Lymphedema and Wound Healing in Rats

The study was approved by the Institutional Animal Care and Use Committee of Taipei Veterans General Hospital. All animal care complied with the Guide for the Care and Use of Laboratory Animals (No. IACUC 2019-108, IACUC 2020-155, IACUC 2020-240). Fifteen male Sprague Dawley rats ((BioLASCO, Yilan, Taiwan)) aged 20 to 24 weeks were used for this research. Animals were divided into three groups randomly, which were as follows: (i) secondary healing (control) (n = 5); (ii) decellularized Wharton’s jelly (dWJ) (n = 5); and (iii) ASC-decellularized Wharton’s jelly (ASC/dWJ) (n = 5). Sprague Dawley rats were anesthetized with an intraperitoneal injection of 50 mg/kg body weight of Zoletil 50 (Virbac, Carros cedex, France). The anesthetized rats were placed in a prone position over a warm pad. The diameter of the tail and the excision wound was calculated by a Vernier scale. Evan’s blue dye (E2129, Sigma-Aldrich, St. Louis, MO, USA) was injected into the tail base before operation in order to identify the lymph vessels. After shaving and sterilization, a circumferential excision (1 cm in width) of skin at a 5 mm distance from the base of the tail was made (Figure 1A). The superficial lymphatic network was removed, and the deep lymphatic system and lateral tail veins were preserved (Figure 1B). The rats recovered from anesthesia in a separate cage after the operation and received food and water ad libitum.
Tail volume and wound width for each group were evaluated to track tail lymphedema and wound-healing. The data for each group were compared from week 1 to week 5 after surgery. The tail volumes were calculated using a truncated cone formula according to previously published methods (Figure 1C) [40 (link)]. For this purpose, tail diameter at 2 cm and 16 cm from the tail’s end were measured by two blinded investigators using a digital caliper. The increase in volume, which represents lymphedema, was defined as post- vs. pre-operative volumes of the same animal for each week.
The digital images of the wound were captured weekly in order to quantify the wound width. Width was determined by the average of three locations (the edge of the wound on both sides and the center of the wound). The values were measured from digital pictures using ImageJ software (version 1.53t).