BDNF Pretreatment of Human Embryonic-Derived Neural Stem Cells Improves Cell Survival and Functional Recovery After Transplantation in Hypoxic-Ischemic Stroke.

Intra-arterial neural stem cell (NSC) therapy has the potential to improve long-term outcomes after stroke. Here we evaluate if pretreatment of NSCs with brain-derived neurotrophic factor (BDNF) prior to transplantation improves cell engraftment and functional recovery following hypoxic-ischemic (HI) stroke. Human embryonic-derived NSCs with or without BDNF pretreatment (1 h, 100 ng/ml) were transplanted 3 days after HI stroke. Functional recovery was assessed using the horizontal ladder test. Cell engraftment was evaluated using bioluminescence imaging (BLI) and histological counts of SC121(+) cells. Fluoro-Jade C (FJC) and NeuN stains were used to evaluate neuroprotection. The effect of BDNF on NSCs was analyzed using a migration assay, immunocytochemistry, Luminex proteomic assay, and RT-qPCR.BLI analysis demonstrated significantly higher photon flux in the BDNF-treated NSC group compared to untreated NSC (p = 0.049) and control groups (p = 0.0021) at 1 week after transplantation. Immunohistochemistry confirmed increased transplanted cell survival in the cortex (p = 0.0126) and hippocampus (p = 0.0098) of animals injected with BDNF-treated NSCs compared to untreated NSCs. Behavioral testing revealed that the BDNF-treated NSC group demonstrated increased sensorimotor recovery compared to the untreated NSC and control groups (p < 0.001) over the 1-month period (p < 0.001) following transplantation. A significant improvement in performance was found in the BDNF-treated NSC group compared to the control group at 14, 21, and 28 (p < 0.05) days after transplantation. The cortex and hippocampus of the BDNF-treated NSC group had significantly more SC121(+) NSCs (p = 0.0125, p = 0.0098), fewer FJC(+) neurons (p = 0.0370, p = 0.0285), and a higher percentage of NeuN(+) expression (p = 0.0354) in the cortex compared to the untreated NSC group. BDNF treatment of NSCs resulted in significantly greater migration to SDF-1, secretion of M-CSF, VEGF, and expression of CXCR4, VCAM-1, Thrombospondins 1 and 2, and BDNF. BDNF pretreatment of NSCs results in higher initial NSC engraftment and survival, increased neuroprotection, and greater functional recovery when compared to untreated NSCs.

The relationship between serial [(18) F]PBR06 PET imaging of microglial activation and motor function following stroke in mice.

PURPOSE: Using [(18) F]PBR06 positron emission tomography (PET) to characterize the time course of stroke-associated neuroinflammation (SAN) in mice, to evaluate whether brain microglia influences motor function after stroke, and to demonstrate the use of [(18) F]PBR06 PET as a therapeutic assessment tool. PROCEDURES: Stroke was induced by transient middle cerebral artery occlusion (MCAO) in Balb/c mice (control, stroke, and stroke with poststroke minocycline treatment). [18 F]PBR06 PET/CT imaging, rotarod tests, and immunohistochemistry (IHC) were performed 3, 11, and 22 days poststroke induction (PSI). RESULTS: The stroke group exhibited significantly increased microglial activation, and impaired motor function. Peak microglial activation was 11 days PSI. There was a strong association between microglial activation, motor function, and microglial protein expression on IHC. Minocycline significantly reduced microglial activation and improved motor function by day 22 PSI. CONCLUSION: [18 F]PBR06 PET imaging noninvasively characterizes the time course of SAN, and shows increased microglial activation is associated with decreased motor function.

Ex vivo Evans blue assessment of the blood brain barrier in three breast cancer brain metastasis models.

The limited entry of anticancer drugs into the central nervous system represents a special therapeutic challenge for patients with brain metastases and is primarily due to the blood brain barrier (BBB). Albumin-bound Evans blue (EB) dye is too large to cross the BBB but can grossly stain tissue blue when the BBB is disrupted. The course of tumor development and the integrity of the BBB were studied in three preclinical breast cancer brain metastasis (BCBM) models. A luciferase-transduced braintropic clone of MDA-231 cell line was used. Nude mice were subjected to stereotactic intracerebral inoculation, mammary fat pad-derived tumor fragment implantation, or carotid artery injections. EB was injected 30 min prior to euthanasia at various timepoints for each of the BCBM model animals. Serial bioluminescent imaging demonstrated exponential tumor growth in all models. Carotid BCBM appeared as diffuse multifocal cell clusters. EB aided the localization of metastases ex vivo. Tumor implants stained blue at 7 days whereas gross staining was not evident until day 14 in the stereotactic model and day 28 for the carotid model. EB assessment of the integrity of the BBB provides useful information relevant to drug testing in preclinical BCBM models.

PET imaging of stroke-induced neuroinflammation in mice using [18F]PBR06.

PURPOSE: The purpose of this study is to evaluate the 18 kDa translocator protein (TSPO) radioligand [(18)F]N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline ([(18)F]PBR06) as a positron emission tomography (PET) imaging biomarker of stroke-induced neuroinflammation in a rodent model. PROCEDURES: Stroke was induced by transient middle cerebral artery occlusion in Balb/c mice. Dynamic PET/CT imaging with displacement and preblocking using PK111195 was performed 3 days later. PET data were correlated with immunohistochemistry (IHC) for the activated microglial markers TSPO and CD68 and with autoradiography. RESULTS: [(18)F]PBR06 accumulation peaked within the first 5 min postinjection, then decreased gradually, remaining significantly higher in infarct compared to noninfarct regions. Displacement or preblocking with PK11195 eliminated the difference in [(18)F]PBR06 uptake between infarct and noninfarct regions. Autoradiography and IHC correlated well spatially with uptake on PET. CONCLUSIONS: [(18)F]PBR06 PET specifically images TSPO in microglial neuroinflammation in a mouse model of stroke and shows promise for imaging and monitoring microglial activation/neuroinflammation in other disease models.

Timing of intra-arterial neural stem cell transplantation after hypoxia-ischemia influences cell engraftment, survival, and differentiation.

BACKGROUND AND PURPOSE: Intra-arterial neural stem cell (NSC) transplantation shows promise as a minimally invasive therapeutic option for stroke. We assessed the effect of timing of transplantation on cell engraftment, survival, and differentiation. METHODS: Mouse NSCs transduced with a green fluorescent protein and renilla luciferase reporter gene were transplanted into animals 6 and 24 hours and 3, 7, and 14 days after hypoxia-ischemia (HI). Bioluminescent imaging was used to assess cell survival at 6 hours and 4 and 7 days after transplantation. Immunohistochemistry was used to assess NSC survival and phenotypic differentiation 1 month after transplantation. NSC receptor expression and brain gene expression were evaluated using real-time reverse transcription-quantitative polymerase chain reaction to elucidate mechanisms of cell migration. Boyden chamber assays were used to assess cell migratory potential in vitro. RESULTS: NSC transplantation 3 days after HI resulted in significantly higher cell engraftment and survival at 7 and 30 days compared with all other groups (P<0.05). Early transplantation at 6 and 24 hours after HI resulted in significantly higher expression of glial fibrillary acidic protein (P=0.0140), whereas late transplantation at 7 and 14 days after HI resulted in higher expression of ß-tubulin (P<0.0001). Corroborating the high cell engraftment 3 days after HI was robust expression of vascular cell adhesion molecule-1, CCL2, and CXCL12 in brain homogenates 3 days after HI. CONCLUSIONS: Intra-arterial transplantation 3 days after HI results in the highest cell engraftment. Early transplantation of NSCs leads to greater differentiation into astrocytes, whereas transplantation at later time points leads to greater differentiation into neurons.

Intravascular stem cell transplantation for stroke.

Stroke is the third leading cause of death and the leading cause of adult disability in North America. Emphasis has been placed on developing treatments that reduce the devastating long-term impacts of this disease, and preclinical research on stem cell therapy has demonstrated promising results. However, questions about the optimal cell delivery method and timing of cell transplantation are not fully answered. Recent findings suggest that intravascular stem cell delivery is a safe and efficacious alternative to stereotactic cell injections. It also offers advantages should repeat treatments prove beneficial. Recent reports further suggest that intra-arterial injection results in a wider distribution of cells throughout the stroked hemisphere with a significantly greater cell engraftment compared to intravenous injection. In this review, we describe the benefits and potential risks associated with intravascular stem cell delivery and compare intra-arterial to intravenous cell transplantation methods. We discuss the importance of cell biodistribution and timing of transplantation in driving cell survival. We examine current proposed mechanisms involved in cell migration and functional recovery and discuss future directions for intravascular stem cell therapy research.