Vagus nerve stimulation inhibits cortical spreading depression via glutamate-dependent TrkB activation mechanism in the nucleus tractus solitarius.

BACKGROUND: Vagus nerve stimulation (VNS) was recently found to inhibit cortical spreading depression (CSD), the underlying mechanism of migraine aura, through activation of the nucleus tractus solitarius (NTS), locus coeruleus (LC) and dorsal raphe nucleus (DRN). The molecular mechanisms underlying the effect of VNS on CSD in these nuclei remain to be explored. We hypothesized that VNS may activate glutamate receptor-mediated tropomyosin kinase B (TrkB) signaling in the NTS, thereby facilitating the noradrenergic and serotonergic neurotransmission to inhibit CSD. METHODS: To investigate the role of TrkB and glutamate receptors in non-invasive VNS efficacy on CSD, a validated KCl-evoked CSD rat model coupled with intra-NTS microinjection of selective antagonists, immunoblot and immunohistochemistry was employed. RESULTS: VNS increased TrkB phosphorylation in the NTS. Inhibition of intra-NTS TrkB abrogated the suppressive effect of VNS on CSD and CSD-induced cortical neuroinflammation. TrkB was found colocalized with glutamate receptors in NTS neurons. Inhibition of glutamate receptors in the NTS abrogated VNS-induced TrkB activation. Moreover, the blockade of TrkB in the NTS attenuated VNS-induced activation of the LC and DRN. CONCLUSIONS: VNS induces the activation of glutamate receptor-mediated TrkB signaling in the NTS, which might modulate serotonergic and norepinephrinergic innervation to the cerebral cortex to inhibit CSD and cortical inflammation.

Neuronal NLRP3 inflammasome mediates spreading depolarization-evoked trigeminovascular activation.

Spreading depolarization (SD), the underlying mechanism of migraine aura, may trigger the opening of the pannexin 1 (PANX1) pore to sustain the cortical neuroinflammatory cascades involved in the genesis of headache. Yet, the mechanism underlying SD-evoked neuroinflammation and trigeminovascular activation remains incompletely understood. We characterized the identity of inflammasome activated following SD-evoked PANX1 opening. Pharmacological inhibitors targeting PANX1 or NLRP3 as well as genetic ablation of Nlrp3 and Il1b were applied to investigate the molecular mechanism of the downstream neuroinflammatory cascades. In addition, we examined whether SD-triggered microglial activation facilitates neuronal NLRP3-mediated inflammatory cascades. Pharmacological inhibition of toll-like receptors TLR2/4, the potential receptors of the damage-associated molecular pattern HMGB1, was further employed to interrogate the neuron-microglia interplay in SD-induced neuroinflammation. We found that NLRP3 but not NLRP1 or NLRP2 inflammasome was activated following PANX1 opening after single or multiple SDs evoked by either KCl topical application or non-invasively with optogenetics. The SD-evoked NLRP3 inflammasome activation was observed exclusively in neurons but not microglia or astrocytes. Proximity ligation assay demonstrated that the assembly of the NLRP3 inflammasome occurred as early as 15 min after SD. Genetic ablation of Nlrp3 or Il1b or pharmacological inhibition of PANX1 or NLRP3 ameliorated SD-induced neuronal inflammation, middle meningeal artery dilatation, calcitonin gene-related peptide expression in trigeminal ganglion and c-Fos expression in trigeminal nucleus caudalis. Moreover, multiple SDs induced microglial activation subsequent to neuronal NLRP3 inflammasome activation, which in turn orchestrated with neurons to mediate cortical neuroinflammation, as demonstrated by decreased neuronal inflammation after pharmacological inhibition of microglia activation or blockade of the TLR2/4 receptors. To conclude, single or multiple SDs evoked activation of neuronal NLRP3 inflammasomes and its downstream inflammatory cascades to mediate cortical neuroinflammation and trigeminovascular activation. In the context of multiple SDs, the cortical inflammatory processes could be facilitated by SD-evoked microglia activation. These findings may implicate the potential role of innate immunity in migraine pathogenesis.

Efficacy profile of noninvasive vagus nerve stimulation on cortical spreading depression susceptibility and the tissue response in a rat model.

BACKGROUND: Noninvasive vagus nerve stimulation (nVNS) has recently emerged as a promising therapy for migraine. We previously demonstrated that vagus nerve stimulation inhibits cortical spreading depression (CSD), the electrophysiological event underlying migraine aura and triggering headache; however, the optimal nVNS paradigm has not been defined. METHODS: Various intensities and doses of nVNS were tested to improve efficacy on KCl-evoked CSD frequency and electrical threshold of CSD in a validated rat model. Chronic efficacy was evaluated by daily nVNS delivery for four weeks. We also examined the effects of nVNS on neuroinflammation and trigeminovascular activation by western blot and immunohistochemistry. RESULTS: nVNS suppressed susceptibility to CSD in an intensity-dependent manner. Two 2-minute nVNS 5 min apart afforded the highest efficacy on electrical CSD threshold and frequency of KCl-evoked CSD. Daily nVNS for four weeks did not further enhance efficacy over a single nVNS 20 min prior to CSD. The optimal nVNS also attenuated CSD-induced upregulation of cortical cyclooxygenase-2, calcitonin gene-related peptide in trigeminal ganglia, and c-Fos expression in trigeminal nucleus caudalis. CONCLUSIONS: Our study provides insight on optimal nVNS parameters to suppress CSD and suggests its benefit on CSD-induced neuroinflammation and trigeminovascular activation in migraine treatment.

Exogenous GDF11 attenuates non-canonical TGF-ß signaling to protect the heart from acute myocardial ischemia-reperfusion injury.

Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor beta 1 (TGF-ß1) superfamily that reverses age-related cardiac hypertrophy, improves muscle regeneration and angiogenesis, and maintains progenitor cells in injured tissue. Recently, targeted myocardial delivery of the GDF11 gene in aged mice was found to reduce heart failure and enhance the proliferation of cardiac progenitor cells after myocardial ischemia-reperfusion (I-R). No investigations have as yet explored the cardioprotective effect of exogenous recombinant GDF11 in acute I-R injury, despite the convenience of its clinical application. We sought to determine whether exogenous recombinant GDF11 protects against acute myocardial I-R injury and investigate the underlying mechanism in Sprague-Dawley rats. We found that GDF11 reduced arrhythmia severity and successfully attenuated myocardial infarction; GDF11 also increased cardiac function after I-R, enhanced HO-1 expression and decreased oxidative damage. GDF11 activated the canonical TGF-ß signaling pathway and inactivated the non-canonical pathways, ERK and JNK signaling pathways. Moreover, administration of GDF11 prior to reperfusion protected the heart from reperfusion damage. Notably, pretreatment with the activin-binding protein, follistatin (FST), inhibited the cardioprotective effects of GDF11 by blocking its activation of Smad2/3 signaling and its inactivation of detrimental TGF-ß signaling. Our data suggest that exogenous GDF11 has cardioprotective effects and may have morphologic and functional recovery in the early stage of myocardial I-R injury. GDF11 may be an innovative therapeutic approach for reducing myocardial I-R injury.

FTY720 inhibits germ cell apoptosis in testicular torsion/detorsion.

BACKGROUND: Testicular torsion/detorsion (T/D) can induce germ cells apoptosis, which may lead to impairment of spermatogenesis. FTY720, an agonist of the sphingosine-1-phosphate receptor 1 (S1PR1), inhibits apoptosis in ischemic stroke. We examined whether FTY720 could mitigate germ cell apoptosis in testicular T/D rats. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were allocated to receive testicular T/D (the T/D group), T/D plus FTY720 (the T/D-FTY group), or T/D plus FTY720 plus the potent S1PR1 antagonist VPC23019 (the T/D-FTY-VPC group; n = 6 in each group). Sham control groups were run simultaneously. At 24 h after detorsion, rats were euthanized. RESULTS: Our data revealed that, in the ipsilateral twisted testes, sperm counts and expression of the S1PR1 of the T/D and the T/D-FTY-VPC groups were significantly lower than those of the T/D-FTY group (all P < 0.001). In contrast, signals of apoptotic cells stained by terminal deoxynucleotidyl transferase dUTP nick end labeling and the proapoptotic protein cleaved caspase-3 of the T/D, and the T/D-FTY-VPC groups were significantly stronger than those of the T/D-FTY group. Moreover, the terminal deoxynucleotidyl transferase dUTP nick end labeling signals mainly localized to germ cells. CONCLUSIONS: FTY720 could mitigate testicular T/D-induced germ cell apoptosis, and the mechanisms may involve the S1PR1.

P2X7 is involved in the anti-inflammation effects of levobupivacaine.

BACKGROUND: We sough to elucidate whether purinergic P2X7 receptor is actively involved in the effects of levobupivacaine on inhibiting microglia activation. MATERIALS AND METHODS: Microglia were treated with lipopolysaccharide (LPS, 50 ng/mL), LPS plus levobupivacaine (50 µM), or LPS plus levobupivacaine plus the P2X7 receptor agonist Bz-ATP (100 µM) and denoted as the LPS, LPS + Levo, and LPS + Levo + Bz-ATP group, respectively. Microglia activation was measured by assaying inflammatory molecules expression. Microglia activation was also measured by assaying neuronal cell viability using coculture of microglia and neurons, as activated microglia may cause neuron injury. We also measured the levels of P2X7 receptor activation in microglia using ethidium uptake assay. RESULTS: Our data confirmed the effects of levobupivacaine on inhibiting inflammatory molecules upregulation in activated microglia, as the concentrations of interleukin (IL)-1ß, tumor necrosis factor α, IL-6, and macrophage inflammatory protein 2, of the LPS + Levo group were significantly lower than those of the LPS group (all P < 0.05). Moreover, Bz-ATP significantly abrogated the inhibitory effects of levobupivacaine, as concentrations of IL-1ß, tumor necrosis factor α, IL-6, and macrophage inflammatory protein 2 of the LPS + Levo + Bz-ATP group were significantly higher than those of the LPS + Levo group (all P < 0.05). In contrast, neuronal cell viability of the LPS + Levo group was significantly higher than those of the LPS and LPS + Levo + Bz-ATP groups (P = 0.012 and 0.002). Moreover, levels of P2X7 receptor activation of the LPS and LPS + Levo + Bz-ATP groups were significantly higher than that of the LPS + Levo group (P = 0.003 and 0.006). CONCLUSIONS: P2X7 receptor is involved in the effects of levobupivacaine on inhibiting microglial activation.

FTY720 mitigates torsion/detorsion-induced testicular injury in rats.

BACKGROUND: FTY720, a sphingosine-1-phosphate (S1P) receptor agonist, possesses potent anti-inflammation capacity. We evaluated the therapeutic potentials of FTY720 against testicular injury induced by testicular torsion and/or detorsion (T/D). MATERIALS AND METHODS: Young adult male Sprague-Dawley rats were allocated to receive T/D (the T/D group) and T/D plus FTY720 (4 mg/kg, the T/D-FTY group, n = 6 in each group). To investigate the possible roles of the S1P receptors, another group of rats received T/D plus FTY720 plus the potent S1P receptor antagonist VPC23019 (1 mg/kg, the T/D-FTY-VPC group, n = 6). FTY720 was administered immediately before testicular detorsion, and VPC23019 was administered 30 min before FTY720. Another set of rats that received sham operation, immediately followed by injection of normal saline, FTY720, or FTY720 plus VPC23019, served as control groups. Sham control groups were run simultaneously. After euthanization, levels of testicular injury were measured. RESULTS: Histologic findings revealed severe testicular injury changes in both the T/D and T/D-FTY-VPC groups and moderate testicular injury changes in the T/D-FTY group. In addition, malondialdehyde activity (oxidative status), concentration of interleukin-1ß (inflammation index), myeloperoxidase activity (neutrophil infiltration index), and wet-to-dry weight ratio (tissue edema index) of both the T/D and T/D-FTY-VPC groups were significantly higher than those of the T/D-FTY group. These data confirmed the protective effects of FTY720 against testicular T/D. Moreover, antagonizing the S1P receptors could reverse the protective effects of FTY720. CONCLUSIONS: FTY720 significantly mitigated testicular injury induced by testicular T/D. The mechanisms may involve activating the S1P receptors.

Voluntary and involuntary running in the rat show different patterns of theta rhythm, physical activity, and heart rate.

Involuntarily exercising rats undergo more physical and mental stress than voluntarily exercising rats; however, these findings still lack electrophysiological evidence. Many studies have reported that theta rhythm appears when there is mental stress and that it is affected by emotional status. Thus we hypothesized that the differences between voluntary and involuntary movement should also exist in the hippocampal theta rhythm. Using the wheel and treadmill exercise models as voluntary and involuntary exercise models, respectively, this study wirelessly recorded the hippocampal electroencephalogram, electrocardiogram, and three-dimensional accelerations of young male rats. Treadmill and wheel exercise produced different theta patterns in the rats before and during running. Even though the waking baselines for the two exercise types were recorded in different environments, there did not exist any significant difference after distinguishing the rats' sleep/wake status. When the same movement-related parameters are considered, the treadmill running group showed more changes in their theta frequency (4-12 Hz), in their theta power between 9.5-12 Hz, and in their heart rate than the wheel running group. A positive correlation between the changes in high-frequency (9.5-12 Hz) theta power and heart rate was identified. Our results reveal various voluntary and involuntary changes in hippocampal theta rhythm as well as divergences in heart rate and high-frequency theta activity that may represent the effects of an additional emotional state or the sensory interaction during involuntary running by rats.

Preventive effect of baicalein on methamphetamine-induced amnesia in the passive avoidance test in mice.

BACKGROUND/AIMS: Methamphetamine abuse may produce cognitive impairment. Baicalein, a bioactive flavonoid, has antioxidative, anti-inflammatory and neuroprotective effects. This study examined the effects of baicalein pretreatment on memory performance in the passive avoidance test after either one dose or an acute binge of methamphetamine in Institute of Cancer Research (ICR) mice. METHODS: Methamphetamine was administered by intraperitoneal (i.p.) injection of either one dose (3 mg/kg) or an acute binge (3 mg/kg, 4 i.p. injections at 2-hour intervals). The effects of baicalein pretreatment (1 mg/kg, i.p.) on methamphetamine-induced changes of locomotor activity and memory performance were compared with those of eticlopride, a selective dopamine D2 receptor antagonist. The effects of baicalein on acute binge methamphetamine-induced oxidative stress (malondialdehyde- and nitrotyrosine-modified protein production) in the mouse hippocampus were also examined. RESULTS: One-dose methamphetamine treatment (i.p., 30 min before or immediately after the training trial) induced hyperlocomotion and amnesia in mice, which were blocked by eticlopride but not by baicalein pretreatment. The memory performance in mice was impaired 5 days after acute binge methamphetamine, which was significantly attenuated by baicalein but not by eticlopride pretreatment. Baicalein pretreatment also attenuated acute binge methamphetamine-induced oxidative stress in the mouse hippocampus. CONCLUSIONS: Baicalein exhibits antioxidative and neuroprotective effects in attenuating acute binge methamphetamine-induced memory deficits and oxidative hippocampal damage.

Suppressive effects of levobupivacaine on endotoxin-induced microglial activation.

BACKGROUND: We sought to elucidate the effects of levobupivacaine on modulating endotoxin-induced upregulation of inflammatory mediators and activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling pathways in activated microglia. MATERIALS AND METHODS: Confluent murine microglia (BV-2) were treated with endotoxin (lipopolysaccharide, 50 ng/mL) or endotoxin plus levobupivacaine (5, 25, or 50 µM) and denoted as the LPS, LPS + L(5), LPS + L(25), and LPS + L(50) groups, respectively. Levobupivacaine was administered immediately after endotoxin. Control groups were run simultaneously. RESULTS: The concentrations of inflammatory mediators, including macrophage inflammatory protein-2 (P = 0.023 and 0.016), tumor necrosis factor-α (P = 0.025 and 0.020), interleukin (IL)-1ß (P = 0.018 and 0.014), IL-6 (P = 0.029 and 0.023), nitric oxide (P = 0.025 and 0.026), and prostaglandin E2 (P = 0.028 and 0.016) of the LPS + L(25) and LPS + L(50) groups were significantly lower than those of the LPS group. The concentrations of macrophage inflammatory protein-2 (P = 0.035), IL-1ß (P = 0.024), nitric oxide (P = 0.031), and prostaglandin E2 (P = 0.036) but not tumor necrosis factor-α and interleukin-6 of the LPS + L(5) group were also significantly lower than those of the LPS group. These data revealed that effects of endotoxin on upregulating inflammatory mediators were inhibited by levobupivacaine. Moreover, effects of endotoxin on activating NF-κB, including inhibitor-κB degradation, NF-κB nuclear translocation, and NF-κB-DNA binding, were also inhibited by levobupivacaine. Similarly, effects of endotoxin on activating MAPKs, including extracellular signal-regulated kinase, c-jun N-terminal kinase, and p38 MAPK, were also significantly inhibited by levobupivacaine. CONCLUSIONS: Levobupivacaine significantly inhibited endotoxin-induced upregulation of inflammatory mediators and activation of NF-κB and MAPKs signaling pathways in activated microglia.

Ameliorative effects of caffeic acid phenethyl ester on an eccentric exercise-induced skeletal muscle injury by down-regulating NF-κb mediated inflammation.

BACKGROUND AND PURPOSE: Caffeic acid phenethyl ester (CAPE), a phenolic compound isolated from propolis, displays a variety of biological activities. The aim is to examine the protective effect and mechanisms of CAPE on an eccentric exercise-induced muscle injury model. EXPERIMENTAL APPROACH: An intermittent downhill eccentric exercise protocol was used. The oxidative tissue injury and expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), interleukin-1ß (IL-1ß), monocyte chemotactic protein-1 (MCP-1), and activation of nuclear factor-κB (NF-κB) were examined. CAPE was applied in a dose of 5 and 10 mg/kg/day, p.o. KEY RESULTS: The eccentric exercise induced remarkable skeletal muscle damage uncovered by a dramatic elevation of creatine kinase in the serum and severe degenerative myopathy. These pathophysiological changes were accompanied by an upregulation of the inflammatory responses including protein nitrotyrosylation, poly-ADP-ribose-polymerase (PARP) upregulation, lipid peroxidation as measured by malondialdehyde (MDA) formation, and leukocyte infiltration as measured by myeloperoxidase (MPO). The inflammatory responses primarily resulted from enhanced expression of COX2, iNOS, and production of IL-1ß and MCP-1, possibly through activation of NF-κB. All these pathological changes were suppressed by treatment of CAPE. CONCLUSIONS AND IMPLICATIONS: Our results indicate that CAPE exhibits protective effects against eccentric exercise-induced skeletal muscle damage in rats by blocking the NF-κB-dependent activation of the inflammatory responses.

Schizandrin protects primary rat cortical cell cultures from glutamate-induced apoptosis by inhibiting activation of the MAPK family and the mitochondria dependent pathway.

Glutamate-induced excitotoxicity has been implicated in a variety of neuronal degenerative disorders. In the present study, we investigated the possible neuroprotective effects of schizandrin against apoptosis of primary cultured rat cortical cells induced by glutamate. Glutamate (10 µM) administered for 24 h decreased the expression of Bcl-2 and Bcl-X(L) protein, whereas increased the expression of Bax, Bak, apoptosis inducing factor (AIF), endonuclease G (Nodo G) and endoplasmic reticulum (ER) stress of caspase-12. Pretreatment with schizandrin (100 µM) before glutamate treatment increased the Bcl-X(L) and Bcl-2 expression and decreased Bax, Bak, AIF, Nodo G and caspase-12 compared with those only treated with glutamate. Furthermore, glutamate-induced phosphorylation of JNK, p38 and ERK mitogen-activated protein kinases (MAPK), and these effects were attenuated by schizandrin (100 µM) treatment. These results suggest that schizandrin possesses the neuroprotective effects. The molecular mechanisms of schizandrin against glutamate-induced apoptosis may involve the regulation of Bcl-2 family proteins expression, and ER stress through blocking the activation of JNK, ERK and p38 MAPK.

Midostaurin Modulates Tumor Microenvironment and Enhances Efficacy of Anti-PD-1 against Colon Cancer.

Immunotherapy modulating the tumor microenvironment (TME) immune function has a promising effect on various types of cancers, but it remains as a limited efficacy in colon cancer. Midostaurin (PKC412) has been used in the clinical treatment of fms-like tyrosine kinase 3 (FLT3)-mutant acute myeloid leukemia and has demonstrated immunomodulatory activity. We aimed to evaluate the effect of midostaurin on the modulation of TME and the efficacy of anti-programmed cell death protein 1 (PD-1) against colon cancer. Midostaurin inhibited the growth of murine CT26 and human HCT116 and SW480 cells with multinucleation and micronuclei formation in morphology examination. The cell cycle arrested in the G2/M phase and the formation of the polyploid phase was noted. The formation of cytosolic DNA, including double-strand and single-strand DNA, was increased. Midostaurin increased mRNA expressions of cGAS, IRF3, and IFNAR1 in colorectal adenocarcinoma cells and mouse spleen macrophages. The protein expressions of Trex-1, c-KIT, and Flt3, but not PKCα/ß/γ and VEGFR1, were down-regulated in midostaurin-treated colorectal adenocarcinoma cells and macrophages. Trex-1 protein expression was abrogated after FLT3L activation. In vivo, the combination of midostaurin and anti-PD-1 exhibited the greatest growth inhibition on a CT26-implanted tumor without major toxicity. TME analysis demonstrated that midostaurin alone decreased Treg cells and increased neutrophils and inflammatory monocytes. NKG2D+ and PD-1 were suppressed and M1 macrophage was increased after combination therapy. When combined with anti-PD-1, STING and INFß protein expression was elevated in the tumor. The oral administration of midostaurin may have the potential to enhance anti-PD-1 efficacy, accompanied by the modulation of cytosolic DNA-sensing signaling and tumor microenvironment.

Protein kinase A-dependent neuronal nitric oxide synthase activation mediates the enhancement of baroreflex response by adrenomedullin in the nucleus tractus solitarii of rats.

BACKGROUND: Adrenomedullin (ADM) exerts its biological functions through the receptor-mediated enzymatic mechanisms that involve protein kinase A (PKA), or neuronal nitric oxide synthase (nNOS). We previously demonstrated that the receptor-mediated cAMP/PKA pathway involves in ADM-enhanced baroreceptor reflex (BRR) response. It remains unclear whether ADM may enhance BRR response via activation of nNOS-dependent mechanism in the nucleus tractus solitarii (NTS). METHODS: Intravenous injection of phenylephrine was administered to evoke the BRR before and at 10, 30, and 60 min after microinjection of the test agents into NTS of Sprague-Dawley rats. Western blotting analysis was used to measure the level and phosphorylation of proteins that involved in BRR-enhancing effects of ADM (0.2 pmol) in NTS. The colocalization of PKA and nNOS was examined by immunohistochemical staining and observed with a laser confocal microscope. RESULTS: We found that ADM-induced enhancement of BRR response was blunted by microinjection of NPLA or Rp-8-Br-cGMP, a selective inhibitor of nNOS or protein kinase G (PKG) respectively, into NTS. Western blot analysis further revealed that ADM induced an increase in the protein level of PKG-I which could be attenuated by co-microinjection with the ADM receptor antagonist ADM22-52 or NPLA. Moreover, we observed an increase in phosphorylation at Ser1416 of nNOS at 10, 30, and 60 min after intra-NTS administration of ADM. As such, nNOS/PKG signaling may also account for the enhancing effect of ADM on BRR response. Interestingly, biochemical evidence further showed that ADM-induced increase of nNOS phosphorylation was prevented by co-microinjection with Rp-8-Br-cAMP, a PKA inhibitor. The possibility of PKA-dependent nNOS activation was substantiated by immunohistochemical demonstration of co-localization of PKA and nNOS in putative NTS neurons. CONCLUSIONS: The novel finding of this study is that the signal transduction cascade that underlies the enhancement of BRR response by ADM in NTS is composed sequentially of cAMP/PKA and nNOS/PKG pathways.

Andrographolide inhibits PI3K/AKT-dependent NOX2 and iNOS expression protecting mice against hypoxia/ischemia-induced oxidative brain injury.

This study aimed to explore the mechanisms by which andrographolide protects against hypoxia-induced oxidative/nitrosative brain injury provoked by cerebral ischemic/reperfusion (CI/R) injury in mice. Hypoxia IN VITRO was modeled using oxygen-glucose deprivation (OGD) followed by reoxygenation of BV-2 microglial cells. Our results showed that treatment of mice that have undergone CI/R injury with andrographolide (10-100 µg/kg, i. v.) at 1 h after hypoxia ameliorated CI/R-induced oxidative/nitrosative stress, brain infarction, and neurological deficits in the mice, and enhanced their survival rate. CI/R induced a remarkable production in the mouse brains of reactive oxygen species (ROS) and a significant increase in protein nitrosylation; this primarily resulted from enhanced expression of NADPH oxidase 2 (NOX2), inducible nitric oxide synthase (iNOS), and the infiltration of CD11b cells due to activation of nuclear factor-kappa B (NF- κB) and hypoxia-inducible factor 1-alpha (HIF-1 α). All these changes were significantly diminished by andrographolide. In BV-2 cells, OGD induced ROS and nitric oxide production by upregulating NOX2 and iNOS via the phosphatidylinositol-3-kinase (PI3K)/AKT-dependent NF- κB and HIF-1 α pathways, and these changes were suppressed by andrographolide and LY294002. Our results indicate that andrographolide reduces NOX2 and iNOS expression possibly by impairing PI3K/AKT-dependent NF- κB and HIF-1 α activation. This compromises microglial activation, which then, in turn, mediates andrographolide's protective effect in the CI/R mice.

In situ slow-release recombinant growth differentiation factor 11 exhibits therapeutic efficacy in ischemic stroke.

Systemic growth differentiation factor 11 (GDF11) treatment improves the vasculature in the hippocampus and cortex in mice in recent studies. However, systemic application of recombinant GDF11 (rGDF11) cannot cross the brain blood barrier (BBB). Thus, large doses and long-term administration are required, while systemically applied high-dose rGDF11 is associated with deleterious effects, such as severe cachexia. This study tested whether in situ low dosage rGDF11 (1 µg/kg) protects the brain against ischemic stroke and it investigated the underlying mechanisms. Fibrin glue mixed with rGDF11 was applied to the surgical cortex for the slow release of rGDF11 in mice after permanent middle cerebral artery occlusion (MCAO). In situ rGDF11 improved cerebral infarction and sensorimotor function by upregulating Smad2/3 and downregulating FOXO3 expression. In situ rGDF11 was associated with reductions in protein and lipid oxidation, Wnt5a, iNOS and COX2 expression, at 24 h after injury. In situ rGDF11 protected hippocampal neurons and subventricular neural progenitor cells against MCAO injury, and increased newborn neurogenesis in the peri-infarct cortex. Systematic profiling and qPCR analysis revealed that Pax5, Sox3, Th, and Cdk5rap2, genes associated with neurogenesis, were increased by in situ rGDF11 treatment. In addition, greater numbers of newborn neurons in the peri-infarct cortex were observed with in situ rGDF11 than with systemic application. Our evidence indicates that in situ rGDF11 effectively decreases the extent of damage after ischemic stroke via antioxidative, anti-inflammatory and proneurogenic activities. We suggest that in situ slow-release rGDF11 with fibrin glue is a potential therapeutic approach against ischemic stroke.

The efficacy of first-line tyrosine kinase inhibitors combined with co-medications in Asian patients with EGFR mutation non-small cell lung cancer.

The real-world efficacy of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) in patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR-activating mutations remains unclear. We conducted a retrospective cohort study using data from the claims database of Taipei Veterans General Hospital to perform direct comparisons of these three EGFR-TKIs (gefitinib, erlotinib, and afatinib) combined with co-medications (metformin, statins, antacids, and steroids). Stage IIIB and IV NSCLC patients with EGFR mutations receiving EGFR-TKIs as first-line treatment for > 3 months between 2011 and 2016 were enrolled. The primary endpoint was time to treatment failure (TTF). Patients who had received co-medications (≥ 28 defined daily doses) in the first 3 months of EGFR-TKI therapy were assigned to co-medications groups. A total of 853 patients treated with gefitinib (n = 534), erlotinib (n = 220), and afatinib (n = 99) were enrolled. The median duration of TTF was 11.5 months in the gefitinib arm, 11.7 months in the erlotinib arm, and 16.1 months in the afatinib arm (log-rank test, P < 0.001). After adjustments, afatinib showed lower risk of treatment failure compared with gefitinib (hazard ratio [HR] 0.54, 95% confidence interval [CI] 0.41-0.71) and erlotinib (HR 0.62, 95% CI 0.46-0.83). The risk of treatment failure in patients treated with EGFR-TKIs who received concomitant systemic glucocorticoid therapy was higher than in those treated with EGFR-TKI monotherapy (HR 1.47, 95% CI 1.08-2.01). Afatinib or erlotinib use was associated with a lower risk of treatment failure in patients with advanced NSCLC harboring EGFR mutations compared to gefitinib use. Concurrent use of systemic glucocorticoids was linked to higher risk of treatment failure.

Efficacy and safety of rituximab in autoimmune and microangiopathic hemolytic anemia: a systematic review and meta-analysis.

BACKGROUND: The efficacy and safety of rituximab (RTX) on hemolytic anemia (HA) is unknown. Therefore we retrospectively analyze the efficacy and safety of RTX in autoimmune hemolytic anemia (AIHA) and microangiopathic hemolytic anemia (MAHA) from the previous literature. METHODS: Data in clinical trials and observational studies were collected from PubMed, Cochrane, Embase, and Google Scholar until Oct 15, 2018. The efficacy and safety of RTX in patients with AIHA or MAHA were assessed and overall response rates (ORRs), complete response rates (CRRs), adverse events (AEs) and relapse rates (RRs) were extracted if available. A meta-analysis was performed with a random-effects model, estimating mean proportions in all studies, and relative rates in comparative studies. RESULTS: After quality assessment, a total of 37 investigations encompassing 1057 patients eligible for meta-analysis were included. Pooled mean proportion of ORR was 0.84 (95% confidence interval [CI] 0.80-0.88), and that of CRR was 0.61 (95% CI 0.49-0.73). Mean AE rate was 0.14 (95% CI 0.10-0.17), and mean RR was 0.21 (95% CI 0.15-0.26). Relative ORR was 1.18 (95% CI 1.02-1.36), and relative CRR was 1.17 (95% CI 0.98-1.39) fold more than the respective non-RTX counter parts. Relative AE rate was 0.77 (95% CI 0.36-1.63), and relative RR was 0.93 (95% CI 0.56-1.55) fold less than the respective non-RTX counter parts. CONCLUSION: RTX is more effective than the treatments without RTX for AIHA and MAHA and is well-tolerated.

Vagus nerve stimulation inhibits cortical spreading depression exclusively through central mechanisms.

Experimental and clinical data strongly support vagus nerve stimulation (VNS) as a novel treatment in migraine. Vagus nerve stimulation acutely suppresses cortical spreading depression (CSD) susceptibility, an experimental model that has been used to screen for migraine therapies. However, mechanisms underlying VNS efficacy on CSD are unknown. Here, we interrogated the central and peripheral mechanisms using VNS delivered either invasively (iVNS) or noninvasively (nVNS) in male Sprague-Dawley rats. Cortical spreading depression susceptibility was evaluated 40 minutes after the stimulation. iVNS elevated the electrical CSD threshold more than 2-fold and decreased KCl-induced CSD frequency by 22% when delivered to intact vagus nerve. Distal vagotomy did not alter iVNS efficacy (2-fold higher threshold and 19% lower frequency in iVNS vs sham). By contrast, proximal vagotomy completely abolished iVNS effect on CSD. Pharmacological blockade of nucleus tractus solitarius, the main relay for vagal afferents, by lidocaine or glutamate receptor antagonist CNQX also prevented CSD suppression by nVNS. Supporting a role for both norepinephrine and serotonin, CSD suppression by nVNS was inhibited by more than 50% after abrogating norepinephrinergic or serotonergic neurotransmission alone using specific neurotoxins; abrogating both completely blocked the nVNS effect. Our results suggest that VNS inhibits CSD through central afferents relaying in nucleus tractus solitarius and projecting to subcortical neuromodulatory centers providing serotonergic and norepinephrinergic innervation to the cortex.

Caffeine does not affect susceptibility to cortical spreading depolarization in mice.

Several factors that modulate migraine, a common primary headache disorder, also affect susceptibility to cortical spreading depolarization (CSD). CSD is a wave of neuronal and glial depolarization and thought to underlie the migraine aura and possibly headache. Here, we tested whether caffeine, known to alleviate or trigger headache after acute exposure or chronic use/withdrawal, respectively, modulates CSD. We injected C57BL/6J mice with caffeine (30, 60, or 120 mg/kg; i.p.) once ( acute) or twice per day for one or two weeks ( chronic). Susceptibility to CSD was evaluated by measuring the electrical CSD threshold and by assessing KCl-induced CSD. Simultaneous laser Doppler flowmetry was used to assess CSD-induced cortical blood flow changes. Recordings were performed 15 min after caffeine/vehicle administration, or 24 h after the last dose of chronic caffeine in the withdrawal group. The latter paradigm was also tested in mice carrying the familial hemiplegic migraine type 1 R192Q missense mutation, considered a valid migraine model. Neither acute/chronic administration nor withdrawal of caffeine affected CSD susceptibility or related cortical blood flow changes, either in WT or R192Q mice. Hence, adverse or beneficial effects of caffeine on headache seem unrelated to CSD pathophysiology, consistent with the non-migrainous clinical presentation of caffeine-related headache.