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Nakamoto et al. Plast Aesthet Res 2024;11:54 https://dx.doi.org/10.20517/2347-9264.2024.82 Page 3 of 14

the efficacy of topical application of beta-blockers in a mesh skin grafted full-thickness burn wound has not
been reported previously. Since endogenous adrenaline is elevated in burn patients and wound healing is
[7]
delayed , topical application of beta-blockers could more effectively promote the epithelialization of burn
graft wounds. Therefore, in the present study, we aimed to demonstrate the safety and efficacy of timolol in
a clinically relevant large animal model that closely mimics a clinical scenario, i.e., eschar removal within
24 h followed by autologous meshed skin graft, adequate wound dressing, and proper daily wound care and
assessment.

METHODS
Animal care
We followed the guidelines of the National Institutes of Health and the American Physiological Society
regarding the care and use of laboratory animals, and the protocol of this study was approved by the
University of Texas Medical Branch Animal Care and Use Committee (protocol #2003030). Adult Merino
female sheep with body weights of 35 to 40 kg were purchased, and all sheep for this study were screened by
a veterinarian and housed at the Animal Research Center, where they had free access to food and water.

Surgical procedure and treatment protocol
For surgical procedures, sheep housed in individual cages were transferred to the Translational Intensive
Care Unit after more than 14 days of quarantine. Sheep were sedated with an intramuscular injection of
500 mg of ketamine, followed by an intravenous injection (300 mg). They were then intubated under mask
inhalation anesthesia with 2%-5% isoflurane. Then, anesthesia was further maintained by 2%-5% isoflurane
inhalation via the endotracheal tube. The catheters were placed through skin incision in the groin into the
femoral artery and vein for continuous monitoring of systemic arterial blood pressure, heart rate, and
resuscitation and intermittent blood sampling, respectively. The analgesia was provided with a
subcutaneous injection of 0.1 mg/kg long-acting (72 h) buprenorphine. After catheter placement, three
25 cm full-thickness flame burn sites were made on each side of the dorsum (a total of six sites) [Figure 1],
2
2
with 5 cm of space between the sites and 3 cm from the spine. A burn-resistant cloth with a 25 cm open
space in the middle was installed to prevent burns to the adjacent tissues. A metal frame enclosing an area
2
of 25 cm was put on the open space of the cloth. The burn was induced by flame until skin shrinkage
stopped. This method was previously established in our laboratory to create full-thickness skin burns that
can be confirmed through histological analysis . No dressing was applied to the burn wounds during the
[14]
first 24 h after burn creation. After wound creation, anesthesia was discontinued and the sheep were
awakened and monitored for 24 h. For pain management, long-acting buprenorphine (72 h) was injected
subcutaneously and additional short-acting buprenorphine was injected intravenously per need.

Twenty-four hours after burn creation, sheep were again anesthetized with ketamine and intubated with
endotracheal tube for the anesthesia with inhaled isoflurane. The eschar was excised, under analgesia and
continued anesthesia, down to the superficial fascia. Split-thickness skin grafts, measuring 0.03 inches in
thickness, were harvested from both sides of the buttocks remote from the wounds. The harvested skins
were meshed at a ratio of 4:1 and grafted to the wounds [Figure 2]. The day of skin graft surgery was set as
Day 0. Wounds were randomly assigned to as either treatment or control. The groups were assigned to
allow comparison of contralateral sites as pairs in order to avoid anatomic location-dependent wound
healing variations. Wounds in the treatment group received topical application of timolol (0.02 mL/cm of
2
0.5%), and wounds in the control group received saline in a similar manner. After the application, the
wounds were covered with non-adherent strips (Curity, COVIDIEN, MA), and the non-adhesive
hydrocellular foam dressing (ALLEVYN, Smith&Nephew, England) was placed on top and secured with
skin staples. To apply proper pressure to the grafted skin, sterile gauze was placed on top of the
hydrocellular foam, followed by a second layer of large hydrocellular foam that was secured with skin