Ldpi analyzer

The laser Doppler perfusion imaging (LDPI) analyzer (Moor Instruments, Axminster, UK) was used to record blood flow measurements on days 0, 7, 14, 21, and 28 post operation. Blood perfusion of the calf region (from ankle to the knee) of both limbs was selected as the regions of interest to facilitate LDPI analysis. The recovery of perfusion was calculated as the ratio of ischemic hindlimb blood perfusion to nonischemic hindlimb blood perfusion. A ratio of 1 before surgery indicated equal blood perfusion of both legs. Tissue necrosis was scored as previously described [34 (link)]: 0, normal/no necrosis; 1, mild necrosis and/or deep cyanosis of toes; 2, necrosis/amputation of toes (two or more); 3, foot amputation; and 4, severe limb loss.

C57BL/6J (WT), 5-LO-deficient and BLTR1-deficient male mice (7 wk-old) were subjected to right femoral artery injury using a 0.25 mm diameter angioplasty guidewire under chloral hydrate (450 mg/kg, intraperitoneal injection) anesthesia and aseptic conditions, as previously described (26 (link)). The adequacy of anesthesia was confirmed by response to toe pinch. Wire-injured femoral arteries were harvested from mice euthanized by carbon dioxide insufflations and cervical dislocation, and then cross sectioned (4 μm). Tissue sections were stained with hematoxylin and eosin (H&E) and immunohistological marker antibodies. Femoral arterial blood flow was measured using a laser Doppler perfusion imaging (LDPI) analyzer (Moor Instruments, Devon, UK) at 0, 1, 2, 3, and 4 wks after femoral artery injury. The changes in blood flow were calculated using the colors of histogram pixels.

Six-week-old male KSN nude mice were anesthetized with pentobarbital, and the right common iliac artery was resected. After surgery, amnion MSCs (1×10⁶ cells/50 µL PBS), chorion MSCs (1×10⁶ cells/50 µL PBS), or PBS (50 µL PBS) was injected into the ischemic muscle with a 30-gauge needle at five different sites (n = 15 in each group). A laser Doppler perfusion image (LDPI) analyzer (Moor Instruments, Devon, UK) was used to measure serial hindlimb blood flow for 7 days, as previously described [12] (link).
Five and seven days after MSC transplantation, ischemic hindlimb tissues were obtained and snap-frozen. Frozen tissue sections were stained with anti-mouse CD31 antibody (BD Biosciences) to detect capillary endothelial cells. Ten fields were randomly selected to count the number of capillaries. The adjusted capillary number per muscle fiber was used to compare the differences in capillary density between the three groups.

Blood perfusion was assessed with a laser Doppler perfusion image (LDPI) analyzer (Moor Instruments, Axminster, UK) 7, 14, 21, and 28 days after the transplantation. A dark-blue color on the images represents low or no blood perfusion; a red color represents high blood flow. Both the ischemic and the nonischemic limbs were scanned twice, and the average blood flow values were calculated. The LDPI index was then calculated as the ratio of the perfusion of ischemic hindlimb to the perfusion of the nonischemic hindlimb [22 (link)]. The mobility damage of the ischemic limb was evaluated before the surgery and 4 weeks after the surgery according to the following scoring criteria: score 3 represents that the ischemic limb was dragged during crawling movement and showed ischemic gangrene; score 2 represents that the ischemic limb showed mild dragging and moderate gangrene but without reflex response; score 1 represents that the ischemic limb showed good reflex response and mild gangrene; and score 0 represents no obvious difference in the ischemic and the nonischemic limbs.