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