Laser doppler flowmeter

Procedures for the use of laboratory animals were approved by the Shanghai Jiao Tong University Institutional Animal Care and Use Committee, Shanghai, China. Adult male CD-1 mice (n = 12) weighing 25–30 grams were anesthetized with ketamine/xylazine (100 mg/10 mg/kg, Sigma, San Louis, MO). Body temperature was controlled and maintained at 37 ± 0.3°C using a heating pad (RWD Life Science, Shenzhen, China) during the anesthesia period. After isolation of left common carotid artery (CCA), external and internal carotid artery (ECA, ICA), a silicone-coated 6–0 suture (Covidien, Mansfield, MA) was gently inserted from the ECA stump to the ICA, and stopped at the opening of the MCA. The distance from the bifurcation of ICA/ECA to MCA was 10 ± 0.5 mm [24 (link)]. Successful occlusion was verified by a laser Doppler flowmeter (Moor Instruments, Devon, UK). Mice with surface cerebral blood flow that was more than 15% of baseline were excluded from the experiment. Sham-operated mice underwent the same surgery procedure except inserting the suture into the ICA.

Registration of microcirculation of the lower limb skeletal muscle was performed by laser Doppler flowmeter (MOOR Instruments Ltd, London, UK; DRT4). The probe of the device was placed on the surface of the left femoral biceps muscle. To characterize the microcirculation, reperfusion area (RA) was used, based on the mathematical calculations of our research group. The mathematical transformations required for correct interpretation of the circulation data were described previously [20] (link).

CBF assessment via the left and right middle cerebral artery was performed with a laser Doppler flowmeter (Moor Instruments, Axminster, Devon, United Kingdom) during the entire tMCAO procedure and treatment to ensure appropriate middle cerebral artery occlusion and reperfusion. The laser Doppler probes were placed on the animal’s skull approximately 1 mm posterior to the bregma and 5 mm lateral to the midline. Baseline measurement was taken 5 min after tail artery catheter insertion.

This study was conducted on adult male IL-1Ra transgenic (Tg) mice (hemizygous mice carrying the transgene encoding secreted IL-1Ra mRNA under the control of its endogenous promoter) and LM controls [11 (link),28 (link)]. Transient (t)MCAo was performed under 1–2% isoflurane anesthesia. Mice received 1 mL (s.c.) 0.9% saline before the transient intraluminal filament technique (45 min) [29 (link)]. Blood flow and body temperature were monitored using an optical fiber probe (T6a and VP10 M200st, Moor Instruments, UK) connected to a laser Doppler flowmeter (Moor Instruments, UK). Postsurgery, the mice received 0.1 mL (s.c.) 5% glucose and 0.1 mL Temgesic (buprenorphium 0.3 mg/mL; pharmaceuticals, USA). Temgesic was given at 8 h intervals for the first 24 h poststroke [11 (link)].
BM harvesting, characterization, and treatment have previously been detailed [8 (link),11 (link)]. Approximately 1 × 10⁷ BM cells were injected into the tail vein of recipient mice 30 min after tMCAo [11 (link)]. In this study, we include samples from LM mice subjected to tMCAo (LM) (n = 10), LM mice subjected to tMCAo, followed by LM BM treatment (LM–LM) (n = 12), or IL-1Ra BM treatment (Tg–LM mice) (n = 12), and unlesioned controls (Ctl) (n = 11) [11 (link)].

Mice were anesthetized (body temperature maintained at 37 ± 0.3ºC) using only 30% and 69% 1–2% isoflurane-oxygen/nitrous oxide mixtures. The left common carotid artery, external carotid artery, and internal carotid artery were isolated and guided with a silicone-coated 6–0 suture from the external carotid stump to the internal carotid artery until reaching the middle cerebral artery lumen. Vascular embolism was assessed with a laser Doppler flowmeter (Moor Instruments, UK). Sham-operated animals used the same procedure except that no embolization was performed [17 (link)].

Adult male ICR mice were randomly assigned to the tMCAO group and sham group. Transient MCAO model in mice was performed as previously described (Yang et al., 1994 (link); Liu et al., 2014 (link)). Briefly, adult male ICR mice weighing 25–30 g were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) intraperitoneally. Then the surgical procedure of tMCAO was performed as follows: 6-O suture (Dermalon, 1741-11, Covidien, Cincinnati, OH, USA) coated with silicone was inserted from the left external carotid artery into the internal carotid artery and reached to the origin of middle cerebral artery. With the help of laser Doppler flowmeter (Moor Instruments, Devon, England), successful occlusion was characterized as a reduction of cerebral blood flow (CBF) down to 10% of baseline. After 1.5 h, the suture was removed for reperfusion. Sham operation group (n = 5) underwent the same procedure except suture insertion. All of the animals were sacrificed 24 h after reperfusion.

Each group (n = 3) of C57BL/6 female mice was used in the hemorheology study. The mice were anesthetized with 10% chloral hydrate at 3.5 mL/kg and then were fixed using stereotaxic apparatus. A midline incision (approximately 1 cm) was made with scissors on the skull of mice subject, and a laser Doppler flowmeter (Moor Instruments Ltd., Wilmington, United Kingdom) was perpendicularly attached to the parietal bone surface at a position of 2 mm posterior and 2 mm lateral from the bregma to monitor CBF in the left middle cerebral artery region (Hedna et al., 2015 (link)). After the surgery, the baseline of mice brain CBF was confirmed for 30 min. NTG was then subcutaneously injected for the establishment of the migraine model. Afterward, hemorheology parameters such as CBF, blood cell concentrations, and blood cell speed were continuously recorded for 60 min. A heating pad was used to maintain the normal temperature of laboratory mice.