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Background Aluminum can induce irreversible structural and synaptic functional damage, and the associated mechanism may be related to the neurite damage regulated by glycogen synthase kinase-3β (GSK-3β)/collapsin response mediator protein 2 (CRMP2). Objective This experiment is conducted to investigate the effect of aluminum-maltolate [Al(mal)3] on primary hippocampal neuron neurites in mice, and reveal the role of GSK-3β-CRMP2 in this process. Methods The hippocampus of newborn ICR mice (≤ 24 h old) was used for primary neuronal cultures. On the 5th day in vitro (DIV5), neuron purity detection were performed by confocal laser scanning microscopy. On DIV7, the neurons were transfected with lentiviral vector-mediated mNeonGreen. On DIV10, the neurons with mNeonGreen fluorescence in good growth state were treated with Al(mal)3. The stage I experimental groups were blank control group, maltol group, 10 µmol·L−1 Al group, 20 µmol·L−1 Al group, and 40 µmol·L−1 Al group. Then 20 µmol·L−1 Al was used to establish a model of neurite injury and for the intervention. The stage II experimental groups were blank control group, dimethyl sulfoxide (DMSO) group, Al (20 µmol·L−1) group, SB (GSK-3β inhibitor, 1 µmol·L−1), and SB (1 µmol·L−1)+Al (20 µmol·L−1) group. CCK-8 method was used to detect the viability of neurons. The primary hippocampal neurons of mice were scanned with high content analysis system at 0 h and 48 h after Al or SB treatment, and the density and length of neurites were analyzed. Western blotting was used to detect the expression and phosphorylation levels of CRMP2 and GSK-3β in primary hippocampal neurons of mice. Results The immunofluorescence results showed that the purity of primary neurons was more than 90%. Compared with the blank control group in stage I, the cell viability rates of the 10, 20, and 40 µmol·L−1 Al groups were decreased after 48h of Al(mal)3 treatment (P<0.05), while the cell viability rate of the maltol group had no significant change. There was no significant difference in cell viability rate among the DMSO group, the SB group, and the control group after 48h of SB treatment, and the viability rate of neurons in the SB+Al group was higher than that in the Al group (P<0.05) in stage II. The 48 h/0 h ratios of average number and length of neurites in the control group were 90.13%±11.70% and 113.24%±8.34%, respectively. The 48 h/0 h ratios in the Al group were 56.47%±16.36% and 62.06%±6.75%, respectively, which were lower than those in the control group (P<0.05). The 48 h/0 h ratios of average number of neurites in the SB group (99.03%±21.83%) was not significantly different from that in the control group, but the 48 h/0 h ratio of average length of neurites in the SB group (128.72%±15.39%) was higher than that in the control group (P<0.05). The 48 h/0 h ratios of average number (72.59%±10.89%) and length of neurites (93.84%±14.65%) in the SB+Al group were significantly increased compared with those in the Al group (P<0.05). Western blotting results showed that: There was no significant difference in GSK-3β protein level among all groups; compared with the control group (1.00±0.18), the protein level of p-GSK-3β in the Al group (0.45±0.05) was significantly decreased, and that in the SB group (1.32±0.23) was significantly increased; the protein level of p-GSK-3β in the SB+Al group (0.80±0.05) was significantly higher than that in the Al group (P<0.05). Compared with the control group (1.00±0.07), the CRMP2 protein level in the Al group (0.66±0.11) was significantly decreased (P<0.05), while that in the SB group (1.01±0.02) was not significantly changed. Compared with the control group (1.00±0.13), the p-CRMP2 protein level in the Al group (1.50±2.18) was significantly increased, and that in the SB group (0.62±0.09) was significantly decreased (P<0.05); the protein level of p-CRMP2 in the SB+Al group (1.28±0.24) was lower than that in the Al group (P<0.05). Conclusion Aluminum may activate GSK-3β, increase CRMP2 phosphorylation level, and damage neurite growth.
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Objective To investigate the regulation of microtubules by Nogo-A in the dorsal root ganglia during the inflammatory pain. Methods The ipsilateral paw withdrawal latency (PWL) was measured in wild type rats(WT, n = 12) and Nogo-A konck-out (Nogo-A KO) rats (n = 14) after complete Freunds adjuvant (CFA) injection. Western blotting and immunofluorescent staining were used to study the expression of microtubules and phosphorylated collapsin response mediator protein 2(p-CRMP2)in the dorsal root ganglion (DRG) of both groups. Results Knock out of Nogo-A in rats had'attenuated the CFA-induced inflammatory hyperalgesia. The acetylated tubulin was reduced, and the expression of p-CRMP2 was increased in the DRG of the Nogo-A KO rats. Conclusion Nogo-A is involved in the regulation of inflammatory heat hyperalgesia by promoting the microtubule polymerization via CRMP2 pathway.
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Objective To study the expression of collapsin response mediator protein 2 (CRMP-2) in the visual cortex of monocular form deprivation amblyopia rats.Methods Sixty-four 14-day-old rats were randomly divided into monocular deprivation amblyopia group and normal control group by random number table method.Right eyelid margin suture was performed at 14 days after birth in the monocular deprivation amblyopia group.Eight rats in the monocular deprivation amblyopia group and the normal control group were observed at 14,21,45 and 120 days after birth,respectively.Flash visual evoked potential (F-VEP) was used to dectect the latency and amplitude of P1 wave.The expression of CRMP-2 in visual cortex was observed by immunohistochemical method.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.This study protocol was approved by Ethic Committee of the University of South China (No.20140228).Results F-VEP results showed that the amplitudes of P1 were decreased and latent periods of P1 were prolonged in the monocular deprivation amblyopia group compared with the normal control group (t=16.760,P =0.000;t =-22.919,P =0.000).CRMP-2 expression levels in the visual cortex of monocular deprivation amblyopia groups and normal control groups were compared at different time points after birth,and the differences were statistically significant (Fgroup =8.855,P =0.010;Ftime =63.091,P =0.000).Compared with normal control groups,the expressions of CRMP-2 at the postnatal 21,45 and 120 days were obviously decreased in the monocular deprivation amblyopia groups,the differences were statistically significant (all at P＜0.05).Conclusions CRMP-2 may be involved in the occurrence and development of amblyopia.
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Background Collapsin response mediator protein-2 (CRMP-2) can promote the growth of axons,but CRMP-2 occurs hyperphosphorylation under the induction of cyclin-dependent kinase-5 (CDK5) after central nervous system injury,which leads to the collapse of the growth cone and hinders the repair of nervous system.Being a central nervous system tissue,whether the expressions of CRMP-2 and its phosphorylated protein (p-CRMP-2) change after optic nerve injury are rarely studied.Objective This study was to investigate the dynamic changes of CRMP-2 and p-CRMP-2 expressions in injured optic nerve tissue.Methods Forty-eight 8-or 9-week-old BALB/c mice were randomly divided into the sham operation group and postoperative 3-,7-and 14-day group.Optic nerves were exposed and clamped at retrobulbar 2 mm for 10 seconds in the right eyes during the surgery in the postoperative 3-,7-and 14-day groups,and the same operation was performed except the clamp of optic nerve in the sham operation group.The optic nerve tissue was obtained from the eyes 3,7 and 14 days after surgery.The relative expression levels of CRMP-2 mRNA and CRMP-2,p-CRMP-2 and CDK5 proteins in the tissue were detected by real-time fluorescence quantitative PCR and Western blot,respectively.The use and care of the experimental animals complied with the Regulation for the Administration of Affair Concerning Experimental Animals of Third Military Medical University.Results No significant differences were found in the expression levels of CRMP-2 mRNA and CRMP-2 protein among the sham operation group and postoperative 3-,7-and 14-day groups (CRMP-2 mRNA:F =2.971,P =0.097;C RMP-2 protein:F=1.202,P =0.370).The relative expression levels of p-CRMP-2 protein in the optical nerve were 0.001±0.000,0.064±0.003,0.136±0.005 and 0.346±0.012,and those of CDK5 protein were 0.440±0.009,0.723±0.011,0.874±0.015 and 0.952±0.019 in the sham operation group and postoperative 3-,7-and 14-day groups respectively,showing statistically significant differences among them (p-CRMP-2:F=445.600,P ＜ 0.001;CDK5:F=186.600,P＜0.001),and the relative expression levels of p-CRMP-2 and CDK5 protein were evidently higher in the optical nerve tissue in the postoperative 3-,7-and 14-day groups than those in the sham operation group (all at P＜0.01).Conclusions There are not significant changes in the expression level of CRMP-2 in the BALB/c mice after optic nerve injury.However,the expression levels of p-CRMP-2 and CDK5 proteins are gradually upregulated as the extending of injured time.
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OBJECTIVE: To investigate the effects of lithium chloride (LiCl) on neuroapoptosis in hippocampus induced by isoflurane and the relationships with GSK-3β/CRMP2 pathway in neonatal rats. METHODS: Sixty neonatal rats at postnatal day 7 were assigned randomly into air + NS group, air + LiCl group, Iso + NS group, Iso + LiCl2, Iso + LiCl4 and Iso + LiCl8 group. Rats in groups of air + NS and air + LiCl were exposed to air for 6 h. Rats in groups of Iso + NS, Iso + LiCl2, Iso + LiCl4 and Iso + LiCl8 were exposed to 0.75% isoflurane for 6 h. Rats in groups of Air + NS and Iso + NS were injectedintracerebroventricularlywith NS 5 μL separately 30 min before exposure to air or isoflurane. Rats in groups of air + LiCl, Iso + LiCl2, Iso + LiCl4 and Iso + LiCl8 were injected intracerebroventricularly with LiCl 5 μL separately 30 min before exposureto air or isoflurane. Rats in groups of Iso + LiCl2, Iso + LiCl4, Iso + LiCl8 and Air + LiCl were injected intracerebroventricularly with LiCl5 μL separately 30 min before exposure with the different concentrations(20, 40, 80, 80 mmol·L-1, respectively). At the end of exposure, the hippocampus of some rats were separated and proteins expression of cleaved caspase-3, phospho-GSK-3β(p-GSK-3β), GSK-3β, phospho-CRMP2 (p-CRMP2) and CRMP2 were detected by Western blot (n=5); other rats were perfused and their brain were embedded by paraffin 2 h after exposure, the neuroapoptosis in the hippocampus CA1 area was detected by TUNEL staining (n=5). RESULTS: Lithium chloride (80 mmol·L-1) significantly decreased the cleaved caspase-3 levels(P < 0.01) induced by isoflurane in hippocampus of neonatal rats. TUNEL positive cells in CA1 area were significantly decreased by 41.69%(P < 0.01) in group Iso + LiCl8 when compared with those in group Iso + NS. The protein expressionof p-GSK-3β and p-CRMP2 in group Iso + NS were significantly decreased and increased respectivelycompared to those in group air + NS. The ratio of p-GSK-3β/GSK-3β and p-CRMP2/CRMP2 in the hippocampal increased 41.03% (P < 0.05) and decreased 23.64%(P < 0.05) respectively in Iso + LiCl8 group when compared with Iso + NS group. CONCLUSION: Lithium chloride attenuates isoflurane-induced neurodegeneration through inhibiting hippocampal neuroapoptosis in neonatal rats. Inhibition of the activation of GSK-3β/CRMP2 signaling pathways may involve in the mechanisms.
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OBJECTIVE: To investigate the effects of dexmedetomidine(Dex) preconditioning on neuroapoptosis in hippocampus induced by isoflurane and the expression of CRMP2 proteins in neonatal rats. METHODS: Sixty neonatal rats at postnatal day 7 were assigned randomly into control group, Dex preconditioning control group, isoflurane group and isoflurane with Dex preconditioning groups which at dose of 25, 50 and 75 μg·kg-1. Rats in groups of isoflurane or control were exposed 0.75% isoflurane or air for 6 h. Rats in groups of Dex preconditioning were injected intraperitoneally with different doses of Dex 20 min before exposure and rats in other groups were injected intraperitoneally with 150 μL saline. At the end of exposure, the hippocampus of some rats were separated and proteins expression of cleaved caspase-3, phospho-CRMP2 (p-CRMP2), CRMP2 were detected by Western blot (n=6); other rats were perfused and their brains were embedded by paraffin, the neuronal apoptosis in hippocampus CA1 was detected by TUNEL staining 2 h after exposure (n=4). RESULTS: The number of TUNEL positive cells in hippocampus CA1 of rats in isoflurane group was increased by 434.99% (P<0.001) when compared with control group, Dex at dose of 75 μg·kg-1 significantly inhibited the increase of isoflurane-induced TUNEL positive cells by 71.87% (P<0.001). The expression of cleaved caspase-3 protein in isoflurane group increased by 90.20% (P<0.001) compared with control group, while Dex at dose of 25, 50 and 75 μg·kg-1 reversed isoflurane-induced increase of cleaved caspase-3 by 30.54% and 53.71%(P<0.05)and 96.71% (P<0.001)separately. Isoflurane increase the ratio of p-CRMP2/CRMP2 in the hippocampus by 119.41% (P<0.001), and didn't influence the expression of CRMP2; while Dex at dose of 50 and 75 μg·kg-1 inhibited isoflurane-induced increase of p-CRMP2/CRMP2 ratio by 63.75% (P<0.01) and 77.70% (P<0.01) and increased the expression of CRMP2 by 44.30% (P<0.05) and 62.13% (P<0.01). CONCLUSION: Dex Attenuates isoflurane-induced neurodegeneration through inhibitory effect of hippocampal neuroapoptosis in neonatal rats, decrease the ratio of p-CRMP2/CRMP2 and increase the expression of CRMP2 may be one of the mechanisms for neuroprotection and this Conclusion may provide the theoretic foundation for reasonable selection of anaesthetics.
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Objective To investigate whether conventional protein kinase C (cPKC ) βⅡ-interacting collapsin response mediator protein-2 (CRMP-2) provides neuroprotection against cerebral ischemic (I) injuries. Methods Male BALB/c mice were randomly divided into normoxic control (Nor) , HPC, Nor + Sham, HPC + Sham, Nor + I and HPC + I groups (n = 6 per group). Using our HPC and MCAO mouse models, we applied immunoprecipita-tion, two-dimensional electrophoresis and mass spectrometry to characterize cPKCβⅡ-interacting proteins and combined with SDS-PAGE and Western blot to quantitatively analyze CRMP-2 phosphorylation and degradation levels in the brain of mice after HPC and MCAO. Results The expression level of 10 cPKCβⅡ-interacting proteins changed obviously in cerebral cortex of HPC mice when compared with Nor group. One of these proteins, CRMP-2 protein level increased in particulate fraction and decreased in cytosolic fraction of cerebral cortex of HPC mice. CRMP-2 phosphorylation level in ischemic core (Ic) of cerebral cortex decreased significantly ( P < 0. 05 , n = 6) as compared with that of Nor + sham group, but CRMP-2 phosphorylation level in HPC +I group increased significantly as compared with that of Nor +I group ( P < 0. 05, n = 6). In ischemic cortex, CRMP-2 degradation (proteolysis) was observed as the appearance of 55 ku breakdown products (BDP). However, the CRMP-2 degradation level, BDPs products decreased significantly in penumbra ( P) of ischemic cortex from HPC +I group when we compared with that of Nor +I group (P < 0. 05, n = 6 ). Conclusion CRMP-2 is involved in attenuating the decrease of CRMP-2 phosphorylation in ischemic core and in inhibiting its degradation in penumbra of cerebral cortex of mice thereby to lessen the ischemic injuries.