Journal: Neural Regeneration Research

Article Title: Overexpression of the inwardly rectifying potassium channel Kir4.1 or Kir4.1 Tyr 9 Asp in Müller cells exerts neuroprotective effects in an experimental glaucoma model

doi: 10.4103/NRR.NRR-D-24-00461

Figure Lengend Snippet: Identification and verification of potential functional phosphorylation sites in Kir4.1. (A) Schematic diagram illustrating the design of the Kir4.1 mutant lentiviral expression system. (B) Mutation sites in the kcnj10 gene. (C) Immunofluorescence staining showing primary cultured Müller cells infected with lentiviruses for overexpressing Kir4.1, the Kir4.1 mutants, and the eGFP control. All lentiviruses successfully infected purified cultured Müller cells. Scale bar: 20 μm. (D, E) Representative immunoblots (D) and quantification (E) demonstrating changes in total Kir4.1 protein expression in DHPG-treated cultured Müller cells infected with lentiviruses overexpressing Kir4.1 and seven different Kir4.1 mutants. All data are normalized to GAPDH levels and then to controls. One-way analysis of variance (Tukey–Kramer multiple comparisons test) was performed (* P < 0.05, *** P < 0.001, vs . Ctr group). (F, G) Representative immunoblots (F) and densitometric quantification (G) showing changes in membrane Kir4.1 protein expression in DHPG-treated cultured Müller cells infected with lentiviruses overexpressing Kir4.1, Kir4.1 Tyr 9 Asp, and Kir4.1 Ser 370 Arg. All data are normalized to Na-K-ATP levels and then to the Kir4.1 group. One-way analysis of variance (Tukey–Kramer multiple comparisons test) was used (** P < 0.01, vs . Kir4.1 group). CMV: Cytomegalovirus ; Ctr: control; DHPG: (S)-3,5-dihydroxyphenylglycine; eGFP: enhanced green fluorescent protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; SV40: simian vacuolating virus 40.

Article Snippet: Briefly, Müller cells were pre-activated with 100 μM DHPG for 12 hours and then co-cultured with microglia in the presence or absence of the P2X7R antagonist A-740003 (1 μM, MedChemExpress, Shanghai, China, Cat# HY-50697) and the P2X4R antagonist 5-BDBD (5 μM, MedChemExpress, Cat# HY101911 ) for 72 hours. mRNA levels of the inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in microglia were assessed by qPCR.

Techniques: Functional Assay, Phospho-proteomics, Mutagenesis, Expressing, Immunofluorescence, Staining, Cell Culture, Infection, Control, Purification, Western Blot, Membrane, Virus

Journal: Neural Regeneration Research

Article Title: Overexpression of the inwardly rectifying potassium channel Kir4.1 or Kir4.1 Tyr 9 Asp in Müller cells exerts neuroprotective effects in an experimental glaucoma model

doi: 10.4103/NRR.NRR-D-24-00461

Figure Lengend Snippet: Effects of Kir4.1 overexpression and Kir4.1Tyr 9 Asp overexpression on GFAP expression in cultured Müller glial cells. (A, B) Representative immunoblots (A) and densitometric quantification (B) showing changes in GFAP expression in non-treated and DHPG-treated cultured Müller cells infected with lentiviruses overexpressing the eGFP control (LV-NC), Kir4.1, and Kir4.1 Tyr 9 Asp. All data are normalized to GADPH and then to Ctr. One-way analysis of variance (Tukey–Kramer multiple comparisons test) was performed (** P < 0.01, vs . Ctr group). (C) Double immunofluorescence staining showing changes in GFAP (Cy3, red) expression in non-treated (c1–c12) and DHPG-treated (c13–24) cultured Müller cells infected with lentiviruses overexpressing the eGFP Ctr (LV-NC) (c4–c6 and c16–c18), Kir4.1 (c7–c9 and c19–c21), and Kir4.1 Tyr 9 Asp (c10–c12 and c22–c24) mutation. Kir4.1 overexpression and Kir4.1 Tyr 9 Asp overexpression reduced the DHPG-induced increase in GFAP expression in Müller cells. GFP (indicated by Alexa488) and DAPI are shown in green and blue, respectively. Scale bars: 20 µm. Ctr: Control; DHPG: (S)-3,5-dihydroxyphenylglycine; eGFP: enhanced green fluorescent protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; GFP: green fluorescent protein; LV-NC: eGFP control lentiviruses.

Article Snippet: Briefly, Müller cells were pre-activated with 100 μM DHPG for 12 hours and then co-cultured with microglia in the presence or absence of the P2X7R antagonist A-740003 (1 μM, MedChemExpress, Shanghai, China, Cat# HY-50697) and the P2X4R antagonist 5-BDBD (5 μM, MedChemExpress, Cat# HY101911 ) for 72 hours. mRNA levels of the inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in microglia were assessed by qPCR.

Techniques: Over Expression, Expressing, Cell Culture, Western Blot, Infection, Control, Double Immunofluorescence Staining, Mutagenesis

Journal: Neural Regeneration Research

Article Title: Overexpression of the inwardly rectifying potassium channel Kir4.1 or Kir4.1 Tyr 9 Asp in Müller cells exerts neuroprotective effects in an experimental glaucoma model

doi: 10.4103/NRR.NRR-D-24-00461

Figure Lengend Snippet: Kir4.1 and Kir4.1Tyr 9 Asp overexpression attenuate retinal Müller cell activation and inhibit DHPG-induced Kir4.1 downregulation. (A) Representative immunoblots showing changes in GFAP and Kir4.1 expression in retinas from uninjected rats and rats subjected to DHPG intravitreal injection at 1 and 2 weeks post-injection. (B–E) Densitometric quantification of changes in GFAP (B, C) and Kir4.1 (D, E) expression under the conditions described in panel A. The retinas were infected with lentiviruses encoding eGFP (LV-NC), Kir4.1, and Kir4.1 Tyr 9 Asp. All data were normalized to GAPDH/β-actin levels and then to control values. One-way analysis of variance (Tukey–Kramer multiple comparisons test) was used for panel D, while the Kruskal–Wallis test (Dunn’s multiple comparisons test) was applied for panel E. * P < 0.05, ** P < 0.01, vs . Ctr group; # P < 0.05, vs . DHPG 2w LV-NC group. (F) Representative immunoblots showing changes in GFAP expression in normal or COH retinas at G2w in Müller cells infected with LV-NC, Kir4.1, and Kir4.1 Tyr 9 Asp lentiviruses. (G, H) Bar charts summarizing the average densitometric quantification of the GFAP bands shown in panel F. All data were normalized to GAPDH levels and then to control values. Unpaired two-tailed t -test was performed for panel B, and one-way analysis of variance (Tukey–Kramer multiple comparisons test) was used for panel C. * P < 0.05, ** P < 0.01, vs . Ctr group; # P < 0.05, vs . LV-NC group. Ctr: Control; DHPG: (S)-3,5-dihydroxyphenylglycine; eGFP: enhanced green fluorescent protein; G2w: chronic ocular hypertension at 2 weeks; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; LV-NC: eGFP control lentiviruses.

Article Snippet: Briefly, Müller cells were pre-activated with 100 μM DHPG for 12 hours and then co-cultured with microglia in the presence or absence of the P2X7R antagonist A-740003 (1 μM, MedChemExpress, Shanghai, China, Cat# HY-50697) and the P2X4R antagonist 5-BDBD (5 μM, MedChemExpress, Cat# HY101911 ) for 72 hours. mRNA levels of the inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in microglia were assessed by qPCR.

Techniques: Over Expression, Activation Assay, Western Blot, Expressing, Injection, Infection, Control, Two Tailed Test

Journal: Neural Regeneration Research

Article Title: Overexpression of the inwardly rectifying potassium channel Kir4.1 or Kir4.1 Tyr 9 Asp in Müller cells exerts neuroprotective effects in an experimental glaucoma model

doi: 10.4103/NRR.NRR-D-24-00461

Figure Lengend Snippet: Kir4.1 overexpression and Kir4.1 Tyr 9 Asp overexpression alleviate the Müller cell activation–induced increase in inflammatory factor mRNA expression levels. Bar charts summarizing the relative mRNA levels of IL-1 β in normal retinas (A) and in retinas from eyes injected intravitreally with DHPG at 1 and 2 weeks post-injection (B), following infection with the LV-NC, Kir4.1, and Kir4.1 Tyr 9 Asp lentiviruses. (C, D) Bar charts summarizing relative TNF-α mRNA levels in normal retinas (C) and in retinas from eyes injected intravitreally with DHPG at 1 and 2 weeks post-injection (D), infected with the same lentiviruses. # P < 0.05 vs . LV-NC group. Kruskal–Wallis test (Dunn’s multiple comparisons test) was performed. Ctr: Control; DHPG: (S)-3,5-dihydroxyphenylglycine; eGFP: enhanced green fluorescent protein; IL-1β: interleukin-1β; LV-NC: eGFP control lentiviruses; TNF-α: tumor necrosis factor-α.

Article Snippet: Briefly, Müller cells were pre-activated with 100 μM DHPG for 12 hours and then co-cultured with microglia in the presence or absence of the P2X7R antagonist A-740003 (1 μM, MedChemExpress, Shanghai, China, Cat# HY-50697) and the P2X4R antagonist 5-BDBD (5 μM, MedChemExpress, Cat# HY101911 ) for 72 hours. mRNA levels of the inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in microglia were assessed by qPCR.

Techniques: Over Expression, Activation Assay, Expressing, Injection, Infection, Control

Journal: Neural Regeneration Research

Article Title: Overexpression of the inwardly rectifying potassium channel Kir4.1 or Kir4.1 Tyr 9 Asp in Müller cells exerts neuroprotective effects in an experimental glaucoma model

doi: 10.4103/NRR.NRR-D-24-00461

Figure Lengend Snippet: Identification and verification of potential functional phosphorylation sites in Kir4.1. (A) Schematic diagram illustrating the design of the Kir4.1 mutant lentiviral expression system. (B) Mutation sites in the kcnj10 gene. (C) Immunofluorescence staining showing primary cultured Müller cells infected with lentiviruses for overexpressing Kir4.1, the Kir4.1 mutants, and the eGFP control. All lentiviruses successfully infected purified cultured Müller cells. Scale bar: 20 μm. (D, E) Representative immunoblots (D) and quantification (E) demonstrating changes in total Kir4.1 protein expression in DHPG-treated cultured Müller cells infected with lentiviruses overexpressing Kir4.1 and seven different Kir4.1 mutants. All data are normalized to GAPDH levels and then to controls. One-way analysis of variance (Tukey–Kramer multiple comparisons test) was performed (* P < 0.05, *** P < 0.001, vs . Ctr group). (F, G) Representative immunoblots (F) and densitometric quantification (G) showing changes in membrane Kir4.1 protein expression in DHPG-treated cultured Müller cells infected with lentiviruses overexpressing Kir4.1, Kir4.1 Tyr 9 Asp, and Kir4.1 Ser 370 Arg. All data are normalized to Na-K-ATP levels and then to the Kir4.1 group. One-way analysis of variance (Tukey–Kramer multiple comparisons test) was used (** P < 0.01, vs . Kir4.1 group). CMV: Cytomegalovirus ; Ctr: control; DHPG: (S)-3,5-dihydroxyphenylglycine; eGFP: enhanced green fluorescent protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; SV40: simian vacuolating virus 40.

Article Snippet: Briefly, Müller cells were pre-activated with 100 μM DHPG for 12 hours and then co-cultured with microglia in the presence or absence of the P2X7R antagonist A-740003 (1 μM, MedChemExpress, Shanghai, China, Cat# HY-50697) and the P2X4R antagonist 5-BDBD (5 μM, MedChemExpress, Cat# HY101911 ) for 72 hours. mRNA levels of the inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in microglia were assessed by qPCR.

Techniques: Functional Assay, Phospho-proteomics, Mutagenesis, Expressing, Immunofluorescence, Staining, Cell Culture, Infection, Control, Purification, Western Blot, Membrane, Virus

Journal: Neural Regeneration Research

Article Title: Overexpression of the inwardly rectifying potassium channel Kir4.1 or Kir4.1 Tyr 9 Asp in Müller cells exerts neuroprotective effects in an experimental glaucoma model

doi: 10.4103/NRR.NRR-D-24-00461

Figure Lengend Snippet: Effects of Kir4.1 overexpression and Kir4.1Tyr 9 Asp overexpression on GFAP expression in cultured Müller glial cells. (A, B) Representative immunoblots (A) and densitometric quantification (B) showing changes in GFAP expression in non-treated and DHPG-treated cultured Müller cells infected with lentiviruses overexpressing the eGFP control (LV-NC), Kir4.1, and Kir4.1 Tyr 9 Asp. All data are normalized to GADPH and then to Ctr. One-way analysis of variance (Tukey–Kramer multiple comparisons test) was performed (** P < 0.01, vs . Ctr group). (C) Double immunofluorescence staining showing changes in GFAP (Cy3, red) expression in non-treated (c1–c12) and DHPG-treated (c13–24) cultured Müller cells infected with lentiviruses overexpressing the eGFP Ctr (LV-NC) (c4–c6 and c16–c18), Kir4.1 (c7–c9 and c19–c21), and Kir4.1 Tyr 9 Asp (c10–c12 and c22–c24) mutation. Kir4.1 overexpression and Kir4.1 Tyr 9 Asp overexpression reduced the DHPG-induced increase in GFAP expression in Müller cells. GFP (indicated by Alexa488) and DAPI are shown in green and blue, respectively. Scale bars: 20 µm. Ctr: Control; DHPG: (S)-3,5-dihydroxyphenylglycine; eGFP: enhanced green fluorescent protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; GFP: green fluorescent protein; LV-NC: eGFP control lentiviruses.

Article Snippet: Briefly, Müller cells were pre-activated with 100 μM DHPG for 12 hours and then co-cultured with microglia in the presence or absence of the P2X7R antagonist A-740003 (1 μM, MedChemExpress, Shanghai, China, Cat# HY-50697) and the P2X4R antagonist 5-BDBD (5 μM, MedChemExpress, Cat# HY101911 ) for 72 hours. mRNA levels of the inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in microglia were assessed by qPCR.

Techniques: Over Expression, Expressing, Cell Culture, Western Blot, Infection, Control, Double Immunofluorescence Staining, Mutagenesis

Journal: Neural Regeneration Research

Article Title: Overexpression of the inwardly rectifying potassium channel Kir4.1 or Kir4.1 Tyr 9 Asp in Müller cells exerts neuroprotective effects in an experimental glaucoma model

doi: 10.4103/NRR.NRR-D-24-00461

Figure Lengend Snippet: Kir4.1 and Kir4.1Tyr 9 Asp overexpression attenuate retinal Müller cell activation and inhibit DHPG-induced Kir4.1 downregulation. (A) Representative immunoblots showing changes in GFAP and Kir4.1 expression in retinas from uninjected rats and rats subjected to DHPG intravitreal injection at 1 and 2 weeks post-injection. (B–E) Densitometric quantification of changes in GFAP (B, C) and Kir4.1 (D, E) expression under the conditions described in panel A. The retinas were infected with lentiviruses encoding eGFP (LV-NC), Kir4.1, and Kir4.1 Tyr 9 Asp. All data were normalized to GAPDH/β-actin levels and then to control values. One-way analysis of variance (Tukey–Kramer multiple comparisons test) was used for panel D, while the Kruskal–Wallis test (Dunn’s multiple comparisons test) was applied for panel E. * P < 0.05, ** P < 0.01, vs . Ctr group; # P < 0.05, vs . DHPG 2w LV-NC group. (F) Representative immunoblots showing changes in GFAP expression in normal or COH retinas at G2w in Müller cells infected with LV-NC, Kir4.1, and Kir4.1 Tyr 9 Asp lentiviruses. (G, H) Bar charts summarizing the average densitometric quantification of the GFAP bands shown in panel F. All data were normalized to GAPDH levels and then to control values. Unpaired two-tailed t -test was performed for panel B, and one-way analysis of variance (Tukey–Kramer multiple comparisons test) was used for panel C. * P < 0.05, ** P < 0.01, vs . Ctr group; # P < 0.05, vs . LV-NC group. Ctr: Control; DHPG: (S)-3,5-dihydroxyphenylglycine; eGFP: enhanced green fluorescent protein; G2w: chronic ocular hypertension at 2 weeks; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; LV-NC: eGFP control lentiviruses.

Article Snippet: Briefly, Müller cells were pre-activated with 100 μM DHPG for 12 hours and then co-cultured with microglia in the presence or absence of the P2X7R antagonist A-740003 (1 μM, MedChemExpress, Shanghai, China, Cat# HY-50697) and the P2X4R antagonist 5-BDBD (5 μM, MedChemExpress, Cat# HY101911 ) for 72 hours. mRNA levels of the inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in microglia were assessed by qPCR.

Techniques: Over Expression, Activation Assay, Western Blot, Expressing, Injection, Infection, Control, Two Tailed Test

Journal: Neural Regeneration Research

Article Title: Overexpression of the inwardly rectifying potassium channel Kir4.1 or Kir4.1 Tyr 9 Asp in Müller cells exerts neuroprotective effects in an experimental glaucoma model

doi: 10.4103/NRR.NRR-D-24-00461

Figure Lengend Snippet: Kir4.1 and Kir4.1 Tyr 9 Asp overexpression attenuate retinal Müller glial cell activation and ganglion cell apoptosis in a rat model of chronic ocular hypertension. (A) Representative images of TUNEL staining detecting apoptotic RGCs in whole flat-mounted retinas from control (a1–a3) and COH (a4–a6) rats at G2w. The number of TUNEL-positive RGCs was significantly increased in COH retinas. Scale bar: 20 μm. (B) Bar charts summarizing the changes in the average number of TUNEL-positive signals. n = 6 for each group. *** P < 0.001, vs . Ctr. Unpaired two-tailed t -test. (C) Representative images of TUNEL staining of normal and COH retinas infected with LV-NC, Kir4.1, and Kir4.1 Tyr 9 Asp lentiviruses at G2w. Kir4.1 overexpression and Kir4.1 Tyr 9 Asp overexpression in Müller cells significantly reduced the number of TUNEL-positive signals in COH retinas at G2w. Scale bar: 20 μm. (D) Bar charts summarizing the changes in the average number of TUNEL-positive signals under the different conditions shown in panel C. n = 6 for each group. * P < 0.05, ** P < 0.01, *** P < 0.001, vs . Ctr group; ### P < 0.001, vs . LV-NC group. One-way analysis of variance (Tukey–Kramer multiple comparisons test) was performed. COH: Chronic ocular hypertension; Ctr: control; DAPI: 4′,6-diamidino-2-phenylindole; eGFP: enhanced green fluorescent protein; G2w: chronic ocular hypertension at 2 weeks; LV-NC: eGFP control lentiviruses; TUNEL: terminal deoxynucleotidy transferase-mediated dUTP nick end labeling.

Article Snippet: Briefly, Müller cells were pre-activated with 100 μM DHPG for 12 hours and then co-cultured with microglia in the presence or absence of the P2X7R antagonist A-740003 (1 μM, MedChemExpress, Shanghai, China, Cat# HY-50697) and the P2X4R antagonist 5-BDBD (5 μM, MedChemExpress, Cat# HY101911 ) for 72 hours. mRNA levels of the inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in microglia were assessed by qPCR.

Techniques: Over Expression, Activation Assay, TUNEL Assay, Staining, Control, Two Tailed Test, Infection, End Labeling

Journal: Neural Regeneration Research

Article Title: Overexpression of the inwardly rectifying potassium channel Kir4.1 or Kir4.1 Tyr 9 Asp in Müller cells exerts neuroprotective effects in an experimental glaucoma model

doi: 10.4103/NRR.NRR-D-24-00461

Figure Lengend Snippet: Kir4.1 overexpression and Kir4.1 Tyr 9 Asp overexpression alleviate the Müller cell activation–induced increase in inflammatory factor mRNA expression levels. Bar charts summarizing the relative mRNA levels of IL-1 β in normal retinas (A) and in retinas from eyes injected intravitreally with DHPG at 1 and 2 weeks post-injection (B), following infection with the LV-NC, Kir4.1, and Kir4.1 Tyr 9 Asp lentiviruses. (C, D) Bar charts summarizing relative TNF-α mRNA levels in normal retinas (C) and in retinas from eyes injected intravitreally with DHPG at 1 and 2 weeks post-injection (D), infected with the same lentiviruses. # P < 0.05 vs . LV-NC group. Kruskal–Wallis test (Dunn’s multiple comparisons test) was performed. Ctr: Control; DHPG: (S)-3,5-dihydroxyphenylglycine; eGFP: enhanced green fluorescent protein; IL-1β: interleukin-1β; LV-NC: eGFP control lentiviruses; TNF-α: tumor necrosis factor-α.

Article Snippet: Briefly, Müller cells were pre-activated with 100 μM DHPG for 12 hours and then co-cultured with microglia in the presence or absence of the P2X7R antagonist A-740003 (1 μM, MedChemExpress, Shanghai, China, Cat# HY-50697) and the P2X4R antagonist 5-BDBD (5 μM, MedChemExpress, Cat# HY101911 ) for 72 hours. mRNA levels of the inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in microglia were assessed by qPCR.

Techniques: Over Expression, Activation Assay, Expressing, Injection, Infection, Control

Journal: Neural Regeneration Research

Article Title: Overexpression of the inwardly rectifying potassium channel Kir4.1 or Kir4.1 Tyr 9 Asp in Müller cells exerts neuroprotective effects in an experimental glaucoma model

doi: 10.4103/NRR.NRR-D-24-00461

Figure Lengend Snippet: Kir4.1 overexpression and Kir4.1 Tyr 9 Asp overexpression in Müller cells attenuate ATP release, microglia activation, and release of inflammatory factors. (A) Representative immunoblots showing changes in TSPO protein levels in microglia co-cultured with normal or activated Müller cells infected with LV-NC, Kir4.1, and Kir4.1 Tyr9Asp lentiviruses. (B) Bar charts summarizing the average densitometric quantification of TSPO bands under different conditions. (C) Bar chart showing changes in ATP secretion by Müller cells under various conditions. n = 5–6. One-way analysis of variance (Tukey–Kramer multiple comparisons test). * P < 0.05, vs . LV-NC group. (D, E) Bar charts showing changes in TNF-α mRNA (D) and protein (E) levels in microglia co-cultured with normal or activated Müller cells infected with LV-NC, Kir4.1, and Kir4.1 Tyr9Asp lentiviruses. (F, G) Bar charts showing changes in IL-1β mRNA (F) and protein (G) levels in microglia co-cultured with normal or activated Müller cells infected with LV-NC, Kir4.1, and Kir4.1 Tyr9Asp lentiviruses. (H, I) Bar charts showing changes in IL-1β mRNA (H) and protein (I) levels in microglia co-cultured with Müller cells in which P2X7/P2X4 receptor signaling was inhibited. n = 5–9. * P < 0.05, vs . LV-NC group. Kruskal–Wallis test (Dunn’s multiple comparisons test) for panels F and H; one-way analysis of variance (Tukey–Kramer multiple comparisons test) for panels D and I; repeated measures one-way analysis of variance (Tukey–Kramer multiple comparisons test) for panels E and G. ATP: Adenosine triphosphate; eGFP: enhanced green fluorescent protein; IL-1β: interleukin-1β; LV-NC: eGFP control lentiviruses; TNF-α: tumor necrosis factor-α; TSPO: translocator protein.

Article Snippet: Briefly, Müller cells were pre-activated with 100 μM DHPG for 12 hours and then co-cultured with microglia in the presence or absence of the P2X7R antagonist A-740003 (1 μM, MedChemExpress, Shanghai, China, Cat# HY-50697) and the P2X4R antagonist 5-BDBD (5 μM, MedChemExpress, Cat# HY101911 ) for 72 hours. mRNA levels of the inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in microglia were assessed by qPCR.

Techniques: Over Expression, Activation Assay, Western Blot, Cell Culture, Infection, Control

Journal: Neural Regeneration Research

Article Title: Overexpression of the inwardly rectifying potassium channel Kir4.1 or Kir4.1 Tyr 9 Asp in Müller cells exerts neuroprotective effects in an experimental glaucoma model

doi: 10.4103/NRR.NRR-D-24-00461

Figure Lengend Snippet: Kir4.1 overexpression and Kir4.1 Tyr 9 Asp overexpression in Müller cells suppress the MYD88/IRAK1/TRAF6/NF-κB p65 inflammatory signaling pathway. (A) Representative immunoblots showing changes in MYD88, IRAK1, TRAF6, and NF-κB p65 expression levels in microglia co-cultured with normal or activated Müller cells infected with LV-NC, Kir4.1, and Kir4.1 Tyr 9 Asp lentiviruses. (B, D, F, H) Bar charts summarizing the average densitometric quantification of immunoreactive bands for MYD88 (B), IRAK1 (D), TRAF6 (F), and NF-κB p65 (H) in microglia co-cultured with normal or activated Müller cells. n = 5 for each group. * P < 0.05, ** P < 0.01, vs. the + normal Müller cells group. Unpaired two-tailed t -test. (C, E, G, I) Bar charts summarizing the average densitometric quantification of immunoreactive bands for MYD88 (C), IRAK1 (E), TRAF6 (G), and NF-κB p65 (I) in microglia co-cultured with normal or activated Müller cells. n = 5 for each group. * P < 0.05, ** P < 0.01, vs. LV-NC + normal Müller cells group. One-way analysis of variance (Tukey–Kramer multiple comparisons test) was performed. eGFP: Enhanced green fluorescent protein; IRAK1: IL-1 receptor associated kinase 1; LV-NC: eGFP control lentiviruses; MYD88: myeloid differentiation primary response protein 88; NF-κB P65: nuclear factor kappa B P65; TRAF6: TNF receptor associated factor 6.

Article Snippet: Briefly, Müller cells were pre-activated with 100 μM DHPG for 12 hours and then co-cultured with microglia in the presence or absence of the P2X7R antagonist A-740003 (1 μM, MedChemExpress, Shanghai, China, Cat# HY-50697) and the P2X4R antagonist 5-BDBD (5 μM, MedChemExpress, Cat# HY101911 ) for 72 hours. mRNA levels of the inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in microglia were assessed by qPCR.

Techniques: Over Expression, Western Blot, Expressing, Cell Culture, Infection, Two Tailed Test, Control

Journal: Neural Regeneration Research

Article Title: Overexpression of the inwardly rectifying potassium channel Kir4.1 or Kir4.1 Tyr 9 Asp in Müller cells exerts neuroprotective effects in an experimental glaucoma model

doi: 10.4103/NRR.NRR-D-24-00461

Figure Lengend Snippet: Effects of Kir4.1 overexpression and Kir4.1 Tyr 9 Asp overexpression in Müller cells on microglial proliferation. (A) Representative images showing changes in the number of EdU-positive microglia (indicated by arrowheads) when microglia were co-cultured with an empty Transwell chamber (no Müller cells) (a1 and a2), normal Müller cells (a4 and a5), and activated Müller cells (a7 and a8). Panels a3, a6, and a9 are enlarged images of the areas outlined in white. The number of EdU-positive microglia increased significantly when microglia were co-cultured with activated Müller cells. (B) Bar chart illustrating the changes in the number of EdU-positive microglia under the different conditions shown in panel A. The data were normalized to the no Müller cells group. n = 5 for each group. ** P < 0.01, vs . no Müller cells group. One-way analysis of variance followed by the Tukey–Kramer multiple comparisons test was performed. (C) Representative images showing changes in the number of EdU-positive microglia (indicated by arrowheads) when microglia were co-cultured with LV-NC + normal Müller cells (c1 and c2), LV-NC + activated Müller cells (c4 and c5), Kir4.1 + activated Müller cells (c7 and c8), and Kir4.1 Tyr 9 Asp + activated Müller cells (c10 and c11). Panels c3, c6, c9, and c12 are enlarged images of the areas outlined in white. The increased numbers of EdU-positive microglia induced by activated Müller cells were reduced by Kir4.1 overexpression, but not by Kir4.1 Tyr 9 Asp overexpression, in Müller cells. (D) Bar chart showing changes in the number of EdU-positive microglia under the different conditions depicted in panel C. The data were normalized to the LV-NC + normal Müller cells group. n = 5 for each group. * P < 0.05, vs . LV-NC + normal Müller cells group; # P < 0.05, vs . LV-NC + activated Müller cells group. One-way analysis of variance (Tukey–Kramer multiple comparisons test) was performed. Scale bars: 20 μm and 10 μm (for the enlarged panels). EdU: 5-Ethynyl-2′-deoxyuridine; eGFP: enhanced green fluorescent protein; Iba1: ionized calcium-binding adapter molecule 1; LV-NC: eGFP control lentiviruses.

Article Snippet: Briefly, Müller cells were pre-activated with 100 μM DHPG for 12 hours and then co-cultured with microglia in the presence or absence of the P2X7R antagonist A-740003 (1 μM, MedChemExpress, Shanghai, China, Cat# HY-50697) and the P2X4R antagonist 5-BDBD (5 μM, MedChemExpress, Cat# HY101911 ) for 72 hours. mRNA levels of the inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in microglia were assessed by qPCR.

Techniques: Over Expression, Cell Culture, Binding Assay, Control

Journal: Neural Regeneration Research

Article Title: Overexpression of the inwardly rectifying potassium channel Kir4.1 or Kir4.1 Tyr 9 Asp in Müller cells exerts neuroprotective effects in an experimental glaucoma model

doi: 10.4103/NRR.NRR-D-24-00461

Figure Lengend Snippet: Effects of Kir4.1 overexpression and Kir4.1 Tyr 9 Asp overexpression in Müller cells on microglial migration. (A) Representative images showing changes in microglial migration when microglia were co-cultured with an empty Transwell chamber (no Müller cells) (a1), normal Müller cells (a2), and activated Müller cells (a3). The number of migrated microglia increased significantly when co-cultured with activated Müller cells. (B) Bar chart illustrating the average number of migrated microglia under the different conditions depicted in panel A. The data were normalized to the no Müller cells group. n = 5 for each group. *** P < 0.001, vs . the no Müller cells group. One-way analysis of variance followed by the Tukey–Kramer multiple comparisons test was performed. (C) Representative images showing changes in microglial migration when microglia were co-cultured with LV-NC + normal Müller cells (c1), LV-NC + activated Müller cells (c2), Kir4.1 + activated Müller cells (c3), and Kir4.1 Tyr 9 Asp + activated Müller cells (c4). The increase in the number of migrated microglia induced by activated Müller cells was reduced by Kir4.1 overexpression, but not by the Kir4.1 Tyr 9 Asp overexpression, in Müller cells. Scale bars: 20 μm. (D) Bar chart showing the average number of migrated microglia under the different conditions depicted in panel C. The data were normalized to the LV-NC + normal Müller cells group. n = 5 for each group. * P < 0.05, vs . LV-NC + normal Müller cells group. ## P < 0.01, vs . LV-NC + activated Müller cells group. One-way analysis of variance (Turkey–Kramer multiple comparisons test) was performed. eGFP: Enhanced green fluorescent protein; LV-NC: eGFP control lentiviruses.

Article Snippet: Briefly, Müller cells were pre-activated with 100 μM DHPG for 12 hours and then co-cultured with microglia in the presence or absence of the P2X7R antagonist A-740003 (1 μM, MedChemExpress, Shanghai, China, Cat# HY-50697) and the P2X4R antagonist 5-BDBD (5 μM, MedChemExpress, Cat# HY101911 ) for 72 hours. mRNA levels of the inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in microglia were assessed by qPCR.

Techniques: Over Expression, Migration, Cell Culture, Control

Journal: bioRxiv

Article Title: Astrocytic RNA degradation suppresses calcium signaling to support synapse function and restrain anxiety

doi: 10.1101/2025.11.18.689044

Figure Lengend Snippet: ( A ) Schematic of tamoxifen dosing and electrophysiology in hippocampal area CA1 of acute brain slices. ( B ) Input-output curve assessment demonstrated reduced synaptic strength in Upf2 cKO mice as compared to CTRL (CTRL: Linear fit 2.04 ± 0.3, r = 0.99; cKO: Linear fit 0.96 ± 0.02, r = 0.99, n = 8 slices, 4 animals per genotype, linear fit slope values: p = 0.011, Unpaired t -test). ( C ) Presynaptic function assessed by the paired-pulse ratio showed no differences across genotypes (CTRL, 200 ms: 1.3 ± 0.07; cKO, 200 ms: 1.35 ± 0.04, n = 8 slices, 4 animals per genotype, p = 0.4, Unpaired t -test). ( D ) LTP induced by theta-burst stimulation led to absent potentiation in the cKO condition (CTRL: 138 ± 15%, n = 6 mice, 12 slices; cKO: 78.8 ± 9%, n = 5 mice, 9 slices; p = 0.003, Unpaired t -test). ( E ) LTD induced by low-frequency stimulation was similar in both genotypes (CTRL: 72.9 ± 5%; cKO: 78.5 ± 4%, n = 5 slices, 3 animals per genotype, p = 0.41, Mann-Whitney test). ( F ) Chemical-LTD induced by DHPG (50 μM, 5 min) revealed dampened synaptic depression in Upf2 cKO brain slices (CTRL: 55.9 ± 6%, n = 4 mice, 6 slices; cKO: 84.7 ± 9%, n = 5 mice, 7 slices, p = 0.02, Unpaired t -test). Data are presented as mean ± S.E.M. * denotes p < 0.05, *** denotes p < 0.005, ns = not significant. This figure illustrates that Upf2 deficiency in astrocytes alters synaptic function.

Article Snippet: A 15 min baseline period was recorded prior to LTD induction and synaptic inputs were stimulated every 20 s. For chemical LTD, mGluR-LTD was elicited by bath-application of DHPG (50 μM, 5 min, No. 0342, Tocris Bioscience) after obtaining a 10 min baseline period.

Techniques: MANN-WHITNEY