Neuroprotection of Low-Frequency Repetitive Transcranial Magnetic Stimulation after Ischemic Stroke in Rats.

OBJECTIVE: Stroke is a leading cause of human death and disability. Effective early treatments with reasonable therapeutic windows remain critically important to improve the outcomes of stroke. Transcranial magnetic stimulation (TMS) is an established noninvasive technique that has been applied clinically and in animal research for multiple brain disorders, but few studies have examined acute neuroprotection against ischemic stroke. The present investigation tested the novel approach of low-frequency repetitive TMS (rTMS) as an acute treatment after ischemic stroke. METHODS: Adult male rats received focal ischemic surgery through occlusion of the right middle cerebral artery for 60 minutes. The rats received either rTMS or sham treatment with 1.5-, 3-, 4-, or 7-hour delay after the onset of stroke. Low-frequency and low-intensity rTMS was applied to the rat brain for two 30-minute episodes separated by a 1-hour interval. RESULTS: Three days after stroke, compared to stroke controls, rats receiving rTMS treatment with a 1.5-hour delay showed a 35% reduction of infarct volume. Protective effects were also seen with 3- or 4-hour-delayed treatments by rTMS, shown as reduced infarct volume and cell death. rTMS treatment upregulated the antiapoptotic factor Bcl-2 and downregulated the proapoptotic caspase-3 cleavage, expressions of Bax and matrix metallopeptidase-9. In sensorimotor functional assessments 3 to 21 days after stroke, rats receiving rTMS treatment with a 1.5- or 3-hour delay showed significantly better performance compared to stroke controls. INTERPRETATION: These results support the inference that low-frequency rTMS may be feasible as a neuroprotective acute treatment after ischemic stroke. ANN NEUROL 2023;93:336-347.

Neurotrophic signaling deficiency exacerbates environmental risks for Alzheimer's disease pathogenesis.

The molecular mechanism of Alzheimer's disease (AD) pathogenesis remains obscure. Life and/or environmental events, such as traumatic brain injury (TBI), high-fat diet (HFD), and chronic cerebral hypoperfusion (CCH), are proposed exogenous risk factors for AD. BDNF/TrkB, an essential neurotrophic signaling for synaptic plasticity and neuronal survival, are reduced in the aged brain and in AD patients. Here, we show that environmental factors activate C/EBPß, an inflammatory transcription factor, which subsequently up-regulates δ-secretase that simultaneously cleaves both APP and Tau, triggering AD neuropathological changes. These adverse effects are additively exacerbated in BDNF+/- or TrkB+/- mice. Strikingly, TBI provokes both senile plaque deposit and neurofibrillary tangles (NFT) formation in TrkB+/- mice, associated with augmented neuroinflammation and extensive neuronal loss, leading to cognitive deficits. Depletion of C/EBPß inhibits TBI-induced AD-like pathologies in these mice. Remarkably, amyloid aggregates and NFT are tempospatially distributed in TrkB+/- mice brains after TBI, providing insight into their spreading in the progression of AD-like pathologies. Hence, our study revealed the roles of exogenous (TBI, HFD, and CCH) and endogenous (TrkB/BDNF) risk factors in the onset of AD-associated pathologies.

Pathogenesis of sporadic Alzheimer's disease by deficiency of NMDA receptor subunit GluN3A.

The Ca2+ hypothesis for Alzheimer's disease (AD) conceives Ca2+ dyshomeostasis as a common mechanism of AD; the cause of Ca2+ dysregulation, however, is obscure. Meanwhile, hyperactivities of N-Methyl-D-aspartate receptors (NMDARs), the primary mediator of Ca2+ influx, are reported in AD. GluN3A (NR3A) is an NMDAR inhibitory subunit. We hypothesize that GluN3A is critical for sustained Ca2+ homeostasis and its deficiency is pathogenic for AD. Cellular, molecular, and functional changes were examined in adult/aging GluN3A knockout (KO) mice. The GluN3A KO mouse brain displayed age-dependent moderate but persistent neuronal hyperactivity, elevated intracellular Ca2+ , neuroinflammation, impaired synaptic integrity/plasticity, and neuronal loss. GluN3A KO mice developed olfactory dysfunction followed by psychological/cognitive deficits prior to amyloid-ß/tau pathology. Memantine at preclinical stage prevented/attenuated AD syndromes. AD patients' brains show reduced GluN3A expression. We propose that chronic "degenerative excitotoxicity" leads to sporadic AD, while GluN3A represents a primary pathogenic factor, an early biomarker, and an amyloid-independent therapeutic target.

[Acceptance Test of Physical Properties of Perfexion Gamma Knife].

Objective The acceptance test of the physical performance for the newly installed PFX Gamma knife was carried out to provide a reference for the quality assurance of the equipment. Method According to the manufacturer's acceptance manual and Chinese health industry standards, the dose rate of the PFX Gamma knife with maximum collimators in the calibration center point, the focal spot dose distribution with all three collimator sizes, the distance between the RFP and the PPS calibration center point, and the multi-target position accuracy were measured by means of the acceptance tools. Results The average absorbed dose rate, as measured in April 29, 2019, was 3.295 Gy/min in the calibration center point by the maximum collimators, which was better than acceptance standard of 2.5 Gy/min. The focal spot dosimetry data measured by the film were basically the same as the TMR-10 data stored in the LGP. The difference in FWHM was 0.18±0.13 mm, and the difference in penumbra was 0.15±0.25 mm, which was normal and as expected. The distance between the RFP and PPS calibration center point in the three-dimensional space was 0.24 mm, which was better than the recommended value of the factory standard and the national standard. The maximum position deviation of multiple targets was 0.16 mm, which was also better than the factory standard of 0.5 mm. Conclusion The various physical performance indicators of the PFX Gamma knife met the acceptance standards of manufacturers and industries.

Optochemogenetic Stimulation of Transplanted iPS-NPCs Enhances Neuronal Repair and Functional Recovery after Ischemic Stroke.

Cell transplantation therapy provides a regenerative strategy for neural repair. We tested the hypothesis that selective excitation of transplanted induced pluripotent stem cell-derived neural progenitor cells (iPS-NPCs) could recapitulate an activity-enriched microenvironment that confers regenerative benefits for the treatment of stroke. Mouse iPS-NPCs were transduced with a novel optochemogenetics fusion protein, luminopsin 3 (LMO3), which consisted of a bioluminescent luciferase, Gaussia luciferase, and an opsin, Volvox Channelrhodopsin 1. These LMO3-iPS-NPCs can be activated by either photostimulation using light or by the luciferase substrate coelenterazine (CTZ). In vitro stimulations of LMO3-iPS-NPCs increased expression of synapsin-1, postsynaptic density 95, brain derived neurotrophic factor (BDNF), and stromal cell-derived factor 1 and promoted neurite outgrowth. After transplantation into the ischemic cortex of mice, LMO3-iPS-NPCs differentiated into mature neurons. Synapse formation between implanted and host neurons was identified using immunogold electron microscopy and patch-clamp recordings. Stimulation of transplanted cells with daily intranasal administration of CTZ enhanced axonal myelination, synaptic transmission, improved thalamocortical connectivity, and functional recovery. Patch-clamp and multielectrode array recordings in brain slices showed that CTZ or light stimulation facilitated synaptic transmission and induced neuroplasticity mimicking the LTP of EPSPs. Stroke mice received the combined LMO3-iPS-NPC/CTZ treatment, but not cell or CTZ alone, showed enhanced neural network connections in the peri-infarct region, promoted optimal functional recoveries after stroke in male and female, young and aged mice. Thus, excitation of transplanted cells via the noninvasive optochemogenetics treatment provides a novel integrative cell therapy with comprehensive regenerative benefits after stroke.SIGNIFICANCE STATEMENT Neural network reconnection is critical for repairing damaged brain. Strategies that promote this repair are expected to improve functional outcomes. This study pioneers the generation and application of an optochemogenetics approach in stem cell transplantation therapy after stroke for optimal neural repair and functional recovery. Using induced pluripotent stem cell-derived neural progenitor cells (iPS-NPCs) expressing the novel optochemogenetic probe luminopsin (LMO3), and intranasally delivered luciferase substrate coelenterazine, we show enhanced regenerative properties of LMO3-iPS-NPCs in vitro and after transplantation into the ischemic brain of different genders and ages. The noninvasive repeated coelenterazine stimulation of transplanted cells is feasible for clinical applications. The synergetic effects of the combinatorial cell therapy may have significant impacts on regenerative approach for treatments of CNS injuries.

Protective effects of GPR37 via regulation of inflammation and multiple cell death pathways after ischemic stroke in mice.

GPCR 37 (GPR37) is a GPCR expressed in the CNS; its physiological and pathophysiological functions are largely unknown. We tested the role of GPR37 in the ischemic brain of GPR37 knockout (KO) mice, exploring the idea that GPR37 might be protective against ischemic damage. In an ischemic stroke model, GPR37 KO mice exhibited increased infarction and cell death compared with wild-type (WT) mice, measured by 2,3,5-triphenyl-2H-tetrazolium chloride and TUNEL staining 24 h after stroke. Moreover, more severe functional deficits were detected in GPR37 KO mice in the adhesive-removal and corner tests. In the peri-infarct region of GPR37 KO mice, there was significantly more apoptotic and autophagic cell death accompanied by caspase-3 activation and attenuated mechanistic target of rapamycin signaling. GPR37 deletion attenuated astrocyte activation and astrogliosis compared with WT stroke controls 24-72 h after stroke. Immunohistochemical staining showed more ionized calcium-binding adapter molecule 1-positive cells in the ischemic cortex of GPR37 KO mice, and RT-PCR identified an enrichment of M1-type microglia or macrophage markers in the GPR37 KO ischemic cortex. Western blotting demonstrated higher levels of inflammatory factors IL-1ß, IL-6, monocyte chemoattractant protein, and macrophage inflammatory protein-1α in GPR37-KO mice after ischemia. Thus, GPR37 plays a multifaceted role after stroke, suggesting a novel target for stroke therapy.-McCrary, M. R., Jiang, M. Q., Giddens, M. M., Zhang, J. Y., Owino, S., Wei, Z. Z., Zhong, W., Gu, X., Xin, H., Hall, R. A., Wei, L., Yu, S. P. Protective effects of GPR37 via regulation of inflammation and multiple cell death pathways after ischemic stroke in mice.

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

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.

Optogenetic stimulation of glutamatergic neuronal activity in the striatum enhances neurogenesis in the subventricular zone of normal and stroke mice.

Neurogenesis in the subventricular zone (SVZ) of the adult brain may contribute to tissue repair after brain injuries. Whether SVZ neurogenesis can be upregulated by specific neuronal activity in vivo and promote functional recovery after stroke is largely unknown. Using the spatial and cell type specific optogenetic technique combined with multiple approaches of in vitro, ex vivo and in vivo examinations, we tested the hypothesis that glutamatergic activation in the striatum could upregulate SVZ neurogenesis in the normal and ischemic brain. In transgenic mice expressing the light-gated channelrhodopsin-2 (ChR2) channel in glutamatergic neurons, optogenetic stimulation of the glutamatergic activity in the striatum triggered glutamate release into SVZ region, evoked membrane currents, Ca2+ influx and increased proliferation of SVZ neuroblasts, mediated by AMPA receptor activation. In ChR2 transgenic mice subjected to focal ischemic stroke, optogenetic stimuli to the striatum started 5days after stroke for 8days not only promoted cell proliferation but also the migration of SVZ neuroblasts into the peri-infarct cortex with increased neuronal differentiation and improved long-term functional recovery. These data provide the first morphological and functional evidence showing a unique striatum-SVZ neuronal regulation via a semi-phasic synaptic mechanism that can boost neurogenic cascades and stroke recovery. The benefits from stimulating endogenous glutamatergic activity suggest a novel regenerative strategy after ischemic stroke and other brain injuries.

Improved Therapeutic Benefits by Combining Physical Cooling With Pharmacological Hypothermia After Severe Stroke in Rats.

BACKGROUND AND PURPOSE: Therapeutic hypothermia is a promising strategy for treatment of acute stroke. Clinical translation of therapeutic hypothermia, however, has been hindered because of the lack of efficiency and adverse effects. We sought to enhance the clinical potential of therapeutic hypothermia by combining physical cooling (PC) with pharmacologically induced hypothermia after ischemic stroke. METHODS: Wistar rats were subjected to 90-minute middle cerebral artery occlusion by insertion of an intraluminal filament. Mild-to-moderate hypothermia was induced 120 minutes after the onset of stroke by PC alone, a neurotensin receptor 1 (NTR1) agonist HPI-201 (formally ABS-201) alone or the combination of both. The outcomes of stroke were evaluated at 3 and 21 days after stroke. RESULTS: PC or HPI-201 each showed hypothermic effect and neuroprotection in stroke rats. The combination of PC and HPI-201 exhibited synergistic effects in cooling process, reduced infarct formation, cell death, and blood-brain barrier damages and improved functional recovery after stroke. Importantly, coapplied HPI-201 completely inhibited PC-associated shivering and tachycardia. CONCLUSIONS: The centrally acting hypothermic drug HPI-201 greatly enhanced the efficiency and efficacy of conventional PC; this combined cooling therapy may facilitate clinical translation of hypothermic treatment for stroke.

Regulation of therapeutic hypothermia on inflammatory cytokines, microglia polarization, migration and functional recovery after ischemic stroke in mice.

Stroke is a leading threat to human life and health in the US and around the globe, while very few effective treatments are available for stroke patients. Preclinical and clinical studies have shown that therapeutic hypothermia (TH) is a potential treatment for stroke. Using novel neurotensin receptor 1 (NTR1) agonists, we have demonstrated pharmacologically induced hypothermia and protective effects against brain damages after ischemic stroke, hemorrhage stroke, and traumatic brain injury (TBI) in rodent models. To further characterize the mechanism of TH-induced brain protection, we examined the effect of TH (at ±33°C for 6h) induced by the NTR1 agonist HPI-201 or physical (ice/cold air) cooling on inflammatory responses after ischemic stroke in mice and oxygen glucose deprivation (OGD) in cortical neuronal cultures. Seven days after focal cortical ischemia, microglia activation in the penumbra reached a peak level, which was significantly attenuated by TH treatments commenced 30min after stroke. The TH treatment decreased the expression of M1 type reactive factors including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-12, IL-23, and inducible nitric oxide synthase (iNOS) measured by RT-PCR and Western blot analyses. Meanwhile, TH treatments increased the expression of M2 type reactive factors including IL-10, Fizz1, Ym1, and arginase-1. In the ischemic brain and in cortical neuronal/BV2 microglia cultures subjected to OGD, TH attenuated the expression of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α), two key chemokines in the regulation of microglia activation and infiltration. Consistently, physical cooling during OGD significantly decreased microglia migration 16h after OGD. Finally, TH improved functional recovery at 1, 3, and 7days after stroke. This study reveals the first evidence for hypothermia mediated regulation on inflammatory factor expression, microglia polarization, migration and indicates that the anti-inflammatory effect is an important mechanism underlying the brain protective effects of a TH therapy.

A neuroprotective role of the NMDA receptor subunit GluN3A (NR3A) in ischemic stroke of the adult mouse.

GluN3A or NR3A is a developmentally regulated N-methyl-d-aspartate receptor (NMDAR) subunit, showing a unique inhibitory role that decreases NMDAR current and the receptor-mediated Ca(2+) influx. In the neonatal brain, GluN3A is shown to associate with synaptic maturation and spine formation and plays a neuroprotective role. Its functional role in the adult brain, however, is largely unknown. We tested the hypothesis that, disrespecting the relatively lower expression level of GluN3A in the adult brain, this inhibitory NMDAR subunit shows a protective action against ischemia-induced brain injury. In littermate wild-type (WT) and GluN3A knockout (KO) mice, focal cerebral ischemia was induced by permanent occlusion of right distal branches of the middle cerebral artery (MCA) plus 10-min ligation of both common carotid arteries (CCAs). Twenty-four hours after focal cerebral ischemia, the infarction volume assessed using 2,3,5-triphenyltetrazolium chloride (TTC) staining was significantly larger in GluN3A KO mice compared with WT mice. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining demonstrated enhanced cell death in GluN3A KO mice. Moreover, the deletion of GluN3A hindered sensorimotor functional recovery after stroke. It is suggested that, although the expression level is relatively lower in the adult brain, GluN3A is still a noteworthy regulator in ischemia-induced excitotoxicity and brain injury.

iPSC Transplantation increases regeneration and functional recovery after ischemic stroke in neonatal rats.

Limited treatments are available for perinatal/neonatal stroke. Induced pluripotent stem cells (iPSCs) hold therapeutic promise for stroke treatment, but the benefits of iPSC transplantation in neonates are relatively unknown. We hypothesized that transplanted iPSC-derived neural progenitor cells (iPSC-NPCs) would increase regeneration after stroke. Mouse pluripotent iPSCs were differentiated into neural progenitors using a retinoic acid protocol. Differentiated neural cells were characterized by using multiple criteria and assessments. Ischemic stroke was induced in postnatal day 7 (P7) rats by occluding the right middle cerebral artery and right common carotid artery. iPSC-NPCs (400,000 in 4 µl) were transplanted into the penumbra via intracranial injection 7 days after stroke. Trophic factor expression in the peri-infarct tissue was measured using Western blot analysis. Animals received daily bromodeoxyuridine (BrdU) injections and were sacrificed 21 days after stroke for immunohistochemistry. The vibrissae-elicited forelimb placement test was used to evaluate functional recovery. Differentiated iPSCs expressed mature neuronal markers, functional sodium and potassium channels, and fired action potentials. Several angiogenic and neurogenic trophic factors were identified in iPSC-NPCs. Animals that received iPSC-NPC transplantation had greater expression of stromal cell-derived factor 1-α (SDF-1α) and vascular endothelial growth factor (VEGF) in the peri-infarct region. iPSC-NPCs stained positive for neuronal nuclei (NeuN) or glial fibrillary acidic protein (GFAP) 14 days after transplantation. iPSC-NPC-transplanted animals showed greater numbers of BrdU/NeuN and BrdU/Collagen IV colabeled cells in the peri-infarct area compared with stroke controls and performed better in a sensorimotor functional test after stroke. iPSC-NPC therapy may play multiple therapeutic roles after stroke by providing trophic factors, increasing angiogenesis and neurogenesis, and providing new cells for tissue repair.

Honokiol for the Treatment of Neonatal Pain and Prevention of Consequent Neurobehavioral Disorders.

This study examined the short- and long-term neuroprotective and analgesic activity of honokiol (a naturally occurring lignan isolated from Magnolia) on developing brains in neonates exposed to inflammatory pain, known to cause neuronal cell death. Postnatal day 4 (P4) neonatal rat pups were subjected to intraplantar formalin injection to four paws as a model of severe neonatal pain. Intraperitoneal honokiol (10 mg/kg) or corn oil vehicle control was administered 1 h prior to formalin insult, and animals were maintained on honokiol through postnatal day 21 (P21). Behavioral tests for stress and pain were performed after the painful insult, followed by morphological examinations of the brain sections at P7 and P21. Honokiol significantly attenuated acute pain responses 30 min following formalin insult and decreased chronic thermal hyperalgesia later in life. Honokiol-treated rats performed better on tests of exploratory behavior and performed significantly better in tests of memory. Honokiol treatment normalized hippocampal and thalamic c-Fos and hippocampal alveus substance P receptor expression relative to controls at P21. Together, these findings support that (1) neonatal pain experiences predispose rats to the development of chronic behavioral changes and (2) honokiol prevents and reduces both acute and chronic pathological pain-induced deteriorations in neonatal rats.

Reprogramming Glioblastoma Cells into Non-Cancerous Neuronal Cells as a Novel Anti-Cancer Strategy.

Glioblastoma Multiforme (GBM) is an aggressive brain tumor with a high mortality rate. Direct reprogramming of glial cells to different cell lineages, such as induced neural stem cells (iNSCs) and induced neurons (iNeurons), provides genetic tools to manipulate a cell's fate as a potential therapy for neurological diseases. NeuroD1 (ND1) is a master transcriptional factor for neurogenesis and it promotes neuronal differentiation. In the present study, we tested the hypothesis that the expression of ND1 in GBM cells can force them to differentiate toward post-mitotic neurons and halt GBM tumor progression. In cultured human GBM cell lines, including LN229, U87, and U373 as temozolomide (TMZ)-sensitive and T98G as TMZ-resistant cells, the neuronal lineage conversion was induced by an adeno-associated virus (AAV) package carrying ND1. Twenty-one days after AAV-ND1 transduction, ND1-expressing cells displayed neuronal markers MAP2, TUJ1, and NeuN. The ND1-induced transdifferentiation was regulated by Wnt signaling and markedly enhanced under a hypoxic condition (2% O2 vs. 21% O2). ND1-expressing GBM cultures had fewer BrdU-positive proliferating cells compared to vector control cultures. Increased cell death was visualized by TUNEL staining, and reduced migrative activity was demonstrated in the wound-healing test after ND1 reprogramming in both TMZ-sensitive and -resistant GBM cells. In a striking contrast to cancer cells, converted cells expressed the anti-tumor gene p53. In an orthotopical GBM mouse model, AAV-ND1-reprogrammed U373 cells were transplanted into the fornix of the cyclosporine-immunocompromised C57BL/6 mouse brain. Compared to control GBM cell-formed tumors, cells from ND1-reprogrammed cultures formed smaller tumors and expressed neuronal markers such as TUJ1 in the brain. Thus, reprogramming using a single-factor ND1 overcame drug resistance, converting malignant cells of heterogeneous GBM cells to normal neuron-like cells in vitro and in vivo. These novel observations warrant further research using patient-derived GBM cells and patient-derived xenograft (PDX) models as a potentially effective treatment for a deadly brain cancer and likely other astrocytoma tumors.

Inhibition of prolyl hydroxylases by dimethyloxaloylglycine after stroke reduces ischemic brain injury and requires hypoxia inducible factor-1α.

Pathological oxygen deprivation inhibits prolyl hydroxylase (PHD) activity and stimulates a protective cellular oxygen-sensing response in part through the stabilization and activation of the Hypoxia Inducible Factor (HIF) 1α transcription factor. The present investigation tested the therapeutic potential of enhanced activation of oxygen-sensing pathways by competitive pharmacologic PHD inhibition after stroke, hypothesizing that post-ischemic PHD inhibition would reduce neuronal cell death and require the activation of HIF-1α. The PHD inhibitor dimethyloxaloylglycine (DMOG, 100 µM) reduced cell death by oxygen glucose deprivation (OGD), an in vitro model of ischemia, and the protection required HIF-1α. In vivo, DMOG (50 mg/kg, i.p.) administered 30 or 60 min after distal occlusion of the middle cerebral artery (MCA) in mice enhanced the activation of HIF-1α protein, enhanced transcription of the HIF-regulated genes vascular endothelial growth factor, erythropoietin, endothelial nitric oxide synthase, and pyruvate dehydrogenase kinase-1, reduced ischemic infarct volume and activation of the pro-apoptotic caspase-3 protein, reduced behavioral deficits after stroke, and reduced the loss of local blood flow in the MCA territory after stroke. Inhibition of HIF-1α in vivo by Digoxin or Acriflavine abrogated the infarct sparing properties of DMOG. These data suggest that supplemental activation of oxygen-sensing pathways after stroke may provide a clinically applicable intervention for the promotion of neurovascular cell survival after ischemia.

Glycosaminoglycan scaffolding and neural progenitor cell transplantation promotes regenerative immunomodulation in the mouse ischemic brain.

Ischemic stroke is a leading cause of morbidity and mortality, with limited treatments that can facilitate brain regeneration. Neural progenitor cells (NPCs) hold promise for replacing tissue lost to stroke, and biomaterial approaches may improve their efficacy to overcome hurdles in clinical translation. The immune response and its role in stroke pathogenesis and regeneration may interplay with critical mechanisms of stem cell and biomaterial therapies. Cellular therapy can modulate the immune response to reduce toxic neuroinflammation early after ischemia. However, few studies have attempted to harness the regenerative effects of neuroinflammation to augment recovery. Our previous studies demonstrated that intracerebrally transplanted NPCs encapsulated in a chondroitin sulfate-A hydrogel (CS-A + NPCs) can improve vascular regeneration after stroke. In this paper, we found that CS-A + NPCs affect the microglia/macrophage response to promote a regenerative phenotype following stroke in mice. Following transplantation, PPARγ-expressing microglia/macrophages, and MCP-1 and IL-10 protein levels are enhanced. Secreted immunomodulatory factor expression of other factors was altered compared to NPC transplantation alone. Post-stroke depression-like behavior was reduced following cellular and material transplantation. Furthermore, we showed in cultures that microglia/macrophages encapsulated in CS-A had increased expression of angiogenic and arteriogenic mediators. Neutralization with anti-IL-10 antibody negated these effects in vitro. Cumulatively, this work provides a framework for understanding the mechanisms by which immunomodulatory biomaterials can enhance the regenerative effects of cellular therapy for ischemic stroke and other brain injuries.

Combinatorial intranasal delivery of bone marrow mesenchymal stem cells and insulin-like growth factor-1 improves neurovascularization and functional outcomes following focal cerebral ischemia in mice.

Bone marrow mesenchymal stem cell (BMSC) transplantation is a promising treatment for ischemic stroke that carries a severe mortality and disability burden amongst the adult population globally. Thus far, BMSC transplantation has been insufficient for ameliorating neurological deficits resulting from cerebral ischemia. This shortcoming may be an outcome due to poor homing and viability of grafted cells in ischemic brain that limit the potential therapeutic benefits of BMSC transplantation. Insulin-like growth factor-1 (IGF-1), a potent anti-apoptotic agent, exerts neuroprotective effects in ischemic stroke as well as rescuing neuronal death in vitro. We hypothesized that IGF-1 could also protect BMSCs from apoptotic death, and examined whether the combination of BMSCs with IGF-1 can enhance functional recovery outcomes in mice following cerebral ischemia. Intranasal administration of BMSCs with IGF-1 was applied in a mouse focal ischemic stroke model. Our in vitro results indicated that BMSCs treated with IGF-1 exhibited less apoptotic death induced by oxygen-glucose deprivation (OGD), and an improved migratory capacity. At 14 days after ischemic insult, the combination of BMSCs with IGF-1 resulted in a larger number of NeuN/BrdU and Glut-1/BrdU co-labeled cells in the areas contiguous to the ischemic core than IGF-1 or BMSC treatment alone. Western blot assays demonstrated that the protein levels of BDNF, VEGF and Ang-1 were significantly upregulated in the peri-infarct region in the combination treatment group compared with single IGF- 1 or BMSC treatment. Co-administration of BMSCs and IGF-1 markedly increases local cerebral blood flow and promoted better functional behavior outcomes. These data suggest that intranasal delivery of BMSCs in conjunction with IGF-1 strengthened functional recovery following ischemia via increasing neurogenesis and angiogenesis, providing a novel optimized strategy for improving the therapeutic efficacy of BMSC transplantation for ischemia.

DL-3-n-butylphthalide Increases Collateriogenesis and Functional Recovery after Focal Ischemic Stroke in Mice.

Recent evidence indicates that collateral circulation is critical for the outcome of ischemic stroke. DL-3-n-butylphthalide (NBP), a synthesized compound based on an extract from seeds of celery Apium graveolens Linn, has been used as a therapeutic drug, showing multiple neuroprotective and regenerative activities. A potential effect of NBP on collateral arterial regulation is unknown. We examined the effects of NBP on arteriogenesis of collateral arteries in vitro and a mouse ischemic stroke model. In cultures of mouse iPS cell-derived vascular progenitors, NBP (10 µM) significantly increased α-smooth muscle actin (αSMA)/CD-31 co-labeled cells and the expression of newly formed vasculature marker PDGFRα. A sensorimotor cortex ischemia was induced in transgenic mice expressing αSMA-GFP that allowed direct observation of arterial vasculatures in brain regions. NBP (80 mg/kg) was intranasally delivered 1 hr after stroke and once daily for 14 days. To label proliferating cells, 5-Bromo-2'-deoxyuridine (BrdU, 50 mg/kg, i.p.) was administrated every day from 3 days after stroke. Western blotting of peri-infarct tissue detected increased expressions of VEGF, Ang-1 and reduced nNOS level in NBP-treated mice. The NBP treatment significantly increased αSMA/BrdU co-labeled cells, the diameter of ipsilateral collaterals, and arterial area in ischemic and peri-infarct regions examined 14 days after stroke. Examined 3 days after stroke, NBP prevented functional deficits in the cylinder test and corner test. The NBP treatment of 14 days improved the local cerebral blood flow (LCBF) and functional performance in multiple tests. Thus, NBP promotes collateriogenesis, short and long-term structural and functional improvements after ischemic stroke.

Glial Cell-Based Vascular Mechanisms and Transplantation Therapies in Brain Vessel and Neurodegenerative Diseases.

Neurodevelopmental and neurodegenerative diseases (NDDs) with severe neurological/psychiatric symptoms, such as cerebrovascular pathology in AD, CAA, and chronic stroke, have brought greater attention with their incidence and prevalence having markedly increased over the past few years. Causes of the significant neuropathologies, especially those observed in neurological diseases in the CNS, are commonly believed to involve multiple factors such as an age, a total environment, genetics, and an immunity contributing to their progression, neuronal, and vascular injuries. We primarily focused on the studies of glial involvement/dysfunction in part with the blood-brain barrier (BBB) and the neurovascular unit (NVU) changes, and the vascular mechanisms, which have been both suggested as critical roles in chronic stroke and many other NDDs. It has been noted that glial cells including astrocytes (which outnumber other cell types in the CNS) essentially contribute more to the BBB integrity, extracellular homeostasis, neurotransmitter release, regulation of neurogenic niches in response to neuroinflammatory stimulus, and synaptic plasticity. In a recent study for NDDs utilizing cellular and molecular biology and genetic and pharmacological tools, the role of reactive astrocytes (RACs) and gliosis was demonstrated, able to trigger pathophysiological/psychopathological detrimental changes during the disease progression. We speculate, in particular, the BBB, the NVU, and changes of the astrocytes (potentially different populations from the RACs) not only interfere with neuronal development and synaptogenesis, but also generate oxidative damages, contribute to beta-amyloid clearances and disrupted vasculature, as well as lead to neuroinflammatory disorders. During the past several decades, stem cell therapy has been investigated with a research focus to target related neuro-/vascular pathologies (cell replacement and repair) and neurological/psychiatric symptoms (paracrine protection and homeostasis). Evidence shows that transplantation of neurogenic or vasculogenic cells could be achieved to pursue differentiation and maturation within the diseased brains as expected. It would be hoped that, via regulating functions of astrocytes, astrocytic involvement, and modulation of the BBB, the NVU and astrocytes should be among major targets for therapeutics against NDDs pathogenesis by drug and cell-based therapies. The non-invasive strategies in combination with stem cell transplantation such as the well-tested intranasal deliveries for drug and stem cells by our and many other groups show great translational potentials in NDDs. Neuroimaging and clinically relevant analyzing tools need to be evaluated in various NDDs brains.

Conversion of Reactive Astrocytes to Induced Neurons Enhances Neuronal Repair and Functional Recovery After Ischemic Stroke.

The master neuronal transcription factor NeuroD1 can directly reprogram astrocytes into induced neurons (iNeurons) after stroke. Using viral vectors to drive ectopic ND1 expression in gliotic astrocytes after brain injury presents an autologous form of cell therapy for neurodegenerative disease. Cultured astrocytes transfected with ND1 exhibited reduced proliferation and adopted neuronal morphology within 2-3 weeks later, expressed neuronal/synaptic markers, and extended processes. Whole-cell recordings detected the firing of evoked action potentials in converted iNeurons. Focal ischemic stroke was induced in adult GFAP-Cre-Rosa-YFP mice that then received ND1 lentivirus injections into the peri-infarct region 7 days after stroke. Reprogrammed cells did not express stemness genes, while 2-6 weeks later converted cells were co-labeled with YFP (constitutively activated in astrocytes), mCherry (ND1 infection marker), and NeuN (mature neuronal marker). Approximately 66% of infected cells became NeuN-positive neurons. The majority (~80%) of converted cells expressed the vascular glutamate transporter (vGLUT) of glutamatergic neurons. ND1 treatment reduced astrogliosis, and some iNeurons located/survived inside of the savaged ischemic core. Western blotting detected higher levels of BDNF, FGF, and PSD-95 in ND1-treated mice. MultiElectrode Array (MEA) recordings in brain slices revealed that the ND1-induced reprogramming restored interrupted cortical circuits and synaptic plasticity. Furthermore, ND1 treatment significantly improved locomotor, sensorimotor, and psychological functions. Thus, conversion of endogenous astrocytes to neurons represents a plausible, on-site regenerative therapy for stroke.