Lumbar Region

Postnatal day 30 (P30) TWI mice and their WT littermates (5 for each experimental group processed in 5 different experimental sessions, every TWI with its WT littermate) and one P15 TWI mouse versus its WT littermate were perfused with a fixative solution (4% paraformaldehyde and 0.1%–1%–2.5% glutaraldehyde in phosphate buffer, pH 7.4). Sciatic nerves, spinal cords and gastrocnemius muscles were dissected and post-fixed for 4 hours at room temperature in the same fixative solution.
Spinal cords were dissected in the lumbar region, isolating four 1-mm-thick sections in the lumbar enlargement region and the gastrocnemius muscles were cut in small portions, approximately 1 mm³ in volume. Sciatic nerves were processed without further sectioning.
The selected tissues were further treated for epoxy resin embedding as previously described⁴³ . Briefly, the samples were deeper fixed in 2–2.5% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.4). After rinsing, specimens were post-fixed with osmium tetroxide (1%)-potassium ferricyanide (1%) in cacodylate buffer, rinsed again, en bloc stained with 3% uranyl acetate in ethanol, dehydrated and embedded in epoxy resin, that was baked for 48 h at 60 °C. Thin sections were obtained with an ultramicrotome (UC7, Leica Microsystems, Vienna, Austria) and collected on G300Cu grids (EMS). Finally, sections were examined with a Zeiss LIBRA 120 plus transmission electron microscope equipped with an in-column omega filter.

For in situ hybridization analysis, cryostat sections were hybridized using digoxigenin-labeled probes [45 (link)] directed against mouse TrkA or TrkB, or rat TrkC (gift from L. F. Parada). Antibodies used in this study were as follows: rabbit anti-Er81 [14 (link)], rabbit anti-Pea3 [14 (link)], rabbit anti-PV [14 (link)], rabbit anti-eGFP (Molecular Probes, Eugene, Oregon, United States), rabbit anti-Calbindin, rabbit anti-Calretinin (Swant, Bellinzona, Switzerland), rabbit anti-CGRP (Chemicon, Temecula, California, United States), rabbit anti-vGlut1 (Synaptic Systems, Goettingen, Germany), rabbit anti-Brn3a (gift from E. Turner), rabbit anti-TrkA and -p75 (gift from L. F. Reichardt), rabbit anti-Runx3 (Kramer and Arber, unpublished reagent), rabbit anti-Rhodamine (Molecular Probes), mouse anti-neurofilament (American Type Culture Collection, Manassas, Virginia, United States), sheep anti-eGFP (Biogenesis, Poole, United Kingdom), goat anti-LacZ [14 (link)], goat anti-TrkC (gift from L. F. Reichardt), and guinea pig anti-Isl1 [14 (link)]. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) to detect apoptotic cells in E13.5 DRG on cryostat sections was performed as described by the manufacturer (Roche, Basel, Switzerland). Quantitative analysis of TUNEL⁺ DRG cells was performed essentially as described [27 (link)]. BrdU pulse-chase experiments and LacZ wholemount stainings were performed as previously described [46 (link)]. For anterograde tracing experiments to visualize projections of sensory neurons, rhodamine-conjugated dextran (Molecular Probes) was injected into single lumbar (L3) DRG at E13.5 or applied to whole lumbar dorsal roots (L3) at postnatal day (P) 5 using glass capillaries. After injection, animals were incubated for 2–3 h (E13.5) or overnight (P5). Cryostat sections were processed for immunohistochemistry as described [14 (link)] using fluorophore-conjugated secondary antibodies (1:1,000, Molecular Probes). Images were collected on an Olympus (Tokyo, Japan) confocal microscope. Images from in situ hybridization experiments were collected with an RT-SPOT camera (Diagnostic Instruments, Sterling Heights, Michigan, United States), and Corel (Eden Prairie, Minnesota, United States) Photo Paint 10.0 was used for digital processing of images.

Radiographic data consisted of full-length coronal and sagittal radiographs were obtained in free- standing posture with the upper limbs resting on a support, the shoulders at 30° forward flexion, and the elbows slightly flexed [19 (link)]. All of the radiographic parameters were measured with Surgimap Software (version: 2.3.2.1; Spine Software, New York, NY).
All of the radiographic parameters concerned in this current study were shown in the Fig. 1A-B, which included thoracic kyphosis (TK), lumbar lordosis (LL), sagittal vertical axis (SVA), sacral slope (SS), pelvic tilt (PT) and pelvic incidence (PI). All of those radiographic measurements were performed by a dedicated team independent from the operating surgeons.Fig. 1

A Sagittal radiologic parameters: Thoracic Kyphosis (TK) measured from the superior endplate of T4 to the inferior endplate of T12 by Cobb method; Lumbar Lordosis (LL) measured from the superior endplate of L1 to the inferior endplate of S1 by Cobb method. Sagittal vertical axis (SVA) defined as the horizontal offset from the posterosuperior corner of S1 to the plumb line going through the vertebral body of C7. B Pelvic parameters: Sacral slope (SS): the angle between the horizontal line and the sacarl endplate; Pelvic tilt (PT): the angle between the vertical and the line through the midpoint of the sacral endplate to the femoral heads axis; Pelvic Incidence (PI): the angle between the perpendicular to the sacral plate at its midpoint and the line connecting this point to the femoral heads axis

Kyphosis was recorded as positive value ( +), and lordosis as negative value (-). The spinopelvic index (SPI) was calculated by the equation: SPI = SS/PT.

Thoracolumbar spinal MRI examinations were performed at the Department of Radiology, Carlanderska Hospital using a 1.5 T scanner (Signa, GE Healthcare, Chicago, IL, USA). The MRI protocol included sagittal T1-and T2-weighted sequences (Th1-S1). In the thoracic spine, a field of view of 360 × 360mm² and slice thickness of 3 mm was used. In the lumbar spine a field of view of 320 × 320mm² and slice thickness of 3.5 mm was utilized.
The MRI images were classified by a senior radiologist (> 15 years of experience) according to a predetermined standardized protocol. Disc degeneration was classified according to the Pfirrmann classification [23 (link)]. In the thoracic spine, no distinction between Pfirrmann grade 1 and grade 2 was made since the resolution of the images was not considered adequate for reliable differentiation between these grades. Vertebral and endplate changes were classified according to the Modic classification [24 (link)] and a modified Endplate defect score, adapted to our MRI protocol. The Endplate defect score [25 (link)] was modified where Type I-III (representing no degeneration) were pooled (Table 1). Schmorl’s nodes were classified as present or not present and defined as a vertebral endplate irregularity associated with intraspongious disc herniation, irrespective of the size, at either the cranial or caudal endplate, or at both endplates relative to the lumbar disc level. Spondylolisthesis was assessed as either present or not [26 , 27 (link)]. Similarly, vertebral apophyseal injury, defined as any irregularity or signal changes in the apophyseal region, was categorized as either present or not.Table 1

Modified endplate score, based on the original endplate defect score [25 (link)]

Modified endplate defect score	Original endplate defect score
1	Type I—Normal endplate with no interruption
	Type II—Thinning of the endplate, no obvious break
	Type III—Focal endplate defect with established disc marrow contact but with maintained endplate contour
2	Type IV—Endplate defects < 25% of the endplate area
3	Type V—Endplate defects up to 50% of the endplate area
4	Type VI—Extensive damaged endplates up to total destruction

Intra-observer and inter-observer reliability measures were carried out on a set of 15 individuals (5 of the climbers and 10 back pain patients not included in the current study) by the senior radiologist and an additional radiologist (5 years of experience). The latter repeated the evaluation after one month, blinded to previous result.

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)].