Page 141 - ITPS-7-2

INNOSC Theranostics and
Pharmacological Sciences Dapsone enhance skin flap survival

from the caudal section. Flaps were maintained in moist, obtaining the specimens, the rats were sacrificed using a carbon
sterile gauzes during the operation. Subsequently, each monoxide (CO) chamber. The first half of the specimens
flap was sutured back into its innate position using 4-0 were processed, embedded in paraffin blocks, sectioned
reversed-cut nylon sutures. Corner stitches were employed into 5 µm thick slices, and prepared for hematoxylin and
to suture the cranial angles, while simple cutaneous eosin (H&E) staining. An expert pathologist, blinded to the
stitches were used for all other margins. The surgical site groups and study design, examined all H&E-stained sections
was covered with sterile wound dressing, and each rat was for neutrophil infiltration, blood vessel distribution and
housed individually in clean cages to facilitate recovery. All angiogenesis, and ulcer thickness using an optical microscope.
of the rats survived the operation and lived through 7 days The second half of the specimen was homogenized using lysis
of recovery. buffer (2X Lysis Buffer; RayBio , USA) and then centrifuged
®
for 15 min (13,000 rpm, 4 °C). Subsequently, the IL-8 levels
2.5. Macroscopic evaluation
were measured using the enzyme-linked immunosorbent
Each day, dorsal skin flaps underwent careful post- assay (Abcam, UK) method, adhering to the manufacturer’s
nd
operative inspection and examination. On the 2 day guidelines.
following the surgery, distal free edge necrosis was
observed in a rat treated with dapsone 5 mg/kg/day. The 2.7. Immunohistochemistry (IHC)
necrotic area extended more than 1 mm from the surgical The paraffin-embedded flap specimens were processed
line, potentially attributed to inadvertent trauma during for IHC staining. These sections were then incubated
the procedure. Moreover, on the 5 post-operative day, with anti-TNF-α (ab6671, Abcam , USA, 1:100) and
th
®
infection-related necrosis was observed in another rat anti-VEGF antibodies (ab46154, Abcam , USA, 1:100).
®
from the control group, likely stemming from inadequate After washing with phosphate-buffered serum (PBS),
sterilization control during the operation. No signs of specific binding to primary antibodies was visualized
circulatory failure were observed among the rats. To ensure by enzymatic conversion of the chromogenic substrate
the evaluation of skin flaps based on a comparable equal 3,3-diaminobenzidine (DAB Substrate Kit, ab64238,
quality of procedure, these rats were excluded from the Abcam , USA) into a brown precipitate using horseradish
®
study. On the 7 post-operative day, rats were anesthetized, peroxidase (ab6721, Abcam , USA), in accordance
th
®
and the flaps were assessed by an author blinded to the with established protocols. Subsequently, the sections
treatment condition. Macroscopic changes, including were mounted, cleared, and dehydrated. The slides were
appearance, skin color, texture, and hair condition of each subjected to double staining using annexin-V fluorescein
flap, were carefully noted. Digital images were captured isothiocyanate (FITC) (Abcam, USA) and were evaluated
from the dorsal skin flaps to evaluate flap viability. Necrotic using a fluorescence microscope. Expression levels of the
areas were characterized by dark color, edema, and eschar desired markers were assessed and analyzed using ImageJ
formation, while the total flap area was delineated by the software (U.S. National Institutes of Health, USA).
surgical borders. ImageJ software (U.S. National Institutes
of Health, USA) was used to demarcate and analyze the 2.8. Statistical analysis
viable surface area. Flap survival was calculated and
reported as follows: Data are expressed as mean ± standard deviation (SD)
and were analyzed using one-way and two-way analyses
Flap survival (%) = (Area of necrotic tissue/Total area of of variance (ANOVA), followed by Tukey’s post hoc test
the flap) × 100 (I) for multiple comparisons between groups. All statistical

2.6. Histopathological analysis and enzyme-linked analyses were performed using GraphPad Prism software
immunosorbent assay version 6.07. P < 0.05 was considered statistically
significant.
Histopathological evaluations were performed to examine the
effect of dapsone on flap survival rate at a microscopic level. 3. Results
After the macroscopic assessments on the 7 post-operative 3.1. Assessment of skin flap survival
th
day, the rats were anesthetized through intraperitoneal
administration of ketamine HCl (90 mg/Kg) and xylazine Figure 2 illustrates the percentage of necrosis as the survival
HCl (10 mg/Kg). The necrotic tissue from the flap’s cranial rate of the skin flap among the rats. A one-way ANOVA
part and the viable tissue from the caudal part were dissected, confirmed a significant difference between study groups
with the obtained specimens divided into two halves: one half (F[3, 13] = 28.01, P < 0.001). Tukey’s test revealed that
was fixed in 10% formalin solution for 24 h, while the other treatment with dapsone at 12.5 mg/kg significantly reduced
half was placed in liquid nitrogen at −70°C. Immediately after the necrosis percentage compared with the control group

Volume 7 Issue 2 (2024) 4 doi: 10.36922/itps.2241