The Effects Of Intravenous Transplantation Of Bone Marrow Stem Cells On Cell Autophagy In Rats With Traumatic Brain Injury

Object: To explore the effects of intravenous transplantation of bonemarrow stem cells (BMSC) on cell autophagy in rats with traumatic braininjury.Methods:①T he isolation and culture of bone marrowstem cells: Thewhole bone marrow adherent screening method was used to get BMSC in5weeks old Sprague Dawley (SD) rats, then the BMSCs were cultured andproliferated in viro. Moreover, cell growth and morphological changes wereobserved using inverted phase contrast microscope.②Rat models withmoderate diffuse brain injury were made using Marmarous method. A copperRod weighting450g fell freely from a height of1.2m to the head of rats tomake the traumatic brain injury (TBI) models.③One hundred and twenty SDmale rats weighting280±20g were divided randomly into four groups: Shamgroup, TBI group, TBI+PBS group and TBI+BMSCs group. Each of the fourgroups had30rats. Equal volume of PBS and BMSCs (3×106/ml) weretransplanted into the tail vein of rats respectively24hours after the modelswere successfully done. This was not done to the Sham and TBI groups. Allthe four groups were tested in five time points, such as the1,3,5,7and14day after the transplantation. Moreover, another40SD female rats wereequally divided into4groups, then Morris water maze test were taken out onthe7,8and9day after the transplantation.④The alteration of BMSCmorphology was observed using inverted phase contrast microscope.Neurological deficit scores were used to assess the function of nerve in rats.Immunofluorescence assay was used to test the sex-determining gene SRY toconfirm whether BMSCs migrate to the brain. Brain structure and pathologicalchanges were observed using HE staining. Western-blot was adopted to testthe expression of LC3and Beclin-1which were the indicators of autophagy. Immunohistochemistry was used to detect the immunoreactivity and tissuelocalization of Beclin-1. Morris water maze test was adopted to test the abilityof memory and learning in rats.Results:1The culture results of BMSCs: The results of whole bone marrowadherent screening in vitro are satisfactory, cells were in the third generation12-14days after passing on from generation to generation, and fused into asingle layer, swirling arranged with obvious directionality and a single form.2The NSS results: With the extension of time after transplantation, therats in each group general condition gradually improved NSS scores graduallydecreases, and The Sham group rats neurological were normal scores were0.The NSS in each group of TBI at each time point were significantly higherthan sham group, and the difference was significant (P<0.01);1d after thetransplanted, in TBI group, PBS group and BMSCs group, the NSS was nosignificant difference (P>0.05); from3d after transplantation, the NSS ofBMSCs group was lower than the TBI and PBS group, the difference wassignificant (P<0.05), and the difference were more significant at5d and later(P<0.01); in both TBI group and PBS group, the NSS was no significantdifference (P>0.05)3Morris water maze results:7，8and9day after transplantation, Thesearch refuge islands incubation period in TBI group were significantlyprolonged compared to the Sham group(p<0.05), the incubation period ofBMSCs group was shorter than the TBI and PBS group, the difference wassignificant (P<0.05). In addition, search moving tracks revealed that BMSCsgroup search strategy better than the TBI group and PBS group.4The confocal laser markers of the Y chromosome results: under afluorescence microscope, the positive expression of Y chromosome shown ingreen.1d after transplantation, the brain tissue of the BMSCs group rats havecells of Scattered distribution of green-labeled, The positive cells can bedetecded at14days after the transplantation,but the number is less than1d after transplantation；and the positive results were not seen in the other threegroups.5The light microscope morphological results: in the Sham group, a largenumber of brain cells and basic integrity of the structure, cell morphologyrules presents a round or oval, nucleoli and nuclear membrane were clear,nucleoli was uniformly blue, nuclear membrane integrity, and cytoplasmicwas red. Traumatic brain tissue in rats can be seen varying degrees of braininjury, and the emergence of significant congestion/hemorrhage stove, theexpanded capillaries and brain edema, the emergence of neuron degenerationand necrosis, can be seen swelling of nerve cells, nuclei vacuolization andnuclear body shrinkage, cytoplasm lightly stained or in the form of ballooning,cell gap widened. BMSCs group compared the PBS and TBI groups, thechanges were mitigated. There was no significant difference in the TBI groupand PBS group.6The western blot test results: the western blot analysis showed that LC3and beclin-1protein was expressed at a low level in the cortex in the shamgroup, and are weaker than the damaged group, the difference wassignificant(p<0.05)；Each injury group can see the LC3-Ⅱ/LC3-Ⅰ values andBeclin-1expression enhanced and at5d post-transplant，the both expressionwere highest, and then gradually weakened; from3d after transplantation, theboth expression in the BMSCs group were weaker than the TBI and PBS twogroups, the difference was significant(p<0.05). The both protein expressionshowed no different in TBI group and PBS group.7The immunohistochemical detection results: in Sham group at eachdetection time Beclin-1have the weak-positive expression, and each timepoint did not change the trend, Its expression is weaker than the damagedgroup, The difference was significant(p<0.05); Each injury group have clearlypositive cells, and5d after transplantation the expression were the highest,Then gradually weakened; from3d after transplantation, the positiveexpression in the BMSCs group weaker than the TBI and PBS two groups, the difference was significant(p<0.05); the positive expression in both TBI groupand PBS group showed no difference.Conclusion:BMSCs transplanted by tail vein could penetrate theblood-brain barrier, reach to the injuried regions and survive at least14days.Which could improve nerve function and learning and memory abiliy.Depression of the degree of neuronal autophagy maybe is one of themechanisms after TBI.