| Objective: Cerebral vascular disease (CVD) is one of the three diseases leading to death and influences people's expectancy and quality of life seriously. Ischemic cerebral vascular disease (ICVD) is the most common type. Amounts of studies have shown that the pathophysiological mechanisms underlying cerebral ischemic injury are multifactorial and interactional involving free radical damage,excitability,calcium overload,apoptosis,inflammation and so on. Brain edema is an important pathological change, which can induce and aggravate the secondary brain injury,cause cerebral hernia,threaten patients'lives. It plays a very important role on patients'survival through the acute stage. Although lots of improvements have been made on the prevention and treatment of ICVD, the mechanism of brain injury is still not well-understood. Therefore, the researches on ICVD has become the focus of clinical and experimental studies. 5-Lipoxygenase (5-LO) is a key enzyme metabolizing arachidonic acid to produce leukotrienes which are important inflammatory mediators. Accumulating evidence has suggested 5-LO participates in the inflammatory process after cerebral ischemia,traumatic brain injury,neurogliocytoma formation and degeneration of the central neuvous system. The purpose of this study was to develop a reliable and reproducible animal model of focal cerebral ischemia reperfusion (I/R), to observe the changes of neurological deficit scores,brain water content and the 5-LO expression in the brain tissue, to analyze the relations among the neurological dysfunction,brain edema and 5-LO expression on the basis of the focal cerebral I/R rat model.Methods: Adult male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO). 3 h later, the intraluminal filament was withdrawn to allow reperfusion. Sham-operated rats underwent identical surgery, except that the intraluminal filament was not inserted. At 6 h,24 h,48 h and 72 h after operation, behavioral function was evaluated using 3 different tests in each rat. The specific 3 tests included (1) Longa behavioral test; (2) Berderson behavioral test; (3) Beam walking test. After the neurobehavioral tests were done,each rat was sacrificed and the brain was quickly moved with the frontal pole(almost 4 mm thick) ablated. The remained brain tissue was cut into 2 parts.The first part (almost 2 mm thick) was used for brain edema measurement with the ipsilateral and contralateral of the ischemic brain tissue weight respectively. Brain water content was calculated as the percentage change between wet weight and dry weight using the following formula: (WW-DW)/WW×100%. The second part was used for histological examinations after several steps, including fixed in 4% formalin,dehydrated, embedded in paraffin,finally cut into paraffin sections (5μm) coronally. Some of the paraffin sections were stained with hematoxylin and eosin to observed the brain edema, inflammatory infiltration, changes of the neurons and the neurogliocytes. The adjacent sections were stained by routine immunohistochemical methods using primary antibodies against 5-LO to observe the changes of 5-LO expression at different time points after operation.Results:①The neurological deficit scores of all the rats were the highest at 6 h after operation among the 4 time points probably due to the anesthesia and injuries during the operation. At 24～72 h, the scores of the I/R group leveled higher than the sham operation group and the behavioral outcomes were most serious at 48 h.②The brain water content of the I/R group increased obviously at 24 h(82.42±0.118)%, peaked at 48 h(85.40±0.099)%, and continued to 72 h. Compared with the sham operation group, there was no significant differcence at 6 h,P>0.05, while significant differences existed at 24 h,48 h and 72 h, P<0.05. There were also statistically differences between the ipsilateral and contralateral of the ischemic brain tissue at the 3 time points, P<0.05.③At the 6 h after reperfusion, the neurons in the ischemic cortex shrunk and became triangular with the nucelus stained deeply, the capillaries were engorged with vascular endothelial cells swelling, and the perivascular space expanded. At 24 h, the ischemic aera enlarged. The ischemic brain tissue was highly swollen at 48 h and even hemorrhagic transformation was shown in some region. At 72 h, gliacyte proliferation and neovascularization appeared. The infiltrated inflammatory cells were displayed at 6 h, more abundant at 24 h and 48 h. There were no significant pathological changes in the sham operation group.④The immunohistochemistry stain indicated that the 5-LO-positive cells were neurons and neurogliocytes, mainly the neurons in the ischemia region. The up-regulation of 5-LO expression began at 6 h after reperfusion and reached to the maximum at 24 h(31.63±3.3). At 48 h,72 h, the numbers of the 5-LO-positive cells were still at a certain level. Compared with the sham operation group, there were significant differences at all the 4 time points after reperfusion, P<0.05.Conclusion: In the experiment, the focal cerebral ischemia reperfusion model in rats was reliable,reproducible and applicable to the empirical and intervention study of cerebral I/R.The results showed that the changes of neurological behavior scores and the brain water content were consistent, which suggested that brain edema was the most important cause leading to neurological function deficit after I/R. The dynamic expression of 5-LO had close relationship with the changes of brain edema and inflammatory cell infiltration. Up–regulated 5-LO was involved in inflammatory reaction,brain edema formation and the acute secondary neuronal injury after cerebral I/R.
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