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.
KeywordsHindlimb Ischemia Angiogenesis LDPI Mouse Model Peripheral Arterial Disease
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.
- Rose GA: The diagnosis of ischemic heart pain and intermittent claudication in field surveys. BullWorld Health Organ. 1962, 27: 645-658.Google Scholar
- The global burden of disease: 2004 update. World Health Organization. 2008Google Scholar
- Criqui MH, Langer RD, Fronek A, Feigelson HS, Klauber MR, McCann TJ, Browner D: Mortality over a period of 10 years in patients with peripheral arterial disease. N Engl J Med. 1992, 326: 381-386. 10.1056/NEJM199202063260605.View ArticlePubMedGoogle Scholar
- Wilterdink JL, Easton JD: Vascular event rates in patients with atherosclerotic cerebrovascular disease. Arch Neurol. 1992, 49: 857-63.View ArticlePubMedGoogle Scholar
- Subcommittee on Laboratory Animal Nutrition, Committee on Animal Nutrition, Board on Agriculture, National Research Council: Nutrient Requirements of Laboratory Animals Fourth Revised Edition. 1995, National Academies Press. Washington, D.CGoogle Scholar
- Institute of Laboratory Animal Resoreces, Commision on Life Sciences, National Research Council: Guide for the Care and Use of Laboratory Animals. 1996, National Academies Press. Washington, D.CGoogle Scholar
- Moon MH, Kim SY, Kim YJ, Kim SJ, Bae YC, Sung SM, Jung JS: Human adipose tissue-derived mesnchymal stem cells improve postnatal neovascularization in mouse model of hindlimb ischemia. Cell Physiol Biochem. 2006, 17: 279-290. 10.1159/000094140.View ArticlePubMedGoogle Scholar
- Goto T, Fukuyama N, Aki A, Kanabuchi K, Kimura K, Taira H, Tanaka E, Wakana N, Mori H, Inoue H: Search for appropriate experimental methods to create stable hind-limb ischemia in mouse. Tokai J Exp Clin Med. 2006, 31: 128-132.PubMedGoogle Scholar
- Couffinhal T, Silver M, Zheng LP, Keamey M, Witzenbichler B, Isner JM: Mouse model of angiogenesis. Am J Pathol. 1998, 152: 1667-1679.PubMed CentralPubMedGoogle Scholar
- Miranville A, Heeschen C, Sengenès C, Curat CA, Busse R, Bouloumié A: Improvement of postnatal neovascularization by human adipose tissue-derived stem cells. Circulation. 2004, 110: 349-355. 10.1161/01.CIR.0000135466.16823.D0.View ArticlePubMedGoogle Scholar
- Napoli C, Williams-Ignarro S, de Nigris F, de Rosa G, Lerman LO, Farzati B, Matarazzo A, Sica G, Botti C, Fiore A, Byrns RE, Sumi D, Sica V, Ignarro LJ: Beneficial effects of concurrent autologous bone marrow cell therapy and metabolic intervention in ischemia-induced angiogenesis in the mouse hindlimb. Proc Natl Acad Sci USA. 2005, 102: 17202-17206. 10.1073/pnas.0508534102.PubMed CentralView ArticlePubMedGoogle Scholar
- Gianella A, Guerrini U, Tilenni M, Sironi L, Milano G, Nobili E, Vaga S, Capogrossi MC, Tremoli E, Pesce M: Magnetic resonance imaging of human endothelial progenitors reveals opposite effects on vascular and muscle regeneration into ischaemic tissues. Cardiovasc Res. 2010, 85: 503-513. 10.1093/cvr/cvp325.View ArticlePubMedGoogle Scholar
- Ohashi K, Ouchi N, Sato K, Higuchi A, Ishikawa TO, Herschman HR, Kihara S, Walsh K: Adiponectin promotes revascularization of ischemic muscle through a cyclooxygenase 2-dependent mechanism. Mol Cell Biol. 2009, 29: 3487-3499. 10.1128/MCB.00126-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Bir SC, Esaki J, Marui A, Sakaguchi H, Kevil CG, Ikeda T, Komeda M, Tabata Y, Sakata R: Therapeutic Treatment with Sustained-Release Platelet-Rich Plasma Restores Blood Perfusion by Augmenting Ischemia-Induced Angiogenesis and Arteriogenesis in Diabetic Mice. J Vasc Res. 2010, 48: 195-205.View ArticlePubMedGoogle Scholar
- Ohashi K, Ouchi N, Higuchi A, Shaw RJ, Walsh K: LKB1 deficiency in Tie2-Cre-expressing cells impairs ischemia-induced angiogenesis. J Biol Chem. 2010, 285: 22291-22298. 10.1074/jbc.M110.123794.PubMed CentralView ArticlePubMedGoogle Scholar
- Park B, Hoffman A, Yang Y, Yan J, Tie G, Bagshahi H, Nowicki PT, Messina LM: Endothelial nitric oxide synthase affects both early and late collateral arterial adaptation and blood flow recovery after induction of hind limb ischemia in mice. J Vasc Surg. 2010, 51: 165-173. 10.1016/j.jvs.2009.08.045.PubMed CentralView ArticlePubMedGoogle Scholar
- Bhang SH, Cho SW, Lim JM, Kang JM, Lee TJ, Yang HS, Song YS, Park MH, Kim HS, Yoo KJ, Jang Y, Langer R, Anderson DG, Kim BS: Locally delivered growth factor enhances the angiogenic efficacy of adipose-derived stromal cells transplanted to ischemic limbs. Stem Cells. 2009, 27: 1976-1986. 10.1002/stem.115.View ArticlePubMedGoogle Scholar
- Tokudome T, Kishimoto I, Yamahara K, Osaki T, Minamino N, Horio T, Sawai K, Kawano Y, Miyazato M, Sata M, Kohno M, Nakao K, Kangawa K: Impaired recovery of blood flow after hind-limb ischemia in mice lacking guanylyl cyclase-A, a receptor for atrial and brain natriuretic peptides. Arterioscler Thromb Vasc Biol. 2009, 29: 1516-1521. 10.1161/ATVBAHA.109.187526.View ArticlePubMedGoogle Scholar
- Yan J, Tie G, Park B, Yan Y, Nowicki PT, Messina LM: Recovery from hind limb ischemia is less effective in type 2 than in type 1 diabetic mice: roles of endothelial nitric oxide synthase and endothelial progenitor cells. J Vasc Surg. 2009, 50: 1412-1422. 10.1016/j.jvs.2009.08.007.PubMed CentralView ArticlePubMedGoogle Scholar
- Kato S, Amano H, Ito Y, Eshima K, Aoyama N, Tamaki H, Sakagami H, Satoh Y, Izumi T, Majima M: Effect of erythropoietin on angiogenesis with the increased adhesion of platelets to the microvessels in the hind-limb ischemia model in mice. J Pharmacol Sci. 2010, 112: 167-175. 10.1254/jphs.09262FP.View ArticlePubMedGoogle Scholar
- Lian Q, Zhang Y, Zhang J, Zhang HK, Wu X, Zhang Y, Lam FF, Kang S, Xia JC, Lai WH, Au KW, Chow YY, Siu CW, Lee CN, Tse HF: Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice. Circulation. 2010, 121: 1113-1123. 10.1161/CIRCULATIONAHA.109.898312.View ArticlePubMedGoogle Scholar
- Leifheit-Nestler M, Conrad G, Heida NM, Limbourg A, Limbourg FP, Seidler T, Schroeter MR, Hasenfuss G, Konstantinides S, Schäfer K: Overexpression of integrin beta 5 enhances the paracrine properties of circulating angiogenic cells via Src kinase-mediated activation of STAT3. Arterioscler Thromb Vasc Biol. 2010, 30: 1398-1406. 10.1161/ATVBAHA.110.206086.View ArticlePubMedGoogle Scholar
- Huang PH, Tsai HY, Wang CH, Chen YH, Chen JS, Lin FY, Lin CP, Wu TC, Sata M, Chen JW, Lin SJ: Moderate intake of red wine improves ischemia-induced neovascularization in diabetic mice--roles of endothelial progenitor cells and nitric oxide. Atherosclerosis. 2010, 212: 426-435. 10.1016/j.atherosclerosis.2010.06.034.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.