Satoshi Sobajima

A slowly progressive and reproducible animal model of intervertebral disc degeneration characterized by MRI, X-ray, and histology

Study Design. The progression of intervertebral disc degeneration following anterolateral “stab” of adult rabbit lumbar discs by 16-gauge hypodermic needle to a limited (5-mm) depth was studied for up to 24 weeks using magnetic resonance imaging, radiograph, and histologic outcome measures. Objectives. To develop a slowly progressive, reproducible rabbit model of intervertebral disc degeneration suitable for studying pathogenesis and pathophysiology of intervertebral disc degeneration and testing safety and efficacy of novel approaches to the treatment of intervertebral disc degeneration (e.g., growth factors, gene therapy, cell therapy, and tissue engineering). Summary of Background Data. Numerous animal models of intervertebral disc degeneration have been proposed in the literature, each with attendant advantages and disadvantages. The classic “stab model,” involving full-thickness stab of anterior anulus fibrosus of adult rabbit lumbar discs by a number 11 scalpel blade, appears to produce changes in certain biochemical and histologic outcome measures that are similar to changes seen in human intervertebral disc degeneration. However, the immediate herniation of nucleus pulposus on full-thickness stab renders this model less suitable for 1) studying effects of less precipitous changes in nucleus pulposus and anulus fibrosus that may be important in the onset and progression of intervertebral disc degeneration and 2) testing novel therapeutic approaches that target the processes of early intervertebral disc degeneration. Methods. The L2–L3, L3–L4, and L4–L5 lumbar intervertebral discs of 18 skeletally mature female New Zealand White rabbits were stabbed by 16-gauge hypodermic needle to a depth of 5 mm in the left anterolateral anulus fibrosus. Serial magnetic resonance imaging scans of the stabbed discs and intact L1–L2 and L5–L6 control discs were performed at 3, 6, 12, and 24 weeks post surgery and compared with preoperative magnetic resonance images. Supplemental radiograph and histologic analyses were performed. Results. The stabbed discs exhibited a progressive decrease in “magnetic resonance imaging index” (the product of nucleus pulposus area and signal intensity from T2-weighted midsagittal plane images) starting at 3 weeks post stab and continuing through 24 weeks, with no evidence of spontaneous recovery or reversal of magnetic resonance imaging changes. Radiograph findings included early osteophyte formation by 6 weeks post stab and extensive, bridging osteophytes by 24 weeks. Histologic analysis revealed progressive loss of notochordal cells from the nucleus pulposus, filling of the nucleus pulposus space with fibrocartilage, and derangement of anulus fibrosus. Conclusions. Stabbing the anterolateral anulus fibrosus of adult rabbit lumbar discs with a 16-gauge hypodermic needle to a limited (5-mm) depth results in a number of slowly progressive and reproducible magnetic resonance imaging, radiograph, and histologic changes over 24 weeks that show a similarity to changes seen in human intervertebral disc degeneration. This model would appear suitable for studying pathogenesis and pathophysiology of intervertebral disc degeneration and testing safety and efficacy of novel treatments of intervertebral disc degeneration.

Gene transfer of the catabolic inhibitor TIMP-1 increases measured proteoglycans in cells from degenerated human intervertebral discs.

Study Design. Cells from degenerated intervertebral discs were transduced with an adenoviral vector delivering cDNA of the catabolic inhibitor, TIMP-1, and alterations in the measured proteoglycan were assessed. Objectives. To assess the potential of TIMP-1 to favorably modify the proteoglycan content of degenerated intervertebral disc cells. Summary of Background Data. Gene therapy with anabolic factors has resulted in increased proteoglycan synthesis in intervertebral disc cells. Biochemical analysis of degenerated discs has revealed elevated levels of the catabolic enzymes, matrix metalloproteinase, suggesting an intimate role of these factors in the degenerative process. The use of TIMP-1, an endogenous inhibitor of matrix metalloproteinase, via gene therapy may provide an additional method to alter the degenerative processes occurring in the intervertebral disc. Materials and Methods. Degenerated intervertebral disc were isolated from eight patients undergoing elective surgical procedures. Cells were cultured in monolayer and transduced with different concentrations of either an adenoviral-tissue inhibitor of metalloproteinase-1 (Ad-TIMP-1) or adenoviral-bone morphogenic protein-2 (Ad-BMP-2) construct. Cells were cultured in a three-dimensional pellet and proteoglycan synthesis was assessed via 35S-sulfur incorporation. Results. Gene delivery of TIMP-1 and BMP-2 increased measured proteoglycan synthesis at each concentration assessed. IVD cells treated with Ad-TIMP-1 demonstrated an optimal response at a multiplicity of infection (MOI) of 100. Cells treated with Ad-BMP-2 demonstrated a progressive increase in proteoglycan synthesis with increasing viral concentrations. Conclusions. Successful delivery of the anticatabolic gene, TIMP-1, results in increased measured proteoglycan in cultured degenerated disc cells. This finding supports catabolic inhibition as a promising avenue of research for the treatment of degenerative disc disease via gene therapy.

Gene Therapy for Degenerative Disc Disease

Degenerative disc disease (DDD) is a chronic process that can become clinically manifest in multiple disorders such as idiopathic low back pain, disc herniation, radiculopathy, myelopathy, and spinal stenosis. The limited available technology for the treatment of these and other pathologic and disabling conditions arising from DDD is highly invasive (eg, surgical discectomy and fusion), manifesting a certain degree of complications and unsatisfactory clinical outcomes. Although the precise pathophysiology of DDD remains to be clearly delineated, the progressive decline in aggrecan, the primary proteoglycan of the nucleus pulposus, appears to be a final common pathway. It has been hypothesized that imbalance in the synthesis and catabolism of certain critical extracellular matrix components can be mitigated by the transfer of genes to intervertebral disc cells encoding factors that modulate synthesis and catabolism of these components. The successful in vivo transfer of therapeutic genes to target cells within the intervertebral disc in clinically relevant animal models of DDD is one example of the rapid progress that is being made towards the development of gene therapy approaches for the treatment of DDD. This chapter reviews the ability of gene therapy to alter biologic processes in the degenerated intervertebral disc and outlines the work needed to be done before human clinical trials can be contemplated.

Safety Assessment of Intradiscal Gene Therapy II: Effect of Dosing and Vector Choice

Study Design. Clinical, biochemical, and histologic analysis was performed after in vivo delivery of cDNA encoding various anabolic cytokines and marker genes to the lumbar epidural space of New Zealand white rabbits, using both adenoviral and adeno-associated viral vectors. Objective. To mimic errant or misplaced doses of gene therapy to better ascertain the potential risks associated with alternative vectors and transgene products with regard to their application to problems of the intervertebral disc. Summary of Background Data. Work done with several anabolic cytokines including bone morphogenic proteins and transforming growth factors, has demonstrated the potential of gene therapy. Recently, data has been published demonstrating that improperly dosed or delivered adenoviral-mediated gene therapy within the subarachnoid space can result in significant morbidity in rabbits. There are currently no studies examining the effect of these errors within the epidural space or using an adeno-associated viral (AAV) vector. Methods. Using either adenoviral or AAV vectors, complementary DNA (cDNA) encoding anabolic cytokines bone morphogenic protein-2 (BMP-2) and transforming growth factor-beta 1 and marker proteins LacZ and green fluorescent protein were injected into the epidural space of 37 New Zealand white rabbits at the L5/6 level. Rabbits were then observed clinically for up to 6 weeks, after which the rabbits were sacrificed in order to perform a comprehensive biochemical and histologic analysis. Results. Following adenoviral-mediated delivery of anabolic cytokine cDNA, up to eighty percent of rabbits demonstrated significant clinical, biochemical, and histologic morbidity. Conversely, AAV-mediated delivery of any cDNA and adenoviral-mediated delivery of marker protein cDNA resulted in no clinical, histologic, or biochemical morbidity. Conclusion. Properly dosed and directed gene therapy seems to be both safe and potentially efficacious. This study suggests that side effects of gene therapy may be due to a combination of dosing, transgene product, and vector choice, and that newer AAV vectors may reduce these side-effects and decrease the risk of this technology.

Implication for melatonin and its receptor in the spinal deformities of hereditary Lordoscoliotic Rabbits.

Study Design. The association between melatonin system and the spontaneous development of the spinal deformities in the Hereditary Lordoscoliotic Rabbit, the natural animal model for idiopathic scoliosis, was studied. Objectives. To examine the implication for melatonin and its receptor in the spinal deformities of the natural animal model, the Hereditary Lordoscoliotic Rabbit. Summary of Background Data. We previously reported radiologic and histologic studies investigating the etiology of spinal deformities in a breed of Japanese White Rabbit, the Hereditary Lordoscoliotic Rabbit. These animals develop thoracic lordoscoliosis during growth and as such can be used as a model for human idiopathic scoliosis. Although previous studies in chickens have established that pinealectomy produces scoliosis, the cause of the condition is yet to be fully elucidated. Methods. Serum melatonin levels in Hereditary Lordoscoliotic Rabbits were measured by radioimmunoassay and compared with those of Japanese White Rabbits (controls). The expression of melatonin receptor in the rabbit was detected by homology cloning to access the number of the melatonin receptor mRNA in the rabbit spinal cord by quantitative reverse-transcribed polymerase chain reaction. Results. Serum melatonin levels in Hereditary Lordoscoliotic Rabbits were significant higher than those of controls in each period until 20 weeks. We detected the expression of melatonin receptor mRNA in rabbit spinal cord. However, no significant quantitative differences were found in the level of expression of melatonin mRNA in the spinal cord between Hereditary Lordoscoliotic Rabbits and controls. Conclusions. In relation to the present study, we suggest that causes of spinal deformities in the Hereditary Lordoscoliotic Rabbit may be the result of the contribution of melatonin receptors as well as that of altered serum melatonin levels in the Hereditary Lordoscoliotic Rabbit. Further studies will be required to investigate the expression of melatonin receptor in other tissues of the Hereditary Lordoscoliotic Rabbit as well as to delineate the role of melatonin in the pathogenesis of idiopathic scoliosis.