Name: an in vivo estrogen deficiency mouse model for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause
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Description: Watch this Scientific Journal Video about An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause at JoVE.com

This work was supported by the National Natural Science Foundation of China (81202526 to J.X.), the National Natural Science Foundation of China (81302769 to B.S.), the Beijing Municipal Natural Science Foundation (47144226 to B.S.), the Chinese Postdoctoral Science Foundation (20110490325 to J.X.), and the Ph.D. Programs Foundation of Ministry of Education of China (20121106120031 to B.S.).

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/6J9,10.
1. Bilateral Ovariectomy via a Double Dorsal-lateral Incision in apoE-/- Mice
<ol>
	<li>At weaning (age 28 days), anesthetize female apoE-...

<ol>
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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+inhibits+vascular+calcification+via+vascular+RANKL+system:+common+mechanism+of+osteoporosis+and+vascular+calcification.">Estrogen inhibits vascular calcification via vascular RANKL system: common mechanism of osteoporosis and vascular calcification.</a> Circulation Research. 107 (4), 466-475 (2010).</li><li>Ivanova, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+of+some+saponins+and+phenolic+components+of+Tribulus+terrestris+and+Smilax+excelsa+as+MDR+modulators.">Screening of some saponins and phenolic components of Tribulus terrestris and Smilax excelsa as MDR modulators.</a> In vivo. 23 (4), 545-550 (2009).</li><li>Xiao, J., Zhu, T., Yin, Y. 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Lack of an effect of added progesterone.</a> Arteriosclerosis. 10 (6), 1051-1057 (1990).</li><li>Keaney, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=17+beta-estradiol+preserves+endothelial+vasodilator+function+and+limits+low-density+lipoprotein+oxidation+in+hypercholesterolemic+swine.">17 beta-estradiol preserves endothelial vasodilator function and limits low-density lipoprotein oxidation in hypercholesterolemic swine.</a> Circulation. 89 (5), 2251-2259 (1994).</li><li>Nakashima, Y., Plump, A. S., Raines, E. W., Breslow, J. 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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<sup clas...

Epidemiological and clinical studies have shown that postmenopausal women are at considerably greater risk of cardiovascular disease than premenopausal women1,2. 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 screening exogenous estrogen treatments of cardiovascular dysfunction after menopause.
Previous studies show that the application of OVX in atherosclerotic rodents fed a high-cholesterol diet can mimic postmenopausal women suffering from atherosclerosis5,6,7,8. A reproducible and convenient animal model resembling the atherosclerotic state in menopausal women is the basis of exogenous estrogen research. Here, a double dorsal-lateral incision of bilateral ovariectomy was applied in atherosclerosis-prone apolipoprotein E knockout (apoE-/-) mice9,10. Compared with middle abdominal or dorsal incision, double dorsal-lateral incision is an easier, less time-consuming method that can avoid severe abdominal cavity adhesion and inflammation. Peroral administration via hazelnut spread (see Table of Materials) is noninvasive and convenient, rendering it widely applicable as a long-term administration mode11. Slow-release pellet implantation is also popular6. However, implants mayaggravate the incidence of infection especially in mice subjected to OVX. Other noninvasive administration modes, such as oral gavage and water administration, also have many drawbacks. Oral gavage typically stress mice and may cause esophageal injury. Administering the hormone via drinking water is highly beneficial; however, the adding of DMSO as an emulsifier is inevitable as exogenous estrogens are insoluble in water. Here, we chose peroral 17&#946;-estradiol or phytoestrogen hormone replacement via hazelnut spread for long-term administration.
Recently, the beneficial effect of HRT on the cardiovascular system of postmenopausal women has been contested in women&#39;s health initiative (WHI) trials12. On the one hand, exogenous estrogen alone exerts a beneficial effect on the cardiovascular system; on the other hand, it can combine with metohydroxyprogesterone acetate to increase the risk of cardiovascular events. More seriously, HRT may lead to breast and uterine tumor progression, and this effect has markedly limited its use13,14. More interest has been focused on the cardiovascular-protective effects of exogenous estrogens lacking mitotic activity in tumor cells15,16,17. Multiple studies in humans and animals suggest that phytoestrogens with structures similar to that of estrogens can play a beneficial role in cardiovascular protection15,18.
Thus, the aims of the present work are (i) to build an in vivo estrogen deficiency mouse model by bilateral ovariectomy via a double dorsal-lateral incision in apoE-/- mice and (ii) to compare the cardiovascular protective effects of perorally administered 17&#946;-estradiol and pseudoprotodioscin (PPD), via hazelnut spread. 17&#946;-estradiol is one kind of exogenous estrogen that belongs to female sexual hormones6,11,19. PPD, a steroid saponin and phytoestrogen from Dioscorea plants, has been previously reported to exert anti-tumor properties20.

<table>
	<tbody>
		<tr>
			<td>17&#946;-estradiol, &#62;98%</td>
			<td>Sigma-Aldrich</td>
			<td>E8875-250MG</td>
			<td>Estrogen</td>
		</tr>
		<tr>
			<td>Disposable syringes (with 25G needles)</td>
			<td>Hunan Luzhou Huikang Development Co., Ltd</td>
			<td>0.5*19TWLB</td>
			<td>Cardiac bleeding</td>
		</tr>
		<tr>
			<td>High-cholesterol mouse diet</td>
			<td>Huafukang Bio-Technology</td>
			<td>N/A</td>
			<td>1.25% cholesterol, 0% cholate</td>
		</tr>
		<tr>
			<td>High-Resolution In Vivo Micro-Imaging System</td>
			<td>VisualSonics</td>
			<td>Vevo&#174;770</td>
			<td>Measurements of intima-media thickness and cardiac dysfunction</td>
		</tr>
		<tr>
			<td>2-Methyl-2-butanol</td>
			<td>Sigma-Aldrich</td>
			<td>152463-250ML</td>
			<td>Preparation of avertin</td>
		</tr>
		<tr>
			<td>Micro Dissecting forceps, Curved 8mm</td>
			<td>Kanghua Medical Equipment Co., Ltd</td>
			<td></td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>Micro Dissecting forceps, Straight 8mm</td>
			<td>Kanghua Medical Equipment Co., Ltd</td>
			<td></td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>Micro Dissecting Scissors, Curved/Sharp 8mm</td>
			<td>Kanghua Medical Equipment Co., Ltd</td>
			<td></td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>Micro Dissecting Scissors, Straight/Sharp 8mm</td>
			<td>Kanghua Medical Equipment Co., Ltd</td>
			<td></td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>Monofilament suture 4-0 1/2 5*12 19mm</td>
			<td>Shanghai Pudong Jinhuan Medical Supplies Co., Ltd</td>
			<td>R413</td>
			<td>Suture and ligation of the tissues</td>
		</tr>
		<tr>
			<td>Nut cream (Nutella)</td>
			<td>Ferrero</td>
			<td>N/A</td>
			<td>Medium for peroral 17&#946;-estradiol or PPD</td>
		</tr>
		<tr>
			<td>OptiVisor optical glass binocular magnifier</td>
			<td>Dohegan Optical Company Inc.</td>
			<td>N/A</td>
			<td>Assistant of identifying the tissues during ovariectomy</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline at pH 7.4</td>
			<td>SIGMA</td>
			<td>P3813</td>
			<td>Preparing 1 L saline</td>
		</tr>
		<tr>
			<td>Pro MultiLabel Microplate Reader</td>
			<td>Tecan</td>
			<td>Infinite M1000</td>
			<td>Plasma TC and TG determination</td>
		</tr>
		<tr>
			<td>Pseudoprotodioscin</td>
			<td>Shanghai Winherb Medical S &#38; T Development</td>
			<td>W-0427</td>
			<td>CAS registry no. 102115-79-7</td>
		</tr>
		<tr>
			<td>Rimadyl, 50 mg/mL</td>
			<td>Pfizer Pharma GmbH</td>
			<td>462986</td>
			<td>Postoperative analgesia after ovariectomy</td>
		</tr>
		<tr>
			<td>Sesame oil</td>
			<td>Sigma-Aldrich</td>
			<td>S3547-1L</td>
			<td>Dissolving the 17&#946;-estradiol or PPD</td>
		</tr>
		<tr>
			<td>Solcoseryl Eye-Gel</td>
			<td>Menarini, Solco Basle Ltd.</td>
			<td></td>
			<td>Eye protection during anesthesia</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>MCALON</td>
			<td>MCL-6STV</td>
			<td>Image of the intimal region of aorta</td>
		</tr>
		<tr>
			<td>Table model high speed centrifuge</td>
			<td>SIGMA</td>
			<td>1-14K</td>
			<td>Preparation of plasma</td>
		</tr>
		<tr>
			<td>Scissors, slight Curve (14cm)</td>
			<td>Kanghua Medical Equipment Co., Ltd</td>
			<td></td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>Scissors, straight Flat (14cm)</td>
			<td>Kanghua Medical Equipment Co., Ltd</td>
			<td></td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>Tissue forceps, serrated, slight Curve (14cm)</td>
			<td>Kanghua Medical Equipment Co., Ltd</td>
			<td></td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>Tissue forceps, serrated, straight Flat (14cm)</td>
			<td>Kanghua Medical Equipment Co., Ltd</td>
			<td></td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>Tribromoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>T48402-5G</td>
			<td>Preparation of avertin</td>
		</tr>
		<tr>
			<td>Triglycerides (TG) assay kit</td>
			<td>Institute of Nanjing Jiancheng Biology Engineering</td>
			<td>A110-1</td>
			<td>Plasma TG determination</td>
		</tr>
		<tr>
			<td>Total cholesterols (TC) assay kit</td>
			<td>Institute of Nanjing Jiancheng Biology Engineering</td>
			<td>A111-1</td>
			<td>Plasma TC determination</td>
		</tr>
	</tbody>
</table>

an in vivo estrogen deficiency mouse model for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause

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 17&#946;-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 17&#946;-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 17&#946;-estradiol or phytoestrogen hormone replacement via hazelnut spread. Plasma lipid and cardiovascular function analyses using echocardiography follow.

A typical experimental treatment scheme, as used in this study, is illustrated in Figure 1. At weaning (age 28 days), female apoE-/- C57BL/6J mice were anesthetized withavertin (tribromoethanol; 200 mg/kg; intraperitoneally). Mice were bilaterally OVX or sham operated through a 1 cm dorsal incision. One week after bilateral OVX, the mice were fed a high-cholesterol diet (1.25% cholesterol, 0% cholate) for 12 weeks. 17&#946;-Estradiol (0.1 mg&#183;k...

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

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.

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning ...

Laboratory models involving estrogen deficient mice presenting an arteriosclerosis pons datas are lacking. This procedure is especially helpful for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause. The main advantage of this technique is that ovariectomy without double dorsolateral incision is a easier, less time-consuming method that avoids severe abdominal cavity adhesion and inflammation.Demonstrating the procedure will be Yue-Zhang Yin, a graduate student from my laboratory. To perform a bilateral ovariectomy in APOE knockout mice, first confirm a lack of response to the pedal reflex and place the animal in the prone position on a heating pad. Apply ointment to the animal's eyes and cover the animal with a surgical drape.Make a one-centimeter incision lateral to the midline, and one centimeter lateral to the costal ribs, and use forceps to bluntly dissect the subcutaneous tissue. Using dissecting goggles, identify the white adipose tissue in the abdominal cavity. Using micro scissors and micro forceps, make a 0.5-to one centimeter incision through the fascia, until the abdominal cavity is reached, and use the micro forceps to gently retract the white adipose tissue.A pink, mulberry-shaped ovary, wrapped in adipose tissue, should be observed within the abdominal cavity. Use a monofilament suture to ligate the 0.5-to one-centimeter proximal vessel and uterine horn, and use the micro scissors to harvest the ovary. Return the remaining tissue to the abdominal cavity, and use individual monofilament sutures to separately close the muscle and skin layers.Then harvest the ovary on the contralateral side of the animal, as just demonstrated, and allow the mouse to recover on a heating pad with monitoring, until full recumbency. Once a week, from the week before to 12 weeks after the surgery, measure the body weight of each sham and experimental animal on a balance. For plasma total cholesterol and triglyceride measurement, at week 12, prepare the ventral chest area with 70%ethanol, and insert a 25-gauge needle into the right ventricle.Aspirate slowly, until one milliliter of blood flows into the syringe, and dispense the sample into a 1.5 milliliter collection tube containing ten microliters of 0.5 molar EDTA. Invert the tube several times thoroughly to mix the EDTA into the blood, and immediately place the tube on ice. Within 30 minutes of collection, centrifuge the sample for 20 minutes at 400 times G and four degrees Celsius.Carefully collect the plasma supernatant for storage at minus 80 degrees Celsius. To isolate the aorta, at week 12, attach a 25-gauge needle to a 50 milliliter syringe filled with PBS, and insert the needle into the left ventricle of the experimental animal. Cut the right atrium to avoid high pressure from the perfusion, and perfuse the cardiac tissue at a 05 to 08 milliliters of PBS per minute rate of flow.Absorb the perfusion fluid with lab tissues and use scissors and forceps to remove the ribs and lungs. Open the abdominal cavity to remove the internal organs, for a better view of the aorta. Grasp the heart with micro forceps to allow separation of the aorta dorsally, from the spine, until the iliac bifurcation.Then fix the heart and aorta for 48 hours in 4%paraformaldehyde, before storing the tissue sample in saline at room temperature or at two to eight degrees Celsius for a few hours. After twelve weeks of a high-cholesterol diet, ovariectomized mice demonstrate a remarkable increase in plasma total cholesterol and triglyceride concentrations. Ovariectomized mice, per orally administered with 17-beta oestradiol, or PPD via hazelnut spread, exhibit significantly lower plasma total cholesterol concentrations than sham-operated mice.Plasma triglyceride levels decrease in ovariectomized mice, per orally administered with 17-beta oestradiol, but not with PPD. While a tendency toward an increased body weight is observed in ovariectomized mice, compared to sham-operated mice, no significant changes in body weight are observed overall, between the four groups of animals. The maximal plaque of the ascending aorta is increased in ovariectomized mice, compared to all other groups, in conjunction with an overall decrease in cardiac function, observed in these animals.Further, the average percentage of aortic lesion area, relative to the entire aortic area, is significantly increased in ovariectomized mice. As expected, the presence of these lesions is decreased with 17-beta oestradiol, or PPD treatment. The main risk for the ovariectomy operation is ureteral ligation, which results in a high mortality.Also remember to cut deeply near the renal artery branches, to avoid aortic damage.

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