Bone Marrow Mononuclear Cells Orientates Differentiation In The Brain’s Impaired Region After Cerebral Ischemia Rats

Background and ObjectivesCerebral ischemic stroke is caused by occlusion of the cerebral artery, leading to focal ischaemia, destruction of neurons and glial cells, and motor, sensory or cognitive impairments. At present the most potential therapeutic strategy for the treatment of ischemic stroke is the transplantation of exogenous totipotent/pluripotent stem cells with self-renewal and differentiation capacity. BMMNCs is a heterogeneous mixture of cells, which is composed of mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), and progenitor and differentiated cells, endothelial progenitor cells (EPCs), et al. Compared with other types of stem cells, such as the embryonic stem cells and neural stem cells, BMMNCs has the advantages of easy extraction, only needingl.5-3h for obtaining, without culturing, autologous transplantation, no moral and ethical disputes. Therefore, it can be regarded as the most ideal promising multipotent candidate. The latest research demonstrates that BMMNCs in specific microenvironment can be included to differentiate into cardiomyocyte cells, liver cells, adipose cells, Schwann cells in vitro. Accumulating evidences have shown administration of BMMNCs in the treatment of ischemia stroke is safe and effective. The possible mechanism of above effects is that it can release a variety of neurotrophic factor research after BMMNCs transplantation according to some researches. However, the critical mechanisms are not yet completely known, and the BMMNCs’ differentiation fate of the specific microenvironment after cerebral infarction has not been reported. Herein, we will further evaluate behavioral performance, infarct volume, and learning and memory abilities after intravenous transplantation of BMMNCs in a rat model of ischemic stroke. Then it is further to detect oriented differentiation of BMMNCs in the impaired fields including sub-ventricular zone (SVZ), dentate gyrus (DG) of hippocampus and ischemic/penumbra (I/P) region in order to investigate therapeutic effect and its possible mechanism.Material and MethodsThe permanent middle cerebral artery occlusion models (pMCAO) were established by intraluminal vascular suture method, the successful models were randomly divided into the vehicle group (vehicle group) and BMMNCs transplantation group (BMMNCs group). Donor rats were intraperitoneally injected of BrdU prior to obtainment for marking BMMNCs. BMMNCs were separated by Ficoll-Paque density-gradient method. BMMNCs group was injected lml cell suspension containing1×107BMMNCs via the tail vein after24h, vehicle group was injected the same volume of PBS. After treatment with BMMNCs, Neurological severity scores was used to observate neural behavioral function at the1st,3rd,7th,14th,21th, and35th day, TTC staining was used to estimate infarct volume at the7th day, Morris water maze was used to test learning and memory abilities from the30th day to the35th day, Nissl staining was used to assess hippocampal neuron density, double immunofluorescent labeling was utilized to trace the BMMNCs and to observate it weather to orientedly differentiate into neuronal and glial cells in the SVZ, DG and ischemic/penumbra impaired niche at the35th day.Results1. mNSS and Morris Water Maze Compared with vehicle group, mNss of BMMNCs group was significantly lower (P<0.05). The difference in the mNSS between BMMNCs and control groups was significant at the7th,14th,21th, and35th day (P<0.05), but not at the1st and3rd day (P>0.05). There is a significant difference in the learning performance between control and transplanted groups in the4-day training session (P<0.05). The difference in the training session between BMMNCs and vehicle groups was significant at the3rd,4th day (P<0.05), but not at the1st and2nd day (P>0.05). BMMNCs-treated animals showed longer time swimming in the target quadrant (P<0.05) and crossed the imaginary platform more times (P<0.05) when compared to the vehicle group in a60-s probe test.2. TTC Staining There were significant reductions (P<0.01) in total infarct volume following transplantation of BMMNCs in MCAO animals at the7th day compared with the vehicle group by TTC staining.3. Nissl Staining As demonstrated by the quantitative analysis35days after treatment, in the CA1,CA3and dentate grus subfield of the hippocampus, the differences in neuronal density of BMMNCs-treated animals were statistically different than vehicle group (P<0.05).4. Double Immunofluorescent Labeling In the SVZ, DG and ischemic/penumbra impaired niche, some NeuN-positive, GFAP-positive, Ibal-positive cells were also showed BrdU immunoreactivity, suggesting neuronal differentiation from transplanted BMMNCs.Conclusions1. BMMNCs can home, migration to ipsilateral ischemia infarction, survive for35days in the SVZ, DG and I/P impaired region.2. BMMNCs can significantly promote neural functional recovery and reduce infarct volume in the treatment of cerebral ischemia.3. BMMNCs in the treatment of cerebral infarction can restore defective learning and memory abilities.4. BMMNCs can significantly mitigate hippocampal neuronal loss in the hippocampal CA1, CA3and DG subfield. The finding has been considered to be part of mechanisms responsible for restoring defective learning and memory abilities. 5. BMMNCs can give rise to neurons, astrocytes, microglias in the SVZ, DG and I/P impaired region. The finding has been considered to repair impaired brain issue and be part of mechanisms responsible for the therapeutic effectiveness.