Study On Mannitol's Effect Of Bone Marrow Mesenchymal Stem Cell Transplantation On Vascular Dementia Rat Models

[Objective] SD rat bone marrow mesenchymal stem cells (MSCs) were isolated, purified and passaged, identified in vitro, By observing the biological characteristics of MSCs, we cultured MSCs for the further use of MSCs transplantation in the treatment of vascular dementia animal models.[Method] killed by cervical dislocation SD rats'femur bone marrow was collected. We cultured MSCs by whole bone marrow adherent culture. We observed the primary and passaged MSCs'morphology, growth, growth curve of MSCs to understand MSCs'growth characteristics, morphological characteristics, and multiplication capacity. MSCs were identified by flow cytometry of its surface markers and differentiation of MSCs into osteoblasts in vitro.[Results] Using microscope MSCs were in the shape of fusiform grew and adhered to the wall, looked like fibroblas cells. After the first passage MSCs tended to form uniformly, were the whirlpool-like, or daisy-like growth. After passage, in the first 1-6 days MSCs grew quickly, in 7-8 days slowed down because of the high density leading a phenomenon of contact inhibition. The multiplication capacity of MSCs were decreased after passage generations. In the third generation of rat MSCs, flow cytometry found that MSCs almost uniformly expressed CD 44, CD90, did not expressed CD34. MSCs could be induced into osteoblasts in vitro, positive staining for alizarin red and alkaline phosphatase (ALP).[Conclusion] SD rats MSCs can be successfully isolated, cultured and amplified in vitro. MSCs have multilineage differentiation potential, can be induced into osteoblasts.Establishment of the vascular dementia rat model[Objective] To explore the ideal vascular dementia (VD) animal model establishments,We were preparing the VD rat models for the MSCs transplantation.[Methods] At first we used the Morris water maze to screen SD rats, excluded the 5% of the abnormal rats. Divided the rest 79 rats into 2 groups: 10 rats in the sham operation group,69 in model group. Rats in the model group were operated in the modified two vessel occlusion (2-VO),which was respectively ligature bilateral carotid artery in 3 days. The sham group were operated in the same surgical procedures without ligature carotid artery. Kept for 4 weeks (w) after the operation we tested rats ability of learning and memory by using the Morris water maze test. [Results] Totally 84 rats, the water maze screening removed of 4.1 rat died. The rest 79 rats were randomly divided into sham operation group(10 rats), model group (69 rats). Before the operation the sham group and model group were no different in rats ability of learning and memory(p> 0.05). After the operation 1 rat died of infection,2 died improper feeding in the sham group. In total there were 7 rats left in the sham group. In the model group,33 rats died,36 survived, removed 1 rat with movement disorder and 3 without dementia. In total, there were 32 rats in the model group. Comparing to the sham group, the model group decreased the ability of learning and memory(p < 0.05) after the operation. the survival rate 52.17%,91.43% of the rats which survived were made into VD model.[Conclusion] The modified 2-VO has a lower mortality, higher rate of rats could be VD model. It is easy to operate. The modified 2-VO is one of the ideal way to make VD rat model.Observation on mannitol's effect of bone marrow mesenchymal stem cell transplantation on vascular dementia rat model [Objective] Primary study of mannitol's effect of bone marrow mesenchymal stem cell transplantation on vascular dementia rat model.[Methods] After the modeling VD rats in model group were randomly divided into 3 groups:8 rats in group one (injected with 1ml serum-free medium),11 rats in group two (injection of 1×106个MSCslml) and 9 rats in group three (1.5g/kg mannitol injection after injection in 10-30 minutes 1×106个MSCslml), kept in the cage for 4 weeks after transplantation. Then tested the behavior by using the Morris water maze,pathological changes stained by hematoxylin eosin (HE) on frontal lobe and VEGF levels in rats' frontal lobe and cerebrospinal fluid by using ElISA.[Results] After 4-week transplantation of MSCs, the results of behavior showed that the escape latency in mannitol+MSCs group and MSCs group was significantly shorter than the medium group(p<0.05),, and the average lingering time at platform quadrant was longger(p<0.05).the escape latency in mannitol+MSCs group was significantly shorter than the MSCs group (p <0.001); and the average lingering time at platform quadrant was longger(p 0.001). The average VEGF level in rat cortex and CSF of the mannitol+MSCs group and the MSCs group was higher the medium group (p<0.001); The average VEGF level in rat cortex and CSF of the mannitol+MSCs group was higher the MSCs group (p<0.001); Compared with the medium group, the pathologic impairments appeared in the mannitol+MSCs group and the MSCs group were better, less neuron died and the cellular swelling,loss and pyknosis were much better. Compared to the MSCs group, mannitol+MSCs group improved more significantly.[Conclusion] The intravenous injection of MSCs can improve the ability of VD rats model's memory and learning; mannitol pretreatment improved VD rats memory and learning ability more than MSCs group. Possible mechanism that may be mannitol in combination with MSCs intravenous transplantation increased VEGF expression in the brain and enhanced protection of neurons.