Page 82 - JCTR-11-4

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

The NADPH oxidase inhibitor VAS2870, a Act, 1986 (Project Licence No. PP9645035), following
triazolopyrimidine derivative, has been shown to inhibit ethical approval by the University of Nottingham Animal
a selection of NADPH oxidase isoforms, including NOX2 Welfare Ethical Review Body (AWERB) and reported
(inhibitory concentration [IC]50: 1.1µM) and NOX4 in line with Animal Research Reporting of in vivo
53
(IC50: 12.3µM), 45-47 both of which are isoforms present Experiments guidelines. Male Sprague Dawley rats
within the cerebrovasculature and ischaemic brain (Envigo, UK) aged between 52 and 59 days on arrival
regions. 26-28 Kleinschnitz et al. reported a neuroprotective were used. Animals, randomly allocated into groups of
32
outcome comparable to that observed in NOX4 knockout three, were housed according to UK standards of care
-/-
54
mice at 24 h following cerebral ischaemia when intrathecal regulations. All animals underwent 45 min of transient
VAS2870 was administered at 2 h and 12 h post-middle intraluminal filament MCAO with recovery at 78–85 days
cerebral artery occlusion (MCAO), suggesting that old. Animals were randomly allocated (n = 18; vehicle = 9,
32
VAS2870 is effective in attenuating NOX4-driven injury VAS2870 = 9XX) to receive 1 mg/kg of intravenous
progression during the early hours of stroke injury. VAS2870 (SML0273 Sigma Aldrich, UK) or vehicle (10%
More recently, VAS2870—when administered before dimethyl sulfoxide [DMSO] in 0.9% sodium chloride
reperfusion—reduced post-ischaemia injury and mortality, [NaCl] solution) at 30 min post-reperfusion, by tail vein
potentially through miRNA autoregulation of the NOX2/4 injection under general anaesthesia (GA). Sterile VAS2870
isoforms. In addition, pre-reperfusion intravenous stock solution (4 mg/mL in 100% DMSO; aliquoted and
48
administration of VAS2870 suppressed OS, neuronal stored at −80°C) was diluted at the point of injection using
apoptosis and NOX2 and NOX4 upregulation, attenuated 0.9% NaCl solution to obtain a 0.4 mg/mL solution in
brain oedema, reduced BBB permeability and improved 10% DMSO. Both VAS2870 and vehicle injection volumes
neurological function in a rat model of acute ischaemic were <5 µL/g total volume. At 48 h post-MCAO, a subset
stroke with haemorrhagic transformation. Furthermore, of animals underwent T2-weighted (T2w) magnetic
49
VAS2870 suppression of OS in vitro has shown enhanced resonance imaging (MRI; n: vehicle = 4, VAS2870 = 5).
tubulogenic and BBB-forming capacities of endothelial All animals underwent sensorimotor and cognitive testing
progenitor cells. 44,50 at various time points (Figure 1). A total of 18 animals
were used within this study: one animal died within the
However, the post-stroke role of NADPH oxidase first 20 h following MCAO, three animals were humanely
remains unclear. It has the potential to induce injuries killed by overdose of anaesthesia followed by femoral
during the acute phase of ischaemic injury while potentially artery severance due to severity limits within the first 48 h
contributing to recovery during the sub-acute phase following MCAO, and one animal was excluded during
through mechanisms, such as angiogenesis. Previous surgery due to technical complications and humanely
51
studies using VAS2870 have investigated the contribution killed under anaesthesia. On day 11 post-MCAO, all
of NADPH oxidase to ischaemic injury in the short term, remaining animals (n = 11) underwent terminal perfusion
typically at 24 h, and other acute post-stroke time points, fixation under GA before tissue removal. Sample sizes
but information regarding sub-acute neuroprotective were calculated using the resource equation (Equation I) 55
outcomes and downstream cellular changes remains to determine a biologically relevant effect size for a pilot
scarce. Furthermore, a previous work has highlighted design. For data analysis, only animals that survived to the
that NADPH oxidase suppression in the ischaemic setting end of the study were included, unless otherwise stated.
may generate opposing effects when combined with other
treatments, such as endothelial stem cell therapies . Error (E) degrees of freedom (dof) = Total dof −Treatment
44
Consequently, we utilised VAS2870, a covalent inhibitor of dof −(blocks−1); 10<E<20 (I)
NOX2 and NOX4, 45,46,52 to assess the therapeutic potential 2.2. Blinding and randomisation
of modulating these key isoforms during early post-stroke
reperfusion and determine the downstream acute and sub- To ensure consistent environmental conditions across
acute effects on brain injury and functional deficits using treatment groups, each cage was allocated one vehicle
a multimodal approach within an ischaemic stroke rodent and one VAS2870 treatment animal, with the remaining
model. vehicle or VAS2870 allocation randomly assigned across
cages (automated randomiser, random.org). Animals
2. Materials and methods were randomly allocated to cages upon arrival by the
facility staff, who were blinded to the allocations. Animals
2.1. Study design
underwent MCAO and behaviour assessment in random
This study was conducted in accordance with the order (automated list randomiser, random.org). The
United Kingdom (UK) Animals (Scientific Procedures) experimenter was blinded to the treatment group at the

Volume 11 Issue 4 (2025) 76 doi: 10.36922/jctr.25.00018