Measurements were taken in a temperature‐regulated environment with subjects lying in a semisupine position. Subjects were asked to abstain from caffeine consumption and use of over‐the‐counter medications the morning prior to testing. Cutaneous perfusion was measured by laser Doppler flowmetry (Moor Instruments Ltd., Axminster, UK). Two combined temperature and laser Doppler fluximetry probes surrounded by an iontophoresis Perspex chamber were secured to the volar aspect of the forearm. Iontophoresis chambers were adhered 4 cm apart, avoiding areas with broken skin and superficial veins. Continuous recordings of cutaneous perfusion and skin temperature were collected using a laser Doppler flow monitor (moorVMS‐LDF, Moor Instruments Ltd.) with data recorded in arbitrary perfusion units of flux (PU).
Laser doppler flowmetry
Manufactured by Moor Instruments
Sourced in United Kingdom, United States
Laser Doppler flowmetry is a non-invasive technique used to measure blood flow. It utilizes the Doppler effect to detect the movement of red blood cells within the microcirculation. The technique provides real-time, continuous measurements of tissue perfusion.
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Lab products found in correlation
Ketamine xylazine, by Merck Group (6 mentions)
Heating pad, by RWD Life Science (4 mentions)
Silicone coated 6 0 suture, by Medtronic (4 mentions)
Operating microscope, by Leica camera (2 mentions)
6 0 suture, by Medtronic (2 mentions)
Xylazine, by Merck Group (2 mentions)
Stereotaxic frame, by RWD Life Science (2 mentions)
Moorvms ldf, by Moor Instruments (1 mentions)
Isoflurane, by RWD Life Science (1 mentions)
Silicon, by Kulzer (1 mentions)
Laser speckle imaging system, by RWD Life Science (1 mentions)
C57bl 6 mice, by Jackson ImmunoResearch (1 mentions)
6 0 monofilament, by Doccol (1 mentions)
Isofluorane, by Abbott (1 mentions)
3 methyladenine 3 ma, by Merck Group (1 mentions)
Triphenyl tetrazolium chloride (ttc), by Merck Group (1 mentions)
Axio imager d2, by Zeiss (1 mentions)
Cm1950, by Leica camera (1 mentions)
Nicolet 6700 ft ir spectrometer, by Thermo Fisher Scientific (1 mentions)
X pert powder diffractometer, by Malvern Panalytical (1 mentions)
51 protocols using laser doppler flowmetry
1
Cutaneous Perfusion Measurement by Laser Doppler
2
Transient Middle Cerebral Artery Occlusion in Rats
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Adult male Sprague Dawley rats (n = 90) weighing 250‐300 g were used. This study was carried out in accordance with the recommendations of the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines and with government approval by the State Agency for Nature, Environment and Consumer Protection North Rhine‐Westphalia. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University, China.
The surgical procedure for transient middle cerebral artery occlusion (MCAO) was previously described.24 Rats were anesthetized by ketamine/xylazine (100/10 mg/kg, Sigma‐Aldrich, St Louis, MO) and positioned supinely on a heating pad (RWD Life Science, Shenzhen, China) to maintain the temperature at 37 ± 0.5°C. With an operating microscope (Leica, Wetzlar, Germany), the left common carotid artery, internal carotid artery, and external carotid artery were carefully isolated. A nylon monofilament suture of 4‐0 (Covidien, Mansfield, MA) coated with silica was gently inserted from the external carotid artery stump to the internal carotid artery and occluded the origin of the middle cerebral artery for 90 minutes. Successful occlusion was characterized as the reduction in cerebral blood flow down to 20% of baseline, which was verified by laser Doppler flowmetry (Moor Instruments, Axminster, Devon, UK).
The surgical procedure for transient middle cerebral artery occlusion (MCAO) was previously described.
3
Transient Middle Cerebral Artery Occlusion in Mice
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tMCAO was carried out owing to previously published research [53 (link)]. Briefly, adult ICR mice (weighing 27 ± 3 g) were anesthetized with 1.5–2% isoflurane (RWD, Shenzhen, China) in a mixture of 30% oxygen and 70% nitrous oxide. The left common carotid artery, along with the internal carotid artery and the external carotid artery, were temporarily ligated. A cut was made between the two ligations on the external carotid artery. Following this, a 6-0 nylon suture (Covidien, Mansfield, MA, USA) coated with silicon (Heraeus Kulzer, Germany) was introduced through the incision and guided toward the ipsilateral middle cerebral artery. The success of middle cerebral artery occlusion was confirmed using laser Doppler flowmetry (Moor Instruments, Devon, UK) to observe a 10% reduction in the surface cerebral blood flow compared to its baseline level. The suture was withdrawn 1.5 h after the occlusion, and successful reperfusion was confirmed by observing the surface cerebral blood flow recovery back to 70% of its baseline level.
4
Transient Middle Cerebral Artery Occlusion Model in Mice
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In the present study, a tMCAO model was established using the modified Koizumi's method; detailed protocols and materials have been previously reported [19 (link)]. Briefly, inhalation anesthesia was induced with 1.5% isoflurane in N2O/O2 (70%/30%), and mice were then placed on a heating pad (Harvard Instruments, Boston, MA, USA). During surgery, the body temperature of mice was monitored using a rectal temperature probe and maintained at 37 ± 1°C. The left side of the skull was exposed, and an optic fiber was attached to the skull to monitor relative cerebral blood flow (rCBF). A midline incision was made on the neck, and soft tissues over the trachea were gently retracted. The external carotid artery (ECA) and common carotid artery (CCA) were knotted with 4–0 suture silk sutures. The ECA and CCA were ligated before clamping the internal carotid artery. Then, an 8–0 monofilament suture line (Ethicon, CA, USA), coated with silicon, was inserted into the MCA through the perforated CCA. For induction of tMCAO, the monofilament was retracted after the predetermined occlusion time was completed (2 h tMCAO). The incision region was sutured and disinfected to complete the surgical procedure. All surgeries were performed using laser Doppler flowmetry (Moor Instruments Ltd., Devon, UK) to detect rCBF and confirm successful occlusion and reperfusion.
5
Detailed MCAO Stroke Model in Mice
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The detailed mice MCAO model was described previously [35 (link), 36 (link)]. Adult male ICR mice weighting 26-32 grams (SLAC laboratory animal, Shanghai, China) were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) intraperitoneally. The body temperature was maintained at 37.0±0.3°C during surgery by a heating pad (RWD life science, Shenzhen, China). A midline incision was made on the neck under an operating microscope (Leica, Wetzlar, Germany). The left common carotid artery (CCA), the external carotid artery (ECA) and the internal carotid artery (ICA) were isolated. A silicone-coated round top 6-0 suture (Dermalon, 1756-31, Covidien, OH) was gently inserted from the ECA stump to the ICA to induce MCA occlusion. The suture inserted distance from the bifurcation to the opening of MCA was 10.5±0.5 mm. The success of occlusion was characterized as the reduction of cerebral blood down to 20%, which was verified by a laser Doppler flowmetry (Moor Instruments, Devon, UK). After 60 minutes of occlusion, the suture was withdrawal, the CCA was restored and blood flow returned to at least 70% of the baseline blood flow. Sham mice underwent the same procedure without inserting the suture.
6
Transient Middle Cerebral Artery Occlusion in Mice
7
Transient Middle Cerebral Artery Occlusion
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Animals were anesthetized with 1.5% isoflurane in a 30% O2/68.5%NO mixture under spontaneous breathing conditions. A 6‐0 suture (Covidien) coated with silicon was gently inserted from the external carotid artery (ECA) with an advancement of 9‐10 mm until reaching the intersection of the middle cerebral artery (MCA). After 90‐minute ischemia, the suture was withdrawn. A laser Doppler flowmetry (Moor Instruments) was used to monitor the blood flow in the MCA territory before surgery, immediately after occlusion, and reperfusion. Successful occlusion of MCA was confirmed as a decline in the regional blood flow of ipsilateral hemisphere by more than 80% compared to the contralateral hemisphere.
8
Quantifying Lower Limb Blood Flow in Mice
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Note: Imaging of lower limb blood flow is obtained by Laser Doppler Flow Metry (Moor Instruments, Devon, UK) as previously described [9, (link)10] .
1. Anesthetize the mouse by intraperitoneal injection of 50 mg/kg pentobarbital and assess the depth of anesthesia by pinching the mouse's feet to ensure the disappearance of foot retraction reaction. 2. Place the mouse in a supine position on the table. Apply the depilatory paste evenly to the skin of the abdomen and lower extremities and wipe up the removed hair with sterile cotton balls. 3. Use a computer-controlled optical scanner to direct a low-power laser beam over the exposed abdomen and lower extremities. 4. Place the scanning head parallel to the exposed site at a distance of about 20 cm. 5. Evaluate and record blood flow perfusion level by the moor FLPIR view V40 software, which is displayed on the video monitor in the form of a color-coded image.
1. Anesthetize the mouse by intraperitoneal injection of 50 mg/kg pentobarbital and assess the depth of anesthesia by pinching the mouse's feet to ensure the disappearance of foot retraction reaction. 2. Place the mouse in a supine position on the table. Apply the depilatory paste evenly to the skin of the abdomen and lower extremities and wipe up the removed hair with sterile cotton balls. 3. Use a computer-controlled optical scanner to direct a low-power laser beam over the exposed abdomen and lower extremities. 4. Place the scanning head parallel to the exposed site at a distance of about 20 cm. 5. Evaluate and record blood flow perfusion level by the moor FLPIR view V40 software, which is displayed on the video monitor in the form of a color-coded image.
9
Transient Middle Cerebral Artery Occlusion Model
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After 4 weeks of STZ injection, the 90-min transient MCAO (tMCAO) model was established. Transient MCAO was performed as previous described [49 (link)]. Briefly, mice were placed in the supine position after anesthesia with ketamine (100 mg/kg, Fujian Gutian Pharmaceutical Co., Ltd, Gutian, China)/xylazine (10 mg/kg, Sigma-Aldrich). After the midline incision was made on the neck, a silicone-coated 6-0 suture (Covidien, Mansfield, MA, USA) was inserted from a small incision of the left external carotid artery and gently advanced into internal carotid artery to occlude the origin of middle cerebral artery. The total distance from the external carotid artery to middle cerebral artery was approximate 9 ± 0.5 mm. Reperfusion was achieved by withdrawing the suture after 90 min of tMCAO. Both occlusion and reperfusion were confirmed by laser Doppler flowmetry (Moor Instruments, Axminster, UK). The filament was removed immediately after insertion in the middle cerebral artery in the sham mice.
10
Transient Middle Cerebral Artery Occlusion in Mice
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Adult male ICR mice (25–30 g) were anesthetized with 1.5–2% isoflurane and 30%/70% oxygen/nitrous oxide. tMCAO was performed as previously described [32 (link), 33 (link)]. Briefly, the left common carotid artery, internal carotid artery and external carotid artery were separated and ligated temporarily. An incision was made between the two ligations on external carotid artery. Then a 6-0 nylon suture coated with silica gel was inserted through the incision into the ipsilateral middle cerebral artery. A laser Doppler flowmetry (Moor Instruments, Devon, UK) was used to determine the successful occlusion of middle cerebral artery by monitoring the decrease of surface cerebral blood flow (CBF) to 10% of its baseline. Reperfusion was performed by withdrawing the suture 1.5 h after the occlusion and the reperfusion was confirmed by the recovery of surface CBF to 70% of baseline. In the sham group, an identical surgical procedure was performed without ligation of any arteries. A laser speckle imaging system (RWD Life Science, Shenzhen, China) was used to record and display the changes of cerebral blood flow in the ischemic zone of tMCAO mice.
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