Beta-hydroxy-butyrate alters the extracellular content of S100B in astrocyte cultures

Astrocytes have a variety of roles in maintaining neural tissue physiology, including energetic support, uptake and metabolism of glutamate and secretion of neurotrophic factors. Glutamate toxicity has been implicated in neurodegenerative disorders associated with conditions related to energy failure, and to elevation of glutamate extracellular levels in brain. Glucose is the main energetic substrate for brain cells but, in some circumstances, the ketone bodies are used as a supplementary source and have been suggested to be neuroprotective agents against seizure disorders. Here, we investigate some possible biochemical changes in astrocyte cultures induced by beta-hydroxy-butyrate, the predominant blood ketone body. Its effect upon S100B secretion, astrocyte morphology and glutamate uptake was particularly investigated. S100B, a calcium-binding protein expressed and secreted by astrocytes, has neurotrophic activity and a possible role in epileptogenesis. Cell morphology was investigated by phase-contrast microscopy and immunocytochemistry for actin, GFAP and S100B. Our data show that beta-hydroxy-butyrate induces dramatic changes in astrocyte morphology and, independent of this, causes changes in the extracellular content of S100B. We observed an increment in S100B 1 h after beta-hydroxy-butyrate addition and a decrease 24 h later. No changes were observed in glutamate uptake. These astrocytic modifications may be associated with reduced neuronal excitability observed in the ketogenic condition.     

Gap junction inhibitors modulate S100B secretion in astrocyte cultures and acute hippocampal slices

Astrocytes sense, integrate, and respond to stimuli generated by neurons or neural injury; this response involves gap junction (GJ) communication. Neuronal vulnerability to injury increased when cocultures of astrocytes and neurons were exposed to GJ inhibitors. However, GJ uncoupling could limit the extension of a lesion. We investigated a possible link between GJ communication and S100B secretion. S100B is a calcium‐binding protein of 21 kDa that is predominantly expressed and secreted by astrocytes, which has trophic paracrine activity on neurite growth, glial proliferation, and neuronal survival. GJ inhibitors were analyzed in isolated astrocytes in primary cultures from hippocampus, acute hippocampal slices, and C6 glioma cells, which were used as a negative control. Our data indicate that GJ blocking stimulates S100B secretion in astrocyte cultures and acute hippocampal slices. Different assays were used to confirm cell integrity during exposure to GJ inhibitors. S100B secretion was observed with different types of GJ inhibitors; the resulting event was dependent on time, the nature of the inhibitor, its putative molecular target of GJ blocking, and/or the cell preparation used. Only carbenoxolone induced a fast and persistent increase in S100B secretion in both preparations. Endothelin‐1 increased S100B secretion in astrocyte cultures at 1 hr, but a decrease was observed at 6 hr or in acute hippocampal slices. Physiologically, a local GJ closure associated with release of S100B in injury conditions favors the idea of a common mechanism available to limit the extension of lesion and increase the chances of cell survival. © 2009 Wiley‐Liss, Inc.     

Differential regulation of microglial activation by propentofylline via cAMP signaling

A pathological microglial activation is believed to contribute to progressive neuronal damage in neurodegenerative diseases by the release of potentially toxic agents and by triggering reactive astrocytic changes. Using cultured microglia from neonatal rat brains, we investigated the mode of propentofylline action in strengthening cAMP-dependent intracellular signaling. We compared this action with the effects of dibutyryl-cAMP, a cell-permeable cAMP analog. Propentofylline inhibited lipopolysaccharide (LPS)-induced release of both tumor necrosis factor (TNF)-α and interleukin (IL)-1β in a dose-dependent manner within the therapeutic low micromolar range. However, LPS-induced release of IL-6 and NO were not affected by propentofylline. All these differential effects of propentofylline on LPS-induced microglial release were mimicked by the addition of dibutyryl-cAMP. Microglial proliferation and phorbol myristate acetate (PMA)-induced O₂⁻ release were also dose-dependently inhibited by propentofylline as well as dibutyryl-cAMP. These results suggest that propentofylline, probably via reinforcement of cAMP intracellular signaling, alters the profile of the newly adopted immune properties in a way that it inhibits potentially neurotoxic functions while maintaining beneficial functions. This differential regulation of microglial activation may explain the neuroprotective mechanism exerted by propentofylline.     

S100B蛋白生物学研究与临床应用进展

S100蛋白是一种分子量较小（10～12ku）的EF-手型钙结合蛋白，通过对钙离子的调节及与靶蛋白的相互作用，在体内发挥多种生物学作用．在细胞增殖、分化，肌肉收缩、基因表达、分泌及细胞凋亡中发挥重要作用。1965年Moore等首先在牛脑组织中发现S100蛋白，因其在中性饱和硫酸铵中100％溶解而得名。现已发现S100蛋白家族成员20个，S100B蛋白为其中一员，     

NO as an autocrine mediator in the apoptosis of activated microglial cells: correlation between activation and apoptosis of microglial cells

Abnormal activation of microglial cells has been implicated in various neurodegenerative diseases. Microglial activation needs to be tightly regulated for physiological maintenance and normal functioning of the central nervous system. Potential mechanisms for the down-regulation of activated microglial cells are the deactivation or elimination of activated cells. We hypothesized that the elimination of activated microglial cells by apoptosis is one of the key mechanisms of auto-regulation of activated microglial cells. To test this hypothesis, we utilized BV-2 mouse microglial cells and rat primary microglial cultures exposed to activating agents such as lipopolysaccharide and interferon-gamma, and investigated a possible correlation between apoptosis and activation of these cells. We found that the activation of microglial cells led to apoptotic death, and the activation state of microglial cells inversely correlated with cell viability. We have also demonstrated that: (i) NO was produced by activated microglial cells in a manner dependent on time and dose of activating agents; (ii) inhibition of NO synthesis by iNOS inhibitor blocked the apoptosis of activated microglial cells; (iii) an exogenous NO donor induced apoptosis of microglial cells; and (iv) inhibition of TNFalpha or FasL using neutralizing antibodies did not affect activation-induced apoptosis of microglial cells. These results indicated that activation of microglial cells leads to the production of NO, which in turn acts as the major mediator of cellular apoptosis in an autocrine fashion. Our work suggests the presence of auto-regulatory mechanism for microglial activation, which may have relevance in the pathogenesis of various neurodegenerative diseases possibly resulting from 'over-activation' of microglial cells.     

Relationship between S100beta and GFAP expression in astrocytes during infarction and glial scar formation after mild transient ischemia

The expression of astrocyte marker proteins (S100beta and GFAP) during infarction and glial scar formation after transient middle cerebral artery (MCA) occlusion was examined using double immunostaining. S100beta immunoreactivity markedly decreased in the core of the injured area when observed immediately after reperfusion and did not increase again. In the periphery, however, S100beta expression increased, showing that S100beta synthesis was up-regulated. S100beta+/iNOS+ astrocytes in the periphery were observed from day 1, when small infarct areas were detectable, up to day 5, when infarct expansion had almost ended. TUNEL+ cells in the periphery were present from days 1 to 5. S100beta+/TUNEL+ cells were observed centrally and around the periphery of the injured area, predicting that cell death contributes to the increase of S100beta concentration in the injured area. Our results suggest that (1) higher concentration of S100beta in the extracellular space due to S100beta leakage from damaged astrocytes leads to up-regulation of S100beta synthesis and induction of inducible nitric oxide synthase (iNOS) synthesis in astrocytes, contributing to infarct expansion that results in DNA damage or cell death via NO and ROS production, and (2) GFAP, but not S100beta, is a main contributor to glial scar formation. On day 1 postreperfusion, the microdiascopic images of the injured areas from the unstained thick sections or the areas detected by S100beta immunoreactivity were larger than those of the infarct areas detected by hematoxylin--eosin (HE)-staining. The difference between these sizes might be useful to predict infarct expansion.     

Ammonia-induced alteration in S100B secretion in astrocytes is not reverted by creatine addition

Hyperammonemia is a major element in the pathogenesis of hepatic encephalopathy (HE) and ammonia neurotoxicity involves an effect on the glutamatergic neurotransmitter system. Astrocytes are intimately related to glutamatergic neurotransmission and, in fact, many specific glial alterations have been reported as a result of ammonia exposure. S100B protein, particularly extracellular S100B, is used as a parameter of glial activation or commitment in several situations of brain injury. However, there is little information about this protein in ammonia toxicity and none about its secretion in astrocytes under ammonia exposure. In this study, we investigated S100B secretion in rat cortical astrocytes acutely exposed to ammonia, as well astrocyte morphology, glial fibrillary acidic protein (GFAP) content and glutamine synthetase (GS) activity. Moreover, we studied a possible effect of creatine on these glial parameters, since this compound has a putative role against ammonia toxicity in cell cultures. We found an increase in S100B secretion by astrocytes exposed to ammonia for 24h, accompanied by a decrease in GFAP content and GS activity. Since elevated and persistent extracellular S100B plays a toxic effect on neural cells, altered extracellular content of S100B induced by ammonia could contribute to the brain impairment observed in HE. Creatine addition did not prevent this increment in S100B secretion, but was able to prevent the decrease in GFAP content and GS activity induced by ammonia exposure.     

Glial-derived proteins activate cultured astrocytes and enhance beta amyloid-induced glial activation

A prominent feature of Alzheimer's disease (AD) pathology is an abundance of activated glia (astrocytes and microglia) in close proximity to the amyloid plaques. These activated glia overexpress a number of proteins that may participate in the progression of the disease, possibly by propagation of inflammatory and oxidative stress responses. The beta-amyloid peptide 1-42 (Abeta), a major constituent of neuritic plaques, can itself induce glial activation. However, little is known about whether other plaque components, especially the upregulated glial proteins, can induce glial activation or modulate the effects of Abeta on glia. In this study, we focused on four glial proteins that are abundant in amyloid plaques and/or that are known to interact with Abeta: alpha1-antichymotrypsin (ACT), interleukin-1beta (IL-1beta), S100beta, and butyrylcholinesterase (BChE). We examined the ability of these proteins to activate rat cortical astrocyte cultures and to influence the ability of Abeta to activate astrocytes. Treatment of astrocytes with ACT, IL-1beta, or S100beta resulted in glial activation, as assessed by reactive morphology, upregulation of IL-1beta, and production of inducible nitric oxide synthase and nitric oxide. The ability of Abeta to induce astrocyte activation was also enhanced in the presence of each of these three proteins. In contrast, BChE alone did not activate astrocytes and had no effect on Abeta-induced activation. These results suggest that certain proteins produced by activated glia may contribute to the chronic glial activation seen in AD through their ability to stimulate astrocytes directly or through their ability to modulate Abeta-induced activation.     

Increased mucosal nitric oxide production in ulcerative colitis is mediated in part by the enteroglial-derived S100B protein

Abstract  In the central nervous system glial-derived S100B protein has been associated with inflammation via nitric oxide (NO) production. As the role of enteroglial cells in inflammatory bowel disease has been poorly investigated in humans, we evaluated the association of S100B and NO production in ulcerative colitis (UC). S100B mRNA and protein expression, inducible NO synthase (iNOS) expression, and NO production were evaluated in rectal biopsies from 30 controls and 35 UC patients. To verify the correlation between S100B and NO production, biopsies were exposed to S100B, in the presence or absence of specific receptor for advanced glycation end-products (RAGE) blocking antibody, to measure iNOS expression and nitrite production. S100B and iNOS expression were evaluated after incubation of biopsies with lipopolysaccharides (LPS) + interferon-gamma (IFN-γ) in the presence of anti-RAGE or anti-S100B antibodies or budesonide. S100B mRNA and protein expression, iNOS expression and NO production were significantly higher in the rectal mucosa of patients compared to that of controls. Exogenous S100B induced a significant increase in both iNOS expression and NO production in controls and UC patients; this increase was inhibited by specific anti-RAGE blocking antibody. Incubation with LPS + IFN-γ induced a significant increase in S100B mRNA and protein expression, together with increased iNOS expression and NO production. LPS + IFN-γ-induced S100B up-regulation was not affected by budesonide, while iNOS expression and NO production were significantly inhibited by both specific anti-RAGE and anti-S100B blocking antibodies. Enteroglial-derived S100B up-regulation in UC participates in NO production, involving RAGE in a steroid insensitive pathway.     

Shared features of S100B immunohistochemistry and cytochrome oxidase histochemistry in the ventroposterior thalamus and lateral habenula in neonatal rats

The ventroposterior thalamus and the habenular nuclei of the epithalamus are relevant to the monoaminergic system functionally and anatomically. The glia-derived S100B protein plays a critical role in the development of the nervous system including the monoaminergic systems. In this study, we performed an immunohistochemical study of glia-related proteins including S100B, serotonin transporter, and microtubule-associated protein 2, as well as cytochrome oxidase histochemistry in neonatal rats. Results showed the same findings for S100B immunohistochemistry between the ventroposterior thalamus and the lateral habenula at postnatal day 7: intense staining in cell bodies of astrocytes, diffusely spread immunoproduct in the intercellular space, and S100B-free areas as well as a strong reaction to cytochrome oxidase histochemistry. Further common features were the scarcity of glial fibrillary acidic protein-positive astrocytes and the few apoptotic cells observed. The results of the cytochrome oxidase reaction suggested that S100B is released actively into intercellular areas in restricted brain regions showing high neuronal activity at postnatal day 7. Pathology of the ventroposterior thalamus and the habenula is suggested in mental disorders, and S100B might be a key factor for investigations in these areas.     

Aging and glial responses to lipopolysaccharide in vitro: greater induction of IL-1 and IL-6, but smaller induction of neurotoxicity

Glial activation during aging was analyzed in primary glia cultured from brain regions sampled across the life span. An initial study showed that phenotypes of activated astrocytes and microglia from aging rat cerebral cortex persisted in primary cultures (Neurobiol. Aging 19 (1998), 97). We extend these findings by examining effects of age on the activation of glial cultures from adult rat brain in response to lipopolysaccharide (LPS), an inflammatory stimulus. Mixed glia from F344 male rats, aged 3 and 24 months, cultured from cerebral cortex (Cx), hippocampus (Hc), and striatum (St), were assayed for cytokines implicated in Alzheimer's disease: IL-1alpha, IL-1beta, IL-6, and TNF-alpha. Regional differences across all age groups included consistently lower expression of these cytokines in glia derived from Cx than Hc and St. Aging increased basal IL-6 mRNA and secretion by >or=3-fold in glia from Cx and Hc. Aging also increased LPS-induced IL-1 and IL-6 in Hc more than in Cx, whereas no significant effects of age were seen in St-derived glial cytokines. TNF-alpha secretion did not differ by donor age (basal or LPS-induced). Nitric oxide production by microglia from Cx of aging brains showed a smaller induction in response to LPS, with proportionately less neurotoxicity. Thus, glial activation during aging shows regional selectivity in cytokine expression, with opposite effects of aging on the increased inducibility of IL-1 and IL-6 vs the decreased production of nitric oxide.     

脑震荡患者血清S100B蛋白的变化与认知功能障碍的相关性

目的 分析脑震荡患者血清S100B含量变化与认知功能损害的相关性,探讨血清S100B水平在脑震荡认知障碍预后判断中的意义. 方法 采用酶联免疫法分别测定126例脑震荡患者急诊入院后6h、12h和第3天的血清S100B浓度,并和同期轻型单纯脑外伤患者比较.脑震荡患者常规治疗后,于入院后第1天、第14天及3个月后进行简易智能精神状态检查量表(MMSE)评估,将S100B浓度与MMSE评分进行相关性分析. 结果入院6 h、12 h后脑震荡组患者血清S100B浓度均明显高于对照组,差异有统计学意义(P＜0.05).入院6 h血清S100B浓度与各时段MMSE评分均具有相关性(P均＜0.05);入院12 h、第3天血清S100B浓度与各时段MMSE评分均无相关性(P均＞0.05). 结论 脑震荡患者早期(6 h)血清S100B含量异常升高,并与认知功能损害有明显相关性,可作为认知功能损害预后判断的重要指标.     

S100B蛋白与心境障碍的相关性研究进展

心境障碍是由各种原因引起的以显著而持久的心境或情感改变为主要特征的一组疾病,包括重性抑郁障碍和双相障碍两个主要疾病亚型.随着现代生活节奏的加快,其患病率正逐年上升.世界卫生组织(WHO)有关全球疾病总负担的统计显示,1990年重性抑郁障碍和双相障碍分别排在第5位和第18位,预计到2020年,重性抑郁障碍的疾病负担将上升至第二位.目前心境障碍的病因及发病机制仍不明,近来国外学者开始提出心境障碍就是胶质细胞的功能障碍,而S100B蛋白作为一种胶质细胞源性的蛋白质,可能与心境障碍有着密切的联系[1].本文总结国内外相关研究,对S100B蛋白与心境障碍的关系作如下综述.     

Role of nitric oxide and melanogenesis in the accomplishment of anticryptococcal activity by the BV-2 microglial cell line

In the present paper, we investigated the involvement of cryptococcal melanogenesis and macrophage nitric oxide (NO) production in the accomplishment of anticryptococcal activity by microglial effector cells, using the murine cell line BV-2. We demonstrate that the constitutive levels of anticryptococcal activity exerted by BV-2 cells is significantly enhanced upon interferon γ plus lipopolysaccharide treatment. The phenomenon, which occurs with no enhancement of phagocytic activity, is associated with the production of high levels of NO and is abolished by addition of ^G-monomethyl-l-arginine. Comparable patterns of results are observed employing either unopsonized or opsonized microbial targets, the latter microorganisms being markedly more susceptible to BV-2 cell antimicrobial activity. Furthermore, melanization of Cryptococcus neoformans significantly reduces its susceptibility to BV-2 antimicrobial activity, regardless of the fact that activated macrophages or opsonized microorganisms have been employed. In conclusion, our results provide evidence that NO-dependent events are involved in the fulfillment of anticryptococcal activity by activated microglial cells and that fungal melanization is a precious escamotage through which C. neoformans overcomes host defenses.     

Serum S100B protein: a useful marker in obstructive sleep apnea syndrome

Background and purposeWe aimed to underline the importance of serum S100B protein as a useful biochemical marker in patients with obstructive sleep apnea syndrome (OSAS).Material and methodsForty-three newly diagnosed patients with OSAS (median apnea-hypopnea index [AHI, events/hour]: 37.5 [range 11.3–137]) and 25 subjects with AHI < 5 (median AHI: 4.4 [range 0.7–4.8]) were included in the study. Serum S100B protein level was tested in serum samples taken after polysomnography in both groups and the difference between OSAS patients and the control group regarding that level was assessed. In addition, the association of S100B protein serum level with age, body mass index, AHI, mean O₂ saturation percentage during sleep, minimum O₂ saturation value (%) at the end of the apneas, and the time spent at an O₂ saturation less than 90% were analyzed in the OSAS patient group.ResultsMedian serum S100B protein level was 133.7 pg/mL (range 20.97–230.70 pg/mL) in patients with OSAS and 16.1 pg/mL (range 10.1–22.9 pg/mL) in the control group (p < 0.005). Serum S100B protein level did not correlate with any studied variable (p > 0.05 for each correlation coefficient).ConclusionsSerum S100B protein level is increased in patients with OSAS and may be a useful biochemical marker in those patients.     

急性缺血性脑卒中患者治疗前后血清S100B蛋白变化及其意义

目的探讨急性缺血性脑卒中患者治疗前后血清S100B蛋白的变化及其临床意义。方法采用ELISA法检测86例急性缺血性脑卒中患者治疗前后及46例正常人的血清S100B蛋白水平。结果急性缺血性脑卒中患者治疗前的血清S100B蛋白水平显著高于对照组（P〈0．001），差异有统计学意义；急性缺血性脑卒中患者治疗后血清S100B蛋白水平显著下降（P〈0．001），差异有统计学意义。结论急性缺血性脑卒中发生时，患者血清S100B蛋白增高；治疗后检测急性缺血性脑卒中患者血清S100B蛋白水平有助于早期诊断、指导治疗及判断预后。     

急性缺血性脑卒中患者治疗前后血清S100B蛋白变化及其意义

目的探讨急性缺血性脑卒中患者治疗前后血清S100B蛋白的变化及其临床意义。方法采用ELISA法检测86例急性缺血性脑卒中患者治疗前后及46例正常人的血清S100B蛋白水平。结果急性缺血性脑卒中患者治疗前的血清S100B蛋白水平显著高于对照组(P<0.001),差异有统计学意义;急性缺血性脑卒中患者治疗后血清S100B蛋白水平显著下降(P<0.001),差异有统计学意义。结论急性缺血性脑卒中发生时,患者血清S100B蛋白增高;治疗后检测急性缺血性脑卒中患者血清S100B蛋白水平有助于早期诊断、指导治疗及判断预后。     

Neuron-specific enolase and S 100B protein as predictors of outcome in ischaemic stroke

Background and purposeAs neuron-specific enolase (NSE) and S 100B protein are brain origin proteins, the aim of this study was to assess whether a single NSE and S 100B measure may predict clinical outcome of patients with cerebral ischaemic infarct.Material and methodsSeventy-one patients with ischaemic stroke and 41 controls were studied. All patients had computed tomography of the brain performed after admission and on the third day and volume of the infarct was assessed by the volumetric method from the second examination. NSE and S 100B protein were analysed by immunochemiluminescence on the fourth day after admission. Clinical state of the patients was determined with the NIH stroke, Barthel and Rankin scales on admission, discharge from the hospital, and after one and 3 months from the onset of stroke.ResultsNSE levels in blood were significantly higher in stroke patients than in the control group – 36.9 ± 24.0 vs. 14.3 ± 9.7 μg/L. Also, the levels of the S 100B protein were significantly higher in the patient group (0.85 ± 1.74 vs. 0.10 ± 0.03 μg/L) but only the levels of S 100B protein correlated with the calculated size of the infarct (Spearman coefficient = 0.77). No such correlation was identified for NSE level (Spearman coefficient = 0.25).ConclusionsAlthough significant differences in NSE and S 100B levels between stroke patients and the control group were found, only S 100B protein level correlated with stroke volume, neurological status at admission and functional outcome. NSE did not correlate with stroke volume, neurological status or clinical outcome.