Abbiategrasso Brain Bank Protocol for Collecting, Processing and Characterizing Aging Brains

In line with our institution's Human Research Ethics Committee and the BNE Code of Conduct, the ABB performs its activities following ethical standards^39,40. The brain harvesting procedure was submitted to and approved by the Ethics Committee of the University of Pavia in the context of the InveCe.Ab study³⁶. The study procedures were in accordance with the principles outlined in the Declaration of Helsinki of 1964 and the following amendments. The consent form is complete and easily comprehensible. Joining the donation program is a personal decision, and complete awareness is needed. In case a person is not deemed competent to sign the consent form, authorization from legal guardian or next-of-kin (NOK) is warranted. Table 2 reports inclusion and exclusion criteria for brain donation. The research was performed under the supervision of the Federazione Alzheimer Italia.

1. Recruitment to the brain donation program

1. Convene the subjects who had expressed interest in brain donation and their NOK and/or legal guardian to present the project (the scope of the donation program; the process of brain removal, conservation, use and distribution), the right to withdraw from the program at any given time, the protection of anonymity, and the follow-up strategies. Discuss the possibility of additional biomarker investigations and genetic tests. Note the wishes of the potential donor or his NOK to be informed about the results.
2. Explain all the economic aspects including the lack of financial gain for both the Foundation and the donor (brains are donated and distributed altruistically). Inform them that ABB covers the cost of body transportation, while the family covers those of the funeral, burial or cremation.
3. In case of acceptance, obtain the signature of the consent to participate in the donation program. Give the donor an individual numerical code to guarantee anonymity. Write down the identification number of those participating in the longitudinal study and provide another code for the brain bank to easily separate the two subgroups of donors (participants or non-participants in the longitudinal study; see Table 3).
4. Give the donor an identification card that declares his status as a donor and displays the telephone number (available 24/7) to notify the Brain Bank of his death.

2. Donor evaluation and serial follow-ups

NOTE: It is possible to give consent only for some, and not all, of the below mentioned examinations. All clinical assessments are performed by the same team including a neurologist, a geriatrician with expertise in neurology and 3 psychologists. If new symptoms develop or the cognitive decline progresses, the time interval between follow-ups can be shortened.

1. As in the case of the InveCe.Ab study, obtain a lifestyle-social questionnaire, including degree of autonomy (ADL, IADL), personal habits, social and lifestyle factors that may affect mental functions. Collect family, medical and neurological history with information regarding genetic, infective, toxic, metabolic, autoimmune, cardiovascular, traumatic, degenerative, neoplastic, and neurological diseases. Collect previous clinical exams and list pharmacological treatments.
2. Carry out an extensive neuro-motor evaluation including testing for cranial nerves function, fundus oculi, visual field, muscle strength and tone, sensibilities, tendon reflexes, exteroceptive and primitive reflexes, meningeal signs, posture, gait, movements, coordination, sphincteric functions, and any presence of focal or Babinski signs. Evaluate language, mental status and any change in behavior.
3. Carry out an extensive neuro-cognitive evaluation including global cognition and a complete neuropsychological assessment of specific cognitive domains: verbal and visual memory, attention, psychomotor speed, language, semantic memory, executive functions, and visuospatial abilities (Table 4 for details). Consider the presence of depression (CES-D scale).
4. Obtain blood sample which is used for both blood chemistry analysis (Table 5) and isolation and storage of DNA, plasma and peripheral blood mononuclear cells (PBMC). Determine APOE genotyping (rs429358 and rs7412) (a more extensive genetic profiling has been determined in InveCe.Ab participants; see Table 6). Register an ECG (cardiac alterations, atrial fibrillation) and a resting state Electroencephalogram for Quantitative analysis (QEEG).
5. Consider optional biomarker testing including cerebrospinal fluid (CSF) TAU, phospho-TAU and β-amyloid, and brain imaging (MRI to detect in vivo degeneration, ischemia or inflammation; FDG-PET to detect metabolic failure; PIB-PET to detect brain amyloidosis related to Alzheimer's pathophysiology).
6. Plan the brain donor's subsequent check-up program. Set up the time intervals according to different age groups (up to 64 years old: every 10 years, 65–74 years old: every 3 years, from 75 years onwards: every 2 years).
7. Assign a clinical diagnosis and/or a neurocognitive diagnosis according to DSM-V. Classify clinical dementia staging with Clinical Dementia Rating (CDR) score. Update CDR in the last period before death.
8. Prepare a database graphically similar to the paper one. Insert all collected data into the Brain Bank database. Plan a revision by another clerk to check for any mistakes.

3. Time of death and brain removal

NOTE: The Italian law establishes that asystole must last longer than 20 min to confirm death. The registration of a flat electrocardiogram (ECG) for at least 20 min (named thanatography) allows for an autopsy to be carried out within 24 h of death (DPR 285/90 art. 8 and Law n. 578 Dec 29, 1993). A postmortem time of <24 h is a good target to preserve the overall tissue quality. In case of a postmortem time >30 h the autopsy is cancelled (see Table 2). The autopsy team is composed of a pathologist, a neurologist and/or a neurobiologist, a nurse, an anatomical room technician and any trainee students; the first two team members also perform the neuropathological diagnosis. During cadaver handling and dissection, the use of appropriate clothing (coat, gloves, eyeglasses and hairnet) is mandatory. In this section, we describe the main tools and equipment normally used in our laboratory. Readers can choose the instruments to be used at their discretion. For a detailed description of the materials herein utilized, please see Table of Materials.

1. Make sure that the funeral agency chosen by the family brings the body to the ABB facilities. Perform the thanatography during which the cardiac activity must be absent. If so, sign a death certificate and begin the autopsy procedures.
2. Transport the cadaver to the autopsy room. Measure the circumference of the skull at the level of the widest part of the head. Measure from the nasion to the inion to obtain the anteroposterior diameter (APD), while the distance from one ear to the other yields the transverse diameter (TD). Calculate the cephalic index (CI) using the formula: CI = TD/APD x 100.
3. Using a sharp scalpel, make a scalp incision on the coronal plane, from the tip of the mastoid process on one side to the other, passing over the vertex. Cut through hair, skin and subcutaneous tissue and separate the two folds of the scalp carefully from the underneath skull. Perform the incision until the yellow supraorbital fat becomes visible and reflect the scalp anteriorly. Pull the other part posteriorly.
4. Using scalpel and forceps, take a small sample of the temporal muscle at each side, put one in 4% formaldehyde and freeze the other (see section 7).
5. Cut the skull using an electrical saw following a V-cut on the frontal side. Remove the skullcap and cut the meninges. Sample pieces of dura are both fixed in 4% formaldehyde and frozen (see section 7).
6. Obtain about 10 mL of CSF by inserting a 20 G 3.5 in. needle through the corpus callosum to reach the third ventricle. Assess the appearance, color and turbidity of CSF and measure the pH. Then, make 10 aliquots of about 1 mL each and store them at -80 °C.
7. Lift up the brain delicately pulling on both frontal lobes and cut both optic nerves infundibulum, the internal carotid arteries (ICA) and the third, fourth, fifth and sixth cranial nerves. Cut the tentorium to reach the posterior fossa and cut vertebral arteries and lower cranial nerves. Insert the scalpel as deep as possible through the foramen magnum, to cut the most caudal portion of the medulla. At this point, remove the whole brain gently.
8. With the scalpel, pierce the bone over Meckel's cave and obtain the Gasserian ganglion from both sides. Fix one ganglion in 4% formaldehyde and freeze the other (see section 7). Fracture the bony sella turcica using a surgical mallet and chisel to remove the pituitary gland and then fix it in 4% formaldehyde.
9. Inspect the skull and the whole brain for macroscopic alterations and vascular changes. With a measuring tape, measure the brain obtaining the transverse and anteroposterior diameters. Retrieve with care the circle of Willis and assess it macroscopically (Figure 2E) for the presence of anatomic variants, lesions or vessel stenosis.
10. Take 1–2 pieces of leptomeninges of 2–4 cm² from the convexity and preserve them in complete high glucose Dulbecco's modified Eagle media (DMEM) culture medium at 4 °C containing 20% fetal bovine serum (FBS), 1% pen/strep, 1% glutamine and 1% non-essential aminoacids (as previously published, with some modifications)⁴¹.
    1. In order to obtain fibroblasts cultures, place the leptomeninges on a 10 cm Petri dish and wash twice with phosphate buffer saline (PBS), each time discarding the liquid.
    2. Using a scalpel and a pipette tip, cut the leptomeninges into small pieces of about 2 or 3 mm, seed 3–4 pieces in each well of a 6-well plate previously coated with gelatin at 0.5% and fill each well with 2 mL of complete medium supplemented with 1% of amphotericin B (conduct the processing in a biosafety cabinet to guarantee the sterility of the culture).
    3. Place the plate into a 37 °C, 5% CO₂ incubator for at least one week and change spent medium every 3–4 days. Trypsinize cells with sterile 1x trypsin when the well is at about 70% of confluence. Place leptomeningeal fibroblasts into a T75 flask and fill it with 12 mL of complete medium. After 5 days trypsinize and preserve them in 900 μL of FBS and 100 μL of dimethyl sulfoxide (DMSO).
11. Separate and inspect cerebrum, cerebellum and brainstem and weight them individually. Take off the pineal gland and fix it in 4% formaldehyde. Remove olfactory bulbs and optic nerves, and fix or freeze one of each pair, independently of the side. Keep the cerebrum, cerebellum and brainstem in ice at 4 °C for at least 2–4 h until ready for dissection to minimize tissue disruption and reduce the softness of tissue.
12. After the harvesting, pay appropriate attention and care to the cadaver. Reattach the bone using a super adhesive glue, and stitch back the scalp using a surgical needle and non-absorbable sutures.
13. Show respect and gratitude to the deceased person by treating the cadaver gently. Clean and shave the face, and wash and dry the hair, because the cadaver will be put in an open coffin visible to loved ones.
    NOTE: Recomposition of the cadaver is done by a technician. The protocol can be paused here.

4. ABB dissection protocol

NOTE: The same pathologist and/or neurologist slice the brainstem, cerebellum and cerebrum under a fume hood. A neurobiologist arranges the slices after sectioning. A trainee not handling brain sections documents the whole procedure with photos to be uploaded to the database to serve as guide during the subsequent tissue processing phases.

1. Using a dissecting knife, cut the brainstem axially and make the first cut at the level of rostral midbrain, through the superior colliculus, to obtain two slices exposing the substantia nigra (SN).
2. Make the rest of the cuts to acquire 8 mm brainstem slices to obtain about 10 sections. Be careful to include cuts passing through the rostral pons near the superior margins of the fourth ventricle to observe the locus coeruleus (LC) and through the medulla oblongata inferior to the acoustic striae just above the inferior apex of the fourth ventricle to have slices including the dorsal motor nucleus of the vagus (DMNV).
3. Name all slices in a rostrocaudal sense as BS (brainstem) followed by Arabic numbers to identify the sections. After the last brainstem section, use the designation SC (spinal cord) followed by numbers 1–n. About 2–4 SC slices are obtained depending on the number of spinal metameres removed (Figure 2H–I).
4. Label all sections to be fixed or to be frozen alternately and take a picture for the photo archive (Figure 2). Then, fix and freeze all slices as described below (steps 4.7 and 4.8).
5. Cut the cerebellum on the sagittal plane to separate the two cerebellar hemispheres at the level of the vermis. Perform sagittal sectioning to obtain 5 slices from each hemisphere (Figure 2F–G).
6. Name all slices from vermis as CBR or CBL (for right and left cerebellum respectively) and use Arabic numbers to identify the sections. Label all sections to be fixed or frozen alternately and take a photo for the archive (Figure 2). Then, fix and freeze all slices as described below.
7. Separate the two hemispheres of the cerebrum through the corpus callosum (Figure 2A–B) and slice them singularly on the coronal plane. Make the first cut in a plane passing between the optic chiasm and the mammillary bodies, through frontal lobe, temporal pole, anterior cingulate, anterior commissure, nucleus of Meynert and basal ganglia.
8. Perform the second cut about 1 cm posteriorly passing through the mammillary bodies and exposing the basal ganglia, anterior thalamus, subthalamus and amygdala. Continue slicing to dissect the anterior and posterior regions to acquire 1 cm slices in order to obtain 15 to 20 sections for each hemisphere.
9. Set the slices on a flat surface. Lay them following their original position in the anteroposterior direction, from the frontal to the occipital pole, with the portion continuous with the previous slice facing upwards.
10. By comparing the sections from both hemispheres, select alternate sections from each hemisphere to be retained as fixed or frozen material. Recognize the main sections to be fixed and processed for histopathology including frontal and temporal lobes, cingulate, basal ganglia, nucleus basalis of Meynert, amygdala, thalamus, hippocampus and entorhinal cortex, occipito-temporal gyrus, parietal and occipital lobes.
11. Name all slices in anteroposterior sense as L (left) or R (right) followed by Arabic numbers and put a label indicating whether the slice is to be fixed or frozen (Figure 2A–D). To identify the sections, take a picture for the photo archive. Then, fix and freeze all slices as described in sections 7 and 8.

5. Brain and vessels inspection and macroscopic pathological assessment (with the naked eye)

1. Carry out a general macroscopic examination considering meningeal alterations, diffuse or localized cortical atrophy, hippocampal atrophy, ventricular enlargement, cerebellar atrophy, brainstem atrophy, substantia nigra pallor, white matter alterations (specify type and location).
2. Examine the circle of Willis considering atherosclerosis and occlusion. Assign score = 1 in the presence of atheroma without stenosis of more than 50%, score = 2 if at least one artery is 50% or more occluded, score = 3 if two or more arteries are 50% or more occluded⁴². Evaluate the presence or absence of pathology in other intracranial vessels.
3. To detect parenchymal vascular lesions, examine brain hemispheres, cerebellum and brainstem. Consider lacunar lesions (diameter < 10 mm), ischemic or hemorrhagic infarcts and hemorrhage (diameter > 10 mm). If present, specify size in millimeter, number and location. Also, consider the presence or absence of subarachnoid hemorrhage.

6. Tissue quality control

NOTE: Agonal factor score (AFS) ranges between 0 and 2 and is important in the evaluation of tissue quality. To determine AFS, consider clinical conditions occurring around the time of death (particularly conditions determining brain acidosis) and the duration of agonal state (sudden death or prolonged agony). If AFS > 1, the brain tissue might not be of optimal quality⁴³. Also consider brain and CSF pH; if pH < 6, the brain tissue might not be of optimal quality^43,44.

1. Verify if death has been caused by conditions that can damage brain tissue including hypoxia, protracted acidosis, mechanical ventilation, multi-organ failure, high fever, severe cranial trauma, ingestion of neurotoxic substances, severe dehydration, severe hypoglycemia, epileptic state and prolonged coma. If one of these conditions is present, assign 1 point.
2. Verify the duration of agonal state (rapid if death occurs within 1 h, intermediate if death occurs between 1 and 24 h, slow if the agonal state lasts more than 1 day). If an intermediate or a slow death happens, assign 1 point.
3. Calculate AFS score, and measure CSF pH by an electrode pH needle and tissue pH by a surface pH meter on a brain slice from each lobe and on a cerebellar section.

7. Tissue freezing

1. Keep all tissues to be frozen in ice at 4 °C. Then, freeze them quickly. Place them on a prefrozen aluminum tray. Cover with an interlocking aluminum plate to keep them flat. Put them in liquid nitrogen at -120 °C for 3 min.
2. Place the slices inside a plastic bag labeled with their corresponding identification code and slice number. Divide the plastic bags into three cryogenic boxes (for right hemisphere, left hemisphere and brainstem) and store at -80 °C.

8. Tissue fixation

1. Wrap the slices in gauze one by one, and put the slices in 10% phosphate buffered formalin solution. After 1 day, substitute the formalin solution with a fresh solution. Leave them for about 5 days in a cold room at 4 °C.
2. Keep all slices as a whole and split only the slices containing hippocampus and amygdala in two parts (Figure 3). The upper part of both slices will contain the frontal lobe (R10a–L9a in Figure 3); the lower parts will contain respectively: (1) amygdala, temporal lobe and basal ganglia (L9b in Figure 3B), and (2) hippocampus and part of temporal lobe (R10b in Figure 3A). This is made in order to maintain the relationship between the various structures.
3. Put all sections in phosphate buffer for at least 2 days to wash out and remove cross linking products.
   NOTE: The protocol can be paused here.

9. Dehydration, clearing, paraffin embedding and slide preparation

1. Prepare increasing concentrations of ethyl alcohol from 70% to 100%, xylene and two sets of molten paraffin wax. Place the brain sections in the automatic processor for dehydration, clearing procedure and paraffin infiltration (use two different tissue processing protocols for macro (cerebrum and cerebellum slices) and micro samples (brainstem slices and the other small samples); Table 7). Embed the tissue in paraffin wax on a metal or plastic mold.
2. Select the sections to slice for evaluating all the regions of interest applying Montine's index for neuropathological characterization⁴⁵ (Table 8). Then, slice the selected sections.
3. Use a sledge microtome for macrosections and a rotary microtome for small sections. Cut 5 μm slices for Haematoxylin and Eosin (H&E) staining and 8 μm slices for the other stainings and immunohistochemistry. Put the slices on three different kind of histological slides: 8.5 cm x 11 cm for biggest slices, 5 cm x 7.5 cm for medium slices and the classic 2.5 cm x 7.5 cm for smallest ones.
   NOTE: The protocol can be paused here.

10. Deparaffinization, histological staining and immunohistochemistry (IHC)

1. Perform deparaffinization to enable reaction with aqueous dye solutions. Perform it using xylene and decreasing alcohol concentration (5 min for every step). Rehydrate for 5 min with distilled water.
2. Use the following histological stainings to evaluate architectural and structural tissue abnormalities, and cellular morphology: H&E (for vascular microscopic lesions and inflammation), Cresyl Violet (NISSL; for neuronal loss), Luxol Fast Blue (LFB; for demyelination), Gallyas (for neuritic plaques and neuropil threads).
3. Use immunohistochemistry (IHC) in order to highlight the presence of specific target structures. Anti-NeuN and anti-GFAP are used to identify neuronal and glial compartments; 4G8, AT8, α-SYN and TDP-43 are used to identify proteins that aggregate in neurodegenerative diseases (Table of Materials).
   1. Pretreat dewaxed sections with 3% H₂O₂ for 10 min then rinse in PBS. Perform retrieval treatment with citrate buffer 0.01 M pH 6 (microwaved serially for 2, 1 and 2 min) for 4G8, α-SYN, TDP-43 and NeuN antigens; use 70% formic acid for 4G8 and α-SYN. Preincubate for 30 min in 5% normal goat serum.
   2. Incubate overnight at 4 °C with the primary antibody. On the day after, rinse the sections in PBS before incubation with the secondary antibody (Envision+ System-HRP labeled Polymer) at a dilution of 1:2 in PBS for 1 h at room temperature.
   3. Wash several times in PBS and incubate in chromogen system with diaminobenzidine (Liquid DAB+Substrate Chromogen System) looking at reaction development under the microscope (magnification 4–10x). Finally wash the sections in PBS. Counterstain the sections in haematoxylin, dehydrate and coverslip with DPX mounting.
      NOTE: Table 8 summarizes the standard protocol. Perform H&E staining on all sections, while special/specific stains and reactions are used on selected sections. Selected cases require additional areas or reactions. Table of Materials shows the details of antibodies used for IHC.

11. Basic neuropathological characterization

NOTE: Nonspecific brain tissue alterations, vascular pathology, Alzheimer's Disease (AD) pathology, non-AD TAUopathies, synucleinopathies, TAR DNA-binding Protein (TDP-43) pathology and hippocampal lesions are studied. An optical microscope connected to a camera is used to detect parenchymal microscopic lesions. The histological assessment is performed by the same team of trained personnel in neuropathology, including a professor of neurology, a neurologist and a pathologist. It is based on a modified Montine's approach⁴⁵ (Table 8) also including: (1) Heterogeneous non-AD TAUopathies which constitute the pathologic hallmark of the Fronto-Temporal Lobe Degeneration (FTLD) related to TAU deposits: Pick's disease, non-fluent Primary Progressive Aphasia (TAU-nfPPA), Progressive Supranuclear Palsy (PSP)^46,47 and Cortico-Basal Degeneration (CBD)⁴⁸. In addition, non-AD TAUopathies include conditions related to aging and with no definite clinical significance such as Primary Age-Related TAUopathy (PART)⁴⁹, Age-Related TAU Astro-Gliopathy (ARTAG)⁵⁰, argyrophilic grain disease⁴⁸. (2) Lewy Type Synucleinopathy (LTS) that is related to Parkinson's Disease (PD) and Lewy Bodies Dementia (LBD). To search for LTS, apply the following hierarchic steps: at first, olfactory bulb, brainstem, amygdala/temporal cortex; if the previous areas are positive, add limbic structures (hippocampal formation, entorhinal cortex, anterior cingulate), middle frontal gyrus, inferior parietal lobule and occipital cortex⁴⁵. If clinical features of cortical LBD are present (i.e., fluctuations and/or hallucinations), consider limbic structures and isocortex for the first step. (3) TDP-43 deposits, the pathologic hallmark of FTLD related to TDP-43 deposits: behavioral variant of Fronto-Temporal Dementia (bvFTD), Semantic Dementia (SD or svFTD), TDP-nfPPA and FTD-Motor Neuron Disease (FTD-MND). The IHC for TDP-43 is performed on the following sections: amygdala, hippocampus, entorhinal cortex and middle frontal gyrus; in cases with suspected clinical FTLD, consider to examine other sections⁵¹.

1. Evaluate nonspecific brain tissue alterations in selected sections using H&E, NISSL, LFB, and IHC for GFAP and NeuN. Consider neuronal rarefaction, ballooned neurons, spongiosis, gliosis, myelin loss, the presence or absence of inflammatory or tumoral infiltrates. Specify the prevalent location of these alterations.
2. To detect microscopic vascular lesions, examine H&E and LFB slides including at least two hemispheric macro-sections (the first passing through the mammillary bodies (Charcot cut) with frontal and temporal lobes, basal ganglia, anterior thalamus, amygdala, and the second passing through the occipital lobe), the "geniculate" section of the hippocampal formation, one cerebellar section, and all three principal sections of the brainstem (through substantia nigra, locus coeruleus and DMNV).
   1. Detect small vessel disease including arteriolosclerosis, lipohyalinosis, perivascular space dilatation, white matter loss, leptomeningeal and/or parenchymal and/or capillary cerebral amyloid angiopathy. Specify grading (0–3) and prevalent location^42,52. Consider the presence or absence of hemosiderin leakage, microbleeds and microinfarcts.
   2. Evaluate the contribution of vascular damage to cognitive impairment using hierarchic Deramecourt's scheme (vessel wall alterations–small vessel disease–macroscopic infarcts). For this evaluation consider a frontal and temporal lobe section, basal ganglia and hippocampus (score 0–20)³⁰. Also, estimate the probability that cerebral vascular disease contributed to the cognitive impairment using VCING score (low-intermediate-high), obtained by considering hemispheric macroscopic infarcts and small vessel disease of the occipital lobe including moderate to severe arteriolosclerosis and amyloid angiopathy⁵³.
3. Evaluate AD pathology using IHC (4G8) for amyloid spread. Define Thal's stages (1–5)⁵⁴, aggregate Amyloid scoring (0–3)⁴⁵, and morphology per site (diffuse, focal, cored).
   1. Evaluate AD Tau pathology using IHC (AT8) and describe the prevalent morphology per site (neurofibrillary tangles, neuropil threads, neuritic plaques). Define Braak's stage (I–VI +; positive sign indicates the presence of additional areas of hyperphosphorylated Tau pathology not following the Braak's hierarchy)⁵⁵ and aggregate scoring (0–3)⁴⁵.
   2. Evaluate neuritic plaques using Gallyas staining and CERAD score (0–3)⁵⁶. Then, define the probability of the neuropathological features corresponding to an AD clinical syndrome using the Amyloid-Braak-CERAD score (ABC score 0–3): none-low-intermediate-high⁴⁵.
4. Use AT8 IHC to notice the presence or absence of non-AD TAU pathology specifying prevalent location and peculiar morphologic picture. Consider neuronal inclusions such as flame shaped or globose tangles, spherical-globular inclusions (i.e., Pick's bodies)⁵⁷, neuropil threads, dystrophic neurites, argyrophilic grains; and glial inclusions such as tufted astrocytes, thorny astrocytes, astrocytic plaques, coiled bodies, globular inclusions^58,59.
5. Use α-syn IHC to detect LTS (Lewy bodies, pale bodies and Lewy neurites) on the areas in the note above. In case of positivity, apply McKeith's grading (0–4) to evaluate the severity of the lesions⁶⁰; then, use Beach's stages (I–IV) for topographical distribution (olfactory bulb, brainstem predominant, limbic predominant, neocortical)⁶¹. Compare Beach's and Braak's stages to define the relationship between LBD and AD pathology^60,61,62.
6. Consider the presence or absence of Glial Cytoplasmic Inclusions (GCI; non Lewy-type glial synucleinopathy) which are the pathological hallmark of Multiple System Atrophy (MSA) and specify their prevalent location⁶³.
7. Use TDP-43 IHC to detect TDP-43 deposits on the areas in the note above. Identify the prevalent morphology per site: Neuronal Cytoplasmic Inclusions (NCIs), Distrophic Neurites inclusions (DNs), Nuclear Inclusions (NIIs). Define the pattern: A (predominant NCIs and DNs: bvFTD and nfPPA); B (predominant NCIs: FTD-MND); C (predominant long DNs: svPPA and bvFTD)^51,64. Use Nelson's scheme to define the presence of TDP-43 in AD cases^65,66.
8. Evaluate the hippocampus starting from subiculum and Sommer sector (CA1). Use the Rauramaa's score (0–4): 1–2 for microinfarcts and macroinfarcts, respectively; 3–4 corresponding to moderate and severe neuronal loss, respectively, and hippocampal atrophy (hippocampal sclerosis: HS). Point out the presence or absence of pathological protein deposits (in decreasing order of frequency: pTAU, TDP-43, α-syn)⁶⁷.
9. Define the neuropathological diagnosis and enter all the pathological data in the database.
   NOTE: The rooms in which the biological material and sensible data of the donors are stored are always locked and only the authorized personnel are allowed to enter, in order to guarantee both security and privacy. The frozen biological material is stored in locked freezers at -80 °C, while formalin-fixed paraffin-embedded tissues are stored in locked closets at room temperature. Dual motor freezers are used and a back-up freezer is also present. Furthermore, to ensure the freezers are always working, they are provided with a 24/7 monitoring system that sets off an alarm. An emergency generator is also present, in case of power failure. The participants are anonymized with a numerical code to ensure their privacy; it is not possible for non-authorized personnel to go back to the identity of the donors and their sensible data. All information is collected in a password-protected database; likewise, a hard copy of these data is kept in locked archives.