ABSTRACT
Objective To study the effect of neuronal Nogo-66 receptor (NgR1) antagonist,soluble Nogo-66 receptor (sNgR1-Fc),on promoting the endogenous neural precursor cells (NPCs) differentiating into neurons in order to clarify the mechanism.Methods The cortical infarction was induced by photochemistry,named photothrombotic cortical injury (PCI).Twelve Sprague Dawley rats were randomly divided (random number) into three groups:Sham-operated group,PBS group,and sNgR1-Fc group.PBS (PBS group) or sNgR1-Fc (sNgR1-Fc group) was injected into the lateral ventricle of brain with a minipump.BrdU (Bromodeoxyuridine) was injected into the peritoneal cavity 4-6 days after PCI.The subdentate gyrus zone (SGZ) of brain from sacrificed rat was harvested for Immunohistochemistry to observe the ratio of NeuN +/BrdU + cells 35 days after PCI.Proteins including Nestin、Notch1 and Mash1 were detected by Western Blot.Results The cortical infarction in rat was successfully induced by photochemistry.Thirty-five days after PCI,the BrdU + cells number and theratio of NeuN +/BrdU + in the SGZ of the ipsilateral cerebrum hemisphere with PCI were significantly higher in sNgR1-Fc group than those in PBS group (P ＜ 0.05).The levels of Notch1,Mash1 and Neuro D in the sNgR1-Fc group were significantly higher than those in the PBS group (P ＜ 0.05),which were significantly higher than those in the Sham-operated group.Conclusions sNgR1-Fc could promote the endogenous NPCs differentiating into neurons in a cortical infarction model.The mechanisms may be attributed to the Notch/bHLH (proneural basic helix-loop-helix genes) signaling way.
ABSTRACT
Objective To observe the protective effects of soluble Nogo-66 receptor (NgR1 )antagonist (sNgR1-Fc) on cortical axons after cortical infarction in rats,and to study the phenomenon and molecular mechanism of its protective effects on and regeneration of axons.Methods The cortical infarction was induced by photochemistry,termed photothrombotic cortical injury (PCI).Fifteen Sprague Dawley rats were randomly divided into three groups:Sham-operated group,PBS (phosphate buffered solution) group,and s-NgR1-Fc group.In PBS group,PBS was injected into the lateral ventricle of rats; and in sNgR1-Fc group,sNgR1-Fc was injected instead of PBS. The ipsilateral cortex with lesion was harvested for histomorphometry and transmission electron microscope observation 7 days after PCI. Proteins including GTP-RhoA,p-JNK,p-c-JUN and p-ATF-2 were detected by Western blot,as well as Total-J and Total-RhoA.Results The cortical infarction in rats was successfully induced by photochemistry.Compared with sham-operated group,the pathological changes in PBS groups were more serious,including extensive edema or disappearance of axoplasm of fiber without medulla sheath involved and extensive thickening or layer derangement in axoplasm of fiber with medulla sheath involved.These changes were improved significantly after sNgR1-Fc treatment.The levels of GTP-RhoA,p-JNK1,p-JNK2,p-c-JUN and p-ATF-2 in the PBS group were significantly higher than those in the sham-operation group ( P ＜ 0.05 ),whereas the levels of Total-RhoA,Total-JNKl and Total-JNK2 were not different significantly between these two groups (P ＞0.05 ).The sNgR1-Fc treatment up-regulated the levels of these proteins ( P ＜ 0.05 ).Conclusions There is pathological change in axon induced by cerebral hypoxia-ischemia for a long period after cortical infarction.The mechanisms may be associated with RhoA/ROCK/JNK/c-Jun signal way,which is activated by ischemia injury and related to the inhibition of regeneration in axon.Our study shows that NgR1-Fc may inhibit this pathway significantly,and then promote the regeneration of axon partially.
ABSTRACT
Objective To construct and identify of a plasmid mediated high efficiency of RNA interference(RNAi) against Nogo-66 receptor(NgR).Methods After cloning of NgR by RT-PCR,the fragments were inserted into pcDNA3.1/CT-GFP-TOPO to produce plasmid expressing NgR-GFP fusion protein.Four pairs of oligonucleotide were designed according to NgR sequence and annealed.The resulting fragments were ligated into short hairpin RNA(shRNA) expressing plasmid.The plasmids expressing NgR-GFP fusion protein and shRNA were co-transfected into AAV-293 cells.RNAi efficiency against NgR was observed under fluorescent microscope and calculated by Western blotting.Results The interference efficiency of one sequence was above 90%.Conclusion A plasmid mediated high RNAi efficiency against NgR is constructed successfully.