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Magnetic particle imaging is an emerging tomographic technique with the potential for simultaneous high-resolution, high-sensitivity, and real-time imaging. Magnetic particle imaging is based on the unique behavior of superparamagnetic iron oxide nanoparticles modeled by the Langevin theory, with the ability to track and quantify nanoparticle concentrations without tissue background noise. It is a promising new imaging technique for multiple applications, including vascular and perfusion imaging, oncology imaging, cell tracking, inflammation imaging, and trauma imaging. In particular, many neuroimaging applications may be enabled and enhanced with magnetic particle imaging. In this review, we will provide an overview of magnetic particle imaging principles and implementation, current applications, promising neuroimaging applications, and practical considerations.

Background: Hyperglycemia occurs in over 40% of ischemic stroke patients, which induces hemorrhagic transformation (HT) and worsens stroke outcomes. The management of hyperglycemia with insulin did not show favorable outcomes. Thus, strategies for managing hyperglycemia-exacerbated stroke injury are urgently needed. We previously demonstrated that ischemic postconditioning (IPostC) (repeated transient interruption of cerebral blood flow during reperfusion) can reduce brain infarct size and improve neurological outcomes. In this study, we hypothesized that IPostC can reduce HT in ischemic stroke with acute hyperglycemia. Method: Male mice were subjected to middle cerebral artery occlusion (MCAO) for 1 hour, followed by reperfusion to mimic ischemic stroke. Glucose was injected before MCAO to induce hyperglycemia. IPostC was initiated upon reperfusion with 3 cycles of 30-second reperfusion followed by 10 seconds of MCA occlusion. Brain infarct was visualized by TTC staining and quantitated using Image J. Hemorrhagic transformation was evaluated by hemorrhagic scores. Result: Acute hyperglycemia significantly increased the brain infarct size (by 25%, p Conclusion: Our findings suggest that IPostC can counteract the effects of acute hyperglycemia and reduce brain injury, edema and HT after stroke. Grant/Other Support: NIH Grant R01NS064136C

Stroke is a leading cause of long-term disability and death in the united states. The development of new therapies for stroke are sorely needed. There is great hope that stem cell therapy will create a paradigm shift in the treatment of stroke patients. A barrier to ensuring clinical success of stem cell therapy is the paucity of understanding of the mechanisms by which stem cells exert their beneficial effects. Using a novel mRNA purification method, we identified 50 genes encoding extracellular space proteins, expressed by human neural stem cells (hNSCs) whose expression positively correlated with functional recovery. In this study, we focus on one of the paracrine factors from grafted hNSCs that correlated best with functional recovery, to investigate its therapeutic potential in promoting recovery after stroke. Male nude rats underwent stroke using the distal middle cerebral artery occlusion (dMCAo) model. One week following stroke, osmotic pumps were prepared and loaded with recombinant MTN-2. The osmotic pumps were inserted into the peri-infarct area and infused recombinant MTN-2 for 5 days. Post-stroke, animals were assessed for functional recovery for 5 weeks using both the Montoya staircase test and the whisker-paw reflex test to assess for forelimb function, dexterity, side bias, and placing deficits. After 5 weeks, brain tissue was isolated to assess glial cell morphology. Brain sections were stained with GFAP and IBA1 to visualize astrocytes and microglia, respectively. Confocal images were processed and analyzed using the Bitplane Imaris image analysis software. Output measurements of number of cells/mm2, cell volume, cell branching, and process length and thickness were obtained to characterize the changes in astrocytic and microglial response to injury and paracrine factor treatment. By identifying paracrine factors that are responsible for the regeneration of brain tissue following implantation of hNSCs in stroke brain, this work will increase the likelihood of successful clinical translation of stem cell therapy for stroke. Moreover, elucidating these molecular pathways important for brain recovery may ultimately identify novel therapeutic targets and offer hope to millions of Americans who live with the devastating effects of stroke.

Stroke is one of the major causes of chronic disability worldwide and increasing efforts have focused on studying brain repair and recovery after stroke. Following stroke, the primary injury site can disrupt functional connections in nearby and remotely connected brain regions, resulting in the development of secondary injuries that may impede long-term functional recovery. In particular, secondary degenerative injury occurs in the connected ipsilesional thalamus following a cortical stroke. Although secondary thalamic injury was first described decades ago, the underlying mechanisms still remain unclear. We performed a systematic literature review using the NCBI PubMed database for studies that focused on the secondary thalamic degeneration after cortical ischemic stroke. In this review, we discussed emerging studies that characterized the pathological changes in the secondary degenerative thalamus after stroke; these included excitotoxicity, apoptosis, amyloid beta protein accumulation, blood-brain-barrier breakdown, and inflammatory responses. In particular, we highlighted key findings of the dynamic inflammatory responses in the secondary thalamic injury and discussed the involvement of several cell types in this process. We also discussed studies that investigated the effects of blocking secondary thalamic injury on inflammatory responses and stroke outcome. Targeting secondary injuries after stroke may alleviate network-wide deficits, and ultimately promote stroke recovery.

Identifying the secretome of grafted stem cells is an important first step to elucidate how the graft communicates with the host brain, at the molecular level , to enhance post-stroke recovery. Here we use Translating Ribosome Affinity Purification (TRAP) to uncover the secretome of grafted human neural stem cells (hNSCs) and identify potential efficacious stem cell factors. Nude rats were subjected to permanent distal cerebral artery occlusion; 7d later hNSCs expressing GFP under a ribosomal-specific promoter were transplanted into the cortex of stroke (S) and naive (N) rats. The graft site was dissected 7d later and processed using TRAP to isolate ribosome-bound RNA from the grafted hNSCs. TRAP-isolated RNA was subjected to RNA sequencing and 479 differentially expressed genes were found between hNSC grafts in S and N brains. Of these, 21 genes encode secreted proteins, 14 of which were upregulated in hNSCs grafts in the stroke-injured brain. Pearson Correlation analysis revealed that the expression level of 9 of the 14 genes positively correlated with functional recovery. Gene Ontology (GO) analysis of the 14 genes identified the top enriched biological process as Response to Wounding which is related (i.e. a direct ancestor in the GO tree) to inflammatory response and axon injury. Three genes were present in this GO term: NOG, MATN2 and WNT1. MATN2 encodes matrilin-2, a protein known to be involved in axon regeneration and inflammation. In situ hybridization and immunohistochemistry showed high expression of matrilin-2 in grafted hNSCs. To test if matrilin-2 could affect post-stroke recovery we infused human recombinant matrilin-2 or vehicle into the cortex of stroke-injured rats. Seven days post-infusion, rats receiving matrilin-2 showed greater behavior recovery than the vehicle group. As matrilin-2 promotes axon outgrowth we investigated if it activated Rac-1 GTPase, which is associated with axon sprouting. Recombinant matrilin-2 increased activation of Rac-1 in primary mouse neural cultures, compared to the control. Our data show that grafted hNSCs in the stroke-injured brain express MATN2 and that secreted matrilin-2 may be involved in hNSC-induced stroke recovery potentially via activation of Rac-1 GTPase and axon sprouting.

Human neural stem cell (hNSC) transplantation improves recovery in preclinical stroke models. However their effects on surviving sensorimotor circuits are not well understood. Here we performed a comprehensive electrophysiological assessment and RNA-seq analysis of the stroke-injured rat cortex after transplantation of two hNSC lines, G010 (fetal-derived) and NR1 (hES-derived). Vehicle, G010, or NR1 cells were transplanted into the ischemic cortex of Nude rats 1 wk after distal middle cerebral artery occlusion. Neurological recovery was accessed by the Whisker-paw test. Acute brain slices were prepared 1 wk post-transplantation for electrophysiological recording. A linear multichannel recording probe was placed in the peri-infarct motor cortex and local field potentials (LFPs) recorded simultaneously from all cortical layers following circuit activation in layer 2/3. For RNA-seq analysis, the transplantation area was dissected, RNA extracted and cDNA libraries prepared for RNA-seq. G010 and NR1 cells enhanced post-stroke behavioral recovery starting 1 or 3 wks post-transplantation, respectively. Current source density (CSD) analysis of evoked LFPs, a method used to more accurately localize synaptic currents, revealed that both G010 and NR1 cells restored circuit excitability in layer 2/3, through reduction in inhibitory/excitatory (I/E) balance. However, the mechanisms driving the I/E balance shift were different, with NR1 cells enhancing excitation while G010 cells reduced inhibition. RNA-seq analysis of the cortex revealed that stem cell- versus vehicle-treated animals had different gene expression patterns in all cortical layers. Moreover, NR1 and G010 cells affected different genes. Layer 2/3-specific genes significantly affected by G010 grafts include Arpp21, Enpp2, Gbe1, Mylk, Pea15, Pkig, Rcn1, Slit3, Uchl1 and Wfs1; while NR1 grafts altered expression of Hapln4, Kcnc1, Ppp1r1b, Scn1a, Slit3 and Slitrk1. GO analysis revealed that both stem cell treatments activated critical canonical pathways e.g. Actin Cytoskeleton Signaling and Synaptic Long Term Depression. Stem cell transplantation modulates host gene expression and this is associated with increased circuit excitability and motor-sensory function.

Currently, no medical therapies exist to augment stroke recovery. Stem cells are an intriguing treatment option being evaluated, but cell-based therapies have several challenges including developing a stable cell product with long term reproducibility. Since much of the improvement observed from cellular therapeutics is believed to result from trophic factors the stem cells release over time, biomaterials are well-positioned to deliver these important molecules in a similar fashion. Here we show that essential trophic factors secreted from stem cells can be effectively released from a multi-component hydrogel system into the post-stroke environment. Using our polymeric system to deliver VEGF-A and MMP-9, we improved recovery after stroke to an equivalent degree as observed with traditional stem cell treatment in a rodent model. While VEGF-A and MMP-9 have many unique mechanisms of action, connective tissue growth factor (CTGF) interacts with both VEGF-A and MMP-9. With our hydrogel system as well as with stem cell delivery, the CTGF pathway is shown to be downregulated with improved stroke recovery.

Exogenous human neural progenitor cells (hNPCs) are promising stroke therapeutics, but optimal delivery conditions and exact recovery mechanisms remain elusive. To further elucidate repair processes and improve stroke outcomes, we developed an electrically conductive, polymer scaffold for hNPC delivery. Electrical stimulation of hNPCs alters their transcriptome including changes to the VEGF-A pathway and genes involved in cell survival, inflammatory response, and synaptic remodeling. In our experiments, exogenous hNPCs were electrically stimulated (electrically preconditioned) via the scaffold 1 day prior to implantation. After in vitro stimulation, hNPCs on the scaffold are transplanted intracranially in a distal middle cerebral artery occlusion rat model. Electrically preconditioned hNPCs improved functional outcomes compared to unstimulated hNPCs or hNPCs where VEGF-A was blocked during in vitro electrical preconditioning. The ability to manipulate hNPCs via a conductive scaffold creates a new approach to optimize stem cell-based therapy and determine which factors (such as VEGF-A) are essential for stroke recovery.

Stroke is the leading cause of adult disability and the fourth most common cause of death in the United States. Inflammation is thought to play an important role in stroke pathology, but the factors that promote inflammation in this setting remain to be fully defined. An understudied but important factor is the role of meningeal-located immune cells in modulating brain pathology. Although different immune cells traffic through meningeal vessels en route to the brain, mature mast cells do not circulate but are resident in the meninges. With the use of genetic and cell transfer approaches in mice, we identified evidence that meningeal mast cells can importantly contribute to the key features of stroke pathology, including infiltration of granulocytes and activated macrophages, brain swelling, and infarct size. We also obtained evidence that two mast cell-derived products, interleukin-6 and, to a lesser extent, chemokine(C-C motif) lig and 7, can contribute to stroke pathology. These findings indicate a novel role for mast cells in the meninges, the membranes that envelop the brain, as potential gatekeepers for modulating brain inflammation and pathology after stroke Refereed/Peer-reviewed

Stem cell therapies can promote neural repair and regeneration, yet controversy regarding optimal cell source and mechanism of action has slowed clinical translation, potentially due to undefined cellular heterogeneity. Single-cell resolution is needed to identify clinically relevant subpopulations with the highest therapeutic relevance. We combine single-cell microfluidic analysis with advanced computational modeling to study for the first time two common sources for cell-based therapies, human NSCs and MSCs. This methodology has the potential to logically inform cell source decisions for any clinical application.

Stem cell transplantation has evolved as a promising experimental treatment approach for stroke. In this review, we address the major hurdles for successful translation from basic research into clinical applications and discuss possible strategies to overcome these issues. We summarize the results from present pre-clinical and clinical studies and focus on specific areas of current controversy and research: (i) the therapeutic time window for cell transplantation; (ii) the selection of patients likely to benefit from such a therapy; (iii) the optimal route of cell delivery to the ischemic brain; (iv) the most suitable cell types and sources; (v) the potential mechanisms of functional recovery after cell transplantation; and (vi) the development of imaging techniques to monitor cell therapy.

Background and Purpose— Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) play important roles during neurovascular repair after stroke. In this study, we imaged VEGFR expression with positron emission tomography (PET) to noninvasively analyze poststroke angiogenesis. Methods— Female Sprague-Dawley rats after distal middle cerebral artery occlusion surgery were subjected to weekly MRI, 18 F-FDG PET, and 64 Cu-DOTA-VEGF 121 PET scans. Several control experiments were performed to confirm the VEGFR specificity of 64 Cu-DOTA-VEGF 121 uptake in the stroke border zone. VEGFR, BrdU, lectin staining, and 125 I-VEGF 165 autoradiography on stroke brain tissue slices were performed to validate the in vivo findings. Results— T2-weighed MRI correlated with the “cold spot” on 18 F-FDG PET for rats undergoing distal middle cerebral artery occlusion surgery. The 64 Cu-DOTA-VEGF 121 uptake in the stroke border zone peaked at ≈10 days after surgery, indicating neovascularization as confirmed by histology (VEGFR-2, BrdU, and lectin staining). VEGFR specificity of 64 Cu-DOTA-VEGF 121 uptake was confirmed by significantly lower uptake of 64 Cu-DOTA-VEGF mutant in vivo and intense 125 I-VEGF 165 uptake ex vivo in the stroke border zone. No appreciable uptake of 64 Cu-DOTA-VEGF 121 was observed in the brain of sham-operated rats. Conclusions— For the first time to our knowledge, we successfully evaluated the VEGFR expression kinetics noninvasively in a rat stroke model. In vivo imaging of VEGFR expression could become a significant clinical tool to plan and monitor therapies aimed at improving poststroke angiogenesis.

Thrombospondins 1 and 2 (TSP-1/2) belong to a family of extracellular glycoproteins with angiostatic and synaptogenic properties. Although TSP-1/2 have been postulated to drive the resolution of postischemic angiogenesis, their role in synaptic and functional recovery is unknown. We investigated whether TSP-1/2 are necessary for synaptic and motor recovery after stroke. Focal ischemia was induced in 8- to 12-week-old wild-type (WT) and TSP-1/2 knockout (KO) mice by unilateral occlusion of the distal middle cerebral artery and the common carotid artery (CCA). Thrombospondins 1 and 2 increased after stroke, with both TSP-1 and TSP-2 colocalizing mostly to astrocytes. Wild-type and TSP-1/2 KO mice were compared in angiogenesis, synaptic density, axonal sprouting, infarct size, and functional recovery at different time points after stroke. Using the tongue protrusion test of motor function, we observed that TSP-1/2 KO mice exhibited significant deficit in their ability to recover function ( P < 0.05) compared with WT mice. No differences were found in infarct size and blood vessel density between the two groups after stroke. However, TSP-1/2 KO mice exhibited significant synaptic density and axonal sprouting deficits. Deficiency of TSP-1/2 leads to impaired recovery after stroke mainly due to the role of these proteins in synapse formation and axonal outgrowth.

Background: Neural stem cells (NSCs) improve functional recovery after stroke, but the optimal method and conditions for delivery remain elusive. Tissue engineered scaffolds offer a unique method to manipulate and improve NSC function. We have developed a novel NSC delivery system utilizing an electrically conductive polymer scaffold to improve recovery and further elucidate repair mechanisms. Methods: Human NSCs were seeded onto an electrically conductive polymer scaffold. Electrical fields were applied to the NSCs to electrically “precondition” the cells prior to transplantation. One day after in vitro stimulation, the polymer scaffold was implanted into a distal MCA occlusion rat model 1 week post stroke, and functional recovery was assessed weekly for 4 weeks post-transplantation using the vibrissae-paw test. Control groups included unstimulated NSCs on the polymer and polymer without cells. To assess how electrical stimulation altered the cells, we performed qPCR analysis of genes of interest in stimulated and unstimulated cells on the polymer, and on NSCs on a plastic petri dish. Results: AC preconditioned NSCs improved the rate of neurologic functional recovery compared to unstimulated NSCs on the polymer (p Conclusions: Our results demonstrate that electrically preconditioned NSCs enhance stroke recovery. Gene expression of the thrombospondins (a family of matricellular proteins thought to be involved with angiogenesis and inflammation) and VEGF (proteins critical to angiogenesis) were found to be modified by electrically stimulating NSCs, suggesting possible mechanisms for functional recovery.

Stem cell therapy is a promising treatment strategy for stroke that has been shown to improve functional recovery in preclinical stroke models and is currently being investigated in clinical trials. In this chapter, we review the development of intracerebral (IC) transplantation as a strategy for targeted cell delivery to the stroke-injured brain, and discuss the use of neural stem cells (NSCs) as a candidate therapeutic cell type. We explore the different types of NSCs that have been shown to improve stroke outcome, and consider how these cells might act in vivo to alter brain repair and recovery. Finally, we summarize current progress towards clinical translation of IC delivery of NSCs, and discuss remaining challenges for developing this approach as an effective treatment for stroke.

Cell transplantation offers a potential new treatment for stroke. Animal studies using models that produce ischemic damage in both the striatum and the frontal cortex have shown beneficial effects when hNT cells (postmitotic immature neurons) were transplanted into the ischemic striatum. In this study, we investigated the effect of hNT cells in a model of stroke in which the striatum remains intact and damage is restricted to the cortex. hNT cells were transplanted into the ischemic cortex 1 week after stroke induced by distal middle cerebral artery occlusion (dMCAo). The cells exhibited robust survival at 4 weeks posttransplant even at the lesion border. hNT cells did not migrate, but they did extend long neurites into the surrounding parenchyma mainly through the white matter. Neurite extension was predominantly toward the lesion in ischemic animals but was bidirectional in uninjured animals. Extension of neurites through the cortex toward the lesion was also seen when there was some surviving cortical tissue between the graft and the infarct. Prolonged deficits were obtained in four tests of sensory-motor function. hNT-transplanted animals showed a significant improvement in functional recovery on one motor test, but there was no effect on the other three tests relative to control animals. Thus, despite clear evidence of graft survival and neurite extension, the functional benefit of hNT cells after ischemia is not guaranteed. Functional benefit could depend on other variables, such as infarct location, whether the cells mature, the behavioral tests employed, rehabilitation training, or as yet unidentified factors.

Background: Inflammation critically contributes to post-stroke brain damage and consequently there is much interest in factors that influence stroke-induced brain inflammation. Interleukin 6 (IL-6) has been heralded as an important predictor of stroke outcome, however there are conflicting reports describing detrimental, beneficial and even no effects of IL-6 on stroke pathology. This has led to the suggestion that the post-stroke effects of IL-6 may be dependent on its cellular location. We report that IL-6 produced by meningeal resident mast cells (pro-inflammatory effector cells) significantly exacerbates brain inflammation and pathology after stroke. These are the first data to identify the role in stroke pathology of IL-6 derived from a specific cell type in a particular anatomic location in vivo. Methods: A mast cell (MC) ‘knock-in’ mouse model was used where MC-deficient mice (KitW/W-v) were repaired of their MC deficiency by engraftment of in-vitro-derived MCs. MCs from wild-type (WT) or IL-6 knock-out mice were engrafted into the meninges. Mice were subjected to 30 min middle cerebral artery occlusion. Brain swelling and infarct size were assessed by T2-weighted MRI and histology. The immune response was quantified by flow cytometry. Results: CNS distribution analysis of MCs in wild-type and MC-engrafted mice revealed equivalent numbers of MCs in meninges in all groups but almost no MCs in brain parenchyma of the MC-engrafted groups. At 3d post-stroke, wild-type mice and MC-deficient mice engrafted with WT MCs exhibited significantly greater brain swelling, larger infarcts, and more brain granulocytes and activated macrophages than MC-deficient mice. These findings indicate that meningeal MCs exacerbate stroke outcome. In contrast, the pathology in IL6-KO MC-engrafted mice resembled that in MC-deficient mice, with significantly less brain swelling, smaller infarcts and fewer granulocytes and activated macrophages than in the WT MC-engrafted mice. This indicates that MC-secreted IL-6 contributes importantly to the detrimental effects of MCs. Conclusions: IL-6 produced by mast cells in the meninges is detrimental to stroke outcome. Thus, intrathecal targeting of meningeal inflammation may offer a novel therapeutic strategy for stroke.

Background: Stroke is a major cause of disability yet pharmacotherapy targeting the recovery phase is lacking. Cortical circuit reorganization adjacent to the stroke site promotes recovery, thus elucidating mechanisms that promote this plasticity could lead to new therapeutics. Tonic neuronal inhibition, mediated by extrasynaptic GABA A receptors,inhibits post-stroke recovery. However, effects of phasic (synaptic) GABA signaling - which promotes plasticity during development - are unknown. Here we use a combined approach of i) array tomography to determine the composition of GABA synapses in the post-stroke mouse brain, ii) electrophysiology to determine whether stroke leads to functional changes in GABA-mediated phasic inhibition, and (iii) treatment with zolpidem, an FDA-approved GABA agonist, to modulate recovery. Results: We found, using array tomography, a 1.7-fold increase in the number of GABAergic synapses containing the α1 receptor subunit in layer 5 of the peri-infarct cortex (synapse number/μm 3 : 0.039±0.006 (control) vs 0.064±0.006 (stroke); PA receptors. Low dose zolpidem increased GABA A phasic signaling in layer 5 pyramidal cells and notably increased the rate and extent of behavioral recovery without altering infarct size. Conclusions: These data provide the first evidence that enhanced GABA A -mediated synaptic activity during the recovery phase improves stroke outcome. These data identify modulation of phasic GABA signaling as a novel therapeutic strategy for stroke, indicate zolpidem as a potential drug to improve recovery, and underscore the necessity to distinguish the role of tonic and phasic GABA inhibition in stroke recovery.

Glucose is the major energy source during normal adult brain activity. However, it appears that glial-derived lactate is preferred as an energy substrate by neurons following hypoxia-ischemia. We examined factors influencing this switch in energetic bias from glucose to lactate in cultured hippocampal neurons, focusing on the effects of the physiological changes in lactate, glucose and adenosine concentrations seen during hypoxia-ischemia. We show that with typical basal concentrations of lactate and glucose, lactate had no effect on glucose uptake. However, at the concentrations of these metabolites found after hypoxia-ischemia, lactate inhibited glucose uptake. Reciprocally, glucose had no effect on lactate utilization regardless of glucose and lactate concentrations. Furthermore, we find that under hypoglycemic conditions adenosine had a small, but significant, inhibitory effect on glucose uptake. Additionally, adenosine increased lactate utilization. Thus, the relative concentrations of glucose, lactate and adenosine, which are indicative of the energy status of the hippocampus, influence which energy substrates are used. These results support the idea that after hypoxia-ischemia, neurons are biased in the direction of lactate rather than glucose utilization and this is accomplished through a number of regulatory steps.

The determination of neurotoxicity in monolayer mixed cultures has traditionally necessitated the time consuming and subjective procedure of counting neurons. In this paper, we propose a modification of an immunohistochemical staining method with a neuron-specific antibody against MAP2, that allows for quantification of neuron number to be done using an enzyme-linked immunosorbent assay (ELISA) plate reader. This new procedure involves the use of the compound 2,3′-azino-bis(ethylbenzothiazoline-6-sulphonic acid) (ABTS) at the last stage of the staining procedure. We employed two neurotoxicity models (the excitotoxin kainic acid and the interactions between gp120, the glycoprotein of HIV, and the stress hormone corticosterone) to compare the results obtained with this new method and the old method of immunohistochemical staining followed by 3,3′-daminobenzidine (DAB) and the counting of neurons. The ABTS/ELISA method was found to be a fast, reliable and objective procedure for the quantification of neurotoxicity.

Inflammation critically contributes to post-stroke brain damage. Mast cells (MCs), perivascular cells best known as effector cells involved in the development of inflammatory processes, have been reported to exacerbate stroke pathology. Unlike other immune cells, mature MCs do not circulate but are resident in virtually all anatomical sites, including brain parenchyma and meninges. Thus a key question is which tissue-specific MCs are important after stroke. To address this, we used ‘mast cell knock-in’ mouse models whereby genetically MC-deficient mice were selectively repaired of their MC deficiency by engraftment of in vitro grown mast cells. Methods: Two different MC-deficient mouse models (KitW-sh/W-sh and KitW/W-v) were used. For each model, 3 groups were tested: wild-type, MC-deficient, and MC-engrafted. Mice were subjected to 30 min occlusion of the middle cerebral artery. Brain swelling and infarct size were assessed by T2-weighted MRI and histology. The immune response was quantified by flow cytometry. Results: MC-deficient mice had less brain swelling at 3d post-stroke, and smaller lesions at 3d and 2wk post-stroke than their corresponding wild-type or systemically MC-engrafted groups, implying that MCs exacerbate ischemic injury. MC-deficient mice also had fewer brain granulocytes at 3d post-stroke compared to the other two groups. However, no MC-dependent changes in granulocyte numbers in blood and spleen were observed at 3d, suggesting a role for central nervous system (CNS) MCs in modulating granulocyte trafficking to the brain and stroke pathology. Analysis of the CNS MC distribution in wild-type and MC-engrafted mice revealed equivalent numbers of MCs in meninges in both groups but almost no MCs in brain parenchyma of MC-engrafted groups. This suggests that meningeal MCs, rather than parenchymal MCs, are key effectors of stroke pathology. To test this, MCs were engrafted locally into the meninges. These meningeal MC-engrafted mice had significantly more brain swelling, larger infarcts, and more brain granulocytes after stroke than MC-deficient mice. Conclusions: Our results support the conclusion that meningeal MCs can exacerbate stroke pathology. Hence, targeting these cells may be a novel therapeutic strategy for stroke.

Introduction: Stem cell transplantation has emerged as a promising new experimental treatment for stroke; understanding its mechanism of action will facilitate the translation of stem cell therapy to the clinic. Previous work from our lab and others suggests that transplanted stem cells function by enhancing endogenous brain repair processes including structural brain plasticity. The ultimate change in brain plasticity is manifested at the synaptic level and thus we hypothesize that stem cells will enhance synaptic structural remodeling in the post-ischemic brain. To test this we use array tomography, a new high-resolution proteomic imaging method, to determine a) the number and subtype of glutamate and GABA synapses after stroke, and b) how these parameters are affected by transplantation of human neural progenitor cells (hNPCs). Method: Vehicle or hNPCs derived from fetal cortex were transplanted into the ischemic cortex of Nude rats at 7 days after distal middle cerebral artery occlusion. Neurological recovery was assessed weekly using a battery of behavioral tests. Small tissue was removed from the peri-infarct cortex at 4 weeks post-transplantation. The tissue was processed and ribbons, or arrays, of serial ultrathin sections (70 nm) were obtained using an ultramicrotome. Ribbons were stained with antibodies for the synaptic markers Synapsin1, VGlut1, VGlut2, PSD-95, GAD, VGAT, GABAAR-α1, and images taken in cortical layer 2/3 and layer 5. Computational analysis of the resultant staining pattern was used to identify and quantify subtypes of glutamatergic and GABAergic synapses. Results: Transplantation of hNPCs significantly improved behavioral recovery after stroke compared to vehicle-treated rats (4 weeks; p Conclusions: These results suggest that stem cells alter synaptic remodeling after stroke and this is coincident with stem cell-induced functional recovery.

Introduction: The mechanisms of functional recovery after stroke are thought to be based on structural and functional changes in brain circuits adjacent to or connected with the stroke site. Deciphering these changes at the synaptic level is key to understanding the re-organization of the synaptic circuitry. Here we use a combined approach of i) array tomography to determine the composition of GABA synapses in the post-stroke mouse brain, with ii) electrophysiology to determine whether stroke leads to functional changes in GABA A receptor-mediated neurotransmission. Methods: A cortical lesion was induced in 12-week-old C57BL/6J male mice using the distal middle cerebral artery occlusion model of ischemia. For array tomography, small tissue was removed from the peri-infarct cortex and ribbons of serial ultrathin sections were obtained. Ribbons were stained with antibodies for synaptic markers. Analysis of the resultant staining pattern was used to quantify GABAergic synapses. In addition, whole-cell patch clamp recordings from acute neocortical brain slices were performed to evaluate GABA-mediated synaptic signaling in the peri-infarct cortex. Behavior was evaluated weekly. Results: At 1 week post-stroke, the array tomography data revealed an increase in the density and proportion of alpha1 subunit-containing GABAergic synapses in layer 5 of the peri-infarct cortex (Density: 0.064 vs 0.036 synapses/μm3. Proportion: 15.3 vs 9.1 %, pA receptor-mediated currents were enhanced in layer 5, but not in layer 2/3. These changes were specific to the pyramidal neurons. Behavioral impairment after stroke was observed only at 1 week compared to sham mice (p Conclusion: Our results suggest that stroke leads to an increased expression of functional GABA A receptors in peri-infarct neocortex and that these changes are layer- and cell type-specific. These synaptic changes may represent a mechanism of post-stroke functional recovery and remapping of surviving circuits.

Background Post-ischemic inflammation plays an important role in stroke pathology. Understanding the details and mechanisms of post-stroke inflammation is essential to develop therapeutics that can reduce the damage and promote recovery. Macrophages are known to promote both injury and repair in various pathologies, and these divergent properties may be due to different macrophage subtypes. Here, we identify different macrophage subtypes in the brain after ischemic stroke and investigate their effects on stroke outcome. Methods C57BL/6 mice (male, 11-14 weeks old) were subjected to left transient middle cerebral artery occlusion (tMCAo). To deplete blood monocytes, clodronate-filled liposomes (CL) were used. Phosphate-buffered saline (PBS)-filled liposomes (PL) served as controls. Behavior was assessed using a neurological score. Infarct size was assessed by TTC at 2d and silver staining at 7d post-stroke. Immune cells in brain, blood, spleen and bone marrow were analyzed by flow cytometry. Results A time-course analysis of immune cells after stroke identified two macrophage subpopulations in the brain: Ly6Chigh and Ly6Clow. Ly6Chigh macrophages were the dominant cell population early after stroke, peaking by 3d and then decreasing in number by 5d. Conversely, the number of Ly6Clow macrophages were initially low and peaked at 5d. To investigate the effects of these macrophage subpopulations, we used clodronate depletion of monocytes with different injection paradigms. Injecting clodronate just after reperfusion, to target the Ly6Chigh macrophages, had no significant effect on infarct size at 2d and 7d post-stroke, or on functional deficit, compared to the PL group. However, when the injections were given every other day after stroke, from day 0 to day 6, CL-treated mice had significantly larger infarct sizes at 7d post-stroke (48.9±4.3 vs 35.6±5.8, % of contralateral hemisphere, n=10; p Conclusion Macrophage can reduce brain damage and play a role in promoting recovery after stroke.

Conventional therapeutic strategies in stroke, both ischemic and hemorrhagic, have focused on the prevention of further stroke. Stem cell transplantation shifts the paradigm of stroke therapy in that it aims to repair the ischemic brain by facilitating the brain’s plasticity to regenerate synaptic structures and reorganize its functional architecture after injury. Transplanted neural progenitor cells have been shown to migrate to the ischemic penumbra and improve functional recovery. The mechanisms through which transplanted cells exert their effects include promoting dendritic branching, facilitating axonal rewiring and axonal transport, enhancing neovascularization, and modulating the inflammatory response. This chapter discusses the molecular mechanisms that underlie axonal and dendritic regeneration; focuses on the effect of stem cells on brain remodeling following stroke, as plasticity is thought to play a major role in recovery; and examines the logistical considerations of stem cell transplantation in a translational context. Stem cell transplantation for stroke is a nascent field of research, and much work is needed to optimize its therapeutic benefit and to minimize its risks.

Introduction: The mechanisms of functional recovery after stroke are thought to be based on structural and functional changes in brain circuits adjacent to or connected with the stroke site. Deciphering these changes at the synaptic level is key to understanding the re-organization of the synaptic circuitry (i.e. the connectome). Quantitative information about such synapse rearrangements after stroke has been inadequate however, due to the technical limitations of available methodologies. Here we describe the use of array tomography, a new high-resolution proteomic imaging method, to determine the composition of glutamate and GABA synapses in the post-stroke mouse brain. Methods: A cortical lesion was induced in 12-week-old C57BL/6J male mice using the distal middle cerebral artery occlusion model of ischemia. Small tissue sections were removed from the peri-infarct cortex and ribbons of serial ultrathin (70 nm) sections were obtained using an ultramicrotome. Ribbons were stained with antibodies for the synaptic markers SynapsinI, VGlut1, VGlut2, PSD-95, GAD, VGAT. Analysis of the resultant staining pattern was used to identify subtypes of glutamatergic and GABAergic synapses. Results: At 1 week post-stroke, an increase in GABAergic synapses was observed in layer 5 of the peri-infarct cortex. A sub-analysis of the type of inhibitory interneurons (e.g. parvalbumin, somatostatin) expressing these synapses is pending. In addition, a trend for an increase of VGlut1+2 synapses was also observed. However, there were no detectable differences in total synapse number between stroke-injured and naïve animals, thus suggesting that VGluT2 expression may be upregulated in existing glutamatergic VGluT1 synapses after stroke. Further analysis will be extended to cortical layers 2/3 and 4. Conclusion: These results provide new information about the organization of synaptic circuitry and its plasticity after stroke. Furthermore, it demonstrates how array tomography enables a previously unobtainable level of volumetric visualization and quantification of synapses.

Cell transplantation offers a novel therapeutic strategy for stroke; however, how transplanted cells function in vivo is poorly understood. We show for the first time that after subacute transplantation into the ischemic brain of human central nervous system stem cells grown as neurospheres (hCNS-SCns), the stem cell-secreted factor, human vascular endothelial growth factor (hVEGF), is necessary for cell-induced functional recovery. We correlate this functional recovery to hVEGF-induced effects on the host brain including multiple facets of vascular repair and its unexpected suppression of the inflammatory response. We found that transplanted hCNS-SCns affected multiple parameters in the brain with different kinetics: early improvement in blood-brain barrier integrity and suppression of inflammation was followed by a delayed spatiotemporal regulated increase in neovascularization. These events coincided with a bimodal pattern of functional recovery, with, an early recovery independent of neovascularization, and a delayed hVEGF-dependent recovery coincident with neovascularization. Therefore, cell transplantation therapy offers an exciting multimodal strategy for brain repair in stroke and potentially other disorders with a vascular or inflammatory component.

Stem cell transplantation promises new hope for the treatment of stroke although significant questions remain about how the grafted cells elicit their effects. One hypothesis is that transplanted stem cells enhance endogenous repair mechanisms activated after cerebral ischaemia. Recognizing that bilateral reorganization of surviving circuits is associated with recovery after stroke, we investigated the ability of transplanted human neural progenitor cells to enhance this structural plasticity. Our results show the first evidence that human neural progenitor cell treatment can significantly increase dendritic plasticity in both the ipsi- and contralesional cortex and this coincides with stem cell-induced functional recovery. Moreover, stem cell-grafted rats demonstrated increased corticocortical, corticostriatal, corticothalamic and corticospinal axonal rewiring from the contralesional side; with the transcallosal and corticospinal axonal sprouting correlating with functional recovery. Furthermore, we demonstrate that axonal transport, which is critical for both proper axonal function and axonal sprouting, is inhibited by stroke and that this is rescued by the stem cell treatment, thus identifying another novel potential mechanism of action of transplanted cells. Finally, we established in vitro co-culture assays in which these stem cells mimicked the effects observed in vivo. Through immunodepletion studies, we identified vascular endothelial growth factor, thrombospondins 1 and 2, and slit as mediators partially responsible for stem cell-induced effects on dendritic sprouting, axonal plasticity and axonal transport in vitro. Thus, we postulate that human neural progenitor cells aid recovery after stroke through secretion of factors that enhance brain repair and plasticity.

Middle cerebral artery occlusion (MCAO) in rats is a well-studied experimental model for ischemic stroke leading to brain infarction and functional deficits. Many preclinical studies have focused on a small time window after the ischemic episode to evaluate functional outcome for screening therapeutic candidates. Short evaluation periods following injury have led to significant setbacks due to lack of information on the delayed effects of treatments, as well as short-lived and reversible neuroprotection, so called false-positive results. In this report, we evaluated long-term functional deficit for 90 days after MCAO in two rat strains with two durations of ischemic insult, in order to identify the best experimental paradigm to assess injury and subsequent recovery. Behavioral outcomes were measured pre-MCAO followed by weekly assessment post-stroke. Behavioral tests included the 18-point composite neurological score, 28-point neuroscore, rearing test, vibrissae-evoked forelimb placing test, foot fault test and the CatWalk. Brain lesions were assessed to correlate injury to behavior outcomes at the end of study. Our results indicate that infarction volume in Sprague-Dawley rats was dependent on occlusion duration. In contrast, the infarction volume in Wistar rats did not correlate with the duration of ischemic episode. Functional outcomes were not dependent on occlusion time in either strain; however, measureable deficits were detectable long-term in limb asymmetry, 18- and 28-point neuroscores, forelimb placing, paw swing speed, and gait coordination. In conclusion, these behavioral assays, in combination with an extended long-term assessment period, can be used for evaluating therapeutic candidates in preclinical models of ischemic stroke.

A consortium of translational stem cell and stroke experts from multiple academic institutes and biotechnology companies, under the guidance of the government (FDA/NIH), is missing. Here, we build a case for the establishment of this consortium if cell therapy for stroke is to advance from the laboratory to the clinic.

A consortium of translational stem cell and stroke experts from multiple academic institutes and biotechnology companies, under the guidance of the government (FDA/NIH), is missing. Here, we build a case for the establishment of this consortium if cell therapy for stroke is to advance from the laboratory to the clinic.

Cesar V Borlongan1,2†, Michael Chopp3, Gary K Steinberg4, Tonya M Bliss4, Yi Li3, Mei Lu3, David C Hess1 & Douglas Kondziolka5 †Author for correspondence 1Medical College of Georgia and Augusta VA Medical Center, Department of Neurology, 1120 15th Street Augusta, GA 30904, USA 2Tel.: +1 706 721 2145; Fax: +1 706 721 7619; E-mail: cborlongan@ mail.mcg.edu 3Henry Ford Hospital, Department of Neurology, 2799 West Grand Blvd., Detroit, MI 48202, USA 4Stanford University School of Medicine, Department of Neurosurgery, R281, 300 Pasteur Drive, Stanford, CA, 94305, USA 5University of Pittsburgh, Department of Neurological Surgery, Suite B-400, UPMC, 200 Lothrop Street Pittsburgh, PA 15213, USA ‘...it is imperative for clinical translation that these cells be tested in multiple models of focal stroke, in two species, in both genders and in multiple laboratories.’

Endogenous neural stem cells normally reside in their niche, the subventricular zone, in the uninjured rodent brain. Upon stroke, these cells become more proliferative and migrate away from the subventricular zone into the surrounding parenchyma. It is not known whether this stroke-induced behavior is due to changes in the niche or introduction of attractive cues in the infarct zone, or both. A related question is how transplanted neural stem cells respond to subsequent insults, including whether exogenous stem cells have the plasticity to respond to subsequent injuries after engraftment. We addressed this issue by transplanting neural progenitor cells (NPCs) into the uninjured brain and then subjecting the animal to stroke. We were able to follow the transplanted NPCs in vivo by labeling them with superparamagnetic iron oxide particles and imaging them via high-resolution magnetic resonance imaging (MRI) during engraftment and subsequent to stroke. We find that transplanted NPCs that are latent can be activated in response to stroke and exhibit directional migration into the parenchyma, similar to endogenous neural NPCs, without a niche environment.

Noninvasive monitoring of stem cells, using high-resolution molecular imaging, will be instrumental to improve clinical neural transplantation strategies. We show that labeling of human central nervous system stem cells grown as neurospheres with magnetic nanoparticles does not adversely affect survival, migration, and differentiation or alter neuronal electrophysiological characteristics. Using MRI, we show that human central nervous system stem cells transplanted either to the neonatal, the adult, or the injured rodent brain respond to cues characteristic for the ambient microenvironment resulting in distinct migration patterns. Nanoparticle-labeled human central nervous system stem cells survive long-term and differentiate in a site-specific manner identical to that seen for transplants of unlabeled cells. We also demonstrate the impact of graft location on cell migration and describe magnetic resonance characteristics of graft cell death and subsequent clearance. Knowledge of migration patterns and implementation of noninvasive stem cell tracking might help to improve the design of future clinical neural stem cell transplantation.

No treatment currently exists to restore lost neurological function after stroke. A growing number of studies highlight the potential of stem cell transplantation as a novel therapeutic approach for stroke. In this review we summarize these studies, discuss potential mechanisms of action of the transplanted cells, and emphasize the need to determine parameters that are critical for transplantation success.

We characterize the survival, migration, and differentiation of human neurospheres derived from CNS stem cells transplanted into the ischemic cortex of rats 7 days after distal middle cerebral artery occlusion. Transplanted neurospheres survived robustly in naive and ischemic brains 4 wk posttransplant. Survival was influenced by proximity of the graft to the stroke lesion and was negatively correlated with the number of IB4-positive inflammatory cells. Targeted migration of the human cells was seen in ischemic animals, with many human cells migrating long distances (≈1.2 mm) predominantly toward the lesion; in naive rats, cells migrated radially from the injection site in smaller number and over shorter distances (0.2 mm). The majority of migrating cells in ischemic rats had a neuronal phenotype. Migrating cells between the graft and the lesion expressed the neuroblast marker doublecortin, whereas human cells at the lesion border expressed the immature neuronal marker β-tubulin, although a small percentage of cells at the lesion border also expressed glial fibrillary acid protein (GFAP). Thus, transplanted human CNS (hCNS)-derived neurospheres survived robustly in naive and ischemic brains, and the microenvironment influenced their migration and fate.

There is an increasing recognition of the damaging role played by oxygen radicals in mediating necrotic neuronal injury. As such, it becomes important to understand the transport mechanisms that help maintain appropriate levels of small molecule antioxidants such as ascorbate in the brain. It has long been known that the transport of dehydroascorbate (DHA) into a variety of cell types is accomplished through the Glut-1 glucose transporter. In this paper, we characterize interactions among the transports of ascorbate, DHA and glucose in hippocampal cultures. We find: (a) sodium-dependent transport of ascorbate in mixed neuronal/glial, pure glial, and neuron-enriched hippocampal cultures; in contrast, we observed no such transport of DHA; (b) such ascorbate transport appeared to be independent of the glucose transporter, in that glucose did not compete for such transport, and overexpression of the Glut-1 glucose transporter did not alter ascorbate uptake; (c) in contrast, ascorbate, at concentrations ranging from 1 to 20 mM inhibited 2-dexogyglucose transport in mixed, glial and enriched neuronal hippocampal cultures; (d) potentially, ascorbate, by acting as an electron donor, could impair the function of molecules involve in the transport or metabolism of glucose. We observed mild inhibition of glucose transport by one unrelated electron donor (glutathione). Moreover, transport was also inhibited by an ascorbate analog which is not an electron donor. Thus, we conclude that ascorbate transport in hippocampal neurons and glia occurs independent of the glucose transporter but that, nevertheless, ascorbate, at concentrations generally thought to be supraphysiological, has the potential for disrupting glucose transport.

Glucocorticoids (GCs), the adrenal steroids released during stress, can compromise the ability of hippocampal neurons to survive necrotic neurological insults. This GC-induced endangerment has energetic facets, in that it can be attenuated with energy supplementation. In the present report, we studied the effects of GCs on the metabolic response of specific hippocampal cell fields to necrotic insults. We used silicon microphysiometry, which allows indirect measurement of metabolism in real time in tissue explants. Aglycemia caused a significant decline in metabolism in dentate gyrus explants, but not in CA1 or CA3 explants. When coupled with our prior report of cyanide disrupting metabolism only in CA1 explants, and the glutamatergic excitotoxin kainic acid disrupting metabolism only in CA3 explants, this demonstrates that microphysiometry can detect the selective regional vulnerability that characterizes the hippocampal response to these necrotic insults. We then examined the effects of GCs on the response to these insults, monitoring explants taken from rats that were adrenalectomized, intact, or treated with corticosterone (the GC of rats) that produced circulating levels equivalent to those of major stressors. Increased exposure to GCs worsened the decline in metabolism in dentate gyrus explants induced by hypoglycemia, and in CA1 explants induced by cyanide (after eliminating the effects of glial release of lactate for the support of neuronal metabolism). Thus, GCs worsen the metabolic consequences of necrotic insults in hippocampal explants.

Double strand break repair and V(D)J recombination in mammalian cells require the function of the Ku protein complex and the DNA-dependent protein kinase. The DNA-dependent protein kinase is targeted to DNA through its interaction with the Ku protein complex, and thus the specificity of template recognition in the repair and recombination reactions depend on Ku. We have studied Ku binding to DNA using competitive gel shift analysis. We find that Ku bound to one DNA molecule can transfer directly to another DNA molecule when the two DNA molecules have homologous ends containing a minimum of four matched bases. This remarkable reaction can give a false impression of sequence specificity of Ku DNA binding under certain assay conditions. A model is proposed for the DNA binding function of Ku on the basis of these results and the discovery of a novel type of DNA-Ku complex formed at high Ku/DNA ratios is discussed.