Mouse Model Of Intervertebral Disc Degeneration Research Articles

Circular RNA CircFOXO3 Functions as a Competitive Endogenous RNA for Acid-Sensing Ion Channel Subunit 1 Mediating Oxeiptosis in Nucleus Pulposus.

Oxeiptosis is a reactive oxygen species (ROS)-induced pathway of cell death. The involvement of circular RNAs (circRNAs) has been confirmed in the incidence and progression of intervertebral disc degeneration (IVDD). However, whether oxeiptosis occurs in IVDD and how circRNAs regulate oxeiptosis is still unclear. In this study, we discovered that oxeiptosis could be induced in nucleus pulposus cells (NPCs), and circFOXO3 was significantly upregulated after oxeiptosis induction. Transfection using circFOXO3 small interfering RNA (siRNA) significantly inhibited oxeiptosis in NPCs. Mechanistically, circFOXO3 upregulated acid-sensing ion channel subunit 1 (ASIC1) expression by functioning as a molecular sponge for miR-185-3p and miR-939-5p. Subsequent rescue experiments validated that circFOXO3 could regulate oxeiptosis in NPCs via the miR-185-3p/miR-939-5p-ASIC1 axis. Further research on ASIC1 functions indicated that this regulation was achieved by affecting the Calcium ion (Ca2+) influx mediated by ASIC1. A mouse IVDD model was established, and silencing circFOXO3 in vivo was found to inhibit IVDD development and the activation of the oxeiptosis-related pathway. Overall, circFOXO3 is one of the factors contributing to the progression of IVDD by mediating oxeiptosis.

CD206+ M2-like macrophages protect against intervertebral disc degeneration partially by targeting R-spondin-2

This study aimed to explore the specific function of M2 macrophages in intervertebral disc degeneration (IDD). Intervertebral disc (IVD) samples from normal (n = 4) and IDD (n = 6) patients were collected, and the expression of M2-polarized macrophage marker, CD206, was investigated using immunohistochemical staining. Nucleus pulposus cells (NPCs) in a TNF-α environment were obtained, and a mouse caudal IVD puncture model was established. Mice with Rheb deletions, specifically in the myeloid lineage, were generated and subjected to surgery-induced IDD. IDD-induced damage and cell apoptosis were measured using histological scoring, X-ray imaging, immunohistochemical staining, and TUNEL assay. Finally, mice and NPCs were treated with R-spondin-2 (Rspo2) or anti-Rspo2 to investigate the role of Rspo2 in IDD. Accumulation of CD206 in human and mouse IDD tissues was detected. Rheb deletion in the myeloid lineage (RheBcKO) increased the number of CD206+ M2-like macrophages (mean difference 18.6% [15.7-21.6%], P < 0.001), decreased cell apoptosis (mean difference -15.6% [-8.9 to 22.2%], P = 0.001) and attenuated the IDD process in the mouse IDD model. NPCs treated with Rspo2 displayed increased extracellular matrix catabolism and apoptosis; co-culture with a conditioned medium derived from RheBcKO mice inhibited these changes. Anti-Rspo2 treatment in the mouse caudal IVD puncture model exerted protective effects against IDD. Promoting CD206+ M2-like macrophages could reduce Rspo2 secretion, thereby alleviating experimental IDD. Rheb deletion may help M2-polarized macrophages accumulate and attenuate experimental IDD partially by inhibiting Rspo2 production. Hence, M2-polarized macrophages and Rspo2 may serve as therapeutic targets for IDD.

MiR-31 from Mesenchymal Stem Cell-Derived Extracellular Vesicles Alleviates Intervertebral Disc Degeneration by Inhibiting NFAT5 and Upregulating the Wnt/β-Catenin Pathway.

In this study, we explored the regulatory mechanism of intervertebral disc degeneration (IDD) that involves miR-31 shuttled by bone marrow mesenchymal stem cell-derived extracellular vesicles (BMSC-EVs) and its downstream signaling molecules. Nucleus pulposus cells (NPCs) were isolated and treated with TNF-α to simulate IDD in vitro. The TNF-α-exposed NPCs were then cocultured with hBMSCs or hBMSC-EVs in vitro to detect the effects of hBMSC-EVs on NPC viability, apoptosis, and ECM degradation. Binding between miR-31 and NFAT5 was determined. A mouse model of IDD was prepared by vertebral disc puncture and injected with EVs from hBMSCs with miR-31 knockdown to discern the function of miR-31 in vivo. The results demonstrated that hBMSC-EVs delivered miR-31 into NPCs. hBMSC-EVs enhanced NPC proliferation and suppressed cell apoptosis and ECM degradation, which was associated with the transfer of miR-31 into NPCs. In NPCs, miR-31 bound to the 3′UTR of NFAT5 and inhibited NFAT5 expression, leading to activation of the Wnt/β-catenin pathway and thus promoting NPC proliferation and reducing cell apoptosis and ECM degradation. In addition, miR-31 in hBMSC-EVs alleviated the IDD in mouse models. Taken together, miR-31 in hBMSC-EVs can alleviate IDD by targeting NFAT5 and activating the Wnt/β-catenin pathway.

Exogenous Parathyroid Hormone Alleviates Intervertebral Disc Degeneration through the Sonic Hedgehog Signalling Pathway Mediated by CREB.

As an important hormone that regulates the balance of calcium and phosphorus, parathyroid hormone (PTH) has also been found to have an important function in intervertebral disc degeneration (IVDD). Our aim was to investigate the mechanism by which PTH alleviates IVDD. In this study, the PTH 1 receptor was found to be highly expressed in severely degenerated human nucleus pulposus (NP) cells. We found in the mouse model of IVDD that supplementation with exogenous PTH alleviated the narrowing of the intervertebral space and the degradation of the extracellular matrix (ECM) caused by tail suspension (TS). In addition, inflammation, oxidative stress, and apoptosis levels were significantly increased in the intervertebral disc tissues of TS-induced mice, and the activity of NP cells was decreased. TS also led to the downregulation of Sonic hedgehog (SHH) signalling pathway-related signal molecules in NP cells such as SHH, Smoothened, and GLI1. However, supplementation with exogenous PTH can reverse these changes. In vitro, PTH also promotes the activity of NP cells and the secretion of ECM. However, the antagonist of the SHH signalling pathway can inhibit the therapeutic effect of PTH on NP cells. In addition, a cAMP-response element-binding protein, as an important transcription factor, was found to mediate the promotion of PTH on the SHH signalling pathway. Our results revealed that PTH can alleviate IVDD by inhibiting inflammation, oxidative stress, and apoptosis and improving the activity of NP cells via activating the SHH signalling pathway.

Bone Marrow-Derived Mesenchymal Stem Cells Augment Regeneration of Intervertebral Disc in a Reproducible and Validated Mouse Intervertebral Disc Degeneration Model.

Back pain and radicular pain due to disc degeneration are probably the most common problems encountered in neurosurgical practice. The experience and results of stem cell therapy in animal disc degeneration model will help us while doing clinical trials. To study the effect of bone marrow-derived mesenchymal stem cells in an established mouse disc degeneration model. An easily reproducible mouse coccygeal (Co) 4-5 disc degenerated model by CT-guided percutaneous needle injury was established. The mesenchymal stem cells (MSCs) were cultured from mouse bone marrow and validated. By an established technique, 24 mice disc degenerative models were generated and divided equally into 3 groups (test, placebo, and control). The test group received MSCs with fibrin glue scaffold and placebo group received only scaffold after 6 weeks of degeneration. The control group did not receive any injection. The effects of MSCs were analyzed 8 weeks post injection. The test group showed a significant change in disc height index (%) in micro CT, whereas in the placebo and control groups, there was no change. The Safranin O staining showed an increase in glycosaminoglycan content and the polarized imaging of picrosirius red staining showed restoration of the collagen fibers in annulus fibrosus, which was statistically significant. Intradiscal MSC injection restored disc height and promoted regeneration in the discs at the end of 8 weeks. MSC's niche depends on the microenvironment of the host tissue. These findings will be helpful for clinical trials.

A novel in vivo mouse intervertebral disc degeneration model induced by compressive suture

Intervertebral disc degeneration (IDD) is the root cause of many musculoskeletal disorders of the spine. However, the etiology of IDD is complex and still not well understood. Animal models of IDD would be useful in deciphering the underlying mechanisms. But the existing animal models have their limitations. Therefore, to establish a novel mouse model that can simulate the human IDD process in vivo, we proposed to carefully circumcise the 2 mm-wide tail skin and then compressively sutured the defect with a simple end-to-end suture to exert excessive pressure on the disc. After 1-week, 2-week, and 4-week compression, the mice were sacrificed and the intervertebral discs were harvested for tissue analysis. The radiological, morphological, and molecular modifications of intervertebral discs were measured to characterize this model. Radiologically, the water content of the intervertebral disc decreased significantly after 2-week compression. Morphologically, the nucleus pulposus showed a decrease in volume and the number of notochordal cells. The compressive suture also broke the balance between anabolic and catabolic enzymes in nucleus pulposus, which led to the remodeling of the extracellular matrix in nucleus pulposus as the content of aggrecan and collagen II decreased. The compressive suture could induce intervertebral discs degeneration in a more reasonable way, which was solely influenced by mechanical loading, as the mice caudal vertebrae still moved freely after the operation. This kind of animal model could be adapted as a reliable in vivo mouse IDD model for the research regarding the etiology and treatments of IDD.

Dexmedetomidine suppresses oxidative stress and inflammation of nucleus pulposus cells by activating the PI3K/Akt signaling pathway.

Objective: Intervertebral disc degeneration (IVDD) is very common in the elderly, so it is particularly important to find appropriate prevention or treatment. The aim of this study was to explore the effect of dexmedetomidine (DEX) on the degeneration of nucleus pulposus (NP) cells and its mechanism. Methods: We established a mouse model of IVDD and cultured mouse NP cells and treated them with IL-1β and DEX. The effect of DEX on NP cells was determined by detecting the extracellular matrix of NP cells, changes in ROS levels and inflammatory mediators. LY294002, a PI3K inhibitor, is used to inhibit the activity of the PI3K/Akt signaling pathway. The effect of DEX on the PI3K/Akt signaling pathway was determined by studying the effects of DEX on PI3K/ Akt signaling pathway-related molecules and the effect of LY294002 on NP cells degeneration. DEX significantly increased the disc height index and attenuated IVDD in mice. Results: DEX significantly inhibited the expression of MMP3/9 in NP cells, effectively inhibiting the degradation of extracellular matrix. In addition, oxidative stress levels and inflammatory levels in NP cells are also attenuated by DEX. The expression of PI3K, Akt and p-Akt was significantly increased in DEX-stimulated NP cells, indicating that DEX increased the activity of the PI3K/Akt signaling pathway. DEX promotes PI3K/Akt signaling pathway, inhibits oxidative stress and inflammatory of NP cells, thereby slowing the degeneration of NP cells. Conclusion: DEX promotes PI3K/Akt signaling pathway, inhibits oxidative stress and inflammatory of NP cells, thereby slowing the degeneration of NP cells.

Nrf2 drives oxidative stress-induced autophagy in nucleus pulposus cells via a Keap1/Nrf2/p62 feedback loop to protect intervertebral disc from degeneration

Intervertebral disc (IVD) degeneration is known to aggravate with age and oxidative stress is implicated in the pathogenesis of many age-related diseases. Nuclear factor (erythroid-derived-2)-like 2 (Nrf2) can confer adaptive protection against oxidative and proteotoxic stress in cells. In this study, we assessed whether Nrf2 can protect against oxidative stress in nucleus pulposus (NP) cells. In addition, we investigated Nrf2 expression in NP tissue samples from patients with different degrees of IVD degeneration and a mouse model of aging and IVD degeneration and the influence of H2O2-induced oxidative stress on autophagic pathways in NP cells. Autophagy was assessed by measuring levels of autophagy-related protein (ATG) family members and the autophagic markers, p62 and LC3. We found that expression of Nrf2 progressively decreased in human NP tissue samples of patients with increasing degrees of IVD degeneration. Nrf2 deficiency leads to the degeneration of IVDs during aging. Nrf2 knockout also aggravates IVD degeneration and reduces autophagic gene expression in an induced mouse model of IVD degeneration. The detrimental effects of H2O2-induced oxidative stress were increased in autophagy-deficient cells via reduced expression of Atg7 and the Keap1–Nrf2–p62 autophagy pathway. Taken together, these results suggest that excessive oxidative stress causes the upregulation of autophagy, and autophagy acts as an antioxidant feedback response activated by a Keap1-Nrf2-p62 feedback loop in IVD degeneration.

A Mouse Intervertebral Disc Degeneration Model by Surgically Induced Instability.

An experimental study to develop a mouse model of lumbar intervertebral disc degeneration (IDD). The aim of this study was to develop a mouse lumbar IDD model using surgically induced instability and to compare the findings of this model to those in human IDD. Previously, various kinds of inducers have been used to reproduce IDD in experimental animals; however, there is yet no standard mouse lumbar IDD model without direct injury to intervertebral disc. A total number of 59 C57BL/6J male mice at 8 weeks old were used. Instability of lumbar spine was induced by surgical resection of posterior elements, including facet joints, supra- and interspinous ligaments. We then analyzed time course changes in radiographical (n = 17) and histological analyses (n = 42), and compared these findings with those in human IDD. Radiographical analyses showed that the disc height began to decrease in the first 2 weeks after the surgery, and the decrease continued throughout 12 weeks. Bone spurs at the vertebral rims were observed in the late stage of 8 and 12 weeks after the surgery. Histological analyses showed that the disorder of the anterior anulus fibrosus (AF) was initially obvious, followed by posterior shift and degeneration of the nucleus pulposus (NP). Proteoglycan detected in inner layer of AF and periphery of NP was decreased after 8 weeks. Immunohistochemistry displayed the increase of type I and X collagen, and matrix metalloproteinase 13 in the anterior AF. Surgical resection of posterior elements of mouse lumbar spine resulted in reproducible IDD. Because the present procedure does not employ direct injury to intervertebral disc and the radiological and histological findings are compatible with those in human IDD, it may contribute to further understanding of the native pathophysiology of IDD in future. N/A.

In Vivo Mouse Intervertebral Disc Degeneration Model Based on a New Histological Classification.

Although human intervertebral disc degeneration can lead to several spinal diseases, its pathogenesis remains unclear. This study aimed to create a new histological classification applicable to an in vivo mouse intervertebral disc degeneration model induced by needle puncture. One hundred six mice were operated and the L4/5 intervertebral disc was punctured with a 35- or 33-gauge needle. Micro-computed tomography scanning was performed, and the punctured region was confirmed. Evaluation was performed by using magnetic resonance imaging and histology by employing our classification scoring system. Our histological classification scores correlated well with the findings of magnetic resonance imaging and could detect degenerative progression, irrespective of the punctured region. However, the magnetic resonance imaging analysis revealed that there was no significant degenerative intervertebral disc change between the ventrally punctured and non-punctured control groups. To induce significant degeneration in the lumbar intervertebral discs, the central or dorsal region should be punctured instead of the ventral region.

The distinct role of the Runx proteins in chondrocyte differentiation and intervertebral disc degeneration: Findings in murine models and in human disease

Runx2 is a transcription factor that regulates chondrocyte differentiation. This study was undertaken to address the role of the different Runx proteins (Runx1, Runx2, or Runx3) in chondrocyte differentiation using chondrocyte-specific Runx-transgenic mice, and to study the importance of the QA domain of Runx2, which is involved in its transcriptional activation. Runx expression was analyzed in the mouse embryo by in situ hybridization. Overexpression of Runx1, Runx2 (lacking the QA domain [DeltaQA]), or Runx3 was induced in chondrocytes in vivo, to produce alpha(1)II-Runx1, alpha(1)II-Runx2DeltaQA, and alpha(1)II-Runx3 mice, respectively, for histologic and molecular analyses. Runx expression was also examined in an experimental mouse model of mechanical stress-induced intervertebral disc (IVD) degeneration and in human patients with IVD degeneration. Runx1 expression was transiently observed in condensations of mesenchymal cells, whereas Runx2 and Runx3 were robustly expressed in prehypertrophic chondrocytes. Similar to alpha(1)II-Runx2 mice, alpha(1)II-Runx2DeltaQA and alpha(1)II-Runx3 mice developed ectopic mineralization of cartilage, but this was less severe in the alpha(1)II-Runx2DeltaQA mice. In contrast, alpha(1)II-Runx1 mice displayed no signs of ectopic mineralization. Surprisingly, alpha(1)II-Runx1 and alpha(1)II-Runx2 mice developed scoliosis due to IVD degeneration, characterized by an accumulation of extracellular matrix and ectopic chondrocyte hypertrophy. During mouse embryogenesis, Runx2, but not Runx1 or Runx3, was expressed in the IVDs. Moreover, both in the mouse model of IVD degeneration and in human patients with IVD degeneration, there was significant up-regulation of Runx2 expression. Each Runx protein has a distinct, yet overlapping, role during chondrocyte differentiation. Runx2 contributes to the pathogenesis of IVD degeneration.