Animal protocols in "Erythropoietin promotes abdominal aortic aneurysms in mice through angiogenesis and inflammatory infiltration"

Animal Protocol

Abstract

Abdominal aortic aneurysm (AAA) is a potentially fatal vascular disease, but the underlying mechanisms remain unknown. Since Daugherty first used angiotensin II (AngII) infusion to induce AAA in Apoe^-/- mice, this model has been commonly used in experimental studies because it shares several common pathological features with human AAA (1). However, this mouse model has important limitations. Severe hyperlipidemia is necessary to achieve a high incidence of AAA in the mouse model but in the clinical scenario, the association of hyperlipidemia with AAA is weak (OR = 1.34). Previous studies showed that AngII induced AAA in only 10% to 20% normocholesterolemic mice, which constituted a major impediment to exploring molecular mechanisms of AAA (2). We found that EPO dose-dependently promoted the formation of AAA in both Apoe^-/- (66.7%) and wild-type (WT) (60%) mice receiving a high dose of EPO, which was similar to the incidence of AAA of AngII-infused Apoe^-/- mice (60%) (3). EPO injection did not affect blood pressure, lipid profile, and liver and renal function in Apoe^-/- and WT mice. These results demonstrated that EPO injection in Apoe^-/- and WT mice offers a promising animal model of AAA, which may be advantageous over traditional AngII-infused Apoe^-/- mouse model and provides a useful tool for exploring the mechanism of AAA in experimental studies. 

Materials and Reagents

1. Eight-week-old male WT mice (C57BL/6J) and Apoe^-/- mice (Jackson Laboratory, USA) were purchased from the Beijing Huafukang Animal Experimental Center
2. Recombinant human erythropoietin was purchased from Shenyang 3SBIOINC (Concentration: 10000IU/mL)
3. Normal diet was purchased from Jiangsu Xietong Co., Ltd
4. High-fat diet (0.25% cholesterol and 15% cocoa butter, TP28521) was purchased from Trophic Animal Feed High-Tech Co., Ltd, China

Equipment

1. Biosafety cabinet: Heraeus, USA, HF1200
2. Vernier caliper: Mitutoyo Co., LTD., Japan, 1204-70

Procedures 

1. WT and Apoe^-/- mice will be each randomly divided into 4 groups for treatment (n=15 per group): the vehicle group, and the low-, medium- and high-dose EPO groups. All mice should be strictly kept on a 12-hour light/ 12-hour dark cycle in specific pathogen free environment and fed with a normal diet in WT mice and a high-fat diet in Apoe^-/- mice with water freely available. All mice should be adapted for environment for 4 weeks before operation. Body weight will be measured in all mice every week.
2. EPO solution should be diluted with normal saline just before use. The EPO dose in the low-, medium- and high-dose EPO groups is defined as 2,500 IU/kg/day, 5,000 IU/kg/day and 10,000 IU/kg/day, respectively, which should be diluted in normal saline resulting in a total solution volume of 0.2ml for intraperitoneal injection. The EPO solution should be prepared under a sterile and dark condition in a biosafety cabinet.
3. For the vehicle group, 0.2 mL normal saline will be intraperitoneally injected into each mouse. The different dose of EPO diluted in normal saline in a total solution volume of 0.2mL will be intraperitoneally injected into the mice of the low-, medium- and high-dose EPO groups, respectively. The injection process should be performed in the absence of hard light. All mice will be injected with EPO solution or normal saline once a day for 4 weeks.
4. Observe the general state of all mice every day and record the number and the cause of death.
5. All mice will be euthanized after 4 weeks of treatment. The aortic tissues will be extracted for further investigation.
6. The external diameter of the normal and dilated segments of the abdominal aorta will be measured with a Vernier caliper. AAA is defined as the localized dilation of the abdominal aorta with an external diameter exceeding the normal aortic diameter by 50%.

References

1. A. Daugherty, M. W. Manning, L. A. Cassis, Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice. J. Clin. Invest. 105, 1605–1612 (2000).

2. H. Lu, D. A. Howatt, A. Balakrishnan, M. J. Graham, A. E. Mullick, A. Daugherty, Hypercholesterolemia induced by a PCSK9 gain-of-function mutation augments angiotensin II-induced abdominal aortic aneurysms in C57BL/6 mice-brief report. Arterioscler. Thromb. Vasc. Biol. 36, 1753–1757 (2016).

3. B. Trachet, R. A. Fraga-Silva, P. A. Jacquet, N. Stergiopulos, P. Segers, Incidence, severity, mortality, and confounding factors for dissecting AAA detection in angiotensin II-infused mice: A meta-analysis. Cardiovasc. Res. 108, 159–170 (2015).

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