Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

Abstract

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at-80 °C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are coregistered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries. Video Link The video component of this article can be found at https://www.jove.com/video/59511/ Recruitment Donations are obtained through either ante-mortem consent obtained directly from patients (pre-consented) or from next of kin after death. Pre-consented patients are typically identified from the clinical population at the Mellen Center for Multiple Sclerosis Treatment and Research

Intrinsic and Extrinsic Mechanisms of Thalamic Pathology in Multiple Sclerosis

“…Cases with prolonged hypoxia or >72 hours of mechanical ventilation, recent central nervous system (CNS) infection, human immunodeficiency virus, or hepatitis B/C were excluded. The brain donation program is approved by the Cleveland Clinic Institutional Review Board and samples were obtained either after obtaining informed consent from patients before their death or by their next of kin following death (explained in detail in prior publications) . Time from death to start of postmortem in situ MRI was available for 70 of 95 cases and to the start of brain fixation for 95 of 95 cases (Table ).…”

“…Brain and spinal cord were removed as previously described . One cerebral hemisphere was fixed in 4% paraformaldehyde (PFA).…”

“…Postmortem in situ imaging was obtained on 1.5 (n = 31) and 3 Tesla (n = 65) MRI with the following sequences: T1‐weighted 3D Magnetization Prepared Rapid Acquisition of Gradient Echo (MPRAGE), T2‐weighted image, fluid‐attenuated inversion recovery (FLAIR), and paired proton density‐weighted images with and without magnetization transfer pulse . After MRI, subjects were transported to the autopsy suite for brain and spinal cord removal and processing.…”

“…Samples for the study were obtained from the Cleveland Clinic MS Brain Donation Program, as previously described . The program entails a postmortem in situ MRI within 6 hours of death followed by brain and spinal cord removal and fixation.…”

Identifying miRNAs in multiple sclerosis gray matter lesions that correlate with atrophy measures

“…Immunohistochemistry was performed on fresh-frozen and paraffin-embedded human brain sections. 12 Freshfrozen human brain sections were fixed in 4% PFA for 10 minutes at room temperature, followed by peroxidase enzyme deactivation. Human MS brain paraffin sections were de-paraffinized and rehydrated followed by antigen…”