Transient receptor potential vanilloid 4 regulates extracellular matrix composition and mediates load-induced intervertebral disc degeneration in a mouse model.

OBJECTIVE: Transient receptor potential vanilloid 4 (TRPV4) is a multi-modally activated cation channel that mediates mechanotransduction pathways by which musculoskeletal tissues respond to mechanical load and regulate tissue health. Using conditional Trpv4 knockout mice, we investigated the role of Trpv4 in regulating intervertebral disc (IVD) health and injury-induced IVD degeneration. METHODS: Col2-Cre;Trpv4fl/f (Trpv4 KO) mice were used to knockout Trpv4 in all type 2 collagen-expressing cells. Effects of gene targeting alone was assessed in lumbar spines, using vertebral bone length measurement, histological, immunohistochemistry and gene expression analyses, and mechanical testing. Disc puncture was performed on caudal IVDs of wild-type (WT) and Trpv4 KO mice at 2.5- and 6.5-months-of-age. Six weeks after puncture (4- and 8-months-of-age at sacrifice), caudal spines were assessed using histological analyses. RESULTS: While loss of Trpv4 did not significantly alter vertebral bone length and tissue histomorphology compared to age-matched WT mice, Trpv4 KO mice showed decreased proteoglycan and PRG4 staining in the annulus fibrosus compared to WT. At the gene level, Trpv4 KO mice showed significantly increased expression of Acan, Bgn, and Prg4 compared to WT. Functionally, loss of Trpv4 was associated with significantly increased neutral zone length in lumbar IVDs. Following puncture, both Trpv4 KO and WT mice showed similar signs of degeneration at the site of injury. Interestingly, loss of Trpv4 prevented mechanically-induced degeneration in IVDs adjacent to sites of injury. CONCLUSION: These studies suggest a role for Trpv4 in regulating extracellular matrix synthesis and mediating the response of IVD tissues to mechanical stress.

Comparative histopathological analysis of age-associated intervertebral disc degeneration in CD-1 and C57BL/6 mice: Anatomical and sex-based differences.

Background: Intervertebral disc (IVD) degeneration is a major contributor to back pain and disability. The cause of IVD degeneration is multifactorial, with no disease-modifying treatments. Mouse models are commonly used to study IVD degeneration; however, the effects of anatomical location, strain, and sex on the progression of age-associated degeneration are poorly understood. Methods: A longitudinal study was conducted to characterize age-, anatomical-, and sex-specific differences in IVD degeneration in two commonly used strains of mice, C57BL/6 and CD-1. Histopathological evaluation of the cervical, thoracic, lumbar, and caudal regions of mice at 6, 12, 20, and 24 months of age was conducted by two blinded observers at each IVD for the nucleus pulposus (NP), annulus fibrosus (AF), and the NP/AF boundary compartments, enabling analysis of scores by tissue compartment, summed scores for each IVD, or averaged scores for each anatomical region. Results: C57BL/6 mice displayed mild IVD degeneration until 24 months of age; at this point, the lumbar spine demonstrated the most degeneration compared to other regions. Degeneration was detected earlier in the CD-1 mice (20 months of age) in both the thoracic and lumbar spine. In CD-1 mice, moderate to severe degeneration was noted in the cervical spine at all time points assessed. In both strains, age-associated IVD degeneration in the thoracic and lumbar spine was associated with increased histopathological scores in all IVD compartments. In both strains, minimal degeneration was detected in caudal IVDs out to 24 months of age. Both C57BL/6 and CD-1 mice displayed sex-specific differences in the presentation and progression of age-associated IVD degeneration. Conclusions: These results showed that the progression and severity of age-associated degeneration in mouse models is associated with marked differences based on anatomical region, sex, and strain. This information provides a fundamental baseline characterization for users of mouse models to enable effective and appropriate experimental design, interpretation, and comparison between studies.

Glycerol induced paraspinal muscle degeneration leads to hyper-kyphotic spinal deformity in wild-type mice.

Degenerative spinal disorders, including kyphotic deformity, are associated with a range of degenerative characteristics of the paraspinal musculature. It has therefore been hypothesized that paraspinal muscular dysfunction is a causative factor for degenerative spinal deformity; however, experimental studies demonstrating causative relationships are lacking. Male and female mice received either glycerol or saline injections bilaterally along the length of the paraspinal muscles at four timepoints, each separated by 2 weeks. Immediately after sacrifice, micro-CT was performed to measure spinal deformity; paraspinal muscle biopsies were taken to measure active, passive and structural properties; and lumbar spines were fixed for analysis of intervertebral disc (IVD) degeneration. Glycerol-injected mice demonstrated clear signs of paraspinal muscle degeneration and dysfunction: significantly (p < 0.01) greater collagen content, lower density, lower absolute active force, greater passive stiffness compared to saline-injected mice. Further, glycerol-injected mice exhibited spinal deformity: significantly (p < 0.01) greater kyphotic angle than saline-injected mice. Glycerol-injected mice also demonstrated a significantly (p < 0.01) greater IVD degenerative score (although mild) at the upper-most lumbar level compared to saline-injected mice. These findings provide direct evidence that combined morphological (fibrosis) and functional (actively weaker and passively stiffer) alterations to the paraspinal muscles can lead to negative changes and deformity within the thoracolumbar spine.

Stiffness and axial pain are associated with the progression of calcification in a mouse model of diffuse idiopathic skeletal hyperostosis.

BACKGROUND: Diffuse idiopathic skeletal hyperostosis (DISH) is characterized by progressive calcification of spinal tissues; however, the impact of calcification on pain and function is poorly understood. This study examined the association between progressive ectopic spine calcification in mice lacking equilibrative nucleoside transporter 1 (ENT1-/-), a preclinical model of DISH, and behavioral indicators of pain. METHODS: A longitudinal study design was used to assess radiating pain, axial discomfort, and physical function in wild-type and ENT1-/- mice at 2, 4, and 6 months. At endpoint, spinal cords were isolated for immunohistochemical analysis of astrocytes (GFAP), microglia (IBA1), and nociceptive innervation (CGRP). RESULTS: Increased spine calcification in ENT1-/- mice was associated with reductions in flexmaze exploration, vertical activity in an open field, and self-supporting behavior in tail suspension, suggesting flexion-induced discomfort or stiffness. Grip force during the axial stretch was also reduced in ENT1-/- mice at 6 months of age. Increased CGRP immunoreactivity was detected in the spinal cords of female and male ENT1-/- mice compared to wild-type. GFAP- and IBA1-immunoreactivity were increased in female ENT1-/- mice compared to wild-type, suggesting an increase in nociceptive innervation. CONCLUSION: These data suggest that ENT1-/- mice experience axial discomfort and/or stiffness and importantly that these features are detected during the early stages of spine calcification.

The Role of Panx3 in Age-Associated and Injury-Induced Intervertebral Disc Degeneration.

Pannexin 3 (Panx3) is a mechanosensitive, channel-forming glycoprotein implicated in the progression of post-traumatic osteoarthritis. Despite evidence for Panx3 expression in the intervertebral disc (IVD), its function in this cartilaginous joint structure remained unknown. Using Panx3 knockout mice, this study investigated the role of Panx3 in age-associated IVD degeneration and degeneration induced by annulus fibrosus (AF) needle puncture. Loss of Panx3 did not significantly impact the progression of age-associated histopathological IVD degeneration; however, loss of Panx3 was associated with decreased gene expression of Acan, Col1a1, Mmp13 and Runx2 and altered localization of COLX in the IVD at 19 months-of-age. Following IVD injury in the caudal spine, histological analysis of wild-type mice revealed clusters of hypertrophic cells in the AF associated with increased pericellular proteoglycan accumulation, disruptions in lamellar organization and increased lamellar thickness. In Panx3 knockout mice, hypertrophic AF cells were rarely detected and AF structure was largely preserved post-injury. Interestingly, uninjured IVDs adjacent to the site of injury more frequently showed evidence of early nucleus pulposus degeneration in Panx3 knockout mice but remained healthy in wild-type mice. These findings suggest a role for Panx3 in mediating the adaptive cellular responses to altered mechanical stress in the IVD, which may buffer aberrant loads transferred to adjacent motion segments.

Targeting the annulus fibrosus of the intervertebral disc: Col1a2-Cre(ER)T mice show specific activity of Cre recombinase in the outer annulus fibrosus.

Degeneration of the intervertebral disc (IVD) is a major underlying contributor to back pain-the single leading cause of disability worldwide. However, we possess a limited understanding of the etiology underlying IVD degeneration. To date, there are a limited number of mouse models that have been used to target proteins in specific compartments of the IVD to explore their functions in disc development, homeostasis and disease. Furthermore, the majority of reports exploring the composition and function of the outer encapsulating annulus fibrosus (AF) of the IVD have considered it as one tissue, without considering the numerous structural and functional differences existing between the inner and outer AF. In addition, no mouse models have yet been reported that enable specific targeting of genes within the outer AF. In the current report, we discuss these issues and demonstrate the localized activity of Cre recombinase in the IVD of Col1a2-Cre(ER)T;ROSA26mTmG mice possessing a tamoxifen-dependent Cre recombinase driven by a Cola2 promoter and distal enhancer and the mTmG fluorescent reporter. Following tamoxifen injection of 3-week-old Col1a2-Cre(ER)T;ROSA26mTmG mice, we show Cre activity specifically in the outer AF of the IVD, as indicated by expression of the GFP reporter. Thus, Col1a2-Cre(ER)T;ROSA26mTmG mice may prove to be a valuable tool in delineating the function of proteins in this unique compartment of the IVD, and in further exploring the compositional differences between the inner and outer AF in disc homeostasis, aging and disease.

Disruption of biomineralization pathways in spinal tissues of a mouse model of diffuse idiopathic skeletal hyperostosis.

Equilibrative nucleoside transporter 1 (ENT1) mediates passage of adenosine across the plasma membrane. We reported previously that mice lacking ENT1 (ENT1(-/-)) exhibit progressive ectopic mineralization of spinal tissues resembling diffuse idiopathic skeletal hyperostosis (DISH) in humans. Here, we investigated mechanisms underlying aberrant mineralization in ENT1(-/-) mice. Micro-CT revealed ectopic mineralization of spinal tissues in both male and female ENT1(-/-) mice, involving the annulus fibrosus of the intervertebral discs (IVDs) of older mice. IVDs were isolated from wild-type and ENT1(-/-) mice at 2months of age (prior to disc mineralization), 4, and 6months of age (disc mineralization present) and processed for real-time PCR, cell isolation, or histology. Relative to the expression of ENTs in other tissues, ENT1 was the primary nucleoside transporter expressed in wild-type IVDs and mediated the functional uptake of [(3)H]2-chloroadenosine by annulus fibrosus cells. No differences in candidate gene expression were detected in IVDs from ENT1(-/-) and wild-type mice at 2 or 4months of age. However, at 6months of age, expression of genes that inhibit biomineralization Mgp, Enpp1, Ank, and Spp1 were reduced in IVDs from ENT1(-/-) mice. To assess whether changes detected in ENT1(-/-) mice were cell autonomous, annulus fibrosus cell cultures were established. Compared to wild-type cells, cells isolated from ENT1(-/-) IVDs at 2 or 6months of age demonstrated greater activity of alkaline phosphatase, a promoter of biomineralization. Cells from 2-month-old ENT1(-/-) mice also showed greater mineralization than wild-type. Interestingly, altered localization of alkaline phosphatase activity was detected in the inner annulus fibrosus of ENT1(-/-) mice in vivo. Alkaline phosphatase activity, together with the marked reduction in mineralization inhibitors, is consistent with the mineralization of IVDs seen in ENT1(-/-) mice at older ages. These findings establish that both cell-autonomous and systemic mechanisms contribute to ectopic mineralization in ENT1(-/-) mice.

ECM-dependent HIF induction directs trophoblast stem cell fate via LIMK1-mediated cytoskeletal rearrangement.

The Hypoxia-inducible Factor (HIF) family of transcriptional regulators coordinates the expression of dozens of genes in response to oxygen deprivation. Mammalian development occurs in a hypoxic environment and HIF-null mice therefore die in utero due to multiple embryonic and placental defects. Mouse embryonic stem cells do not differentiate into placental cells; therefore, trophoblast stem cells (TSCs) are used to study mouse placental development. Consistent with a requirement for HIF activity during placental development in utero, TSCs derived from HIF-null mice exhibit severe differentiation defects and fail to form trophoblast giant cells (TGCs) in vitro. Interestingly, differentiating TSCs induce HIF activity independent of oxygen tension via unclear mechanisms. Here, we show that altering the extracellular matrix (ECM) composition upon which TSCs are cultured changes their differentiation potential from TGCs to multinucleated syncytiotropholasts (SynTs) and blocks oxygen-independent HIF induction. We further find that modulation of Mitogen Activated Protein Kinase Kinase-1/2 (MAP2K1/2, MEK-1/2) signaling by ECM composition is responsible for this effect. In the absence of ECM-dependent cues, hypoxia-signaling pathways activate this MAPK cascade to drive HIF induction and redirect TSC fate along the TGC lineage. In addition, we show that integrity of the microtubule and actin cytoskeleton is critical for TGC fate determination. HIF-2α ensures TSC cytoskeletal integrity and promotes invasive TGC formation by interacting with c-MYC to induce non-canonical expression of Lim domain kinase 1-an enzyme that regulates microtubule and actin stability, as well as cell invasion. Thus, we find that HIF can integrate positional and metabolic cues from within the TSC niche to regulate placental development by modulating the cellular cytoskeleton via non-canonical gene expression.

Loss of equilibrative nucleoside transporter 1 in mice leads to progressive ectopic mineralization of spinal tissues resembling diffuse idiopathic skeletal hyperostosis in humans.

Diffuse idiopathic skeletal hyperostosis (DISH) is a noninflammatory spondyloarthropathy, characterized by ectopic calcification of spinal tissues. Symptoms include spine pain and stiffness, and in severe cases dysphagia and spinal cord compression. The etiology of DISH is unknown and there are no specific treatments. Recent studies have suggested a role for purine metabolism in the regulation of biomineralization. Equilibrative nucleoside transporter 1 (ENT1) transfers hydrophilic nucleosides, such as adenosine, across the plasma membrane. In mice lacking ENT1, we observed the development of calcified lesions resembling DISH. By 12 months of age, ENT1(-/-) mice exhibited signs of spine stiffness, hind limb dysfunction, and paralysis. Micro-computed tomography (µCT) revealed ectopic mineralization of paraspinal tissues in the cervical-thoracic region at 2 months of age, which extended to the lumbar and caudal regions with advancing age. Energy-dispersive X-ray microanalysis of lesions revealed a high content of calcium and phosphorus with a ratio similar to that of cortical bone. At 12 months of age, histological examination of ENT1(-/-) mice revealed large, irregular accumulations of eosinophilic material in paraspinal ligaments and entheses, intervertebral discs, and sternocostal articulations. There was no evidence of mineralization in appendicular joints or blood vessels, indicating specificity for the axial skeleton. Plasma adenosine levels were significantly greater in ENT1(-/-) mice than in wild-type, consistent with loss of ENT1--a primary adenosine uptake pathway. There was a significant reduction in the expression of Enpp1, Ank, and Alpl in intervertebral discs from ENT1(-/-) mice compared to wild-type mice. Elevated plasma levels of inorganic pyrophosphate in ENT1(-/-) mice indicated generalized disruption of pyrophosphate homeostasis. This is the first report of a role for ENT1 in regulating the calcification of soft tissues. Moreover, ENT1(-/-) mice may be a useful model for investigating pathogenesis and evaluating therapeutics for the prevention of mineralization in DISH and related disorders.

Vertebroplastia percutánea / Percutaneous vertebral reconstruction

En este articulo se realiza una revisión de los conceptos actuales sobre la utilización de la vertebroplastia en el tratamiento de diferentes patologías vertebrales. Aplicando los conocimientos adquiridos durante los últimos años y realizando una revisión bibliográfica de todo lo publicado hasta el momento, se describen las indicaciones, la técnica y las complicaciones de este procedimiento (AU)