بسم الله الرحمن الرحیم


Stem cell (SC) transplantation including both neural (NSCs) and mesenchymal (MSCs) shows the feasibility of the treatment of stroke by their survival and migration evidences and brain functional improvement; however their functional mechanism is still unknown.  One hypothesis is that transplanted hNSCs enhance normal repair mechanism of brain to minimise the ischemic effect of stroke through spontaneous brain plasticity by increasing of dendritic plasticity, axonal sprouting & rewiring caused with brain remapping due to secretion of some local & long distance  mediators. Another theory is the effect of human NSCs on axonal transport which means restoration of disrupted axonal circuits to transport the molecule or particles between bodies of the cells to the axon terminal synapsing other axon or gland during neurotransmission. Human neural progenitor cells (NPCs) were isolated from 13.5 weeks gestational age of aborted embryo cortical brain, sub cultured as neurosphers with enriched neural stem cell medium, passage producing   and dissociated to a single cell suspension by incubation at 37°C with Accutase, trypsin inhibitor and DNase followed by gentle trituration. In parallel, T cell-deficient rats were treated by antibiotics 3 days before to 7 days after surgery, anesthesied and its distal middle cerebral artery was blocked permanently with 0.5 hour bilateral common carotid occlusion. Later, human NPCs with initial dose 1x105 cells/µl were injected into ipsilesional cortex of rats locally in day 7 after stroke induction, and then they were divided into 2 groups of case and control (injecting saline instead of NPCs) with free access to food and water. To consider functional ability of rat’s brain, their behaviour was tested before for baseline performance and after surgery for 6 weeks post stroke weekly by an investigator blinded with  vibrissae-elicited forelimb placing test, cylinder test, elevated body swing test and postural reflex test. After Rat’ sacrifice, brain and cervical cord removed and 20-30µm coronal sections cut followed by immunostaining assay using primary & secondary antibodies and confocal imaging. Through Z-stacks capturing, nuclear antigen positive cells in peri-infarct area for NPCs differentiation markers including Nestin, ß tubulin III, glial fibrillary acidic protein, NG2 & Olig2 were scored in a blinded condition (tissue processing and immunostaining in vitro). At week 3 & 12 after transplantation, the reaction between mouse anti-neural filament (anti-SMI312) antibody and rabbit anti-amyloid precursor protein was measured through Immunohistochemistry for axonal amyloid precursor protein performing, and confocal imaging was captured in 2 regions of ipsi and contralesional genu of the corpus callosum (axonal transport analysis in vivo). For dendritic analysis in vitro, 2 & 4 weeks after transplantation the brains were removed, stained with Rapid GolgiStainTM kit in order to evaluation of layer V pyramidal neurons between the lesion and the human NPCs grafted region. Also dendritic length and number of dendritic branches were calculated. At the same time, antrograde axonal tracer biotinylated dextran amine (BDA) was injected into the contralesional regions, and after 5 min lefting the needle in situ it was removed slowly followed by an immunohistochemistry processing after 1 week in order to BDA detection with streptavidin-conjugated antibody and confocal imaging (Axonal tracing in vivo).                                                      Brain 2011: 134; 1777–1789