(E) Methods
Archival ET Cases (N = 6):
Postmortem brain tissue from six archival ET cases (Cases 2,3,5,7,8, and 10) was identified, including tissue from Columbia University Medical Center, New York, NY (n = 2); University of Kansas Medical Center, Kansas City, KS (n = 2); and the Honolulu-Asia Aging Study, Honolulu, HI (n = 2). Each ET case had been followed by their treating or study neurologist during life and clinical data were routinely collected, including demographics, family history, age at tremor onset, medications (including medications that cause cerebellar toxicity [e.g., cytosine arabinoside, cyclosporine]), ethanol consumption, and family history information. Five of six cases had been diagnosed with ET during life by their neurologist; one from the Honolulu-Asia Aging Study was included because, based on the motor portion of the Unified Parkinson’s Disease Rating Scale (UPDRS),1 he fulfilled all clinical diagnostic criteria for ET (see clinical criteria below), despite not having received this diagnosis during life. This case had not received any neurological diagnosis (e.g., PD) during life. The clinical diagnosis of ET was confirmed (n = 5) or assigned after death (n = 1) in each ET case using ETCBR diagnostic criteria, which were modeled after the criteria proposed in the Consensus Statement of the Movement Disorder Society.2 ETCBR criteria were as follows: (1) bilateral action tremor of the arms for five or more years, with a diagnosis of ET during life being preferable, (2) either head tremor or action tremor of at least one hand that was moderate or severe (i.e., it resulted in difficulty with two or more activities of daily living or it required medication), and (3) action tremor not the result of other movement disorders, hyperthyroidism, medical conditions or medications.
For each case, formalin-fixed paraffin-embedded tissue blocks from each region of interest were transferred to the ETCBR for analysis.
Prospectively-Enrolled ET Cases:
To date, four prospectively-enrolled ET cases (cases 1,4,6, and 9) have died and become brain donors. All four were diagnosed as ET by their treating neurologists. Prospective enrollees with ET were recruited through advertisements in the quarterly IETF newsletter. IETF members who expressed an interest in brain donation were mailed a set of standardized clinical questionnaires that included questions on demographics, tremor characteristics, medications (including medications that cause cerebellar toxicity [e.g., cytosine arabinoside, cyclosporine]), ethanol intake, a screening questionnaire for PD and dystonia, and a family history questionnaire. They also received written instructions to assist them in the preparation of a videotaped neurological examination; similar examinations are routinely used in clinical research studies to diagnose ET, other forms of tremor, and PD.3-7 The standardized examination included postures and maneuvers to bring out tremor (rest, postural, re-emergent, and kinetic tremors) and the motor portion of the UPDRS (excluding an assessment of rigidity). Previous studies have demonstrated that videotape-based diagnoses of PD have an overall sensitivity of 85 - 91%,3,7 and a higher sensitivity (96.7%) in the presence of action tremor,3 indicating that this method is likely to identify most cases of PD and minimize the chances that an ET case had unrecognized clinical PD. Once the diagnosis of ET was confirmed (based on a review of questionnaires and videotaped materials and using ETCBR criteria), an IETF member became a prospective enrollee. Four such enrollees have died and undergone a postmortem examination of the brain.
Controls:
Controls were obtained through the New York Brain Bank, which is affiliated with ColumbiaUniversityMedicalCenter. These were individuals who during life had been enrolled as normal elderly control subjects in the Alzheimer’s Disease Research Center or the Washington Heights Inwood Columbia Aging Project, where they were followed prospectively with serial neurological assessments, remaining free of a diagnosis of Alzheimer’s disease, ET or PD during life, and not having a neuropathological diagnosis after autopsy. Ten controls were selected for this study based on their age (i.e., they were frequency matched to ET cases based on age). Later, two additional age-matched normal elderly controls were obtained from the Honolulu-Asia Aging Study to serve as controls for the two Asian ET cases from that study; although neither of these two was demented by clinical history, one met pathological criteria for Alzheimer’s disease. All of the controls were followed prospectively during life and, based on clinical history and serial neurological examinations, none of the 12 had ET, PD, dystonia, spinocerebellar ataxia or other disorders of the central nervous system (epilepsy, stroke, multiple sclerosis, dementia) during life; one had a pathological diagnosis of AD. Their tissue was processed for the current study using the protocol detailed below.
Processing Tissue:
The cerebellum was our designated primary region of interest because in several studies, it has been implicated in the pathogenesis of ET.8-10 Additional (secondary) regions of interest were the red nucleus, thalamus, inferior olivary nucleus, basal ganglia (caudate, putamen, globus pallidum and substantia nigra pars compacta), brainstem nuclei (dorsal vagal nucleus and locus ceruleus), and motor cortex. Whenever possible, 17 standardized blocks were harvested from each brain and processed and 7µm thick, 20 x 25 mm paraffin sections were stained with Luxol fast blue/hematoxylin and eosin (LH&E).11 In addition, selected sections were stained with the Bielschowsky method (for evaluation of axons, neuritic plaques and neurofibrillary tangles), monoclonal antibodies to glial fibrillary acidic protein (GFAP, to examine gliosis), alpha-synuclein (to discern Lewy bodies), AT8 (to discern tangles consisting of hyperphosphorylated tau), and beta-amyloid (to visualize plaques). All tissue was examined microscopically by Dr. Vonsattel, blinded to clinical information including age and diagnosis, using a Zeiss Axioskop 2+.
Microscopic Examination:
Lewy Bodies and Lewy neurites: LH&E-stained sectionsand alpha- synuclein-stained sections were used to assess Lewy bodies. Lewy neurites were assessed with alpha-synuclein-stained sections. Sections included three levels of midbrain showing the substantia nigra pars compacta (rostral, middle and caudal), two levels of the pons showing the locus ceruleus, the medulla with dorsal vagal nucleus,hippocampus, cingulate gyrus, temporal cortex, prefrontal cortex, and motor cortex. A semi-quantitative scale was used for Lewy bodies and Lewy neurites: - (absent or not discernable), + (slight), ++ (moderate), +++ (severe). For Lewy bodies, 34 alpha- synuclein-stained sections, with ratings ranging from – to +++ were rated one month apart by Dr. Vonsattel and the agreement was excellent (weighted kappa statistic = 0.80). For Lewy neurites, 10 alpha- synuclein-stained sections, with ratings ranging from – to +++ were rated one month apart by Dr. Vonsattel and the agreement was excellent (weighted kappa statistic = 0.91).
Purkinje Cells: Using a 20 x 25 mm LH&E-stained section that included portions of the cerebellar cortex, white matter, and dentate nucleus (“standard cerebellar section”), Purkinje cells in five 100x fields were counted. The density of Purkinje cells varies by the location in the cerebellar cortical fold; in several mammalian species including man, the numbers of cells at the bases of the fissure are lower than those at the apices of the folia.12 In choosing the five 100x fields, the blinded assistant: (1) selected fields to represent different regions of the section (i.e., attempted to chose fields that were not in close proximity to one another), and (2) selected fields that were between but not inclusive of the base of the fissure and the apex of the folium. A Purkinje cell was counted only when its cell body was visible (including a portion of the nucleus). The number of Purkinje cells summed across the five 100x fields was the value we used in our analyses.
Torpedoes: A torpedo (“retraction bulb”) is a fusiform swelling of the proximal, unmyelinated segment of the Purkinje cell axon.13,14 Using the standard cerebellar section, the torpedoes in the entire section were counted. Torpedoes were assessed using LH&E-stained sections (100x).
Bergmann Astrogliosis: Bergmann cells are Golgi-type glial cells in the Purkinje layer.13 Using a GFAP-stained standard cerebellar section, the GFAP-positive cells in the Purkinje layer were counted in five 400x fields; these five fields were selected at random by a blinded assistant. The number of Bergmann cells in the five 400x fields was summed.
Cell Loss and Gliosis: In the primary (cerebellum) and each secondary (red nucleus, thalamus, inferior olivary nucleus, and motor cortex) region of interest, LH&E-stained and GFAP-labeled sections were examined using semi-quantitative scales for cell loss (0 – 4 [near complete or complete regional loss of cells]) and gliosis (0 – 3 [diffuse and heavy burden of gliotic cells]).
Data Analyses:
Analyses were performed in SPSS (Version 11). Continuous variables were compared using Student’s t tests and Pearson’s correlation coefficients (r). Logistic regression analyses were performed to assess the association between Lewy bodies (dependent variable, present vs. absent) and diagnosis, resulting in odds ratios (OR) with 95% confidence intervals (CI). Linear regression analyses were performed to assess the association between cerebellar pathologies (e.g., number of torpedoes) and diagnosis. In adjusted logistic and linear regression analyses, we controlled for the effects of possible confounders, as outlined below. All tests were two-sided with type I error = 0.05. None of the cases or controls had lifetime exposure to any medication (cytosine arabinoside, cyclosporine, terfenadine, lithium, diphenylhydantoin, vigabatrin) known to cause cerebellar damage,15-17 and none had a history of heavy ethanol use at any time in their lives (defined as a man who consumed an average of four or more standard drinks per day and a woman who consumed an average of three or more standard drinks per day);18 in fact, 90% consumed less than one drink per day. Other potential confounding variables that we considered were age in years, gender, race, and postmortem interval in hours. A Gliosis-Torpedo index (GTI) was created, which was the number of torpedoes multiplied by the number of Bergmann glia. Tests for trend were performed to determine whether the number of torpedoes on LH&E stained sections, the number of Bergmann glia on GFAP stained sections, or the GTI was lowest in controls, intermediate in ET cases with Lewy bodies and highest in ET cases without Lewy bodies. We repeated all analyses, excluding the one case with a diagnosis of PD during life; the results were similar (data not presented).
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