| Background and ObjectiveCerebral infarction, characterized by high morbidity, mortality, disability and recurrence rate, as a common disease, has seriously threatened the health of human being. Because ischemic cerebral diseases have brought heavy economical and spiritual burden to their families as well as the whole society, it is important and urgent to find effective treatment now. Thrombolytic therapy is proved to be an effective method for some hyperacute cerebral infarction patients, because it can restore cerebral blood flow and mitigate ischemic penumbra damage in time. However it also brings serious reperfusion injuries. Nowadays the role of the blood-brain barrier (BBB) in the pathogenesis of cerebral ischemia reperfusion injury becomes more and more prominent.BBB locates between brain tissues and their blood capillaries. It is consisted of capillary endothelial cells, their tight junction proteins which bond the endothelial cells together, multilayered basal lamina and the numerous astrocyte expansions end feet. In addition the vascular basal lamina is composed of extracellular matrix proteins (ECM), such as type IV collagen, laminin, fibronectin, and heparan sulfate, which plays an important role in maintaining the integrity of BBB structure. However ECM is the main substrate of matrix metalloproteinases (MMPs). Recent studies have shown the increased expression of MMPs after cerebral ischemia reperfusion, particularly matrix metalloproteinase-9 (MMP-9), affect the permeability of blood brain barrier greatly. MMP-9 gene knockout of mice are associated with a reduced infarct volume after cerebral ischemia and reperfusion. MMP-9 not only degrades the extracellular matrix protein molecules, which results in a breakdown in the blood-brain barrier and causes vasogenic edema formation or hemorrhagic transformation, but also promotes neutrophil infiltration causing local inflammation response and aggravating tissue damage. So it is very important to explore proper neuroprotective agents targeted at MMP-9.Urinary trypsin inhibitor (Ulinastatin, UTI), extracted and purified from human urine, can inhibit some serine proteases such as trypsin, chymotrypsin, plasmin, human leukocyte elastase, and hyaluronidase. Many studies have confirmed UTI can antagonize ischemia-reperfusion injury in many other organs such as liver, kidney, heart, intestine, lung, etc. But few studies have focused on brain, especially the roles of UTI in inflammatory and scavenging the damage of free radicals are still unclear, in addition it is still unclear whether UTI can protect the BBB by influencing the activity of MMP-9 after cerebral ischemia-reperfusion. We established reversible middle cerebral artery occlusion (MCAO) with reperfusion rat model to explore effect of UTI on the permeability of the blood-brain barrier and the activity of MMP-9.Materials and Methods72 clean grade healthy Sprague Dawley rats, weighting 250-300g, from the Experimental Animal Center of Zhengzhou University, were randomly divided into three groups:sham-operated group (sham, n=24), middle cerebral artery occlusion and reperfusion group (MCAO/R, n=24), UTI-treated group (UTI, n=24). Each group was further separated into 6h,24h 48h,72h, four time points, and each time point had 6 rats. The model of focal cerebral ischemia-reperfusion injury was made by occluding middle cerebral artery and reperfusing (MCAO/R) referenced from Zea Longa's with improvement. The UTI-treated group received an initial intraperitoneal injection of UTI (10000U/kg) within reperfusion five minutes and later once a day. The other two groups were delivered equivalent normal saline (NS,1 ml/kg) by the same way. Evans blue (2%,2ml/kg weight) was injected through the sublingual intravenous 30min before they were sacrificed. The BBB permeability damage was measured by the different degree of Evans Blue (EB), and the MMP-9 activity was evaluated by gelatin zymography during a time course ranging from 6 hours to 72 hours (n=6) of reperfusion following two hours of cerebral ischemia. All dates were expressed as the mean±standard deviation. Statistical analyses were handled with SPSS 17.0 software, and One-way ANOVA and Bonferroni method were taken for data processing. Significance level was judged by a=0.05.Results1 the extravasations of EB:Rats turned into blue thoroughly shortly after Evans blue injection. Damage to the BBB permeability was judged by extravasations of Evans Blue (EB). EB extravasations weren't apparent in the infarction tissue at 6h, but reached maximum levels at 24h, and afterwards began to decrease at 48h and 72h. The EB extravasations in the UTI-treated group decreased significantly compared with the ischemia/reperfusion (I/R) group (p<0.05).2 the expression of MMP-9:The levels of MMP-9 were low at all the time points in sham-operated group. At 6h, a dramatic increase in MMP-9 activity was detected but there was no significant difference between sham and I/R groups. The activity of MMP-9 significantly elevated at 24h (p<0.05), and reached the peak at 48h (p<0.05) in I/R group. The MMP-9 activity at 72h (p<0.05) began decrease, but was still higher than sham-operated group. The activity of MMP-9 in the UTI-treated 6h,24h 48h, were significantly reduced compared with the I/R group (p<0.05).Conclusions1. There were both EB extravasations and the MMP-9 activity increase in ischemia-reperfusion rats, and both had dynamic changes. These illustrate that BBB and MMP-9 relates to brain ischemia reperfusion injury.2. UTI can reduce the permeability of the blood-brain barrier by suppressing the activity of MMP-9, which may be one of neuroprotective mechanisms in cerebral ischemia/reperfusion injury. |
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