A pilot investigation of chemonucleolysis-induced intervertebral disc degeneration in the ovine lumbar spine

Intervertebral disc (IVD) degeneration (IVDD) initiates in the nucleus pulposus (NP) and is marked by elevated levels of pro-inflammatory cytokines and matrix-degrading proteases, leading to structural and functional disruption. IVDD therapeutics are currently being investigated; however, such approaches require validation using large animal models that recapitulate clinical, biochemical, and biomechanical hallmarks of the human pathology. Others have previously utilized intradiscal administration of chondroitinase-ABC (C-ABC) to initiate IVDD in the NP of sheep lumbar IVDs. While these studies examined changes in IVD height, hydration, and tissue micro-architecture, changes in biochemical content and mechanical properties were not assessed. Thus, the objective herein was to comprehensively characterize this ovine model IVDD for salient features reported in human degenerate IVDs by evaluating biochemical, biomechanical, and histological changes. Briefly, C-ABC (1U) was administered via intradiscal injection into the L1/2, L2/3, and L3/4 IVDs, and degeneration was assessed at 6- and 10-weeks via longitudinal magnetic resonance (MR) imaging. After 6 weeks, degenerative samples showed significant reductions in IVD heights (p=0.048) and MR imaging index (p=0.048), which worsened at 10 weeks. Post-mortem degenerate and controls IVDs were evaluated for differences in interleukin-1β concentration, axial and torsional functional spinal unit kinematics, and histological microarchitecture. Degenerate IVDs demonstrated significantly elevated concentrations of interleukin-1β (p=0.002). Additionally, degenerative samples showed increased creep displacement (p=0.022) and compressive stiffness’s (p=0.007) concurrent with decreased long-term elastic (p=0.007) and viscous dampening coefficients (p=0.002). Histological analysis of degenerative IVDs showed changes in microarchitecture, including derangement of the nucleus pulposus and annulus fibrosus tissue as well as cartilaginous end-plate irregularities. This pilot study demonstrated that intradiscal injection of 1U C-ABC induces significant and progressive degeneration of sheep lumbar IVDs over the time course investigated. The changes observed in this pilot’s study small sample size resemble the hallmarks of moderate to severe IVD degeneration observed in humans. Further study is warranted on a larger sample size to further validate these findings.

The symptoms of IVDD are currently addressed by surgical discectomy to remove IVD 85 tissue, joint fusion, or total disc replacement. These strategies suffer from significant limitations 86 and may lead to degeneration of adjacent levels. To address these issues and restore IVD 87 microarchitecture and function, regenerative medicine approaches are being investigated, 88 including biologic administration and tissue engineering using novel biomaterial scaffolds and/or or researcher preference, may influence model selection in the absence of empirical differences. 100 The authors concluded that current limitations of data concerning large animal models 101 minimized the impact of currently published literature, warranting further comprehensive 102 characterization of in vivo models to determine their suitability for IVD research. Of those 103 reviewed, caprine (goat) and ovine (sheep) models were found to be the most common 104 quadrupeds used to study IVD pathology and repair.

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In goats, Hoogendoorn and colleagues extensively characterized the ability of intradiscal 106 injection of chondroitinase-ABC (C-ABC) to initiate and consistently produce progressive, indoor/outdoor pen with access to a three-sided shelter and evaluated daily by a veterinarian for 130 signs of pain, behavior changes, or gait abnormalities for the duration of the study.

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Surgical procedure and intradiscal administration of C-ABC. 132 Peri-operatively, a transdermal fentanyl patch (150 mcg/hr/sheep) was applied to each 133 animal for five days starting one day prior to surgery. Twenty-four hours prior to surgery, five PBS -Fisher Scientific, Hampton, NH) into the L 1/2 , L 2/3 , L 3/4 IVDs (n=9 IVDs) (Fig 1A). To 141 confirm accurate needle position prior to injection, needle visualization was achieved using 142 lateral and anterior-posterior fluoroscopy (Fig 1B). The L 4/5 (n=3) and L 5/6 (n=3) IVDs served as 143 vehicle and uninjured controls, respectively. Post-operatively, sheep were monitored until 144 ambulatory, and then returned to standard housing conditions. Following the progression of     Following mechanical testing, samples were excised from their pots and fixed for 7 days 226 in 10% neutral-buffered formalin, followed by decalcification in 12% formic acid. Samples were 227 cut into 3-mm sagittal slices and images were captured for macroscopic evaluation by 3 blinded  (Fig 2A). Conversely, uninjured IVDs and those 274 injected with vehicle demonstrated no such changes (Fig 2A). Of note, darkening in adjacent 275 vertebral bodies adjacent to degenerate sheep IVDs were also observed on MR images indicative 276 of endplate abnormalities (Fig 2A). Normalized MR imaging index of degenerate IVDs was 277 significantly lower at week 6 (65.63±4.01%; p=0.024) and week 10 (57.10%; p=0.023) values. 278 Moreover, these values were significantly lower compared to respective uninjured controls at 279 week 6 (p=0.049) and continued to progress at week 10 (Fig 2B). No significant changes in MR  39.7±34.1 [pg/mL]/mg, respectively) (Fig 3). IL-1β was found to be increased in both the NP-303 and AF-regions of degenerate IVDs compared to uninjured IVDs; however, this was significant 304 (p=0.019) only in the NP (Fig 3).  Kinematic loading (Fig 4A)  respectively) (Fig 4C) (Fig 4F). A similar trend was 330 observed for cyclic compressive stiffness of FSUs containing degenerate IVDs (Fig 4G)

Intradiscal C-ABC injection induced significant changes in IVD
338 morphology and microarchitecture. 339 Macroscopic evaluation of IVDs using the Thompson scale (Fig 5) showed substantial  Semi-quantitative histological scoring showed excellent agreement among observers 347 (ICC: 0.975) and significant differences for uninjured and degenerate scores. (Fig 6) 348 Qualitatively, degenerate IVDs revealed significant alterations and disruption of tissue 349 architecture compared to control IVDs (Figs 6A-F). Prominent intravertebral herniations were 350 observed in all evaluated degenerate IVDs (Figs 6D-F), and these consistently exhibited several showed normal, convex AF orientation (Fig 6). Furthermore, gross morphological changes in 17 356 degenerate IVDs seemed to have pronounced effects on AF structure. Specifically, loss of IVD 357 height seemed to compress AF lamellae while CEP displacement resulted in reduced convexity.

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Higher magnifications revealed changes in distinct regions of the IVD (Fig 7). The ability of the 359 CEP and NP to retain stain was severely diminished by C-ABC injection. Additionally, thinning