RELATIONSHIP BETWEEN SPROUTING AXONS, PROTEOGLYCANSAND GLIAL CELLS FOLLOWING UNILATERAL NIGROSTRIATAL

AXOTOMY IN THE ADULT RAT

L. D. F. MOON,* R. A. ASHER, K. E. RHODES and J. W. FAWCETTPhysiological Laboratory, University of Cambridge, Downing Site, Tennis Court Road, Cambridge CB2 3EG, UK

AbstractöProteoglycans may modulate axon growth in the intact and injured adult mammalian CNS. Here we inves-tigate the distribution and time course of deposition of a range of proteoglycans between 4 and 14 days followingunilateral axotomy of the nigrostriatal tract in anaesthetised adult rats. Immunolabelling using a variety of antibodieswas used to examine the response of heparan sulphate proteoglycans, chondroitin sulphate proteoglycans and keratansulphate proteoglycans. We observed that many proteoglycans became abundant between 1 and 2 weeks post-axotomy.Heparan sulphate proteoglycans were predominantly found within the lesion core (populated by blood vessels, amoeboidmacrophages and meningeal ¢broblasts) whereas chondroitin sulphate proteoglycans and keratan sulphate proteoglycanswere predominantly found in the lesion surround (populated by reactive astrocytes, activated microglia and adultprecursor cells). Immunolabelling indicated that cut dopaminergic nigral axons sprouted proli¢cally within the lesioncore but rarely grew into the lesion surround.

We conclude that sprouting of cut dopaminergic nigral axons may be supported by heparan sulphate proteoglycans butrestricted by chondroitin sulphate proteoglycans and keratan sulphate proteoglycans. ß 2002 IBRO. Published byElsevier Science Ltd. All rights reserved.

Key words: heparan sulphate, chondroitin sulphate, keratan sulphate, nigra, regeneration, nigrostriatal.

Following CNS injury in the adult mammal, axons failto regrow through regions of scar formation containing anumber of di¡erent chondroitin sulphate (CS), heparansulphate (HS) and, possibly, keratan sulphate (KS) pro-teoglycans (PGs) (Bovolenta et al., 1992, 1993; McKeonet al., 1995; Grill et al., 1998; Asher et al., 1999, 2000a;McKeon et al., 1999). Numerous experiments indicatethat these PGs are growth modulatory and may contrib-ute towards the failure of spontaneous CNS axon regen-eration. For example, CSPGs are present in boundaryand guidance regions during development and are usu-ally growth inhibitory (Asher et al., 2000b). KSPGs arealso present in boundary and guidance structures indevelopment (Snow et al., 1990b; Geisert et al., 1992;Geisert and Bidanset, 1993; Gonzalez et al., 1993;McAdams and McLoon, 1995) although these have var-

iable e¡ects upon neurite growth in vitro (Snow et al.,1990a; Maeda and Noda, 1996). In contrast, develop-mental studies (Halfter, 1993) and cell culture experi-ments (Wang and Denburg, 1992; Faissner et al.,1994; Walz et al., 1997) indicate that HSPGs are oftengrowth-permissive molecules.

Prior to developing interventionist experiments toexamine the e¡ect of PGs on CNS axon regenerationin vivo, we set out to determine the distribution andtime course of appearance of various CSPGs, KSPGsand HSPGs following unilateral axotomy of the nigro-striatal tract in anaesthetised adult rats. Brains wererecovered between 4 and 14 days post-axotomy, sec-tioned and immunolabelled to visualise the response of,and relationships between PGs, glia and dopaminergicnigral axons.

EXPERIMENTAL PROCEDURES

Animal care

All procedures involving animals were carried out in accor-dance with the UK Animals (Scienti¢c Procedures) Act 1986and associated guidelines. E¡orts were made to minimise thenumber of animals used in these experiments. The followingmeasures were taken to minimise pain and discomfort. Animalswere housed in groups with playthings on a 12-h light^darkcycle and were allowed to feed and drink freely. Animals werehandled, inspected and weighed daily. Postoperative diet wassupplemented with dog food and wet mash, and where necessaryto counteract dehydration, animals were given 10 ml 4% glucose

101

*Correspondence to: L.D.F. Moon, The Miami Project to CureParalysis, P.O. Box 16960, Mail Locator R-48, Miami, FL 33101,USA. Tel. : +1-305-243-7137; fax: +1-305-243-3923.E-mail address: lmoon@miamiproject.med.miami.edu(L. D. F. Moon).Abbreviations: bFGF, basic ¢broblast growth factor; BSA, bovine

serum albumin; CR3, complement receptor 3; CS, chondroitinsulphate; DRG, dorsal root ganglion; DSHB, DevelopmentalStudies Hybridoma Bank; FGF, ¢broblast growth factor;GAG, glycosaminoglycan; GFAP, glial ¢brillary acidic protein;HS, heparan sulphate; Ig, immunoglobulin; KS, keratan sul-phate; NCAM, neural cell adhesion molecule; PBS, phosphatebu¡ered saline; PG, proteoglycan; TH, tyrosine hydroxylase.

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in 0.18% saline subcutaneously during surgery and thereafter asrequired. For analgesia, animals were given soluble paracetamol(1 mg/ml) in their drinking water for at least 3 days postoper-atively.

General surgical details

Twelve female adult Sprague^Dawley rats (bred in-house),weighing between 200 and 250 g were given knife cut lesionsof the right nigrostriatal tract (Fig. 1). Rats were anaesthetisedusing halothane (5%) in carrier (oxygen, 2 l/min) and transferredto a stereotaxic frame (David Kopf Instruments, USA) with theincisor bar set 2.3 mm below the interaural line. Anaesthesiawas maintained thereafter with halothane (1.5^2.0%) in carrier(oxygen, 0.6 l/min) with inhalational analgesic (nitrous oxide,0.6 l/min). The preshaved scalp was washed with 70% ethanoland painted with antiseptic ointment (Betadine) prior to making

a midline incision, retracting the skin and clearing the perios-teum from the cranium. A dental drill with size 3 drill bit wasused to remove small pieces of skull where necessary. All stereo-taxic coordinates are measured in millimetres with anterior andlateral coordinates made relative to bregma and vertical coor-dinates made relative to dura. Following surgery, the woundwas closed with absorbable sutures (Vicryl 4/0, Ethicon, UK)and antiseptic powder was applied.

Unilateral nigrostriatal axotomy

The right medial forebrain bundle (including the nigrostriataltract) was transected using an extruding wire `Scouten' knife(David Kopf Instruments, USA) as follows. The tip was loweredto A =33.0, L = +3.0, V =38.0 and the wire blade was extrudedsuch that it formed a smooth curve in the coronal plane reach-ing medially to the midline and ventrally to the base of thebrain. The assembly was withdrawn vertically by 4 mm andthe blade retracted and reextruded. Finally, the assembly wasrelowered by 4 mm, the blade was retracted and the entireassembly was withdrawn from the brain. This procedure twicetransects the right nigrostriatal tract approximately 650 Wmanterior to the substantia nigra and 4 mm posterior to the prox-imal striatal border (Brecknell et al., 1995).

Histology

Animals were killed by overdose of anaesthesic (2 ml/kg i.p.,Euthatal, Roche Meriaux) either 4, 7 or 14 days post-axotomy(n = 4 per time point). At each time point, two animals wereperfused using 4% paraformaldehyde in phosphate bu¡ered sa-line (PBS) and brains were post¢xed overnight prior to immers-ing in cryoprotectant (30% sucrose in PBS) until sunk. Tenconsecutive series of one-in-ten 40 Wm-thick parasagittal sectionsof brain were then cut on a sledge microtome, prior to immu-noperoxidase labelling as free-£oating sections. The remaining

Fig. 1. Schematic of parasagittal section through adult rat brainshowing site of the unilateral knife cut (kc) placed the medial fore-brain bundle between the substantia nigra (sn) and the ipsilateral

striatum (str). Scale bar = 2 mm.

Table 1. Primary antibodies : speci¢city, type, concentration and source

Antibody name Speci¢city Type Concentration Source

2B6 Epitope created following chondroitinase-ABCmediated degradation of chondroitin-4 sulphateglycosaminoglycans

mouse monoclonal 1:200 Seikagaku1:1000

3B3 Epitope created following chondroitinase-ABCmediated degradation of chondroitin-6 sulphateglycosaminoglycans

mouse monoclonal 1:200 Seikagaku1:1000

CS-56 Epitope present in some intact chondroitin sulphateglycosaminoglycans

mouse monoclonal 1:500 Sigma1:100

1D1 Neurocan CSPG core protein mouse monoclonal 1:200 DSHB1G2 Neurocan CSPG core protein mouse monoclonal 1:1 Gift of A.

Oohira3F8 Phosphacan CSPG core protein mouse monoclonal 1:200 DSHBD31-10 NG2 CSPG core protein mouse monoclonal 1:1 Gift of J. Levine

1:45D4 Keratan sulphate glycosaminoglycans mouse monoclonal 1:100 Seikagaku3H1 Phosphacan-KS mouse monoclonal 1:1 DSHB12C5 Versican CSPG core protein mouse monoclonal 1:1 R. Asher^ Tyrosine hydroxylase (synthetic enzyme found in

catecholaminergic neurones)rabbit polyclonal 1:4000 Jacques Boy

InstitutRat-401 Nestin (intermediate ¢laments in astrocytes) mouse monoclonal 1:400 DSHBOX-42 Complement receptor 3 (microglia, macrophages) mouse monoclonal 1:200 Serotec^ Glial ¢brillary acidic protein (intermediate ¢laments in

astrocytes)rabbit polyclonal 1:10 000 Dako

^ Raised against fusion protein corresponding to theextracellular plus transmembrane domain of ratsyndecan-2

chicken monoclonal 1:50 Gift of J.Couchman

Where two working concentrations are given, the ¢rst refers to the concentration used in immuno£uorescence labelling and the second refersto the concentration used in immunoperoxidase labelling.

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L. D. F. Moon et al.102

two animals from each time point were not perfused ¢xed, butinstead, un¢xed brains were rapidly removed, frozen on dry iceand continuous series of one-in-ten 10 Wm-thick parasagittalsections of brain were cut on a cryostat, prior to immuno£uo-rescence labelling un¢xed sections on slides.

Immuno£uorescence labelling

Prior to immuno£uorescence labelling, to create epitopes ofinterest or to con¢rm speci¢city of antibody binding, parallelseries of un¢xed sections were incubated in a humidi¢er for3 h at 37³C, either in PBS or in PBS containing speci¢c glycos-aminoglycan lyases. Immunolabelling using 2B6, 3B3 and CS-56antibodies was performed following incubation in PBS or inPBS containing chondroitinase ABC (300 mU/ml, BoehringerMannheim, UK). Immunolabelling using 10E4 and 3G10 wasperformed following incubation in PBS or PBS containing hep-aritinase (10 mU/ml, Seikagaku, Japan). Immunolabelling using5D4 and 3H1 was performed following incubation in bu¡er (50mM sodium acetate, 20 mM sodium chloride, pH 5.8) or bu¡ercontaining endo-L-galactosidase (20 mU/ml, Roche, UK).

For subsequent immuno£uorescence histology, all washeswere performed four times using PBS containing 0.05%Tween-20 (BDH, UK) and all incubations were carried outfor 60 min in a dark, humidi¢ed chamber at room temperature.First, sections were washed and incubated in 3% bovine serumalbumin (BSA) in PBS. Next, sections were incubated in 1%BSA containing primary antibody (Table 1): a mixture of 2B6and 3B3 monoclonal antibodies (1:200, Seikagaku), mousemonoclonal antibody CS-56 (1:500, Sigma, UK) or mousemonoclonal antibodies against neurocan (1D1, 1:200, Develop-mental Studies Hybridoma Bank (DSHB), University of Iowa,IA, USA; 1G2, 1:1, Gift of A. Oohira), phosphacan (3F8,1:200, DSHB), NG2 (D31-10, 1:1, gift of J. Levine), versican(12C5, 1:1), KS (5D4, 1:100, Seikagaku), phosphacan-KS (3H1,1:1, DSHB), or chicken monoclonal antibodies against a fusionprotein corresponding to the extracellular plus transmembranedomain of rat syndecan-2 (1:50, gift of J. Couchman). Sectionswere washed prior to incubating in 1% BSA containing theappropriate biotinylated secondary antibody: either sheep anti-mouse immunoglobulin (Ig) (1:200, Amersham, UK) or donkeyanti-chicken IgY (1:200, Jackson ImmunoResearch, UK). Next,sections were washed and incubated in 1% BSA containingstreptavidin conjugated Cy3 (1.0 Wg/ml, Amersham) containing5 WM bisbenzamide nuclear dye (Hoechst 33342, Sigma). Sec-tions were washed and coverslipped using PBS (pH 8.5) contain-ing 90% glycerol and 2.5% 1,4-diazobicyclo-[2,2,2]octane(DABCO, Sigma).

Immunoperoxidase labelling

A standard immunoperoxidase protocol was followed (Moonet al., 2000) using these primary antibodies: rabbit anti-tyrosinehydroxylase (anti-TH) (1:4000, Jacques Boy Institut, France),rabbit anti-glial ¢brillary acidic protein (anti-GFAP, 1:10 000,Dako, UK), mouse monoclonal antibody CS-56 (1:100, Sigma),2B6 and 3B3 (1:1000, Seikagaku) and mouse monoclonal anti-bodies against nestin (clone Rat-401, 1:400, DSHB), againstcomplement receptor 3 (CR3, antibody clone MRC-OX42,1:200, Serotec, UK), against NG2 (antibody clone D31-10,1:4, gift of J. Levine), against neurocan (1D1, 1:200, DSHB)and phosphacan (3F8, 1:200, DSHB). Biotinylated secondaryantibodies were either horse anti-mouse IgG (rat adsorbed,1:200, Vector, UK) or goat anti-rabbit IgG (1:200, Dako),used in conjunction with a streptavidin/biotinylated horseradishperoxidase kit (Dako) with diaminobenzidine as the chromogen.Sections were mounted on presubbed (1% gelatin in PBS) glassslides, dehydrated in an ascending series of ethanols, cleared inxylene and coverslipped using DPX.

In all cases, non-speci¢c binding of the primary antibody wascontrolled for using an isotype-, species- and concentration-matched antibody (mouse IgG1 or IgM, Sigma; chicken IgY,Jackson Immunoresearch).

All sections were examined by light microscopy.

RESULTS

De¢nitions

Ten consecutive series of one-in-ten parasagittal sec-tions were immunolabelled to visualise glial cells, PGsand axons expected to be present at the site of unilateralnigrostriatal transection. GFAP immunolabelling (seebelow) of free-£oating sections ¢xed by perfusion with4% paraformaldehyde allowed two regions of interest tobe distinguished: a `lesion core' and a `lesion surround'.At all times examined, the lesion core was de¢ned by alack of GFAP immunoreactivity. Between 4 and 14 dayspost-axotomy, the lesion core measured approximately

Fig. 2. GFAP immunoreactivity in the lesion surround followingnigrostriatal axotomy examined (A) 4, (B) 7 and (C) 14 days post-axotomy. Lesion core is to left of each image. Scale bars =200 Wm. Digital images were adjusted for brightness and contrast.

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300 Wm in the rostrocaudal axis and reached from themidline to 2.5 mm laterally. There were small regions ofcavitation but in general there was continuity of tissuethrough the lesion core. The lesion surround was de¢nedby the presence of large numbers of hypertrophic stellateGFAP immunoreactive cells, particularly within 200 Wmof the lesion core perimeter.

When immunolabelling series of sections using anti-bodies other than anti-GFAP antibodies, the lesioncore and lesion surround could be accurately delineatedwithout having to double label sections using GFAPantibodies. For example, the series of sections immuno-labelled using CS-56 antibodies (see below) was adjacent(on the medial side) to the series immunolabelled forGFAP. On an animal-by-animal basis, the shape, dimen-sions and location of the lesion core, as de¢ned byGFAP immunolabelling, exactly matched the shape,dimensions and location of the lesion core indicated byCS-56 immunolabelling. This precise mapping was alsoseen in other series of sections immunolabelled using theother anti-PG antibodies. Thus, the lesion core andlesion surround could be de¢ned by either GFAP orimmunolabelling using antibodies against various PGs.

As stated, the lesion core and lesion surround werede¢ned by immunolabelling both for GFAP and CS-56only in free-£oating sections perfused ¢xed using 4%paraformaldehyde. In contrast, when immunolabellingseries of un¢xed cryosections, the lesion core and lesionsurround were de¢ned only by CS-56 immunolabelling.

Glial cell response to axotomy

Astrocyte response to axotomy. The astrocyticresponse to injury was assessed using antibodies againstGFAP and nestin between 4 and 14 days post-axotomy.Four days post-axotomy, discrete, stellate, intenselyGFAP immunoreactive cells were present in the lesionsurround (Fig. 2A). Seven days post-axotomy, increasingnumbers of stellate GFAP immunoreactive cells weredetected throughout the ipsilateral hemisphere, and par-ticularly within 200 Wm of the lesion core. In the lesionsurround, hypertrophic GFAP immunoreactive processeshad begun to form a continuous network around thelesion core (Fig. 2B). By 14 days, GFAP immunoreac-tivity was particularly intense in the lesion surround,walling the lesion core o¡ completely with a dense net-work of cell bodies whose processes were often orientedperpendicular to the lesion border (Fig. 2C). GFAP im-munoreactivity was also present surrounding regions ofcavitation. At all times, nestin immunoreactivity wasabsent from the lesion core.

The distributions of GFAP and nestin immunoreactiv-ities were similar in many respects. Four days post-axot-omy, discrete nestin immunoreactive cell bodies andprocesses were visible in the lesion surround (Fig. 3A).Seven days post-axotomy, nestin immunoreactive pro-cesses appeared hypertrophied in the lesion surround,predominantly within 200 Wm of the lesion core(Fig. 3B). Fourteen days post-axotomy, there werelarge numbers of nestin immunoreactive processes inthe lesion surround, within 200 Wm of the lesion core.

These had formed a dense network in the lesion sur-round entirely surrounding the lesion core (Fig. 3C).Nestin immunoreactivity was also present surroundingregions of cavitation.

Response of adult oligodendrocyte precursor cells toaxotomy. The D31-10 monoclonal antibody againstNG2 CSPG was used to detect stellate adult oligoden-drocyte precursor cells which are present throughout theintact rat CNS (Nishiyama et al., 1999). The distributionof D31-10 immunoreactivity was similar in manyrespects to that of GFAP and nestin.

Four days post-axotomy, stellate, intensely D31-10 im-munoreactive cells were observed in the lesion surroundwithin 200 Wm of the lesion core (Fig. 4A). Seven days

Fig. 3. Nestin immunoreactivity in the lesion surround followingnigrostriatal axotomy examined (A) 4, (B) 7 and (C) 14 days post-axotomy. Lesion core is to left of each image. Scale bars =200 Wm. Digital images were adjusted for brightness and contrast.

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L. D. F. Moon et al.104

post-axotomy, large numbers of D31-10 immunoreactivecells were present in the lesion surround, particularlywithin 200 Wm of the lesion core. D31-10 immunoreactiveprocesses were hypertrophied and had begun to wall o¡the lesion core (Fig. 4B). By 14 days post-axotomy, largenumbers of D31-10 immunoreactive cells were observedin the lesion surround, particularly within 200 Wm of thelesion core, with hypertrophied processes forming adense network entirely surrounding the lesion core(Fig. 4C). D31-10 immunoreactivity was also present sur-rounding regions of cavitation. However, at all times,stellate D31-10 immunoreactive cells were absent fromthe lesion core itself, although D31-10 immunoreactiveblood vessels were observed within this region (Fig. 4B).

Response of microglia and macrophages to axoto-my. Antibodies against CR3 (clone MRC-OX42) wereused to identify microglia and macrophages. CR3 immu-noreactive cells with a stellate or rami¢ed morphologywere assumed to be microglia (resting or, where hyper-trophied, activated) while CR3 immunoreactive cells withan amoeboid morphology were assumed to be macro-phages.

The distribution of rami¢ed, CR3 immunoreactivecells was similar in many respects to that of GFAP,nestin and D31-10. Four days post-axotomy, large num-bers of discrete rami¢ed, CR3 immunoreactive cells wereobserved in the lesion surround (Fig. 5A). Seven days

Fig. 4. Immunolabelling for NG2 CSPG showing adult oligoden-drocyte precursors in the lesion surround following nigrostriatalaxotomy examined (A) 4, (B) 7 and (C) 14 days post-axotomy.Lesion core is to left of each image. Scale bars = 200 Wm. Digital

images were adjusted for brightness and contrast.

Fig. 5. CR3 immunoreactivity showing rami¢ed microglia withinthe lesion surround following nigrostriatal axotomy examined(A) 4, (B) 7 and (C) 14 days post-axotomy. Lesion core is to leftof each image. Scale bars = 200 Wm. Digital images were adjusted

for brightness and contrast.

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post-axotomy, large numbers of intensely CR3 immuno-reactive rami¢ed cells were present in the lesion sur-round, particularly within 200 Wm of the lesion core;these extended only very short processes towards thelesion border (Fig. 5B). By 14 days post-axotomy, largenumbers of CR3 immunoreactive rami¢ed cells werepresent within the lesion surround, particularly within200 Wm of the lesion core, with their cell bodies andshort processes being densely packed (Fig. 5C). CR3 im-munoreactivity was also present surrounding regions ofcavitation. However, at all times, rami¢ed CR3 immuno-reactive cells were absent from the lesion core itself.

Between 4 and 14 days post-axotomy, increasing num-bers of amoeboid CR3 immunoreactive cells weredetected within the lesion core (Fig. 6). At all timesexamined, amoeboid CR3 immunoreactive cells wererarely detected in the lesion surround.

Summary of glial response to axotomy

Large numbers of reactive astrocytes, oligodendrocyteprecursors and rami¢ed microglia were present in thelesion surround between 4 and 14 days post-axotomy.In contrast, these cells were rarely present within thelesion core at these times. Amoeboid macrophages dis-played the complementary distribution, being increas-ingly abundant within the lesion core between 4 and 14days post-axotomy but scarce in the lesion surround atall times examined.

Proteoglycan response to axotomy

The PG response to axotomy was investigated by im-muno£uorescence labelling series of one-in-ten 10 Wm-thick un¢xed frozen cryostat sections. Where stated,results were con¢rmed by immunoperoxidase labellingseries of one-in-ten free-£oating sections obtained follow-ing paraformaldehyde perfusion.

Response of heparan sulphate proteoglycans to axoto-my. The HSPG response was examined using a numberof di¡erent antibodies against HS GAG and HS coreproteins. Mouse monoclonal antibody 10E4 recognisesintact HS GAG (David et al., 1992). At 4 days post-axotomy, very little 10E4 immunoreactivity was observedwithin the lesion core or the lesion surround. However,between 7 and 14 days post-axotomy, extracellular 10E4immunoreactivity was increasingly detected within thelesion core being either di¡use or present as smallpatches or as large sheets of matrix (Fig. 7A). 10E4 im-munoreactivity was also observed within those meningespenetrated during axotomy. In contrast, signi¢cant 10E4immunoreactivity was not observed in the lesion sur-round at any time examined. These patterns of immuno-reactivity were not observed in parallel sectionspredigested with heparitinase (Fig. 7B) and immuno-stained using 10E4 antibodies nor in undigested sectionsimmunostained using an isotype- and species-matchedprimary antibody (mouse IgM); these results indicatethat 10E4 immunoreactivity is speci¢c for HS GAG.

Mouse monoclonal antibody 3G10 recognises `stub'epitopes created following digestion of HS GAG withheparitinase (David et al., 1992). 3G10 immunoreactivitywas absent in non-digested control sections (Fig. 7C) andin digested sections immunolabelled with an isotype- andspecies-matched primary antibody (mouse IgG). How-ever, following digestion with heparitinase, the patternof 3G10 immunoreactivity was similar to that describedfor undigested sections immunolabelled with the 10E4antibody. Thus, at 4 days post-axotomy, very little3G10 immunoreactivity was observed within the lesioncore or the lesion surround. However, between 7 and 14days post-axotomy, extracellular 3G10 immunoreactivitywas increasingly detected within the lesion core (but notthe lesion surround), being either di¡use or present assmall patches or large sheets of matrix (Fig. 7D). 3G10immunoreactivity was also observed within those

Fig. 6. CR3 immunoreactivity showing amoeboid macrophages (arrowheads) within the lesion core, examined 14 days follow-ing nigrostriatal axotomy. Scale bar = 100 Wm. Digital images were adjusted for brightness and contrast.

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meninges penetrated during axotomy. 3G10 immunore-activity was also observed associated with blood vesselswithin 200 Wm of the lesion borders.

Syndecan-2 HSPG core protein was visualised using achicken monoclonal antibody raised against a fusionprotein corresponding to the extracellular and transmem-brane domain of this PG. Four days post-axotomy, faint

di¡use syndecan-2 immunoreactivity was present withinthe lesion core. Between 7 and 14 days post-axotomy,intense syndecan-2 immunoreactivity was detectable,being present extracellularly as large sheets or smallpatches (Fig. 8). In contrast, at all times examined, syn-decan-2 immunoreactivity was absent from the lesionsurround. These patterns of immunoreactivity were not

Fig. 7. Immunoreactivity for HS GAG, showing the lesion core 7 days following nigrostriatal axotomy. (A, B) 10E4 immu-noreactivity, and (C, D) 3G10 immunoreactivity. To con¢rm that reactivity was for HS GAG, sections were preincubatedeither in (A, C) PBS, or (B, D) PBS containing heparitinase. Scale bars = 50 Wm. Digital images were adjusted for brightness

and contrast.

Fig. 8. Immunolabelling using an antibody raised against a domain present in syndecan-2 HSPG core protein. Lesion coreoccupies right three quarters of image, lesion surround occupies left quarter of image. Scale bar = 50 Wm. Digital images were

adjusted for brightness and contrast.

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Proteoglycans and CNS sprouting 107

observed in control sections immunolabelled using anisotype-, species- and concentration-matched antibody(chicken IgY) in place of the primary.

Response of chondroitin sulphate proteoglycans to axot-omy. CSPGs were detected using antibodies againstCSPG core proteins (neurocan, phosphacan, versican,NG2) and against intact and digested CS GAG (CS-56and 2B6/3B3, respectively).

CS GAGs were detected using the CS-56 monoclonalantibody which recognises many intact chondroitin-4and chondroitin-6 sulphate GAGs (Avnur and Geiger,1984; Sorrell et al., 1993). At all times, CS-56 immuno-reactivity was absent from the lesion core. However,4 days post-axotomy, intense CS-56 immunoreactivitywas present in the lesion surround, particularly within200 Wm of the lesion core. Tissue further from the lesiondid not display CS-56 immunoreactivity. Seven dayspost-axotomy, CS-56 immunoreactivity became morewidespread and by 14 days post-axotomy, CS-56 immu-noreactivity was detectable throughout the ipsilateralhemisphere; in particular, by 14 days post-axotomy,CS-56 immunoreactivity was present in a 200 Wm-thickrim surrounding the lesion core, walling it o¡ (Fig. 9A).These patterns of immunoreactivity were not observed inparallel sections predigested with chondroitinase ABC

and immunostained using CS-56 antibodies (Fig. 9B)nor in undigested sections immunolabelled using an iso-type-, species- and concentration-matched primary anti-body (mouse IgM): these results indicate that CS-56immunoreactivity is speci¢c for CS GAG. Immunoper-oxidase labelling series of one-in-ten paraformaldehyde-perfused sections con¢rmed these results and indicated,additionally, the presence of CS-56 immunoreactive stel-late cells and blood vessels surrounding the site of axot-omy at all times examined.

CS GAGs were also detected using a mixture of the2B6 and 3B3 monoclonal antibodies which recognise`stub' epitopes created following digestion of CSPGswith chondroitinase ABC of chondroitin-4 and chondroi-tin-6 sulphate GAGs respectively (Hascall et al., 1972;Baker et al., 1991). Thus, the following patterns of im-munoreactivity were obtained following digestion withchondroitinase ABC (Fig. 9C) but not in undigested con-trol sections nor in digested control sections immunola-belled using an isotype-, species- and concentration-matched antibody (mouse IgG) in place of the primary.Four days post-axotomy, faint, di¡use, extracellular stubimmunoreactivity was observed in the lesion surround.Seven days post-axotomy, stub immunoreactivity wasmore widespread and was detectable throughout the ipsi-lateral hemisphere. Fourteen days post-axotomy, intense

Fig. 9. Immunoreactivity for CS GAG 7 days following nigrostriatal axotomy. (A, B) Immunolabelling using the CS-56 anti-body, and (C, D) immunolabelling using a mixture of 2B6 and 3B3 antibodies. To con¢rm that reactivity was for CS GAG,sections were preincubated either in (A, C) PBS, or (B, D) PBS containing chondroitinase ABC. Lesion core occupies lefthalf of image, lesion surround occupies right half of image. Scale bars = 50 Wm. Digital images were adjusted for brightness

and contrast.

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L. D. F. Moon et al.108

stub immunoreactivity was widespread and particularlyassociated with a 200 Wm-thick rim in the lesion sur-round (Fig. 9D). Stub immunoreactivity was also fre-quently associated with blood vessels between thelesion core and the lesion surround. In contrast, at alltimes examined, stub immunoreactivity was essentiallyabsent from the lesion core itself.

The CSPG response was also examined using antibod-ies against the following core proteins; neurocan, phos-phacan, NG2 and versican.

Neurocan CSPG core protein was detected using the1D1 and 1G2 monoclonal antibodies while phosphacanCSPG core protein was detected using the 3F8 monoclo-nal antibody. Immunoreactivities within the cerebellumwere as described previously (Oohira et al., 1994;Meyer-Pullitz et al., 1995, 1996). Four days post-axotomy,faint extracellular 1D1, 1G2 and 3F8 immunoreactivitieswere present in the lesion surround. Between 7 and 14 dayspost-axotomy, 1D1, 1G2 and 3F8 immunoreactivitiesbecame more widespread, being particularly intense withina 200 Wm-thick rim surrounding the lesion core (Fig. 10A,B). In contrast, at all times examined, 1D1, 1G2 and 3F8immunoreactivities were absent from the lesion core.Immunoperoxidase staining using the 1D1 and 3F8 anti-bodies con¢rmed these results (data not shown).

NG2 CSPG core protein was detected using the D31-10 monoclonal antibody. The spatiotemporal pattern ofD31-10 immunoreactivity following immunoperoxidaselabelling of free-£oating sections of paraformaldehyde-perfused tissue is described above (see results for adultoligodendrocyte precursors). Immuno£uorescent label-ling of un¢xed cryostat sections did not allow visualisa-tion of blood vessels or stellate D31-10 immunoreactivecells (i.e. oligodendrocyte precursors). Rather, 4 dayspost-axotomy, di¡use, extracellular D31-10 immunoreac-tivity was present in the lesion surround. Between 7 and14 days post-axotomy, D31-10 immunoreactivity intensi-¢ed and was particularly prevalent within 200 Wm of thelesion core (Fig. 10C). In contrast, at all times examined,D31-10 immunoreactivity was absent from the lesioncore.

Versican CSPG core protein was detected using 12C5monoclonal antibodies. In cerebellum and spinal cord,di¡use 12C5 immunoreactivity was predominantly asso-ciated with white matter (Bignami et al., 1992). However,in brain, we detected extracellular 12C5 immunoreactiv-ity in both grey and white matter in the lesion surround.Four days post-axotomy, di¡use extracellular 12C5 im-munoreactivity was detected in the lesion surround butonly within 200 Wm of the lesion core. Between 7 and 14

Fig. 10. Immunoreactivity for CSPG core proteins within the lesion surround 7 days following nigrostriatal axotomy.(A) Immunolabelling using 1D1 antibodies indicated the lesion surround (but not the lesion core) was immunoreactive forneurocan. (B) Immunolabelling using 3F8 antibodies indicated the lesion surround (but not the lesion core) was immunoreac-tive for phosphacan. (C) Immunolabelling using D31-10 antibodies indicated the lesion surround (but not the lesion core)was immunoreactive for NG2. (D) Immunolabelling using 12C5 antibodies indicated the lesion surround (but not the lesioncore) was immunoreactive for versican. Lesion core occupies left half of image, lesion surround occupies right half of image.

Scale bars = 50 Wm. Digital images were adjusted for brightness and contrast.

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days post-axotomy, 12C5 immunoreactivity was presentthroughout the ipsilateral hemisphere, being particularlyintense within 200 Wm of the lesion core (Fig. 10D). Incontrast, at all times examined, 12C5 immunoreactivitywas sparse within the core of the site of axotomy itself,although small, discrete patches were infrequentlyobserved.

Response of keratan sulphate proteoglycans to axoto-my. The KSPG response was examined using the3H1 monoclonal antibody against phosphacan-KSand using the 5D4 monoclonal antibody against KSGAG. The patterns of immunoreactivity for 3H1 and5D4 were very similar. At all times examined, neither3H1 nor 5D4 immunoreactivities were detectable withinthe lesion core. Further, 4 days post-axotomy, verylittle 3H1 or 5D4 immunoreactivities were detectablewithin the lesion surround. However, 7 days post-axot-omy, di¡use extracellular 3H1 and 5D4 immunoreactiv-ities were present in the lesion surround. By 14 days3H1 and 5D4 immunoreactivities were widespread andwere particularly intense in the lesion surround withina 200 Wm-thick rim surrounding the lesion core(Fig. 11A, C). The intensity of these patterns of immu-noreactivities was partially (but not completely) attenu-ated in control sections predigested with endo-L-

galactosidase, con¢rming their identities as KS GAG(Fig. 11B, D).

Summary of proteoglycan response to axotomy

Four days post-axotomy, little immunoreactivity forHS GAG and syndecan-2 HSPG was detected at thelesion site but between 7 and 14 days post-axotomy,immunoreactivities for HS GAGs and syndecan-2HSPG core protein were detected within the lesioncore. At all times, the lesion core lacked immunoreactiv-ity for CS GAGs, KS GAGs and CSPG core proteinsincluding neurocan, phosphacan, NG2 and versican. Incontrast, between 4 and 14 days post-axotomy, the lesionsurround contained increasing levels of immunoreactivityfor CS GAGs, KS GAGs and CSPG core proteinsincluding neurocan, phosphacan, NG2 and versican. Atall times, the lesion surround lacked immunoreactivitiesfor HS GAGs and syndecan-2 HSPG core protein.

Axonal response to axotomy

Catecholaminergic neurones including dopaminergicnigral neurones were visualised by immunoperoxidaselabelling paraformaldehyde-¢xed parasagittal sectionsusing antibodies against TH. Two factors allowed us to

Fig. 11. Immunoreactivity for KS GAG 7 days following nigrostriatal axotomy. (A, B) Immunolabelling using the 5D4 anti-body, and (C, D) immunolabelling using the 3H1 antibody. To con¢rm that reactivity was for KS GAG, sections werepreincubated either in (A, C) PBS, or (B, D) PBS containing endo-L-galactosidase. Lesion core occupies left half of image,

lesion surround occupies right half of image. Scale bars = 50 Wm. Digital images were adjusted for brightness and contrast.

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determine with precision whether TH immunoreactiveaxons grew solely in the lesion core, or whether theypenetrated the lesion surround, as de¢ned by GFAPlabelling in parallel series of one-in-ten sections. First,the series of one-in-ten sections immunolabelled for THwas adjacent (on the lateral side) to the series immuno-labelled for GFAP. Second, the polyclonal antibody toTH non-speci¢cally immunolabelled the lesion core morelightly than the lesion surround.

Previous results demonstrate that unilateral axotomyof the medial forebrain bundle results in degeneration ofthe distal portion of the dopaminergic nigrostriatal tractwith loss of innervation of the ipsilateral striatum andwithout spontaneous long distance axon regenerationbeyond the site of axotomy (Brecknell et al., 1995,1996a,b; Moon et al., 2000). Four days post-axotomy,small numbers of TH immunoreactive nigral axons wereobserved sprouting within the lesion core, growing up to200 Wm (Fig. 12A), often being oriented parallel to theplane of axotomy, i.e. perpendicular to the originalnigrostriatal tract. Axons did not grow signi¢cant distan-ces out of the lesion core into the lesion surround. Abun-dant TH immunoreactive debris were present anterior tothe site of axotomy, often presenting as degeneratingprocesses terminating in a 20 Wm-wide ball. Seven dayspost-axotomy, TH immunoreactive debris was less abun-dant anterior to the site of axotomy and increased num-bers of axons were observed sprouting up to 200 Wmwithin the lesion core, but not into the lesion surround(Fig. 12B). Fourteen days post-axotomy, TH immunore-active debris was scarce and axons continued to sproutwithin the lesion core, but not into the lesion surround(Fig. 12C). Thus, following axotomy, dopaminergic ni-gral axons continue to sprout for at least 2 weeks, butaxons do not grow out of the lesion core.

DISCUSSION

We have charted the response of glia, production ofPGs and sprouting of dopaminergic nigral axons follow-ing unilateral axotomy of the adult rat nigrostriatal tract.We were able to distinguish two regions at the site ofaxotomy: a lesion core and a lesion surround. The lesioncore, de¢ned as the region lacking astrocytes, measuredapproximately 300 Wm in the rostrocaudal axis andreached from the midline to 2.5 mm laterally. The lesionsurround was de¢ned as the region containing reactiveastrocytes, particularly within 200 Wm of the lesion core.

Four days post-axotomy, the lesion core containedamoeboid macrophages and was also characterised by

Fig. 12. Immunolabelling for catecholaminergic neurones usingantibodies against TH. Image shows dopaminergic nigral axonssprouting within the lesion core examined (A) 4, (B) 7, or (C) 14days post-axotomy. Arrows indicate lesion borders; left arrowindicates level of axotomy. Nigra is beyond the left of each image,striatum is beyond the right. Scale bars = 100 Wm. Digital images

were sharpened and adjusted for brightness and contrast.

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a lack of astrocytes, oligodendrocyte precursors or rami-¢ed microglia. The lesion core is also known to containmeningeal ¢broblasts, shown by immunolabelling usingan antibody against retinal dehydrogenase (Shearer, M.and Rhodes, K., personal communication). At this time,there was little detectable increase in PG abundancewithin the lesion core or lesion surround. Moderatesprouting of severed dopaminergic nigral axons occurredwithin the lesion core but axons did not grow more than200 Wm anteriorly into the lesion surround.

Seven days post-axotomy, the lesion core containedincreased numbers of amoeboid macrophages and moreabundant HSPGs but astrocytes, oligodendrocyte pre-cursors, rami¢ed microglia, CSPGs and KSPGsremained absent from the lesion core. However, in thelesion surround, increased numbers of reactive astro-cytes, oligodendrocyte precursors and rami¢ed microgliawere detected, particularly within 200 Wm of the lesioncore. CSPGs and KSPGs were also detected in the lesionsurround, also, predominantly within 200 Wm of thelesion core. At this time, severed dopaminergic nigralaxons continued to sprout within the lesion core butnot into the lesion surround.

Fourteen days post-axotomy, the lesion core containedlarge numbers of densely packed amoeboid macrophagesand large patches of HSPGs but very few astrocytes,oligodendrocyte precursors or rami¢ed microglia. Thelesion core also contained some HSPGs but lackeddetectable CSPGs and KSPGs. In contrast, the lesionsurround, particularly that within 200 Wm of the lesioncore, contained reactive astrocytes, oligodendrocyte pre-cursors and rami¢ed microglia as well as CSPGs andKSPGs. Severed dopaminergic nigral axons continuedto sprout at this time but did not grow beyond the lesioncore into the lesion surround.

Thus, in this model of CNS injury, as in others, glia(including reactive astrocytes, oligodendrocyte precursorsand rami¢ed microglia) and PGs including CSPGs (neu-rocan, phosphacan, versican and NG2), HSPGs (includ-ing syndecan-2) and KSPGs become increasinglyabundant in the ¢rst two weeks following CNS damage(Levine, 1994; Asher et al., 1999, 2000a,b; McKeon etal., 1999; Jaworski et al., 1999). Our results show a pos-itive correlation between the location of sprouting of cutCNS axons and the presence of HSPGs, amoeboid mac-rophages and blood vessels (and, probably, meningeal¢broblasts), indicating that these may be growth-permis-sive. Our results also show a negative correlationbetween the location of sprouting of cut CNS axonsand reactive astrocytes, oligodendrocyte precursors,rami¢ed microglia, CSPGs and KSPGs, indicating thatthese may be growth inhibitory.

How might proteoglycans a¡ect axon regeneration in theadult mammalian CNS?

Potential roles for reactive astrocytes, oligodendrocyteprecursors, microglia and amoeboid macrophages inmodulating axon regeneration following unilateralnigrostriatal transection have been reviewed previously(Moon et al., 2000). Here we consider potential roles

and sources of di¡erent PGs in limiting spontaneousCNS axon regeneration in the adult mammal.

Chondroitin sulphate proteoglycans

The distributions of all the studied CSPG core pro-teins (NG2, neurocan, phosphacan and versican) andCS GAGs were consistent with the hypothesis thatCSPGs limit regrowth of cut CNS axons: CSPG immu-noreactivities were predominantly detected in the lesionsurround but not in the lesion core between 4 and 14days post-axotomy. In addition, NG2 immunoreactivitywas associated with blood vessels in the lesion core.Other studies have similarly demonstrated the presenceof CSPGs at sites of CNS injury, including NG2 (Levine,1994), neurocan (McKeon and Buck, 1997), phosphacan(Barker et al., 1996), versican (Asher et al., 1999;McKeon et al., 1999) and brevican (Jaworski et al.,1995; Haas et al., 1999; Thon et al., 2000). In vitro,and at sites of CNS injury, CSPGs are generally pro-duced by reactive glia: astrocytes are thought to makeneurocan and phosphacan while oligodendrocyte precur-sors make NG2, neurocan, and phosphacan (Maurel etal., 1994; Faissner et al., 1994; Engel et al., 1996;Meyer-Pullitz et al., 1996; Nishiyama et al., 1999;Asher et al., 2000a). Additionally, in situ hybridisationsuggests that neurocan may also be produced by neu-rones (Rauch et al., 1992; Engel et al., 1996; Meyer-Pullitz et al., 1996). Currently, the source of versican inthe intact adult mammalian CNS remains controversial.One study (Niedero«st et al., 1999) showed that highlybranched oligodendrocytes in vitro were immunoreactiveonly for the V2 isoform of versican while another studyindicated that oligodendrocytes in vitro were immunore-active for the V2 and V3 isoforms of versican (Le Baron,1996). However, in other studies, CSPG was not beendetected in oligodendrocytes (Margolis and Margolis,1974) while recent work suggests that versican is onlyexpressed by O-2A cells earlier in the oligodendrocytelineage (Asher et al., 1999). The apparently contradictoryresult for highly branched oligodendrocytes might beexplained if versican was synthesised and laid downaround immature O-2A cells and then retained in theperi- and extracellular space post-di¡erentation.

In vitro experiments indicate that CSPGs are oftengrowth-limiting molecules. NG2 limits growth boththrough its core protein and CS GAG side chains(Dou and Levine, 1994; Asher et al., 2000a; Fidler etal., 1999) while neurocan predominantly limits neuritegrowth through interactions with its core protein(Grumet et al., 1993; Milev et al., 1994; Friedlander etal., 1994; Margolis et al., 1996; Margolis and Margolis,1997; Asher et al., 2000a). Phosphacan modulatesgrowth of CNS axons in a context-dependent fashion,being variously growth inhibitory or growth-promoting(Verna et al., 1989; Maeda and Noda, 1996; Grumet etal., 1993; Canoll et al., 1993; Friedlander et al., 1994;Margolis et al., 1996; Margolis and Margolis, 1997;Sakurai et al., 1997; Asher et al., 2000a), these e¡ectsprincipally being mediated through phosphacan's coreprotein (Milev et al., 1994; Maeda et al., 1996; Maeda

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and Noda, 1996). It is not yet clear whether versicancontributes towards axon regenerative failure in vivoalthough various experiments indicate that versican is agrowth modulatory molecule. First, versican appears tomodulate cell migration and neurite growth during devel-opment (Landolt et al., 1995; Henderson et al., 1997)and in vitro (Perris et al., 1996). Second, versican appearsto contribute towards non-permissiveness associated witha fraction of bovine CNS myelin, since immunodepletionof versican increased the number of neurite-bearing post-natal rat cerebellar granule cells in vitro (Niedero«st et al.,1999). In contrast, neonatal rat dorsal root ganglion(DRG) neurites grow across patches of versican immu-noreactivity in monolayers of Neu7 cells (Fidler et al.,1999).

In vivo experiments also indicate that PGs bearing CSGAG do indeed limit CNS axon regeneration followinginjury (Yick et al., 2000; Chung et al., 2000; Moon et al.,2001). The role of particular CSPG core proteins is notyet known, although this could be tested in knockoutmice, for example in neurocan knockout mice, whichare viable, fertile and appear to behave relatively nor-mally (Rauch, 1997).

The mechanisms whereby CSPGs modulate neuritegrowth are not well understood. A putative 280 kDareceptor for NG2 has recently been identi¢ed on thecell surface of cerebellar granule cells in vitro, and bind-ing of NG2 to this receptor activated G-proteins andelevated intracellular calcium and cyclic adenosinemonophosphate (Dou and Levine, 1997). However, thisputative receptor was not detected upon DRGs and thisindicates that NG2 may exert its e¡ects in di¡erent ways.CSPGs may also act indirectly by binding other growthmodulatory molecules. NG2 binds collagens type II, Vand VI, laminin, tenascin and the platelet-derived growthfactor (PDGF) K receptor (Levine and Nishiyama, 1996;Burg et al., 1996; Nishiyama et al., 1996a,b) while neuro-can interacts with neural cell adhesion molecule(NCAM), L1 and TAG-1/axonin-1 (Milev et al., 1994;Friedlander et al., 1994; Meyer-Pullitz et al., 1995),amphoterin and pleiotrophin (Li et al., 1990; Hori etal., 1995; Maeda et al., 1996; Milev et al., 1998) andthe glycoprotein tenascin (although not any other testedcell surface or extracellular matrix molecules) (Grumet etal., 1994; Aspberg et al., 1997; Rauch et al., 1997). Phos-phacan lacks a transmembrane or cytoplasmic domainand must therefore modulate growth indirectly. Likeneurocan, phosphacan interacts with a range of di¡erentgrowth modulatory molecules including NCAM, L1 andTAG-1/axonin-1 and tenascin-C but not to laminin,¢bronectin, collagens, epidermal growth factor or ¢bro-blast growth factor (FGF) receptor or myelin-associatedglycoprotein (Milev et al., 1993, 1996; Grumet et al.,1994; Sakurai et al., 1997). Phosphacan also bindsamphoterin, pleiotrophin/heparin binding growth-associ-ated molecule through its CS GAG chains (Li et al.,1990; Hori et al., 1995; Maeda et al., 1996; Milev etal., 1998). Further, phosphacan may bind the NCAMcontactin/F3/F11 (Peles et al., 1995; Sakurai et al.,1997). The mechanism whereby versican might modulateneurite growth is not clear although it may act as a

physiological ligand for tenascin-R which it binds withhigh a¤nity (Aspberg et al., 1995).

Phosphacan keratan sulphate proteoglycan

Phosphacan-KS immunoreactivity was predominantlydetected in the lesion surround but not the lesion core.This has also been reported elsewhere (Grill et al., 1998).The distribution of phosphacan-KS is consistent with ithaving a growth-limiting role following CNS injury andwith it being derived from astrocytes, oligodendrocyteprecursors or rami¢ed microglia (but not amoeboid mac-rophages or ¢broblasts). It is not yet clear which celltypes make phosphacan-KS although it may be producedas a glycosylation variant of phosphacan-CS by astro-cytes and oligodendrocyte precursors (Meyer-Pullitz etal., 1995; Sakurai et al., 1997). There is some evidencethat phosphacan-KS is a growth modulatory molecule:whereas it has no growth-promoting e¡ect on thalamicneurones cultured on poly-L-lysine or ¢bronectin, phos-phacan-KS promotes neurite outgrowth from corticalneurones (Maeda and Noda, 1996).

Heparan sulphate proteoglycans

Immunoreactivities for HS GAG and for syndecan-2HSPG core protein were found in the lesion core but notin the lesion surround, between 4 and 14 days post-axot-omy. Thus, the distribution of HSPGs is consistent withthe hypothesis that HSPGs in the lesion core might pro-mote local sprouting of cut dopaminergic nigral axons.Further, the absence of HSPGs from the lesion surroundmight contribute towards the failure of sprouting dopa-minergic nigral axons to grow into the lesion surround.

Several lines of evidence show that HSPGs modulateneurite growth in vivo and in vitro. HSPG is present indeveloping axonal tracts in the chick brain (Halfter,1993) while in culture, interference with HS GAGs mod-ulates neurite growth. For example, addition of solubleHS disturbs growth of cockroach pioneer axons (Wangand Denburg, 1992) while scission with HS lyases a¡ectsretinotectal path¢nding in Xenopus embryos (Walz et al.,1997). HSPG^laminin complexes also modulate neuriteoutgrowth in vitro including growth promotion andgrowth inhibition (Lander et al., 1985a,b; Chiu et al.,1986; Hantaz-Ambroise et al., 1987; Mathiessen et al.,1989). For example, whereas monoclonal antibodies toHSPG reduce neurite outgrowth on sciatic nerve in vitro(Sandrock and Matthew, 1985) a Schwannoma-derivedHSPG also blocks the neurite-promoting activity of lam-inin (Muir et al., 1989). Interestingly, HSPGs do notappear necessary for neurite outgrowth on astrocytes(Ard and Bunge, 1988). HSPGs also bind growth-pro-moting molecules including NCAM, L-selectin, PECAM-1 (Bansal et al., 1996). Indeed, homophilic NCAM bind-ing requires participation of HSPG (Cole et al., 1986).There is therefore some limited evidence that HSPGsmight be growth-limiting molecules present at sites ofCNS injury.

Mechanisms whereby HSPGs might modulate neuritegrowth are not well understood. However, most known

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HSPG functions involve binding of a trophic molecule toHS GAG in order for that molecule to exert its function.For example, in Drosophila, e¡ective signalling mediatedby the growth factors wingless and decapentaplegicdepends closely upon dally HSPG core protein (Selleck,2000). Similarly, HS GAGs are important for FGF sig-nalling. An active trimer consisting of HSPG, FGF andFGF receptor allows subsequent FGF receptor dimerisa-tion, activation and subsequent cell signalling; thus, HSGAG lysis (with heparitinase) or inhibition of GAG sul-phation (with chlorate) reduces the signalling activity ofFGFs (Rapraeger et al., 1991) indicating that PGs areimportant for growth factor signalling. Indeed, bindingof basic ¢broblast growth factor (bFGF) to its receptorrequires prior binding of bFGF either to free HS GAGchains or to an HSPG (Yayon et al., 1991). However,HSPG alone can internalise FGF and induce intracellu-lar signalling in the absence of FGF receptor. Further,HS can interfere with FGF signalling (Bansal et al.,1996).

This work also provides evidence that syndecan-2HSPG core protein may be present within sites of CNSinjury. Syndecan-2 immunoreactivity was present inextracellular sheets and patches within the lesion corebut not in the lesion surround. This was similar to thepattern obtained using antibodies against HS GAG.Thus the distribution of the immunoreactivity using theanti-syndecan-2 antibody was consistent with it recognis-ing a molecule-bearing HS GAG. The syndecan-2 immu-noreactivity was extracellular and not obviouslyassociated with any cell type; this is not consistentwith syndecan-2 being a transmembrane HSPG andthus leaves unknown the source of the syndecan-2HSPG core protein. In vitro, syndecan-2 mRNA is pre-dominantly expressed by ¢broblasts (Elenius andJalkanen, 1994) and by oligodendrocyte precursors,mature oligodendrocytes and astrocytes (Bansal et al.,1996) while activated human macrophages express syn-decan-2 mRNA and protein (Clasper et al., 1999). Sincesyndecan-2 immunoreactivity was found in the lesioncore but not the lesion surround, it is possible that it isproduced by macrophages and meningeal ¢broblasts inthe lesion core. It is less likely that syndecan-2 is pro-duced by astrocytes or oligodendrocyte precursors sincethese are not found in the lesion core but in the lesionsurround.

The function of syndecan-2 is not known but synde-cans may play a role in wound £uid homeostasis(Subramanian et al., 1997) and wound healing (Eleniuset al., 1991; Wight et al., 1992). We here report a spatialcorrelation between syndecan-2 immunoreactivity andlocal sprouting of cut dopaminergic nigral axons. Thissuggests that syndecan-2 might act in a growth-promot-ing manner. Indeed, syndecans bind a large variety ofgrowth modulatory molecules including ¢bronectin,

thrombomodulin and collagens types I, III and V andbFGF (Elenius and Jalkanen, 1994; Perrimon andBern¢eld, 2000). However, syndecan-2 immunoreactivitywas not detectable within the lesion core 4 days post-axotomy, indicating that syndecan-2 is unlikely to playa role in axon sprouting at this earlier time.

Other factors

There is the additional enigma that following axot-omy, cut dopaminergic nigral axons frequently fail toenter the rostral half of the lesion core despite the factthat this region lacks reactive astrocytes, oligodendrocyteprecursors and rami¢ed microglia, CSPGs and KSPGs.We detected no di¡erence in the cellular and molecularcomposition of the caudal and rostral halves of the lesioncore. Deposition of basal lamina within the lesion coremight be partially responsible for the restricted growthrostrally. It is known that interference with the integrityof basal lamina, particularly with collagen (Reier et al.,1983; Berry et al., 1983) enhances CNS axon regenera-tion in vivo (Stichel et al., 1999a,b). It is also possiblethat the orientation of the cells and matrix within thelesion core restrict growth anteriorly by redirectinggrowth obliquely. Indeed, we often saw neurites growingperpendicularly to the original pathway, parallel to theplane of transection. Perhaps ¢broblasts invading per-pendicularly from the meninges reorient the matrix sothat the sca¡old lies nonparallel to the original axonaltract.

CONCLUSION

The failure of spontaneous long distance axon regen-eration in the adult mammalian CNS is usually attrib-uted to poor neuronal competence combined with theabsence of growth-promoting factors and the presenceof growth inhibitory factors. We here show that cutdopaminergic nigral axons sprout proli¢cally in a regioncontaining HSPGs but do not grow into the surroundingregion containing CSPGs and KSPGs. The distributionsof the CSPGs and KSPGs in the lesion surround maponto the distribution of the reactive astrocytes, oligoden-drocyte precursors and rami¢ed microglia. In contrast,the distribution of HSPGs in the lesion core maps ontothe distribution of amoeboid macrophages, meningeal¢broblasts and vascular endothelial cells. We concludethat regrowth of cut CNS axons may be promotedlocally by HSPGs but restricted by CSPGs and KSPGs.

AcknowledgementsöThis work was supported by ActionResearch, the Medical Research Council, the Wellcome Trustand the International Spinal Research Trust. We are also grate-ful to Dr Rosie Cooney for additional proofreading.

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(Accepted 28 August 2001)

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