Oxidative DNA injury after experimental intracerebral hemorrhage

Our previous studies have demonstrated that DNA injury occurs in the brain after intracerebral hemorrhage (ICH). DNA damage can result from at least two pathways, either endonuclease-mediated DNA fragmentation or oxidative injury. The present study investigated the occurrence of the latter after ICH and the role of iron in such injury. Male Sprague-Dawley rats received an infusion of autologous whole blood or ferrous iron into the right basal ganglia. Control rats just had a needle insertion (sham). The rats were sacrificed 1, 3, or 7 days later. 8-Hydroxyl-2'-deoxyguanosine (8-OHdG) was analyzed by immunohistochemistry while the number of apurnic/apyrimidinic abasic sites (AP sites) was also quantified. 8-OHdG and AP sites are two hallmarks of DNA oxidation. Dinitrophenyl (DNP) was measured by Western blotting to compare the time course of protein oxidative damage to that of DNA. DNA repair Ku proteins were measured by Western blot analysis. DNA damage was also examined using DNA polymerase I-mediated biotin-dATP nick translation (PANT) labeling. An increase of 8-OHdG, AP sites and DNP levels and a decrease of Ku levels were observed. Abundant PANT-positive cells were also observed in the perihematomal area 3 days after ICH. In addition, intracerebral infusion of iron increased brain DNP levels and resulted in DNA injury. These results suggest that oxidative stress contributes to DNA damage and brain injury after ICH. Reducing DNA oxidative damage (for example, through iron chelation) may be a therapeutic target for ICH.     

Molecular signatures of brain injury after intracerebral hemorrhage

BACKGROUND: The mechanisms of cellular death in the tissue surrounding an intracerebral hemorrhage (ICH) are not defined. OBJECTIVE: To investigate the relationship of markers of excitotoxicity and inflammation to brain injury after ICH. METHODS: A total of 124 consecutive patients with spontaneous ICH admitted within 24 hours of stroke onset were prospectively investigated. The volumes of the initial ICH, peripheral edema on days 3 to 4, and the residual cavity at 3 months were measured on CT scan. Glutamate, cytokines, and adhesion molecules were measured in blood samples obtained on admission. Stroke severity and neurologic outcome were evaluated with the Canadian Stroke Scale. RESULTS: Poor neurologic outcome at 3 months (Canadian Stroke Scale < 7) was observed in 53 patients (43%). Stroke severity and glutamate concentrations (by each increment of 10 micromol/L, odds ratio 1.23; 95% CI 1.09 to 1.41), but not the initial volume of ICH, were independent predictors of poor outcome. In the multiple linear regression analyses, tumor necrosis factor-alpha concentration was correlated (r = 0.83, p < 0.0001) with the volume of perihematoma edema, and glutamate concentrations were correlated (r = 0.78, p < 0.0001) with the volume of the residual cavity. These same results were observed when lobar (n = 58) and deep (n = 66) ICH were analyzed separately. CONCLUSIONS: High plasma levels of proinflammatory molecules within 24 hours of intracerebral hemorrhage onset are correlated with the magnitude of the subsequent perihematoma brain edema, whereas poor neurologic outcome and the volume of the residual cavity are related to increased plasma glutamate concentrations.     

补体在脑出血后脑组织损伤机制中的作用

目的研究补体C9在大鼠实验性脑出血(ICH)后血肿周围组织中的表达情况,探讨补体C9在ICH后脑水肿中的作用以及应用眼镜蛇毒因子(CVF)干预后对血肿周围组织C9表达及脑组织含水量变化的影响。方法采用立体定向技术,将自体不凝血注入大鼠尾状核制备ICH模型,将动物分为假手术组、出血组和CVF干预组,分别在不同时间断头取脑,连续切片分别作补体C9免疫组化染色和HE染色,并进行脑组织含水量测定(干湿重法)。结果ICH后2h血肿周围脑组织开始表达C9,24h达高峰。血肿周围脑组织含水量在ICH后2h开始增加(P<0.05),6h明显增加,24～72h达高峰(P<0.01),此后逐渐回落,1周基本恢复正常水平,脑组织含水量与C9的表达呈正相关关系(r=0.938,P<0.01);对侧半球相应部位及假手术对照组脑组织含水量没有明显变化;经CVF干预后,血肿周围组织C9表达明显下降,干预组与出血组之间比较有显著差异(P<0.01)。CVF干预组脑组织含水量明显低于常规ICH组(P<0.01)。结论脑出血后补体级联激活C9表达明显增加,并证明通过CVF干预后,C9表达下降,脑水肿减轻,能达到神经保护作用。     

Role of lipocalin-2 in brain injury after intracerebral hemorrhage

Lipocalin-2 (LCN2) is a siderophore-binding protein involved in cellular iron transport and neuroinflammation. Both iron and inflammation are involved in brain injury after intracerebral hemorrhage (ICH) and this study examined the role of LCN2 in such injury. Male adult C57BL/6 wild-type (WT) or LCN2-deficient (LCN2^−/−) mice had an intracerebral injection of autologous blood or FeCl₂. Control animals had a sham operation or saline injection. T2-weighted magnetic resonance imaging and behavioral tests were performed at days 1, 3, 7, 14, and 28 after injection. In WT mice, brain LCN2 levels were increased in the ipsilateral basal ganglia after ICH or iron injection. Lipocalin-2-positive cells were astrocytes, microglia, neurons, and endothelial cells. Intracerebral hemorrhage resulted in a significant increase in ferritin expression in the ipsilateral basal ganglia. Compared with WT mice, ICH caused less ferritin upregulation, microglia activation, brain swelling, brain atrophy, and neurologic deficits in LCN2^−/− mice (P<0.05). The size of the lesion induced by FeCl₂ injection as well as the degree of brain swelling and blood–brain barrier disruption were also less in LCN2^−/− mice (P<0.05). These results suggest a role of LCN2 in enhancing brain injury and iron toxicity after ICH.     

H-7在脑出血后脑损伤中作用机制的研究

目的探讨蛋白激酶C(PKC)抑制剂异喹啉磺酰类(H-7)在脑出血后脑损伤中的作用机制。方法SD雄性大鼠随机分为对照组、假手术组、脑出血组、H-7治疗组,采用自体血注入法建立大鼠脑出血模型,治疗组应用H-7每12 h腹腔注射一次。分别用伊文思蓝(EB)测血脑屏障(BBB)通透性,干-湿重法测脑组织含水量,同时进行组织病理学观察。结果H-7治疗组可以明显改善大鼠脑出血后BBB通透性(P<0.05),血肿周围脑水肿明显减轻(P<0.05),并且减少脑出血后血肿周围炎症细胞浸润。结论H-7通过抑制PKC,减轻脑出血后脑水肿?血脑屏障的开放和炎症细胞浸润,揭示PKC参与了脑出血后脑损伤。     

红细胞在实验性脑出血后脑水肿形成中的作用

目的研究红细胞在脑出血后脑水肿形成中的作用。方法向大鼠尾状核分别注入浓缩红细胞（PRBC）、溶解红细胞（LRBC）及不同浓度的大鼠血红蛋白（Hb）,并比较鼠脑不同部位脑水肿形成的特点。结果红细胞对脑水肿的影响要在脑出血后第3d,其水肿的严重程度与Hb的浓度呈正相关。结论红细胞对脑出血后脑水肿形成的影响主要在脑出血后第3d,可能与红细胞溶解释放血红蛋白有关。     

Reduced brain injury in CD18-deficient mice after experimental intracerebral hemorrhage

Many studies have indicated leukocytes are a major contributor to brain injuries caused by intracerebral hemorrhage (ICH). Leukocyte-expressed CD18 is important for neutrophil-endothelial interactions in the vasculature, and CD18 deficiency protects against ischemia-reperfusion injury. We investigated whether CD18 deficiency provides protection against ICH-induced brain injury. Male wild-type (WT) CD18(+/+) mice and CD18(-/-) -knockout mice were used in this study. ICH was induced by a collagenase injection. Mortality, neurological function, brain edema, and myeloperoxidase (MPO) activity as well as tissue expression of nitrotyrosine and MPO were evaluated 24 hr after ICH. We discovered significantly reduced brain edema and diminished mortality with a concomitant decrease in MPO and nitrotyrosine immunoreactivity in brains of CD18-knockout mice.     

依达拉奉激活Nrf2通路减轻脑出血后继发性损伤

目的探讨自由基清除剂依达拉奉对大鼠脑出血神经功能与脑水肿的疗效,并通过检测核因子相关因子2（Nrf2）通路及其下游抗氧化酶的表达变化,探索依达拉奉在脑出血中的抗氧化治疗机制。方法采用自体血立体定向注射建立大鼠脑出血模型,依达拉奉干预后24h分别行神经行为学评分及脑含水量测定,并检测Nrf2通路表达变化,脑组织切片行免疫荧光双标染色,判定Nrf2表达的细胞来源。结果依达拉奉干预脑出血大鼠后24h,前肢抬起及前肢对称实验较对照组均明显改善,脑含水量亦明显降低。依达拉奉组Nrf2及其下游抗氧化酶血红素加氧酶1（HO-1）、硫氧还蛋白（TRX）、谷胱甘肽-S-转移酶a1（GST-a1）、醌氧化还原酶1（NQ01）转录水平较对照组明显上调,依达拉奉组Nrf2及HO-1蛋白表达较对照组明显升高。脑组织切片免疫荧光双标染色表明,Nrf2主要在神经元中特异性表达。结论依达拉奉可明显减轻大鼠脑出血后神经功能障碍及脑水肿程度,其机制可能与激活内源性抗氧化系统Nrf2通路发挥对神经元的保护作用有关。     

Activation of P2X4 receptor exacerbates acute brain injury after intracerebral hemorrhage

IntroductionIntracerebral hemorrhage (ICH) accounts for 10%–15% of all strokes and culminates in high mortality and disability. After ICH, brain injury is initiated by the mass effect of hematoma, followed by secondary cytotoxic injury from dying brain cells, hematoma disintegration, and cascading brain immune response. However, the molecular mechanism of secondary cytotoxic brain injury in ICH is not completely understood. The sensitive purinergic receptor, P2X4 receptor (P2X4R), was known to recognize extracellular free ATP released by dying cells during tissue injury.AimsIn this study, we aim to understand the role of P2X4R in acute brain injury triggered by ICH.ResultsIn this study, we found that the sensitive purinergic receptor, P2X4R, was upregulated in the brain of patients with ICH as well as in a mouse model of ICH induced by collagenase injection. P2X4R blockage with the specific inhibitor 5‐BDBD attenuated brain injury in ICH mice by significantly reducing brain edema, blood–brain barrier leakage, neural death, and ultimately acute neurodeficits. Further study indicated that the protective effect of P2X4R inhibition is related to decreased pro‐inflammatory activity of microglia and recruitment of peripheral immune cells into the hemorrhagic brain.ConclusionsThese results suggest that the P2X4 receptor is activated by ICH stimuli which worsen brain injury following ICH.     

中性粒细胞明胶酶相关载脂蛋白在大鼠脑出血后继发性脑损伤中的作用研究

目的探讨中性粒细胞明胶酶相关载脂蛋白(NGAL)在脑出血(ICH)后继发性脑损伤中的作用。方法采用Ⅶ胶原酶制作ICH模型,将90只雄性SD大鼠随机分为3组:正常对照组(10只)、假手术组(40只)和ICH模型组(40只)。分别在ICH后6 h、24 h、72 h和7 d四个时间点对大鼠进行神经功能缺损评分;用免疫组化及RT-q PCR来观察不同时间点脑组织NGAL及MMP-9表达情况。结果免疫组化及RT-q PCR结果显示ICH后模型组各时间点均可见大量NGAL、MMP-9阳性细胞及NGAL mRNA表达,且明显高于正常组及假手术组(P0.05)。ICH模型组NGAL与MMP-9蛋白表达呈正相关(P0.01)。ICH模型组NGAL、MMP-9蛋白表达均与大鼠神经功能缺损评分呈正相关(P0.05)。结论 ICH后NGAL表达明显增加,提示NGAL可能参与了脑出血后继发性脑损伤,并可能通过调节MMP-9的活性从而发挥作用,但NGAL是否为MMP-9的上游调控因子尚有待进一步研究。     

Mechanisms of brain injury after intracerebral haemorrhage

The past decade has resulted in a rapid increase in knowledge of mechanisms underlying brain injury induced by intracerebral haemorrhage (ICH). Animal studies have suggested roles for clot-derived factors and the initial physical trauma and mass effect as a result of haemorrhage. The coagulation cascade (especially thrombin), haemoglobin breakdown products, and inflammation all play a part in ICH-induced injury and could provide new therapeutic targets. Human imaging has shown that many ICH continue to expand after the initial ictus. Rebleeding soon after the initial haemorrhage is common and forms the basis of a current clinical trial using factor VIIa to prevent rebleeding. However, questions about mechanisms of injuries remain. There are conflicting data on the role of ischaemia in ICH and there is uncertainty over the role of clot removal in ICH therapy. The next decade should bring further information about the underlying mechanisms of ICH-induced brain injury and new therapeutic interventions for this severe form of stroke. This review addresses our current understanding of the mechanisms underlying ICH-induced brain injury.     

Serum biomarkers of spontaneous intracerebral hemorrhage induced secondary brain injury

Intracerebral hemorrhage (ICH) is a devastating form of stroke associated with a high rate of morbidity and mortality. It is now believed that much of this damage occurs in the subacute period following the initial insult via a cascade of complex pathophysiologic pathways that continues to be investigated. Increased levels of certain serum proteins have been identified as biomarkers that may reflect or directly participate in the inflammation, blood brain barrier disruption, endothelial dysfunction, and neuronal and glial toxicity that occur during this secondary period of cerebral injury. Some of these biomarkers have the potential to serve as therapeutic targets or surrogate endpoints for future research or clinical trials. Others may someday augment current clinical techniques in diagnosis, risk-stratification, prognostication, treatment decision and measurement of therapeutic efficacy. While much work remains to be done, biomarkers show significant potential to expand clinical options and improve clinical management, thereby reducing mortality and improving functional outcomes in ICH patients.     

水蛭素对抗脑出血后脑水肿作用机制的研究

目的探讨凝血酶在脑出血后脑水肿形成中的作用和水蛭素抗水肿的作用.方法健康wistar大鼠80只,随机分为4组,每组20只.A组生理盐水+生理盐水;B组生理盐水+水蛭素;C组血+生理盐水;D组血+水蛭素.采用立体定向注射方法,用微量注射器在右侧尾状核部位注入自体未凝血(C、D组)或等量生理盐水(A、B组)50μl,留置20min后同部位2次注射10μl生理盐水(A、C组)或等量水蛭素溶液(B、D组,浓度2u/μl).术后第3d断头取脑,测定脑水含量.结果 A、B组比较无明显差异外,其余各组间均有统计学意义(P＜0.05).结论凝血酶可以引起脑出血后脑组织水肿,水蛭素可以抑制凝血酶所引起的脑水肿,且水蛭素本身不对脑组织产生任何负面影响.     

脑出血后周边组织继发性脑损伤炎性机制的研究

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脑出血后肺组织炎症损伤情况的研究

目的 研究脑出血后肺组织炎症损伤的发病机制。方法 用病理学常规HE染色法观察脑出血组和对照组肺标本的出血、渗出和炎症细胞浸润改变情况,在光镜下对白细胞、巨噬细胞进行记数。结果 与对照组比较,脑出血组肺组织存在明显渗出、出血及大量白细胞、巨噬细胞浸润,炎症细胞记数有显著意义P〈0．05。结论 脑出血可以通过引发炎症反应导致急性肺损伤;炎症反应在3-5d时最明显。