Leif A. Havton - US grants

DESCRIPTION (provided by applicant): Patients with cauda equina and conus medullans lesions represent about twenty percent of all traumatic spinal cord injuries. They develop a permanently a reflexic bladder, urinary retention and a flaccid paraparesis or paraplegia. No effective treatments are available. Avulsions of spinal ventral roots cause an injury similar to what is encountered in some patients with cauda equina / conus medullaris injury. Previous studies in animals and in patients with acute brachial plexus injuries have shown that restitution of motor function is possible after ventral root avulsions by implanting the avulsed roots into the spinal cord. The long-term goal of this project is to achieve neural repair of lower urinary tract function after cauda equina / conus medullaris injuries. We will test three hypotheses: 1) Lumbosacral ventral root avulsions lead to a progressive neuronal death and characteristic patterns of neurotrophic factor receptor expression in preganglionic parasympathetic neurons and motoneurons; 2) Implantation of an avulsed lumbosacral ventral root into the spinal cord exerts neuroprotective effects upon injured spinal cord neurons; 3) Implantation of an avulsed lumbosacral ventral root into the spinal cord promotes functional reinnervation of the lower urinary tract. Morphological methods, including retrograde labeling techniques and quantitative light microscopic studies, immunocytochemistry, in situ hybridization and electron microscopy, will be used to investigate the neuroprotection of motoneurons and preganglionic parasympathetic neurons by implantation of avulsed ventral roots into the lumbosacral spinal cord. Urodynamic studies will be used to investigate the functional reinnervation of the lower urinary tract with simultaneous intravesical bladder recordings and electromyography of the external urethral sphincter. The proposed study will address basic mechanisms regarding biologic responses of lumbosacral preganglionic parasympathetic neurons and motoneurons after ventral root avulsion injury and repair. Furthermore, this work may have important clinical implications, possibly leading to a surgical intervention for patients with cauda equina / conus medullaris injuries.

Both humans and animals can regain the ability to stand or step after a complete spinal cord transection injury. The ability to execute these tasks depends upon specific training regimens, illustrating the importance of motor learning in the spinal cord. A thoracic spinal cord transection in both cats and rats leads to a persistent increase in the inhibitory capacity within the lumbar portion of the spinal cord, and step training returns the inhibitory capacity towards normal levels. However, the degree of plasticity and reorganization of inhibitory and excitatory synapses upon motoneurons, the final common pathway in motor control, is not known. The central hypothesis of this proposal is that complete spinal cord transection results in a selective proliferation of inhibitory synapses upon lumbar flexor and extensor motoneurons, while subsequent repetitive step training selectively decreases the number and capacity of inhibitory synapses within these motor pools. We will use a step-training paradigm and retrograde labeling techniques to study quantitatively the synaptology of soleus and tibialis anterior motoneurons in the electron microscope. We will also use electron microscopic immunogold techniques to study quantitatively the number and ratios of GABAergic and glycinergic terminals forming synaptic contacts with soleus and tibialis anterior motoneurons. The proposed studies will provide fundamental and critical data to assist in our understanding of cellular mechanisms of neural plasticity after spinal cord injury and locomotor training. The proposed studies can therefore contribute significantly in developing strategies designed to improve motor recovery after spinal cord injury.

DESCRIPTION (provided by applicant): A conus medullaris syndrome results from trauma to the sacral portion of the spinal cord and associated lumbosacral roots. Such injuries cause paralysis and sensory impairment of the lower extremities, pain, as well as bladder, bowel, and sexual dysfunctions. No successful treatments are presently available for patients with these injuries. We have developed a clinically relevant model for the study of conus medullaris/ cauda equina injury and repair in the rat. In this model, lumbosacral ventral roots are avulsed from the surface of the spinal cord and subsequently surgically implanted into the conus medullaris. During the initial research period, we demonstrated that this ventral root implantation strategy is neuroprotective, promotes axonal regeneration, and results in functional reinnervation of the lower urinary tract. The renewal of this project has three new aims: 1) to determine cell death mechanisms of autonomic and motor neurons after a lumbosacral ventral root avulsion (VRA) injury;2) to determine potential neuroprotective effects of minocycline and complement inhibition;3) determine whether minocycline and/or complement inhibition may augment functional reinnervation of the lower urinary tract after a lumbosacral VRA injury followed by acute and delayed surgical implantations of avulsed roots into the conus medullaris. We will use a combined therapeutic strategy approach to study neuronal death mechanisms, neuroprotection, and functional reinnervation of the lower urinary tract after lumbosacral VRA injury and repair. We will perform surgical root avulsions and implantations, pharmacological treatment, immunohistochemistry, electron microscopy, retrograde tracing techniques, stereology, and functional urodynamic studies of the lower urinary tract. Our proposed studies will provide a better understanding of death mechanisms for autonomic and motor neuron death in the spinal cord after proximal cauda equina injuries. We will also investigate new pharmacological strategies to protect these neurons against motor root injury-induced cell death in combination with root implantation to augment the function of the lower urinary tract. We believe that our proposal has translational research potential for the development of new treatments for patients with conus medullaris/cauda equina injuries.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The objective of this subproject is to evaluate a new surgical strategy for the repair of lumbosacral nerve roots after cauda equina forms of spinal cord injury. The findings will guide the planning of a phase 1-2 clinical trial in humans with cauda equina injury.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Use of GDNF-Releasing Nanofiber Nerve Guide Conduits for the Repair of Conus Medullaris/Cauda Equina Injury in the Non-Human Primate. All animals will undergo Xray and MRI imaging, behavioral recordings, treadmill training, a surgery to cause a lumosacral nerve root avulsion as well as post-surgical tracer and anatomical marker injections. After the lumbosacral nerve root avulsion, four of the five groups will also undergo a surgical therapy to hopefully ameliorate the expected hindlimb paresis caused by the surgery.

This is a collaboration between experts at UCSD, UCLA, UCSF, UC Davis, UCI and the Ecole Polytechnique Federale de Lausanne (EPFL) to examine plasticity and regeneration in the non-human primate spinal cord. Our goal is to enhance knowledge and translational relevance of research on spinal cord injury (SCI). This project will map the motor cortex ?connectome? to better understand the primate motor network in both the intact state and after recovery from SCI, and will focus on promoting therapeutic growth of the most important motor control system in primates, the corticospinal projection. Aim 1: The Intact Primate Corticospinal Connectome Aim 1 will map the intact connectome for motor control of the hand using new generation, highly specific and highly sensitive viral tools. These findings will be correlated with motor cortex recordings and forelimb EMG activity in awake, behaving subjects. These tools will allow an unprecedented understanding of hand motor control in the primate, thereby revealing novel mechanisms of motor control and identifying new targets for therapy. Aim 2: The Lesioned Primate Corticospinal Connectome How does injury affect the corticospinal connectome? How does the corticospinal system adapt to injury and alter its set of outputs, and how does this influence functional recovery? We will use the elegant and novel tools of Aim 1 to map the injured, reorganized corticospinal connectome, motor cortex dynamics, and forelimb EMG after C7 hemisection lesions. Aim 3: Spinalized Neural Stem Cell Grafts to Enhance Corticospinal Repair Work in Aim 3 will build on recent progress in neural stem cell technology. We will use ?spinalized? neural stem cells to augment growth of the injured corticospinal tract and form trans- lesion relays that enhance recovery of forelimb function after SCI. Further, we will compare the connectome of the corticospinal system after therapeutic stimulation to its intact and lesioned state. This work will reveal how new corticospinal growth after injury impacts the projections and connections of this vitally important motor system in humans. 1

PROJECT SUMMARY/ABSTRACT Organ function may be modulated by stimulating peripheral activity and targeting, for instance, peripheral nerves and autonomic ganglia. However, the detailed functional and anatomical organization is not well understood for autonomic nerves, ganglia and organs involved with visceral functions. Significant knowledge gaps exist with regards to the neural control of many organ functions, including those of the heart, respiratory system, lower urinary tract, and gastro-intestinal system. An improved understanding of the various components of neural circuitries and innervation of peripheral organs is needed in order to provide improved modeling and design of devices to allow for optimal modulation of select organ functions. Transmission electron microscopy (TEM) allows for the identification of the fine structure of neural tissues to characterize, for instance, axonal composition and myelination, organization of neural circuitry in autonomic ganglia, and innervation of autonomic targets. The proposed studies will aim at establishing a TEM service for investigators supported by the Stimulating Peripheral Activity to Relieve Conditions (SPARC) consortium. A total of 6 SPARC-funded projects, which include 7 SPARC-affiliated investigator laboratories, with defined needs for TEM studies have been identified. The projects include studies on the autonomic control of the heart, brain-gut interactions, hypoglossal nerve-influences on respiration, lower urinary tract function, neuro-endocrine regulation of glucose, and modeling of vagal nerve functions. TEM studies will include detailed characterization of myelinated and unmyelinated axons in visceral and somatic nerves as well as organization of autonomic ganglia, and potential synaptic regulation of endocrine functions. Studies will be performed using rodent, large mammal, and human tissues to allow for interspecies comparisons. Sex as a biological variable will also be included in the studies. The TEM service will work closely with each participating SPARC laboratory and provide advice on tissue harvesting and preservation as well as perform tissue processing and embedding of tissues in plastic resin, semi-thin and ultrathin sectioning for ultrastructural studies, TEM of nervous tissues, pre- and post-embedding immuno-EM studies, quantitative analysis of nerves, ganglia, neural circuits, and synaptic structures, customization of TEM protocols to accommodate project specific needs, and interpretation of ultrastructure and quantitative TEM data. It is expected that the proposed TEM studies will add value to SPARC-funded investigations and augment interpretability of functional and modeling studies as well as provide critical ultrastructural data for an evidence-driven approach to the development of new and improved neuro-modulatory strategies.

Summary Throughout its history, the Department of Neurology at Mount Sinai has excelled in training academic neurologists, and is historically #3 by total number and #1 by percentage of trainees among US programs in generating academic neurologists (Arch Neurol. 2011;68(8):999-1004). Over the past decade, the Department has directed itself towards facilitating and improving the training of outstanding neurologist-researchers. Our R25 research residency, first established in 2012, is a burgeoning element for the development of future neurologist-scientists. The present application to renew the NINDS- supported research- resident training program is a cornerstone of an effort that involves all aspects of the department and all stages of career development. Over six years we have recruited sixty new faculty members, including many neurologist-scientists, redesigned the preclinical neuroscience course, developed a 12 week neurology/psychiatry/neuroradiology/neurosurgery/research third year clerkship, increased the size of our residency from 18 to 24 residents, designed a new neuroscience-at-noon neurology residents program taught by faculty from half a dozen departments, established formal residency research and junior faculty mentoring programs, received approval for four new fellowship programs and recruited outstanding academic and research-oriented resident cohorts, two of whom are currently in the program. The PI, Dr. Sealfon, has been continuously funded by NIH for 28 years, has previously directed a T32 program and is wholeheartedly committed to the training of neurologist- scientists. As is typical of our collegial and dynamic institution, our experienced research training faculty is selected from several departments in addition to Neurology. The training program will provide a formal closely-mentored clinical or basic research experience during residency and fellowship to develop the skills, data and publications to submit a career development award and to succeed in a research- intensive department like ours. Success of the program will be judged by the trainees rate of obtaining K08 and K23 awards, their academic placement and their researchcontributions.