Molecular mechanism of microRNA-21 promoting Schwann cell proliferation and axon regeneration during injured nerve repair.

At present, the functional recovery after nerve injury is not satisfactory in clinical practice. The aim of this study was to explore the molecular mechanism of miR-21 promoting Schwann cells (SC) proliferation and axon regeneration after peripheral nerve injury, providing a theoretical basis for injured nerve repair. Nerve injury models were constructed to determine the expression of miR-21 in the injured nerve by Quantitative Real-Time PCR (qRT-PCR). After miR-21 over-expression SC (mimic-miR-21) group, control SC (control-miR-21) group and blank SC (RSC96) group were constructed, SC proliferation was determined by CCK-8, cell cycle was analysed by flow cytometry, dorsal root ganglion neuron (DRGn) axon regeneration was observed after DRGn was cultured with SCs for 7 days, the expressions of TGFßI, TIMP3, EPHA4 as well as apoptosis-related proteins caspase-3 and caspase-9 were detected by qRT-PCR and Western blot in the three groups, respectively. Target genes were confirmed by dual-luciferase reporter gene assay. The expressions of TGFßI, TIMP3 and EPHA4 were assessed by immunofluorescence in vivo. qRT-PCR indicated that miR-21 expression was significantly higher in the model group than in the sham operation and blank groups. SC proliferation index (PI) was significantly higher, the apoptosis rate was significantly lower, the axon was significantly longer, and mRNA and protein expressions of TGFßI, TIMP3, EPHA4 as well as apoptosis-related proteins caspase-3 and caspase-9 were significantly lower in the mimic-miR-21 group than in the control-miR-21 and RSC96 groups. The double luciferase assay confirmed that TGFßI, TIMP3 and EPHA4 were potential target genes of miR-21. In vivo immunofluorescence also indicated that expressions of TGFßI, TIMP3, EPHA4 were lower in the mimic-miR-21 group than in the control-miR-21 and RSC96 groups. We conclude that during injured peripheral nerve repair, miRNA-21 plays an important role in promoting SC proliferation and axon regeneration by regulating TGFßI, TIMP3 and EPHA4 target genes.

Surgical treatment of foreign body embolus in the Middle cerebral artery secondary to neck injury.

Background: We report a case of a foreign body embolus to the middle cerebral artery and reviewed similar cases previously reported. Methods: A 30-year-old man was seen 72 days after a penetrating neck injury with a 1-month history of numbness in the left limb and impairment of the fine movement in the left hand. Radiological examination revealed a foreign body in the M2 portion of the right middle cerebral artery (MCA). The patient received arteriotomy and in situ suturing. Results: During the operation, we found a metallic foreign body at the bifurcation of the M2 upper trunk of the right MCA, narrowed distal blood vessels and thinned vessel walls. The foreign body was surrounded by granulation tissue. Both foreign body and granulation tissue were removed slowly followed by in situ suturing. Indocyanine green angiography confirmed arterial patency. Three days after the surgery, the patient developed numbness and weakness in the left arm, with a muscle strength of grade 4. Computed tomography showed partial infarction in the right temporal lobe. Then, antispasmodic drugs were used. Muscle strength recovered by 14 days after the operation. Conclusions: In the subacute stage, surgery can be conducted to remove intra-arterial foreign bodies along with their surrounding granulation tissue if computed tomography perfusion suggests a decreased blood flow reserve capacity.

Effects of superficial temporal artery-middle cerebral artery bypass on hemodynamics and clinical outcomes in the patients with atherosclerotic stenosis in the intracranial segment of internal carotid artery and middle cerebral artery.

OBJECTIVE: To observe the effects of superficial temporal artery-middle cerebral artery bypass (STA-MCA bypass) on hemodynamics and clinical outcomes in the patients with atherosclerotic stenosis in the intracranial segment of internal carotid artery and (or) middle cerebral artery. PATIENTS AND METHODS: The data of 63 patients who had the symptoms of cerebral ischemia in recent 3 months, intracranial segment of internal carotid artery (ISICA) and (or) middle cerebral artery (MCA) stenoses or occlusion showed by digital subtraction angiography (DSA), and reduced cerebral perfusion displayed by CT perfusion (CTP) imaging were retrospectively collected in this study. According to the patient's choice of different treatment methods (STA-MCA bypass and drugs), these patients were allocated into two groups: Bypass group (30 cases) and Drug group (33 cases). Postoperative symptoms, anastomotic patency and hemodynamics were observed in the Bypass group. Post-treatment ischemic events and clinical outcomes were recorded in the two groups and were compared between the two groups. RESULTS: In the Bypass group, DSA all showed anastomotic patency in 28 patients (93.3%, 28/30), and the improvement rate of CTP was all significantly higher in the patients with stage-III CTP than in the patients with stage-II CTP at post-operative 3 days and 6 months (95% vs 60%). Post-treatment ischemic event incidence (13.3% vs 48.5%) and annual stroke rate (6.7% vs 25.6%) were significantly lower in the Bypass group than in the Drug group (All P < 0.05). Pre-treatment National Institutes of Health Stroke Scale (NIHSS) score and Modified Rankin Scale (MRS) score were not significantly different between the two groups, but the NIHSS (2.87±0.19 and 2.4±0.19 vs 4.03±0.47 and 3.97±0.49) and MRS (1.13±0.09 and 1.0±0.07 vs 1.55±0.14 and 1.52±0.15) all were significantly lower in the Bypass group than in the Drug group at post-treatment 6 and 24 months (all P < 0.05). CONCLUSION: STA-MCA bypass can improve cerebral blood perfusion and reduce the incidence of stroke in the patients who have ISICA and (or) MCA-related symptoms, 70%-100% of stenosis, and above stage-ⅠCTP. However, this conclusion remains to be further confirmed.

NF-κB induces miR-148a to sustain TGF-ß/Smad signaling activation in glioblastoma.

BACKGROUND: Inflammatory cytokines and transforming growth factor-ß (TGF-ß) are mutually inhibitory. However, hyperactivation of nuclear factor-κB (NF-κB) and TGF-ß signaling both emerge in glioblastoma. Here, we report microRNA-148a (miR-148a) overexpression in glioblastoma and that miR-148a directly suppressed Quaking (QKI), a negative regulator of TGF-ß signaling. METHODS: We determined NF-κB and TGF-ß/Smad signaling activity using pNF-κB-luc, pSMAD-luc, and control plasmids. The association between an RNA-induced silencing complex and QKI, mitogen-inducible gene 6 (MIG6), S-phase kinase-associated protein 1 (SKP1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was tested with microribonucleoprotein immunoprecipitation and real-time PCR. Xenograft tumors were established in the brains of nude mice. RESULTS: QKI suppression induced an aggressive phenotype of glioblastoma cells both in vitro and in vivo. Interestingly, we found that NF-κB induced miR-148a expression, leading to enhanced-strength and prolonged-duration TGF-ß/Smad signaling. Notably, these findings were consistent with the significant correlation between miR-148a levels with NF-κB hyperactivation and activated TGF-ß/Smad signaling in a cohort of human glioblastoma specimens. CONCLUSIONS: These findings uncover a plausible mechanism for NF-κB-sustained TGF-ß/Smad activation via miR-148a in glioblastoma, and may suggest a new target for clinical intervention in human cancer.

Construction and identification of human glial cell-derived neurotrophic factor gene-modified Schwann cells from rhesus monkeys.

The objective of this study was to construct stable rhesus monkey Schwann cells (SCs) modified with the human glial cell-derived neurotrophic factor (hGDNF) gene. hGDNF gene amplification was performed with pUC19-hGDNF as templates, and then the coding sequence of hGDNF was inserted into the eukaryotic expression vector pBABE-puro to obtain the recombinant vector pBABE-puro-hGDNF. The recombinant vector pBABE-puro-hGDNF was identified with restriction enzyme, and then underwent DNA sequencing. SCs from rhesus monkeys were transfected with the recombinant vector pBABE-puro-hGDNF, and then the expression levels of mRNA and protein of the hGDNF gene were determined with real-time fluorescence quantitative PCR and Western blot, respectively, in the transfected SCs. The biological activity of GDNF gene-modified SCs (GDNF-SCs) was assessed by MTT assay. The length of the hGDNF coding sequence of PCR products was 569 bp. After the recombinant eukaryotic expression vectors were digested with restriction enzyme, there was a specific segment of 596 bp. The results of DNA sequencing of the specific segment of 596 bp were the same as that of hGDNF in GenBank, suggesting that the hGDNF gene was successfully inserted into the recombinant retrovirus vectors. The expression levels of mRNA and protein were significantly higher in transfected SCs as compared to nontransfected SCs (p<0.05). MTT assay indicated that the OD value was significantly higher in GDNF-SCs group than in SCs and DMEM groups (p<0.05). hGDNF-SCs can steadily and efficiently release hGDNF. This study provides a basis for cell therapy of nerve injury.