There is rapidly increasing interest in developing molecular imaging approaches that enable traditional radiological imaging techniques to obtain a wide range of information about molecular and cellular processes that occur in normal and diseased tissue. A range of information is considered important such as the ability to monitor cell migration, the development of reporters that enable imaging of gene expression, the development of robust strategies to image receptors, and the development of environmentally sensitive agents that can be used to detect the presence of specific enzymes or monitor changes in ion status. The long term goals of this work are to develop strategies that enable MRI contrast that is sensitive to a wide range of molecular and cellular processes. This work builds on over 15 years of work where we have demonstrated the first MRI strategy for detecting gene expression, the first MRI approach for monitoring a surrogate of calcium influx, the first MRI approach for performing neuronal track tracing, and the first MRI approach for monitoring the migration of single cells in vivo. These all represented initial reports by any radiological imaging technique which enabled these processes to be measured. These techniques are finding widespread application to imaging pre-clinical models of a broad range of diseases. Over the past year we have made progress in all of the specific aims.
Aim 1 : Develop iron oxide based contrast for labeling and imaging the migration of endogenous neural stem cells. Over the past few years we have demonstrated the unique advantages of micron sized iron oxide particles for MRI of specific cells. Single cells can be detected and indeed, single particles within single cells can be detected. The main paradigm for MRI of cell migration is to label cells ex vivo and monitor migration after transplantation into an animal. These studies have traditionally required very efficient labeling using nano sized particles. The ability to detect a single particle enables inefficient labeling strategies. In particular, over the past few years we have demonstrated that injection of particles into the ventricles of the rat brain enables particles to be taken up by neural precursors in the subventricular zone and MRI can monitor the migration of cells to the oflactory bulb. Over the past year we have completed a study to determine if daily exposure to odor for two weeks affects the migration of these new cells. The only significant effect was an increase in number of new neurons in the mitral cell layer of the olfactory bulb. These cells have been shown to be simular to granule layer interneurons. We have begun to monitor the response to odor deprivation using naris occlusion. In addition, we are measuring the migratory rates of cells to determine if these rates are ever modulated. We are extending our ability to image the migration of single cells through the entire brain to study immune brain interactions in a model of virus infection. This work has required working out effective strategies to label T cells, a population of cell that has been very difficult to label. This offers the unique potential to follow the low level peripheral immune surveillance that occurs in the normal adult brain as well as any changes due to inflammation or degeneration.
Aim 2 : Apply microfabrication techniques to manufacture unique metal structures that may be valuable for MRI contrast. Iron oxide particles commonly used for MRI are very potent contrast agents enabling detection of single mciron sized particles. However, due to bulk phase manufacture of particles they are not very uniform and they do not contain very high content of metal. A solution to this problem is to use modern microfabrication techniques to manufacture metal based, micron sized contrast agents. To begin this work we have explored a variety of approachs to microfabrication oof MRI contrast agents. Over the past few years we have shown that double dougnut and cylinder structures offer unique advantages for distinguishing particles. Microfabriaction of simple iron discs lead to 10 times more potent contrast than presently available particles. Over the past year wein order to translate this work to tracking cells we have developed strategies to effectively gold coat the particles enbaling stability and biocompatibility in living cells. Furthermore, we have demonstrated that we can accurately locate these microfabricated particles to accuracy abotu a facotr of two higher than than the pixel resolution enabling very high determination of the location. This will enable tracking of particles or cell loaded particles to higher resolution than is available from standard MRI. Finally, we have explored new shapes that give interesting MRI properties.
Aim 3 : Develop novel delivery mechanisms to extend the applicability of manganese enhanced MRI. Over the past ten years we have demonstrated the remarkable utility of the manganese ion for MRI contrast. Manganese ion enters cells on ligand or voltage gated calcium channels and so can be used as an MRI agent to monitor calcium influx. Once inside of neurons, manganese will move in an anterograde direction and cross functional synapses enabling neuronal networks to be imaged with MRI. Finally, manganese given systemically gives cytoarchitectural information about the rodent brain. These successes have us interested in broadening the ways in which manganese ion can be delivered to cells. Over the past couple of years we have made transferrin-manganese complexes. When bound to transferrin manganese is a poor MRI contrast agent. However, when transferrin is taken up by cells it can release manganese which is then trapped intracellularly. Thus, transferrin manganese is an agent that monitors the successful endocytosis of the transferrin by its receptor. We have demonstrated the same effects with MnOxide based nanoparticles. At pH 7 MnO is insoluble and a very weak contrast agent. At low pH, as found in endosomes/lysosomes these particles dissolve greatly increasing MRI relaxation effects. We have completed studies that show that a silica coat on these particles delays dissolution for up to four hours both in vitro and in vivo. Particles injected into the brain had slower rates of contrast development and neuronal tracing then did injection of MnCl2. This opens the possibility of making coatings that can be enzymatically degraded enabling specific in vivo assay of these enzymes. We have demonstrated another approach to makin Mn nanoparticles using block co-polymer synthesis. The first generation of these agents have very high relaxivities and the relaxivity can be modulated. Finally, we have begun to explore ways to translate the advantages of Manganese enhanced MRI to human use one makes use of manganese postiron emitting isotopes that will enable PET to obtain similar information that can be obtained with manganese enhanced MRI.
Aim 4 : Develop strategies that enable cellular processes to alter the relaxivity of MRI contrast agents.
In specific aim 3 we demonstrated a way in which a normal biological process (endocytosis of transferrin-Mn or MnO particles) can alter the effectiveness of an MRI contrast agent. It would be very exciting to find ways in which this can occur which are sensitive to other biological processes. To this end we have begun to explore ways in which the microfabricated particles produced under Aim 2 can be modulated. Over the past year we have confirmed that the microfabricated particles can be made into a pH sensor . The strategy used is generalizable to sense many other processes. The block co-polymer agents offer many possibilities for making environmentally sensitive MRI agents and these will be explored over the next year.
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