Neurologic Symptoms

As a safety precaution against infection, our institute developed a COVID‐19‐optimised autopsy protocol in line with recently published recommendations.²² Two hours prior to autopsy, 4% phosphate‐buffered formalin was instilled into the mouth, nose, and pharynx. Autopsies were performed in a room with adequate airflow (more than six air changes per hour of total room volume) under conditions similar to those recommended for autopsies of those who have died from suspected Creutzfeld–Jakob disease (i.e. hazmat suits, boots, goggles, and FFP2/3 masks) with an in‐corpore technique analogous to that used in forensic institutions. Thoracic organs were eviscerated (see below), and the heart was separately dissected in the direction of blood flow. Parenchymal organs (liver, spleen, kidney, and pancreas) were dissected within the body. After mobilisation of the small and large intestines, their exterior surfaces were inspected; if there were any outstanding findings, tissue was excised for further macroscopic and histological examination. In all cases, tissue samples from the liver, heart and kidney were histologically examined. Other organs, as well as bone marrow and the brain, were analysed upon specific clinical indication (younger age; neurological symptoms). To avoid aerosolisation, the brain was removed by opening the skull with a handsaw; bone marrow from the iliac crest was also procured with a handsaw. A detailed description of this in‐corpore protocol is provided in Doc. S1.
The lungs, trachea and larynx were exenterated intact and perfused via the trachea with 4% refrigerated (4°C) phosphate‐buffered formalin. The trachea was then closed with a clamp, and the specimens immersed in formalin at room temperature for 72 h before dissection. The lungs were subsequently cut into 5–10‐mm parasagittal slices and examined macroscopically. Two sections of each lobe, as well as the trachea, were submitted for histological examination.

The study design and protocol have been approved by the ethics committees for human research at our institute (IRB number: R2019-227). This study followed a prospective and observational design. The study was performed in accordance with the approved guidelines of the Declaration of Helsinki. From November 2020 to February 2022, 133 healthy volunteers aged ≥ 20 years underwent MRI after providing written informed consent explaining the potential for detection of brain disease. Volunteers were recruited from medical staff and students, and their families by open recruitment. Inclusion criteria for this study were those who had no history of brain injury, brain tumor or cerebrovascular disease on previous brain MRI, or those who had never undergone brain MRI and no neurological symptoms including cognitive function. One volunteer aged 84 years old was excluded from this study because of a history of head surgery due to a head injury over 30 years ago. In addition, three volunteers were incidentally found small unruptured intracranial aneurysms with a maximum diameter of < 2 mm on this MRI. They were included in this study, because small unruptured aneurysms might not affect CSF motion.
Patients’ MRI data was used in an opt-out method, after their personal information was anonymized in a linkable manner. Among 44 patients suspected with NPH, 5 patients diagnosed with secondary NPH [29 (link)] that developed after subarachnoid hemorrhage [3 (link)], intracerebral hemorrhage [1 (link)], and severe meningitis [1 (link)], and 3 patients diagnosed with congenital/developmental etiology NPH [30 (link)] were excluded from this study. Finally, 36 patients diagnosed with iNPH who had radiological findings of disproportionately enlarged subarachnoid space hydrocephalus (DESH) [31 (link)], specifically ventricular dilatation, enlarged Sylvian fissure, and narrow sulci at the high convexity, and triad symptoms of gait disturbance, cognitive impairment, and/or urinary incontinence were included in this study, according to the Japanese guidelines for management of iNPH [32 (link)]. Of them, 18 patients (50%) underwent CSF removal in 30–35 ml via a lumbar tap and were evaluated for changes in their symptoms before, one day and two days after the CSF tap test. In addition, 21 patients (86%) underwent CSF shunt surgery and their symptoms improved by ≥ 1 point on the modified Rankin Scale and/or the Japanese iNPH grading scale [32 (link)].

A total of 33 plaques were obtained from 31 patients (21 Males/10 Females) undergoing endarterectomy in the Department of Vascular Surgery and Transplantation, Rambam Medical Centre, using standard surgical techniques (15 (link)). Surgery was performed if carotid stenosis was 50% or more in clinically symptomatic patients, in those who had either ipsilateral cerebrovascular event (CVA) or transient ischemic attack (TIA) or amaurosis fugax in the last 180 days, and in clinically asymptomatic patients if the stenosis was 70% or higher. Asymptomatic patients included those with carotid artery stenosis without a history of ischemic stroke, TIA or other neurologic symptoms referable to the carotid arteries as defined by NASCET-North American Symptomatic carotid Endarterectomy Trial (16 (link)). Demographic data, comorbidities, risk factors, medications, and blood chemistry were recorded. The protocol was approved by The Local Humans Rights Committee of RAMBAM Medical Center (RMB-0175-14), according to the declaration of Helsinki, and all patients signed an informed consent form.

We prospectively and consecutively recruited patients with an acute attack or a history of TM who visited a tertiary referral center (Asan Medical Center, Seoul, South Korea) between July 2018 and April 2020. TM attacks were determined when the following criteria were fulfilled^{1 (link)}: (1) presence of signs or symptoms of sensory, motor, or autonomic dysfunction attributable to the spinal cord; (2) documentation of T2 high signal intensity on spinal MRI. Patients who had experienced clinical attacks within the last two months were regarded as being in an acute attack phase, while all others were regarded as being in the remission phase. For acute attack phase, we only included patients whose last attack was exclusively TM.
A diagnosis of ITM was made based on the previously suggested criteria^{30 (link)}. All patients underwent a detailed diagnostic workup including brain MRI, spinal MRI, cell-based assaying for anti-aquaporin-4 antibody (AQP4-Ab) and anti-myelin oligodendrocyte glycoprotein antibody (MOG-Ab)^{13 (link)}, anti-nuclear antibody (ANA), anti-SS-A/SS-B antibody, anti-neutrophil cytoplasmic (ANCA), screening test for HIV and syphilis and routine laboratory test. Patients with evidence of active infections, active malignancy, a history of systemic autoimmune diseases or signs of other various conditions mimicking ITM, such as mechanical compression, spinal cord infarction or spinal arteriovenous fistula were not diagnosed as ITM.
Among the patients with confirmed etiologies, we included AQP4 + NMOSD and RRMS patients for comparison. The diagnoses of AQP4 + NMOSD and RRMS were based on the 2015 International Panel for NMO diagnostic criteria for NMOSD^{31 (link)} and the 2017 revised McDonald criteria^{32 (link)}, respectively. We did not include patients with other etiologies due to small sample sizes. Additionally, healthy controls (HCs), defined as those who complained of mild neurologic symptoms such as headache or dizziness but had normal brain MRI findings, were recruited for further comparison.
All participants were over 18 years of age. Patients were sampled for blood and evaluated for the EDSS score on the day of enrollment.