Journal: JCI Insight

Article Title: RAGE activation in macrophages and development of experimental diabetic polyneuropathy

doi: 10.1172/jci.insight.160555

Figure Lengend Snippet: ( A ) Kymographs of LysoTracker-labeled organelles in axons from dorsal root ganglia (DRG) neurons with 1.0 U/mL insulin. The horizontal and vertical arrows indicate retrograde direction and recording time (4 minutes), respectively. ( B – E ) The percentage of organelles in 100 μm axon segments that moved anterogradely ( B ), retrogradely ( C ), bidirectionally ( D ), or were stationary ( E ). n = 18–21 axons from 3 independent experiments. ( F ) The velocity of retrograde movements (RV) in 100 μm axon segments. The data consisted of 200–300 movements. ( G ) Kymographs in axons from DRG neurons treated with vehicle and insulin receptor antagonist (BMS-754807, 300 or 500 nmol/L). The stimulation time was 60 minutes. The horizontal and vertical arrows indicate retrograde direction and recording time (4 minutes), respectively. ( H – K ) The percentage of organelles in 100 μm axon segments that moved anterogradely ( H ), retrogradely ( I ), or bidirectionally ( J ), or were stationary ( K ). n = 18–21 axons from 3 independent experiments. ( L ) RV in 100 μm axon segments in each treatment condition. ( M ) Kymographs in axons from DRG neurons treated with vehicle, TNF-α, and TNF-α + JNK inhibitor (SP600125). The stimulation time was 20 minutes. The vertical arrow indicates recording time (4 minutes). ( N – Q ) The percentage of organelles in 100 μm axon segments that moved anterogradely ( N ), retrogradely ( O ), bidirectionally ( P ), or were stationary ( Q ). n = 18–21 axons from 3 independent experiments. ( R ) RV in 100 μm axon segments. The data consisted of 200–300 movements. The data are presented as the mean ± SD. Because the experiments of G – L and M – R were performed contemporaneously, statistical analysis was done using same vehicle control. Statistical analysis was performed by Student’s 2-tailed unpaired t test for B – F and by 1-way ANOVA with Tukey’s multiple-comparison test for H – L and N – R . ** P < 0.01, *** P < 0.001.

Article Snippet: In the experiments with an insulin/IGF-1 receptor antagonist shown in , or with TNF-α with or without a JNK inhibitor shown in , neurons were maintained in DMEM/F12 with 2% B27 and were pretreated with PBS containing 0.1% BSA (vehicle), 300 nmol/L, or 500 nmol/L BMS-754807 (MedChemExpress); 20 ng/mL TNF-α (410-MT, R&D Systems); or 20 ng/mL TNF-α + 50 nmol/L JNK inhibitor (SP600125) (1496, TOCRIS) as indicated in the text.

Techniques: Labeling, Control, Comparison

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Tissue-type plasminogen activator has a neuroprotective effect in the ischemic brain mediated by neuronal TNF- α

doi: 10.1038/jcbfm.2011.106

Figure Lengend Snippet: Tumor necrosis factor (TNF)-α mediates the neuroprotective effect of tissue-type plasminogen activator (tPA). (A) TNF-α mRNA expression in wild-type (Wt) neurons incubated with active tPA or inactive tPA (itPA) 1 to 10 nmol/L; n=12 for each observation. Data represent mean fold increase in TNF-α mRNA compared with control cells±s.d. *P<0.01 versus neurons treated with tPA 1 nmol/L. **P<0.01 versus neurons treated with tPA 5 nmol/L. (B) TNF-α concentration (pg/ml) in the culture media of Wt neurons incubated with tPA 5 nmol/L or plasmin (Pl) 10 nmol/L either alone or in combination with MK-801 10 μmol/L; n=12 for each observation. *P<0.01 versus neurons either left untreated or cotreated with tPA and MK-801. **P<0.01 versus neurons either left untreated or cotreated with Pl and MK-801. (C) Mean TNF-α concentration (pg/ml) in the media of Wt and tPA−/− neurons exposed to 5 minutes of oxygen–glucose deprivation (OGD) conditions (hypoxic preconditioning) alone or in the presence of either α2-antiplasmin 100 nmol/L (AP) or aprotinin 1.4 μmol/L (Apro) or tPA 5 nmol/L or Pl 10 nmol/L or MK-801 10 μmol/L; n=12 for each observation. Lines denote s.d. *P<0.01 versus neurons treated with either AP or Apro or MK-801. ** and ^P<0.01 versus untreated tPA−/− neurons. (D) Mean cell survival in Wt and TNF-α−/− neurons incubated with tPA 1 to 10 nmol/L, or with a combination of tPA 1 to 10 nmol/L and either anti-TNF-α-blocking antibodies 0.4 μg/ml (a-TNF-α) or an isotype immunoglobulin G (IgG) control (dark gray bars), or with Pl alone 5 nmol/L, followed 5 minutes later by exposure to lethal OGD conditions; n=12 for each observation. Lines denote s.d. *P<0.01 versus neurons cotreated with tPA 1 nmol/L and a-TNF-α antibodies. **P<0.01 versus neurons cotreated with tPA 5 nmol/L and a-TNF-α antibodies. ***P<0.01 versus neurons cotreated with tPA 10 nmol/L and a-TNF-α antibodies. Prec. indicates preconditioning and denotes the moment when cells where treated. (E) Mean cell survival in Wt neurons exposed to 5 minutes of OGD (hypoxic preconditioning, HP) alone or in the presence of either anti-TNF-α-neutralizing antibodies or an IgG isotype control, followed 5 minutes later by exposure to lethal OGD conditions; n=12 for each observation. *P<0.01 versus neurons treated with a-TNF-α antibodies during the preconditioning phase. Lines denote mean±s.d. (F) Mean cell survival in tPA−/− and TNF-α−/− neurons exposed to 5 minutes of OGD conditions (HP) either alone or in combination with tPA 5 nmol/L or TNF-α 20 ng/ml, followed 5 minutes later by exposure to 55 minutes OGD; n=10 for each observation. *P<0.01 versus preconditioned and non-preconditioned tPA−/− neurons. **P<0.01 versus non-preconditioned and preconditioned TNF-α−/− neurons and versus TNF-α−/− neurons treated with tPA during the preconditioning phase. Lines denote mean±s.d. (G) Mean percentage decrease in the volume of the ischemic lesion in Wt and TNF-α−/− mice exposed to ischemic preconditioning and transient occlusion of the middle cerebral artery (tMCAO) 1 hour later; n=12; P<0.01 versus non-preconditioned Wt mice and preconditioned and non-preconditioned TNF-α−/− mice. Ns, nonsignificant

Article Snippet: Other reagents were human recombinant tPA (Genentech, San Francisco, CA, USA), aprotinin (1.4 μ mol/L; Calbiochem, Gibbstown, NJ, USA), kainic acid (KA; 250 μ mol/L) and the NMDAR antagonist MK-801 (10 μmol/L; Tocris Bioscience, Ellisville, MO, USA), murine anti-TNF- α neutralizing antibodies (40 ng/mL) and recombinant TNF- α (1.2 nmol/L; R&D Systems; Minneapolis, MN, USA), 4′,6-diamidino-2-phenylindole and triphenyltetrazolium chloride (Sigma-Aldrich, St Louis, MO, USA), and p21 short hairpin RNA (shRNA) and shRNA lentiviral particles (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Incubation, Control, Concentration Assay, Blocking Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Tissue-type plasminogen activator has a neuroprotective effect in the ischemic brain mediated by neuronal TNF- α

doi: 10.1038/jcbfm.2011.106

Figure Lengend Snippet: p21 mediates the neuroprotective effect of tissue-type plasminogen activator (tPA). (A) Mean fold increase in p21 mRNA expression in wild-type (Wt) and tumor necrosis factor-α deficient (TNF-α−/−) neurons incubated with either active tPA 5 nmol/L or inactive tPA (itPA) 5 nmol/L or plasmin 10 nmol/L or TNF-α 20 ng/ml. Values represent mean±s.d; n=8 per group. *P<0.01 compared with p21 mRNA expression in neurons treated with itPA. (B) Mean fold increase in p21 mRNA expression in wild-type (Wt) and tPA-deficient (tPA−/−) neurons exposed to 5 minutes of oxygen–glucose deprivation (OGD) conditions. A subgroup of Wt neurons was incubated with anti-TNF-α antibodies 40 ng/ml. *P<0.01 versus Wt neurons incubated with anti-TNF-α antibodies and tPA−/− neurons; n=8 per group. Values represent mean±s.d. (C) Mean cell survival in Wt neurons infected with a lentiviral vector encoding a scrambled short hairpin RNA (shRNA) sequence that does not lead to the specific degradation of any known cellular mRNA or with a silencing p21 shRNA and treated with tPA 5 nmol/L followed 5 minutes later by exposure to 55 minutes of OGD conditions (lethal hypoxia). *P<0.01 versus non-preconditioned neurons; n=10 observations. Values represent mean±s.d. (D) Mean cell survival in Wt neurons infected with a lentiviral vector encoding a scrambled shRNA sequence or a silencing p21 shRNA and exposed to OGD conditions for 5 minutes (hypoxic preconditioning, HP), followed 5 minutes later by exposure to 55 minutes of OGD conditions (lethal hypoxia). *P<0.01 versus non-preconditioned neurons; n=12 observations. Values represent mean±s.d. Ns, nonsignificant

Article Snippet: Other reagents were human recombinant tPA (Genentech, San Francisco, CA, USA), aprotinin (1.4 μ mol/L; Calbiochem, Gibbstown, NJ, USA), kainic acid (KA; 250 μ mol/L) and the NMDAR antagonist MK-801 (10 μmol/L; Tocris Bioscience, Ellisville, MO, USA), murine anti-TNF- α neutralizing antibodies (40 ng/mL) and recombinant TNF- α (1.2 nmol/L; R&D Systems; Minneapolis, MN, USA), 4′,6-diamidino-2-phenylindole and triphenyltetrazolium chloride (Sigma-Aldrich, St Louis, MO, USA), and p21 short hairpin RNA (shRNA) and shRNA lentiviral particles (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Incubation, Infection, Plasmid Preparation, shRNA, Sequencing