M. Sean Grady - US grants

The developing nervous system undergoes major shifts in neuron number during the period of synaptogenesis. The target tissue appears to have a major influence on the final number of neurons innervating the target region itself. While the mechanism underlying this interaction is not resolved, one strong hypothesis postulates the existence of trophic factors produced in the target region and retrogradely transported back to the projecting neuron. Deprivation of this trophic factor leads to neuron death. The biology of neural trophic factors has been best established in the peripheral nervous system, but has also been extensively investigated in the central nervous system of developing animals and, very recently, in the adult central nervous system. Fetal neural tissue may have significant quantities of trophic substances that might affect adult neuron death. This proposal seeks to define the effects of fetal neural tissue grafts on adult rats using a model of axotomy induced neuron death in the medial forebrain region. Specifically, the temporal and regional parameters of fetal grafts that are important in preventing cholinergic and GABAergic host neuron loss will be investigated. Furthermore, the importance of long term graft survival on host medial forebrain neurons will be assessed. Finally, the cellular components of the graft will be separated to quantify the individual effects of donor glia and donor neurons on host neuron death. By refining the parameters of the graft's effect on the host, it is hoped that the mechanisms underlying axotomy induced neuron death can be studied from a different perspective. The impact this research plan has on health care is derived from recent better understanding of neural events in chronic degenerative neurological disorders such as Parkinson's disease, Huntington's chorea and Alzheimer's disease. Loss of specific neuron populations in those diseases may be linked to loss of trophic substances. Furthermore, degree of recovery from acute neurologic diseases such as head trauma may be based on the extent of delayed neuron death can lead to manipulation of those mechanisms and improve outcome in a wide range of neurologic diseases.

Traumatic brain injury (TBI) occurs to US citizens at the rate of 500,000 cases/yr., with 50,000 deaths and 50,000 survivors with severe residual deficits. Learning and memory are commonly impaired after TBI in humans. These higher cognitive functions play a key role i the successful recovery and rehabilitation from TBI. In humans, the ventral forebrain cholinergic neurons appear to play a significant role in memory, as suggested by the pathophysiology of Alzheimer's Disease. In a similar wy, the pathophysiology of the memory and learning deficits following TBI may be caused by damage to these neurons. There are three specific aims; 1. Characterization of noncholinergic ventrobasal forebrain neuron response to TBI; 2. Further characterization of cholinergic ventrobasal forebrain neuron response to TBI; and 3. Mechanisms of repair and recovery after TBI. These specific aims are focused towards addressing the central hypothesis: TBI causes quantifiable neuronal damage in populations known to be involved in learning and memory. The first specific aim is formulated to answer the hypothesis that noncholinergic neurons in the ventral forebrain region may respond differently than cholinergic neurons to experimental TBI. The second specific aim is targeted towards answering two hypotheses; 1) Experimental TBI causes a loss of acetyl choline in the terminal projection fields of the septal neurons; and 2) Experimental TBI causes a permanent loss of septal neurons. The third specific aim addresses two hypotheses: 1) The extent of cholinergic neuron damage and subsequent degree of recovery in dependent on age of the experimental subject; and 2) neuron protection drugs (21 amino steroids) are capable of modifying the response to experimental TBI and may give some indirect insight into the molecular mechanisms underlying this particular group of neurons susceptibility to injury. TBI is modelled by using the well described rodent fluid percussion injury system. Techniques include use of quantitative immunocytochemistry of specific neuron populations and histochemistry and neurochemistry of cholinergic neurotransmitters. Memory capability is determined using the water maze. Overall, the methodology has a long and accepted record in the study of cholinergic neuron populations. These techniques have created a better pathophysiologic description of neurodegenerative disorders and can be directly applied to the study of the pathophysiology of head injury. The result of the proposed experiments should be a better description of the pathophysiologic mechanisms in head injury, as well s suggesting a rational basis for treatment.

[unreadable] DESCRIPTION (provided by applicant): The proposed program will train M.D., Ph.D. and medical student investigators in state-of-the-art techniques to investigate the molecular and cellular mechanisms underlying central nervous system (CNS) injury. The trainees will be: 1) Physicians involved in the University of Pennsylvania Neurosurgical residency-training program, 2) Ph.D. scientists with prior training in molecular biology, or 3) selected medical students who are participating in Penn's unique Clinical Neuroscience Track program. This program will provide background and training in molecular neurobiology and an introduction to CNS disorders where these new techniques can be applied. This program will be a laboratory-based research-training program that includes experience in novel molecular biology techniques in an interactive and supportive setting. The institution has an extensive didactic program in basic and clinical neurosciences, neurodegeneration, CNS injury, and molecular biology including gene therapy, which will be individually designed for each trainee in order to supplement the laboratory experience. Each trainee will design and complete an independent project providing experience in design and analysis of experiments and in the presentation and publication of results. Weekly trainee research seminars and monthly faculty/trainee mini-symposia provide constant interchange and exchange between faculty trainers and trainees. This program will be run by individual faculty trainers who are the leaders in their field at the University of Pennsylvania, representing disciplines such as CNS ischemia and trauma, neurodegenerative diseases, inheritable neurological disorders, demyelinating diseases, epilepsy, neuroanesthesiology, molecular virology, gene therapy, and molecular pharmacology. Each trainee selects one laboratory for the primary research project but will have complete access to the other trainers for advise, technical assistance, and collaboration. Each trainer has independent NIH funding and all have active and internationally-recognized research programs. Core services available within the University of Pennsylvania, Wistar Institute, Children's Seashore House, and the Institute of Human Genome Therapy include protein sequencing, oligonucleotide synthesis, transgenic knock out/ knock in technology, automated DNA sequencing, viral vector construction, and gene therapy techniques. No funding mechanism currently exists at the University of Pennsylvania to support these trainees. The funding of this Brain Injury Training Grant (BITG) application will establish a novel and important infrastructure involving a highly collaborative faculty for the purpose of training future research scientists and clinicians in the state-of-the-art techniques related to an important clinical disease. [unreadable] [unreadable]

Traumatic brain injury (TBI) in humans as an incredibly heterogeneous disease and the significant effort involved in developing and characterizing reproducible and clinically-relevant models of specific types of TBI has enabled the neurotrauma research community to begin to identify important and injury-specific mechanisms of cell death and dysfunction. This application revolves around the general hypotheses that selective molecular and cellular pathways are activated or initiated which are injury-specific mechanisms of cell death and are activated or initiated which are injury-specific. Through the sue of selective experimental models that closely simulate the major classes of human TBI, we can better understand and characterize the pathological sequelae of clinical head injury and explore novel mechanistic-based therapies. We have chosen to focus on several exciting areas, including (1) the relationship between TBI and the cellular events associated with Alzheimer's and other neurodegenerative disease, (2) the role of the dysfunctional cytoskeleton and calpain-mediated cytoskeletal proteolysis in post- traumatic neuronal death and damage, and (3) the activation of cell death enzymes such as MAP kinases and caspases and their role in post- traumatic cell death. We will continue to forge mechanistic links between these pathological cascades and outcome following TBI. We will also evaluate (4) the important role of secondary hypoxia/ischemia in the exacerbation of cellular injury and the association with one or more of the pathologic mechanisms studied in these Projects. Finally, we will use the accumulated molecular and cellular information to design and drive novel neuroprotective strategies to enhance recovery of function and attenuate neuronal cell death. These projects will be significantly supported by human/histopathology, biomechanics, molecular biology, and administrative cores. We believe that the understanding of the specific cellular and molecular mechanisms underlying cell death and dysfunction is critically important to the continuation of scientific progress in this field and to the development of novel therapeutic strategies targeted to treat human brain injury.

[unreadable] DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is the leading cause of death and disability in children and young adults. Despite initial cognitive deficits, many individuals experience significant recovery. This proposal investigates how the ability to learn and recall information recovers. The limbic system plays a prominent role in memory formation, consolidation, and recall. Using the clinically relevant mouse lateral fluid percussion injury (FPI) model of TBI, preliminary data show time-dependent recovery in an anterograde cognitive task. Permanent negative alterations in neuronal number and circuitry occur in the ipsilateral hippocampus. The central hypothesis of this proposal contends that the time-dependent functional improvement following FPI is due to the reorganization of anatomic and physiologic circuits within the contralateral hippocampus. Experiments will test three hypotheses: 1) mouse lateral FPI transiently alters neuronal function, but not the anatomy of the contralateral hippocampus, 2) multiple sub-cellular events occur in the contralateral hippocampus enabling functional improvement and 3) the contralateral hippocampus plays the major role in cognitive recovery. A systems approach is used, incorporating design-based stereology to quantify neuronal and synapse number by light and electron microscopy, determination of dendritic re-modeling using dil label and confocal microscopy, field and single cell electrophysiology in hippocampal slices to analyze circuitry, and performance in the conditioned-fear paradigm to test cognitive function after manipulations of the contralateral hippocampus. The compensatory mechanisms demonstrated in the contralateral hippocampus may help us better understand cognitive recovery in human TBI and lead to novel strategies for improving speed and extent of recovery. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The proposed program will train M.D., Ph.D. and medical student investigators in state-of-the-art techniques to investigate the molecular and cellular mechanisms underlying central nervous system (CNS) injury. The trainees will be: 1) Physicians involved in the University of Pennsylvania Neurosurgical residency-training program, 2) Ph.D. scientists with prior training in molecular biology, or 3) selected medical students who are participating in Penn's unique Clinical Neuroscience Track program. This program will provide background and training in molecular neurobiology and an introduction to CNS disorders where these new techniques can be applied. This program will be a laboratory-based research-training program that includes experience in novel molecular biology techniques in an interactive and supportive setting. The institution has an extensive didactic program in basic and clinical neurosciences, neurodegeneration, CNS injury, and molecular biology including gene therapy, which will be individually designed for each trainee in order to supplement the laboratory experience. Each trainee will design and complete an independent project providing experience in design and analysis of experiments and in the presentation and publication of results. Weekly trainee research seminars and monthly faculty/trainee mini-symposia provide constant interchange and exchange between faculty trainers and trainees. This program will be run by individual faculty trainers who are the leaders in their field at the University of Pennsylvania, representing disciplines such as CNS ischemia and trauma, neurodegenerative diseases, inheritable neurological disorders, demyelinating diseases, epilepsy, neuroanesthesiology, molecular virology, gene therapy, and molecular pharmacology. Each trainee selects one laboratory for the primary research project but will have complete access to the other trainers for advise, technical assistance, and collaboration. Each trainer has independent NIH funding and all have active and internationally-recognized research programs. Core services available within the University of Pennsylvania, Wistar Institute, Children's Seashore House, and the Institute of Human Genome Therapy include protein sequencing, oligonucleotide synthesis, transgenic knock out/ knock in technology, automated DNA sequencing, viral vector construction, and gene therapy techniques. No funding mechanism currently exists at the University of Pennsylvania to support these trainees. The funding of this Brain Injury Training Grant (BITG) application will establish a novel and important infrastructure involving a highly collaborative faculty for the purpose of training future research scientists and clinicians in the state-of-the-art techniques related to an important clinical disease. [unreadable] [unreadable]