Canine models of spine disorders

Abstract Neck and low back pain are common among the adult human population and impose large social and economic burdens on health care and quality of life. Spine‐related disorders are also significant health concerns for canine companions with etiopathogeneses, clinical presentations, and diagnostic and therapeutic options that are very similar to their human counterparts. Historically, induced and spontaneous pathology in laboratory rodents, dogs, sheep, goats, pigs, and nonhuman primates have been used for study of human spine disorders. While each of these can serve as useful preclinical models, they all have inherent limitations. Spontaneously occurring spine disorders in dogs provide highly translatable data that overcome many of the limitations of other models and have the added benefit of contributing to veterinary healthcare as well. For this scoping review, peer‐reviewed manuscripts were selected from PubMed and Google Scholar searches using keywords: “intervertebral disc,” “intervertebral disc degeneration,” “biomarkers,” “histopathology,” “canine,” and “mechanism.” Additional keywords such as “injury,” “induced model,” and “nucleus degeneration” were used to further narrow inclusion. The objectives of this review were to (a) outline similarities in key features of spine disorders between dogs and humans; (b) describe relevant canine models; and (c) highlight the applicability of these models for advancing translational research and clinical application for mechanisms of disease, diagnosis, prognosis, prevention, and treatment, with a focus on intervertebral disc degeneration. Best current evidence suggests that dogs share important anatomical, physiological, histological, and molecular components of spinal disorders in humans, such that induced and spontaneous canine models can be very effective for translational research. Taken together, the peer‐reviewed literature supports numerous advantages for use of canine models for study of disorders of the spine when the potential limitations and challenges are addressed.


| INTRODUCTION
Disorders of the spine comprise a major global healthcare concern in terms of pain, disability, and associated costs. While tremendous efforts and funding have been poured into spine research, and advances in basic and clinical science have been realized, translational animal models that effectively bridge the gap between bench and bedside appear to be underused. Induced and spontaneous canine models can be very effective in providing preclinical evidence to address this unmet need. Importantly, disorders of the spine affect canine patients with similar prevalence and impact to that seen in human patients, such that translational potential is high, and results can be applied to clinical veterinary medicine as well. Therefore, the objectives of the present review are to outline the applicable similarities in the key features of spine disorders between dogs and humans, describe relevant canine models, and highlight the applicability of these models for advancing understanding in mechanisms of disease, diagnosis, prognosis, prevention, and treatment of spine pathology.

| INTERVERTEBRAL DISC DISEASE IN HUMANS AND DOGS
In humans, symptomatic disorders of the spine are typically classified into one of four categories: axial back/neck pain syndromes, stenosis syndromes, instabilities, and deformities. While there are distinct features of each, there is also considerable overlap. Axial pain syndromes have had several distinct etiologies implicated to include paraspinal muscle dysfunction, 1 facet joint arthrosis, 2 inflammatory arthritides (including enthesitis), 3 and intervertebral disc degeneration (IVDD). 4 Stenosis syndromes in humans typically take the form of compressive myelopathies, postural dysfunction of the cauda equina ("neurogenic claudication"), or focal radiculopathies resulting from acute disc herniation or impingement from osteophytes. 5 More acute compressive pathologies are typically due to traumatic injury, infection, or tumor. These disorders can generate a very wide range of clinical symptoms, from episodic, mildly bothersome paresthesias and aches to very debilitating nervous system dysfunction as in paraplegia and quadriplegia.
Instabilities likewise can have a variety of etiologies, from acute traumatic fractures and/or dislocations to pathologic destruction due to tumor or infection 6 to gradual degenerative spondylolisthesis or cranial settling as seen in inflammatory arthritides. All instabilities result from loss of normal musculoskeletal restrains on spinal segment motion that then allow for segmental motions that could be injurious to the contained neurological structures.
Deformities represent deviations in the normal three-dimensional shape of the spine such as scoliosis or hyperkyphosis. In the most serious cases, deformities lead to an imbalance of the spine in which the patient's head is no longer centered over the sacrum, making ambulation much less energy efficient and causing significant disability.
Potential causes for deformities are developmental, degenerative, or following destruction due to trauma, tumor, or infection. 7 A similar spectrum of pathology has been reported for canine patients with similar categorization algorithms. Categorization may also be based on the anatomic structure(s) considered to be the primary source of pathology and/or pain generators, including disc, endplate, facet joint, and muscle-tendon (Table 1). 8 While disc-driven IVDD is far and away the most prevalent and most studied, endplatedriven, facet-driven, and muscle-driven disorders of the spine have been reported as well. Endplate-driven disorders in dogs include discospondylitis, fatty infiltration, dysplasia/remodeling, osteochondrosis, and Schmorl's nodes. Based on diagnostic imaging studies, 9,10 the lumbosacral (LS;L7-S1 in dogs) region has a predilection for endplate pathology. Fatty infiltration of endplates predominantly occurs in small breed dogs, especially chondrodystrophic (CD) breeds, and may be found anywhere along the spine. All other types of endplate lesions are more prevalent in medium and large breed dogs with discospondylitis being most common followed by sclerotic/reactive/ degenerative changes, osteochondrosis, and Schmorl's nodes. 9 Endplate dysplasia, sclerosis, remodeling, and/or degeneration are associated with vertebral instability in the canine LS region (LS instability) and caudal cervical region (caudal cervical spondylomyelopathy [CCSM] or Wobblers syndrome), both of which typically include some degree of IVDD. [11][12][13] CCSM is most common in Great Danes and oriented facet joints at L7-S1, which are associated with increased LS flexion and extension, 33 and both LS instability and CCSM can also be considered in the facet-driven category based on concurrent dysplasia, remodeling, and degeneration of affected facet joints. [11][12][13][14][15][16] Other facet-driven disorders in dogs include hypoplasia/aplasia 34 and osteoarthritis. 35 In terms of muscle-driven disorders of the canine spine, muscular dystrophy in Golden Retrievers 36 spinal muscular atrophy in Brittany Spaniels 37 have been reported. Spondylosis deformans, diffuse idiopathic skeletal hyperostosis (DISH), and scoliosis may also involve muscle-driven mechanisms.
It is likely that there is a large degree of crossover with respect to the anatomic "drivers" of spine disorders in both canine and human patients. As such, it is important to consider the whole organ or functional spinal unit (FSU) and whole body when treating patients and modeling disease. Since the majority of clinical disease and related research have centered on the intervertebral disc (IVD) and endplatedriven, facet-driven, and muscle-driven disorders typical involved or affect the disc, the present review focuses on IVD disease and degeneration.
IVDD is often characterized by loss of water from the nucleus pulposus (NP) with associated alterations in disc composition and structure, reducing its ability to function as a hydraulic cushion in vertebral column loading bearing and motion. [38][39][40] As degeneration progresses in dogs and humans, NP cells form large clusters and shift from collagen II to collagen I synthesis further compromising the critical biomechanical balance that determines its material properties and function. 41,42 Annulus fibrosus (AF) cells and matrix also undergo degenerative changes in unstable or weak regions of the disc. There is evidence that inflammatory and degradative processes drive IVDD in both dogs and humans. [42][43][44][45] As IVDD progresses, significant changes in articular facets, vertebral endplates and bodies, ligaments, and musculature ensue. 46,47 As with any animal model, there are associated limitations that should be considered when using and translating data from canine studies toward understanding human disease. Anatomically, the canine has seven lumbar IVDs while the human only has five.

| EX VIVO MODELS
Models using cells, single tissues, or whole organs provide a controlled method for investigating mechanisms of disc degeneration. 48 Cell culture models allow for control of certain variables and are typically less complex and expensive to employ than other options. 49 However, extracellular matrix (ECM) is altered or absent, which commonly results in cell dedifferentiation and does not allow for valid assessment of biomechanics or morphological integrity. 50 Tissue cultures of IVDs without adjacent endplates allow for better maintenance of cell distribution and differentiation, ECM integrity, and material properties, but biologic and biomechanical influences of endplate cartilage and vertebral bone are lost, and the NP is allowed to freely swell in culture. [51][52][53] Based on these limitations, whole organ IVD explant models have been developed in several species and used to study biologic and biomechanical components of the FSU in health and disease. Canine ex vivo models have been used effectively to address questions regarding nutrient and oxygen supply, osmolarity, cell phenotype, gene expression, cell signaling pathways, and biomarkers for diagnosis, staging, and therapeutic targets. 29,[54][55][56][57] Used in these ways, these models can serve as excellent screening tools for focused, efficient, and ethical use of animal models for translational studies toward clinical application.

| CANINE MODELS
Numerous animal models have been developed to investigate specific questions about IVDD across the spectrum of disease mechanisms, diagnosis, staging, prevention, treatment, and prognostication. Animal models range from rodents to primates, induced to spontaneous, and acute to chronic with spontaneous disc degeneration in nonhuman primates, age-related disc degeneration in mice, and geneticallyengineered spontaneous disc degeneration in mice having attractive modeling characteristics. When considering all of the factors involved in selecting an animal model including availability, ethics, cost, and translational applicability, canine models can also be considered strong candidates. [58][59][60] Spontaneous and induced canine IVDD models have been used to investigate a wide spectrum of biologic, biomechanical, and clinical components of spine disorders in their human counterparts (Table 3). C5-T1 L6-S1 C2-C3 T12-L1

| Induced models
Induced models provide a method for creating standardized pathology to consistently initiate desired disease processes while mitigating confounding variables and associated variability. The primary types of induced models in dogs include surgical or chemical focal annular injury, removal of disc material, or a combination of these insults.
Annular injury is the most common induced IVDD model across species, having been used for nearly a century to consistently initiate degeneration of IVDs in dogs. 61,62 Annular injuries are induced by incision, puncture, or direct disruption of the AF and/or its attachment to the endplate. These models are intended to mimic IVDD resulting from annular tears in humans by introducing a small AF injury that leads to the known sequelae that result in symptomatic disc disease.
These sequelae include structural compromise of the annulus, loss of resistance to hydrostatic forces within the disc, abnormal loading, apoptosis, necrosis, and cell phenotype shifts, NP protrusion/extrusion, extradiscal exposure of NP causing impingement and/or inflammatory responses, loss and remodeling of ECM, and ultimately, IVD failure. 63 As such, annular injury models can allow for assessments of In an attempt to address these limitations, endplate models, 64,65 biomechanical injury models, 66 and discectomy models 45 In order to induce more expedient and severe inflammatory and degradative changes, surgically and chemically induced partial discectomy models have been employed in dogs. Surgical excision of a portion of the disc (subtotal discectomy) is the most common means of creating this model and has been used to study mechanisms and timing for disease processes, correlations among diagnostic and staging modalities, and safety and efficacy of potential therapies. 58,67,68 Recently, percutaneous laser discectomies have become more widely performed to create discectomy models in order to avoid confounding variables associated with more invasive surgical models and be more directly translational in nature. 59 To determine the effect of physiotherapy on surgery outcome and material properties. 69 Agents commonly used for IVD chemonucleolysis include chondroitinase or papain. 70 To initiate disc degeneration, chemonucleolysis is dose-dependent such that chemically induced models frequently require high concentrations of these agents to effectively result in relevant degenerative changes, which may limit translational potential. 71 The benefits of chemically induced models include their capabilities for targeted damage without associated fibrosis and other confounding variables associated with surgically induced models. 72 These models may also have therapeutic relevance in that chemonucleolysis has been successfully used as a treatment option in select human and veterinary patients. 69,70,[72][73][74] Surgically and chemically induced models have also been used in CD breeds of dogs in order to include the spontaneous-disease components to methods for initiating and perpetuating insults. 45 increased intrinsic and extrinsic tissue inflammation, 92 and increased degradative enzyme production and activity. 43,93 However, the precise roles, interactions, links, and correlations among these mechanistic components of disease and their contributions to the various forms of symptomatic IVDD have not been fully characterized.
The IVDs of CD dogs undergo many of the changes that occur in human IVDs at an early age. 86 Calcification of cartilage endplates and NP can occur as early as 5 months of age in CD dogs, and is observed in 31.2% of cervical and 43% of lumbar discs by 1 year of age. 86 Relative within-animal differences in degree and timing of calcification and associated pathology can be used to characterize drivers of IVD calcification and related clinical disease while reducing the number of animals needed for valid study.
In the NP, the notochordal cell population is lost in humans and CD dogs and replaced with a chondrocyte-like cell population. This transition in cell population is associated with a shift in biochemical composition of the NP from a gel-like tissue with a high proteoglycanto-collagen ratio to a more fibrous and/or chondroid tissue with reduced proteoglycan and water content. Calcification of the cartilage endplates and a resultant reduction in nutrient delivery to and waste removal from the NP is believed to be a primary contributor to these degenerative changes in the NP. In NCD dogs, these changes in the NP occur less consistently and later in life compared to CD dogs.
Therefore, comparative studies that use CD and NCD dogs can be designed to elucidate factors driving the age-and disease-related changes that occur in the NP of dogs and humans.
Another key mechanism in IVD degeneration is cell death due to apoptosis and autophagy. 90  While obesity is accepted as a significant risk factor for symptomatic disc disease in humans, [106][107][108][109][110][111][112] this association is less clear in canine patients. In general, obesity is considered a relative risk factor for IVDD in dogs, 79,113 however, in CD breeds, specifically Dachshunds, body condition score has not been reported to have a strong correlation with prevalence of IVDD. 114,115 This may be a true lack of higher risk or it may be that other risk factors for IVDD-such as disc calcification and spine biomechanics-predominate in CD dogs.
To the authors' knowledge, there are no data reporting the effects of cigarette smoking (second-hand smoke) on IVDD in dogs.
However, other animal models report that exposure to components of tobacco is associated with decreased nutrient transport, altered cell morphology and function, increased oxidative stress and cell death, decreased ECM content and synthesis, and structural changes in IVDs. [116][117][118][119][120][121][122][123][124][125][126][127] Tobacco use is clearly implicated in symptomatic disc disease in human patients. 110,111,128 As such, research aimed at the effects of second-hand smoke on canine companions could provide important insight into mechanisms for IVDD associated with tobacco use, as well as disc degeneration pathways, in general.
Diabetes mellitus (DM) is a chronic metabolic disorder that has been indicated as a risk factor for accelerating IVD degeneration in human patients. [128][129][130][131][132][133][134][135][136][137] DM is thought to accelerate IVD degeneration by increasing advanced glycation end-product (AGE) accumulation in discs. [138][139][140][141][142] Studies examining the degenerative effects of AGEs on IVDs have been performed in murine models primarily. Dogs are affected by DM and require monitoring and insulin therapies such that diabetic dogs could serve as a valid large animal model for study of DM-associated disc degeneration.

| DIAGNOSIS OF IVDD IN DOGS
Symptoms associated with IVDD in dogs closely mimic those seen in human patients. Evidence of pain and a "hunched" or "roaching" appearance are common complaints for owners of dogs with IVDD.
Other early signs include difficulty rising, getting into car, or going up stairs and/or weakness during recreational, performance, or workrelated activities or even those of daily living. These signs may be episodic, may resolve, and/or may progress to ataxia or even paralysis.
For CD dogs with acute disc herniation, ataxia, or paralysis are often the first symptoms noticed by owners. After neurologic assessment and localization, diagnostic imaging is indicated to provide further detail regarding location, extent, and severity of the lesion(s) and to determine treatment options and prognosis.
For dogs with spine disorders, radiographic assessment is a mainstay of diagnostic imaging in order to provide a comprehensive assessment of the patient, and radiographs alone may be sufficient for diagnosis of some disorders. When plain radiographic studies are insufficient for definitive diagnosis, advanced imaging should be performed. Magnetic resonance imaging (MRI) is considered to be the preferred diagnostic imaging modality for IVDD in dogs, when available.

T machines are most common in veterinary medicine with 3 T magnets becoming more commonly available at academic and large specialty centers. 3 T magnets offer improved neurologic imaging with
an increase signal-to-noise ratio and thus improved spatial resolution.
MRI spine protocols may vary depending on the manufacturer, system, and preference of the radiologist or neurologist, but a standard hyperintense CSF and fat signal around the cord and diffuse to patchy hyperintensity within the cord on T2W images. 146,[156][157][158] To the authors' knowledge, the only diagnostic imaging grading system used for canine IVD disease to date is the Pfirrmann system based on MRI. This system uses a grading scale from 1 to 5. Grade 1 is the normal, homogenous, hyperintense disc on T2 spin-echo weighted sequences, while grade 5 is an inhomogenous, hypointense disc signal with no distinction between the nucleus and annulus and collapse of the disc space. 30,159 There was high correlation between the Thompson system of disc degeneration and the Pfirrmann scoring system using low-field MRI in both small and large breed dogs, although there was a group of dogs that were scored higher when the presence of spondylosis was seen. Spondylosis can be seen in dogs with mild disc degeneration and even normal discs on MRI. There are several other factors that may influence the correlation of these two systems. There is variation in the size, shape, and age of the dogs; the resolution in small breed dogs is lower than in large breed dogs; and the coil effect of the MRI has brighter signal of the discs within the focus area of the MRI and decreasing signal of the disc outside of this area. This may falsely affect the grade of the disc at the periphery of the MRI focus. 30 Lumbosacral disease, or cauda equina syndrome, in dogs is typically due to stenosis and/or instability and may be associated with genetic, degenerative (IVDD), overuse, traumatic, infectious/inflammatory, or neoplastic conditions. Radiographic features of LS disease may include subluxation ("stairstepping") of L7-S1 vertebrae, laminar and/or pedicular impingement of canal and/or foramen, disc herniation, osseous dysplasia, proliferation, and remodeling of vertebral bodies and/or articular facets, and/or sclerosis of the endplates. 146,160,161 Each of these findings will be apparent on CT, which can also provide imaging data for loss of epidural fat, increased soft tissue attenuating material within the canal and foramen, thecal sac displacement, narrowed intervertebral foramen, narrowed canal, and articular facet remodeling, subluxation, and osteophytosis. Sagittal or three-dimensional reconstruction images can be particularly useful for revealing subtler "stairstep" lesions and facet remodeling associated with LS instability (Figure 3). CT following intravenous contrast administration or CT myelography may be beneficial for ruling out trauma, infection, or neoplasia in these cases. 10,146,147,160,162 MRI may also be additive for some cases of LS disease in dogs by providing imaging data regarding disc degeneration, disc protrusion, facet capsule and cartilage pathology, endplate, subchondral bone, and marrow lesions, and paraspinal and pelvic musculature alterations. 146,150,163,164 Other features that are common include spondylosis deformans, transitional lumbosacral vertebra (most commonly lumbarization of S1), swelling of the spinal nerve roots, and possible sacral osteochondrosis. 150 The performance of "dynamic" imaging can evaluate the lumbosacral area on neutral, flexion and extension, using any of the modalities (ie, radiographic, CT, and/or MRI), to assist in the diagnosis of lumbosacral instability. 147,150,165 In CCSM, diagnostic imaging features vary between Doberman There is "stairstepping" (arrow heads) of the vertebral canal at the L7 to S1 junction with laminar impingement (arrow) causing deviation of the thecal sac within the canal. There is narrowing of the disc space with disc protrusion, wedging of the disc space (W), and incomplete osseous spondylosis deformans ventrally (*) muscle spasm without significant neurologic deficits are typically treated with oral nonsteroidal anti-inflammatory drugs or corticosteroids (eg, prednisone), analgesics (eg, tramadol), muscle relaxants, and/ or gabapentin along with activity modification and physical therapy.
Corticosteroid, opioid, and/or local anesthetic epidural, sacroiliac, and facet joint injections have also been performed with success. Acupuncture and chiropractic treatments have been advocated by some, but evidence for safety and efficacy is currently lacking in veterinary medicine.
When nonsurgical treatment has failed, significant neurologic deficits are present, and/or the pathology necessitates, surgical treatment for symptomatic IVDD is indicated. The most common indication for surgical treatment of canine IVDD is acute disc extrusion in CD dogs. However, biomechanical studies on human and canine spines have revealed that a significant amount of IVD compression can be attributed to the paraspinal musculature, suggesting that spine biomechanics are comparable between the two species. 76,188 Together with the knowledge IVDD occurs with similar frequency, mechanisms, and causes in dogs, best current evidence indicates that disc degeneration is not solely a product of human bipedal spine biomechanics 44 and supports the use of canine models for study of the biomechanical components of IVDD as well. Ideally, the biomechanical properties of the FSUs should be evaluated in bending, compression, and rotation for pivotal preclinical studies using canine models. 96,189 Each of these tests mimics natural movements of the spine that have been validated in canine and human FSUs. These biomechanical data can then be correlated to clinical, diagnostic imaging, biomarker, macroscopic, and histologic data in order to characterize the effects of degenerative changes on function and differentiate tissue involvement, roles and mechanisms in disc health, and disease.
Incremental loading tests are designed to measure material responses to forces applied in an increasing or decreasing stepwise manner. 190 For IVD testing, incremental loading can be applied in compression to a single disc or FSU or in compression, bending, and/ F I G U R E 4 Lateral cervical spine radiographic views of a Doberman Pinscher with neurologic signs localized to the caudal cervical spine. There are subtle changes at the ventral margin of the cranial endplates at C5, C6, and C7. Lateral myelogram showing compression of the subarachnoid space/contrast column at C5 to C6 and C6 to C7 (arrow heads), and dorsal compression at C4 to C5, C5 to C6, and C6 to C7 (arrows) or rotation to a spinal segment. Incremental loading tests are used to create force-response profiles to characterize tissue properties. 191 When considering biomechanical testing, the state of the sample after said testing is relevant. Compression tests are commonly performed on IVDs, FSUs, and spinal segments. As IVD compression is essentially constant due to muscle forces and gravity, and resistance to compression is a key feature of disc health, various forms of compression testing can be used to assess the compressive modulus, elasticity, creep, stress-relaxation, and permeability of the IVD in order to characterize its functional composition, integrity, and viscoelasticity. 53,64,[190][191][192][193][194][195] In theory, compressive, bending, rotational, biaxial, and multiaxial biomechanical tests can be incremental, single or cyclic or both, and nondestructive or destructive. If loading stays within physiologic ranges and the tissues retain their properties following testing, it can be considered nondestructive such that other assessments can be performed on the same tissues. Destructive, or load-to-failure, testing may be necessary based on experimental design or intended purpose of the study.

| Histology
Healthy IVDs are confined by two adjacent vertebrae lined by cartilaginous end plates with the interposed disc composed of a gelatinous core (NP) surrounded by rings of collagenous tissue bundles arranged in parallel (AF). 88,196,197 ( Figure 5) The region that interconnects AF to NP is the transitional zone (TZ). 88 The healthy NP is composed of large quantities of basophilic ECM populated by sheets or clusters of large, irregularly-shaped cells with a physaliferous appearance (notochordal cells). 88,198 ( Figure 5) The TZ contains chondrocyte-like cells embedded in a loose, collagenous tissue. 88 Fibrochondrocytic cells are embedded within the normal AF; the cells of the outer AF are predominantly fibroblasts/fibrocytes, while the cells of the inner AF are a mixture of fibroblasts/fibrocytes and chondrocyte-like cells. 88 The cartilaginous endplates resemble hyaline cartilage. 38 The cartilaginous endplates are thicker in human IVDs compared to canine IVDs. 45 This difference is thought to exist because vertebral growth in dogs is primarily regulated by separate epiphyseal growth plates located at both the cranial and caudal ends within the vertebrae, while in humans the vertebral growth mainly occurs in the interface between the cartilaginous endplates and the subchondral bone. 45 Grossly, degenerative NP is more solid (less gelatinous) and opaque, tears are often visible in the AF and/or NP, the AF-NP demarcation is lost, and there are irregularities in the endplates. 199 cell death, loss of AF-NP demarcation, granular debris, fibrosis, and neovascularization from the outer AF inward. 31,88,197,202 (Figure 6) Decreasing proteoglycan content of NP is associated with an increased severity of IVD degeneration. 88 Gradual loss of collagenous meshwork and replacement by increasingly hyalinized collagen fibers is observed in degenerative AF. 202 ( Figure 6) Changes in the cartilagi- In humans and dogs, IVDD is associated with inflammation, altered matrix synthesis, catabolic metabolism, cell death, and neural and vascular ingrowth in the disc and surrounding tissues. 206

| CONCLUSIONS
Dogs provide powerful models for disorders of the spine. Pathogenesis, causes, clinical presentations, and diagnostic tools for IVDD are highly similar between human and canine patients. In particular, spontaneously occurring IVD degeneration in CD and NCD breeds of dogs provide highly translatable preclinical data for symptomatic disc degeneration disorders seen across the spectrum of age-, cause-, and pathology-associated patient cohorts. Measurable data obtained through scientific studies in dogs provide insights into histopathology, biomechanics, and various biomarkers with high clinical relevance, but that cannot be practically or ethically obtained from human patients.