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Effects of taurine on reperfusion injury
Journal of Plastic, Reconstructive & Aesthetic Surgery (2011) 64, 921e928
Effects of taurine on reperfusion injury Ovunc Akdemir a, Yan Hede a, Feng Zhang a,*, William C. Lineaweaver b, Zikri Arslan c, Ecmel Songur d a
Department of Plastic Surgery, University of Mississippi Medical Center, Jackson, MS, USA Joseph M. Still Burn and Reconstructive Center, 346 Crossgates Blvd, Suite 202, Brandon, MS, USA c Department of Chemistry, Jackson State University, Jackson, MS, USA d Department of Plastic Surgery, University of Ege, Izmir, Turkey b
Received 15 July 2010; accepted 7 December 2010
KEYWORDS Ischaemia/reperfusion injury; Taurine; Gracilis muscle flap; Malondialdehyde (MDA); Nitroblue tetrazolium (NBT)
Summary Taurine is an organic acid, which has a very important function in the human body. Recently, the antioxidant property of taurine has been much emphasised. In this study, the gracilis muscle flap model was used to investigate the effect of taurine in ischaemia/reperfusion injury in rats. Totally 32 Sprague-Dawley rats were divided into two groups: control group (n Z 16) and the treatment group with taurine (n Z 16). After elevation of the gracilis muscle flap, 4 h of ischaemia was performed in both groups. Thirty min before the reperfusion, taurine (200 mg kg1) was injected intravenously. After 24 h of reperfusion, the amount of malondialdehyde (MDA), tissue water content and flap viability were evaluated. After 72 h of reperfusion, histological findings were evaluated. Amount of MDA and tissue water content were significantly lower (p < 0.005), and the flap viability was significantly higher (p < 0.005) in the treatment group 24 h after reperfusion. On comparing the outcomes of histological analysis between control and treatment groups, the amounts of collagen, fibroblast and angiogenesis in treatment group were significantly higher than those in the control group. However, the amount of polymorphonuclear leucocyte and tissue necrosis in the treatment group were significantly lower than in the control group. Our results showed that taurine played an important role in the process of ischaemia/reperfusion injury and presented certain protective effects with the improvement in flap survival after ischaemia/reperfusion injury. ª 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. The University of Mississippi Medical Center, 2500 North State St. Jackson, MS 39216, USA. Tel.: þ1 601 815 11 47. E-mail address: [email protected] (F. Zhang). 1748-6815/$ - see front matter ª 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.bjps.2010.12.007
922 The success rate of the free flaps at the present time varies between 94% and 98%.1e5 It is a known fact that ischaemia/ reperfusion damage plays an important role in the loss of free flaps. Ischaemia/reperfusion damage includes, acutely, cell apoptosis related to the interaction between the leucocyte and endothelium cells after the cessation of microvascular bleeding and the release of reactive oxygen types.6 The natural antioxidants protect the human body from free radicals and prevent oxidative stress and related illnesses. Thus, they have an important role in human health.7 In recent years, the cause of the damage is claimed to be neutrophils stimulating free oxygen derivatives (ROS), several enzymes and cytokines causing damage in the endothelium and capillary obstruction.8e11 Thus, to protect from ischaemia/reperfusion damage, the antioxidant, anticoagulant, anti-inflammatory and anti-apoptosis agents are beginning to be tried for treatment. Taurine, which cleans the reactive oxygen types, also prevents the changes in the permeability of membrane after oxidant damage.12 Taurine and chlorotaurine neutralise hypochlorosis acid and therefore demonstrate antioxidant effects. It has been seen, in vitro, that this decreases the CD11b receptor in neutrophilia thereby preventing the creation of high-level induced glucose formation and apoptosis in endothelium, that is, protecting the tissues.13e15 In the light of aforementioned information from the literature, we have examined the effects of taurine on reperfusion damages using the gracilis muscle flap model in rats.
Material and method Thirty-two male Sprague-Dawley rats, weighing between 380 and 420 g, were used in this study. The National Research Council’s guidelines for the care and use of laboratory animals were followed. General anaesthesia was applied on all the subjects using isoflurane providing respiratory track anaesthesia (Abbot Laboratories, Chicago, IL, USA).
Surgical procedure A transverse incision was applied in the inguinal zone to expose the gracilis muscle. The small branches to the skin from the gracilis muscle were ligated. It was proceeded to the depth and distal to the inguinal ligament as opening the femoral artery and vein. Dissection was performed carefully until the gracilis pedicle was seen and freed. Then, the proximal borders of the anterior and posterior parts of the gracilis muscle, in the depth of the inguinal ligament, were dissected. The two parts were separated from the abductor muscles. The distal border of the gracilis muscle was dissected from the medial side of saphenous veins so as to be separated from the flap base. After lifting the gracilis muscle completely with only the pedicle intact, the pedicle was clamped for 4 h using Acland V2 microclamp16 (Figure 1). The rats in the control group, group 1, was injected with only 0.5 ml physiological serum 30 min before reperfusion from the saphenous vein, while in the treatment group, group 2, 200 mg kg1 taurine, which had been dissolved in 0.5 ml physiological serum, was administered in the same manner as in the control group. After 4-h ischaemia, the clamp was released for reperfusion. Then, the muscle flaps
O. Akdemir et al.
Figure 1 The images of gracilis muscle, femoral veins and branches after transverse incision. Freed gracilis muscle flap.
were sutured back in situ using 4/0 absorbable suture and the skin incision was closed using 5/0 non-absorbable suture.
Analysis of the subjects After reperfusion, both the group 1 (n Z 16) and group 2 (n Z 16) subjects were placed in cages and separated randomly into two groups, each consisting of eight subjects. We waited for 24 h to assess the reperfusion damage of eight subjects from each group and the gracilis muscles were removed under anaesthesia. The flap survivals and water contents of the removed muscles were examined and biochemical malondialdehyde (MDA) measurements were performed. The gracilis muscles of the other eight subjects were removed after 72 h under anaesthesia. All the removed muscle tissues were washed with physiological serum and immediately frozen in liquid nitrogen and kept at 80 C in a deep freeze (Legaci/Copeland, NC, USA) till the day of analysis. Histopathology scoring was applied to evaluate the reperfusion damage after 72 h (Figure 2).
Assessment at 24th hour Biochemical analysis The working method of Esterbauer et al. was preferred.17 This method depends on the principle of spectrophotometric observation of the complex formed by MDA (which is
Figure 2
Analysis of the Subjects.
Effects of taurine on reperfusion injury a lipid peroxidation product) with thiobarbituric acid (TBA) at 532 nm. The conduct of the test A total of 100 mg ml1 tissue was homogenised in buffer at a pH of 7.4. Artefactual production of additional MDA during processing was eliminated by the addition of 2% butylated hydroxytoluene to homogenised tissue. To this was added 20% trichloroacetic acid in 0.6 N hydrochloric acid. The mixture was centrifuged at 10 000 g for 10 min at 4 C. A total of 0.12 mol l1 TBA in buffer (pH 7.0) was added to the supernatant. The pigment was measured spectrophotometrically at 532 nm. Calculation of MDA level The results were calculated as nmol g1 tissue based on the optical density difference. The water content analysis in flap The muscle sample was dried with a stencil for analysis and the wet weight of the muscle was measured with precision balance (Ohaus Adventurer precision balance). The muscle with known wet weight was placed in a drying oven and kept for 24 h at 85 F. Afterwards, the dry weight of the muscle was measured using the same precision balance. The formula for calculating the water content18: Wet weight Dry weight Water contentð%ÞZ 100 Wet weight Flap survival The gracilis muscles, which had been subjected to 24 h of reperfusion after 4 h of ischaemia, were harvested and their flap survival percentages were analysed.19 To observe the living cell ratio in the gracilis muscles, the histochemical painting method using nitroblue tetrazolium (NBT) was conducted. Coronal sections 5 mm wide were obtained from four different zones of the muscle samples, using a microtome. These sections were painted by placing them into a pH 7.4 solution including 0.05% NBT and 0.2% Tris buffer at room temperature (24 1 C) for 30 min. As shown in the literature with this method, the living cells can be dyed in blue while the necrosed cells cannot. The living cell percentage in the tissue sections was measured by using the standard-point-count method under 40 microscopic magnification. The ratio of the living cells to the total area was calculated.19
923 parameters as polymorphonuclear leucocyte (PMNL), lymphocyte density, capillary vein, fibroblast proliferation, oedema amount, collagen density and necrosis (Table 1). Statistical assessment The statistical analysis of the obtained data was performed using Statistical Package for Social Sciences (SPSS) 14.0 (SPSS, Inc., Chicago, IL, USA) package program. The ManneWhitney U test was used for assessment. The p values <0.05 were regarded as statistically significant.
Results All flaps were bleeding after 4 h of global ischaemia.
The results of 24th-hour assessment Of the eight subjects from each group, the flap colour of three subjects (37.5%) from the control group was observed to be visibly pale by means of macroscopic examination. The flap colour of the treatment group did not demonstrate any change by means of macroscopic examination. Results of biochemical analysis (MDA) The average value of MDA of the control group was 105.53 22.18 nmol g1, while the average value of the treatment group was only 17.68 5.00 nmol g1. The difference of the average value of MDA was statistically significant between the two groups (p < 0.005). The results of flap water content The average value of the control group was 86.6 2.4, while the average value of the treatment group was measured as 74.2 2.8. The water content in the treatment group was slightly lower than that in the control group; however, the difference between the two groups was statistically significant (p < 0.005). Flap survival results The samples taken from the control group (n Z 8) and treatment group (n Z 8) after 24 h of reperfusion were painted with the NBT histochemical painting method and their flap survival results were observed under a microscope (Figure 3). The average value of the control group was 27.7 9.1, while the average value of the treatment group was found to be as high as 82.6 7.4 (Table 2). The difference between the two groups was statistically significant (p < 0.005).
Assessment results at the 72nd hour Assessment at the 72nd hour Histopathology analysis The distal parts of the gracilis muscles, as subjected to 4 h of ischaemia after surgical application, were taken after 72 h of reperfusion. The 1 1-cm tissue biopsies were placed in 10% formalin and embedded in paraffin blocks. Four-micron sections were sliced using a microtome. The sections were painted with haematoxylin and examined under high-performance light microscope (100 and 200 magnification). According to the modified Verhofstad scoring,20 the samples were examined based on the seven
Results of histopathology analysis PMNL density, oedema and necrosis amounts of the samples taken from 16 subjects, as eight from both the treatment and control groups, after 4 h of ischaemia and subsequent 72 h of reperfusion are measured. It has been understood that the aforementioned parameters are higher in the control group. It is also seen that the fibroblast and capillary vein proliferation and collagen density was higher in the treatment group, whereas the necrosis amount is lesser in the same (Figure 4). The lymphocyte scores obtained from both groups are understood to be close to each other.
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O. Akdemir et al.
Table 1
Histopathologic evaluation Verhofstad modified scoring table.
Score
Necrosis
PMNLa
lymphocytes
Oedema
Collagen density
Fibroblast proliferation
vascular density
0 1 2 3
None Superficial Pronounced Massive
Normal Light Pronounced Massive
Normal Light Pronounced Massive
None Light Pronounced Dense
None Light Pronounced Dense
None Light Pronounced Dense
None Light Pronounced Dense
a
PMNL-polymorph nuclear leukocytes.
The average of the necrosis values for 16 subjects varied between 0 and 3, being 2.25 1.164 for the control group and 0.375 0.744 for the treatment group. Thus, it is understood that a significant difference existed for the necrosis values of the control group and the treatment group with taurine administration. This difference was found to be also statistically significant (p < 0.01). The average of the PMNL values was 2 1.069 for the control group and 0.75 1.165 for the treatment group. This difference was found to be also statistically significant (p < 0.05). The average of the lymphocyte values was 0.625 0.744 for the control group and 0.5 0.755 for the treatment group. This difference was not found significant statically (p Z 0.680) (p > 0.05). The average of the oedema values was 1.875 0.640 for the control group and 0.25 0.462 for the treatment group. This difference was found to be significant also statistically (p < 0.005). The average of the collagen density values was 0.5 0.755 for the control group and 2.25 1.165 for the treatment group. This difference was found to be significant also statistically (p < 0.01). The average of the fibroblast proliferation values was 0.625 0.744 for the control group and 1.75 0.707 for the treatment group. This difference was found to be significant also statistically (p < 0.05). The average of the vein proliferation values was 0.875 1.125 for the control group and 2.125 0.834 for the treatment group (Table 3). This difference was found to be significant also statistically (p < 0.05).
Discussion The success rate of free tissue applications, a rather general application in the recent years, varies between 97% and 87%;
however, it has been recorded that the partial or complete flap losses of pedicle flaps, and their complications, may reach 25%.21,22 The insufficient perfusion and ischaemia damage are accepted to cause flap necrosis due to many respective changes in the tissues and veins.23 The known main mechanism in ischaemia/reperfusion damage is that when reperfusion occurs after ischaemia, oxygen reaches the tissues and free oxygen radicals are generated. The free oxygen radical interacts with the lipid acid radicals in the damaged cell membrane and results in a lipid peroxidation reaction.24 The increase of lipid peroxidation causes MDA accumulation, oedema and cell enlargement. Thus, breakages in DNA chain and cell necrosis occur.25 At the capillary level, subsequent to the PMNL infiltration, hypoxic vasoconstriction, increase in capillary stress and endothelium structural changes are proved to exist. It has been understood that PMNL activation plays an active role in reperfusion damage. However, it is also being discussed if the insufficiency of the antioxidant defence system due to increase in free radical reactions has a role in post-ischaemia tissue damage.26 Many contemporary researchers have examined endogenic or exogenic agents, which may eradicate or decrease the oxidative damage and respective injuries caused by free radicals on DNA, proteins, lipids and other components of the cell. The free radical cleaners may not be used clinically, as the respective enzyme levels of rat and humans are different.27e29 These may be summed as free radical cleaners27,28,30e32 (superoxide dysmutase, catalase, allopurinol, deferoxamine, etc.), thrombolytic agents33,34 (urokinase tissue and plasminogen activator (tissue plasminogen activator,TPA)), prostaglandin E1 (PG-E1),33 heparin,33 vitamin C,35 acellular physiological washing
Figure 3 Under the microscope 24 h after reperfusion of the implementation of the gracilis muscle nitroblue tetrazolium (NBT) staining with histochemical staining methods; a) In the control group, necrosis and living areas in muscle b) living and necrosis cells in the treatment group (blue arrow: necrosis of muscle cell, black arrow: live muscle cell, magnification 40).
Effects of taurine on reperfusion injury
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Table 2 Average water content, flap survival and MDA results of control and treatment groups. Control Water Content Flap Survival (NBT) MDA
Treatment
86.6 2.4 27.7 9.1
74.2 2.8* 82.6 7.4*
105.53 22.18
17.68 5.00*
* Statistically (p < 0.005). NBT, painted with the nitroblue tetrazolium; MDA, Malondialdehyde.
solution,36,37 erythropoietin,38,39 anti-inflammatory 33,40 agents, calcium canal blockers,35,41 nitric oxide (NO) donors,42 immunosuppresives40 (FK-506, cyclosporine and dexamethasone), the antibodies of neutrophil, adhesion and function receptors43,44 ((CD11/18, inter-cellular adhesion molecule-1 (ICAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), E-selectin and P-selectin), hyperbaric oxygen,45 hormones(dehydroepiandrosterone and progesterone)46,47 and growth factors48e50 (basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF) and vascular endothelial growth factor (VEGF)). The free radical cleaners may not be used clinically, as the respective enzyme levels of rat and humans are different.27e29 The thrombolytic agents PG-E1, heparin, aspirin and steroid are used in clinical
studies. Many of such agents could not be commonly applicable for clinical purposes due to their additional treatment costs, possible side effects and complications.51 Practical and simple protection methods gain importance for to protect the ischaemic tissue.52,53 In the light of such information, our study examined the effects of taurine on the ischaemia/reperfusion damages on gracilis muscle flap. Researchers have shown many benefits and functions of taurine; however, many of its benefits are now clearly well established.54 The sulphonate group in taurine is decomposed to sulphate in mammals during the oxidation without the use of enzymatic mechanisms and, accordingly, the body is protected against oxygen. Taurine is known to demonstrate antioxidant effects in tissues with peroxidative damage and decrease lipid peroxidation. Green et al. (1991) and Dogru, Abbasoglu et al. (2001) have shown that taurine has antioxidative effects.55 No side effects have been recorded in the literature related to the use of taurine. Our study has chosen the gracilis muscle flap and a 4-h global ischaemia model as had been described by Zhan et al.16 Oedema is an important symptom of reperfusion and we have seen that taurine lessens the oedema significantly after we have measured the water content in flap in the first 24 h (p < 0.005). The experiments have shown that angiogenesis and collateral veins begin to enlarge after 24 h and 3e7 days are required for the smallest capillary enlargement. Therefore, histopathological assessment is performed in our study to examine
Figure 4 With haematoxylin and eosin stained sections were examined under high power light microscope; a) this figure shows concentration of collagen and vascular in treatment group, magnification 100 (red arrow: collagen, black arrow: vessel, blue arrow: fibroblast). b) This figure shows per vascular infiltration by neutrophils.in control group, magnification 20 (black arrow: vessel, red arrow: neutrophil density), c) this figure shows per vascular infiltration by neutrophils.in control group, magnification 200 (red arrow: endothelial cell, black arrow: neutrophils cell).
926 Table 3
O. Akdemir et al. Average values of histopathological scores.
Necrosis Polymorph Nuclear Leukocyte Lymphocyte Edema Collagen Density Fibroblast Proliferation Vascular Density
Control
Treatment
2.250 1.164 2.000 1.069
0.375 0.744* 0.75 1.165**
0.625 1.875 0.500 0.625 0.875
0.744 0.640 0.755 0.744 1.125
0.5 0.25 2.25 1.75 2.125
0.755 0.462*** 1.165* 0.707** 0.834**
* Statistically significant (p < 0.01), ** Statistically significant (p < 0.05), *** Significant statistically (p < 0.005).
vein density and PMNL inflammatory activity, which have a great importance for reperfusion damage. Taurine definitely increased vein density, collagen and fibroblast amounts and lessened PMNL amount; this, in turn, has shown that taurine not only stops reperfusion damage but also speeds up the healing. Moreover, the increase of oedema and PMNL in direct proportion to the necrosis numbers in the control group has shown us that the inflammation due to reperfusion was higher in the control group. Non-increase of the lymphocytes, which are known as late inflammation cells in classical muscle damage, shows that reperfusion damages do not suit to the classic injury hearing. The demonstration of the free oxygen radicals is very difficult, as they have short lives. Particularly, the biological free oxygen radicals depend on the observation of the final product of lipid peroxidation, that is, MDA.56 Nakashima et al. have shown that taurine decreases carbon tetrachloride in the liver and accordingly decreases MDA.57 We have also performed MDA measurement to understand the reperfusion damages. The MDA levels of the taurine group were significantly lesser than in the control group (p < 0.005). The NBT painting method, which is an indicator of flap survival in a muscle flap, shows maximum correctness after 12e16 h of reperfusion, that is, after complete termination of cytoplasmic enzyme activity. The muscle samples obtained earlier may demonstrate erroneous positive rates.58 Thus, we painted our muscle flaps with NBT after 24 h of reperfusion and examined them under the microscope. It has been seen that survival significantly increased in the group with taurine administration (p < 0.005). Sılaeva et al. have shown that the effective use of taurine shall be a 200 mg kg1 dose through intra-peritoneal or intravenous administration.59 Basher et al. have given taurine orally to the randomly removed skin flaps of rats and have analysed the effects of the same to distal flap necrosis in doses 0, 10, 30 and 100 mg kg1.60 They have not observed any difference in effects to the distal flap necrosis by means of these doses. We assume this arises due to the administering of taurine orally and in low doses. Doddakula et al. have conducted a prospective randomised trial to investigate the effect of taurolidine, an anti-endotoxin agent with antioxidant and membrane-stabilising properties, on a total of 60 patients undergoing coronary artery bypass grafting. They believed that taurolidine potentially reduces ischaemia/reperfusion injury through its metabolite taurine. Arrhythmias induced by pro- and anti-inflammatory cytokines (interleukin (IL)-6 and IL-10), and ischaemia/ reperfusion were assessed perioperatively. They found that
administration of taurolidine in coronary artery bypass grafting patients induces a potent anti-inflammatory response that is associated with a significant decrease in arrhythmias.61 Petrosian et al. showed that taurine is characterised as a non-specific growth- or blood-clotting factor, an antioxidant, membrane protector or a regulator of calcium ion homeostasis, just as vitamins A, D, E, F and K are similarly characterised. Therefore, it appears likely that taurine and the water-insoluble vitamins A, D, E, F and K have definite interrelationship, as, at first glance, it is observed that both taurine and water-insoluble vitamins have almost the same spectrum of action.62 In our study, we used perforator gracilis flap, and we believe that taurine will effect all perforator muscle flap viability and ischaemia/reperfusion injury. Our study has shown that taurine prevents neutrophil infiltration in the muscle flaps after ischaemia and reperfusion, decreases oxygen radicals and increases angiogenesis and collagen production. The most important effect is seen in that taurine seriously increases muscle survival. In the light of these findings, it can be said that taurine should be used clinically, as it has strong positive effects on ischaemia/reperfusion damage, requires no secondary surgical intervention, does not effect the time of surgical operation, has no known side effects, costs lesser than many known treatment agents and is easy to apply.
Ethical approval All procedures were conducted in accordance with the Declaration of Helsinki and the Guide for the Care and Use of Laboratory Animals.
Funding None.
Conflict of interest None.
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Effects of taurine on reperfusion injury 7. Yao LH, Jiang YM, Shi J, Toma ´s-Barbera ´n FA, Datta N, Singanusong R, Chen SS, et al. Flavonoids in food and their health benefits. Plant Foods Hum Nutr 2004;59:113e22. 8. Buras J. Basic mechanisms of hyperbaric oxygen in the treatment of ischemia-reperfusion injury. Int Anesthesiol Clin 2000;38:91. 9. Centinkale O, Bilgic L, Bolayirli M, Sengul R, Ayan F, Burcak G. Involvement of neutrophils in ischemia/reperfusion injury of inguinal island flap in rats. Plast Reconstr Surg 1998;102:153. 10. Kerrigan CL, Stotland MA. Ischemia-reperfusion injury: a review. Microsurgery 1993;14:165. 11. Zimmerman GA, Prescott SM, McIntyre TM. Endothelial cell interaction with granulocytes: Tethering and signaling molecules. Immunol Today 1992;13:93. 12. Neal R, Cooper K, Kellogg G, Gurer H, Ercal N, et al. Effects of some sulfur-containing antioxidants on lead-exposed lenses. Free Radic Biol Med 1999;26:239e43. 13. Watson RWG, Redmond HP, Bouchier-Hayes D. Taurine upregulates antimicrobial function of human inflammatory cells through a calcium dependent mechanism. Surg Forum 1994; 45:679. 14. Stapleton PP, Redmond HP, Bouchier-Hayes DJ. Myeloperoxidase (MPO) may mediate neutrophil adherence to the endothelium through upregulation of CD11b expression: an effect downregulated by taurine. Adv Exp Med Biol 1998;442:183. 15. Wu QD, Wang JH, Fennessy F, et al. Taurine prevents highglucose- induced human vascular endothelial cell apoptosis. Am J Physiol 1999;46:1229. 16. Zhang Feng, Hu Eric C, Gerzenshtein Jacob, Lei Man-Ping, Lineaweaver William C. The expression of proinflammatory cytokines in the rat muscle flap with ischemia-reperfusion injury. Ann Plast Surg 2005;54:313e7. 17. Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malondialdehyde and related aldehydes. Free Rad Biol Med 1991;11:81e128. 18. Wang H, Li Z, Liu X. Effects of various protocols of ischemic preconditioning on rat tram flaps. Microsurgery 2008;28(1): 37e43. 19. Mowlavi A, Reynolds C, Neumeister MW, Wilhelmi BJ, Song Y-H, Naffziger R, Glatz FR, Russell RC. Age-related differences of neutrophil activation in a skeletal muscle ischemiae reperfusion model. Ann Plast Surg 2003;50:403e11. 20. Hasanoglu A, Erkan S, Temel S, Urhan MK, Guler O. Effect of sildenafil citrate on healing of colonic anastomoses. Turkish Med J 2007;1:66e75. 21. Schusterman MA, Kroll SS, Eldon ME. Immediate breast reconstruction: why the free TRAM over the conventional TRAM flap? Plast Reconstr Surg 1992;90:255e69. 22. Atterson PA, Bostwick J, Taylor GI. TRAM flap anatomy correlated with a 10-year clinical experience with 556 patients. Plast Reconstr Surg 1995;95:1185e94. 23. Zhang F, Waller W, Lineaweaver WC. Growth factor and flap survival. Microsurgery; 2004;Apr:162e7. 24. Hinder RA, Stein HJ. Oxygen derived free radicals. Arch Surg 1991;126:104e5. 25. Kasprzak KS. Oxidative DNA and protein damage in metalinduced toxicity and carcinogenesis. Free Radic Biol Med 2002; 32(10):958e67. 26. Aebı K, Satoshi Y, Kogure K. Strong attenuation of ischemic and postischemic brain edema in rats by a novel free radical scavenger. Stroke 1988;19:480e5. 27. Angel MF, Ramasastry SS, Swartz WM, et al. Augmentation of skin flap with allopurinol. Ann Plast Surg 1987;18:494e8. 28. Green CJ, Dhami L, Prasad S, Healing G, Shurey C. The effect of desferrioxamine on lipid peroxidation and survival of ischemic island skin flaps in rats. Br J Plast Surg 1989;42:565e9. 29. Wilkins EG, Rees RS, Cashmer B, et al. Identification of xanthine oxidase activityfollowing reperfusion in human tissue. Ann Plast Surg 1993; Jul;31(1):60e5.
927 30. Knight KR, MacPhadyen K, Lepore D, et al. Enhancement of ischemic rabbit skin flap survival with the antioxidant and freeradical scavenger N-acetylcysteine. Clin Sci (Lond) 1991; Jul; 81(1):31e6. 31. Manson PN, Anthenelli RM, Im MJ, Bulkley GB, Hoopes JE. The role of oxygen free radicals in ischemic tissue injury in island skin flaps. Ann Surg 1983; Jul;198(1):87e90. 32. Suzuki S, Matsushita Y, Isshiki N, et al. Salvage of distal flap necrosis by topical superoxide dismutase. Ann Plast Surg Sep. 1991;27(3):253e7. 33. Hashimoto I, Nakanishi H, Shono Y, et al. The features of thrombus in a microvessel injury model and the antithrombotic efficacy of heparin, urokinase, and prostaglandin E1. Plast Reconstr Surg 2003; Jun;111(7):2307e13. 34. Maeda M, Fukui A, Tamai S. Combined therapy with antithrombotic agents and radical scavengers for reperfusion injury of flaps. J Reconstr Microsurg 1991;7:233e43. 35. Galich SP, Druik NF, Stechenko LA, et al. Experimental studying of the possible pharmacological defense of muscular flap tissues from ischemic and reperfusion injury during its free transplantation. Klin Khir 1999;1:35e8. 36. Calhoun KH, Tan L, Seikaly H. An integrated theory of the no-reflow phenomenon and the beneficial effect of vascular washout on no-reflow. Laryngoscope 1999; Apr;109(4): 528e35. 37. Tsuchida T, Kato T, Takagi K, et al. Effect of perfusion during ischemia on skeletal muscle. J Surg Res 2001;101:238e41. 38. Buemi M, Galeano M, Sturiale A, et al. Recombinant human erythropoietin stimulates angiogenesis and healing of ischemic skin wounds. Shock 2004;22(2):169e73. 39. Buemi M, Vaccaro M, Struriale A, et al. Recombinant human erythropoietin influences revascularization and healing in a rat model of random ischaemic flaps. Acta Derm Venereol 2002; 82(6):411e7. 40. Hosnuter M, Babucc ¸u O, Kargı E, Altınyazar C. Dual preconditioning: effects of pharmocological plus ischemic preconditioning on skin flap survival. Ann Plast Surg 2003;; Apr; 50(4):398e402. ¨ zden A, et al. The effect of trimetazidine 41. Kara IG, Kara CO, O on the survival of rat island skin flaps subjected to ischemiareperfusion injury. Ann Plast Surg 2001; Aug;47(2):168e71. 42. Lille S, Boyle EM, Schoeller T, et al. Augmentation of cAMP improves muscle-flap survival and tissue inflammation in response to ischemia/reperfusion injury. J Reconstr Microsurg 1999; Jan;15(1):37e45. 43. Hong JP, Chung YK, Chung SH. The effect of prostoglandin E1 versus ischemia reperfusion injury of musculocutaneous flaps. Ann Plast Surg 2001; Sept;47(3):316e21. 44. Khiabani KT, Stephenson L, Zamboni WA, et al. A quantitative method for determining polarization of neutrophil adhesion molecules associated with ischemia reperfusion. Plast Reconst Surg 2004;Dec;114(7):1846e50. 45. Hong JP, Kwon H, Chung YK, et al. The effect of hyperbaric oxygen on ischemiareperfusion injury. Ann Plast Surg 2003; Nov;51(5):478e87. 46. Asim K, Ashrafpour H, Huang N, et al. Acute local subcutaneous VEGF165 injection for augmentation of skin flap viability:efficacy and mechanism. Am J Physiol Regul Integr Comp Physiol 2004; Nov;287:219e29. 47. Uysal OA. The effect of progesteron hormone on the viability of flaps. Eur J Plast Surg 1991;14:192e6. 48. Lineaweaver WC, Lei MP, Mustain W, et al. Vascular endothelium growth factor, surgical delay, and skin flap survival. Ann Surg 2004; Jun;239(6):866e75. 49. Machens HG, Morgan JR, Bertiaume F, et al. Platelet-derived growth factor-Aamediated functional angiogenesis in the rat epigastric island flap after genetic modification of fibroblasts is ischemic dependent. Surg 2002; Apr;131:393e400.
928 50. Machens HG, Salehi J, Weich H, et al. Angiogenic effects of injected VEGF165 and SVEGFR-1 (sFLT-1) in a rat flap model. J Surg Res 2003;111:136e42. 51. Vedder NB. Flap physiology. mathes plastic surgery. 2nd ed., vol. 1. Philadelphia: Saunders Elsevier Inc; 2006. pp. 483e506. 52. Ege A, Turhan E, Bektas S, Pamuk K, Bayar A, Keser S. In which period of skeletal muscle ischemia-reperfusion injury is local hypothermia more effective? Acta Orthop Traumatol Turc 2008; MayeJul;42(3):193e200. 53. Wei FC, Mardini S. Free-style free flaps. Plast Reconstr Surg Sep; 2004;114(4):910e6. 54. Huxtable RJ. Physiological actions of taurine. Physiol Rev 1992; 72(1):101e63. 55. Pushpakiran G, Mahalakshmi K, Anuradha CV, et al. Taurine restores ethanol-induced depletion of antioxidants and attenuates oxidative stress in rat tissues. Amino Acids 2004;27(1):91e6. 56. Bishop ML, Janet LP. Free radicals in clinical chemistry: principles, procedures, correlations. 3rd ed.; 1996. Philadelphia, New Yorkpp. 765e777. 57. Nakashıma T, Takino T, Kuriyama K. Therapeutic and prophylactic effects of taurine administration on experimental liver
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injury. In: Kuriyama K, Huxtable RJ, Iwata H, editors. Sulfur amino acids: biochemical and clinical aspects. New York: Liss; 1983. p. 449e60. Labbe R, Lindsay T, Gatley R, Romaschin A, Mickle D, Wilson G, Houle S, Walker P. Quantitation of postischemic skeletal muscle necrosis: histochemical and radioisotope techniques. J Surg Res 1988; Jan;44(1):45e53. TIu Silaeva, Dokshina GA, Iartsev EI, Iakovleva VV, Arkhangel’skaia TE. The effect of taurine on the carbohydrate metabolism of diabetic animals. Probl Endokrinol (Mosk) 1976; May- Jun;22(3):99e103. Basher AW, Novotny MJ, Hanna PE. Failure of oral taurine supplementation to influence skinflap survival in rats. Plast Reconstr Surg 1995; Apr;95(5):888e93. Doddakula KK, Neary PM, Wang JH, Sookhai S, O’Donnell A, Aherne T, Bouchier-Hayes DJ, Redmond HP. The antiendotoxin agent taurolidine potentially reduces ischemia/reperfusion injury through its metabolite taurine. Surgery 2010; Sep; 148(3):567e72. Petrosian AM, Haroutounian JE. Taurine as a universal carrier of lipid soluble vitamins: a hypothesis. Amino Acids 2000; 19(2):409e21.
Effects of taurine on reperfusion injury Ovunc Akdemir a, Yan Hede a, Feng Zhang a,*, William C. Lineaweaver b, Zikri Arslan c, Ecmel Songur d a
Department of Plastic Surgery, University of Mississippi Medical Center, Jackson, MS, USA Joseph M. Still Burn and Reconstructive Center, 346 Crossgates Blvd, Suite 202, Brandon, MS, USA c Department of Chemistry, Jackson State University, Jackson, MS, USA d Department of Plastic Surgery, University of Ege, Izmir, Turkey b
Received 15 July 2010; accepted 7 December 2010
KEYWORDS Ischaemia/reperfusion injury; Taurine; Gracilis muscle flap; Malondialdehyde (MDA); Nitroblue tetrazolium (NBT)
Summary Taurine is an organic acid, which has a very important function in the human body. Recently, the antioxidant property of taurine has been much emphasised. In this study, the gracilis muscle flap model was used to investigate the effect of taurine in ischaemia/reperfusion injury in rats. Totally 32 Sprague-Dawley rats were divided into two groups: control group (n Z 16) and the treatment group with taurine (n Z 16). After elevation of the gracilis muscle flap, 4 h of ischaemia was performed in both groups. Thirty min before the reperfusion, taurine (200 mg kg1) was injected intravenously. After 24 h of reperfusion, the amount of malondialdehyde (MDA), tissue water content and flap viability were evaluated. After 72 h of reperfusion, histological findings were evaluated. Amount of MDA and tissue water content were significantly lower (p < 0.005), and the flap viability was significantly higher (p < 0.005) in the treatment group 24 h after reperfusion. On comparing the outcomes of histological analysis between control and treatment groups, the amounts of collagen, fibroblast and angiogenesis in treatment group were significantly higher than those in the control group. However, the amount of polymorphonuclear leucocyte and tissue necrosis in the treatment group were significantly lower than in the control group. Our results showed that taurine played an important role in the process of ischaemia/reperfusion injury and presented certain protective effects with the improvement in flap survival after ischaemia/reperfusion injury. ª 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. The University of Mississippi Medical Center, 2500 North State St. Jackson, MS 39216, USA. Tel.: þ1 601 815 11 47. E-mail address: [email protected] (F. Zhang). 1748-6815/$ - see front matter ª 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.bjps.2010.12.007
922 The success rate of the free flaps at the present time varies between 94% and 98%.1e5 It is a known fact that ischaemia/ reperfusion damage plays an important role in the loss of free flaps. Ischaemia/reperfusion damage includes, acutely, cell apoptosis related to the interaction between the leucocyte and endothelium cells after the cessation of microvascular bleeding and the release of reactive oxygen types.6 The natural antioxidants protect the human body from free radicals and prevent oxidative stress and related illnesses. Thus, they have an important role in human health.7 In recent years, the cause of the damage is claimed to be neutrophils stimulating free oxygen derivatives (ROS), several enzymes and cytokines causing damage in the endothelium and capillary obstruction.8e11 Thus, to protect from ischaemia/reperfusion damage, the antioxidant, anticoagulant, anti-inflammatory and anti-apoptosis agents are beginning to be tried for treatment. Taurine, which cleans the reactive oxygen types, also prevents the changes in the permeability of membrane after oxidant damage.12 Taurine and chlorotaurine neutralise hypochlorosis acid and therefore demonstrate antioxidant effects. It has been seen, in vitro, that this decreases the CD11b receptor in neutrophilia thereby preventing the creation of high-level induced glucose formation and apoptosis in endothelium, that is, protecting the tissues.13e15 In the light of aforementioned information from the literature, we have examined the effects of taurine on reperfusion damages using the gracilis muscle flap model in rats.
Material and method Thirty-two male Sprague-Dawley rats, weighing between 380 and 420 g, were used in this study. The National Research Council’s guidelines for the care and use of laboratory animals were followed. General anaesthesia was applied on all the subjects using isoflurane providing respiratory track anaesthesia (Abbot Laboratories, Chicago, IL, USA).
Surgical procedure A transverse incision was applied in the inguinal zone to expose the gracilis muscle. The small branches to the skin from the gracilis muscle were ligated. It was proceeded to the depth and distal to the inguinal ligament as opening the femoral artery and vein. Dissection was performed carefully until the gracilis pedicle was seen and freed. Then, the proximal borders of the anterior and posterior parts of the gracilis muscle, in the depth of the inguinal ligament, were dissected. The two parts were separated from the abductor muscles. The distal border of the gracilis muscle was dissected from the medial side of saphenous veins so as to be separated from the flap base. After lifting the gracilis muscle completely with only the pedicle intact, the pedicle was clamped for 4 h using Acland V2 microclamp16 (Figure 1). The rats in the control group, group 1, was injected with only 0.5 ml physiological serum 30 min before reperfusion from the saphenous vein, while in the treatment group, group 2, 200 mg kg1 taurine, which had been dissolved in 0.5 ml physiological serum, was administered in the same manner as in the control group. After 4-h ischaemia, the clamp was released for reperfusion. Then, the muscle flaps
O. Akdemir et al.
Figure 1 The images of gracilis muscle, femoral veins and branches after transverse incision. Freed gracilis muscle flap.
were sutured back in situ using 4/0 absorbable suture and the skin incision was closed using 5/0 non-absorbable suture.
Analysis of the subjects After reperfusion, both the group 1 (n Z 16) and group 2 (n Z 16) subjects were placed in cages and separated randomly into two groups, each consisting of eight subjects. We waited for 24 h to assess the reperfusion damage of eight subjects from each group and the gracilis muscles were removed under anaesthesia. The flap survivals and water contents of the removed muscles were examined and biochemical malondialdehyde (MDA) measurements were performed. The gracilis muscles of the other eight subjects were removed after 72 h under anaesthesia. All the removed muscle tissues were washed with physiological serum and immediately frozen in liquid nitrogen and kept at 80 C in a deep freeze (Legaci/Copeland, NC, USA) till the day of analysis. Histopathology scoring was applied to evaluate the reperfusion damage after 72 h (Figure 2).
Assessment at 24th hour Biochemical analysis The working method of Esterbauer et al. was preferred.17 This method depends on the principle of spectrophotometric observation of the complex formed by MDA (which is
Figure 2
Analysis of the Subjects.
Effects of taurine on reperfusion injury a lipid peroxidation product) with thiobarbituric acid (TBA) at 532 nm. The conduct of the test A total of 100 mg ml1 tissue was homogenised in buffer at a pH of 7.4. Artefactual production of additional MDA during processing was eliminated by the addition of 2% butylated hydroxytoluene to homogenised tissue. To this was added 20% trichloroacetic acid in 0.6 N hydrochloric acid. The mixture was centrifuged at 10 000 g for 10 min at 4 C. A total of 0.12 mol l1 TBA in buffer (pH 7.0) was added to the supernatant. The pigment was measured spectrophotometrically at 532 nm. Calculation of MDA level The results were calculated as nmol g1 tissue based on the optical density difference. The water content analysis in flap The muscle sample was dried with a stencil for analysis and the wet weight of the muscle was measured with precision balance (Ohaus Adventurer precision balance). The muscle with known wet weight was placed in a drying oven and kept for 24 h at 85 F. Afterwards, the dry weight of the muscle was measured using the same precision balance. The formula for calculating the water content18: Wet weight Dry weight Water contentð%ÞZ 100 Wet weight Flap survival The gracilis muscles, which had been subjected to 24 h of reperfusion after 4 h of ischaemia, were harvested and their flap survival percentages were analysed.19 To observe the living cell ratio in the gracilis muscles, the histochemical painting method using nitroblue tetrazolium (NBT) was conducted. Coronal sections 5 mm wide were obtained from four different zones of the muscle samples, using a microtome. These sections were painted by placing them into a pH 7.4 solution including 0.05% NBT and 0.2% Tris buffer at room temperature (24 1 C) for 30 min. As shown in the literature with this method, the living cells can be dyed in blue while the necrosed cells cannot. The living cell percentage in the tissue sections was measured by using the standard-point-count method under 40 microscopic magnification. The ratio of the living cells to the total area was calculated.19
923 parameters as polymorphonuclear leucocyte (PMNL), lymphocyte density, capillary vein, fibroblast proliferation, oedema amount, collagen density and necrosis (Table 1). Statistical assessment The statistical analysis of the obtained data was performed using Statistical Package for Social Sciences (SPSS) 14.0 (SPSS, Inc., Chicago, IL, USA) package program. The ManneWhitney U test was used for assessment. The p values <0.05 were regarded as statistically significant.
Results All flaps were bleeding after 4 h of global ischaemia.
The results of 24th-hour assessment Of the eight subjects from each group, the flap colour of three subjects (37.5%) from the control group was observed to be visibly pale by means of macroscopic examination. The flap colour of the treatment group did not demonstrate any change by means of macroscopic examination. Results of biochemical analysis (MDA) The average value of MDA of the control group was 105.53 22.18 nmol g1, while the average value of the treatment group was only 17.68 5.00 nmol g1. The difference of the average value of MDA was statistically significant between the two groups (p < 0.005). The results of flap water content The average value of the control group was 86.6 2.4, while the average value of the treatment group was measured as 74.2 2.8. The water content in the treatment group was slightly lower than that in the control group; however, the difference between the two groups was statistically significant (p < 0.005). Flap survival results The samples taken from the control group (n Z 8) and treatment group (n Z 8) after 24 h of reperfusion were painted with the NBT histochemical painting method and their flap survival results were observed under a microscope (Figure 3). The average value of the control group was 27.7 9.1, while the average value of the treatment group was found to be as high as 82.6 7.4 (Table 2). The difference between the two groups was statistically significant (p < 0.005).
Assessment results at the 72nd hour Assessment at the 72nd hour Histopathology analysis The distal parts of the gracilis muscles, as subjected to 4 h of ischaemia after surgical application, were taken after 72 h of reperfusion. The 1 1-cm tissue biopsies were placed in 10% formalin and embedded in paraffin blocks. Four-micron sections were sliced using a microtome. The sections were painted with haematoxylin and examined under high-performance light microscope (100 and 200 magnification). According to the modified Verhofstad scoring,20 the samples were examined based on the seven
Results of histopathology analysis PMNL density, oedema and necrosis amounts of the samples taken from 16 subjects, as eight from both the treatment and control groups, after 4 h of ischaemia and subsequent 72 h of reperfusion are measured. It has been understood that the aforementioned parameters are higher in the control group. It is also seen that the fibroblast and capillary vein proliferation and collagen density was higher in the treatment group, whereas the necrosis amount is lesser in the same (Figure 4). The lymphocyte scores obtained from both groups are understood to be close to each other.
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Table 1
Histopathologic evaluation Verhofstad modified scoring table.
Score
Necrosis
PMNLa
lymphocytes
Oedema
Collagen density
Fibroblast proliferation
vascular density
0 1 2 3
None Superficial Pronounced Massive
Normal Light Pronounced Massive
Normal Light Pronounced Massive
None Light Pronounced Dense
None Light Pronounced Dense
None Light Pronounced Dense
None Light Pronounced Dense
a
PMNL-polymorph nuclear leukocytes.
The average of the necrosis values for 16 subjects varied between 0 and 3, being 2.25 1.164 for the control group and 0.375 0.744 for the treatment group. Thus, it is understood that a significant difference existed for the necrosis values of the control group and the treatment group with taurine administration. This difference was found to be also statistically significant (p < 0.01). The average of the PMNL values was 2 1.069 for the control group and 0.75 1.165 for the treatment group. This difference was found to be also statistically significant (p < 0.05). The average of the lymphocyte values was 0.625 0.744 for the control group and 0.5 0.755 for the treatment group. This difference was not found significant statically (p Z 0.680) (p > 0.05). The average of the oedema values was 1.875 0.640 for the control group and 0.25 0.462 for the treatment group. This difference was found to be significant also statistically (p < 0.005). The average of the collagen density values was 0.5 0.755 for the control group and 2.25 1.165 for the treatment group. This difference was found to be significant also statistically (p < 0.01). The average of the fibroblast proliferation values was 0.625 0.744 for the control group and 1.75 0.707 for the treatment group. This difference was found to be significant also statistically (p < 0.05). The average of the vein proliferation values was 0.875 1.125 for the control group and 2.125 0.834 for the treatment group (Table 3). This difference was found to be significant also statistically (p < 0.05).
Discussion The success rate of free tissue applications, a rather general application in the recent years, varies between 97% and 87%;
however, it has been recorded that the partial or complete flap losses of pedicle flaps, and their complications, may reach 25%.21,22 The insufficient perfusion and ischaemia damage are accepted to cause flap necrosis due to many respective changes in the tissues and veins.23 The known main mechanism in ischaemia/reperfusion damage is that when reperfusion occurs after ischaemia, oxygen reaches the tissues and free oxygen radicals are generated. The free oxygen radical interacts with the lipid acid radicals in the damaged cell membrane and results in a lipid peroxidation reaction.24 The increase of lipid peroxidation causes MDA accumulation, oedema and cell enlargement. Thus, breakages in DNA chain and cell necrosis occur.25 At the capillary level, subsequent to the PMNL infiltration, hypoxic vasoconstriction, increase in capillary stress and endothelium structural changes are proved to exist. It has been understood that PMNL activation plays an active role in reperfusion damage. However, it is also being discussed if the insufficiency of the antioxidant defence system due to increase in free radical reactions has a role in post-ischaemia tissue damage.26 Many contemporary researchers have examined endogenic or exogenic agents, which may eradicate or decrease the oxidative damage and respective injuries caused by free radicals on DNA, proteins, lipids and other components of the cell. The free radical cleaners may not be used clinically, as the respective enzyme levels of rat and humans are different.27e29 These may be summed as free radical cleaners27,28,30e32 (superoxide dysmutase, catalase, allopurinol, deferoxamine, etc.), thrombolytic agents33,34 (urokinase tissue and plasminogen activator (tissue plasminogen activator,TPA)), prostaglandin E1 (PG-E1),33 heparin,33 vitamin C,35 acellular physiological washing
Figure 3 Under the microscope 24 h after reperfusion of the implementation of the gracilis muscle nitroblue tetrazolium (NBT) staining with histochemical staining methods; a) In the control group, necrosis and living areas in muscle b) living and necrosis cells in the treatment group (blue arrow: necrosis of muscle cell, black arrow: live muscle cell, magnification 40).
Effects of taurine on reperfusion injury
925
Table 2 Average water content, flap survival and MDA results of control and treatment groups. Control Water Content Flap Survival (NBT) MDA
Treatment
86.6 2.4 27.7 9.1
74.2 2.8* 82.6 7.4*
105.53 22.18
17.68 5.00*
* Statistically (p < 0.005). NBT, painted with the nitroblue tetrazolium; MDA, Malondialdehyde.
solution,36,37 erythropoietin,38,39 anti-inflammatory 33,40 agents, calcium canal blockers,35,41 nitric oxide (NO) donors,42 immunosuppresives40 (FK-506, cyclosporine and dexamethasone), the antibodies of neutrophil, adhesion and function receptors43,44 ((CD11/18, inter-cellular adhesion molecule-1 (ICAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), E-selectin and P-selectin), hyperbaric oxygen,45 hormones(dehydroepiandrosterone and progesterone)46,47 and growth factors48e50 (basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF) and vascular endothelial growth factor (VEGF)). The free radical cleaners may not be used clinically, as the respective enzyme levels of rat and humans are different.27e29 The thrombolytic agents PG-E1, heparin, aspirin and steroid are used in clinical
studies. Many of such agents could not be commonly applicable for clinical purposes due to their additional treatment costs, possible side effects and complications.51 Practical and simple protection methods gain importance for to protect the ischaemic tissue.52,53 In the light of such information, our study examined the effects of taurine on the ischaemia/reperfusion damages on gracilis muscle flap. Researchers have shown many benefits and functions of taurine; however, many of its benefits are now clearly well established.54 The sulphonate group in taurine is decomposed to sulphate in mammals during the oxidation without the use of enzymatic mechanisms and, accordingly, the body is protected against oxygen. Taurine is known to demonstrate antioxidant effects in tissues with peroxidative damage and decrease lipid peroxidation. Green et al. (1991) and Dogru, Abbasoglu et al. (2001) have shown that taurine has antioxidative effects.55 No side effects have been recorded in the literature related to the use of taurine. Our study has chosen the gracilis muscle flap and a 4-h global ischaemia model as had been described by Zhan et al.16 Oedema is an important symptom of reperfusion and we have seen that taurine lessens the oedema significantly after we have measured the water content in flap in the first 24 h (p < 0.005). The experiments have shown that angiogenesis and collateral veins begin to enlarge after 24 h and 3e7 days are required for the smallest capillary enlargement. Therefore, histopathological assessment is performed in our study to examine
Figure 4 With haematoxylin and eosin stained sections were examined under high power light microscope; a) this figure shows concentration of collagen and vascular in treatment group, magnification 100 (red arrow: collagen, black arrow: vessel, blue arrow: fibroblast). b) This figure shows per vascular infiltration by neutrophils.in control group, magnification 20 (black arrow: vessel, red arrow: neutrophil density), c) this figure shows per vascular infiltration by neutrophils.in control group, magnification 200 (red arrow: endothelial cell, black arrow: neutrophils cell).
926 Table 3
O. Akdemir et al. Average values of histopathological scores.
Necrosis Polymorph Nuclear Leukocyte Lymphocyte Edema Collagen Density Fibroblast Proliferation Vascular Density
Control
Treatment
2.250 1.164 2.000 1.069
0.375 0.744* 0.75 1.165**
0.625 1.875 0.500 0.625 0.875
0.744 0.640 0.755 0.744 1.125
0.5 0.25 2.25 1.75 2.125
0.755 0.462*** 1.165* 0.707** 0.834**
* Statistically significant (p < 0.01), ** Statistically significant (p < 0.05), *** Significant statistically (p < 0.005).
vein density and PMNL inflammatory activity, which have a great importance for reperfusion damage. Taurine definitely increased vein density, collagen and fibroblast amounts and lessened PMNL amount; this, in turn, has shown that taurine not only stops reperfusion damage but also speeds up the healing. Moreover, the increase of oedema and PMNL in direct proportion to the necrosis numbers in the control group has shown us that the inflammation due to reperfusion was higher in the control group. Non-increase of the lymphocytes, which are known as late inflammation cells in classical muscle damage, shows that reperfusion damages do not suit to the classic injury hearing. The demonstration of the free oxygen radicals is very difficult, as they have short lives. Particularly, the biological free oxygen radicals depend on the observation of the final product of lipid peroxidation, that is, MDA.56 Nakashima et al. have shown that taurine decreases carbon tetrachloride in the liver and accordingly decreases MDA.57 We have also performed MDA measurement to understand the reperfusion damages. The MDA levels of the taurine group were significantly lesser than in the control group (p < 0.005). The NBT painting method, which is an indicator of flap survival in a muscle flap, shows maximum correctness after 12e16 h of reperfusion, that is, after complete termination of cytoplasmic enzyme activity. The muscle samples obtained earlier may demonstrate erroneous positive rates.58 Thus, we painted our muscle flaps with NBT after 24 h of reperfusion and examined them under the microscope. It has been seen that survival significantly increased in the group with taurine administration (p < 0.005). Sılaeva et al. have shown that the effective use of taurine shall be a 200 mg kg1 dose through intra-peritoneal or intravenous administration.59 Basher et al. have given taurine orally to the randomly removed skin flaps of rats and have analysed the effects of the same to distal flap necrosis in doses 0, 10, 30 and 100 mg kg1.60 They have not observed any difference in effects to the distal flap necrosis by means of these doses. We assume this arises due to the administering of taurine orally and in low doses. Doddakula et al. have conducted a prospective randomised trial to investigate the effect of taurolidine, an anti-endotoxin agent with antioxidant and membrane-stabilising properties, on a total of 60 patients undergoing coronary artery bypass grafting. They believed that taurolidine potentially reduces ischaemia/reperfusion injury through its metabolite taurine. Arrhythmias induced by pro- and anti-inflammatory cytokines (interleukin (IL)-6 and IL-10), and ischaemia/ reperfusion were assessed perioperatively. They found that
administration of taurolidine in coronary artery bypass grafting patients induces a potent anti-inflammatory response that is associated with a significant decrease in arrhythmias.61 Petrosian et al. showed that taurine is characterised as a non-specific growth- or blood-clotting factor, an antioxidant, membrane protector or a regulator of calcium ion homeostasis, just as vitamins A, D, E, F and K are similarly characterised. Therefore, it appears likely that taurine and the water-insoluble vitamins A, D, E, F and K have definite interrelationship, as, at first glance, it is observed that both taurine and water-insoluble vitamins have almost the same spectrum of action.62 In our study, we used perforator gracilis flap, and we believe that taurine will effect all perforator muscle flap viability and ischaemia/reperfusion injury. Our study has shown that taurine prevents neutrophil infiltration in the muscle flaps after ischaemia and reperfusion, decreases oxygen radicals and increases angiogenesis and collagen production. The most important effect is seen in that taurine seriously increases muscle survival. In the light of these findings, it can be said that taurine should be used clinically, as it has strong positive effects on ischaemia/reperfusion damage, requires no secondary surgical intervention, does not effect the time of surgical operation, has no known side effects, costs lesser than many known treatment agents and is easy to apply.
Ethical approval All procedures were conducted in accordance with the Declaration of Helsinki and the Guide for the Care and Use of Laboratory Animals.
Funding None.
Conflict of interest None.
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