Treatment Of Traumatic Brain Injury In Rats With Intravenous Administration Of Bone Marrow Stromal Cells

OBJECTIVE1. This study of the first part was designed to investigate how to separate and culturein vitro bone marrow stromal cells (BMSCs).2. This study of the second part was designed to observe whether BMSCs promotecell regeneration and cell differentiation, up-regulate BNP levels and improve thefunctional outcome of rats subjected to traumatic brain injury (TBI).METHODS1. Wistar rats (60～90g) of clean degree were given intraperitoneal injections of5-fluorouracil (150 mg/kg) 3 days before bone marrow was harvested to killperipheral blood cells and to stimulate the proliferation of bone marrow cells.After the donor rats were killed and the marrow was obtained, the treated cellswere cultured in the flasks containing the Dulbecco's minimum essential medium.Cells were counted by use of a haemacytometer and a counter under a phasecontrast microscope. Cell surface markers were assessed using flow cytometer.2. Before transplantation, the third generation BMSCs was incubated withbromodeoxyuridine (BrdU, 3μg/ml) to label the nuclei for 3 days, then lifted by0.25% trypsin.After centrifugation,the cell sediment was suspended by PBS.BMSCs were counted by use of a haemacytometer and a counter under a phasecontrast microscope. Cells of 3×10~6/ml were prepared for transplantation.3. Totally 72 3-month-old adult Wistar rats were randomly divided into: Sham group:rats (n=24) were not subjected to injury except cutting of the scalp and removal ofthe same amount of skull as in the experimental group; TBI control group: rats (n=24) were subjected to TBI and lml phosphate-buffered saline (PBS) wasinjected into a tail vein at 1 day after TBI; BMSCs treatment group: after washingwith PBS and centrifugation, BMSCs (3×10~6) in 1 ml of PBS were injected into atail vein at 1 day after TBI (N=24). Before transplantation, BrdU (3μg/ml) wasadded into the medium for 3 days before BMSCs administration and aftertransplantation, all groups received an intraperitoneal injection of BrdU 50 mg/kgtwice a day for 15 days in order to label the newly generated cells. To study thedifferentiation of newly generating cells, Rats treated with BMSCs or PBS wereeuthanized at 15d after TBI. Coronal brain Sections were used forimmunohistochemical staining. Single staining was used for identification ofnewly generated BrdU-positive cells with diaminobenzidine (DAB). Doublefluorescent staining was performed in the coronal cerebral sections for phenotypicidentification of the newly generated cells with fluorescein isothiocyanate(FITC)and Cy5.At the same time, preinjury and at 3d, 7d (n=6 per time point pertreatment group) after TBI, total RNA was isolated from the brain to measure theexpression of brain natriuretic peptide (BNP). preinjury and at 3d, 7d after TBI,the plasma concentration of BNP was measured using RIA. Neurological functionof the rats were evaluated using a modified neurological severity score preinjuryandat2d, 5d, 8d, lldand 15dafterTBI.RESULTS1. When cultured to the third generation, the BMSC marker CD90-positieve cellsreached to 91.5%±1.87% and CD45-positieve cells decreased from 73.3%±2.16%to 5.8%±1.47%.2. Treatment with 3×10~6 BMSCs significantly improved the functional outcome ofrats Statistically, the modified neurological severity score (mNSS) for theBMSC-treated group were significantly decreased at day 8 (p＜0.05),day 11 (p＜0.05) and day 15 (p＜0.05) compared with PBS-treated group.3. In the BMSC-treated group the newly generated cells in boundary zone(BZ), hippocampal zone (HZ) and subventricular zone(SVZ) of both hemispheres weresignificantly increased compared with PBS-treated group. In the PBS-treatedgroup,the BrdU-positive cells were also increased significantly compared withsham group.4. Compared with PBS-treated group, the number of newly generated cells expressedcell surface marker such as Tuj 1,Doublecortin and NeuN was significantlyincreased (p＜0.05).5. Compared with the sham group, the BNP mRNA level of both the BMSC-treatedgroup and PBS-treated group were up-regulated significantly at day 3 (p＜0.05)and day 7 (p＜0.05). The BNP mRNA level of the BMSC-treated group wasincreased obviously compared PBS-treated group at day 3 (p＜0.05) and day 7(p＜0.05).6. At day 3 and day 7 the plasma BNP concentration of the BMSC-treated group wasincreased significantly compared with the PBS-treated group (p＜0.05); At thesame time, the plasma BNP concentration of the PBS-treated group was alsoincreased significantly compared with the sham group at day 3and day 7 (p＜0.05).CONCLUSIONS1. The third generation of the BMSCs can be used as the seed-cell resource for celltransplantation;2. Intravenous transplantation of BMSCs into rats after TBI induces cell proliferationand cell differentiation, which may enhance neurological function recovery. At thesame time, some anti-edema and vasoactive factors promoted by BMSCs, such asBNP may contributed to the treatment effects.