A comprehensive tool box for large animal studies of intervertebral disc degeneration

Abstract

Preclinical studies involving large animal models aim to recapitulate the clinical situation as much as possible and bridge the gap from benchtop to bedside. To date, studies investigating intervertebral disc (IVD) degeneration and regeneration in large animal models have utilized a wide spectrum of methodologies for outcome evaluation. This paper aims to consolidate available knowledge, expertise, and experience in large animal preclinical models of IVD degeneration to create a comprehensive tool box of anatomical and functional outcomes. Herein, we present a Large Animal IVD Scoring Algorithm based on three scales: macroscopic (gross morphology, imaging, and biomechanics), microscopic (histological, biochemical, and biomolecular analyses), and clinical (neurologic state, mobility, and pain). The proposed algorithm encompasses a stepwise evaluation on all three scales, including spinal pain assessment, and relevant structural and functional components of IVD health and disease. This comprehensive tool box was designed for four commonly used preclinical large animal models (dog, pig, goat, and sheep) in order to facilitate standardization and applicability. Furthermore, it is intended to facilitate comparison across studies while discerning relevant differences between species within the context of outcomes with the goal to enhance veterinary clinical relevance as well. Current major challenges in pre-clinical large animal models for IVD regeneration are highlighted and insights into future directions that may improve the understanding of the underlying pathologies are discussed. As such, the IVD research community can deepen its exploration of the molecular, cellular, structural, and biomechanical changes that occur with IVD degeneration and regeneration, paving the path for clinically relevant therapeutic strategies.

Keywords: biomechanical testing; clinical assessment; disc disease; dog; goat; histopathology; intervertebral disc; low back pain; neck pain; pig; sheep; spine disorders; spine research.

FIGURE 1

Variety of experimental methodologies reported for outcome evaluation. A sample of recent peer reviewed manuscripts employing four common large animal models (ie, canine, caprine, ovine, porcine; n = 10 per species) to study IVD degeneration or therapeutic strategies in the past two decades. The number of six main outcome measures concomitantly used (macroscopic, histologic, radiologic, biochemical, biomechanical, pain), A, and the detailed use of each type of outcome, B, were registered and demonstrated the majority of the studies employed 3‐4 outcome measures; with limited concomitant biomechanical analysis and absent pain assessment. C, When IVD degeneration was induced (“Deg. Induction”; 92% of all studies), healthy and degenerate controls (“Deg. Control” may be either induced or naturally occurring disc degeneration) were regularly, but not consistently, reported. Spontaneous degeneration was only reported for canine species. Adherence to the ARRIVE guidelines was mentioned in one study. D, Available scoring systems in histological and radiological outcomes (“evaluation” refers to yes/no evaluation and “scoring” specifies within those studies whether or not a scoring scheme was employed), including quantitative MR imaging were seldom employed. The studies included are provided in the Supporting Information, Appendix S1

FIGURE 2

A, Comparative Anatomy of biped and quadruped spines. B, Flowchart of possible port‐mortem procedures for evaluation of all read out parameters from each functional spinal unit (FSU). A, Anatomic analogy between humans and quadrupeds. B, Once the spinal column is extracted, the spinal cord can be removed en bloc (a). Transverse processes can be removed by trimming the lateral aspects of the spine (b). To isolate FSUs, cuts are made transversely through the mid‐vertebrae (c). Dorsal aspects can be removed by cutting sagittally through the spinal canal (d) resulting in individual ventral column units (VCUs) (e). The isolated VCU can then be transversely transected into two identical parts (e). Thereafter, both parts can be used to take digital photographs for macroscopic evaluation and subsequently, one part may be fixed for histopathology (f). From the second part, the nucleus pulposus (NP) and annulus fibrosus (AF) tissue can be isolated from the cartilaginous end plates (CEPs) and vertebra with a surgical blade for biochemical analysis (g‐i). Note that prior to the described post‐mortem procedures (advanced) imaging and non‐destructive biomechanical analysis can be conducted as described in this manuscript

FIGURE 3

Gross Morphology. Exemplary gross morphological grading images for discs from goats (caprine), sheep (ovine), pigs (porcine) and dogs (canine). Human IVDs are included for comparison. Grades (1–5) have been assigned according to the criteria outlined by Thompson et al, where Grade 1 corresponds to healthy IVDs and Grade 5 the most severely degenerated. Noteworthy characteristics specific to each image series include: the translucent, notochordal NP region present in healthy dog and pig IVDs, compared to the more cartilaginous NP in healthy sheep, goat and human IVDs; and the presence of growth plates in sexual and skeletal maturity goat and sheep and in the immature pigs. Gross morphological grading includes assessments of the major substructures of the IVD (NP, AF, and CEPs) in addition to the adjacent vertebral body margins and as such, the gross changes may differ depending on the method of disc degeneration and the therapeutic approach tested. Examples in this figure are from induced disc degeneration models via chondroitinase ABC (goat) and partial nuclectomy (ovine) and from naturally occurring disc degeneration (canine). The worst grade of the different substructures is used to define the final score. Animal IVDs are oriented with the ventral side facing down. Human IVDs are oriented with the anterior side facing to the right and are from naturally occurring disc degeneration. n.a.: not available; Relative size differences need to be considered between the different species; grading is independent of the relative IVD size. Goat, dog, and pig images were kindly provided by Prof. Dr. Theo Smit, Dr. Niklas Bergknut, Prof. Dr. Hans‐Joachim Wilke respectively. Images of human IVDs were adapted from Wilke et al and Galbusera et al

FIGURE 4

The disc height index. It is suggested to quantify changes in IVD height on plain radiographs or high field MR images. According to Masuda et al

FIGURE 5

Healthy and degenerated IVDs (Pfirrmann graded) in large animal species and human. Typical examples of T2‐weighted MR images from human (3 T); goat, sheep and pig with experimentally induced IVD degeneration (3 T), and dog with naturally occurring IVD degeneration (1.5 T). Note that the intensity of the IVD signal is compared to the intensity of the cerebrospinal fluid (Table 6) Grade 1: The healthy IVD shows a homogenous structure with a hyperintense signal intensity and normal IVD height. Grade 2: The structure of the IVD is no longer homogeneous and the signal is still intense. Horizontal gray bands may be present in the IVD that is related to a beginning unclear distinction between NP and AF. Grade 3: Signal intensity is intermediate and the height of the IVD is slightly but visibly decreased with unclear distinction between NP and AF. Grade 4: Signal intensity is hypointense and there is no longer a distinction between NP and AF, the IVD height is moderately decreased. Grade 5: Inhomogeneous structure of the IVD with a hypointense signal intensity and a collapsed IVD space. Note that in naturally occurring IVD disease at these stages spondylosis occurs eventually, potentially fusing the segment with progression (eg, dog, Grade 5). NP: nucleus pulposus, AF: annulus fibrosus. Human and sheep MRIs were kindly provided by Frank Niemeyer and Marion Fussilier, respectively

FIGURE 6

The range of motion (ROM) of healthy lumbar IVDs. The ROM of individual healthy lumbar IVD levels of sheep and pig as well as the average ROM of dog (average from L4‐L5 to L6‐L7, unpublished preliminary data from Prof. Dr Hans‐Joachim Wilke) and goat (average from T13‐L1 to L5‐L6) compared to human specimens (unpublished ROM data, described in Kettler et al ²⁰³ ). Note that the sheep spine contains 6 or 7, the dog 7, the goat and the pig 6 lumbar vertebrae compared to 5 in human. Values are mean (SD) ROM in ° for each species; the moment and number of IVDs studied for the large animal models is given in the figure caption. Pig: 15 spines from 6‐month‐old cross of Pie'train boar with hybrid pig. Sheep: 14 spines from 3 to 4‐year‐old female merino sheep. Goat: 8 spine from 3‐ to 4‐year‐old female Dutch milk goats. Dog: 2 spines from ~2 years old non‐chondrodystrophic dogs. Human: 111 adult donors

FIGURE 7

Composite depicting progressive stages of induced IVD degeneration in goat (left images, H&E) and sheep (right images bright‐field microscopy AB/PSR); mid‐sagittal lumbar spine sections, dorsal aspects of discs oriented to the left). IVD degeneration was enzymatically induced by chondroitinase‐ABC^† disc injection in 3‐year‐old, skeletally mature goats 12‐14 weeks prior to harvest. Nuclectomy was performed in 2‐4 year‐old, skeletally mature sheep 4‐5 months prior to harvest. ^†Amsbio, Cambridge, MA. Grade 0 (normal, healthy controls) IVDs have a distinct nucleus pulposus (NP) that stains blue on H&E and deep turquoise on AB/PSR with a well‐defined NP‐annulus fibrosus (AF) interface. Hemi‐concentric well‐defined lamellae of the AF stain eosinophilic with H&E. The cartilage endplates (CEP) are thin uniform contiguous bands. The bony endplates (BEPs; stained pink on H&E and dark red on AB‐PSR) are comprised of uniform, regularly spaced arrays of bone trabeculae that are often flanked by persistent cartilage growth plates of the vertebral bodies (arrowheads). Section artifacts include clefts (short arrows) within the NP, AF or CEP interface that have sharp margins with abrupt transition to clear space devoid of degenerative matrix or cells (see Figure S2). Grade 1 (mild disc degeneration) IVDs show loss of basophilic/turquoise staining and reduced definition between the NP and AF that corresponds to reduced NP glycosaminoglycan (GAG) content and chondroid metaplasia of the AF, respectively. Inner to mid lamellae of the AF in this region (arrows) contain fine clefts spanned by proteoglycan‐rich fibrillated collagen corresponding to microtears (black frame, see Figure S2). CEPs retain their discrete, uniform contour but the trabecular bone of flanking BEPs has compacted (ie, endplate sclerosis). Grade 2 (moderate disc degeneration) changes show almost complete loss of NP basophilia (H&E) with poor definition of NP‐AF interface. Inner to mid lamellae of the AF in this region (arrows) show larger, more extensive clefts in the AF (black frames, see Figure S2) and progressive compaction of flanking trabecular bone. In the sheep, although proteoglycan staining of NP persists, there is loss of the inner to mid AF layers with NP protrusion into this region (arrows) that coincides to narrowing of the disc space and regional endplate thickening. Grade 3 (severe disc degeneration) changes include complete loss of NP basophilia (H&E) and more severely depleted NP GAG staining (AB‐PSR) with loss of NP architecture and poor discernment of NP‐AF interface. There is a collapse of IVD space and clefts within the distorted, degenerate NP (black frame, see Figure S2) and extrusion of degenerate chondroid IVD material beyond the CEPs that extends to the flanking cartilage growth plates of the vertebral bodies (arrows). The chondroid material (white arrow) stains dark blue on AB/PSR (see Figure S2). A triangular cleft at the dorsal aspect of the endplate (asterisk) is an artifact of sectioning. In the sheep, there is herniation of the ventral annulus with osteophytes that span cranial and caudal vertebral bodies (arrowheads). ^†Amsbio, Cambridge, MA

FIGURE 8

Low magnification composite of naturally occurring IVD degeneration. H&E and AB‐PSR stained bright‐field photomicrographs of mid‐sagittal sections of dog IVDs in various stages of naturally occurring degeneration. Mild shows degeneration and loss of glycosaminoglycan (GAG) staining within the nucleus pulposus (NP; asterisks) with increased GAG staining (chondroid metaplasia) of the annulus fibrosus (AF) and loss of definition. Moderate shows progressive NP and AF matrix degeneration with the production of small nodular exostoses (ie, syndesmophytes) at the dorsal margins of the AF (arrows); the ventral aspect of the H&E panel contains section artifact (arrowheads) and cannot be evaluated. Severe shows collapse of the IVD with partial dorsal and ventral extrusion of degenerate NP and AF matrix (arrowheads) and ventral bridging exostoses (arrows) compatible with intervertebral ankylosis (eg, self‐fusion). End‐stage IVDs show more complete extrusion of degenerate IVD matrix dorsally (arrowheads) and ventrally (asterisks) with complete collapse of IVD space and foci of bone‐to‐bone contact; ventrally, a large exostosis (arrows) surrounds the extruded IVD material (arrows). Reprinted with permission from Spine and further modified to serve the needs of demonstrating naturally occurring IVD degeneration changes. Note that these are representative images from a naturally occurring disc degeneration model and not from a large animal model where disc degeneration is induced either chemically or surgically. In the canine species the growth plates close and are as such absent in these sections indicating that they are from skeletally mature dogs

FIGURE 9

Nucleus pulposus notochordal cell depletion. H&E stained photomicrographs of progressive notochordal cell (NC) depletion within the nucleus pulposus (NP) in the dog IVD. Grade 0 shows that >90% of cells within the NP comprise a mixture of individualized and clusters of small and large vacuolated NCs (arrows). Grade 1 shows 30%‐40% NC depletion replaced by a population of nonvacuolated cells having large, eccentrically located, spindle‐shaped nuclei (arrow). Grade 2 shows degeneration and loss of nearly all vacuolated notochordal cells with remaining cells having centralized round hyperchromatic nuclei within lacunae (arrows). Grade 3 shows the complete loss of vacuolated NCs replaced by similar cells (arrows) as described for Grade 2

FIGURE 10

Nucleus pulposus cell clusters and matrix staining. H&E‐stained (left) and AB‐PSR (right, bright‐field microscopy) stained photomicrographs of grades corresponding to progressively increasing nucleus pulposus (NP) cell clusters (arrows) and loss of proteoglycan matrix staining in the goat and sheep IVD. Grade 0 shows individual NP cells (arrows) evenly dispersed in a homogeneously blue‐gray (H&E, sheep) and dark turquoise (AB‐PSR, goat) extracellular matrix. Grade 1 shows small, scattered NP cell clusters of 2 nuclei (arrows) embedded in a light gray‐pink extracellular matrix (H&E, sheep) and heterogenous light‐dark blue matrix staining (AB‐PSR, goat). Grade 2 shows more frequent NP cell clusters of >8 nuclei (arrows) and increased heterogeneity of matrix staining that is hypereosinophilic (H&E, sheep) to dark red (AB‐PSR, goat) between 25‐50% of the total NP area. Grade 3 show presence of huge (>15 nuclei) NP cell clusters (arrows) that contain pyknotic and viable nuclei (H&E, goat) and predominance of dark red matrix staining (AB‐PSR, goat)

FIGURE 11

Nucleus pulposus cell clusters with cell necrosis. H&E stained photomicrographs of cell clusters within the nucleus pulposus in the dog IVD. Grade 0 shows individualized vacuolated notochordal cells. Grade 1 shows conversion of vacuolated notochordal cells into nonvacuolated cells resembling chondrocytes within lacunae with occasional two‐cell clusters (arrows). Grade 2 shows presence of larger cell clusters (double arrows) having >8 nuclei per cluster in addition to scattered cell necrosis (arrowheads) characterized by nuclear fading (karyolysis), pyknosis, and fragmentation (karyolysis). Grade 3 shows the presence of huge cell clusters (double arrows) having >15 nuclei per cluster along with cell necrosis (arrowhead). Typical cell loss and necrosis is further illustrated in Figure 12. Of note, cell clusters are typically considered a hallmark of degeneration but have also been related to cellular proliferation. As such, within the context of therapeutic strategies studied, cell clustering may reflect an attempt for regeneration rather than a degenerative reactive response

FIGURE 12

Nucleus pulposus cell death characterized by karyolysis (empty lacunae) and pyknosis (shrunken nuclei). H&E‐stained photomicrographs in goats showing progressive cell loss (arrowheads) and necrosis (arrows) for Grade 0 (none), Grade 1 (<25% cells), Grade 2 (25%‐50% cells), and Grade 3 (>50% cells). Karyolysis: High (40×) magnification H&E stained photomicrographs feature karyolysis characterized by lacunae devoid of nuclei (white arrowhead) in contrast to the adjacent viable cell (white arrow) containing a distinct round nuclear membrane with single nucleolus. Pyknosis is characterized by cells having shrunken hyperchromatic nuclei (white arrowheads) in contrast to viable cells (white arrows) having open nuclei containing discrete nucleoli; karyorrhexis (ie, nuclear fragmentation) another morphologic manifestation of cell death is not shown

FIGURE 13

Annulus fibrosus (AF) morphology and tears and cleft formation in the AF/nucleus pulposus (NP). AB/PSR stained low magnification bright‐field photomicrographs of progressively severe AF tears in sagittally sectioned goat IVDs from the same model featured in Figure 7. Outer AF of the ventral aspect of the disc is oriented to the left and inner AF/NP to the right. Grade 0 AF fibrous lamellae are intact, uniformly aligned and stained; dark blue linear streaks (arrow) are staining artifacts. Grade 1 AF tears form irregular clefts within the inner annulus in areas of mild matrix degeneration (arrows). Grade 2 AF tears show clefts extending from the outer NP through inner (black arrow) to mid (white arrow) annulus. Grade 3 AF tears comprise large irregularly divergent clefts (asterisk) within a large region of degenerate matrix that extends from inner to outer lamellae. One cleft extends along the interface with the outer endplate (EP) and is partially filled with proteoglycan‐rich fibrillated matrix (white arrow). High (10×) magnification inset of H&E stained AF from the framed area show remnants of AF lamellae (arrowheads) separated by a fibrous stroma containing many vascular profiles (arrows); scale bar = 100 μm

FIGURE 14

Cellular (H&E) and matrix metaplasia (AB‐PSR, bright‐field microscopy) of the annulus fibrosus (AF)/distinction between annulus fibrosus and nucleus pulposus (NP). Alcian Blue‐Picrosirius Red (AB‐PSR)‐stained goat IVD low magnification photomicrographs taken at similar locations of the disc demonstrate progressive loss of AF lamellar organization and definition of AF‐NP interface. Corresponding high magnification H&E stained photomicrographs showing fibrous to chondroid cell metaplasia. Note that scores for AF lamellar architecture vs AF‐NP matrix definition/cell metaplasia may segregate independently. Grade 0 shows well‐defined red collagen lamellae with a sharp transition to turquoise glycosaminoglycan (GAG) staining at the AF‐NP interface; AF fibrocytes are uniformly spindle to stellate shaped. Grade 1 shows some loss of definition of collagen lamellae with less clear staining distinction at the AF‐NP interface; AF cells demonstrate more rounded profiles, but retain single nuclei within lacunae. Grade 2 shows distortion of AF collagen lamellae with turquoise GAG staining extending into outer lamellae; 25%‐50% AF cells contain 2 nuclei within lacunae. Grade 3 shows complete disorganization with collapse of the AF and disc space (arrowheads) demarcate remnant lamellae of outer annulus and arrows demarcate markedly distorted endplate margins). The mid to inner AF lamellae have been replaced by collagen‐rich fibrovascular tissue admixed with islands of turquoise GAG‐rich material containing cells that form multinucleate clusters within lacunae (H&E, arrows), compatible with chondroid cellular and matrix metaplasia

FIGURE 15

Cellular and matrix metaplasia of the annulus fibrosus (AF) with tears and cleft formation in naturally occurring IVD disease. Low and high magnification AB‐PSR‐stained bright‐field photomicrographs of AF degeneration and tearing within a spontaneous IVD disease model of skeletally mature non‐chondrodystrophic dogs. The top left panel (Grade 0/1) shows an intact AF without tears (Grade 0), although there are small scattered cleft‐like artifacts of processing (asterisks); there is mild matrix degeneration based on the increased AB staining of the mid‐to‐outer annulus layers (Grade 1 cellular and matrix metaplasia). Higher magnification of Grade 0 cellular and matrix metaplasia shows predominant fibrillar collagen matrix containing single spindle‐shaped cells (arrows). The top right panel (Grade 3/3) shows large tears extending through multiple layers of the AF (Grade 3 tears) surrounded by degenerate (ie, dark blue, GAG‐rich) matrix with strong AB staining in the outer AF layers (arrowheads) and occasional bridging of the clefts by frayed collagen fibers; the majority of cells have nucleus‐pulposus cell (NPC) morphology with nuclear clusters (Grade 3 cellular and matrix metaplasia). Higher magnification (Grade 3) demonstrates the loss of fibrillar collagen replaced by proteoglycan‐rich matrix with predominant NPC‐like cells within large round lacunae and frequent clusters with >4 nuclei (yellow arrows; Grade 3 cellular and matrix degeneration)

FIGURE 16

Sheep‐cartilage end‐plate (CEP) morphology (AB‐PSR). AB‐PSR stained bright‐field photomicrographs of progressive CEP disruption in the goat IVD. Grade 0 shows intact CEP of uniform contour and thickness. Grade 1 shows regional thinning or the CEP (arrow). Grade 2 shows multifocal disruption of the CEP <10% of the total area (arrows) with limited extrusion of IVD matrix into the bony endplate. Grade 3 shows a focal small disruption (arrowhead) adjacent to a large regional disruption of the CEP involving >30% of total area with extrusion of IVD matrix deep within the bony endplate (arrows). Note the Grade 3 image was obtained at half magnification of Grades 0‐2 in order to demonstrate the extent of the endplate disruption

FIGURE 17

Cartilaginous endplate (CEP) and bony endplate (BEP) morphology (Safranin O/Fast Green). Safranin‐O/Fast Green stained low magnification photomicrographs of progressive CEP disruption in the sheep model where a surgically induced annulus fibrosus (AF) defect was implanted with a composite tissue engineering repair device (asterisks) and harvested at 3 months postsurgery. Grade 0 shows the intact CEP of uniform contour and thickness. The vertebral growth plate (black arrowheads) flanks the bony endplate, which in goats and sheep tends to persist past the age of sexual and skeletal maturity. Grade 1 shows multifocal irregularities in the thickness and contour of the CEP (yellow arrowheads). Grade 2 shows focal disruption of the CEP <10% of the total area (black arrows). Grade 3 shows regional CEP disruption >30% of total area with extrusion of blue‐green IVD matrix deep within the bony endplate (black arrows)

FIGURE 18

Annulus fibrosus (AF) tears and cleft formation with endplate changes in the canine model with spontaneous IVD degeneration (AB‐PSR stain). High magnification AB‐PSR stained bright‐field photomicrographs highlighting spontaneous degenerative changes and tearing at the interface between the annulus fibrosus (AF) and bony endplate (BEP) in the dog IVD. Normal (healthy) shows a proteoglycan‐rich segment of the nucleus pulposus and inner AF without tears and an intact cartilage endplate (CEP), although there are small scattered cleft‐like artifacts of processing (asterisks). Moderate changes include linear clefts arising within a region of reduced proteoglycan matrix staining, compatible with matrix degeneration, that extend from the AF through the CEP (white arrow). Severe changes in the left lower panel shows longitudinal tears forming wide clefts (double‐headed arrows) within the outer fibrous portion of the AF centered within a focus of proteoglycan‐rich degenerative matrix with irregular thinning of the CEP. Severe changes in the lower right panel show numerous coalescing linear fissures within the AF that form free fragments (arrowheads) within a region of inconsistent proteoglycan matrix staining; fissures extend from the AF into the BEP (yellow arrows)

FIGURE 19

Bone modeling at external annulus fibrosus (AF)‐bone interface. Low magnification bright‐field photomicrographs of AB‐PSR stained hemisections of intervertebral IVDs from goat (induced IVD degeneration) and dog specimens (naturally occurring IVD degeneration). Grade 0 shows a smooth bony contour (white arrows) between the AF‐bone interface without peripheral nodular exostoses. Grade 1 shows a focal nodular exostosis (black arrows) at the periphery of the AF‐bone interface. Grade 2 shows regionally extensive nodular exostosis that protrudes from the bony endplate into the AF (white arrows). Grade 3 shows a large nodular exostosis surrounding the extruded IVD. Similar histopathological changes have also been extensively described in the annular lesion ovine model of IVD degeneration, including bony remodeling of the annular rim and are also depicted in Figure 7, right panel