Effect of idazoxan on permeability of inflammatory blood-brain barrier model in vitro

AIM: To study the effect of idazoxan on the permeability of inflammatory blood-brain barrier (BBB) model in vitro and the expression of tight junction protein ZO-1.METHODS: In vitro BBB model was established by murine brain endothelial cell line bEnd.3 incubated for 7 d. The cells were treated with TNF-α (10 nmol/L) for additional 24 h to establish the inflammatory BBB model, which was pretreated with IDA at doses of 50, 100 and 200 μmol/L, respectively. The permeability was measured using fluorescein isothiocyanate-conjugated dextran (FD-40, MW 40,000), the expression of ZO-1 was detected by Western blot analysis, the distribution of ZO-1 was observed by immunofluorescence, and the mRNA expression of MMP-9/TIMP-1 was measured by RT-PCR.RESULTS: After incubated for 7 d, b.End3 cells converged to be confluent monolayer with low permeability. The inflammatory BBB model induced by TNF-α treatment displayed much higher permeability with decreased expression of tight junction protein ZO-1, destroyed distribution of ZO-1 and increased mRNA expression of MMP-9. When pretreated with IDA, the permeability was greatly decreased, the expression of ZO-1 was greatly increased, the abnormal distribution of ZO-1 was greatly ameliorated and the mRNA expression of MMP-9 was obviously reduced. The effect was most significant in IDA (200 μmol/L)-pretreated group (P<0.01).CONCLUSION: IDA directly acts on brain endothelial cells to reduce the expression of MMP-9, increase the expression of tight junction protein ZO-1 and ameliorate the destroyed distribution of ZO-1 in the inflammatory BBB, thus reversing the abnormally elevated permeability in a inflammatory BBB model in vitro induced by TNF-α.     

Effect of idazoxan on permeability of blood-brain barrier and expression of MMP-9/TIMP-1 in mouse experimental autoimmune encephalomyelitis

AIM：To study the effect of idazoxan (IDA) on the permeability of blood-brain barrier (BBB) and the expression of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) in mouse experimental autoimmune encephalomyelitis (EAE).METHODS：Female C57BL/6 mice (n=36) were randomly divided into control group, EAE group and IDA group, with 12 mice in each group. EAE was induced by myelin oligodendrocyte glycoprotein 35-55 (MOG35-55). IDA (2 mg/kg, ip, bid) was administered for 15 d after immunization. The neurological defects of the mice were observed daily and scored. The pathological changes were observed under microscope with HE staining and LFB myelin staining. The BBB permeability was detected by Evans blue extravasation. The expression of MMP-9 and TIMP-1 in the brain of EAE mice was determined by Western blotting.RESULTS：Compared with EAE group, the score of neurological defects in IDA group was decreased, the inflammation was relieved, the BBB permeability was reduced, and the expression MMP-9 and the ratio of MMP-9/TIMP-1 were decreased (P<0.05).CONCLUSION：The neuroprotective effect of IDA on mouse EAE might be related to the down-regulation of MMP-9 and the ratio of MMP-9/TIMP-1, thus reducing the degradation of BBB and the permeability of BBB, and ameliorating the pathologic process of EAE.     

All-trans retinoic acid attenuates blood-brain barrier injury induced by cerebral ischemia-reperfusion in rats through JNK/P38 MAPK signaling pathway

AIM: To investigate the effect of all-trans retinoic acid (ATRA) on blood-brain barrier after cerebral ischemia-reperfusion (CIR) injury in rats and its possible role mechanism.METHODS: Male SD rats were randomly divided into sham group, model (CIR) group and CIR+ATRA (10, 30 and 90 mg/kg) groups. The rat model of CIR injury was established by MCAO thread occlusion method. After ischemia for 1.5 h and reperfusion for 24 h, the neurological functional behavioral score, cerebral infarction volume, brain water content and Evans blue content were determined. The activity of matrix metalloprotein-9 (MMP-9) was measured by gelatin zymography. The protein levels of claudin-5, occludin, ZO-1, JNK, p-JNK, P38, p-P38 and MMP-9 in the brain tissues were determined by Western blot.RESULTS: Compared with CIR model group, ATRA at 30 mg/kg significantly improved neurological function, and decreased cerebral infarction volume, brain water content, Evans blue content and the degradation of tight junction proteins in ischemic area (P<0.01). The activity and protein expression of MMP-9 in ischemic brain tissue were decreased (P<0.01). The phosphorylation of JNK and P38 was inhibited and the protein levels of p-JNK and p-P38 were decreased (P<0.01).CONCLUSION: ATRA reduces the damage of brain tissue and the destruction of blood-brain barrier induced by CIR in rats. The protective effect may be related to inhibiting the activation of JNK/P38 MAPK signaling pathway and MMP-9.     

Expression and histogenesis of matrix metalloproteinase-9 and transforming growth factor-beta 1 in acute cerebral ischemia model of rats

AIM:To observe the expression and tissue localization of matrix metalloproteinase 9 (MMP-9) and transforming growth factor beta 1 (TGF-β1) in the rat acute cerebral ischemia model. METHODS:Male Wistar rats were used to establish acute cerebral ischemia model by a suturing method. The rats were divided into normal control group, sham group and ischemia 6 h, 12 h, 1 d, 2 d, 6 d and 14 d groups. The rat cerebral cortex and hippocampus of the brain were collected at different time points.The mRNA and protein levels of MMP-9 and TGF-β1 in the brain tissues were detected by real-time PCR and in situhistochemistry staining, respectively. The levels of MMP-9 and TGF-β1 in the plasma were also measured by ELISA. RESULTS:The results of real-time PCR showed that the mRNA levels of MMP-9 began to increase 6 h after acute ischemia and reached to a peak 2 d after acute ischemia. Similarly, the mRNA level of TGF-β1began to rise 12 h after acute ischemia and reached to the highest level 6 d after acute ischemia. Compared with the sham rats, the mRNA levels of MMP-9 and TGF-β1 in the rat brains that collected at ischemic time of 12 h, 1 d, 2 d, 6 d and 14 d were significantly increased. Moreover, results of in situhistochemical staining showed that the expression of MMP-9 was detected at cerebral cortex and hippocampus 1 d after acute cerebral ischemia.Further studies showed that MMP-9 dyeing of the rat cerebral cortex was most obvious 2 d after the acute cerebral ischemia. Similarly, the rat cortex and hippocampus began to express TGF-β1 2 d after acute ischemia and TGF-β1 staining at rat cerebral cortex was most obvious 6 d after the acute cerebral ischemia. In addition, ELISA showed that the increase in MMP-9 and TGF-β1 was detected in the plasma 12 h after ischemia. Compared with the sham rats, the level of these 2 factors significantly upregulated since 1 d after ischemia. CONCLUSION: The brain tissue itself contributes to the upregulation of MMP-9 and TGF-β1 post acute cerebral ischemia, which shed light on the related research in the field.