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: Neurobiology of Stress

Article Title: Female-specific role of ciliary neurotrophic factor in the medial amygdala in promoting stress responses

doi: 10.1016/j.ynstr.2022.100435

Figure Lengend Snippet: CNTF in the MeA promotes immobility during acute inescapable swim stress in female but not male mice. A) Injection of CNTF Ab into the MeA of C57BL/6 mice reduced the levels of pStat3 705 24 and 48 h later in female mice as shown by double fluorescence Western blot and quantification. Quantification of pStat3 705 was performed using the same areas in the blots as the total Stat3 bands, which was especially necessary for pStat3 705 at 24 and 48 h when signals were very weak. N = 4,6,6 female mice, *p < 0.05 (one-way ANOVA followed by Bonferroni test. B) Confirmation of intra-MeA injection. In b1-b2, crystal violet-stained coronal brain sections indicate the location of the MeA, BLA and CeA. ctx-cortex, cc-corpus callosum, hip-hippocampus, ot-optic tract. In b3, the arrow indicates an intra-MeA injection tract. The most ventral position of the needle was purposefully kept just dorsal to the nucleus to prevent its injury. In b4, immunostaining for IgG in an IgG-injected mouse validates the spread of injected IgG within the MeA. A high magnification image of the MeA in b4 is shown in b5. C) Intra-MeA injection of CNTF Ab reduced immobility time in the forced swim tested at 24 h post-injection in females but not males. D ) The CNTF Ab did not affect locomotor activity tested in an open field at 48 h post-injection in either sex. CNTF Ab injection into the E ) BLA or F ) CeA did not affect the immobility time or locomotor activity tested at 24 and 48 h post-injection, respectively. N = 7,8 females and 10,10 males in C-D, N = 9,9 females in E and 11,8 females in F, *p < 0.05 (Two-tailed t -test). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: A total of 0.3 μl of purified goat IgG (1 μg/μl, PP40, EMD Millipore, RRID: AB_97837 ) or goat anti-mouse/rat CNTF neutralizing IgG antibody (1 μg/μl, AB-557-NA, R & D Systems, RRID: AB_354368 , ( )) was bilaterally injected using a 26 gauge Hamilton syringe.

Techniques: Injection, Fluorescence, Western Blot, Staining, Immunostaining, Activity Assay, Two Tailed Test

Journal: Neurobiology of Stress

Article Title: Female-specific role of ciliary neurotrophic factor in the medial amygdala in promoting stress responses

doi: 10.1016/j.ynstr.2022.100435

Figure Lengend Snippet: CNTF−/− female, but not male, mice have reduced chronic stress-induced immobility . Mice were subjected to 4 weeks of control handling or CUS. A ) 24 h afterwards, CUS increased immobility time in the forced swim test in female wildtype CNTF+/+ (99.9% C57BL/6 background) but not CNTF−/− females. In males, immobility times were higher in both control- and CUS-treated CNTF−/− mice compared to their respective CNTF+/+ controls. B ) 48 h after CUS, locomotor activity in an open field was not affected in either sexes or genotypes. N = 10 mice/group in females, N = 13,11,10,10 males, *p < 0.05, **p < 0.01 (Two-way ANOVA followed by Newman-Keuls multiple comparisons). C ) Knockout of CNTF did not affect muscle strength and coordination tested by wire hanging in naïve 10–14 week old female and male mice. N = 12,9 females and 9,5 males.

Article Snippet: A total of 0.3 μl of purified goat IgG (1 μg/μl, PP40, EMD Millipore, RRID: AB_97837 ) or goat anti-mouse/rat CNTF neutralizing IgG antibody (1 μg/μl, AB-557-NA, R & D Systems, RRID: AB_354368 , ( )) was bilaterally injected using a 26 gauge Hamilton syringe.

Techniques: Control, Activity Assay, Knock-Out

Journal: Neurobiology of Stress

Article Title: Female-specific role of ciliary neurotrophic factor in the medial amygdala in promoting stress responses

doi: 10.1016/j.ynstr.2022.100435

Figure Lengend Snippet: Knockout of CNTF blocks chronic stress-induced reduction of sucrose preference only in female mice. Sucrose preference was performed at 4 h after control handling or CUS to evaluate behavioral anhedonia. A) After 4 weeks of CUS, CNTF+/+, but not CNTF−/−, females had reduced sucrose preference compared to their own genotype controls. N = 10 mice/group. B ) In male mice, CUS reduced sucrose preference in CNTF+/+ and CNTF−/− mice without genotype difference. N = 13,11,10,10 mice, *, p < 0.05, **p < 0.01 (Two-way ANOVA followed by Newman-keuls multiple comparisons).

Article Snippet: A total of 0.3 μl of purified goat IgG (1 μg/μl, PP40, EMD Millipore, RRID: AB_97837 ) or goat anti-mouse/rat CNTF neutralizing IgG antibody (1 μg/μl, AB-557-NA, R & D Systems, RRID: AB_354368 , ( )) was bilaterally injected using a 26 gauge Hamilton syringe.

Techniques: Knock-Out, Control

Journal: Neurobiology of Stress

Article Title: Female-specific role of ciliary neurotrophic factor in the medial amygdala in promoting stress responses

doi: 10.1016/j.ynstr.2022.100435

Figure Lengend Snippet: CNTF−/− female, but not male, mice have a reduced restraint-induced neuroendocrine response following CUS. Plasma levels of corticosterone were measured at 24 h after control handling or CUS, with or without a subsequent 10 min of restraint challenge. A) In female mice, basal levels of corticosterone were not affected by CUS or genotype. Restraint challenge increased plasma corticosterone in CNTF+/+, but not CNTF−/−, females. B) In males, restraint challenge increased plasma corticosterone in both CNTF+/+ and CNTF−/− mice without genotype difference. N = 5,5,10,8,6,12 female mice and 6,6,7,7,12,9 male mice, *p < 0.05, **p < 0.01, ****p < 0.0001 (Two-way ANOVA followed by Newman-Keuls multiple comparisons).

Article Snippet: A total of 0.3 μl of purified goat IgG (1 μg/μl, PP40, EMD Millipore, RRID: AB_97837 ) or goat anti-mouse/rat CNTF neutralizing IgG antibody (1 μg/μl, AB-557-NA, R & D Systems, RRID: AB_354368 , ( )) was bilaterally injected using a 26 gauge Hamilton syringe.

Techniques: Clinical Proteomics, Control

Journal: Neurobiology of Stress

Article Title: Female-specific role of ciliary neurotrophic factor in the medial amygdala in promoting stress responses

doi: 10.1016/j.ynstr.2022.100435

Figure Lengend Snippet: CNTF knockout does not affect chronic stress-induced anxiety-like behavior in both sexes. Elevated T-maze and open field tests were performed at 24 and 48 h after 4 weeks of control handling or CUS. A) In the elevated T maze test, CUS increased latencies to enter the open arm in both sexes and genotypes, without genotype difference. B) CUS reduced latency to escape the open arm in female CNTF+/+ mice. N = 11, 11, 7, 11 females and 11, 9, 9, 8 males, *p < 0.05, **p < 0.01 (Two-way ANOVA followed by Newman-Keuls multiple comparisons). In the open field test, CUS increased time spent in the center area ( C ) of the open arena and reduced time spent in the peripheral area ( D ). N = 10, 10, 10, 10 females and 13, 11, 10, 10 males, *p < 0.05, **p < 0.01, ***p < 0.001 (Two-way ANOVA followed by Newman-Keuls multiple comparisons).

Article Snippet: A total of 0.3 μl of purified goat IgG (1 μg/μl, PP40, EMD Millipore, RRID: AB_97837 ) or goat anti-mouse/rat CNTF neutralizing IgG antibody (1 μg/μl, AB-557-NA, R & D Systems, RRID: AB_354368 , ( )) was bilaterally injected using a 26 gauge Hamilton syringe.

Techniques: Knock-Out, Control

Journal: Neurobiology of Stress

Article Title: Female-specific role of ciliary neurotrophic factor in the medial amygdala in promoting stress responses

doi: 10.1016/j.ynstr.2022.100435

Figure Lengend Snippet: Knockout of CNTF does not affect sensorimotor gating function. Pre-pulse inhibition was performed at 24, 48 and 72 h after 4 weeks of control handling or CUS. A) In female mice, CUS did not affect pre-pulse inhibition and startle response in both genotypes. B ) In males, CUS increased pre-pulse inhibition without altering the startle response in both genotypes. N = 10,11,10,12 females and 11,10,12,11 males, *p < 0.05, ***p < 0.001, ****p < 0.0001 (Two-way ANOVA followed by Newman-Keuls comparison tests).

Article Snippet: A total of 0.3 μl of purified goat IgG (1 μg/μl, PP40, EMD Millipore, RRID: AB_97837 ) or goat anti-mouse/rat CNTF neutralizing IgG antibody (1 μg/μl, AB-557-NA, R & D Systems, RRID: AB_354368 , ( )) was bilaterally injected using a 26 gauge Hamilton syringe.

Techniques: Knock-Out, Inhibition, Control, Comparison

Journal: Neurobiology of Stress

Article Title: Female-specific role of ciliary neurotrophic factor in the medial amygdala in promoting stress responses

doi: 10.1016/j.ynstr.2022.100435

Figure Lengend Snippet: Chronic stress upregulates CNTF expression in female, but not male, MeA. Female and male wildtype CNTF+/+ mice treated with 4 weeks of control handling or CUS, without subsequent restraint, in were used to measure gene expression. A ) In the female MeA, CUS increased CNTF mRNA measured by RT-qPCR and protein by densitometry of Western blots. B) CUS did not alter CNTFRα, or IL-6, LIF and TNF mRNA in the female MeA. N = 5,10 mice for mRNA analysis and N = 5 mice/group for protein analysis. *p < 0.05, **p < 0.01 (Two-tailed t -test). C) CUS did not affect CNTF mRNA expression in the hypothalamic PVN (Hyp), hippocampus (Hip) and BNST of the same females. N = 5,5 mice. D) In the male MeA, CUS had no effect on CNTF, CNTFRα, IL-6 or TNF, but increased LIF mRNA expression. N = 6,7 mice. *p < 0.05, **p < 0.01 (Two-tailed t -test).

Article Snippet: A total of 0.3 μl of purified goat IgG (1 μg/μl, PP40, EMD Millipore, RRID: AB_97837 ) or goat anti-mouse/rat CNTF neutralizing IgG antibody (1 μg/μl, AB-557-NA, R & D Systems, RRID: AB_354368 , ( )) was bilaterally injected using a 26 gauge Hamilton syringe.

Techniques: Expressing, Control, Gene Expression, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Investigative Ophthalmology & Visual Science

Article Title: Long-Term Durability of Ciliary Neurotrophic Factor–Releasing Revakinagene Taroretcel-lwey in Individuals With Retinal Degenerative Disorders

doi: 10.1167/iovs.66.11.3

Figure Lengend Snippet: Ciliary neurotrophic factor (CNTF) pharmacokinetics from explanted revakinagene taroretcel-lwey devices. Secretion of CNTF (ng/day) by revakinagene taroretcel-lwey devices explanted over 14.5 years of implant durations, grouped to the nearest half-year for durations <3 years. The solid line indicates the weighted mean CNTF output level, and the dotted lines indicate the range of the 95% confidence interval of the slope. CNTF, ciliary neurotrophic factor; ng, nanogram.

Article Snippet: An ELISA was also used to quantify specific anti-human CNTF antibody titers after incubating participant serum on a plate coated with rhCNTF (R&D Systems).

Techniques: Drug discovery

Journal: Investigative Ophthalmology & Visual Science

Article Title: Long-Term Durability of Ciliary Neurotrophic Factor–Releasing Revakinagene Taroretcel-lwey in Individuals With Retinal Degenerative Disorders

doi: 10.1167/iovs.66.11.3

Figure Lengend Snippet: Units of activity of CNTF produced by revakinagene taroretcel-lwey explants over 14.5 years of implant duration. Seven samples were analyzed, 2 of which were analyzed twice, and the results were averaged. The solid line indicates the slope of units of activity, and the dotted lines indicate the range of the 95% confidence interval of the slope. CNTF, ciliary neurotrophic factor; EC 50 , half-maximal effective concentration.

Article Snippet: An ELISA was also used to quantify specific anti-human CNTF antibody titers after incubating participant serum on a plate coated with rhCNTF (R&D Systems).

Techniques: Activity Assay, Produced, Concentration Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Long-Term Durability of Ciliary Neurotrophic Factor–Releasing Revakinagene Taroretcel-lwey in Individuals With Retinal Degenerative Disorders

doi: 10.1167/iovs.66.11.3

Figure Lengend Snippet: Electropherograms of CNTF from explanted revakinagene taroretcel-lwey. ( A ) Electropherograms and ( B ) corresponding bands from CNTF-conditioned media collected from devices explanted from 6 participants over 14.5 years. The explant-derived CNTF is a mixture of 29 kDa human CNTF and the 33 kDa glycosylated CNTF unique to NTC-201-6A cells. Electropherograms of ( C ) controls and ( D ) corresponding bands are shown. The controls used in this analysis include purified CNTF produced in bacteria, purified revakinagene taroretcel-lwey–secreted CNTF, and culture media. Purified CNTF protein produced in bacteria consistently has a high MW shoulder. CNTF, ciliary neurotrophic factor; kDa, kilodalton; MW, molecular weight.

Article Snippet: An ELISA was also used to quantify specific anti-human CNTF antibody titers after incubating participant serum on a plate coated with rhCNTF (R&D Systems).

Techniques: Derivative Assay, Purification, Produced, Bacteria, Molecular Weight

Journal: Brain : a journal of neurology

Article Title: Astrocyte-derived CNTF switches mature RGCs to a regenerative state following inflammatory stimulation.

doi: 10.1093/brain/awm257

Figure Lengend Snippet: Fig. 3 LI activates JAK/STAT3 pathway in RGCs. Immunohistochemical staining of retina of (A) untreated control; (B) 5 days after optic nerve crush (ONC); (C) 5 days after lens injury (LI); (D) 5 days after ONC+LI using a specific antibody against phospho-STAT3. (G, H) Phospho-STAT3 levels were strongly increased in RGC nuclei after LI alone or LI+ONC, as shown by double-staining with an anti-bIII-tubulin antibody (G) and DAPI staining (H). (E, F) Western blot analysis of retinal lysates 5 days after ONC, LI, LI+ONC, or no previous treatment (con) using a specific antibody against phospho-STAT3 (E) or an antibody against total STAT3 (F). (I^P) Immunohistochemical staining of retinas 5 days after ONC (J, N), LI (K,O), ONC + LI (L, P) or no previous treatment (I, M) using an antibody against total STAT3 (red) (I^L) and double-staining with an anti-bIII- tubulin antibody staining RGCs (green) (M^P).Total STAT3 levels remained unaffected in RGCs after ONC, LI, ONC and LI. Fig. 2 LI- or zymosan-induced CNTF upregulation is not dependent on the presence of activated macrophages or other peripherally circulating cells (A) Western blot. After LI, or no LI but intravitreal injection of PBS (con) retinas and vitreous bodies were carefully separated. CNTF expression was strongly increased in the retina 5 days following LI. CNTF was detectable in the vitreous body of LI-treated eyes but not of PBS-treated controls, suggesting that CNTF is released from retinal astrocytes into the vitreous body. Peritoneal macrophages (MP) did not express CNTF protein. Recombinant CNTF (rCNTF) served as positive control.(B) RT-PCR. Retinas and vitreous bodies were isolated 5 days after various treatments in vivo. CNTF upregulation in retinas was measured after LI treatment. RT-PCR of cells derived from the vitreous body (vit) that was carefully separated from the retina after LI treatment showed a strong signal for CD68 (macrophages) but not for CNTF, indicating that vitreal cells did not contribute to the CNTF upregulation. Also peritoneal macro- phages (MP) strongly expressed CD68 but not CNTF, whereas cultured Mu« ller cells (MC) expressed CNTF but not CD68. (C) Western blot. Untreated retinas were isolated and cultured for 2 days in the presence of increasing concentrations of BSA, lens proteins (derived from the water soluble fraction of lens homogenate) or zymosan. In contrast to BSA, exposure of retinas to lens proteins or zymosan increased retinal CNTF expression in a dose-dependent manner. (D) Immunohistochemistry. Astrocytes in the fibre layer were positively stained with an anti-CNTF antibody (red). Activated macrophages that infiltrated the inner eye after LI (indicated by arrows) and that were stained

Article Snippet: Other groups (n= 4 for each group) received ONC, LI + intravitreal injections of BSA (1.5 mg/10 ml), 5 ml of an anti-oncomodulin-antiserum (Swant, Switzerland, order number OM3, lot no.: 1x1, Swant distributes only this batch), suitable to neutralize oncomodulin and to be used for functional assays (Yin et al., 2006) in 10 ml with PBS, 2.5 mg of an anti-CNTF antibody (AAR21, Serotec, suitable for functional assays) diluted to 10ml PBS, 2.5 mg of an anti-parvalbumin antibody (Santa-Cruz) in 10 ml of PBS or 10 ml (17 mM) of the Janus-kinase-2 (JAK) inhibitor AG490 (Calbiochem).

Techniques: Immunohistochemical staining, Staining, Control, Double Staining, Western Blot, Injection, Expressing, Recombinant, Positive Control, Reverse Transcription Polymerase Chain Reaction, Isolation, In Vivo, Derivative Assay, Cell Culture, Immunohistochemistry

Journal: Brain : a journal of neurology

Article Title: Astrocyte-derived CNTF switches mature RGCs to a regenerative state following inflammatory stimulation.

doi: 10.1093/brain/awm257

Figure Lengend Snippet: Fig. 6 Axon regeneration and RGC survival in vivo after repeated intravitreal CNTF application and potentiation of LI effects by increasing intraocular cAMP levels. Longitudinal sections through the optic nerve showing GAP-43-positive axons distal to the injury site (asterisk) 2 weeks after ONC and two intravitreal injections of a solution containing (A) BSA; (B) LI+intravitreal injections of PBS simultaneously to ONC and again after 3 and 7 days; (C) intravi- treal injections of recombinant CNTF at the same time as ONC and again after 3 days; (D) LI and repeated intravitreal injections of dibutyryl-cAMP (db-cAMP) simultaneously to ONC+LI and again after 3 and 7 days. Scale bar=100mm. (E) Quantitation of regen- eration (number of axons growing 5250 and 5500mm beyond the injury site per optic nerve) 2 weeks after surgery. (F) Quantitation of surviving RGCs (bIII-tubulin-positive RGCs per retinal cross section) 2 weeks after ONC. P _ 0.001 compared with BSA-treated controls; yyP _ 0.001 compared with LI-treated control.Total=number of RGCs in naive retina.

Article Snippet: Other groups (n= 4 for each group) received ONC, LI + intravitreal injections of BSA (1.5 mg/10 ml), 5 ml of an anti-oncomodulin-antiserum (Swant, Switzerland, order number OM3, lot no.: 1x1, Swant distributes only this batch), suitable to neutralize oncomodulin and to be used for functional assays (Yin et al., 2006) in 10 ml with PBS, 2.5 mg of an anti-CNTF antibody (AAR21, Serotec, suitable for functional assays) diluted to 10ml PBS, 2.5 mg of an anti-parvalbumin antibody (Santa-Cruz) in 10 ml of PBS or 10 ml (17 mM) of the Janus-kinase-2 (JAK) inhibitor AG490 (Calbiochem).

Techniques: In Vivo, Recombinant, Quantitation Assay, Control

Journal: Frontiers in Pharmacology

Article Title: Synergic Neuroprotection Between Ligusticum Chuanxiong Hort and Borneol Against Ischemic Stroke by Neurogenesis via Modulating Reactive Astrogliosis and Maintaining the Blood–Brain Barrier

doi: 10.3389/fphar.2021.666790

Figure Lengend Snippet: Expressions of BDNF, bFGF, CNTF, NGF, NT-3, TrkB, VEGF, and VEGFR2 within DG and SVZ of MCAO rats. (A) Blot images of the proteins in the DG area. (B) Relative expression of the proteins to GAPDH in the DG area ( n = 3). (C) Blot images of the proteins in the SVZ area. (D) Relative expressions of the proteins to GAPDH in the SVZ area ( n = 3). # p < 0.05 compared to the sham group; * p < 0.05, ** p < 0.01 compared to the model group; & p < 0.05 compared to the LCH + BO group.

Article Snippet: After being blocked by 5% bovine serum albumin (BSA), the membranes were incubated with the following primary antibodies overnight at 4°C: BDNF (1:5,000, catalog number: ab108319, Abcam, Cambridge, MA, United States), bFGF (1:2000, catalog number: PA5–95284, Invitrogen, Carlsbad, CA, United States), CNTF (1:5,000, catalog number: 10,796–1–AP, Proteintech, Rosemont, IL, United States), NGF (1:1,000, catalog number: MA5–32067, Invitrogen, Carlsbad, CA, United States), NT-3 (1:2000, catalog number: 18,084–1–AP, Proteintech, Rosemont, IL, United States), TrkB (1:1,000, catalog number: 13,129–1–AP, Proteintech, Rosemont, IL, United States), VEGF (1:2000, catalog number: 19,003–1–AP, Proteintech, Rosemont, IL, United States), VEGFR2 (1:1,000, catalog number: ab39256, Abcam, Cambridge, MA, United States), C3 (1:2000, catalog number: ab200999, Abcam, Cambridge, MA, United States), PTX3 (1:1,000, catalog number: ab134920, Abcam, Cambridge, MA, United States), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:10,000, catalog number: 10,494–1–AP, Proteintech, Rosemont, IL, United States).

Techniques: Expressing