Delayed and repeated intranasal delivery of bone marrow stromal cells increases regeneration and functional recovery after ischemic stroke in mice

Abstract

Background: Stroke is a leading cause of death and disability worldwide, yet there are limited treatments available. Intranasal administration is a novel non-invasive strategy to deliver cell therapy into the brain. Cells delivered via the intranasal route can migrate from the nasal mucosa to the ischemic infarct and show acute neuroprotection as well as functional benefits. However, there is little information about the regenerative effects of this transplantation method in the delayed phase of stroke. We hypothesized that repeated intranasal deliveries of bone marrow stromal cells (BMSCs) would be feasible and could enhance delayed neurovascular repair and functional recovery after ischemic stroke.

Results: Reverse transcription polymerase chain reaction and immunocytochemistry were performed to analyze the expression of regenerative factors including SDF-1α, CXCR4, VEGF and FAK in BMSCs. Ischemic stroke targeting the somatosensory cortex was induced in adult C57BL/6 mice by permanently occluding the right middle cerebral artery and temporarily occluding both common carotid arteries. Hypoxic preconditioned (HP) BMSCs (HP-BMSCs) with increased expression of surviving factors HIF-1α and Bcl-xl (1 × 10⁶ cells/100 μl per mouse) or cell media were administered intranasally at 3, 4, 5, and 6 days after stroke. Mice received daily BrdU (50 mg/kg) injections until sacrifice. BMSCs were prelabeled with Hoechst 33342 and detected within the peri-infarct area 6 and 24 h after transplantation. In immunohistochemical staining, significant increases in NeuN/BrdU and Glut-1/BrdU double positive cells were seen in stroke mice received HP-BMSCs compared to those received regular BMSCs. HP-BMSC transplantation significantly increased local cerebral blood flow and improved performance in the adhesive removal test.

Conclusions: This study suggests that delayed and repeated intranasal deliveries of HP-treated BMSCs is an effective treatment to encourage regeneration after stroke.

Keywords: BMSC; Hypoxic preconditioning; Intranasal; Ischemic stroke; Trophic factors.

Fig. 1

Experimental timeline. Focal ischemic stroke was induced in adult male mice on “day 0”. Three days after stroke, a total volume of 100 μl of BMSC suspension (~ 1 × 10⁶ cells) or control media was administered via the intranasal route at 3, 4, 5, and 6 days after stroke. Starting on the same day as BMSC administration, all mice received daily BrdU injections intraperitoneally (i.p) until the day of euthanasia. Laser-Doppler scanning was performed 21 days after stroke to measure changes in the local blood flow in penumbra. The adhesive removal test of sensorimotor function was performed at 7 and 14 days after stroke. Immunohistochemistry was performed at 14 days after stroke to analyze regeneration

Fig. 2

Regenerative factors expressed in BMSCs. Immunocytochemistry was performed in cultured BSMCs for the expression of several regenerative factors. A–D Expressions of SDF-1α, VEGF, CXCR4, and FAK were detected in BMSCs. Scale bars represents 20 μm. E Reverse transcription PCR analysis confirms the expression of SDF-1α, VEGF, CXCR4, and FAK in four different batch of BMSCs

Fig. 3

Hypoxic-preconditioning increases survival factors in BMSCs. BMSCs were subjected to 24 h hypoxic preconditioning (0.1–0.3% oxygen) and 1 h of reoxygenation. RT-PCR analysis was used to detect the mRNA level of two key genes for cell survival, HIF-1α and Bcl-xL. a HIF-1α in HP-BMSCs was increased compared to control BMSCs. N = 3, p = 0.013; student’s t test. b Increased Bcl-xL after HP treatment compared to control BMSCs. N = 3, *p < 0.05, student’s t test

Fig. 4

Intranasally delivered BMSCs migrated to the peri-infarct region of the ischemic brain. A–C Brain sections at the stroke penumbra region were collected and analyzed to examine cell presence of BMSCs delivered intranasally. Hoescht-positive BMSCs (blue) counterstained with Propidium Iodide (PI, red) were detected in the peri-infarct area of the cortex at 6 and 24 h after BMSC delivery at 3 days after stroke. Scale bars = 20 μm. D An illustration of the mouse ischemic brain where the cells were found in the cortex

Fig. 5

Intranasally delivered BMSC increased neurogenesis and angiogenesis in the ischemic Brain. A, B Animals were euthanized 14 days after stroke with and without BMSC treatment. Immunohistochemistry stained for BrdU (red), NeuN (green), Glut-1 (blue). Arrows point to the presence of co-labeled cells in the peri-infarct region 14 days post stroke. BrdU/NeuN co-labeled cells indicate the presence of proliferating neuronal cells. BrdU/Glut-1 co-labeled cells indicate the presence of proliferating blood vessel cells. Scale bars = 40 μm. C, D Enlarged image to show a colabeled NeuN and BrdU cell and the counting result of these cells is shown in the bar graph. There was a significant increase in the total number of NeuN/BrdU co-labeled cells in the BMSC treatment group compared to control. N = 5–6, p = 0.024; student’s t test. Scale bar = 10 μm. E, F Enlarged image shows Glut-1 and BrdU double positive endothelia cells. There was an increase in Glut-1/BrdU co-labeled cells in the HP-BMSC treatment compared to control. N = 5–6, *p < 0.05, student’s t test. Scale bar = 10 μm

Fig. 6

BMSC transplantation increased local cerebral blood flow. Laser-Doppler blood perfusion monitor (PeriFlux System 5000-PF5010 LDPM unit, Perimed, Stockholm, Sweden) was used to measure changes of local cerebral blood flow in the penumbra. a Laser scanned images of the stroke penumbra before, during and 21 days after cerebral ischemia in control and BMSC treatment groups. b Quantified data of flow measurement 21 days after stroke show similar flow reduction during the ischemic surgery but better restoration of the local cerebral blood flow in BMSC treatment animals compared to controls. N = 5–10, *p < 0.05, two-way ANOVA

Fig. 7

BMSC transplantation increased functional recovery after stroke. The adhesive removal test was used to assess the sensorimotor impairment after ischemic stroke. The test on both control and treatment groups was performed at 3 days before stroke, and 7 and 14 days after stroke. The BMSC treatment group at 14 days displayed significant improvement (shorter time) to remove the sticky dot compared to control mice. N = 8, *p = 0.0503, two-way ANOVA