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Effects of gamma-low dose irradiation on skin flap survival in rats
Physica Medica xxx (2017) xxx–xxx
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Original paper
Effects of gamma-low dose irradiation on skin flap survival in rats Mojtaba Karimipour a, Vahid Amanzade a, Nasrollah Jabbari b,⇑, Gholam Hossein Farjah a a b
Department of Anatomy and Histology, Urmia University of Medical Sciences, Urmia, Iran Solid Tumor Research Center, Department of Medical Physics and Imaging, Urmia University of Medical Sciences, Urmia, Iran
a r t i c l e
i n f o
Article history: Received 16 January 2017 Received in Revised form 16 July 2017 Accepted 22 July 2017 Available online xxxx Keywords: Random skin flap Gamma ray Low dose radiation Survival
a b s t r a c t Purpose: Skin flap necrosis due to inadequate blood supply has remained a common postoperative problem in constructive surgery. As low-dose irradiation (LDI) has been shown to promote the wound-healing process, this study aims to investigate whether LDI could increase neovascularization and skin flap survival in rats. Methods: McFarlane flaps were created in 21 male rats, which were divided into one control and two treatment groups (Ta and Tb). The treatment groups received a whole body single dose of 100 cGy gamma ray irradiation before (Tb) and after (Ta) flap surgery. The flap survival area was evaluated after seven days. The skin samples were collected for histological analysis and determining the vascular endothelial growth factor (VEGF) using the immunohistochemical method. Serum malondialdehyde (MDA) was examined with the kit. Results: The mean areas of flap survival were 56.7 ± 3.24, 61.7 ± 2.6, and 66.5 ± 3.82 in the control, Tb, and Ta groups, respectively. There were significant differences between the Tb and Ta groups in comparison with the control group (P < 0.05 and P < 0.01, respectively). Compared with the control group (8.0 ± 0.73), the mean numbers of the blood vessels in the Ta group (22 ± 1.24) and the Tb group (14 ± 1.29) were significantly higher (P < 0.001 and P < 0.01). Moreover, the mean numbers of the VEGF-positive cells in the Ta group (4.5 ± 1.04) were significantly higher (P < 0.05) than the control group (2.5 ± 0.83). However, no significant differences in the MDA levels were observed among the groups. Conclusion: The findings of this study suggest that LDI has the potential to promote neovascularization to improve flap survival. Ó 2017 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.
1. Introduction Skin flap is an important and common surgical technique that is widely used in plastic and reconstructive surgery to heal major tissue defects resulting from congenital problems and trauma [1,2]. Postoperative flap damages due to ischemia-reperfusion injury and inadequate blood perfusion are clinical problems that lead to partial or complete skin flap necrosis [2,3]. This complication may require reoperation, delay hospital discharge, and the outpatient visits’ extension [4]. As documented by extensive previous research using skin flaps, the rat has been used as an animal model to evaluate the effect of treatments on flap viability [3,5,6]. To enhance skin flap survival in rats, several pharmacological agents such as antioxidants [7] and anti-inflammatory drugs [8], or even growth factors [9] and other devices, including low-level laser therapy [10] and ultrasound [11] have been used. However, ⇑ Corresponding author. E-mail address: [email protected] (N. Jabbari).
the results obtained in most studies are controversial and require further investigation. The evidence indicates that the whole-body irradiation to low levels of ionizing radiation stimulates many physiological functions, such as increased immune competence, lower morbidity and mortality, longer lifespan, and enhanced reproduction [12]. The biological effects of ionizing radiation VII (BEIR VII) committee has defined ‘low-dose’ as dose levels of less than 100 cGy (100 mSv) of low-linear energy transfer (LET) radiation [13]. Previous studies using animal models have revealed that lowdose irradiation (LDI) accelerated the wound healing process through the up-regulation of VEGF, stimulation of cell proliferation in wound tissue and bone marrow stem cells, and mobilized them into blood circulation [14,15]. Moreover, LDI can protect cells and tissues against oxidant damages mediated by their antioxidant property [16]. An increase of tissue glutathione by LDI has been shown. LDI is considered to have an important role in inducing protective effects in tissues and cells [17–20]. Overall, several studies have demonstrated that low-dose X-irradiation (100 cGy) promotes fracture healing [21,22]. In
http://dx.doi.org/10.1016/j.ejmp.2017.07.019 1120-1797/Ó 2017 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Karimipour M et al. Effects of gamma-low dose irradiation on skin flap survival in rats. Phys. Med. (2017), http://dx.doi. org/10.1016/j.ejmp.2017.07.019
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addition, it has been indicated that a low dose of irradiation promotes tissue revascularization through VEGF release from mast cells and matrix metalloproteinase-9 (MMP-9) mediated progenitor cell mobilization [23]. Thus, we were encouraged to evaluate the effects of low-dose ionizing radiation on skin flap survival using a single gamma-ray dose (100 cGy) irradiation. Therefore, this study explored the therapeutic effect of LDI on the survival of random skin flaps in a rat model. 2. Methods and materials This study was performed in accordance with the guidance for care and use of laboratory animals. It was approved by the Animal Ethics Committee of the Urmia University of Medical Sciences. Twenty one adult male Wistar rats, weighing 220–240 g, were obtained from the animal house of the Urmia University of Medical Sciences and randomly divided into three groups. They were maintained on a 12 h light-dark cycle at a room temperature of 22°– 24 °C with free access to food and water. After performing random-pattern skin flap in the animals, they were randomly divided into three groups, based on the low-dose radiation exposure time. Group 1: Control (non-irradiated) group (n = 7) placed in the treatment room without irradiation similar to the treatment groups Group 2: Treatment group I (n = 7) undergone whole body irradiation before flap surgery (Tb) Group 3: Treatment group II (n = 7) exposed to whole body radiation after flap surgery (Ta) 2.1. Random pattern skin flap model After anesthesia with an intraperitoneal injection of 60 mg/kg ketamine and 10 mg/kg xylazine, the rats were immobilized in a prone position, their dorsal hair was shaved, and then the skin was disinfected with a povidone iodine solution. A caudallybased 7 2 cm random skin flap was created on the dorsum of each rat. The flaps were designed according to the procedures described by McFarlane [24]. The flaps were raised and cut superficial fascia, panniculus carnosus, subcutaneous tissue, and skin and then, sutured back to their original position with a 4-0 silk suture. 2.2. Flap survival assessment At seven days after flap elevation, the rats were re-anesthetized and subsequently, the survival surface area was demarcated and then cut and weighed using a precision electronic scale. The survival area was calculated using the following formula:
Weight of surv iv al area Percentage of skin flap sur v iv al ¼ Total weight of flap template 100 The living tissue was easily identified by gross observation and characterized by being warm and soft to the touch and hairbearing skin, while hairless, stiff, dark, and colder skin was considered necrotic tissue [1,25,26].
tance (SSD) of 78.5 cm. The Tb and Ta groups were irradiated to gamma ray before and after the flap surgical procedure respectively. The control group remained untreated and was kept in the restrainers with the same conditions as those in the irradiation groups. 2.4. Histology At seven days’ post-operation, the rats were re-anaesthetized and blood was taken from their portal veins for biochemistrical analysis. Then they were all sacrificed, and the tissue specimens were collected from the same position of the surviving portion of the flap. The specimens were fixed in 10% paraformaldehyde, embedded in paraffin, sectioned to 6 mm slices, and prepared for hematoxylin and eosin (H&E) staining, and anti-VEGF. For assessment of angiogenesis, the vessels were counted in five fields on H&E stained slides (at 40) [1]. To determine the immunohistochemical localization of the VEGF, tissue sections from the paraffin-embedded blocks were deparaffinized and rehydrated in decreasing concentrations of alcohol, and then washed in a phosphate buffered saline (PBS) pH 7.4. Antigen retrieval was performed by incubating the sections in 10 mM sodium citrate at 36 °C for 30 min. The sections were treated with hydrogen peroxidase (3%) to block endogenous peroxidase and then washed with the PBS. The sections were incubated overnight at 4 °C with an anti-VEGF antibody (Abcam). The slides were washed in the PBS and then incubated with a secondary antibody for 45 min at 37 °C. After washing, the slides were treated with diaminobenzidine for 10 min at room temperature. Finally, they were counterstained with hematoxylin. The sections were observed by a light microscope, and the VEGF-positive cells with brown color were counted in two fields by a person blinded to treatment [27]. 2.5. Measurement of MDA concentration MDA was used as the marker of oxidative stress and analysed based on the kit manufacturer’s instructions (SHANGHAI CRISTAL DAY BIOTHEH, Co, China) using the samples of the blood serum collected on day seven after the flap surgery. 2.6. Statistical analysis The mean and standard deviation values from the different groups were expressed and compared using analysis of variance (ANOVA). One-way ANOVA was followed by the Tukey’s multiple comparison post hoc test for comparing different treatment groups. The statistical significance was set at p < 0.05. 3. Results 3.1. General observation On day seven after the flap surgery, the surviving area of the flap was soft with warm skin and fine hair, while the necrotic area was hard and dark with cold hairless skin, and there was no bleeding when cut with a scalpel. 3.2. Survival assessment
2.3. Exposure to low-dose radiation The two irradiated groups (Ta and Tb) received the whole-body single dose of 100 cGy of gamma-ray using a Cobalt-60 source (Theratron Phoenix, Theratronics, Inc., Ottawa, Canada) with the approximate dose rate of 39.24 cGy/min at the source-to-skin dis-
Digital photographs show the regions of survival and necrosis of the flaps from the different groups on the seventh postoperative day (Fig. 1). Seven days after the surgery, the mean ± standard deviation (SD) of the skin flap survival percentage in the control, Tb, and Ta groups were 56.7 ± 3.24, 61.7 ± 2.6, and 66.5 ± 2.82,
Please cite this article in press as: Karimipour M et al. Effects of gamma-low dose irradiation on skin flap survival in rats. Phys. Med. (2017), http://dx.doi. org/10.1016/j.ejmp.2017.07.019
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Fig. 1. Digital photographs showing the regions of survival and necrosis of the flaps from different groups at seventh postoperative day; the control group (left); the Tb group (center); and the Ta group (right).
respectively. The mean was significantly higher in the animals that received radiation after the flap surgery compared with the control (p < 0.01) and those that received radiation before the surgery (p < 0.05); (Fig. 2). 3.3. Histology
Fig. 2. The mean and standard deviations of the survival percentage of the flaps among the three groups. The flap survival in the Ta was significantly higher than that of both other groups (p < 0.05).
New vessel regeneration in the flap tissues of different groups is shown in Fig. 3. Seven days post-operation, the histological evaluation of four fields of random pattern skin flaps in each rat indicated that the mean ± SD of vascular density, an index of neovascularization, were 9.28 ± 0.95, 11.57 ± 0.97, and 12.71 ± 1.6 in the control, Tb, and Ta groups, respectively. It was significantly increased in the Tb and Ta groups (P < 0.01 and P < 0.001), as compared to the control group. The mean number of the blood vessels in the Ta group was higher than the Tb, but there were no significant differences between them (Fig. 4).
Fig. 3. New vessel regeneration in the flap tissues of different groups (A: control; B: Tb and C: Ta) by the H&E staining, arrows showing blood vessels, original magnification 100.
Please cite this article in press as: Karimipour M et al. Effects of gamma-low dose irradiation on skin flap survival in rats. Phys. Med. (2017), http://dx.doi. org/10.1016/j.ejmp.2017.07.019
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4. Discussion
Fig. 4. The mean number of blood vessels between the different groups. The mean number of the blood vessels in the Ta and Tb groups was significantly higher than that of the control group (p < 0.01).
Based on immunohistochemical evaluation (Fig. 5), the mean numbers of the VEGF-positive cells were 2.5 ± 0.83, 3.83 ± 0.75, and 4.5 ± 1.04 in the control, Tb, and Ta groups respectively. A significant difference was observed between the Ta and the control group (P < 0.05). 3.4. Malondialdehyde (MDA) concentration The mean and standard deviations of the MDA level in the control, Tb, and Ta groups were 9.5 ± 1.28, 9.42 ± 2.08, and 9.32 ± 2.42, respectively. However, no significant difference was observed between the groups (Fig. 6).
In the present study, we set out to investigate whether the whole body LDI would improve survival in random pattern skin flaps. The results demonstrated an increased viability of random pattern skin flaps through using LDI. In addition, the quantitative measurements of the vascular density were consistent with the general observation. Moreover, it was observed that enhancing the viability of random pattern skin flaps depended on increased neovascularization, which occurred through two mechanisms, angiogenesis and vasculogenesis [2]. These processes stimulate tissue survival after ischemia, and they help in the repair of injured tissues [28]. Angiogenesis or sprouting angiogenesis is the formation of new blood vessels from preexisting vessels and is an important feature of tissue healing at the site of injury, which delivers oxygen and nutrients to tissue. Defects and delays in this process are associated with impairment in wound healing [29]. Vasculogenesis is the process by which bone marrow-derived endothelial progenitor cells (EPCs) form blood vessels. In this process, a complex of vascular network forms, which is vital for embryonic development [29]. Vasculogenesis also occurs in postnatal, especially in ischemic conditions [30,31]. EPCs are mobilized from the bone marrow; and through systemic circulation, they are home to ischemic tissues to cooperate endothelial cell repair [32,33]. Angiogenesis and vasculogenesis both occur during the wound healing process, but angiogenesis is investigated more frequently and is known to be responsible for neovascularization in wounds [29]. In our experiment, increased vascular density was proven by the histological assessment. This increased density might have
Fig. 5. Immunohistochemically analysis of VEGF. (A) Photomicrographs of skin tissue sections for VEGF-positive cells in the control group, (B) Radiation before surgery group (Tb), (C) Radiation after surgery group (Ta), and (D) Assessment of VEGF-positive cells. *P < 0.05 vs control. Arrows show VEGF-positive cells.
Please cite this article in press as: Karimipour M et al. Effects of gamma-low dose irradiation on skin flap survival in rats. Phys. Med. (2017), http://dx.doi. org/10.1016/j.ejmp.2017.07.019
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One limitation in this experiment was that we did not use other antioxidants assays such as superoxide dismutase and catalase. It must be noted that there is no previous study on the effects of LDI on random-pattern skin flaps survival. Therefore, we cannot explain why the LDI therapy was proven to be better after flap surgery than prior to the surgery.
5. Conclusion
Fig. 6. The serum MDA level in different groups.
directly accounted for the increased flap viability. We also hypothesized that the LDI might have stimulated the production of angiogenesis growth factors such as VEGF, which has been shown to induce the neovascularization process and increase vascular density in the ischemic area [34]. The VEGF is an important proangiogenic molecule in the skin. Many studies have shown that VEGF is important for wound healing. It acts as a strong positive regulator of angiogenesis and stimulates the endothelial cell functions required for the formation of new blood vessels [35,36]. Previous studies have indicated that the VEGF is produced by a variety of cell types such as keratinocytes, mast cells, and macrophages in injured skin [37,38]. In this study, the VEGF expression level was markedly higher in both the LDI groups than in the control group. Furthermore, the vascular density in the LDI groups was significantly greater than that in the control. These results suggest that LDI may promote neovascularization and microcirculation in ischemic flaps by increasing the VEGF expression, ultimately improving flap viability. Further study is needed to obtain more detailed insight in the underlying mechanisms. LDI was previously revealed to induce the expression of VEGF in bone fracture and wound healing [39,14]. Guo et al. (2010) found that LDI increases blood vessels regeneration and cell proliferation in the wound tissue, along with enhancing the VEGF and MMP-9 expression [14,40]. MMP-9 has been proven to play an important role in angiogenesis and wound healing; and degrades extracellular matrix to permit the migration of new cells in the process of revascularization [41,42]. MMP-9 extends the bioavailability of the VEGF and also increases the mobilization of the progenitor cells from the bone marrow into circulation [41,43]. The other protective mechanism by which the LDI promotes the wound healing process is related to the up-regulation of antioxidant activity. Zhao et al. (2010) indicated that the antioxidants superoxide dismutase and catalase were up-regulated in the blood and testicular tissue of diabetic rats exposed to LDI [16]. Oxygen deficiency and venous flow insufficiency are important causes of necrosis on random skin flaps. These conditions lead to an increase of free radicals that cause peroxidation of lipids and proteins, and damage cell and organelle membranes, thus destroying tissue structure and function [25,44]. MDA is a product of lipid peroxidation and its level indirectly indicates the degree of tissue damage [44]. In the present study, we found that the MDA level in the LDI groups was lower compared with the control group, but not significant. So, we could not confirm that the LDI had a protective effect on the development of flap necrosis by reducing lipid peroxidation activity. The disagreement between the MDA results of the present study and those from the study by Zhao et al. (2010) may be due to the fact that they delivered a fractionated radiation dose in their study, while we used a single irradiation [16].
The results of the current study indicate that the LDI has the potential to improve skin flap survival. Nevertheless, further studies are necessary to determine other mechanisms of the efficacy of LDI for improving flap viability as well as to determine the optimal dose of ionizing radiation, evaluate fractionated doses, and determine the threshold range of LDI to induce flap survival.
Acknowledgement This study was performed as part of the requirements towards MSc in Anatomy. We are especially thankful to the Vice Chancellor for Research (VCR) of Urmia University of Medical Sciences for the financial supporting of this project.
References [1] Lin B, Lin Y, Lin D, Cao B. Effects of bezafibrate on the survival of random skin flaps in rats. J Reconstr Microsurg 2016;32:395–401. [2] Lu F, Mizuno H, Uysal CA, Cai X, Ogawa R, Hyakusoku H. Improved viability of random pattern skin flaps through the use of adipose-derived stem cells. Plast Reconstr Surg 2008;121:50–8. [3] Yang M, Sheng L, Li H, Weng R, Li QF. Improvement of the skin flap survival with the bone marrow-derived mononuclear cells transplantation in a rat model. Microsurgery 2010;30:275–81. [4] Taleb S, Moghaddas P, Rahimi Balaei M, Taleb S, Rahimpour S, Abbasi A, et al. Metformin improves skin flap survival through nitric oxide system. J Surg Res 2014;192:686–91. [5] Minh TC, Ichioka S, Nakatsuka T, Kawai J, Shibata M, Ando J, et al. Effect of hyperthermic preconditioning on the survival of ischemia-reperfused skin flaps: a new skin-flap model in the mouse. J Reconstr Microsurg 2002;18:115–9. [6] Jones M, Zhang F, Blain B, Guo M, Cui D, Dorsett-Martin W, et al. Influence of recipient-bed isolation on survival rates of skin-flap transfer in rats. J Reconstr Microsurg 2001;17:653–8. [7] Cai L, Huang W, Lin D. Effects of traditional Chinese medicine Shuxuetong injection on random skin flap survival in rats. Sci World J 2014;26:816545. http://dx.doi.org/10.1155/2014/816545. [8] Shalom A, Friedman T, Westreich M. Effect of aspirin and heparin on random skin flap survival in rats. Dermatol Surg 2008;34:785–90. [9] Chen GJ, Chen YH, Yang XQ, Li ZJ. Nano-microcapsule basic fibroblast growth factor combined with hypoxia-inducible factor-1 improves random skin flap survival in rats. Mol Med Rep 2016;13:1661–6. [10] Baldan CS, Masson IF, Esteves Júnior I, Baldan AM, Machado AF, Casaroto RA, et al. Inhibitory effects of low-level laser therapy on skin-flap survival in a rat model. Plast Surg (Oakv) 2015;23:35–9. [11] Tacani PM, Liebano RE, Pinfildi CE, Gomes HC, Arias VE, Ferreira LM. Mechanical stimulation improves survival in random-pattern skin flaps in rats. Ultrasound Med Biol 2010;36:2048–56. [12] Luckey TD. Nurture with ionizing radiation: a provocative hypothesis. Nutr Cancer 1999;34:1–11. [13] National Research Council. Health risks from exposure to low levels of ionizing radiation: BEIR VII, Phase 2/ committee: Committee to assess health risks from exposure to low levels of ionizing radiation. National Research Council; 2006. p. 2. [14] Guo WY, Wang GJ, Wang P, Chen Q, Tan Y, Cai L. Acceleration of diabetic wound healing by low-dose radiation is associated with peripheral mobilization of bone marrow stem cells. Radiat Res 2010;174:467–79. [15] Li W, Wang G, Cui J, Xue L, Cai L. Low-dose radiation (LDR) induces hematopoietic hormesis: LDR-induced mobilization of hematopoietic progenitor cells into peripheral blood circulation. Exp Hematol 2004;32:1088–96. [16] Zhao H, Xu S, Wang Z, Li Y, Guo W, Lin C, et al. Repetitive exposures to lowdose X-rays attenuate testicular apoptotic cell death in streptozotocin-induced diabetes rats. Toxicol Lett 2010;192:356–64. [17] Bose Girigoswami K, Bhaumik G, Ghosh R. Induced resistance in cells exposed to repeated low doses of H2O2 involves enhanced activity of antioxidant enzymes. Cell Biol Int 2005;29:761–7.
Please cite this article in press as: Karimipour M et al. Effects of gamma-low dose irradiation on skin flap survival in rats. Phys. Med. (2017), http://dx.doi. org/10.1016/j.ejmp.2017.07.019
6
M. Karimipour et al. / Physica Medica xxx (2017) xxx–xxx
[18] Kawakita Y, Ikekita M, Kurozumi R, Kojima S. Increase of intracellular glutathione by low-dose gamma-ray irradiation is mediated by transcription factor AP-1 in RAW 264.7 cells. Biol Pharm Bull 2003;26:19–23. [19] Kojima S, Matsuki O, Nomura T, Yamaoka K, Takahashi M, Niki E. Elevation of antioxidant potency in the brain of mice by low-dose gamma-ray irradiation and its effect on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)induced brain damage. Free Radic Biol Med 1999;26:388–95. [20] Pathak CM, Avti PK, Kumar S, Khanduja KL, Sharma SC. Whole body exposure to low-dose gamma radiation promotes kidney antioxidant status in Balb/c mice. J Radiat Res 2007;48:113–20. [21] Zhou XZ, Zhang G, Dong QR, Chan CW, Liu CF, Qin L. Low-dose X-irradiation promotes mineralization of fracture callus in a rat model. Arch Orthop Trauma Surg 2009;129:125–32. [22] Song XS, Zhou XZ, Zhang G, Dong QR, Qin L. Low-dose X-ray irradiation promotes fracture healing through up-regulation of vascular endothelial growth factor. Med Hypotheses 2010;75:522–4. [23] Heissig B, RaWi S, Akiyama H, Ohki Y, Sato Y, Rafael T, et al. Low-dose irradiation promotes tissue revascularization through VEGF release from mast cells and MMP-9-mediated progenitor cell mobilization. J Exp Med 2005;202:739–50. [24] McFarlane RM, Deyoung G, Henry RA. The design of a pedicle flap in the rat to study necrosis and its prevention. Plast Reconstr Surg 1965;35:177–82. [25] Akgül GG, Yenidogan E, Gülçelik MA, Sahin D, Çolakoglu MK, Alagöl H. Effects of N-acetylcysteine on random skin flaps in rats. Wounds 2013;25:68–74. [26] Baldan CS, Marques AP, Schiavinato AM, Casarotto RA. The effects of different doses of 670 nm diode laser on skin flap survival in rats. Acta Cir Bras 2012;27:155–61. [27] Bagdas D, Cam Etoz B, Inan Ozturkoglu S, Cinkilic N, Ozyigit MO, Gul Z, et al. Effects of systemic chlorogenic acid on random-pattern dorsal skin flap survival in diabetic rats. Biol Pharm Bull 2014;37:361–70. [28] Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature 2011;473:298–307. [29] Johnson KE, Wilgus TA. Vascular endothelial growth factor and angiogenesis in the regulation of cutaneous wound repair. Adv Wound Care (New Rochelle) 2014;3:647–61. [30] Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M, et al. Ischemiaand cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med 1999;5:434–8. [31] Tepper OM, Capla JM, Galiano RD, Ceradini DJ, Callaghan MJ, Kleinman ME, et al. Adult vasculogenesis occurs through in situ recruitment, proliferation, and tubulization of circulating bone marrow-derived cells. Blood 2005;105:1068–77.
[32] Asahara T, Masuda H, Takahashi T, Kalka C, Pastore C, Silver M, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatalvasculogenesis in physiological and pathological neovascularization. Circ Res 1999;85:221–8. [33] Martins S, Quelhas-Santos J. Circulating endothelial progenitor cells: a review of definition, characterization and identification. RRJOB 2016;4:42–6. [34] Zhang F, Fischer K, Komorowska-Timek E, Guo M, Cui D, Dorsett-Martin W, et al. Improvement of skin paddle survival by application of vascular endothelial growth factor in a rat TRAM flap model. Ann Plast Surg 2001;46:314–9. [35] Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989;246:1306–9. [36] Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, et al. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science 1989;246:1309–12. [37] Brown LF, Yeo KT, Berse B, Yeo TK, Senger DR, Dvorak HF, et al. Expression of vascular permeability factor (vascular endothelial growth factor) by epidermal keratinocytes during wound healing. J Exp Med 1992;176:1375–9. [38] Shiota N, Nishikori Y, Kakizoe E, Shimoura K, Niibayashi T, Shimbori C, et al. Pathophysiological role of skin mast cells in wound healing after scald injury: study with mast cell-deficient W/W(V) mice. Int Arch Allergy Immunol 2010;151:80–8. [39] Chen M, Huang Q, Xu W, She C, Xie ZG, Mao YT, et al. Low-dose X-ray irradiation promotes osteoblast proliferation, differentiation and fracture healing. PLoS One 2014;9:e104016. http://dx.doi.org/10.1371/journal. pone.0104016. [40] Thanik VD, Chang CC, Lerman OZ, Greives MR, Le H, Warren SM, et al. Cutaneous low-dose radiation increases tissue vascularity through upregulation of angiogenic and vasculogenic pathways. J Vasc Res 2010;47:472–80. [41] Heissig B, Rafii S, Akiyama H, Ohki Y, Sato Y, Rafael T, et al. Low-dose irradiation promotes tissue revascularization through VEGF release from mast cells and MMP-9–mediated progenitor cell mobilization. J Exp Med 2005;202:739–50. [42] Nelson AR, Fingleton B, Rothenberg ML, Matrisian LM. Matrix metalloproteinases: biologic activity and clinical implications. J Clin Oncol 2000;18:1135–49. [43] Heissig B, Werb Z, Rafii S, Hattori K. Role of c-kit/Kit ligand signaling in regulating vasculogenesis. Thromb Haemost 2003;90:570–6. [44] Sun L, Li Y, Shi J, Wang X, Wang X. Protective effects of ligustrazine on ischemia-reperfusion injury in rat kidneys. Microsurgery 2002;22:343–6.
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Physica Medica journal homepage: http://www.physicamedica.com
Original paper
Effects of gamma-low dose irradiation on skin flap survival in rats Mojtaba Karimipour a, Vahid Amanzade a, Nasrollah Jabbari b,⇑, Gholam Hossein Farjah a a b
Department of Anatomy and Histology, Urmia University of Medical Sciences, Urmia, Iran Solid Tumor Research Center, Department of Medical Physics and Imaging, Urmia University of Medical Sciences, Urmia, Iran
a r t i c l e
i n f o
Article history: Received 16 January 2017 Received in Revised form 16 July 2017 Accepted 22 July 2017 Available online xxxx Keywords: Random skin flap Gamma ray Low dose radiation Survival
a b s t r a c t Purpose: Skin flap necrosis due to inadequate blood supply has remained a common postoperative problem in constructive surgery. As low-dose irradiation (LDI) has been shown to promote the wound-healing process, this study aims to investigate whether LDI could increase neovascularization and skin flap survival in rats. Methods: McFarlane flaps were created in 21 male rats, which were divided into one control and two treatment groups (Ta and Tb). The treatment groups received a whole body single dose of 100 cGy gamma ray irradiation before (Tb) and after (Ta) flap surgery. The flap survival area was evaluated after seven days. The skin samples were collected for histological analysis and determining the vascular endothelial growth factor (VEGF) using the immunohistochemical method. Serum malondialdehyde (MDA) was examined with the kit. Results: The mean areas of flap survival were 56.7 ± 3.24, 61.7 ± 2.6, and 66.5 ± 3.82 in the control, Tb, and Ta groups, respectively. There were significant differences between the Tb and Ta groups in comparison with the control group (P < 0.05 and P < 0.01, respectively). Compared with the control group (8.0 ± 0.73), the mean numbers of the blood vessels in the Ta group (22 ± 1.24) and the Tb group (14 ± 1.29) were significantly higher (P < 0.001 and P < 0.01). Moreover, the mean numbers of the VEGF-positive cells in the Ta group (4.5 ± 1.04) were significantly higher (P < 0.05) than the control group (2.5 ± 0.83). However, no significant differences in the MDA levels were observed among the groups. Conclusion: The findings of this study suggest that LDI has the potential to promote neovascularization to improve flap survival. Ó 2017 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.
1. Introduction Skin flap is an important and common surgical technique that is widely used in plastic and reconstructive surgery to heal major tissue defects resulting from congenital problems and trauma [1,2]. Postoperative flap damages due to ischemia-reperfusion injury and inadequate blood perfusion are clinical problems that lead to partial or complete skin flap necrosis [2,3]. This complication may require reoperation, delay hospital discharge, and the outpatient visits’ extension [4]. As documented by extensive previous research using skin flaps, the rat has been used as an animal model to evaluate the effect of treatments on flap viability [3,5,6]. To enhance skin flap survival in rats, several pharmacological agents such as antioxidants [7] and anti-inflammatory drugs [8], or even growth factors [9] and other devices, including low-level laser therapy [10] and ultrasound [11] have been used. However, ⇑ Corresponding author. E-mail address: [email protected] (N. Jabbari).
the results obtained in most studies are controversial and require further investigation. The evidence indicates that the whole-body irradiation to low levels of ionizing radiation stimulates many physiological functions, such as increased immune competence, lower morbidity and mortality, longer lifespan, and enhanced reproduction [12]. The biological effects of ionizing radiation VII (BEIR VII) committee has defined ‘low-dose’ as dose levels of less than 100 cGy (100 mSv) of low-linear energy transfer (LET) radiation [13]. Previous studies using animal models have revealed that lowdose irradiation (LDI) accelerated the wound healing process through the up-regulation of VEGF, stimulation of cell proliferation in wound tissue and bone marrow stem cells, and mobilized them into blood circulation [14,15]. Moreover, LDI can protect cells and tissues against oxidant damages mediated by their antioxidant property [16]. An increase of tissue glutathione by LDI has been shown. LDI is considered to have an important role in inducing protective effects in tissues and cells [17–20]. Overall, several studies have demonstrated that low-dose X-irradiation (100 cGy) promotes fracture healing [21,22]. In
http://dx.doi.org/10.1016/j.ejmp.2017.07.019 1120-1797/Ó 2017 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Karimipour M et al. Effects of gamma-low dose irradiation on skin flap survival in rats. Phys. Med. (2017), http://dx.doi. org/10.1016/j.ejmp.2017.07.019
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addition, it has been indicated that a low dose of irradiation promotes tissue revascularization through VEGF release from mast cells and matrix metalloproteinase-9 (MMP-9) mediated progenitor cell mobilization [23]. Thus, we were encouraged to evaluate the effects of low-dose ionizing radiation on skin flap survival using a single gamma-ray dose (100 cGy) irradiation. Therefore, this study explored the therapeutic effect of LDI on the survival of random skin flaps in a rat model. 2. Methods and materials This study was performed in accordance with the guidance for care and use of laboratory animals. It was approved by the Animal Ethics Committee of the Urmia University of Medical Sciences. Twenty one adult male Wistar rats, weighing 220–240 g, were obtained from the animal house of the Urmia University of Medical Sciences and randomly divided into three groups. They were maintained on a 12 h light-dark cycle at a room temperature of 22°– 24 °C with free access to food and water. After performing random-pattern skin flap in the animals, they were randomly divided into three groups, based on the low-dose radiation exposure time. Group 1: Control (non-irradiated) group (n = 7) placed in the treatment room without irradiation similar to the treatment groups Group 2: Treatment group I (n = 7) undergone whole body irradiation before flap surgery (Tb) Group 3: Treatment group II (n = 7) exposed to whole body radiation after flap surgery (Ta) 2.1. Random pattern skin flap model After anesthesia with an intraperitoneal injection of 60 mg/kg ketamine and 10 mg/kg xylazine, the rats were immobilized in a prone position, their dorsal hair was shaved, and then the skin was disinfected with a povidone iodine solution. A caudallybased 7 2 cm random skin flap was created on the dorsum of each rat. The flaps were designed according to the procedures described by McFarlane [24]. The flaps were raised and cut superficial fascia, panniculus carnosus, subcutaneous tissue, and skin and then, sutured back to their original position with a 4-0 silk suture. 2.2. Flap survival assessment At seven days after flap elevation, the rats were re-anesthetized and subsequently, the survival surface area was demarcated and then cut and weighed using a precision electronic scale. The survival area was calculated using the following formula:
Weight of surv iv al area Percentage of skin flap sur v iv al ¼ Total weight of flap template 100 The living tissue was easily identified by gross observation and characterized by being warm and soft to the touch and hairbearing skin, while hairless, stiff, dark, and colder skin was considered necrotic tissue [1,25,26].
tance (SSD) of 78.5 cm. The Tb and Ta groups were irradiated to gamma ray before and after the flap surgical procedure respectively. The control group remained untreated and was kept in the restrainers with the same conditions as those in the irradiation groups. 2.4. Histology At seven days’ post-operation, the rats were re-anaesthetized and blood was taken from their portal veins for biochemistrical analysis. Then they were all sacrificed, and the tissue specimens were collected from the same position of the surviving portion of the flap. The specimens were fixed in 10% paraformaldehyde, embedded in paraffin, sectioned to 6 mm slices, and prepared for hematoxylin and eosin (H&E) staining, and anti-VEGF. For assessment of angiogenesis, the vessels were counted in five fields on H&E stained slides (at 40) [1]. To determine the immunohistochemical localization of the VEGF, tissue sections from the paraffin-embedded blocks were deparaffinized and rehydrated in decreasing concentrations of alcohol, and then washed in a phosphate buffered saline (PBS) pH 7.4. Antigen retrieval was performed by incubating the sections in 10 mM sodium citrate at 36 °C for 30 min. The sections were treated with hydrogen peroxidase (3%) to block endogenous peroxidase and then washed with the PBS. The sections were incubated overnight at 4 °C with an anti-VEGF antibody (Abcam). The slides were washed in the PBS and then incubated with a secondary antibody for 45 min at 37 °C. After washing, the slides were treated with diaminobenzidine for 10 min at room temperature. Finally, they were counterstained with hematoxylin. The sections were observed by a light microscope, and the VEGF-positive cells with brown color were counted in two fields by a person blinded to treatment [27]. 2.5. Measurement of MDA concentration MDA was used as the marker of oxidative stress and analysed based on the kit manufacturer’s instructions (SHANGHAI CRISTAL DAY BIOTHEH, Co, China) using the samples of the blood serum collected on day seven after the flap surgery. 2.6. Statistical analysis The mean and standard deviation values from the different groups were expressed and compared using analysis of variance (ANOVA). One-way ANOVA was followed by the Tukey’s multiple comparison post hoc test for comparing different treatment groups. The statistical significance was set at p < 0.05. 3. Results 3.1. General observation On day seven after the flap surgery, the surviving area of the flap was soft with warm skin and fine hair, while the necrotic area was hard and dark with cold hairless skin, and there was no bleeding when cut with a scalpel. 3.2. Survival assessment
2.3. Exposure to low-dose radiation The two irradiated groups (Ta and Tb) received the whole-body single dose of 100 cGy of gamma-ray using a Cobalt-60 source (Theratron Phoenix, Theratronics, Inc., Ottawa, Canada) with the approximate dose rate of 39.24 cGy/min at the source-to-skin dis-
Digital photographs show the regions of survival and necrosis of the flaps from the different groups on the seventh postoperative day (Fig. 1). Seven days after the surgery, the mean ± standard deviation (SD) of the skin flap survival percentage in the control, Tb, and Ta groups were 56.7 ± 3.24, 61.7 ± 2.6, and 66.5 ± 2.82,
Please cite this article in press as: Karimipour M et al. Effects of gamma-low dose irradiation on skin flap survival in rats. Phys. Med. (2017), http://dx.doi. org/10.1016/j.ejmp.2017.07.019
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Fig. 1. Digital photographs showing the regions of survival and necrosis of the flaps from different groups at seventh postoperative day; the control group (left); the Tb group (center); and the Ta group (right).
respectively. The mean was significantly higher in the animals that received radiation after the flap surgery compared with the control (p < 0.01) and those that received radiation before the surgery (p < 0.05); (Fig. 2). 3.3. Histology
Fig. 2. The mean and standard deviations of the survival percentage of the flaps among the three groups. The flap survival in the Ta was significantly higher than that of both other groups (p < 0.05).
New vessel regeneration in the flap tissues of different groups is shown in Fig. 3. Seven days post-operation, the histological evaluation of four fields of random pattern skin flaps in each rat indicated that the mean ± SD of vascular density, an index of neovascularization, were 9.28 ± 0.95, 11.57 ± 0.97, and 12.71 ± 1.6 in the control, Tb, and Ta groups, respectively. It was significantly increased in the Tb and Ta groups (P < 0.01 and P < 0.001), as compared to the control group. The mean number of the blood vessels in the Ta group was higher than the Tb, but there were no significant differences between them (Fig. 4).
Fig. 3. New vessel regeneration in the flap tissues of different groups (A: control; B: Tb and C: Ta) by the H&E staining, arrows showing blood vessels, original magnification 100.
Please cite this article in press as: Karimipour M et al. Effects of gamma-low dose irradiation on skin flap survival in rats. Phys. Med. (2017), http://dx.doi. org/10.1016/j.ejmp.2017.07.019
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4. Discussion
Fig. 4. The mean number of blood vessels between the different groups. The mean number of the blood vessels in the Ta and Tb groups was significantly higher than that of the control group (p < 0.01).
Based on immunohistochemical evaluation (Fig. 5), the mean numbers of the VEGF-positive cells were 2.5 ± 0.83, 3.83 ± 0.75, and 4.5 ± 1.04 in the control, Tb, and Ta groups respectively. A significant difference was observed between the Ta and the control group (P < 0.05). 3.4. Malondialdehyde (MDA) concentration The mean and standard deviations of the MDA level in the control, Tb, and Ta groups were 9.5 ± 1.28, 9.42 ± 2.08, and 9.32 ± 2.42, respectively. However, no significant difference was observed between the groups (Fig. 6).
In the present study, we set out to investigate whether the whole body LDI would improve survival in random pattern skin flaps. The results demonstrated an increased viability of random pattern skin flaps through using LDI. In addition, the quantitative measurements of the vascular density were consistent with the general observation. Moreover, it was observed that enhancing the viability of random pattern skin flaps depended on increased neovascularization, which occurred through two mechanisms, angiogenesis and vasculogenesis [2]. These processes stimulate tissue survival after ischemia, and they help in the repair of injured tissues [28]. Angiogenesis or sprouting angiogenesis is the formation of new blood vessels from preexisting vessels and is an important feature of tissue healing at the site of injury, which delivers oxygen and nutrients to tissue. Defects and delays in this process are associated with impairment in wound healing [29]. Vasculogenesis is the process by which bone marrow-derived endothelial progenitor cells (EPCs) form blood vessels. In this process, a complex of vascular network forms, which is vital for embryonic development [29]. Vasculogenesis also occurs in postnatal, especially in ischemic conditions [30,31]. EPCs are mobilized from the bone marrow; and through systemic circulation, they are home to ischemic tissues to cooperate endothelial cell repair [32,33]. Angiogenesis and vasculogenesis both occur during the wound healing process, but angiogenesis is investigated more frequently and is known to be responsible for neovascularization in wounds [29]. In our experiment, increased vascular density was proven by the histological assessment. This increased density might have
Fig. 5. Immunohistochemically analysis of VEGF. (A) Photomicrographs of skin tissue sections for VEGF-positive cells in the control group, (B) Radiation before surgery group (Tb), (C) Radiation after surgery group (Ta), and (D) Assessment of VEGF-positive cells. *P < 0.05 vs control. Arrows show VEGF-positive cells.
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One limitation in this experiment was that we did not use other antioxidants assays such as superoxide dismutase and catalase. It must be noted that there is no previous study on the effects of LDI on random-pattern skin flaps survival. Therefore, we cannot explain why the LDI therapy was proven to be better after flap surgery than prior to the surgery.
5. Conclusion
Fig. 6. The serum MDA level in different groups.
directly accounted for the increased flap viability. We also hypothesized that the LDI might have stimulated the production of angiogenesis growth factors such as VEGF, which has been shown to induce the neovascularization process and increase vascular density in the ischemic area [34]. The VEGF is an important proangiogenic molecule in the skin. Many studies have shown that VEGF is important for wound healing. It acts as a strong positive regulator of angiogenesis and stimulates the endothelial cell functions required for the formation of new blood vessels [35,36]. Previous studies have indicated that the VEGF is produced by a variety of cell types such as keratinocytes, mast cells, and macrophages in injured skin [37,38]. In this study, the VEGF expression level was markedly higher in both the LDI groups than in the control group. Furthermore, the vascular density in the LDI groups was significantly greater than that in the control. These results suggest that LDI may promote neovascularization and microcirculation in ischemic flaps by increasing the VEGF expression, ultimately improving flap viability. Further study is needed to obtain more detailed insight in the underlying mechanisms. LDI was previously revealed to induce the expression of VEGF in bone fracture and wound healing [39,14]. Guo et al. (2010) found that LDI increases blood vessels regeneration and cell proliferation in the wound tissue, along with enhancing the VEGF and MMP-9 expression [14,40]. MMP-9 has been proven to play an important role in angiogenesis and wound healing; and degrades extracellular matrix to permit the migration of new cells in the process of revascularization [41,42]. MMP-9 extends the bioavailability of the VEGF and also increases the mobilization of the progenitor cells from the bone marrow into circulation [41,43]. The other protective mechanism by which the LDI promotes the wound healing process is related to the up-regulation of antioxidant activity. Zhao et al. (2010) indicated that the antioxidants superoxide dismutase and catalase were up-regulated in the blood and testicular tissue of diabetic rats exposed to LDI [16]. Oxygen deficiency and venous flow insufficiency are important causes of necrosis on random skin flaps. These conditions lead to an increase of free radicals that cause peroxidation of lipids and proteins, and damage cell and organelle membranes, thus destroying tissue structure and function [25,44]. MDA is a product of lipid peroxidation and its level indirectly indicates the degree of tissue damage [44]. In the present study, we found that the MDA level in the LDI groups was lower compared with the control group, but not significant. So, we could not confirm that the LDI had a protective effect on the development of flap necrosis by reducing lipid peroxidation activity. The disagreement between the MDA results of the present study and those from the study by Zhao et al. (2010) may be due to the fact that they delivered a fractionated radiation dose in their study, while we used a single irradiation [16].
The results of the current study indicate that the LDI has the potential to improve skin flap survival. Nevertheless, further studies are necessary to determine other mechanisms of the efficacy of LDI for improving flap viability as well as to determine the optimal dose of ionizing radiation, evaluate fractionated doses, and determine the threshold range of LDI to induce flap survival.
Acknowledgement This study was performed as part of the requirements towards MSc in Anatomy. We are especially thankful to the Vice Chancellor for Research (VCR) of Urmia University of Medical Sciences for the financial supporting of this project.
References [1] Lin B, Lin Y, Lin D, Cao B. Effects of bezafibrate on the survival of random skin flaps in rats. J Reconstr Microsurg 2016;32:395–401. [2] Lu F, Mizuno H, Uysal CA, Cai X, Ogawa R, Hyakusoku H. Improved viability of random pattern skin flaps through the use of adipose-derived stem cells. Plast Reconstr Surg 2008;121:50–8. [3] Yang M, Sheng L, Li H, Weng R, Li QF. Improvement of the skin flap survival with the bone marrow-derived mononuclear cells transplantation in a rat model. Microsurgery 2010;30:275–81. [4] Taleb S, Moghaddas P, Rahimi Balaei M, Taleb S, Rahimpour S, Abbasi A, et al. Metformin improves skin flap survival through nitric oxide system. J Surg Res 2014;192:686–91. [5] Minh TC, Ichioka S, Nakatsuka T, Kawai J, Shibata M, Ando J, et al. Effect of hyperthermic preconditioning on the survival of ischemia-reperfused skin flaps: a new skin-flap model in the mouse. J Reconstr Microsurg 2002;18:115–9. [6] Jones M, Zhang F, Blain B, Guo M, Cui D, Dorsett-Martin W, et al. Influence of recipient-bed isolation on survival rates of skin-flap transfer in rats. J Reconstr Microsurg 2001;17:653–8. [7] Cai L, Huang W, Lin D. Effects of traditional Chinese medicine Shuxuetong injection on random skin flap survival in rats. Sci World J 2014;26:816545. http://dx.doi.org/10.1155/2014/816545. [8] Shalom A, Friedman T, Westreich M. Effect of aspirin and heparin on random skin flap survival in rats. Dermatol Surg 2008;34:785–90. [9] Chen GJ, Chen YH, Yang XQ, Li ZJ. Nano-microcapsule basic fibroblast growth factor combined with hypoxia-inducible factor-1 improves random skin flap survival in rats. Mol Med Rep 2016;13:1661–6. [10] Baldan CS, Masson IF, Esteves Júnior I, Baldan AM, Machado AF, Casaroto RA, et al. Inhibitory effects of low-level laser therapy on skin-flap survival in a rat model. Plast Surg (Oakv) 2015;23:35–9. [11] Tacani PM, Liebano RE, Pinfildi CE, Gomes HC, Arias VE, Ferreira LM. Mechanical stimulation improves survival in random-pattern skin flaps in rats. Ultrasound Med Biol 2010;36:2048–56. [12] Luckey TD. Nurture with ionizing radiation: a provocative hypothesis. Nutr Cancer 1999;34:1–11. [13] National Research Council. Health risks from exposure to low levels of ionizing radiation: BEIR VII, Phase 2/ committee: Committee to assess health risks from exposure to low levels of ionizing radiation. National Research Council; 2006. p. 2. [14] Guo WY, Wang GJ, Wang P, Chen Q, Tan Y, Cai L. Acceleration of diabetic wound healing by low-dose radiation is associated with peripheral mobilization of bone marrow stem cells. Radiat Res 2010;174:467–79. [15] Li W, Wang G, Cui J, Xue L, Cai L. Low-dose radiation (LDR) induces hematopoietic hormesis: LDR-induced mobilization of hematopoietic progenitor cells into peripheral blood circulation. Exp Hematol 2004;32:1088–96. [16] Zhao H, Xu S, Wang Z, Li Y, Guo W, Lin C, et al. Repetitive exposures to lowdose X-rays attenuate testicular apoptotic cell death in streptozotocin-induced diabetes rats. Toxicol Lett 2010;192:356–64. [17] Bose Girigoswami K, Bhaumik G, Ghosh R. Induced resistance in cells exposed to repeated low doses of H2O2 involves enhanced activity of antioxidant enzymes. Cell Biol Int 2005;29:761–7.
Please cite this article in press as: Karimipour M et al. Effects of gamma-low dose irradiation on skin flap survival in rats. Phys. Med. (2017), http://dx.doi. org/10.1016/j.ejmp.2017.07.019
6
M. Karimipour et al. / Physica Medica xxx (2017) xxx–xxx
[18] Kawakita Y, Ikekita M, Kurozumi R, Kojima S. Increase of intracellular glutathione by low-dose gamma-ray irradiation is mediated by transcription factor AP-1 in RAW 264.7 cells. Biol Pharm Bull 2003;26:19–23. [19] Kojima S, Matsuki O, Nomura T, Yamaoka K, Takahashi M, Niki E. Elevation of antioxidant potency in the brain of mice by low-dose gamma-ray irradiation and its effect on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)induced brain damage. Free Radic Biol Med 1999;26:388–95. [20] Pathak CM, Avti PK, Kumar S, Khanduja KL, Sharma SC. Whole body exposure to low-dose gamma radiation promotes kidney antioxidant status in Balb/c mice. J Radiat Res 2007;48:113–20. [21] Zhou XZ, Zhang G, Dong QR, Chan CW, Liu CF, Qin L. Low-dose X-irradiation promotes mineralization of fracture callus in a rat model. Arch Orthop Trauma Surg 2009;129:125–32. [22] Song XS, Zhou XZ, Zhang G, Dong QR, Qin L. Low-dose X-ray irradiation promotes fracture healing through up-regulation of vascular endothelial growth factor. Med Hypotheses 2010;75:522–4. [23] Heissig B, RaWi S, Akiyama H, Ohki Y, Sato Y, Rafael T, et al. Low-dose irradiation promotes tissue revascularization through VEGF release from mast cells and MMP-9-mediated progenitor cell mobilization. J Exp Med 2005;202:739–50. [24] McFarlane RM, Deyoung G, Henry RA. The design of a pedicle flap in the rat to study necrosis and its prevention. Plast Reconstr Surg 1965;35:177–82. [25] Akgül GG, Yenidogan E, Gülçelik MA, Sahin D, Çolakoglu MK, Alagöl H. Effects of N-acetylcysteine on random skin flaps in rats. Wounds 2013;25:68–74. [26] Baldan CS, Marques AP, Schiavinato AM, Casarotto RA. The effects of different doses of 670 nm diode laser on skin flap survival in rats. Acta Cir Bras 2012;27:155–61. [27] Bagdas D, Cam Etoz B, Inan Ozturkoglu S, Cinkilic N, Ozyigit MO, Gul Z, et al. Effects of systemic chlorogenic acid on random-pattern dorsal skin flap survival in diabetic rats. Biol Pharm Bull 2014;37:361–70. [28] Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature 2011;473:298–307. [29] Johnson KE, Wilgus TA. Vascular endothelial growth factor and angiogenesis in the regulation of cutaneous wound repair. Adv Wound Care (New Rochelle) 2014;3:647–61. [30] Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M, et al. Ischemiaand cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med 1999;5:434–8. [31] Tepper OM, Capla JM, Galiano RD, Ceradini DJ, Callaghan MJ, Kleinman ME, et al. Adult vasculogenesis occurs through in situ recruitment, proliferation, and tubulization of circulating bone marrow-derived cells. Blood 2005;105:1068–77.
[32] Asahara T, Masuda H, Takahashi T, Kalka C, Pastore C, Silver M, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatalvasculogenesis in physiological and pathological neovascularization. Circ Res 1999;85:221–8. [33] Martins S, Quelhas-Santos J. Circulating endothelial progenitor cells: a review of definition, characterization and identification. RRJOB 2016;4:42–6. [34] Zhang F, Fischer K, Komorowska-Timek E, Guo M, Cui D, Dorsett-Martin W, et al. Improvement of skin paddle survival by application of vascular endothelial growth factor in a rat TRAM flap model. Ann Plast Surg 2001;46:314–9. [35] Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989;246:1306–9. [36] Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, et al. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science 1989;246:1309–12. [37] Brown LF, Yeo KT, Berse B, Yeo TK, Senger DR, Dvorak HF, et al. Expression of vascular permeability factor (vascular endothelial growth factor) by epidermal keratinocytes during wound healing. J Exp Med 1992;176:1375–9. [38] Shiota N, Nishikori Y, Kakizoe E, Shimoura K, Niibayashi T, Shimbori C, et al. Pathophysiological role of skin mast cells in wound healing after scald injury: study with mast cell-deficient W/W(V) mice. Int Arch Allergy Immunol 2010;151:80–8. [39] Chen M, Huang Q, Xu W, She C, Xie ZG, Mao YT, et al. Low-dose X-ray irradiation promotes osteoblast proliferation, differentiation and fracture healing. PLoS One 2014;9:e104016. http://dx.doi.org/10.1371/journal. pone.0104016. [40] Thanik VD, Chang CC, Lerman OZ, Greives MR, Le H, Warren SM, et al. Cutaneous low-dose radiation increases tissue vascularity through upregulation of angiogenic and vasculogenic pathways. J Vasc Res 2010;47:472–80. [41] Heissig B, Rafii S, Akiyama H, Ohki Y, Sato Y, Rafael T, et al. Low-dose irradiation promotes tissue revascularization through VEGF release from mast cells and MMP-9–mediated progenitor cell mobilization. J Exp Med 2005;202:739–50. [42] Nelson AR, Fingleton B, Rothenberg ML, Matrisian LM. Matrix metalloproteinases: biologic activity and clinical implications. J Clin Oncol 2000;18:1135–49. [43] Heissig B, Werb Z, Rafii S, Hattori K. Role of c-kit/Kit ligand signaling in regulating vasculogenesis. Thromb Haemost 2003;90:570–6. [44] Sun L, Li Y, Shi J, Wang X, Wang X. Protective effects of ligustrazine on ischemia-reperfusion injury in rat kidneys. Microsurgery 2002;22:343–6.
Please cite this article in press as: Karimipour M et al. Effects of gamma-low dose irradiation on skin flap survival in rats. Phys. Med. (2017), http://dx.doi. org/10.1016/j.ejmp.2017.07.019