**Background:** Angiogenesis and lymphangiogenesis, the formation of new blood and lymphatic vessels from pre-existing vasculature, are essential in embryonic development and occur in pathological conditions such as cancer, ocular disease, and inflammatory disorders. The mouse cornea is a valuable model for studying these processes because it is normally avascular, allowing easy monitoring of induced vascularization. Among various corneal assays, thermal cauterization offers advantages of low cost, speed, and technical simplicity, and it induces an inflammatory response that mimics pathological (lymph-)angiogenesis. However, no standardized protocol existed for this assay, limiting reproducibility and cross-study comparisons. This paper aims to fill that gap by providing a detailed, stepwise protocol for corneal thermal cauterization in mice, including procedures for cauterization, cornea dissection, immunofluorescence staining, and morphometric analysis.
**Methods:** The protocol uses 8- to 12-week-old C57BL/6 mice. Mice are anesthetized with intraperitoneal ketamine (100 mg/kg) and xylazine (10 mg/kg), and a local anesthetic (Unicaine 0.4%) is applied to the eye. Thermal cauterization is performed using a low-temperature cautery kit (World Precision Instruments, 500391) with a monopolar tip heated to approximately 700°C. The cauterization involves a central burn followed by three concentric circumferences of edge-to-edge burns, covering no more than 80% of the cornea. After surgery, antibiotic ointment (Terramycin + Polymyxine B) and analgesic (Vetergesic, 0.05 mg/kg) are applied every 12 hours for 2 days. Mice are monitored daily for 7 days, during which new vessels grow from the limbus toward the injury. On day 7, mice are euthanized, and corneas are dissected: eyeballs are enucleated, muscle tissue removed, sclera and choroid cut off, and retina and lens separated from the cornea. Corneas are fixed in 70% ethanol for 1 hour, then blocked with 3% BSA and 3% Gloria milk in PBS. Whole-mount immunofluorescence staining is performed using rat anti-mouse CD31 antibody (1:200) and rabbit anti-LYVE-1 antibody (1:100) overnight at room temperature, followed by secondary antibodies (donkey anti-rat Cyanine 3 and donkey anti-rabbit Alexa Fluor 488, both 1:200) for 2 hours. Corneas are flat-mounted on slides with ProLong Gold antifade medium. Images are captured using a fluorescence microscope with a 10X objective and mosaic imaging. Quantification of vascularization is done using ImageJ software: the corneal area is measured, and the CD31+ (blood vessel) and LYVE-1+ (lymphatic vessel) areas are determined by thresholding, expressed as a percentage of total corneal area. Branch point density is also quantified.
**Key Results:** The protocol provides representative images of CD31+ and LYVE-1+ staining in cauterized corneas, showing neovascularization from the limbus. Based on previously published data (reference 12), the mean lymphangiogenic vascularized area in control C57BL/6 mice is approximately 25% of total corneal area, with a standard deviation of 5.5. The paper includes a sample size calculation example: for a two-sided t-test with 80% power, alpha error 0.05, and a 40% reduction in vascularized area (from 25% to 15%), the calculated effect size is 1.818, and the required group size is 6 mice per group. The protocol emphasizes that the assay is reproducible and allows for various treatment strategies, such as topical siRNA, ophthalmic drops with inhibitors, intrastromal injections, or use of transgenic mice.
**Clinical Implications:** The corneal thermal cauterization assay is a valuable preclinical tool for studying inflammation-induced (lymph-)angiogenesis and evaluating potential therapeutic targets. The protocol's standardization enhances reproducibility and facilitates comparison across studies. Notably, insights from this model have been translated to clinical contexts: the role of ketone bodies in lymphangiogenesis was discovered using this assay and subsequently replicated in myocardial infarction and lymphoedema models, leading to a clinical trial (NCT03991897) using ketone bodies to treat lymphoedema in breast cancer patients. This demonstrates the translational potential of findings from this assay. The protocol also discusses ethical considerations, including minimizing animal numbers through power calculations, using unilateral injury to preserve eyesight, and providing postoperative care (analgesics, antibiotics, and easy access to food and water). Overall, this protocol supports research into (lymph-)angiogenesis mechanisms and the development of therapies for conditions such as cancer, ocular diseases, and inflammatory disorders.