Journal: Frontiers in Zoology

Article Title: Sensory epithelia of the fish inner ear in 3D: studied with high-resolution contrast enhanced microCT

doi: 10.1186/1742-9994-10-63

Figure Lengend Snippet: High-resolution contrast enhanced microCT imaging allows accurate reconstruction of the maculae in the black molly. Accuracy of reconstruction of the macula lagenae was especially improved by using the image stack scanned with higher resolution (B vs. A) . Comparison with the dissected right ear (D) shows that end organs, maculae (especially the macula sacculi), and otoliths were reliably reconstructed. Note that in (B) the semicircular canals and the utricle are incompletely reconstructed because the field of view during scanning had to be restricted to achieve the higher resolution. For a better comparison with (B) , semicircular canals including the ampullae were removed from the iodine stained, dissected ear (D) . (C) shows the reconstructed otoliths of the specimen prior to staining. c, cristae of the anterior (ca, green), horizontal (ch, yellow), and posterior (cp, blue) semicircular canals; lot, lagenar otolith (yellow); ml, macula lagenae (dark brown); ms, macula sacculi (yellow orange); mu, macula utriculi (light brown); sot, saccular otolith (purple); uot, utricular otolith (red). Iodine stained otoliths all have a yellowish appearance while maculae and the overlying as well as surrounding otolithic membranes are orange red. Scale bars, 500 μm.

Article Snippet: Scans of inner ears with special focus on sensory epithelia were performed with a MicroXCT high-resolution microCT system (Xradia, MicroXCT-200, Zeiss X-Ray Microscopy, Pleasanton, CA) with a tungsten microfocus X-ray source and variable secondary optical magnification.

Techniques: Imaging, Comparison, Staining

Journal: Frontiers in Zoology

Article Title: Sensory epithelia of the fish inner ear in 3D: studied with high-resolution contrast enhanced microCT

doi: 10.1186/1742-9994-10-63

Figure Lengend Snippet: High-resolution contrast enhanced microCT imaging allows accurate reconstruction of the maculae in S. tinanti . Comparison with the dissected left saccule, lagena (B ; anteromedial view ) , and the utricle (C ; ventral view ) and the phalloidin-stained maculae (D-F) shows that end organs, maculae (especially the macula utriculi), and otoliths were reliably reconstructed (A) . For a better comparison, semicircular canals including the ampullae were removed from the iodine stained, dissected saccule and lagena (B) . ca, anterior semicircular canal; ch, horizontal semicircular canal; cp, posterior semicircular canal; lot, lagenar otolith; ml, macula lagenae; ms, macula sacculi; mu, macula utriculi; sot, saccular otolith; uot, utricular otolith. (A-B) : Scale bars, 500 μm; (C-F) : Scale bars, 250 μm.

Article Snippet: Scans of inner ears with special focus on sensory epithelia were performed with a MicroXCT high-resolution microCT system (Xradia, MicroXCT-200, Zeiss X-Ray Microscopy, Pleasanton, CA) with a tungsten microfocus X-ray source and variable secondary optical magnification.

Techniques: Imaging, Comparison, Staining

Journal: Frontiers in Zoology

Article Title: Sensory epithelia of the fish inner ear in 3D: studied with high-resolution contrast enhanced microCT

doi: 10.1186/1742-9994-10-63

Figure Lengend Snippet: Validation of the 3D reconstructed maculae based on microCT imaging . 3D reconstructed macula sacculi (A) and macula lagenae (B) of the herein studied black molly are compared with a corresponding reconstruction from the Atlantic molly Poecilia mexicana in a previous study . Reconstructions of the maculae of P. mexicana (C-E) based on histological serial sections (thickness of sections: 1 μm). The shape of the respective macula types of black molly and Atlantic molly show a high degree of similarity, especially with regard to the macula sacculi, indicating the reliability of 3D reconstructed sensory epithelia based on microCT imaging. Similar features of the maculae sacculi are marked by the arrow and circle. The yellow area in (C) represents reconstructed non-sensory epithelium surrounding the macula sacculi. All maculae are shown in lateral view with their dorsal side up and anterior to the right. Scale bars, 100 μm.

Article Snippet: Scans of inner ears with special focus on sensory epithelia were performed with a MicroXCT high-resolution microCT system (Xradia, MicroXCT-200, Zeiss X-Ray Microscopy, Pleasanton, CA) with a tungsten microfocus X-ray source and variable secondary optical magnification.

Techniques: Biomarker Discovery, Imaging

Journal: Neural regeneration research

Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model.

doi: 10.4103/1673-5374.391193

Figure Lengend Snippet: Figure 2 ｜ p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced cell death. (A) The effect of different concentrations of glutamate on R28 cell viability over 24 hours (n = 5). (B) Light microscopy was used to examine the effect of SB202190 on 10 mM glutamate-treated R28 cells. (C) Effect of SB202190 on the cell viability of R28 cells treated with 10 mM glutamate for 24 hours (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, vs. control group (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. (D) The effect of SB202190 on the morphology of R28 cells was observed under a light microscope. Arrows indicate dead cells. Scale bars: 20 µm.

Article Snippet: The following primary antibodies were used for staining: p38 MAPK rabbit polyclonal antibody (1:200, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), FTL rabbit polyclonal antibody (1:200, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673) and SAT1 rabbit polyclonal antibody (1:200, Proteintech, Cat# 10708- 1-AP, RRID: AB_2877739); phospho-p38 MAPK Thr180/ Tyr182 rabbit mAb (1:200, Cell Signaling Technology, Cat# 4511); rabbit recombinant anti-GPx4 antibody (1:200, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, Cat# PA1-16893).

Techniques: Light Microscopy, Control, Comparison

Journal: Neural regeneration research

Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model.

doi: 10.4103/1673-5374.391193

Figure Lengend Snippet: Figure 3 ｜ p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced ferroptosis. (A) Propidium iodide (PI; red) and Hoechst 33342 (blue) staining of R28 cells treated with glutamate, SB202190 and ferrostatin-1 as indicated for 24 hours. Scale bar: 100 µm. (B) Quantification of the results shown in A (n = 5). (C) Transmission electron microscope images of R28 cells treated with glutamate and SB202190. Scale bar: 1 µm. (D) R28 cells treated as indicated were stained with the fluorescent probe Mito -FerroOrange to determine ferrous ion levels. FerroOrange undergoes an irreversible reaction with intracellular Fe2+ in live cells, resulting in orange fluorescence emission. Ex: 543 nm and Em: 580 nm. Scale bar: 50 µm. (E) Quantification of mean density of FerroOrange fluorescence in the groups (n = 5). (F) R28 cells treated as indicated were stained with Liperfluo for measurement of lipid peroxides. Liperfluo is selectively oxidized by lipid peroxides, and the oxidized form of Liperfluo exhibits strong green fluorescence at the cell membrane. Scale bar: 50 µm. (G) Comparison of mean density of Liperfluor fluorescence in the groups (n = 5). (H, I) Malondialdehyde (MDA; H) and glutathione (GSH; I) levels in R28 cells after glutamate and SB202190 treatment (n = 3). **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen-activated protein kinase.

Article Snippet: The following primary antibodies were used for staining: p38 MAPK rabbit polyclonal antibody (1:200, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), FTL rabbit polyclonal antibody (1:200, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673) and SAT1 rabbit polyclonal antibody (1:200, Proteintech, Cat# 10708- 1-AP, RRID: AB_2877739); phospho-p38 MAPK Thr180/ Tyr182 rabbit mAb (1:200, Cell Signaling Technology, Cat# 4511); rabbit recombinant anti-GPx4 antibody (1:200, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, Cat# PA1-16893).

Techniques: Staining, Transmission Assay, Microscopy, Fluorescence, Membrane, Comparison

Journal: Neural regeneration research

Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model.

doi: 10.4103/1673-5374.391193

Figure Lengend Snippet: Figure 4 ｜ p38 MAPK inhibitor SB202190 regulates ferroptosis in a glutamate R28 cell model by regulating FTL and SAT1. (A) p38 MAPK, p-p38 MAPK, FTL and SAT1 protein expression in R28 cells treated with glutamate and SB202190 for 24 hours (n = 3). (B–D) Quantification analysis of p-p38 MAPK/ p38 MAPK, FTL and SAT1 protein expression in R28 cells (n = 3). (E–I) Three days after intravitreal injection of NMDA and SB202190, paraffin sections were collected and processed for immunofluorescence experiments to measure the fluorescence intensity of p-p38 MAPK (E, F), FTL (G, H), and SAT1 (I, J) of the retinal sections in each group under a fluorescence microscope (n = 3). Scale bars: 10 µm. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were repeated. FTL: Ferritin light chain; MAPK: mitogen activated protein kinases.

Article Snippet: The following primary antibodies were used for staining: p38 MAPK rabbit polyclonal antibody (1:200, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), FTL rabbit polyclonal antibody (1:200, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673) and SAT1 rabbit polyclonal antibody (1:200, Proteintech, Cat# 10708- 1-AP, RRID: AB_2877739); phospho-p38 MAPK Thr180/ Tyr182 rabbit mAb (1:200, Cell Signaling Technology, Cat# 4511); rabbit recombinant anti-GPx4 antibody (1:200, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, Cat# PA1-16893).

Techniques: Expressing, Injection, Immunofluorescence, Fluorescence, Microscopy, Comparison

Journal: Neural regeneration research

Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model.

doi: 10.4103/1673-5374.391193

Figure Lengend Snippet: Figure 6 ｜ p38 MAPK inhibitor SB202190 attenuates N-methyl-D-aspartate (NMDA)- induced damage in the rat retina. (A, B) Qualitative observation (A) and quantitative analysis (B) of the effects of SB202190 on NMDA-induced retinal ganglion cell (RGC) injury in rat retina (n = 5). Three days after intravitreal injection of NMDA and SB202190, retinal plating was performed. RGCs were fluorescently labeled with Brn3a antibody and surviving RGC exhibited strong green fluorescence under a fluorescence microscope. Scale bar: 50 μm. (C) Effects of SB202190 on retinal morphology in NMDA model rats. Hematoxylin and eosin staining was performed 3 days after intravitreal injection of NMDA and SB202190. Scale bar: 50 µm. (D) Effect of SB202190 on RGC density (cells/100 µm) in NMDA model rats 3 days after intravitreal injection (n = 4). (E, F) Thickness of the retinal ganglion cell body complex (GCC) at 500, 1000, 1500, and 2000 µm from the optic disc 3 days after intravitreal injection of NMDA and SB202190. (G–I) Flash visual evoked potentials of rats 3 days after intravitreal injection of NMDA and SB202190 (n = 4). GCL: Ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; ONL: outer nuclear layer; RGC: retinal ganglion cell. Inner retina consists of GCL and IPL. **P < 0.01, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen- activated protein kinase.

Article Snippet: The following primary antibodies were used for staining: p38 MAPK rabbit polyclonal antibody (1:200, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), FTL rabbit polyclonal antibody (1:200, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673) and SAT1 rabbit polyclonal antibody (1:200, Proteintech, Cat# 10708- 1-AP, RRID: AB_2877739); phospho-p38 MAPK Thr180/ Tyr182 rabbit mAb (1:200, Cell Signaling Technology, Cat# 4511); rabbit recombinant anti-GPx4 antibody (1:200, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, Cat# PA1-16893).

Techniques: Injection, Labeling, Fluorescence, Microscopy, Staining, Comparison

Journal: Neural regeneration research

Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model.

doi: 10.4103/1673-5374.391193

Figure Lengend Snippet: Figure 5 ｜ p38 MAPK inhibitor SB202190 alleviates lipid peroxidation by regulating the SLC7A11/GSH/GPx4 pathway. (A, B) Flow cytometry was used to detect the effect of SB202190 on lipid reactive oxygen species production in R28 cells treated as indicated for 24 hours (n = 4). (C–E) SLC7A11 and GPx4 protein expressions in R28 cells were detected by western blotting (n = 3). (F–I) Fluorescence microscopy was performed to evaluate the fluorescence intensity changes of and GPx4 (F, G) and SLC7A11 (H, I) in rat retinal sections after intraluminal injection of NMDA and SB202190 for 3 days (n = 3). Scale bars in F and H: 10 µm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. GPx4: Glutathione peroxidase 4; GSH: glutathione; MAPK: mitogen-activated protein kinase.

Article Snippet: The following primary antibodies were used for staining: p38 MAPK rabbit polyclonal antibody (1:200, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), FTL rabbit polyclonal antibody (1:200, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673) and SAT1 rabbit polyclonal antibody (1:200, Proteintech, Cat# 10708- 1-AP, RRID: AB_2877739); phospho-p38 MAPK Thr180/ Tyr182 rabbit mAb (1:200, Cell Signaling Technology, Cat# 4511); rabbit recombinant anti-GPx4 antibody (1:200, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, Cat# PA1-16893).

Techniques: Flow Cytometry, Western Blot, Fluorescence, Microscopy, Injection, Comparison