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Objective To simulate the chemical microenvironment of traumatic brain injury (TBI) under mild hypothermia, and investigate the effect of such microenvironment on umbilical cord mesenchymal stem cells (UCMSCs) in vitro.Methods Eighteen SD rats were allocated to shamoperated group, TBI group and mild hypothermia group according to the random number table, with 6 rats per group.Rat models of TBI were made by electric cortical contusion impactor.After systemic mild hypothermia (33℃) for 4 h, brain tissue homogenate extracts were harvested.Polyacrylamide gels mimicking the elastic modulus of brain were manufactured.Human UCMSCs were isolated and cultured on the gels, added with brain tissue extracts from each group.After 24 h, the apoptosis level of UCMSCs was checked, and the medium was changed with normal one.Cell growth and morphological changes in each group were given dynamic observation.Seven days later, cell immunofluorescence was implemented, with the differentiation level of each group estimated.Results Apoptotic rate in TBI group was 73.47％,significantly higher than 10.42％ in sham-operated group (P ＜0.01).While the apoptotic rate was 28.57％ in mild hypothermia group, indicating mild hypothermia significantly reversed the apoptosis of cells in TBI group (P ＜ 0.01).Cell immunofluorescence demonstrated rate of neuronal differentiation of UCMSCs in sham-operated group, TBI group and mild hypothermia group was 16.48％, 2.59％ and 11.83％ respectively.Mild hypothermia resulted in significantly improved neuronal differentiation of UCMSCs after TBI (P ＜ 0.05).Conclusions More apoptosis and lower neuronal differentiation ability are observed in UCMSCs in the chemical microenvironment after TBI.However, mild hypothermia significantly reverses the elevation of apoptosis and restores the neuronal differentiation capacity of UCMSCs after TBI.
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In recent years, the rapid development of stem cell transforming technology and regeneration pose great re?search value. With maturation of transformation induction, other cell lineages were shown to be able to transform into neu?rons. In addition, astrocytes are widely distributed in the gray and white matter of the central nervous system, whose exces?sive proliferation contribute to glial fibrillary scar formation, which is the key obstacle in recovery of neurological function. Therefore, the study of transforming astrocytes into neurons draw attention from many scientists. Astrocytes transformation not only prevent the formation of glial scar, but also generate new neurons. This article summarized relevant studies that re?port function of astrocytes and its transformation into neurons.
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Objective To investigate the effect of the overexpression of NeuroD1 on mediating transdifferentiation of spinal reactive astrocytes into neurons. Methods Spinal cord astrocytes were cultured from the SD rat, and reactive astrocytes were prepared by scratches treatment. Cells were divided into blank groups (NV group), control virus group (GFP group) and NeuroD1 virus group (NeuroD1 group). At 7 d after scratches treatment, GFP and NeuroD1 groups were infected with retroviruses carrying the GFP gene and and GFP gene plus NeuroD1 gene, respectively,whereas NV group was not infected with the virus. Twenty-four hours late, the culture medium were replaced by neuron conditioned medi?um. Cell morphology was examined at 1, 2, 3, 5, 7 and 14 d. DCX positive and NeuN positive cells were detected at 7 d and 14 d after infection by using immunofluorescence staining method, respectively. Results After replacement with the neuron conditioned medium, the nucleus was obviously plump, the cytoplasm was thin and neurites was reduced and ex?tended. Compared with the NeuroD1 group, neurites of NV group and GFP group were shorter with many branches and the nucleus was smaller. At 7 d after infection, cell morphology of NV group and GFP group gradually recovered, but cell morphology of NeuroD1 group did not. Compared with NV group and GFP group, NeuroD1 group had more DCX(9.84 ± 2.06%)and NeuN(8.25±2.78%)positive cells [F values 40.107 for DCX and 21.73 for NeuN (P<0.05)]. Conclusion The overexpression of NeuroD1 can mediate the transdifferentiation of spinal reactive astrocytes into neurons.
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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 investigate the effect of mild hypothermia on proliferation and differentiation of neural stem cells (NGCs) in hippocampal subgranular zone after traumatic brain injury (TBI) and the underlying mechanism.Methods SD rats were divided into sham-injured group (only left dura mater exposed),hypothermia group (sham injury + mild hypothermia therapy for 72 hours),TBI group (unilateral fluid percussion was used to generate severe TBI),and TBI + hypothermia group (TBI + mild hypothermia therapy for 72 hours) according to the random number table,with 8 rats per group.Hippocampal homogenates or brain tissues were harvested after BrdU (100 mg/kg) was intraperitoneally administered to rats once a day for 7 days postTBI.Expressions of BrdU and double cortin in hippocampal subgranular zone were respectively detected by immunohistochemical or immunofiuorescence staining.Level of Sirt1 (silence information regulatory proteins,Sirt1) in hippocampus was detected by Western blot.Results BrdU-and double cortin-positive cells in rat hippocampal subgranular zone greatly increased at 7 days after TBI in comparison with sham-injured group (P ＜ 0.01).Moreover,BrdU and double cortin in rat hippocampal subgranular zone in TBI + hypothermia group was significantly higherthan that in TBI group [(257.4 ± 34.3) vs (196.4 ± 23.8) ; (346.4 ± 42.2) vs (245.7 ± 33.2),P ＜0.01].Moreover,mild hypothermia reversed TBI-induced over-expression of Sirt1 [(0.62 ± 0.075) vs(1.18 ± 0.11),P ＜ 0.01].Conclusion Mild hypothermia therapy can promote proliferation andneuronal differentiation of NSCs in hippocampal subgranular zone after TBI and the possible mechanismmay relate to the inhibition of over-expression of Sia1.
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Objective To estimate whether the ligation of bilateral internal carotid artery in combination with one vertebral artery can lead to chronic cerebral hypoperfusion. Methods The sham operation, 2?VO and 3?VO rat models were subjected to the matching operation. Four weeks after operation,the cortical blood flow was determined. The learning and memory abilities were measured with Morris water maze test eight weeks later,then the rats were sacrificed to observe the morphological change of hippocampal CA1 region. Results Compared with the sham operation group((47±8.797)ml·min-1·100 g-1),the cerebral blood flow of 2?VO((24.30±8.999)ml ·min-1·100 g-1) and 3?VO((9.870±2.208)ml·min-1·100 g-1) were significantly decreased (P<0.01). Compared with the sham operation group((8.33±4.88)s),escape latencies of Morris water maze of 2?VO group ((14.78±7.84)s) and 3?VO group((14.86±7.96)s) in the fifth days also presented significantly increased (P<0.01),but rare difference between the two groups. Compared with the sham operation group[ (37.20±9.21) s, (10.01.±2.91)times],the target quadrant swimming time and crossing times of 2?VO group((20.13±5.80)s, (6.60±3.19)times) and 3?VO group((20.05±5.76)s,(6.55±2.59)times) in the fifth days also presented signifi?cantly decreased (P<0.01). There were distinct pathomorphology changes in hippocampal CA1 region of the two groups. Conclusion The ligation of bilateral internal carotid artery in combination with one vertebral artery can lead to chronic cerebral hypoperfusion,and can make the similar ethology representation with the 2?VO models.
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10.3969/j.issn.1008-9691.2013.03.005