Production and Purification of Antibody to Bovine White Matter Proteolipid Apoprotein

Circulating antibody to the bovine white matter proteolipid apoprotein was detected in rabbits 1 month after a single injection of the water-soluble form of the apoprotein. By double immunodiffusion, the antiserum reacted specifically with the delipidated proteolipid apoprotein and the crude proteolipid fraction containing complex lipids; after exposure of the proteolipid apoprotein to sodium dodecyl sulfate (SDS), no reactivity was observed. The antiserum did not react with other myelin components, i.e., basic protein, cerebroside or G_M1 ganglioside, nor was there reactivity with non-neural proteolipids. The anti-apoprotein antibody was purified by affinity chromatography. The antibody-antigen interaction is apparently very hydrophobic, since elution of the antibody from the affinity column requires buffer containing 0.5% Triton X-100-4 M-urea.     

脑梗死患者的脑白质病变危险因素分析

目的：探索脑梗死患者中脑白质病变的危险因素。方法：以2012 年1 月至2014 年12 月我院神经内科收治的837 例脑梗死 患者为研究对象,采用Fazekas量表评价核磁共振T2WI脑室旁白质和皮质下白质病变严重程度。分别比较不同程度脑室旁白质 病变、皮质下白质病变患者间各个危险因素的差异,采用Logistic 回归分析脑白质病变的危险因素。结果：年龄、高血压是脑室旁 白质（OR 年龄=1.090,95%CI:0.073-0.099,P＜0.05;OR 高血压=1.699,95%CI:0.242-0.818,P＜0.05）和皮质下白质病变（OR 年龄 =1.074,95%CI:0.059-0.083,P＜0.05;OR高血压=1.353,95%CI:0.017-0.588,P＜0.05）共同的危险因素。收缩压（OR=1.008,95%CI: 0.001-0.015,P＜0.05）是皮质下白质病变的危险因素。结论：在脑梗死患者中,年龄、高血压是脑室旁白质病变、皮质下白质病变共 同的危险因素,收缩压是皮质下白质病变的危险因素。     

Induction of cerebroside synthesis in oligodendroglia

Oligodendroglia function to produce myelin membranes which surround axons, enhancing saltatory conduction. Myelin consists of a multitude of condensed membranes which are rich in lipids with the major glycolipids, cerebrosides, being 25% of the total lipid. Thus a fully differentiated oligodendroglial cell that is producing myelin membranes would be actively synthesizing cerebrosides. Our laboratory has prepared and analyzed oligodendroglia from mature bovine brain, from neonatal rat brain, and from actively myelinating rat brain. Our studies suggest that the rat oligodendroglia in our culture systems are less differentiated than bovine cells in that they produce lower levels of cerebrosides. Addition of glucocorticoids, thyroid hormone, or retinoic acid all increased synthesis of cerebrosides in rat oligodendroglia. Ketone bodies were also somewhat stimulatory. Having no effect or causing dedifferentiation of the cells were 5-azacytidine and phorbol esters. Thus induction of cerebroside synthesis in oligodendroglia is complex and may involve many factors.     

Identification of Plasmolipin as a Major Constituent of White Matter Clathrin-Coated Vesicles

We have isolated and characterized coated vesicles from bovine white matter and compared them to those isolated from gray matter. The virtual absence of synaptic vesicle antigens in the white matter coated vesicles indicates they are distinct from those found in gray matter and from vesicles derived from synaptic membranes. The white matter coated vesicles also lack compact myelin components, e.g., the myelin proteolipid, galactocerebroside, and sulfatides, as well as the periaxolemmal myelin marker myelin-associated glycoprotein. On the other hand, these vesicles contain 2',3'-cyclic nucleotide phosphohydrolase. The vesicles also contain high levels of plasmolipin, a protein present in myelin and oligodendrocytes. Plasmolipin was found to be four to five times higher in white matter coated vesicles than in gray matter coated vesicles. Based on western blot quantitation, the concentration of plasmolipin in white matter coated vesicles is 3-4% of the vesicle bilayer protein. These studies indicate that a significant proportion of coated vesicles from white matter may be derived from unique membrane domains of the myelin complex or oligodendroglial membrane, which are enriched in plasmolipin.     

Lectin-Reactive Components in White Matter Membranes from Normal and Multiple Sclerosis Brains

Abstract: Polypeptides derived from human white matter membranes reacted with the radioiodinated lectins concanavalin A, Lens culinaris phytohemagglutinin, Ricinus communis agglutinin and wheat germ agglutinin after electrophoresis in polyacrylamide pore gradient gels. The molecular weights of these lectin-reactive bands were estimated by comparison with radioiodinated protein standards by using the linear relationship between log of the molecular weight and log of the gel concentration reached by the protein after electrophoresis in a polyacrylamide gradient gel. The molecular weight estimates for components reactive with concanavalin A were 176,800, 141,200, 72,800, 52,800, 44,700, 40,000, 24,800 and 23,900. The molecular weights of the bands reactive with both wheat germ agglutinin and Lens culinaris phytohemagglutinin were 138,000, 113,500, 92,100, 52,800, 44,700, 24,800 and 23,900. Wheat germ agglutinin was bound also to a band with a molecular weight of 72,800. Ricinus communis agglutinin bound to bands with estimated molecular weights of 138,000, 72,800, 52,800, 44,700, 24,800 and 23,900. The electrophoretic pattern of lectin-reactive polypeptides derived from normal-appearing white matter of multiple sclerosis brains was not qualitatively different from the lectin-binding pattern of control brain membrane polypeptides.     

Melatonin Promotes Oligodendroglial Maturation of Injured White Matter in Neonatal Rats

Objective

To investigate the effects of melatonin treatment in a rat model of white matter damage (WMD) in the developing brain. Additionally, we aim to delineate the cellular mechanisms of melatonin effect on the oligodendroglial cell lineage.

Methods

A unilateral ligation of the uterine artery in pregnant rat at the embryonic day 17 induces fetal hypoxia and subsequent growth restriction (GR) in neonatal pups. GR and control pups received a daily intra-peritoneal injection of melatonin from birth to post-natal day (P) 3.

Results

Melatonin administration was associated with a dramatic decrease in microglial activation and astroglial reaction compared to untreated GR pups. At P14, melatonin prevented white matter myelination defects with an increased number of mature oligodendrocytes (APC-immunoreactive) in treated GR pups. Conversely, melatonin was not found to be associated with an increased density of total oligodendrocytes (Olig2-immunoreactive), suggesting that melatonin is able to promote oligodendrocyte maturation but not proliferation. These effects appear to be melatonin-receptor dependent and were reproduced in vitro.

Interpretation

These data suggest that melatonin has a strong protective effect on developing damaged white matter through decreased microglial activation and oligodendroglial maturation leading to a normalization of the myelination process. Consequently, melatonin should be a considered as an effective neuroprotective candidate not only in perinatal brain damage but also in inflammatory and demyelinating diseases observed in adults.     

Free Sterols of the Rabbit Optic Nerve and Cerebral White Matter During Ontogenic Development

Abstract: Free sterol composition of the developing rabbit optic nerve was compared with that of the homologous cerebral white matter at corresponding stages of ontogeny. The sterols were detected and identified by means of combined gas-chromatography and mass spectrometry. The following free sterols were found in both the optic nerve and cerebral white matter: cholesterol, desmosterol, lanosterol, two dimethylsterols, which are probably 4,4-dimethyl-5α-cholest-8,24-diene-3β-ol, with a molecular weight of 412, and 4α,14α-dirnethyl-5α-cholest-7-ene-3β-ol, with a molecular weight of 414 and probably cholestene, with a molecular weight of 368. The sterol spectrum of the developing optic nerve differed not only from that of the mature nerve but also from that of age-matched white matter of the rabbit brain. The tri- and dimethyl-sterols, detected for the first time in the rabbit optic nerve and cerebral white matter, are natural components of the developing nervous tissue but they were not found in the mature nerve nor in cerebral white matter.     

利用激光显微切割与microarray 技术对恒河猴脑中白质与 灰质基因表达的差异性的研究

目的:利用激光显微切割技术和microarray技术比较恒河猴脑组织中前额叶皮质(prefontal cortex,PFC)与小脑皮质(cere-bellar cortex,CBC)的灰质与白质基因表达的差异。方法:利用激光显微切割技术(laser capture dissection,LCM)与microarray技术的有效结合,提取恒河猴PFC与CBC的白质与灰质,分别提取RNA,合成cDNA文库。最后利用GeneChip 1.0 ST芯片技术,分析得出大脑与小脑中灰质与白质的表达差异性。结果:无论是灰质还是白质,在PFC中的高表达基因都要远远多于在CBC中的高表达基因。结论:使用LCM可以提取单一的细胞群,从而用于要求更为精确的实验当中。     

脑出血后白质微观结构损伤与血管性认知功能障碍的相关性研究

目的:探讨脑出血后白质微观结构损伤情况和血管性认知功能功能障碍(VCI)的相关性。方法:选择2012年1月至2017年12月我院收治的脑出血患者50例作为研究对象,所有患者按照神经心理学评估结果分为认知功能障碍组(A组)29例和认知正常组(B组)21例,观察和比较两组患者病灶情况和白质微观结构的改变情况。结果:A组患者额区、基底核区病灶数量,病灶直径、白质病变、美国国立卫生院卒中量表(NIHSS)_评分等指标均明显高于B组,改良巴氏指数(MBI)评分低于B组(P0.05);将蒙特利尔认知评估(MoCA)量表评分作为因变量,MBI评分和NIHSS量表评分、病灶直径、病灶数量、白质病变程度等作为自变量进行多元Logistic回归分析,结果显示白质病变程度是脑出血患者VCI独立危险因素(P0.05)。结论:脑出血患者白质缺血性病变程度可能用于评估其发生VCI的风险。     

Ischemia-Induced Accumulation of Extracellular Amino Acids in Cerebral Cortex, White Matter, and Cerebrospinal Fluid

Abstract: In a global model of brain ischemia, accumulation of amino acids was studied in the extracellular space of the auditory cortex and the internal capsule using microdialysis, and in CSF of halothane anesthetized cats. In both brain regions, blood flow determined by hydrogen clearance decreased below 10 ml/100 g/min after extracranial multiple-vessel occlusion, and extracellular potassium activity ( K _e) measured in the dialysate increased significantly. A delayed rise in K _e was observed in CSF. In contrast, ischemic amino acid accumulation differed markedly between the two brain regions investigated. In cortex, transmitter amino acids glutamate, aspartate, and γ-aminobutyric acid (GABA) rose almost immediately after onset of ischemia, and increased 30-, 25-, and 250-fold, respectively, after 2 h of ischemia. The nontransmitter amino acids taurine, alanine, and serine increased 10-, seven-, and fourfold, respectively, whereas glutamine and essential amino acids (valine, phenylalanine, isoleucine, and leucine) increased only 1.5-fold. In the internal capsule, increases in amino acids, if any, were delayed and much smaller than in cortex. The largest alteration was a fivefold elevation of GABA. In CSF, changes in amino acids were small and comparable to those in the internal capsule. Our results demonstrate that ischemia-induced extracellular amino acid accumulation is a well localized phenomenon restricted to gray matter structures that possess release and reuptake systems for these substances. We assume that amino acids diffuse slowly into adjacent white matter structures, and into CSF.     

血清硫氧还蛋白1与脑外伤患者的白质恢复的相关性分析

为探讨血清硫氧还蛋白1 (thioredoxin 1, Trx1)含量与脑外伤后白质恢复的相关性关系,本研究选取60例脑外伤患者,根据格拉斯哥昏迷指数(Glasgow coma scale, GCS)将患者分为3组(轻度,中度和重度脑外伤患者),每组20例,采用核磁共振成像(magnetic resonance imaging, MRI)检测各组患者头颅白质情况,并通过ELISA方法检测各组患者血清Trx1水平,最后利用SPSS软件Pearson方法检测血清硫氧还蛋白1含量与脑外伤后白质恢复的相关性。结果显示,轻度脑损伤患者的GCS分值明显高于中度和重度脑外伤患者;轻度脑外伤患者的Trx1含量明显高于中度和重度患者;轻度和中度脑外伤患者的白质恢复情况明显优于重度患者;脑损伤患者的血清Trx1含量和白质恢复程度呈正相关。本研究初步结论表明脑外伤患者的白质恢复情况与血清Trx1存在正相关性关系,这将为脑外伤的治疗提供新思路。     

Organic Matter Preservation and Microbial Community Accumulations in Deep-Hypersaline Anoxic Basins

The Eastern Mediterranean Sea hosts several deep hypersaline anoxic basins (DHABs) such as the Bannock, L'Atalante, Discovery, and Urania which, due to strong salinity gradients, have a limited exchange with the overlying seawater. In the present study, a series of environmental variables associated with the origin and quality of organic matter were thoroughly investigated in an attempt to understand the function of these unique ecosystems. The redox potential of sediments collected from the brines as well as from reference sites varied from ?136 to 543 mV and salinity varied from 38 to 380 psu. Principal component analysis of chemical characteristics, including salinity, redox potential, organic carbon and nitrogen content, and C/N ratio grouped the sediments into two major clusters according to their redox state. Aliphatic hydrocarbon analysis revealed that the organic matter in the DHABs was predominantly of terrestrial origin but there was also evidence for petroleum inputs and for organic matter of phototrophic origin. Phospholipid linked fatty acids (PLFA) which were employed to assess the composition of microbial communities were found in greater abundance in stations situated inside the anoxic basins providing also strong evidence for the presence of methanotrophs and sulfate reducers. These results may represent an enhanced preservation of organic matter and an accumulation of microorganisms in these extreme environments. Heterogeneity in microbial community fatty acid profiles was documented between the anoxic sediments and the oxic and suboxic stations. However there were no significant correlations between PLFA and organic matter parameters. Redox conditions appear to influence microbial community composition, highlighting the role of the redox state as a regulator of organic matter preservation and microbial community accumulations in these ancient hypersaline anoxic lakes.     

脉压差预测中老年脑白质病变的临床价值

目的:研究脉压差等危险因素在中老年脑白质病变(WMLs)中的作用及临床意义。方法:入选213例无神经病学症状及体征的中老年病人(年龄>50岁),根据MRI影像学诊断标准分为中重度WMLs组(125例)及轻无WMLs组(88例),通过回顾性分析,记录年龄、性别、高血压史、糖尿病史、吸烟史、脉压差、收缩压、舒张压、血脂水平、左房内径,比较两组间各项指标差异,并做Binary Logistic回归分析,筛选出WMLs的独立危险因素。结果:在控制了高血压、年龄及其他混杂因素后,脉压差仍与WMLs密切相关,且独立于年龄和高血压,是WMLs的独立危险因素,OR值为2.954,95%CI 1.032~8.453。结论:压差在预测中老年人亚临床脑血管事件中可能起到重要作用。     

Relation of Cellular Phospholipid Composition to Oligodendroglial Differentiation in C-6 Glial Cells

The relation of the polar head group composition of cellular phospholipids to a biochemical expression of oligodendroglial differentiation was studied in cultured C-6 glial cells. Induction of the oligodendroglial enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), was determined after alteration of the polar head group composition of phospholipids by exposure of the cells to choline analogues, especially N,N'-dimethylethanolamine. To accomplish the phospholipid alteration, cells were grown in the presence of the analogue in medium free of exogenous lipid, i.e., first for 24 h in 10% delipidated serum and then for 48 h in serum-free medium. The 48-h exposure to serum-free medium resulted in untreated C-6 cells in a several fold increase in CNP activity, but in cells treated with 2.5 mM N,N'-dimethylethanolamine, total inhibition of this induction was observed. A graded, concentration-dependent inhibitory effect of the analogue on the induction of CNP was defined. The effect of the analogue was relatively specific, e.g., the activity of another plasma membrane enzyme of C-6 cells, (Na+ + K+)-activated ATPase, was not affected. Morever, there was no evidence of a toxic effect of the analogue; thus, total protein synthesis and cell growth were not altered, and the induction of CNP in serum-free medium recurred after removal of the analogue. N,N'-Dimethylethanolamine was shown to be incorporated into cellular phospholipids, primarily at the expense of phosphatidylcholine. The data define an important role for the polar head group composition of membrane phospholipids in oligodendroglial differentiation in this model system.     

The Presence of Aldehyde-Reacted Proteins in Normal and Multiple Sclerosis White Matter

The incorporation of tritium from NaB3H4 into the major protein components of myelin and the presence of weak fluorescence emission bands at wavelengths of approximately 440 and 500 nm from sodium dodecyl sulfate-solubilized, delipidated white matter are indicative of the presence of the products of aldehyde reactions with proteins. The incorporation of tritium from NaB3H4 into myelin proteins was confirmed by reaction with purified components of myelin basic protein or with lipophilin, a purified fraction of proteolipid protein. From the extent of tritium incorporation into the purified proteins, it is estimated that approximately 0.2 mol of tritium is incorporated/mol of myelin basic protein and approximately 0.4 mol of tritium/mol of proteolipid protein. There is approximately 50% greater incorporation of tritium into a more degraded, less positively charged form of the basic protein. The incorporation of tritium into normal and multiple sclerosis white matter was compared. There is a small but statistically significant difference in the percentage of the total counts incorporated into the major protein fractions for the two groups, with the multiple sclerosis samples showing a higher percentage of the counts in the Wolfgram protein and a lower percentage in the myelin basic protein compared with the normal samples.     

Nighttime Blood Pressure and White Matter Hyperintensities in Patients With Parkinson Disease

Increasing evidence indicates that nocturnal blood pressure level and/or loss of nocturnal blood pressure dips are sensitive markers of cardiovascular morbidity and mortality. Several studies have suggested that blunted heart rate variability and nocturnal decline in heart rate are also associated with target organ damage. These phenomena occur relatively commonly in patients with Parkinson disease (PD); however, few studies have assessed the consequences of these abnormalities in patients with PD. We investigated the influence of circadian changes in blood pressure and heart rate on white matter hyperintensities (WMHs) in patients with PD. The presence of nocturnal hypertension was associated with increased WMH score, and nighttime systolic pressure was closely related with white matter changes. Blunted heart rate variability and nocturnal decline in heart rate were also related to increasing WMH scores. The non-dipping phenomenon did not influence WMHs. These findings suggest that white matter changes are related to circadian autonomic dysfunction, particularly nocturnal hypertension in patients with PD. Therefore, it is important to monitor nocturnal blood pressure status, because modifying these circadian regulatory disturbances can be beneficial to protect against vascular brain damage in patients with PD. (Author correspondence: neuronet@catholic.ac.kr)     

Oligodendroglial Glycerophospholipid Synthesis: Incorporation of Radioactive Precursors into Ethanolamine Glycerophospholipids by Calf Oligodendroglia Prepared by a Percoll Procedure and Maintained in Suspension Culture

Abstract: Oligodendroglia prepared from minced calf cerebral white matter by trypsinization at pH 7.4, screening, and isosmotic Percoll (polyvinylpyr-rolidone-coated silica gel) density gradient centrifugation survived in culture on polylysine-coated glass, extending processes and maintaining phenotypic characteristics of oligodendroglia. In the present study, ethanolamine glycerophospholipid (EGP) metabolism of the freshly isolated cells was examined during short-term suspension culture by dual label time course and substrate concentration dependence experiments with [2-³H]glycerol and either [1,2-¹⁴C]ethanolamine or L-[U-¹⁴C]serine. Rates of incorporation of ³H from the glycerol and of ¹⁴C from the ethanolamine into EGP were constant for 14 h. In medium containing 3 mM-[1,2-¹⁴C]ethanolamine and 4.8 mM-[2-³H]glycerol, rates of incorporation of ¹⁴C and ³H into diacyl glycerophosphoethanolamine (diacyl GPE) were similar. Under the same conditions, ³H specific activities of alkylacyl GPE and alkenylacyl GPE were much lower than ¹⁴C specific activities, likely as a result of the loss of tritium during synthesis of these forms of EGP via dihydroxyacetone phosphate. L-[U-¹⁴C]serine was incorporated into serine glycerophospholipid (SGP) by base exchange rather than de novo synthesis. ¹⁴C from L-[U-¹⁴C]serine also appeared in EGP after an initial lag period of several hours. Methylation of oligodendroglial EGP to choline glycerophospholipid (CGP) was not detected.     

综述与专论: 白质消融性白质脑病研究进展

白质消融性白质脑病（leukoencephalopathy with vanishing white matter,VWM）是一种常染色体隐性遗传性脑白质病,其致病基因EIF2B 1~5分别编码真核细胞蛋白质翻译起始因子2B（eukaryotic initiation factor 2B,eIF2B）的5个亚基α~ε,其中任一编码基因突变均可引起发病。起病多见于婴幼儿及儿童期,临床表型差异大,典型表现为进行性运动功能退行,可伴共济失调和癫痫。应激（发热、外伤等）可导致发作性加重。影像学显示大脑白质进行性液化。尸解神经病理学特征主要表现为广泛性白质稀疏和囊性变性,无神经胶质细胞反应性增生,星形胶质细胞形态异常,过表达祖细胞标志物巢蛋白（Nestin）和胶质纤维酸性蛋白δ（GFAPδ）,少突前体细胞数量增加和成熟少突胶质细胞减少、泡沫化且凋亡增加。VWM致病基因EIF2B 1~5是管家基因,但多数患者通常仅脑白质受累。少数胎儿期及婴儿早期发病的患者可出现多系统受累,成年女性患者可有卵巢功能障碍。目前认为,星形胶质细胞在其致病机制中起着核心作用,病理性星形胶质细胞继发性引起少突胶质细胞成熟障碍和髓鞘形成异常,进而导致脑白质病变。其他疾病机制包括内质网应激后未折叠蛋白反应（UPR）过度激活、线粒体功能障碍、自噬抑制等,尚不完全明确。