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Laboratories and Projects
Applicants for Sabbaticals will apply to the PI of CENT and their proposals will be evaluated by the Executive Committee (see section of Administative Structure). All of the researchers indicated below have indicated their willingness to mentor and instruct trainees of CENT. They will also have incentives to do so because support for the projects will be paid by CENT on a $9,000 per/month basis. This will cover prorated portions of technicians’ salaries, supplies, small equipment and other costs or research.
Research Opportunities at PENN
Steven Pickup, Ph.D. is Director of Small Animal Imaging at PENN. This now includes MRI, PET, SPECT and CT. Dr. Pickup has considerable experience in physical chemical applications to radiological imaging, especially MRI, and has published extensively on MR imaging of several systems including in vivo and ex vivo MRI detection of localized and disseminated neural stem cell grafts in the mouse brain brain.
Rita J. Balice-Gordon, Ph.D. is Professor of Neuroscience at PENN. She studies activity-dependent synaptic plasticity during neural development, using neuromuscular and CNS synapses in mice and zebrafish as model systems; neurotrophic and other cell-cell signaling mechanisms underlying synapse formation and maintenance; axon outgrowth and synapse reinnervation after neural injury. Research techniques include: Molecular and cellular biology; electrophysiology; in vivo imaging in mice and zebrafish; confocal microscopy. She is studying the cellular and molecular interactions between neurons and their targets that underlie synapse formation and maintenance during neural development. Her laboratory uses neuromuscular synapses in mice and zebrafish in vivo, and CNS synapses among hippocampal neurons in vitro, as relatively simple, accessible and easily manipulated model systems. Imaging and electrophysiology are used to study how synaptic structure and function are affected by neural activity in mice and zebrafish. Cre/lox genetic deletion strategies are used to study neurotrophin signaling mechanisms that underlie synapse formation, maintenance and axon regeneration in mice. Cellular, molecular and genetic approaches are used to dissect the mechanisms underlying synapse formation in wild type and mutant zebrafish.
R. Nick Bryan, MD, PhD, Eugene P. Pendergrass Professor and Chair Department of Radiology University of Pennsylvania School of Medicine. Dr. Bryan is PI on 2 NIH contracts for Multi-Institutional Brain MRI Studies, Co-investigator in Drs. Davatzikos and Herskovitz’s labs on brain image analysis, and Group leader on Unfounded Functional Neuroimaging Study of Brain Metabolism. Major Findings of Past Six Years have included: 1. Subcortial white matter changes in the elderly are correlated with cognitive decline. 2. Subcortial white matter changes in the elderly are predictive of future stroke. 3. The combination of high resolution MRI and quantitative morphological image analysis can identify early changes of cerebrovascular and neurodegenerative diseases that are related to cognitive decline. 4. BRAID, a human brain structure/function discovery algorithm based on Talairach mapping of MRI defined lesions and Bayesian analysis.
John A. Detre, M.D. is Associate Professor of Neurology and Radiology at PENN. His research focuses on the physiology of functional activation, the development of methods for imaging regional brain function, and clinical applications of functional neuroimaging. Dr. Detre founded and directs the Center for Functional Neuroimaging, which provides training, administrative oversight, and infrastructure support for neuroimaging resources at Penn. An NINDS Center Core grant (P30 NS045839) also supports this effort. Dr. Detre has multidisciplinary expertise in biophysics, physiology, and rodent modeling. He collaborates widely with other investigators working in brain research. Dr. Detre has extensive research interests in neuroimaging applications to cognitive rehabilitation research. Dr. Detre is a faculty member on several NIH training grants, a member of the Neuroscience Graduate Group at the University of Pennsylvania, and currently mentors a number of junior faculty members. He has also organized and participated in numerous meeting workshops and symposia on functional imaging.
Marc A. Dichter, MD, PhD. CNS cell culture. Dr. Dichter’s laboratory has pioneered the development of mammalian CNS cell culture since the first cultures of mammalian cerebral cortex were developed by them in 1972. His laboratory maintains, or has maintained, dissociated cell cultures of cortex, hippocampus, spinal cord, sensory ganglia, brainstem and thalamus. In addition, the laboratory has maintained long term slice cultures using both membrane culturing techniques and roller tubes. Dr. Dichter has implemented a variety of techniques for studying these cultures including immunohistochemistry, electrophysiological recordings with sharp electrodes and patch electrodes, analyses of drug actions, assays of axon and dendritic growth, analyses of neuronal differentiation and synapse formation, and assays for excitotoxicity and neuroprotection. Dr. Dichter has now developed his laboratory as a core resource for investigators at PENN who need to incorporate neural cell cultures into their research.
M. Sean Grady, MD is Charles Harrison Frazier Professor and Chairman, Dept. of Neurological Surgery at PENN. Dr. Grady's current bench research focuses on the consequences of traumatic brain injury on the hippocampus. Using mice and the fluid percussion injury model, he is exploring the mechanisms within the hippocampus that underlie cognitive recovery after brain injury using anatomic, electrophysiologic, and molecular techniques. In particular, the work focuses on the role that the contralateral hippocampus plays in functional recovery. Techniques include determination of neuronal survival using design based stereology, analysis of dendrite morphology by selectively filling specific neurons with fluorescent dyes and exploiting specific genetic manipulations within mice to analyze specific inhibitory neuronal populations. The physiologic consequences of injury are analyzed using in vitro hippocampal slice recordings, both field and single cell recording. Finally, the functional outcome is assessed using the hippocampal dependent conditioned fear paradigm.
Joel H. Greenberg, PhD Studies cerebrovascular physiology in normal and pathophysiological states; positron emission tomography; metabolic tissue changes during cerebral ischemia; activation-flow coupling; functional reorganization following ischemia. Research techniques include: quantitative multiple label autoradiography; local cerebral glucose metabolism; regional blood flow utilizing radio labeled microspheres; positron emission tomography (PET); single photon emission tomography; measurement of nitric oxide in vivo; small animal stroke models; optical imaging of blood flow and oxygen. A crucial factor determining the prognosis of patients in cerebral ischemia is the ability of tissue to recover from biochemical and hemodynamic derangements. Dr. Greenberg is investigating the factors that contribute to irreversibility of tissue damage in focal cerebral ischemia. Studies are being conducted in rats in which the middle cerebral artery is temporarily occluded and a variety of metabolic and hemodynamic parameters are measured and correlated with regional histological damage and functional recovery. Activity dependent plasticity studies are being undertaken in the rat to examine the effect of somatosensory deafferentation on the relationship between local cerebral blood flow and local cerebral glucose metabolism. Double label autoradiography is being used. Studies are in progress to examine reorganization of the blood flow, metabolic, and functional response following focal cerebral ischemia of the somatosensory cortex of the rat. A novel compression ischemia model has been developed to facilitate these studies. Optical techniques are used to provide hemodynamic and metabolic images of the brain during reorganization. Optical techniques are being developed to monitor hemodynamic and tissue oxygen concentration changes in man following brain trauma.
Philip G. Haydon, PhD studies the regulation of synaptic transmission. In particular we are interested in the reciprocal signaling between synapses and astrocytes that is mediated by the release of chemical transmitters and how astrocytes regulate synapse development and function. Research techniques include: Fluorescence microscopy, confocal microscopy, near-field microscopy, electrophysiology, patch clamp recording, calcium imaging, photolytic uncaging, cell and tissue culture, cell and molecular biology. Dr. Haydon is interested in understanding the dynamic regulation of synaptic transmission and in elucidating the roles of glial cells in controlling the synapse. In many regions of the CNS it is apparent that synapses are tripartite structures in which, in addition to the pre- and postsynaptic terminal, the astrocyte (a sub-type of glial cell) acts as a third element that wraps around the synaptic structure. Recent studies are changing the view of the role of astrocytes in synaptic transmission. It is now clear that synaptic activity regulates calcium levels in astrocytes, and as a consequence of elevated calcium, we have demonstrated that astrocytes release the chemical transmitter glutamate onto the synapse to modulate synaptic transmission. In order to understand the role of the astrocyte in the control of the synapse we are using photolysis to manipulate calcium levels within astrocytes while monitoring synaptic transmission. As another approach to study this complex system, we have developed selective inhibitors of the astrocytic glutamate release pathway and are using viral vectors to manipulate the release of this transmitter to determine the role of these glial cells in information processing. Using the hippocampal slice preparation (see figure) and optical approaches, we have recently demonstrated that transmitter-evoked calcium elevations in an astrocyte propagating to neighboring astrocytes as a calcium wave. This raises the possibility that a synapse could signal to neighbors by way of calcium signal that spreads through an astrocytic intermediate. In addition to multi-cellular studies of the synapse, we also investigate the functional sub-structure of synapses using scanning probe microscopy. We have previously studied channel organization using atomic force microscopy and are currently using biological near-field microscopy, with optical resolution of 50 nm, to probe the workings of the synapse. Using this high-resolution system we are studying the local microdomains of calcium accumulation beneath activated calcium channels with the long-term objective of visualizing individual molecules within functional synapses to unravel the mysterious workings of this essential structure of the nervous system.
Douglas H. Smith, MD is Professor of Neurosurgery and Director of the Center for Brain Injury and Repair at the University of Pennsylvania. Over the last 12 years, he has devoted his full-time efforts to neurotrauma research following completion of fellowships in both molecular biology and neurotrauma at the University of Connecticut. His laboratory examines mechanisms of nerve fiber (axon) damage and repair in traumatic brain injury and spinal cord injury. Specific research interests include examining the effects of mechanical deformation of axons, tissue engineering approaches for nerve repair, neuroimaging, and the link between neurodegenerative diseases and traumatic brain injury. These efforts have resulted in over 100 published reports from his laboratory.
Richard Salcido, MD is William J. Erdman, II Professor and Chairman, Department of PM&R at PENN with secondary appointment as Senior Fellow, Center on Aging and Associate in the Institute of Medicine and Engineering. Dr. Salcido’s research focus is on chronic wound healing. Dr. Salcido has invested a decade in the development of an animal model to further our understanding about the physiological, physical, cellular and molecular mechanisms associated with experimentally derived pressure ulcers. He has been a recipient of an RO1, NIH and other sources of support over the last 10 years. He moved his laboratory from the University of Kentucky in 1998, and after a transition period re-established a basic science laboratory and has accepted a t-32 fellow Ling Zhou, MD, Ph.D. (physiatrist) and a post doctoral fellow Adrian Popesque, MD, to train in his lab. Dr. Salcido recently was awarded internal support (competitive) from the University of Pennsylvania to support the purchase of monitoring systems for his laboratory. In 2002 Dr. Michael G. Sowa, Ph.D. Associate Research Officer Institute, adopted his pressure ulcer animal model for Biodiagnostics National Research Council of Canada Winnipeg, Canada. Dr. Salcido was recently a visiting professor in Canada, which resulted in collaborations to further validate the models and techniques utilized in both labs. Dr. Salcido is a nationally recognized resource, for the use of animal models in pressure ulcer research. He was a member of the Agency for Health Care Policy and Research Clinical Practice Guidelines development for the Prevention and Treatment of Pressure Ulcers, and in 1998 was named Editor-in-Chief of Advances In Skin and wound Care a peer reviewed journal.
Michael E. Selzer, MD, PhD, the PI of CENT, is Professor of Neurology and Rehabilitation Medicine at PENN. Unlike axons in the central nervous system of mammals, the axons of the lamprey spinal cord regenerate across a spinal transection, and even grow preferentially through the glial/ependymal scar. This regeneration is specific both with regard to the direction of axon growth and synaptic reconnection. We are attempting to determine the mechanisms involved in the elongation and guidance of regenerating axons. Electron microscopic analysis of intracellularly labeled growing axon tips showed that they are densely packed with neurofilaments (NFs). This is different from growth cones of embryonic neurons studied in dissociated cell culture, which grow much faster and contain no NFs. Thus one of Dr. Selzer’s goals is to determine whether NFs play a role in the axonal elongation during regeneration in the central nervous system. Monoclonal antibodies and sense and antisense riboprobes are being used to study the expression of lamprey NF in injured neurons by immunohistochemistry, in situ hybridization and in vivo transfection targeted at manipulating transcription, translation or assembly of NF.
EM analysis also shows that the growing tips of regenerating spinal axons are in disproportionate contact with glial processes. Lamprey glia differ from mammalian astrocytes in containing keratin intermediate filaments instead of glial fibrillary acidic protein and in being interconnected by desmosomes. This phenotype is found in other systems showing axonal regeneration. Dr. Selzer has been studying the molecular biology and proliferative activity of these glial cells to determine their role in axon regeneration.
Finally, Dr. Selzer’s laboratory has found evidence for the existence of guidance molecules in the lamprey CNS, including semaphorins 3 and 4, netrin 1and the netrin receptors UNC-5 and DCC. The lab is now attempting to determine the cellular localization of these molecules, how their expression is modulated following spinal cord transection, and how manipulation of their expression will alter regeneration.
A major goal of the laboratory is to be able to image the regeneration of axons in the living animal. Toward that end, Dr. Selzer’s laboratory is collaborating with Drs. Balice-Gordon and Haydon in imaging the axons after microinjecting them with fluorescent tracers. The lab is also collaborating with the Division of Neuroradiology to image the spinal cord by MRI and to perform physiological measurements using diffusion weighted imaging and MR spectroscopy. To date they are able to achieve a resolution of 9μm in the isolated spinal cord and thereby image individual large axons.
Research Opportunities at UCLA
Human Brain Imaging
Bruce H. Dobkin, MD, is the coPI of CENT. His main interests include the translation of basic neuroscience research into rehabilitation measures and interventions. His laboratory specializes in the development of locomotor interventions to improve motor skills for walking in patients with stroke, SCI, and after hemispherectomy for epilepsy (treadmill training with partial weight support, robotic assistive devices, pharmacologic interventions that may enhance motor learning, dose-response assessments of the intensity and duration of a therapy) and for upper extremity motor skills (TMS measures of cortical excitability induced by a treatment; longitudinal fMRI studies funded by NIH over the course of specific therapies now in progress with Carolee Winstein, PhD, at the University of Southern California).
Associate faculty include John Mazziotta, MD, PhD (fMRI, probabilistic brain mapping), Susan Bookheimer, PhD (memory and language, neuropsychological rehabilitation), Roger Woods, MD (motor control, imaging analysis and statistics) Marco Iacoboni, MD, PhD (motor skills learning, imagery) Jeff Alger, PhD (fMRI, MRS), and Barbara Knowlton, PhD and Russ Poldrack, PhD (learning systems).
Potential development and validation projects
Dr. Dobkin continues to further the interactions of the First International Workshop on Neuroimaging and Stroke Recovery (JC Baron, LG Cohen, SC Cramer, BH Dobkin, H Johansen-Berg, I Loubinoux, RS Marshall, and NS Ward; Cerebrovasc Dis, Jan 2004; 18(3): 260-7). This group meets to improve the standardization of activation paradigms and behavioral measures for neurorehabilitation studies using fMRI, TMS, near-infrared spectroscopy and other methodologies. One goal is to have a growing database of completed imaging studies available to all over the Internet. Collaborations within this network offer a great potential resource for training of fellows, faculty for symposia, and for Web-based continuing interactions with leaders around the world. We would aim to develop existing software for conferencing across sites with former fellows and with world experts in imaging. Data analysis could be carried out among sites and with experts from outside Penn and UCLA. This aim fits well with a funded project, in which Dr. Mazziotta is currently the principal investigator in the International Consortium for Brain Mapping (ICBM) to develop a probabilistic atlas for the human brain. This involves collaborations with eight laboratories in seven countries on four continents. This important project will incorporate, into the normal human probabilistic atlas, functional relationships for each voxel in the brain. If all of the cerebral functions can be assigned in a probabilistic fashion by location, normal brain function will be better defined and injuries to the brain can be assessed in terms of their functional prognosis. With such a system, one can then evaluate plasticity and recovery of function that alters these probabilities on a voxel by voxel basis, thereby defining the system reorganizations that occur as the brain recovers from acute or ongoing injury or with rehabilitation interventions. Additional functional tasks would be applied in normal subjects and in patients with brain injury. The International Collaboration is developing a quantitative neurological scale assessing the major brain functional systems. It would then apply it to individuals with brain injury. When this scale is compared to normal probabilistic maps, probabilistic deficit atlases could be developed and compared to changes over the period of gains with rehabilitation strategies.
Animal Imaging and Plasticity
S. Thomas Carmichael, MD, PhD, Assistant Professor of Neurology at UCLA is defining the molecular and cellular mechanisms of neural repair after stroke and TBI, and the effect of behavioral training on these repair mechanisms. His studies use an animal model that is highly relevant to human stroke and to repair in its creation, volume of infarction, and location. Stroke triggers two cellular process of neural repair: Axonal sprouting mediates the formation of new connections after stroke, and stem cell responses produce new neurons and glia near the site of stroke. If properly harnessed, both axonal sprouting and post-injury stem cell responses provide mechanisms for neuronal regeneration and reconnection after stroke.
The laboratory has particular expertise in using an anatomically well-defined region of the rodent brain as a novel model system to study the molecular mechanisms of neural repair after stroke. Small cortical strokes are placed within the somatosensory cortex (and adjacent to the forelimb motor cortex), in the representation of the mouse or rat facial whiskers (the barrel field). This area has a well-defined anatomical and physiological structure that allows detailed molecular, cellular and functional imaging study of neural repair after stroke. The detailed anatomy, response to peripheral stimulation and large size of the barrel cortex in the rodent brain have generated the first descriptions of axonal sprouting after stroke (Carmichael et al Neurobiol Dis. 8:910), identified tissue microenvironments of gliosis, neural injury and neural repair after stroke (Katsman et al., J Cereb Blood Flow Met 23:997), provided novel metabolic and optical imaging of zones of injury and diaschisis after stroke (Carmichael et al Neurobiol Dis. 8:910; Carmichael et al. Stroke 35:758), and generated a detailed molecular and cellular analysis of a neuronal growth program and glial inhibitory molecules during axonal sprouting after stroke.
Associate faculty include Arthur Toga, PhD (optical imaging), Jeff Alger, PhD (MRI/MRS), Harley Kornblum, MD, PhD (stem cells and micro-PET), David Hovda, PhD (models of TBI and plasticity), and Joshua Tractenberg, PhD (time-lapse growth of dendritic spines during learning).
The lab is engaged in two core projects and four applied research projects. Core project #1 defines the molecular systems that mediate post-stroke axonal sprouting. Core project #2 defines the ischemic stimulus that induces post-stroke stem cell responses and the molecular guidance cues that direct neural progenitors to areas of cerebral injury. He published a new model of stroke in the rodent somatosensory and motor cortex and used these anatomically well-described brain areas to define the circuitry and functional metabolism formed by axonal sprouting after stroke. He has defined the genetic components of a neuronal growth program that mediates axonal sprouting, and the key cellular components of the glial inhibitory environment that inhibit post-stroke axonal sprouting in this model. The laboratory identified key components of a hypoxia-inducible molecular cascade that stimulates neurogenesis after stroke and identified a re-activation of early developmental molecules that mediate neuronal migration after stroke. Clinical trials suggest that constraint-induced movement in patients promotes improved functional recovery after stroke. Four additional projects build on these molecular findings in the two core projects to study the molecular mechanisms and functional imaging effects of constraint-induced movement. Allied project #1 studies the molecular effect of forced use of the impaired limb on neuronal growth programs and the overall extent of axonal sprouting after stroke, using lentivirus mapping of neuronal connections and gene expression analysis in movement-constrained and unconstrained stroke animals. Allied project #2 studies the effect of constraint-induced overuse of the impaired limb on the overall survivability and connections of new neurons generated after stroke, and the hypoxia-inducible stem cell system after stroke. Allied project #3 directly images post-stroke functional reorganization in motor systems adjacent to the injury using microPET and optical intrinsic signal in collaboration with Drs. Harley Kornblum and Arthur Toga. This work has been partially published. Allied project #4 uses the above-defined regions and time course of post-stroke axonal sprouting and stem cells responses to define instructive zones for embryonic and adult stem cell transplantation and differentiation into neurons, in collaboration with Dr. Harley Kornblum. Finally, in conjunction with Drs. David Hovda and Neil Harris, we have modified a traumatic brain injury model to produce partial damage to rat somatosensory cortex, and are determining the degree, time course and functional imaging effects of axonal sprouting using lentivirus transfection and microPET in this form of cerebral injury. These research projects involve collaborators within the UCLA Neural Repair Community along distinct scientific themes of molecular and cellular analysis of neural repair after cerebral injury and functional imaging of reorganizing neural circuits in animal models of disease. The experiments utilize newly developed techniques (lentivirus gene transfection, microarray analysis) in cutting edge combination with behavioral modification and functional imaging (microPET, OIS) to define the basic molecular principles behind rehabilitative therapies, and to generate novel pharmacological targets to improve functional recovery.
Techniques employed in this research include animal modeling, immunohistochemistry, in situ hybridization histochemistry, real-time quantitative RT-PCR, cDNA microarray analysis, in vivo lentivirus gene transfection, FDG microPET, optical intrinsic signal imaging and behavioral modification. These techniques have been taught to 1 UCLA faculty member, 1 visiting scientist, 3 postdoctoral fellows, 2 graduate students, and 7 technicians or medical students. It takes approximately 6 weeks to teach the stroke model and an associated analytical technique. The lab has recently used this approach of studying damage in the highly defined neural circuits of the rodent barrel field to develop a model of traumatic brain injury in this region with other UCLA faculty members.
Leif Havton, MD, PhD, Assistant Professor of Neurology at UCLA studies neural repair after SCI with a particular interest in neural repair after conus medullaris/ cauda equina injuries. A lumbosacral ventral root avulsion model for the study of conus medullaris/ cauda equina injuries has been developed in my laboratory (Hoang et al, J Comp Neurol, 2003). We have also developed a repair strategy where avulsed ventral roots are surgically implanted into the conus to protect injured neurons against retrograde death and to promote regeneration of axons into the implanted roots for ultimate reinnervation of peripheral autonomic and somatic targets, including the lower urinary tract (Hoang et al., 2004, submitted). In addition, he has begun pharmacological studies in attempts to identify compounds/ agents that may exert neuroprotective effects in attempts to increase the therapeutic time window for root implantation after injury. In a chronic injury model, he implants adult human neural stem cells into the conus medullaris in attempts to replace lost neurons with new motoneurons and autonomic neurons. A variety of functional outcomes with a variety of outcome measures – these included open field locomotor tests, including the BBB scale, urodynamic studies (cystometrogram with simultaneous urethral EMG recordings), pain behavioral studies (threshold responses to non-noxious mechanical stimulation, heat stimulation, cold stimulation, pressure stimulation). The lab also performs functional imaging studies, including optical imaging of intrinsic signals and spectroscopy of the spinal cord. Additional outcome measures include morphological studies to quantify various aspects of sprouting, and neuronal death. Electron microscopy with a camera lucida technique is used to determine the fine structure of synaptic contacts with spinal cord neurons and regenerating arbors after injury and repair. Cell and molecular studies include gene expression analyses, including in situ hybridization.
Ongoing collaborations are in place with researchers at UCLA and other sites who will serve as associate faculty: Dr. Arthur Toga (UCLA, small animal imaging, optical imaging), Dr. V. Reggie Edgerton (UCLA, spinal cord locomotor anatomical studies), Fernando Gomez-Pinilla (neurotrophins and rehab interventions in sci), Dr. Rhonda Voskuhl (UCLA, multiple sclerosis), Dr. Harley Kornblum (UCLA, stem cells), Dr. Daniel Geschwind (UCLA, gene microarrays), Dr. Victor Pikov (Huntington Medical Research Center, urodynamics in rodents), Dr. Mark Tuszynski (UCSD, non-human primate spinal cord injury model), and Jonas Broman (Lund, Sweden) and Mikael Svensson (Stockholm, Sweden, stem cell implant studies). He also collaborates with the California National Primate Research Center at UC Davis for the translation of the rat conus medullaris/ cauda equina injury and repair model to the primate. The outcome from the primate studies will guide decisions on a possible clinical trial in humans involving root implantation after acute cauda equina/ conus medullaris injuries.
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