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SUMMARY: Intervertebral disc degeneration (IVDD) is induced by nucleus pulposus (NP) dysfunction as a result of massive loss of NP cells. It has been reported that the acidic microenvironment of the intervertebral disc (IVD) can induce NP cell pyroptosis, and that up-regulation of periostin (POSTN) expression has a negative effect on NP cell survival. However, the relationship between the acidic environment, POSTN expression level and NP cell pyroptosis is unclear. Therefore, the aim of this study was to explore the relationship between acidic environment and POSTN expression level in NP cells, as well as the effect of POSTN in acidic environment on NP cell pyroptosis. NP cells were obtained from the lumbar vertebrae of Sprague Dawley (SD) male rats. These cells were divided into normal and acidic groups according to whether they were exposed to 6 mM lactic acid solution. And NP cells in the acidic group were additionally divided into three groups: (1) Blank group: no transfection; (2) NC group: cells transfected with empty vector plasmid; (3) sh-POSTN group: cells transfected with sh-POSTN plasmid to knock down the expression level of POSTN. Quantitative real-time PCR (qRT-PCR) and western blot was performed to assess the expression of POSTN at the mRNAand protein levels. CCK8 was used to evaluate cell survival. Western blot, in addition, was performed to examine acid-sensing ion channels (ASIC)-related proteins. And pyroptosis was detected by ELISA and western blot. The expression level of POSTN was significantly increased in NP cells in acidic environment. Knockdown of POSTN expression promoted the survival of NP cells in acidic environment and reduced the protein levels of ASIC3 and ASIC1a in NP cells. Moreover, knockdown of POSTN expression decreased the pyroptosis proportion of NP cells and the levels of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. The levels of pyroptosis-related proteins NLRP3, ASC, cleaved-Caspase-1, and cleaved-GSDMD were also affected by the decreased POSTN expression. The extracellular acidic environment created by lactic acid solution activated NLRP3 inflammatory vesicle-induced caspase-1 to get involved in NP cell pyroptosis by up-regulating POSTN expression.

La degeneración del disco intervertebral (DDIV) es inducida por una disfunción del núcleo pulposo (NP) como resultado de una pérdida masiva de células NP. Se ha informado que el microambiente ácido del disco intervertebral (DIV) puede inducir la piroptosis de las células NP y que la regulación positiva de la expresión de periostina (POSTN) tiene un efecto negativo en la supervivencia de las células NP. Sin embargo, la relación entre el ambiente ácido, el nivel de expresión de POSTN y la piroptosis de las células NP es poco clara. Por lo tanto, el objetivo de este estudio fue explorar la relación entre el ambiente ácido y el nivel de expresión de POSTN en células NP, así como el efecto de POSTN en ambiente ácido sobre la piroptosis de las células NP. Las células NP se obtuvieron de las vertebras lumbares de ratas macho Sprague Dawley (SD). Estas células se dividieron en grupos normales y ácidos según se expusieron a una solución de ácido láctico 6 mM. Las células NP en el grupo ácido se dividieron adicionalmente en tres grupos: (1) Grupo en blanco: sin transfección; (2) grupo NC: células transfectadas con plásmido vector vacío; (3) grupo sh-POSTN: células transfectadas con plásmido sh-POSTN para reducir el nivel de expresión de POSTN. Se realizó una PCR cuantitativa en tiempo real (qRT-PCR) y una transferencia Western para evaluar la expresión de POSTN en los niveles de ARNm y proteína. Se utilizó CCK8 para evaluar la supervivencia celular. Además, se realizó una transferencia Western para examinar las proteínas relacionadas con los canales iónicos sensibles al ácido (ASIC). La piroptosis se detectó mediante ELISA y Western blot. El nivel de expresión de POSTN aumentó significativamente en células NP en ambiente ácido. La eliminación de la expresión de POSTN promovió la supervivencia de las células NP en un ambiente ácido y redujo los niveles de proteína de ASIC3 y ASIC1a en las células NP. Además, la eliminación de la expresión de POSTN disminuyó la proporción de piroptosis de las células NP y los niveles de citocinas proinflamatorias interleucina (IL) - 1β e IL-18. Los niveles de proteínas relacionadas con la piroptosis NLRP3, ASC, Caspasa-1 escindida y GSDMD escindida también se vieron afectados por la disminución de la expresión de POSTN. El ambiente ácido extracelular creado por la solución de ácido láctico activó la caspasa-1 inducida por vesículas inflamatorias NLRP3 para involucrarse en la piroptosis de las células NP mediante la regulación positiva de la expresión de POSTN.

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Ozone therapy has been used to treat disc herniation for more than four decades. There are several papers describing results and mechanism of action. However, it is very important to define the characteristics of extruded disc herniation. Although ozone therapy showed excellent results in the majority of spinal diseases, it is not yet fully accepted within the medical community. Perhaps it is partly due to the fact that, sometimes, indications are not appropriately made. The objective of our work is to explain the mechanisms of action of ozone therapy on the extruded disc herniation. Indeed, these mechanisms are quite different from those exerted by ozone on the protruded disc herniation and on the degenerative disc disease because the inflammatory response is very different between the various cases. Extruded disc herniation occurs when the nucleus squeezes through a weakness or tear in the annulus. Host immune system considers the nucleus material to be a foreign invader, which triggers an immune response and inflammation. We think ozone therapy modulates this immune response, activating macrophages, which produce phagocytosis of extruded nucleus pulposus. Ozone would also facilitate the passage from the M1 to M2 phase of macrophages, going from an inflammatory phase to a reparative phase. Further studies are needed to verify the switch of macrophages.

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Lumbar disc herniation is a common disease characterized by the degeneration of intervertebral discs (IVDs), accompanied by imbalance of metabolic and inflammatory homeostasis. Current studies establish that IVD degeneration is induced by increased apoptosis of nucleus pulposus (NP) cells. However, the underlying mechanisms of NP cell survival/apoptosis are not well elucidated. Here, we reveal a novel mechanism by which mTORC1 signaling controls NP cell survival through regulating metabolic homeostasis. We demonstrated that hyperactivated mTORC1 activity induced by inflammatory cytokines engenders the apoptosis of NP cells, whereas pharmacological inhibition of mTORC1 activity promotes NP cell survival. Using an integrative approach spanning metabolomics and biochemical approaches, we showed that mTORC1 activation enhanced glucose metabolism and lactic acid production, and therefore caused NP cell apoptosis. Our study identified mTORC1 in NP cells as a novel target for IVD degeneration, and provided potential strategies for clinical intervention of lumbar disc herniation.

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BACKGROUND: The cellular basis of adult growth in cephalochordates (lancelets or amphioxus) has received little attention. Lancelets and their constituent organs grow slowly but continuously during adult life. Here, we consider whether this slow organ growth involves tissue-specific stem cells. Specifically, we focus on the cell populations in the notochord of an adult lancelet and use serial blockface scanning electron microscopy (SBSEM) to reconstruct the three-dimensional fine structure of all the cells in a tissue volume considerably larger than normally imaged with this technique. RESULTS: In the notochordal region studied, we identified 10 cells with stem cell-like morphology at the posterior tip of the organ, 160 progenitor (Müller) cells arranged along its surface, and 385 highly differentiated lamellar cells constituting its core. Each cell type could clearly be distinguished on the basis of cytoplasmic density and overall cell shape. Moreover, because of the large sample size, transitions between cell types were obvious. CONCLUSIONS: For the notochord of adult lancelets, a reasonable interpretation of our data indicates growth of the organ is based on stem cells that self-renew and also give rise to progenitor cells that, in turn, differentiate into lamellar cells. Our discussion compares the cellular basis of adult notochord growth among chordates in general. In the vertebrates, several studies implied that proliferating cells (chordoblasts) in the cortex of the organ might be stem cells. However, we think it is more likely that such cells actually constitute a progenitor population downstream from and maintained by inconspicuous stem cells. We venture to suggest that careful searches should find stem cells in the adult notochords of many vertebrates, although possibly not in the notochordal vestiges (nucleus pulposus regions) of mammals, where the presence of endogenous proliferating cells remains controversial.

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RESUMEN La vértebra limbus es una rara condición poco descrita en la literatura médica. Se caracteriza por una herniación marginal intracorporal del núcleo pulposo que resulta en la separación de un fragmento óseo de forma triangular. Usualmente se presenta en niños o adolescentes, principalmente en la columna lumbar, y ocasiona dolor que, en la mayoría de los casos, mejora con el tratamiento con antiinflamatorios.

A B S T R A C T The limbus vertebra is a rare condition poorly described in the medical literature. Marginal intrabody herniation of the nucleus pulposus resulting in the separation of a triangular bone fragment. It usually occurs in children or adolescents mainly in the lumbar spine and causes pain that in most cases improves with treatment with anti-inflammatories.

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SUMMARY OBJECTIVE Nuclear factor erythroid-2 related factor 2 (Nrf2)/ antioxidant response element (ARE) is a novel defensive pathway involved in the oxidative and chemical stress of cells. The aim of the study was to explore the role of Nrf2 on the apoptosis of human disc nucleus pulpous cells induced by hydrogen peroxide (H2O2). METHODS The degeneration model of human intervertebral disc nucleus pulpous cells was established. The expression of Nrf2 was interfered with using sulforaphane (SFN); for that end, three groups were established: a blank group (H2O2-/SFN-), control group (H2O2+/SFN-), and an experimental group (H2O2+/SFN+). CCK8, Hoechst 33258 living cell staining was used to detect reactive oxygen species (ROS) content. RESULTS The apoptotic rates of the three groups were [(0.40±0.46)%], [(25.98±11.28)%], and [(3.83±2.06)%, respectively. The difference was statistically significant (p<0.05). The relative content of ROS in the three groups was [(100±7)%], [(1538±91)%], and [(818±63)%]; the difference was statistically significant (p<0.05). In Western blotting, Nrf2 content in the experimental group was higher than that in the control group. CONCLUSION Nrf2 exists in the nucleus pulpous cells of human intervertebral discs, which is related to the degeneration of the intervertebral disc. It has negative feedback regulation and can prevent the degeneration of the intervertebral disc by inhibiting the apoptosis of nucleus pulpous cells of human intervertebral discs caused by excessive ROS, which provides a new intervention strategy for the prevention and treatment of the degeneration of intervertebral discs.

RESUMO OBJETIVO O fator 2 relacionado a NF-E2 (Nrf2)/elemento de resposta antioxidante (ARE) é uma nova via defensiva envolvida no estresse oxidativo e químico das células. O objetivo deste estudo foi explorar o papel do Nrf2 na apoptose das células do núcleo pulposo do disco humano induzida pelo peróxido de hidrogênio (H2O2). MÉTODOS O modelo de degeneração das células do núcleo pulposo do disco intervertebral humano foi estabelecido. A expressão do Nrf2 foi interferida utilizando-se sulforafano (SFN). Para isso foram estabelecidos três grupos: um grupo vazio (H2O2-/SFN-), um grupo de controle (H2O2+/SFN-), e um grupo experimental (H2O2+/SFN+). Utilizando CCK8 e Hoechst 33258, o conteúdo de espécies reativas de oxigênio (ERO) foi detectado. RESULTADOS As taxas de apoptose dos três grupos foram [(0,40 ± 0,46)%], [(25,98 ± 11,28%)] e [(3,83 ± 2,06)%], respectivamente. A diferença apresentou significância estatística (p < 0,05). O conteúdo relativo de ERO nos três grupos foi [(100±7)%], [(1538±91%)], e [(818±63%); a diferença foi estatisticamente significativa (p < 0,05). O método de Western blotting indicou um maior conteúdo de Nrf2 no grupo experimental do que no grupo de controle. CONCLUSÃO O Nrf2 existe em células do núcleo pulposo do disco intervertebral humano, que estão relacionadas à degeneração do disco intervertebral. Ele apresenta regulação por feedback negativo e pode evitar a degeneração do disco intervertebral inibindo a apoptose de células do núcleo pulposo do disco causada por excesso de ERO. Essa informação proporciona uma nova estratégia de intervenção para a prevenção e o tratamento da degeneração do disco intervertebral.

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ABSTRACT The intervertebral disc (IVD) is one of the parts of the body most commonly affected by disease, and it is only recently that we have come closer to understanding the reasons for its degeneration, in which nutrient supply plays a crucial role. In this literature review, we discuss the basic principles and characteristics of energy supply and demand to the IVD. Specifically, we review how different metabolites influence IVD cell activity, the effects of mechanical loading on IVD cell metabolism, and differences in energy metabolism of the annulus fibrous and nucleus pulposus cell phenotypes. Determining the factors that influence nutrient supply and demand in the IVD will enhance our understanding of the IVD pathology, and help to elucidate new therapeutic targets for IVD degeneration treatment.

RESUMO O disco intervertebral (IVD) é uma das partes mais comuns do corpo e apenas recentemente nos aproximamos de compreender as razões da sua degeneração, em que o suprimento de nutrientes desempenha um papel crucial. Nesta revisão da literatura, discutimos os princípios básicos e as nuances do fornecimento e da demanda de energia para o IVD. Específicamente, analisamos como os diferentes metabólitos influenciam na atividade das células IVD, os efeitos da carga mecânica no metabolismo das células IVD, a diferença no metabolismo energético dos fenótipos das células fibrosas e do núcleo do pulposus anelar. A determinação de fatores que influenciam o suprimento e a demanda de nutrientes no IVD aumentará nossa compreensão da patologia IVD e ajudará a elucidar novos alvos terapêuticos para o tratamento da degeneração IVD.

RESUMEN El disco intervertebral (IVD, por sus siglas en inglés) es una de las partes más comúnmente enfermas del cuerpo y solo recientemente nos acercamos a la comprensión de los motivos de su degeneración, de los cuales el suministro de nutrientes juega un papel crucial. En esta revisión de la literatura discutimos los principios básicos y los matices de la oferta y demanda de energía para el IVD. Específicamente, revisamos cómo los diferentes metabolitos influyen en la actividad de las células IVD, los efectos de la carga mecánica sobre el metabolismo de las células IVD y las diferencias en el metabolismo energético de los fenotipos de las células del anillo fibroso y el núcleo pulposo. La determinación de los factores que influyen en la oferta y demanda de nutrientes en el IVD mejorará nuestra comprensión de la patología IVD y ayudará a dilucidar nuevos objetivos terapéuticos para el tratamiento de la degeneración IVD.

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A extrusão discal aguda e não compressiva é caracterizada pela extrusão de caráter agudo/hiperagudo e não compressivo do núcleo pulposo de um disco intervertebral não degenerado. Pode ser chamada de hérnia de disco de baixo volume e alta velocidade ou explosões discais e geralmente está associado a exercícios intensos ou episódios traumáticos. O núcleo pulposo é fortemente forçado através de uma pequena fissura no ânulo fibroso dorsal, provocando uma contusão espinhal. Este relato tem como objetivo apresentar um caso de provável extrusão aguda de núcleo pulposo não compressiva. Foi atendido um cão macho, três anos e seis meses de idade, maltês, pesando 4,1kg. Como queixa principal, o proprietário relatou dificuldade locomotora e dor à manipulação há um dia, sem histórico de trauma. Foi constatada paraparesia não ambulatória de início agudo com ausência de propriocepção e dor superficial em membros pélvicos e dor à palpação epaxial da coluna toracolombar. A ressonância magnética (RM) evidenciou extensa área de hipersinal em segmento toracolombar da medula espinhal, sem sinais de compressão medular e de atenuação da intensidade do núcleo pulposo do disco intervertebral L1-L2. Foi feito diagnóstico presuntivo de mielopatia focal não compressiva com edema medular de todo segmento toracolombar, característico de uma extrusão aguda de núcleo pulposo não compressiva. Foi prescrito tratamento com anti-inflamatório esteroidal, analgésico, repouso absoluto e protocolo de reabilitação com acupuntura e fisioterapia. Após sete dias de tratamento, o animal recuperou a sensibilidade dolorosa superficial em membros pélvicos e evoluiu para paraparesia ambulatória. Os resultados deste relato sugerem que a RM pode ser útil para fazer um diagnóstico presuntivo em cães com histórico e sinais clínicos compatíveis. Além disso, o tratamento conservativo em extrusões discais não compressivas é preconizado e o paciente pode apresentar boa recuperação.(AU)

Acute and non-compressive disc extrusion is characterized by the acute character of extrusion of the nucleus pulposus without real compression of the spine. It has been called low-volume and high speed disc herniation or disc explosions, and usually is associated with an intense exercise or traumatic episode. This report aims to present a case of an acute extrusion of nucleus pulposus with no compression of the spinal cord. A 3.5 year-old male dog of the Maltes breed, weighing 4.1kg was presented at the Veterinary Hospital with locomotion disorders and pain during manipulation with no history of trauma. At the physical and neurological examination, non-ambulatory paraparesis of acute onset with absence of proprioception and superficial pain in hind limbs was found, as well as pain on palpation of epaxial thoracolumbar spine. Magnetic resonance imaging (MRI) showed extensive hyper intense area in the thoracolumbar spinal cord, with no signs of spinal cord compression, and decreased intensity of the nucleus pulposus of the L1-L2 intervertebral disc. Additionally, a spinal cord edema in all thoracolumbar segments was seen that is characteristic of an acute extrusion of non-compressive nucleus pulposus. A presumptive diagnosis of non-compressive myelopathy was assumed. The dog was prescribed steroidal anti-inflammatory, analgesic, absolute rest and rehabilitation protocol, including acupuncture and physiotherapy. The patient recovered superficial pain in the pelvic limbs and evolved into ambulatory paraparesis after seven days. The results of this report suggested that MRI can be useful for making a presumptive diagnosis in dogs with a history of compatible clinical signs. Moreover, the conservative treatment in non-compressive disc extrusions can be feasible.(AU)

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A extrusão discal aguda e não compressiva é caracterizada pela extrusão de caráter agudo/hiperagudo e não compressivo do núcleo pulposo de um disco intervertebral não degenerado. Pode ser chamada de hérnia de disco de baixo volume e alta velocidade ou explosões discais e geralmente está associado a exercícios intensos ou episódios traumáticos. O núcleo pulposo é fortemente forçado através de uma pequena fissura no ânulo fibroso dorsal, provocando uma contusão espinhal. Este relato tem como objetivo apresentar um caso de provável extrusão aguda de núcleo pulposo não compressiva. Foi atendido um cão macho, três anos e seis meses de idade, maltês, pesando 4,1kg. Como queixa principal, o proprietário relatou dificuldade locomotora e dor à manipulação há um dia, sem histórico de trauma. Foi constatada paraparesia não ambulatória de início agudo com ausência de propriocepção e dor superficial em membros pélvicos e dor à palpação epaxial da coluna toracolombar. A ressonância magnética (RM) evidenciou extensa área de hipersinal em segmento toracolombar da medula espinhal, sem sinais de compressão medular e de atenuação da intensidade do núcleo pulposo do disco intervertebral L1-L2. Foi feito diagnóstico presuntivo de mielopatia focal não compressiva com edema medular de todo segmento toracolombar, característico de uma extrusão aguda de núcleo pulposo não compressiva. Foi prescrito tratamento com anti-inflamatório esteroidal, analgésico, repouso absoluto e protocolo de reabilitação com acupuntura e fisioterapia. Após sete dias de tratamento, o animal recuperou a sensibilidade dolorosa superficial em membros pélvicos e evoluiu para paraparesia ambulatória. Os resultados deste relato sugerem que a RM pode ser útil para fazer um diagnóstico presuntivo em cães com histórico e sinais clínicos compatíveis. Além disso, o tratamento conservativo em extrusões discais não compressivas é preconizado e o paciente pode apresentar boa recuperação.(AU)

Acute and non-compressive disc extrusion is characterized by the acute character of extrusion of the nucleus pulposus without real compression of the spine. It has been called low-volume and high speed disc herniation or disc explosions, and usually is associated with an intense exercise or traumatic episode. This report aims to present a case of an acute extrusion of nucleus pulposus with no compression of the spinal cord. A 3.5 year-old male dog of the Maltes breed, weighing 4.1kg was presented at the Veterinary Hospital with locomotion disorders and pain during manipulation with no history of trauma. At the physical and neurological examination, non-ambulatory paraparesis of acute onset with absence of proprioception and superficial pain in hind limbs was found, as well as pain on palpation of epaxial thoracolumbar spine. Magnetic resonance imaging (MRI) showed extensive hyper intense area in the thoracolumbar spinal cord, with no signs of spinal cord compression, and decreased intensity of the nucleus pulposus of the L1-L2 intervertebral disc. Additionally, a spinal cord edema in all thoracolumbar segments was seen that is characteristic of an acute extrusion of non-compressive nucleus pulposus. A presumptive diagnosis of non-compressive myelopathy was assumed. The dog was prescribed steroidal anti-inflammatory, analgesic, absolute rest and rehabilitation protocol, including acupuncture and physiotherapy. The patient recovered superficial pain in the pelvic limbs and evolved into ambulatory paraparesis after seven days. The results of this report suggested that MRI can be useful for making a presumptive diagnosis in dogs with a history of compatible clinical signs. Moreover, the conservative treatment in non-compressive disc extrusions can be feasible.(AU)

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This study aims to explore the effect of microRNA-21 (miR-21) on the proliferation of human degenerated nucleus pulposus (NP) by targeting programmed cell death 4 (PDCD4) tumor suppressor. NP tissues were collected from 20 intervertebral disc degeneration (IDD) patients, and from 5 patients with traumatic spine fracture. MiR-21 expressions were tested. NP cells from IDD patients were collected and divided into blank control group, negative control group (transfected with miR-21 negative sequences), miR-21 inhibitor group (transfected with miR-21 inhibitors), miR-21 mimics group (transfected with miR-21 mimics) and PDCD4 siRNA group (transfected with PDCD4 siRNAs). Cell growth was estimated by Cell Counting Kit-8; PDCD4, MMP-2,MMP-9 mRNA expressions were evaluated by qRT-PCR; PDCD4, c-Jun and p-c-Jun expressions were tested using western blot. In IDD patients, the expressions of miR-21 and PDCD4 mRNA were respectively elevated and decreased (both P<0.05). The miR-21 expressions were positively correlated with Pfirrmann grades, but negatively correlated with PDCD4 mRNA (both P<0.001). In miR-21 inhibitor group, cell growth, MMP-2 and MMP-9 mRNA expressions, and p-c-Jun protein expressions were significantly lower, while PDCD4 mRNA and protein expressions were higher than the other groups (all P<0.05). These expressions in the PDCD4 siRNA and miR-21 mimics groups was inverted compared to that in the miR-21 inhibitor group (all P<0.05). MiR-21 could promote the proliferation of human degenerated NP cells by targeting PDCD4, increasing phosphorylation of c-Jun protein, and activating AP-1-dependent transcription of MMPs, indicating that miR-21 may be a crucial biomarker in the pathogenesis of IDD.

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Low back pain is a major physical and socioeconomic problem. Degeneration of the intervertebral disc and especially that of nucleus pulposus (NP) has been linked to low back pain. In spite of much research focusing on the NP, consensus among the research community is lacking in defining the NP cell phenotype. A consensus agreement will allow easier distinguishing of NP cells from annulus fibrosus (AF) cells and endplate chondrocytes, a better gauge of therapeutic success, and a better guidance of tissue-engineering-based regenerative strategies that attempt to replace lost NP tissue. Most importantly, a clear definition will further the understanding of physiology and function of NP cells, ultimately driving development of novel cell-based therapeutic modalities. The Spine Research Interest Group at the 2014 Annual ORS Meeting in New Orleans convened with the task of compiling a working definition of the NP cell phenotype with hope that a consensus statement will propel disc research forward into the future. Based on evaluation of recent studies describing characteristic NP markers and their physiologic relevance, we make the recommendation of the following healthy NP phenotypic markers: stabilized expression of HIF-1α, GLUT-1, aggrecan/collagen II ratio >20, Shh, Brachyury, KRT18/19, CA12, and CD24.

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