| Background and ObjectivesSince, for most mammals, adult nerve cells lack regenerative ability, the centralneural system (CNS) could not recover after encountering some extremecircumstances, like dramatic damage. Inspired by integrating stem cellstransplantation technics into the therapy of brain-and-spinal-cord related diseases,embryonic stem cells transplantation technic has become one of prospectivetherapeutic approach aiming at CNS, even though with various practical difficultiesincluding difficulty of material selection, immunological rejection, moral philosophy,etc. While bone mesenchymal stem cells (BMSCs) extensively used in bothfundamental and clinic works because of the amazing variety of advantages they haveshown. To begin with, these cells can highly regenerate, proliferate actively, and havepotentials of multi-directional differentiation. Subsequently, they can be extractedfrom themselves, with low-expression of antigen, and then lead to low immunologicalrejection and graft versus host (GVH) reaction. Apparently, BMSCs can also be takeneasily from abundant resources, and isolated, purified and cultivated in vitro, withhigh-speed of proliferation to meet the quantity of cell transplantation. Research hasindicated that, after being cultivated under suitable conditions, BMSCs can inducehighly expressed specific markers of nerve cells such as nestin, β-tubulinⅢ, MAP2, GFAP, NeuN, etc., followed by differentiating directionally into nerve cells and glialcells. Moreover, other research works associated with some serious diseases (likebrain damage, cerebrovascular diseases, and degenerative diseases of CNS) alsodemonstrated that BMSCs have positive impact on the treatment of these diseases. Tothe best of our knowledge, vascular dementia (VD), a cognitive dysfunctionalsyndrome which is caused by encephalopathy arising from cerebrovascular factors,could be significantly damaging to the elder citizen’s well-being with the patientnumber increasing year by year, but no effective and efficient measure has been foundto address this disease. Consequently, both fundamental and clinic research onBMSCs transplantation could throw insight into the therapy of VD.In terms of this research project, we observed survival, migration anddistribution of BMSCs from blood brain barrier (BBB) in brain parenchyma, as wellas the behaviours, re-cognitive functions and structural changes of brain tissues ofVD rats, after injecting BrdU marked BMSCs into these rats through their internalcarotid artery. Meanwhile, we also examed influence of BMSCs on the expression ofGrowth associated protein-43(GAP-43) in the brain, apoptosis in hippocampus cells,cholinergic system and Neuron-specific enolase (NSE) in serum and S-100proteinlevel. Through these approaches, we tried to make a clear description about thefeasibility and effectiveness of recovering neural functions in VD rats by BMSCstransplantation from internal carotid artery.Methods1. We used density gradient centrifugation and attachment culture of lymphocytefor the extraction, isolation, cultivation and amplification of BMSCs.2. On the basis of20ng/mL NGF and RA, we added BrdU to make concentrationto5μmol/L, and then BMSCs were incubated and differentiated in the incubation boxunder the condition of37and saturation humidity of5%CO2.3. The animal experimental groups were randomly divided into control groups(n=126), model groups (n=126) and treatment groups (n=126). And then the groupswere separated to3phase points including2W,4W and8W. 4. The modified Pulsinellis4-vessel occlusion could establish VD model in rats.After24hours, we injected Cs to internal carotid artery, with concentration of1.2×107/mL and injection dose of0.5ml.5. S-P immunohistochemistry was used to examine BMSCs in survival,migration and distribution in the brain.6. Similarly, we used S-P immunohistochemistry to examine levels of GAP-43inbrain tissues and Choline acetyltransferase (ChAT) in hippocampus cells.7. TdT-mediated dUTP terminal deoxynucleotidyl transferase mediated dUPTnick end-labelling (TUNEL) was carried out to test the level of apoptosis in rathippocampus.8. We used Hestrin alkaline hydroxylamine colorimetric method to test thecontent of Acetyl choline (Ach) and bioactivity of Acetylcholinesterase (AchE).9. Enzyme-linked immunosorbent assay (ELISA) was conducted to examineNSE in serum and the protein level of S-100.10. For behavioural detection, we took advantage of active avoidance reaction(AAR) to test the ability of studying and memorizing.11. We used optical microscope and transmission electron microscopy to observechanges of rat brain tissue pathology.ResultsAccording to this research project, the following findings were gained.1. After cultivating for24hours, BMSCs attached the wall of test tubes, and thenwent into the increased logarithmic phase in3-4days, with spindle-shapedproliferation capacity to form structures of multiple cloning groups. In10days,80%-90%BMSCs were emerged,90%of whose substratum were fusiform andpolygonal fibroblast with huge volume while the rest were globular cells andpleomorphism with high refractive index. The passage cells had shorter latent periods,quicker proliferative speed, fusiform and tabular shape, as well as uniformarrangement, which grew in streamline or swirl manner. After the induction ofBMSCs for30minutes, the soma got smaller, and cytoplasm could shrink to the surrounding of nucleus; thereby the shape of cells turned circular from fusiform withhigher refractive index. Then cells protruded the axon synapses and stretched outsecondary branch of dendritic cells similar to neuron-like cells, which could connectwith the soma or synapses of surrounding cells, thereby establishing network-likerelations. With the extension of induction time,96.7%BMSCs could differentiate intoneuron-like cells.2. BMSCs had a wide distribution in the brain parenchyma, abundant in theblood vessel region, and then they could transfer to and gather in hippocampus andcerebral cortex. After the surgery, for2W treatment group there were more BrdUimmunological active endocrine cells (4151±320), and fewer in4W group(2818±186), while in the8W only few can exist (92±19).3. After the surgery, in all2W,4W and8W groups, the number of GAP-43positive cells was much larger in experimental rats compared with the model group.(P>0.01)4. After the surgery, in all2W,4W and8W groups, the percentage of TUNELpositive cells in hippocampus of the model group was much higher in that of thecontrol group (P<0.01). In2W group, the percentage of TUNEL positive cells inhippocampus was higher than in the control group (P<0.05). Meanwhile in4W and8W groups there was no significant difference compared to the control group(P>0.05). But percentage in all groups was much lower than that in the model group(P<0.01).5. After the surgery, in2W,4W, and8W, the number of ChAT positive cells inhippocampus was much lower than its counterpart in the control group (P<0.05) butmore than that in the model group (P<0.05),6. Compared with the control group, the content of Ach in hippocampus andactivity of AchE were much lower in the model group (P<0.01), while compared withthe model group, both of them were higher in treatment groups (P<0.01).7. After the surgery, in2W, the content of NSE in serum is much higher than thatin the control group (P<0.01), which fell down in the4W with no difference with thecontrol group (P>0.05), and then in8W, it was definitely lower compared to thecontrol group (P<0.01). But compared with the model group, the content was lower in 2W (P<0.05) while higher in4W and8W (P<0.05), having no significant differencefrom the control group.8. In all2W,4W and8W, the content of S-100protein of the rat serum was muchhigher than that in the control group (P<0.05), but much lower than that in the modelgroup (P<0.01or P>0.05) without big difference compared to the control group(P>0.05).9. In all2W,4W, and8W, the rate of AAR in the model group was lower thanthat in the control group (P<0.01), while the treatment groups had lower rate than thecontrol group but higher rate than the model group (P<0.01).10. The pathology changes in all groups of rat brain tissues included at least twoparts. In the model group, there would be diffuse ischemia anoxic changes withconcentration on hippocampus and cerebral cortex, karyopyknosis, fragmentation,diffusion, denaturation, necrosis or disappearance of visible cells, dense cytoplasm,inflammatory cells infiltration. In the treatment group, neuron cells denaturation,descending number of necrosis cells and significantly reduced degree of necrosis cellsmight occur.ConclusionBased on current research findings, we could clarify that the followingcorresponding points.BMSCs transplanted through internal carotid artery could transfer from VD ratBBB to gather or survive in the brain region which was prone to ischemia and injury,resulting in significantly increased expression levels of GAP-43and hippocampusChAT positive cells. And this indicates that BMSCs can promote the recovery andregeneration of neuron axons, beneficial for the differentiation and growth ofcholinergic neuron as well as the reconstruction of synapses.Then BMSCs could protect hippocampus neuron cells in VD rats and reduce cellapoptosis, illustrated by the phenomenon that, after transplanting BMSCs, apoptosisproportion of VD rats in treatment group was considerably higher than its counterpartin control group. Together with the examination results of Ach in hippocampus, AchE activitiesand serum NSE\S-100protein levels, the evidence that, after BMSCs injection,denaturation and necrosis of neuron cells in VD rats were dramatically decreased,suggested that BMSCs may improve the function of cholinergic system and reducethe injury degree of nerve cells and glial cells; thereby putting good protection overthe connection among neuron cells repair, proliferation and synapses.Last but not least, a significant improvement on VD rats’ study ability wasobserved after BMSCs transplantation through internal carotid artery. |
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