Jennifer Flexman

Statistical mapping of functional olfactory connections of the rat brain in vivo

The olfactory pathway is a unique route into the brain. To better characterize this system in vivo, rat olfactory functional connections were mapped using magnetic resonance (MR) imaging and manganese ion (Mn2+) as a transport-mediated tracer combined with newly developed statistical brain image analysis. Six rats underwent imaging on a 1.5-T MR scanner at pre-administration, and 6, 12, 24, 36, 48, and 72 h and 5.5, 7.5, 10.5, and 13.5 days post-administration of manganese chloride (MnCl2) into the right nasal cavity. Images were coregistered, pixel-intensity normalized, and stereotactically transformed to the Paxinos and Watson rat brain atlas, then averaged across subjects using automated image analysis software (NEUROSTAT). Images at each time point were compared to pre-administration using a one-sample t statistic on a pixel-by-pixel basis in 3-D and converted to Z statistic maps. Statistical mapping and group averaging improved signal to noise ratios and signal detection sensitivity. Significant transport of Mn2+ was observed in olfactory structures ipsilateral to site of Mn2+ administration including the bulb, lateral olfactory tract (lo) by 12 h and in the tubercle, piriform cortex, ventral pallidum, amygdala, and in smaller structures such as the anterior commissure after 24 h post-administration. MR imaging with group-wise statistical analysis clearly demonstrated bilateral transsynaptic Mn2+ transport to secondary and tertiary neurons of the olfactory system. The method permits in vivo investigations of functional neuronal connections within the brain.

Transfection of Neuroprogenitor Cells with Iron Nanoparticles for Magnetic Resonance Imaging Tracking: Cell Viability, Differentiation, and Intracellular Localization

PurposeMagnetic resonance imaging (MRI) can track labeled cells in the brain. The use of hemagglutinating virus of Japan envelopes (HVJ-Es) to effectively introduce the contrast agent to neural progenitor cells (NPCs) is limited to date despite their high NPC affinity.ProceduresHVJ-Es and Lipofectamine 2000 were compared as transfection vehicles of superparamagnetic iron oxide (SPIO). Labeled NPCs were examined for iron content, MRI signal change, and fundamental cell characteristics. Prussian Blue staining was used after differentiation to determine SPIO localization.ResultsHVJ-Es transfected up to 12.5 ± 8.8 times more SPIO into NPCs. HVJ-Es do not affect cell viability or differentiation capability. Superparamagnetic iron oxide was disseminated in both the soma and neurites.ConclusionsThese findings indicate that HVJ-Es are an effective vehicle for SPIO transfection of NPCs. The intracellular localization after differentiation raises the question as to the capability of MRI to distinguish cell migration from axonal or dendritic growth in vivo.

Age-related decrease in axonal transport measured by MR imaging in vivo

Axonal transport is a crucial process for neuronal homeostasis and cell functions. In vitro studies have indicated transport rates decrease with age. Disruption of axonal transport has been implicated in age-associated neurodegenerative disorders. We hypothesized that aged rats would show decreased transport in the brain, which could be measured using in vivo manganese-enhanced MR imaging (Mn-MRI) and parametric estimation. Serial T1-weighted images were obtained at pre- and post-administration of MnCl(2) in rats scanned longitudinally (n=4) and in a separate aged group (n=3). Subtraction analysis was performed for group-wise statistical comparison on a pixel-by-pixel basis. Change in intensity over time was plotted for the olfactory bulb and anterior and posterior olfactory tract. Bulk transport of material was estimated over an initial 72 h. Tracer kinetic estimation of time-intensity data, based on a mass transport model, used intensity change in the bulb as input function for subsequent changes in the tract. Time to the peak of Mn(2+) flow was estimated for both anterior and posterior tracts. Results indicated age-related decreases in axonal transport rate and bulk transport of material in the olfactory tract of living rat brains. Longitudinally scanned, mid-age group was decreased by 58% and the aged group by 71% of young rate (neuronal transport=4.07+/-1.24 mm/h, 1.72+/-0.89 mm/h, and 1.16+/-0.18 mm/h for young, mid-age, and aged, respectively). Neuronal transport rate decreases correlated with increased age. The use of kinetic analysis combined with dynamic manganese enhanced MR imaging provides a unique opportunity to study this important neuronal process.

In vivo manganese MR imaging of calcium influx in spontaneous rat pituitary adenoma.

BACKGROUND AND PURPOSE: Rapid uptake of the calcium analog manganese (Mn2+) into spontaneous pituitary adenoma during MR imaging of aged rats generated the hypothesis that neuroendocrine tumors may have a corresponding increase in calcium influx required to trigger hormonal release. A goal of this study was to investigate the potential for clinical evaluation of pituitary adenoma by MR imaging combined with administration of Mn2+ (Mn-MR imaging). MATERIALS AND METHODS: Mn-MR imaging was used to characterize the dynamic calcium influx in normal aged rat pituitary gland as well as spontaneous pituitary adenoma. To confirm the validity of Mn2+ as a calcium analog, we inhibited Mn2+ uptake into the olfactory bulb and pituitary gland of normal rats by using the calcium channel blocker verapamil. Rats with adenomas received fluorodeoxyglucose–positron-emission tomography (FDG-PET) scanning for characterization of tumor metabolism. Mn2+ influx was characterized in cultured pituitary adenoma cells. RESULTS: Volume of interest analysis of the normal aged pituitary gland versus adenoma indicated faster and increased calcium influx in adenoma at 1, 3, 11, and 48 hours. Mn2+ uptake into the olfactory bulb and pituitary gland of normal rats was inhibited by calcium channel blockers and showed dose-dependent inhibition on dynamic MR imaging. FDG-PET indicated correlation between tumor energy metabolism and Mn2+ influx as well as tumor size. CONCLUSION: These results indicate that adenomas have increased activity-dependent calcium influx compared with normal aged pituitary glands, suggesting a potential for exploitation in the clinical work-up of pituitary and other neuroendocrine tumors by developing Mn-MR imaging for humans.

In vivo imaging of functional disruption, recovery and alteration in rat olfactory circuitry after lesion

Compensatory changes following disruption of neuronal circuitry have been indicated by previous imaging studies of stroke and other brain injury, but evidence of the pathways involved in such dynamic changes has not been shown in vivo. We imaged rats before and after lesion-induced disruption of the lateral olfactory tract to investigate the subsequent recovery and/or reorganization of functional neuronal circuitry. Serial magnetic resonance imaging was performed following intranasal administration of a paramagnetic track tracer Mn(2+). Images were analyzed using statistical mapping techniques in the stereotactic coordinate system. At 1 week post-lesion, Mn(2+) transport caudal to lesion was reduced as expected, and more importantly, increased transport through the anterior commissure was seen. At 4 weeks post-lesion, there was recovery of transport caudal to lesion, and increased transport through the anterior commissure extended to the contralateral olfactory cortex. Correlation analysis of regional Mn(2+) transport indicated that contralateral enhancement was not simply due to septal window spillover. This study demonstrates for the first time in vivo evidence of compensatory changes in functional neuronal activity to a contralateral pathway through the commissure following brain injury.

Magnetically Targeted Viral Envelopes: A PET Investigation of Initial Biodistribution

Gene and drug therapy for organ-specific diseases in part depends on the efficient delivery to a particular region of the body. We examined the biodistribution of a viral envelope commonly used as a nanoscale gene delivery vehicle using positron emission tomography (PET) and investigated the magnetic alteration of its biodistribution. Iron oxide nanoparticles and 18F-fluoride were encapsulated by hemagglutinating virus of Japan envelopes (HVJ-Es). HVJ-Es were then injected intravenously in the rat and imaged dynamically using high-resolution PET. Control subjects received injections of encapsulated materials alone. For magnetic targeting, permanent magnets were fixed on the head during the scan. Based on the quantitative analysis of PET images, HVJ-Es accumulated in the liver and spleen and activity remained higher than control subjects for 2 h. Histological sections of the liver confirmed imaging findings. Pixel-wise activity patterns on coregistered PET images of the head showed a significantly different pattern for the subjects receiving magnetic targeting as compared to all control groups. Imaging demonstrated the initial biodistribution of a viral envelope within the rodent by providing quantitative behavior over time and in specific anatomical regions. Magnetic force altered the biodistribution of the viral envelope to a target structure, and could enable region-specific delivery of therapeutic vehicles noninvasively.

Assessment of vessel size by MRI in an orthotopic model of human pancreatic cancer

Pancreatic cancer is a devastating disease with no cure. Therapies that target the tumor vasculature are promising new treatment strategies. Magnetic resonance imaging (MRI) can non-invasively determine a vessel size index and a blood volume fraction to characterize the vascular compartment in a tumor. The changes in the T2 and T2* relaxation rate constants after the administration of superparamagnetic iron oxide (SPIO) particles are dependent on the size and morphology of tissue blood vessels. In this study, MRI was used to investigate changes in the tumor vesculature in an orthotopic primary human pancreatic cancer xenograft model during tumor progression. The SPIO contrast agent Feridex I.V. was first validated as an intravascular contrast agent over the course of the imaging session, and shown to remain in the blood for at least 1.5 h. The average vessel size index was not correlated to the tumor area within an image slice, but the average blood volume fraction was significantly and negatively correlated to the tumor area (p<0.05). Blood volume fraction may serve as a non-invasive biomerker for changes in the tumor vasculature due to tumor growth Further investigation is needed to evaluate this promising technique as a tool to monitor tumor vascular changes in response to sntiengiogenic therapies in pancreatic cancer.

Magneto-Optical Labeling of Fetal Neural Stem Cells for in vivo MRI Tracking

Neural stem cell therapy for neurological pathologies, such as Alzheimers and Parkinsons disease, may delay the onset of symptoms, replace damaged neurons and/or support the survival of endogenous cells. Magnetic resonance imaging (MRI) can be used to track magnetically labeled cells in vivo to observe migration. Prior to transplantation, labeled cells must be characterized to show that they retain their intrinsic properties, such as cell proliferation into neurospheres in a supplemented environment. In vivo images must also be correlated to sensitive, histological markers. In this study, we show that fetus-derived neural stem cells can be co-labeled with superparamagnetic iron oxide and PKH26, a fluorescent dye. Labeled cells retain the ability to proliferate into neurospheres in culture, but labeling prevents neurospheres from merging in a non-adherent culture environment. After labeled NSCs were transplantation into the rat brain, their location and subsequent migration along the corpus callosum was detected using MRI. This study demonstrates an imaging paradigm with which to develop an in vivo assay for quantitatively evaluating fetal neural stem cell migration

Biomedical Engineering and Society: Policy and Ethics

Biomedical engineering impacts health care and contributes to fundamental knowledge in medicine and biology. Policy, such as through regulation and research funding, has the potential to dramatically affect biomedical engineering research and commercialization. New developments, in turn, may affect society in new ways. The intersection of biomedical engineering and society and related policy issues must be discussed between scientists and engineers, policy-makers and the public. As a student, there are many ways to become engaged in the issues surrounding science and technology policy. At the University of Washington in Seattle, the Forum on Science Ethics and Policy (FOSEP, www.fosep.org) was started by graduate students and post-doctoral fellows interested in improving the dialogue between scientists, policymakers and the public and has received support from upper-level administration. This is just one example of how students can start thinking about science policy and ethics early in their careers.

Morphological and Parametric Estimation of Fetal Neural Stem Cell Migratory Capacity in the Rat Brain

Magnetic resonance imaging (MRI) can non- invasively monitor the migratory behavior of magnetically labeled stem cells after transplantation. Signal changes associated with the clearance of the contrast agent due to cell death and leaked tracer in the interstitial space must be better understood in order to accurately interpret imaging results. In this study, fetal neural stem cells were labeled with superparamagnetic iron oxide (SPIO) particles and transplanted into the corpus callosum of the adult rat. MRI was performed on the day of transplantation and at one week. Control subjects received injections of either non-viable, labeled cells or loose SPIO particles. Two quantitative image analysis algorithms were developed to evaluate imaging results: 1) signal intensity drop-out areas were segmented and compared on a pixel-wise basis between initial and one week images; and 2) signal intensity profiles of transplanted materials at one week were parametrically modeled to estimate migration speed. Segmentation results showed that the number of pixels segmented at one week was significantly greater than the initial number of segmented pixels for subjects receiving injections of viable cells as compared to controls (p<0.05). The average speed of migration of viable cells along the corpus callosum was 69.2plusmn41.1 mum/d and was significantly higher than controls (p<0.05). This study demonstrates an in vivo assay to quantitatively evaluate stem cell migration that can be used in different experimental paradigms.