At day 0, 1, 3, 5, 7, 10 and 14 after operation, limb morphology was visually scored to evaluate the degree of ischemia-induced damage. Ten grades were used to measure the degree of limb ischemic injury (Table S1 , Supporting Information). Both limbs blood flow were evaluated by Laser Doppler perfusion imager (Moor Instruments, United Kingdom). Mean values of perfusion were calculated from the stored digital color-coded images. The blood flow level of the ischemic (left) limb was normalized to the non-ischemic (right) limb to avoid data variations caused by ambient light and temperature. Meanwhile, limbs temperature was also recorded by infrared thermal imaging system (FLIR E6 System, USA) and the temperature ratio of ischemic to normal hindlimbs was calculated. Besides, microcirculation imaging of mouse limb was demonstrated with Micro-CT (Quantum GX, PerkinElmer, USA). In addition, the mice were weighed during the experiment, and the optics images of hindlimbs were acquired by a digital camera on day 3, 7 and 14 after operation.
Laser doppler perfusion imager
Manufactured by Moor Instruments
Sourced in United Kingdom
The Laser Doppler perfusion imager is a lab equipment device that utilizes laser technology to measure and visualize blood flow within tissue. It operates by directing a low-power laser beam onto the skin or tissue and analyzing the Doppler shift in the reflected light to provide real-time data on microvascular perfusion.
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Lab products found in correlation
Quantum gx, by PerkinElmer (1 mentions)
7 0 prolene, by Johnson & Johnson (1 mentions)
C57bl 6j mice, by Jackson ImmunoResearch (1 mentions)
Cm dii, by Thermo Fisher Scientific (1 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions)
Cfx connect real time system, by Bio-Rad (1 mentions)
8 protocols using laser doppler perfusion imager
1
Ischemia-Induced Limb Damage Assessment
2
Murine Hindlimb Ischemia Model
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Unilateral high femoral artery ligation and superficial femoral artery (SFA) excision was performed on 6- to 8-week-old male C57BL/6J mice (Jackson Laboratory, Bar Harbor, Me). Hindlimb ischemia surgery was performed as described previously (Niiyama et al., 2009 ). Briefly, mice were anesthetized with 2% isoflurane in 100% oxygen at a flow rate of 1 L/min. Ligations were placed at the femoral artery and its large branches in C57BL6 mice using 7-0 Prolene (Ethicon, Somerville, NJ) and the femoral artery was excised from its proximal origin as a branch of the external iliac artery to the distal point where it bifurcates into the saphenous and popliteal arteries. Immediately after surgery, the mice were randomly assigned into four groups: the MSC + HLI group, the PBS + HLI group, the MSC + Sham group, the PBS + Sham group. In MSC group, a total of 1.0 × 106 cells in 100 μL PBS was injected intramuscularly into 4 sites of the gastrocnemius muscle. Assessment of limb function and ischemic damage were performed as described previously. Tissue perfusion of the hind limbs was assessed with a laser Doppler perfusion imager (Moor Instruments, Devon, United Kingdom) on days 0, 1, 3, 7, and 14 days after treatment. The digital color-coded images were analyzed to quantify blood flow in the region from the knee joint to the toe.
3
Histological and Functional Evaluation of Limb Ischemia
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Ischemic limb muscles retrieved 21 days post-treatment were fixed with a formaldehyde solution, dehydrated with a graded ethanol series, and embedded in paraffin. Next, 4-µm sections obtained from the specimens were stained with hematoxylin and eosin (H&E) to examine muscle degeneration and tissue inflammation. Laser Doppler imaging analysis (LDPI) was performed as described previously [29 (link)]. A laser Doppler perfusion imager (Moor Instruments, Devon, UK) was used for serial noninvasive physiological evaluation of neovascularization. Mice were monitored by serial scanning of surface blood flow in hindlimbs on days 0, 7, 14, and 21 after treatment. Digital color-coded images were scanned and analyzed to quantify a blood flow in ischemic regions from the knee joint to the toe. Mean values of perfusion were subsequently calculated.
4
Laser Doppler Limb Blood Perfusion
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For the blood perfusion assessment, the ischemic limb-to-nonischemic limb blood perfusion ratio was measured weekly up to 3 weeks using a Laser Doppler perfusion imager (Moor Instruments), which provides noninvasive measurement of the blood flow by determining the Doppler frequency shift for light reflected off the moving red blood cells. Briefly, the mice under anesthesia were secured on a monochromatic surface, and an area was scanned from the lower abdomen to the end of the toes. Color images were obtained, and the hindlimb perfusion ratios were determined by comparing the perfusion of the injured hind limbs and the contralateral normal hind limbs. The outcomes were blindly assessed.
5
Hind Limb Ischemia Model in Mice
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The hind limb ischemia model was made in 6-week-old male athymic nude mice. Briefly, the left femoral artery was ligated and excised below the inguinal ligament and above the bifurcation of the popliteal artery. Twenty-four hours later, mice were intramuscularly injected with 1×106 1,1′-dioctadecyl-3,3,3′,3′-tetra-methylindocarbocyanine dye (CM-DiI; Invitrogen)-labeled cells in 100 µl PBS into the ischemic limbs. Cells from high density and regular density culture at day 15 were injected respectively. Mice that received the same volume of PBS served as a control. Ten animals were injected in each group. Blood perfusion of the hind limbs was detected by a laser Doppler perfusion imager (Moor Instruments, Devon, UK) on days 1 and 21 post-treatment. The recovery of perfusion was calculated as the ratio of ischemic to non-ischemic hind limb blood perfusion.
6
Noninvasive Angiogenesis Quantification
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A laser Doppler perfusion imager (Moor Instruments, Devon, UK) was used for serial noninvasive physiological evaluation of neovascularization. Mice were monitored by serial scanning of surface blood flow in hindlimbs on days 0, 7, 14, 21, and 28 after treatment. Digital color-coded images were scanned and analyzed to quantify blood flow in ischemic regions from the knee joint to the toe. Mean values of perfusion were subsequently calculated.
For qRT-PCR, total RNA was extracted from the retrieved ischemic limb tissue (n = 4 per group). RNA was reverse-transcribed into cDNA. The expression of mouse platelet endothelial cell adhesion molecule (Pecam-1) and vascular cell adhesion molecule (Vcam) mRNA was evaluated by qRT-PCR. qRT-PCR was performed using a CFX connectTM real-time system (Bio-Rad Laboratories, Hercules, CA, USA) with SsoAdvancedTM Universal SYBR® Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA). Each cycle consisted of the following times and temperatures: 95 °C for 10 s and 60 °C for 30 s. The primer sequences are shown inTable 1 .
For qRT-PCR, total RNA was extracted from the retrieved ischemic limb tissue (n = 4 per group). RNA was reverse-transcribed into cDNA. The expression of mouse platelet endothelial cell adhesion molecule (Pecam-1) and vascular cell adhesion molecule (Vcam) mRNA was evaluated by qRT-PCR. qRT-PCR was performed using a CFX connectTM real-time system (Bio-Rad Laboratories, Hercules, CA, USA) with SsoAdvancedTM Universal SYBR® Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA). Each cycle consisted of the following times and temperatures: 95 °C for 10 s and 60 °C for 30 s. The primer sequences are shown in
7
Laser Doppler Blood Flow Monitoring
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Blood flow was monitored using a laser Doppler perfusion imager (Moor Instruments Ltd, Devon, UK). Blood flow was expressed as a ratio of the ischemic and non-ischemic hind limbs.
8
Hind-Limb Blood Flow Assessment
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Non-invasive measurements of hind-limb blood flow were performed by a Laser Doppler perfusion imager (Moor Instruments, Devon, UK) at 7, 14, and 21 days after femoral artery excision. Digital colorcoded images were used to quantify hind-limb tissue perfusion. The difference in blood flow between the ischemic and non-ischemic limbs was expressed as the ratio of blood flow in the ischemic limb to blood flow in the non-ischemic limb. Moreover, an ambulation score (0 = flex toes to grasp cage; 1 = plantar flexion; 2 = no foot dragging but no plantar flexion; 3 = foot dragging) and necrosis score (1 to 5 = one to five fingernails damaged, 6 to 10 = one to five fingers fully damaged, 11 = paw fully damaged) were used to grade the degrees of hind-limb impairment and ischemic damage, as described previously [28, (link)29] .
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