Ecological factors associated with persistent circulation of multiple highly pathogenic avian influenza viruses among poultry farms in Taiwan during 2015-17.

Emergence and intercontinental spread of highly pathogenic avian influenza A (HPAI) H5Nx virus clade 2.3.4.4 has resulted in substantial economic losses to the poultry industry in Asia, Europe, and North America. The long-distance migratory birds have been suggested to play a major role in the global spread of avian influenza viruses during this wave of panzootic outbreaks since 2013. Poultry farm epidemics caused by multiple introduction of different HPAI novel subtypes of clade 2.3.4.4 viruses also occurred in Taiwan between 2015 and 2017. The mandatory and active surveillance detected H5N3 and H5N6 circulation in 2015 and 2017, respectively, while H5N2 and H5N8 were persistently identified in poultry farms since their first arrival in 2015. This study intended to assess the importance of various ecological factors contributed to the persistence of HPAI during three consecutive years. We used satellite technology to identify the location of waterfowl flocks. Four risk factors consistently showed strong association with the spatial clustering of H5N2 and H5N8 circulations during 2015 and 2017, including high poultry farm density (aOR:17.46, 95%CI: 5.91-74.86 and 8.23, 95% CI: 2.12-54.86 in 2015 and 2017, respectively), poultry heterogeneity index (aOR of 12.28, 95%CI: 5.02-31.14 and 2.79, 95%CI: 1.00-7.69, in 2015 and 2017, respectively), non-registered waterfowl flock density (aOR: 6.8, 95%CI: 3.41-14.46 and 9.17, 95%CI: 3.73-26.20, in 2015 and 2017, respectively) and higher percentage of cropping land coverage (aOR of 1.36, 95%CI: 1.10-1.69 and 1.04, 95%CI: 1.02-1.07, in 2015 and 2017, respectively). Our study highlights the application of remote sensing and clustering analysis for the identification and characterization of environmental factors in facilitating and contributing to the persistent circulation of certain subtypes of H5Nx in poultry farms in Taiwan.

MicroRNA-mediated interacting circuits predict hypoxia and inhibited osteogenesis of stem cells, and dysregulated angiogenesis are involved in osteonecrosis of the femoral head.

PURPOSE: MicroRNAs (miRNAs) are associated with various pathologic conditions and can serve as diagnostic or therapeutic biomarkers. This study tried to identify the differentially expressed miRNAs to predict the possible pathomechanisms involved in osteonecrosis of the femoral head (ONFH). METHODS: We compared the peripheral blood miRNAs in 46 patients with ONFH and 85 healthy controls by microarray and droplet digital polymerase chain reaction (ddPCR). Putative interacted networks between the differentially responded miRNAs were analyzed by web-based bioinformatics prediction tools. RESULTS: Microarray identified 51 differentially expressed miRNAs with at least twofold change (upregulation in 34 and downregulation in 17), and the results were validated by ddPCR using six selected miRNAs. Bioinformatics genetic network analysis focusing on the six miRNAs found the upregulated miR-18a and miR-19a are associated with angiogenesis after induction of ischemia; the upregulated miR-138-1 can inhibit osteogenic differentiation of mesenchymal stem cells; the most targeted genes, p53 and SERBP1, are associated with hypoxia and hypofibrinolysis. CONCLUSIONS: This study combined the miRNA analysis with the bioinformatics and predicts that hypoxia, inhibited osteogenesis of stem cells, and dysregulated angiogenesis might be orchestrated through the miRNA interacting circuits in the pathogenesis of ONFH.

Melatonin treatment enhances therapeutic effects of exosomes against acute liver ischemia-reperfusion injury.

This study tests the hypothesis that combined melatonin and exogenic adipose mesenchymal stem cell (ADMSC)-derived exosome treatment offers superior protection against liver ischemia-reperfusion (LIR) injury compared to either alone. In vitro studies utilized a macrophage cell line (RAW) pretreated with lipopolysaccharide and hepatocytes pretreated with melatonin or exosomes before hypoxia treatment, while in vitro experiments involved analyses of liver specimens from male adult Sprague-Dawley rats (n = 50) equally categorized into sham controls (SC), LIR only, LIR-exosome (100 µg, 30 minute post-LIR), LIR-melatonin (20 mg/kg, 30 minute post-LIR and 50 mg/kg at 6 and 18 hours post-LIR), and LIR-exosome-melatonin groups. In vitro studies showed suppression of inflammation (MIF, MMP-9, IL-1ß, TNF-α, COX-2) and oxidative stress (NOX-1, NOX-2, oxidized protein)/apoptosis (cleaved caspase 3 and PARP) by exosome and exosome/melatonin treatment, respectively (all P<0.001). In vivo data demonstrated lowest liver injury score and plasma AST concentrations in LIR-exosome-melatonin group compared with other groups (P<0.001). Besides, expressions of inflammatory markers at protein (ICAM-1, IL-1ß, MMP-9, TNF-α, NF-κB, RANTES) and cellular (CD3+, CD4+, CD8+, CD161+, CD11+, CD14+, F4/80) levels, and protein expressions of apoptosis (cleaved caspase-3, PARP), oxidative stress (NOX-1, NOX-2), DNA damage (γ-H2AX) and mitochondrial damage (cytosolic cytochrome-C) markers displayed a pattern similar to that of liver injury score, whereas protein expression of anti-oxidants (HO-1, NQO-1) showed progressive increase from SC to the combined treatment group (all P<0.001). In conclusion, combined exosome-melatonin regimen was superior to either alone in protecting the liver against ischemia-reperfusion injury.

EPO-cyclosporine combination therapy reduced brain infarct area in rat after acute ischemic stroke: role of innate immune-inflammatory response, micro-RNAs and MAPK family signaling pathway.

This study tested the hypothesis that erythropoietin (EPO) and cyclosporine (CsA) could effectively reduce brain infarct area (BIA) in rat after acute ischemic stroke (AIS) through regulating inflammation, oxidative stress, MAPK family signaling and microRNA (miR-223/miR-30a/miR-383). Adult male Sprague-Dawley rats (n = 48) were equally divided into group 1 (sham control), group 2 (AIS), group 3 [AIS+EPO (5,000 IU/kg at 0.5/24/48 h, subcutaneous)] and group 4 [AIS+CsA (20.0 mg/kg at 0.5/24/48 h, intra-peritoneal)]. By 72 h, histopathology showed that BIA was largest in group 2 and smallest in group 1, and significantly larger in group 4 than group 3 (all P<0.0001). The three microRNAs expressed were higher in group 2 than in the other three groups (all P<0.04); between these three latter groups there were no significant differences. The protein expressions of MAPK family [phosphorylated (p)-ERK1/2, p-p38/p-JNK], inflammatory (iNOS/MMP-9/TNF-α/NF-κB/IL-12/MIP-1α/CD14/CD68/Ly6g), apoptotic (caspase-3/PARP/mitochondrial-Bax), oxidative-stress (NOX-1/NOX-2/oxidized protein) and mitochondrial-damaged (cytosolic cytochrome-C) biomarkers exhibited an identical pattern to BIA findings (all P<0.0001). The cellular expressions of brain edema (AQP4+), inflammation (CD11+/glial-fibrillary-acid protein+), and cellular damage (TUNEL assay/positive Periodic acid-Schiff stain) biomarkers exhibited an identical pattern, whereas the cellular-integrity markers (neuN+/MAP2+/doublecorin+) exhibited an opposite pattern to BIA (all P value <0.001). EPO-CsA therapy markedly reduced BIA mainly by suppressing the innate immune response to inflammation, oxidative stress, microRNAs (miR-223/miR-30a/miR-383) and MAPK family signaling.

The therapeutic effect of rosuvastatin and propylthiouracil on ameliorating high-cholesterol diet-induced rabbit aortic atherosclerosis and stiffness.

BACKGROUND: We tested the hypothesis that arteriosclerosis-augmented aortic pulse wave velocity (PWV) and -impaired vasorelaxation were attenuated by rosuvastatin (Rosu) and propylthiouracil (PTU) therapy. METHODS AND RESULTS: Thirty-two New Zealand rabbits were equally divided into group 1 (sham-control), group 2 [high-cholesterol-diet (HCD) for 8weeks], group 3 [HCD-Rosu (20mg/kg/day administration after 4-week HFD for 4weeks)], and group 4 [HCD-PTU (0.1% PTU in drinking water), the treatment course as group 3]. KCl-induced vasoconstriction of carotid artery (CA) was significantly higher in group 2 than in other groups (all p<0.01), but showed no differences among groups 1, 3 and 4, whereas acetylcholine-induced vasorelaxation exhibited an opposite pattern of KCl-induced vasoconstriction among the four groups (p<0.001). Basic nitric-oxide release from endothelial cells of CA was highest in group 1, lowest in group 2, but showed no difference between groups 3 and 4 (all p<0.001). PWV value was highest in group 2, lowest in group 1, and significantly higher in group 4 than in group 3 (all p<0.001). Serum levels of total-cholesterol, LDL and TG showed an identical pattern to PWV (all p<0.001), whereas the levels of free T4, sugar, and body weight did not differ among the four groups (all p>0.4). Aortic inflammatory biomarkers in cellular (CD68+/IL-1ß+/CD14+) and protein (TNF-α/NF-κB/IL-1ß/MMP-9/MCP-1/ICAM-1/PDGF) levels, and aortic oxidative-stress biomarkers in cellular (8-OHdG) and protein (NOX-1/NOX-2/oxidized protein) levels showed an identical pattern to PWV among the four groups (all p<0.001). CONCLUSION: Rosu-PTU therapy ameliorated aortic stiffness and inflammation/oxidative-stress, and improved endothelial-cell function after HCD challenge in rabbit.

Circulating microparticles enhanced rat vascular wall remodeling following endothelial denudation.

This study tested the hypothesis that circulating microparticles (MPs) exacerbated vascular wall (VW) remodeling after endothelial denudation by 0.014 wire in a rat model. Adult male Sprague Dawley rats (n = 40) were equally categorized into group 1 [sham-control (SC); 3.0 mL saline intravenous injection], group 2 [SC + intravenous MPs (1.0 × 107) derived from patients with carotid artery stenosis (CAS)], group 3 [femoral arterial endothelial denudation (FAED)], group 4 (FAED + MPs derived from healthy subjects), and group 5 (FAED + CAS-derived MPs). Animals were euthanized by day 28 after FAED procedure. The results demonstrated that neointimal area (NIA) (mm2), medial area, and number of infiltrated cells in medial layer were highest in group 5 and lowest in groups 1 and 2, and significantly higher in group 4 than those in group 3 (all P<0.0001), but no differences were noted between groups 1 and 2. However, the ratio of luminal area to VW area showed an opposite pattern compared to that of NIA among five groups (P<0.0001). Immunofluorescent study showed an identical pattern of changes in the numbers of inflammatory (F4/80, CD14, CD40, IL-ß) and proliferative (Ki-67, Cx43) cells in VW compared to that of NIA among the five groups (all P<0.00). The mNRA expressions of inflammatory (MMP-9, NF-κB, TNF-α, IL-1ß, iNOS, PDGF) and cell activation (c-Fos, c-Myc, osteopontin, PCNA) biomarkers showed an identical pattern compared to that of NIA among all groups (all P<0.001). Take altogether, CAS-derived MPs further aggravated MP-mediated VW remodeling after endothelial damage compared to that observed after administration of MPS derived from healthy subjects.

Intravenous administration of xenogenic adipose-derived mesenchymal stem cells (ADMSC) and ADMSC-derived exosomes markedly reduced brain infarct volume and preserved neurological function in rat after acute ischemic stroke.

We tested the hypothesis that combined xenogenic (from mini-pig) adipose-derived mesenchymal stem cell (ADMSC) and ADMSC-derived exosome therapy could reduce brain-infarct zone (BIZ) and enhance neurological recovery in rat after acute ischemic stroke (AIS) induced by 50-min left middle cerebral artery occlusion. Adult-male Sprague-Dawley rats (n = 60) were divided equally into group 1 (sham-control), group 2 (AIS), group 3 [AIS-ADMSC (1.2×106 cells)], group 4 [AIS-exosome (100µg)], and group 5 (AIS-exosome-ADMSC). All therapies were provided intravenously at 3h after AIS procedure. BIZ determined by histopathology (by day-60) and brain MRI (by day-28) were highest in group 2, lowest in group 1, higher in groups 3 and 4 than in group 5, but they showed no difference between groups 3 and 4 (all p < 0.0001). By day-28, sensorimotor functional results exhibited an opposite pattern to BIZ among the five groups (p < 0.005). Protein expressions of inflammatory (inducible nitric oxide synthase/tumor necrosis factor-α/nuclear factor-κB/interleukin-1ß/matrix metalloproteinase-9/plasminogen activator inhibitor-1/RANTES), oxidative-stress (NOX-1/NOX-2/oxidized protein), apoptotic (caspase-3/ Poly-ADP-ribose polymerase), and fibrotic (Smad3/transforming growth factor-ß) biomarkers, and cellular expressions of brain-damaged (γ-H2AX+/ XRCC1-CD90+/p53BP1-CD90+), inflammatory (CD11+/CD68+/glial fibrillary acid protein+) and brain-edema (aquaporin-4+) markers showed a similar pattern of BIZ among the groups (all n < 0.0001). In conclusion, xenogenic ADMSC/ADMSC-derived exosome therapy was safe and offered the additional benefit of reducing BIZ and improving neurological function in rat AIS.

The cardioprotective effect of melatonin and exendin-4 treatment in a rat model of cardiorenal syndrome.

We investigated the cardioprotective effect of melatonin (Mel) and exendin-4 (Ex4) treatment in a rat model of cardiorenal syndrome (CRS). Adult male SD rats (n=48) were randomly and equally divided into sham control (SC), dilated cardiomyopathy (DCM) (doxorubicin 7 mg/kg i.p. every five days/4 doses), CRS (defined as DCM+CKD) only, CRS-Mel (20 mg/kg/d), CRS-Ex4 (10 µg/kg/d), and CRS-Mel-Ex4 groups. In vitro results showed protein expressions of oxidative stress (NOX-1/NOX-2/oxidized protein), DNA/mitochondrial damage (γ-H2AX/cytosolic cytochrome c), apoptosis (cleaved caspase-3/PARP), and senescence (ß-galactosidase cells) biomarkers were upregulated, whereas mitochondrial ATP level was decreased in doxorubicin/p-cresol-treated H9c2 cells that were revised by Mel and Ex4 treatments (all P<.001). By day 60, LVEF was highest in the SC and lowest in the CRS, significantly lower in the DCM than in other treatment groups, lower in the CRS-Mel and CRS-Ex4 than in the CRS-Mel-Ex4, and lower in the CRS-Mel than in the CRS-Ex4, whereas LV chamber size and histopathology score showed a pattern opposite to that of LVEF among all groups (all P<.001). Plasma creatinine level was highest in the CRS and lowest in the SC and progressively decreased from the CRS-Mel, CRS-Ex4, CRS-Mel-Ex4 to DCM (P<.0001). Protein expressions of inflammation (TNF-α/NF-κB/MMP-2/MMP-9/IL-1ß), apoptosis/DNA damage (Bax/c-caspase-3/c-PARP/γ-H2AX), fibrosis (Smad3/TGF-ß), oxidative stress (NOX-1/NOX-2/NOX-4/oxidized protein), cardiac hypertrophy/pressure overload (BNP/ß-MHC), and cardiac integrity (Cx43/α-MHC) biomarkers in LV myocardium showed an opposite pattern compared to that of LVEF among all groups (all P<.001). Fibrotic area, DNA damage (γ-H2AX+ /53BP1+ CD90+ /XRCC1+ CD90+ ), and inflammation (CD14+ /CD68+ ) biomarkers in LV myocardium displayed a pattern opposite to that of LVEF among all groups (all P<.001). Combined melatonin and exendin-4 treatment suppressed CRS-induced deterioration of LVEF and LV remodeling.

Human lung cancer-derived microparticles enhanced angiogenesis and growth of hepatoma cells in rodent lung parenchyma.

This study tested the hypothesis that human lung cancer-derived microparticles (LcD-MPs) played an important role in tumor angiogenesis and growth. Fischer 344 rats (F344, n=18) were equally categorized into group 1 [Sham Control (3.0 mL normal saline intravenous injection (IV))], group 2 [hepatoma cell line (2.0 x 10(6) cells, IV)], and group 3 [hepatoma cell line + LcD-MPs (3.0 x 10(6), IV)]. Animals were euthanized by day 28 after hepatoma cells transfusion. Our result showed that the gross pathology confirmed growth of hepatoma cell line in lung parenchyma. The size and weight of the lungs were significantly increased in group 2 and further elevated in group 3 than in group 1 (all p<0.001). Histopathological analysis demonstrated that the lung crowded score and number of small vessel exhibited an identical pattern, whereas the number of alveolar sacs showed an opposite pattern compared to that of total lung weight among the three groups (all p<0.0001). The cellular expressions of CD34(+), CXCR4(+), c-Kit(+), CK19(+), VEGF(+) and vimentin+ cells in lung parenchyma exhibited an identical pattern compared to those of total lung weight among all groups (all p<0.001). The protein expressions of apoptotic (Bax, cleaved caspase-3 and c-PARP), fibrotic (Smad3, TGF-ß), and tumor suppression (PTEN) biomarkers showed an identical pattern, whereas that of anti-apoptotic (Bcl-2) and anti-fibrotic (Smad1/5, BMP-2) biomarkers were displayed an opposite pattern compared to that of total lung weight among all groups (all p<0.001). The MPs could enhance angiogenesis and accelerated hepatoma cell growth in rodent lung parenchyma.

Combination of adipose-derived mesenchymal stem cells (ADMSC) and ADMSC-derived exosomes for protecting kidney from acute ischemia-reperfusion injury.

BACKGROUND: In this study, we tested the hypothesis that a combined adipose-derived mesenchymal stem cell (ADMSC) and ADMSC-derived exosome therapy protected rat kidney from acute ischemia-reperfusion (IR) injury (i.e., ligation of both renal arteries for 1h and reperfusion for 72h prior to euthanization). METHODS AND RESULTS: Adult-male SD rats (n=40) were equally categorized into group 1 (sham control), group 2 (IR), group 3 [IR+exosome (100µg)], group 4 [IR+ADMSC (1.2×10(6) cells)], and group 5 (IR-exosome-ADMSC). All therapies were performed at 3h after IR procedure from venous administration. By 72h, the creatinine level and kidney injury score were the lowest in group 1 and the highest in group 2, significantly higher in group 3 than in groups 4 and 5, and significantly higher in group 4 than in group 5 (all P<0.0001). The protein expression of inflammatory (TNF-α/NF-κB/IL-1ß/MIF/PAI-1/Cox-2), oxidative-stress (NOX-1/NOX-2/oxidized protein), apoptotic (Bax/caspase-3/PARP), and fibrotic (Smad3/TGF-ß) biomarkers showed an identical pattern, whereas the anti-apoptotic (Smad1/5, BMP-2) and angiogenesis (CD31/vWF/angiopoietin) biomarkers and mitochondrial cytochrome-C showed an opposite pattern of creatinine level among the five groups (all P<0.001). The microscopic findings of glomerular-damage (WT-1), renal tubular-damage (KIM-1), DNA-damage (γ-H2AX), inflammation (MPO/MIF/CD68) exhibited an identical pattern, whereas the podocyte components (podocin/p-cadherin/synaptopodin) displayed a reversed pattern of creatinine level (all P<0.0001). CONCLUSION: Combined exosome-ADMSC therapy was superior to either one for protecting kidney from acute IR injury.

Enhanced protection against renal ischemia-reperfusion injury with combined melatonin and exendin-4 in a rodent model.

We tested the hypothesis that combined treatment with melatonin, an anti-oxidant, and exendin-4, an anti-inflammatory agent, was superior to either alone for protecting the kidney from ischemia-reperfusion (IR) injury. Male adult Sprague-Dawley rats (n=40) were equally divided into group 1 (sham-operated control), group 2 (IR only, IR=1h/72h), group 3 (IR-exendin-4, 10 µg/kg at 30 min, 24 h, 48 h after IR procedure), group 4 (IR-melatonin, i.p. 50 mg at 30 min, then 20 mg at 6 and 18 h after IR procedure), and group 5 (combined IR-exendin-4-melatonin). All animals were sacrificed by 72 h after IR/sham procedure. The results showed that the kidney injury score, plasma creatinine, and blood urea nitrogen (BUN) levels were highest in group 2 and lowest in group 1, significantly higher in groups 3 and 4 than those in group 5 and significantly higher in group 3 than those in group 4 (all p < 0.001). The protein expressions of inflammatory (toll-like receptor 4, inducible nitric oxide synthase, interleukin-1ß), apoptotic (mitochondrial Bax, cleaved caspase-3 and poly(ADP-ribose) polymerase, p53), podocyte integrity (E-cadherin, P-cadherin), and cell survival (phosphatidylinositol-3-kinase/AKT/mammalian target of rapamycin) biomarkers, as well the podocyte dysfunction biomarkers (Wnt1/Wnt4/ß-catenin) displayed a pattern identical to that of creatinine level among the five groups (all p < 0.001). Microscopic findings demonstrated that podocyte dysfunction (Wnt1/Wnt4/ß-catenin expression) and inflammatory (CD14 and F4/80-positively stained cells) biomarkers exhibited an identical pattern, whereas that of antioxidant (HO-1(+), NQO-1(+) cells) biomarkers showed an opposite pattern compared to that of creatinine level among the five groups (all p < 0.001). Combined melatonin-exendin-4 therapy offered an additional benefit in protecting the kidney from acute IR injury.

Melatonin pretreatment enhances the therapeutic effects of exogenous mitochondria against hepatic ischemia-reperfusion injury in rats through suppression of mitochondrial permeability transition.

We tested the hypothesis that melatonin (Mel) enhances exogenous mitochondria (Mito) treatment against rodent hepatic ischemia-reperfusion (IR) injury. In vitro study utilized three groups of hepatocytes (i.e. nontreatment, menadione, and menadione-melatonin treatment, 4.0 × 10(5) each), while in vivo study used adult male Sprague Dawley rats (n = 40) equally divided into sham-control (SC), IR (60-min left-lobe ischemia + 72-hr reperfusion), IR-Mel (melatonin at 30 min/6/8 hr after reperfusion), IR-Mito (mitochondria 15,000 µg/rat 30 min after reperfusion), and IR-Mel-Mito. Following menadione treatment in vitro, oxidative stress (NOX-1/NOX-2/oxidized protein), apoptotic (cleaved caspase-3/PARP), DNA damage (γ-H2AX/CD90/XRCC1), mitochondria damage (cytosolic cytochrome c) biomarkers, and mitochondrial permeability transition were found to be lower, whereas mitochondrial cytochrome c were found to be higher in hepatocytes with melatonin treatment compared to those without (all P < 0.001). In vivo study demonstrated highest liver injury score and serum AST in IR group, but lowest in SC group and higher in IR-Mito group than that in groups IR-Mel and IR-Mel-Mito, and higher in IR-Mel group than that in IR-Mel-Mito group after 72-hr reperfusion (all P < 0.003). Protein expressions of inflammatory (TNF-α/NF-κB/IL-1ß/MMP-9), oxidative stress (NOX-1/NOX-2/oxidized protein), apoptotic (caspase-3/PARP/Bax), and mitochondria damage (cytosolic cytochrome c) biomarkers displayed an identical pattern, whereas mitochondria integrity marker (mitochondrial cytochrome c) showed an opposite pattern compared to that of liver injury score (all P < 0.001) among five groups. Microscopically, expressions of apoptotic nuclei, inflammatory (MPO(+) /CD68(+) /CD14(+) cells), and DNA damage (γ-H2AX(+) cells) biomarkers exhibited an identical pattern compared to that of liver injury score (all P < 0.001) among five groups. Melatonin-supported mitochondria treatment offered an additional benefit of alleviating hepatic IR injury.

Tissue plasminogen activator deficiency preserves neurological function and protects against murine acute ischemic stroke.

BACKGROUND: We tested the hypothesis that tissue plasminogen activator (tPA) deficiency protected against acute ischemic stroke (AIS)-induced brain injury. METHODS AND RESULTS: Wild-type mice (n=54) were categorized into group 1 (sham control, n=18) and group 3 [AIS by permanent ligation of left common carotid artery (CCA) and cramping right CCA for 1h and then reperfusion followed by hypoxia (11% of oxygen supply for 2h), n=36]. Similarly, tPA knockout (tPA(-/-)) mice (n=54) were randomized into group 2 (sham control, n=18) and group 4 (AIS, n=36). By day 28 after AIS procedure, mortality rate was higher in group 3 (77.8%) than in group 4 (38.9%) and lowest in groups 1 (0%) and 2 (0%) (p<0.001). By days 3 and 28, MRI demonstrated a pattern of changes in brain-infarct volume identical to that of mortality among four groups (p<0.001). By day 28, protein expressions of inflammatory (MMP-9, TNF-α, NF-κB, iNOS, PAI-1, RANTES), oxidative (NOX-1, NOX-2, oxidized protein), apoptotic (cleaved caspase-3 & PARP, Bax), and fibrotic (Smad3, TGF-ß) biomarkers and cellular expressions of inflammation (CD11, F4/80, GFAP), DNA-damage (γ-H2AX) and brain-edema (AQP4) markers exhibited an identical pattern compared to that of mortality (all p<0.001), whereas protein expressions of endothelial (eNOS, CD31), anti-fibrotic (Smad1/5, BMP-2) biomarkers, and number of small vessels displayed an opposite pattern (all p<0.001) among four groups. Expressions of protein and cellular angiogenesis markers (VEGF, SDF-1α, CXCR4) were progressively increased from groups 1 and 2 to group 4 (all p<0.0001). CONCLUSION: tPA deficiency protected the brain from AIS injury.

Adipose-Derived Stem Cells Accelerate Diabetic Wound Healing Through the Induction of Autocrine and Paracrine Effects.

Cell-based therapy is an attractive approach for the treatment of chronic nonhealing wounds. This study investigated whether adipose-derived stem cells (ASCs) can accelerate diabetic wound healing and traffic in the engraftment of ASCs. Dorsal full-thickness skin wound defects (6 × 5 cm) were created in a streptozotocin (STZ)-induced diabetes rodent model. Group I served as a nondiabetic normal control, group II served as a diabetic control without ASCs, and group III included rats that were injected subcutaneously in the wound margin twice with nondiabetic ASCs (1 × 10(7) ASCs/dose). The wound healing was assessed clinically. Histological examination and immunohistochemical analyses of periwound tissue were performed. Green fluorescence protein (GFP)(+)-ASCs were used to examine the engraftment of these cells after injection. XenoLight DiR-labeled ASCs were implanted to detect migration ability using an IVIS imaging system. Results revealed that complete wound healing time statistically decreased in the ASC-treated group compared to the controls (p < 0.001). Histological examination revealed the ASC-treated group showed a significant reduction in the proinflammatory reaction, with significantly increased levels of EGF, VEGF, rPH, and Ki-67 expression compared to the controls. The populations of GFP(+)-ASCs in circulating blood significantly increased after ASC injection compared to those of controls. Immunofluorescence staining showed GFP(+)-ASCs significantly accumulated in the subdermal layer of the wound margin and increased angiogenesis via vWF and VEGF expression after injection. IVIS analysis revealed ASCs could exist and home into the periwound area up to 8 weeks postimplantation. In conclusion, ASCs significantly enhanced diabetic wound healing, engrafted into the local wound tissue, and implanted into circulating blood. ASC treatment stimulated neoangiogenesis and increased tissue regeneration through paracrine and autocrine mechanisms.

D-cycloserine, sarcosine and D-serine diminish the expression of cocaine-induced conditioned place preference.

Reactivation of cocaine-associated memories plays a critical role in reinstating the cocaine-seeking behavior and causing relapse. Cocaine-induced conditioned place preference (CPP) was used as a behavioral paradigm indicative of cocaine-associated memory and repeated cocaine-free preference tests served as a behavioral procedure to retrieve such a memory in this study. Since D-cycloserine was reported to eradicate drug-associated memories, two other N-methyl-D-aspartate (NMDA) receptor agonists were assessed for their efficacy on facilitating the extinction of cocaine-induced CPP. Although D-cycloserine (30 mg/kg) abolished cocaine (10 mg/kg)-induced CPP, sarcosine (300 and 600 mg/kg) and D-serine (600 mg/kg) diminished the expression of such a cocaine memory. Sarcosine (600 mg/kg) and D-serine (600 mg/kg) did not affect the storage of this cocaine memory. It was of interest to note that D-cycloserine facilitated the extinction of cocaine-induced CPP in a fast and early-onset manner, while sarcosine and D-serine decreased cocaine-induced CPP expression in a delay-onset manner. D-cycloserine (30 mg/kg), D-serine (600 mg/kg) and sarcosine (600 mg/kg) did not affect the consolidation of cocaine (5 mg/kg)-induced CPP. Finally, sarcosine (at 600 mg/kg/day for 3 consecutive days) and D-serine (at 600 mg/kg/day for 3 consecutive days) did not produce observable aversive effect associated with their administration in a conditioned place aversion paradigm. Likewise, a similar dosing regimen of sarcosine or D-serine did not cause evident activity-impairing effect. In addition to D-cycloserine treatment, our results indicate that long-term treatment with D-serine and sarcosine may afford a therapeutic advance in suppressing the expression of cocaine-associated memory.

Neonatal isolation decreases cued fear conditioning and frontal cortical histone 3 lysine 9 methylation in adult female rats.

Early life stress is thought to enhance adult susceptibility to stress and stress-related mood disorders. In this study, fear-potentiated startle was used to model the acquisition of a traumatic event-related memory in female rats experiencing early life stress. Daily 1-hr maternal and sibling separation throughout day 2-9 postpartum (D2-9 PP) caused a decrease in the fear-potentiated startle, but not acoustic startle baseline, in adult female rats. The separation procedure did not affect corticosterone secretion but produced an increase in serum estradiol concentration. Moreover, the separation procedure did not affect histone 3 lysine 9 (H3K9) acetylation but decreased H3K9 mono- and tri-methylation in frontal cortices. Treatment with 5-aza-2'-deoxycytidine (AZA) (5mg/kg at alternative days from D2PP to D9PP or 10mg/kg at D5PP and D9PP), a DNA methylation inhibitor, did not affect the separation-decreased fear-potentiated startle. Treatment with valproic acid (VPA), a histone deacetylase inhibitor, at 3 dosing regimens (300mg/kg at D2-9PP; 100mg/kg at D2-4PP, 200mg/kg at D5-7PP, 300mg/kg at D8-9PP; 100mg/kg at D2-5PP, 200mg/kg at D6-9PP) prior to daily separation reversed such a decrease in fear-potentiated startle. The lowest effective VPA dosing regimen used (100mg/kg at D2-5PP, 200mg/kg at D6-9PP) reversed the separation-decreased H3K9 mono- and tri-methylation in frontal cortices. Eight-day VPA (300mg/kg/day) and AZA (5mg/kg/day) administrations starting at D28PP were ineffective in altering the separation-decreased fear-potentiated startle. We, hereby, suggest that decreased frontal cortical H3K9 mono- and tri-methylation may be involved in early life separation-decreased fear memory of adult rats.

Accumbal 14-3-3ζ protein downregulation is associated with cocaine-conditioned memory.

Cocaine-conditioned memory has been known to cause cocaine craving and relapse, while its underlying mechanisms remain unclear. We explored accumbal protein candidates responsible for a cocaine-conditioned memory, cocaine-induced conditioned place preference (CPP). Two-dimensional gel electrophoresis in conjunction with liquid chromatography mass spectrometry analysis was utilized to identify accumbal protein candidates involved in the retrieval of cocaine-induced CPP. Among the identified candidate proteins, a downregulated 14-3-3ζ protein was chosen and confirmed by Western immunoblotting. A polymer-mediated plasmid DNA delivery system was then used to overexpress 14-3-3 protein in mouse nucleus accumbens before the CPP retrieval tests. Overexpression of accumbal 14-3-3ζ protein was found to diminish conditioned cue/context-mediated cocaine-induced CPP. In contrast, another isoform of 14-3-3 protein, 14-3-3ε protein, did not affect conditioned cue/context-mediated cocaine-induced CPP. Overexpression of accumbal 14-3-3ζ protein did not produce motor activity-impairing effect or alter local dopamine metabolism. Moreover, overexpression of accumbal 14-3-3ζ protein did not affect food-induced CPP. These results, taken together, indicated that overexpressed accumbal 14-3-3ζ protein specifically decreased conditioned cue/context-mediated cocaine memory. Further understanding of the function of accumbal 14-3-3ζ protein may shed light on the treatment of cocaine craving and relapse.

Repeated co-administrations of alcohol- and methamphetamine-produced anxiogenic effect could be associated with the neurotoxicity in the dentate gyrus.

To date, joint use of alcohol (EtOH) and methamphetamine (MA) represents a specific combination of polydrug abuse. Repeated administrations of EtOH, MA, and combined EtOH and MA were assessed for their effects on brain cell toxicity, cell mitosis and anxiety/depression-like behavior. We found that repeated co-administrations of EtOH and MA produced profound anxiogenic effects. Specifically, combined EtOH and MA decreased open arm exploratory responses in the elevated plus maze test. Moreover, combined EtOH and MA significantly decreased immobile responses in the tail suspension test. MA, EtOH, and their combination all reduced the number of NeuN-positive cells in amygdala (Amg), while neither of them altered the number of NeuN-positive cells in striatum (St) or prefrontal cortex (PFC). Combined EtOH and MA decreased the number of NeuN-positive cells in dentate gyrus (DG). EtOH, MA, and combined EtOH and MA all diminished comparable number of GFAP-positive cells in Amg, DG, and St. Neither of these treatment affected the number of GFAP-positive cells in PFC. EtOH, MA, and combined EtOH and MA generated comparable inhibiting effects on cell proliferation in the subventricular zone (SVZ) and DG. These results, taken together, suggest that repeated co-administrations of MA and EtOH may produce an observable anxiogenic effect. This combination-produced anxiogenic effect could be associated with neuronal loss in the dentate gurus. In contrast, such an anxiogenic effect is less likely related to this combination-caused glial toxicity in limbic regions or cell proliferation-inhibiting effect in the SVZ or DG.

Presence of conspecifics and their odor-impregnated objects reverse stress-decreased neurogenesis in mouse dentate gyrus.

Stress and corticosterone level are thought to negatively associate with neurogenesis in mammalian brains. Social support can diminish many adverse effects of stress. The present study examined the modulating effect of social support on stress-decreased cell proliferation and neuronal differentiation in a mouse model. A randomly-scheduled foot shock followed by restraint in water was used as a profound stress-provoking regimen. Bromodeoxyuridine (BrdU) staining was used to indicate newly mitotic cells and doublecortin (DCx) staining was used to reveal immature neurons. This stress-provoking regimen rapidly decreased BrdU- and BrdU/DCx-labeled cells in the dentate gyrus. However, such a stress-provoking regimen did not affect the number of these labeled cells in the subventricular zone. Familiar and unfamiliar mice' company throughout the stress regimen completely reversed the stress-decreased cell proliferation and neuronal genesis in the dentate gyrus. Likewise, both odor-familiar (from their home cages) and -unfamiliar (from cages other than their home cages) wooden blocks completely reversed the stress-decreased BrdU/DCx-labeled cells in the dentate gyrus. In contrast, wooden blocks free of any odor and camphor odor alone failed to affect the stress-decreased BrdU- or BrdU/DCx-labeled cells. Finally, we showed that conspecifics or their odors during the stress regimen reversed the stress-decreased cell proliferation and neuronal differentiation in the dentate gyrus via a corticosterone-independent mechanism. We conclude that stress and familiarity distinctively affect neurogenesis in the dentate gyrus and subventricular zone. Conspecific companions or presence of their odors reverse stress-decreased neurogenesis in the dentate gyrus, suggesting that social support during stress exposure may improve neurogenesis-related psychological functions.