Magnetic labeling of activated microglia in experimental gliomas

Abstract

Microglia, as intrinsic immunoeffector cells of the central nervous system (CNS), play a very sensitive, crucial role in the response to almost any brain pathology where they are activated to a phagocytic state. Based on the characteristic features of activated microglia, we investigated whether these cells can be visualized with magnetic resonance imaging (MRI) using ultrasmall superparamagnetic iron oxides (USPIOs). The hypothesis of this study was that MR microglia visualization could not only reveal the extent of the tumor, but also allow for assessing the status of immunologic defense. Using USPIOs in cell culture experiments and in a rat glioma model, we showed that microglia can be labeled magnetically. Labeled microglia are detected by confocal microscopy within and around tumors in a typical border-like pattern. Quantitative in vitro studies revealed that microglia internalize amounts of USPIOs that are significantly higher than those incorporated by tumor cells and astrocytes. Labeled microglia can be detected and quantified with MRI in cell phantoms, and the extent of the tumor can be seen in glioma-bearing rats in vivo. We conclude that magnetic labeling of microglia provides a potential tool for MRI of gliomas, which reflects tumor morphology precisely. Furthermore, the results suggest that MRI may yield functional data on the immunologic reaction of the CNS.

Figure 1

Activated microglia and invading macrophages infiltrate the tumor region in rat gliomas and show rapid uptake of USPIO in a slice model. Brain slices (150 µm thick) of the tumor region were incubated with 3 mM TexasRed USPIO for 30 minutes, and investigated in the confocal microscope with varying magnifications. (A) OX-42 staining (blue) reveals the proportion of microglia and invading macrophages within the tumor. The inset displays green eGFP-transfected tumor cells in an unlabeled slice. (B) After incubation of slices with TexasRed-labeled USPIO, the magnetite was clearly localized intracellularly, and predominantly within OX-42-positive ameboid cells (inset in B; USPIO red, OX-42 blue). OX-42-positive microglia with a resting ramified appearance (blue) showed no significant USPIO uptake. (C) Visualization of microglia and macrophages by intracellular labeling with TexasRed-stained USPIO showed diffuse infiltration of the tumor, as seen in (A). (D) Formation of a border around the tumor, differentiating the tumor from healthy brain tissue, was also detectable by USPIO labeling. A single tumor cell encircled by USPIO-labeled macrophages could be detected at a distance from the main tumor mass (inset in D).

Figure 2

Visual comparison of USPIO uptake by microglia and astrocytes under identical conditions. Confocal microscopy of a microglia-astrocyte co-culture. All photomicrographs display the same field of view, but are focused on two different cell levels. (A) and (B) are focused on microglia attached to the astrocytes. (C) and (D) are focused on the astrocytic monolayer beneath the microglia. Phase-contrast photographs focusing on each cell type show cell morphology (A and C). After incubation with 3 mM TexasRed-labeled USPIO for 30 minutes, (B) and (D) display intracellular uptake predominantly by microglia.

Figure 3

Labeling of co-cultured microglia and F98 tumor cells with TexasRed-USPIO confirmed the results of glioma slice experiments. (A) Due to the incorporated iron oxide, microglial cells can be detected with confocal microscopy after incubation with 3 mM USPIO for 30 minutes. F98 cells do not incorporate USPIO. (B) Corresponding phase-contrast photograph showing the F98 cells (arrows) between the microglia. (C) Labeling with Isolectin-B4 after incubation identifies phagocytosing cells to be microglia.

Figure 4

Intracellular distribution of TexasRed-labeled USPIO incorporated by microglia. After incubation of cultured microglia with 3 mM TexasRed-labeled USPIO for 30 minutes, an endosome-like distribution pattern of the internalized contrast agent can be observed by confocal microscopy. (A) Phase-contrast, (B) fluorescent USPIO. Control cells not incubated with USPIO showed weak autofluorescence (insets).

Figure 5

USPIO incorporation characteristics of cultured microglia. (A) Concentration dependency: Microglia: cells were incubated at 37°C in different concentrations (15 µM to 18 mM) of TexasRed-labeled USPIO for 30 minutes, and cellular uptake was determined by fluorescence scanning. Inset displays uptake by identically treated peritoneal macrophages. (B) Time dependency: incorporation was quantified after microglia had been incubated for varying time periods (15 to 120 minutes) in 3 mM TexasRed-labeled USPIO.

Figure 6

Concentration dependency of USPIO incorporation in different cultured cell types involved in glioma pathology. Cultured cell lines [C6, 9L, F98 (glioma), and P-388D1 (monocytic)] and primary astrocytes were incubated in unlabeled USPIO for 30 minutes at varying concentrations (1.5 to 6 mM), and cellular uptake was determined as the intracellular iron level by means of ICP-AES.

Figure 7

Effect of microglia USPIO labeling on MRI signal intensity, visualized in phantom models. Signal intensity correlates with intracellular USPIO concentration in cell and standard phantoms. 3D FLASH sequence (TR/TE 34/20 msec, flip angle 15°) provides high spatial resolution. (A) Varying concentrations (0.25x10⁶, 0.5x10⁶, 1x10⁶, 1.5x10⁶, 2x10⁶, and 3x10⁶) of microglia and P-388D1 cells were incubated for 1.5 hours with 3 mM USPIO, and embedded in 100 µl of 4% gelatin. (B) Analysis of the standardized MRI signal revealed similar USPIO uptake by cultured microglia and macrophages (P-388D1).

Figure 8

In vivo distribution of USPIO in glioma-bearing rats. MRI (A) before (I, II) and after (III, IV) administration of USPIO reveals a massive accumulation of iron oxides within the tumor, seen as a signal intensity decrease. Immunohistology [B(III and IV)] confirms predominant USPIO uptake (red) by macrophages and microglia (blue), also under in vivo conditions. T2*-weighted 3D FLASH sequence (TR/TE 34/20 msec, flip angle 15°) is displayed in I and III; T2-weighted turbo spin echo sequence (TR/TE 4500/96) in II and IV. Imaging was performed before and 24 hours after administration of 300 µmol/kg USPIO. Through DAB staining, image [B(II)] confirms iron deposition within and around the tumor. HE staining of corresponding slice [B(I)]. OX-42 staining detects high proportion of microglia and macrophages (red) within the tumor (green) [B(III)]. Also after in vivo administration, USPIO (red) can predominantly be found in the macrophages and microglia [B(IV)].