Journal: Gene therapy

Article Title: AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.

doi: 10.1038/sj.gt.3302791

Figure Lengend Snippet: Figure 1 Retinal wholemounts and sections from non-injured rats that received intravitreal AAV vector injections. (a and b) AAV-GFP injected rats. These rats also received multiple fluorogold (FG) injections into contralateral superior colliculus 5 weeks after the AAV-GFP eye injection. (a) Close correspondence between bIII-tubulin immunostaining (red) and retrograde FG label (gold) in RGCs. (b) Retinal wholemount immunostained for both GFP (green) and bIII-tubulin (red). Examples of retinotectally projecting ganglion cells triple labeled with FG, GFP and bIII-tubulin are shown by the arrowheads; occasional GFP+ cells in the ganglion cell layer were not labelled with FG or bIII- tubulin (arrows) and were presumably displaced amacrine cells. Note in this particular retinal region not all RGCs were FG+. (c–e) Transduced RGCs in retinas 11 weeks after intravitreal AAV-CNTF-GFP injection. (c) Wholemount stained with GFP (green) and bIII-tubulin (red). (d) Section immunostained with CNTF antibody (immunoreactive RGCs are arrowed). (e) Section immunostained for both CNTF (red) and GFP (green). Examples of (f) BDNF and (g) GAP-43 immunoreactivity in RGCs in retinas from AAV-BDNF-GFP and AAV-GAP43-GFP injected eyes respectively. Scale bars: a and c ¼ 25 mm (bar shown on a) b ¼ 25 mm; e–g ¼ 20 mm (bar shown on g).

Article Snippet: Series of retinal sections were immunostained using monoclonal antibodies to bIIItubulin (1:1000, TUJ-1 Covance), rat GAP43 (1:2000, Chemicon) and/or polyclonal antibodies to GFP (1:300, Chemicon), CNTF (recognizes mouse and rat, 1:100, R&D Systems, MN, USA) or BDNF (recognizes rat and human, 1:300, Chemicon).

Techniques: Plasmid Preparation, Injection, Eye Injection, Immunostaining, Labeling, Staining

Journal: Gene therapy

Article Title: AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.

doi: 10.1038/sj.gt.3302791

Figure Lengend Snippet: Figure 2 Retinal tissue from (a and e) AAV-CNTF-GFP, (b, c and f) AAV-BDNF-GFP, (d) AAV-GFP, or (g) AAV-GAP43-GFP treated animals, 7 weeks following optic nerve crush. To identify surviving and transduced RGCs, retinal wholemounts (a and b) and sections (c) were immunostained for both bIII-tubulin (red) and GFP (green). (d–g) fields from representative bIII-tubulin stained wholemounts after different AAV vector injections. These fields were taken from similar retinal eccentricities, about 1–1.5 mm from the optic disk. For each AAV vector, the mean numbers (7s.e.m.) of all surviving (bIII-tubulin+) RGCs and viable bIII-tubulin+ RGCs that were also GFP+ are shown in h. Abbreviations: , ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL). Scale bars: a–c ¼ 50 mm (bar shown on c); d–g ¼ 100 mm (bar shown on g).

Article Snippet: Series of retinal sections were immunostained using monoclonal antibodies to bIIItubulin (1:1000, TUJ-1 Covance), rat GAP43 (1:2000, Chemicon) and/or polyclonal antibodies to GFP (1:300, Chemicon), CNTF (recognizes mouse and rat, 1:100, R&D Systems, MN, USA) or BDNF (recognizes rat and human, 1:300, Chemicon).

Techniques: Staining, Plasmid Preparation

Journal: Gene therapy

Article Title: AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.

doi: 10.1038/sj.gt.3302791

Figure Lengend Snippet: Figure 3 Longitudinal cryosections of optic nerves 7 weeks after optic nerve crush and 8 weeks after intravitreal injection of AAV-GFP (a–c), AAV-BDNF-GFP (d–f) or AAV-CNTF-GFP (g–i). Fields in column one (a, d, g) are shown at higher magnification in column two (b, e, h). Columns two and three are the same fields viewed through different filter blocks. Each section was immunoreacted with bIII-tubulin and GFP antibodies to identify RGC axons proximal to the crush site. Arrowheads in a, d and g show the location of the crush. The bIII-tubulin+ axons arrowed in b, e, h are also visible in c, f, and i, indicating that these particular GFP+ axons originated from AAV-transduced RGCs. Scale bars: a, d, g ¼ 100 mm; b, c, e, f, h, i ¼ 50 mm (bar shown on b and c).

Article Snippet: Series of retinal sections were immunostained using monoclonal antibodies to bIIItubulin (1:1000, TUJ-1 Covance), rat GAP43 (1:2000, Chemicon) and/or polyclonal antibodies to GFP (1:300, Chemicon), CNTF (recognizes mouse and rat, 1:100, R&D Systems, MN, USA) or BDNF (recognizes rat and human, 1:300, Chemicon).

Techniques: Injection

Journal: Gene therapy

Article Title: AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.

doi: 10.1038/sj.gt.3302791

Figure Lengend Snippet: Figure 4 Longitudinal optic nerve sections from an AAV-CNTF-GFP injected rat, immunostained for bIII-tubulin (a–c) or GAP43 (d–e). (a) Many bIII-tubulin+ RGC axons traverse the crush site (arrowheads) and regenerate through the white matter of the distal optic nerve. (b) An adjacent section to A showing regrown bIII-tubulin+ axons at higher magnification. (c) Small fascicle of bIII-tubulin+ axons (arrowhead) with abnormal axonal branching (arrows) towards the middle of the optic nerve, 6.5 mm distal to the crush. (d) GAP43+ axons distal to the crush site. (e) GAP43+ axons at the optic chiasm. Outlined area in e is shown under higher magnification as an insert within e; a few regenerate RGC axons (arrows) are visible. LON, left (crushed) optic nerve; RON, right (uninjured) optic nerve. Scale bars: a ¼ 200 mm; b, d ¼ 100 mm (bar shown on b); c ¼ 50 mm; e ¼ 100 mm.

Article Snippet: Series of retinal sections were immunostained using monoclonal antibodies to bIIItubulin (1:1000, TUJ-1 Covance), rat GAP43 (1:2000, Chemicon) and/or polyclonal antibodies to GFP (1:300, Chemicon), CNTF (recognizes mouse and rat, 1:100, R&D Systems, MN, USA) or BDNF (recognizes rat and human, 1:300, Chemicon).

Techniques: Injection

Journal: Gene therapy

Article Title: AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.

doi: 10.1038/sj.gt.3302791

Figure Lengend Snippet: Figure 5 (a and b) Retinal wholemounts from PN grafted rats, immunostained for bIII-tubulin. There were fewer surviving RGCs in AAV- GFP (a) Compared to AAV-CNTF-GFP (b) injected eyes. (c) Retina from AAV-CNTF-GFP eye. RGCs with regenerating axons were retrogradely labeled after fluorogold (FG) injection into the PN graft. Note the variation in soma size of FG+ RGCs. (d) GFP and FG label in retinal wholemount from another PN grafted animal. The three AAV-CNTF-GFP transduced RGCs (arrows) are not retrogradely labeled with FG. (e) Longitudinal section of PN graft from a rat that received an intravitreal injection of AAV-CNTF-GFP. Many bIII-tubulin+ axons can be seen (Cy3 – red), a few of which are co-stained for GFP (FITC, axons appear yellow). Scale bars: a, b ¼ 100 mm; c, e ¼ 50 mm; d ¼ 25 mm.

Article Snippet: Series of retinal sections were immunostained using monoclonal antibodies to bIIItubulin (1:1000, TUJ-1 Covance), rat GAP43 (1:2000, Chemicon) and/or polyclonal antibodies to GFP (1:300, Chemicon), CNTF (recognizes mouse and rat, 1:100, R&D Systems, MN, USA) or BDNF (recognizes rat and human, 1:300, Chemicon).

Techniques: Injection, Labeling, Staining

Journal: Gene therapy

Article Title: AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.

doi: 10.1038/sj.gt.3302791

Figure Lengend Snippet: Figure 7 Linear maps of the AAV vector plasmids (based on pTRUF12.1) used in these studies. With the exception of the inverted terminal repeats (ITR) all viral genes have been removed and replaced with (a) GFP, or (b) CNTF (with NGF secretory sequence, NGFss-CNTF), BDNF or GAP43 (all represented by X) and GFP, all under the control of a cyglomegalovirus/chicken b-actin hybrid promoter. Abbreviation: CMV (cyglomegalovirus promoter); IRES (internal ribosomal entry site) from poliovirus; SV40polyA (SV40 virus polyadenylation signal).

Article Snippet: Series of retinal sections were immunostained using monoclonal antibodies to bIIItubulin (1:1000, TUJ-1 Covance), rat GAP43 (1:2000, Chemicon) and/or polyclonal antibodies to GFP (1:300, Chemicon), CNTF (recognizes mouse and rat, 1:100, R&D Systems, MN, USA) or BDNF (recognizes rat and human, 1:300, Chemicon).

Techniques: Plasmid Preparation, Sequencing, Control, Virus

Journal: British journal of pharmacology

Article Title: Neurotrophic interactions between neurons and astrocytes following AAV1-Rheb(S16H) transduction in the hippocampus in vivo.

doi: 10.1111/bph.14882

Figure Lengend Snippet: FIGURE 4 Elevation of CNTF production in hippocampal astrocytes via activation of the BDNF/TrkB/mTORC1 signalling pathway. (a) Immunohistochemical staining showing increased CNTF expression in glia‐like cells of the hippocampal CA1 region of CON, AAV1‐GFP‐ injected, and AAV1‐Rheb(S16H)‐injected rats (black arrows). Scale bars, 500 μm (inset 20 μm). (b) Double immunofluorescence staining against GFAP (green) and CNTF (red) showing the co‐localization of these two markers in the CA1 region of the hippocampus of CON and AAV1‐Rheb (S16H)‐injected rats. Scale bars, 100 μm (inset 10 μm). (c, d) Western blot analysis of the levels of CNTF expression in the hippocampus of AAV1‐ GFP‐injected or AAV1‐Rheb(S16H)‐injected rats, with or without BDNF neutralizing antibody (B.NA) or TrkB neutralizing antibody (T.NA). Differences among groups were evaluated by Kruskal–Wallis test (c) or one‐way ANOVA (d) and Tukey's post hoc analysis. *P < .05 versus CON and #P < .05 versus Rheb(S16H) alone (n = 5 for each group). (e) Immunohistochemical staining showing increased CNTF expression in glia‐like cells of the hippocampal CA1 region of recombinant BDNF‐injected rats (black arrows). Double immunofluorescence staining against GFAP (green) and CNTF (red) showing the co‐localization of these two markers in the CA1 region of the hippocampus of recombinant BDNF‐injected rats. Scale bar, 50 μm. (f) Western blot analysis of CNTF. Differences between groups were evaluated by Student's unpaired t test. *P < .05 versus CON (n = 5 for each group). (g–i) Measurement of CNTF concentration in the conditioned medium (CM) of recombinant BDNF‐treated astrocyte cultures using ELISA kits. (g) Schematic of the experimental design for measuring the levels of CNTF in hippocampal astrocyte cultures. (h) Histogram showing the dose‐dependent effects of recombinant BDNF on CNTF release in astrocyte cultures. Differences among groups were evaluated by Kruskal–Wallis test and Tukey's post hoc analysis. *P < .05 versus CON (n = 5 for each group). (i) Quantitative results showing the CNTF concentration after the treatment of astrocyte cultures with recombinant BDNF (80 ng·ml−1), with or without GNF‐5837 (10 μM) or rapamycin (80 nM). Differences among groups were evaluated by one‐way ANOVA and Tukey's post hoc analysis. *P < .05 versus CON and #P < .05 versus recombinant BDNF alone (n = 5 for each group)

Article Snippet: To quantify the CNTF released in the BDNF‐treated astrocyte culture medium, we used commercially available ELISA kits according to the manufacturer's protocol (Cat No. DY557; R&D Systems).

Techniques: Activation Assay, Immunohistochemical staining, Staining, Expressing, Injection, Double Immunofluorescence Staining, Western Blot, Recombinant, Concentration Assay, Enzyme-linked Immunosorbent Assay