脑出血后血肿状态对神经功能损伤的动物实验性研究

目的探讨大鼠脑出血后早期血肿状态对神经功能损伤的影响。方法采用预置管二次注射法,分别用加入促凝剂(6-氨基己酸)、抗凝剂(低分子肝素钠)以及无促凝剂和抗凝剂的大鼠自体动脉血建立尾状核脑出血模型,不同时间点(1h、6h、12h、24h、3d)对比实验组大鼠血肿容积、脑组织水肿病理变化与神经功能损伤的关系。结果血肿容积比较:1h时间点(P0.05);6h、12h、24h时间点,抗凝组血肿容积大于其他2组,差异有统计学意义(P0.01);神经功能损伤比较:6h、12h、24h时间点,促凝组神经损伤评分大于其他2组(P0.01),1h、3d时间点差异均无统计学意义(P0.05);脑组织水肿比较:6h、12h、24h时间点,促凝组大鼠脑组织水肿程度重于其他2组(P0.01)。结论大鼠脑出血后急性期血肿不同状态对神经功能损伤有影响,液化血肿易于血液扩散,益于减轻神经功能损伤。     

Acupuncture through Baihui(DU20) to Qubin(GB7) mitigates neurological impairment after intracerebral hemorrhage

Inflammation plays an important role in nerve defects caused by intracerebral hemorrhage. Repairing brain damage by inhibiting the macrophage-inducible C-type lectin/spleen tyrosine kinase(Mincle/Syk) signaling pathway is a potential new target for treating cerebral hemorrhage. In this study, we aimed to determine whether acupuncture through Baihui(DU20) to Qubin(GB7) is an effective treatment for intracerebral hemorrhage through the Mincle/Syk signaling pathway. An intracerebral hemorrhage rat model was established by autologous blood infusion into the caudate nucleus. Acupuncture through Baihui to Qubin was performed for 30 minutes, once every 12 hours, for a total of three times. Piceatannol(34.62 mg/kg), a Syk inhibitor, was intraperitoneally injected as a control. Modified neurological severity score was used to assess neurological function. Brain water content was measured. Immunohistochemistry and western blot assay were used to detect immunoreactivity and protein expression levels of Mincle, Syk, and CARD9. Real-time polymerase chain reaction was used to determine interleukin-1β m RNA levels. Hematoxylin-eosin staining was performed to observe histopathological changes. Our results showed that acupuncture through Baihui to Qubin remarkably improved neurological function and brain water content, and inhibited immunoreactivity and expression of Mincle, Syk, CARD9, and interkeukin-1β. Moreover, this effect was similar to piceatannol. These findings suggest that acupuncture through Baihui to Qubin can improve neurological impairment after cerebral hemorrhage by inhibiting the Mincle/Syk signaling pathway.     

Early neurological change in patients with spontaneous supratentorial intracerebral hemorrhage

The frequency and causes of neurological change that occurs in patients within 24 hours after the onset of intracerebral hemorrhage (ICH), as well as their relationship to outcome, have seldom been reported. This study evaluated 184 patients with supratentorial ICH and neurological deterioration or improvement; measuring their level of consciousness (LOC) and motor skills the day after admission using the National Institutes of Health Stroke Scale. Nineteen patients (10%) deteriorated and 114 (62%) improved. Patient age, hematoma volume, and change in hematoma volume were independent predictors of early neurological improvement (p < 0.05). Independent predictors of 1-month functional outcome were age, LOC score at admission, motor score at admission, and change in motor score the day after admission (p < 0.05). Approximately 70% of the patients showed early neurological change. Observing early changes in hemiparesis was important for predicting functional outcome.     

Nec-1在小鼠脑出血中通过抑制程序性坏死发挥神经保护作用

目的研究程序性坏死的特异性抑制剂Nec-1对小鼠脑出血神经功能的保护作用,并探讨其机制。方法选取健康雄性ICR小鼠,体重25~30 g,采用在纹状体部位注射胶原酶Ⅳ或生理盐水的方法建立小鼠脑出血组或者脑出血对照组,在脑出血前15 min分别在侧脑室注射Nec-1溶液或vehicle溶液。将实验随机分为:对照组、脑出血+vehicle处理组、脑出血+Nec-1处理组。分别利用干湿重法测定脑水肿程度,神经功能评分检测神经运动功能,Western blotting检测cleaved caspase-3,Bcl-2蛋白表达情况。结果脑出血加剧脑水肿程度,而Nec-1处理组减轻脑水肿程度(P0.05)。经Nec-1处理后,可以提高小鼠脑出血神经运动功能(P0.05)。脑出血可以增加cleaved caspase-3蛋白表达,抑制Bcl-2蛋白表达,经Nec-1处理后,抑制cleaved caspase-3的表达水平(P0.05),增加Bcl-2的表达(P0.05)。结论 Nec-1对脑出血发挥重要神经保护作用,这种保护作用可能是通过抑制凋亡通路实现的,提示程序性坏死在脑出血中具有重要作用,将为脑出血的治疗提供新的思路。     

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.     

Cell-specific activation of RIPK1 and MLKL after intracerebral hemorrhage in mice

Receptor-interacting protein kinase-1 (RIPK1) is a master regulator of cell death and inflammation, and mediates programmed necrosis (necroptosis) via mixed-lineage kinase like (MLKL) protein. Prior studies in experimental intracerebral hemorrhage (ICH) implicated RIPK1 in the pathogenesis of neuronal death and cognitive outcome, but the relevant cell types involved and potential role of necroptosis remain unexplored. In mice subjected to autologous blood ICH, early RIPK1 activation was observed in neurons, endothelium and pericytes, but not in astrocytes. MLKL activation was detected in astrocytes and neurons but not endothelium or pericytes. Compared with WT controls, RIPK1 kinase-dead (RIPK1^D138N/D138N) mice had reduced brain edema (24 h) and blood-brain barrier (BBB) permeability (24 h, 30 d), and improved postinjury rotarod performance. Mice deficient in MLKL (Mlkl^-/-) had reduced neuronal death (24 h) and BBB permeability at 24 h but not 30d, and improved post-injury rotarod performance vs. WT. The data support a central role for RIPK1 in the pathogenesis of ICH, including cell death, edema, BBB permeability, and motor deficits. These effects may be mediated in part through the activation of MLKL-dependent necroptosis in neurons. The data support development of RIPK1 kinase inhibitors as therapeutic agents for human ICH.     

Pulmonary complications after intracerebral hemorrhage

Objective  

There are no data on pulmonary complications in an selected population of patients with intracerebral hemorrhages (ICH). At present, most data is extrapolated from series of patients with stroke that combine patients with both ischemic and hemorrhagic strokes. The purpose of this study was to determine the frequency, types of pulmonary complications in patients with ICH; their association with clinical variables, and contribution to hospital stay.     

Inflammation after intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a devastating clinical event without effective therapies. Increasing evidence suggests that inflammatory mechanisms are involved in the progression of ICH-induced brain injury. Inflammation is mediated by cellular components, such as leukocytes and microglia, and molecular components, including prostaglandins, chemokines, cytokines, extracellular proteases, and reactive oxygen species. Better understanding of the role of the ICH-induced inflammatory response and its potential for modulation might have profound implications for patient treatment. In this review, a summary of the available literature on the inflammatory responses after ICH is presented along with discussion of some of the emerging opportunities for potential therapeutic strategies. In the near future, additional strategies that target inflammation could offer exciting new promise in the therapeutic approach to ICH.     

亚低温对脑出血大鼠神经细胞凋亡及神经功能缺损的影响

目的 研究延迟性亚低温对大鼠脑出血后神经细胞凋亡及神经功能缺损的动态影响.方法 将大鼠随机分为正常组(N),假手术组(S),脑出血组(M),延迟12h亚低温组(HT12)和延迟24h亚低温组(HT24).应用立体定向技术将Ⅳ型胶原酶注人大鼠尾壳核制作脑出血模型,模型制备后于12h及24h点将大鼠放入冷室降温,维持肛温在(33±1)℃,均持续12h,各组在模型制作后12h、24h、36h、3d、7d记录大鼠神经功能缺损评分及死亡率.大鼠脑组织做HE染色及TUNEL染色,流式细胞技术观察神经细胞凋亡数目的变化.结果 亚低温(HT12,HT24)可减少脑出血后大鼠各时间点的TUNEL染色的阳性细胞数(P＜0.05);与模型组相比,3d时亚低温组( HT12,HT24)神经细胞凋亡率及大鼠7d的死亡率明显降低(P＜0.05),差别有统计学意义;延迟24h的亚低温可降低大鼠7d的NDS评分(P＜0.05),而12h的延迟治疗无此作用(P＞0.05).结论 延迟的亚低温治疗可减少大鼠脑出血后神经细胞的凋亡,降低死亡率,发挥脑保护作用.     

老龄与年轻大鼠脑出血后脑损伤神经缺失的研究

目的 研究观察年龄对脑出血后神经功能缺失的影响.方法 年轻(3个月龄)和老龄(16个月龄)雄性S-D(Sprague-Dawley)大鼠(河南省实验动物中心提供)脑内注入100 μL自体全血,观察与年龄相关的神经缺失的变化.所有的动物均由不知情的实验者在手术前后对神经症状进行评分.结果 神经行为测评:经过前肢放置得分评分、前肢使用不对称得分进行评价初期的(1 d)动作行为缺失,老龄与年轻大鼠比较差异明显.老龄组这一差异持续脑出血后2周,脑出血不影响同侧的(未受损的)前肢放置.与另外的两项测试对比,脑出血后转角试验得分神经功能恢复的证据很少,得分接近百分之百(最大极限的缺失).除14 d之外在老龄组之间得分没有显著的差异,此间老龄组大鼠有显著的神经缺失.结论 脑出血后时间依赖性的神经功能恢复很可能源于其可塑性,老龄大鼠脑出血的效应可能由早期损伤和神经功能恢复能力衰减的复合作用而加重.     

急性脑出血后中性粒细胞与淋巴细胞比值在预测患者神经功能恶化中的价值研究

目的探讨外周免疫细胞亚群计数及中性粒细胞与淋巴细胞比值(NLR)在预测急性脑出血(AICH)后神经功能恶化(ND)的价值。方法分析本院收治的87例AICH患者的临床资料。根据AICH后1w内有无ND进展将患者分为神经功能恶化组(ND组24例,占27.6%)和无神经功能恶化组(非ND组63例,占72.4%),并比较2组患者在入院时的外周免疫细胞亚群计数、NLR比值的差异性及与ND发生的相关性。结果 ND组患者的白细胞计数、中性粒细胞计数均高于非ND组患者,分别为10.80±4.44vs8.02±2.24(P0.001)和9.23±4.97vs5.02±1.87(P0.001),而淋巴细胞计数低于非ND组患者1.45±0.58vs2.43±1.87(P=0.025),故ND组患者的NLR比值明显高于非ND组患者9.45±5.40vs3.28±1.94(P0.001)。通过回归分析发现AICH后外周血细胞计数、NLR比值与ND的发生密切相关(OR:1.87,95%CI:1.45~2.23),P0.001)。根据ROC曲线下面积证实NLR比值(面积为0.872,P0.001)对ND发生的预测价值高于白细胞计数、中性粒细胞和淋巴细胞计数(面积分别为0.749,0.823和0.721,均P0.001)。结论 NLR比值的升高对预测AICH后ND的发生具有重要的临床价值,能够为AICH患者的预后管理提供理论依据。     

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.     

Autophagy after experimental intracerebral hemorrhage.

Autophagy contributes to ischemic brain injury, but it is not clear if autophagy occurs after intracerebral hemorrhage (ICH). This study examined whether ICH-induced cell death is partly autophagic. It then examined the role of iron in inducing this form of cell death after ICH. Male, adult Sprague-Dawley rats received an infusion of autologous whole blood or ferrous iron into the right basal ganglia. Control rats (sham) had a needle insertion. The rats were killed at 1, 3, 7, or 28 days later. Some rats were treated with either deferoxamine or vehicle after ICH. Microtubule-associated protein light chain-3 (LC3), a biomarker of autophagosome, and cathepsin D, a lysosomal biomarker, were measured by Western blot analysis and immunohistochemistry. Immunofluorescent double-labeling was used to identify the cell types expressing cathepsin D. Electron microscopy was performed to examine the cellular ultrastructure changes after ICH. We found that conversion of LC3-I to LC3-II, cathepsin D expression, and vacuole formation are increased in the ipsilateral basal ganglia after ICH. Intracerebral infusion of iron also resulted in enhanced conversion of LC3-I to LC3-II and increased cathepsin D levels. Deferoxamine (an iron chelator) treatment significantly reduced the conversion of LC3-I to LC3-II and cathepsin D levels after ICH. Our results demonstrated that autophagy occurs after ICH, and iron has a key role in ICH-induced autophagy. This also suggests that iron-induced autophagy may play a role in brain injury in other diseases associated with iron overload.