Xuexiao Ma

Effect of lentivirus-mediated survivin transfection on the morphology and apoptosis of nucleus pulposus cells derived from degenerative human disc in vitro

Lower back pain is a common concern, and 40% of all cases involve the degeneration of the intervertebral disc (IVD). However, the excessive apoptosis of disc cells plays an important role in IVD degeneration, particularly in the nucleus pulposus (NP). Thus, anti-apoptotic gene therapy to attenuate or reverse the degenerative process within the NP is being developed. Survivin is a unique inhibitor of apoptosis (IAP) and has been extensively investigated in cancer cells. However, little is known of the effects of survivin transfection on NP cells derived from degenerative human disc. In this study, we aimed to investigate the effects of lentivirus (LV)-mediated survivin transfection on the morphology and apoptosis of NP cells derived from degenerative human disc in vitro. NP cells were transfected with LV-mediated survivin. Subsequently, cell morphology was observed and the survivin mRNA expression levels were measured by RT-qPCR. Apoptosis was analyzed by flow cytometry and by measuring caspase-3 activity. The results revealed that the morphology of the NP cells derived from degenerative human disc transfected with LV-mediated survivin was significantly altered as evidenced by cytomorphosis, the reduction of the cytoplasm and cell shrinkage. Following transfection, survivin gene expression significantly increased in the transfected cells and subsequent generation cells; however, no significant differences in the cell apoptotic rate and caspase-3 activity were observed. We found that transfection of the survivin gene into NP cells led to the stable expression of survivin and induced marked changes in cell morphology. Furthermore, no significant anti-apoptotic effects were observed following LV-mediated survivin transfection. Overall, our findings demonstrate that LV carrying surviving may be used to successfully enforce the expression of survivin in NP cells. However, cell morphology was evidently altered, whereas the apoptotic rate did not decrease. Comprehensive studies on the feasibility of using survivin in gene therapy in an aim to attenuate disc degeneration are warranted. Further research on the mechanisms responsible for the changes in cell morphology and cell function are also required.

Adipose-derived mesenchymal stem cells promote osteosarcoma proliferation and metastasis by activating the STAT3 pathway

Osteosarcoma is the most common primary bone malignancy in children and young adults, but the role of adipose-derived mesenchymal stem cells (ADSCs) in the rapid progression of osteosarcoma is still unclear. Here, we found that ADSCs promoted tumour growth and invasion by increasing matrix metalloproteinase 2/9 (MMP2/9) expression in tumour cells. The persistent activation of signal transducer and activator of transcription 3 (STAT3) has been shown to directly promote tumour growth by mediating a wide spectrum of cellular responses, and STAT3 activation was detected in osteosarcoma cells co-cultured with ADSCs or treated with ADSC-conditioned medium. Furthermore, siRNA-mediated STAT3 inhibition in osteosarcoma cells decreased cell proliferation and invasion and down-regulated MMP2/9 expression. In addition, a nude mouse model of osteosarcoma was established by injecting luciferase-labelled MG63 cells into the tibia. As shown in in vivo bioluminescence images, ADSCs promoted tumour cell proliferation, invasion progression and metastasis. STAT3 inhibition attenuated tumour growth and metastasis and prolonged the survival of these mice. After the siRNA treatment, the MMP2, MMP9 and Ki67 levels decreased. Based on these data, stromal ADSCs promote osteosarcoma progression by increasing STAT3 signalling-mediated MMP2/9 expression.

Survivin-TGFB3-TIMP1 Gene Therapy via Lentivirus Vector Slows the Course of Intervertebral Disc Degeneration in an In Vivo Rabbit Model.

Study Design. The ability of lentivirus vector (LV) survivin-transforming growth factor beta 3 (TGFB3)-tissue inhibitor of metalloproteinases 1 (TIMP1) on slowing disc degeneration was evaluated by an animal experiment. Objective. The aim of the study was to investigate the effect of LV survivin-TGFB3-TIMP1 on slowing disc degeneration in an in vivo rabbit model. Summary of Background Data. Cell apoptosis, increase of catabolic activity, and decrease of anabolic activity were the mechanisms of disc degeneration. Meanwhile, survivin, TGFB3, and TIMP1 can influence above process, respectively. However, there were no researches conducted to evaluate the effect of an LV containing all three proteins (referred to as LV-survivin-TGFB3-TIMP1) on slowing disc degeneration in vivo. Methods. Twenty skeletally mature female New Zealand White rabbits were randomly divided into four groups: nonpunctured sham surgical group (group A, n = 5), punctured blank control group (group B, n = 5), punctured empty vector control group (group C, n = 5), and the treatment group (group D, n = 5). Computed tomography–guided puncture was performed at the L3–L4 and L4–L5 discs, in accordance with a previously validated rabbit annulotomy model for intervertebral disc degeneration. After 3 weeks, LV-carrying survivin, TGFB3, and TIMP1 were injected into the nucleus pulposus. Serial magnetic resonance imaging studies at 0, 3, and 12 weeks were performed. The rabbits were sacrificed at 12 weeks, and the histology, immunofluorescence, quantitative real-time polymerase chain reaction, Western blot, and caspase-3 activity was used for evaluation. Results. Magnetic resonance imaging, histology, gene expression, protein content, and apoptosis analyses of group A showed no disc degeneration. Groups B and C showed disc degeneration, which increased over time, and no significant difference was observed between the two groups (P > 0.05). In group D, there was less disc degeneration compared to the punctured control groups and the difference was statistically significant (P < 0.05). Conclusion. The injection of LV-carrying survivin-TGFB3-TIMP1 into punctured rabbit intervertebral discs helps delay degenerative disc changes. Although data from animal models should be extrapolated to the human condition with caution, this study shows promise for gene therapy to decelerate disc degeneration. Level of Evidence: N/A

Survivin is expressed in degenerated nucleus pulposus cells and is involved in proliferation and the prevention of apoptosis in vitro

Survivin is a unique inhibitor of apoptosis, which is frequently present within degenerated human nucleus pulposus (NP) cells. Survivin has been extensively investigated using proliferation and apoptosis assays in tumor cells; however, studies conducted on survivin in degenerative NP cells remain limited to date. The aim of the present study was to investigate survivin expression and its effects on the proliferation and apoptosis of degenerated NP cells in vitro. The expression levels of survivin in the NP cells of patients (>45 years) with lumbar disc degenerative disease and the NP cells of patients (<25 years) with lumbar vertebra fracture were assessed by reverse transcription-quantitative polymerase chain reaction. The effects on in vitro proliferation and apoptosis were investigated through transfection with a specific small interfering (si)RNA. The results of the present study demonstrated that survivin was expressed in the degenerated NP cells, but was undetectable in normal NP cells at the mRNA level. Survivin suppression following transfection with a specific survivin-siRNA reduced the proliferation rate of NP cells and enhanced sensitization to pro-apoptotic stimuli. Therefore, survivin was shown to be expressed and exhibit an important role in the proliferation and prevention of apoptosis of degenerated NP cells. Studies on survivin in NP cells may aid in increasing the understanding of the complex processes underlying NP cell degeneration, and could provide fundamental information for gene therapy to inhibit this degeneration in vitro.

A radical procedure of circumferential spinal cord decompression through a modified posterior approach for thoracic myelopathy caused by severely impinging anterior ossification

BACKGROUND CONTEXT Thoracic myelopathy caused by an anterior, massive ossified plaque is often progressive and responds poorly to conservative treatment. Direct removal of the compressing ossification is the optimal procedure for a spinal cord that is severely impinged anteriorly. However, both anterior and posterior decompressive manipulations have caused catastrophic iatrogenic spinal cord injuries. A comprehensive treatment method for severe thoracic myelopathy that enables a sufficient and safe decompression of the spinal cord is needed. PURPOSE The purpose of this study is to demonstrate the efficacy, safety, and results of a one-stage circumferential decompressive procedure using a modified posterior approach in patients with severe thoracic myelopathy resulting from anterior spinal compression. STUDY DESIGN A modified procedure of circumferential spinal cord decompression for thoracic myelopathy is described. A retrospective study was conducted to investigate the clinical outcomes of 23 sequentially treated patients. PATIENT SAMPLE Twenty-three patients were treated sequentially with a modified procedure for circumferential spinal cord decompression for thoracic myelopathy. OUTCOME MEASURES Outcomes were assessed using the Japanese Orthopedic Association (JOA) score, modified Frankel classification, Hirabayashi recovery rate, and a general assessment of complications. METHODS Twenty-three patients with thoracic myelopathy caused by a massive, anterior ossified structure were treated with an extensive posterior laminectomy, anterior removal of the ossification, and interbody fusion with kyphosis-reversing stabilization through a modified posterolateral approach. The neurologic outcomes are evaluated according to the JOA and the modified Frankel classification before surgery, 2 weeks after surgery, 1 year after surgery, and at the final follow-up visit. The surgical outcomes are also described using the Hirabayashi recovery rate. Radiographs, computed tomography (CT), and magnetic resonance imaging were performed before and after surgery. A postoperative CT scan was obtained to determine the efficacy of the decompression. Operative time, intraoperative blood loss, and complications were reviewed from the medical records. In addition, a 48-year-old man who presented with severe thoracic myelopathy resulting from anterior impingement with multiple osteophytes is described as an illustrative patient. RESULTS The sites of ossification in this series were distributed widely, from T4-T12. The anterior ossified plaques of all patients were resected completely. Five patients who had intraoperative evidence of dural ossification required resection of the ossified dura matter. The average operating time was 276 minutes. Mean intraoperative blood loss was 1,350 mL. The postoperative follow-up ranged from 2.5 to 6 years, with an average of 4.6 years. The average preoperative JOA score was 4.3±1.5 points, and it improved to 6.1±1.9 points 2 weeks postoperatively, to 8.1±1.8 points 1 year postoperatively, and to 8.5±1.9 points at the most recent follow-up. The overall Hirabayashi recovery rate at the final examination averaged 63.6±22.4%. Eight patients were graded as excellent, 10 as good, 4 as fair, and 1 as unchanged. No patient was graded as deteriorated. The paralysis improved by at least 1 grade in 22 patients (95.7%). Transient deterioration of thoracic myelopathy occurred immediately after surgery in three patients (13%). Cerebrospinal fluid leakage occurred in six patients (26.1%). One patient sustained severe bilateral groin pain, three had unilateral intercostal neuralgia, and pleura tear occurred in one patient. CONCLUSION One-stage posterior decompression, anterior extirpation of the ossification, and interbody fusion with instrumentation via a modified posterior approach is a safe and effective treatment for severe thoracic myelopathy resulting from prominent anterior impingement. This procedure is technically demanding, and the indications are limited to thoracic myelopathy caused by severe anterior impingement of various etiologies from T4-T12.

Acute intracranial hematoma formation following excision of a cervical subdural tumor: a report of two cases and literature review

Abstract An intracranial hematoma is a rare, yet significant, complication following spinal surgery. The authors describe two cases with acute intracranial hematoma formation after excision of a cervical subdural schwannoma. One was a 14-year-old girl who developed bilateral intracranial extradural hematomas immediately following excision of the C4 subdural schwannoma. The other was a 59-year-old woman who had an acute cerebellar hematoma after removal of the C2–C5 subdural schwannoma. During the surgeries of both cases, spinal dura was partially removed together with the tumor and the dural sac could not be repaired, resulting in large amounts of intraoperative CSF loss and persistent postoperative CSF leakage. Both patients failed to regain consciousness from anesthesia after surgery, and a cranial CT scan identified large intracranial hematomas. Urgent hematoma evacuation was ultimately performed to save the patients. Based on the authors’ experience and literature review, a conclusion was drawn that considerable CSF leakage and a sharp decrease of CSF pressure are common features during the excision of a spinal subdural tumor, which may lead to acute intracranial hematomas. Continual postoperative monitoring in patients with this condition should be of a very high priority. A CT or MRI should be immediately investigated to exclude intracranial hematomas for any patient with delayed emergence from anesthesia following spinal surgery. Hematoma evacuation is indispensable once an intracranial hematoma is identified in the patient who fails to regain consciousness from anesthesia post surgery. Furthermore, the possible pathophysiological mechanisms responsible for the formation of an intracranial hematoma after spinal procedures, particularly after manipulations of a cervical subdural tumor, are discussed.

Morphological observation of sympathetic nerve fibers in the human posterior longitudinal ligament.

Study Design. Histological study of human living tissue. Objective. To determine sympathetic fiber in the cervical posterior longitudinal ligaments (PLL) obtained from the patients undergoing anterior cervical decompression surgery, and speculate the implication of their presence and distribution. Summary of Background Data. The pathogenic mechanism responsible for cervical spondylosis remains unclear. Cervical vertigo is often confused with aural vertigo, and central vertigo, and et al. It has been gradually realized that mechanical interference to the vertebral artery is not the only way to explain the pathogenic mechanism of cervical vertigo. It should be noted that the sympathetic factor may also involve it because some sympathetic nerves were found in the PLL in an animal study of intervertebral discs. Although it is unclear whether there is a similar phenomenon in adult human PLL. Methods. Forty-six patients who received anterior cervical decompression surgery in The Affiliated Hospital of Qingdao University from January 2013 to December 2013 were classified into 2 groups: with cervical spondylosis and with cervical trauma. Cervical PLL tissues of all the participants were obtained during operation. The paraffin slices of the ligament were stained according to glyoxylic acid-induced fluorescent method. The morphology and distribution of sympathetic nerve fibers were observed by measuring and analyzing fluorescent units expressed on different sections. The positive rates expressed by fluorescent staining were statistically analyzed. Results. Different forms of sympathetic nerve fibers distribution were observed in the 3-dimensional slices in each group selected from 46 cases of specimens. The positive rate of fluorescent units detected from the cervical PLL in patients experiencing cervical spondylosis was not significantly different from that in cervical trauma group (x2 = 0.969, P > 0.05). Conclusion. Sympathetic nerve fibers were confirmed to distribute in the human cervical posterior longitudinal ligament. Level of Evidence: 2

Holocord spinal epidural abscess: Case report and literature review.

Holocord spinal epidural abscess (SEA) is a rare condition. To our knowledge, five cases of SEA have been reported so far, and no consensus has been made on the treatment yet. In this article, we report a case of holocord SEA and review literature to further understanding of SEA. The advent of antibiotic treatment and the recognition of surgical debridement have been important in searching for alternatives to recovery, so the patient was treated surgically together with systemic antibiotics. The patient remained neurologically stable and continued to be clinically in good condition without any low back pain after 1 year. Surgical drainage, together with systemic antibiotics, is the main treatment choice for extensive SEAs. Although treatment should be considered that highlights the importance of examining the factors related to the health and condition of the patients and the anatomy and extent of the abscess, early surgical treatment associated with prolonged antibiotic treatment is necessary.

Effect of Survivin gene therapy via lentivirus vector on the course of intervertebral disc degeneration in an in vivo rabbit model

The aim of the current study was to use gene therapy to attenuate or reverse the degenerative process within the intervertabral disc. The effect of survivin gene therapy via lentiviral vector transfection on the course of intervertebral disc degeneration was investigated in the current study in an in vivo rabbit model. A total of 15 skeletally mature female New Zealand White rabbits were randomly divided into three groups: Punctured blank control group (group A, n=5), punctured empty vector control group (group B, n=5) and the treatment group (group C, n=5). Computed tomography-guided puncture was performed at the L3-L4 and L4-L5 discs, in accordance with a previously validated rabbit annulotomy model for intervertebral disc degeneration. After 3 weeks, a lentiviral vector (LV) carrying survivin was injected into the nucleus pulposus. The results demonstrated that through magnetic resonance imaging, histology, gene expression, protein content and apoptosis analyses, group A and B were observed to exhibit disc degeneration, which increased over time, and no significant difference was observed between the two groups (P>0.05). However, there was reduced disc degeneration in group C compared with the punctured control groups, and the difference was statistically significant (P<0.05). Overall, the results of the present study demonstrated that injection of the LV carrying survivin into punctured rabbit intervertebral discs acted to delay changes associated with the degeneration of the discs. Although data from animal models should be extrapolated to the human condition with caution, the present study suggests potential for the use of gene therapy to decelerate disc degeneration.