Remote limb ischemic post-conditioning attenuates ischemia-reperfusion injury in rat skin flapby limiting oxidative stress

PURPOSE: To investigate the effect of remote ischemic post-conditioning (RIPoC) against ischemia-reperfusion (I/R) injury on flaps of rats. METHODS: Sprague-Dawley rats were randomized into the Sham, Control, RIPoC1 and RIPoC2 groups. All the animals were submitted to a 5×4 cm superficial inferior epigastric artery flap. Eight hours of flap ischemia was induced and two protocols of limb RIPoC were applied. Tissue MDA level and SOD activity in 24-h reperfusion were assessed. Flap survival was assessed 7 days postoperatively. RESULTS: Compared to the Control group, the RIPoC1 group showed statistically decreased MDA level at 6-, 12-, and 24-h reperfusion (P = 0.01, P < 0.01 and P < 0.01, respectively), and statistically increased SOD activity at 12and 24-h reperfusion (P < 0.05 and P < 0.01, respectively). Flap survival rate on the 7th day was significantly higher in the RIPoC1 group than the control group (47.9 ± 6.4 vs. 29.4 ± 7.1 %, P < 0.01). CONCLUSION: Three cycles of 5-min Limb remote ischemic post-conditioning rather than a single cycle of 15-min limb RIPoC has protective effect on flaps against ischemia-reperfusion injury by attenuating oxidative stress.


Introduction
Free flap transplantation is frequently used in plastic and reconstructive surgery.In spite of the high success rate, reperfusion injury by abrupt restoration of circulation after prolonged ischemia has been remained an unsolved problem associated with partial to total flap loss.
Excessive production of free radicals and/or reactive oxygen species during reperfusion triggers lipid peroxidation and initiates ischemia reperfusion (I/R) damage 1 .Malondialdehyde (MDA) is one of intermediate products of lipid peroxidation, and its level is commonly used as a biomarker of oxidative stress 2 .On the contrary, endogenous antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, act as "free radical scavengers" which can transform superoxide anion to the less reactive species H 2 O 2 3 .
In recent years, numerous labs have demonstrated that ischemic post-conditioning (IPostC), defined as brief intermittent episodes of ischemia at the onset of reperfusion, is an endogenous protective strategy against I/R injury for multiple organs [4][5][6] .The underling mechanism of IPostC involves limiting the oxidative stress by reducing reactive oxygen metabolites and increasing antioxidant enzymes' activity in tissues after prolonged ischemic insult 7 .However, repetitive ischemic insult and hypoxia added directly on the primary ischemic tissue are still unacceptable Male Sprague-Dawley (SD) rats, weighing between 290g to 350g, were randomized into4four groups (n=16 in each group): the Sham, Control, RIPoC1 and RIPoC2 groups.The animals were kept in a room with a controlled environment with a 12-h day/ night lighting cycle, and allowed access to standard rodent chow and tap water.General anesthesia was induced with intraperitoneal injections of ketamine 80 mg/kg and maintained with a booster injection of one third of initial dose.

Surgicaleprocedure
The abdomen was shaved with an electric hair clipper, and asepsis was maintained by providing a local sterile.A 5×4 cm island pedicle flap was elevated in all animals with the base at left superficial inferior epigastric (SIE) vessels (Figure 1).The flap consisted of skin, subcutaneous tissue, and intimately attached panniculus carnosus.The SIE pedicle was carefully isolated down to the left femoral vessels, and de-nervated under a microscope to accurately mimic a free tissue transfer.In the Control group, an 8-h flap ischemia was induced using an S&T microvascular clip with 15g compression on the pedicle.During the period of ischemia, the rats were kept

SOD activity and MDAtcontent
A full-thickness, 2-mm punch biopsy of the flap skin wasecollected at the distal end of the flap, after 3h, 6h, 12h, 24h after reperfusion, respectively.The samples were rinsed, weighed, and homogenized in 9 volumes of ice-cold buffers.Supernatant

Flap survival analysis
At the 7 th day after reperfusion, the animal was reanesthetized and the appearance of the skin flap was recorded and photographed in a standardized fashion.The flap viable area was determined based on color, capillary refill, and the pin-prick test.Ratios of viable area to original flap area were calculated by digital planimetry software (Image-Pro Plus Version 7.0), expressed as a percentage (percent survival).

Statisticalsanalysis
The results were expressed as mean + standard deviation (SD).Student t-test was used for comparisons between two means.
All data were analyzed using SPSS software 19.0.Values <0.05 were considered to be statistically significant.

MDA content and SOD activity
Ischemia and the subsequent reperfusion resulted in significantly higher level of MDA content and lower level of SOD activity in flap tissue from both the Control and RIPoC groups than that in tissue from the Sham group, throughout the 24-h reperfusion period (Figure 4).Compared with the controls, induction of RIPoC1 significantly attenuated I/R-induced elevation in MDA at 6-, 12-, and 24-h reperfusion timepoints (P = 0.01, P < 0.01 and P < 0.01, respectively).In contrast, differences in MDA level between the Control and RIPoC2 groups at each timepoint were not significant (P > 0.05).Compared with the controls, RIPoC1 significantly attenuated the decrease in SOD activity at 12-and 24-h reperfusion timepoints (P < 0.05 and P < 0.01, respectively) while RIPoC2 only attenuated the decrease in SOD activity at 24-h reperfusion timepoints (P < 0.05).Detailed statistical evaluation of the MDA levels and SOD activity Tables 1 and 2).

Flap survival rate
The viable region of the flap was measured 7 days after reperfusion (Figure 5).Compared to the Sham group, significant decrease in ratios of the survival area to original flap area was  12 showed that IPostC applied by means of 6 cycles of 30 seconds yields an optimal anti-ischemia protection in a rat skin flap model.In the present study, we expand further on this concept by applying post-conditioning at a distance (hind limbs) in a rat skin flap model, and showed that RIPoC was an effective therapeutic intervention against flap I/R injury.
The protective effect of RIPoC was initially described in the heart 13 , and applied to other organs [14][15][16] .RIPoC conducted in the limbs is most commonly used in the previous studies because of its convenience and safety for clinical application 17 .However, varying protocols of limb RIPoC were selected in different studies, while the optimal one remains to be determined.In the present study, two experimental protocols of limb RIPoC were evaluated.
Limb RIPoC1 applied as 3 cycles of 5-minute ischemia/5minute reperfusion has been proven as a valid protection against I/R injury in brain 8,19 ., while Limb RIPoC2 with 1 cycle of 15-minute ischemia has been proven to confer a strong degree of cardioprotection against I/R injury 20 .Our findings indicated that limb RIPoC1 significantly reduced the flap necrosis area at the 7 h days after reperfusion compared to the controls.On the contrary, limb Rchemiadid not provide equihemia protection for the flap.
, our results provide promising evidence for its effect on decreasing the production of peroxidation products and increasing free radical scavenging capacity in the early stage of flap reperfusion injury.
There's limitation that should be mentioned.Since flap necrosis was the primary endpoint, based on our experience and the previous literature 23 , 8-hour ischemia was conducted to establish a stable model of I/R injury with partial flap loss.
Therefore, our findings might focus on flaps with severe ischemia induced by vaso-occlusive crisis, when RIPoC could be applied as an add-on therapy to re-explorations.Its effect on routine free flap transplantation, in which warm ischemia time usually does not exceed 2 hours, was nevertheless not well discussed in the current study.Thus, further studies based on a model with different ischemia time are needed, and assessments of acute biomarkers of I/R such as neutrophil infiltration are recoended.

Conclusions
The remote ischemic post-co thatRIPoC applied as 3 cycles of 5-min limb ischemia followed by 5-min reperfusion limits I/R injury ineprotective and IPoC may beis mediated by limiting oxidatap tissues in the early stage of reon.Thus, iIntervention with RIPoC, which targets the first few minutes of reperfusion, may be a valuable clinical ''after-injury strategy' for free flap research.
concept and impracticable.Remote ischemic post-conditioning (RIPoC) refers to ischemic tolerance induced by transient ischemia at a site distant to the ischemic target organs immediately at the time of its reperfusion 8 .In contrast to the clinical limitation of IPostC, RIPoC could be a non-invasive clinical intervention.Recent preclinical papers show that RIPoC performed in the limbs can generate effective cardioprotection and neuroprotection against I/R injury similar to IPostC 9,10 , yet its protective effect on skin flap has not yet been well investigated.In the present study, we aimed to evaluate the antiischemic properties of two protocols of limb RIPoC in a rat model of epigastric island flaps subjected to I/R injury, and the possible effect of RIPoC on reduction of I/R-induced oxidative stress.Methods All procedures were conducted in compliance with NIH Guiding Principles for Research Involving Animals and were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine.

FIGURE 1 -
FIGURE 1 -An inferior epigastric flap (5×4 cm) as a skin flap reperfusion injury model.The left superficial inferior epigastric pedicle (horizontal arrow) was carefully isolated down to the femoral vessels (vertical arrow).Flap ischemia was induced using an S&T microvascular clip Ooblique arrow) with 15g compression on the flap pedicle.
anesthetized and the flap was placed in gauze moistened with warm saline solution.After the clip was removed, the vascular patency of the pedicles was confirmed under an operating microscope before the flap was repositioned and sutured.The Sham group underwent the same flap elevation and vessel dissection as the Control group, but flap I/R was not induced (Figure2).

FIGURE 2 -
FIGURE 2 -Experimental protocol.Rats were randomized into 4 groups (n=16 in each group): (1) In the Sham group, rats underwent 8 hrs of general anesthesia (open bar) and a sham operation; (2) In the Control group, rats underwent 8 hrs of flap ischemia (dark bar) followed by reperfusion (gray bar) with no conditioning therapy; In the RIPoC groups, limb RIPOC was administered at the onset of flap reperfusion after 8 hrs of ischemia.(3) RIPoC1 was achieved by 3 cycles of 5-min limb ischemia (dark bar) followed by 5-min reperfusion (open bar); (3) RIPoC2 was achieved by 1 cycles of 15-min limb ischemia (dark bar) followed by reperfusion (open bar).Tissue MDA content and SOD activity within 24 hours after reperfusion was examined, and the flap survival area was evaluated at the 7 th day.

FIGURE 3 -
FIGURE 3 -Remote ischemic post-conditioning induced by placing a tourniquet around the base of the right hind limb. A. Normal limb; B. Ischemic limb; C. Reperfused limb.
homogenate was collected after centrifugation at 5000 rpm/min for 15 min at 4 C.The SOD activity of homogenates was determined by the xanthine oxidase (hydroxylamine) method, using a commercial kit (A001; Nanjing jiancheng Bioengineering Institute, China), and the results are expressed as nmol/mg wet weight.The MDA content of homogenates was determined spectrophotometrically by thiobarbituric acid colorimetric method, using a commercial kit (A003; Nanjing jiancheng Bioengineering Institute, China), and the results are expressed as µmol/mg wet weight.

TABLE 1 -
MDA levels at each timepoint in each group.
P 1 : Control group vs. Sham group; P 2 : Control group vs. RIPoC1 group; P 3 : Control group vs. RIPoC2 group; p value in bold when less than 0.05.

TABLE 3 -
Flap survival rates in each group at the 7th day after reperfusion.Sham group; P 2 : Control group vs. RIPoC1 group; P 3 : Control group vs. RIPoC2 group; p value in bold when less than 0.05.
P 1 : Control group vs.