Expression Of Matrix Metalloproteinases And Its Role In Blood-brian Barrier Disruption After Intracerebral Hemorrhage

BACKGROUND:Homeostasis of the central nervous system (CNS) microenvironment is essential for its normal function and is maintained by the blood-brain barrier (BBB). BBB is built up by endothelial cells lining the cerebral capillaries whereby the physical diffusion barrier is formed by tight junctions sealing the intercellular clefts. Tight junctions between endothelial cells of brain capillaries are the most important structural elements of BBB. The tight junctions is an intricate complex of transmembrane (claudins, occludin and junctional adhesion molecules) and cytoplasmic (zonula occludens) proteins. An elaborate network of complex tight junctions between the endothelial cells forms the structural basis of the BBB and restricts the paracellular diffusion of hydrophilic molecules. The tight junction proteins usually are strictly regulated under physical circumstance and are rapidly modulated by disease pathologies such as trauma, infection, ischemia, hyperpyrexia, tumor to produce changes in BBB permeability indicating it is implicated in pathophysiologic progress, leading to brain edema and brain damage.Intracerebral hemorrhage (ICH) results from rupture of a blood vessel in the brain. ICH accounts for10to15percent of all cases of stroke and is associated with high rates of death and disability. After ICH, the blood-brain barrier (BBB) is disrupted, resulting in uncontrolled fluid leakage from the blood to the brain and vasogenic brain edema. The underling cause of BBB disruption after ICH has not been really been examined. The disruption of tight juctions ultramicrostructure was observed after ICH. Because BBB tight junctions are composed by a series of tight junction proteins and play a crucial role in the regulation of BBB permeability under physiological conditions, abnormal alteration of tight junction proteins may take a key role in BBB breakdown and brain damage after ICH.Matrix metalloproteinases (MMPs) containing a disintegrin and metalloproteinase domains are important in neuroinflammation, and recent studies have linked their actions to degradation of tight junction proteins after ischemic stroke. Matrix metalloproteinases are a family of zinc-dependent endopeptidase enzymes which can degrade all components of the extracellular matrix and are implicated in diverse functions including the regulation of signaling, angiogenesis, inflammation, cell differentiation, migration, proliferation and survival, as well as developmental processes and wound healing. MMPs can cause BBB disruption and even hemorrhage by degrading endothelial basement membrane. There is evidence that MMPs are increased after ICH in man and in animal models, and a MMP inhibitor, tissue inhibitor of metalloproteinase-2(TIMP-2) can reduce BBB disruption in rats. A recent study have shown that activation of MMPs (especially MMP-2,9) opens the BBB by degrading tight junction proteins (claudin-5and occludin) and increases BBB permeability after ischemic stroke, and that an MMP inhibitor BB-1101prevents degradation of tight junction proteins and attenuates BBB disruption.Therefore, this study have speculated that after ICH the hematoma or its decomposition products may result in alteration of tight junction proteins expression by a variety of ways (especially MMPs pathway), thereby leading to BBB structural damage and brain edema.OBJECT:In this study, a rat ICH model was produced by intracerebral injection of autologous blood. The expression of MMP-2,9and related BBB tight junction proteins (claudin-5, of occludin and JAM-1) was detected at different time points to explore the relationship of the expression of tight junction proteins and BBB breakdown, and further analyze the expression of MMPs and it role in BBB damage after ICH.METHODS:1. Experimental groups and ICH model128adult male Sprague-Dawley rats, weighted250-300g, were randomly divided into8groups of ICH6h,12h,24h,48h,3d,7d,14d and control group with16in each group.75μl autologous whole blood was infused stereotaxically into the right caudate nucleus of rats to produce ICH model. Normal rats had been used for controls in the experiment.2. Evans blue permeabilityThe permeability of the BBB was investigated using Evans-Blue extravasation. Evans blue (EB,2%in saline,4ml/kg) was injected intravenously at either6h,12h,24h,48h,3d,7d,14d after blood infusion. After decapitation, the brains were weighted and placed in2ml50%trichloroacetic acid solution. Following homogenization and centrifugation, the extracted dye was diluted with ethanol (1:3), and its fluorescence was determined with Fluorescence spectrophotometer. The brain EB concentration was quantified from a linear standard curve derived from known amounts of EB and was expressed per gram of tissue (μg/g).3. Paraffin tissue embedding and slicingRat were sacrificed by an overdose of chloral hydrate and perfused transcardially with ice-cold saline and followed by4%paraformaldehyde. Their brains were then harvested and2mm coronal segments and were further fixed with4%paraformaldehyde for up2days at4℃. After that, according to the standard procedures, those segments were embedded in paraffin. The blocks of tissues were sliced into5-um sections with a paraffin histotome.4. HE stainingParaffin sections were de-waxed and rehydrated, and stained with hematoxylin for3min and2%eosin for2min. Images were captured with a microscope and the brain damages were evaluated.5. ImmunohistochemistryChanges in MMP-2and MMP-9expression were detected by immunohistochemistry after ICH. Antigen retrieval of paraffin sections was completed using microwave with antigen retrieval solution of Tris-EDTA buffer (0.05M Tris-base,0.001M of EDTA with PH8.0). All sections were heated in a microwave to boil for two times with an interval of15min. At last, after DAB coloration, MMP-2and MMP-9expression was analyzed under a microscope.6. ImmunofluorescenceThe distribution and expression changes of tight junction proteins of claudin-5, occludin, JAM-1were analyzed with immunofluorescence. With mouse anti-claudin-5and rabbit anti-GFAP, double label experiment was carried out as well as single label experiments with rabbit anti-occludin or rabbit anti-JAM-1. With laser scanning confocal microscope under high power field, images were captured and the tight junction protein expression in the small blood vessels was analyzed.7. Quantitative real-time polymerase chain reactionAccording to the manufacturer instructions, purification of total RNA from brain tissue was accomplish with the GeneJETM RNA Purification Kit and the cDNA synthesis was carried out using the Maxima(?) First Strand cDNA Synthesis Kit. Quantitative real-time PCR reactions of tight junction proteins (claudin-5, occludin, JAM-1) mRNA were carried out with an ABI PRISM7500Sequence Detection System using SYBR Green to monitor in real-time the amplification of the target gene. Relative target gene mRNA concentration was detected based on the standard curve that was generated from serial dilutions of standards of PCR amplification products of target genes. The cDNA for the housekeeping gene β-actin was amplified from all samples to normalize expression levels of targets between different samples.8. Statistical analysisSPSS13.0software was used for statistical analysis. All data in this study are presented as mean±SE. Groups of data were compared with ANOVA followed by Dunnett T3or LSD test. Value of P≤0.05were regarded as significant.RESULTS:1. BBB permeability alterations were analyzed by Evans blue test. It shows that the Evans blue contents at the time points of24h,48h,3d and7d are higher than that of control group. The differences are of statistic significance (P<0.01), and the most evident increase takes place at ICH48h, where the Evans blue of the ICH rats is2.23times as that of controls. At other time points, the quantities of Evans blue show no statistic difference compared with that of the control group.2. Histopathologic changes were observed by HE staining. An evident stripe of edema and the loosed extracellular matrix around hematoma was observed at24h following ICH. At48h following ICH the hematoma edema becomes heavier and more extensive, and the number of normal neurons is reduced. A stripe of edema is clearly seen around the hematoma at day3following ICH, distinctly characterized by severe inflammatory reactions, in which a great deal of inflammatory cells effuse, forming cuff-like structures outside the blood vessels. At day7after ICH the hematoma is partly absorbed, and the edema stripe around it becomes less distinct. Until day14the edema is not distinct; although the hematoma is not totally absorbed, and where it is, there is a deposit of hemosiderins.3. After ICH, increased expression of MMP-2and MMP-9was seen in the perihematomal brain tissue by immunohistochemistry. MMP-2distributed at a variety types of cells including glial cells especially endothelial cell after ICH. At48h and3d following ICH significantly increased number of MMP-2positive cells was observed around blood vessels, and endothelial cells also expressed different levels of MMP-2. Accordingly MMP-9expression also significantly increased after ICH. However within24h following ICH a few cells showing positive expression of MMP-9was detected in brain tissue around hematoma, from48h to7d MMP-9expression was significantly increased and MMP-9mainly distributed in the white blood cells and endothelial cells.4. The tight junction protein claudin-5expression was decreased after ICH. By confocal microscope, claudin-5was intensively expressed in a linear fashion at the cell bounds of capillary endothelial cells in the normal brain tissues. Compared to the control groups, the claudin-5expressions was decreased at6h following ICH, showing discontinue and dispersive distribution along capillary endothelial cells. At48h and3d after ICH the perihematomal claudin-5expressions was decreased to the lowest level, then restored slightly at7d, and recovered completely at14d. Accordingly real-time quantitative PCR results showed that claudin-5mRNA expression quickly reduced to the lowest level at6h after ICH and maintained this low lever for several days. From7d claudin-5mRNA expression gradually restored, but until the14d was still below normal levels. The difference was statistically significant (P<0.01). 5. The expression of occludin was decreased after ICH. As like claudin-5, occludin localized at the normal capillary endothelial cells in a continued linear pattern. Compared with control groups, decreased occludin expression with discontinued linear formation around the hematoma occurred from6h following ICH and was decreased to the lowest level at48h and3d. This lower occludin expression was restored slightly at7d, and recovered completely by14d. The real-time quantitative PCR results showed that compared with control group the expression of occludin mRNA decreased from6h to3d following ICH, the difference was statistically significant (P<0.01).6. After ICH the expression of tight junction protein JAM-1firstly significantly reduced and rapid recovered for several days. Confocal microscopy analysis indicated that JAM-1, similar to claudin-5and occludin, intensively distributed along capillary endothelial cells. Compared with control group, JAM-1expression began to decrease with discontinuous or weak distribution in endothelial cells from12h after ICH. At24h and48h lower decreased JAM-1expression was observed in cerebral edema region around the hematoma. Unlike claudin-5and occludin, JAM-1expression on blood vessels returned to normal at3d following ICH and was more intense than that of other time points, and even presented in a number of inflammatory cells. At7d and14d a large number of inflammatory cells expressing JAM-1around the hematoma were detected. Accordingly real-time quantitative PCR results showed that compared with the control group, JAM-1mRNA expression significantly decreased from12h to48h following ICH (P<0.05), and significantly increased at7d (P <0.05). The difference was not statistically significant at other time points.CONCLUSION:1. ICH leads to BBB disruption and secondary brain edema. Decreased expression of transmembrane tight junction proteins (claudin-5, occludin and JAM-1) and increased expression of MMP-2and MMP-9are closely related to the BBB disruption.2. Among these tight junction proteins, claudin-5play a critical role in maintain BBB permeability, whereas occludin may play a regulatory role and JAM-1facilitates adhesion and exudation of inflammatory cells which express excessive MMPs and further aggravate BBB disruption and brain edema after ICH.3. MMPs-mediated change of expression of endothelial cell tight junction proteins may be an important molecular mechanism of BBB disruption and secondary brain edema after ICH.