Schwann Cell Grafts Research Articles

Chapter 18 Transplants and neurotrophic factors increase regeneration and recovery of function after spinal cord injury

Earlier studies suggested that while after spinal cord lesions and transplants at birth, the transplants serve both as a bridge and as a relay to restore supraspinal input caudal to the injury (Bregman, 1994), after injury in the adult the spinal cord transplants serve as a relay, but not as a bridge. We show here, that after complete spinal cord transection in adult rats, delayed spinal cord transplants and exogenous neurotrophic factors, the transplants can also serve as a bridge to restore supraspinal input (Fig. 9). We demonstrate here that when the delivery of transplants and neurotrophins are delayed until 2 weeks after spinal cord transection, the amount of axonal growth and the amount of recovery of function are dramatically increased. Under these conditions, both supraspinal and propriospinal projections to the host spinal cord caudal to the transection are reestablished. The growth of supraspinal axons across the transplant and back into the host spinal cord caudal to the lesion was dependent upon the presence of exogenous neurotrophic support. Without the neurotrophins, only propriospinal axons were able to re-establish connections across the transplant. Studies using peripheral nerve or Schwann cell grafts have shown that some anatomical connectivity can be restored across the injury site, particularly under the influence of neurotrophins (Xu et al., 1995a,b; Cheng et al., 1996; Ye and Houle, 1997). Without neurotrophin treatment, brainstem axons do not enter [figure: see text] the graft (Xu et al., 1995a,b; Cheng et al., 1996; Ye and Houle, 1997). Similarly, cells genetically modified to secrete neurotrophins and transplanted into the spinal cord influence the axonal growth of specific populations of spinally projecting neurons (Tuszynski et al., 1996, 1997; Grill et al., 1997; Blesch and Tuszynski, 1997). Taken together, these studies support a role for neurotrophic factors in the repair of the mature CNS. The regrowth of supraspinal and propriospinal input across the transection site was associated with consistent improvements in hindlimb locomotor function. Animals performed alternating and reciprocal hindlimb stepping with plantar foot contact to the treadmill or stair during ascension. Furthermore, they acquired hindlimb weight support and demonstrated appropriate postural control for balance and equilibrium of all four limbs. After spinal cord injury in the adult, the circuitry underlying rhythmic alternating stepping movements is still present within the spinal cord caudal to the lesion, but is now devoid of supraspinal control. We show here that restoring even relatively small amounts of input allows supraspinal neurons to access the spinal cord circuitry. Removing the re-established supraspinal input after recovery (by retransection rostral to the transplant) abolished the recovery and abolished the serotonergic fibers within the transplant and spinal cord caudal to the transplant. This suggests that at least some of the recovery observed is due to re-establishing supraspinal input across the transplant, rather than a diffuse influence of the transplant on motor recovery. It is unlikely, however, that the greater recovery of function in animals that received delayed transplant and neurotrophins is due solely to the restoration of supraspinal input. Recent work by Ribotta et al. (2000) suggests that segmental plasticity within the spinal cord contributes to weight support and bilateral foot placement after spinal cord transection. This recovery of function occurs after transplants of fetal raphe cells into the adult spinal cord transected at T11. Recovery of function appears to require innervation of the L1-L2 segments with serotonergic fibers, and importantly, animals require external stimulation (tail pinch) to elicit the behavior. In the current study, animals with transection only did not develop stepping overground or on the treadmill without tail pinch, although the transplant and neurotrophin-treated groups did so without external stimuli. Therefore both reorganization of the segmental circuitry and partial restoration of supraspinal input presumably interact to yield the improvements in motor function observed. It is unlikely that the recovery of skilled forelimb movement observed can be mediated solely by reorganization of segmental spinal cord circuitry. We suggest that the restoration of supraspinal input contributes to the recovery observed. It is likely that after CNS injury, reorganization occurs both within the spinal cord and at supraspinal levels, and together contribute to the recovery of automatic and skilled forelimb function and of locomotion. In summary, the therapeutic intervention of tissue transplantation and exogenous neurotrophin support leads to improvements in supraspinal and propriospinal input across the transplant into the host caudal cord and a concomitant improvement in locomotor function. Paradoxically, delaying these interventions for several weeks after a spinal cord transection leads to dramatic improvements in recovery of function and a concomitant restoration of supraspinal input into the host caudal spinal cord. These findings suggest that opportunity for intervention after spinal cord injury may be far greater than originally envisioned, and that CNS neurons with long-standing injuries may be able to re-initiate growth leading to improvement in motor function.

Optic nerve regeneration within artificial Schwann cell graft in the adult rat

We investigate whether an artificial graft made by cultured Schwann cell, extracellular matrix (ECM) and trophic factors can provide the environment for the regeneration of retinal ganglion cell (RGC) axons in adult rats. Six kinds of artificial grafts were used: ECM (control); ECM and Schwann cells; ECM, Schwann cells and either nerve growth factor, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4); ECM, Schwann cells, BDNF and NT-4, combined with intravitreal injection of BDNF. The grafts were transplanted onto the transected optic nerve. RGC regeneration was evaluated by diI retrograde labeling, immunohistochemistry, and electron microscopy at 3 weeks post-operation. The degree of diI labeled RGC was approximately 2% for ECM alone, and 10% for ECM and Schwann cells (p < 0.01). The labeling increased to approximately 20% by administration of neurotrophins. The addition of intravitreous BDNF injection resulted in highest labeling percentage of 30%. Immunohistochemical study showed that axons were association with GAP-43 and cell adhesion molecules. Neurotrophin receptors (Trk-A and Trk-B) were detected in nerve fibers both in the retina and in the graft. Remyelination was seen by electron microscopic observation. These results demonstrate that the regeneration of RGC axons is induced with the use of cultured Schwann cells and ECM as promoting factors for regrowth. The degree of regeneration was significantly increased by neurotrophins in the grafts and in the vitreous.

Conduction of impulses by axons regenerated in a Schwann cell graft in the transected adult rat thoracic spinal cord.

Central nervous system axons regenerate into a Schwann cell implant placed in the transected thoracic spinal cord of an adult rat. The present study was designed to test whether these regenerated axons are capable of conducting action potentials. Following the transection and removal of a 4- to 5-mm segment of the thoracic spinal cord (T8-T9), a polymer guidance channel filled with a mixture of adult rat Schwann cells and Matrigel was grafted into a 4- to 5-mm-long gap in the transected thoracic spinal cord. The two cut ends of the spinal cord were eased into the guidance channel openings. Transected control animals received a channel containing Matrigel only. Three months after implantation, electrophysiological studies were performed. Tungsten microelectrodes were used for monopolar stimulation of regenerated axons within the Schwann cell graft. Glass microelectrodes were used to record responses in the spinal cord rostral to the stimulation site. Evoked responses to electrical stimulation of the axon cable were found in two out of nine Schwann cell-grafted animals. These responses had approximate latencies in the range of those of myelinated axons. No responses were seen in any of the Matrigel-grafted animals. Histological analysis revealed that the two cases that showed evoked potentials had the largest number of myelinated axons present in the cable. This study demonstrates that axons regenerating through Schwann cell grafts in the complete transected spinal cord can produce measurable evoked responses following electrical stimulation.

Neurotrophic factors, cellular bridges and gene therapy for spinal cord injury.

Injury to the adult mammalian spinal cord results in extensive axonal degeneration, variable amounts of neuronal loss, and often severe functional deficits. Restoration of controlled function depends on regeneration of these axons through an injury site and the formation of functional synaptic connections. One strategy that has emerged for promoting axonal regeneration after spinal cord injury is the implantation of autologous Schwann cells into sites of spinal cord injury to support and guide axonal growth. Further, more recent experiments have shown that neurotrophic factors can also promote axonal growth, and, when combined with Schwann cell grafts, can further amplify axonal extension after injury. Continued preclinical development of these approaches to neural repair may ultimately generate strategies that could be tested in human injury.

Axonal regeneration into Schwann cell grafts within resorbable poly( α-hydroxyacid) guidance channels in the adult rat spinal cord

Axonal growth and myelination in a SC graft contained in a resorbable tubular scaffold made of poly( d, l-lactic acid) (PLA 50) or high molecular weight poly( l-lactic acid) mixed with 10% poly( l-lactic acid) oligomers (PLA 100/10) were studied for up to 4 months after implantation in the completely transected adult rat thoracic spinal cord. The PLA 50 tubes collapsed soon after implantation and, consequently, compressed the graft inside, leading to only occasional thin cables with SCs and a low number of myelinated axons: 17±6 at 1 and 158±11 at 2 months post-grafting. The cable contained 32±23 blood vessels at 2 weeks, 55±33 at 1 month and 46±30 at 2 months after implantation. PLA 100/10 tubes, on the other hand, were found to break up into large pieces, which compressed and sometimes protruded into the tissue cable inside. At all time points studied, however, cables contained SCs and were well vascularized with 414±47 blood vessels at 2 weeks, 437±139 at 1, 609±134 at 2 and 396±95 at 4 months post-grafting. The number of myelinated axons was 712±509 at 1 month, 1819±837 at 2 months and 609±132 at 4 months post implantation. These results demonstrated that fiber growth and myelination into a SC graft contained in a resorbable PLA 100/10 tube increases over the first 2 months post-implantation but decreases thereafter. Changes in geometry of both types of polymer tubes were detrimental to axonal regeneration. Future research should explore the use of polymers that better retain the appropriate mechanical, geometrical and permeability properties over time.

Inhibitory Proteoglycan Immunoreactivity Is Higher at the Caudal Than the Rostral Schwann Cell Graft-Transected Spinal Cord Interface

To begin to evaluate the influence that proteoglycans may have on the success of Schwann cell (SC) transplants to induce axonal regrowth across a complete transection lesion and beyond, we determined the pattern of expression of inhibitory chondroitin sulfate proteoglycans (CSPGs) 3 weeks after transplantation into completely transected adult rat thoracic spinal cord. Using immunohistochemistry, we observed that: (1) CSPGs recognized by CS-56 antibody are present on astrocytes, fibroblasts, and SCs in the distal graft, and at lesion and cystic cavity borders; (2) CS-56 immunoreactivity (IR) is greater at the caudal SC graft-host cord interface than the rostral interface; (3) phosphacan-IR, also greater at the caudal interface, is associated with astrocytes, fibroblasts, as yet unidentified cells, and extracellular matrix; (4) neurocan-IR is present on astrocytes and as yet unidentified cells in grey and white matter; and (5) NG2-IR is associated with matrix near SC grafts, unidentified cells mainly in white matter, and lesion borders and cysts. Neither oligodendrocytes nor activated macrophages/microglia were immunostained. In sum, the CSPGs studied are increased at 3 weeks, especially at the caudal SC graft–cord interface, possibly contributing to an inhibitory molecular barrier that precludes regrowing descending axons from entering the caudal host cord.

Neurotrophins BDNF and NT‐3 promote axonal re‐entry into the distal host spinal cord through Schwann cell‐seeded mini‐channels

AbstractTo promote axonal regeneration in the injured adult spinal cord, a two‐phase repair strategy was employed to (i) bridge a spinal cord hemilesion cavity with a grafted Schwann cell (SC)‐seeded mini‐channel, and (ii) promote axonal re‐entry into the distal cord by infusing two neurotrophins, BDNF and/or NT‐3, directly into the distal cord parenchyma. Here we report that infusion of two neurotrophins, delivered alone or in combination, effectively promotes axonal outgrowth from SC‐seeded mini‐channels into the distal host spinal cord. When an anterogradely transported marker, PHA‐L or BDA, was injected into the spinal cord 3 mm rostral to the graft, a large number of axons was observed to regenerate from the SC graft into the distal cord in neurotrophin‐treated groups. A subpopulation of these axons was found to grow up to 6 mm within the distal spinal cord. These axons, which were confined mainly within the grey matter, arborized and formed structures which resemble terminal boutons. In channels containing no SCs, the infusion of neurotrophins did not promote axonal ingrowth from the proximal cord stump. In cases which received SC grafts but no neurotrophin infusion, axonal re‐entry into the distal cord was limited. Thus, the present study demonstrates that regenerating axons not only cross a lesion site when a permissive cellular bridge is provided but also penetrate into the distal host spinal cord and elongate for a distance of several cord segments after the infusion of two neurotrophins. The latter event is prerequisite for establishment of appropriate connections between regenerating axons and target neurons and thus, functional recovery.

Neurotrophins BDNF and NT-3 promote axonal re-entry into the distal host spinal cord through Schwann cell-seeded mini-channels

To promote axonal regeneration in the injured adult spinal cord, a two-phase repair strategy was employed to (i) bridge a spinal cord hemilesion cavity with a grafted Schwann cell (SC)-seeded mini-channel, and (ii) promote axonal re-entry into the distal cord by infusing two neurotrophins, BDNF and/or NT-3, directly into the distal cord parenchyma. Here we report that infusion of two neurotrophins, delivered alone or in combination, effectively promotes axonal outgrowth from SC-seeded mini-channels into the distal host spinal cord. When an anterogradely transported marker, PHA-L or BDA, was injected into the spinal cord 3 mm rostral to the graft, a large number of axons was observed to regenerate from the SC graft into the distal cord in neurotrophin-treated groups. A subpopulation of these axons was found to grow up to 6 mm within the distal spinal cord. These axons, which were confined mainly within the grey matter, arborized and formed structures which resemble terminal boutons. In channels containing no SCs, the infusion of neurotrophins did not promote axonal ingrowth from the proximal cord stump. In cases which received SC grafts but no neurotrophin infusion, axonal re-entry into the distal cord was limited. Thus, the present study demonstrates that regenerating axons not only cross a lesion site when a permissive cellular bridge is provided but also penetrate into the distal host spinal cord and elongate for a distance of several cord segments after the infusion of two neurotrophins. The latter event is prerequisite for establishment of appropriate connections between regenerating axons and target neurons and thus, functional recovery.

Spinal cord repair strategies: Schwann cells, neurotrophic factors, and biodegradable polymers

Injury to the adult mammalian spinal cord leads to permanent loss of controlled neurological function. Endogenous repair mechanisms fail to reestablish functional synoptic connections. Moreover, neurological outcome usually gets worse in time, due to neurodestructive processes inherent to the adult spinal cord. Surgical repair strategies need to focus on replacing damaged/lost nervous tissue, promoting axonal regeneration and reestablishing functional synaptic contacts. This review will discuss the current understanding of the potential beneficial role of Schwann cells, neurotrophic factors, biodegradable polymers or combinations thereof in spinal cord injury. Replacement, of injured spinal tissue with a Schwann cell graft promotes axonal regeneration and myelination. Neurotrophic factors initiate and/or enhance events that are crucial for functional recovery, such as cell survival and axonal regeneration. Biodegradable polymers can be used as a scaffold for cell implantation and/or as a drug delivery vehicle. The complex nature of spinal cord injury demands a combinatorial restorative approach. For the future, the challenge will be to combine individual growth-promoting properties such that neurological recovery in spinal cord injured humans can be achieved. Biomedical Reviews 1999; 10: 75-88.

Nerve growth factor-hypersecreting Schwann cell grafts augment and guide spinal cord axonal growth and remyelinate central nervous system axons in a phenotypically appropriate manner that correlates with expression of L1.

Schwann cells contribute to efficient axonal regeneration after peripheral nerve injury and, when grafted to the central nervous system (CNS), also support a modest degree of central axonal regeneration. This study examined (1) whether Schwann cells grafted to the CNS exhibit normal patterns of differentiation and association with spinal axons and what signals putatively modulate these interactions, and (2) whether Schwann cells overexpressing neurotrophic factors enhance axonal regeneration. Thus, primary Schwann cells were transduced to hypersecrete human nerve growth factor (NGF) and were grafted to spinal cord injury sites in adult rats. Comparisons were made to nontransfected Schwann cells. From 3 days to 6 months later, grafted Schwann cells exhibited a phenotypic and temporal course of differentiation that matched patterns normally observed after peripheral nerve injury. Schwann cells spontaneously aligned into regular spatial arrays within the cord, appropriately remyelinated coerulospinal axons that regenerated into grafts, and appropriately ensheathed but did not myelinate sensory axons extending into grafts. Coordinate expression of the cell adhesion molecule L1 on Schwann cells and axons correlated with establishment of appropriate patterns of axon-Schwann cell ensheathment. Transduction of Schwann cells to overexpress NGF robustly increased axonal growth but did not otherwise alter the nature of interactions with growing axons. These findings suggest that signals expressed on Schwann cells that modulate peripheral axonal regeneration and myelination are also recognized in the CNS and that the modification of Schwann cells to overexpress growth factors significantly augments their capacity to support extensive axonal growth in models of CNS injury.

Regrowth of axons into the distal spinal cord through a Schwann-cell-seeded mini-channel implanted into hemisected adult rat spinal cord.

Schwann cells (SCs) have been shown to be a key element in promoting axonal regeneration after being grafted into the central nervous system (CNS). In the present study, SC-supported axonal regrowth was tested in an adult rat spinal cord implantation model. This model is characterized by a right spinal cord hemisection at the eighth thoracic segment, implantation of a SC-containing mini-channel and restoration of cerebrospinal fluid circulation by suturing the dura. We demonstrate that a tissue cable containing grafted SCs formed an effective bridge between the two stumps of the hemicord 1 month after transplantation. Approximately 10 000 myelinated and unmyelinated axons (1 : 9) per cable were found at its midpoint. In addition to propriospinal axons and axons of peripheral nervous system (PNS) origin, axons from as many as 19 brainstem regions also grew into the graft without additional treatments. Most significantly, some regenerating axons in the SC grafts were able to penetrate through the distal graft-host interface to re-enter the host environment, as demonstrated by anterograde axonal labelling. These axons coursed toward, and then entered the grey matter where terminal bouton-like structures were observed. In channels containing no SCs, limited axonal growth was seen within the graft and no axons penetrated the distal interface. These findings further support the notion that SCs are strong promotors of axonal regeneration and that the mini-channel model may be appropriate for further investigation of axonal re-entry, synaptic reconnection and functional recovery following spinal cord injury.

Long-Distance Axonal Regeneration in the Transected Adult Rat Spinal Cord Is Promoted by Olfactory Ensheathing Glia Transplants

The lack of axonal regeneration in the injured adult mammalian spinal cord leads to permanent functional impairment. To induce axonal regeneration in the transected adult rat spinal cord, we have used the axonal growth-promoting properties of adult olfactory bulb ensheathing glia (EG). Schwann cell (SC)-filled guidance channels were grafted to bridge both cord stumps, and suspensions of pure (98%) Hoechst-labeled EG were stereotaxically injected into the midline of both stumps, 1 mm from the edges of the channel. In EG-transplanted animals, numerous neurofilament-, GAP-43-, anti-calcitonin gene-related peptide (CGRP)-, and serotonin-immunoreactive fibers traversed the glial scars formed at both cord-graft interfaces. Supraspinal serotonergic axons crossed the transection gap through connective tissue bridges formed on the exterior of the channels, avoiding the channel interior. Strikingly, after crossing the distal glial scar, these fibers elongated in white and periaqueductal gray matter, reaching the farthest distance analyzed (1.5 cm). Tracer-labeled axons present in SC grafts were found to extend across the distal interface and up to 800 microm beyond in the distal cord. Long-distance regeneration (at least 2.5 cm) of injured ascending propriospinal axons was observed in the rostral spinal cord. Transplanted EG migrated longitudinally and laterally from the injection sites, reaching the farthest distance analyzed (1.5 cm). They moved through white matter tracts, gray matter, and glial scars, overcoming the inhibitory nature of the CNS environment, and invaded SC and connective tissue bridges and the dorsal and ventral roots adjacent to the transection site. Transplanted EG and regenerating axons were found in the same locations. Because EG seem to provide injured spinal axons with appropriate factors for long-distance elongation, these cells offer new possibilities for treatment of CNS conditions that require axonal regeneration.

Grafts of Genetically Modified Schwann Cells to the Spinal Cord: Survival, Axon Growth, and Myelination

Schwann cells naturally support axonal regeneration after injury in the peripheral nervous system, and have also shown a significant, albeit limited, ability to support axonal growth and remyelination after grafting to the central nervous system (CNS). It is possible that Schwann cell-induced axonal growth in the CNS could be substantially increased by genetic manipulation to secrete augmented amounts of neurotrophic factors. To test this hypothesis, cultured primary adult rat Schwann cells were genetically modified using retroviral vectors to produce and secrete high levels of human nerve growth factor (NGF). These cells were then grafted to the midthoracic spinal cords of adult rats. Findings were compared to animals that received grafts of nontransduced Schwann cells. Spinal cord lesions were not placed prior to grafting because the primary aim of this study was to examine features of grafted Schwann cell survival, growth, and effects on host axons. In vitro prior to grafting, Schwann cells secreted 1.5 ± 0.1 ng human NGF/ml/10 6 cells/day. Schwann cell transplants readily survived for 2 wk to 1 yr after in vivo placement. Some NGF-transduced grafts slowly increased in size over time compared to nontransduced grafts; the latter remained stable in size. NGF-transduced transplants were densely penetrated by primary sensory nociceptive axons originating from the dorsolateral fasciculus of the spinal cord, whereas control grafts showed significantly fewer penetrating sensory axons. Over time, Schwann cell grafts also became penetrated by TH- and DBH-labeled axons of putative coerulospinal origin, unlike control cell grafts. Ultrastructurally, axons in both graft types were extensively myelinated by Schwann cells. Grafted animals showed no changes in gross locomotor function. In vivo expression of the human NGF transgene was demonstrated for periods of at least 6 m. These findings demonstrate that primary adult Schwann cells 1) can be transduced to secrete augmented levels of neurotrophic factors, 2) survive grafting to the CNS for prolonged time periods, 3) elicit robust growth of host neurotrophin-responsive axons, 4) myelinate CNS axons, and 5) express the transgene for prolonged time periods in vivo. Some grafts slowly enlarge over time, a feature that may be attributable to the propensity of Schwann cells to immortalize after multiple passages. Transduced Schwann cells merit further study as tools for promoting CNS regeneration.

Grafts of genetically modified Schwann cells to the spinal cord: survival, axon growth, and myelination.

Schwann cells naturally support axonal regeneration after injury in the peripheral nervous system, and have also shown a significant, albeit limited, ability to support axonal growth and remyelination after grafting to the central nervous system (CNS). It is possible that Schwann cell-induced axonal growth in the CNS could be substantially increased by genetic manipulation to secrete augmented amounts of neurotrophic factors. To test this hypothesis, cultured primary adult rat Schwann cells were genetically modified using retroviral vectors to produce and secrete high levels of human nerve growth factor (NGF). These cells were then grafted to the midthoracic spinal cords of adult rats. Findings were compared to animals that received grafts of nontransduced Schwann cells. Spinal cord lesions were not placed prior to grafting because the primary aim of this study was to examine features of grafted Schwann cell survival, growth, and effects on host axons. In vitro prior to grafting, Schwann cells secreted 1.5+/-0.1 ng human NGF/ml/10(6) cells/day. Schwann cell transplants readily survived for 2 wk to 1 yr after in vivo placement. Some NGF-transduced grafts slowly increased in size over time compared to nontransduced grafts; the latter remained stable in size. NGF-transduced transplants were densely penetrated by primary sensory nociceptive axons originating from the dorsolateral fasciculus of the spinal cord, whereas control grafts showed significantly fewer penetrating sensory axons. Over time, Schwann cell grafts also became penetrated by TH- and DBH-labeled axons of putative coerulospinal origin, unlike control cell grafts. Ultrastructurally, axons in both graft types were extensively myelinated by Schwann cells. Grafted animals showed no changes in gross locomotor function. In vivo expression of the human NGF transgene was demonstrated for periods of at least 6 m. These findings demonstrate that primary adult Schwann cells 1) can be transduced to secrete augmented levels of neurotrophic factors, 2) survive grafting to the CNS for prolonged time periods, 3) elicit robust growth of host neurotrophin-responsive axons, 4) myelinate CNS axons, and 5) express the transgene for prolonged time periods in vivo. Some grafts slowly enlarge over time, a feature that may be attributable to the propensity of Schwann cells to immortalize after multiple passages. Transduced Schwann cells merit further study as tools for promoting CNS regeneration.

Schwann cells genetically modified to secrete human BDNF promote enhanced axonal regrowth across transected adult rat spinal cord.

The infusion of BDNF and NT-3 into Schwann cell (SC) grafts promotes regeneration of brainstem neurones into the grafts placed in adult rat spinal cord transected at T8 (Xu et al., 1995b). Here, we compared normal SCs with SCs genetically modified to secrete human BDNF, grafted as trails 5 mm long in the cord distal to a transection site and also deposited in the transection site, for their ability to stimulate supraspinal axonal regeneration beyond the injury. SCs were infected with the replication-deficient retroviral vector pL(hBDNF)RNL encoding the human preproBDNF cDNA. The amounts of BDNF secreted (as detected by ELISA) were 23 and 5 ng/24 h per 106 cells for infected and normal SCs, respectively. Biological activity of the secreted BDNF was confirmed by retinal ganglion cell bioassay. The adult rat spinal cord was transected at T8. The use of Hoechst prelabelled SCs demonstrated that trails were maintained for a month. In controls, no SCs were grafted. One month after grafting, axons were present in SC trails. More 5-HT-positive and some DbetaH-positive fibres were observed in the infected vs. normal SC trails. When Fast Blue was injected 5 mm below the transection site (at the end of the trail), as many as 135 retrogradely labelled neurones could be found in the brainstem, mostly in the reticular and raphe nuclei (normal SCs, up to 22, mostly in vestibular nuclei). Numerous neurones were labelled in the ventral hypothalamus (normal SCs, 0). Also, following Fast Blue injection, a mean of 138 labelled cells was present in dorsal root ganglia (normal SCs, 46) and spinal cord (39 vs. 32) rostral to the transection. No labelled spinal neurones rostral to the transection were seen when SCs were not transplanted. Thus, the transplantation of SCs secreting increased amounts of BDNF improved the regenerative response across a transection site in the thoracic cord. Moreover, the enhanced regeneration observed with infected SCs may be specific as the largest response was from neurones known to express trkB.

Influence of IN-1 antibody and acidic FGF-fibrin glue on the response of injured corticospinal tract axons to human Schwann cell grafts.

Two strategies have been shown by others to improve CST regeneration following thoracic spinal cord injury: 1) the administration of a monoclonal antibody, IN-1, raised against a myelin-associated, neurite growth inhibitory protein, and 2) the delivery of acidic fibroblast growth factor (aFGF) in fibrin glue in association with peripheral nerve grafts. Because autologous transplantation of human Schwann cells (SCs) is a potential strategy for CNS repair, we evaluated the ability of these two molecular agents to induce CST regeneration into human SC grafts placed to span a midthoracic spinal cord transection in the adult nude rat, a xenograft tolerant strain. IN-1 or control (HRP) antibodies were delivered to the injury/graft region by encapsulated hybridoma cells ("IN-1 ravioli") or daily infusion of hybridoma culture supernatant; aFGF-fibrin glue was placed in the same region in other animals. Anterograde tracing from the motor cortex using the dextran amine tracers, Fluororuby (FR) and biotinylated dextran amine (BDA), was performed. Thirty-five days after grafting, the CST response was evaluated qualitatively by looking for regenerated CST fibers in or beyond grafts and quantitatively by constructing camera lucida composites to determine the sprouting index (SI), the position of the maximum termination density (MTD) rostral to the GFAP-defined host/graft interface, and the longitudinal spread (LS) of bulbous end terminals. The latter two measures provided information about axonal die-back. In control animals (graft only), the CST did not enter the SC graft and underwent axonal die-back [SI = 1.4 +/- 0.1, MTD = 2.0 +/- 0.2, LS = 1.3 +/- 0.3, (n = 3)]. Results of IN-1 delivery from ravioli did not differ from controls, but injections of IN-1-containing supernatant resulted in a significant degree of sprouting but did not prevent axonal die-back [SI = 1.9 +/- 0.1, MTD = 1.5 +/- 0.2, LS = 1.1 +/- 0.1, (n = 7)] and traced fibers did not enter grafts. Acidic FGF dramatically reduced axonal die-back and caused sprouting [SI = 2.0 +/- 0.1 (n = 5), MTD = 0.5 +/- 0.04 (n = 6), LS = 0.4 +/- 0.1 (n = 6)]. Some traced fibers entered SC grafts and in 2/6 cases entered the distal interface. We conclude that 1) human SC grafts alone do not support the regeneration of injured CST fibers and do not prevent die-back, 2) grafts plus IN-1 antibody-containing supernatant support some sprouting but die-back continues, and 3) grafts plus aFGF-fibrin glue support regeneration of some fibers into the grafts and reduce die-back.

Olivocerebellar axon regeneration and target reinnervation following dissociated Schwann cell grafts in surgically injured cerebella of adult rats.

The ability of Schwann cells to induce the regeneration of severed olivocerebellar and Purkinje cell axons across an injury up to their deafferented targets was tested by transplanting freshly dissociated cells from newborn rat sciatic nerves into surgically lesioned adult cerebella. The grafted glial cells consistently filled the lesion gap and migrated into the host parenchyma. Transected olivocerebellar axons vigorously regenerated into the graft, where their growth pattern and direction followed the arrangement of Schwann cell bundles. Although some of these axons terminated within the transplant, many of them rejoined the cerebellar parenchyma beyond the lesion. Here, their fate depended on the territory encountered. No growth occurred in the white matter. Numerous fibres penetrated into the granular layer and formed terminal branches that remained confined within this layer. A few of them, however, regenerated up to the molecular layer and formed climbing fibres on Purkinje cell dendrites. By contrast, the growth of transected Purkinje cell axons into the grafts was very poor. These results underscore the different intrinsic responsiveness of Purkinje cell and olivocerebellar axons to the growth-promoting action of Schwann cells, and show that the development and outcome of the regenerative phenomena is strongly conditioned by the spatial organization and specific features of the environmental cues encountered by the outgrowing axons along the course they follow. However, Schwann cells effectively bridge the lesion gap, induce the regeneration of olivocerebellar axons, and direct their growth up to the deafferented host cortex, where some of them succeed in reinnervating their natural targets.

The ability of human Schwann cell grafts to promote regeneration in the transected nude rat spinal cord.

Advances in the purification and expansion of Schwann cells (SCs) from adult human peripheral nerve, together with biomaterials development, have made the construction of unique grafts with defined properties possible. We have utilized PAN/PVC guidance channels to form solid human SC grafts which can be transplanted either with or without the channel. We studied the ability of grafts placed with and without channels to support regeneration and to influence functional recovery; characteristics of the graft and host/graft interface were also compared. The T9-T10 spinal cord of nude rats was resected and a graft was placed across the gap; methylprednisolone was delivered acutely to decrease secondary injury. Channels minimized the immigration of connective tissue into grafts but contributed to some necrotic tissue loss, especially in the distal spinal cord. Grafts without channels contained more myelinated axons (x = 2129 +/- 785) vs (x = 1442 +/- 514) and were larger in cross-sectional area ( x = 1.53 +/- 0.24 mm2) vs (x = 0.95 +/- 0.86 mm2). The interfaces formed between the host spinal cord and the grafts placed without channels were highly interdigitated and resembled CNS-PNS transition zones; chondroitin sulfate proteoglycans was deposited there. Whereas several neuronal populations including propriospinal, sensory, motoneuronal, and brainstem neurons regenerated into human SC grafts, only propriospinal and sensory neurons were observed to reenter the host spinal cord. Using combinations of anterograde and retrograde tracers, we observed regeneration of propriospinal neurons up to 2.6 mm beyond grafts. We estimate that 1% of the fibers that enter grafts reenter the host spinal cord by 45 days after grafting. Following retrograde tracing from the distal spinal cord, more labeled neurons were unexpectedly found in the region of the dextran amine anterograde tracer injection site where a marked inflammatory reaction had occurred. Animals with bridging grafts obtained modestly higher scores during open field [(x = 8.2 +/- 0.35) vs (x = 6.8 +/- 0.42), P = 0.02] and inclined plane testing (x = 38.6 +/- 0. 542) vs (x = 36.3 +/- 0.53), P = 0.006] than animals with similar grafts in distally capped channels. In summary, this study showed that in the nude rat given methylprednisolone in combination with human SC grafts, some regenerative growth occurred beyond the graft and a modest improvement in function was observed.

Influence of IN‐1 antibody and acidic FGF‐fibrin glue on the response of injured corticospinal tract axons to human Schwann cell grafts

Influence of IN‐1 antibody and acidic FGF‐fibrin glue on the response of injured corticospinal tract axons to human Schwann cell grafts

Targeted Overexpression of the Neurite Growth-Associated Protein B-50/GAP-43 in Cerebellar Purkinje Cells Induces Sprouting after Axotomy But Not Axon Regeneration into Growth-Permissive Transplants

B-50/GAP-43 is a nervous tissue-specific protein, the expression of which is associated with axon growth and regeneration. Its overexpression in transgenic mice produces spontaneous axonal sprouting and enhances induced remodeling in several neuron populations (; ). We examined the capacity of this protein to increase the regenerative potential of injured adult central axons, by inducing targeted B-50/GAP-43 overexpression in Purkinje cells, which normally show poor regenerative capabilities. Thus, transgenic mice were produced in which B-50/GAP-43 overexpression was driven by the Purkinje cell-specific L7 promoter. Uninjured transgenic Purkinje cells displayed normal morphology, indicating that transgene expression does not modify the normal phenotype of these neurons. By contrast, after axotomy numerous transgenic Purkinje cells exhibited profuse sprouting along the axon and at its severed end. Nevertheless, despite these growth phenomena, which never occurred in wild-type mice, the severed transgenic axons were not able to regenerate, either spontaneously or into embryonic neural or Schwann cell grafts placed into the lesion site. Finally, although only a moderate Purkinje cell loss occurred in wild-type cerebella after axotomy, a considerable number of injured transgenic neurons degenerated, but they could be partially rescued by the different transplants placed into the lesion site. Thus, B-50/GAP-43 overexpression substantially modifies Purkinje cell response to axotomy, by inducing growth processes and decreasing their resistance to injury. However, the presence of this protein is not sufficient to enable these neurons to accomplish a full program of axon regeneration.