Dilatation

The candidates for this study were patients with suspected iNPH. After obtaining written informed consent, the eligible patients were pre-registered and received lumbar puncture. The inclusion criteria were (1) age between 60 and 85 years, (2) presence of one or more symptom(s) of the triad (gait disturbance, cognitive impairment, and urinary symptoms), which were measurable on the iNPH Grading Scale (iNPHGS) [14 (link)], (3) MRI features of iNPH, i.e., both ventriculomegaly of Evans' index > 0.3 and tight high-convexity and medial subarachnoid spaces on coronal T1-weighted MRI (Figure 1) [10 (link)], (4) absence of known disorders causing ventriculomegaly, and (5) normal cerebrospinal fluid (CSF) content (protein ≤ 50 mg/dl and cell count ≤ 3 μm³) and pressure (≤ 20 cmH₂O). Exclusion criteria were (1) presence of musculoskeletal, cardiopulmonary, renal, hepatic, or mental disorders that would make it difficult to evaluate changes of symptoms, (2) obstacles to one-year follow-up, and (3) hemorrhagic diathesis or anticoagulant medication. For the evaluation of the MRIs, Evans' index, size of the Sylvian fissures rated according to the protocol of Kitagaki et al. [10 (link)], presence or absence of focal dilatation of the cerebral sulci, and white-matter changes according to scale of Fazekas et al. [15 (link)], were assessed on each site and recorded.
The candidates were pre-registered before CSF examination via a web-based case report system. MRI was reviewed by each site in the pre-registration phase, and the final eligibility of the subjects was judged by the central MRI review committee, which consist of neurosurgeons, neurologists, and a neuroradiologist. The central MRI review committee excluded those whose MRI did not fulfil the inclusion criteria. After the confirmation of normal CSF content and pressure, the investigator was notified of registration via the web system. Tap test was carried out in all subjects with 30 ml CSF removal via lumbar puncture. CT cisternography was carried out 1 week after the tap test with iohexol (Omnipaque^®: 180 mg/ml) 30 mg/kg. Cerebral blood flow was measured using ¹²³I-Iodoamphetamine and single photon emission computed tomography at baseline. However, the results of these measures were not considered for the eligibility.

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)].

To assess terminal cell phenotypes, maximum intensity projection GFP images and the corresponding minimum intensity projection brightfield images of dorsal branch terminal cells were manually assessed and scored for dilation in the transition zone or other phenotypes in the terminal cells (refer to Fig. 1a, and Supplementary Tables 2 and 3 for results). Dilation rate was assessed as the percentage of terminal cell transition zones that had at least one dilation. If multiple dilations were observed in a terminal cell transition zone, it was not scored any differently to a terminal cell transition zone with a single dilation. Each data point graphed represents the mean dilation rate of the indicated genotypes.