Parkinson disease and incidental Lewy body disease

Standard protocol approvals, registrations, and patient consents.

All participants signed a written informed consent approved by the Banner Sun Health Research Institute Institutional Review Board to use their medical data and donated body specimens for research purposes.

Clinical and pathologic data.

For each participant, the following clinical and pathologic diagnoses were excluded: Alzheimer disease (AD), vascular dementia, DLB, progressive supranuclear palsy, corticobasal degeneration, multiple system atrophy, hippocampal sclerosis, motor neuron disease, Pick disease, Huntington disease, and frontotemporal lobar dementia with TDP-43 proteinopathy. Data on clinical and pathologic diagnoses, age at death, sex, race, cause of death, postmortem interval (PMI), brain weight (BW), and APOE genotype were available.

For each examined case, LB, AP, and NFT densities were available. LB densities (phosphorylated α-synuclein immunohistochemistry) were measured in SN, locus ceruleus (LC), olfactory bulb, brainstem at level of the IX and X cranial nerve, amygdala, and transentorhinal, cingulate, temporal, parietal, and frontal cortex. AP and NFT densities were assessed in frontal, temporal, parietal, and entorhinal cortex, and CA1 region of the hippocampus. The staging for each type of pathology was performed according to the Unified Staging System for Lewy Body Disorders (for LTS),⁷ Consortium to Establish a Registry for Alzheimer's Disease criteria (for AP),²³ and NFT-Braak system (for NFT).²⁴ Data on depigmentation of SN,²⁵ vascular pathologies, infarct volumes, and cerebral amyloid angiopathy (CAA) were also available.

As documentation for the motor and cognitive status before death, the following data were available: Unified Parkinson's Disease Rating Scale (UPDRS)–motor scale (off medication),²⁶ Hoehn & Yahr (H&Y) stage,²⁷ time interval between last UPDRS-motor scale and death, time interval between last Mini-Mental State Examination (MMSE)²⁸ and death, cognitive impairment assessment (i.e., cognitively normal, mild cognitive impairment, or dementia), and levels of probability that dementia, if present, was due to AD pathology, as based on National Institute on Aging (NIA)–Reagan criteria.²⁹

Neuropathologic material and stereology methods.

Eighteen sets of 40-μm-thick serially cut sections (1 every 24 from the entire SN) for each participant was received at the Neuropathology Research Labs, Biomedical Research Institute of New Jersey (BRInj). Histologic and staining procedures, stereologic protocols and measurements, and statistical analyses were performed at BRInj.

Each 40-μm-thick section was stained with a 1.0% solution of cresyl violet (CV) and used for the unbiased neuronal counting and volumetric measurements. Each section was randomly coded by an investigator blinded for the clinical and pathologic diagnoses (M.G.-E.) prior to any stereologic measurements, which were performed by a second clinical and pathologic diagnoses–blinded independent investigator (D.I.). Each section included both anatomical regions of SN: the pars compacta and pars reticulata.

We applied the following histologic criteria for neuronal counting:

1. Inclusion of any neuron, of any size, which contained neuromelanin detected by CV stain, within the marked SN anatomical contours.

2. Exclusion of all non-neuronal cells within the marked anatomical SN contours, such as macrophages.

We applied the following adjunctive criteria for neuronal volumetric measurements:

1. Individuation of a well-defined non-neuromelanin-covered nucleolus for each randomly sampled nigral pigmented neuron.

2. Establishing the nucleolus as the only and constant point of spatial reference for any volumetric measurement (cell body, nucleus, and nucleolus) in each randomly sampled nigral pigmented neuron.

Measurements were performed using Stereo-Investigator system, version 10.0 (MBF Bioscience, Williston, VT). The sampling grid area was 500 × 500 μm, with a counting frame of 40 × 40 μm, disector height of 25 μm, and guard zones of ±2 μm. The total number of CV-stained nigral pigmented neurons was estimated using the Optical Fractionator probe.³⁰ The one-side SN neuronal counting was doubled to obtain estimation of the total neuronal population in SN (assuming no pathologic/cellular asymmetry between SN sides in PD, ILBD, and C).

Volumetric measurements were performed using the Nucleator probe. Cell body, nuclear, and nucleolar volumes of each nigral pigmented neuron were measured by placing 6 rays automatically centered on the nucleolus and randomly intersecting the cell membrane of each single randomly sampled pigmented neuron (figure 1). All volumetric measurements were performed employing a 100× oil-immersion objective.

Statistical analyses.

Analyses of variance (ANOVAs) were performed to detect group differences for mean age at death, PMI, BW, last MMSE and UPDRS-motor (off medication) scores, time intervals between last MMSE/UPDRS scores and death, and H&Y scores.

Before statistical analyses for each type of measurement (neuronal counting, cell body, nuclear and nucleolar volume), a series of Kolmogorov-Smirnov tests were performed. Kolmogorov-Smirnov tests confirmed that all data were normally distributed. ANOVAs were followed by 2 post hoc analyses: Tukey and Bonferroni tests for multiple comparisons. Adjustments for multiple comparisons established the statistical significance at p ≤ 0.01.

Correlation analyses were performed between neuronal counting and nigral LB, extranigral LB (LC, olfactory bulb, brainstem at level of the IX and X cranial nerves, amygdala, and transentorhinal, cingulate, temporal, parietal, and frontal cortex), AP, NFT, mean cell body, nuclear and nucleolar volume, last MMSE/UPDRS-motor scores, and H&Y stages for all groups.

Stereologic findings.

Marked NNL was found in PD and ILBD vs C (p < 0.0001). The mean ± SD neuronal count was 120,015 ± 41,051 for PD, 402,045 ± 199,163 for ILBD, and 664,308 ± 103,541 for C. In PD and ILBD, the nigral neuronal counting was, respectively, 82% and 40% lower than C (figure 1).

ANOVAs did not show differences for the mean nigral cell body volume between ILBD and C. Moreover, while the mean cell body, nuclear, and nucleolar volumes were lower (as for a possible subjacent atrophic process) in PD vs ILBD and C (p < 0.0001), the mean nuclear and nucleolar volumes were lower in both PD and ILBD vs C (p < 0.0001). These quantitative volumetric measurements seem to exclude cellular compensatory phenomena, for example a neuronal hypertrophy, in nigral pigmented neurons of ILBD in comparison to C.

The PD group showed correlations between NNL and LB in extranigral regions of the brain, such as olfactory bulb, cingulate, temporal, and parietal cortex. In the ILBD group, by contrast, no correlations were found between NNL and LB in any examined brain region. Furthermore, in both PD and ILBD groups, there were no correlations between NNL and AP or NFT densities, infarct volumes, and CAA.

In the ILBD group, correlations between mean cell body/nucleolar volume and LTS were found in amygdala and transentorhinal cortex, while in the PD group those correlations were found only in transentorhinal, cingulate, and temporal cortex.

No correlations were found in the ILBD or PD group between NNL and neuronal volumes, AP, and NFT densities; the only exception was found in the PD group, where the mean cell body volume and AP densities were correlated in the frontal cortex. No correlations were found between NNL and last UPDRS-motor scores and H&Y stages in either PD or ILBD.

Table 3 shows the single correlation values (Pearson r) among neuronal counting, mean cell body, nuclear, and nucleolar volume with LB, AP, and NFT densities for each assessed brain region.

Figure 1 shows histograms for the estimated neuronal counting, mean cell body, nuclear, and nucleolar volumes of nigral pigmented neurons in PD, ILBD, and C. Figure 2 shows graphic relationships between NNL and their cell body volume, as between NNL and the LB sum densities in PD, ILBD, and C. Table e-1 on the Neurology® Web site at Neurology.org, upper part, using a color-based coding system, describes the neuroanatomical distribution of LB pathology and its level of severity (none, mild, moderate, severe, very severe) across all groups. Table e-1, lower part, using a color-based coding system as well, describes the presence (yes) or the absence (no) of LB across all examined cerebral regions.

Larger autopsy quantitative stereology-based studies need to confirm our novel findings.

We support that ILBD is a genuine pathologic status, and that it can represent a preclinical phase of PD/DLB, since we found significant NNL in those brains in comparison to C. Furthermore, the lack of hypertrophy in nigral pigmented neurons does not support the presence of cellular compensatory mechanisms in the SN of ILBD brains. Moreover, cortical AP and NFT densities, as infarct volumes and CAA, do not seem to have a major contributing role in determining NNL.

Alternative hypotheses remain open:

1. ILBD is not the preclinical phase of PD/DLB per se, but an anatomically parallel and pathogenetically separated process overlapping other pathologic events causing PD/DLB.³⁵ Those PD/DLB-specific pathologic events could simply enhance degenerative phenomena in already predisposed or particularly vulnerable brain areas. An intriguing similitude here could be evoked with the recently defined condition of primary age-related tauopathy.³⁶ Could ILBD be a condition of primary age-related synucleinopathy? Is it just by chance that the olfactory dysfunction normally present during aging and ILBD is accentuated in PD?³⁷

2. ILBD is indeed a preclinical phase of PD/DLB but it requires a longer gestation period to be clinically manifested in some predisposed participants.³⁸

3. ILBD is not a preclinical phase at all, despite a 40% NNL vs C, but simply represents a status of asymptomaticity, perhaps lasting until death and based on unknown protective factors^39,40 limiting the progression of NNL.