Journal: Journal of Neuroinflammation

Article Title: Neuroprotective effect of astrocyte-derived IL-33 in neonatal hypoxic-ischemic brain injury

doi: 10.1186/s12974-020-01932-z

Figure Lengend Snippet: Astrocytic IL-33 is robustly upregulated after hypoxic-ischemia in neonatal mouse brains. a Western blot of IL-33 was performed using protein extracted from the mouse brain at 1, 3, 7, and 14 days after HI or sham operation. Data are mean ± SEM ( n = 6 in each group). # P < 0.01 compared to sham. b Representative images of IL-33 (green) and GFAP or Olig2 (red) labeling in the cerebral cortex of WT mice at 1 day after HI injury. No IL-33 staining was observed in Neun or Iba1 cells 1 d after HI. Scale bar, 25 μm. Nuclei were stained blue with 4,6-diamidino-2-phenylindole (DAPI). c Semi-quantitative analysis of glia or neuron-type cell contributions to the IL-33-positive cell population in vehicle group 1 day after HI. Data are mean ± SEM ( n = 6 in each group). * P < 0.05 compared to sham. d Representative IL-33 expression on astrocytes in WT mice at 1 day after HI injury. The numbers shown indicate the percentage of IL-33 expression on the indicated cell population. Data are mean ± SEM ( n = 6 in each group). * P < 0.05 compared to sham

Article Snippet: The sections were stained overnight at 4 °C with either anti-ST2 (1:200, ab25877, Abcam), anti-IL-33 (1:500, AF3626, R&D), anti-GFAP (1:500, 104805-T08, Sino Biological Inc.), anti-Olig2 (1:500, EPR2673, Abcam), anti-Iba1(1:50, 20A12.1, Sigma-Aldrich), anti-Neun (1:50, A60, Sigma-Aldrich), or anti-Ki67 (1:500, ab15580, Abcam) followed by PE- or FITC-conjugated secondary antibodies (eBioscience).

Techniques: Western Blot, Labeling, Staining, Expressing

Journal: Journal of Neuroinflammation

Article Title: Neuroprotective effect of astrocyte-derived IL-33 in neonatal hypoxic-ischemic brain injury

doi: 10.1186/s12974-020-01932-z

Figure Lengend Snippet: ST2 expression on astrocytes is elevated after HI. a Histograms showing ST2 expression on astrocytes, oligodendrocytes, microglia, and neurons in the brain 3 days after HI or sham operation. The blue and red lines represent staining in sham and HI brains, respectively, at 3 days after surgery. The gray area represents isotype control staining. b–e Percentages of ST2 expression on astrocytes ( b ), oligodendrocytes ( c ), microglia ( d ), and neurons ( e ) in sham brain and HI brain at 3 days after surgery. Data are mean ± SEM ( n = 5–7 per group), * P < 0.05 compared to sham. f Representative images of GFAP (green) and ST2 (red) labeling in the cerebral cortex 3 days after HI. Scale bar, 25 μm. Nuclei were stained blue with DAPI

Article Snippet: The sections were stained overnight at 4 °C with either anti-ST2 (1:200, ab25877, Abcam), anti-IL-33 (1:500, AF3626, R&D), anti-GFAP (1:500, 104805-T08, Sino Biological Inc.), anti-Olig2 (1:500, EPR2673, Abcam), anti-Iba1(1:50, 20A12.1, Sigma-Aldrich), anti-Neun (1:50, A60, Sigma-Aldrich), or anti-Ki67 (1:500, ab15580, Abcam) followed by PE- or FITC-conjugated secondary antibodies (eBioscience).

Techniques: Expressing, Staining, Labeling

Journal: Journal of Neuroinflammation

Article Title: Neuroprotective effect of astrocyte-derived IL-33 in neonatal hypoxic-ischemic brain injury

doi: 10.1186/s12974-020-01932-z

Figure Lengend Snippet: IL-33 promotes astrocyte survival and proliferation after HI injury. a Representative FACS analysis of ST2 + astrocytes at 24 h after 6 h OGD. Right, numbers indicate the percentage of ST2 + astrocytes at 24 h after 6 h OGD. Data are mean ± SEM ( n = 3 in each group). * P < 0.05 compared to untreated controls. b CCK-8 assay in astrocyte-enriched cultures subjected to 6 h OGD or sham conditions followed by treatment with PBS or a range of concentrations of IL-33 for another 24 h. Data are mean ± SEM ( n = 3 in each group). # P < 0.01 compared to untreated controls. c Representative images of Ki-67 (red) and GFAP (green) in the cerebral cortex of IL-33- and PBS-treated mice at 7 days post HI (left). Scale bar, 25 μm. Right, quantification of Ki-67 and GFAP dual-labeled cells at 7 days post HI ( n = 9 per group). d , e Percentages of phosphorylated AKT (pAKT) ( d ) and Ki-67 + ( e ) in astrocytes at 24 h after 6 h OGD and the culture conditions shown. Data are mean ± SEM ( n = 3 in each group). * P < 0.05, # P < 0.01. f Representative FACS analysis of cell-cycle status of astrocytes at 24 h after 6 h OGD and the culture conditions shown. Data are mean ± SEM ( n = 3 in each group). * P < 0.05

Article Snippet: The sections were stained overnight at 4 °C with either anti-ST2 (1:200, ab25877, Abcam), anti-IL-33 (1:500, AF3626, R&D), anti-GFAP (1:500, 104805-T08, Sino Biological Inc.), anti-Olig2 (1:500, EPR2673, Abcam), anti-Iba1(1:50, 20A12.1, Sigma-Aldrich), anti-Neun (1:50, A60, Sigma-Aldrich), or anti-Ki67 (1:500, ab15580, Abcam) followed by PE- or FITC-conjugated secondary antibodies (eBioscience).

Techniques: CCK-8 Assay, Labeling

Journal: Genetics and Molecular Research

Article Title: Olfactory mucosal microstructural changes in a rat model of acute rhinosinusitis with dysosmia

doi: 10.4238/2014.may.16.11

Figure Lengend Snippet: Figure 4. p75NTR and glial fibrillary acidic protein (GFAP) double-labeling immunofluorescence staining. A. Control. The lamina propria of the olfactory mucosa, close to the basement membrane, exhibited a large number of yellow fluorescence-lined fusiform or oblate olfactory ensheathing cells (OEC) (thick arrow), and spindle cells were the majority. B. One week post-inoculation. The olfactory epithelium was thinning, and the OECs were reduced significantly (thick arrow) in the lamina propria. In the olfactory epithelium (thin arrow), OECs grew occasionally. C. Two weeks post-inoculation. The olfactory epithelium was significantly thinner. OECs were increased more than the previous week (thick arrow) in the lamina propria. In the olfactory epithelium, there was a small amount of OEC (thin arrow). D. Three weeks post-inoculation. The thickness of the olfactory epithelium was increased. A part of the increased OEC formed a colony (thick arrow) in the lamina propria. There was a small amount of OECs (thin arrow) in the olfactory epithelium. E. Four weeks post-inoculation. The olfactory epithelium was generally recovered to normal. OECs were almost restored (thick arrow) in the lamina propria. In the olfactory epithelium, OECs grew occasionally (thin arrow). F. p75NTR immunofluorescence labeling protocol. G. GFAP immunofluorescence labeling protocol (scale bar = 40 µm).

Article Snippet: Rabbit anti-rat GFAP (OriGene Technologies, Inc.) (1:200) was added to the sections at room temperature.

Techniques: Labeling, Immunofluorescence, Staining, Control, Membrane, Fluorescence

Journal: Nature medicine

Article Title: Spinal cord reconstitution with homologous neural grafts enables robust corticospinal regeneration

doi: 10.1038/nm.4066

Figure Lengend Snippet: ( a ) Low magnification sagittal overview of CST axons (red) and a rat E14 spinal cord NPC graft (green) placed at the focal CST lesion site six weeks after injury. Scale bar, 200 μm. Dashed lines indicate host/graft interface throughout. Rostral is to the left throughout. ( b ) High magnification views of boxed area in a . Scale bar, 200 μm. ( c ) High magnification view of boxed area in b including rostral host/graft interface. Scale bar, 100 μm. ( d ) High magnification view of boxed area in b . Arrowheads indicate CST axons extending beyond the caudal host/graft interface. Scale bar, 50 μm. ( e – f ) High magnification views of the boxed areas in b . Arrowheads indicate CST axons penetrating host gray matter ( f , NeuN-positive) but not white matter ( g , NeuN-negative) caudal to the graft/lesion site. Scale bars, e , 50 μm; f , 100 μm. ( g ) High magnification view of the area just ventral to the graft/lesion site. Arrowheads indicate spared ventral CST axons located close to the NPC graft. Scale bar, 50 μm. ( h ) Quantification of the proportion of regenerating CST axons found at different locations across the rostro-caudal axis of the NPC graft ( n = 4 rats). ( j ) Example images of GFAP immunoreactivity in the vicinity of the host/graft interface of focal CST lesioned rats after receiving (top images) or not receiving (lower left) NPC graft. Scale bars, 100 μm. Quantification of GFAP immunoreactivity at the lesion boundary ( n = 4 rats lesion alone, n = 4 rats NPC graft). Throughout, error bars represent mean ± s.e.m. Circles indicate data from individual rats. *P<0.05; Wilcoxon test.

Article Snippet: Sections were incubated with primary antibodies against GFP (rabbit from Invitrogen at 1:1000 or chicken from Abcam at 1:1000); GFAP (mouse from Chemicon at 1:1000 or rabbit from Dako at 1:750 to label astrocytes); human GFAP (rabbit from Origene at 1:500 to label human specific astrocytes); NeuN (mouse from Chemicon at 1:500 to label mature neurons); ChAT (goat from Chemicon at 1:250 to label spinal cord motor neurons); MAP2 (mouse from Chemicon at 1:2,000 to label dendrites); vesicular glutamate transporters 1 (vGlut1, mouse from Chemicon at 1:1,000 to label glutamatergic terminals); synaptophysin (mouse from Chemicon at 1:1,000 to label presynaptic terminals); adenomatous polyposis coli (APC, mouse from Oncogene at 1:400 to label oligodendrocytes); NG2 (rabbit from Millipore at 1:400 to label oligodendrocyte precursors); Hu (human at 1:1000 to label neurons, generous gift from Robert Darnell, The Rockefeller University, NY); doublecortin (DCX, goat from Santa Cruz at 1: 250 to label immature neurons); nestin (mouse from BD at 1:200 to label neural progenitor cells); neurofilament (NF200; mouse from Millipore at 1:250 to label axons); 27C7 (mouse at 1:200 to label Schwann cells, generous gift from K. Wewetzer, University of Freiburg, Freiburg, Germanyat); RFP (mouse from Abcam at 1:200); human nuclei (mouse from Millipore at 1:200); PAX6 (rabbit from Covance at 1:1000); Pax6 (mouse from DHSB at 1:5000); Sox1 (Goat from R&B at 1:1000); HoxB4 (Rat from DHSB at 1:50); FoxG1 (rabbit from Abcam at 1:100); OTX2 (goat from R and D at 1:2000); EN1 (mouse from DHSB at 1:800); Camk2 (rabbit from Genetex at 1:200), and Dyelight 405 or 594-conjugated streptavidin (from Invitrogen at 1:250 to label BDA traced CST axons).

Techniques:

Journal: Nature medicine

Article Title: Spinal cord reconstitution with homologous neural grafts enables robust corticospinal regeneration

doi: 10.1038/nm.4066

Figure Lengend Snippet: ( a ) Sagittal views of CST axons (red) and GFP-expressing NPC grafts (green) placed in C4 CST lesions in rats six weeks after injury. Dashed lines indicate rostral host/graft border. Rostral is to the left throughout. Lower panels are high magnification views of boxed areas in upper right panel. Scale bars, 200 μm (upper panels); 50 μm (lower panels). ( b ) Sagittal views of the caudal graft/host interface (dashed lines) in a C4 CST lesioned rat depicting CST axons (red) and GFP-expressing NPC grafts (green). Scale bars, 50 μm. ( c ) Quantification of the proportion of regenerating CST axons (normalized to the total number of CST axons 0.5mm rostral to the graft/lesion site) found at different locations across the rostro-caudal axis of the NPC graft ( n = 9 rats). Circles indicate data from individual rats. (d) Double immunolabeling of GFP-labeled NPC grafts (green) with neuronal (NeuN, red), astrocytic (GFAP, red) and mature oligodendrocytic (adenomatous polyposis coli, APC, red) cell type markers. Boxed region in the low magnification image on the left is shown at higher magnification immediately to the right. Scale bars, 500 μm (left); 20 μm (all panels to the right). ( e ) Quantification of cell type marker immunoreactivity in grafts. ( f ) Experimental paradigm to record optogenetically-evoked synaptic responses. AAV2 vectors expressing ChR2 and GFP were injected into motor cortices, followed by NPCs grafts into lesion cavities two weeks later. Four weeks after NPC grafts, ChR2+ CST axons were stimulated with blue light ( n = 3 rats) and whole-cell recordings of grafted neurons were performed. ( g ) Double immunolabeling for CST axons (green) and neurons (NeuN, blue). Scale bar, 20 μm. ( h ) EPSCs recorded from a grafted neuron evoked with 5 ms of 470 nm light (blue line). Red trace indicates the average of individual EPSCs (gray traces). Averaged peak EPSC amplitude from three responding cells is plotted to the right ( i ) Experimental paradigm to record electrically-evoked synaptic responses. During whole-cell recordings of grafted neurons, CST axons were stimulated by bipolar electrodes positioned 1mm rostral to graft. ( j ) Example of an evoked EPSC (arrow, left) recorded from a grafted neuron. Application of the AMPAR antagonist DNQX (20μM) abolished responses (arrow, right), indicative of a glutamatergic synapse. Averaged peak EPSC amplitude from seven responding cells is plotted to the right. Error bars represent mean ± s.e.m throughout.

Article Snippet: Sections were incubated with primary antibodies against GFP (rabbit from Invitrogen at 1:1000 or chicken from Abcam at 1:1000); GFAP (mouse from Chemicon at 1:1000 or rabbit from Dako at 1:750 to label astrocytes); human GFAP (rabbit from Origene at 1:500 to label human specific astrocytes); NeuN (mouse from Chemicon at 1:500 to label mature neurons); ChAT (goat from Chemicon at 1:250 to label spinal cord motor neurons); MAP2 (mouse from Chemicon at 1:2,000 to label dendrites); vesicular glutamate transporters 1 (vGlut1, mouse from Chemicon at 1:1,000 to label glutamatergic terminals); synaptophysin (mouse from Chemicon at 1:1,000 to label presynaptic terminals); adenomatous polyposis coli (APC, mouse from Oncogene at 1:400 to label oligodendrocytes); NG2 (rabbit from Millipore at 1:400 to label oligodendrocyte precursors); Hu (human at 1:1000 to label neurons, generous gift from Robert Darnell, The Rockefeller University, NY); doublecortin (DCX, goat from Santa Cruz at 1: 250 to label immature neurons); nestin (mouse from BD at 1:200 to label neural progenitor cells); neurofilament (NF200; mouse from Millipore at 1:250 to label axons); 27C7 (mouse at 1:200 to label Schwann cells, generous gift from K. Wewetzer, University of Freiburg, Freiburg, Germanyat); RFP (mouse from Abcam at 1:200); human nuclei (mouse from Millipore at 1:200); PAX6 (rabbit from Covance at 1:1000); Pax6 (mouse from DHSB at 1:5000); Sox1 (Goat from R&B at 1:1000); HoxB4 (Rat from DHSB at 1:50); FoxG1 (rabbit from Abcam at 1:100); OTX2 (goat from R and D at 1:2000); EN1 (mouse from DHSB at 1:800); Camk2 (rabbit from Genetex at 1:200), and Dyelight 405 or 594-conjugated streptavidin (from Invitrogen at 1:250 to label BDA traced CST axons).

Techniques: Expressing, Immunolabeling, Labeling, Marker, Injection