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Updated: Jan 2, 2026
Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia
Published on: November 17, 2014
Rip 1-dependent endothelial necroptosis participates in ischemia-reperfusion injury of mouse flap
Lele Geng1, Gai Zhang1, Min Yao1
1Department of Plastic Surgery, Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
Background:
Ischemia reperfusion injury plays an important role in free flap necrosis. However, the detailed mechanism is not clear, and effective methods for improving the survival rate of skin flap are still lacking.
Objective:
To investigate the regulation and functional link between necroptosis and ischemia-reperfusion injury of mouse flap.
Methods:
We established a mouse ischemia-reperfusion injury flap model and a cell Oxygen Glucose Deprivation (OGD) model intervened with Necrostatin-1. The mouse flap tissues were harvested in vivo for histological immunofluorescence analysis and western blotting analyses. The HUVECs cells with various treatments in vitro were assessed by using Transwell assay, tube formation assay, cell counting kit-8 analysis and flow cytometry. A Rip3-knockout cell line and a TNFR1-knockout cell line were generated from HUVEC cells using the CRISPR-Cas9 technology and were subsequently used to explore the related mechanisms.
Results:
The expression of p-Rip3 is positive in both mouse and cell culture models. When necroptosis is completely or partially inhibited in vivo, damaged tissues are repaired with better efficiency. The cells treated with Necrostatin-1 in vitro exhibit faster migration, proliferation and better tube formation. Deficiency of TNFR1 can block the necroptosis pathway by blocking the phosphorylation of Rip3 in HUVEC OGD/ROG model. Meanwhile, the levels of APJ, HIF-1α, and VEGF are reduced when necroptosis is inhibited by Necrostatin-1.
Conclusion:
TNFR1 mediates Rip1/Rip3 in ischemia-reperfusion injury. Inhibition of necroptosis attenuates the ischemia-reperfusion injury of flap and may enhance hypoxic tolerance of HUVECs and vascular homeostasis through regulation of the HIF-1α signaling pathways.
Insights
Necroptosis inhibition improves flap survival after ischemia reperfusion injury. Tumor Necrosis Factor Receptor 1 (TNFR1) mediates this process, enhancing hypoxic tolerance and vascular homeostasis via HIF-1α signaling.
Area of Science:
- Cellular Biology
- Tissue Engineering
- Pathophysiology
Background:
- Ischemia reperfusion injury (IRI) is a significant cause of free flap necrosis.
- The underlying mechanisms of IRI in flap survival remain unclear, necessitating novel therapeutic strategies.
Purpose of the Study:
- To elucidate the regulatory role and functional connection of necroptosis in mouse flap IRI.
- To investigate the therapeutic potential of inhibiting necroptosis in flap survival.
Main Methods:
- Established mouse IRI flap and cell Oxygen Glucose Deprivation (OGD) models, treated with Necrostatin-1.
- Utilized histological, immunofluorescence, and Western blot analyses in vivo.
- Employed Transwell, tube formation, CCK-8, and flow cytometry assays in vitro.
- Generated Rip3- and TNFR1-knockout HUVEC cell lines using CRISPR-Cas9 for mechanistic studies.
Main Results:
- p-Rip3 expression was observed in both in vivo and in vitro models.
- Necroptosis inhibition significantly improved tissue repair and flap survival.
- Necrostatin-1 treatment enhanced HUVEC migration, proliferation, and tube formation.
- TNFR1 deficiency blocked necroptosis signaling by inhibiting Rip3 phosphorylation.
- Inhibition of necroptosis reduced APJ, HIF-1α, and VEGF levels.
Conclusions:
- TNFR1 plays a critical role in mediating Rip1/Rip3 signaling during IRI.
- Inhibiting necroptosis attenuates flap IRI and enhances hypoxic tolerance in HUVECs.
- The protective effects involve the regulation of HIF-1α signaling pathways, promoting vascular homeostasis.
