An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause

Summary

Clinically, estrogen deficiency in menopausal women may aggravate the incidence of lipid disruption and atherosclerosis. We established an in vivo estrogen deficiency model by bilateral ovariectomy via a double dorsal-lateral incision in apoE^-/- mice. The mouse model is applicable for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause.

Abstract

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning display increased atherosclerotic lesions in the aorta compared with female mice with intact ovarian function. However, laboratory models involving estrogen-deficient mice with atherosclerosis-prone status are lacking. This deficit is crucial because clinical estrogen deficiency in menopausal women may aggravate the incidence of pre-existing or ongoing lipid disruption and atherosclerosis. In this study, we establish an in vivo estrogen-deficient mouse model by bilateral ovariectomy via a double dorsal-lateral incision in apolipoprotein E (apoE)^-/- mice. We then compare the effects of 17β-estradiol and pseudoprotodioscin (PPD) (a phytoestrogen) perorally administered via hazelnut spread. We find that although PPD exerts some effect on reducing final body weight and plasma TG in OVX apoE^-/- mice, it has anti-atherosclerotic and cardiac-protective capacities comparable with its 17β-estradiol counterpart. PPD is a phytoestrogen that has been reported to exert anti-tumor properties. Thus, the proposed method is applicable for screening phytoestrogens via peroral administration to substitute for traditional hormone replacement therapy in postmenopausal women, which has been reported to have potentially deleterious tumorigenetic capacity. Peroral administration via hazelnut spread is noninvasive, rendering it widely applicable to many patients. This article contains step-by-step demonstrations of bilateral ovariectomy via the double dorsal-lateral incision in apoE^-/- mice and peroral 17β-estradiol or phytoestrogen hormone replacement via hazelnut spread. Plasma lipid and cardiovascular function analyses using echocardiography follow.

Introduction

Epidemiological and clinical studies have shown that postmenopausal women are at considerably greater risk of cardiovascular disease than premenopausal women^1,2. Hormone replacement therapy (HRT) may reduce the relative risk of cardiovascular disease to 0.37-0.79³. Among other complications, atherosclerosis caused by cardiovascular diseases is the leading cause of death worldwide⁴. However, laboratory models involving estrogen-deficient mice presenting atherosclerosis prone status are lacking. This protocol provides an in vivo estrogen deficiency mouse model for ....

Protocol

All animal care and experimental protocols were approved by the Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College (Permission No.: SYXK (Beijing) 2013-0023). The origin of apoE^-/- mice is C57BL/6J^9,10.

1. Bilateral Ovariectomy via a Double Dorsal-lateral Incision in apoE^-/- Mice

1. At weaning (age 28 days), anesthetize female apoE^-.......

Discussion

The methodology described here is a mouse model resembling lipid disruption and atherosclerosis seen in menopausal women. It is well-documented that estrogen deficiency in postmenopausal women can aggravate the incidence of pre-existing or ongoing hypercholesterolemia with progressively complex and widespread atherosclerostic lesions¹. To mimic the atherosclerosis-prone status in clinic, apoE-deficient mice, a reproducible and convenient source of animals with which to study atherogenesis

Materials

Name	Company	Catalog Number	Comments
17β-estradiol, >98%	Sigma-Aldrich	E8875-250MG	Estrogen
Disposable syringes (with 25G needles)	Hunan Luzhou Huikang Development Co., Ltd	0.5*19TWLB	Cardiac bleeding
High-cholesterol mouse diet	Huafukang Bio-Technology	N/A	1.25% cholesterol, 0% cholate
High-Resolution In Vivo Micro-Imaging System	VisualSonics	Vevo®770	Measurements of intima-media thickness and cardiac dysfunction
2-Methyl-2-butanol	Sigma-Aldrich	152463-250ML	Preparation of avertin
Micro Dissecting forceps, Curved 8mm	Kanghua Medical Equipment Co., Ltd		Surgical tools
Micro Dissecting forceps, Straight 8mm	Kanghua Medical Equipment Co., Ltd		Surgical tools
Micro Dissecting Scissors, Curved/Sharp 8mm	Kanghua Medical Equipment Co., Ltd		Surgical tools
Micro Dissecting Scissors, Straight/Sharp 8mm	Kanghua Medical Equipment Co., Ltd		Surgical tools
Monofilament suture 4-0 1/2 5*12 19mm	Shanghai Pudong Jinhuan Medical Supplies Co., Ltd	R413	Suture and ligation of the tissues
Nut cream (Nutella)	Ferrero	N/A	Medium for peroral 17β-estradiol or PPD
OptiVisor optical glass binocular magnifier	Dohegan Optical Company Inc.	N/A	Assistant of identifying the tissues during ovariectomy
Phosphate-buffered saline at pH 7.4	SIGMA	P3813	Preparing 1 L saline
Pro MultiLabel Microplate Reader	Tecan	Infinite M1000	Plasma TC and TG determination
Pseudoprotodioscin	Shanghai Winherb Medical S & T Development	W-0427	CAS registry no. 102115-79-7
Rimadyl, 50 mg/mL	Pfizer Pharma GmbH	462986	Postoperative analgesia after ovariectomy
Sesame oil	Sigma-Aldrich	S3547-1L	Dissolving the 17β-estradiol or PPD
Solcoseryl Eye-Gel	Menarini, Solco Basle Ltd.		Eye protection during anesthesia
Stereo microscope	MCALON	MCL-6STV	Image of the intimal region of aorta
Table model high speed centrifuge	SIGMA	1-14K	Preparation of plasma
Scissors, slight Curve (14cm)	Kanghua Medical Equipment Co., Ltd		Surgical tools
Scissors, straight Flat (14cm)	Kanghua Medical Equipment Co., Ltd		Surgical tools
Tissue forceps, serrated, slight Curve (14cm)	Kanghua Medical Equipment Co., Ltd		Surgical tools
Tissue forceps, serrated, straight Flat (14cm)	Kanghua Medical Equipment Co., Ltd		Surgical tools
Tribromoethanol	Sigma-Aldrich	T48402-5G	Preparation of avertin
Triglycerides (TG) assay kit	Institute of Nanjing Jiancheng Biology Engineering	A110-1	Plasma TG determination
Total cholesterols (TC) assay kit	Institute of Nanjing Jiancheng Biology Engineering	A111-1	Plasma TC determination