Nitric oxide (NO) is a critical regulator of vascular tone and cerebral blood flow, and reduced NO bioavailability is implicated in both cardiovascular and neurodegenerative disease. Although inhibition of nitric oxide synthase (NOS) is known to alter cerebral haemodynamics and vasomotion, its effects are typically assessed using individual physiological measures in isolation. Here, we applied a multivariate systems-level approach to characterise how NOS inhibition reshapes coordinated cerebrovascular and systemic physiology.

Multiple indices of systemic and cerebrovascular function were recorded simultaneously in awake mice under control conditions and following administration of the non-specific NOS inhibitor L-NG-nitroarginine methyl ester (L-NAME; 75 mg kg⁻¹, s.c.). Cerebral blood flow, blood oxygenation, total haemoglobin and derived oxygen metabolism (CMRO₂) were measured in visual cortex and hippocampus alongside peripheral heart rate, respiratory rate, arterial oxygen saturation, pulse distension and blood pressure. Vasomotion and neurovascular coupling were quantified from these signals.

L-NAME reduced heart and respiratory rates and increased blood pressure, with minimal effects on peripheral oxygen saturation. In contrast, cerebral blood flow, oxygenation and CMRO₂ were markedly reduced in both regions, while blood volume was relatively preserved. Vasomotion power and frequency increased substantially, particularly in the hippocampus, whereas microvascular pulsatility increased similarly across regions and neurovascular coupling remained largely intact. Multivariate analyses combining principal component analysis with mixed-effects modelling revealed dissociable physiological axes reflecting direct effects of NOS inhibition and secondary effects mediated by reduced cerebral oxygenation. Notably, changes in vasomotion were more strongly associated with reductions in cerebral oxygenation than with drug condition itself, indicating that some effects commonly attributed to loss of NO signalling may instead arise from secondary hypoxia. These findings suggest that NOS inhibition produces complex cerebrovascular changes that cannot be assumed to reflect direct NO-dependent mechanisms alone.