Effects of ischemic preconditioning and cilostazol on muscle ischemia-reperfusion injury in rats1

PURPOSE: To evaluate effects of ischemic preconditioning and Cilostazol on muscle ischemia-reperfusion injury. METHODS: Male Wistar rats were submitted to muscle ischemic and reperfusion injury (4h of the left common iliac artery occlusion followed by 1h of reperfusion). Five experimental groups were constituted: Control group (n=4); Ischemia-Reperfusion (IR, n=5); Ischemic preconditioning group (IP, n=6); Ischemia-Reperfusion group treated with cilostazol (IRCi, n=6) and Ischemic preconditioning group treated with cilostazol (IPCi, n=6). At the end, left gracile muscle was removed and embedded in paraffin. Histopathology, neutrophil infiltration, myocyte necrosis and edema were analyzed. RESULTS: When compared with the control group, IR group showed increased neutrophil infiltration, severe necrosis and edema. There was significant difference between myocytes necrosis of IR group and IP group. There was no difference between the histopathological changes between IP, IRCi and IPCi groups. CONCLUSIONS: The model of IR caused severe muscle injury in the rat hind limb and ischemic preconditioning has a protective effect, reducing myocyte necrosis, however, treatment with cilostazol and also the association between cilostazol and preconditioning has no protective effect on the skeletal muscle subjected to ischemia and reperfusion injury.


Introduction
Despite the development of reconstructive surgery techniques have led the possibility to preservation of members function affected by severe trauma or resection of malignant musculoskeletal tumors, ischemia and reperfusion (IR) injury remain as the major cause of failure of transfer of tissues, leading to extensive surgical revisions or amputation of the extremity 1,2 . IR injury happens when ,during ischemia, blood flow interruption creates local lesions in proportion to oxygen and nutriment privation time, urging reperfusion to be performed as soon as possible. However, reperfusion itself induces more cellular alterations, likely through mitochondrial dysfunction, increased reactive oxygen species production, and inflammation 3,4,5 .
Several methods, such as thrombolytic agents, hyperbaric oxygen treatment, and anti-inflammatory agents have been tested to prevent or lessen the harmful effects of IR injury 6 . It has been shown that Ischemic Preconditioning (IP) confers natural tissue protection to prolonged ischemic stress, involving multiple mediators of ischemic damage such as adenosine, norepinephrine, bradykinin, and unidentified opioids 7 . IP in skeletal muscle has showed a protective effect in vascular reperfusion, cellular membrane structure and function, muscle preservation, decreasing inflammatory infiltrate and vascular stasis [8][9][10][11][12] .
On the other hand, cilostazol is a potent and selective inhibitor of phosphodiesterase III. This enzyme increases cyclic adenosine 3 ', 5'-monophosphate (c AMP) in thrombocytes and smooth muscle cells decreasing intracellular calcium with consequent relaxation and vasodilation. AMPc, in turn, is a regulator of immune and inflammatory responses 13 .
The protective action of cilostazol in IR injury has been demonstrated in the central nervous system, 14, 15 renal tissue 6 , heart 17 and in chronic arterial disease 16 . But the protective role of this drug in acute IR injury of skeletal muscle is not yet established. Studies have indicated the protective role of this drug in skeletal muscle, through the effect on endothelium dependent vascular reactivity 19 , however, no decrease in muscle damage as a biomarker of serum myoglobin muscle and other histopathological changes and apoptosis using cilostazol was observed in IR muscle models 20.21 . The effects of IP associated with cilostazol treatment in muscle ischemia-reperfusion injury did not studied yet. The aim of this study was evaluate the effects of IP associated with cilostazol in experimental model of muscle ischemia-reperfusion injury.

Methods
All animals were handled according to the ethical principles of laboratory animal care according to law 11.794, October 8, 2008, which governs the ethical use of animals for experimentation and followed the guidelines by the Research Ethics Committee of the UNIFESP.
Adult male Wistar rats (Rattus norvegicus albinus), aged between 90 and 120 d and weighing between 250 and 300 g, were used. The animals were kept six days for observation and adaptation, in appropriate cages (40 X 30 X 25 cm), with a maximum of five animals per cage, under controlled conditions for light, temperature, and daily hygiene. They received water and balanced chow ad libitum.

Induction of IR injury of skeletal muscle
The animals were anesthetized with a combination of ketamine (50 mg/kg) and xylazine (15 mg/kg) via intramuscularly injection. The animals were considered anesthetized when they were unresponsive to mechanical stimuli, being unable to withdraw the hind limb after pain stimulus and also presented absence of palpebral reflexes. Additional doses of anesthesia (half of the initial dose) were administered approximately every 50 min.
The animals were also kept well ventilated at room temperature.
The animals were placed on a constantly heated plate (37°C) in supine position with hind limbs immobilized and adhesive tape across the chest. Trichotomy was performed on the abdomen region with a razor blade followed by antisepsis of the operative area with iodine polyvinylpyrrolidone tincture.
Using a no. 11 scalpel blade, a skin incision was made, with dissection and isolation of the left common iliac artery. This artery was clamped during 4 hours and then, the clamp was removed.
The animals were observed for more 1 hour, at which time they were euthanized by lethal dose of anesthetic.

Cilostazol and Ischemic preconditioning
Cilostazol were dissolved in dimethyl sulphoxide and injected intraperitoneally (30mg/kg) 20 minutes before of prolonged ischemia.
The Ischemic preconditioning consisted of 10 minutes occlusion of the left common iliac artery occlusion followed by 10 minutes of reperfusion, before prolonged ischemia.

Collecting gracile muscle and histopathology
After euthanasia, left inferior gracile muscle was excised, fixed in formalin solution, embedded in paraffin. Five micrometer sections were stained with hematoxylin-eosin (HE) and Neutrophil infiltration, muscular edema and signs of myocyte necrosis were evaluated under 100x of magnification and blinded manner by score method: absent (0), rare (1), moderate (2) and severe (3).
There were considered signs of myocyte necrosis: cellular vacuolization, band contraction and/or cellular destruction.

Statistical analysis
The data were analyzed using the SigmaStat statistical program, version 3.1 (Systat Software, SanJose, CA). The groups were compared by Kruskal-Wallis 1-way analysis of variance on ranks, after testing for normality and variance equality, and complemented by post hoc test (Dunn's test). The groups were also compared in pairs using the Mann Whitney test. Data were expressed median (25%-75%). Difference was considered statistically significant when P < 0.05. Figure  1 is depict representative photomicrographs.  The IP group appeared to reduce myocyte necrosis in muscle tissue, but cilostazol or the association between preconditioning and cilostazol did not protect skeletal muscle from IR injury.

In table 1 are depict histological evaluation and in
accordance with these studies, we also observed the protective effect of IP in this experimental model of IR Injury, especially with the reduction of myocyte necrosis.
Previously studies suggest that cilostazol could have a protective effect by perfusion increasing in ischemic areas and anti-apoptotic properties 23,24 . In addition, other studies have observed that cilostazol has a protective effect against ischemic injury in animal models, when observed in other organs 25,26 . However, in this study, histopathological changes due to ischemia and reperfusion in animals treated with cilostazol in striated muscle were observed at a lower intensity, but without statistical difference when compared with the IR group animals.
These results are consistent with the study of Moreira Neto et al. 28 who found no protective effects of cilostazol in IR injury of skeletal muscle in similar experimental models with a reperfusion periods of 2 hours and 6 hours.
Cilostazol treatment alone or in combination with IP showed no significant effect on muscle injury protection in IR and suggests that more experimental studies are required to elucidate the role of these strategies in the treatment of muscle injury IR.

Conclusion
The model of IR caused severe muscle injury in the rat hind limb and ischemic preconditioning has a protective effect, reducing myocyte necrosis, however, despite signs that treatment with cilostazol and also the association between cilostazol and preconditioning could have a protective effect on this injury, this was not observed in this study.  There was no difference between histopathological changes of IPCi and IP group as there was no difference between the histopathological changes between IPCi and IRCi groups.

Discussion
Ischemia means absence of blood flow and consequent reduction or absence of oxygen offer and substrates to the tissue besides excess of metabolites in human being. Acute ischemia can cause, if there is no recovery of blood flow, from small areas of necrosis to member amputations. However, recovery of arterial flow after acute ischemia results, generally, in the morpho-functional restoration, but in some cases a complex post revascularization syndrome appears, causing some times loss of members or life 7 .
One of the first signs that occur after reperfusion is the interstitial edema, caused by mechanical changes, such as increase in intracapillary pressure and in capillary permeability 22 . The ischemic process could promote important inflammatory reaction with neutrophil infiltration in the first 24 hours.
Ischemic preconditioning promotes protection to ischemiareperfusion injury in muscle, showing the muscular fiber morphological preservation 10 . Other authors also observed that preconditioning had a protective effect by avoiding glycogen depletion in skeletal muscle in rats submitted to IR. In