The Study Of Delayed Wound Healing In Mice With Combined Radiation And Wound Injury

Combined radiation-trauma injury are mainly observed in radiation treatment of cancer and radiation injury with traumatic patients. The significant issue is delayed wound healing. Existing research shows that: delayed wound healing caused by total body irradiation injury is the result of multi-healing-factor cooperation with reduction and functional impairment of hematopoietic cells and repair cells. The changes of the damage may be closely related to injury severity, radiation dosage, individual differences and other factors. Its mechanism is not clear at present. Cytokines are a class of small molecule peptide which have significant regulatory functions in cell growth and differentiation. They are important signal transmitters between cell and the extracellular matrix. VEGF (Vascular Endothelial Growth Factor), PDGF(Platelet-derived Growth Factor), TNF-α(Tumor Necrosis Factor) are important messenger molecules and organizers in wound healing. Studies have found that in different periods of combined radiation-trauma injury, PDGF , VEGF and TNF-αexpression decreased significantly. The existing researches in combined radiation-trauma injury repair were focused on function of cytokines in the process of wound healing. Although more complex animal models of injury were reported in the literature, awareness was quite different in many fields as radiation dosage, animal mortality statistics and level of delayed wound healing. Because there is no exclusive standard model, we construct a high stable, good repeatability, standardized animal model of combined radiation injury, analyze the function and significance of PDGF, VEGF and TNF-αin wound healing of combined radiation injury, and explore the relationship between cytokines and inflammatory cells. It is necessary for the study of mechanism of total body irradiation in wound healing.ObjectiveBuild models with different doses of total body radiation with skin defect / incised wound by mice. Test the indexes which can reflect the level of injury as inflammatory cells, wound repair cells and new blood capillaries, and the expression of PDGF,VEGF,TNF-αin wounds of combined radiation injury( Within 14 days after injury ), study the different dosages of total body radiation effects on skin, explore the function of PDGF, VEGF and TNF-αin wound healing. Provide new ideas for in-depth understanding of release mechanisms of combined radiation injury.MethodsUse 250 healthy female Kunming mice( SPF ), weight( 20±2 g). 210 mice were randomly selected to a one-time uniform 60Coγray irradiation 4, 6, 8Gy Within 30min after irradiation, 60 mice were made full thickness skin incisions on the middle back to build different dosages combined radiation incision injury models( n=20 ). Mice were only made incision without radiation as the control group1 ( n=10 ). 150 mice were made 1.5×1.5cm full thickness square defects to build combined radiation injury models( n=50 ). Mice were only made simple defects without radiation as the control group 2 ( n = 30 ). Mice in combined radiation incision injury groups were sacrificed at 10 days after injury, taking full thickness skin wounds. Analyze wound tear strength of wounds by biomechanical method, and evaluate the situation of wound healing by pathological method. Mice in combined radiation injury groups were sacrificed at 3, 5, 7, 10,14 days after injury, 6 mice of each, and taking full thickness skin square wounds. Observe the wound tissue by pathology and inflammatory cells, fibroblasts, changes in the number of new blood capillaries. View the expression of PDGF-BB, VEGF, TNF-αproteins and immunohistochemical in square wounds. Analyze the residual area of the unhealed wound by quantitative image analysis system. Stats the body weight and survival rate of mice.Results1. Tear strength were significantly lower when the radiation dosage was increased. 10 d time, 4 Gy group wound tear strength ( 114.26±0.29 ) g, compared with control group1 ( 117.12±1.86 ) g have no significant difference, and 6 Gy group ( 91.87±1.96 ) g and 8 Gy group wound tear strength ( 55.26±2.64 ) g were lower than control group1 ( p<0.05 ).2. Wound healing time were significantly icreased when the radiation dosage was increased. The survival rates of 6 Gy group in 8 days and 14 days were 73% and 55%. The survival rate of 8 Gy group was only 23% at 8 days ,and the mice all died in 10 days. Within 14 days after injury, with the radiation dosage increased, weight of the combined radiation groups were lower than the level before the injury even more obvious. compared with the control group 2, the residual area of 6 and 8 Gy groups increased significantly( P<0.01 ). 8 days after injury, 4 Gy group compared with the control group, the residual area was significantly increased ( P<0.05 ).3. The number of inflammatory cells, fibroblasts, new blood capillaries in the wound of combined radiation groups are reduced to different extents than single injury groups. In 6 and 8 Gy Groups, 3-5 days after injury, the wound inflammatory cells, fibroblasts and new capillaries were significantly lower than the number of single injury group. 3-7 days after injury, the number of inflammatory cells compared with each other were significantly different in 4, 6, 8 Gy groups( P<0.01 ).4. HE staining showed that: (1) Compared with the simple incision group, arrangement of collagen fibers of total body irradiation combined incision injury mice were disordered, tissue structure were looser, and the proliferation of fibroblasts were less. (2) Compared with simple defect group, wounds of total body irradiation mice with skin defect, the early inflammatory response was inhibited, the number of fibroblast cells and new capillaries, granulation tissue formation, and epithelial coverage were delayed.5. Immunohistochemistry results of the skin defect group showed that: Exposure to total body irradiation,the wound healing were delayed or extended in various stages.(1) From day3 after injury,the increasing of PDGF-BB, VEGF, TNF-αexpression were observed in control group and 6,8 Gy group,peaked on day 7. The control group exceeded 6,8 Gy group. Compared with the skin defect group,PDGF-BB, VEGF expression levels of control group were significantly lower on day 10.(2) In day3-5 after injury ,the increasing of TNF-αexpression were observed in control group and 6,8 Gy group,The control group exceeded 6,8 Gy group. On day 5 after injury TNF-αexpression of the control group reached the peak,then dropped out. The increasing of TNF-αexpression of 6,8 Gy group were observed on day 7, expression phase delaeyed than the control group. (3) Quantitative results showed: Compared with the control group, the integral optical density(IOD)of PDGF-BB, VEGF significantly reduced in 6,8 Gy group( P<0.05 or P<0.01 ) in 3-7 days after injury ; the integral optical density(IOD)of TNF-αsignificantly reduced in 6,8 Gy group( P<0.05 or P<0.01 ) in 3-5 days after injury ; the integral optical density(IOD)of TNF-αsignificantly increased in 6,8 Gy group( P<0.05 or P<0.01 ) in 7-10 days after injury ; the integral optical density(IOD)of PDGF-BB, VEGF significantly increased in 6 Gy group( P<0.05 or P<0.01 ) on day 10 after injury.Conclusion1. Wound healing in combined radiation injury has an obvious dosage-response relationship. In total body irradiation combined incision injury, the wound tear strength decreased when radiation dosage increased. In combined radiation injury with skin defect, the delayed wound healing got worse with increasing radiation dosage.2. Total body irradiation in mice by 6 Gy 60Coγray defect / incision injury model can be build as a platform for the mechanism of refractory combined radiation-trauma injury and early treatment research.3. At early stage of wound healing in skin defect with total body irradiation mice,in wound area , PDGF-BB, VEGF expression levels significantly reduced and expression phase delaeyed in day 0-7 after injury ,TNF-αexpression significantly reduced and expression phase delaeyed in day 0-5 after injury.These findings strongly suggest that they were the candidate reasons and may play the crucial role in the delayed wound healing.4. Total body radiation break the normal physiologic process. PDGF,VEGF and TNF-αexpression increase or decrease regularly in different stages which may be related in the radiation damage level and the periods of wound healing.