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Table of Contents
Year : 2019  |  Volume : 27  |  Issue : 4  |  Page : 156-159

Combined use of ischemic preconditioning and postconditioning in a skin flap model in rats

1 Department of Plastic and Reconstructive Surgery, Antalya Training and Research Hospital, Antalya, Turkey
2 Department of Plastic and Reconstructive Surgery, Memorial Antalya Hospital, Antalya, Turkey

Date of Submission09-Oct-2018
Date of Acceptance06-Jan-2019
Date of Web Publication26-Sep-2019

Correspondence Address:
Dr. Asim Uslu
Department of Plastic and Reconstructive Surgery, Antalya Training and Research Hospital, Varlik, Kazim Karabekir Cd., 07100 Antalya
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/tjps.tjps_70_18

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Purpose: Reperfusion injury (RI) by abrupt restoration of circulation after prolonged ischemia remains an unsolved problem in reconstructive microsurgery. We hypothesized that the combination of ischemic preconditioning (IPreC) and ischemic postconditioning (IPostC) would result in additional preservation of epigastric skin flaps in rats after the ischemic period. Materials and Methods: We used epigastric artery skin flaps measuring 6 cm × 3 cm in 40 Wistar rats. The animals were allocated randomly into four groups: (1) Control, (2) IPreC, (3) IPostC, and (4) IPreC + IPostC group. Flap viability was assessed 1 week after the surgical procedure, and surviving flap area was recorded as a percentage of the whole flap area.Results: Animals in the IPreC, IPostC, and IPreC + IPostC groups showed significantly smaller areas of flap necrosis than those in the control group (P = 0.001). Statistical analyses indicated significant differences between the IPreC group and IPostC group (P = 0.008). However, there were no significant differences between the IPreC group and IPreC + IPostC group (P = 0.453) or between the IPostC group and the IPreC + IPostC group (P = 0.141). Conclusions: IPreC and IPostC showed protective effects against ischemia-reperfusion injury in the epigastric skin flap model. IPostC showed a greater protective effect than IPostC. The combination of preconditioning and postconditioning provided no additional benefit over either intervention performed alone in the skin flap model.

Keywords: Ischemia-reperfusion, ischemia-reperfusion injury, ischemic postconditioning, ischemic preconditioning, skin flap

How to cite this article:
Uslu A, Coskunfirat OK. Combined use of ischemic preconditioning and postconditioning in a skin flap model in rats. Turk J Plast Surg 2019;27:156-9

How to cite this URL:
Uslu A, Coskunfirat OK. Combined use of ischemic preconditioning and postconditioning in a skin flap model in rats. Turk J Plast Surg [serial online] 2019 [cited 2020 Sep 26];27:156-9. Available from: http://www.turkjplastsurg.org/text.asp?2019/27/4/156/267930

  Introduction Top

Ischemic flap complications can occur because of two types of necrosis. In the first type, to reconstruct a very wide defect, a very large sized flap is harvested and the blood circulation cannot reach all over the flap. Necrosis can occur in distal parts of the flap as a result of poor circulation.[1] In the second type, abrupt reperfusion after a prolonged time of ischemia can result in total necrosis of the flap, which is also referred to as reperfusion injury (RI).[2]

The surgical delay is the most effective method for increasing flap survival and has often been used to decrease ischemic flap complications.[3],[4] Because a second surgery is necessary for the surgical delay procedure, there has been a great deal of interest in developing novel methods to decrease ischemic flap complications. Ischemic preconditioning (IPreC) was first described by Murry et al.[5] in myocardium, where short periods of ischemia and reperfusion occurred before a longer ischemic period, which increased the ischemic tolerance of the tissue.

Ischemic postconditioning (IPostC) was defined as a repeated short series of reperfusion and reocclusion periods after a prolonged ischemic period. IPostC was described by Zhao et al.,[6],[7] and Moon et al.[8] first reported that IPostC could decrease ischemia-reperfusion (IR) injury in a skin flap model. Since then, IPreC and IPostC have been shown experimentally to prevent IR injury in muscle and skin flaps.[9],[10],[11]

In this study, we compared IPreC and IPostC procedures and their effects on flap survival. In addition, we examined whether the combined use of these two methods could show additional benefits with regard to survival of the same flaps.

  Materials and Methods Top

All protocols were approved by the Animal Care and Use Committee of Akdeniz University and were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Forty male Wistar rats weighing between 300 g and 450 g were used in the experiments. The animals were kept in individual cages under standard conditions at a mean room temperature of 24°C–25°C under a 12 h light/12 h dark cycle. The animals were allowed free access to standard rat chow and water. General anesthesia was induced by intramuscular injection of 35 mg/kg xylazine and intraperitoneal injection of 10 mg/kg ketamine. Additional doses were given as needed to maintain anesthesia. The left epigastric artery was pedicled and a skin flap measuring 6 cm × 3 cm was harvested from the abdominal region [Figure 1]. The animals were randomly assigned to the control group or one of three experimental groups, with 10 animals in each group. In the control group, epigastric skin flaps were harvested under general anesthesia, and then the flaps were subjected to 6 h of ischemia using a microvascular clamp. In the IPreC group, flaps were harvested in the same way as described above. Before ischemia, a total of three cycles of IPreC were applied with ischemia for 10 min and reperfusion for 10 min [Figure 2]. Then, the flaps were subjected to 6 h of ischemia. In the IPostC group 6 hours of complete ischaemia was performed and the clamp was completely released at the end of ischaemia and the postconditioning procedure was started immediately after the end of ischaemia. A cycle of 15 seconds of full reperfusion, followed by 15 seconds of complete reocclusion was repeated 6-times (total intervention time, 3 minutes). In the IPreC + IPostC group, IPreC was first applied, after which the flaps were subjected to ischemia for 6 h, and then IPostC was applied as in the IPostC group. The animals were sacrificed on day 7 after reperfusion.
Figure 1: (a) Flap design. (b) Harvesting of left inferior epigastric artery skin flap

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Figure 2: Ischemia and reperfusion procedure. (a) Ischemia. (b) Reperfusion

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Flap survival

Necrosis was defined as typical signs of tissue injury including black coloration, dehydration, eschar formation, loss of skin elasticity, and absence of bleeding. The results are expressed as the percentage of the vital area relative to the total flap surface area (tissue survival = vital area/total surface area × 100%). The entire surface area of the flap including the necrotic flap area was traced onto transparent acrylic foil, which was photographed. The ratio of vital area to original flap area was calculated using digital planimetry software (Image-Pro Plus Version 7.0; Media Cybernetics, Bethesda, MD, USA), and expressed as a percentage.

Statistical analysis

Comparisons between groups were performed by calculating vital area relative to total flap surface area as a percentage. SPSS (version 17.0; SPSS Inc., Chicago, IL, USA) was used for the statistical analyses. The Mann–Whitney U test was used to analyze differences between pairs of groups, and the Kruskal–Wallis test was used for analyses among all four groups. In all analyses, α = 0.05 and P < 0.05 were taken to indicate statistical significance.

  Results Top

The average vital flap areas were 11.56% ±4.83% for the control group, 79.78% ± 3.65% for the IPreC group, 62.80% ± 3.39% for the IPostC group, and 71.55% ± 6.36% for the IPreC + IPostC group. The areas were significantly greater in all three experimental conditions compared to controls (P = 0.001). In addition, the areas were significantly greater in the IPreC group than in the IPostC group (P = 0.008). However, there were no significant differences between the IPreC group and IPreC + IPostC group (P = 0.453) or between the IPostC group and the IPreC + IPostC group (P = 0.141) [Figure 3] and [Figure 4].
Figure 3: Necrosis of the skin flap after 7 days of ischemia. (a) Control group. (b) Ischemic preconditioning group. (c) Ischemic postconditioning group. (d) Ischemic preconditioning + ischemic postconditioning group

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Figure 4: Viable surface area on postoperative day 7

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  Discussion Top

The effects of IPreC and IPostC procedures on flap survival were examined using the same flap model as in our previous research.[12] In recent studies, three cycles of 10 min of ischemia and 10 min of reperfusion have been used for IPreC. IPreC begins to show protective effects on skin flaps at 6 h of ischemia.[13] We used the same IPreC procedure and ischemia time as in the above study. Similarly, for the IPostC procedure, we used a similar procedure as reported by Moon et al.,[8] consisting of 15 s ischemia and 15 s reperfusion repeated for six cycles.

The vital flap ratio was greater in the IPreC group than in the IPostC group (P = 0.008). However, IR injury may occur as an unexpected complication under clinical conditions. The first few minutes of reperfusion after prolonged ischemia are the most critical for tissue survival after IR injury. The generation of reactive oxygen species, acute inflammatory reactions, and neutrophil adhesion reach peak levels during the early phase of reperfusion.[14],[15] IPreC has almost no practical use to reduce the risk of IR injury. However, IPostC is feasible under clinical conditions, for example, after replantation and vascular surgery.

According to experimental studies, the application of IPreC and IPostC together in muscle and renal tissues shows no additional protective effects compared to use of either procedure alone.[16],[17] However, skin flap and brain studies have indicated that application of these two methods together has greater protective effects against tissue injury than either procedure alone.[18],[19] In this study, to increase flap survival, both IPreC and IPostC procedures were applied to the same flaps in the IPreC + IPostC group. However, this did not confer any additional benefit to application of either alone.

IPreC has protective effects in two phases. The early phase, which begins minutes after onset of ischemia and ends within 2–6 h, is due to the release of different mediators and activation of complex signal pathways.[20],[21] The late phase begins 12–24 h after IPreC and ends within 3–4 days.[21] The main difference between the two phases is that there are some changes in protein expression in the early phase, whereas some cell protective proteins are synthesized in the late phase.[22]

In this study, we applied IPostC to flaps under the early protective effects of the IPreC procedure. The early effects of IPreC and IPostC are mediated through the same mechanisms.[23],[24],[25] This may explain why the application of these two methods together did not result in increased survival of vital flap area compared to either procedure alone. Our results indicate that IPreC is more effective than IPostC for preventing ischemic flap complications. Previous experimental studies have shown that IPreC has positive effects on skin and musculocutaneous flap survival.[13],[26] Experimental studies have indicated that the effects of IPreC are comparable to or better than surgical delay.[27],[28] However, limited data have been reported regarding the practical application of the IPreC procedure.[29],[30]

  Conclusions Top

IPreC and IPostC procedures increase flap survival in ischemic flaps. The IPreC procedure is much more effective than the IPostC procedure. The application of these two methods together did not show a greater effect on flap survival than applying either alone. This may have been because the effects of IPostC and the early phase of IPreC use the same mediators.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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Zelt RG, Olding M, Kerrigan CL, Daniel RK. Primary and secondary critical ischemia times of myocutaneous flaps. Plast Reconstr Surg 1986;78:498-503.  Back to cited text no. 2
Myers MB, Cherry G. Mechanism of the delay phenomenon. Plast Reconstr Surg 1969;44:52-7.  Back to cited text no. 3
Myers MB. Attempts to Augment Survival in Skin Flaps: Mechanism of the Delay Phenomenon. In: Grabb WC, Myers MB, editors. Skin Flaps. Boston: Little, Brown; 1975. p. 65-79.  Back to cited text no. 4
Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: A delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124-36.  Back to cited text no. 5
Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA, et al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: Comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 2003;285:H579-88.  Back to cited text no. 6
Kin H, Zhao ZQ, Sun HY, Wang NP, Corvera JS, Halkos ME, et al. Postconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting events in the early minutes of reperfusion. Cardiovasc Res 2004;62:74-85.  Back to cited text no. 7
Moon JG, Lim HC, Gye MR, Oh JS, Park JW. Postconditioning attenuates ischemia-reperfusion injury in rat skin flap. Microsurgery 2008;28:531-7.  Back to cited text no. 8
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Hosnuter M, Babucçu O, Kargi E, Altinyazar C. Dual preconditioning: Effects of pharmacological plus ischemic preconditioning on skin flap survival. Ann Plast Surg 2003;50:398-402.  Back to cited text no. 10
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Zahir KS, Syed SA, Zink JR, Restifo RJ, Thomson JG. Ischemic preconditioning improves the survival of skin and myocutaneous flaps in a rat model. Plast Reconstr Surg 1998;102:140-50.  Back to cited text no. 13
Zweier JL, Flaherty JT, Weisfeldt ML. Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proc Natl Acad Sci U S A 1987;84:1404-7.  Back to cited text no. 14
Lefer AM, Tsao PS, Lefer DJ, Ma XL. Role of endothelial dysfunction in the pathogenesis of reperfusion injury after myocardial ischemia. FASEB J 1991;5:2029-34.  Back to cited text no. 15
Costa FL, Yamaki VN, Teixeira RK, Feijó DH, Valente AL, Carvalho LT, et al. Perconditioning combined with postconditioning on kidney ischemia and reperfusion. Acta Cir Bras 2017;32:599-606.  Back to cited text no. 16
Lintz JA, Dalio MB, Joviliano EE, Piccinato CE. Ischemic pre and postconditioning in skeletal muscle injury produced by ischemia and reperfusion in rats. Acta Cir Bras 2013;28:441-6.  Back to cited text no. 17
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Akcal A, Sirvan SS, Karsidag S, Görgülü T, Akcal MA, Ozagari A, et al. Combination of ischemic preconditioning and postconditioning can minimise skin flap loss: Experimental study. J Plast Surg Hand Surg 2016;50:233-8.  Back to cited text no. 19
Hausenloy DJ, Tsang A, Yellon DM. The reperfusion injury salvage kinase pathway: A common target for both ischemic preconditioning and postconditioning. Trends Cardiovasc Med 2005;15:69-75.  Back to cited text no. 20
Bolli R. Cardioprotective function of inducible nitric oxide synthase and role of nitric oxide in myocardial ischemia and preconditioning: An overview of a decade of research. J Mol Cell Cardiol 2001;33:1897-918.  Back to cited text no. 21
Bolli R, Li QH, Tang XL, Guo Y, Xuan YT, Rokosh G, et al. The late phase of preconditioning and its natural clinical application – Gene therapy. Heart Fail Rev 2007;12:189-99.  Back to cited text no. 22
Heusch G. Molecular basis of cardioprotection: Signal transduction in ischemic pre-, post-, and remote conditioning. Circ Res 2015;116:674-99.  Back to cited text no. 23
McAllister SE, Ashrafpour H, Cahoon N, Huang N, Moses MA, Neligan PC, et al. Postconditioning for salvage of ischemic skeletal muscle from reperfusion injury: Efficacy and mechanism. Am J Physiol Regul Integr Comp Physiol 2008;295:R681-9.  Back to cited text no. 24
Parratt JR, Vegh A. Endothelial cells, nitric oxide and ischaemic preconditioning. Basic Res Cardiol 1996;91:27-30.  Back to cited text no. 25
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Zahir KS, Syed SA, Zink JR, Restifo RJ, Thomson JG. Comparison of the effects of ischemic preconditioning and surgical delay on pedicled musculocutaneous flap survival in a rat model. Ann Plast Surg 1998;40:422-8.  Back to cited text no. 27
Ceylan R, Kaya B, Çaydere M, Terzioğlu A, Aslan G. Comparison of ischaemic preconditioning with surgical delay technique to increase the viability of single pedicle island venous flaps: An experimental study. J Plast Surg Hand Surg 2014;48:368-74.  Back to cited text no. 28
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]


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