Abstract: Hemi-body irradiation in multiple fractionated doses is frequently used alone or in combination with surgery for the treatment of cancer. It produces both acute and late effects on the skin that have profound effects on surgical wounds. Because of the crucial practical importance of radiation exposure associated with skin wounds, it is imperative to investigate the efficacy of cost-effective nutritional factors in the reconstruction of irradiated wounds. Therefore, the effect of ascorbic acid treatment was studied on the healing of excision wounds in mice exposed to 2, 4, 6, or 8 Gy hemi-body gamma radiation. A full-thickness skin wound was created on the dorsum of 8- to 10-week-old Swiss albino mice after hemi-body exposure to 2, 4, 6, or 8 Gy. The progression of wound contraction was monitored periodically by capturing video images of wounds. Collagen, hexosamine, deoxyribonucleic acid (DNA), nitric oxide (NO), and histological profiles of excision wounds were also evaluated and either treated or not treated with ascorbic acid before exposure to 0 or 6 Gy. Irradiation caused a dose-dependent delay in wound contraction and wound healing time, while ascorbic acid pretreatment resulted in a significant elevation in the rate of wound contraction and a decrease in mean wound healing time. Treatment with ascorbic acid before irradiation enhanced the synthesis of collagen, hexosamine, DNA, and NO, while histological assessment revealed an improved collagen deposition and an increase in fibroblast and vascular densities. The present study demonstrates that ascorbic acid pretreatment has a beneficial effect on irradiated wounds and could be part of a strategy to ameliorate radiation-induced delay in wound repair.

Continued from Part I. To read Part I, go to WOUNDS home page, Volume 15, No. 11: click on "PART I: Modulation of Radiation-Induced Delay in the Wound Healing by Ascorbic Acid in Mice
Exposed to Different Doses of Hemi-Body Gamma Radiation"

Discussion

The response of normal tissue to radiation can be viewed as comprising two partially interacting components. The first is a process that resembles the healing of traumatic wounds, perturbed by radiation treatment. The second is a set of specific injuries that affect virtually all cellular and extracellular components within irradiated volume and that may be responsible for the progression of injury over a period of time. The detrimental consequences of ionizing radiation on wound healing is multifaceted: it has direct cytotoxic effects on various cellular/molecular components of wound repair, and it has indirect effects through the production of a burst of free radicals, which rearranges tissue components immediately, causing DNA damage and altering other complex molecules involved in tissue repair and regeneration. Irradiation of skin results in slower healing of open wounds and provides a proper in-vivo system for evaluation of natural metabolites/antioxidants in radiation-impaired wounds. There is a need for a multifunction drug that conforms to all criteria of an optimal radioprotector, including effectiveness, toxicity, availability, wider biological functions, and tolerance. The use of natural radioprotectors/antioxidants to overcome direct negative effects of ionizing radiation and for targeting reactive oxygen species could be an important therapeutic strategy to improve healing of irradiated wounds. AA is an important constituent of the daily human diet and has been proven to promote wound healing in various wound models.[15–17] Furthermore, AA has been found to be a good antioxidant[29,30] and radioprotective agent.[18–20] AA has beneficial effect on the course of radiation-induced skin injuries.[21] Therefore, the present study was undertaken to obtain an insight into the effect of AA on wound healing in mice exposed to different doses of hemi-body gamma radiation.

Wound contraction can be defined as the centripetal movement of the edges of a full-thickness wound in order to facilitate closure of the defect.[31] The progression of wound healing can be judged by the periodic assessment of the contraction of excision wounds. The exposure of mice to different doses of hemi-body radiation delayed wound contraction in a dose-dependent manner, and the greatest delay was observed for 8 Gy irradiation. A similar delay in wound contraction after exposure to gamma radiation has been observed earlier, indicating that irradiation alters the local conditions of wounds, which is not conducive to wound repair.[22,32,33] Treatment of mice with 250mg/kg body weight of AA before exposure to different doses of hemi-body irradiation resulted in an enhancement of wound healing as is evident by greater degrees of wound contraction and reduction in mean wound healing time. Moreover, the quality of wound healing was far better than MQW+irradiation group (scab formation was less in the AA-treated group). Studies on the use of vitamins in conjunction with radiation on enhancement of wound healing are scanty. However, Vitamin A supplementation has been reported to ameliorate the acute radiation-induced delay in wound healing.[10] Vitamin E treatment has also been reported to normalize the breaking strength of wounds that received preoperative irradiation.[34] Recently AA administered once daily before exposure of mice to fractionated radiation enhanced the healing of wounds.[22] The similar effect of AA in improving the irradiated wound cannot be ruled out if AA treatment is continued even after irradiation.

Collagen occupies a central role in the healing of wounds, as it is a principal component of connective tissue and provides a structural framework, strength, and proper milieu for the regenerating tissue. Collagen is produced by fibroblasts and assists the wound in gaining tensile strength during repair.[7] Irradiation of animals caused a significant reduction in collagen synthesis, as is evident from the estimation of hydroxyproline content in the granulation tissue. Grant, et al.,[35] demonstrated that with increasing doses of gamma rays there was progressive destruction of the native collagen fibrils. AA inhibited the radiation-induced decrease in collagen synthesis at all post-irradiation days. A resembling effect has been reported earlier in mice exposed to different doses of fractionated gamma radiation.[22]

Hexosamine, ground substratum for collagen synthesis, is known to increase during early stages of wound healing and decrease thereafter.[36] A similar trend has been observed in the present study in hexosamine content of wounds. The increase in DNA content of treated wounds indicated hyperplasia (cellular proliferation) of cells. The hemi-body irradiation decreased hexosamine and DNA content, while AA treatment significantly increased hexosamine and DNA contents at Days 4 and 8 post-irradiation. The topical application and oral administration of Aloe vera has been reported to increase significantly both DNA and hexosamine contents in wounds of diabetic rats.[37] Wound repair results from a series of well-coordinated cellular and biochemical events, including increased synthesis of the bioregulatory molecule NO. Most evidence suggests that adequate rate of NO production promotes processes central to wound healing, such as inflammation, angiogenesis, fibroblast synthetic function, epithelial cell proliferation, regulation of collagen formation, and wound contraction in distinct ways.[37,38] Levels of nitrite and nitrate, the stable end-products of NO synthesis, are elevated early and transiently in fluid obtained from sponges implanted in subcutaneous wounds.[39] A similar trend has been observed in the granulation tissues of mice exposed to 6 Gy hemi-body gamma radiation or pretreated with AA in the present study. This early increase may be due to the onset of inflammatory reaction in the wound micro-environment. The decrease in NO expression has been correlated with radiation-induced impairment in wound healing.[40,41]

Histological observations support the decrease in collagen content where a decline in collagen deposition has been observed. This is further supported by a decrease in the proliferation of fibroblasts, which are responsible for collagen synthesis. A direct negative impact of ionizing radiation on wound fibroblast proliferation, neovascularization, and collagen deposition has been reported.[42] Similarly, total-body and hemi-body irradiation decreased cellular influx, vascularization, and collagen deposition in mice.[43] The decrease in fibroblast proliferation and vascularization is in agreement with earlier reports where a similar decrease in fibroblast proliferation, retardation of collagen maturation, and overall delay in wound repair has been observed.[22,42,44] Cell culture studies of fibroblasts exposed to ionizing radiation have also demonstrated that irradiated fibroblasts have a significant prolonged generation time when compared to normal fibroblasts.[5] The pretreatment of mice with AA improved collagen deposition, reduced the hyalinization, and increased the vascularity and fibroblast density in the AA+irradiation group. AA has been reported to increase collagen deposition in normal animals[15–17] and to improve the overall histological picture in mice exposed to fractionated irradiation.[22]

There are several possible explanations for alterations in wound healing after irradiation. Ionizing radiation has been reported to cause severe damage to vital tissues, especially those with a high rate of cell division, such as the hematopoietic system.[10,45] The loss of a significant number of bone marrow cells can lead to an immunosuppressed state in which the individuals become highly susceptible to bacterial infections leading to complications in the healing of wounds. Shielding of bone marrow during acute whole body X-irradiation has been reported to lower mortality and increase the closure of open dorsal skin wounds of rats.[46] These studies suggest a requirement of radiation-sensitive, bone marrow-derived cells in tissue repair. Another possibility is that a delay may occur in the fixation of the wound edge to underlying tissue, which may be due to a lack of fibroblast proliferation and a decrease in fibroblast synthetic function in the granulation bed. The contraction of open excised wounds has been found to be a function of contractile fibroblasts, known as myofibroblasts.[47] Irradiation is thought to impair wound healing in skin through its cytotoxic effect on fibroblasts. This impairment may be due to the delay in the progression of cells through the cell cycle induced by radiation.[5,48] Radiation may have adverse effects on fibroblasts through bone marrow depression, since some fibroblasts of the normal subcutaneous connective tissue participating in wound healing were shown to take their origin from the bone marrow.[49,50] Recent studies suggest that vascular cell progenitors are resident in bone marrow and contribute to blood vessel formation during tissue repair and in other pathological conditions.[51,52] A similar effect cannot be ruled out in the present study, since the long bones of the hind limbs were irradiated during the hemi-body irradiation. Furthermore, radiation-induced cytotoxicity is mediated through the production of oxygen-derived free radicals. When reactive oxygen species are overproduced, oxidative stress results with detrimental cytotoxic effects causing delayed wound healing.[53]

The inhibition of delay in contraction of irradiated wounds by AA may have several possible causes. Numerous studies have indicated the beneficial effect of AA on wound healing through changes in cell regeneration and collagen synthesis.[15–17,22,54] In addition, AA has been found to act as an antioxidant and a radioprotective agent.[18–20,29,30] A scavenging of radiation-induced free radicals by AA cannot be overlooked in the present study. AA pretreatment has been reported to elevate glutathione and certain antioxidant enzymes and reduce lipid peroxidation in irradiated wounds.[55] Heller, et al.,[56] suggested that intracellular AA enhances NO synthesis in endothelial cells and that this may explain, in part, the beneficial vascular effects of AA. A similar effect has been observed in the present study where pretreatment of mice with AA prior to irradiation enhanced NO levels significantly in the wound bed. Similarly, increased synthesis of hexosamine and DNA contents may also have been responsible for the accelerated healing of irradiated wounds in AA pretreated groups. AA also may have provided strength to the regenerating wound by improving the hematopoietic system causing early closure of the wound as revealed by wound contraction studies.

Investigations on the effect of localized/hemi-body radiation on wound repair are so infrequent that management policies must be derived in part from different but analogous clinical situations and/or from studies in experimental animals. An attempt has been made in the present study to evaluate the efficacy of AA treatment on wound healing in hemi-body irradiated mice, where it has been demonstrated that single administration of AA prior to hemi-body gamma radiation expedites wound healing in mice, as is evident from an improved contraction of the wound, increased synthesis of collagen, hexosamine, DNA, and NO, enhanced proliferation of fibroblasts, and increased vasculature to the wound bed. Observations suggest that strategies to manipulate pathophysiological environment of irradiated wounds by AA are likely to be of outstanding significance in radiation-impaired healing of wounds. Additional studies will be needed to better understand the mechanism of ameliorating action of AA in initiating and supporting the cascade of tissue repair processes in irradiated wounds.

Acknowledgment

We thank Dr. M. S. Vidyasagar, Prof. and Head, and Dr. J. Velumurugan, Department of Radiotherapy and Oncology, Kasturba Medical College, Manipal, India, for providing the necessary irradiation facilities and help in dosimetry. Our thanks are due to Prof. B. Srinivas, Head, Department of Mechanical Engineering, Manipal Institute of Technology, Manipal, India, for his expert help in calculating the wound area. The financial assistance provided by the Defense Research and Development Organization (DRDO), Ministry of Defense, Government of India, New Delhi, to carry out the above study is gratefully acknowledged.