CELL AND MOLECULAR APPROACHES OF PLASTICITY

Director: Dr. Lawrence Kromer and Dr. Barbara Bregman

The Molecular and Cellular Approaches to Neural Plasticity core provides resources and training in cellular therapies (neural tissue transplantation, stem cell / neural progenitor cells) and pharmacological approaches (neurotrophins, modulators of intraneuronal cAMP, modulators of the inhibitory environment (chondroitinase, inhibitors to myelin-associated inhibitors, ephrins and eph receptors) to increase regeneration and recovery of function after spinal cord injury. This core also provides expertise, resources and training in neuroanatomical tracing, quantitative image analysis, and cellular and molecular approaches to evaluate the nature and extent of plasticity associated with these interventions.

The Neuroscience laboratories at Georgetown have extensive research experience using these approaches and extensive training experience in teaching collaborators, students and fellows in these approaches. This core is designed to operate in close association with the animal models core, above. The Department of Neuroscience will make the core equipment in our Morphology and Imaging shared resources available to all NCARRN participants.

Cellular therapies and pharmacological approaches: There are a number of ?windows of opportunity? to influence the outcome of CNS injury. It is now clear that repair and recovery of function after spinal cord injury will involve multiple intervention strategies. Activity and agents that modulate neuronal signal transduction alter neural plasticity by increasing dendritic complexity, increasing synaptogenesis and activating intrinsic neuronal genetic programs associated with growth. This occurs both at supraspinal and spinal levels. In this core, participants will be able to learn cellular replacement strategies (neural tissue transplantation, Schwann cell, neural and glial progenitors (stem cells) for example) and pharmacological treatment with neurotrophic factors and agents that increase neuronal cAMP (rolipram) in conjunction with agents to alter the inhibitory influence of the environment at the injury site (chondroitinase, Nogo). It is likely that in developing treatment strategies to improve recovery of function, cellular and pharmacological approaches will be combined with rehabilitation strategies to alter activity to increase neuronal plasticity and recovery of function after spinal cord injury. We have extensive experience in using cellular and pharmacological approaches to increase the intrinsic capacity of mature CNS neurons for regrowth and for modifying the environment at the injury site to make it more favorable for growth.

Neuroanatomical tracing in spinal cord injury and repair: The Neuroscience Department at Georgetown University has the state-of-the-art anterograde and retrograde neuroanatomical tracing approaches in place to examine the reorganization of the pathways in question. Participants will understand the experimental advantages, applications and limitations of methods to identify regeneration of specific CNS pathways and any potential reorganization that may occur in those pathways after spinal cord injury. The participants will also learn methods of quantitative analysis of neuroanatomical tracing. For example, there are facilities for stereotaxic injections and labeling of corticospinal pathways, rubrospinal pathways and other descending pathways that allow the tracing and comparison of the reorganized pathways with that in normal animals. In addition, tracing techniques are available for analyzing the reorganization of somatosensory pathways in addition to the motor pathways. For example, transganglionic tracing using biotinilated dextranamine can be used to examine the reorganization of primary sensory pathways within the spinal cord and within the brain stem nuclei. In addition to the specific tracing pathways, immunocytochemical analysis of specific neurotransmitter-containing pathways is well established. There are provisions to conduct tracing experiments, and faculty and staff expertise to ensure the accurate and timely analysis of reorganization of CNS pathways after injury.

Quantitative image analysis: The Microscopy and Imaging shared resource provides instrumentation, training and expert advice to investigators undertaking light and electron microscopy as well as image analysis of CNS tissue. The resource supports investigators pursuing routine photomicroscopy and those applying sophisticated, multidisciplinary approaches including quantitative image analysis. These applications include immuno-light and electron microscopy, confocal microscopy, microinjection, videomicroscopy and image analysis. Over the past several years, Georgetown University has developed an outstanding core base of equipment for morphology and image analysis. Most of the approaches available through this core can be transferred to the investigator?s home institution. The Microscopy and Imaging core encourages and supports investigators using microscopic approaches to CNS plasticity, repair and neuropathology. It does so by training users, assisting in experiment design, providing pre-tested secondary antibodies for fluorescence microscopy and by providing extensive assistance for electron microscopy including sample preparation. This staff also designs and implements image analysis programs for specific applications as the need arises.

The state-of-the art imaging facilities in the Department of Neuroscience will be fully available to all NCARRN participants. Because of the great increase in demand for confocal microscopy, the Department of Neuroscience has added an additional Zeiss confocal microscope to its core facilities available to the investigators. The Microscopy and Imaging core expanded its image analysis capabilities recently by adding ImagePro2.0 software and a new computer system coupling a motorized prior stage to the Olympus Vanox upright microscope. This equipment enables us to collect tiled images and subsequently perform image analysis on a large area at high resolution simplifying quantification of image features that would otherwise have been collated from many separate images. This program is useful for bright field histochemistry or fluorescence microscopy and can be generalized for other measurement applications.

The histology/morphology core facilities focus on histology and imaging. This resource is equipped with a Leica 2800E Frigocut cryostat for frozen histopathology and research sectioning, as well as a Vibratome 3000, a refrigerated fresh tissue sectioning system. Workstations, including two chemical-fume hoods, for immunocytochemical and histochemical staining are available. For tissue sections, there is a Zeiss Axioplan 2 fluorescent microscope equipped with 35 mm film system and an Optronics thermoelectrically cooled 3 CCD video camera, ideal for working with extremely low light levels. These cameras send images to a Micron PC computer using a Coreco Ultra II frame grabber for color acquisition and display. This microscope has also been equipped with a MAC 2002 precision stage system, and MicroBrightField?s software and hardware systems for brain mapping, neuron tracing and unbiased stereology. This includes the Lucivid eyepiece monitor and Neurolucida and Stereo Investigator applications. Image and data files from both microscopes can be backed up on Iomega Zip drives or on a LaCie CD-writer. Image processing is supported by a full complement of digital imaging hardware.