Treatment with Anti-EGF Ab Ameliorates Experimental Autoimmune Encephalomyelitis via Induction of Neurogenesis and Oligodendrogenesis

Research ArticleTreatment with Anti-EGF Ab AmelioratesExperimental Autoimmune Encephalomyelitis via Induction ofNeurogenesis and Oligodendrogenesis

Yifat Amir-Levy Karin Mausner-Fainberg and Arnon Karni

Neuroimmunology Laboratory Department of Neurology Tel Aviv Sourasky Medical Center Sackler Faculty of MedicineTel Aviv University 6 Weizmann Street Tel Aviv 64239 Israel

Correspondence should be addressed to Arnon Karni arnonktlvmcgovil

Received 8 October 2014 Revised 11 December 2014 Accepted 12 December 2014 Published 30 December 2014

Academic Editor Gianvito Martino

Copyright copy 2014 Yifat Amir-Levy et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

BackgroundThe neural stem cells (NSCs)migrate to the damaged sites inmultiple sclerosis (MS) and in experimental autoimmuneencephalomyelitis (EAE) However the differentiation into neurons or oligodendrocytes is blocked Epidermal growth factor (EGF)stimulates NSC proliferation and mobilization to demyelinated lesions but also induces astrogenesis and glial scar ObjectiveTo examine the clinical and histopathological effects of EGF neutralization on EAE Methods EAE-induced SJL mice wereintravenously treated with either anti-EGF neutralizing antibody (Ab) or isotype control or PBS On day 9 after immunization3 mice of each group were daily treated for 9 days with BrdU and then sacrificed for immunohistochemical analysis ResultsTreatment with anti-EGFAb significantly ameliorated EAE symptoms during the second relapse Anti-EGFAb induced a shift fromBrdU+GFAP+ NSCs to BrdU+DCX+ neuroblasts in the subventricular zone (SVZ) increased BrdU+NeuN+ neurons in the granularcell layer of the dentate gyrus and increased BrdU+O4+ oligodendrocytes in the SVZ There was no change in the inflammatoryinfiltrates in response to anti-EGF Ab Conclusions Therapy with anti-EGF Ab ameliorates EAE via induction of neurogenesis andoligodendrogenesis No immunosuppressive effect was found Further investigation is needed to support these notions of beneficialeffect of anti-EGF Ab in MS

1 Introduction

The recognition that there is neurodegeneration manifestedby demyelination oligodendrocytes apoptosis axonal tran-section and neuronal loss in multiple sclerosis (MS) [1ndash4] has led to attempts to promote neurogenesis and oligo-dendrogenesis in models of MS [5 6] A proliferation ofsubventricular zone- (SVZ-) derived progenitor cells andtheir migration to white matter lesions was found in experi-mental autoimmune encephalomyelitis (EAE) [7] Moreoverone postmortem study showed that cellular density andproliferation were enhanced in the SVZ of patients with MScompared to nonneurological controls [8] suggesting thatprogenitor cells possess the potential of repopulating sitesof injury Indeed both neuronal progenitor cells (NPCs)and oligodendrocytes progenitor cells (OPCs) derived fromneuroproliferative niches have been observed in MS lesions[9ndash11] However the differentiation of these precursor cells

into functionally active neurons and oligodendrocytes wasdemonstrated to be mostly blocked leading to regenerationfailure [12 13] Instead the sites of lesions are repopulated byreactive astrocytes (RAs) in a process of astrogliosis whichforms glial scars These RAs create a physical barrier andsecrete molecules that inhibit the regeneration of NPCs andOPCs [14 15]

Transit-amplifying cells or type C cells which comprisethe intermediate stage between the neural stem cells (NSCs)and neuroblasts located in the SVZ are known to expressepidermal growth factor receptors (EGFRs) [16] suggest-ing that EGF plays a role in adult NSC proliferation andmaturation Indeed EGF reportedly induced adult NSCsproliferation both in vitro and in vivo and was shown toinduce the proliferation ofNSCs in cultures of embryonic andadult mouse striatum which gave rise to spheres of undif-ferentiated cells [17 18] Moreover infusion of EGF into thelateral ventricle of adult rats led to a dramatic amplification of

Hindawi Publishing CorporationMultiple Sclerosis InternationalVolume 2014 Article ID 926134 9 pageshttpdxdoiorg1011552014926134

2 Multiple Sclerosis International

endogenous SVZ precursor cells but it had no proliferativeeffect on hippocampal progenitor cells [19] Several studieshave demonstrated that EGF induces astrogenesis at theexpense of neurogenesis Infusion of EGF into the murinebrain increased the number of astrocytes at the expense ofneurons in the olfactory bulb and the dentate gyrus of thehippocampus [19] In animal model in which demyelinatinglesions were induced by lysolecithin an intranasal heparin-binding EGF administration induced a significant increase inSVZ cell proliferation and mobilization toward the lesionsconcomitant with a shift of SVZ-derived progenitor cell dif-ferentiation toward the astrocytic lineage [20] Furthermorethe addition of EGF to cultured SVZ-derived type B NSCsinduced their differentiation into highly migratory Olig2+NG2 cells but these cells differentiated into S100120573+O4+oligodendrocytes only after EGF withdrawal [21] EGFR wasalso found to play a role in nerve growth inhibitory signalingin the CNS via a transactivating mechanism and signalingcascade involving the Nogo receptor (NgR) [22 23] suggest-ing that blocking EGFR signalingmay induce nerve regenera-tion Indeed both PD168393 (4-[3(bromophenyl)-amino]-6-acrylamidoquinazoline) an irreversible EGFR inhibitor andAG1478 [4-(3-chloroanilino)-67-dimethoxyquinazoline] areversible EGFR inhibitor were shown to promote neuriteoutgrowth from cerebellar granule cells and from dorsal rootganglion neurons [23]

We have recently demonstrated that peripheral bloodmononuclear cells (PBMCs) of patients with relapsing-remittingMS (RR-MS) secrete higher levels of EGF comparedto matched healthy controls indicating a potential effectof immune cells on the insufficient neuronal and oligo-dendroglial regeneration in MS Furthermore incubationof PC-12 cells in the presence of supernatants from RR-MS patients PBMCs which were previously treated withanti-EGF neutralizing antibody (Ab) significantly elevatedneuronal survival and neurite formation compared to isotypecontrol (IC) treatment [24] Taken together these findingssuggest that although EGF plays a pivotal role in NSCamplification its signaling should be inhibited in order toallow the desired maturation of NSCs towards a neuronaland oligodendroglial lineage We therefore examined theeffect of systemic blockade of EGF signaling via treatmentwith anti-EGF neutralizing Ab on relapsing-EAE symptomsand on neurogenesis and oligodendrogenesis processes inneuroproliferative niches

2 Materials and Methods

21 Induction of Relapsing-EAE and Treatment with Neutral-izing Anti-EGF Ab EAE was induced in 36 SJL female mice(6- to 8-week-old) by subcutaneous immunization (day 0)with 100 120583gmouse proteolipid protein peptide (PLP

139-151synthesized by BioSight Ltd) in 01mL PBS The peptide wasemulsified in an equal volume of complete Freundrsquos adjuvant(CFA fromDIFCO) containing 500120583gMycobacterium tuber-culosis H37RA (MT from DIFCO) The mice also receivedan intraperitoneal injection of 300 ng pertussis toxin (PTXfrom Sigma-Aldrich) in 02mL PBS A second injection of

PTX (300 ngmouse) was given 48 h later The mice wererandomly divided into 3 groups (119899 = 12 each) On day9 after immunization one group of the EAE-induced micewas intravenously injected with a single dose of 60120583gmouseof anti-human EGF antigen affinity-purified polyclonal anti-body (AF236 from RampD systems) another group was intra-venously injected with 60 120583gmouse of the corresponding IC(normal goat IgG control AB-108-C from RampD systems)and the third group was intravenously injected with PBSalone (vehicle) and it served as negative controls In order todetect de novo neural cells in the neuroproliferative niches 3mice of each group were also daily intraperitoneally injectedwith 1mgmouse 5-bromo-21015840-deoxyuridine (BrdU Sigma-Aldrich) starting from treatment on day 9 for the following 9days and they were sacrificed on day 18 after immunizationfor immunohistochemical analysis of brain sections Theanimals were monitored until day 48 after induction forsymptoms of EAE and scored as follows 0 = no disease 1 =tail paralysis 2 = hind limbweakness 3 = hind limb paralysis4 = hind limb plus forelimb paralysis and 5 = moribundThescorer was unaware of the type of therapy allocation sincethe type of therapy was coded and not posted on the cagesAll procedures involving animals were performed accordingto the guidelines of the Animal Ethical Committee of ourinstitute

22 Immunohistochemistry The mice were sacrificed (tran-scardially punctured and saline-perfused) and their brainswere rapidly excised and frozen at minus80∘C Coronal serial 10120583m sections were collected at minus20∘C and were kept frozen(minus80∘C) until the histological examination was performedSections were fixed in 4 paraformaldehyde (PFA Bar-NaorLtd Israel) for 15min at room temperature (RT) denaturedin 2NHCl in distilled water at 37∘C for 30min preincubatedin blocking solution which contained 02 Triton X-100(Sigma-Aldrich) 1 bovine serum albumin (BSA Sigma-Aldrich) and 3 horse serum (Gibco USA) for 1 h and thenincubated overnight at 4∘C with primary Abs followed byincubation with a secondary Ab for 1 h at RTThe primary Abrat anti-BrdU (1 200 AbD Serotec USA) and the secondaryAb Alexa Fluor 594 donkey anti-rat IgG (1 200 Molecu-lar Probes) were used to detect BrdU-incorporated cellsTo detect specific cell types sections were costained withone of the following primary Abs rabbit anti-doublecortin(DCX 4604 1 400 Cell Signaling) mouse anti-neuronal-specific nuclear protein (NeuN MAB377 1 100 MilliporeUSA) mouse anti-oligodendrocyte marker O4 (MAB3451 100Millipore) and rabbit anti-glial fibrillary acidic protein(GFAP G9269-80 1 100 Sigma-Aldrich) The second Abstep was performed by labeling with Alexa Fluor 488-conjugated IgG to mouse or rabbit (1 200 Molecular ProbesUSA) Control slides were incubated with secondary Abalone Stained sections were examined and photographed byLSM 700 confocal microscope (Zeiss) Digital images werecollected and the percentage of double-positive cells werequantified using ZEN 2011 software on 3 sections from eachmouse (3 mice from each group total 119899 = 9) Inflammatoryinfiltrates were detected by hematoxylin and eosin stainingusing a hematoxylin and eosin stain kit (HAE-1-1FU from

Multiple Sclerosis International 3

ScyTek laboratories Inc) and photographed by a light micro-scope

23 Statistics Comparisons between groups were madeusing the Mann-Whitney 119880 statistics The null hypothesisasserted that the medians of the two groups of samples wereidentical The 119880 values were calculated for the two groupsand for the conditions that refute the null hypothesis when119875 le 005 or 119875 le 001 The results are presented as mean plusmnstandard error of the mean (SEM)

3 Results

31 Treatment with Anti-EGF Ab Ameliorates EAE RelapsingEAE-induced mice were treated intravenously with either60 120583gmouse of anti-human EGF with 60 120583gmouse of thecorresponding IC or with PBS (vehicle) alone on day 9 afterimmunization (119899 = 12 in each group) All the animalsin all the groups had EAE (100 incidence) 100 of theanimals in the IC- and PBS-treated group and 90 of theanimals in the anti-EGF Ab-treated groups had a secondrelapse during a follow-up of 48 days As demonstrated inFigure 1 clinical symptoms started to appear on day 9 afterimmunization in all groups A reduced EAE score in responseto treatment with anti-EGF Ab was observed throughoutthe whole experimental period Comparison of the averagescores of each group at 23 time points between day 9 andday 48 after induction revealed that the scores were lower inthe group of anti-EGF Ab-treated mice compared with theIC-treated group Specifically the 119880-value was 37 which waslower than the critical 119880 = 45 at 119875 le 001 Therefore thedifference between the groups was significant at 119875 le 001No significant differences were found between the IC- andPBS-treated groups The maximal scores ranged between 1and 3 (average 171 plusmn 029) in the anti-EGF-treated groupbetween 2 and 4 (average 24 plusmn 028) in the IC-treated groupand between 1 and 3 (average 20 plusmn 014) in the PBS-treatedgroup These differences did not reach a level of significanceThe cumulative scores on day 48 of follow-up in each of themice of the anti-EGF-treated group ranged between 1 and245 (average 1095 plusmn 212) and they were significantly lowerthan the scores of the IC-treatedmice (range 16ndash535 average2550 plusmn 374) (119875 le 001) and the scores of the PBS-treatedmice (range 12ndash31 average 2075 plusmn 154) (119875 le 001) Nosignificant differences were found between the IC- and PBS-treated groups

A significant effect was mainly observed during thesecond relapse A significant reduction in disease severitybetween days 25 and 36 was detected in the anti-EGF Ab-treatedmice compared to the IC-treatedmice and to the PBS-treated mice (average 774 plusmn 46 and 692 plusmn 49 resp)The clinical scores on day 25 were lower in the anti-EGF Ab-treated groups (020 plusmn 013) compared to the IC-treated group(110 plusmn 035 119875 le 005) and compared to the PBS-treatedgroup (060 plusmn 009 119875 le 005) Similarly the clinical scoreson day 29 were lower in the anti-EGF Ab-treated groups(015 plusmn 011) compared to the IC-treated group (155 plusmn 028119875 le 001) and compared to the PBS-treated group (096 plusmn

Days after immunization

RR-E

AE cl

inic

al sc

ore

218161412

108060402

08 10 12 14 16 2320 27 31 36 41 45

P lt 005

Anti-EGF mAbICVehicle

Figure 1 Treatment with anti-EGF Ab ameliorated EAE severityand delayed the onset of the second relapse Relapsing experimentalautoimmune encephalomyelitis (EAE) was induced in 36 SJL femalemice that were subcutaneously injected with PLP

139-151 peptide onday 0 in Freundrsquos adjuvant and were injected intraperitoneally with300 ng pertussis toxin on day 0 and 48 hours after induction ofEAE The mice were divided in 3 groups (119899 = 12 each) On day9 after induction the mice were intravenously injected with either60 120583gmouse anti-human EGFAb or PBS alone (the vehicle) or with60 120583gmouse normal goat IgG and they served as controls (119899 = 12 ineach group) A therapy with anti-EGF Ab significantly amelioratedEAE severity during the second relapse and postponed its onset aneffect that was significant between days 25 and 36 after induction

015 119875 le 001) The same applied to the clinical scores onday 36 the anti-EGF Ab-treated groups scored 065 plusmn 028the IC-treated group scored 165 plusmn 023 (119875 le 005) and thePBS-treated group scored 132 plusmn 018 (119875 le 005) Moreoverthe average day of onset of the second relapse was delayed inthe anti-EGF Ab-treatedmice (day 3311plusmn208) compared tothe IC-treatedmice (day 2590plusmn138119875 le 001) and comparedto the PBS-treatedmice (2577plusmn150119875 le 001) Similarly themaximal average score of the second relapse was delayed inthe anti-EGF Ab-treatedmice (day 4110plusmn177) compared tothe IC-treated mice (day 3630plusmn177 119875 le 005) and the PBS-treated mice (day 3438 plusmn 142 119875 le 005) Examination of thenumber of mice with an EAE score ge2 demonstrated lowercases with such scores in the group of anti-EGF treatmentduring both relapses At the peak of the first relapse therewere only 4 mice with severe scores in the anti-EGF Ab-treated group compared to 8 and 9 mice in the IC and PBSgroups respectively The maximal number of animals withsevere EAE during the second relapse was 3 in the anti-EGFAb-treated group compared to 6 in both the IC- and PBS-treated groups

32 Treatment with Anti-EGF Ab Promotes NSCs Shift toNeuroblasts in the SVZ In order to examine the effectof treatment with anti-EGF Ab on the extent of neuro-genesis and oligodendrogenesis in EAE-induced mice we

4 Multiple Sclerosis InternationalLa

tera

l ven

tric

le

IC

SVZ

LV

BrdUGFAP

Merged

(a)

Anti-EGF

SVZLV

BrdUGFAP

Merged

(b)

IC Anti-EGF0

05

1

15

2

25

3

P = 004

BrdU

+G

FAP+

cells

()

(c)

Late

ral v

entr

icle LV

SVZ

BrdUDCX

Merged

(d)

LV

SVZ

BrdUDCX

Merged

(e)

IC Anti-EGF02

4

6

8

10

12

14

16

18

P = 0006

BrdU

+D

CX+

cells

()

(f)

Figure 2 Reduced numbers of NSCs and increased numbers of neuroblasts in the SVZ in response to therapy with anti-EGF AbImmunohistochemical labeling of BrdU+GFAP+ cells and BrdU+DCX+ cells in the SVZ of IC-treated mice (a and d resp) and of anti-EGF Ab-treated mice (b and e resp) on day 18 after immunization Quantification of BrdU+GFAP+ cells (c) and of BrdU+DCX+ cells (f)revealed reduced numbers of BrdU+GFAP+ cells and increased numbers of BrdU+DCX+ cells in the SVZ of anti-EGF Ab-treated EAE micecompared to IC-treated mice Images were obtained using a confocal microscopy coronal sections Quantification was performed using ZEN2011 software on 3 sections from each mouse (3 mice from each group total 119899 = 9) LV lateral ventricle SVZ subventricular zone Scale bar100120583m

intraperitoneally injected 3 mice in the anti-EGF Ab-treatedgroup and 3 mice in the IC-treated group with 1mgmouseBrdU every day for 9 days starting from day 9 after immuni-zation These mice were sacrificed on day 18 after immuniza-tion for immunohistochemical analysis of brain sections

As demonstrated in Figures 2(a) 2(b) and 2(c) thepercentage of BrdU+GFAP+ NSCs in the SVZ were lower inthe anti-EGF Ab-treated group compared to the IC-treatedgroup (11 plusmn 01 versus 24 plusmn 02 119875 = 004 resp) We alsodetected a substantial elevation in the percentage of prolif-erating neuroblasts expressing doublecortin (BrdU+DCX+)in the SVZ in response to EGF blockade (161 plusmn 01 inthe anti-EGF group versus 22 plusmn 07 in the IC group119875 = 0006 Figures 2(d) 2(e) and 2(f)) suggesting that theEGF blockade promoted the differentiation of SVZ NSCsto DCX+ neuroblasts Interestingly we did not detect anygroup differences in the numbers of BrdU+GFAP+ or ofBrdU+DCX+ in the hippocampus subgranular zone (SGZ) ofthe dentate gyrus (data not shown)

33 Treatment with Anti-EGF Ab Increases the Numbers ofDe Novo Mature Neurons in the Granular Cell Layer of theDentate Gyrus We next examined the effect of anti-EGF Ab

treatment on the number of BrdU+NeuN+ mature neuronsin the neuroproliferative niches We detected a significantincrease in the percentage of newborn cells expressing aneuronal nuclear (NeuN) marker that is BrdU+NeuN+cells in theGCL of the dentate gyrus in response to treatmentwith anti-EGF Ab (05 plusmn 006 in anti-EGF group versus02 plusmn 001 in the IC group 119875 = 002 Figures 3(a) 3(b)and 3(c)) suggesting that an EGF signaling blockade inducedneuroblast differentiation into mature neurons within theGCL of the dentate gyrus at this time point of interven-tion

34 Anti-EGF AbTherapy Promotes Oligodendrogenesis in theSVZ In order to examine the effect of anti-EGFAb treatmenton the extent of oligodendrogenesis within the neuroprolifer-ative niches we comparedBrdU+O4+ cells in the SVZ and theSGZ of the anti-EGF Ab- and IC-treated groups Althoughwe observed a slight trend towards increased numbers ofBrdU+O4+ cells in the SGZ of anti-EGF Ab-treated EAEmice this trend did not reach a level of significance (06 plusmn01 in the anti-EGF Ab-treated group versus 04 plusmn 002in the IC-treated group 119875 = NS Figures 4(a) 4(b) and4(c)) However therapy with anti-EGF Ab led to a significant


Hip

poca

mpu

s

IC

GCL

BrdUNeuN

Merged

(a)

Anti-EGF

BrdUNeuN

Merged

GCL

(b)

IC Anti-EGF

07

06

05

04

03

02

01

0

P = 002

BrdU

+N

euN+

cells

()

(c)

Figure 3 Elevated numbers of de novo mature neurons in the GCL of anti-EGF Ab-treated EAE mice Immunohistochemical labeling ofBrdU+NeuN+ cells in the GCL of IC-treated mice (a) and of anti-EGF Ab-treated mice (b) on day 18 after immunization Quantification ofBrdU+NeuN+ cells (c) revealed increased numbers of BrdU+NeuN+ in the GCL of anti-EGF Ab-treated EAE mice compared to IC-treatedmice Images were obtained using a confocal microscopy coronal sections Quantification was performed using ZEN 2011 software on 3sections from each mouse (3 mice from each group total 119899 = 9) GCL granular cell layer Scale bar 10 120583m (magnification = times63)

induction in BrdU+O4+ cells within the SVZ (19 plusmn 01in the anti-EGF Ab-treated group versus 04 plusmn 001 in theIC-treated group 119875 = 001 Figures 4(d) 4(e) and 4(f))

35 No Decrease in Inflammatory Infiltrates in the Brains ofAnti-EGF Ab-Treated Mice Finally we sought to examinewhether the beneficial clinical effect of treatment with anti-EGFAbmay also bemediated via the suppression of immuneresponses Towards this end we examined inflammatoryinfiltrates in the mouse brains by hematoxylin and eosinstaining on day 18 after immunization and detected infiltratesin the cortex and striatum of vehicle-treated EAE and inthe cortex and fimbria of IC-treated EAE mice (Figures 5(a)and 5(b)) Inflammatory infiltrates were also detected inthe cortex and fimbria of anti-EGF Ab-treated EAE mice(Figure 5(c)) suggesting that the EGF blockade did notsuppress immune activity in the setting of relapsing EAE

4 Discussion

Our objective was to study the effect of EGF blockade asa therapy to promote neurogenesis and oligodendrogenesisin an animal model of MS Our results demonstrated that asingle intravenous administration of 60 120583gmouse anti-EGF

Ab on day 9 after induction of EAE significantly amelioratedrelapsing-EAE severity during the second relapse and delayedits onset

Accumulating evidence suggests that EGF plays a dualrole in the context of CNS injury in general and in MSEAEin particular In addition to basic fibroblast growth factor it iswell known to participate in SVZ-derived NSC amplification[19 20] Moreover EGF induced SVZ precursor migration tothe surrounding parenchyma mainly striatum in physiolog-ical conditions [19] whereas heparin-binding EGF inducedSVZ precursor mobilization specifically to the demyelinatinglesions in the corpus callosum that had been induced bylysolecithin [20] Taking into consideration our previousfindings on the enhanced secretion of EGF from peripheralimmune cells of patients with relapsing-remitting MS [24]and the existence of CNS-infiltrating immune cells (mainlyCD45+ cells) in the SVZ of EAE-inducedmice already on day3 after immunization [25] it can be assumed that the immunesystem contributes to the increased EGF levels within theSVZ and the consequent SVZ-derivedNSC amplification andmobilization Indeed increase of BrdU+ proliferating cellswithin the SVZ of EAE-inducedmice can be detected as earlyas day 7 after immunization [25] In order to allow this initialSVZ-derived NSC amplification we chose to add the anti-EGF neutralizing Ab on day 9 after immunization the time


IC

Hip

poca

mpu

s

BrdUO4

Merged

hilus

DG

(a)

Anti-EGF

BrdUO4

Merged

hilus

DG

(b)

IC Anti-EGF

090807060504030201

0

P = NS

BrdU

+O4+

cells

in S

GZ

()

(c)

Late

ral v

entr

icle

LV

CC

SVZ

BrdUO4

Merged

(d)

LV

CC

BrdUO4

Merged

(e)

IC Anti-EGF

25

2

15

1

05

0

P = 001

BrdU

+O4+

cells

in S

VZ

()

(f)

Figure 4 Increased numbers of de novo oligodendrocytes in the SVZ in response to therapy with anti-EGF Ab Immunohistochemicallabeling of BrdU+O4+ cells in the SGZ and SVZ of IC-treated mice (a and d resp) and of anti-EGF Ab-treated mice (b and e resp) on day18 after immunization Quantification of BrdU+O4+ cells in the SGZ and in the SVZ (c and f correspondingly) revealed a nonsignificanttrend for induction of BrdU+O4+ in the SGZ along with a significant induction in BrdU+O4+ cells in the SVZ Images were obtained usinga confocal microscopy coronal sections Quantification was performed using ZEN 2011 software on 3 sections from each mouse (3 micefrom each group total 119899 = 9) DG dentate gyrus LV lateral ventricle SVZ subventricular zone and cc corpus callosum Scale bar 10 120583m(magnification = times63)

between the initial proliferation of NSCs and the appearanceof EAE symptoms on days 10ndash12 after immunization

At later stages EGF was shown to play a pivotal role inastrogenesis processes at the expense of oligodendrogenesisand neurogenesis Chronical infusion of EGF for 2 weeksinto the lateral ventricle of adult rats was found to reduce thetotal number of newborn neurons reaching the olfactory bulband to substantially enhance the generation of astrocytes inboth the olfactory bulb and the hippocampus [19] MoreoverEGFR expression was upregulated in both reactive astrocytesand scar astrocytes in chronic MS lesions suggesting thatEGF signaling is associated with astrogliosis and glial scarformation [26]

GFAP is generally regarded as being a marker of matureastrocytes and of slowly proliferating type B cells of the SVZ[27 28] and as the corresponding type 1 cells of the SGZ[29] DCX which encodes a microtubule-associated proteinis expressed by type A migrating neuroblasts of the SVZ[30 31] and by type 2 cells of the SGZ [32] We found thattherapy with anti-EGF Ab led to a 21-fold decrease in thelevel of BrdU+GFAP+ NSCs concomitant with a 73-foldincrease in the number of BrdU+DCX+ neuroblasts withinthe SVZ suggesting that an EGF blockade induced type Cdifferentiation into type A neuroblasts We did not detect

this effect of anti-EGF Ab in the SGZ This finding may besupported by a previous observation that EGF infusion didnot alter the total number of newborn cells in the hilus andgranular cell layer (GCL) at the end of EGF for at least 4 weeks[19]

We used the NeuN marker in order to detect matureneurons NeuN-positive cells can be detected in the GCLsince NSCs in the SGZ migrate into the GCL of the dentategyrus where they acquire a mature phenotype [33] Indeedwe detected a 25-fold increase in BrdU+NeuN+ in theGCL of anti-EGF Ab-treated mice suggesting that an EGFblockade augments the hippocampal neuroblast differentia-tion into mature neurons These findings may correlate withthe previously reported shift in response to EGF infusionbetween S100120573 astrocytes and NeuN neurons in the GCL(from 1 astrocytes and 92 neurons to 39 astrocytes and52 neurons) [19]

Oligodendrocyte marker O4 is an antigen on the surfaceof both late progenitor cells and mature oligodendrocytes[34] It can be detected in oligodendrocytes progenitor cells ofthe SGZ and inmature oligodendrocytes in the hilus adjacentto the SGZ which are considered to be differentiated fromSGZ progenitor cells [35] Some type B cells in the SVZand a small subpopulation of actively dividing type C cells


cc

Vehicle

Ctx

Anti-EGF

Ctx

IC

Ctx

St fifi

LV

(a) (c) (e)

(b) (d) (f)

Figure 5 Treatment with anti-EGF Ab did not affect inflammatory cell infiltration into the CNS Estimation of inflammatory infiltrates onday 18 after immunization by hematoxylin and eosin staining Infiltrates (arrows) were detected in the cortex and striatum of vehicle-treatedEAE mice (a and b) in the cortex and fimbria of IC-treated EAE mice (c and d) and in the cortex and fimbria of anti-EGF Ab-treated EAEmice (e and f) Images were obtained by a light microscope Ctx cortex cc corpus callosum St striatum fi fimbria and LV lateral ventricleScale bar 50 120583m

were found to express oligodendrocyte lineage transcriptionfactor 2 indicating that oligodendrocytes differentiationmayalso occur in the SVZ [7] We observed a significant 47-fold increase in the number of de novoO4+ oligodendrocytesin the SVZ but not in the SGZ in response to EGFneutralization This finding suggests that an EGF blockadeinduces oligodendrogenesis in addition to neurogenesissimilar to the results of an earlier in vitro study whichdemonstrated that EGF should be eliminated in order toallow the differentiation of EGF-stimulated cells derived fromthe SVZ into O4+ oligodendrocytes [21] The differences inthe effect of anti-EGF Ab on the SVZ and SGZ in our studyraise the possibility of different degrees of penetration of thistherapy into various stem cell niches and that possibilitywarrants further exploration

Finally we sought to examine whether the beneficialeffect of EGF neutralization may be mediated via immuno-suppression mechanism Functional EGF receptors (EGFRErbB1HER-1) were previously reported to be expressed

by peripheral blood monocytes and monocyte-derivedmacrophages [36] but we did not detect altered numbersof inflammatory infiltrate foci in the anti-EGF Ab-treatedmice suggesting that the beneficial effect of an EGF blockadeis probably not mediated via restriction of the immuneresponse

Taken together our results suggest a therapeutic potentialfor EGF blockade with anti-EGF Ab in EAE via inductionof neurogenesis and oligodendrogenesis processes at theexpense of astrogenesis Further studies on the effect of EGFblockade on EAE are needed in order to replicate our obser-vations and to extend the evidence on the effect of this therapyon spinal cord pathology on the various NSC niches and onimmune activity Our study raises the possibility that com-bined treatment of anti-EGF Ab with immunomodulatorytherapy may have an additivesynergistic effect Our findingsare compatible with previous reports which demonstratedimproved functional outcome in response to inhibition ofEGFR in experimental spinal cord injury [37 38]


Conflict of Interests

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

Authorsrsquo Contribution

Yifat Amir Levy and Karin Mausner Fainberg contributedequally to the paper

Acknowledgment

Esther Eshkol the institutional medical and scientific copy-editor is thanked for editorial assistance

References

[1] B Ferguson M K Matyszak M M Esiri and V H PerryldquoAxonal damage in acute multiple sclerosis lesionsrdquo Brain vol120 no 3 pp 393ndash399 1997

[2] M H Barnett and J W Prineas ldquoRelapsing and remittingmultiple sclerosis pathology of the newly forming lesionrdquoAnnals of Neurology vol 55 no 4 pp 458ndash468 2004

[3] C Matute and F Perez-Cerda ldquoMultiple sclerosis novel per-spectives on newly forming lesionsrdquo Trends in Neurosciencesvol 28 no 4 pp 173ndash175 2005

[4] B D Trapp and K-A Nave ldquoMultiple sclerosis an immune orneurodegenerative disorderrdquo Annual Review of Neurosciencevol 31 pp 247ndash269 2008

[5] D C Lie H Song S A Colamarino G-L Ming and F HGage ldquoNeurogenesis in the adult brain new strategies for cen-tral nervous system diseasesrdquo Annual Review of Pharmacologyand Toxicology vol 44 pp 399ndash421 2004

[6] R Arnon and R Aharoni ldquoNeurogenesis and neuroprotectionin the CNSmdashfundamental elements in the effect of glatirameracetate on treatment of autoimmune neurological disordersrdquoMolecular Neurobiology vol 36 no 3 pp 245ndash253 2007

[7] N Picard-Riera L Decker C Delarasse et al ldquoExperimentalautoimmune encephalomyelitis mobilizes neural progenitorsfrom the subventricular zone to undergo oligodendrogenesisin adult micerdquo Proceedings of the National Academy of Sciencesof the United States of America vol 99 no 20 pp 13211ndash132162002

[8] B Nait-Oumesmar N Picard-Riera C Kerninon and ABaron-Van Evercooren ldquoThe role of SVZ-derived neural pre-cursors in demyelinating diseases from animal models tomultiple sclerosisrdquo Journal of the Neurological Sciences vol 265no 1-2 pp 26ndash31 2008

[9] G Wolswijk ldquoChronic stage multiple sclerosis lesions containa relatively quiescent population of oligodendrocyte precursorcellsrdquo The Journal of Neuroscience vol 18 no 2 pp 601ndash6091998

[10] A Chang W W Tourtellotte R Rudick and B D Trapp ldquoPre-myelinating oligodendrocytes in chronic lesions of multiplesclerosisrdquo The New England Journal of Medicine vol 346 no3 pp 165ndash173 2002

[11] H Snethen S Love and N J Scolding ldquoDisease-responsiveneural precursor cells are present in multiple sclerosis lesionsrdquoRegenerative Medicine vol 3 no 6 pp 835ndash847 2008

[12] M R Kotter W-W Li C Zhao and R J M Franklin ldquoMyelinimpairs CNS remyelination by inhibiting oligodendrocyte pre-cursor cell differentiationrdquoThe Journal of Neuroscience vol 26no 1 pp 328ndash332 2006

[13] T Kuhlmann V Miron Q Cuo C Wegner J Antel and WBruck ldquoDifferentiation block of oligodendroglial progenitorcells as a cause for remyelination failure in chronic multiplesclerosisrdquo Brain vol 131 no 7 pp 1749ndash1758 2008

[14] J Silver and J H Miller ldquoRegeneration beyond the glial scarrdquoNature Reviews Neuroscience vol 5 no 2 pp 146ndash156 2004

[15] G Yiu and Z He ldquoGlial inhibition of CNS axon regenerationrdquoNature Reviews Neuroscience vol 7 no 8 pp 617ndash627 2006

[16] R Machold S Hayashi M Rutlin et al ldquoSonic hedgehog isrequired for progenitor cell maintenance in telencephalic stemcell nichesrdquo Neuron vol 39 pp 937ndash950 2003

[17] R M Anchan T A Reh J Angello A Balliet and MWalker ldquoEGF and TGF-120572 stimulate retinal neuroepithelial cellproliferation in vitrordquo Neuron vol 6 no 6 pp 923ndash936 1991

[18] A L Vescovi B A Reynolds D D Fraser and S Weiss ldquobFGFregulates the proliferative fate of unipotent (neuronal) andbipotent (neuronalastroglial) EGF-generated CNS progenitorcellsrdquo Neuron vol 11 no 5 pp 951ndash966 1993

[19] H G Kuhn J Winkler G Kempermann L J Thal and F HGage ldquoEpidermal growth factor and fibroblast growth factor-2 have different effects on neural progenitors in the adult ratbrainrdquoThe Journal of Neuroscience vol 17 no 15 pp 5820ndash58291997

[20] C Cantarella M Cayre KMagalon and P Durbec ldquoIntranasalHB-EGF administration favors adult SVZ cell mobilization todemyelinated lesions in mouse corpus callosumrdquo Developmen-tal Neurobiology vol 68 no 2 pp 223ndash236 2008

[21] O Gonzalez-Perez and A Quinones-Hinojosa ldquoDose-depend-ent effect of EGF on migration and differentiation of adultsubventricular zone astrocytesrdquo GLIA vol 58 no 8 pp 975ndash983 2010

[22] J M Schwab S K Tuli and V Failli ldquoThe Nogo receptor com-plex confining molecules to molecular mechanismsrdquo Trends inMolecular Medicine vol 12 no 7 pp 293ndash297 2006

[23] V Koprivica K-S Cho J B Park et al ldquoEGFR activationmedi-ates inhibition of axon regeneration by myelin and chondroitinsulfate proteoglycansrdquo Science vol 310 no 5745 pp 106ndash1102005

[24] Y A Levy K M Fainberg T Amidror K Regev E Auriel andAKarni ldquoHigh and dysregulated secretion of epidermal growthfactor from immune cells of patients with relapsing-remittingmultiple sclerosisrdquo Journal of Neuroimmunology vol 257 no 1-2 pp 82ndash89 2013

[25] V Tepavcevic F Lazarini C Alfaro-Cervello et al ldquoInflamma-tioninduced subventricular zone dysfunction leads to olfactorydeficits in a targetedmousemodel of multiple sclerosisrdquo Journalof Clinical Investigation vol 121 no 12 pp 4722ndash4734 2011

[26] J E Holley D Gveric J Newcombe M L Cuzner and N JGutowski ldquoAstrocyte characterization in the multiple sclerosisglial scarrdquoNeuropathology andAppliedNeurobiology vol 29 no5 pp 434ndash444 2003

[27] A Alvarez-Buylla B Seri and F Doetsch ldquoIdentification ofneural stem cells in the adult vertebrate brainrdquo Brain ResearchBulletin vol 57 no 6 pp 751ndash758 2002

[28] J M Garcıa-Verdugo F Doetsch H Wichterle D A Limand A Alvarez-Buylla ldquoArchitecture and cell types of the adultsubventricular zone in search of the stem cellsrdquo Journal ofNeurobiology vol 36 no 2 pp 234ndash248 1998


[29] B Seri J M Garcıa-Verdugo B S McEwen and A Alvarez-Buylla ldquoAstrocytes give rise to new neurons in the adultmammalian hippocampusrdquoThe Journal of Neuroscience vol 21no 18 pp 7153ndash7160 2001

[30] J G Gleeson L Peter T L A Flanagan and C AWalsh ldquoDou-blecortin is a microtubule-associated protein and is expressedwidely by migrating neuronsrdquo Neuron vol 23 no 2 pp 257ndash271 1999

[31] F Francis A Koulakoff D Boucher et al ldquoDoublecortin isa developmentally regulated microtubule-associated proteinexpressed in migrating and differentiating neuronsrdquo Neuronvol 23 no 2 pp 247ndash256 1999

[32] S Fukuda F Kato Y Tozuka M Yamaguchi Y Miyamoto andT Hisatsune ldquoTwo distinct subpopulations of nestin -positivecells in adult mouse dentate gyrusrdquo Journal of Neuroscience vol23 no 28 pp 9357ndash9366 2003

[33] H G Kuhn H Dickinson-Anson and F H Gage ldquoNeurogen-esis in the dentate gyrus of the adult rat age-related decrease ofneuronal progenitor proliferationrdquoThe Journal of Neurosciencevol 16 no 6 pp 2027ndash2033 1996

[34] J A Nielsen D Maric P Lau J L Barker and L D HudsonldquoIdentification of a novel oligodendrocyte cell adhesion proteinusing gene expression profilingrdquo The Journal of Neurosciencevol 26 no 39 pp 9881ndash9891 2006

[35] R Sasaki AMatsumoto K Itoh et al ldquoTarget cells of apoptosisin the adult murine dentate gyrus and O4 immunoreactivityafter ionizing radiationrdquoNeuroscience Letters vol 279 no 1 pp57ndash60 2000

[36] D J Lamb HModjtahedi N J Plant and G A A Ferns ldquoEGFmediates monocyte chemotaxis and macrophage proliferationand EGF receptor is expressed in atherosclerotic plaquesrdquoAtherosclerosis vol 176 no 1 pp 21ndash26 2004

[37] Z-W Li R-H Tang J-P Zhang et al ldquoInhibiting epidermalgrowth factor receptor attenuates reactive astrogliosis andimproves functional outcome after spinal cord injury in ratsrdquoNeurochemistry International vol 58 no 7 pp 812ndash819 2011

[38] M Erschbamer K Pernold and LOlson ldquoInhibiting epidermalgrowth factor receptor improves structural locomotor sensoryand bladder recovery from experimental spinal cord injuryrdquoTheJournal of Neuroscience vol 27 no 24 pp 6428ndash6435 2007


endogenous SVZ precursor cells but it had no proliferativeeffect on hippocampal progenitor cells [19] Several studieshave demonstrated that EGF induces astrogenesis at theexpense of neurogenesis Infusion of EGF into the murinebrain increased the number of astrocytes at the expense ofneurons in the olfactory bulb and the dentate gyrus of thehippocampus [19] In animal model in which demyelinatinglesions were induced by lysolecithin an intranasal heparin-binding EGF administration induced a significant increase inSVZ cell proliferation and mobilization toward the lesionsconcomitant with a shift of SVZ-derived progenitor cell dif-ferentiation toward the astrocytic lineage [20] Furthermorethe addition of EGF to cultured SVZ-derived type B NSCsinduced their differentiation into highly migratory Olig2+NG2 cells but these cells differentiated into S100120573+O4+oligodendrocytes only after EGF withdrawal [21] EGFR wasalso found to play a role in nerve growth inhibitory signalingin the CNS via a transactivating mechanism and signalingcascade involving the Nogo receptor (NgR) [22 23] suggest-ing that blocking EGFR signalingmay induce nerve regenera-tion Indeed both PD168393 (4-[3(bromophenyl)-amino]-6-acrylamidoquinazoline) an irreversible EGFR inhibitor andAG1478 [4-(3-chloroanilino)-67-dimethoxyquinazoline] areversible EGFR inhibitor were shown to promote neuriteoutgrowth from cerebellar granule cells and from dorsal rootganglion neurons [23]

We have recently demonstrated that peripheral bloodmononuclear cells (PBMCs) of patients with relapsing-remittingMS (RR-MS) secrete higher levels of EGF comparedto matched healthy controls indicating a potential effectof immune cells on the insufficient neuronal and oligo-dendroglial regeneration in MS Furthermore incubationof PC-12 cells in the presence of supernatants from RR-MS patients PBMCs which were previously treated withanti-EGF neutralizing antibody (Ab) significantly elevatedneuronal survival and neurite formation compared to isotypecontrol (IC) treatment [24] Taken together these findingssuggest that although EGF plays a pivotal role in NSCamplification its signaling should be inhibited in order toallow the desired maturation of NSCs towards a neuronaland oligodendroglial lineage We therefore examined theeffect of systemic blockade of EGF signaling via treatmentwith anti-EGF neutralizing Ab on relapsing-EAE symptomsand on neurogenesis and oligodendrogenesis processes inneuroproliferative niches

2 Materials and Methods

21 Induction of Relapsing-EAE and Treatment with Neutral-izing Anti-EGF Ab EAE was induced in 36 SJL female mice(6- to 8-week-old) by subcutaneous immunization (day 0)with 100 120583gmouse proteolipid protein peptide (PLP

139-151synthesized by BioSight Ltd) in 01mL PBS The peptide wasemulsified in an equal volume of complete Freundrsquos adjuvant(CFA fromDIFCO) containing 500120583gMycobacterium tuber-culosis H37RA (MT from DIFCO) The mice also receivedan intraperitoneal injection of 300 ng pertussis toxin (PTXfrom Sigma-Aldrich) in 02mL PBS A second injection of

PTX (300 ngmouse) was given 48 h later The mice wererandomly divided into 3 groups (119899 = 12 each) On day9 after immunization one group of the EAE-induced micewas intravenously injected with a single dose of 60120583gmouseof anti-human EGF antigen affinity-purified polyclonal anti-body (AF236 from RampD systems) another group was intra-venously injected with 60 120583gmouse of the corresponding IC(normal goat IgG control AB-108-C from RampD systems)and the third group was intravenously injected with PBSalone (vehicle) and it served as negative controls In order todetect de novo neural cells in the neuroproliferative niches 3mice of each group were also daily intraperitoneally injectedwith 1mgmouse 5-bromo-21015840-deoxyuridine (BrdU Sigma-Aldrich) starting from treatment on day 9 for the following 9days and they were sacrificed on day 18 after immunizationfor immunohistochemical analysis of brain sections Theanimals were monitored until day 48 after induction forsymptoms of EAE and scored as follows 0 = no disease 1 =tail paralysis 2 = hind limbweakness 3 = hind limb paralysis4 = hind limb plus forelimb paralysis and 5 = moribundThescorer was unaware of the type of therapy allocation sincethe type of therapy was coded and not posted on the cagesAll procedures involving animals were performed accordingto the guidelines of the Animal Ethical Committee of ourinstitute

22 Immunohistochemistry The mice were sacrificed (tran-scardially punctured and saline-perfused) and their brainswere rapidly excised and frozen at minus80∘C Coronal serial 10120583m sections were collected at minus20∘C and were kept frozen(minus80∘C) until the histological examination was performedSections were fixed in 4 paraformaldehyde (PFA Bar-NaorLtd Israel) for 15min at room temperature (RT) denaturedin 2NHCl in distilled water at 37∘C for 30min preincubatedin blocking solution which contained 02 Triton X-100(Sigma-Aldrich) 1 bovine serum albumin (BSA Sigma-Aldrich) and 3 horse serum (Gibco USA) for 1 h and thenincubated overnight at 4∘C with primary Abs followed byincubation with a secondary Ab for 1 h at RTThe primary Abrat anti-BrdU (1 200 AbD Serotec USA) and the secondaryAb Alexa Fluor 594 donkey anti-rat IgG (1 200 Molecu-lar Probes) were used to detect BrdU-incorporated cellsTo detect specific cell types sections were costained withone of the following primary Abs rabbit anti-doublecortin(DCX 4604 1 400 Cell Signaling) mouse anti-neuronal-specific nuclear protein (NeuN MAB377 1 100 MilliporeUSA) mouse anti-oligodendrocyte marker O4 (MAB3451 100Millipore) and rabbit anti-glial fibrillary acidic protein(GFAP G9269-80 1 100 Sigma-Aldrich) The second Abstep was performed by labeling with Alexa Fluor 488-conjugated IgG to mouse or rabbit (1 200 Molecular ProbesUSA) Control slides were incubated with secondary Abalone Stained sections were examined and photographed byLSM 700 confocal microscope (Zeiss) Digital images werecollected and the percentage of double-positive cells werequantified using ZEN 2011 software on 3 sections from eachmouse (3 mice from each group total 119899 = 9) Inflammatoryinfiltrates were detected by hematoxylin and eosin stainingusing a hematoxylin and eosin stain kit (HAE-1-1FU from




3 Results




RR-E

AE cl

inic

al sc

ore

218161412

108060402

08 10 12 14 16 2320 27 31 36 41 45

P lt 005







tera

l ven

tric

le

IC

SVZ

LV

BrdUGFAP

Merged

(a)

Anti-EGF

SVZLV

BrdUGFAP

Merged

(b)

IC Anti-EGF0

05

1

15

2

25

3

P = 004

BrdU

+G

FAP+

cells

()

(c)

Late

ral v

entr

icle LV

SVZ

BrdUDCX

Merged

(d)

LV

SVZ

BrdUDCX

Merged

(e)

IC Anti-EGF02

4

6

8

10

12

14

16

18

P = 0006

BrdU

+D

CX+

cells

()

(f)








Hip

poca

mpu

s

IC

GCL

BrdUNeuN

Merged

(a)

Anti-EGF

BrdUNeuN

Merged

GCL

(b)

IC Anti-EGF

07

06

05

04

03

02

01

0

P = 002

BrdU

+N

euN+

cells

()

(c)




4 Discussion





IC

Hip

poca

mpu

s

BrdUO4

Merged

hilus

DG

(a)

Anti-EGF

BrdUO4

Merged

hilus

DG

(b)

IC Anti-EGF

090807060504030201

0

P = NS

BrdU

+O4+

cells

in S

GZ

()

(c)

Late

ral v

entr

icle

LV

CC

SVZ

BrdUO4

Merged

(d)

LV

CC

BrdUO4

Merged

(e)

IC Anti-EGF

25

2

15

1

05

0

P = 001

BrdU

+O4+

cells

in S

VZ

()

(f)









cc

Vehicle

Ctx

Anti-EGF

Ctx

IC

Ctx

St fifi

LV

(a) (c) (e)

(b) (d) (f)











Acknowledgment


References











































3 Results




RR-E

AE cl

inic

al sc

ore

218161412

108060402

08 10 12 14 16 2320 27 31 36 41 45

P lt 005







tera

l ven

tric

le

IC

SVZ

LV

BrdUGFAP

Merged

(a)

Anti-EGF

SVZLV

BrdUGFAP

Merged

(b)

IC Anti-EGF0

05

1

15

2

25

3

P = 004

BrdU

+G

FAP+

cells

()

(c)

Late

ral v

entr

icle LV

SVZ

BrdUDCX

Merged

(d)

LV

SVZ

BrdUDCX

Merged

(e)

IC Anti-EGF02

4

6

8

10

12

14

16

18

P = 0006

BrdU

+D

CX+

cells

()

(f)








Hip

poca

mpu

s

IC

GCL

BrdUNeuN

Merged

(a)

Anti-EGF

BrdUNeuN

Merged

GCL

(b)

IC Anti-EGF

07

06

05

04

03

02

01

0

P = 002

BrdU

+N

euN+

cells

()

(c)




4 Discussion





IC

Hip

poca

mpu

s

BrdUO4

Merged

hilus

DG

(a)

Anti-EGF

BrdUO4

Merged

hilus

DG

(b)

IC Anti-EGF

090807060504030201

0

P = NS

BrdU

+O4+

cells

in S

GZ

()

(c)

Late

ral v

entr

icle

LV

CC

SVZ

BrdUO4

Merged

(d)

LV

CC

BrdUO4

Merged

(e)

IC Anti-EGF

25

2

15

1

05

0

P = 001

BrdU

+O4+

cells

in S

VZ

()

(f)









cc

Vehicle

Ctx

Anti-EGF

Ctx

IC

Ctx

St fifi

LV

(a) (c) (e)

(b) (d) (f)











Acknowledgment


References









































tera

l ven

tric

le

IC

SVZ

LV

BrdUGFAP

Merged

(a)

Anti-EGF

SVZLV

BrdUGFAP

Merged

(b)

IC Anti-EGF0

05

1

15

2

25

3

P = 004

BrdU

+G

FAP+

cells

()

(c)

Late

ral v

entr

icle LV

SVZ

BrdUDCX

Merged

(d)

LV

SVZ

BrdUDCX

Merged

(e)

IC Anti-EGF02

4

6

8

10

12

14

16

18

P = 0006

BrdU

+D

CX+

cells

()

(f)








Hip

poca

mpu

s

IC

GCL

BrdUNeuN

Merged

(a)

Anti-EGF

BrdUNeuN

Merged

GCL

(b)

IC Anti-EGF

07

06

05

04

03

02

01

0

P = 002

BrdU

+N

euN+

cells

()

(c)




4 Discussion





IC

Hip

poca

mpu

s

BrdUO4

Merged

hilus

DG

(a)

Anti-EGF

BrdUO4

Merged

hilus

DG

(b)

IC Anti-EGF

090807060504030201

0

P = NS

BrdU

+O4+

cells

in S

GZ

()

(c)

Late

ral v

entr

icle

LV

CC

SVZ

BrdUO4

Merged

(d)

LV

CC

BrdUO4

Merged

(e)

IC Anti-EGF

25

2

15

1

05

0

P = 001

BrdU

+O4+

cells

in S

VZ

()

(f)









cc

Vehicle

Ctx

Anti-EGF

Ctx

IC

Ctx

St fifi

LV

(a) (c) (e)

(b) (d) (f)











Acknowledgment


References









































Hip

poca

mpu

s

IC

GCL

BrdUNeuN

Merged

(a)

Anti-EGF

BrdUNeuN

Merged

GCL

(b)

IC Anti-EGF

07

06

05

04

03

02

01

0

P = 002

BrdU

+N

euN+

cells

()

(c)




4 Discussion





IC

Hip

poca

mpu

s

BrdUO4

Merged

hilus

DG

(a)

Anti-EGF

BrdUO4

Merged

hilus

DG

(b)

IC Anti-EGF

090807060504030201

0

P = NS

BrdU

+O4+

cells

in S

GZ

()

(c)

Late

ral v

entr

icle

LV

CC

SVZ

BrdUO4

Merged

(d)

LV

CC

BrdUO4

Merged

(e)

IC Anti-EGF

25

2

15

1

05

0

P = 001

BrdU

+O4+

cells

in S

VZ

()

(f)









cc

Vehicle

Ctx

Anti-EGF

Ctx

IC

Ctx

St fifi

LV

(a) (c) (e)

(b) (d) (f)











Acknowledgment


References









































IC

Hip

poca

mpu

s

BrdUO4

Merged

hilus

DG

(a)

Anti-EGF

BrdUO4

Merged

hilus

DG

(b)

IC Anti-EGF

090807060504030201

0

P = NS

BrdU

+O4+

cells

in S

GZ

()

(c)

Late

ral v

entr

icle

LV

CC

SVZ

BrdUO4

Merged

(d)

LV

CC

BrdUO4

Merged

(e)

IC Anti-EGF

25

2

15

1

05

0

P = 001

BrdU

+O4+

cells

in S

VZ

()

(f)









cc

Vehicle

Ctx

Anti-EGF

Ctx

IC

Ctx

St fifi

LV

(a) (c) (e)

(b) (d) (f)











Acknowledgment


References









































cc

Vehicle

Ctx

Anti-EGF

Ctx

IC

Ctx

St fifi

LV

(a) (c) (e)

(b) (d) (f)











Acknowledgment


References













































Acknowledgment


References



















































Documents

Treatment with Anti-EGF Ab Ameliorates Experimental Autoimmune Encephalomyelitis via Induction of Neurogenesis and Oligodendrogenesis