| Objective:To replicate the moderate traumatic brain injury in rats according toMarmarou’s falling model，(1) prove that whether transplant BMSCs enter thelesion brain, survive, and improve neurological functional recovery;(2)investigate the mechanism of BMSCs transplantation for TBI rats, includingthe secretion of neurotrophic factor and nerve tissue differentiation;(3)compare the difference between intracerebrally and intravenously BMSCstransplantation.Methods:1. Experimental animal and group：120SPF female SD rats (10weeksaging,280±20g weight)were divided into4groups randomly, including shamoperated group (sham group,n=30); TBI only (TBI group,n=30); transplantwith BMSCs intracerebrally (intracerebral treatment group,n=30); transplantwith BMSCs intravenously (intravenous treatment group,n=30). BMSC werewere obtained from donor SPF male SD-rats (3-4weeks aging), and the thirdgeneration were used in the experiment. Identification: under a microscope toobserve the morphology and arranged form of the cells.2. Model and Transplantation：The rats were established moderate traumaticbrain injury models according to Marmarou’s falling model. intracerebraltreatment group, rats injected with BMSCs (n=1×106,vol=10ul)intracerebrally(via the lateral ventricle)1day after TBI; intravenous treatmentgroup, rats injected with BMSCs (1×106,vol=0.5ml) intravenously (via the tailvein)1day after TBI.3. Detection of index and method：Randomly selected10rats from each group,Rotarod test and modified Neurological Severity Score [mNSS] were performed at1,2,3and4weeks after TBI.The other rats of each group weresacrificed at2and4weeks after TBI, The morphology changes of braintissues were investigated by HE staining; Western Blot and immunochemistrywere used to evaluate the expression level and regional distribution of Nervegrowth factor (NGF) and Brain-derived neurotrophic factor (BDNF) in thebrain tissues; To study the regional distribution and the differentiation ofnewly generating cells into neurons and astrocytes, Immunohistochemicaldouble-staining was used for Sry, MAP-2and GFAP.4. Quantitative assay and Statistical method：The immunohistochemicalstained sections were analyzed with the average positive cells detected byImage-ProPlus6.0digital medical image analysis system. The films ofwestern blot were analyzed with OD values using Image J image analysissoftware. For all statistical calculations, we used SPSS13.0for windows andone-way ANOVA. There was statistically significant if lateral p value <0.05.All data were presented as mean±SD.Results:1. Culture and identification of BMSCsPrimary adherent cells proliferated by a decentralized clone colony, and95%confluenced about10days later, then passaged. After passage, cells pastthe inhibition times and grew quickly, then each passage taked4-5days, andcell morphology became more and more uniform, like spindle, the cells werearranged parallel to the growth or swirling growth. Third generation ofBMSCs were used. Through cell growth characteristics and morphologicalidentificated BMSCs.2. Pathological changesHE staining showed a normal cortical brain in sham group, blood vessellumen was normal, blood vessel wall was smooth, nerve cells were completeform; In TBI group, it could be seen different degrees of subarachnoidhemorrhage, extensive range of edema in the cerebral cortex, lumen expansion,capillary congestion, nerve cells were swelling, messy distributed, ande even cells size shrunken and deep karyopyknosis dyed; compare with TBI group,the pathological changes were both slightly redused in transplant groups.3. The results of mNSS and Rotarod testThe results of mNSS and Rotarod test in sham group were normal;compared with sham group, in all the same time point of the other modelinggroups, the point of mNSS were increased and the time of Rotarod test wereshorter(P<0.05); compared with TBI group, in all the same time point of thetransplant groups, the point of mNSS were decreased and the time of Rotarodtest were extended(P<0.05); compared with intravenous treatment group, thepoint of mNSS of intracerebral treatment group were decreased at2,3and4weeks after TBI, the time of Rotarod test of intracerebral treatment groupwas extended at3weeks after TBI(P<0.05).4. NGF results in immunochemistry and western blotThe immunochemistry production of NGF located in the cytoplasm ofcortex neural cells and was stained yellow and brown. There was a smallamount of NGF expression in sham group; compared with sham group, in allthe same time point of the other modeling groups, the immunoreactivity ofNGF were significantly increased(P<0.05); compared with TBI group, in allthe same time point of the transplant groups, the immunoreactivity of NGFwere statistically increased(P<0.05); there was significant difference at everytime between intracerebral treatment group and intravenous treatmentgroup(P<0.05).Western blot indicated that the sham group has only little expression ofNGF, there were no changes in two point. The other western blot results ofNGF were similar to the results of immunochemistry(P<0.05).5. DNF results in immunochemistry and western blotThe immunochemistry production of BDNF located in the cytoplasm ofcortex neural cells and was stained yellow and brown too. There was a smallamount of NGF expression in sham group; compared with sham group, in allthe same time point of the other modeling groups, the immunoreactivity ofBDNF were significantly increased(P<0.05); compared with TBI group, in all the same time point of the transplant groups, in all the same time point ofthe transplant groups, the immunoreactivity of BDNF were statisticallyincreased(P<0.05); there was significant difference between intracerebraltreatment group and intravenous treatment group at2weeks afterTBI(P<0.05).Western blot indicated that the sham group has only little expression ofBDNF, there were no changes in two point. There was significant difference atevery time between intracerebral treatment group and intravenous treatmentgroup(P<0.05)；the other western blot results of BDNF were similar to theresults of immunochemistry(P<0.05).6. The results of double-label immunofluorescent stainingThe green fluorescent of Sry positive cells labeled by FICT accumulatedin the nucleus and the red fluorescent of MAP-2and GFAP labeled by TRITCaccumulated in the cytoplasm. We could not see any positive cell in the shamand TBI group; Sry positive cells were observed in all the same time point ofthe transplant groups, and some of Sry positive cells also expressed MAP-2orGFAP; the number of Sry positive cells were decreased at4week, and alsocould see some MAP-2or GFAP colocalization with them; compared withintravenous treatment group, there are more positive cells in intracerebraltreatment group; it could be observed that in the4week′s section ofintracerebral treatment group, there are some MAP-2positive cells alonewithout any Sry colocalization with them; and in the4week′s section ofintravenous treatment group, there are some GFAP positive cells alone withoutany Sry colocalization with them; some Sry positive cells alone were observedin the4week′s section of intravenous treatment group.Conclusion1. Through the way of lateral ventricle and tail vein transplanted BMSCstreatment of rats with TBI, the administered BMSCs could migrate to thelesion brain, survive at least4weeks, differentiate into neurons and astrocytesand promote neurological functional recovery. 2. The mechanism of BMSCs transplantation for TBI rats, including paracrineand autocrine some neurotrophic factor, like NGF and BDNF; differentiateinto neurons and astrocytes by themselves, promote endogenous neuralprecursor cells differentiate into neurons and astrocytes.3. Evaluation from neurological functional recovery, the secretion ofneurotrophic factor and neuronal differentiation, relative to the tail vein grafts,the therapy effect of lateral ventricle transplantation BMSCs is stronger. |
PDF Full Text Request