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-/- C57BL/6J mice with avertin (tribromoethanol; 200 mg/kg; intraperitoneally). 
	NOTE: 32 apoE-/- mice were randomly divided into 4 groups: SHAM, OVX, OVX/E2, and OVX/PPD group (n = 8 per group).</li>
	<li>Place the animal in prone position on a heating pad. Apply eye lubricant for eye protection during anesthesia.</li>
	<li>Maintain body temperature within 36 &#177; 0.5 &#176;C. Administer 5 mg/kg body weight of the analgesic carprofen subcutaneously to the lateral aspect of the mouse&#8217;s neck.</li>
	<li>Shave a 3 x 5 cm2 mouse area cephalic from the iliac crest.&#160;Prior to covering the animal with a 3 x 5 cm2 aperture surgical sheet, clean the shaved area thoroughly with iodine and then 70% ethanol. Use sterile instruments and gloves during the experiment.&#160;</li>
	<li>Use scissors and forceps to make an incision 1 cm lateral to the midline and 1 cm lateral to the costal ribs.</li>
	<li>Bluntly dissect the subcutaneous tissue using forceps.</li>
	<li>Use dissecting goggles (see Table of Materials) to identify the white adipose tissue in the abdominal cavity.</li>
	<li>Use microscissors and microforceps to make a 0.5-1 cm incision through the fascia until the abdominal cavity is reached. 
	NOTE: For the sham-operated group, close the wounds directly. Suture the muscle layer and skin separately using a monofilament suture.</li>
	<li>When the white adipose tissue in the abdominal cavity can be seen, grab the adipose tissue using microforceps and gently pull it out. A pink mulberry-shaped ovary wrapped by adipose tissue in the abdominal cavity can be seen.</li>
	<li>Ligate the 0.5-1 cm proximal vessel and the uterine horn using a monofilament suture. Remove the ovary using microscissors and place the remaining tissue back into the abdominal cavity. 
	NOTE: The main adverse symptoms for the OVX operation is ureteral ligation which leads to high mortality in OVX-operated mice. This can be avoided by identifying the tissues using a dissecting goggle.</li>
	<li>Close the wounds. Suture the muscle layer and skin separately using a monofilament suture.</li>
	<li>Use scissors and forceps to make another incision 1 cm lateral to the midline and 1 cm lateral to the costal ribs on the other side. Repeat the above procedure (1.5 to 1.11).</li>
	<li>Let the animal wake from anesthesia. Separately keep the mouse on the first day after surgery.</li>
	<li>Clean or replace the cage frequently during the recovery phase.</li>
	<li>Approximately 24 h after surgery, administer another 5 mg/kg body weight of the analgesic carprofen subcutaneously.</li>
</ol>
2. Peroral Administration of 17&#946;-estradiol or PPD via Hazelnut Spread
<ol>
	<li>Thoroughly dissolve 17&#946;-estradiol or PPD in sesame oil, and then mix the sesame oil with hazelnut spread (see Table of Materials). A daily portion for each 30 g mouse contains 3 &#956;g of 17&#946;-estradiol or 15 &#956;g of PPD, 4 &#956;L of sesame oil, and 60 mg of hazelnut spread. Prepare a placebo for each 30 g mouse contains 4 &#956;L of sesame oil and 60 mg of hazelnut spread. 
	NOTE: The daily administration portion of 17&#946;-estradiol or PPD was based on previous studies6,11 and preliminary experiments.</li>
	<li>One week after OVX, feed the mice with a high-cholesterol diet (1.25% cholesterol, 0% cholate) for 12 weeks. A typical experimental treatment scheme, as used in this study, is illustrated in Figure 1.</li>
	<li>5 days before the peroral administration of hazelnut spread at week 4, train the mice to eat the placebo containing approximately 30 mg hazelnut spread for 2-5 mice for 5 days. Train the mice in groups in their home cages during the first 3 days. Place the mice in separate cages on the fourth and fifth days of training and serve the daily portion to resemble the experimental situation.</li>
	<li>During the last 9 weeks, place the mice in separate cages and then serve a daily portion the hazelnut spread portion for every feeding occasion. 
	NOTE: Serve a daily portion containing 17&#946;-estradiol (0.1 mg&#183;kg-1) or PPD (0.5 mg&#183;kg-1) via hazelnut spread in OVX/E2 and OVX/PPD group respectively; serve a daily portion containing hormone-free hazelnut spread in SHAM and OVX group.</li>
</ol>
3. Determination of Intima-media Thickness and Cardiac Dysfunction Using a Microultrasound System
<ol>
	<li>Ultrasonographic biomicroscopy
	<ol>
		<li>One day before termination, examine intima-media thickness and cardiac dysfunction using a microultrasound system (see Table of Materials) as previously described21.</li>
		<li>Before examination, give each mouse a 200 mg/kg intraperitoneal injection of avertin (tribromoethanol) as anesthesia (n = 8 mice per group).</li>
		<li>Shave the neck hair of each mouse carefully. Apply warm ultrasound transmission gel liberally to ensure optimal image quality.</li>
		<li>Obtain baseline ultrasonographic images of the aortic root and ascending aorta with the 30 MHz scan head at a 12.7 mm focus and a resolution of 40 &#956;m.</li>
		<li>Use electrocardiography with a lead II configuration for monitoring.</li>
		<li>Capture right parasternal long-axis images of the ascending aorta, aortic arch, and brachiocephalic artery branch in one plane in systole (Figure 3).</li>
	</ol>
	</li>
	<li>Measurements of intima-media and maximal plaque thickness
	<ol>
		<li>Adjust the distance between the transducer and the arterial site readily to obtain clear images.</li>
		<li>Store a 10 s cine loop digitally for offline examination on an image analysis system.</li>
		<li>Choose an optimal freeze-frame ultrasonographic image manually for further measurements. Check the images in the minor curvature of the ascending aorta. If plaque in the ascending aorta can be seen, measure the maximal plaque thickness. If plaque in the ascending aorta cannot be seen, measure the maximal IMT.</li>
		<li>Measure the IMT (distance between the vascular luminal-intimal interface and the medial-adventitial interface). Measure the maximal plaque thickness (the thickest distance between the border of the vascular lumen and adventitial layer).</li>
		<li>Average data from three lesion sites (Figure 3).</li>
	</ol>
	</li>
	<li>Determination of cardiac dysfunction using echocardiography 
	NOTE: Examine cardiac function through echocardiography using a microultrasound system, as previously described22.
	<ol>
		<li>Direct an ultrasound beam toward the heart, near the papillary muscles.</li>
		<li>Achieve two-dimensional electrocardiogram-based kilohertz visualization.</li>
		<li>Perform in vivo transthoracic echocardiography of the left ventricle using a 30 MHz scan head.</li>
		<li>Measure parameters associated with cardiac function digitally from M-mode tracings.</li>
		<li>Average the data from three to five cardiac cycles (Table 1).</li>
	</ol>
	</li>
	<li>Intra- and interobserver variability
	<ol>
		<li>For validation of intraobserver variability, analyze the data by one operator on two different occasions.</li>
		<li>For evaluation of interobserver variability, analyze the data by a different operator.</li>
	</ol>
	</li>
</ol>
4. Weekly Body Weight Measurement and Plasma Total Cholesterol (TC) and Triglyceride (TG) Determination
<ol>
	<li>Weekly body weight measurement
	<ol>
		<li>Measure body weights once a week from week -1 to week 12. 
		NOTE: n = 8 mice per group.</li>
	</ol>
	</li>
	<li>Plasma preparation
	<ol>
		<li>Before collecting blood samples through intracardiac puncture, prepare syringes and tubes. Use EDTA as an anticoagulant. Add 10 &#181;L of 0.5 M EDTA to each 2 mL syringe, and add 8 &#181;L of 0.5 M EDTA to each 1.5 mL tube.</li>
		<li>At week 12, after an overnight fast, anesthetize the mice with avertin (tribromoethanol; 200 mg/kg; intraperitoneally). 
		NOTE: n = 3 mice per group.</li>
		<li>Prepare the ventral chest area with 70% ethanol.</li>
		<li>Use scissors and forceps to open the thoracic cavity and cut the ribs until the beating heart is exposed.</li>
		<li>Insert the 25 G needle into the right ventricle. Aspirate slowly until blood starts to flow into the syringe. 
		NOTE: We use disposable syringes in sterile condition with 25 G needles (see Table of Materials).</li>
		<li>Continue to aspirate with steady, even pressure. If no blood is seen, reposition the needle and repeat aspiration.</li>
		<li>Keep mice deeply anesthetized before collecting the required blood volume. Normally, up to 1 mL of blood can be collected. Euthanize the mice by cervical dislocation under this deep anesthetic condition.</li>
		<li>Pipette blood samples into 1.5 mL tubes and invert the blood thoroughly to ensure mixing EDTA into the blood. Then place blood samples on ice immediately.</li>
		<li>Centrifuge samples for 20 min at 400 x g at 4 &#176;C within 30 min of collection.</li>
		<li>Collect the supernatant carefully. Aliquot and store plasma samples at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Construct standard curves for TC or TG content measurement
	<ol>
		<li>For the TC standard curve, prepare various concentrations of cholesterol standards: 0 mmol/L, 0.52 mmol/L, 1.03 mmol/L, 2.07 mmol/L, 4.14 mmol/L, 6.20 mmol/L, 8.27 mmol/L and 10.34 mmol/L. Measure O.D. for each cholesterol standard. Set average O.D. for each cholesterol standard. As the vertical (Y) axis value, set concentration as the horizontal (X) axis value. Create a standard curve by using a statistical software.</li>
		<li>For the TG standard curve, prepare various concentrations of TG standards: 0 mmol/L, 0.45 mmol/L, 0.90 mmol/L, 1.81 mmol/L, 3.62 mmol/L, 5.42 mmol/L, 7.23 mmol/L and 9.04 mmol/L. Measure O.D. for each TG standard. Set average O.D. for each TG standard as the vertical (Y) axis value; set concentration as the horizontal (X) axis value. Create a standard curve by using a statistical software. 
		NOTE: A four-parameter Logistic curve fitting (4-pl) was used for the standard curve construction in the present study. Check the standard curve before measuring the plasma samples and ensure that r2 is greater than 0.995.</li>
	</ol>
	</li>
	<li>TC content measurement
	<ol>
		<li>Label the bottle of color reagent (25 mL) from TC assay kit as &#8220;TC Working Solution&#8221;.</li>
		<li>Vortex the refrigerated specimens briefly. Prepare dilutions: 20 &#956;L of plasma in 80 &#956;L of distilled water. Briefly vortex dilutions.</li>
		<li>Add 2.5 &#956;L of cholesterol standards (5.17 mmol/L) or 2.5 &#956;L of diluted plasma or 2.5 &#956;L of distilled water (blank) to the appropriate wells of a 96-well plate. Triplication is recommended.</li>
		<li>To all wells, add 250 &#956;L of the color reagent.</li>
		<li>Incubate at 37 &#176;C for 10 min.</li>
		<li>Turn the microplate reader on and allow a 10 min warm up.</li>
		<li>Remove the plate(s) from the incubator and read the microplate reader at 510 nm. Ensure that no bubbles or dust are present in the microtiter wells or at the bottom of the plate, respectively.</li>
		<li>Calculate the TC concentration as follows: 
		&#8203;TC cont. = cholesterol standards cont. &#215; (plasma sample O.D.-blank O.D)/(cholesterol standards O.D.-blank O.D)</li>
	</ol>
	</li>
	<li>TG content measurement
	<ol>
		<li>Label the bottle of color reagent (25 mL) from TG assay kit as &#8220;TG Working Solution&#8221;.</li>
		<li>Vortex the refrigerated specimens briefly.</li>
		<li>Add 2.5 &#956;L of TG standards (2.26 mmol/L) to or 2.5 &#956;L of diluted plasma or 2.5 &#956;L of distilled water (blank) to the appropriate wells of a 96-well plate. Triplication is recommended.</li>
		<li>To all wells, add 250 &#956;L of the color reagent.</li>
		<li>Incubate at 37 &#176;C for 10 min.</li>
		<li>Turn the microplate reader on and allow a 10 min warm up.</li>
		<li>Remove the plate(s) from the incubator and read the microplate reader at 510 nm. 
		NOTE: Ensure that are no bubbles or dust are present in the microtiter wells or on the bottom of plate.</li>
		<li>Calculate the TG concentration as follows: 
		TG cont. = TG standards cont. &#215; (plasma sample O.D.-blank O.D)/(TG standards O.D.-blank O.D)</li>
	</ol>
	</li>
</ol>
5. En Face Analysis of Aortic Atherosclerotic Lesions
<ol>
	<li>Aorta isolation and excision
	<ol>
		<li>At week 12, after an overnight fast, anesthetize the mice with avertin (tribromoethanol; 200 mg/kg; intraperitoneally). Euthanize the mice by cervical dislocation under this deep anesthetic condition. 
		NOTE: We used 3 mice per group.</li>
		<li>Prepare the ventral chest area with 70% ethanol. Use scissors and forceps to open the thoracic cavity and cut the ribs until the beating heart is exposed.</li>
		<li>Fill a 50 mL syringe with phosphate buffered saline at pH 7.4 (see Table of Materials). Insert a 25 G of needle into the left ventricle and cut the right atrium to avoid high pressure from perfusion.</li>
		<li>Perform in situ perfusion at a flow rate of 0.05-0.08 mL/min. Absorb perfusion fluid with tissues.</li>
		<li>Remove ribs and lungs in thoracic cavity with scissors and forceps. Then, open the abdominal cavity and remove the organs inside for a better view of the aorta.</li>
		<li>Remove the aorta by holding the heart with the microforceps and separating the aorta from spine dorsally with microscisssors until the iliac bifurcation. 
		NOTE: When dissecting near the renal atrial branches, cut deeply using microscissors to avoid aorta damage.</li>
		<li>Fix the heart and aorta for 48 h in 4% paraformaldehyde. Store the aortas in saline at room temperature or at 2-8 &#176;C for a few hours. 
		NOTE: This procedure will facilitate cleaning.</li>
	</ol>
	</li>
	<li>Preparation of aorta
	<ol>
		<li>Remove the heart. Carefully remove the adventitial tissues from the aortas using microforceps and microscisssors under a stereomicroscope. Use saline to keep the tissue moist during cleaning. 
		NOTE: Be careful to not tear or nick the aorta and some important branches, such as the innominate artery, left common carotid artery, and left subclavian artery.</li>
		<li>Leave 1 mm of the innominate and left common carotid arteries and cut off the entire left subclavian artery.</li>
		<li>Cut open the outer curvature through the innominate artery, then to the left common carotid artery, and then to the left subclavian artery.</li>
		<li>Cut open along the inner curvature of the ascending portion to the bottom of the abdominal portion.</li>
		<li>Pin the aorta flat onto a black plastic sheet and apply saline to keep aortas from drying.</li>
	</ol>
	</li>
	<li>Image of the intimal region of aorta
	<ol>
		<li>Take pictures of en face aortas with a stereo microscope. Include a millimeter scale ruler in the images to calibrate measurements.</li>
		<li>Include the arch and thoracic regions in the same image and the abdominal region in another. Save images as JPEG or TIFF. 
		NOTE: The arch region is from the junction of the myocardium to 3 mm distal from the left subclavian artery, the thoracic region is 3 mm distal to the left subclavian artery to the last intercostal artery, and the abdominal region is the last intercostal artery to the iliac bifurcation.</li>
	</ol>
	</li>
	<li>Quantification of atherosclerotic lesion-en face method
	<ol>
		<li>Calibration
		<ol>
			<li>Open the image with image analysis software (see Table of Materials), go to Spatial Calibration and follow the instructions.</li>
			<li>Change reference units to mm by positioning the ruler over the line.</li>
		</ol>
		</li>
		<li>Measurement
		<ol>
			<li>Set correct calibration for each image.</li>
			<li>Measure 3 mm on the ruler.</li>
			<li>Arch and thoracic region measurement: Outline the arch region from the junction of the myocardium to 3 mm distal from the left subclavian artery and the thoracic region from 3 mm distal to the left subclavian artery to the last intercostal artery. Trace lesions in the arch and thoracic region and look at the aorta through the microscope.</li>
			<li>Abdominal region measurement: Outline the abdominal region from the end of thoracic region to the iliac bifurcation. Trace lesions in the abdominal region and look at the aorta through the microscope.</li>
			<li>Calculate the lesion area relative to the inner surface of aorta.</li>
			<li>Verify quantification through a second observer who is blind to the study groups.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Mozaffarian, D., 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=Heart+Disease+and+Stroke+Statistics-2016+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association.</a> Circulation. 133 (4), e38-e360 (2016).</li><li>Packard, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+health+and+disease+in+women.">Cardiovascular health and disease in women.</a> The New England Journal of Medicine. 329 (4), 247-256 (1993).</li><li>Samaan, S. A., Crawford, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+and+cardiovascular+function+after+menopause.">Estrogen and cardiovascular function after menopause.</a> Journal of the American College of Cardiology. 26 (6), 1403-1410 (1995).</li><li>Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+in+atherosclerosis.">Inflammation in atherosclerosis.</a> Nature. 420 (6917), 868-874 (2002).</li><li>Tremollieres, F. 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=Effect+of+hormone+replacement+therapy+on+age-related+increase+in+carotid+artery+intima-media+thickness+in+postmenopausal+women.">Effect of hormone replacement therapy on age-related increase in carotid artery intima-media thickness in postmenopausal women.</a> Atherosclerosis. 153 (1), 81-88 (2000).</li><li>Bourassa, P. A., Milos, P. M., Gaynor, B. J., Breslow, J. L., Aiello, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+reduces+atherosclerotic+lesion+development+in+apolipoprotein+E-deficient+mice.">Estrogen reduces atherosclerotic lesion development in apolipoprotein E-deficient mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (3), 10022-10027 (1996).</li><li>Squadrito, 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=Genistein+supplementation+and+estrogen+replacement+therapy+improve+endothelial+dysfunction+induced+by+ovariectomy+in+rats.">Genistein supplementation and estrogen replacement therapy improve endothelial dysfunction induced by ovariectomy in rats.</a> Cardiovascular Research. 45 (2), 454-462 (2000).</li><li>Wang, D., 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=Endothelial+dysfunction+and+enhanced+contractility+in+microvessels+from+ovariectomized+rats:+roles+of+oxidative+stress+and+perivascular+adipose+tissue.">Endothelial dysfunction and enhanced contractility in microvessels from ovariectomized rats: roles of oxidative stress and perivascular adipose tissue.</a> Hypertension. 63 (5), 1063-1069 (2014).</li><li>Zhang, S. H., Reddick, R. L., Piedrahita, J. A., Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+Hypercholesterolemia+and+Arterial+Lesions+in+Mice+Lacking+Apolipoprotein+E.">Spontaneous Hypercholesterolemia and Arterial Lesions in Mice Lacking Apolipoprotein E.</a> Science. 258 (5081), 468-471 (1992).</li><li>Nakashima, Y., Plump, A. S., Raines, E. W., Breslow, J. L., Ross, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ApoE-Deficient+Mice+Develop+Lesions+of+All+Phases+of+Atherosclerosis+Throughout+the+Arterial+Tree.">ApoE-Deficient Mice Develop Lesions of All Phases of Atherosclerosis Throughout the Arterial Tree.</a> Arteriosclerosis and Thrombosis. 14 (1), 133-140 (1994).</li><li>Str&#246;m, J. O., Theodorsson, A., Ingberg, E., Isaksson, I. M., Theodorsson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovariectomy+and+17-estradiol+Replacement+in+Rats+and+Mice:+A+Visual+Demonstration.">Ovariectomy and 17-estradiol Replacement in Rats and Mice: A Visual Demonstration.</a> Journal of Visualized Experiments. 64 (e4013), 1-4 (2012).</li><li>Rossouw, J. E., 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=Risks+and+benefits+of+estrogen+plus+progestin+in+healthy+postmenopausal+women:+principal+results+From+the+Women's+Health+Initiative+randomized+controlled+trial.">Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women's Health Initiative randomized controlled trial.</a> The Journal of the American Medical Association. 288 (3), 321-333 (2002).</li><li>Kumle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Declining+breast+cancer+incidence+and+decreased+HRT+use.">Declining breast cancer incidence and decreased HRT use.</a> Lancet. 372 (9639), 608-610 (2008).</li><li>Jager, W., 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=A+randomized+comparison+of+triptorelin+and+tamoxifen+as+treatment+of+progressive+ovarian+cancer.">A randomized comparison of triptorelin and tamoxifen as treatment of progressive ovarian cancer.</a> Anticancer Research. 15 (6B), 2639-2642 (1995).</li><li>Bhathena, S. J., Velasquez, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+role+of+dietary+phytoestrogens+in+obesity+and+diabetes.">Beneficial role of dietary phytoestrogens in obesity and diabetes.</a> The American Journal of Clinical Nutrition. 76 (6), 1191-1201 (2002).</li><li>Wang, L., Qiu, X. M., Hao, Q., Li, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+effects+of+a+Chinese+herbal+medicine+in+atherosclerosis+via+estrogen+receptor+beta+mediating+nitric+oxide+production+and+NF-kappaB+suppression+in+endothelial+cells.">Anti-inflammatory effects of a Chinese herbal medicine in atherosclerosis via estrogen receptor beta mediating nitric oxide production and NF-kappaB suppression in endothelial cells.</a> Cell Death and Disease. 4 (e551), 1-13 (2013).</li><li>Xiao, J., Wang, N. L., Sun, B., Cai, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+receptor+mediates+the+effects+of+pseudoprotodiocsin+on+adipogenesis+in+3T3-L1+cells.">Estrogen receptor mediates the effects of pseudoprotodiocsin on adipogenesis in 3T3-L1 cells.</a> American Journal of Physiology-Cell Physiology. 299 (1), C128-C138 (2010).</li><li>Guivarc'h, E., 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=Predominant+Role+of+Nuclear+Versus+Membrane+Estrogen+Receptor+alpha+in+Arterial+Protection:+Implications+for+Estrogen+Receptor+alpha+Modulation+in+Cardiovascular+Prevention/Safety.">Predominant Role of Nuclear Versus Membrane Estrogen Receptor alpha in Arterial Protection: Implications for Estrogen Receptor alpha Modulation in Cardiovascular Prevention/Safety.</a> Journal of the American Heart Association. 7 (13), 1-17 (2018).</li><li>Osako, M. 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. Z., Sun, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notoginsenoside+R1,+a+unique+constituent+of+Panax+notoginseng,+blinds+proinflammatory+monocytes+to+protect+against+cardiac+hypertrophy+in+ApoE(-/-)+mice.">Notoginsenoside R1, a unique constituent of Panax notoginseng, blinds proinflammatory monocytes to protect against cardiac hypertrophy in ApoE(-/-) mice.</a> European Journal of Pharmacology. 833 (15), 441-450 (2018).</li><li>Sun, B., Xiao, J., Sun, X. B., Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notoginsenoside+R1+attenuates+cardiac+dysfunction+in+endotoxemic+mice:+an+insight+into+oestrogen+receptor+activation+and+PI3K/Akt+signalling.">Notoginsenoside R1 attenuates cardiac dysfunction in endotoxemic mice: an insight into oestrogen receptor activation and PI3K/Akt signalling.</a> British Journal of Pharmacology. 168 (7), 1758-1770 (2013).</li><li>Adams, M. R., 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=Inhibition+of+coronary+artery+atherosclerosis+by+17-beta+estradiol+in+ovariectomized+monkeys.+Lack+of+an+effect+of+added+progesterone.">Inhibition of coronary artery atherosclerosis by 17-beta estradiol in ovariectomized monkeys. 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. L., Ross, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ApoE-deficient+mice+develop+lesions+of+all+phases+of+atherosclerosis+throughout+the+arterial+tree.">ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 14 (1), 133-140 (1994).</li><li>Saleh, T. M., Cribb, A. E., Connell, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen-induced+recovery+of+autonomic+function+after+middle+cerebral+artery+occlusion+in+male+rats.">Estrogen-induced recovery of autonomic function after middle cerebral artery occlusion in male rats.</a> American Journal of Physiology- Regulatory, Integrative and Comparative Physiology. 281 (5), R1531-R1539 (2001).</li><li>Bingham, D., Macrae, I. M., Carswell, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detrimental+effects+of+17beta-oestradiol+after+permanent+middle+cerebral+artery+occlusion.">Detrimental effects of 17beta-oestradiol after permanent middle cerebral artery occlusion.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 25 (3), 414-420 (2005).</li><li>Pignoli, P., Tremoli, E., Poli, A., Oreste, P., Paoletti, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intimal+plus+medial+thickness+of+the+arterial+wall:+a+direct+measurement+with+ultrasound+imaging.">Intimal plus medial thickness of the arterial wall: a direct measurement with ultrasound imaging.</a> Circulation. 74 (6), 1399-1406 (1986).</li><li>Hodgin, J. B., Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+estrogen+and+mouse+models+of+atherosclerosis.">Minireview: estrogen and mouse models of atherosclerosis.</a> Endocrinology. 143 (12), 4495-4501 (2002).</li><li>Bush, T. L., 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=Cardiovascular+mortality+and+noncontraceptive+use+of+estrogen+in+women:+results+from+the+Lipid+Research+Clinics+Program+Follow-up+Study.">Cardiovascular mortality and noncontraceptive use of estrogen in women: results from the Lipid Research Clinics Program Follow-up Study.</a> Circulation. 75 (6), 1102-1109 (1987).</li><li>Marsh, M. M., Walker, V. R., Curtiss, L. K., Banka, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+against+atherosclerosis+by+estrogen+is+independent+of+plasma+cholesterol+levels+in+LDL+receptor-deficient+mice.">Protection against atherosclerosis by estrogen is independent of plasma cholesterol levels in LDL receptor-deficient mice.</a> Journal of Lipid Research. 40 (5), 893-900 (1999).</li></ol>

The authors declare no conflicts of interest.

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&amp;beta;-estradiol, &gt;98%</td><td>Sigma-Aldrich</td><td>E8875-250MG</td><td>Estrogen</td></tr><tr><td>Disposable syringes (with 25 G 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&amp;reg;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 8 mm</td><td>Kanghua Medical Equipment Co., Ltd</td><td></td><td>Surgical tools</td></tr><tr><td>Micro Dissecting Scissors, Curved/Sharp 8 mm</td><td>Kanghua Medical Equipment Co., Ltd</td><td></td><td>Surgical tools</td></tr><tr><td>Micro Dissecting Scissors, Straight/Sharp 8 mm</td><td>Kanghua Medical Equipment Co., Ltd</td><td></td><td>Surgical tools</td></tr><tr><td>Monofilament suture 4-0 1/2 5 x 12 19 mm</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&amp;beta;-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 &amp;amp; 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&amp;beta;-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 (14 cm)</td><td>Kanghua Medical Equipment Co., Ltd</td><td></td><td>Surgical tools</td></tr><tr><td>Scissors, straight Flat (14 cm)</td><td>Kanghua Medical Equipment Co., Ltd</td><td></td><td>Surgical tools</td></tr><tr><td>Tissue forceps, serrated, slight Curve (14 cm)</td><td>Kanghua Medical Equipment Co., Ltd</td><td></td><td>Surgical tools</td></tr><tr><td>Tissue forceps, serrated, straight Flat (14 cm)</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...

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

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 ...

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Universidad San Sebastian

Research

JoVE Journal

Biochemistry

12.1K Views.  Chinese Academy of Medical Sciences & Peking Union Medical College. 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.

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

Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is being used to investigate the health-protective effects of young ovaries in young females. The current procedures employ an orthotopic transplantation method, where allogenic ovaries are transplanted back to their original position in the ovarian bursa. This is in contrast to the more commonly used heterotopic transplantation of ovaries/ovarian tissue subcutaneously or under the kidney capsule. All three locations provide efficient revascularization of the transplanted tissues. However, orthotopic transplantation provides the ovary with the most natural signaling environment and is the only procedure that provides the opportunity for the animal to reproduce naturally post-operatively. One must take care to remove all endogenous ovarian tissue during the ovariectomy procedure. If any endogenous tissue remains or if only one ovary is removed, the transplanted tissue will remain dormant until the existing tissue becomes senescent. While revascularization of the transplanted ovaries occurs very quickly, the transplant recipient can take a considerable amount of time to adapt to a new hypothalamic/pituitary/gonadal/adrenal (HPG/A) axis signaling regime associated with the transplanted tissue. This normally takes about 100 days in the mouse. Therefore, transplantation experiments should be designed to accommodate this adaptation period. Typical results with ovarian transplantation will include changes in the health of the recipient that reflect the age of the transplanted ovary, rather than the chronological age of the recipient.

Described here are orthotopic ovarian transplantation protocols for modifying ovarian influence on the physiology of the recipient mouse. This surgical procedure is used to expose female mice to various types of ovarian influence and may include heterochronic transplantations and transplantation of ovaries that have undergone various ex vivo treatments.

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is ...

Developmental Biology

A Method to Study the Impact of Chemically-induced Ovarian Failure on Exercise Capacity and Cardiac Adaptation in Mice

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in post-menopausal women has not been adequately studied. Accordingly, we investigated the impact of voluntary cage-wheel exercise and forced treadmill exercise on cardiac adaptation in menopausal mice. The most commonly used inducible model for mimicking menopause in women is the ovariectomized (OVX) rodent. However, the OVX model has a few dissimilarities from menopause in humans. In this study, we administered 4-vinylcyclohexene diepoxide (VCD) to female mice, which accelerates ovarian failure as an alternative menopause model to study the impact of exercise in menopausal mice. VCD selectively accelerates the loss of primary and primordial follicles resulting in an endocrine state that closely mimics the natural progression from pre- to peri- to post-menopause in humans. To determine the impact of exercise on exercise capacity and cardiac adaptation in VCD-treated female mice, two methods were used. First, we exposed a group of VCD-treated and untreated mice to a voluntary cage wheel. Second, we used forced treadmill exercise to determine exercise capacity in a separate group VCD-treated and untreated mice measured as a tolerance to exercise intensity and endurance.

Two exercise paradigms were tested on a newly developed chemically induced menopausal mouse model to examine the impact of menopause on exercise capacity and cardiac adaption to exercise.

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in ...

Medicine

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a lean PCOS-like mouse model that mimics women with PCOS is clinically meaningful. In this protocol, we describe such a model. By inserting a 4 mm length of DHT (dihydrotestosterone) crystal powder pellet (total length of pellet is 8 mm), and replacing it monthly, we are able to produce a PCOS-like mouse model with serum DHT levels 2 fold higher than mice not implanted with DHT (no-DHT). We observed reproductive and metabolic dysfunction without changing body weight and body composition. While exhibiting a high degree of infertility, a small subset of these PCOS-like female mice can get pregnant and their offspring show delayed puberty and increased testosterone as adults. This PCOS-like lean mouse model is a useful tool to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

We describe the development of a lean PCOS-like mouse model with&#160;dihydrotestosterone pellet to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a ...

Using 16&#945;-[18F]-Fluoro-17&#946;-Estradiol PET to Visualize Estrogen Receptor &#945; Expression in Human Breast Cancer Xenografts in Female Ovariectomized Mice

To demonstrate how estrogen receptor alpha (ER&#945;) positive breast cancer xenografts may be visualized in BALB/c nude mice using 16&#945;-[18F]-fluoro-17&#946;-estradiol (18F-FES) positron emission tomography (PET), ovariectomized BALB/c nude mice were injected with ER&#945;-positive breast cancer cells (MCF-7, 3 &#215; 106 cells; shoulder [n = 10] or 4th inguinal mammary fat pad [n = 10]) or ER&#945;-negative breast cancer cells (MDA-MB-231, 1 &#215; 106 cells; mammary fat pad [n = 5]). Mice harboring MCF-7 cells received subcutaneous injections of 20 &#181;g of 17&#946;-estradiol (20 &#181;g/20 &#181;L; corn oil:ethanol, 9:1) in the nape of their necks 2 days prior to cell injection, followed by daily injections five times per week for 5 weeks. Tumor volumes were measured according to the formula: (L*W2)/2 (L; length, W; width). Once tumor volumes reached approximately 100 mm3, 17&#946;-estradiol injections were halted 2 days prior to mice receiving 18F-FES for PET imaging to avoid competitive binding with ER&#945;. Upon 18F-FES administration via the lateral tail vein, PET/MRI was performed for 15 min at 1 h to 1.5 h post-injection. 18F-FES uptake was not observed in ER&#945;-negative, MDA-MB-231 tumor-bearing mice. 18F-FES uptake was most pronounced in mice harboring MCF-7 tumors in the shoulder. In MCF-7 tumors grown in the inguinal mammary fat pad, 18F-FES uptake was less visible, as the intestinal excretion pattern of 18F-FES obscured the radioactivity detectable in these tumors. To use 18F-FES PET as a tool to visualize ER&#945; expression in ER&#945;-positive breast xenografts, we demonstrate that the visibility of 18F-FES uptake is clear in tumors located away from the abdominal region of mice, such as in the shoulder.

Here, we demonstrate a protocol to use 16&#945;-[18F]-fluoro-17&#946;-estradiol (18F-FES) positron emission tomography (PET) as a tool to visualize ER&#945; expression in ER&#945;-positive breast xenografts.

To demonstrate how estrogen receptor alpha (ER&#945;) positive breast cancer xenografts may be visualized in BALB/c nude mice using ...

Cancer Research

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17&beta;-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17&beta;-estradiol level/administration method profoundly influences the experimental results1-3. We have in a series of studies characterized the different modes of 17&beta;-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17&beta;-estradiol4-6. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration7, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17&beta;-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.

Ovariectomy and 17&#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Ovariectomy and subsequent replacement of 17&beta;-estradiol by means of silastic capsules and peroral Nutella are demonstrated in rats and mice.

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

Biology

A Syngeneic Murine Model of Endometriosis using Naturally Cycling Mice

Endometriosis is a leading cause of pelvic pain and infertility. It is defined by the presence of endometrial tissue in extrauterine locations. The development of novel therapies and diagnostic tools for endometriosis has been limited due in part to challenges in studying the disease. Outside of primates, few mammals menstruate, and none develop spontaneous endometriosis. Rodent models are popular but require artificial induction of endometriosis, with many utilizing either immunocompromised mice or surgically induced disease. Recently, more attention has been given to models involving intraperitoneal injection. We present a murine model of endometriosis that integrates several features of existing endometriosis models into a novel, simplified system that relies on microscopic quantification in lieu of subjective grading. In this model, we perform hormonal stimulation of donor mice, intraperitoneal injection, systematic abdominal survey and tissue harvest, and histologic quantification that can be performed and verified at any time after necropsy. This model requires minimal resources and training; does not require expertise by lab technicians in murine survival surgery or in the identification of gross endometriotic lesions; can be used in immunocompromised, immunocompetent, and/or mutant mice; and reliably creates endometriotic lesions that are histologically consistent with human endometriotic disease.

Many rodent models of endometriosis are limited by technical complexity, reproducibility, and/or need for immunocompromised animals or special reporter mice. We present a simplified system of lesion induction using any experimental mouse with an independently verifiable, objective scoring system and with no requirement for ovariectomy or survival surgery.

Endometriosis is a leading cause of pelvic pain and infertility. It is defined by the presence of endometrial tissue in extrauterine locations. The ...

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.
Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real time

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time ...

Murine Orchiectomy and Ovariectomy to Reduce Sex Hormone Production

Sex hormone signaling plays a critical role in multiple organ systems as well as in the progression of various diseases, including neurodegenerative disease. The manipulation of sex hormone levels in the murine model system allows for the study of their impact on organs/tissues and within disease progression. Orchiectomy - the surgical removal of the testes - and ovariectomy - the surgical removal of the ovaries - provide a method to deplete the endogenous sex hormones so that the precise hormone levels can be provided through drug or other delivery methods. Here, we provide rapid and minimally invasive methods for both orchiectomy and ovariectomy in the murine model system for the reduction of sex hormones. This protocol details the surgical preparation and excision of the testes through the scrotal sac, and excision of the ovaries via two incisions in the right and left lateral dorsum.

This manuscript describes a consistent way to quickly perform survival rodent orchiectomies and ovariectomies.

Sex hormone signaling plays a critical role in multiple organ systems as well as in the progression of various diseases, including neurodegenerative ...

Developing an Ovariectomized Mouse Model with Elevated FSH Level

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological changes, including decreased bone mass, increased blood lipids, and increased visceral adiposity. Levels of follicle-stimulating hormone (FSH) rise during the menopausal transition. Many studies have shown that FSH in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important. In this study, C57BL/6 female mice were ovariectomized and supplemented with estradiol valerate (OVX + E2) to eliminate the effect of the hypothalamic-pituitary-gonadal axis. The OVX + E2 mice received solvent (N.S.) or different doses of recombinant FSH via intraperitoneal injection to create a mouse model (OVF) characterized by relatively stable estrogen and rising FSH levels. Thus, we successfully generated an experimental mouse model to mimic the early stage of menopause transition, characterized by elevated serum FSH levels. The OVF model has the advantages of being stable, low cost, and easy to operate, which is suitable for studies to explore the extragonadal actions of FSH. Here, we describe detailed protocols for the mouse OVF model.

Author Spotlight: Investigating the Relationship Between FSH and Pathophysiological Changes in Perimenopausal Women - Insights from a Mouse Model 

Follicle-stimulating hormone (FSH) in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. The ovariectomized and FSH-treated mouse model (OVF) can be used to explore the extragonadal actions of FSH.

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological ...

Optimized Thin Endometrium Mouse Model with Low Mortality and Minimal Adhesions

Establishing a Mouse Model of Thin Endometrium

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory treatment options are still urgently needed. Several animal models of TE have been developed, but the mouse model involving abdominal surgery and injection of 95% ethanol presents a formidable challenge due to the high mortality rate and risk of intrauterine adhesions if not performed correctly. Here, we describe a detailed protocol that produces reliable and reproducible TE with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus with varying infusion times. The results showed that all of the mice successfully developed TE with infusion times ranging from 1-3 min, characterized by a typical reduction in endometrial thickness and the number of glands, as well as excessive endometrial fibrosis. These findings suggest that this mouse model is suitable for studying thin endometrium and can serve as a platform for developing future TE treatments.

This article describes a detailed protocol for producing a reliable and reproducible thin endometrium with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus within 1-3 min.

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory ...

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

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