THE ANATOMICAL RECORD (PART B: NEW ANAT.) 271B:49 – 60, 2003 SPECIAL ARTICLE Olfactory Ensheathing Cells: Historical Perspective and Therapeutic Potential J.G. BOYD, V. SKIHAR, M. KAWAJA, AND R. DOUCETTE* Olfactory ensheathing cells (OECs) are the glial cells that ensheath the axons of the first cranial nerve. They are attracting increasing attention from neuroscientists as potential therapeutic agents for use in the repair of spinal cord injury and as a source of myelinating glia for use in remyelinating axons in demyelinating diseases such as multiple sclerosis. This review mainly addresses the cell biological aspects of OECs pertinent to addressing two questions. Namely, where do OECs fit into the groupings of central nervous system (CNS)/peripheral nervous system (PNS) glial cells and should OECs be viewed as a clinically relevant alternative to Schwann cells in the treatment of spinal cord injury? The evidence indicates that OECs are indeed a clinically relevant alternative to Schwann cells. However, much more work needs to be done before we can even come close to answering the first question as to the lineage and functional relationship of OECs to the other types of CNS and PNS glial cells. Anat Rec (Part B: New Anat) 271B:49 – 60, 2003. © 2003 Wiley-Liss, Inc. KEY WORDS: olfactory ensheathing cells; OEC; regeneration; remyelination; spinal cord; Schwann cells; neuroscience INTRODUCTION Although there are exceptions to the rule (Doucette, 2001), in general, the regeneration of axons within the central nervous system (CNS) is not successful. In contrast, axons regenerating within the peripheral nervous system (PNS) are often successful in reinnervating sensory and effector organs so that almost normal function is restored (Kiernan, 1979; Baher and Bonhoeffer, 1994; Brecknell and Fawcett, 1996; Fawcett, 1998; Fawcett and Asher, 1999; Fraher, 1999; Behar et al., 2000). Both oligodendrocytes and astrocytes undoubtedly play a Dr. Boyd and Dr. Kawaja are with the Department of Anatomy and Cell Biology, Queen’s University, Kingston, Ontario, Canada. Dr. Skihar and Dr. Doucette are with the College of Medicine, University of Saskatchewan, Saskatoon. *Correspondence to: Dr. R. Doucette, Department of Anatomy and Cell Biology, College of Medicine, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada. Fax: 306-966-4298; E-mail: rondouc@duke. usask.ca DOI 10.1002/ar.b.10011 Published online in Wiley InterScience (www.interscience.wiley.com). © 2003 Wiley-Liss, Inc. major role in this abortive regeneration of CNS axons due to the presence of inhibitory molecules as well as to the lack of sufficient growth promoting molecules (Reier and Houle, 1988; Hoke and Silver, 1994; Moore and Thanos, 1996; Gebicke-Haerter et al., 1996; Frisen, 1997; Ridet et al., 1997). For example, the cell surface of astrocytes and the extracellular matrix in which they are embedded are believed to be more of a hindrance than a help during the growth of axons in vivo in the adult mammalian CNS (Reier, 1986; Reier and Houle, 1988; Reier et al., 1989; Ridet et al., 1997; Fraher, 1999). Myelin also, at least partially, contributes to the decreased permissiveness of adult mammalian CNS tissue for supporting axonal growth (Caroni et al., 1988; Cadelli and Schwab, 1991; Schwab, 1991; McLaurin and Yong, 1995; Fawcett and Asher, 1999). Microglia, which are extraordinarily sensitive to microenvironmental changes within the CNS (Barron, 1995; Gebicke-Haerter et al., 1996; Kreutzberg, 1996; Moore and Thanos, 1996), also appear to play a prominent role in creating a nonpermissive environment for axonal growth after CNS injury. Neuroscientists working on brain and spinal cord injury (see Santos-Benito and Ramon-Cueto, 2003; De Lucia et al., 2003), as well as on demyelinating diseases such as multiple sclerosis (see Franklin, 2003), have developed a considerable interest in another glial cell that is found only in the first cranial nerve. These glial cells, which are referred to as olfactory ensheathing cells (OECs), provide ensheathment in vivo for the unmyelinated olfactory axons within both the CNS and PNS portions of the olfactory nerve (Doucette, 1984, 1986, 1993; Raisman, 1985). Two reasons why these cells have become so popular are their ability to promote long distance growth in vivo of regenerating axons (Ramon-Cueto and NietoSampedro, 1994; Smale et al., 1996; Li et al., 1997; Perez-Bouza et al., 1998; Ramon-Cueto et al., 1998; Nash et al., 2002; Pascual et al., 2002;) and to remyelinate spinal cord axons (Franklin et al., 1996; Imaizumi et al., 1998) in the adult mammalian CNS. The present review will mainly address the cell biological aspects of OECs pertinent to addressing two questions. Namely, where do OECs fit into the groupings of CNS/PNS glial cells, and 50 THE ANATOMICAL RECORD (PART B: NEW ANAT.) should OECs be viewed as a clinically relevant alternative to Schwann cells in the treatment of spinal cord injury? For a general overview of the cell biology of these glial cells the reader is referred to several reviews on the subject (Doucette, 1990, 1995; Franklin and Barnett, 1997; Ramon-Cueto and Avila, 1998; Franklin, 2002a,b). WHO ARE OLFACTORY ENSHEATHING CELLS? OECs (Figure 1) provide ensheathment for olfactory axons, all of which are unmyelinated (Doucette, 1984, 1993; Raisman, 1985). Due to their ability both to support CNS axon regeneration and to remyelinate the regenerated axons, these cells have attracted attention as potential therapeutic agents for the treatment of demyelinating diseases and for repairing the damaged spinal cord. Recent studies, however, have questioned the axonal growth promoting (Takami et al., 2002) and remyelinating (Plant et al., 2002) ability of adult CNS-derived OECs. Thus, despite enthusiasm surrounding the therapeutic potential of OECs, it is important to emphasize the need for caution in moving too quickly from animal models to clinical trials. This need for caution is further exemplified by our inability to identify unequivocally the relationship of OECs to the currently accepted groupings of CNS and PNS glial cells. It is pertinent, therefore, to ask the question: Who are OECS? OECs Normally Differentiate into Nonmyelinating Glia In vivo, OECs express a nonmyelinating phenotype (Figure 2), providing ensheathment for small C fibers (i.e., the olfactory axons; Doucette, 1984, 1990; Raisman, 1985). They also express a nonmyelinating phenotype in neuron-free cell cultures, as demonstrated by Barnett et al. (1993) and by Doucette and Devon (1994, 1995). This phenotype is the one OECs should express in vitro, because they would not have expressed any myelinassociated molecules at the time of plating. OECs express a curious mixture of astrocyte-specific and Schwann cell–specific phenotypic features (Doucette, 1990, 1995; RamonCueto and Valverde, 1995; Franklin and Barnett, 1997; Ramon-Cueto and Avila, 1998; Franklin, 2002a) and have been reported to perform the roles of both astrocytes and Schwann cells (Doucette, 1990, 1993; Doucette and Devon, 1993). For example, they contribute to the formation of the glia limitans of the olfactory bulb (Berger, 1971; Barber and Lindsay, 1982; Doucette, 1984, 1990, 1993; Valverde and Lopez-Mascaraque, 1991), a role that elsewhere in the mammalian CNS is the exclusive domain of astrocytes (Peters et al., 1990). They also have the ability to assemble a PNStype myelin sheath around nerve fibers in vitro (Devon and Doucette, 1992, 1995) as well as in vivo (Franklin et al., 1996; Imaizumi et al., 1998; Smith et al., 2002), thus being induced by axons to express a phenotype more like that of Schwann cells. In vivo, the differentiation of OECs is intimately tied to the development Two reasons why OECs have become so popular are their ability to promote long distance growth in vivo of regenerating axons and to remyelinate spinal cord axons in the adult mammalian CNS. of the first cranial nerve. The progenitor cells giving rise to OECs are derived from the olfactory placode. During embryonic and fetal development, these cells migrate away from the placode toward the rostral wall of the telencephalic vesicle where they eventually contribute to the formation of the nerve fiber layer (NFL) of the presumptive olfactory bulb (Doucette, 1989; Marin-Padilla and Amieva, 1989). Upon entering the mesenchyme separating the placode from the telencephalic vesicle during early embryonic development, these immature OECs begin expressing the N-CAM and L1 cell adhesion molecules (Miragall et al., 1989), both of which may contribute to making the OECs an adhesive surface for the SPECIAL ARTICLE growth of olfactory axons from the olfactory epithelium into the developing olfactory bulb (Doucette, 1993). These OECs also begin to express the p75 neurotrophin receptor (Gong et al., 1994), the brain-fatty acid binding protein (B-FABP; Kurtz et al., 1994), and the diazepam binding inhibitor (Yanase et al., 2002). Although the function of p75 expression by OECs remains to be determined, it could potentially play a role in providing chemotactic guidance to growing olfactory axons (Doucette, 1993). In OECs, B-FABP is coexpressed with the diazepam binding inhibitor; these molecules may play a role in lipid metabolism, including perhaps even in retinoid metabolism. As OECs continue to differentiate during fetal stages of development, they begin to express the L14 lectin (Mahanthappa et al., 1994) and the O4 antigenic epitope (Doucette and Devon, 1994; Franceschini and Barnett, 1996). The L14 lectin binds to molecules containing polylactosamine chains (Zhou and Cummings, 1993; Cooper et al., 1991) and was shown by Mahanthappa et al. (1994) to promote the adhesion of primary olfactory neurons to laminin in vitro in an integrin-independent manner. The O4 mouse monoclonal antibody (Sommer and Schachner, 1981) recognizes a cell surface epitope that is also expressed by glial cells in the O2A lineage (Gard and Pfeiffer, 1989; Trotter and Schachner, 1989) and by Schwann cells (Mirsky et al., 1990; Morgan et al., 1991). Postnatally (in rats and mice) OECs begin to express glial fibrillary acidic protein (GFAP) and connexin 43 (Cx43; Miragall et al., 1992), but neither Cx26 nor Cx32 are expressed by OECs in vivo in mice of any age (Miragall et al., 1992). Cx43 is also expressed by astrocytes, thus raising the question of whether OECs and astrocytes might communicate in the NFL by means of gap junctions. OECs also express DM-20 (Griffiths et al., 1995), which is an isoform of proteolipid protein (a CNS-specific myelin protein) and the PNS-specific myelin protein Po (Lee et al., 2001). Although the exact role of the DM-20 isoform remains to be determined, it may play a role in the development and differentiation of myelinating glia that is completely unrelated to myelin formation SPECIAL ARTICLE THE ANATOMICAL RECORD (PART B: NEW ANAT.) 51 phenotype by the olfactory axons. It should be noted, however, that olfactory axons may still contribute to the OEC’s expression of a nonmyelinating phenotype since Barber (1982b) showed these axons could induce Schwann cells to ensheath many of them in a common mesaxon, a type of ensheathment normally only seen in developing peripheral nerves. Inducing OECs to Express a Myelinating Phenotype Figure 1. Olfactory ensheathing cells (OECs) provide ensheathment for the primary olfactory axons from their exit from the olfactory epithelium (lower right) to their termination within the glomeruli (open circles) of the olfactory bulb. The OECs form the glia limitans at the point where the olfactory axons leave the peripheral nervous system to enter the central nervous system (the PNS–CNS transitional zone) and, along with astrocytes of the nerve fiber layer (NFL), also contribute to the glia limitans along more distal portions of the pial surface of the bulb. In this diagram, the astrocytes of the NFL are distinguished from the astrocytes of deeper layers to emphasize that the former cells express protease nexin-1, a chemotropic factor that is not expressed by astrocytes elsewhere within the adult mammalian CNS. (Griffiths et al., 1995). Similarly, although Po is a PNS-specific myelin protein, its expression long before myelination begins is suggestive of additional functions besides the assembly of a myelin sheath (Lee et al., 2001), perhaps even in axon– glial cell interactions during the growth of nerve fibers in embryos. OECs also express 3-phosphoglycerate dehydrogenase (Yamasaki et al., 2001), which is a key enzyme in the synthesis of L-serine; it was suggested by Yamasaki et al. (2001) that the L-serine made by OECs could be supplied to immature neurons (e.g., newly formed olfactory receptor neurons) in support of local membrane synthesis. Both OECs and astrocytes within the NFL of the olfactory bulb also express protease nexin-1 (PN-1; Reinhard et al., 1988; Scotti et al., 1994), whereas astrocytes elsewhere in the CNS do not express this enzyme. PN-1 has been demonstrated to function as a chemotropic factor for neurites in vitro (Monard et al., 1973; Guenther et al., 1985; Zurn et al., 1988) and could potentially be functioning in the same manner within the primary olfactory pathway. Most OECs eventually down-regulate their expression of the p75 neurotrophin receptor (Gong et al., 1994), whereas others, specifically those OECs contributing to the formation of the glia limitans of the olfactory bulb, continue to express the p75 receptor along the basal lamina-apposed portions of their plasma membrane (Vickland et al., 1991). In addition, OECs residing within the PNS but not the CNS portion of the primary olfactory pathway begin to express the tight junctional protein ZO-1 during early postnatal development (Miragall et al., 1994). The function of ZO-1 in OECs remains to be determined, but its differential expression by OECs of the PNS and CNS is supportive of the concept that these glial cells may well be heterogeneous not only in phenotype but also in function. At no time during development in vivo, however, have OECs ever been observed to express a myelinating phenotype. This latter observation perhaps has more to do with the absence of an instructive myelinating signal from the axons than it does with the OECs being induced to express a nonmyelinating Devon and Doucette (1992, 1995) provided the first evidence that OECs could assemble a myelin sheath when they cocultured these glial cells with dorsal root ganglion (DRG) neurons. These DRG cultures were first cleared of all non-neuronal cells by using fluorodeoxyuridine, with the purity of the cell cultures being confirmed by microscopy before the addition of OECs. The OECs added to the purified DRG cultures were prelabelled with a lipophilic fluorescent dye (PKH26) before coculture. The data unequivocally demonstrated that PKH26⫹ve OECs could express a myelinating phenotype; an electron microscopic examination of sister cultures demonstrated the OECs assembled PNS-like myelin around the DRG neurites. A few years later, Franklin et al. (1996) used an O4⫹ve clonal ensheathing cell line, derived from cells of the newborn rodent olfactory bulb and showed these cells would remyelinate spinal cord axons in vivo after transplantation into an ethidium bromide/ x-irradiated demyelinated area of the adult rat spinal cord, thus providing the first in vivo evidence of the myelinating ability of OECs. Subsequently, Imaizumi et al. (1998) replicated these in vivo experiments. More importantly, this report showed that the OEC remyelination of axons in the posterior columns enhanced axonal conduction across the previously demyelinated area, thus demonstrating a functionally significant degree of remyelination. Li et al. (1998) provided evidence that OECs also remyelinate corticospinal axons that have regenerated into and through the site of an OEC graft subsequent to an electrolytic lesion of the corticospinal tract. Curiously, however, an attempt by Plant et al. (2002) 52 THE ANATOMICAL RECORD (PART B: NEW ANAT.) SPECIAL ARTICLE to study the myelinating ability of OECs obtained by using the technique of Ramon-Cueto and Nieto-Sampedro (1992) failed to identify any OEC myelination of neurites when OECs were cocultured with DRG neurons. To what extent the purification procedure used by Plant et al. (2002) contributed to the lack of myelination in vitro by OECs, perhaps by selecting for cells with the least myelinating ability, remains to be determined. It is also known that OECs have similar, but not identical, requirements to Schwann cells for the expression of a myelinating phenotype (Barnett et al., 1993; Devon and Doucette, 1995; Doucette and Devon, 1994, 1995) so the discrepant results could be due as well to methodologic differences rather than to the actual myelinating ability of the OECs. Nevertheless, the contrasting results do highlight the paucity of information on the molecular mechanisms controlling the expression of a myelinating phenotype by OECs and underscore the need for more work in this area. Smith et al. (2001) made an initial attempt to identify which transcription factors might be expressed by OECs engaged in remyelinating spinal cord axons, but they did not present data at the cellular level to confirm that the cells expressing each of the transcription factors were indeed the same ones remyelinating the axons. The expression of a myelinat- Figure 2. Olfactory ensheathing cells (OECs) of the peripheral nervous sytem (A) and the central nervous system (B,C). A: An example of an olfactory nerve fascicle travelling within the subarachnoid space of the cranial cavity. Note the relative paucity of OEC perikarya with respect to the abundance of unmyelinated olfactory axons; the OECs ensheath large numbers of olfactory axons within the same mesaxon. OECs sometimes can be seen lying within such fascicles (as shown here) but are more commonly seen along the periphery of the fascicle. B: Both astrocytes (As) and OECs contribute to the glia limitans of the olfactory bulb. This photomicrograph shows how the cytoplasmic process of an astrocyte interdigitates with those of two adjacent OECs with no intervening basal lamina. C: The plasma membranes of astrocytes and OECs are also intimately apposed in areas where the OECs do not contribute to the formation of a glia limitans. Scale bars ⫽ 10 m in A, 5 m in B, 2 m in C. SPECIAL ARTICLE ing phenotype by OECs is heavily dependent on axonal contact (Devon and Doucette, 1992, 1995; Barnett et al., 1993; Doucette and Devon, 1994, 1995), a requirement that also applies to Schwann cells (Brunden and Brown, 1990; Brunden et al., 1990). Although myelinating OECs (in vivo as well as in vitro) assembled peripheral type myelin around those neurites large enough to be myelinated, in comparison to Schwann cells they have different growth media requirements for the assembly of a basal lamina and a myelin sheath. As reported by Devon and Doucette (1995), OECs, unlike Schwann cells, myelinated DRG neurites and assembled a basal lamina in vitro regardless of whether ascorbic acid was included in the growth medium. When neuron-free cultures of OECs are fed with a serum-free growth medium containing insulin, hydrocortisone, or triiodothyronine, these glial cells were not induced to express either galactocerebroside (Gal-C) or myelin basic protein (MBP; Barnett et al., 1993; Doucette and Devon, 1994, 1995). Gal-C and MBP are two molecules that are expressed by myelinating glia as they assemble a myelin sheath. Oligodendrocytes, on the other hand, are known to synthesize significant levels of myelin-associated molecules in vitro, even in the absence of axonal contact, provided the growth medium contains insulin, hydrocortisone, or triiodothyronine (McMorris et al., 1986, 1990; Lopes-Cardozo et al., 1989; Ved et al., 1989; Poduslo et al., 1990; Mozell and McMorris, 1991). These results point toward a different regulatory control over the promoter region of the MBP gene in oligodendrocytes and OECs (Doucette and Devon, 1994, 1995) in much the same way as this promoter region is believed to be controlled differently in oligodendrocytes and Schwann cells (Foran and Peterson, 1992; Gow et al., 1992). Doucette and Devon (1994, 1995) found that a more effective inducer of Gal-C expression by OECs in neuron-free cultures was increasing the intracellular level of cAMP (Doucette and Devon, 1994, 1995), which nevertheless still failed to induce these cells to express MBP (Doucette and Devon, 1995). Thus, although much has been learned as to how ensheathing cells THE ANATOMICAL RECORD (PART B: NEW ANAT.) 53 differ from both oligodendrocytes and Schwann cells, very little is actually known about the factors regulating the OEC’s expression of a myelinating phenotype or even whether all OECs possess the ability to myelinate. This latter point is important to keep in mind, because OECs are a heterogeneous population of cells. In vivo, some of them contribute to the formation of a basal lamina covering (Doucette, 1984, 1990, 1991, 1993); some continue to express the p75 receptor, whereas others do not (Vickland et al., 1991); and the OECs in the PNS portion of the pathway express ZO-1, whereas those centrally do not (Miragall et al., 1994). Heterogeneity of the OEC phenotype is also observed in vitro (Pixley, 1992; Franceschini and Barnett, 1996; Ramon-Cueto and Avila, 1998). Thus, any technique designed to separate OECs from other mucosal or CNS cells to obtain purified cell cultures runs the risk of selecting for specific functional abilities during the purification process. Can We Say Who OECs Are? Because they share a common phenotype, Nieto-Sampedro and his collaborators (Gudino-Cabrera and NietoSampedro, 2000) have suggested that OECs, pituicytes, and tanycytes are three members of a common family of Schwann-like macroglia of the CNS. Specifically, Gudino-Cabrera and Nieto-Sampedro (2000) have suggested these three glial cell types belong to a separate family of CNS macroglia, which they referred to as aldynoglia, a term derived from a Greek word meaning “to make grow.” OECs, tanycytes, and pituicytes all appear to make the CNS microenvironment more hospitable to growing axons, thus making them ideal candidates for glial cell transplantation into areas of CNS injury. Tanycytes (Prieto et al., 2000) and olfactory ensheathing cells (Ramon-Cueto and Nieto-Sampedro, 1994; Smale et al., 1996; Li et al., 1997, 1998; Ramon-Cueto et al., 1998, 2000; Navarro et al., 1999) have both been shown to promote the growth of CNS axons after grafting into the CNS of adult rats. Furthermore, all three putative Schwann cell–like macroglia possess the ability to myelinate DRG neurites in vitro (Gudino-Cabrera and Nieto-Sampedro, 2000). Are OECs one member of a family of aldynoglia? It is too early to draw a definitive conclusion, but it would also be premature to rule out the possibility of OECs, tanycytes, and pituicytes comprising members of a new family of glial cells. Other clues also point toward there being something different, although we hesitate to say unique, about the glial cells of the primary olfactory pathway. Within the NFL of the olfactory bulb (i.e., the CNS portion of the primary olfactory pathway), the only glial cells that are consistently present, in the absence either of experimental injury or of inflammation affecting the olfactory mucosa or nerve, are astrocytes and OECs. Personal and published ultrastructural observations (Doucette, 1984, 1991, 1993, 1995) made over a period of 20 years by one of the authors (R.D.) consistently support the view that these glial cells are the only ones comprising the glial cell environment of the unmyelinated NFL. It has been suggested that unmyelinated pathways in the adult mammalian CNS may also contain precursor cells with the potential to differentiate into oligodendrocytes (Levine, 1989). Because the origin of the progenitor cells giving rise to the astrocytes of the NFL remains to be determined, one intriguing possibility with respect to the NFL is that these glial progenitor cells, perhaps in response to an instructive signal from the differentiating OECs, differentiate exclusively into astrocytes as opposed to a mixture of astrocytes and oligodendrocytes, as typically happens elsewhere in the CNS. There is circumstantial evidence supporting a functionally relevant interaction between OECs and astrocytes in the NFL because these astrocytes are, to a certain extent, phenotypically distinct from astrocytes elsewhere in the adult mammalian CNS. The expression of PN-1 by astrocytes of the NFL raises the question of whether the intimate contact with OECs during the differentiation of these two cell types might be responsible for the expression of this chemotropic factor by a select group of astrocytes. The cellular interactions, if any, between OECs and astro- 54 THE ANATOMICAL RECORD (PART B: NEW ANAT.) cytes is a relatively unexplored area (Pollock et al., 1999; Lakatos et al., 2000). It is entirely natural to assume, as suggested above, that the astrocytes of the NFL are derived from CNS progenitors in much the same way as astrocytes are formed in other parts of the nervous system. However, it is also plausible the PN-1 expressing astrocytes of the NFL of the bulb are derived from OEC progenitors, rather than from the CNS progenitors giving rise to astrocytes elsewhere in the brain. Pixley (1992) reported the appearance of astrocyte-like and Schwann cell-like glial cells in cultures of olfactory mucosa, thus providing support for the possibility that OECs could differentiate into astrocyte-like cells. This entire area of OEC biology is in dire need of investigation, because the answers will undoubtedly provide clues to the identity of OECs. At the present time, however, it is not possible to make a definitive statement as to where OECs fit into the currently accepted groupings of CNS and PNS glial cells. ARE OECs A CLINICALLY RELEVANT ALTERNATIVE TO SCHWANN CELLS? In the absence of experimental intervention, olfactory axons are usually successful at reinnervating the olfactory bulb (Graziadei and Monti Graziadei, 1978; Monti Graziadei and Graziadei, 1979; Barber, 1982a; Doucette et al., 1983; Doucette, 1990), but the regenerative effort of most other types of axons within the CNS fails to support more than a minimal amount of growth (Kiernan, 1979; Reier et al., 1989; Fawcett, 1998). This dichotomy in the success of axonal growth appears due, at least in part, to the different glial cell environments through which olfactory and nonolfactory axons must grow (Doucette, 1990, 1995; Ramon-Cueto and Valverde, 1995; Franklin and Barnett, 1997; Fawcett, 1998; Ramon-Cueto and Avila, 1998). Macroglia of the CNS play a major role in the normally abortive regeneration of nonolfactory CNS axons, due most likely both to the presence of inhibitory molecules as well as to the lack of sufficient growth promoting molecules (Reier and Houle, 1988; Hoke and Silver, 1994; GebickeHaerter et al., 1996; Moore and Thanos, 1996; Frisen, 1997; Ridet et al., 1997). It is commonly believed that the cell surface of astrocytes and the extracellular matrices in which they are embedded are more likely to hinder rather than facilitate the growth of axons in vivo in the adult mammalian CNS (Reier, 1986; Reier and Houle, 1988; Reier et al., 1989; Ridet et al., 1997; Fraher, 1999). However, it is also clear that astrocytes are not always nonpermissive for axonal growth. In fact, there appear to be differences in the ability of astrocytes to support axonal growth, depending on the degree of differentiation of the cells (Baehr and Bunge, 1989; Hatten et al., 1991; Lucius et al., 1996); on Over the past several years, a number of studies have demonstrated the effectiveness of OECs in supporting the growth of nonolfactory CNS axons in vivo, although there have also been a couple of negative reports. whether the astrocytes have formed a two- or three-dimensional substratum for the growing axons (Fawcett et al., 1989); on the part of the CNS that is injured (Alonso and Privat, 1993a,b; Chauvet et al., 1998; Prieto et al., 2000); and on the presence or absence of macrophages (David et al., 1990), Schwann cells (Berry et al., 1992; Dezawa et al., 1999) or OECs (Li et al., 1997, 1998; Ramon-Cueto et al., 1998, 2000). Over the past several years, a number of studies have demonstrated the effectiveness of OECs in supporting the growth of nonolfactory CNS axons in vivo (Lustgarten et al., 1991; RamonCueto and Nieto-Sampedro, 1994; Smale et al., 1996; Li et al., 1997, 1998; Perez-Bouza et al., 1998; Ramon-Cueto SPECIAL ARTICLE et al., 1998; Navarro et al., 1999), although there have also been a couple of negative reports (Gudino-Cabrera et al., 2000; Takami et al., 2002). The driving force behind these experiments was to manipulate the cellular environment of the lesion site in the brain or spinal cord so as to create one more conducive to supporting axonal growth across the lesion and into the tissue on the distal side. Even though, as mentioned above, macroglia of the CNS appear to play a major role in the abortive regeneration of axons in the brain and spinal cord (Liuzzi and Lasek, 1987; Reier and Houle, 1988; Reier et al., 1989; Fawcett, 1991; Hatten et al., 1991), it was hypothesized that the presence of OECS would create a more favourable and supportive environment for axonal growth (Doucette, 1990, 1995; Wozniak, 1993; Ramon-Cueto and Valverde, 1995; Franklin and Barnett, 1997). The data on the growth of primary sensory afferents across the dorsal root entry zone of the spinal cord support this hypothesis because, in the presence of grafted OECs, the regenerating fibers even crossed the midline to enter the contralateral dorsal horn (Ramon-Cueto and Nieto-Sampedro, 1994; Navarro et al., 1999). OECs also promoted cholinergic fibers of the fimbria/fornix pathway to grow into OEC-containing collagen gels that were placed into a cavity left behind when a large portion of the fiber pathway was destroyed by aspiration (Smale et al., 1996). Raisman and his colleagues (Li et al., 1997, 1998) and Nash et al. (2002) lesioned the corticospinal tract in adult rats and showed that an OEC graft promoted the growth of corticospinal fibers through the lesion and into the distal portion of the pathway. Ramon-Cueto et al. (1998) showed a quite remarkable regrowth of axons within the adult rat spinal cord when they removed an entire spinal cord segment before grafting OECs into the proximal and distal stumps of the transected cord; their study demonstrated long-distance regrowth of the damaged fibers, with some axons growing as far as 12–14 spinal cord segments. Schwann cells are good promoters of axonal growth, and under certain conditions, even astrocytes have been SPECIAL ARTICLE shown to possess growth-promoting properties (Smith et al., 1986; Ard et al., 1987; Ard and Bunge, 1988; Neugebauer et al., 1988; Silver 1988; Bahr and Bunge, 1989; Hatten et al., 1991; Kawaja and Gage, 1991). However, promoting axonal growth in the injured spinal cord of adult mammals is only one of many problems that must be overcome in the treatment of spinal cord injury (Collins and West, 1989). Although it is unlikely that any one glial cell type can correct all of these problems, what is sorely needed is a cell that can adopt several different roles as the need arises. This feature is exactly what OECs appear to do (Doucette and Devon, 1993), being able for example to switch their phenotype from that resembling an astrocyte to one more like that of a myelinating Schwann cell (Devon and Doucette, 1992, 1995). It is very likely that OECs will be able to combine the roles of astrocytes and Schwann cells when transplanted into a lesion cavity, due to their having such a highly malleable phenotype (Doucette and Devon, 1993). These findings are mentioned not to suggest that OECs will promote axonal growth better than either Schwann cells or immature astrocytes. Rather, it is hypothesized that they will differ from Schwann cells in reconstituting the glia limitans and blood brain barrier and in allowing maximal re-entry of axons into the host tissue on the caudal side of the lesion. Admittedly, these roles can also be performed by immature astrocytes when transplanted into a lesion cavity, but such glial cells, being obtained from aborted human fetuses, would be more difficult to obtain. Schwann cells, on the other hand, not only fail to become integrated into the CNS, but astrocytes somehow perturb the neuron–Schwann cell interactions that lead to myelination (Blakemore and Crang, 1989; Franklin et al., 1992; Guenard et al., 1994b). Cellular migration of Schwann cells within the CNS parenchyma is also limited. For example, when they are transplanted into the cerebral hemisphere of newborn shiverer mice, the migration is mostly confined to the vicinity of the graft; the grafted Schwann cells are also eventually extruded from the CNS parenchyma (Baron-Van Everco- THE ANATOMICAL RECORD (PART B: NEW ANAT.) 55 oren et al., 1992). It has been suggested that astrocytes inhibit the migration of Schwann cells within the parenchyma of the CNS (Fawcett and Asher, 1999). Lakatos et al. (2000) have shown that Schwann cells and astrocytes avoid one another when they are present in the same culture dish. This dismal ability of Schwann cells to integrate into and to migrate within the CNS may be responsible, in part, for the poor regenerative growth of CNS axons distal to the site of injury. OECs, in contrast, are able to migrate within the CNS parenchyma of adult animals (Skihar and Doucette, 2001), thus highlighting another important distinction from Schwann cells. Successful regeneration along the distal portions of the fiber tracts There are certain conditions in which the CNS microenvironment can be made very supportive of axonal growth, effectively enabling sufficient numbers of axons to regenerate that some functional recovery may even be observed. may well require the presence of cells that provide a microenvironment more conducive to growth. In regard to the latter point, Ramon-Cueto et al. (1998) found in their spinal cord transection study that axons entered the distal stump of the cut cord only in those animals in which OECs had been grafted adjacent to the cut surface. Furthermore, they will differ from astrocytes in being able to remyelinate the regenerating axons, as demonstrated by Li et al. (1998). We hypothesize that OECs, by virtue of their ability to integrate and migrate within the CNS microenvironment, effectively modify the response of astrocytes and microglia to create a more supportive environment for the regeneration of axons. The microglial cell response to tissue injury has been shown to be modified by the presence of Schwann cells (Zeev-Brann et al., 1998; Dezawa et al., 1999), and some studies have found that CNS axons can regenerate through areas of tissue injury containing microglial cells/ macrophages (Prewitt et al., 1997; Rabchevsky and Streit, 1997; LazarovSpiegler et al., 1998; Stichel et al., 1999). Although it is known that OECs possess the ability to migrate within the adult mammalian CNS in animal models of spinal cord injury (RamonCueto and Nieto-Sampedro, 1994; Li et al., 1998; Ramon-Cueto et al., 1998), there has been no systematic examination of the factors inducing or guiding this migration. The reason this is important to consider is because it is still an open question as to whether grafted OECs are “leaders” or “followers” with respect to regenerating axons. Thus, the regeneration of primary sensory afferents across the midline into the contralateral dorsal horn (Ramon-Cueto and NietoSampedro, 1994; Navarro et al., 1999), the regeneration of corticospinal axons into the distal portion of the respective fiber tract (Li et al., 1997, 1998), and the multisegmental growth of axons after spinal cord transection in the study of Ramon-Cueto et al. (1998) were all associated with the migration of OECs away from the site of the graft. In each study, the regenerating axons were also always seen in areas that also contained migrating OECs. Could it be that the OECs are creating a more favourable and supportive environment for axonal growth? An intriguing question that, for the moment at least, cannot be answered. There are published data, however, to support the idea of grafted glial cells modifying the microenvironment to facilitate the growth of axons. For example, there are certain conditions in which the CNS microenvironment can be made very supportive of axonal growth, effectively enabling sufficient numbers of axons to regenerate that some functional recovery may even be observed (Smale et al., 1996; Beattie et al., 1997; Li et al., 1997, 1998; Berry et al., 1998; Ramon-Cueto et al., 1998, 2000; Huang et al., 1999). For exam- 56 THE ANATOMICAL RECORD (PART B: NEW ANAT.) ple, grafts of embryonic astrocytes (Smith et al., 1986; Kliot et al., 1990; Wunderlich et al., 1994; Sievers et al., 1995), which are known to reduce the inflammatory response and gliosis after CNS injury (Smith et al., 1986; Kliot et al., 1990), are known to support the growth of regenerating axons in the adult mammalian CNS. Silver and colleagues (Smith et al., 1986; Silver, 1988) were one of the first groups to explore the use of embryonic astrocytes to promote axon regeneration in adult animals. They showed that transplants of “immature” astrocytes, so called because they were obtained from a wound cavity in the cerebral cortex of neonatal rats, supported the regeneration of axons across the lesioned corpus callosum, even when transplanted into the CNS of adult animals. When given a choice, the regenerating callosal fibers preferred to grow directly on the plasma membrane of these immature astrocytes rather than on the basal lamina that the transplanted cells had formed in vivo (Smith et al., 1986). In the absence of experimental intervention, it is also known that axons in the adult mammalian CNS grow within areas containing Schwann cells, but poorly or not at all within areas lacking Schwann cells (Berry et al., 1992; Dezawa et al., 1999). Host Schwann cells migrate toward lesion cavities in the adult mammalian spinal cord where they appear to support the regrowth of axons (Matthews et al., 1979; Ohi et al., 1989; West and Collins, 1989; Li and Raisman, 1995). It has even been suggested that Schwann cells can regulate the gliotic response to CNS trauma (Senoo et al., 1998). Thus, in the presence of Schwann cells, the astrocytes and other cells repopulating and sealing the wound in the adult mammalian CNS appear to be more supportive of axonal growth, or at least less of an obstruction, than they are in the absence of Schwann cells (Matthews et al., 1979; Ohi et al., 1989; Dezawa and Nagano, 1996; Senoo et al., 1998; Dezawa et al., 1999). In fact, PNS axons will even regenerate into an optic nerve graft provided Schwann cells are present, as occurs in the optic nerve of the Browman-Wyse mutant rat (Hall et al., 1992). Axons of the sciatic nerve of adult rats also regen- erate into astrocyte-seeded semipermeable guidance channels provided Schwann cells are present in sufficient numbers (Guenard et al., 1994a). It remains to be determined whether Schwann cells directly interact with regenerating CNS axons and/or promote their growth by modifying the CNS microenvironment. We hypothesize that the in vivo axonal growth promoting ability of OECs is intimately tied to at least two features of the cells, which collectively with their support of axonal growth can be considered a triad of interconnected functions: (1) their ability to integrate into the CNS microenvironment, and (2) their ability to migrate long distances within the neuropil of the CNS. In light of these features, it is In light of the enthusiasm generated by these findings, it is somewhat disheartening that we still know so little about the molecular mechanisms driving the growth promoting and remyelinating ability of OECs or where they fit into the glial cell family. surprising that, in the recent study of Takami et al. (2002), grafted OECs simply disappeared from the lesion cavity with no evidence that they had migrated away into the surrounding neuropil. In the study by Gudino-Cabrera et al. (2000), the only other study in which grafted OECs failed to support the regeneration of CNS axons, it was noted that the OEC migration was solely in the proximal direction back toward the neuronal cell bodies, which could possibly explain why the axons failed to regenerate. But in the Takami et al. (2002) study, the cells seemed simply to disappear. CONCLUSIONS Research performed over the past decade demonstrated that OECs are SPECIAL ARTICLE a clinically relevant alternative to Schwann cells for promoting neural repair in the CNS of adult mammals (Doucette, 1990, 1995; Franklin and Barnett, 1997; Franklin, 2002a,b). The potential therapeutic usefulness of OECs is presently being explored in two major research areas, and the evidence thus far is very encouraging! OECs promote long-distance regeneration of axons in the adult mammalian spinal cord and remyelinate CNS axons such that the conduction velocities of previously demyelinated axons are restored to normal levels. OECs are also becoming increasingly easier to obtain from extracranial sources, such as from human peripheral olfactory tissue or from similar tissue in the nasal cavity of rodents (Lu et al., 2001, 2002; Franklin, 2002b), thus providing the attractive proposition of a patient having the opportunity to provide their own olfactory tissue for autologous transplantation. Furthermore, the available evidence supports the view that OECs are more likely to integrate into the CNS parenchyma after grafting to repair the injured or demyelinated spinal cord (Li et al., 1998; Lakatos et al., 2000) than are Schwann cells (Baron-Van Evercooren et al., 1992; Blakemore, 1992; Duncan and Milward, 1995), as suggested by Doucette (1990, 1995) several years ago. 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