John D. Houle - US grants

Intraspinal transplantation of fetal central nervous system (CNS) tissue or segments of peripheral nerve (PNS grafts) has demonstrated the ability of these implants to sustain axonal growth from CNS and PNS neurons following acute injury. The chronically injured spinal cord is a second lesion model with obvious clinical relevance, yet it is not known if neurons associated with longstanding CNS lesions retain the capacity for axonal growth or whether the encapsulating glial scar characteristics of a chronic lesion site impedes the course of regrowing axons. Since our previous studies have shown that fetal spinal cord (FSC) tissue survives transplantation into chronic lesion cavities, the exciting possibility that a neural tissue transplantation approach could provide an appropriate model system to study the regenerative potential of neurons in the chronically injured spinal cord can now be addressed. The proposed research will use quantitative morphometric techniques, immunocytochemistry and electron microscopy to test the hypothesis that implanted FSC tissue has the ability (1) to modify the encapsulating glial scar associated with a chronic lesion cavity and (2) to prevent the reformation of a partitioning glial scar following re-injury of the spinal cord (Specific Aim I). Experiments using immunocytochemical and neuroanatomical tracing techniques will map the pattern and extent of graft-host axonal integration to determine if chronically injured neurons retain the potential for axonal growth and if this potential can be enhanced by grafts of fetal CNS or PNS tissue (Specific Aim II) or by a conditioning lesion and removal of glial scar tissue prior to transplantation (Specific Aim III). The importance of this research is at least threefold: (1) it will establish whether the dense glial scarring that results after CNS injury can be modified to facilitate apposition and integration of graft and host tissues; (2) it will help define the substratum requirements necessary for the initiation and maintenance of axonal growth across an extensive lesion site; and (3) it will indicate the ability of neurons in the chronically injured spinal cord to regrow their axons and determine if this capacity for growth can be stimulated by transplantation paradigms involving either fetal CNS or PNS tissues. The experiments outlined in this proposal will define the potential for axonal regeneration and structural repair of the chronically injured spinal cord, thus forming the basis for future experiments related to the long term goal of re-establishing structural and functional continuity across a longstanding lesion in the spinal cord by the use of neural tissue transplantation techniques.

The long-term objectives of this study are to define effective interventions to the cascade of structural, biochemical and functional alterations that occur in myofibers following a severe spinal cord injury (SCI) in the rat. Experiments are designed to evaluate the temporal sequence of changes that occur in intact muscles and myofibers after SCI in adult rats and to determine how the presence of transplanted fetal spinal cord (FSC) tissue or an exercise regimen of the hindlimbs affects these temporal changes. The specific aims of this component are focused on testing of muscle strength and endurance (fatigability), immunocytochemical identification of myofiber type based on myosin heavy chain (MyHC) composition and determination of possible reversion to more immature MyHC types, identification of changes in expression of genes thought to regulate contractile protein content of myofibers and measurement of myofiber size. Comparison of the changes in the various parameters will establish correlations between effects on myofiber size and myofiber function, metabolic nature and possible genetic regulation. Information from these studies will provide vital knowledge about the potential use and efficient design of an interventive program(s) to alter effectively the extent of disuse atrophy and loss of mobility that occurs after SCI. Aim I of this proposal will establish the acute and chronic effects of SCI on the hindlimbs, to provide a baseline against which all interventive approaches will be tested. Experiments will establish the pattern of changes in muscle size and function in conjunction with changes in gene expression and MyHC isoforms within myofibers. For Aim II, using a similar transection lesion model, we will test the hypothesis that intraspinal transplants of FSC tissue can effectively disrupt the pattern of myofiber changes that occur after SCI. Animals that receive a transplant immediately after injury will be sacrificed at different stages of transplant development to demonstrate the short- and long-term effectiveness of the transplant to limit further muscle alterations. In a second experiment, a delay in transplantation after sa will test whether FSC tissue can reverse or stop progression of muscle changes that would occur without transplant intervention. In Aim III, experiments are designed to test the hypothesis that a long duration, low intensity exercise program for hindlimb muscles following spinal cord injury can intervene in the progressive change that these muscles undergo normally. Exercise will be initiated either immediately after injury or after a short delay of less than 30 days to establish what effects intervention at different post-injury intervals might have on muscle changes. Whether reflex activation during hindlimb exercise is involved in successful intervention will be tested in separate experiments. Finally, we will determine whether there are additive effects from combined treatment of exercise and FSC tissue transplantation on characteristic changes in muscle size, metabolic capacity and muscle function after SCI. One major obstacle for the successful rehabilitation of SCI patients is the restoration of muscle strength and endurance. These experiments have direct relevance to the design and implementation of treatment programs for dealing with this aspect of the spinal cord injured individual.

DESCRIPTION: (Verbatim from the Applicant's Abstract) The long term objectives of this study are to define effective interventions to the cascade of structural, biochemical and functional alterations that occur in myofibers following a severe spinal cord injury (SCI) and to determine if the maintenance of the mass and biochemical features of muscles by different therapies can expand functional capabilities following SCI. The present proposal focuses on defining the potential for activity dependent plasticity within the adult rat spinal cord and hind limb muscles and on determining how structural changes in response to an intervention can translate into functional repair. The Specific Aims address the hypothesis that motor-assisted cycling exercise of paralyzed hind limbs, alone or in combination with a fetal tissue transplant, leads to structural and functional changes in the spinal cord caudal to a complete transection lesion which in turn affect muscle structure and function. Aim I will use an established SCI and hind limb exercise model to address the potential for plasticity of motoneuron properties and spinal cord circuitry caudal to the lesion site. The importance of sensory input to the spinal cord for plasticity will be determined by deafferentation of the caudal spinal cord followed by exercise of the hind limbs. The second Aim will test whether the degree of sensory and motor stimulation that occurs during hind limb exercise can promote and/or guide regenerative axonal growth through a fetal spinal cord tissue transplant. Experiments of Aim III will define the interactive effects of exercise and transplantation on the size, metabolic capacity and contractile features of paralyzed hind limb muscles. Behavioral (overground locomotion) and physiological (H-reflex) measures of muscle activity will be combined with a molecular (cDNA arrays) analysis of changes in gene expression post-injury/intervention to provide a comprehensive view of the benefits of a combined therapy approach. We also will determine if beneficial effects of exercise and transplantation can be attained if interventions are delayed for 1 month after SCI. This study will begin to identify the morphological basis for physiological evidence of activity dependent plasticity within the spinal cord and hind limb muscles. Important information about reorganization within the spinal cord and in affected muscles after injury/intervention will be collected. These experiments have direct relevance to the design and implementation of treatment programs for spinal cord injured individuals.

ABSTRACT NOT PROVIDED

Core A: Administrative Core - John D. Houle, Ph.D., Director This Core is charged with the logistics of managing the demands placed on each of the other proposed Core facilities, with maintaining a database of all animals used in the Program Projects and in providing quality control and Project management. This will be accomplished by weekly meetings of the entire Program faculty, staff, postdoctoral fellows, graduate students and technical assistants; by monthly meetings of the Project Pis and Core Directors and by interaction with the External Advisory Panel. Data management is greatiy facilitated by an established database which logs information on Project number, individuals involved, protocol number, animal history and analyses to be performed. This provides an ongoing record of the progress of individual projects and Core effort devoted to each project. This Core will oversee the distribution of funds for travel and for publication costs for each of the Projects As is customary with our group, all important decisions are made by the Pis after discussion, but in the case of disagreement the final determination of Core use will be made by the Program PI, Dr. Houle. RELEVANCE (See instructions): It is the function of this Administration Core to oversee and manage the use of other Cores by the individual Projects animal models of spinal cord injury. Because all of the Projects will rely upon Core facilities and personnel to maintain uniformity of procedures among the Projects it is important to have centralized oversight of all Core activities.

Experimental spinal cord injury (SCI) models have helped define levels of structural and functional plasticity within the spinal cord and affected muscles. Exercise of hind limb muscles maintains the membrane properties of motoneurons affected by SCI, diminishes muscle atrophy and restores the H-reflex to near normal activity. Exercise-induced increase of neurotrophic factors (NTFs) in both spinal cord and muscle is correlated with neuromuscular plasticity. Intraspinal transplants of BDNF-producing fibroblasts indicate that therapeutic interventions also act at regions distant from the injury site. This study seeks to define structural and functional changes in spinal cord circuitry that occur after SCI and their modifications by exercise and/or transplantation to identify conditions that will lead to local and long distance anatomical and physiological recovery. Aim 1 will address the hypothesis that exercise and/or transplants promote plasticity within the spinal cord that can be harnessed to enhance functional repair. We will use an adult rat transection injury to define histological, cellular and molecular changes in spinal cord after removal of all descending motor input and after single or combined treatments. Aim 2 will determine if acute interventions affecting changes in spinal cord circuitry are effective when treatment is delayed. Aim 3 will examine the possibility that exercise will promote axonal regrowth into the spinal cord distal to an injury and that neurotrophic factor upregulation is involved. A peripheral nerve graft will be used to direct regenerating axons to a distant target and evaluation of functional recovery will be followed by tract tracing procedures to define an anatomical basis for improvement. In Aim 4 we will test the hypothesis that defects in neuromuscular connections post-SCI can be limited by exercise and/or transplantation. Anatomical and physiological measures of stability of neuromuscular junctions (NMJs) will be employed to assess the effectiveness of these 2 interventions. Differences of NMJ stability with short (2 week) and long (8 week) delay between SCI and treatment will demonstrate possible "windows of opportunity" for reversing injury-induced changes in neuromuscular organization. Overall, these experiments will provide fundamental information about cellular and molecular aspects of spinal cord and muscle reorganization in acute and chronic stages of SCI that will be instrumental in designing strategies for repair.

Description as provided by applicant: Axons provide long-range communication in the nervous system. Regeneration of axons in the injured spinal cord brings the potential to reconnect the caudal spinal cord to rostral brain stem and cerebrum and restore sensory and motor function. Significant advances have been made in the field of neural repair that hold promise for restoring function in spinal cord injury, particularly when interventions can be combined to target multiple repair mechanisms. The studies proposed in this project will explore the intracellular mechanisms underlying improved functional recovery in spinal cord injury interventions, focusing on novel interactions in the axonal compartment. We will test the hypothesis that the microenvironment of the injured spinal cord and interventions aimed at overcoming the inhibitory microenvironment can modulate intraaxonal signaling events that converge on the local protein synthesis machinery and this contributes to axonal growth and maturation. We will test this hypothesis with two specific aims that bring together expertise of the principal investigator in axonal growth and intra-axonal signaling with expertise from Project III (Houle) in regenerative therapies for spinal cord injury and Project II (Fischer) in progenitor cell therapies for spinal cord injury. The first aim of this project asks if exercise/training regiens that have been shown to improve recovery from spinal cord injury regulate axonal growth potential through post-transcriptional mechanisms. Both overall and intra-axonal translational control mechanisms will be tested using primary neuronal cultures and peripheral nerve grafting into the transected spinal cord. The second aim will ask if precursor cells used for spinal cord injury can directly modulate intra-axonal signaling to regulate the intrinsic growth potential and maturation of axons through axonal mRNA transport and translational control mechanisms. We will integrate these data with Project II to address mRNA translation in host axons as they interact with grafted precursor cells in SCI. The overall objective of these experiments is to uncover mechanisms underlying enhanced axonal growth and signaling that can be used to rationally fine tune future neural repair strategies. RELEVANCE: Axons have the ability to generate their own proteins needed for regeneration, but it is not clear if this occurs in the spinal cord or if neural repair strategies developed for spinal cord injury target this intra-axonal signaling mechanism. We will determine how growth supportive environments for spinal cord regeneration and training regimens that can improve functional recovery impact on axonal signal transduction and axon regrowth.

DESCRIPTION (provided by applicant): Peripheral nerve grafts (PNGs) provide an excellent substratum for axonal regrowth;they can direct regenerating axons towards a specific target and they facilitate electrophysiological experimentation to detect synaptic connectivity between regenerating axons and distal spinal cord neurons. A major impediment to this and all other transplantation approaches after spinal cord injury is the poor growth of axons out of the graft back into the host spinal cord. We have combined Chondroitinase (ChABC, to digest inhibitory chondroitin sulfate proteoglycan molecules) with PN grafting in rats and have anatomical and electrophysiological evidence for functional synapse formation by injured, regenerating axons in both acute and delayed (chronic injury) treatment paradigms. Recently we replicated this rat acute PNG approach in cats where we observed thousands of axons regenerating into the graft, a small percentage of which extended from the graft into the spinal cord distal to the injury, and spinal neurons synaptically activated (determined by c-Fos immunoreactivity) after electrical stimulation of the nerve graft. While we will continue to use rat models for expanding our treatment repertoire, the objective of this study is to focus on application of our treatment strategies to chronically injured cats as a necessary preclinical step before translation into human research. The cat model permits us to investigate issues related to the scaling up of a transplantation model, cats are easily trained to perform locomotor tasks, and recovery of function can be assessed by kinematic and electrophysiological measures. The biomechanics of locomotion are better defined in cats and cats have a hindlimb gait that is close to human than is the rat. The proposed work also will provide information about the ability to effectively treat glial scarring in a large animal, the ability to promote structural and functional regeneration in a large animal with a chronic injury and the potential for rehabilitation training to foster regeneration and functional recovery. There are 2 Specific Aims for this project. 1) We will identify the source and extent of axonal regeneration into a PNG after chronic injury and test whether these axons form functional connections across the lesion. 2) We will test whether the start time of physical rehabilitation affects outgrowth, integration and/or synaptic activity of regenerating axons. A combination of treatment strategies will be used, including transplantation, ChABC treatments and treadmill training to promote activity dependent plasticity. Structural repair will be assessed by anatomical tract tracing and immunocytochemical labeling;forelimb-hindlimb coordination will be assessed by kinematic and electromyogram (EMG) analysis;functional reconnection will be measured during electrophysiological stimulation of the graft and by c-fos expression in synaptically activated neurons. Surgical intervention after SCI usually is not an option until the patient is stabilized, thus the majority of individuals with SCI likely will be chronically injured before a treatment strategy for repair is initiated. Our work with chronically injured rats demonstrates the ability to promote long distance regeneration with formation of functionally active synapses distal to an injury. The proposed study will take advantage of the treatment approaches that have been (and are being) developed with chronically injured rats, but will apply them to a large animal model of SCI. This preclinical advancement is a crucial step towards translation to a clinical application. We propose a unique approach to address a very important aspect of SCI, i.e. chronic injury in a large animal model. Locomotor training of injured cats has been carried out by numerous labs, but not in a situation where axon regeneration is facilitated. This will be a novel application of neuroregeneration and neurorehabilitation techniques to increase our understanding of the potential for repair after SCI. PUBLIC HEALTH RELEVANCE: Different transplantation models have been used to demonstrate that under the right conditions neurons in spinal cord injured adult rats will regenerate their nerve processes (axons) into and through the transplant, forming a bridge across the lesion to restore lines of communication between the brain and spinal cord. Part of the success that has been demonstrated with these models involves additional treatment of the spinal cord tissue adjacent to the injury, to either increase the presence of growth promoting molecules or to decrease the presence of growth inhibitory molecules. An important issue to address concerns the time after injury when a treatment might be most effective and we have explored this question by delaying treatment for weeks to months after injury. We have demonstrated that chronically injured neurons in rats retain the capacity for regeneration for long (at least 12 months) post injury periods. This observation directly impacts the overwhelming number of spinal cord injured patients because of the perception that most surgical interventions should be delayed until the individual is stable and opportunities for spontaneous recovery have subsided. As a prelude to work with human patients we have pursued our peripheral nerve graft studies in a larger animal model, spinal cord injured cats. The purpose of this study was to determine if multiple surgical procedures could be performed on the cat spinal cord without causing significant pain or discomfort to the animal and if peripheral nerve grafts used to bridge the lesion in rats would be equally effective in cats, providing a channel for regenerating axons to form functional synaptic contacts with spinal cord neurons. We have data demonstrating the successful application of our combination of treatments to the adult cat spinal cord when carried out immediately after injury. The present proposal will extend this observation to the chronically injured cat model where treatment strategies will be delayed for several months, with the objective of advancing the use of a combination of treatment strategies towards translation into human application. The proposed work will provide valuable information about the ability to perform surgical reconstruction of spinal cord circuitry in a large animal, to determine if size might be a hindrance to successful regeneration. It also will provide an understanding of the potential to promote structural and functional recovery in a large animal with a longstanding (chronic) injury and of the ability to foster greater recovery through aggressive physical rehabilitation training. We feel that results of this study would have direct clinical relevance with the major risk being in not pursuing this line of investigation.

CORE C: Cell and Molecular Biology - Ying Jin, Ph.D. - PI This Core has existed for 10 years within the Department of Neurobiology and Anatomy to facilitate the molecular analysis of cells and tissues in the SCI site and within transplants. Most importantly, the core prepares cells for grafting experiments in the individual projects. All projects will use the Cell and Molecular Core. The facility isolates, expands and stores primary fibroblasts, neural stem cells and cell lines as required. We genetically modify cells by viral transduction or transfection and provide quality control for all cells and reagents used in the core. The staff of the Cell and Molecular Core provides assistance for experimental design and data analysis. The core has developed a computerized ordering system in which individual investigators & staff request control, labeled or genetically modified cells for transplantation. Our records show that over the past 5 years the core has provided over 3 x 1 0 ^ cells, including unmodified and modified fibroblasts, neuronal and glial restricted precursors (NRP and GRP), neuroepithelial cells (NEP) and cells from immortalized cell lines. The core maintains viruses and transduced cell stocks in long-term storage and serves as a database and repository for cDNA clones, vectors, viruses and cells. Efficient and cost-effective ordering of tissue culture and molecular biology supplies as well as large scale testing of new sera and improved growth media, enzymes, kits or equipment needed for molecular analysis (thermocyclers, Q-PCR) are key core functions. We are also continuously developing new improved plasmids and viruses and have recently added and adeno-associated virus vector (AAV) as an option to our program. This will allow us to specifically transfect graft neural stem cells with growth promoting and therapeutic factors to be delivered at or near the site of injury. RELEVANCE (See instructions): The complexity of the proposed projects using genetically modified cells for transplantation and delivery of therapeutic genes requires a central facility with technical expertise, equipment and ongoing training. The core provides uniform populations of cells with minimal variations and molecular analysis to allow for inore direct comparisons betiA/een different injury models and treatment strategies.

Experimental spinal cord injury (SCI) models have helped define levels of structural and functional plasticity within the spinal cord and affected muscles. Peripheral nerve grafts (PNGs) support the regeneration of acute and chronically injured neurons although growth beyond the graft, back into the spinal cord, is limited in terms of the number and length of axonal extension. Digestion of inhibitory proteoglycans with Chondroitinase is partially effective in increasing axonal outgrowth and there is evidence of functional synaptic connection between regenerated axons and spinal cord neurons. Exercise-induced increase of neurotrophic factors in thoracic and lumbar spinal cord is correlated with the restoration of motoneuron excitability (spinal reflexes) to near normal activity. Despite these successes there remain thousands of injured neurons that are not involved in reorganization and repair of the injured spinal cord. Our objectives are to address mechanistic questions related to the potential for exercise to provide trophic factor cues to potentially promote the regenerative response of injured neurons and/or to activate spinal networks to facilitate receptivity of regenerating axons. Aim 1 will address the hypothesis that exercise will promote regeneration of acute and/or chronically injured axons into a PNG, using an adult rat lower thoracic level transection injury, separate PNGs to support growth of descending vs. ascending axons and treadmill step training. Tract tracing methods will define the regenerative effort of motor and sensory neurons. Aim 2 will test whether exercise increases axonal outgrowth from a PNG and determine possible functional improve- ment related to regenerated axons by performing sensorimotor behavior, kinematic and electrophysiological analyses. In separate groups we will test whether activity-dependent plasticity is achieved with either/or an acute or delayed treatment approach. To advance the preclinical translation of our treatment strategy, results from SCI rats will be applied to a spinalized cat preparation to test whether exercise and transplantation promote regeneration-based functional recovery in a large animal model. Overall, these experiments will provide fundamental information about cellular and functional aspects of spinal cord reorganization in acute and chronic stages of SCI that will be instrumental in designing strategies for repair. RELEVANCE (See instructions): Here we will combine transplantation and exercise treatment strategies to determine if the regenerative effort of injured neurons can be enhanced in acute and chronically injured animals. This observation directiy impacts the overwhelming number of spinal cord injured patients because of the perception that most surgical interventions should be delayed until the individual is stable and opportunities for spontaneous recovery have subsided

CORE D - Histology and Image Analysis Core - Theresa Connors - PI The goal of the Histology and Image Analysis Core is to identify and quantify the anatomical substrates of functional outcomes tested in the Electrophysiological and Behavior Cores and in the individual projects. This core has evolved over 20+ years to insure standardized throughput of the large quantity of specimens generated by individual projects of the PPG. We have found that training staff from each project and providing a well organized and stocked facility where work can be performed with guidance from core staff is the most efficient way to utilize resources. The Histology and Image Analysis Core maintains a database of all tissue processing, histological and immunohistochemical (IHC) procedures used by PPG personnel. In addition, the core staff oversees training on and maintenance of our image acquisition and analysis systems. The core director and technician are skilled in working with a wide variety of antibodies and procedures used to identify neurotransmitters and neuromodulators, cytoskeleton components, extracellular matrix and adhesion molecules, growth factors, and non-neuronal cells as well as fiuorescence and DAB labeling techniques. Core personnel are also well versed in the operation and application of the image analysis microscopes and softiA/are. The core strives to maintain quality control of materials and procedures used in all PPG projects by: requiring training prior to using core resources, providing stocks of all routinely used buffers and solutions, regularly updating the database of antibodies and procedures and providing guidance in the planning and execution of projects in the core lab. Testing and implementation of new techniques is an essential Core function. This has increased the efficiency of the PPG Histology and Image Analysis Core, reducing start-up time for new procedures while also reducing duplication and waste. With centralized training, equipment, supplies and a database for methods, the Histology and Image Analysis Core has evolved as the model for other Core Labs in the Program Proiect RELEVANCE (See instructions): The core works to develop new methods for anatomical study of the effects of innovative therapies promoting repair, recovery and regeneration after SCI. Extensive experience of Pis and core personnel enable state of the art analysis crucial to the ongoing progress of this PPG. Project investigators with expertise in other fields can take advantage of anatomical analyses that may not be available in their labs.