Journal: Neural Regeneration Research

Article Title: Intrathecal liproxstatin-1 delivery inhibits ferroptosis and attenuates mechanical and thermal hypersensitivities in rats with complete Freund’s adjuvant-induced inflammatory pain

doi: 10.4103/1673-5374.346547

Figure Lengend Snippet: Anti-acyl-CoA synthetase long-chain family member 4 (ACSL4) decreases and glutathione peroxidase 4 (GPX4) increases in CFA rats following intrathecal liproxstatin-1 administration . (A–C) The representative bands and quantitative evaluations of ACSL4 and GPX4 protein levels demonstrated that liproxstatin-1 restored the abnormal levels of ACSL4 and GPX4 in the spinal cord (SC) of CFA rats. (D–F) The representative bands and quantitative evaluations of ACSL4 and GPX4 protein levels demonstrated that liproxstatin-1 restored the abnormal levels of ACSL4 and GPX4 in the dorsal root ganglion tissue of CFA rats. Data in bar charts are expressed as the mean ± SD and represent results of three independent experiments in three rats per group. Data were analyzed using one-way analysis of variance followed by Bonferroni post hoc tests. * P < 0.05, ** P < 0.01, *** P < 0.001. ACSL4: Anti-acyl-coenzyme A synthetase long-chain family member 4; CFA: complete Freund’s adjuvant; GPX4: glutathione peroxidase 4; Lip: liproxstatin-1; Mino: minocycline; Veh: vehicle.

Article Snippet: To determine the specific cellular distribution of the ferroptosis marker ACSL4 in the spinal cord and DRG tissues, we performed double staining using a rabbit polyclonal antibody to ACSL4 (1:200 for the spinal cord and 1:500 for the DRG, ABclonal, Cat# A6826, RRID: AB_2767400) and one of the following cell markers: neuronal nucleus (NeuN) as a specific neuronal marker (mouse monoclonal antibody, 1:200 for the spinal cord and 1:500 for the DRG, Abcam, Cat# ab104224, RRID: AB_10711040), glial fibrillary acidic protein (GFAP) as a hallmark of astrocyte or satellite glial cell (mouse monoclonal antibody, 1:200 for the spinal cord and 1:500 for the DRG, Abcam, Cat# ab10062, RRID: AB_296804), ionized calcium-binding adapter molecule 1 (Iba-1) as a hallmark of microglia (mouse monoclonal antibody, 1:200 for the spinal cord, Abcam, Cat# ab15690, RRID: AB_2224403), 2’3’-cyclic nucleotide 3’-phosphodiesterase (CNPase) as a hallmark of mature oligodendrocytes (mouse monoclonal antibody, 1:100 for the spinal cord, Abcam, Cat# ab6319, RRID: AB_2082593), and S100 calcium binding protein B (S100β) as a hallmark of Schwann cells (mouse monoclonal antibody, 1:200 for the DRG, Immunoway Biotechnology, Plano, TX, USA, Cat# YM0572) at 4°C overnight in a humid box.

Techniques: Adjuvant

Journal: Neural Regeneration Research

Article Title: Intrathecal liproxstatin-1 delivery inhibits ferroptosis and attenuates mechanical and thermal hypersensitivities in rats with complete Freund’s adjuvant-induced inflammatory pain

doi: 10.4103/1673-5374.346547

Figure Lengend Snippet: Distribution of anti-acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) in distinct cell types in the spinal cord . Representative double-immunofluorescence staining images show the distribution of ACSL4 in the spinal cord tissue. The white arrows indicate spinal cells stained with distinct cell markers. Scale bar: 200 μm (50 μm in the magnified boxes). Quantification analyses of cells colocalized by ACSL4 revealed that CFA injection into the hind paw caused widespread ferroptosis in spinal astrocytes, microglia, and oligodendrocytes. Intrathecal treatment of liproxstatin-1 dramatically decreased the ratio of ACSL4 immunoreactivity area to astrocyte/microglia/oligodendrocyte marker immunoreactivity area in spinal cord. Data in bar charts are expressed as the mean ± SD and represent results of five sections per rat with three rats per group. Data were analyzed using one-way analysis of variance, followed by Bonferroni post hoc tests. * P < 0.05, ** P < 0.01, *** P < 0.001. ACSL4: Anti-acyl-coenzyme A synthetase long-chain family member 4; CFA: complete Freund’s adjuvant; Lip: liproxstatin-1; Mino: minocycline; Veh: vehicle.

Article Snippet: To determine the specific cellular distribution of the ferroptosis marker ACSL4 in the spinal cord and DRG tissues, we performed double staining using a rabbit polyclonal antibody to ACSL4 (1:200 for the spinal cord and 1:500 for the DRG, ABclonal, Cat# A6826, RRID: AB_2767400) and one of the following cell markers: neuronal nucleus (NeuN) as a specific neuronal marker (mouse monoclonal antibody, 1:200 for the spinal cord and 1:500 for the DRG, Abcam, Cat# ab104224, RRID: AB_10711040), glial fibrillary acidic protein (GFAP) as a hallmark of astrocyte or satellite glial cell (mouse monoclonal antibody, 1:200 for the spinal cord and 1:500 for the DRG, Abcam, Cat# ab10062, RRID: AB_296804), ionized calcium-binding adapter molecule 1 (Iba-1) as a hallmark of microglia (mouse monoclonal antibody, 1:200 for the spinal cord, Abcam, Cat# ab15690, RRID: AB_2224403), 2’3’-cyclic nucleotide 3’-phosphodiesterase (CNPase) as a hallmark of mature oligodendrocytes (mouse monoclonal antibody, 1:100 for the spinal cord, Abcam, Cat# ab6319, RRID: AB_2082593), and S100 calcium binding protein B (S100β) as a hallmark of Schwann cells (mouse monoclonal antibody, 1:200 for the DRG, Immunoway Biotechnology, Plano, TX, USA, Cat# YM0572) at 4°C overnight in a humid box.

Techniques: Double Immunofluorescence Staining, Staining, Injection, Marker, Adjuvant

Journal: Neural Regeneration Research

Article Title: Intrathecal liproxstatin-1 delivery inhibits ferroptosis and attenuates mechanical and thermal hypersensitivities in rats with complete Freund’s adjuvant-induced inflammatory pain

doi: 10.4103/1673-5374.346547

Figure Lengend Snippet: Distribution of anti-acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) in distinct cell types in the dorsal root ganglion (DRG) cells . Representative double-immunofluorescence staining images show the distribution of the ACSL4 protein in the DRG tissues. The white arrows indicate DRG cells stained with distinct cell markers. Scale bar: 200 μm (50 μm in the magnified boxes). Quantification analyses of cells colocalized by ACSL4 revealed that CFA injection into the hind paw caused widespread ferroptosis in the satellite glial and Schwann cells of the rat DRG. Intrathecal treatment of liproxstatin-1 dramatically decreased the ratio of ACSL4 immunoreactivity to satellite glial cell/Schwann cell marker immunoreactivity area in DRG. Data in bar charts are expressed as the mean ± SD and represent results from five sections per rat with three rats per group. Data were analyzed using one-way analysis of variance followed by Bonferroni post hoc tests. * P < 0.05, ** P < 0.01, *** P < 0.001. ACSL4: Anti-acyl-coenzyme A synthetase long-chain family member 4; CFA: complete Freund’s adjuvant; DRG: dorsal root ganglion; Lip: liproxstatin-1; Mino: minocycline; Veh: vehicle.

Article Snippet: To determine the specific cellular distribution of the ferroptosis marker ACSL4 in the spinal cord and DRG tissues, we performed double staining using a rabbit polyclonal antibody to ACSL4 (1:200 for the spinal cord and 1:500 for the DRG, ABclonal, Cat# A6826, RRID: AB_2767400) and one of the following cell markers: neuronal nucleus (NeuN) as a specific neuronal marker (mouse monoclonal antibody, 1:200 for the spinal cord and 1:500 for the DRG, Abcam, Cat# ab104224, RRID: AB_10711040), glial fibrillary acidic protein (GFAP) as a hallmark of astrocyte or satellite glial cell (mouse monoclonal antibody, 1:200 for the spinal cord and 1:500 for the DRG, Abcam, Cat# ab10062, RRID: AB_296804), ionized calcium-binding adapter molecule 1 (Iba-1) as a hallmark of microglia (mouse monoclonal antibody, 1:200 for the spinal cord, Abcam, Cat# ab15690, RRID: AB_2224403), 2’3’-cyclic nucleotide 3’-phosphodiesterase (CNPase) as a hallmark of mature oligodendrocytes (mouse monoclonal antibody, 1:100 for the spinal cord, Abcam, Cat# ab6319, RRID: AB_2082593), and S100 calcium binding protein B (S100β) as a hallmark of Schwann cells (mouse monoclonal antibody, 1:200 for the DRG, Immunoway Biotechnology, Plano, TX, USA, Cat# YM0572) at 4°C overnight in a humid box.

Techniques: Double Immunofluorescence Staining, Staining, Injection, Marker, Adjuvant