Page 90 - BH-2-4

Brain & Heart Human DPSCs attenuated amyotrophic lateral sclerosis in mice

2.3. Rotarod test sections were observed under the PerkinElmer Launches
™
®
Mice were placed on a fixed-speed rotating rod (3 cm in Vectra Polaris Automated Quantitative Pathology
diameter and rotating at 40 rpm). All mice were trained Imaging System. Cells were quantified for each spinal cord
twice daily for 3 days after the injection at day 80. At each section using the ImageJ software (National Institutes of
experimental time point, the time the mice spent on the Health, MD, USA).
rotating rod was calculated up to a maximum of 180 s. Each 2.6. Flow cytometry analysis
mouse was tested 3 times at the same time each week, and
the results were calculated as the average of the three trials. Fresh mice brain and spinal cord were separated on day
150 and analyzed using flow cytometry. First, 500 µL of
2.4. 7-T magnetic resonance imaging (MRI) collagenase I (1 mg/mL, Solarbio, C8140) was added
MRI was performed using a 7.0-T MRI scanner to fresh mouse brain tissues, which were then cut into
(BioClinScan, Bruker, Germany) equipped with a pieces using pre-cooled ophthalmic scissors on ice. Next,
31-cm aperture Ultra Shield Refrigerated Magnet, with an additional 500 µL of collagenase I was added and
a gradient field strength of 290 mT/m and a switching gently mixed. The mixture was digested for 30 min in an
rate of 1160 T/m/s at days 120 and 150. T2-weighted incubator at 37°C. Then, 2 mL of 1 × PBS (Solarbio, P1020)
imaging (T2WI) – turbo spin echo images were acquired was added to stop the digestion process, followed by
by a spin-echo echo-planar imaging sequence with centrifugation (4°C, 10 min, and 1000 rpm). The resulting
15 slices (repetition time/echo time = 2240/38 ms; matrix supernatant was discarded, and 10 mL of 30% percoll per
size = 192 × 320; field of view = 24 × 30 mm; and slice tube was added, followed by centrifugation (4°C, 10 min,
thickness = 1.0 mm). The T2-weighted images were used and 2,000 rpm) to eliminate myelin. After discarding the
to obtain the cross-sectional area of the lumbar spinal cord supernatant, the pellet was mixed and washed with 1 mL
without any contrast agent. The MRI scans were analyzed of 1× PBS. After another round of centrifugation to remove
by experts blinded to the experimental group using an the supernatant, a single-cell suspension was obtained by
anatomical atlas for guidance. Freehand region-of-interest resuspending the cells in 1 mL of 1 × PBS. The cells were
measurements were conducted using the ImageJ software then suspended in 100 µL of 2% bovine serum albumin
™
(National Institutes of Health, MD, USA). The cross- for blocking. Zombie Violet (BioLegend, 423114)
sectional area of the lumbar spinal cord was measured staining was performed at room temperature for 20 min to
manually by outlining relevant anatomical structures. Six distinguish between live and dead cells. Next, the cells were
slices were analyzed per mouse and averaged to obtain a incubated with conjugated antibodies for surface staining
mean value for each animal. 21 at 4°C for 30 min, followed by fixing using a fixation
buffer (BioLegend, 420801) at 4°C for 20 min and two
2.5. Immunofluorescence staining washing steps in a 1× permeabilization buffer (BioLegend,
The brain and spinal cord of mice were separated at day 150 421002) for 30 min for intracellular staining. The following
and fixed overnight in 4% paraformaldehyde at 4°C. The antibodies were used: NeuN (Abcam, ab223994), CD45
brain was dehydrated in 15% and 30% sucrose sequentially. (BioLegend, 103116), and CD11b (BioLegend, 101216).
™
After embedding in Optimal cutting temperature compound Data and images were acquired using a FACSAria II
(OCT), brain tissues were cut into 8-µm-thick slices using flow cytometer (BD, Biosciences) and analyzed using the
a cryostat. The sections were washed 3 times (5 min each) FlowJo software (version 10.5.3).
with PBS and blocked using 5% normal donkey serum and 2.7. Statistical analysis
0.3% Triton X-100 in PBS for 1 h. Then, the sections were
incubated overnight at 4°C with the following primary All statistical details are provided in figure legends.
antibodies: rabbit anti-Iba1 (1:200; Abcam, ab178846), Animals were randomly assigned to the experimental
mouse anti-NeuN (1:400; Abcam, ab104224), rabbit anti- groups using the random number generator function in the
glial fibrillary acidic protein (1:400; Abcam, ab7260), and computer. All data analyses were performed independently
rabbit brain-derived neurotrophic factor (anti-BDNF) by investigators who were blinded to the experimental
(1:100; Abcam, ab108319). The next day, the sections were groups. Data are expressed as mean ± standard error of
rinsed 3 times with PBS for 10 min and then incubated the mean. p < 0.05 was considered statistically significant.
with corresponding secondary antibodies (Alexa Fluor ® Statistical significance was determined using the unpaired
488 donkey anti-rabbit Immunoglobulin G [IgG], 1:1000; t-test for two groups and two-way analysis of variance for
Thermo Fisher Scientific, USA, R37118; Alexa Fluor 488 three groups. All statistical analyses were conducted using
®
donkey anti-mouse IgG, 1:1000; Thermo Fisher Scientific, GraphPad Prism version 10.0 (GraphPad Software, La
USA, R37114) for 1 h at room temperature. Finally, the Jolla, CA).

Volume 2 Issue 4 (2024) 3 doi: 10.36922/bh.3996