Olfactory Ensheathing Glia: Their Application to Spinal Cord

Home , Mary Bartlett Bunge

Olfactory Ensheathing Glia: Their Application to Spinal Cord Regeneration and Remyelination Strategies Downloaded from http://meridian.allenpress.com/tscir/article-pdf/6/2/65/1985063/5wwp-t27d-lbuu-x24d.pdf by guest on 30 September 2021 Naomi Kleitman and Mary Bartlett Bunge

Grafts of peripheral nerves or peripheral nervous system (PNS) Schwann cells were among the first successful strategies applied to promote regeneration in the central nervous system (CNS). However, glial cells of the PNS and CNS (Schwann cells and astrocytes, respectively) establish borders where they meet, preventing functional reconnection between regenerating axons and CNS targets. Olfactory ensheathing glia (OEG) are cells that share characteristics with both Schwann cells and astrocytes and support the growth of olfactory nerve axons into the CNS throughout life. Application of these cells to promote regeneration and remyelination in the spinal cord is reviewed. Key words: astrocyte, autotransplantation, demyelination, dorsal root entry zone, myelin, olfactory ensheathing glia, regeneration, Schwann cells, spinal cord injury, transplantation

njury to the spinal cord causes the loss of cellular environment near the site of injury. neuronal cells at the level of injury, We will emphasize recent research applica- I disruption of descending and ascend- tions of a support cell population, olfactory ing axonal tracts, and, in some cases, demy- ensheathing glia (OEG), unique to the first elination of surviving tracts that renders cranial nerve. them nonfunctional. Of these, the disruption The receptor cells of the olfactory epithe- of the long descending and ascending tracts lium, whose axons make up the olfactory accounts for the majority of functional loss nerve, undergo continual cell death and re- after spinal cord injury (SCI). Descending newal throughout life. Their regenerative (supraspinal) tracts carry information from capacity is remarkable in that these processes the brain to spinal cord motor or relay neu- involve axonal growth into and new synapse rons, and ascending tracts transmit sensory formation within the adult CNS. It is thought information to the brain. Restoration of nerve that this regenerative success depends in part impulse transmission past the site of injury to on the specific properties of the OEG, cells target neurons beyond is the goal of regeneration strategies for functional repair of SCI. Long considered unattainable, re- Naomi Kleitman, PhD, is Adjunct Associate Profes- growth of damaged spinal axons in the adult sor, The Miami Project to Cure Paralysis, Department CNS is now known to be possible. Cell of Neurological Surgery, University of Miami School of Medicine, Florida. transplantation strategies have yielded many promising results in animal models of SCI, Mary Bartlett Bunge, PhD, is Professor, The Miami although inhibition of growth within spinal Project to Cure Paralysis, Department of Cell Biology and Anatomy and Department of Neurological Sur- cord tissue beyond the grafts still poses a gery, The Chambers Family Electron Microscopy significant barrier to functional regenera- Laboratory, University of Miami School of Medicine, tion. This article will review current informa- Florida. tion on how intrinsic barriers to regeneration Top Spinal Cord Inj Rehabil 2000;6(2):65–81 in the CNS can be overcome by altering the © 2000 Thomas Land Publishers, Inc.

65 66 TOPICS IN SPINAL CORD INJURY REHABILITATION/FALL 2000

which share phenotypic characteristics with ning with Ramon y Cajal and his co-workers both central glia (astrocytes) and peripheral in the early 1900s. With the advent of meth-

nerve Schwann cells, but differ from both in ods to procure and purify populations of Downloaded from http://meridian.allenpress.com/tscir/article-pdf/6/2/65/1985063/5wwp-t27d-lbuu-x24d.pdf by guest on 30 September 2021 ways that have a profound influence on re- Schwann cells in culture, their use to promote generation into the CNS. CNS regeneration was proposed by Richard Bunge in 1975.2 Aguayo and co-workers3 Cellular Therapies for Spinal Cord showed that peripheral nerve bridges support Regeneration—Strengths and regeneration of injured axons from nerve Weaknesses cells situated near implants into spinal cord or optic nerve. Schwann cells appear to be the Although sprouting of injured axons oc- active component of these grafts, likely due curs during the first several weeks after in- to their production of numerous growth-pro- jury, minimal successful regeneration occurs moting surface proteins, extracellular matrix in untreated spinal cord. This is due to the (ECM) molecules including the powerful lack of adequate trophic support (i.e., nutri- growth promoter, laminin, and numerous ents that stimulate nerve growth), the pres- neurotrophic factors. Populations of ence of inhibitory molecules in the CNS, and/ Schwann cells stimulate axonal growth from or other disruptive immunological or sec- cut spinal cord axons and in cavities within ondary injury processes at the site of lesion. spinal tissue.4 Addition of exogenous neu- Attempting to make the cellular environment rotrophic factors to Schwann cell grafts near such injuries conducive to regeneration, stimulates regeneration also from supraspi- researchers have selected cellular implants nal pathways. that reproduce environments where growth It has been disappointing that axonal re- occurs normally, such as embryonic spinal entry into CNS tissue from Schwann cell cord, peripheral nerve, or Schwann cells, the grafts and embryonic spinal tissue trans- support cells of the peripheral nervous system plants has been minimal or unsuccessful. (PNS). Many excellent reviews have de- Probable reasons for this failure include the scribed these strategies, as summarized previ- presence of naturally occurring growth in- ously.1 Embryonic spinal cord transplants sur- hibitors in CNS tissue, such as those associ- vive grafting and integrate well into host cord. ated with myelin. Also problematic is the Ingrowth and egress is very limited and, in development of scar tissue in reaction to the mature hosts, these grafts do not serve as injury and/or to the grafted cells them- bridges across gaps in spinal tissue as regen- selves. This scar is thought to contain eration of long axon tracts requires. Addition proteoglycans, which arrest axonal growth. of trophic factors to embryonic spinal trans- Proteoglycans also constitute regional bor- plants improves axonal elongation into the ders during development, help sculpt ax- grafts and may improve the usefulness of the onal trajectories,5–7 and are thought to pre- grafts as relays for ascending and descending vent entry of Schwann cells into the CNS at signal transmission. spinal nerve roots.5 After traumatic injury, Peripheral nerve grafts that contain however, these natural borders become bar- growth-promoting Schwann cells have a riers to recovery. These issues have led to the long history in regeneration research, begin- exploration of cell types, OEG in particular, Olfactory Ensheathing Glia 67 Downloaded from http://meridian.allenpress.com/tscir/article-pdf/6/2/65/1985063/5wwp-t27d-lbuu-x24d.pdf by guest on 30 September 2021

Fig 1. Schematic diagram indicating the location of olfactory receptor neurons in the nasal epithelium (nasal cavity), whose axons are bundled by olfactory ensheathing glia (OEG) as they cross through the cribriform plate into the cranium. The nerve rootlets containing these bundles join to form the olfactory nerve layer, and their individual basal laminae become continuous with the glia limitans formed by astrocytes and OEG at the margin of the olfactory bulb. Axons cross the nerve layer, enter the glomerular zone, and synapse with dendrites of mitral, tufted, and periglomerular cells without directly contacting astrocytes. On the right is a tracing enlarged to illustrate the segregation of axon bundles by an ensheathing cell. that might navigate the barrier regions and (Fig. 1). Within the epithelium, olfactory chaperone growing axons through hostile receptor neurons undergo constant turnover; CNS territory. their functional mature lifetime is approximately 25 days in the rodent.10 Generated Properties of OEG and Relationships to from a basal cell population, the newly grow- Other Glial Cell Types ing receptor axons extend within olfactory nerve, amidst axon fascicles ensheathed by Olfactory receptor neurons are situated in OEG. Unlike Schwann cells, OEG do not the olfactory epithelium within the nasal cav- enfold nonmyelinated axons individually in ity and the vomeronasal organ. These sen- furrows of cytoplasm. Rather, thin OEG sory neurons extend axons though the olfac- sheets surround and separate olfactory nerve tory and vomeronasal nerves to the olfactory axons into large bundles of densely packed bulb, where they synapse with dendrites of fibers8,9 (Figs. 1 and 2). When axons in the mitral, tufted, and periglomerular cells8,9 nerve reach the surface of the olfactory bulb, 68 TOPICS IN SPINAL CORD INJURY REHABILITATION/FALL 2000 Downloaded from http://meridian.allenpress.com/tscir/article-pdf/6/2/65/1985063/5wwp-t27d-lbuu-x24d.pdf by guest on 30 September 2021

Fig 2. (Top) Electron micrograph of an ensheathing glial cell in the primate olfactory nerve layer. Its cytoplasmic processes (black in inset) partition bundles of 10–50 small diameter axons. (Micrograph generously contributed by James Guest, MD, PhD.) (Bottom) Electron micrograph of adult rat corticospinal axons, 3 months after grafting of a suspension of adult olfactory nerve fiber layer cells precultured for 2 weeks. Depicted are two glial cells. One cell, cut through its lobular nucleus, has cytoskeletal filaments (arrowhead) and a complete basal lamina (arrows); it ensheathes a single axon (a). The second has formed PNS-like myelin around another axon (a). The cells are surrounded by extracellular collagen fibrils and a layer of perineurial-like cell processes (p). (Micrograph generously contributed by Geoffrey Raisman, PhD.) Olfactory Ensheathing Glia 69

these bundles penetrate into the olfactory that newly growing olfactory axons maintain nerve layer along the surface of the bulb. The contact along their entire length with the

basal lamina that surrounds the nerve rootlets peripheral ensheathing glial cytoplasmic Downloaded from http://meridian.allenpress.com/tscir/article-pdf/6/2/65/1985063/5wwp-t27d-lbuu-x24d.pdf by guest on 30 September 2021 (containing these bundles) becomes continu- sleeves and do not directly contact astrocytes ous with that encompassing the outer margin within the olfactory nerve layer. It is the of the olfactory bulb, the glia limitans (Fig. coexistence of the OEG with astrocytes in 1). Whereas astrocytes exist within the olfac- this unusual transition area that appears to tory nerve layer, they are found along the glia account for the successful reinnervation. limitans and in spaces between nerve fas- Early researchers hypothesized that this cicles. The axons contact only OEG (and coexistence related to the astrocytic nature of each other) all the way to the olfactory glom- OEG. The location of OEG within the glia eruli, synaptic regions surrounded by astro- limitans, cytoskeletal expression of the CNS cytes and OEG processes that do not enter the form of glial fibrillary acidic protein glomeruli.8,9 (GFAP),11,13 and their lack of basal lamina In areas such as the dorsal root entry zone, inside the olfactory nerve layer were used as where peripheral sensory nerves enter the arguments for the OEG being modified as- spinal cord, the CNS/PNS border is charac- trocytes. Others argued that OEG’s periph- terized by a transition zone where glia of the eral origin (from olfactory placodes), their two regions (astrocytes and oligodendro- resemblance to nonmyelinating Schwann cytes of the CNS and Schwann cells of the cells, their ability to ensheathe axons, and, PNS) come into contact. There is a smooth more recently, their apparent ability to transition of Schwann cell and oligodendro- myelinate axons supported the contention cyte myelin sheaths along individual axons, that they are a form of Schwann cell. Re- but a barrier is established between areas searchers who focus on OEG have argued containing astrocytes and Schwann cells. that this is a unique cell type14 because of The olfactory nerve layer is unique in that their origin and because the profile of mol- growing axons successfully reach adult CNS ecules they express differs from that of other target cells because the incursion of OEG glial populations. In defining these cells by shifts the CNS border all the way to the level their staining with antibodies to various of the glomeruli.8,9,11,12 Raisman8 described cytoskeletal or surface molecules, some re- ensheathing cells in the vomeronasal nerve ports underestimate the ability of these cells as sequestering bundles of axons along their to express different phenotypes under differ- course through the olfactory nerve layer and ing conditions. This becomes particularly providing a conduit past astrocytes rather important when certain antibodies are used than “handing over” axons to CNS glia. The to purify populations (or select subpopula- axons in olfactory and vomeronasal nerves tions) of OEG for transplantation or tissue ultimately synapse with CNS dendrites in- culture studies and in the evaluation of cell side glia-free glomeruli, and replacement lines that are developed to study or replace axons extending from newly generated ol- OEG populations.15 Moreover, emphasizing factory nerve cells are never confronted with the uniqueness of OEG may also underesti- an OEG/astrocyte barrier.8,9 If this success is mate their similarity to other specialized glial to be reproduced in regions of CNS damage, populations in the CNS (tanycytes, pineal we must begin with the basic phenomenon gland glia, cerebellar Bergmann glia, and 70 TOPICS IN SPINAL CORD INJURY REHABILITATION/FALL 2000

retinal Muller glia)16 that appear to share versus psa-NCAM expression, but clonal many characteristics with OEG, including lines (progeny of single cells) always con-

the ability to establish regeneration-permis- tained mixtures of these cell types. These Downloaded from http://meridian.allenpress.com/tscir/article-pdf/6/2/65/1985063/5wwp-t27d-lbuu-x24d.pdf by guest on 30 September 2021 sive environments within the CNS. OEG are researchers argued, therefore, that these are also similar to glia in the gut.11,17 different states of a multipotent cell. In con- The term OEG is applied broadly to cells trast, they observed that O4 (a monoclonal that express different phenotypes.12,18 In antibody against glycolipids on glial cell vivo, OEG populations have been described surfaces) staining was present throughout the as containing both stellate and fusiform cells. bulb and chose, therefore, to purify OEG In some regions of the olfactory nerve layer populations based on O4 expression. and bulb,15,18 OEG characteristically contain Purifying populations of OEG on the basis of filaments that are immunoreactive for the molecular expression is further complicated by astrocytic form of GFAP, although, unlike observations that expression of various mol- astrocytes, their filaments are scattered ecules in vitro changes with time and culture rather than bundled. Pixley17 defined conditions.15,17,23 In particular, the OEG expres- Schwann cell-like and astrocyte-like OEG, sion of O4 falls and that of NGFr rises with time the latter expressing significantly more in culture15; GFAP content may change as GFAP immunoreactivity. In addition to well.15,17,23 OEG expression of these molecules, GFAP, adult OEG express other structural their cell morphology, migration, and possibly proteins, including vimentin (usually found their ability to promote axonal growth are af- only in immature glia).14,16 Growth-promot- fected by the presence or concentration of se- ing cell surface adhesion/recognition mol- rum in culture media or by contact with differ- ecules such as L1, N-cadherin, and NCAM ent ECM molecules.15,23 These tissue culture (including the embryonic axonal growth-as- phenomena may elucidate important OEG sociated form, psa-NCAM),15,19,20 the ECM properties or lead to apparent discrepancies molecules laminin and tenascin,18,20 as well between results in different laboratories unless as trophic factors and their receptors14,21 have they are taken into account. been described in these cells under some One important aspect of a cell’s ability to conditions. Conversely, OEG fail to express promote regeneration is its production and many markers that are characteristic of other secretion of growth factors. Although one CNS cell types.14,20,21 OEG expression of the group reported that OEG do not secrete dif- low-affinity form of nerve growth factor re- fusible growth factors in vitro,20 expression ceptor (NGFr), called p75, has been exam- of different growth factors by in vitro OEG ined in detail and has also been used to populations, including several members of identify/select for populations of OEG.22 the neurotrophin family and neuregulin (a Franceschini and Barnett15 analyzed OEG family of proteins that regulates Schwann expression of several molecules, both in vivo cell proliferation and maturation), has been and in vitro. They defined regions where described recently.21,23 OEG have long been NGFr, psa-NCAM, and other markers were known to express p75, the low affinity NGFr, and were not expressed in the nerve fiber and and recently have been shown to express glomerular layers. They proposed a classifi- high-affinity neurotrophin receptors as well cation of two types of OEG based on NGFr as receptors for neuregulin.21 Olfactory Ensheathing Glia 71

discussion is the interaction between grow- One important aspect of a cell’s ability ing olfactory axons and OEG; it appears that

to promote regeneration is its the outstanding growth capacity of the olfac- Downloaded from http://meridian.allenpress.com/tscir/article-pdf/6/2/65/1985063/5wwp-t27d-lbuu-x24d.pdf by guest on 30 September 2021 production and secretion of growth tory system depends heavily on OEG. factors. Olfactory receptor neuron pioneer axons grow from the epithelial layer through connective tissue to the developing brain, guided by the presence of ECM proteins that either Another feature of OEG that may set them establish (laminin, collagen IV) or delimit apart, from Schwann cells in particular, is (chondroitin sulfate proteoglycan, their apparent ability to migrate within the fibronectin) pathways conducive for axonal CNS, especially within astrocyte-rich ar- growth.6,7 It is not proven, but is likely, that eas.12 It has been hypothesized that OEG these terrains are established at least in part expression of p75 or exposure to growth by the OEG population that develops at ap- factors or ECM components can induce mi- proximately the same time as pioneering gration. Interestingly, Li and Raisman24 ob- axons. Axons leave the epithelium already in served that Schwann cell migration within close contact with and fasciculated by OEG the CNS is greater in those Schwann cells processes,18 which appear to extend ahead of that retain their expression of p75 than in growing axons.31 In the mature rodent sys- those that lose it. The former intercalate tem, the olfactory receptor neuron popula- within rows of CNS glia, while the latter tion turns over and is constantly renewed myelinate axons. Thus, expression of p75 for from precursors in the olfactory epithelium.10 extended periods by OEG or possibly a delay The axons of these newly generated neurons in the shift to a myelinating phenotype rela- grow individually or in small fascicles within tive to Schwann cells under similar condi- terrains established by ensheathing cell pro- tions could account for significant differ- cesses.28 In vitro evidence supports the con- ences between these cell types that may clusion that OEG cells are a favorable sub- impact on their ability to promote regenera- strate for olfactory receptor axonal tion after transplantation into the CNS. growth.20,23 If axons of olfactory neurons are cut, the Olfactory Nerve Growth in cells die, whereas support cells remain29 and Development, Normal Turnover, and may guide newly growing axons to their Regeneration targets.9 It is more remarkable that after complete removal of the olfactory bulb in new- The development of the olfactory nerve born mice, newly generated olfactory recep- and bulb have been well described.6,10,25,26 tor neurons extend axons within the olfactory Additionally, the ongoing turnover of sen- nerve to contact the cerebral cortex (which sory nerve cells within the olfactory system, expands into the space near the cribriform and the remarkable ability of olfactory nerve plate). The axons penetrate the surface of this to regenerate after transection or even re- ectopic CNS tissue and induce the formation moval of the olfactory bulb, has been well of glomerular structures, ultimately attract- documented.27–30 Of relevance to the present ing dendritic growth and synapse formation 72 TOPICS IN SPINAL CORD INJURY REHABILITATION/FALL 2000

from large cortical neurons.27 Wright et al.30 cells used in this experiment were relatively elegantly showed that by 100–150 days after homogeneous in that they were selected by

complete bilateral bulbectomy in adult mice, their expression of p75. Injected OEG mi- Downloaded from http://meridian.allenpress.com/tscir/article-pdf/6/2/65/1985063/5wwp-t27d-lbuu-x24d.pdf by guest on 30 September 2021 trained odor discrimination and performance grated within the dorsal horn and were found on an operant odor aversion test were rees- to be codistributed with the area where re- tablished through these newly regenerated generating sensory axons were found pathways. New odor discrimination tasks (lamina I-III). This was an improvement could be learned that were dependent on upon other repair strategies where sensory sensory input from the olfactory receptor axons initiated growth but did not reach their cells. Whereas it is unclear to what extent target cells. Neither OEG nor regenerating OEG specifically are responsible for this axons entered the heavily myelinated and remarkable capacity for functional restora- largely undamaged ascending sensory tracts. tion, many investigators have hypothesized Although immunostaining for growing that the unusual properties of OEG could be axons or sensory fibers was shown, the most useful in promoting regeneration of periph- compelling evidence that the axons observed eral axons into the CNS or of CNS axons in the region of the implanted cells were within their naturally inhibitory milieu for regenerating, rather than branching from therapeutic purposes. nearby uninjured axons, was provided by dye injections and axons traced from the injured OEG Promote Axonal Regeneration in nerve itself. Whereas the mechanism for this Ectopic CNS Sites ingrowth and its limitation to the region of the dorsal horn were discussed by these au- The properties of OEG discussed earlier, thors as being determined by “specific cues” along with their function in the constantly guiding either axons or OEG migration, the growing olfactory nerve system, have in- actual regulation of migration of the axons or spired investigators to test the ability of these cells and the mechanisms by which OEG cells to promote regeneration in the injured exceed other cells in promoting this growth CNS. Early successes have led to the use of remain to be elucidated. these cells in a number of different lesions in More recently, Navarro et al.33 extended the brain and spinal cord, with results indicat- this work by demonstrating the restoration of ing great promise for stimulating axonal re- stimulus-evoked conduction of signals generation. through the regenerated sensory fibers. This After avulsion or other damage to the study expanded on the previous one in a sensory nerves of the dorsal roots, functional number of ways. First, the surgeries were regeneration is prevented by the transition carried out at L3-L6, closer to hindlimb mo- area between the PNS and CNS in the dorsal tor neuron pools (as depicted in Fig. 3A) than root entry zone.5 Ramón-Cueto and co-work- previously tested. Second, the cellular grafts, ers reported32 that reapposing cut dorsal roots though not a purified population, were frozen to the thoracic (T10) spinal cord and inject- and thawed before being successfully ing OEG into the dorsal horn of rats (Fig. 3A) grafted. Electrophysiological measures of a improved sensory axon regeneration into the number of leg reflexes indicated that func- cord. The populations of adult-derived glial tional synapses had been formed between 2 Olfactory Ensheathing Glia 73 Downloaded from http://meridian.allenpress.com/tscir/article-pdf/6/2/65/1985063/5wwp-t27d-lbuu-x24d.pdf by guest on 30 September 2021

Fig 3. Cartoon of different experimental strategies used to test the potential of olfactory ensheathing glia (OEG) (*) to promote regeneration and myelination. (A) Section of the dorsal root near its entry into the lumbar spinal cord.33 OEG were injected into the dorsal root entry zone; (B) injections into a circumscribed unilateral lesion of the cervical corticospinal tract35,36; (C) injection of OEG into thoracic spinal cord stumps at both ends of a Schwann cell bridge enclosed within a polymer channel4; (D) injection into spinal cord stumps of transected41 or dorsal hemisected40 spinal cord without removal of spinal tissue; (E) grafting of OEG and fibrin into cavities caused by contusion of the cord, a model of clinical SCI42; (F) injection into demyelinated dorsal columns.48,49 weeks and 2 months after surgery. Whereas regeneration within the long tracts of the the responses were not as strong as normal, spinal cord has also been reported, using two they were consistent with the level of regen- quite different paradigms. Li et al.35,36 studied eration observed anatomically within lami- injection of OEG into small unilateral elec- nae I-IV of the spinal cord of the grafted trolytic corticospinal tract lesions (CST) be- animals. Surprisingly similar levels of regen- tween the first and second cervical segment eration and physiological function were re- (Fig. 3B). Ramón-Cueto et al.37 tested the cently reported,34 stimulated not by cellular ability of OEG to foster long axonal growth implantation but by infusion of neurotrophic and reentry of regenerating axons into the factors near the cord. These similarities distal spinal cord in completely transected could suggest that expression and secretion thoracic segments bridged by a cable of of neurotrophic factors by OEG are respon- Schwann cells (Fig. 3C). Both studies re- sible, in part, for the ability of these cells to ported significant advances in promoting promote regeneration in this region of the axonal growth through and beyond these spinal cord. disparate lesions and regeneration for sig- The ability of OEG to promote axonal nificant distances within spinal cord tissue 74 TOPICS IN SPINAL CORD INJURY REHABILITATION/FALL 2000

beyond the lesion site. Despite the differ- gested that the CST axons successfully found ences in the transplantation paradigms used, and innervated CNS targets that mediated the 35

these studies shared another important find- recovery of function. Although represent- Downloaded from http://meridian.allenpress.com/tscir/article-pdf/6/2/65/1985063/5wwp-t27d-lbuu-x24d.pdf by guest on 30 September 2021 ing: OEG used instead of, or in addition to, ing great progress in promoting regenera- Schwann cell grafts enhanced the success of tion, these studies raise important questions regeneration over that seen with Schwann about why the OEG grafts are so successful, cell grafts alone in comparable systems.12 particularly in comparison with Schwann Raisman and co-workers have produced cells. It is not yet known whether host circumscribed unilateral lesions of the CST Schwann cells or fibroblasts, which readily in the adult rat and implanted Schwann cell24 enter these CNS injury sites, contribute to the or OEG35,36 suspensions within the lesion, regeneration and have their function modi- producing minimal damage to the cellular fied somehow by the OEG grafts. Nor is it architecture surrounding the area of damage clear what guides regeneration and whether (Fig. 3B). After electrolytic lesions alone, normal or aberrant synaptic connections are host astrocytes became reactive and walled formed. Whereas these studies present re- off the region of damage, and axonal regen- generating CST axons with a single degener- eration did not occur. Schwann cell grafts ating pathway after injury relatively close to decreased this scarring, stimulated blood their cell bodies, SCI contusion or transec- vessel growth, aligned within the graft, and tion involves many injured tracts. Massive supported axonal growth into the region and disruption of distal pathways and injury of myelination of the regenerated axons. axons at much greater distances from their Schwann cells did not migrate beyond the cell bodies may present more difficult and lesion site, however, and axons did not reen- clinically relevant scenarios for successful ter the host CST; axons branched erratically regeneration. within the site, as if trapped within the PNS A bridging strategy that confronts many of milieu.24 In contrast, OEG suspensions in- these issues has been developed by Bunge jected into such lesions aligned within the and co-workers.37,38 Utilizing a thoracic degenerating CST and migrated beyond the transection model of SCI, this group has area of damage. Axons grew unbranched into studied extensively the ability of Schwann and beyond the injury site and were myeli- cell/Matrigel cables enclosed within semi- nated by PNS-like myelin for approximately permeable polymer tubes to bridge up to 8- a centimeter, at which point they re-entered mm gaps between completely severed cord the CNS milieu as indicated by transition stumps. Whereas Matrigel grafts with no from Schwann cell-like myelin to oligoden- Schwann cells elicit little axonal growth, drocyte myelin (Fig. 2B).36 Growth cones of Schwann cell grafts promote the regenera- regenerated axons were shown to be sur- tion of propriospinal (neurons within the rounded by thin sheets of cytoplasm indicat- spinal cord above and below the injury) ing that glial cells accompanied the regrow- axons and substantial myelination of these ing axons and could have influenced their fibers (previously reviewed4). Neuroprotec- growth. Based upon functional improvement tion or neurotrophic factor infusion adminis- in behavioral tests of directed reaching with tered in addition to the grafts promoted the affected forelimb, these researchers sug- growth from distant brain stem neurons into Olfactory Ensheathing Glia 75

the Schwann cell bridges. There is little evi- are readily available in sufficient numbers dence to date for reentry of axons into the for potential autologous transplantation 38

CNS region below the grafts as required for strategies (previously reviewed ), the ques- Downloaded from http://meridian.allenpress.com/tscir/article-pdf/6/2/65/1985063/5wwp-t27d-lbuu-x24d.pdf by guest on 30 September 2021 functional restoration or for migration of tion remains as to the interaction between Schwann cells out of the grafts. Recent work Schwann cells and OEG and whether com- with incomplete lesions of the cord (lateral bined strategies hold any mechanistic advan- hemisections)39 indicates that smaller tage over OEG suspensions alone. Schwann cell cables do allow some egress of Two recent observations argue that OEG axons into host CNS, possibly due to im- suspensions are sufficient to induce robust provements in spinal column stability or regeneration and engender significant recov- cerebrospinal fluid circulation. Growth of ery of rapid and secure conduction in ascend- axons out of Schwann cell-only grafts re- ing sensory fibers through a T11 dorsal col- mains limited, however, and little functional umn transection site40 or hindlimb function recovery has been reported. after complete T8 transection.41 In both Collaborating with Ramón-Cueto, the cases, suspensions of OEG were injected on Bunge group asked whether OEG suspen- each side of a transection without a gap (Fig. sions injected into both cord stumps in con- 3D); these transplanted cells were sufficient junction with Schwann cell bridges (i.e., at to bridge the transection site. Imaizumi et the experimentally induced PNS/CNS bor- al.40 reported that either neonatal olfactory ders) would help usher the regenerating nerve cells or freshly dissociated adult axons back into host spinal cord (Fig. 3C).37 Schwann cells injected on both sides of cut Using cultured populations of adult OEG dorsal column sensory tracts induced regen- selected for their expression of p75, these eration and myelination of similar numbers researchers reported profuse axonal growth of axons (about 500 myelinated axons, three- within the bridges and axonal regeneration fold more than in nontransplanted controls) beyond the graft–host interfaces for substan- and restored rapid electrical conduction tial distances (more than 1.5 cm descending across the lesion site. Morphological and and 2.5 cm ascending). Prelabeled OEG cells physiological measures showed characteris- were observed in areas where regeneration tically large caliber and rapid conduction was seen, suggesting that OEG accompanied velocities in the regenerated fibers. Although growing fibers as they navigated the white minor differences were noted between OEG- and gray matter of the CNS, which is remi- and Schwann cell-grafted animals, the niscent of OEG-axon colocalization in the amount of regeneration and myelination, as olfactory system itself. It is interesting to well as migration of the transplanted cells, note that whereas most regenerating axons were remarkably similar. grew through the Schwann cell cables, some, Ramón-Cueto et al.41 reported that rats particularly serotonergic, axons showed a recovered voluntary hindlimb movement, preference for a relatively Schwann cell-free plantar hindpaw placement, body weight path that developed unexpectedly outside the support, and proprioception and light touch guidance channels and was composed prima- by 3–7 months after injection of OEG on rily of OEG and connective tissue. Whereas either side of a complete T8 transection (Fig. Schwann cells, including adult human cells, 3D). Serotonergic, noradrenergic, and CST 76 TOPICS IN SPINAL CORD INJURY REHABILITATION/FALL 2000

fibers crossed the lesion, entered the distal in these systems and the effect of grafted cord, and extended for up to 3 cm (to L6); the OEG on host cells. The ability of olfactory

fibers did not extend as far in those animals cell grafts to engender regeneration in each Downloaded from http://meridian.allenpress.com/tscir/article-pdf/6/2/65/1985063/5wwp-t27d-lbuu-x24d.pdf by guest on 30 September 2021 that demonstrated less behavioral recovery. of the models discussed earlier, or to produce As in the earlier study,36 only in the presence changes in the lesion environments in which of OEG were regenerating axons able to endogenous cells are somehow stimulated to cross the transection area, grow past GFAP- promote recovery, suggests that new thera- reactive astrocytes, and enter the distal peutic approaches using these cells to pro- stump. Again, labeled grafted cells were mote SCI regeneration should be pursued. found in areas containing regenerating fibers. CST fibers did not grow through gray or Potential for Remyelination white matter but extended along the pia mater; some reentered the cord after lengthy In the olfactory nerve, OEG ensheathe growth. Serotonergic and noradrenergic fi- large bundles of small (0.2 µm) axons and do bers, on the other hand, grew in spinal cord not produce myelin (although CNS myelin tissue and invaded regions they normally sheaths are found in the olfactory nerve and innervate. There was a positive correlation glomerular layers of some species, including between functional recovery and the distance the hedgehog9 and primate [Dr. J. Guest, axons regenerated, suggesting that regenera- personal communication]). Yet, in all of the tion of the severed spinal cord fibers may OEG grafting experiments discussed earlier, account at least in part for recovered func- regenerating axons myelinated by peripheral tion. This study, unlike Imaizumi et al.,40 did myelin sheaths have been reported. These are not directly compare OEG and Schwann cell characterized as resembling Schwann cell populations. It is therefore unknown whether myelin by (1) immunostaining for the PNS- this level of recovery might also be observed specific myelin protein, P-zero (P0), but not with Schwann cell injections alone in this for CNS myelin markers; (2) the 1:1 associa- model. tion of myelinating cells to single axon seg- Recent publications and presentations at ments including close association of nuclei scientific meetings indicate that interest in with myelin sheaths; and (3) the presence of OEG grafts is increasing exponentially. basal lamina and collagen fibrils in the extra- These cells are being tested in contusion cellular space, which is bounded by perineur- injuries, which more closely resemble most ial-like cellular elements highly reminiscent clinical SCI and allow for delayed transplan- of peripheral nerve fascicles (Fig. 2, bottom). tation of cells (Fig. 3E).42 Pérez-Bouza et al.43 Several investigators have explored OEG addressed behavior of olfactory cell suspen- populations in vitro, or after transplantation sions injected into the thalamus and choroid into demyelinated spinal cord tracts, specifi- fissure. They observed the spontaneous de- cally to determine whether these cells velopment of an aligned “glial bridge” across myelinate. this natural border, but the cells and growing The first evidence that isolated OEG axons had disappeared 3 weeks after graft- populations can produce myelin came from ing. Much work is still needed to understand tissue culture studies. Using tissue culture the mechanisms for improved regeneration systems well characterized for the study of Olfactory Ensheathing Glia 77

tion of OEG in models for spinal cord demy- The first evidence that isolated OEG elination. Using the ethidium bromide-X

populations can produce myelin came (EB-X) irradiation paradigm, several groups Downloaded from http://meridian.allenpress.com/tscir/article-pdf/6/2/65/1985063/5wwp-t27d-lbuu-x24d.pdf by guest on 30 September 2021 from tissue culture studies. have demonstrated remyelination by populations of cells of olfactory nerve origin, including rodent48 and human49 olfactory nerve cells and an OEG clonal cell line50 (Fig. 3F). Schwann cell myelination of dorsal root gan- In this model system, which has also been glion axons, Devon and Doucette44 showed used to document remyelination of CNS that embryonic OEG produce PNS-type axons by Schwann cells, the demyelinated myelin. They further demonstrated45 that region is rendered devoid of host glial cells, OEG populations myelinated axons under unlike MS plaques that are densely packed culture conditions (low vitamin C) where with astrocytes. The myelin observed after DRG-derived Schwann cells were unable to injections of olfactory-derived cells was 12,40,48,50 form myelin. Vitamin C is required as a clearly PNS-like, and contained P0. cofactor in the formation of triple helical Franklin et al.50 noted that small numbers of collagen fibrils, and Schwann cells do not other cell types of unknown origin were myelinate without sufficient amounts of it to observed within the affected region, which allow collagen formation and basal lamina would indicate either differentiation of the deposition.46 Devon and Doucette45 reported grafted cells into different phenotypes or the that embryonic OEG populations grown in incursion of host cells into the transplanted unsupplemented human placental serum- region. Whereas the latter has been shown containing media, which is known to contain not to be a major consideration in variable amounts of vitamin C, assembled nontransplanted EB-X animals and it is gen- some basal lamina and collagen and at least erally accepted that remyelination is accom- some cells formed myelin. This suggests that plished primarily by the grafted cells, the either these cells make their own vitamin C possibility that transplanted cells might in- (which most rodent and human cells do not) duce the migration of host cells (including or their requirement for it is less than that of Schwann cells) into the area has not been Schwann cells. Although these investigators systematically ruled out in cell graft experi- carefully tried to exclude Schwann cell con- ments. tamination in their cultures, it is impossible Remyelination of EB-X lesions in rats has to rule out this possibility without specific been obtained48,49 using cell populations identification of the cells. In contrast, recent from neonatal rats and adult human olfactory work using a population of adult OEG nerves. Imaizumi et al.48 showed anatomical immunopurified for expression of p75 failed and physiological evidence for to demonstrate myelination under even vita- remyelination by 21–25 days after transplan- min C-supplemented culture conditions.47 tation. The myelin was PNS-like and was The compelling clinical need for popula- observed 2–3 mm rostral and caudal to the tions of cells that might be able to injection site, indicating migration of the remyelinate CNS axons in multiple sclerosis implanted cells. Physiological measures in- (MS) or after SCI have led to the investiga- dicated that approximately 17% of the total 78 TOPICS IN SPINAL CORD INJURY REHABILITATION/FALL 2000

axons were able to conduct signals after demyelinated and regenerating axons, and remyelination. Although the distribution in their association with long axonal growth

this remyelinated population was skewed into spinal cord white matter tracts and gray Downloaded from http://meridian.allenpress.com/tscir/article-pdf/6/2/65/1985063/5wwp-t27d-lbuu-x24d.pdf by guest on 30 September 2021 toward larger axons with high conduction matter has shown great promise. The asso- velocities, conduction was still slower than ciation of much of this regenerative growth control values, and slower than that seen in with either physiological evidence of func- regenerating axons in a more recent study.40 tional conduction of axonal impulses or be- The reason for the latter, very intriguing havioral recovery of seemingly volitional difference between remyelinated and regen- use of fore- and hindlimbs has raised great erating axons is not known. Anatomical evi- hope that these cells might be used in clinical dence of remyelination by adult human-de- strategies to repair SCI. Several questions rived olfactory cell preparations was recently and technical hurdles remain. reported49 and included staining for human The question of whether OEG can be specific markers. Observation of extensive shown to definitively improve upon axonal amounts of human-derived myelin after only regeneration and functional outcome when 3 weeks postgrafting is somewhat surprising. used instead of or in addition to Schwann Kato et al.49 found human-specific staining in cells is an important one. Techniques for regions coinciding with remyelinating obtaining adult-derived human Schwann axons, although colocalization with cells that could be used for autologous trans- myelinating cells was not shown. plantation are now available (recently re- Taken together, the studies discussed pre- viewed38), whereas procurement of human viously constitute rapidly accumulating indi- OEG is not. Schwann cells from sural nerve cations that OEG can remyelinate axons in biopsies that would induce minimal func- vitro and in vivo. It would be advantageous to tional loss in the patients could be extensively clearly mark the transplanted cells and fully expanded by exposure to mitogens in vitro in characterize the extent of cell migration of quantities that would be needed to bridge any host cells into the lesion area as well as of regions of SCI. OEG, alternatively, would grafted cells within it. More important, have to be obtained from donors or after though, to the design of viable clinical strat- substantial facial surgery if autologous graft- egies would be (1) a definitive comparison of ing were to be attempted. The ultimate number grafted Schwann cell versus OEG migration of OEG needed for any proposed transplanta- and remyelination within this system and (2) tion strategy might be minimized if it were an assessment of whether OEG actually inte- found that they had a unique beneficial effect grate better than Schwann cells into regions when used in small numbers in association rich in reactive astrocytes, such as those seen with other, more readily available, cell types in MS plaques.12 (as shown by Ramón-Cueto et al.37). If OEG populations were to be a major Procurement of Cells and Therapeutic component of SCI grafting strategies, once Potential OEG biopsies were obtained, the cell populations would almost certainly have to be Clearly, recent work showing that OEG expanded. Pixley17 observed that laminin- grafts promote regeneration, myelination of coated surfaces appeared to be mitogenic for Olfactory Ensheathing Glia 79

rodent OEG. Recently, Yan et al.51 compared able experimentally. Given the rapid a number of different factors for their mito- progress in this area in the past decade and

genicity for rat OEG. They found that the intensive interest in OEG shown in recent Downloaded from http://meridian.allenpress.com/tscir/article-pdf/6/2/65/1985063/5wwp-t27d-lbuu-x24d.pdf by guest on 30 September 2021 growth factors neuregulin and fibroblast years, these questions are likely to be an- growth factor-2 as well as the cAMP-activat- swered in the near future. This could result in ing agent, forskolin, were mitogenic for p75- the rapid translation of these basic findings to expressing cells in populations selected by clinical trials of OEG transplantation in pa- immunopanning. An alternative might be the tients for the repair and functional restoration development of human OEG cell lines, al- of spinal circuitry. though developing lines of human cells ap- propriate for grafting is technically difficult Acknowledgments and such grafts would require immune sup- pression. The potential tumorogenicity and We thank Margaret Bates for technical phenotypic stability of any expanded popula- assistance, Maria Amador, RN, for insightful tion or cell line must be carefully explored comments on the manuscript, Theresa before such cells can be considered for clini- Robinson-Walters for help with its prepara- cal use. Finally, it should be established tion, and Drs. Geoffrey Raisman (National whether selection of populations of OEG Institute for Medical Research, London, UK) expressing certain markers, such as p75, and James Guest (The Miami Project to Cure which might decrease the number of avail- Paralysis, University of Miami School of able cells, is actually advantageous. Given Medicine, Miami, Florida) for generously that the state of OEG expression of different contributing electron micrographs. This ar- markers varies in vivo and in vitro,15 better ticle was supported by the Miami Project to determination of optimal preparation of Cure Paralysis, National Institute of Neuro- populations for transplantation will be logical Disorders and Stroke (NINDS) grant needed. 09923, and the Christopher Reeve Paralysis All of these issues are readily approach- Foundation.

REFERENCES

1. Sagen J, Bunge MB, Kleitman N. Transplanta- Plasticity. New York, NY: Guilford; 1985: tion strategies for treatment of spinal cord 457–484. dysfunction and injury. In: Lanza R, Langer R, 4. Bunge MB, Kleitman N. Neurotrophins and Vacanti J, eds. Principles of Tissue Engineer- neuroprotection improve axonal regeneration ing. San Diego, CA: Academic Press; into Schwann cell transplants placed in 2000:799–820. transected adult rat spinal cord. In: Tuszynski 2. Bunge RP. Changing uses of nerve tissue cul- MH, Kordower J, eds. CNS Regeneration. San ture 1950-1975. In: Tower DB, ed. The Ner- Diego, CA: Academic Press; 1999:631–646. vous System, Vol. I: The Basic Neurosciences. 5. Pindzola RR, Doller C, Silver J. Putative inhibi- New York: Raven Press; 1975:31–42. tory extracellular matrix molecules at the dor- 3. Aguayo AJ. Axonal regeneration from injured sal root entry zone of the spinal cord during neurons in the adult mammalian central ner- development and after root and sciatic nerve vous system. In: Cotman CW, ed. Synaptic lesions. Devel Biol. 1993;156:34–48. 80 TOPICS IN SPINAL CORD INJURY REHABILITATION/FALL 2000

6. Whitesides JG III, LaMantia A-S. Differential molecules in the olfactory system of the adult adhesion and the initial assembly of the mam- mouse: Presence of the embryonic form of N- malian olfactory nerve. J Comp Neurol. CAM. Devel Biol. 1988;129:516–531.

1996;373:240–254. 20. Sonigra RJ, Brighton PC, Jacoby J, Hall S, Downloaded from http://meridian.allenpress.com/tscir/article-pdf/6/2/65/1985063/5wwp-t27d-lbuu-x24d.pdf by guest on 30 September 2021 7. Treloar HB, Nurcombe V, Key B. Expression of Wigley CB. Adult rat olfactory nerve ensheath- extracellular matrix molecules in the embry- ing cells are effective promoters of adult cen- onic rat olfactory pathway. J Neurobiol. tral nervous system neurite outgrowth in 1996;31:41–55. coculture. Glia. 1999;25:256–269. 8. Raisman G. Specialized neuroglial arrange- 21. Boruch AV, Conners JJ, Pipitone M, et al. ment may explain the capacity of vomeronasal Neurotrophic properties of an olfactory en- axons to reinnervate central neurons. sheathing cell line. Glia. In press Neurosci. 1985;14:237–254. 22. Ramón-Cueto A, Nieto-Sampedro M. Glial 9. Valverde F, Lopez-Mascaraque L. Neuroglial cells from adult rat olfactory bulb: Immunocy- arrangements in the olfactory glomeruli of the tochemical properties of pure cultures of en- hedgehog. J Comp Neurol. 1991;307:658– sheathing cells. Neurosci.1992;47:213–220. 674. 23. Kafitz KW, Greer CA. Olfactory ensheathing 10. Graziadei PPC, Monti Graziadei GA. cells promote neurite extension from embry- Neurogenesis and neuron regeneration in the onic olfactory receptor cells in vitro. Glia. olfactory system of mammals. I. Morphologi- 1999;25:99–110. cal aspects of differentiation and structural 24. Li Y, Raisman G. Integration of transplanted organization of the olfactory sensory neurons. cultured Schwann cells into the long myeli- J Neurocytol. 1979;8:1–18. nated fiber tracts of the adult spinal cord. Exper 11. Barber PC, Lindsay RM. Schwann cells of the Neurol. 1997;145:397–411. olfactory nerves contain glial fibrillary acidic 25. Doucette R. Development of the nerve fiber protein and resemble astrocytes. Neurosci. layer in the olfactory bulb of mouse embryos. 1982;7:3077–3090. J Comp Neurol. 1989;285:514–527. 12. Franklin RJM, Barnett SC. Do olfactory glia 26. Bailey MS, Puche AC, Shipley MT. Develop- have advantages over Schwann cells for CNS ment of the olfactory bulb: Evidence for glia- repair? J Neurosci Res. 1997;50:665–672. neuron interactions in glomerular formation. J 13. Doucette, JR. The glial cells in the nerve fiber Comp Neurol. 1999;415:423–448. layer of the rat olfactory bulb. Anat Rec. 27. Graziadei PPC, Levine RR, Monti Graziadei 1984;210:385–391. GA. Regeneration of olfactory axons and syn- 14. Ramón-Cueto A, Valverde F. Olfactory bulb apse formation in the forebrain after ensheathing glia: A unique cell type with ax- bulbectomy in neonatal mice. Proc Natl Acad onal growth-promoting properties. Glia. Sci USA. 1978;75:5230–5234. 1995;14:163–173. 28. Graziadei PPC, Monti Graziadei GA. 15. Franceschini IA, Barnett SC. Low-affinity Neurogenesis and neuron regeneration in the NGF-receptor and E-N-CAM expression de- olfactory system of mammals. III. Deafferenta- fine two types of olfactory nerve ensheathing tion and reinnervation of the olfactory bulb cells that share a common lineage. Devel Biol. following section of the fila olfactoria in rat. J 1996;173:327–343. Neurocytol. 1980;9:145–162. 16. Gudiño-Cabrera G, Nieto-Sampedro M. Estro- 29. Barber PC, Raisman G. Continuous formation gen receptor immunoreactivity in Schwann- of vomeronasal neurosensory cells in adult like brain macroglia. J Neurobiol. 1999;40: mice and its importance in regeneration. In: 458–470. Meisami E, Brazier MAB, eds. Neural Growth 17. Pixley SK. The olfactory nerve contains two and Differentiation. New York: Raven Press; populations of glia, identified both in vivo and 1979:365–371. in vitro. Glia. 1992;5:269–284. 30. Wright JW, Harding JW. Recovery of olfactory 18. Astic L, Pellier-Monnin V, Godinot F. Spatio- function after bilateral bulbectomy. Science. temporal patterns of ensheathing cell differen- 1982;216:322–324. tiation in the rat olfactory system during devel- 31. Tennent R, Chuah MI. Ultrastructural study of opment. Neurosci. 1998;84:295–307. ensheathing cells in early development of ol- 19. Miragall F, Kadmon G, Husmann M, factory axons. Dev Brain Res. 1996;95:135– Schachner M. Expression of cell adhesion 139. Olfactory Ensheathing Glia 81

32. Ramón-Cueto A, Nieto-Sampedro M. Regen- Bunge MB. Schwann cell and ensheathing glia eration into the spinal cord of transected dorsal transplantation into the contusion injured root axons is promoted by ensheathing glia adult rat spinal cord. Soc Neurosci Abstr.

transplants. Exp Neurol. 1994;127:232–244. 1999;25:748. Downloaded from http://meridian.allenpress.com/tscir/article-pdf/6/2/65/1985063/5wwp-t27d-lbuu-x24d.pdf by guest on 30 September 2021 33. Navarro X, Valero A, Gudiño G, et al. En- 43. Pérez-Bouza A, Wigley CB, Nacimiento W, sheathing glia transplants promote dorsal root Noth J, Brook GA. Spontaneous orientation of regeneration and spinal reflex restitution after transplanted olfactory glia influences axonal multiple lumbar rhizotomy. Ann Neurol. regeneration. NeuroReport. 1998;9:2971– 1999;45:207–215. 2975. 34. Ramer MS, Priestley JV, McMahon SB. Func- 44. Devon R, Doucette R. Olfactory ensheathing tional regeneration of sensory axons into the cells myelinate dorsal root ganglion neurites. adult spinal cord. Nature. 2000;403:312–316. Brain Res. 1992;589:175–179. 35. Li Y, Field PM, Raisman G. Repair of adult rat 45. Devon R, Doucette R. Olfactory ensheathing corticospinal tract by transplants of olfactory cells do not require L-ascorbic acid in vitro to ensheathing cells. Science. 1997;277:2000– assemble a basal lamina or to myelinate dorsal 2002. root ganglion neurites. Brain Res. 36. Li Y, Field PM, Raisman G. Regeneration of 1995;688:223–229. adult rat corticospinal axons induced by trans- 46. Bunge RP, Bunge MB. Interrelationship be- planted olfactory ensheathing cells. J tween Schwann cell function and extracellular Neurosci. 1998;18:10514–10524. matrix production. Trends Neurosci. 37. Ramón-Cueto A, Plant GW, Avila J, Bunge 1983;6:499–505. MB. Long-distance axonal regeneration in the 47. Plant GW, Wood PM, Bunge MB. P75 purified transected adult rat spinal cord is promoted by ensheathing glia from adult rats fail to olfactory ensheathing glia transplants. J myelinate rat dorsal root ganglion axons in Neurosci. 1998;18:3803–3815. vitro. J Neurotrauma. 1999;16:1012. 38. Plant GW, Ramón-Cueto A, Bunge MB. Trans- 48. Imaizumi T, Lankford KL, Waxman SC, Greer plantation of Schwann cells and ensheathing CA, Kocsis JD. Transplanted olfactory en- glia to improve regeneration in adult spinal sheathing cells remyelinate and enhance ax- cord. In: Ingoglia NA, Murray M, eds. Regen- onal conduction in the demyelinated dorsal eration in the Central Nervous System. New columns of the rat spinal cord. J Neurosci. York, NY: Marcel Dekker; 2000:529–561. 1998;18:6176–6185. 39. Xu XM, Zhang S-X, Li H, Aebischer P, Bunge 49. Kato T, Honmou O, Uede T, Hashi K, Kocsis MB. Regrowth of axons into the distal spinal JD. Transplantation of human olfactory en- cord through a Schwann cell-seeded mini- sheathing cells elicits remyelination of demy- channel implanted into hemisected adult rat elinated rat spinal cord. Glia. 2000;30:209– spinal cord. Eur J Neurosci. 1999;11:1723– 218. 1740. 50. Franklin RJM, Gilson JM, Franceschini IA, 40. Imaizumi T, Lankford KL, Kocsis JD. Trans- Barnett SC. Schwann cell-like myelination fol- plantation of olfactory ensheathing cells or lowing transplantation of an olfactory bulb- Schwann cells restores rapid and secure con- ensheathing cell line into areas of demyelina- duction across the transected spinal cord. tion in the adult CNS. Glia. 1996;17:217–224. Brain Res. 2000;854:70–78. 51. Yan HL, Bunge MB, Plant GW. Heregulin and 41. Ramón-Cueto A, Cordero MI, Santos-Bentio FGF-2 stimulate proliferation of olfactory en- FF, Avila J. Functional recovery of paraplegic sheathing glia from adult rat in vitro. Soc rats and motor axon regeneration in their spi- Neurosci Abstr. 1999;25:2044. nal cords by olfactory ensheathing glia. Neu- ron. 2000;25:425–435. 42. Oudega M, Plant GW, Katz J, Marcillo A,