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

and isolating from its small artery branches. The ECA was filter of 540–580 nm and a long-pass emission filter
ligated 3 mm distal to its origin, and an arteriotomy was of >600 nm.
then performed on the ECA. Following that, a silicone-
rubber-coated nylon filament (602356PK5Re, Doccol 2.10. Western blot (WB) analysis
Co., USA) was inserted into the ECA and advanced for Proteins were extracted in a radioimmunoprecipitation
9 – 10 mm to the carotid bifurcation along the internal assay (RIPA) lysis buffer (P0013B, Beyotime, China),
carotid artery (ICA) and the origin of the middle cerebral separated on sodium dodecyl sulfate-polyacrylamide gels,
artery (MCA). The incision in the neck was subsequently and electrophoretically transferred onto polyvinylidene
sutured. After 1 h of occlusion, the silicone-rubber-coated fluoride (PVDF) membranes according to our previously
nylon filament was removed to restore blood flow. In sham described methods . The membranes were blocked with
[23]
mice, the filament was not inserted. 5% of non-fat dry milk in TBST, probed with antibodies
Using the Moor full-field laser perfusion imager recognizing glial fibrillary acidic protein (GFAP,
(moorFLPI-2, Moor Instruments, Axminster, UK), the 16825-1-AP, 1:1000, Proteintech), Nestin (66259-1-Ig,
cortical cerebral blood flow was measured in both cere- 1:1000, Proteintech), TUJ1 (ab18207, 1:1000, Abcam),
bral hemispheres and recorded 15 min before MCAO and β-actin (60008-1-Ig, 1:1000, Proteintech) overnight
and throughout the ischemic period until 15 min after at 4℃ before incubation with a horseradish peroxidase-
the onset of reperfusion. Animals that did not show a conjugated goat anti-mouse/rabbit immunoglobulin (Ig)
reduction in cerebral blood flow (CBF) of at least 75% of G secondary antibody (SA00001-1/SA00001-2, 1:2000,
the baseline level or died after ischemic induction (<10%) Proteintech). To detect the signals, the Microchemi 4.2 ®
were excluded from further experimentation . A total of (DNR Bio-Imaging Systems, Jerusalem, Israel) digital
[24]
33 mice were included in this study; however, based on the image scanner was used. Individual protein bands were
aforementioned criteria, seven mice were excluded due to quantified by densitometry using Image J.
death (n = 4) or failed stroke induction (n = 3).
2.11. Prussian blue staining
2.7. Microinjection of neural stem cells The brain tissue was cut into 30 μm-thick sections. The
The lateral ventricles of mice were microinjected with tissue sections were washed every 5 min with PBS (3 times).
NSCs at 7 days after tMCAO at the following coordinates: Prussian blue (PB) staining was performed using a Prussian
0.3 mm behind the bregma and 1.0 mm lateral from Blue Staining Kit (Sigma-Aldrich) according to the user
the sagittal midline, at a depth of 2.2 mm from the skull manual. In brief, tissue sections were incubated with Perls
surface. A total of 2 μL of cell suspension (1 × 10 cells) was stain for 30 min at room temperature. Then, the tissue
6
injected into the left lateral ventricle at a rate of 0.1 μL/min sections were washed with distilled water for 5 min. The
by microinjection pump. sections were covered with counterstaining solution for
10 min and subsequently washed with distilled water for
2.8. Magnetic resonance imaging scanning 5 min. The tissue sections were sealed with neutral resin.
In vivo MRI was performed using a 7-Tesla small animal 2.12. Bromodeoxyurdine (BrdU) labeling
MRI scanner (BrukerPharmaScan, Karlsruhe, Germany).
The mice were anesthetized with 2% of isoflurane NSCs were prepared on poly-L-lysine-coated glass coverslips.
delivered through a nose cone. The respiratory rate and 10 μL of Dulbecco’s PBS containing 10 mM BrdU was added
body temperature of the mice were monitored through a in the 1 mL cell culture medium. After 4 h, the cells were
physiology monitor. All sequences were performed on the fixed overnight in 4% of paraformaldehyde (PFA) at 4°C.
murine cerebrum in axial/coronal orientation with the The cells were then denatured with HCl for 30 min at room
following parameters: Multigradient-echo sequences (12 temperature and washed with PBS 2 times. Subsequently,
echos; repetition time [TR], 1,500 ms; first echo at 4 ms; the cells were blocked for 1 h in PBS containing 10% normal
2
echo spacing 7 ms; field of view [FOV], 20 × 20 mm ; goat serum and 0.1% Triton X-100 at room temperature.
matrix, 256 × 256; slice thickness, 0.6 mm, 8 average) were The blocked cells were incubated with mouse anti-BrdU
used for T2-star mapping. antibody (1:200; SC-32323, Santa Cruz, CA) that were
diluted in PBS containing 10% normal goat serum and 0.1%
2.9. Near-infrared fluorescence (NIF) imaging Triton X-100 at 4°C overnight. The samples were washed
The mice were microinjected with SPIO-labeled NSCs with PBS 3 times before being incubated with donkey anti-
at 7 d after tMCAO. NIF images were obtained with the mouse (conjugated to Alexa-Fluor 576) secondary antibody
Maestro In Vivo Imaging System (Cri Inc., USA). Near- for 1 h at room temperature. After washing with PBS for
infrared light was collected with an excitation bandpass another 3 times, the samples were mounted using ProLong

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