Evaluation of the effects of differences in silicone hardness on rat model of lumbar spinal stenosis

Abstract

Lumbar spinal stenosis (LSS), one of the most commonly reported spinal disorders, can cause loss of sensation and dyskinesia. In currently used animal models of LSS, the spinal cord is covered entirely with a silicone sheet, or block-shaped silicone is inserted directly into the spinal canal after laminectomy. However, the effects of differences between these implant materials have not been studied. We assessed the degree of damage and locomotor function of an LSS model in Sprague-Dawley rats using silicone blocks of varying hardness (70, 80, and 90 kPa) implanted at the L4 level. In sham rats, the spinal cord remained intact; in LSS rats, the spinal cord was increasingly compressed by the mechanical pressure of the silicone blocks as hardness increased. Inflammatory cells were not evident in sham rats, but numerous inflammatory cells were observed around the implanted silicone block in LSS rats. CD68+ cell quantification revealed increases in the inflammatory response in a hardness-dependent manner in LSS rats. Compared with those in sham rats, proinflammatory cytokine levels were significantly elevated in a hardness-dependent manner, and locomotor function was significantly decreased, in LSS rats. Overall, this study showed that hardness could be used as an index to control the severity of nerve injury induced by silicone implants.

Fig 1. Characteristics of silicone blocks used for the induction of LSS.

(A) Schematic of the experimental design for establishing a LSS model using silicone blocks with graded hardness. (B) Characterization of the silicone components. (C) SEM images of the surface morphology of silicone blocks with differing hardness. (D) Elastic modulus values from nanoindentation.

Fig 2. Inflammation assessment using the LSS model.

(A) Representative immunofluorescence images of CD68⁺ macrophages in the spinal cord. Yellow scale bars = 1 mm and white scale bars = 100 μm. (B, C) Quantitative analyses of the (B) intensity and (C) number of CD68⁺ macrophages in the sham and LSS groups (n = 4 per group). Data are expressed as the means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the sham group analyzed by a one-way ANOVA with Tukey’s post-hoc test.

Fig 3. Demyelination assessment using the LSS model.

(A) Representative LFB-stained images of the myelin sheath in the sham and LSS groups. Scale bars = 1 mm and 100 μm. (B, C) Quantitative analyses of (B) pixel intensity and (C) density in LFB-stained myelin sheaths (n = 4 per group). Data are expressed as the means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the sham group analyzed by a one-way ANOVA with Tukey’s post-hoc test.

Fig 4. Inflammation-related gene expression in the LSS model.

(A–F) Relative mRNA levels of pro- and anti-inflammatory enzymes and cytokines: (A) iNOS, (B) Cox2, (C) TNF-α, (D) IL-1β, (E) IL-6, and (F) IL-10 in spinal cords implanted with a silicone block (n = 6 per group). (G, H) Enzyme-linked immunoassay results for inflammatory cytokine production. (G) IL-6, (H) TNF-α in each group 1 week after implantation of the silicone block (n = 6 per group). Data are expressed as the means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the sham group analyzed by a one-way ANOVA with Tukey’s post-hoc test.

Fig 5. Functional assessment of the LSS model.

(A) Basso, Beattie, and Bresnahan (BBB) score, (B) ladder score, and (C, D) Von Frey test (n = 6 per group). Data are expressed as the means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the sham group analyzed by a one-way ANOVA with Tukey’s post-hoc test.