2.1.

Dye Coating

MPIOs were purchased commercially (Bangs Laboratories, Incorporated, Fishers, Indiana). These iron oxide particles have a hydrophobic, polystyrene coat that was on average $1.63\mathrm{\mu }\mathrm{m}$ in diameter and contained 42.5% iron oxide. A fluorescein derivative, dragon green (ex: $488\mathrm{nm}$ , em: $520\mathrm{nm}$ ) is trapped in the polystyrene coat. A lipophilic tracer $(25\mathrm{mg}∕\mathrm{mL})$ , DiD (ex: $630\mathrm{nm}$ , em: $670\mathrm{nm}$ ; Invitrogen, Carlsbad, California), was added to $2.8\times {10}^9\mathrm{particles}∕\mathrm{mL}$ suspended in deionized water to make a 0.5 to $5\%$ (v/v) dye-to-water suspension. The suspension was mixed 2 to $24\mathrm{h}$ , then rinsed three times, and the particles were resuspended in phosphate buffered saline (PBS) prior to labeling cells or stereotactic injection. The particle concentration was kept constant at $2.8\times {10}^9\mathrm{particles}∕\mathrm{mL}$ throughout. The calcium dyes, Rhod 2 (ex: $550\mathrm{nm}$ , em: $580\mathrm{nm}$ ) and Fura 2 (ex: $335\mathrm{nm}$ , em: $505\mathrm{nm}$ ; Invitrogen) were coated onto the MPIOs similarly. In addition to $1.63\text{-}\mathrm{\mu }\mathrm{m}$ MPIOs, Rhod 2 was coated onto $0.5\text{-}\mathrm{\mu }\mathrm{m}$ iron oxide silica particles (G.Kisker GbR, Steinfurt, Germany).

2.2.

Cell Culture

Cell lines were purchased from ATCC (Manassas, Virginia). C6 glioma and PC-12 pheochromocytomas were grown in Ham’s F12K medium and supplemented with $2\text{-}\mathrm{mM}$ glutamine, $1.5\text{-}\mathrm{g}∕\mathrm{L}$ sodium bicarbonate, 15% horse serum, and 2.5% fetal bovine serum. B35 neuroblastomas were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with $4\text{-}\mathrm{mM}$ L-glutamine, $1.5\text{-}\mathrm{g}∕\mathrm{L}$ sodium bicarbonate, $4.5\text{-}\mathrm{g}∕\mathrm{L}$ glucose, and 10% fetal bovine serum. Cells were incubated at $37°\mathrm{C}$ and 5% ${\mathrm{CO}}_2$ . Approximately 50,000 dye-coated particles were added to 250,000 cells 2 to $24\mathrm{h}$ prior to imaging. After imaging, cells were fixed using 4% paraformaldehyde/PBS solution (Fisher Scientific, Fairlawn, New Jersey). For calcium dye studies, images were taken every $200\mathrm{\mu }\mathrm{s}$ as a $100\text{-}\mathrm{mM}$ glutamate solution was added to the media (DMEM/F12). Fluorescence images were obtained on a Zeiss LSM 510 confocal microscope (Carl Zeiss Incorporated, Germany).

2.3.

Animal Injections

19, 6-week old Sprague-Dawley rats (Charles River Laboratories, Incorporated, Wilmington, Massachusetts) were stereotactically injected with the DiD-coated MPIOs. Coordinates for injections into the lateral ventricle were based on work by Shapiro ⁷ and were $2\text{-}\mathrm{mm}$ caudal from Bregma, $2\mathrm{mm}$ to the right, and $-3\mathrm{mm}$ from the dura. $50\text{-}\mathrm{\mu }\mathrm{L}$ $(1.4\times {10}^8\mathrm{MPIOs})$ were injected into the ventricle of each rat. All the experiments with animals were in compliance with protocols approved by the National Institutes of Neurological Disorders and Stroke Animal Care and Use Committee.

2.4.

Magnetic Resonance Imaging

Two weeks following postinjection, MRI data were acquired on an $11.7\mathrm{T}$ animal MRI system (30-cm $11.7\mathrm{T}$ horizontal magnet, Magnex Scientific, Oxford, England, MRI Electronics, Bruker Biospin, Billerica, Massachusetts, and $9\text{-}\mathrm{cm}$ 3-D gradients, Resonance Research Incorporated, Billerica, Massachusetts) using a volume transmit coil and a custom-built, $2.5\text{-}\mathrm{cm}$ -diam, receive-only surface coil. Animals were placed in a MRI compatible cradle with a stereotactic head frame. The animals were continually under anesthesia using 2 to 3% isofluorane in 75% oxygen/25% medical air throughout the duration of the experiment. Animals were mechanically ventilated at a rate of $66\mathrm{breaths}∕\min$ after intubation, and their steady-state ${\mathrm{CO}}_2$ was monitored. Body temperature was maintained at $37°\mathrm{C}$ by using a circulating water bath with a rectal temperature feedback probe. The following parameters were used to acquire 3-D gradient echo images: TE $8\mathrm{ms}$ , TR $30\mathrm{ms}$ , FOV $1.92{\mathrm{cm}}^3$ , and ${256}^3$ matrix ( $75\mathrm{\mu }\mathrm{m}$ isotropic resolution).

After the in-vivo MRI acquisitions, animals were transcardially perfused with PBS, followed with 10% formalin that contained $1\text{-}\mathrm{mM}$ Gd-DTPA. Brains were removed with the olfactory bulb intact and post-fixed for at least $6\mathrm{h}$ in the same formalin/Gd-DTPA solution. Brains were placed in saline and imaged using a $35\text{-}\mathrm{mm}$ volume coil with the following parameters: FOV $3.08{\mathrm{cm}}^3$ , ${256}^3$ matrix, TE $8\mathrm{ms}$ , TR $30\mathrm{ms}$ , and 20 averages.

2.5.

Histology

Brains were placed in a 30% sucrose solution. After 2 to 3 days, samples were processed for frozen sections (Histoserv, Incorporated, Germantown, Maryland). $16\text{-}\mathrm{\mu }\mathrm{m}$ sagittal sections were cut that included the olfactory bulb, RMS, and SVZ. No further processing was done to the tissue sections.

3.1.

Fluorescence of 1, $1^{\prime }$ -Dioctadecyl-3,3, $3^{\prime }$ , $3^{\prime }$ -Tetramethylindodicarbocyanine Perchlorate/Micron-Sized Particles of Iron Oxide

MPIOs, with a polystyrene matrix, were coated with a lipophilic tracer, DiD, to test whether passively delivering MPIOs to cells could result in fluorescent agents being transferred to the cell. In PBS, greater than 90% of the DiD remained coated on the polystyrene coated particle after $5\mathrm{h}$ of suspension (data not shown). The coated MPIOs were added to cell cultures where the particles were allowed to incubate with the cells for several hours in a manner that only labeled approximately 30% of the cells. One to five particles were found to be internal to the cells, presumably due to endocytosis by the cells after the incubation period. Cells that contained the dye-coated MPIOs are shown in Fig. 1 ; MPIOs appear as green fluorescence and DiD shows up as red fluorescence. For the cells that are labeled, the entire cell is labeled with DiD, enabling the boundaries and morphology of the labeled cells to be visualized. To investigate the cell specificity of this colabeling process, a few different cell lines were investigated. Rows 1 and 2 in Fig. 1 are images of B35 neuroblastomas and PC-12 pheochromocytoma cells, respectively, that have been colabeled with the MPIO and DiD. These data indicate that the mechanism for colabeling is cell line independent.

Fig. 1

(a), (b), and (c) B35 neuroblastoma cells and (d), (e), and (f) PC-12 pheochromocytoma cells labeled in vitro with MPIOs that have been coated with a lipophilic tracer, DiD. (a) and (d) Dragon Green fluorescence from the MPIOs. (b) and (e) Fluorescence from the DiD that has stained the cell morphology. (c) and (f) The fluorescent images overlaid with the bright field image. The scale is constant throughout the panels with the scale bar in (f) representing $20\mathrm{\mu }\mathrm{m}$ .

Neighboring cells that had not incorporated MPIOs showed no evidence of fluorescence from the DiD, even at greater than 90% confluence. This indicates that MPIOs can act as a dye carrier, which is specific to the individual cells that get labeled with MPIOs. The specificity of DiD labeling is illustrated graphically in Fig. 2 . The intensity of the MPIO containing cells was compared to the intensity of the neighboring cells. The DiD fluorescence intensity is at background intensity levels for cells that surround the MPIO labeled cells. Based on these results, it is clear that DiD comes off the MPIO only into the specific cell that has been labeled with the MPIO. Therefore, DiD labeling is specific for MPIO labeled cells.

Fig. 2

Change in intensity compared to background (no cells) of both B35 cells and PC12 cells $(n=3)$ . Inset: plot of the gray value profile from DiD-labeled cells in Fig. 1b, which passes over five cells, two of which were colabeled.

3.2.

Magnetic Resonance Imaging and Fluorescence of 1, $1^{\prime }$ -Dioctadecyl-3,3, $3^{\prime }$ , $3^{\prime }$ -Tetramethylindodicarbocyanine Perchlorate/Micron-Sized-Particles of Iron Oxide

Migrating rodent neuroblasts have recently been labeled with MPIOs for MRI.⁷ These cells are known to change their morphology from a spindle-shaped cell to a fully differentiated neuron as they migrate.¹⁸ DiD-coated MPIOs were injected into the rat lateral ventricle to test whether the particles would be endocytosed by the migrating neural progenitors in vivo and whether the neuroblast morphology could be identified by the DiD in subsequent histology. Serial MRI of a rat brain that was injected with dye-coated MPIOs is shown in Fig. 3 . The hypointense signal in the MRI is a well-known effect of the iron oxide particles and is exploited to observe the neural progenitor cell migration in vivo. The large dark region in the ventricles is a result of the large number of MPIOs remaining around the ventricular region. The hypointensities detected from the subventricular zone to the olfactory bulb along the rostral migratory stream are due to migrating neural progenitors that have been labeled with MPIOs (arrows in Fig. 3). MRI of fixed brain tissue confirmed the presence of MPIOs in the RMS (data not shown). These data are consistent to those previously reported, which used standard MPIOs that were not adhered with DiD.⁷ Therefore, DiD labeling of MPIOs did not block their uptake into neural progenitors and subsequent migration.

Fig. 3

MRI through the RMS in an animal that was injected with the dye-coated MPIOs. (a) through (e) are $75\text{-}\mathrm{\mu }\mathrm{m}$ serial images encompassing the RMS. (f) is the merge of serial images. MRI was performed at $11.7\mathrm{T}$ using 3-D gradient echo images at $75\text{-}\mathrm{\mu }\mathrm{m}$ isotropic resolution. Images were taken two weeks after injection of DiD-labeled MPIOs. Arrows indicate loss of signal due to migration of the MPIO-labeled neural precursors throughout the RMS.

Figure 4 shows the merger of MRI slices through the RMS taken two weeks postinjection, and the corresponding fluorescent images that were taken at separate points along the migration pathway of the neuroblasts. Figure 4b confirms that there is a high density of MPIOs around the ventricle. Many cells in this region are colabeled with DiD. In this figure, there appears to be many DiD-labeled cells that do not contain MPIOs; however, many of the MPIOs are out of plane and visualized with deeper $z$ sectioning with the confocal microscope (data not shown). Nonetheless, this does not discount the possibility of having DiD-only cells. In this region, the cell morphology is difficult to delineate, because many cell types are labeled including neural precursors; however, only neural progenitor cells will migrate rostrally from the ventricle to the olfactory bulb.¹⁹ The well-defined lines of hypointense signal in the MRI adjacent to the ventricle are a result of MPIO-labeled neuroblasts in the white matter track known as the rostral migratory stream (RMS). Lois, Garcia-Verdugo, and Alvarez-Buylla have described these neuroblasts in RMS as having a spindle shape and moving in closely spaced chains.¹⁸ Fluorescent images taken from sections of the RMS are shown in Fig. 4c. This figure shows colabeled cells that are in close proximity to one another and have the characteristic closely spaced, spindle shape. Additionally, this figure shows that the DiD and MPIOs are confined to the RMS pathway. Once the neuroblasts reach the olfactory bulb, they migrate radially and become fully differentiated neurons.²⁰ Figure 3d shows a fluorescent image taken in the olfactory bulb. This cell’s morphology is defined by the DiD and is colabeled with the MPIOs. This distinct morphology is that of a neuron with many processes being extended, indicating that labeling with MPIOs did not affect morphological transitions in neural progenitors.

Fig. 4

MRI of endogenous neural progenitors in the RMS and olfactory bulb, and the corresponding fluorescent images from the histology slices. (a) Adjacent MR slices along the right RMS were merged to create this figure. (b), (c), and (d) are fluorescent images from histological sections showing cells in the rat brain labeled with the MPIOs (green) and stained from the DiD (red). Fluorescent images show (b) the SVZ, (c) spindle-shaped cells in the RMS, and (d) an individual neuron in the olfactory bulb. Scale bars represent $20\mathrm{\mu }\mathrm{m}$ .

3.3.

Calcium Indicator Delivery with Micron Sized Particles of Iron Oxide

Fura-2 and Rhod-2 are commonly used ${\mathrm{Ca}}^{2+}$ indicators, and these were tested to see if the process of passive dye delivery to cells could be mediated with MPIOs. Both indicators were applied using the same method as the DiD experiments. The Fura-2-coated MPIOs and the Rhod-2-coated MPIOs were separately incubated with C6 glioma cells. Only the culture incubated with the Fura-2-coated MPIOs labeled the cells, as shown in row 1 of Fig. 5 . There was no evidence of Rhod-2 labeling the cells; instead, the dye remained on the MPIOs (data not shown). A silica-coated iron oxide particle was used in place of the polystyrene particles to test whether the MPIO coating material would make a difference in the ability of Rhod-2 to disassociate from the MPIOs. Figure 5 row 2 shows that the silica MPIOs were able to colabel C6 gliomas with Rhod-2.

Fig. 5

Fluorescent images of C6 glioma cells that contain calcium-sensitive dyes that were delivered by MPIOs. (a) and (b) show response of Fura-2 labeled cells to a glutamate-induced calcium wave, and (c) and (d) show the response of Rhod-2 labeled cells to a glutamate-induced calcium wave. The polystyrene particles were coated with Fura-2, and silica particles were coated with Rhod-2. The image pairs were acquired using the same field of view and acquisition times.

The response of the calcium dyes was tested to determine whether the MPIO-mediated delivery enabled monitoring a cellular calcium transient. C6 cells were incubated with the ${\mathrm{Ca}}^{2+}$ dye-coated MPIOs long enough for the calcium dye to diffuse off the particle to stain the cell (2 to $6\mathrm{h}$ ). A calcium wave was invoked by applying $100\text{-}\mathrm{mM}$ glutamate²¹ to the cell culture. The first column of Fig. 5 shows the fluorescent image prior to the addition of glutamate. There is little fluorescence from the cells at this gain setting, and only particles are visible because they still contained a high concentration of dye. Once the glutamate is added, the fluorescence increases (Fig. 5 column 2) as the calcium wave propagates across the sample. Therefore, MPIO mediated dye delivery does not interfere with monitoring a cellular response to calcium.