Proc. Natl. Acad. Sci. USAVol. 92, pp. 4323-4327, May 1995Neurobiology

Neural cell adhesion molecule (N-CAM) inhibits astrocyteproliferation after injury to different regions of the adultrat brain

(reactive glia/regeneration/S100 protein/gliosis/homophilic binding)

LESLIE A. KRUSHEL*t, OLAF SPORNSt, BRUCE A. CUNNINGHAM*, KATHRYN L. CROSSIN*,AND GERALD M. EDELMAN*t*Department of Neurobiology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037; and tThe Neurosciences Institute, 3377 NorthTorrey Pines Court, La Jolla, CA 92037

Contributed by Gerald M. Edelman, January 6, 1995

ABSTRACT After a penetrating lesion in the centralnervous system, astrocytes enlarge, divide, and participate increating an environment that adversely affects neuronal re-generation. We have recently shown that the neural celladhesion molecule (N-CAM) partially inhibits the division ofearly postnatal rat astrocytes in vitro. In the present study, wedemonstrate that addition of N-CAM, the third immunoglob-ulin-like domain of N-CAM, or a synthetic decapeptide cor-responding to a putative homophilic binding site in N-CAMpartially inhibits astrocyte proliferation after a stab lesion inthe adult rat brain. Animals were lesioned in the cerebralcortex, hippocampus, or striatum with a Hamilton syringeand needle at defined stereotaxic positions. On one side, thelesions were concomitantly infused with N-CAM or with oneof the N-CAM-related molecules. As a control, a peptide of thesame composition as the N-CAM decapeptide but of randomsequence was infused on the contralateral side ofthe brain.Weconsistently found that the population of dividing astrocyteswas significantly smaller on the side in which N-CAM or oneof the N-CAM-related molecules was infused than on theopposite side. The inhibition was greatest in the cortical lesionsites ('50%) and was less pronounced in the hippocampus(-25%) and striatum ("20%). Two weeks after the lesion, thecerebral cortical sites infused with N-CAM continued toexhibit a significantly smaller population of dividing astro-cytes than the sites on the opposite side. When N-CAM andbasic fibroblast growth factor, which is known to stimulateastrocyte division in vitro, were coinfused into cortical lesionsites, astrocyte proliferation was still inhibited. These resultssuggest the hypothesis that, by reducing glial proliferation,N-CAM or its peptides may help create an environment thatis more suitable for neuronal regeneration.

After a penetrating injury in the central nervous system (CNS),astrocytes transform into reactive glia, whose major morpho-logical changes include an increase in cell volume, an increasein the number and extension of cell processes, and a dramaticupregulation in the expression of glial fibrillary acidic protein(GFAP) (1, 2). Reactive glia divide, migrate, surround thelesion, and contribute to the formation of a glial scar. Modelsystems of glial scarring in vitro reveal that reactive gliaproduce both growth-promoting and growth-inhibiting mole-cules (3, 4). These events tend to inhibit mature neurons fromextending neurites (2, 5, 6). Neurons from the CNS retain theirability to extend neurites (7) despite the fact that reactive glia(or even mature glia) in vitro are poor substrates for axonalgrowth (8, 9). This suggests that it is the environment local tothe lesion that is inhibitory to axon regeneration and that

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reducing the formation of a glial scar resulting from thebuild-up of astrocytes may render the environment less hostileto neurite outgrowth.

Initiation of astrocyte proliferation appears to result fromthe action of cytokines released by invading macrophages andby the glia themselves (10-12). Astrocyte cell division peaks1-2 days after a CNS lesion (13, 14). It is unknown what factorsdownregulate glial proliferation in vivo, although various mol-ecules have been implicated as potential inhibitors in vitro (15,16).The interactions of neurons and glia appear to depend on

cell and substrate adhesion molecules (CAMs and substrateadhesion molecules). These molecules are dynamically regu-lated in distinct spatiotemporal patterns during developmentand their appearance is correlated with cell division, migra-tion, and differentiation (17). Alterations in the expression ofCAMs and substrate adhesion molecules, including the up-regulation of the neural cell adhesion molecule (N-CAM), areseen after neural injury (refs. 18-20 and L.A.K., G.M.E., andK.L.C., unpublished observations). These observations suggestthat CAMs may be involved in the various cellular responsesthat accompany neural injury. It is known that astrocytesdisplay N-CAM on the cell surface (17). Prompted by theseobservations, we have shown (21) that N-CAM inhibits pro-liferation of early postnatal rat astrocytes in vitro.

In the present study, we investigated whether the addition ofN-CAM at the time of a stab lesion in an adult rat brain could alsoinhibit glial proliferation in vivo. N-CAM, one of its domains, anda peptide derived from a putative homophilic binding site ofN-CAM were all able partially to inhibit astrocyte proliferation invivo in three brain regions: cerebral cortex, hippocampus, andstriatum. These results raise the possibility that N-CAM or itspeptides may prove useful for inhibiting gliosis in vivo.

MATERIALS AND METHODSPurification of Proteins and Peptides. The purification of

N-CAM from early postnatal rat brains and the synthesis ofpeptides were done as described (21). To produce the thirdimmunoglobulin-like (Ig) domain of chicken N-CAM, a seg-ment including nt 259-540 of clone pEC 208 (22) was gener-ated by PCR amplification, and its DNA sequence was con-firmed and inserted into the Nco I-BamHI sites of pET3d(Novagen). The recombinant protein was affinity-purified ona column of monoclonal antibody CAM1-Sepharose (23).N-CAMs from different species bind with each other (24) andthe ready availability of the chicken clone recommended itsuse.

Abbreviations: GFAP, glial fibrillary acidic protein; N-CAM, neuralcell adhesion molecule; Ig, immunoglobulin-like; BrdU, bromode-oxyuridine; bFGF, basic fibroblast growth factor; CNS, central ner-vous system.

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Stereotaxic Surgery. Adult male Wistar albino rats (CharlesRiver Breeding Laboratories) were anesthetized with xylazine (12mg/kg) and ketamine (90 mg/kg). Bilateral infusions with a26-gauge needle and Hamilton syringe were done at the followingcoordinates: Hippocampus: anterior-posterior (A-P), -3.30mm; lateral (L), ±+1.60 mm; ventral (V), -3.20 mm. Cerebralcortex: A-P, +0.70 mm; L, ±2.70 mm; V, -1.60 mm, at an angleof 20° to the mediosagittal plane. Striatum: A-P, +0.70 mm; L,±2.60 mm; V, -4.70 mm. Each animal received an infusion of thepurified rat N-CAM (250 ,tg/ml), the third Ig domain of chickenN-CAM (5 mg/ml), or the rat N-CAM peptide KHIFSDDSSE(1 mg/ml) in PBS into one hemisphere; the other hemisphere wasinfused with the random peptide (SFSISDEDHK, 1 mg/ml inPBS) as a control. The experimental and control sides variedbetween animals and choice of side had no significant effect onthe results. Three microliters of each protein or peptide wasinfused at a rate of 0.2 ,l/min after which the needle was left inplace for 5 min and then removed. In one experiment, a group ofanimals received basic fibroblast growth factor (bFGF, 450 ng) insolutions that contained either the random peptide or the purifiedN-CAM.

Labeling ofDividing Cells and Immunocytochemistry. Eachanimal received five intraperitoneal injections of bromode-oxyuridine (BrdU)/0.007 M NaOH (100 mg/kg) at -2, 16,28,42, and 54 h after the surgery. Heavily anesthetized animalswere perfused 72 h after surgery with ice-cold 4% (wt/vol)paraformaldehyde/PBS. The brains were removed, post-fixed,placed in sucrose, and sectioned on a cryostat. Immunocyto-chemistry of tissue sections with antibodies against GFAP,S100, and BrdU was done as described (25-27).

Cellular Analysis. Coronal sections (10 ,um) were madethrough the entire lesion site. Three of these sections 40 ,tmapart from each other and containing the lesion site werechosen for analysis (Fig. 1). Individual sections included bothcontrol and experimental lesion sites, ensuring consistentcomparisons of staining between these two conditions. Inaddition to the region encompassing the needle track, tissuealong the plane of section was examined for 300 Am on eitherside of the track (Fig. 1). Within this demarcated area, the total

10OLm

FIG. 1. Schematic representation (not to scale) of the lesion siteshowing the coronal tissue sections that were used and the area withineach section that was examined to determine the distribution ofmitotically active astrocytes.

number of astrocytes (S100 or GFAP positive), and astrocyteslabeled for BrdU were counted. Approximately 150-300 as-trocytes were counted per section. The percentage of totallabeled astrocytes (S100 or GFAP positive) that were alsolabeled with BrdU was taken to be the proportion of dividingastrocytes at the time of the BrdU injections and is referred toas the labeling index.

RESULTS

Double-Labeling of Cells with BrdU and S100 or GFAP.Three days after lesioning, numerous nonglial cells (BrdUpositive/S100 negative) were seen at the lesion site in all areas.The density of these labeled cells decreased dramatically awayfrom the site. An example from the cerebral cortex is seen inFig. 2A. The majority of these cells were likely invadingmacrophages and resident microglia (10, 28, 29); this conclu-sion was consistent with the labeling results described below.Table 1 summarizes the cumulative observations made for allareas.

Astrocytes in the area of the lesion were detected by S100 andGFAP antibody staining. Immunofluorescent labeling with theS100 antibody showed that all astrocytes stained with equalintensity, including reactive astrocytes, which were detected bytheir increased cell area (Fig. 2 A and B; refs. 26 and 30).Ependymal cells adjacent to the ventricles were also positive forS100 (26), but the lesion sites were placed at distances far fromthe ventricles so these cells were not included in the analysis.Colorimetric staining of astrocytes for GFAP, the expression ofwhich is dramatically increased in reactive astrocytes, showedintense staining around the site of the lesion (data not shown).Although lightly stained GFAP cells could be seen throughoutthe brain, most of the heavily stained GFAP cells were within'300 ,am on either side of the lesion site. Because both immu-nocytochemical staining methods (colorimetric and fluorescentstaining) showed equivalent numbers of double-labeled cells, theresults of both methods were combined. However, astrocyteperikarya were found to be more prominent with S100 fluores-cent labeling, producing easily detectable double-labeled glialcells.

After cumulative BrdU injections, '20% of the astrocyteswere also labeled with BrdU (Fig. 2A and B and Table 1). Thisobservation suggested that a subpopulation of astrocytes wasactively dividing at some time during the 72-h period aftersurgery. These dividing astrocytes were restricted mainly to theregion surrounding but not directly inside the needle track andwere markedly reduced in number at a distance of 300 ,mfrom the lesion site (Fig. 2A). It was for this reason that aregion extending for 300 ,tm around the lesion site (Fig. 1) waschosen for analysis of the number of astrocytes that werepositive or negative for BrdU. Only cells whose entire nucleuswas labeled were counted. This was due to the sensitivity of theBrdU labeling that may lead to false positives because BrdUcan be incorporated into mitochondrial DNA and during DNArepair.N-CAM Effects on Glial Proliferation in Various Brain

Regions. The number of BrdU-labeled astrocytes was signif-icantly less in structures that received N-CAM or some portionof N-CAM compared to the same structures on the side thatreceived the random peptide (Table 1 and Fig. 3). This effectwas consistent for all the groups of animals studied. The effectof N-CAM was most dramatic in the cerebral cortex (Table 1).In the cortex, all three N-CAM proteins were able to inhibitthe number of BrdU-labeled astrocytes by -30-50%, with thewhole N-CAM molecule having the most potent effect. Infu-sions of the whole N-CAM molecule into the cortex at a lowerdose of 100 ,tg/ml inhibited astrocyte proliferation by 0-10%;at 150 ,ug/ml, the inhibition ranged between 4 and 20%.

In the hippocampus, -25-27% fewer BrdU-labeled astro-cytes were found at the site infused with the whole N-CAM

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molecule or its peptide compared to controls; in this brainregion, the third Ig domain recombinant protein decreased thenumber of dividing astrocytes by only 15%. The N-CAMmolecule had the least effect in the striatum: 15-20% fewerBrdU-labeled astrocytes were seen on the side that hadreceived N-CAM infusions.Longer-Term Effects of N-CAM on Cortical Astrocyte

Response. The effects ofN-CAM on the population of dividingastrocytes were examined after a more extended survivalperiod. When animals lesioned in the cerebral cortex andconcomitantly infused with N-CAM were examined 2 weeksafter the lesion, the needle track was reduced in size comparedto the same site 3 days after the lesion. A slender glial scardelineated the needle track as shown by fibrous S100 stainingin the lesioned regions that had received N-CAM or therandom peptide (Fig. 2C). After 2 weeks, there were fewerBrdU-positive/S100-negative cells remaining within andaround the needle track. Consistent with the responses ob-served after 72 h the number of BrdU-labeled astrocytes wasreduced by 37.4% on the side that had received N-CAMcompared to the side that had received the random peptide.

Effects of N-CAM in the Presence of Exogenous bFGF.bFGF is a potent mitogen for astrocytes in vitro (21, 31), andafter a lesion in vivo, production of bFGF and its receptor isdramatically upregulated in glia (12). However, other studiesshowed little or no increase in the number of dividing astro-cytes in response to a single infusion of bFGF in vivo in theadult rat (29). To confirm this and to determine whether theaddition of bFGF could counteract the ability of N-CAM toinhibit astrocyte division, we added the growth factor with andwithout N-CAM. Bilateral lesions were made in the cerebralcortex and a 3-,ul solution containing bFGF (450 ng) and eitherN-CAM or the random peptide was infused. A 72-h survivalperiod was used in these experiments.

In the cortices that had received bFGF plus the randompeptide or N-CAM (Fig. 4B), there was an increase of 114%in the number of nonastrocytes (BrdU positive/S100 negative)compared to the cortices that received only N-CAM or therandom peptide (Fig. 4A), suggesting increased recruitment orcell division. These cells were likely blood-borne macrophagesor resident microglia (10, 28, 29). However, there was only asmall increase in the number of dividing astrocytes in thecortices that received bFGF plus the random peptide com-pared to random peptide alone (24.3 vs. 21.7%). Addition ofN-CAM plus bFGF reduced the proportion of dividing astro-cytes by 52 ± 5% as compared to the region that receivedbFGF and the random peptide. This reduction is comparableto that observed when N-CAM was added in the absence ofbFGF.

DISCUSSIONThis study has shown that a single application at the time of astab lesion of the entire rat N-CAM molecule, a bacterialrecombinant protein corresponding to the third Ig domain ofchicken N-CAM, or a peptide corresponding to a segment inthe third Ig domain of rat N-CAM can partially inhibit theastrocyte proliferative response 72 h after the lesion. Theeffect was observed in three regions of the adult rat brain, the

FIG. 2. Low-power (A) and high-power (B) photomicrographs ofa coronal section of the rat cerebral cortex 3 days after a stab lesionand concomitant infusion of the random peptide. (C) High-powerphotomicrograph of a coronal section of the rat cerebral cortex 14 daysafter such a lesion. The tissue was labeled with anti-S100 (Texas red)and anti-BrdU (fluorescein) antibodies and visualized with a filterwhose excitation and emission wavelengths encompassed both Texasred and fluorescein. Double-labeled cells representing dividing astro-cytes are seen as yellow or very bright green cells. (Bar in C: A, 190,/m; B, 43 ,gm; C, 43 ,um.)

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Table 1. Labeling index after various treatments following a stab injury

Infusion Labeling index %Brain region (experimental) Control Experimental Difference

Cerebral cortex N-CAM 21.3 ± 1.2 10.2 ± 1.5 -52.1 ± 4.5Third Ig domain 24.9 ± 2.9 13.9 ± 1.9 -44.3 ± 9.4N-CAM peptide 18.9 ± 4.8 13.2 + 3.8 -30.4 ± 7.8

Hippocampus N-CAM 19.6 ± 3.3 14.6 ± 2.3 -25.2 + 1.6Third Ig domain 18.4 ± 2.6 15.6 + 2.2 -15.0 ± 3.1N-CAM peptide 19.9 + 2.1 14.5 ± 2.0 -27.1 ± 2.9

Striatum N-CAM 26.1 ± 0.7 20.7 ± 0.7 -20.8 ± 2.1Third Ig domain 24.3 ± 5.2 19.6 ± 3.9 -19.5 ± 5.1N-CAM peptide 19.1 ± 0.9 16.3 ± 0.9 -14.5 ± 2.3

Data are presented as mean ± SEM (n = 3 for each condition). All comparisons of the labeling indicesbetween the N-CAM or N-CAM-related molecules and the random peptide within each brain region werefound to be significant (P < 0.05; paired sample Student's t test).

cerebral cortex, the hippocampus, and the striatum. Infusion ofN-CAM into the cortex produced the most robust inhibition ofastrocyte cell division and the inhibitory effects of a singleinfusion remained evident 2 weeks after the lesion. The partialinhibition of astrocyte proliferation was not reversed whenbFGF was coinfused with purified N-CAM.Although N-CAM had a significant inhibitory effect on

astrocyte cell division, the infusion of N-CAM did not appearto alter the morphological changes that also accompany reac-tive gliosis. These changes include an increase in astrocyte cellvolume, in extension and number of cellular processes, and inGFAP expression (1, 2). Reactive glia in the N-CAM-treatedregions appeared to increase their levels of GFAP and sur-round the lesion site in a manner similar to that which wasobserved in the control lesion, although, on the side receivingN-CAM and its related peptides, the number of BrdU-labeledastrocytes was reduced.The finding that '20% of the astrocyte population was

dividing during the 72-h period after surgery is similar to theobservations of other workers (13, 14), although some groupshave seen a smaller dividing cell population (32-34). Thevariation may be due to methodological differences, includingthe employment of fewer injections of a birthdate marker andthe use of [3H]thymidine, which is only detected in the mostsuperficial portion of the sectioned tissue after autoradiogra-phy, in contrast to BrdU, which is visualized throughout the

Cerebral Cortex Hippocampus Striatum100-

X 75

O3 50 T

_I25.

Random Whole 3rd Ig NCAM Whole 3rd Ig NCAM Whole 3rd Ig NCAMPeptide NCAM Domain Peptide NCAM Domain Peptide NCAM Domain Peptide

NCAM NCAM NCAM

FIG. 3. Labeling index of dividing astrocytes infused with ratN-CAM, the third Ig domain of chicken N-CAM, or the peptidederived from the third Ig domain of rat N-CAM in the cerebral cortex,hippocampus, or striatum 3 days after the lesion (n = 3 for eachexperimental condition; data are presented as mean + SEM). Thelabeling index is defined as the percentage of labeled astrocytes (S100or GFAP positive) that were also BrdU-positive. For each brainregion, the labeling index for the control condition (infusion of therandom peptide) was set to 100%. In all cases, there were significantlyfewer (P < 0.05; paired sample Student's t test) dividing astrocytesafter infusion of N-CAM or related molecules as compared to thecontrol condition.

tissue (25, 35). A number of other parameters that mayinfluence the variance in the data include the area analyzed fordividing astrocytes, the form and size of the lesion, the use ofS100 vs. GFAP as an astrocyte marker, and the length ofsurvival after the lesion.Our central finding was that N-CAM partially inhibited

astrocyte cell division in the three forebrain regions examined.Nevertheless, there was regional variability in the extent towhich N-CAM or its related peptides can inhibit glial prolif-eration. The strongest effects ('50% inhibition) were seen inthe cerebral cortex with smaller effects in the hippocampusand striatum. Astrocytes from different regions of the brainhave been shown to have variable responses to injury (14, 36).In addition, differences in the ability of the injected moleculesto diffuse within a structure may contribute to this variation.Overall, the entire N-CAM molecule was the most efficaciousin its ability to inhibit astrocyte proliferation even though it wasinjected at a lower concentration than the N-CAM peptide andthe recombinant third Ig domain. This is consistent with ourprevious studies on the inhibition of astrocyte proliferation byN-CAM in vitro (21). More extensive studies of these regionsand comparisons of detailed dose-response curves will berequired to explain the differences observed with differentN-CAM reagents.A number of factors are known to affect glial proliferation

after injury. Gonadal hormones, including both testosteroneand estrogen, have been shown to inhibit glial proliferationafter a stab lesion (27). It is not known whether the intracel-

FIG. 4. Photomicrographs of a coronal section of the rat cerebralcortex 3 days after a stab lesion that was infused with the randompeptide (A) or the random peptide plus bFGF (B). The tissue waslabeled with anti-BrdU antibodies and a fluorescein-conjugated sec-ondary antibody. (Bar in B = 43 ,Lm.)

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lular signaling pathway affected by N-CAM has elements incommon with that of the gonadotropins. In any case, it wouldbe revealing to ascertain whether the effects of N-CAM andthe hormones are additive. In the present experiments, theaddition of another agent, exogenous bFGF, did not signifi-cantly increase the percentage of dividing astrocytes and theinhibitory effects of N-CAM were seen despite its presence.Although bFGF is a potent mitogen for astrocytes in vitro (21,31), there may be other constraints regulating astrocyte pro-liferation in vivo, including a paucity of bFGF receptors onastrocytes at the time of the lesion (12). Indeed, consistent withthe present findings, other studies have shown that a singleinfusion of bFGF at the time of a stab lesion did not increasethe number of astrocytes (29).

It is clear from these examples that a large variety of factorsare involved in the response to CNS injury and a particular celltype may simultaneously have both detrimental and supportiveeffects on the neuronal environment. For example, althoughthe transformation into reactive astrocytes appears to beinhibitory to neurite outgrowth, these cells produce trophicfactors that enhance neuronal survival (3, 37). Furthermore,these cells may help reestablish the blood-brain barrier (38),as some BrdU-labeled astrocytes were seen contacting bloodvessels (29, 39). Similarly, while activation of microglia leads tothe secretion of factors involved in the local inflammatoryresponse and the promotion of reactive gliosis, these factorscan also enhance neuronal survival (37, 40). Oligodendrocyteshave been shown to have cell surface components that inhibitneurite outgrowth and isolated oligodendrocyte proteins in-cluding those associated with myelin will not support neuriteoutgrowth in vitro (41-43).

Given the multiplicity of cellular responses to neural injury,long-term studies will be required to assess whether exogenousN-CAM has a useful role in promoting a more permissiveenvironment for axonal regeneration. In particular, experi-ments are required to show differences in long-term glialscarring between animals receiving N-CAM or the randompeptide. It should be stressed that the effects of a singleinfusion observed here reduced astrocyte proliferation by atmost 50% (Table 1). Manipulation of timing and of reagentsmay enhance this effect. A useful paradigm would includeinsertion of a permanent canula infusing N-CAM at differenttimes after the lesion. This may help to reveal the optimal timeafter a lesion at which N-CAM is able most effectively toinhibit astrocyte cell division and whether the constant pres-ence of exogenous N-CAM would increase the inhibition.Whatever the case, further biochemical studies of the effectsof N-CAM on astrocytes both in vivo and in vitro are neededto explore the possibility that exogenous N-CAM or its deriv-atives may help to prevent gliosis.We thank Dr. John Hemperly for the N-CAM monoclonal antibody

and Mr. Nguyen Tran, Ms. Olga Ryabinin, and Mr. Herbert Scherl forexcellent technical assistance. This work was supported by U.S. PublicHealth Service Grants HD-09635 (G.M.E.) and HD-16550 (B.A.C.)and a grant from the G. Harold and Leila Y. Mathers CharitableFoundation (G.M.E.). O.S. is a W. M. Keck Foundation Fellow at TheNeurosciences Institute.

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