| BACKGROUND:Intracerebral hemorrhage (ICH) is a cerebrovascular disease with high morbidity, mortality and disability. The morbidity of it accounts for 10～15% of strokes all over the world, but it’s the severest subtype. The 30 day mortality of ICH has been reported to be 43～51% and the disability rate was 80%～95%. Only a minority of patients could return to daily life. The occurrence and development of perihematomal edema is the main cause of neurologic impairment, death and disability after ICH and the degree of brain edema are closely related with prognosis. Brain edema after ICH is mainly composed of vasogenic cerebral edema and cytotoxic cerebral edema.The BBB is an important structure to maintain the homeostasis of the central nervous system and mainly composed of four cellular elements:endothelial cells (ECs), astrocyte end-feet, extracellular matrix, and perycites. Tight junctions (TJs) between the endothelial cells are key to maintaining the BBB structure integrity and normal function. TJ protein molecules mainly include:transmembrane protein (occludins, claudins, junction associated molecules (JAM) and cytosolic protein zonulaoccludens (ZO)). Abnormalities of TJ proteins can directly cause the increase of BBB permeability, thereby leading to the occurrence of cerebral edema.Erythrocytes are the main components of intracranial hematoma after ICH. The lysis product (Hemoglobin, Hb) mainly composed of globin and heme is a potent mediator of oxidative stress after ICH. It is confirmed that, in the early stage of ICH, Hb was released into the surrounding brain parenchyma which is closely related to the increase of BBB permeability and the occurrence of brain edema. However, the specific mechanism that Hb mediated BBB damage and brain edema formation remains unclear.Nitric oxide (NO), a dual bioregulatory molecular, is continuously synthesized by nitric oxide synthases (including neuronal NOS (nNOS), inducible NOS (iNOS), endothelial NOS (eNOS)) with L-arginine as substrate. Under pathological conditions, such as intracranial infection and subarachnoid hemorrhage, cerebral ischemia and so on, large amounts of NO may be involved in the disruption of BBB. More importantly, peroxynitrite (ONOO-), formed by NO and the superoxide anion (O2-), is a highly reactive or metabolite. In a variety of central nervous system diseases, such as multiple sclerosis, acute bacteial meningitis, traumatic brain injury, cerebral cortical cold injury, brain embolism and cerebral ischemia and reperfusion, abundant ONOO- formation could mediate the BBB disruption directly. What’s more, large amount of evidences demonstrated that the cytotoxicity of NO mainly depends on the ONOO- generation. By nitration of tyrosine residue or S-nitrosation, ONOO" reacts and oxidizes many cellular components such as lipids, proteins, nucleic acids and triggers the activation of poly(ADP-ribose) polymerase, thereby mediating endothelial cell damage, BBB disregulation, brain edema formation and neuronal necrosis and apoptosis.Matrix metalloproteinases (MMPs) comprise a large family of zinc-endopeptidase, have multiple biological activities. There are more than 24 isoforms of mammalian MMPs, of which MMP-9 is closely related to the neurological defects and brain edema formation after ICH. Hb, thrombin and oxidative stress as well as other factors all can activate MMP-9. Activated MMP-9 could degrade vascular basement membrane and endothelial cell tight junction protein, thereby leading to the increase of BBB permeability and brain edema formation.Currently, several metalloporphyrins reported react catalytically to decompose ONOO- and attenuate the toxic effects of ONOO" in vivo and in vitro. Due to their high values, ONOO" decomposition catalysts have been widely used in the field of heart, lung, liver, kidney, intestinal, and other splanchnic artery occlusion and reperfusion injury. FeTPPS, the typical member of metalloporphyrins, can isomerize ONOO- to the harmless nitrate anion, thereby reducing the adverse effects of ONOO-.OBJECT:By establishing Hb-simulated ICH model in male Sprague Dawley rats:1. we investigated the effect of Hb on the expression of three kind of NOS and formation of ONOO-, changes of TJ proteins (claudin-5 and ZO-1), brain edema content and behavior. The role and cell location of NOS and ONOO- in this process were also further investigated; 2. To reduce the cytotoxicity of ONOO-, we examined the effect of FeTPPS, a ONOO" scavenger, on the expression of ZO-1, the activity of MMP-9 and the changes of neurological function, providing a new theraputic strategy for ICH.METHODS:1. Experimental groups and surgical procedureExperiment oneMale Sprague Dawley rats, SPF grade, weighing approximately 280-300g, were randomly divided into three groups:normal group, sham-operated group and Hb-injected group. According to the time points after operation, sham-operated group and Hb group were divided into 6 subgroups (6h,12h,24h,48h,3d and 7d).Experiment twoRats with the same weight and source were randomly divided into sham operated group, control group (Hb+ saline group) and experiment group (Hb+FeTPPS treatment group). After anesthetized and positioned,20ul hemoglobin at a concentration of 150 mg/ml was injected stereotactically into the right caudate nucleus. Rats, in the sham group, were subjected to only a needle insertion in the same way.2. Measurements of brain water contentBrain water content was measured at diverse time points after Hb injection. By anesthesia and decapitation, the brains were removed immediately and divided into two hemispheres along the midline, and the cerebella and brain stem were removed. Ipsilateral and contralateral hemispheres were placed on a pre-weighed piece of aluminum foil to give the wet weight and then dried in an electric oven at 100℃ for 24 h. The brain water percentage was calculated as follows:(wet weight-dry weight)/(wet weight).3. Behavioral testingBehavioral testing was conducted before Hb injection and at 6 h,12 h,1 day,2 day,3 day,5 day,7 day and 14 day after Hb injection using modified Neurological Severity Score (mNSS) tests, which were monitored by blinded investigators for group allocation. The mNSS test includes motor, sensory, reflex and balance tests and is graded on a scale of 0-18, where 13-18 points indicates severe injury,7-12 indicates mean-moderate injury, and 1-6 indicates mild injury.4. Western blot analysisTotal protein was isolated from ipsilateral lesional brain tissues using ice-cold RIPA buffer. Protein concentrations were measured with the BCA Protein Assay Kit (Forevergen Biosciences). Protein amples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. Proteins were detected by incubation with primary antibodies (nNOS, iNOS, eNOS,3-nitrotyrosine, claudin-5, ZO-1) followed by secondary antibodies (goat anti-mouse IgG; goat anti-rabbit IgG; rabbit anti-mouse IgG). Immunoblots were visualized using a ECL Plus Western Blotting Detection Reagents. GAPDH was employed as the loading control. Densitometry analysis was performed with the use of the ImageJ software with normalization to GAPDH.5. Enzyme-linked immunosorbent assay (ELISA) for 3-NT in blood serum and brain tissuesBlood was collected by cardiac puncture and kept static for 2 hours at room temperature. Following centrifugation (4,000 rpm for 10 min), the supernatant serum was collected immediately and stored at -80℃ until use. Simultaneouly, brain tissues around the lesional sites were obtained. By homogenization anud centrifugation (3,000 rpm for 30 min), the supernatants were extracted and then stored at the same condition.3-NT concentrations, both in blood serum and brain tissues, were assayed using commercial ELISA kits and following the manufacturer’s instructions. The intensities of the color of samples were measured at 450 nm using an automatic microplate reader.6. Preparation of paraffin sectionsAfter anaesthetized, rats were perfused transcardially with saline followed by 4% paraformaldehyde solution. Brain tissues were then removed and fixed by immersion in the same solution for 24 hours. After dehydrated and vitrified, they were embedded in paraffin, and 3-um sections were prepared for immunohistochemistry and immunofluorescence.7. Immunohistochemistry (IHC)After dewaxed and rehydrated, slides were boiled in a microwave oven (antigen retrieval) for 30 minutes in 0.01mol/L citrate buffer solution (PH 6.0). Sections were incubated in 0.3% H2O2, goat serum, primary antibody, biotinylated secondary antibody and HRP-Streptavidin reagent. At last, immunoreactivity was detected using DAB. Images were obtained with the use of Leica Application Suite.8. Immunofluorescence (IF)Sections were de-waxed and rehydrated and then performed by heat treatment in a microwave oven in Tris-EDTA buffer solution (PH 8.5) for antigen retrieval. After incubated by serum, sections were incubated overnight at 4℃ with primary antibody. After washed with PBS, sections were then incubated with the second antibody for 1 hours at 37℃. For double staining experiments, primary antibodies were mixed and incubated overnight at 4℃. Images were obtained with confocal fluorescence microscope.9. NO concentration in serumBlood serum was collected as above-mentioned (5). For the examination of the concentration of NO in serum, nitrate reductase method was adopted. NO concentration was represented as umol/L.10. In situ zymography and double labelling with fluorescent probesIn situ gelatinolytic activity was detected on frozen brain sections with a thickness of 10 um using a commercial kit and following the manufacturer’s instructions. Fresh sections were incubated with gelatin conjugate, a fluorogenic substrate, at 4℃ for 10min and then 37℃ for 1 h. Cleavage of gelatin by MMPs results in a green fluorescent product (excitation,480 nm; emission,530 nm). After the gelatinolytic activity was assessed, tissue sections were rinsed with PBS and incubated overnight at 4℃ with primary antibodies specific for neurons (NeuN), astrocytes (GFAP), blood vessel basal lamina (fibronectin); neutrophils (MPO), macrophages/microglia (ED-1). After washed with PBS, the sections were incubated for 1 hours at room temperature with the corresponding second antibodies (Alexa Fluor 594 donkey anti-mouse IgG; Alexa Fluor 568 donkey anti-rabbit IgG). Sections were examined with a confocal microscope. Gelatinolytic activity-positive cells and blood vessels were counted in four fields that were immediately adjacent to the hematoma and were expressed as number/mm2 areas. The number of gelatinolytic-positive blood vessels and cells were quantified.11. Statistical analysisAll data in the text were presented as mean±SD. Statistic analysis was performed using SPSS 19.0 statistical programs. Comparison between groups was determined by Student’s T test or one-way analysis of variance (ANOVA) and followed by LSD test for the two groups’ vcomparison within the multiple groups. Correlation analysis between 3-NT formation and brain edema content or neurological deficts were performed by Spearman test. Differences were considered to be statistically significant at probability values less than 0.05.RESULTS:Experiment one1. Alterations in brain edema contentIn the ipsilateral hemispheres of Hb groups, brain water content increased progressively from 6 h and showed significant differences compared with the contralateral or normal groups at all presetted time points (P< 0.05). The ipsilateral edema reached the maximum at 24 h (P< 0.001) and maintained at a high level till 3 days P<0.001) and then gradually decreased.2. Changes in neurological deficits evaluated by behavioral testingBehavioral testing is of vital importance to evaluate the success of the ICH model and monitor the severity of neurological deficits. Compared with the sham-operated group, rats that received an intracaudate injection of Hb exhibited significantly functional deficits over the first 7 days measured by mNSS tests (P< 0.05). Hb-induced neurological deficits were most severe from day 1 to day 3 (P< 0.001). By 14 days or thereafter, neurological tests of the rats in both groups did not show a significant difference (P>0.05).3. The expression and cell location of nNOS, iNOS and eNOSBy westernblot analysis, nNOS protein was detectable after Hb injection but showed high variability, whereas a significant expression was observed at 3 days, which was threefold higher than sham groups (P<0.001). The iNOS protein was damatically increased after the injection of Hb and reached the peak at 6 h and remained at a high levels till 24 h (P< 0.001), and then diminished with varying degrees. In contrast to nNOS and iNOS, eNOS protein expresson also had a markedly increase at initial time points (from 6 h to 24 h) and reach the peak at 12 h (P< 0.001) and then decreased with diverse degrees.4. Changes in the expression and distribution of claudin-5/ZO-1To determine the effect of Hb on cerebral TJs, we examined the expression of claudin-5 by Western blot. In Hb-injected rats, claudin-5 expression was significantly reduced at 12 h (P<0.05),3 days (P<0.001) and 7 days (P<0.001) compared with nontreated sham controls. In the remaining time points, no significant difference in claudin-5 was detected between both groups. To further examine the distribution of TJ proteins, claudin-5 and ZO-1 were analyzed by fluorescence microscopy. Blood vessels from nontreated sham controls showed intense and continuous reactivity for claudin-5 and ZO-1. Conversely, in Hb-injected animals, claudin-5 and ZO-1 immunoreactivity was comparatively lower and appeared more diffuse and discontinuous.5. Content of NO in SerumWe here examined the production of NO by measurement of nitrite/nitrate in serum. In Hb-injected rats, NO production in serum began to decrease by 6 h, minimal at 12 h, and with a moderate rise at 2 days but still lower than the normal controls. At the 3 and 7 days, the NO decreased again and then recover to the normal levels till 14 days or longer. It showed significant reduction at 12 h (P<0.05),24 h (P<0.05),3 days (P<0.05),7days (P<0.05) and 14 days (P<0.05) compared to normal controls.6. ONOO" formation measured by Westernblot and detected by IHC as well as the cell locationDue to the instability of peroxynitrite, its detection through 3-NT formation is a hallmark of the levels of ONOO-. The expression of 3-NT in the ipsilateral injured hemisphere at indicated times were detected by westernblot and IHC. At normal controls and 6 h of sham groups, there was few 3-NT formation. Correspondingly, after the injection of Hb, the 3-NT progressively increased from 6 h and then maintained at a high level and reached the peak at 3 days, and then decreased gradually. Significant differences exist between Hb groups and normal controls or sham groups at the indicated time points (P<0.05). A similar increase of the expression of 3-NT in the penumbra region was also observed using IHC. In order to confirm the spatial localization, by double-staining, we furtherly identified that ONOO- was mainly present in the neutrophils (MPO+) and microglia/macrophages (ED-1+), indicating a close link exists between ONOO" production and inflammation in Hb-induced injury.7. ONOO-production detected by ELISA in blood serum and brain tissueThe expression of 3-NT in blood serum and brain tissues at indicated times were detected by ELISA. In line with the result of westernblot, the protein levels of 3-NT also has a moderate increase from 6 h, reaching the peak at 3 days and then decreased gradually. Significant differences exist between Hb groups and normal controls or sham groups at the indicated time points (P<0.05). Meaningfully,3-NT production was correlated significantly with the degree of brain edema (r=0.689, P<0.01 analysis with 3-NT in rat brain; r=0.782, P<0.01 analysis with 3-NT in serum) and neurological deficits evaluated by mNSS scores (r=0.844, P<0.01 analysis with 3-NT in rat brain; r=0.851, P<0.01 analysis with 3-NT in serum) after Hb injection.8. Relation between up-regulation of three isoforms of NOS and expression of ZO-1To assess whether up-regulated NOS expression co-localized with sites of BBB disruption, sections were double labeled for ZO-1 and nNOS, iNOS or eNOS. Sham control vessels with no or minimal NOS reactivity showed intense or intact ZO-1 distribution. In Hb-injectioned animals, vessels with intense perivascular or luminal reactivity of NOS consistently showed discontinuous or diffused ZO-1 immunoreactivity, indicating a close-knit correlation beween NOS up-regulation and sites of BBB alterations.9. Relation between ONOO- formation and expression of TJ proteinsTo further investigate the role of ONOO- in BBB disruption, brain sections were double labeled for 3-NT and claudin-5 or ZO-1. In sham control animals, vessels with no or minimal 3-NT reactivity exhibited intense and intact claudin-5 or ZO-1 staining. However, in the Hb-injected rats, enhanced expression of 3-NT was detected in and around lots of blood vessels. The blood vessels with enhanced 3-NT immunoreactivity consistently showed diminished or diffused claudin-5 or ZO-1 staining, suggesting a close correlation between ONOO- production and sites of BBB alterations. Thus, it is credible that ONOO- synthesized by the activaton of three kinds of NOS may have an important role in the process of Hb-induced BBB breakdown.Experiment two1. Cellular localization of gelatinolytic activity after Hb injectionTo confirm the cellular localization of gelatinolytic activity after Hb injection, we performed double staining for in situ zymography and different cellular markers. Obviously, most of the gelatinolytic activity was detectable in FN+ vessel walls and NeuN+ neurons. Colocalization between in situ gelatinolytic activity and astrocytic end-feet at the vessel wall was observed, whereas no significant gelatinolytic activity was detectable in the cellular body of the astrocytes. Though few in number, co-localization was also observed with MPO and ED-1. These results indicate that gelatinolytic activity is triggered in a wide variety of cell types after Hb injection, which are approximately in agreement with published reports.2. FeTPPS attenuates the levels of ONOO- after Hb injectionTo test the effect of FeTPPS on ONOO- accumulation, the levels of 3-NT were measured using IHC and Western blot. The 3-NT was significantly increased at 24 h after Hb injection in the injured region as compared with sham operated groups (P<0.01). FeTPPS treatment significantly reduced the Hb-mediated enhanced levels of 3-NT compared to the Hb-injected+ vehicle group (P<0.01).3. FeTPPS reduces gelatinolytic activity of MMPs in Hb-injected ratsTo investigate whether treatment with FeTPPS could reduce the activity of MMP-9, we detected the gelatinolytic activity shown by in situ zymography (Figure 4A) and gelatin gel zymography (Figure 4B and C). The activity of MMP-9 was significantly increased at 24 h after Hb injection in the peri-hematomal region as compared with sham controls (P<0.01). Treatment with FeTPPS significantly reduced the Hb-mediated increased activity of MMP-9 (P<0.01).4. FeTPPS protects the expression of ZO-1 after Hb injectionOur previous study demonstrated that the expression of ZO-1 is reduced after Hb injection and ONOO- production significantly involves in the BBB disruption. Therefore, using IF and Westernblot, we investigated whether FeTPPS protects BBB via the regulation of ZO-1. Vessels from sham groups showed intense and continuous staining for ZO-1. In contrast, diminished and discontinuous reactivity was detected at 24 h after Hb injection (in white arrows). The ZO-1 was evidently decreased in the injured region as compared with sham operated groups (P<0.01). Administration with FeTPPS significantly ameliorated the Hb-mediated decreased expression of ZO-1 compared to the Hb-injected+ vehicle group (P<0.05).5. FeTPPS treatment ameliorated the neurological deficits following Hb injectionAs shown in the mNSS test, Hb-injected rats exhibited significant behavioral deficits at 24 and 72 h. FeTPPS treatment moderately reduced the neurological impairments at 24 h but present no significant difference (P= 0.065). At 72 h, the neurological deficits measured by mNSS test were significantly ameliorated by drug treatment (P<0.05).CONCLUSION:1. Hb may induce changes of endothelial cell tight junction proteins (claudin-5 ZO-1) and cause the destruction of BBB permeability, thereby resulting in the brain edema formation and neurological deficits.2. Enhanced expression of three kinds of NOS and excessive ONOO" production were observed after Hb injection. Besides, increased diverse NOS expression as well as excessive ONOO- formation all had close relationship with the decrease or destruction of claudin-5/ZO-l protein.3. Hb-induced BBB dysregulation appeared to be involved in the NOS overexpression and excessive ONOO- formation. Abundant ONOO- may participate in the development process of brain edema and neurological deficits after ICH.4. ONOO- may be a promising biomarker for the judgement or prediction of brain injury and clinical prognosis after ICH.5. Scavenging ONOO- by FeTPPS can significantly attenuate the BBB disruption and neurological deficits, which possibly in part through inhibiting the activation of MMP-9. |
PDF Full Text Request