Post-mortem brain tissue
Formalin-fixed postmortem brain tissue from 27 donors with PD(D) and DLB (range = 60–93 years) was collected in close collaboration with the Netherlands Brain Bank (NBB, Amsterdam, The Netherlands; www.brainbank.nl). In compliance with ethical and legal guidelines, all donors or next of kin had provided written informed consents for donation of brain tissue and access to their clinical and neuropathological reports for research purposes. The brain donor program of the NBB were approved by the local medical ethics committee of the VU University Medical Center, Amsterdam. The main inclusion criteria for the current study were: (i) clinical diagnosis of PD, PD with dementia (PDD), or DLB according to revised MDS and McKeith diagnostic criteria [25, 26] and (ii) neuropathological confirmation of diagnosis [27]. Subjects were excluded if they had a long history of neuropsychiatric disorders or concomitant neurological disorders, and/or infarcts and other abnormalities within the insular cortex (Table 1).
Brain dissection was performed according to international guidelines of Brain Net Europe II (BNE) consortium (http://www.brainnet-europe.org) and NIA-AA by an experienced neuropathologist (NBB: AMR). The entire insula was dissected into 0.5–1 cm thick blocks according to its anatomical borders with surrounding brain regions. Using the central sulcus as a landmark separating the anterior from the posterior insula, the anterior insular cortex (AIC) was then dissected further [28]. Tissue blocks were cryo‐protected with 30% sucrose, frozen and sectioned using a sliding microtome into 60μm-thick sections and these were stored at −30°C until further processing. Sections were first incubated in various gradients of alcohol followed by xylene to induce de-fatting then stained with Nissl. This allowed the identification of the agranular and dysgranular sub-regions based on the absence or presence of layers II and IV by two experienced researchers (YF and LJ) as previously described [19, 29].
Neuropathological assessment
For neuropathological diagnosis and staging, 6 μm paraffin sections from brainstem, limbic and neocortical brain tissue blocks of all donors were immunostained for α‐synuclein (clone KM51, 1:500, Monosan Xtra, The Netherlands), amyloid-b (clone 6F/3D, 1:500, Dako, Denmark) , phosphorylated tau (p-tau, clone AT8, 1:500, Thermo Fisher Scientific, USA), haematoxylin and eosin (H&E), TDP‐43 and congo red according to current diagnostic guidelines of BrainNet Europe [27]. Pathological staging protocols were based on Braak α‐synuclein (Braak α‐syn 0–6), Braak staging for neurofibrillary tangles (Braak NFT 0–6), Thal phase for amyloid-β (0–5), and CERAD score for neuritic plaques [30-34]. Glial tauopathy such as age-related tauopathy of the astroglia (ARTAG) and primary age-related tauopathy (PART) were assessed primarily in the temporal cortex, olfactory cortex and amygdala by an experienced neuropathologist (AMR) [35, 36]. Clinical symptoms and diagnosis ante-mortem, along with neuropathological scores, were used to provide a final pathological diagnosis (Table 1).
Evaluation of axonal degeneration using modified Bielschowsky silver staining
A modified Bielschowsky silver staining [37] was used for the evaluation of axonal degeneration in the anterior insula. Prior to staining, all glassware was thoroughly rinsed with purified water for several hours (h) or over-night and metals were avoided. All incubations were performed at cold temperature (5°C) [37]. Free-floating 60-μm tissue sections (interval: 1 in 20) were rinsed in distilled water followed by Tris buffered saline (TBS, pH 7.6) and incubated in 20% silver nitrate solution (AgNO3; Sigma, CAS No: 7761-88-8) for 30 minutes at 5°C in the dark. Subsequently, non-evaporated ammonium hydroxide 30-33% (NH3) solution was slowly added to the sections until the color turned black, then incubated for 30 minutes at 5°C in the dark (Honeywell, CAS No: 1336-21-6). Sections were then rinsed with evaporated ammonia-water for 10 minutes (100 µl ammonia in 50 ml distilled water). Subsequently, a developer solution was freshly prepared and consisted of 100 ml distilled water, 20 ml 37% formalin (Sigma, CAS No: 50-00-0), 1 drop nitric acid (Fisher, CAS No: 7697-37-2), 0.5 gr citric acid (Sigma, CAS No: 77-92-9). Sections were incubated in the developer solution at 5°C in the dark for visualization of the reduced metallic silver. Staining was monitored carefully and the incubation was stopped within 10-15 minutes. This was followed by rinsing in Hypo (sodium thiosulfate 5% in distilled water; Honeywell, CAS No: 7772-98-7) to stabilize the developing reaction and remove free AgNO3 deposits from the tissue. Sections were then rinsed, mounted with 0.3% gelatin (Oxoid), dried and cover-slipped using Entellan (Sigma).
Evaluation of α‐synuclein, p-tau, and amyloid-β pathology in the anterior insular subregions
For α‐synuclein immunostaining, consecutive free‐floating 60-μm thick sections of the anterior insula were pre-treated with 98% formic acid (Sigma‐Aldrich, Darmstadt, Germany) and incubated with primary antibody mouse anti‐α‐synuclein (syn-1, 1:2000, 610786, BD Biosciences, Berkshire, UK) in TBS and 0.5% TritonX-100 solution overnight at room temperature, as previously described by Braak and colleagues [38]. For immunostaining with antibodies against p-tau and amyloid-β, free-floating sections were rinsed with TBS and pre-treated with citrate buffer (pH 6.0) in a steamer (95°C) for antigen-retrieval, followed by 80% formic acid for amyloid-β. Non-specific staining was then blocked with 0.3% H2O2 and 0.1% sodium azide in TBS followed by 2% normal goat serum. Subsequently, sections were incubated in primary antibody mouse anti-p-tau (1:1000, MN1020, AT8, Thermofisher-scientific, Netherlands) and adjacent sections were incubated in mouse anti-amyloid-β (1:1000, M08720, clone 6f/3d, Dako, Denmark) diluted in TBS and 0.1% Triton-X overnight at 4°C. All sections were incubated with biotinylated secondary antibody IgG (1:200, Vector Laboratories, Burlingame, CA, USA) followed by standard avidin‐biotin complex (1:200, Vectastatin ABC kit, Standard; Vector Laboratories) in TBS for 2h. Tissue samples were incubated in 3,3′‐diaminobenzidine (DAB) to visualize staining and were mounted and counter‐stained with thionin (0.13%, Sigma‐Aldrich, Darmstadt, Germany).
Load of a-synuclein, p-tau and amyloid-b pathology was calculated as percentage immunoreactivity per surface area in each region of interest (ROI) using area fraction plugin in ImageJ (1.52n) [39]. Anterior insular sub-regions (agranular and dysgranular) were identified on adjacent Nissl-stained sections, and the same borders were used to define ROIs on sections immuno-stained for pathological aggregates [19].
Multi-labelling immunofluorescence for evaluation of axonal cytoskeletal abnormalities
We included multi-labelling immunofluoresecent staining with axonal and myelin markers to visualize the axonal morphology and cytoskeletal abnormalities in 3D with CSLM. Adjacent 60-μm thick sections from PD(D) and DLB cases were rinsed in TBS, pre-treated with EDTA-Tris (pH 9.0) in a steamer (95°C) and incubated in a cocktail of the following primary antibodies: 1) mouse-anti myelin proteolipid protein (PLP, 1:500, MCA839G, plpc1, Bio-Rad, Netherlands) and 2) chicken anti-neurofilament heavy chain (NfH, 1:500, AB5539, heavy-chain, Millipore) diluted in TBS, 2% normal donkey serum, and 0.5% Triton-X. Immunostaining was performed for 48h at 4°C followed by incubation with secondary antibody goat anti‐chicken coupled with Alexa Fluor 594 (1:400; Molecular Probes, Waltham, MA, USA), donkey anti‐mouse coupled with Alexa Fluor 647, and diamidino‐2‐phenylindole(4,6)dihydrochloride (DAPI; Sigma) for nuclear staining for 2h in the dark. Tissue samples were subsequently rinsed in TBS and blocked in 5% normal mouse serum for 1hr followed by incubation with Alexa488-conjugated mouse anti-phosphorylated-Serine129 (pSer129) α‐synuclein antibody (1:100,11A5, gift from Prothena Biosciences Inc., USA) for 2h at 4°C in the dark. The tissue sections were then mounted on glass slides and cover‐slipped with mowiol-DABCO as a mounting medium (4‐88 Calbiochem).
Microscopic imaging
Digital images of the immunostained slides were made with a photomicroscope (Leica DM5000) equipped with color camera (DFC450), Leica LASV4.4 software and 63× oil objective lens. Immunofluorescent labelling was visualized using CLSM LEICA TCS SP8 (Leica Microsystems, Jena, Germany). Adjacent Nissl-stained sections were used for the delineation of subregions and the ROIs were superimposed on the images of the immunofluorescent stained sections. This was followed by sampling of all axons (Bielschowsky staining) in the superficial and deep layers of each sub-region. Image acquisition was done using 100×/1.4 NA objective lens, 405 nm diode, and pulsed white light laser (80 Hz) with excitation wavelengths 405, 499, 598, and 653 nm, scanned using frame/stack sequential mode. For optimal resolution, z-step size was calculated for each scan based on the Nyquist-Shannon sampling theorem [40] and line accumulation/averaging were used as deemed appropriate per channel. Deconvolution of image stacks was performed using Huygens Professional software (Scientific Volume Imaging, Hilversum, the Netherlands). Images are shown as maximum projections of all channels combined and all figures were composed using Adobe Photoshop (Adobe Systems Incorporated).
Stereological analysis of axonal loss
Axonal length density, based on Bielschowsky silver impregnated sections, was quantified using the stereological space balls probe from stereoinvestigator software (MBF Bioscience, Williston, VT, USA) and Leica microscope DMR HC (v2019.01.4) [41-43]. Serial sections (1:20, Range of sections: 3-8) were used for quantification, and counting parameters were chosen to allow counting ≥200 axon intersections for each sub-region. A sphere (radius=10μm) was used along with a sampling grid 2700 x 2700μm. ROIs corresponding to the grey matter of the agranular and dysgranular sub-regions were drawn at a low magnification (using a 2.5x objective) and axonal quantification was completed at higher magnification (100x oil objective). Only nerve fibers were counted and when a counting frame/sphere contained tangles or other pathologies, they were not counted and the following counting frame was used. Moreover, a fiber was counted only when it fully intersected with the sphere at least once. Tissue thickness was measured manually at each sampling frame (mean= 22 mm ± 1.42) and coefficient of error (CE) was calculated for each sub-region (mean CE agranular=0.13 ± 0.06; CE dysgranular = 0.12 ± 0.05).
To calculate estimated axonal length (L), total number of intersections of fibers with space balls (Qi) throughout all sections is multiplied by the volume of sampling frame. The volume (V= grid X * grid Y * section thickness) is divided by the surface area of sphere (a=) multiplied by the reciprocal of sampling fraction of the section (1:20) [41, 43]. To calculate density (mm/mm3), total axonal length (L) was divided by sampled reference volume per ROI. Reference volume was derived through planimetry; calculated as a measure of total area of the ROI multiplied by section height [41, 43].
Statistical Analysis
Statistical analyses were performed using SPSS (version 26.0, IBM) and graphs were made using graph prism, version 9. When tissue or staining quality was insufficient, cases were omitted from analysis. Demographics of controls, PD(D) and DLB were compared using chi-square test for categorical data and ANOVA, with age corrections, for numerical data. To analyze differences in axonal length density between insular sub-regions (agranular and dysgranular) across all three patient groups (PD, PDD, DLB), an ANCOVA, with adjustment for age, and post-hoc paired t-test were used. Quantitative scores for p-tau and a-synuclein pathology load showed non-normal distribution and were log transformed followed by parametric analysis using ANCOVA and age as covariate with post-hoc paired t-test for group differences. For amyloid-b, due to the presence of multiple zero scores from pathology quantification, scores were dichotomized (0 or 1) and analyzed using logistic regression. To assess the pathology and group effects on axonal density, variables were pooled into the model and a nested linear mixed model analysis was performed using backward elimination. Axonal length density was the dependent variable, area% load of p-tau, a-synuclein, and amyloid-b were main effects and age was a covariate. Correction for multiple comparison was not performed. Statistical significance was set at p<0.05.