| Background and ObjectiveBrain edema is the accumulation of water in the brain parenchyma, which mainly consisted of cytotoxic and vasogenic brain edema. Brain edema may exist in many kinds of central nervous system diseases and be correlated with the processes and prognosis of diseases. The infectious brain edema in severe infection is a mixed type of brain edema, which is not only cytotoxic brain edema, but also vasogenic brain edema. CNS infectious diseases caused by G~- bacteria are common in infants, and LPS, which derived from G~- bacteria is fundamental in formation and development of IBE. A number of hypothesis of BE have been put forwarded, but its exact mechanisms are not yet determined so far.Aquaporins ( AQPs ), homologous to the major intrinsic protein super family of integrated membrane proteins, is a major channel for water flux though plasma membranes of many cell types. AQP9, a novel member of AQPs, has been reported to involve in edema caused by cerebral ischemia or hemorrhage, however, the role of AQP-9 in infectious brain edema has not yet explored.MMP-9, a very important member of MMPs, is associated with ECM degraded, and TIMP-1 is its tissue inhibitor. The balance of MMP-9 and TIMP-1 may play crucial roles in stabilizing the BBB. It has been conformed that MMP-9 and TIMP-1 were involved in brain edema produced by brain tumor, cerebral ischemia, hemorrhage and traumatic injuries, however, little is known about it in IBE. In our study, the infectious brain edema model of rats was established by inject lipopolysaccharide (LPS) to study the rules of infectious brain edema according to pathologic changes of brain in different time points, the changes of brain water content(BWC), Evans blue (EB) content. At the same time the dynamic changes of AQP-9, MMP-9 and TIMP-1 protein and their mRNA of two groups are also monitored with immunohistochemistry and RT-PCR technology respectively and the relationship of them were analyzed respectively to explore their functions in forming and developing infectious brain edema and provide a better therapentic strategy of brain edema.Materials and MethodsOne hundred and twenty eight normal healthy 1 -month-old Spragne-Dawley (SD) rats whose weight varied from 70g～100g, provided by the animal center of Zhengzhou university, with the gender ignored, were randomly divided into two groups: Control group (NS group, n=64), normal saline(1mg/kg) was injected via carotid for each rat; LPS group (LPS group, n=64), each rat was given LPS(1mg/kg) (from Escherichia coli 055:B5, production of Sigma company) by the same methods as NS group. Each group was subdivided into four groups: 6h, 12h, 24h and 48h with sixteen rats at any time points. Half of them in all subgroups weren't injected EB to be dyed by immunohistochemistry technology. The others were given 2% EB via right external jugular vein with 2ml/kg within 1—2 minutes to detect EB content. Rats were decapitated at different time points to take brain tissue samples. By measuring both wet and dry weight, the water content of brain tissue were recorded. Meanwhile the EB content of brain tissue is meteraged by the formamide methods. Immunohistochemistry technology was used to investigate the expression of AQP-9, MMP-9 and TIMP-1 protein, and RT-PCR technology were applied to study the expression level of AQP-9, MMP-9 and TIMP-1 mRNA in the brain tissue at different time point of two groups. Meanwhile the ratio of MMP-9/TIMP-1 protein and mRNA were analyzed respectively. Then all the data were analyzed with SPSS 13.0 software, the significant level was assigned atα=0.05 .Results 1 Histopathologic: In LPS group, the clearance around vessels became broader and inflammatory cells infiltrated out of vessels. Gliocyte became swelled and their cubage augmented than before. Vacuolar degeneration occurred and nuclear appeared apoptosis and necrosis among nerve cells.2 BWC and Brain EB content: In LPS group, 6h after LPS was given the BWC and Brain EB content started to rise, reaching peak at 12h, and still higher than that of control groups at 48h after LPS were injected. The levels of two indexes were all higher in LPS groups at various time points comparing with those in corresponding NS subgroup, the difference was significant (p<0.05).3 AQP-9 protein and mRNA: The expression levels of AQP-9 protein and mRNA in brain tissue were markedly increased 6h after LPS was given and reached the peak at 12h, and then decreased gradually. Compared with control group at any time point , the difference was significant (p<0.05).4 MMP-9 and TIMP-1 protein and mRNA: The expression level of MMP-9 and TIMP-1 protein and their mRNA in brain tissue were obviously up-regulated at 6h after LPS was injected and came their peak at 12h～24h point respectively, and descended little by little afterwards, the expression level of all the index were all higher than that of control groups (p<0.05).5 The ratio of MMP-9/TIMP-1 protein and their mRNA: the ratio of MMP-9/TIMP-1 protein and their mRNA in brain tissue were obviously up-regulated at 6h after LPS was injected and came their peak at 12h～24h point respectively, and descended gradually afterwards, the two ratios were both higher than that of control groups (p< 0.05).6 Relative correlation analysis: positive correlation were gotten between Brain water content and EB content, AQP-9 protein and BWC, AQP-9mRNA and EB content, AQP-9mRNA and BWC, AQP-9 protein and AQP-9mRNA, the ratio of MMP-9mRNA/TIMP-1mRNA and EB content, the ratio of MMP-9mRNA/TIMP-1mRNA and BWC, MMP-9mRNA and MMP-9 protein, TIMP-1 mRNA and TIMP-1 protein and the ratio of MMP-9 /TIMP-1 protein and BWC. and the rvalue was 0.455, 0.475, 0.363, 0.807, 0.530, 0.352, 0.396,0.617, 0.509 and 0.536 respectively (p<0.05).Conclusions1 Infectious brain edema animal model could be established steadily and reliably with injecting LPS via infant rats' carotid.2 Up-regulate expression of AQP-9 protein and mRNA in the IBE animal model of rats induced by LPS indicated that AQP-9 might play roles in the formation and development of IBE.3 MMP-9, TIMP-1 protein and their mRNA were all up-regulated in the IBE animal model of rats induced by LPS and the ratio of MMP-9/TIMP-1 protein and MMP-9/TIMP-1mRNA increased obviously, which indicated that MMP-9 and TIMP-1, especially the unbalance of the two index might be involved in forming and developing IBE.4 The positive correlation between BWC and EB content in the IBE animal model of rats induced by LPS indicated the damage of BBB might lead to the formation of IBE. The positive correlation among AQP-9, the ratio of MMP-9/TIMP-1, BWC and EB content indicated that they might aggravate IBE, probably by impairing BBB. |
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