Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

This protocol has been approved by the Cleveland Clinic Institutional Review Board and follows guidelines of the Cleveland Clinic human research ethics committee.

1. In Situ MRI

1. Take the donor body to the MRI suite and conduct a 2 h MRI imaging protocol at the MS Imaging Facility. Conduct MRI on a 3 T or 7 T imager.
   NOTE: Priority is given to 3T as the majority of legacy data has been performed on 3 T, but when not available, imaging is conducted with 7T. Designated core sequences are performed for all cases (Table 1) and additional sequences that depend upon current research interests are performed if time permits (constrained by achieving tissue fixation less than 12 h after death). Table 1 describes the core sequences.

2. Autopsy

NOTE: Following the in situ MRI, the body is transported to the morgue for brain and spinal cord extraction by a diener and tissue processing by lab members.

1. Perform the following steps prior to body arrival at the morgue. Two hours prior, prepare 3 L of 4% paraformaldehyde (PFA) and label containers and bags for tissue storage. Prepare 3 L of 8% PFA and dilute 1.5 L of 8% PFA to 4% PFA. Place the remaining 8% PFA in 4 °C for day 2.
2. Prior to traveling to the morgue, fill 2 traveling coolers to 50% capacity with dry ice (large blocks broken to fit and small pellets).
3. At the morgue, fill a stainless-steel container halfway with 2-methylbutane and dry ice and cover with a lid in preparation for snap freezing the tissue.
4. Weigh and photograph the brain once removed by the diener.
5. Place any attached dura in a container filled with PFA.
6. Separate the cerebellum and brainstem from the cerebrum and photograph the cerebrum.
7. Identify the optic nerves, chiasm, and tracts and separate using a probe and tweezers. Resect the structure with a scalpel.
   NOTE: The distal segment of one side of the optic nerve is marked using Higgins ink for identification.
8. Separate the cerebral hemispheres longitudinally and photograph each hemisphere individually.
9. Ink the primary motor cortex (PMC) for the left hemisphere, re-photograph it, and place it in a 3.3 L container for long-fixation. Document the start time for brain fixation.
10. The PMC for the right hemisphere may be inked or excised.
    1. If being excised, first remove the covering meninges.
    2. Re-photograph inked or excised PMC.
    3. If PMC is excised, cut into 6 equally-sized sections.
    4. Ink the rostral aspect of each section.
    5. Place odd-numbered sections into PFA-filled containers for short-fixation.
    6. Snap-freeze even-numbered sections and place in sealed freezer bags into cooler #1.
11. Cut the right hemisphere anterior to posterior into 1 cm-thick coronal sections.
    1. Document gross abnormalities (e.g., cutting artifact, hemorrhage, and lesions).
    2. Place odd-numbered sections into containers filled with PFA for short-fixation.
    3. Snap-freeze even-numbered sections and place in sealed freezer bags.
    4. Document end of brain fixation time.
12. Separate the brainstem from the cerebellum and place in a PFA-filled container for short-fixation.
    1. Separate cerebellar hemispheres longitudinally.
    2. Cut each hemisphere into 4 equally thick sagittal sections.
    3. Photograph medial and lateral views.
    4. Place left cerebellar hemispheric slices into a container filled with PFA for short-fixation.
    5. Snap-freeze right cerebellar hemispheric slices and place in sealed freezer bags into cooler #1.
13. Obtain the spinal cord with nerve roots from the diener.
    1. Remove spinal cord dura mater and store dura in a container with PFA.
    2. Separate left and right anterior and posterior nerve roots. Cut the left anterior and posterior nerve roots cut from the spinal cord and place in a PFA filled container for short-fixation.
    3. Cut the right anterior and posterior nerve roots from the spinal cord, snap-freeze, place in sealed freezer bags, and then place in cooler #2.
    4. Photograph the caudal-most 20 cm of the spinal cord. Document the location of the lumbar enlargement.
    5. Cut 2 cm transverse sections of the cord proceeding from caudal to rostral.
    6. Ink the rostral aspect of each cut section.
    7. Place odd-numbered sections into containers filled with PFA for short-fixation.
    8. Snap-freeze even-numbered sections, place into sealed freezer bags, and then place in cooler #2.
    9. Document the start time for spinal cord fixation and any gross abnormalities.
    10. Photograph the remaining rostral portion of the spinal cord.
    11. Document the position of the cervical enlargement. Follow steps 2.13.5–2.13.8 for the remaining spinal cord.
14. Following the morgue place frozen tissue in labeled boxes in -80 °C freezers. Store fixed tissue at 4 °C.
15. At 24 h post-autopsy (day 2) dilute the remaining 8% PFA to 4%.
16. Replace 4% PFA in fixation containers with freshly diluted 4% PFA.
17. At 60 h post-autopsy prepare solutions of 2.5% glutaraldehyde in 4% PFA from glutaraldehyde, PFA, dH₂O and Sorenson’s buffer (prepared by mixing in sequence: 0.2 M phosphate buffer pH 7.4, polyvinylpyrolidone 1% w/v, sucrose 30% w/v, and ethylene glycol 30% v/v).
18. Remove used 4% PFA from short-fixation containers.
19. Rinse tissue in Sorenson’s buffer and place it in cryoprotection solution (glycerol 20%, 0.4 M Sorenson’s buffer 20%, and 0.02% sodium azide in dH₂O).
20. Photograph short-fixed brain slices, cerebellum, brainstem and motor cortex (if applicable).
21. With a scalpel blade cut 2 mm thick transverse sections from each 2 cm short-fixed spinal cord section.
22. Place sections in 2 mL scintillation vials and fill with solution of 2.5% glutaraldehyde in 4% PFA.
23. Return the remaining section to the original 20 mL scintillation vial. Rinse section with Sorenson’s buffer and replace with cryoprotection solution.

3. Pathology

NOTE: Short-fixed slices of the right hemisphere as well as the long-fixed left hemisphere (placed in 4% PFA for several months) are either cut into 30 µm sections (referred to as free-floating) or embedded in paraffin and cut as 12–14 µm sections (referred to as paraffin-embedded). These sections are processed usually with proteolipid protein (PLP) for detecting demyelinating lesions and major histocompatibility complex II (MHC-II) for immune activity using the diaminobenzidine (DAB) method. These protocols have been standardized and used in several publications^{2,3,4,5,6,7,8,9,10,11,12}.

1. Free-Floating (30 µm) DAB-Avidin-Biotin Complex (ABC) Tissue Staining
   1. Remove sections from cryostorage solution, transfer sections to a six-well plate, and wash them 3x for 5 min each in 2 mL of 1x phosphate buffered saline pH 7.0 (PBS). When transferring to the next well in the six-well plate use care not to tear the tissue. During each wash and incubation step, place the six-well plate on a shaker and allow tissue to gently shake.
      NOTE: Tissue sections incubated in smaller volumes and in larger plates (i.e., 12- and 24-well plates) tend to exhibit surface and edge tearing.
   2. Perform antigen retrieval by microwaving sections in a glass beaker containing roughly 30 mL of 10 mM citrate buffer (pH 6.0). Ensure tissues are not folded by manipulating with a paintbrush and microwave sections for 2–3 min or until citrate buffer comes begins to boil. Allow sections to cool to room temperature (~20 min).
   3. Transfer sections back to a six-well plate and wash sections 3x for 5 min each in 2 mL of PBS/0.3% Triton X-100. Block endogenous peroxidases by incubating sections in 2 mL of 3% H₂O₂/0.3% Triton X-100/PBS for 30 min at room temperature (RT).
   4. Wash sections 3x for 5 min each in 2 mL of PBS/0.3% Triton X-100. Block sections in 2 mL of 3% normal goat serum/0.3% Triton X-100/1x PBS for 1 h at RT.
   5. Incubate sections overnight to 5 days (depending on the antibody) in primary antibodies directed against microglia and myelin epitopes to detect inflammation (MHCII) and demyelination (PLP) (see the Table of Materials) at 4 °C.
      NOTE: Ensure that sections are not folded when incubating in this step or subsequent steps as this will lead to areas within the sections being void of stain.
   6. Wash sections 3x for 5 min each in 2 mL of 1x PBS. Then incubate sections in secondary biotinylated antibodies (see the Table of Materials) for 1 h at RT. Prepare Avidin-Biotin Complex (ABC) solution during incubation roughly 45 min prior to the next wash step to allow ABC complexes to form.
   7. Wash sections 3x for 5 min each in 2 mL of 1x PBS. Then incubate sections in ABC for 1 h at RT.
   8. Wash sections in 2 mL of 1x PBS three times for 5 min each. Incubate sections in filtered DAB (2 mL/well/section) containing H₂O₂ (1:500 dilution of 30% H₂O₂ in DAB) until color develops adequately (~3–8 min).
   9. Wash sections 3x for 5 min each in 2 mL of 1x PBS. To enhance signal (optional), osmicate using 0.04% OsO₄ (~30 s).
   10. Wash sections three times for 5 min each in 1x PBS. Individually, transfer each section from the six-well plate to a small container full of 1x PBS and position the tissue section on a glass slide as flat as possible.
   11. Gently lift the slide out of the PBS while ensuring the tissue section is as flat as possible. Using two paint brushes, gently flatten and stretch the tissue out on the slide and dab away the excess water with paper towel. Mount tissue sections with glycerol (or an equivalent mounting media) and seal the coverslip with clear nail polish.

4. Paraffin-embedded Hemispheric Sections: DAB Staining

1. Melt paraffin off the slides in a 60 °C oven for 5–10 min.
2. De-paraffinize sections by incubating in xylene 3x for 5 min each.
3. Rehydrate tissue in graded ethanol at 100% (2x for 5 min each), 95% (2x for 5 min), 70% (2x for 5 min each), and 50% (1x for 5 min each). Cover slides in PBS.
4. Antigen retrieve by microwaving slides in a beaker of 10 mM citrate buffer (pH 6.0).
5. Wash slides in 1x PBS (3x for 5 min each) once cooled to room temperature. Block endogenous peroxidases by incubating tissue in 3% H₂O₂/1% Triton-X 100/PBS for 30 min.
6. Wash tissue in 1x PBS three times for 5 min each. Block tissue with 6% normal goat serum in PBS for 1 h.
7. Incubate sections in primary antibody (see the Table of Materials) in PBS at room temperature (RT) overnight (max 20 h).
8. Wash sections 3x for 5 min each in 1x PBS. Then incubate sections in corresponding secondary antibody (see the Table of Materials) in PBS for 1 h at RT.
9. Prepare ABC solution roughly 45 min prior to the next wash step during incubation.
10. Wash sections 3x for 5 min each in 1x PBS and then incubate in ABC for 1 h at RT.
11. Wash section 3x for 5 min each in 1x PBS.
12. Incubate sections in filtered (0.45 µm filter pore size) DAB containing H₂O₂ (1:500 dilution of 30% H₂O₂ in DAB) until color develops adequately (~3-8 min).
13. Wash sections (3x 5 min) in 1x PBS. To enhance signal, osmicate using 0.04% osmium tetroxide (OsO₄; approximately 30 s).
14. Wash sections (3x 5min) in 1x PBS.
15. Dehydrate tissue in a graded series of ethanol 50% (1x 5min), 70% (1x 5min), 95% (2x 5min), 100% (2x 5 min), and 100% xylene (1x 5min). Allow xylenes to evaporate.
16. Mount sections with toluene-based rapid dry mounting media. Formulations that contain anti-oxidants are recommended to prevent stain bleaching. Remove excess mounting media from slide edges with a razor for subsequent storage.

5. MRI/Pathology Correlations

NOTE: For correlating MRI with pathology, we first perform ex vivo MRI of the long-fixed intact brain hemisphere (step 2.9 above) in an adjustable box with MRI-visible markers indicating slicing slots. We then slice the brain and photograph the 1 cm slices to enable co-registration of in situ MRIs to the individual brain slices. We can then perform MRI-guided analyses, where regions of interest (ROIs) are identified on MRI to direct tissue analysis. We can also perform histopathology-guided analyses, where ROIs are identified on tissue (e.g., white matter lesions, white matter without demyelination, etc.) and then characterized by the co-localized MRI measures (Table 1).

1. Identification of MRI-based ROIs
   NOTE: In previous studies, we have identified ROIs in white matter^{11,12,13,14,15} and gray matter^16,17,18. The example below is for white-matter analysis.
   1. Segment T2 hyperintense lesions on in situ MRIs (from step 1.1), initially processed by an automatic algorithm, and then corrected manually by experienced users.
   2. Segment T1 hypointense lesions within T2 lesions as voxels with a signal intensity less than or equal to 80% of the signal intensity of the surrounding normal-appearing brain tissue.
   3. Segment hypointense areas in magnetization transfer ratio (MTR) maps with an 80% threshold.
   4. Create three classifications using the above segmentations: (a) T2-only lesions which are abnormal on T2-weighted/FLAIR scans but normal on T1-weighted or MTR scans, (b) T2T1MTR lesions, which are abnormal on all T2-weighted/FLAIR, T1-weighted, and MTR scans; and (c) normal-appearing white matter (NAWM), which are normal on all T2-weighted/FLAIR, T1-weighted, and MTR scans.
      NOTE: Our selection of slices is based on the existence of all three regions-of-interest types (T2-only, T2T1MTR, and NAWM) on the same slices.
   5. Calculate normalized intensities for each ROI to minimize variability arising from different brains and different brain locations.
2. Co-registration of in situ MRI to brain slices
   1. Scan the fixed brain hemisphere in a custom slicing box with four rows of MRI-sensitive markers localizing the slots where a knife can be inserted to slice the brain. Perform a T1-weighted 3D MPRAGE acquisition with 1 mm isotropic resolution covering the fixed brain and all of the markers.
      NOTE: This is called post-fixation MRI and is only used as an intermediate step for co-registration of the in situ MRIs to the brain slices.
   2. Immediately after scanning, slice the brain hemisphere in slots located 1 cm apart, resulting in approximately 15 slices.
   3. Photograph the brain slices on both the anterior and posterior sides.
   4. Co-register the in situ MRI and photographs of brain slices with the following steps.
      1. For segmentation of post-fixation and in situ MPRAGE, pre-process both post-fixation and in situ MPRAGE MRIs for intensity non-uniformity¹⁹.
         1. Segment the brain and four rows of markers from preprocessed post-fixation MPRAGE.
         2. Segment the hemisphere corresponding to the post-fixation MRI^20,21 from the pre-processed in situ MPRAGE.
      2. Co-register the brain-extracted in situ and post-fixation MRIs through a series of linear registration processes up to 12 degrees-of-freedom (affine registration) using FSL FLIRT²². The scaling and shearing components account for the effect of fixation shrinkage.
      3. Find the slicing plane’s normal direction by minimizing the sum of maximum projected intensities using the segmented markers. These re-orientation angles are incorporated in the transformation matrix obtained from the previous step.
      4. Visually match MRI images to photographs of fixed posterior brain slices using an in-house image viewer that allows for changing the depth and orientation of the normal vectors. Small modifications are needed because the brain slicing is imperfect.
      5. Apply AFFINE image transformation¹³ for each slice to transform the in situ MRIs to the same slicing locations as the photographs of the brain slices.