Page 43 - JCTR-11-1

Journal of Clinical and
Translational Research Hesperidin enhances repair of γ-irradiated wounds

described. 8-10 Briefly, the animals were anesthetized using 2.4.3.1.1. Collagen
diethyl ether, and the entire body was thoroughly cleaned To assess the total collagen content, hydroxyproline
and decontaminated using sterillium (Bode Chemie, concentration was determined, as described by Woessner.
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Germany) disinfectant solution, followed by fur removal. The weighed granulation tissue was hydrolyzed in 6 N HCl
The cleared dorsal surface of the skin was marked with a for 3 h at 130°C, neutralized (pH 7) with 2.5 N sodium
sterile circular stainless steel stencil (15 mm diameter). hydroxide, and diluted with Milli-Q water (18 MΩ).
A full-thickness wound was created by removing the skin The diluted solution was mixed with chloramine-T
flap in an aseptic environment using sterile scissors and reagent and incubated for 20 min at room temperature,
forceps. Each wounded animal was housed individually in followed by the addition of freshly prepared Ehrlich’s
a sterile polypropylene cage. reagent solution. The mixture was incubated for 15 min

2.4.1.3. Wound contraction at 60°C. The absorbance of each sample was measured
at 550 nm using an Ultraviolet (UV)-visible double-
Wound contraction was monitored by capturing video beam spectrophotometer (Shimadzu UV-260, Shimadzu
images of each full-thickness wound using a charge- Corp., Japan). The concentration of hydroxyproline was
coupled device camera connected to a computer. 8-10 The determined by comparing the absorbance of the samples
first image of each wound from different groups was to a standard curve. Total collagen content was determined
obtained 1 day after wounding, which was designated as by multiplying the hydroxyproline concentration with a
day 1. Subsequent images were captured on days 3, 6, 9, factor of 6.94. The collagen content in granulation tissue
12, and 15 post-wounding. The wound area was measured was expressed as mg/g dry tissue weight.
using Auto CAD R14 (Autodesk Inc., United States)
software. Ten animals were used in each concurrent group 2.4.3.1.2. DNA
for each radiation dose; however, for the 40 Gy radiation The DNA content in the granulation tissue/s was measured
group, a minimum of 12 animals were used to account for using the method described by Burton. Briefly, a known
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potential radiation-related mortality. A total of 168 animals amount of dry granulation tissue was homogenized in
were utilized for this experiment. 5% TCA, followed by centrifugation. The resulting pellets
2.4.2. Experiment 2: MHT were washed with 10% TCA, resuspended in 5% TCA, and
incubated at 90°C for 15 min. Following this incubation,
A separate experiment was performed to evaluate the the contents were centrifuged again, and the resultant
effect of hesperidin on MHT after exposure to 0, 10, 20, supernatant was used to determine DNA content. The DNA
or 40 Gy of fractionated γ-radiation. The grouping and was hydrolyzed with 60% perchloric acid at 80°C for 20 min.
other conditions were essentially similar to that described After hydrolysis, Burton’s diphenylamine reagent was
above. All the animals in each group were monitored until added and the mixture was incubated at room temperature
complete healing of wounds and the day each wound healed overnight. Subsequently, 95% ethanol was added to the
was recorded. The mean of all healing days was calculated solution, and absorbance was recorded at 600 nm using a
and expressed as the MHT in days. Each concurrent UV-visible double-beam spectrophotometer. The amount
group consisted of 10 animals for each radiation dose, of DNA was determined by comparing the absorbance of
except 40 Gy, where a minimum of 12 animals was used to the samples to a standard curve and was expressed as mg/g
account for potential radiation-related mortality. A total of dry tissue weight.
104 animals were used for this experiment.
2.4.3.1.3. NO
2.4.3. Experiment 3: Biochemical estimations
The stable end products of NO biosynthesis were
2.4.3.1. Granulation tissue determined by measuring nitrite levels in the granulation
A separate set of experiments was carried out to study tissue of wounds. Granulation tissues were homogenized in
the effect of hesperidin on various biochemical profiles hypotonic saline and centrifuged to obtain the supernatant.
of irradiated wounds after exposure to 0, 10, 20, or 40 Gy Nitrite concentrations were determined with the Griess
of fractionated γ-radiation. The grouping of animals reagent. Briefly, the supernatant was mixed with freshly
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and production of full-thickness excision wounds were prepared Griess reagent (0.1% NEDD, 1% sulphanilamide,
similar to the procedures described for the wound and 5% phosphoric acid in a 1:1:1 ratio), incubated at 37°C
contraction experiment. However, in this experiment, for 30 min, and the absorbance was recorded at 543 nm
biopsies of regenerating wounds were collected on days using a double beam UV-visible spectrophotometer.
4, 8, and 12 post-irradiation and stored at −70°C until Sodium nitrite was used as the standard for comparison.
analysis. Nitrite levels were expressed as µM/100 mg dry tissue

Volume 11 Issue 1 (2025) 37 doi: 10.36922/jctr.24.00049