158. The effects of a single injection of NTG-101 upon neurotrophin expression in a canine model of degenerative disc disease

Abstract

BACKGROUND CONTEXT In normal intervertebral discs the outer annulus has rich innervation that is necessary to inform reflexive postural muscular activity and to serve as response elements in the event of disease/injury. However, it has been shown that in degenerative, painful discs there is an increased amount of nerve in-growth into the annulus and even into the nucleus pulposus. This ingrowth and associated neovascularization have been hypothesized to be associated with increased sensitivity and therefore pain generation in the case of significant DDD. The expression of the neurotrophins such as brain-derived neurotrophic factor (BDNF), the BDNF receptor ‘TrkB’, and Calcitonin Gene Related Peptide (CGRP) plus nerve growth factor have been shown to increase in severely degenerative discs and are thought to promote neural ingrowth into the IVD. Recently we have developed and demonstrated the anti-degenerative effects of a novel, molecular therapy NTG-101 in a large animal model of DDD. PURPOSE To evaluate the expression of the CGRP receptor (CALCRL), BDNF, the BDNF receptor TrkB and the nerve growth factor receptor TrkA in needle puncture injured chondrodystrophic canines treated with saline or NTG-101. STUDY DESIGN/SETTING In vivo laboratory experimentation involving chondrodystrophic canines injured, injected one month later and then assayed 14 weeks post injection PATIENT SAMPLE No human subjects. Animal Care approved study involving chondrodystrophic canines. OUTCOME MEASURES Immunohistochemistry and HALO immunohistochemically stained quantification methods. METHODS We induced DDD using image-guided needle puncture injury of the IVD nucleus pulposus (NP) in 3-yr old chondrodystrophic canines. One month after needle puncture injury the 3 lumbar IVDs/animal were injected (with image guidance) with 350 µl phosphate buffered saline (PBS) or NTG-101. Intervening IVDs were used as no treatment controls. The animals were humanely euthanized 14-weeks post injection, (18-weeks post injury) and IVDs cleaned of extraneous tissues and excessive bone, fixed in 10% buffered formalin, decalcified using Cal-Ex for 6-8 weeks, embedded in paraffin, sectioned at 5µm, mounted on glass slides, stained with relevant antibodies, counterstained with Haematoxylin and cover slipped. Assays consisted of immunohistochemical staining for relevant proteins followed by quantification using cell counting with HALO automated cell-counting scoring (Indica labs). RESULTS We found that in discs injected with NTG-101 relative to PBS injections, there was significantly reduced expression of: CALCRL (Calcitonin Gene Related Peptide receptor) (P CONCLUSIONS We have previously demonstrated that a single injection of NTG-101 markedly inhibits degeneration and confers improved biomechanical properties in a needle puncture injury model of chondrodystrophic canine DDD. Here we further show that the same intervention under identical conditions significantly inhibits the expression of neurotrophins as compared to PBS injections. These results strongly suggest that not only does NTG-101 inhibit the development of DDD but may also inhibit pain associated with such degenerative disc disease. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.