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Kjell Olmarker - One of the best experts on this subject based on the ideXlab platform.
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Open Access Puncture of a Disc and Application of Nucleus Pulposus Induces Disc Herniation-Like Changes and Osteophytes. An Experimental Study in Rats
2013Co-Authors: Kjell OlmarkerAbstract:Abstract: It has been observed that puncture of a lumbar disc may induce formation of a nodule on the surface of the disc and osteophytes. It is not known if this is based on the presence of a foreign tissue or specifically by the presence of Nucleus Pulposus or on the disc injury. In this study these mechanisms were separated by comparing disc puncture with application of Nucleus Pulposus without disc injury, with superficial disc injury without Nucleus Pulposus and with application of fat. Fifty rats underwent facetectomy of the left L4-5 facet. Ten additional rats were used as donor rats. The rats were exposed to disc puncture (n=10), application of homologous Nucleus Pulposus (n=10), application of homologous fat tissue (n=10), superficial disc injury (n=10) and ten rats served as control. After 3 weeks the rats were examined macroscopically regarding presence of disc nodules and osteophytes. A limited histological analysis was performed to obtain a microscopic overview of any observed changes. In rats with application of fat, superficial disc injury and in sham controls there were almost no changes observed. However, in rats with disc puncture and applied Nucleus Pulposus there were clear disc nodules and osteophytes noted. Microscopically the nodules comprised granulation tissue and the osteophytes cortical bone. In conclusion, the data indicate that the presence of Nucleus Pulposus is more likely to be responsible for the formation of disc nodules and osteophytes than disc injury or the presence of a foreign tissue. This may provide new insights in the mechanisms regarding the formation of disc herniations and osteophytes
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Open Access Pain Behavior Changes Following Disc Puncture Relate to Nucleus Pulposus Rather than to the Disc Injury Per Se: An Experimental Study in Rats
2013Co-Authors: Elin Nilsson, Toshio Nakamae, Kjell OlmarkerAbstract:Abstract: It has previously been demonstrated that disc puncture in the rat induced changes in grooming and wet dog shakes, two behavioral changes that may be linked to discomfort and neuropathic pain. In this study the aim was to separate the effects of disc injury and the epidural presence of Nucleus Pulposus. Following anesthesia, the L4-5 disc was exposed using a dorsal approach. Ten rats received a superficial disc injury without Nucleus Pulposus leakage and ten rats received Nucleus Pulposus from a donor rat without disc injury. In ten animals the L4-5 disc was punctured using a ventral approach, with 10 corresponding controls. Spontaneous behavior was assessed after surgery. The data was matched to historical control of dorsal sham surgery and disc puncture. The study showed that the effects of Nucleus Pulposus were more pronounced than the effects induced by the disc injury. Ventral disc puncture did not induce any behavioral changes different from sham exposure. In conclusion, the data from the study indicate that behavioral changes induced by disc puncture are more likely to relate to the epidural presence of Nucleus Pulposus than the disc injury per se
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macrophage appearance in the epineurium and endoneurium of dorsal root ganglion exposed to Nucleus Pulposus
Journal of The Peripheral Nervous System, 2004Co-Authors: Yasuaki Murata, Björn Rydevik, Kazuhisa Takahashi, Ichiro Takahashi, Kjell OlmarkerAbstract:Recently, it was observed that local application of Nucleus Pulposus may induce a characteristic reaction at the surface of the dorsal root ganglion (DRG). This change was inflammatory in nature and occurred as early as 1 day after the application of Nucleus Pulposus. Herniation of the Nucleus Pulposus was surgically induced in the L4-5 disc in rats. The L4 DRGs were resected 3, 24, and 72 h after surgery and sectioned. The sections were processed for immunohistochemistry using antisera to the macrophage marker ED1 and observed using light microscopy. The appearance of macrophages was confirmed 3, 24, and 72 h after the surgery. Macrophages were mainly distributed in the epineurial space of the DRG 3 h after disc incision and also in the endoneurial tissue 24 and 72 h after disc incision. The immunoreactivity was significantly stronger at 24 and 72 h than at 3 h in the parts of the DRG without apparent changes in the disc incision series (p < 0.01). Within the epineurium adjacent to application of Nucleus Pulposus, the number was significantly higher at 3 h than at 24 and 72 h (p < 0.05). We conclude that experimental disc herniation with leakage of Nucleus Pulposus results in macrophage recruitment to the epineurium of the DRG 3 h after disc incision and to the endoneurium 24 and 72 h after disc incision.
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Nucleus Pulposus-induced Nerve Root Injury: Relationship between Blood Flow and Motor Nerve Conduction Velocity
Neurosurgery, 1999Co-Authors: Koji Otani, Kjell Olmarker, Itaru Arai, Guang-ping Mao, Shinichi Konno, Shinichi KikuchiAbstract:OBJECTIVE: It is well known that Nucleus Pulposus induces nerve root injury. The aim of this study was to assess the relationship between intraneural blood flow and motor nerve conduction velocity (NCV) after incision of the adjacent disc. METHODS: A total of 65 dogs were used. A left hemilaminotomy was performed, the annulus fibrosus of the L6-L7 intervertebral disc was incised, and Nucleus Pulposus was gently pushed into the epidural space by saline solution injection. A left hemilaminotomy without disc incision was used as the sham operation. Seven dogs were used for incision and five dogs for sham treatment for each of the following time points: 1 day, 3 days, 1 week, 1 month, and 2 months of exposure. Five additional dogs were used to establish baseline data. Blood flow in the nerve root was measured in the left L7 nerve root with a tissue blood flowmeter, using an electrolytic hydrogen clearance method. Motor NCV over the exposed area of the nerve root was measured using a neurophysiological technique. RESULTS: There was a reduction in blood flow in the nerve root after disc incision that began after 1 day and was maximal after 1 week. This reduction had resolved by 1 month, however. The motor NCV showed a reduction pattern similar to that for blood flow in the nerve root, but reduction did not begin until 3 days after disc incision and was not fully resolved until 2 months. CONCLUSION: This study demonstrates that the reduction and recovery of motor NCV are related to, and preceded by, a reduction in blood flow in the nerve root. The data might provide important information regarding the basic pathophysiological mechanisms of Nucleus Pulposus-induced nerve root injury.
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tumor necrosis factor α and Nucleus Pulposus induced nerve root injury
Spine, 1998Co-Authors: Kjell Olmarker, Karin LarssonAbstract:STUDY DESIGN The effects of Nucleus Pulposus and various treatments to block tumor necrosis factor alpha activity were evaluated in an experimental set-up using immunohistochemistry and nerve conduction velocity recordings. OBJECTIVES To assess the presence of tumor necrosis factor alpha in pig Nucleus Pulposus cells, and to see if block of tumor necrosis factor alpha also blocks the Nucleus-Pulposus-induced reduction of nerve root conduction velocity. SUMMARY AND BACKGROUND DATA A meta-analysis of observed effects induced by Nucleus Pulposus revealed that these effects might relate to one specific cytokine-tumor necrosis factor alpha. METHODS Series-1: Cultured Nucleus Pulposus cells were stained immunohistologically with a monoclonal antibody for tumor necrosis factor alpha. Series-2: Nucleus Pulposus was harvested from lumbar discs and applied to the sacrococcygeal cauda equina in 13 pigs autologously. Four pigs received 100 mg of doxycycline intravenously; five pigs had a blocking monoclonal antibody to tumor necrosis factor alpha applied locally in the Nucleus Pulposus, and four pigs remained nontreated, forming a control group. Three days after the application, the nerve root conduction velocity was determined over the application zone by local electrical stimulation. RESULTS Series-1: Tumor necrosis factor alpha was found to be present in the Nucleus Pulposus cells. Series-2: The selective antibody to tumor necrosis factor alpha limited the reduction of nerve conduction velocity, although in comparison with the control group this was not statistically significant. However, treatment with doxycycline significantly blocked the Nucleus-Pulposus-induced reduction of conduction velocity. CONCLUSION For the first time, a specific substance, tumor necrosis factor alpha, has been linked to the Nucleus-Pulposus-induced effects of nerve roots after local application. Although the effects of this substance may be synergistic with those of other similar substances, the data of the current study may be of significant importance for the continued understanding of Nucleus Pulposus' biologic activity, and of possible potential use for future strategies in managing sciatica.
Daisuke Sakai - One of the best experts on this subject based on the ideXlab platform.
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angiopoietin 1 receptor tie2 distinguishes multipotent differentiation capability in bovine coccygeal Nucleus Pulposus cells
Stem Cell Research & Therapy, 2016Co-Authors: Adel Tekari, Daisuke Sakai, Sibylle Grad, Samantha C W Chan, Benjamin GantenbeinAbstract:Background The intervertebral disc (IVD) has limited self-healing potential and disc repair strategies require an appropriate cell source such as progenitor cells that could regenerate the damaged cells and tissues. The objective of this study was to identify Nucleus Pulposus-derived progenitor cells (NPPC) and examine their potential in regenerative medicine in vitro.
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angiopoietin 1 receptor tie2 distinguishes multipotent differentiation capability in bovine coccygeal Nucleus Pulposus cells
Stem Cell Research & Therapy, 2016Co-Authors: Adel Tekari, Daisuke Sakai, Sibylle Grad, Samantha Chan, Benjamin GantenbeinAbstract:The intervertebral disc (IVD) has limited self-healing potential and disc repair strategies require an appropriate cell source such as progenitor cells that could regenerate the damaged cells and tissues. The objective of this study was to identify Nucleus Pulposus-derived progenitor cells (NPPC) and examine their potential in regenerative medicine in vitro. Nucleus Pulposus cells (NPC) were obtained from 1-year-old bovine coccygeal discs by enzymatic digestion and were sorted for the angiopoietin-1 receptor Tie2. The obtained Tie2– and Tie2+ fractions of cells were differentiated into osteogenic, adipogenic, and chondrogenic lineages in vitro. Colony-forming units were prepared from both cell populations and the colonies formed were analyzed and quantified after 8 days of culture. In order to improve the preservation of the Tie2+ phenotype of NPPC in monolayer cultures, we tested a selection of growth factors known to have stimulating effects, cocultured NPPC with IVD tissue, and exposed them to hypoxic conditions (2 % O2). After 3 weeks of differentiation culture, only the NPC that were positive for Tie2 were able to differentiate into osteocytes, adipocytes, and chondrocytes as characterized by calcium deposition (p < 0.0001), fat droplet formation (p < 0.0001), and glycosaminoglycan content (p = 0.0095 vs. Tie2– NPC), respectively. Sorted Tie2– and Tie2+ subpopulations of cells both formed colonies; however, the colonies formed from Tie2+ cells were spheroid in shape, whereas those from Tie2– cells were spread and fibroblastic. In addition, Tie2+ cells formed more colonies in 3D culture (p = 0.011) than Tie2– cells. During expansion, a fast decline in the fraction of Tie2+ cells was observed (p < 0.0001), which was partially reversed by low oxygen concentration (p = 0.0068) and supplementation of the culture with fibroblast growth factor 2 (FGF2) (p < 0.0001). Our results showed that the bovine Nucleus Pulposus contains NPPC that are Tie2+. These cells fulfilled formally progenitor criteria that were maintained in subsequent monolayer culture for up to 7 days by addition of FGF2 or hypoxic conditions. We propose that the Nucleus Pulposus represents a niche of precursor cells for regeneration of the IVD.
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exhaustion of Nucleus Pulposus progenitor cells with ageing and degeneration of the intervertebral disc
Nature Communications, 2012Co-Authors: Daisuke Sakai, Tomoko Nakai, Makarand V Risbud, Sibylle Grad, Mauro Alini, Yoshihiko Nakamura, Taishi Mishima, Shunichi Kato, Danny Chan, Kathryn S E CheahAbstract:Despite the high prevalence of intervertebral disc disease, little is known about changes in intervertebral disc cells and their regenerative potential with ageing and intervertebral disc degeneration. Here we identify populations of progenitor cells that are Tie2 positive (Tie2+) and disialoganglioside 2 positive (GD2+), in the Nucleus Pulposus from mice and humans. These cells form spheroid colonies that express type II collagen and aggrecan. They are clonally multipotent and differentiated into mesenchymal lineages and induced reorganization of Nucleus Pulposus tissue when transplanted into non-obese diabetic/severe combined immunodeficient mice. The frequency of Tie2+ cells in tissues from patients decreases markedly with age and degeneration of the intervertebral disc, suggesting exhaustion of their capacity for regeneration. However, progenitor cells (Tie2+GD2+) can be induced from their precursor cells (Tie2+GD2-) under simple culture conditions. Moreover, angiopoietin-1, a ligand of Tie2, is crucial for the survival of Nucleus Pulposus cells. Our results offer insights for regenerative therapy and a new diagnostic standard.
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feasibility of using a human Nucleus Pulposus cell line as a cell source in cell transplantation therapy for intervertebral disc degeneration
Spine, 2006Co-Authors: Tsukasa Iwashina, Yukihiro Yamamoto, Joji Mochida, Kiyoshi Ando, Daisuke Sakai, Takeshi Miyazaki, Tomomitsu HottaAbstract:Study Design. Assessment of the potential use of an immortalized human Nucleus Pulposus cell line as an alternative cell source in cell transplantation therapy for intervertebral disc degeneration. Objectives. To evaluate the effect of transplanting the human Nucleus Pulposus cell line into a disc degeneration model in rabbits and to define whether it is capable of becoming an alternative cell source for cell transplantation therapy for disc degeneration. Summary of Background Data. Interest in cell transplantation therapy for disc degeneration has been growing for several years, and a range of different cell types have been examined as possible donor cells. In addition, the establishment of a novel cell line that possesses some of the major characteristics of a normal human Nucleus Pulposus cells has been reported. Methods. Human Nucleus Pulposus cell line was established, and cells were transplanted into a rabbit disc degeneration model. At 4, 8, and 24 weeks after transplantation, inhibition of intervertebral disc degeneration was assessed by examining the disc height, macroscopic appearance, histologic findings, and immunohistochemistry. In addition, aggrecan, versican, and Type II collagen gene expression in the Nucleus Pulposus were measured semiquantitatively at the mRNA level. Furthermore, the survival of transplanted cells was examined using immunohistochemistry for Simian Virus 40 T antigen, and the presence of graft-versus-host reaction was assessed by immunohistochemistry for CD4 and CD58. Results. The disc height was significantly greater in the transplanted group than in the degenerative group’s disc from 4 weeks’ posttransplantation. Macroscopically, the Nucleus Pulposus was absent and there was loss ofdisc height in the degenerative group at 24 weeks after transplantation, whereas the Nucleus Pulposus was preserved in the transplanted group. Histologic examination showed that the structure of the inner anulus fibrosus was significantly preserved in the transplanted group, and the boundary between the Nucleus and anulus could be clearly visualized. Expression of mRNAs of the Nucleus Pulposus matrix, aggrecan, and Type II collagen was significantly greater in the transplanted group than in the degenerative group. This indicates that transplantation of human Nucleus Pulposus cell line helped to preserve the matrix of the Nucleus Pulposus. Thus, transplantation of a human Nucleus Pulposus cell line was shown to delay disc degeneration in this rabbit model. Conclusion. The human Nucleus Pulposus cell line may become an alternative cell source for cell transplantation therapy of intervertebral disc degeneration.
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atelocollagen for culture of human Nucleus Pulposus cells forming Nucleus Pulposus like tissue in vitro influence on the proliferation and proteoglycan production of hnpsv 1 cells
Biomaterials, 2006Co-Authors: Daisuke Sakai, Joji Mochida, Takuya Watanabe, Kiyoshi Ando, Tsukasa Iwashina, Kaori Suyama, Tomomitsu HottaAbstract:Nucleus Pulposus (NP) is responsible for maintaining function and structure of the disc. Scaffolds to culture disc cells three-dimensionally are emphasized in recent reports on development of a new method for treating disc degeneration using cell transplantation and tissue engineering. Among artificial scaffolds and cell carrying materials, Atelocollagen is a collagen gel that has an advantage in safety issues over others. However, to date there has been no study that investigated culture of human Nucleus Pulposus cells in Atelocollagen. To investigate whether Atelocollagen could be used as a culture scaffold and if it has any effect on cell proliferation and proteoglycan (PG) production, as well as to find the optimal commercially available Atelocollagen for NP cell transplantation and tissue engineering, we cultured human NP cell line HNPSV-1, in three different Atelocollagen and compared with alginate. Furthermore, NP-like tissues were generated using these cells and different Atellocollagen solutions. Results showed that both DNA synthesis and content is significantly greater when cultured in Atelocollagen than in alginate. On the other hand, proteoglycan synthesis and accumulation was significantly greater in alginate compared with the 0.3% Atelocollagen scaffolds; with 3% Atelocollagen, however, results were similar. NP-like tissue generated by Atelocollagen showed good water and proteoglycan preservation. The current study demonstrates that the use of Atelocollagen as an in vitro culture scaffold for three-dimensional culture of human NP cell lines is indeed feasible and moreover, Atelocollagen possesses the potential to become a candidate scaffold for cell transplantation or tissue engineering for the treatment of intervertebral disc degeneration.
Robert R Myers - One of the best experts on this subject based on the ideXlab platform.
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2000 volvo award winner in basic science studies exogenous tumor necrosis factor alpha mimics Nucleus Pulposus induced neuropathology molecular histologic and behavioral comparisons in rats
Spine, 2000Co-Authors: Tamaki Igarashi, Shinichi Kikuchi, Veronica I Shubayev, Robert R MyersAbstract:STUDY DESIGN This study tested the hypothesis that the 17-kDa form of tumor necrosis factor-alpha is the pathophysiologic agent expressed by herniated Nucleus Pulposus in vivo that is primarily responsible for the histologic and behavioral manifestations of experimental sciatica associated with herniated lumbar discs. OBJECTIVE The authors determined the molecular weight and concentration of active tumor necrosis factor-alpha in rat herniated disc and used exogenous tumor necrosis factor-alpha at the same molecular weight to study its neuropathologic effect on rat nerve root and dorsal root ganglion preparations in vivo. SUMMARY OF BACKGROUND DATA Expressed by herniated Nucleus Pulposus in culture, tumor necrosis factor-alpha causes neuropathologic injury in nerve roots and neuropathic pain states in which mechanical allodynia is seen in response to peripheral stimuli. METHODS Western blotting was used to identify the molecular weight of the operative tumor necrosis factor-alpha protein form, and measures of optical density were used for semiquantitative determination of concentration. Plastic-embedded nerve roots and dorsal root ganglion were used for neuropathologic evaluation, and von Frey stimulation was used to quantify mechanical allodynia. RESULTS The 17-kDa form of tumor necrosis factor-alpha is expressed by herniated Nucleus Pulposus at a concentration of approximately 0.48 ng per herniated rat lumbar disc. Exogenous tumor necrosis factor-alpha applied in vivo to rat nerve roots produced neuropathologic changes and behavior deficits that mimicked experimental studies with herniated Nucleus Pulposus applied to nerve roots. CONCLUSIONS The data reinforce other evidence that tumor necrosis factor-alpha is involved in mechanisms of neuropathic pain.
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acute effects of Nucleus Pulposus on blood flow and endoneurial fluid pressure in rat dorsal root ganglia
Spine, 1998Co-Authors: Shoji Yabuki, Kjell Olmarker, Shinichi Kikuchi, Robert R MyersAbstract:Study design An experimental study to elucidate the initial factors in the pathogenesis of lumbar pain caused by disc herniation. Objective To evaluate the effects of autologous Nucleus Pulposus on blood flow and endoneurial fluid pressure in dorsal root ganglia. Summary of background data Human sciatica is known to be associated with compression of lumbar nerve roots and dorsal root ganglia by herniated intervertebral discs. Recently, it has been shown that application of Nucleus Pulposus to nerve roots induces injury and pain-related behavior in experimental animals. In this study, the authors hypothesized that Nucleus Pulposus applied to a nerve root would cause increased intraneural edema and reduced blood flow in the corresponding dorsal root ganglia. Studies in peripheral nerves have shown that these initial pathophysiologic disturbances initiate complex events that exacerbate nerve injury and cause pain. Methods A total of 29 adult female Sprague-Dawley rats weighing 200 to 250 g had their left L5 nerve roots and associated dorsal root ganglia exposed. Autologous Nucleus Pulposus was harvested from the tail and applied to the L5 nerve root just proximally to the dorsal root ganglia (Nucleus Pulposus group). For control, the same volume of muscle was harvested from the surgical area in the back and applied similarly to the neural tissue (control group). Blood flow was continuously monitored using a laser Doppler flow probe for 3 hours (n = 10) or 4 hours (n = 8) in animals with indwelling cannulas for measurement of systemic arterial pressure. Endoneurial fluid pressures were recorded with a servonull micropipette system using glass micropipettes with tip diameters of 4 microns. Endoneurial fluid pressure in the dorsal root ganglia was measured before and 3 hours after application of Nucleus Pulposus (n = 7) or muscle (n = 4). After measurement of blood flow and endoneurial fluid pressure, the nerve root and dorsal root ganglia were processed for histology and evaluated by light microscope. Results Blood flow in the Nucleus Pulposus group was reduced by 10% to 20% from the initial value after 3 to 4 hours. This reduction was statistically significant compared with that of the control group (P 0.05), and 2.0 +/- 0.8 in the control group (P > 0.05). Edema was the principal pathologic finding seen consistently in the nerve roots and in many of the associated dorsal root ganglia from animals treated with Nucleus Pulposus. Conclusion Application of Nucleus Pulposus to nerve root increased endoneurial fluid pressure and decreased blood flow in the dorsal root ganglia. This study's acute observations in the dorsal root ganglia may thus help to explain why disc herniations without compression of neural tissue are sometimes painful because similar pathologic findings are observed after only Nucleus Pulposus application to the nerve root. The authors further suggest that exposure of nerve roots to Nucleus Pulposus may establish a "compartment syndrome" in the dorsal root ganglia.
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pathogenesis of sciatic pain role of herniated Nucleus Pulposus and deformation of spinal nerve root and dorsal root ganglion
Pain, 1998Co-Authors: Kjell Omarker, Robert R MyersAbstract:The basic pathophysiologic mechanisms related to disc herniation and sciatica are poorly understood. Recently it was demonstrated that Nucleus Pulposus from an intervertebral disc could induce structural and functional changes in adjacent nerve roots when applied epidurally, however, it is not known if such changes are painful. In a model for inducing disc herniation in the rat, we found that puncture of a lumbar disc with subsequent herniation of Nucleus Pulposus without nerve root compression, or chronic displacement of the 4th lumbar nerve root and ganglion, did not individually induce significant changes in thresholds for mechanical or thermal stimulation compared to sham-operated animals. However, the combination of disc puncture and displacement induced a reduction of the threshold for thermal stimulation, indicating hyperalgesia, that was present 2 days after surgery and gradually recovered during a 14-day period. These data and the associated description of this new model for experimental disc herniation may increase our understanding of the pathophysiologic events leading to sciatica and help in evaluating new modalities for diagnosis and treatment of disc herniation and sciatica.
Shinichi Kikuchi - One of the best experts on this subject based on the ideXlab platform.
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participation of 5 hydroxytryptamine in pain related behavior induced by Nucleus Pulposus applied on the nerve root in rats
Spine, 2008Co-Authors: Kinshi Kato, Shinichi Kikuchi, Shinichi Konno, Miho SekiguchiAbstract:STUDY DESIGN: The role of 5-hydroxytryptamine (5-HT) in sciatica in lumbar disc herniation (LDH) in rats was investigated. OBJECTIVE: We evaluated the effects of exogenous 5-HT applied on the nerve root on pain-related behavior, the release of endogenous 5-HT in plasma, and the expression of 5-HT2A receptors in dorsal root ganglion (DRG) in a rat LDH model. SUMMARY OF BACKGROUND DATA: In previous studies, 5-HT2A receptor antagonists improved sciatica in patients with LDH and attenuated pain-related behavior induced by Nucleus Pulposus applied to the nerve root in rats. METHODS: Adult female Sprague-Dawley rats were divided into four experimental groups [control group; low-dose (10 microg) 5-HT-group; high-dose (30 microg) 5-HT-group; and autologous Nucleus Pulposus (NP) and saline group] and each drug was applied to the L5 nerve root. Von Frey tests were used for pain-behavior testing. To assess levels of endogenous 5-HT released in capillaries surrounding inflamed nerve roots, we measured 5-hydroxyindole acetic acid (5-HIAA), a metabolite of 5-HT. Expression of 5-HT2A receptors in the left L5 DRG was examined by immunohistochemical and immunoblotting analyses in the control and NP groups. RESULTS: Mechanical withdrawal thresholds of the high-dose 5-HT and the NP groups were significantly decreased after surgery compared with the control group and recovered after 14 days in the high-dose 5-HT group. 5-HIAA in plasma was increased by Nucleus Pulposus applied on the nerve root for 7 days after surgery. The expression of 5-HT2A receptors was enhanced in a time-dependent manner by Nucleus Pulposus. CONCLUSION: Exogenous 5-HT to the nerve root induced pain-related behavior with short-lasting effects compared with the Nucleus Pulposus application. 5-HIAA content in plasma and expression of 5-HT2A receptors in DRG neurons increased early time points after the Nucleus Pulposus application. These results suggest that 5-HT plays a role in the early phase of the chemical pathogenesis of sciatica in LDH in rats.
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2000 volvo award winner in basic science studies exogenous tumor necrosis factor alpha mimics Nucleus Pulposus induced neuropathology molecular histologic and behavioral comparisons in rats
Spine, 2000Co-Authors: Tamaki Igarashi, Shinichi Kikuchi, Veronica I Shubayev, Robert R MyersAbstract:STUDY DESIGN This study tested the hypothesis that the 17-kDa form of tumor necrosis factor-alpha is the pathophysiologic agent expressed by herniated Nucleus Pulposus in vivo that is primarily responsible for the histologic and behavioral manifestations of experimental sciatica associated with herniated lumbar discs. OBJECTIVE The authors determined the molecular weight and concentration of active tumor necrosis factor-alpha in rat herniated disc and used exogenous tumor necrosis factor-alpha at the same molecular weight to study its neuropathologic effect on rat nerve root and dorsal root ganglion preparations in vivo. SUMMARY OF BACKGROUND DATA Expressed by herniated Nucleus Pulposus in culture, tumor necrosis factor-alpha causes neuropathologic injury in nerve roots and neuropathic pain states in which mechanical allodynia is seen in response to peripheral stimuli. METHODS Western blotting was used to identify the molecular weight of the operative tumor necrosis factor-alpha protein form, and measures of optical density were used for semiquantitative determination of concentration. Plastic-embedded nerve roots and dorsal root ganglion were used for neuropathologic evaluation, and von Frey stimulation was used to quantify mechanical allodynia. RESULTS The 17-kDa form of tumor necrosis factor-alpha is expressed by herniated Nucleus Pulposus at a concentration of approximately 0.48 ng per herniated rat lumbar disc. Exogenous tumor necrosis factor-alpha applied in vivo to rat nerve roots produced neuropathologic changes and behavior deficits that mimicked experimental studies with herniated Nucleus Pulposus applied to nerve roots. CONCLUSIONS The data reinforce other evidence that tumor necrosis factor-alpha is involved in mechanisms of neuropathic pain.
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Nucleus Pulposus-induced Nerve Root Injury: Relationship between Blood Flow and Motor Nerve Conduction Velocity
Neurosurgery, 1999Co-Authors: Koji Otani, Kjell Olmarker, Itaru Arai, Guang-ping Mao, Shinichi Konno, Shinichi KikuchiAbstract:OBJECTIVE: It is well known that Nucleus Pulposus induces nerve root injury. The aim of this study was to assess the relationship between intraneural blood flow and motor nerve conduction velocity (NCV) after incision of the adjacent disc. METHODS: A total of 65 dogs were used. A left hemilaminotomy was performed, the annulus fibrosus of the L6-L7 intervertebral disc was incised, and Nucleus Pulposus was gently pushed into the epidural space by saline solution injection. A left hemilaminotomy without disc incision was used as the sham operation. Seven dogs were used for incision and five dogs for sham treatment for each of the following time points: 1 day, 3 days, 1 week, 1 month, and 2 months of exposure. Five additional dogs were used to establish baseline data. Blood flow in the nerve root was measured in the left L7 nerve root with a tissue blood flowmeter, using an electrolytic hydrogen clearance method. Motor NCV over the exposed area of the nerve root was measured using a neurophysiological technique. RESULTS: There was a reduction in blood flow in the nerve root after disc incision that began after 1 day and was maximal after 1 week. This reduction had resolved by 1 month, however. The motor NCV showed a reduction pattern similar to that for blood flow in the nerve root, but reduction did not begin until 3 days after disc incision and was not fully resolved until 2 months. CONCLUSION: This study demonstrates that the reduction and recovery of motor NCV are related to, and preceded by, a reduction in blood flow in the nerve root. The data might provide important information regarding the basic pathophysiological mechanisms of Nucleus Pulposus-induced nerve root injury.
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acute effects of Nucleus Pulposus on blood flow and endoneurial fluid pressure in rat dorsal root ganglia
Spine, 1998Co-Authors: Shoji Yabuki, Kjell Olmarker, Shinichi Kikuchi, Robert R MyersAbstract:Study design An experimental study to elucidate the initial factors in the pathogenesis of lumbar pain caused by disc herniation. Objective To evaluate the effects of autologous Nucleus Pulposus on blood flow and endoneurial fluid pressure in dorsal root ganglia. Summary of background data Human sciatica is known to be associated with compression of lumbar nerve roots and dorsal root ganglia by herniated intervertebral discs. Recently, it has been shown that application of Nucleus Pulposus to nerve roots induces injury and pain-related behavior in experimental animals. In this study, the authors hypothesized that Nucleus Pulposus applied to a nerve root would cause increased intraneural edema and reduced blood flow in the corresponding dorsal root ganglia. Studies in peripheral nerves have shown that these initial pathophysiologic disturbances initiate complex events that exacerbate nerve injury and cause pain. Methods A total of 29 adult female Sprague-Dawley rats weighing 200 to 250 g had their left L5 nerve roots and associated dorsal root ganglia exposed. Autologous Nucleus Pulposus was harvested from the tail and applied to the L5 nerve root just proximally to the dorsal root ganglia (Nucleus Pulposus group). For control, the same volume of muscle was harvested from the surgical area in the back and applied similarly to the neural tissue (control group). Blood flow was continuously monitored using a laser Doppler flow probe for 3 hours (n = 10) or 4 hours (n = 8) in animals with indwelling cannulas for measurement of systemic arterial pressure. Endoneurial fluid pressures were recorded with a servonull micropipette system using glass micropipettes with tip diameters of 4 microns. Endoneurial fluid pressure in the dorsal root ganglia was measured before and 3 hours after application of Nucleus Pulposus (n = 7) or muscle (n = 4). After measurement of blood flow and endoneurial fluid pressure, the nerve root and dorsal root ganglia were processed for histology and evaluated by light microscope. Results Blood flow in the Nucleus Pulposus group was reduced by 10% to 20% from the initial value after 3 to 4 hours. This reduction was statistically significant compared with that of the control group (P 0.05), and 2.0 +/- 0.8 in the control group (P > 0.05). Edema was the principal pathologic finding seen consistently in the nerve roots and in many of the associated dorsal root ganglia from animals treated with Nucleus Pulposus. Conclusion Application of Nucleus Pulposus to nerve root increased endoneurial fluid pressure and decreased blood flow in the dorsal root ganglia. This study's acute observations in the dorsal root ganglia may thus help to explain why disc herniations without compression of neural tissue are sometimes painful because similar pathologic findings are observed after only Nucleus Pulposus application to the nerve root. The authors further suggest that exposure of nerve roots to Nucleus Pulposus may establish a "compartment syndrome" in the dorsal root ganglia.
Makarand V Risbud - One of the best experts on this subject based on the ideXlab platform.
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exhaustion of Nucleus Pulposus progenitor cells with ageing and degeneration of the intervertebral disc
Nature Communications, 2012Co-Authors: Daisuke Sakai, Tomoko Nakai, Makarand V Risbud, Sibylle Grad, Mauro Alini, Yoshihiko Nakamura, Taishi Mishima, Shunichi Kato, Danny Chan, Kathryn S E CheahAbstract:Despite the high prevalence of intervertebral disc disease, little is known about changes in intervertebral disc cells and their regenerative potential with ageing and intervertebral disc degeneration. Here we identify populations of progenitor cells that are Tie2 positive (Tie2+) and disialoganglioside 2 positive (GD2+), in the Nucleus Pulposus from mice and humans. These cells form spheroid colonies that express type II collagen and aggrecan. They are clonally multipotent and differentiated into mesenchymal lineages and induced reorganization of Nucleus Pulposus tissue when transplanted into non-obese diabetic/severe combined immunodeficient mice. The frequency of Tie2+ cells in tissues from patients decreases markedly with age and degeneration of the intervertebral disc, suggesting exhaustion of their capacity for regeneration. However, progenitor cells (Tie2+GD2+) can be induced from their precursor cells (Tie2+GD2-) under simple culture conditions. Moreover, angiopoietin-1, a ligand of Tie2, is crucial for the survival of Nucleus Pulposus cells. Our results offer insights for regenerative therapy and a new diagnostic standard.
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tonicity enhancer binding protein tonebp and hypoxia inducible factor hif coordinate heat shock protein 70 hsp70 expression in hypoxic Nucleus Pulposus cells role of hsp70 in hif 1α degradation
Journal of Bone and Mineral Research, 2012Co-Authors: Shilpa S Gogate, Irving M Shapiro, Nobuyuki Fujita, Renata Skubutyte, Makarand V RisbudAbstract:The objective of our study was to examine the regulation of hypoxic expression of Hsp70 in Nucleus Pulposus cells and to determine if Hsp70 promoted HIF-1α degradation. Rat Nucleus Pulposus cells were maintained in culture in either 21% or 1% oxygen. To determine the regulation of Hsp70 expression by Tonicity enhancer binding protein (TonEBP) and HIF-1/2, loss- and gain-of-function experiments and mutational analysis of Hsp70 promoter were performed. Hypoxia increased Hsp70 expression in Nucleus Pulposus cells. Noteworthy, hypoxia increased TonEBP transactivation and mutation of TonE motifs blocked hypoxic induction of Hsp70 promoter. In contrast, mutation of HRE motifs coupled with loss of function experiments suggested that HIF-1 and HIF-2 suppressed Hsp70 promoter activity and transcription. Interestingly, HIF-α interferes with TonEBP function and suppresses inductive effect of TonEBP on Hsp70 promoter. In terms of Hsp70 function, when treated with Hsp70 transcriptional inhibitor, KNK437, there was an increase in HIF-1α protein stability and transcriptional activity. Likewise, when Hsp70 was overexpressed, the stability of HIF-1α and its transcriptional activity decreased. Hsp70 interacted with HIF-1α under hypoxic conditions and evidenced increased binding when treated with MG132, a proteasomal inhibitor. These results suggest that Hsp70 may promote HIF-1α degradation through proteasomal pathway in Nucleus Pulposus cells. In hypoxic and hyperosmolar Nucleus Pulposus cells, Hsp70, TonEBP and HIFs form a regulatory loop. We propose that the positive regulation by TonEBP and negative regulation of Hsp70 by HIF-1 and HIF-2 may serve to maintain Hsp70 levels in these cells, whereas Hsp70 may function in controlling HIF-1α homeostasis.
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regulation of ccn2 connective tissue growth factor expression in the Nucleus Pulposus of the intervertebral disc role of smad and activator protein 1 signaling
Arthritis & Rheumatism, 2010Co-Authors: Cassie M Tran, Irving M Shapiro, Dessislava Markova, Harvey E Smith, Bala T S Susarla, Ravi K Ponnappan, Greg D Anderson, Aviva J Symes, Makarand V RisbudAbstract:Objective To investigate transforming growth factor β (TGFβ) regulation of connective tissue growth factor (CTGF) expression in cells of the Nucleus Pulposus of rats, mice, and humans. Methods Real-time reverse transcription–polymerase chain reaction and Western blot analyses were used to measure CTGF expression in the Nucleus Pulposus. Transfections were used to measure the effects of Smads 2, 3, and 7 and activator protein 1 (AP-1) on TGFβ-mediated CTGF promoter activity. Results CTGF expression was lower in neonatal rat discs than in skeletally mature rat discs. An increase in CTGF expression and promoter activity was observed in rat Nucleus Pulposus cells after TGFβ treatment. Deletion analysis indicated that promoter constructs lacking Smad and AP-1 motifs were unresponsive to treatment. Analysis showed that full-length Smad3 and the Smad3 MH-2 domain alone increased CTGF activity. Further evidence of Smad3 and AP-1 involvement was seen when DN-Smad3, SiRNA-Smad3, Smad7, and DN-AP-1 suppressed TGFβ-mediated activation of the CTGF promoter. When either Smad3 or AP-1 sites were mutated, CTGF promoter induction by TGFβ was suppressed. We also observed a decrease in the expression of CTGF in discs from Smad3-null mice as compared with those from wild-type mice. Analysis of human Nucleus Pulposus samples indicated a trend toward increasing CTGF and TGFβ expression in the degenerated state. Conclusion TGFβ, through Smad3 and AP-1, serves as a positive regulator of CTGF expression in the Nucleus Pulposus. We propose that CTGF is a part of the limited reparative response of the degenerated disc.
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normoxic stabilization of hif 1α drives glycolytic metabolism and regulates aggrecan gene expression in Nucleus Pulposus cells of the rat intervertebral disk
American Journal of Physiology-cell Physiology, 2007Co-Authors: Amit Agrawal, Irving M Shapiro, Todd J Albert, Asha Guttapalli, Srinivas Narayan, Makarand V RisbudAbstract:The Nucleus Pulposus is an aggrecan-rich, avascular tissue that permits the intervertebral disk to resist compressive loads. In the disk, Nucleus Pulposus cells express hypoxia-inducible factor (HIF)-1alpha, a transcription factor that responds to oxygen tension and regulates glycolysis. The goal of the present study was to examine the importance of HIF-1alpha in rat Nucleus Pulposus cells and to probe the function of this transcription factor in terms of regulating aggrecan gene expression. We found that HIF-1alpha protein levels and mRNA stability were similar at 20 and 2% O(2); there was a small, but significant increase in HIF-1alpha transactivation domain activity in hypoxia. With respect to HIF-1alpha target genes GAPDH, GLUT-1, and GLUT-3, mRNA and protein levels were independent of the oxygen tension. Other than a modest increase in 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase reporter activity, the oxemic state did not change GAPDH, GLUT-1, and GLUT-3 promoter activities. Treatment of cells with 2-deoxyglucose (2-DG), a glycolytic inhibitor, resulted in a significant suppression in ATP synthesis in normoxia, whereas treatment with mitochondrial inhibitors did not affect ATP production and cell viability. However, measurement of the rate of fatty acid oxidation indicated that these cells contained functioning mitochondria. Finally, we showed that when HIF-1alpha was suppressed, irrespective of the oxemic state, there was a partial loss of aggrecan expression and promoter activity. Moreover, when cells were treated with 2-DG, there was inhibition in aggrecan promoter activity. Results of this study indicate that oxygen-independent stabilization of HIF-1alpha in Nucleus Pulposus cells is a metabolic adaptation that drives glycolysis and aggrecan expression.
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fibroblast growth factor 2 maintains the differentiation potential of Nucleus Pulposus cells in vitro implications for cell based transplantation therapy
Spine, 2007Co-Authors: Tsungting Tsai, Irving M Shapiro, Alexander R Vaccaro, Todd J Albert, Asha Guttapalli, Erbil Oguz, Lihhuei Chen, Makarand V RisbudAbstract:STUDY DESIGN: To investigate effects of FGF-2 on Nucleus Pulposus cell growth and differentiation. OBJECTIVES: To elucidate the phenotypic changes that occur during expansion of Nucleus Pulposus cells in monolayer culture, and to investigate the effects of fibroblast growth factor (FGF)-2 on cell growth and differentiation. SUMMARY OF BACKGROUND DATA: Nucleus Pulposus cells would have a limited application for autologous cell transplantation if phenotypic dedifferentiation takes place during culture expansion. FGF-2 has been shown to retain the differentiation potential of monolayer expanded chondrocytic cells. However, its effect on Nucleus Pulposus cells is not known. METHODS: Bovine Nucleus Pulposus cells were serially passaged in the presence or absence of FGF-2 (1 and 10 ng/mL). After passage numbers 1 and 7, cells were immobilized in alginate beads and treated with transforming growth factor (TGF)-beta1 for 1 week to assess their differentiation. RESULTS: During culture expansion in monolayer, Nucleus Pulposus cells maintained the expression of aggrecan messenger ribonucleic acid (mRNA). However, mRNA levels of collagen type I, collagen type II, Sox-9, and versican decreased with increasing passage number for both control (untreated) cells and FGF-2 treated cells. When grown in alginate with TFG-beta1, passage 7 cells that received FGF-2 during culture expansion restored the mRNA expression of type II collagen, Sox-9, COMP, chondroadherin, and fibromodulin. Moreover, FGF-2 treatment resulted in increased sulfated proteoglycan synthesis and lower aggrecan turnover compared to untreated controls under identical culture conditions. FGF-2 treated cells continued to express HIF-1alpha protein till passage 7, while MMP-2 expression was evident in cells treated with TGF-beta1. In addition, cells pretreated with FGF-2 showed higher induction of phospho ERK1/2 after treatment with TGF-beta1. Also, FGF-2 maintained smad 2/smad 3 mediated signaling in cells after TGF-beta treatment. FGF-2 action resulted in reduced actin stress fiber formation and migratory cell morphology, with no effect on cell proliferation. CONCLUSIONS: The presence of FGF-2 during culture expansion of Nucleus Pulposus cells in monolayer can sustain a differentiated cell phenotype by maintaining responsiveness to TGF-beta1. Our results suggest that FGF-2 should be tested for its ability to maintain the reactivity of the Nucleus Pulposus cells to other morphogenic factors that may be used for cell-based transplantation therapy.
