Effect Of Human Umbilical Cord Mensenchymal Stem Cells Transplantation In Rats With Closed Traumatic Cerebral Injury

Objectives: Rat model of closed traumatic brain injury (TBI) wasestablished and accepted human cord mesenchymal stem cells (hUC-MSCs)transplantation to observe its effect on cell survival, migration, differentiationas well as recovery of neurological defect. Explore the mechanism ofhUC-MSCs in treating with TBI and lay a basis for its further application.Methord: The umbilical cords (UCs) from healthy neonatal withfull-term pregnancy cesarean section were used. The UCs were internallywashed with D-Hank's solution. The umbilical arteries and veins wereremoved, and the remaining tissure was diced into small fragments about1mm~3. After collagenase-Ⅱdigestion the explants were transferred intocontainer containing dulbecco's modified essential media/nutrient mixturef-12(DMEM/F12) with20%fetal bovine serum (FBS),100U/ml penicillinand100μg/ml streptomycin. Observed the morphology of primary cell culture,then replanted hUC-MSCs into culture flasks after trypsinization when theyreached80%-90%confluence. The harvesting cells were collected and flowcytometry was adopted to analyze the expression of CD45-PE, CD90-PE,CD105-PE, CD73(SH3)-PE, CD11b-PE, CD34-FITC, CD19-FITC, HLA-DR(HLA-II)-FITC and mouse IgG1-PE/mouse IgG1-FITC as controls.Adult male Sprague-Dawley (SD) rats in good condition (the weight isbetween270and320gram) were selected and made TBI models by the wayof fluid-percussion impact. Estimated by modified neurological severity score(mNSS)24hours later (Table1),50rats with a score of7to12(moderateinjury) were randomly divided into three groups:(1). hUC-MSCsgroup(n=20): Rats accepted P5hUC-MSCs transplantation labeled by1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) inthe area of injury, where we selected two points and each point injected5μl solution, totally1,000,000cells were used in each rat.(2). Phosphate BufferedSaline (PBS) group (n=20): Each rat received twice5μl-injection into thedamaged brain.(3). Simple TBI group(n=10): Each rat only accepted braininjury treatment, neither hUC-MSCs nor PBS solution was injected into thedamaged eare. Another10rats were put into the sham operation grouprandomly, just the cranial bone was removed compared with the TBI group,and neither giving hUC-MSCs transplantation nor PBS solution injection.The neural function was estimated by the mNSS at1,7,14,21and28days after transplantation. After functional outcome measurement the ratswere sacrificed and anatomized. Their brains were prepared for frozen sectionabout5micrometers thick. immunofluorescence is used to detect the cellssurvival, migration, differentiation as well as the expression of glialfibrillary acidic protein (GFAP), neuron specific enolase (NSE),microtubule-associated protein2(MAP-2), factor VIII related antigen (Ⅷ-RAg), neurofilament (NF) and Nestin.Results:A few of cells began to be stuck to a surface24hours after primaryculture and more were found8~10days later. When reached to80%-90%confluent after2weeks of culture, hUC-MSCs were subculture at the rate of1:2or1:3. The cells proliferated quickly and were fairly uniform5~6dayslater, it can be subcultured again. Flow cytometry showed that hUC-MSCsexpressed surface antigen of MSCs include CD73,CD90,CD105, did notexpress surface antigen of Hematopoietic precursor cells (HPCs) as CD34, yetnot express surface antigen CD19, CD11b, CD45or HLA-DR. It indicatedthat these cells were MSCs. Cell vitality was examined with3-(4,5)-dimethylthiahiazol-2-y1-2,5-diphenytetrazolium bromide (MTT)before migration, and the vitality is more than95%.28days after transplantation, only1rat died in the hUC-MSCs group andthe mortality was4.8%;10rats died in the PBS group, the mortality was33.3%; and6rats died in simple TBI group that the death rate was37.5%.compared the mortality, the hUC-MSCs group was lower both than the PBS group and the simple TBI group, the difference was statistically significant(P<0.05). None of the rat in the hUC-MSCs group showed abdominaldistention, while there were5in the PBS group and3in the simple TBI group.The difference was statistically significant (P<0.05) compared the hUC-MSCsgroup with the simple TBI group, but the difference between the hUC-MSCsgroup and the PBS group was statistically insignificant (P>0.05).The outcomes of mNSS (as table3showed) demonstrated that the scorechanged with the time, neural functional recovery in the hUC-MSCs groupwas better than both the PBS group and the simple TBI group, the differencewas statistically significant (P<0.05).Immunofluorescence is performed28days after hUC-MSCs graft. Thecells survived and migrated along the white matter, some of hUC-MSCs hadbeen settled around the vessels. A part of the grafted cells expressed Nestin asa marker of neural stem cells, MAP-2, NF, NSE as neuronal makers, and alsoexpressed astrocytoma cell marker GFAP and the Vascular endothelialcellmarker Ⅷ-R Ag. A lot of nucleus either in the PBS group or in the simpleTBI group were disintegrated and dissolved, but in the hUC-MSCs group itsnumber was limited.Conclusion:1hUC-MSCs transplantation following traumatic brain injury improvedthe neural functional recovery in rats.2hUC-MSCs could survive, migrated to the injured brain and expressedGFAP, NSE, MAP2, NF, Nestin and Ⅷ-R Ag.3hUC-MSCs may play a role in improving the function of digestion aftertraumatic brain injury.