J Plast Surg Hand Surg, 2015; Early Online: 1–6 ISSN: 2000-656X print / 2000-6764 online DOI: 10.3109/2000656X.2015.1047452
ORIGINAL ARTICLE
Effects of ozone pretreatment on viability of random pattern skin flaps in rats Mustafa Öksüz1, Serdar Yüce1, Ömer Faruk Koçak1, Yasin Canbaz1, Murat Cetin ¸ Ragbetli2 & Tolga Mercantepe3
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1 Department of Plastic, Reconstructive and Aesthetic Surgery, 2Department of Medical Histology and Embryology, Faculty of Medicine, Yüzüncü Yıl University, Van, Turkey and 3Department of Medical Histology and Embryology, Faculty of Medicine, Recep Tayyip Erdo gan University, Rize, Turkey
Abstract Background: Medical ozone is a chemical agent that consists of three oxygen atoms and has antioxidant, angiogenic and vasodilator effects. This study evaluated the effects of medical ozone pre-treatment on flap survival. Materials and methods: Rats were divided into four groups of 10 rats each and a 9 3 cm McFarlane flap was used. Sham group: Neither surgical nor ozone pretreatment was used. Control group: No pretreatment was used after surgery. Preoperative ozone group: Preoperative 1 mg/kg ozone was given intraperitoneally for 7 days. No pretreatment was used after surgery. Postoperative ozone Group: Postoperative 1 mg/kg ozone was given intraperitoneally for 7 days. After postoperative 1 week, all groups were evaluated by surface area measurement, histopathology and electron microscopy. Results: With the experimental McFarlane flap model, the experimental groups had better surface area measurements, along with histopathological and electron microscopic results when compared with the control group. Conclusion: Medical ozone had positive effects on flap survival due to its antioxidant, angiogenic and vasodilator qualities. Key Words: Ozone pre-treatment, flap necrosis, McFarlane flap
Introduction Flap survival is linked with its vascularisation. After surgery, some flaps live totally, but some may show distal necrosis. According to the severity of ischaemia, necrosis occurs on flaps [1,2]. To improve flap survival and prevent necrosis or ischaemia, hyperbaric oxygen, ischaemic preconditioning and various pharmacological agents have been used. Sympatholytics, vasodilators, Ca channel blockers, anti-haemorrhagics, prostaglandin inhibitors, anticoagulants, glucocorticoids and free oxygen radical-preventing antioxidants have been tested with differing degrees of success [3,4]. Reperfusion-induced free radicals are effective on the pathophysiology of ischaemic breakdown after flap surgery. Antioxidants, such as ozone, prevent tissue injury by decreasing free radicals and their effects [5]. In addition to being an antioxidant, ozone also decreases oxidant stress by improving hypoxia with its peripheral vasodilator effect and activation of superoxide dismutase [6]. With its antioxidant and angiogenic qualities, medical ozone pretreatment may improve flap survival. Thus, this study investigated the effects of ozone pretreatment on flap survival. Materials and methods Adult female Wistar albino rats (n = 40) weighing 250–290 g were used in this study. The rats were randomly divided into four groups of 10 animals each. No enteral or parenteral antibiotics were administered. Rats were individually housed in wire cages at constant room temperature (21 ± 2 C) with a 12 hour light/dark cycle. Standard laboratory food and water were provided to the rats ad libitum. This study was approved by
the Animal Ethics Committee, Faculty of Medicine, Yüzüncü Yıl University. All surgical procedures were performed by the same surgeon. The rats were anaesthetised with 100 mg/kg ketamine hydrochloride (Ketalar, Eczacıbası, Turkey) and 5 mg/kg xylazine (Rompun, Bayer, Germany). After anaesthesia, rats were laid on an operating table and the hair on their backs was shaved. After that, a 3 9 cm flap with caudal pedicle was planned on the back of the rat. After incision of the margins of the flap, a random pattern skin flap, including skin and panniculuscarnosus, was removed with blunt dissection. The flap was then sutured again to its donor site. . . .
Sham group (Group S): Neither surgery nor ozone treatment was used. Control group (Group C): No other procedure was used after surgery. Experimental groups: Preoperative ozone group (Group Pre-O): Using a 28 G injector tip and 10 cc injector, 50 mg/ml concentrated 1 mg/kg ozone derived from Humanoza Ozone Generator (Humares, Germany) and linked to O2 source was given once a day intraperitoneally for 7 days preoperatively. At the end of the 7th day, surgery was done with the control and postoperative ozone groups. No other treatment was used after surgery. Postoperative ozone group (Group Post-O): Using a 28 G injector tip and 10 cc injector, 50 mg/ml of the concentrated 1 mg/kg ozone was given once a day intraperitoneally for 7 days postoperatively.
Correspondence: Serdar Yüce, MD, Yüzüncü Yıl Üniversitesi, Tıp Fakültesi, Plastik Cer. A.D. 65100, Van, Turkey. Tel: 0090 432 215 04 71. E-mail: [email protected] (Received 22 October 2014; accepted 30 March 2015) 2015 Informa Healthcare.
2 M. Öksüz et al.
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area measurements, the proportion of necrotic area to full flap area was determined. For the histopathology, three parameters (vascular density, collagen density and oedema) were evaluated. For the electron microscopy, mitochondrial injury was investigated. Surface area investigation Photos taken at the end of the 7th day were transformed to graphics with Photoshop CS4 (Adobe Systems, San Jose, CA, USA) and Windows 8 (Microsoft Corporation, Redmond, WA, USA). First, necrotic area and then full flap area drawings were made and the pixel counts were saved with Photoshop. The numbers were compared and written as a percentage area. Suspicious areas on the flaps were accepted as necrotic (Figure 2). Figure 1. Flap photos of postoperative 7th days. Control group (Group C) (Left), Preoperative ozone group (Group Pre-O) (Middle), Postoperative ozone group (Group Post-O) (Right).
At the end of the postoperative 7th day, the C, Pre-O and Post-O groups were photographed under anaesthesia with a Fuji Finepix S1500 (Fujifilm Corporation, Tokyo, Japan) digital camera on a tripod from the same distance (20 cm) (Figure 1). The flap and necrosis areas were measured on the photos. Then tissue biopsies were taken for histopathological and electron microscopic evaluation from the flap-necrosis transit zone on Groups C, Pre-O and Post-O and from the caudal side distal 1/ 3 area on Group S. After this procedure on the 7th day, the rats were sacrificed with cervical dislocation. Evaluation Surface area measurements, histopathology and electron microscopy results were evaluated. On the photos taken for surface
Histopathological investigation Skin biopsies (1 1 cm) taken from the transit zone of necrotic and healthy tissue on the 7th day were set into paraffin blocks after being fixed in 10% formalin (Leica, RM-2125-RT, Nussloch, Germany). For every 55 steps, 4-mm thick slices were taken and one slice was chosen. For each subject, 10 preparations were made. The slices were dyed with hematoxylineosin and analyzed under a light microscope by a histopathology technician who did not know the group of materials. Vascular density, oedema and collagen density per unit volume were determined for the specimens. Shtereom 1.4 software (Yüzüncü Yıl University, Van, Turkey) was used for the histological study. Calculations were made stereologically with the Cavalieri principle [7]. The volume fraction (volume density) ratio obtained using surface area and tissue thickness on preparations between the control group and experimental groups was detected via marking according to descriptive characteristics searched for on H&E dye, 10. The coefficient of error for all
Figure 2. Flap surface area measurement with Photoshop CS4 (Adobe Systems, USA).
Effects of ozone on viability of skin flaps in rats
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subjects determined whether slice number and calculations were enough. It was used to prevent this ratio from passing 5%. In addition, it was used to prevent the coefficient of variation from passing 10%, which arises from biological characteristics of subject for detecting sufficiency of subject numbers. Electron microscopic examination Skin tissue was cut into small pieces of 1 mm3. The 1 mm3 pieces of skin were fixed in 100 mM phosphate buffer containing 2.5% glutaraldehyde for 2 hours at 4 C. The skin was then washed in phosphate buffer and stored at 4 C for later processing. For the study, the prepared skin samples were post-fixed in 1% osmium tetroxide, dehydrated in an ethanol series and then embedded with an epoxy resin Kit (Araldite CY212, Agar Scientific, Essex, UK). Sagittal sections (750 nm thick) were cut and toluidine-blue stained for light microscopy analysis. Stained sections were visualised with a Leica DM 6400 (Leica Microsystems, Wetzlar, Germany). For electron microscopy analysis, ultrathin sections, 60–70 nm thick, were cut in ultramicrotome (LKB Nova, Sweden) set on 200-mesh copper or nickel grids. Ultrathin sections at the top of the copper grid were stained with uranyl acetate and Reynolds’s lead citrate for analysis with a JEOL 100SX transmission electron microscope (JEOL Ltd., Akishima, Tokyo, Japan) and photographic image capture (Kodak 4489, Eastman Kodak Company, Rochester, NY). Statistical analysis The Kruskal-Wallis test was used to compare groups. The Spearman correlation coefficient was calculated separately for every group to define links between variants. Statistical significance levels were accepted as 5% and SPSS was used for calculations. Results At the end of 7 days, surface area, histopathology and electron microscopy measurements were obtained in a blinded fashion. Surface area investigation Mean necrosis ratios on flaps were found as follows: 35.2% for Group C, 21% for Group Pre-O and 27% for Group Post-O. Statistical significance of necrosis percentage was found between groups (p = 0.001).
Figure 3. Histopathological images of groups. Sham group (Group S) (above left), Control group (Group C) (above right), Preoperative ozone group (Group Pre-O) (below left), Postoperative ozone group (Group Post-O) (right) (below right).
Histopathological investigation With regard to vascular density, scores were statistically significant between Groups C and Pre-O, Groups C and S, Groups Pre-O and Post-O and Groups S and Post-O (p = 0.011). The highest ratio for vascular density was in Group Pre-O and no statistical significance was found with Group S. Nevertheless, statistical significance was found with Group C. For oedema, scores were statistically significant between Groups C and Pre-O (p = 0.001). The lowest oedema ratio in the surgically treated groups was detected in the pre-O group. The highest oedema ratio was detected in the control group, but it was not statistically significant from the Post-O group (Figure 3). Nearly the same findings were present for collagen density in all groups and statistical significance was not present (p > 0.05) (Table I). Electron microscopic examination For Group S, mitochondrial fine structure and mitochondrial inner and outer membrane margins were very well defined and mitochondrial matrix granules were apparent. Tubularstructured tips of mitochondrial crista were distinguished (Figure 4). According to the electron microscopic findings, slices taken from the Group C blocks showed necrotic cells. Large cytoplasmic losses and endoplasmic reticulum dilatations
Table I. Histopathological scoring and statistical results for the sham group, control group and experimental groups.
Vascular density
Oedema
Collagen density
Groups
Median
Average
SD
Min.
Max.
p
Group Group Group Group Group Group Group Group Group Group Group Group
4,603.00 4,232.00 5,148.00 4,281.50 1,188.00 5,271.50 4,257.00 4,948.50 108,504.00 97,812.00 97,812.00 97,812.00
4,943.63b 4,312.63a 5,019.30b 4,120.88a 1,213.88c 5,778.75a 4,355.60b 5,252.50a 111,177.00 78,141.75 99,901.40 91,513.00
964.80 499.89 418.36 518.39 199.77 1,357.55 394.09 1,027.23 17,310.16 39,627.87 42,470.26 38,817.73
4,356 3,712 4,356 3,316 945 4,801 3,712 4,207 93,456 32,818 32,818 32,818
7,128 5,197 5,398 4,603 1,485 8,761 5,195 6,781 130,680 135,432 154,440 131,472
0.011
S C Pre-O Post-O S C Pre-O Post-O S C Pre-O Post-O
The average difference between the groups was a significantly different letter.
0.001
0.335
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4 M. Öksüz et al.
Figure 4. Sham group (Group S), micrograph normal mitochondria in skin tissue being monitored. Intensive type of matrix is observed in mitochondria (arrowhead). 25,000.
were seen in the cell cytoplasms and the mitochondrial inner and outer membranes margins had lost their integrity (Figure 5). When micrographs of Group Pre-O were investigated, the mitochondrial matrix had a less dense structure. Mitochondrial inner and outer membranes were seen and tubular-structured tips of mitochondrial crista were distinguished (Figure 6). For Group Post-O, normal apparent mitochondria were seen. When mitochondrial fine structure was investigated, the matrix had a dense structure, inner and outer membranes were apparent and the cristae had a tubular structure (Figure 7). Discussion After repair of tissue defects with flaps, feeding problems, occurring especially in the distal parts, sometimes emerge. This can be a difficult problem to cope with. Many researchers have reported circulatory failure as the primary aetiology of postoperative skin flap necrosis [1,2,8-10]. The trigger event that causes flap necrosis is the separation of sympathetic nerves and vessels supplying the flap after elevation. The main factors responsible for the pathogenesis of the necrosis occurring in the distal part after flap elevation are decreased blood flow and reperfusion injury. The decreased blood flow in the distal part of the flap is a result of the sympathetic activation process. Then
Figure 5. Control group (Group C), micrograph of necrotic cells in skin tissue observed. Observed in the Fibroblasts cytoplasm of dilated rough endoplasmic reticulum (spiral arrow). Collagen fibrils (arrows). 20,000.
Figure 6. Preoperative ozone group (Group Pre-O), micrograph of normal mitochondria in skin tissue observed. Mitochondrial matrices are observed to be of a less dense structure. In the inner and outer membrane with cristae structure monitored (arrowhead). 20,000.
the release of vasoconstrictor substances causes a further decrease in blood flow. The alpha-adrenergic system-based vasoconstriction causes ischaemia mostly in the distal part of the flap. Sympathetic neurotransmitters are consumed within 12–24 hours. If ischaemia in the distal flap persists for 6–12 hours, reperfusion injury occurs when circulation is provided. Then microvascular occlusion occurs and necrosis develops in the tissues [11]. Glycosylation with anaerobic metabolism begins in ischaemic tissues. Reactive oxygen species (ROS) increase with anaerobic glycosylation. Increased ROS results in toxic action by causing lipid peroxidation in cell membranes. This leads to local acute inflammation, leukocytes accumulation, adhesion and, ultimately, endothelial damage, by causing microvascular occlusion [4]. Ozone is a chemical compound consisting of three oxygen atoms. This is a very high-energy carrying form of normal atmospheric oxygen, which consists of two atoms. Ozone is used as an O2/O3 mixture in medical applications. After reinfusion of blood mixed with ozone, the increase in nitric oxide levels reduces hypoxia, superoxide dismutase activity and
Figure 7. Postoperative ozone group (Group Post-O), Micrograph of normal mitochondria monitored in Group 4. Crista structure and mitochondrial dense matrix-type structure being monitored (arrowhead). 25,000.
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Effects of ozone on viability of skin flaps in rats glutathione level by vasodilation in the ischaemic area. Thus, it reduces oxidative stress [6]. In light of pharmacological studies, ozone can be considered as a prodrug. Certain non-toxic doses of ozone may induce the rearrangement of biochemical pathways and may cause activation of secondary messengers in many systems. In this context, it constitutes the best example of ischaemic preconditioning. Low doses of ozone have been seen to regulate antioxidant enzymes, the nitric oxide pathway and other cellular activities and support surprising effects of ozone in many pathological conditions [12]. Ozone effect of induced adaptation against chronic oxidative stress was defined in 1996 [13]. Other studies have shown that controlled ozone administration can reduce damage induced by ROS by oxidative preconditioning, providing adaptation to oxidative stress [14]. Ozone can provide oxidative preconditioning by protecting and activating the endogenous antioxidant system [12]. Preconditioning with ozone increases the manganese superoxide dismutase isoform and prevents mitochondrial damage in hepatic ischaemiareperfusion model rats. In contrast to the protection provided by ozone, cycloheximide prevents this and cellular redox balance is disturbed when cycloheximide is given. Thus, ozone pretreatment maintains mitochondrial function and cellular redox balance [15]. In a study by Calunga et al. [16] in rats exposed to warm ischaemia, biological responses of the kidney to ozone were tested and the results showed increased antioxidant enzyme activity and an effective defense mechanism against the toxic effects of reactive oxygen species ROS in the kidney. In this study, based on Guven et al.’s [17] study, a 1 mg/kg dose of ozone was administered intraperitoneally. In addition, a 1 mg/kg dose of ozone was administered intraperitoneally to another surgery group postoperatively to see whether it would have a positive effect. The injections were administered for a week to benefit from the antioxidant effects of the agents. When comparing the rate of necrosis, flap necrosis rates in the control group were significantly higher than that of the other groups (p < 0.01). The lowest rate of necrosis was found in the Group Pre-O within the treatment groups. A 99% statistical difference was observed between the experimental groups and the control group. The experimental groups showed a significant difference among themselves (p < 0.05). These findings show that preoperative and postoperative ozone administration has a positive effect in reducing flap necrosis. The effect of ozone pretreatment on reducing skin flap necrosis compares favourably to studies using different agents. It showed less tissue necrosis decreasing effects than VEGF (Vascular Endothelial Growth Factor), but more than FGF (Fibroblastic Growth Factor) [18,19]. In rats with experimentally created diabetes mellitus, alpha-tocopherol has been shown to increase flap survival rate by ~13–15% [20]. Sarıfakioglu et al. [21] showed that, with their study, giving 10 mg/kg/day sildenafil citrate orally for 1 week increased the feeding of dorsal flaps by 12.3%. When compared with our study, the effect of ozone pretreatment on flap circulation was greater. Ozone pretreatment prevents tissue necrosis by getting rid of the free radicals with its antioxidant effect. Free radicals cause the formation of tissue oedema and microhaemorrhages by increasing vascular permeability [22]. The antioxidant feature of ozone pretreatment was expected to reduce oedema caused by free radicals. The histopathological results
5
supported this notion. The amount of tissue oedema in Group C was found more in slices. A significant difference was detected between Groups Pre-O and C. Lipid peroxidation, vessel wall damage and, as a result, increased vascular wall permeability generated by free oxygen radicals. In addition, the chemotactic agents that trigger oedema and inflammation can be reduced by administration of antioxidant agents. Group Pre-O had less oedema and this is linked to the inhibition of free radicals by the ozone pre-treatment. Angiogenesis is a biological mechanism characterised by the formation of new capillaries, accomplished by the activation, migration and proliferation of endothelial cells. The potential of growth factors to stimulate angiogenesis is inhibited by free radicals. Antioxidants prevent this situation and may contribute to the proliferation of vessels. Vessel volume was evaluated in order to measure angiogenesis in the study’s histopathology samples. Vascular volume measurements showed a significant increase in Group Pre-O compared to Group C (p = 0.035). Groups Pre-O and S had similar values with no statistical significance. Groups Post-O and C had similar values with no statistical significance; however, both groups had statistically significantly lower values than did Groups S and Pre-O. Ozone pretreatment assisted angiogenesis by preventing free radicals from reducing the effects of angiogenic growth factors. In the electron microscopic examination of the fine structure of the mitochondria, Group C was observed to have different properties than did Groups Pre-O and Post-O. Necrotic cells and quite damaged mitochondria were seen in Group C; however, less damage in the inner and outer membranes of the mitochondria and its cristae were seen in Groups Pre-O and Post-O. The surface area measurements, histopathology and electron microscopy performed in this study showed the flap necrosisreducing effects of the preoperative and postoperative use of ozone. The reduction in surface area of flap necrosis was especially improved with the administration of ozone preoperatively. These results suggest ozone caused systemic oxidative preconditioning and triggered other similar mechanisms. As a result, we believe that, with its antioxidant, angiogenic and vasodilator effects, ozone is capable of preventing necrosis by affecting steps in the formation of ischaemic-reperfusion injury during flap surgery and after it. Acknowledgement This study was approved by the Animal Ethics Committee, Faculty of Medicine, Yüzüncü Yıl University. Declaration of interest: This study was supported by the office of scientific research projects of Yüzüncü Yıl University. References
[1] Daniel RK, Kerrigan CL. Principles and physiology of skin flap surgery. In Mc Carthy, JG. editor. Plastic surgery. Philadelphia, PA: W.B. Saunders; 1990. p 275–328. [2] Fisher J, Gingrass MK. Basic principles of skin flaps. In Georgiade GS, Riefkohl R, Levin LS, editors. Georgiade plastic, maxillofacial and reconstructive surgery. 3rd ed. Baltimore, MD: Williams & Wilkins; 1997. p 19–28. [3] Kayser MR, Hodges PL. Surgical flaps. In Barton Jr FE, editor. Selected readings in plastic surgery. Vol. 8, No. 3. Dallas, TX: Baylor University Medical Center; 1995. p 1–58.
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6 M. Öksüz et al. [4] Vedder NB. Flap physiology. Mathes plastic surgery. 2nd ed. Philadelphia, PA: Saunders Elsevier; 2006. p 483–506. [5] Basu TK. Potential role of antioxidant vitamins. In Basu TK, Temple NJ, Garg ML, editors. Antioxidants in human health and disease. New York: CABI Publishing; 1999. p 15–17. [6] De Monte A, van der Zee H, Bocci V. Major ozonatedautohemotherapy in chronic limb ischemia with ulcerations. J Altern Complement Med 2005;11:363–7. [7] Gundersen HJ, Bendtsen TF, Korbo L, et al. Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 1988;96:379–94. [8] Smith JD, Pribaz JJ. Flaps. Plastic surgery. Indications, operations, and outcomes. In Achauer BM, Eriksson E, Wilkins EG, Vandekam VM, editors. St. Louis, MO: Mosby; 2000. p 261–90. [9] Hosnuter M, Babuccu O, Kargi E, Altinyazar C. Dual preconditioning: effects of pharmacological plus ischemic preconditioning on skin flap survival. Ann Plast Surg 2003;50:398–402. [10] Eroglu L, Orak I, Turhan Haktanir N. Effect of short-term use of oral smokeless tobacco on random- pattern skin flap survival in rats. Scand J Plast Reconstr Surg Hand Surg 2005;39: 272–6. [11] Stein HJ, Fayman MS, Oosthuizen MM, Hinder RA. Verapamil improves survival of rat hyperemic island skin flaps. Surgery 1989;106:617–22. [12] Re L, Mawsouf MN, Menendez S, et al. Ozone therapy: clinical and basic evidence of its therapeutic potential. Arch Med Res 2008;39:17–26. [13] Bocci V. Does ozone therapy normalize the cellular redox balance? Implications for therapy of human immunodeficiency
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virus infection and several other diseases. Med Hypotheses 1996;46:150–4. Leon OS, Menendez S, Merino N, et al. Ozone oxidative preconditioning: a protection against cellular damage by free radicals. Mediators Inflamm 1998;7:289–94. Ajamieh HH, Berlanga J, Merino N, et al. Role of protein synthesis in the protection conferred by ozone-oxidativepreconditioning in hepatic ischaemia/reperfusion. Transpl Int 2005;18:604–12. Calunga JL, Menendez S, Barber E, et al. Ozone biological response in kidneys of rats submitted to warm ischemia. Ozone Sci Eng 2003;25:233–40. Guven A, Gundogdu G, Sadir S, et al. The efficacy of ozone therapy in experimental caustic esophageal burn. J Pediatr Surg 2008;43:1679–84. Fujihara Y, Koyama H, Nishiyama N, et al. Gene transfer of bFGF to recipient bed improves survival of ischemic skin flap. Br J Plast Surg 2005;58:511–17. Murohara T, Horowitz JR, Silver M, et al. Vascular endothelial growth factor/vascular permeability factor enhances vascular permeability via nitric oxide and prostacyclin. Circulation 1998; 7:99–107. Eryaman E, Is¸ıksaçan V, S¸ aro glu M, et al. Free oxygen radicals and flap survival. Turk Arch Otolaryngol 1999;37:79–83. Sarifakio glu N, Gokrem S, Ates¸ L, et al. The influence of sildenafil on random skin flap survival in rats: an experimental study. Br Assoc Plast Surg 2004;57:769–72. Wenbo Q, Dun-Xian T, Reiter JR, et al. Melatonin reduces lipid peroxidation and tissue edema in cerulean-induced acute pancreatitis in rats. Dig Dis Sci 1999;44:2257–62.
ORIGINAL ARTICLE
Effects of ozone pretreatment on viability of random pattern skin flaps in rats Mustafa Öksüz1, Serdar Yüce1, Ömer Faruk Koçak1, Yasin Canbaz1, Murat Cetin ¸ Ragbetli2 & Tolga Mercantepe3
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1 Department of Plastic, Reconstructive and Aesthetic Surgery, 2Department of Medical Histology and Embryology, Faculty of Medicine, Yüzüncü Yıl University, Van, Turkey and 3Department of Medical Histology and Embryology, Faculty of Medicine, Recep Tayyip Erdo gan University, Rize, Turkey
Abstract Background: Medical ozone is a chemical agent that consists of three oxygen atoms and has antioxidant, angiogenic and vasodilator effects. This study evaluated the effects of medical ozone pre-treatment on flap survival. Materials and methods: Rats were divided into four groups of 10 rats each and a 9 3 cm McFarlane flap was used. Sham group: Neither surgical nor ozone pretreatment was used. Control group: No pretreatment was used after surgery. Preoperative ozone group: Preoperative 1 mg/kg ozone was given intraperitoneally for 7 days. No pretreatment was used after surgery. Postoperative ozone Group: Postoperative 1 mg/kg ozone was given intraperitoneally for 7 days. After postoperative 1 week, all groups were evaluated by surface area measurement, histopathology and electron microscopy. Results: With the experimental McFarlane flap model, the experimental groups had better surface area measurements, along with histopathological and electron microscopic results when compared with the control group. Conclusion: Medical ozone had positive effects on flap survival due to its antioxidant, angiogenic and vasodilator qualities. Key Words: Ozone pre-treatment, flap necrosis, McFarlane flap
Introduction Flap survival is linked with its vascularisation. After surgery, some flaps live totally, but some may show distal necrosis. According to the severity of ischaemia, necrosis occurs on flaps [1,2]. To improve flap survival and prevent necrosis or ischaemia, hyperbaric oxygen, ischaemic preconditioning and various pharmacological agents have been used. Sympatholytics, vasodilators, Ca channel blockers, anti-haemorrhagics, prostaglandin inhibitors, anticoagulants, glucocorticoids and free oxygen radical-preventing antioxidants have been tested with differing degrees of success [3,4]. Reperfusion-induced free radicals are effective on the pathophysiology of ischaemic breakdown after flap surgery. Antioxidants, such as ozone, prevent tissue injury by decreasing free radicals and their effects [5]. In addition to being an antioxidant, ozone also decreases oxidant stress by improving hypoxia with its peripheral vasodilator effect and activation of superoxide dismutase [6]. With its antioxidant and angiogenic qualities, medical ozone pretreatment may improve flap survival. Thus, this study investigated the effects of ozone pretreatment on flap survival. Materials and methods Adult female Wistar albino rats (n = 40) weighing 250–290 g were used in this study. The rats were randomly divided into four groups of 10 animals each. No enteral or parenteral antibiotics were administered. Rats were individually housed in wire cages at constant room temperature (21 ± 2 C) with a 12 hour light/dark cycle. Standard laboratory food and water were provided to the rats ad libitum. This study was approved by
the Animal Ethics Committee, Faculty of Medicine, Yüzüncü Yıl University. All surgical procedures were performed by the same surgeon. The rats were anaesthetised with 100 mg/kg ketamine hydrochloride (Ketalar, Eczacıbası, Turkey) and 5 mg/kg xylazine (Rompun, Bayer, Germany). After anaesthesia, rats were laid on an operating table and the hair on their backs was shaved. After that, a 3 9 cm flap with caudal pedicle was planned on the back of the rat. After incision of the margins of the flap, a random pattern skin flap, including skin and panniculuscarnosus, was removed with blunt dissection. The flap was then sutured again to its donor site. . . .
Sham group (Group S): Neither surgery nor ozone treatment was used. Control group (Group C): No other procedure was used after surgery. Experimental groups: Preoperative ozone group (Group Pre-O): Using a 28 G injector tip and 10 cc injector, 50 mg/ml concentrated 1 mg/kg ozone derived from Humanoza Ozone Generator (Humares, Germany) and linked to O2 source was given once a day intraperitoneally for 7 days preoperatively. At the end of the 7th day, surgery was done with the control and postoperative ozone groups. No other treatment was used after surgery. Postoperative ozone group (Group Post-O): Using a 28 G injector tip and 10 cc injector, 50 mg/ml of the concentrated 1 mg/kg ozone was given once a day intraperitoneally for 7 days postoperatively.
Correspondence: Serdar Yüce, MD, Yüzüncü Yıl Üniversitesi, Tıp Fakültesi, Plastik Cer. A.D. 65100, Van, Turkey. Tel: 0090 432 215 04 71. E-mail: [email protected] (Received 22 October 2014; accepted 30 March 2015) 2015 Informa Healthcare.
2 M. Öksüz et al.
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area measurements, the proportion of necrotic area to full flap area was determined. For the histopathology, three parameters (vascular density, collagen density and oedema) were evaluated. For the electron microscopy, mitochondrial injury was investigated. Surface area investigation Photos taken at the end of the 7th day were transformed to graphics with Photoshop CS4 (Adobe Systems, San Jose, CA, USA) and Windows 8 (Microsoft Corporation, Redmond, WA, USA). First, necrotic area and then full flap area drawings were made and the pixel counts were saved with Photoshop. The numbers were compared and written as a percentage area. Suspicious areas on the flaps were accepted as necrotic (Figure 2). Figure 1. Flap photos of postoperative 7th days. Control group (Group C) (Left), Preoperative ozone group (Group Pre-O) (Middle), Postoperative ozone group (Group Post-O) (Right).
At the end of the postoperative 7th day, the C, Pre-O and Post-O groups were photographed under anaesthesia with a Fuji Finepix S1500 (Fujifilm Corporation, Tokyo, Japan) digital camera on a tripod from the same distance (20 cm) (Figure 1). The flap and necrosis areas were measured on the photos. Then tissue biopsies were taken for histopathological and electron microscopic evaluation from the flap-necrosis transit zone on Groups C, Pre-O and Post-O and from the caudal side distal 1/ 3 area on Group S. After this procedure on the 7th day, the rats were sacrificed with cervical dislocation. Evaluation Surface area measurements, histopathology and electron microscopy results were evaluated. On the photos taken for surface
Histopathological investigation Skin biopsies (1 1 cm) taken from the transit zone of necrotic and healthy tissue on the 7th day were set into paraffin blocks after being fixed in 10% formalin (Leica, RM-2125-RT, Nussloch, Germany). For every 55 steps, 4-mm thick slices were taken and one slice was chosen. For each subject, 10 preparations were made. The slices were dyed with hematoxylineosin and analyzed under a light microscope by a histopathology technician who did not know the group of materials. Vascular density, oedema and collagen density per unit volume were determined for the specimens. Shtereom 1.4 software (Yüzüncü Yıl University, Van, Turkey) was used for the histological study. Calculations were made stereologically with the Cavalieri principle [7]. The volume fraction (volume density) ratio obtained using surface area and tissue thickness on preparations between the control group and experimental groups was detected via marking according to descriptive characteristics searched for on H&E dye, 10. The coefficient of error for all
Figure 2. Flap surface area measurement with Photoshop CS4 (Adobe Systems, USA).
Effects of ozone on viability of skin flaps in rats
3
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subjects determined whether slice number and calculations were enough. It was used to prevent this ratio from passing 5%. In addition, it was used to prevent the coefficient of variation from passing 10%, which arises from biological characteristics of subject for detecting sufficiency of subject numbers. Electron microscopic examination Skin tissue was cut into small pieces of 1 mm3. The 1 mm3 pieces of skin were fixed in 100 mM phosphate buffer containing 2.5% glutaraldehyde for 2 hours at 4 C. The skin was then washed in phosphate buffer and stored at 4 C for later processing. For the study, the prepared skin samples were post-fixed in 1% osmium tetroxide, dehydrated in an ethanol series and then embedded with an epoxy resin Kit (Araldite CY212, Agar Scientific, Essex, UK). Sagittal sections (750 nm thick) were cut and toluidine-blue stained for light microscopy analysis. Stained sections were visualised with a Leica DM 6400 (Leica Microsystems, Wetzlar, Germany). For electron microscopy analysis, ultrathin sections, 60–70 nm thick, were cut in ultramicrotome (LKB Nova, Sweden) set on 200-mesh copper or nickel grids. Ultrathin sections at the top of the copper grid were stained with uranyl acetate and Reynolds’s lead citrate for analysis with a JEOL 100SX transmission electron microscope (JEOL Ltd., Akishima, Tokyo, Japan) and photographic image capture (Kodak 4489, Eastman Kodak Company, Rochester, NY). Statistical analysis The Kruskal-Wallis test was used to compare groups. The Spearman correlation coefficient was calculated separately for every group to define links between variants. Statistical significance levels were accepted as 5% and SPSS was used for calculations. Results At the end of 7 days, surface area, histopathology and electron microscopy measurements were obtained in a blinded fashion. Surface area investigation Mean necrosis ratios on flaps were found as follows: 35.2% for Group C, 21% for Group Pre-O and 27% for Group Post-O. Statistical significance of necrosis percentage was found between groups (p = 0.001).
Figure 3. Histopathological images of groups. Sham group (Group S) (above left), Control group (Group C) (above right), Preoperative ozone group (Group Pre-O) (below left), Postoperative ozone group (Group Post-O) (right) (below right).
Histopathological investigation With regard to vascular density, scores were statistically significant between Groups C and Pre-O, Groups C and S, Groups Pre-O and Post-O and Groups S and Post-O (p = 0.011). The highest ratio for vascular density was in Group Pre-O and no statistical significance was found with Group S. Nevertheless, statistical significance was found with Group C. For oedema, scores were statistically significant between Groups C and Pre-O (p = 0.001). The lowest oedema ratio in the surgically treated groups was detected in the pre-O group. The highest oedema ratio was detected in the control group, but it was not statistically significant from the Post-O group (Figure 3). Nearly the same findings were present for collagen density in all groups and statistical significance was not present (p > 0.05) (Table I). Electron microscopic examination For Group S, mitochondrial fine structure and mitochondrial inner and outer membrane margins were very well defined and mitochondrial matrix granules were apparent. Tubularstructured tips of mitochondrial crista were distinguished (Figure 4). According to the electron microscopic findings, slices taken from the Group C blocks showed necrotic cells. Large cytoplasmic losses and endoplasmic reticulum dilatations
Table I. Histopathological scoring and statistical results for the sham group, control group and experimental groups.
Vascular density
Oedema
Collagen density
Groups
Median
Average
SD
Min.
Max.
p
Group Group Group Group Group Group Group Group Group Group Group Group
4,603.00 4,232.00 5,148.00 4,281.50 1,188.00 5,271.50 4,257.00 4,948.50 108,504.00 97,812.00 97,812.00 97,812.00
4,943.63b 4,312.63a 5,019.30b 4,120.88a 1,213.88c 5,778.75a 4,355.60b 5,252.50a 111,177.00 78,141.75 99,901.40 91,513.00
964.80 499.89 418.36 518.39 199.77 1,357.55 394.09 1,027.23 17,310.16 39,627.87 42,470.26 38,817.73
4,356 3,712 4,356 3,316 945 4,801 3,712 4,207 93,456 32,818 32,818 32,818
7,128 5,197 5,398 4,603 1,485 8,761 5,195 6,781 130,680 135,432 154,440 131,472
0.011
S C Pre-O Post-O S C Pre-O Post-O S C Pre-O Post-O
The average difference between the groups was a significantly different letter.
0.001
0.335
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4 M. Öksüz et al.
Figure 4. Sham group (Group S), micrograph normal mitochondria in skin tissue being monitored. Intensive type of matrix is observed in mitochondria (arrowhead). 25,000.
were seen in the cell cytoplasms and the mitochondrial inner and outer membranes margins had lost their integrity (Figure 5). When micrographs of Group Pre-O were investigated, the mitochondrial matrix had a less dense structure. Mitochondrial inner and outer membranes were seen and tubular-structured tips of mitochondrial crista were distinguished (Figure 6). For Group Post-O, normal apparent mitochondria were seen. When mitochondrial fine structure was investigated, the matrix had a dense structure, inner and outer membranes were apparent and the cristae had a tubular structure (Figure 7). Discussion After repair of tissue defects with flaps, feeding problems, occurring especially in the distal parts, sometimes emerge. This can be a difficult problem to cope with. Many researchers have reported circulatory failure as the primary aetiology of postoperative skin flap necrosis [1,2,8-10]. The trigger event that causes flap necrosis is the separation of sympathetic nerves and vessels supplying the flap after elevation. The main factors responsible for the pathogenesis of the necrosis occurring in the distal part after flap elevation are decreased blood flow and reperfusion injury. The decreased blood flow in the distal part of the flap is a result of the sympathetic activation process. Then
Figure 5. Control group (Group C), micrograph of necrotic cells in skin tissue observed. Observed in the Fibroblasts cytoplasm of dilated rough endoplasmic reticulum (spiral arrow). Collagen fibrils (arrows). 20,000.
Figure 6. Preoperative ozone group (Group Pre-O), micrograph of normal mitochondria in skin tissue observed. Mitochondrial matrices are observed to be of a less dense structure. In the inner and outer membrane with cristae structure monitored (arrowhead). 20,000.
the release of vasoconstrictor substances causes a further decrease in blood flow. The alpha-adrenergic system-based vasoconstriction causes ischaemia mostly in the distal part of the flap. Sympathetic neurotransmitters are consumed within 12–24 hours. If ischaemia in the distal flap persists for 6–12 hours, reperfusion injury occurs when circulation is provided. Then microvascular occlusion occurs and necrosis develops in the tissues [11]. Glycosylation with anaerobic metabolism begins in ischaemic tissues. Reactive oxygen species (ROS) increase with anaerobic glycosylation. Increased ROS results in toxic action by causing lipid peroxidation in cell membranes. This leads to local acute inflammation, leukocytes accumulation, adhesion and, ultimately, endothelial damage, by causing microvascular occlusion [4]. Ozone is a chemical compound consisting of three oxygen atoms. This is a very high-energy carrying form of normal atmospheric oxygen, which consists of two atoms. Ozone is used as an O2/O3 mixture in medical applications. After reinfusion of blood mixed with ozone, the increase in nitric oxide levels reduces hypoxia, superoxide dismutase activity and
Figure 7. Postoperative ozone group (Group Post-O), Micrograph of normal mitochondria monitored in Group 4. Crista structure and mitochondrial dense matrix-type structure being monitored (arrowhead). 25,000.
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Effects of ozone on viability of skin flaps in rats glutathione level by vasodilation in the ischaemic area. Thus, it reduces oxidative stress [6]. In light of pharmacological studies, ozone can be considered as a prodrug. Certain non-toxic doses of ozone may induce the rearrangement of biochemical pathways and may cause activation of secondary messengers in many systems. In this context, it constitutes the best example of ischaemic preconditioning. Low doses of ozone have been seen to regulate antioxidant enzymes, the nitric oxide pathway and other cellular activities and support surprising effects of ozone in many pathological conditions [12]. Ozone effect of induced adaptation against chronic oxidative stress was defined in 1996 [13]. Other studies have shown that controlled ozone administration can reduce damage induced by ROS by oxidative preconditioning, providing adaptation to oxidative stress [14]. Ozone can provide oxidative preconditioning by protecting and activating the endogenous antioxidant system [12]. Preconditioning with ozone increases the manganese superoxide dismutase isoform and prevents mitochondrial damage in hepatic ischaemiareperfusion model rats. In contrast to the protection provided by ozone, cycloheximide prevents this and cellular redox balance is disturbed when cycloheximide is given. Thus, ozone pretreatment maintains mitochondrial function and cellular redox balance [15]. In a study by Calunga et al. [16] in rats exposed to warm ischaemia, biological responses of the kidney to ozone were tested and the results showed increased antioxidant enzyme activity and an effective defense mechanism against the toxic effects of reactive oxygen species ROS in the kidney. In this study, based on Guven et al.’s [17] study, a 1 mg/kg dose of ozone was administered intraperitoneally. In addition, a 1 mg/kg dose of ozone was administered intraperitoneally to another surgery group postoperatively to see whether it would have a positive effect. The injections were administered for a week to benefit from the antioxidant effects of the agents. When comparing the rate of necrosis, flap necrosis rates in the control group were significantly higher than that of the other groups (p < 0.01). The lowest rate of necrosis was found in the Group Pre-O within the treatment groups. A 99% statistical difference was observed between the experimental groups and the control group. The experimental groups showed a significant difference among themselves (p < 0.05). These findings show that preoperative and postoperative ozone administration has a positive effect in reducing flap necrosis. The effect of ozone pretreatment on reducing skin flap necrosis compares favourably to studies using different agents. It showed less tissue necrosis decreasing effects than VEGF (Vascular Endothelial Growth Factor), but more than FGF (Fibroblastic Growth Factor) [18,19]. In rats with experimentally created diabetes mellitus, alpha-tocopherol has been shown to increase flap survival rate by ~13–15% [20]. Sarıfakioglu et al. [21] showed that, with their study, giving 10 mg/kg/day sildenafil citrate orally for 1 week increased the feeding of dorsal flaps by 12.3%. When compared with our study, the effect of ozone pretreatment on flap circulation was greater. Ozone pretreatment prevents tissue necrosis by getting rid of the free radicals with its antioxidant effect. Free radicals cause the formation of tissue oedema and microhaemorrhages by increasing vascular permeability [22]. The antioxidant feature of ozone pretreatment was expected to reduce oedema caused by free radicals. The histopathological results
5
supported this notion. The amount of tissue oedema in Group C was found more in slices. A significant difference was detected between Groups Pre-O and C. Lipid peroxidation, vessel wall damage and, as a result, increased vascular wall permeability generated by free oxygen radicals. In addition, the chemotactic agents that trigger oedema and inflammation can be reduced by administration of antioxidant agents. Group Pre-O had less oedema and this is linked to the inhibition of free radicals by the ozone pre-treatment. Angiogenesis is a biological mechanism characterised by the formation of new capillaries, accomplished by the activation, migration and proliferation of endothelial cells. The potential of growth factors to stimulate angiogenesis is inhibited by free radicals. Antioxidants prevent this situation and may contribute to the proliferation of vessels. Vessel volume was evaluated in order to measure angiogenesis in the study’s histopathology samples. Vascular volume measurements showed a significant increase in Group Pre-O compared to Group C (p = 0.035). Groups Pre-O and S had similar values with no statistical significance. Groups Post-O and C had similar values with no statistical significance; however, both groups had statistically significantly lower values than did Groups S and Pre-O. Ozone pretreatment assisted angiogenesis by preventing free radicals from reducing the effects of angiogenic growth factors. In the electron microscopic examination of the fine structure of the mitochondria, Group C was observed to have different properties than did Groups Pre-O and Post-O. Necrotic cells and quite damaged mitochondria were seen in Group C; however, less damage in the inner and outer membranes of the mitochondria and its cristae were seen in Groups Pre-O and Post-O. The surface area measurements, histopathology and electron microscopy performed in this study showed the flap necrosisreducing effects of the preoperative and postoperative use of ozone. The reduction in surface area of flap necrosis was especially improved with the administration of ozone preoperatively. These results suggest ozone caused systemic oxidative preconditioning and triggered other similar mechanisms. As a result, we believe that, with its antioxidant, angiogenic and vasodilator effects, ozone is capable of preventing necrosis by affecting steps in the formation of ischaemic-reperfusion injury during flap surgery and after it. Acknowledgement This study was approved by the Animal Ethics Committee, Faculty of Medicine, Yüzüncü Yıl University. Declaration of interest: This study was supported by the office of scientific research projects of Yüzüncü Yıl University. References
[1] Daniel RK, Kerrigan CL. Principles and physiology of skin flap surgery. In Mc Carthy, JG. editor. Plastic surgery. Philadelphia, PA: W.B. Saunders; 1990. p 275–328. [2] Fisher J, Gingrass MK. Basic principles of skin flaps. In Georgiade GS, Riefkohl R, Levin LS, editors. Georgiade plastic, maxillofacial and reconstructive surgery. 3rd ed. Baltimore, MD: Williams & Wilkins; 1997. p 19–28. [3] Kayser MR, Hodges PL. Surgical flaps. In Barton Jr FE, editor. Selected readings in plastic surgery. Vol. 8, No. 3. Dallas, TX: Baylor University Medical Center; 1995. p 1–58.
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