Roles Of Hemoglobin In Rho Kinase And Matrix Metallopeptidase-9in Endothelial Cells Of Blood-brain Barrier

BACKGROUND:Intracerebral hemorrhage (ICH) is the most devastating subtype of stroke. It is associated with high morbidity, disability and mortality. It accounts for8-30%of all strokes worldwide,30-day mortality is30-55%, and only20%of patients remain independent at6months. Perihematomal cerebral edema is one of the major causes of death in the first few days after ICH. The degree of cerebral edema is associated with early neurological deterioration and poor outcome. Vasogenic edema is the predominant type of cerebral edema after ICH.Vasogenic edema primarily arises from disruption of the blood-brain barrier (BBB) after ICH. The BBB is an important physiologic barrier that maintains the homeostasis of the central nervous system (CNS). The BBB is composed of: endothelial cells (ECs), astrocyte end-feet,extracellular matrix, and perycites.The tight junction is believed to be the key structure of BBB. Tight junction is a complexus that located at the border of two adjacent endothelial cells. Endothelial TJs consist of cytoplasmic proteins known as zonula occludens (ZO) as well as transmembrane proteins such as occludin, claudins and junction adhesion molecules (JAMs). Claudin-5is considered to be the most important endothelial TJ protein. It constitutes the "backbone" of TJ strands and determines the specificity and selectivity of paracellular barriers. Once the integrity of TJs is weakened, the permeability of the BBB is increased. Hemoglobin (Hb) is an essential degradation product of erythrocytes,it plays a crucial part in disruption of the BBB as well as formation of cerebral edema after ICH.Hb is the major component of hematoma, it is composed of globin and heme. Hb are observed in the perihematomal zone as early as24h after ICH,and it is associated with increase of BBB permeability and formation of cerebral edema.However,the precise mechanism of Hb-induced BBB disruption is not known.Rho also know as Rho GTPases, it is a congener of Ras and have GTP enzymatic activity.The family of Rho GTPases include three subtypes, that is:Rho A, RhoB and RhoC. Rho kinase (Rho-associated coiled-coil containing protein kinase, ROCK) is a serine-threonine kinase. It has been identified as the most important downstream effector of the small guanosine triphosphate (GTP)-binding protein Rho. Rho kinase have two subtypes:ROCK1and ROCK2. But only RhoA and ROCK2are expressed primarily in the brain. Myosin light chain (MLC) is a crucial substrates of Rho kinase. MLC can be phosphorylated by Rho kinase, thus, become phosphorylated myosin light chain (p-MLC). Accumulation of p-MLC facilitate formation of stress fibers and enhancement of actin-myosin contractility, which in turn lead to an increase of inter-endothelial gaps, disruption of tight junction and finally increase the permeability of BBB.Matrix metallopeptidases (MMPs) is a important "cleaver" of the extracellular matrix and it has multiple biological functions. There are more than20subtypes of MMPs in vovl, but only MMP-9is closely related to cerebrovascular diseases. The expression of MMP-9can be up-regulated by various adverse factors such as thrombin, hemoglobin,cytokines, oxidative stress and anoxia. Up-regulatioin of MMP-9expression can lead to an imbalance of metabolization of extracellular matrix, which degraded the basement membrane and tight junction in capillary, finally caused increase in BBB permeability and formation of cerebral edema. OBJECT:This study infused the hemoglobin with stereotactic guidance into the right caudate nucleus of SD rats. Then we evaluated the alterations in the BBB permeability and water content, changes of TJ protein claudin-5, expression of RhoA, ROCK2, MMP-9and the effect on ROCK activity in perihematomal tissue as well as the changed behavior after hemoglobin injection. Discussed the effect of hemoglobin on ROCK and MMP-9, the mechanism among the course was also further examined.METHODS:1. Experimental groups and animal modelMale normal Sprague Dawley rats(weighing280±20g) were randomly divided into three groups:Hb-injected group, saline-injected group and normal control group. Hb group and sham-operated group were divided into6subgroups (6h,12h,24h,15rats for each subgroup). As a control experiment, same volume of saline was injected in saline-injected rats in the same way.20μl hemoglobin at a concentration of150mg/ml was injected into the right caudate nucleus after inserted stereotactically.5rats at6h in Hb group was selected randomly to perform HE staining after evaluation of behavior, the observed the location of hematoma and estimated the reliability of this animal model.2. Assess of blood-brain barrier permeabilityAfter anesthesia,2%EB-dye solution was injected (4mL/kg, i.v.) via femoral vein before2hours of killing. normal saline transcardially perfused until the drainage was colorless. Removed the ipsilateral hemisphere and weighted carefully. Then incubated the samples in trichloroacetic acid solution. After homogenization and centrifugation (15,000rpm×20minutes), diluted the supernatants with ethanol (1:3), and determined its fluorescence intensity (excitation at620nm and emission at680nm) using an automatic microplate reader. The EB leakage was represented as μg/g brain weight.3. Alterations in water content in the brain Water content in the brain was determined as described previously. Animals were decapitated6,12, a sharp blade. Then, the ipsilateral cortex was dissected to obtain a sample of basal ganglia. Each sample was weighed immediately after removal (wet weight) and after drying at100°for24h (dry weight) using an electronic analytical balance. The percentage of water content in the brain was calculated using the following equation:Water content in the brain=[(wet weight-dry weight)/wet weight]×100%4. Real-time quantitative polymerase chain reaction (qPCR)After anesthetized and decapitated, tissues (about30mg) around the lesion sites in brain were obtained and total RNA was extracted from the tissue with GeneJETTM RNA Purification Kit, cDNA was synthesized using a Maxima(?) First Strand cDNA Synthesis Kit. And PCR reactions were run using Power SYBR(?) Green PCR Master Mix. PCR incubation and cycling parameters:were95℃for10minutes, then at95℃for15seconds and an annealing cycle of60℃for1minutes, repeated40times. The standard curve method was used to calculate the expression levels of target genes RhoA、ROCK2、MMP-9.5. Western blotting and assay to measure ROCK activityAfter anesthetized and decapitated, tissues (about30mg) around the lesion sites in brain were obtained and tissues were homogenized in liquid nitrogen and diluted in RIP A Lysis Buffer (Beyotime Institute of Biotechnology, Jinan, China). The protein concentration of the supernatant was measured using a Bicinchoninic Acid Protein Assay kit (Thermo Scientific) after centrifugation at12,000rpm for10min at5℃. Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies:RhoA, ROCK2,claudin-5, p-MLC,MLC,p-MYPT1,MYPT1and MMP-9. Secondary antibodies were used to detect the primary antibodies. GAPDH was employed as an internal control to determine and normalize the relative intensity of each band. Secondary antibodies (goat anti-mouse IgG, horseradish peroxidase (HRP) conjugate;1:1000; Sigma-Aldrich; goat anti-rabbit IgG, HRP conjugate,1:1000; Sigma-Aldrich) were used to detect the primary antibodies. GAPDH was employed as an internal control to determine and normalize the relative intensity of each band.6. Preparation of paraffin sections and HE stainingAfter anaesthetized, rats were perfused transcardially with saline followed by4%paraformaldehyde solution. Brain tissues were then removed and fixed by immersion in the same solution for24hours. After dehydrated and vitrified, they were embedded in paraffin, and5-um sections were prepared. Sections were de-waxed in xylene and rehydrated in graded ethanol and deionized water and then stained by hematoxylin for2.5min and eosin for2.5min. Images were captured with a microscope.7. Immunohistochemical analysesSections were de-waxed in xylene and graded ethanol, and rehydrated in deionized water. Antigen retrieval was conducted in citrate buffer solution (0.01mol/L, pH6.0) by heat treatment in a microwave oven for30min followed by incubation in3%H2O2, normal5%goat serum, then incubated with primary antibodies, biotinylated goat anti-rabbit IgG secondary antibody and HRP streptavidin reagent. Finally,3,3’-diaminobenzidine was used to detect immunoreactivity, followed by staining with hematoxylin. Images were obtained using microscope.8. ImmunofluorescenceAfter being de-waxed and rehydrated using deionized water, antigen retrieval was carried out suing Tris-EDTA buffer followed by incubation with5%donkey serum, primary antibodies at4℃overnight. After washing with PBS, incubation with secondary antibody at37℃for1h was carried out. Immunostained images were acquired with a fluorescence microscope and analysised.9. Statistical analysesStatistical analyses were undertaken using SPSS ver19.0(SPSS, Chicago, IL, USA). Data are the mean±SD. Differences among groups were assessed by one-way ANOVA with the least square difference test. Pearson correlated analyses were undertaken to examine the correlation between ROCK activity and MMP-9expression. p<0.05was considered significant.RESULTS:1. HE staining assay to assess the location of Hb injectionAll the5rats that randomly selected at6h in Hb group were observed a fall down to the paretic side or circling toward the contralateral side, this result indicated a significant neurological function deficiency. HE staining showed that the Hb injection site was in the area of right caudate nucleus. Therefore, our establishment of animal model was successful and effective.2. Increase in permeability of the BBBEBD leakage in brain tissues was used to assess alterations in BBB permeability. Compared with normal and saline groups, BBB permeability was significantly increased from6h to72h in the Hb group (p<0.05). EBD content in the saline group remained relatively constant. Compared with the other groups, the maximum permeability of the BBB was at12h in the Hb group (p<0.05).3. Alterations in water content in the brainTo evaluate alterations in cerebral edema, water content in the brain was measured. Unlike EBD leakage, water content in the brain in the Hb group began to increase significantly at12h, and increased persistently until24h. There was a significant difference between the Hb group and other groups at24h (p<0.05).4. Enhanced expression of RhoA, ROCK2and reduction of the level of claudin-5Claudin-5expression decreased gradually to a minimal value at12h (the time of most severe disruption of the BBB) and began to recover by24h in the Hb group. The intensity of the stain of claudin-5was weaker compared with that seen in the saline group. Unlike claudin-5, compared with the other groups, a significant increase in the expression of RhoA and ROCK2mRNA emerged at12h in the Hb group (p<0.05). The trends in change of protein expression of RhoA and ROCK2were in accordance with their mRNA level at all time-points. Similar changes were also detected by immunohistochemical analyses. Compared with Hb group, there was a stronger expression of p-MLC in the endothelia of microvessels after Hb injection.5. Increase in MMP-9expressionContrary to the weak expression in the saline group, the expression of MMP-9protein increased to a peak value at12h, then began to decrease24h after Hb injection similar trend was observed for MMP-9mRNA levels. Compared with the saline group, stronger detection of MMP-9was observed by immunohistochemistry in the Hb group.6. Increased ROCK activityWestern blotting showed that Hb administration led to activation of ROCK with up-regulated expression of p-MLC and p-MYPTl at12h (p<0.05). Similar to alterations of ROCK2expression and BBB permeability, the maximum activity of ROCK were at12h in the Hb group. Pearson correlated analyses indicated that ROCK activity (p-MYPT1/MYPT1and p-MLC/MLC) had a positive correlation with the protein expression of MMP-9(r=0.729, p=0.007and r=0.640, p=0.025, respectively).7. Immunohistochemical analysis of RhoA, RoCK2and p-MLCDAB staining showed a feeble expression of RhoA, RoCK2and p-MLC in saline group. But a significant increase in the staining of RhoA, ROCK2and p-MLC emerged in the Hb group with a peak value at12h. RhoA, ROCK2were increased in the Hb group, that they were detected mainly in glial cells and endothelial cells after Hb injection, but that stronger p-MLC was expressed primarily by endothelial cells.8. Immunohistochemical analysis of MMP-9DAB staining showed a weak detection of MMP-9in saline group.A increased immunoreactivity of MMP-9was detected mainly in glial cells, endothelial cells and extracellular matrix after Hb injection,9. Immunofluorescence assay of claudin-5and p-MLCThe density of claudin-5was strong at interendothelial borders, but there was a weak staining of p-MLC on the wall of capillaries in saline group. However, compared with saline group, significantly reduced immunoreactivity of claudin-5and enhanced p-MLC staining were detected at12h in Hb group (p<0.05).10. Alterations in behavior of rats after Hb injectionNSS scores suggested that comparing with saline group, the neurological function deficiency of rats was significantly increased at12h to7d after Hb injection (p<0.05). Compared with other time points, significantly increased NSS scores reached peak value at24h in Hb group (p<0.05). Compared with saline group and normal group, the neurological function deficiency of rats returned to a normal level at lOd after Hb injection (p>0.05)CONCLUSION:1. Hb induced alteration in expression and distribution of endothelial tight junction claudin-5can caused disruption of BBB and formation of cerebral edema.2. Hb induced activation of ROCK can caused the phosphorylation of MLC on the wall of capillary, which demage the tight junction to caused disruption of BBB and formation of cerebral edema.3. Hb can degrade tight junction by up-regulation of the expression of MMP-9, therefore, damage the structure of BBB.