PART I: Modulation of Radiation-Induced Delay in the Wound Healing by Ascorbic Acid in Mice Exposed to Different Doses of Hemi

Introduction Acute radiation exposure is of interest to defense and civil administrations concerned about atomic weapons. However, it is also of concern to radiation safety personnel due to the possibility of accidents involving large doses of radiation, and to radiotherapists performing more localized hemi-body irradiation for cancer treatment. Hemi-body irradiation in multiple fractionated doses is frequently used alone or in combination with surgery or other modalities for the treatment of various solid tumors. It produces both acute and late effects on the skin and subcutaneous tissues that have profound effects on the healing of surgical wounds.[1,2] Interaction of ionizing radiation with wounded tissue will create a situation where normal response to injury will be disrupted, leading to a protracted recovery period. Irradiation has been reported to produce multiple negative effects on wound healing processes: it inhibits inflammatory reactions, connective tissue proliferation, tissue formation, maturation of granulation tissue, transcription of collagen mRNAs, secretion of collagen, and neovascularization.[3,4] Skin wounds after irradiation may be complicated by infections encouraged by bone marrow injury. Fibroblasts in irradiated tissue either have decreased ability to replicate or there is a selective ablation of faster growing fibroblasts by irradiation.[5,6] Successful healing of wounds represents the sum of a sequence of well organized basic processes including inflammation, cell proliferation, matrix formation, remodeling, wound contraction, and epithelization.[7] Interaction of ionizing radiation with the normal wound healing process leads to delayed healing or chronic nonhealing wounds. An adequate knowledge of the altered pathophysiology of irradiated wounds is necessary to make a proper judgement to select potential therapeutic modalities and prophylaxis for irradiated wounds. Several attempts have been made to identify potential therapeutic approaches to augment healing in this setting. Hydrogel and hydrocolloid gel dressings have been used to decrease wound discomfort and wound healing time in radiation ulcers.[8] Phenytoin sodium has been reported to enhance the stimulating action of wound fluid on proliferation of fibroblasts and synthesis of collagen after irradiation.[9] There is evidence that supplemental vitamin A prevents the acute radiation-induced defects in wound healing, probably by enhancing the early inflammatory reaction to the wound and increasing the number of monocytes and macrophages at the wound site.[10] Certain radioprotective compounds like mercaptoethylamine, serotonin, and WR2721 (amifostine) have also been found to be useful in combined injuries.[11] Several growth factors and antimicrobial agents have been explored in animal models as potential options to improve wound healing in radiation-damaged skin.[12–14] Although basic research has suggested many potential therapies and prophylaxis for irradiated wounds, little attention has been given to the effects of dietary ingredients on the radiation response of healing wounds, which emphasizes a need for continued research in the area of medical management of irradiated wounds. Since wound healing abnormalities cause great physical and psychological stress to affected patients and are extremely expensive, the use of nutritional factors in the reconstruction of irradiated wounds is an attractive proposition because they have wide acceptability, better tolerance, do not have side effects, and can be safely manipulated for human use. Ascorbic acid is an essential ingredient of the daily human diet, and its supplementation has been proven to promote wound healing in animals and humans.[15–17] Ascorbic acid treatment has also been reported to confer protection against radiation in vitro and in vivo[18–20] and has beneficial effect on the course of radiation-induced skin injuries.[21] The present study was undertaken to investigate the efficacy of ascorbic acid treatment in mice exposed to different doses of hemi-body gamma radiation and stressed with additional trauma of open excision wounds on the skin. Materials and Methods Animals. The animal care and handling were carried out according to the guidelines set by the World Health Organization, Geneva, and the INSA (Indian National Science Academy, New Delhi). 8- to 10-week-old Swiss albino mice of either sex weighing 30 to 36g were selected from an inbred colony maintained under the controlled conditions of temperature (23±2 degrees C), humidity (50±5%), and light (10 and 14 hours of light and dark, respectively). The animals had free access to sterile food and water. The food consisted of 50-percent cracked wheat, 40-percent bengal gram, four-percent milk powder, four-percent yeast powder, 0.75-percent sesame oil, 0.25-percent cod liver oil, and one-percent salt. The levels of ascorbic acid were considered to be normal. Four animals were housed in a polypropylene cage containing sterile paddy husk (procured locally) as bedding throughout the experiment. The study was approved by the institutional animal ethical committee. Chemicals. Ascorbic acid (catalog No: A4544), hydroxyproline (catalog No: H5534), chloramine T (catalog No: C9887), glucosamine (catalog No: G44875), acetylacetone (catalog No: A3511), deoxyribonucleic acid (catalog No: D4522), diphenylamine (catalog No: D2385), b-nicotinamide-adenine dinucleotide phosphate (catalog No: N1630), N-(1-Naphthyl)ethylenediamine dihydrochloride (catalog No: N5889), and sulfanilamide (catalog No: S9251) were procured from Sigma Chemical Co., St. Louis, Missouri, USA, while trichloroacetic acid (catalog No: 15213-5000) and p-dimethylamino-benzaldehyde (catalog No: 42363-0250) were requisitioned from Across Organics, Geel, Belgium. Imidazole buffer (catalog No: 38546) was procured from S.D. Fine-Chemicals Ltd., Bangalore, India, while nitrate reductase (catalog No: 981 249) was purchased from Roche Applied Science, Mannheim, Germany. Ethanol (catalog No: SIN 1170) was purchased from Hayman Ltd., England, and sterillium disinfectant solution was procured from Bode Chemie, Hamburg, Germany. Perchloric acid, diethyl ether, formalin, sodium hydroxide, sodium nitrate, sodium nitrite, phosphoric acid, hydrochloric acid, and sodium chloride were requisitioned from Ranbaxy Fine Chemical, New Delhi, India. Preparation of drug and mode of administration. Ascorbic acid (AA) was procured as fine crystals, and the required amount was dissolved in sterile Milli-Q water (Millipore India Ltd., Bangalore), henceforth MQW. The animals were injected with MQW or AA intraperitoneally. Experiment 1 protocol: Wound contraction studies. The animals were divided into the two following groups: 1) MQW+irradiation: The animals in this group received 0.01mL/g body weight of MQW before irradiation; and 2) AA+irradiation: The animals in this group received 250mg/kg body weight of AA before irradiation. Irradiation. Forty-five minutes after administration of MQW or AA, each animal was placed in a specially designed, well ventilated, acrylic restrainer, and the lower halves (below the rib cages) of the animals were exposed to 0, 2, 4, 6, or 8 Gy, given at a dose rate of 1.35 Gy/min from a 60Co Teletherapy source (Theratron, Atomic Energy Agency, Ontario, Canada). Production of full-thickness skin wounds. The fur of the dorsum (below the rib cage) of each animal was removed with a cordless electric mouse clipper (Wahl Clipper Corporation, Illinois, USA) before exposure to radiation, and a full-thickness skin wound was produced on the dorsum (below the rib cage) of the animal as described by Jagetia, et al.,[22] within 10 minutes of irradiation. Briefly, the animals were anesthetized using diethyl ether, and the skin of entire body was cleaned and decontaminated by wiping the whole body with sterillium disinfectant solution. The cleared dorsal surface of skin was marked with a sterile circular (15mm-diameter) stainless steel stencil. A full-thickness wound was created by excising the skin flap in an aseptic environment using sterile scissors and forceps. Each wounded animal was housed in a separate sterile polypropylene cage. Measurement of wound contraction. Wound contraction was monitored as described earlier[22] by capturing the video images of each full-thickness wound with a CCD camera connected to a computer. The first image of each wound from different groups was obtained one day after wounding and that day was considered as Day 1. The subsequent images were captured on 3, 6, 9, 12, and 15 days post-irradiation. The wound area was calculated using Auto CAD R14 (Autodesk Inc., San Rafael, California) software. Eight animals were used in each group at each exposure dose. Experiment 2 protocol: Mean wound healing time. A separate experiment was performed to evaluate the effect of AA on mean healing time after exposure 0, 2, 4, 6, or 8 Gy hemi-body gamma radiation. All animals in each group were monitored until complete healing of wounds, and the day at which each wound healed was recorded. Mean of all healed wounds was determined and has been expressed as mean wound healing time in days. Eight animals were used in each group at each exposure dose. Experiment 3 protocol: Biochemical studies. A separate experiment was carried out to study the alteration in the various biochemical profiles of excision wounds after exposure to 0 or 6 Gy hemi-body gamma radiation and its modulation by AA. Grouping of animals and production of wounds were essentially similar to experiment 1, except the lower half of the animal body (below the rib cage) was exposed to 0 or 6 Gy gamma radiation. Wound biopsies were collected on Days 4, 8, and 12 post-irradiation and stored at -70?C until analysis. As an indication of total collagen content, hydroxyproline concentration was determined as described by Woessner.[23] The weighed granulation tissues were hydrolyzed in 6 N HCl for three hours at 130 degrees C, neutralized to pH 7 with 2.5 N NaOH, and diluted with MQW. The diluted solution was mixed with chloramine-T reagent and incubated for 20 minutes at room temperature. Thereafter, freshly prepared p-dimethylamino-benzaldehyde (Ehrlich’s reagent) solution was added and incubated for 15 minutes at 60 degrees C. The absorbance was measured at 550nm using a double beam ultraviolet (UV)-visible spectrophotometer (Shimadzu UV-260, Shimadzu Corp., Tokyo, Japan). The amount of hydroxyproline was determined by comparing with a standard curve. Thereafter, a factor of 6.94 was applied (used for most mammalian species) to calculate total collagen from hydroxyproline analysis.[24] Collagen content of granulation tissues has been expressed as mg/g dry tissue weight. Six animals were used in each group at each interval. For estimation of hexosamine, the granulation tissues were hydrolyzed in 6 N HCl for eight hours at 98 degrees C, neutralized to pH 7 with 4 N NaOH, and diluted with MQW. Hexosamine contents of granulation tissues were estimated by the method of Elson and Morgan[25] with minor modifications. The diluted solution was mixed with acetylacetone solution and heated to 96 degrees C for 40 minutes. The mixture was cooled, and 96-percent ethanol was added, followed by p-dimethylamino-benzaldehyde solution (Ehrlich’s reagent). The solution was thoroughly mixed and kept at room temperature for one hour, and the absorbance was measured at 530nm using a double beam UV-visible spectrophotometer. The amount of hexosamine was determined by comparing with a standard curve. Hexosamine content has been expressed as mg/g dry tissue weight. Six animals were used in each group at each interval. Deoxyribonucleic acid (DNA) content was measured by homogenizing the vacuum-dried granulation tissues in five-percent trichloroacetic acid (TCA) followed by centrifugation. The pellets were washed with 10-percent TCA, resuspended in five-percent TCA, and incubated at 90 degrees C for 15 minutes. The contents were centrifuged again, and the resultant supernatant was used for the determination of DNA content by the method of Burton.[26] The DNA was hydrolyzed with 60-percent perchloric acid at 80 degrees C for 20 minutes followed by the addition of Burton’s diphenylamine reagent and incubation at room temperature overnight. Thereafter, 95-percent ethanol was added, and absorbance was measured at 600nm using a double-beam UV-visible spectrophotometer. The amount of DNA was determined by comparing with a standard curve and has been expressed as mg/g dry tissue weight. Six animals were used in each group at each interval. Nitrate and nitrite, the stable end products of nitric oxide (NO), were measured in the granulation tissue of wounds. The granulation tissues were homogenized in hypotonic saline and centrifuged. Nitrite concentrations were determined with the Griess reagent.[27] Briefly, the supernatant was mixed with Griess reagent (0.1% NEDD, 1% sulfanilamide, and 5% phosphoric acid in a 1:1:1 ratio, prepared freshly) and incubated at 37 degrees C for 30 minutes, and the absorbance was recorded at 543nm using a double-beam UV-visible spectrophotometer. Sodium nitrite was used as standard. Nitrite levels were expressed in terms of µM/100mg dry tissue weight. Nitrate concentrations were quantified with nitrate reductase assay.[28] Briefly, 0.275mg/mL of b-NADPH in imidazole buffer (pH 6.8), 0.41U/mL nitrate reductase, and test solutions were mixed. Griess reagent was added to this mixture, incubated at 37 degrees C for 30 minutes, and the absorbance was measured at 543nm. Sodium nitrate was used as standard. Nitrate levels were expressed in terms of mM/g dry tissue weight. Six animals were used in each group at each interval. Experiment 4 protocol: Histological studies. A separate experiment was conducted to evaluate the histological alterations during wound healing after exposure to 0 or 6 Gy hemi-body gamma radiation. Grouping of animals and production of wounds were carried out as described earlier, except hemi-body of the animals was exposed to 0 or 6 Gy gamma radiation. The cross-sectional, full-thickness skin biopsies from each group were collected on Days 4, 8, and 12 post-irradiation. The samples were fixed in 10-percent buffered formalin, passed through different grades of alcohol, and were embedded in paraffin wax. Medial samples were sectioned (5µm) and stained with hematoxylin and eosin. Sections were assessed in a blinded fashion under light microscope for fibroblast proliferation, neovascularization, and collagen deposition. For collagen deposition studies, faintest traces of staining reaction, hyalinization, and irregular arrangement of collagen bundles were considered as +, while the most intense reaction and compactly arranged collagen bundles were considered as +++. Two areas in each section were counted for neovascularization and fibroblast proliferation. The elongated or spindle-shaped cells with purple nuclei and pink cytoplasm were identified as fibroblasts and scored. Blood vessels that were conspicuous with the stain were scored for vascular repopulation studies. A total of three animals were used in each group at each interval. Analysis of Data Statistical significance between the treatments was determined using one-way ANOVA. The Solo 4 Statistical Package (BMDP Statistical Software Inc., Los Angles, California, USA) was used for data analysis. All data are expressed as mean ± SEM (standard error of the mean). Results Experiment 1: Wound contraction studies. Progression of the healing of excision wounds can be assessed by periodic computation of wound contraction. The area of each wound at a specific time has been expressed as the percentage of its original size on Day 1. The mean corresponding area of wound for each group was plotted as a function of days after wounding. Wound contraction progressed with time, and a steady contraction of the excision wound was noticed in both MQW+sham-irradiation and AA+sham-irradiation groups (0 Gy). The maximum wound contraction was observed at 6 to 12 days post-irradiation in both MQW and AA+sham-irradiation groups. AA treatment resulted in a significant enhancement of wound contraction on Days 3 (pExperiment 2: Mean wound healing time. The complete closure of wounds was observed at 17.3±0.29 days post-irradiation in MQW+sham-irradiation group, while treatment of mice with AA resulted in a significant decline in healing time (Day 16.47±0.26 post-irradiation) in AA+sham-irradiation group. The hemi-body exposure of mice to different doses of gamma radiation resulted in a dose-dependent delay in the complete closure of wounds; as a result, the mean wound healing time was also increased in MQW+irradiation group when compared with the MQW+sham-irradiation group. A mean wound healing time of 17.8±0.29, 18.7±0.29, 19.6±0.29, and 21.0±0.29 was observed for 2, 4, 6, and 8 Gy, respectively, in MQW+irradiation group (Figure 2B). Treatment of mice with 250mg/kg of AA before irradiation to different doses of gamma radiation inhibited the radiation-induced delay in healing of excision wounds; as a result, there was a decline in mean wound healing time. The mean wound healing time of 17.0±0.28, 17.8±0.28, 18.7±0.29, and 20.2±0.29 was observed for 2, 4, 6, and 8 Gy, respectively, in AA+irradiation group (Figure 2B). This reduction in wound healing time was statistically significant (pExperiment 3: Biochemical studies. Hydroxyproline content is an index of collagen and measures the synthesis of neocollagen. Estimation of hydroxyproline content revealed the greatest synthesis of collagen in MQW or AA+sham-irradiation groups at Day 8 post-irradiation; thereafter, synthesis of collagen remained unaltered in both the groups. Irradiation of animals to 6 Gy resulted in a drastic decline in collagen synthesis at all post-irradiation time periods (Figure 3A), and this decrement was significant only at Day 4 post-irradiation (pExperiment 4: Histological studies. Histological evaluation of wound biopsies at various post-irradiation time periods revealed that AA alone did not alter the histology except for an increase in collagen deposition and fibroblast and vasculature densities compared to the sham-irradiation, non-drug-treated controls. Exposure of mice to 6 Gy radiation caused mild degeneration of collagen bundles. Very few isolated ‘‘fragments’’ of collagen surrounded by unstained spaces were seen at this radiation dose. Treatment of irradiated mice with AA caused an increase and restoration of collagen bundles. The density of fibroblasts also declined in MQW+radiation group when compared with MQW+sham-irradiation group (Figure 5A). Sparse numbers of large and stellate cells or ‘‘radiation fibroblasts’’ were discernible after irradiation at Day 8 post-irradiation. A similar trend was observed for vascularization. Very few irregularly shaped blood vessels were seen in the MQW+irradiation group. Treatment with AA protected mice against radiation-induced damage to fibroblasts and vasculature as revealed by an increase in the density of fibroblasts and vasculature (Figure 5A and B). However, normal histological picture was not restored. Discussion continued in Part II. To read part II, Go to WOUNDS home page, find Volume 15, Issue 11, and Click on "PART II: Modulation of Radiation-Induced Delay in the Wound Healing by Ascorbic Acid in Mice Exposed to Different Doses of Hemi-Body Gamma Radiation."