Cxcl10 enhances blood cells migration in the sub-ventricular zone of mice affected by experimental autoimmune encephalomyelitis

Molecular and Cellular Neuroscience 43 (2010) 268–280

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Molecular and Cellular Neuroscience

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Cxcl10 enhances blood cells migration in the sub-ventricular zone of mice affected byexperimental autoimmune encephalomyelitis

Luca Muzio a,⁎,1, Francesca Cavasinni a,1,2, Cinzia Marinaro a, Andrea Bergamaschi a, Alessandra Bergami a,Cristina Porcheri a, Federica Cerri b, Giorgia Dina b, Angelo Quattrini b, Giancarlo Comi c,Roberto Furlan a,1, Gianvito Martino a,1

a Neuroimmunology Unit, DIBIT-2, Institute of Experimental Neurology (INSPE), Division of Neuroscience, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milano, Italyb Neuropathology Unit DIBIT-2, Institute of Experimental Neurology (INSPE), Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italyc Neurology Unit, DIBIT-2, Institute of Experimental Neurology (INSPE), Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy

⁎ Corresponding author.E-mail address: [email protected] (L. Muzio).

1 These authors contributed equally to this work.2 Present address: Neurologic Clinic, Department

University, 00133 Rome, Italy.

1044-7431/$ – see front matter © 2009 Elsevier Inc. Adoi:10.1016/j.mcn.2009.11.008

a b s t r a c t
a r t i c l e i n f o
Article history:Received 30 April 2009Revised 12 November 2009Accepted 17 November 2009Available online 4 December 2009

Keywords:Cxcl10SvZNeural stem cell nichesMacrophagesEAE

The peri-ventricular area of the forebrain constitutes a preferential site of inflammation in multiple sclerosis,and the sub-ventricular zone (SvZ) is functionally altered in its animal model experimental autoimmuneencephalomyelitis (EAE). The reasons for this preferential localization are still poorly understood. We showhere that, in EAE mice, blood-derived macrophages, T and B cells and microglia (Mg) from the surroundingparenchyma preferentially accumulate within the SvZ, deranging its cytoarchitecture. We found that thechemokine Cxcl10 is constitutively expressed by a subset of cells within the SvZ, constituting a primarychemo-attractant signal for activated T cells. During EAE, T cells and macrophages infiltrating the SvZ in turnsecrete pro-inflammatory cytokines such as TNFα and IFNγ capable to induce Mg cells accumulation and SvZderangement. Accordingly, lentiviral-mediated over-expression of IFNγ or TNFα in the healthy SvZ mimicsMg/microglia recruitment occurring during EAE, while Cxcl10 over-expression in the SvZ is able to increasethe frequency of peri-ventricular inflammatory lesions only in EAE mice. Finally, we show, by RT-PCR and insitu hybridization, that Cxcl10 is expressed also in the healthy human SvZ, suggesting a possible molecularparallelism between multiple sclerosis and EAE.

© 2009 Elsevier Inc. All rights reserved.

Introduction

The identification of a number of mechanisms responsible forbrain repair has stimulated a new avenue of research aimed atidentifying the precise reciprocal relationships between the differentoperating parties. The functional response of adult neural stem/precursor cells (aNPCs) (self-renewal and multipotency) to inflam-mation has gained particular attention. The first evidence stronglysupporting the concept that aNPCs are capable to engage a finalisticdialogue with inflammatory cells has been shown in transplantationexperiments aimed at using aNPCs as therapeutic weapons againstcentral nervous system (CNS ) inflammation (Martino and Pluchino,2006, 2007; Pluchino et al., 2008; Pluchino et al., 2003; Pluchino et al.,2005). It was, in fact, shown that aNPCs express immune-relevantmolecules (e.g. cell adhesion molecules, integrins, chemokine recep-tors) enabling them to functionally interact with an inflamed CNSmicroenvironment (Monje et al., 2003; Ziv et al., 2006). More

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recently, it was shown that inflammatory signals, via activatedmicroglia (Mg) and/or antigen-specific T cells, may regulate neuro-genesis and gliogenesis within germinal CNS areas (Butovsky et al.,2006). Altogether these data support the concept that aNPCs may beconsidered also as bona fide immune-relevant cells in the brain. It isthen important to further explore the interaction between aNPCs andimmune-relevant cells not only to study the basic biological questionsrelated to the proliferative and differentiation capacities of aNPCs butalso because these cells – acting as patrolling and defending agents ofthe brain – may turn useful to translational medicine approachesaiming at treating acute and chronic CNS inflammatory disease via celltransplantation.

Here, we studied the interaction between blood-borne inflammatorycells, CNS-resident inflammatory cells such as Mg cells and sub-ventricular zone (SvZ) aNPCs in a mouse model of CNS-compartmental-ized inflammation,namelyexperimental autoimmuneencephalomyelitis(EAE) (Pluchino et al., 2003). Different timepoints after disease induction[20 days post-immunization (dpi), 45 and 60 dpi] were analyzed toreflect the different phases of the inflammatory process (Owens et al.,2001). As such, CNS inflammation peaks at 20 dpi (effector phase) andthen decreases by 60–70% from 60 dpi on (neurodegenerative phase)(Amadio et al., 2006;Owens et al., 2001; Politi et al., 2007).We found thataNPCs located within SvZ germinal niches are preferentially targeted by

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Fig. 1. The SvZ is morphologically deranged during acute and the chronic inflammation.Panel A shows a whole mount of the lateral ventricle, asterisk indicated the regionsampled in each experiment. (B) Confocal image of HC SvZ showing GFAP (green)putative type-B1 cells. These cells extend long bundles from the cell somas which areoriented toward the anterior pole of the forebrain. Vessels (CD31+ in red) run parallelto the ventricular surface and Mg (blue) cells appear ramified and homogenouslydistributed along the ventricular lining. (C) The SvZ of EAE 20-dpi mice appearsseverely deranged; indeed GFAP+ cells lose their oriented bundles and up-regulateGFAP expression. These cells acquire often the morphology of activated astrocytes andcluster around vessels. Amoeboid Iba1+ cells are often clustered around vessels. (D) InEAE 45-dpi mice, the vast majority of Iba-1+ cells return to their ramified morphology,but many GFAP+ cells remain associated to vessels (arrows). At 60 dpi, several GFAP+

cells remain in contact with vessels (arrows in panel E). These cells are characterized bythick and short somatic extensions that do not show any preferential orientation. Scalebar, 200 μm.

269L. Muzio et al. / Molecular and Cellular Neuroscience 43 (2010) 268–280

inflammatory blood-borne inflammatory cells respect to other regionsof the brain and that this is possibly due to the fact that cells of themouse SvZ constitutively express the chemokine Cxcl10/IP-10 whichis normally involved in controlling T cells migration and Th1polarization (Salomon et al., 2002; Wildbaum and Karin, 1999) ininflamed tissues and lymphoid organs (Luster et al., 1985; Taub et al.,1993). Cxcl10/IP-10 expression by aNPCs is further increased by pro-inflammatory cytokines released in situ by CNS-infiltrating blood-borne inflammatory cells and by resident microglial cells which, as a

results of this pro-inflammatory trigger, migrate toward the inflamedSvZ. Thus, the endogenous Cxcl10 secreted at the SvZ may play a keyrole in the mechanism involved in cells trafficking at the SvZ duringthe CNS inflammation.

Results

Inflammation leads to stem cells niches derangement

Previous studies have demonstrated that peri-ventricular bloodvessels, ependymal cells and adult neural precursors cells (aNPCs)are integrated in a precise functional architecture within neuralstem cells niches (Mirzadeh et al., 2008; Shen et al., 2008; Tavazoieet al., 2008). Given the complexity of this cytoarchitecture, we tookadvantage of whole mounts of the lateral ventricle use, in which itis possible to reconstruct the entire SvZ by orthogonal confocalstacks (Fig. 1A) (Mirzadeh et al., 2008). The effects of chronicneuroinflammation on this architecture were assayed on SvZexplants derived from healthy controls (HC) and MOG-immunizedEAE mice at 20, 45 and 60 dpi. Whole mounts were stained forGFAP, CD31 and Iba-1. As previously described (Mirzadeh et al.,2008), GFAP+ cells placed within the SvZ of HC explants (putativetype-B1 stem cells) extend tangentially oriented processes runningtowards the anterior pole, often taking contact with blood vessels,while Iba-1+ peri-ventricular Mg cells, showing the typicalmorphology of resting microglia, are homogenously placed through-out the ventricular surface (Fig. 1B). In contrast, SvZ whole mountsfrom EAE mice at 20 dpi exhibit a substantial disruption of thisarchitecture. Indeed, GFAP+ cells lose the oriented cell extensionsand often many of them are clustered around CD31+ blood vessels(Fig. 1C). Moreover, Iba-1+ Mg/macrophage cells change theirramified morphology acquiring the activated Mg/macrophagesamoeboid shape. These cells accumulate often around CD31+

vessels (Fig. 1C). Although inflammation fades out after 45 daysfrom the initial immunization (Pluchino et al., 2008), the SvZappears to be still substantially deranged at this time point. Indeed,we found many GFAP+ cells wrapped around CD31+ vessels, someof them showing a substantial alteration of their cell polarization(Fig. 1D). Iba1+ cells, however, at this time point loose theamoeboid morphology re-acquiring a ramified morphology. Similarresults were obtained when we studied whole mounts from EAEmice at 60 dpi (Fig. 1E), thus suggesting that GFAP+ cells cannotreturn to their normal morphology even long after active inflam-mation has vanished or indicating that smoldering inflammatorycues are still present.

The adult SvZ is extensively infiltrated by inflammatory cells duringacute EAE

To asses whether the SvZ may be a site of preferential inflammatorycells accumulation, we compared this region with peri-ventricularregions of the 3rd and 4th ventricle, i.e. the thalamus and the pons,respectively (Figs. 2A, A′ and A″). Coronal brain sections of both HC andEAE mice, at different time points, were probed for CD45 and vonWillebrand Factor (vWf). Infiltrating CD45+ cells are preferentiallyclustered at the SvZ,whilewedetected very few cells in theparenchymaaround to the 3rd and 4th ventricles (Figs. 2C, C′ and C″) and none in thehealthy brain (Figs. 2B, B′ and B″). SvZ-infiltrating cells are especiallyclustered at the ventricular lining and the vast majority of them are notconfined to peri-vascular spaces but appear to have crossed the brainblood barrier (BBB) (Fig. 2C). Although the number of inflammatorylesions is significantly reduced in EAE 60-dpi brains, we found thatseveral SvZs contain scattered CD45+ cells inside (Fig. 2D). In contrast,at this time point, few or any CD45+ cells were detected around the 3rdand 4th ventricles (Figs. 2D′ and D″).

Fig. 2. CD45, CD3 and Iba-1 cells accumulate at the SvZ during EAEmediated inflammation. Lowmagnifications of, respectively, dorsal SvZ (A), thalamus, adjacent to the 3rd ventricle(A′) and pons, adjacent to the 4th ventricle (A″). (B) While healthy brains did not show any CD45+ (red) cells in the SvZ, during acute EAE, these cells exit the vessels (stained withvWf, green) and many of them accumulated at the ventricular lining (arrowheads in C). However, their number was reduced at 60 dpi (arrowheads in D). Sections stained for CD3(red) and vWf (green) show similar results. Indeed, CD3+ cells are not detectable in the healthy SvZ (E), but accumulated in the EAE 20-dpi SvZ (arrows in panel F) and few of themare detectable in the EAE 60-dpi SvZ (arrow in G). Panel H shows restingmicroglia displaying well-ramifiedmorphology in the healthy SvZ (H), but in EAE 20-dpi brains many Iba-1+

cells accumulated in the SvZ changed their morphology and, overall, up-regulated the Iba-1 expression levels (I). Panel J shows the partial reversion of the phenotype at 60 dpi.Neither CD45+ nor CD3+ cells are detectable within regions adjacent to the 3rd or 4th ventricles of healthy brains (B′, B″, E′ and E″). EAE 20-dpi brains, however, show few CD45+ orCD3+ cells located around the 3rd ventricle of the thalamus (arrowheads in C′ and arrow in F′) and very few around the 4th ventricle in the pons (arrowhead in C″ and arrow in F″).These cells completely disappeared at 60 dpi in both thalamus and pons (D′, D″, G′ and G″). Iba-1 expression is only slightly up-regulated in EAE 20-dpi thalamus (I′) and pons (I″).Moreover, this phenotype completely reverted at 60 dpi (J′ and J″). Scale bar, 50 μm.

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To further characterize this phenotype, sections from EAE and HCmicewere also stained for CD3 and vWf.While CD3+ cells are virtuallyundetectable in the healthy brain (Figs. 2E, E′ and E″), many CD3+ cellsinfiltrate the dorsal SvZ of EAEmice at 20 dpi (Fig. 2F), but very few aredetectable within the parenchyma of the 3rd and the 4th ventricles(Figs. 2Fs. ′ and F″). The SvZ-infiltrating CD3+ cells are mainly placedfar from the vessels, often accumulating at the ventricular lining. Theirnumber is reduced at 60 dpi; however, several CD3+ cells are stilldetectable within the SvZ (Fig. 2G) but not within regions adjacent tothe 3rd and 4th ventricles (Figs. 2G′ and G″).

Since microglia activation has been extensively described in manyCNS pathologies (Kreutzberg, 1996), we studied the distribution ofIba-1+ cells along the anterior–posterior axes of the brain. As

previously described (Schwartz et al., 2006), Iba-1+ cells located inthe healthy tissue show a ramified morphology which has beenassociated to resting microglial cells (Figs. 2 H, H′ and H″). In contrast,EAE20-dpi SvZs showa tight accumulation of Iba-1+ cells displaying theamoeboid morphology that has been associated to activatedmicroglia/macrophages. Moreover, these cells display up-regulated Iba-1 expres-sion levels (Fig. 2I). In contrast, regions of the brain lining the 3rd and4th ventricles show reduced microglia activation and only few cellsover-expressing Iba-1 (Figs. 2I′ and I″). Also in this case, this phenotypepartially reverted at 60 dpi. Nevertheless, several cells located within ofthe SvZ maintain high expression levels of Iba-1 (Fig. 2J).

Blood-derived macrophages and CNS-resident microglial cells areindistinguishable within the inflamed tissue; thus, we used bone


marrow chimeric mice, in which blood-derived cells are constitutivelyexpressing GFP, to study the distribution of these cells within the SvZ.Bone marrow cells from donors UBI-GFP/BL6 mice (Schaefer et al.,2001)were transplanted into lethally irradiated C57BL/6Jmice (Simardand Rivest, 2004; Simard et al., 2006). Since irradiation, per se, mayinduce transient alteration of the BBB permeability (Yuan et al., 2003),EAEwas induced2months after irradiation/transplantation treatments.Mice were then sacrificed at 20 and 60 dpi and EAE brains comparedwith healthy controls transplanted mice, hereafter referred as HC-BMTmice. We detected a large number of GFP+ cells within the SvZs of EAEmice at 20dpi (Figs. 3A–D), fewGFP+ cells in the SvZ of EAE 60-dpimice(not shown) and none in the SvZs of HC-BMT (Fig. 3E).We confirmedthat several of these GFP+ infiltrating cells are CD3+ T cells (Fig. 3A),very few are B220+ B cells (Fig. 3B) and the vast majority of themexpress Iba-1 and CD45 antigens (Figs. 3C and D). A substantial fractionof Iba-1+ cells placed in the SvZ, however, are not GFP+, thus suggestingthat many of them may derive from the adjacent parenchyma. Duringinflammation, however, blood cells can enter into the CNS through thechoroid plexus (Engelhardt et al., 2001; Ransohoff et al., 2003). Thus,BMT mice were also analyzed for the presence of GFP+ cells in thechoroidplexuses.WhileHC-BMTmice show few, if any, GFP+cells in theplexus (Fig. 3F), some GFP+ cells are detectable within this structure inmice affected by EAE at 20 dpi. These cells express CD45 (Fig. 3G) andIba-1 (Fig. 3H), but their very low abundance is hardly compatible withthe plexus being the primary entry site for SvZ-infiltrating cells.

Since in EAE inflammatory cells accumulation in the brain isassociated to demyelination, we studied the ultrastructure of the dorsalSvZ by transmission electron microscopy (TEM). Semi-thin sections ofthe dorsal SvZ allowed the visualization of inflammatory lesions(Figs. 4A and B). TEM highmagnification analysis revealed the presenceof many axons that were either naked or surrounded by poorlycompacted myelin sheets (Figs. 4C–I). We found, often, large inflam-matory cells closely located to the deranged myelin (Figs. 4C, E, F, G).

Parenchymal Mg cells are attracted within the inflamed SvZ

Since we found that a large number of Mg/macrophagesaccumulating in the SvZ of EAE mice at 20 dpi are GFP−, we

Fig. 3. Blood stream inflammatory cells infiltrate the SvZ during acute EAE. Panel A shows conlymphocytes infiltrates the SvZ. Panel B shows few b220+ cells (red) in the inflamed SvZ (arrothe Mg/macrophages lineage and are Iba1+ (red) (C) or CD45+ (red) (D). Healthy controlsSvZ. Healthy choroid plexus present few or any GFP+ cells (F). EAE 20-dpi brains show(arrowheads in H). Scale bar, 50 μm.

investigated if they derived from cell proliferation or migration fromadjacent brain areas. Mice were injected with the thymidine analog5′-Iododeoxyuridine (IddU), which is incorporated into proliferatingcells DNA (Takahashi et al., 1992, 1993, 1994).We found no CD3/IddU(Figs. 5A and B) and few Iba1/IddU (Figs. 5C and D) double positivecells, thus ruling out in situ proliferation of lymphocytes and Mg/macrophages. To investigate if the inflamed SvZ may attract theparenchymal Mg cells, we generated primaryMg cell cultures from P2C57BL/6 brains. Cells cultures were assayed for purity by flowcytometry (Fig. 5E) and subsequently infected with a lentivirusexpressing the GFP gene. After testing the GFP expression, they werestereotaxically injected into the striatal parenchyma of both HC andEAE mice at 20 dpi (Fig. 5E). Forty-eight hours later, mice weresacrificed, and one section every 30 μm from each brainwas processedfor GFP detection. Since GFP fills cell processes, we identified manyramified GFP+ cells at the site of injection of both HC and EAE mice at20 dpi (Fig. 5F). Only in EAEmice, many GFP+ cells were recruited alsowithin the dorsal SvZ .Thus, these cells are able to spread out along theanterior–posterior axis and to reach the dorsal SvZ located severalhundred μm far from the injection point (Figs. 5G–J). Taken together,these results suggest the presence of some chemo-attractant cueswhich are able to recruit Mg cells and, possibly, blood cells intothe SvZ.

Pro-inflammatory cytokines and chemokines are over-expressedwithin the inflamed SvZ

Microglial cells attraction and activation at the dorsal ventricularliningmay be a consequence of pro-inflammatory cytokines secretion,i.e. TNFα and IFNγ, by early infiltrating inflammatory T cells andmacrophages. Since SvZ infiltration by inflammatory cells during EAEis discontinuous, we measured these pro-inflammatory cytokines inBMT-EAE chimeric mice comparing SvZ areas displaying, or not,inflammatory cells infiltration. Infiltrated vs. not infiltrated SvZ areasare identified by the presence or absence of GFP+ cells (Fig. 6A). EAE-BMT 20-dpi SvZs were microdissected (Fig. 6B) and GFP+ and GFP−

microdissections were subsequently analyzed by real-time PCR. Eachmicrodissection pool was validated for the presence of the neural

focal images of double immuno-staining for CD3 (red) and GFP (green). A large influx ofw in B). The vast majority of the GFP+ cells locatedwithin the inflamed SvZ belonged to(E) and EAE mice at 60 dpi (not shown) did not show any infiltrating GFP+ cells in thefew GFP+ cells in the plexus that expressed either CD45 (arrowhead in G) or Iba-1

Fig. 4. EM analysis of the EAE SvZ revealed the presence of several demyelinating fibers.Panels A and B show transverse coronal sections and panels C–I the ultrastructure of thedorsal SvZ (D), where many inflammatory cells are evident (arrows in panels A and B).At highmagnification, most axons are normally myelinated but a consistent number arenaked (arrowheads in C, F, G and H). Note a thinly myelinated axon (arrow in I).Inflammatory cells are closely located to damaged fibers (asterisks in C, D, E and G).Scale bars: 10 μm for semi thin sections and 1 μm for ultra-thin sections.


stem cell marker Prom1 (Barraud et al., 2007) and GFP mRNA by RT-PCR (Figs. 6C and D). As expected, TNFα and IFNγ mRNAs aresignificantly up-regulated within the infiltrated SvZs (Fig. 6C).Interestingly, the transcripts of Cxcl10 and Ccl2, two chemokinesknown to be involved in the recruitment of lymphocytes and Mg/

macrophages during EAE (Mennicken et al., 1999), are significantlyover-expressed in these same SvZ extracts (Fig. 6C). Since TNFα(Yeruva et al., 2008) and IFNγ mediated CXCL10 and CCL2transcription is mediated by STAT1 phosphorylation (Stark et al.,1998), we determined by immunoblot analysis on dorsal SVZ explants(n=3 per group) that STAT1 was indeed phosphorylated within SvZsof EAE mice at 20 dpi (Fig. 6D).

TNFα and IFNγ expression at the SvZ are sufficient forMg/macrophages recruitment

To confirm that the release of TNFα or IFNγ within the SvZ is ableto drive the expression of chemo-attractant cues and in turn to recruitMG/macrophages at this site, we stereotaxically injected healthyC57BL/6 mice into the ventricular cavity with lentiviral vectorsexpressing TNFα, IFNγ or GFP (Fig. 7A). Ten days after the injections,efficient infection of the ependymal layer adjacent to SvZs wasconfirmed by radioactive in situ hybridization for the respectivetransgenes (Figs. 7B–D). As predicted, by probing adjacent sections,we found that Cxcl10 mRNA levels were up-regulated within theTNFα-infected (Fig. 7F) and, especially, in IFNγ-infected SvZs(Fig. 7G), as compared to brains infected with the GFP-expressingcontrol virus (Fig. 7E). Concordantly, TNFα (Fig. 7I) or IFNγ-infectedSvZs (Fig. 7J) displayed dramatic accumulation of Iba1+ Mg/macrophages. Over-expression of TNFα also induced some Iba1/IddU double positive cells, thus suggesting this cytokine capable toprompt in situ Mg proliferation (Fig. 7I). We microdissected, from1 mm coronal slices (0 mm to −1 mm relative to Bregma), the SvZsfrom TNFα, IFNγ, GFP and sham-injected mice after 4 and 10 daysfrom the lentivirus injections. We measured, by real-time RT-PCR, theexpression of a panel of chemokines. As expected, in sham- and GFP-injected SvZs, we detected basal levels of Cxcl12 (Tran et al., 2004)but, surprisingly, also of Cxcl10 (Fig. 7K). Four days after TNFα or IFNγinjections, Cxcl10 rapidly increased its expression levels, and westarted to detect also Cxcl9 and Cxcl11 (Fig. 7K). Ten days afterlentivirus injections, Cxcl10 exhibited the highest expression levels ofall analyzed chemokines. At this time point, however, both TNFα andIFNγ are able to induce a substantial up-regulation of Cxcl9 andCxcl11 and a slight up-regulation of CCL5 and CCl1 (Fig. 7K). Cxcl10may be rapidly induced by pro-inflammatory cytokines, thussuggesting that it may participate to the early recruitment/activationof the endogenous microglia at the SvZ.

The healthy SVZ expresses basal levels of Cxcl10 and attractsinflammatory cells

Thus, once the inflammation is robustly activated within the SvZ,macrophages and lymphocytes exert a crucial role in attractingparenchymal Mg because they secrete chemo-attractant cues at thesesites. The reasons why the dorsal SvZ is a preferential site of earlyinflammatory cells penetration are, however, still unknown. Wehypothesized that during EAE, activated inflammatory cells, the onlycells able to cross the BBB (Engelhardt, 2008), are selectively attractedinto the SvZ by chemo-attractant cues expressed by local residentcells. Since the SvZ contains aNPCs, it is possible that cues released bythese cells may act as recruiting signals since the early phases ofinflammation. In order to test this hypothesis, HC mice were injectedwith activated T cells purified from ubiquitously GFP-expressingtransgenic mice. Twelve hours later, mice were sacrificed andforebrain sections were probed for CD3 (Supplementary Fig. 1A)and GFP (not shown). We found several CD3+ cells scatteredthroughout the entire SvZ, while none or very few were detected inthe striatum and thalamus. However, SvZs of mice treated withunstimulated splenocytes showed only very few CD3+ cells (Supple-mentary Fig. 1B). Taken together these results suggest the presence ofchemo-attractant signals in the healthy SvZ.

Fig. 5. Parenchymal Mg cells migrate toward the inflamed SvZs. Panels A–D show confocal images of double immuno-fluorescence for IddU/CD3 (A and B) and Iddu/Iba1 (B and C).None CD3+ (red) cells are detected within HC SvZs (A). Many CD3+ cells are found in EAE 20-dpi SvZs (arrows in C) but rarely co-localized with IddU+ (green) cells. Few or anyIba1+ cells (red) incorporate the IddU tracer in the HC SvZ (C) and only a small number of them incorporated the IddU tracer in the EAE 20-dpi SvZ (arrowheads in panel C). PrimaryMg cells obtained from P2 C57BL6/J were transplanted in the striatal parenchyma. Before use, cells were assayed for CD11b expression by flow cytometry (inset in panel E shows thatmore than 80% of these cells were Cd11b positive) and labeled with a lentivirus expressing the GFP. Approximately 5×104–1×105 mg cells were stereotaxically injected in brains ofHC and EAE 20-dpi mice at the following coordinates: B, +1; L, +2.2; and D, −3. Forty-eight hours later, GFP+ Mg cells are easily detectable at the site of injection in both HC andEAEmice (high and lowmagnification images showing the injection site are reported in F). GFP+ cells are also detected within the SvZ of EAEmice (H–J). (G) Representative sagittalsections showing three different planes along the anterior–posterior axes of an EAE 20-dpi brain, which containsmany GFP+ cell in the SvZ. Panels H–J show themicrographs relativeto these planes and arrows indicate GFP+ cells. Scale bar, 50 μm.


Indeed, as anticipated above, when we examined LCM micro-dissected SvZs (Supplementary Fig. 2A) by RT-PCR, we confirmedCxcl10 basal expression (Fig. 8A and Supplementary Fig. 2B), whileadjacent striata were negative. Further, we found that Cxcl10 is alsoexpressed by cultured SvZ-derived adult neural precursor cells(aNPCs) (n=3 independent cell lines) both as transcript and assecreted protein (Figs. 8A and B). In situ hybridization experimentsconfirmed that Cxcl10 expression is confined to a subpopulation ofcells scattered through the healthy SvZ, but not in the striatum,cerebral cortex or caudal brain regions (Figs. 8C and D and notshown). We then looked at the SvZ of EAE mice at 20 dpi and wefound that Cxcl10 expression was dramatically increased within theSvZ both by RT-PCR and by in situ hybridization paralleling the up-regulation mediated by IFNγ and TNFα lentiviruses (Figs. 8A and F).Cxcl10 expression within the SvZ, however, reverted to basal levelsat later time points (60 dpi), when inflammation fades out (notshown). Moreover, also aNPCs are capable to up-regulate the Cxcl10mRNA expression and secretion if stimulated with pro-inflammatorycytokines (Fig. 8B).

To test if Cxcl10 produced at the SvZ may recruit inflammatorycells during CNS inflammation, we generated a bidirectional lentivirus

expressing both Cxcl10 and GFP genes (named Cxcl10/GFP). Micewere injected with the Cxcl10/GFP or with the GFP lentiviruses andthen fivemice from each groupwere subsequently immunized. Brainsfrom EAE mice were collected at 20 dpi and compared with infectedhealthy brains. Lentivirus infections were validated by probingcoronal sections for GFP (Figs. 9A–C). Healthy mice infected withboth Cxcl10/GFP and GFP lentiviruses exhibited normal brainmorphology and they show only few CD45+ cells located at theventricular lining possibly due to the injection injury (Fig. 9D and datanot shown). On the other hand, in mice affected by EAE, the presenceof an exogenous source of Cxcl10 resulted in a significant increment ofCD45+ peri-vascular cuffs (Figs. 9F and I). CD45+ inflammatory cellswere preferentially located around the infected ependymal layer andseveral Iba-1+ Mg/macrophages inflammatory cuffs were closelylocated to Cxcl10/GFP+ ependymal cells (Supplementary Fig. 3C).Moreover, Cxcl10 over-expression triggers a significant increment ofthe corpus callosum demyelination. In fact, the average number persection of peri-ventricular demyelinating plaques was significantlyincreased when mice were injected with Cxcl10/GFP lentiviruses(Figs. 9G, H and J). However, these lesions appeared as previouslydescribed, i.e. round in shape with the typical peri-vascular

Fig. 6. Inflamed EAE 20-dpi SvZ cells over-express both TNFα and IFNγ. (A and B) HC and EAE bone marrow chimeric mice, expressing the GFP in virtually all blood-derived cells, aresacrificed at 20 dpi and used to generate two serial coronal sections of the anterior forebrain. (A) One representation is used to identify SvZs areas targeted by GFP+ cells (green). (B)Parallel section that are used for laser capture microdissection of the SvZ (box indicates the sampled area). Histogram in panel C showing both pools of microdissections, containingor not GFP+ cells that are used for a real-time PCR assay. Data express the fold changes (log2) of GFP, IFNg, TNFa, Cxcl10 and Ccl2, Cxcl10 and Ccl2 transcripts (±SEM) detected in GFP+

pools over GFP− pools. Data are normalized on the total amount of the housekeeping histone H3 mRNA. Pro-inflammatory cytokines IFNγ and TNFα transcripts were strongly up-regulated within the GFP+ pools. Panel D shows the RT-PCR for Prom1on LCM captured SvZs, total RNA from aNPCs cell cultures was used as positive control. Panel D showsimmunoblot analysis for the phosphorylation of the STAT1 protein at the Tyr-701 site on HC and EAE 20-dpi SvZs.While Phospho-Stat1 (Tyr-701) is undetectable in SvZ extracts fromHCmice, we found a robust expression in all extracts derived from EAEmice at 20 dpi. For each experiment, the total amount of protein blotted in each lanewas normalized by anti-β-actin antibody. Significant differences were marked as follows: ⁎Pb0.05; ⁎⁎Pb0.01; ⁎⁎⁎Pb0.001, t-test. Scale bar, 100 μm.


accumulation of cells (Merkler et al., 2006). Taken together, theseresults show that Cxcl10 can trigger activated blood-derived cells toenter the CNS during an active inflammation as in the case of EAE.However, in the absence of peripherally activated inflammatory cells,neither CD45+ cells infiltration nor Mg/macrophages activation/migration were detected. This result implies that the endogenousCxcl10 expression levels detected in the SvZ may sustain only theneural stem cells niches homeostasis.

CXCL10 is expressed by human peri-ventricular cells

In human multiple sclerosis, peri-ventricular areas are a typical sitefor inflammatory lesions. To verify whether SvZ recruitment of Mg/macrophages during human neuroinflammation may be driven byCXCL10 expression in the SvZ, we dissected human brain specimensobtained postmortem, from non-neurological patients, and we gener-ated coronal sections (Supplementary Fig. 4A).Haematoxylin stainingofthese sections confirmed the previously described cyto-organization ofthe human SvZ (Sanai et al., 2004) (Supplementary Fig. 4B). Radioactivein situ hybridization for the housekeeping gene βACTIN, performed toverify tissue preservation, showed intense signals in virtually all SvZ

Fig. 7. IFNγ and TNFα over-expressions in the SvZ induce the Cxcl10 up-regulation andexperimental plan, IFNγ, TNFα and GFP lentiviruses were injected within healthy brains bylater, mice were IddU administered (10 h) before the sacrifice. Panels B–D show coronal sectriboprobes (insets show high magnifications of the lateral ventricles). Panels E–G show in sithe Cxcl10 slightly up-regulation in TNFα SvZs. Panel F shows its strongly up-regulation in IFIba-1(red) and IddU (green). While Iba 1+Mg/macrophages cells do not accumulate at the vIFNγ (J)-infected SvZs. Iba1+ cells rarely incorporate the IddU tracer in GFP-infected br(arrowheads in I). A global down-regulation of the SvZ proliferation is seen in IFNγ-infectedpanel H shows a real-time PCR analysis of a panel of chemokines in mice injected with: vehic10 days from the lentivirus injection. Each data is normalized on the total amount of the hactivated with ConA. Scale bar, 50 μm.

cells (Supplementary Fig. 4C). Adjacent sections probed for CXCL10mRNA showed scattered CXCL10+ cells mainly located beneath thehypo-cellular gap. Positive cells appeared sometimes organized inclusters (Supplementary Figs. 4D and E), closely resembling thoseobserved within the mouse brain and suggesting a molecularparallelism between mice and human SvZs. Moreover, RT-PCR on totalRNA extracted from microdissections of the peri-ventricular regionobtained from non-neurological patients confirmed CXCL10 expressionwithin the SvZ (Supplementary Fig. 4G).

Discussion

Several studies have demonstrated the pivotal role of chemokinesand their receptors during embryonic life, indeed chemokinesignaling is involved in controlling neuronal migration during braindevelopment (Zou et al., 1998). As a matter of fact, during forebraindevelopment, migrating cells need to travel for a long distance andsecreted chemokines play a crucial role in driving this migration.Recent studies suggest that SDF-1 and its receptor CxcR4 are cruciallyinvolved in routing interneurons during their pallial migration (TranandMiller, 2003; Tran et al., 2004). Although few data are available on

the Mg cells accumulation at the SvZ. Panel A shows the schematic paradigm of thestereotaxic device at the following coordinates: B, +0; L, +0.8; and D, −2.5. Ten daysions from GFP-, IFNγ- and TNFα-infected brains, respectively, probed with their specifictu hybridization for Cxcl10 on GFP (E), TNFα (F) and IFNγ infected SvZs. Panel G showsNγ infected SvZs. Panels H–J show confocal images of double immuno-fluorescence forentricular lining of GFP-infected brains (H), many Iba1+ cells infiltrate the TNFα (I)- andains but several Iba1/IddU double positive cells are detected in TNFα-infected SvZsSvZs (J), although few Iba1+ cells captured the IddU tracer (arrows in J). Histogram inle (sham group), GFP, TNFα and IFNγ lentiviruses. Total RNAs was collected after 4 andousekeeping gene Histone H3 and fold changes±SD are calculated respect splenocytes


chemokines expression within the healthy adult brain neurogenicareas (Imitola et al., 2004; Robin et al., 2006), it may very well be thatthis signaling machinery is maintained by SvZ precursors throughout

adult life. As a partial confirmation of this, it has been shown thatCxcR4, CcR2, CcR5 and the Cxcl10 receptor CxcR3 are expressed byaNPCs descendants into the SvZ (Tran et al., 2007) and that Cxcl10 as

Fig. 8. The healthy SvZ contains cells that constructively express Cxcl10. Panel A shows a real-time RT-PCR for Cxcl10 on total RNA extracted from: SvZs and striata from healthy andEAE 20-dpi mice, untreated aNPCs and aNPCs stimulated with TNFα 200 U/ml or IFNγ (500 U/ml). Data presented are expressed as relative fold changes±SEM over SvZ fromhealthy mice. Data are normalized on the total amount of the housekeeping Histone H3 mRNA. Panel B shows ELISA assay for CXCL10 on aNPCs supernatants obtained from cellstreated for 24 hwith: vehicle, TNFα or IFNγ as above. Data are expressed as pg/ml±SEM. Cytokine stimulation significantly up-regulates CXCl10 release from aNPCs. Panels C, D andF show in situ hybridization on HC and EAE 20-dpi sections probed for Cxcl10 detection. Parallel sections from each sample hybridized with the Cxcl10 sense probe did not reveal anyhybridization pattern as shown in panel E. Panels C and D show several Cxcl10+ cells confined within the healthy SvZ (arrowheads in C and D). Panel F shows Cxcl10 mRNAexpression in EAE 20-dpi brain. Many Cxcl10+ cells are located around the ventricle possibly deriving from the blood stream. However, some cells in the SvZ strongly up-regulatedtheir Cxcl10 mRNA levels (arrow in F). Scale bar, 100 μm. Significant differences were marked as follows: ⁎Pb0.05; ⁎⁎Pb0.01; ⁎⁎⁎Pb0.001, t-test.


well as other chemokines has a chemo-attractant effect on aNPCscultured from postnatal SvZ (Tran et al., 2007). Using in situhybridization experiments, here we show several Cxcl10+ cellslocated beneath the ependymal layer of the SvZ in both human andmouse healthy brains. Upon on that, we found that Cxcl10 isexpressed also in vitro by resting aNPCs thus supporting the conceptthat there are chemokines physiologically produced by a subset ofSvZ-resident aNPCs.

Is this a relevant phenomenon during primary inflammatory eventspecifically targeting the CNS? The current view suggests that duringCNS neuroinflammation, brain-damaged areas become a source ofchemokines and that chemokines over-production triggers ‘regener-ative’ neural progenitor cells to migrate toward these lesions(Belmadani et al., 2006). However, the constitutive expression ofchemokines by aNPCs resident within the SvZ, and in particularof those chemoattracting blood-borne inflammatory cells such asCxcl10, may suggest that aNPCs might, per se, represent a preferentialtarget of inflammatory processes occurring within the CNS. We andothers recently showed that inflammatory signals drive aNPCs into aquiescent state so to block their proliferation and differentiationcapacity (Pluchino et al., 2008; Wang et al., 2008). The data reportedhere further support this latter hypothesis and provide a mechanisticexplanation of such phenomenon. We, in fact, showed that dorsallySvZ aNPCs expressing Cxcl10 are capable to attract activatedinflammatory CD45/CD3 cells from the blood. Once inflammatorycells are recruited within the SVZ, they start secreting in situ largeamounts of pro-inflammatory cytokines, such as IFNγ and TNFα,

which, in turn, not only further increase Cxcl10 expression by dorsallySvZ aNPCs but also attract toward the SvZ parenchymal areas,activated microglia capable per se, to secrete pro-inflammatorycytokines. As a matter of fact, the final result of this auto-excitatoryloop is the morphological derangement of the SvZ cytoarchitecturewhich severely compromise the ‘regenerative’ properties of residentstem cell elements. In our mice, the CNS parenchyma surroundinggerminal areas was particularly damaged (e.g. demyelination of thedorsal corpus callosum). All these data are further corroborated bypassive transfer experiments and by experiments aimed at over-expressing Cxcl10, by lentiviral vector technology, within the peri-ventricular areas. The fact that Cxcl10 over-expression in healthybrains does not induce resting Mg accumulation at the SvZ confirmstheir unresponsiveness and further stresses the concept of the pivotalrole of the auto-excitatory loop that is ignited by CNS-confined (Tcells) and CNS-resident (e.g. Mg) inflammatory cells (Luster et al.,1985).

During EAE, we found the expression in the SvZ of Ccl2, achemokine that has been implicated in the regulation of the BBBendothelial cells permeability by altering tight junctions (Stamatovicet al., 2003; Stamatovic et al., 2005), thuspotentially leading to the site-specific increase of the inflammatory cell influx. Although we cannotexclude the contribution of other attracting signals (Bartholomaus etal., 2009) or other local conditions favoring inflammatory cellsdiapedesis in the SvZ, we showed that a considerable number of SvZ-infiltrating Mg/macrophages is not of haematogenous origin butoriginates from the surrounding parenchyma. This event further

Fig. 9. Cxcl10 over-expression increased both inflammatory lesions and demyelination of peri-ventricular areas in mice affected by EAE. A bidirectional lentivirus codifying for bothGFP and Cxcl10 genes and a lentivirus codifying only for the GFP gene are injected in the ventricular cavity of the forebrain of naïve mice by stereotaxic device at the followingcoordinates: B, +0; L, +0.8; and D,−2.5. Panels A–C show immuno-fluorescence for the GFP detection in healthy GFP-infected brain (A), EAE at 20-dpi GFP-infected brain (B) andEAE 20 -dpi Cxcl10/GFP-infected brain (C). GFP+ cells are easily detectable in the ependymal layer of both GFP and Cxcl10/GFP-injected mice (arrowheads in B and C). Panels D–Fshow an immuno-histochemistry for CD45 (red) in healthy GFP (D), EAE 20-dpi GFP-infected and EAE 20-dpi Cxcl10/GFP-infected brains. Very few CD45 expressing cells are foundin healthy GFP SvZs (D), some CD45+ cuffs are detected in EAE 20 -dpi GFP-infected mice (arrows in E); however, their number significantly increased in EAE 20 -dpi Cxcl10/GFP-infected mice (arrows in F) and histogram in panel I. Panels G and H show double immuno-fluorescence for MBP (red) and neurofilament (green) in EAE 20 -dpi GFP-infected (G)and EAE 20 -dpi Cxcl10/GFP-infected (H) brains. Demyelination was detected for the presence of neurofilament staining and for the absence of the MBP staining. Both EAE 20-dpiinfected mice showed demyelinating plaques; however, demyelination was significantly increased in EAE mice that received the Cxcl10/GFP lentiviruses as shown in histogram (J).Scale bar, 200 μm. ⁎Pb0.05; ⁎⁎⁎Pb0.001, t-test.


contributes to the cell congestion of this brain area and to the severemorphological derangement of neural stem cells niches.

Although it is still far from being elucidated, our data suggest thataNPCs might became a target of CNS-compartmentalized inflamma-tion because of their constitutive expression of chemo attractantsmolecules as a relic of an early developmental mechanism thatregulates tissue (re)generation in the embryo. This not only stressesthe immune relevance of aNPCs but also supports the concept that aninflammatory CNS microenvironment might shape aNPC behaviorand provide an explanation of tissue distribution of inflammatorylesions in multifocal diseases of the CNS such as multiple sclerosis.

While, at first glance, the immune and the neural stem cell systemsappear quite separate in their aims and operations, deeper reflectionleads to the realization that interactions between the two systemsmight actually have important consequences for evolution andhealth.

Thus, this study provides evidence of a basal Cxcl10 expression inthe SvZ thatmight represent an important element of a self-sustainingloop contributing to explain the preferential peri-ventricular locali-zation of forebrain lesions in EAE. The relevance for human MS ofthese findings is underlined by the overlapping SvZs CXCL10expression pattern that we found in human brain specimens,


suggesting that a similar molecular and cellular chain of events mighttake place in the SvZs of MS patients.

Experimental methods

EAE induction

Chronic EAE was induced in C57BL/6 mice and bone marrowchimeric mice, by subcutaneous immunization with 300 μl of 200 μgMOG35-55 (Espikem, Florence) in incomplete Freund's adjuvantsupplemented with 8 mg/ml Mycobacterium tuberculosis (strainH37Ra; Difco). Pertussis toxin (Sigma) (500 ng) was injected on theday of the immunization and again two days later, as described(Amadio et al., 2006; Pluchino et al., 2003). Body weight and clinicalscore (0=healthy; 1=limp tail; 2=ataxia and/or paresis ofhindlimbs; 3=paralysis of hind limbs and/or paresis of forelimbs;4=tetraparalysis; 5=moribund or death) were recorded daily. EAEmice were compared with control groups composed of untreatedmice after 20, 45 and 60 days post-immunization (dpi). Mice werekilled at each time point by anesthetic overdose and transcardiallyperfused with 4% paraformaldehyde in PBS pH 7.2. Dissected brainswere post-fixed in the same solution for 12 h at +4 °C and thencryoprotected for at least 24 h in 30% Sucrose (Sigma) in PBS at +4 °C.All efforts were made to minimize animal suffering and to reduce thenumber of mice used, in accordance with the European CommunitiesCouncil Directive of November 24, 1986 (86/609/EEC). All proceduresinvolving animals were performed according to the guidelines of theInstitutional Animal Care and Use Committee of the San RaffaeleScientific Institute.

In situ hybridization

In situ hybridization were performed as previously described(Centonze et al., 2009; Pluchino et al., 2008). The following riboprobeswere used: IFNγ was generated from nt 10 to nt 1200 based onGenBank data NM_008337, TNFα was generated from nt 15 to nt 600based on GenBank data NM_013693, GFP was generated from nt 5 tont 600 based on GenBank accession no. U55762, mouse Cxcl10 wasgenerated from nt to nt 76 to nt 582 based on Genebank NM_021274,human CXCL10 was generated from nt 387 to nt 1063 based onGenebank NM_001565 and human βACTINwas a gift of V. Capra (U.O.Neurochirurgia, Istituto G. Gaslini, Genoa, Italy). Microphotographs ofone section every 280 μm, ranging from bregma +1.3 to bregma −6,were digitalized in dark field lightmicroscopy (Olympus BX51, and 4×objective) by using a CCD camera (Leica). Images manipulations wereperformed by using Adobe Photoshop CS. To confirm the specificity ofthe different RNA probes, sense strand RNA probes (showing nosignal) were used as negative controls.

Bone marrow chimeric mice

C57BL/6 female mice were used as recipients for all bone marrowtransplant (BMT) experiments. Donor mice were transgenic UBI-GFP/BL6 (Schaefer et al., 2001) female. The recipient mice were 6-8 weeksold and the donors' age was 4–6 weeks. Recipient mice to betransplanted were sublethally exposed to a single dose of 1100 R froma Cesium source. Mouse bone marrow cells were harvested from UBI-GFP/BL6 (Schaefer et al., 2001) mice by flushing femurs and tibiae.Then, cells were injected (2×106 cells/mouse) into the tail vein ofrecipient female. Twomounts after, 100 μl of total blood was collectedfrom the tail vein of BMT mice. Then cells were analyzed for GFPdetection by flow cytometry. Only those mice with a percentage ofchimerism N70% were immunized for EAE induction (three indepen-dent experiments, n=4 mice for each group) as described above.Brains were collected either at 20 or 60 dpi. For each experiment,healthy BMT mice were used as control. Forebrains were entirely cut

as above described and one section every 100 μmwas used for doubleimmuno-fluorescence. For laser capturer experiments, brains wereserially cut and one series was stained to identify SVZ regionsinfiltrated by GFP+ cells.

Splenocytes transplant experiments

Splenocytes from UBI-GFP/BL6 mice (Schaefer et al., 2001) werestimulated in vitro with αCD3 (R&D, 5 μg/ml) and αCD28 (R&D, 1 μg/ml) for 3 days and subsequently injected in healthy control mice inwhich Pertussis toxin (Sigma, 500 ng) was i.p. administered the sameday of the transplant (n=3,40×106 cells/mouse). As negative control,freshly isolated GFP+ splenocyteswere injected in healthy control micetreated with the Pertussis toxin (n=3,40×106 cells/mouse). Twelvehours after the transplants, brains were fixed as above described andcoronal sections were used for GFP and CD3 detections by immuno-fluorescence. Cells were counted in a region spanning from the anteriorbregma+1.3 to theposterior bregma−5. Themean value of CD3+ cellsper SVZ±S.E.M was plotted on histogram.

Laser capturer microdissection, real-time PCR and RT-PCR

At sacrifice, mice were deeply anesthetized via an intra-peritonealinjection of chloralium hydrate 8% and then were rapidly perfusedtranscardially with RNase free 0.9% saline containing 10 U/ml ofheparin. Brains were removed from the skulls, OCT embedded andrapidly frozen by immersion in liquid nitrogen. Then, laser microdis-section was performed as previously described (Pluchino et al., 2008).Detailed protocols and primers sequences are provided in thesupplementary method sections.

Lentiviruses injections for in vivo studies

Vesicular stomatitis virus-pseudotyped lentivirus (LV) generation:the entire coding region of mouse IFNγwas obtained by PCR based onpublished GenBank data NM_008337. The TNFα coding region was agift of A. Corti (San Raffaele Hospital, Milan, Italy). We cloned the twocoding regions into the BamHI and SalI site of pCCL.sin.cPPT.PGK.GFP.WPRE gently provided by L. Naldini (San Raffaele Hospital, Milan,Italy) to create LV-PGK-IFNγ and LV-PGK-TNFα lentiviruses. MouseCxcl10 was amplified by PCR based on GenBank data NM_021274and cloned in #945pCCL.sin.CPPT.SV40polyA.eGFPminCMV-hPGKWpre (provided by Dr. L. Naldini, San Raffaele Hospital, Milan,Italy) lentivirus to create the Cxcl10/GFP lentivirus. Detailed methodsfor both lentiviral stocks preparation and delivery are provided in thesupplementary method sections.

Immunofluorescence and immuno-histochemistry

Detailed protocols for both immuno-fluorescence and immuno-histochemistry are provided in the supplementary method sections.The following antibodies were used: rabbit α Iba-1 (WakoChemicals), rabbit α-von Willebrand Factor (Abcam), rabbit α-glialfibrillary acidic protein (GFAP, Dako), mouse α-glial fibrillary acidicprotein (GFAP, Chemicon), rabbit α-CD3 (Abcam), rat α-CD3(Serotec), rat α-b220 (Becton Dickinson), chicken α-GFP (Chemi-con), rabbit α-GFP (Molecular probes), rat α-CD31 (BD), rat α CD45(Abcam), rabbit α MBP and mouse α NF (Millipore). Appropriatefluorophore-conjugated (Alexa Fluor 488, 546 and 633, MolecularProbes) or biotin-conjugated (e.g., biotin-streptavidin amplificationkit, Vector-Labs) secondary antibodies were used. Nuclei werestained with 4′-6-diamidino-2-phenylindole (DAPI, Roche). Light(Olympus, BX51 with 4× and 20× objectives) and confocal (Leica,SP5 with 40× objective) microscopy was performed to analyze tissuestaining. Analyses were performed by using Leica LCS lite and AdobePhotoshop CS software.


Human fixed specimens were obtained from non-neurologicalpatients (from the Pathology Department, San Raffaele Hospital,Milan, Italy), rinsed in PBS, fixed in paraformaldehyde 4% andcryoprotected in PBS, sucrose 30% solution for cryostat sectioning.Then, tissue was frozen in OCT compound and 10 μm sections weregenerated.

SvZ whole-mount dissection

EAE 20, 45 60 dpi and HC mice (n=4 per group) were killed andsaline perfused as above described. After that, the hippocampus wasdiscarded, brain was cut along the rostro-caudal axes and the SvZwas removed by microdissecting scissor. Dissected lateral hemi-spheres were fixed 3 h in 4% paraformaldehyde on ice. Then,explants were washed three times in PBS 1×, 0.5% Triton X-100.Explants were incubated in PBX 1×, BSA 1 mg/ml, FBS 10% andTriton X-100 0.3% for 1 h at +4 °C. Primary antibodies were dilutedin the same buffer and incubated over night a +4 °C. The next day,explants were washed in PBS 1× Triton X-100 0.3% and propersecondary antibodies conjugated with Alexa Fluor 488, 546 and 633dyes were applied in PBX 1×, BSA 1 mg/ml, FBS 10%, Triton X-1000.3% for 3 h at room temperature. After staining, these explants werefurther dissected to remove the neural parenchyma and mounted oncoverslip. Confocal reconstructions of the SvZ were taken on LeicaSP5 microscope.

Primary microglia cell cultures and stereotaxic injections

Primary Mg cultures were obtained from C57Bl/6 mice (P0-P2).In brief, meninges were removed from the forebrain and tissue wasminced with scissors in cold KRB medium 1× containing albumin0.3% (Sigma) and MgCl2 0.04% (Sigma). Cortices were incubated in10 ml of complete KRB medium supplemented with Trypsin0.25 mg/ml at 37 °C for 15 min, then 10 ml of complete KRBmedium supplemented with DNase I 0.05 mg/ml (Sigma) and SBTrypsin inhibitor 0.08 mg/ml (Sigma) to stop the reaction. Then, cellsuspension was washed in glial culture medium DMEM (Gibco)supplemented with 10% FBS (Gibco), L-glutamine (1 mM) (Gibco),penicillin (100 U/ml) (Gibco) and streptomycin (100 mg/ml)(Gibco), and 3×106/flask cells were plated in the same medium ina humidified incubator with 5% CO2 in poly lysine treated 75 cm2

Corning tissue flasks. Soon after plating, the primary mixed glial cellcultures were infected with pCCL.sin.cPPT.PGK.GFP.WPRE lentivirus(a gift of Dr. L. Naldini, San Raffaele Hospital, Milan, Italy). Mediumwas changed after 3 days in culture. Microglia cells were shaken offthe glial mixed cell culture after 12 days. For each experiment, cellswere stained using anti-CD11b fluorescent antibodies (BectonDickinson). Then, cells were analyzed on a FacsCanto (BD) and50,000 events were acquired.

EAE 20 dpi and HC mice (n=3 for each group) were anesthetizedwith 2,2,2-tribromoethanol (10mg/ml; 1/27 of body weight) and thehead was placed in a stereotaxic apparatus (mod 900, Kopf). Then,5×104 to105 GFP+ microglial cells were suspended in 1 μl of salineand stereotaxically engrafted within the right striatum at thefollowing coordinates: A, +1; L, +2.2; and D,−3. The cell suspensionwas delivered at a rate of 0.1 μl/min by using an infusion pump(Harvard apparatus). Two days later, brains were processed asfollows: brains were removed and fixed as previously described andserial 10-μm coronal cryosections were generated starting from theolfactory bulbs. One section every 30 μm was processed for GFPdetection by immuno-fluorescence.

Adult NPCs in vitro experiments

Primary aNPCs cell lines were established from the lateralventricles wall of 4-week-old C57BL/6 mice as previously described

(Reynolds and Weiss, 1992). Detailed protocols are provided in thesupplementary method sections.

Electron microscopy

Semi-thin and ultra-thin morphological analyses were conductedas previously described (Consiglio et al., 2001). Mice were anesthe-tized intraperitoneally as above described and perfused with 4%paraformaldehyde in PBS. Cross-sections were obtained from brain,then post-fixed in 0.12 M phosphate buffer supplemented with 2%glutaraldehyde and divided in three parts. These parts were thensectioned transversely into 2-mm blocks, post-fixed with osmiumtetroxide and embed in Epon (Fluka, Buchs, Switzerland). Cross-sections were cut from the anterior to the posterior part.

Statistics

Bar graphs represent the mean value±SEM or the mean value±SD. Data were analyzed using Student's test (GraphPad PRISM, USA).Significance was accepted when Pb0.05.

Disclosure of potential conflicts of interest

The authors indicate no potential conflict of interest.

Acknowledgments

We thank Dr. F. Sanvito and Prof. Doglioni for their technicalassistance. This study was supported by the Fondazione Italiana SclerosiMultipla and BMW Italia. This investigation was supported in part by agrant from the National Multiple Sclerosis Society award no. rg4016a2/1.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.mcn.2009.11.008.

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Cxcl10 enhances blood cells migration in the sub-ventricular zone of mice affected by experimental autoimmune encephalomyelitis