Rflsi 3

Left middle cerebral artery occlusion followed by reperfusion was conducted as we previously reported [29 (link)]. In brief, the mice were anesthetized with 2.5% isoflurane. A midline neck incision was used to expose the left external carotid artery (ECA), internal carotid artery (ICA), and common carotid artery (CCA). The ECA is ligated using two surgical 7–0 threads. One was toward the distal end of the ECA, while the other was near the proximal end, as close to the bifurcation of the ECA and ICA as feasible, followed by an incision between the ECA ligatures. Finally, a 2-cm long nylon filament with a diameter of 0.25 ± 0.03 mm was introduced into the ICA through the external carotid stump and progressed to occlude the origin of the middle cerebral artery (MCA). Cerebral blood flow (CBF) was monitored using a Laser Speckle Imaging System (RFLSI III, RWD Life Science, Shenzhen, China). The nylon filament was gently extracted from the ECA after an hour of ischemia for reperfusion. The incision at the neck was then closed. All of the animals were resuscitated on heat pads for two hours before being kept separately for additional investigation. The sham group mice underwent the same surgical operation technique as the control mice but without vaso-occlusive.

Cerebral blood flow (CBF) of each group was monitored 24 h after cerebral I/R, using a laser speckle Doppler flowmeter (RFLSI III, RWD Life Science Co, Shenzhen, China). The images and data analysis were processed with the RFLSI analysis software (RWD Life Science). The change in CBF was quantitated and expressed as a relative blood flow that was calculated as the ratio of the region of interest (ROI) areas between the ipsilateral and contralateral hemispheres [23 (link)].

The mice were anesthetized with isoflurane and fixed on the stereotaxic apparatus. Before tMCAO, before reperfusion, immediately after reperfusion and 14 days after stroke, the cerebral blood flow of the mouse was measured with a laser speckle imaging machine (RFLSI III, RWD, Shanghai, China). The exposure time is 2 ms, the shooting time is 3 s, frame rate is 10 frames/second, the magnification is 3 times, and the laser intensity is 110 mW. The average blood flow was measured on the left of frontal suture and calculated the change rate base on the blood flow before tMCAO.

Cortical cerebral blood flow (CBF) was monitored by a laser speckle flow imaging technique 3 days after reperfusion. All procedures were performed under double-blind conditions. Briefly, rats were anesthetized and disinfected using iodophor, and the skull was exposed. The fascia attached to the skull was removed as much as possible and 0.9% saline was added to maintain the liquid level. Images and quantification of cerebral blood flow in the penumbra can be accessed by the laser speckle flow imaging technique (RFLSI III, RWD, China).

Adult male Sprague–Dawley rats (SD, 250–280 g, 8 weeks old) were group-housed with a 12-h light‑dark cycle and provided chow and water ad libitum. They were randomly divided into sham-operated + saline (sham), middle cerebral artery occlusion (MCAO) + saline (MCAO), and MCAO + scutellarin (MCAO + S) groups. In the MCAO group, anaesthesia was induced by an intraperitoneal injection of sodium pentobarbital (50 mg/kg). A dental drill was used to create a circular aperture in the right parietal bone to expose the underlying main trunk of the MCA. The right MCA was occluded by electrocoagulation. The rats in the MCAO + S groups were administered an intraperitoneal injection of scutellarin (100 mg/kg, as described in our previous study^{15 (link)}) dissolved in saline 2 h before and 12, 24, 48, and 60 h after MCAO; the other two groups were injected with saline. The rats were sacrificed 1, 3, and 7 d after MCAO. Laser speckle imaging system (RFLSI III; RWD) to measure cerebral blood perfusion.

The cerebral I/R model was induced by extracranial intraluminal middle cerebral artery occlusion (MCAO), as previously described (37 ). Briefly, 8 weeks old mice were anesthetized with 5% isoflurane and maintained with 1% isoflurane (RWD Life Science, R511-22) in an oxygen/air mixture by using a gas anesthesia mask (RWD Life Science, R580SRWD). Under the operating stereo microscope, the left common carotid artery, the external carotid artery (ECA), and the internal carotid artery were sequentially exposed. The ECA was ligated with a 5–0 silk suture, and a 2 cm length of silicon-rubber-coated monofilament (RWD Life Science, MSMC24B104PK50) was inserted from the ECA through the internal carotid artery up to the bifurcation of the left middle cerebral artery and anterior cerebral artery to block the circulation in the left middle cerebral artery territory. After 90 min of occlusion, blood flow was restored by the withdrawal of the inserted filament (Reperfusion). The sham-operated mice underwent the same experimental procedures except for the filament insertion. Cerebral blood flow was monitored using a Laser Speckle Imaging System (RWD Life Science, RFLSI III) during MCAO and reperfusion. Mice were sacrificed 24 h post-reperfusion.