Intervertebral disc degeneration induced by long-segment in-situ immobilization: a macro, micro, and nanoscale analysis

Abstract

Background: Cervical spine fixation or immobilization has become a routine treatment for spinal fracture, dislocation, subluxation injuries, or spondylosis. The effects of immobilization of intervertebral discs of the cervical spine is unclear. The goal of this study was to evaluate the effects of long-segment in-situ immobilization of intervertebral discs of the caudal vertebra, thereby simulating human cervical spine immobilization.

Methods: Thirty-five fully grown, male Sprague-Dawley rats were used. Rats were randomly assigned to one of five groups: Group A, which served as controls, and Groups B, C, D, and E, in which the caudal vertebrae were in-situ immobilized using a custom-made external device that fixed four caudal vertebrae (Co7-Co10). After 2 weeks, 4 weeks, 6 weeks, and 8 weeks of in-situ immobilization, the caudal vertebrae were harvested, and the disc height, the T2 signal intensity of the discs, disc morphology, the gene expression of discs, and the structure and the elastic modulus of discs was measured.

Results: The intervertebral disc height progressively decreased, starting at the 6th week. At week 6 and week 8, disc degeneration was classified as grade III, according to the modified Pfirrmann grading system criteria. Long-segment immobilization altered the gene expression of discs. The nucleus pulposus showed a typical cell cluster phenomenon over time. The annulus fibrosus inner layer began to appear disordered with fissure formation. The elastic modulus of collagen fibrils within the nucleus pulposus was significantly decreased in rats in group E compared to rats in group A (p < 0.05). On the contrary, the elastic modulus within the annulus was significantly increased in rats in group E compared to rats in group A (p < 0.05).

Conclusion: Long-segment in-situ immobilization caused target disc degeneration, and positively correlated with fixation time. The degeneration was not only associated with changes at the macroscale and microscale, but also indicated changes in collagen fibrils at the nanoscale. Long-segment immobilization of the spine (cervical spine) does not seem to be an innocuous strategy for the treatment of spine-related diseases and may be a predisposing factor in the development of the symptomatic spine.

Keywords: Biomechanics; Cervical spine; Fixation; Immobilization; Intervertebral disc degeneration; Rat model.

Fig. 1

Animal model. a. the caudal vertebrae were instrumented with K-wires only, which served as controls. b. the caudal vertebrae were immobilized using a custom-made external device to fix four caudal vertebrae (Co7-Co10). Four K-wires (50 mm in length and 1.2 mm in diameter) were fixed in parallel using two aluminum alloy cuboids (43 mm in length, 4 mm in width, net weight 5.0 g, the hole spacing is 12 mm), which do not compress or stretch the experimental discs

Fig. 2

A. The disc space and T2 signal intensity were measured using radiography and MRI Scans. Radiographs: (a~e) were obtained under anesthesia using a digital, self-contained cabinet x-ray machine (exposure time: 10 s, 26 kV). a (Group A) which served as controls, b~e (Group B~E) as shown in the figure, over time, progressive loss of disc height, d and e more obvious. MRI Scans: (f~j) (Scanning Sequence: FRFSE-XL, Slice thickness:1.4 mm, Interlayer Spacing: 5 mm) uses magnetic waves to create pictures to determine nucleus pulposus size and hydration status according to T2 signal intensity. Over time, the T2 signal intensity progressive decrease, as described above, i and j more serious. The IVD degeneration was classified as grade III. Imm indicates immobilization. B. The intervertebral disc height assessment based on radiographs. In control group (group A), imm-2 weeks (group B), and imm-4 weeks (group C), the intervertebral disc space height was slightly decreased, postoperatively, and this reduction was significant starting at the 6th week (group D). (*) indicates significant difference from other groups discs (p < 0.05)

Fig. 3

Histological assessments (Hematoxylin/Eosin stain). Histological section demonstrating early degenerative changes of in-situ immobilization intervertebral disc after 2~ 8 weeks. 1. (a~e), Intervertebral disc at low magnification (50×). The most prominent change in this specimen (b~e) is the cluster formation of the nucleus pulposus cells becomes obvious compared to control group A (a). Cells within these clusters kept their typical morphological structure with stellar-shape dnuclei and a vacuolated cytoplasm. 2. Higher magnification of IVD section shown in figure f~j (100×), the inner layer of AF appear to be progressive disorders and hyperplasia (j). Close to the NP border, the AF becomes infiltrated with chondrocyte-resembling cells. AF indicates annulus fibrosus; NP indicates nucleus pulposus; Imm indicates immobilization

Fig. 4

A. AFM was used to observe the microstructure of AF and NP collagen fibers. The representative AFM images of collagen fibrils in AF and NP of the control group and the experimental group after bearing immobilization for 2, 4, 6 and 8 weeks, respectively. The top row represents the AFM image of the NP (a-e), and the image in the lower row represents the AF AFM image (f-j). AFM indicates Atomic force microscopy; AF indicates annulus fibrosus; NP indicates nucleus pulposus. B. The average elastic modulus of collagen fibrils with different immobilization duration. a).The elastic modulus of NP were analyzed. b).The elastic modulus of AF were analyzed. (*) indicates significant difference from other groups discs (p ≤ 0.05), (+)indicates significant difference from other groups (p ≤ 0.001)

Fig. 5

A. The anabolic and catabolic gene expression of NP. mRNA levels of NP in the disc normalized to endogenous control (GAPDH) and internal controls. Relative transcript levels were calculated as χ = 2-ΔΔCt, in which ΔΔCt = ΔE - ΔC, ΔE = Ctexp - CtGAPDH, and ΔC = Ctct1 – CtGAPDH. A~C are shown for anabolic genes in the NP, catabolic genes as shown in the D~F. (⊗) indicates a significant difference from the internal controls (p ≤ 0.05) and (#) indicates that from other groups (one-way ANOVA with Fischer PLSD post hoc p ≤ 0.05), and (*)indicates a significant difference (p ≤ 0.05), and (+) indicates a significant difference (p ≤ 0.001). B. The anabolic and catabolic gene expression of AF. mRNA levels of AF in the disc normalized to endogenous control (GAPDH) and internal controls. Relative transcript levels were calculated as χ = 2-ΔΔCt, in which ΔΔCt = ΔE - ΔC, ΔE = Ctexp - CtGAPDH, and ΔC = Ctct1 – CtGAPDH. A~C are shown for anabolic genes in the AF, catabolic genes as shown in the D~F. (⊗) indicates a significant difference from the internal controls (p ≤ 0.05) and (*)indicates a significant difference (p ≤ 0.05), and (+) indicates a significant difference (p ≤ 0.001)