Paul Reier - US grants

This research application focuses on repair of the injured spinal cord. Our experiments are based on the hypothesis that the cellular environment of the injured CNS limits the extent to which regrowth of damaged axons is initiated and maintained. Accordingly, the long-term objectives of this research program are to determine whether a more favorable milieu for regeneration can be established in the injured spinal cord and to gain a better understanding of the interaction between environmental factors and intrinsic neuronal growth properties. The aims of this proposal are: (1) To determine whether immediate or delayed transplants of embryonic spinal cord tissue can restore anatomical continuity in the injured spinal cords of adult rats. Anterograde and retrograge neuroanatomical tracing techniques and immunocytochemistry will be used to map the extent of axonal connectivity between host and donor tissues. The regenerative responses of injured fibers of specific long tracts ot embryonic CNS tissue will be determined by light and electron microscopy. We will also investigate whether axonal connections between host and donor tissues can mediate any recovery of locomotor and reflex function. (2) To determine whether the glial environment of The CNS inhibits elongation of axons from neurons that have considerable inherent growth potential. Ventral root-dorsal root anastomoses will be performed to test the ability of adult motoneurons to regenerate through the PNS-CNS interface at the dorsal root entry zone. Whether neurons with neogenic growth properties can traverse the glial limiting membrane of the adult CNS will also be determined. (3) To resolve whether the distance over which fiber elongation occurs in the injured spinal cord is a function of available postsynaptic sites either rostral or caudal to the level of injury. Specifically, we will use grafts of peripheral nerve segments to test whether CNS fibers elongating through such grafts can continue to advance for long distances after re-entering a spinal cord segment that had been extensively denervated by serial dorsal rhizotomies or completely isolated by transections above and below the graft insertion site. Together, these investigations should provide a foundation of basic information for developing strategies for promoting axonal regeneration and functional recovery in the injured spinal cord.

The object is to evaluate the extent to which implanted neural tissues will repair injury to the contused spinal cord. Implants will be either cellular suspensions or whole segments of fetal cord. Mild and severe contusion models will be evaluated using morphological, physiological, and behavioral parameters for both types of implant. The extent of repair or connectivity between implants and host will be investigated with electron microscopy and electrophysiology. These findings will be correlated to observed functional status.

This Program on Spinal Cord Injury represents a bending of interdisciplinary approaches to explore the potential for restoring function in the injured spinal cord. With this long-term objective in mind, various anatomical, behavioral, electrophysiological, neurophysiological, and microsurgical methods will be used to achieve the following immediate goals: (i) to examine the capacity of fetal CNS and peripheral nerve (PNS) grafts to mediate anatomical and functional repair in acute and chronic injuries, (ii) to develop models that will ultimately permit definitive correlative analyses of therapeutic strategies aimed at restoring sensory, motor, and/or autonomic function, (iii) to test new approaches that may permit in-depth studies of behavior and cellular neurophysiology, and (iv) to demonstrate fundamental events underlying functional recovery in the amphibian spinal cord. Accordingly, Project 1 will examine the ability of fetal CNS grafts to establish host- graft synaptic interactions in the chronically injured spinal cord, as well as the capacity of these grafts to prevent the death of certain spinal neurons following cord damage in the adult rodent: methods will also be developed for intraspinal transplantation into the adult cat in conjunction with our Core laboratory. Project 2 focuses on the problem of spasticity, as manifested in the cat, and seeks to establish approaches that will permit direct correlations between non-invasive physiological evaluations and electrophysiological recordings. Project 3 will test the efficacy of PNS grafts in restoring somatosensation and segmental reflex activity in the cat and primate. Project 4 will study the neurophysiology and synaptic organization of the cat sacrocaudal cord - a region which may serve as a novel model for studies of spinal cord plasticity and regeneration. Project 5 will explore sensory physiology and the ascending pathways that subserve cortical perception of respiration in various animal models, as well as in humans with spinal cord injuries. Collectively, these subproject will provide a comprehensive and interactive investigation of various aspects of spinal cord motor, sensory, and autonomic function that are of fundamental scientific and clinical interest.

New insights regarding the cellular biology associated with neurotrauma and neurodegenerative disease have led to several proposed therapeutic interventions directed at functional recovery after spinal cord injury (SCI). It is now more conceivable than before that some useful improvements could be obtained through a convergence of cellular, pharmacological, molecular and/or rehabilitative treatment modalities. However, before any innovative strategies can be judiciously advanced to the clinical setting, there ideally needs to be rigorous analysis of functional impact at both the behavioral and cellular levels in animal models of SCI that approximate the human condition from neuropathological and pathophysiological perspectives. Various features of SCI also should be considered since the efficacy of a potential treatment might vary according to different lesion conditions such as: the spinal level of injury, the type of trauma sustained, the distance of the lesion from neuronal pools in which functional improvement would be most desired, the type of recovery being sought, and the time after injury that a particular therapeutic protocol is instituted. It also would be of benefit to be able to anticipate how a treatment approach could interface with intrinsic repair processes (i.e., neuroplasticity). The three component subprojects of this Program, along with the two laboratory Cores described, will address these issues by exploring the effects of fetal neural tissue transplants in relation to loss of interlimb coordination, occurrence of spasticity, and deficits in respiratory- associated phrenic motoneuron activity after midthoracic, upper lumbar, and cervical spinal cord injuries, respectively. These studies will particularly emphasize spontaneous and graft-mediated functional changes in relation to clinically-relevant contusion/compression injuries and will entail a fusion of qualitative and quantitative neuroanatomical, neurophysiological, magnetic resonance imaging, and behavioral methods. The emphasis on fetal cell transplantation reflects an operational bias of this proposal -namely, that some form of cellular grafting strategy will ultimately constitute part of the overall therapeutic formula directed at functional improvement. At the preclinical level, fetal tissue grafting continues to provide a legitimate and compelling experimental tool whereby a better understanding can be obtained concerning the potential for engineering behavioral recovery after SCI. The coordination of these investigations via the Program Project construct also will provide a valuable template upon which to base future analyses of other viable therapeutic interventions complementary to cellular transplantation.

This subproject will test the feasibility and efficacy of gene therapy for promoting neuronal survival and regeneration in the injured adult rat spinal cord either alone or in combination with a cellular transplantation intervention. Recombinant adeno-associated virus (rAAV) constructs will be employed to determine whether: (i) the efficacy of in vivo gene delivery to neurons can vary relative to post-injury times and the probability of either retrograde cell death or atrophy; (ii) in vivo gene delivery can promote rescue of embryonic and adult neuronal populations susceptible to post-axotomy cell death; and (iii) an effective delivery and expression of transgenes can be used to enhance axonal outgrowth from intraspinal grafts of fetal raphe tissue. Thus, the central hypothesis to be addressed is that either ex vivo and/or in vivo gene delivery can foster the synthesis and release of neurotrophic actors that in turn can serve to promote sparing of neuronal populations and stimulate or enhance axonal regeneration /sprouting following spinal cord injury (SCI) or peripheral nerve damage. Using sciatic nerve lumbar neuron pools (L4-L6) as a model, the efficacy of gene delivery will be compared at acute and chronic post- injury periods in Aim I. The selected post-injury times will relate to different epochs associated with initial retrograde (e.g., chromatolytic) responses and subsequent induced atrophy. Likewise, other experiments will examine the efficacy of gene delivery in two other intrinsic neuronal populations, rubrospinal and dorsal nucleus of Clarke, with contrasting cell survivals after SCI. Aim II will determine whether a model population of neurons (i.e., motoneurons) can be rescued in both the adult spinal cord and grafts of fetal spinal cord (FSC) tissue by in vivo delivery of rAAVs expressing GDNF. The ability of transgene expression to rescue DNC neurons also will be examined. Lastly, Aim III will test the hypothesis that in vivo gene delivery, involving the expression of neurotrophins in conjunction with intraspinal grafts of fetal CNS tissue, can be used to augment host-graft connectivity by enhancing donor neuron-derived axonal sprouting and/or elongation. These experiments will investigate (i) whether the pattern of outgrowth exhibits any preferential disbursement relative to the distribution of transduced cells and (ii) to what extent the presence of NT-3 can promote growth of axons through a cellular milieu (e.g., astrogliotic scar) that can be inhibitory or non permissive to axonal regrowth. This subproject builds on a base of established expertise in SCI neurobiology/neuropathology, neurotransplantation, and AAV development to investigate fundamental issues related to gene transfer and repair of the damaged spinal cord.

[unreadable] DESCRIPTION (provided by applicant): Multiple forms of spontaneous respiratory neuroplasticity occur at spinal and brainstem levels in adult rats after lesions to the cervical spinal cord. The goal of this proposal is to enhance naturally occurring processes that can lead to partial improvement of phrenic motoneuron (PhMN) functions that were initially lost or modified after high cervical spinal cord injury (SCI) and to define underlying changes in the neural substrate. Current understanding of neuroplasticity in the phrenic motor system (PhMtrS) derives from studies of spinal hemilesions made above the phrenic nucleus (e.g., at C2). That injury abolishes descending inspiratory drive to ipsilateral PhMNs and renders the corresponding ipsilateral diaphragm (ipsiDIA) hemiparetic. We and others have recently described spontaneously emerging changes in PhMN function after C2 HMx - two of which are emphasized in the proposal. One entails a partial spontaneous recovery of ipsilateral diaphragm activity. The second involves a specific change in contralateral PhMN responses to elevated respiratory drive (hypercapnia). Aim 1 will test the hypothesis that spontaneous, partial recovery of ipsiPhMN function following a C2 HMx entails neural circuit remodeling via the recruitment of interneurons. Aim 2 will test the hypothesis that stimulation of axonal growth by blockade of the Nogo receptor (NgR), using the NgR antagonist, NEP1-40, will enhance spontaneously evolving ipsilateral PhMN functional recovery and possibly amplify the novel circuit to be defined in Aim 1, as well as contribute to modification of contralateral PhMN neuroplastic responses to respiratory challenge. The third Aim will test the hypothesis that introduction of a novel short propriospinal circuit via neural tissue transplantation can modify, alone or in combination with NEP1-40 delivery, ipsi- and contralateral PhMN neuroplastic responses following C2 HMx injury. We also will assess whether engraftment and therapeutic efficacy of FSC tissue can be complemented by NgR blockade. A multidisciplinary approach will be used involving transneuronal tracing, quantitative neurophysiological indices, and an innovative approach for obtaining repeated measures of breathing in freely-behaving rats. These aims identify with the long-range hypothesis that segmental gray matter repair and neural circuit remodeling provide an effective complement or alternative to long-tract regeneration via enhancement or modification of post-injury neuroplastic events. [unreadable] [unreadable] [unreadable]