The Therapeutic Effect And Mechanisms Of Bone Marrow Mesenchymal Stem Cells Transplantation On Rats With Alcoho-associated Dementia

Objective:1. To explore the best conditions of building rat model with AAD.2.To explore the therapeutic effect and mechanisms of bone marrowmesenchymal stem cells (BMMSCs) transplantation on rats with alcohol-associateddementia (AAD).Methods:1. In order to explore the best conditions of building rat model with AAD, SDrats were divided into①20%alcohol (4ml/kg/d) intragastric administration for28dgroup(G-Eth);②20%alcohol (4ml/kg/d) intraperitoneal injection for28dgroup(I-Eth);③20%alcohol (4ml/kg/d) intragastric administration for14dgroup(L-Eth);④20%alcohol (8ml/kg/d) intragastric administration for14dgroup(M-Eth);⑤20%alcohol (12ml/kg/d) intragastric administration for14dgroup(H-Eth);⑥normal saline (4ml/kg/d) intragastric administration for28dgroup(G-Con);⑦n ormal saline (4ml/kg/d)intraperitoneal injection for28dgroup(I-Con);⑧normal saline (4ml/kg/d) intragastric administration for14dgroup(Con) according to the random number method;.2. Dementia in rats was determinated by escape Latency (EL) in Morris WaterMaze (MWM).3. The morphology change was detected in hippocampal dentate gyrus of ratswith alcohol-associated dementia (AAD) by hemotoxylin and eosin (HE)histochemistry staining.4. The neurons apoptosis was detected in hippocampal dentate gyrus of rats withalcohol-associated dementia (AAD) by Hoechst33342fluorescence staining.5. Bone marrow mesenchymal stem cells (BMMSCs) were cultured, purifiedand amplified by adherent culture method in liquid.6. Cell surface markers (CD90、CD29、CD34、CD45) were identified by flowcytometry. 7. After building AAD model for1week, the3×106BMMSCs or PBS weretransplanted to rats with AAD group in20%alcohol (4ml/kg/d) intragastricadministration for28d by vein or intracerebral stereotaxis.8. The morphology change and neurons apoptosis were detected in hippocampaldentate gyrus of rats of all groups after BMMSCs transplatation by hemotoxylin andeosin (HE) histochemistry staining.9. The BDNF expression was examined in hippocampal dentate gyrus, CA1andCA3of rats with alcohol-associated dementia (AAD) by immunohistochemistry.10. Total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-PX)enzymatic activity in serum of rats in normal saline administration group, AAD groupand BMMSCs or PBS transplantion groups were examined by colorimetry.Result:1. Compared to the control groups of normal saline, the EL was significantlyincreased in G-Eth group (20%,4ml/(kg·d)·28d) at14d and28d after building model(P<0.05), however the EL in L-Eth group、M-Eth group and H-Eth groups had nosignificantly increased (P>0.05). In addition, there were no significant difference ofEL between G-Eth group and I-Eth group. Compared to L-Eth group, there were alsohad no significant difference between M-Eth group and H-Eth group.2. In brain tissues, HE histochemistry staining has shown that all models of ratswith AAD have been significantly changed in morphology of hippocampal DG ofrats.3. In brain tissues, Hoechst33342fluorescence staining has shown that thenumber of apoptosis cells in hippocampal DG of rats with G-Eth group and L-Ethgroup were increased significantly as comparaed to control group (P<0.05). The morelong time was, the more apoptosis cell was (F=20.74, P<0.001). However, the cellapoptosis rate was not influenced by alcohol dose (P>0.05).4. In culture, the BMMSCs was adherent growth and the morphology ofBMMSCs was fusiformis shape. The BMMSCs expressed CD29(98.6%),CD90(80.4%), CD34（0.1%）and CD45(5.8%).5. Compared to control group with normal saline intervention, EL of rats in AADgroup(20%alcohol,4ml/kg/d intragastric administration for28d) had significantlyincreased（P<0.05）. In groups of BMMSCs transplantion via vein or intracerebral stereotaxis, there was a significant recovery with special learning and memory byMWM test as compared to PBS group (p<0.05).6. After BMMSCs transplanting via vein or intracerebral stereotaxis for2weeks,EL of rats in AAD group(20%alcohol,4ml/kg/d intragastric administration for28d)was decreased significantly(P<0.05)and pathologic cells and apoptosis cells inhippocampal DG were decreased significantly as compared to PBS group (P<0.05).7. Compared to control group(normal saline,4ml/kg/d intragastric administrationfor28d), the expressions of BDNF in hippocampal DG、CA1and CA3with AADgroup(20%alcohol,4ml/kg/d intragastric administration for28d) have decreasedsignificantly. After BMMSCs transplanting via vein or intracerebral stereotaxis for2weeks, the expressions of BDNF in hippocampal DG、CA1and CA3were allincreased as compared to PBS transplantion groups. There were significantdifferences in hippocampal CA1of vein transplantion group and in hippocampalDG/CA3of intracerebral stereotaxis group(P <0.05).8. Compared to normal saline administration group, the enzymatic activity oftotal superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-PX) in serumof rats in AAD group were greatly decreased. The enzymatic activity of GSH-PX wasgreatly increased after BMMSCs transplanting via vein or intracerebral stereotaxis for2weeks (P<0.05) and the enzymatic activity of TT-SOD activity was greatlyincreased after BMMSCs transplanting via intracerebral stereotaxis for2weeks(P<0.05) as compared to PBS transplantion groups.Conclutions：1. Low dose(20%alcohol,4ml/kg) alcohol intragastric administration for14-28dis the optimum condition in building rat-model with alcohol-associated dementia.2. The BMMSCs transplantation via intravenous and intracerebral routessignificantly improved the neural function of learning and memory in rat withalcohol-associated dementia is may be due to increase BDNF expression inhippocampus and T-SOD and GSH-PX activities in serum to suppress hippocampalnerve cell apoptosis.