| PART I The Expression of miR-130a in the Serum of Acute Spontaneous Intracerebral Hemorrhage Patients and its Relation with Perihematomal Edema Volume and PrognosisObjective To investigate the level of miR-130a in the serum of acute spontaneous intracerebral hemorrhage (ICH) patients and its relation with perihematomal edema volume (PHE) and prognosis.Methods Patients with acute spontaneous ICH were enrolled from multiple settings between 2012 and 2013. Eligible patients were aged>18 years and admitted within 72 h after onset. Exclusion criteria were infratentorial ICH, intraventricular hemorrhage, hematoma expansion, secondary ICH (hemorrhage resulting from underlying structural lesion, hemorrhage infarction, or the use of a thrombolytic agent), undergoing surgical hematoma evacuation, a recent stroke (within 3 months), or refusal of participation. Demographic and clinical data were collected at admission.To sample the serum of acute spontaneous ICH patients and RT-PCR was performed to detect the level of miR-130a at the time point of days 0-1,2-3,5-8 and 9-14 after symptom onset. Noncontrast CT was performed to detect the patients'hemorrhagic mass and perihemorrhagic edema volume at days 0-1,2-3,5-8, and 9-14 after symptom onset, and the images were evaluated by three doctors who were blind to the experimental design. The National Institute of Health Stroke Scale (NIHSS) and the scores of the modified Rankin scale (mRS) were used at days 14 and 90 respectively after onset to evaluate the patients'function outcome.Results Compared with controls, miR-130a expression was elevated in response to ICH injury, which reached a peak within 1 day after ICH (P<0.01), and then decreased over time. The median hematoma volume was 7.33 cm3 (interquartile range(IQR),3.87-20.94) at days 0-1,8.71cm3 (IQR,5.53-14.43) at days 2-3,10.21 cm3 (IQR,2.92-18.34) at days 5-8, and 6.38 cm3 (IQR,4.19-9.26) at days 9-14. Accordingly, the median PHE volume was 17.41 cm3 (IQR,7.56-27.51) at days 0-1,17.81 cm3 (IQR,7.56-30.83) at days 2-3, 17.24 cm3 (IQR,10.21-28.98) at days 5-8, and 17.26 cm3 (IQR,16.70-27.85) at days 9-14. Meanwhile, miR-130a level was significantly correlated with PHE volume at days 0-1 (R2=0.62, P<0.01). Similarly, high-level correlation was observed between miR-130a content and PHE volume at days 2-3 (R2=0.70, P<0.01) but not at the other two time points (R2days 5-8=0.41, P<0.05; R2days 9-14=0.16, P>0.05). The association remained significant after adjustment for baseline hematoma volume (?days 0-3=0.541, P<0.01). The correlation between hematoma and PHE volume was increasing over time after syndrome onset at days 1-8 (R2days 0-1=0.56, P<0.01; R2days 2-3=0.65, P<0.01; R2days 5-8=0.89, P<0.01; R2days 9-14=0.26, P>0.05). R2 value was higher as revealed by the multiple linear regression with two independent variables (hematoma volume and miR-130a content) within 3 days after the initial bleeding event (R2days0-1=0.73, P<0.01; R2days 2-3=0.89, P<0.01). A positive correlation was found between baseline miR-130a level and NIHSS score (R2= 0.56, P<0.01). This correlation was also significant in both deep (20/30) and lobar (10/30) subgroups (R2=0.62 and 0.57, respectively, both P<0.01). Baseline miR-130a content remained independently correlated with NIHSS score when baseline hematoma volume was integrated into the regression (?=0.703, P<0.01;?deep=0.479, P<0.05; ?lobar= 0.756, P<0.05). But this correlation was not strong enough in the lobar group with the multiple linear regression analysis (R2=0.433, P<0.05). However, good linear relationship was revealed between baseline miR-130a level and mRS score only in patients with deep hematoma (R2=0.602, P<0.01), even after adjustment for baseline hematoma volume (?=0.533, P<0.05).Conclusions Serum miR-130a level was increased in patients with acute spontaneous ICH, which was significantly correlated with the volume of perihematomal edma. and only the patients with deep hematoma could predict the prognosis by serum miR-130a level.PART ? The Effect of miR-130a on Brain Edema by Compromising Blood-Brain Barrier Integrity Following Acute Intracerebral HemorrhageObjective To explore the relation between miR-130a level of serum and perihematomal brain tissues with brain edema in collagenase-induced rat intracerebral hemorrhage model.Methods Collagenase-induced rat intracerebral model was performed in vivo study. The miR-130a level of serum and perihematomal brain tissues was assayed by RT-PCR. The brain water content was detected by wet/dry weight method, while Evans Blue leakage assay was performed to examine the rat BBB permeability. miR-130a inhibitorss was infused to the right lateral ventricle before performing rat ICH model, and the rat brain edema, BBB permeability and behavioral function was evaluated at day 1.Results The miR-130a level in perihematomal tissues was significantly increased and arrived at a peak, which was almost 2.5 times that of the sham-operated animals within 1 day after ICH (Pday1<0.05). Afterwards, the level of miR-130a declined with time (Pday 3<0.05), returning to normal by day 7. Then, a similar profile of miR-130a was also observed in the serum (Pday 1<0.01; Pday 3<0.05). These trends were consistent with the water content in the ipsilateral hemisphere of ICH rats (Pday 1<0.01; Pday3<0.01). miR-130a inhibitors could significantly reduce endogenous expression of miR-130a (P><0.01). Meanwhile, miR-130a inhibitorss significantly alleviated BBB disruption (P<0.05), and decreased brain water content (P<0.05) in ICH rats. As expected, we also exhibited that neurological function was significantly improved after inhibition of miR-130a in ICH rats (P<0.05).Conclusions In the rat model of collagenase-induced intracerebral hemorrhage, the level of miR-130a was elevated both in the serum and perihematomal brain tissues. Upregulated miR-130a was closely associated with brain edema severity in intracerebral hemorrhage rat. miR-130a inhibition in rat ICH model could preserve BBB integrity, attenuate brain edema and improve neurological behavioral function.PART ? The Mechanism of miR-130a in Aggravating BBB Disruption Following Acute Intracerebral HemorrhageObjective To explore the underlying molecular mechanism between high miR-130a level and BBB hyperpermeability.Methods Primary brain microvascular cells was transfected by miR-130a mimic, then BMECs monolayer Permeability Assay was performed to evaluated the permeability of monolayer endothelial cells. Thrombin, FeCl2 and hemin was added into the BMECs culture respectively, then miR-130 level was profiled by RT-PCR. After miR-130a overexpression in BMECs by miR-130a mimic transfection, the level of cav-1 and MMP-2,-9 was examined with RT-PCR and Western-Blot. Through lateral ventricular miR-130a inhibitors injection in ICH rat model, the level of cav-1 and MMP-2,-9 of perihematomal tissues was examined by RT-PCR and Western-Blot.Results Increased miR-130a level by miR-130a mimic transfection significantly promoted the passage of FITCdextran across the BMEC monolayer (P<0.05). Thrombin induced the expression of miR-130a in a dose-dependent manner (P<0.01), whereas FeC12 upregulated the expression of miR-130a only in high-concentration group (cells were virtually killed at high concentration; data not show), and hemin caused no significant change in miR-130a expression. miR-130a expression was not significantly changed as compared with controls when thrombin was applied upon astrocytes. When BMECs were cultured with argatroban, a thrombin inhibitor, miR-130a expression upregulated by thrombin was partially antagonized (P<0.05). The mRNA levels of MMP-2 and MMP-9 were significantly increased after ICH (P<0.01, compared with the sham-operated group), and this upragulation was reversed by miR-130a inhibitorss (P<0.05, compared with the ICH group). The protein of cav-1 level was significantly reduced 1 day post-ICH, and then it was increased with time (P<0.05), while the miR-130a expression exhibited an opposite trend (P<0.05). Overexpression of miR-130a in BMECs by miR-130a mimic transfection could reduce significantly the protein expression of cav-1, while the level of MMP-2/9 was increased (P<0.05). Cavtratin, a cav-1 scaffolding domain peptide, could decrease miR-130a-induced overexpression of MMP-2 and MMP-9 in BMECs (P<0.05). Treatment of BMECs with thrombin could reduce cav-1 level and raise MMP-2/9 level, and such changes could be reversed by miR-130a inhibitorss or argatroban.Conclusions Increased miR-130a level could significantly promote the passage of FITC dextran across the BMEC monolayer. Thrombin induced the expression of miR-130a in a dose-dependent manner in BMECs. BBB disruption by miR-130a could be mediated by activation of Cav-1/MMP-2 and MMP-9 pathway. |
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