Human dorsal root ganglion neurons from embryonic ... - Springer Link

Journal of Neurocytology 26, 811—822 (1997)

Human dorsal root ganglion neurons from embryonic donors extend axons into the host rat spinal cord along laminin-rich peripheral surroundings of the dorsal root transitional zone E L E N A N . K O Z L O V A 1 , 2 * , As K E S E I G E R 3 , and H As K A N A L D S K O G I U S 2 1 Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia; 2 Department of Anatomy, Biomedical Center, Uppsala University, P.O. Box 571, S-751 23 Uppsala, Sweden; 3 Department of Clinical Neuroscience and Family Medicine, Karolinska Institute, Stockholm, Sweden Received 30 June 1997; revised 3 September 1997; accepted 17 September 1997

Summary Following dorsal root crush, the lesioned axons regenerate in the peripheral compartment of the dorsal root, but stop at the boundary between the peripheral and the central nervous system, the dorsal root transitional zone. We have previously shown that fibres from human fetal dorsal root ganglia grafted to adult rat hosts are able to grow into the spinal cord, but were not able to specify the route taken by the ingrowing fibres. In this study we have challenged the dorsal root transitional zone astrocyte boundary with human dorsal root ganglion transplants from 5–8-week-old embryos. By tracing immunolabelled human fibres in serial sections, we found that fibres consistently grow around the dorsal root transitional zone astrocytes in laminin-rich peripheral surroundings, and extend into the host rat spinal cord along blood vessels, either into deep or superficial laminae of the dorsal horn, or into the dorsal funiculus. Human fibres that did not have access to blood vessels grew on the spinal cord surface. These findings indicate, that in spite of a substantial growth capacity by axons from human embryonic dorsal root ganglion cells as well as their tolerance to non-permissive factors in the mature mammalian CNS, these axons are still sensitive to the repellent effects of astrocytes of the mature dorsal root transitional zone. Furthermore, this axonal ingrowth is consistently associated with laminin-expressing structures until the axons reach the host spinal cord.

Introduction The fundamental differences in the capacity of axons to regenerate in the PNS versus the mature CNS of mammals are highlighted by injuries to primary sensory neurons of the spinal somatosensory system. The perikarya of these neurons are collected in segmental dorsal root ganglia (DRG) from which one process projects via peripheral nerve branches to a peripheral target, and one via the dorsal root to the spinal cord. The peripheral processes are associated with the cellular and extracellular environment of the PNS throughout their course. The central processes are initially localized in the PNS environment, and continue into the spinal cord to terminate in different areas of the spinal cord grey matter. A minority of these axons have collaterals which ascend in the dorsal funiculus to the dorsal column nuclei in the caudal brain stem.

* To whom correspondence should be addressed.

0300–4864/97 ( 1997 Chapman and Hall

In the rat, primary sensory fibres grow into the spinal cord and form their pattern of termination long before the boundary between the PNS and CNS is established in the early postnatal period (Berthold et al., 1984; Pindzola et al., 1993). This boundary is located outside the spinal cord, because the CNS tissue extends distally into the rootlets of the dorsal root in a dome-shaped fashion some 100 lm from the spinal cord surface (cf. Fig. 2C). This CNS extension is the central tissue projection (CTP), which is distinguished by a compact layer of astrocytes. The CTP is surrounded by PNS tissue, the peripheral surrounding (PS). Together the CTP and PS form the dorsal root transitional zone (DRTZ). If the central processes of DRG neurons are injured in the PNS compartment, the injured axons regrow

812 towards the spinal cord, but stop abruptly when encountering the astrocytes of the DRTZ (Cajal, 1928; Carlstedt, 1985a; Liuzzi & Lasek, 1987; Stensaas et al., 1987). Numerous attempts have been made over the years to overcome the challenge presented by the DRTZ astrocytes to axonal regeneration. Thus, transected postganglionic sympathetic axons, ventral root motor axons or peripheral axons of DRG neurons, all known to have powerful regeneration capacity, have been anastomosed to the cut proximal portion of a dorsal root (Carlstedt, 1983, 1985b). All these attempts showed that fibres ceased to grow as soon as they reached the DRTZ astrocytes. The first demonstration that fibres were able to grow from the periphery into the adult mammalian spinal cord came with a novel experimental approach, transplantation of human foetal neurons to the cavity of extirpated, native, adult rat DRG (Kozlova et al., 1995). Fibres from human fetal DRG cells of 8–11 weeks gestational age were able to grow into the spinal cord of adult rats. These fibres were sensitive to non-permissive DRTZ astrocytes and were associated with meninges and blood vessels in the host spinal cord. The route of their ingrowth was not defined, however. The DRTZ is characterized by a sharp discontinuity in tissue type at the CNS–PNS interface. The basal lamina associated with the Schwann cells of the PNS covers the superficial surface of the astrocytic processes which form the specialized glia limitans in this area (Fraher, 1992), but is absent central to this site. The DRTZ boundary can therefore be precisely delineated with antibodies to laminin (Bignami et al., 1984). In the present study we have analysed the relationship between this boundary and the growing human axons by combining laminin immunostaining with antibodies specific for human 70 kD neurofilament protein. Materials and methods Experiments were carried out on 15 adult, female SpragueDawley rats (150–250 g body weight), anaesthetized with a mixture of Ketalar, Rompun and Acepromazin intramuscularly. The left fourth and fifth lumbar DRG were exposed and removed via a partial laminectomy. The ensuing cavity was filled with a collection of 6–10 human embryonic DRGs. The use of human tissue was approved by the research ethics committee at Karolinska Institute and the material was obtained from routine abortions after informed consent by the women. The embryonic age was determined by certain anatomical landmarks and by measuring crown-rump length at the time of dissecting the DRGs (England, 1990). Grafts were made of DRGs from embryos with an estimated age of 5–5.5 weeks (n\7), 6–6.5 weeks (n\4), 7–7.5 weeks (n\3), and 8 weeks (n\1). The host animals were maintained continuously on cyclosporin A and vibramycin intraperitoneally. Ten to 15 weeks after grafting the animals were reanaesthetized and perfused

K O Z L O V A , S E I G E R and A L D S K O G I U S via the left ventricle with normal saline (38 °C) followed by a cold (4–6 °C, pH 7.4) fixative containing 4% (w/v) formaldehyde and 14% saturated picric acid (v/v) in 300 mOsm phosphate buffered saline (PDS). The graft sites and spinal cord segments L2–S1, including the proximal parts of dorsal roots, were removed and postfixed for about 2 h in the fixative solution. Thereafter the tissue was kept overnight in cold PBS containing 10% (w/v) sucrose. Serial 14 lm thick sections were cut from the graft site and the spinal cord on a cryostat. The spinal cord sections were cut in the horizontal plane. Sections were mounted on gelatine-coated slides, preincubated for 1 h in 1% phosphate-buffered bovine serum albumin, and incubated overnight with mouse monoclonal antibodies to an epitope specific for human neurofilament protein (hNF) 70 kD (1 : 200, Serotec, UK). Horizontal sections from the spinal cord were incubated with antibodies to laminin (rabbit polyclonyl, 1 : 200, Gibco Labs). The immune complexes were visualized with Texas Red conjugated donkey anti-mouse IgG (1 h, 1 : 40, Jackson ImmunoResearch), and fluorescein (FITC) conjugated swine anti-rabbit IgG (1 h, 1 : 40, Dakopatts). The sections were examined in a Nikon epifluorescence microscope, and hNF-labelled fibres in association with laminin labelled DRTZ were traced in serial sections through the DRTZ.

Results All 15 rats showed human neurofilament immunoreactivity (hNF-IR) in the L4–L5 graft sites and 12 of them had hNF-positive fibres in the host rat spinal cord 10–15 weeks after grafting. In these 12 rats hNF-positive nerve fibres were observed to penetrate the host DRTZ. In two rats from 5–5.5 weeks and one rat from 7–7.5 weeks donor groups, with surviving grafted DRG neurons, numerous fibres were observed in the rootlets, but no fibres were found in the host spinal cord. Graft area The graft site was usually larger than intact L4–L5 ganglia, and appeared to be heavily infiltrated with connective tissue. Grafted cells were organized in clusters and had the typical shape of DRG neurons (Fig. 1A). In some instances these clusters were separated by bundles of fibres, resembling the situation in a normal DRG. Grafted cells displayed different hNFIR intensity, and smaller size than that of mature rat ganglion cells. Immunostaining of adult rat DRG with anti-hNF showed no cross-reactivity (Fig. 1B). DRTZ area L4–L5 DRTZ consists of 8–12 separate rootlets. Each has its own CTP and PS (cf. Fig. 2C). Using laminin antibodies the boundary between PNS and CNS was clearly visualized in every rootlet since laminin was expressed in dorsal rootlets around the DRTZ astrocytes (Fig. 2A and B). We observed a dramatic upregulation of laminin expression throughout the peripheral compartment of the dorsal roots on the operated side (Fig. 2B). Between adjacent rootlets,

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Fig. 1. Staining with antibodies to human neurofilament protein (hNF). (A) Graft site of a 6.5-week-old embryonic donor 3.5 months after transplantation. A cluster of numerous hNF immunoreactive ganglion cells, some of which have the appearance of typical pseudo-unipolar neurons (arrows). (B) Dorsal root ganglion from the contralateral side of the same recipient rat, demonstrating that the anti-hNF antibody does not cross-react with rat antigens. Note that the unlabelled ganglion cells in the adult recipient rat are larger than the human cells in the graft (arrows). Scale bar: 50 lm.

the laminin-positive PS always terminated at blood vessels (Fig. 2A and B). There were two types of blood vessels in contact with the end of the PS. The first type extended parallel to the surface of the spinal cord (Fig. 3). The second type was oriented perpendicular to the spinal cord surface and extended into the deep laminae of the dorsal horn (Figs 5 and 6). Both types of blood vessels served as bridges for growth of

embryonic nerve fibres into the host spinal cord, and determined the pattern of ingrowth displayed by the human fibres. Their direction of growth was dependent on the arrangement of the blood vessels. Growth of human fibres through the DRTZ All cases demonstrated extensive growth of human axons in the dorsal roots and rootlets. The density

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Fig. 3. The same case as in Fig. 2(A, B). Horizontal section through L4 dorsal rootlets, 80 lm from the dorsal surface of the spinal cord. Double staining with antibodies to hNF (red) and laminin (green). PNS, peripheral compartment of dorsal rootlet; SC, spinal cord. Human NF-positive fibres (first type of growth; cf. Fig. 7B) grow into the spinal cord along horizontally oriented blood vessels (larger arrows) in the end of the peripheral surrounding (asterisk), and continue to grow in the spinal cord without association with blood vessels (medium-sized arrows). Some fibres continue their growth along the surface of the spinal cord (small arrows; third type of growth; cf. Fig. 7B). Note that the density of hNF-immunoreactivity fibres is reduced before they encounter the central tissue projection. Arrowheads, end of central tissue projection. Scale bar: 50 lm.

of axons in the dorsal root abruptly decreased some distance before they reached the DRTZ (Fig. 3). Many of the human fibres continued their growth around the CTP in the laminin-rich PS. There, fibres coursed together in tight bundles and grew into the spinal cord as soon as the PS ended. These fibres were always observed in association with blood vessels at the termination of the PS. When blood vessels were running in parallel with the spinal cord surface, axons accompanied them for a short distance and then spread out in the superficial area of the grey matter to a depth of 100–200 lm from the surface of the spinal cord, corresponding to laminae I and II. In horizontal spinal cord sections these fibres appeared to grow straight through the DRTZ (Figs 3 and 4), and could be traced in the horizontal plane for a distance of 500–600 lm from their companion blood vessel in the DRTZ. Some

fibres from these bundles grew to the lateral part of the dorsal column. When blood vessels were running perpendicular to the spinal cord surface, human axons accompanied them into deep laminae of the spinal cord dorsal horn (Figs 5 and 6). By tracing fibres in association with blood vessels in serial sections, it turned out that fibres grew in bundles into the spinal cord in association with blood vessels to a depth of about 600 lm, after which they left the blood vessels and spread out in a flame-shaped pattern in different directions (Fig. 6) over a radius of 500–600 lm around the blood vessels and gradually became extremely thin and difficult to observe. Fibres which did not get access to blood vessels continued to grow on the surface of the spinal cord (Fig. 3). We distinguished human fibres at all levels of the L4 and L5 spinal cord segment, and traced them for about 1 mm in the spinal cord.

Fig. 2. (A, B) Horizontal section through some of the rootlets of the L4 segment of the recipient spinal cord. about 30 lm from the dorsal surface, stained with an antibody to laminin. Donor age 8 weeks, postgrafting survival time 10 weeks, PNS, peripheral compartment of the dorsal root; SC, spinal cord; open arrows, peripheral surroundings, which always end with blood vessels (arrows); arrowheads, distal end of the central tissue projection. Scale bar: 50 lm. (A) Unoperated side showing smooth outline of the dorsal root transitional zone in the rootlets. (B) Operated side. Note the marked upregulation of laminin expression, as well as the irregular outline of the dorsal root transitional zone. (C) Schematic diagram of the organization of the dorsal root transitional zone (DRTZ). The central tissue projection (CTP) extends distally in every rootlet. PS, peripheral surrounding. The two compartments CTP and PS form the DRTZ.

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Fig. 4. Horizontal section through some of the rootlets of the L4 spinal cord, 100 lm from the dorsal surface, stained with antibodies to human NF. Donor age 5 weeks, postgrafting survival time 15 weeks. PNS, peripheral compartment of the dorsal root. Note horizontally growing fibres along horizontally oriented blood vessels (large arrows; first type of growth; cf. Fig. 7B), as well as extensive, fine calibre arborizations (arrows) in the spinal cord (SC). Scale bar: 50 lm.

Figure 7 summarizes the pattern of axonal growth in the normal situation (Fig. 7A), compared to that observed in the present study (Fig. 7B). Thus, human embryonic fibres followed horizontally arranged blood vessels (1) into superficial laminae, perpendicularly arranged blood vessels (2) into deep laminae, or meninges (3). Discussion Axons of adult mammals fail to regenerate in the CNS because the mature CNS environment is inhibitory to axon growth, and the vigour of growth displayed by regenerating mature axons is less than that of neurite outgrowth during development. The non-permissive nature of the mature CNS was overcome, however, by a novel approach, i.e. transplantation of embryonic tissue into the CNS of adult mammals. Axons from grafted rat neurons were found to extend extensively within the host grey, but not in the white matter (Bjo¨ rklund & Lindvall, 1979; Wiklund & Bjo¨ rklund, 1980). These results were consistent with the notion that the white matter is inhibitory to axonal growth in the adult CNS (Schnell & Schwab, 1990; Fawcett et al., 1992; Schwab et al., 1993). This situation was changed by using human neural tissue for transplantation. Thus, grafted human embryonic neurons successfully grow for 6–20 mm in the white matter of adult rat. This was found to be true for

different types of neurons, such as dissociated striatal primordia from 6–8-week-old embryos (Wictorin et al., 1991), human mesencephalic neuroblasts (Wictorin et al., 1992), and human embryonic spinal cord tissue (Wictorin & Bjo¨ rklund, 1992). The results from these experiments suggested that human immature CNS neurons may be relatively insensitive to the myelin- and oligodendrocyte-associated inhibitory factors of the rodent CNS. Subsequently, similar findings were made with mouse embryonic hippocampal neurons transplanted to different myelinated fibre tracts of adult rats. These transplanted neurons were found to extend axons in the host white matter for up to 10 mm (Li & Raisman, 1993; Davis et al., 1994). In our previous study we were able to show that human fetal DRG cells give rise to axons which grow into the host spinal cord when the ganglia were transplanted into the L4–5 host DRG cavities. These fibres first had to grow for 35 mm in the peripheral part of the dorsal root, and then continued their growth in the spinal cord. Since we observed human fibres throughout the L4 and L5 spinal cord segments, we tentatively concluded that these fibres grew in the spinal cord altogether up to 5–6 mm. In the present study we were able to trace bundles of fibres. We then observed that individual bundles extended for about 400–500 lm in association with blood vessels, and then over an area with a radius of an additional 500–600 lm, i.e. a total distance of axonal elongation of about 1 mm in the

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Fig. 5. Horizontal section 40 lm from the dorsal surface of the spinal cord, in the dorsal area of L4 dorsal rootlets. Donor age 5 weeks, postgrafting survival time 15 weeks. Double staining with antibodies to human neurofilament protein (A) and laminin (B). PNS, peripheral nervous system compartment; SC, spinal cord; arrowheads, distal end of central tissue projection. Human fibres grow into the spinal cord in association with perpendicularly oriented blood vessels in the most proximal end of the PS (arrows: second type of growth; cf. Fig. 7B). Scale bar: 50 lm.

spinal cord. An explanation for the limited growth by human fibres within the host spinal cord may be their contact with denervated postsynaptic neurons close to the area of the grey matter where these fibres enter. In our previous study we showed that human embryonic fibres were sensitive to DRTZ astrocytes. The results of our present experiments show that even

young embryonic DRG neurons are sensitive to the inhibitory influence of the DRTZ glial cells. This influence is more effective than the inhibitory influence of the adult spinal cord, since human fibres were able to elongate and arborize in this CNS environment. In all 12 cases we observed fibres in PS growing around the DRTZ astrocytes and passing the PNS–CNS

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Fig. 6. Horizontal sections from the same case as in Fig. 5. Staining with antibodies to human NF. Immunolabelled fibres are traced ventrally in the spinal cord grey matter (SC) along the blood vessels indicated in Fig. 5 (second type of growth, cf. Fig. 7B). (A) Section 160 lm from the dorsal surface of the spinal cord. Labelled fibres course in association with perpendicularly oriented blood vessels (arrows). PNS, peripheral compartment of the dorsal root. (B) Section 400 lm from the dorsal surface of the spinal cord, tracing the same blood vessels as in (A). Human fibres have left their association with blood vessels and give rise to extensively arborizing fine calibre fibres (arrows). Scale bar: 50 lm.

boundary, at the termination of the PS, along blood vessels. Thereafter, they continued to grow in the superficial laminae of the dorsal horn, or in the white matter of the lateral part of the dorsal column, following the ‘‘longitudinally’’ oriented blood vessels, or in deeper laminae, following the ‘‘perpendicularly’’

arranged blood vessels. However, no fibres crossed the DRTZ directly throgh its astrocytic lining. Results from studies in vitro indicate that the inhibitory effects on growing axons by astrocytes depend on their threedimensional arrangement and relationship to the extracellular matrix (Fawcett, 1994; Smith-Thomas et al.,

Human prenatal DRG axons grow into rat spinal cord

Fig. 7. Schematic diagram showing the normal situation in which dorsal root fibres cross the astrocytic lining of the dorsal root transitional zone (DRTZ) (A), and the patterns of growth by human fibres from grafted embryonic dorsal root ganglion cells (B). Human fibres follow horizontally (1), or perpendicularly (2) arranged blood vessels into the rat spinal cord, or grow along the meninges (3).

1994; Fok-Seang et al., 1995). Cell lines forming dense aggregates with large areas of close membrane appositions and abundant extracellular matrix showed the strongest inhibitory effect. In our experiments, the DRTZ astrocytes showed dense growth of processes to the periphery (Kozlova et al., 1995), that might prevent growth of human fibres. Furthermore, we observed that the number of human fibres sharply declined before they came in contact with the astrocytic surface limiting layer of the CTP. The DRTZ astrocytes there appear to have properties similar to those of the astrocytes which form the major component of the glial scar following CNS trauma (see Reier & Houle, 1988 for review). DRTZ astrocytes can prevent growth not because of their particular structural arrangement, but because of the presence of non-permissive molecules. It was shown that the upregulation of immunoreactivity for repellent molecules like tenascin and chondroitin sulphate proteoglycan occurs in the DRTZ among

819 reactive astrocytes after a direct lesion of the dorsal root (Pindzola et al., 1993; Zhang et al., 1995). The removal of the DRG prior to the transplantation in our experiments can have a similar effect on DRTZ astrocytes. The inhibitory astrocyte cell lines produce similar proteoglycans in vitro (Fawcett, 1994). An alternative explanation why fibres grow around the DRTZ astrocytes may be that the human embryonic fibres growing in a laminin-rich environment are resistant to a change in their substrate. Previous studies in vitro have shown collapse of growth cones at the interface between two substrates, even if they are both growthpermissive (Burden-Gulley et al., 1995). When human fibres reach the spinal cord, they are able to continue their growth, perhaps because an alternative substrate for their continued extension does not exist. After removal of the host DRG, L4 and L5 dorsal root axons undergo Wallerian degeneration and the associated Schwann cells proliferate. These events are accompanied by a marked upregulation of laminin in the peripheral compartment of the dorsal root (Bignami et al., 1984). Previous investigators have reported regeneration of dorsal root axons in response to a peripheral nerve environment or Schwann cell transplant. After complete spinal cord transection, including dorsal column axons, in adult rats, dorsal root axons were able to regenerate through a sciatic nerve graft (Richardson & Issa, 1984; Richardson & Verge, 1986). After crushing the dorsal root, the lesioned axons regenerate readily to the PNS–CNS interface (Carlstedt, 1985a, b). Schwann cells implanted into the dorsal column of the adult spinal cord have been shown to promote sprouting by dorsal column axons (Li & Raisman, 1994). Finally, regeneration of dorsal root axons is promoted when the axons are allowed to grow into a peripheral nerve graft (Chong et al., 1996). Laminin, which is a constituent of the Schwann cell basal lamina, has been shown to have exclusively positive effects on growing peripheral and central axons. Laminin is able to mask the inhibitory activity of myelin purified from the PNS or CNS (David et al., 1995). Laminin is abundantly expressed in the developing CNS and has been shown to be concentrated in regions in which extracellular cues have been postulated to guide growing neurites (Hunter et al., 1992). In the adult CNS laminin is associated with blood vessels and reactive astrocytes (Hagg et al., 1989). The close association between laminin-like immunoreactivity and axonal sprouts in the lesioned spinal cord, indicates a role for laminin in axonal growth and/or guidance following spinal cord injury (Frise´ n et al., 1995). Finally, DRG neurons have been shown to express integrin receptors to laminin (Tomaselli et al., 1993). The fibres from the grafted human DRGs that we observed central to the host DRTZ all showed a strong association with laminin by using blood vessels as bridges for their growth into the host spinal cord.

820 In three cases we did not find growth by human fibres into the host spinal cord, although such fibres were present in the dorsal root. The reason for this is unclear, but could be related to insufficient growth capacity by the fibre population originating from the grafted DRG cells in these particular cases. The age of the donor ganglia in the present study was 5–8 weeks, compared to 8–11 in our previous report (Kozlova et al., 1995). This difference in age could influence the survival of the transplanted DRG neurons as well as their ability to find a pathway into the host spinal cord. Studies in vitro of the growth of neurites from DRG cells of different developmental ages on cryocultures of the DRTZ demonstrate that the extent with which neurites pass through this region is great with embryonic compared to neonatal DRGs (Golding et al., 1996). Since human embryonic fibres grew in bundles, it was not possible in our experiments to analyse rigorously the proportion that entered the host spinal cord by 5–8 weeks DRGs. The possibility that axons from young embryonic human DRGs grow more extensively in vivo into the recipient spinal cord can not be excluded. Most of the human axons in the host spinal cord were located in the grey matter, and only a small number was found in the medial part of the dorsal funiculus. This could reflect the influence from nonpermissive factors in the white matter (Schwab et al., 1993) and/or the fact that most of the target neurons for dorsal root axons are located at the segmental levels, where we observed extensive growth. We observed fibres leaving deeply as well as superficially situated blood vessels and arborizing in different grey matter areas from the spinal cord surface and to a depth of about 600 lm. Often, human fibres that followed a blood vessel were found to leave it simultaneously in a particular area. It was not clear what was the ‘signal’ for the fibres to leave their association with blood vessels. Sometimes, it was obviously an abrupt change in the course of the blood vessel itself, but in

K O Z L O V A , S E I G E R and A L D S K O G I U S some cases the blood vessel continued to extend into deeper levels of the spinal cord in the same direction, but without accompanying bundles of nerve fibres. The flame-shaped pattern of extension after the fibres left the blood vessel appeared to be horizontally oriented, i.e. in the coronal plane of the spinal cord, suggesting the existence of specific target attractions at these particular levels. Although the possible inhibitory effect on axonal growth from DRTZ astrocytes was not tested directly, there was a prominent decline in the number of human axons in the rootlet distal to the DRTZ. This observation indicates that despite all the positive influences on axonal growth from the Schwann cells, such as growth factors, adhesion molecules and extracellular matrix components (Gillen et al., 1996; Xu et al., 1997), the DRTZ astrocytes have a strong inhibitory effect on the immature human dorsal root axons. Human fibres never penetrated the DRTZ astrocyte surface layer, but grew around it in the laminin-rich PS, using blood vessels as bridges to grow into the host spinal cord.

Acknowledgements We are very grateful to Professor Gunnar Grant for making it possible to carry out some of our work in his research facilities, and to Professor John P. Fraher for critically reading the manuscript, to Ms Britt Meijer for technical assistance, and to Dr Bengt Fundin for computer assistance with the illustrations. Supported by the Swedish Medical Research Council, project number 5420 and 6555, by Clas Groschinsky Memorial Foundation, Deutsche Stiftung Querschnittla¨ hmung, National Society for Traffic and Polio Injuries, The Spinalis Foundation, The Marianne and Marcus Wallenberg Foundation, and the Karolinska Institute. Dr E. N. Kozlova received a visiting scientist fellowship from the Wenner–Gren Center Foundation.

References BE RT H OL D, C . - H. , C AR L STE DT, T . & C OR N E LIUSSON, O . (1984) Anatomy of the nerve root at the

central–peripheral transitional region. In Peripheral Neuropathy, Vol. 1, 2nd edn (edited by DY C K , P. J. , TH OM AS, P . K. , L AM B ERT, E . H . & BU NG E , R. P .) pp. 150–70. Philadelphia: W. B. Saunders. BIG NA MI, A ., C H I, N . H . & DA H L , D. (1984) Regenerating dorsal roots and the nerve entry zone: an immunofluorescence study with neurofilament and laminin antisera. Experimental Neurology 85, 426–35. BJO® R KL U ND, A . & L IN DVAL L, O. (1979) Regeneration of normal terminal innervation of the globus pallidus by collaterals from the nigro-striatal pathway. Brain Research 171, 271–93.

B UR DE N- GU L L E Y, S. M. , PA YNE , H. R . & L E MMO N , V .

(1995) Growth cones are actively influenced by substrate-bound adhesion molecules. Journal of Neuroscience 15, 4370–81. CAJ A L , S. R . y . (1928) Degeneration and Regeneration of the Nervous System. London: Oxford University Press. CAR L ST E DT , T . (1983) Regrowth of anastomosed ventral root nerve fibers in the dorsal root of rats. Brain Research 272, 162–5. CAR L ST E DT , T . (1985a) Regenerating axons form nerve terminals at astrocytes. Brain Research 347, 188–91. CAR L ST E DT , T. (1985b) Regrowth of cholinergic and catecholaminergic neurons along a peripheral and central nervous pathway. Neuroscience 15, 507–18.

Human prenatal DRG axons grow into rat spinal cord CHONG, M. S., WOOLF, C. J., TURMAINE, M., EMS O N , P . C . & A N D E R S O N , P . N . (1996) Intrinsic ver-

sus extrinsic factors in determining regeneration of the central processes of rat dorsal root ganglion neurons: the influences of a peripheral nerve graft. Journal of Comparative Neurology 370, 97–104. DAVID, S., BRAUN, P. E., JACKSON, D. L., KOTTIS, V. & M c K E R R A C H E R , L . (1995) Laminin overrides

the inhibitory effects of peripheral nervous system and central nervous system myelin-derived inhibitors of neurite growth. Journal of Neuroscience Research 42, 594–602. D A V I E S , S . J . , F I E L D , P . M . & R A I S M A N , G . (1994) Long interfascicular axon growth from embryonic neurons transplanted into adult myelinated tracts. Journal of Neuroscience 14, 1596– 612. E N G L A N D , M . A . (1990) A Colour Atlas of Life Before Birth. 2nd edn. Aylesbury, Bucks: Hazell Books. F A W C E T T , J . (1994) Astrocytes and axon regeneration in the central nervous system. Journal of Neurology 214, S25–8. FAWCETT, J. W., FERSHT, N., HOUSDEN, L., S C H A C H N E R , M . & P E S H E V A , P . (1992) Axonal

growth on astrocytes is not inhibited by oligodendrocytes. Journal of Cell Science 103, 571–9. FOK-SEANG, J., SMITH-THOMAS, L. C., MEINERS, S., MUIR, E., DU, J. S., HOUSDEN, E., JOHNSON, A. R., FAISSNER, A., GELLER, H. M., KEYNES, R. J . , R O G E R S , J . H . & F A W C E T T , J . W . (1995)

An analysis of astrocytic cell lines with different abilities to promote axon growth. Brain Research 689, 207–23. F R A H E R , J . P . (1992) The CNS–PNS transitional zone of the rat. Morphometric studies at cranial and spinal levels. Progress in Neurobiology 38, 261–316. F R I S E¨ N , J . H A E G E R S T R A N D , A . , R I S L I N G , M . , FRIED, K., JOHNSSON, C. B., HAMMARBERG, H., E L D E , R . , H O® K F E L T , T . T . & C U L L H E I M , S . (1995)

Spinal axons in central nervous system scar tissue are closely related to laminin-immunoreactive astrocytes. Neuroscience 65, 293–304. G I L L E N , C . , K O R F H A G E , C . & M U® L L E R , H . W . (1997) Gene expression in nerve regeneration. The Neuroscientist 3, 112–22.

821 circumventing dorsal root entry zone astrocytes. NeuroReport 6, 269–72. L I , Y . & R A I S M A N , G . (1993) Long axon growth from embryonic neurons transplanted into myelinated tracts of the adult rat spinal cord. Brain Research 629, 115–27. L I , Y . & R A I S M A N , G . (1994) Schwann cells induce sprouting in motor and sensory axons in the adult rat spinal cord. Journal of Neuroscience 14, 4050–63. L I U Z Z I , F . J . & L A S E K , R . J . (1987) Astrocytes block axonal regeneration in mammals by activating the physiological stop pathway. Science 237, 642–4. P I N D Z O L A , R . R . , D O L L E R , C . & S I L V E R , J . (1993) Putative inhibitory extracellular matrix molecules at the dorsal root entry zone of the spinal cord during and after root and sciatic nerve lesions. Experimental Neurology 156, 34–48. R E I E R , P . J . & H O U L E , J . (1998) The glial scar: its bearing on axonal elongation and transplantation approaches to CNS repair. In Advances in Neurology, Vol. 47, Functional Recovery in Neurological Disease (edited by W A X M A N , S . G .) pp. 87–138. R I C H A R D S O N , P . M . & I S S A , V . M . K . (1984) Peripheral injury enhances central regeneration of primary sensory neurones. Nature 309, 791–3. R I C H A R D S O N , P . M . & V E R G E , V . M . (1986) The induction of a regenerative propensity in sensory neurons following peripheral axonal injury. Journal of Neurocytology 15, 585–94. S C H N E L L , L . & S C H W A B , M . E . (1990) Axonal regeneration in the rat spinal cord produced by an antibody against myelin associated neurite growth inhibitors. Nature 343, 269–72. SCHWAB, M. E., KAPFHAMMER, J. P. & BANDTLOW, C . E . (1993) Inhibitors of neurite growth. Annual Review

of Neuroscience 16, 565–95. SMITH-THOMAS, C. L. FOK-SEANG, J., STEVENS, J., DU, J. S., MUIR, E., FAISSNER, A., GELLER, H. M . , R O G E R S , J . H . & F A W C E T T , J . W . (1994) An

inhibitor of neurite outgrowth produced by astrocytes. Journal of Cell Science 107, 1687–95. STENSAAS, L. J., PARTLOW, L. M., BURGESS, P. R. & H O R C H , K . W . (1987) Inhibition of regeneration: the

GOLDING, J. P., SHEWAN, D., BERRY, M. & COHEN, J . (1996) An in vitro model of the rat dorsal root entry

ultrastructure of reactive astrocytes and abortive axon terminals in the transition zone of the dorsal root. Progress in Brain Research 71, 457–68.

zone reveals developmental changes in the extent of sensory axon growth into the spinal cord. Molecular and Cellular Neuroscience 7, 191–203.

TOMASELLI, K. J., DOHERTY, P., EMMETT, C. J., DAMSKY, C. H., WALSH, F. S. & REICHARDT, L. F . (1993) Expression of b1 integrins in sensory neurons

HAGG, T., MUIR, D., ENGVALL, E., VARON, S. & M A N T H O R P E , M . (1989) Laminin-like antigen in

of the dorsal root ganglion and their function in neurite outgrowth on two laminin isoforms. Journal of Neuroscience 13, 4880–8.

rat CNS neurons: distribution and changes upon brain injury and nerve growth factor treatment. Neuron 3, 721–32. HUNTER, D. D., LLINAS, R., ARD, M., MERLIE, J. P. & S A N E S , J . R . (1992) Expression of s-laminin and

laminin in the developing rat central nervous system. Journal of Comparative Neurology 323, 238–51. K O Z L O V A , E . N . , S T R O® M B E R G , I . , B Y G D E M A N , M . & A L D S K O G I U S , H . (1995) Peripherally

grafted human foetal dorsal root ganglion cells extend axons into the spinal cord of adult host rats by

WICTORIN, K., LAGENAUER, C. F., LUND, R. D. & B J O® R K L U N D , A . (1991) Efferent projections to the

host brain from intrastriatal striatal mouse-to-rat grafts: time course and tissue-type specificity as revealed by a mouse specific neuronal marker. European Journal of Neuroscience 3, 86–101. W I C T O R I N , K . & B J O® R K L U N D , A . (1992) Axon outgrowth from grafts of human embryonic spinal cord in the lesioned adult rat spinal cord. NeuroReport 3, 1045–8.

822

K O Z L O V A , S E I G E R and A L D S K O G I U S

WICTORIN, K., BRUNDIN, P., SAUER, H., LINDV A L L , O . & B J O® R K L U N D , A . (1992) Long distance

XU, X. M., CHEN, A., GUENARD, V., KLEITMAN, N. & B U N G E , M . B . (1997) Bridging Schwann cell trans-

axonal growth from human dopaminergic mesencephalic neuroblasts implanted along the nigrostriatal pathway in 6-hydroxydopamine lesioned adult rats. Journal of Comparative Neurology 323, 475–94. W I K L U N D , L . & B J O® R K L U N D , A . (1980) Mechanisms of regrowth in the bulbospinal system following 5,6-dihydroxytryptamine induced axotomy. II. Fluorescence histochemical observations. Brain Research 191, 129–60.

plants promote axonal regeneration from both the rostral and caudal stumps of transected adult rat spinal cord. Journal of Neurocytology 26, 1–16. ZHANG, Y., ANDERSON, P. N., CAMPBELL, G., MOHAJERI, H., SCHACHNER, M. & LIEBERMAN, A. R . (1995) Tenascin-C expression by neurons and glial

cells in the rat spinal cord: changes during postnatal development and after dorsal root or sciatic nerve injury. Journal of Neurocytology 24, 585–601.

.