Hsa-circ-0007292 promotes the osteogenic differentiation of posterior longitudinal ligament cells via regulating SATB2 by sponging miR-508-3p.

Ossification of the posterior longitudinal ligament (OPLL) is a disorder with multiple pathogenic mechanisms and leads to different degrees of neurological symptoms. Recent studies have revealed that non-coding RNA (ncRNA), including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), could influence the development of OPLL. Nevertheless, the molecular mechanisms linking circular RNAs (circRNAs) and the progression of OPLL is still unknown. The current research explored the expression profiles of OPLL-related circRNAs by microarray analysis, and applied qRT-PCR to validate the results. Subsequently, we confirmed the upregulation of hsa_circ_0007292 in OPLL cells by qRT-PCR and validated the circular characteristic of hsa_circ_0007292 by Sanger sequencing. Fluorescence in situ hybridization (FISH) unveiled that hsa_circ_0007292 was predominantly located in the cytoplasm. Functionally, gain-of-function and loss-of-function experiments showed that hsa_circ_0007292 promoted the osteogenic differentiation of OPLL cells. Mechanistically, the interaction of hsa_circ_0007292 and miR-508-3p was predicted and validated by bioinformatics analysis, dual-luciferase reporter assays, and Ago2 RNA immunoprecipitation (RIP). Similarly, we validated the correlation between miR-508-3p and SATB2. Furthermore, rescue experiments were performed to prove that hsa_circ_0007292 acted as a sponge for miR-508-3p, and SATB2 was revealed to be the target gene of miR-508-3p. In conclusion, our research shows that hsa_circ_0007292 regulates OPLL progression by the miR-508-3p/SATB2 pathway. Our results indicate that hsa_circ_0007292 can be used as a promising therapeutic target for patients with OPLL.

Autophagy in spinal ligament fibroblasts: evidence and possible implications for ossification of the posterior longitudinal ligament.

BACKGROUND: The molecular mechanisms of ossification of the posterior longitudinal ligament (OPLL) remain to be elucidated. The aim of the present study was to investigate the autophagy of spinal ligament fibroblasts derived from patients with OPLL and to examine whether autophagy-associated gene expression was correlated with the expression of osteogenic differentiation genes. METHODS: Expression of autophagy-associated genes was detected in 37 samples from 21 OPLL patients and 16 non-OPLL patients. The correlation of autophagy-associated gene expression and the expression of osteogenic differentiation genes was analyzed by Pearson's correlation. The expression of autophagy-associated genes of ligament fibroblasts was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting, and immunofluorescence. The incidence of autophagy was assessed by flow cytometry. After knockdown using small interfering RNA targeting Beclin1, the expression of osteogenic differentiation genes were compared in spinal ligament fibroblasts. RESULTS: In clinical specimens, mRNA expression levels of microtubule-associated protein 1 light chain 3 and Beclin1 were higher in the OPLL group compared with the non-OPLL group. Pearson correlation analysis demonstrated that Beclin1 expression was positively correlated with expression of osteocalcin (OCN) (r = 0.8233, P < 0.001), alkaline phosphatase, biomineralization associated (ALP) (r = 0.7821, P < 0.001), and collagen type 1 (COL 1) (r = 0.6078, P = 0.001). Consistently, the upregulation of autophagy-associated genes in ligament fibroblasts from patients with OPLL were further confirmed by western blotting and immunofluorescence. The incidence of autophagy was also increased in ligament fibroblasts from patients with OPLL. Furthermore, knockdown of Beclin1 led to a decrease in the expression of OCN, ALP, and COL 1 by 63.2% (P < 0.01), 52% (P < 0.01), and 53.2% (P < 0.01) in ligament fibroblasts from patients with OPLL, respectively. CONCLUSIONS: Beclin1-mediated autophagy was involved in the osteogenic differentiation of ligament fibroblasts and promoted the development of OPLL.

MicroRNA-181 regulates the development of Ossification of Posterior longitudinal ligament via Epigenetic Modulation by targeting PBX1.

Objectives: Ossification of the posterior longitudinal ligament (OPLL) presents as the development of heterotopic ossification in the posterior longitudinal ligament of the spine. The etiology of OPLL is genetically linked, as shown by its high prevalence in Asian populations. However, the molecular mechanism of the disease remains obscure. In this study, we explored the function and mechanism of OPLL-specific microRNAs. Methods: The expression levels of the ossification-related OPLL-specific miR-181 family were measured in normal or OPLL ligament tissues. The effect of miR-181a on the ossification of normal or pathogenic ligament cells was tested using real-time polymerase chain reaction (PCR), Western blot, alizarin red staining and alkaline phosphatase (ALP) staining. The candidate targets of miR-181 were screened using a dual luciferase reporter assay and functional analysis. The link between miR-181a and its target PBX1 was investigated using chromatin immunoprecipitation, followed by real-time PCR detection. Histological and immunohistochemical analysis as well as micro-CT scanning were used to evaluate the effects of miR-181 and its antagonist using both tip-toe-walking OPLL mice and in vivo bone formation assays. Results: Using bioinformatic analysis, we found that miR-181a-5p is predicted to play important roles in the development of OPLL. Overexpression of miR-181a-5p significantly increased the expression of ossification-related genes, staining level of alizarin red and ALP activity, while the inhibition of miR-181a-5p by treatment with an antagomir had the opposite effects. Functional analysis identified PBX1 as a direct target of miR-181a-5p, and we determined that PBX1 was responsible for miR-181a-5p's osteogenic phenotype. By chromatin immunoprecipitation assay, we found that miR-181a-5p controls ligament cell ossification by regulating PBX1-mediated modulation of histone methylation and acetylation levels in the promoter region of osteogenesis-related genes. Additionally, using an in vivo model, we confirmed that miR-181a-5p can substantially increase the bone formation ability of posterior ligament cells and cause increased osteophyte formation in the cervical spine of tip-toe-walking mice. Conclusions: Our data unveiled the mechanism by which the miR-181a-5p/PBX1 axis functions in the development of OPLL, and it revealed the therapeutic effects of the miR-181a-5p antagomir in preventing OPLL development both in vivo and in vitro. Our work is the first to demonstrate that microRNA perturbation could modulate the development of OPLL through epigenetic regulation.

The roles of autophagy in osteogenic differentiation in rat ligamentum fibroblasts: Evidence and possible implications.

Autophagy, a macromolecular degradation process, plays a pivotal role in cell differentiation and survival. This study was designed to investigate the role of autophagy in the osteogenic differentiation in ligamentum fibroblasts. Rat ligamentum fibroblasts were isolated from the posterior longitudinal ligament and cultured in osteogenic induction medium. Ultrastructural analysis, immunofluorescence assay, western blot, flow cytometry, and lysosomal activity assessment were performed to determine the presence and activity of autophagy in the cells. The mineralization deposit and osteogenic gene expressions were evaluated to classify the association between autophagy activity and the bone formation ability of the spinal ligament cells. The influence of leptin and endothelin-1 on the autophagy activity was also evaluated. Our study demonstrated that autophagy was present and increased in the ligament cells under osteogenic induction. Inhibition of autophagy with either pharmacologic inhibitors (Bafilomycin A and 3-methyladenine) or Belcin1 (BECN1) knocking down weakened the mineralization capacity, decreased the gene expressions of COL1A1, osteocalcin (Ocn), and runt-related transcription factor 2 (Runx2) in the ligamentum fibroblasts and increased cell apoptosis. The Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)-BECN1 autophagic pathway was activated in the osteogenic differentiating ligamentum fibroblasts. Leptin significantly increased the autophagy activity in the ligament cells under osteogenic induction. These discoveries might improve our understanding for the mechanism of ossification of the posterior longitudinal ligament (OPLL) and provide new approaches on the prevention and treatment of this not uncommon disease.

Combined use of leptin and mechanical stress has osteogenic effects on ossification of the posterior longitudinal ligament.

PURPOSE: To evaluate the effects of leptin/leptin receptor (LepR) combined with mechanical stress on the development of ossification of the posterior longitudinal ligament (OPLL), which is a disease characterized by ectopic bone formation of the posterior longitudinal ligament (PLL) and can lead to radiculopathy and myelopathy. METHODS: Six human samples of the PLL were analyzed for the expression of leptin and LepR by RT-PCR and western blotting. PLL cells were stimulated with leptin and mechanical stress delivered via a Flexcell tension system, and osteogenic differentiation was evaluated by RT-PCR and western blotting analysis of osteogenic marker expression as well as by alkaline phosphatase (ALP) staining and alizarin red S staining. Activation of mitogen-activated protein kinase (MAPK), Janus kinase (JAK) 2-signal transducer, activator of transcription (STAT) 3 and phosphatidylinositol 3-kinase (PI3K)-Akt was evaluated by western blotting. RESULTS: Samples from the OPLL group had higher LepR mRNA and protein levels and lower leptin levels than those from healthy controls. Exposure to leptin and Flexcell increased the number of ALP-positive cells and calcium nodules in a dose-dependent manner; this effect was accompanied by upregulation of the osteogenic markers osteocalcin, runt-related transcription factor 2 (RUNX2) and osteopontin. Extracellular signal-regulated kinase, P38 MAPK, JAK2, STAT3, PI3K and Akt signaling, was also activated by the combined effects of leptin and mechanical stress. CONCLUSIONS: Leptin and LepR are differentially expressed in OPLL tissues, and the combined use of leptin/LepR and mechanical stress promotes osteogenic differentiation of PLL cells via MAPK, JAK2-STAT3 and PI3K/Akt signaling. These slides can be retrieved under Electronic Supplementary Material.

Suppression of osteogenic differentiation in mesenchymal stem cells from patients with ossification of the posterior longitudinal ligament by a histamine-2-receptor antagonist.

Mesenchymal stem cells (MSCs) in ossification of the posterior longitudinal ligament (OPLL) patients have a high propensity toward osteogenesis. Histamine receptor H2 (H2R) antagonists (H2 blockers) like famotidine decrease ossification in patients, by an unclear mechanism. To confirm that MSCs express H2R and to clarify how H2 blockers suppress osteogenic differentiation, we used spinal-ligament MSCs from patients with OPLL or with cervical spondylotic myelopathy (CSM) (control). The MSCs were treated with 10, 30, or 100nM famotidine for 7 or 21 days. Flow cytometry revealed that cells from both groups expressed MSC surface markers CD44, CD90, and CD105 (> 97.5%) but not CD34 or CD45 (< 2.5%). Immunoblotting showed that the MSCs from both groups expressed H2R, but those from OPLL patients expressed it at higher levels. Real-time qPCR indicated the H2R expression was significantly suppressed by 30nM famotidine for 7 days or by 30 or 100nM for 21 days. However, histidine decarboxylase, a key enzyme in histamine production, did not change significantly after famotidine addition. Famotidine treatment at 100nM for 21 days significantly suppressed mRNA expression of the osteogenic markers osteocalcin (OCN), bone morphogenetic protein 2 (BMP2), and runt-related transcription factor 2 (RUNX2) only in OPLL-derived MSCs. Immunoblots showed that famotidine suppressed BMP2 and OCN in the OPLL group and H2R and RUNX2 in both groups. These results suggest famotidine inhibits osteogenic differentiation in OPLL-derived MSCs by acting as an H2R antagonist, but also by decreasing H2R expression, and support the clinical use of famotidine to treat OPLL.

Connexin 43 Affects Osteogenic Differentiation of the Posterior Longitudinal Ligament Cells via Regulation of ERK Activity by Stabilizing Runx2 in Ossification.

AIMS: Connexin 43 is one of the most potent gap junction proteins related to osteoblast differentiation and bone formation. We hypothesized that Connexin 43 is a significant factor in osteogenic differentiation in the posterior longitudinal ligament through the regulation of extracellular signal-regulated kinases (ERK) activity by converging on Runt-related transcription factor 2 (Runx2) activity. In this study, we mapped the activity of Connexin 43 to ERK and Runx2 by extracting longitudinal ligament cell for culture and silencing Connexin expression in addition to dexamethasone treatment in vitro. METHODS: qRT-PCR, Western Blot, and Runx2-responsive Luciferase Reporter Assay were performed to detect the activity of ERK, Runx2 and the expression levels of osseous genes under Connexin 43 modification. RESULTS: Downregulation of Connexin 43 resulted in suppression of dexamethasone-induced osteogenic differentiation, inhibition of the ERK and Runx2 activity, and reduction of osseous gene expression. CONCLUSION: these data support that Connexin 43 significantly regulates osteogenic differentiation in the cells from posterior longitudinal ligament by altering the activity of ERK, and subsequently causing the modification of Runx2.

A comprehensive review of the sub-axial ligaments of the vertebral column: part II histology and embryology.

BACKGROUND: As important as the vertebral ligaments are in maintaining the integrity of the spinal column and protecting the contents of the spinal canal, a single detailed review of their histology and embryology is missing in the literature. METHODS: A literature search using online search engines was conducted. RESULTS: Single comprehensive reviews of the histology and embryology of the spinal ligaments are not found in the extant medical literature. CONCLUSIONS: This review will be useful to those who study or treat patients with pathology of the spine.

Upregulated expression of PERK in spinal ligament fibroblasts from the patients with ossification of the posterior longitudinal ligament.

PURPOSE: Molecular mechanism of ossification of the posterior longitudinal ligament (OPLL) remains unclear. This study was to investigate different expressions of PERK between the spinal ligament fibroblasts from OPLL patients and non-OPLL patients, and demonstrate knockdown of PERK protein expression by RNA interference inhibiting expression of osteocalcin (OCN), alkaline phosphatase (ALP), and type I collagen (COL I) in the cells from OPLL patients. METHODS: Spinal ligament cells were cultured using tissue fragment cell culture and identified by immunocytochemistry and immunofluorescence. The mRNA expression of osteoblast-specific genes of OCN, ALP and COL I was detected in the cells from OPLL and non-OPLL patients by semiquantitative reverse transcription-polymerase chain reaction. The protein expression of PERK was detected by Western blotting. And then, after 72 h, when RNA interference against PERK was performed on the cells from OPLL patients, expression of the osteoblast-specific genes was compared again between the transfection group and non-transfection group. RESULTS: Spinal ligament fibroblasts were observed 7-10 days after cell culture. Immunocytochemistry and immunofluorescence exhibited positive results of vimentin staining. The mRNA expressions of OCN, ALP and COL I and protein expression of PERK in the cells from OPLL patients were significantly greater than those from non-OPLL patients. In addition, knockdown of PERK protein expression inhibited the mRNA expressions of OCN, ALP and COL I remarkably in the transfection group compared with the non-transfection group, at 72 h after RNA interference targeting PERK was performed on the cells from OPLL patients. CONCLUSIONS: The cultured fibroblasts from OPLL patients exhibited osteogenic characteristics, and PERK-mediated ER stress might be involved in development of OPLL.

[Attachment points of the posterior longitudinal ligament and their importance for thoracic and lumbar spine fractures]. / Anheftungspunkte des Lig. longitudinale posterius und deren Bedeutung für Wirbelkörperfrakturen.

Spine fractures with damage of the posterior wall of the vertebra often can be anatomically reconstructed by indirect reduction. Whether the posterior longitudinal ligament (PLL) is responsible for the reduction is still subject to debate. The aim of our investigation was to ascertain the role of the PLL in closed reduction of spine fractures by identifying the bony attachment points of this ligament. We performed a gross anatomical dissection, a light- and polarized microscopic investigation on 22 human cadaverous thoracic and lumbar spines to determine the points of attachment of the PLL. We found two layers of the PLL. The superficial layer runs from the first thoracic down to the third lumbar vertebra with a width of 0.4-1.0 cm and from there descends as a thin rudiment to the sacrum. The deep layer shows a segmental rhomboid structure. Lateral fibers are attached to the annulus fibrosus and at the rim of the adjacent vertebrae. Medial fibers are attached additionally to the posterior wall of the vertebral bodies by bridging the foramina basivertebralia. Since these foramina become enlarged in the caudal parts of the vertebral column, the number of attachment points at the posterior wall of the vertebral bodies decreases caudally. Good results for reconstruction of the posterior wall in vertebral fractures of the thoracic and upper lumbar spine can be explained by the anatomical situation of the PLL and stress the important role of the PLL in indirect reduction of spine fractures.

High serum levels of menatetrenone in male patients with ossification of the posterior longitudinal ligament.

STUDY DESIGN: This work was performed to investigate the role of vitamin K (VK) in the pathogenesis of ossification of posterior longitudinal ligament (OPLL), by analyzing the biochemical markers of the blood samples of OPLL patients and responses of ligament cells derived from OPLL lesion to VK2. OBJECTIVES: The pathogenesis of OPLL, classified as a form of diffuse idiopathic skeletal hyperostosis, is still unclear. In this study, we investigated the role of menaquinone (VK2) in patients with OPLL (OPLL patients) and the effects of VK2 on ligament cells isolated from OPLL lesion. METHODS: Serum levels of intact osteocalcin, glu-osteocalcin, MK-4, -7 (VK2 variants) and other minerals in spot blood samples were measured in 24 OPLL patients and in 24 age-matched control patients (non-OPLL patients). The cultured cells isolated from an OPLL patient were treated with MK-4. Alkaline phosphatase (Al-p) activity and osteocalcin release were measured after 2 weeks of culture. RESULTS: In the clinical study, the serum MK-4 in male OPLL patients was significantly higher than that in male non-OPLL patients. However, among female patients, the difference was not significant. Although the serum osteocalcin in females was significantly higher than that in males, there was no significant difference between the OPLL and non-OPLL groups. In in vitro study, MK-4 did not increase Al-p activity in the ligament cells isolated from nonossified region of OPLL patient. Osteoblastic activity of the cultured cells was not stimulated by MK-4. CONCLUSION: From these results and previous reports, we propose the possibility of the impediment in VK2 metabolism in OPLL patients. The results also implicate the gender tendency in OPLL, because the difference of serum level of MK-4 in OPLL patients was significant only in male.

Possible roles of CTGF/Hcs24 in the initiation and development of ossification of the posterior longitudinal ligament.

STUDY DESIGN: A biochemical and histochemical study investigating the role of CTGF/Hcs24 in the ossification of the posterior longitudinal ligament (OPLL) was conducted. OBJECTIVE: To clarify the involvement of CTGF/Hcs24 in ectopic bone formation in OPLL through endochondral ossification using human tissue. SUMMARY OF BACKGROUND DATA: Previous studies have shown that various cytokines are involved in the occurrence or development of ectopic bone formation in OPLL. Recently, the authors cloned an mRNA predominantly expressed in chondrocytes by differential display PCR and found that its gene, hcs24, is identical to that of connective tissue growth factor. It has been shown that CTGF/Hcs24 plays a major role in endochondral ossification. METHODS: Ossified ligament tissues were taken from seven male OPLL patients during surgery. Immunohistochemical staining was performed using an antibody specific for CTGF/Hcs24. Spinal ligament cells were isolated from five OPLL patients as well as five non-OPLL patients. The cells were incubated with recombinant human CTGF/Hcs24 or TGFbeta. The expression of ALP was analyzed by RT-PCR. For the effects of TGFbeta, the expression of CTGF/Hcs24 mRNA was analyzed. RESULTS: Immunohistochemical staining showed that chondrocytes in the transitional region from nonossified to ossified ligament were stained with an antibody against CTGF/Hcs24. It was found that CTGF/Hcs24 enhanced the expression ALP mRNA in OPLL cells, whereas the expression remained unchanged in non-OPLL cells. The expression of CTGF/Hcs24 mRNA in OPLL and non-OPLL cell lines was increased by TGFbeta, and there was no significant difference between the two groups. However, TGFbeta and CTGF/Hcs24 enhanced the expression of ALP mRNA only in OPLL cells. CONCLUSIONS: According to the study results, CTGF/Hcs24 may not only be an important factor in the development of endochondral ossification in OPLL, but may also be responsible for initiating osteogenesis in spinal ligament cells.

In vitro characteristics of cultured posterior longitudinal ligament tissue.

STUDY DESIGN: To determine the osteogenicity of posterior longitudinal ligament ossification, the posterior longitudinal ligament obtained during anterior cervical surgery from patients with the disorder was analyzed with in vitro cultures. OBJECTIVES: To determine the osteogenicity of the posterior longitudinal ligament. SUMMARY OF BACKGROUND DATA: The osteogenicity of posterior longitudinal ligament ossification in North America requires better documentation. METHODS: The posterior longitudinal ligament obtained during anterior cervical corpectomy with fusion from seven patients, three with ossification of the posterior longitudinal ligament documented by magnetic resonance imaging and computed tomography and four with spondylosis, was blindly submitted for in vitro culture. Explants of the posterior longitudinal ligament were placed in Dulbecco modified Eagle medium with 10% fetal calf serum, antibiotics, 4 mmol/L x L-proline, and 50 mg/L ascorbic acid. After reaching confluency, cells were trypsinized, and first-passage cells were used for all osteocalcin measurements to establish their osteoblastic phenotype. Periosteal cells, previously shown to synthesize osteocalcin, were used as a positive control. The cells were incubated with 1,25(OH)2 vitamin D3 at 10E-8 M for 72 hours in serum-free medium. The supernatants were collected and frozen, after which the quantity of osteocalcin induced by exposure to 1,25(OH)2 vitamin D3 was determined using enzyme-linked immunoassay. Control replicate cultures were measured without incubation using vitamin D3. RESULTS: Ossification of the posterior longitudinal ligament cell lines responded positively with osteocalcin synthesis in the 0.1 to 0.4 ng/M range. The cell line of the patient with spondylosis alone did not respond to vitamin D3 priming. CONCLUSIONS: Posterior longitudinal ligament cells from the three North American white patients with ossification of the posterior longitudinal ligament, when cultured in vitro, synthesized osteocalcin on vitamin D3 priming, confirming their osteoblastic phenotype, whereas posterior longitudinal ligament cells from four white patients with isolated spondylosis did not.

Bone morphogenetic protein-2 stimulates differentiation of cultured spinal ligament cells from patients with ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament (OPLL) of the spine is characterized by heterotopic bone formation occurring in spinal ligament, causing severe compression myelopathy. In order to investigate the mechanism of OPLL development, we isolated spinal ligament cells from OPLL patients as well as non-OPLL patients, and established 10 OPLL cell lines and 7 non-OPLL cell lines, respectively. We analyzed the effects of bone morphogenetic protein-2 (BMP-2) on these cells with respect to alkaline phosphatase (AP) activity, DNA synthesis, and collagen production. BMP-2 caused a significant increase of AP activity in 4 OPLL cell lines, whereas the activity did not change in any non-OPLL cells. Among OPLL cells, BMP-2 stimulated DNA synthesis in four cell lines and procollagen type I carboxyl-terminal peptide (PICP) synthesis in five cell lines. Some non-OPLL cells also responded to BMP-2, as there was an increase of DNA synthesis in three cell lines and PICP synthesis in one cell line. These data collectively indicate that BMP-2 preferentially induces osteogenic differentiation in OPLL cells rather than in non-OPLL cells. OPLL cells, therefore, exhibit a different response to BMP-2 than non-OPLL cells, suggesting that the expression of BMP receptor(s) and/or the signal transduction initiated by BMP-2 in the spinal ligament cells of OPLL patients somewhat deviate from those in normal spinal ligament cells. Such abnormal characteristics of OPLL cells as described here provide some clues to the clarification of the pathogenesis of OPLL.

Innervation of the spinal dura. Myth or reality?

STUDY DESIGN: Cranial and spinal dura from nine Sprague Dawley male rats were examined immunocytochemically for the presence of nerve fibers and mast cells and for innervation. The posterior longitudinal ligament and peridural membrane were also examined for these elements. OBJECTIVE: To examine the pattern of sensory innervation and the presence of mast cells in rat spinal dura. SUMMARY OF BACKGROUND DATA: The cranial dura is richly innervated and has a robust population of mast cells, which have been implicated in the pathogenesis of vascular headache. Moskowitz's explanation for vascular headache focused on the dura mater and neurogenic inflammation. Essential to his model are dural trigeminovascular fibers and mast cells. Previous studies provide contradictory and inconclusive results regarding spinal dural innervation. METHODS: Immunocytochemical techniques using antibodies to calcitonin gene-related peptide and substance P were used to identify sensory nerve fibers and antibodies to serotonin were used to identify mast cells. Specimens dissected included dura of the cranial vault in continuity with the dorsal cervical dura, dura of the skull base in continuity with the ventral cervical dura, lumbar dura, and posterior longitudinal ligament from the cervical and lumbar regions. RESULTS: A rich neural network and an abundant mast cell population were identified in the supratentorial and infratentorial cranial dura, both dorsally and ventrally. A paucity of nerve fibers and mast cells was observed in the cervical and lumbar dura; in contrast, these elements were prominent in the posterior longitudinal ligament and peridural membrane. CONCLUSIONS: Spinal dura does not have a rich innervation of calcitonin gene-related peptide- and substance P-positive nerve fibers or a robust population of mast cells. The spinal dura may serve as a protective covering. Unlike the cranial dura, it may not be implicated in the pathogenesis of pain. Additional studies on primate and human spinal dura are warranted to corroborate findings that the spinal dura may be relatively insensitive.