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Hindawi Publishing CorporationISRN ImmunologyVolume 2013 Article ID 205431 10 pageshttpdxdoiorg1011552013205431

Research ArticleCytomorphological and Cytochemical Identification of Microglia

Subhajit Das Sarma Koushik Chatterjee Himadri Dinda Dhriti Chatterjee andJayasri Das Sarma

Department of Biological Sciences Indian Institute of Science Education and Research-Kolkata (IISER-K) Mohanpur CampusPO BCKV Campus Main Office Mohanpur West Bengal Nadia Mohanpur 741252 India

Correspondence should be addressed to Jayasri Das Sarma dassarmajiiserkolacin

Received 7 May 2013 Accepted 18 July 2013

Academic Editors R El Ridi and S M Varga

Copyright copy 2013 Subhajit Das Sarma et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Microglia is one of the major resident immune cells in the central nervous system and is considered to be the key cellularmediator of neuroinflammatory processes Identification of different Microglial states of activation by morphologic means hasbeen one of the major challenges in the field of neurobiology of diseases Therefore microglial biology demands techniquesto identify differing stages of microglia in different neuroanatomic locations as well as understanding the role of Microglia indifferent Neurological diseasesThis present study is aimed towards summarizing the literature and for understanding the progressmade in different Cytomorphological and Cytochemical techniques of identifying Microglia This study also review recently usedImmunohistochemistry techniques along with Ultrastructural studies determining different morphological features of restingto activated phagocytic Microglia in a viral induced experimental animal model of neuroinflammation Results revealed thatchronic Microglial activation is considered to be an important component of neuronal dysfunction injury and loss (and henceto disease progression) Thus Microglial research with special emphasis on identification of different activation states of Microgliahas gradually become significant

1 Introduction

Microglia the resident macrophages of the Central NervousSystem (CNS) is known to support and sustain proper Neu-ronal functions Existence of this Glial cell in CNS has beenreported a century ago [1] Nissl was the first to recognizeMicroglia and name it as ldquostabchenzellenrdquo (rod cells) andconsidered it as a reactive neuroglia He also suggested thatMicroglia has the capacity of migration and Phagocytosis[1] Regarding the origin of Microglia a complete frameworkwas provided for defining this particular cell type by delRio-Hortega in 1932 but still many of the features remainedcontroversial [2] Microglia transits through different stagesof development to attain its maturity and functionality in theCNS The first stage is of Ameboid Microglia and it sharescommon Immunological Histochemical and Morphologicalfeatures with Macrophages outside the CNS [2] Hence itis also sometimes termed as the Macrophages of the BrainThe Ameboid microglia is considered to be round in shapeor have short and broad processes It is assumed that cellswith Dendrite and elongated morphology also belong to this

particular type of Microglia (Ameboid) [3] In due courseof time and under certain circumstances some parts ofthe Embryonic Ameboid Microglia gets degenerated andsome develop thin processes to become Ramified Microgliain an adult Brain [4] This transitory phase leads to totaldisappearance of the preliminary form of Microglia whichis ldquoAmeboid Microgliardquo thereby providing space for theRamified Microglia In response to variety of brain injuriesand inflammation this Ramified Microglia is capable of dra-matically changing its structural and chemical morphologyinto a reactive or an Amedboid Microglia Along with thatit is also capable of rapidly upregulating a large number ofproteins which are receptor types and myriad of secretoryproducts (Figure 1) which acts for defense and potentiallycauses perturbation to the infected and injured CNS [5]

2 Mechanism of Activation

Regarding the mechanism of activation of Microglial cells itis to convey that very little is known about themetabolic state

2 ISRN Immunology

Sl No Specifications

1 Proliferation234 Vimentin56789 CD4 antigen (W325)

1011

B4-isolectinGriffonia simplicifolia

Macrophages markers (ED1 ED2 OX-41)

Resting Activated Phagocytic

CR3 complement receptor (OX-42)MHC class I antigen (OX-18)MHC class II (OX-6)

CD8 antigen (OX-8)Leukocyte common antigen (OX-1)

minus+

minus+ minus+

minus+

minus+

minus+

+

+

+

+

+

+

+

+

+ +

+

+

+

+

+

+

+

+

+

minus

minus

minus

minus

minus

Restingmicroglia

Activatedmicroglia

Phagocyticmicroglia

Figure 1 Different states of Microglia and associated phenotypic characteristics (adopted fromNeuroglia Second Edition Edited by HelmutKettenmann and Bruce R Ransom)

of Resting Microglia Even then there are lines of evidenceof slow but steady rate of proliferation it is confirmed fromincorporated [3H] thymidine in situ in the healthy Brain[6] The Microglial activation for a long period of time hasbeen evaluated based on the phenomenon of proliferationand expression of immunoregulatory molecules [7] Somecell surface markers like Major Histocompatibility Complex-II (MHC-II) are important in Immune Regulation andthese are constitutively expressed in Ramified MicrogliaFor understanding the activation mechanism of Microgliaseveral approaches have been made both in in vitro and invivo model of studies Recent advancement over the past 15to 20 years in the field of Microglia reveals that its responseto different ldquotissue insultsrdquo is in the form of complex arrayof Inflammatory Cytokines and actions Furthermore thisaction surpasses the previous historical perspective of Phago-cytosis and Reactive Gliosis Nowadays Microglial researchhas become the main point of focus in the field of cellularNeuroimmunology and also in the field of neuroinflamma-tion One of the major challenges in the field of Microglialresearch is to detect precisely the different states of activationof Microglia by morphologic means Hence the field ofMicroglial Neurobiology demands techniques to identify

differing stages ofMicroglia in differentNeuroanatomic loca-tions The current understanding is based on the expressionof surface molecules in Ramified Microglia Activated andin Phagocytosed Microglia (Figure 1) One contrary of suchsurface expression is that these molecules are more or lessexpressed in different stages of MicrogliaThe only differencelies in their level of expression In this study we have revieweddifferent staining procedure starting from the time of Pio delRioHortega (1921) Alongwith that special emphasis has beenprovided on the recent Immunohistochemical techniquesto identify Microglia in Neuroanatomic location of a viralinduced Neuroinflammation in experimental Mouse model[8ndash11]

21 Methods of Identification Through the understandingof morphological changes the Microglial role in the dis-eased CNS is recognized Hence identification of Microgliathrough different staining procedure provided Cytomor-phological understanding Staining of Microglia with sil-ver carbonate started with Pio del Rio Hortega and wentthrough few modifications where CNS tissues were fixed informalin and yielded successful staining results [13] Laterthere were efforts made with CNS tissues of a wound where

ISRN Immunology 3

the tissue was colored using trypan blue intravitally whichcoloured the Macrophages of that particular area Alongwith it also stained other Neuroglia which made it difficultto differentiate the Macrophages from Neuroglial cells andhence required much specialized procedure of staining fordifferentiation [14] Formalin-dextran-CaCl

2solution was

used as a fixation solution in an attempt of modificationof Weil and Davenportrsquos (1933) silver carbonate method foridentification of Microglia This staining method was alsoable to demonstrate both resting aswell as activatedMicroglia[15] A rapid and simple impregnation method using ammo-niacal silver nitrate in epoxy-embedded tissues demonstratedthe morphological aspects of a Reactive Microglia [16]

Identification of theMicroglial cells beyond theCytomor-phological understanding was enhanced by the developmentof different Cytochemical procedures of staining Cytochem-ical procedures took advantage of the exclusive expression ofcertain molecules in specific cell types (Table 1 and Figure 1)[17] Different Cytochemical methods used acid phosphataselysozyme peroxidase alpha-naphthyl acetate esterase alpha-naphthyl butyrate esterase adenosine triphosphatase and thePeriodic Acid-Schiff reaction (PAS reaction) The distinctactivity of almost all these enzymes and the PAS reactionwereevident in Reactive Microglia [18]

For visualization of cells in vitro and in tissue sectionslectins and antibodies are directly coupled with fluorescentdyes or with avidinstreptavidin peroxidase conjugate forstaining microglia [17] Identification of Microglia withlectins (carbohydrate binding proteins) as a tool is of par-ticular interest as the intensity of staining varies dependingon their functional state based on the interaction of variouslectins with Microglial membrane [12 18 19] In severalstudies it has been shown that Microglia stained intenselywith GS-1 RCA WGA and ConA and slightly with DBAUEA BPA and SBA Later most of the lectin based stainingwas predominantly used for identify Microglial activationstate in culture (in vitro) only

Immunohistochemistry as a technique is based on theexpression of a stage-specific protein and the availability ofthe antibodies against such specific protein as immunogen

22 Identifying Techniques in Use Immunohistochemistryis the major technique which is being used proficientlyto differentiate different stages of CNS Microglia in ourLaboratory In the field of Neuroimmunology and Neu-robiology our laboratory is focused on understanding themechanism of Demyelination in an Experimental Animalmodel and Immunohistochemistry is one of the most rou-tinely used techniques to identify inflammatory cells inthe Virus-induced inflamed CNS tissues as well as isolatedCNS cells in culture This technique is used based onthe mostly available antibody against the antigen CD11Bwhich is a known marker for Histochemical analysis ofMicrogliaMacrophages in mice CD11B was used in our pre-vious studies to differentiate the Neuroinflammatory statusof the CNS in viral infection [12 19] Our further studyusing CD11B is not consistent enough hence Iba1 antibodyis used [20] Iba1 is aMacrophagesMicroglia specific calcium

binding protein which is involved in RacGTPase-dependentmembrane ruffling and Phagocytosis [20ndash22] In our severalstudies we have noticed that Iba1 is a better marker formorphological differentiation of Resting- RamifiedMicrogliaversus Activated-Amoeboid and Phagocytotic Microglia asit is involved in RacGTPase-dependent membrane rufflingand Phagocytosis [20] (unpublished data from Das SarmaLaboratory)

23 Detailed Method of Iba1 Staining Inflamed Brain tissueswere harvested from MHV-A59 or its Isogenic recombinantstrain RSA59 both of which can induce acute encephalitis(inflammation in the brain) Meningitis and Demyelina-tion during chronic stage [8 19] MHV-A59 or RSA59induced Neuropathology mimics certain aspects of HumanDemyelinating disease like Multiple Sclerosis (MS) Mockand infected Mice were perfused transcardially with Phos-phate Buffer Saline (PBS-74 pH) and thereafter fixed with 4PFA (PFA-72 pH) in 01M sodium phosphate buffer (pH 74)Brains were dissected and postfixed in 4 paraformaldehydeovernight at Room Temperature (RT) All tissues were thenprocessed for paraffin embedding and sectioning in theHistology and Microscopy core at Department of BiologyIISER-Kolkata Slides were deparaffinized and sections wereimmersed in Xylene rehydrated in distilled water and there-after treated in high-temperature antigen retrieval or antigenunmasking solution Briefly Vector Antigen UnmaskingSolutions (Cat No H-3301 citrate based) were designedfor use in high-temperature unmasking procedures AntigenUnmasking Solution was shaken well and 9375mL of theconcentrated stock Antigen Unmasking Solution is dilutedin 1 liter of distilled water into a glass beaker and heated in90∘C Slides were positioned into metal staining racks (Notedo not place slides close together uneven stainingmay occur)and boiled for 15 minutes into the diluted antigen unmaskingsolution Then slides were removed and immediately placedunder tap water Thereafter without letting the tissues drysections were washed in PBS buffer (pH 75) for 5 minutesand preceded for Immunohistochemical Labeling Some tis-sues may bind avidin biotinylated horseradish peroxidaseor other BiotinAvidin System components nonspecificallyThis binding may be due to endogenous biotin or biotin-binding proteins lectins or nonspecific binding substancespresent in the section To avoid such nonspecific bindingantigen unmasked tissue sections were incubated for 1 hrwith normal Goat Serum Briefly one to four drops of stocknormal Goat Serum (VECTASTAIN ABC Kit Catalogue No6105 from Vector Laboratories Inc) in onemL PBS (onedrop is approximately 50 120583L) is added and then incubatedovernight with Rabbit anti-Iba1 (1 100 Wako RichmondVA USA) Thereafter tissue sections were incubated withbiotinylated secondary antibodies (1 200 Vector Labora-tories Burlingame CA) at (RT) for 1 hour Primary andsecondary antibody was then diluted in PBS with 1 normalSerum and the Immunoreactivity was visualized with theVectastain ABC system and 331015840-diaminobenzidine (VectorLaboratories Burlingame CA USA) ABC reagent was pre-pared by adding two drops of ABC Reagent A provided with

4 ISRN Immunology

Table 1 Surface antigens and thier detection techniques with functional relevance

Molecular structureantigen Detection agenttechnique Properties and functional relevanceIdentificationlowast

Surface carbohydrates (oligosaccharidescontaining-galactose residues)

Griffonia (Bandeiraea) simplicifoliaisolectin B4 Not known also expressed by blood vessels

Surface carbohydrates (oligosaccharidescontaining N-acetylglucosamineoligomers)

Lycopersicon esculentum (tomato)lectin Not known also expressed by blood vessels

Iba1 (ionized calcium-bindingadaptormolecule 1) Antibodies against Iba1 Protein with suggested role in calcium homeostasis

CD11b18 (120572M1205732 integrin) Antibodies against CD11bCD18(MAC1 OX42)

Complement receptor 3 (CR3) with additional bindingproperties for other molecules for example Fibrinogen

CD45 (leukocyte common antigen LCA)Antibodies against CD45 lowexpression compared withinfiltrating leukocytes

Transmembrane protein tyrosine phosphatasefunctions in signaling also roles for TCR and BCRactivity

CD68 (macrosialin) Antibodies against CD68 Role in Phagocytosis and as oxidized LDL-bindingprotein

F480 antigen F480 antibodyMember of the epidermal growth factor(EGF)-transmembrane 7 (TM7) family suggested rolein immune tolerance

Functional orientation

MHC II

Antibodies against MHC IIcomponents combinable withfunctional assays for antigenpresentation to T cells in vitro

Antigen presentation by APC

CD11c (integrin 120572X) Antibodies against CD11c Cell adhesion marker for dendritic cells as APC

CD34 (mucosialin sialomucin) Antibodies against CD34 Roles in adhesion marker for precursor cells of themyeloid lineage

iNOS (inducible nitric oxidesynthase)

Antibodies against iNOS in situhybridization indirectly bymeasurement of NO production vianitrite in vitro

Enzymatic synthesis of NO from arginine duringinflammation (upon gene induction) resulting also incitrulline generation

Transgenic models

CX3CR1

Transgenic Mice expressingenhanced green fluorescent protein(EGFP) in the Cx3cr1 locus(fractalkine receptor)

Receptor for the chemokine fractalkine (CX3CL1) withimplications for the control of Microglial activity

Iba1

Transgenic mice expressingenhanced green fluorescent protein(EGFP) under control of theIba1promoter

Protein with suggested role in calcium homeostasis

the kit in 10mL of PBS pH 75 containing 01 Tween 20 andsubsequently adding two drops of ABCReagent B to the ABCmixing solution immediately The ABC Reagent was allowedto stand for about 30 minutes before the use of the sectionsAfter the secondary antibody incubation step sections wererinsed in PBS for three times ABC reagent made in PBSwith 01 Tween 20 was added and incubated for 30 minutesthen after rinsed with PBS for five times And then theimmunoreactivity was visualized with the Vectastain ABCsystem and 331015840-diaminobenzidine (Vector LaboratoriesBurlingame CAUSA) Detailed stepwise protocol of Iba1immunostaining is attached in Table 2 All Immunostainedsections were photographed digitally scanned and analyzedon an Aperio Scan scope XT slide scanner and Aperio Imagescope version 100361805 software (Aperio) Images wereprocessed using Adobe PhotoshopCS3 (Figures 2 3 and 4)

231 Ultrastructural Studies To characterize the Phagocy-tiotic Microglia this study took the advantages of ultra-sturctural images available from our studies (Figure 5) Day30 postinfected inflamed Mice were anesthetized perfusedwith 4 PFA and spinal cords were harvested and fixedovernight in 2 glutaraldehyde Samples for transmissionelectron microscopy (TEM) were postfixed with 1 osmiumtetroxide dehydrated and flat embedded in Poly-Bed 812epoxy resin (Polysciences) Half-micrometer thick sectionswere cut from the lesional epicenter stained with toluidineblue and examined by light microscopy Ultrathin TEMsections (600 A) were cut from representative foci of demyeli-nation from the toluidine blue-stained semithin sections andmounted on 200 mesh copper grids stained with uranylacetate and bismuth subnitrate and viewed under a JEOLJEM 1010 (Figure 5)

ISRN Immunology 5

Table 2 Steps of Iba1 immunostaining on paraffin sections by using ldquovectastain ABC kit (Cat No PK-4001)rdquo

Steps Time of incubation TemperatureDeparaffinize slides on a heat blockDeparaffinize with Xylene 5mins lowast 2 times RT100 ethyl alcohol 5mins RT70 ethyl alcohol 5mins RTDistilled water 5mins RTPBS 1x 5mins RTAntigen retrieval Antigen Unmasking solution (Vector Laboratories H3300)(9375mL of antigen unmasking solution in 1 liter of distilled water) 15mins 95∘C

Blocking (Vector laboratories rabbit kit) (15120583L1mL of PBS) 60mins 37∘CAnti-Iba1 antibody (1 100 dilution) 120mins 37∘CPBS 1x wash 3mins times 3 timesSecondary antibody (Vector laboratories rabbit kit) (5 120583LmL of PBS) 60mins 37∘CPBS wash 3mins RTABC reagent (Vector laboratories rabbit kit) (1120583L Reagent A + 1 120583L reagent B +10120583L of PBS1x) 30mins 37∘C

DAB substrate (Vector laboratories rabbit kit) (500120583L distilled water + 100 120583Lbuffer + 200 120583L DAB + 100 120583L H2O2)

5ndash10mins (till a browncolour develops) RT

Distilled water 5minsHematoxylin (Counter staining) 30 secRunning water wash50 Ethanol 10 sec20 dips70 Ethanol 12 sec24 dips100 Ethanol 14 sec28 dipsXylene 3mins times 3 timesMounting with refrax mounting medium and observed under light microscopeRT room temperature

A

(a)

B

(b)

C

(c)

Figure 2 Distribution of ramified microglia in the nonpathological Brain of adult Mouse 5-micron thick sagittal brain section from 4 week-old C57BL6 mice were Immunostained with anti-Iba1 antibody Ramified resting Microglia are shown in olfactory bulb (a) (200x) basalforebrain (b) (200x) and hippocampal region (c) (100x)

6 ISRN Immunology

(a)

(b)

(c)

(d)

(e)

(f)

Figure 3 Accumulation of activated amoeboidmicroglia in the inflamed brain of RSA59 infectedMice 5-micron thick RSA59 infected Brainsections were immunostained with Iba1 and distribution of activatedmicroglia are shown in different region of inflamed brain Olfactory bulb(a) (200x) basal Fore (b) (200x) hippocampal region (c) (100x) corpus callosum (d) (36x) anterior commisure (e) (200x) deep cerebellarwhite matter (f) (40x) All images were taken from unpublished data of Das Sarma Laboratory

(a) (b) (c)

(d) (e) (f)

Figure 4 Comparative morphology of activated amoeboid microglia to resting quiescent ramified microglia was taken from acute inflamedbrain to resting brain tissue Different shape of amoeboid microglia which is embracing neuron immunostained with Iba1 and counterstained with haematoxylin (a)ndash(e) (1000x) (f) quiescent resting Microglia in a nonpathological brain of 4-week-old mice (Data taken fromDas Sarma laboratory unpublished data)

ISRN Immunology 7

(a) (b) (c)

Figure 5 Ultrastructural appearance of a perineuronal Phagocytic Microglial cell in the chronic demyelinating plaque of RSA59 infectedmouse spinal cord Electron micrograph (a) shows Microglial cells surrounding the demyelinated axon in a demyelinating plaque region ofRSA59 infected mouse spinal cord Microglia has a heterochromatic nucleus with multiple vacuoles High-magnification images (b) and (c)demonstrates the presence of Microglia surrounding the unraveling myelin sheath in an axon The Microglia cell membrane is in intimatecontact with the outer portion of themyelin sheath as theMicroglia is stripping away (phagocytizing) themyelin sheath and engulfs themyelinsheath Multiple vacuoles with myelin fragments are seen within the cytoplasm of the activated microglia (phagocytotic in nature)mdashFigure 5adopted from [12]

All the techniques discussed previously were based onfixed tissues after harvesting Whereas in recent period invivo imaging has made feasible the prospects of monitoringcellular events associated with Neurodegenerative diseasesusing noninvasive techniques of monitoring [23] Positronemission tomography (PET) imaging reveals Microglialactivation in patients with Neurodegenerative diseases andin animal models by using a specific radio ligand of theperipheral benzodiazepine receptor ([11C]-PK11195) whichis upregulated in activated Microglia Disease-specific local-ization of Microglial activation can also therefore be detectedwith PET imaging [24]

Several lines of evidence show that Microglial functionis regulated by NADPH oxidase and the production ofintracellular reactive oxygen species (ROS) Additionally theproduction of Neurotoxic extracellular ROS is also shownto occur primarily through NADPH oxidase for multipledistinct stimuli hence making this enzyme complex couldbe an ideal therapeutic target Recently several peptidesantibiotics and small molecules have been identified thatinhibit NADPH oxidase and are neuroprotective Combinedwith early diagnosis through PET-based Microglial imagingthis approach might provide hope for attenuation of theprogression of neuroinflammatroy and neurodegenerativedisease

3 Discussion

The discipline of Pathology makes a fundamental distinctionbetween acute and chronic inflammation Acute inflamma-tion comprises the immediate and early response to aninjurious agent and is basically a defensive response thatpaves the way for repair of the damaged site The concept ofchronic inflammation (as opposed to acute inflammation) ismore relevant in the context of understanding CNS disease(as opposed to CNS injury) as the very term ldquodiseaserdquo

implies chronicity Still the question remains whether inhi-bition of Neuroinflammation can be an effective means oftherapeutic interventions To address these questions it isnecessary to learn more about how directly Neuroglial cellresponses more specifically towards interaction of neuronwith Microglia induced by injury or insult within the CNSand also the mechanism by which these responses ultimatelycontribute to Neuropathology and consequently Neurobiol-ogy of diseases

The term Neuroinflammation classifies where limitedNeuronal insults trigger Glial cell activation without break-down of the Blood Brain barrier and without concomi-tant LeukocyteBlood Monocyte infiltration Several studieshave documented inflammatory components in AlzheimerrsquosDisease (AD) [25] Parkinsonrsquos Disease (PD) [23] Amy-otrophic Lateral Sclerosis (ALS) Multiple Sclerosis (MS)and a growing number of other nervous system Pathologies[26] Although inflammation may not typically denote aninitiating factor in neurodegenerative disease there is emerg-ing evidence in Animal models that sustained inflammatoryresponses involving Microglia and Astrocytes contribute todisease progression [26] Although inducers of inflammationmay be generated in a disease-specific manner there isevidence for a remarkable convergence in the mechanismsresponsible for the sensing transduction and amplificationof inflammatory processes that result in the production ofNeurotoxic mediators The distinct pathways for produc-tion of inducers of inflammationmdashsuch as Amyloid-120573 a-synuclein mutant SOD1 and myelin peptide mimeticmdasharelikely determinants of the specificNeuropathological featuresof AD PD ALS and MS

Even though this paper discusses the Cytomorphologi-cal and Cytochemical identification of Microglial states ofactivation future studies warrant the characterization ofdifferent activation states of microglia by molecular markersMounting lines of evidence of an inflammatory response

8 ISRN Immunology

in AD consist of changes in Microglial morphologymdashfromRamified (resting) to Amoeboid (active)mdashand Gliosis toAstrogliosis (manifested by an increase in the number andsize of Astrocytes) surrounding the senile plaques Microgliasurrounding Plaques stain positive for MHC class II Cox-2MCP-1 TNF-a IL-1b and IL-6 [25] MCP-1 are the activationmarkers and proinflammatory mediators of inflammationAlong with that it is also known to induce the Chemotaxis of Astrocytes and contributes to the recruitment ofAstrocytes around senile plaques [27] Parkinsonrsquos disease isnow accepted to have an inflammatory component thoughoriginally thought to be a disease characterized with loss ofone Neuronal type [28ndash30] Reactive Microglia expressingHuman Leukocyte Antigen (HLA)-DR and CD11b alongwith Lewy bodies are found in the substantia nigra of PDpatients [31] Prominent neuroinflammation can be readilyobserved in pathologically affected areas of the CNS and inspinal cords in both Human ALS patients and mouse modelsof the ALS disease [32]

In addition to the previous markers elevated levels ofchemokine and cytokines and their receptor like IL-1aCXCR2 CCR3 CCR5 and TGF-120573 have been reported inpostmortem of AD patients brains [33] Increased levels ofcolony-stimulating factor (CSF) [30] in the blood have alsobeen reported in PD patients Activation of Glia in ALShas been extensively characterized and marked with elevatedproduction of potentially cytotoxic molecules such as ROSinflammatory mediators like COX-2 and pro-inflammatorycytokines like IL-1120573 TNF-120572 and IL-6

Very few parts of literature discussed the role of Glialcells in Neurological diseases or disorders previously Withthe understanding of the role of Astrocytes and Microgliain maintaining the homeostasis in the healthy CNS hasbeen the main attention of the researcher drawn towardMicroglia-driven Neuroinflammation Mounting evidencesdemonstrate that Chronic Microglial activation is an impor-tant component of Neurodegenerative diseases and thischronic Neuroinflammatory component contributes to Neu-ronal dysfunction injury and loss (and hence to diseaseprogression) The recognition of microglia as the CNSintrinsic immune system and the understanding of thechronic activation of the system lead to pathologic squealand lead towards themodern concept ofNeuroinflammationAdvancement in staining techniques and in microscopes andtechniques of microscopy has extended the older vision ofpassive Glial responses that were known for a long time asldquoReactive Gliosisrdquo to Microglial Neuroinflammation

Though recent evidence demonstrate the beneficial andneuroprotective profile of Microglia the acute Microglialactivation is deleterious and it is considered that Microgliamay also be involved in maintenance repair and possiblyprotection A major unresolved question is whether inhibi-tion to these responses will be a safe and effective meansof reversing or slowing the disease course Therefore theideal therapeutic approach would involve early attenuationof the Microglial responses to levels that are no longerdeleterious rather than the elimination of the Microglialresponse altogether Hence identification of the activationstate of Microglia is very important in the light of therapeutic

intervention Hence Microglial studies emphasizing towardsthe identification of Microglial states gradually have becomesignificant and therefore many researchers contributed inmajor ways of identification of Microglia (both resting andactivated) using different techniques of Cytomorphologicaland Cytochemical staining

Immunostaining is much more efficient over the otherchemical staining method Most commonly used antibody isCD11B which is integrin alpha chain family protein Integrinalpha-Mbeta-2 is implicated in various adhesive interactionsof Monocytes Macrophages and Granulocytes as well as inmediating the uptake of complement-coated particles It isidentical with CR-3 the receptor for the iC3b fragment of thethird complement component It probably recognizes the R-G-D peptide in C3b Integrin alpha-Mbeta-2 is also a recep-tor for Fibrinogen factor X and ICAM1 It recognizes P1 andP2 peptides of Fibrinogen Gamma chain It is predominantlyexpressed in monocytes and granulocytes and not specificfor tissue Macrophages and more specifically to MicrogliaCD11B tends to label endothelial cells in the CNS Bloodmicrovessels which also sometimes make the identificationdifficult and inaccurateWhereas Iba1 allograft inflammatoryfactor-1 family of proteins and are specific to Microlgia andMacrophages but are not cross reactive with Neurons andAstrocytes in the CNS It is also known not to cross react withgranulocytes or blood microvasculature endothelial cellsLevel of Iba1 protein expression increases with infectionand inflammation within the CNS and with altered level ofCalcium ion as it has calcium binding properties Calciumions are known to be the one of the important signal residuesin all the cells including CNS cells Calcium ion exerts theirsignaling activity through association with various calciumbinding proteins many of which are classified into a largeprotein family the EF hand protein family (FEBS Latter)Iba1 is also an actin-binding protein that enhancesmembraneruffling and RAC activation It also enhances the Actin-bundling activity of LCP1 which binds calcium and plays amajor role in RAC signaling and in Phagocytosis It may alsoplay a role in Macrophages activation and function

In summary advances made in Cytomorphological andCytochemical ways of identifying different states ofMicrogliaand other Glial cells have proven to contribute in the field ofNeuroimmunology as a whole

Acknowledgments

This work was supported by a Research Grant fromDepartment of Biotechnology (DBT) (BTPR14260MED304372010) India Indian Institute of Science Educationand Research-Kolkata (IISER-K) India start up Fund andResearch Grant RG3774A21 from the National MultipleSclerosis Society to Das Sarma Koushik Chatterjee andHimadri Dinda are supported by DST funded to RS SubhajitDas Sarma is supported by DBT funded to JDS and DhritiChatterjee is supported by University Grant Commission(UGC) IndiaThe authors thank Central Animal Facility andDr S G Ramachandra IISC Bangalore India for provid-ing experimental mice The authors thank Dr Lawrence CKenyon Thomas Jefferson University USA for his assistance

ISRN Immunology 9

in blind studies of pathological samples The authors have nofinancial conflict of interest to disclose

References

[1] K D Barron ldquoThe microglial cell A historical reviewrdquo Journalof the Neurological Sciences vol 134 no 1 pp 57ndash68 1995

[2] M A Cuadros and J Navascues ldquoThe origin and differentiationof microglial cells during developmentrdquo Progress in Neurobiol-ogy vol 56 no 2 pp 173ndash189 1998

[3] M A Cuadros A Moujahid A Quesada and J NavascuesldquoDevelopment of microglia in the quail optic tectumrdquo Journalof Comparative Neurology vol 348 no 2 pp 207ndash224 1994

[4] D M Reid V H Perry P-B Andersson and S Gordon ldquoMito-sis and apoptosis of microglia in vivo induced by an anti-CR3antibody which crosses the blood-brain barrierrdquo Neurosciencevol 56 no 3 pp 529ndash533 1993

[5] R B Rock G Gekker S Hu et al ldquoRole of microglia in centralnervous system infectionsrdquo Clinical Microbiology Reviews vol17 no 4 pp 942ndash964 2004

[6] L J Lawson V H Perry and S Gordon ldquoTurnover of residentmicroglia in the normal adult mouse brainrdquo Neuroscience vol48 no 2 pp 405ndash415 1992

[7] Y Matsumoto K Ohmori and M Fujiwara ldquoImmune regula-tion by brain cells in the central nervous system microglia butnot astrocytes present myelin basic protein to encephalitogenicT cells under in vivo-mimicking conditionsrdquo Immunology vol76 no 2 pp 209ndash216 1992

[8] E Lavi D H Gilden and ZWroblewska ldquoExperimental demy-elination produced by the A59 strain of mouse hepatitis virusrdquoNeurology vol 34 no 5 pp 597ndash603 1984

[9] J D Sarma L Fu S T Hingley and E Lavi ldquoMouse hepatitisvirus type-2 infection in mice an experimental model systemof acute meningitis and hepatitisrdquo Experimental and MolecularPathology vol 71 no 1 pp 1ndash12 2001

[10] J Das Sarma L Fu S T Hingley M M C Lai and E LavildquoSequence analysis of the S gene of recombinant MHV-2A59coronaviruses reveals three candidate mutations associatedwith demyelination andhepatitisrdquo Journal ofNeuroVirology vol7 no 5 pp 432ndash436 2001

[11] D Chatterjee K Biswas S Nag S G Ramachandra and JD Sarma ldquoMicroglia play a major role in direct viral-induceddemyelinationrdquo Clinical and Developmental Immunology vol2013 Article ID 510396 12 pages 2013

[12] J Das Sarma L C Kenyon S T Hingley and K S ShindlerldquoMechanisms of primary axonal damage in a viral model ofmultiple sclerosisrdquo Journal of Neuroscience vol 29 no 33 pp10272ndash10280 2009

[13] W Penfield ldquoA method of staining oligodendroglia and mi-croglia (combined method)rdquoThe American Journal of Patholo-gy vol 4 no 2 pp 153ndash157 1928

[14] D S Russell ldquoIntravital staining of microglia with trypan bluerdquoThe American Journal of Pathology vol 5 no 5 pp 451ndash4581929

[15] M Gencic and M Oehmichen ldquoA modification of microgliaimpregnationrdquo Microscopica Acta vol 82 no 3 pp 201ndash2061979

[16] T Scott ldquoA silver impregnation method for reactive microgliain 1 120583m epoxy sectionsrdquo Acta Neuropathologica vol 46 no 1-2pp 155ndash158 1979

[17] H Kettenmann U K Hanisch M Noda and A VerkhratskyldquoPhysiology of microgliardquo Physiological Reviews vol 91 no 2pp 461ndash553 2011

[18] M Oehmichen H Wiethoelter and M Gencic ldquoCytochemicalmarkers for mononuclear phagocytes as demonstrated in reac-tive microglia and globoid cellsrdquoActa Histochemica vol 66 no2 pp 243ndash252 1980

[19] K S Shindler L C Kenyon M Dutt S T Hingley and J DasSarma ldquoExperimental optic neuritis induced by a demyelinat-ing strain of mouse hepatitis virusrdquo Journal of Virology vol 82no 17 pp 8882ndash8886 2008

[20] K S Shindler D Chatterjee K Biswas et al ldquoMacrophage-me-diated optic neuritis induced by retrograde axonal transport ofspike gene recombinant mouse hepatitis virusrdquo Journal of Neu-ropathology and Experimental Neurology vol 70 no 6 pp 470ndash480 2011

[21] H Kanazawa K Ohsawa Y Sasaki S Kohsaka and Y ImaildquoMacrophagemicroglia-specific protein Iba1 enhances mem-brane ruffling and Rac activation via phospholipase C-120574-dependent pathwayrdquo Journal of Biological Chemistry vol 277no 22 pp 20026ndash20032 2002

[22] G W Simmons W W Pong R J Emnett et al ldquoNeurofi-bromatosis-1 heterozygosity increases microglia in a spatiallyand temporally restricted pattern relevant to mouse opticglioma formation and growthrdquo Journal of Neuropathology andExperimental Neurology vol 70 no 1 pp 51ndash62 2011

[23] P Damier E C Hirsch P Zhang Y Agid and F Javoy-AgidldquoGlutathione peroxidase glial cells and Parkinsonrsquos diseaserdquoNeuroscience vol 52 no 1 pp 1ndash6 1993

[24] A Cagnin D J Brooks A M Kennedy et al ldquoIn-vivo meas-urement of activated microglia in dementiardquo The Lancet vol358 no 9280 pp 461ndash467 2001

[25] H Akiyama S Barger S Barnum et al ldquoInflammation andAlz-heimerrsquos diseaserdquo Neurobiology of Aging vol 21 no 3 pp 383ndash421 2000

[26] C K Glass K Saijo B Winner M C Marchetto and F HGage ldquoMechanisms Underlying Inflammation in Neurodegen-erationrdquo Cell vol 140 no 6 pp 918ndash934 2010

[27] T Wyss-Coray J D Loike T C Brionne et al ldquoAdult mouseastrocytes degrade amyloid-120573 in vitro and in siturdquo NatureMedicine vol 9 no 4 pp 453ndash457 2003

[28] M L Block and J S Hong ldquoChronic microglial activation andprogressive dopaminergic neurotoxicityrdquo Biochemical SocietyTransactions vol 35 no 5 pp 1127ndash1132 2007

[29] P L McGeer and E G McGeer ldquoGlial reactions in ParkinsonrsquosdiseaserdquoMovement Disorders vol 23 no 4 pp 474ndash483 2008

[30] T Nagatsu and M Sawada ldquoInflammatory process in Parkin-sonrsquos disease role for cytokinesrdquo Current PharmaceuticalDesign vol 11 no 8 pp 999ndash1016 2005

[31] P LMcGeer S Itagaki B E Boyes and EGMcGeer ldquoReactivemicroglia are positive for HLA-DR in the substantia nigra ofParkinsonrsquos and Alzheimerrsquos disease brainsrdquo Neurology vol 38no 8 pp 1285ndash1291 1988

[32] P LMcGeer andEGMcGeer ldquoInflammatory processes in am-yotrophic lateral sclerosisrdquoMuscle and Nerve vol 26 no 4 pp459ndash470 2002

10 ISRN Immunology

[33] L Cartier O Hartley M Dubois-Dauphin and K-H KrauseldquoChemokine receptors in the central nervous system role inbrain inflammation and neurodegenerative diseasesrdquo BrainResearch Reviews vol 48 no 1 pp 16ndash42 2005

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