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Objective To observe the changes of hippocampal NogoA-NgR1 signaling on postoperative cognitive function (POCD) in aged mice, and explore the potential underling mechanism.Methods Isoflurane anesthesia and laparotomy were applied to establish the POCD model.Forty aged male C57BL/6 mice were randomly divided into the following four groups (n=10): group O2+saline (group OS), group O2+NEP1-40 (group ON), group isoflurane anesthesia+laparotomy surgery+saline (group SS), and group isoflurane anesthesia+laparotomy surgery+NEP1-40 (group SN).Cannula placement was performed into lateral ventricle 7 days before the surgery.Animals were subjected to an administration of NEP1-40 (20 μg/2 μl) or isochoric saline via intracerebroventricular injection once daily for 8 consecutive days, injection was given from 2 h before isoflurane anesthesia to the last behavioral test.Open field test was performed at 5th d after operation.Contextual and cued fear conditioning training and testing were exhibited at 6th and 7th d after operation, respectively.The hippocampus was harvested 2 h after the behavioral test.Western blot was used to detect the expressions of NogoA, NgR1, RhoA, ROCK2 and GAP43.Golgi staining was applied to measure the changes of dendritic spines in hippocampal CA1 area.Results Compared with the groups OS and ON, the freezing time in the contextual fear conditioning test was significantly decreased, the contents of NogoA, NgR1, RhoA and ROCK2 were significantly increased, the content of GAP43 and the number of dendritic spine were significantly decreased in group SS (P<0.05).Compared with the group SS, the freezing time in the contextual fear conditioning test was significantly increased, the contents of RhoA and ROCK2 were significantly decreased, the content of GAP43 and the number of dendritic spine were significantly increased in group SN (P<0.05).Conclusion Over-activated of hippocampal NogoA-NgR1 signaling participated in the pathogenesis of POCD in aged mice.
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Objective To investigate the effects of inhibiting NgR on retinal ganglion cells density and synaptophysin expression of diabetic rat.Methods Thirty-two SD male rats were randomly divided into normal control group,diabetic group,siNgR group and scNgR group,8 rats in each group.Normal control group was given no any treatment.Diabetes model was induced by intraperitoneal injection of 50 mg · kg-1 streptozotocin in diabetic group,siNgR group and scNgR group,and the blood giucose more than 16.7 mmol · L-1 at 72 hours was set as the successfully model.The rats of siNgR group were intravitreally administrated with anti-NgR nucleotide and the rats of scNgR group intravitreally administrated with negative nucleotide.Eight weeks later,HE staining was conducted to detect density of retinal ganglion cell (RGC),immunofluorescence was used to evaluate the expression and distribution of synaptophysin (a marker of synaptic number).Relative expression of NgR and synaptophysin in retina were analyzed by Western blot.Results RGC density in normal control group,diabetes group,siNgR group and scNgR group were (624.33 ± 3.51) mm-2,(420.00 ± 2.65) mm-2,(621.67 ± 1.53) mm-2,(416.67 ± 2.52) mm-2,respectively.There was significant difference among four groups (F =5985.37,P ＜ 0.01).Compared with normal control group,RGC density in diabetes group and scNgR group were obviously decreased (all P ＜0.01),but siNgR group had no obviously change (P ＞ 0.05).The synaptophysin mainly expressed in the inner and outer network layer.Compared with normal control group,the positive expression of synaptophysin in diabetes group and scNgR group were decreased,but siNgR group had no obviously change.The relative expression of NgR in normal control group,diabetes group,siNgR group and scNgR group were (11.26 ±0.02) ％,(19.38 ± 0.10) ％,(11.17 ± 0.02) ％,(19.47 ± 0.31) ％,respectively.There was significant difference among four groups (F =2466.09,P ＜ 0.01).Compared with normal control group,the relative expression of NgR in diabetes group and scNgR group were obviously decreased (all P ＜ 0.01),but siNgR group had no obviously change (P ＞0.05).The relative expression of synaptophysin in normal control group,diabetes group,siNgR group and scNgR group were (35.76 ± 0.15) ％,(25.47 ± 0.36) ％,(35.28 ± 0.12) ％,(25.03 ± 0.75) ％,respectively.There was significant difference among four groups (F =583.70,P ＜ 0.01).Compared with the normal control group,the expression of synaptophysin in diabetic group and scNgR group were decreased increased (all P ＜ 0.01),while there was no significant difference in siNgR group (P ＞ 0.05).Conclusion Inhibiting the expression of NgR in the retina of diabetic rats can help to restore the number of synapses and protect the damaged RGC.
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NgR1, a Nogo receptor, is involved in inhibition of neurite outgrowth and axonal regeneration and regulation of synaptic plasticity. P19 embryonal carcinoma cells were induced to differentiate into neuron-like cells using all trans-retinoic acid and the presence and/or function of cellular molecules, such as NgR1, NMDA receptors and STAT3, were examined. Neuronally differentiated P19 cells expressed the mRNA and protein of NgR1, which could stimulate the phosphorylation of STAT3 when activated by Nogo-P4 peptide, an active segment of Nogo-66. During the whole period of differentiation, mRNAs of all of the NMDA receptor subtypes tested (NR1, NR2A-2D) were consistently expressed, which meant that neuronally differentiated P19 cells maintained some characteristics of neurons, especially central nervous system neurons. Our results suggests that neuronally differentiated P19 cells expressing NgR1 may be an efficient and convenient in vitro model for studying the molecular mechanism of cellular events that involve NgR1 and its binding partners, and for screening compounds that activate or inhibit NgR1.
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Axons , Central Nervous System , Embryonal Carcinoma Stem Cells , Mass Screening , N-Methylaspartate , Neurites , Neurons , Phosphorylation , Plastics , Receptors, N-Methyl-D-Aspartate , Regeneration , RNA, Messenger , TretinoinABSTRACT
Objective To observe-the different effects of 2 doses recombinant human granulocyte colony-stimulating factors (rhG-CSF) on Nogo receptor(NgR) expression in the brain tissue of neonatal rats after hypoxic-ischemic brain damage(HIBD) at different times in order to reveal the neuroprotective effects of rhG-CSF.Methods Seven-day neonatal Sprague-Dawley(SD) rats were randomly divided into 4 groups by drawing method:sham operation group,model group,low-dose rhG-CSF group and high-dose rhG-CSF group,24 rats in each group.Then each group was divided into 4 subgroups (6 rats in each subgroup)and all rats were exterminated at different times after HIBD(1 d,3 d,7 d and 14 d).In the low-dose rhG-CSF group and high-dose rhG-CSF group,the rats were given daily doses of rhG-CSF 50 μg/kg,100 μg/kg respectively for 7 days by subcutaneous injection immediately after the molding(total 7 injections).In model group,rats received an injection of same amount of 9 g/L saline.In sham operation group,rats received no special treatment.Brain tissues of rats from each group were collected at different time points.The expressions of NgR protein and NgR mRNA in the left brain tissue were detected by immunohistochemistry and real-time fluorescent quantitative polymerase chain reaction (PCR).Results Immunohistochemistry:NgR proteins were constitutively expressed in the cerebral cortex in sham operation group at each time point;compared with sham operation group,the expressions of NgR in model group were increased markedly at each time point (135.67 ± 16.63,173.98 ± 17.82,234.00 ± 14.70,319.59 ± 25.22),and the differences were statistically significant(all P ＜ 0.01);compared with model group,the expressions of NgR in the cerebral cortex in low-dose rhG-CSF group (134.35 ± 8.89,109.04 ± 12.62,75.99 ± 13.39) and high-dose rhG-CSF group (81.38 ± 12.25,80.14 ± 10.50,72.58 ± 13.66) on the 3rd,7th,14th day were reduced significantly (all P ＜ 0.01).Compared with low-dose rhG-CSF group,the protein expressions of NgR in the high-does rhG-CSF group were decreased faster,and had the marked difference on the 3rd,7th day (P ＜ 0.05).Real-time fluorescent quantitative PCR:compared with the sham operation group,the expressions of NgR mRNA increased gradually in the cerebral cortex in the model group (1.34 ± 0.24,1.88 ± 0.27,2.88 ± 0.84,4.26 ± 0.86),the differences in NgR mRNA expression were statistically significant at different times(all P ＜ 0.05) ; compared with model group,the expressions of NgR mRNA in low-dose rhG-CSF group on the 7th (1.08 ± 0.30),14th day (0.93 ± O.26) and high-dose rhG-CSF group on the 3rd (0.61 ± 0.10),7th (0.56 ± 0.28),14th day (0.47 ± 0.12) were significantly different (all P ＜ 0.05).The expressions of low-dose group and high-dose group were reduced gradually.The NgR mRNA expression reduced more quickly in the high-dose group than in the low-dose rhG-CSF group and had substantial difference between two groups in 3 days (P ＜ 0.05).Conclusions The findings suggest that rhG-CSF intervention can reduce the expressions of NgR in the brain tissues of neonatal rats after HIBD,and low-dose rhG-CSF also has neuroprotective effect,but it could be weaker than high-dose rhG-CSF.
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Objective To observe the dynamic expression of Nogo receptor (NgR) in spinal cord of rats after spinal cord injury. Meth-ods 108 Sprague-Dawley rats were randomly assigned into normal group, sham operated group and model group, with 36 rats in each group. The model of spinal cord injury was established with the modified Allen's method. The rats were killed 24 h, 3 days, 7 days and 14 days re-spectively after intervention (9 rats from each group), and expression of NgR in the spinal cord tissue of the rats was detected with immuno-histochemistry and Western blotting, and expression of NgR mRNA was detected with fluorescence quantitative PCR. Results There was no significant change in the expression of NgR in the normal group and the sham operated group (P>0.05). The expression of protein and mRNA of NgR was less in the model group 24 h after modeling, dropped to the lowest on the 3rd day, then rapidly peaked on the 7th day, and gradually declined on the 14th day after spinal cord injury. Compared with the normal group, there were significant differences in ex-pression of NgR in immunohistochemistry and Western blotting in the model group at each time point after spinal cord injury (P<0.05). Compared with the sham operated group, there were significant differences in expression of NgR mRNA in the model group at each time point after spinal cord injury (P<0.01). Conclusion The expression of NgR and mRNA peaks on the 7th day after spinal cord injury in the rats, and maintains at high level for a long time, which may associated with the difficulty of axonal regeneration after spinal cord injury.
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Objective To investigate the effect of Nogo receptor on the apoptosis of retinal ganglion cell (RGC) in di-abetic rats, and the potential mechanism thereof. Methods Thirty diabetic model rats were induced by intraperitoneal ad-ministration of streptozotocin. Model rats were randomly divided into control group, diabetes mellitus (DM) group, siNgR group and siRNA control group (n=10 for each group). Diabetic rats in siNgR group were intravitreally administrated with No-go receptor antisense nucleotide. Diabetic rats in siRNA control group were intravitreally administrated with negative nucleo-tide. One month after diabetes onset, colocalization of Nogo receptor and Brn3a (marker of RGC) was observed by immunohis-tochemistry. The apoptosis of RGC was detected by TUNEL staining. The level of retinal malondialdehyde (MDA) was ob-served with kit, and the expressions of Nogo receptor and caspase-3 were detected with Western blot assay. Results It was found that the Nogo receptor was highly expressed in RGC. The levels of retinal MDA were (3.68±0.47), (8.07±1.24), (7.54± 1.53) and (5.12 ± 0.62) μmol/g protein for control group, DM group, siRNA control group and siNgR group. The apoptotic rates of RGC were (5.1 ± 0.2)%, (49.3 ± 2.7)%, (45.6 ± 1.8)%and (12.4 ± 0.6)%respectively. The expressions of Nogo receptor were (0.18 ± 0.07)%, (0.45 ± 0.12)%, (0.40 ± 0.09)%and (0.16 ± 0.09)%. The expressions of caspase-3 were (0.16 ± 0.05)%, (0.40±0.18)%, (0.42±0.12)%and (0.17±0.08)%. Compared with control group, there was significant increase in apoptosis of RGC, significantly up-regulated expressions in Nogo receptor and caspase-3, and significantly increased level of MDA in DM group and siRNA control group(P<0.05). Compared with DM group, there were decreased apoptotic rate of RGC, de-creased expressions of Nogo receptor and caspase-3, and decreased level of retinal MDA in siNgR group (P<0.05). Conclu-sion The increased level of Nogo receptor induces oxidative stress and up-regulation of caspase-3 in diabetic retina, play-ing an important role in the apoptosis of RGC.
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Objective: To construct a recombinant lentivirus harboring RNAi sequence targeting rat nogo receptor gene and to observe its infection efficiency of 293T cells. Methods: Lentivirus shuttle plasmid containing siNgR199 cDNA was constructed by gene engineering and was used to transfect 293T cells in the presence of packaging plasmids. Forty-eight hours later the supernatant was collected and the titer of virus was determined. The recombinant lentivirus and the standard lentivirus were used to transfect 293T cells at 1 MOI,3 MOI,5 MOI,10 MOI and 20 MOI. Polymerase chain reaction (PCR) was used to detect the recombinant vector; enhanced green fluorescent protein (EGFP) expression was used to determine the titer and the infection rate of the recombinant lentivirus under fluorescent microscope. Results: Restriction endonuclease and PCR analysis confirmed that the siNgR199 cDNA was successfully inserted into the lentivirus vector. The titer of the recombinant lentivirus harboring siNgR199 was 1×108 ifu/ ml and the best MOI was 3. Conclusion: The recombinant lentivirus containing siNgR199 gene has been successfully constructed, which lays a foundation for future axon regeneration in treatment of spinal cord injury.
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@#Nogo receptor is specific inhibiting factor conjuncted with myelin of central nervous system(CNS).After spinal cord injury,oligodendroglial cell and Nogo-A released from myelin inside cells inhibited the axonal regeneration.To analyze the intervention for Nogo receptor through molecule outside cell,information inside cell and gene,make clearing the inhibiting action of myelin-associated inhibiting factor-1,provide new thoughts and methods about axonal regeneration after spinal cord injury.
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@# Myelin of the adult mammalian central nervous system(CNS)has been attributed to affect nerve structural plasticity and suppress regeneration of nerve fibers.Nogo-A is possibly the best characterized of a variety of neurite growth inhibitors in CNS myelin.Neutralizing its activity results in improved axon regrowth and functional recovery in experimental spinal cord injury(SCI)models of animals.Nogo-A and its receptors,especially Nogo-66 receptor(NgR),p75 neurotrophin receptor(p75NTR),and LINGO-1 increasingly become the hot spot in the study of SCI repair,and have become the major targets for therapeutic intervention to promote axon regeneration after SCI.Inhibition of Nogo-A and its receptors NgR/p75NTR/LINGO-1 may be promote the regeneration of axon and maximize functional recovery after SCI.
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Objective To explore the possible effect of Nogo-A receptor antagonist NEP1-40 on neurite outgrowth in neonatal rats with hypoxic-ischemic brain damage(HIBD).Methods Sixty-four healthy Wistar rats were randomly divided into 8 groups at 2 different time points(6 h,24 h):control group,HIBD group,NEP1-40 group and ganglioside group(GM-1 group).Rats of control group and HIBD group were injected with saline(0.25 mL/kg)by intraperitoneal injection,while rats of NEP1-40 group and GM-1 group were administrated with 10 mg/(kg?d) NEP1-40 and 20 mg/(kg?d) GM-1 on request in each group,respectively.Immunohistochemical staining was adopted to detect the Nogo-A-positive cells,and ultrastructural changes of neuron and axon were observed by transmission electron microscopy.SPSS 13.0 statistical software was used to run One-Way ANOVA tests on the data presented.Results The Nogo-A-positive cells at 2 time points in rats of HIBD group were significantly higher than those of control group(t=7.63,15.13 Pa0.05),while the Nogo-A-positive cells in GM-1 group at 24 h was lower than that of HIBD group(t=4.25 P
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Objective To explore the changes of Nogo-A mRNA and Nogo receptor(Ng-R) mRNA expressions in hippocampus of immature rats after febrile seizures(FS).Methods One hundred and twenty-seven male SD rats(15 days) were randomly divided into control group(immersed in 37 ℃ water,n=40)and hyperthermia treated group(immersed in 44.5 ℃ water,n=87).The latter was further divided into febrile control group(n=42) and FS group(n=45) according to whether seizures occurred or not.Each group was further divi-ded into 5 groups according to different therapies(1,3,5,7,10 times treatment).Then 5 cases of the 8 rats were randomly used to observe the expressions of Nogo-A mRNA,Ng-R mRNA in hippocampus by using reverse transcriptase polymerase chain reaction(RT-PCR).Three of the 8 rats were randomly used to observe the changes of neurons and mossy fiber sprouting(MFS) in hippocampus by Nissl and Timm staining.Results 1.No seizures occurred in normal control group.Seizures occurred in 2 rats of febrile control group.In FS group,various seizures occurred such as nodding spasms,tonic seizures,clonic seizures and tonic-clonic seizures,2 rats died of drowning and 3 rats died of status epilepticus.2.The expressions of Nogo-A mRNA,Ng-R mRNA in the immature rats′ hippocampus became up-regulated after the 7th and 10th seizure,significantly higher than those of the other 2 control groups(Pa
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OBJECTIVE: After ischemic stroke, partial recovery of function frequently occurs and may depend on the plasticity of axonal connections. Here, we examine whether blockade of the Nogo/NogoReceptor(NgR) pathway might enhance axonal sprouting and thereby recovery after focal brain infarction. METHODS: Adult male Sprague Dawley rats weighing 250~350g were used. Left middle cerebral artery occlusion(MCAO) was induced with a intraluminal filament. An osmotic minipump (Alzet 2ML4, Alza Scientific Products, Palo Alto, CA) for the infusion of NgR-Ecto(310)-Fc to block Nogo/NgR pathway was implanted 1 week after cerebral ischemia. Prior to induction of ischemia, all animals received training in the staircase and rotarod test. Two weeks after biotin dextran amine injection, animals were perfused transcardially with PBS, followed by 4% paraformadehyde/PBS solution. Brain and cervical spinal cord were dissected. Eight coronal sections spaced at 1 mm intervals throughout the forebrain of each animal were stained with cresyl violet acetate for determination of infarction size. Images of each section were digitized and the infarct area per section was measured with image analysis software. RESULTS: Histological examination at 11 weeks post-MCAO demonstrates reproducible stroke lesions and no significant difference in the size of the stroke between the NgR(310)Ecto-Fc protein treated group and the control group. Behavioral recovery is significantly better and more rapid in the NgR-Ecto(310)-Fc treated group. Blockade of NgR enhances axonal sprouting from the uninjured cerebral cortex and improves the return of motor task performance. CONCLUSION: Pharmacological interruption of NgR allows a greater degree of axonal plasticity in response to stroke and this is associated with improved functional recovery of complicated motor tasks.