Moorflpi 2

Repeated measurements of hindlimb blood flow perfusion over the region of interest were obtained, ensuring that the scanning area covered both the hindlimb paws. Serial color-coded perfusion images were collected immediately after surgery (post), as well as 14 and 28 days after surgery using a laser speckle imaging system (moor FLPI-2; Moor Instruments, Axminster, UK) (Figure 2A). The perfusion parameters are expressed as the ratio of the left (stenosis) to the right (normal) limb (18 (link)).

Laser Doppler perfusion imaging (LDPI) was used to evaluate the cerebral blood flow (CBF) dynamics in Poldip2^+/+ and Poldip2^+/− mice under normocapnic and hypercapnic conditions. Anesthetized animals were endotracheally intubated and attached to a mechanical ventilator (PhysioSuite, Kent Scientific) delivering gas with 2% of isoflurane. End-tidal CO₂ pressure (PhysioSuite, Kent Scientific) was monitored, and the intact skull was scanned in the prone position using LDPI (MoorFLPI-2; Moor Instruments Inc.). Hypercapnia was induced by 5% CO₂ inhalation (in 21% O₂/74% N₂) for 5 min alternating with normocapnia (21% O₂/79% N₂) for 5 min. Hypercapnia and normocapnia exposures were repeated three times during each experiment, and the highest CBF increase during hypercapnia was considered to be the maximum response for each animal. The baseline CBF was recorded with the MoorFLPI software in arbitrary perfusion units (flux), and the change of CBF (∆CBF%) was normalized to the baseline CBF. The cerebrovascular reactivity (CVR) was calculated as the increase of CBF (%) divided by the maximum increase in end-tidal CO₂ pressure (∆ mmHg) during hypercapnia [18 (link)].

The microcirculation of the skin of the right dorsal hand was assessed with a calibrated laser speckle contrast imaging device (moorFLPI2, Moor instruments, Millwey, United Kingdom). The dorsal part of the hand was used because the skin temperature of the dorsal and palmar part of the hand have been shown to strongly correlate. To obtain standardized and valid perfusion values, the micro-vascularization of the right dorsal hand was investigated up to the wrist. Due to good temporal and spatial resolutions with a high frame rate, laser speckle contrast imaging allows measurements of acute changes in superficial skin blood flow over wide skin areas with very good inter-day reproducibility compared to traditional assessment technologies such as laser Doppler perfusion imaging and laser Doppler flowmetry (Roustit et al., 2010 (link); Cracowski and Roustit, 2016 (link)). Cutaneous vascular conductance (CVC) was calculated at baseline, and after the CST in 5 min intervals (point measurement) from normalized microcirculation (flux) and MAP (mmHg) values. The normalized change in CVC (∆CVC [flux^.MAP⁻¹, flux^.mmHg⁻¹]) from baseline was then used for the analysis. A representative picture series of n = 1 female and n = 1 male participant of the microcirculation assessment in one condition (normobaric normoxia) can be seen in the Supplementary Figure S1.

Blood perfusion of the stenosis or normal hindlimb was monitored with a laser speckle imaging system (moor FLPI-2; Moor Instruments) after surgery (post), 14 and 28 days after surgery (Figure 2A). Mice were anesthetized continuously in a supine position with 1–1.5% isoflurane-mix gas. The region of interest was selected covering both the hindlimb paws. Perfusion was expressed as a ratio of the left (stenosis) to the right (normal) limb (13 (link)).

Skin perfusion was measured with a LSCI system (moorFLPI2, Moor instruments) working with a wavelength of 750 nm in supine position. Calibration of the system was successfully performed 1 day prior to the experiment. To minimize the risk of confounding factors, daylight and other sources of light were diminished as well as movements of the device during the measurements. The participants were instructed to breathe normally and not to talk during the measurements. To obtain reliable data, the distance between the measured skin area and the LSCI device was controlled with the aiming laser function. These two lasers converge into a single point at a distance of 25 cm between the surface and the LSCI device. To receive high‐resolution images (752 × 580 pixels), the temporal filter was set to 25 frames (1 s/frame) with an interval of 5 seconds.

Laser speckle imaging was used to observe cerebral blood flow (CBF) in rats. The rat was placed on the brain stereotactic device. A cranial window measuring 4 × 6 mm² was polished on the right side of the skull while keeping the dura mater intact. Subsequently, the Moor FLPI‐2 (Moor Instruments Ltd., Axminster, UK) blood flow imager was employed to observe CBF before MCAO, after MCAO, and after 24 h reperfusion.