Journal: Investigative Ophthalmology & Visual Science

Article Title: Corneal Nerves Promote Alkali Burn Repair by Modulating Macrophages and Neutrophils via Calcitonin Gene-Related Peptide

doi: 10.1167/iovs.67.4.60

Figure Lengend Snippet: CGRP suppresses corneal tissue inflammation following alkali burn. ( A ) The volcano plot displayed the upregulated and downregulated genes in the corneas of mice from the CGRP-treated group compared with the PBS-treated group at 7 days post-injury ( n = 3). ( B ) The heatmap showed the differentially expressed genes in the corneas of mice from the CGRP-treated group compared with the PBS-treated group at 7 days post-injury ( n = 3). ( C ) The mRNA expression of the inflammatory factors IL-1β, iNOS, MCP-1, MMP3, and MMP9 was detected by qPCR on day 7 post-injury following CGRP treatment ( n = 4). ( D, E ) Representative flow cytometry plots and bar charts of CD45+ cells in the cornea after 7 days of treatment with CGRP following injury. ( F, G ) Representative micrographs and bar charts of macrophages in the cornea after 7 days treatment with topical CGRP or PBS ( n = 4). ( H ) Corneal macrophages were determined by flow cytometry ( n = 4). ( I, J ) Representative flow cytometry plots and bar charts of CD206+ macrophages in the cornea of the CGRP treatment group ( n = 4). ( K, L ) Representative micrographs and bar charts of neutrophils in the cornea after 7 days treatment with topical CGRP or PBS ( n = 4). (M) Corneal neutrophils were determined by flow cytometry ( n = 4). Data are shown as mean ± SD. ns: not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For the culture of neutrophils, the mouse bone marrow neutrophil isolation solution kit (Solarbio) was used to isolate neutrophils according to the manufacturer's instructions.

Techniques: Expressing, Flow Cytometry

Journal: Investigative Ophthalmology & Visual Science

Article Title: Corneal Nerves Promote Alkali Burn Repair by Modulating Macrophages and Neutrophils via Calcitonin Gene-Related Peptide

doi: 10.1167/iovs.67.4.60

Figure Lengend Snippet: Macrophage depletion results in delayed corneal repair after injury and abolishes the therapeutic effect of CGRP. ( A ) Experimental timeline showing the schedule of PLX5622 feeding, corneal alkali burn induction, topical CGRP or vehicle treatment, clinical evaluation, and tissue harvesting. ( B ) Representative images of corneal fluorescein staining in PLX5622-fed mice topically treated with CGRP or vehicle. ( C ) Percentage of injured corneal area in PLX5622-fed mice treated with CGRP or vehicle. Quantitatively analyzed by ImageJ software ( n = 6). ( D ) Representative images of cornea appearance in PLX5622-fed mice treated with CGRP or vehicle. ( E ) Quantification of the clinical score of corneal opacity ( n = 6). ( F, G ) Representative micrographs and quantification of TUNEL+ cells in corneas 7 days after alkali burn in mice fed with PLX5622 or control diet ( n = 4). ( H, I ) Representative micrographs and quantification of neutrophils in corneas 7 days after alkali burn in mice fed with PLX5622 or control diet ( n = 4). Data are shown as mean ± SD. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For the culture of neutrophils, the mouse bone marrow neutrophil isolation solution kit (Solarbio) was used to isolate neutrophils according to the manufacturer's instructions.

Techniques: Staining, Software, TUNEL Assay, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Corneal Nerves Promote Alkali Burn Repair by Modulating Macrophages and Neutrophils via Calcitonin Gene-Related Peptide

doi: 10.1167/iovs.67.4.60

Figure Lengend Snippet: Expression of the CGRP receptor in neutrophils and macrophages, and transcriptomic analysis of macrophages following CGRP treatment in vitro. ( A, B ) Light microscope image of bone marrow-derived macrophages and bone marrow-derived neutrophils ( n = 4). ( C ) CALCRL and RAMP1 expression was detected in bone marrow-derived macrophages and neutrophils using immunostaining. Green = CALCRL, red = RAMP1 ( n = 4). ( D ) Heatmap of selected significantly upregulated and downregulated genes depicting standardized gene expression values in CGRP-treated cells compared to PBS-treated cells. The colored circles next to the heatmap denote gene functions. ( E ) Volcano plot displaying upregulated and downregulated genes in macrophages from the CGRP-treated group versus the PBS-treated group. ( F ) GO enrichment analysis of significantly upregulated ( red ) and downregulated ( blue ) genes in CGRP-treated and saline-treated macrophages.

Article Snippet: For the culture of neutrophils, the mouse bone marrow neutrophil isolation solution kit (Solarbio) was used to isolate neutrophils according to the manufacturer's instructions.

Techniques: Expressing, In Vitro, Light Microscopy, Derivative Assay, Immunostaining, Gene Expression, Saline

Journal: Investigative Ophthalmology & Visual Science

Article Title: Corneal Nerves Promote Alkali Burn Repair by Modulating Macrophages and Neutrophils via Calcitonin Gene-Related Peptide

doi: 10.1167/iovs.67.4.60

Figure Lengend Snippet: CGRP enhances apoptotic, anti-inflammatory, and efferocytic functions of macrophages. ( A, E ) Representative micrographs and quantification of apoptosis in macrophages treated with CGRP in vitro ( n = 4). ( B, F ) Representative micrographs and quantification of apoptotic macrophages in the cornea following 7 days of topical CGRP treatment ( n = 4). ( C, G ) Representative micrographs and quantification of apoptosis in neutrophils treated with CGRP in vitro ( n = 4). ( D, H ) Representative micrographs and quantification of apoptotic neutrophils in the cornea following 7 days of topical CGRP treatment ( n = 4). ( I, J ) CGRP treatment promotes CD206 and Arg-1 mRNA expression in macrophages ( n = 4). ( K, L ) Representative micrographs and quantification of macrophage efferocytosis of apoptotic neutrophils ( n = 4). Data are shown as mean ± SD. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For the culture of neutrophils, the mouse bone marrow neutrophil isolation solution kit (Solarbio) was used to isolate neutrophils according to the manufacturer's instructions.

Techniques: In Vitro, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Corneal Nerves Promote Alkali Burn Repair by Modulating Macrophages and Neutrophils via Calcitonin Gene-Related Peptide

doi: 10.1167/iovs.67.4.60

Figure Lengend Snippet: Schematic illustration of corneal nerve-derived CGRP facilitating alkali burn repair through coordinated actions on neutrophils and macrophages.

Article Snippet: For the culture of neutrophils, the mouse bone marrow neutrophil isolation solution kit (Solarbio) was used to isolate neutrophils according to the manufacturer's instructions.

Techniques: Derivative Assay