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The Sonic hedgehog (SHH) signaling pathway plays a pivotal role in neurogenesis and brain damage repair. Our previous work demonstrated that the SHH signaling pathway was involved in the neuroprotection of cortical neurons against oxidative stress. The present study was aimed to further examine the underlying mechanism. The cortical neurons were obtained from one-day old Sprague-Dawley neonate rats. Hydrogen peroxide (H(2)O(2), 100 μmol/L) was used to treat neurons for 24 h to induce oxidative stress. Exogenous SHH (3 μg/mL) was employed to activate the SHH pathway, and cyclopamine (20 μmol/L), a specific SHH signal inhibitor, to block SHH pathway. LY294002 (20 μmol/L) were used to pre-treat the neurons 30 min before H(2)O(2) treatment and selectively inhibit the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. The cell viability was measured by MTT and apoptosis rate by flow cytometry analysis. The expression of p38, p-p38, ERK, p-ERK, Akt, p-Akt, Bcl-2, and Bax in neurons was detected by immunoblotting. The results showed that as compared with H(2)O(2) treatment, exogenous SHH could increase the expression of p-Akt by 20% and decrease the expression of p-ERK by 33%. SHH exerted no significant effect on p38 mitogen-activated protein kinase (p38 MAPK) pathway. Blockade of PI3K/Akt pathway by LY294002 decreased the cell viability by 17% and increased the cell apoptosis rate by 2-fold. LY294002 treatment could up-regulate the expression of the pro-apoptotic gene Bax by 12% and down-regulate the expression of the anti-apoptotic gene Bcl-2 by 54%. In conclusion, SHH pathway may activate PI3K/Akt pathway and inhibit the activation of the ERK pathway in neurons under oxidative stress. The PI3K/Akt pathway plays a key role in the neuroprotection of SHH. SHH/PI3K/Bcl-2 pathway may be implicated in the protection of neurons against H(2)O(2)-induced apoptosis.
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This study examined the effect of electro-acupuncture (EA) combined with transcranial magnetic stimulation (TMS) therapy at different time windows on learning and memory ability of rats with cerebral infarction and the underlying mechanism. Two hundred SD rats were randomly divided into four groups: normal group, sham-operated group, model group and EA+TMS group, and each group was then divided into five sub-groups in terms of the different time to start treatment post operation: 6, 12, 24, 48 and 72 h. Cerebral infarction models were established in the model and the EA+TMS groups by left middle cerebral artery occlusion/reperfusion (MCAO/R). After treatment for 14 d, the Morris water maze test was applied to examine the spatial learning and memory abilities of rats. In infarcted area, the expression of caspase-3 was immunohistochemically detected, and real-time fluorescent quantitative PCR was used to measure the expression of Bcl-2 mRNA. The results showed that in EA+TMS group compared with model group at the same treatment time windows, the escape latency was substantially shortened, the expression of caspase-3 was considerably decreased and the expression level of Bcl-2 mRNA significantly increased (P<0.05). In the EA+TMS sub-groups, the escape latency was shortest, the expression level of caspase-3 lowest, and the expression level of Bcl-2 mRNA highest at the treatment time window of 24 h. It was concluded that EA combined with TMS can promote neurological function of rats with cerebral infarction by increasing the expression level of Bcl-2 mRNA and decreasing the expression of caspase-3. The best time window is 24 h after perfusion treatment to ischemia.
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This study examined the ability of 1,2,3,4,6-penta-O-galloyl-β-D-glucose (β-PGG) to induce the expression of heme oxygenase-1 (HO-1) in the PC12 cells and its regulation in the PC12 cells. One week before treatment with the drug, nerve growth factor (NGF) was added to the cultures at a final concentration of 50 ng/mL to induce neuronal differentiation. After drug treatment, HO-1 gene transcription was analyzed by reverse transcription polymerase chain reaction (RT-PCR). Expression of HO-1 and NF-E2-related factor2 (Nrf2) and activation of extracellular signal-regulated kinase (ERK) and Akt were detected by Western blotting. The viability of the PC12 cells treated with different medicines was examined by MTT assay. The oxidative stress in the PC12 cells was evaluated qualitatively and quantitatively by DCFH-DA. The results showed that β-PGG up-regulated HO-1 expression and this increased expression provided neuroprotection against MPP(+)-induced oxidative injury. Moreover, β-PGG induced Nrf2 nuclear translocation, which was found to be upstream of β-PGG-induced HO-1 expression, and the activation of ERK and Akt, a pathway that is involved in β-PGG-induced Nrf2 nuclear translocation, HO-1 expression and neuroprotection. In conclusion, β-PGG up-regulates HO-1 expression by stimulating Nrf2 nuclear translocation in an ERK- and Akt-dependent manner, and HO-1 expression by β-PGG may provide the PC12 cells with an acquired antioxidant defense capacity to survive the oxidative stress.
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bThis study explored whether the transplantation of modified marrow stromal cells (MSCs) has angiogenic effects in a left middle cerebral artery occlusion infarction/reperfusion (MCAO I/R) rat model and preliminarily examined the mechanism of angiogenesis following cerebral infarction. MSCs were isolated by using a direct adherent method and cultured. Vascular endothelial growth factor (VEGF) was transfected into MSCs by employing the liposome transfection. The transfection efficiency was measured by the optical density method. The protein expression of VEGF gene before and after transfection was measured by Western blotting. SD rat model of transient occlusion of the left middle cerebral artery was established by using an approach of intra-luminal occlusion. Tetrazolium (TTC) and HE staining were performed to observe the cerebral infarction. ELISAs were used to measure the levels of VEGF in the rat cerebral tissues. The expression patterns of angiopoietin-2 (Ang-2) and CD34 in cells surrounding the area of infarction were immunohistochemistrically observed. Ang-2 protein expression in the tissue surrounding the area of infarction was measured by Western blotting. VEGF expression in the MSCs increased after transfection at a rate of approximately 28%±3.4%. ELISA showed that the expression of VEGF in the cerebral tissue was significantly increased after induction of infarction, peaking on the 4th day and decreasing to the levels of the sham surgery group (normal) within 7 to 10 days. The VEGF level was significantly higher at each time point in the VEGF-MSC and MSC groups compared to the model group. Moreover, the VEGF level was higher in the VEGF-MSC group than in the MSC group and stayed relatively high until the 10th day. The immunohistochemical results showed that 10 days after the infarction, the number of Ang-2 and CD34-expressing cells in the area surrounding the infarction was significantly higher in the VEGF-MSC group and the MSC group compared to the model group. Moreover, the VEGF level was higher in the VEGF-MSC group than the MSC group. A similar trend in Ang-2 protein expression was revealed by Western blotting. In the MCAO rat model transfected with modified MSCs over-expressing VEGF, compared to the MSC transplantation group, the concentration of VEGF was significantly increased in the brain tissue after cerebral infarction. In addition, the level of Ang-2 was up-regulated, with angiogenesis promoted, the blood supply to the areas surrounding the cerebral infarction increased, and neurological function improved. We are led to speculate that the synergistic effects of VEGF and Ang-2 may be responsible for the angiogenesis following cerebral infarction.
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The Sonic hedgehog (SHH) signaling pathway plays a pivotal role in neurogenesis and brain damage repair. Our previous work demonstrated that the SHH signaling pathway was involved in the neuroprotection of cortical neurons against oxidative stress. The present study was aimed to further examine the underlying mechanism. The cortical neurons were obtained from one-day old Sprague-Dawley neonate rats. Hydrogen peroxide (H(2)O(2), 100 μmol/L) was used to treat neurons for 24 h to induce oxidative stress. Exogenous SHH (3 μg/mL) was employed to activate the SHH pathway, and cyclopamine (20 μmol/L), a specific SHH signal inhibitor, to block SHH pathway. LY294002 (20 μmol/L) were used to pre-treat the neurons 30 min before H(2)O(2) treatment and selectively inhibit the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. The cell viability was measured by MTT and apoptosis rate by flow cytometry analysis. The expression of p38, p-p38, ERK, p-ERK, Akt, p-Akt, Bcl-2, and Bax in neurons was detected by immunoblotting. The results showed that as compared with H(2)O(2) treatment, exogenous SHH could increase the expression of p-Akt by 20% and decrease the expression of p-ERK by 33%. SHH exerted no significant effect on p38 mitogen-activated protein kinase (p38 MAPK) pathway. Blockade of PI3K/Akt pathway by LY294002 decreased the cell viability by 17% and increased the cell apoptosis rate by 2-fold. LY294002 treatment could up-regulate the expression of the pro-apoptotic gene Bax by 12% and down-regulate the expression of the anti-apoptotic gene Bcl-2 by 54%. In conclusion, SHH pathway may activate PI3K/Akt pathway and inhibit the activation of the ERK pathway in neurons under oxidative stress. The PI3K/Akt pathway plays a key role in the neuroprotection of SHH. SHH/PI3K/Bcl-2 pathway may be implicated in the protection of neurons against H(2)O(2)-induced apoptosis.
Subject(s)
Animals , Rats , Cerebral Cortex , Metabolism , Hedgehog Proteins , Metabolism , Neurons , Metabolism , Neuroprotective Agents , Metabolism , Oxidative Stress , Physiology , Phosphatidylinositol 3-Kinase , Metabolism , Proto-Oncogene Proteins c-akt , Metabolism , Rats, Sprague-DawleyABSTRACT
BACKGROUND: Transformation growth factor β1 (TGF-β1) can promote bone marrow mesenchymal stem cells (BMSCs) migration and proliferation, but the underlying mechanisms remain unclear.OBJECTIVE: To observe the invasiveness of TGF-β1 on BMSCs cultured in vitro, and to investigate regulatory effect on Snail and matrix metalloproteinase 2 (MMP-2) expression.METHODS: Rat BMSCs were isolated and cultured with density gradient centrifugalization and adherence method. The influence of different concentrations of TGF-β1 on the BMSC migration was detected using the modified Transwell chambers. Small interfering RNA for Snail gene was synthesized and transfected into BMSCs by liposomel before TGF-β1 was treated, and the expression of Snail and MMP-2 before and after transfection were measured by western blot assay.RESULTS AND CONCLUSION: The exogenous TGF-β1 can induce a dose-dependent increase in cell migration, which peaked at 2 μg/L. The expression levels of Snail mRNA and MMP-2 mRNA were significantly increased after 2 μg/L TGF-β1 treatment. Snail gene can effectively inhibit the expression of MMP-2 promoted by TGF-β1. Experimental findings indicate that TGF-β1 could increase the MMP-2 expression and then promote the BMSCs migration through the upregulation of the Snail expression.
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bThis study explored whether the transplantation of modified marrow stromal cells (MSCs) has angiogenic effects in a left middle cerebral artery occlusion infarction/reperfusion (MCAO I/R) rat model and preliminarily examined the mechanism of angiogenesis following cerebral infarction. MSCs were isolated by using a direct adherent method and cultured. Vascular endothelial growth factor (VEGF) was transfected into MSCs by employing the liposome transfection. The transfection efficiency was measured by the optical density method. The protein expression of VEGF gene before and after transfection was measured by Western blotting. SD rat model of transient occlusion of the left middle cerebral artery was established by using an approach of intra-luminal occlusion. Tetrazolium (TTC) and HE staining were performed to observe the cerebral infarction. ELISAs were used to measure the levels of VEGF in the rat cerebral tissues. The expression patterns of angiopoietin-2 (Ang-2) and CD34 in cells surrounding the area of infarction were immunohistochemistrically observed. Ang-2 protein expression in the tissue surrounding the area of infarction was measured by Western blotting. VEGF expression in the MSCs increased after transfection at a rate of approximately 28%±3.4%. ELISA showed that the expression of VEGF in the cerebral tissue was significantly increased after induction of infarction, peaking on the 4th day and decreasing to the levels of the sham surgery group (normal) within 7 to 10 days. The VEGF level was significantly higher at each time point in the VEGF-MSC and MSC groups compared to the model group. Moreover, the VEGF level was higher in the VEGF-MSC group than in the MSC group and stayed relatively high until the 10th day. The immunohistochemical results showed that 10 days after the infarction, the number of Ang-2 and CD34-expressing cells in the area surrounding the infarction was significantly higher in the VEGF-MSC group and the MSC group compared to the model group. Moreover, the VEGF level was higher in the VEGF-MSC group than the MSC group. A similar trend in Ang-2 protein expression was revealed by Western blotting. In the MCAO rat model transfected with modified MSCs over-expressing VEGF, compared to the MSC transplantation group, the concentration of VEGF was significantly increased in the brain tissue after cerebral infarction. In addition, the level of Ang-2 was up-regulated, with angiogenesis promoted, the blood supply to the areas surrounding the cerebral infarction increased, and neurological function improved. We are led to speculate that the synergistic effects of VEGF and Ang-2 may be responsible for the angiogenesis following cerebral infarction.
Subject(s)
Animals , Male , Rats , Angiopoietin-2 , Genetics , Metabolism , Bone Marrow , Metabolism , Pathology , Cerebral Infarction , Genetics , Metabolism , Pathology , Neovascularization, Pathologic , Genetics , Pathology , Rats, Sprague-Dawley , Stromal Cells , Metabolism , Pathology , Vascular Endothelial Growth Factor A , Genetics , MetabolismABSTRACT
This study examined the ability of 1,2,3,4,6-penta-O-galloyl-β-D-glucose (β-PGG) to induce the expression of heme oxygenase-1 (HO-1) in the PC12 cells and its regulation in the PC12 cells. One week before treatment with the drug, nerve growth factor (NGF) was added to the cultures at a final concentration of 50 ng/mL to induce neuronal differentiation. After drug treatment, HO-1 gene transcription was analyzed by reverse transcription polymerase chain reaction (RT-PCR). Expression of HO-1 and NF-E2-related factor2 (Nrf2) and activation of extracellular signal-regulated kinase (ERK) and Akt were detected by Western blotting. The viability of the PC12 cells treated with different medicines was examined by MTT assay. The oxidative stress in the PC12 cells was evaluated qualitatively and quantitatively by DCFH-DA. The results showed that β-PGG up-regulated HO-1 expression and this increased expression provided neuroprotection against MPP(+)-induced oxidative injury. Moreover, β-PGG induced Nrf2 nuclear translocation, which was found to be upstream of β-PGG-induced HO-1 expression, and the activation of ERK and Akt, a pathway that is involved in β-PGG-induced Nrf2 nuclear translocation, HO-1 expression and neuroprotection. In conclusion, β-PGG up-regulates HO-1 expression by stimulating Nrf2 nuclear translocation in an ERK- and Akt-dependent manner, and HO-1 expression by β-PGG may provide the PC12 cells with an acquired antioxidant defense capacity to survive the oxidative stress.
Subject(s)
Animals , Rats , Cell Death , Genetics , Cell Line, Tumor , Heme Oxygenase-1 , Genetics , Hydrolyzable Tannins , Pharmacology , MAP Kinase Signaling System , Genetics , PC12 Cells , Piperidines , Proto-Oncogene Proteins c-akt , Genetics , PyrazolesABSTRACT
This study examined the effect of electro-acupuncture (EA) combined with transcranial magnetic stimulation (TMS) therapy at different time windows on learning and memory ability of rats with cerebral infarction and the underlying mechanism. Two hundred SD rats were randomly divided into four groups: normal group, sham-operated group, model group and EA+TMS group, and each group was then divided into five sub-groups in terms of the different time to start treatment post operation: 6, 12, 24, 48 and 72 h. Cerebral infarction models were established in the model and the EA+TMS groups by left middle cerebral artery occlusion/reperfusion (MCAO/R). After treatment for 14 d, the Morris water maze test was applied to examine the spatial learning and memory abilities of rats. In infarcted area, the expression of caspase-3 was immunohistochemically detected, and real-time fluorescent quantitative PCR was used to measure the expression of Bcl-2 mRNA. The results showed that in EA+TMS group compared with model group at the same treatment time windows, the escape latency was substantially shortened, the expression of caspase-3 was considerably decreased and the expression level of Bcl-2 mRNA significantly increased (P<0.05). In the EA+TMS sub-groups, the escape latency was shortest, the expression level of caspase-3 lowest, and the expression level of Bcl-2 mRNA highest at the treatment time window of 24 h. It was concluded that EA combined with TMS can promote neurological function of rats with cerebral infarction by increasing the expression level of Bcl-2 mRNA and decreasing the expression of caspase-3. The best time window is 24 h after perfusion treatment to ischemia.
Subject(s)
Animals , Rats , Acupuncture Therapy , Methods , Apoptosis , Physiology , Cerebral Infarction , Learning , Physiology , Memory , Physiology , Neurons , Physiology , Rats, Sprague-Dawley , Transcranial Magnetic Stimulation , MethodsABSTRACT
Objective To investigate the effect of Exendin-4 (Ex-4) on ischemia/reperfusion (I/R) injury-induced apoptosis in primary rat cortical neurons and the possible underlying mechanisms.Methods Rat cortical neurons were cultured in vitro,identified by NES-immunohistological staining and immunofluorescence staining,and randomly divided into the following groups: control group,I/R group and Ex-4 group.RT-PCR was performed to establish the existence of active glucagon-like peptide-1 receptor (GLP-1R).ELISA was used to measure the neuronal cytoplasmic cAMP level. MTT was used to detect viability. Fluorescent DNA binding dye Hoechest 33258 was used to reveal apoptosis. C/EBP-homologous protein (CHOP) and growth arrest and DNA-damage-inducible gene 34 (GADD34) mRNAs were detected by real-time PCR. Results The apoptosis rate induced by ischemia 6 h/reperfusion 12 h was 77.0% ±5.3% and was decreased to 27.0% ± 3.5% after Ex-4 ( 0. 4 μg/ml ) treating ( t = 19. 462,P ＜ 0. 01 ).Levels of CHOP and GADD34 mRNA in cortical neurons increased since 4 h and peaked at 12 h after reperfusion. Ex-4 group showed a sharp elevation of levels of CHOP and GADD34 mRNA,peaking at 8 h reperfusion,and then tended to decrease.Conclusions Ex-4 has protective effect against rat cortical neurons injury induced by ischemia/reperfusion. The protective effect may be related to inhibition of ESR-related neuron apoptosis via regulation of unfolded protein response.
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Objective To study the effects of transcranial magnetic stimulation (TMS) on learning and memory, and angiogenesis and the dendritic structure of hippocampal CA3 pyramidal neurons after cerebral infarction. Methods Forty-eight male Sprague-Dawley rats were divided into a sham operated group, a model group and a TMS group (n = 16). Rat models of focal cerebral infarction were established with unilateral middle cerebral artery (MCA) suture occlusion in the model and TMS groups. The rats of the TMS group were given 4 weeks of TMS treatment beginning 1 day after the infarction (2 times per day, 30 pulses per time). Their learning and memory abilities were tested with a Y-maze. Angiogenesis and the dendritic structure of their hippocampal CA3 pyramidal neurons were detected after 4 weeks. Results Compared with the model group, learning and memory improved significantly in the TMS group. The average microvessel density of the hippocampus in the TMS group was significantly more than in the model group. The total length of apical dendrites of hippocampal CA3 pyramidal neurons in TMS group was significantly longer than in the model group. Conclusions The improved learning and memory observed following TMS treatment are likely to be related to changes in angiogenesis, the dendritic.structure of the hippocampal CA3 pyramidal neurons, and enhanced synaptic plasticity.
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Objective To study the effect of transcranial magnetic stimulation (TMS) on the rehabilitation of rats with cerebral infarction. Methods One hundred Sprague-Dawley rats were randomly divided into a normal group, a sham-operated control group, a model group and a TMS group with 25 rats in each group. A cerebral infarction model was established in the latter two groups by left middle cerebral artery occlusion (MCAO). TMS was started at either 12 or 24 hours after reperfusion, and sham-TMS was given to the first two groups at the same time points. The expression of Bcl-2 mRNA and BDNF mRNA were measured by RT-PCR after 14 days. Results Bcl-2 mRNA and BDNF mRNA were detected in all groups. The expression of Bcl-2 mRNA in the TMS-12 h group, and that of BDNF mRNA in the TMS-24 h group were significantly higher than in the other groups. Conclusions The expression of Bcl-2 mRNA and BDNF mRNA in the brains of rats after cerebral infarction peak when TMS is administered 12 h and 24 h after reperfusion, respectively. TMS might have protective and rehabilitative effects on rats after cere-bral infarct.
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Objective To study the protection and its mechanism of carbamylated erythropoietin (CEPO) on ischemic brain injury and to compare its function with erythropoietin (EPO).Methods Focal cerebral ischemia/reperfusion was induced by occlusion of the middle cerebral artery (MCAO) using the intraluminal filament technique.The expression of endothelial NO synthase (eNOS) and activated caspase-3 were detected with Western blot.The inducible NO synthase (iNOS) positive cells were detected by immunohistochemistry staining.The apoptotic cell was detected by TUNEL staining.Results The expression of eNOS, iNOS and activated caspase-3 in cerebral cortex significantly increased after MCAO.The influence of CEPO and EPO on eNOS in ischemic cortex were not significantly different.However, the expression of activated caspase-3 markedly dropped from 95.4%±16.7% in group NS to 43.5%±13.1% in group CEPO and 45.1%±11.2% in group EPO (t=5.99 and 6.13,P<0.01).Immunohistochemistry staining revealed iNOS positive cells in ischemic cortex was (3.1±1.9) cells/square, CEPO and EPO remarkably reduced them to (0.7±0.2) cells/square and (0.8±0.2) cells/square, respectively (t=3.08 and 2.95, P < 0.05).The apoptotic cells in ischemic cortex fell from (94.2±15.2) cells/square in group NS to (40.5±9.8) cells/square in group CEPO (t=7.27, P < 0.01), the anti-apoptosis by EPO was similar to CEPO.Conclusion CEPO and EPO have the similar function of anti-apoptosis by inhibiting expression of activated caspase-3 and iNOS.
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Objective To observe the effects of passive movement on the functional outcome after occlusion of the middle artery in the brain and reperfusion, and to explore the molecular mechanisms involved. Methods Cerebral infarction models were established in rats using left middle cerebral artery occlusion ( MCAO). The survivors were randomly divided into a passive movement group and a natural recovery group. There was also a sham-operated group and a normal group. Passive movement treatment (twice a day, twenty min per time) was started at different times after reperfusion. The expression of brain-derived neurotrophic factor (BDNF) and B cell lymphoma/leukemia-2 gene (Bcl-2) were determined using real-time PCRs. Results Expression of BDNF and Bcl-2 was detected a-round the infarction area in both groups. The expression of BDNF and Bcl-2 was highest in the sub-groups where passive movement was begun 24 or 48 h after the operation. Conclusions The expression of BDNF and Bcl-2 in the brain peaks when daily, moderate intensity passive movement is administered beginning 24 to 48 h after reperfusion. Passive movement might have a protective and rehabilitative effect after cerebral infarction.
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Objective To study the effect of electroacupuncture (EA) combined with transcranial magnetic stimulation (TMS) on the expression of the B-cell lymphoma/leukemia-2 gene (Bcl-2) and brain derived neurotrophic factor (BDNF) after cerebral infarction. Methods One hundred Sprague-Dawley rats were randomly and equally divided into a normal group, a sham-operated control group, a model group and an EA plus TMS group. A cerebral infarction model was established in the latter two groups using left middle cerebral artery occlusion (MCAO). Five-member subgroups of the EA plus TMS group were then treated at 6, 12, 24,48 and 72 hours after reperfusion. Sham EA plus TMS was given to similar sub-groups from the other groups at the same time points. The expression of Bcl-2 mRNA and BDNF mRNA were measured using a RT-PCR at the 14th day. Results Positive expression of Bcl-2 mRNA and BDNF mRNA was detected around the infarction in all groups. The average expression of both was significantly higher in the EA plus TMS group than in the model group. Bcl-2 mRNA peaked when the therapy was administered at 24 hours and BDNF mRNA at 48 hours.Conclusions The expression of Bcl-2 mRNA and BDNF mRNA is maximized when EA plus TMS is administered 24-48 hours after cerebral infarction. EA plus TMS does have protective and rehabilitative effects on rats after cerebral infarction.
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Objective To study the effect of mild brain hyothermia on cerebral ischemic injury. Methods Global cerebral ischemia was established by modified Pulsinelli 4-vessel occlusion model. Forty-eight Sprague-Dawley rats were divided into 4 group: a sham-operated group, a normothermia (37～38℃) ischemic group and a mild is-chemic hypothermia (31～32℃) group; the mild ischemic hypothermia was subdivided into 4 groups with the hypo-thermia lasting for 30 min, 60 min, 120 min and 240 min, respectively. After 240 rain of reperfusion following 20 min cerebral ischemia, the levels of nitric oxide products nitrite (NO2) ,endothelin-1 (ET1) , tumor necrosis fac-tor alpha (TNFα) and interleukin-1 beta (IL-1β) in brain tissue and the lactate dehydrogenase (LDH), aspartate aminotransferase(AST) , creatine kinase(CK) and its brain band isoenzyme (CK-BB) in plasma were measured. Results The levels of IL-1β,TNFα, ET1 and NO2. in brain tissue, and the amounts of LDH, AST, CK and CK-BB in serum were higher in normothermia ischemic group than those in sham-operated group (P <0.05). Mild hypother-mia lasting for 60 min to 240 min markedly decreased the levels of IL-1β, TNF-α, ET1 and NO2 in brain tissue, and the amounts of LDH, AST, CK and CK-BB in serum in normothermia ischemic group, when compared with normo-thermia ischemic group (P < 0.05 or P < 0.01). Mild hypothermia lasting for 30 min did not influence the content of IL-1β, TNF-α, ET1 and NO2 in brain tissue when compared with normothermia isehemia group (P > 0.05). Con-clusion Mild brain hypothermia post-ischemia can significantly suppress the inflammation response in ischemic brain tissue and stabilize the function of cell membrane. The best neuroprotection of mild brain hypothermia must be carried out immediately and last for more than 60 minutes.
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In order to investigate the transfer and expression of Snail gene in human bone mesenchymal stem cells (MSCs) and to study effects of Snail gene modification on the CXCR4 expression of human MSCs and their capacity of migration to SDF-1 in vitro, the plasmid PCAGGSneo-Snail-HA or the control vector of PCAGGSneo was transferred into the cells. Fluorescence activated cell sorting analysis, immunofluorescence staining and RT-PCR were used to study the expression of CXCR4 by MSCs. Chemotaxis assays were performed to evaluate the migratory capacity of MSCs-Sna and MSCs-neo to SDF-1 in vitro. For the blocking assay, CXCR4 blocking antibody was added into cell culture. CXCR4 expression was higher in MSCs-Sna than that in MSCs-neo (P < 0.05). Chemotaxis assays showed that SDF-1alpha stimulated migratory activity of MSCs-Sna more than MSCs-neo in vitro (P < 0.05). Moreover, the SDF-1alpha-induced migratory activity of MSCs-Sna was inhibited in a concentration-dependent manner by a CXCR4-blocking antibody. It was concluded that Snail enhanced expression of CXCR4 in MSCs, providing a plausible mechanism for Snail-mediated MSCs transmigration to damaged tissues in vivo where SDF-1 has been shown to be up-regulated as part of injury responses.
Subject(s)
Humans , Bone Marrow Cells , Cell Biology , Cell Movement , Genetics , Cells, Cultured , Chemokine CXCL12 , Metabolism , Mesenchymal Stem Cells , Cell Biology , Metabolism , Receptors, CXCR4 , Genetics , Metabolism , Snail Family Transcription Factors , Transcription Factors , Genetics , Transduction, GeneticABSTRACT
BACKGROUND:Neurotrophic factors have been conformed to have the effects of promoting the survival, differentiation and axonal regeneration of the injured neurons, so that the structural integrity of the injured spinal cord can be reserved as much as possible.OBJECTIVE: To observe the effects of nerve growth factor (NGF) on the neural regeneration and motor function following spinal cord injury in rats.DESIGN: A randomized controlled observation.SETTING: Department of Neurology, Union Hospital affiliated to Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: Thirty healthy adult male Wistar rats of common degree, were provided by the Experimental Animal Center, Tongji Medical College of Huazhong University of Science and Technology.METHODS:The experiment was carried out in the Department of Neurology, Union Hospital of Tongji Medical College,Huazhong University of Science and Technology. The 30 rats were randomly divided into NGF-treated group (n=15) and control group (n=15). ① T8 spinal injury was induced by the improved Allen method. ③ The improved Rivlin method (namely, slope test) was used to assess the motor function following spinal cord injury in rats before model establishment and at 3 days, 1, 2 and 3 weeks after treatment respectively. ④ At 2 days and 3 weeks postoperatively, 7 rats in the NGF-treated group and 8 rats in the control group were selated and the cross-sectional tissue(0.5 cm) of the injured spinal cord was removed, fixed in neutral formalin, dehydrated, and embedded with paraffin, and then cut into 5 μm sections.MAIN OUTCOME MEASURES:① The results of the slope test before model establishment and at 3 days,1, 2 and 3weeks after treatment respectively; ② Axonal count in white matter at 2 days and 3 weeks after model establishment.RESULTS:① The scores of slope test after spinal cord injury at 1, 2 and 3 weeks postoperatively in the NGF-treated group were obviously higher than those in the control group [(45.2±3.2), (51.2±3.8), (53.4±4.6)°; (45.2±3.2), (51.2±3.8),(53.4±4.6)°, P ＜ 0.05].② At 3 weeks after treatment, a few dispersal and heterogeneous NGF200 spots were observed in the white matter of rats in the control group, but there were plentiful and symmetrical NGF200 spots in the NGF-treated group. The axonal count in white matter at 3 weeks following spinal cord injury in the NGF-treated group was obviously higher than that in the control group (363.6±34.2, 187.5±32.1, P＜ 0.05).CONCLUSION:NGF can promote the neural regeneration following spinal cord injury to a certain extent, and help to im prove the functional recovery.
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The effect of transcranial magnetic stimulation (TMS) on the neurological functional recovery and expression of c-Fos and brain-derived neurotrophic factor (BDNF) of the cerebral cortex in rats with cerebral infarction was investigated. Cerebral infarction models were established by using left middle cerebral artery occlusion (MCAO) and were randomly divided into a model group (n=40) and a TMS group (n=40). TMS treatment (2 times per day, 30 pulses per time) with a frequency of 0.5 Hz and magnetic field intensity of 1.33 Tesla was carried out in TMS group after MCAO. Modified neurological severity score (NSS) were recorded before and 1, 7, 14, 21, and 28 day(s) after MCAO. The expression of c-Fos and BDNF was immunohistochemically detected 1, 7,14, 21, and 28 day(s) after infarction respectively. Our results showed that a significant recovery of NSS (P＜0.05) was found in animals treated by TMS on day 7, 14, 21, and 28 as compared with the animals in the model group. The positive expression of c-Fos and BDNF was detected in the cortex surrounding the infarction areas, while the expression of c-Fos and BDNF increased significantly in TMS treatment group in comparison with those in model group 7, 14, 21, and 28 days (P＜0.05) and 7,14, 21 days (P＜0.01) after infarction, respectively. It is concluded that TMS has therapeutic effect on cerebral infarction and this may have something to do with TMS's ability to promote the expression of c-Fos and BDNF of the cerebral cortex in rats with cerebral infarction.
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To study the protective effect of rosuvastatin on ischemic brain injury and its mechanism, focal cerebral ischemia/reperfusion was induced by occlusion of the middle cerebral artery (MCA) using the intra-luminal filament technique. The cerebral blood flow was monitored with laser-Doppler flowmetry (LDF). The slices of brain tissue were stained with cresyl-violet. The cerebral volume of infarction and edema were quantified with ImageJ software. The expressions of endothelial NO synthase (eNOS) and activated caspase-3 were detected with Western blot. The inducible NO synthase (iNOS) positive cells were immunohistochemically observed. The results demonstrated that rosuvastatin (20 mg/kg) could remarkably decrease infarct volume and cerebral edema after MCAO 90 min/reperfusion 24 h. Western blots showed that the expression of eNOS in cerebral cortex before and after ischemia was (100+/-43.3) %, (1668.9+/-112.2) % respectively (P<0.001), rosuvastatin significantly up-regulated the expression of eNOS in non-ischemic cortex (P<0.001), whereas in ischemic cortex of rosuvastatin group the expression of eNOS was (1678.8+/-121.3) %. There was no expression of activated caspase-3 in non-ischemic cortex, nonetheless the expression of activated caspase-3 increased after ischemia, and rosuvastatin significantly diminished it (P<0.01). Immunohistochemistry revealed no iNOS-positive cells in non-ischemic brain area, while in ischemic brain area the number of iNOS positive cells went up, and rosuvastatin could significantly reduced them. Consequently, the mechanisms of rosuvastatin's neural protection on ischemic brain injury are to enhance expression of eNOS, to inhibit expression of iNOS and activated caspase-3.