Increased EZH2 Levels in Anterior Cingulate Cortex Microglia Aggravate Neuropathic Pain by Inhibiting Autophagy Following Brachial Plexus Avulsion in Rats / 神经科学通报·英文版

After brachial plexus avulsion (BPA), microglia induce inflammation, initiating and maintaining neuropathic pain. EZH2 (enhancer of zeste homolog 2) has been implicated in inflammation and neuropathic pain, but the mechanisms by which it regulates neuropathic pain remain unclear. Here, we found that EZH2 levels were markedly upregulated during BPA-induced neuropathic pain in vivo and in vitro, stimulating pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) secretion in vivo. In rats with BPA-induced neuropathic pain, mechanical and cold hypersensitivities were induced by EZH2 upregulation and inhibited by EZH2 downregulation in the anterior cingulate cortex. Microglial autophagy was also significantly inhibited, with EZH2 inhibition activating autophagy and reducing neuroinflammation in vivo. However, this effect was impaired by inhibiting autophagy with 3-methyladenine, suggesting that the MTOR signaling pathway is a functional target of EZH2. These data suggest that EZH2 regulates neuroinflammation and neuropathic pain via a novel MTOR-mediated autophagy signaling pathway, providing a promising approach for managing neuropathic pain.

Outcome of contralateral C7 nerve transferring to median nerve / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Contralateral C7 (cC7) transfer had been widely used in many organizations in the world, but the outcomes were significantly different. So the purpose of the study was to evaluate the outcome of patients treated with cC7 transferring to median nerve and to determine the factors affecting the outcome of this procedure.</p><p><b>METHODS</b>A retrospective review of 51 patients with total root avulsion brachial plexus injuries who underwent cC7 transfer was conducted. All of the surgeries were performed with two surgery stages and median nerve was the recipient nerve. The cC7 nerve was used in three different ways. The entire C7 root was used in 11 patients; the posterior division together with the lateral part of the anterior division was used in 15 patients; the anterior or the posterior division alone was used in 25 patients. The mean follow-up period was 6.9 years.</p><p><b>RESULTS</b>The efficiency of the surgery in these 51 patients was 49.02% in motor and 62.75% in sensory function. The patients with entire C7 root transfer obtained significantly better recovery in both motor and sensory function than the patients with partial C7 transfer. The best function recovery could be induced if the interval between the two surgery stages was 4-8 months.</p><p><b>CONCLUSIONS</b>cC7 transfer is an effective procedure in repairing median nerve. But using the entire C7 root transfer can obtain better recovery; so we emphasize using the entire root as the donor. The optimal interval between two surgery stages is 4-8 months.</p>

Expression and role of connective tissue growth factor in the peripheral nerve after chronic compression injury / 中华显微外科杂志

Objective To investigate the effects of connective tissue growth factor (CTGF) on the chronic peripheral nerve compression injury and explore the function of CTGF in peripheral nerve compression injury and repair. Methods From July 2010 to September 2010, fifty aduh male SD rats were randomly divided into group A and B: group A (sham-operated group): only exposed the sciatic nerve; group B (compression group): undergone sciatic nerve entrapment operation on the right hind leg according to the method which Mackinnon adopted when he established the model of chronic sciatic nerve compression.Electron microscopy,immunohistochemistry,RT-PCR and Western-blot were performed to observe the morphological changes of the compressed nerve tissue and to determine the level of CTGF,collagen- Ⅰ,Ⅲ (COL- Ⅰ,Ⅲ),2,4,6,8,10 weeks after the surgery,respectively. Results After sciatic nerve compression,the collagen in nerve increased ; The expression of CTGF and COL- Ⅰ, Ⅲ in sciatic nerve of compressed group increased, which was statistically different compared with the sham-operation group (P ＜ 0.05); In the meanwhile,the contents of CTGF and COL- Ⅰ,Ⅲ were positively correlated in a certain period. Conclusion Peripheral nerve fibrosis can be caused by chronic nerve compression.The expression of COL- Ⅰ,Ⅲ in sciatic nerve increased and CTGF get involved in the pathophysiological process, which suggests that CTGF plays an important role in the process of neural injury and fibrosis.

Role of iatrogenic transforming growth factor-β1 antibody in peripheral neural fibrosis after chronic entrapment / 中华创伤杂志

ObjectiveTo investigate the effect of transforming growth factor-β1 (TGF-β1) antibody on peripheral neural fibrosis after chronic entrapment.MethodsA total of 75 rats were randomly divided into three groups, ie, Group A (sham operation, only the sciatic nerve exposed), Group B (compression only, treated with sciatic nerve entrapment) and Group C (compression plus antibody injection).Electron microscopy, immunohistoehemistry, RT-PCR and Western blot were performed to observe the morphological changes of the compressed nerve tissue and to determine the level of TGF-β1 , collagen Ⅰ and collagen Ⅲ at 2, 4, 6, 8, 10 weeks after sciatic nerve compression, respectively.Results The levels of TGF-β1 , types Ⅰ and Ⅲ collagen protein were increased significantly in the Group B compared with that in the Group A, when the expression of TGF-β1 was increased in the early phase of the compression, reached the peak at the 4th week, and then decreased slowly.The expressions of collagenⅠand collagen Ⅲ were increased after compression, reached a peak at the 6th week and then maintained a relatively high level.The number of the fibrous tissues was decreased significantly and the content of types Ⅰ and Ⅲ collagen protein declined in the Group C, with statistical difference compared with the Group B (P ＜ 0.05).ConclusionsPeripheral nerve fibrosis can be caused by chronic nerve compression.TGF-β1 plays an important role in effectively inhibiting the collagen synthesis and ameliorating the nervous fibrosis of the protein following peripheral nervous entrapment.

AKT-modified autologous intracoronary mesenchymal stem cells prevent remodeling and repair in swine infarcted myocardium / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Transplantation of adult bone marrow-derived mesenchymal stem cells (MSCs) has been proposed as a strategy for cardiac repair following myocardial damage. However cell transplantation strategies to replace lost myocardium are limited by the inability to deliver large numbers of cells that resist peritransplantation graft cell death. Accordingly, we set out to isolate and expand adult swine bone marrow-derived MSCs, and to engineer these cells to overexpress AKT1 (protein kinase B), to test the hypothesis that AKT1-engineered MSCs are more resistant to apoptosis and can enhance cardiac repair after transplantation into the ischemic swine heart.</p><p><b>METHODS</b>The CDS (regulation domain of AKT1) AKT1-cDNA fragment was amplified, and MSCs were transfected following synthesis with a pCDH1-AKT1 shuttling plasmid. Western blotting analysis and real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed. Myocardial infarction (MI) models were constructed in Meishan pigs, and cardiac function was evaluated by magnetic resonance imaging (MRI) measurements and echocardiography 4 weeks later. All pigs were assigned to four groups: control (A), DMEM (B), MSC (C), and AKT-transfected (D). MSCs were transfected with the AKT1 gene, and autologous BrdU-labeled stem cells (1 x 10(7)/5 ml) were injected into left anterior descending coronary atery (LAD) of the infarct heart in groups C and D. In group B, DMEM was injected using the same approach. In group A, there was no injection following LAD occlusion. After 4 weeks, cardiac function and regional perfusion measurements were repeated by MRI and echocardiography, and histological characteristics of the hearts were assessed. Connecxin-43 (CX-43), BrdU, and von Willebrand factor (VWF) immunoreactivity was tested using enzyme linked immunosorbent assay (ELISA). Vascular endothelial growth factor (VEGF), transforming growth factor-beta1 (TGF-beta1) were analyzed at the same time.</p><p><b>RESULTS</b>AKT1-cDNA was cloned into pCDH1-MCS1-EF1-copGFP and the sequence was confirmed. AKT mRNA expression was detected at 24 hours after transfection. AKT1 expression in MSCs remained strong after 2 weeks, according to real-time RT-PCR and Western blotting. Prior to cell implantation, end-diastolic left ventricular dimension (EDLVd) increased and stroke volume (SV) decreased in the MI hearts. MRI scans revealed significantly improved cardiac function following implantation, and implanted MSCs prevented thinning and expanding in the infarct region, as well as improved contraction and increased perfusion in all groups compared to control hearts. The left ventricular chamber size was smaller in cell-transplanted hearts than in control hearts. Moreover, group D exhibited significant improvement. The expression of CX-43, BrdU, and VWF could be found in the immunohistochemical pathological sections of group C and group D. The level of VEGF reached a high level 1 week after implanting the MSCs, but the level of TGF-beta1 decreased gradually.</p><p><b>CONCLUSIONS</b>The AKT1-expressing lentiviral vector resulted in stable over-expression of AKT1 in MSCs. MSC engraftment in host myocardium improved cardiac function by attenuating contractile dysfunction and pathological thinning of the infracted left ventricular wall, which likely resulted from myocardial regeneration and angiogenesis.</p>

Growth rule of activated Schwann cells cultured on Chitosan-collagen film / 中国组织工程研究

BACKGROUND: New-type tissue engineering materials and post-proliferation Schwann cells are implanted into biosynthesis tube for repairing peripheral nerve defect, which are two great developments in the field of artificial biomaterial tube.OBJECTIVE: Taking chitosan-collagen as scaffold, activated Schwann cells as seed cells, we are in attempt to observe the affinity between them as well as growth rule of activated Schwann cells on Chitosan-collagen, so as to provide basis for pre-construction of artificial nerve.DESIGN: Open experiment.SETTING: Department of Hand Surgery, Huashan Hospital Affiliated to Fudan University.MATERIALS: This experiment was conducted at the Key Laboratory of Hand Function Reconstruction, Ministry of Public Health from July 2003 to December 2003. Four male SD rats, of clean degree, were used in this experiment. Chitosan-collagen film was made in Qisheng Biomaterial Technique Institute, Shanghai, Schwann cells activator solution was made in our laboratory (self-made).METHODS: After rats were anaesthenia, the sciatic nerve was cut off to perform predegeneration for 7 days. Another anaesthenia later, the rats were euthanized. Both sides of sorciatic nerves were cut off quickly and put in the D-HANK's solution containing penicillin and streptomycin.Epineurium was eliminated and chipped into 1 mm pieces, then put in the centrifuge tube containing 5 g/L trypsinase and 0.6 g/L collagenase. 0.5 mL activator solution every 2 mL liquid was added and the activated Schwann cells were harvested with the way of two-step enzymolysis. 2×107 L-1 activated Schwann cells in 200 μL were inoculated to Chitosan-collagen film and Petri dish . Two weeks later, cellular growth was observed under phase contrast microscope and scanning electron microscope. Cellular purity was identified with S-100 staining.MAIN OUTCOME MEASURES: ① Drawing cell growth curve and confirming in vitro doubling time. ②Observation of activated Schwanri cells under an inverted phase contrast microscope. ③ Observation of activated Schwann cells inoculated on Chitosan-collagen film under scanning electron microscope.RESULTS: ① Confirmation of in vitro doubling time: Concentration of activated Schwann cells inoculated on both Chitosan-collagen and Petri dish was 2×107 L-1, the final concentration was up to 3.0×108 L-1 and 2.0×108 L-1 respectively 2 weeks later. Doubling time of activated Schwann cells cultured on Chitosan-collagen film was 4 days calculated according to DT=(t-t0) lg2/(lgn-lgn0). ②Observation of activated Schwanncells under an inverted microscope: 24 hours later, the activated Schwann cells inoculated to Petri dish mostly changed from spherical to long shuttle-shape,mutation appeared and most were two-pole shape, fewer were three-pole shape; Morphologically, there was no significant difference between activated Schwann cells inoculated on Chitosan-collagen film and on Petri dish. Activated Schwann cells inoculated to Chitosan-collagen film were like "words cayed on the sand" under phase contrast microscope and the purity was over 95%. ③ Observation of activated Schwann cells inoculated to Chitosan-collagen under scanning electron microscope: Most of activated Schwann cells grew in the introcession of Chitosan-collagen or closely to surface of Chitosan-collagen, presenting regular head-to-end connection and adhesion to Chitosan-collagen film. The cell body was fusiform,with diameter of 4-6 μm, 60-80 μm in length. Cells were shuttle-shape with some small branches. Morphology of Chitosan-collagen film was still complete at week 1.CONCLUSION: There exists great affinity between Chitosan-collagen film and high-purity activated Schwann cell; so tissue-engineering scaffold made of the two components probably promote peripheral nerve regeneration.

Bridging peripheral nerve defect with chitosan-collagen film / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To seek new method for the treatment of peripheral nerve injury.</p><p><b>METHODS</b>In rat model with sciatic nerve defect, chitosan-collagen film was sutured into conduit to bridge 5 mm, 10 mm nerve defects. Rats that underwent end-to-end anastomosis were taken as controls. General observation, electrophysiological study, histological study and image analysis were performed at 4, 8, 12 weeks postoperatively.</p><p><b>RESULTS</b>In 5 mm nerve defects, the quality of nerve regeneration was similar to that of the control group. For 10 mm nerve defect, nerve regeneration was inferior to that of the control group. Chitosan-collagen film obviously degraded at 12 weeks postoperatively.</p><p><b>CONCLUSIONS</b>Chitosan-collagen film conduit can be used to bridge peripheral nerve defect.</p>

Homeostasis between survival and apoptosis of Schwann cells after injury of peripheral nerve / 中华创伤骨科杂志

Schwann cells proliferate and the axis cylinder regenerates after the injury of the peripheral nerves. Schwann cells are a vital factor in the process of regeneration of injured nerves. The article summarizes the survival and apoptosis adaptation of Schwann cells after the injury of the peripheral nerves. It discusses massive NGF secreting, P75 expressing, and IGF 2, PDGF BB, NT 3 and CNTF secreting. It also supposes that there might be a homeostasis between survival and apoptosis in the proliferated Schwann cells and brings forward the question regarding reassessment of administration of NGF to enhance regeneration of the injured peripheral nerves.

Gene expression of growth associated protein 43 in activated Schwann cells / 中华骨科杂志

Objective To compare activated and normal Schwann cells in GAP43 gene expression. Methods 10 male SD rats, weighed from 100 g to 120 g. The right median nerve of SD rats was transected at the axillary level and was buried in muscle for predegeneration. 1 week later, the distal segment of the transected right median nerve with 1 cm long was harvested. The untreated left median nerve was harvested as control with same length. The epineurium of nerve was stripped, then the nerve tract was cut to small pieces. Schwann cells were obtained by way of double kinases digestion with 0.25% trypsin and 0.03% collegenase. The right median nerve was activated with additional liquid during digestion so as to obtain the activated Schwann cells. The normal Schwann cells were harvested from left median nerve. rt-PCR was used for GAP43 gene enlargement. mRNA was distilled from activated Schwann cells and untreated Schwann cells respectively. Then the mRNA was reversely transcripted to cDNA with SuperScriptTM, and cDNA worked as template for PCR enlargement. The product of PCR was separated with 1% agarose gel electrophoresis for 40 -50 min and stained with SYBR Green Ⅰnucleic acid gel. Fluorescence intensity of GAP43 PCR products was measured and then compared between the experiment group and control group. Results The Fluorescence intensity of GAP43 PCR product of activated Schwann cells was higher than that of normal Schwann cell. There was significant difference (P=0.003, Paired t test). It indicated that GAP43 mRNA of activated Schwann cells was much more than that of the normal Schwann cells. Conclusion GAP43 gene expression is up regulated in activated Schwann cells in contrast to normal Schwann cells. Activated Schwann cells secreting more GAP43, which may be one of the important mechanisms in promoting nerve regeneration.

The methodological study of Schwann cell culture from sciatic nerve of adult SD rat / 中华显微外科杂志

Objective Attempt to find out the method of isolated purified Schwann cells from sciatic nerve of adult SD rat Methods Three methods of culturing Schwann cells from sciatic nerve of adult rats were compared: (1) Conventional primary explant technique in tissue culture (Method A) (2) Conventional enzymatic disaggregating technique for cell culture (Method B) (3) A modified method of cell culture (Method C): Test group: we dissected very carefully and separated the sciatic nerve into individual fibers under surgical microscpe, individual fibers were cut into 0 5～1 mm pieces and treated with enzymatic disaggregating by twoenzymes (0 5% Trpsin and 0 06% Collagenase) in 37℃ for 80～90 min Control group: a application of conventional enzymatic disaggregating technique for culturing the nerve adventital tissues, which werethe remaining procedures of Test group We applied immunohistochemistry method with S100 antibody and Fibroblast antibody to identify Schwann cells Function of these Schwann cells proliferated were assessed by MTT assay and H 3 Thymidine incorporation assay. Conclusion In method C, a pure culture of Schwann cells was obtained, where the two techniques were used: careful separation of the sciatic nerve into individual fibers and use of higher concentrations of two enzymes for longer time to digest the nerve tissues These two technigues can cleanly remove and inhibit the fibroblasts, and enable healthy and normal proliferation of Schwann cells

Morphological changes of different time of Schwann cells within the predegenerated nerve / 中华骨科杂志

Objective To study the morphological changes of the Schwann cells within the predegenerated nerve. Methods The ulanr and the median nerves were cut bilaterally at the axillary level in 42 SD rats. A nerve segment of 1 cm in length was harvested from the distal segment of each of the cut nerves right after the operation, 3 days, 1, 2, 3, 4 and 8 weeks postoperatively(group 1-7). The methods of Schwann cells culture and S-100 protein standing were used to study the temporal changes of the number of the Schwann cells, and the thickness of myelin of the predegenerated nerve were studied under electromicroscope. Results The number of Schwann cells increased after nerve transection, and reached a peak of [(3.52?0.27)?107]/ml in one week. The thickness of myelin also increased after nerve transection, and was thickest at about (231.2?8.9) nm in one week. The myelin thickness slowly decreased after this time. Conclusion The number of the Schwann cells and the thickness of myelin of the predegenerated nerve were changed with time.