pad-wnt-3a  (Addgene inc)

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Neuroprotective and regenerative roles of intranasal Wnt-3a administration after focal ischemic stroke in mice

doi: 10.1177/0271678X17702669

Figure Lengend Snippet: Wnt-3a treatment was neuroprotective after stroke. (a) Representative Western blots of cell lysates following infection with IRES-mCherry control vector or IRES-Wnt-3a-mCherry vector with or without XAV-939 cotreatment. (b) Quantification of Western blot analyses for Wnt-3a and β-catenin. (c) and (d) Representative images of in vitro primary cortical neurons subjected to 16-h reperfusion following 3-h OGD treatment among different groups. The cell density is higher in (d) due to the Wnt-3a protective effect against OGD. Cell density before OGD was similar between the two groups (data not shown). (e) Quantification of cell viability via MTT assay (24 h after OGD) demonstrated increased neuronal cell viability with Wnt-3a overexpression. Viability data are presented as box and whisker plot (min to max). All data are represented as mean ± SD; *p < 0.05 compared to mCherry; #p < 0.05 compared to IRES-Wnt-3a. (f) Application of recombinant Wnt-3a into media resulted in greater neuronal cell viability following OGD and reperfusion, and inhibition of Wnt signaling with XAV-939 reversed the neuroprotective effects. All data are represented as mean ± SD; *p < 0.05 compared to OGD; #p < 0.05 compared to OGD + Wnt-3a. (g) and (i) Confirmation for in vivo dosages of intranasal Wnt-3a and XAV-939 administration at 3d after stroke. (g) Representative Western blots for peri-infarct tissue lysates. (h) Quantification of protein levels of Wnt-3a and β-catenin. N = 4 for saline and Wnt3 + XAV-939 groups, and 5 for Wnt-3a group. (i) and (j) Representative TTC staining images and quantification of infarct volume at 72 h after stroke. TTC data are represented as mean ± SD; *p < 0.05 compared to saline; #p < 0.05 compared to Wnt-3a. N = 12 for saline group, 16 for Wnt-3a group, and 16 for Wnt-3a + XAV-939 group.

Article Snippet: 24 For expression of Wnt-3a, the mouse DNA sequence of Wnt-3a was amplified from pAd-Wnt-3a and cloned into FUIMW through BamHI and EcoRI cutting sites. pAd-Wnt-3a was a gift from Tong-Chuan He (Addgene plasmid # 12518).

Techniques: Western Blot, Infection, Plasmid Preparation, In Vitro, MTT Assay, Over Expression, Whisker Assay, Recombinant, Inhibition, In Vivo, Staining

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Neuroprotective and regenerative roles of intranasal Wnt-3a administration after focal ischemic stroke in mice

doi: 10.1177/0271678X17702669

Figure Lengend Snippet: Wnt-3a enhanced angiogenesis in the peri-infarct and improved functional recovery after stroke. Angiogenesis was assessed 14 days after stroke in the peri-infarct region using immunohistochemical and LCBF measurements. (a) Representative immunofluorescence image for Col-IV (green), BrdU (red) and NeuN (blue). Right panel demonstrates a high magnification (100×) confocal image to confirm Col-IV+/BrdU+ colocalization. (b) Quantification for BrdU+/ColIV+ colabeled cells. All data represented as mean ± SD; *p < 0.05 compared to saline. N = 7 for each group. (c) Local cerebral blood flow (LCBF) was measured in ipsilateral hemispheres of vehicle (saline)-treated stroke control, Wnt-3a and Wnt-3a plus XAV-939 treated stroke mice using laser Doppler imaging. (d) Quantified data for LCBF measurement, represented as a percentage normalized to the baseline (before stroke). All data represented as mean ± SD; * p < 0.05 compared to saline (for the respective timepoint); #p < 0.05 compared to Wnt-3a group (for the respective timepoint). N = 6 for each group for each timepoint. (e) and (f) Mice receiving Wnt-3a treatment performed better on the adhesive-removal test following stroke, with a decrease in both latency to contact (e) and removal (f). The data represented as box and whisker plots (min to max). (g) In the corner test, mice receiving Wnt-3a treatment demonstrated a significant decrease in left/right turn ratio (closer to the value of 1) after stroke, suggesting restoration of normal turning behavior. The data are represented as scatterplots with mean ± SD; *p < 0.05 compared to sham; #p < 0.05 compared to saline. N = 15 for sham group, 12 for saline group, and 16 for Wnt-3a group.

Article Snippet: 24 For expression of Wnt-3a, the mouse DNA sequence of Wnt-3a was amplified from pAd-Wnt-3a and cloned into FUIMW through BamHI and EcoRI cutting sites. pAd-Wnt-3a was a gift from Tong-Chuan He (Addgene plasmid # 12518).

Techniques: Functional Assay, Immunohistochemical staining, Immunofluorescence, Imaging, Whisker Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Neuroprotective and regenerative roles of intranasal Wnt-3a administration after focal ischemic stroke in mice

doi: 10.1177/0271678X17702669

Figure Lengend Snippet: Wnt-3a supplementation restored Wnt signaling and neurotrophic expression following stroke. (a–d) Endogenous Wnt-3a and β-catenin levels were determined in wildtype animals following stroke by Western blot and densitometry analysis. (a) and (b) Representative Western blot analysis of tissue from the subventricular zone (SVZ, A) and the peri-infarct (b). (c) and (d) In the SVZ (d), β-catenin and Wnt-3a are significantly downregulated during the subacute phase after stroke. No significant differences were observed in the peri-infarct (c). *p < 0.05 compared to hour 0; **p < 0.01 compared to hour 0; ***p < 0.001 compared to hour 0. N = 5 for each group for each timepoint. (e–h). Animals were subjected to focal ischemic stroke and then were given intranasal injections of saline, Wnt-3a, or Wnt-3a + Dkk1. Then, Western blot and densitometry analysis were performed to detect the expression of Wnt-3a, β-catenin and BDNF in peri-infarct regions at 14 days after stroke. Wnt-3a supplementation increased levels of Wnt-3a, its intracellular signaling mediator β-catenin, and the neurotrophin BDNF. All data represented as mean ± SD; *p < 0.05 compared to saline; #p < 0.05 compared to Wnt-3a. N = 7 for saline group, seven for Wnt-3a group, and eight for Wnt-3a + Dkk1 group.

Article Snippet: 24 For expression of Wnt-3a, the mouse DNA sequence of Wnt-3a was amplified from pAd-Wnt-3a and cloned into FUIMW through BamHI and EcoRI cutting sites. pAd-Wnt-3a was a gift from Tong-Chuan He (Addgene plasmid # 12518).

Techniques: Expressing, Western Blot

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Neuroprotective and regenerative roles of intranasal Wnt-3a administration after focal ischemic stroke in mice

doi: 10.1177/0271678X17702669

Figure Lengend Snippet: The role of the Wnt signaling pathway in neurogenesis after stroke. (a–d) Immunohistochemical examinations of newly formed neurons in the peri-infarct region. (a) Left panel: the region of interest for immunofluorescence analysis following stroke is defined as the peri-infarct region surrounding the core region (as indicated by the asterisk). Right panel: cartoon of the coronal section depicting the peri-infarct region (green boxes) that was acquired and quantified (pink: ischemic core). (b) Representative immunofluorescence images for NeuN+ (green), BrdU+ (red), and NeuN+/BrdU+ co-labeled cells (white arrows) among different treatment groups. (c) High-magnification (60×) confocal three-dimensional image confirming colocalization of BrdU and NeuN fluorescence. (d) Quantification of neurogenesis by NeuN+/BrdU+ co-labeled cells in the peri-infarct following administration of either saline (negative control) or the Wnt-signaling inhibitor, XAV-939. All data represented as mean ± SD; *p < 0.05 compared to sham; #p < 0.05 compared to saline. N = 5 for sham group, 10 for saline group, and 4 for XAV-939 group. (e–i) Immunostaining inspection of neuroblasts migration from the SVZ to the ischemic cortex at 14 days after stroke. (e) Representative images of clusters of newly divided migratory neuroblasts (marked by co-labeling of BrdU, red, and DCX, green) emerging from the ipsilateral SVZ. (f) Higher magnification image to demonstrate colocalization of BrdU and DCX fluorescence. (g) Cartoon of the coronal section depicting the SVZ region of interest (green box) that was acquired and quantified. (h) High-magnification confocal three-dimensional image confirming colocalization of BrdU and DCX fluorescence. (i) Wnt-3a injection increased the number of migrating neuroblasts from the SVZ toward the peri-infarct, as determined by analysis of co-labeling of DCX and BrdU. Inhibition of the Wnt pathway with XAV-939 abolished the pro-regenerative effects. All data represented as mean ± SD; *p < 0.05 compared to saline; #p < 0.05 compared to Wnt-3a. N = 6 for saline group, 13 for Wnt-3a group, and 8 for Wnt-3a + XAV-939 group.

Article Snippet: 24 For expression of Wnt-3a, the mouse DNA sequence of Wnt-3a was amplified from pAd-Wnt-3a and cloned into FUIMW through BamHI and EcoRI cutting sites. pAd-Wnt-3a was a gift from Tong-Chuan He (Addgene plasmid # 12518).

Techniques: Immunohistochemical staining, Immunofluorescence, Labeling, Fluorescence, Negative Control, Immunostaining, Migration, Injection, Inhibition

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Neuroprotective and regenerative roles of intranasal Wnt-3a administration after focal ischemic stroke in mice

doi: 10.1177/0271678X17702669

Figure Lengend Snippet: Wnt-3a signaling enhanced neurogenesis in the peri-infarct following ischemic stroke. (a) Representative immunofluorescence images for NeuN+ (green), BrdU+ (red), and NeuN+/BrdU+ co-labeled cells (white arrows) among different treatment groups. (b) Left panel: cartoon of the coronal section depicting the peri-infarct region (green boxes) that were acquired and quantified. Right panel: high magnification (100×) confocal image to confirm colocalization of BrdU and NeuN. (c) Quantification of neurogenesis by NeuN+/BrdU+ co-labeled cells in the peri-infarct following stroke induction and administration of either saline (negative control), Wnt-3a, or Wnt-3a+XAV-939. All data represented as mean ± SD; *p < 0.05 compared to saline; #p < 0.05 compared to Wnt-3a. N = 10 for saline group and Wnt-3a groups, and 4 for Wnt-3a + XAV-939 group.

Article Snippet: 24 For expression of Wnt-3a, the mouse DNA sequence of Wnt-3a was amplified from pAd-Wnt-3a and cloned into FUIMW through BamHI and EcoRI cutting sites. pAd-Wnt-3a was a gift from Tong-Chuan He (Addgene plasmid # 12518).

Techniques: Immunofluorescence, Labeling, Negative Control

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Neuroprotective and regenerative roles of intranasal Wnt-3a administration after focal ischemic stroke in mice

doi: 10.1177/0271678X17702669

Figure Lengend Snippet: Wnt-3a activation promotes hippocampal neurogenesis in the SGZ after cortical stroke. (a) Representative immunofluorescence images for NeuN+ (green), BrdU+ (red), and GLUT1 (blue). (b) Quantification of neurogenesis by NeuN+/BrdU+ co-labeled cells and angiogenesis by GLUT1+/BrdU+ co-labeled cells in the SGZ following stroke induction and administration of either saline (negative control), Wnt-3a, or Wnt-3a+XAV-939. N = 7 for all groups. (c) 40x images demonstrating BrdU + non-colocalized cell (arrowhead), colocalization of NeuN and BrdU (dashed arrow), and colocalization of GLUT1 + and BrdU (solid arrow). (d) Top panel: cartoon of the coronal section depicting the SGZ region of interest (green box) that was acquired and quantified. Bottom panel: representative high magnification (60×) confocal image to confirm colocalization of BrdU and NeuN. All data represented as mean ± SD; *p < 0.05 compared to saline; #p < 0.05 compared to Wnt-3a + XAV-939. N = 5 for saline and Wnt-3a groups, and 4 for Wnt-3a + XAV-939 group.

Article Snippet: 24 For expression of Wnt-3a, the mouse DNA sequence of Wnt-3a was amplified from pAd-Wnt-3a and cloned into FUIMW through BamHI and EcoRI cutting sites. pAd-Wnt-3a was a gift from Tong-Chuan He (Addgene plasmid # 12518).

Techniques: Activation Assay, Immunofluorescence, Labeling, Negative Control