Surgical microscope

Anesthesia was performed by intraperitoneal injection of 50 mg/kg ketamine with zolazepam (Zoletil; Virbac, Carros, France) and 15 mg/kg xylazine hydrochloride (Rompun, Bayer, Leuverkeusen, Germany). Cautery of three episcleral veins were performed using a surgical microscope (Olympus, Tokyo, Japan). Then, normal perfusion of the retina was checked using planar ophthalmoscopy after cauterization. IOP was measured using a Tono-pen (Solan, Florida, USA) after topical anesthetization with Alcane (Alcon Laboratories, Fort Worth, Texas, USA). Eyes that did not present any complications during surgery or scleral burns were used.

All rats were anesthetized using an intraperitoneal (i.p.) administration of a mixture of medetomidine (0.15 mg/kg, i.p.; Domitor; Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), midazolam (2 mg/kg, i.p.; Dormicum Astellas Pharma Inc., Tokyo, Japan) and butorphanol (2.5 mg/kg, i.p: Vetporphale Meiji Seika Pharma CO., Ltd., Tokyo, Japan). Anesthetized rats were maintained at a constant temperature of 37 °C on a warming plate. The neck skin was shaved and opened under a surgical microscope (Olympus, Tokyo, Japan). The SLN was exposed bilaterally and injured with a vascular clip (60 g/mm²; NATSUME SEISAKUSHO Co Ltd., Tokyo, Japan) over a period of 30 min. The muscle and skin layers were closed with 4–0 Vicryl sutures (Ethicon Inc., Somerville, NJ). The animals were randomly assigned to the following four groups: (1) Sham: SLN exposure without any damage to the nerve tissue; (2) Injury: not injected; (3) DMEM (-): 1 ml DMEM (-)-injected into the tail vein for 10 s simultaneously with the SLN damage; (4) SHED-CM: 1 ml SHED-CM-injected into the tail vein for 10 s simultaneously with the SLN damage.

Post‐surgical lymphedema was induced in the tails of SD rats. The rats were anesthetized with sodium pentobarbital (50 mg kg⁻¹ intraperitoneally). Next, 0.1 mL of 2% methylene blue solution was injected intradermally into the end of the rat tail. An annular incision was made at 13 cm from the end of the tail. The skin and subcutaneous tissue were removed, thus removing the superficial lymphatic network. The muscles, tendons, bones, and major blood vessels below the dermis were left untouched. A surgical microscope (Olympus, Japan) was used to visualize the two deep lymphatic vessels stained with methylene blue, and the vessels were sutured with 6–0 silk thread and cut. This study was approved by the Animal Ethics Committee of China. All methods were performed in accordance with the relevant guidelines and regulations.