), TNFalpha (Endogen, Massachusetts, USA) and myelin basic protein (MBP) (Nichirei, Tokyo, Japan) were applied separately to sections at the following concentrations in Trisbuffered saline: 1:1 (MBP), 1:25 (HLA-DR alpha), 1:50 (bcl-2, vimentin, ICAM-1, VCAM-1, CD3, p53), 1:100 (ferritin, KP-1, L26, LCA, UCHL-1, TNF-alpha), 1:250 (NCAM), 1:500 (GFAP, bcl-x) and 1:1500 (bax). The immune reaction was visualized with an ABC/DAB staining kit (Nichirei, Tokyo, Japan).
3. Results 3.1. Gross and microscopic features (Table 2) The brains were atrophic and had ventricular enlargement in three cases. The cerebral white matter was replaced by rubbery hard and translucent tissues in three cases and pseudocysts formed around the anterior horns of lateral ventricles and in the corpus callosum in case 3. Microscopically, in common for the three cases, severe neuronal loss was observed in the thalamus, cerebellar cortex, dentate nucleus and inferior olivary nucleus, and to a lesser extent in the pontine nucleus, while neurons were comparatively preserved in the cerebral cortex, putamen, caudate nucleus and tegmentum of the brainstem. In the cerebellar cortex, axonal torpedo and cactus developed. Severe destruction of myelin sheaths and axons with reduced oligodendrocytes
was observed in the white matter throughout the central nervous system. The white matter lesion was pronounced in the corona radiata, corpus callosum and cerebellar peduncles in three cases, where a spongy state and/or microcyst cavitation developed (Fig. 1). There was marked fibrillary gliosis in the cerebral white matter, globus pallidus, thalamus, cerebellar cortex, dentate nucleus and brainstem. The myelin breakdown and fibrillary gliosis coexisted in most of the white matter lesions except those with a spongy state and/or microcyst formation. The occurrence of globoid cells was widespread in case 1, in which both the large-sized multinucleated cells and small-sized epithelioid cells with two or three nuclei were observed (Fig. 2A). In contrast, the small-sized globoid cells were scattered in the demyelinated white matter and basal ganglia in case 2, while only the large-sized globoid cells were found in the body of corpus callosum and cerebellar peduncles in case 3 (Fig. 2B). It is likely that the globoid cells of either type tended to appear in the corpus callosum and cerebellar peduncles. And small mononuclear cells, mimicking lymphocytes, were increased around the vessels in the white matter, especially in cases 1 and 2. The infiltration of small mononuclear cells and myelin breakdown with gliosis coexisted in the basal ganglia, thalamus and cerebellar dentate nucleus in addition to most of the affected white matter. 3.2. ISEL and immunohistochemical features There was a severe decrease of oligodendrocytes immunoreactive for MBP (data not shown). GFAP-immunopositive astrocytes were increased in both the gray and white matters, while ramified microglia, being immunoreactive for ferritin, KP-1 and HLA-DR alpha (Fig. 3A,B), were scattered in the demyelinated white matter and some of them were also immunoreactive for vimentin and/or TNFalpha (data not shown). Both the small-sized and large-sized globoid cells showed immunoreactivity for ferritin and KP1 (Fig. 3A), while some of the small-sized globoid cells demonstrated immunoreactivity for vimentin, HLA-DR alpha and TNF-alpha (Fig. 3B,C). Immunoreactivity for NCAM was detected in the cytoplasma rather than the cell surface in the globoid cells of both types (Fig. 3D). However, GFAP, MBP, LCA, UCHL-1, ICAM-1 or VCAM-1 could not label the globoid cells (data not shown). Presumed macrophages with foamy cytoplasma, having immunoreactivity for ferritin, KP-1 and HLA-DR alpha, were observed in the perivascular spaces in the gray and white matters (data not shown). T lymphocytes immunoreactive for LCA, UCHL-1 and CD3 were increased predominantly around the vessels in the demyelinated white matter (Fig. 3E). In cases 1 and 2, some mononuclear cells had ISEL-positive nuclei (Fig. 3F), while ISEL reactivity was completely absent in case 3. Since they lost immunoreactivity for both T cell markers and MBP, the cell origin was hard to speculate in these ISEL-reactive mononuclear cells. In addition, ISEL also visualized the nuclei of
Table 2 Neuropathological findings a Case
Cerebrum
Basal ganglia
Cerebellum and brainstem
Subcortical
Corona radiata
Internal capsule
Corpus callosum
Putamen and caudate
Globus pallidus
Thalamus
Cerebellar cortex
Dentate nucleus
White matter
Cerebellar peduncles
Pontine tegmentum
Pontine base
Fibrillary gliosis 1 2 3
(2) (2) (2)
21 21 21
11 21 (2)
11 21 21
11 21 21
(2) 11 (2)
11 21 21
21 21 21
21 11 21
21 21 21
11 21 21
11 21 11
11 11 11
21 11 11
Globoid cells 1 2 3
(2) (2) (2)
21 (2) (2)
11 11 11
21 11 (2)
21 21 11
(2) 11 (2)
11 (2) (2)
21 21 (2)
(2) (2) (2)
21 (2) (2)
21 (2) (2)
11 11 11
(2) (2) (2)
11 (2) (2)
Infiltrative mononuclear cells 1 2 3
(2) (2) (2)
21 21 (2)
21 21 (2)
21 21 (2)
21 21 11
(2) 21 (2)
11 21 (2)
21 21 21
(2) (2) (2)
21 21 21
11 21 (2)
11 11 (2)
11 (2) (2)
11 (2) 11
Spongy state 1 2 3
(2) (2) (2)
(2) (2) (2)
11 11 21
(2) 11 (2)
21 21 11
(2) 21 (2)
(2) (2) (2)
(2) 21 (2)
(2) (2) (2)
(2) (2) (2)
(2) (2) (2)
21 11 21
(2) (2) (2)
(2) (2) (2)
a
M. Itoh et al. / Brain & Development 24 (2002) 284–290
Cerebral cortex
The severity of lesions was evaluated by non-quantitative visual inspection as to 3 degrees: no (2), mild (1 1 ) and severe (2 1 ) changes.
287
288
M. Itoh et al. / Brain & Development 24 (2002) 284–290
were identified by antibodies against p53 or bcl-2 (data not shown).
4. Discussion
Fig. 1. The white matter lesion in case 3, Holzer stain. Myelinolytic cysts were observed, in addition to severe fibrillary gliosis in the cerebral white matter and thalamus.
a few of the small-sized globoid cells in case 1 (Fig. 3F). Nevertheless, the nuclei of neither GFAP-immunoreactive astrocytes nor KP-1-immunoreactive ramified microglia were labeled by ISEL in three cases (data not shown). In common for the three cases, a few of the mononuclear cells in the demyelinated white matter were weakly immunoreactive for bcl-x and bax, while no immunoreactive structures
Fig. 2. Microscopic features of the globoid cells. The small-sized globoid cells were scattered in the cerebellar white matter in case 1 (A, hematoxylin and eosin stain, £ 400), while the large-sized ones were found in the body of corpus callosum in case 3 (B, PAS stain, £ 400).
The myelin breakdown was most abundant in case 3 with long survival, in which there were many myeloclastic clefts or pseudocysts, while the infiltration of lymphocytes and ramified microglia was pronounced in case 2. It is known that the globoid cells consist of the smaller and larger ones, predominantly appear in the regions of active myelin breakdown, and their occurrence is sparse in older late lesions, in which the brain biopsy tends to miss the presence of the globoid cells [1]. Case 1, the youngest subject in this study, demonstrated a widespread occurrence of the globoid cells of both types, while only the large-sized globoid cells were observed in the more localized brain regions in case 3 with long survival. On this point, it should be noted that the corpus callosum and cerebellar peduncle were the common sites for the occurrence of globoid cells in three cases, although the globoid cells may be absent in these regions over the age of 5 years [5]. It is now widely accepted that the globoid cells derive from the mesodermal macrophage-lineage cell line, based on the presence of acid phosphatase activity and the absence of glial fibril [6]. The immunoreactivity of globoid cells for ferritin, vimentin and KP-1 but not for markers for astrocytes, oligodendrocytes or T lymphocytes in this study also confirms their macrophage/microglia cell origin. In addition, it is intriguing that immunohistochemical phenotypes were different between the small-sized and large-sized globoid cells. The large-sized globoid cells might occur in the more advanced stage in GLD, because they lost immunoreactivity for several functional markers such as vimentin and HLA-DR alpha, which the small-sized ones had. It is also possible that some of the small-sized globoid cells may gather, adhere and transform into the large-sized globoid cells. In this context, the expression of adhesion molecules is important. However, in this study, the globoid cells expressed neither ICAM-1 nor VCAM-1, while immunoreactivity for NCAM localized in the cytoplasma (Fig. 3D) may merely reflect the engulfing of NCAM proteins by the globoid cells, which were released in the neuropil from the damaged nervous tissues. It has been assumed in adrenoleukodystrophy, another inheritable leukodystrophy, that the destruction of myelin is also immunologically mediated, because CD4 1 T lymphocytes are predominantly located around the vessels and immunoreactivity for TNF-alpha is detected in macrophages and glial cells [7]. In the twitcher mutant mouse, an authentic murine model of GLD, Ia antigen expressing cells increased in number with the progression of demyelination, reached a plateau and then rapidly decreased despite continuous demyelination in the central nervous system, while CD4 1 helper type T cells were persistently abundant [3]. Similarly, HLA-DR and/or TNF-alpha immunoreactive
M. Itoh et al. / Brain & Development 24 (2002) 284–290
289
Fig. 3. The results of immunohistochemistry. Both the small-sized globoid cells and ramified microglia were immunoreactive for KP-1 (A), HLA-DR alpha (B) and TNF-alpha (C, arrows) in the cerebellar white matter in case 1, £ 400. The cytoplasma in some of the large-sized globoid cells were faintly visualized by anti-NCAM antibody (arrows), although the surrounding fibers also showed immunoreactivity for NCAM in the corpus callosum in case 3 (D, £ 400). Some of the mononuclear cells in the white matter were immunoreactive for UCHL-1 (E, £ 200). The nuclei of the mononuclear cells and a few of the small-sized globoid cells (arrows) were stained by ISEL (F, £ 400).
microglia, macrophages and globoid cells were increased in our cases of GLD, and UCHL-1- and CD3-immunopositive T lymphocytes expressing apoptotic features were observed around the vessels. The occurrence of these activated immune cells was less predominant in case 3 with longer survival (Fig. 3). The expression of HLA-DR alpha in the microglia is pivotal for the initiation of an immune reaction in the central nervous system [8] and TNF-alpha is potentially myeloclastic, causing oligodendrocyte cell death [9]. Therefore, immunoreactivity for HLA-DR alpha and TNFalpha in the ramified microglia, small-sized globoid cells and/or presumed macrophages may suggest involvement of immunological responses in the pathogenesis of GLD, and similarly in adrenoleukodystrophy and the twitcher mouse.
Apoptotic cell death is reported to be involved in neurodegeneration in congenital metabolic disorders. In the brains from patients with late infantile and juvenile neuronal ceroidlipofuscinosis, there were ISEL-positive neurons and marked up-regulation of bcl-2 at the protein and mRNA levels in the cerebral cortex [10]. However, we failed to demonstrate the altered expression of either bcl-2 or bcl-x in the brains in the same disorders, despite the presence of ISEL-positive neuron [11]. Apoptotic cell death can also be induced in oligodendrocytes and infiltrating mononuclear cells in experimental autoimmune encephalomyelitis, and the relationships between this apoptotic process and demyelination have been investigated [12]. Furthermore, in the twitcher mouse, apoptosis was verified to cause gradual depletion of oligodendrocytes [4]. In GLD, two cases with shorter disease
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M. Itoh et al. / Brain & Development 24 (2002) 284–290
duration showed ISEL-positive mononuclear cells, which were speculated to stem from T lymphocytes and/or oligodendrocytes. Furthermore, in case 1 with the shortest disease length, a few of the small-sized globoid cells had ISEL-positive nuclei (Fig. 3F). These results suggest that the apoptotic process can be involved in the reduction of oligodendrocytes and/or development of the globoid cells in GLD. However, since the in situ expression of several apoptosis-related proteins was not altered, the apoptotic mechanism might not significantly affect the neurodegeneration of GLD.
[5]
[6]
[7]
[8]
References [1] Kolodny EH. Globoid leukodystrophy. In: Moser HW, editor. Neurodystrophies and neuropathy, revised ed. Handbook of clinical neurology, 22. Amsterdam: Elsevier, 1996. pp. 187–210. [2] Friede DL. Globoid cell leukodystrophy. In: Friede DL, editor. Developmental neuropathology, 2nd ed. Berlin: Springer, 1989. pp. 470– 477. [3] Ohno M, Komiyama A, Martin PM, Suzuki K. MHC class II antigen expression and T-cell infiltration in the demyelinating CNS and PNS of the twitcher mouse. Brain Res 1993;625:186–196. [4] Taniike M, Mohri I, Eguchi N, Irikura D, Urade Y, Okada S, et al. An apoptotic deletion of oligodendrocytes in the twitcher, a murine
[9] [10]
[11]
[12]
model of globoid cell leukodystrophy. J Neuropathol Exp Neurol 1999;58:644–653. Dunn HG, Dolman CL, Farrell DF, Tischler B, Hasinoff C, Woolf LI. Krabbe’s leukodystrophy without globoid cells. Neurology 1976;26: 1035–1041. Nelson E, Aurebeck G, Osterberg K, Berry J, Jabbour JT, Bornhofen J. Ultrastructural and chemical studies on Krabbe’s disease. J Neuropathol Exp Neurol 1963;22:414–434. Powers JM, Liu Y, Moser AB, Moser HW. The inflammatory myelinopathy of adrenoleukodystrophy: cells, effector molecules, and pathogenetic implications. J Neuropathol Exp Neurol 1992;51:630– 643. Hayashi M, Dorf ME, Abromson-Leeman S. Granulocyte-macrophage colony stimulating factor inhibits class II major histocompatibility complex expression and antigen presentation by microglia. J Neuroimmunol 1993;48:23–32. Ludwin SK. The pathobiology of the oligodendrocyte. J Neuropathol Exp Neurol 1997;56:111–124. Puranam K, Qian WH, Nikbakht K, Venable M, Obeid L, Hannun Y, et al. Upregulation of Bcl-2 and elevation of ceramide in Batten disease. Neuropediatrics 1997;28:37–41. Kurata K, Hayashi M, Satoh J, Kojima H, Nagata J, Tamagawa K, et al. Pathological study on sibling autopsy cases of the late infantile form of neuronal ceroid lipofuscinosis. Brain Dev 1999;21:63–67. Tsunoda I, Fujinami RS. Two models for multiple sclerosis: experimental allergic encephalomyelitis and Theiler’s murine encephalomyelitis virus. J Neuropathol Exp Neurol 1996;55:673–686.
Original article
Immunohistological study of globoid cell leukodystrophy Masahiro Itoh a, Masaharu Hayashi a,*, Yasunori Fujioka b, Kazuo Nagashima b, Yoshio Morimatsu a, Haruo Matsuyama a a
Department of Clinical Neuropathology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashi-dai, Fuchu-shi, Tokyo 183-8526, Japan b Department of Pathology, Hokkaido University School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo 060-8638, Japan Received 20 December 2001; received in revised form 15 March 2002; accepted 5 April 2002
Abstract We examined three autopsy cases of globoid cell leukodystrophy (GLD) with different survival, using immunohistochemistry and in situ nick end labeling (ISEL). The white matter lesion was pronounced in the corona radiata, corpus callosum and cerebellar peduncles in three cases, where a spongy state developed, in addition to the neuronal loss in the thalamus, cerebellum and inferior olivary nucleus. Ramified microglia, being immunoreactive for ferritin and HLA-DR alpha, were scattered in the white matter, and some of them also had immunoreactivity for TNF-alpha. Both the small-sized and large-sized globoid cells showed immunoreactivity for ferritin KP-1 and NCAM, while some of the small-sized globoid cells were also immunoreactive for HLA-DR alpha and TNF-alpha. As the survival became longer, the occurrence of the globoid cells decreased, however, they were commonly observed in the corpus callosum and cerebellar peduncle in three cases. T lymphocytes immunoreactive for LCA, UCHL-1 and CD3 were increased around the vessels in the white matter. ISEL stained nuclei of mononuclear cells in the white matter in two cases with short survival, although the cell origin was not verified. ISEL also visualized a few nuclei of the small-sized globoid cells in one case. On the other hand, immunostainings against cell death proteins such as bcl-2 family members and p53 failed to identify any significant changes. These data suggest that the immunological step and to a lesser extent the apoptotic process may partly be involved in the myelin breakdown and glial pathology in GLD, as reported in the twitcher mouse, a murine model of GLD. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Apoptosis; Globoid cell; Immune reaction; Krabbe disease; Leukodystrophy
1. Introduction Globoid cell leukodystrophy (GLD), also called Krabbe disease, is a progressive heredodegenerative disease caused by deficiency of the lysosomal enzyme galactocerebrosidase [1]. Psychosine (galactosylsphingosine), a toxic metabolite of galactocerebroside, accumulates in GLD causing degeneration of the myelin-producing cells, oligodendrocytes and Schwann cells [1]. Pathological features of the disease include severe axonal loss, myelin degeneration, astrocyte gliosis and formation of periodic acid schiff (PAS)-positive multinucleated giant cells, globoid cells, containing a granular cytoplasm filled with filamentous inclusion and fibrillated bodies [1,2]. The exact pathogenesis of myelin breakdown and the formation of globoid cells remains to be investigated, although immunological responses and apoptosis are speculated to be involved in the demyelination and/or oligodendrocyte depletion in the twitcher mouse, an * Corresponding author. Tel.: 181-42-325-3881, ext. 4703; fax: 181-42321-8678. E-mail address: [email protected] (M. Hayashi).
animal model of GLD [3,4]. We neuropathologically compared three cases of GLD with different survival lengths and tried to characterize the infiltrating glial cells, globoid cells and lymphocytes, using immunohistochemistry and in situ nick end labeling (ISEL).
2. Materials and methods The clinical subjects comprised three patients with clinicopathologically confirmed GLD (Table 1). Enzyme analysis was performed in all cases except case 1. Each brain was fixed in a 10% buffered formalin solution. The formalinfixed brain was cut coronally, embedded in paraffin, and then subjected to hematoxylin and eosin, Klu¨ver–Barrera, Bodian and Holzer staining. ISEL and immunohistochemical analysis was performed on paraffin blocks of the cerebral white matter, cerebellar white matter and pons. For ISEL, sections were stripped of proteins by incubation with 0.1% trypsin (Sigma, Missouri, USA) at 37 8C for 30 min. After rinsing with Tris-buffered saline, the sections were immersed in TdT buffer (30 mM Trizma base
0387-7604/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S03 87- 7604(02)0005 7-8
Case
Age (months)
Sex
Onset (months)
Psychomotor retardation (months)
Hypertonus (months)
Convulsions (months)
Galactocerebrosidase
Cause of death
Postmortem time (h)
Brain weight (g)
1 2 3
12 19 46
Female Male Female
3 3 0
3 11 10
4 16 24
3 16 24
NT a Decreased Decreased
Pneumonia Pneumonia Apnea
18 2 2
600 580 850
a
NT, not tested.
M. Itoh et al. / Brain & Development 24 (2002) 284–290
Table 1 Summary of cases
285
286
M. Itoh et al. / Brain & Development 24 (2002) 284–290
(pH 7.2), 140 mM sodium cacodylate, 1 mM cobalt chloride). TdT (0.3 units/ml) (Life Technologies, Inc., Maryland, USA) and digoxigenin-dUTP (Roche Diagnostics, Mannheim, Germany) in TdT buffer were then added to cover the sections, followed by incubation under a humid atmosphere at 37 8C for 60 min. The reaction was terminated by transferring the slides to TB buffer (300 mM sodium chloride, 30 mM sodium citrate) at room temperature for 15 min. After extensive rinsing in Tris-buffered saline, the sections were treated with a 1:500 dilution of alkaline phosphatase-labeled anti-digoxigenin antibodies (Roche Diagnostics, Mannheim, Germany) at room temperature for 1 h. 4-Nitroblue-tetrazolium-chloride/5bromine-4-chloride-3-indolyl-phosphate (Sigma, Missouri, USA) visualized the reaction. For immunohistochemistry, 6 mm thick sections were cut, deparaffinized, heated by a microwave oven in a citrate solution for 20 min, and then washed three times in Tris-buffered saline (pH 7.6). In order to abolish endogenous peroxidase activity, the sections were quenched with 1% hydrogen peroxide for 15 min, and then rinsed with Tris-buffered saline. Mouse monoclonal antibodies to bcl-2, p53, GFAP, vimentin, HLA-DR alpha, KP-1 (CD68), L26 (CD20), LCA, UCHL-1, VCAM-1 (DAKO, Glostrup, Denmark), ICAM-1 (Zymed Laboratories, Inc., California, USA), and NCAM (Santa Cruz Biotechnology, Inc., California, USA), and rabbit polyclonal antibodies to bcl-x, bax (Santa Cruz Biotechnology, Inc., California, USA), ferritin, CD3 (DAKO, Glostrup, Denmark), TNFalpha (Endogen, Massachusetts, USA) and myelin basic protein (MBP) (Nichirei, Tokyo, Japan) were applied separately to sections at the following concentrations in Trisbuffered saline: 1:1 (MBP), 1:25 (HLA-DR alpha), 1:50 (bcl-2, vimentin, ICAM-1, VCAM-1, CD3, p53), 1:100 (ferritin, KP-1, L26, LCA, UCHL-1, TNF-alpha), 1:250 (NCAM), 1:500 (GFAP, bcl-x) and 1:1500 (bax). The immune reaction was visualized with an ABC/DAB staining kit (Nichirei, Tokyo, Japan).
3. Results 3.1. Gross and microscopic features (Table 2) The brains were atrophic and had ventricular enlargement in three cases. The cerebral white matter was replaced by rubbery hard and translucent tissues in three cases and pseudocysts formed around the anterior horns of lateral ventricles and in the corpus callosum in case 3. Microscopically, in common for the three cases, severe neuronal loss was observed in the thalamus, cerebellar cortex, dentate nucleus and inferior olivary nucleus, and to a lesser extent in the pontine nucleus, while neurons were comparatively preserved in the cerebral cortex, putamen, caudate nucleus and tegmentum of the brainstem. In the cerebellar cortex, axonal torpedo and cactus developed. Severe destruction of myelin sheaths and axons with reduced oligodendrocytes
was observed in the white matter throughout the central nervous system. The white matter lesion was pronounced in the corona radiata, corpus callosum and cerebellar peduncles in three cases, where a spongy state and/or microcyst cavitation developed (Fig. 1). There was marked fibrillary gliosis in the cerebral white matter, globus pallidus, thalamus, cerebellar cortex, dentate nucleus and brainstem. The myelin breakdown and fibrillary gliosis coexisted in most of the white matter lesions except those with a spongy state and/or microcyst formation. The occurrence of globoid cells was widespread in case 1, in which both the large-sized multinucleated cells and small-sized epithelioid cells with two or three nuclei were observed (Fig. 2A). In contrast, the small-sized globoid cells were scattered in the demyelinated white matter and basal ganglia in case 2, while only the large-sized globoid cells were found in the body of corpus callosum and cerebellar peduncles in case 3 (Fig. 2B). It is likely that the globoid cells of either type tended to appear in the corpus callosum and cerebellar peduncles. And small mononuclear cells, mimicking lymphocytes, were increased around the vessels in the white matter, especially in cases 1 and 2. The infiltration of small mononuclear cells and myelin breakdown with gliosis coexisted in the basal ganglia, thalamus and cerebellar dentate nucleus in addition to most of the affected white matter. 3.2. ISEL and immunohistochemical features There was a severe decrease of oligodendrocytes immunoreactive for MBP (data not shown). GFAP-immunopositive astrocytes were increased in both the gray and white matters, while ramified microglia, being immunoreactive for ferritin, KP-1 and HLA-DR alpha (Fig. 3A,B), were scattered in the demyelinated white matter and some of them were also immunoreactive for vimentin and/or TNFalpha (data not shown). Both the small-sized and large-sized globoid cells showed immunoreactivity for ferritin and KP1 (Fig. 3A), while some of the small-sized globoid cells demonstrated immunoreactivity for vimentin, HLA-DR alpha and TNF-alpha (Fig. 3B,C). Immunoreactivity for NCAM was detected in the cytoplasma rather than the cell surface in the globoid cells of both types (Fig. 3D). However, GFAP, MBP, LCA, UCHL-1, ICAM-1 or VCAM-1 could not label the globoid cells (data not shown). Presumed macrophages with foamy cytoplasma, having immunoreactivity for ferritin, KP-1 and HLA-DR alpha, were observed in the perivascular spaces in the gray and white matters (data not shown). T lymphocytes immunoreactive for LCA, UCHL-1 and CD3 were increased predominantly around the vessels in the demyelinated white matter (Fig. 3E). In cases 1 and 2, some mononuclear cells had ISEL-positive nuclei (Fig. 3F), while ISEL reactivity was completely absent in case 3. Since they lost immunoreactivity for both T cell markers and MBP, the cell origin was hard to speculate in these ISEL-reactive mononuclear cells. In addition, ISEL also visualized the nuclei of
Table 2 Neuropathological findings a Case
Cerebrum
Basal ganglia
Cerebellum and brainstem
Subcortical
Corona radiata
Internal capsule
Corpus callosum
Putamen and caudate
Globus pallidus
Thalamus
Cerebellar cortex
Dentate nucleus
White matter
Cerebellar peduncles
Pontine tegmentum
Pontine base
Fibrillary gliosis 1 2 3
(2) (2) (2)
21 21 21
11 21 (2)
11 21 21
11 21 21
(2) 11 (2)
11 21 21
21 21 21
21 11 21
21 21 21
11 21 21
11 21 11
11 11 11
21 11 11
Globoid cells 1 2 3
(2) (2) (2)
21 (2) (2)
11 11 11
21 11 (2)
21 21 11
(2) 11 (2)
11 (2) (2)
21 21 (2)
(2) (2) (2)
21 (2) (2)
21 (2) (2)
11 11 11
(2) (2) (2)
11 (2) (2)
Infiltrative mononuclear cells 1 2 3
(2) (2) (2)
21 21 (2)
21 21 (2)
21 21 (2)
21 21 11
(2) 21 (2)
11 21 (2)
21 21 21
(2) (2) (2)
21 21 21
11 21 (2)
11 11 (2)
11 (2) (2)
11 (2) 11
Spongy state 1 2 3
(2) (2) (2)
(2) (2) (2)
11 11 21
(2) 11 (2)
21 21 11
(2) 21 (2)
(2) (2) (2)
(2) 21 (2)
(2) (2) (2)
(2) (2) (2)
(2) (2) (2)
21 11 21
(2) (2) (2)
(2) (2) (2)
a
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Cerebral cortex
The severity of lesions was evaluated by non-quantitative visual inspection as to 3 degrees: no (2), mild (1 1 ) and severe (2 1 ) changes.
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were identified by antibodies against p53 or bcl-2 (data not shown).
4. Discussion
Fig. 1. The white matter lesion in case 3, Holzer stain. Myelinolytic cysts were observed, in addition to severe fibrillary gliosis in the cerebral white matter and thalamus.
a few of the small-sized globoid cells in case 1 (Fig. 3F). Nevertheless, the nuclei of neither GFAP-immunoreactive astrocytes nor KP-1-immunoreactive ramified microglia were labeled by ISEL in three cases (data not shown). In common for the three cases, a few of the mononuclear cells in the demyelinated white matter were weakly immunoreactive for bcl-x and bax, while no immunoreactive structures
Fig. 2. Microscopic features of the globoid cells. The small-sized globoid cells were scattered in the cerebellar white matter in case 1 (A, hematoxylin and eosin stain, £ 400), while the large-sized ones were found in the body of corpus callosum in case 3 (B, PAS stain, £ 400).
The myelin breakdown was most abundant in case 3 with long survival, in which there were many myeloclastic clefts or pseudocysts, while the infiltration of lymphocytes and ramified microglia was pronounced in case 2. It is known that the globoid cells consist of the smaller and larger ones, predominantly appear in the regions of active myelin breakdown, and their occurrence is sparse in older late lesions, in which the brain biopsy tends to miss the presence of the globoid cells [1]. Case 1, the youngest subject in this study, demonstrated a widespread occurrence of the globoid cells of both types, while only the large-sized globoid cells were observed in the more localized brain regions in case 3 with long survival. On this point, it should be noted that the corpus callosum and cerebellar peduncle were the common sites for the occurrence of globoid cells in three cases, although the globoid cells may be absent in these regions over the age of 5 years [5]. It is now widely accepted that the globoid cells derive from the mesodermal macrophage-lineage cell line, based on the presence of acid phosphatase activity and the absence of glial fibril [6]. The immunoreactivity of globoid cells for ferritin, vimentin and KP-1 but not for markers for astrocytes, oligodendrocytes or T lymphocytes in this study also confirms their macrophage/microglia cell origin. In addition, it is intriguing that immunohistochemical phenotypes were different between the small-sized and large-sized globoid cells. The large-sized globoid cells might occur in the more advanced stage in GLD, because they lost immunoreactivity for several functional markers such as vimentin and HLA-DR alpha, which the small-sized ones had. It is also possible that some of the small-sized globoid cells may gather, adhere and transform into the large-sized globoid cells. In this context, the expression of adhesion molecules is important. However, in this study, the globoid cells expressed neither ICAM-1 nor VCAM-1, while immunoreactivity for NCAM localized in the cytoplasma (Fig. 3D) may merely reflect the engulfing of NCAM proteins by the globoid cells, which were released in the neuropil from the damaged nervous tissues. It has been assumed in adrenoleukodystrophy, another inheritable leukodystrophy, that the destruction of myelin is also immunologically mediated, because CD4 1 T lymphocytes are predominantly located around the vessels and immunoreactivity for TNF-alpha is detected in macrophages and glial cells [7]. In the twitcher mutant mouse, an authentic murine model of GLD, Ia antigen expressing cells increased in number with the progression of demyelination, reached a plateau and then rapidly decreased despite continuous demyelination in the central nervous system, while CD4 1 helper type T cells were persistently abundant [3]. Similarly, HLA-DR and/or TNF-alpha immunoreactive
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Fig. 3. The results of immunohistochemistry. Both the small-sized globoid cells and ramified microglia were immunoreactive for KP-1 (A), HLA-DR alpha (B) and TNF-alpha (C, arrows) in the cerebellar white matter in case 1, £ 400. The cytoplasma in some of the large-sized globoid cells were faintly visualized by anti-NCAM antibody (arrows), although the surrounding fibers also showed immunoreactivity for NCAM in the corpus callosum in case 3 (D, £ 400). Some of the mononuclear cells in the white matter were immunoreactive for UCHL-1 (E, £ 200). The nuclei of the mononuclear cells and a few of the small-sized globoid cells (arrows) were stained by ISEL (F, £ 400).
microglia, macrophages and globoid cells were increased in our cases of GLD, and UCHL-1- and CD3-immunopositive T lymphocytes expressing apoptotic features were observed around the vessels. The occurrence of these activated immune cells was less predominant in case 3 with longer survival (Fig. 3). The expression of HLA-DR alpha in the microglia is pivotal for the initiation of an immune reaction in the central nervous system [8] and TNF-alpha is potentially myeloclastic, causing oligodendrocyte cell death [9]. Therefore, immunoreactivity for HLA-DR alpha and TNFalpha in the ramified microglia, small-sized globoid cells and/or presumed macrophages may suggest involvement of immunological responses in the pathogenesis of GLD, and similarly in adrenoleukodystrophy and the twitcher mouse.
Apoptotic cell death is reported to be involved in neurodegeneration in congenital metabolic disorders. In the brains from patients with late infantile and juvenile neuronal ceroidlipofuscinosis, there were ISEL-positive neurons and marked up-regulation of bcl-2 at the protein and mRNA levels in the cerebral cortex [10]. However, we failed to demonstrate the altered expression of either bcl-2 or bcl-x in the brains in the same disorders, despite the presence of ISEL-positive neuron [11]. Apoptotic cell death can also be induced in oligodendrocytes and infiltrating mononuclear cells in experimental autoimmune encephalomyelitis, and the relationships between this apoptotic process and demyelination have been investigated [12]. Furthermore, in the twitcher mouse, apoptosis was verified to cause gradual depletion of oligodendrocytes [4]. In GLD, two cases with shorter disease
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duration showed ISEL-positive mononuclear cells, which were speculated to stem from T lymphocytes and/or oligodendrocytes. Furthermore, in case 1 with the shortest disease length, a few of the small-sized globoid cells had ISEL-positive nuclei (Fig. 3F). These results suggest that the apoptotic process can be involved in the reduction of oligodendrocytes and/or development of the globoid cells in GLD. However, since the in situ expression of several apoptosis-related proteins was not altered, the apoptotic mechanism might not significantly affect the neurodegeneration of GLD.
[5]
[6]
[7]
[8]
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