Peripheral Nerve Bridge Research Articles

Migrating Schwann cells direct axon regeneration within the peripheral nerve bridge.

Schwann cells within the peripheral nervous system possess a remarkable regenerative potential. Current research shows that peripheral nerve-associated Schwann cells possess the capacity to promote repair of multiple tissues including peripheral nerve gap bridging, skin wound healing, digit tip repair as well as tooth regeneration. One of the key features of the specialized repair Schwann cells is that they become highly motile. They not only migrate into the area of damaged tissue and become a key component of regenerating tissue but also secrete signaling molecules to attract macrophages, support neuronal survival, promote axonal regrowth, activate local mesenchymal stem cells, and interact with other cell types. Currently, the importance of migratory Schwann cells in tissue regeneration is most evident in the case of a peripheral nerve transection injury. Following nerve transection, Schwann cells from both proximal and distal nerve stumps migrate into the nerve bridge and form Schwann cell cords to guide axon regeneration. The formation of Schwann cell cords in the nerve bridge is key to successful peripheral nerve repair following transection injury. In this review, we first examine nerve bridge formation and the behavior of Schwann cell migration in the nerve bridge, and then discuss how migrating Schwann cells direct regenerating axons into the distal nerve. We also review the current understanding of signals that could activate Schwann cell migration and signals that Schwann cells utilize to direct axon regeneration. Understanding the molecular mechanism of Schwann cell migration could potentially offer new therapeutic strategies for peripheral nerve repair.

RETRACTED: Macrophage-Derived Slit3 Controls Cell Migration and Axon Pathfinding in the Peripheral Nerve Bridge

SummarySlit-Robo signaling has been characterized as a repulsive signal for precise axon pathfinding and cell migration during embryonic development. Here, we describe a role for Sox2 in the regulation of Robo1 in Schwann cells and for Slit3-Robo1 signaling in controlling axon guidance within the newly formed nerve bridge following peripheral nerve transection injury. In particular, we show that macrophages form the outermost layer of the nerve bridge and secrete high levels of Slit3, while migratory Schwann cells and fibroblasts inside the nerve bridge express the Robo1 receptor. In line with this pattern of Slit3 and Robo1 expression, we observed multiple axon regeneration and cell migration defects in the nerve bridge of Sox2-, Slit3-, and Robo1-mutant mice. Our findings have revealed important functions for macrophages in the peripheral nervous system, utilizing Slit3-Robo1 signaling to control correct peripheral nerve bridge formation and precise axon targeting to the distal nerve stump following injury.

Guiding the migration of grafted cells to promote axon regeneration.

A promising therapeutic strategy to promote the regeneration of injured axons in the adult central nervous system (CNS) is the transplantation of cells or tissues that can modify the local host environment and support the growth of regenerating axons. Growth-supportive cells that have been successfully used in experimental transplantation therapy of spinal cord injury (SCI) include Schwann cells, mesenchymal stromal cells, olfactory ensheathing cells, genetically modified fibroblasts, and neural stem/progenitor cells (Huang et al., 2010). Cells derived from the embryonic spinal cord and peripheral nerve grafts have been shown to promote the regeneration of injured axons, due largely to the presence of growth-supportive cells such as glial progenitors and Schwann cells, respectively (Cote et al., 2011; Haas and Fischer, 2013). These transplants generate a permissive environment for axon growth by secreting growth factors and forming an adhesive extracellular matrix to overcome the inhibitory environment of the injured tissue. However, the value of these transplants to promote axon regeneration is limited by the fact that most regenerating axons are trapped inside the permissive environment generated by the transplants, failing to grow out of the graft (Figure ​Figure1A,1A, ​,BB) (Haas and Fischer, 2013). While this strategy can be effective for building functional relays via graft-derived neurons (Haas and Fischer, 2014), this approach can not be generalized to other cell types. Therefore, a remaining challenge for therapeutic cell transplantation in CNS injury, in the context of long distance regeneration and connectivity, is to develop strategies to promote axonal growth beyond the graft into putative target areas to form functional synaptic connections. Currently the nature of the “graft trap” of regenerating axons is not fully understood. One possibility is that the regenerating axons stay inside the graft, which expresses much higher levels of attractive guidance factors, i.e., neurotrophic factors, and much lower levels of inhibitory/repulsive factors, i.e., chondroitin sulfate proteoglycan (CSPG), compared to the adjacent host tissue. Another possibility is that although adult CNS axons maintain their growth potential and can regenerate in an optimized environment, their intrinsic growth capability is much lower than axons of embryonic neurons, and thus not suitable for long-distance regeneration. Targeting these mechanisms, several strategies have recently been applied to overcome the “graft trap” in transplantation-based therapy of SCI. One strategy is to further modify host spinal tissue, making the host tissue less inhibitory and thus allowing some of the regenerating axons inside the graft to exit into the host tissue. As an example, Tom et al. (2009) showed that in an experimental model of grafting a peripheral nerve bridge at the site of the injured spinal cord, application of chondroitinase (Chase) at the distal graft/host interface to reduce CSPG-mediated inhibition promoted modest improvement in host-entry of regenerating axons, which would otherwise stop at the distal graft/host junction. Another strategy to promote axonal growth beyond the graft focuses on genetic modification of injured neurons to enhance their intrinsic growth potential using regeneration associated genes (Ma and Willis, 2015). For example, overexpressing the constitutively active form of the Rheb GTPase (downstream of the mTOR pathway) has been shown to enhance the intrinsic growth potential of adult neurons (Wu et al., 2015).

Convection enhanced drug delivery of BDNF through a microcannula in a rodent model to strengthen connectivity of a peripheral motor nerve bridge model to bypass spinal cord injury

Models employing peripheral nerve to bypass spinal cord injury (SCI), although highly promising, may benefit from improved nerve regeneration and motor bridge connectivity. Recent studies have demonstrated that neuronal growth factor-induced enhancement of endogenous neurorestoration may improve neuronal connectivity after severe neurologic injury, particularly if delivered intraparenchymally with zero-order kinetics. We sought to investigate the effect of convection-enhanced delivery of brain-derived neurotrophic factor (BDNF), a neuronal growth factor, on the connectivity of a peripheral motor-nerve bridge in a rodent model using electrophysiology and immunohistochemistry (IHC). Spinal cords of 29 female rats were hemisected at the L1 level. Ipsilateral T13 peripheral nerves were dissected from their muscular targets distally, while maintaining their connections with the spinal cord, and inserted caudal to the injury site to establish the nerve bridge. A microcannula attached to a six-week mini-osmotic pump was used to deliver either BDNF (n=12), saline (n=14), or fluorescein dye (n=3) directly into the spinal cord parenchyma between the site of nerve insertion and hemisection to a depth of 2mm into the area of the lateral motor pool. After four weeks, gastrocnemius muscle activation was assessed electromyographically in five animals from each group. Spinal cords were harvested and analyzed with IHC for cannula-associated injury, and nerve regeneration. Strength of motor bridge connection was illustrated by electrophysiology data. Intraspinal BDNF levels were measured using enzyme-linked immunosorbent assay. IHC revealed increased intraparenchymal BDNF concentration at the nerve bridge insertion site with evidence of minimal trauma from cannulation. BDNF infusion resulted in stronger connections between bridge nerves and spinal motor axons. Bridge nerve electrical stimulation in BDNF-treated rats evoked hind leg electromyogram responses of shorter latency and larger amplitudes than saline-infused controls. Thus, direct convection-assisted delivery provides reliable administration of potent growth factors directly into the spinal cord parenchyma. Delivery of BDNF at the peripheral nerve bridge site results in enhanced connectivity of the peripheral motor bridge in a rodent model of SCI.

Combining Peripheral Nerve Grafts and Chondroitinase Promotes Functional Axonal Regeneration in the Chronically Injured Spinal Cord

Because there currently is no treatment for spinal cord injury, most patients are living with long-standing injuries. Therefore, strategies aimed at promoting restoration of function to the chronically injured spinal cord have high therapeutic value. For successful regeneration, long-injured axons must overcome their poor intrinsic growth potential as well as the inhibitory environment of the glial scar established around the lesion site. Acutely injured axons that regenerate into growth-permissive peripheral nerve grafts (PNGs) reenter host tissue to mediate functional recovery if the distal graft-host interface is treated with chondroitinase ABC (ChABC) to cleave inhibitory chondroitin sulfate proteoglycans in the scar matrix. To determine whether a similar strategy is effective for a chronic injury, we combined grafting of a peripheral nerve into a highly relevant, chronic, cervical contusion site with ChABC treatment of the glial scar and glial cell line-derived neurotrophic factor (GDNF) stimulation of long-injured axons. We tested this combination in two grafting paradigms: (1) a peripheral nerve that was grafted to span a chronic injury site or (2) a PNG that bridged a chronic contusion site with a second, more distal injury site. Unlike GDNF-PBS treatment, GDNF-ChABC treatment facilitated axons to exit the PNG into host tissue and promoted some functional recovery. Electrical stimulation of axons in the peripheral nerve bridge induced c-Fos expression in host neurons, indicative of synaptic contact by regenerating fibers. Thus, our data demonstrate, for the first time, that administering ChABC to a distal graft interface allows for functional axonal regeneration by chronically injured neurons.

Gene therapy and transplantation in CNS repair: The visual system

Normal visual function in humans is compromised by a range of inherited and acquired degenerative conditions, many of which affect photoreceptors and/or retinal pigment epithelium. As a consequence the majority of experimental gene- and cell-based therapies are aimed at rescuing or replacing these cells. We provide a brief overview of these studies, but the major focus of this review is on the inner retina, in particular how gene therapy and transplantation can improve the viability and regenerative capacity of retinal ganglion cells (RGCs). Such studies are relevant to the development of new treatments for ocular conditions that cause RGC loss or dysfunction, for example glaucoma, diabetes, ischaemia, and various inflammatory and neurodegenerative diseases. However, RGCs and associated central visual pathways also serve as an excellent experimental model of the adult central nervous system (CNS) in which it is possible to study the molecular and cellular mechanisms associated with neuroprotection and axonal regeneration after neurotrauma. In this review we present the current state of knowledge pertaining to RGC responses to injury, neurotrophic and gene therapy strategies aimed at promoting RGC survival, and how best to promote the regeneration of RGC axons after optic nerve or optic tract injury. We also describe transplantation methods being used in attempts to replace lost RGCs or encourage the regrowth of RGC axons back into visual centres in the brain via peripheral nerve bridges. Cooperative approaches including novel combinations of transplantation, gene therapy and pharmacotherapy are discussed. Finally, we consider a number of caveats and future directions, such as problems associated with compensatory sprouting and the reformation of visuotopic maps, the need to develop efficient, regulatable viral vectors, and the need to develop different but sequential strategies that target the cell body and/or the growth cone at appropriate times during the repair process.

Combining an Autologous Peripheral Nervous System “Bridge” and Matrix Modification by Chondroitinase Allows Robust, Functional Regeneration beyond a Hemisection Lesion of the Adult Rat Spinal Cord

Chondroitinase-ABC (ChABC) was applied to a cervical level 5 (C5) dorsal quadrant aspiration cavity of the adult rat spinal cord to degrade the local accumulation of inhibitory chondroitin sulfate proteoglycans. The intent was to enhance the extension of regenerated axons from the distal end of a peripheral nerve (PN) graft back into the C5 spinal cord, having bypassed a hemisection lesion at C3. ChABC-treated rats showed (1) gradual improvement in the range of forelimb swing during locomotion, with some animals progressing to the point of raising their forelimb above the nose, (2) an enhanced ability to use the forelimb in a cylinder test, and (3) improvements in balance and weight bearing on a horizontal rope. Transection of the PN graft, which cuts through regenerated axons, greatly diminished these functional improvements. Axonal regrowth from the PN graft correlated well with the behavioral assessments. Thus, many more axons extended for much longer distances into the cord after ChABC treatment and bridge insertion compared with the control groups, in which axons regenerated into the PN graft but growth back into the spinal cord was extremely limited. These results demonstrate, for the first time, that modulation of extracellular matrix components after spinal cord injury promotes significant axonal regeneration beyond the distal end of a PN bridge back into the spinal cord and that regenerating axons can mediate the return of useful function of the affected limb.

Actions of Neurotrophic Factors and Their Signaling Pathways in Neuronal Survival and Axonal Regeneration

Adult axons in the mammalian central nervous system do not elicit spontaneous regeneration after injury, although many affected neurons have survived the neurotrauma. However, axonal regeneration does occur under certain conditions. These conditions include: (a) modification of regrowth environment, such as supply of peripheral nerve bridges and transplantation of Schwann cells or olfactory ensheathing glia to the injury site; (b) application of neurotrophic factors at the cell soma and axon tips; (c) blockade of growth-inhibitory molecules such as Nogo-A, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein; (d) prevention of chondroitin-sulfate-proteoglycans-related scar tissue formation at the injury site using chondroitinase ABC; and (e) elevation of intrinsic growth potential of injured neurons via increasing intracellular cyclic adenosine monophosphate level. A large body of evidence suggests that these conditions achieve enhanced neuronal survival and axonal regeneration through sometimes overlapping and sometimes distinct signal transduction mechanisms, depending on the targeted neuronal populations and intervention circumstances. This article reviews the available information on signal transduction pathways underlying neurotrophic-factor-mediated neuronal survival and neurite outgrowth/axonal regeneration. Better understanding of signaling transduction is important in helping us develop practical therapeutic approaches for encouraging neuronal survival and axonal regeneration after traumatic injury in clinical context.

Early events of peripheral nerve regeneration

Early regeneration of injured peripheral nerves involves a series of events that are important in the success of eventual reconnection. In many nerve injuries, such as transections with gaps, axons and Schwann cells (SCs) penetrate into new microenvironments de novo, not involving zones of Wallerian degeneration. We studied unexplored axon-SC interactions by sampling of newly forming connections through a silicone conduit across transected rat sciatic peripheral nerve gaps. Axon and SC participation in bridge formation was addressed by light microscopy, electron microscopy and by double-labeling immunohistochemistry,including confocal imaging, and several, less appreciated aspects of early regrowth were identified. There are limitations to early and widespread regeneration of axons and SCs into bridges initially formed from connective tissue and blood vessels.Regrowth is 'staggered' such that only a small percentage of parent axons sampled the early bridge. There is an intimate, almost invariable relationship between SCs and extension of axons, which challenges the concept that axons lead and SCs follow.'Naked' axons were infrequent and limited in scope. Axons did not seek out and adhere to vascular laminin but intimately followed laminin deposits associated with apposed SCs. Growth cones identified by labeling of beta III tubulin, PGP(9 x 5) and GAP(43)/B(50) were complex, implying a pause in their regrowth, and were most prominent at the proximal stump-regenerative bridge interface. There is surprising and substantial hostility to local regrowth of axons into newly forming peripheral nerve bridges.Early axon outgrowth, associated with apposed Schwann cell processes, is highly constrained even when not exposed to adjacent myelin and products of Wallerian degeneration.

Inhibition of caspases promotes long-term survival and reinnervation by axotomized spinal motoneurons of denervated muscle in newborn rats

We examined whether (1) a pan-caspase inhibitor, Boc-D-FMK, exerts long-term neuroprotective effects on spinal motoneurons (MNs) after root avulsion in neonatal rats and (2) whether the rescued spinal MNs regenerate their axons into a peripheral nerve (PN) graft and reinnervate a previously denervated target muscle. Eight weeks after root avulsion, 67% of spinal MNs remained in the Boc-D-FMK-treated group, whereas all MNs died in the sham control group. By 12 weeks postinjury, however, all Boc-D-FMK treated MNs died. In the regeneration experiment, a PN graft was implanted at different times after injury. The animals were allowed to survive for 4 weeks following the operation. Without caspase inhibition, MNs did not regenerate at any time point. In animals treated with Ac-DEVD-CHO, a caspase-3-specific inhibitor, and Boc-D-FMK, 44 and 62% of MNs, respectively, were found to regenerate their axons into a PN graft implanted immediately after root avulsion. When the PN graft was implanted 2 weeks after injury, however, MNs failed to regenerate following Ac-DEVD-CHO treatment, whereas 53% of MNs regenerated their axons into the graft after treatment with Boc-D-FMK. No regeneration was observed when a PN graft was implanted later than 2 weeks after injury. In the reinnervation study, injured MNs and the target biceps muscle were reconnected by a PN bridge implanted 2 weeks after root avulsion with administration of Boc-D-FMK. Eight weeks following the operation, 39% of MNs reinnervated the biceps muscle. Morphologically normal synapses and motor endplates were reformed in the muscle fibers. Collectively, these data provide evidence that injured neonatal motoneurons can survive and reinnervate peripheral muscle targets following inhibition of caspases.

Alpha-motoneurons of the injured cervical spinal cord of the adult rat can reinnervate the biceps brachii muscle by regenerating axons through peripheral nerve bridges: combined ultrastructural and retrograde axonal tracing study.

Following our previous studies related to brachial plexus injury and repair, the present experimentation was designed to examine the ultrastructural features of those motoneurons of the locally injured cervical spinal cord of adult rats that were seen to regenerate into peripheral nerve (PN) bridges and to reinnervate nearby skeletal muscles. Here, the peripheral connection of the PN bridge was made with the biceps brachii (BB) muscle. Three months postsurgery, the spinal motoneurons labelled by retrograde axonal transport of horseradish peroxidase (HRP), after its injection into the BB, were selected on thick sections, using light microscopy, for the presence of dark amorphous granules of the HRP reaction product. Serial ultrathin sections were then made from the selected material. For the 10 labelled neurons studied, we examined the synaptic boutons present on the membrane of the neuronal soma. For five of them, we could observe three of the six types of synaptic boutons described for the alpha-motoneurons of the cat (S-type with spherical vesicles, F-types with flattened vesicles, and C-type with subsynaptic cistern). The largest boutons (type C) are specific to alpha-motoneurons. In comparison to normal material, we noticed a decrease in the number of boutons and an increase in the number of glial processes. After a transient phase of trophic changes, the reinnervated BB muscles showed a return of their fibers to nearly normal diameters as well as evidence of fiber type grouping. Simultaneous staining with silver and cholinesterase also revealed the presence of new motor endplates frequently contacted by several motoneurons. The present study indicates that, after a local spinal injury, typical alpha-motoneurons can reinnervate a skeletal muscle by regenerating axons into the permissive microenvironment provided by a PN graft. These data offer prospects for clinical reconstruction of the brachial plexus after avulsion of one or several nerve roots.

Motoneurons of the adult marmoset can grow axons and reform motor endplates through a peripheral nerve bridge joining the locally injured cervical spinal cord to the denervated biceps brachii muscle

Motoneurons of the adult marmoset can grow axons and reform motor endplates through a peripheral nerve bridge joining the locally injured cervical spinal cord to the denervated biceps brachii muscle

Neutralizing antibodies against neurite growth inhibitor ni-35/250 do not promote regeneration of sensory axons in the adult rat spinal cord

Neutralization of the myelin-associated neurite growth inhibitors NI-35 and NI-250 by IN-1 antibodies can promote axonal regeneration of several types of central nervous neurons. Here, we investigated in adult rats whether IN-1 can promote regeneration of ascending sensory axons across a peripheral nerve bridge back into the spinal cord. IN-1 was administered by hybridoma cells injected in the cerebral cortex or thoracic cord, its presence confirmed in tissue sections and cerebrospinal fluid, and its effectiveness demonstrated in co-cultures of oligodendrocytes and sensory neurons. With a two week infusion of control vehicle into the dorsal spinal cord 3 mm rostral to the nerve graft, only 3±2% of the anterogradely labeled sensory fibers present at the rostral end of the nerve graft had grown up to 0.5 mm, but not farther into the spinal cord. A similar limited extent of regeneration was seen with IN-1 or with infusion of Dantrolene, an inhibitor of NI-35/250 activity in vitro. With infusion of nerve growth factor rostral to the nerve graft, 40% of the fibers at the rostral end of the graft were found at 0.5 mm, 34% at 1 mm, 24% at 2 mm and 14% at 3 mm (the infusion site) into the spinal cord. Treatment with IN-l antibodies did not enhance the growth-promoting effects of nerve growth factor. We suggest that the neurite growth inhibitors NI-35 or NI-250 do not play a major inhibitory role in the regeneration of the ascending sensory fibers across a nerve bridge and back into the spinal cord of the adult rat.

Intercostal Nerve Implants Transduced with an Adenoviral Vector Encoding Neurotrophin-3 Promote Regrowth of Injured Rat Corticospinal Tract Fibers and Improve Hindlimb Function

Following injury to central nervous tissues, damaged neurons are unable to regenerate their axons spontaneously. Implantation of peripheral nerves into the CNS, however, does result in axonal regeneration into these transplants and is one of the most powerful strategies to promote CNS regeneration. In the present study implantation of peripheral nerve bridges following dorsal hemisection is combined with ex vivo gene transfer with adenoviral vectors encoding neurotrophin-3 (Ad-NT-3) to examine whether this would stimulate regeneration of one of the long descending tracts of the spinal cord, the corticospinal tract (CST), into and beyond the peripheral nerve implant. We chose to use an adenoviral vector encoding NT-3 because CST axons are sensitive to this neurotrophin and Schwann cells in peripheral nerve implants do not express this neurotrophin. At 16 weeks postimplantation of Ad-NT-3-transduced intercostal nerves, approximately three- to fourfold more of the anterogradely traced corticospinal tract fibers had regrown their axons through gray matter below the lesion site when compared to control animals. Regrowth of CST fibers occurred over more than 8 mm distal to the lesion site. No regenerating CST fibers were, however, observed into the transduced peripheral implant. Animals with a peripheral nerve transduced with Ad-NT-3 also exhibited improved function of the hindlimbs when compared to control animals treated with an adenoviral vector encoding LacZ. Thus, transient overexpression of NT-3 in peripheral nerve tissue bridges is apparently sufficient to stimulate regrowth of CST fibers and to promote recovery of hindlimb function, but does not result in regeneration of CST fibers into such transplants. Taken together, combining an established neurotransplantation approach with viral vector-gene transfer promotes the regrowth of injured CST fibers through gray matter and improves the recovery of hindlimb function.

Motoneurons of the adult marmoset can grow axons and reform motor endplates through a peripheral nerve bridge joining the locally injured cervical spinal cord to the denervatedbiceps brachii muscle

Reconnection of the injured spinal cord (SC) of the marmoset with the denervated biceps brachii muscle (BB) was obtained by using a peripheral nerve (PN) bridge. In 13 adult males, a 45 mm segment of the peroneal nerve was removed: one end was implanted unilaterally into the cervical SC of the same animal (autograft), determining a local injury, although the other end was either directly inserted into the BB (Group A) or, alternatively, sutured to its transected motor nerve, the musculocutaneous nerve (Group B). From 2-4 months post-surgery, eight out of the 10 surviving animals responded by a contraction of the BB to electrical stimulations of the PN bridge. All ten were then processed for a morphological study. As documented by retrograde axonal tracing studies using horse radish peroxidase or Fast Blue (FB), a mean number of 314 (Group A) or 45 (Group B) spinal neurons, mainly located close to the site of injury and grafting, re-expressed a capacity to grow and extend axons into the PN bridge. Most of these regenerated axons were able to grow up to the BB and form or reform functional motor endplates. Many of the spinal neurons that were retrogradely labeled with FB simultaneously displayed immunoreactivity for choline acetyl-transferase and consequently were assumed to be motoneurons. Reinnervation and regeneration of the BB were documented by methods revealing axon terminals, endplates and myofibrillary ATPase activity. Our results indicate that motoneurons of the focally injured SC of a small-sized primate can, following the example of the adult rat, re-establish a lost motor function by extending new axons all the way through a PN bridge connected to a denervated skeletal muscle.

Adenoviral Vector-Mediated Expression of a Foreign Gene in Peripheral Nerve Tissue Bridges Implanted in the Injured Peripheral and Central Nervous System

Axons of the CNS do normally not regenerate after injury, in contrast to axons of the PNS. This is due to a different microenvironment at the site of the lesion as well as a particular intrinsic program of axonal regrowth. Although transplantation of peripheral nerve tissue bridges is perhaps the most successful approach to promoting regeneration in the CNS, ingrowth of CNS nerve fibers with such transplants is limited. Genetic modification of peripheral nerve bridges to overexpress outgrowth-promoting proteins should, in principle, improve the permissive properties of peripheral nerve transplants. The present study shows that pieces of peripheral intercostal nerve, subjected to ex vivo adenoviral vector-mediated gene transfer and implanted as nerve bridges in transected sciatic nerve, avulsed ventral root, hemi-sected spinal cord and intact brain, are capable of expressing a foreign gene. In vitro studies showed expression of the reporter gene LacZ up to 30 days in Schwann cells. After implantation, LacZ expression could be detected at 7 days postimplantation, but had virtually disappeared at 14 days. Schwann cells of the transduced nerve bridges retained the capacity of guiding regenerative peripheral and central nerve fiber ingrowth. Transduction of intercostal nerve pieces prior to implantation should, in principle, enable enhanced local production of neurotrophic factors within the transplant and has the potential to improve the regeneration of injured axons into the graft.

On CNS repair and protection strategies: novel approaches with implications for spinal cord injury and Parkinson's disease

In the adult mammalian central nervous system lost nerve cells are not replaced and there is no regeneration of injured axons in white matter. Together, these two facts mean that there are no spontaneous reparative mechanisms in operation. Instead, the adult central nervous system copes with the risks of injuries and diseases by protective encapsulation in bone, by a multitude of neuroprotective mechanisms, and finally by the fact that many important functions are represented by a much larger number of neurons than minimally needed. The long life expectancy of a human being nevertheless means that the risk that the central nervous system is affected by disease, injury or other forms of insults for which it cannot fully compensate is relatively high. Experimentally, two strategies are being pursued in order to develop ways of minimizing various forms of CNS damage, namely neuroprotective and reparative strategies. Here we present a possible reparative intervention applicable to spinal cord injury based on multiple white-to-gray matter peripheral nerve bridge grafts and work based on the specific role of Nurr1 for dopamine neuron development, suggesting that development of ligands to transcription factor might be a new inroad to neuroprotective treatments in Parkinson's disease.

Gait analysis of adult paraplegic rats after spinal cord repair.

This study presents a novel detailed method of analysis of rat gait and uses this method to demonstrate recovery of forward locomotion patterns in adult rats made paraplegic by surgical spinal cord transection and subjected to a novel strategy for spinal cord repair. Six normal rats were compared to five animals in which the cord was transected at T8-T9, and a 5-mm segment of the spinal cord removed, and to seven animals in which, following spinal cord transection and removal of a spinal cord segment, multiple intercostal peripheral nerve bridges were implanted, rerouting pathways from white to gray matter in both directions. The implanted area was filled with fibrin glue containing acidic fibroblast growth factor. Details of the repair strategy have been published (H. Cheng, Y. Cao, and L. Olson, 1996, Science 273: 510-513). Gait analysis was carried out 3 and 4 months after surgery and once in the normal animals. Animals were allowed to walk across a runway with a transparent floor. Each test consisted of five trials, and each trial was videorecorded from underneath. Using frame-by-frame playback, individual footprints were then recorded regarding location and order of limb use, as well as step quality (degree of weight bearing, etc.). These data allowed measuring runway transit time, five different measures of step numbers, all possible temporal patterns of limb use, stride length, and base of support. Transected controls remained paralyzed in the hindlimbs with only occasional reflex hindlimb movements without weight bearing. Animals subjected to the full repair procedure were significantly faster than the controls, used their hindlimbs for 25-30% of the movements, and regained several of the specific limb recruitment patterns used by normal rats. Taken together, the gait analysis data demonstrate remarkable recovery of coordinated gait in the repaired animals, which was significantly better than controls for all relevant parameters, while at the same time clearly inferior to normal rats for most of the examined parameters. We conclude that normal rats use a multitude of interchangeable step sequence patterns, and that our spinal cord repair strategy leads to recovery of some of these patterns following complete spinal cord transection. These data suggest functionally relevant neuronal communication across the lesion.

1220 Possibility of reconstruction of retino-geniculate pathway with a peripheral nerve bridge in adult cats

The Pdss2 gene product is needed for the isoprenylation of benzoquinone to generate coenzyme Q (CoQ). A fatal kidney disease occurs in mice that are homozygous for a missense mutation in Pdss2, which can be recapitulated in conditional Pdss2 knockouts targeted to glomerular podocytes. We now report that homozygous missense mutants also demonstrate significant neuromuscular deficits, as validated by behavioral and coordination assays, and these deficits are recapitulated in conditional Pdss2 knockouts targeted to dopaminergic neurons. Both conditional knockout and missense mutant mice demonstrate deficiencies in tyrosine hydroxylase-positive neurons in the substantia nigra, implicating a pathology similar to sporadic Parkinson's disease (PD).

Possibility of reconstruction of retino-geniculate pathway with a peripheral nerve bridge in adult cats

Cadmium (Cd) is a heavy metal naturally present in varying amounts in soils. While industrialization helped economic growth, heavy industrialization has left a legacy of metal-contaminated soils. The fate and transport of trace elements in soils are determined by the dynamic equilibrium between those present in various soil components and biogeochemical processes. Many plants accumulate relatively high levels of Cd, which could cause health issues in humans due to its relatively high half-life of approximately 20 years. Phosphate fertilizers are the source of Cd in edible plants. Zinc has been reported to alleviate or aggravate Cd stress based on interaction doses. To investigate these interactions, we chose sorghum, a high biomass-producing crop that grows in semiarid agroclimatic conditions and is well suited for the cultivation of Cd-contaminated substrates. We analyzed the interactive effects of zinc and phosphates on Cd-induced stress in sorghum seedlings.