| Background and PurposeCellular therapy has emerged as a potential novel treatment for ischemic stroke. Promising progress in the safety and efficacy of the therapy has been made in preclinical and clinical studies using bone marrow mesenchymal stem cells (BMSCs) and other cell types. Current experimental and clinical research on BMSC stroke therapy utilizes two approaches:local delivery and systemic administration. Local delivery directly targets cells in the injured brain region but the high degree of invasiveness may cause additional brain damage. Systemic delivery can be achieved by intravenous (i.v.) which results in massive cell entrapment in capillary beds of other periphery organs with only a small amount of cells being able to reach the brain. Injection of stem cells via the intra-arterial route could result in a reduction of cerebral blood flow and microembolization within the cerebrovasculature.Intranasal administration is a noninvasive and convenient drug delivery method that bypasses the blood brain barrier (BBB) and directly guides therapeutics to the central nervous system (CNS). Studies have shown that intranasally administered BMSCs can migrate to different brain regions utilizing pathways along olfactory and trigeminal nerves innervating the nasal passages with the highest cell number in the brain.In the present investigation, we tested a delayed treatment with non-invasive and brain targeted intranasal delivery of BMSCs in a mouse focal cerebral ischemia model. The investigation tested the feasibility and effectiveness of intranasal delivery of BMSCs to alleviate the ischemic cortex. Hypoxic preconditioned (HP) of BMSCs was performed before transplantation in order to promote their survival, migration and homing to the ischemic brain region after intranasal transplantation.MethodsBMSCs were isolated and cultured from the legs of the baby rats and their population were characterized using CD105, CD73, CD34, and CD45 markers. A focal cortex ischemic model was used in this study. Hoechst dye-labeled BMSCs (1×106 cells/animal) were delivered intranasally 24 hrs after stroke. At 1.5 hrs after delivery, brains were sliced into 20μm sections and counterstained with propidium iodide (PI, red) to reveal the whole cell population of the brain. To examine homing capacity, the PI and Hoechst double labeled cells (magenta) were quantified from bregma 1.10 to-2.10 mm. Immunofluorescence staining of collagen Ⅳ for vessels was used in order to determine the location of pre-labeled cells in brain tissue with the respect to brain vessels.Hypoxic precondition (HP) of BMSCs was performed before transplantation in order to promote their survival, migration and homing to the ischemic brain region after intranasal transplantation. Western blot, scratch assay, transwell assay was performed to test BMSCs directional migration ability towards injury induced signals or chemoattrant and possible mechamisms after hypoxia preconditioning in vitro.To test the neuroprotective effects of intranasally delivered BMSCs, infarct size was assessed 72 h after intranasal delivery using Triphenyl Tetrazolium Chloride (TTC) staining. TUNEL staining was used to visualize DNA fragmentation and cell death around the boundary of ischemic core. Adhesive-removal tests were performed before and at 1 and 4 days after stroke to evaluate the neurological function deficits.ResultsCells reached the ischemic cortex and deposited outside of vasculatures as early as1.5 hrs after administration. HP-treated BMSCs (HP-BMSCs) showed a higher level of expression of proteins associated with migration, including CXC chemokine receptor type 4 (CXCR4), matrix metalloproteinase 2 (MMP-2), and MMP-9. HP-BMSCs exhibited enhanced directional migratory capacities in vitro and dramatically enhanced homing efficiency to the infarct cortex when compared with normoxic cultured BMSCs (N-BMSCs). Three days after transplantation and 4 days after stroke, both N-BMSCs and HP-BMSCs decreased cell death in the peri-infarct region; significant neuroprotection of reduced infarct volume was seen in mice that received HP-BMSCs. In adhesive-removal test of sensorimotor functional assay performed 3 days after transplantation, HP-BMSC-treated mice performed significantly better than vehicle-treated animals.ConclusionThese data suggest that, subacute intranasal administration of stem cells is feasible in the treatment of stroke. HP pretreatment and intranasal delivery of BMSCs are two feasible and effective strategies for improving the clinical potential of cell transplantation therapy. |
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