| ObjectiveThe central nervous system diseases, such as brain tumor, cerebral stroke and Parkinson' s disease are very harmful to the health of the people. At present , the treatment of them is still unsatisfied. The reason is that the blood brain barrier (BBB) limits the delivery of the drugs to the brain tissues, resulting in the failure of the treatment. It has been reported that the vasoactive compounds, such as histamine, leukotriene C4 and bradykinin (BK) etc play very important roles in increasing the permeability of BBB. The most effective method is applying BK or its analog, receptor - mediated permeabilizer - 7 ( RMP - 7) , to selectively open the BBB.In recent years, studies have found that low dose BK or RMP - 7 significantly increase the permeability of blood tumor barrier ( BTB) , while the normal brain is unaffected. The mechanism of selectively increasing the permeability of BBB with BK or RMP - 7 has been investigated. Recently, Liu Yunhui reported that low dose BK selectively increased the permeability of BBB in the ischemic area. However, the mechanism of selectively increasing the permeability of BBB in the ischemic area with BK is unknown. Meanwhile, whether or not BK will increase the secondary edema caused by ischemia is still to be investigated. This study applied the ischemia model established by occluding the middle cerebral artery of rats to investigate the effect induced by low dose bradykinin on the ul-trastructure and the permeability of BBB at the earlier period of reperfusion after the focal ischemia, and the water content of the brain at the time 24 hours afterreperfusion.Materials1. Experimental animals:Healthy mature male Wistar rats, weight 250 -300g, total number is 40. (Obtained from the Center of the Experimental Animals in China Medical University )2. Experimental instruments;Perfusor Compact S micro - infusion pump; JEM - 1200EX electron microscope; SPECTRONIC 21 UV - visual spectrophotometer.3. Experimental reagents: Bradykinin( Sigma,U. S. A) ;Evance blue ( Fluka separate packing).Methods1. Establishment of the ischemia model by occluding the middle cerebral artery of rats.2. Grouped the experimental animals.3. Scoring the nervous functions.4. Observation of index.The left middle cerebral artery was occluded 2hr followed by lhr reperfu-sion.(1) The ultrastructure's change of the BBB was observed with electron microscope after the further intracarotid infusion of low dose BK (10 μg/kg/min).(2) The Evance blue (EB) content was analyzed after the further intracarotid infusion of low dose BK (10 μg/kg/min).(3) The water content of the brain was assessed after 24hr reperfusion.5. Statistical analysis.Data were expressed as mean ± standard deviation (SD) with SPSS 11.0statistic software. The differences between groups were determined using unpaired Student t - test. P - value less than 0.05 was considered significant.Results1. Changes of BBB ultrastructureAt the earlier period of the reperfusion following 2hr occlusion, in the control group, the part of the endothelium and end foot in the BBB were swelled slightly. The tight junction and the basal membrane were not destructed. But in the bradykinin infusion group, the patency of the junctions between the endothelial cells was seen clearly.2. Changes of BBB permeabilityThere is no difference in the EB content of the brain between the ischemic side and the opposite side in the control group (the ischemic side vs. the opposite side: 1. 72 ± 0. 21 μg/g vs. 1. 53 ± 0. 15 μg/g, n=8, p > 0. 05 ). Compared with the opposite side, the EB content in the ischemic side increased significantly in the bradykinin infusion group (the ischemic side vs. the opposite side: 5.01 ±0.97 μg/g vs. 1. 56 ±0.20 μg/g, n=8, p<0.01). Compared with the control group, the EB content in the ischemic side increased evidently in the bradykinin infusion group (the bradykinin infusion group vs. the control group: 5.01 ±0.97 μg/g vs. 1.72±0.21 μg/g, n=8, p<0.01).3. Changes of brain water contentWhen the left middle cerebral artery was occluded 2hr followed by 24hr reperfusion, the water content of the brain increased significantly in ischemic side, not only in the bradykinin infusion group but also in the control group {the bradykinin infusion group : the ischemic side vs . the opposite side : 8 1 . 7 4 ± 1.05% vs. 78.28 ±0.73% , n=8, p<0.01; the control group: the ischemic sidevs. the opposite side : 81. 21 ± 1. 23 % vs. 77. 96 ± 0. 86% , n = 8 , p < 0.01). But there is no difference in the water content of the brain in the ischemic side between the bradykinin infusion group and the control group (the bradykinin infusion group vs. the control group : 81. 74 ±1.05% vs. 81. 21 ± 1.23%, n=8,p>0.05 ).DiscussionThis study demonstrated that BK did not increase the permeability of BBB in the non - ischemic area, but significantly increase the permeability of BBB in the ischemic area. Low dose BK selectively increased the permeability of BBB in the ischemic area by loosing the tight junction without affecting the extent of ischemia - induced secondary edema.Now the mechanism of the selective increase of the BBB permeability induced by intracarotid infusion of low dose BK is not clear. The possible mechanism is that the binding of bradykinin with B2 receptor on the capillary endothelial cells cause the transient increase of intracellular Ca2 + , eliciting a cascade of signal transduction reaction, including the rise of NO and cGMP, leading to the increase of the pinocytic vesicles or the opening of tight junction. Hashizume reported that BK selectively increased the permeability of BTB because of an increase in pinocytic vesicles. Elena reported that BK selectively increased the permeability of the normal BBB as of the opening of tight junction. Our study demonstrated that low dose bradykinin selectively increased the permeability of BBB in the ischemic area by loosing the tight junction. The selective action on the BBB by BK or RMP - 7 may be related with the local change of the ultra-structure and the penneability of BBB.This study indicated that low dose BK( l0μg/kg/min) selectively increased the permeability of BBB in the ischemic area without affecting the extent of ischemia - induced secondary edema. BK is a vasoactive compound that results in the tissue edema by dilating the capillary and increasing the vascular permeability. As of the low dose and the different infusion way, bradykinin infused by intracarotid artery only takes effect on a limited scope. So there are no evident side effects. Because of the desensitization function of B2 receptor, the activation of B2 receptors upon binding BK is rapid, transient and reversible. That is to say, BK open the BBB rapidly, also close it quickly. Therefore BK selectively increased the permeability of BBB without affecting the extent of edema.Cerebral ischemia is a common clinical symptom. Based on these experi-...
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