All methods below were carried out in accordance with ARRIVE guidelines and relevant regulations.
2.1 Surgical Procedure
Sheet insertion: asymptomatic spinal canal stenosis model
Thirty adult female Sprague-Dawley rats (8 weeks, 180-200 g; Japan SLC, Inc., Hamamatsu, Japan) were provided with sufficient food and water in individual cages. Rats were anesthetized with sevoflurane and fixed in the prone position. An incision was made in the midline behind the neck. The C4 to C7 laminae were exposed, and the C6 laminae were removed. A 0.3 mm-thick sheet of expandable water-absorbing polyurethane elastomer Aquaprene Dx (Sanyo Chemical Industries, Kyoto, Japan) was inserted under the C4-C5 laminae using a surgical microscope. This polymer expands in volume by 200% after insertion. (Kim et al., 2004) [5] A computed tomography (CT) scan was taken immediately to confirm that stenosis was created after surgery (Fig. 1).
Cervical SCI: contusion model
Eight weeks after the sheet insertion procedure, mild myelopathy or asymptomatic rats with a Basso, Beattie, and Bresnahan (BBB) score [6] of 19 or higher were identified. Animals with a score of 18 or less that developed myelopathy were excluded from the study. The rats were anesthetized with sevoflurane inhalation, and an incision was made in the posterior cervical vertebrae. The C4 vertebral arch was removed, confirming the sheet was just posterior to the spinal cord. A contusion injury (75 Kdyn) to the sheet at the C4-5 level was made using the Infinite Horizon Impactor (Precision Systems & Instrumentation LLC, Notting Hill.) from 1 mm above the spinal cord. This contusion force is recognized as a mild contusion force in the rat spinal cord injury model.
2.2 Group design
Thirty rats were divided randomly into three groups of 10 rats each:
(1) Immediate decompression after SCI (immediate decompression group)
(2) Decompression one week after SCI (sub-acute decompression group)
(3) No decompression after SCI (non-decompression group)
Decompression was defined as the resection of the remaining C5 lamina and the absorbing sheet posterior to the spinal cord. To equalize the surgical invasion in each group, the immediate decompression and non-decompression groups underwent surgery under inhalation anesthesia one week after the SCI.
2.3 Behavioral evaluations
Forelimb function was assessed using the Forelimb Locomotor Scale (FLS).[7] Hindlimb motor function was evaluated using BBB scores composed of 21 different criteria for movement of the hindlimb as previously reported. (Basso et al., 1995) The scale is based on the accurate observation of coordination, joint movements, and hindlimb stepping for two min. by two observers blinded to the experimental conditions. The assessments were performed in an open field at 0, 1, 3, 7, 14, 21, 28, 35, 42, 49, and 56 days after the sheet-insertion procedure, and 0, 1, 3, 7, 14, 21, and 28 days after cervical SCI.
Mechanical withdrawal thresholds were evaluated using the Von Frey hair employing a previously reported protocol. [8] The von Frey hair was applied in ascending order of force, and the median withdrawal threshold was calculated from the values following one descending and two ascending trials.
2.4 Histological analysis
Tissue preparation
Four weeks after SCI, the rats were sacrificed by transperitoneal injection with a triple mixture of 7.5μg /100μl of DomitorTM (medetomidine, Orion Corporation, Espoo, Finland), 40μg /100μl of DormicumTM (midazolam, Maruishi Pharma, Osaka, Japan), and 50μg /100μl of BetolpharTM (Butorphanol Tartrate, Meiji Seika, Tokyo, Japan). The cervical spinal cord was then removed and fixed overnight in PBS with 4% formaldehyde and stored in PBS with 20% sucrose at 4o C. Finally, the cervical spinal cord was encapsulated in an optimal cutting temperature compound (Sakura Fine Technical, Tokyo, Japan). Cryoprotected samples were frozen at -80° C.
Histological evaluation
A 2 cm long section of the cervical spinal cord centered on the C4/C5 spinal cord injury area was sectioned in horizontal (n=5) and sagittal (n=5) planes at a thickness of 16 μm. LFB (Luxol fast blue) staining was performed to measure the percentage of demyelinated cells in the myelin sheath of the pyramidal tract at the C7 level and to calculate the cross-sectional area of the cavity formation at the spinal cord injury. Transferase-mediated dUTP nick end labeling (TUNEL) staining was performed using an in situ Apoptosis Detection kit (Takara, Kusatsu, Japan). The number of TUNEL-positive cells in the bilateral anterior horns at the C4/5 level was counted. Tissue sections were co-incubated with growth-associated protein 43 (GAP-43, sc-10786, Santa Cruz Biotechnology, Inc., Dallas, US) (1:800) at 4o C overnight followed by co-incubation with fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG for one hr. at room temperature. We calculated the density of GAP43-positive cells in the immediate caudal part of the cavity formation normalized by area. All histological images were taken using an all-in-one microscope, BZ-X800, and analyzed using a BZ-X analyzer (Keyence, Osaka, Japan). The hybrid cell count function was used to calculate the number of positive cells and the percentage of demyelination to the cross-sectional area.
2.5 Statistical and data analysis
All computations were done using the statistical package JMP (version 13.2.0.; SAS Institute Inc., Cary, NC). Statistical differences in BBB score, FLS score, number of TUNEL-positive cells in the anterior horn, LFB- positive area, and the number of GAP43-positive cells were compared among the three groups using Tukey’s honest significant difference test. A p-value of < 0.05 was considered significant using two-sided tests of statistical inference.