Biochemical study of the effects of cilostazol in rats subjected to acute ischemia and reperfusion of hind limbs1

PURPOSE: To investigate whether cilostazol has a protective effect on acute ischemia and reperfusion of hind limbs of rats through study of biochemical variables in blood and urine. METHODS: Forty six 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 the reperfusion period, was held to collect urine and blood for biochemical measurements. The biochemical parameters studied were: urea, creatinine, sodium, potassium and myoglobin in blood and urea, creatinine, myoglobin in urine. RESULTS: There was no statistically significant difference between groups. CONCLUSION: Cilostazol had no protective effect on ischemic acute reperfusion of hind limbs of rats 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 .
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 7 intended to reduce the symptoms of peripheral vascular disease, intermittent claudication 8 and prevention of recurrent cerebral stroke 9 .
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 rats submitted to acute ischemia and reperfusion of hind limbs through study of biochemical variables in blood and urine, since no experimental models in animals studies on the effectiveness of cilostazol in acute ischemia and reperfusion have been reported. Forty six male Wistar rats, ten months age and average weight of 300 grams were used.

Surgical technique
The animals were anesthetized and placed an orogastric

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.

Reperfusion time
After removing aortic ligature, started to measure time of reperfusion.
Two animals were excluded from the previous steps of the experiment because they died before the final reperfusion time.
Biochemical study of the effects of cilostazol in rats subjected to acute ischemia and reperfusion of hind limbs Acta Cirúrgica Brasileira -Vol. 28 (5)

Collection of blood and urine and euthanasia
Again the animals were anesthetized and proceeded to puncture the bladder and completing cardiac puncture euthanasia.
The secret allocation was obeyed.
For biochemical analysis were measured to mioglobulina, urea and creatinine, sodium and potassium in the blood and mioglobulina, urea and creatinine in urine.
When the sample showed a normal distribution, shown by the normality of Shapiro-Wilk test was used t Test for sample data from two independent samples, with p ≤ to 0.05 for significance.
When the sample did not show a normal distribution, we used nonparametric test for two independent samples, Mann-Whitney (Wilcoxon rank-sum test), with p ≤ to 0.05 for significance.

Results
The biochemical parameters studied, both in blood and in urine were not statistically significant change compared between groups with two to six hours of reperfusion, animals treated with cilostazol or not (Tables 1, 2 and 3).

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 In this study, biochemical changes due to ischemia and reperfusion were observed in the blood 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.
This may 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.
It was not possible to study the urinary levels of myoglobin and creatinine in groups of six hours of reperfusion due to insufficient amount of urine in the bladder of the animal at the time of collection.

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.