Quercetin ameliorates oxidative stress-induced senescence in rat nucleus pulposus-derived mesenchymal stem cells via the miR-34a-5p/SIRT1 axis.

BACKGROUND: Intervertebral disc degeneration (IDD) is a main contributor to low back pain. Oxidative stress, which is highly associated with the progression of IDD, increases senescence of nucleus pulposus-derived mesenchymal stem cells (NPMSCs) and weakens the differentiation ability of NPMSCs in degenerated intervertebral discs (IVDs). Quercetin (Que) has been demonstrated to reduce oxidative stress in diverse degenerative diseases. AIM: To investigate the role of Que in oxidative stress-induced NPMSC damage and to elucidate the underlying mechanism. METHODS: In vitro, NPMSCs were isolated from rat tails. Senescence-associated ß-galactosidase (SA-ß-Gal) staining, cell cycle, reactive oxygen species (ROS), real-time quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, and western blot analyses were used to evaluated the protective effects of Que. Meanwhile the relationship between miR-34a-5p and Sirtuins 1 (SIRT1) was evaluated by dual-luciferase reporter assay. To explore whether Que modulates tert-butyl hydroperoxide (TBHP)-induced senescence of NPMSCs via the miR-34a-5p/SIRT1 pathway, we used adenovirus vectors to overexpress and downregulate the expression of miR-34a-5p and used SIRT1 siRNA to knockdown SIRT1 expression. In vivo, a puncture-induced rat IDD model was constructed, and X rays and histological analysis were used to assess whether Que could alleviate IDD in vivo. RESULTS: We found that TBHP can cause NPMSCs senescence changes, such as reduced cell proliferation ability, increased SA-ß-Gal activity, cell cycle arrest, the accumulation of ROS, and increased expression of senescence-related proteins. While abovementioned senescence indicators were significantly alleviated by Que treatment. Que decreased the expression levels of senescence-related proteins (p16, p21, and p53) and senescence-associated secreted phenotype (SASP), including IL-1ß, IL-6, and MMP-13, and it increased the expression of SIRT1. In addition, the protective effects of Que on cell senescence were partially reversed by miR-34a-5p overexpression and SIRT1 knockdown. In vivo, X-ray, and histological analyses indicated that Que alleviated IDD in a puncture-induced rat model. CONCLUSION: In summary, the present study provides evidence that Que reduces oxidative stress-induced senescence of NPMSCs via the miR-34a/SIRT1 signaling pathway, suggesting that Que may be a potential agent for the treatment of IDD.

1,25(OH)2D3 Mitigates Oxidative Stress-Induced Damage to Nucleus Pulposus-Derived Mesenchymal Stem Cells through PI3K/Akt Pathway.

Intervertebral disc degeneration (IVDD) is one of the main causes of low back pain. The local environment of the degenerated intervertebral disc (IVD) increases oxidative stress and apoptosis of endogenous nucleus pulposus-derived mesenchymal stem cells (NPMSCs) and weakens its ability of endogenous repair ability in degenerated IVDs. A suitable concentration of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been certified to reduce oxidative stress and cell apoptosis. The current study investigated the protective effect and potential mechanism of 1,25(OH)2D3 against oxidative stress-induced damage to NPMSCs. The present results showed that 1,25(OH)2D3 showed a significant protective effect on NPMSCs at a concentration of 10-10 M for 24 h. Protective effects of 1,25(OH)2D3 were also exhibited against H2O2-induced NPMSC senescence, mitochondrial dysfunction, and reduced mitochondrial membrane potential. The Annexin V/PI apoptosis detection assay, TUNEL assay, immunofluorescence, western blot, and real-time quantitative polymerase chain reaction assay showed that pretreatment with 1,25(OH)2D3 could alleviate H2O2-induced NPMSC apoptosis, including the apoptosis rate and the expression of proapoptotic-related (Caspase-3 and Bax) and antiapoptotic-related (Bcl-2) proteins. The intracellular expression of p-Akt increased after pretreatment with 1,25(OH)2D3. However, these protective effects of 1,25(OH)2D3 were significantly decreased after the PI3K/Akt pathway was inhibited by the LY294002 treatment. In vivo, X-ray, MRI, and histological analyses showed that 1,25(OH)2D3 treatment relieved the degree of IVDD in Sprague-Dawley rat disc puncture models. In summary, 1,25(OH)2D3 efficiently attenuated oxidative stress-induced NPMSC apoptosis and mitochondrial dysfunction via PI3K/Akt pathway and is a promising candidate treatment for the repair of IVDD.

Urolithin a alleviates oxidative stress-induced senescence in nucleus pulposus-derived mesenchymal stem cells through SIRT1/PGC-1α pathway.

BACKGROUND: In degenerative intervertebral disc (IVD), an unfavorable IVD environment leads to increased senescence of nucleus pulposus (NP)-derived mesenchymal stem cells (NPMSCs) and the inability to complete the differentiation from NPMSCs to NP cells, leading to further aggravation of IVD degeneration (IDD). Urolithin A (UA) has been proven to have obvious effects in delaying cell senescence and resisting oxidative stress. AIM: To explore whether UA can alleviate NPMSCs senescence and to elucidate the underlying mechanism. METHODS: In vitro, we harvested NPMSCs from rat tails, and divided NPMSCs into four groups: the control group, H2O2 group, H2O2 + UA group, and H2O2 + UA + SR-18292 group. Senescence-associated ß-Galactosidase (SA-ß-Gal) activity, cell cycle, cell proliferation ability, and the expression of senescence-related and silent information regulator of transcription 1/PPAR gamma coactivator-1α (SIRT1/ PGC-1α) pathway-related proteins and mRNA were used to evaluate the protective effects of UA. In vivo, an animal model of IDD was constructed, and X-rays, magnetic resonance imaging, and histological analysis were used to assess whether UA could alleviate IDD in vivo. RESULTS: We found that H2O2 can cause NPMSCs senescence changes, such as cell cycle arrest, reduced cell proliferation ability, increased SA-ß-Gal activity, and increased expression of senescence-related proteins and mRNA. After UA pretreatment, the abovementioned senescence indicators were significantly alleviated. To further demonstrate the mechanism of UA, we evaluated the mitochondrial membrane potential and the SIRT1/PGC-1α pathway that regulates mitochondrial function. UA protected mitochondrial function and delayed NPMSCs senescence by activating the SIRT1/PGC-1α pathway. In vivo, we found that UA treatment alleviated an animal model of IDD by assessing the disc height index, Pfirrmann grade and the histological score. CONCLUSION: In summary, UA could activate the SIRT1/PGC-1α signaling pathway to protect mitochondrial function and alleviate cell senescence and IDD in vivo and vitro.

[Biological treatment for intervertebral disc degeneration].

Neck shoulder pain or lumbocrural pain caused by intervertebral disc degeneration (IDD) could seriously affect the qualities life of patients. Current treatments mainly focus on alleviating pain and the symptoms of nerve compression, which could not radically stop the process of intervertebral disc degeneration, but conversely lead to high recurrence rate. In recent years, scholars have turned to study the biological treatment for repair and rebuild the intervertebral disc by biological molecular therapy, gene therapy, cell therapy and tissue engineering to solve the problem of intervertebral disc degeneration, while most of the above methods are still in animal experiments or in vitro experiments and the clinical application is still a long way to go.

Single-stage combined decompression for patients with tandem ossification in the cervical and thoracic spine.

STUDY DESIGN: Retrospective study of clinical outcomes of single-staged combined cervical and thoracic decompression for patients with tandem ossification (TO). OBJECTIVE: To describe primary clinical outcomes of this procedure. TO is introduced to described a double ossification lesion of the posterior longitudinal ligament (OPLL) or the ligament flavum (OLF) at the cervical, thoracic and lumbar spine. In clinical practice, cervical OPLL combined with thoracic OPLL or/and OLF are the most common types of TO. However, little is known about the clinical outcomes of surgical treatment and there is no consensus on the optimal treatment to this combined disorder. METHODS: Between January 2005 and December 2008, 15 patients of this complicated phenomenon were treated by single-staged combined cervical and thoracic decompression in conditions where patients' general condition allowed and individuals agreed on. Surgical intervention, perioperative complications, and clinical outcomes were reviewed in these 15 TO patients who were followed up for more than 2 years (range 2-5 years). Clinical symptoms were evaluated using the JOA scoring system and activity of daily life was evaluated by Nurick classification before surgery, at 6 months postoperatively, and at final follow-up. Patient satisfaction was determined at final follow-up. RESULTS: The mean blood loss was 1,553.3 ± 735.7 ml (range 700-2,900 ml) and the mean operation time was 280.7 ± 53.6 min (range 220-370 min). The important intraoperative and postoperative complications recorded in medical documents included CSF leakage, hematoma, C5 palsy and neurological deterioration. The JOA score was significantly higher 6 months after surgery (8.1 ± 1.8 points vs. 11.0 ± 1.6 points, p < 0.0001), and there was no significant change between 6 months after surgery and final follow-up (11.0 ± 1.6 points vs. 11.3 ± 2.1, p = 0.5894). The mean Nurick classification significantly improved from grade 3.6 ± 0.7 before surgery to grade 2.5 ± 0.9 at 6 months after surgery (p < 0.001), and well maintained as grade 2.3 ± 1.0 at final follow-up (p = 0.3343). Three patients had satisfaction scores of 3 points, 5 had scores of 2 or 1 point, and 2 had score of 0 point. Pearson correlation analysis showed a significant positive correlation between satisfaction score and JOA score (r = 0.6493, p = 0.0093), and a significant negative correlation between satisfaction score and Nurick classification (r = -0.5941, p = 0.0195). Besides, perioperative complications and progression of tandem ossification which needed revision surgery had significant adverse effect on patients' satisfaction. CONCLUSIONS: The results showed that single-staged combined decompression could provide comparable clinical outcomes, and patients' satisfaction was significantly related with postoperative neurological function. In addition, satisfaction score could be decreased by perioperative complications and progression of tandem ossification. Thus, this aggressive surgical strategy should be used more carefully with emphasis on preoperative communication with patients.

Mechanical strain induces Cx43 expression in spinal ligament fibroblasts derived from patients presenting ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament (OPLL) is characterized by ectopic bone formation in spinal ligaments. Some evidence indicates that mechanical strain can lead to the development of OPLL, although the signaling mechanism is not fully understood. Connexin43 (Cx43), a gap-junction protein, has been shown to be of particular importance in bone formation. We hypothesized that Cx43 may play an important role in the signal transmission induced by mechanical strain during the development of OPLL. To explore this possibility, we cultured fibroblasts from spinal ligaments of OPLL and non-OPLL patients and preloaded mechanical stretch onto the cells via a Flexercell 4000 Tension Plus system. We evaluated expression changes in osteocalcin (OCN), alkaline phosphatase (ALP), type I collagen (COL I) and Cx43 via semi-quantitative RT-PCR and western blotting at 12 and 24 h after mechanical strain application in contrast to static conditions. We observed a significant gene up-regulation of OCN, ALP and COL I and Cx43 protein in OPLL cells after mechanical strain application, but no changes in non-OPLL cells. Notably, after RNA interference targeting Cx43 was performed in OPLL cells, we found that there were no significant changes in the expressions of OCN, ALP, COL I and Cx43 after the mechanical strain was applied for 24 h. Thus, we propose that the increase in Cx43 expression induced by mechanical strain in OPLL cells plays an important role in the progression of OPLL.

Upregulated expression of connexin43 in spinal ligament fibroblasts derived from patients presenting ossification of the posterior longitudinal ligament.

STUDY DESIGN: A case-control study was conducted. OBJECTIVE: To investigate different expressions of connexin43 (Cx43) between spinal ligament fibroblasts from patients with ossification of the posterior longitudinal ligament (OPLL) and non-OPLL patients and demonstrate knockdown of Cx43 protein expression by RNA interference inhibiting expression of osteoblast-specific genes in OPLL cells. SUMMARY OF BACKGROUND DATA: The OPLL is characterized by ectopic bone formation in spinal ligaments. Some evidence indicates that ligament fibroblasts from OPLL patients have osteogenic characteristics. However, the relevant cellular signaling pathways remain unclear. METHODS: Twenty patients presenting with OPLL and 18 non-OPLL patients underwent anterior decompression between January 2008 and June 2009. Specimens of the posterior longitudinal ligament were collected intraoperatively. Tissue fragment cell culture was performed. Inverted phase contrast microscopy and hematoxylin-eosin staining were used to observe cell morphology. The mouse antivimentin antibody was used to identify the cultured cells via immunocytochemistry and immunofluorescence. The messenger RNA expression of osteoblast-specific genes of osteocalcin (OCN), alkaline phosphatase (ALP), and type I collagen (COL I) were detected in OPLL and non-OPLL cells by semiquantitative reverse transcription-polymerase chain reaction. The protein expression of Cx43 was detected via Western blotting. And then, after 72 hours, when RNA interference against Cx43 was performed in OPLL cells, expression of the indexes mentioned earlier was compared again between the transfection group and the nontransfection group. RESULTS: Cultivated cells were observed 7 to 10 days after cell culture. Hematoxylin-eosin staining showed fusiform and multiangular star morphologies, large and elliptical cell nuclei, and ill-defined cell appearances. Immunocytochemistry and immunofluorescence exhibited positive results of vimentin staining. The messenger RNA expressions of OCN, ALP, and COL I and protein expressions of Cx43 from OPLL fibroblasts were greater than those from non-OPLL cells, and the difference was significant. Furthermore, knockdown of Cx43 protein expression inhibited the messenger RNA expressions of OCN, ALP, and COL I remarkably in the transfection group compared with the nontransfection group, 72 hours after RNA interference targeting Cx43 was performed in OPLL cells. CONCLUSION: Tissue fragment culture of the cervical posterior longitudinal ligament provided a successful fibroblast culture, showing good adherence and subculture. The cultured fibroblasts from OPLL patients exhibited osteogenic characteristics, in which Cx43 played an important role.

Choice of surgical approach for ossification of the posterior longitudinal ligament in combination with cervical disc hernia.

Ossification of the posterior longitudinal ligament (OPLL) is a common spinal disorder that presents with or without cervical myelopathy. Furthermore, there is evidence suggesting that OPLL often coexists with cervical disc hernia (CDH), and that the latter is the more important compression factor. To raise the awareness of CDH in OPLL for spinal surgeons, we performed a retrospective study on 142 patients with radiologically proven OPLL who had received surgery between January 2004 and January 2008 in our hospital. Plain radiograph, three-dimensional computed tomography construction (3D CT), and magnetic resonance imaging (MRI) of the cervical spine were all performed. Twenty-six patients with obvious CDH (15 of segmental-type, nine of mixed-type, two of continuous-type) were selected via clinical and radiographic features, and intraoperative findings. By MRI, the most commonly involved level was C5/6, followed by C3/4, C4/5, and C6/7. The areas of greatest spinal cord compression were at the disc levels because of herniated cervical discs. Eight patients were decompressed via anterior cervical discectomy and fusion (ACDF), 13 patients via anterior cervical corpectomy and fusion (ACCF), and five patients via ACDF combined with posterior laminectomy and fusion. The outcomes were all favorable. In conclusion, surgeons should consider the potential for CDH when performing spinal cord decompression and deciding the surgical approach in patients presenting with OPLL.

[Anterior decompression for the treatment of cervical spondylotic myelopathy associated with ossification of posterior longitudinal ligament].

OBJECTIVE: To study the technique and effect of anterior decompression for the treatment of cervical spondylotic myelopathy associated with ossification of posterior longitudinal ligament (OPLL). METHOD: Sixty-one patients (42 male and 19 female, 45 - 74 years with mean age of 57 years old) underwent anterior decompression for the treatment of cervical spondylotic myelopathy associated with OPLL. Among them, OPLL was definitely diagnosed in 49 patients preoperatively, and was found during the operation in the other 12 patients. The occupying rate of OPLL ranged 32%-70% with an average of 52%. The preoperative JOA scores ranged 4 - 14 points with an average of 9.6 points. In additional to conventional decompression, the ossification was removed completely after discectomy and corpectomy. RESULTS: Corpectomy was performed in 41 cases, discectomy in 6 cases and combination of corpectomy and discectomy in 14 cases. The follow-up of all patients ranged from 6 to 36 months (mean 16 months). The postoperative JOA scores ranged 8-16 points with an average of 12.8 points. The neurological improvement rate ranged from 25.0% to 87.5% with an average of 65.2%. The transient leakage of cerebrospinal fluid (CSF) occurred in 5 cases, and stopped after conservational treatment. No neurological deterioration developed. CONCLUSIONS: The difficulty and risk of anterior decompression are significantly increased in the patients with cervical spondylotic myelopathy associated with OPLL. Remove of ossification after corpectomy and discectomy could provide complete decompression and better results.

Combination of dexamethasone and aminoguanidine reduces secondary damage in compression spinal cord injury.

The study was performed to investigate the effect of combination therapy with aminoguanidine (AG) and dexamethasone (DEX) on the compression spinal cord injury (SCI) in rat. Compared to the control group, the combination therapy group with AG (75 mg/kg) and DEX (0.025 mg/kg) significantly reduced the degree of (1) spinal cord edema, (2) the permeability of blood spinal cord barrier (measured by (99m)Tc-Albumin), (3) infiltration of neutrophils (MPO evaluation), (4) cytokines expression (tumor necrosis factor-alpha and interleukin-1 beta), and (5) apoptosis (measured by Bax and Bcl-2 expression). In addition, we have also clearly demonstrated that the combination therapy significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly indicated for the first time that strategies targeting multiple proinflammatory pathways may be more effective than a single effector molecule for the treatment of SCI.

Ossification of ligamentum flavum related to thoracic kyphosis after tuberculosis: case report and review of the literature.

STUDY DESIGN: Case report and review of the literature. OBJECTIVE: To report 2 patients, with a history of thoracic tuberculosis, in whom ossification of ligamentum flavum (OLF) was found adjacent to progressive kyphosis and resulted in incomplete paralysis. The relevant literature was also to be reviewed. SUMMARY OF BACKGROUND DATA: Progressive kyphosis is the most common cause of late onset paralysis after spinal tuberculosis. However, OLF related to tuberculous kyphosis is rarely observed and this makes surgical treatment become very difficult. METHODS: Clinical history, laboratory examination, and radiographic findings of the 2 patients were described. Posterior laminectomy and instrumented fusion were performed for both 2 patients, but additional osteoectomy was performed through posterolateral zygapophysial approach in only 1 patient in whom the kyphotic deformity was partially corrected. A review of the relevant literature was also done. RESULTS: Two patients were observed up for 2 and 6 years, respectively. Neurologic status was significantly improved after operation and no further kyphosis developed. To our knowledge, only 1 article had reported 2 similar cases of spinal stenosis above a healed tuberculous kyphosis. CONCLUSION: OLF related to tuberculous kyphosis was rarely observed. The local instability and repetitive stimulus of excessive stress could lead to development of OLF. Our experience suggested that decompression of OLF was more important than correction of kyphotic deformity to improve the neurologic status in these patients.

[Anterior multilevel corpectomy for the treatment of severe ossification of posterior longitudinal ligament in the cervical spine].

OBJECTIVE: To discuss the surgical indications, outcomes and complications of anterior multilevel corpectomy for the treatment of severe ossification of posterior longitudinal ligament (OPLL) in the cervical spine. METHODS: Between April 2006 and March 2008, a total of 25 patients (20 males and 5 females, age range: 42 - 75 yr, mean: 53. 2 yr) underwent anterior multilevel corpectomy for severe ossification of posterior longitudinal ligament in the cervical spine. Radiological studies showed that the type of OPLL was distributed as follows: 7 local, 5 segmental, 8 continuous and 5 mixed. The OPLL extended an average of 2. 8 vertebrae (2 - 4) and the stenotic rate of spinal canal was 68.4% (50% - 97%). After corpectomy and removal of OPLL, titanium mesh cage and anterior plate were employed to restore cervical stability in all patients. RESULTS: Among these patients, 16 underwent two-level corpectomy and 9 three-level corpectomy. After a follow-up of 2 - 18 months, the mean JOA score increased from 9. 3 (5 - 12) points pre-operation to 14.2 (11 - 16) points post-operation. The mean improvement rate of neurological status was 63.2% (22.2% - 87.5%). The complications included CSF leakage in 6 cases (intermittent CSF pseudocyst in 4), nerve root palsy in 2, hematoma in 1 and transient neurological deterioration in 1. CONCLUSION: Anterior multilevel corpextomy can achieve a better clinical outcome in the treatment of severe cervical ossification of posterior longitudinal ligament. But it is technically demanding and carries a higher risk.

Bradykinin preconditioning modulates aquaporin-4 expression after spinal cord ischemic injury in rats.

The study investigated whether bradykinin (BK) preconditioning could regulate the expression of aquaporin-4 (AQP4) using an in vivo transient spinal cord ischemia model in rats. BK was infused continuously via the left femoral artery with infusion pump for 15 min (10 microg/kg/min) then we induced ischemia for 20 min and reperfusion for 24 and 72 h respectively. The results demonstrated that the central part of the white matter exhibited loss of perivascular AQP4 and showed a partial recovery toward 72 h of reperfusion. The border zone of white matter was different from the central part of the white matter by showing no loss of perivascular AQP4 at 24 h of reperfusion but rather a slight increase. BK significantly reduced the expression level of AQP4 protein in the white matter, but it had none of this effect in the gray matter region at 72 h post-reperfusion. There was no difference in AQP4 protein levels between BK group and control group at the two above-mentioned spinal cord regions at 24 h after reperfusion. In addition, the changes in AQP4 protein induced by BK preconditioning were obvious at 72 h after reperfusion, which were accompanied by a reduction of spinal cord edema. Our results demonstrated that the expression of AQP4 protein after spinal cord ischemia/reperfusion was region-specific, time-dependent and also indicated that the attenuation of AQP4 expression induced by BK could be one of the important molecular mechanisms in physiopathology of spinal cord ischemic edema.