| Objective:⑴The purposes of this study are to establish a reliable and reproducible animal model of permanent middle cerebral artery occlusion (PMCAO) in rats, by observing the changes of the neurologic function, the infarction volume, brain water content and dynamic expression of aquaporin-9(AQP9)mRNA, to analyze the relations between AQP9mRNA expression and brain edema, and to confirm the function of dynamic AQP9 expression in brain tissue after cerebral ischemic injury in rats.⑵To explore edaravone's brain protection function and possibility of protection mechanism by studying the effect of edaravone on the AQP9mRNA expression and brain edema following focal cerebral ischemia in rats, which maybe provide theoretical foundation and experimental basis for acute ischemic cerebrovascular disease of clinical treatment. Method: 252 Sprague-Dawley adult male rats were randomly divided into three groups as shame-operated group (n=84), physiological saline group (n=84), edaravone-treated group (n=84). Each group was divided into 7 teams according to 6 hours,12 hours,24 hours,48 hours,72 hours,5 days and 7 days after operation, each team was composed of 12 model rats. With the adoption of reforming line bolt's method, the permanent cerebral ischemic model was established by occluding middle cerebral arteries (MCA) with nylon sutures. Model after the establishment, all the rats were tested for the neurologic impairment scores using Longa Score and Berderson Score methods at each time point, the 1～3 score rats were taken as the research objects. Correspondingly the model rats were randomly complemented so that the experiment animals'amount was constant. The shame-operated group underwent the same operation as the model group except nylon suture insertion. Each rat in edaravone treat group was intraperitoneal injected with edaravone 3mg/kg per 12h after occlusion, each rat in physiological saline group was administered the equal sodium Chloride in stead of drug, and nothing was injected in the shame-operated group. According to the time observation point, the 6 models were selected randomly from each team and killed at different time point, the brain tissues were quickly moved from the skull. The olfactory bulb and the frontal part (4mm thick) were coronally cut and deserted. The frontal part (3mm thick) of the other cerebrum was for brain water content measurement, which was calculated as the percentage changes between wet weight(WW) and dry weight(DW) using the following formula: (WW?DW)∕WW×100%. The posterior part was used for detecting the AQP9mRNA expression level using real time fluorescent quantitation polymerase chain reaction (PCR) method. At different time point the other 6 models were killed, exposed the heart, injected sodium chloride 200ml through the left ventricles. Taking the anterior fontanelle as the center, the coronal plane 2.0mm, the six brain slices were sliced. The third slice was used for histological examination, this brain slice was dealled after several procedures, including fixed in 4% formalin, dehydrated by different concentrations of ethanol and then embedded in paraffin. Then the rat brain's coronal plane paraffin section was sliced continuously to make 5μm thick. The infarction volume of the other five brain slices were observed by 2, 3, 5-Triphenyl tetrazolium chloride (TTC) coloring. Result:⑴The model of focal cerebral ischemia made by using the intraluminal suture in this study was reliable and reproducible.⑵The neurological deficit was observed after operation. As the consequences of anesthesia and operation injury, the neurological function was decreased at 24h after operation in shame-operated group. Compared with the shame-operated group, the neurological function in the physiological saline group was decreased during the 6h to 24h (p<0.05). During the 6h to 72h, the neurological function of the edaravone-treated group was lighter than the physiological saline group (p<0.05). At 7d, the neurological function of the physiological saline group and edaravone-treated group almost recovered.⑶At 6h～7d after operation, the brain water content of the shame-operated group almost have no changes at each time point. The brain water content in rats of the physiological saline group increased obviously along with the prolongation of time following cerebral ischemia, and reached its peak at 48 hours postischemia, which was much higher than that of edaravone-treated group (p<0.05).⑷In the physiological saline group, the real time fluorescent quantitation polymerase chain reaction(PCR) indicated that there were up-regulated of AQP9mRNA expression around the ischemic region after brain ischemic compared with that of the shame-operated group at each time point(p<0.05). In the physiological saline group and edaravone-treated group, the expression of AQP9mRNA around the ischemic region gradually increased at 6h postischemic, reached its peak at 72h, and reduced but was slightly higher than the shame-operated group at 7d. Meanwhile, in the physiological saline group and the edaravone-treated group, the expression levels of AQP9mRNA were significantly increased versus the shame-operated group at each time ?point (p<0.05). The change tendency was nearly the same as that of the brain water content. The correlation analysis demonstrated that expression level of AQP9mRNA was correlated apparently with the brain water content (r=0.788, p<0.05).⑸The infarct volume in the edaravone treat group and the physiological saline group reached its peak at 24 hours postischemia. Compared with the physiological saline group, the infarct volume in the edaravone treat group was decreased manifestly during 6～24h after cerebral ischemia in physiological saline group(p<0.05).⑹The HE stain of the physiological saline group indicated that the shape of neurons around the ischemic region had no obvious changes at 6h. At 6h postischemia, brain tissues around the ischemic focus were obviously swollen and necrotic. The pathematology damage such as brain edema and neuron necrosis was ameliorated significantly in the edaravone treat group. In the shame-operated group, there were no significant changes in the brain tissue. Conclusion:⑴The experiment demonstrates that the rat model of focal cerebral ischemia made successfully by using the intraluminal suture in this study was reliable and reproducible. The model is similar to clinical cerebral ischemia. It is applicable to the acute empirical study.⑵The AQP9mRNA expression level in the physiological saline group is also significantly higher than the shame-operated group. Moreover, the up-regulated expression of AQP9mRNA and the brain water content are also consistent after cerebral ischemic at each time point. This implies that Up-regulated AQP9mRNA expression is closely associated with the occurrence and development of brain edema after cerebral ischemic. And AQP9 might play an important role in brain edema formation and the secondary neuronal injury after cerebral ischemic.⑶Edaravone, as a free radical scavenging agent, can inhibit effectively the expression of AQP9mRNA and attenuate brain edema. It is assumed that the inhibition of AQP9mRNA expression to relieve cerebral edema may be the mechanisms of edaravone neuroprotection. Modification of AQP9mRNA activity and regulation of AQP9mRNA expression level would be the new strategy to prevent the cerebral edema and to reduce cerebral injury after stroke. Which imply that the therapeutic strategy targeted to modulation with AQP9 may offer a new perspective for cerebral ischemic treatment. Meanwhile, the mechanism how Edarvaone influences the expression of the AQP9mRNA is not clear and further study is needed.⑷Edaravone can attenuate brain edema effectively, reduce cerebral infarction volume and improve neurological function after cerebral ischemia, and it exhibits an excellent neuroprotective effect against ischemia brain injury.
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