Effects of cilostazol in kidney and skeletal striated muscle of Wistar rats submitted to acute ischemia and reperfusion of hind limbs

PURPOSE : To investigate the effect of cilostazol, in kidney and skeletal muscle of rats submitted to acute ischemia and reperfusion. METHODS : Fourty three animals were randomized and divided into two groups. Group I received a solution of cilostazol (10 mg/ Kg) and group II received saline solution 0.9% (SS) by orogastric tube after ligature of the abdominal aorta. After four hours of ischemia the animals were divided into four subgroups: group IA (Cilostazol): two hours of reperfusion. Group IIA (SS): two hours of reperfusion. Group IB (Cilostazol): six hours of reperfusion. Group IIB (SS) six hours of reperfusion. After reperfusion, a left nephrectomy was performed and removal of the muscles of the hind limb. The histological parameters were studied. In kidney cylinders of myoglobin, vacuolar degeneration and acute tubular necrosis. In muscle interstitial edema, inflammatory infiltrate, hypereosinophilia fiber, cariopicnose and necrosis. Apoptosis was assessed by immunohistochemistry for cleaved caspase-3 and TUNEL. RESULTS : There was no statistically significant difference between groups. CONCLUSION : Cilostazol had no protective effect on the kidney and the skeletal striated muscle in rats submitted to acute ischemia and reperfusion in this model.


Introduction
The reperfusion syndrome is characterized by metabolic acidosis, hyperkalemia by the loss of intracellular potassium, increased serum creatine kinase and myoglobin with myoglobinury 1 . May result in acute renal failure: coagulation disorders, accumulation of extracellular fluid and acute pulmonary distress 2 .
With the acute ischemia, initiating anaerobic metabolism, glycogen with transformation into lactate, leading to decreased production of adenosine triphosphate (ATP), which alters the permeability of the cell muscle, allowing the output of potassium and myoglobin and entry Sodium and calcium célula 3 .
The cell damage occurs only after an interval of thirty minutes of ischemia and irreversible changes in skeletal muscle occur after four to six hours of ischemia. Ischemia can lead to cell death by necrosis or apoptosis 4 .
The caspases are a group of protease essential for apoptosis, which makes irreversible cleavage of DNA. The cascade activation of caspases can be triggered by many factors, such as the presence of superoxide, which is a derivative of the mass muscular ischemia 5 .
After reperfusion muscle, is released into the circulation of acid metabolites and products of cell destruction that cause significant metabolic alterations, such as metabolic acidosis and hyperkalemia 5 . The more severe change is due to precipitation of myoglobin in the renal tubules in acid environment, causing acute tubular necrosis 6 .
Treatment of acute ischemia reperfusion is only the affected territory reperfusion of which may lead to ischemia and reperfusion syndrome.
Many times reperfusion cannot be performed immediately after the onset of ischemia, since there is a very variable period of time between the first symptoms of the disease and emergency medical care.
Cilostazol is an antiplatet drug and vasodilator with antimitogenics and cardiotonic actions intended to reduce the symptoms of peripheral vascular disease, intermittent claudication 7 and prevention of recurrent cerebral stroke 8 .
We hypothesize that cilostazol inhibits platelet aggregation and promoting vasodilation could decrease the deleterious effects of ischemia and reperfusion syndrome. If administered at the onset of ischemia could reduce acute ischemic events, and consequently reduce renal injury after reperfusion.
The objective of this study is to assess the effect of cilostazol in the kidney and skeletal striated muscle of Wistar rats submitted to acute ischemia and reperfusion of hind limbs, since no experimental models in animals studies on the effectiveness of cilostazol in acute ischemia and reperfusion have been reported.

Methods
Forty three male Wistar rats aged ten months and average weight of 300g were used.

Surgical technique
The animals were anesthetized and placed an orogastric tube.
Laparotomy was performed in 4cm long. The abdominal aorta was ligated just below the renal artery with propylene 7.0 9 .
Proceeded to divided into two randomly groups: The solutions were administered immediately after aortic ligature.
The effectiveness of aortic ligature was confirmed by the appearance of pallor, cyanosis and decreased temperature on their hind legs for thermometry. The absence of pulse and flow in the aorta below the ligature was confirmed by intraoperative Dopplermetry.

Ischemic time
After aortic ligature, started to measure ischemia time and proceeded to the closure of the laparotomy.
After four hours of ischemia relaparotomy was performed in order to remove the aortic ligature and then, closed.

Kidney and muscle harvesting and euthanasia
The animals were anesthetized, a left nephrectomy was performed, and the hind limb muscles harvested and proceded the euthanasia.
The kidney and muscles of the left hind limb were divided into two parts, fixed in peraformaldehyde 10%. One part was designed to histological analysis and the other for immunohistochemical study.

Histological and immunohistochemical analysis
The histological changes were examined in muscle:    The results of histological analysis and expression of immunohistochemical markers were tested using non-parametric Mann-Whitney (Wilcoxon Rank-Sum Test) for two independent samples, p ≤0.05 for significance. It was also made a graph of the 95% confidence interval of the mean for immunohistochemistry between groups with six hours of reperfusion.

Histological evaluation
Histological changes observed in muscle and kidney that received cilostazol compared to control group showed no significant differences in two and six hours of reperfusion.

Immunohistochemical evaluation
The expression of cleaved caspase 3 in the kidney and TUNEL expression in the kidney and muscle are shown in Table   1 (next page).
The results showed that the total number of cells undergoing apoptosis quantified by cleaved caspase 3 and TUNEL was lower in the kidneys of animals that received cilostazol with six hours of reperfusion, however these differences were not statistically significant (Figures 4 and 5).

Discussion
The Cilostazol has been widely used in the treatment of chronic peripheral arterial disease and in treatment of ischemic coronary artery disease due to its antiplatelet and vasodilatation properties 7 .
The therapeutic use of cilostazol in acute ischemia and its role in prevention of reperfusion syndrome has not been recommended. The lack of randomized controlled studies, using cilostazol in ischemia and reperfusion in rat kidney and muscle, we have motivated the design of this research. In this study it was hypothesized that cilostazol might have efficacy in treating acute ischemia and would decrease the metabolic syndrome of reperfusion protects target organs such as muscle and kidney.
Experimental studies have demonstrated that cilostazol obtained a protective effect against ischemic injury in animal models when used in another organs than skeletal muscle and kidneys [11][12][13] .
Several experimental studies had model and design similar to our study to investigate the effectiveness of other drugs in ischemia and reperfusion 6,15,16 .
A recent experimental study demonstrated that cilostazol reduces the oxidative stress of ischemia and reperfusion in rats The tissue injury caused by ischemia and reperfusion is described as early onset, and studies have shown that histological changes are observable by microscopy after four hours of ischemia and 15 minutes of reperfusion 14 .
In this study, histological changes due to ischemia and reperfusion were observed in the kidney and striated muscle in similar intensity in the animals receiving cilostazol and those who received only saline solution. These changes were independent of reperfusion time, since it did not differ significantly in the two groups and underwent six hours of reperfusion.
The expression of cleaved caspase 3 18 and TUNEL 19 for quantification of apoptosis currently have been used.
The expression of cleaved caspase 3 and TUNEL was lower in the cilostazol group with six hours of reperfusion, but not statistically significant. Therefore these results suggest that cilostazol could have a positive effect to reduce renal apoptosis in later periods of reperfusion.
Effects of cilostazol in kidney and skeletal striated muscle of Wistar rats submitted to acute ischemia and reperfusion of hind limbs Acta Cirúrgica Brasileira -Vol. 27 (11) 2012 -787 Another hypothesis could be due to the number of subjects used, which the analysis of a few animals per group, had no power to find statistically significant differences.

Conclusion
Cilostazol had no protective effect on the kidney and the skeletal striated muscle in rats submitted to acute ischemia and reperfusion in this model.