| Objective: To abserve the dynamic state changes of nerve function,water content of brain tissue,cerebral infarction size, brain tissue pathodamage, the permeability of BBB, components of microvessel basement membrane, plasminogen activating system and matrix metalloproteinasees at different intervals after focal cerebral ischemia reperfusion in rats.To discuss the injury feature of microvessel after cerebral ischemia reperfusion, and reveal its changeable regulatitye. To study the influence of these change and MSCs'survival and differentiation in brain tissue by using Naomaitong (NMT) assosiated with MSCs transplantation. The protective effect and mechanism of NMT assosiated with MSCs transplantation on brain microvessel damage after focal cerebral ischemia reperfusion in rats is explored.Methods:SD(n=195,3～4months)rats were randomly divided into sham- operated group (n=15) and operated groups witch involved model group (n=45), NMT group (n=45). MSCs transplantation (MSC-T) group(n=45) and NMT associated with MSCs (NAM) group(n=45), then operated groups were randomly divided into 7, 14 and 28d. Each group had 15 rats. All the rats were lavaged NMT 4d before animal trial,one time each day, except for sham-operated group,model group and MSC-T group with equal volume saline.MSCs were isolated and purified by adhering to the culture glassware wall and had been marked with Brdu for 48h before transplantation.Ischemia-perfudion animal model was duplicated in accordance with improved Longa's focal middle cerebral artery occlusion (MCAO) model.With the exception of the insert of nylon thread, same operation was performed on the sham-operated group. After cerebral ischemia for 3h, the nylon thread ispicked out to made the brain reperfused and 24h after reperfusion, 0.2ml suspension containing 2×106 MSCs were respectively administrated via carotid artery in MSC-T group and NAM group. The same volume of Saline were give to model group and NMT group. The rats were tested neural symptoms on 7,14 and 28d.Then the brain specimens were taken for observing the water conternt of the brain tissue, the area of cerebral infarction.pathological changes in ischemic area were observed with electron microscope. Immunohistoche- mistry was used to identify the expression of immunoglobulin G in brain tissue (IgG), collogen-Ⅳ(ColⅣ), laminin(LN), matrix metallo proteinase -9 (MMP-9), tissue inhibitor of metalloproteinase -1(TIMP-1), tissue-type plasminogen activator(t-PA), plasminogen activator inhibitor-1 (PAI -1).Number of Brdu and FⅧdouble- staining positive cells and differentiation ratio of endothelial cells was tested by immunohistochemical double-staining.Results:①The death rate of rats, changes of weight, brain water ratio and infracted area were all higher and the score of neural symptoms was lower in model group of 7, 14 and 28d than those of sham-operated group. The pathological changes and microvessel damage could be found in model group rats brain tissue through electron microscope,but not be found in sham- operated group. Compared with model group, the brain water ratio, infracted area, pathological changes and microvessel damage were lower and the score of neural symptoms was higher in NMT group, MSC-T group and NAM group. After operation, in all treatment groups, the rats'weight was lost less in 7d group and gained more in 14d, 28d group of than that of model group. Compared with NMT group and MSC-T group, NAM group had significant effect, expecially 28d group.The score of neural symptoms was the lowest at 7d with the extension of ischemic reperfusion time, then nerve function resumed gradually. The water content of brain increased and size of cerebral infarction were the highest at 14th day, and lessened at 28th day. The trauma of microvessel structure was the most serious at 14th day, since then 28th day the trauma stilled.②Compared with sham-operated group, the expression of IgG was higher, and the express ofⅣCol and LN were lower in model group. Contrasted with model group, the expression of IgG was lower, and the express of ColⅣand LN were higher in all treatment group. Compared with NMT group and MSC-T group, NAM 28d group was the best one. The expression of LN in NMT and NAM 7d group was higher than that of MSC-T group. In model group, the expression of IgG at 14d and 28d were higher than that of 7d group. the expression of ColⅣat 14d and 28d were lower than at 7d.③Compared with sham-operated group, the expression of MMP-9 was higher in model group. Compared with model group, the expression of MMP-9 was lower, and the expression of TIMP-1 was higher in all treatment groups. Compared with NMT group and MSC-T group, NAM 28d group was the best one. NMT and NAM 7d group had the better effect than MSC-T group. In model group, the expression of MMP-9 was higher at 14d and 28d than at 7d. In NAM group, the expression of TIMP-1 was higher at 14d and 28d than at 7d.④Compared with sham-operated group, the expression of t-PA and PAI -1 were higher in model group. Compared with model group, the expression of t-PA was lower in MSC-T 14d, 28d group, all NMT and MSC-T groups. Compared with NMT group and MSC-T group, the expression of PAI-1 was higher in NAM 28d group. The expression of t-PA in NAM 28d group was lower than that of MSC-T group. In model group, the expression of t-PA at 28d was higher than at 14d and 28d.⑤The cells marked with Brdu and FⅧdouble-staining could be found at brain tissue in transplantation group and associated group. Number of cells marked with Brdu in associated 14d group was more, and the number of Brdu and FⅧdouble-staining positive cells and differentiation ratio of EC in associated 7d group were higher than that in transplantation group. In same group, the number of Brdu cells and FⅧdouble-staining cells at 14d and 28d were more than at 7d.Conclusion:①Cerebral ischemia reperfusion could result in serious pathological injury, including the change of nerve shape, increase of brain water ratio, microvessel damage . The most severe injury was in the 14th day and remaind a high lever of injury in 28th day. NMT, MSCs transplantation and associated could improve the ischemic injury. The protcective effect was obvious along with the days passed by. NAM 28d group was much more effctive than NMT and MSC-T groups.Time of therapy of MSC-T was later than NMT and NMT groups, and the influence of lighting the dropsy was less than NMT.②The damage of microvessel was the key to pathological injury of cerebral ischemia reperfusion, and it was characterized with expression of IgG increase and the expression of LN and ColIV decreasing. NMT, MSCs transplantation and associated could reduce IgG expression and improve LN and ColIV expression in order to reduce BBB'permeability and components of microvessel basement membrane degradation.③The mechanism of cerebral ischemia reperfusion injury was related to the disequilibrium of gelatine system(MMP-9 and TIMP-1)and lyso-enzymatic system(t-PA and PAI-1). NMT, MSCs transplantation and associated could decrease the degradation of components of microvessel basement membrane by decreasing the expression of MMP-9, t-PA and increasing the expression of TIMP-1, PAI-1.④NMT could promote MSCs to suvive in brain tissue and differentiat toward vascular endothelial cell,the most significant effect was at 14th day, when NMT was associated with MSCs transplantation, they influened each other, and the most obvious was at 28th day.
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