Page 84 - JCTR-11-4

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

Instruments, United States) with a respiration pillow and (version 16) software and were randomly allocated to a
rectal thermometer to monitor temperature. Temperature start arm before the trial.
was maintained at 37 ± 1°C using a water-heated MRI bed.
To locate the cortical region, coronal scout imaging was 2.8. Terminal perfusion, blood sampling and tissue
performed. T2w images were acquired using a turboRARE collection
sequence with the following parameters: a repetition On post-MCAO day 11, animals underwent transcardial
time/echo time of 5,000/50 ms, a field of view of 28 × terminal perfusion following GA overdose (induction:
28 mm (256 × 256 matrix), 18 × 1 mm cortical coronal isoflurane 5% in O ; overdose: IP pentobarbital). After
2
slices, four signal averages and a total scan duration of confirming pedal reflex loss, animals were perfused with
10 min and 40 s. Region of interest analysis of infarct a 0.9% NaCl solution followed by 4% paraformaldehyde
volume was performed using T2w images in FIJI ImageJ solution. Cortices were removed and stored in 4%
58
(v1.54f; NIH USA) software, using Bio-formats Plug-in paraformaldehyde solution overnight at 4°C, then
for Neuroimaging Informatics Technology Initiative file dehydrated in 30% sucrose and phosphate buffer saline
visualisation. For all slices, three repeat measurements of and stored at 4°C until the tissue sank (~24–48 h). Cortices
the contralateral hemisphere area, ipsilateral hemisphere were optimal cutting temperature-embedded and stored at
area, and, if identified, infarct area (denoted by regions of −80°C. For plasma samples, blood was collected from the
hyperintense T2 signal) were delimitated manually by an atrium blood pool at the start of transcardial perfusion and
experienced operator, who was blinded to the treatment placed in ethylenediaminetetraacetic acid-coated 1.3 mL
group and animal ID. To calculate volume, the area vials (ID 41.1395.005, Starsdet, UK). The samples were left
means were multiplied by the slice thickness. Total infarct to rest at room temperature for an hour, and then spun at
volume was adjusted for hemispheric swelling per slice 2,000 × g for 15 min. Plasma supernatant was aliquoted
(Equation II). and stored at −80°C.
Infarct volume/(ipsilateral as a % of contralateral) (II) 2.9. Biomarker analysis

2.6. Neurogenesis marker administration Total antioxidant capacity, a measure of OS reducing
Cell proliferation at 48 h post-MCAO was assessed using capacity, was measured using the Trolox equivalent
the cell proliferation marker 5‐bromo‐2’deoxyuridine antioxidant capacity assay kit (MAK187-1KT; Merck, UK),
(BrdU). At 48 h post-MCAO, animals received 50 mg/kg according to the manufacturer’s instructions, with samples
BrdU solution (IP; 10 mg/mL in 10% DMSO and 0.9% run in triplicate. An estimation of total antioxidant capacity
NaCl solution) under GA (isoflurane in O : induction 5%; was assessed using a copper (II) ion-buffered reagent
2
maintenance 2%). Animals that underwent MRI received compared against the Trolox (a water-soluble vitamin E
the BrdU treatment after removal from the MRI scanner, analogue) antioxidant standard through colourimetric
and animals that did not undergo MRI received GA and detection at 570 nm (A ).
570
BrdU at the same time point. Following injection, animals Cytokine analytes interleukin (IL) 1β, tissue inhibitor
were observed and were left to recover within a pre- of metalloproteinase (TIMP) 1 and vascular endothelin
warmed recovery cage, before being returned to the home growth factor (VEGF) levels were measured, in triplicate,
cage. from plasma using a multiplex assay (Luminex Discovery
Assay [Rat A Standards], R&D Systems, UK) with a Bio-
2.7. Behavioural assessment Rad Bio-Plex 200 system (Bio-Rad Laboratories Ltd, UK),
®
®
Sensorimotor impairment was assessed using a modified according to the manufacturer’s instructions. Standard
neurological severity score (mNSS) adapted from Ord curves for each analyte were used to calculate protein
et al. to incorporate assessments relevant to sensorimotor concentrations (pg/mg). Initial fluorescence analysis
59
function. The score has a total range of 0–28, where lower was conducted using the Bio-Rad Bio-Plex Manager
scores indicate greater deficits (Figure A1). Baseline (v.6.2.0.175), with subsequent comparative analysis using
assessment was conducted three days before MCAO, GraphPad PRISM (v10.3.1).
with post-MCAO assessment on days 2 and 10. Cognitive
dysfunction, particularly spatial working memory, was 2.10. Tissue slicing
assessed using spontaneous alternations during an 8-min Five randomly selected brains per treatment group
exploratory activity observation within a Y-maze on post- underwent coronal sectioning using a cryostat (Microm
60
MCAO day 11. During the Y-maze assessment, animals HM505e) at −16°C. Brains were serially sectioned using
were remotely tracked using a ceiling-mounted camera interlocking intermittent series for free-floating double-
(VCB-310SP, Sanyo Panasonic, Japan) with the EthoVision label immunofluorescence (40 µM; 1:10 series) and Nissl

Volume 11 Issue 4 (2025) 78 doi: 10.36922/jctr.25.00018