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A neuropathological study of the disturbance of the nigro-amygdaloid connections in brains from patients with dementia with Lewy bodies
Journal of the Neurological Sciences 185 Ž2001. 129–134 www.elsevier.comrlocaterjns
A neuropathological study of the disturbance of the nigro-amygdaloid connections in brains from patients with dementia with Lewy bodies Eizo Iseki a,) , Masanori Kato b, Wami Marui a , Kenji Ueda ´ c , Kenji Kosaka a a
Department of Psychiatry, Yokohama City UniÕersity School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan b Soga Hospital, 148 Sogagishi, Odawara 250-0203, Japan c Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156-8585, Japan Received 12 October 2000; received in revised form 8 February 2001; accepted 12 February 2001
Abstract We neuropathologically and immunohistochemically investigated characteristics of the central amygdaloid nucleus lesion and its relationship with the substantia nigra lesion in dementia with Lewy bodies ŽDLB. brains. Nine DLB, four Parkinson’s disease ŽPD. and four Alzheimer-type dementia ŽATD. cases were examined. The degree of neuronal loss in the substantia nigra was Žq. – Žqqq . in DLB cases, Žqqq . in PD cases and Žq. in ATD cases. All DLB cases showed spongy change and ubiquitin-positive spheroids in the central nucleus. The degree of spongy change was Žq. – Žqqq . in DLB cases, Žq. in PD cases and Žy. – Žq. in ATD cases, which was correlated with the degree of neuronal loss in the substantia nigra in DLB cases. The number of ubiquitin-positive spheroids was parallel to the degree of spongy change. The central nucleus receives dense dopaminergic fibers from the substantia nigra. Many ubiquitin-positive spheroids were also positive to a-synuclein and tyrosine-hydroxylase, suggesting that they derive from the degeneration of terminal or distal axons of Lewy body-bearing dopaminergic neurons in the substantia nigra. The disturbance of the dopaminergic connections from the substantia nigra to the central nucleus may be responsible for psychotic symptoms in DLB patients. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Dementia with Lewy bodies; Substantia nigra; Central amygdaloid nucleus; Spongy change; Spheroid; Immunohistochemistry; Psychotic symptom
1. Introduction Dementia with Lewy bodies ŽDLB. is the second most frequent neurodegenerative dementing disorder after Alzheimer-type dementia ŽATD. w1x. The clinical features include progressive dementia as a central feature, and fluctuating cognition, visual hallucination and parkinsonism as core features. The presence or absence of each core feature leads to clinical diagnosis of probable DLB or possible DLB w2x. The occurrence of Lewy bodies ŽLB. is the only feature essential for pathological diagnosis of DLB, although Lewy-related neurites, Alzheimer pathology, neuronal loss in the brainstem nuclei and spongy change are also frequently seen in DLB brains w2x. Parkinsonism is mainly caused by neuronal loss in the substantia nigra in DLB patients as well as Parkinson’s ) Corresponding author. Tel.: q81-45-787-2667; fax: q81-45-7832540. E-mail address: [email protected] ŽE. Iseki..
disease ŽPD. patients w3x. However, it remains unclear what lesions cause psychotic symptoms such as fluctuating cognition and visual hallucination in DLB patients. These psychotic symptoms are similar to those seen after administrating L-DOPA to PD patients w1,4,5x. The amygdala, a major constituent of the limbic system, has the fiber connections with the cerebral cortex, hippocampus, olfactory system, thalamus, hypothalamus, brainstem nuclei, etc. w6,7x, and is thought to be related to emotional, sensory and cognitive functions w7–9x. In DLB brains, the amygdala is the most preferential area of the cortical LB w10,11x. The central amygdaloid nucleus among seven nuclei constituting the amygdala w12x receives the most dense afferent fibers from the substantia nigra and locus ceruleus w6,7,13x. The central nucleus shows many ubiquitin-positive spheroids, which derive from the degeneration of terminal or distal axons in DLB brains w10x. Therefore, the disturbance of dopaminergic connections from the substantia nigra to the central nucleus may be responsible for psychotic symptoms in DLB patients.
0022-510Xr01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 5 1 0 X Ž 0 1 . 0 0 4 8 1 - 6
E. Iseki et al.r Journal of the Neurological Sciences 185 (2001) 129–134
130
In this study, we neuropathologically and immunohistochemically investigated characteristics of the central nucleus lesion and its relationship with the sustantia nigra lesion in DLB brains, and discussed the relationship between these lesions and psychotic symptoms in DLB patients.
2. Materials and methods This study was performed in nine pathologically verified DLB cases w2x. For comparison, four PD cases and four ATD cases were examined. Clinical data of the examined cases are shown in Table 1. In DLB cases, case 1 corresponded to the neocortical type, pure form without Alzheimer pathology, and cases 2–9 corresponded to the neocortical type, common form with Alzheimer pathology w14x. Brains were fixed in 4% paraformaldehyde in 0.1 molrl phosphate buffer ŽpH 7.4.. A coronal slice of the cerebral hemisphere through the level of the mammilary body, and two horizontal slices of the midbrain through the red nucleus and the pons through the oculomotor nucleus, were embedded in paraffin. The 6-mm-thick sections were stained with hematoxylin–eosin ŽHE., Kluver–Barrera ¨ ŽKB. and methenamine–silver ŽMS.. The sections were also immunostained with anti-GFAP Žpolyclonal, rabbit,
1:8000; Dako, Denmark., anti-ubiquitin ŽMAB1510: monoclonal, mouse, 1:8000; Chemicon, USA., anti-a-synuclein ŽMDV2: polyclonal, rabbit, 1:4000; PQE3: polyclonal, rabbit, 1:4000. w15x and anti-tyrosine hydroxylase ŽMAB318: monoclonal, mouse, 1:100; Chemicon. antibodies. Immunolabeling was detected using the avidin-biotinylated HRP complex ŽABC. method ŽElite Kit, Vector, USA. and visualized with DAB ŽSigma, USA.. The degree of spongy change was determined on the HE section by counting the number of vacuoles in microscope fields Žfield size: 0.66 mm 2 . at a 400 = magnification, and was graded from Žy. to Žqqq .: Žy. absent, Žq. - 5, Žqq . 5–10, Žqqq . ) 10. The degree of gliosis was determined on the GFAP-immunostained section by counting the number of GFAP-positive astroglia at a 200 = magnification, and was graded from Ž". to Žqqq .: Ž". - 5, Žq. 5–10, Žqq . 10–15, Žqqq . ) 15. The number of spheroids was determined on the ubiquitin-immunostained section by counting the number of ubiquitin-positive spheroids at a 200 = magnification, and was graded from Žy. to Žqqq .: Žy. absent, Žq. - 5, Žqq . 5–10, Žqqq . ) 10. Spongy change and spheroids were evaluated in the amygdala, and gliosis in the substantia nigra, locus ceruleus and amygdala. The degree of neuronal loss in the substantia nigra was determined by counting the total number of pigmented neurons in each quadrant of one side of the KB section, and was
Table 1 Clinical data of the examined cases Case number
Sex
Age Žyears old.
DLB 1 2 3 4 5 6 7 8 9 Mean
Duration Žyears.
M M M F F M M M M
50 67 73 74 79 79 81 84 88 75.0
7 5 3 2 3 3 6 4 2 3.9
PD 1 2 3 4 Mean
F M F M
57 76 81 90 76.0
18 9 15 2 11.0
ATD 1 2 3 4 Mean
M F F F
82 85 89 94 87.5
3 4 9 4 5.0
Dementia
Parkinsonism
Psychotic symptoms
Brain weight Žg.
qq qq qq qq qq qq qq qq q
qqq qq qq qq q qq q qq q
y y q q q q q q q
1050 1030 900 1300 1170 1165 1120 1060 1145 1104.4
y y y y
qqq qqq qqq qq
y y y y
1270 1140 1050 1150 1152.5
qqq qqq qqq qq
y y y y
y y y y
1200 900 1040 1070 1052.5
DLB: dementia with Lewy bodies; PD: Parkinson’s disease; ATD: Alzheimer-type dementia. The degree of dementia or parkinsonism: absent Žy., mild Žq., moderate Žqq., severe Žqqq.. Psychotic symptoms represent the presence or absence of fluctuating cognition and visual hallucination.
E. Iseki et al.r Journal of the Neurological Sciences 185 (2001) 129–134
graded from Žq. to Žqqq .: Žq. ) 200, Žqq . 100– 200, Žqqq . - 100. The degree of neuronal loss in the locus ceruleus was determined by counting the number of pigmented neurons on one side of the KB section, and was graded from Žq. to Žqqq .: Žq. ) 20, Žqq . 10–20, Žqqq . - 10.
3. Results Neuropathological findings in the examined cases are shown in Table 2. 3.1. The substantia nigra and locus ceruleus lesions In DLB cases, the degree of neuronal loss in the substantia nigra ranged from Žq. to Žqqq .. The mean number of pigmented neurons in DLB cases was 110.2, corresponding to Žqq . in the degree of neuronal loss. In PD cases, all cases showed Žqqq . in the degree of neuronal loss, and the mean number of pigmented neurons was 59.5. In ATD cases, all cases showed Žq. in the degree of neuronal loss, and the mean number of pigmented neurons was 294.8. The degree of neuronal loss in the locus ceruleus was Žqqq . in all DLB cases and Žqq . in all PD cases, and the mean number of pigmented neurons was 6.9 in DLB cases and 11.0 in PD cases. In ATD cases, all cases
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showed Žq. in the degree of neuronal loss, and the mean number of pigmented neurons was 30.3. With regard to the relationship between the degrees of neuronal loss and gliosis, the degree of gliosis in the substantia nigra was Žq. – Žqq . in DLB cases, Žqq . – Žqqq . in PD cases and Ž". in ATD cases, which was approximately parallel to the degree of neuronal loss. The degree of gliosis in the locus ceruleus was Žq. – Žqq . in DLB cases, Žq. – Žqq . in PD cases and Ž". in ATD cases, showing no apparent relationship with the degree of neuronal loss. 3.2. The amygdala lesion All DLB cases showed spongy change in the central nucleus on HE sections ŽFig. 1.. The degree of spongy change ranged from Žq. to Žqqq . in DLB cases. In contrast, the degree of spongy change was Žq. in PD cases and Žy. – Žq. in ATD cases. With regards to the relationship between the degrees of spongy change and gliosis, the degree of gliosis ranged from Žq. to Žqqq . in DLB cases, exhibiting a reciprocal relationship with the degree of spongy change. The degree of gliosis was Žqq . – Žqqq . in PD cases and Žq. in ATD cases. The cortical and accessory basal nuclei among the other six amygdaloid nuclei also showed Žq. degree of spongy change in five of DLB cases, while there was no spongy change in the remaining DLB cases, PD cases and ATD cases.
Table 2 Neuropathological findings of the examined cases Case number
AM Spongy
Spheroid
Gliosis
SN pig. neuron
Gliosis
LC pig. neuron
Gliosis
DLB 1 2 3 4 5 6 7 8 9 Mean
q q qqq qqq qq qq qqq qq qqq
qq qq qqq qqq qq qqq qqq qq qq
qqq qqq q q q q q q qq
12 Žqqq. 79 Žqqq. 120 Žqq. 64 Žqqq. 153 Žqq. 157 Žqq. 94 Žqqq. 197 Žqq. 116 Žqq. 110.2
qq qq q q q qq q q qq
5 Žqqq. 6 Žqqq. 9 Žqqq. 9 Žqqq. 2 Žqqq. 8 Žqqq. 8 Žqqq. 9 Žqqq. 6 Žqqq. 6.9
q qq q q q q q q q
PD 1 2 3 4 Mean
q q q q
q q q q
qq qqq qqq qq
58 Žqqq. 50 Žqqq. 49 Žqqq. 81 Žqqq. 59.5
qq qqq qqq qq
18 Žqq. 3 Žqqq. 10 Žqq. 13 Žqq. 11.0
q q qq q
ATD 1 2 3 4 Mean
y y y y
y y y y
q q q q
261 Žq. 232 Žq. 290 Žq. 396 Žq. 294.8
" " " "
22 Žq. 23 Žq. 26 Žq. 50 Žq. 30.3
" " " "
AM: the central amygdaloid nucleus; SN: the substantia nigra; LC: the locus ceruleus; Spongy: spongy change; pig. neuron: pigmented neurons.
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E. Iseki et al.r Journal of the Neurological Sciences 185 (2001) 129–134
Fig. 1. Spongy change in the central amygdaloid nucleus; HE stain.
Fig. 3. a-Synuclein-positive spheroidsŽarrows. in the central amygdaloid nucleus; MDV2-immunostaining.
On ubiquitin-immunostained sections, all DLB cases revealed ubiquitin-positive spheroids composed of spherical or fusiform structures as well as a few LB in the central nucleus ŽFig. 2.. In DLB cases, the number of spheroids was Žqq . – Žqqq ., and was approximately parallel to the degree of spongy change. In contrast, the number of spheroids was Žq. in PD cases and Žy. in ATD cases. These ubiquitin-positive spheroids were also positively immunostained with anti-a-synuclein antibodies in DLB and PD cases ŽFig. 3.. On tyrosine hydroxylase
ŽTH.-immunostained sections, DLB cases revealed an uneven TH-immunoreactivity and some TH-positive spheroids in the central nucleus ŽFig. 4.. PD cases showed a diffuse decrease of TH-immunoreactivity, whereas ATD cases showed an even TH-immunoreactivity. The cortical and accessory basal nuclei of the above five DLB cases revealed Žq. number of ubiquitin-positive spheroids as well as many LB, but only slight TH-immunoreactivity without TH-positive spheroids.
Fig. 2. Ubiquitin-positive spheroids composed of spherical or fusiform structures Žarrows. in the central amygdaloid nucleus; MAB1510immunostaining.
Fig. 4. Tyrosine hydroxylaseŽTH.-positive spheroidsŽarrows. with an uneven TH-immunoreactivity in the central amygdaloid nucleus; MAB318-immunostaining.
E. Iseki et al.r Journal of the Neurological Sciences 185 (2001) 129–134
3.3. The relationship between the substantia nigra and locus ceruleus lesions and the amygdala lesion in DLB cases With regards to the relationship between the degree of neuronal loss in the substantia nigra and the degree of spongy change in the central nucleus, cases with Žqq . – Žqqq . neuronal loss showed Žqq . – Žqqq . spongy change. However, cases 1 and 2 showed Žq. spongy change, despite Žqqq . neuronal loss. There was no relationship between the degree of neuronal loss in the locus ceruleus and the degree of spongy change in the central nucleus and between the degree of neuronal loss in the substantia nigra or the locus ceruleus and the degree of spongy change in the cortical and accessory basal nuclei. 4. Discussion DLB is frequently accompanied by parkinsonism in addition to progressive dementia. Parkinsonism develops after dementia, frequently at the late clinical stage, in patients with the neocortical type, common form, of DLB, whereas parkinsonism is an initial symptom in patients with the neocortical type, pure form, of DLB as it is in PD patients w3,14x. These clinical findings may correspond to neuropathological findings shown in this study that most cases of the neocortical type, common form, of DLB revealed milder neuronal loss with gliosis in the substantia nigra than that of case of the neocortical type, pure form, of DLB and that of PD cases. We previously showed that spongy change or ubiquitin-positive spheroids in the II–III layers of the entorhinal cortex and in the cortico-medial nuclei of the amygdala derived from the degeneration of terminal or distal axons of the large pyramidal neurons in the entorhinal cortex w16–18x. Ubiquitin-positive spheroids described here represented spherical or fusiform structures, which were cross- or longitudinal-sections of degenerated neurites. In addition, the occurrence of spongy change or ubiquitin-positive spheroids, without significant gliosis in DLB brains, suggested that these large pyramidal neurons degenerated more rapidly in DLB brains than in ATD brains w17,18x. In this study, spongy change or ubiquitinpositive spheroids were found in the central amygdaloid nucleus in all DLB cases, and in the cortical and accessory basal amygdaloid nuclei in five DLB cases. The central nucleus showed mild gliosis in DLB cases with severe spongy change, whereas it showed severe gliosis in DLB and PD cases showing mild spongy change. There was no spongy change or gliosis found in the central nucleus in ATD cases. The number of ubiquitin-positive spheroids was parallel to the degree of spongy change. The central nucleus receives dense afferent fibers from the substantia nigra and locus ceruleus w6,7,13x. Ubiquitin-positive spheroids in the central nucleus were also a-synucleinpositive and tyrosine hydroxylase-positive, suggesting that
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ubiquitin-positive spheroids derive from the degeneration of terminal axons of LB-bearing dopaminergic or noradrenergic neurons in the substantia nigra or locus ceruleus w10,19x. In cases of the neocortical type, common form, of DLB, the degree of spongy change in the central nucleus correlated with the degree of neuronal loss in the substantia nigra, but not with the degree of neuronal loss in the locus ceruleus. However, the case of the neocortical type, pure form, of DLB as well as PD cases showed mild spongy change with severe gliosis in the central nucleus regardless of severe neuronal loss in the substantia nigra. These findings suggest that the central nucleus lesion in cases of the neocortical type, common form, of DLB is caused by more rapid neuronal degeneration in the substantia nigra at later clinical stage than that in the case of the neocortical type, pure form, of DLB and PD cases. In contrast, spongy change or ubiquitin-positive spheroids in the cortical and accessory basal nuclei appeared to represent the degeneration of terminal or distal axons from the regions other than the brainstem nuclei in DLB brains, especially the entorhinal cortex w16–18x, because the cortical and accessory basal nuclei revealed only slight TH-immunoreactivity without TH-positive spheroids, and there was no relationship between the degree of neuronal loss in the substantia nigra and the degree of spongy change in these amygdaloid nuclei. PD patients occasionally develop psychotic symptoms, such as visual hallucination and delusion, after the administration of L-DOPA w20,21x. Agid et al. w22x pointed out that dopamine deficiency due to the degeneration of dopaminergic neurons in the substantia nigra induced hypersensitivity of the dopamine receptor in the mesocortico-limbic dopaminergic system, which caused psychotic symptoms after administrating L-DOPA. These psychotic symptoms are similar to those in DLB patients, although psychotic symptoms persistently or recurrently develop without the administration of L-DOPA and delusion in DLB patients w1,4,5x. In this study, disturbance of the nigro-amygdaloid connections was considered to cause the central nucleus lesion. The central nucleus showed TH-positive spheroids with an uneven TH-immunoreactivity in DLB cases, whereas it showed a diffuse decrease in TH-immunoreactivity in PD cases, suggesting that fluctuating dopamine secretions in the nigro-amygdaloid connections in DLB brains may induce the fluctuation of psychotic symptoms in DLB patients. In a future study, we will examine the course of the central amygdaloid nucleus lesion as a sequence of damage to the substantia nigra in animals and the relationship between disturbance of nigro-amygdaloid connections and cognitive and behavioral changes. Acknowledgements This research was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan.
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References w1x Byrne E, Lennox G, Lowe J, Godwin-Austen RB. Diffuse Lewy body disease: clinical features in 15 cases. J Neurol, Neurosurg Psychiatry 1989;52:709–17. w2x McKeith IG, Galasko D, Kosaka K, et al. Consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy bodies ŽDLB.: report of the consortium on DLB international workshop. Neurology 1996;47:1113–24. w3x Kosaka K. Diffuse Lewy body disease in Japan. J Neurol 1990;237:197–204. w4x Perry RH, Irving D, Blessed G, et al. Senile dementia of Lewy body type. J Neurol Sci 1990;95:119–39. w5x Kratka L, Louis D, Schiffer RB. Psychiatric features in diffuse Lewy body disease: a clinicopathological study using Alzheimer’s disease and Parkinson’s disease comparison groups. Neurology 1996; 47:1148–52. w6x de Olmos JS. Amygdaloid nuclear complex. In: Paxinos G, editor. The Human Nervous system. San Diego: Academic Press, 1990: 583–710. w7x Parent A. Amygdala. Carpenter’s Human Neuroanatomy. 9th edn. Baltimore: Williams&Wilkins, 1996:773–80. w8x Sanghera MK, Rolls ET, Roper-Hall A. Visual responses of neurons in the dorsolateral amygdala of the alert monkey. Exp Neurol 1979;63:610–26. w9x Rolls ET. Neurophysiology and functions of the primate amygdala. In: Aggleton JP, editor. The Amygdala: Neurobiological Aspects of Emotion, Memory, and Mental Dysfunction. New York: Wiley-Liss, 1992:143–65. w10x Iseki E, Odawara T, Suzuki K, et al. A pathological study of Lewy bodies and senile changes in the amygdala in diffuse Lewy body disease. Neuropathology 1995;15:112–6. w11x Rezaie P, Cairns NJ, Chadwick A, Lantos PL. Lewy bodies are located preferentially in limbic areas in diffuse Lewy body disease. Neurosci Lett 1996;212:111–4.
w12x Crosby EC, Humphrey T. Studies of the vertebrate telencephalon: II. The nuclear pattern of the anterior olfactory nucleus, tuberculum olfactorium and the amygdaloid complex in adult man. J Comp Neurol 1941;74:309–52. w13x Lindvall O, Bjorklung A. Dopamine and norepinephrine-containing neuron system: their anatomy in the rat brain. In: Emson PC, editor. Chemical Neuroanatomy. New York: Raven Press, 1983:229–55. w14x Iseki E, Marui W, Kosaka K, et al. Clinocopathological multiplicity of dementia with Lewy bodies. Neuropathology 1999;19:386–94. w15x Arima K, Ueda K, Sunohara N, et al. Immunoelectron-microscopic demonstration of NACPra-synuclein-epitopes on the filamentous component of Lewy bodies in Parkinson’s disease and in dementia with Lewy bodies. Brain Res 1998;808:93–100. w16x Iseki E, Odawara T, Li F, et al. Age-related ubiquitin-positive granular structures in non-demented subjects and neurodegenerative disorders. J Neurol Sci 1996;142:25–9. w17x Iseki E, Li F, Kosaka K. Close relationship between spongiform change and ubiquitin-positive granular structures in diffuse Lewy body disease. J Neurol Sci 1997;146:53–7. w18x Iseki E, Li F, Odawara T, Kosaka K. Hippocampal pathology in diffuse Lewy body disease using ubiquitin-immunohistochemistry. J Neurol Sci 1997;149:165–9. w19x Iseki E, Marui W, Kosaka K, et al. Degenerative terminals of the perforant pathway are human a-synuclein-immunoreactive in the hippocampus of patients with diffuse Lewy body disease. Neurosci Lett 1998;258:81–4. w20x Moskovits C, Moses H, Klawans HL. Levodopa-induced psychosis: a kindling phenomenon. Am J Psychiatry 1978;135:669–75. w21x Rondot R, de Recondo J, Coignet A, et al. Mental disorders in Parkinson’s disease after treatment with L-DOPA. Adv Neurol 1984;40:259–69. w22x Agid Y, Ruberg M, Dubois B, Javoy-Agid F. Biochemical substrates of mental disturbance in Parkinson’s disease. In: Hassler RG, Christ JF, editors. Advances in Neurology, vol. 40. New York: Raven Press, 1984:211–8.
A neuropathological study of the disturbance of the nigro-amygdaloid connections in brains from patients with dementia with Lewy bodies Eizo Iseki a,) , Masanori Kato b, Wami Marui a , Kenji Ueda ´ c , Kenji Kosaka a a
Department of Psychiatry, Yokohama City UniÕersity School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan b Soga Hospital, 148 Sogagishi, Odawara 250-0203, Japan c Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156-8585, Japan Received 12 October 2000; received in revised form 8 February 2001; accepted 12 February 2001
Abstract We neuropathologically and immunohistochemically investigated characteristics of the central amygdaloid nucleus lesion and its relationship with the substantia nigra lesion in dementia with Lewy bodies ŽDLB. brains. Nine DLB, four Parkinson’s disease ŽPD. and four Alzheimer-type dementia ŽATD. cases were examined. The degree of neuronal loss in the substantia nigra was Žq. – Žqqq . in DLB cases, Žqqq . in PD cases and Žq. in ATD cases. All DLB cases showed spongy change and ubiquitin-positive spheroids in the central nucleus. The degree of spongy change was Žq. – Žqqq . in DLB cases, Žq. in PD cases and Žy. – Žq. in ATD cases, which was correlated with the degree of neuronal loss in the substantia nigra in DLB cases. The number of ubiquitin-positive spheroids was parallel to the degree of spongy change. The central nucleus receives dense dopaminergic fibers from the substantia nigra. Many ubiquitin-positive spheroids were also positive to a-synuclein and tyrosine-hydroxylase, suggesting that they derive from the degeneration of terminal or distal axons of Lewy body-bearing dopaminergic neurons in the substantia nigra. The disturbance of the dopaminergic connections from the substantia nigra to the central nucleus may be responsible for psychotic symptoms in DLB patients. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Dementia with Lewy bodies; Substantia nigra; Central amygdaloid nucleus; Spongy change; Spheroid; Immunohistochemistry; Psychotic symptom
1. Introduction Dementia with Lewy bodies ŽDLB. is the second most frequent neurodegenerative dementing disorder after Alzheimer-type dementia ŽATD. w1x. The clinical features include progressive dementia as a central feature, and fluctuating cognition, visual hallucination and parkinsonism as core features. The presence or absence of each core feature leads to clinical diagnosis of probable DLB or possible DLB w2x. The occurrence of Lewy bodies ŽLB. is the only feature essential for pathological diagnosis of DLB, although Lewy-related neurites, Alzheimer pathology, neuronal loss in the brainstem nuclei and spongy change are also frequently seen in DLB brains w2x. Parkinsonism is mainly caused by neuronal loss in the substantia nigra in DLB patients as well as Parkinson’s ) Corresponding author. Tel.: q81-45-787-2667; fax: q81-45-7832540. E-mail address: [email protected] ŽE. Iseki..
disease ŽPD. patients w3x. However, it remains unclear what lesions cause psychotic symptoms such as fluctuating cognition and visual hallucination in DLB patients. These psychotic symptoms are similar to those seen after administrating L-DOPA to PD patients w1,4,5x. The amygdala, a major constituent of the limbic system, has the fiber connections with the cerebral cortex, hippocampus, olfactory system, thalamus, hypothalamus, brainstem nuclei, etc. w6,7x, and is thought to be related to emotional, sensory and cognitive functions w7–9x. In DLB brains, the amygdala is the most preferential area of the cortical LB w10,11x. The central amygdaloid nucleus among seven nuclei constituting the amygdala w12x receives the most dense afferent fibers from the substantia nigra and locus ceruleus w6,7,13x. The central nucleus shows many ubiquitin-positive spheroids, which derive from the degeneration of terminal or distal axons in DLB brains w10x. Therefore, the disturbance of dopaminergic connections from the substantia nigra to the central nucleus may be responsible for psychotic symptoms in DLB patients.
0022-510Xr01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 5 1 0 X Ž 0 1 . 0 0 4 8 1 - 6
E. Iseki et al.r Journal of the Neurological Sciences 185 (2001) 129–134
130
In this study, we neuropathologically and immunohistochemically investigated characteristics of the central nucleus lesion and its relationship with the sustantia nigra lesion in DLB brains, and discussed the relationship between these lesions and psychotic symptoms in DLB patients.
2. Materials and methods This study was performed in nine pathologically verified DLB cases w2x. For comparison, four PD cases and four ATD cases were examined. Clinical data of the examined cases are shown in Table 1. In DLB cases, case 1 corresponded to the neocortical type, pure form without Alzheimer pathology, and cases 2–9 corresponded to the neocortical type, common form with Alzheimer pathology w14x. Brains were fixed in 4% paraformaldehyde in 0.1 molrl phosphate buffer ŽpH 7.4.. A coronal slice of the cerebral hemisphere through the level of the mammilary body, and two horizontal slices of the midbrain through the red nucleus and the pons through the oculomotor nucleus, were embedded in paraffin. The 6-mm-thick sections were stained with hematoxylin–eosin ŽHE., Kluver–Barrera ¨ ŽKB. and methenamine–silver ŽMS.. The sections were also immunostained with anti-GFAP Žpolyclonal, rabbit,
1:8000; Dako, Denmark., anti-ubiquitin ŽMAB1510: monoclonal, mouse, 1:8000; Chemicon, USA., anti-a-synuclein ŽMDV2: polyclonal, rabbit, 1:4000; PQE3: polyclonal, rabbit, 1:4000. w15x and anti-tyrosine hydroxylase ŽMAB318: monoclonal, mouse, 1:100; Chemicon. antibodies. Immunolabeling was detected using the avidin-biotinylated HRP complex ŽABC. method ŽElite Kit, Vector, USA. and visualized with DAB ŽSigma, USA.. The degree of spongy change was determined on the HE section by counting the number of vacuoles in microscope fields Žfield size: 0.66 mm 2 . at a 400 = magnification, and was graded from Žy. to Žqqq .: Žy. absent, Žq. - 5, Žqq . 5–10, Žqqq . ) 10. The degree of gliosis was determined on the GFAP-immunostained section by counting the number of GFAP-positive astroglia at a 200 = magnification, and was graded from Ž". to Žqqq .: Ž". - 5, Žq. 5–10, Žqq . 10–15, Žqqq . ) 15. The number of spheroids was determined on the ubiquitin-immunostained section by counting the number of ubiquitin-positive spheroids at a 200 = magnification, and was graded from Žy. to Žqqq .: Žy. absent, Žq. - 5, Žqq . 5–10, Žqqq . ) 10. Spongy change and spheroids were evaluated in the amygdala, and gliosis in the substantia nigra, locus ceruleus and amygdala. The degree of neuronal loss in the substantia nigra was determined by counting the total number of pigmented neurons in each quadrant of one side of the KB section, and was
Table 1 Clinical data of the examined cases Case number
Sex
Age Žyears old.
DLB 1 2 3 4 5 6 7 8 9 Mean
Duration Žyears.
M M M F F M M M M
50 67 73 74 79 79 81 84 88 75.0
7 5 3 2 3 3 6 4 2 3.9
PD 1 2 3 4 Mean
F M F M
57 76 81 90 76.0
18 9 15 2 11.0
ATD 1 2 3 4 Mean
M F F F
82 85 89 94 87.5
3 4 9 4 5.0
Dementia
Parkinsonism
Psychotic symptoms
Brain weight Žg.
qq qq qq qq qq qq qq qq q
qqq qq qq qq q qq q qq q
y y q q q q q q q
1050 1030 900 1300 1170 1165 1120 1060 1145 1104.4
y y y y
qqq qqq qqq qq
y y y y
1270 1140 1050 1150 1152.5
qqq qqq qqq qq
y y y y
y y y y
1200 900 1040 1070 1052.5
DLB: dementia with Lewy bodies; PD: Parkinson’s disease; ATD: Alzheimer-type dementia. The degree of dementia or parkinsonism: absent Žy., mild Žq., moderate Žqq., severe Žqqq.. Psychotic symptoms represent the presence or absence of fluctuating cognition and visual hallucination.
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graded from Žq. to Žqqq .: Žq. ) 200, Žqq . 100– 200, Žqqq . - 100. The degree of neuronal loss in the locus ceruleus was determined by counting the number of pigmented neurons on one side of the KB section, and was graded from Žq. to Žqqq .: Žq. ) 20, Žqq . 10–20, Žqqq . - 10.
3. Results Neuropathological findings in the examined cases are shown in Table 2. 3.1. The substantia nigra and locus ceruleus lesions In DLB cases, the degree of neuronal loss in the substantia nigra ranged from Žq. to Žqqq .. The mean number of pigmented neurons in DLB cases was 110.2, corresponding to Žqq . in the degree of neuronal loss. In PD cases, all cases showed Žqqq . in the degree of neuronal loss, and the mean number of pigmented neurons was 59.5. In ATD cases, all cases showed Žq. in the degree of neuronal loss, and the mean number of pigmented neurons was 294.8. The degree of neuronal loss in the locus ceruleus was Žqqq . in all DLB cases and Žqq . in all PD cases, and the mean number of pigmented neurons was 6.9 in DLB cases and 11.0 in PD cases. In ATD cases, all cases
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showed Žq. in the degree of neuronal loss, and the mean number of pigmented neurons was 30.3. With regard to the relationship between the degrees of neuronal loss and gliosis, the degree of gliosis in the substantia nigra was Žq. – Žqq . in DLB cases, Žqq . – Žqqq . in PD cases and Ž". in ATD cases, which was approximately parallel to the degree of neuronal loss. The degree of gliosis in the locus ceruleus was Žq. – Žqq . in DLB cases, Žq. – Žqq . in PD cases and Ž". in ATD cases, showing no apparent relationship with the degree of neuronal loss. 3.2. The amygdala lesion All DLB cases showed spongy change in the central nucleus on HE sections ŽFig. 1.. The degree of spongy change ranged from Žq. to Žqqq . in DLB cases. In contrast, the degree of spongy change was Žq. in PD cases and Žy. – Žq. in ATD cases. With regards to the relationship between the degrees of spongy change and gliosis, the degree of gliosis ranged from Žq. to Žqqq . in DLB cases, exhibiting a reciprocal relationship with the degree of spongy change. The degree of gliosis was Žqq . – Žqqq . in PD cases and Žq. in ATD cases. The cortical and accessory basal nuclei among the other six amygdaloid nuclei also showed Žq. degree of spongy change in five of DLB cases, while there was no spongy change in the remaining DLB cases, PD cases and ATD cases.
Table 2 Neuropathological findings of the examined cases Case number
AM Spongy
Spheroid
Gliosis
SN pig. neuron
Gliosis
LC pig. neuron
Gliosis
DLB 1 2 3 4 5 6 7 8 9 Mean
q q qqq qqq qq qq qqq qq qqq
qq qq qqq qqq qq qqq qqq qq qq
qqq qqq q q q q q q qq
12 Žqqq. 79 Žqqq. 120 Žqq. 64 Žqqq. 153 Žqq. 157 Žqq. 94 Žqqq. 197 Žqq. 116 Žqq. 110.2
qq qq q q q qq q q qq
5 Žqqq. 6 Žqqq. 9 Žqqq. 9 Žqqq. 2 Žqqq. 8 Žqqq. 8 Žqqq. 9 Žqqq. 6 Žqqq. 6.9
q qq q q q q q q q
PD 1 2 3 4 Mean
q q q q
q q q q
qq qqq qqq qq
58 Žqqq. 50 Žqqq. 49 Žqqq. 81 Žqqq. 59.5
qq qqq qqq qq
18 Žqq. 3 Žqqq. 10 Žqq. 13 Žqq. 11.0
q q qq q
ATD 1 2 3 4 Mean
y y y y
y y y y
q q q q
261 Žq. 232 Žq. 290 Žq. 396 Žq. 294.8
" " " "
22 Žq. 23 Žq. 26 Žq. 50 Žq. 30.3
" " " "
AM: the central amygdaloid nucleus; SN: the substantia nigra; LC: the locus ceruleus; Spongy: spongy change; pig. neuron: pigmented neurons.
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Fig. 1. Spongy change in the central amygdaloid nucleus; HE stain.
Fig. 3. a-Synuclein-positive spheroidsŽarrows. in the central amygdaloid nucleus; MDV2-immunostaining.
On ubiquitin-immunostained sections, all DLB cases revealed ubiquitin-positive spheroids composed of spherical or fusiform structures as well as a few LB in the central nucleus ŽFig. 2.. In DLB cases, the number of spheroids was Žqq . – Žqqq ., and was approximately parallel to the degree of spongy change. In contrast, the number of spheroids was Žq. in PD cases and Žy. in ATD cases. These ubiquitin-positive spheroids were also positively immunostained with anti-a-synuclein antibodies in DLB and PD cases ŽFig. 3.. On tyrosine hydroxylase
ŽTH.-immunostained sections, DLB cases revealed an uneven TH-immunoreactivity and some TH-positive spheroids in the central nucleus ŽFig. 4.. PD cases showed a diffuse decrease of TH-immunoreactivity, whereas ATD cases showed an even TH-immunoreactivity. The cortical and accessory basal nuclei of the above five DLB cases revealed Žq. number of ubiquitin-positive spheroids as well as many LB, but only slight TH-immunoreactivity without TH-positive spheroids.
Fig. 2. Ubiquitin-positive spheroids composed of spherical or fusiform structures Žarrows. in the central amygdaloid nucleus; MAB1510immunostaining.
Fig. 4. Tyrosine hydroxylaseŽTH.-positive spheroidsŽarrows. with an uneven TH-immunoreactivity in the central amygdaloid nucleus; MAB318-immunostaining.
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3.3. The relationship between the substantia nigra and locus ceruleus lesions and the amygdala lesion in DLB cases With regards to the relationship between the degree of neuronal loss in the substantia nigra and the degree of spongy change in the central nucleus, cases with Žqq . – Žqqq . neuronal loss showed Žqq . – Žqqq . spongy change. However, cases 1 and 2 showed Žq. spongy change, despite Žqqq . neuronal loss. There was no relationship between the degree of neuronal loss in the locus ceruleus and the degree of spongy change in the central nucleus and between the degree of neuronal loss in the substantia nigra or the locus ceruleus and the degree of spongy change in the cortical and accessory basal nuclei. 4. Discussion DLB is frequently accompanied by parkinsonism in addition to progressive dementia. Parkinsonism develops after dementia, frequently at the late clinical stage, in patients with the neocortical type, common form, of DLB, whereas parkinsonism is an initial symptom in patients with the neocortical type, pure form, of DLB as it is in PD patients w3,14x. These clinical findings may correspond to neuropathological findings shown in this study that most cases of the neocortical type, common form, of DLB revealed milder neuronal loss with gliosis in the substantia nigra than that of case of the neocortical type, pure form, of DLB and that of PD cases. We previously showed that spongy change or ubiquitin-positive spheroids in the II–III layers of the entorhinal cortex and in the cortico-medial nuclei of the amygdala derived from the degeneration of terminal or distal axons of the large pyramidal neurons in the entorhinal cortex w16–18x. Ubiquitin-positive spheroids described here represented spherical or fusiform structures, which were cross- or longitudinal-sections of degenerated neurites. In addition, the occurrence of spongy change or ubiquitin-positive spheroids, without significant gliosis in DLB brains, suggested that these large pyramidal neurons degenerated more rapidly in DLB brains than in ATD brains w17,18x. In this study, spongy change or ubiquitinpositive spheroids were found in the central amygdaloid nucleus in all DLB cases, and in the cortical and accessory basal amygdaloid nuclei in five DLB cases. The central nucleus showed mild gliosis in DLB cases with severe spongy change, whereas it showed severe gliosis in DLB and PD cases showing mild spongy change. There was no spongy change or gliosis found in the central nucleus in ATD cases. The number of ubiquitin-positive spheroids was parallel to the degree of spongy change. The central nucleus receives dense afferent fibers from the substantia nigra and locus ceruleus w6,7,13x. Ubiquitin-positive spheroids in the central nucleus were also a-synucleinpositive and tyrosine hydroxylase-positive, suggesting that
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ubiquitin-positive spheroids derive from the degeneration of terminal axons of LB-bearing dopaminergic or noradrenergic neurons in the substantia nigra or locus ceruleus w10,19x. In cases of the neocortical type, common form, of DLB, the degree of spongy change in the central nucleus correlated with the degree of neuronal loss in the substantia nigra, but not with the degree of neuronal loss in the locus ceruleus. However, the case of the neocortical type, pure form, of DLB as well as PD cases showed mild spongy change with severe gliosis in the central nucleus regardless of severe neuronal loss in the substantia nigra. These findings suggest that the central nucleus lesion in cases of the neocortical type, common form, of DLB is caused by more rapid neuronal degeneration in the substantia nigra at later clinical stage than that in the case of the neocortical type, pure form, of DLB and PD cases. In contrast, spongy change or ubiquitin-positive spheroids in the cortical and accessory basal nuclei appeared to represent the degeneration of terminal or distal axons from the regions other than the brainstem nuclei in DLB brains, especially the entorhinal cortex w16–18x, because the cortical and accessory basal nuclei revealed only slight TH-immunoreactivity without TH-positive spheroids, and there was no relationship between the degree of neuronal loss in the substantia nigra and the degree of spongy change in these amygdaloid nuclei. PD patients occasionally develop psychotic symptoms, such as visual hallucination and delusion, after the administration of L-DOPA w20,21x. Agid et al. w22x pointed out that dopamine deficiency due to the degeneration of dopaminergic neurons in the substantia nigra induced hypersensitivity of the dopamine receptor in the mesocortico-limbic dopaminergic system, which caused psychotic symptoms after administrating L-DOPA. These psychotic symptoms are similar to those in DLB patients, although psychotic symptoms persistently or recurrently develop without the administration of L-DOPA and delusion in DLB patients w1,4,5x. In this study, disturbance of the nigro-amygdaloid connections was considered to cause the central nucleus lesion. The central nucleus showed TH-positive spheroids with an uneven TH-immunoreactivity in DLB cases, whereas it showed a diffuse decrease in TH-immunoreactivity in PD cases, suggesting that fluctuating dopamine secretions in the nigro-amygdaloid connections in DLB brains may induce the fluctuation of psychotic symptoms in DLB patients. In a future study, we will examine the course of the central amygdaloid nucleus lesion as a sequence of damage to the substantia nigra in animals and the relationship between disturbance of nigro-amygdaloid connections and cognitive and behavioral changes. Acknowledgements This research was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan.
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