Degeneration of an IVD results in disc-related disorders, Chemokine profile of disc degeneration with acute or chronic pain. Laboratory investigation

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1 J Neurosurg Spine 18: , 2013 AANS, 2013 Chemokine profile of disc degeneration with acute or chronic pain Laboratory investigation *Malte Schroeder, M.D., 1 Lennart Viezens, M.D., 1 Christian Schaefer, M.D., 2 Barbara Friedrichs, M.D., 2 Petra Algenstaedt, M.D., Ph.D., 3 Wolfgang Rüther, M.D., 4 Lothar Wiesner, M.D., 2 and Nils Hansen-Algenstaedt, M.D., Ph.D. 2 1 Department of Trauma, Hand and Reconstructive Surgery, 2 Department of Orthopaedic Spine Surgery, Spine Center, 3 Department of Internal Medicine, and 4 Department of Orthopaedic Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Object. Disc-related disorders such as herniation and chronic degenerative disc disease (DDD) are often accompanied by acute or chronic pain. Different mediators have been identified in the development of radicular pain and DDD. Previous studies have not analyzed individual cytokine profiles discriminating between acute sciatic and chronic painful conditions, nor have they distinguished between different anatomical locations within the disc. The aim of this study was to elucidate the protein biochemical mechanisms in DDD. Methods. The authors determined expression levels of matrix metalloproteinase 3, transforming growth factor b (TGF-b), tumor necrosis factor a, interleukin-1a, and pro-substance P using enzyme-linked immunosorbent assay and Western blot analyses in patients suffering from DDD (n = 7), acute back pain due to herniated discs with radiculopathy (n = 7), and a control group (n = 7). Disc tissue samples from the anulus fibrosus (AF) and nucleus pulposus (NP) were analyzed. Statistical analysis was performed using nonparametric tests. Results. A distinct distribution of cytokines was found in different anatomical regions of intervertebral discs in patients with DDD and herniated NP. Increased TGF-b levels were predominantly found in DDD. Matrix metalloproteinase 3 was increased in acute herniated disc material. Increased levels of substance P were found in patients suffering from DDD but not in patients with disc herniation. The data showed significantly higher levels of proinflammatory cytokines in the AF and NP of patients with DDD, and the expression levels in the AF were even higher than in the NP, suggesting that the inflammatory response initiates from the AF. Conclusions. These results highlight the complex mechanisms involved during disc degeneration and the need to distinguish between acute and chronic processes as well as different anatomical regions, namely the AF and NP. They also highlight potential problems in disc nucleus replacement therapies because the results suggest a biochemical link between AF and NP cytokine expression. ( Key Words intervertebral disc degeneration chronic back pain lumbar substance P nucleus pulposus anulus fibrosus Degeneration of an IVD results in disc-related disorders, including herniation and chronic DDD. These disorders are often accompanied by acute or chronic pain. 9,40 The most common causes of disc degeneration are compositional changes of the IVD that compromise the biomechanical disc properties, thus impairing its function. 45,49 The understanding of the pathophysiological Abbreviations used in this paper: AF = anulus fibrosus; DDD = degenerative disc disease; ELISA = enzyme-linked immunosorbent assay; IL = interleukin; IVD = intervertebral disc; MMP-3 = matrix metalloproteinase 3; NP = nucleus pulposus; TGF = transforming growth factor; TNF = tumor necrosis factor. * Drs. Schroeder and Viezens contributed equally to this work. development of radicular pain has changed, from the assumption of a pure mechanical compression of the nerve root, to the notion of a more complex mechanism involving both mechanical and biochemical mechanisms. 31,32 Cytokines and neurokinins such as TNF-a, IL-1a, and substance P appear to play an important role in the development of radicular (sciatic) pain and chronic low-back pain. 2,4,21,25,44 Previous studies focused selectively on single cytokines in herniated disc tissue, 2,4,24,42,44 compared only proinflammatory cytokine levels in patients with sciatica or low-back pain, 10 or did not consider individual cytokine profiles of different anatomical regions when discriminating between acute sciatic and chronic painful conditions. In particular, substance P appears to be an interesting mol- 496 J Neurosurg: Spine / Volume 18 / May 2013

2 Chemokines in disc degeneration ecule because its multifunctional properties might help to distinguish between acute and chronic painful conditions. 20,22,29 Substance P is a neuropeptide released from afferent nerve endings and acts as a neurotransmitter. 16 As a member of the tachykinins, substance P may contribute to neurogenic inflammation and sensitization of peripheral nociceptors. 7 Proinflammatory cytokines, such as TNF-a and IL- 1a, stimulate the synthesis of MMP-3, a protease, which degrades the extracellular matrix of IVDs. 18 Induction of MMP-3 can be inhibited by TGF-b, 15 a multifunctional growth factor involved in angiogenesis, fibrogenesis, chondrogenesis, and osteogenesis. 39,47 However, until now the exact expression patterns and activity profiles of MMP-3 and TGF-b were not well understood for acute sciatica and DDD. The aim of this study was to elucidate the protein biochemical mechanisms, expression patterns of proinflammatory cytokines, and signal transducers that are involved in the process of painful degeneration of IVDs. The determination of expression patterns of MMP-3, TGF-b, TNF-a, IL-1a, and pro-substance P (the precursor form of substance P) could help to differentiate between chronic and acute pain. Methods Patients and Specimens Vertebral disc tissue was obtained from patients who underwent anterior lumbar interbody fusion for DDD (Group A) and microsurgical sequestrectomy for herniated discs with radiculopathy (Group B). Patients with scoliosis receiving ventral derotation spondylodesis for deformity correction served as the control group (Group C; Table 1). None of the patients with scoliosis presented with clinical signs of discogenic back pain, such as persistent, nociceptive low-back, groin, and/or leg pain that worsens with axial loading and improves with recumbence. In Group A, the dissection included the posterior anulus while the posterior longitudinal ligament was spared. Written consent was obtained from all patients enrolled in this study, and the procedures were performed under the surveillance of the local ethics committee. Pain levels were quantified using a visual analog scale (0 = no pain, 10 = maximum pain). Group A patients underwent MRI and lumbar discography to evaluate painful discs prior to surgery. Degenerated discs were characterized by decreased T2 signal intensity and decreased disc height as indicators of disc degeneration, 30 and a sensation of pain during discography. 11 None of these patients presented with extruded or sequestrated disc tissue. The specimens in Group B only included sequestrated and extruded material. The disc specimens were separated into AF and NP with the aid of a surgical microscope (maximum magnification 12.5; Zeiss OPMI Vario S88). The material was immediately frozen in liquid nitrogen and stored at 80 C until further analysis. Western Blot Analysis Total protein was extracted from the discs as previously described. 19 Protein samples were solubilized and J Neurosurg: Spine / Volume 18 / May 2013 TABLE 1: Patient details including pain history, age, sex, and spinal segment from which the specimens were obtained* Case No. Group Age (yrs), Sex BMI Spinal Segment Pain History (mos) 1 A 19, M 21.6 L5 S A 37, F 21.9 L A 37, M 25.2 L A 51, M 33.1 L A 44, M 26.0 L4 5, L5 S A 64, M 36.1 L A 60, M 31.1 L3 S B 38, F 19.3 L B 45, F 28.8 L B 34, M 25.7 L5 S B 30, M 27.1 L5 S B 73, F 30.5 L B 29, M 22.7 L5 S B 32, M 22.8 L C 25, F 20.2 thoracolumbar 0 16 C 19, F 19.6 thoracolumbar 0 17 C 15, F 25.2 thoracolumbar 0 18 C 23, F 25.2 thoracolumbar 0 19 C 23, F 18.4 thoracolumbar 0 20 C 22, F 18.2 thoracolumbar 0 21 C 16, F 18.4 thoracolumbar 0 * BMI = body mass index. Group A = DDD, Group B = sequestrectomy, Group C = controls. subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (15% resolving gel). Immunoblots were incubated with a primary antibody against human TNFa; MMP-3; TGF-b 1, 2, and 3; substance P; and IL-1a (Santa Cruz Biotechnology, Inc.), and after washing were treated with a secondary antibody (anti rabbit immunoglobulin G for TGF-b or anti goat immunoglobulin G for TNF-a, MMP-3, and substance P; Amersham). Chemiluminescence was detected by exposure to enhanced chemiluminescence reagent (Amersham) followed by autoradiography. Enzyme-Linked Immunosorbent Assay Samples were prepared as lysates. The immunoassays for substance P (R&D Systems), TNF-a (Bender MedSystem), and IL-1a (R&D Systems) were used according to the manufacturer s instructions, and the absorbance of the resulting yellow product was measured at 450 nm with a microplate reader. Samples and standards were tested in duplicate. Data Analysis The intensity of the protein bands was quantified by scanning densitometry using imaging software (NIH Image, version 1.63). Values were normalized to a control band on each blot. The results are presented as means ± SEM. Statistical analysis was performed using the non- 497

3 M. Schroeder et al. parametric Mann-Whitney U-test to compare parameters between the groups and the Wilcoxon signed-rank test to compare samples within 1 group (SPSS version 16, IBM Corp.). Data were considered statistically significant for p values less than 5%. Results Substance P Western blot analysis of Group B (sequestrectomy) revealed a 0.7-fold decreased expression of pro-substance P compared with the control group (Fig. 1). More importantly, for active substance P, ELISA of NPs from Group B patients showed a 0.6-fold decrease in protein compared with NPs of control subjects ( ± 18 pg/mg vs ± 32 pg/mg protein, respectively; p = ). Compared with controls, the relative expression of pro-substance P in the AF of chronic degenerated discs was increased 1.4-fold (p = 0.024). Similar to results in the sequestrectomy group, we found a 0.7-fold decreased expression of pro-substance P in the NP (p = 0.03). Divergent from the Western blot analysis, we could not determine a significant increase of active substance P in the AF by ELISA in Group A compared with controls ( ± 25 pg/mg vs ± 33 pg/mg protein, respectively, but did find a significant 1.2-fold increase to 470 ± 20 pg/mg protein in the NP of DDD samples (p = 0.031). The NP of the DDD samples contained 2.1-fold more active substance P than NP from the sequestrectomy group (p = ; Fig. 1). Tumor Necrosis Factor α Compared with controls, relative TNF-a expression increased significantly by 2.5-fold (p = 0.008) in the AF and decreased 0.9-fold in the NP of patients with DDD (Fig. 2). Expression of the TNF-a precursor showed a significant increase in both AF (2.5-fold; p = ) and NP (2.2-fold; p = 0.023) of DDD samples. Using ELISA, we found a 1.4-fold increase in TNF-a in both AF (55.47 ± 7 pg/mg protein) and NP (46.72 ± 8 pg/mg protein) from DDD samples compared with controls (AF ± 6 pg/ mg protein, NP ± 6 pg/mg protein. The ELISA analysis of active TNF-a in the sequestrectomy group (28.91 ± 4 pg/mg protein) revealed a slight, although not significant, reduction compared with controls (p = 0.6). Western blot analyses of the sequestrated tissue showed almost identical decreases in the expression levels for active TNF-a. Conversely, the pro form of TNF-a increased significantly (p = 0.041) in the sequestrectomy group. The expression of TNF-a in the sequestrectomy group was similar to the expression of NP in the DDD group (Fig. 2). Interleukin-1α Protein content of IL-1a was poor for all tissues examined. We found a tendency toward increased expression levels of IL-1a in the AF of patients with DDD. The ELISA analysis showed increased levels of IL-1a in the AF (5.0 ± 0.7 pg/mg protein) and in the NP (5.1 ± 1.9 pg/ mg protein) of DDD samples. No significant difference was found in Group B (2.4 ± 0.3 pg/mg protein) compared with controls (AF 2.6 ± 0.8 pg/mg protein, NP 2.4 ± 0.5 pg/mg protein). Matrix Metalloproteinase 3 Expression of MMP-3 was elevated in both DDD samples and samples of sequestrated tissue compared Fig. 1. Graph of the relative protein expression of pro-substance P (prosp) compared with the controls and corresponding Western blot images (A) and graph of ELISA measurements of active substance P (pg/mg; B). Compared with controls we found a significant increase of pro-substance P in the probe of the DDD and a significant decrease of substance P in the probe of the sequestrated tissue. Interestingly, we observed an opposed expression of pro-substance P and the active form of substance P in the AF and NP in cases of DDD. Values are presented as mean ± SEM. * Significant (p < 0.05) compared with controls. Significant compared with the corresponding probes of DDD and the sequestrated tissue. 498 J Neurosurg: Spine / Volume 18 / May 2013

4 Chemokines in disc degeneration Fig. 2. Graph of the relative protein expression and corresponding Western blot images of TNF-a and pro-tnf-a compared with controls (A), and graph of ELISA measurements of TNF-a (B). We found a significant increase of pro-tnf-a in the probes of DDD and the sequestrated tissue compared with controls, and a significant increase of TNF-a only in the probes of AF from patients with DDD. The ratios of TNF-a and pro-tnf-a in DDD and in the sequestrated tissue also showed a significant difference. Enzyme-linked immunosorbent assay measurements of TNF-a showed increased levels in both AF and NP in patients with DDD. Values are presented as mean ± SEM. * p < 0.05, ** p < 0.01, significant compared with controls. Significant compared with corresponding probes of TNF-a and pro-tnf-a. pc = positive control. with controls (Fig. 3). The expression levels of both the active and pro forms of MMP-3 were increased in the sequestrum (Group B) and degenerated NP (Group A), yet sequestrated disc tissue showed the highest expression levels of the pro form of MMP-3 with a significant (p = 0.025) 3-fold increase compared with controls. Transforming Growth Factor β We found a significant 2.2-fold increase in TGF-b in degenerated AF (Group A) compared with controls (p = 0.028). Conversely, compared with control samples, the expression of TGF-b was significantly decreased in the NP of DDD samples (0.8-fold) and sequestra (0.6-fold; p = for both; Fig. 4). Pain History Patients with chronic DDD suffered from low-back pain for several months (range months) with an increase of the pain level in the last months (range 4 48 months). The mean visual analog scale value for back pain was 7 (range 0 10); it was 6 for leg pain (range 2 10). The patients with sequestrated discs suffered from low-back pain for a shorter period of time (range months) with an acute increase of pain and development of radiculopathy a few days (range 1 21 days) before they underwent surgery (Table 1). A positive correlation between the TNF-a expression in AF and NP and the length and severity of pain could be established. J Neurosurg: Spine / Volume 18 / May 2013 Discussion The pathogenic mechanisms of disc degeneration with acute or chronic pain are a matter of ongoing debate. Mechanical and biochemical mechanisms contribute to the development of chronic low-back pain. 25 Cytokines such as substance P, TNF-a, and IL-1a are involved in transmitting pain 4,13 and have been identified in disc tissue. 16,42 Furthermore, MMP-3 as a relevant protease and TGF-b as a growth factor have been identified as contributors to disc degeneration. 2,18,33 The differences in the profile of these chemokines between NP and AF and between chronic versus acute states of pain are not well understood. Our data show significantly higher levels of proinflammatory cytokines in the AF and NP of patients with DDD, with the expression levels in the AF even higher than those in the NP, suggesting that the inflammatory response initiates from the AF. The expression levels of MMP-3 and TGF-b were also higher in chronic degenerated discs. However, the expression of 57 kd MMP-3 in sequestrated disc tissue was greater than its expression in both the AF and NP of chronic degenerated discs. In contrast, TGF-b was elevated only in the AF of chronic degenerated discs, suggesting that it functions not so much as an acute phase protein but as a key player in chronic DDD. Substance P and Proinflammatory Cytokines in Chronic Disc Disease The increased levels found in chronic degenerated 499

5 M. Schroeder et al. Fig. 3. Graph of the relative protein expression of active MMP-3 (28 kd) and pro-mmp-3 (57 kd) compared with controls (left), and the corresponding Western blot images (right). We found a significant increase of active MMP-3 (28 kd) and pro-mmp-3 (57 kd) in the probes of DDD and the sequestrated tissue compared with controls. The ratios of active MMP-3 and pro-mmp-3 in DDD NP also showed a significant difference. Values are presented as mean ± SEM. * p < 0.05, significant compared with controls. Significant compared with corresponding probes of active MMP-3 (28 kd) and pro-mmp-3 (57 kd). discs corroborate the assumption that substance P plays an important role in painful chronic disc disease. 1,5,13 Others reported increased levels of substance P in dorsal root ganglia and the thalamus in a rat model of DDD, 23 which suggests a direct association between peripheral inflammation (in the IVD) and central sensitization. The increased expression of pro-substance P in the AF of patients with DDD might therefore result from an increased release from afferent nerve fibers in the AF. 20 The observation of an increased content of the cleaved active substance P in the NP indicates an ingrowth of new nerve endings containing substance P in degenerated discs of patients with DDD. 16 An explanation for the missing increase of active substance P in the chronic degenerative AF could be the rapid cleavage of active substance P into the more important biological active fragments (substance P 7 11). 5 Although previous studies described few nerve fiber endings at the outer surface of prolapsed discs, 38 we found reduced levels of substance P in sequestrated tissue. We interpret this as the result of several factors: the acute occurrence, the fact that the prolapsed disc tissue includes AF, and the fact that detachment of the sequestrated disc increased the diffusion capability tremendously. Tumor Necrosis Factor α and Chronic Back Pain Previous animal studies drew attention to the role of proinflammatory cytokines (such as TNF-a) in nerve root dysfunction and pain after epidural application of NP tissue. 4 Previous in vitro studies detected several proin- Fig. 4. Graph of the relative protein expression of TGF-b compared with the controls (left) and corresponding Western blot images (right). We found significant differences of TGF-b in the probes of DDD (increase) and the sequestrated tissue (decrease) compared with controls. Interestingly, there were opposing levels of TGF-b and the active MMP-3 in AF and NP in cases of chronic disease. Values are presented as mean ± SEM. * p < 0.05, significant compared with controls. Significant compared with corresponding probes of active MMP-3 (12.5 kd). 500 J Neurosurg: Spine / Volume 18 / May 2013

6 Chemokines in disc degeneration flammatory cytokines (TNF-a, IL-6, IL-8, and IL-1b) in degenerated disc tissue. 10 For the first time, we present in vivo data of increased TNF-a levels in the AF and NP in chronic degenerated discs and in sequestrated disc tissue, which confirm the data from an in vitro study of Burke et al. 10 and highlight the principal role of TNF-a in the development of neuropathic pain. 21 Furthermore, a link between high-intensity zones on MRI (representing a local inflammation process) and TNF-a positive cell proliferation has recently been described. 14 By analysis of sequestrated disc tissue and chronic degenerated discs in which the AF and NP were separated, we found distinct differences in the distribution pattern of TNF-a in the two regions, which further corroborates the previous findings by other groups. The higher expression of TNFa in chronic degenerated discs compared with sequestered disc tissue emphasizes the possible role of therapies targeting TNF-a expression in the treatment of chronic back pain. 26 The increased vascularization of the AF in chronic degenerated discs 27 might explain the higher expression of TNF-a, because the blood vessels are necessary for the supply of activated phagocytes that release proinflammatory mediators. Although the precursor of TNF-a is predominantly expressed in the AF, the activated form of TNF-a can reach the NP by diffusion. 21 Additionally, the higher content of active substance P, which we observed in tissues from chronic degenerated discs, could explain the higher content of TNF-a and IL-1a, because substance P can enhance the release of these cytokines from macrophages. 6 Recent research has shown that NP tissue may induce structural and functional changes in the adjacent nerve roots when placed epidurally and can cause a painful neuropathy if applied externally to an intact nerve bundle. 37 In our study, the levels of TNF-a and IL-1a in sequestra (Group B) were not increased when compared with disc tissue in controls. However, our patients with herniated discs suffered from severe radiculopathy. The data presented here do not confirm the thesis that the mere presence of NP and proinflammatory cytokines (TNF-a and IL-1a) induces severe pain. Because the level of the proinflammatory cytokines is not increased in the sequestra, this assumption does not appear favorable. A combination of innervation of the chronic degenerative disc and increased production of proinflammatory mediators suggests that the mechanism for discogenic low-back pain may be the induction of hyperalgesia in the newly innervated IVD. An antiinflammatory therapy would not only reduce the inflammatory and degenerative process in the IVDs, but would also abate neuropathic pain, including nerve root injury and radicular pain. 4 Several previous studies showed promising in vitro and in vivo results using different therapeutic agents (betamethasone, methylprednisolone, cyclosporin A, indomethacin, and lidocaine). 35,36,44 Therapeutic Modulation of MMP Function and Expression The control of MMP synthesis and activity is an interesting target for the treatment of DDDs. Suppression of MMP synthesis by suppressing transcription can be J Neurosurg: Spine / Volume 18 / May 2013 achieved by naturally expressed inhibitors of synthesis (TGF-b and all-transretinoic acid) and synthetic compounds (synthetic derivates of retinoic acid and glucocorticoid hormones). 18 Highly specific and biologically active MMP inhibitors have been engineered; 17 these potent inhibitors are effective in vitro in preventing the destruction of cartilage in animal models. 18 Because multiple MMPs are present in IVDs and the enzymes that are most critical for the destruction of disc tissue have not yet been identified, direct inhibition of the damaging MMPs can be challenging. Thompson et al. 46 suggested that TGF-b might be used in the management of disc degeneration. However, locally injected growth factors have a relatively short biological half-life, whereas chronic types of disease, such as disc degeneration, would require a sustained delivery of exogenous growth factors to the disc. 33 Surgical Treatment Options for DDD Although surgical interbody fusion, using a variety of approaches and techniques, is a recognized treatment option for DDD, its long-term success is controversial. 43 Because this surgery solely addresses the symptoms of DDD without treating the underlying pathology and fusion might lead to alterations of the adjacent segment, motion-preserving procedures have been introduced. With regard to disc arthroplasty, there have been no general recommendations up to now; this is mainly due to the lack of evidence of long-term effectiveness and safety of the current therapies. 48 Early results after posterior dynamic stabilization indicated significant reduction in pain levels, while longer-term studies have demonstrated a considerable amount of adjacent-segment disease. 34,41 Treatment based on the local application of growth factors and cell-based therapies have shown some early results indicating a reversal of the degenerative cascade, but their clinical effects and long-term results are uncertain. 3 Intervertebral disc regeneration offers promising perspectives for patients treated for degeneration or herniation of lumbar IVDs. Thus far, studies have mainly been directed toward replacement or regeneration of the nucleus. 28 Only recently have alternative treatment options for the AF been described, and combination therapies with disc nucleus replacements have been suggested as possible long-term goals. 8 The data presented in this study strengthen the hypothesis that degenerative processes in the IVD are multifactorial. Local differences in biochemical profiles exist between the AF and NP and these differences have to be considered in the development of specific targeted therapies. Limitations of the Study While we carefully dissected the AF and NP using the maximum magnification of the operating microscope, no histological studies were performed to further verify the separation of both tissue types. Due to the small sample size, no exact matching of patients was possible and a certain overlap in the duration of symptoms in patients with DDD and herniated NP was present. Possible confounders of the study are differences in patient age, duration of symptoms, and spinal levels affected. These 501

7 M. Schroeder et al. differences in the patient groups could possibly influence the production of the mediators studied and are not controlled for. The discography performed prior to anterior lumbar interbody fusion could by itself have led to degenerative processes in IVDs, as described previously by Carragee et al. 12 However, in humans, the observed accelerated progression of disc degeneration after discography was only described after a latent period of several years. To subject those patients who did not present with clinical signs of disc degeneration to discography is ethically questionable. Therefore, this variable cannot be controlled for. Because we focused on chemokine profiles in painful intervertebral discs and did not take preoperative functional parameters into account, no functional followup data were documented. However, none of the operated patients underwent revision surgery in the first 12 months. Conclusions We found a distinct distribution of cytokines in different anatomical regions of IVDs in patients with DDD and herniated NP. We confirmed that alterations of the relative expression of TGF-b and MMP-3 play a major role in the development of IVD diseases as shown in previous studies. The lack of inflammatory cytokines and substance P in the disc tissue of patients with acute disc herniation suggests that proteins (such as TNF-a, IL-1a, and substance P) are not responsible for the development of pain in these patients. Thus, an antiinflammatory therapy or inhibition of substance P and the ingrowth of nerves does not appear to be an appropriate treatment for avoiding pain in these patients. In contrast, patients suffering from painful chronic DDD showed high levels of inflammatory cytokines and substance P in both the AF and NF region. Furthermore, our results suggest that there are interactions between the anabolic and catabolic enzymes in IVDs. In the development of targeted therapies directed at disc-related disorders, these diseasespecific differences and the individual profile of distinct anatomical regions should be taken into account. Local differences in biochemical profiles between the AF and NP should be taken into account in the development of novel disc repair therapies. Disclosure Dr. Hansen-Algenstaedt serves as a consultant for Stryker, Globus Medical, and Synthes. Author contributions to the study and manuscript preparation include the following. Conception and design: Hansen-Algenstaedt. Acquisition of data: Friedrichs, Wiesner, Hansen-Algenstaedt. Analysis and interpretation of data: Schroeder, Viezens, Schaefer. Drafting the article: Schroeder, Viezens. Critically revising the article: all authors. Reviewed submitted version of manuscript: all authors. Approved the final version of the manuscript on behalf of all authors: Schroeder. Statistical analysis: Viezens. Administrative/ technical/material support: Algenstaedt. Study supervision: Rüther, Hansen-Algenstaedt. Acknowledgments The authors would like to thank Chieko Koike, Ph.D., (Department of Developmental Biology, Osaka Bioscience Institute, Osaka, Japan) for her valuable contribution and insights into the applied biochemical techniques, and Kristin Westphalen, M.D., (Department of Medicine, Columbia University Medical Center, New York, New York) for comments on the manuscript. References 1. Afrah AW, Fiskå A, Gjerstad J, Gustafsson H, Tjølsen A, Olgart L, et al: Spinal substance P release in vivo during the induction of long-term potentiation in dorsal horn neurons. Pain 96:49 55, Ahn SH, Cho YW, Ahn MW, Jang SH, Sohn YK, Kim HS: mrna expression of cytokines and chemokines in herniated lumbar intervertebral discs. Spine (Phila Pa 1976) 27: , An HS, Masuda K: Relevance of in vitro and in vivo models for intervertebral disc degeneration. 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Eur Spine J 10: , van den Eerenbeemt KD, Ostelo RW, van Royen BJ, Peul WC, van Tulder MW: Total disc replacement surgery for symptomatic degenerative lumbar disc disease: a systematic review of the literature. Eur Spine J 19: , Vernon-Roberts B, Pirie CJ: Degenerative changes in the intervertebral discs of the lumbar spine and their sequelae. Rheumatol Rehabil 16:13 21, 1977 Manuscript submitted May 17, Accepted January 15, Please include this information when citing this paper: published online March 8, 2013; DOI: / SPINE Address correspondence to: Malte Schroeder, M.D., Department of Trauma, Hand and Reconstructive Surgery, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Hamburg 20246, Germany. m.schroeder@uke.de. J Neurosurg: Spine / Volume 18 / May