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BACKGROUND: Previous studies have observed the expression of formyl peptide receptor 2 in newly differentiated neurons from neural stem cells and confirmed that formyl peptide receptor 2 can promote the migration of neural stem/progenitor cells and induce them to differentiate into neurons. Formyl peptide receptor 2 ligands are present in damaged spinal cord tissues, but the binding of different ligands with FPR2 may lead to different and even opposite biological effects. OBJECTIVE: To investigate the neurite outgrowth after the binding of the ligands produced following spinal cord injury with formyl peptide receptor 2. METHODS: The fetal rat cerebral cortical neurons were extracted by enzymatic digestion. Spinal cord injury models were established in Sprague-Dawley rats, and the injured spinal cord homogenate was extracted. (1) Experiment 1: To observe the effect of the activation of formyl peptide receptor 2 on neurite outgrowth, the cells were divided into control group, formyl peptide receptor 2 blocker group (addition of WRW4), spinal cord homogenate group, spinal cord homogenate+WRW4 group. (2) Experiment 2: To observe the effect of blockade of AKT and ERK signaling pathways on neurite outgrowth after activation of formyl peptide receptor 2, the cells were divided into control group, AKT and ERK signaling pathway blocker group (addition of Ly294002+PD98059), spinal cord homogenate group, spinal cord homogenate+ Ly294002+PD98059 group. After 24 hours of culture, adherent neurons were treated with above-mentioned regimens for 7 days. Immunofluorescence staining with confocal microscope detection was used to observe the effect of spinal cord homogenate on neurite outgrowth via the activation of formyl peptide receptor 2. The cells were treated by the above-mentioned regimens for 30 minutes and phosphorylated protein levels were detected by western blot. The cells were treated with the above-mentioned regimens for 24 hours, and western blot assay was used to detect F-actin levels and observe the phosphorylation of key proteins in MAPK and PI3K/Akt pathways with the presence of formyl peptide receptor 2 specific blocker WRW4. RESULTS AND CONCLUSION: WRW4 could eliminate the effects of injured spinal cord homogenates on neurite outgrowth, including neurite length, the number of primary neurites, and the number of branch points. Spinal cord homogenate increased the phosphorylation of ERK1/2 and Akt in neurons, whereas this effect could be blocked by WRW4. Ly294002 and PD98059 could also eliminate the effects of homogenates on the neurite outgrowth. Spinal cord homogenate significantly increased the expression of F-actin in neurons, but this effect was blocked by WRW4. These results suggest that spinal cord homogenates can expedite neurite outgrowth by activating formyl peptide receptor 2, which may be related to the increased phosphorylation of ERK1/2 and Akt.
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Objective:To investigate the effects of Buyang Huanwu Tang （BHT） on axonal regeneration and neurological rehabilitation of the rats suffering ischemic stroke （IS）. Method:A total of 180 SD rats were used to establish a middle cerebral artery infarction （MCAO） model. The animals that were successfully modeled were randomly divided into model group， BHT group （12 g·kg-1） and nimodipine group （20 mg·kg-1）， and a sham group was established， with 28 rats in each group. After seven-days intragastric administration of BHT， the animals were sacrificed. TTC staining was used to test cerebral infarction. Brain water content was measured to observe cerebral edema. Bielschowsky's silver staining and immunofluorescence were performed to observe axonal degeneration and the protein expression of neurofilament protein-200（NF-200）. Quantitative real-time polymerase chain reaction （PCR） was used to analyze the mRNA expression of repulsion oriented molecule a （RGMa）， Ras homologous enzyme （Rho）， Rho kinase （ROCK）， and collapsion response regulatory protein 2 （CRMP2）. Neurological function scores assay was used to examine neurological recovery. Result:Compared with sham group， the cerebral infarction volume and brain water content increased significantly（P<0.01）， and motor function was markablely decreased in the model group. Axonal degeneration and nerve fiber damage were obviously observed. Also， gene expression of axon growth-related protein was deviation from normal （P<0.01）. Compared with model group， the cerebral infarction rate （P<0.01）， brain water content （P<0.01） and axonal degeneration of BHT group and nimodipine group were significantly reduced. The expression of NF-200 was increased. Also， the mRNA expression of RGMa， Rho and ROCK was lower （P<0.05） while the mRNA expression of CRMP2 was higher （P<0.01）. And the neurological function was significantly improved （P<0.05）. Conclusion:BHT can promote axon regeneration after ischemic stroke injury by regulating the mRNA expression of axon growth-related protein， thereby improving nerve function.
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Aim To investigate the anti-aging effect of catalpol on normal cortical neurons and its possible mechanism. Methods Primary cultured cortical neu-rons from 24 h newly born rats were dissociated and cultured. The cells were divided into normal group, catalpol (0. 1 mg · L - 1 )group,catalpol (1 mg · L - 1 )group and catalpol (10 mg·L - 1 )group. Neu-rons were cultured for 13d,and the cell morphology was observed by microscope. Cell activity was detected by MTT assay. p-S6 and Map-2 of 13d neurons were detected by immunofluorescence to reveal cell viability and the axon growth. GAP-43,p-S6,PI3K,p-PI3K, Akt,p-Akt,mTOR and p-mTOR protein expression of 13d neurons were detected by Western blot. Results Compared with normal group,different concentrations of catalpol could delay the aging of neurons,promote their survival and increase cell viability. The nμmber of p-S6 positive cells increased. Different concentra-tions of catalpol could promote axon growth. GAP-43, p-S6,PI3K,p-PI3K,Akt,p-Akt,mTOR and p-mTOR protein expression of 13d neurons increased. Conclusions Catalpol has anti-aging effects on cere-bral cortical neurons,and its mechanism may be relat-ed to regulating PI3K/ Akt/ mTOR signaling pathway and enhancing neuronal growth activity.
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Objective To investigate the effect of silence regulatory protein 1 (Sirt1) on axonal outgrowth. Methods The hippocampal neurons was first isolated in vitro from rat embryos. The distribution and expression of Sirt1 were then detected 72 h later. The down-regulation of Sirt1 was induced by RNAi technology and up-regulation of Sirt1 was in-duced by overexpression of Sirt1 and resveratrol (RES). Immunofluorescence staining was used to examine the axon length. Results Immunofluorescence staining showed that Sirt 1 was located in neuronal cell body and neurite, especially in the distal axons. Down-regulation of Sirt1 significantly decreased axonal length compared with siRNA control group [(178.3 ± 3.2) μm vs. (110.2 ± 18.30) μm, P< 0.01 ]; Overexpression of Sirt1 significantly increased axonal length com-pared with eGFP control group [(178.3±3.2)μm vs (310.6±39.5)μm, P<0.01 ];Activation of Sirt1 by RES treatment al-so significantly increased axonal length compared with vehicle control group (DMSO treated group) [(291.7±13.2)μm vs. (525.1±49.1)μm, P<0.01 ]. Conclusions Sirt1 plays a key role in axonal growth which may be used as a potential thera-peutic target of axon regeneration.
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Objective To explore the effect on effect of combined use of Y27632 (ROCK II inhibitor)and TDZD-8(GSK-3β inhibitor)on axonal regeneration of dorsal root ganglion (DRG) neurons in neogenetic rats. Methods All the thoracolumbar DRGs of two neogenetic Sprague-Dawley (SD)rats(<5 days)were harvested under the stereopsis raicrostat,and then the DRG neurons were cultured,purified and indentified.Fifteen adult female SD rats were randomly divided into three groups,ie,complete paraplegia group(5 rats),sham operation control group(5 rats)and normal group(5 rats)respectively.The T8-10 spinal cord extracts (SCEs) were harvested in the complete paraplegia group,sham operation control group and normal group respectively at day 7 after spinal cord injury.The experiment was divided into group A(DRG neurons + PBS),group B(DRG neurons + complete paralysis SCE),group C(DRG neurons + complete paralysis SCE + different concentration Y27632),group D(DRG neurons + complete paralysis SCE + different concentration TDZD-8)and group E(DRG neurons + complete paralysis SCE + proper concentration Y27632 and TDZD-8).The average axonal length and expression intensity of Tubulin βⅢ at distal end of neuronal axons were observed after two days of co-culture respectively in intro. Results (1)The average axonal length and expression intensity of Tubulin βⅢ at axon shaft and growth cone in the group B were significantly shorter and weaker than that in the group A,with statistical difference.(2)In the group C,the average axonal length and expression intensity of Tubulln βⅢ at axon shaft and growth cone in 5-10 μmol/L Y27632 treatment groups were more than that in the group B but lower than that in the group A.The average axonal length and expression intensity of Tubuhn βⅢ at axon shaft and growth cone in 20-50 μmol/L Y27632 treatment group were longer and stronger than that in the group A and the group B,especially the group B.Among different concentration Y27632 treatment groups,there was a longest average axonal length and strongest expression intensity of Tubulin βⅢ in 30 μmol/L treatment group.(3) In the group D,there was a longer average axonal length in 0.5-3 μmol/L TDZD-8 treatment group than that in the group A and the group B,with the longeat average axonal length in l μmool/L TDZD-8 treatment group.In 5-25 μmol/L TDZD-8 treatment groups,the average axonal length showed no difference compared with the group B but wns shorter than that in the group A.In all different concentration TDZD-8 treatment groups,the expression intensity of Tubulin βⅢ at axon shaft and growth cone was significantly stronger than that in the groups A and B.(4) In the group E,although the average axonal length was increased in the group E,there was no statisilcal difference compared with the group A,30 μmol/L Y27632 treatment group and l μmol/L TDZD-8treatment group.There was a significantly longer average axonal length in the group E than it in the group B and the expression intensity of Tubulin βⅢ at axon shaft and growth cone was stronger in the group E compared to the group A,30 μmol/L Y27632 treatment group and l μmol/L TDZD-8 treatment group.Conclusion The complete paralysis SCEs obviously inhibits DRG axonal growth,induces axonal retraction and growth cone collapse.High concentration of Y27632 can more obviously promote the axon growth compared with the low concentration,while the low concentration of TDZD-8 can obviously promote the axon growth.Combined use of appropriate concentration of TDZD-8 and Y27632 can promote the axon growth and induce the axons branching,as facilitates the formation of the axon circuit.
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Transplantation of neural stem/progenitor cells (NPC) holds potential to improve functional outcomes in various neurological disorders. It seems more difficult than previously envisioned, however, to functionally replace the lost neural cells by grafted NPCs. A lack of appropriate developmental cues in the injured tissue contributes to the failure to guide the NPCs to survive, differentiate, grow axons, and functionally integrate to the host neural circuit. Therefore, we need to design possible strategies to recapitulate the developmental processes for the grafted NPCs to fully mature into functional neural cells. To enhance survival of NPCs following transplantation, pharmacological treatments targeting apoptosis and inflammation can be combined with transplantation. Genetic overexpression of prosurvival genes or growth factors can also improve survival. In vitro predifferentiation not only provides neural cells of a specific lineage in high purity but also greatly reduces chances of a tumor formation. Genetic overexpression of various transcription factors or manipulating molecular microenvironment of the host can also be tried to force differentiation of NPCs to a desired lineage. Pharmacological application to overcome myelin inhibition or enzymatic degradation of the inhibitory extracellular matrix will enhance axonal growth of NPC-derived neurons. Increasing synaptic activity by behavioral training or patterned electrical stimulation may promote proper development of synaptic integration and myelination of the axon. A thorough understanding of cellular and molecular aspects of neural development will help design more sophisticated strategies to enhance therapeutic capacity of NPC transplantation to reconstruct the damaged neural circuit.
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Apoptose , Axones , Signaux , Stimulation électrique , Matrice extracellulaire , Inflammation , Protéines et peptides de signalisation intercellulaire , Gaine de myéline , Maladies du système nerveux , Neurones , Facteurs de transcription , Transplantation , TransplantsRésumé
Objective To detect the expression of BAT2L protein in developmental brain of rats.Methods Expression,distribution and location of BAT2L protein in developmental brain rats on embryonic day 18,postnatal days 1,7 and 15 and in adult at different time points were detected by Western blotting analysis,immunohistochemistry and immunofluorescence,respectively.Results Western blotting showed that the BAT2L protein expression level was higher in brain of rats on embryonic day 18,postnatal days 1 and 7 than on postnatal day 15 and in brain of adult rats.Immunohistochemistry and immunofluorescence showed that the BAT2L protein was distributed and located in cell membrane and cytoplasm of neurons but not in nuclei and extra-cellular space.Positive neurons were widely distributed in cerebral and cerebellar cortex,hippocampus and cerebral ganglion.The morphology of neurons was significantly different in newborn and adult rats.Positive prominences and branches of certain neurons were found in brain tissues of newborn rats but hardly in those of adult rats.Conclusion BAT2L protein is specifically expressed in cell body,membrane and prominence of neurons in brain of rats.