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Bridging Lesions in Cord Volume 7, Number 4, 2001THE NEUROSCIENTIST REVIEW Bridging Areas of Injury in the Spinal Cord MARY BARTLETT BUNGE The Miami Project to Cure Paralysis University of Miami School of Medicine There is a devastating loss of function when substantial numbers of axons are interrupted by injury to the spinal cord. This loss may be eventually reversed by providing bridging prostheses that will enable axons to regrow across the injury site and enter the spinal cord beyond. This review addresses the bridging strategies that are being developed in a number of spinal cord lesion models: complete and partial transection and cavities arising from contusion. Bridges containing peripheral nerve, Schwann cells, olfactory ensheathing glia, fetal tissue, stem cells/neuronal precursor cells, and macrophages are being evaluated as is the administration of neurotrophic factors, administered by infusion or secreted by genetically engineered cells. Biomaterials may be an important factor in developing successful strategies. Due to the complexity of the sequelae following spinal cord injury, no one strategy will be effective. The compelling question today is: What combinations of the strategies discussed, or new ones, along with an initial neuroprotective treatment, will substantially improve outcome after spinal cord injury? NEUROSCIENTIST 7(4):325–339, 2001 KEY WORDS Cellular bridges, CNS regeneration, CNS injury, Neurotrophins, Transplantation Laceration injuries or those that lead to cavitation in the spinal cord interrupt descending and ascending axons. Whereas loss of gray matter may be small, it is the interruption of fiber tracts that leads to devastating sequelae for the injured person. How can we bridge these interrupted areas with permissive cells or materials to enable severed axons to once again extend to appropriate areas on either side of the lesion? A number of investigators have conducted experiments to test cellular and noncellular components to achieve this goal of eliciting growth across the lesion. Of course, this is but the first step. Strategies to save as much tissue as possible directly following injury and to support growth beyond the bridge to reach areas for reconnection will be different and additive. Thus, it will be important to combine neuroprotective agents and guidance techniques with bridging to accomplish the long-term goal of improving outcome after injury. This overview will discuss only bridging strategies in the adult spinal cord. Additional strategies utilizing cell/polymer scaffolds to Work in the Bunge laboratory has been supported by NINDS grant NS09923, The Miami Project to Cure Paralysis, The Christopher Reeve Paralysis Foundation, and the Heumann, Hollfelder, and Rudin Foundations. I thank Dr. N. Kleitman for helpful comments on the manuscript and C. Rowlette, S. Knockee, and J. Cox for word processing. I also appreciate the contributions of illustrations by Drs. J. Guest (The Miami Project; Fig. 5), A. Ramón-Cueto (Centro de Biologia Molecular “Severo Ochoa,” Universidad Autónoma, Madrid; Fig. 9), and X. M. Xu (color renditions of figures in Xu, Chen, and others 1997; Xu and others 1999; Figs. 2 and 11). Dr. C. W. Christman (Richmond, VA) finalized the drawings in Fig. 1. Address correspondence to: Mary Bartlett Bunge, Ph.D., The Miami Project to Cure Paralysis, University of Miami School of Medicine, P.O. Box 016960 (R-48), Miami, FL 33101 (e-mail: [email protected]). bridge CNS injuries are described in a review by Harvey (2000) (see also Plant and others 2000). Also, the review by Houweling and others (1998) contains supplementary information about bridging experiments. The Complete Transection Model Peripheral Nerve Transplantation A landmark article published in 1980 (Richardson and others 1980) that importantly influenced subsequent spinal cord injury (SCI) research involved bridging a complete transection gap in the spinal cord with peripheral nerve. Why complete transection and why peripheral nerve? Complete transection lesions yield unambiguous results about axonal regeneration compared with sprouting from noninjured axons and sparing of fibers. Peripheral nerve was tested because the rationale was to place an environment known to support axonal regeneration into a milieu in which adequate regeneration did not occur. This concept had been embraced by Spanish workers in the early 1900s (Ramón y Cajal 1928). The growth of fibers into peripheral nerve implanted in the central nervous system (CNS) prompted Ramón y Cajal to speculate that if the environment is suitable, axons from central neurons are able to respond and regrow. In fact, that most perspicacious of investigators further hinted at a combination strategy by hypothesizing that “nutritive” and “orienting” substances will be required to achieve axonal regeneration in the adult. By the time the Aguayo team (Richardson and others 1980) investigated the efficacy of peripheral nerve transplantation into the spinal cord, tracing techniques had become available, allowing clear demonstration of the source of Volume 7, Number 4, 2001 Copyright © 2001 Sage Publications ISSN 1073-8584 Downloaded from nro.sagepub.com at PENNSYLVANIA STATE UNIV on September 17, 2016 THE NEUROSCIENTIST 325 Box 1: Spinal Cord Injury Models: Transection Complete transection Over hemisection Dorsal hemisection Lateral hemisection Tract lesion regenerated fibers in this and related studies (rev. in Aguayo 1985). Peripheral nerve was placed into a complete transection gap of 5 or 10 mm (Richardson and others 1980). After 3 to 4 months, tracing demonstrated that fibers grow into and across the implant from both stumps. A mean of 5850 myelinated axons are found in the graft if the nearby dorsal roots are avulsed. Regenerated fibers do not leave the graft, and corticospinal tract (CST) fibers do not enter these grafts (Richardson and others 1982). Of course, another possibility is to skirt the lesion area completely. Other work by the Aguayo team (rev. in Aguayo 1985) showed that ends of a piece of peripheral nerve inserted into the medulla and the spinal cord results in fiber ingrowth as much as 30 mm along the graft. Efforts to achieve functional improvement after peripheral nerve grafting have been made by the Olson team (Cheng and others 1996). Their strategy combined compressive wiring of the spinal column, transplantation of 18 thin peripheral nerves oriented to extend from white matter to gray matter, and the use of fibrin glue to which fibroblast growth factor-1 was added. If one of these steps is omitted, the improvement in locomotion and evidence of growth of axons from corticospinal and a variety of brainstem neurons into the grafts and beyond are not found. The investigators reported movements in the three joints of the hind limbs, partial support of body weight, and contact placing of the hind limbs, suggestive of CST involvement. Reports confirming these studies have not yet appeared in the literature. Possible reasons for this are the surgical expertise (causing minimal damage) and care with which this study was executed and the way in which the nerves were inserted into the spinal cord. Further discussion may be found in another review (Bunge 2000). Schwann Cell/Ensheathing Glia Transplantation Because Schwann cells (SCs) are considered to be responsible for the supportive milieu provided by peripheral nerve, they have been substituted for nerve in bridges (Fig. 1). SCs possess many attributes such as extracellular matrix production, surface adhesion and integrin molecules, and growth factor secretion that are favorable for axonal growth (Plant and others 2000). Purified populations can be prepared in numbers adequate to construct bridges. An attractive concept is that 326 Box 2: Bridges across Transection Gap Peripheral nerve Rat Schwann cells (SCs) Human SCs Olfactory ensheathing glia Peripheral nerve-activated macrophages SCs from peripheral nerve of a spinal cord–injured person could be placed in culture for expansion. Not only rat but also human SCs can be rapidly expanded, nearly 10,000 to 100,000 times, with a final purity of 95% to 98% (Bonamichi and others 1997; G. Casella and P. Wood, unpublished observations). From a 1-cm piece of human nerve, a graft 30 m long and 2.6 mm in diameter can be prepared in 6 weeks. This, then, could be transplanted into a spinal cord lesion in the same person. Human SCs have been found to be as effective as rat SCs in bridges; they support axonal regrowth and ensheathment and myelination of regenerated central nerve fibers (Guest and others 1997). Finally, the SCs can be genetically modified during their culture period. Bridges of purified SCs (4–5 mm long) unite transected adult rat spinal cord stumps (Xu, Chen, and others 1997) (Fig. 2); fibers grow into both ends of the graft, as in the peripheral nerve work cited above. Much of the SC bridge work tested a cable of SCs in Matrigel (a commercial preparation of basal lamina components) inside a PAN/PVC polymer channel (Fig. 3), with the stumps inserted 1 mm into each end of the channel to be in apposition to the cable. A channel is advantageous in some ways. In the nude rat transection/human SC transplantation paradigm, the effectiveness of an SC/Matrigel cable with or without the surrounding channel has been compared (Guest and others 1997). The SCs, once compacted into a cable inside the channel, can be removed for transplantation without the channel (Fig. 4). Whereas the spinal cord stumps deteriorate less and there tend to be more regenerated fibers in the graft without a channel, in the presence of a channel, nerve fibers are in more parallel array (Fig. 5) and there is less invasion of scar tissue into the graft-cord interfaces. But no substantial difference in neuroanatomic or functional outcome is observed. When polymer-enclosed SCs are transplanted into adult Fischer rats, spinal neurons as far away as levels C3 and S4 extend ascending and descending fibers into the graft, where myelinated axons and 8 times as many unmyelinated but ensheathed axons are found (Xu, Chen, and others 1997) (Table 1) (Figs. 6, 7). It is only when SCs are present in the graft that considerable n u m b e r s o f a x o n s a r e f o u n d w e e k s l a t e r. Immunoreactive serotonergic and noradrenergic axons grow for a short distance into the rostral end of the graft, indicating a modest brainstem response; CST regeneration into the graft and tracer-labeled fiber THE NEUROSCIENTIST Bridging Lesions in Cord Downloaded from nro.sagepub.com at PENNSYLVANIA STATE UNIV on September 17, 2016 Fig. 2. A Schwann cell (SC) graft inside a polymer channel placed between completely transected stumps of adult Fischer rat thoracic spinal cord 1 month before perfusion. The channel has been cut open to reveal the SC bridge. The ends of the bridge are fused well to the stumps. Bar, 1 mm. (Courtesy of Dr. X. M. Xu, color rendition of Fig. 3 in Xu, Chen, and others 1997.) Fig. 1. Cartoons of experimental strategies in adult rat thoracic spinal cord. A, A Schwann cell (SC) bridge implanted in a complete transection gap. SCs and stumps of transected spinal cord are enclosed within a polymer channel (Xu, Chen, and others 1997). B, Injection of olfactory ensheathing glia (OEG) (dark blue) into the spinal cord stumps near the ends of the polymer-enclosed SC bridge. Tracer (orange) was delivered 6 weeks later at level C7. Tracer-labeled axons are present beyond the distal interface, and lumbar cord nerve cell bodies are labeled, suggesting that their severed axons regrow across both graft-cord interfaces (Ramón-Cueto and others 1998). C, Injection of OEG (dark blue) into the spinal cord stumps after complete transection (Ramón-Cueto and others 2000). D, Infusion of neurotrophins (light blue) into the distal end of a polymerenclosed SC cable (Xu, Guénard, Kleitman, Aebischer, and Bunge 1995). E, Injection of SCs into the distal spinal cord to create a trail 5 mm long beyond the transection site and also into the transection site itself. Tracer (orange) was injected into the distal end of the trail later to identify nerve cells that had extended axons the length of the trail (Menei and others 1998). F, Insertion of an SC-containing hemi-channel into the right side of the spinal cord (Xu and others 1999). All cartoons except F are sagittal section representations; F depicts a horizontal view. (Drawings finalized by Dr. C. W. Christman.) egress from the SC graft into the distal spinal cord are not found. The failure of axons to leave SC grafts is of obvious concern. A gauntlet of chondroitin sulfate proteoglycan (CSPG) at the distal SC graft-host cord may be, in part, responsible. CSPGs, up-regulated in CNS injury, have been implicated in failure of neurite growth in many in vitro and in vivo studies (reviewed in Fawcett and Asher 1999 and Fitch and Silver 1999). In the complete transection/SC transplantation paradigm, the proteoglycans, CS-56 antigen, neurocan, phosphacan, and NG2, are expressed near the proximal and distal interfaces (Plant and others 2001). Moreover, the CS-56 antigen and phosphacan are more heavily expressed at the distal than the proximal interface only when SCs are present in the channel. We do not yet understand in what ways SCs interact differently with the distal than the proximal stump. Proximal stumps may differ from distal ones in tissue viability, types of degenerating and regenerating axons, blood-brain barrier deficiency, number of macrophages/microglia, and blood circulation, as examples. New initiatives have begun to reduce CSPG in areas of CNS injury to potentially improve axonal regeneration (Lemons and others 1999; Moon and others 1999; Yick and others 2000). There are three strategies that lead to at least modest growth from the SC graft into the distal spinal cord. Human SCs can be grafted into nude rats, which accept cells from other species. The regenerative response is improved compared with the Fischer rat model in some ways, but this reflects the use of the nude rat, not the human SCs (J. Guest, unpublished observations). When grafting is done in a similar way, a comparable number of myelinated axons is found in the human SC graft (Guest and others 1997) (Table 1). Despite the distant location of the nerve cell bodies in the brain stem, serotonergic and noradrenergic fibers are detected within the grafts. A small number of propriospinal and sensory fibers extend throughout the graft and into the distal spinal cord for a maximum distance of 2.6 mm. Inclined plane and BBB open field locomotion evaluation (Basso and others 1995) has revealed that in both Volume 7, Number 4, 2001 THE NEUROSCIENTIST Downloaded from nro.sagepub.com at PENNSYLVANIA STATE UNIV on September 17, 2016 327 Fig. 3. Scanning electron micrographs of a PAN/PVC channel and the contained Schwann cell (SC) graft. After maintenance in culture overnight, the SC-Matrigel mix that initially fills the channel contracts to yield a solid cable of SCs, smaller in diameter than that of the channel (left). The inner diameter of the channel is 2.6 mm, the width of the thoracic spinal cord of a 170 g Fischer rat. The SCs become aligned during the syneresis process (right). Bar, right panel, 20 µ m. (From Guénard and others 1992.) Box 3: Axonal Growth from Graft into Cord Peripheral nerve bridge + additional strategies Human Schwann cell (SC) bridge in nude rat Rat SC bridge + ensheathing glia Neurotrophin-engineered SCs Rat SC hemibridge Olfactory ensheathing glia Peripheral nerve-activated macrophages Fig. 4. A 3 to 4 mm bridge of human Schwann cells placed in a complete transection gap of an adult nude rat thoracic spinal cord 6 weeks earlier (upper panel ). This graft was inserted without an enclosing channel. In the higher magnification (lower panel ), a ventral view (dura intact) is illustrated. The union of the bridge and stumps appears seamless (arrows), and scarring and cavitation are not obvious. Bar, 5 mm. (From Guest and others 1997.) cases there is a modest but statistically significant improvement in behavioral scores when human SCgrafted animals are compared with animals transplanted with similar cables but capped at the distal end to prevent outgrowth into the distal spinal cord (Guest and others 1997); also, during the open field testing, animals with open-ended grafts exhibit more frequent and longer episodes of alternating stepping than those with capped grafts. In a few animals with bridging grafts, there is evidence of some form of contact-placing response, but, because the animals lacked retrograde tracing evidence of CST regeneration, Guest and colleagues (1997) 328 assumed that these responses represent propriospinal placing mediated by local neuronal circuits. A treadmill test, developed at The Miami Project (Broton, Nikolic, and others 1996), enables kinematic analysis of limb positions. When transected-only (Broton, Xu, and others 1996) and human SC-transplanted rats are compared in this way, the former exhibit rhythmic alternating stepping 20% of the time and the latter exhibit stepping 60% of the time they are on the treadmill (J. Guest and J. Broton, unpublished observations). An interpretation is that the graft increases excitability in the distal stump, possibly by influencing the activity of the lumbar-stepping pattern-generating neurons. There is no evidence of fore- to hind-limb coordination. A second strategy in which some outgrowth of regenerated fibers from the SC graft into the spinal cord is observed is the combination with methylprednisolone THE NEUROSCIENTIST Bridging Lesions in Cord Downloaded from nro.sagepub.com at PENNSYLVANIA STATE UNIV on September 17, 2016 Box 4: Brainstem Axonal Growth into Distant Thoracic Implant Peripheral nerve bridge + additional strategies Human Schwann cell (SC) bridge in nude rat Rat SC bridge + neuroprotection Rat SC bridge + neurotrophins Rat SC hemibridge Neurotrophin-engineered fibroblasts or SCs Olfactory ensheathing glia Fetal CNS + neurotrophins Fig. 5. The same section of an interface between host nude rat spinal cord and a human Schwann cell graft, immunostained for neurofilaments (upper panel ) and glial fibrillary acidic protein (lower panel ). The convex surface of the host spinal cord is well demarcated by the staining for astrocytic filaments. The upper panel shows that the nerve fibers leaving the host spinal cord become disorganized at the interface but assume a longitudinal course once in the graft. Bar, 1 mm. (From Zwimpfer and Guest 1999.) administration at the time of transection and Fischer rat SC transplantation (Chen and others 1996). Methylprednisolone is now used routinely in the United States within 8 h after human SCI to reduce secondary injury. With methylprednisolone, the SC bridge contains more myelinated axons and more spinal neurons extend axons into the graft (Table 1) (Fig. 8, top). Also, brainstem neurons respond (mean = 57); serotonergic and noradrenergic axons are found 2.0 to 2.5 mm into the graft. This is a significant finding because when peripheral nerve is implanted at the same level as the SC graft a response of brainstem neurons is not seen (Richardson and others 1984; Houle and others 1994). Thus, the administration of methylprednisolone overcomes, to some degree, the distance between graft and brainstem. In addition, a striking finding is that more spinal cord tissue inserted into each end of the polymer channel survives; the inserted tissue largely deteriorates without this treatment. The egress of fibers from the graft may be related to the reduction in secondary spinal cord tissue loss adjacent to the graft. In more recent work, Oudega and colleagues (1999) showed that die-back of one of the tracts, the vestibulospinal tract, is significantly diminished when methylprednisolone is administered after completely transecting the spinal cord (with no SC graft). A third strategy, combining transection/Fischer rat SC transplantation with injection of olfactory ensheathing glia (OEG) into the stumps beside the graft (Fig. 1), leads to substantial growth of regenerated fibers from the graft into the distal spinal cord. Why ensheathing glia? They are found in that part of the olfactory system where nerve fiber growth continues throughout adulthood (reviewed in Ramón-Cueto and Avila 1998; Kleitman and Bunge 2000). This was the rationale for testing them earlier to see if the dorsal root entry zone, usually a barrier to regenerating sensory neurites, would allow axons to enter the spinal cord after cutting and reapposing dorsal roots to the thoracic spinal cord. After injecting OEG beside the entry zone, regrowing axons are not only able to enter the spinal cord but also extend within the dorsal horn to appropriate laminae (RamónCueto and Nieto-Sampedro 1994). These axons are accompanied by OEG, highly migratory cells. Stimulusevoked conduction of signals through the regenerated sensory fibers is restored to some extent, an indicator that functional synapses are formed (Navarro and others 1999). Ramón-Cueto and Nieto-Sampedro’s 1994 work prompted Ramón-Cueto and others (1998) to determine if the presence of OEG promotes growth across SC graft-cord interfaces. Preparations of 200,000 OEG from adult rats, purified on the basis of their expression of p75, were injected into each stump adjacent to the SC bridge. This strategy does, indeed, improve the ability of regenerating fibers to cross both interfaces and grow into the spinal cord, more than 2.5 cm for ascending fibers (Figs. 1, 9, 10). The Hoechst-labeled OEG are found consistently in areas where regeneration is seen. Serotonergic fibers, detected by immunostaining, regenerate not through the channel but in a connective tissueOEG milieu on the outside of the channel and reenter the distal spinal cord where they extend for at least 1.5 cm. Following this work, OEG were injected on each side of a transection without an implanted SC bridge (Ramón-Cueto and others 2000) (Fig. 1); they migrated Volume 7, Number 4, 2001 THE NEUROSCIENTIST Downloaded from nro.sagepub.com at PENNSYLVANIA STATE UNIV on September 17, 2016 329 Table 1. Numbers of myelinated axons in the SC graft and neurons in the spinal cord that extend axons into the graft in differing transplantation procedures Myelinated Axons Strategy Capped Channel Open Channel Capped Channel Open Channel Rat SC/Matrigel (Matrigel only) 501 ± 83 (71 ± 25) 1990 ± 594 (3 ± 0.9) 306 ± 69 1064 ± 145 a Reference Xu, Chen, and others (1997); Xu, Guénard, Kleitman, and Bunge (1995) Guest and others (1997) Chen and others (1996); Bunge and Kleitman (1999) Xu, Guénard, Kleitman, Aebischer, and Bunge (1995) Oudega and others (1997) Xu and others (1999) Responding Cord Neurons Human SC/Matrigel (Matrigel only) Rat SC/Matrigel + methylprednisolone (SC/Matrigel + vehicle) Rat SC/Matrigel + BDNF, NT-3 (SC/Matrigel + vehicle) Rat SC/Matrigel + IGF-1, PDGF (Rat SC/Matrigel only) Rat SC/Matrigel in hemi-channel (Matrigel only) a 1442 ± 514 1159 ± 308 3237 ± 2478 1116 ± 113 2083 ± 321 (335 ±108) (1324 ± 342) (284 ± 88) (1064 ± 145) 1523 ± 292 967 ± 104 (882 ± 287) 316 ± 42 (504 ± 78) 1004 ± 126 (185 ± 72.3) 550 ± 89 (126 ± 18) The numbers are means ± SEM. The information in parentheses pertains to control animals. SC = Schwann cells. a. Initial experiments were conducted with distally capped channels to focus on responses of descending tracts. from both stumps into the transection site. The rats so treated showed improved voluntary hind-limb movement, plantar hind paw placement, body weight support, and proprioception and light touch by 3 to 7 months. Serotonergic, noradrenergic, and CST fibers crossed the lesion, entered the distal spinal cord, and extended for up to 3 cm (to L6); the animals showing the best behavioral recovery exhibited fibers that extended for greater distances. Whereas serotonergic and noradrenergic fibers grew in spinal cord tissue and invaded regions they normally innervate, CST fibers extended along the pia mater and reentered the spinal cord after lengthy growth. In general, CST growth is not seen in complete transection models. They consistently regenerate only in surviving spinal cord tissue (Schwab and Bartholdi 1996; Grill and others 1997). They do not respond to the peripheral nerve or SC environment, with the one exception of the complex peripheral nerve bridging strategy (Cheng and others 1996). Schwann Cell/Neurotrophin Transplantation That neurotrophins are salutary in the SC grafting paradigm was discovered by Xu, Guénard, Kleitman, Aebischer, and Bunge (1995), who studied the regeneration response to SC grafts and neurotrophins delivered into the polymer channel, not in this case a bridge because the distal end was blocked to concentrate on de330 scending fibers (Fig. 1). BDNF and NT-3 were delivered together for 14 days after transplantation; the animals were maintained for an additional 14 days. A month later, myelinated axon number in the graft and retrogradely labeled neurons in the rostral spinal cord were both increased with neurotrophin administration (Table 1, Fig. 8). Also, in SC/neurotrophin but not SC/ vehicle grafts, some nerve fibers were immunoreactive for serotonin in the graft at least 5 mm from the rostral cord-graft interface. A mean of 92 neurons in the brainstem extended axons at least 5 mm into the bridge; this is in contrast to SC bridges without neurotrophins in which there was essentially no response from the brainstem because of the distance involved. Thus, regeneration of some neuronal populations distant from the spinal cord transection and implant can be elicited by a combination of trophic factors and a favorable cellular substrate. But not all growth factors have the same effect in this paradigm. The inclusion of neurotrophic factors, IGF-1 and PDGF, in the SC transplant does not promote axonal regeneration into the bridge; axonal regrowth into the transplant is diminished up to 63% although SC myelination of ingrowing axons is promoted (Oudega and others 1997) (Table 1). In another complete transection study (Menei and others 1998), SCs were infected with a retroviral vector carrying the human prepro BDNF/cDNA and then transplanted into the spinal cord. The engineered SCs were THE NEUROSCIENTIST Bridging Lesions in Cord Downloaded from nro.sagepub.com at PENNSYLVANIA STATE UNIV on September 17, 2016 Fig. 6. To assess the response of neurons to a Schwann cell graft positioned between completely transected stumps of adult Fischer rat spinal cord, Fast Blue is injected into the middle of the 4 to 5 mm long graft. Axons from neurons A and D pick up tracer because they grow to or beyond (B, E) the injection site; neurons such as C do not regenerate far enough to become labeled. Neurons grow into the graft from both stumps. More neurons are labeled close to the graft, and labeling is seen primarily in laminae VII and VIII where most propriospinal nerve cells are located. The brackets contain numbers of labeled neurons found at the spinal cord level indicated in one of the traced animals studied. (From Xu, Chen, and others 1997.) deposited in a 5 mm long trail extending into the distal spinal cord from the transection site and into the transection site itself (Fig. 1). No polymer channel was used in these experiments. The trails were largely intact for at least a month, as determined by examining SCs prelabeled with Hoechst dye. Cues for this strategy were taken from two studies, one by Brook and others (1994), who transplanted SCs in columns extending through the thalamus and across the choroid fissure into the hippocampus where they acted as bridges that enable directed axonal growth across boundary membranes of the brain. The second study by Oudega and Hagg (1996) will be mentioned later. When animals with engineered SCs were compared with those receiving untreated SCs, more serotonergic and some adrenergic axons were seen in the trail beyond the transection site (Menei and others 1998). After injecting a retrograde tracer, Fast Blue, at the distal end of the SC trail, more retrogradely traced neurons were found in the brainstem, mostly in reticular and raphe nuclei Fig. 7. This survey electron micrograph portrays the content of a Schwann cell (SC) graft placed in a complete transection gap. Scattered SC-myelinated axons occur throughout. Numerous nonmyelinated axons are present as well but are not readily visible at this low magnification. Well-developed fascicles of SC-myelinated and -ensheathed axons (surrounded by differentiated perineurium in some cases) are present in other areas of the graft as illustrated earlier in Xu, Chen, and others (1997). Bar, 5 µ m. (From the study by Xu, Chen, and others 1997.) Volume 7, Number 4, 2001 THE NEUROSCIENTIST Downloaded from nro.sagepub.com at PENNSYLVANIA STATE UNIV on September 17, 2016 331 Fig. 8. These panels illustrate the injection sites (*) of Fast Blue, into a Schwann cell (SC) graft about 5 mm from the rostral spinal cord stump, and the nearest labeled nerve cell bodies in the Fischer rat spinal cord. The host-graft interfaces are indicated by arrowheads. The rostral end of the channels is designated by large arrows. The injected Fast Blue has not reached the interfaces; the Fast Blue that is deposited along the epineurial-like covering of the SC graft does not enter the graft or host spinal cord. The nearest labeled cell bodies of the spinal cord are within the channel in the animal given methylprednisolone at the time of SC transplantation (top), indicating improved survival of the spinal cord stump inserted into the channel compared with animals not receiving this compound. In contrast, despite improved regeneration following the administration of BDNF and NT-3, increased tissue survival is not seen, because the nearest labeled cell bodies are rostral to the channel (bottom). The insets show labeled neuronal somata at higher magnification. Sagittal sections; bars, 300 µ m (upper panel ) and 200 µ m (lower panel ). (From Chen and others 1996 and Xu, Guénard, Kleitman, Aebischer, and Bunge 1995.) (means, 135 vs. 22, mostly in vestibular nuclei for untreated SC transplants). When the animals did not receive SCs, no serotonergic or noradrenergic axons were seen beyond the transection, and no labeled neurons were found rostral to the transection. Thus, transplantation of SCs secreting human BDNF improves the regenerative response across the transection site and into the thoracic spinal cord. Behavioral testing was not done in this study. Other Transplantation Strategies When bundles of 10,000 carbon filaments were implanted into a complete thoracic transection lesion, fi332 bers labeled following intracortical injections of tracer were visualized among the filaments and up to 5.6 mm distal to the implanted fibers (Khan and others 1991). In a lumbar model, Houle and Ziegler (1994) employed NGF-treated nitrocellulose strips as bridges 5 weeks after complete transection. Fetal spinal cord tissue was positioned between the strips. Six weeks later, this combination led to growth of numerous sensory fibers rostralward along the strips and into the rostral spinal cord; in the absence of NGF, fewer fibers extended beyond the caudal host-graft interface and along the strips. Polylactic/polyglycolic acid has been fashioned into channels to implant SCs into complete spinal cord gaps THE NEUROSCIENTIST Bridging Lesions in Cord Downloaded from nro.sagepub.com at PENNSYLVANIA STATE UNIV on September 17, 2016 Fig. 9. This photomicrograph illustrates the numerous fibers that regenerate into a Schwann cell graft from the rostral Fischer rat spinal cord stump in the presence of injected olfactory ensheathing glia. The interface between the spinal cord (left) and the graft (right) lies between the arrowheads. This section was stained with GAP-43 antibody to enable visualization of axons. (Courtesy of Dr. A. Ramón-Cueto, from the investigation reported in Ramón-Cueto and others 1998.) (Oudega and others 2001). The interest in testing this material is that it is biodegradable and would, over time, avoid the continued irritation of the spinal cord and possible disruption of the host-graft junction by remaining channels (Guest and others 1997). This material is well tolerated by the spinal cord (Gautier and others 1998). Rapalino and others (1998) transplanted autologous macrophages, first exposed ex vivo to peripheral nerve segments, into completely transected thoracic rat spinal cord. The rats were evaluated with a number of techniques: BBB open field testing, cortically evoked hind-limb muscle responses, and immunochemistry and tracing to detect growth of fibers across the transection site. Behavioral recovery was seen in the form of increased movement of the hind limbs, plantar placement of the hind paws, and weight support by 8 weeks. Retransection led to complete loss of recovered locomotor activity, including the improved evoked potentials. Nerve fibers were present across the transection site in recovered animals only. The Partial Transection Model Partial transection injuries consist of dorsal or lateral hemisections, highly focal damage, or “over-hemisections” in which more than half the spinal cord is removed. Advantages are that the animals are not as paralyzed and there is remaining tissue to serve as a bridge. In comprehensive studies of neutralization of myelin-related neurite growth inhibitory substances by the antibody, IN-1, CST fibers unfailingly extend through the remaining ventral spinal cord tissue (reviewed in Schwab and Bartholdi 1996). In fact, in a study of CST fiber regeneration in dorsally hemisected thoracic spinal cord, they consistently avoided transplants of embryonic spinal cord, newborn pons, collagen, amnion extracellular matrix, gelfoam, laminin-coated nitrocellulose filters, carbon filaments, and glass fibers (Schnell and Schwab 1993). In this study, no fibers entered any of the implanted tissue or materials. In a recent study investigating OEG in which there was no remaining tissue bridge (Ramón-Cueto and others 2000), some CST fibers chose to regenerate along the pia for some distance below the complete transection before entering the gray matter. Ensheathing Glia/Schwann Cell Transplantation The efficacy of OEG in axonal regeneration was tested in very localized lesions of the CST between levels C1 and C2 by Raisman’s group (Li and others 1997, 1998). Suspensions containing OEG injected into these lesions aligned with the degenerating CST and migrated beyond the area of damage. Axons extended into and 2 to 3 mm beyond the injury site and were myelinated with Schwann-like myelin until they reentered the CNS where myelin was formed by oligodendrocytes. A thin layer of cytoplasm abutting on regenerating axons suggested that the glia escorted regenerating fibers across the lesion. In four animals whose forelimbs functioned normally following complete lesioning, transplanted cells were present along the entire extent of the lesion; in three animals in which forelimb function did not return to normal, OEG did not bridge the entire lesion rostrocaudally. Thus, improved function correlated with the presence of a continuous bridge of glia across the lesion. Imaizumi and others (2000) reported both improved conduction and sensory axonal regeneration foll ow i n g OE G ( a n d S C , b o t h f r e s h l y i s o l a t e d ) transplantation on either side of severed T11 dorsal col- Volume 7, Number 4, 2001 THE NEUROSCIENTIST Downloaded from nro.sagepub.com at PENNSYLVANIA STATE UNIV on September 17, 2016 333 Box 5: Improvement in Locomotion with Transplantation Peripheral nerve graft + additional strategies Neurotrophin-engineered fibroblasts Human Schwann cell bridge in nude rat Olfactory ensheathing glia Peripheral nerve-activated macrophages Embryonic stem cells Collagen bridge + neurotrophin Fetal CNS Fig. 10. By presenting tracer to fibers in the cervical spinal cord region, the extension of these fibers into the Schwann cell (SC) graft and beyond into the caudal spinal cord stump may be detected. This is shown in the top panel; traced fibers extend through the distal interface (arrows) between the graft (left) and the spinal cord (right). This is seen when olfactory ensheathing glia (OEG) are injected into the spinal cord near the ends of the SC bridge. When OEG are not injected, there is no evidence of fibers leaving the SC graft (bottom panel, distal interface indicated by arrows). Horizontal sections; the bar represents 160 µ m (top) or 50 µ m (bottom). (From Ramón-Cueto and others 1998.) umns. The transplants apparently induced a new rapidly conducting pathway across the transection site. Polymer (PAN/PVC) channels enclosing rat SCs but only half the diameter of the spinal cord (Fig. 1) lead to improved regeneration (Xu and others 1999) compared with SC-filled channels into which the entire spinal cord stumps are inserted (described above; Xu, Chen, and others 1997). The model consists of placement of the channel on the right side of the spinal cord at T8 (Fig. 11). A mean of 1000 myelinated axons and 9 times more unmyelinated axons was found at the graft midpoint, as in the Xu, Chen, and others (1997) work. But, in addition to propriospinal and sensory axons, neurons (mean = 125) from a number of brainstem regions extended fibers into the graft without additional treatment. Moreover, some regenerating axons exited the graft to extend into the distal spinal cord as far as 3.5 mm. These axons grew toward the gray matter where they formed bouton-like structures. The response of brainstem neurons and growth of fibers into the distal spinal cord may result, in part, from restoration of 334 cerebrospinal fluid circulation and relatively more stable cord-graft interfaces due to the more limited laminectomy. As in the larger channel paradigm (Xu, Guénard, Kleitman, Aebischer, and Bunge 1995), infusion of BDNF and NT-3 improves regeneration; a larger number of axons leave the graft to enter the spinal cord and grow as much as 6 mm beyond (Xu, Bamber, and others 1997). Montgomery and others (1996) placed SC-filled polycarbonate channels into the dorsal half of the spinal cord. Typical SC/myelinated and ensheathed axon fascicles were found in the interior of these tubes. Axons grew in from both rostral and caudal locations. No quantitation was performed, and whether the regenerated axons leave the graft was not tested. Genetically Modified Cell Transplantation A very important pioneering effort has been made by the Gage-Tuszynski team to genetically modify fibroblasts to provide neurotrophic factors to spinal cord (reviewed in Tuszynski and others 1999). The cells embedded in a collagen gel are positioned in thoracic dorsal hemisection lesion cavities. There is robust growth of sensory and coerulospinal axons into the graft if the fibroblasts secrete NGF, for example. These grafts could potentially serve as bridges for certain fiber populations, but sensory and coerulospinal axons have not yet been observed to exit the graft. In one of the most recent studies, Grill and others (1997) severed the CST bilaterally or the entire dorsal half of the mid-thoracic spinal cord to remove additional tracts. The more extensive lesioning led to functional deficits lasting for 2 months, as observed with a locomotion grid task that requires sensory motor integration, thus partially reflecting the function of supraspinal motor projections to the spinal cord. When fibroblasts genetically modified to secrete NT-3 were grafted into the more extensive lesion, significant sensorimotor functional improvement occurred and a significant increase in CST axon growth at and up to 8 mm distal to the injury site was observed. In no case did CST axons enter grafts, confirming that CST fibers regenerate only into surviving ventral gray matter. SCs have also been genetically modified to produce NGF and tranplanted into uninjured midthoracic adult rat spinal cord for 2 weeks to 1 year (Tuszynski and THE NEUROSCIENTIST Bridging Lesions in Cord Downloaded from nro.sagepub.com at PENNSYLVANIA STATE UNIV on September 17, 2016 Fig. 11. Implantation of a Schwann cell (SC) graft-containing polymer hemi-channel that is one-half the diameter of the spinal cord. Panel A illustrates a dorsal view of an adult Fischer rat spinal cord following this procedure. The bridge between the two laterally hemisected stumps inside the channel is shown in panel B (arrows). Panel C presents a ventral view. Panel D is a toluidine blue-stained plastic cross section of the intact spinal cord (left) and implanted channel (arrows) and SC bridge (F ). The dorsal (DH ) and ventral (VH ) horns are visible in the intact left hemi-cord. The contents of boxes labeled E and F are not included in this assemblage. Bars, A through C, 2.0 mm and D, 100 µ m. (Courtesy of Dr. X. M. Xu, color rendition of Fig. 4 in Xu and others 1999.) others 1998). In vivo expression of the human NGF transgene lasts for at least 6 months. At 3 months and later, the NGF-secreting grafts slowly increase in size in contrast to nontransduced SC grafts. After 2 weeks, the transduced transplants contain numerous sensory axons and, after 4 months, dopaminergic and noradrenergic axons, which continue to increase to 6 months. Control grafts contain far fewer axons. No graft, transduced or not, contains serotonergic or CST axons. Again, genetic manipulation of SCs to increase their production of a neurotrophin improves their ability to support axonal regeneration in the adult rat spinal cord. Another team (Liu and others 1999) transplanted fibroblasts genetically engineered to produce BDNF into partial cervical hemisection cavities created to interrupt the rubrospinal tract on one side. One to two months later, 7% of rubrospinal tract neurons possessed axons that were present three to four segments caudal to the transplants. These axons, in and around the transplants and in white matter caudal to the transplant, terminated in appropriate gray matter regions. These fibers were not seen when unmodified fibroblasts were transplanted. Behavioral testing showed that recipients of BDNF-producing fibroblasts exhibited significant recovery of forelimb function, which was abolished by a second lesion. Neurotrophin Administration Cervical axotomy leads to atrophy of rubrospinal neurons in the adult rat. This can be prevented if BDNF is infused near the cell bodies 7 to 14 days later; in addition, expression of “regeneration genes,” GAP-43 and T∝ 1-tubulin, is stimulated, receptor expression for BDNF is maintained, and the number of axons that regenerate into a peripheral nerve graft placed into the lateral hemisection cavity is increased (Kobayashi and others 1997). The stimulation of these genes is correlated with the increase in rubrospinal axon regeneration into the graft. The graft was not tested as a bridge because only the proximal end of the nerve was in apposition to the spinal cord. Whereas peripheral nerve grafts also have not been tested as bridges by Houle and colleagues (Ye and Houle 1997), valuable characterization of neurotrophic factor influence on axonal regeneration into these grafts has been accomplished. The neurotrophin NGF has been tested as a supplement to peripheral nerve grafts in dorsal columns. Infusion of NGF into peripheral nerve grafts inserted into adult rat dorsal columns causes a greater number of spinal cord neurons to regenerate axons into the graft, and some of these neurons are farther from the graft site than when NGF is not present (Fernandez and others 1990). Oudega and Hagg (1996) found that infusion of NGF into the spinal cord beyond a peripheral nerve graft in the dorsal columns leads to increased numbers of regenerating sensory fibers that exit the graft into the spinal cord. This work is important in suggesting ways in which regenerated fibers can be lured from bridges into the spinal cord. Neurotrophic factors, possibly in gradients, may be a key to overcoming barriers to axonal growth at the SC graft-distal cord interface. It is not yet known whether the use of IN-1 antibody, efficacious in neutralizing myelin-related growth inhibitory molecules (Schnell and Schwab 1990), will enhance growth through such an interface as well as along the Volume 7, Number 4, 2001 THE NEUROSCIENTIST Downloaded from nro.sagepub.com at PENNSYLVANIA STATE UNIV on September 17, 2016 335 Box 6: Spinal Cord Injury Models: Cavitation Impaction instrument Weight-drop device Inflatable balloon Photochemical technique (Bunge and others 1994) Compression clip (Fehlings and Tator 1995) (Oudega and others 1999; Bunge and others 2000). The animals were maintained for 2 to 12 weeks. In control rats, not injected with cells, a large cavity was evident. All three types of cellular implants were well integrated with the spinal cord, reduced injury-induced tissue loss, improved axonal sparing/regeneration of propriospinal projections (by 3 times) and of brainstem axons (by 1.4 times), and decreased CST die-back (by 40%). Potential Bridging Strategies spinal cord. NT-3, a neurotrophin to which CST fibers respond, improves growth of these fibers into a collagen matrix transplanted in a lesion of the CST (Houweling and others 1998). No CST fibers leave the collagen implant despite functional recovery (as assessed by a grid walk test) in rats receiving the NT-3-containing collagen implants. Joosten and others (1995) found earlier that whether the collagen is introduced in a fluid or gel state makes a difference in promoting CST growth. Only when collagen is added in the fluid state are tracer-labeled CST axons present in the implant, perhaps because apposition to the spinal cord is better or because an astroglial scaffolding structure forms only in the initially fluid collagen. Transplantation into Models of Cavitation Because studying cysts that develop from contusion is highly clinically relevant (Bunge and others 1997), transplantation into the spinal cord to bridge these cavities has been initiated. In 1991 and 1996, Martin and others published accounts of injecting SC suspensions into a spinal cord cavity created by an inflatable balloon. SC survival is better when transplantation is done immediately or at 10 days than at 3 days. Also, the gliotic reaction is less with immediate injection. The grafts contain numerous axons, mainly from nearby dorsal roots; neither CST nor monoaminergic fibers are detectable. Paino and others (Paino and Bunge 1991; Paino and others 1994) prepared SCs, either in bands of Bungner on the original collagen substratum or added as suspensions later to collagen, and transplanted them into photochemical lesion-induced dorsal column cavities (Bunge and others 1994) after rolling the cell-carrying collagen into jelly rolls. Profuse growth of axons into the implants occurred but their source was not known. Injection of fibroblasts, genetically modified to produce NGF or BDNF, into an acute contusion injury, accelerates locomotor recovery (BBB testing; Basso and others 1995) and leads to larger cross-sectional areas of the spinal cord at the epicenter than when nontransduced cells are implanted (Kim and others 1996). More axons, enhanced myelinogenesis, and new oligodendrocytes are found in transplants of BDNF- or NT-3-producing fibroblasts at 10 weeks after contusion of adult rat spinal cord (McTigue and others 1998). More recently, SCs and/or OEG have been injected into thoracic cavities 1 week after contusion by the NYU impactor 336 Transplantation of fetal CNS tissue into adult spinal cord has been studied extensively (reviewed in Reier and others 1992 and Bregman 1994). Fetal tissue survives well, differentiates, forms synapses, becomes vascularized, fills the lesion cavity, becomes integrated with the spinal cord, reduces gliosis around the lesion, and may provide trophic factors. At present, this type of transplant may be more appropriate to provide immature neuronal populations that become relay stations (Bregman and others 1993) rather than bridges in the adult. Host axons grow into this type of transplant, and neurons in the transplanted tissue extend axons into the host tissue, but generally fibers have not been traced all the way across. Host CST and brainstem-spinal axons project into fetal transplants, but their distribution is restricted to within 200 µm of the host-transplant border. CNS tissue transplants enable some degree of functional recovery, however (Bregman 1994). When transplants of E14 fetal spinal cord tissue are placed into cervical or thoracic spinal cord hemisection lesions in adult rats in combination with the administration of BDNF, NT-3, or NT-4/5 at the site of injury, there is an increase in the extent of CST, serotonergic, and noradrenergic growth into the transplants after 1 to 2 months (Bregman and others 1997). In this case, neurotrophins promote more extensive axonal growth into the fetal transplants. The application of stem cells or neural progenitor cells (e.g., Shihabuddin and others 1999) to devise therapeutic strategies for the CNS by replenishing neurons and/or glia may lead to bridging SCI lesions. In a new article testing the efficacy of neural differentiated embryonic stem cells transplanted into a 9-day contusion cavity in the rat spinal cord, the cells survived and differentiated into astrocytes (19% ± 4%), oligodendrocytes (43% ± 6%), and neurons (8% ± 5%) (McDonald and others 1999). The cells migrated as far as 8 mm away from the rostral or caudal lesion edge. Hind-limb weight support and coordination (as assessed by BBB open field testing; Basso and others 1995) partially improved by 2 weeks with the transplantation of these cells. Gelfoam impregnated with cultured microglia placed into a thoracic dorsolateral cavity led to the presence of neurites (along with blood vessels and SCs) in the implant (Rabchevsky and Streit 1997). Injection of embryonic serotonergic neurons near a complete thoracic transection did not serve as a bridge. But the dense meshwork of graft-derived 5HT+ fibers in the ventral horn enabled recovery of bilateral alternating, rhythmic THE NEUROSCIENTIST Bridging Lesions in Cord Downloaded from nro.sagepub.com at PENNSYLVANIA STATE UNIV on September 17, 2016 locomotor-like activity, possibly resulting from activation of the central pattern generator (Feraboli-Lohnherr and others 1997). Prospectus The many new advances in basic neuroscience research are providing more and more opportunities to devise promising therapeutic strategies to improve outcome after SCI. The numerous causes of restricted axonal regeneration after injury in the CNS will require a multifaceted approach to advance successful treatment. Much more needs to be learned about mechanisms that protect spinal cord tissue after injury; that govern appropriate presentation, dosage, transport, and signaling of neurotrophic molecules; that diminish the influence of inhibitory molecules; that differentiate between destructive and beneficial effects of macrophages/microglia; that reveal effective ways of genetically modifying cells for transplantation; that improve the efficacy of biomaterials; that instruct in the use of guidance molecules; and that indicate the most appropriate choice for cellular bridges. It will be a challenging but exciting journey for the scientist and an inspiring and eventually successful one for those in wheelchairs. References Aguayo AJ. 1985. Axonal regeneration from injured neurons in the adult mammalian central nervous system. In: Cotman CW, editor. 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