| Backgrounds and objectsPatients survivorship quality was severely influenced by ischemic cerebrovasculardisease, because it could lead to high mutilation. Functional impairment could be caused byneuron disorganization and depletion which performed important pathological significance incerebral ischemic injury. The strategies aimed directly at brain biochemical and metabolicalteration possessed considerable effects which could protect neuron from damage andpromote neurocyte regeneration in cerebral ischemia injury, then to intervene in impairmentand make the function restored. At present, it was not satisfied with the nervousness medicinein study and clinical application. Bone marrow stromal stem cells (BMSCs) possessed thepotentiality of reproductive activity and multi-directional differentiation, which coulddifferentiate into neuron, astrocyte and oligodendrocyte, then become the ideal seed sourcesand were used in cerebral ischemia injury. Thinking of the toxicity and side effect of drugsused in deriving in vitro and poor survival potentia of derived mesenchymal stem cells, theinvestigations were developed in transplantation after purification, amplification andcultivation of mesenchymal stem Cells in vitro in recent years. Therefore, studies whichfocused on the regeneration of neurocyte and recovery of function by using implantation ofmesenchymal stem cells had the considerable significance in cerebral ischemia injury.Researches about Traditional Chinese medicine used in implantation of mesenchymal stemcells were reported gradually in recent years, but more studies were focused on the inductionand differentiation of mesenchymal stem cells in vitro, and most of the drugs selected in thoseresearches were simplex or its extractive, while a few compound recipes in TraditionalChinese Medicine were selected. Moreover, researches developed in vivo about compoundrecipes of Traditional Chinese Medicine using in survival and differentiation of mesenchymalstem cells after transplantation were rarely reported. Cerebral infarction was categorized inapoplexy in Traditional Chinese Medicine, and the pathogenesis was complic, especially theturbid, toxic materials and bloodstasis were more significant. According to the pathogenesis ofdeficiency of vital energy and stagnation of blood, as well as the damage of toxin in apoplexy,methods of.detoxicating, descending the turbid and promoting blood flow performed theprincipal role in therapy. On these grounds, compound recipe named NaoMaiTong was prepared (rhubarb, ligustici, ginseng and radix) and possessed the effect of promoting bloodcirculation to remove meridian obstruction, then protected brain from ischemic injury. Studiesof animal experiments and clinical study indicated that the effect of NaoMaiTong on cerebralinfarction was identified. NaoMaiTong could improve the survivorship quality of patientssignificantly. Based on the backgrounds above-mentioned, investigation of Traditional ChineseMedicine associatied with implantation of mesenchymal stem cells was significant in therapyof cerebral ischemic injury. In this Study, the effect of NaoMaiTong associated withimplantation of mesenchymal stem cells on neural regeneration in rats with cerebral ischemiareperfusion was be evaluated, then the mechanisms were explored from aspects of influencingthe levels of nerve nutrient factors and increasing the differentiation ratio of BMSCs inclinedto nerve cell in brain tissue, as well as the contribution to reconstruction of synapse byup-regulating the expression of growth associated protein(GAP).MethodsSD rats were used in this study, and BMSCs were acquired through the operations ofadherence and cultivation using entire bone marrow. BMSCs were labelled by Brdu at 48 h ofpretransplant. MCAO was duplicated by occluding rats middle cerebral artery with nylonthread. At the time of 3 h in ischemia and 24 h after reperfusion, BMSCs suspension wasinjected through rats internal carotid artery. Rats neural function and survivorship conditionwere observed in earlier period (7d), metaphase (14d) and anaphase (28d) oftransplantation. Methods of pathomorphologic and immunohistochemical operation wereemployed to observe the protection of NaoMaiTong associated with implantation ofmesenchymal stem cells from the aspects of neural function comprehend evaluation, braintissue water ratio, cerebral infarction size and ultramicrostructure of neurocyte, and theimmunohistochemical double markng method was used to explore the effect of NaoMaiTongfrom influencing on BMSCs survival, immigration and differentiating into neuron orneurogliocyte and regulating the expression of nerve nutrient factors, then mechanisms ofNaoMaiTong associated with BMSCs in contributing the reestablishment of synapse wereexplored. The main results were as follows.Results 1 The effect of NaoMaiTong associated with implantation of BMSCs on cerebral ischemicreperfusion injury in rats1.1 BMSCs were acquired through the operations of adherence and cultivation using entirebone marrow, and performances of the method was feasible, then lots of BMSCs wereprovided in short period. BMSCs were injected through rats internal carotid artery, and lesscell suspcnsion outleakagcd, which showed that the transplantation approach selected in thestudy was acceptable.1.2 Rats weight showed decreased in carlicr period, then increased in metaphase (14d) andanaphasc (28d). Following the change of weight, rats systematic condition appeared to bc inconcord with that tendency. Rats neural function restored gradually in the period from 7d to28d. Degrce of rats brain tissue edema, sizc infarction focus and nerve cell ultrastructureshowed the changes from exacerbation to abatement following the prolongation of ischcmicreperfusion.1.3 The effect of NaoMaiTong associated with implantation of BMSCs on ischemic injury wasrelated to the time after transplantation. In earlier period (7d), the effect wasn't obvious, and inintermediate stage (14d), rats recovery of function, brain tissue edema and infarction focusshowed tendency of amelioration, then in later period (28d), the protection was significant incompared with that of earlier and intermediate period.1.4 NaoMaiTong showed the protection against cerebral ischemic injury in different stages.Effet of NaoMaiTong associated with BMSCs implantation appeared to be advanced incomparison with that of simple implantation of BMSCs, and the role in association groupshowed that the improvement in pathological injury of nerve cell were more obvious. Theprotection of NaoMaiTong associated with BMSCs implantation was more significantfollowing the prolongation of postgraft.2 The influence of NaoMaiTong associated with BMSCs implantation on regeneration ofneurocyte and nerve nutrient factors in rats with cerebral ischemic injury2.1 Specific identification markers of neuron and neurogliocyte show that the expressionattenuated following the prolongation of postgraft. Nerve growth factor and glial cell linederived neurotrophic factor increased in 7d and protected nerve cells from injury, while thelevel decreased in 14d and 28d, such changes was disadvantagous to neuronalcyte regeneration and functional recovery of rats in later stages.2.2 In earlier stage of BMSCs transplantation, NSE and GFAP showed no obvious increase.However, expression of NSE and GFAP enhanced significantly following the prolongation ofpostgraft, especially in later stage, the expression of NSE increased more obviously. The levelsof NGF and GDNF showed less increase in earlier stage of BMSCs implantation, while theupregulations of NGF and GDNF were significant in intermediate and later period.2.3 Effect of NaoMaiTong associated with BMSCs implantation appeared to be more obviousthan that of simple BMSCs implantation in earlier stage. Meanwhile, NaoMaiTong couldenhance the expressions of NSE and GFAP in later period. Employment of NaoMaiTongassociated with BMSCs implantation could increase the level of GDNF in intermediate stageand make the expression of NGF enhanced in later period.3. The influence of NaoMaiTong associated with BMSCs implantation on differentiationinto nerve cells in rats with cerebral ischemic injury.3.1 BMSCs distribution ratio of brain parenchymatous tissue to cortex intraluminal increasedaccording to the prolongation of postgraft. In earlier stage of BMSCs transplantation, BMSCsmainly distributed in blood vessels and accreted to endothelium, only a few BMSCs enteredbrain tissue, and the distribution didn't show obvious regular pattern. In metaphase, BMSCspast through vessel wall(blood-brain barrier, BBB) and implanted in brain tissue, especiallyimmigrated towards ischemia cortex, and a few BMSCs could be observed in blood vessels. Inlater stage, survived BMSCs were all in brain tissue, few lied in blood vessels. Employment ofNaoMaiTong associated with BMSCs implantation could increase the number in brain tissue,but the distribution ratio of brain parenchymatous tissue to cortex intraluminal and migratorydirection showed no obvious changes.3.2 Differentiation ratio and direction of BMSCs towards nerve cells changed following theprolongation of postgraft. Differentiation ratio of BMSCs was lower in earlier stage andshowed no variability in direction. Differentiation ratio became higher in intermediate stage,and the differentiation mainly towards the glial neuronalcyte, while the differentiation towardsneuron was slight. Differentiation of BMSCs mainly towards neuron in later period. The studyindicated that NaoMaiTong performed several effects in different stages, that meansNaoMaiTong could enhance the BMSCs differentiating into neurogliocyte in metaphase and neuron in later stage. Meanwhile, NaoMaiTong could make the differentiation of BMSCsenhanced in later stage.4. The influence of NaoMaiTong associated with BMSCs implantation on thereestablishment of synapse in rats with cerebral ischemic injury4.1 Neuron organization was impaired by cerebral ischcmic reperfusion injury, and thecharacteristics showed that neurofilaments(NF) were turbid in line, and became shorter andrare, the number reduced, as well as the distribution was irregular. The changes became moreobvious following the prolongation of cerebral ischemic repeffusion injury. Expression ofGAP-43 which could contribute the reestablishment of synapse weakened gradually accordingto the prolongation of cerebral ischcmic rcpcrfusion injury. NaoMaiTong could protect neuronfrom injury in earlier stage, but showed no obvious influence on the expression of GAP-43.4.2 BMSCs showed no obvious effect on neuron organization in earlier stage, while inmetaphase and later period, expression of Synincreascd gradually. Meanwhile, expressions ofNF, Syn and GAP-43 increased in later period. The contribution effect of BMSCs onreestablishment of synapse was related to the time of postgraft, which means the role enhancedfollowing the prolongation of postgraft.4.3 The study indicated that NaoMaiTong could increase the expressin of NF and reduce thedamage of neuron organization. Meanwhile, NaoMaiTong could make the expression ofGAP-43 advanced and enhance the level in later period, as well as increased the expression ofSyn.ConclusionsThe study indicated that BMSCs were acquired through the operations of adherence andcultivation using entire bone marrow, and performances of the method was feasible. BMSCscould survive and immigrate in brain tissue, then perform the effect on injury caused bycerebral ischemic reperfusion. Neural function, brain water edema and ischemic infarctionimproved gradually according to the prolongation of postinjury. NaoMaiTong possesseddifferent roles in three stages, and the effect seemed more obvious in earier stage. Theprotection of BMSCs enhanced gradually following the prolongation of postgraft. The studyalso identified that the number of neuron reduced following the postinjury, and the changes ofnerve nutrient factors showed increased at first, and then decreased. The increase in earlier stage could protect neuron from injury, while the decrease was disadvatageous to theneuronalcyte regeneration and funcional recovery of rats in later stages. NaoMaiTong couldupregulate the levels of GDNF in earlier stage and metaphase, while increase the expression ofNGF in later period. On the contrary, the effect of BMSCs increase grandually according to theprolongation of postgraft. NaoMaiTong could make the protection advanced, and make theeffect of BMSCs enhanced in later period, which was thought to be related to the upregulationof nerve nutrient factors. BMSCs could survive and immigrate towards damaged cortex, andthen differentiate into nerve cells, but the direction of differentiation had no variability.NaoMaiTong could make the survival of BMSCs enhanced, and intervene with the direction ofdifferentiation, that was to enhance the differentiation into neurogliocyte in metaphase and intoneuron in later stage. Reestablishment of synapse was the fundmental of recovery of neuronalorganization and function, and showed the reduction tendency in this study, which wasthought to be related to the down-regulation of GAP-43. NaoMaiTong could protect neuronfrom injury in earlier stage, and contribute reestablishment of synapse in later period. BMSCscould also perform the effect of contribution to reestablishment of synapse and be thought tobe related to up-regulation of the GAP-43, which enhanced following the prolongation ofpostgraft. NaoMaiTong could make the effect of BMSCs on reestablishment of synapseadvanced and make the reestablishment of synapse enhanced in later period by regulating theexpression of GAP-43. Therefore, NaoMaiTong associated with BMSCs implantationpossessed the considerable significance in protecting brain from ischemic injury. Certainevidence could be provided from this study for the therapy of cerebral ischemic injury and thefurther study of Traditional Chinese Medicine. Moreover, the investigation possessed theimportant significance for employment of Traditional Chinese medicine and cellular therapy. |
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