| Objective To explore whether Salvia Miltorrhiza can induce mesenchymal stem cells (MSC) with optimized proposal to differentiate into neuron-like cells characterized with neurophysiological function or not.Method Passage 4~5 MSC in good shape were cultured for preinduction for 24 h in culture media containing 10% Fetal Bovine Serum (FBS) and 10 ng/ml basic Fibroblast Growth Factor (bFGF) , then induced with FBS-free culture media but containing 60 mg/L Salvia Miltorrhiza and 10ng/ml bFGF (induction media) for 2 h. After that 10% FBS was added to induce the cells to dedifferentiate, about 24 h later, the culture media was replaced with induction media to induce the MSC again for 3 h, the procedure: differentiation-dedifferentiation was repeated for several times, the inducing time was prolonged for one more hour each time than the last time, observation under inverted phase contrast microscope, immunofluorocytochemistry and measurement of neurophysiological function were carried out after 5 h of the 4th time, respectively. Membrane potential (MP), Ca2+ influx, synapse function were detected with laser-scanning confocal microscope (LSCM), 50 mmol/L KCl acted as stimuli to evoke action potential, fluorescence excitation of DiBAC4(3), Fluo-3/AM and SynaptoRed C-2 was challenged by LSCM for the measurement of MP, Ca2+ influx and synapse cycle function.Result There was no significant change in MSC within the 24 h of preinduction, after 2 h of the first induction, MSC began contracting and extending processes, changing its form into neurons'. However most of cells were monopole and dipolar. After 6 h of the 5th induction, the cells became multipolar and the processes stretched out and extended to form complicated net. Immunocytofluorochemistry presented that the ratio of Nestin expression in MSC without any treatment was 41.6±3.3% ((x|-)±s, n=3), after 2 h of the first induction, the ratio of Nestin expression in the neuron-like cells was raised up to 98.1±1.5% ((x|-)±s, n=3), but there was no any expression of TUJ-1 and Neurofilament (NF). After 1 h of the 4th induction, the cells began expressed Synaptophysin, and the ratio of expression was (95.3±1.6) % ((x|-)±s, n=3), 5 h later, synaptophysin expression was more extensive. After 6 h of the 5th induction, the ratio of Nestin expression was 0, TUJ-1 was 96.1±2.5% ((x|-)±s, n=3), NF 96.6±2.4% ((x|-)±s, n=3), Synaptophysin 95.2±3.1%((x|-)±s, n=3), but there was no expression of GFAP. The study of MP demonstrated the voltage-sensitive fluorescence of cells enhanced as soon as the cells were stimulated with high concentration KCl solution, and physiology chart showed the curve rose shapely, but MSC not treated had no any response and the physiology curve kept at horizon. The study of Ca2+ influx showed before high K+ solution was added, there was almost no change in the basal fluorescence intensity, the curve chart showed the amplitude of [Ca2+]i changed little, when additional 50 mM K+ presented, the fluorescence intensity increased rapidly and dramatically, the [Ca2+]i curve raised sharply, the difference of the amplitude of [Ca2+]i was significant. The study of synapse cycle showed When the neuron-like cells were stimulated by 50 mM K+ at first time, synaptoRed-C2 anchored onto the membrane, the cells presented red fluorescence, then after the second excitation by high concentration K+, the fluorescence intensity of the cells decreased quickly, the curve of fluorescence mean intensity descended sharply.Conclusion Salvia Miltorrhiza can induce MSCs to differentiate into neuron-like cells efficiently and rapidly with optimized proposal and the induced cells have neurophysiological function. Objective To investigate the effect of salvia miltiorrhiza on migration and differentiation of endogenous neural stem cells (NSC).Method Each 6-day old rat was injected with bromodeoxyuridine (BrdU) intraperitoneally, then the animal model of newborn rat HIBD was established at the second day, and the rats with HIBD were injected with 60 mg/kg salvia miltiorrhiza for 5 days, the control group was injected with the same dose of normal saline (NS). The damage severity of the brain was detected by TTC staining after 3 d of HIBD. The expression of BrdU was determined by immunohistochemistry to mark the migration of neural stem cell after 2 weeks of HIBD. Double-label immunofluorohistochemistry of BrdU/Nestin, BrdU/GFAP, BrdU/TUJ1 was carried out to assess the neural differentiation of BrdU+ cells after 4 weeks of HIBD.Result TTC staining shew the necrosis degree in the rats with HIBD treated with salvia miltiorrhiza was slighter than that in the rats with HIBD treated with NS. The number of BrdU+ cells in the left and right hemicerebrum of the sham operated group was 214.86±14.07 ((x|-)±s, n=8) and 215.63±15.98 ((x|-)±s, n=8), respectively, there was no significant difference between two sides of hemicerebrum (P>0.05), that in the left and right hemicerebrum of the rats with HIBD treated with NS was 260.88±18.89 ((x|-)±s, n=8) and 44.38±7.65 ((x|-)±s, n=8), respectively, there was significant difference between two sides of hemicerebrum (P<0.05), that in the left and right hemicerebrum of the rats with HIBD treated with salvia miltiorrhiza was 317.25±22.66 ((x|-)±s, n=8) and 44.38±7.65 ((x|-)±s, n=8), there was significant difference between two sides of hemicerebrum (P<0.05), the total number of BrdU+ cells in the brain of the three groups was 430.50±25.12 ((x|-)±s, n=8), 335.75±24.12 ((x|-)±s, n=8), 361.63±27.02 ((x|-)±s, n=8), repectively, there was significant difference between the three groups (P<0.05), the proportion of BrdU+ cells in the left and right hemicerebrum of the sham operated group was 0.50±0.02 ((x|-)±s, n=8) and 0.50±0.02 ((x|-)±s, n=8), there was no significant difference (P>0.05), that in the left and right hemicerebrum of the rats with HIBD treated with NS was 0.22±0.03 ((x|-)±s, n=8) and 0.78±0.03 ((x|-)±s, n=8), there was significant difference (P<0.05), that in the left and right hemicerebrum of the rats with HIBD treated with salvia miltiorrhiza was 0.12±0.01 ((x|-)±s, n=8) and 0.88±0.01 ((x|-)±s, n=8), there was significant difference (P<0.05). The ratio of the expression of Nestin, GFAP and TUJ-1 of BrdU+ cells in the sham operated group was (77.75±5.65)% ((x|-)±s, n=8), (12.17±3.32)% ((x|-)±s, n=8) and (4.14±1.36)% ((x|-)±s, n=8), respectively, that in the rats with HIBD treated with NS was (63.19±5.37)% ((x|-)±s, n=8), (19.75±3.49)% ((x|-)±s, n=8) and (8.12±2.01)% ((x|-)±s, n=8), respectively, that in the rats with HIBD treated with salvia miltiorrhiza was (44.81±5.02)% ((x|-)±s, n=8), (23.93±2.88)% ((x|-)±s, n=8) and (22.12±2.50)% ((x|-)±s, n=8), respectively, there was significant difference between the three group (P<0.05). .Conclusion Salvia miltiorrhiza can protect the brain from the progressing hypoxic ischemia damage, and can promote the BrdU+ cells migrate to the zone of necrosis and enhance the ratio of neural differentiation. Objective To investigate a suitable time window of mesenchymal stem cells (MSC) transplantation for neonate rats with hypoxic ischemic brain damage (HIBD).Method The animal model of newborn rat HIBD was established, then the rats with hypoxic ischemic encephalopathy (HIE) were sacrificed at 4 time points (2 h,1 d,3 d,6 d) after HIBD, the fresh brain was taken out and olfactory bulb and cerebellum were removed rapidly, then the rest was homogenated. The content of basic fibroblast growth factor (bFGF) of the supernatant was tested with enzyme linked immunosorbent assay (ELISA). The rats of the same day age from sham operated group were consided as control. Rat MSC was cultured in vitro, and the passage 4～5 MSC in good shape was selected to transplant into the rats with HIBD by intraperitoneal injection at the 4 time points, 4 weeks later, behavior reactivity of the rat was evaluated with Morri water maze.Result The bFGF content (pg/ml) in the brain of the rats that have suffered from HIBD for 2 h, 24 h, 3 d, 6 d was 66.25±36.13 ((x|-)±s, n=8), 71.25±31.88 ((x|-)±s, n=8), 113.13±31.41 ((x|-)±s, n=8), 105.63±43.28 ((x|-)±s, n=8), there was no significant difference between 4 time points, however, there still existed rising tendency; the bFGF content (pg/ml) in the brain of the sham operated group was 214.38±37.03 ((x|-)±s, n=8), 131.25±32.50 ((x|-)±s, n=8), 99.38±40.47 ((x|-)±s, n=8), 96.25±49.06 ((x|-)±s, n=8), there existed decreasing tendency. The place navigation test in the Morris water maze showed the escape latent time (s) of the model group treated with MSC transplantation after 2 h of HIBD (group A), the model group treated with MSC transplantation after 24 h of HIBD (group B), the model group treated with MSC transplantation after 3 d of HIBD (group C) , the model group treated with MSC transplantation after 6 d of HIBD (group D), the sham operated group (group E) and the HIBD model group treated with PBS (group F) was 18.29±3.10 ((x|-)±s, n=8), 46.63±6.48 ((x|-)±s, n=8), 44.28±6.09 ((x|-)±s, n=8), 22.07±4.39 ((x|-)±s, n=8), 16.22±3.14 ((x|-)±s, n=8), 67.49±4.15 ((x|-)±s, n=8), the escape latent time of the group E and other HIBD groups treated with MSC transplantation was shorter than that of group F (p<0.05); there was no significant difference between group A and group E (p>0.05), and the escape latent time of group A and group E was shorter than that of other groups (p<0.05); the escape latent time of group D was shorter than that of group B and group C (p<0.05), there was no difference between group B and group C (p>0.05). The spatial probe test in the Morris water maze showed the proportion of the quadrantal swimming distance in the platform quadrant of group A, group B, group C, group D, group E, group F was 0.57±0.04 ((x|-)±s, n=8), 0.43±0.03 ((x|-)±s, n=8), 0.41±0.03 ((x|-)±s, n=8), 0.55±0.03 ((x|-)±s, n=8), 0.60±0.04 ((x|-)±s, n=8), 0.30±0.02 ((x|-)±s, n=8), the proportion of the quadrantal swimming distance in the platform quadrant of group E and other HIBD groups treated with MSC transplantation was longer than that of group F (p<0.05); there was no significant difference between group A and group E (p>0.05), and there was no significant difference between group A and group D (p>0.05); the proportion of the quadrantal swimming distance in the platform quadrant of group D was shorter than that of group E (p<0.05), however, longer than that of group B and group C (p<0.05).Conclusion The capability of spatial learning of the rats with HIBD can be improved after treated with MSC transplantation, the suitable time point of transplantation was 2 h after HIBD in the study. Objective To explore whether Salvia Miltorrhiza can promote neural differentiation of transplanted MSC in vivo, study whether the differentiated cells own neurophysiological function, and investigate whether the transplanted MSC has any effect on neural differentiation of endogenous NSC.Method Each 6-day old rat was injected with 5-bromo-2 -deoxyuridine (BrdU) intraperitoneally, then the animal model of newborn rat HIBD was established at the second day. MSC was cultured in vivo,and the passage 4～5 MSC in good shape was labeled with DAPI and transplanted into the rats with HIBD by intraperitoneal injection after 2 h of HIBD, 4 weeks later, behavior reactivity of the rat was evaluated with Morri water maze, after that, triple-label immunofluorohistochemistry of BrdU/DAPI/Nestin, BrdU/DAPI/TUJ1, BrdU/DAPI/GFAP was carried out to access the neural differentiation of MSC and BrdU+ cells, at the same time, the synapse cycle was detected with laser-scanning confocal microscope (LSCM), 50 mmol/L K+ acted as stimuli to evoke action potential, fluorescence excitation of SynaptoRed C-2 was challenged by LSCM for the measurement of synapse cycle function.Result The place navigation test in the Morris water maze showed the escape latent time (s) of the sham operated group (group A), the model group treated with MSC after 2 h of HIBD and salvia miltiorrhiza after 24 h of HIBD (group B), the model group treated with MSC after 2 h of HIBD and PBS after 24 h of HIBD (group C), the model group treated with salvia miltiorrhiza after of 2 h HIBD and MSC at the same time (group D), the model group treated with salvia miltiorrhiza after 2 h of HIBD (group E), and the HIE model group treated with PBS (group F) was 18.16±1.90 ((x|-)±s, n=8), 19.43±2.07 ((x|-)±s, n=8), 22.84±1.97 ((x|-)±s, n=8), 31.05±4.09 ((x|-)±s, n=8), 33.60±3.30 ((x|-)±s, n=8), 60.63±5.66 ((x|-)±s, n=8), respectively, the escape latent time (EL) of group A was shorted than that of group C, group D, group E, group F, P<0.05, there was no significant difference between group A and group B, P>0.05; the EL of group B was shorter than that of grouop D, group E, group F, P<0.05; the EL of group C was shorter than that of group D, group E, group F, P<0.05, but there was no significant difference between group C and group B, P>0.05; the EL of group D was shorter than that of group F, P<0.05, but longer than that of group A, group B, group C, P<0.05, and there was no difference between group D and group E, P>0.05; the EL of group E was shorter than that of group F, P<0.05, but longer than that of group A, group B, group C, P<0.05; the EL of group F was longer than any other groups, P<0.05. The spatial probe test in the Morris water maze showed the proportion of the quadrantal swimming distance in the platform quadrant of group A, group B, group C, group D, group E, group F was 0.62±0.03 ((x|-)±s, n=8), 0.63±0.04 ((x|-)±s, n=8), 0.57±0.07 ((x|-)±s, n=8), 0.48±0.03 ((x|-)±s, n=8), 0.45±0.02 ((x|-)±s, n=8), 0.31±0.03 ((x|-)±s, n=8), respectively, the proportion of the quadrantal swimming distance in the platform quadrant of group A and group B was longer than that of other groups, P<0.05, but there was no significant difference between group A and group B, P>0.05; the proportion of the quadrantal swimming distance in the platform quadrant of group C was longer than that of group D, group E, group F, P<0.05; the proportion of the quadrantal swimming distance in the platform quadrant of group D and group E was longer than that of group F, P<0.05, but there was no significant difference between group D and group E, P>0.05. The triple-label immunofluorohistochemistry of BrdU/DAPI/Nestin, BrdU/DAPI/TUJ1, BrdU/DAPI/GFAP showed the ratio of expression of Nestin of BrdU+ cells in the group A, group B, group C, group D, group E, group F was (78.04±6.03)% ((x|-)±s, n=8), (23.43±3.57)% ((x|-)±s, n=8), (33.73±3.69)% ((x|-)±s, n=8), (43.01±5.45)% ((x|-)±s, n=8), (48.59±5.45)% ((x|-)±s, n=8), (65.63±7.20)% ((x|-)±s, n=8), respectively, the ratio of expression of Nestin of BrdU+ cells in the group B was lower than that in the other groups, P<0.05; the ratio of expression of Nestin of BrdU+ cells in the group A and group F was higher than that in the other groups, P<0.05, but that in the group F was lower than that in the group A, P<0.05; the ratio of expression of Nestin of BrdU+ cells in the group C was lower than that in the group A, group D, group E, group F, P<0.05; there was no difference between group D and group E, P>0.05; the ratio of expression of GFAP of BrdU+ cells in the group A, group B, group C, group D, group E, group F was (11.71±3.28)% ((x|-)±s, n=8), (26.85±3.22)% ((x|-)±s, n=8), (24.87±3.17)% ((x|-)±s, n=8), (23.62±2.74)% ((x|-)±s, n=8), (21.70±3.08)% ((x|-)±s, n=8), (18.27±3.86)% ((x|-)±s, n=8), respectively, the ratio of expression of GFAP of BrdU+ cells in the group B, group C, group D, group E, group F was higher than that in the group A, P<0.05; the ratio of expression of TUJ1 of BrdU+ cells in the group A, group B, group C, group D, group E, group F was 4.68±1.67 ((x|-)±s, n=8),35.04±3.65 ((x|-)±s, n=8),27.48±4.25 ((x|-)±s, n=8),22.61±2.74 ((x|-)±s, n=8),19.18±1.96 ((x|-)±s, n=8),7.72±2.10 ((x|-)±s, n=8), respectively, the ratio of expression of TUJ1 of BrdU+ cells in the group B> that in the group C> that in the group D> that in the group E> that in the group G> that in the grouop A, P<0.05; the ratio of expression of TUJ1 of transplanted MSC in the group B, group C, group D was 0.59±0.07 ((x|-)±s, n=8), 0.28±0.07 ((x|-)±s, n=8), 0.23±0.03 ((x|-)±s, n=8), respectively, the ratio of expression of TUJ1 of transplanted MSC in the group B> that in group C> that in group D, P<0.05, the ratio of expression of GFAP of transplanted MSC in the group B, group C, group D was 0.12±0.03 ((x|-)±s, n=8), 0.23±0.04 ((x|-)±s, n=8), 0.17±0.03 ((x|-)±s, n=8), respectively, the ratio of expression of GFAP of transplanted MSC in the group C> that in the group D> that in the group B, P<0.05, the ratio of expression of Nestin of transplanted MSC in the group B, group C, group D was 0.12±0.03 ((x|-)±s, n=8), 0.23±0.06 ((x|-)±s, n=8), 0. 23±0.04 ((x|-)±s, n=8), respectively, the ratio of expression of Nestin of transplanted MSC in the group C> that in the group D> that in the group B, P<0.05. The study of synapse cycle showed When the brain slice was stimulated by 50 mM K+ at first time, synaptoRed-C2 anchored onto the cell membrane, the cells presented red fluorescence, then after the second excitation by high concentration K+, the fluorescence intensity of the cells decreased quickly, the curve of fluorescence mean intensity descended sharply.Conclusion salvia miltiorrhiza can promote neural differentiation of transplanted MSC in vivo, the differentiated cells posses synapse cycle function, the transplanted MSC can enhance the neural differentiation of BrdU+ cells.
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