- Email: [email protected]
Thursday, October 31, 2001 4:18–4:44 pm Focused Review: Disc Metabolism
Proceedings of the NASS 17th Annual Meeting / The Spine Journal 2 (2002) 47S–128S
66S
Overall significance of findings: Pathological compression by the ossified ligament above a certain critical point may be the most significant factor in inducing myelopathy, whereas below that point dynamic factors may be largely involved in inducing myelopathy. Disclosures: No disclosures. Conflict of interest: Shunji Matsunaga, grant research support, Ministry of Health and Welfare, Japan. PII: S1529-9430(02)00307-8
Thursday, October 31, 2001 4:18–4:44 pm Focused Review: Disc Metabolism 4:18 Epidural glutamate from herniated disc material as a nociceptive stimulus in lumbar radiculopathy Fred Harrington, MD1, Arthur Meffier2, Elizabeth Wy3; 1Brown University, Providence, RI, USA; 2Rhode Island Hospital, Providence, RI, USA; 3 Brown University School of Medicine, Providence, RI, USA Purpose of study: To determine whether levels of free glutamate previously measured from human herniated disc specimens could lead to physiological or behavioral symptoms consistent with a hyperesthetic state in glutamate-infused rats. Methods used: Sprague-Dawley rats were unilaterally infused with epidural glutamate at the L5–L6 levels for 72 hours by a subcutaneously implanted miniosmotic pump. Because herniated disc material shows an average concentration of 0.5 mM, we infused with concentrations of 0.002 to 2.0 mM. Animals were studied behaviorly by 49 C hot plate withdrawal tests, Von Frey fiber withdrawal tests for left-right hindpaw reactivities and 53 C tail flick assays. These were performed 24 hours before and 24, 72 and 144 hours after onset of glutamate infusion. Other animals were terminated 72 hours after onset of infusion and were evaluated immunohistochemically for NMDA, AMPA and kainate receptor expression in dorsal horn regions by quantitative means. Summary of findings: Hot plate withdrawal showed significance at 72 hours and concentrations of 0.002, 0.02 and 0.2 mM. Tail flick tests showed similar differences in latencies compared with saline controls (p.02; two-tailed t test). Von Frey fiber tests showed decreased latencies on the ipsilateral side to the infusion in glutamate-infused animals compared with saline controls (p.05; two-tailed t test). Immunohistochemical evaluation showed increasing levels of glutamate expression over salineinfused controls ipsilateral to the side of glutamate infusion at 2.0 mM concentration. Results at lower infusion levels are pending (will be available at North American Spine Society meeting in October 2002). Relationship between findings and existing knowledge: These findings suggest that the concentrations of glutamate found in human disc material in the epidural space are large enough to have biologic effects on nociceptive responses. Overall significance of findings: Lumbar radiculopathy resulting from disc herniation may be amenable to treatment with glutamate antagonists. Cartilage may promote pain whenever degraded cartilage is in the vicinity of glutamate receptors in other joints as well. Disclosures: No disclosures. Conflict of interest: No conflict.
Reference [1] Spine 25:925–36. PII: S129-9430(02)00308-X
4:21 Gene delivery of the catabolic inhibitor tissue inhibitor of metalloproteinase–1 increases proteoglycan synthesis in human intervertebral disc cells Corey J. Wallach, MD1, Yasu Watanabe, MD1, Lars G. Gilbertson, PhD1, James D. Kang, MD1; 1University of Pittsburgh, Pittsburgh, PA, USA Purpose of study: Intervertebral disc degeneration is characterized by a progressive loss of proteoglycan content and subsequent disc dehydration. Previous studies demonstrate increased matrix metalloproteinases in degenerated discs compared with controls and propose an intimate role of these catabolic proteins in the degenerative process. Gene therapy has been investigated as a method to induce endogenous synthesis of therapeutic growth factors to stimulate proteoglycan synthesis. Although many anabolic growth factors increase matrix synthesis, the use of catabolic inhibitors has yet to be explored. The objective of this study was to evaluate the effect of adenoviral-mediated delivery of the catabolic inhibitor tissue inhibitor of metalloproteinase–1 (TIMP–1), as well as the anabolic potential of bone morphogenic protein–2 (BMP–2), on proteoglycan synthesis. Methods used: Human nucleus pulposus cells were isolated by means of enzymatic digestion and cultured in monolayer. Patient samples were pooled and experimental groups normalized for cell number before transduction with either Ad-TIMP-1 or Ad-BMP-2 at 50, 75, 100 or 150 multiplicity of infection (MOI). After 48 hours of incubation, cells were incorporated into a threedimensional “pellet” culture system. After an additional 48 hours of incubation, active proteoglycan synthesis was assessed with 35S radioactive sulfate incorporation using chromatography and scintillation count. Summary of findings: Both Ad-TIMP-1 and Ad-BMP-2 increase proteoglycan synthesis compared with controls (p.05). Cultures treated with Ad-BMP2 demonstrated a progressive increase in synthesis with increasing viral MOI (190%, 212%, 310% and 466%). Cells treated with Ad-TIMP-1 exhibited a maximum response at a MOI of 100 (65%, 289%, 378% and 249%). Relationship between findings and existing knowledge: Although anabolic factors have previously been shown to increase proteoglycan synthesis in cultured disc cells, this is the first report demonstrating a similar upregulating capacity with the use of catabolic inhibitors. Overall significance of findings: This study demonstrates that both catabolic inhibitors (TIMP–1) and anabolic growth factors (BMP-2) are successful at increasing measured proteoglycan synthesis in cultured human intervertebral disc cells. The optimal regulation of matrix synthesis may be achievable with gene therapy using a combination of both anabolic factors and catabolic inhibitors. Further studies are warranted to assess the potential of combination therapy with these growth factors to upregulate proteoglycan synthesis, as well as to determine if such combinations can minimize the total vector load required to elicit a beneficial, and potentially therapeutic, response. Disclosures: No disclosures. Conflict of interest: No conflict. Table 1 Stimulatory effect on proteoglycan synthesis TIMP–1 BMP–2
50 MOI
75 MOI
100 MOI
150 MOI
65% 190%
289% 212%
378% 310%
249% 466%
PII: 1529-9430(02)00309-1
4:24 Human annulus cell response to vibratory loading and modulation of the effect by interleukin-1B Anthony Russo, MD1, Al Banes, PhD1, Michelle Elfervig1, Mari Tsuzaki, PhD1, Saturo Yamazaki1, Joe Minchew, MD1; 1University of North Carolina, Chapel Hill, NC, USA
66S
Overall significance of findings: Pathological compression by the ossified ligament above a certain critical point may be the most significant factor in inducing myelopathy, whereas below that point dynamic factors may be largely involved in inducing myelopathy. Disclosures: No disclosures. Conflict of interest: Shunji Matsunaga, grant research support, Ministry of Health and Welfare, Japan. PII: S1529-9430(02)00307-8
Thursday, October 31, 2001 4:18–4:44 pm Focused Review: Disc Metabolism 4:18 Epidural glutamate from herniated disc material as a nociceptive stimulus in lumbar radiculopathy Fred Harrington, MD1, Arthur Meffier2, Elizabeth Wy3; 1Brown University, Providence, RI, USA; 2Rhode Island Hospital, Providence, RI, USA; 3 Brown University School of Medicine, Providence, RI, USA Purpose of study: To determine whether levels of free glutamate previously measured from human herniated disc specimens could lead to physiological or behavioral symptoms consistent with a hyperesthetic state in glutamate-infused rats. Methods used: Sprague-Dawley rats were unilaterally infused with epidural glutamate at the L5–L6 levels for 72 hours by a subcutaneously implanted miniosmotic pump. Because herniated disc material shows an average concentration of 0.5 mM, we infused with concentrations of 0.002 to 2.0 mM. Animals were studied behaviorly by 49 C hot plate withdrawal tests, Von Frey fiber withdrawal tests for left-right hindpaw reactivities and 53 C tail flick assays. These were performed 24 hours before and 24, 72 and 144 hours after onset of glutamate infusion. Other animals were terminated 72 hours after onset of infusion and were evaluated immunohistochemically for NMDA, AMPA and kainate receptor expression in dorsal horn regions by quantitative means. Summary of findings: Hot plate withdrawal showed significance at 72 hours and concentrations of 0.002, 0.02 and 0.2 mM. Tail flick tests showed similar differences in latencies compared with saline controls (p.02; two-tailed t test). Von Frey fiber tests showed decreased latencies on the ipsilateral side to the infusion in glutamate-infused animals compared with saline controls (p.05; two-tailed t test). Immunohistochemical evaluation showed increasing levels of glutamate expression over salineinfused controls ipsilateral to the side of glutamate infusion at 2.0 mM concentration. Results at lower infusion levels are pending (will be available at North American Spine Society meeting in October 2002). Relationship between findings and existing knowledge: These findings suggest that the concentrations of glutamate found in human disc material in the epidural space are large enough to have biologic effects on nociceptive responses. Overall significance of findings: Lumbar radiculopathy resulting from disc herniation may be amenable to treatment with glutamate antagonists. Cartilage may promote pain whenever degraded cartilage is in the vicinity of glutamate receptors in other joints as well. Disclosures: No disclosures. Conflict of interest: No conflict.
Reference [1] Spine 25:925–36. PII: S129-9430(02)00308-X
4:21 Gene delivery of the catabolic inhibitor tissue inhibitor of metalloproteinase–1 increases proteoglycan synthesis in human intervertebral disc cells Corey J. Wallach, MD1, Yasu Watanabe, MD1, Lars G. Gilbertson, PhD1, James D. Kang, MD1; 1University of Pittsburgh, Pittsburgh, PA, USA Purpose of study: Intervertebral disc degeneration is characterized by a progressive loss of proteoglycan content and subsequent disc dehydration. Previous studies demonstrate increased matrix metalloproteinases in degenerated discs compared with controls and propose an intimate role of these catabolic proteins in the degenerative process. Gene therapy has been investigated as a method to induce endogenous synthesis of therapeutic growth factors to stimulate proteoglycan synthesis. Although many anabolic growth factors increase matrix synthesis, the use of catabolic inhibitors has yet to be explored. The objective of this study was to evaluate the effect of adenoviral-mediated delivery of the catabolic inhibitor tissue inhibitor of metalloproteinase–1 (TIMP–1), as well as the anabolic potential of bone morphogenic protein–2 (BMP–2), on proteoglycan synthesis. Methods used: Human nucleus pulposus cells were isolated by means of enzymatic digestion and cultured in monolayer. Patient samples were pooled and experimental groups normalized for cell number before transduction with either Ad-TIMP-1 or Ad-BMP-2 at 50, 75, 100 or 150 multiplicity of infection (MOI). After 48 hours of incubation, cells were incorporated into a threedimensional “pellet” culture system. After an additional 48 hours of incubation, active proteoglycan synthesis was assessed with 35S radioactive sulfate incorporation using chromatography and scintillation count. Summary of findings: Both Ad-TIMP-1 and Ad-BMP-2 increase proteoglycan synthesis compared with controls (p.05). Cultures treated with Ad-BMP2 demonstrated a progressive increase in synthesis with increasing viral MOI (190%, 212%, 310% and 466%). Cells treated with Ad-TIMP-1 exhibited a maximum response at a MOI of 100 (65%, 289%, 378% and 249%). Relationship between findings and existing knowledge: Although anabolic factors have previously been shown to increase proteoglycan synthesis in cultured disc cells, this is the first report demonstrating a similar upregulating capacity with the use of catabolic inhibitors. Overall significance of findings: This study demonstrates that both catabolic inhibitors (TIMP–1) and anabolic growth factors (BMP-2) are successful at increasing measured proteoglycan synthesis in cultured human intervertebral disc cells. The optimal regulation of matrix synthesis may be achievable with gene therapy using a combination of both anabolic factors and catabolic inhibitors. Further studies are warranted to assess the potential of combination therapy with these growth factors to upregulate proteoglycan synthesis, as well as to determine if such combinations can minimize the total vector load required to elicit a beneficial, and potentially therapeutic, response. Disclosures: No disclosures. Conflict of interest: No conflict. Table 1 Stimulatory effect on proteoglycan synthesis TIMP–1 BMP–2
50 MOI
75 MOI
100 MOI
150 MOI
65% 190%
289% 212%
378% 310%
249% 466%
PII: 1529-9430(02)00309-1
4:24 Human annulus cell response to vibratory loading and modulation of the effect by interleukin-1B Anthony Russo, MD1, Al Banes, PhD1, Michelle Elfervig1, Mari Tsuzaki, PhD1, Saturo Yamazaki1, Joe Minchew, MD1; 1University of North Carolina, Chapel Hill, NC, USA