Peri-infarct Area Research Articles

Effects of exercise and bryostatin-1 on serotonin dynamics after cerebral infarction.

Although it has been suggested that the combination of exercise and bryostatin-1 administration may induce greater functional recovery than exercise alone, the detailed molecular mechanisms are not well known. Here, we examined the relationship between this combination treatment and monoamine dynamics in the cerebral cortex peri-infarction area to promote our understanding of these molecular mechanisms. Experimental cerebral cortex infarctions were produced by photothrombosis in rats. Voluntary exercise was initiated 2 days after surgery. Motor performance was then measured using the rotarod test. Monoamine concentrations in the perilesional cortex were analyzed by high-performance liquid chromatography. In behavioral evaluations, performance in the rotarod test was significantly increased by exercise. Moreover, performance in the rotarod test after the combination of exercise and bryostatin-1 administration was significantly greater than that after exercise alone. In the analysis of monoamines, serotonin (5-HT) concentrations were significantly higher in the groups treated with exercise and bryostatin-1. In addition, 5-HT turnover was significantly lower in the groups treated with exercise and bryostatin-1. Furthermore, the mean latency in the rotarod test showed a significant positive correlation with 5-HT levels. In immunohistochemical analysis, 5-HT immunoreactivity in the dorsal raphe nucleus was shown to be higher in the groups treated with exercise. In the present study, we detected changes in the levels of monoamines associated with the combined treatment of exercise and bryostatin-1 administration in the perilesional cortex. It has been suggested that this combination of therapies may affect 5-HT turnover and serve to increase local 5-HT concentrations in the perilesional area.

BMP Antagonist Gremlin 2 Limits Inflammation After Myocardial Infarction.

We have recently shown that the bone morphogenetic protein (BMP) antagonist Gremlin 2 (Grem2) is required for early cardiac development and cardiomyocyte differentiation. Our initial studies discovered that Grem2 is strongly induced in the adult heart after experimental myocardial infarction (MI). However, the function of Grem2 and BMP-signaling inhibitors after cardiac injury is currently unknown. To investigate the role of Grem2 during cardiac repair and assess its potential to improve ventricular function after injury. Our data show that Grem2 is transiently induced after MI in peri-infarct area cardiomyocytes during the inflammatory phase of cardiac tissue repair. By engineering loss- (Grem2(-/-)) and gain- (TG(Grem2)) of-Grem2-function mice, we discovered that Grem2 controls the magnitude of the inflammatory response and limits infiltration of inflammatory cells in peri-infarct ventricular tissue, improving cardiac function. Excessive inflammation in Grem2(-/-) mice after MI was because of overactivation of canonical BMP signaling, as proven by the rescue of the inflammatory phenotype through administration of the canonical BMP inhibitor, DMH1. Furthermore, intraperitoneal administration of Grem2 protein in wild-type mice was sufficient to reduce inflammation after MI. Cellular analyses showed that BMP2 acts with TNFα to induce expression of proinflammatory proteins in endothelial cells and promote adhesion of leukocytes, whereas Grem2 specifically inhibits the BMP2 effect. Our results indicate that Grem2 provides a molecular barrier that controls the magnitude and extent of inflammatory cell infiltration by suppressing canonical BMP signaling, thereby providing a novel mechanism for limiting the adverse effects of excessive inflammation after MI.

SCF/c-kit transactivates CXCR4-serine 339 phosphorylation through G protein-coupled receptor kinase 6 and regulates cardiac stem cell migration.

C-kit positive cardiac stem cells (CSCs) have been shown to contribute to myocardial regeneration after infarction. Previously, we have shown that the c-kit ligand stem cell factor (SCF) can induce CSC migration into the infarcted area during myocardial infarction (MI). However, the precise mechanism involved is not fully understood. In this study, we found that CSCs also express C-X-C chemokine receptor type 4 (CXCR4), which is a typical member of the seven transmembrane-spanning G protein-coupled receptor (GPCR). In vitro, activation of c-kit signalling by SCF promotes migration of CSCs with increased phosphorylation of CXCR4-serine 339, p38 mitogen-activated protein kinase (p38 MAPK) and extracellular regulated protein kinases 1/2 (ERK1/2). Knockdown of CXCR4 expression by siRNA reduces SCF/c-kit-induced migration and downstream signalling. As previously reported, CXCR4-serine 339 phosphorylation is mainly regulated by GPCR kinase 6 (GRK6); thus, silencing of GRK6 expression by siRNA impairs CXCR4-serine 339 phosphorylation and migration of CSCs caused by SCF. In vivo, knockdown of GRK6 impairs the ability of CSCs to migrate into peri-infarcted areas. These results demonstrate that SCF-induced CSC migration is regulated by the transactivation of CXCR4-serine 339 phosphorylation, which is mediated by GRK6.

MiR-211/STAT5A Signaling Modulates Migration of Mesenchymal Stem Cells to Improve its Therapeutic Efficacy.

Our previous study showed that the therapeutic effects of mesenchymal stem cells (MSCs) transplantation were improved by enhancing migration. MicroRNA-211 (miR-211) can modulate the migratory properties of some cell types by mechanisms that are not fully understood. This study was designed to investigate a possible role for miR-211 in MSC migration, and whether genetic manipulation of miR-211 in MSCs could be used to enhance its beneficial effects of cell transplantation. Transwell assays confirmed that MSCs migration of was significantly impaired by miR-211 knockdown but enhanced by miR-211 overexpression. MiR-211 overexpressing MSCs also exhibited significantly increased cell engraftment in the peri-infarct areas of female rat hearts 2 days after intravenous transplantation of male MSCs as shown by GFP tracking and SYR gene quantification. This conferred a significant decrease in infarct size and improved cardiac performance. By using a loss or gain of gene function approach, we demonstrated that miR-211 targeted STAT5A to modulate MSCs migration, possibly by interacting with MAPK signaling. Furthermore, the beneficial effects of miR-211 overexpression in MSCs were abolished by simultaneous overexpression of STAT5A whereas the negative effects of miR-211 silencing on MSC migration were rescued by simultaneous downregulation of STAT5A. Finally, using ChIP-PCR and luciferase assays, we provide novel evidence that STAT3 can directly bind to promoter elements that activate miR-211 expression. STAT3/miR-211/STAT5A signaling plays a key role in MSCs migration. Intravenous infusion of genetically modified miR-211 overexpressing MSCs conveys enhanced protection from adverse post-MI remodeling compared with unmodified MSCs. Stem Cells 2016;34:1846-1858.

Retraction Note To: Expression of SOCS2 mRNA and protein in the ischemic core and penumbra after transient focal cerebral ischemia in rats.

The suppressor of cytokine signaling 2 (SOCS2) has been reported to be involved in astroglial reactions and adult neurogenesis in the ischemic hippocampus. To elucidate whether SOCS2 is implicated in the pathophysiology of stroke, we investigate spatiotemporal regulation and identification of cell phenotypes expressing SOCS2 after transient focal cerebral ischemia. Weak hybridization signals for SOCS2 mRNA were constitutively observed in striatal neurons and upregulation of SOCS2 mRNA was induced in association with nestin-positive cells in stroke-lesioned rats. Analysis of the characteristics and phenotypes of SOCS2/nestin double-labeled cells revealed spatial differences between infarct and peri-infarct areas. SOCS2/nestin double-labeled cells in the infarct area were associated with the vasculature and were highly proliferative. In contrast, the double-labeled cells in the peri-infarct area were indeed glial fibrillary acidic protein (GFAP)-positive reactive astrocytes forming the glial scar, although nestin-negative reactive astrocytes also exhibited weak SOCS2 expression. In addition, induction of SOCS2 expression was observed in Iba1-positive cells showing a macrophage-like phenotype with amoeboid morphology; these cells were predominantly localized in the infarct area. In the peri-infarct area, only a small proportion of Iba1-positive cells with the morphology of brain macrophages expressed SOCS2 and most activated stellate microglial cells with thick and short processes exhibited weak or negligible SOCS2 expression. Thus, our results revealed the phenotypic and functional heterogeneity of SOCS2-expressing cells within infarct and peri-infarct areas, suggesting the involvement of SOCS2 in astroglial reactions and activation/recruitment of brain macrophages and its potential role in perivascular progenitors/stem cells after ischemic stroke.

Bone Fracture Pre-Ischemic Stroke Exacerbates Ischemic Cerebral Injury in Mice.

Ischemic stroke is a devastating complication of bone fracture. Bone fracture shortly after stroke enhances stroke injury by augmenting inflammation. We hypothesize that bone fracture shortly before ischemic stroke also exacerbates ischemic cerebral injury. Tibia fracture was performed 6 or 24 hours before permanent middle cerebral artery occlusion (pMCAO) on C57BL/6J mice or Ccr2RFP/+Cx3cr1GFP/+ mice that have the RFP gene knocked into one allele of Ccr2 gene and GFP gene knocked into one allele of Cx3cr1 gene. Behavior was tested 3 days after pMCAO. Infarct volume, the number of CD68+ cells, apoptotic neurons, bone marrow-derived macrophages (RFP+), and microgila (GFP+) in the peri-infarct region were quantified. Compared to mice subjected to pMCAO only, bone fracture 6 or 24 hours before pMCAO increased behavioral deficits, the infarct volume, and the number of CD68+ cells and apoptotic neurons in the peri-infarct area. Both bone marrow-derived macrophages (CCR2+) and microglia (CX3CR1+) increased in the peri-infarct regions of mice subjected to bone fracture before pMCAO compared to stroke-only mice. The mice subjected to bone fracture 6 hours before pMCAO had more severe injury than mice that had bone fracture 24 hours before pMCAO. Our data showed that bone fracture shortly before stroke also increases neuroinflammation and exacerbates ischemic cerebral injury. Our findings suggest that inhibition of neuroinflammation or management of stroke risk factors before major bone surgery would be beneficial for patients who are likely to suffer from stroke.

RETRACTED: Secretory pathway Ca2+-ATPase isoform 1 knockdown promotes Golgi apparatus stress injury in a mouse model of focal cerebral ischemia–reperfusion: In vivo and in vitro study

The present study was designed to investigate the potential role of secretory pathway Ca(2+)-ATPase isoform 1(SPCA1) in experimental focal cerebral ischemia-reperfusion injury. Cerebral ischemia-reperfusion was induced by transient middle cerebral artery occlusion (MCAO) for 2h s in Sprague-Dawley rats, and then the expression levels of SPAC1 mRNA and protein were determined. Results showed that SPCA1 level was transiently increased 1 day after reperfusion in peri-infarction area, while markedly increased in infarction core on 3day and 7 day after reperfusion. Then a SPCA1 lentivirus was used to achieve knockdown of SPCA1 gene: Ca(2+) transporting type 2C, member 1 (ATP2C1) gene. It has been observed that SPCA1 knockdown by lentivirus markedly increased cerebral infarction volume in vivo. Meanwhile, SPCA1 knockdown also facilitated per-oxidative production, including nitric oxide (NO) and 3-nitrotyrosine (3-NT) and decreased the expression of total superoxide dismutase (SOD) and manganese superoxide dismutase (MnSOD). Moreover, in vitro study showed that SPCA1 knockdown increased hydrogen peroxide (H2O2)-induced lactate dehydrogenase (LDH) leakage dose-dependently, and elevated caspase3 level in neuro-2a (N2a) cells. In addition, SPCA1 knockdown increased H2O2-induced production of nitric oxide and 3-NT dose-dependently, and reversed the increased activity of total SOD and MnSOD in neuro-2a cells. In conclusion, the present study indicated that SPCA1 could suppress over active Golgi apparatus (GA) stress thus attenuate cerebral ischemia-reperfusion injury.

Up-regulation of serotonin receptor 2B mRNA and protein in the peri-infarcted area of aged rats and stroke patients.

Despite the fact that a high proportion of elderly stroke patients develop mood disorders, the mechanisms underlying late-onset neuropsychiatric and neurocognitive symptoms have so far received little attention in the field of neurobiology. In rodents, aged animals display depressive symptoms following stroke, whereas young animals recover fairly well. This finding has prompted us to investigate the expression of serotonin receptors 2A and 2B, which are directly linked to depression, in the brains of aged and young rats following stroke. Although the development of the infarct was more rapid in aged rats in the first 3 days after stroke, by day 14 the cortical infarcts were similar in size in both age groups i.e. 45% of total cortical volume in young rats and 55.7% in aged rats. We also found that the expression of serotonin receptor type B mRNA was markedly increased in the perilesional area of aged rats as compared to the younger counterparts. Furthermore, histologically, HTR2B protein expression in degenerating neurons was closely associated with activated microglia both in aged rats and human subjects. Treatment with fluoxetine attenuated the expression of Htr2B mRNA, stimulated post-stroke neurogenesis in the subventricular zone and was associated with an improved anhedonic behavior and an increased activity in the forced swim test in aged animals. We hypothesize that HTR2B expression in the infarcted territory may render degenerating neurons susceptible to attack by activated microglia and thus aggravate the consequences of stroke.

Matrix metalloproteinase-13 participates in neuroprotection and neurorepair after cerebral ischemia in mice

New neuroreparative and neuroprotective therapies are being sought to treat stroke patients. One approach is the remodeling of extracellular matrix, which participates in both brain injury and neurovascular repair when matrix metalloproteinases (MMPs) are thought to be key players. Our aim was to investigate the role of MMP-13 (collagenase-3) in the acute (24h and 3days) and delayed (2weeks) phases of stroke. Permanent and transient cerebral ischemia models involving the cortex were induced in MMP-13 knock-out (KO) and wild-type (WT) mice. In the transient model, MMP-13 deficiency reduced the amount of TTC-stained infarct tissue, reduced hemorrhagic events and improved functional outcomes (p<0.01). At two weeks, normal neuroblast (DCX+) migration from the subventricular zone toward the peri-infarct area was observed. However, MMP-13 deficiency significantly reduced the number of newborn neuroblasts (DCX+/BrdU+) in the cortical peri-infarct area (p<0.01). This result occurred in parallel with aberrant cortical vascular remodeling: post-stroke peri-infarct vessel density increased in the WT mice (p<0.01) but this increase was blocked in the MMP-13 KO mice. Prior to these vascular alterations, the levels of pro-angiogenic factors, including G-CSF, VEGF-A and angiopoietin-2, were lower in the ischemic cortex of MMP-13 KO mice than in WT mice (p<0.05). In vitro, gene-silencing of MMP-13 in endothelial progenitor cells (EPCs) confirmed the reduced ability of these cells to build tubulogenic networks in Matrigel™ substrate. Together, our results indicate that MMP-13 is a central protease in infarct development and cortical remodeling during post-stroke neurorepair, which is critical for optimal angiogenic and neurogenic responses.

Astrocyte-Derived Pentraxin 3 Supports Blood-Brain Barrier Integrity Under Acute Phase of Stroke.

Pentraxin 3 (PTX3) is released on inflammatory responses in many organs. However, roles of PTX3 in brain are still mostly unknown. Here we asked whether and how PTX3 contributes to blood-brain barrier dysfunction during the acute phase of ischemic stroke. In vivo, spontaneously hypertensive rats were subjected to focal cerebral ischemia by transient middle cerebral artery occlusion. At day 3, brains were analyzed to evaluate the cellular origin of PTX3 expression. Correlations with blood-brain barrier breakdown were assessed by IgG staining. In vitro, rat primary astrocytes and rat brain endothelial RBE.4 cells were cultured to study the role of astrocyte-derived PTX3 on vascular endothelial growth factor-mediated endothelial permeability. During the acute phase of stroke, reactive astrocytes in the peri-infarct area expressed PTX3. There was negative correlation between gradients of IgG leakage and PTX3-positive astrocytes. Cell culture experiments showed that astrocyte-conditioned media increased levels of tight junction proteins and reduced endothelial permeability under normal conditions. Removing PTX3 from astrocyte-conditioned media by immunoprecipitation increased endothelial permeability. PTX3 strongly bound vascular endothelial growth factor in vitro and was able to decrease vascular endothelial growth factor-induced endothelial permeability. Astrocytes in peri-infarct areas upregulate PTX3, which may support blood-brain barrier integrity by regulating vascular endothelial growth factor-related mechanisms. This response in astrocytes may comprise a compensatory mechanism for maintaining blood-brain barrier function after ischemic stroke.

Attenuation of Postischemic Genomic Alteration by Mesenchymal Stem Cells: a Microarray Study.

Intravenous administration of mesenchymal stem cells (IV-MSC) protects the ischemic rat brain in a stroke model, but the molecular mechanism underlying its therapeutic effect is unclear. We compared genomic profiles using the mRNA microarray technique in a rodent stroke model. Rats were treated with 1 × 106 IV-MSC or saline (sham group) 2 h after transient middle cerebral artery occlusion (MCAo). mRNA microarray was conducted 72 h after MCAo using brain tissue from normal rats (normal group) and the sham and MSC groups. Predicted pathway analysis was performed in differentially expressed genes (DEGs), and functional tests and immunohistochemistry for inflammation-related proteins were performed. We identified 857 DEGs between the sham and normal groups, with the majority of them (88.7%) upregulated in sham group. Predicted pathway analysis revealed that cerebral ischemia activated 10 signaling pathways mainly related to inflammation and cell cycle. IV-MSC attenuated the numbers of dysregulated genes in cerebral ischemia (118 DEGs between the MSC and normal groups). In addition, a total of 218 transcripts were differentially expressed between the MSC and sham groups, and most of them (175/218 DEGs, 80.2%) were downregulated in the MSC group. IV-MSC reduced the number of Iba-1+ cells in the peri-infarct area, reduced the overall infarct size, and improved functional deficits in MCAo rats. In conclusion, transcriptome analysis revealed that IV-MSC attenuated postischemic genomic alterations in the ischemic brain. Amelioration of dysregulated inflammation- and cell cycle-related gene expression in the host brain is one of the molecular mechanisms of IV-MSC therapy for cerebral ischemia.

Abstract WP108: Co-transplantation of NPCs and EPCs Synergistically Promotes Brain Recovery After Ischemia-reperfusion Injury in Mice

Introduction: The promising of neuron progenitor cells (NPCs) or endothelial progenitor cells (EPCs) for treating ischemic stroke has been recognized. In this study, we determined the therapeutic effects of NPC and EPC co-transplantation and the underlying mechanisms in a mouse model of ischemia-reperfusion (I-R) stroke. Methods: NPCs and EPCs were generated from human inducible pluripotent stem cells. C57BL/6 adult mice were subjected to middle cerebral artery occlusion (MCAO; 90 min) followed by reperfusion (30 min), and treated with (n=10/group): 1) PBS; 2) EPCs; 3) NPCs; 4) EPCs+NPCs (1:1 ratio); 5) EPCs+NPCs (1:1 ratio)+LY294002 (1μM). Cells (3x105/2μl PBS) were injected into ipsilateral striatum at 2 sites (1μl/site). Bromodeoxyuridine (BrdU, 65 mg/g/day, i.p.) was injected to label the new generated cells. Mice were sacrificed at days 2 and 10. Motor function (Rotarod test and neurologic deficit score), infarct volume, cerebral microvascular density (cMVD), neurogenesis and angiogenesis, and gene expressions of the PI3K/Akt pathway were evaluated. Results: Co-transplantation of EPCs and NPCs exhibited synergistic effects on improving motor function, increasing cMVD in the peri-infarct area, and decreasing infarct volume at days 2 and 10 (refer to table). Moreover, neurogenesis (Brdu+NeuN+) and angiogenesis (Brdu+CD31+) in the peri-infarct area were largely enhanced in the co-transplantation group at day 10 (refer to table). In addition, the protein ratio of p-Akt/Akt was increased in the brain in the co-transplantation group (p&lt;0.05). These effects were significantly reduced by LY294002 administration. Conclusion: Co-transplantation of NPCs and EPCs synergistically increases cMVD, promotes angiogenesis and neurogenesis, and improves functional outcome in I-R injured mice. Activation of the PI3K/Akt signal pathway contributes to the synergistic effects of NPCs and EPCs.

Abstract TP99: In vivo Tracking of Bone Marrow Stromal Cells Using 111 In-oxine Labeling and SPECT Following Transplantation for Cerebral Infarct of Rats

Background: This study investigates the effect of 111In-oxine incorporation on the viability of bone marrow stromal cells (BMSCs) in vitro, and the applicability of 111In-oxine labeling and single-photon emission computed tomography (SPECT) imaging of the BMSCs transplanted in rat model of cerebral infarct in vivo. Methods: Rat BMSCs were labeled with 111In-oxine (10 Bq/cell). Cellular retention of radioactivity, cell survival, and proliferation capacity were evaluated for 7 days. The rats were subjected to permanent middle cerebral artery occlusion. The 111In-oxine labeled BMSCs were stereotactically (1.0 х 1000000 cells) or intravenously (3.0 х 1000000 cells) transplanted at 7 days after the insult. Using SPECT, the behaviors of BMSCs were serially monitored during 14 days after transplantation. The distribution of engrafted cells was examined, using fluorescence immunohistochemistry. Results: Labeling efficiency was 35.8 ± 18.6 %, with 36.5 ± 20.6 % of 111In remaining in the BMSCs at 7 day. The radiolabeling did not affect cell viability, whereas proliferation capacity was significantly reduced. In vivo SPECT imaging clearly visualized that the transplanted BMSCs gradually migrated toward the peri-infarct area during 14 days after transplantation in the intracerebral group, but not in the intravenous group. The BMSCs were widely distributed in the peri-infarct area and some of them expressed neural cell markers in the intracerebral group, but not in the intravenous group. Conclusions: 111In-oxine labeling preserved viability of the BMSCs, while moderately impairing their proliferation capacity. SPECT imaging of 111In-oxine labeled BMSCs was valuable in monitoring the behaviors of donor cells into the rodent brain, and the results would also suggest that the intracerebral administration is more effective to deliver the BMSCs to the cerebral ischemic lesions than the intravenous administration. SPECT imaging of 111In-oxine labeled BMSCs may be a valuable modality to track the transplanted cells in the host brain non-invasively and serially in clinical situation.

Abstract WP110: The Next Generation of Autologous Bone Marrow Stromal Cell Transplantation Against Stroke

Background and purpose: Recent studies have elucidated that the bone marrow stromal cells (BMSCs) have therapeutic potential against stroke. Some clinical trials have been starting up in practice. We aimed to evolve the autologous BMSC transplantation for stroke into the next generation. Materials and methods: Human BMSCs were cultured with human platelet lysate (hPL) instead of fetal calf serum (FCS). They were labeled with superparamagnetic iron oxide (SPIO). Rat ischemic stroke models were made and 5x105 cells were injected into the ipsilateral striatum stereotactically 7 days post-insult. Behavioral analysis, MRI for cell tracking, (18)F-FDG PET, and (123)I-Iomazenil SPECT were performed. The animals were sacrificed 5 to 8 weeks post-transplantation and histological analysis was performed. Results: There was no difference in the surface markers and cell proliferation between hPL and FCS. Although rotarod test showed that motor function deteriorated in rats suffered from permanent MCAo, BMSC-hPL transplantation enhanced recovery of the motor function, significantly. MRI demonstrated that SPIO-BMSCs aggressively migrated towards the lesion. Moreover, (18)F-FDG PET and (123)I-Iomazenil SPECT showed that BMSC transplantation promoted recovery of the glucose utilization and the binding potential of iomazenil in the peri-infarct area, respectively. Histological analysis supported the findings on MRI and showed the inclination for neural differentiation of donor cells. Conclusion: The hPL may be valuable and safe in expanding BMSCs. The application of bio-imaging techniques is also valuable for BMSC transplantation for stroke. Now we prepare the novel clinical trial against stroke, Research on Advanced Intervention using Novel Bone marrOW stem cell (RAINBOW) study. The present results are translated into the optimal design of the trial.

Abstract 133: Donor Age Drastically Affects Structural and Functional Recovery After Cell Therapy Following Ischemic Stroke

Background and purpose: Cell transplantation therapy hold great potential to improve impairments after cerebral ischemia. However, it is still unclear whether donor age affects structural and functional recovery after cell therapy. Here, we investigate the hypothesis that donor age (Young vs. Old) drastically affects the repair mechanism after cell transplantation for ischemic stroke. Methods: Male Sprague Dawley rats were subjected to transient middle cerebral artery occlusion. At 24 hours after stroke, rats were randomly assigned to receive PBS (Control), human mesenchymal stem cells derived from 24 years old man (Young) and MSC derived from 64 years old man (Old) via internal carotid artery. Behavioral recovery was assessed with modified Neurological severity score (mNSS) until 21 days (D21) after stroke. Animals were euthanized at D21, and histological analysis was performed regarding neovascularization and neurogenesis. Results: Young showed a significant recovery in mNSS (3.7±0.6) compared with Control (6.1±0.5) or Old (5.2±0.7) at D21 (P&lt;0.01). Immunohistological analysis revealed that the number of RECA-1 and PDGFR-1β double positive vessels much increased in Young (113±48.6/mm2) compared with that in Control (61.5±35.9/mm2) or Old (76.9±36.9/mm2) suggesting vessel maturation (P &lt;0.01). Interestingly, direction of the astrocyte process, defined as direction index, significantly correlated with the number of Msi-1 positive neural stem cells (P&lt;0.01, r=0.27; Spearman), and direction index with many neural stem cells was significantly higher in Young compared with Old (P &lt;0.05). Conclusions: Donor age drastically affects the host environment and functional recovery after cell transplantation for ischemic stroke. This study revealed that direction of the astrocyte contributed to neural stem cell migration into the periinfarct area. Young hMSC was superior to old hMSC in structural and functional recovery after ischemic stroke.

Abstract WP112: Trans-arterial Regional Hypothermia Provides Robust Neuroprotection in Rat Permanent Middle Cerebral Artery Occlusion Model

Background and Purpose: We previously reported the neuroprotective effects of trans-arterial regional hypothermia in rat transient middle cerebral artery occlusion (tMCAO) model (ISC 2014, ISC 2015). The tMCAO model is representative of the clinical setting of successful recanalization in acute ischemic stroke. However, it is not always possible to achieve successful recanalization. Herein, we investigated the neuroprotective effects of trans-arterial regional hypothermia in permanent MCAO (pMCAO) model as well. Methods: Three groups were provided to investigate the neuroprotective effects. Regional hypothermia group had a single infusion of 20ml/kg 10°C cold saline via the ipsilateral internal carotid artery for 15 min at 0 hr or 1 hr after the onset of pMCAO. Vehicle group had a 37°C warm saline infusion in the same manner. Control group had no infusion after pMCAO. The rats were tested for neurological score after 48-hour pMCAO and then sacrificed to evaluate infarction size and pathological condition. Immunohistochemical analysis was performed to investigate the neuronal apoptosis (NeuN, cleaved caspase 3), glial reaction (GFAP), and microglial activation (Iba-1). Results: The regional hypothermia group showed significantly better neurological score compared with the other groups (p&lt;0.01). Infarct volume was significantly smaller in the regional hypothermia group (8.1±4.7% at 0 hr and 15.7±5.9% at 1 hr) than in the vehicle group (35.8±5.6% at 0 hr and 34.0±3.6% at 1 hr) and also in the control group (35.1±8.9%) (p&lt;0.01). Neuronal apoptosis (p&lt;0.01), reactive gliosis (p&lt;0.01), and microglial activation (p&lt;0.05) in the peri-infarct area were significantly inhibited in the regional hypothermia group compared with the other groups. The degree of the inhibition was somewhat more evident at 0 hr than at 1 hr. Conclusions: Our result demonstrated that trans-arterial regional hypothermia provided robust neuroprotection in pMCAO model as well as tMCAO model, suggesting that this therapy may have a wide clinical application in ischemic stroke treatment.

Thromboxane A2 Receptor Stimulation Enhances Microglial Interleukin-1β and NO Biosynthesis Mediated by the Activation of ERK Pathway.

Background and Purpose: Thromboxane A2 (TXA2) receptors (TP) interact with the ligand TXA2 to induce platelet aggregation and regulate hemostasis. Recently TP-mediated signaling has been suggested to function in multiple cell types in the brain. In this report, we aim to study the expression and physiological role of TP in microglia, in particular after brain ischemia.Methods: Ischemic brain sections were analyzed for TP expression. Microglial cell line and primary microglia were cultured, or neuronal cell line co-culture system was used to determine the TP mediated signaling in inflammation and microglia activation.Results: We found that the TP level was significantly increased in ipsilateral mouse brain tissue at 24 h after ischemia-reperfusion, which was also found to partly co-localize with CD11b, a marker for microglial and infiltrated monocyte/macrophage, in peri-infarct area. Immunofluorescence staining of primary microglia and microglial cell line BV2 revealed the predominant membrane distribution of TP. Conditioned culture media from TP agonist U46619-treated BV2 cells decreased neuronal SH-SY5Y cell viability and induced apoptotic morphological changes. Furthermore, U46619 enhanced IL-1β, IL-6, and iNOS mRNA expression as well as IL-1β and NO releases in BV2 cells or primary microglia. Such stimulation could be attenuated by TP antagonist SQ29548 or MEK inhibitor U0126. The dose- and time-dependent extracellular-signal-regulated kinase (ERK) phosphorylation induced by U46619 further demonstrated ERK signaling-mediated microglia activation by TP agonist.Conclusion: This study has shown a novel role of TP in microglia activation via the ERK signaling pathway, which provides insights for the management of neuroinflammation in diseases like cerebral infarction.

Increase in cholinergic modulation with pyridostigmine induces anti-inflammatory cell recruitment soon after acute myocardial infarction in rats.

We tested the hypothesis that an increase in the anti-inflammatory cholinergic pathway, when induced by pyridostigmine (PY), may modulate subtypes of lymphocytes (CD4+, CD8+, FOXP3+) and macrophages (M1/M2) soon after myocardial infarction (MI) in rats. Wistar rats, randomly allocated to receive PY (40 mg·kg(-1)·day(-1)) in drinking water or to stay without treatment, were followed for 4 days and then were subjected to ligation of the left coronary artery. The groups-denominated as the pyridostigmine-treated infarcted (IP) and infarcted control (I) groups-were submitted to euthanasia 3 days after MI; the heart was removed for immunohistochemistry, and the peripheral blood and spleen were collected for flow cytometry analysis. Noninfarcted and untreated rats were used as controls (C Group). Echocardiographic measurements were registered on the second day after MI, and heart rate variability was measured on the third day after MI. The infarcted groups had similar MI areas, degrees of systolic dysfunction, blood pressures, and heart rates. Compared with the I Group, the IP Group showed a significant higher parasympathetic modulation and a lower sympathetic modulation, which were associated with a small, but significant, increase in diastolic function. The IP Group showed a significant increase in M2 macrophages and FOXP3(+)cells in the infarcted and peri-infarcted areas, a significantly higher frequency of circulating Treg cells (CD4(+)CD25(+)FOXP3(+)), and a less extreme decrease in conventional T cells (CD25(+)FOXP3(-)) compared with the I Group. Therefore, increasing cholinergic modulation with PY induces greater anti-inflammatory cell recruitment soon after MY in rats.

Amelioration of acute myocardial infarction by saponins from flower buds of Panax notoginseng via pro-angiogenesis and anti-apoptosis

Ethnopharmacological relevanceThe root of Panax notoginseng is traditionally used as an anti-hemorrhagic agent to promote blood circulation without causing “congealed” blood. Furthermore, the flower of P. notoginseng is a popular, traditional medicine taken daily for the preventing of hypertension and for reducing blood cholesterol profiles. Besides, the flower of P. notoginseng contains a higher level of saponins, particularly protopanaxadiol-type ginsenosides, as compared to the root. However, detailed pharmacological studies on this flower have rarely been conducted. Material and methodsIn this study, the saponins extracted from the flower of P. notoginseng (FS) were examined on the endothelial cell migration assay, chemically induced vascular insufficiency model in zebrafish larvae and myocardial infraction (MI) model in rats, for determination of their pro-angiogenic and therapeutic effects on MI treatment. ResultsOur results demonstrate that FS significantly promoted VEGF-induced migration of human umbilical vein endothelial cells (HUVECs) and partially restored defective intersegmental vessels (ISV) in a chemically induced vascular insufficiency model of zebrafish larvae. When compared to MI group, two weeks post-treatment of FS (25–50mg/kg/day) induced approximately 3-fold upregulation of VEGF mRNA expression and a concomitant increase in blood vessel density in the peri-infarct area of the heart. Moreover, TUNEL analysis indicates a reduction in the mean apoptotic nuclei per field in peri-infarct myocardium upon FS treatment. ConclusionsThe pro-angiogenic effects of FS demonstrated in in vitro and in vivo experimental models suggest that the purified saponin preparation from flowers of P. notoginseng may potentially provide preventive and therapeutic agent for cardiovascular diseases.

Endothelial fibroblast growth factor receptor signaling is required for vascular remodeling following cardiac ischemia-reperfusion injury

Fibroblast growth factor (FGF) signaling is cardioprotective in various models of myocardial infarction. FGF receptors (FGFRs) are expressed in multiple cell types in the adult heart, but the cell type-specific FGFR signaling that mediates different cardioprotective endpoints is not known. To determine the requirement for FGFR signaling in endothelium in cardiac ischemia-reperfusion injury, we conditionally inactivated the Fgfr1 and Fgfr2 genes in endothelial cells with Tie2-Cre (Tie2-Cre, Fgfr1(f/f), Fgfr2(f/f) DCKO mice). Tie2-Cre, Fgfr1(f/f), Fgfr2(f/f) DCKO mice had normal baseline cardiac morphometry, function, and vessel density. When subjected to closed-chest, regional cardiac ischemia-reperfusion injury, Tie2-Cre, Fgfr1(f/f), Fgfr2(f/f) DCKO mice showed a significantly increased hypokinetic area at 7 days, but not 1 day, after reperfusion. Tie2-Cre, Fgfr1(f/f), Fgfr2(f/f) DCKO mice also showed significantly worsened cardiac function compared with controls at 7 days but not 1 day after reperfusion. Pathophysiological analysis showed significantly decreased vessel density, increased endothelial cell apoptosis, and worsened tissue hypoxia in the peri-infarct area at 7 days following reperfusion. Notably, Tie2-Cre, Fgfr1(f/f), Fgfr2(f/f) DCKO mice showed no impairment in the cardiac hypertrophic response. These data demonstrate an essential role for FGFR1 and FGFR2 in endothelial cells for cardiac functional recovery and vascular remodeling following in vivo cardiac ischemia-reperfusion injury, without affecting the cardiac hypertrophic response. This study suggests the potential for therapeutic benefit from activation of endothelial FGFR pathways following ischemic injury to the heart.