Hong Joo Moon

Annulus Fibrosus Cells Interact With Neuron-Like Cells to Modulate Production of Growth Factors and Cytokines in Symptomatic Disc Degeneration

Study Design. We hypothesized that AF/neuron interactions during annular injury were involved in neovascularization and nerve ingrowth, the pathologic hallmarks of symptomatic disc degeneration. Objective. To identify growth factors and inflammatory cytokines related to AF/neuron interactions using in vitro model. Summary of Background Data. Discogenic pain is the chronic intractable pain initiated by tears in the outer annulus fibrosus (AF); this is a unique structure with free nerve endings at outer one-third, located beside dorsal root ganglia. The relationship between AF and neuron cells in annular injury has not been extensively investigated. Methods. Human AF cells were cocultured with a retinoic acid (RA)-treated SH-SY5Y human neuroblastoma cell line (neuron-like cells). Conditioned media from cells cultured alone or in coculture were assayed for growth factors and inflammatory cytokines using enzyme-linked immunosorbent assays. The responses of the neuron-like cells, the AF cells, and the cocultured group to IL-1&bgr;/TNF-&agr; were compared using the same outcome measures. Results. RA-treated SH-SY5Y cells showed significant neurite outgrowth on the 7th day; this is a typical morphologic finding of neuron-like cells. Neuron-like cells produced vascular endothelial growth factor (VEGF) and IGF-1 under basal conditions and dose-dependently secreted small amounts of IL-8 in response to TNF-&agr;. Coculturing enhanced the secretion of VEGF, TGF-&bgr;1, and &bgr;-NGF, and suppressed the production of IGF-1. VEGF in the coculture group and the AF cells was downregulated by IL-1&bgr;/TNF-&agr; stimulation. IL-1&bgr;/TNF-&agr; stimulation enhanced the production of large amounts of IL-6 and IL-8 from AF cells; IL-1&bgr; produced a greater response than TNF-&agr;. The neuron-like cells did not produce detectable amounts of IL-6 or IL-8. Conclusion. These studies suggest that AF cells are involved in an inflammatory reaction and that the interactions between AF and neuron-like cells enhance the production of growth factors responsible for neovascularization and nerve ingrowth. AF injury has the potential to initiate neovascularization/nerve ingrowth and an inflammatory reaction through the interactions of AF and neural tissues.

Effects of secreted factors in culture medium of annulus fibrosus cells on microvascular endothelial cells: elucidating the possible pathomechanisms of matrix degradation and nerve in-growth in disc degeneration

OBJECTIVE To test whether the interaction between annulus fibrosus cells (AFCs) and endothelial cells (ECs) disrupts matrix homeostasis and stimulates production of innervation mediators. METHODS Human microvascular ECs were cultured in the conditioned media of AF cell culture derived from degenerated human surgical specimen. Matrix-metalloproteinases (MMPs) and platelet-derived growth factor (PDGF) of ECs of this culture were analyzed by qRT-PCR, Western, and immunofluorescence. Vascular endothelial growth factor (VEGF), Interleukin-8 (IL-8), and nerve growth factor (NGF) in the media of this cell culture were assayed by ELISA. To determine the effects of ECs on AFCs, qRT-PCR was performed to determine mRNA levels of collagen I, II and aggrecan in AFCs cultured in EC conditioned media. RESULTS Compared to ECs cultured in naïve media, ECs exposed to AFC conditioned media expressed higher mRNA and protein levels of key biomarkers of invasive EC phenotype, MMP-2 (2×), MMP-13 (4×), and PDGF-B (1.5-2×), and NGF (24.9 ± 15.2 pg/mL vs 0 in naïve media). Treatment of AF cells with EC culture conditioned media decreased collagen type II expression two fold. Considerable quantities of pro-angiogenic factors IL-8 (396.7 ± 302.0 pg/mL) and VEGF (756.2 ± 375.9 pg/mL) were also detected in the conditioned media of untreated AF cell culture. DISCUSSION AFCs from degenerated discs secreted factors which stimulated EC production of factors known to induce matrix degradation, angiogenesis, and innervation. IL-8 and VEGF maybe the secreted factors from AFCs which mediate a pro-angiogenic stimulus often implicated in the development of disc degeneration.

The angiogenic capacity from ligamentum flavum subsequent to inflammation: a critical component of the pathomechanism of hypertrophy.

Study Design. In vitro study about angiogenic potentiality of ligamentum flavum (LF) cells using coculture of human lumbar LF cells and activated macropage-like THP-1 cells. Objective. To test our hypothesis that activated LF, which was exposed to inflammation, induces angiogenesis, thus resulting in hypertrophy. Summary of Background Data. Inflammatory reactions after mechanical stress produce fibrosis and scarring of the LF that result in hypertrophy, a major pathological feature of spinal stenosis. This study evaluated the roles of LF cells in the pathomechanism of hypertrophy, focusing on angiogenesis. Methods. To determine their response to the inflammatory reaction, human LF cells were cocultured with phorbol myristate acetate-stimulated macrophage-like THP-1 cells. The conditioned media were assayed for tumor necrosis factor (TNF)-&agr;, interleukin (IL)-1&bgr;, IL-6, IL-8, vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-&bgr;1. Naïve and macrophage-exposed LF cells that responded to TNF-&agr;/IL-1&bgr; were compared using the same outcome measures. Hypertrophied LF tissue was stained by TGF-&bgr;1 primary antibody using immunohistochemical method. Results. Larger quantities of IL-6, IL-8, and VEGF were secreted by cocultured cells than by macrophages alone and LF cells alone combined. Prior macrophage exposure increased the secretion of IL-8 and VEGF in response to TNF-&agr;/IL-1&bgr; stimulation whereas IL-6 production was increased in response to IL-1&bgr;. The coculture appeared to increase TGF-&bgr;1 secretion but the level was lower than that for macrophage-like cells alone and LF cells alone combined. Conclusion. LF cells interact with macrophage-like cells to produce angiogenesis-related factors except TGF-&bgr;1. Activated LF cells that have been exposed to macrophage, can impact the inducement of angiogenesis-related factors, suggesting that fibrosis and scarring during inflammatory reaction is the major pathomechanism of LF hypertrophy.

Rabbit notochordal cells modulate the expression of inflammatory mediators by human annulus fibrosus cells cocultured with activated macrophage-like THP-1 cells.

Study Design. We evaluated the influence of rabbit notochordal cells on the expression of inflammatory mediators by human annulus fibrosus (AF) cells cocultured with macrophage-like cells. Objective. To identify the protective effect of rabbit notochordal cells on AF during in vitro inflammation. Summary of Background Data. Discogenic pain, which is an important cause of intractable lower back pain, is associated with macrophage-mediated inflammation in the AF. Although rabbit notochordal cells prevent intervertebral disc degeneration, their effects on human AF inflammation remain unknown. Methods. Human AF pellets were cocultured for 48 hours with notochordal cell clusters from adult New Zealand White rabbits and phorbol myristate acetate (PMA)-stimulated human macrophage-like THP-1 cells. Conditioned media (CM) from the cocultures were assayed by enzyme-linked immunosorbent assay. The expression of inflammatory mediators in the AF pellets was evaluated by real-time reverse-transcription polymerase chain reaction. Results. The levels of mRNA for interleukin (IL)-6, IL-8, and inducible nitric oxide synthase (iNOS) in the AF pellets cocultured with notochordal cells and macrophages (hAF[rNC-M]) were significantly lower than those in the AF pellets cultured with macrophages alone (hAF[M]) (P < 0.05). The levels of IL-6 and IL-8 proteins in the CM of hAF(rNC-M) were significantly lower than those in the CM of hAF(M) (P < 0.05). Coculturing with notochordal cells significantly decreased the levels of mRNA for IL-6, IL-8, and iNOS in the macrophage-exposed AF pellets (P < 0.05). After 1 ng/mL IL-1&bgr; stimulation, the levels of IL-6 and IL-8 mRNA and the level of IL-8 protein production were significantly decreased in the AF pellets with notochordal cells compared with naïve AF pellets (P < 0.05). Conclusion. In an in vitro coculture system, rabbit notochordal cells reduced the levels of main inflammatory mediators and gene expression in the human AF during inflammation. Therefore, rabbit notochordal cells may constitute an important protective tool against symptomatic disc development.

Analysis of Associating Factors With C2-7 Sagittal Vertical Axis After Two-level Anterior Cervical Fusion: Comparison Between Plate Augmentation and Stand-alone Cages

Study Design. A retrospective review. Objective. We investigated the longitudinal change of cervical alignment parameters including C2-7 lordosis, C2-7 sagittal vertical axis (SVA), T1 slope, and segmental angle (SA) after two-level anterior cervical discectomy and fusion (ACDF). Summary of Background Data. Cervical alignment may influence postoperative clinical outcomes. Several studies have suggested that cervical alignment may serve as a parameter for assessing cervical deformities similar to those used to assess thoracolumbar spine deformities. However, to our knowledge, no studies have investigated the effect of ACDF on cervical sagittal alignment. Methods. We enrolled patients whom had ACDF, 23 patients with stand-alone cages and 22 with plate augmentation. Radiologic parameters including C2-7 lordosis, C2-7 SVA, T1 slope, and SA at the operated level were evaluated preoperatively and at 1 week and 6 months postoperatively. The differences between preoperative and 6-month postoperative parameter values were designated as &Dgr;values. T1S-CL was calculated as the T1 slope minus C2-7 lordosis. Clinical outcome were obtained by the Visual Analog Scale (VAS) and the Neck Disability Index (NDI). Results. &Dgr;C2-7 SVA was significantly correlated with &Dgr;T1S-CL and &Dgr;C2-7 lordosis. &Dgr;C2-7 lordosis was significantly correlated with &Dgr;SA. &Dgr;C2-7 lordosis had a significantly greater impact on &Dgr;T1S-CL than did &Dgr;T1 slope. The &Dgr;SA and &Dgr;C2-7 lordosis in the ACDF-plate were significantly higher than those in the in ACDF-cage. &Dgr;T1S-CL and &Dgr;C2-7 SVA in the ACDF-plate were significantly lower than those in the ACDF-cage. Conclusion. C2-7 SVA after two-level ACDF was affected more significantly by the SA and C2-7 angle than by the T1 slope. Two-level ACDF with plate restored more cervical lordosis by obtaining more segmental lordosis at the operated level and was more effective in terms of cervical alignment compared with ACDF using stand-alone cages. Level of Evidence: 3

Induction of proinflammatory cytokine production in intervertebral disc cells by macrophage-like THP-1 cells requires mitogen-activated protein kinase activity

OBJECTIVE To determine the role played by mitogen-activated protein kinase (MAPK) signaling in the interactions between macrophages and intervertebral disc (IVD) cells, it was hypothesized that MAPK inhibition would modulate the production of the proinflammatory cytokines associated with inflammatory reaction in IVD cells. METHODS Human annulus fibrosus (AF) and nucleus pulposus (NP) cells were cocultured with phorbol myristate acetate-stimulated macrophage-like THP-1 cells, with and without SB202190 (a p38-α and -β inhibitor), SP600125 (a c-Jun N-terminal kinase [JNK] inhibitor), and PD98059 (an extracellular signal-regulated kinase [ERK] 1/2 inhibitor). The cytokines in conditioned media from cocultured and macrophage-exposed (nemotic) cells were assayed using enzyme-linked immunosorbent assays (ELISAs). RESULTS Interleukin (IL)-6 and IL-8 were secreted in greater quantities by the cocultured cells compared with naive IVD cells and macrophages (MΦ) cultured alone. The tumor necrosis factor (TNF)- α and IL-6 levels produced by the NP cells cocultured with MΦs (NP-MΦ) were significantly lower than those produced by AF cells cocultured with MΦs (AF-MΦ). SB202190 dose-dependently suppressed IL-6 secretion by AF-MΦ and NP-MΦ cocultures, and 10 μM SB202190 significantly decreased IL-6 and IL-8 production in nemotic AF and NP pellets. SP600125 at 10 μM significantly suppressed the production of TNF α IL-6. and IL-8 in AF-MΦ and NP-MΦ cocultures and significantly suppressed IL-1β production in the NP-MΦ coculture. Administration of 10 μM PD98059 significantly decreased IL-6 levels in the AF-MΦ coculture, and decreased the levels of TNF α and IL-8 in both the AF-MΦ and NP-MΦ cocultures. CONCLUSIONS The present study shows that inhibitors of p38 MAPK effectively controlled IL-6 production during inflammatory reactions and that JNK and ERK1/2 inhibitors successfully suppressed the production of major proinflammatory cytokines during interactions between macrophages and IVD cells. Therefore, selective blockade of these signals may serve as a therapeutic approach to symptomatic IVD degeneration.

Expression of matrix metalloproteinase-2 and -9 in human ligamentum flavum cells treated with tumor necrosis factor-α and interleukin-1β

OBJECT An in vitro study was performed to understand the potential roles of matrix metalloproteinase (MMP)-2 and MMP-9 in the elastin degradation of human ligamentum flavum (LF) cells via treatment with tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β). Previous studies have identified a decreased elastin to collagen ratio in hypertrophic LF. Among the extracellular matrix remodeling endopeptidases, MMP-2 and MMP-9 are known to have elastolytic activity. The hypothesis that activated LF cells exposed to inflammation would secrete MMP-2 and MMP-9, thereby resulting in elastin degradation, was examined. METHODS To examine MMP-2 and MMP-9 expression in human LF, cells were isolated and cultured from LF tissues that were obtained during lumbar disc surgery. Isolated LF cells were equally divided into 3 flasks and subcultured. Upon cellular confluency, the LF cells were treated with TNFα, IL-1β, or none (as a control) and incubated for 48 hours. The conditioned media were collected and assayed for MMP-2 and MMP-9 using gelatin zymography and Western blot analysis. The electrophoresis bands were compared on densitometric scans using ImageJ software. RESULTS The conditioned media from the isolated human LF cells naturally expressed 72-kD and 92-kD gelatinolytic activities on gelatin zymography. The IL-1β-treated LF cells presented sustained increases in the proenzyme/zymogen forms of MMP-2 and -9 (proMMP-2 and proMMP-9), and activeMMP-9 expression (p = 0.001, 0.022, and 0.036, respectively); the TNFα-treated LF cells showed the most elevated proMMP9 secretion (p = 0.006), as determined by Western blot analyses. ActiveMMP-2 expression was not observed on zymography or the Western blot analysis. CONCLUSIONS TNFα and IL-1β promote proMMP-2 and proMMP-9 secretion. IL-1β appears to activate proMMP-9 in human LF cells. Based on these findings, selective MMP-9 blockers or antiinflammatory drugs could be potential treatment options for LF hypertrophy.

The Korean Spinal Neurosurgery Society ; Are We Reimbursed Properly for Spinal Neurosurgical Practices under the Korean Resource Based Relative Value Scale Service?

Objectives The Korean Resource Based Relative Value Scale (K-RBRVS) was introduced in 2001 as an alternative of the previous medical fee schedule. Unfortunately, most neurosurgeons are unfamiliar with the details of the K-RBRVS and how it affects the reimbursement rates for the surgical procedures we perform. We summarize the K-RBRVS in brief, and discuss on how the relative value (RV) of the spinal neurosurgical procedures have changed since the introduction in 2001. Methods We analyzed the change of spinal procedure RVs since 2001, and compared it with the change of values in the brain neurosurgical procedures. RVs of 88 neurospinal procedures on the list of K-RBRVS were analyzed, while 24 procedures added during annual revisions were excluded. Results During the past 15 years, RVs for spinal procedures have increased 62.8%, which is not so different with the cumulative increase of consumer prices during this time period or the increase rate of 92.3% for brain surgeries. When comparing the change of RVs in more complex procedures between spinal and brain neurosurgery, the increase rate was 125.3% and 133%, respectively. Conclusion More effort of the society of spinal surgeons seems to be needed to get adequate reimbursement, as there have been some discrimination compared to brain surgeons in the increase of RVs. And considering the relative underestimation of spinal neurosurgeons’ labor, more objective measures of neurospinal surgeons’ work and productivity should be developed for impartial reimbursement.

Metastases to Pituitary: A Case Report and Review of Literature

Pituitary metastasis (PM) secondary to systemic malignancies has been reported in the literature. Variety of clinical and neuroimaging presentation has been reported; however the diagnosis of PM is challenging. We report a case of a 44-year-old male with PM from non–small cell lung cancer (stage IV). He presented with sudden onset polyuria, polydypsia, and visual disturbance. Laboratory evaluation revealed pan-hypopituitarism and visual field test showed bitemporal lower quadrantanopsia. Brain magnetic resonance imaging demonstrated a suprasellar mass with focal hemorrhage and thickening of infundibular stalk. Surgical resection of the tumor followed by chemoradiotherapy was employed. Histopathologic examination of the tumor specimen revealed metastatic adenocarcinoma and immunostaning demonstrated findings consistent with lung carcinoma. Visual disturbances improved postoperatively and the patient is tumor free with no clinical or radiologic evidence of recurrence at 19 months of follow-up. The review of literature is included with the goal of elucidating the clinical presentation, imaging diagnosis, histogenesis, and the treatment strategies associated with the PM.