Animal Model Of Disc Degeneration Research Articles

Original animal model of lumbar disc degeneration

ABSTRACT Purpose: Disc degeneration (DD) is a common cause of low back pain, which represents one of the most widespread public health problems in the world. Therefore, the establishment of a reproducible animal model is indispensable to understand the pathogenic mechanisms of DD and to test new therapeutic strategies. From this perspective, the fundamental objective of this study was to elucidate the effect of ovariectomy in establishing a new animal model of DD in rats. Methods: 36 female Sprague-Dawley rats were divided into four groups of 9 rats: Group 1: Negative control (Sham): Only an abdominal skin incision and sutures were performed. Group 2: Ovariectomy (OVX): Removal of two ovaries through a transverse incision in the middle of the abdomen. Group 3: Puncture (Punct): Puncture of lumbar intervertebral discs (L3/4, L4/5, and L5/6) by a 21 G needle. Group 4: Puncture+ovariectomy (Punct+OVX): Removal of two ovaries and puncture of L3/4, L4/5, and L5/6 discs. The rats were euthanized 1, 3, and 6 weeks post-surgery, and the discs were harvested. Validity was assessed by radiography, histology, and biochemistry (water content). Results: Disc height, water content, and histologic score decreased significantly in the last 3 groups and at all three-time points (P < 0.05). DD progressed over time in the Punct and Punct+OVX groups (P < 0.05). The changes were more severe in the Punct+OVX group compared to the Punct group and the OVX group. Conclusion: The combination of puncture and ovariectomy induced rapid and progressive DD in the lumbar discs of rats without spontaneous recovery.

Evaluation of the Efficacy of Stem Cell Therapy in Animal Models of Intervertebral Disc Degeneration Based on Imaging Indicators: A Systematic Review and Meta-Analysis.

Objective The purpose of this study is to make a systematic review of the therapeutic effect of stem cells in animal models of disc degeneration from an imaging point of view. Methods Data were extracted by searching electronic databases for RCTs that met the inclusion criteria. Data analysis was performed using RevMan 5.3 and STATA 15.1 software. This meta-analysis was registered with INPLASY, registration number INPLASY202240148. Results A total of 34 studies were included, covering four species of animals, rabbits, sheep, rats, and mice, with a total of 1163 intervertebral discs. In terms of DHI, the efficacy of stem cell group in rabbits (P < 0.001), mice (P < 0.001), sheep (P < 0.001), and rats (P = 0.001) was better than that in control group. In terms of disc height, the efficacy of stem cell group in rats (P < 0.001) was better than that in control group, while in sheep (P = 0.355), there was no statistical difference between two groups. In terms of MRI index, the efficacy of stem cell group in rats (P < 0.001), mice (P < 0.001), and rabbits (P = 0.016) was better than that in control group. In terms of MRI signal score, the efficacy of stem cell group in rabbits (P < 0.001) was better than that of control group. In terms of T2 signal intensity, stem cell group was more effective than control group in rabbits (P < 0.001), mice (P < 0.001), and rats (P = 0.003).Conclusion Stem cell therapy can improve intervertebral disc-related imaging parameters in animal models of disc degeneration, indicating that stem cell therapy has a repairing effect on intervertebral discs. However, given the heterogeneity and limitations of this study, this conclusion still needs to be tested by a large number of studies.

The role of microenvironment in stem cell-based regeneration of intervertebral disc

Regenerative medicine for intervertebral disc (IVD) disease, by utilizing chondrocytes, IVD cells, and stem cells, has progressed to clinical trials in the treatment of back pain, and has been studied in various animal models of disc degeneration in the past decade. Stem cells exist in their natural microenvironment, which provides vital dynamic physical and chemical signals for their survival, proliferation and function. Long-term survival, function and fate of mesenchymal stem cells (MSCs) depend on the microenvironment in which they are transplanted. However, the transplanted MSCs and the endogenous disc cells were influenced by the complicated microenvironment in the degenerating disc with the changes of biochemical and biophysical components. It is important to understand how the MSCs and endogenous disc cells survive and thrive in the harsh microenvironment of the degenerative disc. Furthermore, materials containing stem cells and their natural microenvironment have good clinical effects. However, the implantation of tissue engineering IVD (TE-IVD) cannot provide a complete and dynamic microenvironment for MSCs. IVD graft substitutes may need further improvement to provide the best engineered MSC microenvironment. Additionally, the IVD progenitor cells inside the stem cell niches have been regarded as popular graft cells for IVD regeneration. However, it is still unclear whether actual IVD progenitor cells exist in degenerative spinal conditions. Therefore, the purpose of this review is fourfold: to discuss the presence of endogenous stem cells; to review and summarize the effects of the microenvironment in biological characteristics of MSC, especially those from IVD; to explore the feasibility and prospects of IVD graft substitutes and to elaborate state of the art in the use of MSC transplantation for IVD degeneration in vivo as well as their clinical application.

Inflammatory cytokine and catabolic enzyme expression in a goat model of intervertebral disc degeneration.

Intervertebral disc degeneration is implicated as a leading cause of low back pain. Persistent, local inflammation within the disc nucleus pulposus (NP) and annulus fibrosus (AF) is an important mediator of disc degeneration and negatively impacts the performance of therapeutic stem cells. There is a lack of validated large animal models of disc degeneration that recapitulate clinically relevant local inflammation. We recently described a goat model of disc degeneration in which increasing doses of chondroitinase ABC (ChABC) were used to reproducibly induce a spectrum of degenerative changes. The objective of this study was to extend the clinical relevance of this model by establishing whether these degenerative changes are associated with the local expression of inflammatory cytokines and catabolic enzymes. Degeneration was induced in goat lumbar discs using ChABC at different doses. After 12 weeks, degeneration severity was determined histologically and using quantitative magnetic resonance imaging (MRI). Expression levels of inflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β], and IL-6) and catabolic enzymes (matrix metalloproteinases-1 [MMPs-1] and 13, and a disintegrin and metalloproteinase with thrombospondin type-1 motifs-4 [ADAMTS-4]) were assessed as the percentage of immunopositive cells in the NP and AF. With the exception of MMP-1, cytokine, and enzyme expression levels were significantly elevated in ChABC-treated discs in the NP and AF. Expression levels of TNF-α, IL1-β, and ADAMTS-4 were positively correlated with histological grade, while all cytokines and ADAMTS-4 were negatively correlated with MRI T2 and T1ρ scores. These results demonstrate that degenerate goat discs exhibit elevated expression of clinically relevant inflammatory mediators, and further validate this animal model as a platform for evaluating new therapeutic approaches for disc degeneration.

Efficiency of dual siRNA-mediated gene therapy for intervertebral disc degeneration (IVDD)

BACKGROUND CONTEXTOne of the common causes of low back pain is intervertebral disc degeneration. The pathophysiology of disc degeneration involves apoptosis of nucleus pulposes cells and degradation of extra cellular matrix (ECM). Caspase 3 plays a central role in apoptosis and the ADAMTS5 (A Disintegrin and Metalloproteinase with Thrombospondin motifs 5) gene plays a critical role in ECM degradation. Hence, we hypothesized that if one can silence these two genes, both apoptosis and ECM degradation can be prevented, thereby preventing the progression and even reverse disc degeneration. PURPOSEThe purpose of this study is to demonstrate the regenerative potential of small interfering RNA (siRNA) designed against Caspase 3 and ADAMTS5 genes in an in vitro and animal model of disc degeneration. STUDY DESIGNIn vitro study followed by in vivo study in a rabbit model. METHODSIn vitro studies were done using the human hepatocellular carcinoma (Hep G2) cell line for validating the efficacy of liposomal siRNA in controlling the expression of genes (Caspase 3 and ADAMTS5). Later, siRNA's validation was done in a rabbit annular punctured model by administering siRNA's individually (Caspase 3 and ADAMTS5) and in combination Caspase3-ADAMTS5) for assessing their synergistic effect in down regulating the gene expression in the degenerative discs. Annular punctured intervertebral discs of the rabbit were injected with siRNA formulations (single and dual) and phosphate buffer saline, one week after initial puncture. Magnetic resonance imaging (MRI) scans were done before and after siRNA treatment (1, 4 and 8 weeks) for assessing the progression of disc degeneration. The histopathology and real time polymerase chain reaction (RT-PCR) studies were done for evaluating their efficacy. We did not receive any funding for conducting the study, and we do not have a conflict of interest with any researchers or scientific groups. RESULTSThe observations made from both in vitro and in vivo studies indicate the beneficial effects of siRNA formulation in down regulating the expression of Caspase 3 and ADAMTS5 genes. The MRI and histopathological evaluation showed that the disc degeneration was progressive in phosphate buffer saline and AT5-siRNA injected discs but the discs that received Caspase 3-siRNA and dual siRNA (Cas3-AT5-siRNA) formulation showed signs of recovery and regeneration 4 and 8 weeks after injection. The efficacy of siRNA designed against Cas3 and AT5 was also assessed in both in vitro and in vivo experiments by using RT-PCR analysis and the results showed downregulation of Caspase 3 gene in Caspase 3-siRNA group, but there was no significant downregulation of ADAMTS5 gene in ADAMTS5-siRNA group (ie, indicated by fold change). Synergistic effect was observed in the group that received dual siRNA (Cas3-AT5 siRNA) formulation. CONCLUSIONSThis experiment suggests that intervention by siRNA treatment significantly reduced the extent of apoptosis in the discs. CLINICAL SIGNIFICANCEDelivery of siRNA directly into spinal discs has a potential in treating disc degeneration nonsurgically.

A large animal model that recapitulates the spectrum of human intervertebral disc degeneration

The objective of this study was to establish a large animal model that recapitulates the spectrum of intervertebral disc degeneration that occurs in humans and which is suitable for pre-clinical evaluation of a wide range of experimental therapeutics. Degeneration was induced in the lumbar intervertebral discs of large frame goats by either intradiscal injection of chondroitinase ABC (ChABC) over a range of dosages (0.1U, 1U or 5U) or subtotal nucleotomy. Radiographs were used to assess disc height changes over 12 weeks. Degenerative changes to the discs and endplates were assessed via magnetic resonance imaging (MRI), semi-quantitative histological grading, microcomputed tomography (μCT), and measurement of disc biomechanical properties. Degenerative changes were observed for all interventions that ranged from mild (0.1U ChABC) to moderate (1U ChABC and nucleotomy) to severe (5U ChABC). All groups showed progressive reductions in disc height over 12 weeks. Histological scores were significantly increased in the 1U and 5U ChABC groups. Reductions in T2 and T1ρ, and increased Pfirrmann grade were observed on MRI. Resorption and remodeling of the cortical boney endplate adjacent to ChABC-injected discs also occurred. Spine segment range of motion (ROM) was greater and compressive modulus was lower in 1U ChABC and nucleotomy discs compared to intact. A large animal model of disc degeneration was established that recapitulates the spectrum of structural, compositional and biomechanical features of human disc degeneration. This model may serve as a robust platform for evaluating the efficacy of therapeutics targeted towards varying degrees of disc degeneration.

A Review of Animal Models of Intervertebral Disc Degeneration: Pathophysiology, Regeneration, and Translation to the Clinic

Lower back pain is the leading cause of disability worldwide. Discogenic pain secondary to intervertebral disc degeneration is a significant cause of low back pain. Disc degeneration is a complex multifactorial process. Animal models are essential to furthering understanding of the degenerative process and testing potential therapies. The adult human lumbar intervertebral disc is characterized by the loss of notochordal cells, relatively large size, essentially avascular nature, and exposure to biomechanical stresses influenced by bipedalism. Animal models are compared with regard to the above characteristics. Numerous methods of inducing disc degeneration are reported. Broadly these can be considered under the categories of spontaneous degeneration, mechanical and structural models. The purpose of such animal models is to further our understanding and, ultimately, improve treatment of disc degeneration. The role of animal models of disc degeneration in translational research leading to clinical trials of novel cellular therapies is explored.

A New Porcine Animal Model of Cervical Disc Degeneration

Introduction Previous in vivo animal model for disc degeneration was predominantly smaller animals. But such small discs were inappropriate to investigate the cell transplantation therapy highlighted recently. A porcine disc degeneration model in lumbar spine was introduced; however, the lumbar spine was not influenced by the gravity. Hence, the authors performed porcine animal model of disc degeneration in cervical spine. Materials and Methods Three domestic minipigs (aged, 5 months; weight, 40-50 kg) were used. After anesthesia, right transverse 5 cm-sized incision was made over the three fingers above the clavicle on the anterior neck. Similar to a standard anterior cervical approach of Smith-Robinson method, anatomic dissection was performed to expose anterior surface of the cervical spine. The partial nucleotomy by a Jamshidi bone marrow biopsy needle was performed in one intervertebral disc, and the other disc spaces served as control. Radiologic study using lateral plain radiograph (X-ray) and magnetic resonance image (MRI) was evaluated at preoperation and postoperative 4 and 8 weeks. Results Two of three minipigs were confirmed disc degeneration at C4-5 and C6-7, respectively. The disc height was reduced by 20% at nucleotomy level. And these discs with previous Pfirrmann grade I were changed to grade IV degeneration at postoperative 4 and 8 weeks. The other disc spaces were maintained previous disc height and Pfirrmann grade I condition. Conclusion This was a pilot study to introduce a new disc degeneration model with relatively large animal at cervical spine. The authors will conduct a further experiment to confirm the cervical disc degeneration and plan the cell therapy for treatment of disc degeneration. Disclosure of Interest None declared

Glucosamine Supplementation Demonstrates a Negative Effect on Intervertebral Disc Matrix in an Animal Model of Disc Degeneration

Laboratory based controlled in vivo study. To determine the in vivo effects of oral glucosamine sulfate on intervertebral disc degeneration. Although glucosamine has demonstrated beneficial effect in articular cartilage, clinical benefit is uncertain. A Centers for Disease Control report from 2009 reported that many patients are using glucosamine supplementation for low back pain, without significant evidence to support its use. Because disc degeneration is a major contributor of low back pain, we explored the effects of glucosamine on disc matrix homeostasis in an animal model of disc degeneration. Eighteen skeletally mature New Zealand White rabbits were divided into 4 groups: control, annular puncture, glucosamine, and annular puncture + glucosamine. Glucosamine treated rabbits received daily oral supplementation with 107 mg/d (weight based equivalent to human 1500 mg/d). Annular puncture surgery involved puncturing the annulus fibrosus of 3 lumbar discs with a 16-gauge needle to induce degeneration. Serial magnetic resonance images were obtained at 0, 4, 8, 12, and 20 weeks. Discs were harvested at 20 weeks for determination of glycosaminoglycan content, relative gene expression measured by real time polymerase chain reaction, and histological analyses. The magnetic resonance imaging index and nucleus pulposus area of injured discs of glucosamine treated animals with annular puncture was found to be lower than that of degenerated discs from rabbits not supplemented with glucosamine. Consistent with this, decreased glycosaminoglycan was demonstrated in glucosamine fed animals, as determined by both histological and glycosaminoglycan content. Gene expression was consistent with a detrimental effect on matrix. These data demonstrate that the net effect on matrix in an animal model in vivo, as measured by gene expression, magnetic resonance imaging, histology, and total proteoglycan is antianabolic. This raises concern about this commonly used supplement, and future research is needed to establish the clinical relevance of these findings.

Needle puncture injury causes acute and long-term mechanical deficiency in a mouse model of intervertebral disc degeneration

Low back pain is a significant socioeconomic burden and intervertebral disc degeneration has been implicated as a cause. A reliable animal model of disc degeneration is necessary to evaluate therapeutics, and functional metrics are essential to quantify their benefit. To this end, needle puncture injuries were created in the caudal intervertebral discs of mice to induce disc degeneration. Compression, torsion, and creep mechanics were assessed both immediately and after eight weeks to distinguish between the effects of injury and the subsequent reparative or degenerative response. Two needle sizes (29 and 26 gauge) were used to determine injury size-dependence. Compressive stiffness (62%), torsional stiffness (60%), and early damping stiffness (84%) decreased immediately after injury with the large needle (26G). These mechanical properties did not change over time despite structural and compositional changes. At 8 weeks following large needle injury, disc height decreased (37%), nucleus pulposus (NP) glycosaminoglycan content decreased (41%), and NP collagen content increased (45%). The small needle size had no significant effect on mechanics and did not initiate degenerative changes in structure and composition. Thus, the injection of therapeutics into the NP with a minimal needle size may limit damage due to the needle insertion. These findings, along with the wide commercial availability of mouse-specific biological probes, indicate that the mouse caudal disc model can be a powerful tool for investigating disc degeneration and therapy.

Experimental model of intervertebral disc degeneration by needle puncture in Wistar rats

Animal models of intervertebral disc degeneration play an important role in clarifying the physiopathological mechanisms and testing novel therapeutic strategies. The objective of the present study is to describe a simple animal model of disc degeneration involving Wistar rats to be used for research studies. Disc degeneration was confirmed and classified by radiography, magnetic resonance and histological evaluation. Adult male Wistar rats were anesthetized and submitted to percutaneous disc puncture with a 20-gauge needle on levels 6-7 and 8-9 of the coccygeal vertebrae. The needle was inserted into the discs guided by fluoroscopy and its tip was positioned crossing the nucleus pulposus up to the contralateral annulus fibrosus, rotated 360° twice, and held for 30 s. To grade the severity of intervertebral disc degeneration, we measured the intervertebral disc height from radiographic images 7 and 30 days after the injury, and the signal intensity T2-weighted magnetic resonance imaging. Histological analysis was performed with hematoxylin-eosin and collagen fiber orientation using picrosirius red staining and polarized light microscopy. Imaging and histological score analyses revealed significant disc degeneration both 7 and 30 days after the lesion, without deaths or systemic complications. Interobserver histological evaluation showed significant agreement. There was a significant positive correlation between histological score and intervertebral disc height 7 and 30 days after the lesion. We conclude that the tail disc puncture method using Wistar rats is a simple, cost-effective and reproducible model for inducing disc degeneration.

MMP-2 mediates local degradation and remodeling of collagen by annulus fibrosus cells of the intervertebral disc

IntroductionDegeneration of the intervertebral disc (IVD) is characterized by marked degradation and restructuring of the annulus fibrosus (AF). Although several matrix metalloproteinases (MMPs) have been found to be more prevalent in degenerate discs, their coordination and function within the context of the disease process are still not well understood. In this study, we sought to determine whether MMP-2 is associated with degenerative changes in the AF and to identify the manner by which AF cells use MMP-2.MethodsTwo established animal models of disc degeneration, static compression and transannular needle puncture of rodent caudal discs, were examined for MMP-2 immunopositivity. With lentiviral transduction of an shRNA expression cassette, we screened and identified an effective shRNA sequence for generating stable RNA interference to silence MMP-2 expression in primary rat AF cells. Gelatin films were used to compare gelatinase activity and spatial patterns of degradation between transduced cells, and both noninfected and nonsense shRNA controls. The functional significance of MMP-2 was determined by assessing the ability for cells to remodel collagen gels.ResultsBoth static compression and 18-g annular puncture of rodent caudal discs stimulated an increase in MMP-2 activity with concurrent lamellar disorganization in the AF, whereas 22-g and 26-g needle injuries did not. To investigate the functional role of MMP-2, we established lentivirus-mediated RNAi to induce stable knockdown of transcript levels by as much as 88%, and protein levels by as much as 95% over a 10-day period. Culturing transduced cells on gelatin films confirmed that MMP-2 is the primary functional gelatinase in AF cells, and that MMP-2 is used locally in regions immediately around AF cells. In collagen gels, transduced cells demonstrated an inability to remodel collagen matrices.ConclusionsOur study indicates that increases in MMP-2 observed in human degenerate discs are mirrored in experimentally induced degenerative changes in rodent animal models. AF cells appear to use MMP-2 in a very directed fashion for local matrix degradation and collagen remodeling. This suggests that MMP-2 may have a functionally significant role in the etiology of degenerative disc disease and could be a potential therapeutic target.

Mechanical Deformation and Glycosaminoglycan Content Changes in a Rabbit Annular Puncture Disc Degeneration Model

Evaluation of degenerated intervertebral discs from a rabbit annular puncture model by using specialized magnetic resonance imaging (MRI) techniques, including displacement encoding with stimulated echoes and a fast-spin echo (DENSE-FSE) acquisition and delayed gadolinium-enhanced MRI of cartilage (dGEMRIC). To evaluate a rabbit disc degeneration model by using various MRI techniques. To determine the displacements and strains, spin-lattice relaxation time (T1), and glycosaminoglycan (GAG) distribution of degenerated discs as compared to normal and adjacent level discs. Annular puncture of the intervertebral disc produces disc degeneration in rabbits. DENSE-FSE has been previously demonstrated in articular cartilage for the measurement of soft tissue displacements and strains. MRI also can measure the T1 of tissue, and dGEMRIC can quantify GAG concentration in cartilage. METHODS.: In eight New Zealand white rabbits, the annulus fibrosis of a lumbar disc was punctured. After 4 weeks, the punctured and cranially adjacent motion segments were isolated for MRI and histology. MRI was used to estimate the disc volume and map T1. DENSE-FSE was used to determine displacements for the estimation of strains. dGEMRIC was then used to determine GAG distributions. Histology and standard MRI indicated degeneration in punctured discs. Disc volume increased significantly at 4 weeks after the puncture. Displacement of the nucleus pulposus was distinct from that of the annulus fibrosis in most untreated discs but not in punctured discs. T1 was significantly higher and GAG concentration significantly lower in punctured discs compared with untreated adjacent level discs. Noninvasive and quantitative MRI techniques can be used to evaluate the mechanical and biochemical changes that occur with animal models of disc degeneration. DENSE-FSE, dGEMRIC, and similar techniques have potential for evaluating the progression of disc degeneration and the efficacy of treatments.

Innervation of Anulus Tears

A prospective immunohistological study in an animal model. To identify and describe the phenotype of neoinnervation in experimental anular tears. Controversy surrounds neoinnervation of degenerate discs which has been proposed as the anatomic basis for discogenic pain. Ablation of neoinnervation has been postulated as the theoretical basis for the claimed successes of procedures such as intradiscal electrotherapy. The animal model of disc degeneration previously developed in our research center provides an opportunity to investigate the innervation of anular tears in an extensively characterized lesion. A surgical anular tear was created in 5 lumbar discs in 11 sheep which were killed at 1, 2, 3, and 12 months. Each spine was x-rayed and divided into motion segments for histologic analysis. Serial sections through the tear were immunostained for protein gene product 9.5, tyrosine hydroxylase, and calcitonin gene receptor protein. Neoinnervation of the periphery of the anular tear was observed. Ingrowing nerves penetrated marginally deeper than the normal anular innervation but no nerves were identified in the inner anulus or nucleus. A minority of the new axons were calcitonin gene receptor protein or tyrosine hydroxylase positive. The anulus tears in this model are innervated only peripherally to a depth only marginally greater than that of the normal anulus.

In vivo intervertebral disc regeneration using stem cell–derived chondroprogenitors

There is currently no biologic therapy to repair or restore a degenerated intervertebral disc. A potential solution may rest with embryonic stem cells (ESCs), which have a potential to grow indefinitely and differentiate into a variety of cell types in vitro. Prior studies have shown that ESCs can be encouraged to differentiate toward specific cell lineages by culture in selective media and specific growth environment. Among these lineages, there are cells capable of potentially producing nucleus pulposus (NP) in vivo. In this investigation, the authors studied ESCderived chondroprogenitors implanted into a degenerated disc in a rabbit. For this purpose, a rabbit model of disc degeneration was developed. A percutaneous animal model of disc degeneration was developed by needle puncture of healthy intact discs in 16 New Zealand white rabbits. Series of spine MR imaging studies were obtained before disc puncture and after 2, 6, and 8 weeks. Prior to implantation, murine ESCs were cultured with cis-retinoic acid, transforming growth factor beta, ascorbic acid, and insulin-like growth factor to induce differentiation toward a chondrocyte lineage. After confirmation by MR imaging, degenerated disc levels were injected with chondrogenic derivatives of ESCs expressing green fluorescent protein. At 8 weeks post-ESC implantation, the animals were killed and the intervertebral discs were harvested and analyzed using H & E staining, confocal fluorescent microscopy, and immunohistochemical analysis. Three intervertebral disc groups were analyzed in 16 rabbits, as follows: 1) Group A, control: naïve, nonpunctured discs (32 discs, levels L4-5 and L5-6); 2) Group B, experimental control: punctured disc (16 discs, level L2-3); and 3) Group C, experimental: punctured disc followed by implantation of chondroprogenitor cells (16 discs, level L3-4). The MR imaging studies confirmed intervertebral disc degeneration at needle-punctured segments starting at approximately 2 weeks. Postmortem H & E histological analysis of Group A discs showed mature chondrocytes and no notochordal cells. Group B discs displayed an intact anulus fibrosus and generalized disorganization within fibrous tissue of NP. Group C discs showed islands of notochordal cell growth. Immunofluorescent staining for notochordal cells was negative for Groups A and B but revealed viable notochordal-type cells within experimental Group C discs, which had been implanted with ESC derivatives. Notably, no inflammatory response was noted in Group C discs. This study illustrates a reproducible percutaneous model for studying disc degeneration. New notochordal cell populations were seen in degenerated discs injected with ESCs. The lack of immune response to a xenograft of mouse cells in an immunocompetent rabbit model may suggest an as yet unrecognized immunoprivileged site within the intervertebral disc space.

A porcine model of intervertebral disc degeneration induced by annular injury characterized with magnetic resonance imaging and histopathological findings

Appropriate animal models of disc degeneration are critical for the study of proposed interventions as well as to further delineate the degenerative process. The purpose of this study was to characterize a porcine model for disc degeneration confirmed on magnetic resonance (MR) imaging studies and histological analysis. Twelve miniature pigs were used (weight 48-65 kg) to study degeneration in the lumbar spine. Under fluoroscopic guidance, the disc was percutaneously punctured with a 3.2-mm-diameter trephine to a 5-mm depth into the annulus fibrosus. Control and experimental levels were randomized among 6 levels in the lumbar spine. The unlesioned spinal levels were used as controls and were compared with lesioned levels. Magnetic resonance imaging grading and disc height were serially recorded preoperatively, and at 5, 8, 19, 32, and 39 weeks postoperatively. The animals were killed in groups of 3 at 7, 18, 32, and 41 weeks postinjury, and the discs were examined histopathologically. Consistent, sequential, and progressive degeneration of the annular injury was observed on MR imaging and histopathological studies from the time of injury to the final time point. The disc height and the disc height index also sequentially decreased from the time of the injury in a consistent manner. The uninjured control levels did not show any progressive degeneration and maintained their normal state. Based on MR imaging and histopathological findings, the authors demonstrated and characterized a reliable model of sequential disc degeneration in miniature pigs with percutaneous injury to the annulus fibrosus. In the early stages, as soon as 5 weeks after injury, significant disc degeneration was seen on MR imaging grading with decreases in disc height. This degeneration did not improve by the final time point of 39 weeks.

Anti‐catabolic effect of OP‐1 in chronically compressed intervertebral discs

Experimental animal models of disc degeneration have been used to assess the biomechanical behavior, biochemical composition, and biological changes in the intervertebral discs. The objective of our study was to evaluate the anabolic and anti-catabolic effects of intradiscal injection of Osteogenic Protein-1 (OP-1) by histology and immunohistochemistry in disc degeneration model. Thirty-four rats were divided into five groups: intact control; sham control; compressed nucleus pulposus (NP) injected with saline; and two OP-1 groups: COP-1 group (compression was continued after intradiscal OP-1 injection) and ROP-1 group (compression was released at the time of OP-1 injection). Anabolic and anti-catabolic effects of OP-1 were evaluated by histology and immunohistochemistry with the following antibodies: anti-pro- and anti-mature OP-1, anti-MMP-13, anti-aggrecanase, anti-substance P, anti-tumor necrosis factor-alpha (TNF-alpha), and anti-interleukin-1beta (IL-1beta). The OP-1 injection to the degenerative disc stimulated an anabolic response characterized by the restoration of the normal morphology of the disc, increased Safranin O staining in the NP, extention of the extracellular matrix, and stimulation of endogenous OP-1 synthesis in the NP, annulus fibrosis (AF), and end-plate. The anti-catabolic effect of OP-1 was documented by reduced immunostaining for aggrecanase, MMP-13, substance P, TNF-alpha, and IL-1beta. This study confirmed the anti-catabolic activity of OP-1 as demonstrated previously in human articular cartilage and provided critical evidence for the potential of OP-1 therapy in the treatment of disc degeneration. Because substance P is a neuropeptide linked with inflammation and pain, a reduction in the level of this protein may support our previously reported results on the effect of OP-1 on pain-related behavior.

Reliability of radiographic intervertebral disc height measurement for in vivo rat-tail model

Quantification of changes in intervertebral disc height is critical for studying intervertebral disc degeneration. Reliability of disc height measurement is therefore especially important for in vivo studies using animal models of disc degeneration. In this study, the effects of image intensity and percentage of disc width used for disc height measurement from radiographic images were evaluated in a rat-tail model. Radiographs were taken for 10 Sprague–Dawley rats using a standardized protocol. Average disc heights of the caudal 8–9 discs were determined using original and intensity adjusted images with different percentages of disc width. The average disc height was found to be significantly affected by both the image intensity and the percentage of disc width measured. A higher reliability was found in the measurement for image with adjusted intensity and using smaller disc width. Image intensity is suggested to be controlled and the disc width should be taken into account in quantifying the disc height.

BIOLOGIC MODIFICATION OF ANIMAL MODELS OF INTERVERTEBRAL DISC DEGENERATION

Intervertebral disc degeneration is a chronic process that can become manifest in clinical disorders such as idiopathic low back pain, sciatica, disc herniation, spinal stenosis, and myelopathy. The limited available treatment options (including discectomy and spinal fusion) for these and other disabling conditions that arise from intervertebral disc degeneration are highly invasive, achieve limited success, and only address acute symptoms while doing nothing to halt the process of degeneration. Although the precise pathophysiology of intervertebral disc degeneration has yet to be clearly delineated, the progressive decline in aggrecan, the primary proteoglycan of the nucleus pulposus, appears to be a final common pathway. Animal models as well as in vitro studies of the process of disc degeneration have yielded many potentially useful targets for the reversal of disc degeneration. One current research trend is the use of established animal models of disc degeneration to study the role of therapeutic modalities in reversing the process of degeneration, often with use of the delivery of genes or gene products that influence the anabolic and catabolic pathways of the disc. This article reviews the ability of gene-product delivery systems and gene therapy to alter biologic processes in animal models of disc degeneration and examines future trends in this field.

Biologic Modification of Animal Models of Intervertebral Disc Degeneration

Intervertebral disc degeneration is a chronic process that can become manifest in clinical disorders such as idiopathic low back pain, sciatica, disc herniation, spinal stenosis, and myelopathy. The limited available treatment options (including discectomy and spinal fusion) for these and other disabling conditions that arise from intervertebral disc degeneration are highly invasive, achieve limited success, and only address acute symptoms while doing nothing to halt the process of degeneration. Although the precise pathophysiology of intervertebral disc degeneration has yet to be clearly delineated, the progressive decline in aggrecan, the primary proteoglycan of the nucleus pulposus, appears to be a final common pathway. Animal models as well as in vitro studies of the process of disc degeneration have yielded many potentially useful targets for the reversal of disc degeneration. One current research trend is the use of established animal models of disc degeneration to study the role of therapeutic modalities in reversing the process of degeneration, often with use of the delivery of genes or gene products that influence the anabolic and catabolic pathways of the disc. This article reviews the ability of gene-product delivery systems and gene therapy to alter biologic processes in animal models of disc degeneration and examines future trends in this field.