Page 86 - JCTR-11-4

Journal of Clinical and
Translational Research NADPH oxidase inhibition in a rodent stroke model

dried and stored at room temperature until imaging. Slides groups at any time post-MCAO (Figure 2D). A significant
were imaged using a light box with a digital single-lens interaction between time and treatment was observed
reflex camera (Canon M50II, Canon, Japan). (F [11,99 = 1.98, p=0.038); however, treatment was not a
Slices depicting an area of decreased/absent CV significant source of variation (F [1,9 = 1.95, p=0.196). In
staining, indicative of ischaemic damage, were analysed. contrast, time post-MCAO had a significant overall effect
Three repeat measurements of the contralateral on body weight (F [2.43, 21.9 = 34.6, p<0.001; Figure 2D).
hemisphere, ipsilateral hemisphere and infarct (denoted Post hoc analysis revealed significant weight loss in the
by regions of decreased CV Nissl stain intensity in the vehicle-treated group at day 3 post-MCAO compared to
region of the MCA territory, indicating neuronal loss) areas the baseline (p=0.07; Figure 2D), which was not observed
were delimitated manually by an experienced operator. in the VAS2870-treated group.
To calculate volume, the area means were multiplied by 3.2. Post-ischaemic functional and cognitive
slice thickness and series slicing distance (400 µM). Total impairments following NADPH oxidase inhibition
infarct volume was adjusted for hemispheric swelling/
shrinking per slice (infarct volume/[ipsilateral as a % of To assess post-stroke functional deficits, mNSS scores
contralateral]). Image analysis was performed using FIJI were measured. Results showed no significant difference
ImageJ (v1.54f; NIH, United States). was observed for the overall effect of treatment (vehicle
vs. VAS2870) on mNSS scores (F [1,9 = 3.622, p=0.09),
2.13. Statistical analysis or between treatment and time (F [2,18 = 3.173, p=0.07).

Statistical analysis was performed using GraphPad However, post-MCAO time had a significant effect on
PRISM (v10.3.1). All data were assessed for normality mNSS score (F [2,18 = 23.72, p<0.001; Figure 3A). Post
before statistical analysis, using the Shapiro–Wilk test. hoc analysis revealed that both vehicle- and VAS2870-
For two-group comparisons, an unpaired t-test with treated groups had significantly reduced mNSS scores at
Welch’s correction was performed. For comparisons of day 2 post-MCAO compared to the baseline (p=0.02 and
multiple groups, two-way repeated measures analysis p<0.001, respectively; Figure 3A). In addition, at day 2
of variance followed by a multiple comparison test with post-MCAO, the VAS2870-treated group had significantly
Šídák correction was used for post hoc analysis. Repeated lower mNSS scores (p=0.02) compared to the vehicle-
measure matching levels were brain region, hemisphere, or treated group. By day 10 post-MCAO, mNSS scores in
time, depending on the experimental design. For survival both groups returned to baseline levels (Figure 3A).
analysis, the Mantel-Cox test was used. A significance level Cognitive deficits in spatial working memory, measured
of p<0.05 was considered statistically significant. All data by percentage spontaneous alternations using the Y-maze,
were expressed as mean ± standard deviation. did not differ significantly between VAS2870- and vehicle-
treated groups (t [9 = 0.183, p=0.859; Figure 3B). Similarly,
3. Results there were no significant differences in total arm entries
(t [5.91 = 0.708, p=0.51; Figure 3C) and total distance
3.1. Post-ischaemic NADPH oxidase inhibition does travelled (t [9 = 0.96, p=0.362; Figure 3D), suggesting
not attenuate brain injury or general wellbeing but that VAS2870 treatment did not affect cognitive function,
improves survival probability motor activity, or exploratory behaviour.
To evaluate post-ischaemic VAS2870-induced NADPH
oxidase inhibition on brain injury, infarct volume was 3.3. Pro-inflammatory cytokine levels and
assessed at 48 h post-MCAO using a T2-weighted MRI, circulating antioxidant capacity post- NADPH
and at day 11 post-MCAO through Nissl quantification oxidase inhibition
(Figure 2). Representative brain slice images from both The effect of post-ischaemic NADPH oxidase inhibition
modalities are shown in Figure 2A. VAS2870 treatment on circulating inflammatory markers and antioxidant
did not significantly alter infarct volume at 48 h capacity was evaluated at day 11 post-MCAO. Circulating
(t [4.123] = 1.972, p=0.118), nor at day 11 (t [4.02] = 2.03, total antioxidant capacity, expressed as the concentration
p=0.111) post-MCAO (Figure 2B). Survival analysis, of Trolox equivalents, did not significantly differ between
which included animals removed from the study due VAS2870 and vehicle-treated groups (t [6.22 = 0.23,
to severity limits, showed that VAS2870 treatment p=0.83; Figure 4A). Similarly, circulating concentrations of
significantly improved survival by 50% by day 2 post- inflammatory cytokines IL-1β (t [6.28 = 0.301], p=0.77),
MCAO (χ² [1 = 3.939, p=0.047; Figure 2C). No significant TIMP1 (t [8.67] = 0.123, p=0.9), and VEGF (t [5.847] = 1.09,
difference in weight loss, an indicator of general well-being, p=0.317) were not significantly different between the two
was observed between vehicle- and VAS2870-treated groups at day 11 post-MCAO (Figure 4B-D).

Volume 11 Issue 4 (2025) 80 doi: 10.36922/jctr.25.00018