| Objective:The objective of the present study generally related to the treatment effect and the mechanism of intravenously administered Bone marrow mesenchymal stem cells (BMSCs ) on the cerebral infarction in rats, and more specifically to the following aspects: BMSCs of rats would be separated, cultured, and amplified in vitro, and further differentiated into neuron tissues with BME induction, and the differentiation direction would be identified by means of the immunocytochemical stain and the transmission electron microscope;the model of middle cerebral artery occlusion (MCAO) of rats would be established with modified Zea Longa method;the treatment effect of intravenously administered 4th-generation BMSCs on the model rats would be observed;the possibility that BMSCs may home into damaged encephalic region via blood circulation would be confirmed;the possibility that BMSCs may survive, and may be differentiated into neural cells in encephalon would be verified;the possibility that BMSCs may promote the secretion of neurotrophic factors and cell factors,such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and vascular endothelial growth factor (VEGF), etc., would be confirmed; and the possibility that BMSCs may reduce apoptosis in the ischemic penumbra would be confirmed.Methods:1 Providing healthy male rats of 3-4 weeks old, separating and culturing BMSCs by means of the Percoll density gradient centrifugation method combined with the attachment culture method, amplifying with generations, performing observation on cell morphology under the inverted phase contrast microscope, and differentiating 4th-generation BMSCs with BME induction;2 Performing preinduction in preinduction media consisting of DMEM , 1mmol/L BME, and 20%FBS for 24h, and induction in neuronal induction media consisting of DMEM and 5mmol/L BME for 5hours in accordance with Woodbury method, placing the neuronal induction medium by the routine medium and culturing for 2 weeks;3 Performing cell identification, observing cell morphology change under the inverted phase contrast microscope, observing cell ultrastructure change under the transmission electron microscope, identifying surface markers of the neural precursor cells (Nestin), neuron cells(neuron specific enolase, NSE), and neuroglial cells (glial fibrillary acidic protein, GFAP) with respect to the induced cells in light of immunocytochemistry method;4 Performing Brdu marking for about 3 days after 3rd-generation BMSCs passage and the relevant medium was replaced, and gathering the 4th-generation BMSCs to be administered when the growed 4th-generation BMSCs suffused on the bottom of a vessel;5 Selecting healthy male adult SD rat whose weight was about 270-300g, establishing the model of the middle cerebral artery occlusion (MCAO) in the right cerebral side with modified Zea Longa method, providing 50 model rats and randomly dividing into a BMSCs treatment group and a comparison group, each of which had 25 model rats;6 At 24hr after the MCAO model was established, injecting 1mL PBS containing 3x106 4th-gernation BMSCs with Brdu marker through caudal vein with respect to the BMSCs treatment group, and injecting the same amount of PBS as the prescribed PBS through caudal vein with respect to the comparison group; respectively performing mNSS on the rats of each group on 1d after the MCAO model was established and before the administration was performed, on 7d, 14d and 21d after administration; performing TTC staining on five rates randomly selected from each group on 7d after administration and detecting infarct volume of these rats; preparing paraffin slices of cerebral tissue of the each five rats randomly selected from each group on 7d,14d,21d,28d after administration in a manner that those rats were put to death , brain was removed with the perfusion method, brain tissues were fixed by paraformaldehyde, dehydrated by a graded series of alcohol, made transparent by imethylbenzene, and embedded into paraffin; performing HE staining and TUNEL staining on paraffin slices; and identifying administered BMSCs, differentiation thereof and secretion of NGF, BDNF, and VEGF factors.7 Statistical Analyses:The data was evaluated using SPSS11.5 software.All data were expressed as mean±SEM. Between-groups comparisons were made using a repeated mea-sures one-way analysis of variance (ANOVA). When the F values were significant (p < 0.05), the Tukey HSD multiple range tests were followed. Differences were deemed significant when values were p < 0.05.Results:1 In initial stage of the innoculation of BMSCs, the cells were in the form of circular shape in the state of suspension, and most of the cells were attached on the wall after 24 hours, the attached cells either were individually and dispersedly or were formed into several cell clone, the cells gradually extended horns so as to form various shape, such as circular, polygon, fusiform, etc.. The cells were gradually purified after 3rd-generation cells passage to the 4th-generation cells which had unitary fusiform shape and grow in vortex and in parallel with each other.2 At 24 hour after preinduction, the individual BMSCs were fusiform when observed under the inverted phase contrast microscope, which had not obvious change in comparison with the uninduced cells. The shape of the cell would be changed rapidly as such that, the cell gradually became a sphere shape which had increased refractivity as the cytoplasm in the BMSCS retracts towards the nucleus thereof, dendrite was extended from cell membrane toward outer periphery of cell membrane, the dendrite continuously growed and formed primary and secondary branches on the tip end thereof. The number of the differentiated cell was obviously increased after 1 hours induction, and many dendrites of the cell were interlaced each other into a network , the shape of the cell would no longer change obviously after 5th hour. After 5th hour of the induction, the induction medium was replaced of the routine culture medium; one week later, most of cells still had neuron-like morphology; and two weeks later, the cell gradually restored to morphology as flat fusiform before the induction.3 The following results were presented according to the immunocytochemistry detection on the cell neuro marker after the induction: Nestin,NSE and GFAP were not expressed in the blank comparison group, GFAP was negative in the other groups. The number of Nestin positive cells was relatively high until reaches a peak value of 29.35% at 1th hour of the induction , but was few at 5th hour of the induction. NSE positive cell ratio increased with the time of the induction, and would be 57.53% at the 5th hour of the induction. Induction medium was replaced by routine medium and the cells restored to the morphology as before the induction, NSE and GFAP detected herein were negative.4 The following results was presented according to the observation under the transmission electron microscope: in comparison with one before the induction, after the cell was performed with the induction, the cell nucelus was big and round, nucleoli was obvious, cellular organelle was much more inside the nucleoli, Golgi complex existed aboundly around cells nucelus, a lots of rough endoplasmic reticulum expanded into circular shape and some of rough endoplasmic reticulum expanded into flat shape (in static state) were observed, round and elliptical bioblast and a few free ribosome were observed. As for the uninduced BMSCs, the cell nucelus was anomaly, cytoplasm was a little, nucleus versus cytoplasm ratio was large, cellular organelle was undeveloped, such cells in cell division process could be occasionally observed, which exhibited the characteristics of juvenile cells in the initial stage.5 The rat left upper limb was weak, the rat turned right or fell down towards right side during walks after right MCAO. The results from TTC staining on 1d after the operation showed that infarct focus was white and located in right striatum and temporoparietal cortex; the results from HE staining on 1d after the operation showed that neural cells in right striatum and temporoparietal cortex vanishes heavily, nucleus which had suffered from pyknosis, fragmentation and deformation, hyperchromatic kytoplasm, and infiltrated granular leukocytes are found; interstitial edema was obvious. All these suggested that middle cerebral artery infarction was formed.6 The mNSS in the BMSCs in the both groups was raised obviously on 1d after the operation, and there was no difference between the both groups (P>0.05). The mNSS for each group was gradually lower over the time; and the mNSS in the treatment group after administration was significantly lower than that of the comparison group on the same time points and the difference between the both groups was significant (p<0.05).7 Based on the presentation by TTC staining, the infarct volume, 21.21%土1.58%, in the BMSCs treatment group was relatively reduced on 7d after the administration in comparison with that, 25.75%土2.08%, of the comparison group, and the difference was significant (p<0.05).8 Based on the presentation by HE staining, with respect to the both groups on 7d after the administration, neuron loss in the necrosis center was great, hyperplasia of gliocyte occurs, and neutrophile granulocyte was infiltrated; but the extent in treatment BMSCs group was ligher than that in the comparison group. With respect to the both groups on 21d after the administration, hyperplasia of gliocyte was observed in the ischemic peripheral region; but the extent in treatment BMSCs group was lower than that in the comparison group. In comparison with the comparison group, the capillary vessel hyperplasia was more obvious and in ischemic damage was alleviated.9 Based on the presentation by TUNEL staining, apoptotic cells, each nucelus of which was stained as dark brown,were observed in the ischemic peripheral region with respect to the both groups. However, the number of apoptotic cells in the BMSCs treatment group, i.e. 24.6±3.2/HP, was lower obviously than that, i.e. 64.2±4.1/HP, in the comparison group (P=1.53403E-07<0.01).10 Brdu positive cells had been observed on 7d,14d,21d,28d after administration, most of Brdu positive cells was located on the ischemic peripheral region and the ischemic hemisphere, each nucelus of the Brdu positive cells was stained as yellowish-brown or dark-brown in according to DAB, and the number of Brdu positive cells were gradually reduced with time (P<0.05). Brdu/GFAP double positive cells were observed in the cerebral slice on 21d, which was not counted; at the same time, Brdu/NSE double positive cells were not observed. The number of GFAP positive cells on the ischemic peripheral region with respect to the BMSCs treatment group was lower than with respect to the comparison group, which was not counted yet. 11 Based on the presentation of cerebral slices suffered from BDNF, NGF, and VEGF immunostaining on 7d with respect to the both groups, positive results exhibit yellowish-brown kytoplasm. As for the BDNF expression, the mean optical density values were 0.47±0.05 for the BMSCs treatment group and 0.31±0.03 for the comparison group. As for the NGF expression, the mean optical density values were 0.49±0.10for the BMSCs treatment group and 0.28±0.11 for the comparison group. As for the VEGF expression, the mean optical density values were 0.28±0.03 for the BMSCs treatment group and 0.13±0.05 for the comparison group. The accumulated optical density value of BDNF, NGF, and VEGF positive cells for the BMSCs treatment group was obviously higher than that for the comparison group. For two independent sample Test with parameters t and P, they were respectively 0.000596,0.001877,1.92151E-05, all of them was lower than 0.01.Conclusion:1 Better purified BMSCs are obtained by means of the Percoll density gradient centrifugation method combined with the attachment culture method.2 BMSCs may be transferred toward neuron-like cells in morphology after BMSCs are induced, GFAP expression is negative before Nestin is expressed by NSE. This means that BMSCs may be differentiated into neuron-like cells instead of neuroglial cell, that is, BMSCs may be differentiated into neural stem cells at first and then neural stem cells may be further differentiated into the neuron-like cells. At the same time, the process of this above differentiation process may be performed rapidly and reversibly in very short time, however may be not stable and durable.3 The model of MCAO of rats may be stably established with modified Zea Longa method and easily replicated. The infarction portion is located in the middle cerebral artery and this location is unchangeable. This model may be a desired animal model for study on local cerebral ischemic focus.4 After BMSCs are intravenously administered into cerebral ischemic rats, the significant improvement on eurological function may be derived the following reasons: most of the administered BMSCs home into ischemic focus and the periphery thereof, a few of the administered BMSCs survive and are differentiated so as to possibly replace the damaged neural cells in part; secretion of trophic factor may be increased and apoptosis of cells in ischemic penumbra is decrease so as to protect nerve; secretion of vascular endothelial growth factor may be increased and angiogenesis may be promoted; the thickness of scar wall around periphery of ischemic focus may be lowered; and so on.5 The present experimental study may be provided as an experimental fundament and theory evidence on treatment of cerebral infarction by means of neuronal differentiation of BMSCs in vitro with induction and administration of BMSCs in vivo.
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