presentation on microglia

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微观形貌观察英文作文高中

微观形貌观察英文作文高中英文：Microscopic observation is an essential part of scientific research in various fields, including biology, chemistry, and material science. In my opinion, it is fascinating to observe the microscopic morphology of different objects and materials, as it reveals the hidden structure and properties that cannot be seen by the naked eye.For instance, when I observed the surface of a butterfly wing under a scanning electron microscope, I was amazed by the intricate patterns and textures that were invisible to the human eye. The wing surface was covered with tiny scales, which were arranged in a precise and orderly manner, creating a beautiful and unique pattern. The microscopic observation of the butterfly wing not only revealed its aesthetic value but also provided insights into its functional properties, such as water repellencyand light reflection.Similarly, when I examined the surface of a metal alloy using transmission electron microscopy, I was able to see the crystal structure and defects at the atomic level. This allowed me to understand the mechanical properties and deformation behavior of the material, which is crucial for designing and optimizing its performance in various applications.Overall, microscopic observation is a powerful tool for scientific research, enabling us to explore and understand the hidden world of microstructures and properties. It is a fascinating and rewarding experience that requires patience, skill, and curiosity.中文：微观形貌观察是各个领域科学研究中不可或缺的一部分，包括生物学、化学和材料科学等。

仿生科技演讲稿英语范文

仿生科技演讲稿英语范文Bionics, as an emerging interdisciplinary field, has been making significant strides in recent years. It integrates biology and engineering to develop innovative technologies that mimic natural systems. Today, I am honored to speak to you about the exciting advancements in bionic technology and its potential impact on the future.First and foremost, bionic technology has revolutionized the medical field. Prosthetic limbs, for example, have become more advanced and functional, allowing amputees to regain mobility and independence. The development of bionic organs and tissues also holds great promise for the treatment of various medical conditions. By replicating the structure and function of natural biological systems, bionic technology has the potential to improve the quality of life for countless individuals.In addition to its medical applications, bionic technology has also made significant contributions to the field of robotics. By drawing inspiration from nature, engineers have been able to design robots with enhanced agility, dexterity, and adaptability. These bio-inspired robots are capable of navigating complex environments, performing delicate tasks, and even interacting with humans in more natural ways. As a result, bionic technology is reshaping the landscape of automation and has the potential to revolutionize industries such as manufacturing, healthcare, and agriculture.Furthermore, bionic technology has paved the way for the development of innovative materials and structures. By studying natural biological systems, researchers have been able to create materials that are stronger, more flexible, and more durable than traditional synthetic materials. These biomimetic materials have the potential to transform various industries, from construction and aerospace to fashion and consumer goods. Additionally, the integration of bionic principles into architecture and design has led to the creation of more sustainable and environmentally friendly solutions.Looking ahead, the future of bionic technology is filled with endless possibilities. As our understanding of biology and engineering continues to advance, we can expect to see even more groundbreaking innovations in the field of bionics. From the development ofadvanced neural interfaces to the creation of fully autonomous bio-inspired systems, the potential applications of bionic technology are truly limitless.In conclusion, bionic technology represents a convergence of biology and engineering that has the power to transform the world as we know it. From its impact on healthcare and robotics to its influence on materials and design, bionic technology is reshaping the way we approach innovation and problem-solving. As we continue to push the boundaries of what is possible, the future of bionics holds tremendous promise for improving the human experience and advancing society as a whole. Thank you.。

脸上的微生物英文演讲稿

脸上的微生物英文演讲稿以下是一篇关于“脸上的微生物”的英文演讲稿，供您参考：Ladies and gentlemen,Today, I am going to talk about the microorganisms on our faces. Our faces are constantly covered by a variety of microorganisms, some of which are beneficial while others may cause harm. Understanding the composition and function of these microorganisms is crucial for maintaining our health and skin well-being.Firstly, let’s take a look at the types of microorganisms found on our faces. There are over 100 species of bacteria, fungi, and viruses that call our faces their home. The distribution of these microorganisms varies from person to person and depends on a range of factors such as genetics, environment, and personal hygiene habits.One of the most common types of bacteria found on the face is Propionibacterium acnes. This bacteria is known to play a role in the development of acne vulgaris, commonly known as “acne”. However, Propionibacterium acnes is not the only bacteria that can cause acne. Other species such as Staphylococcus aureus and Streptococcus pyogenes can also be involved in the development of this skin condition.On the other hand, there are also beneficial microorganisms on our faces. One example is the yeast strain known as Malassezia globosa. This yeast is believed to have anti-inflammatory properties and can help protect the skin from harmful microorganisms.Now that we have a better understanding of the types of microorganisms on our faces, let’s move on to discuss their function and how they impact our health. Microorganisms on our faces can influence our skin in a number of ways. For example, some bacteria can help to protect our skin from harmful external factors such as UV rays and environmental toxins. In addition, some species can also help to maintain the acidity of the skin surface, which helps to keep the skin healthy and hydrated.Moreover, the presence of certain microorganisms can also trigger an immune response in our bodies, helping us to fight off harmful invaders. However, if the balance of these microorganisms is disturbed, it can lead to various skin problems such as acne, rosacea, or psoriasis.To maintain a healthy balance of microorganisms on our faces, it is important to practice good hygiene habits. Regularly washing your face with warm water and mild soap can help to remove excess oil, dirt, and dead skin cells that can clog pores and lead to the development of acne.In conclusion, our faces are home to a diverse community of microorganisms that play both harmful and beneficial roles in our health. Understanding the types and functions of these microorganisms is crucial for maintaining a healthy complexion. By practicing good hygiene habits and adopting an anti-inflammatory diet, we can help to support the balance of microorganisms on our faces and promote healthy, glowing skin. Thank！。

micro-ch17-presentation

OLIGOPOLY
2
Concentration Ratios in Selected U.S. Industries
Industry Video game consoles Tennis balls Credit cards Batteries Soft drinks Web search engines Breakfast cereal Cigarettes Greeting cards Beer Cell phone service Autos Concentration ratio 100% 100% 99% 94% 93% 92% 92% 89% 88% 85% 82% 79%
CHAPTER
17
Oligopoly
Microeonomics
N. Gregory Mankiw
Premium PowerPoint Slides by Ron Cronovich
2009 South-Western, a part of Cengage Learning, all rights reserved
OLIGOPOLY

EXAMPLE: Cell Phone Duopoly in Smalltown
P $0 5 10 15 20 25 30 35 40 45 Q 140 130 120 110 100 90 80 70 60 50
5
Smalltown has 140 residents The “good”: cell phone service with unlimited anytime minutes and free phone Smalltown’s demand schedule Two firms: T-Mobile, Verizon (duopoly: an oligopoly with two firms) Each firm’s costs: FC = $0, MC = $10

presentation部分的作用和如何实现这部分的教学

presentation部分的作用和如何实现这部分的教学一、引言在当今的教育环境中，Presentation已经成为一项重要的学习技能。

它不仅是传达信息的一种方式，更是培养学生沟通、协作和公众演讲能力的手段。

因此，了解Presentation的作用并掌握其实现方法，对于提高学生的综合素质具有重要意义。

二、Presentation的作用1. 信息共享：Presentation为学生提供了一个平台，让他们可以与他人分享自己的研究、理解和发现。

通过有效的口头表达，学生能够更好地组织并传达自己的思想。

2. 沟通能力：一个成功的Presentation需要良好的沟通技巧。

学生需要理解观众的需求，清晰地表达自己的观点，并回答可能的问题。

这有助于提高学生的沟通技巧。

3. 自信心建立：公众演讲往往让人感到紧张，但通过练习和经验积累，学生可以克服这种紧张感，增强自信心。

4. 团队协作：在小组项目中，学生需要与队友协作，分配任务，这有助于培养学生的团队协作能力。

三、如何实现Presentation的教学1. 明确教学目标：教师需明确Presentation的教学目标，是提高学生的沟通能力、公众演讲技巧还是信息组织能力。

2. 制定教学计划：根据教学目标，制定详细的教学计划，包括教学内容、教学方法和评估标准。

3. 激活学生的前知：了解学生已有的知识和经验，以便更好地引导他们进行Presentation。

4. 教学策略选择：选择适合的教学策略，如小组讨论、案例分析或角色扮演等。

5. 练习与反馈：为学生提供充足的练习机会，并给予及时反馈，帮助他们改进。

6. 评估与反思：制定评估标准，对学生的Presentation进行评估，并反思教学方法，以便进一步提高教学效果。

四、结论Presentation在教育中的作用不容忽视。

它不仅能够提高学生的沟通能力、自信心和团队协作能力，还有助于培养学生的综合素质。

为了实现良好的Presentation教学效果，教师需要明确教学目标、制定教学计划、选择合适的教学策略、提供充足的练习机会和及时的反馈，以及进行有效的评估和反思。

显微镜的英语作文

显微镜的英语作文Microscopes have been a cornerstone of scientific discovery since their invention. They have allowed us to explore the microscopic world that is invisible to the naked eye. In this essay, we will delve into the history of microscopy, its various types, and the impact it has had on our understanding of the natural world.The first microscopes were simple, with limited magnification power. However, they laid the groundwork for the sophisticated instruments we have today. As technology advanced, so did the complexity and magnificationcapabilities of microscopes. The electron microscope, for example, can magnify objects up to two million times their original size, revealing atomic and molecular structures.There are several types of microscopes, each serving a specific purpose. The compound light microscope is the most common and uses visible light to magnify samples. The scanning electron microscope (SEM) uses electron beams to create detailed images of surfaces, while the transmission electron microscope (TEM) is used to study thin samples at an atomic level.Microscopes have revolutionized fields such as biology, medicine, and materials science. In biology, they have enabled us to study cells and microorganisms, leading to breakthroughs in understanding life processes. In medicine,they have been instrumental in the diagnosis of diseases by allowing doctors to observe pathogens and cellular changes. Materials science has also benefited from microscopy, as it allows for the examination of material structures and defects at a microscopic level.Moreover, the use of microscopes extends beyond the laboratory. They are used in forensic science to analyze evidence, in environmental science to study microorganisms and their impact on ecosystems, and in education to teach students about the unseen world.In conclusion, microscopes are not just scientific tools; they are windows into a world that is as complex and diverse as the macroscopic world we inhabit. They have expanded our knowledge and continue to be essential in the quest for understanding the intricate details of life and matter. As technology progresses, we can expect microscopes to become even more powerful, unveiling even more secrets of the microscopic universe.。

Microbial Biofilm Sample

Microbial Biofilm SampleMicrobial biofilms are complex communities of microorganisms that grow on surfaces and are embedded in a self-produced extracellular matrix. These biofilms play a crucial role in various fields such as medicine, industry, and environmental engineering. However, they also pose a significant challenge to human health, as they can cause chronic infections that are difficult to treat. In this essay, I will discuss the importance ofstudying microbial biofilms, their impact on human health, and potential strategies to prevent and control their growth.Microbial biofilms are important to study because they are ubiquitous in nature and have a significant impact on various ecosystems. They can be found in aquatic environments, soil, and on the surfaces of plants and animals. Biofilms also play a crucial role in biogeochemical cycles, suchas the nitrogen and carbon cycles. Therefore, understanding the structure, function, and dynamics of biofilms is essential to gain insights into the ecological processes that drive microbial communities.However, biofilms can also have detrimental effects on human health. They are responsible for a wide range of infections, including chronic wound infections, dental plaque, and infections associated with medical devices. Biofilms are particularly difficult to treat because the extracellular matrix provides protection against antibiotics and the immune system. Moreover, biofilms can act as a reservoir for antibiotic-resistant bacteria, making infections even harder to treat.To prevent and control biofilm growth, various strategies have been developed. One approach is to use antimicrobial agents that target the biofilm matrix. For example, enzymes that degrade the extracellular matrix have been shown to disrupt biofilm formation and enhance the efficacy of antibiotics. Another strategy is to use physical methods, such asultrasound or photodynamic therapy, to disrupt biofilms. These approacheshave shown promising results in laboratory studies, but their effectiveness in clinical settings needs further investigation.Additionally, understanding the mechanisms that drive biofilm formation and persistence is crucial for developing new strategies to prevent and control biofilms. For example, quorum sensing, a mechanism by which bacteria communicate with each other, plays a critical role in biofilm formation. Targeting quorum sensing pathways has been proposed as a potential strategy to prevent biofilm formation. Moreover, understanding the interactions between different microbial species within biofilms can provide insights into the dynamics of microbial communities and their impact on human health. In conclusion, microbial biofilms are complex communities of microorganisms that play a crucial role in various ecosystems, but also pose a significant challenge to human health. Studying biofilms is essential to gain insights into the ecological processes that drive microbial communities and to develop strategies to prevent and control their growth. While various approaches have been developed to prevent and control biofilms, further research is needed to understand the mechanisms that drive biofilm formation and persistence and to develop new strategies to combat biofilm-associated infections.。

Microglia express distinct M1 and M2 phenotypic markers in nervous system in mice

Microglia Express Distinct M1and M2Phenotypic Markers in the Postnatal and Adult Central Nervous System in Male and Female MiceJessica M.Crain,1,2Maria Nikodemova,3*and Jyoti J.Watters 1,2,31Program in Cellular and Molecular Biology,University of Wisconsin,Madison,Wisconsin 2Center for Women’s Health Research,University of Wisconsin,Madison,Wisconsin 3Department of Comparative Biosciences,University of Wisconsin,Madison,WisconsinAlthough microglial activation is associated with all CNS disorders,many of which are sexually dimorphic or age-dependent,little is known about whether microglial basal gene expression is altered with age in the healthy CNS or whether it is sex dependent.Analysis of microglia from the brains of 3-day (P3)-to 12-month-old male and female C57Bl/6mice revealed distinct gene expression proﬁles during postnatal development that differ signiﬁ-cantly from those in adulthood.Microglia at P3are char-acterized by relatively high iNOS,TNF a and arginase-I mRNA levels,whereas P21microglia have increased expression of CD11b,TLR4,and FcR g I.Adult microglia (2–4months)are characterized by low proinﬂammatory cytokine expression,which increases by 12months of age.Age-dependent differences in gene expression sug-gest that microglia likely undergo phenotypic changes during ontogenesis,although in the healthy brain they did not express exclusively either M1or M2phenotypic markers at any time.Interestingly,microglia were sexually dimorphic only at P3,when females had higher expres-sion of inﬂammatory cytokines than males,although there were no sex differences in estrogen receptor expression at this or any other time evaluated pared with microglia in vivo ,primary microglia prepared from P3mice had considerably altered gene expression,with higher levels of TNF a ,CD11b,arginase-I,and VEGF ,sug-gesting that culturing may signiﬁcantly alter microglial properties.In conclusion,age-and sex-speciﬁc variances in basal gene expression may allow differential microglial responses to the same stimulus at different ages,perhaps contributing to altered CNS vulnerabilities and/or diseasecourses.VC 2013Wiley Periodicals,Inc.Key words:microglia;development;aging;sexualdimorphism;M1/M2phenotypeMicroglia,the resident innate immune cells in the central nervous system (CNS),are associated with the pathogenesis of virtually all CNS disorders or injuries.One important characteristic of these cells is high morpho-logical and functional plasticity.They acquire an activated,amoeboid morphology in response to invading pathogens and/or CNS damage.At the same time,they increase their production of a wide array of chemokines,cytokines,ni-tric oxide,and reactive oxygen species that mediate neu-roinﬂammation (Hoek et al.,2000;Streit et al.,2005;Graeber et al.,2011).In contrast,microglia in the healthy adult CNS are characterized by a quiescent morphology with numerous thin,ramiﬁed processes.Although com-monly considered “resting,”emerging evidence suggests that quiescent microglia are highly motile and are actively involved in many physiological processes that include making dynamic contacts with neurons (Wake et al.,2009;Graeber,2010;Parkhurst and Gan,2010;Ketten-mann et al.,2011;Paolicelli et al.,2011;Tremblay and Majewska,2011;Tremblay et al.,2011).However,the gene expression proﬁles of “resting”microglia in the healthy CNS are not well characterized,and even less is known about whether microglia undergo changes in gene expression that accompany their functional alterations from postnatal development to aging.In the postnatal brain,microglia are important for synaptic pruning (Paoli-celli et al.,2011),and they have an activated,amoeboid morphology with high phagocytic activity (Schwarz et al.,2012).Microglial changes toward an amoeboid morphol-ogy are also associated with aging (Conde and Streit,2006;von Bernhardi et al.,2010),suggesting that their gene expression proﬁles may also be altered at these times.Therefore,for the present study,we hypothesized that,in the normal CNS,microglia undergo age-dependent geneJ.M.Crain and M.Nikodemova contributed equally to this work.Contract grant sponsor:NINDS;Contract grant number:R01NS049033;Contract grant sponsor:NIH;Contract grant number:R25GM083252(to J.M.C.)*Correspondence to:Maria Nikodemova,PhD,Department of Compar-ative Biosciences,University of Wisconsin,2015Linden Drive,Madison,WI 53706.E-mail:nikodemova@Received 2January 2013;Revised 20February 2013;Accepted 29March 2013Published online 17May 2013in Wiley Online Library().DOI:10.1002/jnr.23242VC 2013Wiley Periodicals,Inc.Journal of Neuroscience Research 91:1143–1151(2013)expression changes that reﬂect the morphologic and func-tional plasticity that they exhibit during development and aging.We focused on key genes associated with the classi-cal proinﬂammatory(M1)and alternative anti-inﬂamma-tory(M2)phenotypes,hypothesizing that microglia will express more M1markers in the postnatal and aging CNS when they display an activated morphology,whereas,in the young adult CNS,microglia will be polarized toward the M2phenotype.Another aspect of microglial biology that is rarely studied is whether microglial gene expression is sex de-pendent(Sierra et al.,2007).Many neurodegenerative diseases characterized by neuroinﬂammation are sexually dimorphic.For example,women are at higher risk for developing Alzheimer’s disease and multiple sclerosis, whereas men are more likely to develop amyotrophic lat-eral sclerosis and Parkinson’s disease(Payami et al.,1996; Logroscino et al.,2010;Wirdefeldt et al.,2011;Voskuhl and Gold,2012).Although the causes of these sex differ-ences remain poorly understood,potential sexual dimor-phisms in microglia may play a role.Estrogen receptors (ER)in the CNS mediate the effects of estrogens in females as well as the effects of testosterone in males, which is converted in the brain to estrogen by aromatase (Balthazart and Ball,1998).ERs underlie sex-dependent differences in neurons(Bloch et al.,1992;Simerly et al., 1997;Shughrue et al.,2002;Flores et al.,2003)and sup-press inﬂammatory responses of microglia and macro-phages(Vegeto et al.,2006;Smith et al.,2011;Arevalo et al.,2012).Therefore,we hypothesized that microglial inﬂammatory gene expression would be sex dependent and that alterations in ER expression would accompany these changes.A major goal of this study was to deter-mine the age at which potential sexual dimorphisms in microglial gene expression would be evident.To address our hypotheses,we evaluated ER and M1 and M2marker gene expression in microglia from healthy C57Bl/6male and female mice ranging in age from3days to12months.Primary microglial cultures derived from neonatal animals are an invaluable tool to study many aspects of microglial activities.Therefore,we also com-pared their gene expression proﬁles with those of microglia in vivo from neonates of the same age,to determine whether and how in vitro culturing alters their properties.MATERIALS AND METHODSAnimalsC57Bl/6mice were purchased from Charles River.All animals were maintained in an AALAC-accredited animal facil-ity with a12-hr light/dark cycle regime and access to food and water ad libitum.The7–8-week-old and4-month-old females were virgins.The12-month-old mice were retired breeders, with females not having borne a litter for at least2months prior to their use to minimize the possibility that hormones associated with pregnancy/lactation would interfere with microglial activ-ities.All experiments were approved by the University of Wis-consin Madison Institutional Animal Care and Use Committee.We examined microglial gene expression at different ages,selected based on important developmental milestones. Postnatal day3(P3)is a time following the testosterone surge in males(that occurs on the day of birth)that is responsible for masculinization of the still developing CNS.In addition,pri-mary microglial cultures are usually prepared from mice of this age.P21is a time proximal to weaning and represents an im-portant transition before the onset of puberty that begins during the fourth week of age in this mouse strain(Witham et al., 2012).Seven-to eight-week-old mice are young adults that have acquired full reproductive capacity,and4-month-old mice represent sexually mature adults.These adult ages are also the most commonly used ages in most studies.Finally,12-month-old mice represent older animals at a time when both male and female reproductive potential and gonadal hormone levels are beginning to decline.C57Bl/6mice usually do not produce litters after1year of age(Liu et al.,2013).CD11b1Cell IsolationCD11b1cells were isolated as previously described (Crain et al.,2009;Nikodemova and Watters,2012). Brieﬂy,mice were euthanized and perfused with cold phos-phate-buffered saline(PBS).Whole brains(including cere-bellum and brainstem)were dissected and dissociated into a single-cell suspension using the Neural Tissue Dissociation Kit containing papain(Miltenyi Biotec,Bergisch Gladbach, Germany).Myelin was removed by centrifugation in0.9M sucrose in Hank’s buffered salt solution.Cells were stained with phycoerythrin(PE)-conjugated anti-CD11b antibodies, followed by magnetic bead-conjugated secondary antibodies against PE.Magnetically tagged CD11b1cells were then separated using MS columns according to the manufacturer’s protocol(Miltenyi Biotec).Reagents were used at4 C,and the cells were kept on ice during the isolation process.The average purity of isolated cells having the characteristics of microglia was>97%as determined byﬂow cytometry for CD11b/CD45staining(Crain et al.,2009;Nikodemova and Watters,2012).We recently showed that the isolation efﬁciencies of microglia expressing low and high CD11b levels were equal;therefore,microglial isolation is not expected to be affected by variations in CD11b expression at different time points(Nikodemova and Watters,2012). GenotypingThe sex of the3-day-old mice was veriﬁed by genotyp-ing for the sex-determining region Y(SRY)gene,which is located on the Y chromosome,as previously described(Crain et al.,2009).Genomic DNA was isolated by digestion of a small section of tail and then used in PCR for SRY.Primary Microglial CulturesPrimary neonatal microglial cultures were prepared as previously described,from approximately50%female and50% male litters(Nikodemova et al.,2007).Brieﬂy,3-day-old C57Bl/6mice were euthanized,and brains were dissected and cleaned of meninges and visible blood vessels and then dissoci-ated by incubation in0.25%trypsin and DNase I,followed by1144Crain et al.Journal of Neuroscience Researchtrituration with a Pasteur pipette.Cells were plated in T75ﬂasks containing Dulbecco’s modiﬁed Eagle’s medium supple-mented with10%fetal bovine serum and penicillin/streptomy-cin.Microglia were harvested by shaking10–14days later and cultured for2days in the medium described above.The purity of microglial cultures was>96%as assessed by CD11b1stain-ing,as described previously(Nikodemova et al.,2007).RNA Extraction and Quantitative PCRRNA was extracted from either primary microglial cul-tures or freshly isolated microglia using Tri reagent(Sigma-Aldrich,St.Louis,MO).cDNA was synthesized from1l g total RNA and MMLV reverse transcriptase(Invitrogen,Carlsbad, CA)as previously described(Crain et al.,2009).Quantitative PCR was performed on an ABI7300system using Power SYBR green(Applied Biosystems,Carlsbad,CA).The primer sequences are given in Table I and were designed to span introns whenever possible.Primer efﬁciency was tested by serial dilu-tion.ER b expression was tested by using three independent primer sets whose efﬁciency was veriﬁed with cDNA from ovary as a positive control.The Ct values for ER b were 20in the ovaries(highly expressed),26in whole brain tissue homoge-nates,and>33in isolated microglia(deﬁned as undetectable).Ct values from duplicate measurements of each sample were averaged,and relative expression levels were determined by the DD Ct method.The expression of each gene was nor-malized to the levels of18s and/or b-actin within each sample as previously described(Crain et al.,2009).Statistical AnalysisData are expressed as mean6SEM of n58–14mice in each group.Results for primary microglial cultures are from three independent culture preparations.Statistical analyses were performed in Sigma Stat3.1software.One-way ANOVA was used to determine statistically signiﬁcant differences in gene expression over time in the same sex,and two-way ANOVA followed by the Holm-Sidak test was used to determine differ-ences in age-dependent gene expression between females and males.Statistical signiﬁcance was set at P<0.05.Levels of gene expression are displayed relative to3-day-old males,which allowed comparison over time and between sexes.In some cases,a Student’s t-test was used to determine differences between primary cultures and P3microglia or differences in expression between males and females at the same time point, as indicated in Results.Gene expression in microglial cultures was compared with both P3male and female microglia.RESULTSAge-and Sex-Dependent M1Gene Expression We examined basal expression levels of key proin-ﬂammatory genes typically associated with the M1pheno-type:iNOS,TNF a,IL-1b,and IL-6in na€ıve mice at P3, P21,7–8weeks,4months,and12months of age.Nota-bly,the expression of each gene displayed a unique time course,suggesting their independent regulation with age. The levels of mRNA for each gene are expressed relative to3-day-old males.iNOS mRNA levels were highest at P3,followed by a signiﬁcant70%downregulation by P21(Fig.1A).In adult mice,iNOS expression was only10–20%of that seen at P3(two-way ANOVA,P<0.001).We did not detect any sex differences in the expression of iNOS at any age.TNF a was highly expressed at P3,but it was signiﬁ-cantly lower between P21and4months of age(Fig.1B).A second peak of TNF a mRNA levels occurred at12months in both sexes.A two-way ANOVA revealed signiﬁcant age-dependent changes in TNF a expression(P<0.001), without signiﬁcant interaction with sex(P<0.2).Although a two-way ANOVA analysis did not reveal signiﬁcant sex-dependent TNF a expression,females had60%higher TNF a mRNA levels at P3than males,a difference that was statistically signiﬁcant by Student’s t-test(P<0.007).In males,there were no signiﬁcant age-dependent changes in the expression of IL-1b(Fig.1C),and,in females,IL-1b decreased by50%at7weeks of age com-pared with P3.By12months,IL1-b appeared to be up-regulated in both sexes,but this increase did not reach statistical signiﬁcance as determined by one-way or two-way ANOVA.A sex difference was observed in IL-1bTABLE I.Primer Sequences Used for qPCRGene Forward primer Reverse primerVEGF TTGAGACCCTGGTGGACATCT CACACAGGACGGCTTGAAGA ER a TGCGCAAGTGTTACGAAGTGG TCATGTCTCCTGAAGCACCCA ER b GCTGGCTGACAAGGAACTGGT CGAGGTCTGGAGCAAAGATGA Arginase-I AGCCAATGAAGAGCTGGCTGGT AACTGCCAGACTGTGGTCTCCA IL-10GCCTTATCGGAAATGATCCA TCTCACCCAGGGAATTCAAA iNOS TGACGCTCGGAACTGTAGCAC TGATGGCCGACCTGATGTT TNF a TGTAGCCCACGTCGTAGCAA AGGTACAACCCATCGGCTGG IL-6ACTTCCATCCAGTTGCCTTC GTCTCCTCTCCGGACTTGTG IL-1b TGTGCAAGTGTCTGAAGCAGC TGGAAGCAGCCCTTCATCTT TLR4GAGGCAGCAGGTGGAATTGTAT TTCGAGGCTTTTCCATCCAA TLR2CGAGTGGTGCAAGTACGAACTG TGGTGTTCATTATCTTGCGCAG FcR g I TGCTACTTTGGGTTCCAGTCGGT TACTGACCCATGGAGGCTGCA CD11b AAGGATTCAGCAAGCCAGAA GGAGGGATGAGAGTCCACAT 18S CGGGTGCTCTTAGCTGAGTGTCCCG CTCGGGCCTGCTTTGAACAC b-Atin ACCCTAAGGCCAACCGTGAA AGAGCATAGCCCTCGTAGATGGMicroglial Gene Expression in Healthy Brain1145 Journal of Neuroscience ResearchmRNA levels at P3,when expression was signiﬁcantly higher in females than in males (t -test,P <0.001).Contrary to other proinﬂammatory genes that have high expression levels at P3,IL-6expression was lowest at P3compared with adult animals,which had three to four times greater IL-6mRNA levels (two-way ANOVA,P <0.002).Interestingly,whereas females had higher IL-6mRNA levels at P3(t -test,P <0.001),this sexual dimorphism did not persist in adulthood (Fig.1D).Age-and Sex-Dependent M2Gene ExpressionWe investigated the expression of genes often used to indicate the M2phenotype:the anti-inﬂammatory cytokine IL-10,arginase-I,and the growth factor VEGF.We detected no signiﬁcant age-dependent changes in the expression of IL-10in males (one-way ANOVA,P 50.12;Fig.2A);however,females showed decreased expression at 7weeks of age (one-way ANOVA,P <0.04).Females alsohad almost twofold higher levels of IL-10mRNA at P3than males (two-way ANOVA,P <0.03).The time course of arginase-I was very similar to that of iNOS.The highest expression was observed at P3,followed by downregulation by P21to 30%of P3levels (Fig.2B).In adulthood,the levels of arginase-I mRNA were <10%of P3expression (two-way ANOVA,P <0.001).No differences were observed between males and females.The expression of VEGF,a growth factor that supports neuronal survival,was unchanged at all time points evaluated (Fig.2C),and no differences between males and females were observed.Age-and Sex-Dependent Expression of Membrane ProteinsToll-like receptors (TLRs)play an important role in the activation of innate immune cells,including microglia.We analyzed the expression of TLR4and TLR2becauseFig.1.Basal expression of proinﬂammatory genes in microglia.The expression of iNOS (A )and TNF a (B ),but not of IL-1b (C ),was signiﬁcantly higher in microglia isolated from whole brains of 3-day-old mice.On the contrary,IL-6(D )expression was lowest at P3compared with other ages.Females had higher expression of TNF a ,IL-1b ,and IL-6than males at P3.Gene expression in primary micro-glia was signiﬁcantly affected by culturing in vitro .Gene expressiondata are expressed as fold change relative to 3-day-old males.+Signiﬁ-cant age-dependent differences vs.3-day-old males.*Signiﬁcant age-dependent differences vs.3-day-old females.#Signiﬁcant differences between males and females of the same age.“a”indicates signiﬁcant difference in gene expression in primary microglia vs.3-day-old males.One symbol,P <0.05;two symbols,P <0.01;three symbols,P <0.001;N.D.,not determined.1146Crain et al.Journal of Neuroscience Researchthey are associated with CNS disorders such as ischemia,infections,multiple sclerosis,and others,and males and females are differentially predisposed to these disorders.The highest expression of TLR4was observed at P21in both sexes (Fig.3A;two-way ANOVA,P <0.02).TLR2expression also exhibited age-dependent changes (two-way ANOVA,P <0.016),the lowest mRNA levels being observed at 7weeks of age (Fig.3B).There were no statis-tically signiﬁcant sex-dependent differences in the expres-sion of TLR2or TLR4.Fc receptors mediate antibody-dependent phagocy-tosis,and morphological studies indicate differences in the prevalence of amoeboid microglia in postnatal males and females (Schwarz et al.,2012).We evaluated the expres-sion of FcR g I that binds IgG,the most common class of antibodies (Fig.3C).FcR g I mRNA levels were signiﬁ-cantly upregulated at P21compared with all other ages (two-way ANOVA,P <0.002),but there were no differ-ences between males and females.Finally,we examined the expression of CD11b,an integrin involved in cell adhesion,phagocytosis,chemo-taxis,and inﬂammation.CD11b is often upregulated upon microglial activation.CD11b mRNA levels were lowest at P3,followed by the highest expression levels at P21(one-way ANOVA,P <0.001,for males;P <0.01,for females;Fig.3D).Although CD11b expression was downregulated after P21,it still remained higher than at P3.We found no signiﬁcant differences in CD11b expression between males and females at any age.Age-and Sex-Dependent Expression of ERsWe also evaluated ER a and ER b expression in microglia,given the sexual dimorphisms in several neuro-logic disorders.ER a mRNA expression was very low at P3but increased at P21.Its expression further increased by 7–8weeks of age,after which time its levels remained constant until 12months of age in both sexes (Fig.4).Compared with P3,ER a mRNA levels were approxi-mately fourfold higher at 21days and six-to sevenfold higher at the other time points.Importantly,no differen-ces were observed in microglial ER a expression between males and females at any age.ER b mRNA expression was not detectable at any age evaluated,in either male or female mice,suggesting that this gene is not expressed in microglia from healthy animals.Basal Gene Expression in Primary MicrogliaBecause mixed-sex primary microglial cultures are commonly used to study microglia in vitro ,we compared gene expression in cultured cells to that of microglia freshly isolated from animals of the same age (P3)from which the primary cultures had been prepared.We found that gene expression in primary microglia was signiﬁcantly different from that of male and female P3microglia in vivo .Moreover,primary microglial gene proﬁles did not match the proﬁle of microglia at any age evaluated here.TNF a mRNA levels were highly upregulated in neonatal microglial cultures compared with male butnotFig.2.Basal expression of anti-inﬂammatory and trophic factor genes in microglia.P3females had higher microglial expression of IL-10(A )than males.Arginase-I (B )expression was highest at P3both in males and in females compared with other ages.There were no sex-or age-dependent changes in VEGF (C )expression.Primary microglia cultures had signiﬁ-cantly lower expression of IL-10compared with males or females in vivo ,whereas VEGF was signiﬁcantly upregulated compared with any age or sex in vivo .Gene expression data are expressed as fold change relative to 3-day-old males.+Signiﬁcant age-dependent differences vs.3-day-old males.*Sig-niﬁcant age-dependent differences vs.3-day-old females.#Signiﬁcant differ-ences between males and females of the same age.“a”indicates signiﬁcant difference in gene expression in primary microglia vs.3-day-old males.One symbol,P <0.05;two symbols,P <0.01;three symbols,P <0.001.Microglial Gene Expression in Healthy Brain 1147Journal of Neuroscience Researchfemale P3microglia (Fig.1B),whereas iNOS expression was signiﬁcantly downregulated,with levels comparable to those observed in male and female adult microglia (Fig.1A).IL-10mRNA levels were also signiﬁcantly lower in primary cultures compared with male and female micro-glia of any age (Fig.2A).Arginase-I mRNA showed lev-els comparable to levels in male and female P3microglia (Fig.2B).Interestingly,the expression of VEGF was increased by sevenfold in primary cultures compared with freshly isolated male and female microglia from mice of any age.CD11b mRNA levels in primary cultures were 16–18times higher than at P3in males and females (Fig.3),whereas the expression of TLR2,TLR4,and FcR g I was comparable to that of P3mice.Finally,ER a mRNA levels in primary microglial cells were signiﬁcantly down-regulated compared with male and female microglia in vivo from any age,whereas ER b mRNA levels remained undetectable (Fig.4).DISCUSSIONMicroglia possess great morphological and functional plas-ticity that allow their rapid response to speciﬁc physiolog-ical or pathological signals.However,it is not known whether microglial properties differ in the healthy CNS of postnatal,adult,and aged mice,since no studies have systematically evaluated microglia over time.Our data demonstrate that basal microglial gene expression signiﬁcantly varies in the postnatal and the adult brain,perhaps allowing microglial acquisition of speciﬁc age-dependent phenotypes.Interestingly,however,microglia in the healthy CNS are not fully committed to either an inﬂammatory or an anti-inﬂammatory phenotype at any age but rather display some M1and M2markers with variable age-dependent expression levels.At P3,microglia were characterized by high expres-sion of iNOS,TNF a ,and arginase-I mRNAlevelsFig. 3.Basal expression of membrane proteins in microglia.P21microglia were characterized by elevated expression of TLR4(A ),FcR g I (C ),and CD11b (D ),but not of TLR2(B ),compared with other ages.Interestingly,primary microglial cells had elevated CD11b levels compared with male or female P3pups in vivo .Gene expression data are expressed as fold change relative to 3-day-old males.+Signiﬁcant age-dependent differences vs.3-day-old males.*Signiﬁ-cant age-dependent differences vs.3-day-old females.#Signiﬁcant differences between males and females of the same age.“a”indicates signiﬁcant difference in gene expression in primary microglia vs.3-day-old males.One symbol,P <0.05;two symbols,P <0.01;three symbols,P <0.001.1148Crain et al.Journal of Neuroscience Researchrelative to other ages.Thus,during the early postnatal pe-riod,microglia express concomitant M1(iNOS,TNF a )and M2(arginase-I)markers,suggesting either that they acquire a unique phenotype related to speciﬁc develop-mental needs at this age or that there are several microglial subpopulations that may be region speciﬁc.The latter is supported by the presence of at least three different microglial morphologies found in many CNS regions at this age,with the amoeboid morphology being the most prevalent (Schwarz et al.,2012).Both TNF a and nitric oxide (produced by iNOS)exert pleiotropic effects.In addition to their well-known role in inﬂammation,both are involved in neuronal apoptosis and synaptic plasticity and pruning,frequent processes during early CNS devel-opment in which microglia are actively involved (McCoy and Tansey,2008;Zhou and Zhu,2009).The signiﬁ-cance of arginase-I expression at this age is not yet clear.Both iNOS and arginase-I use arginine as a substrate for their enzymatic activities,thus competing for arginine availability.Some studies suggest that arginase-I may function as a modulator of iNOS activity to prevent over-production of nitric oxide in immune cells (Chang et al.,1998;Mori,2007).On the other hand,in macrophages,arginase-I activity is important for extracellular matrix production,facilitating wound healing (Bansal and Ochoa,2003).At P3the brain is still developing and growing,so it is possible that microglia participate in extracellular matrix building through arginase-I activities.At P21,iNOS,TNF a ,and arginase-I are downre-gulated,whereas IL-6,CD11b,TLR4,and FcR g ImRNAs are signiﬁcantly increased,suggesting that micro-glia at P21are phenotypically and functionally distinct from both P3and adult microglia.The functional signiﬁ-cance of elevated CD11b and TLR4expression at P21is not yet clear and warrants further study.Fc receptors mediate antibody-dependent phagocytosis and are impor-tant modulators of microglial activities.Although increased IgG and Fc receptor levels are evident in the aged CNS and during neurodegenerative disease in ani-mal models and humans (Lira et al.,2011;Lunnon et al.,2011;Cribbs et al.,2012),the role of microglial Fc recep-tors during CNS development is unknown.Our data sug-gest that they may play a role in the postnatal period,when increased phagocytosis may be necessary for clear-ing debris from neuronal remodeling processes.Between 2and 4months of age,microglia express low levels of the M1markers iNOS and TNF a mRNA.IL-10and arginase-I expressions,markers of two different M2subtypes,are also low,suggesting that microglia in the healthy adult brain are not polarized to either the M1or M2phenotype.However,by 12months of age,microglial TNF a mRNA had increased to the levels found during early postnatal development.IL-1b mRNA was also increased at 12months,although not signiﬁcantly.Similar results have been reported from other studies (Godbout et al.,2005;Sierra et al.,2007)and suggest that,at this older age,microglia may be polarizing toward the M1phenotype.The only signiﬁcant sexual dimorphisms we observed in microglial gene expression were in the early postnatal period (P3).Microglia from female mice had higher mRNA levels for TNF a ,IL-1b ,IL-6,and IL-10than those from males.The testosterone surge occurring in male mice shortly after birth may underlie this sex-related differences,as androgens reportedly reduce the expression of proinﬂammatory cytokines in macrophages (Brown et al.,2007;Vignozzi et al.,2012),and they are converted to estrogens in the CNS which also exert anti-inﬂammatory effects.ER a levels were lowest at P3rela-tive to other ages,and no differences between males and females were found at this age or at any other tested.In addition,we did not detect ER b mRNA in microglia at all,consistent with a report by Sierra et al.(2008).Previ-ous studies have shown effects of ER b in activated micro-glial cell lines and primary cultures and in ischemically injured nonhuman primates (Mor et al.,1999;Baker et al.,2004;Takahashi et al.,2004;Lewis et al.,2008),but no reports to our knowledge have demonstrated effects of ER b activation on inﬂammatory gene expres-sion in quiescent microglia.Together these data suggest that ER b is not expressed in microglia in the healthy CNS and that neither ER a nor ER b underlies the sexual dimor-phisms observed in early postnatal microglial gene expres-sion.However,a recent study by Sato et al.(2004)showed that some effects of male sex hormones in the CNS are mediated via androgen receptors,so they may be responsi-ble for some sex-dependent differences in microglial gene expression,although androgen receptors have not been detected in microglia (Sierra et al.,2008).Regardless,theFig.4.Basal expression of estrogen receptors in microglia.The expres-sion levels of ER a and ER b were evaluated by qRT-PCR and are expressed as fold change relative to 3-day-old males.P3male and female microglia had the lowest expression of ER a compared with other ages.No sex-dependent differences were detected in ER a expression at any age.Primary microglia had downregulated ER a levels compared with P3male and female microglia in vivo .ER b was unde-tectable in all ages.+Signiﬁcant age-dependent differences vs.3-day-old males.*Signiﬁcant age-dependent differences vs.3-day-old females.#Signiﬁcant differences between males and females of the same age.“a”indicates signiﬁcant difference in gene expression in primary microglia vs.3-day-old males.Two symbols,P <0.01;three symbols,P <0.001.Microglial Gene Expression in Healthy Brain 1149Journal of Neuroscience Research。

The function of microglia

Associate editor:M.EndohThe function of microglia through purinergic receptors:Neuropathic pain and cytokine releaseKazuhide Inoue *Department of Molecular and System Pharmacology,Graduate School of Pharmaceutical Sciences,Kyushu University,Maidashi 3-1-1,Higashi-ku,Fukuoka 812-8582,JapanAbstractMicroglia play an important role as immune cells in the central nervous system (CNS).Microglia are activated in threatened physiological homeostasis,including CNS trauma,apoptosis,ischemia,inflammation,and infection.Activated microglia show a stereotypic,progressive series of changes in morphology,gene expression,function,and number and produce and release various chemical mediators,including proinflammatory cytokines that can produce immunological actions and can also act on neurons to alter their function.Recently,a great deal of attention is focusing on the relation between activated microglia through adenosine 5V -triphosphate (ATP)receptors and neuropathic pain.Neuropathic pain is often a consequence of nerve injury through surgery,bone compression,diabetes,or infection.This type of pain can be so severe that even light touching can be intensely painful and it is generally resistant to currently available treatments.There is abundant evidence that extracellular ATP and microglia have an important role in neuropathic pain.The expression of P2X4receptor,a subtype of ATP receptors,is enhanced in spinal microglia after peripheral nerve injury model,and blocking pharmacologically and suppressing molecularly P2X4receptors produce a reduction of the neuropathic pain.Several cytokines such as interleukin-1h (IL-1h ),interleukin-6(IL-6),and tumor necrosis factor-a (TNF-a )in the dorsal horn are increased after nerve lesion and have been implicated in contributing to nerve-injury pain,presumably by altering synaptic transmission in the CNS,including the spinal cord.Nerve injury also leads to persistent activation of p38mitogen-activated protein kinase (MAPK)in microglia.An inhibitor of this enzyme reverses mechanical allodynia following spinal nerve ligation (SNL).ATP is able to activate MAPK,leading to the release of bioactive substances,including cytokines,from microglia.Thus,diffusible factors released from activated microglia by the stimulation of purinergic receptors may have an important role in the development of neuropathic pain.Understanding the key roles of ATP receptors,including P2X4receptors,in the microglia may lead to new strategies for the management of neuropathic pain.D 2005Elsevier Inc.All rights reserved.Keywords:ATP;P2X4;Microglia;Neuropathic pain;Allodynia;Spinal cord;p38Abbreviations:ADP,adenosine 5V -diphosphate;ATP,adenosine 5V -triphosphate;ATP g S,adenosine 5V -O -(3-thiotriphosphate);BDNF,brain-derived neurotrophic factor;BzATP,2V -and 3V -O -(4-benzoylbenzoyl)adenosine 5V -triphosphate;[Ca 2+]i,intracellular Ca 2+concentration;CD11b,clusterdeterminant 11b;CNS,central nervous system;CR3,complement receptor 3;ERK,extracellular signal-regulated protein kinase;Iba1,ionized calcium binding adaptor molecule 1;ICE,IL-1h -converting enzyme;IL-1h ,interleukin-1h ;IL-6,interleukin-6;iNOS,inducible nitric oxide synthase;InsP3,inositol 1,4,5-trisphosphate;JNK,c-Jun N-terminal kinase;LPS,lipopolysaccharide;MAPK,mitogen-activated protein kinase;MEK,mitogenactivated protein kinase kinase;MHC,histocompatibility complex;oATP,oxidized ATP;PK11195,[1-(2-chlorophenyl)-N -methyl-N -(1-methylpropyl)-3-isoquinolineisoquinoline carboxamide];PKC,protein kinase C;PLC,phospholipase C;PPADS,pyridoxalphosphate-6-azophenyl-2V ,4V -disulphonic acid;PTK,protein tyrosine kinase;PTX,pertussis toxin;SB203580,4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)I H -imidazole;SOC,store-operated Ca 2+entry;SP600125,anthra[1,9-cd]pyrazol-6(2H )-one;TNF-a ,tumor necrosis factor-a ;TNP-ATP,2V ,3V -O -(2,4,6-trinitrophenyl)adenosine 5V -triphosphate;U0126,1,4-diamino-2,3-dicyano-1,4-bis [2-amino-phenylthio]butadiene;UTP,uridine 5V -triphosphate.0163-7258/$-see front matter D 2005Elsevier Inc.All rights reserved.doi:10.1016/j.pharmthera.2005.07.001*Tel./fax:+81926424729.E-mail address:inoue@phar.kyushu-u.ac.jp.Pharmacology &Therapeutics 109(2006)210–226/locate/pharmtheraContents1.Introduction (211)2.Purinergic receptors expressing in microglia (212)3.Activated microglia in neuropathic pain (213)3.1.Microglia activation in patients bearing a kind of neuropathic pain (213)3.2.Microglia activation in neuropathic pain model rats (213)3.3.High expression of P2X4in spinal microglia in neuropathic pain model (213)3.4.P2X4stimulation causes and maintains allodynia (214)4.Microglia activation through purinergic receptors (215)4.1.Chemotaxis following membrane ruffling (215)4.2.Function and release of plasminogen (215)4.3.Function and the release of tumor necrosis factor-a (217)4.4.Function and the release of interleukin-6 (218)4.5.Function and the release of interleukin-1h (219)4.6.Function and the release of ionized calcium binding adaptor molecule1/microglialresponse factor-1/allograft inflammatory factor-1 (220)5.Conclusion and consideration (221)References (222)1.IntroductionMicroglia are often considered to be resident macro-phages and to play an important role as immune cells in the central nervous system(CNS;Kreutzberg,1996;Stoll &Jander,1999;Nakajima&Kohsaka,2001).In adults, microglia are distributed throughout CNS and represent5–10%of glia.They have a small soma bearing thin and branched processes under normal conditions.Such micro-glia are said to be F resting_,but acting as sensors for a range of stimuli that threaten physiological homeostasis, that is,CNS trauma,apoptosis,ischemia,inflammation, and infection.Once activated by these stimuli,that is, bioactive substances,cytokines and neurotransmitters, including adenosine5V-triphosphate(ATP),microglia show a stereotypic,progressive series of changes in morphology, gene expression,function,and number(Perry,1994; Kreutzberg,1996;Stoll&Jander,1999;Streit et al., 1999;Nakajima&Kohsaka,2001).Activated microglia change their morphology from a resting,ramified shape into an active,amoeboid shape(Perry,1994;Kreutzberg, 1996;Stoll&Jander,1999;Streit et al.,1999;Nakajima& Kohsaka,2001).They up-regulate expression of a variety of cell-surface molecules,including the complement receptor3(CR3;also known as clusterdeterminant[CD] 11b(integrin a M)/CD18(integrin h2),or as Mac-1; Kreutzberg,1996;Stoll&Jander,1999;Ehlers,2000; Nakajima&Kohsaka,2001),which is recognized by the antibody OX-42(Robinson et al.,1986).Activated micro-glia also express immunomolecules such as major histo-compatibility complex(MHC)class I and II(Kreutzberg, 1996;Stoll&Jander,1999;Streit et al.,1999),which have a role in antigen presentation to T lymphocytes.Activated microglia produce and release various chemical mediators, including proinflammatory cytokines that can produce immunological actions and can also act on neurons to alter their function(Kreutzberg,1996;Stoll&Jander, 1999;Nakajima&Kohsaka,2001;Hanisch,2002). Recently,a great deal of attention has been focused on the relation between neuropathic pain and microglia activated through ATP receptors(Tsuda et al.,2005).ATP is released from damaged cells as a result of ischemia or inflammation and serves as a cell-to-cell mediator through cell surface P2receptors,which are widely distributed throughout the nervous system,including microglia(Inoue, 2002).P2receptors are divided into2subtypes:P2X and P2Y(Abbracchio&Burnstock,1994;Fig.1).P2X receptors (P2X1–P2X7)are coupled to nonselective cation channels, allowing the influx of Na+and Ca2+(North,2002),whereas Fig.1.P2receptors.P2receptors are divided into2subtypes:P2X and P2Y. P2X receptor subtypes(P2X1–P2X7)are40–50%identical in amino acid sequence.Each subtype has2transmembrane domains.Nonselective cation channels form as multimers(presumably3multimers)of several subunits. Homomeric P2X1,P2X2,P2X3,P2X4,P2X5,and P2X7channels and heteromeric P2X2/3and P2X1/5channels have been most fully charac-terized following heterologous expression.P2Y receptors(P2Y1,P2Y2, P2Y4,P2Y6,P2Y11,P2Y12,P2Y13,and P2Y14)are G-protein coupled, and their activation leads to inositol lipid hydrolysis,intracellular Ca2+ mobilization,or modulation of adenylate cyclase activation,through G q/11 (P2Y1,P2Y2,P2Y4,P2Y6),G s(P2Y6)and G i/o(P2Y11,P2Y12,P2Y13, and P2Y14),respectively.K.Inoue/Pharmacology&Therapeutics109(2006)210–226211P2Y receptors (P2Y1,P2Y2,P2Y4,P2Y6,P2Y11,P2Y12,P2Y13,and P2Y14)are G-protein coupled,and their activation leads to inositol lipid hydrolysis,intracellular Ca 2+mobilization,or modulation of adenylate cyclase activation (Inoue,2002).ATP strongly activates microglial to show chemotaxis via the Gi-and Go-coupled P2Y12receptor (Honda et al.,2001)and stimulates the release of plasminogen,interleukin (IL)-6,tumor necrosis factor-a (TNF-a ),and IL-1h (Ferrari et al.,1997a,1997b;Inoue et al.,1998;Hide et al.,2000;Shigemoto-Mogami et al.,2001;Inoue,2002;Suzuki et al.,2004)by means of different types of P2receptor and intracellular signals.Neuropathic pain is a type of pathological pain that often develops when nerves are damaged through surgery,bone compression,diabetes or infection,also which does not resolve even when the overt tissue damage has healed (Aldskogius &Kozlova,1998;Carson,2002;Eikelen-boom et al.,2002).Neuropathic pain can be so severe that even light contact with clothing can be intensely painful (tactile allodynia:an abnormal hypersensitivity to innocuous stimuli)and is often resistant to most current treatments,including a narcotic analgesia,although a number of drugs produce some relief.Accumulating evidence concerning how peripheral nerve injury creates neuropathic pain has suggested that molecular and cellular alterations in primary sensory neurons and in the spinal dorsal horn after nerve injury have an important role in the pathogenesis of neuropathic pain (Aldskogius &Kozlova,1998;Carson,2002;Eikelenboom et al.,2002).While there is an increasing body of evidence suggesting that P2X3Rs,a subtype of ionotropic ATP receptors,in primary sensory neurons have a role in neuropathic pain (Colburn et al.,1999;Banati,2002;Garden,2002),other P2XR and P2YR subtypes of ATPreceptors are also beginning to be investigated in terms of their changes in expression using cDNA microarray (Visentin et al.,1999;Inoue,2002;Suzuki et al.,2004).Recently,it was reported that astrocyte and microglia are activated strongly in neuropathic model animals,suggest-ing the role of glial cells in the pain sensation (Watkins et al.,2001).However,there was no direct evidence supporting this suggestion.More recently,we have revealed that the P2X4R subtype in the activated spinal microglia is required for the expression of neuropathic pain after nerve injury (Tsuda et al.,2003).This review shows the progress in the current understanding of how the ATP receptor participates in the activation of microglia leading into neuropathic pain.2.Purinergic receptors expressing in microglia ATP evokes currents in rat microglia (No ¨renberg et al.,1994;Illes et al.,1996)and increases in intracellular calcium ([Ca 2+]i)in mouse and human microglia (Walz et al.,1993;Toescu et al.,1998;Moller et al.,2000).ATP induces the release of IL-1h (Ferrari et al.,1996,1997b )and IL-6(Shigemoto-Mogami et al.,2001)from mice microglia.ATP causes chemotaxis (Honda et al.,2001)and the release of plasminogen (Inoue et al.,1998)and TNF-a (Hide et al.,2000;Morigiwa et al.,2000)from rat microglia.ATP activates nuclear factor of activated T-cells (NFAT;Ferrari et al.,1999),which modulates the early inflammatory gene expression and transcriptional activator NF-n B,which controls cytokine expression and apoptosis (Ferrari et al.,1997a ).ATP also stimulates the phosphor-ylation of mitogen-activated protein kinase (MAPK;Hide et al.,2000;Honda et al.,2001;Shigemoto-Mogami et al.,P2XP2YM X1 X2 X3 X4 X5 X 6 X7M X1 X2 X3 X4 X5 X 6 X7M Y1 Y2 Y4 Y6 Y12M Y1 Y2 Y4 Y6 Y12-RT-RT200015001000700500400300200200015001000700500400300200Fig.2.mRNA expression of P2purinoceptors receptors in primary culture microglia from rat brain.Upper :Electrophoresis photograph after a quantitative RT-PCR using specific primers for P2X1,P2X2,P2X3,P2X4,P2X5,P2X6,and P2X7.Clear bands were detected in the P2X4and P2X7lanes.Lower :Electrophoresis photograph after a quantitative RT-PCR using specific primers for P2Y1,P2Y2,P2Y4,P2Y6,and P2Y12receptors.Clear bands were detected in the P2Y2,P2Y6,and P2Y12lanes.K.Inoue /Pharmacology &Therapeutics 109(2006)210–2262122001).These data suggest that microglia possess functional receptors for purines and pyrimidines,that is,P2X receptors,ligand-gated ion-channels(Cook et al.,1998; Li et al.,1999;Ueno et al.,1999;Tsuda et al.,2000;Chizh &Illes,2001;Dunn et al.,2001),and P2Y receptors,G protein-coupled receptors(Svichar et al.,1997;Koizumi et al.,2001;Tominaga et al.,2001;Molliver et al.,2002; Sanada et al.,2002;Moriyama et al.,2003).There, however,are very few reports available indicating the mRNA expression of P2receptor subtypes in microglia. We examined using a quantitative RT-PCR method and found that microglia in a primary culture from rat brain express mainly mRNAs of P2X4and P2X7,and P2Y2, P2Y6,and P2Y12(Shigemoto-Mogami et al.,personal communication),as shown in Fig.2.3.Activated microglia in neuropathic pain3.1.Microglia activation inpatients bearing a kind of neuropathic painSince the peripheral benzodiazepine binding site practi-cally absent in the normal brain parenchyma is strongly and preferentially expressed by activated microglia around the soma of the injured neuron,[1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline isoquinoline carboxa-mide](PK11195),a ligand for the peripheral benzodiazepine binding site,binds with relative cellular selectivity to activated microglia,not to residential microglia.Thus,(R)-PK11195labeled with carbon-11and positron emission tomography(PET)have been used for the study of inflammatory and neurodegenerative brain disease in vivo, even in human.This technology is highly useful to reveal the retrograde and anterograde projection areas(Banati et al., 1997,2000).For example,increased microglial(R)-PK11195 binding is seen in the motor facial nucleus after peripheral facial nerve transection(Banati et al.,1997),in the gracile nucleus after sciatic nerve lesion(Banati et al.,1997),in the ipsilateral thalamus after cerebral cortical ischemic stroke (Pappata et al.,2000),and in the lateral geniculate bodies in multiple sclerosis patients with optic neuritis(Banati et al., 2000).The potentially useful clinical application of the specific ligand PK11195is based on3observations(Banati et al.,2001):(1)normal brain shows only minimal binding of PK11195;(2)in CNS pathology,in vivo PK11195binding is predominantly found on activated microglia;and(3)when labeled with carbon-11,PK11195can be used as a ligand for PET(Benavides et al.,1988;Junck et al.,1989;Myers et al., 1991a,1991b,1999;Cremer et al.,1992;Ramsay et al.,1992; Sette et al.,1993;Banati et al.,1999).The cortical plasticity developed after the amputation of a limb may be associated with the development of abnormal sensations,such as phantom pain,a kind of neuropathic pain, and referred cutaneous sensations(Banati et al.,2001).It was reported that cortical reorganization may be the consequence of a reorganization of the thalamus following changes of afferent inputs from the amputated limb(Jones,2000).The cause of the sustained representational plasticity in the thalamus has recently been suggested to be transneuronal atrophy in the thalamus that,in turn,would mediate cortical plasticity(Woods et al.,2000).Acute or chronic neuronal injury after the amputation of a limb evokes a rapid,transient and localized activation of microglia(Kreutzberg,1996). Banati et al.(2001)reported that limb amputation induces a trans-synaptic increase in[11C](R)-PK11195binding in the human thalamus but not somatosensory cortex,suggesting the activation of microglia in the thalamus of a patient with phantom pain.The increased thalamic signal is detectable many years after nerve injury,and this means persistent reorganization of the thalamus.The microglial activation, beyond the first-order projection area of the injured neurons, may reflect continually altered afferent activity.The activa-tion of microglia can therefore be used as a sensor of neuronal injury.3.2.Microglia activation in neuropathic pain model ratsClinical evidence that neuropathic pain results from damage to peripheral nerves in humans led to the develop-ment of a variety of models for studying neuropathic pain in laboratory animals.Evidence from studies using such models has revealed that peripheral nerve injury leads to a dramatic change in microglia within the spinal dorsal horn(Eriksson et al.,1993;Colburn et al.,1997,1999;Coyle,1998;Stuesse et al.,2000).Spinal microglia become hypertrophic in their short and thick processes within24hr after peripheral nerve injury(Eriksson et al.,1993;Tsuda et al.,2003).This is followed by a burst proliferation of microglia with a peak at around2–3days after the nerve injury(Gehrmann&Banati, 1995).Activated microglia exhibit up-regulated OX42label-ing(Eriksson et al.,1993;Liu et al.,1995;Coyle,1998; Colburn et al.,1999;Stuesse et al.,2000;Tsuda et al.,2003), which starts to increase as early as1day after nerve injury and peaks at around14days(Coyle,1998).The temporal pattern of OX42up-regulation in the dorsal horn correlated with that of the development of tactile allodynia(Coyle,1998), suggesting the role of microglia in neuropathic pain. Although there have been many studies showing that the activation of microglia in the dorsal horn is correlated with the development of pain hypersensitivity in a wide variety of nerve injury models(Eriksson et al.,1993;Liu et al.,1995; Coyle,1998;Colburn et al.,1999;Stuesse et al.,2000; Watkins et al.,2001),it remained an open question whether spinal microglia play a causal role in neuropathic pain until the report by Tsuda et al.(2003).3.3.High expression of P2X4inspinal microglia in neuropathic pain modelA clue to identifying P2X4Rs in the spinal microglia required for neuropathic pain first came from pharmaco-K.Inoue/Pharmacology&Therapeutics109(2006)210–226213logical investigations of pain behaviour after nerve injury using the P2XR antagonists 2V ,3V -O -(2,4,6-trinitrophenyl)a-denosine 5V -triphosphate (TNP-ATP)and pyridoxalphos-phate-6-azophenyl-2V ,4V -disulphonic acid (PPADS;Tsuda et al.,2003).It was found that the marked tactile allodynia that develops following the injury of a spinal nerve is reversed by acutely administering TNP-ATP intrathecally but is unaffected by administering PPADS (Fig.3A).TNP-ATP has no effect on acute pain behaviour in the uninjured state nor on motor behaviour.TNP-ATP at high concentration shows the antagonistic effect on P2X1,P2X2,P2X3,P2X4,P2X5,and P2X7.PPADS inhibits the action of all these subtypes,excluding P2X4.From these pharmacological profiles of TNP-ATP and PPADS,it was inferred that tactile allodynia depends upon P2X4Rs in the spinal cord.The expression of P2X4R protein,normally low in the naivespinal cord,progressively increases in the days following nerve injury with a time-course parallel to that of the development of tactile allodynia (Fig.3B and C).Immuno-histochemical analysis demonstrated that many small cells in the dorsal horn on the side of the nerve injury are intensely positive for P2X4R protein.These cells are identified as microglia rather than neurons or astrocytes by double immunolablelling using cell-specific markers (Fig.5A).The cells expressing P2X4R in the nerve-injured side of the dorsal horn are more numerous than under control conditions and showed high levels of OX42labeling and morphological hypertrophy,all of which are character-istic markers of activated microglia.Moreover,intrathecal administration with antisense oligodeoxynucleotide (AS)targeting P2X4R reduces the up-regulation of P2X4R protein in spinal microglia (Fig.4A)and prevents the development of the nerve injury-induced tactile allodynia (Fig.4B).The treatment with a mismatch ODN (MM)as a control does not reduce the expression of P2X4nor prevent the tactile allodynia (Fig.4A and B).The evidence implies that P2X4R’s activation is necessary for pain hypersensi-tivity following nerve injury,and that microglia are required for this hypersensitivity since the expression of these receptors in the dorsal horn is restricted to this type of cell.3.4.P2X4stimulation causes and maintains allodyniaTo demonstrate the sufficiency of P2X4R activation in microglia for the development of allodynia,Tsuda et al.(2003)performed the intrathecal administration of primary cultured microglia a stimulated in vitro by ATP.In normal rats,intrathecal administration of cultured microglia that were preincubated with ATP to activate P2X4Rs on microglia produces tactile allodynia progressively over the 3–5hr following the administration.In contrast,intrathecal administration of unstimulated microglia does not cause allodynia,nor does administering vehicle or ATP alone.Microglia also express another subtype of P2XR,P2X7R,but this receptor subtype appears not to be involved because the activation of P2X7Rs typically requires a higher concentration (more than 1mM)of ATP (Surprenant et al.,1996;Khakh et al.,2001).Moreover,in the tactile allodynia caused by the admin-istration of ATP-stimulated microglia,this allodynia is reversed by administering TNP-ATP (Tsuda et al.,2003).Thus,the stimulation of P2X4Rs is required in the tactile allodynia caused by ATP-stimulated microglia,and this tactile allodynia therefore resembles that caused by nerve injury.These findings indicate that P2X4R stimulation of microglia is not only necessary for tactile allodynia,but is also sufficient to cause the allodynia.Furthermore,this finding makes a strong case that microglia activation is not simply correlated with neuropathic pain behaviour.Rather,microglia within the dorsal horn play an active and ongoing role in the tactile allodynia produced by injury to peripheral nerves.For revealing the exact mechanism ofPost i.th. injection (min)P W T (g r a m )Day 0(Naive)Post-operation Post operation (day)P W T(g r a m )Neuropathic Pain after Nerve Injury11015ABCDay 3Day 14Day 1Day 7Fig.3.Effect of TNP-ATP on tactile allodynia and expression of P2X4R in the dorsal horn after nerve injury.(A )Tactile allodynia that develops following the injury of a spinal nerve was reversed by acutely administering TNP-ATP intrathecally but was unaffected by administering PPADS.TNP-ATP had no effect on acute pain behaviour in the uninjured state or on motor behaviour.(B ,C )The expression of P2X4R protein and the development of tactile allodynia after nerve injury.The expression of P2X4R protein progressively increased in the days following nerve injury with a time course parallel to that of the development of tactile allodynia.K.Inoue /Pharmacology &Therapeutics 109(2006)210–226214the P2X4-microglia-involving neuropathic pain,more deep research efforts should be endeavored.4.Microglia activation through purinergic receptorsThe variety of biological effects produced by ATP in microglia may provide hints towards clarifying the mech-anisms of neuropathic pain.4.1.Chemotaxis following membrane rufflingThe initial microglial responses that occur after brain injury and in various neurological diseases are characterized by microglial accumulation in the affected sites of the brain, which results from the migration and proliferation of these cells.The early-phase signal responsible for this accumu-lation is likely to be transduced by rapidly diffusible factors. Honda et al.(2001)examined the possibility that ATP released from injured neurons and nerve terminals affects the cell motility in rat primary cultured microglia.They found that extracellular ATP and adenosine5V-diphosphate (ADP)induces membrane ruffling and markedly enhances chemokinesis in a Boyden chamber assay.Further analyses using the Dunn chemotaxis chamber assay,which allows direct observation of the cell movement,revealed that both ATP and ADP induce chemotaxis of microglia.The elimination of extracellular calcium or treatment with PPADS or suramin does not inhibit ATP-or ADP-induced membrane ruffling,whereas AR-C69931MX,a P2Y12and P2Y13receptor blocker(Hollopeter et al.,2001;Fumagalli et al.,2004),or pertussis toxin(PTX)treatments clearly inhibit the ruffling.As an intracellular signaling moleculeunderlying these phenomena,the small G-protein Rac is activated by ATP and ADP stimulation,and its activation is also inhibited by pretreatment with PTX.These findings suggested that the membrane ruffling and chemotaxis of microglia induced by ATP or ADP are mediated by G(i/o)-coupled P2Y receptors(P2Y12and/or P2Y13).4.2.Function and release of plasminogenIt was shown that ATP stimulates the release of plasminogen from primary cultured rat microglia in a concentration-dependent manner from10to100A M,with a peak response at5–10min after the stimulation(Inoue et al.,1998).A1-hr pretreatment with BAPTA-AM com-pletely inhibits the plasminogen release evoked by ATP.The Ca2+ionophore A23187induces plasminogen release in a concentration-dependent manner(0.3to10A M).ATP induces a transient increase in the[Ca2+]i in a concentration-dependent manner,which is very similar to the ATP-evoked plasminogen release.A second application of ATP(100A M) stimulates an increase in[Ca2+]i similar to that of the first application(21out of21cells).The ATP-evoked increase in [Ca2+]i is totally dependent on extracellular Ca2+.2-Methylthio ATP is effective(7out of7cells),but a,h-methylene ATP was ineffective(7out of7cells)at inducing an increase in[Ca2+]i.Suramin(100A M)is shown not to inhibit the ATP-evoked increase in[Ca2+]i(20out of20 cells).2V-and3V-O-(4-benzoylbenzoyl)adenosine5V-triphosphate(BzATP)evokes a long-lasting increase in [Ca2+]i,even at1A M,a concentration at which ATP does not evoke the increase.One hour pretreatment with oxidized ATP(oATP;100A M),a selective antagonist of P2X7 receptors,blocks the increase in[Ca2+]i induced by ATP(10 and100A M).These data suggest that ATP may transit information from neurones to microglia,resulting in an increase in[Ca2+]i via the ionotropic P2X7receptor which stimulates the release of plasminogen from the microglia. However,the possible involvement of P2X4in this release cannot be excluded because BzATP can affect on P2X4.It has been found that uridine5V-triphosphate(UTP)also stimulates plasminogen release from a subpopulation of microglia(about20%of total cells),presumably through store-operated Ca2+entry(SOC)activated by ATP stim-ulation of G protein-coupled receptors,since the release evoked by UTP is also dependent on extracellular Ca2+ (Inoue et al.,unpublished data).ASMMP2X4RASMM***B LNerve injury51015PWT(gram)##ABFig.4.Effects of antisense oligodeoxynucleotide(AS)targeting P2X4R on the expression of P2X4protein and the development of tactile allodynia after nerve injury.(A)Western blotting analysis of the expression of P2X4receptor protein in the spinal dorsal horn7days after nerve injury.The animals were treated with intrathecal administration of antisense oligodeoxynucleotide (AS)targeting P2X4R for7days,beginning on the day of the nerve lesion. Intrathecal administration of antisense oligodeoxynucleotide(AS)targeting P2X4R reduced the up-regulation of P2X4R protein in spinal microglia.The treatment with a mismatch ODN(MM)as a control did not reduce the expression of P2X4.(B)AS treatment prevented the development of the nerve injury-induced tactile allodynia.The paw withdrawal threshold in animals treated with MM was not different from that of untreated controls, suggesting that MM did not prevent the tactile allodynia.K.Inoue/Pharmacology&Therapeutics109(2006)210–226215。

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01 How to Make a Mini Presentation in English 怎样做英语迷你演讲？

1. 列举法 2. 对比法 3. 时间顺序法 4. 空间顺序法 5. 数据分析法
3. Persuasive Presentation 说服型演讲

Objectives目的：
1）Change views 改变听众观念 2）Call for actions 号召听众采取行动
说服型演讲“四要诀”
1. 论点鲜明 —— 忌模糊不清 2. 论据给力 —— 忌不确凿不充分 3. 论证合理 —— 忌不条理不简洁 4. 结论可靠 —— 忌不可靠不可信
2. 准确 —— 忌模糊不清 3. 科学 —— 忌杂乱无章
信息型演讲用途
1. 发布信息 e.g. Weather forecast Stock exchange prices 获取信息，掌握先机。 2. 传播知识 e.g. Lectures in school 获取知识，增强力量。
信息型演讲组织方法
信息型演讲与说服型演讲的关系
1. 信息是说服的基础；说服是信息的归宿。
2.
陈述事实要像科学家；说服劝导要像政治家。
3 Levels of Oral Presentation
1. Speak correctly -- pronunciation, grammar 2. Speak clearly -- structure, content 3. Speak convincingly -- proposition, evidence
How to Make a Mini Presentation in English 怎样做迷你英语演讲？
Hu Yinping The College of Foreign Languages, USST huyinping66@ Aug. 31, 2012

Microglia - Wikipedia, the free encyclopedia

MicrogliaAnatomical terminology MicrogliaFrom Wikipedia, the free encyclopediaMicroglia are a type of glial cell thatare the resident macrophages of the brainand spinal cord, and thus act as the firstand main form of active immune defense inthe central nervous system (CNS).Microglia constitute 10-15% of all cells found within the brain.[1] Microglia (and astrocytes) are distributed in large non-overlapping regions throughout the brain and spinal cord.[2][3] Microglia are constantly scavenging the CNS for plaques, damaged neurons and infectious agents.[4] The brain and spinal cord are considered "immune privileged" organs in that they are separated from the rest of the body by a series of endothelial cells known as the blood–brain barrier, which prevents most infections from reaching the vulnerable nervous tissue. In the case where infectious agents are directly introduced to the brain or cross the blood–brain barrier, microglial cells must react quickly to decrease inflammation and destroy the infectious agents before they damage the sensitive neural tissue. Due to the unavailability of antibodies from the rest of the body (few antibodies are small enough to cross the blood–brain barrier), microglia must be able to recognize foreign bodies, swallow them, and act as antigen-presenting cells activating T-cells. Since this process must be done quickly to prevent potentially fatal damage, microglia are extremely sensitive to even small pathological changes in the CNS.[5] They achieve this sensitivity in part by having unique potassium channels that respond to even small changes in extracellular potassium.[4]Microglia -ramified form fromrat cortex beforetraumatic braininjury (lectinstaining with HRP)Microglia/Macrophage - activatedform from ratcortex aftertraumatic braininjury (lectinstaining with HRP)Contents1 Origin2 History3 Types3.1 Amoeboid3.2 Ramified3.3 Activated3.3.1 Non-phagocytic3.3.2 Phagocytic3.4 Gitter cells3.5 Perivascular3.6 Juxtavascular4 Functions4.1 Scavenging4.2 Phagocytosis4.3 Cytotoxicity4.4 Antigen presentation4.5 Synaptic stripping4.6 Promotion of repair4.7 Extracellular signaling5 Role in chronic neuroinflammation5.1 Cytokines5.2 Chemokines5.3 Proteases5.4 Amyloid precursor protein6 Aging7 Role of microglia in neurodegeneration7.1 Alzheimer's disease7.1.1 Treatment7.2 Parkinson's disease7.3 Cardiovascular Diseases8 Role in viral infections8.1 Human immunodeficiency virus8.2 Herpes simplex virus9 Role in bacterial infections9.1 Streptococcus pneumoniae10 Role in parasitic infections10.1 Plasmodium falciparum11 Role in neuropathic pain12 As a target to treat neuroinflammation12.1 Inhibition of activation12.2 Regulation of chemokine receptor12.3 Inhibition of amyloid deposition12.4 Inhibition of cytokine synthesis13 References14 External linksOriginMicroglial cells differentiate in the bone marrow from hematopoietic stem cells, the progenitors of all blood cells. During hematopoiesis, some of these stem cells differentiate into monocytes and travel from the bone marrow to the brain, wherethey settle and further differentiate into microglia. However, recent studies indicate microglia originate in the yolk sac during a remarkably restricted period and populate the brain mesenchyme. [6]Monocytes can also differentiate into myeloid dendritic cells and macrophages in the peripheral systems. Like macrophages in the rest of the body, microglia use phagocytic and cytotoxic mechanisms to destroy foreign materials. Microglia and macrophages both contribute to the immune response by acting as antigen presenting cells, as well as promoting inflammation and homeostatic mechanisms within the body by secreting cytokines and other signaling molecules.In their downregulated form, microglia lack the MHC class I/MHC class II proteins, IFN-γ cytokines, CD45 antigens, and many other surface receptors required to act in the antigen-presenting, phagocytic, and cytotoxic roles that hallmark normal macrophages. Microglia also differ from macrophages in that they are much moretightly regulated spatially and temporally in order to maintain a precise immune response.[7]Another difference between microglia and other cells that differentiate from myeloid progenitor cells is the turnover rate. Macrophages and dendritic cells are constantly being used up and replaced by myeloid progenitor cells whichdifferentiate into the needed type. Due to the blood–brain barrier, it would be fairly difficult for the body to constantly replace microglia. Therefore, instead of constantly being replaced with myeloid progenitor cells, the microglia maintaintheir status quo while in their quiescent state, and then, when they are activated, they rapidly proliferate in order to keep their numbers up. Bone chimera studies have shown, however, that in cases of extreme infection the blood–brain barrierwill weaken, and microglia will be replaced with haematogenous, marrow-derived cells, namely myeloid progenitor cells and macrophages. Once the infection has decreased the disconnect between peripheral and central systems is reestablished and only microglia are present for the recovery and regrowth period.[8]HistoryThe ability to view and characterize different neural cells including microglia began in 1880 when Nissl staining was developed by Franz Nissl. Franz Nissl and F. Robertson first described microglial cells during their histology experiments. The cell staining techniques in the 1880s showed that microglia are related to macrophages. The activation of microglia and formation of ramified microglial clusters was first noted by Victor Babeş while studying a rabies case in 1897. Babeşnoted the cells were found in a variety of viral brain infections but did not know what the clusters of microglia he saw were.[9] Pío del Río Hortega, a student of Santiago Ramón y Cajal, first called the cells "microglia" around 1920. He went on to characterize microglial response to brain lesions in 1927 and note the "fountains of microglia" present in the corpus callosum and other perinatal white matter areas in 1932. After many years of research Rio-Hortega became generally considered as the "Father of Microglia."[10][11] For a long period of time little improvement was made in our knowledge of microglia. Then, in 1988, Hickey and Kimura showed that perivascular microglial cells are bone-marrow derived, and express high levels ofMHC class II proteins used for antigen presentation. This confirmed Pio Del Rio-Hortega's postulate that microglial cells functioned similarly to macrophages by performing phagocytosis and antigen presentation.TypesMicroglial cells are extremely plastic, and undergo a variety of structural changes based on their location and current role. This level of plasticity is required to fulfill the vast variety of immunological functions that microglia perform, as well as maintaining homeostasis within the brain. If microglia were not capable of this they would need to be replaced on a regular basis like macrophages, and would not be available to the CNS immune defense on extremely short notice without causing immunological imbalance under normal conditions.[4]AmoeboidThis form of microglial cell is found mainly within the perinatal white matter areas in the corpus callosum known as the "Fountains of Microglia." This shape allows the microglial free movement throughout the neural tissue, which allows it to fulfillits role as a scavenger cell. Amoeboid microglia are able to phagocytose debris, but do not fulfill the same antigen-presenting and inflammatory roles as activated microglia. Amoeboid microglia are especially prevalent during the development and rewiring of the brain, when there are large amounts of extracellular debris and apoptotic cells to remove.[4][12][13]RamifiedThis form of microglial cell is commonly found at specific locations throughout the entire brain and spinal cord in the absence of foreign material or dying cells. This "resting" form of microglia is composed of long branching processes and a small cellular body. Unlike the ameboid forms of microglia, the cell body of the ramified form remains fairly motionless, while its branches are constantly moving and surveying the surrounding area. The branches are very sensitive to small changes in physiological condition and require very specific culture conditions to observe in vitro. Unlike activated or ameboid microglia, ramified microglia are unable to phagocytose cells and display little or no immunomolecules. This includes the MHC class I/II proteins normally used by macrophages and dendritic cells to present antigens to t-cells, and as a result ramified microglia function extremely poorly as antigen presenters. The purpose of this state is to maintain a constant level of available microglia to detect and fight infection, while maintaining an immunologically silent environment.[7][13]ActivatedActivated microglia can be stained via the marker Iba1, which is upregulated during activation.Non-phagocyticThis state is actually part of a graded response as microglia move from their ramified form to their fully active phagocytic form. Microglia can be activated by a variety of factors including: glutamate receptor agonists, pro-inflammatory cytokines, cell necrosis factors, lipopolysaccharide, and changes in extracellular potassium (indicative of ruptured cells). Once activated the cells undergo several key morphological changes including the thickening and retraction of branches, uptake of MHC class I/II proteins, expression of immunomolecules, secretion of cytotoxic factors, secretion of recruitment molecules, and secretion of pro-inflammatory signaling molecules (resulting in a pro-inflammation signal cascade). Activated non-phagocytic microglia generally appear as "bushy," "rods," or small ameboids depending on how far along the ramified to full phagocytic transformation continuum they are. In addition, the microglia also undergo rapid proliferation in order to increase their numbers. From a strictly morphological perspective, the variation in microglial form along the continuum is associated with changing morphological complexity and can be quantitated using the methods of fractal analysis, which have proven sensitive to even subtle, visually undetectable changes associated with different morphologies in different pathologicalstates.[4][7][13][14]PhagocyticActivated phagocytic microglia are the maximally immune responsive form of microglia. These cells generally take on a large, ameboid shape, although some variance has been observed. In addition to having the antigen presenting, cytotoxic and inflammatory mediating signaling of activated non-phagocytic microglia, they are also able to phagocytose foreign materials and display the resulting immunomolecules for T-cell activation. Phagocytic microglia travel to the site of the injury, engulf the offending material, and secrete pro-inflammatory factors to promote more cells to proliferate and do the same. Activated phagocytic microglia also interact with astrocytes and neural cells to fight off the infection as quickly as possible with minimal damage to the healthy brain cells.[4][7]Gitter cellsGitter cells are the eventual result of microglial cell's phagocytosis of infectious material or cellular debris. Eventually, after engulfing a certain amount of material, the phagocytic microglia becomes unable to phagocytose any further materials. The resulting cellular mass is known as a granular corpuscle, named for its ‘grainy' appearance. By looking at tissues stained to reveal gitter cells, pathologists can see post-infection areas that have healed.[15]PerivascularUnlike the other types of microglia mentioned above, "perivascular" microglia refers to the location of the cell rather than its form/function. Perivascular microglia are mainly found encased within the walls of the basal lamina. They perform normal microglial functions, but unlike normal microglia they are replaced by bone marrow derived precursor cells on a regular basis and express MHC class II antigens regardless of the outside environment. Perivascular microglia also react strongly to macrophage differentiation antigens.[4] These microglia have been shown to beActivation of microglia via purinergic signalling essential to repair of vascular walls, as shown by Ritter's experiments andobservations on ischemic retinopathy. Perivascular microglia promote endothelial cell proliferation allowing new vessels to be formed and damaged vessels to be repaired. During repair and development, myeloid recruitment and differentiation into microglial cells is highly accelerated to accomplish these tasks.[6]JuxtavascularLike perivascular microglia, juxtavascular microglia can be distinguished mainly by their location. Juxtavascular microglia are found making direct contact with the basal lamina wall of blood vessels but are not found within the walls. Likeperivascular cells, they express MHC class II proteins even at low levels ofinflammatory cytokine activity. Unlike perivascular cells, but similar to resident microglia, juxtavascular microglia do not exhibit rapid turnover or replacement with myeloid precursor cells on a regular basis.[4]FunctionsMicroglial cells fulfill a variety of differenttasks within the CNS mainly related to both immuneresponse and maintaining homeostasis. The followingare some of the major known functions carried out bythese cells.ScavengingIn addition to being very sensitive to small changesin their environment, each microglial cell also physically surveys its domain on a regular basis.This action is carried out in the ameboid andresting states. While moving through its set region,if the microglial cell finds any foreign material, damaged cells, apoptotic cells,neurofibrillary tangles, DNA fragments, or plaques it will activate and phagocytose the material or cell. In this manner microglial cells also act as "housekeepers"cleaning up random cellular debris.[7] During developmental wiring of the brain,microglial cells play a large role removing unwanted excess cellular matter. Post development, the majority of dead or apoptotic cells are found in the cerebral cortex and the subcortical white matter. This may explain why the majority ofameboid microglial cells are found within the "fountains of microglia" in thecerebral cortex.[12]PhagocytosisThe main role of microglia, phagocytosis, involves the engulfing of variousmaterials. Engulfed materials generally consist of cellular debris, lipids, and apoptotic cells in the non-inflamed state, and invading virus, bacteria, or otherforeign materials in the inflamed state. Once the microglial cell is "full" it stops phagocytic activity and changes into a relatively non-reactive gitter cell.CytotoxicityIn addition to being able to destroy infectious organisms through cell to cell contact via phagocytosis, microglia can also release a variety of cytotoxic substances. Microglia in culture secrete large amounts of H2O2 and NO in a process known as ‘respiratory burst'. Both of these chemicals can directly damage cells and lead to neuronal cell death. Proteases secreted by microglia catabolise specific proteins causing direct cellular damage, while cytokines like IL-1 promote demyelination of neuronal axons. Finally, microglia can injure neurons through NMDA receptor-mediated processes by secreting glutamate and aspartate. Cytotoxic secretion is aimed at destroying infected neurons, virus, and bacteria, but can also cause large amounts of collateral neural damage. As a result, chronic inflammatory response can result in large scale neural damage as the microglia ravage the brainin an attempt to destroy the invading infection.[4]Antigen presentationAs mentioned above, resident non-activated microglia act as poor antigen presenting cells due to their lack of MHC class I/II proteins. Upon activation they rapidly uptake MHC class I/II proteins and quickly become efficient antigen presenters. In some cases, microglia can also be activated by IFN-γ to present antigens, but do not function as effectively as if they had undergone uptake of MHC class I/II proteins. During inflammation, T-cells cross the blood–brain barrier thanks to specialized surface markers and then directly bind to microglia in order to receive antigens. Once they have been presented with antigens, T-cells go on to fulfill a variety of roles including pro-inflammatory recruitment, formation of immunomemories, secretion of cytotoxic materials, and direct attacks on the plasma membranes of foreign cells.[4][7]Synaptic strippingIn a phenomenon first noticed in spinal lesions by Blinzinger and Kreutzberg in 1968, post-inflammation microglia remove the branches from nerves near damaged tissue. This helps promote regrowth and remapping of damaged neural circuitry.[4]Promotion of repairPost-inflammation, microglia undergo several steps to promote regrowth of neural tissue. These include synaptic stripping, secretion of anti-inflammatory cytokines, recruitment of neurons and astrocytes to the damaged area, and formation of gitter cells. Without microglial cells regrowth and remapping would be considerably slower in the resident areas of the CNS and almost impossible in many of the vascular systems surrounding the brain and eyes.[4][6]Extracellular signalingA large part of microglial cell's role in the brain is maintaining homeostasis in non-infected regions and promoting inflammation in infected or damaged tissue. Microglia accomplish this through an extremely complicated series of extracellularsignaling molecules which allow them to communicate with other microglia, astrocytes, nerves, T-cells, and myeloid progenitor cells. As mentioned above the cytokine IFN-γ can be used to activate microglial cells. In addition, after becoming activated with IFN-γ, microglia also release more IFN-γ into the extracellular space. This activates more microglia and starts a cytokine induced activation cascade rapidly activating all nearby microglia. Microglia-produced TNF-α causes neural tissue to undergo apoptosis and increases inflammation. IL-8 promotes B-cell growth and differentiation, allowing it to assist microglia in fighting infection. Another cytokine, IL-1, inhibits the cytokines IL-10 and TGF-β, which downregulate antigen presentation and pro-inflammatory signaling. Additional dendritic cells and T-cells are recruited to the site of injury through the microglial production of the chemotactic molecules like MDC, IL-8, and MIP-3β. Finally, PGE2 and other prostanoids prevent chronic inflammation by inhibiting microglial pro-inflammatory response and downregulating Th1 (T-helper cell) response.[7]Role in chronic neuroinflammationThe word neuroinflammation has come to stand for chronic, central nervous system (CNS) specific, inflammation-like glial responses that may produce neurodegenerative symptoms such as plaque formation, dystrophic neurite growth, and excessive tau phosphorylation.[16] It is important to distinguish between acute and chronic neuroinflammation. Acute neuroinflammation is generally caused by some neuronal injury after which microglia migrate to the injured site engulfing dead cells and debris.[16] The term neuroinflammation generally refers to more chronic, sustained injury when the responses of microglial cells contribute to and expand the neurodestructive effects, worsening the disease process.[16]When microglia are activated they take on an amoeboid shape and they alter their gene expression. Altered gene expression leads to the production of numerous potentially neurotoxic mediators. These mediators are important in the normal functions of microglia and their production is usually decreased once their task is complete.[17] In chronic neuroinflammation, microglia remain activated for an extended period during which the production of mediators is sustained longer than usual.[17] This increase in mediators contributes to neuronal death.[17]Neuroinflammation is distinct from inflammation in other organs, but does include some similar mechanisms such as the localized production of chemoattractant molecules to the site of inflammation.[17] The following list contains a few of the numerous substances that are secreted when microglia are activated:CytokinesMicroglia activate the proinflammatory cytokines IL-1α, IL-1β and TNF-α in the CNS.[17] Cytokines play a potential role in neurodegeneration when microglia remain in a sustained activated state.[17] Direct injection of the cytokines IL-1α, IL-1βand TNF-α into the CNS result in local inflammatory responses and neuronal degradation.[17] This is in contrast with the potential neurotrophic (inducing growth of neurons) actions of these cytokines during acute neuroinflammation.[17]ChemokinesChemokines are cytokines that stimulate directional migration of inflammatory cells in vitro and in vivo.[17] Chemokines are divided into four main subfamilies: C, CC, CXC, and CX3C. Microglial cells are sources of some chemokines and express themonocyte chemoattractant protein-1 (MCP-1) chemokine in particular.[17] Other inflammatory cytokines like IL-1β and TNF-α, as well as bacterial-derived lipopolysaccharide (LPS) may stimulate microglia to produce MCP-1, MIP-1α, and MIP-1β.[17] Microglia can express CCR3, CCR5, CXCR4, and CX3CR1 in vitro.[17] Chemokines are proinflammatory and therefore contribute to the neuroinflammation process.[17]ProteasesWhen microglia are activated they induce the synthesis and secretion of proteolytic enzymes that are potentially involved in many functions.[17] There are a number of proteases that possess the potential to degrade both the extracellular matrix and neuronal cells that are in the neighborhood of the microglia releasing these compounds.[17] These proteases include; cathepsins B, L, and S, the matrix metalloproteinases MMP-1, MMP-2, MMP-3, and MMP-9, and the metalloprotease-disintegrin ADAM8 (plasminogen) which forms outside microglia and degrades the extracellular matrix.[17] Both Cathepsin B, MMP-1 and MMP-3 have been found to be increased in Alzheimer's disease (AD) and cathepsin B is increased in multiple sclerosis (MS).[17] Elastase, another protease, could have large negative effects on the extracellular matrix.[17]Amyloid precursor proteinMicroglia synthesize amyloid precursor protein (APP) in response to excitotoxic injury.[17] Plaques result from abnormal proteolytic cleavage of membrane bound APP.[17] Amyloid plaques can stimulate microglia to produce neurotoxic compounds such as cytokines, excitotoxin, nitric oxide and lipophylic amines, which all cause neural damage.[18] Plaques in Alzheimer's disease contain activated microglia.[17] A study has shown that direct injection of amyloid into brain tissue activates microglia, which reduces the number of neurons.[18] Microglia have also been suggested as a possible source of secreted β amyloid.[17]AgingMicroglia undergo a burst of mitotic activity during injury; this proliferation is followed by apoptosis to reduce the cell numbers back to baseline.[16] Activation of microglia places a load on the anabolic and catabolic machinery of the cells causingactivated microglia to die sooner than non-activated cells.[16] To compensate for microglial loss over time, microglia undergo mitosis and bone marrow derived progenitor cells migrate into the brain via the meninges and vasculature.[16]Accumulation of minor neuronal damage that occurs during normal aging can transform microglia into enlarged and activated cells.[19] These chronic, age-associated increases in microglial activation and IL-1 expression may contribute to increased risk of Alzheimer's disease with advancing age through favoring neuritic plaque formation in susceptible patients.[19] DNA damage might contribute to age-associated microglial activation. Another factor might be the accumulation of advancedglycation endproducts, which accumulate with aging.[19] These proteins are strongly resistant to proteolytic processes and promote protein cross-linking.[19]Research has discovered dystrophic (defective development) human microglia. "These cells are characterized by abnormalities in their cytoplasmic structure, such as deramified, atrophic, fragmented or unusually tortuous processes, frequently bearing spheroidal or bulbous swellings."[16] The incidence of dystrophic microglia increases with aging.[16] Microglial degeneration and death have been reported in research on Prion disease, Schizophrenia and Alzheimer's disease, indicating that microglial deterioration might be involved in neurodegenerative diseases.[16] A complication of this theory is the fact that it is difficult to distinguish between "activated" and "dystrophic" microglia in the human brain.[16]Role of microglia in neurodegenerationNeurodegenerative disorders are characterized by progressive cell loss in specific neuronal populations.[17] "Many of the normal trophic functions of glia may be lost or overwhelmed when the cells become chronically activated in progressive neurodegenerative disorders, for there is abundant evidence that in such disorders, activated glia play destructive roles by direct and indirect inflammatory attack."[17] The following are prominent examples of microglial cells' role in neurodegenerative disorders.Alzheimer's diseaseAlzheimer's disease (AD) is a progressive, neurodegenerative disease where the brain develops abnormal clumps (amyloid plaques) and tangled fiber bundles(neurofibrillary tangles).[20]There are many activated microglia over-expressing IL-1 in the brains of Alzheimer patients that are distributed with both Aβ plaques and neurofibrillary tangles.[19] This over expression of IL-1 leads to excessive tau phosphorylation that is related to tangle development in Alzheimer's disease.[19]Many activated microglia are found to be associated with amyloid deposits in the brains of Alzheimer's patients.[17] Microglia interact with β-amyloid plaques through cell surface receptors that are linked to tyrosine kinase based signalingcascades that induce inflammation.[17] When microglia interact with the deposited fibrillar forms of β-amyloid it leads to the conversion of the microglia into an activated cell and results in the synthesis and secretion of cytokines and other proteins that are neurotoxic.[17]One preliminary model as to how this would occur involves a positive feedback loop. When activated, microglia will secrete proteases, cytokines, and reactive oxygen species. The cytokines may induce neighboring cells to synthesize amyloid precursor protein. The proteases then possibly could cause the cleaving required to turn precursor molecules into the beta amyloid that characterizes the disease. Then, the oxygen species encourage the aggregation of beta amyloid in order to form plaques. The growing size of these plaques then in turn triggers the action of even more microglia, which then secrete more cytokines, proteases, and oxygen species, thus amplifying the neurodegeneration.[21]TreatmentNon-steroidal anti-inflammatory drugs (NSAIDs) have proven to be effective in reducing the risk of AD.[17] "Sustained treatment with NSAIDs lowers the risk of AD by 55%, delays disease onset, attenuates symptomatic severity and slows the loss of cognitive abilities. The main cellular target for NSAIDs is thought to be microglia. This is supported by the fact that in patients taking NSAIDs the number of activated microglia is decreased by 65%."[17]Parkinson's diseaseParkinson's disease is a movement disorder in which the dopamine-producing neuronsin the brain do not function as they should, the neurons of the Substantia Nigra become dysfunctional and eventually die, leaving a lack of dopamine input into the striatum. This causes the symptoms of Parkinson's disease.[22]Cardiovascular DiseasesRecently microglial activation has been reported in rats with myocardial infarction (Rana et al.,2010). This activation was specific to brain nuclei involved in cardiovascular regulation suggesting possible role of microglial activation in pathogenesis of heart failure.Role in viral infectionsHuman immunodeficiency virusThe infection of mononuclear phagocytes with HIV-1 is an important element in the development of HIV-associated dementia complex (HAD).[23] The only brain cell type that is "productively" infected with the virus are microglial cells.[23] It has also become clear that neurotoxic mediators released from brain microglia play an important role in the pathogenesis of HIV-1.[23]。

介绍芯片英语演讲稿范文

Ladies and gentlemen,Good morning/afternoon/evening. It is my great pleasure to stand before you today to discuss a topic that is as essential as it is fascinating: the microchip. In this speech, I will take you on a journey through the history, the impact, and the future of microchips, the tiny devices that have revolutionized our world.Introduction:Microchips, often referred to as integrated circuits, are marvels of modern technology. These compact, yet powerful devices have transformed the way we live, work, and communicate. From the smallest electronic devices to the most complex systems, microchips are the unsung heroes that make it all possible. Today, I invite you to join me as we delve into the world of microchips.I. The Birth of MicrochipsA. The ConceptualizationThe idea of an integrated circuit was first conceptualized in the 1950s by Jack Kilby, an engineer at Texas Instruments. Kilby's vision was to create a single semiconductor device that could perform multiple functions, thus reducing the size and complexity of electronic systems.B. The InventionIn 1958, Kilby successfully demonstrated the first working integrated circuit. This breakthrough marked the birth of microchips and laid the foundation for the digital age.II. The Evolution of MicrochipsA. The Early YearsIn the 1960s and 1970s, microchips began to gain traction in the consumer market. The development of microprocessors, which are essentially central processing units (CPUs) on a single chip, paved the way for the creation of personal computers.B. The Rise of Personal ComputersThe 1980s saw the rapid growth of personal computers, thanks to the increasing power and decreasing size of microchips. Companies like Intel and AMD played a crucial role in the development of microprocessors that would power these devices.C. The MiniaturizationThe 1990s and 2000s brought about the miniaturization of microchips. The introduction of nanotechnology allowed for the creation of smaller, more efficient, and more powerful chips. This led to advancements in mobile devices, such as smartphones and tablets.III. The Impact of MicrochipsA. Revolutionizing IndustriesMicrochips have had a profound impact on various industries, including telecommunications, healthcare, transportation, and entertainment. They have enabled the development of new technologies and the enhancement of existing ones.B. Improving Quality of LifeMicrochips have made our lives more convenient, efficient, and enjoyable. From the automation of everyday tasks to the ability to connect with people across the globe, microchips have become an integral part of our daily lives.C. Economic GrowthThe microchip industry has contributed significantly to economic growth. It has created millions of jobs, fostered innovation, and driven technological advancements that have far-reaching effects on the global economy.IV. The Future of MicrochipsA. Emerging TechnologiesAs we move forward, the future of microchips looks promising. Emerging technologies, such as quantum computing and artificial intelligence,will rely heavily on the development of advanced microchips.B. Environmental ConcernsWith the increasing demand for microchips, environmental concerns are also on the rise. Efforts are being made to develop more sustainable and energy-efficient chip manufacturing processes.C. Ethical ConsiderationsAs microchips become more powerful and ubiquitous, ethical considerations come into play. Questions about privacy, security, and the potential misuse of microchips need to be addressed.Conclusion:Ladies and gentlemen, microchips have come a long way since their inception. From a simple idea to a global phenomenon, these tiny devices have shaped the modern world. As we continue to innovate and push the boundaries of technology, microchips will undoubtedly play a crucialrole in our future. Let us embrace the power of microchips and use them to create a better, more connected, and more sustainable world.Thank you for your attention.。

《小型演示最重要的是》英语作文

《小型演示最重要的是》英语作文全文共3篇示例，供读者参考篇1The Most Important Aspects of a Small-Scale PresentationWhen it comes to giving a small-scale presentation, whether it be in a business setting or an academic one, there are several key aspects that are crucial to its success. In this article, we will discuss why each of these aspects is important and offer some tips on how to ensure that your presentation is effective and engaging.First and foremost, one of the most important aspects of a small-scale presentation is preparation. Before you even begin to create your slides or write your script, you need to have a clear understanding of what your presentation is about and who your audience is. This will help you to tailor your content and delivery to their needs and interests, ensuring that they remain engaged throughout.Secondly, the way you present your information is also crucial. You need to be confident, authoritative, and engaging in your delivery, using a clear and concise style that is easy for youraudience to follow. Remember to speak clearly and project your voice, maintaining eye contact with your audience to keep them engaged and interested in what you have to say.In addition to this, the visual aspects of your presentation are also important. While the content of your slides is of course important, the design and layout of them can also have a big impact on how your audience perceives your message. Make sure that your slides are visually appealing, with a clean and professional design that complements your content rather than distracts from it.Another important aspect of a small-scale presentation is timing. You need to make sure that you have enough time to cover all of your key points without rushing through them, but also that you are not taking up too much time and losing your audience's interest. It can be helpful to practice your presentation several times beforehand to ensure that you can deliver it within the allotted time frame.Finally, it is important to remember that a small-scale presentation is not just about conveying information, but also about engaging and connecting with your audience. Try to make your presentation interactive, involving your audience in the discussion and asking for their feedback and input. This will helpto keep them engaged and interested in what you have to say, making your presentation more memorable and impactful.In conclusion, there are several key aspects that are important when giving a small-scale presentation. By preparing carefully, delivering your content confidently and engagingly, paying attention to the visual aspects of your presentation, managing your time effectively, and engaging with your audience, you can ensure that your presentation is successful and leaves a lasting impression.篇2The Most Important Aspect of a Small-Scale PresentationWhen it comes to delivering a small-scale presentation, it is crucial to focus on the most important aspects to ensure that your message is understood and well-received by your audience. In this article, we will discuss the key elements that make a small-scale presentation successful and effective.First and foremost, the most important aspect of asmall-scale presentation is clarity. It is essential to clearly convey your message and ideas to your audience in a concise and straightforward manner. Make sure to eliminate any unnecessary information or jargon that may confuse or overwhelm yourlisteners. Use simple language and visuals to enhance understanding and engagement.Secondly, organization is key to a successful presentation. Make sure to structure your content in a logical and coherent manner, with a clear introduction, main points, and conclusion. Create an outline or storyboard to help you stay on track and ensure that your presentation flows smoothly from start to finish.Another crucial aspect of a small-scale presentation is engagement. It is important to actively engage your audience and capture their interest from the beginning. Use interactive elements such as polls, quizzes, or Q&A sessions to encourage participation and create a two-way dialogue with your listeners. Remember to maintain eye contact, vary your tone and pace, and use gestures to keep your audience engaged throughout the presentation.Furthermore, visual aids play a significant role in enhancing the effectiveness of a small-scale presentation. Utilize slides, videos, images, or props to support your key points and make your presentation more visually appealing and memorable. Keep your visuals simple, relevant, and uncluttered to avoid distractions and maintain focus on your message.In addition, practice and preparation are essential for a successful small-scale presentation. Rehearse your presentation multiple times to familiarize yourself with the content, timing, and delivery. Anticipate potential questions or challenges that may arise during the presentation and prepare responses in advance. Confidence and poise are key to establishing credibility and trust with your audience.Lastly, feedback and reflection are crucial for continuous improvement and growth. Seek feedback from your audience or peers to identify areas for improvement and learn from your experiences. Reflect on your strengths and weaknesses as a presenter and strive to enhance your communication skills and presentation techniques.In conclusion, the most important aspect of a small-scale presentation is clarity, organization, engagement, visual aids, practice, and feedback. By focusing on these key elements, you can deliver a successful and effective presentation that resonates with your audience and leaves a lasting impact. Remember to tailor your presentation to the needs and preferences of your audience and remain authentic and relatable as a presenter. With dedication, passion, and attention to detail, you can master theart of small-scale presentations and achieve your communication goals effectively.篇3The Most Important Aspect of a Small DemoWhen it comes to showcasing a product or service, small demos play a vital role in attracting potential customers and clients. Whether it's a startup looking to impress investors or a company launching a new product, a small demo can make all the difference. In this article, we will discuss the most important aspects of a small demo and how to make it stand out.First and foremost, the most important aspect of a small demo is clarity. It is crucial to convey the key features and benefits of your product or service in a concise and understandable manner. Your audience should be able to grasp the value proposition within the first few minutes of the demo. Avoid jargon and technical language that may confuse or alienate viewers. Keep it simple and easy to digest.Secondly, a small demo should be visually appealing. The design and interface of your demo should be clean, modern, and intuitive. Use graphics, animations, and videos to enhance the presentation and make it more engaging. Visual elements canhelp convey complex ideas and concepts in a simple and effective way. Remember, a picture is worth a thousand words.Another important aspect of a small demo is interactivity. Allow your audience to interact with the demo and explore its features on their own. This not only keeps viewers engaged but also gives them a sense of ownership and control. Incorporate interactive elements such as clickable buttons, sliders, anddrag-and-drop features to make the demo more dynamic and immersive.Furthermore, a small demo should be tailored to the needs and preferences of your target audience. Understand who your viewers are and what they are looking for in a product or service. Customize the demo to address their pain points, solve their problems, and meet their expectations. Personalization is key to creating a memorable and impactful demo that resonates with your audience.Last but not least, the most important aspect of a small demo is storytelling. Use storytelling techniques to create a narrative that captivates and inspires viewers. Take them on a journey that highlights the journey of your product or service, from conception to execution. Share success stories, testimonials, and case studies that demonstrate the tangible benefits andresults of your offering. Engage your audience emotionally and make them feel connected to your brand.In conclusion, the most important aspect of a small demo is a combination of clarity, visual appeal, interactivity, personalization, and storytelling. By focusing on these key elements, you can create a compelling and persuasive demo that leaves a lasting impression on your audience. Remember, the goal of a small demo is not just to showcase your product or service, but to inspire and engage viewers in a meaningful and memorable way.。

天文科普演讲英文作文

天文科普演讲英文作文Title: Unveiling the Wonders of the Cosmos: An Astronomical Science Popularization Speech。

Ladies and gentlemen,。

Welcome to an exploration of the cosmos, where we embark on a journey through the vast expanse of the universe, unraveling its mysteries and marvels. Today, I have the privilege of sharing with you the wonders of astronomy, a science that not only unveils the beauty ofthe night sky but also ignites our curiosity about the cosmos.Let us begin our journey by contemplating the stars, those distant suns that adorn the night sky. Each star is a cosmic beacon, shining billions of miles away, yet theirlight travels through space and time to reach us here on Earth. Through telescopes, we peer into the depths of space, witnessing the birth and death of stars, the formation ofgalaxies, and the dance of celestial bodies across the heavens.One of the most fascinating phenomena in astronomy is the life cycle of stars. From the fiery infernos of their birth in nebulae to the serene glow of their old age as white dwarfs or the cataclysmic explosion of a supernova, stars captivate us with their splendor and diversity. They are the engines of the universe, synthesizing elements like carbon, oxygen, and iron, which form the building blocks of planets, moons, and even life itself.But stars are not the only inhabitants of the cosmos; there are also galaxies, vast island universes containing billions or even trillions of stars, along withinterstellar gas, dust, and dark matter. Our own Milky Way is but one of billions of galaxies in the observable universe, each with its own unique structure and history. From the majestic spirals to the enigmatic ellipticals, galaxies come in a variety of shapes and sizes, offering a glimpse into the cosmic tapestry of our universe.As we venture further into the cosmos, we encounterother celestial phenomena that challenge our understandingof the universe. Black holes, for example, are regions of space where gravity is so intense that nothing, not even light, can escape their grasp. These cosmic behemoths lurkin the depths of space, distorting spacetime itself and devouring anything that ventures too close.On the opposite end of the cosmic scale, we find the vast expanses of empty space punctuated by filaments, voids, and superclusters of galaxies. These cosmic structures form the cosmic web, the scaffolding upon which the universe is built. By studying the distribution of galaxies and thelarge-scale structure of the universe, astronomers can unravel the history of cosmic expansion and uncover the secrets of dark energy and dark matter, the invisibleforces that shape the cosmos.Yet, for all the wonders we have discovered, there is still much we do not know about the universe. The quest to unravel its mysteries continues, driving astronomers topush the boundaries of human knowledge and explore thecosmos with ever-greater precision and insight. From the humble beginnings of ancient stargazers to the cutting-edge observatories of today, astronomy has been a journey of discovery, wonder, and awe.In conclusion, astronomy is more than just a science;it is a window into the soul of the universe, a testamentto the boundless beauty and complexity of the cosmos. By studying the stars, galaxies, and other celestial phenomena, we not only expand our understanding of the universe but also deepen our appreciation for the wonders that surround us. So let us continue to gaze at the stars, to ponder the mysteries of the cosmos, and to marvel at the beauty of the universe in all its glory.Thank you.。

智能隐形眼镜想像作文

智能隐形眼镜想像作文英文回答：In the realm of wearable technology, smart contact lenses have emerged as a transformative concept, reshaping the boundaries of vision correction and human augmentation. These cutting-edge devices seamlessly integrate advanced sensors and microelectronics into the tiny form factor of a contact lens, granting wearers an array of unprecedented capabilities.One of the most compelling applications of smart contact lenses lies in the seamless integration of augmented reality (AR). By projecting digital information onto the wearer's retina, these devices can provide real-time data, enhance navigational capabilities, and offer immersive experiences. Imagine navigating unfamiliarstreets with turn-by-turn directions projected directlyinto your line of sight, or accessing vital healthstatistics with a simple glance.Beyond AR, smart contact lenses also hold immense potential for enhancing human vision. By incorporating features such as adjustable focus, color correction, and night vision, these devices can address a wide range of visual impairments, offering wearers the freedom to fully experience the world around them.Furthermore, smart contact lenses can serve as a non-invasive gateway for monitoring key health parameters. By continuously measuring metrics such as glucose levels, intraocular pressure, and tear film dynamics, these devices can provide valuable insights into the wearer's overall health and well-being.The development of smart contact lenses faces several technological hurdles that need to be overcome. Miniaturization of electronics, efficient energy management, and ensuring biocompatibility are among the key challenges that researchers are actively addressing. However, as technological advancements continue to break new ground,the vision of smart contact lenses as a ubiquitous realitydraws closer.中文回答：想象一下，佩戴上智能隐形眼镜，它将开辟一个全新的世界，彻底改变我们的视觉体验。

Microglia cell culture,a primer for the novice

49Lucio G. Costa et al. (eds.), In Vitro Neurotoxicology: Methods and Protocols , Methods in Molecular Biology, vol. 758,DOI 10.1007/978-1-61779-170-3_4, © Springer Science+Business Media, LLC 2011Chapter 4Microglia Cell Culture: A Primer for the NoviceAnke Witting and Thomas MöllerAbstractMicroglial cells are the resident immune cells of the central nervous system. Progress in the recent decade has clearly established that microglial cells participate or even actively drive neurological disease. Much of our current knowledge has been generated by investigating microglial cells in cell culture. The aim of this chapter is to give the uninitiated a basic and adaptable protocol for the culturing of microglial cells. We discuss the challenges of microglial cell culture and provide a collection of tips which reflect our 25+ years of collective experience.Key words: Microglia, Cell culture, Medium, Serum, Growth factor, M-CSF, GM-CSF, Transfection, Endotoxin, Cell yieldThis chapter only provides a brief introduction into microglial cells as a point of reference. For more detailed information on the topic, the reader is respectfully referred to a number of excellent and comprehensive reviews on microglial biology (1–7). Microglia are the resident immune cells of the CNS. They resemble periph-eral tissue macrophages and are the primary mediators of neuroin-flammation (1, 8). Studies in the last two decades have demonstrated the involvement of microglia in many acute and chronic neuro-logical diseases (2, 9). In the healthy adult brain, microglia exist as so-called “resting” or “surveilling” microglia, characterized by a small cell body with fine, ramified processes and minimal expres-sion of surface antigens. Upon CNS injury, these cells are rapidly activated and participate in the pathogenesis of n eurological disor-ders. They secrete various inflammatory molecules, including TNF-a , IL-6, and nitric oxide (8). When CNS cells die, microglia 1. Introduction1.1. Microglia50 A. Witting and T. Möllerare further activated and become phagocytes. It is widely believed that substances released from damaged cells within the brain trig-ger microglial activation, consequently leading to the long-term changes of gene expression and reorganization of the cell pheno-type (2, 8).Activated microglia exert their effects on neurons and macroglia (astrocytes and oligodendrocytes) through the release of cytotoxic substances such as oxygen radicals, nitric oxide, glutamate, proteases, and neurotoxic cytokines, as well as cytoprotective agents such as growth factors, plasminogen, and neuroprotective cytokine (8). The effects of microglia are themselves modulated by astrocytes and neurons through cytokines and neurotransmitters, thus giving rise to complex interactions between microglia, neurons, and astrocytes. Evidence suggests that microglial cells play a central role in HIV encephalopathy and multiple sclerosis (10, 11). In addi-tion to infectious or inflammatory diseases, there is accumulat-ing evidence that microglia play a significant role in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lat-eral sclerosis (ALS) (12–15).EARLY evidence that microglial cells play a role in neurological diseases emerged from histological studies. With the advent of cell culture and its successful application to neurons and astro-cytes, it was only a question of time until ingenious “glioscien-tists” developed protocols to isolate microglia. A seminal paper by Giulian and Baker in the mid-1980s describing the culture of microglial cells from neonatal mice jump started the field and with roughly 800 citations it might be the most cited primary research paper on microglial cells (16). Much has been learnedfrom these cells in a culture dish; from their complement of receptors and ion channels, to their ability to proliferate and migrate, as well as their annotated genomic profile. However, one lingeringconcern has always been this: what happens to immune cells, which are supposed to monitor CNS integrity after you put a brain through a wire mash and bath the cells in 10% serum? The answer seems blatantly clear, the cells get acti-vated. This has been ignored at the peril of the field for some-time, until it became clear that microglial cells in vitro not always (and this might be an understatement) resemble microglial cells in vivo. This controversial topic has been covered in recent years by several reviews and primary papers and is far outside the scope of this chapter (1, 17, 18). Advances in imaging technology and smart genetic approaches however have considerably improved our understanding of microglia in vitro (19–22) and the field isnow embarking on reconciling the in vitro and in vitro findings. 1.2. Microglia in CellCulture: A Catch 22514 Microglia Cell Culture: A Primer for the Novice It is clear that mechanistic s tudies will necessitate the in vitro approach for the time being. However, no self-respecting micro-gliologist will attempt to explain a complex neurological disease from data solely derived from a Petri dish.While there might be as many specific microglial cell culture p rotocols as there are laboratories working with microglia, most protocols in use are based on the above-mentioned publication of Giulian and Baker (16). The overriding principle is to first gener-ate mixed CNS cultures from late embryonic to postnatal day 5 brains and then repeatedly isolate microglial cells from these mixed cultures. However, other approaches exist, for example the isolation and (long-term) culture of adult microglia (23); the generation of larger amounts of microglia from the subventricular zone (24); or the derivation of microglia precursors from embry-onic stem cells (25). Microglial cells have not only been isolated from mouse and rat, but also from fetal human tissue (26); adult human surgically resected tissue (27); human postmortem tissue (28), human retinal tissue (29); and porcine CNS (30) and even gold fish (31).In this chapter, we provide a basic protocol for the initial mixed CNS cultures similar to what can be found in many CNS cell culture books (32). The described protocol for microglia iso-lation has several advantages. It will generate reasonable amounts of microglia; microglia can be kept for an extended time in cul-ture to do stimulation experiments and lastly, in all its variations, it is time proven. While the overall properties of microglia gener-ated this way seem to be the same, it is difficult to judge whether reported differences are species-dependent or cell culture protocol-dependent.For many procedures, written instructions are similar to instruction on how to tie shoe laces. In such a case, a picture is worth a thousand words, and a video of the procedures is invalu-able. The reader is referred to the Journal of Visualized Experiments at , which has several instruc-tional videos on the general procedures on how to obtain CNS cell cultures. The added value in our protocol are the notes, which highlight some of the common challenges and are usually not spelled out in detail. We are almost certain that most of our esteemed colleagues will have a divergent opinion on one issue or the other. However, we would like to point out that for the most part there is no single right or single wrong in how to pre-pare microglial cultures. There are actually many right and unfortunately many wrong ways. We intend to provide one pos-sible way which has successfully worked for us and warn of many wrong turns one can take along the arduous road to microglial cultures.1.3. Microglia CellCulture: A GenericProtocol52 A. Witting and T. Möller1. Dissecting microscope.2. Sterile dissection tools (scalpels, scissors, forceps, etc.).3. Hanks’ Balanced Salt Solutions (HBSS) (see Note 1).4. 0.25% Trypsin in HBSS (cell culture grade, not trypsin/EDTA).5. Petri dishes, 15-ml tubes, 50-ml tubes, cell strainer (100 m m; BD Falcon).6. DNase I (not the expensive molecular grade). A stock solution at 10 mg/ml in HBSS should be aliquoted and frozen.7. Dulbecco’s Modified Eagle’s Medium (DMEM, high glu-cose, with glutamine) supplemented with 100 U/ml of peni-cillin and 100 m g/ml streptomycin (see Note 2).8. DMEM/10% FBS: DMEM supplemented with 10% heat inactivated fetal bovine serum (FBS) and 100 U/ml of peni-cillin and 100 m g/ml streptomycin (P/S) (see Note 3). Of note, one should avoid HEPES containing medium. HEPES is used to increase the pH buffer capacity of the medium once removed from the CO 2 atmosphere of the incubator. However, it as a negative influence on microglia yield in primary mixed CNS culture (see Note 1).9. Poly-l -ornithine-coated flasks for primary mixed cultures: flasks are coated with 1 mg/ml poly-l -ornithine dissolved in tissue-culture water for at least 30 min at 37°C or over night at 4°C. The coating solution should cover the whole surface of the flasks. After the coating, flasks are washed two times with tissue culture grade water and one time with HBSS (see Note 4). For three brains, one 75 cm 2-coated cell culture flask will be needed, or for one brain, one 25 cm 2-coated cell culture flask. For higher throughput cultures, ratios can be scaled to 175-cm 2 culture flasks. The actual number of brains is flexible and can be empirically determined. However, it is imperative that for cultures which will be compared, the same number of brains is used. Otherwise differences in the growth phase will lead to nonlinear differences with might distort results.10. Culture dishes or well plates for microglia cells are either coated with poly-l -lysine or poly-l -ornithine or specialized cell culture ware can be used (e.g., PRIMARIA from BD Falcon).2. Materials534 Microglia Cell Culture: A Primer for the Novice The extraction of the brains from the animals (P1–P5 mouse or rat) and the removal of the meninges should be preferably done in a sterile work bench. The “cleaner” the work at this step, the less likely is a (low-level) contamination in the later steps, which most likely will be cleared by the antibiotics in the medium (see Note 2). However, the presence of microbial products will lead to an undefined activation state of the cultures (see Note 5). Please note that the sterile work bench for animal preparation should be different from the actual cell culture hood. Avoid bringing ani-mals into your cell culture facility. If there is not a sterile work bench for animal preparation, the preparation can be done on a laboratory bench, which gets sanitized beforehand and is prefer-ably located in a low-traffic area.1. Sterilize the surface of the bench and the dissection- m icroscope with 70% ethanol. Lay out the sterile dissection tools on a sterile surface (e.g., sterile Petri dish). To sterilize the prepa-ration tools directly after each usage, a bead sterilizer is opti-mal; however, a beaker filled with 70% ethanol can work as well.2. For the collection of brains, put Petri dishes and 15-ml cen-trifugation tubes filled with 10 ml HBSS on ice to prechill the solution. To keep the Petri dishes cold, while under the dis-ecting microscope a rectangular cold pack as frequently used for shipping has been proven useful. To increase contrast in the dissection microscope, a black sheet might be put between the cold pack and the Petri dish.3. Decapitate animals, spray the head with 70% ethanol, and then place it in a sterile Petri dish. Please check institutional animal care and use rule for decapitation of animals. CO 2 nar-cosis prior to decapitation is not desirable, and usually omis-sion can be justified.4. Remove the skin from the skull and cut the skull on the right and left side from the entry of the spinal cord toward the eyes. For an easy removal of the brain, it is recommended to also cut the skull along the midline of the brain, which is eas-ily visible as a meandering line in the skull. To cut only the skull and avoid cutting into the brain tissue, a scissor with one blunt blade is helpful. Once the incision is done along-side the base of the brain and along the midline the skull bone can be removed by gripping them with forceps and lifting up and outward. An alternative approach is to half the skull along the midline and carefully scoop out the hemisphere. However, this approach is only advisable for very young animals.3. Methods54 A. Witting and T. MöllerExtreme care has to be taken not to squish the brain with adull blade. Nevertheless, if executed with care, this procedurecan be very efficient and safe valuable time.5. Remove the brain as a whole after cutting the olfactory nerves.Place the brain into a Petri dish filled with ice-cold HBSS.Once the brains are on ice (HBSS in a 50-ml Falcon tube),they can be stored for up to 4 h with little effect on glial cellviability. Nevertheless, for consistent results, it is advisable toperform the preparation as fast as possible.6. Remove the olfactory bulb, cerebellum, and midbrain bytweezing them off with forceps. Remove the meninges underthe dissection microscope: first cut the brain along the hemi-spheres, detach the meninges along the inner side of thehemispheres, and carefully pull off the meninges. They mightcome off in one piece; however, they may also rip and onlycome off in little pieces. It is very important to check for leftover meninges and remove them carefully, as they might con-taminate the culture with fibroblasts. This might not seem abig issue if one plans to isolate microglia from the mixed cul-tures. However, one needs to keep in mind that the fibroblastwill release a different set of growth factors and cytokines intothe mixed culture and therefore might lead to a different dif-ferentiation pattern of microglia than in cultures withoutfibroblasts. In general, the meninges are easily removed froma carefully disected brain. A brain bruised while extracted willhave sticky meninges.7. Transfer the meninges-free brain to a 15-ml centrifugationtube filled with 10 ml HBSS and store on ice. Up to six brainsper tube can be pooled together.The next steps are performed in the cell culture hood.8. Wash the brain tissue at least three times with 7 ml of HBSS.This step will remove contaminations such as blood, pieces ofmeninges, etc., and reduce microbial load by dilution. If thepreparation took longer and was performed in an unsterileenvironment, more washes are recommended. To wash,remove the supernatant and add 7 ml of HBSS. Close thecentrifugation tube and reverse it twice. When the brains havesettled down, remove the supernatant, and repeat the nextwashing step as desired. After the last wash, leave 1–2 mlsupernatant to cover the brains.9. Trypsinize the brain tissue by adding 1 ml of 0.25% trypsin inHBSS and 50 m l of the DNase solution. Incubate the brainswith trypsin and DNase for 5–10 min at room temperature.During the incubation agitate, the centrifugation tubes a fewtimes to ensure good trypsinization. Several other enzymepreparations have been used such as collagenase or dispase.554 Microglia Cell Culture: A Primer for the NoviceSome protocols actually perform the dissociation step enzyme-free, and some combine a mincing step with enzymatic diges-tions. There are pros and cons for each approach. However, we have found that the procedure we describe is a good compro-mise between speed and consistent results.10. Stop the trypsinization by adding 10 ml of DMEM/10%FBS. At this point, a jelly-like band might enwrap some of the tissue. This is DNA from broken up cells. This is more fre-quently the case, if a strong mechanical force is used, and can be countered by adding more DNAse. However, generally this should not be necessary, and it is usually a sign that either the trypsinization was too long or the DNase had expired. 11. Wash the tissue twice with HBSS as stated in step 9. After thelast wash, keep 5 ml of supernatant. This amount of volume can be adjusted to match the number of brains used.12. Homogenize the brains by pipetting with either serologicalor fire-polished Pasteur pipettes of decreasing opening dia-meters. Care has to be taken to avoid bubbles, as this will reduce cell viability and yield. A fine line has to been walked between pipetting often enough to break up the tissue and pipetting too often, thus killing cells because of the shear forces in the pipette tip. This is the most critical part of the preparation and is indeed more an art than a science. We rec-ommend limiting the homogenization to only two fire-pol-ished Pasteur pipettes for a maximum of five pipetting steps each time. It is better to err on the side of still having some undissociated tissue, than to homogenize too much and kill liberated cells. Again, if in this step jelly-like bands or clumps are observed, more DNase can be added.13. Filter the cell suspension is through a 100-m m cell strainer.This step will remove larger undigested clumps. After trans-ferring the cell suspension, the cell strainer can be washed with additional DMEM + 10% FBS to increase cell recovery from the cell strainer.14. Centrifuge the cell suspension for 10 min at 200 × g. Discardthe supernatant and resuspend the cell pellet in 10 ml of DMEM + 10% FBS + P/S. Add the cell homogenate to the flasks in an amount that correspond to three whole brains for one 75-cm2 flask.15. Incubate the cell culture flasks at 5% CO2and 37°C. Thecultures will look rather cloudy because of a lot of debris in the supernatant.16. Live cells will attach within hours. One considerable differ-ence among different protocols is what happens next. One school of thought suggests removing debris after overnight incubation, arguing that the myelin in the debris is toxic to56 A. Witting and T. Möllerthe cells and should be removed as soon as all cells have safelyattached. Others argue that leaving debris around will stimu-late microglial proliferation. Both arguments are most likelycorrect. The important point, as actually in everything relatedto microglia culture, is consistency. If you decide to removedebris at, let us say DIV 2, then always remove it on DIV 2.Changing these seemingly unimportant, early parameters canlead to variable result later on.17. After 24 h, wash the attached cells three times with PBS, thenadd 12 ml of DMEM + 10% FBS + P/S. After about 7 days inculture, an astrocyte monolayer with a few microglia on topwill form. Frequency of medium change is also at varianceamong different protocols. We found that keeping the initial12 ml of medium until DIV 7 yields good results. However,other laboratories immediately start with a 3–4 day mediumchange cycle. In general, the longer you maintain the cells inthe same medium (without depletion of nutrients!), the morethe cultures get conditioned by factors secreted from the astro-cytic monolayer. This seems to have beneficial effects on micro-glial cell yield. One frequently employed alternative is the useof growth factors to increase microglia yield (see Note 6).18. Depending on the details of the preparation (number of brainsper flask, medium change frequency) 10–20 days after thepreparation, a larger number of microglia appear on top ofthe astrocytic monolayer. In mouse-derived cultures, micro-glia usually remain attached to the astrocytes, in rat-derivedcultures the microglia tend to float in the supernatant.19. To collect the microglia cells from a primary culture flask, col-lect the loosely attached microglial cells by gently shaking theflasks for 30 min on an orbital shaker at 37°C. Alternatively,the flask can be tapped on the side with the flat hand withmedium intensity for 5–10 times in a way that does not inducefoaming of the medium. A quick inspection with a micro-scope will indicate whether microglial cells are successfullydislodged and can be collected from the supernatant. Similarto the trituration of the CNS, this process is more art thanscience and needs to be perfected by iteration. Too lights haking will cause microglial cells to stay attached, while toovigorous shaking will also dislodge oligodendrocytes. A fur-ther consideration is the need to strike a balance for currentmicroglial yield and leaving enough cells remaining in theflask to repopulate the primary culture. While there is ongo-ing discussion whether the actual microglial cells proliferateor if the cell culture contains an actively proliferating micro-glial precursor, it is clear that removing too many cells willreduce the repopulation rate (see Notes 6 and 7).574 Microglia Cell Culture: A Primer for the Novice20. Collect the supernatant in a centrifugation tube, and c entrifugefor 10 min at 200 × g. Because microglial cells reattach veryquickly, it is important to collect the supernantant immedi-ately after shaking the flasks. Primary mixed CNS cultures arerefed with 12 ml DMEM + 10% FBS and the flasks are placedback in the incubator. Microglial cells will continue to prolif-erate and the flasks can be shaken off again after 5–7 days.The yield of microglia will decrease significantly with everyshake off and after the forth shake off the microglia yield willbe usually very low.21. After the centrifugation, carefully remove the supernatant.Resuspend the microglial cells in the pellet with a fire-polishedPasteur pipette in DMEM/10% FBS, and plate them into theappropriate cell culture dish or multi-well plate by addingthe appropriate amount of cells (see Note 8) and the mediumof choice (see Note 3). Cells can be used for stimulationexperiments (see Notes 9 and 10) or can be transfected foroverexpression or knock-down studies (see Note 11).4. Notes1. Many protocols use phosphate-buffered saline (PBS) orDulbecco’s Phosphate-Buffered Saline (DPBS) as basis forthe digestion solutions. We prefer Hank’s buffered salt solu-tion (HBSS). While different in salts, the main difference isthe presence of glucose in HBSS, therefore providing anenergy source for the cells. Trypsinization is affected by thepresence of divalent cations and the use of buffers withoutcalcium and magnesium might increase trypsinization. Onthe other hand, optimal DNase activity requires buffers withcalcium and magnesium. Again there is no right and wrong.However, being cognizant of these underlying mechanismswill allow for efficient troubleshooting.2. The use of penicillin and streptomycin in cell cultures is stan-dard operating procedure (SOP). It prevents the hostile take-over of the cultures by microbes carried in from the preparationor due to unsterile work practices. This SOP indeed seems likea good thing. However, even low levels of bacterial contami-nation will activate the microglial cells in culture. So, a con-tamination kept in check by antibiotics might give theimpression of a healthy culture, while the cells are actuallyactivated by bacterial products. This would indeed confoundresults. Therefore some laboratories, prefer to work withoutantibiotics. However, this is not without risk either. Low-levelcontamination could be cleared by the cells themselves (after58 A. Witting and T. Möllerall, they are phagocytes) appear healthy but are activated by bacterial products as well. Additionally, cultures where a con-tamination could have been easily eliminated by antibiotics could be lost due to unchecked bacterial growth. Our approach is to work in the cleanest possible conditions, thus reducing the risk of contamination from the preparation, and the need to “clean” the cultures with antibiotics. If a given primary culture leads to experimental outcomes, which substantially differ from the data usually produced in the laboratory, a low-level contamination might be a potential explanation.3. One important difference between microglia culture proto-cols, and most likely the one which might be at the root of the large body of divergent data on microglia responses to a given stimulus, is the medium microglial cultures are main-tained in-culture medium with or without serum. Serum was the decisive factor which actually enabled the successful cul-turing of cells. It is the major source of nutrients. However, based on its biological nature, it is also the most variable com-ponent in a cell culture medium. The virtues of testing and banking serum lots are discussed at length in any basic cell culture hand book and will not be repeated here. And while the actual source, lot and repeated freeze/thaw cycles of a given FBS bottle will certainly influence the culture condi-tions, it is self-evident that the absence or presence of serum will have a much more profound effect on cells. The most commonly acknowledged reason to culture cells serum free is to synchronize cells and arrest them in G 0/G 1 phase (33). This is virtually true for all cells. However, there are other issues worth contemplating when culturing micro-glia. Microglia reside behind the blood brain barrier. The only time these cells are exposed to serum components is dur-ing break down of the blood brain barrier, for example, dur-ing stroke, trauma, or in active multiple sclerosis regions. Indeed several serum components have been identified as microglia-activating signals (1, 34–36). This could lead to an increased baseline activation of the cells. Proponents of serum in the medium would argue that cell culture is artificial in the first place and that there are no resting microglia in a culture dish anyway (see below for more detailed discussion on the microglial “activation state” in vitro). To our mind, there is no right or wrong in this discussion. As with everything we stressed before, consistency is the most important message. However, when interpreting own results in context of the literature it is very important to pay attention to this detail. For example, there are broadly diverging data on the sensitiv-ity of microglial cells to lipopolysaccharide (LPS), the arche-typical activator of toll-like receptor (TLR4). While it is now appreciated that not all LPS is created equally potent (seeNotes 5 and 9), it is now also well understood that serum contains LPS binding protein (LBP). LBP, together with CD14 on the cell surface, leads to optimal presentation of LPS to TLR4 and increases cell sensitivity to LPS by 2–3 orders of magnitude (37). Similarly, serum might contain cofactors for other agonists and/or receptor systems or rather unselectively serve as a priming signal for an unrelated trigger.Because of the many potential pitfalls with serum-contain-ing medium, many laboratories and commercial sources have developed serum-free alternatives. For microglia, approaches developed for peripheral macrophages have been successful adapted. While it is certainly possible to culture microglia in unsupplemented DMEM, cells do not survive long. Serum withdrawal is also a common way to induce autophagy (38) and the induction of autophagocitic pathways in serum starved microglial is a concern. Most laboratory-derived serum-free media start with DMEM, supplemented with serum albumin (bovine or human, usually 1 mg/ml), insulin, transferrin, and selenite. The latter three are readily available as an ITS supplement, making this a convenient and inexpen-sive alternative to commercial media.Of the many commercial serum-free media, the Macrophage Serum-free Medium from Invitrogen and Mediatech’s Cellgro COMPLETE TM Serum-Free/Low-Protein Medium are of note. While the former is specially formulated for mac-rophages both allow for ready growth of microglial cells without further supplementation. The compositions are pro-prietary, however, one can enter in an agreement with the respective supplier to get information on the ingredients (not concentration) of the media, (e.g., if one needs to determine the presence of a specific factor). We have had excellent expe-rience with Invitrogen’s Macrophage Serum-free Medium and it is our (pricey) medium of choice.4. Polystyrol culture flasks or plates should be coated with sub-strates that facilitate the attachment of cells. Usually poly-d-lysine (MW 30,000–70,000; 70,000–150,000; >300,000) or poly-l-lysine (MW 70,000–150,000; 150,000–300,000) are used for the attachment of the primary mixed CNS cultures. When it is required that poly-lysine is not metabolized by the cells, poly-d-lysine is preferred. Instead of poly-d/l-lysin, poly-l-ornithine (MW 30,000–70,000) can be used. Our observation is that with a poly-ornithin more microglial cells float in the supernantant of the primary mixed cultures. Pure microglia cultures can be plated without coating on pre-treated polystyrol flasks or specialty cell culture ware such as PRIMARIA TM, (Becton Dickinson) or Cell+(Sarstedt).。

Lecture1presentationsmall

Lecture1 presentation smallECONOMETRIC MODELLING: THE 'GENERAL TO SPECIFIC' PROCEDURE.(A)Some “facts of life”:∙Economic theory does not tell us much about form & content of empirical model∙Dynamics matter: adjustments are not instantaneous∙Want to include “relevant” and exclude “irrelevant” variables: but how do we choose? (B)Key themes∙Principle of parsimony∙Any model we wish to use must be statistically well-specified.(C) ECONOMIC THEORY(D) OUR OBJECTIVES1.To establish whether the hypothesised economic relationship is supported by empiricalevidence.2.If such support is found:∙To obtain 'good' estimates of the unknown parameters β1 and β2∙To be able to test hypotheses about the unknown parameters∙To use our estimated regression model for(i)forecasting(ii)policy analysis/simulation modelling.[E] THE REQUIREMENTS OF A GOOD ECONOMETRIC MODELWhat are the fundamental requirements of an 'adequate' econometric model?(1) THE MODEL MUST BE DATA ADMISSIBLE.It must be logically possible for the data to have been generated by the model.(2) THE MODEL MUST BE CONSISTENT WITH SOME ECONOMIC THEORY.(3) REGRESSORS SHOULD BE (AT LEAST) WEAKLY EXOGENOUS WITHRESPECT TO THE PARAMETERS OF INTEREST.(4) THE MODEL SHOULD EXHIBIT PARAMETER CONSTANCY.(5) THE MODEL SHOULD BE DATA COHERENT.The residuals should be unpredictable from their past history.(6) THE MODEL SHOULD BE ABLE TO ENCOMPASS A RANGE OFALTERNATIVE MODELS.12(F) STATICS AND DYNAMICS: EQUILIBRIUM AND ADJUSTMENTS TO EQUILIBRIUMThe regression model counterpart to (1) (assuming time-series data)∙ We need to take account of the possibility of dynamic adjustment processes.∙ Dynamic models are those which contain lagged or leading values of variables, as well as current-dated ones.There are many forms such models can take.Y t = β1 + β2X t + β3X t-1 + u t is a distributed lag modelY t = β1 + β2X t + β4Y t-1 + u t is an autoregressive modelY t = β1 + β2X t + β3X t-1 + β4Y t-1 + u t is an autoregressive-distributed lag modelY t = μ + β0X t + β1X t-1 +... + βq X t-q + χ1Y t-1 + ... + χp Y t-p+ u t is an ADL(p,q)) model.(G) THE ERROR CORRECTION MODELThe long run (equilibrium) relationship between Y and XtWhat kind of behaviour might one expect Y to exhibit over time?period (t+1) consumption will change by the amount δ(Y * - Y)tFigure 1:(i) The change from a to a'(ii) The change from a' to a''stable.As it stands, equation (9) cannot be estimated by Ordinary Least Squares (OLS) as the variable in parentheses cannot be formed without knowledge of β1 and β2.1θ2, δ, β1 and β2.This is the ECM model.(H) DYNAMIC ESTIMATION PROCEDURES IN PRACTICEreparameterised form of (10), an autoregressive-distributed lagWe could also estimate aA number of decisions have to be made at this starting point:31.Functional form: should the model be linear in levels or in logs, or have some otherfunctional form?2.Which variables do we believe determine Y? (Note that it is possible that some variable(s)may not enter the long run equilibrium equation for Y but do affect the disequilibrium adjustment process for Y).3.What should be the 'order' of lags in the general model? Usually, an economic researcherhas no prior information about the required lag lengths, and this decision has to be data-based, using a 'general-to-specific' modelling strategy.(I): OBTAINING A PARSIMONIOUS RESTRICTED MODEL: THE 'GENERAL-TO-SPECIFIC MODELLING APPROACH.It is difficult to give any simple rules about how to proceed next. The model we start with - the 'general' model - will typically be an ARDL(p,q) model.It should be a statistically adequate representation of the data.The general model is likely to be heavily 'overparameterised', and it is not in a form that has any explicit economic interpretation.It is now necessary to simplify this into a final 'specific' or parsimonious model that overcomes these weaknesses.This is done through a process of testing restrictions on parameters in the general model, and then imposing the restrictions if we cannot reject them on statistical grounds.Among other things, we try to identify 'irrelevant' variables (or irrelevant lags on variables) and exclude these from our model. (This is what Hendry calls 'marginalising irrelevant variables').We should be guided here by the goal of obtaining a final specification that is 'simple', consistent with the data, and has a ready interpretation in terms of economic theory.It will be helpful to reparameterise the general model into an error correction model (ECM) form at some stage in this sequence. There are two reasons for doing so; firstly, the ECM model is more easy to interpret in economic terms, as the distinction between short term adjustment responses and long term relationships between the variables is easier to see in this form. Secondly, there are some statistical advantages in working with the ECM form.Our final model should:(i) satisfy all misspecification test criteria to be discussed later;(ii) be "parsimonious";(iii)be parameterised in a form that has an intuitive economic interpretation.(G) THE LONG RUN (EQUILIBRIUM) SOLUTION TO A DYNAMIC ECONOMETRIC MODEL4In long run (static) equilibrium, all variables remain at constant levels through time. So for any variable Z in static long run equilibriumSuppose we have the following econometric model:in this expression by their estimated values.REQUIRED READING:Thomas, Ch 11: Estimating dynamic modelsThomas, Ch 12: Choosing the appropriate modelThomas, Ch 13 (section 13.3 and 13.4 only)Gilbert (1986) [easy]5。

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