Reduced Number of Pigmented Neurons in the Substantia Nigra of Dystonia Patients? Findings from Extensive Neuropathologic, Immunohistochemistry, and Quantitative Analyses

Abstract

Background: Dystonias (Dys) represent the third most common movement disorder after essential tremor (ET) and Parkinson's disease (PD). While some pathogenetic mechanisms and genetic causes of Dys have been identified, little is known about their neuropathologic features. Previous neuropathologic studies have reported generically defined neuronal loss in various cerebral regions of Dys brains, mostly in the basal ganglia (BG), and specifically in the substantia nigra (SN). Enlarged pigmented neurons in the SN of Dys patients with and without specific genetic mutations (e.g., GAG deletions in DYT1 dystonia) have also been described. Whether or not Dys brains are associated with decreased numbers or other morphometric changes of specific neuronal types is unknown and has never been addressed with quantitative methodologies.

Methods: Quantitative immunohistochemistry protocols were used to estimate neuronal counts and volumes of nigral pigmented neurons in 13 SN of Dys patients and 13 SN of age-matched control subjects (C).

Results: We observed a significant reduction (∼20%) of pigmented neurons in the SN of Dys compared to C (p<0.01). Neither significant volumetric changes nor evident neurodegenerative signs were observed in the remaining pool of nigral pigmented neurons in Dys brains. These novel quantitative findings were confirmed after exclusion of possible co-occurring SN pathologies including Lewy pathology, tau-neurofibrillary tangles, β-amyloid deposits, ubiquitin (ubiq), and phosphorylated-TAR DNA-binding protein 43 (pTDP43)-positive inclusions.

Discussion: A reduced number of nigral pigmented neurons in the absence of evident neurodegenerative signs in Dys brains could indicate previously unconsidered pathogenetic mechanisms of Dys such as neurodevelopmental defects in the SN.

Keywords: Substantia nigra; neurodegenerative disorder; neurodevelopmental disorder; neuronal loss; neuronal reduction; pigmented neurons.

Figure 1A/1B. Quantification of SN Pigmented Neurons. The quadrants in the figure show how a randomly sampled pigmented neuron (first upper-right quadrant, 20× objective), and 6 randomly projected rays on that pigmented neuron intersect its cell body, nucleus, and nucleolus (respectively, upper-right, lower-left, and lower-right quadrant). A 100× oil immersion objective was used for all volumetric measurements and for each pigmented neuron. The green markers in each quadrant indicate the point of measurement from which the areas of the cell body, nucleus, and nucleolus were calculated. The colored square upper-right quadrant represents the “counting frame” used for the Optical Fractionator probe. The Stereo-investigator software automatically calculates the volumes of each single neuron computing various parameters such as the thickness of the tissue, the disector height, and guard zones. Figure 1B. Tissue thickness, cell counting, volumetric measurability of nigral pigmented neurons. SNc from a C brain (case#16), cut at different levels of thicknesses (40 µm and 10 µm), stained with CV, and inspected at two different magnifications (10× and 20× objectives). The green circles in the inferior parts of the figure indicate the number of pigmented neurons measurable when pigmented neurons were sampled using 40 µm-thick and 10 µm-thick tissue sections, respectively, with a 20× objective. Notably, the number of measurable pigmented neurons in SN cut at 10 µm of thickness (right of figure) is markedly higher due to the increased number of clear visible CV-stained nucleoli. The nucleolus was the established point of spatial reference on this study. All measurements were performed using a 100× oil-immersion, NA 1.30, neofluor ∞/0.17 objective. Abbreviations: C, Control; CV, Cresyl Violet; SN, Substantia Nigra; SNc, Pars Compacta of Substantia Nigra.

Figure 2. Histograms of the Estimated Mean Number of SN Pigmented Neurons in 13 Dys Patients and 13 C Subjects. The histograms on the left side include all Dys and C brains, while those on the right show Dys and C data after exclusion of all brains with immunohistochemical evidence of co-occurring pathologies. The lower part of the figure shows histograms of cell body, nuclear, and nucleolar mean volumes of pigmented neurons in the SN. The y-axis indicates mean volumetric values expressed in µm³. *p<0.01. Abbreviations: C, Control; Dys, Dystonia; SN, Substantia Nigra.

Figure 3. Co-occurring Pathologies in the SN of Dys Patients. Abbreviations: CD, Cervical Dystonia (subject Dys#6); Ch-on, Childhood-onset; Gen., Generalized (Subject Dys#13); LB, α-synuclein-positive Lewy Body; MB, Marinesco Body (intranuclear eosinophilic body); Tau-NFT, Tau-positive Neurofibrillary Tangle; Tau-th, Tau-positive Thread; Ubiq+, Ubiquitin Positivity.

Figure 4. Histograms of Estimated Mean Numbers of Pigmented Neurons. We quantified neurons in the substantia nigra (SN) of dystonia (Dys) vs. age-matched control subjects (C) and compared counts in types of dystonia (adulthood-onset, childhood-onset, generalized, focal/segmental [foc/seg] dystonias). For each measurement there are two histograms showing the obtained estimated number of neurons before and after exclusion of all nigral co-occurring pathologies (β-amyloid, tau, Lewy bodies, and ubiquitin). All co-occurring nigral pathologies were assessed using immunohistochemistry protocols.