CircRNA and miRNA expression profiles during remote ischemic postconditioning attenuate brain ischemia/reperfusion injury.

Remote ischemic postconditioning (RIPostC) is a protective procedure for brain damage caused by ischemia/reperfusion (IR), yet the mechanism of this treatment remains to be elucidated. Circular RNAs (circRNAs) are endogenous non-coding RNAs that have recently been recognized to play vital roles in ischemic brain injury. The aim of this study was to explore the role of circRNAs in the protective mechanism of RIPostC and to analyze the circRNA-microRNA (miRNA) regulation network in RIPostC. Nine rats were assigned randomly into three groups (three rats per group): sham, IR, and RIPostC. Their brain tissues were extracted for next-generation RNA sequencing and bioinformatics analysis was performed for two comparisons: sham vs. IR and IR vs. RIPostC. The expression patterns of selected circRNAs and miRNAs were validated by quantitative real-time PCR (qPCR). We detected 82 upregulated and 51 downregulated circRNAs and 137 upregulated and 127 downregulated miRNAs in the IR group compared with the sham group, and 41 upregulated and 100 downregulated circRNAs and 45 upregulated and 64 downregulated miRNAs in the RIPostC group compared with the IR group. The proposed competitive endogenous RNA (ceRNA) network, which included 24 circRNAs, 20 miRNAs, and 145 mRNAs, indicated that the dysregulated circRNAs played important roles in brain IR injury. On the basis of the expression patterns of selected circRNAs, miRNAs, and mRNAs obtained by qPCR, we proposed a circRNA_0002286-miR-124-3p-VLCAD pathway. In PC12 cell, the expression level of miR-124-3p was significantly upregulated when the expression of circRNA_0002286 was repressed and the expression level of VLCAD (very-long chain acyl-CoA dehydrogenase) was significantly downregulated, which suggested that circRNA_0002286 may act as a miRNA sponge for miR-124-3p to regulate the expression of VLCAD. We found that upregulation of circRNA_0002286 attenuated IR injury and was associated with downregulation of miR-124-3p and upregulation of VLCAD. This is the first time that circRNAs have been shown to be closely related to brain IR injury and RIPostC and suggests that targeting the circRNA_0002286-miR-124-3p-VLCAD pathway might attenuate brain IR injury.

Neuroprotective effects of long noncoding RNAs involved in ischemic postconditioning after ischemic stroke.

During acute reperfusion, the expression profiles of long noncoding RNAs in adult rats with focal cerebral ischemia undergo broad changes. However, whether long noncoding RNAs are involved in neuroprotective effects following focal ischemic stroke in rats remains unclear. In this study, RNA isolation and library preparation was performed for long noncoding RNA sequencing, followed by determining the coding potential of identified long noncoding RNAs and target gene prediction. Differential expression analysis, long noncoding RNA functional enrichment analysis, and co-expression network analysis were performed comparing ischemic rats with and without ischemic postconditioning rats. Rats were subjected to ischemic postconditioning via the brief and repeated occlusion of the middle cerebral artery or femoral artery. Quantitative real-time reverse transcription-polymerase chain reaction was used to detect the expression levels of differentially expressed long noncoding RNAs after ischemic postconditioning in a rat model of ischemic stroke. The results showed that ischemic postconditioning greatly affected the expression profile of long noncoding RNAs and mRNAs in the brains of rats that underwent ischemic stroke. The predicted target genes of some of the identified long noncoding RNAs (cis targets) were related to the cellular response to ischemia and stress, cytokine signal transduction, inflammation, and apoptosis signal transduction pathways. In addition, 15 significantly differentially expressed long noncoding RNAs were identified in the brains of rats subjected to ischemic postconditioning. Nine candidate long noncoding RNAs that may be related to ischemic postconditioning were identified by a long noncoding RNA expression profile and long noncoding RNA-mRNA co-expression network analysis. Expression levels were verified by quantitative real-time reverse transcription-polymerase chain reaction. These results suggested that the identified long noncoding RNAs may be involved in the neuroprotective effects associated with ischemic postconditioning following ischemic stroke. The experimental animal procedures were approved by the Animal Experiment Ethics Committee of Kunming Medical University (approval No. KMMU2018018) in January 2018.

Negative regulation by proBDNF signaling of peripheral neurogenesis in the sensory ganglia of adult rats.

Neurogenesis in the adult brain is well recognized and plays a critical role in the maintenance of brain function and homeostasis. However, whether neurogenesis also occurs in the adult peripheral nervous system remains unknown. Here, using sensory ganglia (dorsal root ganglia, DRGs) as a model, we show that neurogenesis also occurs in the peripheral nervous system, but in a manner different from that in the central nervous system. Satellite glial cells (SGCs) express the neuronal precursor markers Nestin, POU domain, class 4, transcription factor 1, and p75 pan-neurotrophin receptor. Following sciatic nerve injury, the suppression of endogenous proBDNF by proBDNF antibodies resulted in the transformation of proliferating SGCs into doublecortin-positive cells in the DRGs. Using purified SGCs migrating out from the DRGs, the inhibition of endogenous proBDNF promoted the conversion of SGCs into neuronal phenotypes in vitro. Our findings suggest that SGCs are neuronal precursors, and that proBDNF maintains the SGC phenotype. Furthermore, the suppression of proBDNF signaling is necessary for neuronal phenotype acquisition by SGCs. Thus, we propose that peripheral neurogenesis may occur via the direct conversion of SGCs into neurons, and that this process is negatively regulated by proBDNF.

[Emission Concentration and Characteristics of Particulate Matter and Water-Soluble Ions in Exhaust Gas of Typical Combustion Sources with Ultra-Low Emission].

A self-developed direct condensation sampling system and monitoring method for total particulate matter (TPM) in ultra-low-emission and high-humidity exhaust gas were applied to the emission monitoring of particulate matter in flue gas from three typical combustion sources with ultra-low emissions in Beijing. The emission levels and composition characteristics of particulate matter and water-soluble ions in the exhaust gas of typical combustion sources with ultra-low emissions were analyzed and evaluated. The interaction and influencing factors of filterable particulate matter (FPM) and condensable particulate matter (CPM) and their water-soluble ions were explored. The results showed that the emission concentration of FPM in the exhaust gas of the coal-fired boiler with ultra-low emissions was between 1.04 mg·m-3 and 1.11 mg·m-3 in standard smoke oxygen content, and that of TPM was between 3.82 mg·m-3 and 8.69 mg·m-3, which all met the national ultra-low emission limit (10 mg·m-3). However, the TPM emission concentration of the coal-fired power plant exceeded the emission limit of Beijing (5 mg·m-3). The emission concentrations of CPM and its total water-soluble ions from the coal-fired heating boiler were 3.05 mg·m-3 and 1.30 mg·m-3, respectively, which were significantly lower than those of the coal-fired power plant, and were related to the higher load and flue gas temperature of the coal-fired power plant. Furthermore, the emission concentrations of CPM and its total water-soluble ions from the coal-fired power plant boiler were 2.2 to 2.4 times and 1.7 to 2.2 times greater than those of the coal-fired heating boiler, respectively. The emission concentrations of TPM and its total water-soluble ions from the gas power plant were 1.99 mg·m-3 and 1.44 mg·m-3, respectively, which were significantly lower than those from the coal-fired boiler. CPM was the main form of particulate matter in the exhaust gas of the combustion source. The contribution of CPM to TPM in the ultra-low-emission boiler flue gas increased significantly, and increased with the increase in the flue gas temperature, ranging from 72.6% to 88.1% for the coal-fired boiler and 93.1% for the gas power plant. Total water-soluble ions made up 66.1% to 94.2% of the CPM. The flue gas temperature had a significant impact on the existing forms, removal efficiencies, and emission concentrations of particulate matter and water-soluble ions. SO42- was the main characteristic water-soluble ion of particulate matter in the coal-fired boiler, and its emission concentration ranged from 0.98 mg·m-3 to 1.18 mg·m-3, accounting for 27.7% to 49.6% of the total water-soluble ion emissions, which originated from flue gas desulfurization. F- was another characteristic water-soluble ion of particulate matter in the coal-fired power plant, and its emission concentration ranged from 1.91 mg·m-3 to 2.32 mg·m-3, accounting for 54.4% to 56.1% of the total water-soluble ion emissions, which might have been related to the high F content of fuel coal. NH4+ was the main characteristic water-soluble ion of particulate matter in the gas power plant, and its emission concentration was 0.92 mg·m-3, accounting for 64.2% of the total water-soluble ion emissions, which originated from the escape of NH3 in the process of selective catalytic reduction. The emission concentration of NH4+ was significantly higher than that of the coal-fired boiler; this might have been related to the synergistic removal effect of the gas-fired power plant, which lacked other purification facilities.

Salidroside inhibits NLRP3 inflammasome activation and apoptosis in microglia induced by cerebral ischemia/reperfusion injury by inhibiting the TLR4/NF-κB signaling pathway.

BACKGROUND: The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is an important mediator of neuroinflammatory responses that regulates inflammatory injury following cerebral ischemia and may be a potential target. Salidroside (Sal) has good anti-inflammatory effects; however, it remains unclear whether Sal can regulate NLRP3 inflammasome activation through the Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathway after cerebral ischemia to alleviate inflammatory injury. METHODS: We established an oxygen-glucose deprivation and reoxygenation (OGD/R) model of BV2 cells and a middle cerebral artery occlusion/reperfusion (MCAO/R) rat model. Cell Counting Kit-8 (CCK-8), flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay were used to detect the viability and apoptosis of BV2 cells. Enzyme-linked immunosorbent assay (ELISA) was used to detect the level of inflammatory factors. 2,3,5-triphenyltetrazolium chloride (TTC) staining and modified Neurological Severity Score (mNSS) were used to detect cerebral infarction volume and neurological deficit in rats. Western blot, immunohistochemistry and immunofluorescence staining were used to detect the protein expression levels. RESULTS: Our results showed that Sal increased viability, inhibited lactate dehydrogenase (LDH) release, and reduced apoptosis in OGD/R-induced BV2 cells. Sal reduced the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-8. Following induction by OGD/R, BV2 cells exhibited NLRP3 inflammasome activation and increased protein levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, IL-1ß, and IL-18. Protein levels of key TLR4 signaling pathway elements, such as TLR4, myeloid differentiation primary response 88 (MyD88), and phosphorylated nuclear factor kappa B p65 (p-NF-κB p65)/NF-κB p65 were upregulated. Interestingly, it was revealed that Sal could reverse these changes. In addition, TAK242, a specific inhibitor of TLR4, had the same effect as Sal treatment on BV2 cells following induction by OGD/R. In the MCAO/R rat model, Sal was also observed to inhibit NLRP3 inflammasome activation in microglia, reduce cerebral infarction volume, and inhibit apoptosis. CONCLUSIONS: In summary, we found that Sal inhibited NLRP3 inflammasome activation and apoptosis in microglia induced by cerebral ischemia/reperfusion injury by inhibiting the TLR4/NF-κB signaling pathway, thus playing a protective role. Therefore, Sal may be a promising drug for the clinical treatment of ischemic stroke.

Advances in intervention methods and brain protection mechanisms of in situ and remote ischemic postconditioning.

Ischemic postconditioning (PostC) conventionally refers to a series of brief blood vessel occlusions and reperfusions, which can induce an endogenous neuroprotective effect and reduce cerebral ischemia/reperfusion (I/R) injury. Depending on the site of adaptive ischemic intervention, PostC can be classified as in situ ischemic postconditioning (ISPostC) and remote ischemic postconditioning (RIPostC). Many studies have shown that ISPostC and RIPostC can reduce cerebral IS injury through protective mechanisms that increase cerebral blood flow after reperfusion, decrease antioxidant stress and anti-neuronal apoptosis, reduce brain edema, and regulate autophagy as well as Akt, MAPK, PKC, and KATP channel cell signaling pathways. However, few studies have compared the intervention methods, protective mechanisms, and cell signaling pathways of ISPostC and RIPostC interventions. Thus, in this article, we compare the history, common intervention methods, neuroprotective mechanisms, and cell signaling pathways of ISPostC and RIPostC.

Different ischemic duration and frequency of ischemic postconditioning affect neuroprotection in focal ischemic stroke.

BACKGROUND: Many studies have confirmed that "in situ ischemia postconditioning" (ISPostC) and "remote ischemic postconditioning" (RIPostC) can reduce cerebral ischemia/reperfusion injury, but there is no comparison was made on the consistency of neuroprotection in ISPostC and RIPostC to different ischemic duration and number of cycles. NEW METHOD: We used a transient middle cerebral artery occlusion model to compare the neuroprotection of ISPostC and RIPostC. We conducted ISPostC and RIPostC via brief and repeated MCA and Femoral artery occlusion followed by different ischemic duration and number of cycles. Infarct volume, brain edema, Neurological deficit scores and Apoptosis were evaluated. RESULTS: First, the ISPostC with three cycles of 10-s occlusion/30-s release of both carotid arteries and the RIPostC with three cycles of 10-min occlusion/10-min release of the left and right femoral arteries can obviously reduce cerebral infarction size, brain edema, apoptosis, and improve behavioral deficits than other approaches. Second, three cycles of ischemia/reperfusion may be the best for RIPostC. COMPARISON WITH EXISTING METHOD(S): In this paper, we compared different ischemic duration and frequency of ISPostC and RIPostC models to determine the best method. This conclusion helps to unify the experimental methods. CONCLUSIONS: Different ischemic duration and frequency of ischemic postconditioning affect neuroprotection. three cycles of 10-s occlusion/30-s release of both carotid arteries and three cycles of 10-min occlusion/10-min release of both femoral arteries could be the first choice to study mechanisms of ischemic postconditioning and be conducive to the unification of research results.

[Pollution Characteristics and Potential Ecological Risks of Heavy Metals in Road Dust in Beijing].

Based on the concentrations of 21 inorganic elements in particulate matter with diameters less than 10 µm (PM10) in 2004, and PM2.5 in 2004 and 2013 of representative road dust in Beijing, the pollution characteristics and potential ecological risks of heavy metals in this dust were analyzed and discussed. The results showed that the six main elements in road dust in Beijing were Si, Ca, Al, Fe, Mg, and K, and the proportions of the total content of the six elements in PM10 in 2004, PM2.5 in 2004, and PM2.5 in 2013 accounted for 96.51%, 96.42%, and 96.53% of the total content of all elements tested, respectively. The elemental enrichment level and the pollution degree and the potential ecological risk of heavy metal in road dust in Beijing in 2004 were PM2.5>PM10. Se, a characteristic element of coal dust, was highly enriched in PM2.5 in 2004, and Cd was high in PM10 and PM2.5 in 2004 with enrichment factors of 1024.03, 68.15, and 871.55, respectively. Co, Zn, Ca, and Cu were significantly enriched in PM10 and PM2.5 in 2004 with enrichment factors of 12.93, 12.33, 8.30, and 8.07 in PM10 and 17.41, 21.80, 12.83, and 19.73 in PM2.5, respectively; Na and Si were not enriched in the road dust. The pollution load index (PLI) of heavy metals was 3.95 in PM10 and 7.71 in PM2.5 in 2004. Owing to the implementation of dust, motor vehicles, and combustion source control measures in Beijing and the relocation of the Shougang corporation, the elemental enrichment level, pollution degree, and potential ecological risk of heavy metals in road dust PM2.5 in 2013 were significantly lower than those in 2004. The enrichment factors of Cd and Se in PM2.5 in 2013 decreased to 98.47 and 0.95, respectively; those of Cu, Ca, and Zn decreased to 11.90, 8.84, and 8.20, respectively; and PLI decreased to 2.56. The results showed that the total potential ecological risk of heavy metals in road dust in Beijing was extremely strong. Heavy metal Cd was the most significant pollution factor and the main potential ecological risk source; its potential ecological risk index (RI) contribution to the total RI of heavy metals was more than 85%. In 2004, the pollution degree of heavy metals in road dust of main roads was significantly higher than that for other road types. The pollution degree of heavy metals in PM10 was main road > expressway entrance to Beijing > secondary main road > ring road; that for PM2.5 was main road > ring road > expressway entrance to Beijing > secondary main road. For PM2.5 in 2013, however, the order was expressway entrance to Beijing > main road > ring road > secondary main road. The pollution degree of heavy metals in road dust of secondary main roads was significantly lower than that for other road types. In 2013, for road dust PM2.5 in Beijing, the correlation of heavy metals Ti, Zn, V, Cr, Cu, Pb, and Ni was significant owing mainly to traffic-related emissions.

Preparation of Cellulose/Graphene Oxide Composite Membranes and Their Application in Removing Organic Contaminants in Wastewater.

Cellulose/graphene oxide composite membranes (CGCMs) were prepared using a vacuum-filtration method. The CGCMs were then used as filters to remove organic pollutants from wastewater. It was found that the CGCM filters could efficiently and simultaneously achieve wastewater treatment and adsorbent separation. Their adsorption of Rhodamine B (RhB, an organic dye) varied with varying cellulose/graphene oxide mass ratios. The CGCM obtained at a cellulose/graphene oxide mass ratio of 8:1 exhibited the maximum removal efficiency for RhB. The maximum adsorption capacity of the CGCMs for RhB was found to be 86.4 mg/g. In addition, the CGCMs were easily regenerated and the regenerated CGCMs retained good abilities to remove contaminants, which could be significant for their application in wastewater treatment.

A novel primary culture method for high-purity satellite glial cells derived from rat dorsal root ganglion.

Satellite glial cells surround neurons within dorsal root ganglia. Previous studies have focused on single-cell suspensions of cultured neurons derived from rat dorsal root ganglia. At present, the primary culture method for satellite glial cells derived from rat dorsal root ganglia requires no digestion skill. Hence, the aim of the present study was to establish a novel primary culture method for satellite glial cells derived from dorsal root ganglia. Neonatal rat spine was collected and an incision made to expose the transverse protrusion and remove dorsal root ganglia. Dorsal root ganglia were freed from nerve fibers, connective tissue, and capsule membranes, then rinsed and transferred to 6-well plates, and cultured in a humidified 5% CO2 incubator at 37°C. After 3 days in culture, some cells had migrated from dorsal root ganglia. After subculture, cells were identified by immunofluorescence labeling for three satellite glial cell-specific markers: glutamine synthetase, glial fibrillary acidic protein, and S100ß. Cultured cells expressed glutamine synthetase, glial fibrillary acidic protein, and S100ß, suggesting they are satellite glial cells with a purity of > 95%. Thus, we have successfully established a novel primary culture method for obtaining high-purity satellite glial cells from rat dorsal root ganglia without digestion.

Notoginsenoside R1 Promotes the Growth of Neonatal Rat Cortical Neurons via the Wnt/ß-catenin Signaling Pathway.

BACKGROUND & OBJECTIVE: Notoginsenoside R1 (NGR1) is one of the main effective components of Panax notoginseng. METHOD: Primary cortical neurons were harvested from neonatal rats and cultured to analyze the role of NGR1 in neuronal growth and the effects of NGR1 on the Wnt/ß-catenin signaling pathway. Following treatment with NGR1, immunocytochemistry was used to detect expression of Tuj1 and MAP2, and RT-qPCR was used to measure mRNA levels of key factors in the Wnt signaling pathway. RESULTS: Results showed that NGR1 promotes growth of cultured neurons and significantly upregulates mRNA levels of ß-catenin, Dishevelled, and Frizzled. To further confirm whether NGR1 promoted cortical neuron growth via the Wnt/ß-catenin signaling pathway, we knocked down ß- catenin mRNA by siRNA interference; following NGR1 treatment of ß-catenin-knockdown neurons, ß-catenin mRNA levels increased significantly. CONCLUSION: In conclusion, these results demonstrate that NGR1 promotes growth of cultured cortical neurons from the neonatal rat, possibly via the Wnt/ß-catenin signaling pathway.

The prognostic role of Gas6/Axl axis in solid malignancies: a meta-analysis and literature review.

BACKGROUND: Axl is a receptor tyrosine kinase that is involved in many pathological conditions and carcinogenesis. Gas6 is the major ligand of Axl. Activation of Gas6/Axl pathway is essential for cancer development. However, its prognostic significance in solid tumors remains unclear. Therefore, we performed this meta-analysis to elucidate the prognostic impact of Axl. METHODS: Published studies on Axl or Gas6 expression and overall survival (OS) and/or disease-free survival (DFS) were searched from databases. The outcome measurement is hazard ratio (HR) for OS or DFS related to Axl/Gas6 expression. Meta-analysis was performed using RevMan. The pooled HR was calculated by fixed-/random-effect models. RESULTS: A total of 3,344 patients from 25 studies were included. The results of meta-analysis showed that Axl overexpression was correlated with shorter OS (HR: 2.03, p<0.0001) and DFS (HR: 1.85, p<0.0001). In subgroup analysis, Axl expression was significantly correlated with poor prognosis in hepatocellular, esophageal and lung cancer. Axl expression was associated with differentiation grade, TNM stage, lymph node and distant metastasis. CONCLUSION: These results suggest that Axl overexpression is correlated with poor prognosis in solid tumors. This correlation varies among different types of cancers. More studies are needed to further investigate the prognostic value of Axl.

Brain-derived Neurotrophic Factor Promotes Growth of Neurons and Neural Stem Cells Possibly by Triggering the Phosphoinositide 3-Kinase/ AKT/Glycogen Synthase Kinase-3ß/ß-catenin Pathway.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) plays a crucial role in promoting survival and differentiation of neurons and neural stem cells (NSCs), but the downstream regulating mechanisms remain poorly understood. OBJECTIVE: We investigated whether BDNF exerts its effect by triggering the phosphoinositide 3-kinase (PI3K), protein kinase B, PKB (AKT), glycogen synthase kinase-3ß (GSK-3ß) and ß-catenin signaling pathway in cultured neurons and NSCs derived from the rat embryonic spinal cord. METHOD: Immunocytochemistry was used to detect neuronal and NSCs characteristics. RT-PCR was used to detect PI3K/AKT/GSK3ß/ß-catenin pathway expression. RESULTS: Neurons and NSCs were successfully separated and cultured from Sprague-Dawley rat embryonic spinal cord and were respectively labeled using immunocytochemistry. Neuron-specific nuclear protein, neuronal class III ß-tubulin, and neurofilament expression were detected in neurons; nestin, glial fibrillary acidic protein, microtubule-associated protein 2 and chondroitin sulfate glycosaminoglycan expression were detected in the NSCs. BDNF promoted significant neuronal growth (number, soma size, and average neurite length), as well as NSCs proliferation and differentiation, but BDNF antibody decreased neuronal growth and NSCs proliferation and differentiation. RT-PCR was used to detect changes in BDNF signal pathway components, showing that BDNF upregulated tropomyosin receptor kinase B, phosphoinositide 3-kinase (PI3K), AKT and ß-catenin, but downregulated GSK-3ß in the neurons and NSCs. BDNF antibody downregulated BDNF, tropomyosin receptor kinase B, PI3K, AKT, ß-catenin and cellular-myelocytomatosis viral oncogene, but upregulated GSK- 3ß, in the neurons and NSCs. CONCLUSION: Our findings suggested that BDNF contributed to neuronal growth and proliferation and differentiation of NSCs in vitro by stimulating PI3K/AKT/GSK3ß/ß-catenin pathways.

Effects of multiwalled carbon nanotubes on electrospun poly(lactide-co-glycolide)-based nanocomposite scaffolds on neural cells proliferation.

The repair of nerves remains a major challenge in neuron-regeneration. In this study, poly(lactic-co-glycolic acid)/multi-walled carbon nanotubes (PLGA/MWCNTs) nanofibrous scaffolds were fabricated by electrospinning method. The surface morphology, physical, and mechanical properties were characterized through scanning electron microscopy (SEM), transmission electron microscopy, and tensile tests, respectively. SEM analysis, Live/Dead staining, immunostaining assays were performed to evaluate neural cells growth. Blending PLGA with MWCNTs resulted in increase diameter and porosity of the scaffolds, and exhibited better mechanical properties. The results demonstrated that the scaffolds with higher MWCNTs concentration provided better survival for neural cells after 8 days of culture, especially for astrocytes growth. This could be useful in treating the disease like multiple sclerosis that causing central nervous system demyelination and axonal injury. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 934-943, 2017.

Region-specific expression of precursor and mature brain-derived neurotrophic factors after chronic alcohol exposure.

BACKGROUND: Alcohol abuse is a serious health problem worldwide that causes a variety of physical and mental disorders. Research has shown that the brain-derived neurotrophic factor (BDNF) plays an important role in alcohol addiction. The BDNF precursor (proBDNF) exhibits different actions than BDNF through separate receptors and pathways in the central nervous system. However, the effects of proBDNF and BDNF in alcohol addiction are not fully known. OBJECTIVES: The objective was to identify the expression patterns and effects of proBDNF and BDNF after chronic alcohol exposure. METHODS: A total of 40 male adult mice were studied. A mouse psychomotor sensitization (PS) model was established to explore the effects of BDNF and proBDNF treatment following chronic alcohol exposure. Reverse transcription PCR (RT-PCR) was performed to measure mRNA levels for BDNF, TrkB, P75NTR, and sortilin in the prefrontal cortex, hippocampus, and dorsal striatum of Kunming mice after chronic alcohol exposure. RESULTS: In Kunming mice, chronic alcohol exposure up-regulated BDNF and TrkB mRNA levels in the prefrontal cortex, but decreased sortilin and P75 mRNA levels in the dorsal striatum. No changes in mRNA levels were found in other measured brain regions in the alcohol and control groups. CONCLUSION: Chronic alcohol exposure induced the region-specific expression of BDNF and proBDNF and their respective receptors in the brain. These results suggest that BDNF and proBDNF signaling pathways may play major roles in alcohol preference and addiction.

BDNF promotes human neural stem cell growth via GSK-3ß-mediated crosstalk with the wnt/ß-catenin signaling pathway.

Brain-derived neurotrophic factor (BDNF) plays important roles in neural stem cell (NSC) growth. In this study, we investigated whether BDNF exerts its neurotrophic effects through the Wnt/ß-catenin signaling pathway in human embryonic spinal cord NSCs (hESC-NSCs) in vitro. We found an increase in hESC-NSC growth by BDNF overexpression. Furthermore, expression of Wnt1, Frizzled1 and Dsh was upregulated, whereas GSK-3ß expression was downregulated. In contrast, hESC-NSC growth was decreased by BDNF RNA interference. BDNF, Wnt1 and ß-catenin components were all downregulated, whereas GSK-3ß was upregulated. Next, we treated hESC-NSCs with 6-bromoindirubin-3'-oxime (BIO), a small molecule inhibitor of GSK-3ß. BIO reduced the effects of BDNF upregulation/downregulation on the cell number, soma size and differentiation, and suppressed the effect of BDNF modulation on the Wnt signaling pathway. Our findings suggest that BDNF promotes hESC-NSC growth in vitro through crosstalk with the Wnt/ß-catenin signaling pathway, and that this interaction may be mediated by GSK-3ß.

Expression pattern of ß-catenin during the development of human fetal spinal cord.

Development of the human fetal spinal cord is a very complicated process involving numerous signaling pathways including Wnt signaling pathways. These pathways are critical for the development and function of the mammalian nervous system. ß-Catenin is a key molecule in the canonical Wnt signaling pathway. However, the distributions of ß-catenin during development of the human fetal spinal cord have not been well characterized. Therefore, in this study, we performed immunohistochemical analysis of the ß-catenin distribution in the developing human spinal cord from 35 fetuses at three weeks to eight months of gestation. As early as E3W and E4W, ß-catenin was mainly expressed in the internal limiting membrane of the neural tube and neuroepithelium (E: Embryos; W: Weeks). During developmental stages, ß-catenin was widely expressed in various structures and cells including the neuroepithelium, internal limiting membrane, mantle layer, marginal layer, basal plate, alar plate, ependyma, gray matter, white matter, neurons with multiple processes, glial cells, and nerve fibers. This study clarifies the morphological developmental characteristics of the human fetal spinal cord as well as the distribution and expression pattern of ß-catenin in chronological and spatial aspects. Our results suggest that the Wnt÷ß-catenin signaling pathway might play a crucial role in various stages of the formation and differentiation of the human fetal spinal cord.

BDNF promotes the growth of human neurons through crosstalk with the Wnt/ß-catenin signaling pathway via GSK-3ß.

Brain-derived neurotrophic factor (BDNF) plays an important role in neuronal growth; however, the downstream regulatory mechanisms remain unclear. In this study, we investigated whether BDNF exerts its neurotrophic effects through the Wnt/ß-catenin signaling pathway in human embryonic spinal cord neurons in vitro. We found that neuronal growth (soma size and average neurite length) was increased by transfection with a BDNF overexpression plasmid. Western blotting and real-time quantitative PCR showed that expression of the BDNF pathway components TrkB, PI3K, Akt and PLC-γ was increased by BDNF overexpression. Furthermore, the Wnt signaling factors Wnt, Frizzled and Dsh and the downstream target ß-catenin were upregulated, whereas GSK-3ß was downregulated. In contrast, when BDNF signaling was downregulated with BDNF siRNA, the growth of neurons was decreased. Furthermore, BDNF signaling factors, Wnt pathway components and ß-catenin were all downregulated, whereas GSK-3ß was upregulated. This suggests that BDNF affects the growth of neurons in vitro through crosstalk with Wnt signaling, and that GSK-3ß may be a critical factor linking these two pathways. To evaluate this possibility, we treated neurons with 6-bromoindirubin-3'-oxime (BIO), a small molecule GSK-3ß inhibitor. BIO reduced the effects of BDNF upregulation/downregulation on soma size and average neurite length, and suppressed the impact of BDNF modulation on the Wnt signaling pathway. Taken together, our findings suggest that BDNF promotes the growth of neurons in vitro through crosstalk with the Wnt/ß-catenin signaling pathway, and that this interaction may be mediated by GSK-3ß.

Expression pattern of NeuN and GFAP during human fetal spinal cord development.

PURPOSE: The development of the human embryonic spinal cord is very complicated, and many cell types are involved in the process. However, the morphological characteristics of neuronal and glial cells during the development of the human fetal spinal cord have not been described. We investigated the systemic distributions and expression pattern of the cell type-specific markers Neuron-specific nuclear protein (NeuN) and glial fibrillary acidic protein (GFAP) during the development of the human fetal spinal cord, in order to clarify the detailed developmental changes of neuronal and glial cells in chronological and spatial aspects. METHODS: A total of 35 fetuses, aged 3 weeks to 8 months of gestation (E3W-E8M), were studied. The markers used for immunohistochemical study were NeuN and GFAP. RESULTS: The intracellular makers NeuN and GFAP were widely detected expression in different structures and cells during the development of the human fetal spinal cord, including the following: central canal, neuroepithelial layer, internal limiting membrane, mantle layer, marginal layer, basal plate, alar plate, ependymal layer, gray matter, white matter, neuron, astrocytes, and nerve fibers. However, there was an absence of GFAP in astrocytes during early fetal spinal cord development until E9W, and the appearance of GFAP-positive reactivity was later than that of neurons. CONCLUSIONS: We consider that NeuN and GFAP can be used to identify neuronal and glial cells during the development of the human fetal spinal cord, and their distribution differs both chronologically and spatially. These characteristic expression patterns would give us a clue to better understand the developmental characteristics of the human spinal cord.

Monitoring the Effects of Acupoint Antioxidant Intervention by Measuring Electrical Potential Difference along the Meridian.

Previous studies suggest that superoxide anions are possibly traveling along acupuncture meridians. The electrical potential difference (EPD) between acupoints may be related to the movement. To test the above hypothesis, we conducted a study investigating the effects of acupoint antioxidant interventions on the meridian EPD. Firstly, ST39 (L) and ST44 (L) were screened out for the EPD detection along the stomach meridian, and ST36 (L) was selected for interventions including acumassage with the control cream, as well as the TAT-SOD cream for 30 minutes, or injection with reduced glutathione sodium. The EPD between ST39 and ST44 was recorded for 80 minutes and measured again 48 h later. While the EPD increased during the acumassage, the acumassage with TAT-SOD cream and the glutathione injection generated waves of EPD increased, indicating the migration or removal from the visceral organ of a greater quantity of superoxide. Remarkably lower EPD readings 48 h later with both antioxidant acupoint interventions than the mere acumassage imply a more complete superoxide flushing out due to the restored superoxide pathway at the acupoint after interventions. The results confirm superoxide transportation along the meridians and demonstrate a possibility of acupoint EPD measurement as a tool to monitor changes in the meridians and acupoints.