Effects on duration of post-operative ischemia and patterns of blood flow recovery in different conditions of mouse hind limb ischemia
© Al-Mubarak et al; licensee BioMed Central Ltd. 2011
Received: 7 February 2011
Accepted: 14 June 2011
Published: 14 June 2011
Current limitations to the experimentation on patients with peripheral arterial disease push the development of different preclinical strategies. We investigated both duration of ischemia and blood flow recovery in mouse models of partial femoral artery ligation.
Male BALB/c mice were used. The ligation over needle method involved placing a suture needle over the femoral artery, ligating over it and then removing the needle. The transfixation method involved transfixing the approximate center of the femoral artery and then tying the suture. Laser Doppler Perfusion Imaging was used to assess perfusion every 3rd day until 42 days after the procedure.
Ligation over needle method: Immediately post procedure, mean perfusion was -71.87% ± 4.43. Then mean difference in perfusion remained below the base line reading on days 3, 6, 9, and 12. From day 15 on wards mean perfusion progressively improved remaining near base line. Transfixation Method: Immediately post procedure mean perfusion was -70.82% ± 4.73. Mean perfusion improved following the procedure on days 3 and 6; a plateau followed this on days 9, 12 and 15. From day 15 onwards perfusion progressively improved remaining well below base line until crossing it on day 36.
The currently described models do not pose major improvements over previously described methods.
Peripheral arterial disease (PAD) is a clinical manifestation of atherosclerosis affecting the aorto-iliac and infra-inguinal arterial trees. Intermittent claudication is the classical symptom of PAD [1, 2]; patients may be asymptomatic or present with non-healing ulcers and tissue loss. More relevantly, PAD can also be considered as a marker of systemic atherosclerosis, as patients with PAD are at greater risk of having myocardial infarction  and ischemic stroke .
Translational approaches using mouse models of hind limb ischemia have advantages over other animal models. Mice require less food , housing space , and time to acquire Laser Doppler Perfusion Imaging scans as compared to other rodents.
In general, previously described mouse models of ischemia revolve around the same scheme [7–11]. After exposure, the femoral artery of the hind limb is dissected free from the surrounding tissue. Then ligations are made at various points with or without removing part of the artery with or without the vein. The end result is complete obstruction of the blood supply to the limb. The abrupt and absolute nature of the ischemia induced by these methods is similar to what occurs in acute arterial occlusion. The objective of this study was to develop a murine model of surgically induced ischemia via partial femoral artery ligation that remains ischemic for a minimum period of 27 days.
Methods and Results
All procedures and protocols were approved by the Institutional Review Committee of The College of Medicine, King Saud University, Riyadh, Saudi Arabia. Animal experiments were performed at the animal housing facilities of The College of Medicine, King Saud University, Riyadh, Saudi Arabia. Male BALB/c mice aged between 11-12 weeks and weighing 23-34 g were used. The animals were anesthetized with Ketamine-Xylazine intraperitonially (IP) for the surgical procedures and for the laser Doppler measurements of limb perfusion.
On day 42 of the study, the mice were euthanized with an overdose of anesthetic and cervical dislocation after performing LDPI scans. Histological experiments were done at The Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University, Riyadh, Saudi Arabia. Samples of skeletal muscle were taken from the calf and thigh of both limbs and placed in 10% formaldehyde till processing. After fixation in paraffin wax, histological sections 5 μm thick were stained with Hematoxylin and Eosin stains and were examined under a light microscope. Skin samples from the medial aspect of both limbs were placed in 10% formaldehyde until fixation in paraffin blocks.
The surgical procedures were performed on the right hind limb. Exposure of the femoral artery was obtained by a skin incision parallel to the inguinal ligament approximately the width of the thigh. After dissecting the proximal portion femoral artery just distal to the inguinal ligament, one of two procedures was performed to induce unilateral hind limb ischemia.
Results are presented as mean ± SE.
Ligation over needle group
The major findings of our paper are: 1) recovery pattern of ischemia is independent of initial postoperative decrease in perfusion and 2) calf muscle is the sample of choice for obtaining representative results. The limitations of our study include: 1) relatively low success rate, 50% in both methods and 2) limited number of mice that completed the study duration, n = 4.
Previously described models of ischemia completely impede blood flow [7–11], causing an acute ischemic episode. Variations seen between the different methods employed include: use of suture material or electro-cautery , inclusion of the accompanying vein [13–15] and extent of excision which ranges from the external iliac artery to the popliteal vessels [16–23]. The end result of using such procedures results in an immediate post procedure drop in perfusion ranging from -90% to -75% and a perfusion ranging from - 5% to - 60% twenty eight days after the procedure [13–20]. The post procedure decrease in perfusion of the ligation over needle and transfixation methods is comparable (-71.87% and -70.82%, respectively) to previously described methods. However, when the perfusion on the 27th day after the procedure is well above what can be achieved in previously described methods (-12.25% and -26.24%, ligation over needle and transfixation methods respectively).
In conclusion, both methods described were successful in inducing ischemia. The major differences between them is the duration of the ischemia postoperatively and the pattern of recovery. The optimum area to obtain skeletal muscle samples is from the calf. The currently described models do not pose major improvements over previously described methods.
We thank the international scientist exchange program promoted by postgraduate and continuing medical education of King Saud University, Riyadh, Kingdom of Saud Arabia.
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