Genetic control of MRI contrast using the manganese transporter Zip14.

PURPOSE: Gene-expression reporter systems, such as green fluorescent protein, have been instrumental to understanding biological processes in living organisms at organ system, tissue, cell, and molecular scales. More than 30 years of work on developing MRI-visible gene-expression reporter systems has resulted in a variety of clever application-specific methods. However, these techniques have not yet been widely adopted, so a general-purpose expression reporter is still required. Here, we demonstrate that the manganese ion transporter Zip14 is an in vivo MRI-visible, flexible, and robust gene-expression reporter to meet this need. METHODS: Plasmid constructs consisting of a cell type-specific promoter, gene coding for human Zip14, and a histology-visible tag were packaged into adeno-associated viruses. These viruses were intracranially injected into the mouse brain. Serial in vivo MRI was performed using a vendor-supplied 3D-MPRAGE sequence. No additional contrast agents were administered. Animals were sacrificed after the last imaging timepoint for immunohistological validation. RESULTS: Neuron-specific overexpression of Zip14 produced substantial and long-lasting changes in MRI contrast. Using appropriate viruses enabled both anterograde and retrograde neural tracing. Expression of Zip14 in astrocytes also enabled MRI of glia populations in the living mammalian brain. CONCLUSIONS: The flexibility of this system as an MRI-visible gene-expression reporter will enable many applications of serial, high-resolution imaging of gene expression for basic science and therapy development.

Dynamic 3D imaging of cerebral blood flow in awake mice using self-supervised-learning-enhanced optical coherence Doppler tomography.

Cerebral blood flow (CBF) is widely used to assess brain function. However, most preclinical CBF studies have been performed under anesthesia, which confounds findings. High spatiotemporal-resolution CBF imaging of awake animals is challenging due to motion artifacts and background noise, particularly for Doppler-based flow imaging. Here, we report ultrahigh-resolution optical coherence Doppler tomography (µODT) for 3D imaging of CBF velocity (CBFv) dynamics in awake mice by developing self-supervised deep-learning for effective image denoising and motion-artifact removal. We compare cortical CBFv in awake vs. anesthetized mice and their dynamic responses in arteriolar, venular and capillary networks to acute cocaine (1 mg/kg, i.v.), a highly addictive drug associated with neurovascular toxicity. Compared with awake, isoflurane (2-2.5%) induces vasodilation and increases CBFv within 2-4 min, whereas dexmedetomidine (0.025 mg/kg, i.p.) does not change vessel diameters nor flow. Acute cocaine decreases CBFv to the same extent in dexmedetomidine and awake states, whereas decreases are larger under isoflurane, suggesting that isoflurane-induced vasodilation might have facilitated detection of cocaine-induced vasoconstriction. Awake mice after chronic cocaine show severe vasoconstriction, CBFv decreases and vascular adaptations with extended diving arteriolar/venular vessels that prioritize blood supply to deeper cortical capillaries. The 3D imaging platform we present provides a powerful tool to study dynamic changes in vessel diameters and morphology alongside CBFv networks in the brain of awake animals that can advance our understanding of the effects of drugs and disease conditions (ischemia, tumors, wound healing).

Optical imaging of stimulation-evoked cortical activity using GCaMP6f and jRGECO1a.

BACKGROUND: Genetically encoded calcium indicators (GECIs), especially the GCaMP-based green fluorescence GECIs have been widely used for in vivo detection of neuronal activity in rodents by measuring intracellular neuronal Ca2+ changes. More recently, jRGECO1a, a red shifted GECI, has been reported to detect neuronal Ca2+ activation. This opens the possibility of using dual-color GECIs for simultaneous interrogation of different cell populations. However, there has been no report to compare the functional difference between these two GECIs for in vivo imaging. Here, a comparative study is reported on neuronal responses to sensory stimulation using GCaMP6f and jRGECO1a that were virally delivered into the neurons in the somatosensory cortex of two different groups of animals, respectively. METHODS: GCaMP6f and jRGECO1a GECI were virally delivered to sensory cortex. After 3-4 weeks, the animals were imaged to capture the spatiotemporal changes of neuronal Ca2+ and the hemodynamic responses to forepaw electrical stimulation (0.3 mA, 0.3 ms/pulse, 0.03 Hz). The stimulation-evoked neuronal Ca2+ transients expressed with GCaMP6f or jRGECO1a were recorded during the baseline period and after an acute cocaine administration (1 mg/kg, i.v.). RESULTS: Histology confirmed that the efficiency of jRGECO1a and GCaMP6f expression into the cortical neurons was similar, i.e., 34%±3% and 32.7%±1.6%, respectively. Our imaging in vivo showed that the hemodynamic responses to the stimulation were the same between jRGECO1a and GCaMP6f expressed groups. Although the stimulation-evoked fluorescence change (∆F/F) and the time-to-peak of the neuronal Ca2+ transients were not significantly different between these two indicators, the full-width-half-maximum (FWHM) duration of the ∆F/F rise in the jRGECO1a-expressed group (0.16±0.02 s) was ~50 ms or 46% longer than that of the GCaMP6f group (0.11±0.003 s), indicating a longer recovery time in jRGECO1a than in GCaMP6f transients (P<0.01). This is likely due to the longer off rate of jRGECO1a than that of GCaMP6f. After cocaine, the time-to-peak of Ca2+ transients was delayed and their FWHM duration was prolonged for both expression groups, indicating that these are cocaine's effects on neuronal Ca2+ signaling and not artifacts due to the property differences of the GCEIs. CONCLUSIONS: This study shows that both jRGECO1a and GCaMP6f have sufficient sensitivity for tracking single-stimulation-evoked Ca2+ transients to detect neuronal activities from the brain. Since these GECIs are emitted at the different wavelengths, it will be possible to use them together to characterize the activity of different cell types (e.g., neurons and astrocytes) to study brain activation and brain functional changes in normal or diseased brains.

Ex vivo MR microscopy of a human brain with multiple sclerosis: Visualizing individual cells in tissue using intrinsic iron.

PURPOSE: To perform magnetic resonance microscopy (MRM) on human cortex and a cortical lesion as well as the adjacent normal appearing white matter. To shed light on the origins of MRI contrast by comparison with histochemical and immunostaining. METHODS: 3D MRM at a nominal isotropic resolution of 15 and 18 µm was performed on 2 blocks of tissue from the brain of a 77-year-old man who had MS for 47 years. One block contained normal appearing cortical gray matter (CN block) and adjacent normal appearing white matter (NAWM), and the other also included a cortical lesion (CL block). Postmortem ex-vivo MRI was performed at 11.7T using a custom solenoid coil and T2*-weighted 3D GRE sequence. Histochemical and immunostaining were done after paraffin embedding for iron, myelin, oligodendrocytes, neurons, blood vessels, macrophages and microglia, and astrocytes. RESULTS: MRM could identify individual iron-laden oligodendrocytes with high sensitivity (70% decrease in signal compared to surrounding) in CN and CL blocks, as well as some iron-laden activated macrophages and microglia. Iron-deficient oligodendrocytes seemed to cause relative increase in MRI signal within the cortical lesion. High concentration of myelin in the white matter was primarily responsible for its hypointense appearance relative to the cortex, however, signal variations within NAWM could be attributed to changes in density of iron-laden oligodendrocytes. CONCLUSION: Changes in iron accumulation within cells gave rise to imaging contrast seen between cortical lesions and normal cortex, as well as the patchy signal in NAWM. Densely packed myelin and collagen deposition also contributed to MRM signal changes. Even though we studied only one block each from normal appearing and cortical lesions, such studies can help better understand the origins of histopathological and microstructural correlates of MRI signal changes in multiple sclerosis and contextualize the interpretation of lower-resolution in vivo MRI scans.

Endosphenoidal coil for intraoperative magnetic resonance imaging of the pituitary gland during transsphenoidal surgery.

OBJECTIVE Pituitary MR imaging fails to detect over 50% of microadenomas in Cushing's disease and nearly 80% of cases of dural microinvasion. Surface coils can generate exceptionally high-resolution images of the immediately adjacent tissues. To improve imaging of the pituitary gland, a receive-only surface coil that can be placed within the sphenoid sinus (the endosphenoidal coil [ESC]) during transsphenoidal surgery (TSS) was developed and assessed. METHODS Five cadaver heads were used for preclinical testing of the ESC. The ESC (a double-turn, 12-mm-diameter surface coil made from 1-mm-diameter copper wire) was developed to obtain images in a 1.5-T MR scanner. The ESC was placed (via a standard sublabial TSS approach) on the anterior sella face. Clinical MR scans were obtained using the 8-channel head coil and ESC as the receiver coils. Using the ESC, ultra-high-resolution, 3D, balanced fast field echo (BFFE) and T1-weighted imaging were performed at resolutions of 0.25 × 0.25 × 0.50 mm3 and 0.15 × 0.15 × 0.30 mm3, respectively. RESULTS Region-of-interest analysis indicated a 10-fold increase in the signal-to-noise ratio (SNR) of the pituitary when using the ESC compared with the 8-channel head coil. ESC-related improvements (p < 0.01) in the SNR were inversely proportional to the distance from the ESC tip to the anterior pituitary gland surface. High-resolution BFFE MR imaging obtained using ESC revealed a number of anatomical features critical to pituitary surgery that were not visible on 8-channel MR imaging, including the pituitary capsule, the intercavernous sinus, and microcalcifications in the pars intermedia. These ESC imaging findings were confirmed by the pathological correlation with whole-mount pituitary sections. CONCLUSIONS ESC can significantly improve SNR in the sellar region intraoperatively using current 1.5-T MR imaging platforms. Improvement in SNR can provide images of the sella and surrounding structures with unprecedented resolution. Clinical use of this ESC may allow for MR imaging detection of previously occult pituitary adenomas and identify microscopic invasion of the dura or cavernous sinus.

Interhemispheric plasticity protects the deafferented somatosensory cortex from functional takeover after nerve injury.

Functional changes across brain hemispheres have been reported after unilateral cortical or peripheral nerve injury. Interhemispheric callosal connections usually underlie this cortico-cortical plasticity. However, the effect of the altered callosal inputs on local cortical plasticity in the adult brain is not well studied. Ipsilateral functional magnetic resonance imaging (fMRI) activation has been reliably detected in the deafferented barrel cortex (BC) at 2 weeks after unilateral infraorbital denervation (IO) in adult rats. The ipsilateral fMRI signal relies on callosal-mediated interhemispheric plasticity. This form of interhemispheric plasticity provides a good chronic model to study the interaction between callosal inputs and local cortical plasticity. The receptive field of forepaw in the primary somatosensory cortex (S1), which is adjacent to the BC, was mapped with fMRI. The S1 receptive field expanded to take over a portion of the BC in 2 weeks after both ascending inputs and callosal inputs were removed in IO rats with ablated contralateral BC (IO+ablation). This expansion, estimated specifically by fMRI mapping, is significantly larger than what has been observed in the IO rats with intact callosal connectivity, as well as in the rats with sham surgery. This work indicates that altered callosal inputs prevent the functional takeover of the deafferented BC from adjacent cortices and may help preserve the functional identity of the BC.

Transcranial amelioration of inflammation and cell death after brain injury.

Traumatic brain injury (TBI) is increasingly appreciated to be highly prevalent and deleterious to neurological function. At present, no effective treatment options are available, and little is known about the complex cellular response to TBI during its acute phase. To gain insights into TBI pathogenesis, we developed a novel murine closed-skull brain injury model that mirrors some pathological features associated with mild TBI in humans and used long-term intravital microscopy to study the dynamics of the injury response from its inception. Here we demonstrate that acute brain injury induces vascular damage, meningeal cell death, and the generation of reactive oxygen species (ROS) that ultimately breach the glial limitans and promote spread of the injury into the parenchyma. In response, the brain elicits a neuroprotective, purinergic-receptor-dependent inflammatory response characterized by meningeal neutrophil swarming and microglial reconstitution of the damaged glial limitans. We also show that the skull bone is permeable to small-molecular-weight compounds, and use this delivery route to modulate inflammation and therapeutically ameliorate brain injury through transcranial administration of the ROS scavenger, glutathione. Our results shed light on the acute cellular response to TBI and provide a means to locally deliver therapeutic compounds to the site of injury.

Manganese graft ionomer complexes (MaGICs) for dual imaging and chemotherapy.

Novel manganese graft ionomer complexes (MaGICs) that contain Mn ions complexed with a polyaminobisphosphonate-g-poly(ethylene oxide) (PEO) copolymer were developed for use as T1-weighted contrast agents for MRI. The complexes exhibited good colloidal stability without release of free manganese and did not result in any in vitro toxicity against mouse hepatocytes. T1 relaxivities of the MaGICs at physiological pH were 2-10 times higher than that of a commercial manganese-based positive contrast agent. Anticancer drugs including doxorubicin, cisplatin and carboplatin were successfully encapsulated into the MaGICs with high efficiency. Drug release behavior was sustained and depended on pH (faster in acidic environments), drug structures and drug concentration (faster with high concentration). The anticancer drug-loaded manganese nanocarriers exhibited excellent anticancer activity against MCF-7 breast cancer cells together with high relaxivity. Thus, these drug-loaded MaGICs could potentially be utilized for simultaneous diagnosis and treatment of cancer.

Orientation-specific responses to sustained uniaxial stretching in focal adhesion growth and turnover.

Cells are mechanosensitive to extracellular matrix (ECM) deformation, which can be caused by muscle contraction or changes in hydrostatic pressure. Focal adhesions (FAs) mediate the linkage between the cell and the ECM and initiate mechanically stimulated signaling events. We developed a stretching apparatus in which cells grown on fibronectin-coated elastic substrates can be stretched and imaged live to study how FAs dynamically respond to ECM deformation. Human bone osteosarcoma epithelial cell line U2OS was transfected with GFP-paxillin as an FA marker and subjected to sustained uniaxial stretching. Two responses at different timescales were observed: rapid FA growth within seconds after stretching, and delayed FA disassembly and loss of cell polarity that occurred over tens of minutes. Rapid FA growth occurred in all cells; however, delayed responses to stretch occurred in an orientation-specific manner, specifically in cells with their long axes perpendicular to the stretching direction, but not in cells with their long axes parallel to stretch. Pharmacological treatments demonstrated that FA kinase (FAK) promotes but Src inhibits rapid FA growth, whereas FAK, Src, and calpain 2 all contribute to delayed FA disassembly and loss of polarity in cells perpendicular to stretching. Immunostaining for phospho-FAK after stretching revealed that FAK activation was maximal at 5 s after stretching, specifically in FAs oriented perpendicular to stretch. We hypothesize that orientation-specific activation of strain/stress-sensitive proteins in FAs upstream to FAK and Src promote orientation-specific responses in FA growth and disassembly that mediate polarity rearrangement in response to sustained stretch.

EPR oxygen imaging and hyperpolarized 13C MRI of pyruvate metabolism as noninvasive biomarkers of tumor treatment response to a glycolysis inhibitor 3-bromopyruvate.

The hypoxic nature of tumors results in treatment resistance and poor prognosis. To spare limited oxygen for more crucial pathways, hypoxic cancerous cells suppress mitochondrial oxidative phosphorylation and promote glycolysis for energy production. Thereby, inhibition of glycolysis has the potential to overcome treatment resistance of hypoxic tumors. Here, EPR imaging was used to evaluate oxygen dependent efficacy on hypoxia-sensitive drug. The small molecule 3-bromopyruvate blocks glycolysis pathway by inhibiting hypoxia inducible enzymes and enhanced cytotoxicity of 3-bromopyruvate under hypoxic conditions has been reported in vitro. However, the efficacy of 3-bromopyruvate was substantially attenuated in hypoxic tumor regions (pO2<10 mmHg) in vivo using squamous cell carcinoma (SCCVII)-bearing mouse model. Metabolic MRI studies using hyperpolarized 13C-labeled pyruvate showed that monocarboxylate transporter-1 is the major transporter for pyruvate and the analog 3-bromopyruvate in SCCVII tumor. The discrepant results between in vitro and in vivo data were attributed to biphasic oxygen dependent expression of monocarboxylate transporter-1 in vivo. Expression of monocarboxylate transporter-1 was enhanced in moderately hypoxic (8-15 mmHg) tumor regions but down regulated in severely hypoxic (<5 mmHg) tumor regions. These results emphasize the importance of noninvasive imaging biomarkers to confirm the action of hypoxia-activated drugs.

Magnetic manipulation of actin orientation, polymerization, and gliding on myosin using superparamagnetic iron oxide particles.

The actin cytoskeleton controls cell shape, motility, as well as intracellular molecular trafficking. The ability to remotely manipulate actin is therefore highly desirable as a tool to probe and manipulate biological processes at the molecular level. We demonstrate actin manipulation by labeling actin filaments with superparamagnetic iron oxide particles (IOPs) and applying a uniform magnetic field to affect actin orientation, polymerization and gliding on myosin. We show for the first time magnetic manipulation of magnetizable actin filaments at the molecular level while gliding on a bed of myosin molecules and during polymerization. A model for the magnetic alignment and guiding mechanism is proposed based on the torque from the induced molecular anisotropy due to interactions between neighboring IOPs distributed along magnetically labeled actin molecules.

Detecting response of rat C6 glioma tumors to radiotherapy using hyperpolarized [1- 13C]pyruvate and 13C magnetic resonance spectroscopic imaging.

We show here that hyperpolarized [1-(13) C]pyruvate can be used to detect treatment response in a glioma tumor model; a tumor type where detection of response with (18) fluoro-2-deoxyglucose, using positron emission tomography, is limited by the high background signals from normal brain tissue. (13) C chemical shift images acquired following intravenous injection of hyperpolarized [1-(13) C]pyruvate into rats with implanted C6 gliomas showed significant labeling of lactate within the tumors but comparatively low levels in surrounding brain.Labeled pyruvate was observed at high levels in blood vessels above the brain and from other major vessels elsewhere but was detected at only low levels in tumor and brain.The ratio of hyperpolarized (13) C label in tumor lactate compared to the maximum pyruvate signal in the blood vessels was decreased from 0.38 ± 0.16 to 0.23 ± 0.13, (a reduction of 34%) by 72 h following whole brain irradiation with 15 Gy.

In vivo detection of individual glomeruli in the rodent olfactory bulb using manganese enhanced MRI.

MRI contrast based on relaxation times, proton density, or signal phase have been applied to delineate neural structures in the brain. However, neural units such as cortical layers and columns have been difficult to identify using these methods. Manganese ion delivered either systemically or injected directly has been shown to accumulate specifically within cellular areas of the brain enabling the differentiation of layers within the hippocampus, cortex, cerebellum, and olfactory bulb in vivo. Here we show the ability to detect individual olfactory glomeruli using manganese enhanced MRI (MEMRI). Glomeruli are anatomically distinct structures ( approximately 150 microm in diameter) on the surface of the olfactory bulb that represent the first processing units for olfactory sensory information. Following systemic delivery of MnCl(2) we used 3D-MRI with 50 microm isotropic resolution to detect discrete spots of increased signal intensity between 100 and 200 microm in diameter in the glomerular layer of the rat olfactory bulb. Inflow effects of arterial blood and susceptibility effects of venous blood were suppressed and were evaluated by comparing the location of vessels in the bulb to areas of manganese enhancement using iron oxide to increase vessel contrast. These potential vascular effects did not explain the contrast detected. Nissl staining of individual glomeruli were also compared to MEMRI images from the same animals clearly demonstrating that many of the manganese enhanced regions corresponded to individual olfactory glomeruli. Thus, MEMRI can be used as a non-invasive means to detect olfactory glomeruli for longitudinal studies looking at neural plasticity during olfactory development or possible degeneration associated with disease.

Targeting neural precursors in the adult brain rescues injured dopamine neurons.

In Parkinson's disease, multiple cell types in many brain regions are afflicted. As a consequence, a therapeutic strategy that activates a general neuroprotective response may be valuable. We have previously shown that Notch ligands support neural precursor cells in vitro and in vivo. Here we show that neural precursors express the angiopoietin receptor Tie2 and that injections of angiopoietin2 activate precursors in the adult brain. Signaling downstream of Tie2 and the Notch receptor regulate blood vessel formation. In the adult brain, angiopoietin2 and the Notch ligand Dll4 activate neural precursors with opposing effects on the density of blood vessels. A model of Parkinson's disease was used to show that angiopoietin2 and Dll4 rescue injured dopamine neurons with motor behavioral improvement. A combination of growth factors with little impact on the vasculature retains the ability to stimulate neural precursors and protect dopamine neurons. The cellular and pharmacological basis of the neuroprotective effects achieved by these single treatments merits further analysis.

In vivo labeling of adult neural progenitors for MRI with micron sized particles of iron oxide: quantification of labeled cell phenotype.

The subventricular zone (SVZ) is a continual source of neural progenitors throughout adulthood. Many of the animal models designed to study the migration of these cells from the ventricle to places of interest like the olfactory bulb or an injury site require histology to localize precursor cells. Here, it is demonstrated that up to 30% of the neural progenitors that migrate along the rostral migratory stream (RMS) in an adult rodent can be labeled for MRI via intraventricular injection of micron sized particles of iron oxide (MPIOs). The precursors migrating from the SVZ along the RMS were found to populate the olfactory bulb with all three types of neural cells; neurons, oligodendrocytes, and astrocytes. In all cases 10-30% of these cells were labeled in the RMS en route to the olfactory bulb. Ara-C, an anti-mitotic agent, eliminated precursor cells at the SVZ, RMS, and olfactory bulb and also eliminated the MRI detection of the precursors. This indicates that the MRI signal detected is due to progenitor cells that leave the SVZ and is not due to non-specific diffusion of MPIOs. Using MRI to visualize neural progenitor cell behavior in individual animals during plasticity or disease models should be a useful tool, especially in combination with other information that MRI can supply.

Delivery of fluorescent probes using iron oxide particles as carriers enables in-vivo labeling of migrating neural precursors for magnetic resonance imaging and optical imaging.

Iron oxide particles are becoming an important contrast agent for magnetic resonance imaging (MRI) cell tracking studies. Simultaneous delivery of fluorescence indicators with the particles to individual cells offers the possibility of correlating optical images and MRI. In this work, it is demonstrated that micron-sized iron oxide particles (MPIOs) can be used as a carrier to deliver fluorescent probes to cells in culture as well as to migrating neural progenitors in vivo. Migrating progenitors were tracked with MRI and easily identified by histology because of the fluorescent probe. These data suggest that using MPIOs to deliver fluorescent probes should make it possible to combine MRI and optical imaging for in vivo cell tracking.

Antibody-mediated cell labeling of peripheral T cells with micron-sized iron oxide particles (MPIOs) allows single cell detection by MRI.

Labeling cells with iron oxide is a useful tool for MRI based cellular imaging. Here it is demonstrated that peripheral rat T cells can be labeled in whole blood, in vitro, with streptavidin-coated micron-sized iron oxide particles (MPIOs), achieving iron concentrations as high as 60 pg iron per cell. This is 30 times the amount of labeling reported with ultrasmall particles of iron oxide (USPIOs). Labeling was mediated by use of a biotinylated anti-CD5 antibody, which is specific for peripheral T cells. Such labeling allowed the in vitro detection of single lymphocytes by MRI, using conditions well suited for in vivo animal work. Electron microscopic analysis demonstrated that MPIOs remained largely extracellular after labeling, with some evidence of intracellular uptake. Cell viability and early and late cytokine release studies showed no significant differences between labeled and unlabeled cells. Therefore, the use of MPIOs for achieving high iron concentrations for cellular MRI is potentially an effective new modality for non-invasive imaging of lymphocytes.

Magnetic resonance imaging of the migration of neuronal precursors generated in the adult rodent brain.

Neural progenitor cells (NPCs) reside within the subventricular zone (SVZ) in rodents. These NPCs give rise to neural precursors in adults that migrate to the olfactory bulb (OB) along a well-defined pathway, the rostral migratory stream (RMS). Here we demonstrate that these NPCs can be labeled, in vivo, in adult rats with fluorescent, micron-sized iron oxide particles (MPIOs), and that magnetic resonance imaging (MRI) can detect migrating neural precursors carrying MPIOs along the RMS to the OB. Immunohistochemistry and electron microscopy indicated that particles were inside GFAP(+) neural progenitor cells in the SVZ, migrating PSA-NCAM(+) and Doublecortin(+) neural precursors within the RMS and OB, and Neu-N(+) mature neurons in the OB. This work demonstrates that in vivo cell labeling of progenitor cells for MRI is possible and enables the serial, non-invasive visualization of endogenous progenitor/precursor cell migration.

High-resolution mapping of tumor redox status by magnetic resonance imaging using nitroxides as redox-sensitive contrast agents.

PURPOSE: There is considerable research directed toward the identification and development of functional contrast agents for medical imaging that superimpose tissue biochemical/molecular information with anatomical structures. Nitroxide radicals were identified as in vivo radioprotectors. Being paramagnetic, they can provide image contrast in magnetic resonance imaging (MRI) and electron paramagnetic resonance imaging (EPRI). The present study sought to determine the efficacy of nitroxide radioprotectors as functional image contrast agents. EXPERIMENTAL DESIGN: Nitroxide radioprotectors, which act as contrast agents, were tested by EPRI and MRI to provide tissue redox status information noninvasively. RESULTS: Phantom studies showed that the nitroxide, 3-carbamoyl-PROXYL (3CP), undergoes time-dependent reduction to the corresponding diamagnetic hydroxylamine only in the presence of reducing agents. The reduction rates of 3CP obtained by EPRI and MRI were in agreement suggesting the feasibility of using MRI to monitor nitroxide levels in tissues. The levels of 3CP were examined by EPRI and MRI for differences in reduction between muscle and tumor (squamous cell carcinoma) implanted in the hind leg of C3H mice simultaneously. In vivo experiments showed a T1-dependent image intensity enhancement afforded by 3CP which decreased in a time-dependent manner. Reduction of 3CP was found to be the dominant mechanism of contrast loss. The tumor regions exhibited a faster decay rate of the nitroxide compared to muscle (0.097 min(-1) versus 0.067 min(-1), respectively). CONCLUSIONS: This study shows that MRI can be successfully used to co-register tissue redox status along with anatomic images, thus providing potentially valuable biochemical information from the region of interest.

Cell labeling for magnetic resonance imaging with the T1 agent manganese chloride.

There is growing interest in using MRI to track cellular migration. To date, most work in this area has been performed using ultra-small particles of iron oxide. Immune cells are difficult to label with iron oxide particles. The ability of adoptively infused tumor specific T cells and N cells to traffic to the tumor microenvironment may be a critical determinant of their therapeutic efficacy. We tested the hypothesis that lymphocytes and B cells would label with MnCl2 to a level that would allow their detection by T1-weighted MRI. Significant signal enhancement was observed in human lymphocytes after a 1 h incubation with 0.05-1.0 mM MnCl2. A flow cytometry-based evaluation using propidium iodide and Annexin V staining showed that lymphocytes did not undergo apoptosis or necrosis immediately after and 24 h following a 1 h incubation with up to 1.0 mM MnCl2. Importantly, NK cells and cytotoxic T cells maintained their in vitro killing capacity after being incubated with up to 0.5 mM MnCl2. This is the first report to describe the use of MnCl2 to label lymphocytes. Our data suggests MnCl2 might be an alternative to iron oxide cell labeling for MRI-based cell migration studies.