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

uate their effect on ischemic stroke is the key to evaluating medium into 24-well microplate. Then, the cells were
the treatment strategy for ischemic stroke. harvested and counted. The cell suspension was dissociated
In recent years, a number of methods have been used to by hydrochloric acid (HCl). The concentration of iron in
track NSCs, such as bromodeoxyuridine (BrdU) . How- cell lysates was measured by ICP-MS according to Perkin
[15]
ever, this method has its limitations because BrdU signal Elmer’s operating procedures.
becomes weak when transplanted NSCs proliferate and dif- 2.3. Ethics approval and consent to participate
ferentiate, making it difficult to monitor the cells over a long
time. Researchers are now making efforts to develop appro- All animal experiments were performed in accordance with
priate methods for long-term tracking of stem cells after the standard guidelines for the care and use of laboratory
transplantation. Here, superparamagnetic iron oxide (SPIO) animals and approved by the Institutional Animal Care
nanoparticles are considered to be the practical application and Use Committee of the Medical School of Southeast
of stem cells because nano iron oxide is the only inorganic University.
nanomaterial that has been approved by the Food and Drug 2.4. Cell cultures
Administration (FDA) as a nanodrug . SPIO can be used
[16]
as a clinical magnetic resonance imaging (MRI) contrast NSCs from C57BL/6J or enhanced green fluorescent protein
agent [17,18] . Studies have shown that SPIO has been success- (eGFP) transgenic mice were isolated from embryonic day
fully used for tracking different cell types by MRI in vivo 14 mice hippocampus and sterilized with 75% of alcohol.
because of its characteristics, which include easy synthesis, Fetal mice were taken out from pregnant mice and placed
superparamagnetic, high saturation magnetization, good in a 50 mL centrifuge tube with sterile phosphate-buffered
biocompatibility, and low toxicity . To monitor the migra- saline (PBS). Brains were removed from the fetal mice
[19]
tion and survival of transplanted NSCs in vivo, SPIO can be and dissociated to obtain the hippocampus. Next, the
used as an MRI tracer to non-invasively monitor in vivo. hippocampus was placed in a medium supplemented with
PBS, and the tissues were digested with trypsin (25200056,
In the present study, SPIO nanoparticles were used Gibco, USA). Then, the cells were plated on cell culture
to detect the migration and distribution of transplanted flasks containing basic fibroblast growth factor (bFGF,
NSCs in (transient middle cerebral artery occlusion 20 ng/mL, Stem Cell, Canada), epidermal growth factor
[tMCAO]) mice. We sought to demonstrate the authentic- (EGF, 20 ng/mL, Stem Cell, Canada), and B-27 supplement
ity of SPIO-labeled NSCs tracking in tMCAO mice and the (2%, Gibco, USA). The culture was maintained in a
capacity of NSCs for migration, thus providing a visual basis humidified chamber (5% carbon dioxide [CO ], 37°C). The
for evaluating their therapeutic effect on ischemic stroke. NSCs (3 – 8 ) were used in the study. 2
th
rd
2. Materials and methods 2.5. Cell Counting Kit-8 (CCK-8) assay
2.1. Synthesis of SPIO nanoparticles NSCs were digested into single-cell suspension by enzyme
SPIO was synthesized based on previous reports [16,20] . First, and cultured at a density of 1 × 10 /well in 100 μL of
4
polyglucose sorbitol carboxymethyl ether (PSC, 200 mg) medium into 96-well microplate for 24 h. Then, the cells
aqueous solution (10 mL) was aerated with nitrogen to were treated with different concentrations of SPIO (0, 25,
remove oxygen for 5 min. Second, ferric chloride (FeCl , 50, 100, 200, and 300 μg/mL). After treatment for 48 h,
3
60 mg, 0.37 mmol) and ferrous chloride (FeCl , 30 mg, 10 μL of CCK-8 reagent was added to each well, and then
2
0.236 mmol) were dissolved in 15 mL of double distilled water cultured for 4 h. The absorbance was analyzed at 450 nm.
(ddH O). The mixture was then added into the PSC solution.
2
Third, 28% of ammonium hydroxide (1 g) was added into 2.6. tMCAO
the solution with the mixture, and then mechanically stirred tMCAO was processed according to a previous study [12,21-23] .
in a water bath for 30 min (80°C). Next, the iron oxide was Briefly, anesthesia was induced with 3% of isoflurane,
collected by an ultrafiltration centrifuge tube (30 kD) and 30% of oxygen, and 70% of nitrous oxide in an anesthetic
washed with ultrapure water 3 times. The technique was chamber and maintained with 1.5% of isoflurane through
based on the preparation process of ferumoxytol, which has a facemask. A temperature-controlled heating pad was
been approved by the FDA as an inorganic nanodrug. used to maintain the temperature at 37.0 ± 0.5°C during
the surgery and recovery period. A 1 cm midline skin
2.2. Inductively coupled plasma mass spectrometry incision was made over the neck, and the right common
(ICP-MS) carotid artery (CCA) was carefully dissected free from the
NSCs were digested into single-cell suspension by enzyme surrounding nerves under a stereo dissecting microscope
and cultured at a density of 5 × 10 /well in 500 μL of and tied off, exposing the right external carotid artery (ECA)
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Volume 1 Issue 3 (2022) 2 https://doi.org/10.36922/an.v1i3.278