Chemically functionalized single-walled carbon nanotubes enhance the glutamate uptake characteristics of mouse cortical astrocytes

Abstract

Using a radioactive glutamate uptake assay and immunolabeling, we report that single-walled carbon nanotubes, chemically functionalized with polyethylene glycol (SWCNT-PEG), delivered as a colloidal solute, cause an increase in the uptake of extracellular glutamate by astrocytes and an increase in the immunoreactivity of the glutamate transporter GLAST on their cell surface, which is likely a consequence of an increase in the immunoreactivity of glial fibrillary acidic protein. Additional corollary is that astrocytes exposed to SWCNT-PEG became larger and stellate, morphological characteristics of maturation and heightened activity of these glial cells. These results imply that SWCNT-PEG could potentially be used as a viable candidate for neural prosthesis applications, perhaps to alleviate the death toll of neurons due to glutamate excitotoxicity, a pathological process observed in brain and spinal cord injuries.

Fig. 1

SWNCT-PEG solute enhances the uptake of [³H]-L-glutamate by mouse cortical astrocytes in culture. a Images of astrocytes in culture, labeled for the glutamate transporters GLAST (left) and GLT-1 (right), using indirect immunocytochemistry. Scale bar, 20 μm. Gray scale is a linear representation of the fluorescence intensities, expressed in fluorescence intensity units (iu), of the pixels in the images. b Summary graph showing the normalized average effect of 1 μg/ml PEG, 5 μg/ml SWCNT-PEG, 100 μM TBOA and 30 μM Y-27632 on the uptake of [³H]-L-glutamate by cultured astrocytes. Bars represent means with standard error of means. 18 wells containing astrocytes were analyzed per condition. Asterisks indicate a statistical difference compared to the control. Other differences are marked by the brackets. One-way ANOVA followed by Fisher’s LSD test; *p < 0.05, **p < 0.01.

Fig. 2

SWCNT-PEG solute increases the surface appearance of GLAST on mouse cortical astrocytes. a Images of astrocytes in culture, a subset of which were treated with 5 μg/ml SWCNT-PEG, labeled for GLAST using indirect immunocytochemistry and the non-permeabilization procedure. Scale bar, 20 μm. Gray scale is a linear representation of the fluorescence intensities, expressed in fluorescence intensity units (iu), of the pixels in the images. b Summary graphs showing the median effect of SWCNT-PEG on the quantitative GLAST immunoreactivity (ir) parameters i.e. density, content and occupancy. Density is shown in fluorescence intensity units (iu) per area (pixel). Boxes represent medians with interquartile range. Number of astrocytes studied in each condition is given in parentheses in the density graph. Asterisks indicate a statistical difference when compared to the untreated/control astrocytes. Mann-Whitney U-test. **p < 0.01

Fig. 3

SWCNT-PEG increases GFAP-ir parameters in mouse cortical astrocytes. a Images of astrocytes in culture, treated with 1 μg/ml PEG, 5 μg/ml SWCNT-PEG or 30 μM Y-27632 and labeled for GFAP using indirect immunocytochemistry. Scale bar, 20 μm. b Summary graphs showing the median effect of the various treatments on the quantitative GFAP-ir parameters. Number of astrocytes studied in each condition is given in parentheses in the occupancy graph. Asterisks indicate a statistical difference when compared to the untreated/control astrocytes. Other differences are marked by the brackets. Kruskal–Wallis one-way ANOVA (KWA) followed by Dunn’s test. *p < 0.05, **p < 0.01.

Fig. 4

SWCNT-PEG solute induces morphological changes in mouse cortical astrocytes. a Images of astrocytes in culture, treated with 1 μg/ml PEG, 5 μg/ml SWCNT-PEG or 30 μM Y-27632, and loaded with calcein, a vital fluorescent dye. Scale bar, 20 μm. b Summary graphs showing the median effect of the various treatments on astrocytic morphology, i.e. area, perimeter and form factor. Number of astrocytes studied in each condition is given in parentheses in the area graph. Asterisks indicate a statistical difference when compared to the untreated/control astrocytes. The other differences are marked by the brackets. Kruskal–Wallis one-way ANOVA (KWA) followed by Dunn’s test. *p < 0.05, **p < 0.01.