Movement Disorders Vol. 5 , No.4, 1990, pp. 286-293 0 1990 Movement Disorder Society
Neuropathological Studies in a Mutant Hamster Model of Paroxysmal Dystonia Ulrich Wahnschaffe, Gabriele Fredow, *Peter Heintz, and Wolfgang Loscher Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, and *Division of Nuclear Medicine and Biophysics, Radiological Center, Medical School, Hannover, F.R.G.
Summary: Dystonic movements in a mutation of the Syrian golden hamster, named d f " , have several features in common with clinically observed paroxysmal dystonic choreoathetosis. In this study the CNS of the mutant hamsters and age-matched nondystonic controls was examined for morphological alterations at the age of 30 days, i.e., when the seventy of the dystonic syndrome is fully developed. Particular interest was directed to those brain regions (caudate nucleus, putamen, globus pallidus, ventrolateral thalamus) that are presumably involved in symptomatic dystonia of humans, as well as to regions (e.g., spinal cord, dorsal root ganglia, nucleus ruber) for which neuropathologically detectable lesions have been found previously in the dystonia musculorum mouse. The neuropathological investigation was carried out on routine paraffin histology on step sections of the whole brain and spinal cord. In addition, a silver impregnation method was used for detection of pre- and/or postsynaptic degeneration. Light microscopic examination, including morphometry, of the nervous tissue failed to reveal any morphological or morphometric differences between control and dystonic hamsters. The only abnormality that was found in several control and dystonic hamsters was hydrocephalus. Breeding studies using magnetic resonance imaging for detection of hydrocephalus showed that hydrocephalus was hereditary but not related to dystonia. Virus infections as a cause of hydrocephalus or dystonia could be excluded by serological analysis with determinations of various virus antibodies in hamster sera. The lack of neuropathological alterations related to dystonic movements in the present study in d P hamsters is comparable to most cases of human hereditary or idiopathic dystonia, which show dystonic movements in the absence of morphological alterations. Key Words: HamsterDystonia-Neuropathology-Silver method.
tonia (2). Symptomatic dystonias are associated with inherited neurological disorders such as Wilson's syndrome, or they are caused by lesions of the putamen, caudate nucleus, globus pallidus, or thalamus. The etiology of these lesions is infarction, hemorrhage, tumor, or arteriovenous malformation (3-5). These pathological findings in patients with symptomatic dystonia have led to the search for abnormalities in the basal ganglia in primary dystonia, although with different results. Micheli et al. (6) described sporadic paroxysmal dystonic choreoathetosis associated with bilateral calcification of
Dystonic movements are classified in three ways: by age at onset, by body distribution, and by etiology (1). The etiologic classification divides the causes of dystonia into two major divisions: primary (hereditary or idiopathic) and secondary (symptomatic) dystonia. Approximately one-third of all patients with dystonia have symptomatic dysAddress correspondence and reprint requests to Dr. U . Wahnschaffe, Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, Biinteweg 17, D-3000 Hannover 71, F.R.G.
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NEUROPATHOLOG Y IN D YSTONIC HAMSTER the globus pallidus. However, in most studies no qualitative abnormalities in cell somata, neuropil, or associated white matter were detected in the basal ganglia [for review see (7)]. With respect to animal models of dystonia, in the mouse mutant dystonia musculorum (8), lesions were found in peripheral and central sensory pathways (8-11), striatum (12), and nucleus ruber (13). In contrast, no obvious central and peripheral neuropathological lesions have been observed in the dt mutant rat (14). In a recent study (15), we described dystonic movements in a mutation of Syrian golden hamsters (Mesocricetus auratus). This mutant, named d f “ (see below), shows several features in common with clinically observed paroxysmal dystonic choreoathetosis, e.g., paroxysmal nature, same type and expression of motor disturbances and electromyogram alterations, lack of electroencephalogram alterations, response to benzodiazepines in a dosedependent fashion, and an autosomal recessive hereditary pattern (15). In the present study we investigated possible degeneration of neurons in the CNS of the d f “ mutant hamster by routine paraffin histology on step sections of the whole brain and spinal cord. A silver impregnation method (16) was used for detection of dendritic and axonal degeneration. Furthermore, we measured neuron density of the striatum, globus pallidus, ventrolateral thalamus, and nucleus ruber with standardized morphometric techniques, to detect cell loss that could have happened at an earlier age without leaving morphological alterations detectable with the abovementioned neuropathological methods. In addition to these traditional light microscopic techniques, magnetic resonance (MR) imaging was used to depict brain pathology in intact animals.
MATERIALS AND METHODS
Animals The mutant hamsters used for the present studies were obtained from breeding pairs of a recombinant subline of dystonic hamster (“UGA 700,” see below) that were provided by Prof. W. B. Iturrian (University of Georgia, College of Pharmacy, Athens, GA, U.S.A.). Originally, motor impairment had been observed in a small number of animals from an inbred line BIO 86.93 of hamsters (17). This trait was designated by the gene symbol sz, because
the abnormal movements were thought to represent epileptic seizures (17). The BIO 86.93s~hamsters arose from the inbred BIO 86.93 strain presumably from a point mutation, and therefore were considered to be coisogenic. Hamsters with such motor disturbances had been maintained by BIO-Research Consultants (Cambridge, MA, U.S.A.). Prof. Iturrian crossed sz hamsters from this BIO 86.93s~ strain with a normal inbred progenitor strain derived from stock LVG hamsters. Chi-square analysis of the phenotypes for each of the segregating generations indicated that the risk of motor disturbances is inherited as an autosomal recessive trait, confirming the original breeding studies of Yoon et al. (17). Homozygous expression of the mutant gene in the hybrid is essentially the same as initially described (17), but is carried on a different genetic background with increased vigor. Uniformity of the motor impairment has been increased in Prof. Iturrian’s department by brother-sister inbreeding of one of the sublines (“UGA 700”) obtained from a recombinant inbreeding study. Breeding pairs of this subline were also used for obtaining the hamsters used for the present studies. As mentioned in the introduction, electrophysiological, clinical, and pharmacological studies have shown that the motor disturbances in these hamsters are not epileptic but are dystonic (15). In this respect it should be noted that the motor disturbances originally described in BIO 86.93s~hamsters were not qualitatively different from those observed in the “UGA 700” hamsters, indicating that the BIO 86.93s~hamsters were probably also dystonic. In agreement with the laboratory that originally described the sz mutant hamsters (17), we therefore have proposed that the hamster mutation, i.e., the BIO 86.93s~trait and hamsters (such as the “UGA 700” line) derived from this trait should be renamed dystonic, and that the new gene symbol for these mutant hamsters should be dr‘“ (15), retaining the old symbol sz as a superscript to maintain continuity with the work published in the past. Nondystonic control hamsters used in this study were obtained by crossing heterozygous BIO 86.93s~hamsters with LVG hamsters and then using the offspring, which did not carry the gene for motor disturbances (as determined by recrossing) for further breeding.
MRI Because several dystonic and nondystonic hamsters exhibited hydrocephalus (see Results), breed-
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ing studies were undertaken to determine whether the hydrocephalus was hereditary and related to dystonia. To view hydrocephalus in breeding pairs and their offspring, MR imaging was used. For this purpose, the hamsters were anesthetized with pentobarbital (60 mg/kg) and scans were made with the MR imager Magnetom (Siemens, Erlangen, F.R.G.) using a field strength of 1 T, a high-resolution extremity coil, a three-dimensional gradient-echo sequence with a volume thickness of 32 mm (slice thickness 2 mm), a zoom factor of 1.3, aflip angle of 40", a time to echo of 11 ms, a repetition time of 40 ms, two data acquisitions, and a measuring time of 5.47 min. A 256 x 256 image matrix was used. The slice orientation was transverse. The cross wires were oriented on a point in the median line halfway between the eyes and the ears of the animal. The film was developed with a Cronet-T6-Processor (Du Pont, daylight developer) and photographs were made with a multispot camera (Siemens). Determination of Virus Antibodies As already mentioned, several dystonic and nondystonic hamsters exhibited hydrocephalus. Because virus-induced forms of hydrocephalus have been reported in hamsters (18), sera from four dystonic hamsters in which hydrocephalus had been detected by MR imaging were examined for the presence of antibodies to nine different viruses, i.e., a reovirus type 3, Theiler's encephalomyelitis virus, pneumonia virus of mice, Sendai virus, minute virus of mice, virus of lymphocytic choriomeningitis, Toolan H-1 virus, Simian virus 5, and mouse cytomegalovirus. In addition, sera were analyzed for antibodies to Bacillus piliformis (which induces Tyzzer's disease). Respective antibodies were determined by indirect immunofluorescence by Prof. Volker Kraft (Department of Virology, Central Institute for Laboratory Animal Breeding, Hannover, F.R.G.). Neuropathoiogical Studies with Light Microscopy Nonhydrocephalic dystonic and nondystonic hamsters were used for all neurohistological studies. Thirty dt"' mutant hamsters of both sexes and 15 control hamsters were used for paraffin histology at the age of 30 days. To test the possible effect of dystonic attacks themselves, dystonic movements were induced by handling and environmental stimuli (IS) in 15 mutant hamsters 7, 5, and 3 days before the neuropathological examinations, whereas the other 15 dystonic hamsters were left at the
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mother without induction of dystonic attacks until neuropathological examination. For neuropathology , all hamsters were anaesthetized with sodium pentobarbital (60 mg/kg) and were perfused intracardially with 0.15 M phosphate buffer (pH 7.35) for 60 s followed by 4% phosphate-buffered formaldehyde (pH 7.35) for 10 min. Two hours later, the brains were removed from the skull, postfixed in the same fixative overnight at 4"C, and processed for parafin embedding. Subsequently, serial sections of the entire brain were cut coronally at 7 pm, and every 20th section was mounted on a glass slide and was stained with cresyl violet or hematoxylin and eosin. In addition, in four dystonic hamsters and four controls, the vertebral columns, together with spinal cord, closely surrounding tissues containing dorsal root and ventral roots, and dorsal root ganglia were removed after perfusion, were postfixed for 24 days at 4"C, were decalcified in 4.5% hydrochloric acid and 9% formic acid for 8 days, and were processed for paraffin embedding. This tissue was cut and stained as described above. For the silver impregnation method of Gallyas et al. (16), eight control hamsters and eight tested df' hamsters of both sexes were perfused at the same age, and brains were postfixed for 3 days, washed with distilled water, and cut coronally in 80-pmthick serial frozen sections. Every third frozen section was stained with the silver method and was examined with a Zeiss light microscope with darkfield illumination. Morphometric Analysis For morphometric analysis, five control hamsters and five tested dtSZhamsters of both sexes were used at the same age. Hamsters were processed as described for paraffin histology and every third section was mounted on a glass slide and stained with cresyl violet. Neuron density was determined unilaterally in the dorsolateral part of the striatum 4.8 mm rostral of the interaural line (19), in the globus pallidum A = 4.1 mm rostral of the interaural line, in the ventrolateral thalamus (A = 3.4 mm), and in the red nucleus (A = 1.6 mm). Using a calibrated grid in the microscope ocular, counts were made at a magnification of x 250 in six nonoverlapping fields (area of one field = 7,500 pm2) in each sample section, and five sections were counted from each region for a total of 30 fields (total, 225,000 pm') per region of each hamster. The unit of count for neurons was based on cell nuclei. The distance between
NEUROPATHOLOGY IN D YSTONLC HAMSTER sections (214 pm) prevented counting the same nucleus twice. With the formula of Floderus (20) the number of neuron nuclei per unit volume (N,) was estimated by N , = N,/(D
+ t - 2h)
In this formula, D = mean nuclear diameter, t = the section thickness, h = the height of the smallest recognizable “cap,” and N A = the number of counted nuclei per unit test area. Because a proportion of nuclei will not have been cut through their maximum diameter, and the mean diameter (d) will be an underestimate of the actual mean diameter, the “true” nuclei diameter D was calculated from the mean diameter (6)and the section thickness ( t )using the formula given by Smolen et al. (21) as follows:
D=d
(
1-
(1 - 4 h ) d t+d
The height of lost caps (h) was determined by 2h
=
D - (D2 - do2)‘”
in which do represents the diameter of the smallest visible nuclear profile (22). Differences in nuclei number between control and dtSZhamsters were tested by using Student’s t test.
RESULTS Gross Morphological Changes and Serology Coronal brain sections of several animals of both the dystonic and the control groups showed hydrocephalus. Lateral ventricles of affected animals were grossly distended. Because of the distension and accompanying pressure of the lateral ventricles, there was an extensive displacement of various brain structures (septum, striatum, hippocampus, and cerebral cortex except lamina I and 11) in hydrocephalic animals. Third and fourth ventricles were not affected. These marked alterations, however, were not associated with any behavioral abnormalities in the nondystonic control hamsters. Furthermore, hydrocephalic dystonic hamsters did not differ in the severity of their dystonia from dystonic hamsters without hydrocephalus. Because hydrocephalus in hamsters can occur either hereditarily or in response to virus infections, subsequent experiments were undertaken to examine the cause(s) of the hydrocephalus.
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Experiments with MR imaging showed that by this technique of brain imaging, hydrocephalic hamsters could be easily separated from nonhydrocephalic animals (Fig. 1). Breeding experiments with hydrocephalic and nonhydrocephalic male and female animals indicated that the hydrocephalus is inherited as an autosomal recessive trait, but is not genetically associated with dystonia. Indeed, the number of hydrocephalic animals in the segregating generations derived from matings between nonhydrocephalic animals with dystonia decreased, but all animals still exhibited dystonia. Serological determinations gave no evidence of virus infections that could be related to hydrocephalus or other brain abnormalities. None of the 10 virus antibody assays used showed a reaction. Because the behavioral and breeding experiments had indicated that hydrocephalus and dystonia were not related, all subsequent histological studies described below were carried out in hamsters without ventricular abnormalities. Neurohistological Alterations Light microscopic examination of paraffinembedded nervous tissue failed to reveal morphological differences between normal and dystonic hamsters. Furthermore, no differences were found between dystonic hamsters in which dystonic attacks had been induced and those that were left at the mother. Specifically, the dystonic hamsters showed none of the degenerative changes seen in the spinal cord, dorsal root ganglia, striatum or nucleus ruber of the mouse mutant dystonia musculorum (1G12). For illustration, pictures of paraffin sections of dorsal root ganglia of control and dystonic hamsters are shown in Fig. 2. Furthermore, no morphological alterations were observed in any part of the extrapyramidal system, e.g., frontal cortex, striatum, globus pallidus, entopenducular nucleus, ventrolateral thalamus, subthalamic nucleus, substantia nigra, and cerebellum. Light microscopy of silver-impregnated frozen sections showed no silver deposits that could indicate degenerative alterations of pre- or postsynaptic structures in any part of the abovementioned localizations. Accumulation of silver deposits observed in several brain regions, e.g., the plexiform layer and glomeruli of the olfactory bulb, and the molecular layer of the cerebellum or ventromedial thalamic nucleus, are naturally occurring argentophilic neuronal structures or the consequence of a perma-
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FIG. 1. Coronal magnetic resonance images of the heads (arrowheads point to the brains) from a dystonic hamster with hydrocephalus (right images) and a dystonic hamster without hydrocephalus (left images). The arrows point to the distended lateral ventricles. Coronal sections shown for both animals are in comparable planes. The bars indicate 10 mm. The animals did not differ from each other in the type and seventy of their dystonic movements.
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FIG. 2. Hematoxylin and eosin-stained parafin sections of the dorsal root ganglia after decalcification.Axons (A) and neurons (arrows) do not show any signs of degeneration and lysis in a dystonic (a) and a control (b) hamster. (a) X225; (b) X 190.
nent synaptic reorganization (23) and were also observed in control hamsters.
showed no significant differences between controls and dystonic df" hamsters (Fig. 3).
Morphometry
DISCUSSION
Morphometric analysis of several brain regions supported the light microscopic observations. Evaluation of neuron densities in the striatum, globus pallidus, ventrolateral thalamus, and nucleus ruber
In the present pathological studies of a hereditary dystonia in hamsters, no differences between nondystonic and dystonic animals were found. In several hamsters with and without dystonia, hydro-
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292 Neurons/0,001 mm3 1
120-
100-
80-
60-
40 -
Str Pal V It FIG.3. Number of neurons per unit volume of 0.001 mm3 in the nucleus ruber (Rub), striaturn (Str), globus pallidum (Pal), and ventrolateral thalamic nucleus (Vlt) of age-matched dystonic and nondystonic hamsters. Results are shown as mean t SD of five animals per group. White bars, control; hatched bars, dystonic.
cephalus with marked distension of the lateral ventricles was observed either during neuropathological examination or by MR imaging in vivo. This hydrocephalus, however, was not associated with abnormal movements in controls, thus indicating that dystonia and hydrocephalus were not related. Indeed, breeding studies showed that the nonhydrocephalic offspring of nonhydrocephalic dystonic parents did not differ in time course or severity of dystonic movements from dystonic hamsters with hydrocephalus. A hereditary form of hydrocephalus has been described previously in a mutation of Syrian golden hamsters (24). In contrast to the hydrocephalic hamsters described by Yoon and Slaney (24), as already mentioned above, nondystonic hamsters with hydrocephalus did not exhibit behavioral abnormalities, showed normal development and fertility, and did not die earlier than nonhydrocephalic hamsters did. Virus infections as a cause of hydrocephalus (18) could be excluded by serological examination and by the breeding experiments.
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Because the breeding studies indicated that hydrocephalus was neither the cause of dystonia nor genetically associated with dystonia, subsequent histological studies were restricted to nonhydrocephalic animals. In these studies no light microscopic morphological alterations could be observed in the df" mutant hamster at the age of 30 days, i.e., when the severity of the dystonic syndrome is fully developed (15). No differences were detected between the dystonic hamster group in which dystonic attacks were repeatedly induced by new environment and handling, and the dystonic hamster group left at the mother until perfusion. If there exist morphological changes besides degenerative changes of neuronal somata that could be observed with usual paraffin histology, these pre- and/or postsynaptic degenerations should be detected with the silver-impregnation method. The sensitivity of this method has been shown in a combined light and electron microscopical study (25). Although these light microscopical examinations indicated absence of obvious degenerative changes in dystonic hamsters at the age of 30 days, it was still possible that cell loss had happened at an earlier age without leaving detectable morphological changes. In this case, differences in neuron density between control and dystonic hamsters should be detected by morphometric analysis, i.e. a unit volume of neuronal tissue should contain fewer neurons and more glial cells. Evaluation of neuron density showed no evidence of such neuron loss in the df" hamster. Particular interest was directed to those brain regions that were involved in symptomatic dystonia of humans (3), e.g. nucleus caudatus, putamen, globus pallidus, and ventrolateral thalamus. However, the light microscopical studies of the nervous system of the df" hamster did not reveal alterations in these regions. Furthermore, neuropathological alterations previously described in the dystonia musculorum mouse, which exhibits neuropathologically detectable lesions in the striatum, nucleus ruber (12), spinal cord, and dorsal root ganglia ( l l ) , were not observed in the dystonic hamster. The morphological integrity of the extrapyramidal system in df" mutant hamsters also distinguishes the disease from degenerative diseases of the basal ganglia, such as Huntington's chorea or Parkinson's disease. The clinical symptoms of the dtSZhamster (15) and the results of the present study suggest that the d f Zhamster and most cases of human hereditary or
NEUROPATHOLOGY IN DYSTONIC HAMSTER
idiopathic dystonia have common findings: presence of altered muscle function in the absence of morphological alterations (7,26). Similar results were documented by Lorden et al. (14) in their study on the dystonic mutant rat. In their study also no morphological lesions of neural tissue have been observed with routine light microscopy. However, neurochemical investigations showed significantly elevated cerebellar norepinephrine levels in the dt rat (14). Similar neurochemical results have been shown in two cases of human idiopathic torsion dystonia (27). Norepinephrine levels were increased in several brainstem regions, whereas no neuropathological alteration could be observed. These results suggest that abnormality in hereditary or idiopathic dystonia may be at a sublightmicroscopic level, e.g., functional loss of axon terminals as determined by ultrastructural immunocytochemistry, or that a biochemical disturbance may be the cause of the disease. Acknowledgment: We thank Prof. Kraft for the serological determinations and nical assistance.
Mrs. C. Bartling for skillful tech-
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neuropathy in mice (dystonia musculorum). Bruin 1972; 95529-536. 11. Al-Ali SY, Al-Zuhair AGH. Fine structural study of the spinal cord and spinal ganglia in mice afflicted with a hereditary sensory neuropathy, dystonia musculorum. J Submicrosc Cytol Pathol 1989;21:737-748. 12. Messer A, Strominger NL. An allele of the mouse mutant dystonia musculorum exhibits lesions in red nucleus and striatum. Neuroscience 1980;5:543-549. 13. Stanley E, Messer A, Strominger NL. Effects of age and strain differences on the red nucleus of the mouse mutant dystonia musculorum. Anat Rec 1983;206:313-318. 14. Lorden JF, McKeon TW, Baker HJ, Cox N, Walkley SU. Characterisation of the rat mutant dystonic (dt): a new animal model of dystonia musculorum deformans. J Neurosci 1984;4:1925-1932. 15. Loscher W. Fisher JE. Schmidt D. Fredow G. Honack D. Ituman WB. The sz mutant hamster: a genetic model of epilepsy or of paroxysmal dystonia? Movement Disorders 1989;4:219-232. 16. Gallyas F, Wolff JR, Bottcher H, Zaborszky L. A reliable and sensitive method to localise terminal degeneration and lysosornes in the central nervous system. Stain Technol 1980;55:299-306. 17. Yoon CH, Peterson JS, Corrow D. Spontaneous seizures: a new mutation in Syrian golden hamster. J Hered 1976;67: 115-1 16. 18. Ennis FA, Hopps HE, Douglas RD, Meyer HM Jr. Hydrocephalus in hamsters: induction by natural and attenuated mumps viruses. J Infect Dis 1969;119:75-79. 19. Knigge KM, Joseph SA. A stereotaxic atlas of the brain of the eolden hamster. In: Hoffmann RA. Robinson PF. Magalhaes H, eds. The golden hamster-its biology and use in medical research. Ames, Iowa: Iowa State University Press, 1968:28&3 13. 20. Floderus S. Untersuchungen uber den Bau der menschlichen Hypophyse mit besonderer Beriicksichtigung der qualitativen mikrobiologischen Verhaltnisse. Acta Pathol Microbiol Scand 1944;53:1-276. 21. Smolen AJ, Wright LL, Cunningham TJ. Neuron numbers in the superior cervical sympathetic ganglion of the rat: a critical comparison of methods for cell counting. J Neurocytol 1983;12:739-750. 22. Weibel ER. Stereological methods, vol 1. Practical methods for biological rnorphometry. New York: Academic Press, ~
Y
REFERENCES 1 . Fahn S. Concept and classification of dystonia. In: Fahn S, Marsden CD, Calne DB, eds. Dystonia 2. New York: Raven Press, 1988:l-9. (Advances in neurology; vol50). 2. Fahn S, Marsden CD, Calne DB. Classification and investigation of dystonia. In: Marsden CD, Fahn S, eds. Movement disorders 2. London: Buttenvorths, 1987:332-358. 3. Obeso JA, GimCnez-Rolditn S. Clinicopathological correlation in symptomatic dystonia. In: Fahn S, Marsden CD, Calne DB, eds. Dystonia 2. New York: Raven Press, 1988:113-122. (Advances in neurology; vol50). 4. Marsden CD, Obeso JA, Zarranz JJ, Lang AE. The anatomical basis of symptomatic hemidystonia. Brain 1985;108:463483. 5 . Zeeman W, Whithlock CC. Symptomatic dystonias. In: Vinken PJ, Bruyn GW, eds. Handbook of neurology, vol6. Amsterdam: North Holland, 1968544566. 6. Micheli F, Pardal MMF, Parera IC, Giannaula R. Sporadic paroxysmal dystonic choreoathetosis associated with basal ganglia calcifications. Ann Neurol 1986;20:750. 7. Hedreen JC, Zweig RM, DeLong MR, Whitehouse PJ, Price DL. Primary dystonias: a review of the pathology and suggestions for new directions of study. In: Fahn S, Marsden CD, Calne DB, eds. Dystonia 2. New York: Raven Press, 1988:123-132. (Advances in neurology; vol 50). 8. Duchen LW, Falconer DS, Strich SJ. Dystonia musculorum. A hereditary neuropathy of mice affecting mainly sensory pathways. J Physiol 1963;165:7-9. 9. Duchen LW, Strich SJ, Falconer DS. Clinical and pathological studies of an hereditary neuropathy in mice (dystonia musculorum). Brain 1964;87:367-378. 10. Janota I. Ultrastructural studies of an hereditary sensory
1 n70 1717.
23. de Olmos JS, Ebbesson OE, Heimer L. Silver methods for the impregnation of degenerating axoplasm. In: Heimer L, Robards MJ, eds. Neuroanatomical tract-tracing methods. New York: Plenum Press, 1981:117-170. 24. Yoon CH, Slaney J. Hydrocephalus: a new mutation in the Syrian golden hamster. J Hered 1972;63:344-346. 25. Wahnschae U, Esslen J. Structural evidence for the neurotoxicity of methylamphetamine in the frontal cortex of gerbils (Meriones unguiculatus): a light and electron microscopical study. Brain Res 1985;337:299-310. 26. Marsden CD. Dystonia: the spectrum of the disease. In: Yahr MD, ed. The basal ganglia. New York: Raven Press, 1976:351-367. (Association for Research in Nervous and Mental Disease [ARNMD], research publication, vol 55). 27. Homykiewicz 0, Kish SJ, Becker LE, Farley I, Shannak K. Biochemical evidence for brain neurotransmitter changes in idiopathic torsion dystonia (dystonia musculorum deformans). In: Fahn S, Marsden CD, Calne DB. Dystonia 2. New York: Raven Press, 1988:157-166. (Advances in Neurology; vol 50).
Movement Disorders, Vol. 5 , No. 4, 1990
Neuropathological Studies in a Mutant Hamster Model of Paroxysmal Dystonia Ulrich Wahnschaffe, Gabriele Fredow, *Peter Heintz, and Wolfgang Loscher Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, and *Division of Nuclear Medicine and Biophysics, Radiological Center, Medical School, Hannover, F.R.G.
Summary: Dystonic movements in a mutation of the Syrian golden hamster, named d f " , have several features in common with clinically observed paroxysmal dystonic choreoathetosis. In this study the CNS of the mutant hamsters and age-matched nondystonic controls was examined for morphological alterations at the age of 30 days, i.e., when the seventy of the dystonic syndrome is fully developed. Particular interest was directed to those brain regions (caudate nucleus, putamen, globus pallidus, ventrolateral thalamus) that are presumably involved in symptomatic dystonia of humans, as well as to regions (e.g., spinal cord, dorsal root ganglia, nucleus ruber) for which neuropathologically detectable lesions have been found previously in the dystonia musculorum mouse. The neuropathological investigation was carried out on routine paraffin histology on step sections of the whole brain and spinal cord. In addition, a silver impregnation method was used for detection of pre- and/or postsynaptic degeneration. Light microscopic examination, including morphometry, of the nervous tissue failed to reveal any morphological or morphometric differences between control and dystonic hamsters. The only abnormality that was found in several control and dystonic hamsters was hydrocephalus. Breeding studies using magnetic resonance imaging for detection of hydrocephalus showed that hydrocephalus was hereditary but not related to dystonia. Virus infections as a cause of hydrocephalus or dystonia could be excluded by serological analysis with determinations of various virus antibodies in hamster sera. The lack of neuropathological alterations related to dystonic movements in the present study in d P hamsters is comparable to most cases of human hereditary or idiopathic dystonia, which show dystonic movements in the absence of morphological alterations. Key Words: HamsterDystonia-Neuropathology-Silver method.
tonia (2). Symptomatic dystonias are associated with inherited neurological disorders such as Wilson's syndrome, or they are caused by lesions of the putamen, caudate nucleus, globus pallidus, or thalamus. The etiology of these lesions is infarction, hemorrhage, tumor, or arteriovenous malformation (3-5). These pathological findings in patients with symptomatic dystonia have led to the search for abnormalities in the basal ganglia in primary dystonia, although with different results. Micheli et al. (6) described sporadic paroxysmal dystonic choreoathetosis associated with bilateral calcification of
Dystonic movements are classified in three ways: by age at onset, by body distribution, and by etiology (1). The etiologic classification divides the causes of dystonia into two major divisions: primary (hereditary or idiopathic) and secondary (symptomatic) dystonia. Approximately one-third of all patients with dystonia have symptomatic dysAddress correspondence and reprint requests to Dr. U . Wahnschaffe, Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, Biinteweg 17, D-3000 Hannover 71, F.R.G.
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NEUROPATHOLOG Y IN D YSTONIC HAMSTER the globus pallidus. However, in most studies no qualitative abnormalities in cell somata, neuropil, or associated white matter were detected in the basal ganglia [for review see (7)]. With respect to animal models of dystonia, in the mouse mutant dystonia musculorum (8), lesions were found in peripheral and central sensory pathways (8-11), striatum (12), and nucleus ruber (13). In contrast, no obvious central and peripheral neuropathological lesions have been observed in the dt mutant rat (14). In a recent study (15), we described dystonic movements in a mutation of Syrian golden hamsters (Mesocricetus auratus). This mutant, named d f “ (see below), shows several features in common with clinically observed paroxysmal dystonic choreoathetosis, e.g., paroxysmal nature, same type and expression of motor disturbances and electromyogram alterations, lack of electroencephalogram alterations, response to benzodiazepines in a dosedependent fashion, and an autosomal recessive hereditary pattern (15). In the present study we investigated possible degeneration of neurons in the CNS of the d f “ mutant hamster by routine paraffin histology on step sections of the whole brain and spinal cord. A silver impregnation method (16) was used for detection of dendritic and axonal degeneration. Furthermore, we measured neuron density of the striatum, globus pallidus, ventrolateral thalamus, and nucleus ruber with standardized morphometric techniques, to detect cell loss that could have happened at an earlier age without leaving morphological alterations detectable with the abovementioned neuropathological methods. In addition to these traditional light microscopic techniques, magnetic resonance (MR) imaging was used to depict brain pathology in intact animals.
MATERIALS AND METHODS
Animals The mutant hamsters used for the present studies were obtained from breeding pairs of a recombinant subline of dystonic hamster (“UGA 700,” see below) that were provided by Prof. W. B. Iturrian (University of Georgia, College of Pharmacy, Athens, GA, U.S.A.). Originally, motor impairment had been observed in a small number of animals from an inbred line BIO 86.93 of hamsters (17). This trait was designated by the gene symbol sz, because
the abnormal movements were thought to represent epileptic seizures (17). The BIO 86.93s~hamsters arose from the inbred BIO 86.93 strain presumably from a point mutation, and therefore were considered to be coisogenic. Hamsters with such motor disturbances had been maintained by BIO-Research Consultants (Cambridge, MA, U.S.A.). Prof. Iturrian crossed sz hamsters from this BIO 86.93s~ strain with a normal inbred progenitor strain derived from stock LVG hamsters. Chi-square analysis of the phenotypes for each of the segregating generations indicated that the risk of motor disturbances is inherited as an autosomal recessive trait, confirming the original breeding studies of Yoon et al. (17). Homozygous expression of the mutant gene in the hybrid is essentially the same as initially described (17), but is carried on a different genetic background with increased vigor. Uniformity of the motor impairment has been increased in Prof. Iturrian’s department by brother-sister inbreeding of one of the sublines (“UGA 700”) obtained from a recombinant inbreeding study. Breeding pairs of this subline were also used for obtaining the hamsters used for the present studies. As mentioned in the introduction, electrophysiological, clinical, and pharmacological studies have shown that the motor disturbances in these hamsters are not epileptic but are dystonic (15). In this respect it should be noted that the motor disturbances originally described in BIO 86.93s~hamsters were not qualitatively different from those observed in the “UGA 700” hamsters, indicating that the BIO 86.93s~hamsters were probably also dystonic. In agreement with the laboratory that originally described the sz mutant hamsters (17), we therefore have proposed that the hamster mutation, i.e., the BIO 86.93s~trait and hamsters (such as the “UGA 700” line) derived from this trait should be renamed dystonic, and that the new gene symbol for these mutant hamsters should be dr‘“ (15), retaining the old symbol sz as a superscript to maintain continuity with the work published in the past. Nondystonic control hamsters used in this study were obtained by crossing heterozygous BIO 86.93s~hamsters with LVG hamsters and then using the offspring, which did not carry the gene for motor disturbances (as determined by recrossing) for further breeding.
MRI Because several dystonic and nondystonic hamsters exhibited hydrocephalus (see Results), breed-
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ing studies were undertaken to determine whether the hydrocephalus was hereditary and related to dystonia. To view hydrocephalus in breeding pairs and their offspring, MR imaging was used. For this purpose, the hamsters were anesthetized with pentobarbital (60 mg/kg) and scans were made with the MR imager Magnetom (Siemens, Erlangen, F.R.G.) using a field strength of 1 T, a high-resolution extremity coil, a three-dimensional gradient-echo sequence with a volume thickness of 32 mm (slice thickness 2 mm), a zoom factor of 1.3, aflip angle of 40", a time to echo of 11 ms, a repetition time of 40 ms, two data acquisitions, and a measuring time of 5.47 min. A 256 x 256 image matrix was used. The slice orientation was transverse. The cross wires were oriented on a point in the median line halfway between the eyes and the ears of the animal. The film was developed with a Cronet-T6-Processor (Du Pont, daylight developer) and photographs were made with a multispot camera (Siemens). Determination of Virus Antibodies As already mentioned, several dystonic and nondystonic hamsters exhibited hydrocephalus. Because virus-induced forms of hydrocephalus have been reported in hamsters (18), sera from four dystonic hamsters in which hydrocephalus had been detected by MR imaging were examined for the presence of antibodies to nine different viruses, i.e., a reovirus type 3, Theiler's encephalomyelitis virus, pneumonia virus of mice, Sendai virus, minute virus of mice, virus of lymphocytic choriomeningitis, Toolan H-1 virus, Simian virus 5, and mouse cytomegalovirus. In addition, sera were analyzed for antibodies to Bacillus piliformis (which induces Tyzzer's disease). Respective antibodies were determined by indirect immunofluorescence by Prof. Volker Kraft (Department of Virology, Central Institute for Laboratory Animal Breeding, Hannover, F.R.G.). Neuropathoiogical Studies with Light Microscopy Nonhydrocephalic dystonic and nondystonic hamsters were used for all neurohistological studies. Thirty dt"' mutant hamsters of both sexes and 15 control hamsters were used for paraffin histology at the age of 30 days. To test the possible effect of dystonic attacks themselves, dystonic movements were induced by handling and environmental stimuli (IS) in 15 mutant hamsters 7, 5, and 3 days before the neuropathological examinations, whereas the other 15 dystonic hamsters were left at the
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mother without induction of dystonic attacks until neuropathological examination. For neuropathology , all hamsters were anaesthetized with sodium pentobarbital (60 mg/kg) and were perfused intracardially with 0.15 M phosphate buffer (pH 7.35) for 60 s followed by 4% phosphate-buffered formaldehyde (pH 7.35) for 10 min. Two hours later, the brains were removed from the skull, postfixed in the same fixative overnight at 4"C, and processed for parafin embedding. Subsequently, serial sections of the entire brain were cut coronally at 7 pm, and every 20th section was mounted on a glass slide and was stained with cresyl violet or hematoxylin and eosin. In addition, in four dystonic hamsters and four controls, the vertebral columns, together with spinal cord, closely surrounding tissues containing dorsal root and ventral roots, and dorsal root ganglia were removed after perfusion, were postfixed for 24 days at 4"C, were decalcified in 4.5% hydrochloric acid and 9% formic acid for 8 days, and were processed for paraffin embedding. This tissue was cut and stained as described above. For the silver impregnation method of Gallyas et al. (16), eight control hamsters and eight tested df' hamsters of both sexes were perfused at the same age, and brains were postfixed for 3 days, washed with distilled water, and cut coronally in 80-pmthick serial frozen sections. Every third frozen section was stained with the silver method and was examined with a Zeiss light microscope with darkfield illumination. Morphometric Analysis For morphometric analysis, five control hamsters and five tested dtSZhamsters of both sexes were used at the same age. Hamsters were processed as described for paraffin histology and every third section was mounted on a glass slide and stained with cresyl violet. Neuron density was determined unilaterally in the dorsolateral part of the striatum 4.8 mm rostral of the interaural line (19), in the globus pallidum A = 4.1 mm rostral of the interaural line, in the ventrolateral thalamus (A = 3.4 mm), and in the red nucleus (A = 1.6 mm). Using a calibrated grid in the microscope ocular, counts were made at a magnification of x 250 in six nonoverlapping fields (area of one field = 7,500 pm2) in each sample section, and five sections were counted from each region for a total of 30 fields (total, 225,000 pm') per region of each hamster. The unit of count for neurons was based on cell nuclei. The distance between
NEUROPATHOLOGY IN D YSTONLC HAMSTER sections (214 pm) prevented counting the same nucleus twice. With the formula of Floderus (20) the number of neuron nuclei per unit volume (N,) was estimated by N , = N,/(D
+ t - 2h)
In this formula, D = mean nuclear diameter, t = the section thickness, h = the height of the smallest recognizable “cap,” and N A = the number of counted nuclei per unit test area. Because a proportion of nuclei will not have been cut through their maximum diameter, and the mean diameter (d) will be an underestimate of the actual mean diameter, the “true” nuclei diameter D was calculated from the mean diameter (6)and the section thickness ( t )using the formula given by Smolen et al. (21) as follows:
D=d
(
1-
(1 - 4 h ) d t+d
The height of lost caps (h) was determined by 2h
=
D - (D2 - do2)‘”
in which do represents the diameter of the smallest visible nuclear profile (22). Differences in nuclei number between control and dtSZhamsters were tested by using Student’s t test.
RESULTS Gross Morphological Changes and Serology Coronal brain sections of several animals of both the dystonic and the control groups showed hydrocephalus. Lateral ventricles of affected animals were grossly distended. Because of the distension and accompanying pressure of the lateral ventricles, there was an extensive displacement of various brain structures (septum, striatum, hippocampus, and cerebral cortex except lamina I and 11) in hydrocephalic animals. Third and fourth ventricles were not affected. These marked alterations, however, were not associated with any behavioral abnormalities in the nondystonic control hamsters. Furthermore, hydrocephalic dystonic hamsters did not differ in the severity of their dystonia from dystonic hamsters without hydrocephalus. Because hydrocephalus in hamsters can occur either hereditarily or in response to virus infections, subsequent experiments were undertaken to examine the cause(s) of the hydrocephalus.
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Experiments with MR imaging showed that by this technique of brain imaging, hydrocephalic hamsters could be easily separated from nonhydrocephalic animals (Fig. 1). Breeding experiments with hydrocephalic and nonhydrocephalic male and female animals indicated that the hydrocephalus is inherited as an autosomal recessive trait, but is not genetically associated with dystonia. Indeed, the number of hydrocephalic animals in the segregating generations derived from matings between nonhydrocephalic animals with dystonia decreased, but all animals still exhibited dystonia. Serological determinations gave no evidence of virus infections that could be related to hydrocephalus or other brain abnormalities. None of the 10 virus antibody assays used showed a reaction. Because the behavioral and breeding experiments had indicated that hydrocephalus and dystonia were not related, all subsequent histological studies described below were carried out in hamsters without ventricular abnormalities. Neurohistological Alterations Light microscopic examination of paraffinembedded nervous tissue failed to reveal morphological differences between normal and dystonic hamsters. Furthermore, no differences were found between dystonic hamsters in which dystonic attacks had been induced and those that were left at the mother. Specifically, the dystonic hamsters showed none of the degenerative changes seen in the spinal cord, dorsal root ganglia, striatum or nucleus ruber of the mouse mutant dystonia musculorum (1G12). For illustration, pictures of paraffin sections of dorsal root ganglia of control and dystonic hamsters are shown in Fig. 2. Furthermore, no morphological alterations were observed in any part of the extrapyramidal system, e.g., frontal cortex, striatum, globus pallidus, entopenducular nucleus, ventrolateral thalamus, subthalamic nucleus, substantia nigra, and cerebellum. Light microscopy of silver-impregnated frozen sections showed no silver deposits that could indicate degenerative alterations of pre- or postsynaptic structures in any part of the abovementioned localizations. Accumulation of silver deposits observed in several brain regions, e.g., the plexiform layer and glomeruli of the olfactory bulb, and the molecular layer of the cerebellum or ventromedial thalamic nucleus, are naturally occurring argentophilic neuronal structures or the consequence of a perma-
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FIG. 1. Coronal magnetic resonance images of the heads (arrowheads point to the brains) from a dystonic hamster with hydrocephalus (right images) and a dystonic hamster without hydrocephalus (left images). The arrows point to the distended lateral ventricles. Coronal sections shown for both animals are in comparable planes. The bars indicate 10 mm. The animals did not differ from each other in the type and seventy of their dystonic movements.
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FIG. 2. Hematoxylin and eosin-stained parafin sections of the dorsal root ganglia after decalcification.Axons (A) and neurons (arrows) do not show any signs of degeneration and lysis in a dystonic (a) and a control (b) hamster. (a) X225; (b) X 190.
nent synaptic reorganization (23) and were also observed in control hamsters.
showed no significant differences between controls and dystonic df" hamsters (Fig. 3).
Morphometry
DISCUSSION
Morphometric analysis of several brain regions supported the light microscopic observations. Evaluation of neuron densities in the striatum, globus pallidus, ventrolateral thalamus, and nucleus ruber
In the present pathological studies of a hereditary dystonia in hamsters, no differences between nondystonic and dystonic animals were found. In several hamsters with and without dystonia, hydro-
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292 Neurons/0,001 mm3 1
120-
100-
80-
60-
40 -
Str Pal V It FIG.3. Number of neurons per unit volume of 0.001 mm3 in the nucleus ruber (Rub), striaturn (Str), globus pallidum (Pal), and ventrolateral thalamic nucleus (Vlt) of age-matched dystonic and nondystonic hamsters. Results are shown as mean t SD of five animals per group. White bars, control; hatched bars, dystonic.
cephalus with marked distension of the lateral ventricles was observed either during neuropathological examination or by MR imaging in vivo. This hydrocephalus, however, was not associated with abnormal movements in controls, thus indicating that dystonia and hydrocephalus were not related. Indeed, breeding studies showed that the nonhydrocephalic offspring of nonhydrocephalic dystonic parents did not differ in time course or severity of dystonic movements from dystonic hamsters with hydrocephalus. A hereditary form of hydrocephalus has been described previously in a mutation of Syrian golden hamsters (24). In contrast to the hydrocephalic hamsters described by Yoon and Slaney (24), as already mentioned above, nondystonic hamsters with hydrocephalus did not exhibit behavioral abnormalities, showed normal development and fertility, and did not die earlier than nonhydrocephalic hamsters did. Virus infections as a cause of hydrocephalus (18) could be excluded by serological examination and by the breeding experiments.
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Because the breeding studies indicated that hydrocephalus was neither the cause of dystonia nor genetically associated with dystonia, subsequent histological studies were restricted to nonhydrocephalic animals. In these studies no light microscopic morphological alterations could be observed in the df" mutant hamster at the age of 30 days, i.e., when the severity of the dystonic syndrome is fully developed (15). No differences were detected between the dystonic hamster group in which dystonic attacks were repeatedly induced by new environment and handling, and the dystonic hamster group left at the mother until perfusion. If there exist morphological changes besides degenerative changes of neuronal somata that could be observed with usual paraffin histology, these pre- and/or postsynaptic degenerations should be detected with the silver-impregnation method. The sensitivity of this method has been shown in a combined light and electron microscopical study (25). Although these light microscopical examinations indicated absence of obvious degenerative changes in dystonic hamsters at the age of 30 days, it was still possible that cell loss had happened at an earlier age without leaving detectable morphological changes. In this case, differences in neuron density between control and dystonic hamsters should be detected by morphometric analysis, i.e. a unit volume of neuronal tissue should contain fewer neurons and more glial cells. Evaluation of neuron density showed no evidence of such neuron loss in the df" hamster. Particular interest was directed to those brain regions that were involved in symptomatic dystonia of humans (3), e.g. nucleus caudatus, putamen, globus pallidus, and ventrolateral thalamus. However, the light microscopical studies of the nervous system of the df" hamster did not reveal alterations in these regions. Furthermore, neuropathological alterations previously described in the dystonia musculorum mouse, which exhibits neuropathologically detectable lesions in the striatum, nucleus ruber (12), spinal cord, and dorsal root ganglia ( l l ) , were not observed in the dystonic hamster. The morphological integrity of the extrapyramidal system in df" mutant hamsters also distinguishes the disease from degenerative diseases of the basal ganglia, such as Huntington's chorea or Parkinson's disease. The clinical symptoms of the dtSZhamster (15) and the results of the present study suggest that the d f Zhamster and most cases of human hereditary or
NEUROPATHOLOGY IN DYSTONIC HAMSTER
idiopathic dystonia have common findings: presence of altered muscle function in the absence of morphological alterations (7,26). Similar results were documented by Lorden et al. (14) in their study on the dystonic mutant rat. In their study also no morphological lesions of neural tissue have been observed with routine light microscopy. However, neurochemical investigations showed significantly elevated cerebellar norepinephrine levels in the dt rat (14). Similar neurochemical results have been shown in two cases of human idiopathic torsion dystonia (27). Norepinephrine levels were increased in several brainstem regions, whereas no neuropathological alteration could be observed. These results suggest that abnormality in hereditary or idiopathic dystonia may be at a sublightmicroscopic level, e.g., functional loss of axon terminals as determined by ultrastructural immunocytochemistry, or that a biochemical disturbance may be the cause of the disease. Acknowledgment: We thank Prof. Kraft for the serological determinations and nical assistance.
Mrs. C. Bartling for skillful tech-
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neuropathy in mice (dystonia musculorum). Bruin 1972; 95529-536. 11. Al-Ali SY, Al-Zuhair AGH. Fine structural study of the spinal cord and spinal ganglia in mice afflicted with a hereditary sensory neuropathy, dystonia musculorum. J Submicrosc Cytol Pathol 1989;21:737-748. 12. Messer A, Strominger NL. An allele of the mouse mutant dystonia musculorum exhibits lesions in red nucleus and striatum. Neuroscience 1980;5:543-549. 13. Stanley E, Messer A, Strominger NL. Effects of age and strain differences on the red nucleus of the mouse mutant dystonia musculorum. Anat Rec 1983;206:313-318. 14. Lorden JF, McKeon TW, Baker HJ, Cox N, Walkley SU. Characterisation of the rat mutant dystonic (dt): a new animal model of dystonia musculorum deformans. J Neurosci 1984;4:1925-1932. 15. Loscher W. Fisher JE. Schmidt D. Fredow G. Honack D. Ituman WB. The sz mutant hamster: a genetic model of epilepsy or of paroxysmal dystonia? Movement Disorders 1989;4:219-232. 16. Gallyas F, Wolff JR, Bottcher H, Zaborszky L. A reliable and sensitive method to localise terminal degeneration and lysosornes in the central nervous system. Stain Technol 1980;55:299-306. 17. Yoon CH, Peterson JS, Corrow D. Spontaneous seizures: a new mutation in Syrian golden hamster. J Hered 1976;67: 115-1 16. 18. Ennis FA, Hopps HE, Douglas RD, Meyer HM Jr. Hydrocephalus in hamsters: induction by natural and attenuated mumps viruses. J Infect Dis 1969;119:75-79. 19. Knigge KM, Joseph SA. A stereotaxic atlas of the brain of the eolden hamster. In: Hoffmann RA. Robinson PF. Magalhaes H, eds. The golden hamster-its biology and use in medical research. Ames, Iowa: Iowa State University Press, 1968:28&3 13. 20. Floderus S. Untersuchungen uber den Bau der menschlichen Hypophyse mit besonderer Beriicksichtigung der qualitativen mikrobiologischen Verhaltnisse. Acta Pathol Microbiol Scand 1944;53:1-276. 21. Smolen AJ, Wright LL, Cunningham TJ. Neuron numbers in the superior cervical sympathetic ganglion of the rat: a critical comparison of methods for cell counting. J Neurocytol 1983;12:739-750. 22. Weibel ER. Stereological methods, vol 1. Practical methods for biological rnorphometry. New York: Academic Press, ~
Y
REFERENCES 1 . Fahn S. Concept and classification of dystonia. In: Fahn S, Marsden CD, Calne DB, eds. Dystonia 2. New York: Raven Press, 1988:l-9. (Advances in neurology; vol50). 2. Fahn S, Marsden CD, Calne DB. Classification and investigation of dystonia. In: Marsden CD, Fahn S, eds. Movement disorders 2. London: Buttenvorths, 1987:332-358. 3. Obeso JA, GimCnez-Rolditn S. Clinicopathological correlation in symptomatic dystonia. In: Fahn S, Marsden CD, Calne DB, eds. Dystonia 2. New York: Raven Press, 1988:113-122. (Advances in neurology; vol50). 4. Marsden CD, Obeso JA, Zarranz JJ, Lang AE. The anatomical basis of symptomatic hemidystonia. Brain 1985;108:463483. 5 . Zeeman W, Whithlock CC. Symptomatic dystonias. In: Vinken PJ, Bruyn GW, eds. Handbook of neurology, vol6. Amsterdam: North Holland, 1968544566. 6. Micheli F, Pardal MMF, Parera IC, Giannaula R. Sporadic paroxysmal dystonic choreoathetosis associated with basal ganglia calcifications. Ann Neurol 1986;20:750. 7. Hedreen JC, Zweig RM, DeLong MR, Whitehouse PJ, Price DL. Primary dystonias: a review of the pathology and suggestions for new directions of study. In: Fahn S, Marsden CD, Calne DB, eds. Dystonia 2. New York: Raven Press, 1988:123-132. (Advances in neurology; vol 50). 8. Duchen LW, Falconer DS, Strich SJ. Dystonia musculorum. A hereditary neuropathy of mice affecting mainly sensory pathways. J Physiol 1963;165:7-9. 9. Duchen LW, Strich SJ, Falconer DS. Clinical and pathological studies of an hereditary neuropathy in mice (dystonia musculorum). Brain 1964;87:367-378. 10. Janota I. Ultrastructural studies of an hereditary sensory
1 n70 1717.
23. de Olmos JS, Ebbesson OE, Heimer L. Silver methods for the impregnation of degenerating axoplasm. In: Heimer L, Robards MJ, eds. Neuroanatomical tract-tracing methods. New York: Plenum Press, 1981:117-170. 24. Yoon CH, Slaney J. Hydrocephalus: a new mutation in the Syrian golden hamster. J Hered 1972;63:344-346. 25. Wahnschae U, Esslen J. Structural evidence for the neurotoxicity of methylamphetamine in the frontal cortex of gerbils (Meriones unguiculatus): a light and electron microscopical study. Brain Res 1985;337:299-310. 26. Marsden CD. Dystonia: the spectrum of the disease. In: Yahr MD, ed. The basal ganglia. New York: Raven Press, 1976:351-367. (Association for Research in Nervous and Mental Disease [ARNMD], research publication, vol 55). 27. Homykiewicz 0, Kish SJ, Becker LE, Farley I, Shannak K. Biochemical evidence for brain neurotransmitter changes in idiopathic torsion dystonia (dystonia musculorum deformans). In: Fahn S, Marsden CD, Calne DB. Dystonia 2. New York: Raven Press, 1988:157-166. (Advances in Neurology; vol 50).
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