RESUMO
OBJECTIVE: To investigate the effects of exosomes of mouse astrocytes on the viability of neural stem cells. METHODS: Cultured and isolated the mouse astrocytes, and collected the cell supernatant for obtain the exosomes by ultracentrifugation. Neural stem cells that primary cultured for 2 nd to 6 th generation were obtained and treated with medium contained 0, 20, 40, 60 µg/mL of exosomes respectively. Screening the optimal exosome concentration for culturing neural stem cells by CCK-8 method. The optimal exosome concentration for neural stem cells was 40 µg/mL according to CCK-8 results. Then cells were intervened with 40 µg/mL of exosome in experimental group for 72 h, and the control group was added with the same volume of PBS. After intervention, the positive stem cells were labeled with EdU kit. Using the Transwell model, the number of nucleus stained by DAPI in the lower chamber in 40 µg/mL exosome treatment group and the control group were counted under a fluorescence microscope. RESULTS: â Identification of astrocyte exosomes: The successful obtain of exosomes of cell supernatant were confirmed by techniques such as electron microscopy, Western blot, exosome concentration and particle size measurement. â¡ CCK8 experiment: As the increasement of the concentration of exosomes, cell proliferation of primary neural stem cells gradually increased. Compared with the control group, proliferation of the cells in 40 µg/mL and 60 µg/mL exosome treatment groups was significantly enhanced, but there was no significant difference between the two groups. So, 40 µg/mL was selected as the best intervention concentration. ⢠EdU detection: Number of EdU positive labeled cells in the 40 µg/mL exosome group was higher than that in the control group (P<0.05). ⣠Transwell experiment: In the Transwell model, more neural stem cells in the 40 µg/mL exosome group migrated from the upper layer to the lower layer of the Transwell membrane, and the number was higher than that of the control group (P<0.05). CONCLUSION: Mouse astrocyte exosomes can improve the viability of neural stem cells.
Assuntos
Astrócitos , Exossomos , Células-Tronco Neurais , Animais , Astrócitos/fisiologia , Proliferação de Células , CamundongosRESUMO
OBJECTIVE: To study the effect of bone marrow mesenchyml stem cell (BMSC) exosomes (Exo) on the proliferation and migration of brain microvascular endothelial cells in rats. METHODS: BMSCs were extracted from rats and identified. The BMSCs were co-cultured with bEnd.3 cells in Transwell chamber for 24 h (BMSCs group). Extracted and identified the BMSCs exosomes (BMSC-Exo). Observed and qualitatively evaluated the cells' abilities on swallowing the BMSC-Exo under a fluorescence microscope. The optimal work concentration of BMSC-Exo was selected by detecting the cell vitality under different BMSC-Exo concentrations by CCK8 method. bEnd.3 cells were co-cultured with BMSC-Exo for 24 h (BMSC-Exo group). bEnd.3 cells cultured alone was set as control group. The proliferation and migration of bEnd.3 cells in the three groups were respectively detected by EDU and cell scratching experiment after 24 h of culture. RESULTS: Flow cytometry showed that P3 BMSCs were CD90 and CD29 positive and CD45 negative, with osteogenic differentiation and adipogenesis differentiation, indicating the extracted BMSCs high purity. The BMSC-Exo under transmission electron microscopy was round-shaped with a diameter of about 100 nm; NTA analysis found the diameter distribution of BMSC-Exo ranged from 50 to 600 nm, with a peak size of 150 nm. Immunofluorescence showed that the endothelial cells could swallow BMSC-Exo. CCK8 showed that supplement of 20 µg/mL BMSC-Exo had the best effect on cell proliferation. EDU results showed that BMSCs group and BMSC-Exo group could promote the proliferation of bEnd.3 cells compared with the control group (P<0.05), and there was no difference between BMSCs group and BMSC-Exo group (P>0.05). Cell scratch test showed that the cell mobility of the BMSC-Exo group was higher than that of the control group (P<0.05), but there was no significant difference between the BMSC-Exo group and the BMSCs group (P>0.05). CONCLUSION: BMSC-Exo can replace BMSCs in effectively promoting the proliferation and migration of cerebral microvascular endothelial cells, which provide a new potential treatment for angiogenesis after stroke.
Assuntos
Exossomos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Animais , Células da Medula Óssea , Encéfalo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Células Endoteliais , Osteogênese , RatosRESUMO
Modified constraint-induced movement therapy (mCIMT) has shown beneficial effects on motor function improvement after brain injury, but the exact mechanism remains unclear. In this study, amplitude of low frequency fluctuation (ALFF) metrics measured by resting-state functional magnetic resonance imaging was obtained to investigate the efficacy and mechanism of mCIMT in a control cortical impact (CCI) rat model simulating traumatic brain injury. At 3 days after control cortical impact model establishment, we found that the mean ALFF (mALFF) signals were decreased in the left motor cortex, somatosensory cortex, insula cortex and the right motor cortex, and were increased in the right corpus callosum. After 3 weeks of an 8-hour daily mCIMT treatment, the mALFF values were significantly increased in the bilateral hemispheres compared with those at 3 days postoperatively. The mALFF signal values of left corpus callosum, left somatosensory cortex, right medial prefrontal cortex, right motor cortex, left postero dorsal hippocampus, left motor cortex, right corpus callosum, and right somatosensory cortex were increased in the mCIMT group compared with the control cortical impact group. Finally, we identified brain regions with significantly decreased mALFF values at 3 days postoperatively. Pearson correlation coefficients with the right forelimb sliding score indicated that the improvement in motor function of the affected upper limb was associated with an increase in mALFF values in these brain regions. Our findings suggest that functional cortical plasticity changes after brain injury, and that mCIMT is an effective method to improve affected upper limb motor function by promoting bilateral hemispheric cortical remodeling. mALFF values correlate with behavioral changes and can potentially be used as biomarkers to assess dynamic cortical plasticity after traumatic brain injury.
RESUMO
Paired associative stimulation has been used in stroke patients as an innovative recovery treatment. However, the mechanisms underlying the therapeutic effectiveness of paired associative stimulation on neurological function remain unclear. In this study, rats were randomly divided into middle cerebral occlusion model (MCAO) and paired associated magnetic stimulation (PAMS) groups. The MCAO rat model was produced by middle cerebral artery embolization. The PAMS group received PAMS on days 3 to 20 post MCAO. The MCAO group received sham stimulation, three times every week. Within 18 days after ischemia, rats were subjected to behavioral experiments-the foot-fault test, the balance beam walking test, and the ladder walking test. Balance ability was improved on days 15 and 17, and the foot-fault rate was less in their affected limb on day 15 in the PAMS group compared with the MCAO group. Western blot assay showed that the expression levels of brain derived neurotrophic factor, glutamate receptor 2/3, postsynaptic density protein 95 and synapsin-1 were significantly increased in the PAMS group compared with the MCAO group in the ipsilateral sensorimotor cortex on day 21. Resting-state functional magnetic resonance imaging revealed that regional brain activities in the sensorimotor cortex were increased in the ipsilateral hemisphere, but decreased in the contralateral hemisphere on day 20. By finite element simulation, the electric field distribution showed a higher intensity, of approximately 0.4 A/m2, in the ischemic cortex compared with the contralateral cortex in the template. Together, our findings show that PAMS upregulates neuroplasticity-related proteins, increases regional brain activity, and promotes functional recovery in the affected sensorimotor cortex in the rat MCAO model. The experiments were approved by the Institutional Animal Care and Use Committee of Fudan University, China (approval No. 201802173S) on March 3, 2018.