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An immunohistochemical investigation of the human neostriatum in huntington's disease.

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An Immunohstochemical Investigation
of the Human Neostriatum
in Huntington’s Disease
Satoshi Goto, MD,” Asao Hirano, MD,X and Rogelio R. Rojas-Corona, MDt
The neostriatum of 7 autopsied patients with Huntington’s disease (HD) was examined immunohistochemicdly using
purified antibody against calcineurin, which may be present only in the medium-size spinous neurons of the mammalian striatum. This study revealed a marked loss of calcineurin-positivecells in the caudate nucleus and the putamen in
dl HD patients, compared with control subjects, and there was some variation among the HD patients. Four HD
patients showed significantly lower density of calcineurin-positive cells in the caudate nucleus than in the putamen.
The remaining calcineurin-positive cells in the caudate nucleus and the putamen had a mosaic-like pattern, demonstrating a subregional difference in distribution. This finding suggests that there are subregional as well as compartmental differences in the vulnerability of the calcineurin-positivecells in the striatum of patients with HD.
Goto S, Hirano A, Rojas-Corona RR. An immunohistochemical investigation of the human neostriatum in
Huntington’s disease. Ann Neurol 1989;25:298-304
Huntington’s disease (HD) is a well-known hereditary
and neurodegenerative disorder characterized clinically by progressive chorea and dementia 11-31. The
neostriatum is the site most affected, with a greater
loss of medium-size neurons than large neurons 12-41.
Recently it was reported that medium-size spinous
neurons, which act as major projection neurons and
send their axons to the globus pallidus and the substantia nigra 151, might be specifically involved in HD 16,
7). In addition, marked depletion of striatal efferents
in the globus pallidus and the substantia nigra of HD
patients has been demonstrated neurochemically 18101 and immunohistochernically 111, 121. O n the
other hand, a subset of striatal neurons (i.e., a population of medium-size aspinous interneurons) has been 1
reported to be spared in HD patients 16, 13, 141.
These findings may reflect the difference of vulnerabil- I
ity among neuronal subsets in HD patients and may
contribute to an understanding of the pathogenesis of
Calcineurin (CaN) is a multifunctional Ca2+/calmeddin-regulated phosphoprotein phosphatase 115 , 161
that occurs in the highest concentrations in mammalian
brains, especially in the basal ganglia (including the
substantia nigra) 112, 17-191. We previously reported
that in the rat basal ganglia CaN is present only in the
From the Divisions of *Neuropathology and tImmunopathology,
Department of Pathology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY.
Received Jul 5, 1988, and in revised form Sep 14. Accepted for
publication Sep 14, 1988.
striatal neurons and in their projections to the globus
pallidus and the substantia nigra [191. In addition, we
reported findings from the brain of an HD patient,
which showed marked loss of neurons containing CaN
immunoreactivity in the strianun and marked depletion of CaN-immunoreactive nerve fibers in the globus
pallidus and the substantia nigra (i.e., striatal efferents)
1121. Finally, several morphological results indicated
that most CaN-immunoreactive striatal neurons might
belong to the medium-size spinous neurons in the rat
basal ganglia 120).
In this report, we describe the results of a morphometric and topographical study of the neostriatum
from 7 patients with HD. We used the CaN-immunostaining technique. Regional and subregional neuronal
involvement was demonstrated in each.
Materials and Methods
We examined the brains of 7 patients who had HD and the
brains of 4 elderly patients who had no neuropathology, as
summarized in the Table. Brain tissue was obtained at autopsy from these patients, fixed in 10% neutral formalin for
3 weeks, sliced coronally (sliced horizontally only in Patient
5), and embedded in paraffin. Neuropathological examinations using conventional techniques (hematoxylin and eosin,
Address correspondence to Dr Hirano, Division of Neuropathology, Montefiore Medical Center, 11 1 East 210th St, Bronx, NY
298 Copyright 0 1989 by the American Neurological Association
Immunohistochemical Results from the Neostriatum of Postmortem Brain Tissuea
Number of Calcineurin-Immunoreactive Neurons
Control subjects
Patients with
Huntington’s disease
Caudate Nucleus
269 2 20
268 k 11
261 k 16
8 177.3%)
* 8182.8%)
* 4 191.8%)
* 4 193.7%)
2 197.0%)
31 166.2%)
5 195.5%)
* 9E81.2951
* 6183.1%)
43 -t
31 2
15 2
142 2
13 5
21 184.2%)
12 E88.696)
3 194.4%)
27 147.8%)
7 195.2%)
“The numbers of calcineurin (CaN)-positive cells were determined by counting the cells containing brown peroxidase products in the caudate
nucleus and the putamen. In control subjects, the arbitrarily selected areas at the levels of the caudate-putaminal junction (Controls 1 and 2) and
the globus pallidus (Controls 3 and 4) were examined. Values represent mean 2 SD (n = 10). The percent reduction (in brackets) was
calculated as the mean number of CaN-positive cells in the caudate nucleus and the putamen of each patient with Huntington’s disease (HD)
divided by the mean number of them in the caudate nucleus and the putamen of control subjects, respectively. The percent reductions of
Patients 6 and 9 were calculated by the mean numbers of CaN-positive cells in the caudate nucleus (268 cells/mm*)and the putamen (267 cells/
mm2)of Controls 3 and 4. Also, those of other HD patients were calculated by the mean numbers of CaN-positive cells in the caudate nucleus
(269 cells/mm’) and the putamen (272 cells/mm*) of Controls 1 and 2.
Nissl, and Holzer stains) confirmed the clinical diagnosis of
HD. Immunohistochemical studies were done on 5-pm sections from paraffin-embedded tissues.
The antiserum against CaN was produced in a rabbit as described previously 118). Purified antibody by CaN-affinity
column chromatography was used. The monospecificity of
the antibody was assessed and confirmed 118, 21, 22). Immunoperoxidase staining procedures were carried out according to the instructions of the Vectastain ABC kit (Vector Labs, Burlingame, CA) [23), using 3,3’-diaminobenzidine as the chromogen. The staining specificity was assessed
and confirmed, as previously described 118, 201.
The numbers of CaN-positive neurons in the caudate nucleus and putamen of HD patients were determined by
counting the number of cells containing the brown reaction
product in 10 arbitrarily selected 1-mm2 areas using an eyepiece fitted with agrid at x 100 magnification. The examined
sections from Patients 5, 6, 7, 8, and 10 are illustrated in
Figures 4a, la, 4b, 4c, and 4d, respectively. The sections of
the striatum of Patients 9 and 11 were examined at the levels
of the globus pallidus and the caudate-putaminal junction,
respectively. In the control subjects, 10 arbitrarily selected
areas at the level of the caudate-putaminal junction (Controls
1 and 2) and the globus pallidus (Controls 3 and 4) were
examined in the same way.
The two-sample t test was used for the comparison between the stained cells in the caudate nucleus and in the
All counted neurons showed strong CaN immunoreactivity. The results of the immunohistochemical measurements are shown in the Table. In all HD patients,
a marked decrease in the numbers of the CaNimmunoreactive neurons was seen in both the caudate
nucleus and the putamen as compared to the control
subjects. The percent reduction was calculated on the
basis of control mean numbers at the appropriate level
(see the Table). The density of CaN-immunoreactive
cells in the caudate nucleus (n = 10) was significantly
smaller than that in the putamen (n = 10) in 4 H D
patients (Patient 7, p < 0.01; Patient 8, p < 0.001;
Patient 9, p < 0.005; Patient 10, p < 0.002). In the
other 3 HD patients, the differences of cell density
between the caudate nucleus and the putamen were
not statistically significant.
The immunohistochemical macroscopic findings in
the basal gangha of 2 normal control subjects are illustrated in Figures l c and Id. Under the light microscope, CaN-immunoreactive cells are essentially
evenly distributed throughout both the caudate nucleus and the putamen. The large neurons were all
devoid of CaN immunoreactivity. The CaN-positive
cells were all medium-size neurons, as reported previously [20] (Figs 2c and 2d). The CaN immunoreactivity was distributed throughout the neuronal cell bodies
and the processes, including synaptic endings.
Except for Patient 5 , all HD patients had marked
Goto et al: Immunohistochemical Investigation of Human Neostriatum in HD 2 9
300 Annals of Neurology Vol 25 No 3 March 1989
atrophy of the caudate nucleus with a remarkable loss
of medium-size neurons and replacement with astrocytes. The putamen was also atrophic but to a lesser
degree than the caudate nucleus. The remaining CaN
immunoreactivity was not homogeneous and showed a
mosaic-like pattern in both the caudate nucleus and the
putamen (Figs l a and lb). In addition, the decrease of
CaN immunoreactivity in the putamen was more
prominent in the lateral portion than in the medial
portion. Under the low-power light microscope, the
poorly immunoreactive areas contained only a few
CaN-positive cells and were occasionally surrounded
4Fig 1. Immunohistochemicalmacroscopicjindings in the basal
ganglia in Patient 6 at the kvel of the globus pallidus (a) and
the caudzte-putaminaljunction (b) ( x 6 before 12% reduction).
Also, comparable photomicrographs of the control subjects are disclosed (c, x 7 before 2% reduction, and d, x 4 before 2% enlargement). Note that the caudate nucleus and the putamen are
atrophic, and that CaN immunoreactivity is not homogetzeously
distributed and is shawing a mosaic-like pattern in both the
caudzte nucleus and the putamen. CN = caudate nucleus, Pu
= putamen, GP = globus pallidus.
Fig 2. Immunohistocbemical microscopicjindings with low-pawer
mugnifcation in the caudzte nucleus (a) and the putamen (b) in
Patient 6 ( X 60 before 3% reduction). Note that the poorly
CaN-immunoreactive areas are surrounded b./ the grouping of
the CaN-positive cells. Also, the photomicrographs of the caudate nucleus (c, x 45 before 3 % reduction) and the putamen (d,
x 45) in a control subject are provided.
by clusters of CaN-positive cells (Fig 2). Many remaining CaN-positive cells showed dysmorphic cellular features (Fig 3) but the reaction product was distributed
throughout the cell bodies and the processes in all of
the positive cells.
In each case, the distribution of the CaN-positive
cells revealed a mosaic-like arrangement (Fig 4). The
severity of neuronal involvement varied and subregional distribution of the remaining CaN-positive cells
differed from patient to patient.
Using an antibody to CaN,which may serve as a reliable marker for the medium-size spinous neurons
1201, we demonstrated immunohistochemically the
patterns of neuronal loss in the striatum of HD pa-
Goto et d: Immunohistochemical Investigation of Human Neostriatum in HD 301
Fig 3. Patient 6. Morphologicalfeatures of the remaining CaNpositive neurons in the putamen (a, b) and the cauahte nucleus
(c) ( x 480 before 34% reduction).The nearon indicated by
large arrow (a)preserves the normal morphological features of
the medium-size spinous type, whereas some neurons indicated by
small arrows (a, b, and c) show dysmorphicfeatures.
tients. It is noteworthy that the CaN epitopes were
well preserved and the surviving CaN-positive cells
showed strong immunoreactivity in all HD patients, as
well as in the control subjects.
Our findings are, in part, essentially consistent with
those from conventional neuropathological study of
neuronal involvement in HD patients. The loss of
CaN-positive cells in the striatum is not surprising,
since we previously identified them as part of the population of medium-size spinous neurons in the striatum
302 Annals of Neurology Vol 25 No 3 March 1989
[20) and these are known to be among cells lost in HD
patients [6, 7 , 141. The difference in percent reduction
of CaN-positive cells in each HD patient may be consistent with the variations reported in the degree of
severity of neuropathological changes in individual
HD patients 1241. In HD the caudate nucleus is usually reported to be more severely involved than the
putamen, but at least two studies 125, 261 showed the
reverse. The loss of CaN-positive cells displays the
same variation. Indeed, in some patients, no significant
difference could be found between the loss of CaNpositive cells in the caudate nucleus and that in the
putamen. On the other hand, the impression we get
from the gross examination is that in all our HD patients, except for Patient 5 , the caudate nucleus was
more atrophic than the putamen. This may suggest that
the extent of striatal atrophy is not always consistent
with the degree of reduction in the density of CaNpositive cells. In addition, the decrease of CaNpositive cells was more prominent in the lateral portion than in the medial portion of the putamen. This is
considered to reflect the subregional difference in vulnerability among CaN-positive cells in the HD striatum and this may be supported by previously reported
findings 1271.
The major finding in this report is the mosaic-like
involvement of the CaN-positive cells in the striatum
of HD patients. The mosaic-like pattern of involvement of the medium-size spinous neurons has not
been previously revealed. This finding may indicate
that there is a compartmental difference in the way in
which the CaN-positive cells are involved in HD, and
is reminiscent of Graybiel's novel neurochemical compartmentalization 128, 291. Recently, it was reported
that the mammalian neostriatum has 2 neurochemically distinct compartments 128-321. Gerfen 1311 demonstrated the neostriatal mosaic in the rat brain as the
compartmentalization (matrix and patch compartments) of corticostriatal input and striatonigral output
systems. In addition, there is the neurotoxin hypothesis for explanation of neuronal involvement in the striatal degeneration. Neurotoxins such as glutamate and
kainic acid 133, 341 and quinolinic acid 135-37) are
reported to damage the striatal neurons (medium-size
spinous neurons) depending on the corticostriatal
glutamatergic inputs 136, 38). These reported findings
may indicate that corticostriatal glutamatergic inputs,
which have an ability to damage the striatal mediumsize spinous neurons, may be localized, causing a mosaic-like arrangement. This may account for the compartmental distribution of the preserved CaN-positive
cells in the striatum of our HD patients. Unfortunately, we cannot, at this time, state that the CaNpositive cells are more preserved in the matrix or in
the patches in the striatum of HD patients. This is
currently under investigation.
Fig 4. Diagrams of the distribution of the CaN-positive neurons
in the neostriatum of Patient 5 (A), Patient 7 (B), Patient 8
(C), and Patient 10 (0)( x 4.5 before 3% reduction).
Goto et al: Immunohistochemical Investigation of Human Neostriatum in HD 303
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investigation, immunohistochemical, neostriatum, disease, huntington, human
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