Page 100 - GTM-4-1

Global Translational Medicine Comparative study of Schisandra species on VCI

Tecan, Switzerland). While, the concentrations of tumor of motor function (muscle status, abnormal movements),
necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) sensory function (visual, tactile, and proprioceptive), and
were measured using ELISA kits (Cat. no: 431304 [IL-6], reflexes. Scores range from 0 (normal) to 18 (maximal
Cat. no: 430915 [TNF-α], Biolegend, USA) according to deficit), with higher scores indicating greater neurological
the manufacturer’ s protocol. impairment.

2.4. Measurement of mitochondrial membrane 2.7. Open field test
potential
Behavioral activity was assessed on day 29 using the open
The mitochondrial membrane potential was assessed using field test. Mice were placed individually in the center of a
a JC-1 kit (Beyotime, China) under OGD/R conditions. 50 × 50 × 50 cm white box, and their total distance traveled
Cells were incubated with JC-1 working buffer in the dark for (in mm) was recorded over 5 min using a video analysis
30 min, following the manufacturer’s protocol. Fluorescence system (v5.2.0.0, SuperMaze, China).
signals representing mitochondrial membrane potential
levels were detected using a fluorescence microscope at 2.8. Novel object recognition test (NORT)
excitation/emission wavelengths of 514/529 nm (for JC-1 Cognitive abilities were evaluated using the NORT from
monomers) and 585/590 nm (for JC-1 aggregates). days 30 to 33. During the first 3 days, mice acclimated daily

2.5. Animal models and treatment protocols to a 50 × 50 × 50 cm white box containing two identical red
cylinders placed at opposite ends. Tests were conducted on
All animal experiments were approved by the Committee the 4 day in two phases:
th
on Use and Care of Animals of the Institute of Medicinal (i) Phase 1: Mice were allowed to explore two identical
Plant Development. A total of 50 10-week-old male cylinders (F1 and F2) for 5 min. The exploration
C57BL/6J mice (weighing 24 – 29 g) were provided by time for each object was recorded, and the relative
Vital River Laboratory Animal Technology (China) and preference index was calculated as
acclimated to laboratory conditions for 7 days. The mice
were randomly divided into five groups (n = 10 per group): RPI ( ) =% F 1 − 2F ×100 (I)
(i) Control (CON): Sham operation with no treatment. F 1 + 2F
(ii) Model (MOD): Bilateral common carotid artery (ii) Phase 2: After a 30-min rest period, one cylinder was
stenosis (BCAS) surgery with vehicle treatment (0.5% replaced with a new green square of the same height.
sodium carboxymethyl cellulose [CMC-Na]). Mice were allowed to explore the new object (N) and
(iii) Positive control (POS): BCAS surgery with 20 mg/kg the remaining cylinder (F) for 5 min. The exploration
of nimodipine (Bayer, Germany) time was recorded, and the relative discrimination
(iv) SCF group: BCAS surgery with 400 mg/kg SCF index was calculated as follows:
extract.
−
(v) SSF group: BCAS surgery with 400 mg/kg of SSF RDI ( ) = NF ×100 (II)
%
extract. N + F
Mice were anesthetized, and both common carotid Exploration behaviors were recorded and analyzed
arteries (CCAs) were exposed. BCAS was induced using using SuperMaze software.
microcoils (0.18 mm inner diameter; Sawane Spring Co, 2.9. Morris water maze (MWM) test
Japan) following established protocols. The microcoil
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was first applied to the right CCA by rotating it around Spatial learning deficits were assessed twice daily using an
the artery and then to the left CCA after a 30-min interval. enhanced MWM test from days 33 to 37. A 1-m-diameter
Sham-operated control mice underwent the same surgical pool was placed in a room devoid of external orientation
procedure without the placement of microcoils. Seven days cues and divided into four quadrants with four fixed points
post-surgery, BCAS mice in different groups received daily using the SuperMaze system. An invisible, submerged
intragastric administration of the corresponding drug platform was fixed in the northwest quadrant. Mice were
treatments for 30 consecutive days, while the CON and randomly placed at one of the designated starting points
MOD groups received 0.5% CMC-Na as vehicle controls. and were allowed 1.5 min to locate the platform, with a
10-s stay permitted upon successful navigation. Mice
2.6. Neurological severity score test unable to find the platform were passively placed on it
Neurological function was evaluated using the modified for 10 s. On day 38, the platform was removed, and mice
neurological severity score (mNSS) test on days 7, 14, 21, underwent a free swim test for 1.5 min to evaluate spatial
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and 28 post-BCAS. The mNSS is a composite assessment reference memory. The time taken to reach the previous

Volume 4 Issue 1 (2025) 92 doi: 10.36922/gtm.6879