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Postconditioning Ozone Alleviates
Ischemia-Reperfusion Injury and Enhances Flap Endurance
in Rats
Cagdas Elsurer, Merih Onal, Nebil Selimoglu, Omer Erdur, Mustafa Yilmaz,
Ender Erdogan, Oznur Kal, Jale Bengi Celik & Ozkan Onal
To cite this article: Cagdas Elsurer, Merih Onal, Nebil Selimoglu, Omer Erdur, Mustafa Yilmaz, Ender Erdogan, Oznur Kal, Jale Bengi Celik & Ozkan Onal (2020) Postconditioning Ozone Alleviates Ischemia-Reperfusion Injury and Enhances Flap Endurance in Rats, Journal of Investigative Surgery, 33:1, 15-24, DOI: 10.1080/08941939.2018.1473901
To link to this article: https://doi.org/10.1080/08941939.2018.1473901
Published online: 19 Oct 2018.
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ORIGINAL RESEARCH
Postconditioning Ozone Alleviates Ischemia-Reperfusion
Injury and Enhances Flap Endurance in Rats
Cagdas Elsurer
1, Merih Onal
1, Nebil Selimoglu
2, Omer Erdur
1, Mustafa Yilmaz
3,
Ender Erdogan
3, Oznur Kal
4, Jale Bengi Celik
5, Ozkan Onal
51Department of Otorhinolaryngology, Selcuk University Medical Faculty, Konya, Turkey,2Department of Hand
Microsurgery, Konya Educational and Training Hospital, Konya, Turkey,3Department of Histology and Embryology, Selcuk University Medical Faculty, Konya, Turkey,4Department of Nephrology, Baskent University Medical Faculty Konya Hospital, Konya, Turkey,5Department of Anesthesiology and Intensive Care, Selcuk University Medical Faculty,
Konya, Turkey
ABSTRACT
Introduction: Muscle-flap transferring is a routine approach utilized in reconstructive operations; however, flap morbidity is often a source of post-operative difficulty. Ischemia-Reperfusion Injury (IRI) is an import-ant contributor to the viability of flaps after transferring. The goal of this research was for assess the prob-able useful impacts of ozone on flap survival in a rat muscle-flap design. Materials and Methods: We examined the effects of postconditioning ozone administration on viability of pedicled composite flaps. Twenty-eight Wistar rats were randomized into four groups: sham-operated (S), ischemia-reperfusion (IR), sham-operatedþ ozone (O), IR þ ozone (IR þ O), respectively. The animals were sacrificed on the eighth day. In a general histological evaluation, flap tissues were examined with a light microscope, and apoptotic cells were counted. The Apoptotic Index (AI) was then calculated. Flap-tissue samples were sent for analyses of malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and protein carbonyl (PCO), and blood samples were sent for analyses of Total Oxidant Score (TOS), and Total Antioxidant Capacity (TAC). Data were evaluated statistically using the Kruskal–Wallis test. Results: The histomorphometric score was remarkably greater in O (p ¼ .002). The AI was greater in IR (p ¼ .002). The antioxidant parameters values as regards SOD, GSH-Px, CAT, and TAC were found to be greater in O (p< .005). The oxidant parameters values as regards MDA, PCO, TOS were found to be greater in IR (p< .005). Discussion: The current research indicates that ozone application can attenuate the muscle-flap injury brought about by IR through triggering the increase of the antioxidant capacity.
Keywords: antioxidant; apoptosis; ischemia reperfusion injury; ozone; surgical flaps
INTRODUCTION
Ischemia-reperfusion (IR) is a serious condition, and endothelial impairment and arteriolar vasoconstric-tion are the two best-identified pathologies in IR injury (IRI) [1]. Although the reperfusion of ischemic tissue is an important repair mechanism, it also been shown to impair vasodilation in the feeding arteries of rat skeletal muscle, arteriole inaction, and capillary no-reflow in the muscle microcirculation via promoting the production of oxygen-originated
free radicals and the leave of inflammatory media-tors [2].
Ozone has been utilized as a curative factor and its beneficial actions have been seen at experimental and clinical levels in the management of different diseases [3–5]. Oxidative stress can be defined as exposure to oxidants (e.g., reactive oxygen species [ROS]) and/or reduced antioxidant capacity. Ozone protects Ca2þ-ATPase from inactivation through oxi-dative stress, regulating the gathering of adenosine, and lock-up the xanthine/xanthine oxidase pathway
Received 11 February 2018; accepted 03 May 2018.
Color versions of one or more of the figures in the article can be found online atwww.tandfonline.com/icnv.
Address correspondence to Ozkan Onal, Associate Professor, Department of Anesthesiology and Intensive Care, Selcuk University Medical Faculty, Konya, Turkey. E-mail:drozkanonal@selcuk.edu.tr
15 Journal of Investigative Surgery, 33, 15–24, 2020
Copyright # 2018 Taylor & Francis Group, LLC ISSN: 0894-1939 print / 1521-0553 online DOI:10.1080/08941939.2018.1473901
for ROS formation via the protective mechanism of oxidative preconditioning, which results in the stimulation of antioxidant endogenous systems, and the decrease of glycogen depletion, and lactate pro-duction [6,7].
It has been proven that administering small doses of ozone as a predrug could modified antioxidant enzymes, nitric oxide pathways, and other subcellular activities [8]. Skin-flap surgery, the gold standard, is routinely performed to correct the coverage of injury and tissue fault in order to hinder infection and/or renovate form and function to parts of the body where local options are unsuitable or unavailable [9, 10]. IRI is a common cause of local free-flap col-lapse. It is unavoidable in free-tissue transfers, leads to flap collapse, and has no efficient treatment [1].
The elective nature of free-flap surgeries allows the administration of pharmacological agents to pre-condition against IRI, but there are not many reports on postconditioning, which may be used clinically. The purpose of the present research was to evaluate the impact of the possible use of postconditioning ozone on muscle-flap IRI in rats.
MATERIALS AND METHODS
Twenty-eight adult Wistar rats weighing 250–350 g were used for the research. The use and care of ani-mals in this research was supported by the Guidelines for Animal Experimentation of Selcuk University School of Medicine, Konya, Turkey. All rats were housed at a temperature of 24C ± 3C with a 12-h light-dark period and were acclimated for 7 days before the research. The animals were fed with a standard lump regime and provided with water freely. Animals were anesthetized by intramus-cular injection of a mixture of ketamine 80 mg kg1 IP (Ketalar, Pfizer, Istanbul, Turkey) and xylazine 5 mg/kg IP (Rompun 2%, Bayer, Istanbul, Turkey).
The animals were separated into four groups of seven rats randomly. The number of rats in the groups was determined according to previous stud-ies and limited to 28 in order to eliminate excessive sacrification [1]. In all 28 rats, caudally based dorsal random skin flaps were constituted using the pro-cedure described by Zhang et al. [11] (Figure 1):
Group 1 (n¼ 7) (sham-operated group) (S): The pectoral muscle flap was lifted according to the operating technique, and serum physiologic (SF) was given intraperitoneally (IP) for 7 days without inducing any ischemia. The flap was sutured back into place, and the animals were sacrificed on the eighth day.
Group 2 (n¼ 7) (IR group) (IR): The pectoral muscle flap was lifted according to the operating technique. The total ischemia was created via
axillary artery ligation with 6.0 vicryl for 3 h, and then the suture was released for restoring the flow of blood. The muscle flap was then stitched back in situ using absorbable 4/0 sutures, and the skin cut was stitched using nonabsorbable 5/0 sutures not able to be absorbed. Reperfusion was allowed for 24 h and neither ozone nor SF were given.
Group 3 (n¼ 7) (sham þ ozone group) (O): The pectoral muscle flap was removed but IR injury was not applied, and the ozone/oxygen mixture was given in single 1 mg kg1 doses IP each day for 7 days.
Group 4 (n¼ 7) (IR þ ozone group) (IR þ O): 1 mg kg1 ozone was given IP for 7 days following the pectoral muscle flap, according to the operating technique. The axillary artery was ligatured with 6.0 vicryl for 3 h, and then the suture was released. The muscle flap was then stitched back in situ using absorbable 4/0 sutures, and the skin cut was stitched with nonabsorbable 5/0 sutures. Reperfusion was allowed for 24 h, and then intracardiac blood was drawn under anesthesia for sacrificing animals. The pectoralis flap muscles were removed for histopatho-logical and biochemical investigation. Flap survival was assessed with the apoptosis rates of muscle cells using the TUNEL assay.
Ozone gas was constituted with an ozone-gener-ating set machine (Evozone Basic Plus, Reutlingen,
FIGURE 1. Skin flaps were created following the procedure described by Zahng et al.
Germany). This machine controls the gas flux ratio and ozone collection in the actual period with a let-in UV spectrometer. The ozone flux ratio was malet-in- main-tained consistently at 3 L min1 of a gas mixture of 97% O2þ 3% O3, which equals for a concentration of
60mg mL1. The mass of the gas combination given IP to every animal was almost 3.2–4.2 mL. Tygon polymer pipes and one-use silicon-processed polypro-pylene syringes, which are resistant to ozone, were utilized all over the reaction to guarantee subsump-tion of O3 and intensity of collection. The ozone was
administered for 7 days after ischemia, for a total of seven doses. The duration of ozone administration was determined based on our previous studies. According to these previous studies [12, 13], ozone was administered for 7 days before ischemia, which was sufficient. The rats were narcotized with keta-mine 80 mg kg1IP and xylazine 5 mg kg1IP before IR, and supplementary doses of ketamine and xyla-zine were given at appropriate intervals during the ischemia and reperfusion period. A standard model of IR was used, as described previously by Zhang et al. [11]. A transverse incision was made on half of the thorax to allow access to the pectoralis muscle. After the skin and subcutaneous tissue were dis-sected, the pectoral muscle was uncovered. The muscle was entered from under its lateral edge and the dissection proceeded through the medial side. The pedicle was identified in this plane. Fibers attached to the sternum and humerus were cut, and the muscle was released from the lateral and medial sides. The clavicular-holding fibers were cut carefully. After release of the muscle from all areas, it was brought into the island flap with the blunt, dissected axillary artery. After the pectoralis muscle was completely lifted with only the pedicle intact, the pedicle was ligatured in 3 h handling 6.0 vicryl sutures. Following a 3 h ischemic period, the sutures were released and 24 h of reperfusion was allowed. The muscle flap was then stitched back in situ using absorbable 4/0 sutures and the skin cut was stitched with nonabsorbable 5/0 sutures. The pectoral muscle flap was lifted in the sham group, and the flap was once again stitched into its place without provoking any ischemia.
Histopathological Examination
Pectoral muscle samples measuring 1 cm3were fixed in 4% paraformaldehyde solution overnight. They were then immersed in 30% sucrose solution with 0.1% sodium azide. With a cryostat (Leica CM1900, Leica Microsystems GmbH, Vienna, Austria), 4lm portions were taken on poly-L lysine-covered slides.
The portions were stained by Hematoxylin and Eosin (H&E) and evaluated histopathologically for
muscle cell structure, vascularity, fibrotic and deteri-oration alterations, and mononuclear cell infiltration. For histomorphometric evaluation, digital micropho-tographs of the H&E-stained slides were used at 20 magnification. Prepared on a transparent acet-ate paper, equally spaced a point/grid scale (each representing 1 square unit), placed on monitor screen. The widest muscle area was selected on evaluation and the muscle tissue area portion was calculated by counting grid number over the muscles tissues via point/grid scale, semiquantita-tively [14]. Points on the nonmuscular tissue were not counted. Stereological calculations were per-formed by two researchers in a double-blinded man-ner. To evaluate apoptosis of the pectoral muscle at the cellular grade, TUNEL was done as a widely used and acceptable apoptosis marker. The totic index (AI) was also calculated by total apop-totic cell count divided by 100.
Biochemical Analysis
For the biochemical analysis, the tissues were rinsed twice with cold, saline liquor, put into glass bottles, and stored in a deep refrigerant at80C until proc-essing. The frozen flap tissues were homogenized in a phosphate buffer (pH 7.4) by means of a homogeni-zator (Ultra Turrax IKA T18 Basic, IKA Labortecnic, Staufen, Germany) in an ice cube. The cell fragments were centrifuged at 14,000 rpm (7.530 g) at 4C for 10 min, and the supernatant was analyzed. The total protein concentration of the flap-tissue homogenates was first detected using the technique of Lowry et al. [15] with bovine serum albumin as the usual. Next, MDA, CAT, GSH-Px, SOD, and PCO were evaluated in the flap-tissue samples, and TOS and TAC were evaluated in the blood samples. For these analyses, the blood samples were centrifuged at 3,500 rpm for 15 min, and the serum was gathered and stored at 80C until processing.
Measurement of Malondialdehyde (MDA)
The lipid peroxidation product and flap tissues were homogenized in 1.15% KCl liquor. The homogenates were made from 20 mg of dried, frosted flap tissue and were suspended in 1.5 mL of cold, saline liquor, including 0.001% of hydroxytoluene butylate (BHT) and 200mL 8.1% sodium dodecyl sulfate. The MDA was evaluated handling a calorimetric reaction by thiobarbituric acid as reported by Shin et al. [16]. The concentration of MDA levels was stated qua nmol/mg protein.
Ozone Enhances Flap Endurances in Rats 17
Measurement of Catalase (CAT)
The catalase concentration was evaluated owing to the catalytic activity which assists the degradation of hydrogen peroxide (H2O2) to oxygen and water
through the use of Cayman’s catalase assay kit [17].
Measurement of Glutathione Peroxide (GSH-Px)
A Cayman GSH-Px assay kit was employed to esti-mate the activity of GSH-Px. The effectiveness of the treatment was detected by tissue levels of glutathi-one peroxidase, using the technique of Paglia and Valentine [18]. GSH-Px activity was combined with the oxidation of NADPH by glutathione reductase. The oxidation of NADPH was spectrophotometric-ally carried out at 340 nm at 37C. The absorbance at 340 nm was listed and GSH-Px activity was exhib-ited as U/g protein.
Measurement of Superoxide Dismutase (SOD)
The superoxide dismutase concentration was assessed by inhibition of nitro blue tetrazolium degradation by superoxide anion created with the xhanthine/ xhanthineoxide tract with a trading analysis kit (Nanjing Jiancheng Biological Product, Nanjing China) using the technique of Sun et al. [19]. The cal-culated SOD activity was exhibited as U/g protein.
Measurement of Protein Carbonyl (PCO)
Cayman’s protein carbonyl colorimetric analysis kit was utilized. The quantity of protein hydrozone was measured spectrophotometrically at an absorbance between 360 and 385 nm, using the technique of Levine et al. [20]. The consequences were exhibited as millimoles carbonyl per gram protein.
Measurement of Total Oxidant Status (TOS)
Serum TOS levels of blood samples were detected using an automated, colorimetric evaluation tech-nique of Erel [21]. It was carried out with an Aeroset 2.0 analyzer and a commercial Cayman’s TOS kit. The test is attributed to the oxidation of fer-rous ion to ferric ion in the existence of different oxidant types in acidic medium and the indication of the ferric ion with xylenol orange. The color dens-ity, which can be evaluated spectrophotometrically,
is associated with the overall sum of oxidant par-ticles in the sample and the analysis is calibrated with hydrogen peroxide (H2O2). The
consequen-ces are exhibited according to the micromolar hydrogen peroxide equivalent per liter (lmol H2O2Eq/L).
Measurement of Total Antioxidant Capacity (TAC)
Measurement of TAC was performed employing an aero set 2.0 analyzer and a Cayman’s total antioxi-dant score kit, using a novel automated evaluation technique by Erel [22]. It is a novel automated direct evaluation technique for total antioxidant capacity utilizing a novel generation, over steady ABTS rad-ical cation. In this way, the antioxidative influence of the sample versus the strong free-radical effects, which is started by the generated hydroxyl radical, is evaluated. The consequences are exhibited as lmol Trolox Eq/L. The free-radical effect ratio is calibrated with Trolox, which is standard for usage of TAC measurement assays.
Statistical Analysis
Statistical analysis was done employing SPSS 15.0 for Windows (SPSS, IL). The consequences are exhibited as mean ± standard deviation. The differ-ences in pathological detections among the research groups were examined with the Kruskal–Wallis test. While a whole, statistically meaningful difference was found, pairwise comparisons were controlled with the Mann–Whitney U test. Consequences were considered statistically meaningful if the two-tailed p was<.05.
RESULTS Histopathological Results
Histomorphometric evaluation was remarkably dif-ferent among the four groups (ptrend< .001). In the
pairwise analysis, the histomorphometric score was remarkably higher in S than in IR (p¼ .002) and IRþ O (p ¼ .002), and higher in O than in S (p¼ .002), IR þ O (p ¼ .002), and IR (p ¼ .002). The histomorphometric score was significantly higher in IRþ O than in IR (p ¼ .002) (Figures 2and3).
The histopathological results were as follows: Group I: Muscle organization was moderate. Vascularization was increased in a smaller amount. A small amount of fibrotic changes was shown (Figure 3, I). Mononuclear cell infiltration was
shown, especially in restricted, deteriorated muscular areas compared to the protected muscular zones (Figure 3, I).
Group II: Muscle-tissue organization was dra-matically damaged so that some slides had mono-nuclear cell infiltration rather than muscle fibers (Figure 3, II). Vascularization was substantially increased. Fibrotic tissue formation was the highest in this group (Figure 3, II).
Group III: Muscle organization was almost moderate. Muscle-fiber structures were degenerated and there was mononuclear cell infiltration among the fibers (Figure 3, III). Vascularization was increased in a smaller amount. Fibrotic tissue forma-tion was poorly observed (Figure 4, III).
Group IV: Muscle organization was less than in Group III. Muscle-fiber structures had a small amount of degeneration. Mononuclear cell infiltra-tion was seen among some muscle fibers (Figure 3, IV). Little fibrotic tissue formation and vasculariza-tion were observed (Figure 3, IV).
The AI was significantly different between the four groups (ptrend< .001). In the pairwise analysis,
AI was higher in IR than in IRþ O (p ¼ .03), S (p¼ .002), and O (p ¼ .002). AI was higher in IR þ O than in S (p¼ .002) and O (p ¼ .002). AI was higher in S than in O (p¼ .005) The results show that a stat-istically important difference is present among the apoptosis values of the control group and the treat-ment group with ozone application (p< .01) (Figures 4and 5).
Biochemical Results
The results of the oxidant and antioxidant markers in all groups are demonstrated inFigure 6.
SOD
SOD was significantly different among the four groups (ptrend< .0001). In the pairwise comparison, SOD was
remarkably greater in O, according to S, IR, and IRþ O (all p¼ .001). SOD was remarkably greater in IR þ O, according to S and IR (both p¼ .001). In the IR group, SOD was remarkably smaller, according to S (p¼ .001).
GSH-Px
GSH-Px was remarkably different among the four groups (ptrend< .0001). In the pairwise comparison,
GSH-Px was remarkably greater in O, according to S and IR (both p¼ .001). GSH-Px was not signifi-cantly different between S and IRþ O (p ¼ .259). GSH-PX was remarkably smaller in IR, according to S and IRþ O (both p ¼ .001).
CAT
CAT was significantly different between the four groups (ptrend< .0001). In the pairwise comparison,
CAT was remarkably greater in O, according to S, IR,
FIGURE 2. The results of the histomorphometric evaluation.
Ozone Enhances Flap Endurances in Rats 19
and IRþ O (all p ¼ .001). CAT was remarkably smaller in IR, according to S and IRþ O (both p ¼ .001). CAT was smaller in S than in IRþ O (p ¼ .001).
MDA
MDA was significantly different between the four groups (ptrend< .0001). In the pairwise comparison,
MDA was remarkably greater in IR, according to S, O, and IRþ O (all p ¼ .001). MDA seemed to be lower in O than in IRþ O; but the difference was not statistic-ally important (p¼ .053). MDA was higher in S than in O and IRþ O (p ¼ .001 and p ¼ .004, respectively).
PCO
PCO was remarkably different between the four groups (ptrend< .0001). In the pairwise comparison,
PCO was remarkably greater in IR, according to S, O,
and IRþ O (p ¼ .007, p ¼ .001, and p ¼ .001, respect-ively). PCO was remarkably smaller in O, according to S and IRþ O (both p ¼ .001). PCO was remarkably greater in S, according to IRþ O (p ¼ .001).
TAC
TAC was significantly different between the four groups (ptrend< .0001). In the pairwise comparison,
TAC was significantly higher in O than in S, IR, and IRþ O (all p ¼ .001). TAC was significantly lower in IR than in S and IRþ O (both p ¼ .001). TAC was lower in S than in IRþ O (p ¼ .011).
TOS
TOS was remarkably different among the four groups (ptrend< .0001). In the pairwise comparison,
TOS was remarkably greater in IR, according to S,
FIGURE 3. Histomorphometric evaluation staining with H&E. I: Muscle organization, mononuclear cell infiltration in some endomysium and perimysium layers with poor muscle organization (black arrowheads) and few fibrotic changes (white arrowheads) in G1. II: Muscular organization is quite poor. In some areas, mononuclear cell infiltration is greater than muscular tissue (black arrowheads). There is widespread fibrotic tissue development (arrowheads) and increased vascularization (black arrows) in G2. III: Muscular organization is moderate, with mononuclear cell infiltration in some endomysium areas (black arrowheads), impairment in some muscle fibers (white arrows), fibrotic changes in some perimysium areas (white arrowheads), and a small amount of vascularization (black arrows) in G3. IV. Muscular organization shows moderate impairment in some muscular fibers (white arrows), mononuclear cell infiltration in endomysium and perimysium (black arrowheads), fibrous tissue formation in large areas (white arrowheads), and some increased vascularization (black arrows) in G4.
FIGURE 4. The result of apoptotic index which cell was positive marked with TUNEL.
FIGURE 5. Apoptotic cell labeling with TUNEL (white arrowheads: TUNEL positive, apopitotic muscle cell nuclei) in all groups. A few labeled apoptotic cells (white arrowheads) in G1. Many labeled apoptotic cells (white arrowheads) in G2. Labeled apoptotic cells (white arrowheads), more than G1 and less than G2 in G4. Many labeled apoptotic cells (white arrowheads) in G4.
Ozone Enhances Flap Endurances in Rats 21
O, and IRþ O (all p ¼ .001). TOS was remarkably smaller in O, according to S and IRþ O (both p¼ .001). TOS was greater in S according to IRþ O (p ¼ .001).
DISCUSSION
The aim of the research was to determine if the appli-cation of postconditioning ozone could play a role in the viability of skin flaps. Despite several studies using ozone to augment the survival of several organs, there has been no study investigating the effects of ozone on flap viability. The mechanisms whereby ozone acts still remain to be clarified.
A significant number of inflammatory cells were determined in the IR group in the present research, especially in neutrophils combined with fibroblasts in an edematous tissue, while in the IRþ O and S groups, slight edema and neutrophilic infiltration of the ischemic tissue were observed. The objective results of muscle-flap survival after the flap-surgery phase demonstrated that the apoptosis index ratio was 10% in the S group. Rats subjected to 3 h of ischemia and 7 days of reperfusion in the IR group had 17% injury. In the ozone-applied groups (O and IRþ O), the injury rates were 8% and 15%, respectively.
It has been reported that the local or total loss of pedicle flaps and their complications may approach 25%; however, the success rate of free-tis-sue applications varies between 87 and 97% [23,24]. IRI is incriminated as a reason of tissue collapse in 10% of microvascular reparations that fail, in spite of patent vessel anastomosis [25]. Many pathways are present for IR damage; the main known mecha-nisms include cytokines, coagulopathy, a thrombo-genic state, and ROS, which are generated in the reperfusion period after ischemia. This ultimately leads to necrotic and apoptotic cell death by forming
a thrombogenic situation and thrombus creation and by interacting with the lipid acid radicals in the injured cell membrane, resulting in a lipid peroxida-tion reacperoxida-tion [26]. Several endogenic or exogenic fac-tors that may eliminate or reduce the oxidative injury and respective damages induced by free radi-cals on the components of the cell have been studied. These include superoxide dismutase, cata-lase, allopurinol, and deferoxamine; thrombolytic agents [27]; prostaglandin E1 (PG-E1); vitamin C [28]; anti-inflammatory agents [29]; the antibodies of neutrophil, adhesion, and function receptors (CD11/18, intercellular adhesion molecule-1 [ICAM-1], platelet endothelial cell adhesion molecule-1 [PECAM-1], E-selectin, and P-selectin) [30]; hyper-baric oxygen [31]; and growth factors (basic fibro-blast growth factor [bFGF], platelet-derived growth factor [PDGF], transforming growth factor [TGF], and vascular endothelial growth factor [VEGF]) [32]. Many trials have demonstrated the useful efficacy of ozone on IRI in various organs and tissues [13, 33]. However, these studies have generally been carried out during preconditioning. In the present study, ozone was administered as a postconditioning agent and we investigated its probable effects on flap via-bility, which has not so far been reported in the lit-erature. In addition, we believe that the present research is among the first to analyze the impact of ozone on AI and biochemical parameters. Previous studies have indicated that ozone has an antioxida-tive impact [13,33]. No deleterious effects have been reported in the literature, associated with the employment of ozone, except in those with uncon-trolled hyperthyroidism, sepsis, fauvism, severe blood loss and hemophilia, clotting disorders, recent myocardial infarction, brain stroke with active bleeding, and in pregnant women in the first trimes-ter [13]. We chose the pectoralis muscle flap and a 3-h total ischemia model, as reported by Zhang
et al. [11]. Our experiments showed that angiogen-esis and collateral veins started to expand after 24 h, and 3–7 days are warranted for the smallest capil-lary expansion. Thus, histopathological assessments were carried out in our study to investigate vascu-larization and polymorphonuclear leukocyte (PMNL) inflammatory activity, which are very important in reperfusion damage. Fibrotic changes, which are very important in ischemic damage, were also evaluated in our study’s histopathological assessment. Ozone definitely increased muscle tissue organization and lessened collagen, fibroblast, vas-cularization, and PMNL amounts; this, in turn, showed that ozone either ceased reperfusion dam-age or accelerated healing. Furthermore, the increased edema, apoptotic cells, and PMNLs in dir-ect proportion to the necrosis numbers in the S group showed that inflammation as a result of sur-gery was higher than in the O group. Ozone treat-ment decreased the tissue oxidative stress parameters (lipid peroxidation, protein oxidation, and nitrite plus nitrate) and increased the antioxi-dant enzyme activity (superoxide dismutase and GSH-Px) during IR. Although ozone is a potent oxi-dant agent, at low doses it may behave as an anti-oxidant [33]. Because it is a strong oxidizer, it can stimulate the increase of antioxidant systems, even-tually inhibiting the evolution of oxidative stress. Past reports indicate that separate evaluations of dis-similar oxidant and antioxidant molecules are impractical, as there are large numbers of oxidants and antioxidants in the body and their effects are additive [34]. For this reason, MDA, PCO, SOD, CAT, GSH-Px were analyzed in tissue samples, and TAC and TOS were analyzed in blood samples. Our biochemical results were found to be consistent with those of recent studies regarding the effects of anti-oxidants in IRI [35, 36]. Additionally, these results are congruent with our former study in which ozone acted as an antioxidant, decreasing free-radical-mediated injury in tissues and hindering lipid per-oxidation [13]. Investigators have demonstrated many benefits and functions of ozone [37–40]. Given this information, our study investigated the effects of ozone on total IR damage of pectoralis flaps. We hypothesized that this agent could enhance muscle-flap viability in IRI, and we observed that ozone sig-nificantly increased muscle survival. Ozone also resulted in a significant decrease in muscle cell apoptosis. The data from IR studies concerning the reducing and protective effects of ozone on cell death through apoptosis in different tissues are clear [39, 41] However, one study showed that acute ozone exposure severely compromised cell mem-brane integrity, while repetitive, short-duration exposures to ozone increased cellular plasticity by means of the induction of antiapoptotic pathways in
a treatment-regimen-specific fashion [39]. By limit-ing the damage produced durlimit-ing ischemia with ozone administration, we can prolong this reversible period and restrict tissue damage.
This study has some limitations. First, we administered the ozone after the creation of the IR injury. Administering ozone both before and after IR injury would yield more reliable results. The final and most important limitation is that our study is not a clinical trial and further study is needed before use in a clinic.
CONCLUSION
In conclusion, the present study shows that ozone administration alleviates the muscle-flap injury caused by IR. This benefit correlates with elevated cell proliferation and decreased cell death via apop-tosis. Further clinical trials may be required to estab-lish whether an ozone/oxygen mixture may be useful for maintaining muscle-flap viability. Our study showed that ozone decreased neutrophil infil-tration, fibrotic tissue formation, angiogenesis in the muscle flap after ischemia and reperfusion, and free oxygen radicals, while it increased muscle tissue organization. The most important effect was that ozone significantly increased muscle survival. Ozone can be used clinically, since it has strong favorable effects on IR damage, warrants no second-ary surgical intervention, costs less than many known treatment modalities, and is easy to use. In addition, it did not affect the duration of surgical operations in the present study because it was administered as a postconditioning agent for 7 days after surgery, and it markedly decreased IRI.
DECLARATION OF INTEREST
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.
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