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Degeneration of the intervertebral disc (IVD) is associated with significant biochemical and morphological changes that include a loss of disc height, decreased water content and decreased cellularity. Cell delivery has been widely explored as a strategy to supplement the nucleus pulposus (NP) region of the degenerated IVD in both pre-clinical and clinical trials, using progenitor or primary cell sources. We previously demonstrated an ability for a polymer-peptide hydrogel, serving as a culture substrate, to promote adult NP cells to undergo a shift from a degenerative fibroblast-like state to a juvenile-like NP phenotype. In the current study, we evaluate the ability for this peptide-functionalized hydrogel to serve as a bioactive system for cell delivery, retention and preservation of a biosynthetic phenotype for primary IVD cells delivered to the rat caudal disc in an anular puncture degeneration model. Our data suggest that encapsulation of adult degenerative human NP cells in a stiff formulation of the hydrogel functionalized with laminin-mimetic peptides IKVAV and AG73 can promote cell viability and increased biosynthetic activity for this population in 3D culture in vitro. Delivery of the peptide-functionalized biomaterial with primary rat cells to the degenerated IVD supported NP cell retention and NP-specific protein expression in vivo, and promoted improved disc height index (DHI) values and endplate organization compared to untreated degenerated controls. The results of this study suggest the physical cues of this peptide-functionalized hydrogel can serve as a supportive carrier for cell delivery to the IVD. STATEMENT OF SIGNIFICANCE: Cell delivery into the degenerative intervertebral disc has been widely explored as a strategy to supplement the nucleus pulposus. The current work seeks to employ a biomaterial functionalized with laminin-mimetic peptides as a cell delivery scaffold in order to improve cell retention rates within the intradiscal space, while providing the delivered cells with biomimetic cues in order to promote phenotypic expression and increase biosynthetic activity. The use of the in situ crosslinkable material integrated with the native IVD, presenting a system with adequate physical properties to support a degenerative disc.

The nucleus pulposus (NP) of the intervertebral disc plays a critical role in distributing mechanical loads to the axial skeleton. Alterations in NP cells and, consequently, NP matrix are some of the earliest changes in the development of disc degeneration. Previous studies demonstrated a role for laminin-presenting biomaterials in promoting a healthy phenotype for human NP cells from degenerated tissue. Here we investigate the use of laminin-mimetic peptides presented individually or in combination on a poly(ethylene) glycol hydrogel as a platform to modulate the behaviors of degenerative human NP cells. Data confirm that NP cells attach to select laminin-mimetic peptides that results in cell signaling downstream of integrin and syndecan binding. Furthermore, the peptide-functionalized hydrogels demonstrate an ability to promote cell behaviors that mimic that of full-length laminins. These results identify a set of peptides that can be used to regulate NP cell behaviors toward a regenerative engineering strategy.

Cells of the adult nucleus pulposus (NP) are critically important in maintaining overall disc health and function. NP cells reside in a soft, gelatinous matrix that dehydrates and becomes increasingly fibrotic with age. Such changes result in physical cues of matrix stiffness that may be potent regulators of NP cell phenotype and may contribute to a transition toward a senescent and fibroblastic NP cell with a limited capacity for repair. Here, we investigate the mechanosignaling cues generated from changes in matrix stiffness in directing NP cell phenotype and identify mechanisms that can potentially preserve a biosynthetically active, juvenile NP cell phenotype. Using a laminin-functionalized polyethylene glycol hydrogel, we show that when NP cells form rounded, multicell clusters, they are able to maintain cytosolic localization of myocardin-related transcription factor (MRTF)-A, a coactivator of serum-response factor (SRF), known to promote fibroblast-like behaviors in many cells. Upon preservation of a rounded shape, human NP cells similarly showed cytosolic retention of transcriptional coactivator Yes-associated protein (YAP) and its paralogue PDZ-binding motif (TAZ) with associated decline in activation of its transcription factor TEA domain family member–binding domain (TEAD). When changes in cell shape occur, leading to a more spread, fibrotic morphology associated with stronger F-actin alignment, SRF and TEAD are up-regulated. However, targeted deletion of either cofactor was not sufficient to overcome shape-mediated changes observed in transcriptional activation of SRF or TEAD. Findings show that substrate stiffness-induced promotion of F-actin alignment occurs concomitantly with a flattened, spread morphology, decreased NP marker expression, and reduced biosynthetic activity. This work indicates cell shape is a stronger indicator of SRF and TEAD mechanosignaling pathways than coactivators MRTF-A and YAP/TAZ, respectively, and may play a role in the degeneration-associated loss of NP cellularity and phenotype.—Fearing, B. V., Jing, L., Barcellona, M. N., Witte, S. E., Buchowski, J. M., Zebala, L. P., Kelly, M. P., Luhmann, S., Gupta, M. C., Pathak, A., Setton, L. A. Mechanosensitive transcriptional coactivators MRTF-A and YAP/TAZ regulate nucleus pulposus cell phenotype through cell shape.

Degeneration and aging of the nucleus pulposus (NP) of the intervertebral disc (IVD) is accompanied by alterations in NP cell phenotype marked by a shift towards a fibroblast-like, catabolic state. We have recently demonstrated an ability to manipulate the phenotype of human adult degenerative NP cells through 2D culture upon poly(ethylene glycol) (PEG) based hydrogels dually functionalized with integrin- and syndecan-binding laminin-mimetic peptides (LMPs). In the present study, we sought to understand the transcriptomic changes elicited through NP cell interactions with the LMP-functionalized hydrogel system (LMP gel) by examining targets of interesta prioriand by conducting unbiased analysis to identify novel mechanosensitive targets. The results of gene specific analysis demonstrated that the LMP gel promoted adult degenerative NP cells to upregulate 148 genes including several NP markers (e.g. NOG and ITGA6) and downregulate 277 genes, namely several known fibroblastic markers. Additionally, 13 genes associated with G protein-coupled receptors, many of which are known drug targets, were identified as differentially regulated following culture upon the gel. Furthermore, through gene set enrichment analysis we identified over 700 pathways enriched amongst the up- and downregulated genes including pathways related to cell differentiation, notochord morphogenesis, and intracellular signaling. Together these findings demonstrate the global mechanobiological effects induced by the LMP gel and confirm the ability of this substrate to modulate NP cell phenotype.

Cells of the nucleus pulposus (NP) are essential contributors to extracellular matrix synthesis and function of the intervertebral disc. With age and degeneration, the NP becomes stiffer and more dehydrated, which is associated with a loss of phenotype and biosynthetic function for its resident NP cells. Also, with aging, the NP cell undergoes substantial morphological changes from a rounded shape with pronounced vacuoles in the neonate and juvenile, to one that is more flattened and spread with a loss of vacuoles. Here, we make use of the clinically relevant pharmacological treatment verteporfin (VP), previously identified as a disruptor of yes‐associated protein‐TEA domain family member‐binding domain (TEAD) signaling, to promote morphological changes in adult human NP cells in order to study variations in gene expression related to differences in cell shape. Treatment of adult, degenerative human NP cells with VP caused a shift in morphology from a spread, fibroblastic‐like shape to a rounded, clustered morphology with decreased transcriptional activity of TEAD and serum‐response factor. These changes were accompanied by an increased expression of vacuoles, NP‐specific gene markers, and biosynthetic activity. The contemporaneous observation of VP‐induced changes in cell shape and prominent, time‐dependent changes within the transcriptome of NP cells occurred over all timepoints in culture. Enriched gene sets with the transition to VP‐induced cell rounding suggest a major role for cell adhesion, cytoskeletal remodeling, vacuolar lumen, and MAPK activity in the NP phenotypic and functional response to changes in cell shape.

Cells of the nucleus pulposus have been observed to undergo a shift from their notochordal-like juvenile phenotype to a more fibroblast-like state with age and maturation. It has been demonstrated that culture of degenerative adult human nucleus pulposus cells upon soft (< 1 kPa) full length laminin-containing hydrogel substrates promotes increased levels of a panel of markers associated with the juvenile nucleus pulposus cell phenotype. In the current work, we observed an ability to use soft polymeric substrates functionalized with short laminin-mimetic peptide sequences to recapitulate the behaviors elicited by soft, full-length laminin containing materials. Furthermore, our work suggests an ability to mimic features of soft systems through control of peptide density upon stiffer substrates. Specifically, results suggest that stiffer polymer-peptide hydrogel substrates can be used to promote the expression of a more juvenile-like phenotype for cells of the nucleus pulposus by reducing adhesive ligand presentation. Here we show how polymer stiffness combined with adhesive ligand presentation can be controlled to be supportive of nucleus pulposus cell phenotype and biosynthesis.

Nucleus pulposus (NP) cells are derived from the notochord and differ from neighboring cells of the intervertebral disc in phenotypic marker expression and morphology. Adult human NP cells lose this phenotype and morphology with age in a pattern that contributes to progressive disc degeneration and pathology. Select laminin-mimetic peptide ligands and substrate stiffnesses were examined for their ability to regulate human NP cell phenotype and biosynthesis through the expression of NP-specific markers aggrecan, N-cadherin, collagen types I and II, and GLUT1. Peptide-conjugated substrates demonstrated an ability to promote expression of healthy NP-specific markers, as well as increased biosynthetic activity. We show an ability to re-express markers of the juvenile NP cell and morphology through control of peptide presentation and stiffness on well-characterized polyacrylamide substrates. NP cells cultured on surfaces conjugated with α3 integrin receptor peptides P4 and P678, and on α2, α5, α6, β1 integrin-recognizing peptide AG10, show increased expression of aggrecan, N-cadherin, and types I and II collagen, suggesting a healthier, more juvenile-like phenotype. Multi-cell cluster formation was also observed to be more prominent on peptide-conjugated substrates. These findings indicate a critical role for cell-matrix interactions with specific ECM-mimetic peptides in supporting and maintaining a healthy NP cell phenotype and bioactivity. Statement of Significance NP cells reside in a laminin-rich environment that deteriorates with age, including a loss of water content and changes in the extracellular matrix (ECM) structure that may lead to the development of a degenerated IVD. There is great interest in methods to re-express healthy, biosynthetically active NP cells using laminin-derived biomimetic peptides toward the goal of using autologous cell sources for tissue regeneration. Here, we describe a novel study utilizing several laminin mimetic peptides conjugated to polyacrylamide gels that are able to support an immature, healthy NP phenotype after culture on “soft” peptide gels. These findings can support future studies in tissue regeneration where cells may be directed to a desired regenerative phenotype using niche-specific ECM peptides.

Low back pain is associated with degeneration of the intervertebral disc, but specific mechanisms of pain generation in this pathology remain unknown. Sensory afferent nerve fiber growth into the intervertebral disc after injury-induced inflammation may contribute to discogenic pain. We describe a clinically relevant behavioral phenotype in a rodent model of chronic intervertebral disc degeneration which provides a means to map sensory neuron changes to a single affected lumbar intervertebral disc. Unilateral disc puncture of one lumbar intervertebral disc revealed a bilateral behavioral phenotype characterized by gait changes and decreased activity. Moreover, neurons extracted from the dorsal root ganglia in animals with intervertebral disc injury demonstrated altered TRPV1 activation in vitro independent of exogenous NGF administration. Finally, neuronal nuclear hypertrophy and elevated expression of p75NTR provide evidence of active adaptation of innervating sensory neurons in chronic intervertebral disc degeneration. Therefore, this model and findings provide the template for future studies to establish specific mechanisms of nociceptive pain in chronic intervertebral disc degeneration.

Previous study claimed that disc degeneration may be preceded by structure and matrix changes in the intervertebral disc (IVD) which coincide with the loss of distinct notochordally derived nucleus pulposus (NP) cells. However, the fate of notochordal cells and their molecular phenotype change during aging and degeneration in human are still unknown. In this study, a set of novel molecular phenotype markers of notochordal NP cells during aging and degeneration in human IVD tissue were revealed with immunostaining and flow cytometry. Furthermore, the potential of phenotype juvenilization and matrix regeneration of IVD cells in a laminin-rich pseudo-3D culture system were evaluated at day 28 by immunostaining, Safranin O, and type II collagen staining. Immunostaining and flow cytometry demonstrated that transcriptional factor Brachyury T, neuronal-related proteins (brain abundant membrane attached signal protein 1, Basp1; Neurochondrin, Ncdn; Neuropilin, Nrp-1), CD24, and CD221 were expressed only in juvenile human NP tissue, which suggested that these proteins may be served as the notochordal NP cell markers. However, the increased expression of CD54 and CD166 with aging indicated that they might be referenced as the potential biomarker for disc degeneration. In addition, 3D culture maintained most of markers in juvenile NP, and rescued the expression of Basp1, Ncdn, and Nrp 1 that disappeared in adult NP native tissue. These findings provided new insight into molecular profile that may be used to characterize the existence of a unique notochordal NP cells during aging and degeneration in human IVD cells, which will facilitate cell-based therapy for IVD regeneration. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1316-1326, 2016.

Figure S1. Showing immunofluorescence of sorted T-GFP+ cells promoted to differentiate in culture. (A) Immunocytofluorescence showed the positive expression of CD24 (green) and (B) positive expression of CD239 (red) in sorted T-GFP+ cells when differentiated in monolayer for 12 days using step 1b and step 2 of NPDM protocol. (C) Yellow denotes colocalization of the markers. All cells counterstained with DAPI. Scale bar = 20 Îźm. (DOCX 995 kb)

Juvenile nucleus pulposus (NP) cells of the intervertebral disc (IVD) are large, vacuolated cells that form cell clusters with strong cell–cell interactions. With maturation and aging, NP cells lose their ability to form these cell clusters, with aging-associated changes in NP cell phenotype, morphology, and proteoglycan synthesis that may contribute to IVD degeneration. Therefore, it is important to understand the mechanisms governing juvenile NP cell cluster behavior towards the goal of revealing factors that can promote juvenile, healthy NP cell phenotypes. N-cadherin has been identified as a cell–cell adhesion marker that is present in juvenile NP cells, but disappears with age. The goal of this study was to reveal the importance of N-cadherin in regulating cell–cell interactions in juvenile NP cell cluster formation and test for a regulatory role in maintaining a juvenile NP phenotype in vitro. Juvenile porcine IVD cells, of notochordal origin, were promoted to form cell clusters in vitro, and analyzed for preservation of the juvenile NP phenotype. Additionally, cadherin-blocking experiments were performed to prevent cluster formation in order to study the importance of cluster formation in NP cell signaling. Findings reveal N-cadherin-mediated cell–cell contacts promote cell clustering behavior and regulate NP cell matrix production and preservation of NP-specific markers. Inhibition of N-cadherin-mediated contacts resulted in loss of all features of the juvenile NP cell. These results establish a regulatory role for N-cadherin in juvenile NP cells, and suggest that preservation of the N-cadherin mediated cell–cell contact is important for preserving juvenile NP cell phenotype and morphology.

Object Chordoma cells can generate solid-like tumors in xenograft models that express some molecular characteristics of the parent tumor, including positivity for brachyury and cytokeratins. However, there is a dearth of molecular markers that relate to chordoma tumor growth, as well as the cell lines needed to advance treatment. The objective in this study was to isolate a novel primary chordoma cell source and analyze the characteristics of tumor growth in a mouse xenograft model for comparison with the established U-CH1 and U-CH2b cell lines. Methods Primary cells from a sacral chordoma, called “DVC-4,” were cultured alongside U-CH1 and U-CH2b cells for more than 20 passages and characterized for expression of CD24 and brachyury. While brachyury is believed essential for driving tumor formation, CD24 is associated with healthy nucleus pulposus cells. Each cell type was subcutaneously implanted in NOD/SCID/IL2Rγnull mice. The percentage of solid tumors formed, time to maximum tumor size, and immunostaining scores for CD24 and brachyury (intensity scores of 0–3, heterogeneity scores of 0–1) were reported and evaluated to test differences across groups. Results The DVC-4 cells retained chordoma-like morphology in culture and exhibited CD24 and brachyury expression profiles in vitro that were similar to those for U-CH1 and U-CH2b. Both U-CH1 and DVC-4 cells grew tumors at rates that were faster than those for U-CH2b cells. Gross tumor developed at nearly every site (95%) injected with U-CH1 and at most sites (75%) injected with DVC-4. In contrast, U-CH2b cells produced grossly visible tumors in less than 50% of injected sites. Brachyury staining was similar among tumors derived from all 3 cell types and was intensely positive (scores of 2–3) in a majority of tissue sections. In contrast, differences in the pattern and intensity of staining for CD24 were noted among the 3 types of cell-derived tumors (p < 0.05, chi-square test), with evidence of intense and uniform staining in a majority of U-CH1 tumor sections (score of 3) and more than half of the DVC-4 tumor sections (scores of 2–3). In contrast, a majority of sections from U-CH2b cells stained modestly for CD24 (scores of 1–2) with a predominantly heterogeneous staining pattern. Conclusions This is the first report on xenografts generated from U-CH2b cells in which a low tumorigenicity was discovered despite evidence of chordoma-like characteristics in vitro. For tumors derived from a primary chordoma cell and U-CH1 cell line, similarly intense staining for CD24 was observed, which may correspond to their similar potential to grow tumors. In contrast, U-CH2b tumors stained less intensely for CD24. These results emphasize that many markers, including CD24, may be useful in distinguishing among chordoma cell types and their tumorigenicity in vivo.

Intervertebral disc (IVD) disorders and age-related degeneration are believed to contribute to lower back pain. There is significant interest in cell-based strategies for regenerating the nucleus pulposus (NP) region of the disc; however, few scaffolds have been evaluated for their ability to promote or maintain an immature NP cell phenotype. Previous studies have shown that NP cell-laminin interactions promote cell adhesion and biosynthesis, which suggests a laminin-functionalized biomaterial may be useful for promoting or maintaining the NP cell phenotype. Here, a photocrosslinkable poly(ethylene glycol)-laminin 111 (PEG-LM111) hydrogel was developed. The mechanical properties of PEG-LM111 hydrogel could be tuned within the range of dynamic shear moduli values previously reported for human NP. When primary immature porcine NP cells were seeded onto PEG-LM111 hydrogels of varying stiffnesses, LM111-presenting hydrogels were found to promote cell clustering and increased levels of sGAG production as compared to stiffer LM111-presenting and PEG-only gels. When cells were encapsulated in 3-D gels, hydrogel formulation was found to influence NP cell metabolism and expression of proposed NP phenotypic markers, with higher expression of N-cadherin and cytokeratin 8 observed for cells cultured in softer (

Nucleus pulposus (NP) cells of the intervertebral disc are essential for synthesizing extracellular matrix that contributes to disc health and mechanical function. NP cells have a unique morphology and molecular expression pattern derived from their notochordal origin, and reside in N-cadherin (CDH2) positive cell clusters in vivo. With disc degeneration, NP cells undergo morphologic and phenotypic changes including loss of CDH2 expression and ability to form cell clusters. Here, we investigate the role of CDH2 positive cell clusters in preserving healthy, biosynthetically active NP cells. Using a laminin-functionalized hydrogel system designed to mimic features of the native NP microenvironment, we demonstrate NP cell phenotype and morphology is preserved only when NP cells form CDH2 positive cell clusters. Knockdown (CRISPRi) or blocking CDH2 expression in vitro and in vivo results in loss of a healthy NP cell. Findings also reveal that degenerate human NP cells that are CDH2 negative can be promoted to re-express CDH2 and healthy, juvenile NP matrix synthesis patterns by promoting cell clustering for controlled microenvironment conditions. This work also identifies CDH2 interactions with β-catenin-regulated signaling as one mechanism by which CDH2-mediated cell interactions can control NP cell phenotype and biosynthesis towards maintenance of healthy intervertebral disc tissues.

The inflammatory responses of primary human intervertebral disc (IVD) cells to tumor necrosis factor α (TNF-α) and an antagonist were evaluated in vitro.To investigate an ability for soluble TNF receptor type II (sTNFRII) to antagonize TNF-α-induced inflammatory events in primary human IVD cells in vitro.TNF-α is a known mediator of inflammation and pain associated with radiculopathy and IVD degeneration. sTNFRs and their analogues are of interest for the clinical treatment of these IVD pathologies, although information on the effects of sTNFR on human IVD cells remains unknown.IVD cells were isolated from surgical tissues procured from 15 patients and cultured with or without 1.4 nmol/L TNF-α (25 ng/mL). Treatment groups were coincubated with varying doses of sTNFRII (12.5-100 nmol/L). Nitric oxide (NO), prostaglandin E₂ (PGE₂), and interleukin-6 (IL6) levels in media were quantified to characterize the inflammatory phenotype of the IVD cells.Across all patients, TNF-α induced large, statistically significant increases in NO, PGE₂, and IL6 secretion from IVD cells compared with controls (60-, 112-, and 4-fold increases, respectively; P0.0001). Coincubation of TNF-α with nanomolar doses of sTNFRII significantly attenuated the secretion of NO and PGE₂ in a dose-dependent manner, whereas IL6 levels were unchanged. Mean IC₅₀ values for NO and PGE₂ were found to be 35.1 and 20.5 nmol/L, respectively.Nanomolar concentrations of sTNFRII were able to significantly attenuate the effects of TNF-α on primary human IVD cells in vitro. These results suggest this sTNFR to be a potent TNF antagonist with potential to attenuate inflammation in IVD pathology.

National Institutes of Health - R01EB002263, R21AR052745, P01AR050245, K99AR057426, F32AR056190. The North Carolina Biotechnology Center - CFG-8013. An OREF/DePuy Orthopaedic Resident Educational Grant.

Supported by grants from the NIH R01AR047442, P01AR05024506, R01AR057410, R01EB00226307, K99AR057426 and NCBC GRANT #2008-CFG-8013. This study was also supported by the Pratt Undergraduate Research Fellowship and the Howard Clark Pre-Doctoral Fellowship.

Intervertebral disc herniation can cause clinical radiculopathy based on mechanical deformation and biochemical irritation of apposed neural structures,1,2 and subsequent clinical presentation vary greatly in the extent of pain, neurological deficit, and functional disability. A number of studies have applied autologous nucleus pulposus (NP) to the rat DRG as a model of non-compressive disc herniation, inducing inflammatory histopathology and neuronoglial apoptosis, decreases in nerve conduction velocities, and onset of mechanical allodynia and thermal hyperalgesia.2–8 A key mediator of these changes is suggested to be tumor necrosis factor alpha (TNFα),9–11 expressed at higher levels in herniated discs than degeneration-matched controls.12,13 Furthermore, systemic immunomodulatory treatment targeting TNFα activity attenuates these effects in animal models14–17 and may provide clinical relief of sciatica in human studies.18 The impact of these changes on animal locomotor function remains questionable,5,19 despite being of clinical interest.20 Involvement of TNFα in radiculopathy may relate to the presence of macrophages, more numerous and widespread than other inflammatory cells after herniation.21,22 Recruitment of macrophages after disc herniation is incompletely understood, though it may relate to an underlying immune mechanism. The immune-privilege of the NP may lead to extracellular matrix or cellular structures recognition as antigenic upon systemic exposure following herniation, a theory supported by regional lymph node accumulation of lymphocytes after exposure to autologous NP,23 and lymphocyte accumulation in the intervertebral disc after anular injury.24 Further, IgM and IgG immunoglobulin deposition occurs in human herniated disc tissue, suggesting specific immune activation.25 Given this antigenic potential of NP tissue, the immune response to herniated material may include cytokines involved in lymphocyte differentiation and products of their activation. Markers of inflammatory and immune activation in this study are interleukins 23 (IL-23) and 17 (IL-17). Lymphocyte transmigration occurs following peripheral nerve constriction injury, with efficient recruitment of CD4+ lymphocytes and activation of local microglia to inflammatory phenotype.26 Production of IL-23 preceded expression of IL-17 at the injury site, with the more important finding this IL-17 response was absent despite normal IL-23 levels in recombination-activating gene-1 knockout mice deficient in T-lymphocytes.27 This supports the known role for T-lymphocytes producing IL-17; and indeed, IL-23 acts on naive CD4+ T-lymphocytes to facilitate differentiation and expansion into a Th17 lineage, characterized by substantial IL-17 production.28,29 Further, there is evidence of a potential role of IL-23 and IL-17 in regulating an immune response in the peripheral nervous system. Glial cells also produce IL-23, with enhanced T-lymphocyte IL-17 release upon costimulation with a specific antigen, implicating the neuronal support cells with a role in immune activation.30,31 Tissue effects of this mediator include blood-brain-barrier disruption with efficient lymphocyte transmigration32 and further recruitment of CD4+ lymphocytes and activation of local microglia to an inflammatory phenotype.26 This study evaluated an animal model of NP herniation radiculopathy for evidence of functional deficits of mechanical allodynia and altered gait patterns, and local expression of IL-23 and IL-17 for a role in the biochemical inflammatory process. The results demonstrate novel findings of gait asymmetry and imbalance between left and right duty factors, and confirm known findings of mechanical allodynia. Further, the results provide evidence of a role for both local inflammatory and immune activation, but not a systemic inflammatory response, after DRG exposure to autologous NP.

Intervertebral disc (IVD) disorders are believed to be related to aging-related cell loss and phenotypic changes, as well as biochemical and structural changes in the extracellular matrix of the nucleus pulposus (NP) region. Previously, we found that the laminin gamma1 chain was more highly expressed in immature NP porcine tissues, in parallel with the expression pattern for a laminin receptor, integrin alpha6 subunit, as compared to adjacent anulus fibrosus region. This result suggests that cell-matrix interactions may be unique to the immature NP. However, the identity of laminin isoforms specific to immature or mature NP tissues, their associated receptors, and functional significance are still poorly understood. In this study, we evaluated the zonal-specific expression of the laminin chains, receptors (i.e., integrins), and other binding proteins in immature tissue and isolated cells of rat, porcine and human intervertebral disc. Our goal was to reveal features of cellular environment and cell-matrix interactions in the immature NP. Results from both immunohistochemical staining and flow cytometry analysis found that NP cells expressed higher levels of the laminin alpha5 chain, laminin receptors (integrin alpha3, alpha6, beta4 subunit, and CD239), and related binding proteins (CD151), as compared to cells from adjacent anulus fibrosus. These differences suggest that laminin interactions with NP cells are distinct from that of the anulus fibrosus and that laminins may be important contributors to region-specific IVD biology. The revealed laminin isoforms, their receptors, and related binding proteins may be used as distinguishing features of these immature NP cells in the intervertebral disc.

ObjectBiochemical irritation of the dorsal root ganglion (DRG) after intervertebral disc herniation contributes to radiculopathy through tumor necrosis factor–α (TNFα)–mediated inflammation. Soluble TNF receptor Type II (sTNFRII) sequesters this cytokine, providing clinical benefit. Previous work involving conjugation of sTNFRII with thermally responsive elastin-like polypeptide (ELP) yielded a chimeric protein (ELP–sTNFRII) with in vitro anti-TNFα bioactivity. Furthermore, temperature-triggered ELP aggregation into a “depot” prolongs protein residence time following perineural injection. In this study the authors evaluated the inflammatory phenotype of DRG explants after TNFα stimulation, and assessed the abilities of sTNFRII or ELP–sTNFRII to attenuate these neuro-inflammatory changes.MethodsRat lumbar DRGs (35 animals) were treated in 6 groups, as follows: control; TNFα (25 ng/ml); TNFα with low-(0.2 μg/ml) or high-dose (1 μg/ml) sTNFRII; and TNFα with low-(52.5 μg/ml) or high-dose (262.5 μg/ml) ELP–sTNFRII. After 24 hours, supernatant was evaluated for inflammatory cytokines (interleukin [IL]–1, IL-6, and IL-10); prostaglandin E2; and metabolites (glutamate, lactate, and pyruvate). Single-factor analysis of variance with post hoc Dunn analysis (α = 0.05) was used to assess treatment differences.ResultsIncubation of explants with TNFα caused metabolic stress reflected by an increased lactate/pyruvate ratio (1.8 ± 0.5–fold) and extracellular glutamate (79 ± 8% increase). Inflammatory activation was observed with heightened IL-6 release (5.2 ± 1.4–fold) and prostaglandin E2production (14 ± 3–fold). An autoregulatory response occurred with an 11.8 ± 0.6–fold increase in sTNFRI shedding. Treatment with high doses of sTNFRII or ELP–sTNFRII reversed all changes. Values are expressed as the mean ± standard deviation.ConclusionsThese results demonstrate that TNFα stimulation of DRG explants yields a phenotype of neurotoxic metabolite release and inflammatory mediator expression. Coincubation with either sTNFRII or ELP–sTNFRII antagonizes TNFα activity to abrogate these changes, suggesting potential for therapeutic intervention to treat peripheral nerve inflammatory disease.

Objective Type IX collagen is an important component of the intervertebral disc extracellular matrix. Mutations in type IX collagen are associated with premature disc degeneration in mice and a predisposition to disc disorders in humans. The aim of this study was to assess the prevalence and timeline of intervertebral disc degeneration in mice homozygous for an inactivated Col9a1 gene. Methods Intact spine segments were harvested from wild-type (WT) and type IX collagen–knockout (Col9a1−/−) mice at 3, 6, and 12 months of age. Sagittal spine sections were evaluated for evidence of histologic changes, by 2 blinded graders, using a semiquantitative grading method. Results There was evidence of more degeneration of the disc and end plate in the spines of Col9a1−/− mice compared with those of WT controls, at most time points. These findings were significant for the disc region at 3 and 6 months (P < 0.01) and at 12 months (P < 0.10) and for the end plate region only at 6 months (P < 0.10). Degenerative changes in the disc consisted of cellular changes and mucous degeneration. Degeneration in the end plates was associated with more cell proliferation, cartilage disorganization, and new bone formation. Conclusion A deletion mutation for type IX collagen is associated with connective tissue changes characteristic of musculoskeletal degeneration in bony and cartilaginous tissue regions. Some of the observed changes were similar to cartilage changes in osteoarthritis, while others were more similar to disc degenerative changes in humans. The finding of premature onset of intervertebral disc degeneration in this mouse model may be useful in studies of the pathology and treatment of human disc degeneration.

The correct timing of flowering is essential for plants to maximize reproductive success and is controlled by environmental and endogenous signals. We report that nitric oxide (NO) repressed the floral transition in Arabidopsis thaliana . Plants treated with NO, as well as a mutant overproducing NO ( nox1 ), flowered late, whereas a mutant producing less NO ( nos1 ) flowered early. NO suppressed CONSTANS and GIGANTEA gene expression and enhanced FLOWERING LOCUS C expression, which indicated that NO regulates the photoperiod and autonomous pathways. Because NO is induced by environmental stimuli and constitutively produced, it may integrate both external and internal cues into the floral decision.

Chordoma cells can generate solid-like tumors in xenograft models that express some molecular characteristics of the parent tumor, including positivity for brachyury and cytokeratins. However, there is a dearth of molecular markers that relate to chordoma tumor growth, as well as the cell lines needed to advance treatment. The objective in this study was to isolate a novel primary chordoma cell source and analyze the characteristics of tumor growth in a mouse xenograft model for comparison with the established U-CH1 and U-CH2b cell lines.Primary cells from a sacral chordoma, called "DVC-4," were cultured alongside U-CH1 and U-CH2b cells for more than 20 passages and characterized for expression of CD24 and brachyury. While brachyury is believed essential for driving tumor formation, CD24 is associated with healthy nucleus pulposus cells. Each cell type was subcutaneously implanted in NOD/SCID/IL2Rγ(null) mice. The percentage of solid tumors formed, time to maximum tumor size, and immunostaining scores for CD24 and brachyury (intensity scores of 0-3, heterogeneity scores of 0-1) were reported and evaluated to test differences across groups.The DVC-4 cells retained chordoma-like morphology in culture and exhibited CD24 and brachyury expression profiles in vitro that were similar to those for U-CH1 and U-CH2b. Both U-CH1 and DVC-4 cells grew tumors at rates that were faster than those for U-CH2b cells. Gross tumor developed at nearly every site (95%) injected with U-CH1 and at most sites (75%) injected with DVC-4. In contrast, U-CH2b cells produced grossly visible tumors in less than 50% of injected sites. Brachyury staining was similar among tumors derived from all 3 cell types and was intensely positive (scores of 2-3) in a majority of tissue sections. In contrast, differences in the pattern and intensity of staining for CD24 were noted among the 3 types of cell-derived tumors (p0.05, chi-square test), with evidence of intense and uniform staining in a majority of U-CH1 tumor sections (score of 3) and more than half of the DVC-4 tumor sections (scores of 2-3). In contrast, a majority of sections from U-CH2b cells stained modestly for CD24 (scores of 1-2) with a predominantly heterogeneous staining pattern.This is the first report on xenografts generated from U-CH2b cells in which a low tumorigenicity was discovered despite evidence of chordoma-like characteristics in vitro. For tumors derived from a primary chordoma cell and U-CH1 cell line, similarly intense staining for CD24 was observed, which may correspond to their similar potential to grow tumors. In contrast, U-CH2b tumors stained less intensely for CD24. These results emphasize that many markers, including CD24, may be useful in distinguishing among chordoma cell types and their tumorigenicity in vivo.

The intervertebral disc (IVD) is a heterogeneous, fibrocartilaginous tissue that provides load support, energy dissipation, and flexibility in the spine. The IVD, which is composed of the nucleus pulposus (NP), anulus fibrosus (AF), and cartilage endplate (Fig. ​(Fig.1),1), is situated between adjacent vertebral bodies and acts as the main joint of the spinal column, occupying approximately 1/3 of its total height [1,2]. The cells within each of the regions of the IVD are subjected to a variety of signals from both physical and biochemical stimuli from their surrounding extracellular matrix (ECM) microenvironment [3–8]. These cues are believed to play critical roles in regulating development, maintenance, and repair of the IVD, but in ways that are poorly understood. Fig. 1 The intervertebral disc is situated between vertebral bodies in the spinal column, and acts to support loads, provide flexibility, and dissipate energy in the spine. The disc is comprised of distinct anatomic zones: the anulus fibrosus (AF), nucleus pulposus ... During disc degeneration or aging, significant changes are observed in IVD cell phenotype and density in parallel with changes in ECM composition and structure. A dramatic decrease in cell density and multicell clusters in both the NP and AF regions is observed [9–11] with increased prevalence of cells with cytoplasmic projections [12–14]. While the exact factors resulting in decreased NP cell clustering with age in vivo are not fully understood, it is likely that cell death associated with decreased nutrient oxygen and glucose transport to the IVD can contribute to these decreased cell numbers and cell clusters [2]. With decreases in cell density, the large, vacuolated cells in the NP, which are normally arranged in cell clusters, transition to a sparse population of smaller, isolated chondrocyte-like cells [15]. The loss of proteoglycan matrix causes changes in proteoglycan structure [16–18], which results in decreased negative fixed-charge density, decreased water content, and a loss of swelling pressure [19,20], impairing the tissue's ability to resist and redistribute compressive loads. Corresponding with these compositional changes are structural alterations including loss of disc height and increased anulus lamellar disorganization. Changes in ECM composition and structure may also result in substantially altered mechanics and kinematics for the entire IVD motion segment, with decreased internal pressurization and disc height resulting in higher compressive loads transferred to the AF, compromising its structure and function (e.g., overload leading to clefts, buckling, or rupture). Nerve compression, spinal canal impingement, and altered spinal loading configurations can also occur, which can contribute to symptomatic back pain [21]. These dramatic shifts in ECM mechanical environment can be expected to impact NP cell health, metabolism and survival, although the direct links between environmental factors and NP cell behaviors are still under study. The purpose of this article is to review our experience with studies of NP cell interactions with their surrounding ECM, as this knowledge can be useful in the development of treatments for disc-related ailments. The first section of this article covers what we have learned of how NP cells interact with select proteins of the native ECM. We then describe how changes in the surrounding ECM can alter NP cell phenotype and morphology, and summarize recent work performed to reveal how NP cells sense, interpret, and respond to different mechanical and biochemical cues in their ECM.

Cell supplementation to the herniated or degenerated intervertebral disc (IVD) is a potential strategy to promote tissue regeneration and slow disc pathology. Human umbilical cord mesenchymal stromal cells (HUCMSCs) – originating from the Wharton’s jelly – remain an attractive candidate for such endeavors with their ability to differentiate into multiple lineages. Previously, mesenchymal stem cells (MSCs) have been studied as a potential source for disc tissue regeneration. However, no studies have demonstrated that MSCs can regenerate matrix with unique characteristics matching that of immature nucleus pulposus (NP) tissues of the IVD. In our prior work, immature NP cells were found to express specific laminin isoforms and laminin-binding receptors that may serve as phenotypic markers for evaluating MSC differentiation to NP-like cells. The goal of this study is to evaluate these markers and matrix synthesis for HUCMSCs cultured in a laminin-rich pseudo-three-dimensional culture system. HUCMSCs were seeded on top of Transwell inserts pre-coated with Matrigel™, which contained mainly laminin-111. Cells were cultured under hypoxia environment with three differentiation conditions: NP differentiation media (containing 2.5% Matrigel™ solution to provide for a pseudo-three-dimensional laminin culture system) with no serum, or the same media supplemented with either insulin-like growth factor-1 (IGF-1) or transforming growth factor-β1 (TGF-β1). Cell clustering behavior, matrix production and the expression of NP-specific laminin and laminin-receptors were evaluated at days 1, 7, 13 and 21 of culture. Data show that a pseudo-three-dimensional culture condition (laminin-1 rich) promoted HUCMSC differentiation under no serum conditions. Starting at day 1, HUCMSCs demonstrated a cell clustering morphology similar to that of immature NP cells in situ and that observed for primary immature NP cells within the similar laminin-rich culture system (prior study). Differentiated HUCMSCs under all conditions were found to contain glycosaminoglycan, expressed extracellular matrix proteins of collagen II and laminin α5, and laminin receptors (integrin α3 and β4 subunits). However, neither growth factor treatment generated distinct differences in NP-like phenotype for HUCMSC as compared with no-serum conditions. HUCMSCs have the potential to differentiate into cells sharing features with immature NP cells in a laminin-rich culture environment and may be useful for IVD cellular therapy.

A large percentage of the population may be expected to experience painful symptoms or disability associated with intervertebral disc (IVD) degeneration - a condition characterized by diminished integrity of tissue components. Great interest exists in the use of autologous or allogeneic cells delivered to the degenerated IVD to promote matrix regeneration. Induced pluripotent stem cells (iPSCs), derived from a patient's own somatic cells, have demonstrated their capacity to differentiate into various cell types although their potential to differentiate into an IVD cell has not yet been demonstrated. The overall objective of this study was to assess the possibility of generating iPSC-derived nucleus pulposus (NP) cells in a mouse model, a cell population that is entirely derived from notochord. This study employed magnetic activated cell sorting (MACS) to isolate a CD24(+) iPSC subpopulation. Notochordal cell-related gene expression was analyzed in this CD24(+) cell fraction via real time RT-PCR. CD24(+) iPSCs were then cultured in a laminin-rich culture system for up to 28 days, and the mouse NP phenotype was assessed by immunostaining. This study also focused on producing a more conducive environment for NP differentiation of mouse iPSCs with addition of low oxygen tension and notochordal cell conditioned medium (NCCM) to the culture platform. iPSCs were evaluated for an ability to adopt an NP-like phenotype through a combination of immunostaining and biochemical assays. Results demonstrated that a CD24(+) fraction of mouse iPSCs could be retrieved and differentiated into a population that could synthesize matrix components similar to that in native NP. Likewise, the addition of a hypoxic environment and NCCM induced a similar phenotypic result. In conclusion, this study suggests that mouse iPSCs have the potential to differentiate into NP-like cells and suggests the possibility that they may be used as a novel cell source for cellular therapy in the IVD.

Intervertebral disc herniation may contribute to inflammatory processes that associate with radicular pain and motor deficits. Molecular changes at the affected dorsal root ganglion (DRG), spinal cord, and even midbrain, have been documented in rat models of radiculopathy or nerve injury. The objective of this study was to evaluate gait and the expression of key pain receptors in the midbrain in a rodent model of radiculopathy. Radiculopathy was induced by harvesting tail nucleus pulposus (NP) and placing upon the right L5 DRG in rats (NP-treated, n=12). Tail NP was discarded in sham-operated animals (n=12). Mechanical allodynia, weight-bearing, and gait were evaluated in all animals over time. At 1 and 4 weeks after surgery, astrocyte and microglial activation was tested in DRG sections. Midbrain sections were similarly evaluated for immunoreactivity to serotonin (5HT2B), mu-opioid (µ-OR), and metabotropic glutamate (mGluR4 and 5) receptor antibodies. NP-treated animals placed less weight on the affected limb 1 week after surgery and experienced mechanical hypersensitivity over the duration of the study. Astroctye activation was observed at DRGs only at 4 weeks after surgery. Findings for pain receptors in the midbrain of NP-treated rats included an increased expression of 5HT2B at 1, but not 4 weeks; increased expression of µ-OR and mGluR5 at 1 and 4 weeks (periaqueductal gray region only); and no changes in expression of mGluR4 at any point in this study. These observations provide support for the hypothesis that the midbrain responds to DRG injury with a transient change in receptors regulating pain responses.

Galectin-1 (Gal-1), an endogenous β-galactoside-binding protein, binds to laminins, which are highly expressed in the nucleus pulposus (NP) of the intervertebral disc (IVD). The objective of this study is to evaluate the expression of Gal-1 protein in IVD tissues during aging and the effect of Gal-1 on IVD cell adhesion to laminins. Tissues from rat, porcine, and human (scoliosis or disc degeneration) IVDs were used to evaluate Gal-1 expression via immunostaining, RT-PCR, and Western blot analysis. Attachment of isolated IVD cells (porcine and human) on select laminin isoforms (LM-111 and LM-511) was compared with/without pre-incubation with exogenous Gal-1. A biotinylated Gal-1(B-Gal-1) was used to evaluate for binding to IVD cells and to select for IVD cells by magnetic activated cell sorting (MACS). NP cells expressed high levels of Gal-1 protein as compared to anulus fibrosus (AF) cells in immature tissues, while exogenous Gal-1 increased both NP and AF cell attachment to laminins and exhibited a similar binding to both cell types in vitro. With aging, Gal-1 levels in NP tissue appeared to decrease. In addition, incubation with B-Gal-1 was able to promote the retention of more than 50% of IVD cells via MACS. Our results provide new findings for the presence and functional role of Gal-1 within IVDs. Similar staining patterns for Gal-1 and LM-511 in IVD tissue suggest that Gal-1 may serve as an adhesion molecule to interact with both cells and laminins. This MACS protocol may be useful for selecting pure IVD cells from mixed cells of pathological tissue.

Interleukin-17 (IL-17) is a cytokine recently shown to be elevated, along with interferon-γ (IFNγ) and tumor necrosis factor (TNFα), in degenerated and herniated intervertebral disc (IVD) tissues, suggesting a role for these cytokines in intervertebral disc disease. The objective of our study was to investigate the involvement of IL-17 and costimulants IFNγ and TNFα in intervertebral disc pathology. Cells were isolated from anulus fibrosus and nucleus pulposus tissues of patients undergoing surgery for intervertebral disc degeneration or scoliosis. The production of inflammatory mediators, nitric oxide (NOx), prostaglandin E2 (PGE2) and interleukin-6 (IL-6), as well as intercellular adhesion molecule (ICAM-1) expression, were quantified for cultured cells following exposure to IL-17, IFNγ, and TNFα. Intervertebral disc cells exposed to IL-17, IFNγ, or TNFα showed a remarkable increase in inflammatory mediator release and ICAM-1 expression (GLM and ANOVA, p 0.05). Addition of IFNγ or TNFα to IL-17 demonstrated a synergistic increase in inflammatory mediator release, and a marked increase in ICAM-1 expression. These findings suggest that IVD cells not only respond with a catabolic phenotype to IL-17 and costimulants IFNγ and TNFα, but also express surface ligands with consequent potential to recruit additional lymphocytes and immune cells to the IVD microenvironment. IL-17 may be an important regulator of inflammation in the IVD pathologies.

Acute low back pain is among the most common reasons for which patients seek medical care, with estimates of annual economic impact as high as $200 billion in the US (1). Degeneration of the intervertebral disc (IVD) is a common cause of such pain and disability, characterized by anatomic, morphologic, and biochemical changes including altered expression of both matrix metalloproteinases and proinflammatory cytokines (2). Elevated levels of molecular mediators of inflammation have been described in pathologic disc tissue, increasing with grade of degeneration (3,4). Such findings have been observed for both interleukin-1 (IL-1) and tumor necrosis factor α (TNFα), both of which have established roles in regulating nitric oxide (NO) and prostaglandin production, metalloproteinase expression, and apoptosis, all changes that may contribute to progressive pathology of the IVD. The inflammatory and immune activation profile exhibited by a herniated disc may be distinct from that observed in IVD degeneration and may differ further among different extents of herniation from protrusion through sequestration. Histologic evaluation of herniated disc tissue has revealed prominent infiltration of inflammatory cells, most markedly macrophages (5–10). Surgical specimens of herniated disc tissue also exhibit a greater presence of TNFα, IL-1β, and IL-6 compared with autopsy control tissue specimens (3,4), and explant cultures demonstrate a heightened release of IL-6, IL-8, prostaglandin E2 (PGE2), and NO from herniated disc tissue compared with control tissue (11). Recent studies have also revealed greater IL-12 and interferon-γ (IFNγ) expression in herniated disc fragments compared with bulging discs that remain contained within the disc space by an intact anulus fibrosus (12). Most of these cytokines are macrophage products, which is consistent with the greater presence of macrophages documented for herniated disc tissues (9,10). These cytokines can promote lymphocyte activation and differentiation while also recruiting additional macrophages and activating phagocytosis and secretion of proteolytic enzymes. Activated Th1 lymphocytes produce IFNγ that assists in further macrophage recruitment and activation, and the mechanism eliciting the heightened IFNγ expression in herniated disc fragments may represent a specific immune response against herniated nucleus pulposus tissue. The immune privilege of the nucleus pulposus occurs by both vascular isolation and a biochemical phenotype with the Fas ligand causing infiltrating T lymphocyte apoptosis. Support for this theory is found in regional lymph node accumulation of lymphocytes after exposure to autologous nucleus pulposus (13), lymphocyte accumulation in the IVD after anulus fibrosus injury (6), and immunoglobulin and complement membrane attack complex deposition in human herniated disc tissue (14–16). Recent studies have revealed the importance of Th17 lymphocytes in a range of inflammatory pathologies and autoimmune diseases, including rheumatoid arthritis, Crohn’s disease, and multiple sclerosis (17,18). IL-17 is a proinflammatory cytokine produced primarily by Th17 lymphocytes activated in response to IL-23 production. IL-17 can promote inflammation by different signaling cascades, some modulated by other lymphocyte products such as IFNγ (19), and thereby promote the synthesis of other cytokines, proteases, NO, and PGE2. Given the demonstrated presence of infiltrating and activated lymphocytes in herniated disc fragments and the potential importance of immune system activation by herniated disc tissues, it is of interest to determine a potential role of Th17 lymphocytes in mediating this process. The objective of this study was to evaluate differences in the immunophenotype of the IVD fragments obtained from patients undergoing surgery for treatment of degenerative disc disease and lumbar disc herniation. The presence of Th17 lymphocyte markers in IVD pathology has not yet been investigated, and a primary goal of this study was to use immunohistochemistry to evaluate the molecular markers of different inflammatory and immune cells alongside expression of the lymphocyte product IL-17 in this surgically obtained tissue. Macrophage and lymphocyte markers as well as other mediators of inflammation known to be of relevance to IVD disease, including IL-6, IFNγ, IL-12, IL-10, and IL-4, were also evaluated to more comprehensively characterize the inflammation pattern. All markers were evaluated in degenerated and herniated disc fragments as well as in cadaveric nucleus pulposus and anulus fibrosus tissues with little to no evidence of degeneration. Results of this study reveal a high expression level of IL-17 in surgical tissues obtained from both degenerated and herniated IVDs, but not from autopsy tissue, suggesting a role of Th17-mediated inflammatory processes in IVD pathology. Additional results reveal the heightened presence of immune-activating IFNγ in herniated disc fragments, with concomitant greater macrophage infiltration and IL-6 expression as compared with degenerated tissues. These findings illustrate a pattern of immune activation by herniated disc tissue involving macrophage infiltration and activation, and further suggest that a novel lineage of Th17 lymphocytes may also be involved in both forms of IVD pathology.

Biochemical irritation of the dorsal root ganglion (DRG) after intervertebral disc herniation contributes to radiculopathy through tumor necrosis factor-alpha (TNFalpha)-mediated inflammation. Soluble TNF receptor Type II (sTNFRII) sequesters this cytokine, providing clinical benefit. Previous work involving conjugation of sTNFRII with thermally responsive elastin-like polypeptide (ELP) yielded a chimeric protein (ELP-sTNFRII) with in vitro anti-TNFalpha bioactivity. Furthermore, temperature-triggered ELP aggregation into a "depot" prolongs protein residence time following perineural injection. In this study the authors evaluated the inflammatory phenotype of DRG explants after TNFalpha stimulation, and assessed the abilities of sTNFRII or ELP-sTNFRII to attenuate these neuro-inflammatory changes.Rat lumbar DRGs (35 animals) were treated in 6 groups, as follows: control; TNFalpha (25 ng/ml); TNFalpha with low-(0.2 microg/ml) or high-dose (1 microg/ml) sTNFRII; and TNFalpha with low-(52.5 microg/ml) or high-dose (262.5 microg/ml) ELP-sTNFRII. After 24 hours, supernatant was evaluated for inflammatory cytokines (interleukin [IL]-1, IL-6, and IL-10); prostaglandin E2; and metabolites (glutamate, lactate, and pyruvate). Single-factor analysis of variance with post hoc Dunn analysis (alpha = 0.05) was used to assess treatment differences.Incubation of explants with TNFalpha caused metabolic stress reflected by an increased lactate/pyruvate ratio (1.8 +/- 0.5-fold) and extracellular glutamate (79 +/- 8% increase). Inflammatory activation was observed with heightened IL-6 release (5.2 +/- 1.4-fold) and prostaglandin E2 production (14 +/- 3-fold). An autoregulatory response occurred with an 11.8 +/- 0.6-fold increase in sTNFRI shedding. Treatment with high doses of sTNFRII or ELP-sTNFRII reversed all changes. Values are expressed as the mean +/- standard deviation.These results demonstrate that TNFalpha stimulation of DRG explants yields a phenotype of neurotoxic metabolite release and inflammatory mediator expression. Coincubation with either sTNFRII or ELP-sTNFRII antagonizes TNFalpha activity to abrogate these changes, suggesting potential for therapeutic intervention to treat peripheral nerve inflammatory disease.

Objective: The goal of this study was to evaluate gait and behavioral changes in an animal model of disc-herniation induced radiculopathy. A second objective was to correlate these functional changes to evidence of neuroinflammation and autoreactive lymphocyte immune activation. Methods: The animal model of radiculopathy involved harvesting autologous nucleus pulposus (NP) from a tail intervertebral disc, and then exposing the L5 dorsal root ganglion (DRG) by hemilaminectomy and partial facetectomy. Experimental animals (n = 16) received NP placement onto the DRG andcontrol animals (n = 16) underwent exposure only. At weekly time points, animals were evaluated for mechanical allodynia by Von Frey testing and for gait symmetry by digitized video analysis. At sacrifice, serum was evaluated for inflammatory cytokine content. The L5 DRGs were then evaluated by immunohistochemistry for mediators of inflammation and immune activation. Statistical analyses were at the ? = 0.05 level of significance, with Bonferroni corrections for multiple comparisons when appropriate. Results: Sensory testing revealed persistent mechanical allodynia in rats subjected to NP stimulus compared with the sham surgery group (Von Frey testing, p < 0.01). Gait analysis reflected the functional consequence of this altered sensation revealing marked asymmetry and apreference to place load on the contralateral limb (symmetry index, p < 0.01). Serum cytokine expressionwas equivalent between groups, reaffirming that the sensation and behavioral changes observed in these animals results primarily from local inflammatory changes. Immunohistochemicalanalysis of the sectioned DRGs after sacrifice revealed equivalent post-surgical inflammatory activation (IL23, p = 0.47), but substantial immuneactivation in the NP group (IL17, p = 0.01). Conclusion: This model of radiculopathy provides the first evidence of altered gait symmetry and locomotor ability in animals subjected to non-compressive placement of NP tissue. Systemic inflammation was absent as expected, but mechanical allodynia, local inflammation, and autoreactive immune activation were observed. Future work involves therapeutic interventions targeting these changes to rescue animals from the phenotype of inflammatory radiculopathy.

Table S1. Presenting source of probe/primers for real-time PCR (18s RNA is control). (DOCX 14 kb)

Table S1. Presenting source of probe/primers for real-time PCR (18s RNA is control). (DOCX 14 kb)