Evaluating an Interleukin-1β Injection to Induce Degeneration in the Rat Lumbar Spine
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1 Evaluating an Interleukin-1β Injection to Induce Degeneration in the Rat Lumbar Spine Alta Berger George Washington University Amy Orlansky, Dawn M. Elliott McKay Orthopaedic Research Laboratory University of Pennsylvania SUNFEST Symposium August 7th, 2008 McKay Orthopaedic Research Laboratory
2 The Intervertebral Disc Composition: Nucleus Pulposus Gelatinous center, provides osmotic pressure for absorption of force Annulus Fibrosus Highly organized fibers the encircle the NP /IntervertebralDisc.jpg
3 Disc Degeneration Begins in the NP in water content in GAG content Spreads to AF Disorganization Tears Healthy (Adams et al. 2002) Degenerate
4 Previous Studies ChABC used to decrease GAG content Corresponded to loss of mechanical function Side Chain Core Protein Hyaluronic Acid
5 Interleukin-1 Naturally occurring cytokine linked to cartilage degradation Shown to be produced by both degenerate and non-degenerate discs Linked to matrix degrading enzymes, decrease in production of proteoglycans
6 Summer Goal Create in-vivo rat model of intervertebral disc degeneration brought on by IL-1 Hypothesis: IL-1 will cause decrease in GAG content and a loss of mechanical function diac/zodiac-rat-pic.gif
7 Method 3 groups: IL-1, PBS Sham, Control (1 μl injections) 3 discs injected in each rat Euthanized 1 or 4 weeks post injection Motion segments harvested, kept at -20 o C until testing
8 Biochemistry Discs isolated from motion segment Customized punches used to separate NP, IAF, and OAF Spectrophotometer used to determine GAG content based on the color metric scale from DMMB assay
9 Mechanical Testing Axial Compression- Tension cyclic testing: 20 cycles of 4.5 N compression to 3 N Tension 45 minute Creep test Data from final cycle analyzed using trilinear fit model through MATLAB
10 Results - Water Content 90 Water Content in the NP Water Content (%) Week 4 Week 0 IL-1 PBS
11 Results - Water Content 80 Water Content in the AF Water Content (%) Week 4 Week 0 IAF OAF IAF OAF IL-1 PBS
12 Results - GAG content GAG Content in the NP (per Wet Weight) GAG/WW (ug/mg) IL-1 PBS 1 Week 4 Week
13 Results - GAG Content GAG content in the AF of Rat Discs (per Wet Weight) 30 GAG/WW (ug/mg) Week 4 Week 0 IAF OAF IAF OAF IL-1 PBS
14 Results - Mechanics Load Displacement 4 Week Post Injection 6 4 Force (N) IL-1_1 IL-1_2 PBS_1 PBS_ Displacement (mm)
15 Results - Mechanics 0.35 Mechanics Data for Rat Lumbar Disc 4 weeks post injection 0.3 Displacement (mm) NZ Length IL-1 PBS ROM
16 Results - Mechanics Neutral Zone Stiffness for Rat Lumbar Disc 4 Weeks Post Injection Stiffness (N/mm) IL-1 PBS
17 Results - 1 week Histology IL-1 PBS Control
18 Results - 4 week Histology IL-1 PBS Control
19 Results Biochemical results correspond to mechanics Recovery of GAG % difference of GAG content in IL-1 vs. PBS discs per wet weight 1 week: % 4 week: %
20 Increase in GAG Possible that the presence of IL-1 caused an increase in IL-1 RA Further regulation If IL-1 acts as foreign substance, cells may produce antibodies against it to prevent further attack Future studies: observe levels of IL-1 RA in addition to IL-1 in the disc
21 Challenges Difficulties with mechanical testing led to loss of 1 week data Small study (n=2) Discs susceptible to tears during dissection and sectioning
22 Future Work Develop method to provide lasting effect of IL-1: released over time Observe levels of IL-1 RA Monitor activity of IL-1 in the disc Larger study
23 Results: Interleukin-1b GAG Content in the Rat Disc (5 Days Post Surgery) Control (n=3) IL-1b: 200 ng (n=3) GAG/Dry Weight (ug/mg) NP IAF OAF
24 Results: Interleukin-1b Hydroxyproline/Wet Weight (ug/mg) Control (n=3) Hydroyproline Content in Rat Disc (5 Days Post-Surgery) IL-1b: 200 ng (n=3) NP IAF OAF
25 Results: Interleukin-1b Water Content in Rat Discs (5 Days Post-Surgery) Control (n=3) IL-1b: 200 ng (n=3) H2O Content (%) NP IAF OAF
26 Future Work Increase sample size to look at impact of IL-1 on the disc at short, medium, and long time points Establish temporal inter-relationships between mechanics, biochemistry, molecular and cellular events as a results of IL-1beta in the rat disc
27 Thank You McKay Orthopaedic Research Laboratory
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