Tao Sui

EIF2α and caspase-12 activation are involved in oxygen-glucose-serum deprivation/restoration-induced apoptosis of spinal cord astrocytes

Astrocytes play an important role in protecting neurons during ischemia and reperfusion in the central nervous system. Although many studies have shown that oxygen-glucose deprivation (OGD) can induce astrocyte apoptosis, the role of PERK/eIF2 alpha/ATF4 integrated stress response (ISR) in astrocyte apoptosis mediated by oxygen-glucose-serum deprivation (OGSD)/restoration remains uncertain. Astrocytes were subjected to a combination of oxygen, glucose, and serum deprivation for 8h followed by restoration. Hoechst 33342 staining was performed to quantify apoptotic astrocytes and cell viability was assessed with Cell Counting Kit-8 (CCK8). Immunocytochemical analysis and Western blotting for some related molecules, including pancreatic ER stress kinase (PERK), p-PERK, eukaryotic initiation factor 2 alpha (eIF2 alpha), p-eIF2 alpha, activating transcription factor 4 (ATF4), caspase-12, were examined. Caspase activation and apoptosis were detected in neonatal rat astrocytes from spinal cord subjected to OGSD/restoration. We also observed an increase in cytoplasmic staining of p-eIF2 alpha in astrocytes (8h OGSD/15 min restoration) compared with that of non-treated cells. In addition, we found the sequential activation of PERK, eIF2 alpha, and ATF4 during OGSD/restoration by Western blotting. These results indicate that both the PERK/eIF2 alpha/ATF4 ISR and activation of caspase-12 may be involved in apoptosis of spinal cord astrocytes induced by OGSD/restoration.

Effect of topical application of mitomycin-C on wound healing in a postlaminectomy rat model: an experimental study.

The aim of this study was to investigate the effects of topical application mitomycin-C (MMC) on wound healing after laminectomy. 60 adult male SD rats were equally and randomly divided into five groups. Laminectomy was performed at the level of L1 in all rats. After hemostasis was achieved, cotton pads soaked with saline and MMC (0.1mg/ml, 0.3mg/ml, 0.5mg/ml and 0.7mg/ml) were directly subjected to the exposed dura for 5min in each group. Two weeks after laminectomy all the rats were killed. The vertebral column including the back scar tissue and muscles was obtained to make paraffin sections. The hematoxylin-eosin staining and Masson staining were performed with the obtained paraffin sections. The number of the fibroblast and the capillary density were counted by the hematoxylin-eosin staining slice. The extent of epidural fibrosis and the expression of vascular endothelial growth factor (VEGF) were evaluated by the immunohistochemical slice through a computer image analysis system. Our data showed that the number of fibroblast, capillary density and fibrotic tissue in the 0.5 and 0.7mg/ml MMC groups was significantly lower than the control, 0.1 and 0.3mg/ml MMC groups; while the expression of VEGF in control and 0.1mg/ml MMC groups was notably higher than 0.3, 0.5 and 0.7mg/ml MMC groups. Topical application of MMC above the concentration of 0.3mg/ml could affect all steps of the wound healing process via inhibiting the angiogenesis and fibroblast proliferation, thus delayed the wound healing after laminectomy.

Position and complications of pedicle screw insertion with or without image-navigation techniques in the thoracolumbar spine: a meta-analysis of comparative studies

Abstract Computer-navigated pedicle screw insertion is applied to the thoracic and lumbar spine to attain high insertion accuracy and a low rate of screw-related complications. However, some in vivo and in vitro studies have shown that no advantages are gained with the use of navigation techniques compared to conventional techniques. Additionally, inconsistent conclusions have been drawn in various studies due to different population characteristics and methods used to assess the accuracy of screw placement. Moreover, it is not clear whether pedicle screw insertion with navigation techniques decreases the incidence of screw-related complications. Therefore, this study was sought to perform a meta-analysis of all available prospective evidence regarding pedicle screw insertion with or without navigation techniques in human thoracic and lumbar spine. We considered in vivo comparative studies that assessed the results of pedicle screw placement with or without navigation techniques. PubMed, Ovid MEDLINE and EMBASE databases were searched. Three published randomized controlled trials (RCTs) and nine retrospective comparative studies met the inclusion criteria. These studies included a total of 732 patients in whom 4,953 screws were inserted. In conclusion, accuracy of the position of grade I, II, III and IV screws and complication rate related to pedicle screw placement were significantly increased when navigation techniques were used in comparison to conventional techniques. Future research in this area should include RCTs with well-planned methodology to limit bias and report on validated, patient-based outcome measures.

Taurine Reduced Epidural Fibrosis in Rat Models after Laminectomy via Downregulating EGR1

Background/Aims: Epidural fibrosis, a common complication after laminectomy, has been demonstrated to be closely associated with poor surgical outcomes. Previous studies showed that taurine had remarkable anti-fibrotic effects on lung and liver fibrosis. We performed this study to investigate the effects of taurine in rat models of epidural fibrosis after laminectomy and to explore the potential molecular mechanism. Methods: Laminectomy was performed on each rat to establish epidural fibrosis model. After taurine treatment, Massons trichrome and immunohistochemistry staining were used to examine epidural fibrosis. Cell viability was determined using the Cell Counting Kit-8 assay. Annexin V/Propidium Iodide double staining was performed to detect fibroblasts apoptosis. Microarray was adopted to identify significantly changed mRNAs. mRNA expression was measured by qRT-PCR. Lentivirus infection was performed to establish stable knockdown and overexpression cell lines. The expression of fibrosis-related proteins was determined via Western blot. Results: Taurine treatment markedly reduced laminectomy-induced epidural fibrosis in rat models. However, this effect of taurine was independent on TGF-β/Smad pathway, evidenced by no change in the expression of TGF-β and its receptors. Besides, taurine had almost no effect on cell apoptosis. Interestingly, taurine treatment significantly decreased expression of EGR1 (Early growth response protein 1), an enhancer of fibrosis, both in vivo and in vitro. Furthermore, overexpression of EGR1 increased activation of fibroblasts, while EGR1 knockdown achieved an opposite effect, indicating that EGR1 plays a key role in the inhibitory effect of taurine on TGF-β-induced fibrosis. Conclusions: Reduced epidural fibrosis in vivo and decreased activation of fibroblasts in vitro after taurine treatment was mediated by EGR1. Taurine promises to be a potential prevention for epidural fibrosis after laminectomy.

Tacrolimus reduces scar formation and promotes sciatic nerve regeneration

A sciatic nerve transection and repair model was established in Sprague-Dawley rats by transecting the tendon of obturator internus muscle in the greater sciatic foramen and suturing with nylon sutures. The models were treated with tacrolimus gavage (4 mg/kg per day) for 0, 2, 4 and 6 weeks. Specimens were harvested at 6 weeks of intragastric administration. Masson staining revealed that the collagen fiber content and scar area in the nerve anastomosis of the sciatic nerve injury rats were significantly reduced after tacrolimus administration. Hematoxylin-eosin staining showed that tacrolimus significantly increased myelinated nerve fiber density, average axon diameter and myelin sheath thickness. Intragastric administration of tacrolimus also led to a significant increase in the recovery rate of gastrocnemius muscle wet weight and the sciatic functional index after sciatic nerve injury. The above indices were most significantly improved at 6 weeks after of tacrolimus gavage. The myelinated nerve fiber density in the nerve anastomosis and the sciatic nerve functions had a significant negative correlation with the scar area, as detected by Spearman’s rank correlation analysis. These findings indicate that tacrolimus can promote peripheral nerve regeneration and accelerate the recovery of neurological function through the reduction of scar formation.

Confocal imaging reveals three-dimensional fine structure difference between ventral and dorsal nerve roots

Peripheral nerve injury repair is one of the most challenging problems in neurosurgery, partially due to lack of knowledge of three-dimensional (3-D) fine structure and organization of peripheral nerves. In this paper, we explored the structures of nerve fibers in ventral and dorsal nerves with a laser scanning confocal microscopy. Thick tissue staining results suggested that nerve fibers have a different 3-D structure in ventral and dorsal nerves, and reconstruction from serial sectioning images showed that in ventral nerves the nerve fibers travel in a winding form, while in dorsal nerves, the nerve fibers form in a parallel cable pattern. These structural differences could help surgeons to differentiate ventral and dorsal nerves in peripheral nerve injury repair, and also facilitate scientists to get a deeper understanding about nerve fiber organization.

Extradural nerve anastomosis technique for bladder reinnervation in spinal cord injury: anatomical feasibility study in human cadavers.

Study Design. An anatomic study of extradural spinal root in 9 embalmed cadavers. Objective. To ascertain the anatomical parameters of the extradural spinal root and to demonstrate the feasibility of spinal root anastomoses without opening the spinal dura mater. Summary of Background Data. Intradural anastomosis of the spinal root has made breakthrough progress in treating neurogenic bladder in spinal cord injury. However, because of the complex surgical procedures and extensive bony destruction, its clinical use is not widely promoted. Methods. Nine formalin-fixed cadavers were used. The distance between the nerve root outlet and ganglion center, the neighboring nerve root-outlet distance, and the gross anatomy of the extradural spinal root were measured with a surgical microscope. The number of nerve fibers from the T7 to S4 ventral roots (VRs) was calculated by immunohistochemical staining. Results. The longest and shortest lengths of the extradural spinal root were observed at the S4 and T7 levels, with average values of 33.29 and 6.06 mm, respectively. The longest distance between the adjacent nerve root outlets was observed at L1–L2 (mean, 29.16 mm), and shortest at S3–S4 (mean, 11.79 mm). After leaving the dural sac, the spinal root descends in the spinal canal until reaching the corresponding intervertebral foramina, and the motor nerve roots still lie ventrally to the sensory nerve roots. The largest and smallest numbers of nerve fibers were observed at the L3 and S4 levels (mean, 9169 and 1356, respectively). Conclusion. The dorsal roots and VRs can both be successfully harvested and identified outside the dural sac. The S1 VR can be anastomosed to the S2 VR extradurally without nerve grafts. For extradural neuroanastomosis of the thoracic VRs to the S2 VR, a nerve graft is required. In addition, there are a sufficient number of nerve fibers for functional bladder recovery at the T7–T12 and S1 levels. This study supports the feasibility of extradural spinal root anastomosis as a modified surgical method for treating neurogenic bladder. Level of Evidence: N/A

Repair, protection and regeneration of spinal cord injury

First authors and corresponding authors: Xiao-jian Cao Department of Spine Surgery, First Affiliated Hospital of Nanjing Medical University Shi-qing Feng Department of Orthopedics, Tianjin Medical University General Hospital Chang-feng Fu Department of Spine Surgery, First Hospital of Jilin University Kai Gao Department of Orthopedics, First Affiliated Hospital of Liaoning Medical University Jia-song Guo Department of Histology and Embryology, Southern Medical University; Key Laboratory of Tissue Construction and Detection of Guangdong Province; Institute of Bone Biology, Academy of Orthopedics of Guangdong Province Xiao-dong Guo Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology Xi-jing He Department of Spine Surgery, The Second Hospital of Xi’an Jiaotong University Zi-wei Huang Department of Orthopedics, the First Affiliated Hospital, Orthopedic Institute, Soochow University Zhong-hai Li * Department of Orthopedics, the First Affiliated Hospital of the PLA General Hospital, Orthopedics Institute of PLA Ling Liu Institute of Neuroscience, the Fourth Military Medical University of Chinese PLA Rong-han Liu Department of Spine Surgery, Jinan Central Hospital Affiliated to Shandong University He-Zuo Lü * Anhui Key Laboratory of Tissue Transplantation, Bengbu Medical College, Bengbu, Anhui Province, China Xi-fan Mei Department of Orthopedics, First Affiliated Hospital of Liaoning Medical University Bin Ning Department of Spine Surgery, Jinan Central Hospital Affiliated to Shandong University Guang-zhi Ning Department of Orthopedics, Tianjin Medical University General Hospital Chang-hui Qian Department of Histology and Embryology, Southern Medical University Jie Qin Department of Spine Surgery, The Second Hospital of Xi’an Jiaotong University Yan-zhen Qu Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology Saijilafu Department of Orthopedics, the First Affiliated Hospital, Orthopedic Institute, Soochow University Bo Shi Department of Spine Surgery, First Hospital of Jilin University Tao Sui Department of Spine Surgery, First Affiliated Hospital, Nanjing Medical University Tian-sheng Sun Institute of Trauma Orthopedic Surgery of Chinese PLA, General Hospital of Beijing Military Region Jian Wang Institute of Neuroscience, the Fourth Military Medical University Jin-kun Wen Department of Histology and Embryology, Southern Medical University Jian Xiao Molecular Pharmacology Research Center, School of Pharmacy, Wenzhou Medical University Bin Xu Department of Orthopedics, Jinling Hospital, Nanjing University (Nanjing General Hospital of Nanjing Military Command) Hai-dong Xu Department of Orthopedics, Jinling Hospital, Nanjing University (Nanjing General Hospital of Nanjing Military Command) Pan-pan Yu * Guangdong-Hongkong-Macau Institute of CNS Regeneration (GHMICR), Jinan University Zhi-cheng Zhang Institute of Trauma Orthopedic Surgery of Chinese PLA, General Hospital of Beijing Military Region Yong Zhou Department of Orthopedics, Tianjin Medical University General Hospital Yu-long Zhou Department of Orthopedics, the Second Affiliated Hospital, Wenzhou Medical University; Molecular Pharmacology Research Center, School of Pharmacy, Wenzhou Medical University

Mitomycin C induces apoptosis in human epidural scar fibroblasts after surgical decompression for spinal cord injury

Numerous studies have shown that topical application of mitomycin C after surgical decompression effectively reduces scar adhesion. However, the underlying mechanisms remain unclear. In this study, we investigated the effect of mitomycin C on the proliferation and apoptosis of human epidural scar fibroblasts. Human epidural scar fibroblasts were treated with various concentrations of mitomycin C (1, 5, 10, 20, 40 μg/mL) for 12, 24 and 48 hours. Mitomycin C suppressed the growth of these cells in a dose- and time-dependent manner. Mitomycin C upregulated the expression levels of Fas, DR4, DR5, cleaved caspase-8/9, Bax, Bim and cleaved caspase-3 proteins, and it downregulated Bcl-2 and Bcl-xL expression. In addition, inhibitors of caspase-8 and caspase-9 (Z-IETD-FMK and Z-LEHD-FMK, respectively) did not fully inhibit mitomycin C-induced apoptosis. Furthermore, mitomycin C induced endoplasmic reticulum stress by increasing the expression of glucose-regulated protein 78, CAAT/enhancer-binding protein homologous protein (CHOP) and caspase-4 in a dose-dependent manner. Salubrinal significantly inhibited the mitomycin C-induced cell viability loss and apoptosis, and these effects were accompanied by a reduction in CHOP expression. Our results support the hypothesis that mitomycin C induces human epidural scar fibroblast apoptosis, at least in part, via the endoplasmic reticulum stress pathway.

Potential risk of mitomycin C at high concentrations on peripheral nerve structure.

Although the local application of mitomycin C may prevent epidural adhesion after laminectomy, mitomycin C can induce neurotoxicity in optic and acoustic nerves at high concentrations. To determine the safe concentration range for mitomycin C, cotton pads soaked with mitomycin C at different concentrations (0.1, 0.3, 0.5, and 0.7 mg/mL) were immediately applied for 5 minutes to the operation area of rats that had undergone laminectomy at L1. Rat sciatic nerves, instead of dorsal nerves, were used in this study. The results showed that mitomycin C at 0.1-0.5 mg/mL did not damage the structure and function of the sciatic nerve, while at 0.7 mg/mL, mitomycin C significantly reduced the thickness of the sciatic nerve myelin sheath compared with lower concentrations, though no functional change was found. These experimental findings indicate that the local application of mitomycin C at low concentrations is safe to prevent scar adhesion following laminectomy, but that mitomycin C at high concentrations (> 0.7 mg/mL) has potential safety risks to peripheral nerve structures.