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Advanced Neurology Long-term in vivo MRI tracking of SPIO-labeled NSCs

Gold Antifade Reagent with 4’,6-diamidino-2-phenylindole concentration of SPIO (Figure 1B). Subsequently, MRI was
(DAPI) (0100-20, SouthernBiotech, USA). The samples used to test the signal of SPIO nanoparticles at different
were imaged by microscopy (Olympus, Japan) and analyzed concentrations (Figure 1C). With the increase in SPIO
by Image J software. nanoparticle concentration, the MRI signal increased
gradually, and markedly enhanced signals were observed
2.13. Immunofluorescence (IF) staining in samples with concentration over 200 μg/mL. Moreover,
Before staining, for tissue samples, coronal slices (30 μm) we assessed the concentration-related internalization of
were prepared from brain tissues and incubated with Triton SPIO by ICP-MS (Figure 1D). We found that the amount
X-100 (0.25% in PBS) for 15 min, and subsequently blocked of iron increased along with the concentration of SPIO,
for 1 h in PBS containing 10% of normal goat serum and demonstrating that NSCs internalized SPIO nanoparticles
0.1% of Triton X-100 at room temperature. For cell samples, in a dose-dependent manner. As shown in Figure 1E and F,
the NSCs were fixed for 20 min in 4% of PFA in PBS and SPIO-labeled NSCs did not affect cell proliferation. In
washed 3 times with PBS. Then, the cells were incubated conclusion, at a concentration of 200 μg/mL, the uptake
with Triton X-100 (0.25% in PBS) for 15 min and blocked for of SPIO nanoparticles was sufficient and did not have any
1 h in PBS containing 10% of normal goat serum and 0.1% noticeable influence on the viability of NSCs. Therefore, we
of Triton X-100 at room temperature. The blocked sections used 200 μg/mL for all experiments.
and cells were incubated with primary antibodies that were
diluted in PBS containing 10% normal goat serum and 0.1% 3.2. Differentiation of SPIO-labeled neural stem cells
Triton X-100 at 4°C overnight. The primary antibodies used Biosafety is a crucial requirement for nanotracers to label
in this study contained GFAP (16825-1-AP, Proteintech) stem cells. To investigate whether SPIO is involved in
and TUJ1 (ab18207, Abcam). The samples were then washes the regulation of NSC differentiation, NSCs were labeled
with PBS three times before being incubated with secondary with SPIO at a concentration of 200 μg/mL. WB analysis
antibodies, including Alexa Fluor 594 goat anti-mouse IgG showed that the levels of GFAP (the astrocyte marker)
(A-11005, Invitrogen) and Alexa Fluor 594 goat anti-rabbit and TUJ1 (the neuronal marker) increased during NSC
IgG (A-11012, Invitrogen) for 1 h at room temperature. The differentiation, while the expression of Nestin (NSC
samples were washed with PBS three times and mounted marker) significantly decreased. However, there was no
using ProLong Gold Antifade Reagent with DAPI (Southern significant difference in the expression of Nestin, GFAP,
Biotech, USA). The samples were imaged by microscopy and TUJ1 between the SPIO-labeled NSCs (SPIO) group
(Olympus, Japan) and analyzed by Image J software. compared with the no SPIO-labeled NSCs (control) group
2.14. Statistics (Figure 2A and B). The detection of GFAP expression
at 1 day, 7 days, and 14 days after NSC treatment by IF
The data were presented as mean ± standard error of staining showed that there was no significant difference
the mean (SEM). Significance was established using in GFAP expression between the SPIO-labeled group and
t-test for paired values. One-way analysis of variance the control group (Figure 2C and D). Meanwhile, as shown
(ANOVA) followed by Holm-Sidak post hoc test was used in Figure 2E and F, there was no significant difference in
for comparisons of three groups. Statistical analysis was TUJ1 expression between the SPIO-labeled group and the
performed using GraphPad Prism 8.0. control group, indicating that SPIO did not influence the
3. Results differentiation of NSCs.
3.1. Characterization of SPIO -labeled neural stem 3.3. Tracking the signal of SPIO nanoparticles in a
cells tMCAO stroke model
To determine the characterization of transplanted NSCs To investigate the migration of transplanted NSCs labeled
labeled with SPIO, we first evaluated the viability of SPIO- with SPIO, MRI was used to detect signals in the coronal
labeled NSCs. CCK-8 assay was performed with NSCs that plane of mouse brains that were detected by MRI. Signals
were incubated with SPIO nanoparticles at concentrations were detected over the injection site at 7 days and the
of 0, 25, 50, 100, 200, and 300 μg/mL for 48 h. As shown in ischemic area at 14 days, 21 days, and 28 days in the SPIO-
Figure 1A, SPIO nanoparticle concentrations of <300 μg/mL labeled NSCs group after tMCAO (Figure 3A). However,
showed low cytotoxicity (90%). The viability decreased by no signals of SPIO nanoparticles were detected in the
9.64% when the concentration was 300 μg/mL. We then control group. This suggests that NSCs labeled with SPIO
explored the uptake of SPIO nanoparticles, in which a PB nanoparticles could migrate from the injection site to the
staining assay was performed. The results showed that the ischemic area. Besides, signals were also detected in the
intake of SPIO by NSCs was directly proportional to the horizontal plane of mouse brains by MRI (Figure 3B).

Volume 1 Issue 3 (2022) 4 https://doi.org/10.36922/an.v1i3.278