Research Article MeHg Developing Exposure Causes DNA Double...

Research ArticleMeHg Developing Exposure Causes DNA Double-Strand Breaksand Elicits Cell Cycle Arrest in Spinal Cord Cells

Fabiana F Ferreira12 Dib Ammar13 Gilian F Bourckhardt1 Karoline Kobus-Bianchini4

Yara M R Muumlller1 and Evelise M Nazari1

1Departamento de Biologia Celular Embriologia e Genetica Centro de Ciencias Biologicas UFSC Campus UniversitarioTrindade 88040-900 Florianopolis SC Brazil2Instituto de Ciencias Naturais Humanas e Sociais UFMT Avenida Alexandre Ferronato 1200 Setor Industrial78557287 Sinop MT Brazil3Centro Universitario Catolica de Santa Catarina Rua Visconde de Taunay 427 Centro 89203-005 Joinville SC Brazil4Departamento de Fisioterapia Centro de Ciencias da Saude e do Esporte UDESC Rua Pascoal Simone 358Coqueiros 88080-350 Florianopolis SC Brazil

Correspondence should be addressed to Evelise M Nazari evelisenazariufscbr

Received 3 September 2015 Revised 22 November 2015 Accepted 23 November 2015

Academic Editor Lucio Guido Costa

Copyright copy 2015 Fabiana F Ferreira 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

The neurotoxicity caused by methylmercury (MeHg) is well documented however the developmental neurotoxicity in spinal cordis still not fully understood Here we investigated whether MeHg affects the spinal cord layers development Chicken embryos atE3 were treated in ovo with 01 120583g MeHg50 120583L saline solution and analyzed at E10 Thus we performed immunostaining usinganti-120574-H2AX to recognize DNA double-strand breaks and antiphosphohistone H3 anti-p21 and anti-cyclin E to identify cellsin proliferation and cell cycle proteins Also to identify neuronal cells we used anti-NeuN and anti-120573III-tubulin antibodiesAfter the MeHg treatment we observed the increase on 120574-H2AX in response to DNA damage MeHg caused a decrease in theproliferating cells and in the thickness of spinal cord layers Moreover we verified that MeHg induced an increase in the numberof p21-positive cells but did not change the cyclin E-positive cells A significantly high number of TUNEL-positive cells indicatingDNA fragmentation were observed inMeHg-treated embryos Regarding the neuronal differentiationMeHg induced a decrease inNeuN expression and did not change the expression of 120573III-tubulin These results showed that in ovoMeHg exposure alters spinalcord development by disturbing the cell proliferation and death also interfering in early neuronal differentiation

1 Introduction

Mercury is a metal of known toxic properties which occursnaturally and anthropogenically in the environment [1 2]Methylmercury (MeHg) is an organic chemical form ofmercury that has been widely studied due to its neurotoxiceffects in humans and animal models especially when theexposure occurs prenatally [3ndash6]

MeHg is able to cross the blood-brain barrier which isimmature in embryos and fetuses leading to an increasedvulnerability of the fetal brain to the toxic effects of thisorganicmercury [7ndash9]MoreoverMeHg tends to accumulatein fetuses due to their inability to excrete this metal [10]

Development of the central nervous system (CNS) is slowand gradual and involves the differentiation and migrationof neuronal and glial cells to organize cellular layers thatcomprise the brain and spinal cord Thus damage to thedeveloping CNS caused by MeHg can result in irreversiblemorphological and physiological impairments which com-promise postnatal motor coordination learning and mem-ory functions [3 11ndash14]

The effects of MeHg exposure on the CNS are relatedparticularly in adults to neurochemical changes that includedisturbances in the calcium and glutamate homeostasis[15 16] reactive oxygen species generation and oxidativestress in the brain of mammals [17ndash19] Additionally the

Hindawi Publishing CorporationJournal of ToxicologyVolume 2015 Article ID 532691 10 pageshttpdxdoiorg1011552015532691

2 Journal of Toxicology

mitochondrial dysfunction accompanied by the expressionof apoptotic proteins has also been identified as an effect ofMeHg-induced neurotoxicity [20ndash22]

Although neurotoxicity caused by MeHg is well docu-mented developmental neurotoxicity is still not fully under-stood A previous work has shown that in ovo MeHg expo-sure results in behavioral impairments such as anomalousmovements and low exploratory activity as well as mor-phological and biochemical changes including alterationsin the organization of the cerebellar cortical layers and theincrease of the antioxidant enzyme activity in the cerebellumof MeHg-exposed chicks [23] In this study we offer a newapproach to the exploration of developmental neurotoxicityinduced by MeHg using a chicken embryo as a model Theaim of this study was to investigate the impact of MeHgon the cellular layers of the spinal cord mainly focusing oncell proliferation and cell cycle The spinal cord was chosenbecause the cellular organization of its three layers is lesscomplex when compared to the brain and cerebellum whichmakes it easier to characterize the CNS cellular dynamicsand thus developmental neurotoxicity Moreover there is alack of knowledge about the developmental neurotoxicityinduced by MeHg in spinal cord because the most studiedimpairments caused by this metal mainly relate to the brainand cerebellum

2 Materials and Methods

21 Eggs and Embryos Fertilized eggs of Gallus domesticuswere obtained from a commercial hatchery (Tyson FoodsBrasil Ltda Brazil) The eggs were weighed (666 plusmn 47 g)and transferred to an incubator at 375ndash380∘C and 650humidity Prenatal acute MeHg exposure was performed atembryonic day 3 (E3) that is 20 HH stage series [24] Theembryos received a single dose of 01 120583g of methylmercuryII chloride (Sigma-Aldrich USA) diluted in 50120583L of salinesolution administered into the yolk sac near the vitellinvessels Untreated control embryos received only 50 120583L ofsaline solution The dose of MeHg used in this study wasdetermined according to Heinz et al [25 26] and on thebasis of a previous study performed by Carvalho et al [23]After treatment each egg was returned to the incubator andembryos were monitored daily in ovo up to embryonic day 10(E10) that is 36 HH At E10 the embryos were anesthetizedby cooling to 4∘C for 15ndash20min removed from the egg shelland washed in saline solution All experiments were carriedout according to the Ethics Committee for Animal Researchof the Universidade Federal de Santa Catarina FlorianopolisBrazil (Approval number 355CEUAUFSC)

22 Routine Microscopy Techniques and Morphometry of theSpinal Cord Whole embryos at E10 (119899 = 7 embryos pergroup of three independent experiments) were fixed in 4formaldehyde and the trunk region was dissected embeddedin paraffin and cut into serial transversal sections (6120583m)The sections were stained with hematoxylin-eosin (HE) toanalyze the spinal cord morphology and to determine thedistribution and morphology of cells in the ependymal

mantle and marginal layers The thicknesses of the ependy-mal mantle and marginal layers were measured using amorphometric eyepiece (Olympus USA) (200x) in midlineregion of the spinal cord

23 Autometallography Method We evaluated mercurydeposition by the autometallography (AMG) methodin which dewaxed sections were immersed in the AMGdeveloper solution (60 gum arabic 10 potassium citratebuffer 30 hydroquinone and 05 silver nitrate) indarkness for 60min Next the spinal cord sections (119899 = 7embryos per group of three independent experiments) werewashed with tap water and counterstained with hematoxylinMercury deposition was evaluated as brown stained cellswhich represent silver surrounding the deposited mercury[27] and classified as absent (minus) mild (+) moderate (++)and intense (+++) by an investigator who was blind to thetreatment assignments according to Muller et al [28]

24 Immunohistochemistry To evaluate the effect of prenatalacute MeHg exposure we first looked for proliferation cellcycle and DNA damage in the spinal cord using antibodiesrabbit anti-phosphohistone H3 IgG (1 250 Upstate USA)rabbit anti-cyclin E IgG (1 100 Santa Cruz BiotechnologyUSA) mouse anti-p21 IgG (1 100 Santa Cruz Biotech-nology USA) and rabbit anti-120574-H2AX IgG (1 50 SantaCruz Biotechnology USA) Next we investigated neuronaldifferentiation using antibody mouse anti-neuronal nucleiNeuN IgG (1 100 Chemicon International USA) andmouseanti-120573III tubulin IgG (1 100 Promega USA) (119899 = 7 embryosper group of three independent experiments) Endogenousperoxidase activity was stoppedwith 03 hydrogen peroxideinmethanolThe sectionswerewashedwith 01Mphosphate-buffered saline (PBS) pH 74 + 03 Triton X-100 and thenblocked with 5 fetal bovine serum (FBS) in PBS The sec-tions were incubated overnight at 4∘C with primary antibod-ies washed with PBS and then incubated for 90min at roomtemperature with peroxidase-conjugated secondary antibodyanti-rabbit IgG (1 400 Sigma USA) and biotin-conjugatedantibodies including anti-rabbit IgG (1 200 Sigma USA)and anti-mouse IgG (1 200 Sigma USA) Next the sectionswere washed in 01M PBS and binding sites of antibodieswere revealed with 331015840-diaminobenzidine (DAB) (Sigma-Aldrich USA) For immunofluorescence Alexa Fluor 488goat anti-mouse IgG (1 200 Life Technologies USA) andAlexa Fluor 568 goat anti-rabbit IgG (1 100 Life Technolo-gies USA) were used Next the sections were incubatedwith 46-diamidino-2-phenylindole dihydrochloride (DAPI)(Sigma-Aldrich USA) for 3min at room temperature Nega-tive controls of immunohistochemical reaction were treatedin the same way except that the primary antibodies werereplaced with 01M PBS

25 TUNEL Assay We used TdT-mediated dUTP nick endlabeling (TUNEL) staining to identify apoptotic cells inthe dewaxed spinal cord sections (119899 = 7 embryos pergroup of three independent experiments) TUNEL stainingwas conducted with a TdT-FragEL DNA Fragmentation

Journal of Toxicology 3

Detection Kit (Calbiochem USA) according to the manu-facturerrsquos instructions TUNEL-stained cells displayed dark-brown precipitates in their nuclei

26 Quantitative Analysis To quantify immunoreactive cells(phosphohistone H3 and NeuN) and TUNEL-positive cellsstereological analysis was performed using the M-42 testsystem (Weibel number 2 Tonbridge UK) (200x) Thenumerical density per area (NA) of the cells was determinedaccording to Mandarim-de-Lacerda [29] Five random fieldsof the spinal cord were counted for each section (3 sectionsper embryo 7 embryos per group of three independent exper-iments) In order to quantify the 120574-H2AX-positive cells theintegrated density of pixels of the fluorescence digital imageswas determined using Image J software (NIH Image) A scalebar was determined and the measurement of the integrateddensity of pixels was determined in a 359977120583m2 frame

27 Flow Cytometry Dissected and unfixed spinal cords werehomogenized and submitted to consecutive washes with01M PBS pH 78 (119899 = 7 embryos per group of threeindependent experiments) Then the cells were dissociatedusing 025 trypsin for 15min at 37∘C and added to 5 FBSunder agitation Subsequently the samples were centrifugedat 640timesg for 10min and the supernatant was collected [30]incubated with primary antibodies rabbit anti-cyclin E IgG(1 1000 Santa Cruz Biotechnology USA) mouse anti-p21IgG (1 1000 Santa Cruz Biotechnology USA) and mouseanti-120573III tubulin IgG (1 1000 Promega USA) for 1 h andthen incubated for 45min with the secondary antibodiesAlexa Fluor 568 anti-rabbit IgG and Alexa Fluor 488 anti-mouse IgG (1 1000 Life Technologies USA) Analyses wereseparately conducted for each antibody in each treatmentPreviously a run with unstained cells was performed todetermine the gates of cells of interest Additionally propid-ium iodide was used to refine the gates of interest Thusfrom the dot plot with 20000 events and considering theparameters side scatter (SSC-A) and forward scatter (FSC-A)the gates with 1800 events were determined FACSCanto IIflow cytometer (BD Biosciences Canada) was used for theanalysis The values are presented in absolute count

28 Statistical Analysis Quantitative data were analyzedusing Statistica 100 for Windows The differences betweenMeHg-treated and untreated control embryos were evaluatedby Studentrsquos unpaired t-test All data were expressed as meanplusmn SEM and 119875 lt 005 was considered statistically significant

3 Results

31MercuryDeposition in the Spinal Cord TheAMGmethodwas used to show that a single injection of 01 120583g MeHg intothe yolk sac could reach the embryo and its spinal cord Asexpected the mercury deposition was evident in all MeHg-treated embryos butwas not observed in the control embryosThe MeHg deposition was recognized in three spinal cordlayers and a greater intensity of deposition was found in theependymal and mantle layers (Figure 1)

32 Spinal Cord Morphology and Morphometry Consid-ering the fact that the mercury was incorporated in theembryonic tissues we evaluated the general features of thespinal cord Similar features of the cell morphology anddistribution in the ependymal mantle and marginal layerswere observed between MeHg-treated and control embryos(Figure 2) However MeHg treatment caused a significantdecrease in the thickness of the mantle layer (17201 120583m plusmn741) when compared to control embryos (24819 120583mplusmn2148119875 lt 001)The same effect was observed in the marginal layerof MeHg-treated embryos (9203 120583m plusmn 412) in comparisonto controls (12774 120583mplusmn 1474 119875 lt 005) Only the thicknessof the ependymal layer did not change after MeHg treatmentbetween the control (1894 120583m plusmn 114) and MeHg-treatedembryos (2077 120583m plusmn 038 119875 gt 005)

33 Effect of Mercury on Proliferation and Cell Cycle On thebasis of the reduced size of the spinal cord caused by MeHgexposure the next step was evaluating the proliferation andcell cycle which are essential for development progress A fewproliferating cells were observed in all embryos and aftertreatment MeHg caused a decrease in the number of thesecells per area In the ependymal layer the NA of proliferatingcells was significantly lower in MeHg-treated embryos (1375cellsmm2 plusmn 73) when compared to controls (5501 cellsmm2plusmn 137 119875 lt 005) In the mantle layer the highest numberof proliferating cells was observed in the control (6017cellsmm2 plusmn 104) in comparison to MeHg-treated embryos(2182 cellsmm2 plusmn 86 119875 lt 005) No significant differencewas observed in themarginal layer between the control (3264cellsmm2plusmn 102) andMeHg-treated embryos (205 cellsmm2plusmn 105) (Figure 3)

Moreover we examined the proteins p21 and cyclin Ethat play a crucial role in the progression of cell cycle MeHgcaused an increase in the absolute number of p21-positivecells in the spinal cord of treated embryos (47333 cells plusmn6240) when compared to controls (10067 cells plusmn 1747 119875 lt005) However no changes were observed in the number ofcyclin E-positive cells between MeHg-treated (1027 cells plusmn152) and control embryos (1450 cells plusmn 5528 119875 gt 005)Figure 4 display the immunolocalization and percentage ofp21 and cyclin E-positive cells in MeHg-treated and controlembryos

34 DNA Damage and Apoptosis In order to verify if MeHgcauses DNA damage the expression of 120574-H2AX proteinin response to DNA double-strand breaks was examinedAfter treatment MeHg caused an increase in the expressionof 120574-H2AX (5374089 plusmn 783414 120583m2) when compared tothe control embryos (3447389 plusmn 67097 120583m2 119875 lt 005)These anti-120574-H2AX-positive cells were found mainly in thetransition zone between the ependymal and mantle layers ofthe MeHg-treated embryos (Figure 5)

Regarding the increase of DNA damage we investigatedthe occurrence of apoptotic cells after MeHg treatmentFew apoptotic cells were observed in the spinal cord ofthe control and MeHg-treated embryos A significantly highNA of TUNEL-positive cells was observed in the mantle


e mt mg

100120583m

(a)

Con

trol

MeH

g

Ependymal Mantle Marginal

Control

MeHg


minus

++

minus

++

minus

+

10120583m

(b)

Figure 1 Mercury deposition in the spinal cord of E10 control and MeHg-treated embryos examined by the AMG method Spinal cordshowing ependymal (e) mantle (mt) and marginal (mg) layers in low magnification (a) and high magnification (b) The brown colorcorresponds to the mercury deposits in cells (arrows) in MeHg-treated embryos The accompanying table displays the intensity of mercurydeposition in the cells of the spinal cord layers classified as absent (minus) mild (+) and moderate (++)

e mt mg e mt mg

Control MeHg

ControlMeHg

Mantle MarginalEpendymal

lowast

lowastlowast

Thic

knes

s(120583

m)

0

100

200

300

Figure 2 Thickness of the spinal cord layers in embryos at E10 Observe the evident difference in the size of the spinal cord between thecontrol and MeHg-treated embryos The graph displays the effect of MeHg treatment on the ependymal mantle and marginal layers Dataare expressed as mean plusmn SEM lowast119875 lt 005 and lowastlowast119875 lt 001 e ependymal layer mg marginal layer and mt mantle layer Scale bar 10 120583m


ControlMeHg

lowast

lowast

Con

trol

MeH

g



NA

(pH

H3

-pos

itive

cells

) (m

m2)

0

20

40

60

80

Figure 3 Effects of MeHg on the spinal cord layers of embryos at E10 Proliferating cells labeled with anti-phosphohistone H3 (arrows) wereobserved in the ependymal mantle and marginal layers of the control and MeHg-treated embryos The square in the ependymal controlembryo represents the negative control of immunohistochemical reaction The accompanying graph displays the NA of proliferating cellsobtained by stereological analysis in each layer of the spinal cord Data are presented as mean plusmn SEM lowast119875 lt 005 Scale bar 10 120583m

layer of MeHg-treated embryos (1273 cellsmm2 plusmn 36) whencompared to controls (382 cellsmm2 plusmn 21 119875 lt 005)However no significant differences were found between thecontrol (309 cellsmm2 plusmn 15) and MeHg-treated embryos(205 cellsmm2 plusmn 12) in the marginal layer Additionally noTUNEL-positive cells were observed in the ependymal layerof either the control or MeHg-treated embryos (Figure 6)

35 Effect ofMercury onNeuronalDifferentiation We investi-gated whether MeHg compromises the neuronal differentia-tion in the developing spinal cord considering that the majoreffects of MeHg were observed mainly in the mantle layerwhere intense cell differentiation occurs Immunofluores-cence using anti-120573III-tubulin antibody revealed the expres-sion of this protein in the mantle and marginal layers at E10After treatment no changes were observed in the expressionof 120573III-tubulin between the control (4240 cells plusmn 4091)and MeHg-treated embryos (51733 cells plusmn 185 119875 gt 005)(Figure 7) Additionally when we analyze the expressionof NeuN recognized in postmitotic neurons andor duringneuronal differentiation a significantly decrease in the NAof NeuN-positive cells was observed in the mantle layerof MeHg-treated embryos (1432 cellsmm2 plusmn 41) whencompared to controls (6455 cellsmm2 plusmn 55 119875 lt 00001)(Figure 8)

4 Discussion

The neurotoxicity of MeHg is a well-known phenomenonand the present study contributes new data to improve thecurrent understanding of the embryonic cell responses aftera single in ovo injection of 01 120583g MeHg Although this dosedid not affect the overall morphology of the spinal cord wedemonstrated here that it is related to a reduction in thickness

of the spinal cord layers as well as to impairments in cell cycleproteins and in early neuronal differentiation

In fact in ovo development is a good model for toxicitystudies because embryos develop in the absence of maternalfactors which may compromise the assay results The metalinjection in the yolk sac is effective for neurodevelopmentaltoxicology and these assays have been validated in our previ-ous studies with lead acetate andMeHg [23 28 31]Moreoverthe experimental design is also important particularly theestablishment of the exposure time Preliminary tests (datanot shown) using a shorter exposure times than the 7 daysadopted here were not sufficient for the incorporation ofMeHg by embryos Additionally considering the exposuretime the choice of treatment and analysis ages are equallyessential

In this study we exposed embryos in earlier stage ofdevelopment of CNS (E3) soon after the neural tube closureAt this stage neural tube is composed by a nondifferentiatedneuroepithelium Then to understand the neurodevelop-mental toxicity of MeHg cellular analyses were performedat E10 when the layers of the spinal cord are distinguishedand composed by neuronal and glial differentiated cells Theused time gap between exposure and analyses was calculatedin order to assess the period of vulnerability of embryosand then to assess the effects of MeHg on essential cellmechanisms which are inherent to development of spinalcord

Our results showed that MeHg causes a reduction in thethickness of the layers reflecting the neurotoxicity of thismetal in the spinal cord tissue Carvalho et al [23] demon-strated the deposition ofMeHg in the layers of the cerebellumof chicken embryos and also showed morphological changesin the Purkinje layer in the first postnatal week Studiesabout MeHg poisoning have shown the effects of MeHg onthe cytoarchitecture of CNS layers in humans and animals


Ependymal

Fluorescence intensity


Control 559MeHg 2546

Control 806MeHg 570

Mantle MarginalC

ontro

lM

eHg

Con

trol

p21

Cycli

n E

MeH

g

Figure 4 Cell cycle proteins analyzed by immunohistochemistry and flow cytometry Immunohistochemistry revealed p21 and cyclin E-positive cells (arrows) in the control and MeHg-treated embryos The square in the ependymal control embryos represents the negativecontrol of immunohistochemical reaction The graphs display the expression profile and relative frequency of positive cells of p21 and cyclinE in the spinal cord of the control andMeHg-treated embryos For each treatment 1800 events were analyzed per antibody Scale bars 10 120583m

affecting the disposition and number of neurons as well ason the size of the brain and cerebellum [6 7 32ndash35]

Regarding the effect on the morphology of CNS layerswe tested the hypothesis that MeHg causes impairments incell proliferation an essential mechanism of developmentThen we analyzed more specifically some proteins involvedin cell cycle in order to better comprehend the cellular basisof MeHg toxicity Our data showed a significant reductionin the number of proliferating neural cells in the ependymaland mantle layers Neural cell proliferation was disturbedby MeHg as demonstrated by in vitro and in vivo assays[14 35 36] It was also demonstrated that MeHg affects theproliferation in all regions of the developing CNS such as thespinal cord in Xenopus laevis andDanio rerio [37 38] as wellas in the murine brain and cerebellum [34 39 40]

The idea that MeHg compromises the cell proliferationwas explored in classic works that focused on the mitosis

inhibition related to the disruption of G1 and G2 progress[7 34 39] Data about the expression of regulatory moleculesinvolved in cell cycle checkpoints such as p21 and cyclin havebeen demonstratedmainly in the brain [14] and hippocampal[35] and cerebral cortex [40 41] p21 protein plays a centralrole in cell cycle arrest in response to DNA damage byinhibiting the initiation of replication in the S phase Asexpected our results showed an increase in the expressionof p21 in the ependymal and mantle layers after MeHgexposureOu et al [42] and Faustman et al [43] also observedthis behavior of p21 in neural cells after MeHg treatmentHowever here we found the association of a decrease incell proliferation with an increase of p21 expression in thedeveloping spinal cord Taken together these data suggestthe cellular impairment caused by MeHg and the fact thatthis organometal causes the arrest of the cell cycle in G1Interestingly regarding the interactive role of cyclin E and


120574-H2AXDAPI Merge

lowast

0

20000

40000

60000

80000

Con

trol

MeH

g

Control MeHg

(pix

el120583

m2 )

Figure 5 DNA double-strand breaks labeled with 120574-H2AX antibody analyzed by immunohistochemistry 120574-H2AX-positive cells (arrows)were observed inMeHg-treated embryosThe graph displays the expression profile of 120574-H2AX in the spinal cord of control andMeHg-treatedembryos Integrated density of pixels of the fluorescence of the 120574-H2AX was determined and data are expressed as mean plusmn SEM lowast119875 lt 005Scale bar 10 120583m

ControlMeHg



lowast

NA

(TU

NEL

-pos

itive

cell)

(mm

2 )

0

5

10

15

20

Con

trol

MeH

g

Figure 6 Apoptotic cells in the spinal cord of chicken embryos at E10 recognized by the TUNELmethodMeHg-treated and control embryosshowed apoptotic cells (arrows) in the mantle andmarginal layersThe accompanying graph displays the NA of TUNEL-positive cells in eachspinal cord layer Data are presented as mean plusmn SEM lowast119875 lt 005 Scale bar 10 120583m

p21 the decrease of cyclin E was expected after the MeHgtreatment as demonstrated by Burke et al [14] Falluel-Morelet al [35] and Xu et al [40] In fact cyclin E has beenidentified as a target ofMeHg reducing its expression in braindevelopment On the other hand our data showed thatMeHgdid not change the cyclin E expression in the spinal cord ofchicken embryos

The upregulation of p21 is required in response to DNAdamage Indeed using 120574-H2AX as amarker ofDNAdamagewe verified the occurrence of DNA double-strand breaks inthe spinal cord of MeHg-treated embryos demonstratingthe genotoxic effect of this metal The DNA damage maycause sequences of intracellular signaling that contribute tothe upregulation of p21 and unrepaired damage may causesignaling to apoptosis Here we found that MeHg inducesapoptosis recognized byDNA fragmentation Apoptotic cells

were observed mainly in the mantle layer the same layerwhere 120574-H2AX-positive cells were found This combinationof data reinforces the argument that the toxicity of MeHgseems to activate the signaling cascade of programmed celldeath or apoptosis in both adult [44ndash46] and developing CNS[6 14 22 35 38] Moreover we proposed that the associationbetween decreased proliferation and increased apoptosismayact as one cause of the reduction in the thickness of thespinal cord layers Additionally this impairment can progressduring embryonic development as well as in childhood andadulthood phases

Considering the fact that the mantle layer appears bethe more affected by MeHg and also that neurons are wellrecognized in this layer at the embryonic age evaluated weinvestigated whether MeHg interferes in the expression of120573-tubulin III a marker of differentiated neurons In spite


DAPI MergeC

ontro

lM

eHg

120573III-tubulin



Figure 7 120573III-tubulin protein analyzed by immunohistochemistry and flow cytometry 120573III-tubulin-positive cells (white arrows) observedmainly in the mantle layer The graph displays the expression profile and relative frequency of 120573III-tubulin in the spinal cord of the controland MeHg-treated embryos Scale bar 20 120583m

Control MeHg

Control MeHg

lowastlowastlowast

NA

(Neu

N-p

ositi

ve ce

lls) (

mm

2 )

0

20

40

60

80

Figure 8 NeuN a neuron-specific nuclear protein analyzed by immunohistochemistry Positive cells (arrows) were found in the mantlelayerThe square in the control image represents the negative control of immunohistochemical reactionThe graph displays the NA of NeuN-positive cells Bars are represented as mean plusmn SEM lowastlowastlowast119875 lt 00001 Scale bars 10 120583m

of our expectation the dose used did not compromise thisprotein However when we analyze the expression of NeuNa neuron-specific nuclear protein which is recognized inpostmitotic neurons andor during neuronal differentiationwe observed a significant decrease on the expression of thisprotein Our results showed that MeHg affects differentiallythe neuron maturation in the same embryonic stage Thiscan be explained considering that to organize the spinal cordlayers during development the cells need to differentiate andmigrate in different rhythms Thus in the same embryonicstage we found both early and late phases of neurogenesisIn general our results provide new insights in attemptto contribute to better understanding the cellular basis ofcomplex MeHg neurotoxicity in developing spinal cord

5 Conclusion

The basis of how MeHg acts during the spinal cord develop-ment is incompletely described From toxicological point ofview these results are very important because they showedfor the first time that in ovo MeHg exposure alters spinal

cord development by causing DNA double-strand breaksand also disturbing the mechanisms of proliferation and celldeath differentially interfering in early and late neurogenesisphases

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Evelise M Nazari and Yara M R Muller contributed equallyto this work

References

[1] FMMMorel AM L Kraepiel andMAmyot ldquoThe chemicalcycle and bioaccumulation of mercuryrdquo Annual Review ofEcology and Systematics vol 29 no 1 pp 543ndash566 1998


[2] R A Bernhoft ldquoMercury toxicity and treatment a review of theliteraturerdquo Journal of Environmental andPublicHealth vol 2012Article ID 460508 10 pages 2012

[3] P Grandjean P Weihe R F White et al ldquoCognitive deficit in7-year-old children with prenatal exposure to methylmercuryrdquoNeurotoxicology and Teratology vol 19 no 6 pp 417ndash428 1997

[4] A Kakita K Wakabayashi M Su M Sakamoto F Ikutaand H Takahashi ldquoDistinct pattern of neuronal degenerationin the fetal rat brain induced by consecutive transplacentaladministration of methylmercuryrdquo Brain Research vol 859 no2 pp 233ndash239 2000

[5] M Bertossi F Girolamo M Errede et al ldquoEffects ofmethylmercury on the microvasculature of the developingbrainrdquo Neurotoxicology vol 25 no 5 pp 849ndash857 2004

[6] M Sakamoto A Kakita R B De Oliveira H Sheng Panand H Takahashi ldquoDose-dependent effects of methylmercuryadministered during neonatal brain spurt in ratsrdquoDevelopmen-tal Brain Research vol 152 no 2 pp 171ndash176 2004

[7] P M Rodier M Aschner and P R Sager ldquoMitotic arrest inthe developing CNS after prenatal exposure to methylmercuryrdquoNeurobehavioral Toxicology and Teratology vol 6 no 5 pp379ndash385 1984

[8] M Aschner and J L Aschner ldquoMercury neurotoxicity mech-anisms of blood-brain barrier transportrdquo Neuroscience andBiobehavioral Reviews vol 14 no 2 pp 169ndash176 1990

[9] K Yurdakok ldquoEnvironmental pollution and the fetusrdquo Journalof Pediatric and Neonatal Individualized Medicine vol 1 no 1pp 33ndash42 2012

[10] S Bose-OrsquoReilly K MMcCarty N Steckling and B LettmeierldquoMercury exposure and childrenrsquos healthrdquo Current Problems inPediatric and Adolescent Health Care vol 40 no 8 pp 186ndash2152010

[11] M Farina J B T Rocha and M Aschner ldquoMechanisms ofmethylmercury-induced neurotoxicity evidence from experi-mental studiesrdquoLife Sciences vol 89 no 15-16 pp 555ndash563 2011

[12] E Patel and M Reynolds ldquoMethylmercury impairs motorfunction in early development and induces oxidative stress incerebellar granule cellsrdquo Toxicology Letters vol 222 no 3 pp265ndash272 2013

[13] K Sokolowski M Obiorah K Robinson E Mccandlish BBuckley and E Dicicco-Bloom ldquoNeural stem cell apoptosisafter low-methylmercury exposures in postnatal hippocampusproduce persistent cell loss and adolescent memory deficitsrdquoDevelopmental Neurobiology vol 73 no 12 pp 936ndash949 2013

[14] K Burke Y Cheng B Li et al ldquoMethylmercury elicits rapidinhibition of cell proliferation in the developing brain anddecreases cell cycle regulator cyclin ErdquoNeuroToxicology vol 27no 6 pp 970ndash981 2006

[15] M Aschner C P Yao J W Allen and K H Tan ldquoMethylmer-cury alters glutamate transport in astrocytesrdquo NeurochemistryInternational vol 37 no 2-3 pp 199ndash206 2000

[16] T L Limke S R Heidemann andW D Atchison ldquoDisruptionof intraneuronal divalent cation regulation by methylmercuryare specific targets involved in altered neuronal developmentand cytotoxicity in methylmercury poisoningrdquo NeuroToxicol-ogy vol 25 no 5 pp 741ndash760 2004

[17] J L Franco T Posser P R Dunkley et al ldquoMethylmercuryneurotoxicity is associated with inhibition of the antioxidantenzyme glutathione peroxidaserdquo Free Radical Biology andMedicine vol 47 no 4 pp 449ndash457 2009

[18] M Farina M Aschner and J B T Rocha ldquoOxidative stress inMeHg-induced neurotoxicityrdquo Toxicology and Applied Pharma-cology vol 256 no 3 pp 405ndash417 2011

[19] M Polunas A Halladay R B Tjalkens M A Philbert HLowndes and K Reuhl ldquoRole of oxidative stress and themitochondrial permeability transition in methylmercury cyto-toxicityrdquo NeuroToxicology vol 32 no 5 pp 526ndash534 2011

[20] C Tamm J Duckworth O Hermanson and S CeccatellildquoHigh susceptibility of neural stem cells to methylmercurytoxicity effects on cell survival and neuronal differentiationrdquoJournal of Neurochemistry vol 97 no 1 pp 69ndash78 2006

[21] T-H Lu S-Y Hsieh C-C Yen et al ldquoInvolvement ofoxidative stress-mediated ERK12 and p38 activation regulatedmitochondria-dependent apoptotic signals in methylmercury-induced neuronal cell injuryrdquo Toxicology Letters vol 204 no 1pp 71ndash80 2011

[22] K Sokolowski A Falluel-Morel X Zhou and E DiCicco-Bloom ldquoMethylmercury (MeHg) elicits mitochondrial-dependent apoptosis in developing hippocampus and acts atlow exposuresrdquo NeuroToxicology vol 32 no 5 pp 535ndash5442011

[23] M C Carvalho E M Nazari M Farina and Y M R MullerldquoBehavioral morphological and biochemical changes afterin ovo exposure to methylmercury in chicksrdquo ToxicologicalSciences vol 106 no 1 pp 180ndash185 2008

[24] V Hamburger and H L Hamilton ldquoA series of normal stages inthe development of the chick embryordquo Journal of Morphologyvol 88 no 1 pp 49ndash92 1951

[25] G H Heinz D J Hoffman S L Kondrad and C A ErwinldquoFactors affecting the toxicity of methylmercury injected intoeggsrdquoArchives of Environmental Contamination and Toxicologyvol 50 no 2 pp 264ndash279 2006

[26] G H Heinz D J Hoffman J D Klimstra K R Stebbins S LKondrad andC A Erwin ldquoSpecies differences in the sensitivityof avian embryos to methylmercuryrdquoArchives of EnvironmentalContamination and Toxicology vol 56 no 1 pp 129ndash138 2009

[27] G Danscher ldquoAutometallography a new technique for lightand electron microscopic visualization of metals in biologicaltissues (gold silver metal sulphides and metal selenides)rdquoHistochemistry vol 81 no 4 pp 331ndash335 1984

[28] Y M R Muller K Kobus J C Schatz D Ammar and EM Nazari ldquoPrenatal lead acetate exposure induces apoptosisand changesGFAP expression during spinal cord developmentrdquoEcotoxicology and Environmental Safety vol 75 no 1 pp 223ndash229 2012

[29] C A Mandarim-de-Lacerda ldquoStereological tools in biomedicalresearchrdquo Anais da Academia Brasileira de Ciencias vol 75 no4 pp 469ndash486 2003

[30] G F Bourckhardt M S Cecchini D Ammar K Kobus-Bianchini Y M R Muller and E M Nazari ldquoEffects ofhomocysteine on mesenchymal cell proliferation and differen-tiation during chondrogenesis on limb developmentrdquo Journal ofApplied Toxicology vol 35 pp 1390ndash1397 2015

[31] Y M R Muller L B D Rivero M C Carvalho K Kobus MFarina and E M Nazari ldquoBehavioral impairments related tolead-induced developmental neurotoxicity in chicksrdquo Archivesof Toxicology vol 82 no 7 pp 445ndash451 2008

[32] H Matsumoto G Koya and T Takeuchi ldquoFetal Minamatadisease a neuropathological study of two cases of intrauterineintoxication by a methyl mercury compoundrdquo Journal of Neu-ropathology and Experimental Neurology vol 24 no 4 pp 563ndash574 1965


[33] B H Choi L W Lapham L Amin-Zaki and T SaleemldquoAbnormal neuronal migration deranged cerebral corticalorganization and diffuse white matter astrocytosis of humanfetal brain amajor effect of methylmercury poisoning in uterordquoJournal of Neuropathology and Experimental Neurology vol 37no 6 pp 719ndash733 1978

[34] P R Sager R A Doherty and P M Rodier ldquoEffects ofmethylmercury on developing mouse cerebellar cortexrdquo Exper-imental Neurology vol 77 no 1 pp 179ndash193 1982

[35] A Falluel-Morel K Sokolowski H M Sisti X Zhou TJ Shors and E DiCicco-Bloom ldquoDevelopmental mercuryexposure elicits acute hippocampal cell death reductions inneurogenesis and severe learning deficits during pubertyrdquoJournal of Neurochemistry vol 103 no 5 pp 1968ndash1981 2007

[36] S Ceccatelli R Bose K Edoff N Onishchenko and S SpulberldquoLong-lasting neurotoxic effects of exposure to methylmercuryduring developmentrdquo Journal of Internal Medicine vol 273 no5 pp 490ndash497 2013

[37] S A Hassan E A Moussa and L C Abbott ldquoThe effectof methylmercury exposure on early central nervous systemdevelopment in the zebrafish (Danio rerio) embryordquo Journal ofApplied Toxicology vol 32 no 9 pp 707ndash713 2012

[38] R W Huyck M Nagarkar N Olsen S E Clamons and MS Saha ldquoMethylmercury exposure during early Xenopus laevisdevelopment affects cell proliferation and death but not neuralprogenitor specificationrdquo Neurotoxicology and Teratology vol47 pp 102ndash113 2015

[39] R A Ponce T J Kavanagh N K Mottet S G Whittakerand E M Faustman ldquoEffects of methyl mercury on the cellcycle of primary rat CNS cells in vitrordquo Toxicology and AppliedPharmacology vol 127 no 1 pp 83ndash90 1994

[40] M Xu C Yan Y Tian X Yuan and X Shen ldquoEffects of lowlevel of methylmercury on proliferation of cortical progenitorcellsrdquo Brain Research vol 1359 pp 272ndash280 2010

[41] M Fujimura and F Usuki ldquoLow concentrations of methylmer-cury inhibit neural progenitor cell proliferation associated withup-regulation of glycogen synthase kinase 3120573 and subsequentdegradation of cyclin E in ratsrdquo Toxicology and Applied Phar-macology vol 288 no 1 pp 19ndash25 2015

[42] Y C Ou S A Thompson R A Ponce J Schroeder T JKavanagh and EM Faustman ldquoInduction of the cell cycle reg-ulatory gene p21 (waf1 cip1) followingmethylmercury exposurein vitro and in vivordquo Toxicology and Applied Pharmacology vol157 no 3 pp 203ndash212 1999

[43] E M Faustman R A Ponce Y C Ou M A C Men-doza T Lewandowski and T Kavanagh ldquoInvestigations ofmethylmercury-induced alterations in neurogenesisrdquo Environ-mental Health Perspectives vol 110 no 5 pp 859ndash864 2002

[44] M Kunimoto ldquoMethylmercury induces apoptosis of rat cere-bellar neurons in primary culturerdquo Biochemical and BiophysicalResearch Communications vol 204 no 1 pp 310ndash317 1994

[45] A F Castoldi S Barni I Turin C Gandini and L ManzoldquoEarly acute necrosis delayed apoptosis and cytoskeletal break-down in cultured cerebellar granule neurons exposed tomethylmercuryrdquo Journal of Neuroscience Research vol 59 no6 pp 775ndash787 2000

[46] S Ceccatelli E Dare and M Moors ldquoMethylmercury-inducedneurotoxicity and apoptosisrdquo Chemico-Biological Interactionsvol 188 no 2 pp 301ndash308 2010

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


mitochondrial dysfunction accompanied by the expressionof apoptotic proteins has also been identified as an effect ofMeHg-induced neurotoxicity [20ndash22]

Although neurotoxicity caused by MeHg is well docu-mented developmental neurotoxicity is still not fully under-stood A previous work has shown that in ovo MeHg expo-sure results in behavioral impairments such as anomalousmovements and low exploratory activity as well as mor-phological and biochemical changes including alterationsin the organization of the cerebellar cortical layers and theincrease of the antioxidant enzyme activity in the cerebellumof MeHg-exposed chicks [23] In this study we offer a newapproach to the exploration of developmental neurotoxicityinduced by MeHg using a chicken embryo as a model Theaim of this study was to investigate the impact of MeHgon the cellular layers of the spinal cord mainly focusing oncell proliferation and cell cycle The spinal cord was chosenbecause the cellular organization of its three layers is lesscomplex when compared to the brain and cerebellum whichmakes it easier to characterize the CNS cellular dynamicsand thus developmental neurotoxicity Moreover there is alack of knowledge about the developmental neurotoxicityinduced by MeHg in spinal cord because the most studiedimpairments caused by this metal mainly relate to the brainand cerebellum

2 Materials and Methods

21 Eggs and Embryos Fertilized eggs of Gallus domesticuswere obtained from a commercial hatchery (Tyson FoodsBrasil Ltda Brazil) The eggs were weighed (666 plusmn 47 g)and transferred to an incubator at 375ndash380∘C and 650humidity Prenatal acute MeHg exposure was performed atembryonic day 3 (E3) that is 20 HH stage series [24] Theembryos received a single dose of 01 120583g of methylmercuryII chloride (Sigma-Aldrich USA) diluted in 50120583L of salinesolution administered into the yolk sac near the vitellinvessels Untreated control embryos received only 50 120583L ofsaline solution The dose of MeHg used in this study wasdetermined according to Heinz et al [25 26] and on thebasis of a previous study performed by Carvalho et al [23]After treatment each egg was returned to the incubator andembryos were monitored daily in ovo up to embryonic day 10(E10) that is 36 HH At E10 the embryos were anesthetizedby cooling to 4∘C for 15ndash20min removed from the egg shelland washed in saline solution All experiments were carriedout according to the Ethics Committee for Animal Researchof the Universidade Federal de Santa Catarina FlorianopolisBrazil (Approval number 355CEUAUFSC)

22 Routine Microscopy Techniques and Morphometry of theSpinal Cord Whole embryos at E10 (119899 = 7 embryos pergroup of three independent experiments) were fixed in 4formaldehyde and the trunk region was dissected embeddedin paraffin and cut into serial transversal sections (6120583m)The sections were stained with hematoxylin-eosin (HE) toanalyze the spinal cord morphology and to determine thedistribution and morphology of cells in the ependymal

mantle and marginal layers The thicknesses of the ependy-mal mantle and marginal layers were measured using amorphometric eyepiece (Olympus USA) (200x) in midlineregion of the spinal cord

23 Autometallography Method We evaluated mercurydeposition by the autometallography (AMG) methodin which dewaxed sections were immersed in the AMGdeveloper solution (60 gum arabic 10 potassium citratebuffer 30 hydroquinone and 05 silver nitrate) indarkness for 60min Next the spinal cord sections (119899 = 7embryos per group of three independent experiments) werewashed with tap water and counterstained with hematoxylinMercury deposition was evaluated as brown stained cellswhich represent silver surrounding the deposited mercury[27] and classified as absent (minus) mild (+) moderate (++)and intense (+++) by an investigator who was blind to thetreatment assignments according to Muller et al [28]

24 Immunohistochemistry To evaluate the effect of prenatalacute MeHg exposure we first looked for proliferation cellcycle and DNA damage in the spinal cord using antibodiesrabbit anti-phosphohistone H3 IgG (1 250 Upstate USA)rabbit anti-cyclin E IgG (1 100 Santa Cruz BiotechnologyUSA) mouse anti-p21 IgG (1 100 Santa Cruz Biotech-nology USA) and rabbit anti-120574-H2AX IgG (1 50 SantaCruz Biotechnology USA) Next we investigated neuronaldifferentiation using antibody mouse anti-neuronal nucleiNeuN IgG (1 100 Chemicon International USA) andmouseanti-120573III tubulin IgG (1 100 Promega USA) (119899 = 7 embryosper group of three independent experiments) Endogenousperoxidase activity was stoppedwith 03 hydrogen peroxideinmethanolThe sectionswerewashedwith 01Mphosphate-buffered saline (PBS) pH 74 + 03 Triton X-100 and thenblocked with 5 fetal bovine serum (FBS) in PBS The sec-tions were incubated overnight at 4∘C with primary antibod-ies washed with PBS and then incubated for 90min at roomtemperature with peroxidase-conjugated secondary antibodyanti-rabbit IgG (1 400 Sigma USA) and biotin-conjugatedantibodies including anti-rabbit IgG (1 200 Sigma USA)and anti-mouse IgG (1 200 Sigma USA) Next the sectionswere washed in 01M PBS and binding sites of antibodieswere revealed with 331015840-diaminobenzidine (DAB) (Sigma-Aldrich USA) For immunofluorescence Alexa Fluor 488goat anti-mouse IgG (1 200 Life Technologies USA) andAlexa Fluor 568 goat anti-rabbit IgG (1 100 Life Technolo-gies USA) were used Next the sections were incubatedwith 46-diamidino-2-phenylindole dihydrochloride (DAPI)(Sigma-Aldrich USA) for 3min at room temperature Nega-tive controls of immunohistochemical reaction were treatedin the same way except that the primary antibodies werereplaced with 01M PBS

25 TUNEL Assay We used TdT-mediated dUTP nick endlabeling (TUNEL) staining to identify apoptotic cells inthe dewaxed spinal cord sections (119899 = 7 embryos pergroup of three independent experiments) TUNEL stainingwas conducted with a TdT-FragEL DNA Fragmentation






3 Results








e mt mg

100120583m

(a)

Con

trol

MeH

g


Control

MeHg


minus

++

minus

++

minus

+

10120583m

(b)


e mt mg e mt mg

Control MeHg

ControlMeHg


lowast

lowastlowast

Thic

knes

s(120583

m)

0

100

200

300



ControlMeHg

lowast

lowast

Con

trol

MeH

g



NA

(pH

H3

-pos

itive

cells

) (m

m2)

0

20

40

60

80




4 Discussion







Ependymal




Control 806MeHg 570

Mantle MarginalC

ontro

lM

eHg

Con

trol

p21

Cycli

n E

MeH

g








lowast

0

20000

40000

60000

80000

Con

trol

MeH

g

Control MeHg

(pix

el120583

m2 )


ControlMeHg



lowast

NA

(TU

NEL

-pos

itive

cell)

(mm

2 )

0

5

10

15

20

Con

trol

MeH

g







DAPI MergeC

ontro

lM

eHg

120573III-tubulin




Control MeHg

Control MeHg

lowastlowastlowast

NA

(Neu

N-p

ositi

ve ce

lls) (

mm

2 )

0

20

40

60

80



5 Conclusion







References





















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






3 Results








e mt mg

100120583m

(a)

Con

trol

MeH

g


Control

MeHg


minus

++

minus

++

minus

+

10120583m

(b)


e mt mg e mt mg

Control MeHg

ControlMeHg


lowast

lowastlowast

Thic

knes

s(120583

m)

0

100

200

300



ControlMeHg

lowast

lowast

Con

trol

MeH

g



NA

(pH

H3

-pos

itive

cells

) (m

m2)

0

20

40

60

80




4 Discussion







Ependymal




Control 806MeHg 570

Mantle MarginalC

ontro

lM

eHg

Con

trol

p21

Cycli

n E

MeH

g








lowast

0

20000

40000

60000

80000

Con

trol

MeH

g

Control MeHg

(pix

el120583

m2 )


ControlMeHg



lowast

NA

(TU

NEL

-pos

itive

cell)

(mm

2 )

0

5

10

15

20

Con

trol

MeH

g







DAPI MergeC

ontro

lM

eHg

120573III-tubulin




Control MeHg

Control MeHg

lowastlowastlowast

NA

(Neu

N-p

ositi

ve ce

lls) (

mm

2 )

0

20

40

60

80



5 Conclusion







References





















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


e mt mg

100120583m

(a)

Con

trol

MeH

g


Control

MeHg


minus

++

minus

++

minus

+

10120583m

(b)


e mt mg e mt mg

Control MeHg

ControlMeHg


lowast

lowastlowast

Thic

knes

s(120583

m)

0

100

200

300



ControlMeHg

lowast

lowast

Con

trol

MeH

g



NA

(pH

H3

-pos

itive

cells

) (m

m2)

0

20

40

60

80




4 Discussion







Ependymal




Control 806MeHg 570

Mantle MarginalC

ontro

lM

eHg

Con

trol

p21

Cycli

n E

MeH

g








lowast

0

20000

40000

60000

80000

Con

trol

MeH

g

Control MeHg

(pix

el120583

m2 )


ControlMeHg



lowast

NA

(TU

NEL

-pos

itive

cell)

(mm

2 )

0

5

10

15

20

Con

trol

MeH

g







DAPI MergeC

ontro

lM

eHg

120573III-tubulin




Control MeHg

Control MeHg

lowastlowastlowast

NA

(Neu

N-p

ositi

ve ce

lls) (

mm

2 )

0

20

40

60

80



5 Conclusion







References





















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


ControlMeHg

lowast

lowast

Con

trol

MeH

g



NA

(pH

H3

-pos

itive

cells

) (m

m2)

0

20

40

60

80




4 Discussion







Ependymal




Control 806MeHg 570

Mantle MarginalC

ontro

lM

eHg

Con

trol

p21

Cycli

n E

MeH

g








lowast

0

20000

40000

60000

80000

Con

trol

MeH

g

Control MeHg

(pix

el120583

m2 )


ControlMeHg



lowast

NA

(TU

NEL

-pos

itive

cell)

(mm

2 )

0

5

10

15

20

Con

trol

MeH

g







DAPI MergeC

ontro

lM

eHg

120573III-tubulin




Control MeHg

Control MeHg

lowastlowastlowast

NA

(Neu

N-p

ositi

ve ce

lls) (

mm

2 )

0

20

40

60

80



5 Conclusion







References





















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Ependymal




Control 806MeHg 570

Mantle MarginalC

ontro

lM

eHg

Con

trol

p21

Cycli

n E

MeH

g








lowast

0

20000

40000

60000

80000

Con

trol

MeH

g

Control MeHg

(pix

el120583

m2 )


ControlMeHg



lowast

NA

(TU

NEL

-pos

itive

cell)

(mm

2 )

0

5

10

15

20

Con

trol

MeH

g







DAPI MergeC

ontro

lM

eHg

120573III-tubulin




Control MeHg

Control MeHg

lowastlowastlowast

NA

(Neu

N-p

ositi

ve ce

lls) (

mm

2 )

0

20

40

60

80



5 Conclusion







References





















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



lowast

0

20000

40000

60000

80000

Con

trol

MeH

g

Control MeHg

(pix

el120583

m2 )


ControlMeHg



lowast

NA

(TU

NEL

-pos

itive

cell)

(mm

2 )

0

5

10

15

20

Con

trol

MeH

g







DAPI MergeC

ontro

lM

eHg

120573III-tubulin




Control MeHg

Control MeHg

lowastlowastlowast

NA

(Neu

N-p

ositi

ve ce

lls) (

mm

2 )

0

20

40

60

80



5 Conclusion







References





















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


DAPI MergeC

ontro

lM

eHg

120573III-tubulin




Control MeHg

Control MeHg

lowastlowastlowast

NA

(Neu

N-p

ositi

ve ce

lls) (

mm

2 )

0

20

40

60

80



5 Conclusion







References





















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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