Increased vascular stiffness and, consequently, pulsatility increase the risk of developing Alzheimer’s disease and several cardiovascular diseases, but the exact pathways are still a mystery. Most of our knowledge on the in-vivo effects of increased stiffness comes from the large vessels, while capillaries are expected to play the most significant role in cognitive decline. Using Laser Speckle Contrast Imaging (LSCI), we investigated how the microvascular pulsatility changes with age in wild-type mice (C57BL/6) and how it is affected by two of the most commonly used anaesthesias: isoflurane and ketamine/xylazine (k/x).

Twelve mice aged 18 (n=5), 43 (n=3), and 65 (n=4) weeks, respectively, were imaged at 5 separate time points, 4 weeks apart. To access the cortical microvessels, we used LSCI in awake-restrained mice with a chronic cranial window over the left middle cerebral artery (MCA) and its branches. Without changing the field of view, the animals were also imaged under isoflurane anaesthesia followed by k/x anaesthesia. After finishing in-vivo experiments, the MCA was isolated and mounted on a wire myograph and tone was recorded to assess the physiological changes of ageing in the vasculature.

The blood flow and pulsatility index in veins, arteries and parenchyma did not change with age and remained constant throughout the experiment. The pulse-associated relative diameter dilation remained constant in veins, but in arteries, it began to increase after 65 weeks of age, from 0.0986 arb. unit at 65 weeks to 0.1580 a.u. at 81 weeks (60.2 %, p= 0.0521). These results are supported by the myograph results, indicating an age-related increase in compliance of the MCA.
Both anaesthesia caused significant changes to the pulsatility of blood flow velocity in arteries (p=8.5*10^-10 under isoflurane and p=1.4*10^-11 under k/x using a paired t-test) veins (9.4*10^-9 under isoflurane and 1.3*10¹² under k/x using a paired t-test) and parenchyma (9.2*10^-9 under isoflurane and 5.5*10^-12 under k/x using a paired t-test). However, because isoflurane causes a strong vasodilation, the pulse-associated diameter change did not change in arteries (p=0.69, paired t-test) but did in veins (5.4*10^-7, paired t-test). But k/x caused a significant response in both arteries (p=1.1*10^-7, paired t-test) and veins (1.8*10^-8, paired t-test). These results clearly show that while more time-consuming, awake imaging is better when assessing physiological changes in the vasculature and blood flow.

All experimental protocols were approved by the Danish National Animals Experiments Inspectorate and conducted according to their guidelines.