The role of complement C3 in intracerebral hemorrhage-induced brain injury.

Activation of the complement cascade contributes to brain injury after intracerebral hemorrhage (ICH). However, a recent study found that complement C5 deficient mice had enhanced ICH-induced brain injury. The present study, therefore, investigated the role of complement C3 (which is upstream from C5) in ICH. Male complement C3 deficient and sufficient mice had an intracerebral infusion of 30-muL autologous whole blood. The mice were killed and the brains were sampled for edema, Western blotting, immunohistochemistry and histologic analysis. Behavioral tests including forelimb use asymmetry test and corner turn were also performed before and after ICH. Compared to complement C3 sufficient mice, C3 deficient mice had less brain edema, lower hemeoxygenase-1 levels, less microglia activation and neutrophil infiltration around the clot after ICH. In addition, the C3-deficient mice had less ICH-induced forelimb use asymmetry deficits compared with C3-sufficient mice. These results suggest complement activation may affect heme metabolism and the inflammatory response after ICH suggesting that complement C3 is an important factor causing ICH-induced brain injury.     

Acute inflammatory reaction following experimental intracerebral hemorrhage in rat

Previous studies on intracerebral hemorrhage (ICH) indicate that brain edema increases progressively in the first 24 h and remains elevated for several days. The cause of secondary brain injury and edema formation is uncertain. We hypothesized that inflammatory mediators released from the blood after cerebral hemorrhage might cause secondary brain injury and edema formation. This study investigates if, when and where inflammation occurs after ICH in rat. Immunocytochemistry for polymorphonuclear leukocyte marker (myeloperoxidase, MPO), microglia marker (OX42) and intracellular adhesion molecule-1 (ICAM-1) was performed in control, and 1, 3, 7 and 10 days after the injection of 100 microliter autologous blood in the right basal ganglia. Double labeling immunohistochemistry was used to identify ICAM-1 positive cells. The results show that an inflammatory response occurred in and around the blood clot after ICH, characterized by the infiltration of neutrophils and macrophages as well as activation of microglia. ICAM-1 immunoreactivity was observed in blood vessels adjacent to the clot, as well as in activated microglia and neurons in the ipsilateral hemisphere. The present study demonstrates there is an inflammatory response in the brain after ICH. Infiltrating leukocytes and activated microglia may release cytotoxic mediators contributing to secondary brain injury.     

Iron toxicity in mice with collagenase-induced intracerebral hemorrhage

Intracerebral hemorrhage (ICH) is a devastating form of stroke. In this study, we examined the efficacy of deferoxamine (DFX), an iron chelator, after collagenase-induced ICH in 12-month-old mice. Intracerebral hemorrhage was induced by intrastriatal injection of collagenase. Deferoxamine (200 mg/kg, intraperitoneal) or vehicle was administrated 6 hours after ICH and then every 12 hours for up to 3 days. Neurologic deficits were examined on days 1 and 3 after ICH. Mice were killed after 1 or 3 days of DFX treatment for examination of iron deposition, neuronal death, oxidative stress, microglia/astrocyte activation, neutrophil infiltration, brain injury volume, and brain edema and swelling. Collagenase-induced ICH resulted in iron overload in the perihematomal region on day 3. Systemic administration of DFX decreased iron accumulation and neuronal death, attenuated production of reactive oxygen species, and reduced microglial activation and neutrophil infiltration without affecting astrocytes. Although DFX did not reduce brain injury volume, edema, or swelling, it improved neurologic function. Results of our study indicate that iron toxicity contributes to collagenase-induced hemorrhagic brain injury and that reducing iron accumulation can reduce neuronal death and modestly improve functional outcome after ICH in mice.     

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.     

Frameless stereotactic aspiration and thrombolysis of deep intracerebral hemorrhage is associated with reduced levels of extracellular cerebral glutamate and unchanged lactate pyruvate ratios

Introduction  

Intracerebral hemorrhage (ICH) is a devastating form of stroke commonly resulting in severe morbidity and high mortality. Secondary brain injury often occurs in the days following the initial hemorrhage and is associated with significant neurological deterioration. The neurochemistry associated with secondary injury is poorly understood The purpose of this study is to characterize the neurochemical changes, in perihematomal tissue during frameless minimally invasive evacuation of spontaneous hematomas     

Genetic deletion of the prostaglandin E2 E prostanoid receptor subtype 3 improves anatomical and functional outcomes after intracerebral hemorrhage

Intracerebral hemorrhage (ICH) is a stroke subtype associated with high mortality and morbidity. Following ICH, excitotoxicity and inflammation significantly contribute to secondary brain injury and poor outcomes. Prostaglandin E₂ (PGE₂) levels rise locally with insult to the nervous system, and PGE₂ is known to modulate these processes mainly through its E prostanoid (EP) receptors, EP1‐4. EP receptor subtype 3 (EP3) is the most abundant EP receptor in the brain and we have previously shown that signaling through the PGE₂–EP3 axis exacerbates excitotoxicity and ischemic stroke outcomes. This study aimed to investigate the contribution of this pathway in modulating anatomical outcomes and functional recovery following ICH. Genetic deletion of EP3 resulted in 48.2 ± 7.3% less ICH‐induced brain injury (P < 0.005) and improved functional recovery (P < 0.05), as identified by neurological deficit scoring. To start investigating the mechanisms involved in neuroprotection with impaired PGE₂–EP3 signaling, histological staining was performed to evaluate blood and ferric iron accumulation, neuroinflammation, blood–brain barrier dysfunction, and peripheral neutrophil infiltration. After ICH, EP3 knockout mice demonstrated 49.5 ± 8.8% and 42.8 ± 13.1% less blood (P < 0.01) and ferric iron (P < 0.05), respectively. Furthermore, EP3 knockout mice had significantly reduced astrogliosis, microglial activation, blood–brain barrier breakdown, and neutrophil infiltration. Collectively, these results suggest an injurious role for the PGE₂–EP3 signaling axis in modulating brain injury, inflammation, and neurological functional recovery after ICH. Modulation of the PGE₂–EP3 signaling axis may represent a putative therapeutic avenue for the treatment of ICH.     

Protective role of tuftsin fragment 1-3 in an animal model of intracerebral hemorrhage

Intracerebral hemorrhage (ICH) causes morbidity and mortality and commonly follows the reperfusion after an ischemic event. Tissue plasminogen activator (tPA), a fibrinolytic serine protease, is routinely given for the treatment of stroke. However, tPA also can promote neuronal death, suggesting that caution should be exercised when using it. Furthermore, tPA upon brain injury mediates microglial activation and modulates neuronal survival. To investigate the role of tPA and microglia during brain hemorrhage, we induced experimentally ICH by intracerebral injection of collagenase. Seven days after the introduction of ICH, it persisted in tPA-deficient (tPA(-/-)) mice but is drastically reduced in size in wild-type mice. Three weeks after ICH, there are still red blood cells in tPA(-/-) but not in wild-type animals. Activated microglia persist around the injury site. When microglial activation is inhibited by tuftsin fragment 1-3 macrophage/microglial inhibitory factor (MIF), the stroke injury volume is significantly reduced, and the neurobehavioral deficits exhibited by the mice are improved. Our results suggest that endogenous tPA assists in the clearance of intracerebral hemorrhage, presumably by affecting microglial activation, and MIF could be a valuable neuroprotective agent for the treatment of ICH.     

Molecular pathophysiology of cerebral hemorrhage: secondary brain injury

Intracerebral hemorrhage (ICH) is an often fatal type of stroke that kills approximately 30,000 people annually in the United States. If the patient survives the ictus, then the resulting hematoma within brain parenchyma triggers a series of adverse events causing secondary insults and severe neurological deficits. This article discusses selected aspects of secondary brain injury after ICH and outlines key mechanisms associated with hematoma toxicity, oxidative stress, and inflammation. Finally, this review discusses the relevance of hematoma resolution processes as a target for ICH therapy and presents potential clinically relevant molecular targets that could be harnessed to treat secondary injury associated with ICH injury.     

Toxic role of prostaglandin E2 receptor EP1 after intracerebral hemorrhage in mice

Inflammatory mechanisms mediated by prostaglandins may contribute to the progression of intracerebral hemorrhage (ICH)-induced brain injury, but they are not fully understood. In this study, we examined the effect of prostaglandin E₂ receptor EP1 (EP1R) activation and inhibition on brain injury in mouse models of ICH and investigated the underlying mechanism of action. ICH was induced by injecting collagenase, autologous blood, or thrombin into the striatum of middle-aged male and female mice and aged male mice. Effects of selective EP1R agonist ONO-DI-004, antagonist SC51089, and nonspecific Src family kinase inhibitor PP2 were evaluated by a combination of histologic, magnetic resonance imaging (MRI), immunofluorescence, molecular, cellular, and behavioral assessments. EP1R was expressed primarily in neurons and axons but not in astrocytes or microglia after ICH induced by collagenase. In middle-aged male mice subjected to collagenase-induced ICH, EP1R inhibition mitigated brain injury, brain edema, cell death, neuronal degeneration, neuroinflammation, and neurobehavioral deficits, whereas its activation exacerbated these outcomes. EP1R inhibition also was protective in middle-aged female mice and aged male mice after collagenase-induced ICH and in middle-aged male mice after blood- or thrombin-induced ICH. EP1R inhibition also reduced oxidative stress, white matter injury, and brain atrophy and improved functional outcomes. Histologic results were confirmed by MRI. Src kinase phosphorylation and matrix metalloproteinase-9 activity were increased by EP1R activation and decreased by EP1R inhibition. EP1R regulated matrix metalloproteinase-9 activity through Src kinase signaling, which mediated EP1R toxicity after collagenase-induced ICH. We conclude that prostaglandin E₂ EP1R activation plays a toxic role after ICH through mechanisms that involve the Src kinases and the matrix metalloproteinase-9 signaling pathway. EP1R inhibition could be a novel therapeutic strategy to improve outcomes after ICH.     

Efficacy of the lipid-soluble iron chelator 2,2'-dipyridyl against hemorrhagic brain injury

Previous studies have indicated that 2,2′-dipyridyl, a lipid-soluble ferrous iron chelator, can reduce brain injury after cerebral ischemia and reduce cerebral vasospasm after subarachnoid hemorrhage. In this study, we examined the efficacy of 2,2′-dipyridyl after intracerebral hemorrhage (ICH) in 12-month-old mice. ICH was modeled by intrastriatal injection of collagenase or autologous whole blood. 2,2′-Dipyridyl or vehicle was administered intraperitoneally 2 h before ICH (pretreatment) or 6 h after ICH (post-treatment) and then once daily for up to 3 days. Mice in the pretreatment group were sacrificed 1 or 3 days after ICH and examined for iron deposition, neuronal death, oxidative stress, microglial/astrocyte activation, neutrophil infiltration, and white matter damage. Mice in the post-treatment group were examined for brain lesion volume and edema on day 3 and for neurologic deficits on days 1, 3, and 28 after ICH. Pretreatment with 2,2′-dipyridyl decreased iron accumulation and neuronal death, attenuated production of reactive oxygen species, reduced microglial activation without affecting astrocytes or neutrophil infiltration, and attenuated white matter damage. Post-treatment reduced brain lesion volume and edema and improved neurologic function. These results indicate that the lipid-soluble ferrous iron chelator 2,2′-dipyridyl can reduce brain injury and improve functional outcome after ICH.     

Src kinase inhibition decreases thrombin-induced injury and cell cycle re-entry in striatal neurons

Since Src kinase inhibitors decrease brain injury produced by intracerebral hemorrhage (ICH) and thrombin is activated following ICH, this study determined whether Src kinase inhibitors decrease thrombin-induced brain injury. Thrombin injections into adult rat striatum produced focal infarction and motor deficits. The Src kinase inhibitor PP2 decreased thrombin-induced Src activation, infarction in striatum and motor deficits in vivo. Thrombin applied to cultured post-mitotic striatal neurons caused: injury to axons and dendrites; many TUNEL positive neuronal nuclei; and re-entry into the cell cycle as manifested by cyclin D1 expression, induction of several other cell cycle genes and cyclin-dependent kinase 4 activation. PP2 dose-dependently attenuated thrombin-induced injury to the cultured neurons; and attenuated thrombin-induced neuronal cell cycle re-entry. These results are consistent with the hypotheses that Src kinase inhibitors decrease injury produced by ICH by decreasing thrombin activation of Src kinases and, at least in part, by decreasing Src induced cell cycle re-entry.     

Intracerebral hemorrhage in mice: model characterization and application for genetically modified mice.

Gene knockout or transgenic animals may assist in elucidating the mechanisms of brain injury after intracerebral hemorrhage (ICH). However, almost all commercially available transgenic or knockout animals are mice. The purpose of this study was to develop an ICH model in mice and to investigate the influence of gender and complement C5 genetic differences on outcome after ICH. Male and female C57BL/6 mice and C5-deficient and -sufficient control mice were anesthetized and then received an injection of 30 microL autologous whole blood into the right basal ganglia. Brain water content was studied at 1 and 3 days after ICH. Behavioral tests (fore-limb use asymmetry and corner turn test) were performed at 1, 3, 7, 14, 21, or 28 days after ICH. In male mice, brain water content was significantly increased in the ipsilateral basal ganglia 1 and 3 days after ICH, compared with saline injection controls (P < 0.01). There were marked neurological deficits 1 and 3 days after ICH, with progressive recovery over 28 days. In contrast, although brain edema and behavioral deficits were similar at 1 day after ICH in female and male mice, female mice showed reduced edema at 3 days and a faster recovery of behavioral deficits after ICH. 17 beta-estradiol treatment in male mice markedly reduced ICH-induced edema (P < 0.01). Brain water content was significantly increased in C5-deficient mice compared with C5-sufficient at 3 days after ICH (P < 0.05). These findings suggest that the mouse ICH model is a reproducible and feasible model. These results also suggest that gender and complement C5 are factors affecting brain injury after ICH.     

CVF干预对大鼠实验性脑出血后C9表达及脑水肿的影响

目的 研究大鼠实验性脑出血后血肿周围补体成分C9的表达情况及其与脑水肿的关系,探讨补体C9在脑出血后继发性损伤中的作用以及应用眼镜蛇毒因子（CVF）干预后的影响.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脑出血模型,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,连续切片作补体C9免疫组化染色和HE染色.结果 脑出血后2 h血肿周围脑组织开始表达C9,24 h达高峰;各组与假手术组之间比较有差异（P＜0.01）;血肿周围脑组织含水量在ICH后6 h开始增加（P＜0.05）,12 h明显增加,24～72 h达高峰（P＜0.01）,此后逐渐回落,2 w基本恢复正常水平.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;C9的表达脑组织含水量呈正相关关系（r=0.833,P＜0.05）;经CVF干预后,血肿周围组织C9表达量及脑组织含水量明显下降,干预组与出血组之间比较有显著差异（P＜0.01）.结论脑出血后补体级联激活,C9表达明显增加,脑组织含水量增加,经CVF干预后,C9表达下降,脑水肿减轻,能达到神经保护作用.     

脑出血后继发性脑损伤的机制与治疗

脑出血具有高发病率和高死亡率的特点。近年研究表明，脑出血产生的继发性脑损伤机 制主要涉及凝血酶诱导、红细胞裂解、毒性反应、氧化损伤和炎症反应等多个方面。根据不同的损 伤机制，应运而生了多种脑出血的治疗策略，但是否能成功应用于临床还有待进一步研究。本文对脑 出血后继发性损伤的潜在机制和新兴治疗方法进行了综述。     

脑出血损伤相关分子模式研究进展

脑出血后产生的免疫风暴可导致神经元及其支持细胞死亡,红细胞裂解释放的血红蛋白、血红素和铁离子等细胞毒性物质也具有促进神经细胞死亡的作用。神经细胞死亡后释放的损伤相关分子模式(DAMP)激活固有免疫反应,导致炎症反应-细胞死亡-DAMP释放-炎症反应的恶性循环,是脑出血后继发性脑损伤的重要机制。本文旨在对目前开展的一系列探讨DAMP在脑出血继发性脑损伤中作用的研究进展进行概述,以为进一步的基础研究与临床转化研究提供参考。     

NLRP3 is Required for Complement-Mediated Caspase-1 and IL-1beta Activation in ICH

Complement-mediated inflammation plays a vital role in intracerebral hemorrhage (ICH), implicating pro-inflammatory factor interleukin-1beta (IL-1β) secretion. Brain samples and contralateral hemiencephalon were all collected and detected by Western blot. NLRP3 expression was located by dual immunofluorescence staining at 1, 3, and 5 days post-ICH. Brain water content was examined post-ICH. The neural deficit scores were evaluated by observers blindly. ILs were detected by ELISA. SiRNAs targeting NLRP3 (siNLRP3), siASC, and siControl were injected to inhibit NLRP3 function. To test the complement activation via Nod-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), normal rabbit complement (NRC) was injected with lipopolysaccharide (LPS) to facilitate the complement function. As a result, complement 3a (C3a) and complement 5a (C5a) were upregulated during the ICH-induced neuroinflammation, and ablation of C3 attenuates ICH-induced IL-1β release. Though the LPS rescues the neuroinflammation in the ICH model, C3 deficiency attenuates the LPS-induced inflammatory effect. The NLRP3 inflammasome was activated after ICH and was located in the microglial cell of the mouse brain, which exhibits a time-dependent manner. However, the number of NLRP3/Iba-1 dual-labeled cells in the C3^?/? group is less than that in the WT group in each time course, respectively. IL-1β and IL-18 released in perihematoma tissue, caspase-1-p20, brain water content, and behavioral outcomes were attenuated in the siNLRP3 and siASC groups than in the siControl and ICH groups. We also found that 5% of complement supplement enhances ICH-induced IL-1β release, while NLRP3 and ASC inhibition attenuates it. In conclusion, complement-induced ICH neuroinflammation depended on NLRP3 activation, which facilities LPS- and ICH-induced neuroinflammation, and NLRP3 is required for ICH-induced inflammation.     

NF-κB activation and cell death after intracerebral hemorrhage in patients

Nuclear factor-κB (NF-κB) plays an important role in secondary damage after intracerebral hemorrhage (ICH). We explored NF-κB activation and the relationship between NF-κB and cell death in the perihematomal brain tissue of patients after ICH. According to the interval between onset of hemorrhage and specimen collection, 53 cases of patients with basal ganglia hemorrhage were divided into six experimental groups: 0–6, 7–12, 13–24, 25–48, 49–96, and >96 h group. Brain tissues of the experimental groups and control group were collected. IL-1β, TNF-α, and NF-κB p65 expressions at the protein level were detected by immunohistochemistry. Cell death was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. All of the detection items of immunohistochemistry and TUNEL showed significant differences between the experimental groups and control group. At the protein level, nuclear NF-κB p65, IL-1β, and TNF-α achieved maximum values at 13–48, 0–24, and 13–48 h, respectively. Maximum cell death was reached at 13–48 h. NF-κB activation increased dramatically in perihematomal brain tissue after ICH. NF-κB activation was closely related with cell death and had an important function in secondary brain damage after ICH in patients.     

Blast-induced moderate neurotrauma (BINT) elicits early complement activation and tumor necrosis factor alpha (TNFα) release in a rat brain

Blast-induced neurotrauma (BINT) is a major medical concern yet its etiology is largely undefined. Complement activation may play a role in the development of secondary injury following traumatic brain injury; however, its role in BINT is still undefined. The present study was designed to characterize the complement system and adaptive immune-inflammatory responses in a rat model of moderate BINT. Anesthetized rats were exposed to a moderate blast (120 kPa) using an air-driven shock tube. Brain tissue injury, systemic and local complement, cerebral edema, inflammatory cell infiltration, and pro-inflammatory cytokine production were measured at 0.5, 3, 48, 72, 120, and 168 h. Injury to brain tissue was evaluated by histological evaluation. Systemic complement was measured via ELSIA. The remaining measurements were determined by immunohistoflourescent staining. Moderate blast triggers moderate brain injuries, elevated levels of local brain C3/C5b-9 and systemic C5b-9, increased leukocyte infiltration, unregulated tumor necrosis factor alpha (TNFα), and aquaporin-4 in rat brain cortex at 3- and 48-hour post blast. Early immune-inflammatory response to BINT involves complement and TNFα, which correlates with hippocampus and cerebral cortex damage. Complement and TNFα activation may be a novel therapeutic target for reducing the damaging effects of BINT inflammation.