Dystonias (Dys), which manifest either as an isolated abnormal motor phenomenon or in combination with other motor and nonmotor signs of more complex neuropsychiatric syndromes, represent a relatively frequent movement disorder.1,2 Dys, in fact, represent the third most frequent movement disorder after essential tremor (ET) and Parkinson's disease (PD).3
A number of efforts have led to various classification systems of Dys,4–8 attempts to characterize their biological bases,9–13 and multiple proposals of clinical guidelines for their treatment.14–16 However, although important progress has been achieved in understanding some of the neurological bases of Dys, especially in terms of molecular mechanisms17 and genetic causes,18 the distinct characteristic neuropathologic features of Dys remain poorly defined19 with some very rare exceptions.20,21
There have been few clinicopathologic correlation studies of postmortem brain tissues from Dys patients, with most analyses described in sporadic case-reports.22–27 These neuropathologic studies have proven to be mostly inconclusive due to the clinical heterogeneity of Dys cases examined, the different classification systems used, and more importantly, difficulty in obtaining brains from clinically well-characterized Dys patients.28 However, a few neuropathologic studies have begun to shed light on possible neuropathologic and morphometric aspects of specific types of Dys, such as DYT1-dystonia (DYT1),29 cervical dystonia (CD)30, and dopa-responsive dystonia (DRD).31 Rostasy and colleagues assessed brains from five patients with DYT1 (GAG-deletions) and three Dys patients without GAG-deletions and reported larger and more compacted pigmented neurons in the substantia nigra (SN) of both types of Dys compared to control subjects (C).29 In contrast, Göttle et al. described smaller SN neurons in Lesch-Nyhan disease.32 In general, these lines of evidence support the hypothesis that the SN is indeed involved in Dys pathogenesis.33–35
The primary aim of this study was to investigate morphometric aspects of neuromelanin-containing (pigmented) neurons in the SN of Dys patients by performing rigorous neuropathologic quantitative analyses. We aimed to estimate nigral pigmented neuronal counts and cell body, nuclear, and nucleolar volumes using established quantification methods and the largest number of publicly available Dys autopsy brains to date.
We observed a significant decrease in the number of pigmented neurons in the SN of Dys brains versus C. This reduction was present in the SN of patients with both adulthood- and childhood-onset Dys. Interestingly, fewer pigmented neurons in the SN were not associated with significant changes in cellular, nuclear, or nucleolar volumes of the remaining pool of nigral pigmented neurons, or with other major neuropathologic and neurodegenerative signs. These findings might indicate that a reduction in SN pigmented neurons in Dys patients could be due to a neurodevelopmental defect rather than a neurodegenerative process.
A nigral pigmented neuronal reduction in the absence of cellular (cell body) and subcellular (nuclear/nucleolar) volumetric changes in the SN of Dys brains was confirmed by excluding other possible confounding or co-occurring nigral pathologies with immunohistochemistry protocols. In this study, the Dys and C groups did not differ in terms of mean age at death, further minimizing the influence of the important confounding factor of age.36
A total of 13 SN from patients (9 females and 4 males) with various Dys subtypes were available for this study. The mean age at death was 68.8±15.5 years (range: 44–95). These individuals had a history of childhood- or adulthood-onset Dys with focal, segmental, or generalized distribution. Dys cases did not show signs of any other major neurological, psychiatric, or medical disorders except for one patient with diagnosis of possible dementia with Lewy bodies (possDLB), and another with cardiovascular disease. Cases of DYT1, the most frequent genetic form of Dys, were excluded from this investigation to avoid performing measurements on neurons from the brains of patients with Dys attributable to specific genetic causes. With this exclusion, we aimed to obtain general but specific quantitative findings on non-DYT1 or idiopathic Dys only.
The 13 SN from Dys patients were compared to 13 SN from age-matched control subjects (C, 9 females and 4 males). The mean age at death was 64.3±13.8 years (range: 44–88 years). C did not show clinical or pathologic evidence of any major neurological, psychiatric, or medical disorders except for heart disease in some cases.
In addition to the standard microscopic neuropathological assessments, all SNs were immunohistochemically assessed for the presence of: α-synuclein-positive Lewy bodies (LB) and Lewy neurites (LN), hyperphosphorylated-tau neurofibrillary tangles (tau-NFT), hyperphosphorylated-tau threads (tau-th), ubiquitin (ubiq), intraneuronal cytoplasmic inclusions of phosphorylated-TAR-DNA binding protein-43 (pTDP-43), and extracellular deposits of insoluble 1–42 β-amyloid (diffuse-Aβ and Aβ-neuritic plaques). Subjects with neuropathologic findings indicative of any other neurodegenerative disorder or severe cerebrovascular disease were excluded from the study.
All Dys and C brains were collected after obtaining written consent from the next-of-kin or legal representative and after approval from each institution where the case was examined. Tissues were obtained from the University of Maryland Brain and Tissue Bank (UMBTB). The use of these tissues was approved by UMBTB and the local Institutional Research Board. Each hemibrain was fixed in 10% buffered formalin for at least 2 weeks and then grossly and microscopically examined in coronal sections for a general neuropathologic assessment at UMBTB.
Each hemi-SN block was randomly cut at different anatomical levels along the rostro-caudal anatomical axis of the structure. This anatomically random cutting minimized possible anatomical selection biases. We were unable to obtain the entire SN for each subject, but we were confident that at least three quarters of the entire structure was actually sampled for each case. This conclusion was based on our samples measurements (see below) in comparison to previous anatomical studies that assessed the entire length of SN.37,38
The total length of each block of formalin-fixed SN received ranged from 8–10 mm. Tissues were processed at the Neuropathology Research labs at the Biomedical Research Institute of New Jersey (BRInj). Tissue blocks were processed with an automated tissue-processor (Tissue-Tek V.I.P. 1000 Vacuum Infiltration Processor, Ames Division, Miles Laboratories, Inc., Elkhart, IN, USA) using standard protocols. Tissue blocks were then embedded in paraffin, oriented with the rostral area facing the bottom of the paraffin mold, and serially cut along the rostro-caudal direction using a semi-automatic microtome (Leica RM2255, Leica Biosystems, Nussloch, Germany). Each block was cut for its entire length in series of 40 µm-thick consecutive sections, alternating to a series of 16 consecutive 10 µm-thick sections. These serial, consecutive, and alternating sectioning procedures guaranteed the constancy of the established sampling rate for this investigation (one every five 40 µm-thick sections) and simultaneously allowed the use of 10 µm-thick consecutive sections anatomically adjacent to the 40 µm-thick sections for further immunohistochemistry assessments and volumetric measurements.39
The mean length of the sampled SN did not differ between Dys and C brains (mean lengths of 9,392.31±1,518.99 µm and 10,015.38±1,573.2 µm, respectively). The total number of consecutive sections (including 40 µm- and 10 µm-thick sections) obtained for Dys and C were 792.9±129.3 and 845.4±132.3, respectively. No statistical differences in terms of total length of sampled tissue and number of sections obtained were present between the Dys and C groups. Means of 48.6±7.5 and 51.6±7.5 40 µm-thick consecutive sections were respectively obtained for Dys and C.
Each set of 40 µm-thick sections was stained with a 1.0% cresyl violet (CV) solution for neuronal counting.40 Each set of 40 µm-thick sections was separately and randomly recoded by an investigator blinded to the clinical and pathologic diagnoses (M.G.E.). Quantitative measurements using well-established stereological probes (the Optical Fractionator and Nucleator, see below) were performed by a second investigator (D.I.). Slides codes were opened, and statistical analyses were performed only after all measurements were completed.
A single 10 µm-thick tissue section was randomly chosen, stained with hematoxylin and eosin (H&E), and microscopically inspected at low (2.5×) and high (20×) magnifications to assess for possible micro-ischemic vascular pathologies, microhemorrhages, tissue rarefaction, and other possible histologic abnormalities not visible on gross examination.
Each section was stereoscopically inspected (2×) across the entire sectional area to localize SN anatomical borders. The pars compacta and pars reticulata were identified and marked as based on previous detailed anatomical descriptions.41
For neuronal counting, the following histologic criteria were applied:
For neuronal volumetric measurements, the following histologic criteria were applied:
Measurements were performed using the Stereo-Investigator system, Version 10.0 (MBF Bioscience, Williston, VT, USA), equipped with a digital camera (AxioCam MRm, and MRc, Zeiss, Oberkochen, Germany) and multiple objective head (2.5–100× oil-immersion). The sampling grid area was 500×500 µm, the counting frame was 40×40 µm, and the disector height was 25 µm with guard zones of ±2 µm. The total number of SN pigmented neurons was estimated using the Optical Fractionator probe.42,43
Series of 10 µm-thick consecutive sections adjacent to each previously analyzed 40 µm-thick section were stained with CV to measure the cellular, nuclear, and nucleolar volumes of pigmented neurons. Volumetric measurements were performed using the Nucleator probe.38 Pigmented neuron volumes were measured by placing six rays that automatically centered on the nucleolus and randomly intersecting the cell membrane (Figure 1A). Pigmented neurons were measured if their nucleolus was inside the counting frame intersecting or touching the green inclusion line; they were excluded if their nucleolus intersected or touched the red exclusion line (Figure 1A, upper right quadrant). This second-run of volumetric measurements on separate sets of 10 µm-thick consecutive sections was chosen to increase the number of available pigmented neurons showing a well-defined nucleolus, that is, the established point of spatial reference for volumetric measurements in this study. In fact, a consistent number of pigmented neurons that could be clearly counted on 40 µm-thick sections could not be volumetrically measured due to the presence of neuromelanin covering, partially or totally, the nucleolus (Figure 1B).
Each Dys and C case underwent an extensive immunohistochemical assessment. For each case, we examined 16 10-µm thick sections adjacent to the median 40 µm-thick set of sections. The 10 µm-thick tissue sections were deparaffinized, hydrated, and treated to block endogenous peroxidase activity (3% hydrogen peroxide in water). They were then rinsed in buffer (Tris with 0.05% Triton-X), microwaved with antigen retrieval solution (sodium citrate, pH 6.0) for 10 minutes, and cooled. Protein block (1.5% horse serum) was applied for 30 minutes. The following antibodies were used: mouse anti-β-amyloid, 17–24 (dilution 1:500, 4G8; SIG-39220, Covance, Princeton, NJ, USA) with overnight incubation at 4°C (sections were pre-treated with 90% formic acid, 5 minutes); mouse anti-PHF-tau (dilution 1:500, MN1020; Thermo Fisher Scientific, Waltham, MA, USA); mouse anti-α-synuclein (dilution 1:500, Ab27766; Abcam, Cambridge, UK) rabbit anti-ubiq (dilution 1:250, ab7780, Abcam); rabbit anti phospho-TDP-43 (dilution 1:2,000, TIP-PTD-P02; Cosmo Bio Co. LTD, Carlsbad, CA, USA). With the exception of the anti-β-amyloid antibody, sections were incubated overnight at 4°C, rinsed with buffer, incubated with biotinylated horse anti-mouse or anti-rabbit secondary antibodies (30 minutes), Vector Kit reagents (Vector Labs, Inc., Burlingame, CA, USA), then with a 3, 3'-diaminobenzidine substrate system (D3939; Sigma, St. Louis, MO, USA) for 5 minutes. Slides were counterstained with hematoxylin, dehydrated in xylene, and coverslipped.
Statistical analyses for quantification and volumes were performed to compare Dys and C, adulthood-onset Dys and C, childhood-onset Dys and C, childhood-onset Dys and adulthood-onset Dys, and generalized and segmental/focal Dys. All comparisons were computed before and after excluding co-occurring pathology (LB, LN, tau-NFT, tau-th, ubiq, and pTDP43 inclusions, β-amyloid deposits). The childhood-onset Dys group included subjects with early adulthood-onset diagnoses and the only subject with posttraumatic Dys (Dys#10, onset at age 12).
Neuronal counting results revealed a Gaussian distribution, and a t-test was initially performed to compare Dys and C and Dys and C without copathologies. Furthermore, we performed analyses of variance (ANOVAs) with Tukey's posthoc tests across all comparisons. The Tukey's test compares every mean with every other mean and takes into account multiple comparisons and adjusted p-values for each comparison. Statistical significance was established at p<0.01 (adjusted for all five types of comparisons).
The preliminary analyses for the obtained neuronal volumetric measurements did not show a Gaussian curve, so nonparametric Mann-Whitney tests were performed across all comparisons for those measurements. Data refer to the values estimated for the bilateral SN. All computations were performed using GraphPad Prism software, version 5 (GraphPad Software, Inc., La Jolla, CA, USA).
The demographic, medical, and genetic data for both the Dys and C groups are summarized in Table 1. Table 1 shows diagnoses of Dys as received from UMBTB records, while Table 2 classifies each Dys patient based on a review of supplemental medical records according to the newest Dys classification system.4 This most recent classification system was not available for any of the subjects in this cohort at the time of their clinical diagnosis. Table 2 also summarizes all available data in terms of age at onset, body distribution, temporal pattern, variability, associated features, occurrence of other neurological and systemic manifestations, and etiologies (i.e., following the Axis I and II of the newer classification).4 In terms of temporal patterns, 3 out of 13 Dys cases were classified as adulthood onset, 3 as early adulthood onset, 6 as childhood onset, and 1 case of childhood-onset posttraumatic Dys. For body distribution, 6 out of 13 Dys cases were classified as generalized, 6 as segmental/focal, and 1 segmental/focal posttraumatic Dys.
Table 1
Subject#/UMBTB Code | Age at Death | Sex | Diagnosis (As Received) | Genetics | Other Relevant Medical Data |
---|---|---|---|---|---|
1 (Dys)/1458 | 62 | M | Dystonia | Neg for GAG del in DYT1 | None |
2 (Dys)/1484 | 81 | F | Dystonia (started as blepharospasm) | Not tested for DYT1 mutations | None |
3 (Dys)/1850 | 50 | F | Dystonia (started at 42 years old: adulthood onset, focal and segmental) | Not tested for DYT1 mutations | Mother with PD, multiple drugs |
4 (Dys)/4897 | 49 | F | Dystonia (generalized) | Neg for GAG del in DYT1 | None |
5 (Dys)/4880 | 44 | F | Dystonia (generalized dystonia with involvement of trunk, limbs, neck, and flexion contractures in the right leg; severe episodes of jerking [head back and forth] with generalized body contortions; possible seizures generalized) | Neg for GAG del in DYT1 | Brain aneurysm, intracerebral hemorrhage, multiple drugs |
6 (Dys)/5145 | 84 | F | Dystonia (cervical dystonia for >4 decades, head begun to turn 43 years before death with pain 20 years later, mouth involvement 22 years later) | Not tested for DYT1 mutations | Father with tremor (unspecified) |
7 (Dys)/5414 | 95 | F | Dystonia (tremor since the 6th grade, myoclonic jerks and terminal intentional tremor, marked paraspinal muscles spasms with lordosis, spastic dysphonia) | Not tested for DYT1 mutations | None |
8 (Dys)/5421 | 81 | F | Dystonia | Not tested for DYT1 mutations | None |
9 (Dys)/1457 | 72 | M | Dystonia | Neg for GAG del in DYT1 | None |
10 (Dys)/1353 | 78 | M | Dystonia (severe bike-automobile accident at 12 years old caused a fractured skull, tremor and posttraumatic dystonia followed) | Neg for GAG del in DYT1 | None |
11 (Dys)/4635 | 76 | M | Dystonia (generalized in combination with cervical myelopathy, initial cervical dystonia at 27 years old) | Neg for GAG del in DYT1 | History of familial dystonia symptoms (father and sister), possDLB |
12 (Dys)/1481 | 64 | F | Dystonia | Neg for GAG del in DYT1 | Diabetes, congestive heart failure, angina, angioplasty, daughter with dystonia |
13 (Dys)/1554 | 59 | F | Dystonia | Neg for GAG del in DYT1 | Small cell carcinoma, pneumonia, history of dystonia preceding lung carcinoma |
14 (C)/4263 | 61 | M | Control | Not tested | Ischemic cardiomyopathy, heart transplant, left ventricular dysfunction, depression, chronic renal failure, diabetes mellitus |
15 (C)/4921 | 73 | F | Control | Not tested | Gallbladder dysfunction, high blood pressure |
16 (C)/5357 | 51 | F | Control | Not tested | Drug overdose, suicidal attempt |
17 (C)/4788 | 48 | F | Control | Not tested | Multiple injuries resulting from a motorcycle accident |
18 (C)/4228 | 44 | F | Control | Not tested | None |
19 (C)/5219 | 76 | F | Control | Not tested | None |
20 (C)/M3642M | 88 | F | Control | Not tested | Congestive heart disease |
21 (C)/5274 | 64 | F | Control | Not tested | Hypertension, heart disease, skin cancer |
22 (C)/1818 | 76 | M | Control | Not tested | None |
23 (C)/5171 | 79 | M | Control | Not tested | COPD |
24 (C)/M3903M | 71 | M | Control | Not tested | Coronary artery disease, coronary angioplasty (twice), COPD |
25 (C)/1503 | 53 | F | Control | Not tested | Knee surgery after falling |
26 (C)/1379 | 53 | F | Control | Not tested | High blood pressure, asthma |
Abbreviations: C, Control; Cauc, Caucasian; COPD, Chronic Obstructive Pulmonary Disease; del, Deletion; Dys, Dystonia; DYT1, Dystonia Gene; F, Female; GAG, Three-nucleotide Deletion; M, Male; Neg, Negative; PD, Parkinson's Disease; possDLB, Possible Dementia with Lewy Bodies; UMBTB, University of Maryland Brain and Tissue Bank.
Table 2
Subject# | Diagnosis (As Received) | Dystonia Diagnosis based on the Newer Classification | Dystonia Diagnosis based on the Newer Classification |
---|---|---|---|
Axis I | Axis II | ||
(Clinical Features) | (Etiology) | ||
1 (Dys) | Dystonia | Age at onset: not reported (probably childhood-onset as for medical history) | Nervous system pathology: no evidence of degeneration or structural lesions |
Body distribution: not reported (probably generalized as for medical history) | Inherited: possible | ||
Temporal pattern: not reported | Acquired: no | ||
Variability: not reported | Idiopathic: yes | ||
Associated features: unknown/not clinically significant | |||
Occurrence of other neurological/systemic manifestations: unknown/not clinically significant | |||
2 (Dys) | Dystonia (started as blepharospasm in 1983, 22 years of dystonia history until death) | Age at onset: late adulthood | Nervous system pathology: no evidence of degeneration or structural lesions |
Body distribution: initially focal, then segmental (oromandibular dystonia with jaw closure and lip pursing, facial grimacing) | Inherited: no (aunt, mother's sister, with resting tremor) | ||
Temporal pattern: static | Acquired: no | ||
Variability: diurnal | Idiopathic: yes | ||
Associated features: headache (with temporal and orbital pain) | |||
Occurrence of other neurological/systemic manifestations: tardive dyskinesia secondary to adverse effects to metoclopramide, moderate bradykinesia, rigidity, and rest tremor; possible side effects of various drugs used for the treatment of the blepharospasm | |||
3 (Dys) | Dystonia (started at 42 years old: adulthood onset, focal and segmental) | Age at onset: late adulthood | Nervous system pathology: evidence of degeneration (SN) |
Body distribution: focal, segmental (face, neck, shoulders), dysphagia | Inherited: no (mother with PD) | ||
Temporal pattern: static | Acquired: no | ||
Variability: diurnal | Idiopathic: yes | ||
Associated features: unknown/not clinically significant | |||
Occurrence of other neurological/systemic manifestations: unknown/not clinically significant | |||
4 (Dys) | Dystonia (generalized) | Age at onset: childhood | Nervous system pathology: no evidence of degeneration or structural lesion |
Body distribution: generalized (with leg involvement) | Inherited: no | ||
Temporal pattern: progressive | Acquired: no | ||
Variability: diurnal | Idiopathic: yes | ||
Associated features: headache (not clinically significant) | |||
Occurrence of other neurological/systemic manifestations: unknown/not clinically significant | |||
5 (Dys) | Dystonia (generalized) | Age at onset: childhood | Nervous system pathology: cerebellar cortical degeneration |
Body distribution: multifocal neck, trunk, limbs), segmental (leg involvement) | Inherited: no | ||
Temporal pattern: progressive | Acquired: no | ||
Variability: paroxysmal | Idiopathic: yes | ||
Associated features: pain, headache | |||
Occurrence of other neurological/systemic manifestations: frontal lobe intracerebral hemorrhage (after many years of generalized dystonia) | |||
6 (Dys) | Dystonia (cervical dystonia for >4 decades) | Age at onset: early adulthood | Nervous system pathology: no evidence of degeneration or structural lesion |
Body distribution: segmental (started as cervical dystonia, then mouth and lingual involvement) | Inherited: unknown (father with head tremor) | ||
Temporal pattern: progressive (mount involvement, paraspinal muscles spasm, spastic dysphonia) | Acquired: no | ||
Variability: diurnal | Idiopathic: yes | ||
Associated features: facial pain | |||
Occurrence of other neurological/systemic manifestations: resting hand tremor (later, after many years cervical dystonia appearance) | |||
7 (Dys) | Dystonia | Age at onset: childhood | Nervous system pathology: no evidence of degeneration or structural lesion |
Body distribution: segmental (truncal, paraspinal spams with lordosis) | Inherited: no | ||
Temporal pattern: diurnal | Acquired: no | ||
Variability: diurnal | Idiopathic: yes | ||
Associated features: spastic dysphonia, tremor started in 6th grade | |||
Occurrence of other neurological/systemic manifestations: unknown | |||
8 (Dys) | Dystonia | Age at onset: late adulthood | Nervous system pathology: evidence of degeneration (SN), LB pathology |
Body distribution: segmental (started as blepharospasm then spasmodic dysphonia; lip pursing, grimacing, and jaw opening) | Inherited: no | ||
Temporal pattern: static | Acquired: no | ||
Variability: diurnal (getting worse with action); not action-specific but with associated | Idiopathic: yes | ||
features of late-onset tardive dyskinesia (oromandibular, pharyngeal, and facial dystonia) due to possible side effects of neuroleptic drugs (perphenazine/amitriptyline) | |||
Occurrence of other neurological/systemic manifestations: unknown/not clinically significant | |||
9 (Dys) | Dystonia | Age at onset: childhood (probably) | Nervous system pathology: cerebellar atrophy (moderate) |
Body distribution: generalized | Inherited: no | ||
Temporal pattern: progressive (probably) | Acquired: no | ||
Variability: diurnal | Idiopathic: yes | ||
Associated features: unknown/not clinically significant | |||
Occurrence of other neurological/systemic manifestations: unknown/not clinically significant | |||
10 (Dys) | Dystonia (posttraumatic) | Age at onset: childhood | Nervous system pathology: cerebellar atrophy, LBs in the SN and LC |
Body distribution: focal (right hand) | Inherited: no | ||
Temporal pattern: static | Acquired: yes, (posttraumatic) | ||
Variability: persistent | Idiopathic: no | ||
Associated features: tremor (right hand) | |||
Occurrence of other neurological/systemic manifestations: unknown/not clinically significant | |||
11 (Dys) | Dystonia (generalized in combination with cervical myelopathy; initial cervical dystonia at 27 years old) | Age at onset: early adulthood | Nervous system pathology: diffuse LB pathology (brainstem, cortex), cerebellar heterotaxia (white matter) |
Body distribution: generalized | Inherited: possible (reported familiarity for movement disorders) | ||
Temporal pattern: progressive (started as cervical dystonia/torticollis then progressed with arms and legs) | Acquired: no | ||
Variability: diurnal | Idiopathic: yes | ||
Associated features: marked diffuse tremor | |||
Occurrence of other neurological/systemic manifestations: possible dementia | |||
12 (Dys) | Dystonia | Age at onset: early adulthood | Nervous system pathology: no evidence of degeneration or structural lesion |
Body distribution: generalized | Inherited: possible (daughter with dystonia) | ||
Temporal pattern: progressive | Acquired: no | ||
Variability: diurnal | Idiopathic: yes | ||
Associated features: unknown/not clinically significant | |||
Occurrence of other neurological/systemic manifestations: mastectomy for breast cancer | |||
13 (Dys) | Dystonia | Age at onset: childhood (probably) | Nervous system pathology: no evidence of degeneration or structural lesion (metastatic cells in cerebrum, cerebellum, brainstem) |
Body distribution: generalized | Inherited: no | ||
Temporal pattern: progressive | Acquired: no | ||
Variability: diurnal | Idiopathic: yes | ||
Associated features: unknown/not clinically significant | |||
Occurrence of other neurological/systemic manifestations: lung cancer |
Abbreviations: C, Control; Dys, Dystonia; LB, Lewy Body; LC, Locus Coeruleus; PD, Parkinson's Disease; SN, Substantia Nigra.
All main neuropathologic data, postmortem delay (PMD, time between death and autopsy) values, and causes of death are shown in Table 3. The mean PMD was 11.7±6.4 hours, with no significant differences between the Dys (13±6.7 hours) and C (10.4±6.0 hours) groups. The majority of Dys and C did not show signs of major recent or remote ischemic, hemorrhagic lesions, metastatic infiltration, or arteriovenous or congenital malformations. The exception was case#5 (Dys who showed microhemorrhages, probably due to the prolonged periagonal status and history of remote hemorrhage from an aneurysmatic rupture. Intima thickening was observed in most of the arteries and arterioles in case #3, #12, #13, and #23, which was compatible with arteriosclerotic disease (see Table 4).
Table 3
Subjects# | BW (Grams) | PMD (Hours) | Cause of Death | Main Neuropathologic Findings |
---|---|---|---|---|
1 (Dys) | 1,455 | 12 | Natural | No significant neuropathologic findings |
2 (Dys) | N/A | 16 | Natural | Remote infarct in right parieto-occipital region; moderate cerebral artery atherosclerosis |
3 (Dys) | N/A | 3 | Brain ischemic injury | Acute hypoxic-ischemic encephalopathy, idiopathic SN degeneration |
4 (Dys) | N/A | 16 | Complications of the disorder (multiple falls) | No significant neuropathologic findings |
5 (Dys) | N/A | 20 | Complications of the disorder | Encephalomalacia involving frontal lobes with subarachnoid hematoma due to ruptured berry aneurysm and surgical clip placement, cerebellar cortical degeneration |
6 (Dys) | N/A | 20 | Complications of the disorder | Generalized mild atrophy, arteriosclerotic cerebrovascular disease |
7 (Dys) | N/A | 16 | Complications of the disorder | Neocortical gyral atrophy, arteriosclerosis with leukomalacia |
8 (Dys) | N/A | 8 | Complications of the disorder | Diffuse brain atrophy, arteriosclerotic cerebrovascular disease, SN degeneration with LBs in the SN and limbic cortex, AD-type lesions (amyloid neuritic plaques and neurofibrillary tangles) in the hippocampus |
9 (Dys) | N/A | 12 | Natural | Microscopic remote infarct in the left caudate, moderate cerebellar atrophy |
10 (Dys) | N/A | 5 | Septic shock, metabolic acidosis, respiratory failure | Mild cerebral hemispheric atrophy, rare LBs in the SN and LC, mild cerebellar atrophy |
11 (Dys) | N/A | 9 | Complications of the disorder | Amyloid neuritic plaques in the cortex; diffuse LBs in the SN, limbic structures, and cortical regions; incidental cerebellar white matter heterotopia |
12 (Dys) | N/A | 26 | Natural | No significant neuropathologic findings |
13 (Dys) | N/A | 6 | Lung cancer | Metastatic poorly differentiated carcinoma in the cerebrum, cerebellum, and brainstem |
14 (C) | 1,309 | 6 | Cardiac arrest | Small cystic infarcts in the left parieto-occipital and frontal lobes |
15 (C) | N/A | 13 | Peritonitis | No significant neuropathologic findings |
16 (C) | N/A | 8 | Atherosclerosis-Cardiovascular disease | No significant neuropathologic findings |
17 (C) | N/A | 17 | Multiple traumatic injuries | No significant neuropathologic findings |
18 (C) | 1,260 | 16 | Coronary artery atherosclerosis and thrombosis | Acute hypoxic-ischemic encephalopathy in hippocampus |
19 (C) | N/A | 3 | Complications of cancer | No significant neuropathologic findings |
20 (C) | N/A | 8 | Congestive heart failure | No significant neuropathologic findings |
21 (C) | N/A | 20 | Acute cerebrovascular disease | No significant neuropathologic findings |
22 (C) | N/A | 3 | Acute cerebrovascular disease | Mild to moderate atheromatosis of the circle of Willis, arteriosclerosis of the middle- and small-sized intraparenchymal arteries, microinfarct in the right Sommer's sector (cornu ammonis 1 of the hippocampus) |
23 (C) | N/A | 5 | COPD, peripheral vascular disease | No significant neuropathologic findings |
24 (C) | N/A | 16 | Cardiac arrest | Brain edema |
25 (C) | N/A | 5 | Pulmonary thromboembolism | No significant neuropathologic findings |
26 (C) | N/A | 15 | Respiratory distress | No significant neuropathologic findings |
Abbreviations: AD, Alzheimer's Disease; BW, Brain Weight; C, Control; COPD, Chronic Obstructive Pulmonary Disease; Dys, Dystonia; LB, Lewy Body; LC, Locus Coeruleus; N/A, Not Applicable; PMD, Postmortem Delay; SN, Substantia Nigra.
Table 4
Case# | 1–42 β-amyloid | Hyperphosphorylated-tau | α-synuclein | Ubiq-cytopl. incl./MB | Phosphorylated-TDP43 | H&E |
---|---|---|---|---|---|---|
1 (Dys) | Neg | Neg | Neg | Neg/Neg | Neg | Normal |
2 (Dys) | Neg | Neg | Neg | Neg/Pos | Neg | Normal |
3 (Dys) | Neg | Neg | Neg | Neg/Pos | Neg | Intimal thickening |
4 (Dys) | Neg | Neg | Neg | Neg/Neg | Neg | Normal |
5 (Dys) | Neg | Neg | Neg | Neg/Neg | Neg | Microhemorrhages |
6 (Dys) | Neg | NFT and Th (sparse), tau-glia | Neg | Neg/Pos | Neg | Normal |
7 (Dys) | Neg | NFT and Th (moderate) | Neg | Neg/Pos | Neg | Normal |
8 (Dys) | Neg | Th (moderate) | Pos (sparse) | Neg/Neg | Neg | Normal |
9 (Dys) | Neg | Neg | Neg | Neg/Neg | Neg | Normal |
10 (Dys) | Neg | NFT (rare) | Pos (rare) | Neg/Pos | Neg | Normal |
11 (Dys) | Neg | Th (sparse), pre-NFT (rare) | Pos (rare) | Neg/Neg | Neg | Normal |
12 (Dys) | Neg | Neg | Neg | Neg/Neg | Neg | Intimal thickening |
13 (Dys) | Neg | Neg | Neg | Neg/Pos | Neg | Intimal thickening |
14 (C) | Neg | Neg | Neg | Neg/Neg | Neg | Normal |
15 (C) | Neg | Th (sparse) | Neg | Neg/Neg | Neg | Normal |
16 (C) | Neg | Neg | Neg | Neg/Neg | Neg | Normal |
17 (C) | Neg | Neg | Neg | Neg/Neg | Neg | Normal |
18 (C) | Neg | Neg | Neg | Neg/Neg | Neg | Normal |
19 (C) | Neg | Th (rare) | Neg | Neg/Neg | Neg | Normal |
20 (C) | Neg | NFT (rare) | Neg | Neg/Neg | Neg | Hyperemia |
21 (C) | Neg | Th (rare) | Neg | Neg/Neg | Neg | Normal |
22 (C) | Neg | Neg | Neg | Neg/Pos | Neg | Normal |
23 (C) | Neg | Neg | Neg | Neg/Neg | Neg | Intimal thickening |
24 (C) | Neg | Th (sparse) | Neg | Neg/Neg | Neg | Normal |
25 (C) | Neg | Neg | Neg | Neg/Neg | Neg | Normal |
26 (C) | Neg | Neg | Neg | Neg/Neg | Neg | Normal |
Abbreviations: C, Control; Dys, Dystonia; H&E, Hematoxylin and Eosin; Neg, Negative; NFT, Neurofibrillary Tangle; Pos, Positive; SN, Substantia Nigra; TDP43, TAR DNA-binding Protein 43; Th, Thread
Overall, 5/13 Dys and 5/13 C were positive for tau lesions. In the majority of cases, tau lesions were sparse or rare (tau-NFT or tau-th), and only two Dys cases (cases #7 and #8) had moderate levels of tau lesions, mostly tau-th.
No C cases had α-synuclein-positive lesions (LBs or LNs), whereas three Dys cases (#8, #10, and #11) did. These three cases were classified as possible Lewy Body disease (possLBD) of the brainstem.40 None of the SN samples were positive for 1–42 β-amyloid (diffuse amyloid or neuritic plaques), ubiq, or pTDP43-positive cytoplasmic intraneuronal inclusions. Ubiq-positive intranuclear inclusions (Marinesco bodies [MB])44 were observed in some cases, and more often in Dys than C. As a reminder, MB are eosinophilic intranuclear neuronal inclusions observed in human brains that have an uncertain origin and pathologic meaning. This intriguing finding has been reported previously; 30 however, larger studies are necessary to confirm the higher frequency of MB in Dys in general or in a specific Dys subtype. Table 4 summarizes the immunohistochemistry findings in both groups.
A total number of 496,601±130,010 and 648,926±162,475 pigmented neurons were estimated in the SNs of the Dys and C groups, respectively. The estimated nigral neuronal population in C did not differ proportionally from previous stereologic studies that assessed the entire SN length in control subjects.45,46
Histograms depicting the estimated nigral pigmented neuronal counts and volumes are shown in Figure 2. The estimated nigral pigmented neuronal population was calculated using neuronal numbers weighted for each section thickness per each subject. A significant reduction of pigmented neurons in SN of Dys versus C was found (p = 0.01). A separate computation after exclusion of all positive LB/LN and/or tau-NFT/th Dys (#6, #7, #8, #10, and #11) and C (#15, #19, #20, #21, and #24) cases was also performed. This second narrower computation confirmed a significant reduction of pigmented neurons in the SN of Dys (n = 8) versus C (n = 7) (p = 0.01). No significant differences in terms of neuronal counts were observed between childhood-onset Dys and adulthood-onset Dys cases regardless of whether cases with co-occurring nigral pathologies were excluded. Table 5 summarizes all comparisons for neuronal counting between Dys and C with their numerical values and statistical significance. See also Figure 4.
Table 5
Comparisons | Estimated Number of Pigmented Neurons (±SD) | Cell Body | Nuclear | Nucleolar |
---|---|---|---|---|
Volume (µm3) | Volume (µm3) | Volume (µm3) | ||
Dys (n = 13) vs. C (n = 13) | 496,601.6±130,009.4 vs. 648,925.7±169,700.2 (p = 0.0186) | 15,136.1±4,122.2 vs. 15,632.1±2,718.2 (ns) | 1,768.1±505.4 vs. 1,662.2±347.0 (ns) | 26.0±8.2 vs. 25.9±3.1 (ns) |
*Dys (n = 8) vs. *C (n = 8) | 518,991.3± 139,210.6 vs. 725,432.2±16,531.8 (p = 0.0045) | 14,340.1±5,097.2 vs. 15,936.1±2,992.1 (ns) | 1,627.1±560.1 vs. 1,767.2±399.2 (ns) | 25.8±8.9 vs. 26.0±3.8 (ns) |
Adulthood-onset Dys (n = 3) vs. C (n = 13) | 408,748.1±85,747.9 vs. 648,925.7±169,700.2 (p = 0.0366) | 13,669.1±3,557.2 vs. 15,936.1±2,992.1 (ns) | 1,661.1±491.9 vs. 1,662.2±347.0 (ns) | 24.0±6.8 vs. 25.9±3.1 (ns) |
*Adulthood-onset Dys (n = 2) vs. *C (n = 8) | 408,748.1±85,747.9 vs. 725,432.2±16,531.8 (p = 0.0366) | 11,709.5±1,510.5 vs. 15,222.7±766.4 (ns) | 1,491.1±556.6 vs. 1,796.1±372.0 (ns) | 24.1±9.7 vs. 26.0±5.2 (ns) |
Childhood-onset Dys (n = 10) vs. C (n = 13) | 503,648.9±124,734.1 vs. 648,925.7±169,700.2 (p = 0.0436) | 15,764.1±4,569.2 vs. 15,632.1±2,718.2 (ns) | 1,764±533.1 vs. 1,662±347.0 (ns) | 27.1±9.1 vs. 25.9±3.1(ns) |
*Childhood-onset Dys (n = 6) vs. *C (n = 8) | 541,369.8±112,651.7 vs. 725,432.2±16,531.8 (p = 0.0192) | 15,216.7±1,373.2 vs. 15,222.7±766.4 (ns) | 1,671.7±400.9 vs. 1,796.1±372.0 (ns) | 25.6±5.2 vs. 26.0±5.2 (ns) |
Childhood-onset Dys (n = 10) vs. Adulthood-onset Dys (n = 3) | 504,647.0±371,697.8 vs. 463,247.9±97,180.9 (ns) | 15,764.1±4,569.2 vs. 13,669.1±3,557.2 (ns) | 1,764±533.1 vs. 1,662.2±347.0 (ns) | 27.1±9.1 vs. 25.9±3.1 (ns) |
*Childhood-onset Dys (n = 6) vs. *Adulthood-onset Dys (n = 2) | 535,589.0±371,697.8 vs. 512,103.1±67,582.0 (ns) | 15,216.7±1,373.2 vs. 11,709.5±1,510.5 (ns) | 1,671.7±400.9 vs. 1,796.1±372.0 (ns) | 25.6±5.2 vs. 26.0±5.2 (ns) |
Generalized Dys (n = 6) vs. segmental/focal Dys (n = 7) | 514,023.1±98,491.31 vs. 481,668.9± 163,184.3 (ns) | 14,379.1±1,004.9 vs. 15,784.8±3,587.4 (ns) | 1,767.6±409.5 vs. 15,784.8±3,587.4 (ns) | 28.9±3.5 vs. 23.4±4.2 (ns) |
*Generalized Dys (n = 5) vs. *segmental/focal Dys (n = 3) | 513,204.1±139,210.5 vs. 528,636.7±139,513.19 (ns) | 13,984.5±1,373.2 vs. 14,932.3±5,683.2 (ns) | 1,655.5±400.9 vs. 1,982.4±312.7 (ns) | 27.2±1.0 vs. 32.1±5.5 (ns) |
Abbreviations: C, Control; Dys, Dystonia; ns, not significant; SD, Standard Deviations; SN, Substantia Nigra.
The mean for each type of volumetric measurement (cell body, nucleus, and nucleolus) performed on nigral pigmented neurons of Dys and C are shown in Table 5. A mean of 179.7±56.3 nigral pigmented neurons were volumetrically measured (range: 97–297) across all cases. No difference was measured between the mean numbers of pigmented neurons volumetrically measured in Dys (157.5±40.0) and C (201.9±56.3). Importantly, no significant differences in mean cellular, nuclear, or nucleolar volumes were found across all types of comparisons between the Dys and C groups (Table 5). Table 6 shows the coefficient error (CE) values for each performed measurement (counting and volumes) for each subject in the study.
Table 6
Subject# | Mean Pigmented Neuronal Count | CE | Mean Cellular Volume | CE | Mean Nuclear Volume | CE | Mean Nucleolar Volume | CE |
1 (Dys) | 530,990.7 | 0.05 | 18,457.9 | 0.006 | 2,332.3 | 0.006 | 38.1 | 0.007 |
2 (Dys) | 409,690.0 | 0.07 | 12,777.6 | 0.003 | 1,884.7 | 0.003 | 31.0 | 0.004 |
3 (Dys) | 494,021.3 | 0.06 | 10,641.4 | 0.005 | 1,097.5 | 0.006 | 17.2 | 0.022 |
4 (Dys) | 604,631.9 | 0.06 | 12,589.7 | 0.004 | 1,730.0 | 0.005 | 27.3 | 0.005 |
5 (Dys) | 682,198.8 | 0.05 | 21,377.8 | 0.005 | 1,752.9 | 0.005 | 24.4 | 0.006 |
6 (Dys) | 491,138.2 | 0.05 | 17,297.2 | 0.007 | 1,510.4 | 0.007 | 24.8 | 0.006 |
7 (Dys) | 275,364.9 | 0.08 | 16,926.6 | 0.009 | 1,840.5 | 0.009 | 21.6 | 0.011 |
8 (Dys) | 322,533.2 | 0.07 | 17,586.4 | 0.007 | 2,002.1 | 0.007 | 23.7 | 0.062 |
9 (Dys) | 405,516.6 | 0.06 | 5,273.8 | 0.006 | 581.8 | 0.009 | 8.0 | 0.008 |
10 (Dys) | 696,735.9 | 0.05 | 13,886.4 | 0.004 | 2,286.8 | 0.005 | 21.3 | 0.049 |
11 (Dys) | 518,118.1 | 0.06 | 16,352.0 | 0.006 | 2,328.5 | 0.007 | 37.4 | 0.007 |
12 (Dys) | 414,003.9 | 0.06 | 17,771.6 | 0.006 | 1,533.1 | 0.006 | 30.6 | 0.079 |
13 (Dys) | 610,877.4 | 0.05 | 15,829.5 | 0.005 | 2,100.2 | 0.005 | 32.0 | 0.005 |
14 (C) | 787,705.8 | 0.06 | 13,858.9 | 0.004 | 1,115.2 | 0.005 | 22.0 | 0.016 |
15 (C) | 648,925.7 | 0.06 | 11,253.3 | 0.007 | 1,390.6 | 0.008 | 23.6 | 0.045 |
16 (C) | 351,255.9 | 0.05 | 15,631.9 | 0.003 | 1,663.0 | 0.003 | 25.8 | 0.004 |
17 (C) | 856,559.1 | 0.06 | 11,864.0 | 0.003 | 1,600.5 | 0.003 | 24.0 | 0.004 |
18 (C) | 621,172.8 | 0.07 | 15,532.5 | 0.007 | 2,084.9 | 0.008 | 29.0 | 0.053 |
19 (C) | 370,591.1 | 0.06 | 20,711.7 | 0.010 | 1,585.1 | 0.009 | 21.1 | 0.010 |
20 (C) | 571,960.5 | 0.06 | 18,168.9 | 0.003 | 1,704.5 | 0.004 | 27.3 | 0.048 |
21 (C) | 696,553.0 | 0.06 | 15,519.3 | 0.003 | 1,365.8 | 0.003 | 26.8 | 0.034 |
22 (C) | 488,401.4 | 0.05 | 14,754.4 | 0.004 | 1,327.2 | 0.005 | 25.1 | 0.057 |
23 (C) | 800,241.8 | 0.05 | 18,947.8 | 0.004 | 1,660.0 | 0.004 | 28.4 | 0.004 |
24 (C) | 718,722.8 | 0.04 | 15,375.8 | 0.004 | 2,007.7 | 0.003 | 29.7 | 0.004 |
25 (C) | 750,282.5 | 0.05 | 16,342.6 | 0.004 | 1,788.9 | 0.005 | 23.0 | 0.059 |
26 (C) | 773,662.1 | 0.05 | 15,258.7 | 0.003 | 2,315.1 | 0.003 | 30.5 | 0.004 |
Abbreviations: C, Control; CE, Coefficient of Error (of Gunderson); Dys, Dystonia.
To our knowledge, this is the first investigation to apply rigorous neuropathologic quantitative methods to estimate the numbers and volumes of nigral pigmented neurons in a consistent series of Dys and an equivalent number of age-matched control brains that were systematically assessed by specific immunohistochemistry protocols for the individuation and exclusion of possible co-occurring pathologies affecting the SN. Importantly, this is the largest quantitative analyses of Dys brain tissue to date. Our results show that both adulthood- and childhood-onset Dys patients exhibited significant reductions of pigmented SN neurons compared to the C groups. Furthermore, these quantitative analyses showed no significant differences between Dys and C in terms of nigral pigmented neuronal volumes. These findings would seem to exclude phenomena of atrophy or hypertrophy for this type of pigmented neuron in non-DYT1 idiopathic Dys brains. Previous studies describing semiquantitative or pathologic findings on Dys brains mainly focused on genetic Dys, specifically DYT1 cases.20,29 Therefore, most of the findings from those studies do not seem to be directly applicable to the Dys cohort examined here.
The previous report of larger pigmented neurons in the SN of subjects with Dys (with and without GAG deletions)29 was not confirmed in our non-DYT1 idiopathic Dys cohort. However, our findings do not exclude the possibility that certain genetic forms of Dys, such as DYT1 characterized by TorsinA protein dysfunction, could induce specific cellular adaptation phenomena during brain development. Indeed, it could be hypothesized that congenital reductions or enlargements of specific neuronal types are present in some specific sensory-motor cortical or subcortical areas47 as adaptive or compensatory neuronal mechanisms against the subjacent initial pathology. Phenomena of neuroplasticity48 in Dys patients illustrated by some recent functional neuroimaging studies49 might support a neurodevelopmental hypothesis of idiopathic Dys.
Although the quantitative methods used in this study were very similar to those used in unbiased stereology investigations, and we tried to minimize confounding factors, it is important to consider our results in the context of some important limitations:
This investigation, however, also has several strengths:
Our neuropathologic morphometric-quantitative findings, which are similar to the results of other recent pathologic studies,31,32 seem to confirm the direct involvement of the SN in the pathomechanisms of Dys. Importantly, the involvement of the SN in Dys with regard to a reduced number of pigmented neurons (mainly dopaminergic cells) is not necessarily or directly related to dopaminergic dysfunction per se (i.e., dopaminergic function loss), or to a specific clinical phenomenology of Dys. In fact, in a hypothetical and more global context of a neurodevelopmental disorder, it is possible that other nondopaminergic circuits and neurotrasmettitorial abnormalities (i.e., gamma-aminobutyric acid, acetylcholine)50,51 could play important or even major roles. Moreover, results from two clinical and functional neuroimaging series52,53 support a more global view on Dys phenomenology where the motor and nonmotor abnormalities observed in Dys patients are associated with a broad spectrum of abnormalities in multiple cerebral regions, some of which are nondopaminergic. Furthermore, at a higher speculative level, a neurodevelopmental hypothesis for idiopathic Dys (or some specific form of it) that would not primarily involve dopaminergic dysfunction (i.e., dopaminergic function loss) could help to explain the constant and frustrating inefficacy of dopaminergic therapies in most Dys patients, with the remarkable exception of DRD.
Larger studies are needed to confirm our novel findings on idiopathic Dys. However, these preliminary results seem to suggest the presence of a specific neuropathologic substrate in idiopathic Dys in the form of a reduced number of nigral pigmented neurons that is not due to a neurodegenerative process.
Some very important questions remain:
In addition, the absence of a difference in the number of nigral pigmented neurons between the only available posttraumatic dystonia case (#10) and C brains seems to support a neurodevelopmental hypothesis for idiopathic Dys. In the future, larger studies of posttraumatic and nonposttraumatic/idiopathic Dys could provide pathologic findings that support or exclude a neurodevelopmental defect of the SN in idiopathic Dys.
Our preliminary quantitative findings, if confirmed, would offer novel insight into specific neuronal alterations in idiopathic Dys. However, these new findings need to take into account very complex pathophysiologic mechanisms and possible pathologic differences across the various forms of Dys.55–58 Nonetheless, partial overlapping or common mechanisms among groups or subgroups of Dys with similar clinical phenotypes remain possible.59
Future major efforts should include unbiased stereological analyses in larger groups of different genetic and nongenetic forms of Dys to confirm our observation of fewer pigmented neurons in the SN, and if so, clarify whether this decrease is “congenital” or neurodegenerative in nature. Another important issue is whether this cellular abnormality is unique to the SN or specific forms of Dys.
1 Funding: The Parkinson's & Movement Disorder Foundation (PDMD), Fountain Valley, CA, Grant 2012; Biomedical Research Institute of New Jersey, BRInj, NJ, “Dystonia Grant” 2013.
2 Financial disclosures: D.I., M.G.E., H.P. have no disclosures to report. M.L.R. has received honoraria from Teva and Allergan and grants from Ipsen; R.K. reports grants from the NIH, Astra-Zeneca, Psyadon, Rhythm, and Kyowa and honoraria from Teva.
3 Conflict of interests: The authors report no conflict of interest.
We are grateful to all dystonia patients, control subjects, and families for their generous gifts. Human tissue was obtained from the NIH NeuroBioBank at the University of Maryland, Baltimore, MD. We are also thankful to all personnel of the University of Maryland Brain and Tissue Bank (UMBTB), and especially to John Cottrell, M.S., P.A., A.S.C.P. for his technical support.
Defazio, G, Abbruzzese, G, Livrea, P and Berardelli, A (2004). Epidemiology of primary dystonia. Lancet Neurol 3: 673–678. [PubMed]
Steeves, TD, Day, L, Dykeman, J, Jette, N and Pringsheim, T (2012). The prevalence of primary dystonia: a systematic review and meta-analysis. Mov Disord 27: 1789–1796. [PubMed]
Defazio, G, Jankovic, J, Giel, JL and Papapetropoulos, S (2013). Descriptive epidemiology of cervical dystonia.. Tremor Other Hyperkinet Mov (N Y) 3DOI: https://doi.org/10.7916/D80C4TGJ
Albanese, A Bhatia, K Bressman, SB et al. (2013). Phenomenology and classification of dystonia: a consensus update. Mov Disord 28: 863–873. [PubMed]
Marsden, CD (1976). Dystonia: the spectrum of the disease. Res Publ Assoc Res Nerv Ment Dis 55: 351–367. [PubMed]
Fahn, S and Eldridge, R (1976). Definition of dystonia and classification of the dystonic states. Adv Neurol 14: 1–5. [PubMed]
Fahn, S (1988). Concept and classification of dystonia. Adv Neurol 50: 1–8. [PubMed]
Bressman, SB (2004). Dystonia genotypes, phenotypes, and classification. Adv Neurol 94: 101–107. [PubMed]
Ozelius, LJ Hewett, JW Page, CE et al. (1997). The early-onset torsion dystonia gene (DYT1) encodes an ATP-binding protein. Nat Genet 17: 40–48. [PubMed]
Kaji, R, Goto, S, Tamiya, G, Ando, S, Makino, S and Lee, LV (2005). Molecular dissection and anatomical basis of dystonia: X-linked recessive dystonia-parkinsonism (DYT3). J Med Invest 52(Suppl:280–283)
Chen, P Burdette, AJ Porter, JC et al. (2010). The early-onset torsion dystonia-associated protein, torsinA, is a homeostatic regulator of endoplasmic reticulum stress response. Hum Mol Genet 19: 3502–3515. [PubMed]
Bragg, DC, Armata, IA, Nery, FC, Breakefield, XO and Sharma, N (2011). Molecular pathways in dystonia. Neurobiol Dis 42: 136–147. [PubMed]
Hettich, J Ryan, SD de Souza, ON et al. (2014). Biochemical and cellular analysis of human variants of the DYT1 dystonia protein, TorsinA. Hum Mutat 35: 1101–1113. [PubMed]
Jankovic, J (2006). Treatment of dystonia. Lancet Neurol 5: 864–872. [PubMed]
Swope, D and Barbano, R (2008). Treatment recommendations and practical applications of botulinum toxin treatment of cervical dystonia. Neurol Clin 26(Suppl 1): 54–65. [PubMed]
Albanese, A Asmus, F Bhatia, KP et al. (2011). EFNS guidelines on diagnosis and treatment of primary dystonias. Eur J Neurol 18: 5–18. [PubMed]
Tanabe, LM, Kim, CE, Alagem, N and Dauer, WT (2009). Primary dystonia: molecules and mechanisms. Nat Rev Neurol 5: 598–609. [PubMed]
Xiao, J, Vemula, SR and LeDoux, MS (2014). Recent advances in the genetics of dystonia. Curr Neurol Neurosci Rep 14: 462. [PubMed]
Standaert, DG (2011). Update on the pathology of dystonia. Neurobiol Dis 42: 148–151. [PubMed]
McNaught, KS Kapustin, A Jackson, T et al. (2004). Brainstem pathology in DYT1 primary torsion dystonia. Ann Neurol 56: 540–547. [PubMed]
Paudel, R Kiely, A Li, A et al. (2014). Neuropathological features of genetically confirmed DYT1 dystonia: investigating disease-specific inclusions. Acta Neuropathol Commun 2: 159. [PubMed]
Altrocchi, PH and Forno, LS (1983). Spontaneous oral-facial dyskinesia: neuropathology of a case. Neurology 33: 802–805. [PubMed]
Gibb, WR, Lees, AJ and Marsden, CD (1988). Pathological report of four patients presenting with cranial dystonias. Mov Disord 3: 211–221. [PubMed]
Kulisevsky, J, Marti, MJ, Ferrer, I and Tolosa, E (1988). Meige syndrome: neuropathology of a case. Mov Disord 3: 170–175. [PubMed]
Zweig, RM, Hedreen, JC, Jankel, WR, Casanova, MF, Whitehouse, PJ and Price, DL (1988). Pathology in brainstem regions of individuals with primary dystonia. Neurology 38: 702–706. [PubMed]
Zweig, RM and Hedreen, JC (1988). Brain stem pathology in cranial dystonia. Adv Neurol 49: 395–407. [PubMed]
Bhatia, K, Daniel, SE and Marsden, CD (1993). Orofacial dystonia and rest tremor in a patient with normal brain pathology. Mov Disord 8: 361–362. [PubMed]
Holton, JL Schneider, SA Ganesharajah, T et al. (2008). Neuropathology of primary adult-onset dystonia. Neurology 70: 695–699. [PubMed]
Rostasy, K Augood, SJ Hewett, JW et al. (2003). TorsinA protein and neuropathology in early onset generalized dystonia with GAG deletion. Neurobiol Dis 12: 11–24. [PubMed]
Prudente, CN Pardo, CA Xiao, J et al. (2013). Neuropathology of cervical dystonia. Exp Neurol 241: 95–104. [PubMed]
Mencacci, NE Isaias, IU Reich, MM et al. (2014). International Parkinson's Disease Genomics Consortium and UCL-exomes consortium. Parkinson's disease in GTP cyclohydrolase 1 mutation carriers. Brain 137: 2480–2492. [PubMed]
Göttle, M Prudente, CN Fu, R et al. (2014). Loss of dopamine phenotype among midbrain neurons in Lesch-Nyhan disease. Ann Neurol 76: 95–107. [PubMed]
Asanuma, K, Carbon-Correll, M and Eidelberg, D (2005). Neuroimaging in human dystonia. J Med Invest 52(Suppl:272–279)
Neychev, VK, Gross, RE, Lehéricy, S, Hess, EJ and Jinnah, HA (2011). The functional neuroanatomy of dystonia. Neurobiol Dis 42: 185–201. [PubMed]
Stoessl, AJ, Lehericy, S and Strafella, AP (2014). Imaging insights into basal ganglia function, Parkinson's disease, and dystonia. Lancet 18
Rudow, G O′Brien, R Savonenko, AV et al. (2008). Morphometry of the human substantia nigra in ageing and Parkinson's disease. Acta Neuropathol 115: 461–470. [PubMed]
Cabello, CR, Thune, JJ, Pakkenberg, H and Pakkenberg, B (2002). Ageing of substantia nigra in humans: cell loss may be compensated by hypertrophy. Neuropathol Appl Neurobiol 28: 283–291. [PubMed]
Ma, SY, Röytt, M, Collan, Y and Rinne, JO (1999). Unbiased morphometrical measurements show loss of pigmented nigral neurones with ageing. Neuropathol Appl Neurobiol 25: 394–399. [PubMed]
West, MJ (2012). Basic Stereology for Biologists and Neuroscientists. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Luna, G (1968). AFIP Manual of Histologic Staining Techniques. 3rd ed New York, NY: McGraw-Hill Publications.
van Domburg, PH and ten Donkelaar, HJ (1991). The human substantia nigra and ventral tegmental area. A neuroanatomical study with notes on aging and aging diseases. Adv Anat Embryol Cell Biol 121: 1–132. [PubMed]
Gundersen, HJ Bagger, P Bendtsen, TF et al. (1988). The new stereological tools: disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS 96: 857–881. [PubMed]
King, MA, Scotty, N, Klein, RL and Meyer, EM (2002). Particle detection, number estimation, and feature measurement in gene transfer studies: optical fractionator stereology integrated with digital image processing and analysis. Methods 28: 293–299. [PubMed]
Yuen, P and Baxter, DW (1963). The morphology of Marinesco bodies (paranucleolar corpuscles) in the melanin pigmented nuclei of the brain-stem. J Neurol Neurosurg Psychiatry 26: 178–183. [PubMed]
Pakkenberg, B, Møller, A, Gundersen, HJ, Mouritzen Dam, A and Pakkenberg, H (1991). The absolute number of nerve cells in substantia nigra in normal subjects and in patients with Parkinson's disease estimated with an unbiased stereological method. J Neurol Neurosurg Psychiatry 54: 30–33. [PubMed]
Ma, SY, Collan, Y, Röyttä, M, Rinne, JO and Rinne, UK (1995). Cell counts in the substantia nigra: a comparison of single section counts and disector counts in patients with Parkinson's disease and in controls. Neuropathol Appl Neurobiol 21: 10–17. [PubMed]
Liang, CC, Tanabe, LM, Jou, S, Chi, F and Dauer, WT (2014). TorsinA hypofunction causes abnormal twisting movements and sensorimotor circuit neurodegeneration. J Clin Invest 12: 3080–3092. [PubMed]
Peterson, DA, Sejnowski, TJ and Poizner, H (2010). Convergent evidence for abnormal striatal synaptic plasticity in dystonia. Neurobiol Dis 37: 558–573. [PubMed]
Lerner, RP, Niethammer, M and Eidelberg, D (2013). Understanding the anatomy of dystonia: determinants of penetrance and phenotype. Curr Neurol Neurosci Rep 13: 401. [PubMed]
Hallett, M (2011). Neurophysiology of dystonia: The role of inhibition. Neurobiol Dis 42: 177–184. [PubMed]
Eskow Jaunarajs, KL, Bonsi, P, Chesselet, MF, Standaert, DG and Pisani, A (2015). Striatal cholinergic dysfunction as a unifying theme in the pathophysiology of dystonia. Prog Neurobiol,
Stamelou, M, Edwards, MJ, Hallett, M and Bhatia, KP (2012). The non-motor syndrome of primary dystonia: clinical and pathophysiological implications. Brain 135: 1668–1681. [PubMed]
Lehéricy, S, Tijssen, MA, Vidailhet, M, Kaji, R and Meunier, S (2013). The anatomical basis of dystonia: current view using neuroimaging. Mov Disord 28: 944–957. [PubMed]
Niethammer, M, Carbon, M, Argyelan, M and Eidelberg, D (2011). Hereditary dystonia as a neurodevelopmental circuit disorder: evidence from neuroimaging. Neurobiol Dis 42: 202–209. [PubMed]
Simonyan, K, Berman, BD, Herscovitch, P and Hallett, M (2013). Abnormal striatal dopaminergic neurotransmission during rest and task production in spasmodic dysphonia. J Neurosci 33: 14705–14714. [PubMed]
Herzfeld, T, Nolte, D, Grznarova, M, Hofmann, A, Schultze, JL and Müller, U (2013). X-linked dystonia parkinsonism syndrome (XDP, lubag): disease-specific sequence change DSC3 in TAF1/DYT3 affects genes in vesicular transport and dopamine metabolism. Hum Mol Genet 22: 941–951. [PubMed]
Vo, A, Sako, W, Niethammer, M, Carbon, M, Bressman, SB, Uluğ, AM and Eidelberg, D (2014). Thalamocortical connectivity correlates with phenotypic variability in dystonia.. Cereb Cortex, May 23 2014 [Epub ahead of print].
Edwards, MJ, Huang, YZ, Mir, P, Rothwell, JC and Bhatia, KP (2006). Abnormalities in motor cortical plasticity differentiate manifesting and nonmanifesting DYT1 carriers. Mov Disord 21: 2181–2186. [PubMed]
Thompson, VB, Jinnah, HA and Hess, EJ (2011). Convergent mechanisms in etiologically-diverse dystonias. Expert Opin Ther Targets 15: 1387–1403. [PubMed]