Comparison and optimization of sheep in vivo intervertebral disc injury model

Abstract

Background: The current standard of care for intervertebral disc (IVD) herniation, surgical discectomy, does not repair annulus fibrosus (AF) defects, which is partly due to the lack of effective methods to do so and is why new repair strategies are widely investigated and tested preclinically. There is a need to develop a standardized IVD injury model in large animals to enable comparison and interpretation across preclinical study results. The purpose of this study was to compare in vivo IVD injury models in sheep to determine which annulus fibrosus (AF) defect type combined with partial nucleus pulposus (NP) removal would better mimic degenerative human spinal pathologies.

Methods: Six skeletally mature sheep were randomly assigned to one of the two observation periods (1 and 3 months) and underwent creation of 3 different AF defect types (slit, cruciate, and box-cut AF defects) in conjunction with 0.1 g NP removal in three lumbar levels using a lateral retroperitoneal surgical approach. The spine was monitored by clinical CT scans pre- and postoperatively, at 2 weeks and euthanasia, and by magnetic resonance imaging (MRI) and histology after euthanasia to determine the severity of degeneration (disc height loss, Pfirrmann grading, semiquantitative histopathology grading).

Results: All AF defects led to significant degenerative changes detectable on CT and MR images, produced bulging of disc tissue without disc herniation and led to degenerative and inflammatory histopathological changes. However, AF defects were not equal in terms of disc height loss at 3 months postoperatively; the cruciate and box-cut AF defects showed significantly decreased disc height compared to their preoperative height, with the box-cut defect creating the greatest disc height loss, while the slit AF defect showed restoration of normal preoperative disc height.

Conclusions: The tested IVD injury models do not all generate comparable disc degeneration but can be considered suitable IVD injury models to investigate new treatments. Results of the current study clearly indicate that slit AF defect should be avoided if disc height is used as one of the main outcomes; additional confirmatory studies may be warranted to generalize this finding.

Keywords: annulus fibrosus defect; discectomy; intervertebral disc degeneration; intervertebral disc injury; preclinical model; sheep.

FIGURE 1

Sheep lumbar spine across multiple imaging modalities. Normal macroscopic intervertebral sheep disc anatomy in superior cross‐sectional view (left). Magnetic resonance imaging (MRI) in T1 sequence, coronal view, showing the intervertebral disc (IVD) between the 2 adjacent end plates (top right). Histological Safranin O‐stained image, dorsal view, showing the normal transition between the IVD tissues (bottom right)

FIGURE 2

Study design of in vivo sheep lumbar spine model. Schematics and intraoperative images depicting the annulus fibrosus (AF) defects created in conjunction with nucleus pulposus removal. Intraoperative images are oriented with the cranial side of the sheep to the left, caudal to the right, ventral on the bottom, and dorsal on the top. The lumbar spine was visualized through a lateral retroperitoneal surgical approach and the intervertebral discs) were exposed (A), and those receiving the discectomy injury were subjected to a 16 mm AF annulotomy (B), two 8 mm AF annulotomies in a cruciate pattern (C), or a 5 mm by 3 mm box‐cut annulectomy (D)

FIGURE 3

Pfirrmann grading system used to assess lumbar intervertebral disc (IVD) degeneration in a sheep annulus fibrosus (AF) defect and partial nucleus pulposus (NP) removal model. Grading using T2‐weighted midsagittal MR images from a sheep model of injured lumbar intervertebral discs using 3 different annulus fibrosus status (control; slit, cruciate, box‐cut AF defect) followed by a 0.1 g nucleus pulposus removal, performed according to the described grading used for human intervertebral discs from Pfirrmann et al. (A) Grade I: The structure of the disc is homogeneous, with a bright hyperintense white signal intensity and a normal disc height. (B) Grade II: The structure of the disc is inhomogeneous, with a hyperintense white signal. The distinction between nucleus and annulus is clear, and the disc height is normal, with or without horizontal gray bands. (C) Grade III: The structure of the disc is inhomogeneous, with an intermediate gray signal intensity. The distinction between nucleus and annulus is unclear, and the disc height is normal or slightly decreased. (D) Grade IV: The structure of the disc is inhomogeneous, with an hypointense dark gray signal intensity. The distinction between nucleus and annulus is lost, and the disc height is normal or moderately decreased

FIGURE 4

Computed tomography (CT) imaging shows IVD height loss in all 3 defect types. (A) CT image and results from a sheep model of injured lumbar intervertebral discs using 3 different annulus fibrosus (AF) status (control; slit, cruciate, box‐cut AF defect) followed by a 0.1 g nucleus pulposus (NP) removal. Representative coronal CT image of a sheep lumbar spine 3 months after AF defect and partial NP removal showing no to minimal degenerative changes of injured disc with mild periosteal proliferation (arrow). (B) Histogram demonstrating the variation in postoperative total disc height expressed in percentage of height variation (%) compared to respective preoperative disc height according to the AF status (intact control; slit, cruciate, box‐cut AF defect injury) at each observation period (postoperative, 2 weeks, 4 weeks, and 12 weeks postoperatively). Within the same observation period, asterisks indicate a significant difference in height variation of the injured AF (slit, cruciate, box‐cut AF defect) compared to control (p < 0.05). Additionally, double asterisks indicate a significant difference between the AF status at 12 weeks postoperatively. The analysis is based on 6 IVD per AF status (pooling the 3 different IVD levels) for all observation periods except the 12 weeks postoperatively, which was based on 3 IVD per AF status

FIGURE 5

Magnetic resonance (MR) imaging shows increased degeneration grade for all defect types. MR image and results from a sheep model of injured lumbar intervertebral discs using 3 different AF status (control; slit, cruciate, box‐cut AF defect) followed by a 0.1 g NP removal. (A) Representative T2‐weighted midsagittal MR image from the same sheep lumbar spine as from Figure 4 taken 3 months after annulus fibrosus (AF) defect and partial nucleus pulposus (NP) removal. (B) Scatter plot demonstrating the Pfirrmann grading according to the AF status (intact control; slit, cruciate, box‐cut AF defect injury) after 1‐month (red) and 3‐month (black) observation period. The line of each AF status represents the grand median. Asterisks indicate a significant difference between the AF status (p < 0.05). The analysis is based on 6 IVD per AF status (pooling the 3 different IVD levels and both observation periods)

FIGURE 6

Magnetic resonance (MR) imaging identified AF fissures in all defect types. Representative MR T2‐weighted transverse (left) and Short‐Tau Inversion Recovery turbo spin echo coronal (right) MR images changes observed from a sheep model of injured lumbar intervertebral discs (IVD) using 3 different annulus fibrosus (AF) status (control; slit, cruciate, box‐cut AF defect) followed by a 0.1 g nucleus pulposus (NP) removal taken 3 months after surgery. The images are illustrating annular fissure characterized by loss of the morphology of the AF characterized by separation between the annular fibers (arrow and arrowhead) in the injured IVD; scale bar: 1 cm

FIGURE 7

Histopathological grading shows increased IVD degeneration grades in all defect types. Histology results from a sheep model of injured lumbar intervertebral discs (IVD) using 3 different annulus fibrosus (AF) status (control; slit, cruciate, box‐cut AF defect) followed by a 0.1 g nucleus pulposus removal. (A) Control sample showing highly aligned AF lamellae compared to injured samples (intact control; slit, cruciate, box‐cut AF defect injury) showing reduction of staining (region of white asterisk) and discontinuous AF tissue (black arrowhead) (nondecalcified, resin‐embedded, Safranin O‐Fast Green‐stained material; scale bar: 2 mm; images taken in dorsal plane. Orientation: left is left ventrolateral and bottom is caudal). (B–H) Scatter plots demonstrating the semiquantified histological findings according to the AF status (intact control; slit, cruciate, box‐cut AF defect injury) after 1‐month (red) and 3‐month (black) observation period. The line of each AF status represents the grand median. Asterisks indicate a significant difference between the AF status (p < 0.05). The analysis is based on 6 IVD per AF status (pooling the 3 different IVD levels and both observation periods)

FIGURE 8

Microscopical evaluation showed intervertebral discs (IVD) degeneration changes in all defect types. Representative microscopical changes observed during histopathological grading from a sheep model of injured lumbar IVD using 3 different annulus fibrosus (AF) status (slit, cruciate, box‐cut AF defect) followed by a 0.1 g nucleus pulposus (NP) removal. (A‐B) Severe morphological changes in the AF and NP visualized by illumination with polarized light characterized by a disruption and de organization of the structured annular lamellae (delamination; arrowheads). Depletion of proteoglycan content of the AF and part of the NP regions demonstrated by markedly reduced Safranin O staining (asterisk) compared to a normal intensely stained region is also observed; scale bar: 1 mm. (C) Cleft formation visualized by illumination with polarized light characterized by complete discontinuation of fibrils leading to the formation of voids (open arrows); scale bar: 200 μm. (D) Changes in cell morphology characterized by the formation of differently sized clusters of rounded chondrocytic cells; scale bar: 100 μm. (E) Blood vessel ingrowth characterized by small capillaries in close vicinity of an IVD defect (not shown in the picture); scale bar: 50 μm. (F) Influx of inflammatory cells characterized by aggregation of round to oval lymphocytic cells in close vicinity of an IVD defect (not shown in the picture); scale bar: 50 μm. (nondecalcified, resin‐embedded, Safranin O‐Fast Green‐stained thick‐sections)