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.

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.

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.

[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.

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.

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.

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.

Modulation of Stem Cells as Therapeutics for Severe Mental Disorders and Cognitive Impairments.

Severe mental illnesses (SMI) such as schizophrenia and bipolar disorder affect 2-4% of the world population. Current medications and diagnostic methods for mental illnesses are not satisfying. In animal studies, stem cell therapy is promising for some neuropsychiatric disorders and cognitive/social deficits, not only treating during development (targeting modulation and balancing) but also following neurodegeneration (cell replacement and regenerating support). We believe that novel interventions such as modulation of particular cell populations to develop cell-based treatment can improve cognitive and social functions in SMI. With pathological synaptic/myelin damage, oligodendrocytes seem to play a role. In this review, we have summarized oligodendrogenesis mechanisms and some related calcium signals in neural cells and stem/progenitor cells. The related benefits from endogenous stem/progenitor cells within the brain and exogenous stem cells, including multipotent mesenchymal-derived stromal cells (MSC), fetal neural stem cells (NSC), pluripotent stem cells (PSC), and differentiated progenitors, are discussed. These also include stimulating mechanisms of oligodendrocyte proliferation, maturation, and myelination, responsive to the regenerative effects by both endogenous stem cells and transplanted cells. Among the mechanisms, calcium signaling regulates the neuronal/glial progenitor cell (NPC/GPC)/oligodendrocyte precursor cell (OPC) proliferation, migration, and differentiation, dendrite development, and synaptic plasticity, which are involved in many neuropsychiatric diseases in human. On the basis of numerous protein annotation and protein-protein interaction databases, a total of 119 calcium-dependent/activated proteins that are related to neuropsychiatry in human are summarized in this investigation. One of the advanced methods, the calcium/cation-channel-optogenetics-based stimulation of stem cells and transplanted cells, can take advantage of calcium signaling regulations. Intranasal-to-brain delivery of drugs and stem cells or local delivery with the guidance of brain imaging techniques may provide a unique new approach for treating psychiatric disorders. It is also expected that preconditioning stem cell therapy following precise brain imaging as pathological confirmation has high potential if translated to cell clinic use. Generally, modulable cell transplantation followed by stimulations should provide paracrine protection, synaptic modulation, and myelin repair for the brain in SMI.

Cortical Transplantation of Brain-Mimetic Glycosaminoglycan Scaffolds and Neural Progenitor Cells Promotes Vascular Regeneration and Functional Recovery after Ischemic Stroke in Mice.

Stroke causes significant mortality and morbidity. Currently, there are no treatments which can regenerate brain tissue lost to infarction. Neural progenitor cells (NPCs) are at the forefront of preclinical studies for regenerative stroke therapies. NPCs can differentiate into and replace neurons and promote endogenous recovery mechanisms such as angiogenesis via trophic factor production and release. The stroke core is hypothetically the ideal location for replacement of neural tissue since it is in situ and develops into a potential space where injections may be targeted with minimal compression of healthy peri-infarct tissue. However, the compromised perfusion and tissue degradation following ischemia create an inhospitable environment resistant to cellular therapy. Overcoming these limitations is critical to advancing cellular therapy. In this work, the therapeutic potential of mouse-induced pluripotent stem cell derived NPCs is tested encapsulated in a basic fibroblast growth factor (bFGF) binding chondroitin sulfate-A (CS-A) hydrogel transplanted into the infarct core in a mouse sensorimotor cortex mini-stroke model. It is shown that CS-A encapsulation significantly improves vascular remodeling, cortical blood flow, and sensorimotor behavioral outcomes after stroke. It is found these improvements are negated by blocking bFGF, suggesting that the sustained trophic signaling endowed by the CS-A hydrogel combined with NPC transplantation can promote tissue repair.

DPP-4 Inhibitor Linagliptin is Neuroprotective in Hyperglycemic Mice with Stroke via the AKT/mTOR Pathway and Anti-apoptotic Effects.

Dipeptidyl peptidase 4 (DPP-4) inhibitors have been shown to have neuroprotective effects in diabetic patients suffering from stroke, but less research has focused on patients with mild hyperglycemia below the threshold for a diagnosis of diabetes. In this investigation, a hyperglycemic mouse model was generated by intraperitoneal injection of streptozotocin and then subjected to focal cerebral ischemia. We demonstrated that the DPP-4 inhibitor linagliptin significantly decreased the infarct volume, reduced neuronal cell death, decreased inflammation, and improved neurological deficit compared with control mice. Linagliptin up-regulated the expression of p-Akt and p-mTOR and regulated the apoptosis factors Bcl-2, Bax, and caspase 9. Taken together, these results suggest that linagliptin exerts a neuroprotective action likely through activation of the Akt/mTOR pathway along with anti-apoptotic and anti-inflammatory mechanisms. Therefore, linagliptin may be considered as a therapeutic treatment for stroke patients with mild hyperglycemia.

Pharmacological hypothermia induced neurovascular protection after severe stroke of transient middle cerebral artery occlusion in mice.

Therapeutic hypothermia is a potential protective strategy after stroke. The present study evaluated the neurovascular protective potential of pharmacological hypothermia induced by the neurotensin receptor 1 agonist HPI-201 after severe ischemic stroke. Adult C57BL/6 mice were subjected to filament insertion-induced occlusion of the middle cerebral artery (60 min MCAO). HPI-201 was i.p. injected 120 min after the onset of MCAO to initiate and maintain the body temperature at 32-33°C for 6 hrs. The infarct volume, cell death, integrity of the blood brain barrier (BBB) and neurovascular unit (NVU), inflammation, and functional outcomes were evaluated. The hypothermic treatment significantly suppressed the infarct volume and neuronal cell death, accompanied with reduced caspase-3 activation and BAX expression while Bcl-2 increased in the peri-infarct region. The cellular integrity of the BBB and NVU was significantly improved and brain edema was attenuated in HPI-201-treated mice compared to stroke controls. The hypothermic treatment decreased the expression of inflammatory factors including tumor necrosis factor-α (TNF-α), MMP-9, interleukin-1ß (IL-1ß), the M1 microglia markers IL-12 and inducible nitric oxide synthase (iNOS), while increased the M2 marker arginase-1 (Arg-1). Stroke mice received the hypothermic treatment showed lower neurological severity score (NSS), performed significantly better in functional tests, the mortality rate in the hypothermic group was noticeably lower compared with stroke controls. Taken together, HPI-201 induced pharmacological hypothermia is protective for different neurovascular cells after a severely injured brain, mediated by multiple mechanisms.

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.

Enhanced Neurogenesis and Collaterogenesis by Sodium Danshensu Treatment After Focal Cerebral Ischemia in Mice.

Ischemic stroke remains a serious threat to human life. There are limited effective therapies for the treatment of stroke. We have previously demonstrated that angiogenesis and neurogenesis in the brain play an important role in functional recovery following ischemic stroke. Recent studies indicate that increased arteriogenesis and collateral circulation are determining factors for restoring reperfusion and outcomes of stroke patients. Danshensu, the Salvia miltiorrhiza root extract, is used in treatments of various human ischemic events in traditional Chinese medicine. Its therapeutic mechanism, however, is not well clarified. Due to its proposed effect on angiogenesis and arteriogenesis, we hypothesized that danshensu could benefit stroke recovery through stimulating neurogenesis and collaterogenesis in the post-ischemia brain. Focal ischemic stroke targeting the right sensorimotor cortex was induced in wild-type C57BL6 mice and transgenic mice expressing green fluorescent protein (GFP) to label smooth muscle cells of brain arteries. Sodium danshensu (SDS, 700 mg/kg) was administered intraperitoneally (i.p.) 10 min after stroke and once daily until animals were sacrificed. To label proliferating cells, 5-bromo-2'-deoxyuridine (BrdU; 50 mg/kg, i.p.) was administered, starting on day 3 after ischemia and continued once daily until sacrifice. At 14 days after stroke, SDS significantly increased the expression of vascular endothelial growth factor (VEGF), stromal-derived factor-1 (SDF-1), brain-derived neurotrophic factor (BDNF), and endothelial nitric oxide synthase (eNOS) in the peri-infarct region. SDS-treated animals showed increased number of doublecortin (DCX)-positive cells. Greater numbers of proliferating endothelial cells and smooth muscle cells were detected in SDS-treated mice 21 days after stroke in comparison with vehicle controls. The number of newly formed neurons labeled by NeuN and BrdU antibodies increased in SDS-treated mice 28 days after stroke. SDS significantly increased the newly formed arteries and the diameter of collateral arteries, leading to enhanced local cerebral blood flow recovery after stroke. These results suggest that systemic sodium danshensu treatment shows significant regenerative effects in the post-ischemic brain, which may benefit long-term functional recovery from ischemic stroke.

Pyruvate Kinase M2 Increases Angiogenesis, Neurogenesis, and Functional Recovery Mediated by Upregulation of STAT3 and Focal Adhesion Kinase Activities After Ischemic Stroke in Adult Mice.

Ischemic stroke remains a serious threat to human life. Generation of neuronal and vascular cells is an endogenous regenerative mechanism in the adult brain, which may contribute to tissue repair after stroke. However, the regenerative activity is typically insufficient for significant therapeutic effects after brain injuries. Pyruvate kinase isoform M2 (PKM2) is a key regulator for energy metabolism. PKM2 also has nonmetabolic roles involving regulations of gene expression, cell proliferation, and migration in cancer cells as well as noncancerous cells. In a focal ischemic stroke mouse model, recombinant PKM2 (rPKM2) administration (160 ng/kg, intranasal delivery) at 1 h after stroke showed the significant effect of a reduced infarct volume of more the 60%. Delayed treatment of rPKM2, however, lost the acute neuroprotective effect. We then tested a novel hypothesis that delayed treatment of PKM2 might show proregenerative effects for long-term functional recovery and this chronic action could be mediated by its downstream STAT3 signaling. rPKM2 (160 ng/kg) was delivered to the brain using noninvasive intranasal administration 24 h after the stroke and repeated every other day. Western blot analysis revealed that, 7 days after the stroke, the levels of PKM2 and phosphorylated STAT3 and the expression of angiogenic factors VEGF, Ang-1, and Tie-2 in the peri-infarct region were significantly increased in the rPKM2 treatment group compared with those of the stroke vehicle group. To label proliferating cells, 5-bromo-2'-deoxyuridine (BrdU, 50 mg/kg, i.p.) was injected every day starting 3 days after stroke. At 14 days after stroke, immunohistochemistry showed that rPKM2 increased cell homing of doublecortin (DCX)-positive neuroblasts to the ischemic cortex. In neural progenitor cell (NPC) cultures, rPKM2 (0.4-4 nM) increased the expression of integrin ß1 and the activation/phosphorylation of focal adhesion kinase (FAK). A mediator role of FAK in PKM2-promoted cell migration was verified in FAK-knockout fibroblast cultures. In the peri-infarct region of the brain, increased numbers of Glut-1/BrdU and NeuN/BrdU double-positive cells indicated enhanced angiogenesis and neurogenesis, respectively, compared to stroke vehicle mice. Using Laser Doppler imaging, we observed better recovery of the local blood flow in the peri-infarct region of rPKM2-treated mice 14 days after stroke. Meanwhile, rPKM2 improved the sensorimotor functional recovery measured by the adhesive removal test. Inhibiting the STAT3 phosphorylation/activation by the STAT3 inhibitor, BP-1-102 (3 mg/kg/day, o.g.), abolished all beneficial effects of rPKM2 in the stroke mice. Taken together, this investigation provides the first evidence demonstrating that early treatment of rPKM2 shows an acute neuroprotective effect against ischemic brain damage, whereas delayed rPKM2 treatment promotes regenerative activities in the poststroke brain leading to better functional recovery. The underlying mechanism involves activation of the STAT3 and FAK signals in the poststroke brain.

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.

Transplantation of iPS cell-derived neural progenitors overexpressing SDF-1α increases regeneration and functional recovery after ischemic stroke.

Ischemic stroke is a leading cause of human death and disability while clinical treatments are limited. The adult brain possesses endogenous regenerative activities that may benefit tissue repair after stroke. Trophic factors such as stromal cell-derived factor 1 alpha (SDF-1α) are upregulated in the ischemic brain, which promote endogenous regeneration. The regenerative response, however, is normally insufficient. Transplantation of exogenous cells has been explored as regenerative therapies. One promising cell type for transplantation is induced pluripotent stem (iPS) cells which are cells genetically reprogrammed from adult somatic cells. We hypothesized that transplanting neural progenitor cells derived from iPS cells (iPS-NPCs) could provide cell replacement and trophic support. The trophic factor SDF-1α was overexpressed in iPS-NPCs by lentiviral transduction to test if SDF-1α could increase regeneration in the ischemic brain. These SDF-1α-iPS-NPCs were differentiated in vitro to express mature neuronal and synaptic markers. Differentiated cells expressed functional Na+ and K+ channels, and fired action potentials. In the oxygen glucose deprivation (OGD) test, SDF-1α-iPS-NPCs survived significantly better compared to control iPS-NPCs. In mice subjected to focal cerebral ischemia in the sensorimotor cortex, iPS-NPCs and SDF-1α-iPS-NPCs were intracranially transplanted into the ischemic cortex 7 days after stroke. Neuronal differentiation of transplanted cells was identified using NeuN 14 days after transplantation. Mice that received SDF-1α-iPS-NPCs had greater numbers of NeuN/BrdU and Glut-1/BrdU co-labeled cells in the peri-infarct area and improved locomotion compared to the control iPS-NPC transplantation. Thus, SDF-1α upregulation in transplanted cells may be a therapeutic strategy to enhance endogenous neurovascular repair after ischemic stroke in adult mice.

DL-3-n-butylphthalide induced neuroprotection, regenerative repair, functional recovery and psychological benefits following traumatic brain injury in mice.

Previous investigations suggest that DL-3-n-butylphthalide (NBP) is a promising multifaceted drug for the treatment of stroke. It is not clear whether NBP can treat traumatic brain injury (TBI) and what could be the mechanisms of therapeutic benefits. To address these issues, TBI was induced by a controlled cortical impact in adult male mice. NBP (100 mg/kg) or saline was intraperitoneally administered within 5 min after TBI. One day after TBI, apoptotic events including caspase-3/9 activation, cytochrome c release from the mitochondria, and apoptosis-inducing factor (AIF) translocation into the nucleus in the pericontusion region were attenuated in NBP-treated mice compared to TBI-saline controls. In the assessment of the nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) pathway, NBP ameliorated the p65 expression and the p-IκB-α/IκB-α ratio, indicating reduced NF-κB activation. Consistently, NBP reduced the upregulation of proinflammatory cytokines such as tumor necrotizing factor-alpha (TNF-α) and interleukin-1beta (IL-1ß) after TBI. In sub-acute treatment experiments, NBP was intranasally delivered once daily for 3 days. At 3 days after TBI, this repeated NBP treatment significantly reduced the contusion volume and cell death in the pericontusion region. In chronic experiments up to 21 days after TBI, continues daily intranasal NBP treatment increased neurogenesis, angiogenesis, and arteriogenesis in the post-TBI brain, accompanied with upregulations of regenerative genes including brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), endothelial-derived nitric oxide synthase (eNOS), and matrix metallopeptidase 9 (MMP-9). The NBP treatment significantly improved sensorimotor functional recovery and reduced post-TBP depressive behavior. These new findings demonstrate that NBP shows multiple therapeutic benefits after TBI.

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.

Intracranial Transplantation of Hypoxia-Preconditioned iPSC-Derived Neural Progenitor Cells Alleviates Neuropsychiatric Defects After Traumatic Brain Injury in Juvenile Rats.

Traumatic brain injury (TBI) is a common cause of mortality and long-term morbidity in children and adolescents. Posttraumatic stress disorder (PTSD) frequently develops in these patients, leading to a variety of neuropsychiatric syndromes. Currently, few therapeutic strategies are available to treat juveniles with PTSD and other developmental neuropsychiatric disorders. In the present investigation, postnatal day 14 (P14) Wistar rats were subjected to TBI induced by a controlled cortical impact (CCI) (velocity = 3 m/s, depth = 2.0 mm, contact time = 150 ms). This TBI injury resulted in not only cortical damages, but also posttrauma social behavior deficits. Three days after TBI, rats were treated with intracranial transplantation of either mouse iPSC-derived neural progenitor cells under normal culture conditions (N-iPSC-NPCs) or mouse iPSC-derived neural progenitor cells pretreated with hypoxic preconditioning (HP-iPSC-NPCs). Compared to TBI animals that received N-iPSC-NPCs or vehicle treatment, HP-iPSC-NPC-transplanted animals showed a unique benefit of improved performance in social interaction, social novelty, and social transmission of food preference tests. Western blotting showed that HP-iPSC-NPCs expressed significantly higher levels of the social behavior-related genes oxytocin and the oxytocin receptor. Overall, HP-iPSC-NPC transplantation exhibits a great potential as a regenerative therapy to improve neuropsychiatric outcomes after juvenile TBI.

Expression of the NMDA receptor subunit GluN3A (NR3A) in the olfactory system and its regulatory role on olfaction in the adult mouse.

Glutamate is an excitatory neurotransmitter in the olfactory system and its N-methyl-D-aspartate-(NMDA) receptor subunits [GluN1 (NR1), GluN2A (NR2A), and GluN2B (NR2B)] are expressed at synapses in the olfactory bulb and olfactory epithelium. Thus, glutamatergic neurons and NMDA receptors play key roles in olfaction. GluN3A (NR3A) is a unique inhibitory subunit in the NMDA receptor complex; however, the expression and functional role of GluN3A in the olfactory bulb and epithelium remain unclear. The present study examined the expression patterns of GluN3A in the olfactory bulb and epithelium and explored its functional role in the olfactory system. Immunohistochemical and Western blot analyses revealed that GluN3A is abundantly expressed in different cellular layers of the olfactory bulb and epithelium of the adult wild type (WT) mice. In littermate GluN3A knockout (GluN3A(-/-); KO) mice, the expression of olfactory marker protein normally found in mature olfactory sensory neurons was significantly reduced in the olfactory bulb and epithelium. A butyl alcohol stimulus increased immediate-early gene c-Fos expression in the olfactory system of WT mice, while this response was absent in GluN3A KO mice. The level of phosphorylated Ca(2+)/calmodulin-dependent kinase II was significantly lower in GluN3A KO mice compared to WT mice. In buried food finding test, GluN3A mice took significantly longer time to find food compared to WT mice. Consistently, impaired odor distinguishing ability was seen in GluN3A KO mice. These findings suggest that GluN3A, expressed in the adult olfactory system, plays a significant regulatory role in olfactory development and functional activity.