MiR-133b Promotes neurite outgrowth by targeting RhoA expression.

BACKGROUND: MicroRNA-133b (miR-133b) has been shown to play a critical role in spinal cord regeneration. The aim of this study was to investigate the cellular role of miR-133b in neural cells. METHODS: PC12 cells and primary cortical neurons (PCNs) were transfected with lenti-miR-133b, lenti-miR-133b inhibitor, plasmid-shRNA-RhoA, plasmid-RhoA and their negative controls. After 48 hours of transfection, the levels of proteins and mRNA or miRNA were evaluated by Western blotting and qRT-PCR, respectively. Moreover, the neurite outgrowth was analyzed by Image J. For pharmacological experiments, inhibitors of MEK1/2 kinase (PD98059), phosphoinositide-3 kinase (PI3K) (LY294002) and ROCK (Y27632) were added into the culture medium. RESULTS: Overexpression of miR-133b in PC12 cells enhanced neurite outgrowth. Conversely, inhibition of miR-133b reduced neurite length. We further identified RhoA as a target and mediator of mir-133b for neurite extension by Western blot and knockdown experiment. Moreover, overexpression of RhoA could attenuate the neurite growth effects of miR-133b. Also, we observed that miR-133b activated MEK/ERK and PI3K/Akt signaling pathway by targeting RhoA. Finally, in PCNs, miR-133b also increased axon growth and attenuated axon growth restrictions from chondroitin sulfate proteoglycans (CSPG). CONCLUSIONS: In summary, our study suggested that miR-133b regulated neurite outgrowth via ERK1/2 and PI3K/Akt signaling pathway by RhoA suppression.

Neurogenin 2 converts mesenchymal stem cells into a neural precursor fate and improves functional recovery after experimental stroke.

BACKGROUND: Neurogenin2 (Ngn2) is a proneural gene that directs neuronal differentiation of progenitor cells during development. Here, we investigated whether Ngn2 can reprogram MSCs to adopt a neural precursor fate and enhance the therapeutic effects of MSCs after experimental stroke. METHODS: In vitro, MSCs were transfected with lenti-GFP or lenti-Ngn2. Following neuronal induction, cells were identified by immunocytochemistry, Western blot and electrophysiological analyses. In a stroke model induced by transient right middle cerebral artery occlusion (MCAO), PBS, GFP-MSCs or Ngn2-MSCs were injected 1 day after MCAO. Behavioral tests, neurological and immunohistochemical assessments were performed. RESULTS: In vitro, Ngn2-MSCs expressed neural stem cells markers (Pax6 and nestin) and lost the potential to differentiate into mesodermal cell types. Following neural induction, Ngn2-MSCs expressed higher levels of neuron-specific proteins MAP2, Tuj1 and NeuN, and also expressed voltage-gated Na+ channel, which was absent in GFP-MSCs. In vivo, after transplantation, Ngn2-MSCs significantly reduced apoptotic cells, decreased infarct volume, and increased the expression of VEGF and BDNF. Finally, Ngn2-MSCs treated animals showed the highest functional recovery among the three groups. CONCLUSIONS: Ngn2 was sufficient to convert MSCs into a neural precursor fate and transplantation of Ngn2-MSCs was advantageous for the treatment of stroke rats.

L4-to-L4 nerve root transfer for hindlimb hemiplegia after hypertensive intracerebral hemorrhage.

There is no effective treatment for hemiplegia after hypertensive intracerebral hemorrhage. Considering that the branches of L4 nerve roots in the lumbar plexus root control the movement of the lower extremity anterior and posterior muscles, we investigated a potential method of nerve repair using the L4 nerve roots. Rat models of hindlimb hemiplegia after a hypertensive intracerebral hemorrhage were established by injecting autogenous blood into the posterior limb of internal capsule. The L4 nerve root on the healthy side of model rats was transferred and then anastomosed with the L4 nerve root on the affected side to drive the extensor and flexor muscles of the hindlimbs. We investigated whether this method can restore the flexible movement of the hindlimbs of paralyzed rats after hypertensive intracerebral hemorrhage. In a beam-walking test and ladder rung walking task, model rats exhibited an initial high number of slips, but improved in accuracy on the paretic side over time. At 17 weeks after surgery, rats gained approximately 58.2% accuracy from baseline performance and performed ankle motions on the paretic side. At 9 weeks after surgery, a retrograde tracing test showed a large number of fluoro-gold-labeled motoneurons in the left anterior horn of the spinal cord that supports the L4-to-L4 nerve roots. In addition, histological and ultramicrostructural findings showed axon regeneration of motoneurons in the anterior horn of the spinal cord. Electromyography and paw print analysis showed that denervated hindlimb muscles regained reliable innervation and walking coordination improved. These findings suggest that the L4-to-L4 nerve root transfer method for the treatment of hindlimb hemiplegia after hypertensive intracerebral hemorrhage can improve the locomotion of hindlimb major joints, particularly of the distal ankle. Findings from study support that the L4-to-L4 nerve root transfer method can effectively repair the hindlimb hemiplegia after hypertensive intracerebral hemorrhage. All animal experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Nanjing Medical University (No. IACUC-1906009) in June 2019.

Non-receptor tyrosine kinase Src is required for ischemia-stimulated neuronal cell proliferation via Raf/ERK/CREB activation in the dentate gyrus.

BACKGROUND: Neurogenesis in the adult mammalian hippocampus may contribute to repairing the brain after injury. However, Molecular mechanisms that regulate neuronal cell proliferation in the dentate gyrus (DG) following ischemic stroke insult are poorly understood. This study was designed to investigate the potential regulatory capacity of non-receptor tyrosine kinase Src on ischemia-stimulated cell proliferation in the adult DG and its underlying mechanism. RESULTS: Src kinase activated continuously in the DG 24 h and 72 h after transient global ischemia, while SU6656, the Src kinase inhibitor significantly decreased the number of bromodeoxyuridine (BrdU) labeling-positive cells of rats 7 days after cerebral ischemia in the DG, as well as down-regulated Raf phosphorylation at Tyr(340/341) site, and its down-stream signaling molecules ERK and CREB expression followed by 24 h and 72 h of reperfusion, suggesting a role of Src kinase as an enhancer on neuronal cell proliferation in the DG via modifying the Raf/ERK/CREB cascade. This hypothesis is supported by further findings that U0126, the ERK inhibitor, induced a reduction of adult hippocampal progenitor cells in DG after cerebral ischemia and down-regulated phospho-ERK and phospho-CREB expression, but no effect was detected on the activities of Src and Raf. CONCLUSION: Src kinase increase numbers of newborn neuronal cells in the DG via the activation of Raf/ERK/CREB signaling cascade after cerebral ischemia.

Gastric Residual Volume after Split-Dose Bowel Preparation versus Conventional Single-Dose Regimen before Anesthetic Colonoscopy.

The aim of this study was to compare gastric residual volume (GRV) in patients given a split-dose versus a conventional single-dose of polyethylene glycol (PEG) preparation before undergoing anesthetic colonoscopy. Methods. In a prospective observational study, we assessed GRV in outpatients undergoing same-day anesthetic gastroscopy and colonoscopy between October 8 and December 30 of 2016. Outpatients were assigned to the split-dose (1 L PEG in the prior evening and 1 L PEG 2-4 h before endoscopy) or single-dose (ingestion of 2 L PEG ≥ 6 h before endoscopy) regimen randomly. Bowel cleansing quality was assessed with the Boston Bowel Preparation Scale (BBPS). Results. The median GRV in the split-dose group (17 ml, with a range of 0-50 ml; N = 65) was significantly lower than that in the single-dose group (22 ml, with a range of 0-62 ml; N = 64; p = 0.005), with a better bowel cleansing quality (BBPS score 8.05 ± 0.82 versus 7.64 ± 1.21; p = 0.028). GRV was not associated with diabetes or the use of medications. Conclusions. GRV after a split-dose preparation and fasting for 2-4 hours is not larger than that after a conventional single-dose preparation and fasting for 6-8 hours. The data indicates that the split-dose bowel preparation might not increase the risk of aspiration.