Progress 01/01/07 to 09/30/12
Outputs
OUTPUTS: Conducted laboratory experiments and completed analysis of responses of glial cells in rodent optic nerve to N-acetylaspartylglutamate (NAAG), glutamate and ATP and of human adipose-derived stem cells (hADSC) to ATP, histamine and glutamate. Trained and mentored 3 graduate students and approximately 12 undergraduate students who have gone on to research or bio-medicine professional careers. PARTICIPANTS: The Principal Investigator directed the design and implementation of experiments and data analysis, and trained and mentored student participants. The following graduate students participated: Dr. Scott Dobrin, currently Assistant Professor at the U. of Maine-Presque Isle, transferred imaging technology from England to this lab and conducted experiments on optic nerve responses to NAAG and glutamate and on changes in NAAG peptidase activity during optic development and regeneration; Dr. Joshua Cooper, currently medical resident at The Ohio State University Medical Center, conducted data analyses of optic nerve; and Master Philip Hall, currently a medical student in Missouri, conducted initial experiments on responses of hADSC to ATP and glutamate. The following undergraduate students conducted experiments and data analysis on responses of optic nerve glia and/or of hADSC to small signaling agents or of changes in NAAG peptidase activity during optic nerve development and regeneration: Hamish Patel, Kareem Clark, Hitesh Shivalingappa, Cole Andrew, Margaret Carnes (all currently medical students), Christopher Gambino (currently in a bio-agricultural doctoral program at Washington State University, Dr. Jessica Sherman (veterinarian), Dr. Jacob Hampton (optometrist), Sarah Cunningham (genetic counselor), Bradley Poston (research technician at The University of North Carolina), Andrea Dorsch (Biology instructor at a community college), and Samantha Goodwin and Heidi Klumpe (currently undergraduates pursuing careers in biotechnology and bio-engineering, respectively). TARGET AUDIENCES: Target audiences are research scientists, particularly those engaged in research on tissue development, repair and regeneration, and classroom instruction on cell culture and imaging of cellular responses to test compounds in real time. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The research contributed to a change of fundamental knowledge about mechanisms of cellular signaling between functional cells in optic nerve as a well-accepted model for brain and spinal cord and of the possible contributions of the same small signaling agents to the differentiation and viability of stem cells that offer the potential for repair of nervous and musculo-skeletal tissues.
Publications
- No publications reported this period
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: ATP, glutamate and histamine are universal signaling agents that regulate cell development and function across many diverse tissues, including neural and mesenchymal tissues. In vertebrate nervous tissue, glial cells greatly outnumber neurons and modulate neuronal output and blood flow. Understanding normal and abnormal intercellular glial-neuronal interactions in nerves mediated by ATP, glutamate and/or histamine has important basic and therapeutic implications for improving function and health in invertebrate and vertebrate animals. In addition, identifying small molecule transmitters that can activate stem cells can facilitate application of those cells for regenerative medicine. Glial cells of rat optic nerves were selectively labeled with a Ca2+ indicator dye for microscopic imaging of their responses in vitro to treatment of the nerves with ATP or glutamate. Adult adipose-derived stem cells (ADSC) with the potential to differentiate into nerve cells, chondrocytes or osteocytes were treated similarly in culture. PARTICIPANTS: The PI, Robert Grossfeld, supervised the research. In the process, he trained and supervised four undergraduate students in conducting research on responses of optic nerve glial cells and three in conducting research on adipose derived stem cells. Several of these students are planning careers that are primarily invested in research or that combine research with the practice of medicine. TARGET AUDIENCES: The primary target audiences for this research are neuroscientists pursuing basic and translational work on brain and spinal cord injury and repair and scientists and physicians engaged in studies to improve prospects for regenerative medicine for humans and/or animals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Glial cells of neonatal rat optic nerve responded to ATP and glutamate with an increase in cytoplasmic calcium, as did ADSC treated with ATP or histamine. Extracellular calcium was the primary source for the response of ADSC to histamine whereas intracellular calcium was the primary source for the response to ATP. The responses of ADSC were dependent on cell culture conditions. Specific antagonists of these receptors were identified for further use in determining the impact of these transmitters on cellular function and differentiation. Application of this knowledge offers the potential to influence nerve development and differentiation of stem cells for tissue repair.
Publications
- No publications reported this period
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Toward the aims of this project, we have further examined dose-response results for the actions of ATP, glutamate and NAAG on nerve glial cells, and effects of specific receptor blockers on those responses. We also have further examined the relative effects of NAAG vs. glutamate, as these related compounds are reported to have some actions in common owing to the glutamate content of the commercial dipeptide NAAG. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We have completed reevaluation of the impact of NAAG on neuron-glia interactions in rodent optic nerve. A substantial literature developed over the past three decades has implicated NAAG as an important neurotransmitter or modulator in central nervous system that influences pain responses, nerve degeneration, and addictive and other behaviors. During 2009, two publications appeared questioning whether the primary action of administered NAAG is due to the compound itself, either in the intact form or as glutamate formed from its hydrolysis, or to a small amount of glutamate remaining in commercial NAAG stocks. We have now completed additional experiments that indicate that most, if not all,of the activity of the NAAG samples can be accounted for by its small but finite content of glutamate. However, we have not been able to definitively rule out the possibility that NAAG could account for a small fraction of the biological activity of our samples. We are pursuing studies of the action of glutamate on the glial cells as a possible major neuron-glia signaling agent in CNS nerves.
Publications
- No publications reported this period
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Toward the aims of this project, we have examined dose-response results for the action of ATP, glutamate and NAAG on nerve glial cells, and effects of specific receptor blockers on those responses. We also have examined the relative effects of NAAG vs. glutamate, as these compounds are reported to have some actions in common owing to the glutamate content of the dipeptide NAAG. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We have reevaluated the impact of NAAG on neuron-glia interactions in central nervous system nerves. A substantial literature that has developed over the past three decades has implicated NAAG as an important neurotransmitter or neuromodulator in CNS and possibly in peripheral nervous tissue as well. During 2009, two publications appeared questioning whether the primary action of administered NAAG is due to the compound itself, either in the intact form or as glutamate formed from its hydrolysis, or to a small amount of glutamate remaining in commercial NAAG stocks. We also have found that much or all of the activity of the NAAG samples in our experiments could be accounted for by its small but significant content of glutamate. However, that does not rule out the possibility that NAAG itself is biologically active in our preparations. We are currently pursuing further experimentation to answer that question conclusively.
Publications
- No publications reported this period
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: ATP and glutamate have both beneficial and detrimental effects on cellular function throughout the body. Besides serving as essential cellular fuels, they also mediate physiological intercellular communication in nervous and non-nervous tissues. However, at high extracellular concentrations that may occur with injury or vascular compromise, they may cause cell damage or death. N-acetylaspartylglutamate (NAAG), a dipeptide that forms glutamate upon hydrolysis, has been found to have similar neuroprotective and neurotoxic properties. Much remains to be learned about the effects of these substances on development and differentiation of cells, e.g. glial cells of nerve fiber tracts. Glial cells constitute the large majority of cells in the nervous system, where they regulate neuronal and vascular function. Nerve fiber tracts are the cellular highways that enable electrical and chemical communication throughout a nerve cell. Understanding normal and deranged signaling processes in nerves mediated by ATP, glutamate and NAAG has important basic and therapeutic implications for improving function and health in invertebrate and vertebrate animals. Glial cells of rat optic nerves were labeled with a Ca2+ indicator dye for microscopic imaging of their responses in vitro to treatment of the nerves with ATP, glutamate or NAAG. Adult adipose derived stem cells with the potential to differentiate into nerve cells, chondrocytes or osteocytes were treated similarly. Data were analyzed with imaging software that facilitates quantitation of responding cells in a series of digital frames in comparison with a preceding control period. Transfer of the data to a speadsheet allowed examination of the kinetics of the response by individual selected cells. In addition, gelfoam saturated with ATP or glutamate was implanted in neonatal rats to evaluate whether chronic delivery of the test substance in vivo at a critical stage in nerve development affects glial cell viability or proliferation. PARTICIPANTS: Experiments on optic and sciatic nerve responsiveness to ATP and glutamate were conducted by two Biology undergraduate researchers, Deborah L. Smith and William C. Andrew. They are considering a research component to complement their intended professional development in medical school. Experiments on stem cells were performed by an undergraduate researcher, John P. McQuilling in Dr. Grossfeld's laboratory in collaboration with graduate (Seth McCullen) and undergraduate assistants in Dr. Elizabeth G. Loboa's Biomedical Engineering laboratory, also at North Carolina State University. Mr. McQuilling is now planning to pursue biomechanics research in graduate school upon completion of his Bachelor's degree in Biomedical Engineering. TARGET AUDIENCES: The primary target audiences are researchers interested in intercellular chemical communication in the nervous system, nerve development, and pathophysiology of the nervous system, and researchers interested in stem cell applications for tissue engineering and tissue repair. PROJECT MODIFICATIONS: The only major change in the project was to initiate experiments with adult adipose-derived stem cells. The rationale was to determine whether intercellular signaling via small diffusible molecules is a general process by which differentiation and function of a variety of cell types is specified and a potentially important application of the research to live animals.
Impacts
Glial cells of neonatal rat optic nerve responded to ATP and glutamate with an increase in cytoplasmic calcium. Glial cell receptors were identified for several types of ionotropic and metabotropic glutamate receptors. NAAG produced an effect like that to glutamate but at a higher concentration, both under conditions that would minimize its hydrolysis and conditions that would promote its enzymatic hydrolysis to form glutamate in situ. This suggests that both intact NAAG and glutamate released from NAAG are potential neuron-glia signaling agents in the intact nerve. The effect of ATP was largely, but not completely, blocked by antagonists of glutamate receptors, suggesting that much, but not all, of the response to this substance is mediated through cellular release of glutamate. ATP and glutamate had similar effects on adult adipose derived stem cells, suggesting a general link to cellular differentiation among a variety of cell types. These responses were dependent on cell culture conditions and were mimicked by application of a weak electrical field. Application of this knowledge offers the potential to influence nerve development and differentiation of stem cells for tissue repair.
Publications
- McCullen SD, McQuilling JP, Clarke LI, Gorga RE, van Aalst JA, Grossfeld RM, Loboa EG (Nov 2008). Electric field exposure induces frequency dependent Ca2+ oscillations in human adipose-derived adult stem cells. 10th Annual North Carolina Tissue Engineering and Regenerative Medicine (NCTERM) Conference, Raleigh, NC.
- McCullen SD, McQuilling JP, Gorga RE, Grossfeld RM, Clarke LI, Loboa EG (Feb 2009). Electric field exposure induces magnitude-dependent Ca2+ oscillations in human adipose-derived adult stem cells. Proceedings of the 55th Annual Meeting of the Orthopaedic Research Society, Las Vegas, NV.
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