小胶质细胞在蛛网膜下腔出血后脑损伤中作用机制的研究进展

小胶质细胞是位于中枢的单核巨噬细胞系统成员,对维持中枢神经系统的稳态有重要意义。蛛网膜下腔出血（SAH）是脑卒中的一种亚型,其发生后会造成72 h内早期脑损伤,以及SAH后3～15 d迟发性脑损伤。以往认为,血管痉挛在SAH后脑损伤中发挥重要作用,但脑损伤中的炎症反应近年逐渐受到重视,其中小胶质细胞可在SAH后早期和延迟相脑损伤的炎症反应中向不同方向极化,既可发挥促炎作用,也可产生神经保护作用。我们综述近几年小胶质细胞在SAH后脑损伤中发挥的作用及相关治疗靶点的研究进展及相关热点,为临床处理该类疾病提供潜在治疗靶点。     

大鼠肢体缺血再灌注所致脑损伤及其机制探讨

目的:观察肢体缺血-再灌注对脑的损伤作用,并探讨其可能的机制。方法:SD大鼠随机分为正常(N)、假手术(S)、后肢单纯缺血(I)及缺血-再灌注(I-R)各时间点组。通过夹闭腹主动脉末端4h、开放2-24h复制I、I-R组模型。光、电镜观察脑的病理变化;反转录多聚酶链反应及免疫组化染色法,观察脑组织iNOS表达的变化及过氧亚硝基阴离子(ONOO^-)的硝基化产物硝基酪氨酸(NT)的生成与分布;比色法测定脑组织MDA含量及SOD活性。结果:①I-R组脑组织水肿,神经元受损较重,S、I组未见异常;②S、I、I-R组iNOS均有表达,I-R6h表达量最大。I-R组大脑皮质、海马等区可见弥散分布的NT阳性神经元;③I-R6h组MDA含量显著高于N、S、I组,SOD活性显著低于这些组(P＜0.05);而N、S、I组之间无显著差别。结论:肢体I-R能引发脑损伤,脑内高表达的iNOS-NO-ONOO^-可能是脑损伤的重要因素。     

Response of astrocytes and microglia/macrophages to brain injury after bone marrow stromal cell transplantation: a quantitative study

The aim of the present study was to investigate the effects of intracerebral and intravenous administration of bone marrow stromal cells (BMSCs) on cellular activity in the injured brain. Female Wistar rats were subjected to cerebral cortex injury followed by the injection of BMSCs or saline, directly to the injured site or to the tail vein. Lectin histochemistry and glial fibrillary acidic protein immunohistochemistry were used to analyze the number of microglia/macrophages and astrocytes in the injured cerebral cortex, respectively. BMSC treatment affected cell response to brain injury. The effects of BMSC action were dependent on the site of their administration. The intravenous injection of BMSCs noticeably increased the number of microglia/macrophages in the injured brain. Intracerebral transplantation of BMSCs significantly enhanced the number of astrocytes, and in a less degree caused changes in the number of microglia/macrophages. The results suggest that BMSCs can affect the restorative processes in the injured brain through stimulation of the cellular response to injury.     

Constitutive and inducible nitric oxide synthases after dynorphin-induced spinal cord injury

It has recently been demonstrated that selective inhibition of both neuronal constitutive and inducible nitric oxide synthases (ncNOS and iNOS) is neuroprotective in a model of dynorphin (Dyn) A(1-17)-induced spinal cord injury. In the present study, various methods including the conversion of 3H-L-arginine to 3H-citrulline, immunohistochemistry and in situ hybridization are employed to determine the temporal profiles of the enzymatic activities, immunoreactivities, and mRNA expression for both ncNOS and iNOS after intrathecal injection of a neurotoxic dose (20 nmol) of Dyn A(1-17). The expression of ncNOS immunoreactivity and mRNA increased as early as 30 min after injection and persisted for 1-4 h. At 24-48 h, the number of ncNOS positive cells remained elevated while most neurons died. The cNOS enzymatic activity in the ventral spinal cord also significantly increased at 30 min 48 h, but no significant changes in the dorsal spinal cord were observed. However, iNOS mRNA expression increased later at 2 h, iNOS immunoreactivity and enzymatic activity increased later at 4 h and persisted for 24-48 h after injection of 20 nmol Dyn A(1-17). These results indicate that both ncNOS and iNOS are associated with Dyn-induced spinal cord injury, with ncNOS predominantly involved at an early stage and iNOS at a later stage.     

Global hypomethylation defines a sub-population of reactive microglia/macrophages in experimental traumatic brain injury

Global alterations in gene expression have been observed in different traumatic brain injury (TBI) models and are considered of crucial importance to the development of subsequent tissue injury and repair. Cytosine methylation is a well-known process of endogenous DNA modification in mammals and the primary mechanism responsible for changes in epigenetic gene expression. Here we have investigated the early global spatio-temporal changes of the status of cellular DNA methylation in a rat TBI model by immunohistochemistry and analyzed the effects of dexamethasone on these changes. Global cellular hypomethylation was seen as early as day 1 in pannecrosis and day 2 in peripannecrosis following TBI. A sub-population of reactive microglia/macrophages was identified as the major source of hypomethylated cells by double-staining experiments. Further, peripheral administration of dexamethasone suppressed this lesional hypomethylation at day 2 post-injury. In sum, our data suggest that lesional hypomethylation defines a sub-population of activated microglia/macrophages involved in the early processes following traumatic brain injury.     

Sinomenine inhibits microglia activation and attenuates brain injury in intracerebral hemorrhage

Intracerebral hemorrhage (ICH) causes morbidity and mortality and commonly follows the reperfusion after an ischemic event. Microglial activation mediated cytokine and protease secretion contributes to brain injury in ICH. Previous studies have shown that sinomenine possesses potent immunoregulatory properties. However, little is known about its exact role in ICH. In the present study, to investigate the effect of sinomenine on microglial cells inflammation, we treated ICH-challenged BV2 microglial cells with sinomenine in vitro, and explored its neuroprotection role in intracerebral hemorrhage in vivo. Changes in inflammatory cytokines, such as TNF-α, IL-1β and IL-6, reactive oxygen species (ROS) and NF-κB activation NF-κB were observed. In addition, the neurological deficit and cerebral water content of ICH mice were studied. The results demonstrated that sinomenine could inhibit the release of these cytokines and attenuate ROS production in a dose-dependent manner, and reduce NF-κB activation. Furthermore, sinomenine markedly inhibited cerebral water content and neurological deficit. In conclusion, our findings suggest that sinomenine played the protective effects through inhibition of microglial inflammation, and the findings also provided a novel therapy to treat ICH induced brain injury.     

NG-硝基-L-精氨酸对大鼠内毒素性肺线粒体损伤的影响

目的： 观察非选择性一氧化氮合酶(NOS)抑制剂NG-硝基-L-精氨酸(L-NA)对急性肺损伤大鼠肺线粒体功能的影响，并探讨其改善急性肺损伤的作用机制。方法: 将SD大鼠随机分为空白对照组、急性肺损伤组、L-NA治疗组，采用舌静脉注射脂多糖(LPS)复制大鼠急性肺损伤模型，于大鼠急性肺损伤3h后给L-NA治疗3h，断头放血处死大鼠，迅速取出肺脏，匀浆器混匀后，低温差速离心法提取肺线粒体，测定线粒体总ATP酶、超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、总一氧化氮合酶（T-NOS）、诱生型一氧化氮合酶（iNOS）、结构型一氧化氮合酶（cNOS）的活性，以及线粒体肿胀度、膜流动性和线粒体一氧化氮（NO）、丙二醛（MDA）含量；电镜观察大鼠肺线粒体超微结构的改变及治疗药对此改变的影响。结果: 在大鼠内毒素性急性肺损伤后，肺脏组织中线粒体表现为肿胀、膜流动性降低，线粒体中的T-NOS和iNOS活性显著升高，线粒体NO生成明显增加，而cNOS活性无明显变化；线粒体总ATP酶、SOD、GSH-Px活性均明显下降，线粒体MDA含量明显升高。急性肺损伤3h给予L-NA治疗3h,与急性肺损伤组相比，一氧化氮合酶活性有所改变，NO生成显著下降，总ATP酶、SOD、GSH-Px活性均显著升高，MDA含量下降。电镜结果显示内毒素性急性肺损伤后肺脏组织细胞水肿，线粒体肿胀、嵴断裂、溶解、消失；L-NA能改善内毒素性急性肺损伤引起的细胞水肿、线粒体肿胀和空泡化。结论: L-NA能明显抑制急性肺损伤后线粒体一氧化氮合酶活性，减少NO生成，改善线粒体能量供应，增加线粒体抗氧化作用，从而减轻急性肺损伤。     

活化的小胶质细胞在大鼠海马神经元缺氧损伤中的作用

目的 探讨缺氧诱导活化的小胶质细胞在SD大鼠海马神经元缺氧损害中的作用机制.方法 建立共培养体系,应用原位缺口末端标记(TUNEL)法、化学发光法探讨不同组别神经元生长状况以及Caspase-3活性;采用免疫荧光法、格里斯试剂法(Griess Reagent)、还原WST-1法、酶联免疫吸咐测定(ELISA)等方法检测各组培养液中NO、O-2以及TNF-α的表达水平.结果 缺氧12h, N9细胞培养液可抑制常规培养的神经元生长增殖活力,同时可加重由缺氧抑制的共培养体系中神经元活力;既可诱导常规培养的神经元凋亡,又可促进共缺氧培养的神经元凋亡;较之于单纯神经元培养系和常规共培养系,共缺氧培养系的培养液中NO、O-2、TNF-α 3类应激性神经毒性因子产量最高.结论 小胶质细胞活化在缺氧诱发的神经元损害中发挥了重要的作用,其活化后产生的神经毒性分子是效应分子.     

肾缺血-再灌注损伤引起的脑生化改变

目的:观察血清及脑脊液生化成分变化，初步探讨肾缺血-再灌注损伤对脑的影响。 方法: 20只健康新西兰兔随机分为对照组和肾缺血再灌注组（IR组），检测和比较血清和脑脊液中肌酐（Cr）、尿素氮(UN)、Na+ 、Ca2+、一氧化氮（NO）代谢产物含量及脑组织总一氧化氮合酶（NOS）活力。 结果: 在肾缺血再灌注后24 h，IR组血清和脑脊液中的UN、Cr和NO均显著高于对照组（P<0.05），Na+明显低于对照组；IR组血清Ca2+显著低于（P<0.05）、而脑脊液中Ca2+显著高于对照组（P<0.05）。IR组脑组织中NO2-/NO3-含量和NOS活力均明显高于对照组（P<0.05）。 结论: 肾缺血再灌注损伤不仅影响血清成分，而且还可引起脑生化的改变；NO可能参与了肾缺血-再灌注损伤对脑功能的影响。     

White matter-associated microglia: New players in brain aging and neurodegenerative diseases

There has been growing interest in brain aging and rejuvenation. It is well known that brain aging is one of the leading causes of neurodegenerative diseases, such as Alzheimer’s disease, but brain aging alone can cause cognitive decline. Microglia are thought to act as ‘conductors’ of white matter aging by modulating diverse glial cells and phagocytosing white matter-derived myelin debris. A recent study identified a specific subpopulation of microglia in the white matter of aged mice, termed white matter-associated microglia (WAM). Additionally, senescent microglia show impaired phagocytic function and altered lipid metabolism, which cause accumulation of lipid metabolites and eventually lead to myelin sheath degeneration. These results suggest that senescent WAM could be pivotal players in axonal loss during brain aging. The aim of this review is to assess the current state of knowledge on brain aging, with an emphasis on the roles of the white matter and microglia, and suggest potential approaches for rejuvenating the aged brain.     

神经生长因子减轻大鼠脑缺血再灌注耳蜗损伤

目的:研究神经生长因子(NGF)对脑缺血再灌注引起的听力及耳蜗损伤的作用。方法:本研究给实验组大鼠脑缺血再灌注前后肌注NGF,对照组动物肌注等量生理盐水,于处理前后对两组的听力损失及耳蜗结构变化加以比较。结果:缺血30min再灌注60min和24h,实验组的听力损失明显小于对照组(P<001)。耳蜗的扫描和透射电镜观察发现,对照组毛细胞静纤毛粘连、部分脱落,外毛细胞肿胀、散在性坏死缺失,螺旋神经节神经元变性,线粒体水肿、嵴断裂。实验组耳蜗Corti’s器毛细胞及螺旋神经节神经元的损伤明显轻于对照组。结论:NGF能减轻脑缺血再灌注引起的听力及耳蜗损伤。     

一氧化氮和过氧亚硝基阴离子在肢体缺血再灌注致肺损伤中的作用

目的:观察肢体缺血再灌注致肺损伤时肺组织中一氧化氮(NO)及过氧亚硝基阴离子(ONOO^-)的变化,以探讨二者在此种损伤中的作用。方法:采用夹闭大鼠腹主动脉下段造成双下肢缺血和再灌注后肺损伤模型,分别测定假手术组、缺血4h组、缺血4h再灌注1h组及再灌注4h组肺组织匀浆中超氧化物歧化酶(SOD)活性和丙二醛(MDA)、NO₂^-/NO₃^-含量变化;应用免疫组化方法测定上述各组肺组织中诱导型一氧化氮合酶(iNOS)及ONOO^-体内生成标志物硝基酪氨酸(NT)的变化。结果:肢体缺血再灌注后1h和4h肺组织中MDA和NO₂^-/NO₃^-的含量显著高于对照组和单纯缺血组(P＜0.05),而SOD活性则显著低于此两组(P＜0.05),并出现大量iNOS及NT阳性信号。结论:肢体缺血再灌注致肺损伤时肺组织中有大量NO和ONOO^-产生,脂质过氧化增强,提示ONOO^-参与介导此种肺损伤。     

人参二醇组皂苷（PDS）抑制NOS和p38减轻LPS休克脑损伤

目的：探讨人参二醇组皂苷（PDS）对LPS诱导休克脑的保护机制。方法：将大鼠随机分为对照组、LPS组、LPS＋地塞米松组及LPS＋人参二醇皂苷组。大鼠静脉注射LPS（4 mg/kg）4 h后，测定大脑皮质中NOS的活性、NO的含量及磷酸化p38 蛋白的表达。结果：LPS组脑皮质中NOS活性、NO含量及p38的蛋白表达水平明显高于control组，而LPS+Dex组和LPS+PDS组脑皮质中NOS活性、NO含量及p38的蛋白表达水平明显低于LPS组。结论：PDS减轻LPS脑损伤与降低脑皮质中NOS活性、NO含量有关，可能涉及p38MAPKs信号转导通路。     

孕酮抑制大鼠脑损伤后炎症反应诱导的神经细胞凋亡

目的:研究孕酮(progesterone,PROG)对大鼠脑损伤后皮质COX-2、iNOS、NF-κB和caspase-3表达的影响,以探讨PROG脑保护作用的可能机制。方法:雄性SD大鼠45只,随机分为假手术组、脑损伤组和PROG治疗组,按照改良的Feeney自由落体损伤装置制作大鼠脑损伤模型。PROG治疗组伤后1,6h腹腔注射PROG16mg/kg,各组于伤后24h取材。用免疫组织化学法及免疫蛋白印迹法,观察大鼠皮质COX-2、iNOS、NF-κB和caspase-3的免疫阳性细胞数和蛋白表达水平变化。结果:与假手术组大鼠相比,脑损伤组大鼠皮质COX-2、iN-OS、NF-κB和caspase-3阳性细胞数和蛋白表达水平较假手术组COX-2、iNOS、NF-κB和caspase-3显著增加(P0.05);PROG治疗组与脑损伤组比较,大鼠皮质COX-2、iNOS、NF-κB和caspase-3阳性细胞数和蛋白表达水平明显减少(P0.05)。结论:PROG可通过抑制NF-κB的活化,阻断NF-κB和iNOS自身强化的炎症反应过程,降低COX-2、caspase-3的表达,抑制脑损伤后的炎症反应和细胞凋亡,从而发挥脑保护作用。     

四逆汤对缺血-再灌注离体鼠肺的保护作用

目的： 建立离体大鼠肺灌流模型，研究四逆汤（SND）对缺血-再灌注肺的保护作用及可能机制。方法：将SD大鼠随机分成假手术组、模型组和模型+SND组，观察SND对大鼠肺组织形态学改变、平均肺动脉压（MPAP）、肺组织湿/干重比（W/D）、灌流液及肺组织匀浆中超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量以及肺组织匀浆中一氧化氮（NO）含量和一氧化氮合酶（NOS）活力的影响。结果：模型+SND组的肺泡壁增厚程度和肺泡水肿程度显著低于模型组，肺组织W/D值及MPAP显著小于模型组，灌流液和肺组织匀浆中SOD活性显著高于模型组，而MDA含量显著低于模型组。SND可显著抑制缺血-再灌注引起的肺组织NO含量的减少。但3组间比较，NOS含量无显著差异。结论：SND有抗大鼠肺缺血-再灌注损伤作用，其机制可能与清除氧自由基和改善组织灌流有关。     

过氧亚硝基阴离子对气道上皮细胞的损伤作用

目的:探讨过氧亚硝基阴离子(ONOO^-)对气道上皮细胞的损伤作用。方法:在培养的大鼠气道上皮(RTE)细胞观察外源性给予ONOO^-对RTE细胞线粒体呼吸、8-羟基脱氧鸟苷(8-OHdG)水平、乳酸脱氢酶(LDH)释放及凋亡细胞百分率的影响。结果:ONOO^-(0.25-1mmol/L)呈剂量依赖方式抑制RTE细胞线粒体呼吸功能、增高LDH释放率。并呈剂量依赖性引起8-OHdG水平升高。不同浓度(0.25mmol/L、0.5mmol/L及1mmol/L)ONOO^-均以时间依赖方式引起RTE细胞凋亡。结论:ONOO^-可引起培养的RTE细胞发生凋亡和坏死。低浓度的ONOO^-损伤RTE细胞以凋亡为主;高浓度的ONOO^-可能主要引起RTE细胞发生坏死。ONOO^-对RTE细胞线粒体呼吸抑制和DNA的氧化损伤可能是ONOO^-导致RTE细胞坏死和凋亡的主要原因。     

不同药物对烟雾吸入性损伤大鼠肺保护作用的研究

目的探讨乌司他丁、地塞米松和氨溴索对烟雾吸入性损伤大鼠肺脏损害的保护作用及机制。方法SD大鼠随机分为5组，C组为正常对照组，A组、D组和U组大鼠分别在炯雾致伤后给予氨溴索、地塞米松、乌司他丁腹腔注射，M组大鼠致伤后注射等量生理盐水。各组大鼠在致伤6h后测血清肿瘤坏死因子（TNF-α）、白细胞介素6（IL-6）含量。取肺组织匀浆测超氧化物歧化酶（SOD）、丙二醛（MDA）、一氧化氮（NO）、诱导型一氧化氮合酶（iNOS）含量，右下肺组织病理切片光镜观察。结果光镜下见C组大鼠肺组织正常，M组肺间质出血，肺泡腔内可见大量粒细胞聚集、浸润，肺泡间隔增厚，而U、D组上述病理表现明显减轻。与M组相比，U组TNF-α、IL-6及MDA含量明显降低（P〈0．05），D组上二述指标显著降低（P〈0．01）。与C组比，A组NO、iNOS、SOD浓度明显增高（P〈0．05）。D组和U组iNOS与C组比无统计学差异（P〉0．05）。结论乌司他丁和地塞米松可能通过减少炎性介质和自由基的产生对吸入性损伤大鼠肺脏产生保护作用，氨溴索对吸入性损伤大鼠肺脏保护作用不强。     

Insulin-induced oxidative stress in the brain is nitric oxide-dependent

Insulin is known to increase brain nitric oxide (NO) level and to cause oxidative stress but the relationship between these phenomena has not been well elucidated. This study aimed to examine the role of NO in the insulin-NO-oxidative stress axis in the brain. Mice were grouped into four (n = 5) and treated for seven days with 0.2 ml deionized water (control); 10 I.U./kg insulin; 10 I.U./kg insulin + 50 mg/kg L-NAME; and 50 mg/kg L-NAME. The mice were anaesthesized using ketamine + xylazine and sacrificed at the end of the study. Forebrain was immediately harvested from which brain homogenates were prepared in order to determine NO and malondialdehyde (MDA) concentrations as well as glutathione peroxidase (GPx) activity using commercially available kits. Data were processed using IBM SPSS Statistics 20.0. Nitric oxide values were higher in the insulin group (p < 0.05) but not in the insulin+L-NAME (p > 0.05) group when compared with the control. Values of MDA in the insulin and insulin+L-NAME groups were higher (p < 0.05) and the same (p > 0.05), respectively, than those in the control group. The activity of GPx in the insulin group was lower (p < 0.05) than, but that of the insulin+L-NAME was the same (p > 0.05) as in the control group. Insulin increased NO concentration and oxidative stress as indicated by increased MDA concentration and decreased GPx activity in the treated mice. This insulin effect was reversed by L-NAME (a non-specific NO inhibitor). These data suggest that insulin increased oxidative stress in the brain through an NO-dependent process. Insulin treatment may be harmful to the brain.     

miR-203 protects microglia mediated brain injury by regulating inflammatory responses via feedback to MyD88 in ischemia

Much evidence demonstrates that microglia mediated inflammatory responses play an important role in brain injury in ischemia. miRNA is the important factor in regulation of inflammation. However, the effect of miRNA in microglia mediated inflammatory responses has not been well studied. In the study, we demonstrate that miR-203 negatively regulates ischemia induced microglia activation by targeting MyD88, an important adapter protein involved in most Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R) pathways. Through negative feedback, enforced expression of miR-203 or MyD88 siRNA silencing inhibits downstream NF-κβ signaling and microglia activation, thereby alleviating neuronal injury. These findings reveal that miR-203 represents a novel target regulating neuroinflammation and brain injury, thus offering a new therapeutical strategy for cerebral hypoxic diseases.     

自噬在创伤性脑损伤中的作用

自噬(autophagy)是真核细胞在营养与能量缺乏情况下,通过分解亚细胞成分提供生物合成原料,使蛋白质和细胞器得以循环利用的一种降解代谢途径,同时调控线粒体更新及过氧化物酶体等,从而维持细胞稳态.